Articular chondrocyte metabolism and osteoarthritis

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Leipold, H.R.

1989-01-01

The three main objectives of this study were: (1) to determine if depletion of proteoglycans from the cartilage matrix that occurs during osteoarthritis causes a measurable increase of cartilage proteoglycan components in the synovial fluid and sera, (2) to observe what effect intracellular cAMP has on the expression of matrix components by chondrocytes, and (3) to determine if freshly isolated chondrocytes contain detectable levels of mRNA for fibronectin. Canine serum keratan sulfate and hyaluronate were measured to determine if there was an elevation of these serum glycosaminoglycans in a canine model of osteoarthritis. A single intra-articular injection of chymopapain into a shoulder joint increased serum keratan sulfate 10 fold and hyaluronate less than 2 fold in 24 hours. Keratan sulfate concentrations in synovial fluids of dogs about one year old were unrelated to the presence of spontaneous cartilage degeneration in the joints. High keratan sulfate in synovial fluids correlated with higher keratan sulfate in serum. The mean keratan sulfate concentration in sera of older dogs with osteoarthritis was 37% higher than disease-free controls, but the difference between the groups was not statistically significant. Treatment of chondrocytes with 0.5 millimolar (mM) dibutyryl cAMP (DBcAMP) caused the cells to adopt a more rounded morphology. There was no difference between the amount of proteins synthesized by cultures treated with DBcAMP and controls. The amount of fibronectin (FN) in the media of DBcAMP treated cultures detected by an ELISA was specifically reduced, and the amount of {sup 35}S-FN purified by gelatin affinity chromatography decreased. Moreover, the percentage of FN containing the extra domain. A sequence was reduced. Concomitant with the decrease in FN there was an increase in the concentration of keratan sulfate.

Chondrogenic differentiation of human articular chondrocytes differs in biodegradable PGA/PLA scaffolds

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Zwingmann, Joern; Mehlhorn, Alexander T; Südkamp, Norbert

2007-01-01

Cartilage tissue engineering is applied clinically to cover and regenerate articular cartilage defects. Two bioresorbable nonwoven scaffolds, polyglycolic acid (PGA) and poly(lactic-co-glycolic acid) (PLGA) (90/10 copolymer of L-lactide and glycolide), were seeded with human chondrocytes after in...

Role of interleukin-1 in antigen presentation by normal articular chondrocytes

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Tiku, M.L.; Liu, S.; Tiku, K.

1986-01-01

Recently the authors have described that normal articular chondrocytes of rabbits present antigen to immune T cells. In the present study the authors investigated the role of interleukin-1 (IL-1) on antigen presentation by chondrocytes. For these experiments the antigen pulsed chondroyctes were either untreated or fixed with paraformaldehyde and then co-cultured with immune T cells. T cell proliferation was measured by 3 H-thymidine incorporation. Pulsed non-fixed chondrocytes presented antigen, as expected, but pulsed and fixed cells failed to present antigen to T cells. The 3 H-TdR incorporation was partially restored by addition of purified human IL-1. Next, IL-1 activity was measured in primary chondrocyte culture supernatants stimulated with or without lipopolysaccharide (LPS) in comitogen thymocyte assay. No activity was detected in chondrocyte supernatants. Propagated chondrocyte culture supernatants also lacked IL-1 activity when stimulated with LPS in the presence of increasing concentration of indomethacin. On the other hand the authors observed that chondrocyte culture supernatants in a dose dependent manner inhibited human IL-1 induced 3 H-TdR incorporation of murine thymocytes. This suggested that these cells may produce an inhibitor of IL-1 and IL-1 production by chondrocytes may be essential for T cell proliferation by these cells. Inability to detect IL-1 in chondrocyte supernatants may be due to the presence of an inhibitor to IL-1. These findings may help in elucidating the immunological mechanisms in situations where chondrocytes and T cell interact, such as in arthritis

Articular chondrocyte alignment in the rat after surgically induced osteoarthritis

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Takahashi, Hideaki; Tamaki, Hiroyuki; Yamamoto, Noriaki; Onishi, Hideaki

2017-01-01

[Purpose] Chondrocytes in articular cartilage are aligned as columns from the joint surface. Notably, loss of chondrocyte and abnormalities of differentiation factors give rise to osteoarthritis (OA). However, the relationship between chondrocyte alignment and OA progression remains unclear. This study was performed to investigate temporal alterations in surgically-induced OA rats. [Subjects and Methods] Thirteen-week-old Wistar rats (n=30) underwent destabilized medial meniscus surgery in their right knee and sham surgery in their left knee. Specimens (n=5) were collected at 0, 1, 2, 4 and 8 weeks after surgery. Histological analysis with Osteoarthritis Research Society International (OARSI) scores, cell density ratios, cell alignments and correlation between OARSI scores and cell density/alignment was performed. [Results] OARSI scores were significantly higher at 1, 2, 4 and 8 weeks in the DMM group than in the control. Cell density ratios were decreased significantly in the DMM group at 2, 4 and 8 weeks compared with the control. Chondrocyte alignment was decreased significantly in the DMM group at 4 and 8 weeks. There were negative correlations between OA severity and cell density / cell alignment. [Conclusion] The results suggest a relationship between chondrocyte alignment and cartilage homeostasis, which plays an important role in OA progression. PMID:28533592

Nanosized fibers' effect on adult human articular chondrocytes behavior

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Stenhamre, Hanna; Thorvaldsson, Anna; Enochson, Lars; Walkenström, Pernilla; Lindahl, Anders; Brittberg, Mats; Gatenholm, Paul

2013-01-01

Tissue engineering with chondrogenic cell based therapies is an expanding field with the intention of treating cartilage defects. It has been suggested that scaffolds used in cartilage tissue engineering influence cellular behavior and thus the long-term clinical outcome. The objective of this study was to assess whether chondrocyte attachment, proliferation and post-expansion re-differentiation could be influenced by the size of the fibers presented to the cells in a scaffold. Polylactic acid (PLA) scaffolds with different fiber morphologies were produced, i.e. microfiber (MS) scaffolds as well as nanofiber-coated microfiber scaffold (NMS). Adult human articular chondrocytes were cultured in the scaffolds in vitro up to 28 days, and the resulting constructs were assessed histologically, immunohistochemically, and biochemically. Attachment of cells and serum proteins to the scaffolds was affected by the architecture. The results point toward nano-patterning onto the microfibers influencing proliferation of the chondrocytes, and the overall 3D environment having a greater influence on the re-differentiation. In the efforts of finding the optimal scaffold for cartilage tissue engineering, studies as the current contribute to the knowledge of how to affect and control chondrocytes behavior. - Highlights: ► Chondrocyte behavior in nanofiber-coated microfiber versus microfiber scaffolds ► High porosity (> 90%) and large pore sizes (a few hundred μm) of nanofibrous scaffolds ► Proliferation enhanced by presence of nanofibers ► Differentiation not significantly affected ► Cell attachment improved in presence of both nanofibers and serum

Dynamic Mechanical Compression of Chondrocytes for Tissue Engineering: A Critical Review

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Devon E. Anderson

2017-12-01

Full Text Available Articular cartilage functions to transmit and translate loads. In a classical structure–function relationship, the tissue resides in a dynamic mechanical environment that drives the formation of a highly organized tissue architecture suited to its biomechanical role. The dynamic mechanical environment includes multiaxial compressive and shear strains as well as hydrostatic and osmotic pressures. As the mechanical environment is known to modulate cell fate and influence tissue development toward a defined architecture in situ, dynamic mechanical loading has been hypothesized to induce the structure–function relationship during attempts at in vitro regeneration of articular cartilage. Researchers have designed increasingly sophisticated bioreactors with dynamic mechanical regimes, but the response of chondrocytes to dynamic compression and shear loading remains poorly characterized due to wide variation in study design, system variables, and outcome measurements. We assessed the literature pertaining to the use of dynamic compressive bioreactors for in vitro generation of cartilaginous tissue from primary and expanded chondrocytes. We used specific search terms to identify relevant publications from the PubMed database and manually sorted the data. It was very challenging to find consensus between studies because of species, age, cell source, and culture differences, coupled with the many loading regimes and the types of analyses used. Early studies that evaluated the response of primary bovine chondrocytes within hydrogels, and that employed dynamic single-axis compression with physiologic loading parameters, reported consistently favorable responses at the tissue level, with upregulation of biochemical synthesis and biomechanical properties. However, they rarely assessed the cellular response with gene expression or mechanotransduction pathway analyses. Later studies that employed increasingly sophisticated biomaterial-based systems, cells

Dynamic Mechanical Compression of Chondrocytes for Tissue Engineering: A Critical Review.

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Anderson, Devon E; Johnstone, Brian

2017-01-01

Articular cartilage functions to transmit and translate loads. In a classical structure-function relationship, the tissue resides in a dynamic mechanical environment that drives the formation of a highly organized tissue architecture suited to its biomechanical role. The dynamic mechanical environment includes multiaxial compressive and shear strains as well as hydrostatic and osmotic pressures. As the mechanical environment is known to modulate cell fate and influence tissue development toward a defined architecture in situ , dynamic mechanical loading has been hypothesized to induce the structure-function relationship during attempts at in vitro regeneration of articular cartilage. Researchers have designed increasingly sophisticated bioreactors with dynamic mechanical regimes, but the response of chondrocytes to dynamic compression and shear loading remains poorly characterized due to wide variation in study design, system variables, and outcome measurements. We assessed the literature pertaining to the use of dynamic compressive bioreactors for in vitro generation of cartilaginous tissue from primary and expanded chondrocytes. We used specific search terms to identify relevant publications from the PubMed database and manually sorted the data. It was very challenging to find consensus between studies because of species, age, cell source, and culture differences, coupled with the many loading regimes and the types of analyses used. Early studies that evaluated the response of primary bovine chondrocytes within hydrogels, and that employed dynamic single-axis compression with physiologic loading parameters, reported consistently favorable responses at the tissue level, with upregulation of biochemical synthesis and biomechanical properties. However, they rarely assessed the cellular response with gene expression or mechanotransduction pathway analyses. Later studies that employed increasingly sophisticated biomaterial-based systems, cells derived from different

Analysis of human articular chondrocyte CD44 isoform expression and function in health and disease.

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Salter, D M; Godolphin, J L; Gourlay, M S; Lawson, M F; Hughes, D E; Dunne, E

1996-08-01

Interactions between articular chondrocytes and components of the extracellular matrix are of potential importance in the normal function of cartilage and in the pathophysiology of arthritis. Little is known of the basis of these interactions, but cell adhesive molecules such as CD44 are likely to be involved. Immunohistology using six well-characterized anti-CD44 monoclonal antibodies demonstrated standard CD44 isoform (CD44H) expression by all chondrocytes in normal and osteoarthrotic (OA) cartilage but absence of the CD44E variant. Polymerase chain reaction (PCR) of reverse transcribed mRNA from monolayer cultures of normal and OA chondrocytes using primer sequences which span the region containing variably spliced exons produced a predominant band representing the standard form of CD44, which lacks the variable exons 6-15 (v1-v10). No product was seen at the expected size of the epithelial variant of CD44 (CD44v8-10). Use of exon-specific primers, however, showed expression of variant exons resulting in multiple minor isoforms. Standard CD44 was also shown to be the predominantly expressed isoform identified by immunoprecipitation, but human articular chondrocytes did not adhere to hyaluronan in vitro. Chondrocyte CD44 may function as an adhesion receptor for other matrix molecules such as fibronectin or collagen.

Proteoglycon synthesis by articular chondrocytes in agarose culture

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Sweet, M.B.E.; Grisillo, A.; Coehlo, A.; Schnitzler, C.M.

1987-01-01

Articular chondrocytes were isolated from knee joints of full-term bovine foetuses and grown in long-term agarose cultures. At intervals, cultures were labelled with 35 S-[sulphate] or D[6- 3 H] glucosamine. Newly synthesized proteoglycans were extracted with 4 M guanidine HCl and purified by isopycnic density gradient centrifugation or on DEAE cellulose in the presence of 8 M urea. Characterization of the proteoglycans revealed them to be identical in size to those present in the tissue and to be similarly capable of aggregation with hyaluronate. Newly synthesized chondroitin sulphate chains were identical in size, but newly synthesized keratan sulphate chains were somewhat larger than those present in the tissue. The newly synthesized proteoglycans were shown to contain the same range of O-linked oligosaccharides identified in proteoglycans of the Swarm rat chondrosarcoma. Cartilage-specific proteoglycan continued to be synthesized by the chondrocytes for up to 60 days; however, with time, proportionately more of a small non-aggregating proteoglycan appeared

Is the repair of articular cartilage lesion by costal chondrocyte transplantation donor age-dependent? An experimental study in rabbits.

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Janusz Popko

2006-09-01

Full Text Available The repair of chondral injuries is a very important problem and a subject of many experimental and clinical studies. Different techniques to induce articular cartilage repair are under investigation. In the present study, we have investigated whether the repair of articular cartilage folowing costal chondrocyte transplantation is donor age-dependent. Transplantation of costal chondrocytes from 4- and 24-week old donors, with artificially induced femoral cartilage lesion, was performed on fourteen 20-week-old New Zealand White male rabbits. In the control group, the lesion was left without chondrocyte transplantation. The evaluation of the cartilage repair was performed after 12 weeks of transplantation. We analyzed the macroscopic and histological appearance of the newly formed tissue. Immunohistochemistry was also performed using monoclonal antibodies against rabbit collagen type II. The newly formed tissue had a hyaline-like appearance in most of the lesions after chondrocyte transplantation. Positive immunohistochemical reaction for collagen II was also observed in both groups with transplanted chondrocytes. Cartilage from adult donors required longer isolation time and induced slightly poorer repair. However, hyaline-like cartilage was observed in most specimens from this group, in contrast to the control group, where fibrous connective tissue filled the lesions. Rabbit costal chondrocytes seem to be a potentially useful material for inducing articular cartilage repair and, even more important, they can also be derived from adult, sexually mature animals.

Three-dimensional scaffold-free fusion culture: the way to enhance chondrogenesis of in vitro propagated human articular chondrocytes

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M. Lehmann

2013-11-01

Full Text Available Cartilage regeneration based on isolated and culture-expanded chondrocytes has been studied in various in vitro models, but the quality varies with respect to the morphology and the physiology of the synthesized tissues. The aim of our study was to promote in vitro chondrogenesis of human articular chondrocytes using a novel three-dimensional (3-D cultivation system in combination with the chondrogenic differentiation factors transforming growth factor beta 2 (TGF-b2 and L-ascorbic acid. Articular chondrocytes isolated from six elderly patients were expanded in monolayer culture. A single-cell suspension of the dedifferentiated chondrocytes was then added to agar-coated dishes without using any scaffold material, in the presence, or absence of TGF-b2 and/or L-ascorbic acid. Three-dimensional cartilage-like constructs, called single spheroids, and microtissues consisting of several spheroids fused together, named as fusions, were formed. Generated tissues were mainly characterized using histological and immunohistochemical techniques. The morphology of the in vitro tissues shared some similarities to native hyaline cartilage in regard to differentiated S100-positive chondrocytes within a cartilaginous matrix, with strong collagen type II expression and increased synthesis of proteoglycans. Finally, our innovative scaffold-free fusion culture technique supported enhanced chondrogenesis of human articular chondrocytes in vitro. These 3-D hyaline cartilage-like microtissues will be useful for in vitro studies of cartilage differentiation and regeneration, enabling optimization of functional tissue engineering and possibly contributing to the development of new approaches to treat traumatic cartilage defects or osteoarthritis.

Calcitonin directly attenuates collagen type II degradation by inhibition of matrix metalloproteinase expression and activity in articular chondrocytes

DEFF Research Database (Denmark)

Sondergaard, B C; Wulf, H; Henriksen, K

2006-01-01

OBJECTIVE: Calcitonin was recently reported to counter progression of cartilage degradation in an experimental model of osteoarthritis, and the effects were primarily suggested to be mediated by inhibition of subchondral bone resorption. We investigated direct effects of calcitonin on chondrocytes...... by assessing expression of the receptor and pharmacological effects on collagen type II degradation under ex vivo and in vivo conditions. METHODS: Localization of the calcitonin receptor on articular chondrocytes was investigated by immunohistochemistry, and the expression by reverse transcriptase polymerase.......0001-1 microM]. In vivo, cartilage degradation was investigated in ovariectomized (OVX) rats administered with oral calcitonin [2 mg/kg calcitonin] for 9 weeks. RESULTS: The calcitonin receptor was identified in articular chondrocytes by immunohistochemistry and RT-PCR. Calcitonin concentration...

Effects of tofacitinib on nucleic acid metabolism in human articular chondrocytes.

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Koizumi, Hideki; Arito, Mitsumi; Endo, Wataru; Kurokawa, Manae S; Okamoto, Kazuki; Omoteyama, Kazuki; Suematsu, Naoya; Beppu, Moroe; Kato, Tomohiro

2015-07-01

In our previous screening of chondrocyte protein profiles, the amount of adenosine monophosphate deaminase (AMPD) 2 was found to be decreased by tofacitinib. Extending the study, here we confirmed the decrease of AMPD2 by tofacitinib and further investigated effects of tofacitinib on purine nucleotide metabolism. Human articular chondrocytes and a chondrosarcoma cell line: OUMS-27 were stimulated with tofacitinib. Then the levels of AMPD2 and its related enzymes were investigated by Western blot. The levels of AMP and adenosine were assessed by mass spectrometry. We confirmed the significant decrease of AMPD2 by tofacitinib in chondrocytes (p = 0.025). The levels of adenosine kinase and 5'-nucleotidase were decreased in chondrocytes, although they did not meet statistical significance (p = 0.067 and p = 0.074, respectively). The results from OUMS-27 were similar to those from the chondrocytes. The cellular adenosine levels were significantly decreased by tofacitinib in OUMS-27 (p = 0.014). The cellular AMP levels were increased, although they did not meet statistical significance in OUMS-27 (p = 0.066). Our data indicate that tofacitinib increases the cellular levels of adenosine, which is known to have anti-inflammatory activity, through the downregulation of AMPD2. This would be a novel functional aspect of tofacitinib.

Nanosized fibers' effect on adult human articular chondrocytes behavior

Energy Technology Data Exchange (ETDEWEB)

Stenhamre, Hanna [Biopolymer Technology, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg (Sweden); Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Gothenburg (Sweden); Thorvaldsson, Anna, E-mail: anna.thorvaldsson@swerea.se [Biopolymer Technology, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg (Sweden); Swerea IVF, Mölndal (Sweden); Enochson, Lars [Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Gothenburg (Sweden); Walkenström, Pernilla [Swerea IVF, Mölndal (Sweden); Lindahl, Anders [Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Gothenburg (Sweden); Brittberg, Mats [Cartilage Research Unit, University of Gothenburg, Department Orthopaedics, Kungsbacka Hospital, Kungsbacka (Sweden); Gatenholm, Paul [Biopolymer Technology, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg (Sweden)

2013-04-01

Tissue engineering with chondrogenic cell based therapies is an expanding field with the intention of treating cartilage defects. It has been suggested that scaffolds used in cartilage tissue engineering influence cellular behavior and thus the long-term clinical outcome. The objective of this study was to assess whether chondrocyte attachment, proliferation and post-expansion re-differentiation could be influenced by the size of the fibers presented to the cells in a scaffold. Polylactic acid (PLA) scaffolds with different fiber morphologies were produced, i.e. microfiber (MS) scaffolds as well as nanofiber-coated microfiber scaffold (NMS). Adult human articular chondrocytes were cultured in the scaffolds in vitro up to 28 days, and the resulting constructs were assessed histologically, immunohistochemically, and biochemically. Attachment of cells and serum proteins to the scaffolds was affected by the architecture. The results point toward nano-patterning onto the microfibers influencing proliferation of the chondrocytes, and the overall 3D environment having a greater influence on the re-differentiation. In the efforts of finding the optimal scaffold for cartilage tissue engineering, studies as the current contribute to the knowledge of how to affect and control chondrocytes behavior. - Highlights: ► Chondrocyte behavior in nanofiber-coated microfiber versus microfiber scaffolds ► High porosity (> 90%) and large pore sizes (a few hundred μm) of nanofibrous scaffolds ► Proliferation enhanced by presence of nanofibers ► Differentiation not significantly affected ► Cell attachment improved in presence of both nanofibers and serum.

Reduction of Environmental Temperature Mitigates Local Anesthetic Cytotoxicity in Bovine Articular Chondrocytes

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Tarik Onur, Alexis Dang

2014-09-01

Full Text Available The purpose of this study was to assess whether reducing environmental temperature will lead to increased chondrocyte viability following injury from a single-dose of local anesthetic treatment. Bovine articular chondrocytes from weight bearing portions of femoral condyles were harvested and cultured. 96-well plates were seeded with 15,000 chondrocytes per well. Chondrocytes were treated with one of the following conditions: ITS Media, 1x PBS, 2% lidocaine, 0.5% bupivacaine, or 0.5% ropivacaine. Each plate was then incubated at 37°C, 23°C, or 4°C for one hour and then returned to media at 37°C. Chondrocyte viability was assessed 24 hours after treatment. Chondrocyte viability is presented as a ratio of the fluorescence of the treatment group over the average of the media group at that temperature (ratio ± SEM. At 37°C, lidocaine (0.35 ± 0.04 and bupivacaine (0.30 ± 0.05 treated chondrocytes show low cell viability when compared to the media (1.00 ± 0.03 control group (p < 0.001. Lidocaine treated chondrocytes were significantly more viable at 23°C (0.84 ± 0.08 and 4°C (0.86±0.085 than at 37°C (p < 0.001. Bupivacaine treated chondrocytes were significantly more viable at 4°C (0.660 ± 0.073 than at 37°C or 23°C (0.330 ± 0.069 (p < 0.001 and p = 0.002 respectively. Reducing the temperature from 37°C to 23°C during treatment with lidocaine increases chondrocyte viability following injury. Chondrocytes treated with bupivacaine can be rescued by reducing the temperature to 4°C.

The rate of hypo-osmotic challenge influences regulatory volume decrease (RVD) and mechanical properties of articular chondrocytes.

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Wang, Z; Irianto, J; Kazun, S; Wang, W; Knight, M M

2015-02-01

Osteoarthritis (OA) is associated with a gradual reduction in the interstitial osmotic pressure within articular cartilage. The aim of this study was to compare the effects of sudden and gradual hypo-osmotic challenge on chondrocyte morphology and biomechanics. Bovine articular chondrocytes were exposed to a reduction in extracellular osmolality from 327 to 153 mOsmol/kg applied either suddenly (osmotic stress, 66% of chondrocytes exhibited an increase in diameter followed by RVD, whilst 25% showed no RVD. By contrast, cells exposed to gradual hypo-osmotic stress exhibited reduced cell swelling without subsequent RVD. There was an increase in the equilibrium modulus for cells exposed to sudden hypo-osmotic stress. However, gradual hypo-osmotic challenge had no effect on cell mechanical properties. This cell stiffening response to sudden hypo-osmotic challenge was abolished when actin organization was disrupted with cytochalasin D or RVD inhibited with REV5901. Both sudden and gradual hypo-osmotic challenge reduced cortical F-actin distribution and caused chromatin decondensation. Sudden hypo-osmotic challenge increases chondrocyte mechanics by activation of RVD and interaction with the actin cytoskeleton. Moreover, the rate of hypo-osmotic challenge is shown to have a profound effect on chondrocyte morphology and biomechanics. This important phenomenon needs to be considered when studying the response of chondrocytes to pathological hypo-osmotic stress. Copyright © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

The in vitro biocompatibility of d-(+) raffinose modified chitosan: Two-dimensional and three-dimensional systems for culturing of horse articular chondrocytes.

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De Angelis, Elena; Ravanetti, Francesca; Martelli, Paolo; Cacchioli, Antonio; Ivanovska, Ana; Corradi, Attilio; Nasi, Sonia; Bianchera, Annalisa; Passeri, Benedetta; Canelli, Elena; Bettini, Ruggero; Borghetti, Paolo

2017-12-01

The present study investigated the biocompatibility of chitosan films and scaffolds modified with d-(+)raffinose and their capability to support the growth and maintenance of the differentiation of articular chondrocytes in vitro. Primary equine articular chondrocytes were cultured on films and scaffolds of modified d-(+) raffinose chitosan. Their behavior was compared to that of chondrocytes grown in conventional bi- and three-dimensional culture systems, such as micromasses and alginate beads. Chitosan films maintained the phenotype of differentiated chondrocytes (typical round morphology) and sustained the synthesis of cartilaginous extracellular matrix (ECM), even at 4weeks of culture. Indeed, starting from 2weeks of culture, chondrocytes seeded on chitosan scaffolds were able to penetrate the surface pores and to colonize the internal matrix. Moreover they produced ECM expressing the genes of typical chondrocytes differentiation markers such as collagen II and aggrecan. In conclusion, chitosan modified with d-raffinose represents an ideal support for chondrocyte adhesion, proliferation and for the maintenance of cellular phenotypic and genotypic differentiation. This novel biomaterial could potentially be a reliable support for the re-differentiation of dedifferentiated chondrocytes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Isolation and characterization of human articular chondrocytes from surgical waste after total knee arthroplasty (TKA

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Jakob Naranda

2017-03-01

Full Text Available Background Cartilage tissue engineering is a fast-evolving field of biomedical engineering, in which the chondrocytes represent the most commonly used cell type. Since research in tissue engineering always consumes a lot of cells, simple and cheap isolation methods could form a powerful basis to boost such studies and enable their faster progress to the clinics. Isolated chondrocytes can be used for autologous chondrocyte implantation in cartilage repair, and are the base for valuable models to investigate cartilage phenotype preservation, as well as enable studies of molecular features, nature and scales of cellular responses to alterations in the cartilage tissue. Methods Isolation and consequent cultivation of primary human adult articular chondrocytes from the surgical waste obtained during total knee arthroplasty (TKA was performed. To evaluate the chondrogenic potential of the isolated cells, gene expression of collagen type 2 (COL2, collagen 1 (COL1 and aggrecan (ACAN was evaluated. Immunocytochemical staining of all mentioned proteins was performed to evaluate chondrocyte specific production. Results Cartilage specific gene expression of COL2 and ACAN has been shown that the proposed protocol leads to isolation of cells with a high chondrogenic potential, possibly even specific phenotype preservation up to the second passage. COL1 expression has confirmed the tendency of the isolated cells dedifferentiation into a fibroblast-like phenotype already in the second passage, which confirms previous findings that higher passages should be used with care in cartilage tissue engineering. To evaluate the effectiveness of our approach, immunocytochemical staining of the evaluated chondrocyte specific products was performed as well. Discussion In this study, we developed a protocol for isolation and consequent cultivation of primary human adult articular chondrocytes with the desired phenotype from the surgical waste obtained during TKA. TKA is a

IKKα/CHUK regulates extracellular matrix remodeling independent of its kinase activity to facilitate articular chondrocyte differentiation.

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Eleonora Olivotto

Full Text Available BACKGROUND: The non-canonical NF-κB activating kinase IKKα, encoded by CHUK (conserved-helix-loop-helix-ubiquitous-kinase, has been reported to modulate pro- or anti- inflammatory responses, cellular survival and cellular differentiation. Here, we have investigated the mechanism of action of IKKα as a novel effector of human and murine chondrocyte extracellular matrix (ECM homeostasis and differentiation towards hypertrophy. METHODOLOGY/PRINCIPAL FINDINGS: IKKα expression was ablated in primary human osteoarthritic (OA chondrocytes and in immature murine articular chondrocytes (iMACs derived from IKKα(f/f:CreERT2 mice by retroviral-mediated stable shRNA transduction and Cre recombinase-dependent Lox P site recombination, respectively. MMP-10 was identified as a major target of IKKα in chondrocytes by mRNA profiling, quantitative RT-PCR analysis, immunohistochemistry and immunoblotting. ECM integrity, as assessed by type II collagen (COL2 deposition and the lack of MMP-dependent COL2 degradation products, was enhanced by IKKα ablation in mice. MMP-13 and total collagenase activities were significantly reduced, while TIMP-3 (tissue inhibitor of metalloproteinase-3 protein levels were enhanced in IKKα-deficient chondrocytes. IKKα deficiency suppressed chondrocyte differentiation, as shown by the quantitative inhibition of.Alizarin red staining and the reduced expression of multiple chondrocyte differentiation effectors, including Runx2, Col10a1 and Vegfa,. Importantly, the differentiation of IKKα-deficient chondrocytes was rescued by a kinase-dead IKKα protein mutant. CONCLUSIONS/SIGNIFICANCE: IKKα acts independent of its kinase activity to help drive chondrocyte differentiation towards a hypertrophic-like state. IKKα positively modulates ECM remodeling via multiple downstream targets (including MMP-10 and TIMP-3 at the mRNA and post-transcriptional levels, respectively to maintain maximal MMP-13 activity, which is required for ECM

Nasal chondrocyte-based engineered autologous cartilage tissue for repair of articular cartilage defects: an observational first-in-human trial.

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Mumme, Marcus; Barbero, Andrea; Miot, Sylvie; Wixmerten, Anke; Feliciano, Sandra; Wolf, Francine; Asnaghi, Adelaide M; Baumhoer, Daniel; Bieri, Oliver; Kretzschmar, Martin; Pagenstert, Geert; Haug, Martin; Schaefer, Dirk J; Martin, Ivan; Jakob, Marcel

2016-10-22

Articular cartilage injuries have poor repair capacity, leading to progressive joint damage, and cannot be restored predictably by either conventional treatments or advanced therapies based on implantation of articular chondrocytes. Compared with articular chondrocytes, chondrocytes derived from the nasal septum have superior and more reproducible capacity to generate hyaline-like cartilage tissues, with the plasticity to adapt to a joint environment. We aimed to assess whether engineered autologous nasal chondrocyte-based cartilage grafts allow safe and functional restoration of knee cartilage defects. In a first-in-human trial, ten patients with symptomatic, post-traumatic, full-thickness cartilage lesions (2-6 cm 2 ) on the femoral condyle or trochlea were treated at University Hospital Basel in Switzerland. Chondrocytes isolated from a 6 mm nasal septum biopsy specimen were expanded and cultured onto collagen membranes to engineer cartilage grafts (30 × 40 × 2 mm). The engineered tissues were implanted into the femoral defects via mini-arthrotomy and assessed up to 24 months after surgery. Primary outcomes were feasibility and safety of the procedure. Secondary outcomes included self-assessed clinical scores and MRI-based estimation of morphological and compositional quality of the repair tissue. This study is registered with ClinicalTrials.gov, number NCT01605201. The study is ongoing, with an approved extension to 25 patients. For every patient, it was feasible to manufacture cartilaginous grafts with nasal chondrocytes embedded in an extracellular matrix rich in glycosaminoglycan and type II collagen. Engineered tissues were stable through handling with forceps and could be secured in the injured joints. No adverse reactions were recorded and self-assessed clinical scores for pain, knee function, and quality of life were improved significantly from before surgery to 24 months after surgery. Radiological assessments indicated variable degrees of

Reciprocal regulation by hypoxia-inducible factor-2α and the NAMPT-NAD(+)-SIRT axis in articular chondrocytes is involved in osteoarthritis.

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Oh, H; Kwak, J-S; Yang, S; Gong, M-K; Kim, J-H; Rhee, J; Kim, S K; Kim, H-E; Ryu, J-H; Chun, J-S

2015-12-01

Hypoxia-inducible factor-2α (HIF-2α) transcriptionally upregulates Nampt in articular chondrocytes. NAMPT, which exhibits nicotinamide phosphoribosyltransferase activity, in turn causes osteoarthritis (OA) in mice by stimulating the expression of matrix-degrading enzymes. Here, we sought to elucidate whether HIF-2α activates the NAMPT-NAD(+)-SIRT axis in chondrocytes and thereby contributes to the pathogenesis of OA. Assays of NAD levels, SIRT activity, reporter gene activity, mRNA, and protein levels were conducted in primary cultured mouse articular chondrocytes. Experimental OA in mice was induced by intra-articular (IA) injection of adenovirus expressing HIF-2α (Ad-Epas1) or NAMPT (Ad-Nampt). The functions of SIRT in OA were examined by IA co-injection of SIRT inhibitors or adenovirus expressing individual SIRT isoforms or shRNA targeting specific SIRT isoforms. HIF-2α activated the NAMPT-NAD(+)-SIRT axis in chondrocytes by upregulating NAMPT, which stimulated NAD(+) synthesis and thereby activated SIRT family members. The activated NAMPT-SIRT pathway, in turn, promoted HIF-2α protein stability by negatively regulating its hydroxylation and 26S proteasome-mediated degradation, resulting in increased HIF-2α transcriptional activity. Among SIRT family members (SIRT1-7), SIRT2 and SIRT4 were positively associated with HIF-2α stability and transcriptional activity in chondrocytes. This reciprocal regulation was required for the expression of catabolic matrix metalloproteinases (MMP3, MMP12, and MMP13) and OA cartilage destruction caused by IA injection of Ad-Epas1 Ad-Nampt. The reciprocal regulation of HIF-2α and the NAMPT-NAD(+)-SIRT axis in articular chondrocytes is involved in OA cartilage destruction caused by HIF-2α or NAMPT. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Influence of Knee Immobilization on Chondrocyte Apoptosis and Histological Features of the Anterior Cruciate Ligament Insertion and Articular Cartilage in Rabbits.

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Mutsuzaki, Hirotaka; Nakajima, Hiromi; Wadano, Yasuyoshi; Furuhata, Syogo; Sakane, Masataka

2017-01-26

This study examined the influence of immobilization on chondrocyte apoptosis and histological features of the anterior cruciate ligament (ACL) insertion and knee articular cartilage in rabbits. Forty-eight male Japanese white rabbits were assigned to an immobilization ( n = 24) or sham ( n = 24) group. Rabbits in the immobilization group underwent complete unilateral surgical knee immobilization and rabbits in the sham group underwent a sham surgery. The average thickness of the glycosaminoglycan (GAG) stained red area by safranin O staining, the chondrocyte apoptosis rate and the chondrocyte proliferation rate in the cartilage layer in the ACL insertion and the articular cartilage of the medial tibial condyle were measured at one, two, four and eight weeks in six animals from each group. In the ACL insertion, the chondrocyte apoptosis rate was higher in the immobilization group than in the sham group at two and eight weeks after surgery ( p immobilization group. The GAG layer was thinner in the immobilization group than in the sham group at two, four and eight weeks after surgery ( p immobilization group was higher than in the sham group at four and eight weeks after surgery ( p immobilization group than that in the sham group at four and eight weeks after surgery ( p immobilization significantly increased chondrocyte apoptosis at two and eight weeks after surgery in the ACL insertion and at four and eight weeks after surgery in the articular cartilage of the medial tibial condyle, and decreased GAG layer thickness from two to eight weeks after surgery in the ACL insertion and from four to eight weeks after surgery in the articular cartilage.

Gene Modification of Mesenchymal Stem Cells and Articular Chondrocytes to Enhance Chondrogenesis

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Saliya Gurusinghe

2014-01-01

Full Text Available Current cell based treatment for articular cartilage and osteochondral defects are hampered by issues such as cellular dedifferentiation and hypertrophy of the resident or transplanted cells. The reduced expression of chondrogenic signalling molecules and transcription factors is a major contributing factor to changes in cell phenotype. Gene modification of chondrocytes may be one approach to redirect cells to their primary phenotype and recent advances in nonviral and viral gene delivery technologies have enabled the expression of these lost factors at high efficiency and specificity to regain chondrocyte function. This review focuses on the various candidate genes that encode signalling molecules and transcription factors that are specific for the enhancement of the chondrogenic phenotype and also how epigenetic regulators of chondrogenesis in the form of microRNA may also play an important role.

Type II collagen peptide is able to accelerate embryonic chondrocyte differentiation: an association with articular cartilage matrix resorption in osteoarthrosis

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Elena Vasil'evna Chetina

2010-01-01

Conclusion. The effect of CP on gene expression and collagen decomposition activity depends on the morphotype of embryonic chondrocytes. Lack of effect of CP on collagen decomposition activity in both the embryonic hypertrophic chondrocytes and the cartilage explants from OA patients supports the hypothesis that the hypertrophic morphotype is a dominant morphotype of articular chondrocytes in OA. Moreover, collagen decomposition products can be involved in the resorption of matrix in OA and in the maintenance of chronic nature of the pathology.

Effects of low molecular weight hyaluronan combined with carprofen on canine osteoarthritis articular chondrocytes and cartilage explants in vitro.

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Euppayo, Thippaporn; Siengdee, Puntita; Buddhachat, Kittisak; Pradit, Waranee; Viriyakhasem, Nawarat; Chomdej, Siriwadee; Ongchai, Siriwan; Harada, Yasuji; Nganvongpanit, Korakot

2015-09-01

Intra-articular injection with non-steroidal anti-inflammatory drugs (NSAIDs) is used to treat inflammatory joint disease, but the side effects of NSAIDs include chondrotoxicity. Hyaluronan has shown positive effects on chondrocytes by reducing apoptosis and increasing proteoglycan synthesis. The purposes of this study were to evaluate the effects of low molecular weight hyaluronan (low MW HA), carprofen 25 mg/ml, carprofen 12.5 mg/ml, and a combination of HA and carprofen on canine osteoarthritis (OA) articular chondrocytes and a cartilage explant model in terms of cell viability, extracellular matrix remaining, and gene expression after exposure. In chondrocyte culture, MTT assay was used to evaluate the chondrotoxicity of IC50 and IC80 of carprofen with HA. In cartilage explant culture, two kinds of extracellular matrix (uronic acid and collagen) remaining in cartilage were used to evaluate cartilage damage for 14 d after treatment. Expression of COL2A1, AGG, and MMP3 was used to evaluate the synthesis and degradation of the matrix for 7 d after treatment. In chondrocyte culture, low MW HA could preserve OA chondrocyte viability but could not reduce the chondrotoxicity level of carprofen (P carprofen caused less destruction of uronic acid and collagen structure when compared with the control (P carprofen resulted in higher COL2A1 and AGG expression levels than carprofen alone.

Using Costal Chondrocytes to Engineer Articular Cartilage with Applications of Passive Axial Compression and Bioactive Stimuli.

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Huwe, Le W; Sullan, Gurdeep K; Hu, Jerry C; Athanasiou, Kyriacos A

2018-03-01

Generating neocartilage with suitable mechanical integrity from a cell source that can circumvent chondrocyte scarcity is indispensable for articular cartilage regeneration strategies. Costal chondrocytes of the rib eliminate donor site morbidity in the articular joint, but it remains unclear how neocartilage formed from these cells responds to mechanical loading, especially if the intent is to use it in a load-bearing joint. In a series of three experiments, this study sought to determine efficacious parameters of passive axial compressive stimulation that would enable costal chondrocytes to synthesize mechanically robust cartilage. Experiment 1 determined a suitable time window for stimulation by its application during either the matrix synthesis phase, the maturation phase, or during both phases of the self-assembling process. The results showed that compressive stimulation at either time was effective in increasing instantaneous moduli by 92% and 87% in the synthesis and maturation phases, respectively. Compressive stimulation during both phases did not further improve properties beyond a one-time stimulation. The magnitude of passive axial compression was examined in Experiment 2 by applying 0, 3.3, 5.0, or 6.7 kPa stresses to the neocartilage. Unlike 6.7 kPa, both 3.3 and 5.0 kPa significantly increased neocartilage compressive properties by 42% and 48% over untreated controls, respectively. Experiment 3 examined how the passive axial compression regimen developed from the previous phases interacted with a bioactive regimen (transforming growth factor [TGF]-β1, chondroitinase ABC, and lysyl oxidase-like 2). Passive axial compression significantly improved the relaxation modulus compared with bioactive treatment alone. Furthermore, a combined treatment of compressive and bioactive stimulation improved the tensile properties of neocartilage 2.6-fold compared with untreated control. The ability to create robust articular cartilage from passaged costal

Chondrocyte secreted CRTAC1: a glycosylated extracellular matrix molecule of human articular cartilage.

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Steck, Eric; Bräun, Jessica; Pelttari, Karoliina; Kadel, Stephanie; Kalbacher, Hubert; Richter, Wiltrud

2007-01-01

Cartilage acidic protein 1 (CRTAC1), a novel human marker which allowed discrimination of human chondrocytes from osteoblasts and mesenchymal stem cells in culture was so far studied only on the RNA-level. We here describe its genomic organisation and detect a new brain expressed (CRTAC1-B) isoform resulting from alternate last exon usage which is highly conserved in vertebrates. In humans, we identify an exon sharing process with the neighbouring tail-to-tail orientated gene leading to CRTAC1-A. This isoform is produced by cultured human chondrocytes, localized in the extracellular matrix of articular cartilage and its secretion can be stimulated by BMP4. Of five putative O-glycosylation motifs in the last exon of CRTAC1-A, the most C-terminal one is modified according to exposure of serial C-terminal deletion mutants to the O-glycosylation inhibitor Benzyl-alpha-GalNAc. Both isoforms contain four FG-GAP repeat domains and an RGD integrin binding motif, suggesting cell-cell or cell-matrix interaction potential. In summary, CRTAC1 acquired an alternate last exon from the tail-to-tail oriented neighbouring gene in humans resulting in the glycosylated isoform CRTAC1-A which represents a new extracellular matrix molecule of articular cartilage.

Effect of extracellular fatty acids on lipid metabolism in cultured rabbit articular chondrocytes

International Nuclear Information System (INIS)

Nagao, M.; Ishii, S.; Murata, Y.; Akino, T.

1991-01-01

Rabbit articular chondrocytes were cultured for 8 h in the presence of various concentrations (5-500 microM) of 14 C oleic, 14 C linoleic, and 3H arachidonic acids. The radioactive unsaturated fatty acids were incorporated into triacylglycerol (TG) and phosphatidylcholine (PC) in a concentration-dependent manner; more fatty acids were incorporated into TG than into PC, at higher concentrations of extracellular fatty acids. Among these fatty acids, arachidonic acid was incorporated into TG much more than into PC, in spite of a very low concentration of arachidonic acid in TG. After transfer of the labeled cells to maintenance medium, the radioactivity in TG declined rapidly and 3 H arachidonic acid radioactivity in PC increased continuously during the chase time periods. Palmitoyl-unsaturated species were mainly formed in PC when cultured at a concentration of 5 microM of each fatty acid. However, when cultured at 500 microM, unsaturated-unsaturated species, specific for each unsaturated fatty acid were actively formed. These findings indicate that (1) fatty acid composition of TG and PC in articular chondrocytes is influenced by the degree of fatty acid supply, (2) formation and turnover of TG plays a role in fatty acid metabolism of cells, and (3) fatty acid pairing in PC is modulated by extracellular fatty acid concentrations

Deletion of IFT80 Impairs Epiphyseal and Articular Cartilage Formation Due to Disruption of Chondrocyte Differentiation

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Yuan, Xue; Yang, Shuying

2015-01-01

Intraflagellar transport proteins (IFT) play important roles in cilia formation and organ development. Partial loss of IFT80 function leads Jeune asphyxiating thoracic dystrophy (JATD) or short-rib polydactyly (SRP) syndrome type III, displaying narrow thoracic cavity and multiple cartilage anomalies. However, it is unknown how IFT80 regulates cartilage formation. To define the role and mechanism of IFT80 in chondrocyte function and cartilage formation, we generated a Col2α1; IFT80f/f mouse model by crossing IFT80f/f mice with inducible Col2α1-CreER mice, and deleted IFT80 in chondrocyte lineage by injection of tamoxifen into the mice in embryonic or postnatal stage. Loss of IFT80 in the embryonic stage resulted in short limbs at birth. Histological studies showed that IFT80-deficient mice have shortened cartilage with marked changes in cellular morphology and organization in the resting, proliferative, pre-hypertrophic, and hypertrophic zones. Moreover, deletion of IFT80 in the postnatal stage led to mouse stunted growth with shortened growth plate but thickened articular cartilage. Defects of ciliogenesis were found in the cartilage of IFT80-deficient mice and primary IFT80-deficient chondrocytes. Further study showed that chondrogenic differentiation was significantly inhibited in IFT80-deficient mice due to reduced hedgehog (Hh) signaling and increased Wnt signaling activities. These findings demonstrate that loss of IFT80 blocks chondrocyte differentiation by disruption of ciliogenesis and alteration of Hh and Wnt signaling transduction, which in turn alters epiphyseal and articular cartilage formation. PMID:26098911

Influence of bone morphogenetic protein-2 on the extracellular matrix, material properties, and gene expression of long-term articular chondrocyte cultures: loss of chondrocyte stability.

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Krawczak, David A; Westendorf, Jennifer J; Carlson, Cathy S; Lewis, Jack L

2009-06-01

The aim of this study was to determine the effects of bone morphogenetic protein-2 (BMP-2) on articular chondrocyte tissues grown as monolayers in vitro for up to 8 weeks. Articular chondrocytes were isolated from New Zealand White rabbits and plated in monolayer cultures. The cultures were supplemented with 100 ng/mL of BMP-2 for up to 8 weeks and the extracellular matrix (ECM) composition, material properties, and messenger RNA (mRNA) expression were analyzed. mRNA expression of cartilage-specific genes, type II collagen, and aggrecan showed that BMP-2 enhanced chondrocyte stability for up to 3 weeks. After 3 weeks in culture, there was substantially more type I collagen expression and more osteopontin and runt-related transcription factor 2 expression in 5- and 8-week cultures treated with BMP-2 than in controls. Additionally, matrix metalloproteinase-13 and ADAMTS-5 (A disintegrin-like and metalloproteinase with thrombospondin 5) were upregulated in 5- and 8-week cultures treated with BMP-2, coinciding with a loss of ECM density, collagen, and proteoglycan. Eight-week tissue stimulated with BMP-2 was more fragile and tore more easily when removed from the culture dish as compared to controls, suggesting temporal limitations to the effectiveness of BMP-2 in monolayer systems and perhaps other models to enhance the generation of a cartilage-like tissue for tissue engineering purposes.

The synovial microenvironment of osteoarthritic joints alters RNA-seq expression profiles of human primary articular chondrocytes

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Lewallen, Eric A.; Bonin, Carolina A.; Li, Xin; Smith, Jay; Karperien, Marcel; Larson, A. Noelle; Lewallen, David G.; Cool, Simon M.; Westendorf, Jennifer J.; Krych, Aaron J.; Leontovich, Alexey A.; Im, Hee-Jeong; van Wijnen, Andre J.

2018-01-01

Osteoarthritis (OA) is a disabling degenerative joint disease that prompts pain with limited treatment options. To permit early diagnosis and treatment of OA, a high resolution mechanistic understanding of human chondrocytes in normal and diseased states is necessary. In this study, we assessed the biological effects of OA-related changes in the synovial microenvironment on chondrocytes embedded within anatomically intact cartilage from joints with different pathological grades by next generation RNA-sequencing (RNA-seq). We determined the transcriptome of primary articular chondrocytes derived from pristine knees and ankles, as well as from joints affected by OA. The GALAXY bioinformatics platform was used to facilitate biological interpretations. Comparisons of patient samples by k-means, hierarchical clustering and principal component analysis reveal that primary chondrocytes exhibit OA grade-related differences in gene expression, including genes involved in cell-adhesion, ECM production and immune response. We conclude that diseased synovial microenvironments in joints with different histopathological OA grades directly alter gene expression in chondrocytes. One ramification of this finding is that sampling anatomically intact cartilage from OA joints is not an ideal source of healthy chondrocytes, nor should they be used to generate a normal baseline for the molecular characterization of diseased joints. PMID:27378743

Biomechanics of Meniscus Cells: Regional Variation and Comparison to Articular Chondrocytes and Ligament Cells

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Sanchez-Adams, Johannah; Athanasiou, Kyriacos A.

2012-01-01

Central to understanding mechanotransduction in the knee meniscus is the characterization of meniscus cell mechanics. In addition to biochemical and geometric differences, the inner and outer regions of the meniscus contain cells that are distinct in morphology and phenotype. This study investigated the regional variation in meniscus cell mechanics in comparison to articular chondrocytes and ligament cells. It was found that the meniscus contains two biomechanically distinct cell populations,...

Similar properties of chondrocytes from osteoarthritis joints and mesenchymal stem cells from healthy donors for tissue engineering of articular cartilage.

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Amilton M Fernandes

Full Text Available Lesions of hyaline cartilage do not heal spontaneously, and represent a therapeutic challenge. In vitro engineering of articular cartilage using cells and biomaterials may prove to be the best solution. Patients with osteoarthritis (OA may require tissue engineered cartilage therapy. Chondrocytes obtained from OA joints are thought to be involved in the disease process, and thus to be of insufficient quality to be used for repair strategies. Bone marrow (BM derived mesenchymal stem cells (MSCs from healthy donors may represent an alternative cell source. We have isolated chondrocytes from OA joints, performed cell culture expansion and tissue engineering of cartilage using a disc-shaped alginate scaffold and chondrogenic differentiation medium. We performed real-time reverse transcriptase quantitative PCR and fluorescence immunohistochemistry to evaluate mRNA and protein expression for a range of molecules involved in chondrogenesis and OA pathogenesis. Results were compared with those obtained by using BM-MSCs in an identical tissue engineering strategy. Finally the two populations were compared using genome-wide mRNA arrays. At three weeks of chondrogenic differentiation we found high and similar levels of hyaline cartilage-specific type II collagen and fibrocartilage-specific type I collagen mRNA and protein in discs containing OA and BM-MSC derived chondrocytes. Aggrecan, the dominant proteoglycan in hyaline cartilage, was more abundantly distributed in the OA chondrocyte extracellular matrix. OA chondrocytes expressed higher mRNA levels also of other hyaline extracellular matrix components. Surprisingly BM-MSC derived chondrocytes expressed higher mRNA levels of OA markers such as COL10A1, SSP1 (osteopontin, ALPL, BMP2, VEGFA, PTGES, IHH, and WNT genes, but lower levels of MMP3 and S100A4. Based on the results presented here, OA chondrocytes may be suitable for tissue engineering of articular cartilage.

Cell expansion of human articular chondrocytes on macroporous gelatine scaffolds-impact of microcarrier selection on cell proliferation

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Pettersson, Sofia; Kratz, Gunnar [Laboratory for Reconstructive Plastic Surgery, Department of Clinical and Experimental Medicine, Linkoeping University, SE-581 85 Linkoeping (Sweden); Wetteroe, Jonas [Rheumatology/AIR, Department of Clinical and Experimental Medicine, Linkoeping University, SE-581 85 Linkoeping (Sweden); Tengvall, Pentti, E-mail: sofia.pettersson@liu.se [Institute of Clinical Sciences, Department of Biomaterials, The Sahlgrenska Academy at University of Gothenburg, SE-405 30 Gothenburg (Sweden)

2011-12-15

This study investigates human chondrocyte expansion on four macroporous gelatine microcarriers (CultiSpher) differing with respect to two manufacturing processes-the amount of emulsifier used during initial preparation and the gelatine cross-linking medium. Monolayer-expanded articular chondrocytes from three donors were seeded onto the microcarriers and cultured in spinner flask systems for a total of 15 days. Samples were extracted every other day to monitor cell viability and establish cell counts, which were analysed using analysis of variance and piecewise linear regression. Chondrocyte densities increased according to a linear pattern for all microcarriers, indicating an ongoing, though limited, cell proliferation. A strong chondrocyte donor effect was seen during the initial expansion phase. The final cell yield differed significantly between the microcarriers and our results indicate that manufacturing differences affected chondrocyte densities at this point. Remaining cells stained positive for chondrogenic markers SOX-9 and S-100 but extracellular matrix formation was modest to undetectable. In conclusion, the four gelatine microcarriers supported chondrocyte adhesion and proliferation over a two week period. The best yield was observed for microcarriers produced with low emulsifier content and cross-linked in water and acetone. These results add to the identification of optimal biomaterial parameters for specific cellular processes and populations.

Aging and oxidative stress reduce the response of human articular chondrocytes to insulin-like growth factor 1 and osteogenic protein 1.

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Loeser, Richard F; Gandhi, Uma; Long, David L; Yin, Weihong; Chubinskaya, Susan

2014-08-01

To determine the effects of aging and oxidative stress on the response of human articular chondrocytes to insulin-like growth factor 1 (IGF-1) and osteogenic protein 1 (OP-1). Chondrocytes isolated from normal articular cartilage obtained from tissue donors were cultured in alginate beads or monolayer. Cells were stimulated with 50-100 ng/ml of IGF-1, OP-1, or both. Oxidative stress was induced using tert-butyl hydroperoxide. Sulfate incorporation was used to measure proteoglycan synthesis, and immunoblotting of cell lysates was performed to analyze cell signaling. Confocal microscopy was performed to measure nuclear translocation of Smad4. Chondrocytes isolated from the articular cartilage of tissue donors ranging in age from 24 years to 81 years demonstrated an age-related decline in proteoglycan synthesis stimulated by IGF-1 and IGF-1 plus OP-1. Induction of oxidative stress inhibited both IGF-1- and OP-1-stimulated proteoglycan synthesis. Signaling studies showed that oxidative stress inhibited IGF-1-stimulated Akt phosphorylation while increasing phosphorylation of ERK, and that these effects were greater in cells from older donors. Oxidative stress also increased p38 phosphorylation, which resulted in phosphorylation of Smad1 at the Ser(206) inhibitory site and reduced nuclear accumulation of Smad1. Oxidative stress also modestly reduced OP-1-stimulated nuclear translocation of Smad4. These results demonstrate an age-related reduction in the response of human chondrocytes to IGF-1 and OP-1, which are 2 important anabolic factors in cartilage, and suggest that oxidative stress may be a contributing factor by altering IGF-1 and OP-1 signaling. Copyright © 2014 by the American College of Rheumatology.

Laser solder welding of articular cartilage: tensile strength and chondrocyte viability.

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Züger, B J; Ott, B; Mainil-Varlet, P; Schaffner, T; Clémence, J F; Weber, H P; Frenz, M

2001-01-01

The surgical treatment of full-thickness cartilage defects in the knee joint remains a therapeutic challenge. Recently, new techniques for articular cartilage transplantation, such as mosaicplasty, have become available for cartilage repair. The long-term success of these techniques, however, depends not only on the chondrocyte viability but also on a lateral integration of the implant. The goal of this study was to evaluate the feasibility of cartilage welding by using albumin solder that was dye-enhanced to allow coagulation with 808-nm laser diode irradiation. Conventional histology of light microscopy was compared with a viability staining to precisely determine the extent of thermal damage after laser welding. Indocyanine green (ICG) enhanced albumin solder (25% albumin, 0.5% HA, 0.1% ICG) was used for articular cartilage welding. For coagulation, the solder was irradiated through the cartilage implant by 808-nm laser light and the tensile strength of the weld was measured. Viability staining revealed a thermal damage of typically 500 m in depth at an irradiance of approximately 10 W/cm(2) for 8 seconds, whereas conventional histologies showed only half of the extent found by the viability test. Heat-bath investigations revealed a threshold temperature of minimum 54 degrees C for thermal damage of chondrocytes. Efficient cartilage bonding was obtained by using bovine albumin solder as adhesive. Maximum tensile strength of more than 10 N/cm(2) was achieved. Viability tests revealed that the thermal damage is much greater (up to twice) than expected after light microscopic characterization. This study shows the feasibility to strongly laser weld cartilage on cartilage by use of a dye-enhanced albumin solder. Possibilities to reduce the range of damage are suggested. Copyright 2001 Wiley-Liss, Inc.

The effect of a slightly acidic somatomedin peptide (ILAs) on the sulphation of proteoglycans from articular and growth plate chondrocytes in culture

International Nuclear Information System (INIS)

Corvol, M.-T.; Dumontier, M.-F.; Rappaport, R.; Guyda, H.; Posner, B.I.

1978-01-01

Chondrocyte cultures were prepared from rabbit growth plate (GPC) and articular (ARC) chondrocytes. These two cell types have distinct morphological characteristics. The cells reached maximum numbers by days 10 and 21 for ARC and GPC, respectively. The proteoglycans (PG) contained in the cellular pool were extracted and purified by DEAE cellulose chromatography. The effect of a partially purified somatomedin peptide with insulin-like activity on [ 35 S]sulphate incorporation into PG was evaluated. In both ARC and GPC a significant stimulation of [ 35 S]sulphate uptake into PG subunits was obtained with 1 ng Eq./ml of somatomedin peptide. In order to obtain the same stimulatory effect with porcine insulin, a 1000-fold greater concentration was required. The electrophoretic patterns of the PG subunits on acrylamide-agarose electrophoresis were identical on control incubations and after stimulation with the somatomedin peptide. These data demonstrate in vitro biological activity of this peptide on well differentiated articular and epiphyseal growth plate chondrocytes in culture. These cultures appear to provide a sensitive biological assay for somatomedin peptides. (author)

Effects of 99Tc-MDP on synoviocytes and articular chondrocytes apoptosis associated factors on CIA rats

International Nuclear Information System (INIS)

Hu Shaoxian; Kong Fang; Ke Dan; Shu Min; Len Xiaomei; Tu Wei; Shen Guifen; He Peigen

2009-01-01

Objective: Collagen induced arthritis (CIA) rats is an animal model of human rheumatoid arthritis (RA). It is widely used in research of the pathogenesis and the therapeutic targets of RA. This paper was to investigate the therapeutic action of 99 Tc-methylene diphosphonic acid (MDP) on CIA rats and its effects on the expression of apoptosis associated factor bcl-2 and bax in synoviocytes and articular chondrocytes. Methods: CIA rat models were carried out by subcutaneous injection with bovine collagen II and incomplete Freud's adjuvant. Rats were divided into four groups: control group, CIA model group (the CIA rats were infused with physiological saline via tail vein daily), 99 Tc-MDP group (the C1A rats were injected with 99 Tc-Mi)P 0.04 μg 99 Tc/kg via tail vein daily) and methotrexate (MTX) group (the CIA rats were injected with MTX 1 mg/kg via tail vein weekly). The signs of arthritis were evaluated by arthritis index (AI) scores. Immunohistochemistry was performed to detect the expression of bcl-2 and bax in synoviocytes and articular chondrocytes. SPSS 13.0 was used for data analysis. Results: (1) The signs of arthritis, AI scores and pathological changes of arthrosynovitis in CIA rats were significantly improved by 99 Tc-MDP or MTX. (2) The expression of bcl-2 and box in the synoviocytes of CIA model group [(39.30 ± 0.53) %, (27.37 ± 2.45)%] was significantly increased compared with control group [(7.56 ± 1. 18)% , (6.14 ± 1.71) % ; q = 46.27, 24.57, all P 99 Tc-M DP group and MTX group, the level of bcl-2 was remarkably decreased [(30.24 ± 2.09) %, (27.25 ± 3.33) %] compared with CIA model group (q = 13.20, 17.56, all P 99 Tc-MDP group [(26. 58 ± 2. 52) %] and MTX group [(27.06 ± 1.92) %] was remarksbly increased [(24.26 ± 2.75) %, (23.53 ± 0.74) % ; q = 6.53, 7.01, all P 99 Tc-MDP could improve the signs of arthritis, meanwhile regulate the expression of bcl-2 and bax in synoviocytes and articular ehondrocytes, suggesting that one of the

Advances in the Surgical Management of Articular Cartilage Defects: Autologous Chondrocyte Implantation Techniques in the Pipeline.

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Stein, Spencer; Strauss, Eric; Bosco, Joseph

2013-01-01

The purpose of this review is to gain insight into the latest methods of articular cartilage implantation (ACI) and to detail where they are in the Food and Drug Administration approval and regulatory process. A PubMed search was performed using the phrase "Autologous Chondrocyte Implantation" alone and with the words second generation and third generation. Additionally, clinicaltrials.gov was searched for the names of the seven specific procedures and the parent company websites were referenced. Two-Stage Techniques: BioCart II uses a FGF2v1 culture and a fibrinogen, thrombin matrix, whereas Hyalograft-C uses a Hyaff 11 matrix. MACI uses a collagen I/III matrix. Cartipatch consists of an agarose-alginate hydrogel. Neocart uses a high-pressure bioreactor for culturing with a type I collagen matrix. ChondroCelect makes use of a gene expression analysis to predict chondrocyte proliferation and has demonstrated significant clinical improvement, but failed to show superiority to microfracture in a phase III trial. One Step Technique: CAIS is an ACI procedure where harvested cartilage is minced and implanted into a matrix for defect filling. As full thickness defects in articular cartilage continue to pose a challenge to treat, new methods of repair are being researched. Later generation ACI has been developed to address the prevalence of fibrocartilage with microfracture and the complications associated with the periosteal flap of first generation ACI such as periosteal hypertrophy. The procedures and products reviewed here represent advances in tissue engineering, scaffolds and autologous chondrocyte culturing that may hold promise in our quest to alter the natural history of symptomatic chondral disease.

Cell compaction influences the regenerative potential of passaged bovine articular chondrocytes in an ex vivo cartilage defect model.

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Schmutzer, Michael; Aszodi, Attila

2017-04-01

The loss and degradation of articular cartilage tissue matrix play central roles in the process of osteoarthritis (OA). New models for evaluating cartilage repair/regeneration are thus of great value for transferring various culture systems into clinically relevant situations. The repair process can be better monitored in ex vivo systems than in in vitro cell cultures. I have therefore established an ex vivo defect model prepared from bovine femoral condyles for evaluating cartilage repair by the implantation of cells cultured in various ways, e.g., monolayer-cultured cells or suspension or pellet cultures of articular bovine chondrocytes representing different cell compactions with variable densities of chondrocytes. I report that the integrin subunit α10 was significantly upregulated in suspension-cultured bovine chondrocytes at passage P2 compared with monolayer-cultured cells at P1 (p = 0.0083) and P2 (p innovation of this system over in vitro differentiation (e.g., micromass, pellet) assays is the possibility of examining and evaluating cartilage regeneration in an environment in which implanted cells are embedded within native surrounding tissue at the defect site. Such ex vivo explants might serve as a better model system to mimic clinical situations. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Hyaluronic acid-coated bovine serum albumin nanoparticles loaded with brucine as selective nanovectors for intra-articular injection

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Chen Z

2013-10-01

Full Text Available Zhipeng Chen,* Juan Chen,* Li Wu, Weidong Li, Jun Chen, Haibo Cheng, Jinhuo Pan, Baochang CaiDepartment of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, People's Republic of China*These authors contributed equally to this workObjective: To evaluate the potential of hyaluronic acid (HA-coated bovine serum albumin nanoparticles (BSANPs as a novel chondrocyte-targeting drug-delivery nanomedicine.Methods: The HA-BSANPs were characterized by dynamic light scattering, transmission electron microscopy, differential scanning calorimetry, and X-ray diffraction. Fluorescence imaging was used to visualize the distribution of nanoparticles after intra-articular injection. The chondrocyte-targeting efficiency and cellular uptake mechanism of HA-BSANPs were investigated using endocytic inhibitors.Results: HA-BSANPs were successfully prepared with HA coating the surface and amorphous drug in the core. Compared with BSANPs, HA-BSANPs exhibited improved uptake by chondrocytes through a receptor-mediated active uptake mechanism. The endocytosis process of BSANPs and HA-BSANPs involved clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis. No apparent thickening or hyperplasia of the synovium was observed in either BSANPs or HA-BSANPs. The HA-BSANPs could reside in the articular cavity of rats for more than 14 days, which was significantly longer than BSANPs.Conclusion: HA-BSANPs are a promising carrier for articular-related diseases due to elongated articular residence and improved chondrocytic accumulation.Keywords: chondrocyte, intra-articular injection, hyaluronic acid, BSA, nanoparticles

Elastic cartilage reconstruction by transplantation of cultured hyaline cartilage-derived chondrocytes.

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Mizuno, M; Takebe, T; Kobayashi, S; Kimura, S; Masutani, M; Lee, S; Jo, Y H; Lee, J I; Taniguchi, H

2014-05-01

Current surgical intervention of craniofacial defects caused by injuries or abnormalities uses reconstructive materials, such as autologous cartilage grafts. Transplantation of autologous tissues, however, places a significant invasiveness on patients, and many efforts have been made for establishing an alternative graft. Recently, we and others have shown the potential use of reconstructed elastic cartilage from ear-derived chondrocytes or progenitors with the unique elastic properties. Here, we examined the differentiation potential of canine joint cartilage-derived chondrocytes into elastic cartilage for expanding the cell sources, such as hyaline cartilage. Articular chondrocytes are isolated from canine joint, cultivated, and compared regarding characteristic differences with auricular chondrocytes, including proliferation rates, gene expression, extracellular matrix production, and cartilage reconstruction capability after transplantation. Canine articular chondrocytes proliferated less robustly than auricular chondrocytes, but there was no significant difference in the amount of sulfated glycosaminoglycan produced from redifferentiated chondrocytes. Furthermore, in vitro expanded and redifferentiated articular chondrocytes have been shown to reconstruct elastic cartilage on transplantation that has histologic characteristics distinct from hyaline cartilage. Taken together, cultured hyaline cartilage-derived chondrocytes are a possible cell source for elastic cartilage reconstruction. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

Fibrin and poly(lactic-co-glycolic acid) hybrid scaffold promotes early chondrogenesis of articular chondrocytes: an in vitro study.

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Sha'ban, Munirah; Kim, Soon Hee; Idrus, Ruszymah Bh; Khang, Gilson

2008-04-25

Synthetic- and naturally derived- biodegradable polymers have been widely used to construct scaffolds for cartilage tissue engineering. Poly(lactic-co-glycolic acid) (PLGA) are bioresorbable and biocompatible, rendering them as a promising tool for clinical application. To minimize cells lost during the seeding procedure, we used the natural polymer fibrin to immobilize cells and to provide homogenous cells distribution in PLGA scaffolds. We evaluated in vitro chondrogenesis of rabbit articular chondrocytes in PLGA scaffolds using fibrin as cell transplantation matrix. PLGA scaffolds were soaked in chondrocytes-fibrin suspension (1 x 10(6) cells/scaffold) and polymerized by dropping thrombin-calcium chloride (CaCl2) solution. PLGA-seeded chondrocytes was used as control. All constructs were cultured for a maximum of 21 days. Cell proliferation activity was measured at 1, 3, 7, 14 and 21 days in vitro using 3-(4,5-dimethylthiazole-2-yl)-2-, 5-diphenyltetrazolium-bromide (MTT) assay. Morphological observation, histology, immunohistochemistry (IHC), gene expression and sulphated-glycosaminoglycan (sGAG) analyses were performed at each time point of 1, 2 and 3 weeks to elucidate in vitro cartilage development and deposition of cartilage-specific extracellular matrix (ECM). Cell proliferation activity was gradually increased from day-1 until day-14 and declined by day-21. A significant cartilaginous tissue formation was detected as early as 2-week in fibrin/PLGA hybrid construct as confirmed by the presence of cartilage-isolated cells and lacunae embedded within basophilic ECM. Cartilage formation was remarkably evidenced after 3 weeks. Presence of cartilage-specific proteoglycan and glycosaminoglycan (GAG) in fibrin/PLGA hybrid constructs were confirmed by positive Safranin O and Alcian Blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrix. Chondrogenic properties were further demonstrated by the expression of genes encoded for

HIF-1α-induced HSP70 regulates anabolic responses in articular chondrocytes under hypoxic conditions.

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Tsuchida, Shinji; Arai, Yuji; Takahashi, Kenji A; Kishida, Tsunao; Terauchi, Ryu; Honjo, Kuniaki; Nakagawa, Shuji; Inoue, Hiroaki; Ikoma, Kazuya; Ueshima, Keiichiro; Matsuki, Tomohiro; Mazda, Osam; Kubo, Toshikazu

2014-08-01

We assessed whether heat shock protein 70 (HSP70) is involved in hypoxia inducible factor 1 alpha (HIF-1α)-dependent anabolic pathways in articular chondrocytes under hypoxic conditions. Primary rabbit chondrocytes were cultured under normoxia (20% oxygen condition) or hypoxia (1% oxygen condition). Alternatively, cells cultured under normoxia were treated with CoCl2 , which induces HIF-1α, to simulate hypoxia, or transfected with siRNAs targeting HIF-1α (si-HIF-1α) and HSP70 (si-HSP70) under hypoxia. HSP70 expression was enhanced by the increased expression of HIF-1α under hypoxia or simulated hypoxia, but not in the presence of si-HIF-1α. Hypoxia-induced overexpression of ECM genes was significantly suppressed by si-HIF-1α or si-HSP70. Cell viability positively correlated with hypoxia, but transfection with si-HIF-1α or si-HSP70 abrogated the chondroprotective effects of hypoxia. Although LDH release from sodium nitroprusside-treated cells and the proportion of TUNEL positive cells were decreased under hypoxia, transfection with si-HIF-1α or si-HSP70 almost completely blocked these effects. These findings indicated that HIF-1α-induced HSP70 overexpression increased the expression levels of ECM genes and cell viability, and protected chondrocytes from apoptosis. HIF-1α may regulate the anabolic effects of chondrocytes under hypoxic conditions by regulating HSP70 expression. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Bovine lactoferricin induces TIMP-3 via the ERK1/2-Sp1 axis in human articular chondrocytes.

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Yan, Dongyao; Chen, Di; Hawse, John R; van Wijnen, Andre J; Im, Hee-Jeong

2013-03-15

Bovine lactoferricin (LfcinB) is a heparan sulfate-binding peptide with multiple bioactivities. In human articular cartilage, LfcinB antagonizes interleukin-1 β (IL-1β) and fibroblast growth factor 2 (FGF-2) in proteoglycan metabolism, catabolic protease expression, and induction of pro-inflammatory mediators. LfcinB specifically activates ERK1/2, p38 and Akt, but whether these signaling pathways control the expression of LfcinB target genes remained unknown. In this report, we characterized a novel aspect of LfcinB-mediated genetic response in human articular chondrocytes, tissue inhibitor of metalloproteinase 3 (TIMP-3) induction. Inhibition of individual signaling pathways revealed that ERK1/2 functions as the major pathway in TIMP-3 expression, whereas Akt plays a minor role. Further investigation identified Sp1 as a critical transcriptional activator in TIMP-3 regulation, and Sp1 activity is modulated by ERK1/2, not Akt. Comparative quantification indicates that significant downregulation of TIMP-3 occurs in OA chondrocytes, suggesting a beneficial role of LfcinB in OA pathogenesis. Our results collectively provide new insights into the mechanism of action of LfcinB, and support the candidacy of LfcinB as a chondroprotective agent. Copyright © 2013 Elsevier B.V. All rights reserved.

Autologous Chondrocyte Implantation in Osteoarthritic Surroundings

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Ossendorff, Robert; Grad, Sibylle; Stoddart, Martin J

2018-01-01

BACKGROUND: Autologous chondrocyte implantation (ACI) fails in up to 20% of cases. Advanced intra-articular degeneration paired with an inflammatory environment may be closely related to implantation failure. Certain cytokines have been identified to play a major role during early osteoarthritis....... PURPOSE: To investigate the effects of tumor necrosis factor α (TNFα) and its potential inhibition by adalimumab on cartilage regeneration in an in vitro model of ACI. STUDY DESIGN: Controlled laboratory study. METHODS: Bovine articular chondrocytes were cultivated and transferred at passage 3 to fibrin...

Hyaline Articular Matrix Formed by Dynamic Self-Regenerating Cartilage and Hydrogels.

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Meppelink, Amanda M; Zhao, Xing; Griffin, Darvin J; Erali, Richard; Gill, Thomas J; Bonassar, Lawrence J; Redmond, Robert W; Randolph, Mark A

2016-07-01

Injuries to the articular cartilage surface are challenging to repair because cartilage possesses a limited capacity for self-repair. The outcomes of current clinical procedures aimed to address these injuries are inconsistent and unsatisfactory. We have developed a novel method for generating hyaline articular cartilage to improve the outcome of joint surface repair. A suspension of 10(7) swine chondrocytes was cultured under reciprocating motion for 14 days. The resulting dynamic self-regenerating cartilage (dSRC) was placed in a cartilage ring and capped with fibrin and collagen gel. A control group consisted of chondrocytes encapsulated in fibrin gel. Constructs were implanted subcutaneously in nude mice and harvested after 6 weeks. Gross, histological, immunohistochemical, biochemical, and biomechanical analyses were performed. In swine patellar groove, dSRC was implanted into osteochondral defects capped with collagen gel and compared to defects filled with osteochondral plugs, collagen gel, or left empty after 6 weeks. In mice, the fibrin- and collagen-capped dSRC constructs showed enhanced contiguous cartilage matrix formation over the control of cells encapsulated in fibrin gel. Biochemically, the fibrin and collagen gel dSRC groups were statistically improved in glycosaminoglycan and hydroxyproline content compared to the control. There was no statistical difference in the biomechanical data between the dSRC groups and the control. The swine model also showed contiguous cartilage matrix in the dSRC group but not in the collagen gel and empty defects. These data demonstrate the survivability and successful matrix formation of dSRC under the mechanical forces experienced by normal hyaline cartilage in the knee joint. The results from this study demonstrate that dSRC capped with hydrogels successfully engineers contiguous articular cartilage matrix in both nonload-bearing and load-bearing environments.

Treatment of articular cartilage lesions of the knee by microfracture or autologous chondrocyte implantation: a systematic review.

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Oussedik, Sam; Tsitskaris, Konstantinos; Parker, David

2015-04-01

We performed a systematic review of the treatment of articular cartilage lesions of the knee by microfracture or autologous chondrocyte implantation to determine the differences in patient outcomes after these procedures. We searched PubMed/Medline, Embase, and The Cochrane Library databases in the period from January 10 through January 20, 2013, and included 34 articles in our qualitative analysis. All studies showed improvement in outcome scores in comparison with baseline values, regardless of the treatment modality. The heterogeneity of the results presented in the studies precluded a meta-analysis. Microfracture appears to be effective in smaller lesions and is usually associated with a greater proportion of fibrocartilage production, which may have an effect on durability and eventual failure. Autologous chondrocyte implantation is an effective treatment that may result in a greater proportion of hyaline-like tissue at the repair site, which may in turn have a beneficial effect on durability and failure; it appears to be effective in larger lesions. Autologous chondrocyte implantation with periosteum has been shown to be associated with symptomatic cartilage hypertrophy more frequently than autologous chondrocyte implantation with collagen membrane. Matrix-associated autologous chondrocyte implantation is technically less challenging than the other techniques available, and in lesions greater than 4 cm(2), it has been shown to be more effective than microfracture. Level IV, systematic review of Level I-IV studies. Copyright © 2015 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

Reconstruction of Hyaline Cartilage Deep Layer Properties in 3-Dimensional Cultures of Human Articular Chondrocytes.

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Nanduri, Vibudha; Tattikota, Surendra Mohan; T, Avinash Raj; Sriramagiri, Vijaya Rama Rao; Kantipudi, Suma; Pande, Gopal

2014-06-01

Articular cartilage (AC) injuries and malformations are commonly noticed because of trauma or age-related degeneration. Many methods have been adopted for replacing or repairing the damaged tissue. Currently available AC repair methods, in several cases, fail to yield good-quality long-lasting results, perhaps because the reconstructed tissue lacks the cellular and matrix properties seen in hyaline cartilage (HC). To reconstruct HC tissue from 2-dimensional (2D) and 3-dimensional (3D) cultures of AC-derived human chondrocytes that would specifically exhibit the cellular and biochemical properties of the deep layer of HC. Descriptive laboratory study. Two-dimensional cultures of human AC-derived chondrocytes were established in classical medium (CM) and newly defined medium (NDM) and maintained for a period of 6 weeks. These cells were suspended in 2 mm-thick collagen I gels, placed in 24-well culture inserts, and further cultured up to 30 days. Properties of chondrocytes, grown in 2D cultures and the reconstructed 3D cartilage tissue, were studied by optical and scanning electron microscopic techniques, immunohistochemistry, and cartilage-specific gene expression profiling by reverse transcription polymerase chain reaction and were compared with those of the deep layer of native human AC. Two-dimensional chondrocyte cultures grown in NDM, in comparison with those grown in CM, showed more chondrocyte-specific gene activity and matrix properties. The NDM-grown chondrocytes in 3D cultures also showed better reproduction of deep layer properties of HC, as confirmed by microscopic and gene expression analysis. The method used in this study can yield cartilage tissue up to approximately 1.6 cm in diameter and 2 mm in thickness that satisfies the very low cell density and matrix composition properties present in the deep layer of normal HC. This study presents a novel and reproducible method for long-term culture of AC-derived chondrocytes and reconstruction of cartilage

Increasing the Dose of Autologous Chondrocytes Improves Articular Cartilage Repair: Histological and Molecular Study in the Sheep Animal Model.

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Guillén-García, Pedro; Rodríguez-Iñigo, Elena; Guillén-Vicente, Isabel; Caballero-Santos, Rosa; Guillén-Vicente, Marta; Abelow, Stephen; Giménez-Gallego, Guillermo; López-Alcorocho, Juan Manuel

2014-04-01

We hypothesized that implanting cells in a chondral defect at a density more similar to that of the intact cartilage could induce them to synthesize matrix with the features more similar to that of the uninjured one. We compared the implantation of different doses of chondrocytes: 1 million (n = 5), 5 million (n = 5), or 5 million mesenchymal cells (n = 5) in the femoral condyle of 15 sheep. Tissue generated by microfracture at the trochlea, and normal cartilage from a nearby region, processed as the tissues resulting from the implantation, were used as references. Histological and molecular (expression of type I and II collagens and aggrecan) studies were performed. The features of the cartilage generated by implantation of mesenchymal cells and elicited by microfractures were similar and typical of a poor repair of the articular cartilage (presence of fibrocartilage, high expression of type I collagen and a low mRNA levels of type II collagen and aggrecan). Nevertheless, in the samples obtained from tissues generated by implantation of chondrocytes, hyaline-like cartilage, cell organization, low expression rates of type I collagen and high levels of mRNA corresponding to type II collagen and aggrecan were observed. These histological features, show less variability and are more similar to those of the normal cartilage used as control in the case of 5 million cells implantation than when 1 million cells were used. The implantation of autologous chondrocytes in type I/III collagen membranes at high density could be a promising tool to repair articular cartilage.

Fibrin and poly(lactic-co-glycolic acid hybrid scaffold promotes early chondrogenesis of articular chondrocytes: an in vitro study

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Idrus Ruszymah BH

2008-04-01

Full Text Available Abstract Background Synthetic- and naturally derived- biodegradable polymers have been widely used to construct scaffolds for cartilage tissue engineering. Poly(lactic-co-glycolic acid (PLGA are bioresorbable and biocompatible, rendering them as a promising tool for clinical application. To minimize cells lost during the seeding procedure, we used the natural polymer fibrin to immobilize cells and to provide homogenous cells distribution in PLGA scaffolds. We evaluated in vitro chondrogenesis of rabbit articular chondrocytes in PLGA scaffolds using fibrin as cell transplantation matrix. Methods PLGA scaffolds were soaked in chondrocytes-fibrin suspension (1 × 106cells/scaffold and polymerized by dropping thrombin-calcium chloride (CaCl2 solution. PLGA-seeded chondrocytes was used as control. All constructs were cultured for a maximum of 21 days. Cell proliferation activity was measured at 1, 3, 7, 14 and 21 days in vitro using 3-(4,5-dimethylthiazole-2-yl-2-, 5-diphenyltetrazolium-bromide (MTT assay. Morphological observation, histology, immunohistochemistry (IHC, gene expression and sulphated-glycosaminoglycan (sGAG analyses were performed at each time point of 1, 2 and 3 weeks to elucidate in vitro cartilage development and deposition of cartilage-specific extracellular matrix (ECM. Results Cell proliferation activity was gradually increased from day-1 until day-14 and declined by day-21. A significant cartilaginous tissue formation was detected as early as 2-week in fibrin/PLGA hybrid construct as confirmed by the presence of cartilage-isolated cells and lacunae embedded within basophilic ECM. Cartilage formation was remarkably evidenced after 3 weeks. Presence of cartilage-specific proteoglycan and glycosaminoglycan (GAG in fibrin/PLGA hybrid constructs were confirmed by positive Safranin O and Alcian Blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrix. Chondrogenic properties were further

In Vitro Expression of the Extracellular Matrix Components Aggrecan, Collagen Types I and II by Articular Cartilage-Derived Chondrocytes.

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Schneevoigt, J; Fabian, C; Leovsky, C; Seeger, J; Bahramsoltani, M

2017-02-01

The extracellular matrix (ECM) of hyaline cartilage is perfectly suited to transmit articular pressure load to the subchondral bone. Pressure is transferred by a high amount of aggrecan-based proteoglycans and collagen type II fibres in particular. After any injury, the hyaline cartilage is replaced by fibrocartilage, which is low in proteoglycans and contains collagen type I predominantly. Until now, long-term results of therapeutic procedures including cell-based therapies like autologous chondrocyte transplantation (ACT) lead to a replacement tissue meeting the composition of fibrocartilage. Therefore, it is of particular interest to discover how and to what extent isolation and in vitro cultivation of chondrocytes affect the cells and their expression of ECM components. Hyaline cartilage-derived chondrocytes were cultivated in vitro and observed microscopically over a time period of 35 days. The expression of collagen type I, collagen type II and aggrecan was analysed using RT-qPCR and Western blot at several days of cultivation. Chondrocytes presented a longitudinal shape for the entire cultivation period. While expression of collagen type I prevailed within the first days, only prolonged cultivation led to an increase in collagen type II and aggrecan expression. The results indicate that chondrocyte isolation and in vitro cultivation lead to a dedifferentiation at least to the stage of chondroprogenitor cells. © 2016 Blackwell Verlag GmbH.

Formation of Hyaline Cartilage Tissue by Passaged Human Osteoarthritic Chondrocytes.

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Bianchi, Vanessa J; Weber, Joanna F; Waldman, Stephen D; Backstein, David; Kandel, Rita A

2017-02-01

When serially passaged in standard monolayer culture to expand cell number, articular chondrocytes lose their phenotype. This results in the formation of fibrocartilage when they are used clinically, thus limiting their use for cartilage repair therapies. Identifying a way to redifferentiate these cells in vitro is critical if they are to be used successfully. Transforming growth factor beta (TGFβ) family members are known to be crucial for regulating differentiation of fetal limb mesenchymal cells and mesenchymal stromal cells to chondrocytes. As passaged chondrocytes acquire a progenitor-like phenotype, the hypothesis of this study was that TGFβ supplementation will stimulate chondrocyte redifferentiation in vitro in serum-free three-dimensional (3D) culture. Human articular chondrocytes were serially passaged twice (P2) in monolayer culture. P2 cells were then placed in high-density (3D) culture on top of membranes (Millipore) and cultured for up to 6 weeks in chemically defined serum-free redifferentiation media (SFRM) in the presence or absence of TGFβ. The tissues were evaluated histologically, biochemically, by immunohistochemical staining, and biomechanically. Passaged human chondrocytes cultured in SFRM supplemented with 10 ng/mL TGFβ3 consistently formed a continuous layer of articular-like cartilage tissue rich in collagen type 2 and aggrecan and lacking collagen type 1 and X in the absence of a scaffold. The tissue developed a superficial zone characterized by expression of lubricin and clusterin with horizontally aligned collagen fibers. This study suggests that passaged human chondrocytes can be used to bioengineer a continuous layer of articular cartilage-like tissue in vitro scaffold free. Further study is required to evaluate their ability to repair cartilage defects in vivo.

Gel-type autologous chondrocyte (Chondron™ implantation for treatment of articular cartilage defects of the knee

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Chun Chung-Woo

2010-05-01

Full Text Available Abstract Background Gel-type autologous chondrocyte (Chondron™ implantations have been used for several years without using periosteum or membrane. This study involves evaluations of the clinical results of Chondron™ at many clinical centers at various time points during the postoperative patient follow-up. Methods Data from 98 patients with articular cartilage injury of the knee joint and who underwent Chondron™ implantation at ten Korean hospitals between January 2005 and November 2008, were included and were divided into two groups based on the patient follow-up period, i.e. 13~24-month follow-up and greater than 25-month follow-up. The telephone Knee Society Score obtained during telephone interviews with patients, was used as the evaluation tool. Results On the tKSS-A (telephone Knee Society Score-A, the score improved from 43.52 ± 20.20 to 89.71 ± 13.69 (P Conclusion Gel-type autologous chondrocyte implantation for chondral knee defects appears to be a safe and effective method for both decreasing pain and improving knee function.

Treatment of a Focal Articular Cartilage Defect of the Talus with Polymer-Based Autologous Chondrocyte Implantation: A 12-Year Follow-Up Period.

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Kreuz, Peter Cornelius; Kalkreuth, Richard Horst; Niemeyer, Philipp; Uhl, Markus; Erggelet, Christoph

Autologous chondrocyte implantation (ACI) is a first-line treatment option for large articular cartilage defects. Although well-established for cartilage defects in the knee, studies of the long-term outcomes of matrix-assisted ACI to treat cartilage defects in the ankle are rare. In the present report, we describe for the first time the long-term clinical and radiologic results 12 years after polymer-based matrix-assisted ACI treat a full-thickness talar cartilage defect in a 25-year-old male patient. The clinical outcome was assessed using the visual analog scale and Freiburg ankle score, magnetic resonance imaging evaluation using the Henderson-Kreuz scoring system and T2 mapping. Clinical assessment revealed improved visual analog scale and Freiburg ankle scores. The radiologic analysis and T2 relaxation time values indicated the formation of hyaline-like repair tissue. Polymer-based autologous chondrocytes has been shown to be a safe and clinically effective long-term treatment of articular cartilage defects in the talus. Copyright © 2017 American College of Foot and Ankle Surgeons. Published by Elsevier Inc. All rights reserved.

Autologous chondrocytes as a novel source for neo-chondrogenesis in haemophiliacs.

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Stocco, Elena; Barbon, Silvia; Radossi, Paolo; Rajendran, Senthilkumar; Dalzoppo, Daniele; Bortolami, Marina; Bagno, Andrea; Grandi, Francesca; Gamba, Pier Giorgio; Parnigotto, Pier Paolo; Tagariello, Giuseppe; Grandi, Claudio

2016-10-01

Haemophilic arthropathy is the major cause of disability in patients with haemophilia and, despite prophylaxis with coagulation factor concentrates, some patients still develop articular complications. We evaluate the feasibility of a tissue engineering approach to improve current clinical strategies for cartilage regeneration in haemophiliacs by using autologous chondrocytes (haemophilic chondrocytes; HaeCs). Little is known about articular chondrocytes from haemophilic patients and no characterisation has as yet been performed. An investigation into whether blood exposure alters HaeCs should be interesting from the perspective of autologous implants. The typical morphology and expression of specific target genes and surface markers were therefore assessed by optical microscopy, reverse transcription plus the polymerase chain reaction (PCR), real-time PCR and flow-cytometry. We then considered chondrocyte behaviour on a bio-hybrid scaffold (based on polyvinyl alcohol/Wharton's jelly) as an in vitro model of articular cartilage prosthesis. Articular chondrocytes from non-haemophilic donors were used as controls. HaeC morphology and the resulting immunophenotype CD44(+)/CD49c(+)/CD49e(+)/CD151(+)/CD73(+)/CD49f(-)/CD26(-) resembled those of healthy donors. Moreover, HaeCs were active in the transcription of genes involved in the synthesis of the extracellular matrix proteins of the articular cartilage (ACAN, COL1A, COL2A, COL10A, COL9A, COMP, HAS1, SOX9), although the over-expression of COL1A1, COL10A1, COMP and HAS was observed. In parallel, the composite scaffold showed adequate mechanical and biological properties for cartilage tissue engineering, promoting chondrocyte proliferation. Our preliminary evidence contributes to the characterisation of HaeCs, highlighting the opportunity of using them for autologous cartilage implants in patients with haemophilia.

Influence of cytochalasin D-induced changes in cell shape on proteoglycan synthesis by cultured articular chondrocytes

International Nuclear Information System (INIS)

Newman, P.; Watt, F.M.

1988-01-01

There is growing evidence that cell shape regulates both proliferation and differentiated gene expression in a variety of cell types. The authors have explored the relationship between the morphology of articular chondrocytes in culture and the amount and type of proteoglycan they synthesize, using cytochalasin D to induce reversible cell rounding. When chondrocytes were prevented from spreading or when spread cells were induced to round up, 35 SO 4 incorporation into proteoglycan was stimulated. Incorporation into the cell layer was stimulated more than into the medium. When the cells were allowed to respread by removing cytochalasin D, proteoglycan synthesis returned to control levels. Cytochalasin D-induced stimulation of 35 SO 4 incorporation reflected an increase in core protein synthesis rather than lengthening of glycosaminoglycan chains, because [ 3 H]serine incorporation into core protein was also stimulated. Cytochalasm D-treatment of cells in suspension caused no further stimulation of 35 SO 4 incorporation, suggesting that the observed effects were due to cell rounding rather than exposure to cytochalasin D per se

MR imaging of autologous chondrocyte implantation of the knee

Energy Technology Data Exchange (ETDEWEB)

James, S.L.J.; Connell, D.A.; Saifuddin, A.; Skinner, J.A.; Briggs, T.W.R. [RNOH Stanmore, Department of Radiology, Stanmore, Middlesex (United Kingdom)

2006-05-15

Autologous chondrocyte implantation (ACI) is a surgical technique that is increasingly being used in the treatment of full-thickness defects of articular cartilage in the knee. It involves the arthroscopic harvesting and in vitro culture of chondrocytes that are subsequently implanted into a previously identified chondral defect. The aim is to produce a repair tissue that closely resembles hyaline articular cartilage that gradually becomes incorporated, restoring joint congruity. Over the long term, it is hoped that this will prevent the progression of full-thickness articular cartilage defects to osteoarthritis. This article reviews the indications and operative procedure performed in ACI. Magnetic resonance imaging (MRI) sequences that provide optimal visualization of articular cartilage in the post-operative period are discussed. Normal appearances of ACI on MRI are presented along with common complications that are encountered with this technique. (orig.)

Laser biostimulation of articular cartilage: in vitro evaluation

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Jia, Yali; Guo, Zhouyi; Yang, Xiaohong; Zeng, Chang-Chun

2004-07-01

In the orthopaedic field, the repair of ariticular cartilage is still a difficult problem, because of the physiological characters of cartilaginous tissues and chondrocytes. To find an effective method of stimulating their regeneration, this in vitro study focuses on the biostimulation of rabbit articular chondrocytes by low-power He-Ne laser. The articular chondrocytes isolated from the cartilage of the medial condyle of the femur of the rabbit were incubated in HamF12 medium. The second passage culture were spread on 24 petri dishes and were irradiated with laser at power density of 2 - 12 mW/cm2 for 6.5 minutes, corresponding to the energy density of 1-6 J/cm2. Laser treatment was performed three times at a 24-hour interval. After lasering, incubation was continued for 24 hours. Non-irradiated cells were kept under the same conditions as the irradiated ones. The cell proliferation activity was evaluated with a XTT colorimetric method. Irradiation of 4 - 6 J/cm2 revealed a considerably higher cell proliferation activity comparing to control cultures. Thereinto, the energy density of 4 and 5 J/cm2 remarkably increased cell growth (P<0.01). The present study showed that a particular laser irradiation stimulates articular chondrocytes proliferation. These findings might be clinically relevant, indicating that low-power laser irradiation treatment is likely to achieve the repair of articular cartilage in clinic.

Effects of non-steroidal anti-inflammatory drugs on cell proliferation and death in cultured epiphyseal-articular chondrocytes of fetal rats

International Nuclear Information System (INIS)

Chang, J.-K.; Wu, S.-C.; Wang, G.-J.

2006-01-01

Previous reports indicated that non-steroidal anti-inflammatory drugs (NSAIDs) suppress bone repair. Our previous study further found that ketorolac delayed the endochondral bone formation, and the critical effective timing was at the early stage of repair. Furthermore, we found that NSAIDs suppressed proliferation and induced cell death of cultured osteoblasts. In this study, we hypothesized that chondrocytic proliferation and death, which plays an important role at the early stage of endochondral bone formation, might be affected by NSAIDs. Non-selective NSAIDs, indomethacin, ketorolac, diclofenac and piroxicam; cyclooxygenase-2 (COX-2) selective NSAIDs, celecoxib and DFU (an analog of rofecoxib); prostaglandins (PGs), PGE1, PGE2 and PGF2α; and each NSAID plus each PG were tested. The effects of NSAIDs on proliferation, cell cycle kinetics, cytotoxicity and cell death of epiphyseal-articular chondrocytes of fetal rats were examined. The results showed that all the tested NSAIDs, except DFU, inhibited thymidine incorporation of chondrocytes at a concentration range (10 -8 to 10 -4 M) covering the theoretic therapeutic concentrations. Cell cycle was arrested by NSAIDs at the G /G 1 phase. Upon a 24 h treatment, LDH leakage and cell death (both apoptosis and necrosis) were significantly induced by the four non-selective NSAIDs in chondrocyte cultures. However, COX-2 inhibitors revealed non-significant effects on cytotoxicity of chondrocytes except higher concentration of celecoxib (10 -4 M). Replenishments of PGE1, PGE2 or PGF2α could not reverse the effects of NSAIDs on chondrocytic proliferation and cytotoxicity. In this study, we found that therapeutic concentrations of non-selective NSAIDs caused proliferation suppression and cell death of chondrocytes, suggesting these adverse effects may be one of the reasons that NSAIDs delay the endochondral ossification during bone repair found in previous studies. Furthermore, these effects of NSAIDs may act via PG

Epiphyseal abnormalities, trabecular bone loss and articular chondrocyte hypertrophy develop in the long bones of postnatal Ext1-deficient mice.

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Sgariglia, Federica; Candela, Maria Elena; Huegel, Julianne; Jacenko, Olena; Koyama, Eiki; Yamaguchi, Yu; Pacifici, Maurizio; Enomoto-Iwamoto, Motomi

2013-11-01

Long bones are integral components of the limb skeleton. Recent studies have indicated that embryonic long bone development is altered by mutations in Ext genes and consequent heparan sulfate (HS) deficiency, possibly due to changes in activity and distribution of HS-binding/growth plate-associated signaling proteins. Here we asked whether Ext function is continuously required after birth to sustain growth plate function and long bone growth and organization. Compound transgenic Ext1(f/f);Col2CreERT mice were injected with tamoxifen at postnatal day 5 (P5) to ablate Ext1 in cartilage and monitored over time. The Ext1-deficient mice exhibited growth retardation already by 2weeks post-injection, as did their long bones. Mutant growth plates displayed a severe disorganization of chondrocyte columnar organization, a shortened hypertrophic zone with low expression of collagen X and MMP-13, and reduced primary spongiosa accompanied, however, by increased numbers of TRAP-positive osteoclasts at the chondro-osseous border. The mutant epiphyses were abnormal as well. Formation of a secondary ossification center was significantly delayed but interestingly, hypertrophic-like chondrocytes emerged within articular cartilage, similar to those often seen in osteoarthritic joints. Indeed, the cells displayed a large size and round shape, expressed collagen X and MMP-13 and were surrounded by an abundant Perlecan-rich pericellular matrix not seen in control articular chondrocytes. In addition, ectopic cartilaginous outgrowths developed on the lateral side of mutant growth plates over time that resembled exostotic characteristic of children with Hereditary Multiple Exostoses, a syndrome caused by Ext mutations and HS deficiency. In sum, the data do show that Ext1 is continuously required for postnatal growth and organization of long bones as well as their adjacent joints. Ext1 deficiency elicits defects that can occur in human skeletal conditions including trabecular bone loss

Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis.

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David Garciadiego-Cázares

Full Text Available The Integrin β1 family is the major receptors of the Extracellular matrix (ECM, and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA. In this scenario, integrins modify their pattern expression and regulate chondrocyte differentiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β Superfamily, such as Growth differentiation factor 5 (Gdf-5 and Bone morphogenetic protein 7 (Bmp-7, play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedifferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressed αV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of the α5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh, Gdf-5 and α5 integrin to maintain articular cartilage and prevent

Human articular chondrocytes secrete parathyroid hormone-related protein and inhibit hypertrophy of mesenchymal stem cells in coculture during chondrogenesis.

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Fischer, J; Dickhut, A; Rickert, M; Richter, W

2010-09-01

The use of bone marrow-derived mesenchymal stem cells (MSCs) has shown promise in cell-based cartilage regeneration. A yet-unsolved problem, however, is the unwanted up-regulation of markers of hypertrophy, such as alkaline phosphatase (AP) and type X collagen, during in vitro chondrogenesis and the formation of unstable calcifying cartilage at heterotopic sites. In contrast, articular chondrocytes produce stable, nonmineralizing cartilage. The aim of this study was to address whether coculture of MSCs with human articular chondrocytes (HACs) can suppress the undesired hypertrophy in differentiating MSCs. MSCs were differentiated in chondrogenic medium that had or had not been conditioned by parallel culture with HAC pellets, or MSCs were mixed in the same pellet with the HACs (1:1 or 1:2 ratio) and cultured for 6 weeks. Following in vitro differentiation, the pellets were transplanted into SCID mice. The gene expression ratio of COL10A1 to COL2A1 and of Indian hedgehog (IHH) to COL2A1 was significantly reduced by differentiation in HAC-conditioned medium, and less type X collagen protein was deposited relative to type II collagen. AP activity was significantly lower (P chondrogenesis. The main inhibitory effects seen with HAC-conditioned medium were reproducible by PTHrP supplementation of unconditioned medium. HAC-derived soluble factors and direct coculture are potent means of improving chondrogenesis and suppressing the hypertrophic development of MSCs. PTHrP is an important candidate soluble factor involved in this effect.

Importance of Donor Chondrocyte Viability for Osteochondral Allografts.

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Cook, James L; Stannard, James P; Stoker, Aaron M; Bozynski, Chantelle C; Kuroki, Keiichi; Cook, Cristi R; Pfeiffer, Ferris M

2016-05-01

Osteochondral allograft (OCA) transplantation provides a biological treatment option for functional restoration of large articular cartilage defects in multiple joints. While successful outcomes after OCA transplantation have been linked to viable donor chondrocytes, the importance of donor cell viability has not been comprehensively validated. To use a canine model to determine the importance of donor chondrocyte viability at the time of implantation with respect to functional success of femoral condylar OCAs based on radiographic, gross, cell viability, histologic, biochemical, and biomechanical outcome measures. Controlled laboratory study. After approval was obtained from the institutional animal care and use committee, adult female dogs (N = 16) were implanted with 8-mm cylindrical OCAs from male dogs in the lateral and medial femoral condyles of 1 knee. OCAs were preserved for 28 or 60 days after procurement, and chondrocyte viability was quantified before implantation. Two different storage media, temperatures, and time points were used to obtain a spectrum of percentage chondrocyte viability at the time of implantation. A successful outcome was defined as an OCA that was associated with graft integration, maintenance of hyaline cartilage, lack of associated cartilage disorder, and lack of fibrillation, fissuring, or fibrous tissue infiltration of the allograft based on subjective radiographic, gross, and histologic assessments at 6 months after implantation. Chondrocyte viability ranged from 23% to 99% at the time of implantation. All successful grafts had >70% chondrocyte viability at the time of implantation, and no graft with chondrocyte viability <70% was associated with a successful outcome. Live-dead stained sections and histologic findings with respect to cell morphological features suggested that successful grafts were consistently composed of viable chondrocytes in lacunae, while grafts that were not successful were composed of nonviable

Treatment of osteoarthritis using a helper-dependent adenoviral vector retargeted to chondrocytes

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Merry ZC Ruan

2016-01-01

Full Text Available Osteoarthritis (OA is a joint disease characterized by degeneration of the articular cartilage, subchondral bone remodeling, and secondary inflammation. It is among the top three causes of chronic disability, and currently there are no treatment options to prevent disease progression. The localized nature of OA makes it an ideal candidate for gene and cell therapy. However, gene and cell therapy of OA is impeded by inefficient gene transduction of chondrocytes. In this study, we developed a broadly applicable system that retargets cell surface receptors by conjugating antibodies to the capsid of helper-dependent adenoviral vectors (HDVs. Specifically, we applied this system to retarget chondrocytes by conjugating an HDV to an α-10 integrin monoclonal antibody (a10mab. We show that a10mab-conjugated HDV (a10mabHDV-infected chondrocytes efficiently in vitro and in vivo while detargeting other cell types. The therapeutic index of an intra-articular injection of 10mabHDV-expressing proteoglycan 4 (PRG4 into a murine model of post-traumatic OA was 10-fold higher than with standard HDV. Moreover, we show that PRG4 overexpression from articular, superficial zone chondrocytes is effective for chondroprotection in postinjury OA and that α-10 integrin is an effective protein for chondrocyte targeting.

[The concept of cellular immortality, a myth or a reality. Example of "immortalized" articular chondrocytes].

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Adolphe, M; Thenet, S

1990-01-01

The concept of cellular immortality, which arose from the historical studies of A. Carrel, is getting a new start with the progress of virology. However, the definition of cell immortalization is still ambiguous. Although scientists agree that cells regarded as immortal have acquired an infinite growth capacity, the relationship of this change with the first stages of transformation is difficult to clearly define. Immortalized cell lines have already been obtained from numerous cell types by using viral infection or transfection with viral and cellular genes. Immortalization of cells is interesting for three main reasons: it permits study of the steps in progression to transformation, allows establishment of cell lines for producing biological products, and permits various cell types to maintain a part of their differentiated functions. For example, hypothalamic neurosecretory cells, macrophages, astrocytes and intestinal epithelial cells have been immortalized and these lines can be used for understanding the balance between division and differentiation, and also for pharmacotoxicological studies. In our laboratory, we immortalized rabbit articular chondrocytes by transfection with SV40 large T and little t encoding genes. At the 9th subculture, when the control culture was senescent, clones of polygonal cells appeared in the transfected cell cultures. Three clones have been selected and have been maintained in culture for two years. Growth curves of normal and SV40-transfected chondrocytes were compared and displayed similar doubling times (approximately 20 hours). The exponential phase of growth was longer for immortalized cells resulting in a 2-fold higher saturation density. These cells appear to be not fully transformed and maintain some properties of differentiated chondrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

On the genesis of articular cartilage. Embryonic joint development and gene expression - implications for tissue engineering

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Jenner, F

2013-01-01

Articular chondrocytes descend from a distinct cohort of progenitor cells located in the embryonic joint anlagen, termed interzones. Their unique lineage might explain some of the problems encountered using chondrocytes of different lineages for articular cartilage tissue engineering. While it is

Lithium Chloride Dependent Glycogen Synthase Kinase 3 Inactivation Links Oxidative DNA Damage, Hypertrophy and Senescence in Human Articular Chondrocytes and Reproduces Chondrocyte Phenotype of Obese Osteoarthritis Patients.

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Serena Guidotti

Full Text Available Recent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (OA, but at the same time, its inactivation has been proposed as an anti-inflammatory therapeutic option. Here we evaluated the extent of GSK3β inactivation in vivo in OA knee cartilage and the molecular events downstream GSK3β inactivation in vitro to assess their contribution to cell senescence and hypertrophy.In vivo level of phosphorylated GSK3β was analyzed in cartilage and oxidative damage was assessed by 8-oxo-deoxyguanosine staining. The in vitro effects of GSK3β inactivation (using either LiCl or SB216763 were evaluated on proliferating primary human chondrocytes by combined confocal microscopy analysis of Mitotracker staining and reactive oxygen species (ROS production (2',7'-dichlorofluorescin diacetate staining. Downstream effects on DNA damage and senescence were investigated by western blot (γH2AX, GADD45β and p21, flow cytometric analysis of cell cycle and light scattering properties, quantitative assessment of senescence associated β galactosidase activity, and PAS staining.In vivo chondrocytes from obese OA patients showed higher levels of phosphorylated GSK3β, oxidative damage and expression of GADD45β and p21, in comparison with chondrocytes of nonobese OA patients. LiCl mediated GSK3β inactivation in vitro resulted in increased mitochondrial ROS production, responsible for reduced cell proliferation, S phase transient arrest, and increase in cell senescence, size and granularity. Collectively, western blot data supported the occurrence of a DNA damage response leading to cellular senescence with increase in γH2AX, GADD45β and p21. Moreover, LiCl boosted 8-oxo-dG staining, expression of IKKα and MMP-10.In articular chondrocytes, GSK3β activity is required for the maintenance of proliferative potential and phenotype. Conversely, GSK3β inactivation, although preserving chondrocyte survival, results in functional impairment via

Lithium Chloride Dependent Glycogen Synthase Kinase 3 Inactivation Links Oxidative DNA Damage, Hypertrophy and Senescence in Human Articular Chondrocytes and Reproduces Chondrocyte Phenotype of Obese Osteoarthritis Patients.

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Guidotti, Serena; Minguzzi, Manuela; Platano, Daniela; Cattini, Luca; Trisolino, Giovanni; Mariani, Erminia; Borzì, Rosa Maria

2015-01-01

Recent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (OA), but at the same time, its inactivation has been proposed as an anti-inflammatory therapeutic option. Here we evaluated the extent of GSK3β inactivation in vivo in OA knee cartilage and the molecular events downstream GSK3β inactivation in vitro to assess their contribution to cell senescence and hypertrophy. In vivo level of phosphorylated GSK3β was analyzed in cartilage and oxidative damage was assessed by 8-oxo-deoxyguanosine staining. The in vitro effects of GSK3β inactivation (using either LiCl or SB216763) were evaluated on proliferating primary human chondrocytes by combined confocal microscopy analysis of Mitotracker staining and reactive oxygen species (ROS) production (2',7'-dichlorofluorescin diacetate staining). Downstream effects on DNA damage and senescence were investigated by western blot (γH2AX, GADD45β and p21), flow cytometric analysis of cell cycle and light scattering properties, quantitative assessment of senescence associated β galactosidase activity, and PAS staining. In vivo chondrocytes from obese OA patients showed higher levels of phosphorylated GSK3β, oxidative damage and expression of GADD45β and p21, in comparison with chondrocytes of nonobese OA patients. LiCl mediated GSK3β inactivation in vitro resulted in increased mitochondrial ROS production, responsible for reduced cell proliferation, S phase transient arrest, and increase in cell senescence, size and granularity. Collectively, western blot data supported the occurrence of a DNA damage response leading to cellular senescence with increase in γH2AX, GADD45β and p21. Moreover, LiCl boosted 8-oxo-dG staining, expression of IKKα and MMP-10. In articular chondrocytes, GSK3β activity is required for the maintenance of proliferative potential and phenotype. Conversely, GSK3β inactivation, although preserving chondrocyte survival, results in functional impairment via induction of

Considerations on the use of ear chondrocytes as donor chondrocytes for cartilage tissue engineering

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van Osch, Gerjo J. V. M.; Mandl, Erik W.; Jahr, Holger; Koevoet, Wendy; Nolst-Trenité, Gilbert; Verhaar, Jan A. N.

2004-01-01

Articular cartilage is often used for research on cartilage tissue engineering. However, ear cartilage is easier to harvest, with less donor-site morbidity. The aim of this study was to evaluate whether adult human ear chondrocytes were capable of producing cartilage after expansion in monolayer

Expression Profiling and Functional Implications of a Set of Zinc Finger Proteins, ZNF423, ZNF470, ZNF521, and ZNF780B, in Primary Osteoarthritic Articular Chondrocytes

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Maria Mesuraca

2014-01-01

Full Text Available Articular chondrocytes are responsible for the maintenance of healthy articulations; indeed, dysregulation of their functions, including the production of matrix proteins and matrix-remodeling proteases, may result in fraying of the tissue and development of osteoarthritis (OA. To explore transcriptional mechanisms that contribute to the regulation of chondrocyte homeostasis and may be implicated in OA development, we compared the gene expression profile of a set of zinc finger proteins potentially linked to the control of chondrocyte differentiation and/or functions (ZNF423, ZNF470, ZNF521, and ZNF780B in chondrocytes from patients affected by OA and from subjects not affected by OA. This analysis highlighted a significantly lower expression of the transcript encoding ZNF423 in chondrocytes from OA, particularly in elderly patients. Interestingly, this decrease was mirrored by the similarly reduced expression of PPARγ, a known target of ZNF423 with anti-inflammatory and chondroprotective properties. The ZNF521 mRNA instead was abundant in all primary chondrocytes studied; the RNAi-mediated silencing of this gene significantly altered the COL2A/COL1 expression ratio, associated with the maintenance of the differentiated phenotype, in chondrocytes cultivated in alginate beads. These results suggest a role for ZNF423 and ZNF521 in the regulation of chondrocyte homeostasis and warrant further investigations to elucidate their mechanism of action.

Diverse roles of integrin receptors in articular cartilage.

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Shakibaei, M; Csaki, C; Mobasheri, A

2008-01-01

Integrins are heterodimeric integral membrane proteins made up of alpha and beta subunits. At least eighteen alpha and eight beta subunit genes have been described in mammals. Integrin family members are plasma membrane receptors involved in cell adhesion and active as intra- and extracellular signalling molecules in a variety of processes including embryogenesis, hemostasis, tissue repair, immune response and metastatic spread of tumour cells. Integrin beta 1 (beta1-integrin), the protein encoded by the ITGB1 gene (also known as CD29 and VLAB), is a multi-functional protein involved in cell-matrix adhesion, cell signalling, cellular defense, cell adhesion, protein binding, protein heterodimerisation and receptor-mediated activity. It is highly expressed in the human body (17.4 times higher than the average gene in the last updated revision of the human genome). The extracellular matrix (ECM) of articular cartilage is a unique environment. Interactions between chondrocytes and the ECM regulate many biological processes important to homeostasis and repair of articular cartilage, including cell attachment, growth, differentiation and survival. The beta1-integrin family of cell surface receptors appears to play a major role in mediating cell-matrix interactions that are important in regulating these fundamental processes. Chondrocyte mechanoreceptors have been proposed to incorporate beta1-integrins and mechanosensitive ion channels which link with key ECM, cytoskeletal and signalling proteins to maintain the chondrocyte phenotype, prevent chondrocyte apoptosis and regulate chondrocyte-specific gene expression. This review focuses on the expression and function of beta1-integrins in articular chondrocytes, its role in the unique biology of these cells and its distribution in cartilage.

The Results of Fetal Chondrocytes Transplantation in Patients with Rheumatoid Arthritis

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Natalya Krivoruchko

2014-12-01

Full Text Available Introduction. Nowadays anti-inflammatory and immunosuppressive therapy has significantly improved the quality of life and prognosis of rheumatoid arthritis (RA. Nevertheless, there are still many patients with progressive rheumatoid inflammation, resulting in the destruction of joints. Cell therapy seems like a promising direction in rheumatology. The aim of our research was to evaluate the efficacy of fetal chondrocyte transplantation in patients with RA.Methods. We examined 60 patients with rheumatoid arthritis (I - III stages between 20 and 63 years of age. They were divided into 2 groups: the first group underwent the fetal chondrocytes transplantation (n = 40, and the second was a control group who got conservative therapy (n = 20. Donor cells were taken from the chondrogenic layer of the humerus or femur heads and hip condyles of human embryos in gestation for 17-20 weeks. A suspension of fetal chondrocytes injected into affected areas of the articular surfaces under X-ray control. Cell viability was determined before the injection. Efficacy of the therapy was assessed by clinical, instrumental, and laboratory tests. This clinical trial was allowed by The Ministry of Public Health and Ethics Committee. All of our patients gave informed consent for the fetal chondrocytes transplantation.Results. Evaluation of the clinical manifestations of RA in the first group of patients showed 3.7 times decrease in pain and 1.6 times relief of synovitis. Complete reduction of contracture was observed in 82% of patients in the first group. Morphometric changes in X-ray demonstrated inhibition of the destruction in articular cartilage and surfaces of bones after transplantation of fetal chondrocytes. The dynamics of morphological changes in synovium showed 2.5 times reduction of the inflammatory reaction. Transplantation of fetal chondrocytes led to a significant reduction in ESR, CRP, fibrinogen , γ-globulin after a period of 12 months (p < 0

Culture of chondrocytes in alginate surrounded by fibrin gel: characteristics of the cells over a period of eight weeks

NARCIS (Netherlands)

Almqvist, K. F.; Wang, L.; Wang, J.; Baeten, D.; Cornelissen, M.; Verdonk, R.; Veys, E. M.; Verbruggen, G.

2001-01-01

OBJECTIVE: To produce tissue engineered cartilage by human articular chondrocytes in vitro for further use in in vivo manipulations for the treatment of cartilage defects. METHODS: Human articular chondrocytes were cultured in 0.5%, 1.0%, and 2.0% of alginate for up to four weeks. The optimal

Novel bioadhesive polymers as intra-articular agents: Chondroitin sulfate-cysteine conjugates.

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Suchaoin, Wongsakorn; Bonengel, Sonja; Griessinger, Julia Anita; Pereira de Sousa, Irene; Hussain, Shah; Huck, Christian W; Bernkop-Schnürch, Andreas

2016-04-01

The aim of this study was to generate and characterize a chondroitin sulfate-cysteine conjugate (CS-cys) as a novel bioadhesive agent for intra-articular use. Mucoadhesive properties of synthesized CS-cys were investigated by rheological measurement of polymer-mucus mixture and rotating cylinder method, while bioadhesive features of CS-cys on porcine articular cartilage were evaluated via tensile studies. Thiolation was achieved by attachment of l-cysteine to CS via amide bond formation mediated by carbodiimide as a coupling reagent. The conjugate exhibited 421.17±35.14 μmol free thiol groups per gram polymer. The reduced CS-cys displayed 675.09±39.67 μmol free thiol groups per gram polymer after disulfide bonds reduction using tris(2-carboxyethyl)phosphine hydrochloride. The increase in dynamic viscosity of thiolated CS due to oxidative disulfide bond formation was demonstrated using capillary viscometer. The combination of CS-cys and mucus led to 4.57-fold increase in dynamic viscosity in comparison with mucus control. Furthermore, adhesion time to porcine mucosa of CS-cys-based test disk was enhanced by 2.48-fold compared to unmodified CS as measured by rotating cylinder method suggesting the interaction between thiomers and mucus gel layer via disulfide bonds formation. Tensile studies of thiolated CS on porcine articular cartilage showed 5.37- and 1.76-fold increase in the total work of adhesion and the maximum detachment force, respectively, in comparison with unmodified CS indicating bioadhesive features of CS-cys. Cytotoxicity of CS-cys was assessed in Caco-2 cells and rat primary articular chondrocytes using MTT and LDH release assay, thereby showing the safety of CS-cys at a concentration of 0.25% (w/v) in Caco-2 cells. Furthermore, 0.1% of CS-cys was found non-toxic to rat primary articular chondrocytes. According to these results, CS-cys provides improved bioadhesive properties that might be useful as an intra-articular agent for treatment of

Autologous chondrocyte implantation: superior biologic properties of hyaline cartilage repairs.

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Henderson, Ian; Lavigne, Patrick; Valenzuela, Herminio; Oakes, Barry

2007-02-01

Information regarding the quality of autologous chondrocyte implantation repair is needed to determine whether the current autologous chondrocyte implantation surgical technology and the subsequent biologic repair processes are capable of reliably forming durable hyaline or hyaline-like cartilage in vivo. We report and analyze the properties and qualities of autologous chondrocyte implantation repairs. We evaluated 66 autologous chondrocyte implantation repairs in 57 patients, 55 of whom had histology, indentometry, and International Cartilage Repair Society repair scoring at reoperation for mechanical symptoms or pain. International Knee Documentation Committee scores were used to address clinical outcome. Maximum stiffness, normalized stiffness, and International Cartilage Repair Society repair scoring were higher for hyaline articular cartilage repairs compared with fibrocartilage, with no difference in clinical outcome. Reoperations revealed 32 macroscopically abnormal repairs (Group B) and 23 knees with normal-looking repairs in which symptoms leading to arthroscopy were accounted for by other joint disorders (Group A). In Group A, 65% of repairs were either hyaline or hyaline-like cartilage compared with 28% in Group B. Autologous chondrocyte repairs composed of fibrocartilage showed more morphologic abnormalities and became symptomatic earlier than hyaline or hyaline-like cartilage repairs. The hyaline articular cartilage repairs had biomechanical properties comparable to surrounding cartilage and superior to those associated with fibrocartilage repairs.

Naringenin regulates production of matrix metalloproteinases in the knee-joint and primary cultured articular chondrocytes and alleviates pain in rat osteoarthritis model.

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Wang, C C; Guo, L; Tian, F D; An, N; Luo, L; Hao, R H; Wang, B; Zhou, Z H

2017-03-23

Inflammation of cartilage is a primary symptom for knee-joint osteoarthritis. Matrix metalloproteinases (MMPs) are known to play an important role in the articular cartilage destruction related to osteoarthritis. Naringenin is a plant-derived flavonoid known for its anti-inflammatory properties. We studied the effect of naringenin on the transcriptional expression, secretion and enzymatic activity of MMP-3 in vivo in the murine monosodium iodoacetate (MIA) osteoarthritis model. The assessment of pain behavior was also performed in the MIA rats. The destruction of knee-joint tissues was analyzed microscopically. Moreover, the effect of naringenin was also studied in vitro in IL-1β activated articular chondrocytes. The transcriptional expression of MMP-3, MMP-1, MMP-13, thrombospondin motifs (ADAMTS-4) and ADAMTS-5 was also studied in primary cultured chondrocytes of rats. Naringenin caused significant reduction in pain behavior and showed marked improvement in the tissue morphology of MIA rats. Moreover, a significant inhibition of MMP-3 expression in MIA rats was observed upon treatment with naringenin. In the in vitro tests, naringenin caused a significant reduction in the transcriptional expression, secretion and enzymatic activity of the studied degradative enzymes. The NF-κB pathway was also found to be inhibited upon treatment with naringenin in vitro. Overall, the study suggests that naringenin alleviated pain and regulated the production of matrix-metalloproteinases via regulation of NF-κB pathway. Thus, naringenin could be a potent therapeutic option for the treatment of osteoarthritis.

Naringenin regulates production of matrix metalloproteinases in the knee-joint and primary cultured articular chondrocytes and alleviates pain in rat osteoarthritis model

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C.C. Wang

Full Text Available Inflammation of cartilage is a primary symptom for knee-joint osteoarthritis. Matrix metalloproteinases (MMPs are known to play an important role in the articular cartilage destruction related to osteoarthritis. Naringenin is a plant-derived flavonoid known for its anti-inflammatory properties. We studied the effect of naringenin on the transcriptional expression, secretion and enzymatic activity of MMP-3 in vivo in the murine monosodium iodoacetate (MIA osteoarthritis model. The assessment of pain behavior was also performed in the MIA rats. The destruction of knee-joint tissues was analyzed microscopically. Moreover, the effect of naringenin was also studied in vitro in IL-1β activated articular chondrocytes. The transcriptional expression of MMP-3, MMP-1, MMP-13, thrombospondin motifs (ADAMTS-4 and ADAMTS-5 was also studied in primary cultured chondrocytes of rats. Naringenin caused significant reduction in pain behavior and showed marked improvement in the tissue morphology of MIA rats. Moreover, a significant inhibition of MMP-3 expression in MIA rats was observed upon treatment with naringenin. In the in vitro tests, naringenin caused a significant reduction in the transcriptional expression, secretion and enzymatic activity of the studied degradative enzymes. The NF-κB pathway was also found to be inhibited upon treatment with naringenin in vitro. Overall, the study suggests that naringenin alleviated pain and regulated the production of matrix-metalloproteinases via regulation of NF-κB pathway. Thus, naringenin could be a potent therapeutic option for the treatment of osteoarthritis.

Incorporation of hyaluronic acid into collagen scaffolds for the control of chondrocyte-mediated contraction and chondrogenesis

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Tang Shunqing [Department of Biomedical Engineering, Jinan University, Guangzhou 510632 (China); Spector, Myron [Tissue Engineering, VA Boston Healthcare System, Boston, MA 02130 (United States)

2007-09-15

Hyaluronic acid (HA), a principal matrix molecule in many tissues, is present in high amounts in articular cartilage. HA contributes in unique ways to the physical behavior of the tissue, and has been shown to have beneficial effects on chondrocyte activity. The goal of this study was to incorporate graduated amounts of HA into type I collagen scaffolds for the control of chondrocyte-mediated contraction and chondrogenesis in vitro. The results demonstrated that the amount of contraction of HA/collagen scaffolds by adult canine articular chondrocytes increased with the HA content of the scaffolds. The greatest amount of chondrogenesis after two weeks was found in the scaffolds which had undergone the most contraction. HA can play a useful role in adjusting the mechanical behavior of tissue engineering scaffolds and chondrogenesis in chondrocyte-seeded scaffolds.

Evaluation on Cartilage Morphology after Intra-Articular Injection of Titanium Dioxide Nanoparticles in Rats

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Wang, J.; Gao, Y.; Hou, Y.; Zhao, F.; Pu, F.; Liu, X.; Fan, Y.; Wu, Z.

2012-01-01

Nano scale wear particles would generate from orthopedic implants with nano scale surface topography because of residual stress. In this study, the effect of TiO 2 nanoparticles on articular cartilage was investigated by intra-articular injection in rats. Using contrast-enhanced high-resolution micro computed tomography (micro-CT) technology, the decreased thickness of articular cartilage in distal femur was determined at 1, 7, 14, and 30 days after nanoparticle exposure. A strong linear correlation (r=0.928, P 2 nanoparticles, cartilage thickness showed time-dependent decrease, and cartilage volume was decreased too. Further, the histopathological examination showed the edema chondrocyte and shrinked nucleus in the radial and calcified zone of cartilage. The ultrastructure of articular cartilage implied that the chondrocytes was degenerated, expressing as the condensed chromatin, the dilated endoplasmic reticulum, and the rich mitochondria. Even, the fragments of ruptured endoplasmic reticulum were observed in the cytoplasm of chondrocytes at postexposure day 30. Results indicate that potential damage of articular cartilage was induced by particles existed in knee joint and imply that the bio monitoring should be strengthened in patients with prostheses replacement.

Scaffold-assisted cartilage tissue engineering using infant chondrocytes from human hip cartilage.

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Kreuz, P C; Gentili, C; Samans, B; Martinelli, D; Krüger, J P; Mittelmeier, W; Endres, M; Cancedda, R; Kaps, C

2013-12-01

Studies about cartilage repair in the hip and infant chondrocytes are rare. The aim of our study was to evaluate the use of infant articular hip chondrocytes for tissue engineering of scaffold-assisted cartilage grafts. Hip cartilage was obtained from five human donors (age 1-10 years). Expanded chondrocytes were cultured in polyglycolic acid (PGA)-fibrin scaffolds. De- and re-differentiation of chondrocytes were assessed by histological staining and gene expression analysis of typical chondrocytic marker genes. In vivo, cartilage matrix formation was assessed by histology after subcutaneous transplantation of chondrocyte-seeded PGA-fibrin scaffolds in immunocompromised mice. The donor tissue was heterogenous showing differentiated articular cartilage and non-differentiated tissue and considerable expression of type I and II collagens. Gene expression analysis showed repression of typical chondrocyte and/or mesenchymal marker genes during cell expansion, while markers were re-induced when expanded cells were cultured in PGA-fibrin scaffolds. Cartilage formation after subcutaneous transplantation of chondrocyte loaded PGA-fibrin scaffolds in nude mice was variable, with grafts showing resorption and host cell infiltration or formation of hyaline cartilage rich in type II collagen. Addition of human platelet rich plasma (PRP) to cartilage grafts resulted robustly in formation of hyaline-like cartilage that showed type II collagen and regions with type X collagen. These results suggest that culture of expanded and/or de-differentiated infant hip cartilage cells in PGA-fibrin scaffolds initiates chondrocyte re-differentiation. The heterogenous donor tissue containing immature chondrocytes bears the risk of cartilage repair failure in vivo, which may be possibly overcome by the addition of PRP. Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Chondrocyte heterogeneity: immunohistologically defined variation of integrin expression at different sites in human fetal knees.

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Salter, D M; Godolphin, J L; Gourlay, M S

1995-04-01

During development and at maturity different forms of cartilage vary in morphology and macromolecular content. This reflects heterogeneity of chondrocyte activity, in part involving differential interactions with the adjacent extracellular matrix via specialized cell surface receptors such as integrins. We undertook an immunohistological study on a series of human fetal knee joints to assess variation in the expression of integrins by chondrocytes and potential matrix ligands in articular, epiphyseal, growth plate, and meniscal cartilage. The results show that articular chondrocytes (beta 1+, beta 5 alpha V+, alpha 1+, alpha 2+/-, alpha 5+, weakly alpha 6+, alpha V+) differed from epiphyseal (beta 1+, beta 5 alpha V+, alpha 1+/-, alpha 2+/-, alpha 5+, alpha 6+, alpha V+) growth plate (beta 1+, beta 5 alpha V+, alpha 1-, alpha 2-, alpha 5+, alpha 6+, alpha V+), and meniscal cells (beta 1+, beta 5 alpha V+, alpha 1+, strongly alpha 2+, alpha 5+, alpha 6+, alpha V+ in expression of integrin subunits. There was no expression of beta 3, beta 4, beta 6, or alpha 3 by chondrocytes. These results differ from previous reports on the expression of integrins by adult articular cartilage, where alpha 2 and alpha 6 are not seen. Variation in distribution of matrix ligands was also seen. Fibronectin, laminin and Type VI collagen were expressed in all cartilages but there was restricted expression of tenascin, ED-A and ED-B fibronectin isoforms (articular cartilage and meniscus), and vitronectin (absent from growth plate cartilage). Regulated expression of integrins by chondrocytes, associated with changes in the pericellular matrix composition, is of potential importance in control of cartilage differentiation and function in health and disease.

Culture of bovine articular chondrocytes in funnel-like collagen-PLGA hybrid sponges

International Nuclear Information System (INIS)

Lu Hongxu; Ko, Young-Gwang; Kawazoe, Naoki; Chen Guoping

2011-01-01

Three-dimensional porous scaffolds play an important role in tissue engineering and regenerative medicine. Structurally, these porous scaffolds should have an open and interconnected porous architecture to facilitate a homogeneous cell distribution. Moreover, the scaffolds should be mechanically strong to support new tissue formation. We developed a novel type of funnel-like collagen sponge using embossing ice particulates as a template. The funnel-like collagen sponges could promote the homogeneous cell distribution, ECM production and chondrogenesis. However, the funnel-like collagen sponges deformed during cell culture due to their weak mechanical strength. To solve this problem, we reinforced the funnel-like collagen sponges with a knitted poly(D,L-lactic-co-glycolic acid) (PLGA) mesh by hybridizing these two types of materials. The hybrid scaffolds were used to culture bovine articular chondrocytes. The cell adhesion, distribution, proliferation and chondrogenesis were investigated. The funnel-like structure promoted the even cell distribution and homogeneous ECM production. The PLGA knitted mesh protected the scaffold from deformation during cell culture. Histological and immunohistochemical staining and cartilaginous gene expression analyses revealed the cartilage-like properties of the cell/scaffold constructs after in vivo implantation. The hybrid scaffold, composed of a funnel-like collagen sponge and PLGA mesh, would be a useful tool for cartilage tissue engineering.

Culture of bovine articular chondrocytes in funnel-like collagen-PLGA hybrid sponges

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Lu Hongxu; Ko, Young-Gwang; Kawazoe, Naoki; Chen Guoping, E-mail: Guoping.Chen@nims.go.jp [Tissue Regeneration Materials Unit, International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044 (Japan)

2011-08-15

Three-dimensional porous scaffolds play an important role in tissue engineering and regenerative medicine. Structurally, these porous scaffolds should have an open and interconnected porous architecture to facilitate a homogeneous cell distribution. Moreover, the scaffolds should be mechanically strong to support new tissue formation. We developed a novel type of funnel-like collagen sponge using embossing ice particulates as a template. The funnel-like collagen sponges could promote the homogeneous cell distribution, ECM production and chondrogenesis. However, the funnel-like collagen sponges deformed during cell culture due to their weak mechanical strength. To solve this problem, we reinforced the funnel-like collagen sponges with a knitted poly(D,L-lactic-co-glycolic acid) (PLGA) mesh by hybridizing these two types of materials. The hybrid scaffolds were used to culture bovine articular chondrocytes. The cell adhesion, distribution, proliferation and chondrogenesis were investigated. The funnel-like structure promoted the even cell distribution and homogeneous ECM production. The PLGA knitted mesh protected the scaffold from deformation during cell culture. Histological and immunohistochemical staining and cartilaginous gene expression analyses revealed the cartilage-like properties of the cell/scaffold constructs after in vivo implantation. The hybrid scaffold, composed of a funnel-like collagen sponge and PLGA mesh, would be a useful tool for cartilage tissue engineering.

Transforming growth factor β-induced superficial zone protein accumulation in the surface zone of articular cartilage is dependent on the cytoskeleton.

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McNary, Sean M; Athanasiou, Kyriacos A; Reddi, A Hari

2014-03-01

The phenotype of articular chondrocytes is dependent on the cytoskeleton, specifically the actin microfilament architecture. Articular chondrocytes in monolayer culture undergo dedifferentiation and assume a fibroblastic phenotype. This process can be reversed by altering the actin cytoskeleton by treatment with cytochalasin. Whereas dedifferentiation has been studied on chondrocytes isolated from the whole cartilage, the effects of cytoskeletal alteration on specific zones of cells such as superficial zone chondrocytes are not known. Chondrocytes from the superficial zone secrete superficial zone protein (SZP), a lubricating proteoglycan that reduces the coefficient of friction of articular cartilage. A better understanding of this phenomenon may be useful in elucidating chondrocyte dedifferentiation in monolayer and accumulation of the cartilage lubricant SZP, with an eye toward tissue engineering functional articular cartilage. In this investigation, the effects of cytoskeletal modulation on the ability of superficial zone chondrocytes to secrete SZP were examined. Primary superficial zone chondrocytes were cultured in monolayer and treated with a combination of cytoskeleton modifying reagents and transforming growth factor β (TGFβ) 1, a critical regulator of SZP production. Whereas cytochalasin D maintains the articular chondrocyte phenotype, the hallmark of the superficial zone chondrocyte, SZP, was inhibited in the presence of TGFβ1. A decrease in TGFβ1-induced SZP accumulation was also observed when the microtubule cytoskeleton was modified using paclitaxel. These effects of actin and microtubule alteration were confirmed through the application of jasplakinolide and colchicine, respectively. As Rho GTPases regulate actin organization and microtubule polymerization, we hypothesized that the cytoskeleton is critical for TGFβ-induced SZP accumulation. TGFβ-mediated SZP accumulation was inhibited by small molecule inhibitors ML141 (Cdc42), NSC23766 (Rac1

NF-κB Mediates the Stimulation of Cytokine and Chemokine Expression by Human Articular Chondrocytes in Response to Fibronectin Fragments1

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Pulai, Judit I.; Chen, Hong; Im, Hee-Jeong; Kumar, Sanjay; Hanning, Charles; Hegde, Priti S.; Loeser, Richard F.

2010-01-01

Fibronectin fragments (FN-f) that bind to the α5β1 integrin stimulate chondrocyte-mediated cartilage destruction and could play an important role in the progression of arthritis. The objective of this study was to identify potential cytokine mediators of cartilage inflammation and destruction induced by FN-f and to investigate the mechanism of their stimulation. Human articular chondrocytes, isolated from normal ankle cartilage obtained from tissue donors, were treated with a 110-kDa FN-f in serum-free culture, and expression of various cytokine genes was analyzed by cDNA microarray and by a cytokine protein array. Compared with untreated control cultures, stimulation by FN-f resulted in a >2-fold increase in IL-6, IL-8, MCP-1, and growth-related oncogene β (GRO-β). Constitutive and FN-f-inducible expression of GRO-α and GRO-γ were also noted by RT-PCR and confirmed by immunoblotting. Previous reports of IL-1β expression induced by FN-f were also confirmed, while TNF expression was found to be very low. Inhibitor studies revealed that FN-f-induced stimulation of chondrocyte chemokine expression was dependent on NF-κB activity, but independent of IL-1 autocrine signaling. The ability of FN-f to stimulate chondrocyte expression of multiple proinflammatory cytokines and chemokines suggests that damage to the cartilage matrix is capable of inducing a proinflammatory state responsible for further progressive matrix destruction, which also includes the chemoattraction of inflammatory cells. Targeting the signaling pathways activated by FN-f may be an effective means of inhibiting production of multiple mediators of cartilage destruction. PMID:15843581

CARTILAGE CONSTRUCTS ENGINEERED FROM CHONDROCYTES OVEREXPRESSING IGF-I IMPROVE THE REPAIR OF OSTEOCHONDRAL DEFECTS IN A RABBIT MODEL

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Madry, Henning; Kaul, Gunter; Zurakowski, David; Vunjak-Novakovic, Gordana; Cucchiarini, Magali

2015-01-01

Tissue engineering combined with gene therapy is a promising approach for promoting articular cartilage repair. Here, we tested the hypothesis that engineered cartilage with chondrocytes over expressing a human insulin-like growth factor I (IGF-I) gene can enhance the repair of osteochondral defects, in a manner dependent on the duration of cultivation. Genetically modified chondrocytes were cultured on biodegradable polyglycolic acid scaffolds in dynamic flow rotating bioreactors for either 10 or 28 d. The resulting cartilaginous constructs were implanted into osteochondral defects in rabbit knee joints. After 28 weeks of in vivo implantation, immunoreactivity to ß-gal was detectable in the repair tissue of defects that received lacZ constructs. Engineered cartilaginous constructs based on IGF-I-over expressing chondrocytes markedly improved osteochondral repair compared with control (lacZ) constructs. Moreover, IGF-I constructs cultivated for 28 d in vitro significantly promoted osteochondral repair vis-à-vis similar constructs cultivated for 10 d, leading to significantly decreased osteoarthritic changes in the cartilage adjacent to the defects. Hence, the combination of spatially defined overexpression of human IGF-I within a tissue-engineered construct and prolonged bioreactor cultivation resulted in most enhanced articular cartilage repair and reduction of osteoarthritic changes in the cartilage adjacent to the defect. Such genetically enhanced tissue engineering provides a versatile tool to evaluate potential therapeutic genes in vivo and to improve our comprehension of the development of the repair tissue within articular cartilage defects. Insights gained with additional exploration using this model may lead to more effective treatment options for acute cartilage defects. PMID:23588785

Cartilage constructs engineered from chondrocytes overexpressing IGF-I improve the repair of osteochondral defects in a rabbit model

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H Madry

2013-04-01

Full Text Available Tissue engineering combined with gene therapy is a promising approach for promoting articular cartilage repair. Here, we tested the hypothesis that engineered cartilage with chondrocytes overexpressing a human insulin-like growth factor I (IGF-I gene can enhance the repair of osteochondral defects, in a manner dependent on the duration of cultivation. Genetically modified chondrocytes were cultured on biodegradable polyglycolic acid scaffolds in dynamic flow rotating bioreactors for either 10 or 28 d. The resulting cartilaginous constructs were implanted into osteochondral defects in rabbit knee joints. After 28 weeks of in vivo implantation, immunoreactivity to ß-gal was detectable in the repair tissue of defects that received lacZ constructs. Engineered cartilaginous constructs based on IGF-I-overexpressing chondrocytes markedly improved osteochondral repair compared with control (lacZ constructs. Moreover, IGF-I constructs cultivated for 28 d in vitro significantly promoted osteochondral repair vis-à-vis similar constructs cultivated for 10 d, leading to significantly decreased osteoarthritic changes in the cartilage adjacent to the defects. Hence, the combination of spatially defined overexpression of human IGF-I within a tissue-engineered construct and prolonged bioreactor cultivation resulted in most enhanced articular cartilage repair and reduction of osteoarthritic changes in the cartilage adjacent to the defect. Such genetically enhanced tissue engineering provides a versatile tool to evaluate potential therapeutic genes in vivo and to improve our comprehension of the development of the repair tissue within articular cartilage defects. Insights gained with additional exploration using this model may lead to more effective treatment options for acute cartilage defects.

Production of serum amyloid A in equine articular chondrocytes and fibroblast-like synoviocytes treated with proinflammatory cytokines and its effects on the two cell types in culture

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Jacobsen, Stine; Ladefoged, Søren; Berg, Lise Charlotte

2016-01-01

OBJECTIVE: To investigate the role of the major equine acute phase protein serum amyloid A (SAA) in inflammation of equine intraarticular tissues. SAMPLE: Articular chondrocytes and fibroblast-like synoviocytes (FLSs) from 8 horses (4 horses/cell type). PROCEDURES: Chondrocytes and FLSs were...... stimulated in vitro for various periods up to 48 hours with cytokines (recombinant interleukin [IL]-1β, IL-6, tumor necrosis factor-α, or a combination of all 3 [IIT]) or with recombinant SAA. Gene expression of SAA, IL-6, matrix metalloproteinases (MMP)-1 and −3, and cartilage-derived retinoic acid......-sensitive protein were assessed by quantitative real-time PCR assay; SAA protein was evaluated by immunoturbidimetry and denaturing isoelectric focusing and western blotting. RESULTS: All cytokine stimulation protocols increased expression of SAA mRNA and resulted in detectable SAA protein production...

Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro

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S Giovannini

2010-10-01

Full Text Available Cell therapies for articular cartilage defects rely on expanded chondrocytes. Mesenchymal stem cells (MSC represent an alternative cell source should their hypertrophic differentiation pathway be prevented. Possible cellular instruction between human articular chondrocytes (HAC and human bone marrow MSC was investigated in micromass pellets. HAC and MSC were mixed in different percentages or incubated individually in pellets for 3 or 6 weeks with and without TGF-beta1 and dexamethasone (±T±D as chondrogenic factors. Collagen II, collagen X and S100 protein expression were assessed using immunohistochemistry. Proteoglycan synthesis was evaluated applying the Bern score and quantified using dimethylmethylene blue dye binding assay. Alkaline phosphatase activity (ALP was detected on cryosections and soluble ALP measured in pellet supernatants. HAC alone generated hyaline-like discs, while MSC formed spheroid pellets in ±T±D. Co-cultured pellets changed from disc to spheroid shape with decreasing number of HAC, and displayed random cell distribution. In -T-D, HAC expressed S100, produced GAG and collagen II, and formed lacunae, while MSC did not produce any cartilage-specific proteins. Based on GAG, collagen type II and S100 expression chondrogenic differentiation occurred in -T-D MSC co-cultures. However, quantitative experimental GAG and DNA values did not differ from predicted values, suggesting only HAC contribution to GAG production. MSC produced cartilage-specific matrix only in +T+D but underwent hypertrophy in all pellet cultures. In summary, influence of HAC on MSC was restricted to early signs of neochondrogenesis. However, MSC did not contribute to the proteoglycan deposition, and HAC could not prevent hypertrophy of MSC induced by chondrogenic stimuli.

Hyperpolarisation of cultured human chondrocytes following cyclical pressure-induced strain: evidence of a role for alpha 5 beta 1 integrin as a chondrocyte mechanoreceptor.

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Wright, M O; Nishida, K; Bavington, C; Godolphin, J L; Dunne, E; Walmsley, S; Jobanputra, P; Nuki, G; Salter, D M

1997-09-01

Mechanical stimuli influence chondrocyte metabolism, inducing changes in intracellular cyclic adenosine monophosphate and proteoglycan production. We have previously demonstrated that primary monolayer cultures of human chondrocytes have an electrophysiological response after intermittent pressure-induced strain characterised by a membrane hyperpolarisation of approximately 40%. The mechanisms responsible for these changes are not fully understood but potentially involve signalling molecules such as integrins that link extracellular matrix with cytoplasmic components. The results reported in this paper demonstrate that the transduction pathways involved in the hyperpolarisation response of human articular chondrocytes in vitro after cyclical pressure-induced strain involve alpha 5 beta 1 integrin. We have demonstrated, using pharmacological inhibitors of a variety of intracellular signalling pathways, that the actin cytoskeleton, the phospholipase C calmodulin pathway, and both tyrosine protein kinase and protein kinase C activities are important in the transduction of the electrophysiological response. These results suggest that alpha 5 beta 1 is an important chondrocyte mechanoreceptor and a potential regulator of chondrocyte function.

Transcriptional profiling differences for articular cartilage and repair tissue in equine joint surface lesions

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Stromberg Arnold J

2009-09-01

Full Text Available Abstract Background Full-thickness articular cartilage lesions that reach to the subchondral bone yet are restricted to the chondral compartment usually fill with a fibrocartilage-like repair tissue which is structurally and biomechanically compromised relative to normal articular cartilage. The objective of this study was to evaluate transcriptional differences between chondrocytes of normal articular cartilage and repair tissue cells four months post-microfracture. Methods Bilateral one-cm2 full-thickness defects were made in the articular surface of both distal femurs of four adult horses followed by subchondral microfracture. Four months postoperatively, repair tissue from the lesion site and grossly normal articular cartilage from within the same femorotibial joint were collected. Total RNA was isolated from the tissue samples, linearly amplified, and applied to a 9,413-probe set equine-specific cDNA microarray. Eight paired comparisons matched by limb and horse were made with a dye-swap experimental design with validation by histological analyses and quantitative real-time polymerase chain reaction (RT-qPCR. Results Statistical analyses revealed 3,327 (35.3% differentially expressed probe sets. Expression of biomarkers typically associated with normal articular cartilage and fibrocartilage repair tissue corroborate earlier studies. Other changes in gene expression previously unassociated with cartilage repair were also revealed and validated by RT-qPCR. Conclusion The magnitude of divergence in transcriptional profiles between normal chondrocytes and the cells that populate repair tissue reveal substantial functional differences between these two cell populations. At the four-month postoperative time point, the relative deficiency within repair tissue of gene transcripts which typically define articular cartilage indicate that while cells occupying the lesion might be of mesenchymal origin, they have not recapitulated differentiation to

The effect of dexamethasone and triiodothyronine on terminal differentiation of primary bovine chondrocytes and chondrogenically differentiated mesenchymal stem cells.

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Randau, Thomas M; Schildberg, Frank A; Alini, Mauro; Wimmer, Matthias D; Haddouti, El-Mustapha; Gravius, Sascha; Ito, Keita; Stoddart, Martin J

2013-01-01

The newly evolved field of regenerative medicine is offering solutions in the treatment of bone or cartilage loss and deficiency. Mesenchymal stem cells, as well as articular chondrocytes, are potential cells for the generation of bone or cartilage. The natural mechanism of bone formation is that of endochondral ossification, regulated, among other factors, through the hormones dexamethasone and triiodothyronine. We investigated the effects of these hormones on articular chondrocytes and chondrogenically differentiated mesenchymal stem cells, hypothesizing that these hormones would induce terminal differentiation, with chondrocytes and differentiated stem cells being similar in their response. Using a 3D-alginate cell culture model, bovine chondrocytes and chondrogenically differentiated stem cells were cultured in presence of triiodothyronine or dexamethasone, and cell proliferation and extracellular matrix production were investigated. Collagen mRNA expression was measured by real-time PCR. Col X mRNA and alkaline phosphatase were monitored as markers of terminal differentiation, a prerequisite of endochondral ossification. The alginate culture system worked well, both for the culture of chondrocytes and for the chondrogenic differentiation of mesenchymal stem cells. Dexamethasone led to an increase in glycosaminoglycan production. Triiodothyronine increased the total collagen production only in chondrocytes, where it also induced signs of terminal differentiation, increasing both collagen X mRNA and alkaline phosphatase activity. Dexamethasone induced terminal differentiation in the differentiated stem cells. The immature articular chondrocytes used in this study seem to be able to undergo terminal differentiation, pointing to their possible role in the onset of degenerative osteoarthritis, as well as their potential for a cell source in bone tissue engineering. When chondrocyte-like cells, after their differentiation, can indeed be moved on towards terminal

PEDF Is Associated with the Termination of Chondrocyte Phenotype and Catabolism of Cartilage Tissue.

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Klinger, P; Lukassen, S; Ferrazzi, F; Ekici, A B; Hotfiel, T; Swoboda, B; Aigner, T; Gelse, K

2017-01-01

Objective. To investigate the expression and target genes of pigment epithelium-derived factor (PEDF) in cartilage and chondrocytes, respectively. Methods. We analyzed the expression pattern of PEDF in different human cartilaginous tissues including articular cartilage, osteophytic cartilage, and fetal epiphyseal and growth plate cartilage, by immunohistochemistry and quantitative real-time (qRT) PCR. Transcriptome analysis after stimulation of human articular chondrocytes with rhPEDF was performed by RNA sequencing (RNA-Seq) and confirmed by qRT-PCR. Results. Immunohistochemically, PEDF could be detected in transient cartilaginous tissue that is prone to undergo endochondral ossification, including epiphyseal cartilage, growth plate cartilage, and osteophytic cartilage. In contrast, PEDF was hardly detected in healthy articular cartilage and in the superficial zone of epiphyses, regions that are characterized by a permanent stable chondrocyte phenotype. RNA-Seq analysis and qRT-PCR demonstrated that rhPEDF significantly induced the expression of a number of matrix-degrading factors including SAA1, MMP1, MMP3, and MMP13. Simultaneously, a number of cartilage-specific genes including COL2A1, COL9A2, COMP, and LECT were among the most significantly downregulated genes. Conclusions. PEDF represents a marker for transient cartilage during all neonatal and postnatal developmental stages and promotes the termination of cartilage tissue by upregulation of matrix-degrading factors and downregulation of cartilage-specific genes. These data provide the basis for novel strategies to stabilize the phenotype of articular cartilage and prevent its degradation.

The properties of bioengineered chondrocyte sheets for cartilage regeneration

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Ota Naoshi

2009-03-01

Full Text Available Abstract Background Although the clinical results of autologous chondrocyte implantation for articular cartilage defects have recently improved as a result of advanced techniques based on tissue engineering procedures, problems with cell handling and scaffold imperfections remain to be solved. A new cell-sheet technique has been developed, and is potentially able to overcome these obstacles. Chondrocyte sheets applicable to cartilage regeneration can be prepared with this cell-sheet technique using temperature-responsive culture dishes. However, for clinical application, it is necessary to evaluate the characteristics of the cells in these sheets and to identify their similarities to naive cartilage. Results The expression of SOX 9, collagen type 2, 27, integrin α10, and fibronectin genes in triple-layered chondrocyte sheets was significantly increased in comparison to those in conventional monolayer culture and in a single chondrocyte sheet, implying a nature similar to ordinary cartilage. In addition, immunohistochemistry demonstrated that collagen type II, fibronectin, and integrin α10 were present in the triple-layered chondrocyte sheets. Conclusion The results of this study indicate that these chondrocyte sheets with a consistent cartilaginous phenotype and adhesive properties may lead to a new strategy for cartilage regeneration.

Gremlin 1, Frizzled-related protein, and Dkk-1 are key regulators of human articular cartilage homeostasis

NARCIS (Netherlands)

Leijten, Jeroen Christianus Hermanus; Emons, J.; Sticht, C.; van Gool, S.; Decker, E.; Uitterlinden, A.; Rappold, G.; Hofman, A.; Rivadeneira, F.; Scherjon, S.; Wit, J.M.; van Meurs, J.; van Blitterswijk, Clemens; Karperien, Hermanus Bernardus Johannes

2012-01-01

Objective The development of osteoarthritis (OA) may be caused by activation of hypertrophic differentiation of articular chondrocytes. Healthy articular cartilage is highly resistant to hypertrophic differentiation, in contrast to other hyaline cartilage subtypes, such as growth plate cartilage.

Effect of Cyclic Dynamic Compressive Loading on Chondrocytes and Adipose-Derived Stem Cells Co-Cultured in Highly Elastic Cryogel Scaffolds

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Chih-Hao Chen

2018-01-01

Full Text Available In this study, we first used gelatin/chondroitin-6-sulfate/hyaluronan/chitosan highly elastic cryogels, which showed total recovery from large strains during repeated compression cycles, as 3D scaffolds to study the effects of cyclic dynamic compressive loading on chondrocyte gene expression and extracellular matrix (ECM production. Dynamic culture of porcine chondrocytes was studied at 1 Hz, 10% to 40% strain and 1 to 9 h/day stimulation duration, in a mechanical-driven multi-chamber bioreactor for 14 days. From the experimental results, we could identify the optimum dynamic culture condition (20% and 3 h/day to enhance the chondrocytic phenotype of chondrocytes from the expression of marker (Col I, Col II, Col X, TNF-α, TGF-β1 and IGF-1 genes by quantitative real-time polymerase chain reactions (qRT-PCR and production of ECM (GAGs and Col II by biochemical analysis and immunofluorescence staining. With up-regulated growth factor (TGF-β1 and IGF-1 genes, co-culture of chondrocytes with porcine adipose-derived stem cells (ASCs was employed to facilitate chondrogenic differentiation of ASCs during dynamic culture in cryogel scaffolds. By replacing half of the chondrocytes with ASCs during co-culture, we could obtain similar production of ECM (GAGs and Col II and expression of Col II, but reduced expression of Col I, Col X and TNF-α. Subcutaneous implantation of cells/scaffold constructs in nude mice after mono-culture (chondrocytes or ASCs or co-culture (chondrocytes + ASCs and subject to static or dynamic culture condition in vitro for 14 days was tested for tissue-engineering applications. The constructs were retrieved 8 weeks post-implantation for histological analysis by Alcian blue, Safranin O and Col II immunohistochemical staining. The most abundant ectopic cartilage tissue was found for the chondrocytes and chondrocytes + ASCs groups using dynamic culture, which showed similar neo-cartilage formation capability with half of the

Doublecortin May Play a Role in Defining Chondrocyte Phenotype

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Dongxia Ge

2014-04-01

Full Text Available Embryonic development of articular cartilage has not been well understood and the role of doublecortin (DCX in determination of chondrocyte phenotype is unknown. Here, we use a DCX promoter-driven eGFP reporter mouse model to study the dynamic gene expression profiles in mouse embryonic handplates at E12.5 to E13.5 when the condensed mesenchymal cells differentiate into either endochondral chondrocytes or joint interzone cells. Illumina microarray analysis identified a variety of genes that were expressed differentially in the different regions of mouse handplate. The unique expression patterns of many genes were revealed. Cytl1 and 3110032G18RIK were highly expressed in the proximal region of E12.5 handplate and the carpal region of E13.5 handplate, whereas Olfr538, Kctd15, and Cited1 were highly expressed in the distal region of E12.5 and the metacarpal region of E13.5 handplates. There was an increasing gradient of Hrc expression in the proximal to distal direction in E13.5 handplate. Furthermore, when human DCX protein was expressed in human adipose stem cells, collagen II was decreased while aggrecan, matrilin 2, and GDF5 were increased during the 14-day pellet culture. These findings suggest that DCX may play a role in defining chondrocyte phenotype.

The balancing act of transcription factors C-1-1 and Runx2 in articular cartilage development

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Iwamoto, Masahiro; Koyama, Eiki; Enomoto-Iwamoto, Motomi; Pacifici, Maurizio

2005-01-01

In previous studies we found that the ets transcription factor C-1-1 is involved in articular chondrocyte development, and we and others found that the transcription factor Runx2 is required for growth plate chondrocyte maturation and ossification. We determined here whether the two factors exert reciprocal influences on their expression and function and in so doing, steer chondrocyte developmental paths. Virally driven Runx2 over-expression in cultured chick chondrocytes did indeed lead to decreased C-1-1 expression, accompanied by decreased expression of articular cartilage marker tenascin-C, decreased proliferation, and increased expression of maturation marker collagen X. In good agreement, over-expression of a dominant-negative Runx2 form had opposite phenotypic consequences. When C-1-1 itself was over-expressed in chondrocytes already undergoing maturation, maturation was halted and the cells became small, rich in tenascin-C, and mitotically quite active. To extend these observations, we misexpressed C-1-1 in mouse cartilage and found that it caused a severe inhibition of chondrocyte maturation and widespread tenascin-C expression. In sum, C-1-1 and Runx2 do influence their respective expression patterns. The factors are powerful chondrocyte regulators and their functional interrelationships may be important for steering the cells toward alternative developmental paths

Extension of knee immobilization delays recovery of histological damages in the anterior cruciate ligament insertion and articular cartilage in rabbits.

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Mutsuzaki, Hirotaka; Nakajima, Hiromi; Sakane, Masataka

2018-01-01

[Purpose] To investigate the influence of knee immobilization period on recovery of histological damages in the anterior cruciate ligament (ACL) insertion and articular cartilage in rabbits. This knowledge is important for determining the appropriate rehabilitation approach for patients with ligament injuries, fracture, disuse atrophy, and degenerative joint disease. [Materials and Methods] Forty-eight male Japanese white rabbits were divided equally into the remobilization and control groups. The remobilization group had the right knee surgically immobilized, and was divided equally into four subgroups according to the duration of immobilization (1, 2, 4 and 8 weeks). After the immobilization was removed, the rabbits moved freely for 8 weeks. The control group underwent sham operation and followed the same time course as the remobilization group. The chondrocyte apoptosis rate and chondrocyte proliferation rate in the ACL insertion and articular cartilage were analyzed after remobilization. [Results] In the ACL insertion, the remobilization group had a higher chondrocyte apoptosis rate than the control group after 8 weeks of immobilization, and a lower chondrocyte proliferation rate than the control group after 4 and 8 weeks of immobilization. In the articular cartilage, the remobilization group had a lower chondrocyte proliferation rate than the control group after 8 weeks of immobilization. After 8 weeks of remobilization, the ACL insertion and articular cartilage are not completely recovered after 4 and 8 weeks of immobilization, respectively. [Conclusion] Our results suggest that 8 weeks of remobilization will result in recovery of the ACL insertion after 2 weeks of knee immobilization, and recovery of the articular cartilage after 4 weeks of knee immobilization. If 8 weeks of immobilization occurs, a remobilization duration of more than 8 weeks may be necessary.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components

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Chang Qing

2011-04-01

Full Text Available Chondrocytes and bone marrow mesenchymal stem cells (BMSCs are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs. This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components.

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Qing, Chang; Wei-ding, Cui; Wei-min, Fan

2011-04-01

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

Articular chondrocyte network mediated by gap junctions: role in metabolic cartilage homeostasis

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Mayan, Maria D; Gago-Fuentes, Raquel; Carpintero-Fernandez, Paula; Fernandez-Puente, Patricia; Filgueira-Fernandez, Purificacion; Goyanes, Noa; Valiunas, Virginijus; Brink, Peter R; Goldberg, Gary S; Blanco, Francisco J

2017-01-01

Objective This study investigated whether chondrocytes within the cartilage matrix have the capacity to communicate through intercellular connections mediated by voltage-gated gap junction (GJ) channels. Methods Frozen cartilage samples were used for immunofluorescence and immunohistochemistry assays. Samples were embedded in cacodylate buffer before dehydration for scanning electron microscopy. Co-immunoprecipitation experiments and mass spectrometry (MS) were performed to identify proteins that interact with the C-terminal end of Cx43. GJ communication was studied through in situ electroporation, electrophysiology and dye injection experiments. A transwell layered culture system and MS were used to identify and quantify transferred amino acids. Results Microscopic images revealed the presence of multiple cellular projections connecting chondrocytes within the matrix. These projections were between 5 and 150 μm in length. MS data analysis indicated that the C-terminus of Cx43 interacts with several cytoskeletal proteins implicated in Cx trafficking and GJ assembly, including α-tubulin and β-tubulin, actin, and vinculin. Electrophysiology experiments demonstrated that 12-mer oligonucleotides could be transferred between chondrocytes within 12 min after injection. Glucose was homogeneously distributed within 22 and 35 min. No transfer was detected when glucose was electroporated into A549 cells, which have no GJs. Transwell layered culture systems coupled with MS analysis revealed connexins can mediate the transfer of L-lysine and L-arginine between chondrocytes. Conclusions This study reveals that intercellular connections between chondrocytes contain GJs that play a key role in cell-cell communication and a metabolic function by exchange of nutrients including glucose and essential amino acids. A three-dimensional cellular network mediated through GJs might mediate metabolic and physiological homeostasis to maintain cartilage tissue. PMID:24225059

Chondrogenic Differentiation of Human Adipose-Derived Stem Cells: A New Path in Articular Cartilage Defect Management?

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Jan-Philipp Stromps

2014-01-01

Full Text Available According to data published by the Centers for Disease Control and Prevention, over 6 million people undergo a variety of medical procedures for the repair of articular cartilage defects in the U.S. each year. Trauma, tumor, and age-related degeneration can cause major defects in articular cartilage, which has a poor intrinsic capacity for healing. Therefore, there is substantial interest in the development of novel cartilage tissue engineering strategies to restore articular cartilage defects to a normal or prediseased state. Special attention has been paid to the expansion of chondrocytes, which produce and maintain the cartilaginous matrix in healthy cartilage. This review summarizes the current efforts to generate chondrocytes from adipose-derived stem cells (ASCs and provides an outlook on promising future strategies.

POSSIBILITIES OF CURRENT CELLULAR TECHNOLOGIES FOR ARTICULAR CARTILAGE REPAIR (ANALYTICAL REVIEW

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M. S. Bozhokin

2016-01-01

Full Text Available Despite a wide variety of surgical procedures utilized in clinical practice for treatment of articular cartilage lesions, the search for other options of articular reconstruction remains a relevant and open issue at the current stage of medicine and biotechnologies development. The recent years demonstrated a strong belief in cellular methods of hyaline cartilage repair such as implantation of autologous chondrocytes (ACI or cultures of mesenchymal stem cells (MSC including techniques for genetic modification of cells.The purpose of presented review is to summarize the published scientific data on up to date results of perspective cellular technologies for articular cartilage repair that are being developed. Autologous chondrocyte transplantation originally performed by Swedish researchers in 1987 is considered the first clinically applied technique for restoration of hyaline cartilage using cellular technologies. However, the transplanted cell culture featured low proliferative capacity and inability to form a regenerate resistant to high physical activity. Another generation of methods originated at the turn of the century utilized mesenchymal stem cells instead of autologous chondrocytes. Preparation of MSCs is a less invasive procedure compared to chondrocytes harvesting and the culture is featured by a higher proliferative ability. Researchers use various biodegradable carriers (matrices to secure cell fixation. Despite good clinical mid-term outcomes the transplanted tissue-engineering structures deteriorate with time due to cellular de-differentiation. Next generation of techniques being currently under pre-clinical studies is featured by the preliminary chondrogenic modification of transplanted cell culture. Usage of various growth factors, modified cell product and gene-activated matrices allow to gain a stable regulatory and key proteins synthesis and achieve a focused influence on regenerate's chondrogenic proliferation and in result

Synergy between Piezo1 and Piezo2 channels confers high-strain mechanosensitivity to articular cartilage

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Lee, Whasil; Leddy, Holly A.; Chen, Yong; Lee, Suk Hee; Zelenski, Nicole A.; McNulty, Amy L.; Wu, Jason; Beicker, Kellie N.; Coles, Jeffrey; Zauscher, Stefan; Grandl, Jörg; Sachs, Frederick; Liedtke, Wolfgang B.

2014-01-01

Diarthrodial joints are essential for load bearing and locomotion. Physiologically, articular cartilage sustains millions of cycles of mechanical loading. Chondrocytes, the cells in cartilage, regulate their metabolic activities in response to mechanical loading. Pathological mechanical stress can lead to maladaptive cellular responses and subsequent cartilage degeneration. We sought to deconstruct chondrocyte mechanotransduction by identifying mechanosensitive ion channels functioning at injurious levels of strain. We detected robust expression of the recently identified mechanosensitive channels, PIEZO1 and PIEZO2. Combined directed expression of Piezo1 and -2 sustained potentiated mechanically induced Ca2+ signals and electrical currents compared with single-Piezo expression. In primary articular chondrocytes, mechanically evoked Ca2+ transients produced by atomic force microscopy were inhibited by GsMTx4, a PIEZO-blocking peptide, and by Piezo1- or Piezo2-specific siRNA. We complemented the cellular approach with an explant-cartilage injury model. GsMTx4 reduced chondrocyte death after mechanical injury, suggesting a possible therapy for reducing cartilage injury and posttraumatic osteoarthritis by attenuating Piezo-mediated cartilage mechanotransduction of injurious strains. PMID:25385580

Autoradiographic evidence of sup 125 I-. beta. -endorphin binding sites in the articular cartilage of the rat

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Castano, M.T.; Freire-Garabal, M.; Giraldez, M.; Nunez, M.J.; Belmonte, A.; Couceiro, J.; Jorge, J. (Univ. of Santiago (Spain))

1991-01-01

After {sup 125}I-{beta}-endorphin was intravenously injected to rats, an autoradiographic study of distal femur articular cartilage was performed. Results show a specific binding of {sup 125}I-{beta}-endorphin to chondrocytes, suggesting the possible existence of an opiate modulation of articular cartilage.

RHEB: a potential regulator of chondrocyte phenotype for cartilage tissue regeneration.

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Ashraf, S; Ahn, J; Cha, B-H; Kim, J-S; Han, I; Park, H; Lee, S-H

2017-09-01

As articular cartilage has a limited ability to self-repair, successful cartilage regeneration requires clinical-grade chondrocytes with innate characteristics. However, cartilage regeneration via chondrocyte transplantation is challenging, because chondrocytes lose their innate characteristics during in vitro expansion. Here, we investigated the mechanistic underpinning of the gene Ras homologue enriched in brain (RHEB) in the control of senescence and dedifferentiation through the modulation of oxidative stress in chondrocytes, a hallmark of osteoarthritis. Serial expansion of human chondrocytes led to senescence, dedifferentiation and oxidative stress. RHEB maintained the innate characteristics of chondrocytes by regulating senescence, dedifferentiation and oxidative stress, leading to the upregulation of COL2 expression via SOX9 and the downregulation of p27 expression via MCL1. RHEB also decreased the expression of COL10. RHEB knockdown mimics decreased the expression of SOX9, COL2 and MCL1, while abrogating the suppressive function of RHEB on p27 and COL10 in chondrocytes. RHEB-overexpressing chondrocytes successfully formed cartilage tissue in vitro as well as in vivo, with increased expression of GAG matrix and chondrogenic markers. RHEB induces a distinct gene expression signature that maintained the innate chondrogenic properties over a long period. Therefore, RHEB expression represents a potentially useful mechanism in terms of cartilage tissue regeneration from chondrocytes, by which chondrocyte phenotypic and molecular characteristics can be retained through the modulation of senescence, dedifferentiation and oxidative stress. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Towards Regeneration of Articular Cartilage

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Iwamoto, Masahiro; Ohta, Yoichi; Larmour, Colleen; Enomoto-Iwamoto, Motomi

2014-01-01

Articular cartilage is classified into permanent hyaline cartilage and has significant differences in structure, extracelluar matrix components, gene expression profile, and mechanical property from transient hyaline cartilage found in growth plate. In the process of synovial joint development, articular cartilage is originated from the interzone, developing at the edge of the cartilaginous anlagen, it establishes zonal structure over time and supports smooth movement of the synovial joint through life. The cascade actions of key regulators such as Wnts, GDF5, Erg, and PTHLH coordinate sequential steps of articular cartilage formation. Articular chondrocytes are restrictedly controlled not to differentiate into a hypertrophic stage by autocrine and paracrine factors and extracerllular matrix microenvironment, but retain potential to undergo hypertrophy. The basal calcified zone of articular cartilage is connected with subchondral bone, but not invaded by blood vessels nor replaced by bone, which is highly contrasted with the growth plate. Articular cartilage has limited regenerative capacity, but likely possesses and potentially uses intrinsic stem cell source in the superficial layer, Ranvier’s groove, the intra-articular tissues such as synovium and fat pad, and marrow below the subchondral bone. Considering the biological views on articular cartilage, several important points are raised for regeneration of articular cartilage. We should evaluate the nature of regenerated cartilage as permanent hyaline cartilage and not just hyaline cartilage. We should study how a hypertrophic phenotype of transplanted cells can be lastingly suppressed in regenerating tissue. Further, we should develop the methods and reagents to activate recruitment of intrinsic stem/progenitor cells into the damaged site. PMID:24078496

TGF-β1, GDF-5, and BMP-2 stimulation induces chondrogenesis in expanded human articular chondrocytes and marrow-derived stromal cells.

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Murphy, Meghan K; Huey, Daniel J; Hu, Jerry C; Athanasiou, Kyriacos A

2015-03-01

Replacement of degenerated cartilage with cell-based cartilage products may offer a long-term solution to halt arthritis' degenerative progression. Chondrocytes are frequently used in cell-based FDA-approved cartilage products; yet human marrow-derived stromal cells (hMSCs) show significant translational potential, reducing donor site morbidity and maintaining their undifferentiated phenotype with expansion. This study sought to investigate the effects of transforming growth factor β1 (TGF-β1), growth/differentiation factor 5 (GDF-5), and bone morphogenetic protein 2 (BMP-2) during postexpansion chondrogenesis in human articular chondrocytes (hACs) and to compare chondrogenesis in passaged hACs with that of passaged hMSCs. Through serial expansion, chondrocytes dedifferentiated, decreasing expression of chondrogenic genes while increasing expression of fibroblastic genes. However, following expansion, 10 ng/mL TGF-β1, 100 ng/mL GDF-5, or 100 ng/mL BMP-2 supplementation during three-dimensional aggregate culture each upregulated one or more markers of chondrogenic gene expression in both hACs and hMSCs. Additionally, in both cell types, the combination of TGF-β1, GDF-5, and BMP-2 induced the greatest upregulation of chondrogenic genes, that is, Col2A1, Col2A1/Col1A1 ratio, SOX9, and ACAN, and synthesis of cartilage-specific matrix, that is, glycosaminoglycans (GAGs) and ratio of collagen II/I. Finally, TGF-β1, GDF-5, and BMP-2 stimulation yielded mechanically robust cartilage rich in collagen II and GAGs in both cell types, following 4 weeks maturation. This study illustrates notable success in using the self-assembling method to generate robust, scaffold-free neocartilage constructs using expanded hACs and hMSCs. © 2014 AlphaMed Press.

Downregulation of protein kinase CK2 activity facilitates tumor necrosis factor-α-mediated chondrocyte death through apoptosis and autophagy.

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Sung Won Lee

Full Text Available Despite the numerous studies of protein kinase CK2, little progress has been made in understanding its function in chondrocyte death. Our previous study first demonstrated that CK2 is involved in apoptosis of rat articular chondrocytes. Recent studies have suggested that CK2 downregulation is associated with aging. Thus examining the involvement of CK2 downregulation in chondrocyte death is an urgently required task. We undertook this study to examine whether CK2 downregulation modulates chondrocyte death. We first measured CK2 activity in articular chondrocytes of 6-, 21- and 30-month-old rats. Noticeably, CK2 activity was downregulated in chondrocytes with advancing age. To build an in vitro experimental system for simulating tumor necrosis factor (TNF-α-induced cell death in aged chondrocytes with decreased CK2 activity, chondrocytes were co-treated with CK2 inhibitors and TNF-α. Viability assay demonstrated that CK2 inhibitors facilitated TNF-α-mediated chondrocyte death. Pulsed-field gel electrophoresis, nuclear staining, flow cytometry, TUNEL staining, confocal microscopy, western blot and transmission electron microscopy were conducted to assess cell death modes. The results of multiple assays showed that this cell death was mediated by apoptosis. Importantly, autophagy was also involved in this process, as supported by the appearance of a punctuate LC3 pattern and autophagic vacuoles. The inhibition of autophagy by silencing of autophage-related genes 5 and 7 as well as by 3-methyladenine treatment protected chondrocytes against cell death and caspase activation, indicating that autophagy led to the induction of apoptosis. Autophagic cells were observed in cartilage obtained from osteoarthritis (OA model rats and human OA patients. Our findings indicate that CK2 down regulation facilitates TNF-α-mediated chondrocyte death through apoptosis and autophagy. It should be clarified in the future if autophagy observed is a consequence

The influence of “Efial” medicine on the chondrocytes functional state

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N. А. Volkova

2014-12-01

Full Text Available Renewal of articular cartilage is a topical issue of modern orthopedics. High frequency of injuries, complexity of clinical diagnosis and subsequent treatment, and also the delay in recovery lead to the development of osteoarthritis, and in some cases, to disability. Articular cartilage belongs to the highly specialized tissues, which is characterized by the lack of blood supply, the low number of cell elements that are placed in the matrix, include collagen, proteoglycans, non-collagenous proteins and water. For the treatment of articular cartilage lesions the medicine which are tissue specific promoters of regeneration are used. The ability of most reparants to stimulate cartilage regeneration combines with other effects, such as: anti-inflammatory, antioxidant and antibacterial. The purpose of administration of these medicines is to stimulate regeneration of tissue in the area of injury. The aim of research was to investigate the effect of “Efial” medicine on functional state of chondrocytes in cultivation conditions. Materials and methods. The chondrocytes were obtained from articular cartilage of rats by enzymatic disaggregation. In all experiments the seeding concentration of chondrocytes was 1.2 x 104 cells/cm2.The "Efial" medicine in concentration of peptides of 0.137 mg/ml was used. Investigated concentration range was 70; 7.6; 1.5; 0.15µg/ml and 75; 15; 1.5 ng/ml. The medicine was added to the cell culture medium when seeding and on the 3rd cultivation day. The control (comparison group was the cultures of chondrocytes which were cultivated under the same conditions without medicine addition. Functional state of chondrocytes under interaction with investigated "Efial" medicine was evaluated by the presence of glycosaminoglycans after Toluidine blue staining (Fluka, Germany and collagen type II (1:200 and FITC-conjugate, Sigma -Aldrich, USA. For statistical study ANOVA and t-Student tests were used with application of Microsoft

Osteoarthritis: Control of human cartilage hypertrophic differentiation. Research highlight van: Gremlin1, frizzled-related protein, and Dkk-1 are key regulators of human articular cartilage homeostasis

NARCIS (Netherlands)

Buckland, J.; Leijten, Jeroen Christianus Hermanus; van Blitterswijk, Clemens; Karperien, Hermanus Bernardus Johannes

2012-01-01

Disruption of articular cartilage homeostasis is important in osteoarthritis (OA) pathogenesis, key to which is activation of articular chondrocyte hypertrophic differentiation. Healthy articular cartilage is resistant to hypertrophic differentiation, whereas growth-plate cartilage is destined to

Repair of experimentally produced defects in rabbit articular cartilage by autologous chondrocyte transplantation

International Nuclear Information System (INIS)

Grande, D.A.; Pitman, M.I.; Peterson, L.; Menche, D.; Klein, M.

1989-01-01

Using the knee joints of New Zealand White rabbits, a baseline study was made to determine the intrinsic capability of cartilage for healing defects that do not fracture the subchondral plate. A second experiment examined the effect of autologous chondrocytes grown in vitro on the healing rate of these defects. To determine whether any of the reconstituted cartilage resulted from the chondrocyte graft, a third experiment was conducted involving grafts with chondrocytes that had been labeled prior to grafting with a nuclear tracer. Results were evaluated using both qualitative and quantitative light microscopy. Macroscopic results from grafted specimens displayed a marked decrease in synovitis and other degenerative changes. In defects that had received transplants, a significant amount of cartilage was reconstituted (82%) compared to ungrafted controls (18%). Autoradiography on reconstituted cartilage showed that there were labeled cells incorporated into the repair matrix

Review on patents for mechanical stimulation of articular cartilage tissue engineering

NARCIS (Netherlands)

Donkelaar, van C.C.; Schulz, R.M.

2008-01-01

To repair articular cartilage defects in osteoarthritic patients with three-dimensional tissue engineered chondrocyte grafts, requires the formation of new cartilage with sufficient mechanical properties. The premise is that mechanical stimulation during the culturing process is necessary to reach

Optimization of Methods for Articular Cartilage Surface Tissue Engineering: Cell Density and Transforming Growth Factor Beta Are Critical for Self-Assembly and Lubricin Secretion.

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Iwasa, Kenjiro; Reddi, A Hari

2017-07-01

Lubricin/superficial zone protein (SZP)/proteoglycan4 (PRG4) plays an important role in boundary lubrication in articular cartilage. Lubricin is secreted by superficial zone chondrocytes and synoviocytes of the synovium. The specific objective of this investigation is to optimize the methods for tissue engineering of articular cartilage surface. The aim of this study is to investigate the effect of cell density on the self-assembly of superficial zone chondrocytes and lubricin secretion as a functional assessment. Superficial zone chondrocytes were cultivated as a monolayer at low, medium, and high densities. Chondrocytes at the three different densities were treated with transforming growth factor beta (TGF-β)1 twice a week or daily, and the accumulated lubricin in the culture medium was analyzed by immunoblots and quantitated by enzyme-linked immunosorbent assay (ELISA). Cell numbers in low and medium densities were increased by TGF-β1; whereas cell numbers in high-density cell cultures were decreased by twice-a-week treatment of TGF-β1. On the other hand, the cell numbers were maintained by daily TGF-β treatment. Immunoblots and quantitation of lubricin by ELISA analysis indicated that TGF-β1 stimulated lubricin secretion by superficial zone chondrocytes at all densities with twice-a-week TGF-β treatment. It is noteworthy that the daily treatment of TGF-β1 increased lubricin much higher compared with twice-a-week treatment. These data demonstrate that daily treatment is optimal for the TGF-β1 response in a higher density of monolayer cultures. These findings have implications for self-assembly of surface zone chondrocytes of articular cartilage for application in tissue engineering of articular cartilage surface.

A vision on the future of articular cartilage repair

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M Cucchiarini

2014-05-01

Full Text Available An AO Foundation (Davos, Switzerland sponsored workshop "Cell Therapy in Cartilage Repair" from the Symposium "Where Science meets Clinics" (September 5-7, 2013, Davos gathered leaders from medicine, science, industry, and regulatory organisations to debate the vision of cell therapy in articular cartilage repair and the measures that could be taken to narrow the gap between vision and current practice. Cell-based therapy is already in clinical use to enhance the repair of cartilage lesions, with procedures such as microfracture and articular chondrocyte implantation. However, even though long term follow up is good from a clinical perspective and some of the most rigorous randomised controlled trials in the regenerative medicine/orthopaedics field show beneficial effect, none of these options have proved successful in restoring the original articular cartilage structure and functionality in patients so far. With the remarkable recent advances in experimental research in cell biology (new sources for chondrocytes, stem cells, molecular biology (growth factors, genes, biomaterials, biomechanics, and translational science, a combined effort between scientists and clinicians with broad expertise may allow development of an improved cell therapy for cartilage repair. This position paper describes the current state of the art in the field to help define a procedure adapted to the clinical situation for upcoming translation in the patient.

Kaempferol Alleviates the Interleukin-1β-Induced Inflammation in Rat Osteoarthritis Chondrocytes via Suppression of NF-κB.

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Zhuang, Zhengling; Ye, Guangqun; Huang, Bin

2017-08-14

BACKGROUND This study was designed to examine the anti-inflammatory and anti-osteoarthritis (OA) effects of kaempferol in rat articular chondrocytes stimulated with interleukin-1β. MATERIAL AND METHODS Rat articular chondrocytes cultures were treated with interleukin-1β alone or with kaempferol (25, 50, 100, and 200 μM) and interleukin-1β. The effect of kaempferol on chondrocyte cells viability was measured by MTT assay. The effect on prostaglandin E2 (PGE2) and nitric oxide (NO) level were also assessed using the ELISA and Griess reagent, respectively, for kaempferol activity. Moreover, the expression of iNOS, Cox-2 and activation of NF-κB under influence of kaempferol was also assessed by Western blot. RESULTS Kaempferol treatment (up to 100 μM) in a concentration-dependent way caused reduction in the interleukin-1b-stimulated formations of PGE2 and NO. Kaempferol also upregulated the expression of iNOS and Cox-2 in interleukin-1β-stimulated rat OA chondrocytes. Additionally, kaempferol was found to inhibit the IkBa degradation and NF-κB activation in rat chondrocytes stimulated with interleukin-1β. CONCLUSIONS Kaempferol significantly caused reduction in interleukin-1β-stimulated pro-inflammatory mediators in rat OA chondrocytes by inhibiting the NF-κB pathway. These results suggest that kaempferol had significant anti-inflammatory and anti-arthritis effects. Thus, kaempferol, as a novel therapeutic active agent, may prevent, stop, or retard the progression of OA.

Sprifermin (rhFGF18) enables proliferation of chondrocytes producing a hyaline cartilage matrix.

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Gigout, A; Guehring, H; Froemel, D; Meurer, A; Ladel, C; Reker, D; Bay-Jensen, A C; Karsdal, M A; Lindemann, S

2017-11-01

Fibroblast growth factor (FGF) 18 has been shown to increase cartilage volume when injected intra-articularly in animal models of osteoarthritis (OA) and in patients with knee OA (during clinical development of the recombinant human FGF18, sprifermin). However, the exact nature of this effect is still unknown. In this study, we aimed to investigate the effects of sprifermin at the cellular level. A combination of different chondrocyte culture systems was used and the effects of sprifermin on proliferation, the phenotype and matrix production were evaluated. The involvement of MAPKs in sprifermin signalling was also studied. In monolayer, we observed that sprifermin promoted a round cell morphology and stimulated both cellular proliferation and Sox9 expression while strongly decreasing type I collagen expression. In 3D culture, sprifermin increased the number of matrix-producing chondrocytes, improved the type II:I collagen ratio and enabled human OA chondrocytes to produce a hyaline extracellular matrix (ECM). Furthermore, we found that sprifermin displayed a 'hit and run' mode of action, with intermittent exposure required for the compound to fully exert its anabolic effect. Finally, sprifermin appeared to signal through activation of ERK. Our results indicate that intermittent exposure to sprifermin leads to expansion of hyaline cartilage-producing chondrocytes. These in vitro findings are consistent with the increased cartilage volume observed in the knees of OA patients after intra-articular injection with sprifermin in clinical studies. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Synthesis of collagen by bovine chondrocytes cultured in alginate; posttranslational modifications and cell-matrix interaction

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Beekman, B.; Verzijl, N.; Bank, R.A.; Von Der Mark, K.; TeKoppele, J.M.

1997-01-01

The extracellular matrix synthesized by articular chondrocytes cultured in alginate beads was investigated. Collagen levels increased sigmoidally with time and remained constant after 2 weeks of culture. The presence of cartilage-specific type II collagen was confirmed immunohistochemically.

ICAM-1 expression on chondrocytes in rheumatoid arthritis: induction by synovial cytokines

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M. E. Davies

1992-01-01

Full Text Available The intercellular adhesion molecule-1 (ICAM-1 was found by immunostaining chondrocytes in cartilage from three patients with rheumatoid arthritis. Expression of ICAM-1 was restricted to chondrocytes in areas of erodedcartilage adjacent to the invading synovial tissue. Toluidine blue staining of these areas demonstrated severe depletion of the cartilage extracellular matrix. In areas of undamaged cartilage there was no ICAM-1 expression. Since ICAM-1 is not constitutively expressed on normal human articular cartilage, but could be induced in vitro by exogenous IL-1α, TNFα and IFNγ or by co-culturing cartilage with inflammatory rheumatoid synovium, we conclude that the induction of ICAM-1 on rheumatoid chondrocytes results from the synergistic action of a variety of cytokines produced by the inflammatory cells of the invading pannus.

Effects of chondroitin sulfate and sodium hyaluronate on chondrocytes and extracellular matrix of articular cartilage in dogs with degenerative joint disease Efeitos do sulfato de condroitina e do hialuronato de sódio nos condrócitos e na matriz extracelular na cartilagem articular de cães com doença articular degenerativa

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G. Gonçalves

2008-02-01

Full Text Available Samples of articular cartilage of femur, tibia and patella of 15 dogs with experimentally induced degenerative joint disease (DJD were microscopically analyzed. Animals were distributed into three groups (n=5: the control group received no medication; the second group was treated with chondroitin sulfate and the third received sodium hyaluronate. Samples were processed and stained with HE and toluidine blue for morphological evaluation. The metabolic and proliferative activity of the chondrocytes was evaluated by the measurement of nucleolar organizer regions (NORs after impregnation by silver nitrate. Significant differences were not observed (P>0.05 in the morphology among the groups, however, the group treated with sodium hyaluronate had a higher score suggesting a trend to a greater severity of the lesions. Significant differences were not observed (P>0.05 in the measurement of NORs, cells and NORs/cells among the groups. Although differences were not significant, sodium hyaluronate group showed higher NOR and cell counts which suggested an increase of the proliferation rate of chondrocytes. In addition, a higher NOR/cell ratio in the group treated with chondroitin sulfate suggested that this drug may have stimulated the metabolic activity of the chondrocytes, minimizing the lesions resulting from DJD.Foram utilizadas amostras de cartilagem articular do fêmur, tíbia e patela de 15 cães com doença articular degenerativa (DAD, induzida experimentalmente. Foram constituídos três grupos de cinco animais: grupo 1 - controle, não medicado; grupo 2 - tratado com sulfato de condroitina e grupo 3 - tratado com hialuronato de sódio. As amostras foram processadas e coradas pelas técnicas de HE e de azul de toluidina para avaliação das alterações morfológicas, e impregnadas pelo nitrato de prata para análise da atividade metabólica e/ou proliferativa dos condrócitos, por meio da visualização e quantificação de regiões organizadoras

IFT88 influences chondrocyte actin organization and biomechanics.

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Wang, Z; Wann, A K T; Thompson, C L; Hassen, A; Wang, W; Knight, M M

2016-03-01

Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88(orpk)). Confocal microscopy was used to quantify F-actin and myosin IIB organisation. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualised in live cells transfected with LifeACT-GFP. IFT88(orpk) cells exhibited a significant increase in acto-myosin stress fibre organisation relative to wild-type (WT) cells in monolayer and an altered response to cytochalasin D. Rounded IFT88(orpk) cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88(orpk) cells. Following membrane blebbing, IFT88(orpk) cells exhibited slower reformation of the actin cortex. IFT88(orpk) cells showed increased actin deformability and reduced cortical tension confirming that IFT regulates actin cortex mechanics. The reduced cortical tension is also consistent with the reduced bleb formation. This study demonstrates for the first time that the ciliary protein IFT88 regulates fundamental actin organisation and the stiffness of the actin cortex leading to alterations in cell deformation, mechanical properties and blebbing in an IFT88 chondrocyte cell line. This adds to the growing understanding of the role of primary cilia and IFT in regulating cartilage biology. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Inflammation induction of Dickkopf-1 mediates chondrocyte apoptosis in osteoarthritic joint.

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Weng, L-H; Wang, C-J; Ko, J-Y; Sun, Y-C; Su, Y-S; Wang, F-S

2009-07-01

Dysregulated Wnt signaling appears to modulate chondrocyte fate and joint disorders. Dickkopf-1 (DKK1) regulates the pathogenesis of skeletal tissue by inhibiting Wnt actions. This study examined whether DKK1 expression is linked to chondrocyte fate in osteoarthritis (OA). Articular cartilage specimens harvested from nine patients with knee OA and from six controls with femoral neck fracture were assessed for DKK1, interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), Bad, Bax, Bcl2 and caspase-3 expression by real time-polymerase chain reaction (RT-PCR) and immunohistochemistry. Apoptotic chondrocytes were detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labelling (TUNEL) and 4', 6-dianidino-2-phenylindole dihydrochloride (DAPI) staining. Human chondrocyte cultures were treated with recombinant IL-1beta and monoclonal DKK1 antibody to determine whether DKK1 impairs chondrocyte survival. Expression of DKK1 correlated with inflammatory cytokine levels (IL-1beta and TNF-alpha expressions), proapoptosis regulators (Bad and caspase-3 expressions) and TUNEL staining in OA cartilage tissues. The IL-1beta induced expressions of DKK1, Bax, Bad and caspase-3-dependent apoptosis of chondrocyte cultures. Neutralization of DKK1 by monoclonal DKK1 antibody significantly abrogated IL-1beta-mediated caspase-3 cleavage and apoptosis and reversed chondrocyte proliferation. Recombinant DKK1 treatment impaired chondrocyte growth and promoted apoptosis. By suppressing nuclear beta-catenin accumulation and Akt phosphorylation, DKK1 mediated IL-1beta promotion of chondrocyte apoptosis. Chondrocyte apoptosis correlates with joint OA. Expression of DKK1 contributes to cartilage deterioration and is a potent factor in OA pathogenesis. Attenuating DKK1 may reduce cartilage deterioration in OA.

Culture temperature affects human chondrocyte messenger RNA expression in monolayer and pellet culture systems.

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Ito, Akira; Nagai, Momoko; Tajino, Junichi; Yamaguchi, Shoki; Iijima, Hirotaka; Zhang, Xiangkai; Aoyama, Tomoki; Kuroki, Hiroshi

2015-01-01

Cell-based therapy has been explored for articular cartilage regeneration. Autologous chondrocyte implantation is a promising cell-based technique for repairing articular cartilage defects. However, there are several issues such as chondrocyte de-differentiation. While numerous studies have been designed to overcome some of these issues, only a few have focused on the thermal environment that can affect chondrocyte metabolism and phenotype. In this study, the effects of different culture temperatures on human chondrocyte metabolism- and phenotype-related gene expression were investigated in 2D and 3D environments. Human chondrocytes were cultured in a monolayer or in a pellet culture system at three different culture temperatures (32°C, 37°C, and 41°C) for 3 days. The results showed that the total RNA level, normalized to the threshold cycle value of internal reference genes, was higher at lower temperatures in both culture systems. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and citrate synthase (CS), which are involved in glycolysis and the citric acid cycle, respectively, were expressed at similar levels at 32°C and 37°C in pellet cultures, but the levels were significantly lower at 41°C. Expression of the chondrogenic markers, collagen type IIA1 (COL2A1) and aggrecan (ACAN), was higher at 37°C than at 32°C and 41°C in both culture systems. However, this phenomenon did not coincide with SRY (sex-determining region Y)-box 9 (SOX9), which is a fundamental transcription factor for chondrogenesis, indicating that a SOX9-independent pathway might be involved in this phenomenon. In conclusion, the expression of chondrocyte metabolism-related genes at 32°C was maintained or enhanced compared to that at 37°C. However, chondrogenesis-related genes were further induced at 37°C in both culture systems. Therefore, manipulating the culture temperature may be an advantageous approach for regulating human chondrocyte metabolic activity and chondrogenesis.

Culture temperature affects human chondrocyte messenger RNA expression in monolayer and pellet culture systems.

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Akira Ito

Full Text Available Cell-based therapy has been explored for articular cartilage regeneration. Autologous chondrocyte implantation is a promising cell-based technique for repairing articular cartilage defects. However, there are several issues such as chondrocyte de-differentiation. While numerous studies have been designed to overcome some of these issues, only a few have focused on the thermal environment that can affect chondrocyte metabolism and phenotype. In this study, the effects of different culture temperatures on human chondrocyte metabolism- and phenotype-related gene expression were investigated in 2D and 3D environments. Human chondrocytes were cultured in a monolayer or in a pellet culture system at three different culture temperatures (32°C, 37°C, and 41°C for 3 days. The results showed that the total RNA level, normalized to the threshold cycle value of internal reference genes, was higher at lower temperatures in both culture systems. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH and citrate synthase (CS, which are involved in glycolysis and the citric acid cycle, respectively, were expressed at similar levels at 32°C and 37°C in pellet cultures, but the levels were significantly lower at 41°C. Expression of the chondrogenic markers, collagen type IIA1 (COL2A1 and aggrecan (ACAN, was higher at 37°C than at 32°C and 41°C in both culture systems. However, this phenomenon did not coincide with SRY (sex-determining region Y-box 9 (SOX9, which is a fundamental transcription factor for chondrogenesis, indicating that a SOX9-independent pathway might be involved in this phenomenon. In conclusion, the expression of chondrocyte metabolism-related genes at 32°C was maintained or enhanced compared to that at 37°C. However, chondrogenesis-related genes were further induced at 37°C in both culture systems. Therefore, manipulating the culture temperature may be an advantageous approach for regulating human chondrocyte metabolic activity and

In Vivo Articular Cartilage Regeneration Using Human Dental Pulp Stem Cells Cultured in an Alginate Scaffold: A Preliminary Study

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Manuel Mata

2017-01-01

Full Text Available Osteoarthritis is an inflammatory disease in which all joint-related elements, articular cartilage in particular, are affected. The poor regeneration capacity of this tissue together with the lack of pharmacological treatment has led to the development of regenerative medicine methodologies including microfracture and autologous chondrocyte implantation (ACI. The effectiveness of ACI has been shown in vitro and in vivo, but the use of other cell types, including bone marrow and adipose-derived mesenchymal stem cells, is necessary because of the poor proliferation rate of isolated articular chondrocytes. In this investigation, we assessed the chondrogenic ability of human dental pulp stem cells (hDPSCs to regenerate cartilage in vitro and in vivo. hDPSCs and primary isolated rabbit chondrocytes were cultured in chondrogenic culture medium and found to express collagen II and aggrecan. Both cell types were cultured in 3% alginate hydrogels and implanted in a rabbit model of cartilage damage. Three months after surgery, significant cartilage regeneration was observed, particularly in the animals implanted with hDPSCs. Although the results presented here are preliminary, they suggest that hDPSCs may be useful for regeneration of articular cartilage.

In Vivo Articular Cartilage Regeneration Using Human Dental Pulp Stem Cells Cultured in an Alginate Scaffold: A Preliminary Study.

Science.gov (United States)

Mata, Manuel; Milian, Lara; Oliver, Maria; Zurriaga, Javier; Sancho-Tello, Maria; de Llano, Jose Javier Martin; Carda, Carmen

2017-01-01

Osteoarthritis is an inflammatory disease in which all joint-related elements, articular cartilage in particular, are affected. The poor regeneration capacity of this tissue together with the lack of pharmacological treatment has led to the development of regenerative medicine methodologies including microfracture and autologous chondrocyte implantation (ACI). The effectiveness of ACI has been shown in vitro and in vivo , but the use of other cell types, including bone marrow and adipose-derived mesenchymal stem cells, is necessary because of the poor proliferation rate of isolated articular chondrocytes. In this investigation, we assessed the chondrogenic ability of human dental pulp stem cells (hDPSCs) to regenerate cartilage in vitro and in vivo . hDPSCs and primary isolated rabbit chondrocytes were cultured in chondrogenic culture medium and found to express collagen II and aggrecan. Both cell types were cultured in 3% alginate hydrogels and implanted in a rabbit model of cartilage damage. Three months after surgery, significant cartilage regeneration was observed, particularly in the animals implanted with hDPSCs. Although the results presented here are preliminary, they suggest that hDPSCs may be useful for regeneration of articular cartilage.

Cartilage repair: Generations of autologous chondrocyte transplantation

International Nuclear Information System (INIS)

Marlovits, Stefan; Zeller, Philip; Singer, Philipp; Resinger, Christoph; Vecsei, Vilmos

2006-01-01

Articular cartilage in adults has a limited capacity for self-repair after a substantial injury. Surgical therapeutic efforts to treat cartilage defects have focused on delivering new cells capable of chondrogenesis into the lesions. Autologous chondrocyte transplantation (ACT) is an advanced cell-based orthobiologic technology used for the treatment of chondral defects of the knee that has been in clinical use since 1987 and has been performed on 12,000 patients internationally. With ACT, good to excellent clinical results are seen in isolated post-traumatic lesions of the knee joint in the younger patient, with the formation of hyaline or hyaline-like repair tissue. In the classic ACT technique, chondrocytes are isolated from small slices of cartilage harvested arthroscopically from a minor weight-bearing area of the injured knee. The extracellular matrix is removed by enzymatic digestion, and the cells are then expanded in monolayer culture. Once a sufficient number of cells has been obtained, the chondrocytes are implanted into the cartilage defect, using a periosteal patch over the defect as a method of cell containment. The major complications are periosteal hypertrophy, delamination of the transplant, arthrofibrosis and transplant failure. Further improvements in tissue engineering have contributed to the next generation of ACT techniques, where cells are combined with resorbable biomaterials, as in matrix-associated autologous chondrocyte transplantation (MACT). These biomaterials secure the cells in the defect area and enhance their proliferation and differentiation

Cartilage repair: Generations of autologous chondrocyte transplantation

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Marlovits, Stefan [Department of Traumatology, Center for Joint and Cartilage, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria)]. E-mail: stefan.marlovits@meduniwien.ac.at; Zeller, Philip [Department of Traumatology, Center for Joint and Cartilage, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Singer, Philipp [Department of Traumatology, Center for Joint and Cartilage, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Resinger, Christoph [Department of Traumatology, Center for Joint and Cartilage, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Vecsei, Vilmos [Department of Traumatology, Center for Joint and Cartilage, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria)

2006-01-15

Articular cartilage in adults has a limited capacity for self-repair after a substantial injury. Surgical therapeutic efforts to treat cartilage defects have focused on delivering new cells capable of chondrogenesis into the lesions. Autologous chondrocyte transplantation (ACT) is an advanced cell-based orthobiologic technology used for the treatment of chondral defects of the knee that has been in clinical use since 1987 and has been performed on 12,000 patients internationally. With ACT, good to excellent clinical results are seen in isolated post-traumatic lesions of the knee joint in the younger patient, with the formation of hyaline or hyaline-like repair tissue. In the classic ACT technique, chondrocytes are isolated from small slices of cartilage harvested arthroscopically from a minor weight-bearing area of the injured knee. The extracellular matrix is removed by enzymatic digestion, and the cells are then expanded in monolayer culture. Once a sufficient number of cells has been obtained, the chondrocytes are implanted into the cartilage defect, using a periosteal patch over the defect as a method of cell containment. The major complications are periosteal hypertrophy, delamination of the transplant, arthrofibrosis and transplant failure. Further improvements in tissue engineering have contributed to the next generation of ACT techniques, where cells are combined with resorbable biomaterials, as in matrix-associated autologous chondrocyte transplantation (MACT). These biomaterials secure the cells in the defect area and enhance their proliferation and differentiation.

Botanical Extracts from Rosehip (Rosa canina), Willow Bark (Salix alba), and Nettle Leaf (Urtica dioica) Suppress IL-1β-Induced NF-κB Activation in Canine Articular Chondrocytes

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Shakibaei, Mehdi; Allaway, David; Nebrich, Simone; Mobasheri, Ali

2012-01-01

The aim of this study was to characterize the anti-inflammatory mode of action of botanical extracts from rosehip (Rosa canina), willow bark (Salix alba), and nettle leaf (Urtica dioica) in an in vitro model of primary canine articular chondrocytes. Methods. The biological effects of the botanical extracts were studied in chondrocytes treated with IL-1β for up to 72 h. Expression of collagen type II, cartilage-specific proteoglycan (CSPG), β1-integrin, SOX-9, COX-2, and MMP-9 and MMP-13 was examined by western blotting. Results. The botanical extracts suppressed IL-1β-induced NF-κB activation by inhibition of IκBα phosphorylation, IκBα degradation, p65 phosphorylation, and p65 nuclear translocation. These events correlated with downregulation of NF-κB targets including COX-2 and MMPs. The extracts also reversed the IL-1β-induced downregulation of collagen type II, CSPG, β1-integrin, and cartilage-specific transcription factor SOX-9 protein expression. In high-density cultures botanical extracts stimulated new cartilage formation even in the presence of IL-1β. Conclusions. Botanical extracts exerted anti-inflammatory and anabolic effects on chondrocytes. The observed reduction of IL-1β-induced NF-κB activation suggests that further studies are warranted to demonstrate the effectiveness of plant extracts in the treatment of OA and other conditions in which NF-κB plays pathophysiological roles. PMID:22474508

Botanical Extracts from Rosehip (Rosa canina), Willow Bark (Salix alba), and Nettle Leaf (Urtica dioica) Suppress IL-1β-Induced NF-κB Activation in Canine Articular Chondrocytes.

Science.gov (United States)

Shakibaei, Mehdi; Allaway, David; Nebrich, Simone; Mobasheri, Ali

2012-01-01

The aim of this study was to characterize the anti-inflammatory mode of action of botanical extracts from rosehip (Rosa canina), willow bark (Salix alba), and nettle leaf (Urtica dioica) in an in vitro model of primary canine articular chondrocytes. Methods. The biological effects of the botanical extracts were studied in chondrocytes treated with IL-1β for up to 72 h. Expression of collagen type II, cartilage-specific proteoglycan (CSPG), β1-integrin, SOX-9, COX-2, and MMP-9 and MMP-13 was examined by western blotting. Results. The botanical extracts suppressed IL-1β-induced NF-κB activation by inhibition of IκBα phosphorylation, IκBα degradation, p65 phosphorylation, and p65 nuclear translocation. These events correlated with downregulation of NF-κB targets including COX-2 and MMPs. The extracts also reversed the IL-1β-induced downregulation of collagen type II, CSPG, β1-integrin, and cartilage-specific transcription factor SOX-9 protein expression. In high-density cultures botanical extracts stimulated new cartilage formation even in the presence of IL-1β. Conclusions. Botanical extracts exerted anti-inflammatory and anabolic effects on chondrocytes. The observed reduction of IL-1β-induced NF-κB activation suggests that further studies are warranted to demonstrate the effectiveness of plant extracts in the treatment of OA and other conditions in which NF-κB plays pathophysiological roles.

Botanical Extracts from Rosehip (Rosa canina, Willow Bark (Salix alba, and Nettle Leaf (Urtica dioica Suppress IL-1β-Induced NF-κB Activation in Canine Articular Chondrocytes

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Mehdi Shakibaei

2012-01-01

Full Text Available The aim of this study was to characterize the anti-inflammatory mode of action of botanical extracts from rosehip (Rosa canina, willow bark (Salix alba, and nettle leaf (Urtica dioica in an in vitro model of primary canine articular chondrocytes. Methods. The biological effects of the botanical extracts were studied in chondrocytes treated with IL-1β for up to 72 h. Expression of collagen type II, cartilage-specific proteoglycan (CSPG, β1-integrin, SOX-9, COX-2, and MMP-9 and MMP-13 was examined by western blotting. Results. The botanical extracts suppressed IL-1β-induced NF-κB activation by inhibition of IκBα phosphorylation, IκBα degradation, p65 phosphorylation, and p65 nuclear translocation. These events correlated with downregulation of NF-κB targets including COX-2 and MMPs. The extracts also reversed the IL-1β-induced downregulation of collagen type II, CSPG, β1-integrin, and cartilage-specific transcription factor SOX-9 protein expression. In high-density cultures botanical extracts stimulated new cartilage formation even in the presence of IL-1β. Conclusions. Botanical extracts exerted anti-inflammatory and anabolic effects on chondrocytes. The observed reduction of IL-1β-induced NF-κB activation suggests that further studies are warranted to demonstrate the effectiveness of plant extracts in the treatment of OA and other conditions in which NF-κB plays pathophysiological roles.

The distribution of YKL-40 in osteoarthritic and normal human articular cartilage

DEFF Research Database (Denmark)

Volck, B; Ostergaard, K; Johansen, J S

1999-01-01

YKL-40, also called human cartilage glycoprotein-39, is a major secretory protein of human chondrocytes in cell culture. YKL-40 mRNA is expressed by cartilage from patients with rheumatoid arthritis, but is not detectable in normal human cartilage. The aim was to investigate the distribution of YKL......-40 in osteoarthritic (n=9) and macroscopically normal (n=5) human articular cartilage, collected from 12 pre-selected areas of the femoral head, to discover a potential role for YKL-40 in cartilage remodelling in osteoarthritis. Immunohistochemical analysis showed that YKL-40 staining was found...... in chondrocytes of osteoarthritic cartilage mainly in the superficial and middle zone of the cartilage rather than the deep zone. There was a tendency for high number of YKL-40 positive chondrocytes in areas of the femoral head with a considerable biomechanical load. The number of chondrocytes with a positive...

Influence of intermittent pressure, fluid flow, and mixing on the regenerative properties of articular chondrocytes.

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Carver, S E; Heath, C A

1999-11-05

Equine articular chondrocytes, embedded within a polyglycolic acid nonwoven mesh, were cultured with various combinations of intermittent pressure, fluid flow, and mixing to examine the effects of different physical stimuli on neochondrogenesis from young cells. The cell/polymer constructs were cultured first in 125 ml spinner flasks for 1, 2, or 4 weeks and then in a perfusion system with intermittent pressure for a total of up to 6 weeks. Additional constructs were either cultured for all 6 weeks in the spinner flasks or for 1 week in spinners followed by 5 weeks in the perfusion system without intermittent pressure. Tissue constructs cultivated for 2 or 4 weeks in spinner flasks followed by perfusion with intermittent pressure had significantly higher concentrations of both sulfated glycosaminoglycan and collagen than constructs cultured entirely in spinners or almost entirely in the pressure/perfusion system. Initial cultivation in the spinner flasks, with turbulent mixing, enhanced both cell attachment and early development of the extracellular matrix. Subsequent culture with perfusion and intermittent pressure appeared to accelerate matrix formation. While the correlation was much stronger in the pressurized constructs, the compressive modulus was directly proportional to the concentration of sulfated glycosaminoglycan in all physically stressed constructs. Constructs that were not stressed beyond the 1-week seeding period lost mechanical integrity upon harvest, suggesting that physical stimulation, particularly with intermittent pressure, of immature tissue constructs during their development may contribute to their ultimate biomechanical functionality. Copyright 1999 John Wiley & Sons, Inc.

In vitro cell quality of articular chondrocytes assigned for autologous implantation in dependence of specific patient characteristics

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Pestka, Jan M; Schmal, Hagen; Salzmann, Gian

2011-01-01

OBJECTIVE: Autologous chondrocyte implantation (ACI) is a well-established therapeutic option for the treatment of cartilage defects of the knee joint. Since information concerning the cellular aspects of ACI is still limited, the aim of the present study was to investigate relevant differences...... between chondrocyte quality after in vitro cultivation and possible correlations with patient-specific factors. DESIGN: Cell quality of 252 consecutive ACI patients was assessed after chondrocyte in vitro expansion by determination of the expression of cartilage relevant surface marker CD44 and cartilage......, aggrecan or collagen type II nor cell density or viability after proliferation seemed to correlate with the grade of joint degeneration, defect aetiology or patient gender. However, chondrocytes harvested from the knee joints of patients at less than 20 years of age showed significantly higher expression...

Acute and chronic response of articular cartilage to Ho:YAG laser irradiation

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Trauner, Kenneth B.; Nishioka, Norman S.; Flotte, Thomas J.; Patel, Dinesh K.

1992-06-01

A Ho:YAG laser system operating at a wavelength of 2.1 microns has recently been introduced for use in arthroscopic surgery. The acceptability of this new tool will be determined not only by its ability to resect tissue, but also by its long term effects on articular surfaces. In order to investigate these issues further, we performed two studies to evaluate the acute and chronic effects of the laser on cartilaginous tissue. We evaluated the acute, in vitro effects of 2.1 micron laser irradiation on articular and fibrocartilage. This included the measurement of ablation efficiency, ablation threshold and thermal damage in both meniscus and articular cartilage. To document the chronic effects on articular cartilage in vivo, we next performed a ten week healing study. Eight sheep weighing 30 - 40 kg underwent bilateral arthrotomy procedures. Multiple full thickness and partial thickness defects were created. Animals were sacrificed at 0, 2, 4, and 10 weeks. The healing study demonstrated: (1) no healing of full or partial thickness defects at 10 weeks with hyaline cartilage; (2) fibrocartilaginous granulation tissue filling full thickness defects at two and four weeks, but no longer evident at ten weeks; (3) chondrocyte necrosis extending to greater than 900 microns distal to ablation craters at four weeks with no evidence of repair at later dates; and (4) chondrocyte hyperplasia at the borders of the damage zone at two weeks but no longer evident at later sacrifice dates.

Noninvasive assessment of articular cartilage surface damage using reflected polarized light microscopy

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Huynh, Ruby N.; Nehmetallah, George; Raub, Christopher B.

2017-06-01

Articular surface damage occurs to cartilage during normal aging, osteoarthritis, and in trauma. A noninvasive assessment of cartilage microstructural alterations is useful for studies involving cartilage explants. This study evaluates polarized reflectance microscopy as a tool to assess surface damage to cartilage explants caused by mechanical scraping and enzymatic degradation. Adult bovine articular cartilage explants were scraped, incubated in collagenase, or underwent scrape and collagenase treatments. In an additional experiment, cartilage explants were subject to scrapes at graduated levels of severity. Polarized reflectance parameters were compared with India ink surface staining, features of histological sections, changes in explant wet weight and thickness, and chondrocyte viability. The polarized reflectance signal was sensitive to surface scrape damage and revealed individual scrape features consistent with India ink marks. Following surface treatments, the reflectance contrast parameter was elevated and correlated with image area fraction of India ink. After extensive scraping, polarized reflectance contrast and chondrocyte viability were lower than that from untreated explants. As part of this work, a mathematical model was developed and confirmed the trend in the reflectance signal due to changes in surface scattering and subsurface birefringence. These results demonstrate the effectiveness of polarized reflectance microscopy to sensitively assess surface microstructural alterations in articular cartilage explants.

[Comparative effects of vitamin C on the effects of local anesthetics ropivacaine, bupivacaine, and lidocaine on human chondrocytes].

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Tian, Jun; Li, Yan

2016-01-01

Intra-articular injections of local anesthetics are commonly used to enhance post-operative analgesia following orthopedic surgery as arthroscopic surgeries. Nevertheless, recent reports of severe complications due to the use of intra-articular local anesthetic have raised concerns. The study aims to assess use of vitamin C in reducing adverse effects of the most commonly employed anesthetics - ropivacaine, bupivacaine and lidocaine - on human chondrocytes. The chondrocyte viability following exposure to 0.5% bupivacaine or 0.75% ropivacaine or 1.0% lidocaine and/or vitamin C at doses 125, 250 and 500μM was determined by Live/Dead assay and annexin V staining. Expression levels of caspases 3 and 9 were assessed using antibodies by Western blotting. Flow cytometry was performed to analyze the generation of reactive oxygen species. On exposure to the local anesthetics, chondrotoxicity was found in the order ropivacaineC effectively improved the reduced chondrocyte viability and decreased the raised apoptosis levels following exposure to anesthesia. At higher doses, vitamin C was found efficient in reducing the generation of reactive oxygen species and as well down-regulate the expressions of caspases 3 and 9. Vitamin C was observed to effectively protect chondrocytes against the toxic insult of local anesthetics ropivacaine, bupivacaine and lidocaine. Copyright © 2015 Sociedade Brasileira de Anestesiologia. Publicado por Elsevier Editora Ltda. All rights reserved.

Comparative effects of vitamin C on the effects of local anesthetics ropivacaine, bupivacaine, and lidocaine on human chondrocytes.

Science.gov (United States)

Tian, Jun; Li, Yan

2016-01-01

Intra-articular injections of local anesthetics are commonly used to enhance post-operative analgesia following orthopedic surgery as arthroscopic surgeries. Nevertheless, recent reports of severe complications due to the use of intra-articular local anesthetic have raised concerns. The study aims to assess use of vitamin C in reducing adverse effects of the most commonly employed anesthetics - ropivacaine, bupivacaine and lidocaine - on human chondrocytes. The chondrocyte viability following exposure to 0.5% bupivacaine or 0.75% ropivacaine or 1.0% lidocaine and/or vitamin C at doses 125, 250 and 500 μM was determined by LIVE/DEAD assay and annexin V staining. Expression levels of caspases 3 and 9 were assessed using antibodies by Western blotting. Flow cytometry was performed to analyze the generation of reactive oxygen species. On exposure to the local anesthetics, chondrotoxicity was found in the order ropivacaineC effectively improved the reduced chondrocyte viability and decreased the raised apoptosis levels following exposure to anesthesia. At higher doses, vitamin C was found efficient in reducing the generation of reactive oxygen species and as well down-regulate the expressions of caspases 3 and 9. Vitamin C was observed to effectively protect chondrocytes against the toxic insult of local anesthetics ropivacaine, bupivacaine and lidocaine. Copyright © 2015 Sociedade Brasileira de Anestesiologia. Published by Elsevier Editora Ltda. All rights reserved.

Importance of collagen orientation and depth-dependent fixed charge densities of cartilage on mechanical behavior of chondrocytes.

NARCIS (Netherlands)

Korhonen, R.K.; Julkunen, P.; Wilson, W.; Herzog, W.

2008-01-01

The collagen network and proteoglycan matrix of articular cartilage are thought to play an important role in controlling the stresses and strains in and around chondrocytes, in regulating the biosynthesis of the solid matrix, and consequently in maintaining the health of diarthrodial joints.

The major basement membrane components localize to the chondrocyte pericellular matrix--a cartilage basement membrane equivalent?

DEFF Research Database (Denmark)

Kvist, Alexander J.; Nyström, Alexander; Hultenby, Kjell

2007-01-01

In this study, we demonstrate that articular cartilage chondrocytes are surrounded by the defining basement membrane proteins laminin, collagen type IV, nidogen and perlecan, and suggest that these form the functional equivalent of a basement membrane. We found by real-time PCR that mouse...... chondrocytes express these four cardinal components of basement membranes and demonstrated by immunohistochemistry that the proteins are present in bovine and mouse cartilage tissues and are deposited in a thin pericellular structure. Immunoelectron microscopy confirmed high laminin concentration...... becomes less distinct, especially in areas of obvious mechanical attrition. Interestingly, individual laminin subunits were located in different zones of the cartilage, with laminin alpha1 showing preferential localization around a select population of superficial layer chondrocytes. We propose...

Identification and clonal characterisation of a progenitor cell sub-population in normal human articular cartilage.

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Rebecca Williams

Full Text Available BACKGROUND: Articular cartilage displays a poor repair capacity. The aim of cell-based therapies for cartilage defects is to repair damaged joint surfaces with a functional replacement tissue. Currently, chondrocytes removed from a healthy region of the cartilage are used but they are unable to retain their phenotype in expanded culture. The resulting repair tissue is fibrocartilaginous rather than hyaline, potentially compromising long-term repair. Mesenchymal stem cells, particularly bone marrow stromal cells (BMSC, are of interest for cartilage repair due to their inherent replicative potential. However, chondrocyte differentiated BMSCs display an endochondral phenotype, that is, can terminally differentiate and form a calcified matrix, leading to failure in long-term defect repair. Here, we investigate the isolation and characterisation of a human cartilage progenitor population that is resident within permanent adult articular cartilage. METHODS AND FINDINGS: Human articular cartilage samples were digested and clonal populations isolated using a differential adhesion assay to fibronectin. Clonal cell lines were expanded in growth media to high population doublings and karyotype analysis performed. We present data to show that this cell population demonstrates a restricted differential potential during chondrogenic induction in a 3D pellet culture system. Furthermore, evidence of high telomerase activity and maintenance of telomere length, characteristic of a mesenchymal stem cell population, were observed in this clonal cell population. Lastly, as proof of principle, we carried out a pilot repair study in a goat in vivo model demonstrating the ability of goat cartilage progenitors to form a cartilage-like repair tissue in a chondral defect. CONCLUSIONS: In conclusion, we propose that we have identified and characterised a novel cartilage progenitor population resident in human articular cartilage which will greatly benefit future cell

The role of autologous chondrocyte implantation in the treatment of symptomatic chondromalacia patellae.

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Macmull, Simon; Jaiswal, Parag K; Bentley, George; Skinner, John A; Carrington, Richard W J; Briggs, Tim W R

2012-07-01

Chondromalacia patella is a distinct clinical entity of abnormal softening of the articular cartilage of the patella, which results in chronic retropatellar pain. Its aetiology is still unclear but the process is thought to be a due to trauma to superficial chondrocytes resulting in a proteolytic enzymic breakdown of the matrix. Our aim was to assess the effectiveness of autologous chondrocyte implantation on patients with a proven symptomatic retropatellar lesion who had at least one failed conventional marrow-stimulating therapy. We performed chondrocyte implantation on 48 patients: 25 received autologous chondrocyte implantation with a type I/III membrane (ACI-C) method (Geistlich Biomaterials, Wolhusen, Switzerland), and 23 received the Matrix-assisted Chondrocyte Implantation (MACI) technique (Genzyme, Kastrup, Denmark). Over a mean follow-up period of 40.3 months, there was a statistically significant improvement in subjective pain scoring using the visual analogue scale (VAS) and objective functional scores using the Modified Cincinnati Rating System (MCS) in both groups. Chondromalacia patellae lesions responded well to chondrocyte implantation. Better results occurred with MACI than with ACI-C. Excellent and good results were achieved in 40% of ACI-C patients and 57% of MACI patients, but success of chondrocyte implantation was greater with medial/odd-facet lesions. Given that the MACI procedure is technically easier and less time consuming, we consider it to be useful for treating patients with symptomatic chondral defects secondary to chondromalacia patellae.

Deficiency of Thrombospondin-4 in Mice Does Not Affect Skeletal Growth or Bone Mass Acquisition, but Causes a Transient Reduction of Articular Cartilage Thickness.

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Anke Jeschke

Full Text Available Although articular cartilage degeneration represents a major public health problem, the underlying molecular mechanisms are still poorly characterized. We have previously utilized genome-wide expression analysis to identify specific markers of porcine articular cartilage, one of them being Thrombospondin-4 (Thbs4. In the present study we analyzed Thbs4 expression in mice, thereby confirming its predominant expression in articular cartilage, but also identifying expression in other tissues, including bone. To study the role of Thbs4 in skeletal development and integrity we took advantage of a Thbs4-deficient mouse model that was analyzed by undecalcified bone histology. We found that Thbs4-deficient mice do not display phenotypic differences towards wildtype littermates in terms of skeletal growth or bone mass acquisition. Since Thbs4 has previously been found over-expressed in bones of Phex-deficient Hyp mice, we additionally generated Thbs4-deficient Hyp mice, but failed to detect phenotypic differences towards Hyp littermates. With respect to articular cartilage we found that Thbs4-deficient mice display transient thinning of articular cartilage, suggesting a protective role of Thbs4 for joint integrity. Gene expression analysis using porcine primary cells revealed that Thbs4 is not expressed by synovial fibroblasts and that it represents the only member of the Thbs gene family with specific expression in articular, but not in growth plate chondrocytes. In an attempt to identify specific molecular effects of Thbs4 we treated porcine articular chondrocytes with human THBS4 in the absence or presence of conditioned medium from porcine synovial fibroblasts. Here we did not observe a significant influence of THBS4 on proliferation, metabolic activity, apoptosis or gene expression, suggesting that it does not act as a signaling molecule. Taken together, our data demonstrate that Thbs4 is highly expressed in articular chondrocytes, where its

Linkage of chondroitin-sulfate to type I collagen scaffolds stimulates the bioactivity of seeded chondrocytes in vitro.

NARCIS (Netherlands)

Susante, J.L.C. van; Pieper, J.S.; Buma, P.; Kuppevelt, A.H.M.S.M. van; Beuningen, H.M. van; Kraan, P.M. van der; Veerkamp, J.H.; Berg, W.B. van den; Veth, R.P.H.

2001-01-01

An increasing amount of interest is focused on the potential use of tissue-engineered articular cartilage implants, for repair of defects in the joint surface. In this perspective, various biodegradable scaffolds have been evaluated as a vehicle to deliver chondrocytes into a cartilage defect. This

Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Contribute to Chondrogenesis in Coculture with Chondrocytes.

Science.gov (United States)

Li, Xingfu; Duan, Li; Liang, Yujie; Zhu, Weimin; Xiong, Jianyi; Wang, Daping

2016-01-01

Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have been shown as the most potential stem cell source for articular cartilage repair. In this study, we aimed to develop a method for long-term coculture of human articular chondrocytes (hACs) and hUCB-MSCs at low density in vitro to determine if the low density of hACs could enhance the hUCB-MSC chondrogenic differentiation as well as to determine the optimal ratio of the two cell types. Also, we compared the difference between direct coculture and indirect coculture at low density. Monolayer cultures of hUCB-MSCs and hACs were investigated at different ratios, at direct cell-cell contact groups for 21 days. Compared to direct coculture, hUCB-MSCs and hACs indirect contact culture significantly increased type II collagen (COL2) and decreased type I collagen (COL1) protein expression levels. SRY-box 9 (SOX9) mRNA levels and protein expression were highest in indirect coculture. Overall, these results indicate that low density direct coculture induces fibrocartilage. However, indirect coculture in conditioned chondrocyte cell culture medium can increase expression of chondrogenic markers and induce hUCB-MSCs differentiation into mature chondrocytes. This work demonstrates that it is possible to promote chondrogenesis of hUCB-MSCs in combination with hACs, further supporting the concept of novel coculture strategies for tissue engineering.

Spatial regulation of bone morphogenetic proteins (BMPs) in postnatal articular and growth plate cartilage

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Garrison, Presley; Yue, Shanna; Hanson, Jeffrey; Baron, Jeffrey; Lui, Julian C.

2017-01-01

Articular and growth plate cartilage both arise from condensations of mesenchymal cells, but ultimately develop important histological and functional differences. Each is composed of three layers—the superficial, mid and deep zones of articular cartilage and the resting, proliferative and hypertrophic zones of growth plate cartilage. The bone morphogenetic protein (BMP) system plays an important role in cartilage development. A gradient in expression of BMP-related genes has been observed across growth plate cartilage, likely playing a role in zonal differentiation. To investigate the presence of a similar expression gradient in articular cartilage, we used laser capture microdissection (LCM) to separate murine growth plate and articular cartilage from the proximal tibia into their six constituent zones, and used a solution hybridization assay with color-coded probes (nCounter) to quantify mRNAs for 30 different BMP-related genes in each zone. In situ hybridization and immunohistochemistry were then used to confirm spatial expression patterns. Expression gradients for Bmp2 and 6 were observed across growth plate cartilage with highest expression in hypertrophic zone. However, intracellular BMP signaling, assessed by phospho-Smad1/5/8 immunohistochemical staining, appeared to be higher in the proliferative zone and prehypertrophic area than in hypertrophic zone, possibly due to high expression of Smad7, an inhibitory Smad, in the hypertrophic zone. We also found BMP expression gradients across the articular cartilage with BMP agonists primarily expressed in the superficial zone and BMP functional antagonists primarily expressed in the deep zone. Phospho-Smad1/5/8 immunohistochemical staining showed a similar gradient. In combination with previous evidence that BMPs regulate chondrocyte proliferation and differentiation, the current findings suggest that BMP signaling gradients exist across both growth plate and articular cartilage and that these gradients may

THE ACTIVATION OF MATRIX METALLOPROTEINASES AND CHONDROCYTE DIFFERENTIATION, WHICH ACCOMPANIES THE INDUCTION OF COLLAGEN DECOMPOSITION UNDER THE ACTION OF COLLAGEN PEPTIDE IN THE CARTILAGE OFHEALTHY INDIVIDUALS

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Elena Vasil'evna Chetina

2010-01-01

Conclusion. This study has shown that the induction of collagenase activity by CB12-2 in the human articular cartilage chondrocytes is attended by terminal differentiation/hypertrophy of these cells. The terminal differentiation of chondrocytes may be one of the mechanisms of chondrolysis in osteoarthrosis since it naturally occurs not only in endochondrial ossification, but also in the development of pathology.

Effects of Chondroitinase ABC-Mediated Proteoglycan Digestion on Decellularization and Recellularization of Articular Cartilage.

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Catherine A Bautista

Full Text Available Articular cartilage has a limited capacity to heal itself and thus focal defects often result in the development of osteoarthritis. Current cartilage tissue engineering strategies seek to regenerate injured tissue by creating scaffolds that aim to mimic the unique structure and composition of native articular cartilage. Decellularization is a novel strategy that aims to preserve the bioactive factors and 3D biophysical environment of the native extracellular matrix while removing potentially immunogenic factors. The purpose of this study was to develop a procedure that can enable decellularization and recellularization of intact articular cartilage matrix. Full-thickness porcine articular cartilage plugs were decellularized with a series of freeze-thaw cycles and 0.1% (w/v sodium dodecyl sulfate detergent cycles. Chondroitinase ABC (ChABC was applied before the detergent cycles to digest glycosaminoglycans in order to enhance donor chondrocyte removal and seeded cell migration. Porcine synovium-derived mesenchymal stem cells were seeded onto the decellularized cartilage scaffolds and cultured for up to 28 days. The optimized decellularization protocol removed 94% of native DNA per sample wet weight, while collagen content and alignment were preserved. Glycosaminoglycan depletion prior to the detergent cycles increased removal of nuclear material. Seeded cells infiltrated up to 100 μm into the cartilage deep zone after 28 days in culture. ChABC treatment enhances decellularization of the relatively dense, impermeable articular cartilage by reducing glycosaminoglycan content. ChABC treatment did not appear to affect cell migration during recellularization under static, in vitro culture, highlighting the need for more dynamic seeding methods.

FK506 protects against articular cartilage collagenous extra-cellular matrix degradation.

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Siebelt, M; van der Windt, A E; Groen, H C; Sandker, M; Waarsing, J H; Müller, C; de Jong, M; Jahr, H; Weinans, H

2014-04-01

Osteoarthritis (OA) is a non-rheumatologic joint disease characterized by progressive degeneration of the cartilage extra-cellular matrix (ECM), enhanced subchondral bone remodeling, activation of synovial macrophages and osteophyte growth. Inhibition of calcineurin (Cn) activity through tacrolimus (FK506) in in vitro monolayer chondrocytes exerts positive effects on ECM marker expression. This study therefore investigated the effects of FK506 on anabolic and catabolic markers of osteoarthritic chondrocytes in 2D and 3D in vitro cultures, and its therapeutic effects in an in vivo rat model of OA. Effects of high and low doses of FK506 on anabolic (QPCR/histochemistry) and catabolic (QPCR) markers were evaluated in vitro on isolated (2D) and ECM-embedded chondrocytes (explants, 3D pellets). Severe cartilage damage was induced unilaterally in rat knees using papain injections in combination with a moderate running protocol. Twenty rats were treated with FK506 orally and compared to twenty untreated controls. Subchondral cortical and trabecular bone changes (longitudinal microCT) and macrophage activation (SPECT/CT) were measured. Articular cartilage was analyzed ex vivo using contrast enhanced microCT and histology. FK506 treatment of osteoarthritic chondrocytes in vitro induced anabolic (mainly collagens) and reduced catabolic ECM marker expression. In line with this, FK506 treatment clearly protected ECM integrity in vivo by markedly decreasing subchondral sclerosis, less development of subchondral pores, depletion of synovial macrophage activation and lower osteophyte growth. FK506 protected cartilage matrix integrity in vitro and in vivo. Additionally, FK506 treatment in vivo reduced OA-like responses in different articular joint tissues and thereby makes Cn an interesting target for therapeutic intervention of OA. Copyright © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

A study of crystalline biomaterials for articular cartilage bioengineering

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Gross-Aviv, Talia [Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer Sheva, 84105 (Israel)], E-mail: taliag@bgu.ac.il; DiCarlo, Bryan B. [Department of Bioengineering, Rice University, Houston, TX 77003 (United States)], E-mail: bdicarlo@rice.edu; French, Margaret M. [Department of Bioengineering, Rice University, Houston, TX 77003 (United States)], E-mail: mmfrench@rice.edu; Athanasiou, Kyriacos A. [Department of Bioengineering, Rice University, Houston, TX 77003 (United States)], E-mail: athanasiou@rice.edu; Vago, Razi [Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer Sheva, 84105 (Israel)], E-mail: rvago@bgu.ac.il

2008-12-01

This study examines the suitability of marine origin coral species, Porites lutea (POR) and the hydrozoan Millepora dichotoma (MIL), for use as novel three dimensional growth matrices in the field of articular cartilage tissue engineering. Therefore, mesenchymal stem cells (MSCs) and chondrocytes were grown on the skeletal material obtained from each of these two organisms to investigate their potential use as three dimensional scaffolding for cartilage tissue growth. Chondrogenic induction of MSCs was achieved by addition of transforming growth factor-{beta}1 (TGF-{beta}1) and insulin growth factor-I (IGF-I). Cell adherence, proliferation, differentiation and tissue development were investigated through six weeks of culture. Cartilage tissue growth and chondrocytic phenotype maintenance of each cell type were examined by cell morphology, histochemical analyses, expression of collagen type II and quantitative measures of glycosaminoglycan (GAG) content. The MSCs and the chondrocytes were shown good adherence to the scaffolds and maintenance of the chondrocytic phenotype in the initial stages of culture. However after two weeks of culture on MIL and three weeks on POR these cultures began to exhibit signs of further differentiation and phenotypic loss. The shown results indicated that POR was a better substrate for chondrocytes phenotype maintenance than MIL. We believe that surface modification of POR combined with mechanical stimuli will provide a suitable environment for chondrogenic phenotype maintenance. Further investigation of POR and other novel coralline biomatrices is indicated and warranted in the field of cartilage tissue engineering applications.

Evolution of Autologous Chondrocyte Repair and Comparison to Other Cartilage Repair Techniques

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Ashvin K. Dewan

2014-01-01

Full Text Available Articular cartilage defects have been addressed using microfracture, abrasion chondroplasty, or osteochondral grafting, but these strategies do not generate tissue that adequately recapitulates native cartilage. During the past 25 years, promising new strategies using assorted scaffolds and cell sources to induce chondrocyte expansion have emerged. We reviewed the evolution of autologous chondrocyte implantation and compared it to other cartilage repair techniques. Methods. We searched PubMed from 1949 to 2014 for the keywords “autologous chondrocyte implantation” (ACI and “cartilage repair” in clinical trials, meta-analyses, and review articles. We analyzed these articles, their bibliographies, our experience, and cartilage regeneration textbooks. Results. Microfracture, abrasion chondroplasty, osteochondral grafting, ACI, and autologous matrix-induced chondrogenesis are distinguishable by cell source (including chondrocytes and stem cells and associated scaffolds (natural or synthetic, hydrogels or membranes. ACI seems to be as good as, if not better than, microfracture for repairing large chondral defects in a young patient’s knee as evaluated by multiple clinical indices and the quality of regenerated tissue. Conclusion. Although there is not enough evidence to determine the best repair technique, ACI is the most established cell-based treatment for full-thickness chondral defects in young patients.

Comparing effects of perfusion and hydrostatic pressure on gene profiles of human chondrocyte.

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Zhu, Ge; Mayer-Wagner, Susanne; Schröder, Christian; Woiczinski, Matthias; Blum, Helmut; Lavagi, Ilaria; Krebs, Stefan; Redeker, Julia I; Hölzer, Andreas; Jansson, Volkmar; Betz, Oliver; Müller, Peter E

2015-09-20

Hydrostatic pressure and perfusion have been shown to regulate the chondrogenic potential of articular chondrocytes. In order to compare the effects of hydrostatic pressure plus perfusion (HPP) and perfusion (P) we investigated the complete gene expression profiles of human chondrocytes under HPP and P. A simplified bioreactor was constructed to apply loading (0.1 MPa for 2 h) and perfusion (2 ml) through the same piping by pressurizing the medium directly. High-density monolayer cultures of human chondrocytes were exposed to HPP or P for 4 days. Controls (C) were maintained in static cultures. Gene expression was evaluated by sequencing (RNAseq) and quantitative real-time PCR analysis. Both treatments changed gene expression levels of human chondrocytes significantly. Specifically, HPP and P increased COL2A1 expression and decreased COL1A1 and MMP-13 expression. Despite of these similarities, RNAseq revealed a list of cartilage genes including ACAN, ITGA10 and TNC, which were differentially expressed by HPP and P. Of these candidates, adhesion related molecules were found to be upregulated in HPP. Both HPP and P treatment had beneficial effects on chondrocyte differentiation and decreased catabolic enzyme expression. The study provides new insight into how hydrostatic pressure and perfusion enhance cartilage differentiation and inhibit catabolic effects. Copyright © 2015 Elsevier B.V. All rights reserved.

Stimulation of chondrocyte proliferation following photothermal, thermal, and mechanical injury in ex-vivo cartilage grafts

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Pandoh, Nidhi S.; Truong, Mai T.; Diaz-Valdes, Sergio H.; Gardiner, David M.; Wong, Brian J.

2002-06-01

Laser irradiation may stimulate chondrocytes proliferation in the peripheral region surrounding a photothermally-heated area in rabbit nasal septal cartilage. In this study, ex- vivo rabbit nasal septal cartilages maintained in culture were irradiated with an Nd:YAG laser ((lambda) equals1.32 micrometers , 4-16 sec, 10-45 W/cm2) to examine the relationship between the diameter of replicating cells and irradiation time. Also, this study investigated whether proliferation occurs following heating (by immersion in hot saline baths, with a heated metal rod, and a soldering iron) and mechanical modification (crushing with a metal stamp and scoring with a scalpel). Replicating chondrocytes were identified using a Bromodeoxyuridine (BrdU) double antibody detection system in whole mount tissue. Light microscopy was used to confirm the presence of BrdU stained chondrocytes. The mechanical and thermal stressors used failed to produce a proliferative response in chondrocytes as previously seen with laser irradiation. We suspect that chondrocyte proliferation may be induced as a response to alteration in matrix structure produced by photothermal, thermal, or mechanical modification of the matrix. Heat generated by a laser to stimulate chondrocyte proliferation may lead to new treatment options for degenerative articular diseases and disorders. Laser technology can be adapted for use with minimally invasive surgical instrumentation to deliver light into otherwise inaccessible regions of the body.

Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Contribute to Chondrogenesis in Coculture with Chondrocytes

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Xingfu Li

2016-01-01

Full Text Available Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs have been shown as the most potential stem cell source for articular cartilage repair. In this study, we aimed to develop a method for long-term coculture of human articular chondrocytes (hACs and hUCB-MSCs at low density in vitro to determine if the low density of hACs could enhance the hUCB-MSC chondrogenic differentiation as well as to determine the optimal ratio of the two cell types. Also, we compared the difference between direct coculture and indirect coculture at low density. Monolayer cultures of hUCB-MSCs and hACs were investigated at different ratios, at direct cell-cell contact groups for 21 days. Compared to direct coculture, hUCB-MSCs and hACs indirect contact culture significantly increased type II collagen (COL2 and decreased type I collagen (COL1 protein expression levels. SRY-box 9 (SOX9 mRNA levels and protein expression were highest in indirect coculture. Overall, these results indicate that low density direct coculture induces fibrocartilage. However, indirect coculture in conditioned chondrocyte cell culture medium can increase expression of chondrogenic markers and induce hUCB-MSCs differentiation into mature chondrocytes. This work demonstrates that it is possible to promote chondrogenesis of hUCB-MSCs in combination with hACs, further supporting the concept of novel coculture strategies for tissue engineering.

The effect of dexamethasone and triiodothyronine on terminal differentiation of primary bovine chondrocytes and chondrogenically differentiated mesenchymal stem cells

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Randau, T.M.; Schildberg, F.A.; Alini, M.; Wimmer, M.D.; Haddouti, E.-M.; Gravius, S.; Ito, K.; Stoddart, M.J.

2013-01-01

The newly evolved field of regenerative medicine is offering solutions in the treatment of bone or cartilage loss and deficiency. Mesenchymal stem cells, as well as articular chondrocytes, are potential cells for the generation of bone or cartilage. The natural mechanism of bone formation is that of

PPAR-δ Agonist With Mesenchymal Stem Cells Induces Type II Collagen-Producing Chondrocytes in Human Arthritic Synovial Fluid.

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Heck, Bruce E; Park, Joshua J; Makani, Vishruti; Kim, Eun-Cheol; Kim, Dong Hyun

2017-08-01

Osteoarthritis (OA) is an inflammatory joint disease characterized by degeneration of articular cartilage within synovial joints. An estimated 27 million Americans suffer from OA, and the population is expected to reach 67 million in the United States by 2030. Thus, it is urgent to find an effective treatment for OA. Traditional OA treatments have no disease-modifying effect, while regenerative OA therapies such as autologous chondrocyte implantation show some promise. Nonetheless, current regenerative therapies do not overcome synovial inflammation that suppresses the differentiation of mesenchymal stem cells (MSCs) to chondrocytes and the expression of type II collagen, the major constituent of functional cartilage. We discovered a synergistic combination that overcame synovial inflammation to form type II collagen-producing chondrocytes. The combination consists of peroxisome proliferator-activated receptor (PPAR) δ agonist, human bone marrow (hBM)-derived MSCs, and hyaluronic acid (HA) gel. Interestingly, those individual components showed their own strong enhancing effects on chondrogenesis. GW0742, a PPAR-δ agonist, greatly enhanced MSC chondrogenesis and the expression of type II collagen and glycosaminoglycan (GAG) in hBM-MSC-derived chondrocytes. GW0742 also increased the expression of transforming growth factor β that enhances chondrogenesis and suppresses cartilage fibrillation, ossification, and inflammation. HA gel also increased MSC chondrogenesis and GAG production. However, neither GW0742 nor HA gel could enhance the formation of type II collagen-producing chondrocytes from hBM-MSCs within human OA synovial fluid. Our data demonstrated that the combination of hBM-MSCs, PPAR-δ agonist, and HA gel significantly enhanced the formation of type II collagen-producing chondrocytes within OA synovial fluid from 3 different donors. In other words, the novel combination of PPAR-δ agonist, hBM-MSCs, and HA gel can overcome synovial inflammation to form

Inflammation and Immune Response of Intra-Articular Serotype 2 Adeno-Associated Virus or Adenovirus Vectors in a Large Animal Model

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Akikazu Ishihara

2012-01-01

Full Text Available Intra-articular gene therapy has potential for the treatment of osteoarthritis and rheumatoid arthritis. To quantify in vitro relative gene transduction, equine chondrocytes and synovial cells were treated with adenovirus vectors (Ad, serotype 2 adeno-associated virus vectors (rAAV2, or self-complementary (sc AAV2 vectors carrying green fluorescent protein (GFP. Using 6 horses, bilateral metacarpophalangeal joints were injected with Ad, rAAV2, or scAAV2 vectors carrying GFP genes to assess the in vivo joint inflammation and neutralizing antibody (NAb titer in serum and joint fluid. In vitro, the greater transduction efficiency and sustained gene expression were achieved by scAAV2 compared to rAAV2 in equine chondrocytes and synovial cells. In vivo, AAV2 demonstrated less joint inflammation than Ad, but similar NAb titer. The scAAV2 vectors can induce superior gene transduction than rAAV2 in articular cells, and both rAAV2 and scAAV2 vectors were showed to be safer for intra-articular use than Ad vectors.

The relationship between ultra-short telomeres, aging of articular cartilage and the development of human hip osteoarthritis

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Harbo, M; Delaisse, J M; Kjaersgaard-Andersen, P

2013-01-01

Ultra-short telomeres caused by stress-induced telomere shortening are suggested to induce chondrocyte senescence in human osteoarthritic knees. Here we have further investigated the role of ultra-short telomeres in the development of osteoarthritis (OA) and in aging of articular cartilage in human...

Increased adipogenesis in cultured embryonic chondrocytes and in adult bone marrow of dominant negative Erg transgenic mice.

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Sébastien Flajollet

Full Text Available In monolayer culture, primary articular chondrocytes have an intrinsic tendency to lose their phenotype during expansion. The molecular events underlying this chondrocyte dedifferentiation are still largely unknown. Several transcription factors are important for chondrocyte differentiation. The Ets transcription factor family may be involved in skeletal development. One family member, the Erg gene, is mainly expressed during cartilage formation. To further investigate the potential role of Erg in the maintenance of the chondrocyte phenotype, we isolated and cultured chondrocytes from the rib cartilage of embryos of transgenic mice that express a dominant negative form of Erg (DN-Erg during cartilage formation. DN-Erg expression in chondrocytes cultured for up to 20 days did not affect the early dedifferentiation usually observed in cultured chondrocytes. However, lipid droplets accumulated in DN-Erg chondrocytes, suggesting adipocyte emergence. Transcriptomic analysis using a DNA microarray, validated by quantitative RT-PCR, revealed strong differential gene expression, with a decrease in chondrogenesis-related markers and an increase in adipogenesis-related gene expression in cultured DN-Erg chondrocytes. These results indicate that Erg is involved in either maintaining the chondrogenic phenotype in vitro or in cell fate orientation. Along with the in vitro studies, we compared adipocyte presence in wild-type and transgenic mice skeletons. Histological investigations revealed an increase in the number of adipocytes in the bone marrow of adult DN-Erg mice even though no adipocytes were detected in embryonic cartilage or bone. These findings suggest that the Ets transcription factor family may contribute to the homeostatic balance in skeleton cell plasticity.

Interleukin-1 Acts via the JNK-2 Signaling Pathway to Induce Aggrecan Degradation by Human Chondrocytes.

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Ismail, Heba M; Yamamoto, Kazuhiro; Vincent, Tonia L; Nagase, Hideaki; Troeberg, Linda; Saklatvala, Jeremy

2015-07-01

Aggrecan enables articular cartilage to bear load and resist compression. Aggrecan loss occurs early in osteoarthritis and rheumatoid arthritis and can be induced by inflammatory cytokines such as interleukin-1 (IL-1). IL-1 induces cleavage of specific aggrecans characteristic of the ADAMTS proteinases. The aim of this study was to identify the intracellular signaling pathways by which IL-1 causes aggrecan degradation by human chondrocytes and to investigate how aggrecanase activity is controlled by chondrocytes. We developed a cell-based assay combining small interfering RNA (siRNA)-induced knockdown with aggrecan degradation assays. Human articular chondrocytes were overlaid with bovine aggrecan after transfection with siRNAs against molecules of the IL-1 signaling pathway. After IL-1 stimulation, released aggrecan fragments were detected with AGEG and ARGS neoepitope antibodies. Aggrecanase activity and tissue inhibitor of metalloproteinases 3 levels were measured by enzyme-linked immunosorbent assay. Low-density lipoprotein receptor-related protein 1 (LRP-1) shedding was analyzed by Western blotting. ADAMTS-5 is a major aggrecanase in human chondrocytes, regulating aggrecan degradation in response to IL-1. The tumor necrosis factor receptor-associated 6 (TRAF-6)/transforming growth factor β-activated kinase 1 (TAK-1)/MKK-4 signaling axis is essential for IL-1-induced aggrecan degradation, while NF-κB is not. Of the 3 MAPKs (ERK, p38, and JNK), only JNK-2 showed a significant role in aggrecan degradation. Chondrocytes constitutively secreted aggrecanase, which was continuously endocytosed by LRP-1, keeping the extracellular level of aggrecanase low. IL-1 induced aggrecanase activity in the medium in a JNK-2-dependent manner, possibly by reducing aggrecanase endocytosis, because IL-1 caused JNK-2-dependent shedding of LRP-1. The signaling axis TRAF-6/TAK-1/MKK-4/JNK-2 mediates IL-1-induced aggrecanolysis. The level of aggrecanase is controlled by its

Promoted Chondrogenesis of Cocultured Chondrocytes and Mesenchymal Stem Cells under Hypoxia Using In-situ Forming Degradable Hydrogel Scaffolds

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Huang, Xiaobin; Hou, Yong; Zhong, Leilei; Huang, Dechun; Qian, Hongliang; Karperien, Marcel; Chen, Wei

2018-01-01

We investigated the effects of different oxygen tension (21% and 2.5% O2) on the chondrogenesis of different cell systems cultured in pH-degradable PVA hydrogels, including human articular chondrocytes (hACs), human mesenchymal stem cells (hMSCs), and their cocultures with a hAC/hMSC ratio of 20/80.

Melatonin protects chondrocytes from impairment induced by glucocorticoids via NAD+-dependent SIRT1.

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Yang, Wei; Kang, Xiaomin; Qin, Na; Li, Feng; Jin, Xinxin; Ma, Zhengmin; Qian, Zhuang; Wu, Shufang

2017-10-01

Intra-articular injection of glucocorticoids is used to relieve pain and inflammation in osteoarthritis patients, which is occasionally accompanied with the serious side effects of glucocorticoids in collagen-producing tissue. Melatonin is the major hormone released from the pineal gland and its beneficial effects on cartilage has been suggested. In the present study, we investigated the protective role of melatonin on matrix degeneration in chondrocytes induced by dexamethasone (Dex). The chondrocytes isolated from mice knee joint were treated with Dex, melatonin, EX527 and siRNA targeted for SIRT6, respectively. Dex treatment induced the loss of the extracellular matrix, NAD + /NADH ratio and NADPH concentration in chondrocytes. Melatonin alone have no effect on the quantity of proteoglycans and collagen type IIa1, however, the pretreatment of melatonin reversed the negative effects induced by Dex. Meanwhile, the significant decrease in NAD + /NADH ratio and NADPH concentration in Dex group were up-regulated by pretreatment of melatonin. Furthermore, it was revealed that inhibition of SIRT1 blocked the protective effects of melatonin. The enhancement of NAD + -dependent SIRT1 activity contributes to the chondroprotecfive effects of melatonin, which has a great benefit to prevent dexamethasone-induced chondrocytes impairment. Copyright © 2017 Elsevier Inc. All rights reserved.

The Regulatory Role of Signaling Crosstalk in Hypertrophy of MSCs and Human Articular Chondrocytes

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Zhong, Leilei; Huang, X; Karperien, Hermanus Bernardus Johannes; Post, Janine Nicole

2015-01-01

Hypertrophic differentiation of chondrocytes is a main barrier in application of mesenchymal stem cells (MSCs) for cartilage repair. In addition, hypertrophy occurs occasionally in osteoarthritis (OA). Here we provide a comprehensive review on recent literature describing signal pathways in the

The ECM-Cell Interaction of Cartilage Extracellular Matrix on Chondrocytes

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Yue Gao

2014-01-01

Full Text Available Cartilage extracellular matrix (ECM is composed primarily of the network type II collagen (COLII and an interlocking mesh of fibrous proteins and proteoglycans (PGs, hyaluronic acid (HA, and chondroitin sulfate (CS. Articular cartilage ECM plays a crucial role in regulating chondrocyte metabolism and functions, such as organized cytoskeleton through integrin-mediated signaling via cell-matrix interaction. Cell signaling through integrins regulates several chondrocyte functions, including differentiation, metabolism, matrix remodeling, responses to mechanical stimulation, and cell survival. The major signaling pathways that regulate chondrogenesis have been identified as wnt signal, nitric oxide (NO signal, protein kinase C (PKC, and retinoic acid (RA signal. Integrins are a large family of molecules that are central regulators in multicellular biology. They orchestrate cell-cell and cell-matrix adhesive interactions from embryonic development to mature tissue function. In this review, we emphasize the signaling molecule effect and the biomechanics effect of cartilage ECM on chondrogenesis.

Phenotypic variations in chondrocyte subpopulations and their response to in vitro culture and external stimuli.

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Coates, Emily E; Fisher, John P

2010-11-01

Articular cartilage defects have limited capacity to self-repair, and cost society up to 60 billion dollars annually in both medical treatments and loss of working days. Recent developments in cartilage tissue engineering have resulted in many new products coming to market or entering clinical trials. However, there is a distinct lack of treatments which aim to recreate the complex zonal organization of articular cartilage. Cartilage tissue withstands repetitive strains throughout an individual's lifetime and provides frictionless movement between joints. The structure and composition of its intricately organized extracellular matrix varies with tissue depth to provide optimal resistance to loading, ensure ease of movement, and integrate with the subchondral bone. Each tissue zone is specially designed to resist the load it experiences, and maximize the tissue properties needed for its location. It is unlikely that a homogenous solution to tissue repair will be able to optimally restore the function of such a heterogeneous tissue. For zonal engineering of articular cartilage to become practical, maintenance of phenotypically stable zonal cell populations must be achieved. The chondrocyte phenotype varies considerably by zone, and it is the activity of these cells that help achieve the structural organization of the tissue. This review provides an examination of literature which has studied variations in cellular phenotype between cartilage zones. By doing so, we have identified critical differences between cell populations and highlighted areas of research which show potential in the field. Current research has made the morphological and metabolic variations between these cell populations clear, but an ideal way of maintaining these differences in vitro culture is yet to be established. Combinations of delivered growth factors, mechanical loading, and layered three-dimensional culture systems all show potential for achieving this goal. Furthermore, differentiation

Induction of increased cAMP levels in articular chondrocytes blocks matrix metalloproteinase-mediated cartilage degradation, but not aggrecanase-mediated cartilage degradation

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Karsdal, Morten Asser; Sumer, Eren Ufuk; Wulf, Helle

2007-01-01

OBJECTIVE: Calcitonin has been suggested to have chondroprotective effects. One signaling pathway of calcitonin is via the second messenger cAMP. We undertook this study to investigate whether increased cAMP levels in chondrocytes would be chondroprotective. METHODS: Cartilage degradation......-dependently inhibited by forskolin and IBMX. The highest concentration of IBMX lowered cytokine-induced release of sGAG by 72%. CONCLUSION: Levels of cAMP in chondrocytes play a key role in controlling catabolic activity. Increased cAMP levels in chondrocytes inhibited MMP expression and activity and consequently...... strongly inhibited cartilage degradation. Specific cAMP modulators in chondrocytes may be potential treatments for cartilage degenerative diseases....

Molecular changes in articular cartilage and subchondral bone in the rat anterior cruciate ligament transection and meniscectomized models of osteoarthritis.

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Pickarski, Maureen; Hayami, Tadashi; Zhuo, Ya; Duong, Le T

2011-08-24

Osteoarthritis (OA) is a debilitating, progressive joint disease. Similar to the disease progression in humans, sequential events of early cartilage degradation, subchondral osteopenia followed by sclerosis, and late osteophyte formation were demonstrated in the anterior cruciate ligament transection (ACLT) or ACLT with partial medial meniscectomy (ACLT + MMx) rat OA models. We describe a reliable and consistent method to examine the time dependent changes in the gene expression profiles in articular cartilage and subchondral bone. Local regulation of matrix degradation markers was demonstrated by a significant increase in mRNA levels of aggrecanase-1 and MMP-13 as early as the first week post-surgery, and expression remained elevated throughout the 10 week study. Immunohistochemistry confirmed MMP-13 expression in differentiated chondrocytes and synovial fibroblasts at week-2 and cells within osteophytes at week-10 in the surgically-modified-joints. Concomitant increases in chondrocyte differentiation markers, Col IIA and Sox 9, and vascular invasion markers, VEGF and CD31, peaked around week-2 to -4, and returned to Sham levels at later time points in both models. Indeed, VEGF-positive cells were found in the deep articular chondrocytes adjacent to subchondral bone. Osteoclastic bone resorption markers, cathepsin K and TRAP, were also elevated at week-2. Confirming bone resorption is an early local event in OA progression, cathepsin K positive osteoclasts were found invading the articular cartilage from the subchondral region at week 2. This was followed by late disease events, including subchondral sclerosis and osteophyte formation, as demonstrated by the upregulation of the osteoanabolic markers runx2 and osterix, toward week-4 to 6 post-surgery. In summary, this study demonstrated the temporal and cohesive gene expression changes in articular cartilage and subchondral bone using known markers of OA progression. The findings here support genome-wide profiling

Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication

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Eva Skiöldebrand

2018-01-01

Full Text Available Chondrocytes are effectively involved in the pathophysiological processes of inflammation in joints. They form cellular processes in the superficial layer of the articular cartilage and form gap junction coupled syncytium to facilitate cell-to-cell communication. However, very little is known about their physiological cellular identity and communication. The aim with the present work is to evaluate the physiological behavior after stimulation with the inflammatory inducers interleukin-1β and lipopolysaccharide. The cytoskeleton integrity and intracellular Ca2+ release were assessed as indicators of inflammatory state. Cytoskeleton integrity was analyzed through cartilage oligomeric matrix protein and actin labeling with an Alexa 488-conjugated phalloidin probe. Ca2+ responses were assessed through the Ca2+ sensitive fluorophore Fura-2/AM. Western blot analyses of several inflammatory markers were performed. The results show reorganization of the actin filaments. Glutamate, 5-hydoxytryptamine, and ATP evoked intracellular Ca2+ release changed from single peaks to oscillations after inflammatory induction in the chondrocytes. The expression of toll-like receptor 4, the glutamate transporters GLAST and GLT-1, and the matrix metalloproteinase-13 increased. This work demonstrates that chondrocytes are a key part in conditions that lead to inflammation in the cartilage. The inflammatory inducers modulate the cytoskeleton, the Ca2+ signaling, and several inflammatory parameters. In conclusion, our data show that the cellular responses to inflammatory insults from healthy and inflammatory chondrocytes resemble those previously observed in astrocyte and cardiac fibroblasts networks.

Stem cells catalyze cartilage formation by neonatal articular chondrocytes in 3D biomimetic hydrogels.

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Lai, Janice H; Kajiyama, Glen; Smith, Robert Lane; Maloney, William; Yang, Fan

2013-12-19

Cartilage loss is a leading cause of disability among adults and effective therapy remains elusive. Neonatal chondrocytes (NChons) are an attractive allogeneic cell source for cartilage repair, but their clinical translation has been hindered by scarce donor availability. Here we examine the potential for catalyzing cartilage tissue formation using a minimal number of NChons by co-culturing them with adipose-derived stem cells (ADSCs) in 3D hydrogels. Using three different co-culture models, we demonstrated that the effects of co-culture on cartilage tissue formation are dependent on the intercellular distance and cell distribution in 3D. Unexpectedly, increasing ADSC ratio in mixed co-culture led to increased synergy between NChons and ADSCs, and resulted in the formation of large neocartilage nodules. This work raises the potential of utilizing stem cells to catalyze tissue formation by neonatal chondrocytes via paracrine signaling, and highlights the importance of controlling cell distribution in 3D matrices to achieve optimal synergy.

BMP-2, hypoxia, and COL1A1/HtrA1 siRNAs favor neo-cartilage hyaline matrix formation in chondrocytes.

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Ollitrault, David; Legendre, Florence; Drougard, Carole; Briand, Mélanie; Benateau, Hervé; Goux, Didier; Chajra, Hanane; Poulain, Laurent; Hartmann, Daniel; Vivien, Denis; Shridhar, Vijayalakshmi; Baldi, Alfonso; Mallein-Gerin, Frédéric; Boumediene, Karim; Demoor, Magali; Galera, Philippe

2015-02-01

Osteoarthritis (OA) is an irreversible pathology that causes a decrease in articular cartilage thickness, leading finally to the complete degradation of the affected joint. The low spontaneous repair capacity of cartilage prevents any restoration of the joint surface, making OA a major public health issue. Here, we developed an innovative combination of treatment conditions to improve the human chondrocyte phenotype before autologous chondrocyte implantation. First, we seeded human dedifferentiated chondrocytes into a collagen sponge as a scaffold, cultured them in hypoxia in the presence of a bone morphogenetic protein (BMP), BMP-2, and transfected them with small interfering RNAs targeting two markers overexpressed in OA dedifferentiated chondrocytes, that is, type I collagen and/or HtrA1 serine protease. This strategy significantly decreased mRNA and protein expression of type I collagen and HtrA1, and led to an improvement in the chondrocyte phenotype index of differentiation. The effectiveness of our in vitro culture process was also demonstrated in the nude mouse model in vivo after subcutaneous implantation. We, thus, provide here a new protocol able to favor human hyaline chondrocyte phenotype in primarily dedifferentiated cells, both in vitro and in vivo. Our study also offers an innovative strategy for chondrocyte redifferentiation and opens new opportunities for developing therapeutic targets.

The Effect of Chondroitin Sulphate and Hyaluronic Acid on Chondrocytes Cultured within a Fibrin-Alginate Hydrogel

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Christopher J. Little

2014-09-01

Full Text Available Osteoarthritis is a painful degenerative joint disease that could be better managed if tissue engineers can develop methods to create long-term engineered articular cartilage tissue substitutes. Many of the tissue engineered cartilage constructs currently available lack the chemical stimuli and cell-friendly environment that promote the matrix accumulation and cell proliferation needed for use in joint cartilage repair. The goal of this research was to test the efficacy of using a fibrin-alginate hydrogel containing hyaluronic acid (HA and/or chondroitin sulphate (CS supplements for chondrocyte culture. Neonatal porcine chondrocytes cultured in fibrin-alginate hydrogels retained their phenotype better than chondrocytes cultured in monolayer, as evidenced by analysis of their relative expression of type II versus type I collagen mRNA transcripts. HA or CS supplementation of the hydrogels increased matrix glycosaminoglycan (GAG production during the first week of culture. However, the effects of these supplements on matrix accumulation were not additive and were no longer observed after two weeks of culture. Supplementation of the hydrogels with CS or a combination of both CS and HA increased the chondrocyte cell population after two weeks of culture. Statistical analysis indicated that the HA and CS treatment effects on chondrocyte numbers may be additive. This research suggests that supplementation with CS and/or HA has positive effects on cartilage matrix production and chondrocyte proliferation in three-dimensional (3D fibrin-alginate hydrogels.

The inhibitory effect of salmon calcitonin on tri-iodothyronine induction of early hypertrophy in articular cartilage.

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Pingping Chen-An

Full Text Available Salmon calcitonin has chondroprotective effect both in vitro and in vivo, and is therefore being tested as a candidate drug for cartilage degenerative diseases. Recent studies have indicated that different chondrocyte phenotypes may express the calcitonin receptor (CTR differentially. We tested for the presence of the CTR in chondrocytes from tri-iodothyronin (T3-induced bovine articular cartilage explants. Moreover, investigated the effects of human and salmon calcitonin on the explants.Early chondrocyte hypertrophy was induced in bovine articular cartilage explants by stimulation over four days with 20 ng/mL T3. The degree of hypertrophy was investigated by molecular markers of hypertrophy (ALP, IHH, COLX and MMP13, by biochemical markers of cartilage turnover (C2M, P2NP and AGNxII and histology. The expression of the CTR was detected by qPCR and immunohistochemistry. T3-induced explants were treated with salmon or human calcitonin. Calcitonin down-stream signaling was measured by levels of cAMP, and by the molecular markers.Compared with untreated control explants, T3 induction increased expression of the hypertrophic markers (p<0.05, of cartilage turnover (p<0.05, and of CTR (p<0.01. Salmon, but not human, calcitonin induced cAMP release (p<0.001. Salmon calcitonin also inhibited expression of markers of hypertrophy and cartilage turnover (p<0.05.T3 induced early hypertrophy of chondrocytes, which showed an elevated expression of the CTR and was thus a target for salmon calcitonin. Molecular marker levels indicated salmon, but not human, calcitonin protected the cartilage from hypertrophy. These results confirm that salmon calcitonin is able to modulate the CTR and thus have chondroprotective effects.

Age-Related Alterations in Signaling Pathways in Articular Chondrocytes: Implications for the Pathogenesis and Progression of Osteoarthritis - A Mini-Review.

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van der Kraan, Peter; Matta, Csaba; Mobasheri, Ali

2017-01-01

Musculoskeletal conditions are a major burden on individuals, healthcare systems, and social care systems throughout the world, with indirect costs having a predominant economic impact. Aging is a major contributing factor to the development and progression of arthritic and musculoskeletal diseases. Indeed, aging and inflammation (often referred to as 'inflammaging') are critical risk factors for the development of osteoarthritis (OA), which is one of the most common forms of joint disease. The term 'chondrosenescence' has recently been introduced to define the age-dependent deterioration of chondrocyte function and how it undermines cartilage function in OA. An important component of chondrosenescence is the age-related deregulation of subcellular signaling pathways in chondrocytes. This mini-review discusses the role of age-related alterations in chondrocyte signaling pathways. We focus our attention on two major areas: age-dependent alterations in transforming growth factor-β signaling and changes in protein kinase and phosphoprotein phosphatase activities in aging chondrocytes. A better understanding of the basic signaling mechanisms underlying aging in chondrocytes is likely to facilitate the development of new therapeutic and preventive strategies for OA and a range of other age-related osteoarticular disorders. © 2016 The Author(s) Published by S. Karger AG, Basel.

Basic science and surgical treatment options for articular cartilage injuries of the knee.

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Tetteh, Elizabeth S; Bajaj, Sarvottam; Ghodadra, Neil S

2012-03-01

The complex structure of articular cartilage allows for diverse knee function throughout range of motion and weight bearing. However, disruption to the structural integrity of the articular surface can cause significant morbidity. Due to an inherently poor regenerative capacity, articular cartilage defects present a treatment challenge for physicians and therapists. For many patients, a trial of nonsurgical treatment options is paramount prior to surgical intervention. In instances of failed conservative treatment, patients can undergo an array of palliative, restorative, or reparative surgical procedures to treat these lesions. Palliative methods include debridement and lavage, while restorative techniques include marrow stimulation. For larger lesions involving subchondral bone, reparative procedures such as osteochondral grafting or autologous chondrocyte implantation are considered. Clinical success not only depends on the surgical techniques but also requires strict adherence to rehabilitation guidelines. The purpose of this article is to review the basic science of articular cartilage and to provide an overview of the procedures currently performed at our institution for patients presenting with symptomatic cartilage lesions.

Serum Metabonomics of Articular Cartilage Destruction Induced by T-2 Toxin in Wistar Rats.

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Zhu, Lei; Zhao, Zhi Jun; Ren, Xiao Bin; Li, Qiang; Ding, Hua; Sun, Zhou; Kao, Qing Jun; Wang, Li Hua

2018-01-01

The molecular pathogenesis of T-2 toxin-induced cartilage destruction has not been fully unraveled yet. The aim of this study was to detect changes in serum metabolites in a rat anomaly model with articular cartilage destruction. Thirty healthy male Wistar rats were fed a diet containing T-2 toxin (300 ng/kg chow) for 3 months. Histopathological changes in femorotibial cartilage were characterized in terms of chondrocyte degeneration/necrosis and superficial cartilage defect, and the endogenous metabolite profile of serum was determined by UPLC/Q-TOF MS. Treated rats showed extensive areas of chondrocyte necrosis and superficial cartilage defect in the articular cartilage. In addition, 8 metabolites were found to change significantly in these rats compared to the control group, including lysoPE (18:0/0:0), lysoPC(14:0), lysoPC[18:4 (6Z,9Z,12Z,15Z)], lysoPC[(16:1(9Z)], lysoPC(16:0), L-valine, hippuric acid, and asparaginyl-glycine. These 8 metabolites associated with cartilage injury are mainly involved in phospholipid and amino acid metabolic pathways. Copyright © 2018 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Redifferentiation of in vitro expanded adult articular chondrocytes by combining the hanging-drop cultivation method with hypoxic environment.

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Martinez, Inigo; Elvenes, Jan; Olsen, Randi; Bertheussen, Kjell; Johansen, Oddmund

2008-01-01

The main purpose of this work has been to establish a new culturing technique to improve the chondrogenic commitment of isolated adult human chondrocytes, with the aim of being used during cell-based therapies or tissue engineering strategies. By using a rather novel technique to generate scaffold-free three-dimensional (3D) structures from in vitro expanded chondrocytes, we have explored the effects of different culture environments on cartilage formation. Three-dimensional chondrospheroids were developed by applying the hanging-drop technique. Cartilage tissue formation was attempted after combining critical factors such as serum-containing or serum-free media and atmospheric (20%) or low (2.5%) oxygen tensions. The quality of the formed microtissues was analyzed by histology, immunohistochemistry, electron microscopy, and real-time PCR, and directly compared with native adult cartilage. Our results revealed highly organized, 3D tissue-like structures developed by the hanging-drop method. All culture conditions allowed formation of 3D spheroids; however, cartilage generated under low oxygen tension had a bigger size, enhanced matrix deposition, and higher quality of cartilage formation. Real-time PCR demonstrated enhanced expression of cartilage-specific genes such us collagen type II and aggrecan in 3D cultures when compared to monolayers. Cartilage-specific matrix proteins and genes expressed in hanging-drop-developed spheroids were comparable to the expression obtained by applying the pellet culture system. In summary, our results indicate that a combination of 3D cultures of chondrocytes in hanging drops and a low oxygen environment represent an easy and convenient way to generate cartilage-like microstructures. We also show that a new specially tailored serum-free medium is suitable for in vitro cartilage tissue formation. This new methodology opens up the possibility of using autogenously produced solid 3D structures with redifferentiated chondrocytes as an

Subchondral Bone Plate Thickening Precedes Chondrocyte Apoptosis and Cartilage Degradation in Spontaneous Animal Models of Osteoarthritis

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Zaitunnatakhin Zamli

2014-01-01

Full Text Available Osteoarthritis (OA is the most common joint disorder characterised by bone remodelling and cartilage degradation and associated with chondrocyte apoptosis. These processes were investigated at 10, 16, 24, and 30 weeks in Dunkin Hartley (DH and Bristol Strain 2 (BS2 guinea pigs that develop OA spontaneously. Both strains had a more pronounced chondrocyte apoptosis, cartilage degradation, and subchondral bone changes in the medial than the lateral side of the tibia, and between strains, the changes were always greater and faster in DH than BS2. In the medial side, a significant increase of chondrocyte apoptosis and cartilage degradation was observed in DH between 24 and 30 weeks of age preceded by a progressive thickening and stiffening of subchondral bone plate (Sbp. The Sbp thickness consistently increased over the 30-week study period but the bone mineral density (BMD of the Sbp gradually decreased after 16 weeks. The absence of these changes in the medial side of BS2 may indicate that the Sbp of DH was undergoing remodelling. Chondrocyte apoptosis was largely confined to the deep zone of articular cartilage and correlated with thickness of the subchondral bone plate suggesting that cartilage degradation and chondrocyte apoptosis may be a consequence of continuous bone remodelling during the development of OA in these animal models of OA.

Silk fibroin-chondroitin sulfate scaffold with immuno-inhibition property for articular cartilage repair.

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Zhou, Feifei; Zhang, Xianzhu; Cai, Dandan; Li, Jun; Mu, Qin; Zhang, Wei; Zhu, Shouan; Jiang, Yangzi; Shen, Weiliang; Zhang, Shufang; Ouyang, Hong Wei

2017-11-01

The demand of favorable scaffolds has increased for the emerging cartilage tissue engineering. Chondroitin sulfate (CS) and silk fibroin have been investigated and reported with safety and excellent biocompatibility as tissue engineering scaffolds. However, the rapid degradation rate of pure CS scaffolds presents a challenge to effectively recreate neo-tissue similar to natural articular cartilage. Meanwhile the silk fibroin is well used as a structural constituent material because its remarkable mechanical properties, long-lasting in vivo stability and hypoimmunity. The application of composite silk fibroin and CS scaffolds for joint cartilage repair has not been well studied. Here we report that the combination of silk fibroin and CS could synergistically promote articular cartilage defect repair. The silk fibroin (silk) and silk fibroin/CS (silk-CS) scaffolds were fabricated with salt-leaching, freeze-drying and crosslinking methodologies. The biocompatibility of the scaffolds was investigated in vitro by cell adhesion, proliferation and migration with human articular chondrocytes. We found that silk-CS scaffold maintained better chondrocyte phenotype than silk scaffold; moreover, the silk-CS scaffolds reduced chondrocyte inflammatory response that was induced by interleukin (IL)-1β, which is in consistent with the well-documented anti-inflammatory activities of CS. The in vivo cartilage repair was evaluated with a rabbit osteochondral defect model. Silk-CS scaffold induced more neo-tissue formation and better structural restoration than silk scaffold after 6 and 12weeks of implantation in ICRS histological evaluations. In conclusion, we have developed a silk fibroin/ chondroitin sulfate scaffold for cartilage tissue engineering that exhibits immuno-inhibition property and can improve the self-repair capacity of cartilage. Severe cartilage defect such as osteoarthritis (OA) is difficult to self-repair because of its avascular, aneural and alymphatic nature

Effect of Collagen Type I or Type II on Chondrogenesis by Cultured Human Articular Chondrocytes

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Rutgers, M.; Saris, Daniël B.F.; Vonk, L.A.; van Rijen, M.H.P.; Akrum, V.; Langeveld, D.; van Boxtel, A.; Dhert, W.J.A.; Creemers, L.B.

2013-01-01

Introduction: Current cartilage repair procedures using autologous chondrocytes rely on a variety of carriers for implantation. Collagen types I and II are frequently used and valuable properties of both were shown earlier in vitro, although a preference for either was not demonstrated. Recently,

Hydrostatic Pressure Influences HIF-2 Alpha Expression in Chondrocytes

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Hiroaki Inoue

2015-01-01

Full Text Available Hypoxia-inducible factor (HIF-2α is considered to play a major role in the progression of osteoarthritis. Recently, it was reported that pressure amplitude influences HIF-2α expression in murine endothelial cells. We examined whether hydrostatic pressure is involved in expression of HIF-2α in articular chondrocytes. Chondrocytes were cultured and stimulated by inflammation or hydrostatic pressure of 0, 5, 10, or 50 MPa. After stimulation, heat shock protein (HSP 70, HIF-2α, nuclear factor kappa B (NF-κB, matrix metalloproteinase (MMP-13, MMP-3, and vascular endothelial growth factor (VEGF gene expression were evaluated. The levels of all gene expression were increased by inflammatory stress. When chondrocytes were exposed to a hydrostatic pressure of 5 MPa, HIF-2α, MMP-13, and MMP-3 gene expression increased significantly although those of HSP70 and NF-κB were not significantly different from the control group. In contrast, HIF-2α gene expression did not increase under a hydrostatic pressure of 50 MPa although HSP70 and NF-κB expression increased significantly compared to control. We considered that hydrostatic pressure of 5 MPa could regulate HIF-2α independent of NF-κB, because the level of HIF-2α gene expression increased significantly without upregulation of NF-κB expression at 5 MPa. Hydrostatic pressure may influence cartilage degeneration, inducing MMP-13 and MMP-3 expression through HIF-2α.

Hydrostatic pressure influences HIF-2 alpha expression in chondrocytes.

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Inoue, Hiroaki; Arai, Yuji; Kishida, Tsunao; Terauchi, Ryu; Honjo, Kuniaki; Nakagawa, Shuji; Tsuchida, Shinji; Matsuki, Tomohiro; Ueshima, Keiichirou; Fujiwara, Hiroyoshi; Mazda, Osam; Kubo, Toshikazu

2015-01-05

Hypoxia-inducible factor (HIF)-2α is considered to play a major role in the progression of osteoarthritis. Recently, it was reported that pressure amplitude influences HIF-2α expression in murine endothelial cells. We examined whether hydrostatic pressure is involved in expression of HIF-2α in articular chondrocytes. Chondrocytes were cultured and stimulated by inflammation or hydrostatic pressure of 0, 5, 10, or 50 MPa. After stimulation, heat shock protein (HSP) 70, HIF-2α, nuclear factor kappa B (NF-κB), matrix metalloproteinase (MMP)-13, MMP-3, and vascular endothelial growth factor (VEGF) gene expression were evaluated. The levels of all gene expression were increased by inflammatory stress. When chondrocytes were exposed to a hydrostatic pressure of 5 MPa, HIF-2α, MMP-13, and MMP-3 gene expression increased significantly although those of HSP70 and NF-κB were not significantly different from the control group. In contrast, HIF-2α gene expression did not increase under a hydrostatic pressure of 50 MPa although HSP70 and NF-κB expression increased significantly compared to control. We considered that hydrostatic pressure of 5 MPa could regulate HIF-2α independent of NF-κB, because the level of HIF-2α gene expression increased significantly without upregulation of NF-κB expression at 5 MPa. Hydrostatic pressure may influence cartilage degeneration, inducing MMP-13 and MMP-3 expression through HIF-2α.

Freeze-thaw treatment effects on the dynamic mechanical properties of articular cartilage

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Muldrew Ken

2010-10-01

Full Text Available Abstract Background As a relatively non-regenerative tissue, articular cartilage has been targeted for cryopreservation as a method of mitigating a lack of donor tissue availability for transplant surgeries. In addition, subzero storage of articular cartilage has long been used in biomedical studies using various storage temperatures. The current investigation studies the potential for freeze-thaw to affect the mechanical properties of articular cartilage through direct comparison of various subzero storage temperatures. Methods Both subzero storage temperature as well as freezing rate were compared using control samples (4°C and samples stored at either -20°C or -80°C as well as samples first snap frozen in liquid nitrogen (-196°C prior to storage at -80°C. All samples were thawed at 37.5°C to testing temperature (22°C. Complex stiffness and hysteresis characterized load resistance and damping properties using a non-destructive, low force magnitude, dynamic indentation protocol spanning a broad loading rate range to identify the dynamic viscoelastic properties of cartilage. Results Stiffness levels remained unchanged with exposure to the various subzero temperatures. Hysteresis increased in samples snap frozen at -196°C and stored at -80°C, though remained unchanged with exposure to the other storage temperatures. Conclusions Mechanical changes shown are likely due to ice lens creation, where frost heave effects may have caused collagen damage. That storage to -20°C and -80°C did not alter the mechanical properties of articular cartilage shows that when combined with a rapid thawing protocol to 37.5°C, the tissue may successfully be stored at subzero temperatures.

Articular cartilage changes in chondromalacia patellae.

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Bentley, G

1985-11-01

Full thickness samples of articular cartilage were removed from areas of chondromalacia on the medial and "odd" facets of the patellae of 21 adults and examined by histology, autoradiography and electron microscopy. Surface fibrillation, loss of superficial matrix staining and reduced 35SO4 labelling was seen, with little change in the deep zone. Ten cases showed "fibrous metaplasia" of the superficial cartilage with definite evidence of cell division and apparent smoothing of the surface. Scattered chondrocyte replication appeared to occur in the surrounding intact cartilage. The findings suggest that early lesions in chondromalacia patellae may heal either by cartilage or fibrous metaplasia and that this may account for the resolution of clinical symptoms.

Effect of Cell Sheet Manipulation Techniques on the Expression of Collagen Type II and Stress Fiber Formation in Human Chondrocyte Sheets.

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Wongin, Sopita; Waikakul, Saranatra; Chotiyarnwong, Pojchong; Siriwatwechakul, Wanwipa; Viravaidya-Pasuwat, Kwanchanok

2018-03-01

Cell sheet technology is applied to human articular chondrocytes to construct a tissue-like structure as an alternative treatment for cartilage defect. The effect of a gelatin manipulator, as a cell sheet transfer system, on the quality of the chondrocyte sheets was investigated. The changes of important chondrogenic markers and stress fibers, resulting from the cell sheet manipulation, were also studied. The chondrocyte cell sheets were constructed with patient-derived chondrocytes using a temperature-responsive polymer and a gelatin manipulator as a transfer carrier. The properties of the cell sheets, including sizes, expression levels of collagen type II and I, and the localization of the stress fibers, were assessed and compared with those of the cell sheets harvested without the gelatin manipulator. Using the gelatin manipulator, the original size of the chondrocyte cell sheets was retained with abundant stress fibers, but with a decrease in the expression of collagen type II. Without the gelatin manipulator, although the cell shrinkage occurred, the cell sheet with suppressed stress fiber formation showed significantly higher levels of collagen type II. These results support our observations that stress fiber formation in chondrocyte cell sheets affected the production of chondrogenic markers. These densely packed tissue-like structures possessed a good chondrogenic activity, indicating their potential for use in autologous chondrocyte implantation to treat cartilage defects.

Can microcarrier-expanded chondrocytes synthesize cartilaginous tissue in vitro?

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Surrao, Denver C; Khan, Aasma A; McGregor, Aaron J; Amsden, Brian G; Waldman, Stephen D

2011-08-01

Tissue engineering is a promising approach for articular cartilage repair; however, it is challenging to produce adequate amounts of tissue in vitro from the limited number of cells that can be extracted from an individual. Relatively few cell expansion methods exist without the problems of de-differentiation and/or loss of potency. Recently, however, several studies have noted the benefits of three-dimensional (3D) over monolayer expansion, but the ability of 3D expanded chondrocytes to synthesize cartilaginous tissue constructs has not been demonstrated. Thus, the purpose of this study was to compare the properties of engineered cartilage constructs from expanded cells (monolayer and 3D microcarriers) to those developed from primary chondrocytes. Isolated bovine chondrocytes were grown for 3 weeks in either monolayer (T-Flasks) or 3D microcarrier (Cytodex 3) expansion culture. Expanded and isolated primary cells were then seeded in high density culture on Millicell™ filters for 4 weeks to evaluate the ability to synthesize cartilaginous tissue. While microcarrier expansion was twice as effective as monolayer expansion (microcarrier: 110-fold increase, monolayer: 52-fold increase), the expanded cells (monolayer and 3D microcarrier) were not effectively able to synthesize cartilaginous tissue in vitro. Tissues developed from primary cells were substantially thicker and accumulated significantly more extracellular matrix (proteoglycan content: 156%-292% increase; collagen content: 70%-191% increase). These results were attributed to phenotypic changes experienced during the expansion phase. Monolayer expanded chondrocytes lost their native morphology within 1 week, whereas microcarrier-expanded cells were spreading by 3 weeks of expansion. While the use of 3D microcarriers can lead to large cellular yields, preservation of chondrogenic phenotype during expansion is required in order to synthesize cartilaginous tissue.

Synoviocytes, not chondrocytes, release free radicals after cycles of anoxia/re-oxygenation

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Schneider, Nicole; Mouithys-Mickalad, Ange L.; Lejeune, Jean-Philippe; Deby-Dupont, Ginette P.; Hoebeke, Maryse; Serteyn, Didier A.

2005-01-01

By oxymetry and electron paramagnetic resonance (EPR), we investigated the effects of repeated anoxia/re-oxygenation (A/R) periods on the respiration and production of free radicals by synoviocytes (rabbit HIG-82 cell line and primary equine synoviocytes) and equine articular chondrocytes. Three periods of 20 min anoxia followed by re-oxygenation were applied to 10 7 cells; O 2 consumption was measured before anoxia and after each re-oxygenation. After the last A/R, cellular free radical formation was investigated by EPR spectroscopy with spin trapping technique (n = 3 for each cell line). Both types of synoviocytes showed a high O 2 consumption, which was slowered after anoxia. By EPR with the spin trap POBN, we proved a free radical formation. Results were similar for equine and rabbit synoviocytes. For chondrocytes, we observed a low O 2 consumption, unchanged by anoxia, and no free radical production. These observations suggest an oxidant activity of synoviocytes, potentially important for the onset of osteoarthritis

An evaluation of chondrocyte morphology and gene expression on superhydrophilic vertically-aligned multi-walled carbon nanotube films.

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Antonioli, Eliane; Lobo, Anderson O; Ferretti, Mario; Cohen, Moisés; Marciano, Fernanda R; Corat, Evaldo J; Trava-Airoldi, Vladimir J

2013-03-01

Cartilage serves as a low-friction and wear-resistant articulating surface in diarthrodial joints and is also important during early stages of bone remodeling. Recently, regenerative cartilage research has focused on combinations of cells paired with scaffolds. Superhydrophilic vertically aligned carbon nanotubes (VACNTs) are of particular interest in regenerative medicine. The aim of this study is to evaluate cell expansion of human articular chondrocytes on superhydrophilic VACNTs, as well as their morphology and gene expression. VACNT films were produced using a microwave plasma chamber on Ti substrates and submitted to an O2 plasma treatment to make them superhydrophilic. Human chondrocytes were cultivated on superhydrophilic VACNTs up to five days. Quantitative RT-PCR was performed to measure type I and type II Collagen, Sox9, and Aggrecan mRNA expression levels. The morphology was analyzed by scanning electron microscopy (SEM) and confocal microscopy. SEM images demonstrated that superhydrophilic VACNTs permit cell growth and adhesion of human chondrocytes. The chondrocytes had an elongated morphology with some prolongations. Chondrocytes cultivated on superhydrophilic VACNTs maintain the level expression of Aggrecan, Sox9, and Collagen II determined by qPCR. This study was the first to indicate that superhydrophilic VACNTs may be used as an efficient scaffold for cartilage or bone repair. Copyright © 2012 Elsevier B.V. All rights reserved.

Demonstration of variation in chondrocyte activity in different zones of articular cartilage: an assessment of the value of in-situ hybridization.

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Marles, P J; Hoyland, J A; Parkinson, R; Freemont, A J

1991-04-01

Several methods have been described for investigating chondrocyte metabolism in vitro. In this study, in-situ hybridization (ISH) using an oligonucleotide probe (i.e. a poly-d(T) probe) to detect total messenger RNA (mRNA) in cartilage explants has been compared with radiosulphate and radioleucine uptake studies in an attempt to assess the value of ISH in investigating chondrocyte metabolism. The relative results of the three parameters indicate qualitative similarities in cells in the intermediate, deep and calcified zones but differences in the superficial zone. The relative levels of mRNA and leucine and sulphate uptake in the midzone areas could be construed as indicating that the bulk of cellular activity was directed towards the synthesis of proteoglycans. A similar relation between the three parameters, but at a lower level, was seen in chondrocytes in the calcified zone demonstrating that these cells are viable and biosynthetic. Both quantitative and qualitative differences between the three methods were observed in the superficial chondrocytes regarding the amount of mRNA compared to sulphate and leucine uptake. The results suggest that ISH can detect differences in the amount of mRNA present in chondrocytes in differing zones of cartilage and, like the radioleucine and radiosulphate studies, particularly emphasizes their functional heterogeneity.

Molecular changes in articular cartilage and subchondral bone in the rat anterior cruciate ligament transection and meniscectomized models of osteoarthritis

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Zhuo Ya

2011-08-01

Full Text Available Abstract Background Osteoarthritis (OA is a debilitating, progressive joint disease. Methods Similar to the disease progression in humans, sequential events of early cartilage degradation, subchondral osteopenia followed by sclerosis, and late osteophyte formation were demonstrated in the anterior cruciate ligament transection (ACLT or ACLT with partial medial meniscectomy (ACLT + MMx rat OA models. We describe a reliable and consistent method to examine the time dependent changes in the gene expression profiles in articular cartilage and subchondral bone. Results Local regulation of matrix degradation markers was demonstrated by a significant increase in mRNA levels of aggrecanase-1 and MMP-13 as early as the first week post-surgery, and expression remained elevated throughout the 10 week study. Immunohistochemistry confirmed MMP-13 expression in differentiated chondrocytes and synovial fibroblasts at week-2 and cells within osteophytes at week-10 in the surgically-modified-joints. Concomitant increases in chondrocyte differentiation markers, Col IIA and Sox 9, and vascular invasion markers, VEGF and CD31, peaked around week-2 to -4, and returned to Sham levels at later time points in both models. Indeed, VEGF-positive cells were found in the deep articular chondrocytes adjacent to subchondral bone. Osteoclastic bone resorption markers, cathepsin K and TRAP, were also elevated at week-2. Confirming bone resorption is an early local event in OA progression, cathepsin K positive osteoclasts were found invading the articular cartilage from the subchondral region at week 2. This was followed by late disease events, including subchondral sclerosis and osteophyte formation, as demonstrated by the upregulation of the osteoanabolic markers runx2 and osterix, toward week-4 to 6 post-surgery. Conclusions In summary, this study demonstrated the temporal and cohesive gene expression changes in articular cartilage and subchondral bone using known markers of

Effects of platelet rich plasma and chondrocyte co-culture on MSC chondrogenesis, hypertrophy and pathological responses

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Ramezanifard, Rouhallah; Kabiri, Mahboubeh; Hanaee Ahvaz, Hana

2017-01-01

Regarding the inadequate healing capability of cartilage tissue, cell-based therapy is making the future of cartilage repair and regeneration. Mesenchymal stem cells (MSC) have shown great promise in cartilage regeneration. However, a yet-unresolved issue is the emergence of hypertrophic and pathologic markers during in vitro MSC chondrogenesis. Articular chondrocytes (AC) can suppress the undesired hypertrophy when co-cultured with MSC. On the other hand, platelet rich plasma (PRP), is consi...

Topographic variation in redifferentiation capacity of chondrocytes in the adult human knee joint.

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Stenhamre, H; Slynarski, K; Petrén, C; Tallheden, T; Lindahl, A

2008-11-01

The aim of this study was to investigate the topographic variation in matrix production and cell density in the adult human knee joint. Additionally, we have examined the redifferentiation potential of chondrocytes expanded in vitro from the different locations. Full thickness cartilage-bone biopsies were harvested from seven separate anatomical locations of healthy knee joints from deceased adult human donors. Chondrocytes were isolated, expanded in vitro and redifferentiated in a pellet mass culture. Biochemical analysis of total collagen, proteoglycans and cellular content as well as histology and immunohistochemistry were performed on biopsies and pellets. In the biochemical analysis of the biopsies, we found lower proteoglycan to collagen (GAG/HP) ratio in the non-weight bearing (NWB) areas compared to the weight bearing (WB) areas. The chondrocytes harvested from different locations in femur showed a significantly better attachment and proliferation ability as well as good post-expansion chondrogenic capacity in pellet mass culture compared with the cells harvested from tibia. These results demonstrate that there are differences in extra cellular content within the adult human knee in respect to GAG/HP ratio. Additionally, the data show that clear differences between chondrocytes harvested from femur and tibia from healthy human knee joints exist and that the differences are not completely abolished during the process of de- and redifferentiation. These findings emphasize the importance of the understanding of topographic variation in articular cartilage biology when approaching new cartilage repair strategies.

Activation of Indian Hedgehog Promotes Chondrocyte Hypertrophy and Upregulation of MMP-13 in Human Osteoarthritic Cartilage

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Wei, Fangyuan; Zhou, Jingming; Wei, Xiaochun; Zhang, Juntao; Fleming, Braden C.; Terek, Richard; Pei, Ming; Chen, Qian; Liu, Tao; Wei, Lei

2012-01-01

Objective The objectives of this study were to 1) determine the correlation between osteoarthritis (OA) and Ihh expression, and 2) establish the effects of Ihh on expression of markers of chondrocyte hypertrophy and MMP-13 in human OA cartilage. Design OA cartilage and synovial fluid samples were obtained during total knee arthroplasty. Normal cartilage samples were obtained from intra-articular tumor resections, and normal synovial fluid samples were obtained from healthy volunteers and the contralateral uninjured knee of patients undergoing anterior cruciate ligament reconstruction. OA was graded using the Mankin score. Expression of Ihh in synovial fluid was determined by western blot. Ihh, type X collagen and MMP-13 mRNA were determined by real time PCR. Protein expression of type X collagen and MMP-13 in cartilage samples were analyzed with immunohistochemistry. Chondrocyte size was measured using image analysis. Results Ihh expression was increased 2.6 fold in OA cartilage and 37% in OA synovial fluid when compared to normal control samples. Increased expression of Ihh was associated with the severity of OA and expression of markers of chondrocyte hypertrophy: type X collagen and MMP-13, and chondocyte size. Chondrocytes were more spherical with increasing severity of OA. There was a significant correlation between Mankin score and cell size (r2= 0.80) and Ihh intensity (r2 = 0.89). Exogenous Ihh induced a 6.8 fold increase of type X collagen and 2.8 fold increase of MMP-13 mRNA expression in cultured chondrocytes. Conversely, knockdown of Ihh by siRNA and Hh inhibitor Cyclopamine had the opposite effect. Conclusions Ihh expression correlates with OA progression and changes in chondrocyte morphology and gene expression consistent with chondrocyte hypertrophy and cartilage degradation seen in OA cartilage. Thus, Ihh may be a potential therapeutic target to prevent OA progression. PMID:22469853

Reduced primary cilia length and altered Arl13b expression are associated with deregulated chondrocyte Hedgehog signaling in alkaptonuria.

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Thorpe, Stephen D; Gambassi, Silvia; Thompson, Clare L; Chandrakumar, Charmilie; Santucci, Annalisa; Knight, Martin M

2017-09-01

Alkaptonuria (AKU) is a rare inherited disease resulting from a deficiency of the enzyme homogentisate 1,2-dioxygenase which leads to the accumulation of homogentisic acid (HGA). AKU is characterized by severe cartilage degeneration, similar to that observed in osteoarthritis. Previous studies suggest that AKU is associated with alterations in cytoskeletal organization which could modulate primary cilia structure/function. This study investigated whether AKU is associated with changes in chondrocyte primary cilia and associated Hedgehog signaling which mediates cartilage degradation in osteoarthritis. Human articular chondrocytes were obtained from healthy and AKU donors. Additionally, healthy chondrocytes were treated with HGA to replicate AKU pathology (+HGA). Diseased cells exhibited shorter cilia with length reductions of 36% and 16% in AKU and +HGA chondrocytes respectively, when compared to healthy controls. Both AKU and +HGA chondrocytes demonstrated disruption of the usual cilia length regulation by actin contractility. Furthermore, the proportion of cilia with axoneme breaks and bulbous tips was increased in AKU chondrocytes consistent with defective regulation of ciliary trafficking. Distribution of the Hedgehog-related protein Arl13b along the ciliary axoneme was altered such that its localization was increased at the distal tip in AKU and +HGA chondrocytes. These changes in cilia structure/trafficking in AKU and +HGA chondrocytes were associated with a complete inability to activate Hedgehog signaling in response to exogenous ligand. Thus, we suggest that altered responsiveness to Hedgehog, as a consequence of cilia dysfunction, may be a contributing factor in the development of arthropathy highlighting the cilium as a novel target in AKU. © 2017 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.

An evaluation of chondrocyte morphology and gene expression on superhydrophilic vertically-aligned multi-walled carbon nanotube films

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Antonioli, Eliane; Lobo, Anderson O.; Ferretti, Mario; Cohen, Moisés; Marciano, Fernanda R.; Corat, Evaldo J.; Trava-Airoldi, Vladimir J.

2013-01-01

Cartilage serves as a low-friction and wear-resistant articulating surface in diarthrodial joints and is also important during early stages of bone remodeling. Recently, regenerative cartilage research has focused on combinations of cells paired with scaffolds. Superhydrophilic vertically aligned carbon nanotubes (VACNTs) are of particular interest in regenerative medicine. The aim of this study is to evaluate cell expansion of human articular chondrocytes on superhydrophilic VACNTs, as well as their morphology and gene expression. VACNT films were produced using a microwave plasma chamber on Ti substrates and submitted to an O 2 plasma treatment to make them superhydrophilic. Human chondrocytes were cultivated on superhydrophilic VACNTs up to five days. Quantitative RT-PCR was performed to measure type I and type II Collagen, Sox9, and Aggrecan mRNA expression levels. The morphology was analyzed by scanning electron microscopy (SEM) and confocal microscopy. SEM images demonstrated that superhydrophilic VACNTs permit cell growth and adhesion of human chondrocytes. The chondrocytes had an elongated morphology with some prolongations. Chondrocytes cultivated on superhydrophilic VACNTs maintain the level expression of Aggrecan, Sox9, and Collagen II determined by qPCR. This study was the first to indicate that superhydrophilic VACNTs may be used as an efficient scaffold for cartilage or bone repair. Highlights: ► Chondrocytes were cultivated on Superhydrophilic Vertically Aligned Multiwall Carbon Nanotubes (VACNT). ► We have shown a correlation between gene expression and thermodynamics aspects. ► Superhydrhophilic VACNT will be an excellent substrate for cartilage and bone tissue regeneration.

An evaluation of chondrocyte morphology and gene expression on superhydrophilic vertically-aligned multi-walled carbon nanotube films

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Antonioli, Eliane, E-mail: eliane.antonioli@einstein.br [Research and Education Institute, Hospital Israelita Albert Einstein, Sao Paulo, SP (Brazil); Lobo, Anderson O., E-mail: aolobo@univap.br [Laboratory of Biomedical Nanotechnology, Universidade do Vale do Paraiba, Sao Jose dos Campos, Sao Paulo (Brazil); Ferretti, Mario, E-mail: ferretti@einstein.br [Research and Education Institute, Hospital Israelita Albert Einstein, Sao Paulo, SP (Brazil); Ortophedic Division, Federal University of Sao Paulo, SP (Brazil); Cohen, Moises, E-mail: m.cohen@uol.com.br [Research and Education Institute, Hospital Israelita Albert Einstein, Sao Paulo, SP (Brazil); Ortophedic Division, Federal University of Sao Paulo, SP (Brazil); Marciano, Fernanda R., E-mail: femarciano@uol.com.br [Laboratory of Biomedical Nanotechnology, Universidade do Vale do Paraiba, Sao Jose dos Campos, Sao Paulo (Brazil); Corat, Evaldo J., E-mail: corat@las.inpe.br [Laboratorio Associado de Sensores e Materiais, Instituto Nacional de Pesquisas Espaciais, Sao Jose dos Campos, Sao Paulo (Brazil); Trava-Airoldi, Vladimir J., E-mail: vladimir@las.inpe.br [Laboratorio Associado de Sensores e Materiais, Instituto Nacional de Pesquisas Espaciais, Sao Jose dos Campos, Sao Paulo (Brazil)

2013-03-01

Cartilage serves as a low-friction and wear-resistant articulating surface in diarthrodial joints and is also important during early stages of bone remodeling. Recently, regenerative cartilage research has focused on combinations of cells paired with scaffolds. Superhydrophilic vertically aligned carbon nanotubes (VACNTs) are of particular interest in regenerative medicine. The aim of this study is to evaluate cell expansion of human articular chondrocytes on superhydrophilic VACNTs, as well as their morphology and gene expression. VACNT films were produced using a microwave plasma chamber on Ti substrates and submitted to an O{sub 2} plasma treatment to make them superhydrophilic. Human chondrocytes were cultivated on superhydrophilic VACNTs up to five days. Quantitative RT-PCR was performed to measure type I and type II Collagen, Sox9, and Aggrecan mRNA expression levels. The morphology was analyzed by scanning electron microscopy (SEM) and confocal microscopy. SEM images demonstrated that superhydrophilic VACNTs permit cell growth and adhesion of human chondrocytes. The chondrocytes had an elongated morphology with some prolongations. Chondrocytes cultivated on superhydrophilic VACNTs maintain the level expression of Aggrecan, Sox9, and Collagen II determined by qPCR. This study was the first to indicate that superhydrophilic VACNTs may be used as an efficient scaffold for cartilage or bone repair. Highlights: Black-Right-Pointing-Pointer Chondrocytes were cultivated on Superhydrophilic Vertically Aligned Multiwall Carbon Nanotubes (VACNT). Black-Right-Pointing-Pointer We have shown a correlation between gene expression and thermodynamics aspects. Black-Right-Pointing-Pointer Superhydrhophilic VACNT will be an excellent substrate for cartilage and bone tissue regeneration.

Characterization and chondrocyte differentiation stage-specific expression of KRAB zinc-finger protein gene ZNF470

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Hering, Thomas M.; Kazmi, Najam H.; Huynh, Tru D.; Kollar, John; Xu, Laura; Hunyady, Aaron B.; Johnstone, Brian

2004-01-01

As part of a study to identify novel transcriptional regulators of chondrogenesis-related gene expression, we have cloned and characterized cDNA for zinc-finger protein 470 (ZNF470), the human ortholog of which encodes a 717 amino acid residue protein containing 17 Cys 2 His 2 zinc-finger domains, as well as KRAB-A and KRAB-B motifs. The cDNA library used to isolate the initial ZNF470 clone was prepared from human bone marrow-derived mesenchymal progenitor cells at an intermediate stage of chondrogenic differentiation. We have determined the intron-exon structure of the human ZNF470 gene, which has been mapped to a zinc-finger cluster in a known imprinted region of human chromosome 19q13.4. ZNF470 is expressed at high levels in human testis and is expressed at low or undetectible levels in other adult tissues. Human ZNF470 expressed in mammalian cells as an EGFP fusion protein localizes predominantly to the nucleus, consistent with a role in transcriptional regulation. ZNF470, analyzed by quantitative real time PCR, was transiently expressed before the maximal expression of COL2A1 during chondrogenic differentiation in vitro. We have also characterized the bovine ortholog of human ZNF470, which encodes a 508 amino acid residue protein having 10 zinc-finger domains. A bovine ZNF470 cDNA clone was used to examine expression of ZNF470 in bovine articular chondrocytes treated with retinoic acid to stimulate dedifferentiation. Bovine ZNF470 expression was undetectable in freshly isolated bovine articular chondrocytes, but was dramatically upregulated in dedifferentiated retinoic acid-treated chondrocytes. These results, in two model systems, suggest a possible role for ZNF470 in the regulation of chondrogenesis-specific gene expression

24R,25-Dihydroxyvitamin D3 Protects against Articular Cartilage Damage following Anterior Cruciate Ligament Transection in Male Rats.

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Barbara D Boyan

Full Text Available Osteoarthritis (OA in humans is associated with low circulating 25-hydroxyvitamin D3 [25(OHD3]. In vitamin D replete rats, radiolabeled 24R,25-dihydroxyvitamin D3 [24R,25(OH2D3] accumulates in articular cartilage following injection of [3H]-25(OHD3. Previously, we showed that 24R,25(OH2D3 blocks chondrocyte apoptosis via phospholipase D and p53, suggesting a role for 24R,25(OH2D3 in maintaining cartilage health. We examined the ability of 24R,25(OH2D3 to prevent degenerative changes in articular cartilage in an OA-like environment and the potential mechanisms involved. In vitro, rat articular chondrocytes were treated with IL-1β with and without 24R,25(OH2D3 or 1α,25(OH2D3. 24R,25(OH2D3 but not 1α,25(OH2D3 blocked the effects of IL-1β in a dose-dependent manner, and its effect was partially mediated through the TGF-β1 signaling pathway. In vivo, unilateral anterior cruciate ligament transections were performed in immunocompetent rats followed by intra-articular injections of 24R,25(OH2D3 or vehicle (t = 0, 7, 14, 21 days. Tissues were harvested on day 28. Joints treated with vehicle had changes typical of OA whereas joints treated with 24R,25(OH2D3 had less articular cartilage damage and levels of inflammatory mediators. These results indicate that 24R,25(OH2D3 protects against OA, and suggest that it may be a therapeutic approach for preventing trauma-induced osteoarthritis.

Effect of stiffness of chitosan-hyaluronic acid dialdehyde hydrogels on the viability and growth of encapsulated chondrocytes.

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V Thomas, Lynda; Vg, Rahul; D Nair, Prabha

2017-11-01

Substrate elasticity or stiffness can influence the phenotypic and functional characteristics of chondrocytes. This work aimed to study the effect of varying stiffness compositions of a two-component injectable hydrogel based on chitosan (CH) and oxidized hyaluronic acid (HDA) on the growth and functionality of encapsulated chondrocytes. Three different ratios of the gel were prepared (10:1,10:3 and 10:5 CH-HDA) and characterized. The stiffness of the gels was evaluated from the force displacement curves using force spectroscopy AFM analysis. Rabbit articular chondrocytes were harvested and the cells from Passage 2 to 4 were used for the encapsulation study. The viability and ECM production of encapsulated chondrocytes were assessed at 7day, 14day and 28day post culture. The results of the study show that as the ratio of hyaluronic acid dialdehyde component was increased, the stiffness of the gels increased from 130.78±19.83kPa to 181.47±19.77kPa which was also evidenced from the decrease in gelling time. Although there was an increase in the percentage of viable encapsulated cells which also maintained the spherical phenotype in the less stiff gels, decreased expression of ECM markers- Collagen type II and Glycosaminoglycans was observed compared to the stiffer gels. These findings indicate that gel stiffness strongly impacts the chondrocyte microenvironment both in maintenance of phenotypic integrity and ECM production. Copyright © 2017. Published by Elsevier B.V.

Investigation of the direct effects of salmon calcitonin on human osteoarthritic chondrocytes

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Pedersen Christian

2010-04-01

Full Text Available Abstract Background Calcitonin has been demonstrated to have chondroprotective effects under pre-clinical settings. It is debated whether this effect is mediated through subchondral-bone, directly on cartilage or both in combination. We investigated possible direct effects of salmon calcitonin on proteoglycans and collagen-type-II synthesis in osteoarthritic (OA cartilage. Methods Human OA cartilage explants were cultured with salmon calcitonin [100 pM-100 nM]. Direct effects of calcitonin on articular cartilage were evaluated by 1 measurement of proteoglycan synthesis by incorporation of radioactive labeled 35SO4 [5 μCi] 2 quantification of collagen-type-II formation by pro-peptides of collagen type II (PIINP ELISA, 3 QPCR expression of the calcitonin receptor in OA chondrocytes using four individual primer pairs, 4 activation of the cAMP signaling pathway by EIA and, 5 investigations of metabolic activity by AlamarBlue. Results QPCR analysis and subsequent sequencing confirmed expression of the calcitonin receptor in human chondrocytes. All doses of salmon calcitonin significantly elevated cAMP levels (P 35SO4 incorporation, with a 96% maximal induction at 10 nM (P Conclusion Calcitonin treatment increased proteoglycan and collagen synthesis in human OA cartilage. In addition to its well-established effect on subchondral bone, calcitonin may prove beneficial to the management of joint diseases through direct effects on chondrocytes.

Bioluminescence imaging of chondrocytes in rabbits by intraarticular injection of D-luciferin

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Moon, Sung Min; Min, Jung Joon; Kim, Sung Mi; Bom, Hee Seung [Chonnam National University Medical School, Gwangju (Korea, Republic of); Oh, Suk Jung; Kang, Han Saem; Kim, Kwang Yoon [ECOBIO INC., Gwangju (Korea, Republic of); Kim, Young Ho [College of Natural Science, Chosun University, Gwangju (Korea, Republic of)

2007-02-15

Luciferase is one of the most commonly used reporter enzymes in the field of in vivo optical imaging. D-luciferin, the substrate for firefly luciferase has very high cost that allows this kind of experiment limited to small animals such as mice and rats. In this current study, we validated local injection of D-luciferin in the articular capsule for bioluminescence imaging in rabbits. Chondrocytes were cultured and infected by replication-defective adenoviral vector encoding firefly luciferase (Fluc). Chondrocytes expressing Fluc were injected or implanted in the left knee joint. The rabbits underwent optical imaging studies after local injection of D-luciferin at 1, 5, 7, 9 days after cellular administration. We sought whether optimal imaging signals was could be by a cooled CCD camera after local injection of D-luciferin. Imaging signal was not observed from the left knee joint after intraperitoneal injection of D-luciferin (15 mg/kg), whereas it was observed after intraarticular injection. Photon intensity from the left knee joint of rabbits was compared between cell injected and implanted groups after intraarticular injection of D-luciferin. During the period of imaging studies, photon intensity of the cell implanted group was 5-10 times higher than that of the cell injected group. We successfully imaged chondrocytes expressing Fluc after intraarticular injection of D-luciferin. This technique may be further applied to develop new drugs for knee joint disease.

Local intra-articular injection of resveratrol delays cartilage degeneration in C57BL/6 mice by inducing autophagy via AMPK/mTOR pathway.

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Qin, Na; Wei, Liwei; Li, Wuyin; Yang, Wei; Cai, Litao; Qian, Zhuang; Wu, Shufang

2017-07-01

Autophagy is an essential cellular homeostasis mechanism that was found to be compromised in aging and osteoarthritis (OA) cartilage. Previous studies showed that resveratrol can effectively regulate autophagy in other cells. The purpose of this study was to determine whether the chondroprotective effect of resveratrol was related to chondrocyte autophagy and to elucidate underlying mechanisms. OA model was induced by destabilization of the medial meniscus (DMM) in 10-week-old male mice. OA mice were treated with resveratrol with/without 3-MA for 8 weeks beginning 4 weeks after surgery. The local intra-articular injection of resveratrol delayed articular cartilage degradation in DMM-induced OA by OARSI scoring systems and Safranin O-fast green. Resveratrol treatment increased Unc-51-like kinase1, Beclin1, microtubule-associated protein light chain 3, hypoxia inducible factor-1α, phosphorylated AMPK, collagen-2A1, Aggrecan expressions, but decreased hypoxia inducible factor-2α, phosphorylated mTOR, matrix metalloproteinases13 and a disintegrin and metalloproteinase with thrombospondin motifs 5 expressions. The effects of resveratrol were obviously blunted by 3-MA except HIF and AMPK. These findings indicate that resveratrol intra-articular injection delayed articular cartilage degeneration and promoted chondrocyte autophagy in an experimental model of surgical DMM-induced OA, in part via balancing HIF-1α and HIF-2α expressions and thereby regulating AMPK/mTOR signaling pathway. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

Zonal Articular Cartilage Possesses Complex Mechanical Behavior Spanning Multiple Length Scales: Dependence on Chemical Heterogeneity, Anisotropy, and Microstructure

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Wahlquist, Joseph A.

This work focused on characterizing the mechanical behavior of biological material in physiologically relevant conditions and at sub millimeter length scales. Elucidating the time, length scale, and directionally dependent mechanical behavior of cartilage and other biological materials is critical to adequately recapitulate native mechanosensory cues for cells, create computational models that mimic native tissue behavior, and assess disease progression. This work focused on three broad aspects of characterizing the mechanical behavior of articular cartilage. First, we sought to reveal the causes of time-dependent deformation and variation of mechanical properties with distance from the articular surface. Second, we investigated size dependence of mechanical properties. Finally, we examined material anisotropy of both the calcified and uncalcified tissues of the osteochondral interface. This research provides insight into how articular cartilage serves to support physiologic loads and simultaneously sustain chondrocyte viability.

Human platelet lysate successfully promotes proliferation and subsequent chondrogenic differentiation of adipose-derived stem cells: a comparison with articular chondrocytes.

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Hildner, F; Eder, M J; Hofer, K; Aberl, J; Redl, H; van Griensven, M; Gabriel, C; Peterbauer-Scherb, A

2015-07-01

Fetal calf serum (FCS) bears a potential risk for carrying diseases and eliciting immune reactions. Nevertheless, it still represents the gold standard as medium supplement in cell culture. In the present study, human platelet lysate (PL) was tested as an alternative to FCS for the expansion and subsequent chondrogenic differentiation of human adipose-derived stem cells (ASCs). ASCs were expanded with 10% FCS (group F) or 5% PL (group P). Subsequently, three-dimensional (3D) micromass pellets were created and cultured for 5 weeks in chondrogenic differentiation medium. Additionally, the de- and redifferentiation potential of human articular chondrocytes (HACs) was evaluated and compared to ASCs. Both HACs and ASCs cultured with PL showed strongly enhanced proliferation rates. Redifferentiation of HACs was possible for cells expanded up to 3.3 population doublings (PD). At this stage, PL-expanded HACs demonstrated better redifferentiation potential than FCS-expanded cells. ASCs could also be differentiated following extended passaging. Glycosaminoglycan (GAG) quantification and qRT-PCR of 10 cartilage related markers demonstrated a tendency for increased chondrogenic differentiation of PL-expanded ASCs compared to cells expanded with FCS. Histologically, collagen type II but also collagen type X was mainly present in group P. The present study demonstrates that PL strongly induces proliferation of ASCs, while the chondrogenic differentiation potential is retained. HACs also showed enhanced proliferation and even better redifferentiation when previously expanded with PL. This suggests that PL is superior to FCS as a supplement for the expansion of ASCs and HACs, particularly with regard to chondrogenic (re)differentiation. Copyright © 2013 John Wiley & Sons, Ltd.

Engineering zonal cartilage through bioprinting collagen type II hydrogel constructs with biomimetic chondrocyte density gradient.

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Ren, Xiang; Wang, Fuyou; Chen, Cheng; Gong, Xiaoyuan; Yin, Li; Yang, Liu

2016-07-20

Cartilage tissue engineering is a promising approach for repairing and regenerating cartilage tissue. To date, attempts have been made to construct zonal cartilage that mimics the cartilaginous matrix in different zones. However, little attention has been paid to the chondrocyte density gradient within the articular cartilage. We hypothesized that the chondrocyte density gradient plays an important role in forming the zonal distribution of extracellular matrix (ECM). In this study, collagen type II hydrogel/chondrocyte constructs were fabricated using a bioprinter. Three groups were created according to the total cell seeding density in collagen type II pre-gel: Group A, 2 × 10(7) cells/mL; Group B, 1 × 10(7) cells/mL; and Group C, 0.5 × 10(7) cells/mL. Each group included two types of construct: one with a biomimetic chondrocyte density gradient and the other with a single cell density. The constructs were cultured in vitro and harvested at 0, 1, 2, and 3 weeks for cell viability testing, reverse-transcription quantitative PCR (RT-qPCR), biochemical assays, and histological analysis. We found that total ECM production was positively correlated with the total cell density in the early culture stage, that the cell density gradient distribution resulted in a gradient distribution of ECM, and that the chondrocytes' biosynthetic ability was affected by both the total cell density and the cell distribution pattern. Our results suggested that zonal engineered cartilage could be fabricated by bioprinting collagen type II hydrogel constructs with a biomimetic cell density gradient. Both the total cell density and the cell distribution pattern should be optimized to achieve synergistic biological effects.

Sulfato de condroitina e hialuronato de sódio no tratamento da doença articular degenerativa em cães: estudo histológico da cartilagem articular e membrana sinovial Chondroitin sulfate and sodium hyaluronate in the treatment of the degenerative joint disease in dogs: histological features of articular cartilage and synovium

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E.G. Melo

2008-02-01

processo degenerativo da cartilagem articular. Não foi constatada ação favorável das drogas na membrana sinovial.Fifteen mongrel dogs, both genders, weighting from 18 to 25kg were used and Degenerative Joint Disease (DJD was induced through cranial cruciate ligament (CCrL artroscopical section. After three weeks, CCrL was reconstructed by Schawalder's (1989 technique. Then, dogs were distributed in three groups and the following protocols were used: group I, control, no other treatment but the CCrL reconstruction; group II received chondroitin sulfate 24mg per animal every five days, intramuscularly, in a total of six injections; and group III received sodium hyaluronate 20mg per animal every five days, intravenously, in a total of three injections. Clinical observation was done until 90 days after treatments. By that time, the articular cartilage and synovium were collected and their morphology was evaluated. In group I, the degenerative alterations of the DJD were the most intense. Thus, decrease of chondrocytes number, pannus, fibrillations, grooves, erosion, and irregular articular surface were observed on the cartilage. In group II, raise of chondrocytes number was observed, with increase of synthesis activity of matrix and decrease of lesions on the articular surface. There was an increase of chondrocytes in group III, but the cells were morphologically unviable. All the groups showed proliferation of the synovial membrane, with limpho-plasma cells infiltrated in subintim and perivascular. In groups I and III, the proliferation of synovium was abundant, with formation of pannus, flattened synoviocytes or synovium absent with granulation tissue. Those results suggest that the chondroitin sulfate stimulated the articular cartilage; decreasing or delaying the alterations of DJD, as well as, the sodium hyaluronate did not interfere on degenerative process in articular cartilage. No favorable action of these drugs in the synovial membrane was verified.

The structure and function of the pericellular matrix of articular cartilage.

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Wilusz, Rebecca E; Sanchez-Adams, Johannah; Guilak, Farshid

2014-10-01

Chondrocytes in articular cartilage are surrounded by a narrow pericellular matrix (PCM) that is both biochemically and biomechanically distinct from the extracellular matrix (ECM) of the tissue. While the PCM was first observed nearly a century ago, its role is still under investigation. In support of early hypotheses regarding its function, increasing evidence indicates that the PCM serves as a transducer of biochemical and biomechanical signals to the chondrocyte. Work over the past two decades has established that the PCM in adult tissue is defined biochemically by several molecular components, including type VI collagen and perlecan. On the other hand, the biomechanical properties of this structure have only recently been measured. Techniques such as micropipette aspiration, in situ imaging, computational modeling, and atomic force microscopy have determined that the PCM exhibits distinct mechanical properties as compared to the ECM, and that these properties are influenced by specific PCM components as well as disease state. Importantly, the unique relationships among the mechanical properties of the chondrocyte, PCM, and ECM in different zones of cartilage suggest that this region significantly influences the stress-strain environment of the chondrocyte. In this review, we discuss recent advances in the measurement of PCM mechanical properties and structure that further increase our understanding of PCM function. Taken together, these studies suggest that the PCM plays a critical role in controlling the mechanical environment and mechanobiology of cells in cartilage and other cartilaginous tissues, such as the meniscus or intervertebral disc. Copyright © 2014 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Articular cartilage: from formation to tissue engineering.

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Camarero-Espinosa, Sandra; Rothen-Rutishauser, Barbara; Foster, E Johan; Weder, Christoph

2016-05-26

Hyaline cartilage is the nonlinear, inhomogeneous, anisotropic, poro-viscoelastic connective tissue that serves as friction-reducing and load-bearing cushion in synovial joints and is vital for mammalian skeletal movements. Due to its avascular nature, low cell density, low proliferative activity and the tendency of chondrocytes to de-differentiate, cartilage cannot regenerate after injury, wear and tear, or degeneration through common diseases such as osteoarthritis. Therefore severe damage usually requires surgical intervention. Current clinical strategies to generate new tissue include debridement, microfracture, autologous chondrocyte transplantation, and mosaicplasty. While articular cartilage was predicted to be one of the first tissues to be successfully engineered, it proved to be challenging to reproduce the complex architecture and biomechanical properties of the native tissue. Despite significant research efforts, only a limited number of studies have evolved up to the clinical trial stage. This review article summarizes the current state of cartilage tissue engineering in the context of relevant biological aspects, such as the formation and growth of hyaline cartilage, its composition, structure and biomechanical properties. Special attention is given to materials development, scaffold designs, fabrication methods, and template-cell interactions, which are of great importance to the structure and functionality of the engineered tissue.

Adeno-associated virus gene therapy vector scAAVIGF-I for transduction of equine articular chondrocytes and RNA-seq analysis.

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Hemphill, D D; McIlwraith, C W; Slayden, R A; Samulski, R J; Goodrich, L R

2016-05-01

IGF-I is one of several anabolic factors being investigated for the treatment of osteoarthritis (OA). Due to the short biological half-life, extended administration is required for more robust cartilage healing. Here we create a self-complimentary adeno-associated virus (AAV) gene therapy vector utilizing the transgene for IGF-I. Various biochemical assays were performed to investigate the cellular response to scAAVIGF-I treatment vs an scAAVGFP positive transduction control and a negative for transduction control culture. RNA-sequencing analysis was also performed to establish a differential regulation profile of scAAVIGF-I transduced chondrocytes. Biochemical analyses indicated an average media IGF-I concentration of 608 ng/ml in the scAAVIGF-I transduced chondrocytes. This increase in IGF-I led to increased expression of collagen type II and aggrecan and increased protein concentrations of cellular collagen type II and media glycosaminoglycan vs both controls. RNA-seq revealed a global regulatory pattern consisting of 113 differentially regulated GO categories including those for chondrocyte and cartilage development and regulation of apoptosis. This research substantiates that scAAVIGF-I gene therapy vector increased production of IGF-I to clinically relevant levels with a biological response by chondrocytes conducive to increased cartilage healing. The RNA-seq further established a set of differentially expressed genes and gene ontologies induced by the scAAVIGF-I vector while controlling for AAV infection. This dataset provides a static representation of the cellular transcriptome that, while only consisting of one time point, will allow for further gene expression analyses to compare additional cartilage healing therapeutics or a transient cellular response. Copyright © 2015. Published by Elsevier Ltd.

The Involvement of Mutual Inhibition of ERK and mTOR in PLCγ1-Mediated MMP-13 Expression in Human Osteoarthritis Chondrocytes

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Zejun Liu

2015-08-01

Full Text Available The issue of whether ERK activation determines matrix synthesis or degradation in osteoarthritis (OA pathogenesis currently remains controversial. Our previous study shows that PLCγ1 and mTOR are involved in the matrix metabolism of OA cartilage. Investigating the interplays of PLCγ1, mTOR and ERK in matrix degradation of OA will facilitate future attempts to manipulate ERK in OA prevention and therapy. Here, cultured human normal chondrocytes and OA chondrocytes were treated with different inhibitors or transfected with expression vectors, respectively. The levels of ERK, p-ERK, PLCγ1, p-PLCγ1, mTOR, p-mTOR and MMP-13 were then evaluated by Western blotting analysis. The results manifested that the expression level of ERK in human OA chondrocytes was lower than that in human normal articular chondrocytes, and the up-regulation of ERK could promote matrix synthesis, including the decrease in MMP-13 level and the increase in Aggrecan level in human OA chondrocytes. Furthermore, the PLCγ1/ERK axis and a mutual inhibition of mTOR and ERK were observed in human OA chondrocytes. Interestingly, activated ERK had no inhibitory effect on MMP-13 expression in PLCγ1-transformed OA chondrocytes. Combined with our previous study, the non-effective state of ERK activation by PLCγ1 on MMP-13 may be partly attributed to the inhibition of the PLCγ1/mTOR axis on the PLCγ1/ERK axis. Therefore, the study indicates that the mutual inhibition of ERK and mTOR is involved in PLCγ1-mediated MMP-13 expression in human OA chondrocytes, with important implication for the understanding of OA pathogenesis as well as for its prevention and therapy.

The use of fibrin and poly(lactic-co-glycolic acid hybrid scaffold for articular cartilage tissue engineering: an in vivo analysis

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S Munirah

2008-02-01

Full Text Available Our preliminary results indicated that fibrin and poly(lactic-co-glycolic acid (PLGA hybrid scaffold promoted early chondrogenesis of articular cartilage constructs in vitro. The aim of this study was to evaluate in vivo cartilaginous tissue formation by chondrocyte-seeded fibrin/PLGA hybrid scaffolds. PLGA scaffolds were soaked carefully, in chondrocyte-fibrin suspension, and polymerized by dropping thrombin-calcium chloride (CaCl2 solution. PLGA-seeded chondrocytes were used as a control. Resulting constructs were implanted subcutaneously, at the dorsum of nude mice, for 4 weeks. Macroscopic observation, histological evaluation, gene expression and sulphated-glycosaminoglycan (sGAG analyses were performed at each time point of 1, 2 and 4 weeks post-implantation. Cartilaginous tissue formation in fibrin/PLGA hybrid construct was confirmed by the presence of lacunae and cartilage-isolated cells embedded within basophilic ground substance. Presence of proteoglycan and glycosaminoglycan (GAG in fibrin/PLGA hybrid constructs was confirmed by positive Safranin O and Alcian Blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrices. Chondrogenic properties were further demonstrated by the expression of gene encoded cartilage-specific markers, collagen type II and aggrecan core protein. The sGAG production in fibrin/PLGA hybrid constructs was higher than in the PLGA group. In conclusion, fibrin/PLGA hybrid scaffold promotes cartilaginous tissue formation in vivo and may serve as a potential cell delivery vehicle and a structural basis for articular cartilage tissue-engineering.

Repair of articular cartilage defects by tissue-engineered cartilage constructed with adipose-derived stem cells and acellular cartilaginous matrix in rabbits.

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Wang, Z J; An, R Z; Zhao, J Y; Zhang, Q; Yang, J; Wang, J B; Wen, G Y; Yuan, X H; Qi, X W; Li, S J; Ye, X C

2014-06-18

After injury, inflammation, or degeneration, articular cartilage has limited self-repair ability. We aimed to explore the feasibility of repair of articular cartilage defects with tissue-engineered cartilage constructed by acellular cartilage matrices (ACMs) seeded with adipose-derived stem cells (ADSCs). The ADSCs were isolated from 3-month-old New Zealand albino rabbit by using collagenase and cultured and amplified in vitro. Fresh cartilage isolated from adult New Zealand albino rabbit were freeze-dried for 12 h and treated with Triton X-100, DNase, and RNase to obtain ACMs. ADSCs were seeded in the acellular cartilaginous matrix at 2x10(7)/mL, and cultured in chondrogenic differentiation medium for 2 weeks to construct tissue-engineered cartilage. Twenty-four New Zealand white rabbits were randomly divided into A, B, and C groups. Engineered cartilage was transplanted into cartilage defect position of rabbits in group A, group B obtained ACMs, and group C did not receive any transplants. The rabbits were sacrificed in week 12. The restored tissue was evaluated using macroscopy, histology, immunohistochemistry, and transmission electron microscopy (TEM). In the tissue-engineered cartilage group (group A), articular cartilage defects of the rabbits were filled with chondrocyte-like tissue with smooth surface. Immunohistochemistry showed type II-collagen expression and Alcian blue staining was positive. TEM showed chondrocytes in the recesses, with plenty of secretary matrix particles. In the scaffold group (group B), the defect was filled with fibrous tissue. No repaired tissue was found in the blank group (group C). Tissue-engineered cartilage using ACM seeded with ADSCs can help repair articular cartilage defects in rabbits.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is increased in osteoarthritis and regulates chondrocyte catabolic and anabolic activities

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Long, D.L.; Ulici, V.; Chubinskaya, S.; Loeser, R.F.

2015-01-01

Objective We determined if the epidermal growth factor receptor ligand HB-EGF is produced in cartilage and if it regulates chondrocyte anabolic or catabolic activity. Methods HB-EGF expression was measured by quantitative PCR using RNA isolated from mouse knee joint tissues and from normal and OA human chondrocytes. Immunohistochemistry was performed on normal and OA human cartilage and meniscus sections. Cultured chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus and osteogenic protein 1 (OP-1) as an anabolic stimulus. Effects of HB-EGF on cell signaling were analyzed by immunoblotting of selected signaling proteins. MMP-13 was measured in conditioned media, proteoglycan synthesis was measured by sulfate incorporation, and matrix gene expression by quantitative PCR. Results HB-EGF expression was increased in 12-month old mice at 8 weeks after surgery to induce OA and increased amounts of HB-EGF were noted in human articular cartilage from OA knees. FN-f stimulated chondrocyte HB-EGF expression and HB-EGF stimulated chondrocyte MMP-13 production. However, HB-EGF was not required for FN-f stimulation of MMP-13 production. HB-EGF activated the ERK and p38 MAP kinases and stimulated phosphorylation of Smad1 at an inhibitory serine site which was associated with inhibition of OP-1 mediated proteoglycan synthesis and reduced aggrecan (ACAN) but not COL2A1 expression. Conclusion HB-EGF is a new factor identified in OA cartilage that promotes chondrocyte catabolic activity while inhibiting anabolic activity suggesting it could contribute to the catabolic-anabolic imbalance seen in OA cartilage. PMID:25937027

The bio in the ink: cartilage regeneration with bioprintable hydrogels and articular cartilage-derived progenitor cells.

Science.gov (United States)

Levato, Riccardo; Webb, William R; Otto, Iris A; Mensinga, Anneloes; Zhang, Yadan; van Rijen, Mattie; van Weeren, René; Khan, Ilyas M; Malda, Jos

2017-10-01

Cell-laden hydrogels are the primary building blocks for bioprinting, and, also termed bioinks, are the foundations for creating structures that can potentially recapitulate the architecture of articular cartilage. To be functional, hydrogel constructs need to unlock the regenerative capacity of encapsulated cells. The recent identification of multipotent articular cartilage-resident chondroprogenitor cells (ACPCs), which share important traits with adult stem cells, represents a new opportunity for cartilage regeneration. However, little is known about the suitability of ACPCs for tissue engineering, especially in combination with biomaterials. This study aimed to investigate the potential of ACPCs in hydrogels for cartilage regeneration and biofabrication, and to evaluate their ability for zone-specific matrix production. Gelatin methacryloyl (gelMA)-based hydrogels were used to culture ACPCs, bone marrow mesenchymal stromal cells (MSCs) and chondrocytes, and as bioinks for printing. Our data shows ACPCs outperformed chondrocytes in terms of neo-cartilage production and unlike MSCs, ACPCs had the lowest gene expression levels of hypertrophy marker collagen type X, and the highest expression of PRG4, a key factor in joint lubrication. Co-cultures of the cell types in multi-compartment hydrogels allowed generating constructs with a layered distribution of collagens and glycosaminoglycans. By combining ACPC- and MSC-laden bioinks, a bioprinted model of articular cartilage was generated, consisting of defined superficial and deep regions, each with distinct cellular and extracellular matrix composition. Taken together, these results provide important information for the use of ACPC-laden hydrogels in regenerative medicine, and pave the way to the biofabrication of 3D constructs with multiple cell types for cartilage regeneration or in vitro tissue models. Despite its limited ability to repair, articular cartilage harbors an endogenous population of progenitor cells

Tribology approach to the engineering and study of articular cartilage.

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Wimmer, Markus A; Grad, Sibylle; Kaup, Thomas; Hänni, Markus; Schneider, Erich; Gogolewski, Sylwester; Alini, Mauro

2004-01-01

This study has been based on the assumption that articular motion is an important aspect of mechanotransduction in synovial joints. For this reason a new bioreactor concept, able to reproduce joint kinematics more closely, has been designed. The prototype consists of a rotating scaffold and/or cartilage pin, which is pressed onto an orthogonally rotating ball. By oscillating pin and ball in phase difference, elliptical displacement trajectories are generated that are similar to the motion paths occurring in vivo. Simultaneously, dynamic compression may be applied with a linear actuator, while two-step-motors generate the rotation of pin and ball. The whole apparatus is placed in an incubator. The control station is located outside. Preliminary investigations at the gene expression level demonstrated promising results. Compared with free-swelling control and/or simply compression-loaded samples, chondrocyte-seeded scaffolds as well as nasal cartilage explants exposed to interface motion both showed elevated levels of cartilage oligomeric matrix protein mRNA. The final design of the bioreactor will include four individual stations in line, which will facilitate the investigation of motion-initiated effects at the contacting surfaces in more detail.

Chondrocyte-seeded type I/III collagen membrane for autologous chondrocyte transplantation

DEFF Research Database (Denmark)

Niemeyer, Philipp; Lenz, Philipp; Kreuz, Peter C

2010-01-01

PURPOSE: We report the 2-year clinical results and identify prognostic factors in patients treated with autologous chondrocyte transplantation by use of a collagen membrane to seed the chondrocytes (ACT-CS). METHODS: This is a prospective study of 59 patients who were treated with ACT......-CS represents a technical modification of membrane-associated autologous chondrocyte transplantation that combines easy handling and attractive application properties with reliable clinical results 24 months after surgery, especially in patients with isolated cartilage defects. Even though the failure rate...

[Research of repairing rabbit knee joint cartilage defect by compound material of fibrin glue and decalcified bone matrix (DBM) and chondrocytes].

Science.gov (United States)

He, Jie; Yang, Xiang; Yue, Peng-ju; Wang, Guan-yu; Guo, Ting; Zhao, Jian-ning

2009-07-01

To investigate the feasibility and effectivity of using compound material of fibrin glue and DBM as scaffolds for cartilage tissue engineering. Chondrocytes isolated from articular cartilage were seeded into prepared scaffolds, after incubation for 4 weeks in vitro. Chondrocytes and fibrin glue and DBM constructs were implanted in the joint cave of rabbit. The specimens were excised at the 4th, 8th, 12th week, examined grossly analyzed by haematoxylin cosine, toluidine blues staining and type II collagen immunohistochemistry reaction. Wakitani score was counted to evaluate the repairing effect. Grossly analysis showed some ivory tissue filled the caves after 4 weeks and the caves were full filled with smooth surface after 12 weeks. The microscope showed a good deal of chondrocytes appeared after 8 weeks and more type II collagen than 4 weeks. Twelve weeks later, cartilage lacuna could be observed. The cells arrangement and the amount of type II collagen both showed the same as the natural one. Complicated material of fibrin glue and DBM as scaffolds can be used as scaffolds for cartilage tissue engineering.

The role of MCP-1-CCR2 ligand-receptor axis in chondrocyte degradation and disease progress in knee osteoarthritis

Directory of Open Access Journals (Sweden)

Yuan-kun Xu

Full Text Available BACKGROUND: Osteoarthritis (OA is a common arthritic disease and multifactorial whole-joint disease. Interactions of chemokines and OA is inadequately documented RESULTS: In vivo and in vitro studies were conducted to investigate monocyte chemoattractant protein 1 (MCP-1 and receptor chemokine (C-C motif receptor 2 (CCR2 in chondrocyte degradation and cartilage degeneration. Chondrocytes from 16 OA patients and 6 normal controls were involved in this study. After stimulation of MCP-1, the expression of MCP-1 and CCR2 increased significantly (P < 0.001 and the expression of MMP-13 also increased (P < 0.05. MCP-1 stimulation also induced (or enhanced the apoptosis of OA chondrocytes (P < 0.05. Additionally, the degradation of cartilage matrix markers (metalloproteinase 3 and 13, MMP3 and MMP13 in the culture medium of normal chondrocytes was also assessed. Furthermore, intra-articular injection of MCP-1 in mouse knees induced cartilage degradation and the CCR2 antagonist did not impede cartilage destroy in rats knees of monosodium iodoacetate (MIA model CONCLUSIONS: The results of this study demonstrate that the MCP-1-CCR2 ligand-receptor axis plays a special role in the initiation and progression of OA pathology. Patients with ambiguous etiology can gain some insight from the MCP-1-CCR2 ligand-receptor axis

Accumulation of advanced glycation endproducts reduces chondrocyte-mediated extracellular matrix turnover in human articular cartilage

NARCIS (Netherlands)

Degroot, J.; Verzijl, N.; Jacobs, K. M.; Budde, M.; Bank, R. A.; Bijlsma, J. W.; TeKoppele, J. M.; Lafeber, F. P.

2001-01-01

The prevalence of osteoarthritis (OAs) increases with age and coincides with the accumulation of advanced glycation endproducts (AGEs) in articular cartilage, suggesting that accumulation of glycation products may be involved in the development of OA. This study was designed to examine the effects

Spatial and temporal changes of subchondral bone proceed to articular cartilage degeneration in rats subjected to knee immobilization.

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Xu, Lei; Li, Zhe; Lei, Lei; Zhou, Yue-Zhu; Deng, Song-Yun; He, Yong-Bin; Ni, Guo-Xin

2016-03-01

This study was aimed to investigate the spatial and temporal changes of subchondral bone and its overlying articular cartilage in rats following knee immobilization. A total of 36 male Wistar rats (11-13 months old) were assigned randomly and evenly into 3 groups. For each group, knee joints in 6 rats were immobilized unilaterally for 1, 4, or 8 weeks, respectively, while the remaining rats were allowed free activity and served as external control groups. For each animal, femurs at both sides were dissected after sacrificed. The distal part of femur was examined by micro-CT. Subsequently, femoral condyles were collected for further histological observation and analysis. For articular cartilage, significant changes were observed only at 4 and 8 weeks of immobilization. The thickness of articular cartilage and chondrocytes numbers decreased with time. However, significant changes in subchondral bone were defined by micro-CT following immobilization in a time-dependent manner. Immobilization led to a thinner and more porous subchondral bone plate, as well as a reduction in trabecular thickness and separation with a more rod-like architecture. Changes in subchondral bone occurred earlier than in articular cartilage. More importantly, immobilization-induced changes in subchondral bone may contribute, at least partially, to changes in its overlying articular cartilage. © 2016 Wiley Periodicals, Inc.

Biodegradable and injectable cure-on-demand polyurethane scaffolds for regeneration of articular cartilage.

Science.gov (United States)

Werkmeister, J A; Adhikari, R; White, J F; Tebb, T A; Le, T P T; Taing, H C; Mayadunne, R; Gunatillake, P A; Danon, S J; Ramshaw, J A M

2010-09-01

This paper describes the synthesis and characterization of an injectable methacrylate functionalized urethane-based photopolymerizable prepolymer to form biodegradable hydrogels. The tetramethacrylate prepolymer was based on the reaction between two synthesized compounds, diisocyanato poly(ethylene glycol) and monohydroxy dimethacrylate poly(epsilon-caprolactone) triol. The final prepolymer was hydrated with phosphate-buffered saline (pH 7.4) to yield a biocompatible hydrogel containing up to 86% water. The methacrylate functionalized prepolymer was polymerized using blue light (450 nm) with an initiator, camphorquinone and a photosensitizer, N,N-dimethylaminoethyl methacrylate. The polymer was stable in vitro in culture media over the 28 days tested (1.9% mass loss); in the presence of lipase, around 56% mass loss occurred over the 28 days in vitro. Very little degradation occurred in vivo in rats over the same time period. The polymer was well tolerated with very little capsule formation and a moderate host tissue response. Human chondrocytes, seeded onto Cultispher-S beads, were viable in the tetramethacrylate prepolymer and remained viable during and after polymerization. Chondrocyte-bead-polymer constructs were maintained in static and spinner culture for 8 weeks. During this time, cells remained viable, proliferated and migrated from the beads through the polymer towards the edge of the polymer. New extracellular matrix (ECM) was visualized with Masson's trichrome (collagen) and Alcian blue (glycosaminoglycan) staining. Further, the composition of the ECM was typical for articular cartilage with prominent collagen type II and type VI and moderate keratin sulphate, particularly for tissue constructs cultured under dynamic conditions. 2010. Published by Elsevier Ltd. All rights reserved.

Tissue Engineering Based Therapy for Articular Cartilage Defects - A New Approach

Directory of Open Access Journals (Sweden)

Abraham S

2007-01-01

Full Text Available Background: Articular cartilage, the load-bearing tissue in diarthrodial joints, when damaged due to trauma could lead to osteoarthritis. At present Autologous Cartilage Implantation is an established method in which patients own chondrocytes are isolated and then implanted after in vitro expansion over the affected area with bovine or porcine collagen matrix. This procedure results in more of Collagen Type I during in vitro expansion, which eventually becomes fibrocartilage. Also it requires growth factors. We have in this study tried growing human Chondrocytes without growth factors using synthetic scaffolds to grow more Collagen Type II Materials and Methods: Human cartilage specimens were harvested through arthroscopy from the non-weight bearing area of the knee joint from 13 patients who underwent surgical procedures of the knee joint after getting their informed consent. The tissues were transported in saline taking 1 hour to laboratory and subjected to digestion with Collagenase type II for 16~18 Hrs. The chondrocyte cells obtained after dissociation were divided into two groups for culture. Gr. I were embedded in a Thermogelation polymer (TGP and Gr. II in basal culture media (DMEM + Ascorbic Acid without using any growth factors. The Group II cells were viable only for 4 weeks and then started degenerating. The TGP-Chondrocytes scaffolds were grown for 16 weeks and the specimens were harvested at 4, 8, 12 and 16-week intervals and their morphology and molecular characteristics were studied by H&E staining, S-100 protein analysis and RT-PCR.Results: Human chondrocytes could be cultured in both TGP (group I and Basal culture media (group II. The Gr. I cells were viable upto the 16th week while the Group II chondrocytes started degenerating after the 4 week. Both the groups were proven positive for S-100 protein, a Chondrocyte specific marker protein; Gr. II specimens after 4 weeks, and Gr. I specimens after 4, 8, 12 and 16 weeks. RT

Intra-articular enzyme replacement therapy with rhIDUA is safe, well-tolerated, and reduces articular GAG storage in the canine model of mucopolysaccharidosis type I.

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Wang, Raymond Y; Aminian, Afshin; McEntee, Michael F; Kan, Shih-Hsin; Simonaro, Calogera M; Lamanna, William C; Lawrence, Roger; Ellinwood, N Matthew; Guerra, Catalina; Le, Steven Q; Dickson, Patricia I; Esko, Jeffrey D

2014-08-01

Treatment with intravenous enzyme replacement therapy and hematopoietic stem cell transplantation for mucopolysaccharidosis (MPS) type I does not address joint disease, resulting in persistent orthopedic complications and impaired quality of life. A proof-of-concept study was conducted to determine the safety, tolerability, and efficacy of intra-articular recombinant human iduronidase (IA-rhIDUA) enzyme replacement therapy in the canine MPS I model. Four MPS I dogs underwent monthly rhIDUA injections (0.58 mg/joint) into the right elbow and knee for 6 months. Contralateral elbows and knees concurrently received normal saline. No intravenous rhIDUA therapy was administered. Monthly blood counts, chemistries, anti-rhIDUA antibody titers, and synovial fluid cell counts were measured. Lysosomal storage of synoviocytes and chondrocytes, synovial macrophages and plasma cells were scored at baseline and 1 month following the final injection. All injections were well-tolerated without adverse reactions. One animal required prednisone for spinal cord compression. There were no clinically significant abnormalities in blood counts or chemistries. Circulating anti-rhIDUA antibody titers gradually increased in all dogs except the prednisone-treated dog; plasma cells, which were absent in all baseline synovial specimens, were predominantly found in synovium of rhIDUA-treated joints at study-end. Lysosomal storage in synoviocytes and chondrocytes following 6 months of IA-rhIDUA demonstrated significant reduction compared to tissues at baseline, and saline-treated tissues at study-end. Mean joint synovial GAG levels in IA-rhIDUA joints were 8.62 ± 5.86 μg/mg dry weight and 21.6 ± 10.4 μg/mg dry weight in control joints (60% reduction). Cartilage heparan sulfate was also reduced in the IA-rhIDUA joints (113 ± 39.5 ng/g wet weight) compared to saline-treated joints (142 ± 56.4 ng/g wet weight). Synovial macrophage infiltration, which was present in all joints at baseline, was

Total glucosides of paeony prevents juxta-articular bone loss in experimental arthritis.

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Wei, Chen Chao; You, Fan Tian; Mei, Li Yu; Jian, Sun; Qiang, Chen Yong

2013-07-21

Total glucosides of paeony (TGP) is a biologically active compound extracted from Paeony root. TGP has been used in rheumatoid arthritis therapy for many years. However, the mechanism by which TGP prevents bone loss has been less explored. TGP was orally administered for 3 months to New Zealand rabbits with antigen-induced arthritis (AIA). Digital x-ray knee images and bone mineral density (BMD) measurements of the subchondral knee bone were performed before sacrifice. Chondrocytes were observed using transmission electron microscopy (TEM). Histological analysis and mRNA expression of receptor activator of nuclear factor-B ligand (RANKL) and osteoprotegerin (OPG) were evaluated in joint tissues. The BMD value in TGP rabbits was significantly higher compared with that seen in the AIA model rabbits. In addition, the subchondral bone plate was almost completely preserved by TGP treatment, while there was a decrease in bone plate integrity in AIA rabbits. There was less damage to the chondrocytes of the TGP treated group. Immunohistochemical examination of the TGP group showed that a higher percentage of TGP treated chondrocytes expressed OPG as compared to the chondrocytes isolated from AIA treated animals. In contrast, RANKL expression was significantly decreased in the TGP treated group compared to the AIA group. In support of the immunohistochemistry data, the expression of RANKL mRNA was decreased and OPG mRNA expression was enhanced in the TGP group when compared to that of the AIA model group. These results reveal that TGP suppresses juxta-articular osteoporosis and prevents subchondral bone loss. The decreased RANKL and increased OPG expression seen in TGP treated animals could explain how administration of TGP maintains higher BMD.

Vitamin D prevents articular cartilage erosion by regulating collagen II turnover through TGF-β1 in ovariectomized rats.

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Li, S; Niu, G; Wu, Y; Du, G; Huang, C; Yin, X; Liu, Z; Song, C; Leng, H

2016-02-01

To explore the effect of vitamin D on turnover of articular cartilage with ovariectomy (OVX) induced OA, and to investigate transforming growth factor-β1 (TGF-β1) as a possible underlying mechanism mediated by 1α,25(OH)2D3. Sixty-six rats were randomly allocated into seven groups: sham plus control diet (SHAM+CTL), OVX+CTL diet, sham plus vitamin D-deficient (VDD) diet, OVX+VDD diet, and three groups of ovariectomized rats treated with different doses of 1α,25(OH)2D3. The cartilage erosion and the levels of serum 17β-estradiol, 1α,25(OH)2D3 and C-telopeptide of type II collagen (CTX-II) were measured. TGF-β1, type II Collagen (CII), matrix metalloproteinases (MMP)-9,-13 in articular cartilage were assessed by immunohistochemistry. TGF-β1 and CTX-II expression were measured in articular cartilage chondrocytes treated with/without tumor necrosis factor (TNF-α), 1α,25(OH)2D3, and TGF-β receptor inhibitor (SB505124) in vitro. Cartilage erosion due to OVX was significantly reduced in a dose-dependent manner by 1α,25(OH)2D3 supplementation, and exacerbated by VDD. The expressions of TGF-β1 and CII in articular cartilage were suppressed by OVX and VDD, and rescued by 1α,25(OH)2D3 supplementation. The expression of MMP-9,-13 in articular cartilage increased with OVX and VDD, and decreased with 1α,25(OH)2D3 supplementation. In vitro experiments showed that 1α,25(OH)2D3 increased the TGF-β1 expression of TNF-α stimulated chondrocytes in a dose-dependent manner. 1α,25(OH)2D3 significantly counteracted the increased CTX-II release due to TNF-α stimulation, and this effect was significantly suppressed by SB505124. VDD aggravated cartilage erosion, and 1α,25(OH)2D3 supplementation showed protective effects in OVX-induced OA partly through the TGF-β1 pathway. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Transcription factor ERG and joint and articular cartilage formation during mouse limb and spine skeletogenesis.

Science.gov (United States)

Iwamoto, Masahiro; Tamamura, Yoshihiro; Koyama, Eiki; Komori, Toshihisa; Takeshita, Nobuo; Williams, Julie A; Nakamura, Takashi; Enomoto-Iwamoto, Motomi; Pacifici, Maurizio

2007-05-01

Articular cartilage and synovial joints are critical for skeletal function, but the mechanisms regulating their development are largely unknown. In previous studies we found that the ets transcription factor ERG and its alternatively-spliced variant C-1-1 have roles in joint formation in chick. Here, we extended our studies to mouse. We found that ERG is also expressed in developing mouse limb joints. To test regulation of ERG expression, beads coated with the joint master regulator protein GDF-5 were implanted close to incipient joints in mouse limb explants; this led to rapid and strong ectopic ERG expression. We cloned and characterized several mammalian ERG variants and expressed a human C-1-1 counterpart (hERG3Delta81) throughout the cartilaginous skeleton of transgenic mice, using Col2a1 gene promoter/enhancer sequences. The skeletal phenotype was severe and neonatal lethal, and the transgenic mice were smaller than wild type littermates and their skeletons were largely cartilaginous. Limb long bone anlagen were entirely composed of chondrocytes actively expressing collagen IX and aggrecan as well as articular markers such as tenascin-C. Typical growth plates were absent and there was very low expression of maturation and hypertrophy markers, including Indian hedgehog, collagen X and MMP-13. The results suggest that ERG is part of molecular mechanisms leading chondrocytes into a permanent developmental path and become joint forming cells, and may do so by acting downstream of GDF-5.

Return to sports participation after articular cartilage repair in the knee: scientific evidence.

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Mithoefer, Kai; Hambly, Karen; Della Villa, Stefano; Silvers, Holly; Mandelbaum, Bert R

2009-11-01

Articular cartilage injury in the athlete's knee presents a difficult clinical challenge. Despite the importance of returning injured athletes to sports, information is limited on whether full sports participation can be successfully achieved after articular cartilage repair in the knee. Systematic analysis of athletic participation after articular cartilage repair will demonstrate the efficacy of joint surface restoration in high-demand patients and help to optimize outcomes in athletes with articular cartilage injury of the knee. Systematic review. A comprehensive literature review of original studies was performed to provide information about athletic participation after articular cartilage repair. The athlete's ability to perform sports postoperatively was assessed by activity outcome scores, rate of return to sport, timing of the return, level of postoperative sports participation, and the continuation of athletic activity over time. Twenty studies describing 1363 patients were included in the review, with an average follow-up of 42 months. Return to sports was possible in 73% overall, with highest return rates after osteochondral autograft transplantation. Time to return to sports varied between 7 and 18 months, depending on the cartilage repair technique. Initial return to sports at the preinjury level was possible in 68% and did not significantly vary between surgical techniques. Continued sports participation at the preinjury level was possible in 65%, with the best durability after autologous chondrocyte transplantation. Several factors affected the ability to return to sport: athlete's age, preoperative duration of symptoms, level of play, lesion size, and repair tissue morphology. Articular cartilage repair in the athletic population allows for a high rate of return to sports, often at the preinjury level. Return to sports participation is influenced by several independent factors. The findings provide pertinent information that is helpful for the

In-vitro chondrogenic potential of synovial stem cells and chondrocytes allocated for autologous chondrocyte implantation

DEFF Research Database (Denmark)

Kubosch, Eva Johanna; Heidt, Emanuel; Niemeyer, Philipp

2017-01-01

Purpose: The use of passaged chondrocytes is the current standard for autologous chondrocyte implantation (ACI). De-differentiation due to amplification and donor site morbidity are known drawbacks highlighting the need for alternative cell sources. Methods: Via clinically validated flow cytometry...... analysis, we compared the expression of human stem cell and cartilage markers (collagen type 2 (Col2), aggrecan (ACAN), CD44) of chondrocytes (CHDR), passaged chondrocytes for ACI (CellGenix™), bone marrow derived mesenchymal stem cells (BMSC), and synovial derived stem cells (SDSC). Results: Primary...

The epigenetic effect of glucosamine and a nuclear factor-kappa B (NF-kB) inhibitor on primary human chondrocytes - Implications for osteoarthritis

Energy Technology Data Exchange (ETDEWEB)

Imagawa, Kei, E-mail: k.Imagawa@soton.ac.uk [University of Southampton Medical School, Bone and Joint Research Group, Southampton (United Kingdom); Tohoku University School of Medicine, Sendai (Japan); Andres, MC de [University of Southampton Medical School, Bone and Joint Research Group, Southampton (United Kingdom); Hashimoto, Ko [Hospital for Special Surgery, NY (United States); Pitt, Dominic [University of Southampton Medical School, Bone and Joint Research Group, Southampton (United Kingdom); Itoi, Eiji [Tohoku University School of Medicine, Sendai (Japan); Goldring, Mary B. [Hospital for Special Surgery, NY (United States); Roach, Helmtrud I.; Oreffo, Richard O.C. [University of Southampton Medical School, Bone and Joint Research Group, Southampton (United Kingdom)

2011-02-18

Research highlights: {yields} Glucosamine and a NF-kB inhibitor reduce inflammation in OA. {yields} Cytokine induced demethylation of CpG site in IL1{beta} promoter prevented by glucosamine. {yields} Glucosamine and NF-kB inhibitor have epigenetic effects on human chondrocytes. -- Abstract: Objective: Idiopathic osteoarthritis is the most common form of osteoarthritis (OA) world-wide and remains the leading cause of disability and the associated socio-economic burden in an increasing aging population. Traditionally, OA has been viewed as a degenerative joint disease characterized by progressive destruction of the articular cartilage and changes in the subchondral bone culminating in joint failure. However, the etiology of OA is multifactorial involving genetic, mechanical and environmental factors. Treatment modalities include analgesia, joint injection with steroids or hyaluronic acid, oral supplements including glucosamine and chondroitin sulfate, as well as physiotherapy. Thus, there is significant interest in the discovery of disease modifying agents. One such agent, glucosamine (GlcN) is commonly prescribed even though the therapeutic efficacy and mechanism of action remain controversial. Inflammatory cytokines, including IL-1{beta}, and proteinases such as MMP-13 have been implicated in the pathogenesis and progression of OA together with an associated CpG demethylation in their promoters. We have investigated the potential of GlcN to modulate NF-kB activity and cytokine-induced abnormal gene expression in articular chondrocytes and, critically, whether this is associated with an epigenetic process. Method: Human chondrocytes were isolated from the articular cartilage of femoral heads, obtained with ethical permission, following fractured neck of femur surgery. Chondrocytes were cultured for 5 weeks in six separate groups; (i) control culture, (ii) cultured with a mixture of 2.5 ng/ml IL-1{beta} and 2.5 ng/ml oncostatin M (OSM), (iii) cultured with 2 mM N

The epigenetic effect of glucosamine and a nuclear factor-kappa B (NF-kB) inhibitor on primary human chondrocytes - Implications for osteoarthritis

International Nuclear Information System (INIS)

Imagawa, Kei; Andres, MC de; Hashimoto, Ko; Pitt, Dominic; Itoi, Eiji; Goldring, Mary B.; Roach, Helmtrud I.; Oreffo, Richard O.C.

2011-01-01

Research highlights: → Glucosamine and a NF-kB inhibitor reduce inflammation in OA. → Cytokine induced demethylation of CpG site in IL1β promoter prevented by glucosamine. → Glucosamine and NF-kB inhibitor have epigenetic effects on human chondrocytes. -- Abstract: Objective: Idiopathic osteoarthritis is the most common form of osteoarthritis (OA) world-wide and remains the leading cause of disability and the associated socio-economic burden in an increasing aging population. Traditionally, OA has been viewed as a degenerative joint disease characterized by progressive destruction of the articular cartilage and changes in the subchondral bone culminating in joint failure. However, the etiology of OA is multifactorial involving genetic, mechanical and environmental factors. Treatment modalities include analgesia, joint injection with steroids or hyaluronic acid, oral supplements including glucosamine and chondroitin sulfate, as well as physiotherapy. Thus, there is significant interest in the discovery of disease modifying agents. One such agent, glucosamine (GlcN) is commonly prescribed even though the therapeutic efficacy and mechanism of action remain controversial. Inflammatory cytokines, including IL-1β, and proteinases such as MMP-13 have been implicated in the pathogenesis and progression of OA together with an associated CpG demethylation in their promoters. We have investigated the potential of GlcN to modulate NF-kB activity and cytokine-induced abnormal gene expression in articular chondrocytes and, critically, whether this is associated with an epigenetic process. Method: Human chondrocytes were isolated from the articular cartilage of femoral heads, obtained with ethical permission, following fractured neck of femur surgery. Chondrocytes were cultured for 5 weeks in six separate groups; (i) control culture, (ii) cultured with a mixture of 2.5 ng/ml IL-1β and 2.5 ng/ml oncostatin M (OSM), (iii) cultured with 2 mM N-acetyl GlcN (Sigma

Hypoxia Potentiates Anabolic Effects of Exogenous Hyaluronic Acid in Rat Articular Cartilage.

Science.gov (United States)

Ichimaru, Shohei; Nakagawa, Shuji; Arai, Yuji; Kishida, Tsunao; Shin-Ya, Masaharu; Honjo, Kuniaki; Tsuchida, Shinji; Inoue, Hiroaki; Fujiwara, Hiroyoshi; Shimomura, Seiji; Mazda, Osam; Kubo, Toshikazu

2016-06-25

Hyaluronic acid (HA) is used clinically to treat osteoarthritis (OA), but its pharmacological effects under hypoxic conditions remain unclear. Articular chondrocytes in patients with OA are exposed to a hypoxic environment. This study investigated whether hypoxia could potentiate the anabolic effects of exogenous HA in rat articular cartilage and whether these mechanisms involved HA receptors. HA under hypoxic conditions significantly enhanced the expression of extracellular matrix genes and proteins in explant culture, as shown by real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and dimethylmethylene blue (DMMB) assays. Staining with Safranin-O and immunohistochemical staining with antibody to type II collagen were also enhanced in pellet culture. The expression of CD44 was increased by hypoxia and significantly suppressed by transfection with siRNAs targeting hypoxia-inducible factor 1 alpha (siHIF-1α). These findings indicate that hypoxia potentiates the anabolic effects of exogenous HA by a mechanism in which HIF-1α positively regulates the expression of CD44, enhancing the binding affinity for exogenous HA. The anabolic effects of exogenous HA may increase as OA progresses.

Up-regulated expression of cartilage intermediate-layer protein and ANK in articular hyaline cartilage from patients with calcium pyrophosphate dihydrate crystal deposition disease.

Science.gov (United States)

Hirose, Jun; Ryan, Lawrence M; Masuda, Ikuko

2002-12-01

Excess accumulation of extracellular inorganic pyrophosphate (ePPi) in aged human cartilage is crucial in calcium pyrophosphate dihydrate (CPPD) crystal formation in cartilage matrix. Two sources of ePPi are ePPi-generating ectoenzymes (NTPPPH) and extracellular transport of intracellular PPi by ANK. This study was undertaken to evaluate the role of NTPPPH and ANK in ePPi elaboration, by investigating expression of NTPPPH enzymes (cartilage intermediate-layer protein [CILP] and plasma cell membrane glycoprotein 1 [PC-1]) and ANK in human chondrocytes from osteoarthritic (OA) articular cartilage containing CPPD crystals and without crystals. Chondrocytes were harvested from knee cartilage at the time of arthroplasty (OA with CPPD crystals [CPPD], n = 8; OA without crystals [OA], n = 10). Normal adult human chondrocytes (n = 1) were used as a control. Chondrocytes were cultured with transforming growth factor beta1 (TGFbeta1), which stimulates ePPi elaboration, and/or insulin-like growth factor 1 (IGF-1), which inhibits ePPi elaboration. NTPPPH and ePPi were measured in the media at 48 hours. Media CILP, PC-1, and ANK were determined by dot-immunoblot analysis. Chondrocyte messenger RNA (mRNA) was extracted for reverse transcriptase-polymerase chain reaction to study expression of mRNA for CILP, PC-1, and ANK. NTPPPH and ANK mRNA and protein were also studied in fresh frozen cartilage. Basal ePPi elaboration and NTPPPH activity in conditioned media from CPPD chondrocytes were elevated compared with normal chondrocytes, and tended to be higher compared with OA chondrocytes. Basal expression of mRNA for CILP (chondrocytes) and ANK (cartilage) was higher in both CPPD chondrocytes and CPPD cartilage extract than in OA or normal samples. PC-1 mRNA was less abundant in CPPD chondrocytes and cartilage extract than in OA chondrocytes and extract, although the difference was not significant. CILP, PC-1, and ANK protein levels were similar in CPPD, OA, and normal chondrocytes

H2O2 INDUCES APOPTOSIS OF RABBIT CHONDROCYTES VIA BOTH THE EXTRINSIC AND THE CASPASE-INDEPENDENT INTRINSIC PATHWAYS

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CAIPING ZHUANG

2013-07-01

Full Text Available Osteoarthritis (OA, one of the most common joint diseases with unknown etiology, is characterized by the progressive destruction of articular cartilage and the apoptosis of chondrocytes. The purpose of this study is to elucidate the molecular mechanisms of H2O2-mediated rabbit chondrocytes apoptosis. CCK-8 assay showed that H2O2 treatment induced a remarkable reduction of cell viability, which was further verified by the remarkable phosphatidylserine externalization after H2O2 treatment for 1 h, the typical characteristics of apoptosis. H2O2 treatment induced a significant dysfunction of mitochondrial membrane potential (ΔΨm, but did not induce casapse-9 activation, indicating that H2O2 treatment induced caspase-independent intrinsic apoptosis that was further verified by the fact that silencing of AIF but not inhibiting caspase-9 potently prevented H2O2-induced apoptosis. H2O2 treatment induced a significant increase of caspase-8 and -3 activation, and inhibition of caspase-8 or -3 significantly prevented H2O2-induced apoptosis, suggesting that the extrinsic pathway played an important role. Collectively, our findings demonstrate that H2O2 induces apoptosis via both the casapse-8-mediated extrinsic and the caspase-independent intrinsic apoptosis pathways in rabbit chondrocytes.

THE FUNCTIONAL EFFECTIVENESS OF A CELL-ENGINEERED CONSTRUCT FOR THE REGENERATION OF ARTICULAR CARTILAGE

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V. I. Sevastianov

2015-01-01

Full Text Available The aim of this study is an analysis of the functional effectiveness of a biomedical cell product consisting of a biopolymer microheterogeneous collagen-containing hydrogel (BMCH, human adipose-derived mesenchymal stromal cells (hADMSCs, and chondrogenic induction medium in the regeneration of articular cartilage. Materials and methods. The test model of the adjuvant arthritis was used (female Soviet Chinchilla rabbits with the further development into osteoarthrosis (OA combined with the clinical, biochemical, radiological, and histochemical trials. Results. On Day 92 of the OA model it has been found that the intra-articular introduction of a BMCH with hADMSCs into the left knee joint (n = 3 30 days after the OA modeling, as opposed to the right joint (negative control, n = 3, stimulates the regenerative processes of the cartilaginous tissue structure characterized by the formation of chondrocyte «columns», the emergence of isogenic groups in the intracellular matrix and the regeneration of its structure. Upon the intra-articular introduction of a BMCH (n = 3 such effects are markedly less pronounced. Conclusions. A significant regenerative potential of a cell-engineered construct of human articular tissue (CEC ATh has been proven. It is possible to presume that biostimulating properties of CEC ATh are due to the activating effect of a biomedical cell product on the stem cell migration processes from the surrounding tissue into the injured area with their subsequent differentiation. 

miR-139 is up-regulated in osteoarthritis and inhibits chondrocyte proliferation and migration possibly via suppressing EIF4G2 and IGF1R

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Hu, Weihua; Zhang, Weikai; Li, Feng; Guo, Fengjing; Chen, Anmin, E-mail: chenanmin6072@126.com

2016-05-27

Osteoarthritis (OA) is one of the most progressive articular cartilage erosions. microRNAs (miRNAs) play pivotal roles in OA modulation, but the role of miR-139 in OA remains elusive. This study aims to reveal the effects and possible mechanism of miR-139 in OA and chondrocytes. The levels of miR-139 and its possible targets eukaryotic translation initiation factor 4 gamma 2 (EIF4G2) and insulin-like growth factor 1 receptor (IGF1R) were detected by qRT-PCR in the articular cartilages of 20 OA patients and 20 non-OA patients. Human chondrocyte CHON-001 cells were transfected with miR-139 mimic or inhibitor, as well as the siRNAs of EIF4G2 and IGF1R. Cell viability by MTT assay, proliferation by colony formation assay and migration by Transwell assay were performed. Results showed that miR-139 was up-regulated, while EIF4G2 and IGF1R mRNAs down-regulated in OA cartilages (P < 0.001), and negative correlations existed between the level of miR-139 and EIF4G2 or IGF1R. Overexpression of miR-139 in CHON-001 cells suppressed both mRNA and protein levels of EIF4G2 and IGF1R, and inhibited cell viability, colony formation number and cell migration, while miR-139 inhibitor induced the opposite effects. Knockdown of EIF4G2 or IGF1R in CHON-001 cells reversed the effects of miR-139 inhibitor on cell viability, colony formation and cell migration. These results indicate that miR-139 is capable of inhibiting chondrocyte proliferation and migration, thus being a possible therapeutic target for OA. The mechanism of miR-139 in chondrocytes may be related to its regulation on EIF4G2 and IGF1R.

Biochanin-A antagonizes the interleukin-1β-induced catabolic inflammation through the modulation of NFκB cellular signaling in primary rat chondrocytes

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Oh, Ji-Su [Department of Oral and Maxillofacial Surgery, Chosun University, Gwangju, 61452 (Korea, Republic of); Cho, In-A; Kang, Kyeong-Rok [Department of Dental Bioengineering, Chosun University, Gwangju, 61452 (Korea, Republic of); You, Jae-Seek [Department of Oral and Maxillofacial Surgery, Chosun University, Gwangju, 61452 (Korea, Republic of); Yu, Sang-Joun [Department of Periodontology, Chosun University, Gwangju, 61452 (Korea, Republic of); Lee, Gyeong-Je [Department of Prosthodontics, Chosun University, Gwangju, 61452 (Korea, Republic of); Seo, Yo-Seob [Department of Oral and Maxillofacial Radiology, Chosun University, Gwangju, 61452 (Korea, Republic of); Kim, Chun Sung; Kim, Do Kyung [Pre-Dentistry, School of Dentistry, Chosun University, Gwangju, 61452 (Korea, Republic of); Kim, Su-Gwan [Department of Oral and Maxillofacial Surgery, Chosun University, Gwangju, 61452 (Korea, Republic of); Seo, Young-Woo [Korea Basic Science Institute, Gwangju Center, Chonnam National University, Gwangju, 61186 (Korea, Republic of); Im, Hee-Jeong [Department of Biochemistry, Rush University Medical Center, Chicago, IL, 60612 (United States); Kim, Jae-Sung, E-mail: js_kim@chosun.ac.kr [Pre-Dentistry, School of Dentistry, Chosun University, Gwangju, 61452 (Korea, Republic of)

2016-09-02

Biochanin-A, a phytoestrogen derived from herbal plants, protected from the IL-1β-induced loss of proteoglycans through the suppression of matrix degrading enzymes such as matrix metalloproteinase (MMP)-13, MMP-3, MMP-1, and ADAMTS-5 in primary rat chondrocytes and the knee articular cartilage. It also suppressed the expression of IL-1β-induced catabolic factors such as nitric oxide synthase 2, cyclooxygenase-2, prostaglandin E{sub 2}, and inflammatory cytokines. Furthermore, biochanin-A suppressed the IL-1β-induced phosphorylation of NFκB, and inhibited its nuclear translocation in primary rat chondrocytes. These results indicate that biochanin-A antagonizes the IL-1β-induced catabolic effects through its anti-inflammatory activity that involves the modulation of NFκB signaling. - Highlights: • Biochanin-A is a phytoestrogen derived from medicinal plants. • It suppressed the IL-1β-induced matrix degrading enzymes and catabolic factors. • It inhibited IL-1β-induced proteoglycan loss in chondrocytes and cartilage tissues. • Its anti-catabolic effects were mediated by modulation of NFκB signaling. • It may be used as a potential anti-catabolic biomaterial for osteoarthritis.

Stromal cell-derived factor 1 regulates the actin organization of chondrocytes and chondrocyte hypertrophy.

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Murata, Koichi; Kitaori, Toshiyuki; Oishi, Shinya; Watanabe, Naoki; Yoshitomi, Hiroyuki; Tanida, Shimei; Ishikawa, Masahiro; Kasahara, Takashi; Shibuya, Hideyuki; Fujii, Nobutaka; Nagasawa, Takashi; Nakamura, Takashi; Ito, Hiromu

2012-01-01

Stromal cell-derived factor 1 (SDF-1/CXCL12/PBSF) plays important roles in the biological and physiological functions of haematopoietic and mesenchymal stem cells. This chemokine regulates the formation of multiple organ systems during embryogenesis. However, its roles in skeletal development remain unclear. Here we investigated the roles of SDF-1 in chondrocyte differentiation. We demonstrated that SDF-1 protein was expressed at pre-hypertrophic and hypertrophic chondrocytes in the newly formed endochondral callus of rib fracture as well as in the growth plate of normal mouse tibia by immunohistochemical analysis. Using SDF-1(-/-) mouse embryo, we histologically showed that the total length of the whole humeri of SDF-1(-/-) mice was significantly shorter than that of wild-type mice, which was contributed mainly by shorter hypertrophic and calcified zones in SDF-1(-/-) mice. Actin cytoskeleton of hypertrophic chondrocytes in SDF-1(-/-) mouse humeri showed less F-actin and rounder shape than that of wild-type mice. Primary chondrocytes from SDF-1(-/-) mice showed the enhanced formation of philopodia and loss of F-actin. The administration of SDF-1 to primary chondrocytes of wild-type mice and SDF-1(-/-) mice promoted the formation of actin stress fibers. Organ culture of embryonic metatarsals from SDF-1(-/-) mice showed the growth delay, which was recovered by an exogenous administration of SDF-1. mRNA expression of type X collagen in metatarsals and in primary chondrocytes of SDF-1(-/-) mouse embryo was down-regulated while the administration of SDF-1 to metatarsals recovered. These data suggests that SDF-1 regulates the actin organization and stimulates bone growth by mediating chondrocyte hypertrophy.

Stromal cell-derived factor 1 regulates the actin organization of chondrocytes and chondrocyte hypertrophy.

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Koichi Murata

Full Text Available Stromal cell-derived factor 1 (SDF-1/CXCL12/PBSF plays important roles in the biological and physiological functions of haematopoietic and mesenchymal stem cells. This chemokine regulates the formation of multiple organ systems during embryogenesis. However, its roles in skeletal development remain unclear. Here we investigated the roles of SDF-1 in chondrocyte differentiation. We demonstrated that SDF-1 protein was expressed at pre-hypertrophic and hypertrophic chondrocytes in the newly formed endochondral callus of rib fracture as well as in the growth plate of normal mouse tibia by immunohistochemical analysis. Using SDF-1(-/- mouse embryo, we histologically showed that the total length of the whole humeri of SDF-1(-/- mice was significantly shorter than that of wild-type mice, which was contributed mainly by shorter hypertrophic and calcified zones in SDF-1(-/- mice. Actin cytoskeleton of hypertrophic chondrocytes in SDF-1(-/- mouse humeri showed less F-actin and rounder shape than that of wild-type mice. Primary chondrocytes from SDF-1(-/- mice showed the enhanced formation of philopodia and loss of F-actin. The administration of SDF-1 to primary chondrocytes of wild-type mice and SDF-1(-/- mice promoted the formation of actin stress fibers. Organ culture of embryonic metatarsals from SDF-1(-/- mice showed the growth delay, which was recovered by an exogenous administration of SDF-1. mRNA expression of type X collagen in metatarsals and in primary chondrocytes of SDF-1(-/- mouse embryo was down-regulated while the administration of SDF-1 to metatarsals recovered. These data suggests that SDF-1 regulates the actin organization and stimulates bone growth by mediating chondrocyte hypertrophy.

The NAD-Dependent Deacetylase Sirtuin-1 Regulates the Expression of Osteogenic Transcriptional Activator Runt-Related Transcription Factor 2 (Runx2 and Production of Matrix Metalloproteinase (MMP-13 in Chondrocytes in Osteoarthritis

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Koh Terauchi

2016-06-01

Full Text Available Aging is one of the major pathologic factors associated with osteoarthritis (OA. Recently, numerous reports have demonstrated the impact of sirtuin-1 (Sirt1, which is the NAD-dependent deacetylase, on human aging. It has been demonstrated that Sirt1 induces osteogenic and chondrogenic differentiation of mesenchymal stem cells. However, the role of Sirt1 in the OA chondrocytes still remains unknown. We postulated that Sirt1 regulates a hypertrophic chondrocyte lineage and degeneration of articular cartilage through the activation of osteogenic transcriptional activator Runx2 and matrix metalloproteinase (MMP-13 in OA chondrocytes. To verify whether sirtuin-1 (Sirt1 regulates chondrocyte activity in OA, we studied expressions of Sirt1, Runx2 and production of MMP-13, and their associations in human OA chondrocytes. The expression of Sirt1 was ubiquitously observed in osteoarthritic chondrocytes; in contrast, Runx2 expressed in the osteophyte region in patients with OA and OA model mice. OA relating catabolic factor IL-1βincreased the expression of Runx2 in OA chondrocytes. OA chondrocytes, which were pretreated with Sirt1 inhibitor, inhibited the IL-1β-induced expression of Runx2 compared to the control. Since the Runx2 is a promotor of MMP-13 expression, Sirt1 inactivation may inhibit the Runx2 expression and the resultant down-regulation of MMP-13 production in chondrocytes. Our findings suggest thatSirt1 may regulate the expression of Runx2, which is the osteogenic transcription factor, and the production of MMP-13 from chondrocytes in OA. Since Sirt1 activity is known to be affected by several stresses, including inflammation and oxidative stress, as well as aging, SIRT may be involved in the development of OA.

Berberine prevents nitric oxide-induced rat chondrocyte apoptosis and cartilage degeneration in a rat osteoarthritis model via AMPK and p38 MAPK signaling.

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Zhou, Yan; Liu, Shi-Qing; Yu, Ling; He, Bin; Wu, Shi-Hao; Zhao, Qi; Xia, Shao-Qiang; Mei, Hong-Jun

2015-09-01

Chondrocyte apoptosis is an important mechanism involved in osteoarthritis (OA). Berberine (BBR), a plant alkaloid derived from Chinese medicine, is characterized by multiple pharmacological effects, such as anti-inflammatory and anti-apoptotic activities. This study aimed to evaluate the chondroprotective effect and underlying mechanisms of BBR on sodium nitroprusside (SNP)-stimulated chondrocyte apoptosis and surgically-induced rat OA model. The in vitro results revealed that BBR suppressed SNP-stimulated chondrocyte apoptosis as well as cytoskeletal remodeling, down-regulated expressions of inducible nitric oxide synthase (iNOS) and caspase-3, and up-regulated Bcl-2/Bax ratio and Type II collagen (Col II) at protein levels, which were accompanied by increased adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and decreased phosphorylation of p38 mitogen-activated protein kinase (MAPK). Furthermore, the anti-apoptotic effect of BBR was blocked by AMPK inhibitor Compound C (CC) and adenosine-9-β-D-arabino-furanoside (Ara A), and enhanced by p38 MAPK inhibitor SB203580. In vivo experiment suggested that BBR ameliorated cartilage degeneration and exhibited an anti-apoptotic effect on articular cartilage in a rat OA model, as demonstrated by histological analyses, TUNEL assay and immunohistochemical analyses of caspase-3, Bcl-2 and Bax expressions. These findings suggest that BBR suppresses SNP-stimulated chondrocyte apoptosis and ameliorates cartilage degeneration via activating AMPK signaling and suppressing p38 MAPK activity.

Comparison of three types of chondrocytes in collagen scaffolds for cartilage tissue engineering

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Zhang Lu; Spector, Myron

2009-01-01

The objective of this study was to compare the chondrogenesis in type I and II collagen scaffolds seeded with chondrocytes from three types of cartilage, after four weeks of culture: auricular (AU), articular (AR) and meniscal (ME). Related aims were to investigate the expression of a contractile muscle actin isoform, α-smooth muscle actin (SMA), in the cells in the scaffold and to determine the presence of a lubricating glycoprotein, lubricin, in the constructs. Adult goat AU, AR and ME chondrocytes were seeded into two types of collagen scaffolds: type II collagen and type I/III collagen. After four weeks of culture, the constructs were prepared for histochemical and immunohistochemical analysis of the distribution of glycosaminoglycan (GAG), types I and II collagen, elastin, SM and lubricin. AU constructs contained substantially more tissue than the AR and ME samples. The AU constructs exhibited neocartilage, but no elastin. There were no notable differences between the type I and II collagen scaffolds. Novel findings were the expression of SMA by the AU cells in the scaffolds and the presence of lubricin in the AR and AU constructs. AU cells have the capability to produce cartilage in collagen scaffolds under conditions in which there is little histogenesis by AR and ME cells.

Comparison of three types of chondrocytes in collagen scaffolds for cartilage tissue engineering

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Zhang Lu [Department of Plastic and Reconstructive Surgery, Shanghai Tissue Engineering Center, Shanghai 9th People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Spector, Myron, E-mail: luzhangmd@gmail.co [Tissue Engineering, VA Boston Healthcare System, Boston, MA (United States)

2009-08-15

The objective of this study was to compare the chondrogenesis in type I and II collagen scaffolds seeded with chondrocytes from three types of cartilage, after four weeks of culture: auricular (AU), articular (AR) and meniscal (ME). Related aims were to investigate the expression of a contractile muscle actin isoform, alpha-smooth muscle actin (SMA), in the cells in the scaffold and to determine the presence of a lubricating glycoprotein, lubricin, in the constructs. Adult goat AU, AR and ME chondrocytes were seeded into two types of collagen scaffolds: type II collagen and type I/III collagen. After four weeks of culture, the constructs were prepared for histochemical and immunohistochemical analysis of the distribution of glycosaminoglycan (GAG), types I and II collagen, elastin, SM and lubricin. AU constructs contained substantially more tissue than the AR and ME samples. The AU constructs exhibited neocartilage, but no elastin. There were no notable differences between the type I and II collagen scaffolds. Novel findings were the expression of SMA by the AU cells in the scaffolds and the presence of lubricin in the AR and AU constructs. AU cells have the capability to produce cartilage in collagen scaffolds under conditions in which there is little histogenesis by AR and ME cells.

Chondroprotective Effects of Ginsenoside Rg1 in  Human Osteoarthritis Chondrocytes and a Rat Model  of Anterior Cruciate Ligament Transection

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Wendan Cheng

2017-03-01

Full Text Available This study aimed to assess whether Ginsenoside Rg1 (Rg1 inhibits inflammatory responses in human chondrocytes and reduces articular cartilage damage in a rat model of osteoarthritis (OA. Gene expression and protein levels of type II collagen, aggrecan, matrix metalloproteinase (MMP‐13 and cyclooxygenase‐2 (COX‐2 were determined in vitro by quantitative real‐time‐polymerase chain reaction and Western blotting. Prostaglandin E2 (PGE2 amounts in the culture medium were determined by enzyme‐linked immunosorbent assay (ELISA. For in vivo assessment, a rat model of OA was generated by anterior cruciate ligament transection (ACLT. Four weeks after ACLT, Rg1 (30 or 60 mg/kg or saline was administered by gavage once a day for eight consecutive weeks. Joint damage was analyzed by histology and immunohistochemistry. Ginsenoside Rg1 inhibited Interleukin (IL‐1β‐induced chondrocyte gene and protein expressions of MMP‐13, COX‐2 and PGE2, and prevented type II collagen and aggrecan degradation, in a dose‐dependent manner. Administration of Ginsenoside Rg1 to OA rats attenuated cartilage degeneration, and reduced type II collagen loss and MMP‐13 levels. These findings demonstrated that Ginsenoside Rg1 can inhibit inflammatory responses in human chondrocytes in vitro and reduce articular cartilage damage in vivo, confirming the potential therapeutic value of Ginsenoside Rg1 in OA.

Effect of Dynamic Culture and Periodic Compression on Human Mesenchymal Stem Cell Proliferation and Chondrogenesis.

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Guo, Ting; Yu, Li; Lim, Casey G; Goodley, Addison S; Xiao, Xuan; Placone, Jesse K; Ferlin, Kimberly M; Nguyen, Bao-Ngoc B; Hsieh, Adam H; Fisher, John P

2016-07-01

We have recently developed a bioreactor that can apply both shear and compressive forces to engineered tissues in dynamic culture. In our system, alginate hydrogel beads with encapsulated human mesenchymal stem cells (hMSCs) were cultured under different dynamic conditions while subjected to periodic, compressive force. A customized pressure sensor was developed to track the pressure fluctuations when shear forces and compressive forces were applied. Compared to static culture, dynamic culture can maintain a higher cell population throughout the study. With the application of only shear stress, qRT-PCR and immunohistochemistry revealed that hMSCs experienced less chondrogenic differentiation than the static group. The second study showed that chondrogenic differentiation was enhanced by additional mechanical compression. After 14 days, alcian blue staining showed more extracellular matrix formed in the compression group. The upregulation of the positive chondrogenic markers such as Sox 9, aggrecan, and type II collagen were demonstrated by qPCR. Our bioreactor provides a novel approach to apply mechanical forces to engineered cartilage. Results suggest that a combination of dynamic culture with proper mechanical stimulation may promote efficient progenitor cell expansion in vitro, thereby allowing the culture of clinically relevant articular chondrocytes for the treatment of articular cartilage defects.

Study of the collagen structure in the superficial zone and physiological state of articular cartilage using a 3D confocal imaging technique

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Zheng Ming H

2008-07-01

the collagen network in the superficial zone during early physiological alteration of articular cartilage. The fibre confocal imaging technology used in this study has allowed developing confocal arthroscopy for in vivo studying the chondrocytes in different depth of articular cartilage. Therefore, the current study has potential to develop an in vivo 3D histology for diagnosis of early osteoarthritis.

Inhibition of T-Type Voltage Sensitive Calcium Channel Reduces Load-Induced OA in Mice and Suppresses the Catabolic Effect of Bone Mechanical Stress on Chondrocytes.

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Padma P Srinivasan

Full Text Available Voltage-sensitive calcium channels (VSCC regulate cellular calcium influx, one of the earliest responses to mechanical stimulation in osteoblasts. Here, we postulate that T-type VSCCs play an essential role in bone mechanical response to load and participate in events leading to the pathology of load-induced OA. Repetitive mechanical insult was used to induce OA in Cav3.2 T-VSCC null and wild-type control mouse knees. Osteoblasts (MC3T3-E1 and chondrocytes were treated with a selective T-VSCC inhibitor and subjected to fluid shear stress to determine how blocking of T-VSCCs alters the expression profile of each cell type upon mechanical stimulation. Conditioned-media (CM obtained from static and sheared MC3T3-E1 was used to assess the effect of osteoblast-derived factors on the chondrocyte phenotype. T-VSCC null knees exhibited significantly lower focal articular cartilage damage than age-matched controls. In vitro inhibition of T-VSCC significantly reduced the expression of both early and late mechanoresponsive genes in osteoblasts but had no effect on gene expression in chondrocytes. Furthermore, treatment of chondrocytes with CM obtained from sheared osteoblasts induced expression of markers of hypertrophy in chondrocytes and this was nearly abolished when osteoblasts were pre-treated with the T-VSCC-specific inhibitor. These results indicate that T-VSCC plays a role in signaling events associated with induction of OA and is essential to the release of osteoblast-derived factors that promote an early OA phenotype in chondrocytes. Further, these findings suggest that local inhibition of T-VSCC may serve as a therapy for blocking load-induced bone formation that results in cartilage degeneration.

Low‑dose halofuginone inhibits the synthesis of type I collagen without influencing type II collagen in the extracellular matrix of chondrocytes.

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Li, Zeng; Fei, Hao; Wang, Zhen; Zhu, Tianyi

2017-09-01

Full‑thickness and large area defects of articular cartilage are unable to completely repair themselves and require surgical intervention, including microfracture, autologous or allogeneic osteochondral grafts, and autologous chondrocyte implantation. A large proportion of regenerative cartilage exists as fibrocartilage, which is unable to withstand impacts in the same way as native hyaline cartilage, owing to excess synthesis of type I collagen in the matrix. The present study demonstrated that low‑dose halofuginone (HF), a plant alkaloid isolated from Dichroa febrifuga, may inhibit the synthesis of type I collagen without influencing type II collagen in the extracellular matrix of chondrocytes. In addition, HF was revealed to inhibit the phosphorylation of mothers against decapentaplegic homolog (Smad)2/3 and promoted Smad7 expression, as well as decrease the synthesis of type I collagen synthesis. Results from the present study indicated that HF treatment suppressed the synthesis of type I collagen by inhibiting the transforming growth factor‑β signaling pathway in chondrocytes. These results may provide an alternative solution to the problems associated with fibrocartilage, and convert fibrocartilage into hyaline cartilage at the mid‑early stages of cartilage regeneration. HF may additionally be used to improve monolayer expansion or 3D cultures of seed cells for the tissue engineering of cartilage.

Coordinate and synergistic effects of extensive treadmill exercise and ovariectomy on articular cartilage degeneration.

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Miyatake, Kazumasa; Muneta, Takeshi; Ojima, Miyoko; Yamada, Jun; Matsukura, Yu; Abula, Kahaer; Sekiya, Ichiro; Tsuji, Kunikazu

2016-05-31

Although osteoarthritis (OA) is a multifactorial disease, little has been reported regarding the cooperative interaction among these factors on cartilage metabolism. Here we examined the synergistic effect of ovariectomy (OVX) and excessive mechanical stress (forced running) on articular cartilage homeostasis in a mouse model resembling a human postmenopausal condition. Mice were randomly divided into four groups, I: Sham, II: OVX, III: Sham and forced running (60 km in 6 weeks), and IV: OVX and forced running. Histological and immunohistochemical analyses were performed to evaluate the degeneration of articular cartilage and synovitis in the knee joint. Morphological changes of subchondral bone were analyzed by micro-CT. Micro-CT analyses showed significant loss of metaphyseal trabecular bone volume/tissue volume (BV/TV) after OVX as described previously. Forced running increased the trabecular BV/TV in all mice. In the epiphyseal region, no visible alteration in bone morphology or osteophyte formation was observed in any of the four groups. Histological analysis revealed that OVX or forced running respectively had subtle effects on cartilage degeneration. However, the combination of OVX and forced running synergistically enhanced synovitis and articular cartilage degeneration. Although morphological changes in chondrocytes were observed during OA initiation, no signs of bone marrow edema were observed in any of the four experimental groups. We report the coordinate and synergistic effects of extensive treadmill exercise and ovariectomy on articular cartilage degeneration. Since no surgical procedure was performed on the knee joint directly in this model, this model is useful in addressing the molecular pathogenesis of naturally occurring OA.

Chondrocyte-specific ablation of Osterix leads to impaired endochondral ossification

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Oh, Jung-Hoon [Department of Molecular Medicine, Cell and Matrix Research Institute, BK21 Medical Education Program for Human Resources, Kyungpook National University School of Medicine, Daegu (Korea, Republic of); Park, Seung-Yoon [Department of Biochemistry, School of Medicine, Dongguk University, Gyeongju 780-714 (Korea, Republic of); Crombrugghe, Benoit de [Department of Genetics, University of Texas, M.D. Anderson Cancer Center, Houston (United States); Kim, Jung-Eun, E-mail: kjeun@knu.ac.kr [Department of Molecular Medicine, Cell and Matrix Research Institute, BK21 Medical Education Program for Human Resources, Kyungpook National University School of Medicine, Daegu (Korea, Republic of)

2012-02-24

Highlights: Black-Right-Pointing-Pointer Conditional ablation of Osterix (Osx) in chondrocytes leads to skeletal defects. Black-Right-Pointing-Pointer Osx regulates chondrocyte differentiation and bone growth in growth plate chondrocytes. Black-Right-Pointing-Pointer Osx has an autonomous function in chondrocytes during endochondral ossification. -- Abstract: Osterix (Osx) is an essential transcription factor required for osteoblast differentiation during both intramembranous and endochondral ossification. Endochondral ossification, a process in which bone formation initiates from a cartilage intermediate, is crucial for skeletal development and growth. Osx is expressed in differentiating chondrocytes as well as osteoblasts during mouse development, but its role in chondrocytes has not been well studied. Here, the in vivo function of Osx in chondrocytes was examined in a chondrocyte-specific Osx conditional knockout model using Col2a1-Cre. Chondrocyte-specific Osx deficiency resulted in a weak and bent skeleton which was evident in newborn by radiographic analysis and skeletal preparation. To further understand the skeletal deformity of the chondrocyte-specific Osx conditional knockout, histological analysis was performed on developing long bones during embryogenesis. Hypertrophic chondrocytes were expanded, the formation of bone trabeculae and marrow cavities was remarkably delayed, and subsequent skeletal growth was reduced. The expression of several chondrocyte differentiation markers was reduced, indicating the impairment of chondrocyte differentiation and endochondral ossification in the chondrocyte-specific Osx conditional knockout. Taken together, Osx regulates chondrocyte differentiation and bone growth in growth plate chondrocytes, suggesting an autonomous function of Osx in chondrocytes during endochondral ossification.

Postnatal development of collagen structure in ovine articular cartilage

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Kranenbarg Sander

2010-06-01

Full Text Available Abstract Background Articular cartilage (AC is the layer of tissue that covers the articulating ends of the bones in diarthrodial joints. Across species, adult AC shows an arcade-like structure with collagen predominantly perpendicular to the subchondral bone near the bone, and collagen predominantly parallel to the articular surface near the articular surface. Recent studies into collagen fibre orientation in stillborn and juvenile animals showed that this structure is absent at birth. Since the collagen structure is an important factor for AC mechanics, the absence of the adult Benninghoff structure has implications for perinatal AC mechanobiology. The current objective is to quantify the dynamics of collagen network development in a model animal from birth to maturity. We further aim to show the presence or absence of zonal differentiation at birth, and to assess differences in collagen network development between different anatomical sites of a single joint surface. We use quantitative polarised light microscopy to investigate properties of the collagen network and we use the sheep (Ovis aries as our model animal. Results Predominant collagen orientation is parallel to the articular surface throughout the tissue depth for perinatal cartilage. This remodels to the Benninghoff structure before the sheep reach sexual maturity. Remodelling of predominant collagen orientation starts at a depth just below the future transitional zone. Tissue retardance shows a minimum near the articular surface at all ages, which indicates the presence of zonal differentiation at all ages. The absolute position of this minimum does change between birth and maturity. Between different anatomical sites, we find differences in the dynamics of collagen remodelling, but no differences in adult collagen structure. Conclusions The collagen network in articular cartilage remodels between birth and sexual maturity from a network with predominant orientation parallel to the

Hypoxia Potentiates Anabolic Effects of Exogenous Hyaluronic Acid in Rat Articular Cartilage

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Shohei Ichimaru

2016-06-01

Full Text Available Hyaluronic acid (HA is used clinically to treat osteoarthritis (OA, but its pharmacological effects under hypoxic conditions remain unclear. Articular chondrocytes in patients with OA are exposed to a hypoxic environment. This study investigated whether hypoxia could potentiate the anabolic effects of exogenous HA in rat articular cartilage and whether these mechanisms involved HA receptors. HA under hypoxic conditions significantly enhanced the expression of extracellular matrix genes and proteins in explant culture, as shown by real-time reverse transcription-polymerase chain reaction (RT-PCR, Western blotting, and dimethylmethylene blue (DMMB assays. Staining with Safranin-O and immunohistochemical staining with antibody to type II collagen were also enhanced in pellet culture. The expression of CD44 was increased by hypoxia and significantly suppressed by transfection with siRNAs targeting hypoxia-inducible factor 1 alpha (siHIF-1α. These findings indicate that hypoxia potentiates the anabolic effects of exogenous HA by a mechanism in which HIF-1α positively regulates the expression of CD44, enhancing the binding affinity for exogenous HA. The anabolic effects of exogenous HA may increase as OA progresses.

The effect og aging and mechanical loading on the metabolism og articular cartilage

DEFF Research Database (Denmark)

Jørgensen, Adam El Mongy; Kjær, Michael; Heinemeier, Katja Maria

2017-01-01

Objective. The morphology of articular cartilage (AC) enables painless movement. Aging and mechanical loading are believed to influence development of osteoarthritis (OA), yet the connection remains unclear. Methods. This narrative review describes the current knowledge regarding this area......, with the literature search made on PubMed using appropriate keywords regarding AC, age, and mechanical loading. Results. Following skeletal maturation, chondrocyte numbers decline while increasing senescence occurs. Lower cartilage turnover causes diminished maintenance capacity, which produces accumulation...... collagen network damage and proteoglycan loss, leading to irreversible cartilage destruction because of lack of regenerative capacity. Catabolic pathways involve inflammation and the transcription factor nuclear factor-κB. Thus, age seems to be a predisposing factor for OA, with mechanical overload being...

The Effect of Aging and Mechanical Loading on the Metabolism of Articular Cartilage

DEFF Research Database (Denmark)

Jørgensen, Adam El Mongy; Kjaer, Michael; Heinemeier, Katja Maria

2017-01-01

Objective. The morphology of articular cartilage (AC) enables painless movement. Aging and mechanical loading are believed to influence development of osteoarthritis (OA), yet the connection remains unclear. Methods. This narrative review describes the current knowledge regarding this area......, with the literature search made on PubMed using appropriate keywords regarding AC, age, and mechanical loading. Results. Following skeletal maturation, chondrocyte numbers decline while increasing senescence occurs. Lower cartilage turnover causes diminished maintenance capacity, which produces accumulation...... collagen network damage and proteoglycan loss, leading to irreversible cartilage destruction because of lack of regenerative capacity. Catabolic pathways involve inflammation and the transcription factor nuclear factor-κB. Thus, age seems to be a predisposing factor for OA, with mechanical overload being...

Three-year clinical outcome after chondrocyte transplantation using a hyaluronan matrix for cartilage repair

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Nehrer, S. [Department of Orthopedics, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria)]. E-mail: stefan.nehrer@meduniwien.ac.at; Domayer, S. [Department of Orthopedics, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Dorotka, R. [Department of Orthopedics, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Schatz, K. [Department of Orthopedics, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Bindreiter, U. [Department of Orthopedics, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Kotz, R. [Department of Orthopedics, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria)

2006-01-15

Repair of articular cartilage represents a significant clinical problem and although various new techniques - including the use of autologous chondrocytes - have been developed within the last century the clinical efficacy of these procedures is still discussed controversially. Although autologous chondrocyte transplantation (ACT) has been widely used with success, it has several inherent limitations, including its invasive nature and problems related to the use of the periosteal flap. To overcome these problems autologous chondrocytes transplantation combined with the use of biodegradable scaffolds has received wide attention. Among these, a hyaluronan-based scaffold has been found useful for inducing hyaline cartilage regeneration. In the present study, we have investigated the mid-term efficacy and safety of Hyalograft[reg] C grafts in a group of 36 patients undergoing surgery for chronic cartilage lesions of the knee. Clinical Outcome was assessed prospectively before and at 12, 24, and 36 months after surgery. No major adverse events have been reported during the 3-year follow-up. Significant improvements of the evaluated scores were observed (P < 0.02) at 1 year and a continued increase of clinical performance was evident at 2 and 3 years follow-up. Patients under 30 years of age with single lesions showed statistically significant improvements at all follow-up visits compared to those over 30 with multiple defects (P < 0.01). Hyalograft[reg] C compares favorably with classic ACT and is particularly indicated in younger patients with single lesions. The graft can be implanted through a miniarthrotomy and needs no additional fixation with sutures except optional fibrin gluing at the defect borders. These results suggest that Hyalograft[reg] C is a valid alternative to ACT.

Regulation of the friction coefficient of articular cartilage by TGF-beta1 and IL-1beta.

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DuRaine, Grayson; Neu, Corey P; Chan, Stephanie M T; Komvopoulos, Kyriakos; June, Ronald K; Reddi, A Hari

2009-02-01

Articular cartilage functions to provide a low-friction surface for joint movement for many decades of life. Superficial zone protein (SZP) is a glycoprotein secreted by chondrocytes in the superficial layer of articular cartilage that contributes to effective boundary lubrication. In both cell and explant cultures, TGF-beta1 and IL-1beta have been demonstrated to, respectively, upregulate and downregulate SZP protein levels. It was hypothesized that the friction coefficient of articular cartilage could also be modulated by these cytokines through SZP regulation. The friction coefficient between cartilage explants (both untreated and treated with TGF-beta1 or IL-1beta) and a smooth glass surface due to sliding in the boundary lubrication regime was measured with a pin-on-disk tribometer. SZP was quantified using an enzyme-linked immunosorbant assay and localized by immunohistochemistry. Both TGF-beta1 and IL-1beta treatments resulted in the decrease of the friction coefficient of articular cartilage in a location- and time-dependent manner. Changes in the friction coefficient due to the TGF-beta1 treatment corresponded to increased depth of SZP staining within the superficial zone, while friction coefficient changes due to the IL-1beta treatment were independent of SZP depth of staining. However, the changes induced by the IL-1beta treatment corresponded to changes in surface roughness, determined from the analysis of surface images obtained with an atomic force microscope. These findings demonstrate that the low friction of articular cartilage can be modified by TGF-beta1 and IL-1beta treatment and that the friction coefficient depends on multiple factors, including SZP localization and surface roughness.

Improvement of the Chondrocyte-Specific Phenotype upon Equine Bone Marrow Mesenchymal Stem Cell Differentiation: Influence of Culture Time, Transforming Growth Factors and Type I Collagen siRNAs on the Differentiation Index

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Thomas Branly

2018-02-01

Full Text Available Articular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA, a degenerative disease that is currently incurable. Similar musculoskeletal disorders also affect horses and OA incurs considerable economic loss for the equine sector. In the view to develop new therapies for humans and horses, significant progress in tissue engineering has led to the emergence of new generations of cartilage therapy. Matrix-associated autologous chondrocyte implantation is an advanced 3D cell-based therapy that holds promise for cartilage repair. This study aims to improve the autologous chondrocyte implantation technique by using equine mesenchymal stem cells (MSCs from bone marrow differentiated into chondrocytes that can be implanted in the chondral lesion. The optimized protocol relies on culture under hypoxia within type I/III collagen sponges. Here, we explored three parameters that influence MSC differentiation: culture times, growth factors and RNA interference strategies. Our results suggest first that an increase in culture time from 14 to 28 or 42 days lead to a sharp increase in the expression of chondrocyte markers, notably type II collagen (especially the IIB isoform, along with a concomitant decrease in HtrA1 expression. Nevertheless, the expression of type I collagen also increased with longer culture times. Second, regarding the growth factor cocktail, TGF-β3 alone showed promising result but the previously tested association of BMP-2 and TGF-β1 better limits the expression of type I collagen. Third, RNA interference targeting Col1a2 as well as Col1a1 mRNA led to a more significant knockdown, compared with a conventional strategy targeting Col1a1 alone. This chondrogenic differentiation strategy showed a strong increase in the Col2a1:Col1a1 mRNA ratio in the chondrocytes derived from equine bone marrow MSCs, this ratio being considered as an

Improvement of the Chondrocyte-Specific Phenotype upon Equine Bone Marrow Mesenchymal Stem Cell Differentiation: Influence of Culture Time, Transforming Growth Factors and Type I Collagen siRNAs on the Differentiation Index.

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Branly, Thomas; Contentin, Romain; Desancé, Mélanie; Jacquel, Thibaud; Bertoni, Lélia; Jacquet, Sandrine; Mallein-Gerin, Frédéric; Denoix, Jean-Marie; Audigié, Fabrice; Demoor, Magali; Galéra, Philippe

2018-02-01

Articular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Similar musculoskeletal disorders also affect horses and OA incurs considerable economic loss for the equine sector. In the view to develop new therapies for humans and horses, significant progress in tissue engineering has led to the emergence of new generations of cartilage therapy. Matrix-associated autologous chondrocyte implantation is an advanced 3D cell-based therapy that holds promise for cartilage repair. This study aims to improve the autologous chondrocyte implantation technique by using equine mesenchymal stem cells (MSCs) from bone marrow differentiated into chondrocytes that can be implanted in the chondral lesion. The optimized protocol relies on culture under hypoxia within type I/III collagen sponges. Here, we explored three parameters that influence MSC differentiation: culture times, growth factors and RNA interference strategies. Our results suggest first that an increase in culture time from 14 to 28 or 42 days lead to a sharp increase in the expression of chondrocyte markers, notably type II collagen (especially the IIB isoform), along with a concomitant decrease in HtrA1 expression. Nevertheless, the expression of type I collagen also increased with longer culture times. Second, regarding the growth factor cocktail, TGF-β3 alone showed promising result but the previously tested association of BMP-2 and TGF-β1 better limits the expression of type I collagen. Third, RNA interference targeting Col1a2 as well as Col1a1 mRNA led to a more significant knockdown, compared with a conventional strategy targeting Col1a1 alone. This chondrogenic differentiation strategy showed a strong increase in the Col2a1 : Col1a1 mRNA ratio in the chondrocytes derived from equine bone marrow MSCs, this ratio being considered as an index of the

Silencing of microRNA-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes by targeting FOXC1: miR-138 promotes cartilage degradation.

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Yuan, Y; Zhang, G Q; Chai, W; Ni, M; Xu, C; Chen, J Y

2016-10-01

Osteoarthritis (OA) is characterised by articular cartilage degradation. MicroRNAs (miRNAs) have been identified in the development of OA. The purpose of our study was to explore the functional role and underlying mechanism of miR-138-5p in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation of OA cartilage. Human articular cartilage was obtained from patients with and without OA, and chondrocytes were isolated and stimulated by IL-1β. The expression levels of miR-138-5p in cartilage and chondrocytes were both determined. After transfection with miR-138-5p mimics, allele-specific oligonucleotide (ASO)-miR-138-5p, or their negative controls, the messenger RNA (mRNA) levels of aggrecan (ACAN), collagen type II and alpha 1 (COL2A1), the protein levels of glycosaminoglycans (GAGs), and both the mRNA and protein levels of matrix metalloproteinase (MMP)-13 were evaluated. Luciferase reporter assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot were performed to explore whether Forkhead Box C1 (FOCX1) was a target of miR-138-5p. Further, we co-transfected OA chondrocytes with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 and then stimulated with IL-1β to determine whether miR-138-5p-mediated IL-1β-induced cartilage matrix degradation resulted from targeting FOXC1. MiR-138-5p was significantly increased in OA cartilage and in chondrocytes in response to IL-1β-stimulation. Overexpression of miR-138-5p significantly increased the IL-1β-induced downregulation of COL2A1, ACAN, and GAGs, and increased the IL-1β-induced over expression of MMP-13.We found that FOXC1 is directly regulated by miR-138-5p. Additionally, co-transfection with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 resulted in higher levels of COL2A1, ACAN, and GAGs, but lower levels of MMP-13. miR-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes, possibly by targeting FOXC1.Cite this article: Y. Yuan, G. Q. Zhang, W. Chai,M. Ni, C. Xu, J

Characterization of cells from pannus-like tissue over articular cartilage of advanced osteoarthritis.

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Yuan, G-H; Tanaka, M; Masuko-Hongo, K; Shibakawa, A; Kato, T; Nishioka, K; Nakamura, H

2004-01-01

To identify the characteristics of cells isolated from pannus-like soft tissue on osteoarthritic cartilage (OA pannus cells), and to evaluate the role of this tissue in osteoarthritis (OA). OA pannus cells were isolated from pannus-like tissues in five joints obtained during arthroplasty. The phenotypic features of the isolated cells were characterized by safranin-O staining and immunohistochemical studies. Expression of MMP-1, MMP-3 and MMP-13 was also assessed using reverse transcriptase-polymerase chain reactions (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry. Foci and plaque formation of pannus-like tissue over cartilage surface were found in 15 of 21 (71.4%) OA joints macroscopically, and among them, only five samples had enough tissue to be isolated. OA pannus cells were positive for type I collagen and vimentin, besides they also expressed type II collagen and aggrecan mRNA. Spontaneous expression of MMP-1, MMP-3 and MMP-13 was detected in OA pannus cells. Similar or higher levels of MMPs were detected in the supernatant of cultured OA pannus cells compared to OA chondrocytes, and among these MMP-3 levels were relatively higher in OA pannus cells. Immunohistochemically, MMP-3 positive cells located preferentially in pannus-like tissue on the border of original hyaline cartilage. Our results showed that OA pannus cells shared the property of mesenchymal cells and chondrocytes; however, their origin seemed different from chondrocytes or synoviocytes. The spontaneous expression of MMPs suggests that they are involved in the articular degradation in OA.

Gel structure has an impact on pericellular and extracellular matrix deposition, which subsequently alters metabolic activities in chondrocyte-laden PEG hydrogels.

Science.gov (United States)

Nicodemus, G D; Skaalure, S C; Bryant, S J

2011-02-01

While designing poly(ethylene glycol) hydrogels with high moduli suitable for in situ placement is attractive for cartilage regeneration, the impact of a tighter crosslinked structure on the organization and deposition of the matrix is not fully understood. The objectives of this study were to characterize the composition and spatial organization of new matrix as a function of gel crosslinking and study its impact on chondrocytes in terms of anabolic and catabolic gene expression and catabolic activity. Bovine articular chondrocytes were encapsulated in hydrogels with three crosslinking densities (compressive moduli 60, 320 and 590 kPa) and cultured for 25 days. Glycosaminoglycan production increased with culture time and was greatest in the gels with lowest crosslinking. Collagens II and VI, aggrecan, link protein and decorin were localized to pericellular regions in all gels, but their presence decreased with increasing gel crosslinking. Collagen II and aggrecan expression were initially up-regulated in gels with higher crosslinking, but increased similarly up to day 15. Matrix metalloproteinase (MMP)-1 and MMP-13 expression were elevated (∼25-fold) in gels with higher crosslinking throughout the study, while MMP-3 was unaffected by gel crosslinking. The presence of aggrecan and collagen degradation products confirmed MMP activity. These findings indicate that chondrocytes synthesized the major cartilage components within PEG hydrogels, however, gel structure had a significant impact on the composition and spatial organization of the new tissue and on how chondrocytes responded to their environment, particularly with respect to their catabolic expression. Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Applications of Chondrocyte-Based Cartilage Engineering: An Overview

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Abdul-Rehman Phull

2016-01-01

Full Text Available Chondrocytes are the exclusive cells residing in cartilage and maintain the functionality of cartilage tissue. Series of biocomponents such as different growth factors, cytokines, and transcriptional factors regulate the mesenchymal stem cells (MSCs differentiation to chondrocytes. The number of chondrocytes and dedifferentiation are the key limitations in subsequent clinical application of the chondrocytes. Different culture methods are being developed to overcome such issues. Using tissue engineering and cell based approaches, chondrocytes offer prominent therapeutic option specifically in orthopedics for cartilage repair and to treat ailments such as tracheal defects, facial reconstruction, and urinary incontinence. Matrix-assisted autologous chondrocyte transplantation/implantation is an improved version of traditional autologous chondrocyte transplantation (ACT method. An increasing number of studies show the clinical significance of this technique for the chondral lesions treatment. Literature survey was carried out to address clinical and functional findings by using various ACT procedures. The current study was conducted to study the pharmacological significance and biomedical application of chondrocytes. Furthermore, it is inferred from the present study that long term follow-up studies are required to evaluate the potential of these methods and specific positive outcomes.

Matrix-induced autologous chondrocyte implantation for the treatment of chondral defects of the knees in Chinese patients

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Zhang ZW

2014-12-01

Full Text Available Zhongwen Zhang,1 Xin Zhong,2 Huiru Ji,1 Zibin Tang,1 Jianpeng Bai,1 Minmin Yao,1 Jianlei Hou,1 Minghao Zheng,3 David J Wood,3 Jiazhi Sun,4 Shu-Feng Zhou,4,5 Aibing Liu6 1Department of Orthopedics, General Hospital of Chinese People’s Armed Police Forces (CAPF, Beijing; 2Department of MRI Center, General Hospital of CAPF, Beijing, People’s Republic of China; 3Center for Orthopedic Research, School of Surgery and Pathology, University of Western Australia, Perth, Western Australia, Australia; 4Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 5Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino–US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, Guizhou; 6Medical Research Center, General Hospital of Chinese People’s Armed Police Forces (CAPF, Beijing, People’s Republic of China Abstract: Articular cartilage injury is the most common type of damage seen in clinical orthopedic practice. The matrix-induced autologous chondrocyte implant (MACI was developed to repair articular cartilage with an advance on the autologous chondrocyte implant procedure. This study aimed to evaluate whether MACI is a safe and efficacious cartilage repair treatment for patients with knee cartilage lesions. The primary outcomes were the Knee Injury and Osteoarthritis Outcome Score (KOOS domains and magnetic resonance imaging (MRI results, compared between baseline and postoperative months 3, 6, 12, and 24. A total of 15 patients (20 knees, with an average age of 33.9 years, had a mean defect size of 4.01 cm2. By 6-month follow-up, KOOS results demonstrated significant improvements in symptoms and knee-related quality of life. MRI showed significant improvements in four individual graft scoring parameters at 24 months postoperatively. At 24 months, 90% of MACI grafts had filled completely and 10% had good

Chondrocyte behavior on nanostructured micropillar polypropylene and polystyrene surfaces

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Prittinen, Juha [Department of Applied Physics, University of Eastern Finland, Kuopio (Finland); Jiang, Yu [Department of Chemistry, University of Eastern Finland, Joensuu (Finland); Ylärinne, Janne H. [Department of Applied Physics, University of Eastern Finland, Kuopio (Finland); Pakkanen, Tapani A. [Department of Chemistry, University of Eastern Finland, Joensuu (Finland); Lammi, Mikko J., E-mail: mikko.lammi@uef.fi [Department of Applied Physics, University of Eastern Finland, Kuopio (Finland); Qu, Chengjuan [Department of Applied Physics, University of Eastern Finland, Kuopio (Finland)

2014-10-01

This study was aimed to investigate whether patterned polypropylene (PP) or polystyrene (PS) could enhance the chondrocytes' extracellular matrix (ECM) production and phenotype maintenance. Bovine primary chondrocytes were cultured on smooth PP and PS, as well as on nanostructured micropillar PP (patterned PP) and PS (patterned PS) for 2 weeks. Subsequently, the samples were collected for fluorescein diacetate-based cell viability tests, for immunocytochemical assays of types I and II collagen, actin and vinculin, for scanning electronic microscopic analysis of cell morphology and distribution, and for gene expression assays of Sox9, aggrecan, procollagen α{sub 1}(II), procollagen α{sub 1}(X), and procollagen α{sub 2}(I) using quantitative RT-PCR assays. After two weeks of culture, the bovine primary chondrocytes had attached on both patterned PP and PS, while practically no adhesion was observed on smooth PP. However, the best adhesion of the cells was on smooth PS. The cells, which attached on patterned PP and PS surfaces synthesized types I and II collagen. The chondrocytes' morphology was extended, and an abundant ECM network formed around the attached chondrocytes on both patterned PP and PS. Upon passaging, no significant differences on the chondrocyte-specific gene expression were observed, although the highest expression level of aggrecan was observed on the patterned PS in passage 1 chondrocytes, and the expression level of procollagen α{sub 1}(II) appeared to decrease in passaged chondrocytes. However, the expressions of procollagen α{sub 2}(I) were increased in all passaged cell cultures. In conclusion, the bovine primary chondrocytes could be grown on patterned PS and PP surfaces, and they produced extracellular matrix network around the adhered cells. However, neither the patterned PS nor PP could prevent the dedifferentiation of chondrocytes. - Highlights: • Methods to avoid chondrocyte dedifferentiation would be useful for cartilage

Biodistribution and Immunogenicity of Allogeneic Mesenchymal Stem Cells in a Rat Model of Intraarticular Chondrocyte Xenotransplantation

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Maribel Marquina

2017-11-01

Full Text Available Xenogeneic chondrocytes and allogeneic mesenchymal stem cells (MSC are considered a potential source of cells for articular cartilage repair. We here assessed the immune response triggered by xenogeneic chondrocytes when injected intraarticularly, as well as the immunoregulatory effect of allogeneic bone marrow-derived MSC after systemic administration. To this end, a discordant xenotransplantation model was established by injecting three million porcine articular chondrocytes (PAC into the femorotibial joint of Lewis rats and monitoring the immune response. First, the fate of MSC injected using various routes was monitored in an in vivo imaging system. The biodistribution revealed a dependency on the injection route with MSC injected intravenously (i.v. succumbing early after 24 h and MSC injected intraperitoneally (i.p. lasting locally for at least 5 days. Importantly, no migration of MSC to the joint was detected in rats previously injected with PAC. MSC were then administered either i.v. 1 week before PAC injection or i.p. 3 weeks after to assess their immunomodulatory function on humoral and adaptive immune parameters. Anti-PAC IgM and IgG responses were detected in all PAC-injected rats with a peak at week 2 postinjection and reactivity remaining above baseline levels by week 18. IgG2a and IgG2b were the predominant and long-lasting IgG subtypes. By contrast, no anti-MSC antibody response was detected in the cohort injected with MSC only, but infusion of MSC before PAC injection temporarily augmented the anti-PAC antibody response. Consistent with a cellular immune response to PAC in PAC-injected rats, cytokine/chemokine profiling in serum by antibody array revealed a distinct pattern relative to controls characterized by elevation of multiple markers at week 2, as well as increases in proliferation in draining lymph nodes. Notably, systemic administration of allogeneic MSC under the described conditions did not diminish the immune

5-Aza-2'-deoxycytidine acts as a modulator of chondrocyte hypertrophy and maturation in chick caudal region chondrocytes in culture.

Science.gov (United States)

Haq, Samina Hyder

2016-06-01

This study was carried out to explore the effect of DNA hypomethylation on chondrocytes phenotype, in particular the effect on chondrocyte hypertrophy, maturation, and apoptosis. Chondrocytes derived from caudal region of day 17 embryonic chick sterna were pretreated with hypomethylating drug 5-aza-2'-deoxycytidine for 48 hours and then maintained in the normal culture medium for up to 14 days. Histological studies showed distinct morphological changes occurred in the pretreated cultures when compared to the control cultures. The pretreated chondrocytes after 7 days in culture became bigger in size and acquired more flattened fibroblastic phenotype as well as a loss of cartilage specific extracellular matrix. Scanning electron microscopy at day 7 showed chondrocytes to have increased in cell volume and at day 14 in culture the extracellular matrix of the pretreated cultures showed regular fibrillar structure heavily embedded with matrix vesicles, which is the characteristic feature of chondrocyte hypertrophy. Transmission electron microscopic studies indicated the terminal fate of the hypertrophic cells in culture. The pretreated chondrocytes grown for 14 days in culture showed two types of cells: dark cells which had condense chromatin in dark patches and dark cytoplasm. The other light chondrocytes appeared to be heavily loaded with endoplasmic reticulum indicative of very active protein and secretory activity; their cytoplasm had large vacuoles and disintegrating cytoplasm. The biosynthetic profile showed that the pretreated cultures were actively synthesizing and secreting type X collagen and alkaline phosphatase as a major biosynthetic product.

R-spondin 2 facilitates differentiation of proliferating chondrocytes into hypertrophic chondrocytes by enhancing Wnt/β-catenin signaling in endochondral ossification

International Nuclear Information System (INIS)

Takegami, Yasuhiko; Ohkawara, Bisei; Ito, Mikako; Masuda, Akio; Nakashima, Hiroaki; Ishiguro, Naoki; Ohno, Kinji

2016-01-01

Endochondral ossification is a crucial process for longitudinal growth of bones. Differentiating chondrocytes in growth cartilage form four sequential zones of proliferation, alignment into column, hypertrophy, and substitution of chondrocytes with osteoblasts. Wnt/β-catenin signaling is essential for differentiation of proliferating chondrocytes into hypertrophic chondrocytes in growth cartilage. R-spondin 2 (Rspo2), a member of R-spondin family, is an agonist for Wnt signaling, but its role in chondrocyte differentiation remains unknown. Here we report that growth cartilage of Rspo2-knockout mice shows a decreased amount of β-catenin and increased amounts collagen type II (CII) and Sox9 in the abnormally extended proliferating zone. In contrast, expression of collagen type X (CX) in the hypertrophic zone remains unchanged. Differentiating chondrogenic ATDC5 cells, mimicking proliferating chondrocytes, upregulate Rspo2 and its putative receptor, Lgr5, in parallel. Addition of recombinant human Rspo2 to differentiating ATDC5 cells decreases expressions of Col2a1, Sox9, and Acan, as well as production of proteoglycans. In contrast, lentivirus-mediated knockdown of Rspo2 has the opposite effect. The effect of Rspo2 on chondrogenic differentiation is mediated by Wnt/β-catenin signaling, and not by Wnt/PCP or Wnt/Ca 2+ signaling. We propose that Rspo2 activates Wnt/β-catenin signaling to reduce Col2a1 and Sox9 and to facilitate differentiation of proliferating chondrocytes into hypertrophic chondrocytes in growth cartilage. - Highlights: • Rspo2 is a secreted activator of Wnt, and its knockout shows extended proliferating chondrocytes in endochondral ossification. • In proliferating chondrocytes of Rspo2-knockout mice, Sox9 and collagen type 2 are increased and β-catenin is decreased. • Rspo2 and its receptor Lgr5, as well as Sox9 and collagen type 2, are expressed in differentiating ATDC5 chondrogenic cells. • In ATDC5 cells, Rspo2 decreases expressions

R-spondin 2 facilitates differentiation of proliferating chondrocytes into hypertrophic chondrocytes by enhancing Wnt/β-catenin signaling in endochondral ossification

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Takegami, Yasuhiko [Division of Neurogenetics, Center of Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, Nagoya (Japan); Department of Orthopaedic Surgery, Nagoya University School of Medicine, Nagoya (Japan); Ohkawara, Bisei; Ito, Mikako; Masuda, Akio [Division of Neurogenetics, Center of Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, Nagoya (Japan); Nakashima, Hiroaki; Ishiguro, Naoki [Department of Orthopaedic Surgery, Nagoya University School of Medicine, Nagoya (Japan); Ohno, Kinji, E-mail: ohnok@med.nagoya-u.ac.jp [Division of Neurogenetics, Center of Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, Nagoya (Japan)

2016-04-22

Endochondral ossification is a crucial process for longitudinal growth of bones. Differentiating chondrocytes in growth cartilage form four sequential zones of proliferation, alignment into column, hypertrophy, and substitution of chondrocytes with osteoblasts. Wnt/β-catenin signaling is essential for differentiation of proliferating chondrocytes into hypertrophic chondrocytes in growth cartilage. R-spondin 2 (Rspo2), a member of R-spondin family, is an agonist for Wnt signaling, but its role in chondrocyte differentiation remains unknown. Here we report that growth cartilage of Rspo2-knockout mice shows a decreased amount of β-catenin and increased amounts collagen type II (CII) and Sox9 in the abnormally extended proliferating zone. In contrast, expression of collagen type X (CX) in the hypertrophic zone remains unchanged. Differentiating chondrogenic ATDC5 cells, mimicking proliferating chondrocytes, upregulate Rspo2 and its putative receptor, Lgr5, in parallel. Addition of recombinant human Rspo2 to differentiating ATDC5 cells decreases expressions of Col2a1, Sox9, and Acan, as well as production of proteoglycans. In contrast, lentivirus-mediated knockdown of Rspo2 has the opposite effect. The effect of Rspo2 on chondrogenic differentiation is mediated by Wnt/β-catenin signaling, and not by Wnt/PCP or Wnt/Ca{sup 2+} signaling. We propose that Rspo2 activates Wnt/β-catenin signaling to reduce Col2a1 and Sox9 and to facilitate differentiation of proliferating chondrocytes into hypertrophic chondrocytes in growth cartilage. - Highlights: • Rspo2 is a secreted activator of Wnt, and its knockout shows extended proliferating chondrocytes in endochondral ossification. • In proliferating chondrocytes of Rspo2-knockout mice, Sox9 and collagen type 2 are increased and β-catenin is decreased. • Rspo2 and its receptor Lgr5, as well as Sox9 and collagen type 2, are expressed in differentiating ATDC5 chondrogenic cells. • In ATDC5 cells, Rspo2 decreases

Autologous chondrocyte implantation for cartilage injury treatment in Chiang Mai University Hospital: a case report.

Science.gov (United States)

Wongtriratanachai, Prasit; Pruksakorn, Dumnoensun; Pothacharoen, Peraphan; Nimkingratana, Puwapong; Pattamapaspong, Nuttaya; Phornphutkul, Chanakarn; Setsitthakun, Sasiwariya; Fongsatitkul, Ladda; Phrompaet, Sureeporn

2013-11-01

Autologous chondrocyte implantation (ACI) has become one of the standard procedures for articular cartilage defect treatment. This technique provides a promising result. However the procedural process requires an approach of several steps from multidisciplinary teams. Although the success of this procedure has been reported from Srinakharinvirot University since 2007, the application of ACI is still limited in Thailand due to the complexity of processes and stringent quality control. This report is to present the first case of the cartilage defect treatment using the first generation-ACI under Chiang Mai University's (CMU) own facility and Ethics Committee. This paper also reviews the process of biotechnology procedures, patient selection, surgical, and rehabilitation techniques. The success of the first case is an important milestone for the further development of the CMU Human Translational Research Laboratory in near future.

Comparative study of the chondrogenic potential of human bone marrow stromal cells, neonatal chondrocytes and adult chondrocytes

International Nuclear Information System (INIS)

Saha, Sushmita; Kirkham, Jennifer; Wood, David; Curran, Stephen; Yang, Xuebin

2010-01-01

Research highlights: → This study has characterised three different cell types under conditions similar to those used for autologous chondrocyte implantation (ACI) for applications in cartilage repair/regeneration. → Compared for the first time the chondrogenic potential of neonatal chondrocytes with human bone marrow stromal cells (HBMSCs) and adult chondrocytes. → Demonstrated that adult chondrocytes hold greatest potential for use in ACI based on their higher proliferation rates, lower alkaline phosphatise activity and enhanced expression of chondrogenic genes. → Demonstrated the need for chondroinduction as a necessary pre-requisite to efficient chondrogenesis in vitro and, by extrapolation, for cell based therapy (e.g. ACI or cartilage tissue engineering). -- Abstract: Cartilage tissue engineering is still a major clinical challenge with optimisation of a suitable source of cells for cartilage repair/regeneration not yet fully addressed. The aims of this study were to compare and contrast the differences in chondrogenic behaviour between human bone marrow stromal cells (HBMSCs), human neonatal and adult chondrocytes to further our understanding of chondroinduction relative to cell maturity and to identify factors that promote chondrogenesis and maintain functional homoeostasis. Cells were cultured in monolayer in either chondrogenic or basal medium, recapitulating procedures used in existing clinical procedures for cell-based therapies. Cell doubling time, morphology and alkaline phosphatase specific activity (ALPSA) were determined at different time points. Expression of chondrogenic markers (SOX9, ACAN and COL2A1) was compared via real time polymerase chain reaction. Amongst the three cell types studied, HBMSCs had the highest ALPSA in basal culture and lowest ALPSA in chondrogenic media. Neonatal chondrocytes were the most proliferative and adult chondrocytes had the lowest ALPSA in basal media. Gene expression analysis revealed a difference in the

Comparative study of the chondrogenic potential of human bone marrow stromal cells, neonatal chondrocytes and adult chondrocytes

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Saha, Sushmita [Biomaterials and Tissue Engineering Group, Leeds Dental Institute, University of Leeds, LS29LU (United Kingdom); Kirkham, Jennifer [Biomineralisation Group, Leeds Dental Institute, University of Leeds, LS29LU (United Kingdom); NIHR Leeds Musculoskeletal Biomedical Research Unit, University of Leeds, Chapel Allerton Hospital, Leeds LS74SA (United Kingdom); Wood, David [Biomaterials and Tissue Engineering Group, Leeds Dental Institute, University of Leeds, LS29LU (United Kingdom); Curran, Stephen [Smith and Nephew Research Centre, YO105DF (United Kingdom); Yang, Xuebin, E-mail: X.B.Yang@leeds.ac.uk [Biomaterials and Tissue Engineering Group, Leeds Dental Institute, University of Leeds, LS29LU (United Kingdom); NIHR Leeds Musculoskeletal Biomedical Research Unit, University of Leeds, Chapel Allerton Hospital, Leeds LS74SA (United Kingdom)

2010-10-22

Research highlights: {yields} This study has characterised three different cell types under conditions similar to those used for autologous chondrocyte implantation (ACI) for applications in cartilage repair/regeneration. {yields} Compared for the first time the chondrogenic potential of neonatal chondrocytes with human bone marrow stromal cells (HBMSCs) and adult chondrocytes. {yields} Demonstrated that adult chondrocytes hold greatest potential for use in ACI based on their higher proliferation rates, lower alkaline phosphatise activity and enhanced expression of chondrogenic genes. {yields} Demonstrated the need for chondroinduction as a necessary pre-requisite to efficient chondrogenesis in vitro and, by extrapolation, for cell based therapy (e.g. ACI or cartilage tissue engineering). -- Abstract: Cartilage tissue engineering is still a major clinical challenge with optimisation of a suitable source of cells for cartilage repair/regeneration not yet fully addressed. The aims of this study were to compare and contrast the differences in chondrogenic behaviour between human bone marrow stromal cells (HBMSCs), human neonatal and adult chondrocytes to further our understanding of chondroinduction relative to cell maturity and to identify factors that promote chondrogenesis and maintain functional homoeostasis. Cells were cultured in monolayer in either chondrogenic or basal medium, recapitulating procedures used in existing clinical procedures for cell-based therapies. Cell doubling time, morphology and alkaline phosphatase specific activity (ALPSA) were determined at different time points. Expression of chondrogenic markers (SOX9, ACAN and COL2A1) was compared via real time polymerase chain reaction. Amongst the three cell types studied, HBMSCs had the highest ALPSA in basal culture and lowest ALPSA in chondrogenic media. Neonatal chondrocytes were the most proliferative and adult chondrocytes had the lowest ALPSA in basal media. Gene expression analysis revealed

MSX2 stimulates chondrocyte maturation by controlling Ihh expression.

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Amano, Katsuhiko; Ichida, Fumitaka; Sugita, Atsushi; Hata, Kenji; Wada, Masahiro; Takigawa, Yoko; Nakanishi, Masako; Kogo, Mikihiko; Nishimura, Riko; Yoneda, Toshiyuki

2008-10-24

Several studies indicated that a homeobox gene, Msx2, is implicated in regulation of skeletal development by controlling enchondral ossification as well as membranous ossification. However, the molecular basis by which Msx2 conducts chondrogenesis is currently unclear. In this study, we examined the role of Msx2 in chondrocyte differentiation using mouse primary chondrocytes and embryonic metatarsal explants. Treatment with BMP2 up-regulated the expression of Msx2 mRNA along with chondrocyte differentiation in murine primary chondrocytes. Overexpression of wild-type Msx2 stimulated calcification of primary chondrocytes in the presence of BMP2. We also found that constitutively active Msx2 (caMsx2) enhanced BMP2-dependent calcification more efficiently than wild-type Msx2. Consistently, caMsx2 overexpression up-regulated the expression of alkaline phosphatase and collagen type X induced by BMP2. Furthermore, organ culture experiments using mouse embryonic metatarsals indicated that caMsx2 clearly stimulated the maturation of chondrocytes into the prehypertrophic and hypertrophic stages in the presence of BMP2. In contrast, knockdown of Msx2 inhibited maturation of primary chondrocytes. The stimulatory effect of Msx2 on chondrocyte maturation was enhanced by overexpression of Smad1 and Smad4 but inhibited by Smad6, an inhibitory Smad for BMP2 signaling. These data suggest that Msx2 requires BMP2/Smad signaling for its chondrogenic action. In addition, caMsx2 overexpression induced Ihh (Indian hedgehog) expression in mouse primary chondrocytes. Importantly, treatment with cyclopamine, a specific inhibitor for hedgehogs, blocked Msx2-induced chondrogenesis. Collectively, our results indicated that Msx2 promotes the maturation of chondrocytes, at least in part, through up-regulating Ihh expression.

Effect of a Herbal-Leucine mix on the IL-1β-induced cartilage degradation and inflammatory gene expression in human chondrocytes

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Haqqi Tariq M

2011-08-01

Full Text Available Abstract Background Conventional treatments for the articular diseases are often effective for symptom relief, but can also cause significant side effects and do not slow the progression of the disease. Several natural substances have been shown to be effective at relieving the symptoms of osteoarthritis (OA, and preliminary evidence suggests that some of these compounds may exert a favorable influence on the course of the disease. The objective of this study was to investigate the anti-inflammatory/chondroprotective potential of a Herbal and amino acid mixture containing extract of the Uncaria tomentosa, Boswellia spp., Lepidium meyenii and L-Leucine on the IL-1β-induced production of nitric oxide (NO, glycosaminoglycan (GAG, matrix metalloproteinases (MMPs, aggrecan (ACAN and type II collagen (COL2A1 in human OA chondrocytes and OA cartilage explants. Methods Primary OA chondrocytes or OA cartilage explants were pretreated with Herbal-Leucine mixture (HLM, 1-10 μg/ml and then stimulated with IL-1β (5 ng/ml. Effect of HLM on IL-1β-induced gene expression of iNOS, MMP-9, MMP-13, ACAN and COL2A1 was verified by real time-PCR. Estimation of NO and GAG release in culture supernatant was done using commercially available kits. Results HLM tested in these in vitro studies was found to be an effective anti-inflammatory agent, as evidenced by strong inhibition of iNOS, MMP-9 and MMP-13 expression and NO production in IL-1β-stimulated OA chondrocytes (p Leucine mixture (HLM up-regulation of ACAN and COL2A1 expression in IL-1β-stimulated OA chondrocytes was also noted (p Conclusion Our data suggests that HLM could be chondroprotective and anti-inflammatory agent in arthritis, switching chondrocyte gene expression from catabolic direction towards anabolic and regenerative, and consequently this approach may be potentially useful as a new adjunct therapeutic/preventive agent for OA or injury recovery.

Regulative mechanisms of chondrocyte adhesion

DEFF Research Database (Denmark)

Schmal, Hagen; Mehlhorn, Alexander T; Fehrenbach, Miriam

2006-01-01

Interaction between chondrocytes and extracellular matrix is considered a key factor in the generation of grafts for matrix-associated chondrocyte transplantation. Therefore, our objective was to study the influence of differentiation status on cellular attachment. Adhesion of chondrocytes...... to collagen type II increased after removal from native cartilage up to the third day in monolayer in a dose-dependent manner. Following dedifferentiation after the second passage, adhesion to collagen types I (-84%) and II (-46%) decreased, whereas adhesion to fibrinogen (+59%) and fibronectin (+43......%) increased. A cartilage construct was developed based on a clinically established collagen type I scaffold. In this matrix, more than 80% of the cells could be immobilized by mechanisms of adhesion, filtration, and cell entrapment. Confocal laser microscopy revealed focal adhesion sites as points of cell...

Rabbit chondrocytes maintained in serum-free medium. I. Synthesis and secretion of hydrodynamically-small proteoglycans

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Malemud, C.J.; Papay, R.S.

1986-01-01

The biosynthesis of sulfated proteoglycan in vitro by rabbit articular chondrocytes in first passage monolayer culture maintained in fetal bovine serum (FBS) or in serum-free conditions was compared. Neosynthesized proteoglycan in the culture medium in the most dense fraction of an associative CsCl density gradient (fraction dAl) declined with increasing time under serum-free conditions, but not when cells were maintained in the presence of serum. After one day, the major peak of incorporated 35 SO 4 in medium fraction dAl eluted as a retarded peak on Sepharose CL-2B, whether cells were maintained under serum-free or serum-containing conditions. The hydrodynamic size of proteoglycan monomer fraction dAlDl obtained after one day of exposure to serum-free culture media was smaller than dAlDl from serum-containing cultures. The hydrodynamic size of dAlDl obtained from serum-free culture media became even progressively smaller after 2 and 3 days' exposure to these conditions. Hydrodynamically small sulfated proteoglycans were identified in the cell-associated dAlDl fraction as early as one day after switching chondrocytes from serum-containing to serum-free medium. Proteoglycan monomer from serum-containing medium reaggregated more efficiently under both conditions. No change in the size of glycosaminoglycan chains was seen in the smaller proteoglycan subpopulations, nor was there any indication of marked changes in the glycosaminoglycan types

Composition-function relations of cartilaginous tissues engineered from chondrocytes and mesenchymal stem cells isolated from bone marrow and infrapatellar fat pad.

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Vinardell, T; Buckley, C T; Thorpe, S D; Kelly, D J

2011-10-01

The objective of this study was to determine the functional properties of cartilaginous tissues generated by porcine MSCs isolated from different tissue sources, and to compare these properties to those derived from chondrocytes (CCs). MSCs were isolated from bone marrow (BM) and infrapatellar fat pad (FP), while CCs were harvested from the articular surface of the femoro-patellar joint. Culture-expanded CCs and MSCs were encapsulated in agarose hydrogels and cultured in the presence of TGFβ3. Samples were analysed biomechanically, biochemically and histologically at days 0, 21 and 42. After 42 days in free swelling culture, mean GAG content was 1.50% w/w in CC-seeded constructs, compared to 0.95% w/w in FP- and 0.43% w/w in BM-seeded constructs. Total collagen accumulation was highest in FP constructs. DNA content increased with time for all the groups. The mechanical functionality of cartilaginous tissues engineered using CCs was superior to that generated from either source of MSCs. Differences were also observed in the spatial distribution of matrix components in tissues engineered using CCs and MSCs, which appears to have a strong influence on the apparent mechanical properties of the constructs. Therefore, while functional cartilaginous tissues can be engineered using MSCs isolated from different sources, the spatial composition of these tissues is unlike that generated using chondrocytes, suggesting that MSCs and chondrocytes respond differently to the regulatory factors present within developing cartilaginous constructs. Copyright © 2010 John Wiley & Sons, Ltd.

Trophic effects of adipose-tissue-derived and bone-marrow-derived mesenchymal stem cells enhance cartilage generation by chondrocytes in co-culture.

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Pleumeekers, M M; Nimeskern, L; Koevoet, J L M; Karperien, M; Stok, K S; van Osch, G J V M

2018-01-01

Combining mesenchymal stem cells (MSCs) and chondrocytes has great potential for cell-based cartilage repair. However, there is much debate regarding the mechanisms behind this concept. We aimed to clarify the mechanisms that lead to chondrogenesis (chondrocyte driven MSC-differentiation versus MSC driven chondroinduction) and whether their effect was dependent on MSC-origin. Therefore, chondrogenesis of human adipose-tissue-derived MSCs (hAMSCs) and bone-marrow-derived MSCs (hBMSCs) combined with bovine articular chondrocytes (bACs) was compared. hAMSCs or hBMSCs were combined with bACs in alginate and cultured in vitro or implanted subcutaneously in mice. Cartilage formation was evaluated with biochemical, histological and biomechanical analyses. To further investigate the interactions between bACs and hMSCs, (1) co-culture, (2) pellet, (3) Transwell® and (4) conditioned media studies were conducted. The presence of hMSCs-either hAMSCs or hBMSCs-increased chondrogenesis in culture; deposition of GAG was most evidently enhanced in hBMSC/bACs. This effect was similar when hMSCs and bAC were combined in pellet culture, in alginate culture or when conditioned media of hMSCs were used on bAC. Species-specific gene-expression analyses demonstrated that aggrecan was expressed by bACs only, indicating a predominantly trophic role for hMSCs. Collagen-10-gene expression of bACs was not affected by hBMSCs, but slightly enhanced by hAMSCs. After in-vivo implantation, hAMSC/bACs and hBMSC/bACs had similar cartilage matrix production, both appeared stable and did not calcify. This study demonstrates that replacing 80% of bACs by either hAMSCs or hBMSCs does not influence cartilage matrix production or stability. The remaining chondrocytes produce more matrix due to trophic factors produced by hMSCs.

Biomarkers of Chondrocyte Apoptosis and Autophagy in Osteoarthritis

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Musumeci, Giuseppe; Castrogiovanni, Paola; Trovato, Francesca Maria; Weinberg, Annelie Martina; Al-Wasiyah, Mohammad K.; Alqahtani, Mohammed H.; Mobasheri, Ali

2015-01-01

Cell death with morphological and molecular features of apoptosis has been detected in osteoarthritic (OA) cartilage, which suggests a key role for chondrocyte death/survival in the pathogenesis of OA. Identification of biomarkers of chondrocyte apoptosis may facilitate the development of novel therapies that may eliminate the cause or, at least, slow down the degenerative processes in OA. The aim of this review was to explore the molecular markers and signals that induce chondrocyte apoptosis in OA. A literature search was conducted in PubMed, Scopus, Web of Science and Google Scholar using the keywords chondrocyte death, apoptosis, osteoarthritis, autophagy and biomarker. Several molecules considered to be markers of chondrocyte apoptosis will be discussed in this brief review. Molecular markers and signalling pathways associated with chondroycte apoptosis may turn out to be therapeutic targets in OA and approaches aimed at neutralizing apoptosis-inducing molecules may at least delay the progression of cartilage degeneration in OA. PMID:26334269

The inhibitory roles of Ihh downregulation on chondrocyte growth and differentiation.

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Deng, Ang; Zhang, Hongqi; Hu, Minyu; Liu, Shaohua; Wang, Yuxiang; Gao, Qile; Guo, Chaofeng

2018-01-01

The proliferative rate of chondrocytes affects bone elongation. Chondrocyte hypertrophy is required for endochondral bone formation as chondrocytes secrete factors required for osteoblast differentiation and maturation. Previous studies have demonstrated that the Indian hedgehog (Ihh) signaling pathway is a key regulator of skeletal development and homeostasis. The aim of the present study was to investigate the function of Ihh in chondrocyte proliferation and differentiation, as well as the underlying mechanisms. Ihh was knocked down in mouse chondrocyte cells using short hairpin RNA. Chondrocyte apoptosis and cell cycle arrest were assessed using flow cytometry and the results indicated that knockdown of Ihh significantly inhibited cell growth (PIhh also resulted in cell cycle arrest at G1 to S phase in chondrocytes. It was also observed that knockdown of Ihh decreased alkaline phosphatase activity and mineral deposition of chondrocytes. The inhibitory roles of Ihh downregulation on chondrocyte growth and differentiation may be associated with the transforming growth factor-β/mothers against decapentaplegic and osteoprotegerin/receptor activator of nuclear factor κB ligand signaling pathway. The results of the present study suggest that chondrocyte-derived Ihh is essential for maintaining bone growth plates and that manipulation of Ihh expression or its signaling components may be a novel therapeutic technique for the treatment of skeletal diseases, including achondroplasia.

Sphingosine-1-phosphate stimulates rat primary chondrocyte proliferation

International Nuclear Information System (INIS)

Kim, Mi-Kyoung; Lee, Ha Young; Kwak, Jong-Young; Park, Joo-In; Yun, Jeanho; Bae, Yoe-Sik

2006-01-01

Rat primary chondrocytes express the sphingosine-1-phosphate (S1P) receptor, S1P 2 , S1P 3 , S1P 4 , but not S1P 1 . When chondrocytes were stimulated with S1P or phytosphingosine-1-phosphate (PhS1P, an S1P 1 - and S1P 4 -selective agonist), phospholipase C-mediated cytosolic calcium increase was dramatically induced. S1P and PhS1P also stimulated two kinds of mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK) and p38 kinase in chondrocytes. In terms of the two phospholipids-mediated functional modulation of chondrocytes, S1P and PhS1P stimulated cellular proliferation. The two phospholipids-induced chondrocyte proliferations were almost completely blocked by PD98059 but not by SB203580, suggesting that ERK but not p38 kinase is essentially required for the proliferation. Pertussis toxin almost completely inhibited the two phospholipids-induced cellular proliferation and ERK activation, indicating the crucial role of G i protein. This study demonstrates the physiological role of two important phospholipids (S1P and PhS1P) on the modulation of rat primary chondrocyte proliferation, and the crucial role played by ERK in the process

Influence of extremely low frequency, low energy electromagnetic fields and combined mechanical stimulation on chondrocytes in 3-D constructs for cartilage tissue engineering.

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Hilz, Florian M; Ahrens, Philipp; Grad, Sibylle; Stoddart, Martin J; Dahmani, Chiheb; Wilken, Frauke L; Sauerschnig, Martin; Niemeyer, Philipp; Zwingmann, Jörn; Burgkart, Rainer; von Eisenhart-Rothe, Rüdiger; Südkamp, Norbert P; Weyh, Thomas; Imhoff, Andreas B; Alini, Mauro; Salzmann, Gian M

2014-02-01

Articular cartilage, once damaged, has very low regenerative potential. Various experimental approaches have been conducted to enhance chondrogenesis and cartilage maturation. Among those, non-invasive electromagnetic fields have shown their beneficial influence for cartilage regeneration and are widely used for the treatment of non-unions, fractures, avascular necrosis and osteoarthritis. One very well accepted way to promote cartilage maturation is physical stimulation through bioreactors. The aim of this study was the investigation of combined mechanical and electromagnetic stress affecting cartilage cells in vitro. Primary articular chondrocytes from bovine fetlock joints were seeded into three-dimensional (3-D) polyurethane scaffolds and distributed into seven stimulated experimental groups. They either underwent mechanical or electromagnetic stimulation (sinusoidal electromagnetic field of 1 mT, 2 mT, or 3 mT; 60 Hz) or both within a joint-specific bioreactor and a coil system. The scaffold-cell constructs were analyzed for glycosaminoglycan (GAG) and DNA content, histology, and gene expression of collagen-1, collagen-2, aggrecan, cartilage oligomeric matrix protein (COMP), Sox9, proteoglycan-4 (PRG-4), and matrix metalloproteinases (MMP-3 and -13). There were statistically significant differences in GAG/DNA content between the stimulated versus the control group with highest levels in the combined stimulation group. Gene expression was significantly higher for combined stimulation groups versus static control for collagen 2/collagen 1 ratio and lower for MMP-13. Amongst other genes, a more chondrogenic phenotype was noticed in expression patterns for the stimulated groups. To conclude, there is an effect of electromagnetic and mechanical stimulation on chondrocytes seeded in a 3-D scaffold, resulting in improved extracellular matrix production. © 2013 Wiley Periodicals, Inc.

Effect of Enrofloxacin on Histochemistry, Immunohistochemistry and Molecular Changes in Lamb Articular Cartilage.

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Khazaeel, Kaveh; Mazaheri, Yazdan; Hashemi Tabar, Mahmood; Najafzadeh, Hossein; Morovvati, Hassan; Ghadrdan, Alireza

2015-01-01

Enrofloxacin is a synthetic chemotherapeutic agent from the class of the fluoroquinolones that is widely used to treat bacterial infections. It is metabolized to ciprofloxacin in the body as active metabolite. Fluoroquinolones change in the articular cartilage, especially with high doses and more than two weeks use. So, due to relatively excessive use of enrofloxacin in mammals and similarity of lambs to human subjects with respect to skeletal activity cycles, this study was done to investigate the effects of enrofloxacin on some cellular and molecular changes in growing lamb articular cartilage to evaluate some possible mechanisms involved these changes. Twelve, 2-month-old male lambs divided into three groups: control group received only normal saline; therapeutic group received 5mg/kg enrofloxacin subcutaneously, daily, for 15 days and toxic group received 35 mg/kg enrofloxacin in the same manner as therapeutic group. Twenty four hours after the last dose, the animals were sacrificed, and their stifle joints were dissected. Sampling from distal femoral and proximal tibial extremities was done quickly for further histological and molecular studies. Collagen-п content was studied with avidin-biotin immunohistochemistry method in different groups. Expression of Sox9 and caspase-3 was evaluated by Real-time PCR. Immunohistochemical changes were included decreases of matrix proteoglycans, carbohydrates, and Collagen-п in the toxic group. Some of these changes were observed in the therapeutic group with less intensity in comparison to the toxic group. Enrofloxacin were significantly decreased (P≤0.05). Sox9 expression in therapeutic and toxic groups compared to control group. But caspase -3 expressions in the toxic group significantly increased (P≤0.0001) with a comparison to other groups, while, between control and therapeutic groups, there were no significant differences. So, it can be concluded that enrofloxacin increases apoptosis in chondrocytes and

Design of group IIA secreted/synovial phospholipase A(2 inhibitors: an oxadiazolone derivative suppresses chondrocyte prostaglandin E(2 secretion.

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Jean-Edouard Ombetta

Full Text Available Group IIA secreted/synovial phospholipase A(2 (GIIAPLA(2 is an enzyme involved in the synthesis of eicosanoids such as prostaglandin E(2 (PGE(2, the main eicosanoid contributing to pain and inflammation in rheumatic diseases. We designed, by molecular modeling, 7 novel analogs of 3-{4-[5(indol-1-ylpentoxy]benzyl}-4H-1,2,4-oxadiazol-5-one, denoted C1, an inhibitor of the GIIAPLA(2 enzyme. We report the results of molecular dynamics studies of the complexes between these derivatives and GIIAPLA(2, along with their chemical synthesis and results from PLA(2 inhibition tests. Modeling predicted some derivatives to display greater GIIAPLA(2 affinities than did C1, and such predictions were confirmed by in vitro PLA(2 enzymatic tests. Compound C8, endowed with the most favorable energy balance, was shown experimentally to be the strongest GIIAPLA(2 inhibitor. Moreover, it displayed an anti-inflammatory activity on rabbit articular chondrocytes, as shown by its capacity to inhibit IL-1beta-stimulated PGE(2 secretion in these cells. Interestingly, it did not modify the COX-1 to COX-2 ratio. C8 is therefore a potential candidate for anti-inflammatory therapy in joints.

Multi-membrane chitosan hydrogels as chondrocytic cell bioreactors.

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Ladet, S G; Tahiri, K; Montembault, A S; Domard, A J; Corvol, M-T M

2011-08-01

We investigated the bioactivity of new chitosan-based multi-membrane hydrogel (MMH) architectures towards chondrocyte-like cells. The microstructure of the hydrogels constituting the membranes precludes any living cell penetration, whereas their lower scale architecture allows the protein diffusion. The biological behavior of chondrocytes implanted within the MMH inter-membrane spaces was studied for 45 days in culture. Chondrocytes formed cell aggregates and proliferated without loosing their chondrogenic phenotype as illustrated by collagen II and aggrecan expressions at the mRNA and protein levels. Cells produced neo-formed alcyan blue matrix proteins filling MMH interspaces. The HiF-2α/SOX9 pattern of expression suggested that the elevated chondrocytic phenotype in MMH could be related to a better hypoxic local environment than in classical culture conditions. Pro-inflammatory markers were not expressed during the period of culture. The low level of nitric oxide accumulation within the inter-membrane spaces and in the incubation medium implied that chitosan consumed nitrites produced by entrapped chondrocytes, in relation with the decrease of its molecular weight of 50%. Our data suggest that MMH structures may be considered as complex chondrocytic cell bioreactors; "active decoys of biological media", potentially promising for various biomedical applications like the inter-vertebral disk replacement. Copyright © 2011 Elsevier Ltd. All rights reserved.

Viability of chondrocytes seeded onto a collagen I/III membrane for matrix-induced autologous chondrocyte implantation.

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Hindle, Paul; Hall, Andrew C; Biant, Leela C

2014-11-01

Cell viability is crucial for effective cell-based cartilage repair. The aim of this study was to determine the effect of handling the membrane during matrix-induced autologous chondrocyte implantation surgery on the viability of implanted chondrocytes. Images were acquired under five conditions: (i) Pre-operative; (ii) Handled during surgery; (iii) Cut edge; (iv) Thumb pressure applied; (v) Heavily grasped with forceps. Live and dead cell stains were used. Images were obtained for cell counting and morphology. Mean cell density was 6.60 × 10(5) cells/cm(2) (5.74-7.11 × 10(5) ) in specimens that did not have significant trauma decreasing significantly in specimens that had been grasped with forceps (p < 0.001) or cut (p = 0.004). Cell viability on delivery grade membrane was 75.1%(72.4-77.8%). This dropped to 67.4%(64.1-69.7%) after handling (p = 0.002), 56.3%(51.5-61.6%) after being thumbed (p < 0.001) and 28.8%(24.7-31.2%) after crushing with forceps (p < 0.001). When cut with scissors there was a band of cell death approximately 275 µm in width where cell viability decreased to 13.7%(10.2-18.2%, p < 0.001). Higher magnification revealed cells without the typical rounded appearance of chondrocytes. We found that confocal laser-scanning microscope (CLSM) can be used to quantify and image the fine morphology of cells on a matrix-induced autologous chondrocyte implantation (MACI) membrane. Careful handling of the membrane is essential to minimise chondrocyte death during surgery. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Benefits of Ilizarov automated bone distraction for nerves and articular cartilage in experimental leg lengthening.

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Shchudlo, Nathalia; Varsegova, Tatyana; Stupina, Tatyana; Shchudlo, Michael; Saifutdinov, Marat; Yemanov, Andrey

2017-09-18

To determine peculiarities of tissue responses to manual and automated Ilizarov bone distraction in nerves and articular cartilage. Twenty-nine dogs were divided in two experimental groups: Group M - leg lengthening with manual distraction (1 mm/d in 4 steps), Group A - automated distraction (1 mm/d in 60 steps) and intact group. Animals were euthanized at the end of distraction, at 30 th day of fixation in apparatus and 30 d after the fixator removal. M-responses in gastrocnemius and tibialis anterior muscles were recorded, numerical histology of peroneal and tibialis nerves and knee cartilage semi-thin sections, scanning electron microscopy and X-ray electron probe microanalysis were performed. Better restoration of M-response amplitudes in leg muscles was noted in A-group. Fibrosis of epineurium with adipocytes loss in peroneal nerve, subperineurial edema and fibrosis of endoneurium in some fascicles of both nerves were noted only in M-group, shares of nerve fibers with atrophic and degenerative changes were bigger in M-group than in A-group. At the end of experiment morphometric parameters of nerve fibers in peroneal nerve were comparable with intact nerve only in A-group. Quantitative parameters of articular cartilage (thickness, volumetric densities of chondrocytes, percentages of isogenic clusters and empty cellular lacunas, contents of sulfur and calcium) were badly changed in M-group and less changed in A-group. Automated Ilizarov distraction is more safe method of orthopedic leg lengthening than manual distraction in points of nervous fibers survival and articular cartilage arthrotic changes.

Melanocortin 1 receptor-signaling deficiency results in an articular cartilage phenotype and accelerates pathogenesis of surgically induced murine osteoarthritis.

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Lorenz, Julia; Seebach, Elisabeth; Hackmayer, Gerit; Greth, Carina; Bauer, Richard J; Kleinschmidt, Kerstin; Bettenworth, Dominik; Böhm, Markus; Grifka, Joachim; Grässel, Susanne

2014-01-01

Proopiomelanocortin-derived peptides exert pleiotropic effects via binding to melanocortin receptors (MCR). MCR-subtypes have been detected in cartilage and bone and mediate an increasing number of effects in diathrodial joints. This study aims to determine the role of MC1-receptors (MC1) in joint physiology and pathogenesis of osteoarthritis (OA) using MC1-signaling deficient mice (Mc1re/e). OA was surgically induced in Mc1re/e and wild-type (WT) mice by transection of the medial meniscotibial ligament. Histomorphometry of Safranin O stained articular cartilage was performed with non-operated controls (11 weeks and 6 months) and 4/8 weeks past surgery. µCT-analysis for assessing epiphyseal bone architecture was performed as a longitudinal study at 4/8 weeks after OA-induction. Collagen II, ICAM-1 and MC1 expression was analysed by immunohistochemistry. Mc1re/e mice display less Safranin O and collagen II stained articular cartilage area compared to WT prior to OA-induction without signs of spontaneous cartilage surface erosion. This MC1-signaling deficiency related cartilage phenotype persisted in 6 month animals. At 4/8 weeks after OA-induction cartilage erosions were increased in Mc1re/e knees paralleled by weaker collagen II staining. Prior to OA-induction, Mc1re/e mice do not differ from WT with respect to bone parameters. During OA, Mc1re/e mice developed more osteophytes and had higher epiphyseal bone density and mass. Trabecular thickness was increased while concomitantly trabecular separation was decreased in Mc1re/e mice. Numbers of ICAM-positive chondrocytes were equal in non-operated 11 weeks Mc1re/e and WT whereas number of positive chondrocytes decreased during OA-progression. Unchallenged Mc1re/e mice display smaller articular cartilage covered area without OA-related surface erosions indicating that MC1-signaling is critical for proper cartilage matrix integrity and formation. When challenged with OA, Mc1re/e mice develop a more severe OA

Imaging of articular cartilage

Directory of Open Access Journals (Sweden)

Bhawan K Paunipagar

2014-01-01

Full Text Available We tried to review the role of magnetic resonance imaging (MRI in understanding microscopic and morphologic structure of the articular cartilage. The optimal protocols and available spin-echo sequences in present day practice are reviewed in context of common pathologies of articular cartilage. The future trends of articular cartilage imaging have been discussed with their appropriateness. In diarthrodial joints of the body, articular cartilage is functionally very important. It is frequently exposed to trauma, degeneration, and repetitive wear and tear. MRI has played a vital role in evaluation of articular cartilage. With the availability of advanced repair surgeries for cartilage lesions, there has been an increased demand for improved cartilage imaging techniques. Recent advances in imaging strategies for native and postoperative articular cartilage open up an entirely new approach in management of cartilage-related pathologies.

Multiscale Mechanics of Articular Cartilage: Potentials and Challenges of Coupling Musculoskeletal, Joint, and Microscale Computational Models

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Halloran, J. P.; Sibole, S.; van Donkelaar, C. C.; van Turnhout, M. C.; Oomens, C. W. J.; Weiss, J. A.; Guilak, F.; Erdemir, A.

2012-01-01

Articular cartilage experiences significant mechanical loads during daily activities. Healthy cartilage provides the capacity for load bearing and regulates the mechanobiological processes for tissue development, maintenance, and repair. Experimental studies at multiple scales have provided a fundamental understanding of macroscopic mechanical function, evaluation of the micromechanical environment of chondrocytes, and the foundations for mechanobiological response. In addition, computational models of cartilage have offered a concise description of experimental data at many spatial levels under healthy and diseased conditions, and have served to generate hypotheses for the mechanical and biological function. Further, modeling and simulation provides a platform for predictive risk assessment, management of dysfunction, as well as a means to relate multiple spatial scales. Simulation-based investigation of cartilage comes with many challenges including both the computational burden and often insufficient availability of data for model development and validation. This review outlines recent modeling and simulation approaches to understand cartilage function from a mechanical systems perspective, and illustrates pathways to associate mechanics with biological function. Computational representations at single scales are provided from the body down to the microstructure, along with attempts to explore multiscale mechanisms of load sharing that dictate the mechanical environment of the cartilage and chondrocytes. PMID:22648577

Deciphering chondrocyte behaviour in matrix-induced autologous chondrocyte implantation to undergo accurate cartilage repair with hyaline matrix.

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Demoor, M; Maneix, L; Ollitrault, D; Legendre, F; Duval, E; Claus, S; Mallein-Gerin, F; Moslemi, S; Boumediene, K; Galera, P

2012-06-01

Since the emergence in the 1990s of the autologous chondrocytes transplantation (ACT) in the treatment of cartilage defects, the technique, corresponding initially to implantation of chondrocytes, previously isolated and amplified in vitro, under a periosteal membrane, has greatly evolved. Indeed, the first generations of ACT showed their limits, with in particular the dedifferentiation of chondrocytes during the monolayer culture, inducing the synthesis of fibroblastic collagens, notably type I collagen to the detriment of type II collagen. Beyond the clinical aspect with its encouraging results, new biological substitutes must be tested to obtain a hyaline neocartilage. Therefore, the use of differentiated chondrocytes phenotypically stabilized is essential for the success of ACT at medium and long-term. That is why researchers try now to develop more reliable culture techniques, using among others, new types of biomaterials and molecules known for their chondrogenic activity, giving rise to the 4th generation of ACT. Other sources of cells, being able to follow chondrogenesis program, are also studied. The success of the cartilage regenerative medicine is based on the phenotypic status of the chondrocyte and on one of its essential component of the cartilage, type II collagen, the expression of which should be supported without induction of type I collagen. The knowledge accumulated by the scientific community and the experience of the clinicians will certainly allow to relief this technological challenge, which influence besides, the validation of such biological substitutes by the sanitary authorities. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Platelet lysate activates quiescent cell proliferation and reprogramming in human articular cartilage: Involvement of hypoxia inducible factor 1.

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Nguyen, Van Thi; Cancedda, Ranieri; Descalzi, Fiorella

2018-03-01

The idea of rescuing the body self-repair capability lost during evolution is progressively gaining ground in regenerative medicine. In particular, growth factors and bioactive molecules derived from activated platelets emerged as promising therapeutic agents acting as trigger for repair of tissue lesions and restoration of tissue functions. Aim of this study was to assess the potential of a platelet lysate (PL) for human articular cartilage repair considering its activity on progenitor cells and differentiated chondrocytes. PL induced the re-entry in the cell cycle of confluent, growth-arrested dedifferentiated/progenitor cartilage cells. In a cartilage permissive culture environment, differentiated cells also resumed proliferation after exposure to PL. These findings correlated with an up-regulation of the proliferation/survival pathways ERKs and Akt and with an induction of cyclin D1. In short- and long-term cultures of articular cartilage explants, we observed a release of proliferating chondroprogenitors able to differentiate and form an "in vitro" tissue with properties of healthy articular cartilage. Moreover, in cultured cartilage cells, PL induced a hypoxia-inducible factor (HIF-1) alpha increase, its nuclear relocation and the binding to HIF-1 responsive elements. These events were possibly related to the cell proliferation because the HIF-1 inhibitor acriflavine inhibited HIF-1 binding to HIF-1 responsive elements and cell proliferation. Our study demonstrates that PL induces quiescent cartilage cell activation and proliferation leading to new cartilage formation, identifies PL activated pathways playing a role in these processes, and provides a rationale to the application of PL for therapeutic treatment of damaged articular cartilage. Copyright © 2017 John Wiley & Sons, Ltd.

Expansion of bovine chondrocytes on microcarriers enhances redifferentiation

NARCIS (Netherlands)

Malda, J.; van Blitterswijk, Clemens; Grojec, M.; Martens, D.E.; Tramper, J.; Riesle, J.U.

2003-01-01

Functional cartilage implants for orthopedic surgery or in vitro tissue evaluation can be created from expanded chondrocytes and biodegradable scaffolds. Expansion of chondrocytes in two-dimensional culture systems results in their dedifferentiation. The hallmark of this process is the switch of

Indian hedgehog signaling triggers Nkx3.2 protein degradation during chondrocyte maturation

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Choi, Seung-Won; Jeong, Da-Un; Kim, Jeong-Ah; Lee, Boyoung; Joeng, Kyu Sang; Long, Fanxin; Kim, Dae-Won

2015-01-01

The Indian hedgehog (Ihh) pathway plays an essential role in facilitating chondrocyte hypertrophy and bone formation during skeletal development. Nkx3.2 is initially induced in chondrocyte precursor cells, maintained in early-stage chondrocytes, and down-regulated in terminal-stage chondrocytes. Consistent with these expression patterns, Nkx3.2 has been shown to enhance chondrocyte differentiation and cell survival, while inhibiting chondrocyte hypertrophy and apoptosis. Thus, in this work, we investigate whether Nkx3.2, an early stage chondrogenic factor, can be regulated by Ihh, a key regulator for chondrocyte hypertrophy. Here, we show that Ihh signaling can induce proteasomal degradation of Nkx3.2. In addition, we found that Ihh can suppress levels of Lrp (Wnt co-receptor) and Sfrp (Wnt antagonist) expression, which, in turn, may selectively enhance Lrp-independent non-canonical Wnt pathways in chondrocyte. In agreement with these findings, Ihh-induced Nkx3.2 degradation requires Wnt5a, which is capable of triggering Nkx3.2 degradation. Finally, we found that Nkx3.2 protein levels in chondrocytes are remarkably elevated in mice defective in Ihh signaling by deletion of either Ihh or Smoothened. Thus, these results suggest that Ihh/Wnt5a signaling may play a role in negative regulation of Nkx3.2 for appropriate progression of chondrocyte hypertrophy during chondrogenesis. PMID:22507129

Emerging Players at the Intersection of Chondrocyte Loss of Maturational Arrest, Oxidative Stress, Senescence and Low-Grade Inflammation in Osteoarthritis

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Manuela Minguzzi

2018-01-01

Full Text Available The prevalence of Osteoarthritis (OA is increasing because of the progressive aging and unhealthy lifestyle. These risk factors trigger OA by removing constraints that keep the tightly regulated low turnover of the extracellular matrix (ECM of articular cartilage, the correct chondrocyte phenotype, and the functionality of major homeostatic mechanisms, such as mitophagy, that allows for the clearance of dysfunctional mitochondria, preventing increased production of reactive oxygen species, oxidative stress, and senescence. After OA onset, the presence of ECM degradation products is perceived as a “danger” signal by the chondrocytes and the synovial macrophages that release alarmins with autocrine/paracrine effects on the same cells. Alarmins trigger innate immunity in the joint, with important systemic crosstalks that explain the beneficial effects of dietary interventions and improved lifestyle. Alarmins also boost low-grade inflammation: the release of inflammatory molecules and chemokines sustained by continuous triggering of NF-κB within an altered cellular setting that allows its higher transcriptional activity. Chemokines exert pleiotropic functions in OA, including the recruitment of inflammatory cells and the induction of ECM remodeling. Some chemokines have been successfully targeted to attenuate structural damage or pain in OA animal models. This represents a promising strategy for the future management of human OA.

Emerging Players at the Intersection of Chondrocyte Loss of Maturational Arrest, Oxidative Stress, Senescence and Low-Grade Inflammation in Osteoarthritis.

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Minguzzi, Manuela; Cetrullo, Silvia; D'Adamo, Stefania; Silvestri, Ylenia; Flamigni, Flavio; Borzì, Rosa Maria

2018-01-01

The prevalence of Osteoarthritis (OA) is increasing because of the progressive aging and unhealthy lifestyle. These risk factors trigger OA by removing constraints that keep the tightly regulated low turnover of the extracellular matrix (ECM) of articular cartilage, the correct chondrocyte phenotype, and the functionality of major homeostatic mechanisms, such as mitophagy, that allows for the clearance of dysfunctional mitochondria, preventing increased production of reactive oxygen species, oxidative stress, and senescence. After OA onset, the presence of ECM degradation products is perceived as a "danger" signal by the chondrocytes and the synovial macrophages that release alarmins with autocrine/paracrine effects on the same cells. Alarmins trigger innate immunity in the joint, with important systemic crosstalks that explain the beneficial effects of dietary interventions and improved lifestyle. Alarmins also boost low-grade inflammation: the release of inflammatory molecules and chemokines sustained by continuous triggering of NF- κ B within an altered cellular setting that allows its higher transcriptional activity. Chemokines exert pleiotropic functions in OA, including the recruitment of inflammatory cells and the induction of ECM remodeling. Some chemokines have been successfully targeted to attenuate structural damage or pain in OA animal models. This represents a promising strategy for the future management of human OA.

Antioxidant effect of bisphosphonates and simvastatin on chondrocyte lipid peroxidation

International Nuclear Information System (INIS)

Dombrecht, E.J.; De Tollenaere, C.B.; Aerts, K.; Cos, P.; Schuerwegh, A.J.; Bridts, C.H.; Van Offel, J.F.; Ebo, D.G.; Stevens, W.J.; De Clerck, L.S.

2006-01-01

The objective of this study was to evaluate the effect of bisphosphonates (BPs) and simvastatin on chondrocyte lipid peroxidation. For this purpose, a flow cytometrical method using C11-BODIPY 581/591 was developed to detect hydroperoxide-induced lipid peroxidation in chondrocytes. Tertiary butylhydroperoxide (t-BHP) induced a time and concentration dependent increase in chondrocyte lipid peroxidation. Addition of a Fe 2+ /EDTA complex to t-BHP or hydrogen peroxide (H 2 O 2 ) clearly enhanced lipid peroxidation. The lipophilic simvastatin demonstrated a small inhibition in the chondrocyte lipid peroxidation. None of three tested BPs (clodronate, pamidronate, and risedronate) had an effect on chondrocyte lipid peroxidation induced by t-BHP. However, when Fe 2+ /EDTA complex was added to t-BHP or H 2 O 2 , BPs inhibited the lipid peroxidation process varying from 25% to 58%. This study demonstrates that BPs have antioxidant properties as iron chelators, thereby inhibiting the chondrocyte lipid peroxidation. These findings add evidence to the therapeutic potential of bisphosphonates and statins in rheumatoid arthritis

Stimulation of matrix formation in rabbit chondrocyte cultures by ascorbate. 1. Effect of ascorbate analogs and beta-aminopropionitrile.

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Wright, G C; Wei, X Q; McDevitt, C A; Lane, B P; Sokoloff, L

1988-01-01

The most consistent effects of 0.2 mM L-ascorbate on monolayer cultures of rabbit articular chondrocytes were a diversion of incorporated radiosulfate into a pericellular matrix and enhancement of cell proliferation. Only with certain batches of fetal bovine serum (FBS) was there a cell-for-cell increase of proteoglycan synthesis. These actions increased as the cell inoculum rose from 0.5 to 2 x 10(5) cells/T25 flask. Maximal effects of ascorbate and D-isoascorbate were found over a range of 0.05-0.2 mM. L-Dehydroascorbic acid was less effective than either, and no stimulatory action was exerted by L-cysteine, glutathione, dithiothreitol, methylene blue, or phenazine methosulfate. Ascorbate increased the hypro:pro ratio of newly synthesized proteins. beta-Aminopropionitrile (1 mM) reduced the proportion of [3H]hydroxyproline and [35S]O4-proteoglycans in the ascorbate-supplemented matrix 31 and 7%, respectively. In corresponding electronmicrographs, the number of pericellular filaments was reduced. We conclude: (a) Ascorbate has a general anabolic effect on chondrocytes in culture and enhances matrix assembly through mechanisms other than its redox function; (b) deposition of proteoglycans in the matrix is not simply the result of mechanical entrapment by allysine- or hydroxyallysine-derived cross-linking of collagen; and (c) contradictory reports on the subject result from variations in the serum employed, inoculum density, and concentration of ascorbate.

The importance of bicarbonate and nonbicarbonate buffer systems in batch and continuous flow bioreactors for articular cartilage tissue engineering.

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Khan, Aasma A; Surrao, Denver C

2012-05-01

In cartilage tissue engineering an optimized culture system, maintaining an appropriate extracellular environment (e.g., pH of media), can increase cell proliferation and extracellular matrix (ECM) accumulation. We have previously reported on a continuous-flow bioreactor that improves tissue growth by supplying the cells with a near infinite supply of medium. Previous studies have observed that acidic environments reduce ECM synthesis and chondrocyte proliferation. Hence, in this study we investigated the combined effects of a continuous culture system (bioreactor) together with additional buffering agents (e.g., sodium bicarbonate [NaHCO₃]) on cartilaginous tissue growth in vitro. Isolated bovine chondrocytes were grown in three-dimensional cultures, either in static conditions or in a continuous-flow bioreactor, in media with or without NaHCO₃. Tissue constructs cultivated in the bioreactor with NaHCO₃-supplemented media were characterized with significantly increased (p<0.05) ECM accumulation (glycosaminoglycans a 98-fold increase; collagen a 25-fold increase) and a 13-fold increase in cell proliferation, in comparison with static cultures. Additionally, constructs grown in the bioreactor with NaHCO₃-supplemented media were significantly thicker than all other constructs (p<0.05). Further, the chondrocytes from the primary construct expanded and synthesized ECM, forming a secondary construct without a separate expansion phase, with a diameter and thickness of 4 mm and 0.72 mm respectively. Tissue outgrowth was negligible in all other culturing conditions. Thus this study demonstrates the advantage of employing a continuous flow bioreactor coupled with NaHCO₃ supplemented media for articular cartilage tissue engineering.

Mechanical confinement regulates cartilage matrix formation by chondrocytes

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Lee, Hong-Pyo; Gu, Luo; Mooney, David J.; Levenston, Marc E.; Chaudhuri, Ovijit

2017-12-01

Cartilage tissue equivalents formed from hydrogels containing chondrocytes could provide a solution for replacing damaged cartilage. Previous approaches have often utilized elastic hydrogels. However, elastic stresses may restrict cartilage matrix formation and alter the chondrocyte phenotype. Here we investigated the use of viscoelastic hydrogels, in which stresses are relaxed over time and which exhibit creep, for three-dimensional (3D) culture of chondrocytes. We found that faster relaxation promoted a striking increase in the volume of interconnected cartilage matrix formed by chondrocytes. In slower relaxing gels, restriction of cell volume expansion by elastic stresses led to increased secretion of IL-1β, which in turn drove strong up-regulation of genes associated with cartilage degradation and cell death. As no cell-adhesion ligands are presented by the hydrogels, these results reveal cell sensing of cell volume confinement as an adhesion-independent mechanism of mechanotransduction in 3D culture, and highlight stress relaxation as a key design parameter for cartilage tissue engineering.

Cultura de condrócitos em arcabouço tridimensional: hidrogel de alginato Chondrocyte cultures in tridimensional scaffold: alginate hydrogel

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Renata Aparecida de Camargo Bittencourt

2009-01-01

Full Text Available OBJETIVOS: O presente estudo teve como objetivo cultivar condrócitos retirados da articulação do joelho de coelhos encapsulados em hidrogel de alginato (HA e caracterizar a produção de matriz extracelular (ECM. MÉTODOS: A cartilagem articular foi removida do joelho de coelhos, com três a seis meses, fragmentada em pedaços de 1mm e submetida à digestão enzimática. Uma concentração de 1x106 céls/mL foram ressuspensas em uma solução de alginato de sódio a 1,5% (w/v, em seguida fez-se o processo de gelatinização em CaCl2 (102 mM, permitindo a formação do HA e cultivo em meio DMEM-F12 durante quatro semanas. A distribuição das células e a ECM foram acessadas através das secções histológicas coradas com e azul de toluidina hematoxilina e eosina (HE. RESULTADOS: Houve um aumento no número e na viabilidade dos condrócitos durante as quatro semanas de cultura. Através das análises histológicas dos HAs corados com azul de toluidina e HE foi possível observar a distribuição definida dos condrócitos no hidrogel, assemelhando-se a grupos isógenos e formação de matriz territorial. CONCLUSÃO: Este estudo demonstrou a eficiência do HA como arcabouço para ser usado na cultura de condrócitos, constituindo uma alternativa no reparo de lesões na cartilagem articular.OBJECTIVES: The aim of this study was to culture chondrocytes from knee joint cartilage of rabbits encapsulated in alginate hydrogel (HA and to characterize the production of extracelular matrix (ECM. METHODS: Joint cartilage was obtained from rabbits' knees, three to six months old, fragmented into 1-mm pieces and submitted to enzymatic digestion. A concentration of 1x106 cells/mL were re-suspended into a 1.5% (w/v sodium alginate solution, followed by gel formation process with CaCl2 (102 mM, allowing HA to build for culturing it into a DMEM-F12 medium for four weeks. The distribution of cells and ECM were assessed from histological slices stained toluidine

THE EFFECT OF PIROXICAM ON THE METABOLISM OF ISOLATED HUMAN CHONDROCYTES

NARCIS (Netherlands)

BULSTRA, SK; KUIJER, R; BUURMAN, WA; TERWINDTROUWENHORST, E; GUELEN, PJM; VANDERLINDEN, AJ

The effect of piroxicam on the metabolism of healthy and osteoarthrotic (OA) chondrocytes was studied in vitro. The chondrocytes were obtained from five healthy, five moderately OA, and four severely OA hips or knees. The chondrocytes were cultured in a high-density, short-term in vitro model. In

Synergistic chondroprotective effects of curcumin and resveratrol in human articular chondrocytes: inhibition of IL-1beta-induced NF-kappaB-mediated inflammation and apoptosis.

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Csaki, Constanze; Mobasheri, Ali; Shakibaei, Mehdi

2009-01-01

Currently available treatments for osteoarthritis (OA) are restricted to nonsteroidal anti-inflammatory drugs, which exhibit numerous side effects and are only temporarily effective. Thus novel, safe and more efficacious anti-inflammatory agents are needed for OA. Naturally occurring polyphenolic compounds, such as curcumin and resveratrol, are potent agents for modulating inflammation. Both compounds mediate their effects by targeting the NF-kappaB signalling pathway. We have recently demonstrated that in chondrocytes resveratrol modulates the NF-kappaB pathway by inhibiting the proteasome, while curcumin modulates the activation of NF-kappaB by inhibiting upstream kinases (Akt). However, the combinational effects of these compounds in chondrocytes has not been studied and/or compared with their individual effects. The aim of this study was to investigate the potential synergistic effects of curcumin and resveratrol on IL-1beta-stimulated human chondrocytes in vitro using immunoblotting and electron microscopy. Treatment with curcumin and resveratrol suppressed NF-kappaB-regulated gene products involved in inflammation (cyclooxygenase-2, matrix metalloproteinase (MMP)-3, MMP-9, vascular endothelial growth factor), inhibited apoptosis (Bcl-2, Bcl-xL, and TNF-alpha receptor-associated factor 1) and prevented activation of caspase-3. IL-1beta-induced NF-kappaB activation was suppressed directly by cocktails of curcumin and resveratrol through inhibition of Ikappakappa and proteasome activation, inhibition of IkappaBalpha phosphorylation and degradation, and inhibition of nuclear translocation of NF-kappaB. The modulatory effects of curcumin and resveratrol on IL-1beta-induced expression of cartilage specific matrix and proinflammatory enzymes were mediated in part by the cartilage-specific transcription factor Sox-9. We propose that combining these natural compounds may be a useful strategy in OA therapy as compared with separate treatment with each individual

Protocols for the in vitro design of animal articular cartilage based on tissue engineering methods

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Diego Correa

2002-01-01

Full Text Available The articular cartilage is the structure that covers the joint ends. It has some specific tasks crucial to the correct joint physiology. It may experience a large amount of injuries that could generate considerable disabilities. Unfortunately its selfrepair capacity is too limited; therefore, many treatments have been developed with partial success, given the suboptimal biomechanical behavior of the resultant tissue. Given that, Tissue Engineering offers an alternative, based on the design of a new tissue with biological and biomechanical features which resembles the native tissue. In this work, the authors describe the methodologies followed to accomplish that goal, studying the chondrocytes harvesting, the cellular cultures, the scaffold seeding processes, the mechanical stimulation and the structural and biomechanical evaluation. Finally, exposed some of the preliminary results, as a experimental validation of the methods proposed are.

Cell type dependent morphological adaptation in polyelectrolyte hydrogels governs chondrogenic fate.

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Raghothaman, Deepak; Leong, Meng Fatt; Lim, Tze Chiun; Wan, Andrew C A; Ser, Zheng; Lee, Eng Hin; Yang, Zheng

2016-04-04

Repair of critical-size articular cartilage defects typically involves delivery of cells in biodegradable, 3D matrices. Differences in the developmental status of mesenchymal stem cells (MSCs) and terminally differentiated mature chondrocytes might be a critical factor in engineering appropriate 3D matrices for articular cartilage tissue engineering. This study examined the relationship between material-driven early cell morphological adaptations and chondrogenic outcomes, by studying the influence of aligned collagen type I (Col I) presentation on chondrocytes and MSC in interfacial polyelectrolyte complexation (IPC)-based hydrogels. In the absence of Col I, both chondrocytes and MSCs adopted rounded cell morphology and formed clusters, with chondrocyte clusters favoring the maintenance of hyaline phenotype, while MSC clusters differentiated to fibro-superficial zone-like chondrocytes. Encapsulated chondrocytes in IPC-Col I hydrogel adopted a fibroblastic morphology forming fibro-superficial zone-like phenotype, which could be reversed by inhibiting actin polymerization using cytochalasin D (CytD). In contrast, adoption of fibroblastic morphology by encapsulated MSCs in IPC-Col I facilitated superior chondrogenesis, generating a mature, hyaline neocartilage tissue. CytD treatment abrogated the elongation of MSCs and brought about a single cell-like state, resulting in insignificant chondrogenic differentiation, underscoring the essential requirement of providing matrix environments that are amenable to cell-cell interactions for robust MSC chondrogenic differentiation. Our study demonstrates that MSCs and culture-expanded chondrocytes favour differential microenvironmental niches and emphasizes the importance of designing biomaterials that meet cell type-specific requirements, in adopting chondrocyte or MSC-based approaches for regenerating hyaline, articular cartilage.

Bi-articular Knee-Ankle-Foot Exoskeleton Produces Higher Metabolic Cost Reduction than Weight-Matched Mono-articular Exoskeleton

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Malcolm, Philippe; Galle, Samuel; Derave, Wim; De Clercq, Dirk

2018-01-01

The bi-articular m. gastrocnemius and the mono-articular m. soleus have different and complementary functions during walking. Several groups are starting to use these biological functions as inspiration to design prostheses with bi-articular actuation components to replace the function of the m. gastrocnemius. Simulation studies indicate that a bi-articular configuration and spring that mimic the m. gastrocnemius could be beneficial for orthoses or exoskeletons. Our aim was to test the effect of a bi-articular and spring configuration that mimics the m. gastrocnemius and compare this to a no-spring and mono-articular configuration. We tested nine participants during walking with knee-ankle-foot exoskeletons with dorsally mounted pneumatic muscle actuators. In the bi-articular plus spring condition the pneumatic muscles were attached to the thigh segment with an elastic cord. In the bi-articular no-spring condition the pneumatic muscles were also attached to the thigh segment but with a non-elastic cord. In the mono-articular condition the pneumatic muscles were attached to the shank segment. We found the highest reduction in metabolic cost of 13% compared to walking with the exoskeleton powered-off in the bi-articular plus spring condition. Possible explanations for this could be that the exoskeleton delivered the highest total positive work in this condition at the ankle and the knee and provided more assistance during the isometric phase of the biological plantarflexors. As expected we found that the bi-articular conditions reduced m. gastrocnemius EMG more than the mono-articular condition but this difference was not significant. We did not find that the mono-articular condition reduces the m. soleus EMG more than the bi-articular conditions. Knowledge of specific effects of different exoskeleton configurations on metabolic cost and muscle activation could be useful for providing customized assistance for specific gait impairments. PMID:29551959

Bi-articular Knee-Ankle-Foot Exoskeleton Produces Higher Metabolic Cost Reduction than Weight-Matched Mono-articular Exoskeleton

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Philippe Malcolm

2018-03-01

Full Text Available The bi-articular m. gastrocnemius and the mono-articular m. soleus have different and complementary functions during walking. Several groups are starting to use these biological functions as inspiration to design prostheses with bi-articular actuation components to replace the function of the m. gastrocnemius. Simulation studies indicate that a bi-articular configuration and spring that mimic the m. gastrocnemius could be beneficial for orthoses or exoskeletons. Our aim was to test the effect of a bi-articular and spring configuration that mimics the m. gastrocnemius and compare this to a no-spring and mono-articular configuration. We tested nine participants during walking with knee-ankle-foot exoskeletons with dorsally mounted pneumatic muscle actuators. In the bi-articular plus spring condition the pneumatic muscles were attached to the thigh segment with an elastic cord. In the bi-articular no-spring condition the pneumatic muscles were also attached to the thigh segment but with a non-elastic cord. In the mono-articular condition the pneumatic muscles were attached to the shank segment. We found the highest reduction in metabolic cost of 13% compared to walking with the exoskeleton powered-off in the bi-articular plus spring condition. Possible explanations for this could be that the exoskeleton delivered the highest total positive work in this condition at the ankle and the knee and provided more assistance during the isometric phase of the biological plantarflexors. As expected we found that the bi-articular conditions reduced m. gastrocnemius EMG more than the mono-articular condition but this difference was not significant. We did not find that the mono-articular condition reduces the m. soleus EMG more than the bi-articular conditions. Knowledge of specific effects of different exoskeleton configurations on metabolic cost and muscle activation could be useful for providing customized assistance for specific gait impairments.

Bi-articular Knee-Ankle-Foot Exoskeleton Produces Higher Metabolic Cost Reduction than Weight-Matched Mono-articular Exoskeleton.

Science.gov (United States)

Malcolm, Philippe; Galle, Samuel; Derave, Wim; De Clercq, Dirk

2018-01-01

The bi-articular m. gastrocnemius and the mono-articular m. soleus have different and complementary functions during walking. Several groups are starting to use these biological functions as inspiration to design prostheses with bi-articular actuation components to replace the function of the m. gastrocnemius. Simulation studies indicate that a bi-articular configuration and spring that mimic the m. gastrocnemius could be beneficial for orthoses or exoskeletons. Our aim was to test the effect of a bi-articular and spring configuration that mimics the m. gastrocnemius and compare this to a no-spring and mono-articular configuration. We tested nine participants during walking with knee-ankle-foot exoskeletons with dorsally mounted pneumatic muscle actuators. In the bi-articular plus spring condition the pneumatic muscles were attached to the thigh segment with an elastic cord. In the bi-articular no-spring condition the pneumatic muscles were also attached to the thigh segment but with a non-elastic cord. In the mono-articular condition the pneumatic muscles were attached to the shank segment. We found the highest reduction in metabolic cost of 13% compared to walking with the exoskeleton powered-off in the bi-articular plus spring condition . Possible explanations for this could be that the exoskeleton delivered the highest total positive work in this condition at the ankle and the knee and provided more assistance during the isometric phase of the biological plantarflexors. As expected we found that the bi-articular conditions reduced m. gastrocnemius EMG more than the mono-articular condition but this difference was not significant. We did not find that the mono-articular condition reduces the m. soleus EMG more than the bi-articular conditions . Knowledge of specific effects of different exoskeleton configurations on metabolic cost and muscle activation could be useful for providing customized assistance for specific gait impairments.

The Signaling Pathways Involved in Chondrocyte Differentiation and Hypertrophic Differentiation

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Jianmei Li

2016-01-01

Full Text Available Chondrocytes communicate with each other mainly via diffusible signals rather than direct cell-to-cell contact. The chondrogenic differentiation of mesenchymal stem cells (MSCs is well regulated by the interactions of varieties of growth factors, cytokines, and signaling molecules. A number of critical signaling molecules have been identified to regulate the differentiation of chondrocyte from mesenchymal progenitor cells to their terminal maturation of hypertrophic chondrocytes, including bone morphogenetic proteins (BMPs, SRY-related high-mobility group-box gene 9 (Sox9, parathyroid hormone-related peptide (PTHrP, Indian hedgehog (Ihh, fibroblast growth factor receptor 3 (FGFR3, and β-catenin. Except for these molecules, other factors such as adenosine, O2 tension, and reactive oxygen species (ROS also have a vital role in cartilage formation and chondrocyte maturation. Here, we outlined the complex transcriptional network and the function of key factors in this network that determine and regulate the genetic program of chondrogenesis and chondrocyte differentiation.

Chondrocyte survival in osteochondral transplant cylinders depends on the harvesting technique.

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Hafke, Benedikt; Petri, Maximilian; Suero, Eduardo; Neunaber, Claudia; Kwisda, Sebastian; Krettek, Christian; Jagodzinski, Michael; Omar, Mohamed

2016-07-01

In autologous osteochondral transplantation, the edges of the harvested plug are particularly susceptible to mechanical or thermal damage to the chondrocytes. We hypothesised that the applied harvesting device has an impact on chondrocyte vitality. Both knees of five blackhead sheep (ten knees) underwent open osteochondral plug harvesting with three different circular harvesting devices (osteoarticular transfer system harvester [OATS; diameter 8 mm; Arthrex, Munich, Germany], diamond cutter [DC; diameter 8.35 mm; Karl Storz, Tuttlingen, Germany] and hollow reamer with cutting crown [HRCC; diameter 7 mm; Dannoritzer, Tuttlingen, Germany]) from distinctly assigned anatomical sites of the knee joint. The rotary cutters (DC and HRCC) were either used with (+) or without cooling (-). Surgical cuts of the cartilage with a scalpel blade were chosen as control method. After cryotomy cutting, chondrocyte vitality was assessed using fluorescence microscopy and a Live/Dead assay. There were distinct patterns of chondrocyte vitality, with reproducible accumulations of dead chondrocytes along the harvesting edge. No statistical difference in chondrocyte survivorship was seen between the OATS technique and the control method, or between the HRCC+ technique and the control method (P > 0.05). The DC+, HRCC- and DC- techniques yielded significantly lower chondrocyte survival rates compared with the control method (P vitality.

NF-kappaB specifically activates BMP-2 gene expression in growth plate chondrocytes in vivo and in a chondrocyte cell line in vitro.

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Feng, Jian Q; Xing, Lianping; Zhang, Jiang-Hong; Zhao, Ming; Horn, Diane; Chan, Jeannie; Boyce, Brendan F; Harris, Stephen E; Mundy, Gregory R; Chen, Di

2003-08-01

Bone morphogenetic protein-2 (BMP-2) regulates growth plate chondrogenesis during development and postnatal bone growth, but the control mechanisms of BMP-2 expression in growth plate chondrocytes are unknown. Here we have used both in vitro and in vivo approaches to demonstrate that transcription factor, NF-kappaB, regulates BMP-2 gene expression in chondrocytes. Two putative NF-kappaB response elements were found in the -2712/+165 region of the BMP-2 gene. Cotransfection of mutant I-kappaBalpha expression plasmids with BMP-2 promoter-luciferase reporters into TMC-23 chondrocyte cell line suppressed BMP-2 transcription. Mutations in NF-kappaB response elements in the BMP-2 gene lead to decreases in BMP-2 promoter activity. Electrophoretic mobility shift assay using nuclear extracts from TMC-23 chondrocytic cells revealed that the NF-kappaB subunits p50 and p65 bound to the NF-kappaB response elements of the BMP-2 gene. Thus, NF-kappaB may positively regulate BMP-2 gene transcription. Consistent with these findings, expression of BMP-2 mRNA was significantly reduced in growth plate chondrocytes in NF-kappaB p50/p52 dKO mice, which associated with decreased numbers of 5-bromo-2'-deoxyuridine (BrdUrd)-positive cells in the proliferating zone of growth plate in these mice. Therefore, in postnatal growth plate chondrocytes, expression of BMP-2 is regulated by NF-kappaB, which may play an important role in chondrogenesis.

Carnosol Inhibits Pro-Inflammatory and Catabolic Mediators of Cartilage Breakdown in Human Osteoarthritic Chondrocytes and Mediates Cross-Talk between Subchondral Bone Osteoblasts and Chondrocytes.

Directory of Open Access Journals (Sweden)

Christelle Sanchez

Full Text Available The aim of this work was to evaluate the effects of carnosol, a rosemary polyphenol, on pro-inflammatory and catabolic mediators of cartilage breakdown in chondrocytes and via bone-cartilage crosstalk.Osteoarthritic (OA human chondrocytes were cultured in alginate beads for 4 days in presence or absence of carnosol (6 nM to 9 μM. The production of aggrecan, matrix metalloproteinase (MMP-3, tissue inhibitor of metalloproteinase (TIMP-1, interleukin (IL-6 and nitric oxide (NO and the expression of type II collagen and ADAMTS-4 and -5 were analyzed. Human osteoblasts from sclerotic (SC or non-sclerotic (NSC subchondral bone were cultured for 3 days in presence or absence of carnosol before co-culture with chondrocytes. Chondrocyte gene expression was analyzed after 4 days of co-culture.In chondrocytes, type II collagen expression was significantly enhanced in the presence of 3 μM carnosol (p = 0.008. MMP-3, IL-6, NO production and ADAMTS-4 expression were down-regulated in a concentration-dependent manner by carnosol (p<0.01. TIMP-1 production was slightly increased at 3 μM (p = 0.02 and ADAMTS-5 expression was decreased from 0.2 to 9 μM carnosol (p<0.05. IL-6 and PGE2 production was reduced in the presence of carnosol in both SC and NSC osteoblasts while alkaline phosphatase activity was not changed. In co-culture experiments preincubation of NSC and SC osteoblasts wih carnosol resulted in similar effects to incubation with anti-IL-6 antibody, namely a significant increase in aggrecan and decrease in MMP-3, ADAMTS-4 and -5 gene expression by chondrocytes.Carnosol showed potent inhibition of pro-inflammatory and catabolic mediators of cartilage breakdown in chondrocytes. Inhibition of matrix degradation and enhancement of formation was observed in chondrocytes cocultured with subchondral osteoblasts preincubated with carnosol indicating a cross-talk between these two cellular compartments, potentially mediated via inhibition of IL-6 in

A comparison of self-assembly and hydrogel encapsulation as a means to engineer functional cartilaginous grafts using culture expanded chondrocytes.

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Mesallati, Tariq; Buckley, Conor T; Kelly, Daniel J

2014-01-01

Despite an increased interest in the use of hydrogel encapsulation and cellular self-assembly (often termed "self-aggregating" or "scaffold-free" approaches) for tissue-engineering applications, to the best of our knowledge, no study to date has been undertaken to directly compare both approaches for generating functional cartilaginous grafts. The objective of this study was to directly compare self-assembly (SA) and agarose hydrogel encapsulation (AE) as a means to engineer such grafts using passaged chondrocytes. Agarose hydrogels (5 mm diameter × 1.5 mm thick) were seeded with chondrocytes at two cell seeding densities (900,000 cells or 4 million cells in total per hydrogel), while SA constructs were generated by adding the same number of cells to custom-made molds. Constructs were either supplemented with transforming growth factor (TGF)-β3 for 6 weeks, or only supplemented with TGF-β3 for the first 2 weeks of the 6 week culture period. The SA method was only capable of generating geometrically uniform cartilaginous tissues at high seeding densities (4 million cells). At these high seeding densities, we observed that total sulphated glycosaminoglycan (sGAG) and collagen synthesis was greater with AE than SA, with higher sGAG retention also observed in AE constructs. When normalized to wet weight, however, SA constructs exhibited significantly higher levels of collagen accumulation compared with agarose hydrogels. Furthermore, it was possible to engineer such functionality into these tissues in a shorter timeframe using the SA approach compared with AE. Therefore, while large numbers of chondrocytes are required to engineer cartilaginous grafts using the SA approach, it would appear to lead to the faster generation of a more hyaline-like tissue, with a tissue architecture and a ratio of collagen to sGAG content more closely resembling native articular cartilage.

Unphysiologically high magnesium concentrations support chondrocyte proliferation and redifferentiation.

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Feyerabend, Frank; Witte, Frank; Kammal, Michael; Willumeit, Regine

2006-12-01

The effect of unphysiologically high extracellular magnesium concentrations on chondrocytes, induced by the supplementation of magnesium sulfate, was studied using a 3-phase tissue engineering model. The experiments showed that chondrocyte proliferation and redifferentiation, on the gene and protein expression level, are enhanced. A negative influence was found during chondrogenesis where an inhibition of extracellular matrix formation was observed. In addition, a direct impact on chondrocyte metabolism, elevated magnesium concentrations also affected growth factor effectiveness by consecutive influences during chondrogenesis. All observations were dosage dependent. The results of this study indicate that magnesium may be a useful tool for cartilage tissue engineering.

Detecting new microRNAs in human osteoarthritic chondrocytes identifies miR-3085 as a human, chondrocyte-selective, microRNA

OpenAIRE

Crowe, N.; Swingler, T.E.; Le, L.T.T.; Barter, M.J.; Wheeler, G.; Pais, H.; Donell, S.T.; Young, D.A.; Dalmay, T.; Clark, I.M.

2016-01-01

Summary Objective To use deep sequencing to identify novel microRNAs (miRNAs) in human osteoarthritic cartilage which have a functional role in chondrocyte phenotype or function. Design A small RNA library was prepared from human osteoarthritic primary chondrocytes using in-house adaptors and analysed by Illumina sequencing. Novel candidate miRNAs were validated by northern blot and qRT-PCR. Expression was measured in cartilage models. Targets of novel candidates were identified by microarray...

The chondrocytic journey in endochondral bone growth and skeletal dysplasia.

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Yeung Tsang, Kwok; Wa Tsang, Shun; Chan, Danny; Cheah, Kathryn S E

2014-03-01

The endochondral bones of the skeleton develop from a cartilage template and grow via a process involving a cascade of chondrocyte differentiation steps culminating in formation of a growth plate and the replacement of cartilage by bone. This process of endochondral ossification, driven by the generation of chondrocytes and their subsequent proliferation, differentiation, and production of extracellular matrix constitute a journey, deviation from which inevitably disrupts bone growth and development, and is the basis of human skeletal dysplasias with a wide range of phenotypic severity, from perinatal lethality to progressively deforming. This highly coordinated journey of chondrocyte specification and fate determination is controlled by a myriad of intrinsic and extrinsic factors. SOX9 is the master transcription factor that, in concert with varying partners along the way, directs the different phases of the journey from mesenchymal condensation, chondrogenesis, differentiation, proliferation, and maturation. Extracellular signals, including bone morphogenetic proteins, wingless-related MMTV integration site (WNT), fibroblast growth factor, Indian hedgehog, and parathyroid hormone-related peptide, are all indispensable for growth plate chondrocytes to align and organize into the appropriate columnar architecture and controls their maturation and transition to hypertrophy. Chondrocyte hypertrophy, marked by dramatic volume increase in phases, is controlled by transcription factors SOX9, Runt-related transcription factor, and FOXA2. Hypertrophic chondrocytes mediate the cartilage to bone transition and concomitantly face a live-or-die situation, a subject of much debate. We review recent insights into the coordination of the phases of the chondrocyte journey, and highlight the need for a systems level understanding of the regulatory networks that will facilitate the development of therapeutic approaches for skeletal dysplasia. Copyright © 2014 Wiley Periodicals

Influence of cell printing on biological characters of chondrocytes.

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Qu, Miao; Gao, Xiaoyan; Hou, Yikang; Shen, Congcong; Xu, Yourong; Zhu, Ming; Wang, Hengjian; Xu, Haisong; Chai, Gang; Zhang, Yan

2015-01-01

To establish a two-dimensional biological printing technique of chondrocytes and compare the difference of related biological characters between printed chondrocytes and unprinted cells so as to control the cell transfer process and keep cell viability after printing. Primary chondrocytes were obtained from human mature and fetal cartilage tissues and then were regularly sub-cultured to harvest cells at passage 2 (P2), which were adjusted to the single cell suspension at a density of 1×10(6)/mL. The experiment was divided into 2 groups: experimental group P2 chondrocytes were transferred by rapid prototype biological printer (driving voltage value 50 V, interval in x-axis 300 μm, interval in y-axis 1500 μm). Afterwards Live/Dead viability Kit and flow cytometry were respectively adopted to detect cell viability; CCK-8 Kit was adopted to detect cell proliferation viability; immunocytochemistry, immunofluorescence and RT-PCR was employed to identify related markers of chondrocytes; control group steps were the same as the printing group except that cell suspension received no printing. Fluorescence microscopy and flow cytometry analyses showed that there was no significant difference between experimental group and control group in terms of cell viability. After 7-day in vitro culture, control group exhibited higher O.D values than experimental group from 2nd day to 7th day but there was no distinct difference between these two groups (P>0.05). Inverted microscope observation demonstrated that the morphology of these two groups had no significant difference either. Similarly, Immunocytochemistry, immunofluorescence and RT-PCR assays also showed that there was no significant difference in the protein and gene expression of type II collagen and aggrecan between these two groups (P>0.05). Conclusion Cell printing has no distinctly negative effect on cell vitality, proliferation and phenotype of chondrocytes. Biological printing technique may provide a novel approach

Chondrocyte differentiation for auricular cartilage reconstruction using a chitosan based hydrogel.

Science.gov (United States)

García-López, J; Garciadiego-Cázares, D; Melgarejo-Ramírez, Y; Sánchez-Sánchez, R; Solís-Arrieta, L; García-Carvajal, Z; Sánchez-Betancourt, J I; Ibarra, C; Luna-Bárcenas, G; Velasquillo, C

2015-12-01

Tissue engineering with the use of biodegradable and biocompatible scaffolds is an interesting option for ear repair. Chitosan-Polyvinyl alcohol-Epichlorohydrine hydrogel (CS-PVA-ECH) is biocompatible and displays appropriate mechanical properties to be used as a scaffold. The present work, studies the potential of CS-PVA-ECH scaffolds seeded with chondrocytes to develop elastic cartilage engineered-neotissues. Chondrocytes isolated from rabbit and swine elastic cartilage were independently cultured onto CS-PVA-ECH scaffolds for 20 days to form the appropriate constructs. Then, in vitro cell viability and morphology were evaluated by calcein AM and EthD-1 assays and Scanning Electron Microscopy (SEM) respectively, and the constructs were implanted in nu/nu mice for four months, in order to evaluate the neotissue formation. Histological analysis of the formed neotissues was performed by Safranin O, Toluidine blue (GAG's), Verhoeff-Van Gieson (elastic fibers), Masson's trichrome (collagen) and Von Kossa (Calcium salts) stains and SEM. Results indicate appropriate cell viability, seeded with rabbit or swine chondrocyte constructs; nevertheless, upon implantation the constructs developed neotissues with different characteristics depending on the animal species from which the seeded chondrocytes came from. Neotissues developed from swine chondrocytes were similar to auricular cartilage, while neotissues from rabbit chondrocytes were similar to hyaline cartilage and eventually they differentiate to bone. This result suggests that neotissue characteristics may be influenced by the animal species source of the chondrocytes isolated.

Ionizing Radiation Induces Cellular Senescence of Articular Chondrocytes via Negative Regulation of SIRT1 by p38 Kinase

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Hong, Eun Hee; Hwang, Sang Gu [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

2009-05-15

Senescent cells exhibit irreversible growth arrest, large flat morphology, and up-regulated senescence-associated {beta}-galactosidase activity at pH 6.0. Several conditions, including oncogenic stress, oxidative stress, and DNA damage are associated with cellular senescence. Massive acute DNA double-strand breaks occurring as a result of mechanical and chemical stress can be repaired, but some DNA damage persists, eventually triggering premature senescence. Since ionizing radiation directly induces DBS, it is possible that cellular senescence is activated under these conditions. The biological events in chondrocytes following irradiation are poorly understood, and limited information is available on the molecular signal transduction mechanisms of cellular senescence at present. In this study, we identify SIRT1 as a target molecule of p38 kinase and demonstrate that the interactions between p38 kinase and SIRT1 protein play an important role in the regulation of cellular senescence in response to IR.

Biological knee reconstruction for combined malalignment, meniscal deficiency, and articular cartilage disease.

Science.gov (United States)

Harris, Joshua D; Hussey, Kristen; Wilson, Hillary; Pilz, Kyle; Gupta, Anil K; Gomoll, Andreas; Cole, Brian J

2015-02-01

The aim of this study was to analyze patient-reported outcomes in those undergoing the triad of simultaneous osteotomy, meniscal transplantation, and articular cartilage repair. Patients undergoing simultaneous meniscal transplantation, distal femoral or proximal tibial osteotomy, and articular cartilage surgery by a single surgeon (B.J.C.) were analyzed. Meniscal transplantation was performed using bone-in-slot techniques. Distal femoral and high tibial osteotomies were performed for valgus and varus malalignment, respectively. Microfracture, autologous chondrocyte implantation, and osteochondral autograft or allograft were performed for articular cartilage disease. Validated patient-reported and surgeon-measured outcomes were collected. Preoperative and postoperative outcomes and medial versus lateral disease were compared using Student t tests. Eighteen participants (mean age, 34 ± 7.8 years; symptomatic patients, 7.4 ± 5.6 years; 2.4 ± 1.0 surgical procedures before study enrollment; mean follow-up, 6.5 ± 3.2 years) were analyzed. Two thirds of participants had medial compartment pathologic conditions and one third had lateral compartment pathologic processes. At final follow-up, there were statistically significant clinically meaningful improvements in International Knee Documentation Committee (IKDC) subjective classification, Lysholm score, and 4 Knee Injury and Osteoarthritis Outcome Score (KOOS) subscores. Postoperative 12-item short form (SF-12) physical and mental component scores were not significantly different from preoperative scores. The Kellgren-Lawrence classification grade was 1.5 ± 1.1 at 2.5 ± 3.0 years after surgery. There was a significantly higher preoperative SF-12 physical composite score (PCS) in participants with lateral compartment pathologic conditions (v medial compartment conditions) (P = .011). Although there were 13 reoperations in 10 patients (55.5% reoperation rate), only one patient was converted to knee arthroplasty (5

Characterization of collagenase-3 binding and internalization by rabbit chondrocytes

International Nuclear Information System (INIS)

Raggatt, L.J.; Choundhury, I.; Williams, S.

2002-01-01

Full text: Collagenase-3 (MMP-13) is an extracellular matrix metalloproteinase that cleaves type II collagen, the major protein component of cartilage, with high specificity. Several studies have identified increased levels of MMP-13 in arthritic synovial fluid where it may contribute to matrix destruction in this disease. Our laboratory has previously documented a process where by osteoblastic cells remove MMP-13 from the surrounding milieu by binding the enzyme to a specific receptor. The enzyme is then internalized and degraded through the actions of the endocytotic receptor, the low-density lipoprotein receptor-related protein (LRP). Such a mechanism provides for a controlled elimination of a potentially destructive enzyme from the extracellular environment. This process of MMP-13 internalization also occurs in chondrocytes and is significantly reduced in OA chondrocytes. We are currently characterizing the internalization of MMP-13 in normal rabbit chondrocytes. Primary rabbit chondrocytes were harvested and cultured in monolayers for three passages. Reverse transcription polymerase chain reaction (RT-PCR) was used to asses the cell phenotype during the culture period and the rabbit chondrocytes were found to express the cartilage specific genes aggrecan and type II collagen throughout this time. 125I-MMP-13 was used to assess the ability of the rabbit chondrocytes to bind MMP-13. Appreciable specific cell-association of MMP-13 was detected after 10 mm of exposure to the ligand and equilibrium was obtained after 2 h. After identifying the time to equilibrium we determined whether binding was saturable by incubating the chondrocytes with increasing concentrations of 125I-MMP-13 ranging from 0 to 100 nM at 4 deg C for 2h. The amount of specifically associated MMP-13 approached saturation at 75 nM, allowing assessment of the receptor kinetics. Finally, we have assessed the ability of rabbit chondrocytes to internalize a single cohort of 125I-MMP-13 over time at

Chondrocytic Atf4 regulates osteoblast differentiation and function via Ihh.

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Wang, Weiguang; Lian, Na; Ma, Yun; Li, Lingzhen; Gallant, Richard C; Elefteriou, Florent; Yang, Xiangli

2012-02-01

Atf4 is a leucine zipper-containing transcription factor that activates osteocalcin (Ocn) in osteoblasts and indian hedgehog (Ihh) in chondrocytes. The relative contribution of Atf4 in chondrocytes and osteoblasts to the regulation of skeletal development and bone formation is poorly understood. Investigations of the Atf4(-/-);Col2a1-Atf4 mouse model, in which Atf4 is selectively overexpressed in chondrocytes in an Atf4-null background, demonstrate that chondrocyte-derived Atf4 regulates osteogenesis during development and bone remodeling postnatally. Atf4 overexpression in chondrocytes of the Atf4(-/-);Col2a1-Atf4 double mutants corrects the reduction in stature and limb in Atf4(-/-) embryos and rectifies the decrease in Ihh expression, Hh signaling, proliferation and accelerated hypertrophy that characterize the Atf4(-/-) developing growth plate cartilages. Unexpectedly, this genetic manipulation also restores the expression of osteoblastic marker genes, namely Ocn and bone sialoprotein, in Atf4(-/-) developing bones. In Atf4(-/-);Col2a1-Atf4 adult mice, all the defective bone parameters found in Atf4(-/-) mice, including bone volume, trabecular number and thickness, and bone formation rate, are rescued. In addition, the conditioned media of ex vivo cultures from wild-type or Atf4(-/-);Col2a1-Atf4, but not Atf4(-/-) cartilage, corrects the differentiation defects of Atf4(-/-) bone marrow stromal cells and Ihh-blocking antibody eliminates this effect. Together, these data indicate that Atf4 in chondrocytes is required for normal Ihh expression and for its paracrine effect on osteoblast differentiation. Therefore, the cell-autonomous role of Atf4 in chondrocytes dominates the role of Atf4 in osteoblasts during development for the control of early osteogenesis and skeletal growth.

Enhanced chondrocyte culture and growth on biologically inspired nanofibrous cell culture dishes.

Science.gov (United States)

Bhardwaj, Garima; Webster, Thomas J

2016-01-01

Chondral and osteochondral defects affect a large number of people in which treatment options are currently limited. Due to its ability to mimic the natural nanofibrous structure of cartilage, this current in vitro study aimed at introducing a new scaffold, called XanoMatrix™, for cartilage regeneration. In addition, this same scaffold is introduced here as a new substrate onto which to study chondrocyte functions. Current studies on chondrocyte functions are limited due to nonbiologically inspired cell culture substrates. With its polyethylene terephthalate and cellulose acetate composition, good mechanical properties and nanofibrous structure resembling an extracellular matrix, XanoMatrix offers an ideal surface for chondrocyte growth and proliferation. This current study demonstrated that the XanoMatrix scaffolds promote chondrocyte growth and proliferation as compared with the Corning and Falcon surfaces normally used for chondrocyte cell culture. The XanoMatrix scaffolds also have greater hydrophobicity, three-dimensional surface area, and greater tensile strength, making them ideal candidates for alternative treatment options for chondral and osteochondral defects as well as cell culture substrates to study chondrocyte functions.

Human YKL39 (chitinase 3-like protein 2), an osteoarthritis-associated gene, enhances proliferation and type II collagen expression in ATDC5 cells

International Nuclear Information System (INIS)

Miyatake, Kazumasa; Tsuji, Kunikazu; Yamaga, Mika; Yamada, Jun; Matsukura, Yu; Abula, Kahaer; Sekiya, Ichiro; Muneta, Takeshi

2013-01-01

Highlights: ► hYKL-39 expression is increased in osteoarthritic articular chondrocytes. ► To examine the molecular functions of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in chondrocytic ATDC5 cells. ► hYKL-39 enhanced proliferation and colony formation in ATDC5 cells. ► hYKL-39 increased type II collagen expression in ATDC5 cells treated with chondrogenic medium. -- Abstract: Human YKL39 (chitinase 3-like protein 2/CHI3L2) is a secreted 39 kDa protein produced by articular chondrocytes and synoviocytes. Recent studies showed that hYKL-39 expression is increased in osteoarthritic articular chondrocytes suggesting the involvement of hYKL-39 in the progression of osteoarthritis (OA). However little is known regarding the molecular function of hYKL-39 in joint homeostasis. Sequence analyses indicated that hYKL-39 has significant identity with the human chitotorisidase family molecules, although it is considered that hYKL-39 has no enzymatic activity since it lacks putative chitinase catalytic motif. In this study, to examine the molecular function of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in ATDC5 cells. Here we report that hYKL-39 enhances colony forming activity, cell proliferation, and type II collagen expression in these cells. These data suggest that hYKL-39 is a novel growth and differentiation factor involved in cartilage homeostasis

Human YKL39 (chitinase 3-like protein 2), an osteoarthritis-associated gene, enhances proliferation and type II collagen expression in ATDC5 cells

Energy Technology Data Exchange (ETDEWEB)

Miyatake, Kazumasa [Department of Joint Surgery and Sports Medicine, Tokyo Medical and Dental University, Tokyo (Japan); Tsuji, Kunikazu, E-mail: ktsuji.gcoe@tmd.ac.jp [International Research Center for Molecular Science in Tooth and Bone Diseases (Global Center of Excellence Program), Tokyo Medical and Dental University, Tokyo (Japan); Yamaga, Mika; Yamada, Jun; Matsukura, Yu; Abula, Kahaer [Department of Joint Surgery and Sports Medicine, Tokyo Medical and Dental University, Tokyo (Japan); Sekiya, Ichiro [Section of Cartilage Regeneration, Tokyo Medical and Dental University, Tokyo (Japan); Muneta, Takeshi [Department of Joint Surgery and Sports Medicine, Tokyo Medical and Dental University, Tokyo (Japan); International Research Center for Molecular Science in Tooth and Bone Diseases (Global Center of Excellence Program), Tokyo Medical and Dental University, Tokyo (Japan)

2013-02-01

Highlights: ► hYKL-39 expression is increased in osteoarthritic articular chondrocytes. ► To examine the molecular functions of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in chondrocytic ATDC5 cells. ► hYKL-39 enhanced proliferation and colony formation in ATDC5 cells. ► hYKL-39 increased type II collagen expression in ATDC5 cells treated with chondrogenic medium. -- Abstract: Human YKL39 (chitinase 3-like protein 2/CHI3L2) is a secreted 39 kDa protein produced by articular chondrocytes and synoviocytes. Recent studies showed that hYKL-39 expression is increased in osteoarthritic articular chondrocytes suggesting the involvement of hYKL-39 in the progression of osteoarthritis (OA). However little is known regarding the molecular function of hYKL-39 in joint homeostasis. Sequence analyses indicated that hYKL-39 has significant identity with the human chitotorisidase family molecules, although it is considered that hYKL-39 has no enzymatic activity since it lacks putative chitinase catalytic motif. In this study, to examine the molecular function of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in ATDC5 cells. Here we report that hYKL-39 enhances colony forming activity, cell proliferation, and type II collagen expression in these cells. These data suggest that hYKL-39 is a novel growth and differentiation factor involved in cartilage homeostasis.

SHP2 regulates chondrocyte terminal differentiation, growth plate architecture and skeletal cell fates.

Directory of Open Access Journals (Sweden)

Margot E Bowen

Full Text Available Loss of PTPN11/SHP2 in mice or in human metachondromatosis (MC patients causes benign cartilage tumors on the bone surface (exostoses and within bones (enchondromas. To elucidate the mechanisms underlying cartilage tumor formation, we investigated the role of SHP2 in the specification, maturation and organization of chondrocytes. Firstly, we studied chondrocyte maturation by performing RNA-seq on primary chondrocyte pellet cultures. We found that SHP2 depletion, or inhibition of the ERK1/2 pathway, delays the terminal differentiation of chondrocytes from the early-hypertrophic to the late-hypertrophic stage. Secondly, we studied chondrocyte maturation and organization in mice with a mosaic postnatal inactivation of Ptpn11 in chondrocytes. We found that the vertebral growth plates of these mice have expanded domains of early-hypertrophic chondrocytes that have not yet terminally differentiated, and their enchondroma-like lesions arise from chondrocytes displaced from the growth plate due to a disruption in the organization of maturation and ossification zones. Furthermore, we observed that lesions from human MC patients also display disorganized chondrocyte maturation zones. Next, we found that inactivation of Ptpn11 in Fsp1-Cre-expressing fibroblasts induces exostosis-like outgrowths, suggesting that loss of SHP2 in cells on the bone surface and at bone-ligament attachment sites induces ectopic chondrogenesis. Finally, we performed lineage tracing to show that exostoses and enchondromas in mice likely contain mixtures of wild-type and SHP2-deficient chondrocytes. Together, these data indicate that in patients with MC, who are heterozygous for inherited PTPN11 loss-of-function mutations, second-hit mutations in PTPN11 can induce enchondromas by disrupting the organization and delaying the terminal differentiation of growth plate chondrocytes, and can induce exostoses by causing ectopic chondrogenesis of cells on the bone surface. Furthermore, the

Intra-articular osteoid osteoma as a differential diagnosis of diffuse mono-articular joint pain.

Science.gov (United States)

Rolvien, Tim; Zustin, Jozef; Mussawy, Haider; Schmidt, Tobias; Pogoda, Pia; Ueblacker, Peter

2016-11-04

The aim of this retrospective study was to investigate the frequency of intra-articular osteoid osteoma (iaOO) in a large study cohort and to demonstrate its clinical relevance as an important differential diagnosis of non-specific mono-articular joint pain. We searched the registry for bone tumours of the University Medical Centre Hamburg-Eppendorf for osteoid osteomas in the last 42 years. Herein, we present three selected iaOO which were detected in the three major weight-bearing joints. Computed tomography (CT) or magnetic resonance imaging (MRI) scans were performed for initial diagnosis. Out of a total of 367 osteoid osteomas, 19 (5.2 %) tumours were localized intra-articularly. In all three presented tumours, a history of severe mono-articular pain was reported; however, the mean time to correct diagnosis was delayed to 20.7 months. Clearly, the nidus seen in CT and MRI images in combination with inconsistent salicylate-responsive nocturnal pain led to the diagnosis of iaOO. Rarely, osteoid osteoma can occur in an intra-articular location. In cases of diffuse mono-articular pain, iaOO should be considered both in large and smaller joints to avoid delays in diagnosis and therapy of this benign bone tumour.

Rapid Chondrocyte Isolation for Tissue Engineering Applications: The Effect of Enzyme Concentration and Temporal Exposure on the Matrix Forming Capacity of Nasal Derived Chondrocytes

Directory of Open Access Journals (Sweden)

Srujana Vedicherla

2017-01-01

Full Text Available Laboratory based processing and expansion to yield adequate cell numbers had been the standard in Autologous Disc Chondrocyte Transplantation (ADCT, Allogeneic Juvenile Chondrocyte Implantation (NuQu®, and Matrix-Induced Autologous Chondrocyte Implantation (MACI. Optimizing cell isolation is a key challenge in terms of obtaining adequate cell numbers while maintaining a vibrant cell population capable of subsequent proliferation and matrix elaboration. However, typical cell yields from a cartilage digest are highly variable between donors and based on user competency. The overall objective of this study was to optimize chondrocyte isolation from cartilaginous nasal tissue through modulation of enzyme concentration exposure (750 and 3000 U/ml and incubation time (1 and 12 h, combined with physical agitation cycles, and to assess subsequent cell viability and matrix forming capacity. Overall, increasing enzyme exposure time was found to be more detrimental than collagenase concentration for subsequent viability, proliferation, and matrix forming capacity (sGAG and collagen of these cells resulting in nonuniform cartilaginous matrix deposition. Taken together, consolidating a 3000 U/ml collagenase digest of 1 h at a ratio of 10 ml/g of cartilage tissue with physical agitation cycles can improve efficiency of chondrocyte isolation, yielding robust, more uniform matrix formation.

Curcumin Inhibits Chondrocyte Hypertrophy of Mesenchymal Stem Cells through IHH and Notch Signaling Pathways.

Science.gov (United States)

Cao, Zhen; Dou, Ce; Dong, Shiwu

2017-01-01

Using tissue engineering technique to repair cartilage damage caused by osteoarthritis is a promising strategy. However, the regenerated tissue usually is fibrous cartilage, which has poor mechanical characteristics compared to hyaline cartilage. Chondrocyte hypertrophy plays an important role in this process. Thus, it is very important to find out a suitable way to maintain the phenotype of chondrocytes and inhibit chondrocyte hypertrophy. Curcumin deriving from turmeric was reported with anti-inflammatory and anti-tumor pharmacological effects. However, the role of curcumin in metabolism of chondrocytes, especially in the chondrocyte hypertrophy remains unclear. Mesenchymal stem cells (MSCs) are widely used in cartilage tissue engineering as seed cells. So we investigated the effect of curcumin on chondrogenesis and chondrocyte hypertrophy in MSCs through examination of cell viability, glycosaminoglycan synthesis and specific gene expression. We found curcumin had no effect on expression of chondrogenic markers including Sox9 and Col2a1 while hypertrophic markers including Runx2 and Col10a1 were down-regulated. Further exploration showed that curcumin inhibited chondrocyte hypertrophy through Indian hedgehog homolog (IHH) and Notch signalings. Our results indicated curcumin was a potential agent in modulating cartilage homeostasis and maintaining chondrocyte phenotype.

Adipose-Derived Mesenchymal Stem Cells for the Treatment of Articular Cartilage: A Systematic Review on Preclinical and Clinical Evidence

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Francesco Perdisa

2015-01-01

Full Text Available Among the current therapeutic approaches for the regeneration of damaged articular cartilage, none has yet proven to offer results comparable to those of native hyaline cartilage. Recently, it has been claimed that the use of mesenchymal stem cells (MSCs provides greater regenerative potential than differentiated cells, such as chondrocytes. Among the different kinds of MSCs available, adipose-derived mesenchymal stem cells (ADSCs are emerging due to their abundancy and easiness to harvest. However, their mechanism of action and potential for cartilage regeneration are still under investigation, and many other aspects still need to be clarified. The aim of this systematic review is to give an overview of in vivo studies dealing with ADSCs, by summarizing the main evidence for the treatment of cartilage disease of the knee.

Local anaesthetics and chondrotoxicty: What is the evidence?

LENUS (Irish Health Repository)

Baker, Joseph F

2012-11-01

Recent reports have suggested that local anaesthetic agents have a toxic effect on articular chondrocytes. This is despite the widespread intra-articular use of local anaesthetic agents following arthroscopic procedures for a number of years.

Three-dimensional assembly of tissue-engineered cartilage constructs results in cartilaginous tissue formation without retainment of zonal characteristics

NARCIS (Netherlands)

Schuurman, W; Harimulyo, E B; Gawlitta, D; Woodfield, T B F; Dhert, Wouter J A; van Weeren, P. René; Malda, J

Articular cartilage has limited regenerative capabilities. Chondrocytes from different layers of cartilage have specific properties, and regenerative approaches using zonal chondrocytes may yield better replication of the architecture of native cartilage than when using a single cell population. To

Secondary chondrocyte-derived Ihh stimulates proliferation of periosteal cells during chick development.

Science.gov (United States)

Buxton, Paul G; Hall, Brian; Archer, Charles W; Francis-West, Philippa

2003-10-01

The development of the skull is characterised by its dependence upon epigenetic influences. One of the most important of these is secondary chondrogenesis, which occurs following ossification within certain membrane bone periostea, as a result of biomechanical articulation. We have studied the genesis, character and function of the secondary chondrocytes of the quadratojugal of the chick between embryonic days 11 and 14. Analysis of gene expression revealed that secondary chondrocytes formed coincident with Sox9 upregulation from a precursor population expressing Cbfa1/Runx2: a reversal of the normal sequence. Such secondary chondrocytes rapidly acquired a phenotype that is a compound of prehypertrophic and hypertrophic chondrocytes, exited from the cell cycle and upregulated Ihh. Pulse and pulse/chase experiments with BrdU confirmed the germinal region as the highly proliferative source of the secondary chondrocytes, which formed by division of chondrocyte-committed precursors. By blocking Hh signalling in explant cultures we show that the enhanced proliferation of the germinal region surrounding the secondary chondrocytes derives from this Ihh source. Additionally, in vitro studies on membrane bone periosteal cells (non-germinal region) demonstrated that these cells can also respond to Ihh, and do so both by enhanced proliferation and precocious osteogenesis. Despite the pro-osteogenic effects of Ihh on periosteal cell differentiation, mechanical articulation of the quadratojugal/quadrate joint in explant culture revealed a negative role for articulation in the regulation of osteocalcin by germinal region descendants. Thus, the mechanical stimulus that is the spur to secondary chondrocyte formation appears able to override the osteogenic influence of Ihh on the periosteum, but does not interfere with the cell cycle-promoting component of Hh signalling.

Roles of Chondrocytes in Endochondral Bone Formation and Fracture Repair

Science.gov (United States)

Hinton, R.J.; Jing, Y.; Jing, J.; Feng, J.Q.

2016-01-01

The formation of the mandibular condylar cartilage (MCC) and its subchondral bone is an important but understudied topic in dental research. The current concept regarding endochondral bone formation postulates that most hypertrophic chondrocytes undergo programmed cell death prior to bone formation. Under this paradigm, the MCC and its underlying bone are thought to result from 2 closely linked but separate processes: chondrogenesis and osteogenesis. However, recent investigations using cell lineage tracing techniques have demonstrated that many, perhaps the majority, of bone cells are derived via direct transformation from chondrocytes. In this review, the authors will briefly discuss the history of this idea and describe recent studies that clearly demonstrate that the direct transformation of chondrocytes into bone cells is common in both long bone and mandibular condyle development and during bone fracture repair. The authors will also provide new evidence of a distinct difference in ossification orientation in the condylar ramus (1 ossification center) versus long bone ossification formation (2 ossification centers). Based on our recent findings and those of other laboratories, we propose a new model that contrasts the mode of bone formation in much of the mandibular ramus (chondrocyte-derived) with intramembranous bone formation of the mandibular body (non-chondrocyte-derived). PMID:27664203

Evaluation of the Thompson articular index

NARCIS (Netherlands)

van den Brink, H. R.; van der Heide, A.; Jacobs, J. W.; van der Veen, M. J.; Bijlsma, J. W.

1993-01-01

Three articular indices for measuring disease activity are compared. In a cross sectional study the Thompson articular index (a modified Lansbury index) correlated better with laboratory variables than the Ritchie articular index or a swollen joint score (Thompson 0.74-0.77; Ritchie 0.57-0.58;

Roles of Ihh signaling in chondroprogenitor function in postnatal condylar cartilage.

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Kurio, Naito; Saunders, Cheri; Bechtold, Till E; Salhab, Imad; Nah, Hyun-Duck; Sinha, Sayantani; Billings, Paul C; Pacifici, Maurizio; Koyama, Eiki

2018-04-01

Condylar articular cartilage in mouse temporomandibular joint develops from progenitor cells near the articulating surface that proliferate, undergo chondrogenesis and mature into hypertrophic chondrocytes. However, it remains unclear how these processes are regulated, particularly postnatally. Here we focused on the apical polymorphic layer rich in progenitors and asked whether the phenotype and fate of the cells require signaling by Indian hedgehog (Ihh) previously studied in developing long bones. In condyles in newborn mice, the apical polymorphic/progenitor cell layer was ~10 cell layer-thick and expressed the articular matrix marker Tenascin-C (Tn-C), and the underlying thick cell layer expressed Tn-C as well as the chondrogenic master regulator Sox9. By 1 month, condylar cartilage had gained its full width, but became thinner along its main longitudinal axis and displayed hypertrophic chondrocytes. By 3 months, articular cartilage consisted of a 2-3 cell layer-thick zone of superficial cells and chondroprogenitors expressing both Tn-C and Sox9 and a bottom zone of chondrocytes displaying vertical matrix septa. EdU cell tracing in juvenile mice revealed that conversion of chondroprogenitors into chondrocytes and hypertrophic chondrocytes required about 48 and 72 h, respectively. Notably, EdU injection in 3 month-old mice labeled both progenitors and maturing chondrocytes by 96 h. Conditional ablation of Ihh in juvenile/early adult mice compromised chondroprogenitor organization and function and led to reduced chondroprogenitor and chondrocyte proliferation. The phenotype of mutant condyles worsened over time as indicated by apoptotic chondrocyte incidence, ectopic chondrocyte hypertrophy, chondrocyte column derangement and subchondral bone deterioration. In micromass cultures of condylar apical cells, hedgehog (Hh) treatment stimulated chondrogenesis and alkaline phosphatase (APase) activity, while treatment with HhAntag inhibited both. Our findings

Atf4 regulates chondrocyte proliferation and differentiation during endochondral ossification by activating Ihh transcription.

Science.gov (United States)

Wang, Weiguang; Lian, Na; Li, Lingzhen; Moss, Heather E; Wang, Weixi; Perrien, Daniel S; Elefteriou, Florent; Yang, Xiangli

2009-12-01

Activating transcription factor 4 (Atf4) is a leucine-zipper-containing protein of the cAMP response element-binding protein (CREB) family. Ablation of Atf4 (Atf4(-/-)) in mice leads to severe skeletal defects, including delayed ossification and low bone mass, short stature and short limbs. Atf4 is expressed in proliferative and prehypertrophic growth plate chondrocytes, suggesting an autonomous function of Atf4 in chondrocytes during endochondral ossification. In Atf4(-/-) growth plate, the typical columnar structure of proliferative chondrocytes is disturbed. The proliferative zone is shortened, whereas the hypertrophic zone is transiently expanded. The expression of Indian hedgehog (Ihh) is markedly decreased, whereas the expression of other chondrocyte marker genes, such as type II collagen (Col2a1), PTH/PTHrP receptor (Pth1r) and type X collagen (Col10a1), is normal. Furthermore, forced expression of Atf4 in chondrocytes induces endogenous Ihh mRNA, and Atf4 directly binds to the Ihh promoter and activates its transcription. Supporting these findings, reactivation of Hh signaling pharmacologically in mouse limb explants corrects the Atf4(-/-) chondrocyte proliferation and short limb phenotypes. This study thus identifies Atf4 as a novel transcriptional activator of Ihh in chondrocytes that paces longitudinal bone growth by controlling growth plate chondrocyte proliferation and differentiation.

[Construction of porous hydroxyapatite (HA) block loaded with cultured chondrocytes].

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Yan, M; Dang, G

1999-07-01

To construct a kind of bone healing enhancing implant with cultured chondrocytes bound to hydroxyapatite (HA). Chondrocytes were obtained from the costicartilage of rat and were cultured on the porous HA blocks, 3 mm x 3 mm x 4 mm size, for three and seven days. Scanning electron micrograph was taken to show whether the cells grew outside and inside the pore of HA block. The cells cultured on tiny glass sheet for 2 days were used to prove where the cells come from by in situ hybridization technique with alpha1 (II) cDNA probe. Scanning electron micrographs showed that the pores of the HA surface and inside of the blocks are filled with cultured cells, especially the longer cultured block. The cells were chondrocytes confirmed by in situ hybridization. The porous HA can be used as cell cultured substrate and chondrocyte can adhere and proliferate inside the porous HA block.

[3T magnetic resonance T2 mapping for evaluation of cartilage repair after matrix-associated autologous chondrocyte transplantation].

Science.gov (United States)

Zhang, Jun; Xu, Xian; Li, Xue; Chen, Min; Dong, Tian-Ming; Zuo, Pan-Li; An, Ning-Yu

2015-01-01

To assess the value of magnetic resonance imaging (MRI) T2 mapping in quantitative evaluation of cartilage repair following matrix-associated autologous chondrocyte transplantation (MACT). Six patients (with 9 plug cartilages) following MACT underwent MRI on a 3.0 Tesla MR scan system at 3, 6 and 12 months after the surgery. The full-thickness and zonal areas (deep and superficial layers) T2 values were calculated for the repaired cartilage and control cartilage. The mean T2 values of the repaired cartilage after MACT were significantly higher than that of the control cartilages at 3 and 6 months (PT2 values of the superficial layers were significantly higher than those of the deep layers in the repaired cartilages (PT2 values of the repaired cartilages decreased significantly over time at 6 and 12 months as compared to those at 3 months after the surgery (PT2 mapping can serve as an important modality for assessing the repair of the articular cartilage following MACT.

Phlpp1 facilitates post-traumatic osteoarthritis and is induced by inflammation and promoter demethylation in human osteoarthritis

Science.gov (United States)

Bradley, Elizabeth W.; Carpio, Lomeli R.; McGee-Lawrence, Meghan E.; Becerra, Clara Castillejo; Amanatullah, Derek F.; Ta, Lauren E.; Otero, Miguel; Goldring, Mary B.; Kakar, Sanjeev; Westendorf, Jennifer J.

2016-01-01

OBJECTIVE Osteoarthritis (OA) is the most common form of arthritis and a leading cause of disability. OA is characterized by articular chondrocyte deterioration, subchondral bone changes and debilitating pain. One strategy to promote cartilage regeneration and repair is to accelerate proliferation and matrix production of articular chondrocytes. We previously reported that the protein phosphatase Phlpp1 controls chondrocyte differentiation by regulating the activities of anabolic kinases. Here we examined the role of Phlpp1 in osteoarthritis progression in a murine model. We also assessed PHLPP1 expression and promoter methylation. DESIGN Knee joints of WT and Phlpp1−/− mice were surgically destabilized by transection of the medial meniscal ligament (DMM). Mice were assessed for signs of OA progression via radiographic and histological analyses, and pain assessment for mechanical hypersensitivity using the von Frey assay. Methylation of the PHLPP1 promoter and PHLPP1 expression was evaluated in human articular cartilage and chondrocyte cell lines. RESULTS Following DMM surgeries, Phlpp1 deficient mice showed fewer signs of OA and cartilage degeneration. Mechanical allodynia associated with DMM surgeries was also attenuated in Phlpp1−/− mice. PHLPP1 was highly expressed in human articular cartilage from OA patients, but was undetectable in cartilage specimens from femoral neck fractures. Higher PHLPP1 levels correlated with less PHLPP1 promoter CpG methylation in cartilage from OA patients. Blocking cytosine methylation or treatment with inflammatory mediators enhanced PHLPP1 expression in human chondrocyte cell lines. CONCLUSION Phlpp1 deficiency protects against OA progression while CpG demethylation and inflammatory responses promote PHLPP1 expression. PMID:26746148

Conditional expression of constitutively active estrogen receptor α in chondrocytes impairs longitudinal bone growth in mice

International Nuclear Information System (INIS)

Ikeda, Kazuhiro; Tsukui, Tohru; Imazawa, Yukiko; Horie-Inoue, Kuniko; Inoue, Satoshi

2012-01-01

Highlights: ► Conditional transgenic mice expressing constitutively active estrogen receptor α (caERα) in chondrocytes were developed. ► Expression of caERα in chondrocytes impaired longitudinal bone growth in mice. ► caERα affects chondrocyte proliferation and differentiation. ► This mouse model is useful for understanding the physiological role of ERαin vivo. -- Abstract: Estrogen plays important roles in the regulation of chondrocyte proliferation and differentiation, which are essential steps for longitudinal bone growth; however, the mechanisms of estrogen action on chondrocytes have not been fully elucidated. In the present study, we generated conditional transgenic mice, designated as caERα ColII , expressing constitutively active mutant estrogen receptor (ER) α in chondrocytes, using the chondrocyte-specific type II collagen promoter-driven Cre transgenic mice. caERα ColII mice showed retardation in longitudinal growth, with short bone lengths. BrdU labeling showed reduced proliferation of hypertrophic chondrocytes in the proliferating layer of the growth plate of tibia in caERα ColII mice. In situ hybridization analysis of type X collagen revealed that the maturation of hypertrophic chondrocytes was impaired in caERα ColII mice. These results suggest that ERα is a critical regulator of chondrocyte proliferation and maturation during skeletal development, mediating longitudinal bone growth in vivo.

Single-Stage Cell-Based Cartilage Regeneration Using a Combination of Chondrons and Mesenchymal Stromal Cells: Comparison With Microfracture

NARCIS (Netherlands)

Bekkers, J.E.J.; Tsuchida, A.I.; van Rijen, M.H.P.; Vonk, L.A.; Dhert, W.J.A.; Saris, Daniël B.F.

2013-01-01

Background: Autologous chondrocyte implantation (ACI) is traditionally a 2-step procedure used to repair focal articular cartilage lesions. With use of a combination of chondrons (chondrocytes in their own territorial matrix) and mesenchymal stromal cells (MSCs), ACI could be innovated and performed

Efficiency of Human Epiphyseal Chondrocytes with Differential Replication Numbers for Cellular Therapy Products

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Michiyo Nasu

2016-01-01

Full Text Available The cell-based therapy for cartilage or bone requires a large number of cells; serial passages of chondrocytes are, therefore, needed. However, fates of expanded chondrocytes from extra fingers remain unclarified. The chondrocytes from human epiphyses morphologically changed from small polygonal cells to bipolar elongated spindle cells and to large polygonal cells with degeneration at early passages. Gene of type II collagen was expressed in the cells only at a primary culture (Passage 0 and Passage 1 (P1 cells. The nodules by implantation of P0 to P8 cells were composed of cartilage and perichondrium. The cartilage consisted of chondrocytes with round nuclei and type II collagen-positive matrix, and the perichondrium consisted of spindle cells with type I collage-positive matrix. The cartilage and perichondrium developed to bone with marrow cavity through enchondral ossification. Chondrogenesis and osteogenesis by epiphyseal chondrocytes depended on replication number in culture. It is noteworthy to take population doubling level in correlation with pharmaceutical efficacy into consideration when we use chondrocytes for cell-based therapies.

Effects of intermittent versus continuous parathyroid hormone administration on condylar chondrocyte proliferation and differentiation

International Nuclear Information System (INIS)

Liu, Qi; Wan, Qilong; Yang, Rongtao; Zhou, Haihua; Li, Zubing

2012-01-01

Highlights: ► Different PTH administration exerts different effects on condylar chondrocyte. ► Intermittent PTH administration suppresses condylar chondrocyte proliferation. ► Continuous PTH administration maintains condylar chondrocyte proliferating. ► Intermittent PTH administration enhances condylar chondrocyte differentiation. -- Abstract: Endochondral ossification is a complex process involving chondrogenesis and osteogenesis regulated by many hormones and growth factors. Parathyroid hormone (PTH), one of the key hormones regulating bone metabolism, promotes osteoblast differentiation and osteogenesis by intermittent administration, whereas continuous PTH administration inhibits bone formation. However, the effects of PTH on chondrocyte proliferation and differentiation are still unclear. In this study, intermittent PTH administration presented enhanced effects on condylar chondrocyte differentiation and bone formation, as demonstrated by increased mineral nodule formation and alkaline phosphatase (ALP) activity, up-regulated runt-related transcription factor 2 (RUNX2), ALP, collagen type X (COL10a1), collagen type I (COL1a1), osteocalcin (OCN), bone sialoprotein (BSP), bone morphogenetic protein 2 (BMP2) and osterix (OSX) mRNA and/or protein expression. On the contrary, continuous PTH administration promoted condylar chondrocyte proliferation and suppressed its differentiation, as demonstrated by up-regulated collagen type II (COL2a1) mRNA expression, reduced mineral nodule formation and down-regulated expression of the mRNAs and/or proteins mentioned above. Our data suggest that PTH can regulate condylar chondrocyte proliferation and differentiation, depending on the type of PTH administration. These results provide new insight into the effects of PTH on condylar chondrocytes and new evidence for using local PTH administration to cure mandibular asymmetry.

Modulation of Hyaluronan Synthesis by the Interaction between Mesenchymal Stem Cells and Osteoarthritic Chondrocytes

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Eliane Antonioli

2015-01-01

Full Text Available Bone marrow mesenchymal stem cells (BM-MSCs are considered a good source for cellular therapy in cartilage repair. But, their potential to repair the extracellular matrix, in an osteoarthritic environment, is still controversial. In osteoarthritis (OA, anti-inflammatory action and extracellular matrix production are important steps for cartilage healing. This study examined the interaction of BM-MSC and OA-chondrocyte on the production of hyaluronan and inflammatory cytokines in a Transwell system. We compared cocultured BM-MSCs and OA-chondrocytes with the individually cultured controls (monocultures. There was a decrease in BM-MSCs cell count in coculture with OA-chondrocytes when compared to BM-MSCs alone. In monoculture, BM-MSCs produced higher amounts of hyaluronan than OA-chondrocytes and coculture of BM-MSCs with OA-chondrocytes increased hyaluronan production per cell. Hyaluronan synthase-1 mRNA expression was upregulated in BM-MSCs after coculture with OA-chondrocytes, whereas hyaluronidase-1 was downregulated. After coculture, lower IL-6 levels were detected in BM-MSCs compared with OA-chondrocytes. These results indicate that, in response to coculture with OA-chondrocytes, BM-MSCs change their behavior by increasing production of hyaluronan and decreasing inflammatory cytokines. Our results indicate that BM-MSCs per se could be a potential tool for OA regenerative therapy, exerting short-term effects on the local microenvironment even when cell:cell contact is not occurring.

Inhibition of Chondrocyte Hypertrophy of Osteoarthritis by Disruptor Peptide

Science.gov (United States)

2017-07-01

with PTHR and inhibits the pathogenic beta-catenin- mediated PTHR signaling switch. In Aim 2, we will define the role of disruptor peptide in...confirmed that disruptor peptide conjugated to penetratin can enter cells. Importantly, disruptor peptide can reverse the beta-catenin- mediated PTH... mediated PTHR signaling switch in chondrocytes. Mouse primary chondrocytes express both β-catenin and PTHR. Our data showed that Pen-dis- pep

The embryogenesis of the equine femorotibial joint : The equine interzone

NARCIS (Netherlands)

Jenner, F; van Osch, G J V M; Weninger, W; Geyer, S; Stout, T; van Weeren, René; Brama, P; van Weeren, René

REASONS FOR PERFORMING STUDY: Articular cartilage regeneration is the focus and goal of considerable research effort. Since articular chondrocytes descend from a distinct cohort of progenitor cells located in embryonic nascent joints (interzones), establishing the timing of equine interzone

High seeding density of human chondrocytes in agarose produces tissue-engineered cartilage approaching native mechanical and biochemical properties.

Science.gov (United States)

Cigan, Alexander D; Roach, Brendan L; Nims, Robert J; Tan, Andrea R; Albro, Michael B; Stoker, Aaron M; Cook, James L; Vunjak-Novakovic, Gordana; Hung, Clark T; Ateshian, Gerard A

2016-06-14

Animal cells have served as highly controllable model systems for furthering cartilage tissue engineering practices in pursuit of treating osteoarthritis. Although successful strategies for animal cells must ultimately be adapted to human cells to be clinically relevant, human chondrocytes are rarely employed in such studies. In this study, we evaluated the applicability of culture techniques established for juvenile bovine and adult canine chondrocytes to human chondrocytes obtained from fresh or expired osteochondral allografts. Human chondrocytes were expanded and encapsulated in 2% agarose scaffolds measuring ∅3-4mm×2.3mm, with cell seeding densities ranging from 15 to 90×10(6)cells/mL. Subsets of constructs were subjected to transient or sustained TGF-β treatment, or provided channels to enhance nutrient transport. Human cartilaginous constructs physically resembled native human cartilage, and reached compressive Young's moduli of up to ~250kPa (corresponding to the low end of ranges reported for native knee cartilage), dynamic moduli of ~950kPa (0.01Hz), and contained 5.7% wet weight (%/ww) of glycosaminoglycans (≥ native levels) and 1.5%/ww collagen. We found that the initial seeding density had pronounced effects on tissue outcomes, with high cell seeding densities significantly increasing nearly all measured properties. Transient TGF-β treatment was ineffective for adult human cells, and tissue construct properties plateaued or declined beyond 28 days of culture. Finally, nutrient channels improved construct mechanical properties, presumably due to enhanced rates of mass transport. These results demonstrate that our previously established culture system can be successfully translated to human chondrocytes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Study of differential properties of fibrochondrocytes and hyaline chondrocytes in growing rabbits.

Science.gov (United States)

Huang, L; Li, M; Li, H; Yang, C; Cai, X

2015-02-01

We aimed to build a culture model of chondrocytes in vitro, and to study the differential properties between fibrochondrocytes and hyaline chondrocytes. Histological sections were stained with haematoxylin and eosin so that we could analyse the histological structure of the fibrocartilage and hyaline cartilage. Condylar fibrochondrocytes and femoral hyaline chondrocytes were cultured from four, 4-week-old, New Zealand white rabbits. The production of COL2A1, COL1OA1, SOX9 and aggrecan was detected by real time-q polymerase chain reaction (RT-qPCR) and immunoblotting and the differences between them were compared statistically. Histological structures obviously differed between fibrocartilage and hyaline cartilage. COL2A1 and SOX9 were highly expressed within cell passage 2 (P2) of both fibrochondrocytes and hyaline chondrocytes, and reduced significantly after cell passage 4 (P4). The mRNA expressions of COL2A1 (p=0.05), COL10A1 (p=0.04), SOX9 (p=0.03), and aggrecan (p=0.04) were significantly higher in hyaline chondrocytes than in fibrochondrocytes, whereas the expression of COL1A1 (p=0.02) was the opposite. Immunoblotting showed similar results. We have built a simple and effective culture model of chondrocytes in vitro, and the P2 of chondrocytes is recommended for further studies. Condylar fibrocartilage and femoral hyaline cartilage have unique biological properties, and the regulatory mechanisms of endochondral ossification for the condyle should be studied independently in the future. Copyright © 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Culture temperature affects redifferentiation and cartilaginous extracellular matrix formation in dedifferentiated human chondrocytes.

Science.gov (United States)

Ito, Akira; Aoyama, Tomoki; Iijima, Hirotaka; Tajino, Junichi; Nagai, Momoko; Yamaguchi, Shoki; Zhang, Xiangkai; Kuroki, Hiroshi

2015-05-01

To date, there have been few studies on how temperature affects the phenotype and metabolism of human chondrocytes. Thus, the purpose of this study was to elucidate the effects of culture temperature on chondrocyte redifferentiation and extracellular matrix (ECM) formation using dedifferentiated mature human chondrocytes in vitro. Dedifferentiated chondrocytes were cultured in a pellet culture system for up to 21 days. The pellets were randomly divided into three groups with different culture temperature (32, 37, and 41°C). Chondrocyte redifferentiation and ECM formation were evaluated by wet weight, messenger ribonucleic acid (mRNA), histological, and biochemical analyses. The results showed that the wet weight and the mRNA expressions of collagen type II A1 and cartilage oligomeric matrix protein at 37°C were higher than the corresponding values at 32°C. The histological and biochemical analyses revealed that the syntheses of type II collagen and proteoglycan were promoted at 37°C compared to those at 32°C, whereas they were considerably inhibited at 41°C. In conclusion, the results obtained herein indicated that temperature affects chondrocyte redifferentiation and ECM formation, and modulation of temperature might thus represent an advantageous means to regulate the phenotype and biosynthetic activity of chondrocytes. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Definition of pertinent parameters for the evaluation of articular cartilage repair tissue with high-resolution magnetic resonance imaging

International Nuclear Information System (INIS)

Marlovits, Stefan; Striessnig, Gabriele; Resinger, Christoph T.; Aldrian, Silke M.; Vecsei, Vilmos; Imhof, Herwig; Trattnig, Siegfried

2004-01-01

To evaluate articular cartilage repair tissue after biological cartilage repair, we propose a new technique of non-invasive, high-resolution magnetic resonance imaging (MRI) and define a new classification system. For the definition of pertinent variables the repair tissue of 45 patients treated with three different techniques for cartilage repair (microfracture, autologous osteochondral transplantation, and autologous chondrocyte transplantation) was analyzed 6 and 12 months after the procedure. High-resolution imaging was obtained with a surface phased array coil placed over the knee compartment of interest and adapted sequences were used on a 1 T MRI scanner. The analysis of the repair tissue included the definition and rating of nine pertinent variables: the degree of filling of the defect, the integration to the border zone, the description of the surface and structure, the signal intensity, the status of the subchondral lamina and subchondral bone, the appearance of adhesions and the presence of synovitis. High-resolution MRI, using a surface phased array coil and specific sequences, can be used on every standard 1 or 1.5 T MRI scanner according to the in-house standard protocols for knee imaging in patients who have had cartilage repair procedures without substantially prolonging the total imaging time. The new classification and grading system allows a subtle description and suitable assessment of the articular cartilage repair tissue

Fisiopatología celular de la osteoartritis: el condrocito articular como protagonista = Osteoarthritis cellular pathophysiology: The articular chondrocyte as a central player

Directory of Open Access Journals (Sweden)

Sánchez Naranjo, Julio César

2011-06-01

Full Text Available La osteoartritis es una de las enfermedades más prevalentes y que más discapacidad produce en todo el mundo, lo que ocasiona costos altos para el paciente y la sociedad. En años recientes se ha venido obteniendo información importante sobre el funcionamiento normal del condrocito, la única célula presente en el cartílago articular y responsable de la síntesis de matriz extracelular. El condrocito responde a las condiciones fluctuantes del medio, generadas por los cambios de presión, modificando su composición iónica y alterando el transporte de solutos y agua en su membrana. Esta capacidad de respuesta es clave para el mantenimiento de la matriz extracelular y, por ende, de un cartílago funcional. Diversos factores relacionados con enfermedades crónicas metabólicas inician una cascada de eventos que termina con una respuesta inadecuada del condrocito ante la carga mecánica, lo cual lleva a un predominio del catabolismo de la matriz y a un cartílago defectuoso que es la base del desarrollo de la osteoartritis. En este proceso están implicadas diversas citocinas y hormonas que afectan la homeostasis del cartílago y que pueden constituirse en blancos terapéuticos prometedores.

First and second order stereology of hyaline cartilage: Application on mice femoral cartilage.

Science.gov (United States)

Noorafshan, Ali; Niazi, Behnam; Mohamadpour, Masoomeh; Hoseini, Leila; Hoseini, Najmeh; Owji, Ali Akbar; Rafati, Ali; Sadeghi, Yasaman; Karbalay-Doust, Saied

2016-11-01

Stereological techniques could be considered in research on cartilage to obtain quantitative data. The present study aimed to explain application of the first- and second-order stereological methods on articular cartilage of mice and the methods applied on the mice exposed to cadmium (Cd). The distal femoral articular cartilage of BALB/c mice (control and Cd-treated) was removed. Then, volume and surface area of the cartilage and number of chondrocytes were estimated using Cavalieri and optical dissector techniques on isotropic uniform random sections. Pair-correlation function [g(r)] and cross-correlation function were calculated to express the spatial arrangement of chondrocytes-chondrocytes and chondrocytes-matrix (chondrocyte clustering/dispersing), respectively. The mean±standard deviation of the cartilage volume, surface area, and thickness were 1.4±0.1mm 3 , 26.2±5.4mm 2 , and 52.8±6.7μm, respectively. Besides, the mean number of chondrocytes was 680±200 (×10 3 ). The cartilage volume, cartilage surface area, and number of chondrocytes were respectively reduced by 25%, 27%, and 27% in the Cd-treated mice in comparison to the control animals (pcartilage components carried potential advantages for investigating the cartilage in different joint conditions. Chondrocyte clustering/dispersing and cellularity can be evaluated in cartilage assessment in normal or abnormal situations. Copyright © 2016 Elsevier GmbH. All rights reserved.

Effects of intermittent versus continuous parathyroid hormone administration on condylar chondrocyte proliferation and differentiation

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Liu, Qi; Wan, Qilong; Yang, Rongtao; Zhou, Haihua [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); Li, Zubing, E-mail: lizubing0827@163.com [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China)

2012-07-20

Highlights: Black-Right-Pointing-Pointer Different PTH administration exerts different effects on condylar chondrocyte. Black-Right-Pointing-Pointer Intermittent PTH administration suppresses condylar chondrocyte proliferation. Black-Right-Pointing-Pointer Continuous PTH administration maintains condylar chondrocyte proliferating. Black-Right-Pointing-Pointer Intermittent PTH administration enhances condylar chondrocyte differentiation. -- Abstract: Endochondral ossification is a complex process involving chondrogenesis and osteogenesis regulated by many hormones and growth factors. Parathyroid hormone (PTH), one of the key hormones regulating bone metabolism, promotes osteoblast differentiation and osteogenesis by intermittent administration, whereas continuous PTH administration inhibits bone formation. However, the effects of PTH on chondrocyte proliferation and differentiation are still unclear. In this study, intermittent PTH administration presented enhanced effects on condylar chondrocyte differentiation and bone formation, as demonstrated by increased mineral nodule formation and alkaline phosphatase (ALP) activity, up-regulated runt-related transcription factor 2 (RUNX2), ALP, collagen type X (COL10a1), collagen type I (COL1a1), osteocalcin (OCN), bone sialoprotein (BSP), bone morphogenetic protein 2 (BMP2) and osterix (OSX) mRNA and/or protein expression. On the contrary, continuous PTH administration promoted condylar chondrocyte proliferation and suppressed its differentiation, as demonstrated by up-regulated collagen type II (COL2a1) mRNA expression, reduced mineral nodule formation and down-regulated expression of the mRNAs and/or proteins mentioned above. Our data suggest that PTH can regulate condylar chondrocyte proliferation and differentiation, depending on the type of PTH administration. These results provide new insight into the effects of PTH on condylar chondrocytes and new evidence for using local PTH administration to cure mandibular

Smad6/Smurf1 overexpression in cartilage delays chondrocyte hypertrophy and causes dwarfism with osteopenia

Science.gov (United States)

Horiki, Mitsuru; Imamura, Takeshi; Okamoto, Mina; Hayashi, Makoto; Murai, Junko; Myoui, Akira; Ochi, Takahiro; Miyazono, Kohei; Yoshikawa, Hideki; Tsumaki, Noriyuki

2004-01-01

Biochemical experiments have shown that Smad6 and Smad ubiquitin regulatory factor 1 (Smurf1) block the signal transduction of bone morphogenetic proteins (BMPs). However, their in vivo functions are largely unknown. Here, we generated transgenic mice overexpressing Smad6 in chondrocytes. Smad6 transgenic mice showed postnatal dwarfism with osteopenia and inhibition of Smad1/5/8 phosphorylation in chondrocytes. Endochondral ossification during development in these mice was associated with almost normal chondrocyte proliferation, significantly delayed chondrocyte hypertrophy, and thin trabecular bone. The reduced population of hypertrophic chondrocytes after birth seemed to be related to impaired bone growth and formation. Organ culture of cartilage rudiments showed that chondrocyte hypertrophy induced by BMP2 was inhibited in cartilage prepared from Smad6 transgenic mice. We then generated transgenic mice overexpressing Smurf1 in chondrocytes. Abnormalities were undetectable in Smurf1 transgenic mice. Mating Smad6 and Smurf1 transgenic mice produced double-transgenic pups with more delayed endochondral ossification than Smad6 transgenic mice. These results provided evidence that Smurf1 supports Smad6 function in vivo. PMID:15123739

Conditional expression of constitutively active estrogen receptor {alpha} in chondrocytes impairs longitudinal bone growth in mice

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Ikeda, Kazuhiro [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Tsukui, Tohru [Experimental Animal Laboratory, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Imazawa, Yukiko; Horie-Inoue, Kuniko [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Inoue, Satoshi, E-mail: INOUE-GER@h.u-tokyo.ac.jp [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo (Japan); Department of Anti-Aging Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo (Japan)

2012-09-07

Highlights: Black-Right-Pointing-Pointer Conditional transgenic mice expressing constitutively active estrogen receptor {alpha} (caER{alpha}) in chondrocytes were developed. Black-Right-Pointing-Pointer Expression of caER{alpha} in chondrocytes impaired longitudinal bone growth in mice. Black-Right-Pointing-Pointer caER{alpha} affects chondrocyte proliferation and differentiation. Black-Right-Pointing-Pointer This mouse model is useful for understanding the physiological role of ER{alpha}in vivo. -- Abstract: Estrogen plays important roles in the regulation of chondrocyte proliferation and differentiation, which are essential steps for longitudinal bone growth; however, the mechanisms of estrogen action on chondrocytes have not been fully elucidated. In the present study, we generated conditional transgenic mice, designated as caER{alpha}{sup ColII}, expressing constitutively active mutant estrogen receptor (ER) {alpha} in chondrocytes, using the chondrocyte-specific type II collagen promoter-driven Cre transgenic mice. caER{alpha}{sup ColII} mice showed retardation in longitudinal growth, with short bone lengths. BrdU labeling showed reduced proliferation of hypertrophic chondrocytes in the proliferating layer of the growth plate of tibia in caER{alpha}{sup ColII} mice. In situ hybridization analysis of type X collagen revealed that the maturation of hypertrophic chondrocytes was impaired in caER{alpha}{sup ColII} mice. These results suggest that ER{alpha} is a critical regulator of chondrocyte proliferation and maturation during skeletal development, mediating longitudinal bone growth in vivo.

Cyclic Equibiaxial Tensile Strain Alters Gene Expression of Chondrocytes via Histone Deacetylase 4 Shuttling.

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Chongwei Chen

Full Text Available This paper aims to investigate whether equibiaxial tensile strain alters chondrocyte gene expression via controlling subcellular localization of histone deacetylase 4 (HDAC4.Murine chondrocytes transfected with GFP-HDAC4 were subjected to 3 h cyclic equibiaxial tensile strain (CTS, 6% strain at 0.25 Hz by a Flexcell® FX-5000™ Tension System. Fluorescence microscope and western blot were used to observe subcellular location of HDAC4. The gene expression was analyzed by real-time RT-PCR. The concentration of Glycosaminoglycans in culture medium was quantified by bimethylmethylene blue dye; Collagen II protein was evaluated by western blot. Cells phenotype was identified by immunohistochemistry. Cell viability was evaluated by live-dead cell detect kit. Okadaic acid, an inhibitor of HDAC4 nuclear relocation, was used to further validate whether HDAC4 nuclear relocation plays a role in gene expression in response to tension stimulation.87.5% of HDAC4 was located in the cytoplasm in chondrocytes under no loading condition, but it was relocated to the nucleus after CTS. RT-PCR analysis showed that levels of mRNA for aggrecan, collagen II, LK1 and SOX9 were all increased in chondrocytes subjected to CTS as compared to no loading control chondrocytes; in contrast, the levels of type X collagen, MMP-13, IHH and Runx2 gene expression were decreased in the chondrocytes subjected to CTS as compared to control chondrocytes. Meanwhile, CTS contributed to elevation of glycosaminoglycans and collagen II protein, but did not change collagen I production. When Okadaic acid blocked HDAC4 relocation from the cytoplasm to nucleus, the changes of the chondrocytes induced by CTS were abrogated. There was no chondrocyte dead detected in this study in response to CTS.CTS is able to induce HDAC4 relocation from cytoplasm to nucleus. Thus, CTS alters chondrocytes gene expression in association with the relocation of HDAC4 induced by CTS.

Centrifugation assay for measuring adhesion of serially passaged bovine chondrocytes to polystyrene surfaces.

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Kaplan, David S; Hitchins, Victoria M; Vegella, Thomas J; Malinauskas, Richard A; Ferlin, Kimberly M; Fisher, John P; Frondoza, Carmelita G

2012-07-01

A major obstacle in chondrocyte-based therapy for cartilage repair is the limited availability of cells that maintain their original phenotype. Propagation of chondrocytes as monolayer cultures on polystyrene surfaces is used extensively for amplifying cell numbers. However, chondrocytes undergo a phenotypic shift when propagated in this manner and display characteristics of more adherent fibroblastic cells. Little information is available about the effect of this phenotypic shift on cellular adhesion properties. We evaluated changes in adhesion property as bovine chondrocytes were serially propagated up to five passages in monolayer culture using a centrifugation cell adhesion assay, which was based on counting of cells before and after being exposed to centrifugal dislodgement forces of 120 and 350 g. Chondrocytes proliferated well in a monolayer culture with doubling times of 2-3 days, but they appeared more fibroblastic and exhibited elongated cell morphology with continued passage. The centrifugation cell adhesion assay showed that chondrocytes became more adhesive with passage as the percentage of adherent cells after centrifugation increased and was not statistically different from the adhesion of the fibroblast cell line, L929, starting at passage 3. This increased adhesiveness correlated with a shift to a fibroblastic morphology and increased collagen I mRNA expression starting at passage 2. Our findings indicate that the centrifugation cell adhesion assay may serve as a reproducible tool to track alterations in chondrocyte phenotype during their extended propagation in culture.

RAGE, receptor of advanced glycation endoproducts, negatively regulates chondrocytes differentiation.

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Tatsuya Kosaka

Full Text Available RAGE, receptor for advanced glycation endoproducts (AGE, has been characterized as an activator of osteoclastgenesis. However, whether RAGE directly regulates chondrocyte proliferation and differentiation is unclear. Here, we show that RAGE has an inhibitory role in chondrocyte differentiation. RAGE expression was observed in chondrocytes from the prehypertrophic to hypertrophic regions. In cultured cells, overexpression of RAGE or dominant-negative-RAGE (DN-RAGE demonstrated that RAGE inhibited cartilaginous matrix production, while DN-RAGE promoted production. Additionally, RAGE regulated Ihh and Col10a1 negatively but upregulated PTHrP receptor. Ihh promoter analysis and real-time PCR analysis suggested that downregulation of Cdxs was the key for RAGE-induced inhibition of chondrocyte differentiation. Overexpression of the NF-κB inhibitor I-κB-SR inhibited RAGE-induced NF-κB activation, but did not influence inhibition of cartilaginous matrix production by RAGE. The inhibitory action of RAGE was restored by the Rho family GTPases inhibitor Toxin B. Furthermore, inhibitory action on Ihh, Col10a1 and Cdxs was reproduced by constitutively active forms, L63RhoA, L61Rac, and L61Cdc42, but not by I-κB-SR. Cdx1 induced Ihh and Col10a1 expressions and directly interacted with Ihh promoter. Retinoic acid (RA partially rescued the inhibitory action of RAGE. These data combined suggests that RAGE negatively regulates chondrocyte differentiation at the prehypertrophic stage by modulating NF-κB-independent and Rho family GTPases-dependent mechanisms.

Msx2 Stimulates Chondrocyte Maturation by Controlling Ihh Expression*

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Amano, Katsuhiko; Ichida, Fumitaka; Sugita, Atsushi; Hata, Kenji; Wada, Masahiro; Takigawa, Yoko; Nakanishi, Masako; Kogo, Mikihiko; Nishimura, Riko; Yoneda, Toshiyuki

2008-01-01

Several studies indicated that a homeobox gene, Msx2, is implicated in regulation of skeletal development by controlling enchondral ossification as well as membranous ossification. However, the molecular basis by which Msx2 conducts chondrogenesis is currently unclear. In this study, we examined the role of Msx2 in chondrocyte differentiation using mouse primary chondrocytes and embryonic metatarsal explants. Treatment with BMP2 up-regulated the expression of Msx2 mRNA...

Beta1 integrins regulate chondrocyte rotation, G1 progression, and cytokinesis

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Aszodi, Attila; Hunziker, Ernst B; Brakebusch, Cord

2003-01-01

Beta1 integrins are highly expressed on chondrocytes, where they mediate adhesion to cartilage matrix proteins. To assess the functions of beta1 integrin during skeletogenesis, we inactivated the beta1 integrin gene in chondrocytes. We show here that these mutant mice develop a chondrodysplasia...... of various severity. beta1-deficient chondrocytes had an abnormal shape and failed to arrange into columns in the growth plate. This is caused by a lack of motility, which is in turn caused by a loss of adhesion to collagen type II, reduced binding to and impaired spreading on fibronectin, and an abnormal F......-actin organization. In addition, mutant chondrocytes show decreased proliferation caused by a defect in G1/S transition and cytokinesis. The G1/S defect is, at least partially, caused by overexpression of Fgfr3, nuclear translocation of Stat1/Stat5a, and up-regulation of the cell cycle inhibitors p16 and p21...

Histone Deacetylase 3 Suppresses Erk Phosphorylation and Matrix Metalloproteinase (Mmp)-13 Activity in Chondrocytes

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Carpio, Lomeli R.; Bradley, Elizabeth W.; Westendorf, Jennifer J.

2017-01-01

Histone deacetylase inhibitors are emerging therapies for many diseases including cancers and neurological disorders; however, these drugs are teratogens to the developing skeleton. Hdac3 is essential for proper endochondral ossification as its deletion in chondrocytes increases cytokine signaling and the expression of matrix remodeling enzymes. Here we explored the mechanism by which Hdac3 controls Mmp13 expression in chondrocytes. In Hdac3-depleted chondrocytes, Erk1/2 as well as its downstream substrate, Runx2, were hyperphosphorylated as a result of decreased expression and activity of the Erk1/2 specific phosphatase, Dusp6. Erk1/2 kinase inhibitors and Dusp6 adenoviruses reduced Mmp13 expression and partially rescued matrix production in Hdac3-deficient chondrocytes. Postnatal chondrocyte-specific deletion of Hdac3 with an inducible Col2a1-Cre caused premature production of pErk1/2 and Mmp13 in the growth plate. Thus, Hdac3 controls the temporal and spatial expression of tissue-remodeling genes in chondrocytes to ensure proper endochondral ossification during development. PMID:27662443

Passaged adult chondrocytes can form engineered cartilage with functional mechanical properties: a canine model.

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Ng, Kenneth W; Lima, Eric G; Bian, Liming; O'Conor, Christopher J; Jayabalan, Prakash S; Stoker, Aaron M; Kuroki, Keiichi; Cook, Cristi R; Ateshian, Gerard A; Cook, James L; Hung, Clark T

2010-03-01

It was hypothesized that previously optimized serum-free culture conditions for juvenile bovine chondrocytes could be adapted to generate engineered cartilage with physiologic mechanical properties in a preclinical, adult canine model. Primary or passaged (using growth factors) adult chondrocytes from three adult dogs were encapsulated in agarose, and cultured in serum-free media with transforming growth factor-beta3. After 28 days in culture, engineered cartilage formed by primary chondrocytes exhibited only small increases in glycosaminoglycan content. However, all passaged chondrocytes on day 28 elaborated a cartilage matrix with compressive properties and glycosaminoglycan content in the range of native adult canine cartilage values. A preliminary biocompatibility study utilizing chondral and osteochondral constructs showed no gross or histological signs of rejection, with all implanted constructs showing excellent integration with surrounding cartilage and subchondral bone. This study demonstrates that adult canine chondrocytes can form a mechanically functional, biocompatible engineered cartilage tissue under optimized culture conditions. The encouraging findings of this work highlight the potential for tissue engineering strategies using adult chondrocytes in the clinical treatment of cartilage defects.

Visualization of the extra-articular portion of the long head of the biceps tendon during intra-articular shoulder arthroscopy.

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Festa, Anthony; Allert, Jesse; Issa, Kimona; Tasto, James P; Myer, Jonathan J

2014-11-01

To quantify the amount of the extra-articular long head of the biceps tendon (LHBT) seen during intra-articular shoulder arthroscopy by pulling the tendon into the joint with a probe through an anterior portal while viewing through a standard posterior portal. Intra-articular shoulder arthroscopy was performed on 10 forequarter cadaveric specimens. The extra-articular portion of the LHBT was evaluated by pulling the tendon into the joint with an arthroscopic probe inserted through an anterior portal. The tendon was marked at the pulley insertion on the humerus with a vascular clip before and after the tendon was pulled into the joint. An open deltopectoral approach was performed, and the amount of extra-articular tendon visualized was calculated as an absolute amount and in relation to nearby anatomic structures. An additional 1.9 cm (range, 1.4 to 2.6 cm) of extra-articular LHBT was viewed by pulling the tendon into the joint with an arthroscopic probe through an anterior portal during shoulder arthroscopy. This represented 30.8% of the extra-articular portion of the tendon, 47.7% of tendon in the bicipital groove, and 76.3% of the tendon that lies under the area from the pulley insertion to the distal edge of the transverse humeral ligament. During intra-articular shoulder arthroscopy, the extra-articular portion of the LHBT is incompletely visualized by pulling the tendon into the joint with a probe placed through an anterior portal while viewing through a standard posterior portal. An additional extra-articular portion of the LHBT may be viewed by pulling the tendon into the joint with an arthroscopic probe during shoulder arthroscopy. Copyright © 2014 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

Imaging of the cervical articular pillar

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Yeomans, E. [Orange Base Hospital, Orange, NSW (Australia)

1998-12-01

The cervical articular pillar, due to the complex anatomical structure of the cervical spine, is not well demonstrated in routine plain radiographic views. Dedicated views have been devised to demonstrate the pillar, yet their performance has abated considerably since the inception of Computed Tomography (CT) in the 1970`s. It is the consideration that CT does not image the articular pillar with a 10 per cent accuracy that poses the question: Is there still a need for plain radiography of the cervical articular pillar? This paper studies the anatomy, plain radiography, and incidence of injury to the cervical articular pillar. It discusses (with reference to current and historic literature) the efficacy of current imaging protocols in depicting this injury. It deals with plain radiography, CT, complex tomography, and Magnetic Resonance Imaging (MRI) of the cervical spine to conclude there may still be a position in current imaging protocols for plain radiography of the cervical articular pillar. Copyright (1998) Australian Institute of Radiography 43 refs., 5 figs.

Nicotine promotes proliferation and collagen synthesis of chondrocytes isolated from normal human and osteoarthritis patients.

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Ying, Xiaozhou; Cheng, Shaowen; Shen, Yue; Cheng, Xiaojie; An Rompis, Ferdinand; Wang, Wei; Lin, Zhongqin; Chen, Qingyu; Zhang, Wei; Kou, Dongquan; Peng, Lei; Tian, Xin Qiao; Lu, Chuan Zhu

2012-01-01

The aims of the study were to show the direct effect of nicotine with different concentrations (0, 25, 50, and 100 ng/ml) on chondrocytes isolated from normal human and osteoarthritis patients, respectively. Microscopic observation was performed during the culture with an inverted microscope. Methyl thiazolyl tetrazolium (MTT) assay method was adopted to observe the influence of nicotine on the proliferation of chondrocytes, and real-time PCR and ELISA were used to assay the mRNA and protein expression of type II collagen and aggrecan, respectively. We discovered that the OA chondrocytes were similar to fibroblasts in shape and grow slower than normal chondrocytes. The proliferation of the two kinds of chondrocytes was increased in a concentration-dependent manner and in a time-dependent manner (P<0.05). Also, we found that the mRNA level of type II collagen were upregulated under 25-100 ng/ml nicotine doses both in the two kinds of chondrocytes compared with control. The expression of protein levels of type II collagen were synthesized in line with the increase in mRNA. No effect was observed on aggrecan synthesis with any nicotine dose. We concluded that nicotine has the same effect on both chondrocytes, obtained either from osteoarthritis patients or from normal human, and the positive effect of smoking in OA may relate to the alteration in metabolism of chondrocytes.

New bioreactor vessel for tissue engineering of human nasal septal chondrocytes

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Princz Sascha

2016-09-01

Full Text Available Cultivation of human nasal septal chondrocytes in a self-established automated bioreactor system with a new designed reactor glass vessel and the results of a computational fluid dynamics model are presented. The first results show the effect of a homogeneous fluidic condition of the continuous medium flow and the resulting stresses on the scaffolds’ surface and their influence on the migration of the cells into the scaffold matrix under these conditions. For this purpose computational models, generated with the computational fluid dynamics software STAR-CCM+, and the results of alcian blue staining for newly synthesized sulphated glycosaminoglycans have been compared during cultivation in the new and a first version of the glass reactor vessel with inhomogeneous fluidic conditions, with the same automated bioreactor system and under similar cultivation conditions.

IGF1 regulates RUNX1 expression via IRS1/2: Implications for antler chondrocyte differentiation

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Yang, Zhan-Qing; Zhang, Hong-Liang; Duan, Cui-Cui; Geng, Shuang; Wang, Kai; Yu, Hai-Fan; Yue, Zhan-Peng; Guo, Bin

2017-01-01

Although IGF1 is important for the proliferation and differentiation of chondrocytes, its underlying molecular mechanism is still unknown. Here we addressed the physiologic function of IGF1 in antler cartilage and explored the interplay of IGF1, IRS1/2 and RUNX1 in chondrocyte differentiation. The results showed that IGF1 was highly expressed in antler chondrocytes. Exogenous rIGF1 could increase the proliferation of chondrocytes and cell proportion in the S phase, whereas IGF1R inhibitor PQ4...

Efeito do Plasma Rico em Plaquetas na apoptose pós-traumática de condrócitos Effect of Platelet-Rich Plasma on impact-induced chondrocyte apoptosis

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Márcia Uchôa de Rezende

2011-04-01

Full Text Available OBJETIVO: Avaliar se a injeção intra-articular de Plasma rico em plaquetas (PRP pode reduzir a apoptose pós-traumática de condrócitos. MÉTODOS: Foi desenvolvido um estudo experimental duplo-cego com quatro joelhos de coelhos adultos. Após a anestesia, os animais foram submetidos à contusão padronizada dos joelhos. Depois foi injetado 1ml de PRP humano nos dois joelhos esquerdos e 1ml de solução fisiológica (SF nos dois joelhos direitos. Os dois coelhos foram mantidos no mesmo ambiente sob controle de temperatura, de atividades diárias e de alimentação. A eutanásia dos animais ocorreu dez dias após a intervenção e foram realizadas biópsias da cartilagem de cada joelho. As peças foram preparadas para análise em microscopia eletrônica (ME. RESULTADOS: Quatro preparados para ME foram obtidos, cada um correspondendo a um joelho. Os joelhos-PRP apresentaram as taxas de apoptose de 47,62% (50/105 e de 48,36% (59/122, respectivamente. Nos joelhos-SF as taxas de apoptose foram, respectivamente, 56,67% (17/30 e 70,40% (88/125. A diferença do índice de apoptose nos joelhos-PRP (48,02% e nos joelhos-SF (67,74% foi significante (pOBJECTIVE: To evaluate if the injection of intra-articular platelet-rich plasma (PRP can reduce impact-induced chondrocyte apoptosis. METHODS: A double-blind experimental study was developed in four knees of two adult rabbits. Each knee was injured after anesthesia. Subsequently, 1ml PRP was injected in the right knees and 1ml of normal saline (NS in the left knees. The animals were euthanized ten days after the intervention. All cartilage was removed from the 4 knees and prepared for analysis in electron microscopy (EM. RESULTS: Four EM samples were obtained. The PRP-injected knees showed apoptosis rates of 47,62% (50/105 and 48,36% (59/122, respectively. NS-injected knees showed 56.67% (17/30 and 70.40% (88/125 of apoptosis. PRP-injected knees had statistically significant less apoptosis (48.02% than NS

Normal age-related viscoelastic properties of chondrons and chondrocytes isolated from rabbit knee

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DUAN Wang-ping; SUN Zhen-wei; LI Qi; LI Chun-jiang; WANG Li; CHEN Wei-yi; Jennifer Tickner; ZHENG Ming-hao; WEI Xiao-chun

2012-01-01

Background The mechanical microenvironment of the chondrocytes plays an important role in cartilage homeostasis and in the health of the joint.The pericellular matrix,cellular membrane of the chondrocytes,and their cytoskeletal structures are key elements in the mechanical environment.The aims of this study are to measure the viscoelastic properties of isolated chondrons and chondrocytes from rabbit knee cartilage using micropipette aspiration and to determine the effect of aging on these properties.Methods Three age groups of rabbit knees were evaluated:(1) young (2 months,n=10);(2) adult (8 months,n=10);and (3) old (31 months,n=10).Chondrocytes were isolated from the right knee cartilage and chondrons were isolated from left knees using enzymatic methods.Micropipette aspiration combined with a standard linear viscoelastic solid model was used to quantify changes in the viscoelastic properties of chondrons and chondrocytes within 2 hours of isolation.The morphology and structure of isolated chondrons were evaluated by optical microscope using hematoxylin and eosin staining and collagen-6 immunofluorescence staining.Results In response to an applied constant 0.3-0.4 kPa of negative pressure,all chondrocytes exhibited standard linear viscoelastic solid properties.Model predictions of the creep data showed that the average equilibrium modulus (E∞),instantaneous modulus (E0).and apparent viscosity (μ) of old chondrocytes was significantly lower than the young and adult chondrocytes (P＜0.001);however,no difference was found between young and adult chondrocytes (P＞0.05).The adult and old chondrons generally possessed a thicker pericellular matrix (PCM) with more enclosed cells.The young and adult chondrons exhibited the same viscoelastic creep behavior under a greater applied pressure (1.0-1.1kPa) without the deformation seen in the old chondrons.The viscoelastic properties (E∞,E0,and u) of young and adult chondrons were significantly greater than that observed

Single Cell Confocal Raman Spectroscopy of Human Osteoarthritic Chondrocytes: A Preliminary Study

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Rajesh Kumar

2015-04-01

Full Text Available A great deal of effort has been focused on exploring the underlying molecular mechanism of osteoarthritis (OA especially at the cellular level. We report a confocal Raman spectroscopic investigation on human osteoarthritic chondrocytes. The objective of this investigation is to identify molecular features and the stage of OA based on the spectral signatures corresponding to bio-molecular changes at the cellular level in chondrocytes. In this study, we isolated chondrocytes from human osteoarthritic cartilage and acquired Raman spectra from single cells. Major spectral differences between the cells obtained from different International Cartilage Repair Society (ICRS grades of osteoarthritic cartilage were identified. During progression of OA, a decrease in protein content and an increase in cell death were observed from the vibrational spectra. Principal component analysis and subsequent cross-validation was able to associate osteoarthritic chondrocytes to ICRS Grade I, II and III with specificity 100.0%, 98.1%, and 90.7% respectively, while, sensitivity was 98.6%, 82.8%, and 97.5% respectively. The overall predictive efficiency was 92.2%. Our pilot study encourages further use of Raman spectroscopy as a noninvasive and label free technique for revealing molecular features associated with osteoarthritic chondrocytes.

Pronounced biomaterial dependency in cartilage regeneration using nonexpanded compared with expanded chondrocytes

NARCIS (Netherlands)

Tsuchida, A.I.; Bekkers, J.E.J.; Beekhuizen, M.; Vonk, L.A.; Dhert, W.J.A.; Saris, Daniël B.F.; Creemers, L.B.

2013-01-01

We aimed to investigate freshly isolated compared with culture-expanded chondrocytes with respect to early regenerative response, cytokine production and cartilage formation in response to four commonly used biomaterials. Materials & methods: Chondrocytes were both directly and after expansion to

Normal proliferation and differentiation of Hoxc-8 transgenic chondrocytes in vitro

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Mello Maria

2003-04-01

Full Text Available Abstract Background Hox genes encode transcription factors that are involved in pattern formation in the skeleton, and recent evidence suggests that they also play a role in the regulation of endochondral ossification. To analyze the role of Hoxc-8 in this process in more detail, we applied in vitro culture systems, using high density cultures of primary chondrocytes from neonatal mouse ribs. Results Cultured cells were characterized on the basis of morphology (light microscopy and production of cartilage-specific extracellular matrix (sulfated proteoglycans and type II Collagen. Hypertrophy was demonstrated by increase in cell size, alkaline phosphatase activity and type X Collagen immunohistochemistry. Proliferation was assessed by BrdU uptake and flow cytometry. Unexpectedly, chondrocytes from Hoxc-8 transgenic mice, which exhibit delayed cartilage maturation in vivo 1, were able to proliferate and differentiate normally in our culture systems. This was the case even though freshly isolated Hoxc-8 transgenic chondrocytes exhibited significant molecular differences as measured by real-time quantitative PCR. Conclusions The results demonstrate that primary rib chondrocytes behave similar to published reports for chondrocytes from other sources, validating in vitro approaches for studies of Hox genes in the regulation of endochondral ossification. Our analysis of cartilage-producing cells from Hoxc-8 transgenic mice provides evidence that the cellular phenotype induced by Hoxc-8 overexpression in vivo is reversible in vitro.

Effect of chondrocyte-derived early extracellular matrix on chondrogenesis of placenta-derived mesenchymal stem cells.

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Park, Yong-Beom; Seo, Sinji; Kim, Jin-A; Heo, Jin-Chul; Lim, Young-Cheol; Ha, Chul-Won

2015-06-24

The extracellular matrix (ECM) surrounding cells contains a variety of proteins that provide structural support and regulate cellular functions. Previous studies have shown that decellularized ECM isolated from tissues or cultured cells can be used to improve cell differentiation in tissue engineering applications. In this study we evaluated the effect of decellularized chondrocyte-derived ECM (CDECM) on the chondrogenesis of human placenta-derived mesenchymal stem cells (hPDMSCs) in a pellet culture system. After incubation with or without chondrocyte-derived ECM in chondrogenic medium for 1 or 3 weeks, the sizes and wet masses of the cell pellets were compared with untreated controls (hPDMSCs incubated in chondrogenic medium without chondrocyte-derived ECM). In addition, histologic analysis of the cell pellets (Safranin O and collagen type II staining) and quantitative reverse transcription-PCR analysis of chondrogenic markers (aggrecan, collagen type II, and SOX9) were carried out. Our results showed that the sizes and masses of hPDMSC pellets incubated with chondrocyte-derived ECM were significantly higher than those of untreated controls. Differentiation of hPDMSCs (both with and without chondrocyte-derived ECM) was confirmed by Safranin O and collagen type II staining. Chondrogenic marker expression and glycosaminoglycan (GAG) levels were significantly higher in hPDMSC pellets incubated with chondrocyte-derived ECM compared with untreated controls, especially in cells precultured with chondrocyte-derived ECM for 7 d. Taken together, these results demonstrate that chondrocyte-derived ECM enhances the chondrogenesis of hPDMSCs, and this effect is further increased by preculture with chondrocyte-derived ECM. This preculture method for hPDMSC chondrogenesis represents a promising approach for cartilage tissue engineering.

Quantitative evaluation of in vitro effects and interactions of active fractions in a Chinese medicinal formula (Yaotongning Capsule) on rat chondrocytes.

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Zhang, Li-Guo; Ouyang, Xiao-Wen; Wu, Ting-Ting; Ni, Li-Jun; Shi, Wan-Zhong

2014-09-29

Yaotongning Capsule (YTNC) is a Traditional Chinese Medicinal (TCM) formula that has been demonstrated to be effective for osteoarthritis (OA) treatment in clinical use. Many compounds and 10 component medicinal materials (CMMs for short, i.e., the fundamental elements used in TCM formulas) in YTNC are challenging to study the pharmacological effects and interactions of the CMMs. Besides, it is difficult to know whether the YTNC formula is reasonable, and if YTNC formula could be improved without comparing YTNC with other TCM formulas of treating OA. Based on different combinations of the active fractions from the 10 CMMs of YTNC and eight additional herbs frequently used in the TCM formulas of treating OA, the present study evaluated systematically the in vitro effects of these active fractions and the interactions among the active fractions from YTNC on rat chondrocytes to find possible solutions of the above questions. Based on the formulation of YTNC and the concept of combinatorial chemistry, the active fractions were applied to form the whole YTNC prescription (i.e., the combination of all YTNC active fractions and the extract of YTNC׳s vehicle), five disassembled formulas of YTNC (i.e., the combinations of some active fractions in YTNC) and 21 TCM samples consisted of different kinds of active fractions. The degenerated chondrocytes were induced with interleukin-1β (IL-1β), and then the half-effective concentration (EC50) value of the proliferation activity was analyzed to evaluate the 27 TCM samples. Nine samples were screened for the following evaluation on glycosaminoglycan (GAG) synthesis. Rat articular cartilage was obtained from six Sprague-Dawley rats (seven days of age), and then chondrocytes were isolated through enzymatic digestion with 0.2% Collagenase II. Proliferations of chondrocytes were examined through Cell Counting Kit-8 assay, when the intracellular levels of GAG were detected by 1,9-Dimethylmethylene blue staining. The interactions

Anti-inflammatory and anti-osteoarthritis effects of tectorigenin

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Cheng-Long Wang

2017-08-01

Full Text Available Osteoarthritis (OA is a common and dynamic disease of the joints, including the articular cartilage, underlying bones and synovium. In particular, OA is considered as the degeneration of the cartilage. Tectorigenin (Tec is known to affect many biological processes; however, its effects on articular chondrocytes remain unclear. This study aimed to assess the effects of Tec on articular cartilage. In vitro, Tec inhibited the expression levels of type X collagen, cyclooxigenase-2, matrix metalloproteinase (MMP-3 and MMP-13, but enhanced the expression of Runx1, type II collagen and aggrecan in the presence of IL-1β. Meanwhile, Tec inhibited apoptosis through the Bax/Bcl-2/caspase-3 pathway, upregulating p-Bad, downregulating the Bax/Bcl-2 ratio, and activating caspase-3 compared with IL-1β treatment only. Moreover, this process was partially regulated by NF-κB P65. In vivo, the chondroprotective effects of Tec were assessed by establishing a model of surgically induced OA. Tec-treated joints exhibited fewer osteoarthritic changes than saline-treated joints. Meanwhile, 1.5 μg/kg Tec treatment produced a greater protective effect than 0.75 μg/kg Tec. The Osteoarthritis Research Society International (OARSI scoring system, employed to assess histopathological grading of the models, as well immunohistochemistry for Aggrecan Neoepitope and MMP-3, further confirmed the results. In conclusion, this study showed that Tec plays a chondroprotective role in the OA process by preventing articular cartilage degeneration and chondrocyte apoptosis via the NF-κB P65 pathway.

Intra-articular chondroma of the knee

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Talwalkar, S.C.; Kambhampati, S.B.S.; Lang Stevenson, A.I. [Oldchurch Hospital, Romford, Essex (United Kingdom); Whitehouse, R. [Manchester University, Department of Radiology, Manchester (United Kingdom); Freemont, A. [University of Manchester, Department of Osteoarticular Pathology, Manchester (United Kingdom)

2005-06-01

Chondromas are tumours that develop in relation to the periosteum and, although they are common around the knee, most reports deal with soft tissue chondromas in para-articular locations or intracortical tumours in extra-articular regions. We report a rare case of an intra-articular chondroma in a 16-year-old boy of Asian origin developing in the region of the medial femoral condyle of the femur and extending into the femoral sulcus and the patellofemoral joint. (orig.)

Intra-articular chondroma of the knee

International Nuclear Information System (INIS)

Talwalkar, S.C.; Kambhampati, S.B.S.; Lang Stevenson, A.I.; Whitehouse, R.; Freemont, A.

2005-01-01

Chondromas are tumours that develop in relation to the periosteum and, although they are common around the knee, most reports deal with soft tissue chondromas in para-articular locations or intracortical tumours in extra-articular regions. We report a rare case of an intra-articular chondroma in a 16-year-old boy of Asian origin developing in the region of the medial femoral condyle of the femur and extending into the femoral sulcus and the patellofemoral joint. (orig.)

RAGE and activation of chondrocytes and fibroblast-like synoviocytes in joint diseases

NARCIS (Netherlands)

Steenvoorden, Marjan Maria Claziena

2007-01-01

This dissertation describes a new model in which cartilage degradation can be studied. New cartilage is formed by bovine chondrocytes obtained from the slaughterhouse and cocultured with synovial cells from rheumatoid arthritis (RA) patients to study the interaction between the chondrocytes and

KNEE CARTILAGE AND SYNOVIAL MEMBRANE STRUCTURAL CHANGES DURING TIBIA DISTRACTION WITH PLATING

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T. A. Stupina

2017-01-01

Full Text Available Purpose of the study — to analyze the changes in knee articular cartilage and synovial membrane during distraction external fixation of the tibia in combination with plating.Material and methods. Articular cartilage and synovial membrane of the knee joint were studied using histomorphometry methods in 9 mongrel dogs during distraction external fixation of the tibia combined with plating. Tibia and fibula osteotomies were performed at the border of middle and upper third, plate was fixed on tibia diaphysis. Lengthening was achieved at rate of 1 mm per day in four stages during 21–28 days. Animals were withdrawn from experiment in 30 and 90 days. After autopsy of knee joints the authors excised sections of synovial membrane from suprapatellar area, articular cartilage with underlying subchondral bone from loadable surface of femoral condyles. Thickness of articular cartilage, its area and volumetric density of chondrocytes was measured, proportion of chondrocytes within isogenic groups from the overall number of chondrocytes as well as proportion of empty lacunae. In synovial membrane the authors measured thickness of surface layer and numeric density of micro vessels. Articular cartilage of 5 intact animals was used as a control group.Results. After 30 days of plate fixation a hyperplasia of the integument layer, mild synovitis, and hypervascularization were observed in synovial membrane. Density of micro vessels increased to 363.93±33.71 (control group — 335.05±28.88. The authors also observed subperineural and endoneural edema as well as destruction of nerve fibers in subsynovial layer. Articular cartilage retained the zonal structure. Destructive changes were manifested by fibers separation in the superficial part of surface zone and by partial loss of chondrocytes. The following parameters were reduced: cartilage thickness, area and volumetric density of chondrocytes, proportion of isogenic groups; empty lacunae exceeded the values in

Latexin is involved in bone morphogenetic protein-2-induced chondrocyte differentiation

International Nuclear Information System (INIS)

Kadouchi, Ichiro; Sakamoto, Kei; Tangjiao, Liu; Murakami, Takashi; Kobayashi, Eiji; Hoshino, Yuichi; Yamaguchi, Akira

2009-01-01

Latexin is the only known carboxypeptidase A inhibitor in mammals. We previously demonstrated that BMP-2 significantly induced latexin expression in Runx2-deficient mesenchymal cells (RD-C6 cells), during chondrocyte and osteoblast differentiation. In this study, we investigated latexin expression in the skeleton and its role in chondrocyte differentiation. Immunohistochemical studies revealed that proliferating and prehypertrophic chondrocytes expressed latexin during skeletogenesis and bone fracture repair. In the early phase of bone fracture, latexin mRNA expression was dramatically upregulated. BMP-2 upregulated the expression of the mRNAs of latexin, Col2a1, and the gene encoding aggrecan (Agc1) in a micromass culture of C3H10T1/2 cells. Overexpression of latexin additively stimulated the BMP-2-induced expression of the mRNAs of Col2a, Agc1, and Col10a1. BMP-2 treatment upregulated Sox9 expression, and Sox9 stimulated the promoter activity of latexin. These results indicate that latexin is involved in BMP-2-induced chondrocyte differentiation and plays an important role in skeletogenesis and skeletal regeneration.

Acquiring Chondrocyte Phenotype from Human Mesenchymal Stem Cells under Inflammatory Conditions

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Masahiro Kondo

2014-11-01

Full Text Available An inflammatory milieu breaks down the cartilage matrix and induces chondrocyte apoptosis, resulting in cartilage destruction in patients with cartilage degenerative diseases, such as rheumatoid arthritis or osteoarthritis. Because of the limited regenerative ability of chondrocytes, defects in cartilage are irreversible and difficult to repair. Mesenchymal stem cells (MSCs are expected to be a new tool for cartilage repair because they are present in the cartilage and are able to differentiate into multiple lineages of cells, including chondrocytes. Although clinical trials using MSCs for patients with cartilage defects have already begun, its efficacy and repair mechanisms remain unknown. A PubMed search conducted in October 2014 using the following medical subject headings (MeSH terms: mesenchymal stromal cells, chondrogenesis, and cytokines resulted in 204 articles. The titles and abstracts were screened and nine articles relevant to “inflammatory” cytokines and “human” MSCs were identified. Herein, we review the cell biology and mechanisms of chondrocyte phenotype acquisition from human MSCs in an inflammatory milieu and discuss the clinical potential of MSCs for cartilage repair.

Nobiletin Inhibits Expression of Inflammatory Mediators and ...

African Journals Online (AJOL)

Articular cartilage samples from 25 informed and consented patients (age: 57 ± 13) who were undergoing total knee replacement surgery were obtained. The study was approved by the institutional ethical committee. The articular cartilage was harvested from non-lesional areas and primary chondrocytes were isolated as.

CCAAT/Enhancer Binding Protein β Regulates Expression of Indian Hedgehog during Chondrocytes Differentiation

Science.gov (United States)

Ushijima, Takahiro; Okazaki, Ken; Tsushima, Hidetoshi; Ishihara, Kohei; Doi, Toshio; Iwamoto, Yukihide

2014-01-01

Background CCAAT/enhancer binding protein β (C/EBPβ) is a transcription factor that promotes hypertrophic differentiation of chondrocytes. Indian hedgehog (Ihh) also stimulates the hypertrophic transition of chondrocytes. Furthermore, runt-related transcription factor-2 (RUNX2) was reported to regulate chondrocyte maturation during skeletal development and to directly regulate transcriptional activity of Ihh. In this study, we investigated whether the interaction of C/EBPβ and RUNX2 regulates the expression of Ihh during chondrocyte differentiation. Methodology/Results Immunohistochemistry of embryonic growth plate revealed that both C/EBPβ and Ihh were strongly expressed in pre-hypertrophic and hypertrophic chondrocytes. Overexpression of C/EBPβ by adenovirus vector in ATDC5 cells caused marked stimulation of Ihh and Runx2. Conversely, knockdown of C/EBPβ by lentivirus expressing shRNA significantly repressed Ihh and Runx2 in ATDC5 cells. A reporter assay revealed that C/EBPβ stimulated transcriptional activity of Ihh. Deletion and mutation analysis showed that the C/EBPβ responsive element was located between −214 and −210 bp in the Ihh promoter. An electrophoretic mobility shift assay (EMSA) and a chromatin immunoprecipitation (ChIP) assay also revealed the direct binding of C/EBPβ to this region. Moreover, reporter assays demonstrated that RUNX2 failed to stimulate the transcriptional activity of the Ihh promoter harboring a mutation at the C/EBPβ binding site. EMSA and ChIP assays showed that RUNX2 interacted to this element with C/EBPβ. Immunoprecipitation revealed that RUNX2 and C/EBPβ formed heterodimer complex with each other in the nuclei of chondrocytes. These data suggested that the C/EBPβ binding element is also important for RUNX2 to regulate the expression of Ihh. Ex vivo organ culture of mouse limbs transfected with C/EBPβ showed that the expression of Ihh and RUNX2 was increased upon ectopic C/EBPβ expression. Conclusions C

CCAAT/enhancer binding protein β regulates expression of Indian hedgehog during chondrocytes differentiation.

Directory of Open Access Journals (Sweden)

Takahiro Ushijima

Full Text Available CCAAT/enhancer binding protein β (C/EBPβ is a transcription factor that promotes hypertrophic differentiation of chondrocytes. Indian hedgehog (Ihh also stimulates the hypertrophic transition of chondrocytes. Furthermore, runt-related transcription factor-2 (RUNX2 was reported to regulate chondrocyte maturation during skeletal development and to directly regulate transcriptional activity of Ihh. In this study, we investigated whether the interaction of C/EBPβ and RUNX2 regulates the expression of Ihh during chondrocyte differentiation.Immunohistochemistry of embryonic growth plate revealed that both C/EBPβ and Ihh were strongly expressed in pre-hypertrophic and hypertrophic chondrocytes. Overexpression of C/EBPβ by adenovirus vector in ATDC5 cells caused marked stimulation of Ihh and Runx2. Conversely, knockdown of C/EBPβ by lentivirus expressing shRNA significantly repressed Ihh and Runx2 in ATDC5 cells. A reporter assay revealed that C/EBPβ stimulated transcriptional activity of Ihh. Deletion and mutation analysis showed that the C/EBPβ responsive element was located between -214 and -210 bp in the Ihh promoter. An electrophoretic mobility shift assay (EMSA and a chromatin immunoprecipitation (ChIP assay also revealed the direct binding of C/EBPβ to this region. Moreover, reporter assays demonstrated that RUNX2 failed to stimulate the transcriptional activity of the Ihh promoter harboring a mutation at the C/EBPβ binding site. EMSA and ChIP assays showed that RUNX2 interacted to this element with C/EBPβ. Immunoprecipitation revealed that RUNX2 and C/EBPβ formed heterodimer complex with each other in the nuclei of chondrocytes. These data suggested that the C/EBPβ binding element is also important for RUNX2 to regulate the expression of Ihh. Ex vivo organ culture of mouse limbs transfected with C/EBPβ showed that the expression of Ihh and RUNX2 was increased upon ectopic C/EBPβ expression.C/EBPβ and RUNX2 cooperatively stimulate

Engineering Cartilage Tissue by Pellet Coculture of Chondrocytes and Mesenchymal Stromal Cells

NARCIS (Netherlands)

Wu, Ling; Post, Janine Nicole; Karperien, Hermanus Bernardus Johannes; Westendorf, Jennifer J.; van Wijnen, Andre J.

2015-01-01

Coculture of chondrocytes and mesenchymal stromal cells (MSCs) in pellets has been shown to be beneficial in engineering cartilage tissue in vitro. In these cultures trophic effects of MSCs increase the proliferation and matrix deposition of chondrocytes. Thus, large cartilage constructs can be made

The distribution of YKL-40 in osteoarthritic and normal human articular cartilage

DEFF Research Database (Denmark)

Volck, B; Ostergaard, K; Johansen, J S

1999-01-01

YKL-40, also called human cartilage glycoprotein-39, is a major secretory protein of human chondrocytes in cell culture. YKL-40 mRNA is expressed by cartilage from patients with rheumatoid arthritis, but is not detectable in normal human cartilage. The aim was to investigate the distribution of YKL...... in chondrocytes of osteoarthritic cartilage mainly in the superficial and middle zone of the cartilage rather than the deep zone. There was a tendency for high number of YKL-40 positive chondrocytes in areas of the femoral head with a considerable biomechanical load. The number of chondrocytes with a positive...

Exploration of mechanisms underlying the strain-rate-dependent mechanical property of single chondrocytes

Energy Technology Data Exchange (ETDEWEB)

Nguyen, Trung Dung; Gu, YuanTong, E-mail: yuantong.gu@qut.edu.au [School of Chemistry, Physics and Mechanical Engineering, Queensland University of Technology, Brisbane, Queensland (Australia)

2014-05-05

Based on the characterization by Atomic Force Microscopy, we report that the mechanical property of single chondrocytes has dependency on the strain-rates. By comparing the mechanical deformation responses and the Young's moduli of living and fixed chondrocytes at four different strain-rates, we explore the deformation mechanisms underlying this dependency property. We found that the strain-rate-dependent mechanical property of living cells is governed by both of the cellular cytoskeleton and the intracellular fluid when the fixed chondrocytes are mainly governed by their intracellular fluid, which is called the consolidation-dependent deformation behavior. Finally, we report that the porohyperelastic constitutive material model which can capture the consolidation-dependent behavior of both living and fixed chondrocytes is a potential candidature to study living cell biomechanics.

Role of Insulin-Transferrin-Selenium in Auricular Chondrocyte Proliferation and Engineered Cartilage Formation in Vitro

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Xia Liu

2014-01-01

Full Text Available The goal of this study is to determine the effects of Insulin-Transferrin-Selenium (ITS on proliferation of auricular chondrocytes and formation of engineered cartilage in vitro. Pig auricular monolayer chondrocytes and chondrocyte pellets were cultured in media containing 1% ITS at different concentrations of fetal bovine serum (FBS, 10%, 6%, 2%, 0%, or 10% FBS alone as a control for four weeks. Parameters including cell proliferation in monolayer, wet weight, collagen type I/II/X (Col I, II, X and glycosaminoglycan (GAG expression, GAG content of pellets and gene expression associated with cartilage formation/dedifferentiation (lost cartilage phenotype/hypertrophy within the chondrocyte pellets were assessed. The results showed that chondrocytes proliferation rates increased when FBS concentrations increased (2%, 6%, 10% FBS in ITS supplemented groups. In addition, 1% ITS plus 10% FBS significantly promoted cell proliferation than 10% FBS alone. No chondrocytes grew in ITS alone medium. 1% ITS plus 10% FBS enhanced cartilage formation in terms of size, wet weight, cartilage specific matrices, and homogeneity, compared to 10% FBS alone group. Furthermore, ITS prevented engineered cartilage from dedifferentiation (i.e., higher index of Col II/Col I mRNA expression and expression of aggrecan and hypertrophy (i.e., lower mRNA expression of Col X and MMP13. In conclusion, our results indicated that ITS efficiently enhanced auricular chondrocytes proliferation, retained chondrogenic phenotypes, and promoted engineered cartilage formation when combined with FBS, which is potentially used as key supplementation in auricular chondrocytes and engineered cartilage culture.

A Preliminary Study of Human Amniotic Membrane as a Potential Chondrocyte Carrier

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L Boo

2009-11-01

Full Text Available PURPOSE: To investigate the feasibility of using processed human amniotic membrane (HAM to support the attachment and proliferation of chondrocytes in vitro which in turn can be utilised as a cell delivery vehicle in tissue engineering applications. METHODS: Fresh HAM obtained from patients undergoing routine elective caesarean sections was harvested, processed and dried using either freeze drying (FD or air drying (AD methods prior to sterilisation by gamma irradiation. Isolated, processed and characterised rabbit autologous chondrocytes were seeded on processed HAM and cultured for up to three weeks. Cell attachment and proliferation were examined qualitatively using inverted brightfield microscopy. RESULTS: Processed HAM appeared to allow cell attachment when implanted with chondrocytes. Although cells seeded on AD and FD HAM did not appear to attach as strongly as those seeded on glycerol preserved intact human amniotic membrane, these cells to be proliferated in cell culture conditions. CONCLUSION: Preliminary results show that processed HAM promotes chondrocyte attachment and proliferation.

Dietary variation and mechanical properties of articular cartilage in the temporomandibular joint: implications for the role of plasticity in mechanobiology and pathobiology.

Science.gov (United States)

Ravosa, Matthew J; Kane, Robert J

2017-10-01

Due to their nature as tissue composites, skeletal joints pose an additional challenge in terms of evaluating the functional significance of morphological variation in their bony and cartilaginous components in response to altered loading conditions. Arguably, this complexity requires more direct means of investigating joint plasticity and performance than typically employed to analyze macro- and micro-anatomical phenomena. To address a significant gap in our understanding of the plasticity of the mammalian temporomandibular joint (TMJ), we investigated the histology and mechanical properties of condylar articular cartilage in rabbits subjected to long-term variation in diet-induced masticatory stresses, specifically cyclical loading. Three cohorts of male weanlings were raised for six months on different diets until adulthood. Following euthanasia, the TMJ condyles on one side were dissected away, fixed, decalcified, dehydrated, embedded and sectioned. Safranin O staining was employed to identify variation in proteoglycan content, which in turn was used to predict patterns of articular cartilage stiffness in contralateral condylar specimens for each treatment group. Hematoxylin and eosin staining was used to quantify diet-induced changes in chondrocyte hypertrophy and cellularity. Mechanical tests document significant decreases in articular cartilage stiffness corresponding to patterns of extracellular matrix relative protein abundance in rabbits subjected to greater cyclical loading. This indicates that TMJs routinely subjected to higher masticatory stresses due to a challenging diet eventually develop postnatal decreases in the ability to counter compressive loads during postcanine biting and chewing. These findings provide novel information regarding TMJ performance, with broader implications about the costs and benefits of phenotypic plasticity as well as implications for how such biological processes affect connective tissue mechanobiology and pathobiology

Bauhinia championi (Benth.) Benth. polysaccharides upregulate Wnt/β-catenin signaling in chondrocytes.

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Li, Huiting; Li, Xihai; Liu, Guozhong; Chen, Jiashou; Weng, Xiaping; Liu, Fayuan; Xu, Huifeng; Liu, Xianxiang; Ye, Hongzhi

2013-12-01

Bauhinia championi (Benth.) Benth. polysaccharides (BCBPs), extracted from Bauhinia championi (Benth.) Benth., which has been used in traditional Chinese medicine (TCM) for the treatment of osteoarthritis (OA), are the bioactive constituents of Bauhinia championi (Benth.) rattan. However, the molecular mechanisms responsible for their effects on OA are poorly understood. The Wnt/β-catenin signaling pathway plays an important role in the proliferation of chondrocytes. In the present study, the effects of BCBPs on Wnt/β-catenin signaling in chondrocytes were investigated. BCBPs were obtained by hot-water extraction and identified by the modified high performance liquid chromatography (HPLC) method. Chondrocytes were isolated from the knees of Sprague‑Dawley rats and identified by type II collagen immunohistochemistry. The chondrocytes were treated with or without BCBPs for 48 h. Cell viability was evaluated by MTT assay. The mRNA and protein levels of Wnt-4, β-catenin, Frizzled-2, glycogen synthase kinase (GSK)-3β, cyclin D1 and collagen II were detected by western blot analysis and reverse transcription PCR (RT-PCR), respectively. We found that the BCBPs contained at least seven monosaccharides, including D-mannose, rhamnose, D-(+) glucuronic acid, D-(+) galacturonic acid, D-glucose, galactose and arabinose. The cell viability of the chondrocytes treated with 50, 100 and 200 µg/ml BCBPs was significantly higher than that of the chondroctyes in the control group (treated with 0 µg/ml BCBPs). Furthermore, compared with the control group, the mRNA and protein expression of Wnt-4, β-catenin, Frizzled-2 and cyclin D1 in the BCBP-treated groups markedly increased, whereas the mRNA and protein expression of GSK-3β significantly decreased. Of note, the dose of 100 µg/ml BCBPs was more effective than the dose of 50 µg/ml BCBPs and 200 µg/ml BCBPs. In addition, we found that treatment with BCBPs upregulated the protein levels of collagen II in the

Fate of Meckel's cartilage chondrocytes in ocular culture

International Nuclear Information System (INIS)

Richman, J.M.; Diewert, V.M.

1988-01-01

Modulation of the chondrocyte phenotype was observed in an organ culture system using Meckel's cartilage. First branchial arch cartilage was dissected from fetal rats of 16- and 17-day gestation. Perichondrium was mechanically removed, cartilage was split at the rostral process, and each half was grafted into the anterior chamber of an adult rat eye. The observed pattern of development in nonirradiated specimens was the following: hypertrophy of the rostral process and endochondral-type ossification, fibrous atrophy in the midsection, and mineralization of the malleus and incus. A change in matrix composition of the implanted cartilage was demonstrated with immunofluorescence staining for cartilage-specific proteoglycan (CSPG). After 15 days of culture, CSPG was found in the auricular process but not in the midsection or rostral process. In order to mark the implanted cells and follow their fate, cartilage was labeled in vitro with [3H]thymidine [3H]TdR). Immediately after labeling 20% of the chondrocytes contained [3H]TdR. After culturing for 5 days, 20% of the chondrocytes were still labeled and 10% of the osteogenic cells also contained radioactive label. The labeling index decreased in both cell types with increased duration of culture. Multinucleated clast-type cells did not contain label. Additional cartilages not labeled with [3H]TdR were exposed to between 20000 and 6000 rad of gamma irradiation before ocular implantation. Irradiated cartilage did not hypertrophy or form bone but a fibrous region developed in the midsection. Cells of the host animal were not induced to form bone around the irradiated cartilage. Our studies suggest that fully differentiated chondrocytes of Meckel's cartilage have the capacity to become osteocytes, osteoblasts, and fibroblasts

The Ihh signal is essential for regulating proliferation and hypertrophy of cultured chicken chondrocytes.

Science.gov (United States)

Ma, R S; Zhou, Z L; Luo, J W; Zhang, H; Hou, J F

2013-10-01

The Indian hedgehog (Ihh) signal plays a vital role in regulating proliferation and hypertrophy of chondrocytes. To investigate its function in postnatal chicken (Gallus gallus) chondrocytes, cyclopamine was used to inhibit Ihh signaling. The MTT and ALP assays revealed the downgrade-proliferation and upgrade-differentiation of chondrocytes. To further elucidate the mechanism, the mRNA expression levels of Ihh, parathyroid hormone related protein (PTHrP), Gli-2, Bcl-2, Bone Morphogenetic Protein 6 (BMP-6), type X collagen (Col X) and type II collagen (Col II) were detected by quantitative real-time RT-PCR analysis, and the protein expressions of Ihh, Col X, and Col II were determined using Western blot analysis. After the Ihh signal was blocked, chondrocytes demonstrated high expression levels of PTHrP and Col X and low levels of Gli-2, BMP-6, Bcl-2 and Col II although Ihh expression was increased. Based on these results, the Ihh signal is essential for balancing chicken chondrocyte proliferation and hypertrophy, and the regulatory function of PTHrP acts in an Ihh-dependent manner. Furthermore, BMP-6 and Bcl-2 played roles in maintaining the development of chondrocytes and may be downstream regulatory factors of Ihh signaling. © 2013.

Articular manifestations in patients with Lyme disease.

Science.gov (United States)

Vázquez-López, María Esther; Díez-Morrondo, Carolina; Sánchez-Andrade, Amalia; Pego-Reigosa, Robustiano; Díaz, Pablo; Castro-Gago, Manuel

To determine the percentage of Lyme patients with articular manifestations in NW Spain and to know their evolution and response to treatment. A retrospective study (2006-2013) was performed using medical histories of confirmed cases of Lyme disease showing articular manifestations. Clinical and laboratory characteristics, together with the treatment and evolution of the patients, were analysed. Seventeen out of 108 LD confirmed patients (15.7%) showed articular manifestations. Regarding those 17 patients, 64.7%, 29.4% and 5.9% presented arthritis, arthralgia and bursitis, respectively. The knee was the most affected joint. Articular manifestations were often associated to neurological, dermatological and cardiac pathologies. Otherwise, most patients were in Stage III. The 11.8% of the cases progressed to a recurrent chronic arthritis despite the administration of an appropriate treatment. Lyme disease patients showing articular manifestations should be included in the diagnosis of articular affections in areas of high risk of hard tick bite, in order to establish a suitable and early treatment and to avoid sequels. Copyright © 2015 Elsevier España, S.L.U. and Sociedad Española de Reumatología y Colegio Mexicano de Reumatología. All rights reserved.

Fundamental study on articular disc with magnetic resonance imagings

International Nuclear Information System (INIS)

Chiba, Toyokazu

1993-01-01

In order to establish criteria of reading MRI of the temporomandibular joint, a morphological comparison between MRI and the section, and an observation of the articular disc associated with the opening were made. Five temporomandibular joints isolated from 3 human cadavers were subjected to MRI, and sections were prepared to examine criteria of reading MRI. In 20 male adults, 40 temporomandibular joints underwent MRI in three conditions of the intercuspal position, 10 and 20 mm opening positions, and the kinetics of the articular disc were examined. External feature of the head of mandible and that of the articular fossa, the articular tubercule and the postglenoid process were outlined in a row of blacks. The articular disc was outlined in a row of dark ashen areas of the anterior band, the intermediate region, and the posterior band. In the intercuspal position, the head of mandible was rarely covered with the articular disc, and being situated postero-inferiorly, at the most rear point of the posterior band of the articular disc. In the 10 mm-opening position, the head of mandible was practically covered with the articular disc. In the 20 mm-opening position, the intermediate region of the articular disc, and the head of mandible were situated in an approximate position. Quantitative movement of the articular disc was slower than that of the head of mandible. Comparison of various points of the articular disc revealed that movements of the anterior and posterior band varied almost proportionally to the opening distance, but with lesser movement of the intermediate region. (author)

TNF/TNFR1 pathway and endoplasmic reticulum stress are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes

International Nuclear Information System (INIS)

Zhang, Fu-Tao; Ding, Yi; Shah, Zahir; Xing, Dan; Gao, Yuan; Liu, Dong Ming; Ding, Ming-Xing

2014-01-01

Background and purpose: Quinolones cause obvious cartilaginous lesions in juvenile animals by chondrocyte apoptosis, which results in the restriction of their use in pediatric and adolescent patients. Studies showed that chondrocytes can be induced to produce TNFα, and the cisternae of the endoplasmic reticulum in quinolone-treated chondrocytes become dilated. We investigated whether TNF/TNFR 1 pathway and endoplasmic reticulum stress (ERs) are involved in ofloxacin (a typical quinolone)-induced apoptosis of juvenile canine chondrocytes. Experimental approach: Canine juvenile chondrocytes were treated with ofloxacin. Cell survival and apoptosis rates were determined with MTT method and flow cytometry, respectively. The gene expression levels of the related signaling molecules (TNFα, TNFR 1 , TRADD, FADD and caspase-8) in death receptor pathways and main apoptosis-related molecules (calpain, caspase-12, GADD153 and GRP78) in ERs were measured by qRT-PCR. The gene expression of TNFR 1 was suppressed with its siRNA. The protein levels of TNFα, TNFR 1 and caspase-12 were assayed using Western blotting. Key results: The survival rates decreased while apoptosis rates increased after the chondrocytes were treated with ofloxacin. The mRNA levels of the measured apoptosis-related molecules in death receptor pathways and ERs, and the protein levels of TNFα, TNFR 1 and caspase-12 increased after the chondrocytes were exposed to ofloxacin. The downregulated mRNA expressions of TNFR 1 , Caspase-8 and TRADD, and the decreased apoptosis rates of the ofloxacin-treated chondrocytes occurred after TNFR 1 –siRNA interference. Conclusions and implications: Ofloxacin-induced chondrocyte apoptosis in a time- and concentration-dependent fashion. TNF/TNFR 1 pathway and ERs are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes in the early stage. - Highlights: • Chondrocyte apoptosis is induced by ofloxacin in a time- and concentration-dependent manners. �

Articular cartilage repair with recombinant human type II collagen/polylactide scaffold in a preliminary porcine study.

Science.gov (United States)

Muhonen, Virpi; Salonius, Eve; Haaparanta, Anne-Marie; Järvinen, Elina; Paatela, Teemu; Meller, Anna; Hannula, Markus; Björkman, Mimmi; Pyhältö, Tuomo; Ellä, Ville; Vasara, Anna; Töyräs, Juha; Kellomäki, Minna; Kiviranta, Ilkka

2016-05-01

The purpose of this study was to investigate the potential of a novel recombinant human type II collagen/polylactide scaffold (rhCo-PLA) in the repair of full-thickness cartilage lesions with autologous chondrocyte implantation technique (ACI). The forming repair tissue was compared to spontaneous healing (spontaneous) and repair with a commercial porcine type I/III collagen membrane (pCo). Domestic pigs (4-month-old, n = 20) were randomized into three study groups and a circular full-thickness chondral lesion with a diameter of 8 mm was created in the right medial femoral condyle. After 3 weeks, the chondral lesions were repaired with either rhCo-PLA or pCo together with autologous chondrocytes, or the lesion was only debrided and left untreated for spontaneous repair. The repair tissue was evaluated 4 months after the second operation. Hyaline cartilage formed most frequently in the rhCo-PLA treatment group. Biomechanically, there was a trend that both treatment groups resulted in better repair tissue than spontaneous healing. Adverse subchondral bone reactions developed less frequently in the spontaneous group (40%) and the rhCo-PLA treated group (50%) than in the pCo control group (100%). However, no statistically significant differences were found between the groups. The novel rhCo-PLA biomaterial showed promising results in this proof-of-concept study, but further studies will be needed in order to determine its effectiveness in articular cartilage repair. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:745-753, 2016. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Supporting Biomaterials for Articular Cartilage Repair

Science.gov (United States)

Duarte Campos, Daniela Filipa; Drescher, Wolf; Rath, Björn; Tingart, Markus

2012-01-01

Orthopedic surgeons and researchers worldwide are continuously faced with the challenge of regenerating articular cartilage defects. However, until now, it has not been possible to completely mimic the biological and biochemical properties of articular cartilage using current research and development approaches. In this review, biomaterials previously used for articular cartilage repair research are addressed. Furthermore, a brief discussion of the state of the art of current cell printing procedures mimicking native cartilage is offered in light of their use as future alternatives for cartilage tissue engineering. Inkjet cell printing, controlled deposition cell printing tools, and laser cell printing are cutting-edge techniques in this context. The development of mimetic hydrogels with specific biological properties relevant to articular cartilage native tissue will support the development of improved, functional, and novel engineered tissue for clinical application. PMID:26069634

Gli3 acts as a repressor downstream of Ihh in regulating two distinct steps of chondrocyte differentiation.

Science.gov (United States)

Koziel, Lydia; Wuelling, Manuela; Schneider, Sabine; Vortkamp, Andrea

2005-12-01

During endochondral ossification, the secreted growth factor Indian hedgehog (Ihh) regulates several differentiation steps. It interacts with a second secreted factor, parathyroid hormone-related protein (PTHrP), to regulate the onset of hypertrophic differentiation, and it regulates chondrocyte proliferation and ossification of the perichondrium independently of PTHrP. To investigate how the Ihh signal is translated in the different target tissues, we analyzed the role of the zinc-finger transcription factor Gli3, which acts downstream of hedgehog signals in other organs. Loss of Gli3 in Ihh mutants restores chondrocyte proliferation and delays the accelerated onset of hypertrophic differentiation observed in Ihh-/- mutants. Furthermore the expression of the Ihh target genes patched (Ptch) and PTHrP is reactivated in Ihh-/-;Gli3-/- mutants. Gli3 seems thus to act as a strong repressor of Ihh signals in regulating chondrocyte differentiation. In addition, loss of Gli3 in mice that overexpress Ihh in chondrocytes accelerates the onset of hypertrophic differentiation by reducing the domain and possibly the level of PTHrP expression. Careful analysis of chondrocyte differentiation in Gli3-/- mutants revealed that Gli3 negatively regulates the differentiation of distal, low proliferating chondrocytes into columnar, high proliferating cells. Our results suggest a model in which the Ihh/Gli3 system regulates two distinct steps of chondrocyte differentiation: (1) the switch from distal into columnar chondrocytes is repressed by Gli3 in a PTHrP-independent mechanism; (2) the transition from proliferating into hypertrophic chondrocytes is regulated by Gli3-dependent expression of PTHrP. Furthermore, by regulating distal chondrocyte differentiation, Gli3 seems to position the domain of PTHrP expression.

The effects of monosodium urate monohydrate crystals on chondrocyte viability and function: implications for development of cartilage damage in gout.

Science.gov (United States)

Chhana, Ashika; Callon, Karen E; Pool, Bregina; Naot, Dorit; Gamble, Gregory D; Dray, Michael; Pitto, Rocco; Bentley, Jarome; McQueen, Fiona M; Cornish, Jillian; Dalbeth, Nicola

2013-12-01

Cartilage damage is frequently observed in advanced destructive gout. The aim of our study was to investigate the effects of monosodium urate monohydrate (MSU) crystals on chondrocyte viability and function. The alamarBlue assay and flow cytometry were used to assess the viability of primary human chondrocytes and cartilage explants following culture with MSU crystals. The number of dead chondrocytes in cartilage explants cultured with MSU crystals was quantified. Real-time PCR was used to determine changes in the relative mRNA expression levels of chondrocytic genes. The histological appearance of cartilage in joints affected by gout was also examined. MSU crystals rapidly reduced primary human chondrocyte and cartilage explant viability in a dose-dependent manner (p gout, normal cartilage architecture was lost, with empty chondrocyte lacunae observed. MSU crystals have profound inhibitory effects on chondrocyte viability and function. Interactions between MSU crystals and chondrocytes may contribute to cartilage damage in gout through reduction of chondrocyte viability and promotion of a catabolic state.

High throughput generated micro-aggregates of chondrocytes stimulate cartilage formation in vitro and in vivo

Directory of Open Access Journals (Sweden)

LS Moreira Teixeira

2012-06-01

Full Text Available Cell-based cartilage repair strategies such as matrix-induced autologous chondrocyte implantation (MACI could be improved by enhancing cell performance. We hypothesised that micro-aggregates of chondrocytes generated in high-throughput prior to implantation in a defect could stimulate cartilaginous matrix deposition and remodelling. To address this issue, we designed a micro-mould to enable controlled high-throughput formation of micro-aggregates. Morphology, stability, gene expression profiles and chondrogenic potential of micro-aggregates of human and bovine chondrocytes were evaluated and compared to single-cells cultured in micro-wells and in 3D after encapsulation in Dextran-Tyramine (Dex-TA hydrogels in vitro and in vivo. We successfully formed micro-aggregates of human and bovine chondrocytes with highly controlled size, stability and viability within 24 hours. Micro-aggregates of 100 cells presented a superior balance in Collagen type I and Collagen type II gene expression over single cells and micro-aggregates of 50 and 200 cells. Matrix metalloproteinases 1, 9 and 13 mRNA levels were decreased in micro-aggregates compared to single-cells. Histological and biochemical analysis demonstrated enhanced matrix deposition in constructs seeded with micro-aggregates cultured in vitro and in vivo, compared to single-cell seeded constructs. Whole genome microarray analysis and single gene expression profiles using human chondrocytes confirmed increased expression of cartilage-related genes when chondrocytes were cultured in micro-aggregates. In conclusion, we succeeded in controlled high-throughput formation of micro-aggregates of chondrocytes. Compared to single cell-seeded constructs, seeding of constructs with micro-aggregates greatly improved neo-cartilage formation. Therefore, micro-aggregation prior to chondrocyte implantation in current MACI procedures, may effectively accelerate hyaline cartilage formation.

High throughput generated micro-aggregates of chondrocytes stimulate cartilage formation in vitro and in vivo.

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Moreira Teixeira, L S; Leijten, J C H; Sobral, J; Jin, R; van Apeldoorn, A A; Feijen, J; van Blitterswijk, C; Dijkstra, P J; Karperien, M

2012-06-05

Cell-based cartilage repair strategies such as matrix-induced autologous chondrocyte implantation (MACI) could be improved by enhancing cell performance. We hypothesised that micro-aggregates of chondrocytes generated in high-throughput prior to implantation in a defect could stimulate cartilaginous matrix deposition and remodelling. To address this issue, we designed a micro-mould to enable controlled high-throughput formation of micro-aggregates. Morphology, stability, gene expression profiles and chondrogenic potential of micro-aggregates of human and bovine chondrocytes were evaluated and compared to single-cells cultured in micro-wells and in 3D after encapsulation in Dextran-Tyramine (Dex-TA) hydrogels in vitro and in vivo. We successfully formed micro-aggregates of human and bovine chondrocytes with highly controlled size, stability and viability within 24 hours. Micro-aggregates of 100 cells presented a superior balance in Collagen type I and Collagen type II gene expression over single cells and micro-aggregates of 50 and 200 cells. Matrix metalloproteinases 1, 9 and 13 mRNA levels were decreased in micro-aggregates compared to single-cells. Histological and biochemical analysis demonstrated enhanced matrix deposition in constructs seeded with micro-aggregates cultured in vitro and in vivo, compared to single-cell seeded constructs. Whole genome microarray analysis and single gene expression profiles using human chondrocytes confirmed increased expression of cartilage-related genes when chondrocytes were cultured in micro-aggregates. In conclusion, we succeeded in controlled high-throughput formation of micro-aggregates of chondrocytes. Compared to single cell-seeded constructs, seeding of constructs with micro-aggregates greatly improved neo-cartilage formation. Therefore, micro-aggregation prior to chondrocyte implantation in current MACI procedures, may effectively accelerate hyaline cartilage formation.

Quantitative assessment of synovial inflammation by dynamic gadolinium-enhanced magnetic resonance imaging. A study of the effect of intra-articular methylprednisolone on the rate of early synovial enhancement

DEFF Research Database (Denmark)

Østergaard, Mikkel; Stoltenberg, M; Henriksen, O

1996-01-01

The effect of temporary inflammatory suppression on synovial membrane enhancement, as determined by dynamic and static gadolinium-DTPA enhanced magnetic resonance imaging (MRI), was studied. MRI of 18 arthritic knees was performed before and 1, 7, 30 and 180 days after intra-articular methylpredn......The effect of temporary inflammatory suppression on synovial membrane enhancement, as determined by dynamic and static gadolinium-DTPA enhanced magnetic resonance imaging (MRI), was studied. MRI of 18 arthritic knees was performed before and 1, 7, 30 and 180 days after intra...

Chondrogenic properties of collagen type XI, a component of cartilage extracellular matrix.

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Li, Ang; Wei, Yiyong; Hung, Clark; Vunjak-Novakovic, Gordana

2018-08-01

Cartilage extracellular matrix (ECM) has been used for promoting tissue engineering. However, the exact effects of ECM on chondrogenesis and the acting mechanisms are not well understood. In this study, we investigated the chondrogenic effects of cartilage ECM on human mesenchymal stem cells (MSCs) and identified the contributing molecular components. To this end, a preparation of articular cartilage ECM was supplemented to pellets of chondrogenically differentiating MSCs, pellets of human chondrocytes, and bovine articular cartilage explants to evaluate the effects on cell proliferation and the production of cartilaginous matrix. Selective enzymatic digestion and screening of ECM components were conducted to identify matrix molecules with chondrogenic properties. Cartilage ECM promoted MSC proliferation, production of cartilaginous matrix, and maturity of chondrogenic differentiation, and inhibited the hypertrophic differentiation of MSC-derived chondrocytes. Selective digestion of ECM components revealed a contributory role of collagens in promoting chondrogenesis. The screening of various collagen subtypes revealed strong chondrogenic effect of collagen type XI. Finally, collagen XI was found to promote production and inhibit degradation of cartilage matrix in human articular chondrocyte pellets and bovine articular cartilage explants. Our results indicate that cartilage ECM promotes chondrogenesis and inhibits hypertrophic differentiation in MSCs. Collagen type XI is the ECM component that has the strongest effects on enhancing the production and inhibiting the degradation of cartilage matrix. Copyright © 2018 Elsevier Ltd. All rights reserved.

MR imaging of articular cartilage

International Nuclear Information System (INIS)

Schaefer, F.K.W.; Muhle, C.; Heller, M.; Brossmann, J.

2001-01-01

MR imaging has evolved to the best non-invasive method for the evaluation of articular cartilage. MR imaging helps to understand the structure and physiology of cartilage, and to diagnose cartilage lesions. Numerous studies have shown high accuracy and reliability concerning detection of cartilage lesions and early changes in both structure and biochemistry. High contrast-to-noise ratio and high spatial resolution are essential for analysis of articular cartilage. Fat-suppressed 3D-T 1 weighted gradient echo and T 2 -weighted fast spin echo sequences with or without fat suppression are recommended for clinical routine. In this article the anatomy and pathology of hyaline articular cartilage and the complex imaging characteristics of hyaline cartilage will be discussed. (orig.) [de

Chondrocytes and stem cells in 3D-bioprinted structures create human cartilage in vivo.

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Apelgren, Peter; Amoroso, Matteo; Lindahl, Anders; Brantsing, Camilla; Rotter, Nicole; Gatenholm, Paul; Kölby, Lars

2017-01-01

Cartilage repair and replacement is a major challenge in plastic reconstructive surgery. The development of a process capable of creating a patient-specific cartilage framework would be a major breakthrough. Here, we described methods for creating human cartilage in vivo and quantitatively assessing the proliferative capacity and cartilage-formation ability in mono- and co-cultures of human chondrocytes and human mesenchymal stem cells in a three-dimensional (3D)-bioprinted hydrogel scaffold. The 3D-bioprinted constructs (5 × 5 × 1.2 mm) were produced using nanofibrillated cellulose and alginate in combination with human chondrocytes and human mesenchymal stem cells using a 3D-extrusion bioprinter. Immediately following bioprinting, the constructs were implanted subcutaneously on the back of 48 nude mice and explanted after 30 and 60 days, respectively, for morphological and immunohistochemical examination. During explantation, the constructs were easy to handle, and the majority had retained their macroscopic grid appearance. Constructs consisting of human nasal chondrocytes showed good proliferation ability, with 17.2% of the surface areas covered with proliferating chondrocytes after 60 days. In constructs comprising a mixture of chondrocytes and stem cells, an additional proliferative effect was observed involving chondrocyte production of glycosaminoglycans and type 2 collagen. This clinically highly relevant study revealed 3D bioprinting as a promising technology for the creation of human cartilage.

Chondrocytes and stem cells in 3D-bioprinted structures create human cartilage in vivo.

Directory of Open Access Journals (Sweden)

Peter Apelgren

Full Text Available Cartilage repair and replacement is a major challenge in plastic reconstructive surgery. The development of a process capable of creating a patient-specific cartilage framework would be a major breakthrough. Here, we described methods for creating human cartilage in vivo and quantitatively assessing the proliferative capacity and cartilage-formation ability in mono- and co-cultures of human chondrocytes and human mesenchymal stem cells in a three-dimensional (3D-bioprinted hydrogel scaffold. The 3D-bioprinted constructs (5 × 5 × 1.2 mm were produced using nanofibrillated cellulose and alginate in combination with human chondrocytes and human mesenchymal stem cells using a 3D-extrusion bioprinter. Immediately following bioprinting, the constructs were implanted subcutaneously on the back of 48 nude mice and explanted after 30 and 60 days, respectively, for morphological and immunohistochemical examination. During explantation, the constructs were easy to handle, and the majority had retained their macroscopic grid appearance. Constructs consisting of human nasal chondrocytes showed good proliferation ability, with 17.2% of the surface areas covered with proliferating chondrocytes after 60 days. In constructs comprising a mixture of chondrocytes and stem cells, an additional proliferative effect was observed involving chondrocyte production of glycosaminoglycans and type 2 collagen. This clinically highly relevant study revealed 3D bioprinting as a promising technology for the creation of human cartilage.

Effects of NSAIDs on the metabolism of sulphated glycosaminoglycans in healthy and (post) arthritic murine articular cartilage.

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de Vries, B J; van den Berg, W B; Vitters, E; van de Putte, L B

1988-01-01

Several non-steroidal anti-inflammatory drugs (NSAIDs) were studied for their effects on normal and damaged murine articular cartilage, both in vitro and in vivo. In vitro, in the absence of serum, sodium salicylate caused significant suppression of 35S-glycosaminoglycan (GAG) synthesis, whereas tiaprofenic acid, piroxicam, prednisolone sodium phosphate and several other NSAIDs were without effect. Trypsin-mediated proteoglycan depletion did not change the susceptibility of the articular chondrocyte to these drugs. Similarly, no enhancement of drug effect was seen when arthritic cartilage was taken from an acutely inflamed joint, and prenisolone sodium phosphate even seemed to diminish inflammation-mediated suppression of 35S-GAG synthesis. The short term in vivo effects of some of the drugs were tested in mice with unilateral zymosan-induced arthritis. At day 1 after arthritis induction, in vivo 35S-GAG synthesis by the cartilage of the arthritic joint was decreased to 63%. Only sodium salicylate suppressed in vivo 35S-GAG synthesis in the healthy and arthritic joint to the same extent in both. At day 28, GAG synthesis in the postarthritic joint was enhanced to 160%. This type of cartilage appeared to be more susceptible to drug effects, since all drugs tested showed clear suppression of the augmented GAG production in vivo. Finally, in vivo drug effects were tested on normal and enhanced 35S-GAG degradation, the latter in the zymosan-induced arthritic joint. Both tiaprofenic acid and prednisolone sodium phosphate appeared to suppress degradation in healthy and, to some extent, in arthritic cartilage.(ABSTRACT TRUNCATED AT 250 WORDS)

Smad signaling pathway is a pivotal component of tissue inhibitor of metalloproteinases-3 regulation by transforming growth factor beta in human chondrocytes.

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Qureshi, Hamid Yaqoob; Ricci, Gemma; Zafarullah, Muhammad

2008-09-01

Transforming growth factor beta (TGF-beta1) promotes cartilage matrix synthesis and induces tissue inhibitor of metalloproteinases-3 (TIMP-3), which inhibits matrix metalloproteinases, aggrecanases and TNF-alpha-converting enzyme implicated in articular cartilage degradation and joint inflammation. TGF-beta1 activates Akt, ERK and Smad2 pathways in chondrocytes. Here we investigated previously unexplored roles of specific Smads in TGF-beta1 induction of TIMP-3 gene by pharmacological and genetic knockdown approaches. TGF-beta1-induced Smad2 phosphorylation and TIMP-3 protein expression could be inhibited by the Smad2/3 phosphorylation inhibitors, PD169316 and SB203580 and by Smad2-specific siRNA. Specific inhibitor of Smad3 (SIS3) and Smad3 siRNA abolished TGF-beta induction of TIMP-3. Smad2/3 siRNAs also down regulated TIMP-3 promoter-driven luciferase activities, suggesting transcriptional regulation. SiRNA-driven co-Smad4 knockdown abrogated TIMP-3 augmentation by TGF-beta. TIMP-3 promoter deletion analysis revealed that -828 deletion retains the original promoter activity while -333 and -167 deletions display somewhat reduced activity suggesting that most of the TGF-beta-responsive, cis-acting elements are found in the -333 fragment. Chromatin Immunoprecipitation (ChIP) analysis confirmed binding of Smad2 and Smad4 with the -940 and -333 promoter sequences. These results suggest that receptor-activated Smad2 and Smad3 and co-Smad4 critically mediate TGF-beta-stimulated TIMP-3 expression in human chondrocytes and TIMP-3 gene is a target of Smad signaling pathway.

Intra-articular therapies for osteoarthritis.

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Yu, Shirley P; Hunter, David J

2016-10-01

Conventional medical therapies for osteoarthritis are mainly palliative in nature, aiming to control pain and symptoms. Traditional intra-articular therapies are not recommended in guidelines as first line therapy, but are potential alternatives, when conventional therapies have failed. Current and future intra-articular drug therapies for osteoarthritis are highlighted, including corticosteroids, hyaluronate, and more controversial treatments marketed commercially, namely platelet rich plasma and mesenchymal cell therapy. Intraarticular disease modifying osteoarthritis drugs are the future of osteoarthritis treatments, aiming at structural modification and altering the disease progression. Interleukin-1β inhibitor, bone morphogenic protein-7, fibroblast growth factor 18, bradykinin B2 receptor antagonist, human serum albumin, and gene therapy are discussed in this review. The evolution of drug development in osteoarthritis is limited by the ability to demonstrate effect. High quality trials are required to justify the use of existing intra-articular therapies and to advocate for newer, promising therapies. Challenges in osteoarthritis therapy research are fundamentally related to the complexity of the pathological mechanisms of osteoarthritis. Novel drugs offer hope in a disease with limited medical therapy options. Whether these future intra-articular therapies will provide clinically meaningful benefits, remains unknown.

The effects of intra-articular glucocorticoids and exercise on pain and synovitis assessed on static and dynamic magnetic resonance imaging in knee osteoarthritis

DEFF Research Database (Denmark)

Riis, R G C; Henriksen, M; Klokker, L

2017-01-01

) investigate if any of the changes in patient reported outcome measures (PROMs) were associated with changes in MRI-measures of synovitis. DESIGN: We performed a randomized, double-blinded, placebo-controlled clinical trial evaluating the effects of intra-articular corticosteroid vs placebo injections given......-articular corticosteroids over intra-articular saline when combined with an exercise program for reduction of synovitis in KOA. The improvement in pain and function following the intervention with intra-articular corticosteroids/saline and exercise could not be explained by a decrease in synovitis on MRI indicating other...... pain causing/relieving mechanisms in KOA....

TNF/TNFR{sub 1} pathway and endoplasmic reticulum stress are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes

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Zhang, Fu-Tao; Ding, Yi; Shah, Zahir; Xing, Dan; Gao, Yuan; Liu, Dong Ming; Ding, Ming-Xing, E-mail: dmx@mail.hzau.edu.cn

2014-04-15

Background and purpose: Quinolones cause obvious cartilaginous lesions in juvenile animals by chondrocyte apoptosis, which results in the restriction of their use in pediatric and adolescent patients. Studies showed that chondrocytes can be induced to produce TNFα, and the cisternae of the endoplasmic reticulum in quinolone-treated chondrocytes become dilated. We investigated whether TNF/TNFR{sub 1} pathway and endoplasmic reticulum stress (ERs) are involved in ofloxacin (a typical quinolone)-induced apoptosis of juvenile canine chondrocytes. Experimental approach: Canine juvenile chondrocytes were treated with ofloxacin. Cell survival and apoptosis rates were determined with MTT method and flow cytometry, respectively. The gene expression levels of the related signaling molecules (TNFα, TNFR{sub 1}, TRADD, FADD and caspase-8) in death receptor pathways and main apoptosis-related molecules (calpain, caspase-12, GADD153 and GRP78) in ERs were measured by qRT-PCR. The gene expression of TNFR{sub 1} was suppressed with its siRNA. The protein levels of TNFα, TNFR{sub 1} and caspase-12 were assayed using Western blotting. Key results: The survival rates decreased while apoptosis rates increased after the chondrocytes were treated with ofloxacin. The mRNA levels of the measured apoptosis-related molecules in death receptor pathways and ERs, and the protein levels of TNFα, TNFR{sub 1} and caspase-12 increased after the chondrocytes were exposed to ofloxacin. The downregulated mRNA expressions of TNFR{sub 1}, Caspase-8 and TRADD, and the decreased apoptosis rates of the ofloxacin-treated chondrocytes occurred after TNFR{sub 1}–siRNA interference. Conclusions and implications: Ofloxacin-induced chondrocyte apoptosis in a time- and concentration-dependent fashion. TNF/TNFR{sub 1} pathway and ERs are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes in the early stage. - Highlights: • Chondrocyte apoptosis is induced by ofloxacin in a time- and

Biological and Chemical Removal of Primary Cilia Affects Mechanical Activation of Chondrogenesis Markers in Chondroprogenitors and Hypertrophic Chondrocytes.

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Deren, Matthew E; Yang, Xu; Guan, Yingjie; Chen, Qian

2016-02-04

Chondroprogenitors and hypertrophic chondrocytes, which are the first and last stages of the chondrocyte differentiation process, respectively, are sensitive to mechanical signals. We hypothesize that the mechanical sensitivity of these cells depends on the cell surface primary cilia. To test this hypothesis, we removed the primary cilia by biological means with transfection with intraflagellar transport protein 88 (IFT88) siRNA or by chemical means with chloral hydrate treatment. Transfection of IFT88 siRNA significantly reduced the percentage of ciliated cells in both chondroprogenitor ATDC5 cells as well as primary hypertrophic chondrocytes. Cyclic loading (1 Hz, 10% matrix deformation) of ATDC5 cells in three-dimensional (3D) culture stimulates the mRNA levels of chondrogenesis marker Type II collagen (Col II), hypertrophic chondrocyte marker Type X collagen (Col X), and a molecular regulator of chondrogenesis and chondrocyte hypertrophy bone morphogenetic protein 2 (BMP-2). The reduction of ciliated chondroprogenitors abolishes mechanical stimulation of Col II, Col X, and BMP-2. In contrast, cyclic loading stimulates Col X mRNA levels in hypertrophic chondrocytes, but not those of Col II and BMP-2. Both biological and chemical reduction of ciliated hypertrophic chondrocytes reduced but failed to abolish mechanical stimulation of Col X mRNA levels. Thus, primary cilia play a major role in mechanical stimulation of chondrogenesis and chondrocyte hypertrophy in chondroprogenitor cells and at least a partial role in hypertrophic chondrocytes.

Biological and Chemical Removal of Primary Cilia Affects Mechanical Activation of Chondrogenesis Markers in Chondroprogenitors and Hypertrophic Chondrocytes

Directory of Open Access Journals (Sweden)

Matthew E. Deren

2016-02-01

Full Text Available Chondroprogenitors and hypertrophic chondrocytes, which are the first and last stages of the chondrocyte differentiation process, respectively, are sensitive to mechanical signals. We hypothesize that the mechanical sensitivity of these cells depends on the cell surface primary cilia. To test this hypothesis, we removed the primary cilia by biological means with transfection with intraflagellar transport protein 88 (IFT88 siRNA or by chemical means with chloral hydrate treatment. Transfection of IFT88 siRNA significantly reduced the percentage of ciliated cells in both chondroprogenitor ATDC5 cells as well as primary hypertrophic chondrocytes. Cyclic loading (1 Hz, 10% matrix deformation of ATDC5 cells in three-dimensional (3D culture stimulates the mRNA levels of chondrogenesis marker Type II collagen (Col II, hypertrophic chondrocyte marker Type X collagen (Col X, and a molecular regulator of chondrogenesis and chondrocyte hypertrophy bone morphogenetic protein 2 (BMP-2. The reduction of ciliated chondroprogenitors abolishes mechanical stimulation of Col II, Col X, and BMP-2. In contrast, cyclic loading stimulates Col X mRNA levels in hypertrophic chondrocytes, but not those of Col II and BMP-2. Both biological and chemical reduction of ciliated hypertrophic chondrocytes reduced but failed to abolish mechanical stimulation of Col X mRNA levels. Thus, primary cilia play a major role in mechanical stimulation of chondrogenesis and chondrocyte hypertrophy in chondroprogenitor cells and at least a partial role in hypertrophic chondrocytes.

Comparison of Efficacy of Endogenous and Exogenous IGF-I in Stimulating Matrix Production in Neonatal and Mature Chondrocytes.

Science.gov (United States)

Aguilar, Izath N; Trippel, Stephen B; Shi, Shuiliang; Bonassar, Lawrence J

2015-10-01

The goal of this study was to compare the efficacy of endogenous upregulation of IGF-I by gene therapy and exogenous addition of insulin-like growth factor I (IGF-I) in enhancing proteoglycan synthesis by skeletally mature and neonatal chondrocytes. Chondrocyte transplantation therapy is a common treatment for focal cartilage lesions, with both mature and neonatal chondrocytes used as a cell source. Additionally, gene therapy strategies to upregulate growth factors such as IGF-I have been proposed to augment chondrocyte transplantation therapies. Both skeletally mature and neonatal chondrocytes were exposed to either an adeno-associated virus-based plasmid containing the IGF-I gene or exogenous IGF-I. Analysis of IGF-I and glycosaminoglycan production using a 4-parameter dose-response model established a clear connection between the amount of IGF-I produced by cells and their biosynthetic response. Both neonatal and mature chondrocytes showed this relationship, but the sensitivities were quite different, with EC50 of 0.57 ng/mL for neonatal chondrocytes and EC50 of 8.70 ng/mL IGF-I for skeletally mature chondrocytes. These data suggest that IGF-I gene therapy may be more effective with younger cell sources. Both cell types were less sensitive to exogenous IGF-I than endogenous IGF-I.