WorldWideScience

Sample records for chondrogenesis

  1. The control of chondrogenesis.

    Science.gov (United States)

    Goldring, Mary B; Tsuchimochi, Kaneyuki; Ijiri, Kosei

    2006-01-01

    Chondrogenesis is the earliest phase of skeletal development, involving mesenchymal cell recruitment and migration, condensation of progenitors, and chondrocyte differentiation, and maturation and resulting in the formation of cartilage and bone during endochondral ossification. This process is controlled exquisitely by cellular interactions with the surrounding matrix, growth and differentiation factors, and other environmental factors that initiate or suppress cellular signaling pathways and transcription of specific genes in a temporal-spatial manner. Vertebrate limb development is controlled by interacting patterning systems involving prominently the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), and hedgehog pathways. Both positive and negative signaling kinases and transcription factors, such as Sox9 and Runx2, and interactions among them determine whether the differentiated chondrocytes remain within cartilage elements in articular joints or undergo hypertrophic maturation prior to ossification. The latter process requires extracellular matrix remodeling and vascularization controlled by mechanisms that are not understood completely. Recent work has revealed novel roles for mediators such as GADD45beta, transcription factors of the Dlx, bHLH, leucine zipper, and AP-1 families, and the Wnt/beta-catenin pathway that interact at different stages during chondrogenesis. PMID:16215986

  2. Vinculin Functions as Regulator of Chondrogenesis*

    Science.gov (United States)

    Koshimizu, Takao; Kawai, Masanobu; Kondou, Hiroki; Tachikawa, Kanako; Sakai, Norio; Ozono, Keiichi; Michigami, Toshimi

    2012-01-01

    To identify the genes involved in chondrocytic differentiation, we applied gene trap mutagenesis to a murine mesenchymal chondrogenic cell line ATDC5 and isolated a clone in which the gene encoding vinculin was trapped. The trapped allele was assumed to express a fusion protein containing a truncated vinculin lacking the tail domain and the geo product derived from the trap vector. The truncated vinculin was suggested to exert a dominant negative effect. Impaired functioning of vinculin caused by gene trapping in ATDC5 cells or knockdown in primary chondrocytes resulted in the reduced expression of chondrocyte-specific genes, including Col2a1, aggrecan, and Col10a1. The expression of Runx2 also was suppressed by the dysfunctional vinculin. On the other hand, the expression of Sox9, encoding a key transcription factor for chondrogenesis, was retained. Knockdown of vinculin in metatarsal organ cultures impaired the growth of the explants and reduced the expression of Col2a1 and aggrecan. Gene trapping or knockdown of vinculin decreased the phosphorylation of ERK1/2 but increased that of Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2) and Akt during chondrocytic differentiation, suggesting a disturbance of signaling by insulin-like growth factor I (IGF-I). Knockdown of vinculin in the metatarsal organ culture abrogated the IGF-I-induced growth and inhibited the up-regulation of Col2a1 and aggrecan expression by IGF-I. Loss of vinculin function in differentiating chondrocytes impaired the activation of the p38 MAPK pathway also, suggesting its involvement in the regulation of chondrogenesis by vinculin. Our results indicate a tissue-specific function of vinculin in cartilage whereby it controls chondrocytic differentiation. PMID:22416133

  3. Role of c-Myb in chondrogenesis.

    Science.gov (United States)

    Oralová, V; Matalová, E; Janečková, E; Drobná Krejčí, E; Knopfová, L; Šnajdr, P; Tucker, A S; Veselá, I; Šmarda, J; Buchtová, M

    2015-07-01

    The Myb locus encodes the c-Myb transcription factor involved in controlling a broad variety of cellular processes. Recently, it has been shown that c-Myb may play a specific role in hard tissue formation; however, all of these results were gathered from an analysis of intramembranous ossification. To investigate a possible role of c-Myb in endochondral ossification, we carried out our study on the long bones of mouse limbs during embryonic development. Firstly, the c-myb expression pattern was analyzed by in situ hybridization during endochondral ossification of long bones. c-myb positive areas were found in proliferating as well as hypertrophic zones of the growth plate. At early embryonic stages, localized expression was also observed in the perichondrium and interdigital areas. The c-Myb protein was found in proliferating chondrocytes and in the perichondrium of the forelimb bones (E14.5-E17.5). Furthermore, protein was detected in pre-hypertrophic as well as hypertrophic chondrocytes. Gain-of-function and loss-of-function approaches were used to test the effect of altered c-myb expression on chondrogenesis in micromass cultures established from forelimb buds of mouse embryos. A loss-of-function approach using c-myb specific siRNA decreased nodule formation, as well as downregulated the level of Sox9 expression, a major marker of chondrogenesis. Transient c-myb overexpression markedly increased the formation of cartilage nodules and the production of extracellular matrix as detected by intense staining with Alcian blue. Moreover, the expression of early chondrogenic genes such as Sox9, Col2a1 and activity of a Col2-LUC reporter were increased in the cells overexpressing c-myb while late chondrogenic markers such as Col10a1 and Mmp13 were not significantly changed or were downregulated. Taken together, the results of this study demonstrate that the c-Myb transcription factor is involved in the regulation and promotion of endochondral bone formation. PMID

  4. Autologous bone-marrow mesenchymal cell induced chondrogenesis (MCIC).

    Science.gov (United States)

    Huh, Sung Woo; Shetty, Asode Ananthram; Ahmed, Saif; Lee, Dong Hwan; Kim, Seok Jung

    2016-01-01

    Degenerative and traumatic articular cartilage defects are common, difficult to treat, and progressive lesions that cause significant morbidity in the general population. There have been multiple approaches to treat such lesions, including arthroscopic debridement, microfracture, multiple drilling, osteochondral transplantation and autologous chondrocyte implantation (ACI) that are currently being used in clinical practice. Autologous bone-marrow mesenchymal cell induced chondrogenesis (MCIC) is a single-staged arthroscopic procedure. This method combines a modified microfracture technique with the application of a bone marrow aspirate concentrate (BMAC), hyaluronic acid and fibrin gel to treat articular cartilage defects. We reviewed the current literatures and surgical techniques for mesenchymal cell induced chondrogenesis. PMID:27489409

  5. Gene Transfer Strategies to Promote Chondrogenesis and Cartilage Regeneration.

    Science.gov (United States)

    Im, Gun-Il

    2016-04-01

    Gene transfer has been used experimentally to promote chondrogenesis and cartilage regeneration. While it is controversial to apply gene therapy for nonlethal conditions such as cartilage defect, there is a possibility that the transfer of therapeutic transgenes may dramatically increase the effectiveness of cell therapy and reduce the quantity of cells that are needed to regenerate cartilage. Single or combination of growth factors and transcription factors has been transferred to mesenchymal stem cells or articular chondrocytes using both nonviral and viral approaches. The current challenge for the clinical applications of genetically modified cells is ensuring the safety of gene therapy while guaranteeing effectiveness. Viral gene delivery methods have been mainstays currently with enhanced safety features being recently refined. On the other hand, efficiency has been greatly improved in nonviral delivery. This review summarizes the history and recent update on the gene transfer to enhance chondrogenesis from stem cells or articular chondrocytes.

  6. Gene Transfer Strategies to Promote Chondrogenesis and Cartilage Regeneration.

    Science.gov (United States)

    Im, Gun-Il

    2016-04-01

    Gene transfer has been used experimentally to promote chondrogenesis and cartilage regeneration. While it is controversial to apply gene therapy for nonlethal conditions such as cartilage defect, there is a possibility that the transfer of therapeutic transgenes may dramatically increase the effectiveness of cell therapy and reduce the quantity of cells that are needed to regenerate cartilage. Single or combination of growth factors and transcription factors has been transferred to mesenchymal stem cells or articular chondrocytes using both nonviral and viral approaches. The current challenge for the clinical applications of genetically modified cells is ensuring the safety of gene therapy while guaranteeing effectiveness. Viral gene delivery methods have been mainstays currently with enhanced safety features being recently refined. On the other hand, efficiency has been greatly improved in nonviral delivery. This review summarizes the history and recent update on the gene transfer to enhance chondrogenesis from stem cells or articular chondrocytes. PMID:26414246

  7. Induction of mesenchymal stem cell chondrogenesis by polyacrylate substrates

    OpenAIRE

    Glennon-Alty, Laurence; Williams, Rachel; Dixon, Simon; Murray, Patricia

    2013-01-01

    Mesenchymal stem cells (MSCs) can generate chondrocytes in vitro, but typically need to be cultured as aggregates in the presence of transforming growth factor beta (TGF-β), which makes scale-up difficult. Here we investigated if polyacrylate substrates modelled on the functional group composition and distribution of the Arg-Gly-Asp (RGD) integrin-binding site could induce MSCs to undergo chondrogenesis in the absence of exogenous TGF-β. Within a few days of culture on the biomimetic polyacry...

  8. Induction of mesenchymal stem cell chondrogenesis by polyacrylate substrates.

    Science.gov (United States)

    Glennon-Alty, Laurence; Williams, Rachel; Dixon, Simon; Murray, Patricia

    2013-04-01

    Mesenchymal stem cells (MSCs) can generate chondrocytes in vitro, but typically need to be cultured as aggregates in the presence of transforming growth factor beta (TGF-β), which makes scale-up difficult. Here we investigated if polyacrylate substrates modelled on the functional group composition and distribution of the Arg-Gly-Asp (RGD) integrin-binding site could induce MSCs to undergo chondrogenesis in the absence of exogenous TGF-β. Within a few days of culture on the biomimetic polyacrylates, both mouse and human MSCs, and a mesenchymal-like mouse-kidney-derived stem cell line, began to form multi-layered aggregates and started to express the chondrocyte-specific markers, Sox9, collagen II and aggrecan. Moreover, collagen II tended to be expressed in the centre of the aggregates, similarly to developing limb buds in vivo. Surface analysis of the substrates indicated that those with the highest surface amine content were most effective at promoting MSC chondrogenesis. These results highlight the importance of surface group functionality and the distribution of those groups in the design of substrates to induce MSC chondrogenesis. PMID:23237986

  9. Optimizing a novel method for low intensity ultrasound in chondrogenesis induction

    Directory of Open Access Journals (Sweden)

    Hajar Shafaei

    2013-01-01

    Conclusion: Using LIUS resulted in early chondrogenesis in comparison with terminally differentiated chondrocytes by TGFβ. Therefore, LIUS might provide an applicable, safe, efficient, and cheap tool for chondrogenic differentiation of ASCs in cartilage tissue engineering.

  10. Chondroblastoma and chondromyxoid fibroma : disentangling the neoplastic chondrogenesis of two rare cartilaginous tumours

    NARCIS (Netherlands)

    Romeo, Salvatore

    2010-01-01

    The scope of this study was to disentangle neoplastic chondrogenesis in two rare cartilaginous tumours: chondroblastoma and chondromyxoid fibroma. It was addressed: 1 The spectrum of phenotypic differentiation in chondroblastoma and chondromyxoid fibroma, 2 The signalling pathways driving chondrobla

  11. Differential effect of hypoxia on human mesenchymal stem cell chondrogenesis and hypertrophy in hyaluronic acid hydrogels.

    Science.gov (United States)

    Zhu, Meiling; Feng, Qian; Bian, Liming

    2014-03-01

    Photocrosslinked hyaluronic acid (HA) hydrogels provide a conducive 3-D environment that supports the chondrogenesis of human mesenchymal stem cells (hMSCs). The HA macromer concentration in the hydrogels has a significant impact on the chondrogenesis of the encapsulated MSCs due to changes in the physical properties of the hydrogels. Meanwhile, hypoxia has been shown to promote MSC chondrogenesis and suppress subsequent hypertrophy. This study investigates the combinatorial effect of tuning HA macromer concentration (1.5-5%w/v) and hypoxia on MSC chondrogenesis and hypertrophy. To decouple the effect of HA concentration from that of crosslinking density, the HA hydrogel crosslinking density was adjusted by varying the extent of the reaction through the light exposure time while keeping the HA concentration constant (5%w/v at 5 or 15 min). It was found that hypoxia had no significant effect on the chondrogenesis and cartilaginous matrix synthesis of hMSCs under all hydrogel conditions. In contrast, the hypoxia-mediated positive or negative regulation of hMSC hypertrophy in HA hydrogels is dependent on the HA concentration but independent of the crosslinking density. Specifically, hypoxia significantly suppressed hMSC hypertrophy and neocartilage calcification in low HA concentration hydrogels, whereas hypoxia substantially enhanced hMSC hypertrophy, leading to elevated tissue calcification in high HA concentration hydrogels irrespective of their crosslinking density. In addition, at a constant high HA concentration, increasing hydrogel crosslinking density promoted hMSC hypertrophy and matrix calcification. To conclude, the findings from this study demonstrate that the effect of hypoxia on hMSC chondrogenesis and hypertrophy is differentially influenced by the encapsulating HA hydrogel properties. PMID:24342044

  12. DAPT inhibits the chondrogenesis of human umbilical cord mesenchymal stem cells

    OpenAIRE

    Hu Xiaoke; Zhang Jiawen; Shao Xinxin; Luo Ermei; Yu Li

    2015-01-01

    Notch signaling plays a key role in cell proliferation and differentiation, and is important in several biological processes, but its role in the chondrogenesis of human umbilical cord mesenchymal stem cells (UC-MSCs) is still unknown. N-[N-(3,5- difluorophenacetyl-L-alanyl)]-(S)-phenylglycinet-butyl ester (DAPT) is the inhibitor of Notch pathway. The aim of this study is to investgate the influence of DAPT on the chondrogenesis of UC-MSCs. In our study, UC-MSCs wer...

  13. Forkhead transcription factor foxe1 regulates chondrogenesis in zebrafish.

    Science.gov (United States)

    Nakada, Chisako; Iida, Atsumi; Tabata, Yoko; Watanabe, Sumiko

    2009-12-15

    Forkhead transcription factor (Fox) e1 is a causative gene for Bamforth-Lazarus syndrome, which is characterized by hypothyroidism and cleft palate. Applying degenerate polymerase chain reaction using primers specific for the conserved forkhead domain, we identified zebrafish foxe1 (foxe1). Foxe1 is expressed in the thyroid, pharynx, and pharyngeal skeleton during development; strongly expressed in the gill and weakly expressed in the brain, eye, and heart in adult zebrafish. A loss of function of foxe1 by morpholino antisense oligo (MO) exhibited abnormal craniofacial development, shortening of Meckel's cartilage and the ceratohyals, and suppressed chondrycytic proliferation. However, at 27 hr post fertilization, the foxe1 MO-injected embryos showed normal dlx2, hoxa2, and hoxb2 expression, suggesting that the initial steps of pharyngeal skeletal development, including neural crest migration and specification of the pharyngeal arch occurred normally. In contrast, at 2 dpf, a severe reduction in the expression of sox9a, colIIaI, and runx2b, which play roles in chondrocytic proliferation and differentiation, was observed. Interestingly, fgfr2 was strongly upregulated in the branchial arches of the foxe1 MO-injected embryos. Unlike Foxe1-null mice, normal thyroid development in terms of morphology and thyroid-specific marker expression was observed in foxe1 MO-injected zebrafish embryos. Taken together, our results indicate that Foxe1 plays an important role in chondrogenesis during development of the pharyngeal skeleton in zebrafish, probably through regulation of fgfr2 expression. Furthermore, the roles reported for FOXE1 in mammalian thyroid development may have been acquired during evolution.

  14. Quantitative transcriptional profiling of ATDC5 mouse progenitor cells during chondrogenesis

    DEFF Research Database (Denmark)

    Chen, Li; Fink, Trine; Zhang, Xiao-Yan;

    2005-01-01

    During the differentiation of a mouse chondroprogenitor cell line, ATDC5, an analysis of the transcription cartilage-related genes was carried out using real-time RT-PCR in a semiquantitative fashion. A total number of 104 genes both previously linked to chondrogenesis and hitherto not associated...

  15. Hypoxic treatment inhibits insulin-induced chondrogenesis of ATDC5 cells despite upregulation of DEC1

    DEFF Research Database (Denmark)

    Chen, Li; Fink, Trine; Ebbesen, Peter;

    2006-01-01

    Chondrogenesis occurs in vivo in a hypoxic environment, in which the hypoxia inducible factor 1, HIF-1, plays a regulatory role, possibly mediated through the transcription factor DEC1. We have analyzed the effect of hypoxia (1% oxygen) alone and in combination with insulin on the chondrogenic di...

  16. Cellular ATP Synthesis Mediated by Type III Sodium-dependent Phosphate Transporter Pit-1 Is Critical to Chondrogenesis*

    OpenAIRE

    Sugita, Atsushi; Kawai, Shinji; Hayashibara, Tetsuyuki; Amano, Atsuo; Ooshima, Takashi; Michigami, Toshimi; Yoshikawa, Hideki; Yoneda, Toshiyuki

    2010-01-01

    Disturbed endochondral ossification in X-linked hypophosphatemia indicates an involvement of Pi in chondrogenesis. We studied the role of the sodium-dependent Pi cotransporters (NPT), which are a widely recognized regulator of cellular Pi homeostasis, and the downstream events in chondrogenesis using Hyp mice, the murine homolog of human X-linked hypophosphatemia. Hyp mice showed reduced apoptosis and mineralization in hypertrophic cartilage. Hyp chondrocytes in culture displayed decreased ap...

  17. Metabolomic Analysis of Differential Changes in Metabolites during ATP Oscillations in Chondrogenesis

    Directory of Open Access Journals (Sweden)

    Hyuck Joon Kwon

    2013-01-01

    Full Text Available Prechondrogenic condensation is a critical step for skeletal pattern formation. Recent studies reported that ATP oscillations play an essential role in prechondrogenic condensation. However, the molecular mechanism to underlie ATP oscillations remains poorly understood. In the present study, it was investigated how changes in metabolites are implicated in ATP oscillations during chondrogenesis by using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS. CE-TOF-MS detected 93 cationic and 109 anionic compounds derived from known metabolic pathways. 15 cationic and 18 anionic compounds revealed significant change between peak and trough of ATP oscillations. These results implicate that glycolysis, mitochondrial respiration and uronic acid pathway oscillate in phase with ATP oscillations, while PPRP and nucleotides synthesis pathways oscillate in antiphase with ATP oscillations. This suggests that the ATP-producing glycolysis and mitochondrial respiration oscillate in antiphase with the ATP-consuming PPRP/nucleotide synthesis pathway during chondrogenesis.

  18. 17{beta}-Estradiol inhibits chondrogenesis in the skull development of zebrafish embryos

    Energy Technology Data Exchange (ETDEWEB)

    Fushimi, Shigeko, E-mail: fushimi@med.kawasaki-m.ac.jp [Department of Public Health, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 (Japan); Wada, Naoyuki, E-mail: wada@med.kawasaki-m.ac.jp [Department of Molecular and Developmental Biology, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 (Japan); Nohno, Tsutomu, E-mail: nohno@bcc.kawasaki-m.ac.jp [Department of Molecular and Developmental Biology, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 (Japan); Tomita, Masafumi, E-mail: toxicology@med.kawasaki-m.ac.jp [Department of Medical Toxicology, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 (Japan); Saijoh, Kiyofumi, E-mail: saijohk@med.kanazawa-u.ac.jp [Department of Hygiene, Kanazawa University School of Medicine, 13-1 Takaramachi, Kanazawa 920-8564 (Japan); Sunami, Shigeo, E-mail: ssunami@med.kawasaki-m.ac.jp [Department of Clinical Nutrition, Kawasaki University of Medical Welfare, 288 Matsushima, Kurashiki 701-0193 (Japan); Katsuyama, Hironobu, E-mail: katsu@med.kawasaki-m.ac.jp [Department of Public Health, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 (Japan)

    2009-12-13

    17{beta}-Estradiol (E2) plays important roles in the development and differentiation of the gonad and central nervous systems, but little is known regarding the effects of exogenous E2 on chondrogenesis in skeletal development. In the present study, we found that treatment with E2 1-5 days post-fertilization (dpf) at concentrations above 1.5 x 10{sup -5} M increased the mortality rate in zebrafish embryos. Morphological analysis showed that treatment with E2 1-5 dpf caused abnormal cartilage formation in a dose-dependent manner at concentrations above 5 x 10{sup -6} M. E2 1-5 dpf at 1.5 x 10{sup -5} M caused defects of the ethmoid plate, parallel cleft of the trabecular cartilage, and hypoplasia of Meckel's cartilage and the ceratohyal cartilage. The sensitivity of embryos to E2 depended on the developmental stage. In early chondrogenesis (1-2 dpf), the embryos were highly sensitive to E2, leading to hypoplasia of the cartilage. In situ hybridization studies showed that expression levels of patched1 (ptc1) and patched2 (ptc2) receptor mRNAs were markedly decreased by exposure to 2 x 10{sup -5} M E2 1-2 dpf. However, the expression levels of sonic hedgehog (shh) and tiggywinkle hedgehog (twhh) mRNAs were constant in the E2-treated embryos. In addition, the estrogen receptor antagonist ICI 182,780 did not completely abolish the effects of E2, suggesting that E2 may not inhibit chondrogenesis through its nuclear estrogen receptor. These results suggest that exposure to exogenous E2 possibly inhibits chondrogenesis via inhibition of the hedgehog (Hh) signal transduction system.

  19. Temporally degradable collagen-mimetic hydrogels tuned to chondrogenesis of human mesenchymal stem cells.

    Science.gov (United States)

    Parmar, Paresh A; Skaalure, Stacey C; Chow, Lesley W; St-Pierre, Jean-Philippe; Stoichevska, Violet; Peng, Yong Y; Werkmeister, Jerome A; Ramshaw, John A M; Stevens, Molly M

    2016-08-01

    Tissue engineering strategies for repairing and regenerating articular cartilage face critical challenges to recapitulate the dynamic and complex biochemical microenvironment of native tissues. One approach to mimic the biochemical complexity of articular cartilage is through the use of recombinant bacterial collagens as they provide a well-defined biological 'blank template' that can be modified to incorporate bioactive and biodegradable peptide sequences within a precisely defined three-dimensional system. We customized the backbone of a Streptococcal collagen-like 2 (Scl2) protein with heparin-binding, integrin-binding, and hyaluronic acid-binding peptide sequences previously shown to modulate chondrogenesis and then cross-linked the recombinant Scl2 protein with a combination of matrix metalloproteinase 7 (MMP7)- and aggrecanase (ADAMTS4)-cleavable peptides at varying ratios to form biodegradable hydrogels with degradation characteristics matching the temporal expression pattern of these enzymes in human mesenchymal stem cells (hMSCs) during chondrogenesis. hMSCs encapsulated within the hydrogels cross-linked with both degradable peptides exhibited enhanced chondrogenic characteristics as demonstrated by gene expression and extracellular matrix deposition compared to the hydrogels cross-linked with a single peptide. Additionally, these combined peptide hydrogels displayed increased MMP7 and ADAMTS4 activities and yet increased compression moduli after 6 weeks, suggesting a positive correlation between the degradation of the hydrogels and the accumulation of matrix by hMSCs undergoing chondrogenesis. Our results suggest that including dual degradation motifs designed to respond to enzymatic activity of hMSCs going through chondrogenic differentiation led to improvements in chondrogenesis. Our hydrogel system demonstrates a bimodal enzymatically degradable biological platform that can mimic native cellular processes in a temporal manner. As such, this novel

  20. Effect of Fibroblast Growth Factor 2 on Equine Synovial Fluid Chondroprogenitor Expansion and Chondrogenesis

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    Marta Bianchessi

    2016-01-01

    Full Text Available Mesenchymal stem cells have been identified in the synovial fluid of several species. This study was conducted to characterize chondroprogenitor (CP cells in equine synovial fluid (SF and to determine the effect of fibroblast growth factor 2 (FGF-2 on SF-CP monolayer proliferation and subsequent chondrogenesis. We hypothesized that FGF-2 would stimulate SF-CP proliferation and postexpansion chondrogenesis. SF aspirates were collected from adult equine joints. Colony-forming unit (CFU assays were performed during primary cultures. At first passage, SF-cells were seeded at low density, with or without FGF-2. Following monolayer expansion and serial immunophenotyping, cells were transferred to chondrogenic pellet cultures. Pellets were analyzed for chondrogenic mRNA expression and cartilage matrix secretion. There was a mean of 59.2 CFU/mL of SF. FGF-2 increased the number of population doublings during two monolayer passages and halved the population doubling times. FGF-2 did not alter the immunophenotype of SF-CPs during monolayer expansion, nor did FGF-2 compromise chondrogenesis. Hypertrophic phenotypic markers were not expressed in control or FGF-2 groups. FGF-2 did prevent the development of a “fibroblastic” cell layer around pellet periphery. FGF-2 significantly accelerates in vitro SF-CP expansion, the major hurdle to clinical application of this cell population, without detrimentally affecting subsequent chondrogenic capacity.

  1. Microenvironment is involved in cellular response to hydrostatic pressures during chondrogenesis of mesenchymal stem cells.

    Science.gov (United States)

    Ye, Rui; Hao, Jin; Song, Jinlin; Zhao, Zhihe; Fang, Shanbao; Wang, Yating; Li, Juan

    2014-06-01

    Chondrocytes integrate numerous microenvironmental cues to mount physiologically relevant differentiation responses, and the regulation of mechanical signaling in chondrogenic differentiation is now coming into intensive focus. To facilitate tissue-engineered chondrogenesis by mechanical strategy, a thorough understanding about the interactional roles of chemical factors under mechanical stimuli in regulating chondrogenesis is in great need. Therefore, this study attempts to investigate the interaction of rat MSCs with their microenvironment by imposing dynamic and static hydrostatic pressure through modulating gaseous tension above the culture medium. Under dynamic pressure, chemical parameters (pH, pO2, and pCO2) were kept in homeostasis. In contrast, pH was remarkably reduced due to increased pCO2 under static pressure. MSCs under the dynamically pressured microenvironment exhibited a strong accumulation of GAG within and outside the alginate beads, while cells under the statically pressured environment lost newly synthesized GAG into the medium with a speed higher than its production. In addition, the synergic influence on expression of chondrogenic genes was more persistent under dynamic pressure than that under static pressure. This temporal contrast was similar to that of activation of endogenous TGF-β1. Taken altogether, it indicates that a loading strategy which can keep a homeostatic chemical microenvironment is preferred, since it might sustain the stimulatory effects of mechanical stimuli on chondrogenesis via activation of endogenous TGF-β1.

  2. Differential effects of dexamethasone on the chondrogenesis of mesenchymal stromal cells: Influence of microenvironment, tissue origin and growth factor

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    N Shintani

    2011-11-01

    Full Text Available nchymal stromal cells (MSCs, which reside within various tissues, are utilized in the engineering of cartilage tissue. Dexamethasone (DEX – a synthetic glucocorticoid – is almost invariably applied to potentiate the growth-factor-induced chondrogenesis of MSCs in vitro, albeit that this effect has been experimentally demonstrated only for transforming-growth-factor-beta (TGF-β-stimulated bone-marrow-derived MSCs. Clinically, systemic glucocorticoid therapy is associated with untoward side effects (e.g., bone loss and increased susceptibility to infection. Hence, the use of these agents should be avoided or limited. We hypothesize that the influence of DEX on the chondrogenesis of MSCs depends upon their tissue origin and microenvironment [absence or presence of an extracellular matrix (ECM], as well as upon the nature of the growth factor. We investigated its effects upon the TGF-β1- and bone-morphogenetic-protein 2 (BMP-2-induced chondrogenesis of MSCs as a function of tissue source (bone marrow vs. synovium and microenvironment [cell aggregates (no ECM vs. explants (presence of a natural ECM]. In aggregates of bone-marrow-derived MSCs, DEX enhanced TGF-β1-induced chondrogenesis by an up-regulation of cartilaginous genes, but had little influence on the BMP-2-induced response. In aggregates of synovial MSCs, DEX exerted no remarkable effect on either TGF-β1- or BMP-2-induced chondrogenesis. In synovial explants, DEX inhibited BMP-2-induced chondrogenesis almost completely, but had little impact on the TGF-β1-induced response. Our data reveal that steroids are not indispensable for the chondrogenesis of MSCs in vitro. Their influence is context dependent (tissue source of the MSCs, their microenvironment and the nature of the growth-factor. This finding has important implications for MSC based approaches to cartilage repair.

  3. 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.

  4. The Cross-talk Between TGF-β1 and Dlk1 Mediates Early Chondrogenesis During Embryonic Endochondral Ossification

    DEFF Research Database (Denmark)

    Taipaleenmaki, Hanna; M, Linda; Chen, Li;

    2012-01-01

    Dlkl/Pref-1/FA1 (delta like-1/preadipocyte factor-1/Fetal Antigen-1) is a novel surface marker for embryonic chondroprogenitor cells undergoing lineage progression from proliferation to prehypertrophic stages. However, mechanisms mediating control of its expression during chondrogenesis...... are not known. Thus, we examined the effect of a number of signalling molecules and their inhibitors on Dlk1 expression during in vitro chondrogenic differentiation in mouse embryonic limb bud mesenchymal micromass cultures and mouse embryonic fibroblast (MEF) pellet cultures. Dlk1 was initially expressed...... its function in embryonic chondrogenesis. The cross-talk between TGF-β1 and Dlk1/FA1 was shown to promote early chondrogenesis during the embryonic endochondral ossification process....

  5. Retinoic acid receptor gamma-induced misregulation of chondrogenesis in the murine limb bud in vitro.

    Science.gov (United States)

    Galdones, Eugene; Hales, Barbara F

    2008-11-01

    Vitamin A derivatives modulate gene expression through retinoic acid and rexinoid receptor (RAR/RXR) heterodimers and are indispensable for limb development. Of particular interest, RARgamma is highly expressed in cartilage, a target affected following retinoid-induced limb insult. The goal of this study was to examine how selective activation of RARgamma affects limb development. Forelimbs from E12.5 CD-1 mice were cultured for 6 days in the presence of all-trans RA (pan-RAR agonist; 0.1 or 1.0 microM) or BMS-189961 (BMS961, RARgamma-selective agonist; 0.01 or 0.1 microM) and limb morphology assessed. Untreated limbs developed normal cartilage elements whereas pan-RAR or RARgamma agonist-treated limbs exhibited reductive effects on chondrogenesis. Retinoid activity was assessed using RAREbeta2 (retinoic acid response element beta2)-lacZ reporter limbs; after 3 h of treatment, both drugs increased retinoid activity proximally. To elucidate the expression profiles of a subset of genes important for development, limbs were cultured for 3 h and cRNA hybridized to osteogenesis-focused microarrays. Two genes, matrix GLA protein (Mgp; chondrogenesis inhibitor) and growth differentiation factor-10 (Gdf10/Bmp3b) were induced by RA and BMS-189961. Real-time PCR was done to validate our results and whole mount in situ hybridizations against Mgp and Gdf10 localized their upregulation to areas of cartilage and programmed cell death, respectively. Thus, our results illustrate the importance of RARgamma in mediating the retinoid-induced upregulation of Mgp and Gdf10; determining their roles in chondrogenesis and cell death will help further unravel mechanisms underlying retinoid teratogenicity. PMID:18703560

  6. Chondrogenesis of human infrapatellar fat pad stem cells on acellular dermal matrix

    Directory of Open Access Journals (Sweden)

    Ken eYe

    2016-01-01

    Full Text Available Acellular dermal matrix (ADM has been in clinical use for decades in numerous surgical applications. The ability for ADM to promote cellular repopulation and revascularisation, and tissue regeneration is well documented. Adipose stem cells have the ability to differentiate into mesenchymal tissue types, including bone and cartilage. The aim of this study was to investigate the potential interaction between ADM and adipose stem cells in vitro using TGFβ3 and BMP6.Human infrapatellar fat pad derived adipose stem cells (IPFP-ASC were cultured with ADM derived from rat dermis under chondrogenic (TGFβ3 and BMP6 in vitro for 2 and 4 weeks. Histology, qPCR and immunohistochemistry were performed to assess for markers of chondrogenesis (collagen Type II, SOX9 and proteoglycans. At 4 weeks, cell-scaffold constructs displayed cellular changes consistent with chondrogenesis, with evidence of stratification of cell layers and development of a hyaline-like cartilage layer superficially which stained positively for collagen Type II and proteoglycans. Significant cell-matrix interaction was seen between the cartilage layer and the ADM itself with seamless integration between each layer. Real time qPCR showed significantly increases of COL2A1, SOX9, and ACAN gene expression over 4 weeks when compared to control. COL1A2 gene expression remained unchanged over 4 weeks.We believe the principles which make ADM versatile and successful for tissue regeneration are application to cartilage regeneration. This study demonstrates in vitro the ability for IPFP-ASCs to undergo chondrogenesis, infiltrate and interact with ADM. These outcomes serve as a platform for in vivo modelling of ADM for cartilage repair.

  7. Chondrogenesis of Human Infrapatellar Fat Pad Stem Cells on Acellular Dermal Matrix.

    Science.gov (United States)

    Ye, Ken; Traianedes, Kathy; Choong, Peter F M; Myers, Damian E

    2016-01-01

    Acellular dermal matrix (ADM) has been in clinical use for decades in numerous surgical applications. The ability for ADM to promote cellular repopulation, revascularisation and tissue regeneration is well documented. Adipose stem cells have the ability to differentiate into mesenchymal tissue types, including bone and cartilage. The aim of this study was to investigate the potential interaction between ADM and adipose stem cells in vitro using TGFβ3 and BMP6. Human infrapatellar fat pad-derived adipose stem cells (IPFP-ASC) were cultured with ADM derived from rat dermis in chondrogenic (TGFβ3 and BMP6) medium in vitro for 2 and 4 weeks. Histology, qPCR, and immunohistochemistry were performed to assess for markers of chondrogenesis (collagen Type II, SOX9 and proteoglycans). At 4 weeks, cell-scaffold constructs displayed cellular changes consistent with chondrogenesis, with evidence of stratification of cell layers and development of a hyaline-like cartilage layer superficially, which stained positively for collagen Type II and proteoglycans. Significant cell-matrix interaction was seen between the cartilage layer and the ADM itself with seamless integration between each layer. Real time qPCR showed significantly increased COL2A1, SOX9, and ACAN gene expression over 4 weeks when compared to control. COL1A2 gene expression remained unchanged over 4 weeks. We believe that the principles that make ADM versatile and successful for tissue regeneration are applicable to cartilage regeneration. This study demonstrates in vitro the ability for IPFP-ASCs to undergo chondrogenesis, infiltrate, and interact with ADM. These outcomes serve as a platform for in vivo modelling of ADM for cartilage repair. PMID:26858950

  8. In vitro chondrogenesis with lysozyme susceptible bacterial cellulose as a scaffold.

    Science.gov (United States)

    Yadav, Vikas; Sun, Lin; Panilaitis, Bruce; Kaplan, David L

    2015-12-01

    A current focus of tissue engineering is the use of adult human mesenchymal stem cells (hMSCs) as an alternative to autologous chondrocytes for cartilage repair. Several natural and synthetic polymers (including cellulose) have been explored as a biomaterial scaffold for cartilage tissue engineering. While bacterial cellulose (BC) has been used in tissue engineering, its lack of degradability in vivo and high crystallinity restricts widespread applications in the field. Recently we reported the formation of a novel bacterial cellulose that is lysozyme-susceptible and -degradable in vivo from metabolically engineered Gluconacetobacter xylinus. Here we report the use of this modified bacterial cellulose (MBC) for cartilage tissue engineering using hMSCs. MBC's glucosaminoglycan-like chemistry, combined with in vivo degradability, suggested opportunities to exploit this novel polymer in cartilage tissue engineering. We have observed that, like BC, MBC scaffolds support cell attachment and proliferation. Chondrogenesis of hMSCs in the MBC scaffolds was demonstrated by real-time RT-PCR analysis for cartilage-specific extracellular matrix (ECM) markers (collagen type II, aggrecan and SOX9) as well as histological and immunohistochemical evaluations of cartilage-specific ECM markers. Further, the attachment, proliferation, and differentiation of hMSCs in MBC showed unique characteristics. For example, after 4 weeks of cultivation, the spatial cell arrangement and collagen type-II and ACAN distribution resembled those in native articular cartilage tissue, suggesting promise for these novel in vivo degradable scaffolds for chondrogenesis.

  9. Biomimetic alginate/polyacrylamide porous scaffold supports human mesenchymal stem cell proliferation and chondrogenesis

    International Nuclear Information System (INIS)

    We describe the development of alginate/polyacrylamide (ALG/PAAm) porous hydrogels based on interpenetrating polymer network structure for human mesenchymal stem cell proliferation and chondrogenesis. Three ALG/PAAm hydrogels at molar ratios of 10/90, 20/80, and 30/70 were prepared and characterized with enhanced elastic and rubbery mechanical properties, which are similar to native human cartilage tissues. Their elasticity and swelling properties were also studied under different physiological pH conditions. Finally, in vitro tests demonstrated that human mesenchymal stem cells could proliferate on the as-synthesized hydrogels with improved alkaline phosphatase activities. These results suggest that ALG/PAAm hydrogels may be a promising biomaterial for cartilage tissue engineering. - Highlights: • ALG/PAAm hydrogels were prepared at different molar ratios for cartilage tissue engineering. • ALG/PAAm hydrogels feature an interpenetrating polymer network structure. • ALG/PAAm hydrogels demonstrate strengthened elastic and rubbery mechanical properties. • hMSCs could be cultured on the ALG/PAAm hydrogels for proliferation and chondrogenesis

  10. Biomimetic alginate/polyacrylamide porous scaffold supports human mesenchymal stem cell proliferation and chondrogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Peng [Department of ENT-Head and Neck Surgery, EENT Hospital, Shanghai 200031 (China); Shanghai Medical School, Fudan University, 210029 (China); Yuan, Yasheng, E-mail: yuanyasheng@163.com [Department of ENT-Head and Neck Surgery, EENT Hospital, Shanghai 200031 (China); Shanghai Medical School, Fudan University, 210029 (China); Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114 (United States); Chi, Fanglu [Department of ENT-Head and Neck Surgery, EENT Hospital, Shanghai 200031 (China); Shanghai Medical School, Fudan University, 210029 (China)

    2014-09-01

    We describe the development of alginate/polyacrylamide (ALG/PAAm) porous hydrogels based on interpenetrating polymer network structure for human mesenchymal stem cell proliferation and chondrogenesis. Three ALG/PAAm hydrogels at molar ratios of 10/90, 20/80, and 30/70 were prepared and characterized with enhanced elastic and rubbery mechanical properties, which are similar to native human cartilage tissues. Their elasticity and swelling properties were also studied under different physiological pH conditions. Finally, in vitro tests demonstrated that human mesenchymal stem cells could proliferate on the as-synthesized hydrogels with improved alkaline phosphatase activities. These results suggest that ALG/PAAm hydrogels may be a promising biomaterial for cartilage tissue engineering. - Highlights: • ALG/PAAm hydrogels were prepared at different molar ratios for cartilage tissue engineering. • ALG/PAAm hydrogels feature an interpenetrating polymer network structure. • ALG/PAAm hydrogels demonstrate strengthened elastic and rubbery mechanical properties. • hMSCs could be cultured on the ALG/PAAm hydrogels for proliferation and chondrogenesis.

  11. Effects of FGF-2 on human adipose tissue derived adult stem cells morphology and chondrogenesis enhancement in Transwell culture

    International Nuclear Information System (INIS)

    Highlights: ► We investigated effects of FGF-2 on hADSCs. ► We examine changes in the level of gene expressions of SOX-9, aggrecan and collagen type II and type X. ► FGF-2 induces chondrogenesis in hADSCs, which •Increasing information will decrease quality if hospital costs are very different. ► The result of this study may be beneficial in cartilage tissue engineering. -- Abstract: Injured cartilage is difficult to repair due to its poor vascularisation. Cell based therapies may serve as tools to more effectively regenerate defective cartilage. Both adult mesenchymal stem cells (MSCs) and human adipose derived stem cells (hADSCs) are regarded as potential stem cell sources able to generate functional cartilage for cell transplantation. Growth factors, in particular the TGF-b superfamily, influence many processes during cartilage formation, including cell proliferation, extracellular matrix synthesis, maintenance of the differentiated phenotype, and induction of MSCs towards chondrogenesis. In the current study, we investigated the effects of FGF-2 on hADSC morphology and chondrogenesis in Transwell culture. hADSCs were obtained from patients undergoing elective surgery, and then cultured in expansion medium alone or in the presence of FGF-2 (10 ng/ml). mRNA expression levels of SOX-9, aggrecan and collagen type II and type X were quantified by real-time polymerase chain reaction. The morphology, doubling time, trypsinization time and chondrogenesis of hADSCs were also studied. Expression levels of SOX-9, collagen type II, and aggrecan were all significantly increased in hADSCs expanded in presence of FGF-2. Furthermore FGF-2 induced a slender morphology, whereas doubling time and trypsinization time decreased. Our results suggest that FGF-2 induces hADSCs chondrogenesis in Transwell culture, which may be beneficial in cartilage tissue engineering.

  12. Effects of FGF-2 on human adipose tissue derived adult stem cells morphology and chondrogenesis enhancement in Transwell culture

    Energy Technology Data Exchange (ETDEWEB)

    Kabiri, Azadeh, E-mail: z_kabiri@resident.mui.ac.ir [Department of Anatomical Sciences and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences (Iran, Islamic Republic of); Esfandiari, Ebrahim, E-mail: esfandiari@med.mui.ac.ir [Department of Anatomical Sciences and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences (Iran, Islamic Republic of); Hashemibeni, Batool, E-mail: hashemibeni@med.mui.ac.ir [Department of Anatomical Sciences and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences (Iran, Islamic Republic of); Kazemi, Mohammad, E-mail: m_kazemi@med.mui.ac.i [Department of Anatomical Sciences and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences (Iran, Islamic Republic of); Mardani, Mohammad, E-mail: mardani@med.mui.ac.ir [Department of Anatomical Sciences and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences (Iran, Islamic Republic of); Esmaeili, Abolghasem, E-mail: abesmaeili@yahoo.com [Cell, Molecular and Developmental Biology Division, Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan (Iran, Islamic Republic of)

    2012-07-27

    Highlights: Black-Right-Pointing-Pointer We investigated effects of FGF-2 on hADSCs. Black-Right-Pointing-Pointer We examine changes in the level of gene expressions of SOX-9, aggrecan and collagen type II and type X. Black-Right-Pointing-Pointer FGF-2 induces chondrogenesis in hADSCs, which Bullet Increasing information will decrease quality if hospital costs are very different. Black-Right-Pointing-Pointer The result of this study may be beneficial in cartilage tissue engineering. -- Abstract: Injured cartilage is difficult to repair due to its poor vascularisation. Cell based therapies may serve as tools to more effectively regenerate defective cartilage. Both adult mesenchymal stem cells (MSCs) and human adipose derived stem cells (hADSCs) are regarded as potential stem cell sources able to generate functional cartilage for cell transplantation. Growth factors, in particular the TGF-b superfamily, influence many processes during cartilage formation, including cell proliferation, extracellular matrix synthesis, maintenance of the differentiated phenotype, and induction of MSCs towards chondrogenesis. In the current study, we investigated the effects of FGF-2 on hADSC morphology and chondrogenesis in Transwell culture. hADSCs were obtained from patients undergoing elective surgery, and then cultured in expansion medium alone or in the presence of FGF-2 (10 ng/ml). mRNA expression levels of SOX-9, aggrecan and collagen type II and type X were quantified by real-time polymerase chain reaction. The morphology, doubling time, trypsinization time and chondrogenesis of hADSCs were also studied. Expression levels of SOX-9, collagen type II, and aggrecan were all significantly increased in hADSCs expanded in presence of FGF-2. Furthermore FGF-2 induced a slender morphology, whereas doubling time and trypsinization time decreased. Our results suggest that FGF-2 induces hADSCs chondrogenesis in Transwell culture, which may be beneficial in cartilage tissue engineering.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Small Molecule-BIO Accelerates and Enhances Marrow-Derived Mesenchymal Stem Cell in Vitro Chondrogenesis

    Directory of Open Access Journals (Sweden)

    Mohamadreza Baghaban Eslaminejad

    2014-03-01

    Full Text Available Background: Hyaline cartilage defects exhibit a major challenge in the field of orthopedic surgery owing to its limited repair capacity. On the other hand, mesenchymal stem cells (MSCs are regarded as potent cells with a property of cartilage regeneration. We aimed to optimize marrow-derived MSC chondrogenic culture using a small bioactive molecule referred to as BIO. Methods: MSCs from the marrow of NMRI mice were extracted, culture-expanded, and characterized. Micro-mass culture was then established for chondrogenic differentiation (control group. The cultures of MSC in chondrogenic medium supplemented with 0.01, 0.05, 0.1, and 1 µM BIO were taken as the experimental groups. Cartilage differentiation was examined by both histological sections and real-time PCR for Sox9, aggrecan, and collagen II at different time points. Moreover, the involvement of the Wnt pathway was investigated. Results: Based on histological sections, there was seemingly more intense metachromatic matrix produced in the cultures with 0.01 µM BIO. In this experimental group, cartilage-specific genes tended to be upregulated at day 14 compared to day 21 of the control group, indicating the accelerating effect of BIO on cartilage differentiation. Overall, there was statistically a significant increase (P=0.01 in the expression level of cartilage-specific genes in cultures with 0.01 µM BIO (enhancing effects. These upregulations appeared to be mediated through the Wnt pathway evident from the significant upregulation of T-cell factor and beta-catenin molecules (P=0.01. Conclusion: Taken together, BIO at 0.01 µM could accelerate and enhance in vitro chondrogenesis of mouse marrow-derived MSCs. Please cite this article as: Baghaban Eslaminejad MR, Fallah N. Small Molecule-BIO Accelerates and Enhances Marrow-Derived Mesenchymal Stem Cell in Vitro Chondrogenesis. Iran J Med Sci. 2014;39(2:107-116.

  15. Feasibility of arthroscopic placement of autologous matrix-induced chondrogenesis grafts in the cadaver hip joint

    Directory of Open Access Journals (Sweden)

    Fritz Thorey

    2013-09-01

    Full Text Available An assortment of clinical trials have been done presenting the effectiveness of autologous matrix-induced chondrogenesis (AMIC for the regeneration of chondral leasions. The purpose of the study was to underline the accessability of the acetabulum and the femoral head through the known portals and prove i the feasibility of placing the AMIC in the different zones of the hip joint and ii check for dislocation after joint movement. Six human cadavers underwent hip arthroscopy on both hips. Two chondral lesions were set on each femoral head and two in the acetabulum to evaluate a total of 48 defects. After microfracturing an autologous matrix-induced chondrogenesis graft was placed on these lesions arthroscopically. After repeated joint movement the dislocation of the graft was checked. It was possible to place the AMIC graft in all 48 chondral lesions. The time needed for placing the graft was 8±2.9 minutes. A trend of time reduction could be detected throughout this study as the surgeon gained more experience. For the femoral head, after twenty cycles of joint movement 18/24 spots showed no displacement, 4/24 showed minor displacement (<3 mm and 2/24 showed major displacement (>3 mm. None showed total displacement. For the acetabulum 22/24 spots showed no displacement and 2/24 showed minor displacement. A combined microfracturing and placing of an AMIC graft of focal chondral lesions of the hip joint can be done arthroscopically. Prospective randomized in vivo studies should compare the results of arthroscopilally placed AMIC grafts with microfracturing and microfracturing alone.

  16. Non-destructive label-free continuous monitoring of in vitro chondrogenesis via electrical conductivity and its anisotropy.

    Science.gov (United States)

    Oh, Tong In; Kim, Changhwan; Karki, Bishal; Son, Youngsook; Lee, EunAh; Woo, Eung Je

    2015-02-01

    Non-destructive label-free continuous monitoring of in vitro tissue culture is an unmet demand in tissue engineering. Noting that different compositions of cartilage lead to different electrical tissue properties, we propose a new method to measure the electrical conductivity and its anisotropy during in vitro chondrogenesis. We used a conductivity tensor probe with 17 electrodes and a bio-impedance spectroscopy (BIS) device to measure the conductivity values and the anisotropy ratios at the bottom and top surfaces of the tissue samples during the culture period of 6 weeks. Clearly distinguishing glycosaminoglycans (GAGs), collagen, and also various mixtures of them, the measured conductivity value and the estimated tissue anisotropy provide diagnostic information of the depth-dependent tissue structure and compositions. Continuously monitoring the individual tissue during the entire chondrogenesis process without any adverse effect, the proposed method may significantly increase the productivity of cartilage tissue engineering.

  17. Chondrogenesis in scleral stem/progenitor cells and its association with form-deprived myopia in mice

    OpenAIRE

    Wu, Pei-Chang; Tsai, Chia-LIng; Gordon, Gabriel M; Jeong, Shinwu; Itakura, Tatsuo; Patel, Nitin; Shi, Songtao; Fini, M. Elizabeth

    2015-01-01

    Purpose: Previously, we demonstrated that scleral stem/progenitor cells (SSPCs) from mice have a chondrogenic differentiation potential, which is stimulated by transforming growth factor-β (TGF-β). In the present study, we hypothesized that chondrogenesis in the sclera could be a possible mechanism in myopia development. Therefore, we investigated the association of form-deprivation myopia (FDM) with expressions in mice sclera representing the chondrogenic phenotype: collagen type II (Col2) a...

  18. The influence of hyaluronic acid hydrogel crosslinking density and macromolecular diffusivity on human MSC chondrogenesis and hypertrophy

    OpenAIRE

    Bian, Liming; Hou, Chieh; Tous, Elena; Rai, Reena; Mauck, Robert L.; Burdick, Jason A.

    2012-01-01

    Hyaluronic acid (HA) hydrogels formed via photocrosslinking provide stable 3D hydrogel environments that support the chondrogenesis of mesenchymal stem cells (MSCs). Crosslinking density has a significant impact on the physical properties of hydrogels, including their mechanical stiffness and macromolecular diffusivity. Variations in the HA hydrogel crosslinking density can be obtained by either changes in the HA macromer concentration (1, 3, or 5% w/v at 15 min exposure) or the extent of rea...

  19. The immediate early gene product EGR1 and polycomb group proteins interact in epigenetic programming during chondrogenesis.

    Directory of Open Access Journals (Sweden)

    Frank Spaapen

    Full Text Available Initiation of and progression through chondrogenesis is driven by changes in the cellular microenvironment. At the onset of chondrogenesis, resting mesenchymal stem cells are mobilized in vivo and a complex, step-wise chondrogenic differentiation program is initiated. Differentiation requires coordinated transcriptomic reprogramming and increased progenitor proliferation; both processes require chromatin remodeling. The nature of early molecular responses that relay differentiation signals to chromatin is poorly understood. We here show that immediate early genes are rapidly and transiently induced in response to differentiation stimuli in vitro. Functional ablation of the immediate early factor EGR1 severely deregulates expression of key chondrogenic control genes at the onset of differentiation. In addition, differentiating cells accumulate DNA damage, activate a DNA damage response and undergo a cell cycle arrest and prevent differentiation associated hyper-proliferation. Failed differentiation in the absence of EGR1 affects global acetylation and terminates in overall histone hypermethylation. We report novel molecular connections between EGR1 and Polycomb Group function: Polycomb associated histone H3 lysine27 trimethylation (H3K27me3 blocks chromatin access of EGR1. In addition, EGR1 ablation results in abnormal Ezh2 and Bmi1 expression. Consistent with this functional interaction, we identify a number of co-regulated targets genes in a chondrogenic gene network. We here describe an important role for EGR1 in early chondrogenic epigenetic programming to accommodate early gene-environment interactions in chondrogenesis.

  20. Steric Interference of Adhesion Supports In-Vitro Chondrogenesis of Mesenchymal Stem Cells on Hydrogels for Cartilage Repair

    Science.gov (United States)

    Goldshmid, Revital; Cohen, Shlomit; Shachaf, Yonatan; Kupershmit, Ilana; Sarig-Nadir, Offra; Seliktar, Dror; Wechsler, Roni

    2015-01-01

    Recent studies suggest the presence of cell adhesion motifs found in structural proteins can inhibit chondrogenesis. In this context, the current study aims to determine if a polyethylene glycol (PEG)-modified fibrinogen matrix could support better chondrogenesis of human bone marrow mesenchymal stem cells (BM-MSC) based on steric interference of adhesion, when compared to a natural fibrin matrix. Hydrogels used as substrates for two-dimensional (2D) BM-MSC cultures under chondrogenic conditions were made from cross-linked PEG-fibrinogen (PF) and compared to thrombin-activated fibrin. Cell morphology, protein expression, DNA and sulfated proteoglycan (GAG) content were correlated to substrate properties such as stiffness and adhesiveness. Cell aggregation and chondrogenic markers, including collagen II and aggrecan, were observed on all PF substrates but not on fibrin. Shielding fibrinogen’s adhesion domains and increasing stiffness of the material are likely contributing factors that cause the BM-MSCs to display a more chondrogenic phenotype. One composition of PF corresponding to GelrinC™—a product cleared in the EU for cartilage repair—was found to be optimal for supporting chondrogenic differentiation of BM-MSC while minimizing hypertrophy (collagen X). These findings suggest that semi-synthetic biomaterials based on ECM proteins can be designed to favourably affect BM-MSC towards repair processes involving chondrogenesis. PMID:26411496

  1. Chemical modification of extracellular matrix by cold atmospheric plasma-generated reactive species affects chondrogenesis and bone formation.

    Science.gov (United States)

    Eisenhauer, Peter; Chernets, Natalie; Song, You; Dobrynin, Danil; Pleshko, Nancy; Steinbeck, Marla J; Freeman, Theresa A

    2016-09-01

    The goal of this study was to investigate whether cold plasma generated by dielectric barrier discharge (DBD) modifies extracellular matrices (ECM) to influence chondrogenesis and endochondral ossification. Replacement of cartilage by bone during endochondral ossification is essential in fetal skeletal development, bone growth and fracture healing. Regulation of this process by the ECM occurs through matrix remodelling, involving a variety of cell attachment molecules and growth factors, which influence cell morphology and protein expression. The commercially available ECM, Matrigel, was treated with microsecond or nanosecond pulsed (μsp or nsp, respectively) DBD frequencies conditions at the equivalent frequencies (1 kHz) or power (~1 W). Recombinant human bone morphogenetic protein-2 was added and the mixture subcutaneously injected into mice to simulate ectopic endochondral ossification. Two weeks later, the masses were extracted and analysed by microcomputed tomography. A significant increase in bone formation was observed in Matrigel treated with μsp DBD compared with control, while a significant decrease in bone formation was observed for both nsp treatments. Histological and immunohistochemical analysis showed Matrigel treated with μsp plasma increased the number of invading cells, the amount of vascular endothelial growth factor and chondrogenesis while the opposite was true for Matrigel treated with nsp plasma. In support of the in vivo Matrigel study, 10 T1/2 cells cultured in vitro on μsp DBD-treated type I collagen showed increased expression of adhesion proteins and activation of survival pathways, which decreased with nsp plasma treatments. These results indicate DBD modification of ECM can influence cellular behaviours to accelerate or inhibit chondrogenesis and endochondral ossification. Copyright © 2016 John Wiley & Sons, Ltd.

  2. Role of PTHrP(1-34) Pulse Frequency Versus Pulse Duration to Enhance Mesenchymal Stromal Cell Chondrogenesis.

    Science.gov (United States)

    Fischer, Jennifer; Ortel, Marlen; Hagmann, Sebastien; Hoeflich, Andreas; Richter, Wiltrud

    2016-12-01

    Generation of phenotypically stable, articular chondrocytes from mesenchymal stromal cells (MSCs) is still an unaccomplished task, with formation of abundant, hyaline extracellular matrix, and avoidance of hypertrophy being prime challenges. We recently demonstrated that parathyroid hormone-related protein (PTHrP) is a promising factor to direct chondrogenesis of MSCs towards an articular phenotype, since intermittent PTHrP application stimulated cartilage matrix production and reduced undesired hypertrophy. We here investigated the role of frequency, pulse duration, total exposure time, and underlying mechanisms in order to unlock the full potential of PTHrP actions. Human MSC subjected to in vitro chondrogenesis for six weeks were exposed to 2.5 nM PTHrP(1-34) pulses from days 7 to 42. Application frequency was increased from three times weekly (3 × 6 h/week) to daily maintaining either the duration of individual pulses (6 h/day) or total exposure time (18 h/week; 2.6 h/day). Daily PTHrP treatment significantly increased extracellular matrix deposition regardless of pulse duration and suppressed alkaline-phosphatase activity by 87%. High total exposure time significantly reduced cell proliferation at day 14. Pulse duration was critically important to significantly reduce IHH expression, but irrelevant for PTHrP-induced suppression of the hypertrophic markers MEF2C and IBSP. COL10A1, RUNX2, and MMP13 expression remained unaltered. Decreased IGFBP-2, -3, and -6 expression suggested modulated IGF-I availability in PTHrP groups, while drop of SOX9 protein levels during the PTHrP-pulse may delay chondroblast formation and hypertrophy. Overall, the significantly optimized timing of PTHrP-pulses demonstrated a vast potential to enhance chondrogenesis of MSC and suppress hypertrophy possibly via superior balancing of IGF- and SOX9-related mechanisms. J. Cell. Physiol. 231: 2673-2681, 2016. © 2016 Wiley Periodicals, Inc. PMID:27548511

  3. Paraspeckle protein p54nrb links Sox9-mediated transcription with RNA processing during chondrogenesis in mice

    OpenAIRE

    Hata, Kenji; Nishimura, Riko; Muramatsu, Shuji; Matsuda, Akio; Matsubara, Takuma; Amano, Katsuhiko; Ikeda, Fumiyo; Harley, Vincent R.; Yoneda, Toshiyuki

    2008-01-01

    The Sox9 transcription factor plays an essential role in promoting chondrogenesis and regulating expression of chondrocyte extracellular-matrix genes. To identify genes that interact with Sox9 in promoting chondrocyte differentiation, we screened a cDNA library generated from the murine chondrogenic ATDC5 cell line to identify activators of the collagen, type II, α 1 (Col2a1) promoter. Here we have shown that paraspeckle regulatory protein 54-kDa nuclear RNA-binding protein (p54nrb) is an ess...

  4. Chondrogenesis of human bone marrow mesenchymal stromal cells in highly porous alginate-foams supplemented with chondroitin sulfate

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Zhao [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Nooeaid, Patcharakamon [Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg (Germany); Kohl, Benjamin [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Roether, Judith A.; Schubert, Dirk W. [Institute of Polymer Materials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg (Germany); Meier, Carola [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Boccaccini, Aldo R. [Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg (Germany); Godkin, Owen; Ertel, Wolfgang; Arens, Stephan [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Schulze-Tanzil, Gundula, E-mail: gundula.schulze@pmu.ac.at [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Institute of Anatomy, Paracelsus Medical University, Nuremberg (Germany)

    2015-05-01

    To overcome the limited intrinsic cartilage repair, autologous chondrocyte or bone-marrow-derived mesenchymal stromal cell (BM-MSC) was implanted into cartilage defects. For this purpose suitable biocompatible scaffolds are needed to provide cell retention, chondrogenesis and initial mechanical stability. The present study should indicate whether a recently developed highly porous alginate (Alg) foam scaffold supplemented with chondroitin sulfate (CS) allows the attachment, survival and chondrogenesis of BM-MSCs and articular chondrocytes. The foams were prepared using a freeze-drying method; some of them were supplemented with CS and subsequently characterized for porosity, biodegradation and mechanical profile. BM-MSCs were cultured for 1–2 weeks on the scaffold either under chondrogenic or maintenance conditions. Cell vitality assays, histology, glycosaminoglycan (sGAG) assay, and type II and I collagen immunolabelings were performed to monitor cell growth and extracellular matrix (ECM) synthesis in the scaffolds. Scaffolds had a high porosity ~ 93–95% with a mean pore sizes of 237 ± 48 μm (Alg) and 197 ± 61 μm (Alg/CS). Incorporation of CS increased mechanical strength of the foams providing gradually CS release over 7 days. Most of the cells survived in the scaffolds. BM-MSCs and articular chondrocytes formed rounded clusters within the scaffold pores. The BM-MSCs, irrespective of whether cultured under non/chondrogenic conditions and chondrocytes produced an ECM containing sGAGs, and types II and I collagen. Total collagen and sGAG contents were higher in differentiated BM-MSC cultures supplemented with CS than in CS-free foams after 14 days. The cell cluster formation induced by the scaffolds might stimulate chondrogenesis via initial intense cell–cell contacts. - Highlights: • Alginate foam scaffolds revealed a high porosity and mean pore size of 197–237 μm. • Chondroitin sulfate was released over 14 days by the scaffolds. • Chondrocytes

  5. Conditional expression of Wnt4 during chondrogenesis leads to dwarfism in mice.

    Directory of Open Access Journals (Sweden)

    Hu-Hui Lee

    Full Text Available Wnts are expressed in the forming long bones, suggesting roles in skeletogenesis. To examine the action of Wnts in skeleton formation, we developed a genetic system to conditionally express Wnt4 in chondrogenic tissues of the mouse. A mouse Wnt4 cDNA was introduced into the ubiquitously expressed Rosa26 (R26 locus by gene targeting in embryonic stem (ES cells. The expression of Wnt4 from the R26 locus was blocked by a neomycin selection cassette flanked by loxP sites (floxneo that was positioned between the Rosa26 promoter and the Wnt4 cDNA, creating the allele designated R26(floxneoWnt4. Wnt4 expression was activated during chondrogenesis using Col2a1-Cre transgenic mice that express Cre recombinase in differentiating chondrocytes. R26(floxneoWnt4; Col2a1-Cre double heterozygous mice exhibited a growth deficiency, beginning approximately 7 to 10 days after birth, that resulted in dwarfism. In addition, they also had craniofacial abnormalities, and delayed ossification of the lumbar vertebrae and pelvic bones. Histological analysis revealed a disruption in the organization of the growth plates and a delay in the onset of the primary and secondary ossification centers. Molecular studies showed that Wnt4 overexpression caused decreased proliferation and altered maturation of chondrocytes. In addition, R26(floxneoWnt4; Col2a1-Cre mice had decreased expression of vascular endothelial growth factor (VEGF. These studies demonstrate that Wnt4 overexpression leads to dwarfism in mice. The data indicate that Wnt4 levels must be regulated in chondrocytes for normal growth plate development and skeletogenesis. Decreased VEGF expression suggests that defects in vascularization may contribute to the dwarf phenotype.

  6. The influence of IL-10 and TNFα on chondrogenesis of human mesenchymal stromal cells in three-dimensional cultures.

    Science.gov (United States)

    Jagielski, Michal; Wolf, Johannes; Marzahn, Ulrike; Völker, Anna; Lemke, Marion; Meier, Carola; Ertel, Wolfgang; Godkin, Owen; Arens, Stephan; Schulze-Tanzil, Gundula

    2014-01-01

    Chondrogenic differentiated mesenchymal stromal cells (MSCs) are a promising cell source for articular cartilage repair. This study was undertaken to determine the effectiveness of two three-dimensional (3D) culture systems for chondrogenic MSC differentiation in comparison to primary chondrocytes and to assess the effect of Interleukin (IL)-10 and Tumor Necrosis Factor (TNF)α on chondrogenesis by MSCs in 3D high-density (H-D) culture. MSCs were isolated from femur spongiosa, characterized using a set of typical markers and introduced in scaffold-free H-D cultures or non-woven polyglycolic acid (PGA) scaffolds for chondrogenic differentiation. H-D cultures were stimulated with recombinant IL-10, TNFα, TNFα + IL-10 or remained untreated. Gene and protein expression of type II collagen, aggrecan, sox9 and TNFα were examined. MSCs expressed typical cell surface markers and revealed multipotency. Chondrogenic differentiated cells expressed cartilage-specific markers in both culture systems but to a lower extent when compared with articular chondrocytes. Chondrogenesis was more pronounced in PGA compared with H-D culture. IL-10 and/or TNFα did not impair the chondrogenic differentiation of MSCs. Moreover, in most of the investigated samples, despite not reaching significance level, IL-10 had a stimulatory effect on the type II collagen, aggrecan and TNFα expression when compared with the respective controls. PMID:25207597

  7. The Influence of IL-10 and TNFα on Chondrogenesis of Human Mesenchymal Stromal Cells in Three-Dimensional Cultures

    Directory of Open Access Journals (Sweden)

    Michal Jagielski

    2014-09-01

    Full Text Available Chondrogenic differentiated mesenchymal stromal cells (MSCs are a promising cell source for articular cartilage repair. This study was undertaken to determine the effectiveness of two three-dimensional (3D culture systems for chondrogenic MSC differentiation in comparison to primary chondrocytes and to assess the effect of Interleukin (IL-10 and Tumor Necrosis Factor (TNFα on chondrogenesis by MSCs in 3D high-density (H-D culture. MSCs were isolated from femur spongiosa, characterized using a set of typical markers and introduced in scaffold-free H-D cultures or non-woven polyglycolic acid (PGA scaffolds for chondrogenic differentiation. H-D cultures were stimulated with recombinant IL-10, TNFα, TNFα + IL-10 or remained untreated. Gene and protein expression of type II collagen, aggrecan, sox9 and TNFα were examined. MSCs expressed typical cell surface markers and revealed multipotency. Chondrogenic differentiated cells expressed cartilage-specific markers in both culture systems but to a lower extent when compared with articular chondrocytes. Chondrogenesis was more pronounced in PGA compared with H-D culture. IL-10 and/or TNFα did not impair the chondrogenic differentiation of MSCs. Moreover, in most of the investigated samples, despite not reaching significance level, IL-10 had a stimulatory effect on the type II collagen, aggrecan and TNFα expression when compared with the respective controls.

  8. Silk fibroin/gelatin–chondroitin sulfate–hyaluronic acid effectively enhances in vitro chondrogenesis of bone marrow mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Sawatjui, Nopporn [Biomedical Sciences, Graduate School, Khon Kaen University, Khon Kaen 40002 (Thailand); Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002 (Thailand); Damrongrungruang, Teerasak [Department of Oral Diagnosis, Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002 (Thailand); Leeanansaksiri, Wilairat [Stem Cell Therapy and Transplantation Research Group, Suranaree University of Technology, Nakhon Ratchasima 30000 (Thailand); School of Microbiology, Suranaree University of Technology, Nakhon Ratchasima 30000 (Thailand); Jearanaikoon, Patcharee [Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002 (Thailand); Hongeng, Suradej [Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400 (Thailand); Limpaiboon, Temduang, E-mail: temduang@kku.ac.th [Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002 (Thailand)

    2015-07-01

    Tissue engineering is becoming promising for cartilage repair due to the limited self-repair capacity of cartilage tissue. We previously fabricated and characterized a three-dimensional silk fibroin/gelatin–chondroitin sulfate–hyaluronic acid (SF–GCH) scaffold and showed that it could promote proliferation of human bone marrow mesenchymal stem cells (BM-MSCs). This study aimed to evaluate its biological performance as a new biomimetic material for chondrogenic induction of BM-MSCs in comparison to an SF scaffold and conventional pellet culture. We found that the SF–GCH scaffold significantly enhanced the proliferation and chondrogenic differentiation of BM-MSCs compared to the SF scaffold and pellet culture in which the production of sulfated glycoaminoglycan was increased in concordance with the up-regulation of chondrogenic-specific gene markers. Our findings indicate the significant role of SF–GCH by providing a supportive structure and the mimetic cartilage environment for chondrogenesis which enables cartilage regeneration. Thus, our fabricated SF–GCH scaffold may serve as a potential biomimetic material for cartilage tissue engineering. - Highlights: • SF–GCH scaffold enhances proliferation and chondrogenic differentiation of BM-MSCs. • SF–GCH acts as a supportive and biomimetic material for BM-MSC chondrogenesis. • SF–GCH is a potential biomimetic scaffold suitable for cartilage tissue engineering.

  9. Silk fibroin/gelatin–chondroitin sulfate–hyaluronic acid effectively enhances in vitro chondrogenesis of bone marrow mesenchymal stem cells

    International Nuclear Information System (INIS)

    Tissue engineering is becoming promising for cartilage repair due to the limited self-repair capacity of cartilage tissue. We previously fabricated and characterized a three-dimensional silk fibroin/gelatin–chondroitin sulfate–hyaluronic acid (SF–GCH) scaffold and showed that it could promote proliferation of human bone marrow mesenchymal stem cells (BM-MSCs). This study aimed to evaluate its biological performance as a new biomimetic material for chondrogenic induction of BM-MSCs in comparison to an SF scaffold and conventional pellet culture. We found that the SF–GCH scaffold significantly enhanced the proliferation and chondrogenic differentiation of BM-MSCs compared to the SF scaffold and pellet culture in which the production of sulfated glycoaminoglycan was increased in concordance with the up-regulation of chondrogenic-specific gene markers. Our findings indicate the significant role of SF–GCH by providing a supportive structure and the mimetic cartilage environment for chondrogenesis which enables cartilage regeneration. Thus, our fabricated SF–GCH scaffold may serve as a potential biomimetic material for cartilage tissue engineering. - Highlights: • SF–GCH scaffold enhances proliferation and chondrogenic differentiation of BM-MSCs. • SF–GCH acts as a supportive and biomimetic material for BM-MSC chondrogenesis. • SF–GCH is a potential biomimetic scaffold suitable for cartilage tissue engineering

  10. BMP-2-enhanced chondrogenesis involves p38 MAPK-mediated down-regulation of Wnt-7a pathway.

    Science.gov (United States)

    Jin, Eun-Jung; Lee, Sun-Young; Choi, Young-Ae; Jung, Jae-Chang; Bang, Ok-Sun; Kang, Shin-Sung

    2006-12-31

    The bone morphogenetic protein (BMP) family has been implicated in control of cartilage development. Here, we demonstrate that BMP-2 promotes chondrogenesis by activating p38 mitogen-activated protein kinase (MAPK), which in turn downregulates Wnt-7a/b-catenin signaling responsible for proteasomal degradation of Sox9. Exposure of mesenchymal cells to BMP-2 resulted in upregulation of Sox9 protein and a concomitant decrease in the level of b-catenin protein and Wnt-7a signaling. In agreement with this, the interaction of Sox9 with b-catenin was inhibited in the presence of BMP-2. Inhibition of the p38 MAPK pathway using a dominant negative mutant led to sustained Wnt-7a signaling and decreased Sox9 expression, with consequent inhibition of precartilage condensation and chondrogenic differentiation. Moreover, overexpression of b-catenin caused degradation of Sox9 via the ubiquitin/26S proteasome pathway. Our results collectively indicate that the increase in Sox9 protein resulting from downregulation of b-catenin/Wnt-7a signaling is mediated by p38 MAPK during BMP-2 induced chondrogenesis in chick wing bud mesenchymal cells. PMID:17202865

  11. Regulating Chondrogenesis of Human Mesenchymal Stromal Cells with a Retinoic Acid Receptor-Beta Inhibitor: Differential Sensitivity of Chondral Versus Osteochondral Development

    Directory of Open Access Journals (Sweden)

    Solvig Diederichs

    2014-05-01

    Full Text Available Aim: Main objective was to investigate whether the synthetic retinoic acid receptor (RAR-β antagonist LE135 is able to drive in vitro chondrogenesis of human mesenchymal stromal cells (MSCs or improve differentiation by suppressing hypertrophic chondrocyte development. Methods: Chondrogenesis of human bone marrow and adipose tissue-derived MSCs was induced in micromass pellet culture for six weeks. Effects of LE135 alone and in combinatorial treatment with TGF-β on deposition of cartilaginous matrix including collagen type II and glycosaminoglycans, on deposition of non-hyaline cartilage collagens type I and X, and on hypertrophy markers including alkaline phosphatase (ALP, indian hedghehog (IHH and matrix metalloproteinase (MMP-13 were assessed. Results: LE135 was no inducer of chondrogenesis and failed to stimulate deposition of collagen type II and glycosaminoglycans. Moreover, addition of LE135 to TGF-β-treated pellets inhibited cartilaginous matrix deposition and gene expression of COL2A1. In contrast, non-hyaline cartilage collagens were less sensitive to LE135 and hypertrophy markers remained unaffected. Conclusion: This demonstrates a differential sensitivity of chondral versus endochondral differentiation pathways to RARβ signaling; however, opposite to the desired direction. The relevance of trans-activating versus trans-repressing RAR signaling, including effects on activator protein (AP-1 is discussed and implications for overcoming current limits of hMSC chondrogenesis are considered.

  12. Fibronectin- and collagen-mimetic ligands regulate bone marrow stromal cell chondrogenesis in three-dimensional hydrogels

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    JT Connelly

    2011-09-01

    Full Text Available Modification of tissue engineering scaffolds with bioactive molecules is a potential strategy for modulating cell behavior and guiding tissue regeneration. While adhesion to RGD peptides has been shown to inhibit in vitro chondrogenesis, the effects of extracellular matrix (ECM-mimetic ligands with complex secondary and tertiary structures are unknown. This study aimed to determine whether collagen- and fibronectin-mimetic ligands would retain biologic functionality in three-dimensional (3D hydrogels, whether different ECM-mimetic ligands differentially influence in vitro chondrogenesis, and if effects of ligands on differentiation depend on soluble biochemical stimuli. A linear RGD peptide, a recombinant fibronectin fragment containing the seven to ten Type III repeats (FnIII7-10 and a triple helical, collagen mimetic peptide with the GFOGER motif were covalently coupled to agarose gels using the sulfo-SANPAH crosslinker, and bone marrow stromal cells (BMSCs were cultured within the 3D hydrogels. The ligands retained biologic functionality within the agarose gels and promoted density-dependent BMSC spreading. Interactions with all adhesive ligands inhibited stimulation by chondrogenic factors of collagen Type II and aggrecan mRNA levels and deposition of sulfated glycosaminoglycans. In medium containing fetal bovine serum, interactions with the GFOGER peptide enhanced mRNA expression of the osteogenic gene osteocalcin whereas FnIII7-10 inhibited osteocalcin expression. In conclusion, modification of agarose hydrogels with ECM-mimetic ligands can influence the differentiation of BMSCs in a manner that depends strongly on the presence and nature of soluble biochemical stimuli.

  13. Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull.

    Science.gov (United States)

    Wada, Naoyuki; Javidan, Yashar; Nelson, Sarah; Carney, Thomas J; Kelsh, Robert N; Schilling, Thomas F

    2005-09-01

    Neural crest cells that form the vertebrate head skeleton migrate and interact with surrounding tissues to shape the skull, and defects in these processes underlie many human craniofacial syndromes. Signals at the midline play a crucial role in the development of the anterior neurocranium, which forms the ventral braincase and palate, and here we explore the role of Hedgehog (Hh) signaling in this process. Using sox10:egfp transgenics to follow neural crest cell movements in the living embryo, and vital dye labeling to generate a fate map, we show that distinct populations of neural crest form the two main cartilage elements of the larval anterior neurocranium: the paired trabeculae and the midline ethmoid. By analyzing zebrafish mutants that disrupt sonic hedgehog (shh) expression, we demonstrate that shh is required to specify the movements of progenitors of these elements at the midline, and to induce them to form cartilage. Treatments with cyclopamine, to block Hh signaling at different stages, suggest that although requirements in morphogenesis occur during neural crest migration beneath the brain, requirements in chondrogenesis occur later, as cells form separate trabecular and ethmoid condensations. Cell transplantations indicate that these also reflect different sources of Shh, one from the ventral neural tube that controls trabecular morphogenesis and one from the oral ectoderm that promotes chondrogenesis. Our results suggest a novel role for Shh in the movements of neural crest cells at the midline, as well as in their differentiation into cartilage, and help to explain why both skeletal fusions and palatal clefting are associated with the loss of Hh signaling in holoprosencephalic humans.

  14. Optimal Seeding Densities for In Vitro Chondrogenesis of Two- and Three-Dimensional-Isolated and -Expanded Bone Marrow-Derived Mesenchymal Stromal Stem Cells Within a Porous Collagen Scaffold

    OpenAIRE

    Bornes, Troy D.; Jomha, Nadr M; Mulet-Sierra, Aillette; Adesida, Adetola B.

    2016-01-01

    Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects. The objective of this study was to assess the impact of cell seeding density within a collagen I scaffold on in vitro BMSC chondrogenesis following isolation and expansion in two-dimensional (2D) and three-dimensional (3D) environments. It was hypothesized that both expansion protocols would produce BMSCs capable of hyaline-like chondrogenesis with an optimal seedin...

  15. Changes in type II procollagen isoform expression during chondrogenesis by disruption of an alternative 5’ splice site within Col2a1 exon 2

    OpenAIRE

    Hering, Thomas M.; Wirthlin, Louisa; Ravindran, Soumya; McAlinden, Audrey

    2014-01-01

    This study describes a new mechanism controlling the production of alternatively-spliced isoforms of type II procollagen (Col2a1) in vivo. During chondrogenesis, precursor chondrocytes predominantly produce isoforms containing alternatively-spliced exon 2 (type IIA and IID) while Col2a1 mRNA devoid of exon 2 (type IIB) is the major isoform produced by differentiated chondrocytes. We previously identified an additional Col2a1 isoform containing a truncated exon 2 and premature termination codo...

  16. Nicotine-induced retardation of chondrogenesis through down-regulation of IGF-1 signaling pathway to inhibit matrix synthesis of growth plate chondrocytes in fetal rats

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Yu; Cao, Hong; Cu, Fenglong [Department of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China); Xu, Dan [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China); Lei, Youying [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Tan, Yang [Department of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China); Magdalou, Jacques [UMR 7561 CNRS-Nancy Université, Faculté de Médicine, Vandoeuvre-lès-Nancy (France); Wang, Hui [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China); Chen, Liaobin, E-mail: lbchen@whu.edu.cn [Department of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China)

    2013-05-15

    Previous studies have confirmed that maternal tobacco smoking causes intrauterine growth retardation (IUGR) and skeletal growth retardation. Among a multitude of chemicals associated with cigarette smoking, nicotine is one of the leading candidates for causing low birth weights. However, the possible mechanism of delayed chondrogenesis by prenatal nicotine exposure remains unclear. We investigated the effects of nicotine on fetal growth plate chondrocytes in vivo and in vitro. Rats were given 2.0 mg/kg·d of nicotine subcutaneously from gestational days 11 to 20. Prenatal nicotine exposure increased the levels of fetal blood corticosterone and resulted in fetal skeletal growth retardation. Moreover, nicotine exposure induced the inhibition of matrix synthesis and down-regulation of insulin-like growth factor 1 (IGF-1) signaling in fetal growth plates. The effects of nicotine on growth plates were studied in vitro by exposing fetal growth plate chondrocytes to 0, 1, 10, or 100 μM of nicotine for 10 days. Nicotine inhibited matrix synthesis and down-regulated IGF-1 signaling in chondrocytes in a concentration-dependent manner. These results suggest that prenatal nicotine exposure induces delayed chondrogenesis and that the mechanism may involve the down-regulation of IGF-1 signaling and the inhibition of matrix synthesis by growth plate chondrocytes. The present study aids in the characterization of delayed chondrogenesis caused by prenatal nicotine exposure, which might suggest a candidate mechanism for intrauterine origins of osteoporosis and osteoarthritis. - Highlights: ► Prenatal nicotine-exposure could induce delayed chondrogenesis in fetal rats. ► Nicotine inhibits matrix synthesis of fetal growth plate chondrocytes. ► Nicotine inhibits IGF-1 signaling pathway in fetal growth plate chondrocytes.

  17. The effect of 3D nanofibrous scaffolds on the chondrogenesis of induced pluripotent stem cells and their application in restoration of cartilage defects.

    Directory of Open Access Journals (Sweden)

    Ji Liu

    Full Text Available The discovery of induced pluripotent stem cells (iPSCs rendered the reprogramming of terminally differentiated cells to primary stem cells with pluripotency possible and provided potential for the regeneration and restoration of cartilage defect. Chondrogenic differentiation of iPSCs is crucial for their application in cartilage tissue engineering. In this study we investigated the effect of 3D nanofibrous scaffolds on the chondrogenesis of iPSCs and articular cartilage defect restoration. Super-hydrophilic and durable mechanic polycaprolactone (PCL/gelatin scaffolds were fabricated using two separate electrospinning processes. The morphological structure and mechanical properties of the scaffolds were characterized. The chondrogenesis of the iPSCs in vitro and the restoration of the cartilage defect was investigated using scanning electron microscopy (SEM, the Cell Counting Kit-8 (CCK-8, histological observation, RT-qPCR, and western blot analysis. iPSCs on the scaffolds expressed higher levels of chondrogenic markers than the control group. In an animal model, cartilage defects implanted with the scaffold-cell complex exhibited an enhanced gross appearance and histological improvements, higher cartilage-specific gene expression and protein levels, as well as subchondral bone regeneration. Therefore, we showed scaffolds with a 3D nanofibrous structure enhanced the chondrogenesis of iPSCs and that iPSC-containing scaffolds improved the restoration of cartilage defects to a greater degree than did scaffolds alone in vivo.

  18. All-Arthroscopic Autologous Matrix-Induced Chondrogenesis for the Treatment of Osteochondral Lesions of the Talus

    Science.gov (United States)

    Usuelli, Federico Giuseppe; de Girolamo, Laura; Grassi, Miriam; D'Ambrosi, Riccardo; Montrasio, Umberto Alfieri; Boga, Michele

    2015-01-01

    Several surgical techniques have been described for the treatment of talar chondral lesions. Among them, microfracture is well established. Autologous matrix-induced chondrogenesis (AMIC), using microfracture and biomaterials, has shown promising results for the treatment of knee osteochondral lesions and has been proposed for the ankle as an open technique. We describe an all-arthroscopic AMIC technique. The benefits of an all-arthroscopic procedure include smaller incisions with less soft-tissue dissection, better visualization of the joint, and a quicker recovery compared with open surgery. The use of matrix to support cartilage regeneration promotes good-quality cartilage tissue with satisfactory long-term outcomes. Our all-arthroscopic AMIC technique uses a type I–type III porcine collagen matrix (Chondro-Gide; Geistlich Pharma, Wolhusen, Switzerland) and is characterized by 2 different arthroscopic surgical phases. First, adequate exposure is achieved through use of a Hintermann spreader (Integra LifeSciences, Plainsboro, NJ) with sufficient joint distraction and wet lesion preparation. The second surgical step is performed dry, involving matrix placement and fixation. The all-arthroscopic AMIC technique for the treatment of osteochondral lesions of the talus allows a very precise reconstruction in the case of cartilage defects and avoids the need for a more invasive operation associated with higher morbidity and a longer surgical time. PMID:26258040

  19. Small molecule-directed specification of sclerotome-like chondroprogenitors and induction of a somitic chondrogenesis program from embryonic stem cells.

    Science.gov (United States)

    Zhao, Jiangang; Li, Songhui; Trilok, Suprita; Tanaka, Makoto; Jokubaitis-Jameson, Vanta; Wang, Bei; Niwa, Hitoshi; Nakayama, Naoki

    2014-10-01

    Pluripotent embryonic stem cells (ESCs) generate rostral paraxial mesoderm-like progeny in 5-6 days of differentiation induced by Wnt3a and Noggin (Nog). We report that canonical Wnt signaling introduced either by forced expression of activated β-catenin, or the small-molecule inhibitor of Gsk3, CHIR99021, satisfied the need for Wnt3a signaling, and that the small-molecule inhibitor of BMP type I receptors, LDN193189, was able to replace Nog. Mesodermal progeny generated using such small molecules were chondrogenic in vitro, and expressed trunk paraxial mesoderm markers such as Tcf15 and Meox1, and somite markers such as Uncx, but failed to express sclerotome markers such as Pax1. Induction of the osteochondrogenically committed sclerotome from somite requires sonic hedgehog and Nog. Consistently, Pax1 and Bapx1 expression was induced when the isolated paraxial mesodermal progeny were treated with SAG1 (a hedgehog receptor agonist) and LDN193189, then Sox9 expression was induced, leading to cartilaginous nodules and particles in the presence of BMP, indicative of chondrogenesis via sclerotome specification. By contrast, treatment with TGFβ also supported chondrogenesis and stimulated Sox9 expression, but failed to induce the expression of Pax1 and Bapx1. On ectopic transplantation to immunocompromised mice, the cartilage particles developed under either condition became similarly mineralized and formed pieces of bone with marrow. Thus, the use of small molecules led to the effective generation from ESCs of paraxial mesodermal progeny, and to their further differentiation in vitro through sclerotome specification into growth plate-like chondrocytes, a mechanism resembling in vivo somitic chondrogenesis that is not recapitulated with TGFβ. PMID:25294938

  20. Bone morphogenetic protein 2 promotes transforming growth factor β3-induced chondrogenesis of human osteoarthritic synovium-derived stem cells

    Institute of Scientific and Technical Information of China (English)

    RUI Yun-feng; DU Lin; WANG You; WANG Yang; LUI Pauline po-yee; TANG Ting-ting; CHAN Kai-ming; DAI Ke-rong

    2010-01-01

    Background Synovium-derived stem cells (SDSCs) with higher chondrogenic potential are attracting considerable attention as a cell source for cartilage regeneration. We investigated the effect of bone morphogenetic protein 2 (BMP-2) on transforming growth factor beta3 (TGF-β3)-induced chondrogenesis of SDSCs isolated from human osteoarthritic synovium in a pellet culture system. Methods The clonogenicity, stem cell marker expression and multi-differentiation potential of isolated SDSCs were determined by colony forming unit assay, flow cytometry and specific staining including alizarin red S, Oil red O and alcian blue staining, respectively. SDSCs pellet was cultured in chondrogenic medium with or without TGF-β3 or/and BMP-2. At day 21, the diameter and the weight of the pellets were measured. Chondrogenic differentiation of SDSCs was evaluated by Safranin O staining, immunohistochemical staining of collagen type Ⅱ, sulfated glycosaminoglycan (sGAG) synthesis and mRNA expression of collagen type Ⅱ, aggrecan, SOX9, link-protein, collagen type X and BMP receptor Ⅱ. Results Cells isolated under the optimized culturing density (104/60 cm2) showed clonogenicity and multi-differentiation potential. These cells were positive (>99%) for CD44, CD90, CD105 and negative (<10%) for CD34 and CD71. SDSCs differentiated to a chondrocytic phenotype in chondrogenic medium containing TGF-β3 with or without BMP-2. Safranin O staining of the extracellular matrix was positive and the expression of collagen type Ⅱ was detected. Cell pellets treated with TGF-β3 and BMP-2 were larger in diameter and weight, produced more sGAGs, and expressed higher levels of collagen type Ⅱ and other chondrogenic markers, except COL10A1, than medium with TGF-β3 alone. Conclusions SDSCs could be isolated from human osteoarthritic synovium. Supplementation with BMP-2 significantly promoted the in vitro TGF-β3-induced chondrogenic differentiation of SDSCs.

  1. Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: an improved approach in cartilage tissue engineering.

    Science.gov (United States)

    Moroz, Andrei; Bittencourt, Renata Aparecida Camargo; Almeida, Renan Padron; Felisbino, Sérgio Luis; Deffune, Elenice

    2013-01-01

    Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n = 5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1 × 10(5)) were than encapsulated inside 60 µl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI.

  2. The influence of hyaluronic acid hydrogel crosslinking density and macromolecular diffusivity on human MSC chondrogenesis and hypertrophy.

    Science.gov (United States)

    Bian, Liming; Hou, Chieh; Tous, Elena; Rai, Reena; Mauck, Robert L; Burdick, Jason A

    2013-01-01

    Hyaluronic acid (HA) hydrogels formed via photocrosslinking provide stable 3D hydrogel environments that support the chondrogenesis of mesenchymal stem cells (MSCs). Crosslinking density has a significant impact on the physical properties of hydrogels, including their mechanical stiffness and macromolecular diffusivity. Variations in the HA hydrogel crosslinking density can be obtained by either changes in the HA macromer concentration (1, 3, or 5% w/v at 15 min exposure) or the extent of reaction through light exposure time (5% w/v at 5, 10, or 15 min). In this work, increased crosslinking by either method resulted in an overall decrease in cartilage matrix content and more restricted matrix distribution. Increased crosslinking also promoted hypertrophic differentiation of the chondrogenically induced MSCs, resulting in more matrix calcification in vitro. For example, type X collagen expression in the high crosslinking density 5% 15 min group was ~156 and 285% higher when compared to the low crosslinking density 1% 15 min and 5% 5 min groups on day 42, respectively. Supplementation with inhibitors of the small GTPase pathway involved in cytoskeletal tension or myosin II had no effect on hypertrophic differentiation and matrix calcification, indicating that the differential response is unlikely to be related to force-sensing mechanotransduction mechanisms. When implanted subcutaneously in nude mice, higher crosslinking density again resulted in reduced cartilage matrix content, restricted matrix distribution, and increased matrix calcification. This study demonstrates that hydrogel properties mediated through alterations in crosslinking density must be considered in the context of the hypertrophic differentiation of chondrogenically induced MSCs. PMID:23084553

  3. Genipin-crosslinked cartilage-derived matrix as a scaffold for human adipose-derived stem cell chondrogenesis.

    Science.gov (United States)

    Cheng, Nai-Chen; Estes, Bradley T; Young, Tai-Horng; Guilak, Farshid

    2013-02-01

    Autologous cell-based tissue engineering using three-dimensional scaffolds holds much promise for the repair of cartilage defects. Previously, we reported on the development of a porous scaffold derived solely from native articular cartilage, which can induce human adipose-derived stem cells (ASCs) to differentiate into a chondrogenic phenotype without exogenous growth factors. However, this ASC-seeded cartilage-derived matrix (CDM) contracts over time in culture, which may limit certain clinical applications. The present study aimed to investigate the ability of chemical crosslinking using a natural biologic crosslinker, genipin, to prevent scaffold contraction while preserving the chondrogenic potential of CDM. CDM scaffolds were crosslinked in various genipin concentrations, seeded with ASCs, and then cultured for 4 weeks to evaluate the influence of chemical crosslinking on scaffold contraction and ASC chondrogenesis. At the highest crosslinking degree of 89%, most cells failed to attach to the scaffolds and resulted in poor formation of a new extracellular matrix. Scaffolds with a low crosslinking density of 4% experienced cell-mediated contraction similar to our original report on noncrosslinked CDM. Using a 0.05% genipin solution, a crosslinking degree of 50% was achieved, and the ASC-seeded constructs exhibited no significant contraction during the culture period. Moreover, expression of cartilage-specific genes, synthesis, and accumulation of cartilage-related macromolecules and the development of mechanical properties were comparable to the original CDM. These findings support the potential use of a moderately (i.e., approximately one-half of the available lysine or hydroxylysine residues being crosslinked) crosslinked CDM as a contraction-free biomaterial for cartilage tissue engineering.

  4. Master regulator for chondrogenesis, Sox9, regulates transcriptional activation of the endoplasmic reticulum stress transducer BBF2H7/CREB3L2 in chondrocytes.

    Science.gov (United States)

    Hino, Kenta; Saito, Atsushi; Kido, Miori; Kanemoto, Soshi; Asada, Rie; Takai, Tomoko; Cui, Min; Cui, Xiang; Imaizumi, Kazunori

    2014-05-16

    The endoplasmic reticulum (ER) stress transducer, box B-binding factor 2 human homolog on chromosome 7 (BBF2H7), is a basic leucine zipper (bZIP) transmembrane transcription factor. This molecule is activated in response to ER stress during chondrogenesis. The activated BBF2H7 accelerates cartilage matrix protein secretion through the up-regulation of Sec23a, which is responsible for protein transport from the ER to the Golgi apparatus and is a target of BBF2H7. In the present study, we elucidated the mechanisms of the transcriptional activation of Bbf2h7 in chondrocytes. The transcription of Bbf2h7 is regulated by Sex determining region Y-related high-mobility group box 9 (Sox9), a critical factor for chondrocyte differentiation that facilitates the expression of one of the major cartilage matrix proteins Type II collagen (Col2), through binding to the Sox DNA-binding motif in the Bbf2h7 promoter. BBF2H7 is activated as a transcription factor in response to physiological ER stress caused by abundant synthesis of cartilage matrix proteins, and consequently regulates the secretion of cartilage matrix proteins. Taken together, our findings demonstrate novel regulatory mechanisms of Sox9 for controlling the secretion of cartilage matrix proteins through the activation of BBF2H7-Sec23a signaling during chondrogenesis.

  5. Master regulator for chondrogenesis, Sox9, regulates transcriptional activation of the endoplasmic reticulum stress transducer BBF2H7/CREB3L2 in chondrocytes.

    Science.gov (United States)

    Hino, Kenta; Saito, Atsushi; Kido, Miori; Kanemoto, Soshi; Asada, Rie; Takai, Tomoko; Cui, Min; Cui, Xiang; Imaizumi, Kazunori

    2014-05-16

    The endoplasmic reticulum (ER) stress transducer, box B-binding factor 2 human homolog on chromosome 7 (BBF2H7), is a basic leucine zipper (bZIP) transmembrane transcription factor. This molecule is activated in response to ER stress during chondrogenesis. The activated BBF2H7 accelerates cartilage matrix protein secretion through the up-regulation of Sec23a, which is responsible for protein transport from the ER to the Golgi apparatus and is a target of BBF2H7. In the present study, we elucidated the mechanisms of the transcriptional activation of Bbf2h7 in chondrocytes. The transcription of Bbf2h7 is regulated by Sex determining region Y-related high-mobility group box 9 (Sox9), a critical factor for chondrocyte differentiation that facilitates the expression of one of the major cartilage matrix proteins Type II collagen (Col2), through binding to the Sox DNA-binding motif in the Bbf2h7 promoter. BBF2H7 is activated as a transcription factor in response to physiological ER stress caused by abundant synthesis of cartilage matrix proteins, and consequently regulates the secretion of cartilage matrix proteins. Taken together, our findings demonstrate novel regulatory mechanisms of Sox9 for controlling the secretion of cartilage matrix proteins through the activation of BBF2H7-Sec23a signaling during chondrogenesis. PMID:24711445

  6. BMP treatment of C3H10T1/2 mesenchymal stem cells induces both chondrogenesis and osteogenesis.

    Science.gov (United States)

    Shea, Colleen M; Edgar, Cory M; Einhorn, Thomas A; Gerstenfeld, Louis C

    2003-12-15

    The molecular mechanisms by which bone morphogenetic proteins (BMPs) promote skeletal cell differentiation were investigated in the murine mesenchymal stem cell line C3H10T1/2. Both BMP-7 and BMP-2 induced C3H10T1/2 cells to undergo a sequential pattern of chondrogenic followed by osteogenic differentiation that was dependent on both the concentration and the continuous presence of BMP in the growth media. Differentiation was determined by the expression of chondrogenesis and osteogenesis associated matrix genes. Subsequent experiments using BMP-7 demonstrated that withdrawal of BMP from the growth media led to a complete loss of skeletal cell differentiation accompanied by adipogenic differentiation of these cells. Continuous treatment with BMP-7 increased the expression of Sox9, Msx 2, and c-fos during the periods of chondrogenic differentiation after which point their expression decreased. In contrast, Dlx 5 expression was induced by BMP-7 treatment and remained elevated throughout the time-course of skeletal cell differentiation. Runx2/Cbfa1 was not detected by ribonuclease protection assay (RPA) and did not appear to be induced by BMP-7. The sequential nature of differentiation of chondrocytic and osteoblastic cells and the necessity for continuous BMP treatment to maintain skeletal cell differentiation suggests that the maintenance of selective differentiation of the two skeletal cell lineages might be dependent on BMP-7-regulated expression of other morphogenetic factors. An examination of the expression of Wnt, transforming growth factor-beta (TGF-beta), and the hedgehog family of morphogens showed that Wnt 5b, Wnt 11, BMP-4, growth and differentiation factor-1 (GDF-1), Sonic hedgehog (Shh), and Indian hedgehog (Ihh) were endogenously expressed by C3H10T1/2 cells. Wnt 11, BMP-4, and GDF-1 expression were inhibited by BMP-7 treatment in a dose-dependent manner while Wnt 5b and Shh were selectively induced by BMP-7 during the period of chondrogenic

  7. TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants and arrests downstream differentiation at an early stage of hypertrophy.

    Directory of Open Access Journals (Sweden)

    Nahoko Shintani

    Full Text Available BACKGROUND: Synovial explants furnish an in-situ population of mesenchymal stem cells for the repair of articular cartilage. Although bone morphogenetic protein 2 (BMP-2 induces the chondrogenesis of bovine synovial explants, the cartilage formed is neither homogeneously distributed nor of an exclusively hyaline type. Furthermore, the downstream differentiation of chondrocytes proceeds to the stage of terminal hypertrophy, which is inextricably coupled with undesired matrix mineralization. With a view to optimizing BMP-2-induced chondrogenesis, the modulating influences of fibroblast growth factor 2 (FGF-2 and transforming growth factor beta 1 (TGF-ß1 were investigated. METHODOLOGY/PRINCIPAL FINDINGS: Explants of bovine calf metacarpal synovium were exposed to BMP-2 (200 ng/ml for 4 (or 6 weeks. FGF-2 (10 ng/ml or TGF-ß1 (10 ng/ml was introduced at the onset of incubation and was present either during the first week of culturing alone or throughout its entire course. FGF-2 enhanced the BMP-2-induced increase in metachromatic staining for glycosaminoglycans (GAGs only when it was present during the first week of culturing alone. TGF-ß1 enhanced not only the BMP-2-induced increase in metachromasia (to a greater degree than FGF-2, but also the biochemically-assayed accumulation of GAGs, when it was present throughout the entire culturing period; in addition, it arrested the downstream differentiation of cells at an early stage of hypertrophy. These findings were corroborated by an analysis of the gene- and protein-expression levels of key cartilaginous markers and by an estimation of individual cell volume. CONCLUSIONS/SIGNIFICANCE: TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants, improves the hyaline-like properties of the neocartilage, and arrests the downstream differentiation of cells at an early stage of hypertrophy. With the prospect of engineering a mature, truly articular type of cartilage in the context of

  8. Chondrogenesis of periodontal ligament stem cells by transforming growth factor-β3 and bone morphogenetic protein-6 in a normal healthy impacted third molar

    Institute of Scientific and Technical Information of China (English)

    Sunyoung Choi; Tae-Jun Cho; Soon-Keun Kwon; Gene Lee; Jaejin Cho

    2013-01-01

    The periodontal ligament-derived mesenchymal stem cell is regarded as a source of adult stem cells due to its multipotency. However, the proof of chondrogenic potential of the cells is scarce. Therefore, we investigated the chondrogenic differentiation capacity of periodontal ligament derived mesenchymal stem cells induced by transforming growth factor (TGF)-β3 and bone morphogenetic protein (BMP)-6. After isolation of periodontal ligament stem cells (PDLSCs) from human periodontal ligament, the cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 20% fetal bovine serum (FBS). A mechanical force initiated chondrogenic differentiation of the cells. For chondrogenic differentiation, 10 μg ·L-1 TGF-β3 or 100 μg ·L-1 BMP-6 and the combination treating group for synergistic effect of the growth factors. We analyzed the PDLSCs by fluorescence-activated cell sorting and chondrogenesis were evaluated by glycosaminoglycans assay, histology, immunohistochemistry and genetic analysis. PDLSCs showed mesenchymal stem cell properties proved by FACS analysis. Glycosaminoglycans contents were increased 217% by TGF-β3 and 220% by BMP-6. The synergetic effect of TGF-β3 and BMP-6 were shown up to 281% compared to control. The combination treatment increased Sox9, aggrecan and collagen II expression compared with not only controls, but also TGF-β3 or BMP-6 single treatment dramatically. The histological analysis also indicated the chondrogenic differentiation of PDLSCs in our conditions. The results of the present study demonstrate the potential of the dental stem cell as a valuable cell source for chondrogenesis, which may be applicable for regeneration of cartilage and bone fracture in the field of cell therapy.

  9. 3, 3', 5-triiodo-L-thyronine Increases In Vitro Chondrogenesis of Mesenchymal Stem Cells From Human Umbilical Cord Stroma Through SRC2.

    Science.gov (United States)

    Fernández-Pernas, Pablo; Fafián-Labora, Juan; Lesende-Rodriguez, Iván; Mateos, Jesús; De la Fuente, Alexandre; Fuentes, Isaac; De Toro Santos, Javier; Blanco García, Fco; Arufe, María C

    2016-09-01

    Our group focuses on the study of mesenchymal stem cells (MSCs) from human umbilical cord stroma or Warthońs jelly and their directed differentiation toward chondrocyte-like cells capable of regenerating damaged cartilage when transplanted into an injured joint. This study aimed to determine whether lactogenic hormone prolactin (PRL) or 3, 3', 5-triiodo-L-thyronine (T3), the active thyroid hormone, modulates chondrogenesis in our in vitro model of directed chondrogenic differentiation, and whether Wnt signalling is involved in this modulation. MSCs from human umbilical cord stroma underwent directed differentiation toward chondrocyte-like cells by spheroid formation. The addition of T3 to the chondrogenic medium increased the expression of genes linked to chondrogenesis like collagen type 2, integrin alpha 10 beta 1, and Sox9 measured by quantitative real time polymerase chain reaction (qRT-PCR) analysis. Levels of collagen type 2 and aggrecane analyzed by immunohistochemistry, and staining by Safranin O were increased after 14 days in spheroid culture with T3 compared to those without T3 or only with PRL. B-catenin, Frizzled, and GSK-3β gene expressions were significantly higher in spheroids cultured with chondrogenic medium (CM) plus T3 compared to CM alone after 14 days in culture. The increase of chondrogenic differentiation was inhibited when the cells were treated with T3 plus ML151, an inhibitor of the T3 steroid receptor. This work demonstrates, for first time, that T3 promotes differentiation towards chondrocytes-like cells in our in vitro model, that this differentiation is mediated by steroid receptor co-activator 2 (SRC2) and does not induce hypertrophy. J. Cell. Biochem. 117: 2097-2108, 2016. © 2016 Wiley Periodicals, Inc. PMID:26869487

  10. Positive regulation by γ-aminobutyric acid B receptor subunit-1 of chondrogenesis through acceleration of nuclear translocation of activating transcription factor-4.

    Science.gov (United States)

    Takahata, Yoshifumi; Hinoi, Eiichi; Takarada, Takeshi; Nakamura, Yukari; Ogawa, Shinya; Yoneda, Yukio

    2012-09-28

    A view that signaling machineries for the neurotransmitter γ-aminobutyric acid (GABA) are functionally expressed by cells outside the central nervous system is now prevailing. In this study, we attempted to demonstrate functional expression of GABAergic signaling molecules by chondrocytes. In cultured murine costal chondrocytes, mRNA was constitutively expressed for metabotropic GABA(B) receptor subunit-1 (GABA(B)R1), but not for GABA(B)R2. Immunohistochemical analysis revealed the predominant expression of GABA(B)R1 by prehypertrophic to hypertrophic chondrocytes in tibial sections of newborn mice. The GABA(B)R agonist baclofen failed to significantly affect chondrocytic differentiation determined by Alcian blue staining and alkaline phosphatase activity in cultured chondrocytes, whereas newborn mice knocked out of GABA(B)R1 (KO) showed a decreased body size and delayed calcification in hyoid bone and forelimb and hindlimb digits. Delayed calcification was also seen in cultured metatarsals from KO mice with a marked reduction of Indian hedgehog gene (Ihh) expression. Introduction of GABA(B)R1 led to synergistic promotion of the transcriptional activity of activating transcription factor-4 (ATF4) essential for normal chondrogenesis, in addition to facilitating ATF4-dependent Ihh promoter activation. Although immunoreactive ATF4 was negligibly detected in the nucleus of chondrocytes from KO mice, ATF4 expression was again seen in the nucleus and cytoplasm after the retroviral introduction of GABA(B)R1 into cultured chondrocytes from KO mice. In nuclear extracts of KO chondrocytes, a marked decrease was seen in ATF4 DNA binding. These results suggest that GABA(B)R1 positively regulates chondrogenesis through a mechanism relevant to the acceleration of nuclear translocation of ATF4 for Ihh expression in chondrocytes. PMID:22879594

  11. N-cadherin mediated distribution of beta-catenin alters MAP kinase and BMP-2 signaling on chondrogenesis-related gene expression.

    Science.gov (United States)

    Modarresi, Rozbeh; Lafond, Toulouse; Roman-Blas, Jorge A; Danielson, Keith G; Tuan, Rocky S; Seghatoleslami, M Reza

    2005-05-01

    We have examined the effect of calcium-dependent adhesion, mediated by N-cadherin, on cell signaling during chondrogenesis of multipotential embryonic mouse C3H10T1/2 cells. The activity of chondrogenic genes, type II collagen, aggrecan, and Sox9 were examined in monolayer (non-chondrogenic), and micromass (chondrogenic) cultures of parental C3H10T1/2 cells and altered C3H10T1/2 cell lines that express a dominant negative form of N-cadherin (delta390-T1/2) or overexpress normal N-cadherin (MNCD2-T1/2). Our findings show that missexpression or inhibition of N-cadherin in C3H10T1/2 cells results in temporal and spatial changes in expression of the chondrogenic genes Sox9, aggrecan, and collagen type II. We have also analyzed activity of the serum response factor (SRF), a nuclear target of MAP kinase signaling implicated in chondrogenesis. In semi-confluent monolayer cultures (minimum cell-cell contact) of C3H10T1/2, MNCD2-T1/2, or delta390-T1/2 cells, there was no significant change in the pattern of MAP kinase or bone morphogenetic protein-2 (BMP-2) regulation of SRF. However, in micromass cultures, the effect of MAP kinase and BMP-2 on SRF activity was proportional to the nuclear localization of beta-catenin, a Wnt stabilized cytoplasmic factor that can associate with lymphoid enhancer-binding factor (LEF) to serve as a transcription factor. Our findings suggest that the extent of adherens junction formation mediated by N-cadherin can modulate the potential Wnt-induced nuclear activity of beta-catenin. PMID:15723280

  12. Treatment of osteochondral lesions of the talus with autologous collagen-induced chondrogenesis: clinical and magnetic resonance evaluation at one-year follow-up

    Science.gov (United States)

    USUELLI, FEDERICO GIUSEPPE; GRASSI, MIRIAM; MANZI, LUIGI; GUARRELLA, VINCENZO; BOGA, MICHELE; DE GIROLAMO, LAURA

    2016-01-01

    Purpose the aim of this study is to report the clinical and imaging results recorded by a series of patients in whom osteochondral lesions of the talus (OLTs) were repaired using the autologous collagen-induced chondrogenesis (ACIC) technique with a completely arthroscopic approach. Methods nine patients (mean age 37.4±10 years) affected by OLTs (lesion size 2.1±0.9 cm2) were treated with the ACIC technique. The patients were evaluated clinically both preoperatively and at 12 months after surgery using the American Orthopaedic Foot and Ankle Society Ankle-Hindfoot Scale (AOFAS) and a visual analog scale (VAS). For morphological evaluation, the magnetic resonance observation of cartilage repair tissue (MOCART) score was used. Results the AOFAS score improved from 51.4±11.6 preoperatively to 71.8±20.6 postoperatively, while the VAS value decreased from 6.9±1.8 to 3.2±1.9. The mean MOCART score was 51.7±16.6 at 12 months of follow-up; these scores did not directly correlate with the clinical results. Conclusion use of the ACIC technique for arthroscopic repair of OLTs allowed satisfactory clinical results to be obtained in most of the patients as soon as one year after surgery, with no major complications or delayed revision surgery. ACIC is a valid and low-invasive surgical technique for the treatment of chondral and osteochondral defects of the talus. Level of evidence therapeutic case series, level IV. PMID:27602347

  13. In vitro chondrogenesis of the goat bone marrow mesenchymal stem cells directed by chondrocytes in monolayer and 3-dimetional indirect co-culture system

    Institute of Scientific and Technical Information of China (English)

    LI Jian-wei; GUO Xiao-lei; HE Chun-la; TUO Yong-hua; WANG Zhao; WEN Jun; JIN Dan

    2011-01-01

    Background Cartilage injury has a very poor capacity for intrinsic regeneration. The cell-based treatment strategy for the cartilage repair using differentiated bone marrow mesenchymal stem cells (BMSCs) is, however, a promising approach to the chondral repair. This study was aimed to explore the chondrogenic potential of the goat BMSCs in the Transwell co-culture system and the poly-laetide-co-glycolide (PLGA) scaffolds.Methods The BMSCs were isolated from the goat iliac crest while the chondrocytes were obtained from the goat's last costal cartilage. In the Transwell co-culture system, the BMSCs co-cultured with chondrocytes were designed as group A,whereas the goat's BMSCs induced with the chondrogenic medium were group B. Both groups A and B were the experimental groups, while group C that only contained BMSCs was the control group. In the PLGA scaffolds co-culture system, BMSCs were seeded into the PLGA scaffolds, which were suspended in the 24-well plate, and the control group was established by presence or absence of chondrocytes at the bottom of the 24-well plate. Toluidine blue staining,Alcian blue staining, collagen Ⅱ immunofluoresence, collagen Ⅱ immunochemical staining, collagen Ⅰ, collagen Ⅱ, COL2a Q-PCR and osteopontin Q-PCR were used to examine the chondrogenic conditions as well as the expressions of chondrogenic and osteogenic genes.Results Cells isolated from the aspirates of the goat bone marrow proliferated rapidly and gained characteristics of stem cells in Passage 4. However, the differentiations of chondrocytes were not apparent in Passage 3. The results from Toluidine blue staining, collagen Ⅱ immunofluoresence and PCR showed the transformation of BMSCs to chondrocytes in the Transwell co-culture system and PLGA scaffolds. Although the cartilage gene expressions were upgraded in both chondrogenesis group and co-culture system, the osteopontin gene expression, which represents osteogenic level, was also up-regulated.Conclusions The

  14. Optimal Seeding Densities for In Vitro Chondrogenesis of Two- and Three-Dimensional-Isolated and -Expanded Bone Marrow-Derived Mesenchymal Stromal Stem Cells Within a Porous Collagen Scaffold.

    Science.gov (United States)

    Bornes, Troy D; Jomha, Nadr M; Mulet-Sierra, Aillette; Adesida, Adetola B

    2016-03-01

    Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects. The objective of this study was to assess the impact of cell seeding density within a collagen I scaffold on in vitro BMSC chondrogenesis following isolation and expansion in two-dimensional (2D) and three-dimensional (3D) environments. It was hypothesized that both expansion protocols would produce BMSCs capable of hyaline-like chondrogenesis with an optimal seeding density of 10 × 10(6) cells/cm(3). Ovine BMSCs were isolated in a 2D environment by plastic adherence, expanded to passage two in flasks containing an expansion medium, and seeded within collagen I scaffolds at densities of 50, 10, 5, 1, and 0.5 × 10(6) BMSCs/cm(3). For 3D isolation and expansion, aspirates containing known quantities of mononucleated cells (bone marrow-derived mononucleated cells [BMNCs]) were seeded on scaffolds at 50, 10, 5, 1, and 0.5 × 10(6) BMNCs/cm(3) and cultured in the expansion medium for an equivalent duration to 2D expansion. Constructs were differentiated in vitro in the chondrogenic medium for 21 days and assessed with reverse-transcription quantitative polymerase chain reaction, safranin O staining, histological scoring using the Bern Score, collagen immunofluorescence, and glycosaminoglycan (GAG) quantification. Two-dimensional-expanded BMSCs seeded at all densities were capable of proteoglycan production and displayed increased expressions of aggrecan and collagen II messenger RNA (mRNA) relative to predifferentiation controls. Collagen II deposition was apparent in scaffolds seeded at 0.5-10 × 10(6) BMSCs/cm(3). Chondrogenesis of 2D-expanded BMSCs was most pronounced in scaffolds seeded at 5-10 × 10(6) BMSCs/cm(3) based on aggrecan and collagen II mRNA, safranin O staining, Bern Score, total GAG, and GAG/deoxyribonucleic acid (DNA). For 3D-expanded BMSC-seeded scaffolds, increased aggrecan and collagen II m

  15. Hedgehog signal regulates the chondrogenesis from bone marrow mesenchymal stem cells:controlling methods and cross-talking relationship with other signals need further studies%Hedgehog信号调控骨髓间充质干细胞成软骨细胞分化:调控方式及其串话机制尚待研究

    Institute of Scientific and Technical Information of China (English)

    刘宽; 吴兴

    2014-01-01

    BACKGROUND:The hedgehog pathway has paid an important role in the progress of chondrogenesis from bone marrow mesenchymal stem cells. However, the definite signal transduction pathway and cross-talking relationship with other common signal pathways are stil poorly understood and the researches related to this field is to continue as a hotspot in the future study. OBJECTIVE:To investigate the research progress of hedgehog signal pathway on the regulation of the chondrogenesis from bone marrow mesenchymal stem cells and the relationship between hedgehog and other signal pathways in the process. METHODS:A computer-based online search in CNKI, PubMed and Google Scholar databases was performed using key words of“Hedgehog, IHH, SHH, bone marrow mesenchymal stem cells, cartilage, chondrogenesis”in English and Chinese, respectively. Literatures related to the process of chondrogenesis from bone marrow mesenchymal stem cells were included and 36 articles were extensively summarized for review. RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cells are currently accepted optimal cellseeds for the cartilage tissue engineering, and hedgehog is a critical signal molecule in the development of skeletal system. The IHH and SHH in hedgehog signal closely participate in control ing the processes of bone marrow mesenchymal stem cellproliferation and chondrogenesis, chondrocyte phenotype maintenance and cooperation with other common single pathways. However, the specific signal transduction mechanism and cross-talking contact with other signal pathways stil need to be further studied, and it stands for the future research directions.%背景:Hedgehog 信号通路在骨髓间充质干细胞成软骨细胞分化过程中发挥重要的调控作用,但具体的调控机制,以及与其他信号通路的串话机制仍需进一步的研究,是近来研究的热点。目的:介绍hedgehog信号在调控骨髓间充质干细胞成软骨分化过程中信号转导机制的

  16. Poly(dl)lactic acid/polyglycolic acid/iron and poly(dl)lactic acid/polyglycolic acid/samarium cobalt composites for use as a delivery mechanism for magnetically directed chondrogenesis

    Science.gov (United States)

    Oppermann, Dean Alan

    Magnetically directed chondrogenesis (MDC) is a fundamental approach to articular cartilage repair. In MDC a magnet is implanted into the subchondral trabecular bone underlying a cartilage defect and used to attract chondrocytes, magnetically tagged with Fe nanoparticles, to the defect site. Pilot studies by Halpern, Crimp and Grande, using solid neodymium (Nd) magnets, indicated optimistic results by producing a hyaline-like articular cartilage after 8 weeks implantation. Since solid Nd magnets introduce long-term biocompatibility issues, the focus of this dissertation was to develop P(dl)A/PGA/Fe and P(dl)A/PGA/SmCo 5 implants for use in MDC. The effect of implant porosity, implant composition and magnetic material (Fe or SmCo5) on the initial and degraded magnetic properties were evaluated. The biocompatibility of P(dl)A/PGA/Fe implants were investigated by implantation into New Zealand white rabbits for 8 weeks. The effect of hydrogen peroxide (H2O2) and ethylene oxide (EO) sterilization techniques on the molecular weight and chemical structure of P(dl)A/PGA polymers were evaluated using gel permeation chromatography and Fourier transform infrared spectroscopy. The effect of implant morphology, size and number on the von Mises stress in the trabecular bone surrounding the implant was evaluated using a finite element model. In general, SmCo5 implants resulted in higher magnetic fields initially and after 8 weeks of degradation than comparable Fe implants. Increases in magnetic field strength were achieved by increasing the volume fraction of magnetic material and by increasing the PGA concentration. The magnetic field strength degradation rate decreased with increases in volume fraction of magnetic material and increases in PLA concentration. Implantation studies indicated that 50/50 P(dl)A/PGA were more bioactive than 75/25 P(dl)A/PGA with an increased cellular response that is specific to bone growth. The compressive strength and elastic modulus of porous

  17. Fibronectin and stem cell differentiation - lessons from chondrogenesis.

    Science.gov (United States)

    Singh, Purva; Schwarzbauer, Jean E

    2012-08-15

    The extracellular matrix (ECM) is an intricate network of proteins that surrounds cells and has a central role in establishing an environment that is conducive to tissue-specific cell functions. In the case of stem cells, this environment is the stem cell niche, where ECM signals participate in cell fate decisions. In this Commentary, we describe how changes in ECM composition and mechanical properties can affect cell shape and stem cell differentiation. Using chondrogenic differentiation as a model, we examine the changes in the ECM that occur before and during mesenchymal stem cell differentiation. In particular, we focus on the main ECM protein fibronectin, its temporal expression pattern during chondrogenic differentiation, its potential effects on functions of differentiating chondrocytes, and how its interactions with other ECM components might affect cartilage development. Finally, we discuss data that support the possibility that the fibronectin matrix has an instructive role in directing cells through the condensation, proliferation and/or differentiation stages of cartilage formation.

  18. TGFb signalling inhibits DLK1 expression during chondrogenesis in vitro

    DEFF Research Database (Denmark)

    Harkness, Linda; Taipaleenmaki, Hanna; Saamanen, Anna-Marja;

    2011-01-01

    the effect of a number of signalling molecules on DLK1 expression during in vitro chondrogenic differentiation in mouse embryonic limb bud mesenchymal micromass cultures and mouse embryonic fibroblast (MEF) pellet cultures. Dlk1 was initially expressed during mesenchymal condensation and chondrocyte...

  19. A combinatorial approach towards the design of nanofibrous scaffolds for chondrogenesis

    Science.gov (United States)

    Ahmed, Maqsood; Ramos, Tiago André Da Silva; Damanik, Febriyani; Quang Le, Bach; Wieringa, Paul; Bennink, Martin; van Blitterswijk, Clemens; de Boer, Jan; Moroni, Lorenzo

    2015-10-01

    The extracellular matrix (ECM) is a three-dimensional (3D) structure composed of proteinaceous fibres that provide physical and biological cues to direct cell behaviour. Here, we build a library of hybrid collagen-polymer fibrous scaffolds with nanoscale dimensions and screen them for their ability to grow chondrocytes for cartilage repair. Poly(lactic acid) and poly (lactic-co-glycolic acid) at two different monomer ratios (85:15 and 50:50) were incrementally blended with collagen. Physical properties (wettability and stiffness) of the scaffolds were characterized and related to biological performance (proliferation, ECM production, and gene expression) and structure-function relationships were developed. We found that soft scaffolds with an intermediate wettability composed of the highly biodegradable PLGA50:50 and collagen, in two ratios (40:60 and 60:40), were optimal for chondrogenic differentiation of ATDC5 cells as determined by increased ECM production and enhanced cartilage specific gene expression. Long-term cultures indicated a stable phenotype with minimal de-differentiation or hypertrophy. The combinatorial methodology applied herein is a promising approach for the design and development of scaffolds for regenerative medicine.

  20. Identification of SCAN domain zinc-finger gene ZNF449 as a novel factor of chondrogenesis.

    Directory of Open Access Journals (Sweden)

    Keita Okada

    Full Text Available Transcription factors SOX9, SOX5 and SOX6 are indispensable for generation and differentiation of chondrocytes. However, molecular mechanisms to induce the SOX genes are poorly understood. To address this issue, we previously determined the human embryonic enhancer of SOX6 by 5'RACE analysis, and identified the 46-bp core enhancer region (CES6. We initially performed yeast one-hybrid assay for screening other chondrogenic factors using CES6 as bait, and identified a zinc finger protein ZNF449. ZNF449 and Zfp449, a counterpart in mouse, transactivated enhancers or promoters of SOX6, SOX9 and COL2A1. Zfp449 was expressed in mesenchyme-derived tissues including cartilage, calvaria, muscle and tendon, as well as in other tissues including brain, lung and kidney. In limb cartilage of mouse embryo, Zfp449 protein was abundantly located in periarticular chondrocytes, and decreased in accordance with the differentiation. Zfp449 protein was also detected in articular cartilage of an adult mouse. During chondrogenic differentiation of human mesenchymal stem cells, ZNF449 was increased at an early stage, and its overexpression enhanced SOX9 and SOX6 only at the initial stage of the differentiation. We further generated Zfp449 knockout mice to examine the in vivo roles; however, no obvious abnormality was observed in skeletal development or articular cartilage homeostasis. ZNF449 may regulate chondrogenic differentiation from mesenchymal progenitor cells, although the underlying mechanisms are still unknown.

  1. Genetically engineered flavonol enriched tomato fruit modulates chondrogenesis to increase bone length in growing animals.

    Science.gov (United States)

    Choudhary, Dharmendra; Pandey, Ashutosh; Adhikary, Sulekha; Ahmad, Naseer; Bhatia, Chitra; Bhambhani, Sweta; Trivedi, Prabodh Kumar; Trivedi, Ritu

    2016-01-01

    Externally visible body and longitudinal bone growth is a result of proliferation of chondrocytes. In growth disorder, there is delay in the age associated increase in height. The present study evaluates the effect of extract from transgenic tomato fruit expressing AtMYB12 transcription factor on bone health including longitudinal growth. Constitutive expression of AtMYB12 in tomato led to a significantly enhanced biosynthesis of flavonoids in general and the flavonol biosynthesis in particular. Pre-pubertal ovary intact BALB/c mice received daily oral administration of vehicle and ethanolic extract of wild type (WT-TOM) and transgenic AtMYB12-tomato (MYB12-TOM) fruits for six weeks. Animal fed with MYB12-TOM showed no inflammation in hepatic tissues and normal sinusoidal Kupffer cell morphology. MYB12-TOM extract significantly increased tibial and femoral growth and subsequently improved the bone length as compared to vehicle and WT-TOM. Histomorphometry exhibited significantly wider distal femoral and proximal tibial growth plate, increased number and size of hypertrophic chondrocytes in MYB12-TOM which corroborated with micro-CT and expression of BMP-2 and COL-10, marker genes for hypertrophic cells. We conclude that metabolic reprogramming of tomato by AtMYB12 has the potential to improve longitudinal bone growth thus helping in achievement of greater peak bone mass during adolescence. PMID:26917158

  2. Collagen Type II Enhances Chondrogenesis in Adipose Tissue-Derived Stem Cells by Affecting Cell Shape

    NARCIS (Netherlands)

    Lu, ZuFu; Doulabi, Behrouz Zandieh; Huang, ChunLing; Bank, Ruud A.; Helder, Marco N.

    2010-01-01

    Ideally, biomaterials have inductive properties, favoring specific lineage differentiation. For chondrogenic induction, these properties have been attributed to collagen type II. However, the underlying mechanisms are largely unknown. This study aimed to investigate whether collagen type II favors c

  3. Collagen type II enhances chondrogenesis in adipose tissue-derived stem cells by affecting cell shape

    NARCIS (Netherlands)

    Z. Lu; B.Z. Doulabi; C. Huang; R.A. Bank; M.N. Helder

    2010-01-01

    Ideally, biomaterials have inductive properties, favoring specific lineage differentiation. For chondrogenic induction, these properties have been attributed to collagen type II. However, the underlying mechanisms are largely unknown. This study aimed to investigate whether collagen type II favors c

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

    NARCIS (Netherlands)

    Rutgers, M.; Saris, D.B.F.; Vonk, L.A.; Rijen, van M.H.P.; Akrum, V.; Langeveld, D.; Boxtel, van 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, ho

  5. Enhanced chondrogenesis of human nasal septum derived progenitors on nanofibrous scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Shafiee, Abbas [Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Stem Cell biology and Tissue Engineering Departments, Stem Cell Technology Research Center, Tehran (Iran, Islamic Republic of); Institute of Health and Biomedical Innovation, Queensland University of Technology (QUT), Brisbane, QLD (Australia); Seyedjafari, Ehsan [Department of Biotechnology, College of Science, University of Tehran, Tehran (Iran, Islamic Republic of); Sadat Taherzadeh, Elham [Stem Cell biology and Tissue Engineering Departments, Stem Cell Technology Research Center, Tehran (Iran, Islamic Republic of); Dinarvand, Peyman [Stem Cell biology and Tissue Engineering Departments, Stem Cell Technology Research Center, Tehran (Iran, Islamic Republic of); The Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO (United States); Soleimani, Masoud [Hematology Department, Faculty of Medical Science, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Ai, Jafar, E-mail: jafar_ai@tums.ac.ir [Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Brain and Spinal Injury Research Center, Imam Hospital, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

    2014-07-01

    Topographical cues can be exploited to regulate stem cell attachment, proliferation, differentiation and function in vitro and in vivo. In this study, we aimed to investigate the influence of different nanofibrous topographies on the chondrogenic differentiation potential of nasal septum derived progenitors (NSP) in vitro. Aligned and randomly oriented Ploy (L-lactide) (PLLA)/Polycaprolactone (PCL) hybrid scaffolds were fabricated via electrospinning. First, scaffolds were fully characterized, and then NSP were seeded on them to study their capacity to support stem cell attachment, proliferation and chondrogenic differentiation. Compared to randomly oriented nanofibers, aligned scaffolds showed a high degree of nanofiber alignment with much better tensile strength properties. Both scaffolds supported NSP adhesion, proliferation and chondrogenic differentiation. Despite the higher rate of cell proliferation on random scaffolds, a better chondrogenic differentiation was observed on aligned nanofibers as deduced from higher expression of chondrogenic markers such as collagen type II and aggrecan on aligned scaffolds. These findings demonstrate that electrospun constructs maintain NSP proliferation and differentiation, and that the aligned nanofibrous scaffolds can significantly enhance chondrogenic differentiation of nasal septum derived progenitors. - Highlights: • Electrospun nanofiber scaffolds with different topographies were fabricated. • Aligned nanofiber scaffolds had better tensile strength properties. • Nasal septum derived progenitors were cultured on nanofibrous scaffolds. • Both topographies support proliferation and chondrogenic differentiation. • Better chondrogenic differentiation was observed on aligned nanofibers.

  6. Antioxidation of Decellularized Stem Cell Matrix Promotes Human Synovium-Derived Stem Cell-Based Chondrogenesis

    OpenAIRE

    Pei, Ming; Zhang, Ying; Li, Jingting; Chen, Dongquan

    2012-01-01

    Clinical treatment of cartilage defects is challenging due to concomitant post-traumatic joint inflammation. This study was to demonstrate that the antioxidant ability of human adult synovium-derived stem cells (SDSCs) could be enhanced by ex vivo expansion on a decellularized stem cell matrix (DSCM). Microarray was used to evaluate oxidative, antioxidative, and chondrogenic status in SDSCs after expansion on the DSCM and induction in the chondrogenic medium. Hydrogen peroxide (H2O2) was adde...

  7. Effect of a synthetic folic acid analogue, 9-methyl-pteroylglutamic acid, on fetal chondrogenesis: ultrastructural observations.

    Science.gov (United States)

    Schmidt, R R; Slobodian, S A; Chepenik, K P; Cotler, J M

    1983-01-01

    Maternally administered folic acid antagonists (x-methyl-PGA and 9-methyl-PGA) are known to produce various skeletal malformations in the neonate. These defects are thought to be due in part to abnormal metabolism and/or deposition of various extracellular matrix components, i.e., collagen and glycosaminoglycans. Experimental reduction of glycosaminoglycan biosynthesis in vitro has been shown previously to alter the spatial orientation and normal pattern of collagen fibrillogenesis. Furthermore, dietary withdrawal of folic acid concomitant with maternal administration of 9-methyl-PGA has been shown to result in abnormal collagen, uronic acid, and hexosamine metabolism by fetal limbs. In the present study pregnant rats were exposed to a transitory folic acid deficiency from day 11 to 14 of gestation and fetal tibias (mid-diaphyseal region) were examined with the electron microscope on day 18 of gestation. Although we were unable to ascertain any aberrant patterns of fibrillogenesis and orientation with respect to collagen, this particular teratogenic regimen resulted in an altered pattern of chondrocyte development when observed at the ultrastructural level.

  8. Chondrogenesis of Precartilaginous Stem Cells in KLD-12 Self-assembling Peptide Nanofiber Scaffold Loading TGF-β3 Gene

    Institute of Scientific and Technical Information of China (English)

    YOU Hongbo; CHEN Anmin; SUN Kai; LIU Tie

    2011-01-01

    The effect of culture in KLD-12 self-assembling peptide nanofiber scaffold containing TGF-β3 gene on differentiation of precartilaginous stem cells(PSCs)into chondrocytes was studied.KLD-12 was synthesized by solid-state method.After TGF-β3 plasmid was loaded into KLD-12 self-assembling peptide nanofiber scaffold,DNA release ability was investigated.PSCs and hTGF-β3 gene were loaded into KLD-12 3-D scaffold,and MTT assay was performed to investigate the cell proliferation,and ELASA assay was used to investigate the expression of TGF-β3.Specific cartilage matrix was examined by quantitative real-time PCR,immunohistochemistry and Alcian Blue staining.Compared with control group,DNA synthesis level of PSCs reached the peak within 3 days when PSCs were cultured in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid,and maintained this high level within 2 weeks.MTT results showed that the proliferation ability of experimental group was statistically higher than that in control group(P<0.05).Quantitative realtime PCR suggested that the percentage of TGF-β3 positive PSCs in experimental group was higher than that in control group(P<0.01).ELISA assay showed that the TGF-β3 protein level increased in supernatant of experimental group's PSCs,reached the peak after 72 h and then declined a little to the plateau phase.Compared with the control group,the specific gene of chondrocyte typical extracellular matrix significantly up-regulated (P<0.01).The results showed that PSCs differentiated into chondrocytes in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid,which provided a flesh approach to cartilage tissue engineering.

  9. Transcriptional signature of human adipose tissue-derived stem cells (hASCs) preconditioned for chondrogenesis in hypoxic conditions

    Energy Technology Data Exchange (ETDEWEB)

    Pilgaard, L.; Lund, P.; Duroux, M. [Laboratory for Stem Cell Research, Aalborg University, Fredrik Bajers Vej 3B, 9220 Aalborg (Denmark); Lockstone, H.; Taylor, J. [Bioinformatics and Statistical Genetics, Wellcome Trust Centre for Human Genetics, Oxford University, Roosevelt Drive, Oxford, OX3 7BN (United Kingdom); Emmersen, J.; Fink, T. [Laboratory for Stem Cell Research, Aalborg University, Fredrik Bajers Vej 3B, 9220 Aalborg (Denmark); Ragoussis, J. [Genomics, Wellcome Trust Centre for Human Genetics, Oxford University, Roosevelt Drive, Oxford, OX3 7BN (United Kingdom); Zachar, V., E-mail: vlaz@hst.aau.dk [Laboratory for Stem Cell Research, Aalborg University, Fredrik Bajers Vej 3B, 9220 Aalborg (Denmark)

    2009-07-01

    Hypoxia is an important factor involved in the control of stem cells. To obtain a better insight into the phenotypical changes brought about by hypoxic preconditioning prior to chondrogenic differentiation; we have investigated growth, colony-forming and chondrogenic capacity, and global transcriptional responses of six adipose tissue-derived stem cell lines expanded at oxygen concentrations ranging from ambient to 1%. The assessment of cell proliferation and colony-forming potential revealed that the hypoxic conditions corresponding to 1% oxygen played a major role. The chondrogenic inducibility, examined by high-density pellet model, however, did not improve on hypoxic preconditioning. While the microarray analysis revealed a distinctive inter-donor variability, the exposure to 1% hypoxia superseded the biological variability and produced a specific expression profile with 2581 significantly regulated genes and substantial functional enrichment in the pathways of cell proliferation and apoptosis. Additionally, exposure to 1% oxygen resulted in upregulation of factors related to angiogenesis and cell growth. In particular, leptin (LEP), the key regulator of body weight and food intake was found to be highly upregulated. In conclusion, the results of this investigation demonstrate the significance of donor demographics and the importance of further studies into the use of regulated oxygen tension as a tool for preparation of ASCs in order to exploit their full potential.

  10. Ascorbate-dependent impact on cell-derived matrix in modulation of stiffness and rejuvenation of infrapatellar fat derived stem cells toward chondrogenesis.

    Science.gov (United States)

    Pizzute, Tyler; Zhang, Ying; He, Fan; Pei, Ming

    2016-08-10

    Developing an in vitro microenvironment using cell-derived decellularized extracellular matrix (dECM) is a promising approach to efficiently expand adult stem cells for cartilage engineering and regeneration. Ascorbic acid serves as a critical stimulus for cells to synthesize collagens, which constitute the major component of dECM. In this study, we hypothesized that optimization of ascorbate treatment would maximize the rejuvenation effect of dECM on expanded stem cells from human infrapatellar fat pad in both proliferation and chondrogenic differentiation. In the duration regimen study, we found that dECM without L-ascorbic acid phosphate (AA) treatment, exhibiting lower stiffness measured by atomic force microscopy, yielded expanded cells with higher proliferation capacity but lower chondrogenic potential when compared to those with varied durations of AA treatment. dECM with 250 µM of AA treatment for 10 d had better rejuvenation in chondrogenic capacity if the deposited cells were from passage 2 rather than passage 5, despite no significant difference in matrix stiffness. In the dose regimen study, we found that dECMs deposited by varied concentrations of AA yielded expanded cells with higher proliferation capacity despite lower expression levels of stem cell related surface markers. Compared to cells expanded on tissue culture polystyrene, those on dECM exhibited greater chondrogenic potential, particularly for the dECMs with 50 µM and 250 µM of AA treatment. With the supplementation of ethyl-3,4-dihydroxybenzoate (EDHB), an inhibitor targeting procollagen synthesis, the dECM with 50 µM of AA treatment exhibited a dramatic decrease in the rejuvenation effect of expanded cell chondrogenic potential at both mRNA and protein levels despite no significant difference in matrix stiffness. Defined AA treatments during matrix preparation will benefit dECM-mediated stem cell engineering and future treatments for cartilage defects.

  11. Ascorbate-dependent impact on cell-derived matrix in modulation of stiffness and rejuvenation of infrapatellar fat derived stem cells toward chondrogenesis.

    Science.gov (United States)

    Pizzute, Tyler; Zhang, Ying; He, Fan; Pei, Ming

    2016-01-01

    Developing an in vitro microenvironment using cell-derived decellularized extracellular matrix (dECM) is a promising approach to efficiently expand adult stem cells for cartilage engineering and regeneration. Ascorbic acid serves as a critical stimulus for cells to synthesize collagens, which constitute the major component of dECM. In this study, we hypothesized that optimization of ascorbate treatment would maximize the rejuvenation effect of dECM on expanded stem cells from human infrapatellar fat pad in both proliferation and chondrogenic differentiation. In the duration regimen study, we found that dECM without L-ascorbic acid phosphate (AA) treatment, exhibiting lower stiffness measured by atomic force microscopy, yielded expanded cells with higher proliferation capacity but lower chondrogenic potential when compared to those with varied durations of AA treatment. dECM with 250 µM of AA treatment for 10 d had better rejuvenation in chondrogenic capacity if the deposited cells were from passage 2 rather than passage 5, despite no significant difference in matrix stiffness. In the dose regimen study, we found that dECMs deposited by varied concentrations of AA yielded expanded cells with higher proliferation capacity despite lower expression levels of stem cell related surface markers. Compared to cells expanded on tissue culture polystyrene, those on dECM exhibited greater chondrogenic potential, particularly for the dECMs with 50 µM and 250 µM of AA treatment. With the supplementation of ethyl-3,4-dihydroxybenzoate (EDHB), an inhibitor targeting procollagen synthesis, the dECM with 50 µM of AA treatment exhibited a dramatic decrease in the rejuvenation effect of expanded cell chondrogenic potential at both mRNA and protein levels despite no significant difference in matrix stiffness. Defined AA treatments during matrix preparation will benefit dECM-mediated stem cell engineering and future treatments for cartilage defects. PMID:27508528

  12. A Simplified Method for the Aspiration of Bone Marrow from Patients Undergoing Hip and Knee Joint Replacement for Isolating Mesenchymal Stem Cells and In Vitro Chondrogenesis

    OpenAIRE

    Juneja, Subhash C.; Sowmya Viswanathan; Milan Ganguly; Christian Veillette

    2016-01-01

    The procedure for aspiration of bone marrow from the femur of patients undergoing total knee arthroplasty (TKA) or total hip arthroplasty (THA) may vary from an OR (operating room) to OR based on the surgeon’s skill and may lead to varied extent of clotting of the marrow and this, in turn, presents difficulty in the isolation of mesenchymal stem cells (MSCs) from such clotted bone marrow. We present a simple detailed protocol for aspirating bone marrow from such patients, isolation, and chara...

  13. Induction of chondrogenesis and expression of superficial zone protein (SZP)/lubricin by mesenchymal progenitors in the infrapatellar fat pad of the knee joint treated with TGF-beta1 and BMP-7.

    Science.gov (United States)

    Lee, Sang Yang; Nakagawa, Toshiyuki; Reddi, A Hari

    2008-11-01

    Superficial zone protein (SZP) is a key mediator of boundary lubrication of articular cartilage in joints. In this investigation, we made the unexpected discovery that SZP was expressed in infrapatellar fat pad (IFP) from bovine knee. Quantitative analysis of secreted proteins in the medium of the IFP stromal cells demonstrated a significant stimulation by TGF-beta1 and BMP-7. Real-time PCR analysis revealed the SZP expression was up-regulated by TGF-beta1 and BMP-7. Chondrogenically differentiated IFP progenitor cells were stimulated by TGF-beta1 and BMP-7 to synthesize and secrete SZP. SZP mRNA was significantly up-regulated by chondrogenic induction for 21 days. These findings indicate that the stimulation of SZP expression by TGF-beta and BMP-7 may lead to functional improvement of damaged intraarticular tissues and that IFP progenitor cells may be a potential useful source for inducing superficial zone of articular cartilage by tissue engineering for regeneration of damaged articular cartilage due to osteoarthritis.

  14. Nkx3.2 Promotes Primary Chondrogenic Differentiation by Upregulating Col2a1 Transcription

    OpenAIRE

    Kawato, Yoshitaka; Hirao, Makoto; Ebina, Kosuke; Shi, Kenrin; Hashimoto, Jun; Honjo, Yui; Yoshikawa, Hideki; Myoui, Akira

    2012-01-01

    Background The Nkx3.2 transcription factor promotes chondrogenesis by forming a positive regulatory loop with a crucial chondrogenic transcription factor, Sox9. Previous studies have indicated that factors other than Sox9 may promote chondrogenesis directly, but these factors have not been identified. Here, we test the hypothesis that Nkx3.2 promotes chondrogenesis directly by Sox9-independent mechanisms and indirectly by previously characterized Sox9-dependent mechanisms. Methodology/Princip...

  15. Runx1 is critical for PTH-induced onset of mesenchymal progenitor cell chondrogenic differentiation.

    Directory of Open Access Journals (Sweden)

    Jinwu Wang

    Full Text Available Parathyroid hormone (PTH plays a critical role in the regulation of chondrogenesis. In this study, we have found for the first time that Runt-related transcription factor 1 (Runx1 contributes to PTH-induced chondrogenesis. Upon PTH treatment, limb bud mesenchymal progenitor cells in micromass culture showed an enhanced chondrogenesis, which was associated with a significant increase of chondrogenic marker gene expression, such as type II collagen and type X collagen. Runx1 was also exclusively expressed in cells treated with PTH at the onset stage of chondrogenesis. Knockdown of Runx1 completely blunted PTH-mediated chondrogenesis. Furthermore, PTH induced Runx1 expression and chondrogenesis were markedly reduced by inhibition of protein kinase A (PKA signaling. Taken together, our present study indicates that chondrogenesis induced by PTH in mesenchymal progenitor cells is mediated by Runx1, which involves the activation of PKA. These data provide a novel insight into understanding the molecular mechanisms behind PTH-enhanced cartilage regeneration.

  16. Comparative Analysis of Cartilage Marker Gene Expression Patterns during Axolotl and Xenopus Limb Regeneration.

    Science.gov (United States)

    Mitogawa, Kazumasa; Makanae, Aki; Satoh, Ayano; Satoh, Akira

    2015-01-01

    Axolotls (Ambystoma mexicanum) can completely regenerate lost limbs, whereas Xenopus laevis frogs cannot. During limb regeneration, a blastema is first formed at the amputation plane. It is thought that this regeneration blastema forms a limb by mechanisms similar to those of a developing embryonic limb bud. Furthermore, Xenopus laevis frogs can form a blastema after amputation; however, the blastema results in a terminal cone-shaped cartilaginous structure called a "spike." The causes of this patterning defect in Xenopus frog limb regeneration were explored. We hypothesized that differences in chondrogenesis may underlie the patterning defect. Thus, we focused on chondrogenesis. Chondrogenesis marker genes, type I and type II collagen, were compared in regenerative and nonregenerative environments. There were marked differences between axolotls and Xenopus in the expression pattern of these chondrogenesis-associated genes. The relative deficit in the chondrogenic capacity of Xenopus blastema cells may account for the absence of total limb regenerative capacity. PMID:26186213

  17. Comparative Analysis of Cartilage Marker Gene Expression Patterns during Axolotl and Xenopus Limb Regeneration.

    Science.gov (United States)

    Mitogawa, Kazumasa; Makanae, Aki; Satoh, Ayano; Satoh, Akira

    2015-01-01

    Axolotls (Ambystoma mexicanum) can completely regenerate lost limbs, whereas Xenopus laevis frogs cannot. During limb regeneration, a blastema is first formed at the amputation plane. It is thought that this regeneration blastema forms a limb by mechanisms similar to those of a developing embryonic limb bud. Furthermore, Xenopus laevis frogs can form a blastema after amputation; however, the blastema results in a terminal cone-shaped cartilaginous structure called a "spike." The causes of this patterning defect in Xenopus frog limb regeneration were explored. We hypothesized that differences in chondrogenesis may underlie the patterning defect. Thus, we focused on chondrogenesis. Chondrogenesis marker genes, type I and type II collagen, were compared in regenerative and nonregenerative environments. There were marked differences between axolotls and Xenopus in the expression pattern of these chondrogenesis-associated genes. The relative deficit in the chondrogenic capacity of Xenopus blastema cells may account for the absence of total limb regenerative capacity.

  18. Comparative Analysis of Cartilage Marker Gene Expression Patterns during Axolotl and Xenopus Limb Regeneration.

    Directory of Open Access Journals (Sweden)

    Kazumasa Mitogawa

    Full Text Available Axolotls (Ambystoma mexicanum can completely regenerate lost limbs, whereas Xenopus laevis frogs cannot. During limb regeneration, a blastema is first formed at the amputation plane. It is thought that this regeneration blastema forms a limb by mechanisms similar to those of a developing embryonic limb bud. Furthermore, Xenopus laevis frogs can form a blastema after amputation; however, the blastema results in a terminal cone-shaped cartilaginous structure called a "spike." The causes of this patterning defect in Xenopus frog limb regeneration were explored. We hypothesized that differences in chondrogenesis may underlie the patterning defect. Thus, we focused on chondrogenesis. Chondrogenesis marker genes, type I and type II collagen, were compared in regenerative and nonregenerative environments. There were marked differences between axolotls and Xenopus in the expression pattern of these chondrogenesis-associated genes. The relative deficit in the chondrogenic capacity of Xenopus blastema cells may account for the absence of total limb regenerative capacity.

  19. In vitro and in vivo study of chondrogenesis on the hybrid scaffold from fibrin modified PLGA and adipose-derived stem cells%PLGA-Fibrin杂合支架构建及与脂肪源性干细胞复合成软骨的研究

    Institute of Scientific and Technical Information of China (English)

    王君; 周琦; 邓廉夫; 胡蕴玉; 魏义勇

    2010-01-01

    目的:探讨在特定的软骨细胞诱导培养液中,由纤维蛋白(fibrin)修饰的聚乳酸/聚羟基乙酸共聚物(PLGA)杂合支架材料对脂肪源性干细胞成软骨的影响.方法:PLGA支架材料采用快速成形技术制备;PLGA-Fibrin杂和支架材料采用低温冷冻的方法制备.分离兔脂肪源性干细胞,分别种植在未修饰的PLGA支架材料和Fbirin修饰的PLGA上,并在特定软骨诱导液中培养至14 d,分别应用SEM观察杂合支架材料的性能;生化方法检测硫酸软骨素的含量;支架材料的亲水性和细胞黏附能力也被检测.通过体内实验对两种支架材料与干细胞复合修复软骨缺损的性能进行形态学染色和评分.结果:体外研究表明,与未修饰的PLGA支架材料相比,Fibrin修饰的PLGA亲水性明显被提高,同时也提高了干细胞在杂合支架材料上的黏附性能和硫酸软骨素的含量.软骨损伤修复研究表明,脂肪源性干细胞与Fibirn修饰的PLGA支架材料复合后,具有良好的修复软骨缺损的功能,其性能明显优于单纯的PLGA材料复合干细胞.结论:Fibirn修饰的PLGA支架材料可提高脂肪源性干细胞向软骨细胞分化的能力,促进缺损软骨的再生.

  20. Reactive Oxygen Species Generated by NADPH Oxidase 2 and 4 Are Required for Chondrogenic Differentiation*

    OpenAIRE

    Kim, Ki Soon; Choi, Hae Woong; Yoon, Hee Eun; Kim, Ick Young

    2010-01-01

    Although generation of reactive oxygen species (ROS) by NADPH oxidases (Nox) is thought to be important for signal transduction in nonphagocytic cells, little is known of the role ROS plays in chondrogenesis. We therefore examined the possible contribution of ROS generation to chondrogenesis using both ATDC5 cells and primary chondrocytes derived from mouse embryos. The intracellular level of ROS was increased during the differentiation process, which was then blocked by treatment with the RO...

  1. Collagen gene expression during limb cartilage differentiation

    OpenAIRE

    1986-01-01

    As limb mesenchymal cells differentiate into chondrocytes, they initiate the synthesis of type II collagen and cease synthesizing type I collagen. Changes in the cytoplasmic levels of type I and type II collagen mRNAs during the course of limb chondrogenesis in vivo and in vitro were examined using cloned cDNA probes. A striking increase in cytoplasmic type II collagen mRNA occurs coincident with the crucial condensation stage of chondrogenesis in vitro, in which prechondrogenic mesenchymal c...

  2. rBMP Represses Wnt Signaling and Influences Skeletal Progenitor Cell Fate Specification During Bone Repair

    OpenAIRE

    Minear, Steve; Leucht, Philipp; Miller, Samara; Helms, Jill A.

    2010-01-01

    Bone morphogenetic proteins (BMPs) participate in multiple stages of the fetal skeletogenic program from promoting cell condensation to regulating chondrogenesis and bone formation through endochondral ossification. Here, we show that these pleiotropic functions are recapitulated when recombinant BMPs are used to augment skeletal tissue repair. In addition to their well-documented ability to stimulate chondrogenesis in a skeletal injury, we show that recombinant BMPs (rBMPs) simultaneously su...

  3. Thyroid development in zebrafish lacking Taz

    NARCIS (Netherlands)

    Pappalardo, Andrea; Porreca, Immacolata; Caputi, Luigi; De Felice, Elena; Schulte-Merker, S.; Zannini, Mariastella; Sordino, Paolo

    2015-01-01

    Taz is a signal-responsive transcriptional coregulator implicated in several biological functions, from chondrogenesis to regulation of organ size. Less well studied, however, is its role in thyroid formation. Here, we explored the in vivo effects on thyroid development of morpholino (MO)-mediated k

  4. Subglottic stenosis in Shwachman-Diamond syndrome - is there a link?

    Science.gov (United States)

    Keereweer, S; Appel, I M; Hoeve, L J

    2012-10-01

    Clinically relevant cartilaginous subglottic stenosis was found in 2 patients with Shwachman-Diamond syndrome (SDS) for which tracheotomy was required in one case. Considering the pathogenesis of SDS, including deficient chondrogenesis, we hypothesise that subglottic stenosis may be a rare symptom of SDS. Otorhinolaryngologist and paediatricians should be aware of the risk of airway pathology in patients with SDS.

  5. Chondrogenic differentiation of adipose-derived stromal cells in combinatorial hydrogels containing cartilage matrix proteins with decoupled mechanical stiffness.

    Science.gov (United States)

    Wang, Tianyi; Lai, Janice H; Han, Li-Hsin; Tong, Xinming; Yang, Fan

    2014-08-01

    Adipose-derived stromal cells (ADSCs) are attractive autologous cell sources for cartilage repair given their relative abundance and ease of isolation. Previous studies have demonstrated the potential of extracellular matrix (ECM) molecules as three-dimensional (3D) scaffolds for promoting chondrogenesis. However, few studies have compared the effects of varying types or doses of ECM molecules on chondrogenesis of ADSCs in 3D. Furthermore, increasing ECM molecule concentrations often result in simultaneous changes in the matrix stiffness, which makes it difficult to elucidate the relative contribution of biochemical cues or matrix stiffness on stem cell fate. Here we report the development of an ECM-containing hydrogel platform with largely decoupled biochemical and mechanical cues by modulating the degree of methacrylation of ECM molecules. Specifically, we incorporated three types of ECM molecules that are commonly found in the cartilage matrix, including chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS). To elucidate the effects of interactive biochemical and mechanical signaling on chondrogenesis, ADSCs were encapsulated in 39 combinatorial hydrogel compositions with independently tunable ECM types (CS, HA, and HS), concentrations (0.5%, 1.25%, 2.5%, and 5% [w/v]), and matrix stiffness (3, 30, and 90 kPa). Our results show that the effect of ECM composition on chondrogenesis is dependent on the matrix stiffness of hydrogels, suggesting that matrix stiffness and biochemical cues interact in a nonlinear manner to regulate chondrogenesis of ADSCs in 3D. In soft hydrogels (~3 kPa), increasing HA concentrations resulted in substantial upregulation of aggrecan and collagen type II expression in a dose-dependent manner. This trend was reversed in HA-containing hydrogels with higher stiffness (~90 kPa). The platform reported herein could provide a useful tool for elucidating how ECM biochemical cues and matrix stiffness interact together to

  6. Dry arthroscopy with a retraction system for matrix-aided cartilage repair of patellar lesions.

    Science.gov (United States)

    Sadlik, Boguslaw; Wiewiorski, Martin

    2014-02-01

    Several commercially available cartilage repair techniques use a natural or synthetic matrix to aid cartilage regeneration (e.g., autologous matrix-induced chondrogenesis or matrix-induced cartilage implantation). However, the use of matrix-aided techniques during conventional knee joint arthroscopy under continuous irrigation is challenging. Insertion and fixation of the matrix can be complicated by the presence of fluid and the confined patellofemoral joint space with limited access to the lesion. To overcome these issues, we developed a novel arthroscopic approach for matrix-aided cartilage repair of patellar lesions. This technical note describes the use of dry arthroscopy assisted by a minimally invasive retraction system. An autologous matrix-induced chondrogenesis procedure is used to illustrate this novel approach.

  7. The ECM-Cell Interaction of Cartilage Extracellular Matrix on Chondrocytes

    Directory of Open Access Journals (Sweden)

    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.

  8. Photo-Cross-Linked Scaffold with Kartogenin-Encapsulated Nanoparticles for Cartilage Regeneration.

    Science.gov (United States)

    Shi, Dongquan; Xu, Xingquan; Ye, Yanqi; Song, Kai; Cheng, Yixiang; Di, Jin; Hu, Quanyin; Li, Jianxin; Ju, Huangxian; Jiang, Qing; Gu, Zhen

    2016-01-26

    The regeneration of cartilage, an aneural and avascular tissue, is often compromised by its lack of innate abilities to mount a sufficient healing response. Kartogenin (KGN), a small molecular compound, can induce bone marrow-derived mesenchymal stem cells (BMSCs) into chondrocytes. The previous in vitro study showed that kartogenin also had a chondrogenesis effect on synovium derived mesenchymal stem cells (SMSCs). Herein, we present the effect of an ultraviolet-reactive, rapidly cross-linkable scaffold integrated with kartogenin-loaded nanoparticles using an innovational one-step technology. In vivo studies showed its potential role for cell homing, especially for recruiting the host's endogenous cells, including BMSCs and SMSCs, without cell transplantation. Of note, the regenerated tissues were close to the natural hyaline cartilage based on the histological tests, specific markers analysis, and biomechanical tests. This innovative KGN release system makes the chondrogenesis efficient and persistent. PMID:26757419

  9. A stem cell-based approach to cartilage repair.

    Science.gov (United States)

    Johnson, Kristen; Zhu, Shoutian; Tremblay, Matthew S; Payette, Joshua N; Wang, Jianing; Bouchez, Laure C; Meeusen, Shelly; Althage, Alana; Cho, Charles Y; Wu, Xu; Schultz, Peter G

    2012-05-11

    Osteoarthritis (OA) is a degenerative joint disease that involves the destruction of articular cartilage and eventually leads to disability. Molecules that promote the selective differentiation of multipotent mesenchymal stem cells (MSCs) into chondrocytes may stimulate the repair of damaged cartilage. Using an image-based high-throughput screen, we identified the small molecule kartogenin, which promotes chondrocyte differentiation (median effective concentration = 100 nM), shows chondroprotective effects in vitro, and is efficacious in two OA animal models. Kartogenin binds filamin A, disrupts its interaction with the transcription factor core-binding factor β subunit (CBFβ), and induces chondrogenesis by regulating the CBFβ-RUNX1 transcriptional program. This work provides new insights into the control of chondrogenesis that may ultimately lead to a stem cell-based therapy for osteoarthritis. PMID:22491093

  10. The trans-well coculture of human synovial mesenchymal stem cells with chondrocytes leads to self-organization, chondrogenic differentiation, and secretion of TGFβ

    DEFF Research Database (Denmark)

    Kubosch, Eva Johanna; Heidt, Emanuel; Bernstein, Anke;

    2016-01-01

    BACKGROUND: Synovial mesenchymal stem cells (SMSC) possess a high chondrogenic differentiation potential, which possibly supports natural and surgically induced healing of cartilage lesions. We hypothesized enhanced chondrogenesis of SMSC caused by the vicinity of chondrocytes (CHDR). METHODS....... RESULTS: After 7 days, phase-contrast microscopy revealed cell aggregation of SMSC in coculture with CHDR. Afterwards, cells formed spheres and lost adherence. However, this phenomenon was not observed when culturing SMSC alone. Fluorescence labeling showed concurrent collagen type II expression. Addition...

  11. The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

    OpenAIRE

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W. P.; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesi...

  12. Inhibition of β-Catenin Signaling in Chondrocytes Induces Delayed Fracture Healing in Mice

    OpenAIRE

    Huang, Yang; Zhang, Xiaoling; Du, Kewei; Yang, Fei; Shi, Yu; Huang, Jingang; TANG, TINGTING; Chen, Di; DAI, KERONG

    2011-01-01

    Appropriate and controlled chondrogenesis and endochondral ossification play fundamental roles in the fracture healing cascade, a regenerative process involved in highly coordinated biological events, including the Wnt/β-catenin signaling pathway. To examine the role and importance of this pathway in chondrocytes, we studied bone repair of closed tibias fractures in Col2a1-ICAT transgenic mice, in which the Wnt/β-catenin signaling pathway is specially inhibited in chondrocytes. Radiological, ...

  13. Nkx3.2 promotes primary chondrogenic differentiation by upregulating Col2a1 transcription.

    Directory of Open Access Journals (Sweden)

    Yoshitaka Kawato

    Full Text Available BACKGROUND: The Nkx3.2 transcription factor promotes chondrogenesis by forming a positive regulatory loop with a crucial chondrogenic transcription factor, Sox9. Previous studies have indicated that factors other than Sox9 may promote chondrogenesis directly, but these factors have not been identified. Here, we test the hypothesis that Nkx3.2 promotes chondrogenesis directly by Sox9-independent mechanisms and indirectly by previously characterized Sox9-dependent mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: C3H10T1/2 pluripotent mesenchymal cells were cultured with bone morphogenetic protein 2 (BMP2 to induce endochondral ossification. Overexpression of wild-type Nkx3.2 (WT-Nkx3.2 upregulated glycosaminoglycan (GAG production and expression of type II collagen α1 (Col2a1 mRNA, and these effects were evident before WT-Nkx3.2-mediated upregulation of Sox9. RNAi-mediated inhibition of Nkx3.2 abolished GAG production and expression of Col2a1 mRNA. Dual luciferase reporter assays revealed that WT-Nkx3.2 upregulated Col2a1 enhancer activity in a dose-dependent manner in C3H10T1/2 cells and also in N1511 chondrocytes. In addition, WT-Nkx3.2 partially restored downregulation of GAG production, Col2 protein expression, and Col2a1 mRNA expression induced by Sox9 RNAi. ChIP assays revealed that Nkx3.2 bound to the Col2a1 enhancer element. CONCLUSIONS/SIGNIFICANCE: Nkx3.2 promoted primary chondrogenesis by two mechanisms: Direct and Sox9-independent upregulation of Col2a1 transcription and upregulation of Sox9 mRNA expression under positive feedback system.

  14. Expression of Two Novel Alternatively Spliced COL2A1 Isoforms During Chondrocyte Differentiation

    OpenAIRE

    McAlinden, Audrey; Johnstone, Brian; Kollar, John; Kazmi, Najam; Hering, Thomas M.

    2007-01-01

    Alternative splicing of the type II procollagen gene (COL2A1) is developmentally-regulated during chondrogenesis. Type IIA procollagen (+ exon 2) is synthesized by chondroprogenitor cells while type IIB procollagen (- exon 2) is synthesized by differentiated chondrocytes. Here, we report expression of two additional alternatively spliced COL2A1 isoforms during chondrocyte differentiation of bone marrow derived mesenchymal stem cells (MSCs). One isoform, named IIC, contains only the first 34 n...

  15. Alternative Splicing of Type II Procollagen: IIB or not IIB?

    OpenAIRE

    McAlinden, Audrey

    2014-01-01

    Over two decades ago, two isoforms of the type II procollagen gene (COL2A1) were discovered. These isoforms, named IIA and IIB, are generated in a developmentally-regulated manner by alternative splicing of exon 2. Chondroprogenitor cells synthesize predominantly IIA isoforms (containing exon 2) while differentiated chondrocytes produce mainly IIB transcripts (devoid of exon 2). Importantly, this IIA-to-IIB alternative splicing switch occurs only during chondrogenesis. More recently, two othe...

  16. Dry Arthroscopy With a Retraction System for Matrix-Aided Cartilage Repair of Patellar Lesions

    OpenAIRE

    Sadlik, Boguslaw; Wiewiorski, Martin

    2014-01-01

    Several commercially available cartilage repair techniques use a natural or synthetic matrix to aid cartilage regeneration (e.g., autologous matrix–induced chondrogenesis or matrix-induced cartilage implantation). However, the use of matrix-aided techniques during conventional knee joint arthroscopy under continuous irrigation is challenging. Insertion and fixation of the matrix can be complicated by the presence of fluid and the confined patellofemoral joint space with limited access to the ...

  17. Differentiation of hMSC in Micromass Culture

    Institute of Scientific and Technical Information of China (English)

    Natalia; De; ISIA; Céline; HUSELSTEIN; Luc; MARCHAL; Marie-Nathalie; SARDA-KOLOPP; Jing-Ping; OU; YANG; Jean-Franois; STOLTZ; Assia; ELJAAFARI

    2005-01-01

    1 Introduction Mesenchymal stem cells (MSCs) are multipotential stem cells which can be expanded in culture while still maintaining their undifferentiated state. They have the potential to differentiate into distinct mesenchymal tissue cells, including chondrocytes. Thus, they are an attractive cell source for cartilage tissue engineering. In vitro high density micromass culture has been widely used for chondrogenesis induction. The objective of our study was to analyze viability and differentiation of hMSC...

  18. Mouse limb skeletal growth and synovial joint development are coordinately enhanced by Kartogenin

    OpenAIRE

    Decker, Rebekah S.; Koyama, Eiki; Enomoto-Iwamoto, Motomi; Maye, Peter; Rowe, David; Zhu, Shoutian; Schultz, Peter G.; Pacifici, Maurizio

    2014-01-01

    Limb development requires the coordinated growth of several tissues and structures including long bones, joints and tendons, but the underlying mechanisms are not wholly clear. Recently, we identified a small drug-like molecule -we named Kartogenin (KGN)- that greatly stimulates chondrogenesis in marrow-derived mesenchymal stem cells (MSCs) and enhances cartilage repair in mouse osteoarthritis (OA) models. To determine whether limb developmental processes are regulated by KGN, we tested its a...

  19. GEP, a Local Growth Factor, is Critical for Odontogenesis and Amelogenesis

    OpenAIRE

    Zhengguo Cao, Baichun Jiang, Yixia Xie, Chuan-ju Liu, Jian Q. Feng

    2010-01-01

    Granulin epithelin precursor (GEP) is a new growth factor that functions in brain development, chondrogenesis, tissue regeneration, tumorigenesis, and inflammation. The goal of this study was to study whether GEP was critical for odontogenesis and amelogenesis both in vivo and in vitro. The in situ hybridization and immunohistochemistry data showed that GEP was expressed in both odontoblast and ameloblast cells postnatally. Knockdown of GEP by crossing U6-ploxPneo-GEP and Sox2-Cre transgenic ...

  20. Periodic heat shock accelerated the chondrogenic differentiation of human mesenchymal stem cells in pellet culture.

    Directory of Open Access Journals (Sweden)

    Jing Chen

    Full Text Available Osteoarthritis (OA is one of diseases that seriously affect elderly people's quality of life. Human mesenchymal stem cells (hMSCs offer a potential promise for the joint repair in OA patients. However, chondrogenic differentiation from hMSCs in vitro takes a long time (∼ 6 weeks and differentiated cells are still not as functionally mature as primary isolated chondrocytes, though chemical stimulations and mechanical loading have been intensively studied to enhance the hMSC differentiation. On the other hand, thermal stimulations of hMSC chondrogenesis have not been well explored. In this study, the direct effects of mild heat shock (HS on the differentiation of hMSCs into chondrocytes in 3D pellet culture were investigated. Periodic HS at 41 °C for 1 hr significantly increased sulfated glycosaminoglycan in 3D pellet culture at Day 10 of chondrogenesis. Immunohistochemical and Western Blot analyses revealed an increased expression of collagen type II and aggrecan in heat-shocked pellets than non heat-shocked pellets on Day 17 of chondrogenesis. In addition, HS also upregulated the expression of collagen type I and X as well as heat shock protein 70 on Day 17 and 24 of differentiation. These results demonstrate that HS accelerated the chondrogenic differentiation of hMSCs and induced an early maturation of chondrocytes differentiated from hMSCs. The results of this study will guide the design of future protocols using thermal treatments to facilitate cartilage regeneration with human mesenchymal stem cells.

  1. The Synergistic Effects of Matrix Stiffness and Composition on the Response of Chondroprogenitor Cells in a 3D Precondensation Microenvironment.

    Science.gov (United States)

    Carrion, Bita; Souzanchi, Mohammad F; Wang, Victor T; Tiruchinapally, Gopinath; Shikanov, Ariella; Putnam, Andrew J; Coleman, Rhima M

    2016-05-01

    Improve functional quality of cartilage tissue engineered from stem cells requires a better understanding of the functional evolution of native cartilage tissue. Therefore, a biosynthetic hydrogel was developed containing RGD, hyaluronic acid and/or type-I collagen conjugated to poly(ethylene glycol) acrylate to recapitulate the precondensation microenvironment of the developing limb. Conjugation of any combination of the three ligands did not alter the shear moduli or diffusion properties of the PEG hydrogels; thus, the influence of ligand composition on chondrogenesis could be investigated in the context of varying matrix stiffness. Gene expression of ligand receptors (CD44 and the b1-integrin) as well as markers of condensation (cell clustering and N-cadherin gene expression) and chondrogenesis (Col2a1 gene expression and sGAG production) by chondroprogenitor cells in this system were modulated by both matrix stiffness and ligand composition, with the highest gene expression occurring in softer hydrogels containing all three ligands. Cell proliferation in these 3D matrices for 7 d prior to chondrogenic induction increased the rate of sGAG production in a stiffness-dependent manner. This biosynthetic hydrogel supports the features of early limb-bud condensation and chondrogenesis and is a novel platform in which the influence of the matrix physicochemical properties on these processes can be elucidated.

  2. A comparative study of the chondrogenic potential between synthetic and natural scaffolds in an in vivo bioreactor

    Science.gov (United States)

    Huang, Jung-Ju; Yang, Shu-Rui; Chu, I.-Ming; Brey, Eric M.; Hsiao, Hui-Yi; Cheng, Ming-Huei

    2013-10-01

    The clinical demand for cartilage tissue engineering is potentially large for reconstruction defects resulting from congenital deformities or degenerative disease due to limited donor sites for autologous tissue and donor site morbidities. Cartilage tissue engineering has been successfully applied to the medical field: a scaffold pre-cultured with chondrocytes was used prior to implantation in an animal model. We have developed a surgical approach in which tissues are engineered by implantation with a vascular pedicle as an in vivo bioreactor in bone and adipose tissue engineering. Collagen type II, chitosan, poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) were four commonly applied scaffolds in cartilage tissue engineering. To expand the application of the same animal model in cartilage tissue engineering, these four scaffolds were selected and compared for their ability to generate cartilage with chondrocytes in the same model with an in vivo bioreactor. Gene expression and immunohistochemistry staining methods were used to evaluate the chondrogenesis and osteogenesis of specimens. The result showed that the PLGA and PCL scaffolds exhibited better chondrogenesis than chitosan and type II collagen in the in vivo bioreactor. Among these four scaffolds, the PCL scaffold presented the most significant result of chondrogenesis embedded around the vascular pedicle in the long-term culture incubation phase.

  3. Three dimensional alginate-fucoidan composite hydrogel augments the chondrogenic differentiation of mesenchymal stromal cells.

    Science.gov (United States)

    Karunanithi, Puvanan; Murali, Malliga Raman; Samuel, Shani; Raghavendran, Hanumanantha Rao Balaji; Abbas, Azlina Amir; Kamarul, Tunku

    2016-08-20

    Presence of sulfated polysaccharides like heparan sulphate has often been implicated in the regulation of chondrogenesis. However, recently there has been a plethora of interest in the use of non-animal extracted analogs of heparan sulphate. Here we remodeled alginate (1.5%) by incorporating fucoidan (0.5%), a natural sulphated polysaccharide extracted from seaweeds to form a composite hydrogel (Al-Fu), capable of enhancing chondrogenesis of human mesenchymal stromal cells (hMSCs). We confirmed the efficiency of fucoidan incorporation by FTIR and EDX analysis. Further, its ability to support hMSC attachment and chondrogenic differentiation was confirmed by SEM, biochemical glycosaminoglycan quantification, real-time quantitative PCR and immunocytochemical analyses of chondrogenic markers Sox-9, Collagen II, Aggrecan and COMP. Effect of Al-Fu hydrogel on hMSC hypertrophy was also confirmed by the downregulation of hypertrophic genes Collagen X and Runx2. This composite scaffold can hence be used as a cartilage biomimetic biomaterial to drive hMSC chondrogenesis and for other cartilage repair based therapies. PMID:27178935

  4. Significance of soluble growth factors in the chondrogenic response of human umbilical cord matrix stem cells in a porous three dimensional scaffold

    Directory of Open Access Journals (Sweden)

    RS Nirmal

    2013-11-01

    Full Text Available Stem cell based tissue engineering has emerged as a promising strategy for articular cartilage regeneration. Foetal derived mesenchymal stem cells (MSCs with their ease of availability, pluripotency and high expansion potential have been demonstrated to be an attractive cell source over adult MSCs. However, there is a need for optimisation of chondrogenic signals to direct the differentiation of these multipotent MSCs to chondrogenic lineage. In this study we have demonstrated the in vitro chondrogenesis of human umbilical cord matrix MSCs in three dimensional PVA-PCL (polyvinyl alcohol-polycaprolactone scaffolds in the presence of the individual growth factors TGFβ1, TGFβ3, IGF, BMP2 and their combination with BMP2. Gene expression, histology and immunohistology were evaluated after 28 d culture. The induced cells showed the feature of chondrocytes in their morphology and expression of typical chondrogenic extracellular matrix molecules. Moreover, the real-time PCR assay has shown the expression of gene markers of chondrogenesis, SOX9, collagen type II and aggrecan. The expression of collagen type I and collagen type X was also evaluated. This study has demonstrated the successful chondrogenic induction of human umbilical cord MSCs in 3D scaffolds. Interestingly, the growth factor combination of TGF-β3 and BMP-2 was found to be more effective for chondrogenesis as shown by the real-time PCR studies. The findings of this study suggest the importance of using growth factor combinations for successful chondrogenic differentiation of umbilical cord MSCs.

  5. Induction of chondrogenic differentiation of human adipose-derived stem cells by low frequency electric field

    Science.gov (United States)

    Mardani, Mohammad; Roshankhah, Shiva; Hashemibeni, Batool; Salahshoor, Mohammadreza; Naghsh, Erfan; Esfandiari, Ebrahim

    2016-01-01

    Background: Since when the cartilage damage (e.g., with the osteoarthritis) it could not be repaired in the body, hence for its reconstruction needs cell therapy. For this purpose, adipose-derived stem cells (ADSCs) is one of the best cell sources because by the tissue engineering techniques it can be differentiated into chondrocytes. Chemical and physical inducers is required order to stem cells to chondrocytes differentiating. We have decided to define the role of electric field (EF) in inducing chondrogenesis process. Materials and Methods: A low frequency EF applied the ADSCs as a physical inducer for chondrogenesis in a 3D micromass culture system which ADSCs were extracted from subcutaneous abdominal adipose tissue. Also enzyme-linked immunosorbent assay, methyl thiazolyl tetrazolium, real time polymerase chain reaction and flowcytometry techniques were used for this study. Results: We found that the 20 minutes application of 1 kHz, 20 mv/cm EF leads to chondrogenesis in ADSCs. Although our results suggest that application of physical (EF) and chemical (transforming growth factor-β3) inducers at the same time, have best results in expression of collagen type II and SOX9 genes. It is also seen EF makes significant decreased expression of collagens type I and X genes. Conclusion: The low frequency EF can be a good motivator to promote chondrogenic differentiation of human ADSCs. PMID:27308269

  6. Activin Receptor-Like Kinase Receptors ALK5 and ALK1 Are Both Required for TGFβ-Induced Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells.

    Directory of Open Access Journals (Sweden)

    Laurie M G de Kroon

    Full Text Available Bone marrow-derived mesenchymal stem cells (BMSCs are promising for cartilage regeneration because BMSCs can differentiate into cartilage tissue-producing chondrocytes. Transforming Growth Factor β (TGFβ is crucial for inducing chondrogenic differentiation of BMSCs and is known to signal via Activin receptor-Like Kinase (ALK receptors ALK5 and ALK1. Since the specific role of these two TGFβ receptors in chondrogenesis is unknown, we investigated whether ALK5 and ALK1 are expressed in BMSCs and whether both receptors are required for chondrogenic differentiation of BMSCs.ALK5 and ALK1 gene expression in human BMSCs was determined with RT-qPCR. To induce chondrogenesis, human BMSCs were pellet-cultured in serum-free chondrogenic medium containing TGFβ1. Chondrogenesis was evaluated by aggrecan and collagen type IIα1 RT-qPCR analysis, and histological stainings of proteoglycans and collagen type II. To overexpress constitutively active (ca receptors, BMSCs were transduced either with caALK5 or caALK1. Expression of ALK5 and ALK1 was downregulated by transducing BMSCs with shRNA against ALK5 or ALK1.ALK5 and ALK1 were expressed in in vitro-expanded as well as in pellet-cultured BMSCs from five donors, but mRNA levels of both TGFβ receptors did not clearly associate with chondrogenic induction. TGFβ increased ALK5 and decreased ALK1 gene expression in chondrogenically differentiating BMSC pellets. Neither caALK5 nor caALK1 overexpression induced cartilage matrix formation as efficient as that induced by TGFβ. Moreover, short hairpin-mediated downregulation of either ALK5 or ALK1 resulted in a strong inhibition of TGFβ-induced chondrogenesis.ALK5 as well as ALK1 are required for TGFβ-induced chondrogenic differentiation of BMSCs, and TGFβ not only directly induces chondrogenesis, but also modulates ALK5 and ALK1 receptor signaling in BMSCs. These results imply that optimizing cartilage formation by mesenchymal stem cells will depend on

  7. The promotion of osteochondral repair by combined intra-articular injection of parathyroid hormone-related protein and implantation of a bi-layer collagen-silk scaffold.

    Science.gov (United States)

    Zhang, Wei; Chen, Jialin; Tao, Jiadong; Hu, Changchang; Chen, Longkun; Zhao, Hongshi; Xu, Guowei; Heng, Boon C; Ouyang, Hong Wei

    2013-08-01

    The repair of osteochondral defects can be enhanced with scaffolds but is often accompanied with undesirable terminal differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Parathyroid hormone-related protein (PTHrP) has been shown to inhibit aberrant differentiation, but administration at inappropriate time points would have adverse effects on chondrogenesis. This study aims to develop an effective tissue engineering strategy by combining PTHrP and collagen-silk scaffold for osteochondral defect repair. The underlying mechanisms of the synergistic effect of combining PTHrP administration with collagen-silk scaffold implantation for rabbit knee joint osteochondral defect repair were investigated. In vitro studies showed that PTHrP treatment significantly reduced Alizarin Red staining and expression of terminal differentiation-related markers. This is achieved in part through blocking activation of the canonical Wnt/β-catenin signaling pathway. For the in vivo repair study, intra-articular injection of PTHrP was carried out at three different time windows (4-6, 7-9 and 10-12 weeks) together with implantation of a bi-layer collagen-silk scaffold. Defects treated with PTHrP at the 4-6 weeks time window exhibited better regeneration (reconstitution of cartilage and subchondral bone) with minimal terminal differentiation (hypertrophy, ossification and matrix degradation), as well as enhanced chondrogenesis (cell shape, Col2 and GAG accumulation) compared with treatment at other time windows. Furthermore, the timing of PTHrP administration also influenced PTHrP receptor expression, thus affecting the treatment outcome. Our results demonstrated that intra-articular injection of PTHrP at 4-6 weeks post-injury together with collagen-silk scaffold implantation is an effective strategy for inhibiting terminal differentiation and enhancing chondrogenesis, thus improving cartilage repair and regeneration in a rabbit model. PMID:23702148

  8. Development of the turtle plastron, the order-defining skeletal structure.

    Science.gov (United States)

    Rice, Ritva; Kallonen, Aki; Cebra-Thomas, Judith; Gilbert, Scott F

    2016-05-10

    The dorsal and ventral aspects of the turtle shell, the carapace and the plastron, are developmentally different entities. The carapace contains axial endochondral skeletal elements and exoskeletal dermal bones. The exoskeletal plastron is found in all extant and extinct species of crown turtles found to date and is synaptomorphic of the order Testudines. However, paleontological reconstructed transition forms lack a fully developed carapace and show a progression of bony elements ancestral to the plastron. To understand the evolutionary development of the plastron, it is essential to know how it has formed. Here we studied the molecular development and patterning of plastron bones in a cryptodire turtle Trachemys scripta We show that plastron development begins at developmental stage 15 when osteochondrogenic mesenchyme forms condensates for each plastron bone at the lateral edges of the ventral mesenchyme. These condensations commit to an osteogenic identity and suppress chondrogenesis. Their development overlaps with that of sternal cartilage development in chicks and mice. Thus, we suggest that in turtles, the sternal morphogenesis is prevented in the ventral mesenchyme by the concomitant induction of osteogenesis and the suppression of chondrogenesis. The osteogenic subroutines later direct the growth and patterning of plastron bones in an autonomous manner. The initiation of plastron bone development coincides with that of carapacial ridge formation, suggesting that the development of dorsal and ventral shells are coordinated from the start and that adopting an osteogenesis-inducing and chondrogenesis-suppressing cell fate in the ventral mesenchyme has permitted turtles to develop their order-specific ventral morphology. PMID:27114549

  9. Human immunodeficiency virus type 1 enhancer-binding protein 3 is essential for the expression of asparagine-linked glycosylation 2 in the regulation of osteoblast and chondrocyte differentiation.

    Science.gov (United States)

    Imamura, Katsuyuki; Maeda, Shingo; Kawamura, Ichiro; Matsuyama, Kanehiro; Shinohara, Naohiro; Yahiro, Yuhei; Nagano, Satoshi; Setoguchi, Takao; Yokouchi, Masahiro; Ishidou, Yasuhiro; Komiya, Setsuro

    2014-04-01

    Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.

  10. MicroRNA Levels as Prognostic Markers for the Differentiation Potential of Human Mesenchymal Stromal Cell Donors.

    Science.gov (United States)

    Georgi, Nicole; Taipaleenmaki, Hanna; Raiss, Christian C; Groen, Nathalie; Portalska, Karolina Janaeczek; van Blitterswijk, Clemens; de Boer, Jan; Post, Janine N; van Wijnen, Andre J; Karperien, Marcel

    2015-08-15

    The ability of human mesenchymal stromal/stem cells (hMSCs) to differentiate into various mesenchymal cell lineages makes them a promising cell source for the use in tissue repair strategies. Since the differentiation potential of hMSCs differs between donors, it is necessary to establish biomarkers for the identification of donors with high differentiation potential. In this study, we show that microRNA (miRNA) expression levels are effective for distinguishing donors with high differentiation potential from low differentiation potential. Twenty hMSC donors were initially tested for marker expression and differentiation potential. In particular, the chondrogenic differentiation potential was evaluated on the basis of histological matrix formation, mRNA expression levels of chondrogenic marker genes, and quantitative glycosaminoglycan deposition. Three donors out of twenty were identified as donors with high chondrogenic potential, whereas nine showed moderate and eight showed low chondrogenic potential. Expression profiles of miRNAs involved in chondrogenesis and cartilage homeostasis were used for the distinction between high-performance hMSCs and low-performance hMSCs. Global mRNA expression profiles of the donors before the onset of chondrogenic differentiation revealed minor differences in gene expression between low and high chondrogenic performers. However, analysis of miRNA expression during a 7-day differentiation period identified miR-210 and miR-630 as positive regulators of chondrogenesis. In contrast, miR-181 and miR-34a, which are negative regulators of chondrogenesis, were upregulated during differentiation in low-performing donors. In conclusion, profiling of hMSC donors for a specific panel of miRNAs may have a prognostic value for selecting donors with high differentiation potential to improve hMSC-based strategies for tissue regeneration.

  11. Tissue engineering strategies to study cartilage development, degeneration and regeneration.

    Science.gov (United States)

    Bhattacharjee, Maumita; Coburn, Jeannine; Centola, Matteo; Murab, Sumit; Barbero, Andrea; Kaplan, David L; Martin, Ivan; Ghosh, Sourabh

    2015-04-01

    Cartilage tissue engineering has primarily focused on the generation of grafts to repair cartilage defects due to traumatic injury and disease. However engineered cartilage tissues have also a strong scientific value as advanced 3D culture models. Here we first describe key aspects of embryonic chondrogenesis and possible cell sources/culture systems for in vitro cartilage generation. We then review how a tissue engineering approach has been and could be further exploited to investigate different aspects of cartilage development and degeneration. The generated knowledge is expected to inform new cartilage regeneration strategies, beyond a classical tissue engineering paradigm.

  12. Potential benefits and limitations of utilizing chondroprogenitors in cell-based cartilage therapy.

    Science.gov (United States)

    Jayasuriya, Chathuraka T; Chen, Qian

    2015-01-01

    Chondroprogenitor cells are a subpopulation of multipotent progenitors that are primed for chondrogenesis. They are believed to have the biological repertoire to be ideal for cell-based cartilage therapy. In addition to summarizing recent advances in chondroprogenitor cell characterization, this review discusses the projected pros and cons of utilizing chondroprogenitors in regenerative medicine and compares them with that of pre-existing methods, including autologous chondrocyte implantation (ACI) and the utilization of bone marrow derived mesenchymal stem cells (MSCs) for the purpose of cartilage tissue repair. PMID:26075411

  13. Co-delivery of adipose-derived stem cells and growth factor-loaded microspheres in RGD-grafted N-methacrylate glycol chitosan gels for focal chondral repair.

    Science.gov (United States)

    Sukarto, Abby; Yu, Claire; Flynn, Lauren E; Amsden, Brian G

    2012-08-13

    The coencapsulation of growth factor-loaded microspheres with adipose-derived stem cells (ASCs) within a hydrogel matrix was studied as a potential means to enhance ASC chondrogenesis in the development of a cell-based therapeutic strategy for the regeneration of partial thickness chondral defects. A photopolymerizable N-methacrylate glycol chitosan (MGC) was employed to form an in situ gel used to encapsulate microspheres loaded with bone morphogenetic protein 6 (BMP-6) and transforming growth factor-β3 (TGF-β3) with human ASCs. ASC viability and retention were enhanced when the Young's modulus of the MGC ranged between 225 and 380 kPa. Grafting an RGD-containing peptide onto the MGC backbone (RGD-MGC) improved ASC viability within the gels, remaining at greater than 90% over 14 days in culture. The effects of BMP-6 and TGF-β3 released from the polymer microspheres on ASC chondrogenesis were assessed, and the level of differentiation was compared to ASCs in control gels containing nongrowth factor-loaded microspheres cultured with and without the growth factors supplied in the medium. There was enhanced expression of chondrogenic markers at earlier time points when the ASCs were induced with the sustained and local release of BMP-6 and TGF-β3 from the microspheres. More specifically, the normalized glycosaminoglycan and collagen type II protein expression levels were significantly higher than in the controls. In addition, the ratio of collagen type II to type I was significantly higher in the microsphere delivery group and increased over time. End-point RT-PCR analysis supported that there was a more rapid induction and enhancement of ASC chondrogenesis in the controlled release group. Interestingly, in all of the assays, there was evidence of chondrogenic differentiation when the ASCs were cultured in the gels in the absence of growth factor stimulation. Overall, the co-delivery of growth-factor-loaded microspheres and ASCs in RGD-modified MGC gels

  14. Potential benefits and limitations of utilizing chondroprogenitors in cell-based cartilage therapy.

    Science.gov (United States)

    Jayasuriya, Chathuraka T; Chen, Qian

    2015-01-01

    Chondroprogenitor cells are a subpopulation of multipotent progenitors that are primed for chondrogenesis. They are believed to have the biological repertoire to be ideal for cell-based cartilage therapy. In addition to summarizing recent advances in chondroprogenitor cell characterization, this review discusses the projected pros and cons of utilizing chondroprogenitors in regenerative medicine and compares them with that of pre-existing methods, including autologous chondrocyte implantation (ACI) and the utilization of bone marrow derived mesenchymal stem cells (MSCs) for the purpose of cartilage tissue repair.

  15. First evidence of dinosaurian secondary cartilage in the post-hatching skull of Hypacrosaurus stebingeri (Dinosauria, Ornithischia.

    Directory of Open Access Journals (Sweden)

    Alida M Bailleul

    Full Text Available Bone and calcified cartilage can be fossilized and preserved for hundreds of millions of years. While primary cartilage is fairly well studied in extant and fossilized organisms, nothing is known about secondary cartilage in fossils. In extant birds, secondary cartilage arises after bone formation during embryonic life at articulations, sutures and muscular attachments in order to accommodate mechanical stress. Considering the phylogenetic inclusion of birds within the Dinosauria, we hypothesized a dinosaurian origin for this "avian" tissue. Therefore, histological thin sectioning was used to investigate secondary chondrogenesis in disarticulated craniofacial elements of several post-hatching specimens of the non-avian dinosaur Hypacrosaurus stebingeri (Ornithischia, Lambeosaurinae. Secondary cartilage was found on three membrane bones directly involved with masticatory function: (1 as nodules on the dorso-caudal face of a surangular; and (2 on the bucco-caudal face of a maxilla; and (3 between teeth as islets in the alveolar processes of a dentary. Secondary chondrogenesis at these sites is consistent with the locations of secondary cartilage in extant birds and with the induction of the cartilage by different mechanical factors - stress generated by the articulation of the quadrate, stress of a ligamentous or muscular insertion, and stress of tooth formation. Thus, our study reveals the first evidence of "avian" secondary cartilage in a non-avian dinosaur. It pushes the origin of this "avian" tissue deep into dinosaurian ancestry, suggesting the creation of the more appropriate term "dinosaurian" secondary cartilage.

  16. Stimulation of chondrogenic differentiation of adult human bone marrow-derived stromal cells by a moderate-strength static magnetic field.

    Science.gov (United States)

    Amin, Harsh D; Brady, Mariea Alice; St-Pierre, Jean-Philippe; Stevens, Molly M; Overby, Darryl R; Ethier, C Ross

    2014-06-01

    Tissue-engineering strategies for the treatment of osteoarthritis would benefit from the ability to induce chondrogenesis in precursor cells. One such cell source is bone marrow-derived stromal cells (BMSCs). Here, we examined the effects of moderate-strength static magnetic fields (SMFs) on chondrogenic differentiation in human BMSCs in vitro. Cells were cultured in pellet form and exposed to several strengths of SMFs for various durations. mRNA transcript levels of the early chondrogenic transcription factor SOX9 and the late marker genes ACAN and COL2A1 were determined by reverse transcription-polymerase chain reaction, and production of the cartilage-specific macromolecules sGAG, collage type 2 (Col2), and proteoglycans was determined both biochemically and histologically. The role of the transforming growth factor (TGF)-β signaling pathway was also examined. Results showed that a 0.4 T magnetic field applied for 14 days elicited a strong chondrogenic differentiation response in cultured BMSCs, so long as TGF-β3 was also present, that is, a synergistic response of a SMF and TGF-β3 on BMSC chondrogenic differentiation was observed. Further, SMF alone caused TGF-β secretion in culture, and the effects of SMF could be abrogated by the TGF-β receptor blocker SB-431542. These data show that moderate-strength magnetic fields can induce chondrogenesis in BMSCs through a TGF-β-dependent pathway. This finding has potentially important applications in cartilage tissue-engineering strategies. PMID:24506272

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  18. Alendronate Can Improve Bone Alterations in Experimental Diabetes by Preventing Antiosteogenic, Antichondrogenic, and Proadipocytic Effects of AGEs on Bone Marrow Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Sara Rocío Chuguransky

    2016-01-01

    Full Text Available Bisphosphonates such as alendronate are antiosteoporotic drugs that inhibit the activity of bone-resorbing osteoclasts and secondarily promote osteoblastic function. Diabetes increases bone-matrix-associated advanced glycation end products (AGEs that impair bone marrow progenitor cell (BMPC osteogenic potential and decrease bone quality. Here we investigated the in vitro effect of alendronate and/or AGEs on the osteoblastogenic, adipogenic, and chondrogenic potential of BMPC isolated from nondiabetic untreated rats. We also evaluated the in vivo effect of alendronate (administered orally to rats with insulin-deficient Diabetes on long-bone microarchitecture and BMPC multilineage potential. In vitro, the osteogenesis (Runx2, alkaline phosphatase, type 1 collagen, and mineralization and chondrogenesis (glycosaminoglycan production of BMPC were both decreased by AGEs, while coincubation with alendronate prevented these effects. The adipogenesis of BMPC (PPARγ, intracellular triglycerides, and lipase was increased by AGEs, and this was prevented by coincubation with alendronate. In vivo, experimental Diabetes (a decreased femoral trabecular bone area, osteocyte density, and osteoclastic TRAP activity; (b increased bone marrow adiposity; and (c deregulated BMPC phenotypic potential (increasing adipogenesis and decreasing osteogenesis and chondrogenesis. Orally administered alendronate prevented all these Diabetes-induced effects on bone. Thus, alendronate could improve bone alterations in diabetic rats by preventing the antiosteogenic, antichondrogenic, and proadipocytic effects of AGEs on BMPC.

  19. Transforming Growth Factors β Coordinate Cartilage and Tendon Differentiation in the Developing Limb Mesenchyme*

    Science.gov (United States)

    Lorda-Diez, Carlos I.; Montero, Juan A.; Martinez-Cue, Carmen; Garcia-Porrero, Juan A.; Hurle, Juan M.

    2009-01-01

    Transforming growth factor β (TGFβ) signaling has an increasing interest in regenerative medicine as a potential tool to repair cartilages, however the chondrogenic effect of this pathway in developing systems is controversial. Here we have analyzed the function of TGFβ signaling in the differentiation of the developing limb mesoderm in vivo and in high density micromass cultures. In these systems highest signaling activity corresponded with cells at stages preceding overt chondrocyte differentiation. Interestingly treatments with TGFβs shifted the differentiation outcome of the cultures from chondrogenesis to fibrogenesis. This phenotypic reprogramming involved down-regulation of Sox9 and Aggrecan and up-regulation of Scleraxis, and Tenomodulin through the Smad pathway. We further show that TGFβ signaling up-regulates Sox9 in the in vivo experimental model system in which TGFβ treatments induce ectopic chondrogenesis. Looking for clues explaining the dual role of TGFβ signaling, we found that TGFβs appear to be direct inducers of the chondrogenic gene Sox9, but the existence of transcriptional repressors of TGFβ signaling modulates this role. We identified TGF-interacting factor Tgif1 and SKI-like oncogene SnoN as potential candidates for this inhibitory function. Tgif1 gene regulation by TGFβ signaling correlated with the differential chondrogenic and fibrogenic effects of this pathway, and its expression pattern in the limb marks the developing tendons. In functional experiments we found that Tgif1 reproduces the profibrogenic effect of TGFβ treatments. PMID:19717568

  20. Human stem cells and articular cartilage regeneration.

    Science.gov (United States)

    Inui, Atsuyuki; Iwakura, Takashi; Reddi, A Hari

    2012-11-05

    The regeneration of articular cartilage damaged due to trauma and posttraumatic osteoarthritis is an unmet medical need. Current approaches to regeneration and tissue engineering of articular cartilage include the use of chondrocytes, stem cells, scaffolds and signals, including morphogens and growth factors. Stem cells, as a source of cells for articular cartilage regeneration, are a critical factor for articular cartilage regeneration. This is because articular cartilage tissue has a low cell turnover and does not heal spontaneously. Adult stem cells have been isolated from various tissues, such as bone marrow, adipose, synovial tissue, muscle and periosteum. Signals of the transforming growth factor beta superfamily play critical roles in chondrogenesis. However, adult stem cells derived from various tissues tend to differ in their chondrogenic potential. Pluripotent stem cells have unlimited proliferative capacity compared to adult stem cells. Chondrogenesis from embryonic stem (ES) cells has been studied for more than a decade. However, establishment of ES cells requires embryos and leads to ethical issues for clinical applications. Induced pluripotent stem (iPS) cells are generated by cellular reprogramming of adult cells by transcription factors. Although iPS cells have chondrogenic potential, optimization, generation and differentiation toward articular chondrocytes are currently under intense investigation.

  1. Alloxan-induced diabetes delays repair in a rat model of closed tibial fracture

    Directory of Open Access Journals (Sweden)

    S.F. Diniz

    2008-05-01

    Full Text Available A closed fracture was performed on the left tibia of 3-month-old Wistar rats weighing 250 to 350 g that were either healthy (N = 24 or made diabetic with alloxan (N = 24 to investigate the effect of alloxan-induced diabetes on the course of bone fracture healing. Histomorphometric analysis of the fracture site was performed at 7, 14, 25, and 35 days. After 7 days, diabetic rats had significantly less cartilage (P = 0.045 and greater fibrous connective (P = 0.006 tissue formation at the fracture site compared to controls. In contrast, marked callus formation was seen in diabetic rats with significant osteogenesis (P = 0.011, P = 0.010, P = 0.010, respectively, for 14, 25, and 35 days and chondrogenesis (P = 0.028, P = 0.033, P = 0.019 compared to controls. Radiographic analysis revealed a displaced fracture with poor bone fragment alignment and delayed consolidation at these times in the diabetic group. The levels of alkaline phosphatase were significantly higher in diabetic rats at 25 days (P = 0.009. These results suggest that the initial excessive formation of fibrous connective tissue associated with delay in chondrogenesis and osteogenesis may not provide suitable stability of the fractured site, contributing to the inappropriate alignment of fragments and an increase in the volume of callus in later stages of repair. The resulting displaced fracture in diabetic rats requires long periods for remodeling and complete bone consolidation.

  2. Matrix dimensions, stiffness, and structural properties modulate spontaneous chondrogenic commitment of mouse embryonic fibroblasts.

    Science.gov (United States)

    Fernández-Muiños, Teresa; Suárez-Muñoz, Melva; Sanmartí-Espinal, Marta; Semino, Carlos E

    2014-04-01

    Experimental models for cartilage and bone development have been studied in order to understand the biomechanical and biological parameters that regulate skeletal tissue formation. We have previously described that when mouse embryonic fibroblasts (MEFs) were cultured in a three-dimensional (3D)-soft self-assembling peptide nanofiber, the system engaged in a spontaneous process of cartilage-like formation evidenced by the expression of Sox9, Collagen type II, and proteoglycans. In the present work, we studied the influence that matrix mechanical properties have in modulating lineage commitment in an in vitro model of chondrogenesis. This effect was observed only when MEFs were cultured at low elastic modulus values (∼ 0.1 kPa). Interestingly, under these conditions, the system expressed the chondrogenic inductor BMP4 and its antagonist Noggin. On the other hand, at higher elastic modulus values (∼ 5 kPa), the system expressed Noggin but not BMP4, and did not engage in chondrogenesis, which suggest that the balance between bone morphogenetic protein/Noggin could be implicated in the chondrogenic process. Finally, no evidence of hypertrophy was detected under the conditions tested (by assessing expression of Collagen type X and Runx2) unless we challenged the system by co-culturing it with endothelial cells. Importantly, under these new conditions, the system underwent spontaneous matrix calcium mineralization. These results suggest that the 3D-system described here is sensitive to respond to environmental changes such as biomechanical and biological cues.

  3. The Potential Application of Pulsed Ultrasound on Bone Defect Repair via Developmental Engineering: An In Vitro Study.

    Science.gov (United States)

    Wang, Jue; Tang, Na; Xiao, Qiang; Zhao, Lixing; Li, Yu; Li, Juan; Wang, Jun; Zhao, Zhihe; Tan, Lijun

    2016-05-01

    Repairing bone defect by recapitulation of endochondral bone formation, known as developmental engineering, has been a promising approach in bone tissue engineering. The critical issue in this area is how to effectively construct the hypertrophic cartilaginous template in vitro and enhance in vivo endochondral ossification process after implantation. Pulsed ultrasound stimulation has been widely used in the clinic for accelerating bone healing in fractures and nonunions. The aim of this study was to investigate whether ultrasound (US) could accelerate in vitro chondrogenesis and the hypertrophic process in certain microenvironments. Rat bone marrow mesenchymal stem cells were chondrogenic or hypertrophic differentiated in a three-dimensional pellet culture system with different media, and treated with different intensities of US. US exposure promoted chondrogenic differentiation of stem cells and inhibited their transition into the hypertrophic stage in a chondrogenic-friendly microenvironment. US significantly advanced hypertrophic differentiation of bone marrow stem cell pellets in hypertrophic medium after chondrogenesis. Our data indicated that pulsed US promoted in vitro chondrogenic and hypertrophic differentiation of stem cell pellets in specific culture conditions. The present study proves the potential application of US in the in vitro stage of "developmental engineering" for bone development and repair. PMID:26526417

  4. A Hydrogel Model Incorporating 3D-Plotted Hydroxyapatite for Osteochondral Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Michal Bartnikowski

    2016-04-01

    Full Text Available The concept of biphasic or multi-layered compound scaffolds has been explored within numerous studies in the context of cartilage and osteochondral regeneration. To date, no system has been identified that stands out in terms of superior chondrogenesis, osteogenesis or the formation of a zone of calcified cartilage (ZCC. Herein we present a 3D plotted scaffold, comprising an alginate and hydroxyapatite paste, cast within a photocrosslinkable hydrogel made of gelatin methacrylamide (GelMA, or GelMA with hyaluronic acid methacrylate (HAMA. We hypothesized that this combination of 3D plotting and hydrogel crosslinking would form a high fidelity, cell supporting structure that would allow localization of hydroxyapatite to the deepest regions of the structure whilst taking advantage of hydrogel photocrosslinking. We assessed this preliminary design in terms of chondrogenesis in culture with human articular chondrocytes, and verified whether the inclusion of hydroxyapatite in the form presented had any influence on the formation of the ZCC. Whilst the inclusion of HAMA resulted in a better chondrogenic outcome, the effect of HAP was limited. We overall demonstrated that formation of such compound structures is possible, providing a foundation for future work. The development of cohesive biphasic systems is highly relevant for current and future cartilage tissue engineering.

  5. The effect of a chitosan-gelatin matrix and dexamethasone on the behavior of rabbit mesenchymal stem cells

    International Nuclear Information System (INIS)

    Cartilage tissue has poor capability of self-repair, especially in the case of severe cartilage damage due to trauma or age-related degeneration. Cell-based tissue engineering using scaffolds has provided an option for the repair of defects in adult cartilage tissue. Mesenchymal stem cells (MSC) and chondrocytes are the two major cell sources for cartilage tissue engineering. The present study combined culture conditions of MSC in a chitosan-gelatin matrix in chondrogenic media to evaluate their effects on MSC viability and chondrogenesis for cartilage tissue engineering. MSC were harvested from rabbit bone marrows and cultured in chondrogenic media supplemented, or not, with dexamethasone in a chitosan-gelatin film (C-GF). The association of C-GF and dexamethasone promoted significant increase in cell adhesivity, viability and proliferation when compared to MCS cultured in media without dexamethasone or C-GF. In addition, dexamethasone promoted increase in the collagen concentration of MSC cultures. A reduction of alkaline phosphatase activity after three weeks of culture in chondrogenic media was verified. No influence of the C-GF or of dexamethasone was observed in this matter. Therefore, it is reasonable to suggest that biomaterial-based chitosan-gelatin and chondrogenic media supplemented with dexamethasone may stimulate the proliferation and differentiation of MSC according to the complex environmental conditions. The information presented here should be useful for the development of biomaterials to regulate the chondrogenesis of MSC suitable for cartilage tissue engineering

  6. The effect of a chitosan-gelatin matrix and dexamethasone on the behavior of rabbit mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Medrado, G C B [Medicine School, Federal University of Minas Gerais (UFMG), Belo Horizonte, MG (Brazil); Machado, C B [Biochemistry and Immunology Department, Biological Sciences Institute, UFMG - Federal University of Minas Gerais, mailbox 486, zip code 31270-901, Belo Horizonte, MG (Brazil); Valerio, P [Biochemistry and Immunology Department, Biological Sciences Institute, UFMG - Federal University of Minas Gerais, mailbox 486, zip code 31270-901, Belo Horizonte, MG (Brazil); Sanches, M D [Medicine School, Federal University of Minas Gerais (UFMG), Belo Horizonte, MG (Brazil); Goes, A M [Biochemistry and Immunology Department, Biological Sciences Institute, UFMG - Federal University of Minas Gerais, mailbox 486, zip code 31270-901, Belo Horizonte, MG (Brazil)

    2006-09-15

    Cartilage tissue has poor capability of self-repair, especially in the case of severe cartilage damage due to trauma or age-related degeneration. Cell-based tissue engineering using scaffolds has provided an option for the repair of defects in adult cartilage tissue. Mesenchymal stem cells (MSC) and chondrocytes are the two major cell sources for cartilage tissue engineering. The present study combined culture conditions of MSC in a chitosan-gelatin matrix in chondrogenic media to evaluate their effects on MSC viability and chondrogenesis for cartilage tissue engineering. MSC were harvested from rabbit bone marrows and cultured in chondrogenic media supplemented, or not, with dexamethasone in a chitosan-gelatin film (C-GF). The association of C-GF and dexamethasone promoted significant increase in cell adhesivity, viability and proliferation when compared to MCS cultured in media without dexamethasone or C-GF. In addition, dexamethasone promoted increase in the collagen concentration of MSC cultures. A reduction of alkaline phosphatase activity after three weeks of culture in chondrogenic media was verified. No influence of the C-GF or of dexamethasone was observed in this matter. Therefore, it is reasonable to suggest that biomaterial-based chitosan-gelatin and chondrogenic media supplemented with dexamethasone may stimulate the proliferation and differentiation of MSC according to the complex environmental conditions. The information presented here should be useful for the development of biomaterials to regulate the chondrogenesis of MSC suitable for cartilage tissue engineering.

  7. Chondrogenic potential of human mesenchymal stem cells and expression of Slug transcription factor.

    Science.gov (United States)

    Brini, Anna T; Niada, Stefania; Lambertini, Elisabetta; Torreggiani, Elena; Arrigoni, Elena; Lisignoli, Gina; Piva, Roberta

    2015-06-01

    The scientific literature rarely reports experimental failures or inconsistent outcomes in the induction of cell differentiation; however, researchers commonly experience poor or unsuccessful responses to differentiating agents when culturing stem cells. One way of investigating the underlying reasons for such responses is to look at the basal expression levels of specific genes in multipotent stem cells before the induction of differentiation. In addition to shedding light on the complex properties of stem cells and the molecular modulation of differentiation pathways, this strategy can also lead to the development of important time- and money-saving tools that aid the efficient selection of cellular specimens--in this case, stem cells that are more prone to differentiate towards specific lineages and are therefore more suitable for cell-based therapeutic protocols in regenerative medicine. To address this latter aspect, this study focused on understanding the reasons why some human mesenchymal stem cell (hMSC) samples are less efficient at differentiating towards chondrogenesis. This study shows that analysis of the basal expression levels of Slug, a negative regulator of chondrogenesis in hMSC, provides a rapid and simple tool for distinguishing stem cell samples with the potential to form a cartilage-like matrix, and that are therefore suitable for cartilage tissue engineering. It is shown that high basal levels of Slug prevent the chondrogenic differentiation of hMSCs, even in the presence of transforming growth factor-β and elevated levels of Sox9.

  8. First evidence of dinosaurian secondary cartilage in the post-hatching skull of Hypacrosaurus stebingeri (Dinosauria, Ornithischia).

    Science.gov (United States)

    Bailleul, Alida M; Hall, Brian K; Horner, John R

    2012-01-01

    Bone and calcified cartilage can be fossilized and preserved for hundreds of millions of years. While primary cartilage is fairly well studied in extant and fossilized organisms, nothing is known about secondary cartilage in fossils. In extant birds, secondary cartilage arises after bone formation during embryonic life at articulations, sutures and muscular attachments in order to accommodate mechanical stress. Considering the phylogenetic inclusion of birds within the Dinosauria, we hypothesized a dinosaurian origin for this "avian" tissue. Therefore, histological thin sectioning was used to investigate secondary chondrogenesis in disarticulated craniofacial elements of several post-hatching specimens of the non-avian dinosaur Hypacrosaurus stebingeri (Ornithischia, Lambeosaurinae). Secondary cartilage was found on three membrane bones directly involved with masticatory function: (1) as nodules on the dorso-caudal face of a surangular; and (2) on the bucco-caudal face of a maxilla; and (3) between teeth as islets in the alveolar processes of a dentary. Secondary chondrogenesis at these sites is consistent with the locations of secondary cartilage in extant birds and with the induction of the cartilage by different mechanical factors - stress generated by the articulation of the quadrate, stress of a ligamentous or muscular insertion, and stress of tooth formation. Thus, our study reveals the first evidence of "avian" secondary cartilage in a non-avian dinosaur. It pushes the origin of this "avian" tissue deep into dinosaurian ancestry, suggesting the creation of the more appropriate term "dinosaurian" secondary cartilage. PMID:22558351

  9. Human Stem Cells and Articular Cartilage Regeneration

    Directory of Open Access Journals (Sweden)

    A. Hari Reddi

    2012-11-01

    Full Text Available  The regeneration of articular cartilage damaged due to trauma and posttraumatic osteoarthritis is an unmet medical need. Current approaches to regeneration and tissue engineering of articular cartilage include the use of chondrocytes, stem cells, scaffolds and signals, including morphogens and growth factors. Stem cells, as a source of cells for articular cartilage regeneration, are a critical factor for articular cartilage regeneration. This is because articular cartilage tissue has a low cell turnover and does not heal spontaneously. Adult stem cells have been isolated from various tissues, such as bone marrow, adipose, synovial tissue, muscle and periosteum. Signals of the transforming growth factor beta superfamily play critical roles in chondrogenesis. However, adult stem cells derived from various tissues tend to differ in their chondrogenic potential. Pluripotent stem cells have unlimited proliferative capacity compared to adult stem cells. Chondrogenesis from embryonic stem (ES cells has been studied for more than a decade. However, establishment of ES cells requires embryos and leads to ethical issues for clinical applications. Induced pluripotent stem (iPS cells are generated by cellular reprogramming of adult cells by transcription factors. Although iPS cells have chondrogenic potential, optimization, generation and differentiation toward articular chondrocytes are currently under intense investigation.

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

    Directory of Open Access Journals (Sweden)

    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

  11. Oxygen tension regulates the osteogenic, chondrogenic and endochondral phenotype of bone marrow derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Sheehy, Eamon J.; Buckley, Conor T. [Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin, Dublin 2 (Ireland); Kelly, Daniel J., E-mail: kellyd9@tcd.ie [Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin, Dublin 2 (Ireland)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Expansion in low oxygen enhances MSC proliferation and osteogenesis. Black-Right-Pointing-Pointer Differentiation in low oxygen enhances chondrogenesis and suppresses hypertrophy. Black-Right-Pointing-Pointer Oxygen can regulate the MSC phenotype for use in tissue engineering applications. -- Abstract: The local oxygen tension is a key regulator of the fate of mesenchymal stem cells (MSCs). The objective of this study was to investigate the effect of a low oxygen tension during expansion and differentiation on the proliferation kinetics as well as the subsequent osteogenic and chondrogenic potential of MSCs. We first hypothesised that expansion in a low oxygen tension (5% pO{sub 2}) would improve both the subsequent osteogenic and chondrogenic potential of MSCs compared to expansion in a normoxic environment (20% pO{sub 2}). Furthermore, we hypothesised that chondrogenic differentiation in a low oxygen environment would suppress hypertrophy of MSCs cultured in both pellets and hydrogels used in tissue engineering strategies. MSCs expanded at 5% pO{sub 2} proliferated faster forming larger colonies, resulting in higher cell yields. Expansion at 5% pO{sub 2} also enhanced subsequent osteogenesis of MSCs, whereas differentiation at 5% pO{sub 2} was found to be a more potent promoter of chondrogenesis than expansion at 5% pO{sub 2}. Greater collagen accumulation, and more intense staining for collagen types I and X, was observed in pellets maintained at 20% pO{sub 2} compared to 5% pO{sub 2}. Both pellets and hydrogels stained more intensely for type II collagen when undergoing chondrogenesis in a low oxygen environment. Differentiation at 5% pO{sub 2} also appeared to inhibit hypertrophy in both pellets and hydrogels, as demonstrated by reduced collagen type X and Alizarin Red staining and alkaline phosphatase activity. This study demonstrates that the local oxygen environment can be manipulated in vitro to either stabilise a

  12. Zebrafish con/disp1 reveals multiple spatiotemporal requirements for Hedgehog-signaling in craniofacial development

    Directory of Open Access Journals (Sweden)

    Schwend Tyler

    2009-11-01

    Full Text Available Abstract Background The vertebrate head skeleton is derived largely from cranial neural crest cells (CNCC. Genetic studies in zebrafish and mice have established that the Hedgehog (Hh-signaling pathway plays a critical role in craniofacial development, partly due to the pathway's role in CNCC development. Disruption of the Hh-signaling pathway in humans can lead to the spectral disorder of Holoprosencephaly (HPE, which is often characterized by a variety of craniofacial defects including midline facial clefting and cyclopia 12. Previous work has uncovered a role for Hh-signaling in zebrafish dorsal neurocranium patterning and chondrogenesis, however Hh-signaling mutants have not been described with respect to the ventral pharyngeal arch (PA skeleton. Lipid-modified Hh-ligands require the transmembrane-spanning receptor Dispatched 1 (Disp1 for proper secretion from Hh-synthesizing cells to the extracellular field where they act on target cells. Here we study chameleon mutants, lacking a functional disp1(con/disp1. Results con/disp1 mutants display reduced and dysmorphic mandibular and hyoid arch cartilages and lack all ceratobranchial cartilage elements. CNCC specification and migration into the PA primorida occurs normally in con/disp1 mutants, however disp1 is necessary for post-migratory CNCC patterning and differentiation. We show that disp1 is required for post-migratory CNCC to become properly patterned within the first arch, while the gene is dispensable for CNCC condensation and patterning in more posterior arches. Upon residing in well-formed pharyngeal epithelium, neural crest condensations in the posterior PA fail to maintain expression of two transcription factors essential for chondrogenesis, sox9a and dlx2a, yet continue to robustly express other neural crest markers. Histology reveals that posterior arch residing-CNCC differentiate into fibrous-connective tissue, rather than becoming chondrocytes. Treatments with Cyclopamine, to

  13. Pre-metatarsal skeletal development in tissue culture at unit- and microgravity

    Science.gov (United States)

    Klement, B. J.; Spooner, B. S.

    1994-01-01

    Explant organ culture was used to demonstrate that isolated embryonic mouse pre-metatarsal mesenchyme is capable of undergoing a series of differentiative and morphogenetic developmental events. Mesenchyme differentiation into chondrocytes, and concurrent morphogenetic patterning of the cartilage tissue, and terminal chondrocyte differentiation with subsequent matrix mineralization show that cultured tissue closely parallels in vivo development. Whole mount alizarin red staining of the cultured tissue demonstrates that the extracellular matrix around the hypertrophied chondrocytes is competent to support mineralization. Intensely stained mineralized bands are similar to those formed in pre-metatarsals developing in vivo. We have adapted the culture strategy for experimentation in a reduced gravity environment on the Space Shuttle. Spaceflight culture of pre-metatarsals, which have already initiated chondrogenesis and morphogenetic patterning, results in an increase in cartilage rod size and maintenance of rod shape, compared to controls. Older pre-metatarsal tissue, already terminally differentiated to hypertrophied cartilage, maintained rod structure and cartilage phenotype during spaceflight culture.

  14. Cartilage constructs from human cord blood stem cells seeded in structurally-graded polycaprolactone scaffolds

    DEFF Research Database (Denmark)

    Munir, Samir; Koch, Thomas Gadegaard; Foldager, Casper Bindzus;

    Cartilage is an avascular tissue incapable of regeneration. Current treatment modalities for joint cartilage injuries are inefficient in regenerating hyaline cartilage and often leads to the formation of fibrocartilage with undesirable mechanical properties. There is an increasing interest...... stimulation. This study demonstrated the chondrogenic potential of human cord blood-derived Multi-Lineage Progenitor Cells (MLPCs) under normoxic and hypoxic culture conditions. Second, MLPCs were seeded in a novel, structurally graded polycaprolactone (SGS-PCL) scaffold and chondrogenesis was evaluated...... this novel SGS-PCL scaffold supports the chondrogenic differentiation of MLPCs will be interesting to evaluate since this scaffold possesses mechanical properties absent from other “soft” scaffolds currently being investigated for cartilage regeneration and implantation....

  15. Multiscale dynamics of biological cells with chemotactic interactions: from a discrete stochastic model to a continuous description

    CERN Document Server

    Alber, M; Glimm, T; Lushnikov, P M; Alber, Mark; Chen, Nan; Glimm, Tilmann; Lushnikov, Pavel M.

    2006-01-01

    The Cellular Potts Model (CPM) has been used for simulating various biological phenomena such as differential adhesion, fruiting body formation of the slime mold Dictyostelium discoideum, angiogenesis, cancer invasion, chondrogenesis in embryonic vertebrate limbs, and many others. In this paper, we derive continuous limit of discrete one dimensional CPM with the chemotactic interactions between cells in the form of a Fokker-Planck equation for the evolution of the cell probability density function. This equation is then reduced to the classical macroscopic Keller-Segel model. In particular, all coefficients of the Keller-Segel model are obtained from parameters of the CPM. Theoretical results are verified numerically by comparing Monte Carlo simulations for the CPM with numerics for the Keller-Segel model.

  16. Temporal evolution of mechanical properties of skeletal tissue regeneration in rabbits. An experimental study

    CERN Document Server

    Mokoko, Didier; Chabrand, Patrick

    2007-01-01

    Various mathematical models represent the effects of local mechanical environment on the regulation of skeletal regeneration. Their relevance relies on an accurate description of the evolving mechanical properties of the regenerating tissue. The object of this study was to develop an experimental model which made it possible to characterize the temporal evolution of the structural and mechanical properties during unloaded enchondral osteogenesis in the New Zealand rabbit, a standard animal model for studies of osteogenesis and chondrogenesis. A 25mm segment of tibial diaphysis was removed sub-periosteally from rabbits. The defect was repaired by the preserved periosteum. An external fixator was applied to prevent mechanical loading during osteogenesis. The regenerated skeletal tissues were studied by CT scan, histology and mechanical tests. The traction tests between 7 to 21 days post-surgery were done on formaldehyde-fixated tissue allowing to obtain force/displacement curves. The viscoelastic properties of ...

  17. A Direct Sulfation Process of a Marine Polysaccharide in Ionic Liquid.

    Science.gov (United States)

    Chopin, Nathalie; Sinquin, Corinne; Ratiskol, Jacqueline; Zykwinska, Agata; Weiss, Pierre; Cérantola, Stéphane; Le Bideau, Jean; Colliec-Jouault, Sylvia

    2015-01-01

    GY785 is an exopolysaccharide produced by a mesophilic bacterial strain Alteromonas infernus discovered in the deep-sea hydrothermal vents. GY785 highly sulfated derivative (GY785 DRS) was previously demonstrated to be a promising molecule driving the efficient mesenchymal stem cell chondrogenesis for cartilage repair. This glycosaminoglycan- (GAG-) like compound was modified in a classical solvent (N,N'-dimethylformamide). However, the use of classical solvents limits the polysaccharide solubility and causes the backbone degradation. In the present study, a one-step efficient sulfation process devoid of side effects (e.g., polysaccharide depolymerization and/or degradation) was developed to produce GAG-like derivatives. The sulfation of GY785 derivative (GY785 DR) was carried out using ionic liquid as a reaction medium. The successful sulfation of this anionic and highly branched heteropolysaccharide performed in ionic liquid would facilitate the production of new molecules of high specificity for biological targets such as tissue engineering or regenerative medicine.

  18. Gelatin-Based Hydrogels Promote Chondrogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells In Vitro

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    Achim Salamon

    2014-02-01

    Full Text Available Due to the weak regeneration potential of cartilage, there is a high clinical incidence of articular joint disease, leading to a strong demand for cartilaginous tissue surrogates. The aim of this study was to evaluate a gelatin-based hydrogel for its suitability to support chondrogenic differentiation of human mesenchymal stem cells. Gelatin-based hydrogels are biodegradable, show high biocompatibility, and offer possibilities to introduce functional groups and/or ligands. In order to prove their chondrogenesis-supporting potential, a hydrogel film was developed and compared with standard cell culture polystyrene regarding the differentiation behavior of human mesenchymal stem cells. Cellular basis for this study were human adipose tissue-derived mesenchymal stem cells, which exhibit differentiation potential along the adipogenic, osteogenic and chondrogenic lineage. The results obtained show a promotive effect of gelatin-based hydrogels on chondrogenic differentiation of mesenchymal stem cells in vitro and therefore encourage subsequent in vivo studies.

  19. ACTIVITY OF CANONICAL WNT SIGNAL SYSTEM IN HYALINE CARTILAGE ARTICULAR CHONDROCYTES IN PROCESS OF SYNOVIAL JOINT DEVELOPMENT

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    A.O. Molotkov

    2009-03-01

    Full Text Available Canonical and non-canonical Wnt systems are essential regulators of chondrogenesis and bone development. However, the roles of these systems in synovial joint development are not well studied. To determine if canonical Wnt system is active in developing articular chondrocytes we used immunohistochemistry for в-galactosidase and doublecortin (cell-type specific marker for articular chondrocytes to double label sections through joint regions of E14.5, E18.5, P10 and adult mice. Here the following results are presented. Canonical Wnt signal system does not work in developing articular chondrocytes at early embryonic stages (E14.5; it is active in the articular chondrocytes at late embryonic stages (E16.5-E18.5 and during postnatal development (P7-P10, but is turned off again in the adult articular chondrocytes. These results suggest that canonical Wnt signaling is being regulated during articular chondrocytes differentiation and joint formation.

  20. Critical review on the physical and mechanical factors involved in tissue engineering of cartilage.

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    Gaut, Carrie; Sugaya, Kiminobu

    2015-01-01

    Articular cartilage defects often progress to osteoarthritis, which negatively impacts quality of life for millions of people worldwide and leads to high healthcare expenditures. Tissue engineering approaches to osteoarthritis have concentrated on proliferation and differentiation of stem cells by activation and suppression of signaling pathways, and by using a variety of scaffolding techniques. Recent studies indicate a key role of environmental factors in the differentiation of mesenchymal stem cells to mature cartilage-producing chondrocytes. Therapeutic approaches that consider environmental regulation could optimize chondrogenesis protocols for regeneration of articular cartilage. This review focuses on the effect of scaffold structure and composition, mechanical stress and hypoxia in modulating mesenchymal stem cell fate and the current use of these environmental factors in tissue engineering research.

  1. Developmental dysplasia of the hip: usefulness of next generation genomic tools for characterizing the underlying genes - a mini review.

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    Basit, S; Hannan, M A; Khoshhal, K I

    2016-07-01

    Developmental dysplasia of the hip (DDH) is one of the most common skeletal anomalies. DDH encompasses a spectrum of the disorder ranging from minor acetabular dysplasia to irreducible dislocation, which may lead to premature arthritis in later life. Involvement of genetic factors underlying DDH became evident when several studies reported chromosomal loci linked to DDH in families with multiple affected individuals. Moreover, using association studies, variants in genes involved in chondrogenesis and joint formation have been shown to be associated with DDH. At least, one study identified a pathogenic variant in the chemokine receptor gene in DDH. No genetic analysis has been reported or carried out in DDH patients from the Middle East. Here, we review the literature related to genetics of DDH and emphasized the usefulness of new generation technologies in identifying genetic variants underlying DDH in consanguineous families. PMID:26842108

  2. The future of osteoarthritis therapeutics: emerging biological therapy.

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    Mobasheri, A

    2013-12-01

    Biological therapy is a thriving area of research and development, and is well established for chronic forms of rheumatoid arthritis (RA) and ankylosing spondylitis (AS). However, there is no clinically validated biological therapy for osteoarthritis (OA). Chronic forms of OA are increasingly viewed as an inflammatory disease. OA was largely regarded as a "wear and tear disease". However, the disease is now believed to involve "low grade" inflammation and the growth of blood vessels and nerves from the subchondral bone into articular cartilage. This realization has focused research effort on the development and evaluation of biological therapy that targets proinflammatory mediators, angiogenic factors and cytokines in articular cartilage, subchondral bone and synovium in chronic forms of OA. This review article provides an overview of emerging biological therapy for OA, and discusses recent molecular targets implicated in angiogenesis and neurogenesis and progress with antibody-based therapy, calcitonin, and kartogenin, the small molecule stimulator of chondrogenesis. PMID:24170255

  3. GEP, a Local Growth Factor, is Critical for Odontogenesis and Amelogenesis

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    Zhengguo Cao, Baichun Jiang, Yixia Xie, Chuan-ju Liu, Jian Q. Feng

    2010-01-01

    Full Text Available Granulin epithelin precursor (GEP is a new growth factor that functions in brain development, chondrogenesis, tissue regeneration, tumorigenesis, and inflammation. The goal of this study was to study whether GEP was critical for odontogenesis and amelogenesis both in vivo and in vitro. The in situ hybridization and immunohistochemistry data showed that GEP was expressed in both odontoblast and ameloblast cells postnatally. Knockdown of GEP by crossing U6-ploxPneo-GEP and Sox2-Cre transgenic mice led to a reduction of dentin thickness, an increase in predentin thickness, and a reduction in mineral content in enamel. The in vitro application of recombinant GEP up-regulated molecular markers important for odontogenesis (DMP1, DSPP, and ALP and amelogenesis (ameloblastin, amelogenin and enamelin. In conclusion, both the in vivo and the in vivo data support an important role of GEP in tooth formation during postnatal development.

  4. Surface Markers for Chondrogenic Determination: A Highlight of Synovium-Derived Stem Cells

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    Douglas D. Campbell

    2012-11-01

    Full Text Available Cartilage tissue engineering is a promising field in regenerative medicine that can provide substantial relief to people suffering from degenerative cartilage disease. Current research shows the greatest chondrogenic potential for healthy articular cartilage growth with minimal hypertrophic differentiation to be from mesenchymal stem cells (MSCs of synovial origin. These stem cells have the capacity for differentiation into multiple cell lineages related to mesenchymal tissue; however, evidence exists for cell surface markers that specify a greater potential for chondrogenesis than other differentiation fates. This review will examine relevant literature to summarize the chondrogenic differentiation capacities of tested synovium-derived stem cell (SDSC surface markers, along with a discussion about various other markers that may hold potential, yet require further investigation. With this information, a potential clinical benefit exists to develop a screening system for SDSCs that will produce the healthiest articular cartilage possible.

  5. Human soluble delta-like 1 homolog exerts antitumor effects in vitro and in vivo.

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    Lee, Donghee; Yoon, Sun Ha; Lee, Hyun Ju; Jo, Ki Won; Park, Bum-Chan; Kim, In Seop; Choi, Yunseon; Lim, Jung Chae; Park, Young Woo

    2016-06-24

    Proteolysis of delta-like 1 homolog (DLK1), a cell-surface transmembrane protein, produces an active soluble form of DLK1 (sDLK1). Both membrane-bound DLK1 and sDLK1 modulate multiple developmental processes including adipogenesis, osteogenesis, chondrogenesis and myogenesis. However, cancer-related functions of DLK1 have not yet been established. We thus evaluated the roles of extracellular sDLK1, comprising six EGF-like domains and juxtamembrane regions, in human pancreatic cancer MIA PaCa-2 cells in vitro and in vivo. We observed that sDLK1 exerted antitumor effects not only in cancer cell migration and anchorage-independent cell growth but also in in vivo tumor growth. PMID:27191393

  6. REGENERATION OF ARTICULAR CARTILAGE UNDER THE IMPLANTATION OF BONE MATRIX

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    Yuri M. Iryanov, Nikolay A. Kiryanov, Olga V. Dyuriagina , Tatiana Yu. Karaseva, Evgenii A. Karasev

    2015-07-01

    Full Text Available Background: The damage or loss of articular cartilage is costly medical problem. The purpose of this work – morphological analysis of reparative chondrogenesis when implanted in the area of the knee joint cartilage of granulated mineralized bone matrix. Material and Methods: The characteristic features of the knee cartilage regeneration studied experimentally in pubertal Wistar rats after modeling a marginal perforated defect and implantation of granulated mineralized bone matrix obtained according to original technology without heat and demineralizing processing into the injury zone. Results: This biomaterial established to have pronounced chondro- and osteoinductive properties, and to provide prolonged activation of reparative process, accelerated organotypical remodeling and restoration of the articular cartilage injured. Conclusion: The data obtained demonstrate the efficacy of МВМ in clinical practice for the treatment of diseases and injuries of the articular cartilage.

  7. Fibroblast-Derived Extracellular Matrix Induces Chondrogenic Differentiation in Human Adipose-Derived Mesenchymal Stromal/Stem Cells in Vitro.

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    Dzobo, Kevin; Turnley, Taegyn; Wishart, Andrew; Rowe, Arielle; Kallmeyer, Karlien; van Vollenstee, Fiona A; Thomford, Nicholas E; Dandara, Collet; Chopera, Denis; Pepper, Michael S; Parker, M Iqbal

    2016-01-01

    Mesenchymal stromal/stem cells (MSCs) represent an area being intensively researched for tissue engineering and regenerative medicine applications. MSCs may provide the opportunity to treat diseases and injuries that currently have limited therapeutic options, as well as enhance present strategies for tissue repair. The cellular environment has a significant role in cellular development and differentiation through cell-matrix interactions. The aim of this study was to investigate the behavior of adipose-derived MSCs (ad-MSCs) in the context of a cell-derived matrix so as to model the in vivo physiological microenvironment. The fibroblast-derived extracellular matrix (fd-ECM) did not affect ad-MSC morphology, but reduced ad-MSC proliferation. Ad-MSCs cultured on fd-ECM displayed decreased expression of integrins α2 and β1 and subsequently lost their multipotency over time, as shown by the decrease in CD44, Octamer-binding transcription factor 4 (OCT4), SOX2, and NANOG gene expression. The fd-ECM induced chondrogenic differentiation in ad-MSCs compared to control ad-MSCs. Loss of function studies, through the use of siRNA and a mutant Notch1 construct, revealed that ECM-mediated ad-MSCs chondrogenesis requires Notch1 and β-catenin signaling. The fd-ECM also showed anti-senescence effects on ad-MSCs. The fd-ECM is a promising approach for inducing chondrogenesis in ad-MSCs and chondrogenic differentiated ad-MSCs could be used in stem cell therapy procedures. PMID:27527147

  8. Expression of Bone Morphogenetic Protein-2 in the Chondrogenic and Ossifying Sites of Calcific Tendinopathy and Traumatic Tendon Injury Rat Models

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    Chan Lai

    2009-07-01

    Full Text Available Abstract Background Ectopic chondrogenesis and ossification were observed in a degenerative collagenase-induced calcific tendinopathy model and to a lesser extent, in a patellar tendon traumatic injury model. We hypothesized that expression of bone morphogenetic protein-2 (BMP-2 contributed to ectopic chondrogenesis and ossification. This study aimed to study the spatial and temporal expression of BMP-2 in our animal models. Methods Seventy-two rats were used, with 36 rats each subjected to central one-third patellar tendon window injury (C1/3 group and collagenase-induced tendon injury (CI group, respectively. The contralateral limb served as controls. At week 2, 4 and 12, 12 rats in each group were sacrificed for immunohistochemistry and RT-PCR of BMP-2. Results For CI group, weak signal was observed at the tendon matrix at week 2. At week 4, matrix around chondrocyte-like cells was also stained in some samples. In one sample, calcification was observed and the BMP-2 signal was observed both in the calcific matrix and the embedded chondrocyte-like cells. At week 12, the staining was observed mainly in the calcific matrix. Similar result was observed in C1/3 group though the immunopositive staining of BMP-2 was generally weaker. There was significant increase in BMP-2 mRNA compared to that in the contralateral side at week 2 and the level became insignificantly different at week 12 in CI group. No significant increase in BMP-2 mRNA was observed in C1/3 group at all time points. Conclusion Ectopic expression of BMP-2 might induce tissue transformation into ectopic bone/cartilage and promoted structural degeneration in calcific tendinopathy.

  9. Forelimb-hindlimb developmental timing changes across tetrapod phylogeny

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    Selwood Lynne

    2007-10-01

    Full Text Available Abstract Background Tetrapods exhibit great diversity in limb structures among species and also between forelimbs and hindlimbs within species, diversity which frequently correlates with locomotor modes and life history. We aim to examine the potential relation of changes in developmental timing (heterochrony to the origin of limb morphological diversity in an explicit comparative and quantitative framework. In particular, we studied the relative time sequence of development of the forelimbs versus the hindlimbs in 138 embryos of 14 tetrapod species spanning a diverse taxonomic, ecomorphological and life-history breadth. Whole-mounts and histological sections were used to code the appearance of 10 developmental events comprising landmarks of development from the early bud stage to late chondrogenesis in the forelimb and the corresponding serial homologues in the hindlimb. Results An overall pattern of change across tetrapods can be discerned and appears to be relatively clade-specific. In the primitive condition, as seen in Chondrichthyes and Osteichthyes, the forelimb/pectoral fin develops earlier than the hindlimb/pelvic fin. This pattern is either retained or re-evolved in eulipotyphlan insectivores (= shrews, moles, hedgehogs, and solenodons and taken to its extreme in marsupials. Although exceptions are known, the two anurans we examined reversed the pattern and displayed a significant advance in hindlimb development. All other species examined, including a bat with its greatly enlarged forelimbs modified as wings in the adult, showed near synchrony in the development of the fore and hindlimbs. Conclusion Major heterochronic changes in early limb development and chondrogenesis were absent within major clades except Lissamphibia, and their presence across vertebrate phylogeny are not easily correlated with adaptive phenomena related to morphological differences in the adult fore- and hindlimbs. The apparently conservative nature of this

  10. Chondrogenic differentiation of human mesenchymal stem cells on fish scale collagen.

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    Hsu, Han-Hsiu; Uemura, Toshimasa; Yamaguchi, Isamu; Ikoma, Toshiyuki; Tanaka, Junzo

    2016-08-01

    Fish collagen has recently been reported to be a novel biomaterial for cell and tissue culture as an alternative to conventional mammalian collagens such as bovine and porcine collagens. Fish collagen could overcome the risk of zoonosis, such as from bovine spongiform encephalopathy. Among fish collagens, tilapia collagen, the denaturing temperature of which is near 37°C, is appropriate for cell and tissue culture. In this study, we investigated chondrogenic differentiation of human mesenchymal stem cells (hMSCs) cultured on tilapia scale collagen fibrils compared with porcine collagen and non-coated dishes. The collagen fibrils were observed using a scanning electronic microscope. Safranin O staining, glycosaminoglycans (GAG) expression, and real-time PCR were examined to evaluate chondrogenesis of hMSCs on each type of collagen fibril. The results showed that hMSCs cultured on tilapia scale collagen showed stronger Safranin O staining and higher GAG expression at day 6. Results of real-time PCR indicated that hMSCs cultured on tilapia collagen showed earlier SOX9 expression on day 4 and higher AGGRECAN and COLLAGEN II expression on day 6 compared with on porcine collagen and non-coated dishes. Furthermore, low mRNA levels of bone gamma-carboxyglutamate, a specific marker of osteogenesis, showed that tilapia collagen fibrils specifically enhanced chondrogenic differentiation of hMSCs in chondrogenic medium, as well as porcine collagen. Accordingly, tilapia scale collagen may provide an appropriate collagen source for hMSC chondrogenesis in vitro. PMID:26829997

  11. Chondrogenic differentiation of human subchondral progenitor cells is affected by synovial fluid from donors with osteoarthritis or rheumatoid arthritis

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    Krüger Jan

    2012-03-01

    Full Text Available Abstract Background Microfracture is a first-line treatment option for cartilage repair. In microfracture, subchondral mesenchymal cortico-spongious progenitor cells (CSP enter the defect and form cartilage repair tissue. The aim of our study was to investigate the effects of joint disease conditions on the in vitro chondrogenesis of human CSP. Methods CSP were harvested from the subchondral bone marrow. CSP characterization was performed by analysis of cell surface antigen pattern and by assessing the chondrogenic, osteogenic and adipogenic differentiation potential, histologically. To assess the effect of synovial fluid (SF on chondrogenesis of CSP, micro-masses were stimulated with SF from healthy (ND, osteoarthritis (OA and rheumatoid arthritis donors (RA without transforming growth factor beta 3. Results CSP showed the typical cell surface antigen pattern known from mesenchymal stem cells and were capable of osteogenic, adipogenic and chondrogenic differentiation. In micro-masses stimulated with SF, histological staining as well as gene expression analysis of typical chondrogenic marker genes showed that SF from ND and OA induced the chondrogenic marker genes aggrecan, types II and IX collagen, cartilage oligomeric matrix protein (COMP and link protein, compared to controls not treated with SF. In contrast, the supplementation with SF from RA donors decreased the expression of aggrecan, type II collagen, COMP and link protein, compared to CSP treated with SF from ND or OA. Conclusion These results suggest that in RA, SF may impair cartilage repair by subchondral mesenchymal progenitor cells in microfracture, while in OA, SF may has no negative, but a delaying effect on the cartilage matrix formation.

  12. Swim-training changes the spatio-temporal dynamics of skeletogenesis in zebrafish larvae (Danio rerio).

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    Fiaz, Ansa W; Léon-Kloosterziel, Karen M; Gort, Gerrit; Schulte-Merker, Stefan; van Leeuwen, Johan L; Kranenbarg, Sander

    2012-01-01

    Fish larvae experience many environmental challenges during development such as variation in water velocity, food availability and predation. The rapid development of structures involved in feeding, respiration and swimming increases the chance of survival. It has been hypothesized that mechanical loading induced by muscle forces plays a role in prioritizing the development of these structures. Mechanical loading by muscle forces has been shown to affect larval and embryonic bone development in vertebrates, but these investigations were limited to the appendicular skeleton. To explore the role of mechanical load during chondrogenesis and osteogenesis of the cranial, axial and appendicular skeleton, we subjected zebrafish larvae to swim-training, which increases physical exercise levels and presumably also mechanical loads, from 5 until 14 days post fertilization. Here we show that an increased swimming activity accelerated growth, chondrogenesis and osteogenesis during larval development in zebrafish. Interestingly, swim-training accelerated both perichondral and intramembranous ossification. Furthermore, swim-training prioritized the formation of cartilage and bone structures in the head and tail region as well as the formation of elements in the anal and dorsal fins. This suggests that an increased swimming activity prioritized the development of structures which play an important role in swimming and thereby increasing the chance of survival in an environment where water velocity increases. Our study is the first to show that already during early zebrafish larval development, skeletal tissue in the cranial, axial and appendicular skeleton is competent to respond to swim-training due to increased water velocities. It demonstrates that changes in water flow conditions can result into significant spatio-temporal changes in skeletogenesis.

  13. Comparative characteristics of mesenchymal stem cells derived from reamer-irrigator-aspirator, iliac crest bone marrow, and adipose tissue.

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    Toosi, S; Naderi-Meshkin, H; Kalalinia, F; Peivandi, M T; Hossein Khani, H; Bahrami, A R; Heirani-Tabasi, A; Mirahmadi, M; Behravan, J

    2016-01-01

    Mesenchymal stem cells (MSCs) have been considered promising tools for new clinical concepts in supporting cellular therapy and regenerative medicine. More recently, Ream/Irrigator/Aspirator (RIA) was introduced as a source of MSCs. In this study we compared MSCs derived from three different sources (iliac crest bone marrow (ICBM), adipose tissue (AT), and (RIA)) regarding the morphology, the success rate of isolating MSCs, colony frequency, expansion potential, osteogenic and chondrogenic differentiation capacity. MSCs were isolated from three different sources and flow cytometric analyses were performed for cell characterization. Colony-forming unit-fibroblast (CFU-F) assay and population doubling time (PDT) were evaluated for MSCs derived from three different sources and differentiation potential of RIA, ICBM-, and AT-MSCs were determined by staining. Additionally, gene expression profiles for tissue specific markers corresponding to osteogenesis and chondrogenesis were analyzed using real time polymerase chain reaction (RT-PCR). Cultured with the appropriate condition, osteogenic and chondrogenic differentiation could be confirmed in all MSC preparations. Flow cytometry analysis indicated that RIA- and AT-derived MSCs have more homogenous populations than ICBM-MSCs. A comparison of the colonogenic ability in different tissues by CFU-F assay after 10 days showed that more colonies are formed from RIA-MSCs than from ICBM-MSCs, and AT-MSCs. AT-MSCs, were dispersed with no obvious colonies. The RIA-MSCs underwent osteogenesis and chondrogenesis at a faster rate than ICBM and AT-MSCs. Direct comparisons of RIA- to ICBM- and AT-MSCs have shown the RIA-MSCs have higher differentiation toward osteoblast and chondrocytes compared to other sources of MSCs. Hence, RIA-MSCs may be recommended as a more suitable source for treating orthopedic disorders. PMID:27609477

  14. Fibroblast-Derived Extracellular Matrix Induces Chondrogenic Differentiation in Human Adipose-Derived Mesenchymal Stromal/Stem Cells in Vitro

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    Kevin Dzobo

    2016-08-01

    Full Text Available Mesenchymal stromal/stem cells (MSCs represent an area being intensively researched for tissue engineering and regenerative medicine applications. MSCs may provide the opportunity to treat diseases and injuries that currently have limited therapeutic options, as well as enhance present strategies for tissue repair. The cellular environment has a significant role in cellular development and differentiation through cell–matrix interactions. The aim of this study was to investigate the behavior of adipose-derived MSCs (ad-MSCs in the context of a cell-derived matrix so as to model the in vivo physiological microenvironment. The fibroblast-derived extracellular matrix (fd-ECM did not affect ad-MSC morphology, but reduced ad-MSC proliferation. Ad-MSCs cultured on fd-ECM displayed decreased expression of integrins α2 and β1 and subsequently lost their multipotency over time, as shown by the decrease in CD44, Octamer-binding transcription factor 4 (OCT4, SOX2, and NANOG gene expression. The fd-ECM induced chondrogenic differentiation in ad-MSCs compared to control ad-MSCs. Loss of function studies, through the use of siRNA and a mutant Notch1 construct, revealed that ECM-mediated ad-MSCs chondrogenesis requires Notch1 and β-catenin signaling. The fd-ECM also showed anti-senescence effects on ad-MSCs. The fd-ECM is a promising approach for inducing chondrogenesis in ad-MSCs and chondrogenic differentiated ad-MSCs could be used in stem cell therapy procedures.

  15. Dose-response of superparamagnetic iron oxide labeling on mesenchymal stem cells chondrogenic differentiation: a multi-scale in vitro study.

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    Emilie Roeder

    Full Text Available AIM: The aim of this work was the development of successful cell therapy techniques for cartilage engineering. This will depend on the ability to monitor non-invasively transplanted cells, especially mesenchymal stem cells (MSCs that are promising candidates to regenerate damaged tissues. METHODS: MSCs were labeled with superparamagnetic iron oxide particles (SPIO. We examined the effects of long-term labeling, possible toxicological consequences and the possible influence of progressive concentrations of SPIO on chondrogenic differentiation capacity. RESULTS: No influence of various SPIO concentrations was noted on human bone marrow MSC viability or proliferation. We demonstrated long-term (4 weeks in vitro retention of SPIO by human bone marrow MSCs seeded in collagenic sponges under TGF-β1 chondrogenic conditions, detectable by Magnetic Resonance Imaging (MRI and histology. Chondrogenic differentiation was demonstrated by molecular and histological analysis of labeled and unlabeled cells. Chondrogenic gene expression (COL2A2, ACAN, SOX9, COL10, COMP was significantly altered in a dose-dependent manner in labeled cells, as were GAG and type II collagen staining. As expected, SPIO induced a dramatic decrease of MRI T2 values of sponges at 7T and 3T, even at low concentrations. CONCLUSIONS: This study clearly demonstrates (1 long-term in vitro MSC traceability using SPIO and MRI and (2 a deleterious dose-dependence of SPIO on TGF-β1 driven chondrogenesis in collagen sponges. Low concentrations (12.5-25 µg Fe/mL seem the best compromise to optimize both chondrogenesis and MRI labeling.

  16. Overexpression of TGF-β1 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cells

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    Kim, Yong Il; Ryu, Jae-Sung; Yeo, Jee Eun; Choi, Yun Jin; Kim, Yong Sang [Center for Stem Cell and Arthritis Research, Department of Orthopedic Surgery, Yonsei Sarang Hospital, Seoul (Korea, Republic of); Ko, Kinarm [Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 143-701 (Korea, Republic of); Koh, Yong-Gon, E-mail: yonseranglab@daum.net [Center for Stem Cell and Arthritis Research, Department of Orthopedic Surgery, Yonsei Sarang Hospital, Seoul (Korea, Republic of)

    2014-08-08

    Highlights: • Continuous TGF-β1 overexpression in hSD-MSCs did not influence their phenotypes. • Retroviral-mediated transduction of TGFB1 in hSD-MSCs enhances cell proliferation. • TGF-β1 overexpression did not effect to adipo- or osteogenic potential of hSD-MSCs. • TGF-β1 overexpression in hSD-MSCs could stimulate and accelerate chondrogenesis. - Abstract: Transforming growth factor-beta (TGF-β) superfamily proteins play a critical role in proliferation, differentiation, and other functions of mesenchymal stem cells (MSCs). During chondrogenic differentiation of MSCs, TGF-β up-regulates chondrogenic gene expression by enhancing the expression of the transcription factor SRY (sex-determining region Y)-box9 (Sox9). In this study, we investigated the effect of continuous TGF-β1 overexpression in human synovium-derived MSCs (hSD-MSCs) on immunophenotype, differentiation potential, and proliferation rate. hSD-MSCs were transduced with recombinant retroviruses (rRV) encoding TGF-β1. The results revealed that continuous overexpression of TGF-β1 did not affect their phenotype as evidenced by flow cytometry and reverse transcriptase PCR (RT-PCR). In addition, continuous TGF-β1 overexpression strongly enhanced cell proliferation of hSD-MSCs compared to the control groups. Also, induction of chondrogenesis was more effective in rRV-TGFB-transduced hSD-MSCs as shown by RT-PCR for chondrogenic markers, toluidine blue staining and glycosaminoglycan (GAG)/DNA ratio. Our data suggest that overexpression of TGF-β1 positively enhances the proliferation and chondrogenic potential of hSD-MSCs.

  17. Ultra-structural changes and expression of chondrogenic and hypertrophic genes during chondrogenic differentiation of mesenchymal stromal cells in alginate beads

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    Dashtdar, Havva; Selvaratnam, Lakshmi; Balaji Raghavendran, Hanumantharao; Suhaeb, Abdulrazzaq Mahmod; Ahmad, Tunku Sara

    2016-01-01

    Chondrogenic differentiation of mesenchymal stromal cells (MSCs) in the form of pellet culture and encapsulation in alginate beads has been widely used as conventional model for in vitro chondrogenesis. However, comparative characterization between differentiation, hypertrophic markers, cell adhesion molecule and ultrastructural changes during alginate and pellet culture has not been described. Hence, the present study was conducted comparing MSCs cultured in pellet and alginate beads with monolayer culture. qPCR was performed to assess the expression of chondrogenic, hypertrophic, and cell adhesion molecule genes, whereas transmission electron microscopy (TEM) was used to assess the ultrastructural changes. In addition, immunocytochemistry for Collagen type II and aggrecan and glycosaminoglycan (GAG) analysis were performed. Our results indicate that pellet and alginate bead cultures were necessary for chondrogenic differentiation of MSC. It also indicates that cultures using alginate bead demonstrated significantly higher (p < 0.05) chondrogenic but lower hypertrophic (p < 0.05) gene expressions as compared with pellet cultures. N-cadherin and N-CAM1 expression were up-regulated in second and third weeks of culture and were comparable between the alginate bead and pellet culture groups, respectively. TEM images demonstrated ultrastructural changes resembling cell death in pellet cultures. Our results indicate that using alginate beads, MSCs express higher chondrogenic but lower hypertrophic gene expression. Enhanced production of extracellular matrix and cell adhesion molecules was also observed in this group. These findings suggest that alginate bead culture may serve as a superior chondrogenic model, whereas pellet culture is more appropriate as a hypertrophic model of chondrogenesis. PMID:26966647

  18. Effects of cyclophosphamide and acrolein in organoid cultures of mouse limb bud cells grown in the presence of adult rat hepatocytes.

    Science.gov (United States)

    Ghaida, J; Merker, H J

    1992-01-01

    The effects were evaluated of cyclophosphamide (CPA) and its metabolite, acrolein, on chondrogenesis in organoid cultures of mouse limb bud mesenchymal cells co-cultured with non-enzymatically isolated adult rat hepatocytes. The studies were conducted with or without the simultaneous addition of 2-mercaptoethanesulphonic acid sodium (mesna) or glutathione (GSH). Alcian blue binding assay and light and electron microscopic techniques were used. Increasing concentrations of the two compounds (bioactivated CPA, 18-180 mum; acrolein, 50-500 mum) led to a dose-dependent inhibition of chondrogenesis associated with cellular dedifferentiation and/or cytotoxicity. Addition of mesna (1 mm) or GSH (1 mm) partially protected the cultures against CPA and acrolein. However, the protective effect depended on the dose of CPA or acrolein used. A higher protection was observed with mesna than with GSH, and the effect was more pronounced with acrolein than with CPA. The morphological findings suggested that CPA and acrolein acted by different mechanisms. Bioactivated CPA primarily inhibited the differentiation process, whereas acrolein exhibited a high cytotoxic activity affecting particularly monolayer cells that normally grow on the periphery of the cultures. These findings suggest that acrolein possesses a specific mode of action directed towards this type of cell. This could be explained by the specific shape and/or behaviour of the cells (i.e. cytoskeletal arrangement, proliferation rate, migration activity, intercellular communication pattern, etc.). The results demonstrated that the cell system used was suitable for the performance of cytotoxicity and teratogenicity studies such as those conducted with CPA and acrolein.

  19. Identification and characterization of chondrogenic progenitor cells in the fascia of postnatal skeletal muscle

    Institute of Scientific and Technical Information of China (English)

    Guangheng Li; Bo Zheng; Laura B. Meszaros; Joseph B. Vella; Arvydas Usas; Tomoyuki Matsumoto; Johnny Huard

    2011-01-01

    Intramuscular injection of bone morphogenetic proteins (BMPs) has been shown to induce ectopic bone formation.A chondrogenic phase is typically observed in this process,which suggests that there may exist a chondrogenic subpopulation of cells residing in skeletal muscle.Two prospective cell populations were isolated from rat skeletal muscle:fascia-derived cells (FDCs),extracted from gluteus maximus muscle fascia (epimysium) and muscle-derived cells (MDCs) isolated from the muscle body.Both populations were investigated for their cell surface marker profiles (flowcytometry analysis),proliferation rates as well as their myogenic and chondrogenic potentials.The majority of FDCs expressed mesenchymai stromai cell markers but not endothelial cell markers.FDCs underwent chondrogenic differentiation after BMP4 treatment in vitro,but not myogenic differentiation.Although MDCs showed chondrogenic potential,they expressed the myogenic cell marker desmin and readily underwent myogenic differentiation in vitro; however,the chondrogenic potential of the MDCs is confounded by the presence of FDC-like cells residing in the muscle perimysium and endomysium.To clarify the role of the muscle-derived myogenic cells in chondrogenesis,mixed pellets with varying ratios of FDCs and L6 myoblasts were formed and studied for chondrogenic potential.Our results indicated that the chondrogenic potential of the mixed pellets decreased with the increased ratio of myogenic cells to FDCs supporting the role of FDCs in chondrogenesis.Taken together,our results suggest that non-myogenic cells residing in the fascia of skeletal muscle have a strong chondrogenic potential and may represent a novel donor cell source for cartilage regeneration and repair.

  20. Murine Mesenchymal Stem Cell Commitment to Differentiation Is Regulated by Mitochondrial Dynamics.

    Science.gov (United States)

    Forni, Maria Fernanda; Peloggia, Julia; Trudeau, Kyle; Shirihai, Orian; Kowaltowski, Alicia J

    2016-03-01

    Mouse skin mesenchymal stem cells (msMSCs) are dermis CD105(+) CD90(+) CD73(+) CD29(+) CD34(-) mesodermal precursors which, after in vitro induction, undergo chondro, adipo, and osteogenesis. Extensive metabolic reconfiguration has been found to occur during differentiation, and the bioenergetic status of a cell is known to be dependent on the quality and abundance of the mitochondrial population, which may be regulated by fusion and fission. However, little is known regarding the impact of mitochondrial dynamics on the differentiation process. We addressed this knowledge gap by isolating MSCs from Swiss female mice, inducing these cells to differentiate into osteo, chondro, and adipocytes and measuring changes in mass, morphology, dynamics, and bioenergetics. Mitochondrial biogenesis was increased in adipogenesis, as evaluated through confocal microscopy, citrate synthase activity, and mtDNA content. The early steps of adipo and osteogenesis involved mitochondrial elongation, as well as increased expression of mitochondrial fusion proteins Mfn1 and 2. Chondrogenesis involved a fragmented mitochondrial phenotype, increased expression of fission proteins Drp1, Fis1, and 2, and enhanced mitophagy. These events were accompanied by profound bioenergetic alterations during the commitment period. Moreover, knockdown of Mfn2 in adipo and osteogenesis and the overexpression of a dominant negative form of Drp1 during chondrogenesis resulted in a loss of differentiation ability. Overall, we find that mitochondrial morphology and its regulating processes of fission/fusion are modulated early on during commitment, leading to alterations in the bioenergetic profile that are important for differentiation. We thus propose a central role for mitochondrial dynamics in the maintenance/commitment of mesenchymal stem cells. PMID:26638184

  1. Connective-Tissue Growth Factor (CTGF/CCN2 Induces Astrogenesis and Fibronectin Expression of Embryonic Neural Cells In Vitro.

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    Fabio A Mendes

    Full Text Available Connective-tissue growth factor (CTGF is a modular secreted protein implicated in multiple cellular events such as chondrogenesis, skeletogenesis, angiogenesis and wound healing. CTGF contains four different structural modules. This modular organization is characteristic of members of the CCN family. The acronym was derived from the first three members discovered, cysteine-rich 61 (CYR61, CTGF and nephroblastoma overexpressed (NOV. CTGF is implicated as a mediator of important cell processes such as adhesion, migration, proliferation and differentiation. Extensive data have shown that CTGF interacts particularly with the TGFβ, WNT and MAPK signaling pathways. The capacity of CTGF to interact with different growth factors lends it an important role during early and late development, especially in the anterior region of the embryo. ctgf knockout mice have several cranio-facial defects, and the skeletal system is also greatly affected due to an impairment of the vascular-system development during chondrogenesis. This study, for the first time, indicated that CTGF is a potent inductor of gliogenesis during development. Our results showed that in vitro addition of recombinant CTGF protein to an embryonic mouse neural precursor cell culture increased the number of GFAP- and GFAP/Nestin-positive cells. Surprisingly, CTGF also increased the number of Sox2-positive cells. Moreover, this induction seemed not to involve cell proliferation. In addition, exogenous CTGF activated p44/42 but not p38 or JNK MAPK signaling, and increased the expression and deposition of the fibronectin extracellular matrix protein. Finally, CTGF was also able to induce GFAP as well as Nestin expression in a human malignant glioma stem cell line, suggesting a possible role in the differentiation process of gliomas. These results implicate ctgf as a key gene for astrogenesis during development, and suggest that its mechanism may involve activation of p44/42 MAPK signaling

  2. Functional Characterization of the Osteoarthritis Susceptibility Mapping to CHST11—A Bioinformatics and Molecular Study

    Science.gov (United States)

    Reynard, Louise N.; Ratnayake, Madhushika; Santibanez-Koref, Mauro

    2016-01-01

    The single nucleotide polymorphism (SNP) rs835487 is associated with hip osteoarthritis (OA) at the genome-wide significance level and is located within CHST11, which codes for carbohydrate sulfotransferase 11. This enzyme post-translationally modifies proteoglycan prior to its deposition in the cartilage extracellular matrix. Using bioinformatics and experimental analyses, our aims were to characterise the rs835487 association signal and to identify the causal functional variant/s. Database searches revealed that rs835487 resides within a linkage disequilibrium (LD) block of only 2.7 kb and is in LD (r2 ≥ 0.8) with six other SNPs. These are all located within intron 2 of CHST11, in a region that has predicted enhancer activity and which shows a high degree of conservation in primates. Luciferase reporter assays revealed that of the seven SNPs, rs835487 and rs835488, which have a pairwise r2 of 0.962, are the top functional candidates; the haplotype composed of the OA-risk conferring G allele of rs835487 and the corresponding T allele of rs835488 (the G-T haplotype) demonstrated significantly different enhancer activity relative to the haplotype composed of the non-risk A allele of rs835487 and the corresponding C allele of rs835488 (the A-C haplotype) (p < 0.001). Electrophoretic mobility shift assays and supershifts identified several transcription factors that bind more strongly to the risk-conferring G and T alleles of the two SNPs, including SP1, SP3, YY1 and SUB1. CHST11 was found to be upregulated in OA versus non-OA cartilage (p < 0.001) and was expressed dynamically during chondrogenesis. Its expression in adult cartilage did not however correlate with rs835487 genotype. Our data demonstrate that the OA susceptibility is mediated by differential protein binding to the alleles of rs835487 and rs835488, which are located within an enhancer whose target may be CHST11 during chondrogenesis or an alternative gene. PMID:27391021

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

    International Nuclear Information System (INIS)

    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

  4. Versican Expression during Synovial Joint Morphogenesis

    Directory of Open Access Journals (Sweden)

    John B. Shepard, Heidi A. Krug, Brooklynn A. LaFoon, Stanley Hoffman, Anthony A. Capehart

    2007-01-01

    Full Text Available The extracellular matrix (ECM plays a critical role in governing cell behavior and phenotype during limb skeletogenesis. Chondroitin sulfate proteoglycans (Cspgs are highly expressed in the ECM of precartilage mesenchymal condensations and are important to limb chondrogenesis and cartilage structure, but little is known regarding their involvement in formation of synovial joints in the embryonic limb. Matrix versican Cspg expression has previously been reported in the epiphysis of developing long bones and presumptive joint; however, detailed analysis has not yet been conducted. In the present study we immunolocalized versican and aggrecan Cspgs during chick elbow joint morphogenesis between HH st25-41 of development. In this study we show that versican and aggrecan expression initially overlapped in the incipient cartilage model of long bones in the wing, but versican was also highly expressed in the perichondrium and presumptive joint interzone during early stages of morphogenesis (HH st25-34. By HH st36-41 versican localization was restricted to the future articular surfaces of the developing joint and surrounding joint capsule while aggrecan localized in an immediately adjacent and predominately non-overlapping region of chondrogenic cells at the epiphyses. These results suggest a potential role for versican proteoglycan in development and maintenance of the synovial joint interzone.

  5. Bioinformatics and Microarray Analysis of miRNAs in Aged Female Mice Model Implied New Molecular Mechanisms for Impaired Fracture Healing

    Science.gov (United States)

    He, Bing; Zhang, Zong-Kang; Liu, Jin; He, Yi-Xin; Tang, Tao; Li, Jie; Guo, Bao-Sheng; Lu, Ai-Ping; Zhang, Bao-Ting; Zhang, Ge

    2016-01-01

    Impaired fracture healing in aged females is still a challenge in clinics. MicroRNAs (miRNAs) play important roles in fracture healing. This study aims to identify the miRNAs that potentially contribute to the impaired fracture healing in aged females. Transverse femoral shaft fractures were created in adult and aged female mice. At post-fracture 0-, 2- and 4-week, the fracture sites were scanned by micro computed tomography to confirm that the fracture healing was impaired in aged female mice and the fracture calluses were collected for miRNA microarray analysis. A total of 53 significantly differentially expressed miRNAs and 5438 miRNA-target gene interactions involved in bone fracture healing were identified. A novel scoring system was designed to analyze the miRNA contribution to impaired fracture healing (RCIFH). Using this method, 11 novel miRNAs were identified to impair fracture healing at 2- or 4-week post-fracture. Thereafter, function analysis of target genes was performed for miRNAs with high RCIFH values. The results showed that high RCIFH miRNAs in aged female mice might impair fracture healing not only by down-regulating angiogenesis-, chondrogenesis-, and osteogenesis-related pathways, but also by up-regulating osteoclastogenesis-related pathway, which implied the essential roles of these high RCIFH miRNAs in impaired fracture healing in aged females, and might promote the discovery of novel therapeutic strategies. PMID:27527150

  6. The Chondrogenic Induction Potential for Bone Marrow-Derived Stem Cells between Autologous Platelet-Rich Plasma and Common Chondrogenic Induction Agents: A Preliminary Comparative Study

    Directory of Open Access Journals (Sweden)

    Shan-zheng Wang

    2015-01-01

    Full Text Available The interests in platelet-rich plasma (PRP and their application in stem cell therapy have contributed to a better understanding of the basic biology of the prochondrogenesis effect on bone marrow-derived stem cells (BMSCs. We aimed at comparing the effect of autologous PRP with common chondrogenic induction agents (CCIAs on the chondrogenic differentiation of BMSCs. Rabbit BMSCs were isolated and characterized by flow cytometry and differentiated towards adipocytes and osteoblasts. The chondrogenic response of BMSCs to autologous PRP and CCIAs which included transforming growth factor-β1 (TGF-β1, dexamethasone (DEX, and vitamin C (Vc was examined by cell pellet culture. The isolated BMSCs after two passages highly expressed CD29 and CD44 but minimally expressed CD45. The osteogenic and adipogenic differentiation potentials of the isolated BMSCs were also confirmed. Compared with common CCIAs, autologous PRP significantly upregulated the chondrogenic related gene expression, including Col-2, AGC, and Sox-9. Osteogenic related gene expression, including Col-1 and OCN, was not of statistical significance between these two groups. Thus, our data shows that, compared with common chondrogenic induction agents, autologous PRP can be more effective in promoting the chondrogenesis of BMSCs.

  7. Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Z.H. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Li, X.L. [Department of Dermatology, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); He, X.J. [Department of Orthopedics, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Wu, B.J.; Xu, M. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Chang, H.M. [Department of Otolaryngology - Head and Neck Surgery, Affiliated Hospital of Xi' an Medical University, Xi' an (China); Zhang, X.H. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Xing, Z. [Department of Clinical Dentistry, Faculty of Dentistry, Center for Clinical Dental Research, University of Bergen, Bergen (Norway); Jing, X.H.; Kong, D.M.; Kou, X.H.; Yang, Y.Y. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China)

    2014-03-18

    SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.

  8. An Injectable Enzymatically Crosslinked Carboxymethylated Pullulan/Chondroitin Sulfate Hydrogel for Cartilage Tissue Engineering

    Science.gov (United States)

    Chen, Feng; Yu, Songrui; Liu, Bing; Ni, Yunzhou; Yu, Chunyang; Su, Yue; Zhu, Xinyuan; Yu, Xiaowei; Zhou, Yongfeng; Yan, Deyue

    2016-01-01

    In this study, an enzymatically cross-linked injectable and biodegradable hydrogel system comprising carboxymethyl pullulan-tyramine (CMP-TA) and chondroitin sulfate-tyramine (CS-TA) conjugates was successfully developed under physiological conditions in the presence of both horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) for cartilage tissue engineering (CTTE). The HRP crosslinking method makes this injectable system feasible, minimally invasive and easily translatable for regenerative medicine applications. The physicochemical properties of the mechanically stable hydrogel system can be modulated by varying the weight ratio and concentration of polymer as well as the concentrations of crosslinking reagents. Additionally, the cellular behaviour of porcine auricular chondrocytes encapsulated into CMP-TA/CS-TA hydrogels demonstrates that the hydrogel system has a good cyto-compatibility. Specifically, compared to the CMP-TA hydrogel, these CMP-TA/CS-TA composite hydrogels have enhanced cell proliferation and increased cartilaginous ECM deposition, which significantly facilitate chondrogenesis. Furthermore, histological analysis indicates that the hydrogel system exhibits acceptable tissue compatibility by using a mouse subcutaneous implantation model. Overall, the novel injectable pullulan/chondroitin sulfate composite hydrogels presented here are expected to be useful biomaterial scaffold for regenerating cartilage tissue.

  9. Dynamic nanomechanics of individual bone marrow stromal cells and cell-matrix composites during chondrogenic differentiation.

    Science.gov (United States)

    Lee, BoBae; Han, Lin; Frank, Eliot H; Grodzinsky, Alan J; Ortiz, Christine

    2015-01-01

    Dynamic nanomechanical properties of bovine bone marrow stromal cells (BMSCs) and their newly synthesized cartilage-like matrices were studied at nanometer scale deformation amplitudes. The increase in their dynamic modulus, |E(*)| (e.g., 2.4±0.4 kPa at 1 Hz to 9.7±0.2 kPa at 316 Hz at day 21, mean±SEM), and phase angle, δ, (e.g., 15±2° at 1 Hz to 74±1° at 316 Hz at day 21) with increasing frequency were attributed to the fluid flow induced poroelasticity, governed by both the newly synthesized matrix and the intracellular structures. The absence of culture duration dependence suggested that chondrogenesis of BMSCs had not yet resulted in the formation of a well-organized matrix with a hierarchical structure similar to cartilage. BMSC-matrix composites demonstrated different poro-viscoelastic frequency-dependent mechanical behavior and energy dissipation compared to chondrocyte-matrix composites due to differences in matrix molecular constituents, structure and cell properties. This study provides important insights into the design of optimal protocols for tissue-engineered cartilage products using chondrocytes and BMSCs. PMID:25468666

  10. Growth and the Growth Hormone-Insulin Like Growth Factor 1 Axis in Children With Chronic Inflammation: Current Evidence, Gaps in Knowledge, and Future Directions.

    Science.gov (United States)

    Wong, S C; Dobie, R; Altowati, M A; Werther, G A; Farquharson, C; Ahmed, S F

    2016-02-01

    Growth failure is frequently encountered in children with chronic inflammatory conditions like juvenile idiopathic arthritis, inflammatory bowel disease, and cystic fibrosis. Delayed puberty and attenuated pubertal growth spurt are often seen during adolescence. The underlying inflammatory state mediated by proinflammatory cytokines, prolonged use of glucocorticoid, and suboptimal nutrition contribute to growth failure and pubertal abnormalities. These factors can impair growth by their effects on the GH-IGF axis and also directly at the level of the growth plate via alterations in chondrogenesis and local growth factor signaling. Recent studies on the impact of cytokines and glucocorticoid on the growth plate further advanced our understanding of growth failure in chronic disease and provided a biological rationale of growth promotion. Targeting cytokines using biological therapy may lead to improvement of growth in some of these children, but approximately one-third continue to grow slowly. There is increasing evidence that the use of relatively high-dose recombinant human GH may lead to partial catch-up growth in chronic inflammatory conditions, although long-term follow-up data are currently limited. In this review, we comprehensively review the growth abnormalities in children with juvenile idiopathic arthritis, inflammatory bowel disease, and cystic fibrosis, systemic abnormalities of the GH-IGF axis, and growth plate perturbations. We also systematically reviewed all the current published studies of recombinant human GH in these conditions and discussed the role of recombinant human IGF-1. PMID:26720129

  11. β-Catenin Does Not Confer Tumorigenicity When Introduced into Partially Transformed Human Mesenchymal Stem Cells

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    Sajida Piperdi

    2012-01-01

    Full Text Available Although osteosarcoma is the most common primary malignant bone tumor in children and adolescents, its cell of origin and the genetic alterations are unclear. Previous studies have shown that serially introducing hTERT, SV40 large TAg, and H-Ras transforms human mesenchymal stem cells into two distinct sarcomas cell populations, but they do not form osteoid. In this study, β-catenin was introduced into mesenchymal stem cells already containing hTERT and SV40 large TAg to analyze if this resulted in a model which more closely recapitulated osteosarcoma. Results. Regardless of the level of induced β-catenin expression in the stable transfectants, there were no marked differences induced in their phenotype or invasion and migration capacity. Perhaps more importantly, none of them formed tumors when injected into immunocompromised mice. Moreover, the resulting transformed cells could be induced to osteogenic and chondrogenic differentiation but not to adipogenic differentiation. Conclusions. β-catenin, although fostering osteogenic differentiation, does not induce the malignant features and tumorigenicity conveyed by oncogenic H-RAS when introduced into partly transformed mesenchymal stem cells. This may have implications for the role of β-catenin in osteosarcoma pathogenesis. It also may suggest that adipogenesis is an earlier branch point than osteogenesis and chondrogenesis in normal mesenchymal differentiation.

  12. Simulated microgravity alters the expression of key genes involved in fracture healing

    Science.gov (United States)

    McCabe, N. Patrick; Androjna, Caroline; Hill, Esther; Globus, Ruth K.; Midura, Ronald J.

    2013-11-01

    Fracture healing in animal models has been shown to be altered in both ground based analogs of spaceflight and in those exposed to actual spaceflight. The molecular mechanisms behind altered fracture healing as a result of chronic exposure to microgravity remain to be elucidated. This study investigates temporal gene expression of multiple factors involved in secondary fracture healing, specifically those integral to the development of a soft tissue callus and the transition to that of hard tissue. Skeletally mature female rats were subjected to a 4 week period of simulated microgravity and then underwent a closed femoral fracture procedure. Thereafter, they were reintroduced to the microgravity and allowed to heal for a 1 or 2 week period. A synchronous group of weight bearing rats was used as a normal fracture healing control. Utilizing Real-Time quantitative PCR on mRNA from fracture callus tissue, we found significant reductions in the levels of transcripts associated with angiogenesis, chondrogenesis, and osteogenesis. These data suggest an altered fracture healing process in a simulated microgravity environment, and these alterations begin early in the healing process. These findings may provide mechanistic insight towards developing countermeasure protocols to mitigate these adaptations.

  13. Chondrocytes expressing intracellular collagen type II enter the cell cycle and co-express collagen type I in monolayer culture.

    Science.gov (United States)

    Tekari, Adel; Luginbuehl, Reto; Hofstetter, Willy; Egli, Rainer J

    2014-11-01

    For autologous chondrocyte transplantation, articular chondrocytes are harvested from cartilage tissue and expanded in vitro in monolayer culture. We aimed to characterize with a cellular resolution the synthesis of collagen type II (COL2) and collagen type I (COL1) during expansion in order to further understand why these cells lose the potential to form cartilage tissue when re-introduced into a microenvironment that supports chondrogenesis. During expansion for six passages, levels of transcripts encoding COL2 decreased to COL2/COL1-double positive phenotype during expansion, and the COL2 positive cells were able to enter the cell cycle. While the fraction of COL2 positive cells decreased from 70% to 95%. In parallel to the decrease of the fraction of COL2 positive cells, the cells' potential to form cartilage-like tissue in pellet cultures steadily decreased. Intracellular staining for COL2 enables for characterization of chondrocyte lineage cells in more detail with a cellular resolution, and it may allow predicting the effectiveness of expanded chondrocytes to form cartilage-like tissue. PMID:25043137

  14. Sprouty2 regulates endochondral bone formation by modulation of RTK and BMP signaling.

    Science.gov (United States)

    Joo, Adriane; Long, Roger; Cheng, Zhiqiang; Alexander, Courtney; Chang, Wenhan; Klein, Ophir D

    2016-07-01

    Skeletal development is regulated by the coordinated activity of signaling molecules that are both produced locally by cartilage and bone cells and also circulate systemically. During embryonic development and postnatal bone remodeling, receptor tyrosine kinase (RTK) superfamily members play critical roles in the proliferation, survival, and differentiation of chondrocytes, osteoblasts, osteoclasts, and other bone cells. Recently, several molecules that regulate RTK signaling have been identified, including the four members of the Sprouty (Spry) family (Spry1-4). We report that Spry2 plays an important role in regulation of endochondral bone formation. Mice in which the Spry2 gene has been deleted have defective chondrogenesis and endochondral bone formation, with a postnatal decrease in skeletal size and trabecular bone mass. In these constitutive Spry2 mutants, both chondrocytes and osteoblasts undergo increased cell proliferation and impaired terminal differentiation. Tissue-specific Spry2 deletion by either osteoblast- (Col1-Cre) or chondrocyte- (Col2-Cre) specific drivers led to decreased relative bone mass, demonstrating the critical role of Spry2 in both cell types. Molecular analyses of signaling pathways in Spry2(-/-) mice revealed an unexpected upregulation of BMP signaling and decrease in RTK signaling. These results identify Spry2 as a critical regulator of endochondral bone formation that modulates signaling in both osteoblast and chondrocyte lineages. PMID:27130872

  15. A chitosan-hyaluronan-based hydrogel-hydrocolloid supports in vitro culture and differentiation of human mesenchymal stem/stromal cells.

    Science.gov (United States)

    Lindborg, Beth A; Brekke, John H; Scott, Carolyn M; Chai, Yi Wen; Ulrich, Connor; Sandquist, Lee; Kokkoli, Efrosini; O'Brien, Timothy D

    2015-06-01

    Three-dimensional (3D) cell culture platforms are increasingly utilized due to their ability to more closely mimic the in vivo microenvironment compared to traditional two-dimensional methods. Limitations of currently available 3D materials include lack of cell attachment, long polymerization times, and inclusion of undefined xenobiotics, and cytotoxic cross-linkers. Evaluated here is a unique hydrogel comprised of polyelectrolytic complex (PEC) fibers formed by hyaluronic acid and chitosan (CT). When hydrated with fetal bovine serum containing human mesenchymal stem/stromal cells (hMSCs), a hydrogel with an elastic modulus of 264±38 Pa formed in seconds with cells distributed throughout the matrix. Scanning electron microscopy showed a lattice-like meshwork of PEC fibers forming irregular compartments. hMSCs showed 48% viability during the first 24 h, with cell populations thereafter reaching a steady state for 14 days. hMSCs in the matrix were induced to differentiate to chondrogenic, osteogenic, and adipogenic phenotypes. Emergent features, at days 56 and 70, consisted of chondrogenesis on the surface of hydrogels induced to osteogenic and adipogenic phenotypes. Results indicate that this matrix may be useful for tissue engineering and disease modeling applications. PMID:25748146

  16. 3D printing of composite tissue with complex shape applied to ear regeneration

    International Nuclear Information System (INIS)

    In the ear reconstruction field, tissue engineering enabling the regeneration of the ear's own tissue has been considered to be a promising technology. However, the ear is known to be difficult to regenerate using traditional methods due to its complex shape and composition. In this study, we used three-dimensional (3D) printing technology including a sacrificial layer process to regenerate both the auricular cartilage and fat tissue. The main part was printed with poly-caprolactone (PCL) and cell-laden hydrogel. At the same time, poly-ethylene-glycol (PEG) was also deposited as a sacrificial layer to support the main structure. After complete fabrication, PEG can be easily removed in aqueous solutions, and the procedure for removing PEG has no effect on the cell viability. For fabricating composite tissue, chondrocytes and adipocytes differentiated from adipose-derived stromal cells were encapsulated in hydrogel to dispense into the cartilage and fat regions, respectively, of ear-shaped structures. Finally, we fabricated the composite structure for feasibility testing, satisfying expectations for both the geometry and anatomy of the native ear. We also carried out in vitro assays for evaluating the chondrogenesis and adipogenesis of the cell-printed structure. As a result, the possibility of ear regeneration using 3D printing technology which allowed tissue formation from the separately printed chondrocytes and adipocytes was demonstrated. (paper)

  17. Tissue-engineering strategies to repair joint tissue in osteoarthritis: nonviral gene-transfer approaches.

    Science.gov (United States)

    Madry, Henning; Cucchiarini, Magali

    2014-10-01

    Loss of articular cartilage is a common clinical consequence of osteoarthritis (OA). In the past decade, substantial progress in tissue engineering, nonviral gene transfer, and cell transplantation have provided the scientific foundation for generating cartilaginous constructs from genetically modified cells. Combining tissue engineering with overexpression of therapeutic genes enables immediate filling of a cartilage defect with an engineered construct that actively supports chondrogenesis. Several pioneering studies have proved that spatially defined nonviral overexpression of growth-factor genes in constructs of solid biomaterials or hydrogels is advantageous compared with gene transfer or scaffold alone, both in vitro and in vivo. Notably, these investigations were performed in models of focal cartilage defects, because advanced cartilage-repair strategies based on the principles of tissue engineering have not advanced sufficiently to enable resurfacing of extensively degraded cartilage as therapy for OA. These studies serve as prototypes for future technological developments, because they raise the possibility that cartilage constructs engineered from genetically modified chondrocytes providing autocrine and paracrine stimuli could similarly compensate for the loss of articular cartilage in OA. Because cartilage-tissue-engineering strategies are already used in the clinic, combining tissue engineering and nonviral gene transfer could prove a powerful approach to treat OA.

  18. Genome-wide association scan suggests basis for microtia in Awassi sheep.

    Science.gov (United States)

    Jawasreh, K; Boettcher, P J; Stella, A

    2016-08-01

    Hereditary underdevelopment of the ear, a condition also known as microtia, has been observed in several sheep breeds as well as in humans and other species. Its genetic basis in sheep is unknown. The Awassi sheep, a breed native to southwest Asia, carries this phenotype and was targeted for molecular characterization via a genome-wide association study. DNA samples were collected from sheep in Jordan. Eight affected and 12 normal individuals were genotyped with the Illumina OvineSNP50(®) chip. Multilocus analyses failed to identify any genotypic association. In contrast, a single-locus analysis revealed a statistically significant association (P = 0.012, genome-wide) with a SNP at basepair 34 647 499 on OAR23. This marker is adjacent to the gene encoding transcription factor GATA-6, which has been shown to play a role in many developmental processes, including chondrogenesis. The lack of extended homozygosity in this region suggests a fairly ancient mutation, and the time of occurrence was estimated to be approximately 3000 years ago. Many of the earless sheep breeds may thus share the causative mutation, especially within the subgroup of fat-tailed, wool sheep. PMID:26990958

  19. Stem cell application for osteoarthritis in the knee joint: A minireview

    Institute of Scientific and Technical Information of China (English)

    Kristin; Uth; Dimitar; Trifonov

    2014-01-01

    Knee osteoarthritis is a chronic, indolent disease that will affect an ever increasing number of patients, especially the elderly and the obese. It is characterized by degeneration of the cartilage substance inside the knee which leads to pain, stiffness and tenderness. By some estimations in 2030, only in the United States, this medical condition will burden 67 million people. While conventional treatments like physiotherapy or drugs offer temporary relief of clinical symptoms, restoration of normal cartilage function has been difficult to achieve. Moreover, in severe cases of knee osteoarthritis total knee replacement may be required. Total knee replacements come together with high effort and costs and are not always successful. The aim of this review is to outline the latest advances in stem cell therapy for knee osteoarthritis as well as highlight some of the advantages of stem cell therapy over traditional approaches aimed at restoration of cartilage function in the knee. In addition to the latest advances in the field, challenges associated with stem cell therapy regarding knee cartilage regeneration and chondrogenesis in vitro and in vivo are also outlined and analyzed. Furthermore, based on their critical assessment of the present academic literature the authors of this review share their vision about the future of stem cell applications in the treatment of knee osteoarthritis.

  20. Stem cell application for osteoarthritis in the knee joint: A minireview.

    Science.gov (United States)

    Uth, Kristin; Trifonov, Dimitar

    2014-11-26

    Knee osteoarthritis is a chronic, indolent disease that will affect an ever increasing number of patients, especially the elderly and the obese. It is characterized by degeneration of the cartilage substance inside the knee which leads to pain, stiffness and tenderness. By some estimations in 2030, only in the United States, this medical condition will burden 67 million people. While conventional treatments like physiotherapy or drugs offer temporary relief of clinical symptoms, restoration of normal cartilage function has been difficult to achieve. Moreover, in severe cases of knee osteoarthritis total knee replacement may be required. Total knee replacements come together with high effort and costs and are not always successful. The aim of this review is to outline the latest advances in stem cell therapy for knee osteoarthritis as well as highlight some of the advantages of stem cell therapy over traditional approaches aimed at restoration of cartilage function in the knee. In addition to the latest advances in the field, challenges associated with stem cell therapy regarding knee cartilage regeneration and chondrogenesis in vitro and in vivo are also outlined and analyzed. Furthermore, based on their critical assessment of the present academic literature the authors of this review share their vision about the future of stem cell applications in the treatment of knee osteoarthritis.

  1. Zebrafish Fins as a Model System for Skeletal Human Studies

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    Manuel Marí-Beffa

    2007-01-01

    Full Text Available Recent studies on the morphogenesis of the fins of Danio rerio (zebrafish during development and regeneration suggest that a number of inductive signals involved in the process are similar to some of those that affect bone and cartilage differentiation in mammals and humans. Akimenko et al. (2002 has shown that bone morphogenetic protein-2b (BMP2b is involved in the induction of dermal bone differentiation during fin regeneration. Many other groups have also shown that molecules from the transforming growth factor-beta superfamily (TGFβ, including BMP2, are effective in promoting chondrogenesis and osteogenesis in vivo in higher vertebrates, including humans. In the present study, we review the state of the art of this topic by a comparative analysis of skeletal tissue development, regeneration and renewal processes in tetrapods, and fin regeneration in fishes. A general conclusion of this study states that lepidotrichia is a special skeletal tissue different to cartilage, bone, enamel, or dentine in fishes, according to its extracellular matrix (ECM composition. However, the empirical analysis of inducing signals of skeletal tissues in fishes and tetrapods suggests that lepidotrichia is different to any responding features with main skeletal tissues. A number of new inductive molecules are arising from fin development and regeneration studies that might establish an empirical basis for further molecular approaches to mammal skeletal tissues differentiation. Despite the tissue dissimilarity, this empirical evidence might finally lead to clinical applications to skeletal disorders in humans.

  2. A Direct Sulfation Process of a Marine Polysaccharide in Ionic Liquid

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    Nathalie Chopin

    2015-01-01

    Full Text Available GY785 is an exopolysaccharide produced by a mesophilic bacterial strain Alteromonas infernus discovered in the deep-sea hydrothermal vents. GY785 highly sulfated derivative (GY785 DRS was previously demonstrated to be a promising molecule driving the efficient mesenchymal stem cell chondrogenesis for cartilage repair. This glycosaminoglycan- (GAG- like compound was modified in a classical solvent (N,N′-dimethylformamide. However, the use of classical solvents limits the polysaccharide solubility and causes the backbone degradation. In the present study, a one-step efficient sulfation process devoid of side effects (e.g., polysaccharide depolymerization and/or degradation was developed to produce GAG-like derivatives. The sulfation of GY785 derivative (GY785 DR was carried out using ionic liquid as a reaction medium. The successful sulfation of this anionic and highly branched heteropolysaccharide performed in ionic liquid would facilitate the production of new molecules of high specificity for biological targets such as tissue engineering or regenerative medicine.

  3. Induction of systemic bone changes by preconditioning total body irradiation for bone marrow transplantation

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    Miyazaki, Osamu; Okamoto, Reiko; Masaki, Hidekazu [National Centre for Child Health and Development, Department of Radiology, Tokyo (Japan); Nishimura, Gen [Tokyo Metropolitan Kiyose Children' s Hospital, Department of Radiology, Tokyo (Japan); Kumagai, Masaaki; Shioda, Yoko [National Centre for Child Health and Development, Department of Oncology, Tokyo (Japan); Nozawa, Kumiko [Saitama Children' s Medical Centre, Department of Radiology, Saitama (Japan); Kitoh, Hiroshi [Nagoya University Hospital, Department of Orthopaedic Surgery, Nagoya, Aichi (Japan)

    2009-01-15

    Preconditioning total body irradiation (TBI) prior to bone marrow transplantation (BMT) has been believed to be a safe procedure that does not cause late morbidity; yet, a recent report raises the suspicion that TBI-induced chondroosseous abnormalities do occur. To evaluate the radiological manifestations of TBI-induced skeletal alterations and their orthopaedic morbidity. Subjects included 11 children with TBI-induced skeletal changes, including 9 in our hospital and 2 in other hospitals. The former were selected from 53 children who had undergone TBI with BMT. Radiographic examinations (n=11), MRI (n=3), CT (n=2), and medical records in the 11 children were retrospectively reviewed. The skeletal alterations included abnormal epiphyseal ossification and metaphyseal fraying (8/11), longitudinal metaphyseal striations (8/11), irregular metaphyseal sclerosis (6/11), osteochondromas (4/11), slipped capital femoral epiphysis (2/10), genu valgum (3/10), and platyspondyly (2/3). MRI demonstrated immature primary spongiosa in the metaphysis. Of the 11 children, 9 had clinical symptoms. TBI can induce polyostotic and/or generalized bone changes, mainly affecting the epiphyseal/metaphyseal regions and occasionally the spine. The epi-/metaphyseal abnormalities represent impaired chondrogenesis in the epiphysis and growth plate and abnormal remodelling in the metaphysis. Generalized spine changes may lead to misdiagnosis of a skeletal dysplasia. (orig.)

  4. Genome-wide association scan suggests basis for microtia in Awassi sheep.

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    Jawasreh, K; Boettcher, P J; Stella, A

    2016-08-01

    Hereditary underdevelopment of the ear, a condition also known as microtia, has been observed in several sheep breeds as well as in humans and other species. Its genetic basis in sheep is unknown. The Awassi sheep, a breed native to southwest Asia, carries this phenotype and was targeted for molecular characterization via a genome-wide association study. DNA samples were collected from sheep in Jordan. Eight affected and 12 normal individuals were genotyped with the Illumina OvineSNP50(®) chip. Multilocus analyses failed to identify any genotypic association. In contrast, a single-locus analysis revealed a statistically significant association (P = 0.012, genome-wide) with a SNP at basepair 34 647 499 on OAR23. This marker is adjacent to the gene encoding transcription factor GATA-6, which has been shown to play a role in many developmental processes, including chondrogenesis. The lack of extended homozygosity in this region suggests a fairly ancient mutation, and the time of occurrence was estimated to be approximately 3000 years ago. Many of the earless sheep breeds may thus share the causative mutation, especially within the subgroup of fat-tailed, wool sheep.

  5. Impact of tissue-specific stem cells on lineage-specific differentiation: a focus on the musculoskeletal system.

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    Pizzute, Tyler; Lynch, Kevin; Pei, Ming

    2015-02-01

    Tissue-specific stem cells are found throughout the body and, with proper intervention and environmental cues, these stem cells exercise their capabilities for differentiation into several lineages to form cartilage, bone, muscle, and adipose tissue in vitro and in vivo. Interestingly, it has been widely demonstrated that they do not differentiate with the same efficacy during lineage-specific differentiation studies, as the tissue-specific stem cells are generally more effective when differentiating toward the tissues from which they were derived. This review focuses on four mesodermal lineages for tissue-specific stem cell differentiation: adipogenesis, chondrogenesis, myogenesis, and osteogenesis. It is intended to give insight into current multilineage differentiation and comparative research, highlight and contrast known trends regarding differentiation, and introduce supporting evidence which demonstrates particular tissue-specific stem cells' superiority in lineage-specific differentiation, along with their resident tissue origins and natural roles. In addition, some epigenetic and transcriptomic differences between stem cells which may explain the observed trends are discussed.

  6. Reduced reactivation from dormancy but maintained lineage choice of human mesenchymal stem cells with donor age.

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    Verena Dexheimer

    Full Text Available UNLABELLED: Mesenchymal stem cells (MSC are promising for cell-based regeneration therapies but up to date it is still controversial whether their function is maintained throughout ageing. Aim of this study was to address whether frequency, activation in vitro, replicative function, and in vitro lineage choice of MSC is maintained throughout ageing to answer the question whether MSC-based regeneration strategies should be restricted to younger individuals. MSC from bone marrow aspirates of 28 donors (5-80 years were characterized regarding colony-forming unit-fibroblast (CFU-F numbers, single cell cloning efficiency (SSCE, osteogenic, adipogenic and chondrogenic differentiation capacity in vitro. Alkaline phosphatase (ALP activity, mineralization, Oil Red O content, proteoglycan- and collagen type II deposition were quantified. While CFU-F frequency was maintained, SSCE and early proliferation rate decreased significantly with advanced donor age. MSC with higher proliferation rate before start of induction showed stronger osteogenic, adipogenic and chondrogenic differentiation. MSC with high osteogenic capacity underwent better chondrogenesis and showed a trend to better adipogenesis. Lineage choice was, however, unaltered with age. CONCLUSION: Ageing influenced activation from dormancy and replicative function of MSC in a way that it may be more demanding to mobilize MSC to fast cell growth at advanced age. Since fast proliferation came along with high multilineage capacity, the proliferation status of expanded MSC rather than donor age may provide an argument to restrict MSC-based therapies to certain individuals.

  7. Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System

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    Christian Bucher

    2013-01-01

    Full Text Available Intervertebral disc (IVD cell therapy with unconditioned 2D expanded mesenchymal stem cells (MSC is a promising concept yet challenging to realize. Differentiation of MSCs by nonviral gene delivery of growth and differentiation factor 5 (GDF5 by electroporation mediated gene transfer could be an excellent source for cell transplantation. Human MSCs were harvested from bone marrow aspirate and GDF5 gene transfer was achieved by in vitro electroporation. Transfected cells were cultured as monolayers and as 3D cultures in 1.2% alginate bead culture. MSC expressed GDF5 efficiently for up to 21 days. The combination of GDF5 gene transfer and 3D culture in alginate showed an upregulation of aggrecan and SOX9, two markers for chondrogenesis, and KRT19 as a marker for discogenesis compared to untransfected cells. The cells encapsulated in alginate produced more proteoglycans expressed in GAG/DNA ratio. Furthermore, GDF5 transfected MCS injected into an IVD papain degeneration organ culture model showed a partial recovery of the GAG/DNA ratio after 7 days. In this study we demonstrate the potential of GDF5 transfected MSC as a promising approach for clinical translation for disc regeneration.

  8. 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.

  9. Identification of a novel population of human cord blood cells with hematopoietic and chondrocytic potential

    Institute of Scientific and Technical Information of China (English)

    Karen E JAY; Anne ROULEAU; T Michael UNDERHILL; Mickie BHATIA

    2004-01-01

    With the exception of mature erythrocytes, cells within the human hematopoietic system are characterized by the cell surface expression of the pan-leukocyte receptor CD45. Here, we identify a novel subset among mononuclear cord blood cells depleted of lineage commitment markers (Lin-) that are devoid of CD45 expression. Surprisingly, functional examination of Lin-CD45- cells also lacking cell surface CD34 revealed they were capable of multipotential hematopoietic progenitor capacity. Co-culture with mouse embryonic limb bud cells demonstrated that Lin-CD45-CD34- cells were capable of contributing to cartilage nodules and differentiating into human chondrocytes. BMP-4, a mesodermal factor known to promote chondrogenesis, significantly augmented Lin-CD45-CD34- differentiation into chondrocytes.Moreover, unlike CD34+ human hematopoietic stem cells, Lin-CD45-CD34- cells were unable to proliferate or survive in liquid cultures, whereas single Lin-CD45-CD34- cells were able to chimerize the inner cell mass (ICM) of murine blastocysts and proliferate in this embryonic environment. Our study identifies a novel population of Lin-CD45-CD34-cells capable of commitment into both hematopoietic and chondrocytic lineages, suggesting that human cord blood may provide a more ubiquitous source of tissue with broader developmental potential than previously appreciated.

  10. Effects of Eucommia ulmoides extract on longitudinal bone growth rate in adolescent female rats.

    Science.gov (United States)

    Kim, Ji Young; Lee, Jeong-Il; Song, MiKyung; Lee, Donghun; Song, Jungbin; Kim, Soo Young; Park, Juyeon; Choi, Ho-Young; Kim, Hocheol

    2015-01-01

    Eucommia ulmoides is one of the popular tonic herbs for the treatment of low back pain and bone fracture and is used in Korean medicine to reinforce muscles and bones. This study was performed to investigate the effects of E. ulmoides extract on longitudinal bone growth rate, growth plate height, and the expressions of bone morphogenetic protein 2 (BMP-2) and insulin-like growth factor 1 (IGF-1) in adolescent female rats. In two groups, we administered a twice-daily dosage of E. ulmoides extract (at 30 and 100 mg/kg, respectively) per os over 4 days, and in a control group, we administered vehicle only under the same conditions. Longitudinal bone growth rate in newly synthesized bone was observed using tetracycline labeling. Chondrocyte proliferation in the growth plate was observed using cresyl violet dye. In addition, we analyzed the expressions of BMP-2 and IGF-1 using immunohistochemistry. Eucommia ulmoides extract significantly increased longitudinal bone growth rate and growth plate height in adolescent female rats. In the immunohistochemical study, E. ulmoides markedly increased BMP-2 and IGF-1 expressions in the proliferative and hypertrophic zones. In conclusion, E. ulmoides increased longitudinal bone growth rate by promoting chondrogenesis in the growth plate and the levels of BMP-2 and IGF-1. Eucommia ulmoides could be helpful for increasing bone growth in children who have growth retardation. PMID:25087723

  11. Changes in the extracellular matrix and glycosaminoglycan synthesis during the initiation of regeneration in adult newt forelimbs

    Energy Technology Data Exchange (ETDEWEB)

    Mescher, A.L.; Munaim, S.I.

    1986-04-01

    The extracellular matrix (ECM) of the distal tissues in a newt limb stump is completely reorganized in the 2-3-week period following amputation. In view of numerous in vitro studies showing that extracellular material influences cellular migration and proliferation, it is likely that the changes in the limb's ECM are important activities in the process leading to regeneration of such limbs. Using biochemical, autoradiographic, and histochemical techniques we studied temporal and spatial differences in the synthesis of glycosaminoglycans (GAGs) during the early, nerve-dependent phase of limb regeneration. Hyaluronic acid synthesis began with the onset of tissue dedifferentiation, became maximal within 1 weeks, and continued throughout the period of active cell proliferation. Chondroitin sulfate synthesis began somewhat later, increased steadily, and reached very high levels during chondrogenesis. During the first 10 days after amputation, distributions of sulfated and nonsulfated GAGs were both uniform throughout dedifferentiating tissues, except for a heavier localization near the bone. Since nerves are necessary to promote the regenerative process, we examined the neural influence on synthesis and accumulation of extracellular GAGs. Denervation decreased GAG production in all parts of the limb stump by approximately 50%. Newt dorsal root ganglia and brain-derived fibroblast growth factor each produced twofold stimulation of GAG synthesis in cultured 7-day regenerates. The latter effect was primarily on synthesis of hyaluronic acid. The results indicate that the trophic action of nerves on amphibian limb regeneration includes a positive influence on synthesis and extracellular accumulation of GAGs.

  12. DEC2 is a negative regulator for the proliferation and differentiation of chondrocyte lineage-committed mesenchymal stem cells.

    Science.gov (United States)

    Sasamoto, Tomoko; Fujimoto, Katsumi; Kanawa, Masami; Kimura, Junko; Takeuchi, Junpei; Harada, Naoko; Goto, Noriko; Kawamoto, Takeshi; Noshiro, Mitsuhide; Suardita, Ketut; Tanne, Kazuo; Kato, Yukio

    2016-09-01

    Differentiated embryo chondrocyte 2 (DEC2) is a basic helix-loop-helix-Orange transcription factor that regulates cell differentiation in various mammalian tissues. DEC2 has been shown to suppress the differentiation of mesenchymal stem cells (MSCs) into myocytes and adipocytes. In the present study, we examined the role of DEC2 in the chondrogenic differentiation of human MSCs. The overexpression of DEC2 exerted minimal effects on the proliferation of MSCs in monolayer cultures with the growth medium under undifferentiating conditions, whereas it suppressed increases in DNA content, glycosaminoglycan content, and the expression of several chondrocyte-related genes, including aggrecan and type X collagen alpha 1, in MSC pellets in centrifuge tubes under chondrogenic conditions. In the pellets exposed to chondrogenesis induction medium, DEC2 overexpression downregulated the mRNA expression of fibroblast growth factor 18, which is involved in the proliferation and differentiation of chondrocytes, and upregulated the expression of p16INK4, which is a cell cycle inhibitor. These findings suggest that DEC2 is a negative regulator of the proliferation and differentiation of chondrocyte lineage-committed mesenchymal cells. PMID:27430159

  13. Derivation of mesenchymal stromal cells from canine induced pluripotent stem cells by inhibition of the TGFβ/activin signaling pathway.

    Science.gov (United States)

    Whitworth, Deanne J; Frith, Jessica E; Frith, Thomas J R; Ovchinnikov, Dmitry A; Cooper-White, Justin J; Wolvetang, Ernst J

    2014-12-15

    In this study we have generated canine mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, from canine induced pluripotent stem cells (ciPSCs) by small-molecule inhibition of the transforming growth factor beta (TGFβ)/activin signaling pathway. These ciPSC-derived MSCs (ciPSC-MSCs) express the MSC markers CD73, CD90, CD105, STRO1, cPDGFRβ and cKDR, in addition to the pluripotency factors OCT4, NANOG and REX1. ciPSC-MSCs lack immunostaining for H3K27me3, suggesting that they possess two active X chromosomes. ciPSC-MSCs are highly proliferative and undergo robust differentiation along the osteo-, chondro- and adipogenic pathways, but do not form teratoma-like tissues in vitro. Of further significance for the translational potential of ciPSC-MSCs, we show that these cells can be encapsulated and maintained within injectable hydrogel matrices that, when functionalized with bound pentosan polysulfate, dramatically enhance chondrogenesis and inhibit osteogenesis. The ability to efficiently derive large numbers of highly proliferative canine MSCs from ciPSCs that can be incorporated into injectable, functionalized hydrogels that enhance their differentiation along a desired lineage constitutes an important milestone towards developing an effective MSC-based therapy for osteoarthritis in dogs, but equally provides a model system for assessing the efficacy and safety of analogous approaches for treating human degenerative joint diseases. PMID:25055193

  14. Direct influence of culture dimensionality on human mesenchymal stem cell differentiation at various matrix stiffnesses using a fibrous self-assembling peptide hydrogel.

    Science.gov (United States)

    Hogrebe, Nathaniel J; Gooch, Keith J

    2016-09-01

    Much is unknown about the effects of culture dimensionality on cell behavior due to the lack of biomimetic substrates that are suitable for directly comparing cells grown on two-dimensional (2D) and encapsulated within three-dimensional (3D) matrices of the same stiffness and biochemistry. To overcome this limitation, we used a self-assembling peptide hydrogel system that has tunable stiffness and cell-binding site density as well as a fibrous microarchitecture resembling the structure of collagen. We investigated the effect of culture dimensionality on human mesenchymal stem cell differentiation at different values of matrix stiffness (G' = 0.25, 1.25, 5, and 10 kPa) and a constant RGD (Arg-Gly-Asp) binding site concentration. In the presence of the same soluble induction factors, culture on top of stiff gels facilitated the most efficient osteogenesis, while encapsulation within the same stiff gels resulted in a switch to predominantly terminal chondrogenesis. Adipogenesis dominated at soft conditions, and 3D culture induced better adipogenic differentiation than 2D culture at a given stiffness. Interestingly, initial matrix-induced cell morphology was predictive of these end phenotypes. Furthermore, optimal culture conditions corresponded to each cell type's natural niche within the body, highlighting the importance of incorporating native matrix dimensionality and stiffness into tissue engineering strategies. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2356-2368, 2016. PMID:27163888

  15. PTH stimulated growth and decreased Col-X deposition are phosphotidylinositol-3,4,5 triphosphate kinase and mitogen activating protein kinase dependent in avian sterna.

    Science.gov (United States)

    Harrington, Erik Kern; Coon, David J; Kern, Matthew F; Svoboda, Kathy K H

    2010-02-01

    Type X collagen (Col-X) deposition is a marker of terminal differentiation during chondrogenesis, in addition to appositional growth and apoptosis. The parathyroid hormone/parathyroid hormone related peptide (PTH/PTHrP) receptor, or PPR, is a G-Protein coupled receptor (GPCR), which activates several downstream pathways, moderating chondrocyte differentiation, including suppression of Col-X deposition. An Avian sterna model was used to analyze the PPR GPCR downstream kinase role in growth rate and extracellular matrix (ECM) including Col-II, IX, and X. Phosphatidylinositol kinase (PI3K), mitogen activating protein kinase (MAPK) and protein kinase A (PKA) were inhibited with specific established inhibitors LY294002, PD98059, and H89, respectively to test the hypothesis that they could reverse/inhibit the PTH/PTHrP pathway. Excised E14 chick sterna were PTH treated with or without an inhibitor and compared to controls. Sternal length was measured every 24 hr. Cultured sterna were immuno-stained using specific antibodies for Col-II, IX, or X and examined via confocal microscopy. Increased growth in PTH-treated sterna was MAPK, PI3K, and PKA dose dependent, suggesting growth was regulated through multiple pathways. Col-X deposition was rescued in PTH-treated sterna in the presence of PI3K or MAPK inhibitors, but not with the PKA inhibitor. All three inhibitors moderately disrupted Col-II and Col-IX deposition. These results suggest that PTH can activate multiple pathways during chondrocyte differentiation.

  16. Fibroblast-Like Synoviocytes Induce Calcium Mineral Formation and Deposition

    Directory of Open Access Journals (Sweden)

    Yubo Sun

    2014-01-01

    Full Text Available Calcium crystals are present in the synovial fluid of 65%–100% patients with osteoarthritis (OA and 20%–39% patients with rheumatoid arthritis (RA. This study sought to investigate the role of fibroblast-like synoviocytes (FLSs in calcium mineral formation. We found that numerous genes classified in the biomineral formation process, including bone gamma-carboxyglutamate (gla protein/osteocalcin, runt-related transcription factor 2, ankylosis progressive homolog, and parathyroid hormone-like hormone, were differentially expressed in the OA and RA FLSs. Calcium deposits were detected in FLSs cultured in regular medium in the presence of ATP and FLSs cultured in chondrogenesis medium in the absence of ATP. More calcium minerals were deposited in the cultures of OA FLSs than in the cultures of RA FLSs. Examination of the micromass stained with nonaqueous alcoholic eosin indicated the presence of birefringent crystals. Phosphocitrate inhibited the OA FLSs-mediated calcium mineral deposition. These findings together suggest that OA FLSs are not passive bystanders but are active players in the pathological calcification process occurring in OA and that potential calcification stimuli for OA FLSs-mediated calcium deposition include ATP and certain unidentified differentiation-inducing factor(s. The OA FLSs-mediated pathological calcification process is a valid target for the development of disease-modifying drug for OA therapy.

  17. Mammalian limb loading and chondral modeling during ontogeny.

    Science.gov (United States)

    Hammond, Ashley S; Ning, Jie; Ward, Carol V; Ravosa, Matthew J

    2010-04-01

    The adaptive growth response of cartilage, or chondral modeling, can result in changes in joint and limb proportions during ontogeny and ultimately contribute to the adult form. Despite Hamrick's (1999) reevaluation of the mechanisms of chondral modeling, the process of chondral modeling remains poorly studied in animal models. Here, we characterize the macro- and microanatomical responses of the femoral growth plate, articular cartilage, and bone in 15 juvenile Sus scrofa domestica subjected to different locomotor activity patterns. The exercised animals exhibit thinner cartilage zones, greater cellularity and larger proliferative chondrocyte areas in the growth plate, as well as larger femoral dimensions and a more elongate femoral head compared with sedentary controls. In general, the growth plate demonstrates greater adaptive changes than articular cartilage. Moreover, chondrocyte hypertrophy and proliferation were found to be responsive to locomotor loading and thus more important factors in chondral modeling than the extracellular matrix variables that were examined herein. In sum, the underlying mechanisms of adaptive chondrogenesis and bone plasticity are key to informing evolutionary and translational studies regarding determinants of variation in joint form and function. Given the disparity between the predictions of chondral modeling theory and our experimental findings, this suggests a need for further evaluation of chondral modeling responses during ontogeny.

  18. On the development of Cetacean extremities: II. Morphogenesis and histogenesis of the flippers in the spotted dolphin (Stenella attenuata).

    Science.gov (United States)

    Sedmera, D; Misek, I; Klima, M

    1997-04-01

    Externally, the flippers of Cetacea resemble fish fins, but their internal structure is entirely mammalian. They show, however, some adaptative deviations from the typical pattern of the mammalian extremities, the most striking of which is an increased number of phalanges. The aim of this study is to describe the course of the development of flippers in the spotted dolphin (Stenella attenuata) and compare its features with other similar species from an evolutionary perspective. Early stages of flipper development were studied histologically. Differentiation of cartilaginous anlagens of the skeleton progresses proximodistally, condensation in digital rays being evident sooner than chondrogenesis in the carpal region. In one specimen, the temporary presence of cartilaginous rudiments of two carpal elements, which are not found in adults, was observed. At all examined stages, phalangeal number progressively increases up to (radial to ulnar) 3, 7, 7, 5, 3 in the most advanced stage. The reason for this condition is the specialised function of these limb-like structures. It is a classical example of convergence, in which mammalian extremities change their form to emulate the fin function. A similar condition is found in another group of originally terrestrial animals secondarily fully adapted to the aquatic mode of life-Ichyosauria (Reptilia). PMID:9253589

  19. ELK3 Expression Correlates With Cell Migration, Invasion, and Membrane Type 1-Matrix Metalloproteinase Expression in MDA-MB-231 Breast Cancer Cells.

    Science.gov (United States)

    Heo, Sun-Hee; Lee, Je-Yong; Yang, Kyung-Min; Park, Kyung-Soon

    2015-01-01

    ELK3 is a member of the Ets family of transcription factors. Its expression is associated with angiogenesis, vasculogenesis, and chondrogenesis. ELK3 inhibits endothelial migration and tube formation through the regulation of MT1-MMP transcription. This study assessed the function of ELK3 in breast cancer (BC) cells by comparing its expression between basal and luminal cells in silico and in vitro. In silico analysis showed that ELK3 expression was higher in the more aggressive basal BC cells than in luminal BC cells. Similarly, in vitro analysis showed that ELK3 mRNA and protein expression was higher in basal BC cells than in normal cells and luminal BC cells. To investigate whether ELK3 regulates basal cell migration or invasion, knockdown was achieved by siRNA in the basal BC cell line MDA-MB-231. Inhibition of ELK3 expression decreased cell migration and invasion and downregulated MT1-MMP, the expression of which is positively correlated with tumor cell invasion. In silico analysis revealed that ELK3 expression was associated with that of MT1-MMP in several BC cell lines (0.98 Pearson correlation coefficient). Though MT1-MMP expression was upregulated upon ELK3 nuclear translocation, ELK3 did not directly bind to the 1.3-kb promoter region of the MT1-MMP gene. These results suggest that ELK3 plays a positive role in the metastasis of BC cells by indirectly regulating MT1-MMP expression. PMID:26637400

  20. Matricellular proteins of the Cyr61/CTGF/NOV (CCN family and the nervous system

    Directory of Open Access Journals (Sweden)

    Anna R Malik

    2015-06-01

    Full Text Available Matricellular proteins are secreted proteins that exist at the border of cells and the extracellular matrix (ECM. However, instead of playing a role in structural integrity of the ECM, these proteins, that act as modulators of various surface receptors, have a regulatory function and instruct a multitude of cellular responses. Among matricellular proteins are members of the Cyr61/CTGF/NOV (CCN protein family. These proteins exert their activity by binding directly to integrins and heparan sulfate proteoglycans and activating multiple intracellular signaling pathways. CCN proteins also influence the activity of growth factors and cytokines and integrate their activity with integrin signaling. At the cellular level, CCN proteins regulate gene expression and cell survival, proliferation, differentiation, senescence, adhesion, and migration. To date, CCN proteins have been extensively studied in the context of osteo- and chondrogenesis, angiogenesis, and carcinogenesis, but the expression of these proteins is also observed in a variety of tissues. The role of CCN proteins in the nervous system has not been systematically studied or described. Thus, the major aim of this review is to introduce the CCN protein family to the neuroscience community. We first discuss the structure, interactions, and cellular functions of CCN proteins and then provide a detailed review of the available data on the neuronal expression and contribution of CCN proteins to nervous system development, function, and pathology.

  1. Up-to-date Review and Cases Report on Chondral Defects of Knee Treated by ACI Technique: Clinical-instrumental and Histological Results.

    Science.gov (United States)

    Dell'Osso, Giacomo; Ghilardi, Marco; Bottai, Vanna; Bugelli, Giulia; Guido, Giulio; Giannotti, Stefano

    2015-05-01

    The limited regenerative potential of a full thickness defect of the knee joint cartilage has certainly conditioned the development of therapeutic strategies that take into account all the aspects of the healing process. The most common treatments to repair chondral and osteochondral lesions are bone marrow stimulation, osteochondral autograft transplantation, autologous matrix-induced chondrogenesis, and autologous chondrocyte implantation. We like to emphasize the difference between a chondral and an osteochondral lesion because the difference is sometimes lost in the literature. In the context of treatment of injuries of the knee joint cartilage, the second-generation autologous chondrocyte transplant is a consolidated surgical method alternative to other techniques. Our experience with the transplantation of chondrocytes has had exceptional clinical results. We report 2 complete cases of a group of 22 in knee and ankle. These 2 cases had histological and instrumental evaluation. We cannot express conclusions, but can only make considerations, stating that, with the clinical functional result being equal, we obtained an excellent macroscopic result in both cases of second look. Autologous chondrocyte implantation (ACI) is a multiple surgical procedure with expensive chondrocyte culture, but even with this limitation, we think that it must be the choice in treating chondral lesions, especially in young patients. PMID:26055026

  2. Direct influence of culture dimensionality on human mesenchymal stem cell differentiation at various matrix stiffnesses using a fibrous self-assembling peptide hydrogel.

    Science.gov (United States)

    Hogrebe, Nathaniel J; Gooch, Keith J

    2016-09-01

    Much is unknown about the effects of culture dimensionality on cell behavior due to the lack of biomimetic substrates that are suitable for directly comparing cells grown on two-dimensional (2D) and encapsulated within three-dimensional (3D) matrices of the same stiffness and biochemistry. To overcome this limitation, we used a self-assembling peptide hydrogel system that has tunable stiffness and cell-binding site density as well as a fibrous microarchitecture resembling the structure of collagen. We investigated the effect of culture dimensionality on human mesenchymal stem cell differentiation at different values of matrix stiffness (G' = 0.25, 1.25, 5, and 10 kPa) and a constant RGD (Arg-Gly-Asp) binding site concentration. In the presence of the same soluble induction factors, culture on top of stiff gels facilitated the most efficient osteogenesis, while encapsulation within the same stiff gels resulted in a switch to predominantly terminal chondrogenesis. Adipogenesis dominated at soft conditions, and 3D culture induced better adipogenic differentiation than 2D culture at a given stiffness. Interestingly, initial matrix-induced cell morphology was predictive of these end phenotypes. Furthermore, optimal culture conditions corresponded to each cell type's natural niche within the body, highlighting the importance of incorporating native matrix dimensionality and stiffness into tissue engineering strategies. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2356-2368, 2016.

  3. Biological Therapies for Cartilage Lesions in the Hip: A New Horizon.

    Science.gov (United States)

    Chahla, Jorge; LaPrade, Robert F; Mardones, Rodrigo; Huard, Johnny; Philippon, Marc J; Nho, Shane; Mei-Dan, Omer; Pascual-Garrido, Cecilia

    2016-07-01

    Treatment of hip cartilage disease is challenging, and there is no clear algorithm to address this entity. Biomarkers are arising as promising diagnostic tools because they could play a role in the early assessment of the prearthritic joint and as a prognostic factor before and after treatment. The potential effect of biomarkers may be used to categorize individuals at risk of evolving to severe osteoarthritis, to develop new measures for clinical progression of the disease, and to develop new treatment options for the prevention of osteoarthritis progression. A trend toward a less invasive biological treatment will usher in a new treatment era. With the growth of surgical skills in hip arthroscopy, cartilage restoration techniques are evolving in a fast and exponential manner. Biological and surgical treatments have been proposed to treat these pathologies. Biological treatments include platelet-rich plasma, stem cells or bone marrow aspirate concentration, hyaluronic acid, losartan, and fish oil. Surgical treatments include microfracture alone or augmented, direct repair, autologous chondrocyte implantation, matrix-induced chondrocyte implantation, autologous matrix-induced chondrogenesis, mosaicplasty, osteochondral allograft transplantation, and stem cells implanted in matrix (stem cells in membranes/expanded stem cells). This article reviews new evidence available on treatment options for chondral lesions and early osteoarthritis of the hip. [Orthopedics. 2016; 39(4):e715-e723.]. PMID:27359284

  4. Articular Cartilage Repair Using Marrow Stimulation Augmented with a Viable Chondral Allograft: 9-Month Postoperative Histological Evaluation

    Directory of Open Access Journals (Sweden)

    James K. Hoffman

    2015-01-01

    Full Text Available Marrow stimulation is frequently employed to treat focal chondral defects of the knee. However, marrow stimulation typically results in fibrocartilage repair tissue rather than healthy hyaline cartilage, which, over time, predisposes the repair to failure. Recently, a cryopreserved viable chondral allograft was developed to augment marrow stimulation. The chondral allograft is comprised of native viable chondrocytes, chondrogenic growth factors, and extracellular matrix proteins within the superficial, transitional, and radial zones of hyaline cartilage. Therefore, host mesenchymal stem cells that infiltrate the graft from the underlying bone marrow following marrow stimulation are provided with the optimal microenvironment to undergo chondrogenesis. The present report describes treatment of a trochlear defect with marrow stimulation augmented with this novel chondral allograft, along with nine month postoperative histological results. At nine months, the patient demonstrated complete resolution of pain and improvement in function, and the repair tissue consisted of 85% hyaline cartilage. For comparison, a biopsy obtained from a patient 8.2 months after treatment with marrow stimulation alone contained only 5% hyaline cartilage. These outcomes suggest that augmenting marrow stimulation with the viable chondral allograft can eliminate pain and improve outcomes, compared with marrow stimulation alone.

  5. Effects of Bone Morphogenic Proteins on Engineered Cartilage

    Science.gov (United States)

    Gooch, Keith, J.; Blunk, Torsten; Courter, Donald L.; Sieminski, Alisha; Vunjak-Novakovic, Gordana; Freed, Lisa E.

    2007-01-01

    A report describes experiments on the effects of bone morphogenic proteins (BMPs) on engineered cartilage grown in vitro. In the experiments, bovine calf articular chondrocytes were seeded onto biodegradable polyglycolic acid scaffolds and cultured in, variously, a control medium or a medium supplemented with BMP-2, BMP-12, or BMP-13 in various concentrations. Under all conditions investigated, cell-polymer constructs cultivated for 4 weeks macroscopically and histologically resembled native cartilage. At a concentration of 100 ng/mL, BMP-2, BMP-12, or BMP-13 caused (1) total masses of the constructs to exceed those of the controls by 121, 80, or 62 percent, respectively; (2) weight percentages of glycosaminoglycans in the constructs to increase by 27, 18, or 15, respectively; and (3) total collagen contents of the constructs to decrease to 63, 89, or 83 percent of the control values, respectively. BMP-2, but not BMP-12 or BMP-13, promoted chondrocyte hypertrophy. These observations were interpreted as suggesting that the three BMPs increase the growth rates and modulate the compositions of engineered cartilage. It was also concluded that in vitro engineered cartilage is a suitable system for studying effects of BMPs on chondrogenesis in a well-defined environment.

  6. Cloning and study of adult-tissue-specific expression of Sox9 in Cyprinus carpio

    Indian Academy of Sciences (India)

    Du Qi-Yan; Wang Feng-Yu; Hua Hui-Ying; Chang Zhong-Jie

    2007-08-01

    The Sox9 gene is one of the important transcription factors in the development of many tissues and organs, particularly in sex determination and chondrogenesis. We amplified the genomic DNA of Cyprinus carpio using degenerate primers, and found that there were two versions of Sox9 in this species: Sox9a and Sox9b, that differ in having an intron of different length (704 bp and 616 bp, respectively) in the conserved HMG box region that codes for identical amino acid sequences. We used a two-phase rapid amplification of cDNA ends (RACE) for the isolation of full-length cDNA of Sox9b. Sequence analyses revealed a 2447-bp cDNA containing 233-bp 5′ untranslated region, a 927-bp 3′ untranslated region, including poly(A), and a 1287 bp open reading frame (ORF) encoding a protein of 428 amino acids. The HMG box of 79 amino acid motif was confirmed from positions 96–174. Sequence alignment showed that the identity of amino acids of Sox9 among ten animal species, including C. carpio, is 75%, indicating that the Sox9 gene is evolutionarily quite conserved. The expression level of Sox9b gene varied among several organs of adult C. carpio, with the level of expression being highest in the brain and testis.

  7. Insights from amphioxus into the evolution of vertebrate cartilage.

    Directory of Open Access Journals (Sweden)

    Daniel Meulemans

    Full Text Available Central to the story of vertebrate evolution is the origin of the vertebrate head, a problem difficult to approach using paleontology and comparative morphology due to a lack of unambiguous intermediate forms. Embryologically, much of the vertebrate head is derived from two ectodermal tissues, the neural crest and cranial placodes. Recent work in protochordates suggests the first chordates possessed migratory neural tube cells with some features of neural crest cells. However, it is unclear how and when these cells acquired the ability to form cellular cartilage, a cell type unique to vertebrates. It has been variously proposed that the neural crest acquired chondrogenic ability by recruiting proto-chondrogenic gene programs deployed in the neural tube, pharynx, and notochord. To test these hypotheses we examined the expression of 11 amphioxus orthologs of genes involved in neural crest chondrogenesis. Consistent with cellular cartilage as a vertebrate novelty, we find that no single amphioxus tissue co-expresses all or most of these genes. However, most are variously co-expressed in mesodermal derivatives. Our results suggest that neural crest-derived cartilage evolved by serial cooption of genes which functioned primitively in mesoderm.

  8. Acquiring Chondrocyte Phenotype from Human Mesenchymal Stem Cells under Inflammatory Conditions

    Directory of Open Access Journals (Sweden)

    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.

  9. Identification of rabbit annulus fibrosus-derived stem cells.

    Directory of Open Access Journals (Sweden)

    Chen Liu

    Full Text Available Annulus fibrosus (AF injuries can lead to substantial deterioration of intervertebral disc (IVD which characterizes degenerative disc disease (DDD. However, treatments for AF repair/regeneration remain challenging due to the intrinsic heterogeneity of AF tissue at cellular, biochemical, and biomechanical levels. In this study, we isolated and characterized a sub-population of cells from rabbit AF tissue which formed colonies in vitro and could self-renew. These cells showed gene expression of typical surface antigen molecules characterizing mesenchymal stem cells (MSCs, including CD29, CD44, and CD166. Meanwhile, they did not express negative markers of MSCs such as CD4, CD8, and CD14. They also expressed Oct-4, nucleostemin, and SSEA-4 proteins. Upon induced differentiation they showed typical osteogenesis, chondrogenesis, and adipogenesis potential. Together, these AF-derived colony-forming cells possessed clonogenicity, self-renewal, and multi-potential differentiation capability, the three criteria characterizing MSCs. Such AF-derived stem cells may potentially be an ideal candidate for DDD treatments using cell therapies or tissue engineering approaches.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Chondrogenic potential of bone marrow–derived mesenchymal stem cells on a novel, auricular-shaped, nanocomposite scaffold

    Directory of Open Access Journals (Sweden)

    Kavi H Patel

    2013-12-01

    Full Text Available Reconstruction of the human auricle remains a challenge to plastic surgeons, and current approaches are not ideal. Tissue engineering provides a promising alternative. This study aims to evaluate the chondrogenic potential of bone marrow–derived mesenchymal stem cells on a novel, auricular-shaped polymer. The proposed polyhedral oligomeric silsesquioxane-modified poly(hexanolactone/carbonateurethane/urea nanocomposite polymer has already been transplanted in patients as the world’s first synthetic trachea, tear duct and vascular bypass graft. The nanocomposite scaffold was fabricated via a coagulation/salt-leaching method and shaped into an auricle. Adult bone marrow–derived mesenchymal stem cells were isolated, cultured and seeded onto the scaffold. On day 21, samples were sent for scanning electron microscopy, histology and immunofluorescence to assess for neocartilage formation. Cell viability assay confirmed cytocompatability and normal patterns of cellular growth at 7, 14 and 21 days after culture. This study demonstrates the potential of a novel polyhedral oligomeric silsesquioxane-modified poly(hexanolactone/carbonateurethane/urea scaffold for culturing bone marrow–derived mesenchymal stem cells in chondrogenic medium to produce an auricular-shaped construct. This is supported by scanning electron microscopy, histological and immunofluorescence analysis revealing markers of chondrogenesis including collagen type II, SOX-9, glycosaminoglycan and elastin. To the best of our knowledge, this is the first report of stem cell application on an auricular-shaped scaffold for tissue engineering purposes. Although many obstacles remain in producing a functional auricle, this is a promising step forward.

  12. Neocartilage formation from mesenchymal stem cells grown in type II collagen-hyaluronan composite scaffolds.

    Science.gov (United States)

    Yeh, Hsi-Yi; Lin, Ting-Yu; Lin, Chen-Huan; Yen, B Linju; Tsai, Ching-Lin; Hsu, Shan-Hui

    2013-01-01

    Three-dimensional (3D) collagen type II-hyaluronan (HA) composite scaffolds (CII-HA) which mimics the extracellular environment of natural cartilage were fabricated in this study. Rheological measurements demonstrated that the incorporation of HA increased the compression modulus of the scaffolds. An initial in vitro evaluation showed that scaffolds seeded with porcine chondrocytes formed cartilaginous-like tissue after 8 weeks, and HA functioned to promote the growth of chondrocytes into scaffolds. Placenta-derived multipotent cells (PDMC) and gingival fibroblasts (GF) were seeded on tissue culture polystyrene (TCPS), CII-HA films, and small intestinal submucosa (SIS) sheets for comparing their chondrogenesis differentiation potentials with those of adipose-derived adult stem cells (ADAS) and bone marrow-derived mesenchymal stem cells (BMSC). Among different cells, PDMC showed the greatest chondrogenic differentiation potential on both CII-HA films and SIS sheets upon TGF-β3 induction, followed by GF. This was evidenced by the up-regulation of chondrogenic genes (Sox9, aggrecan, and collagen type II), which was not observed for cells grown on TCPS. This finding suggested the essential role of substrate materials in the chondrogenic differentiation of PDMC and GF. Neocartilage formation was more obvious in both PDMC and GF cells plated on CII-HA composite scaffolds vs. 8-layer SIS at 28 days in vitro. Finally, implantation of PDMC/CII-HA constructs into NOD-SCID mice confirmed the formation of tissue-engineered cartilage in vivo.

  13. Thyroid development in zebrafish lacking Taz.

    Science.gov (United States)

    Pappalardo, Andrea; Porreca, Immacolata; Caputi, Luigi; De Felice, Elena; Schulte-Merker, Stephan; Zannini, Mariastella; Sordino, Paolo

    2015-11-01

    Taz is a signal-responsive transcriptional coregulator implicated in several biological functions, from chondrogenesis to regulation of organ size. Less well studied, however, is its role in thyroid formation. Here, we explored the in vivo effects on thyroid development of morpholino (MO)-mediated knockdown of wwtr1, the gene encoding zebrafish Taz. The wwtr1 gene is expressed in the thyroid primordium and pharyngeal tissue of developing zebrafish. Compared to mammalian cells, in which Taz promotes expression of thyroid transcription factors and thyroid differentiation genes, wwtr1 MO injection in zebrafish had little or no effect on the expression of thyroid transcription factors, and differentially altered the expression of thyroid differentiation genes. Analysis of wwtr1 morphants at later stages of development revealed that the number and the lumen of thyroid follicles, and the number of thyroid follicle cells, were significantly smaller. In addition, Taz-depleted larvae displayed patterning defects in ventral cranial vessels that correlate with lateral displacement of thyroid follicles. These findings indicate that the zebrafish Taz protein is needed for the normal differentiation of the thyroid and are the first to suggest that Taz confers growth advantage to the endocrine gland.

  14. Characteristics of stem cells derived from the degenerated human intervertebral disc cartilage endplate.

    Directory of Open Access Journals (Sweden)

    Lan-Tao Liu

    Full Text Available Mesenchymal stem cells (MSCs derived from adult tissues are an important candidate for cell-based therapies and regenerative medicine due to their multipotential differentiation capability. MSCs have been identified in many adult tissues but have not reported in the human intervertebral disc cartilage endplate (CEP. The initial purpose of this study was to determine whether MSCs exist in the degenerated human CEP. Next, the morphology, proliferation capacity, cell cycle, cell surface epitope profile and differentiation capacity of these CEP-derived stem cells (CESCs were compared with bone-marrow MSCs (BM-MSCs. Lastly, whether CESCs are a suitable candidate for BM-MSCs was evaluated. Isolated cells from degenerated human CEP were seeded in an agarose suspension culture system to screen the proliferative cell clusters. Cell clusters were chosen and expanded in vitro and were compared with BM-MSCs derived from the same patient. The morphology, proliferation rate, cell cycle, immunophenotype and stem cell gene expression of the CESCs were similar to BM-MSCs. In addition, the CESCs could be induced into osteoblasts, adipocytes, chondrocytes, and are superior to BM-MSCs in terms of osteogenesis and chondrogenesis. This study is first to demonstrate the presence of stem cells in the human degenerated CEP. These results may improve our understanding of intervertebral disc (IVD pathophysiology and the degeneration process, and could provide cell candidates for cell-based regenerative medicine and tissue engineering.

  15. Expression of TGF-β in Fractures Fixed by Nitinol Swan-like Memory Compressive Connectors

    Science.gov (United States)

    Li, M.; Zhang, C. C.; Xu, S. G.; Fu, Q. G.

    2011-07-01

    In this article, the effect of internal fixation of a Nitinol swan-like memory compressive connector (SMC) on the temporal expression of transforming growth factor-β (TGF-β) at fracture sites is evaluated. Specimens were collected from 35 New Zealand rabbits modeled for bilateral humeral fracture fixed with either SMC or stainless dynamic compression plate (DCP). Five rabbits each were killed at day 1, 3, 7, 14, 21, 28, and 56. The local positive staining potency, positive area ratio, and positive index of TGF-β were measured using an immunohistochemistry approach (EnVision) in combination with a computerized image analysis system. TGF-β staining was seen in mesenchymal cells, osteoblasts, chondrocytes, and in the extracellular matrix of fractures fixed in both the SMC and the DCP samples without a significant difference in staining at both the early stages (days 1 and 3) and day 56. A higher TGF-β content was observed in the fractures fixed with SMC when compared to that of DCP from day 7 to 28. As a conclusion, TGF-β is highly expressed in fractures fixed with SMC during chondrogenesis stage and entochondrostosis stage. Finally, the mechanism of how SMC promoting synthesis and secretion of TGF-β in the process of fracture healing has been discussed.

  16. Diverse Effects of Lead Nitrate on the Proliferation, Differentiation, and Gene Expression of Stem Cells Isolated from a Dental Origin

    Directory of Open Access Journals (Sweden)

    Mariam Abdullah

    2014-01-01

    Full Text Available Lead (Pb2+ exposure continues to be a significant public health problem. Therefore, it is vital to have a continuous epidemiological dataset for a better understanding of Pb2+ toxicity. In the present study, we have exposed stem cells isolated from deciduous and permanent teeth, periodontal ligament, and bone marrow to five different types of Pb2+ concentrations (160, 80, 40, 20, and 10 µM for 24 hours to identify the adverse effects of Pb2+ on the proliferation, differentiation, and gene expression on these cell lines. We found that Pb2+ treatment altered the morphology and adhesion of the cells in a dose-dependent manner. There were no significant changes in terms of cell surface phenotypes. Cells exposed to Pb2+ continued to differentiate into chondrogenesis and adipogenesis, and a severe downregulation was observed in osteogenesis. Gene expression studies revealed a constant expression of key markers associated with stemness (Oct 4, Rex 1 and DNA repair enzyme markers, but downregulation occurred with some ectoderm and endoderm markers, demonstrating an irregular and untimely differentiation trail. Our study revealed for the first time that Pb2+ exposure not only affects the phenotypic characteristics but also induces significant alteration in the differentiation and gene expression in the cells.

  17. Study of chondrogenic potential of stem cells in co-culture with chondrons

    Directory of Open Access Journals (Sweden)

    Parisa Nikpou

    2016-06-01

    Full Text Available Objective(s: Three-dimensional biomimetic scaffolds have widespread applications in biomedical tissue engineering due to similarity of their nanofibrous architecture to native extracellular matrix. Co-culture system has stimulatory effect on chondrogenesis of adult mesenchymal stem cells. This work presents a co-culture strategy using human articular chondrons and adipose-derived stem cells (ASCs from infrapatellar fat pad (IPFP for cartilage tissue production. Materials and Methods: Isolated stem cells were characterized by flowcytometry. Electrospun and polycaprolactone (PCL scaffolds (900 nm fiber diameter was obtained from Bon Yakhteh (Tehran- Iran and human infrapatellar fat pad-derived stem cells (IPFP-ASCs were seeded on them. IPFP- ASCs on scaffolds were co-cultured with articular chondrons using transwell. After 21 day, chondrogenic differentiation of stem cell was evaluated by determining the genes expression of collagen2, aggrecan and Indian hedgehog using real- time RT-PCR. Results: Genes expression of collagen2, aggrecan by IPFP-ASCs did not alter significantly in comparison with control group. Howevers, expression of Indian hedgehog decreased significantly compared to control group (P˂ 0.05. Conclusion: These findings indicate that chondrons obtained from osteoarthritic articular cartilage did not stimulate chondrogenic differentiation of IPFP-ASCs in co-culture.

  18. Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage.

    Science.gov (United States)

    Brunger, Jonathan M; Huynh, Nguyen P T; Guenther, Caitlin M; Perez-Pinera, Pablo; Moutos, Franklin T; Sanchez-Adams, Johannah; Gersbach, Charles A; Guilak, Farshid

    2014-03-01

    The ability to develop tissue constructs with matrix composition and biomechanical properties that promote rapid tissue repair or regeneration remains an enduring challenge in musculoskeletal engineering. Current approaches require extensive cell manipulation ex vivo, using exogenous growth factors to drive tissue-specific differentiation, matrix accumulation, and mechanical properties, thus limiting their potential clinical utility. The ability to induce and maintain differentiation of stem cells in situ could bypass these steps and enhance the success of engineering approaches for tissue regeneration. The goal of this study was to generate a self-contained bioactive scaffold capable of mediating stem cell differentiation and formation of a cartilaginous extracellular matrix (ECM) using a lentivirus-based method. We first showed that poly-L-lysine could immobilize lentivirus to poly(ε-caprolactone) films and facilitate human mesenchymal stem cell (hMSC) transduction. We then demonstrated that scaffold-mediated gene delivery of transforming growth factor β3 (TGF-β3), using a 3D woven poly(ε-caprolactone) scaffold, induced robust cartilaginous ECM formation by hMSCs. Chondrogenesis induced by scaffold-mediated gene delivery was as effective as traditional differentiation protocols involving medium supplementation with TGF-β3, as assessed by gene expression, biochemical, and biomechanical analyses. Using lentiviral vectors immobilized on a biomechanically functional scaffold, we have developed a system to achieve sustained transgene expression and ECM formation by hMSCs. This method opens new avenues in the development of bioactive implants that circumvent the need for ex vivo tissue generation by enabling the long-term goal of in situ tissue engineering. PMID:24550481

  19. Structural determinants of heparin-transforming growth factor-β1 interactions and their effects on signaling.

    Science.gov (United States)

    Lee, Jonathan; Wee, Sheena; Gunaratne, Jayantha; Chua, R J E; Smith, Raymond A A; Ling, Ling; Fernig, David G; Swaminathan, Kunchithapadam; Nurcombe, Victor; Cool, Simon M

    2015-12-01

    Transforming growth factor-β1 (TGF-β1, Uniprot: P01137) is a heparin-binding protein that has been implicated in a number of physiological processes, including the initiation of chondrogenesis by human mesenchymal stem cells (hMSCs). Here, we identify the molecular features in the protein and in heparin required for binding and their effects on the potentiation of TGF-β1's activity on hMSCs. Using a proteomics "Protect and Label" approach, lysines K291, K304, K309, K315, K338, K373, K375 and K388 were identified as being directly involved in binding heparin (Data are available via ProteomeXchange with identifier PXD002772). Competition assays in an optical biosensor demonstrated that TGF-β1 does require N- and 6-O-sulfate groups for binding but that 2-O-sulfate groups are unlikely to underpin the interaction. Heparin-derived oligosaccharides as short as degree of polymerization (dp) 4 have a weak ability to compete for TGF-β1 binding to heparin, which increases with the length of the oligosaccharide to reach a maximum between dp18 and dp24. In cell-based assays, heparin, 2-O-, 6-O- and N-desulfated re-N-acetylated heparin and oligosaccharides 14-24 saccharides (dp14-24) in length all increased the phosphorylation of mothers against decapentaplegic homolog 2 (SMAD2) after 6 h of stimulation with TGF-β1. The results provide the structural basis for a model of heparin/heparan sulfate binding to TGF-β1 and demonstrate that the features in the polysaccharide required for binding are not identical to those required for sustaining the signaling by TGF-β1 in hMSCs.

  20. Chordoma-derived cell line U-CH1-N recapitulates the biological properties of notochordal nucleus pulposus cells.

    Science.gov (United States)

    Fujita, Nobuyuki; Suzuki, Satoshi; Watanabe, Kota; Ishii, Ken; Watanabe, Ryuichi; Shimoda, Masayuki; Takubo, Keiyo; Tsuji, Takashi; Toyama, Yoshiaki; Miyamoto, Takeshi; Horiuchi, Keisuke; Nakamura, Masaya; Matsumoto, Morio

    2016-08-01

    Intervertebral disc degeneration proceeds with age and is one of the major causes of lumbar pain and degenerative lumbar spine diseases. However, studies in the field of intervertebral disc biology have been hampered by the lack of reliable cell lines that can be used for in vitro assays. In this study, we show that a chordoma-derived cell line U-CH1-N cells highly express the nucleus pulposus (NP) marker genes, including T (encodes T brachyury transcription factor), KRT19, and CD24. These observations were further confirmed by immunocytochemistry and flow cytometry. Reporter analyses showed that transcriptional activity of T was enhanced in U-CH1-N cells. Chondrogenic capacity of U-CH1-N cells was verified by evaluating the expression of extracellular matrix (ECM) genes and Alcian blue staining. Of note, we found that proliferation and synthesis of chondrogenic ECM proteins were largely dependent on T in U-CH1-N cells. In accordance, knockdown of the T transcripts suppressed the expression of PCNA, a gene essential for DNA replication, and SOX5 and SOX6, the master regulators of chondrogenesis. On the other hand, the CD24-silenced cells showed no reduction in the mRNA expression level of the chondrogenic ECM genes. These results suggest that U-CH1-N shares important biological properties with notochordal NP cells and that T plays crucial roles in maintaining the notochordal NP cell-like phenotype in this cell line. Taken together, our data indicate that U-CH1-N may serve as a useful tool in studying the biology of intervertebral disc. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 34:1341-1350, 2016.

  1. The actin binding protein adseverin regulates osteoclastogenesis.

    Science.gov (United States)

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W P; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

  2. The actin binding protein adseverin regulates osteoclastogenesis.

    Directory of Open Access Journals (Sweden)

    Siavash Hassanpour

    Full Text Available Adseverin (Ads, a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG. Ads is induced during OCG downstream of RANK-ligand (RANKL stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion.

  3. An injectable hydrogel incorporating mesenchymal precursor cells and pentosan polysulphate for intervertebral disc regeneration.

    Science.gov (United States)

    Frith, Jessica E; Cameron, Andrew R; Menzies, Donna J; Ghosh, Peter; Whitehead, Darryl L; Gronthos, Stan; Zannettino, Andrew C W; Cooper-White, Justin J

    2013-12-01

    Intervertebral disc (IVD) degeneration is one of the leading causes of lower back pain and a major health problem worldwide. Current surgical treatments include excision or immobilisation, with neither approach resulting in the repair of the degenerative disc. As such, a tissue engineering-based approach in which stem cells, coupled with an advanced delivery system, could overcome this deficiency and lead to a therapy that encourages functional fibrocartilage generation in the IVD. In this study, we have developed an injectable hydrogel system based on enzymatically-crosslinked polyethylene glycol and hyaluronic acid. We examined the effects of adding pentosan polysulphate (PPS), a synthetic glycosaminoglycan-like factor that has previously been shown (in vitro and in vivo) to this gel system in order to induce chondrogenesis in mesenchymal precursor cells (MPCs) when added as a soluble factor, even in the absence of additional growth factors such as TGF-β. We show that both the gelation rate and mechanical strength of the resulting hydrogels can be tuned in order to optimise the conditions required to produce gels with the desired combination of properties for an IVD scaffold. Human immunoselected STRO-1+ MPCs were then incorporated into the hydrogels. They were shown to retain good viability after both the initial formation of the gel and for longer-term culture periods in vitro. Furthermore, MPC/hydrogel composites formed cartilage-like tissue which was significantly enhanced by the incorporation of PPS into the hydrogels, particularly with respect to the deposition of type-II-collagen. Finally, using a wild-type rat subcutaneous implantation model, we examined the extent of any immune reaction and confirmed that this matrix is well tolerated by the host. Together these data provide evidence that such a system has significant potential as both a delivery vehicle for MPCs and as a matrix for fibrocartilage tissue engineering applications. PMID:24050877

  4. Substrate stiffness and oxygen as regulators of stem cell differentiation during skeletal tissue regeneration: a mechanobiological model.

    Directory of Open Access Journals (Sweden)

    Darren Paul Burke

    Full Text Available Extrinsic mechanical signals have been implicated as key regulators of mesenchymal stem cell (MSC differentiation. It has been possible to test different hypotheses for mechano-regulated MSC differentiation by attempting to simulate regenerative events such as bone fracture repair, where repeatable spatial and temporal patterns of tissue differentiation occur. More recently, in vitro studies have identified other environmental cues such as substrate stiffness and oxygen tension as key regulators of MSC differentiation; however it remains unclear if and how such cues determine stem cell fate in vivo. As part of this study, a computational model was developed to test the hypothesis that substrate stiffness and oxygen tension regulate stem cell differentiation during fracture healing. Rather than assuming mechanical signals act directly on stem cells to determine their differentiation pathway, it is postulated that they act indirectly to regulate angiogenesis and hence partially determine the local oxygen environment within a regenerating tissue. Chondrogenesis of MSCs was hypothesized to occur in low oxygen regions, while in well vascularised regions of the regenerating tissue a soft local substrate was hypothesised to facilitate adipogenesis while a stiff substrate facilitated osteogenesis. Predictions from the model were compared to both experimental data and to predictions of a well established computational mechanobiological model where tissue differentiation is assumed to be regulated directly by the local mechanical environment. The model predicted all the major events of fracture repair, including cartilaginous bridging, endosteal and periosteal bony bridging and bone remodelling. It therefore provides support for the hypothesis that substrate stiffness and oxygen play a key role in regulating MSC fate during regenerative events such as fracture healing.

  5. Histone demethylase KDM2B inhibits the chondrogenic differentiation potentials of stem cells from apical papilla.

    Science.gov (United States)

    Wang, Jing-Jing; Dong, Rui; Wang, Li-Ping; Wang, Jin-Song; Du, Juan; Wang, Song-Lin; Shan, Zhao-Chen; Fan, Zhi-Peng

    2015-01-01

    Mesenchymal stem cells (MSCs) are a reliable resource for tissue regeneration, but the molecular mechanism underlying directed differentiation remains unclear; this has restricted potential MSC applications. Histone methylation, controlled by histone methyltransferases and demethylases, may play a key role in MSCs differentiation. Previous studies determined that KDM2B can regulate the cell proliferation and osteo/dentinogenic differentiation of MSCs. It is not known whether KDM2B is involved in the other cell lineages differentiation of MSCs. Here we used the stem cells from apical papilla (SCAPs) to study the role of KDM2B on the chondrogenic differentiation potentials in MSCs. In this study, Gain- and loss-of-function assays were applied to investigate the role of KDM2B on the chondrogenic differentiation. Alcian Blue Staining and Quantitative Analysis were used to investigate the synthesis of proteoglycans by chondrocytes. Real-time RT-PCR was used to detect the expressions of chondrogenesis related genes. The Alcian Blue staining and Quantitative Analysis results revealed that overexpression of KDM2B decreased the proteoglycans production, and real-time RT-PCR results showed that the expressions of the chondrogenic differentiation markers, COL1, COL2 and SOX9 were inhibited by overexpression of KDM2B in SCAPs. On the contrary, depletion of KDM2B increased the proteoglycans production, and inhibited the expressions of COL1, COL2 and SOX9. In conclusion, our results indicated that KDM2B is a negative regulator of chondrogenic differentiation in SCAPs and suggest that inhibition of KDM2B might improve MSC mediated cartilage regeneration. PMID:25932147

  6. Clonal analysis of the differentiation potential of human adipose-derived adult stem cells.

    Science.gov (United States)

    Guilak, Farshid; Lott, Kristen E; Awad, Hani A; Cao, Qiongfang; Hicok, Kevin C; Fermor, Beverley; Gimble, Jeffrey M

    2006-01-01

    Pools of human adipose-derived adult stem (hADAS) cells can exhibit multiple differentiated phenotypes under appropriate in vitro culture conditions. Because adipose tissue is abundant and easily accessible, hADAS cells offer a promising source of cells for tissue engineering and other cell-based therapies. However, it is unclear whether individual hADAS cells can give rise to multiple differentiated phenotypes or whether each phenotype arises from a subset of committed progenitor cells that exists within a heterogeneous population. The goal of this study was to test the hypothesis that single hADAS are multipotent at a clonal level. hADAS cells were isolated from liposuction waste, and ring cloning was performed to select cells derived from a single progenitor cell. Forty-five clones were expanded through four passages and then induced for adipogenesis, osteogenesis, chondrogenesis, and neurogenesis using lineage-specific differentiation media. Quantitative differentiation criteria for each lineage were determined using histological and biochemical analyses. Eighty one percent of the hADAS cell clones differentiated into at least one of the lineages. In addition, 52% of the hADAS cell clones differentiated into two or more of the lineages. More clones expressed phenotypes of osteoblasts (48%), chondrocytes (43%), and neuron-like cells (52%) than of adipocytes (12%), possibly due to the loss of adipogenic ability after repeated subcultures. The findings are consistent with the hypothesis that hADAS cells are a type of multipotent adult stem cell and not solely a mixed population of unipotent progenitor cells. However, it is important to exercise caution in interpreting these results until they are validated using functional in vivo assays.

  7. Controlling the Fibroblastic Differentiation of Mesenchymal Stem Cells Via the Combination of Fibrous Scaffolds and Connective Tissue Growth Factor

    Science.gov (United States)

    Tong, Zhixiang; Sant, Shilpa

    2011-01-01

    Controlled differentiation of multi-potent mesenchymal stem cells (MSCs) into vocal fold-specific, fibroblast-like cells in vitro is an attractive strategy for vocal fold repair and regeneration. The goal of the current study was to define experimental parameters that can be used to control the initial fibroblastic differentiation of MSCs in vitro. To this end, connective tissue growth factor (CTGF) and micro-structured, fibrous scaffolds based on poly(glycerol sebacate) (PGS) and poly(ɛ-caprolactone) (PCL) were used to create a three-dimensional, connective tissue-like microenvironment. MSCs readily attached to and elongated along the microfibers, adopting a spindle-shaped morphology during the initial 3 days of preculture in an MSC maintenance medium. The cell-laden scaffolds were subsequently cultivated in a conditioned medium containing CTGF and ascorbic acids for up to 21 days. Cell morphology, proliferation, and differentiation were analyzed collectively by quantitative PCR analyses, and biochemical and immunocytochemical assays. F-actin staining showed that MSCs maintained their fibroblastic morphology during the 3 weeks of culture. The addition of CTGF to the constructs resulted in an enhanced cell proliferation, elevated expression of fibroblast-specific protein-1, and decreased expression of mesenchymal surface epitopes without markedly triggering chondrogenesis, osteogenesis, adipogenesis, or apoptosis. At the mRNA level, CTGF supplement resulted in a decreased expression of collagen I and tissue inhibitor of metalloproteinase 1, but an increased expression of decorin and hyaluronic acid synthesase 3. At the protein level, collagen I, collagen III, sulfated glycosaminoglycan, and elastin productivity was higher in the conditioned PGS-PCL culture than in the normal culture. These findings collectively demonstrate that the fibrous mesh, when combined with defined biochemical cues, is capable of fostering MSC fibroblastic differentiation in vitro. PMID

  8. An injectable hydrogel incorporating mesenchymal precursor cells and pentosan polysulphate for intervertebral disc regeneration.

    Science.gov (United States)

    Frith, Jessica E; Cameron, Andrew R; Menzies, Donna J; Ghosh, Peter; Whitehead, Darryl L; Gronthos, Stan; Zannettino, Andrew C W; Cooper-White, Justin J

    2013-12-01

    Intervertebral disc (IVD) degeneration is one of the leading causes of lower back pain and a major health problem worldwide. Current surgical treatments include excision or immobilisation, with neither approach resulting in the repair of the degenerative disc. As such, a tissue engineering-based approach in which stem cells, coupled with an advanced delivery system, could overcome this deficiency and lead to a therapy that encourages functional fibrocartilage generation in the IVD. In this study, we have developed an injectable hydrogel system based on enzymatically-crosslinked polyethylene glycol and hyaluronic acid. We examined the effects of adding pentosan polysulphate (PPS), a synthetic glycosaminoglycan-like factor that has previously been shown (in vitro and in vivo) to this gel system in order to induce chondrogenesis in mesenchymal precursor cells (MPCs) when added as a soluble factor, even in the absence of additional growth factors such as TGF-β. We show that both the gelation rate and mechanical strength of the resulting hydrogels can be tuned in order to optimise the conditions required to produce gels with the desired combination of properties for an IVD scaffold. Human immunoselected STRO-1+ MPCs were then incorporated into the hydrogels. They were shown to retain good viability after both the initial formation of the gel and for longer-term culture periods in vitro. Furthermore, MPC/hydrogel composites formed cartilage-like tissue which was significantly enhanced by the incorporation of PPS into the hydrogels, particularly with respect to the deposition of type-II-collagen. Finally, using a wild-type rat subcutaneous implantation model, we examined the extent of any immune reaction and confirmed that this matrix is well tolerated by the host. Together these data provide evidence that such a system has significant potential as both a delivery vehicle for MPCs and as a matrix for fibrocartilage tissue engineering applications.

  9. Generation, Characterization, and Multilineage Potency of Mesenchymal-Like Progenitors Derived from Equine Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Lepage, Sarah I; Nagy, Kristina; Sung, Hoon-Ki; Kandel, Rita A; Nagy, Andras; Koch, Thomas G

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are more and more frequently used to treat orthopedic injuries in horses. However, these cells are limited in their expandability and differentiation capacity. Recently, the first equine-induced pluripotent stem cell (iPSC) lines were reported by us [ 1 ]. In vitro differentiation of iPSCs into MSC-like cells is an attractive alternative to using MSCs derived from other sources, as a much larger quantity of patient-specific cells with broad differentiation potential could be generated. However, the differentiation capacity of iPSCs to MSCs and the potential for use in tissue engineering have yet to be explored. In this study, equine iPSCs were induced to differentiate into an MSC-like population. Upon induction, the iPSCs changed morphology toward spindle-shaped cells similar to MSCs. The ensuing iPSC-MSCs exhibited downregulation of pluripotency-associated genes and an upregulation of MSC-associated genes. In addition, the cells expressed the same surface markers as MSCs derived from equine umbilical cord blood. We then assessed the multilineage differentiation potential of iPSC-MSCs. Although chondrogenesis was not achieved after induction with transforming growth factor-beta 3 (TGFβ3) and/or bone morphogenic protein 4 (BMP-4) in 3D pellet culture, mineralization characteristic of osteogenesis and lipid droplet accumulation characteristic of adipogenesis were observed after chemical induction. We demonstrate a protocol for the derivation of MSC-like progenitor populations from equine iPS cells.

  10. Longitudinal analysis of osteogenic and angiogenic signaling factors in healing models mimicking atrophic and hypertrophic non-unions in rats.

    Directory of Open Access Journals (Sweden)

    Susann Minkwitz

    Full Text Available Impaired bone healing can have devastating consequences for the patient. Clinically relevant animal models are necessary to understand the pathology of impaired bone healing. In this study, two impaired healing models, a hypertrophic and an atrophic non-union, were compared to physiological bone healing in rats. The aim was to provide detailed information about differences in gene expression, vascularization and histology during the healing process. The change from a closed fracture (healing control group to an open osteotomy (hypertrophy group led to prolonged healing with reduced mineralized bridging after 42 days. RT-PCR data revealed higher gene expression of most tested osteogenic and angiogenic factors in the hypertrophy group at day 14. After 42 days a significant reduction of gene expression was seen for Bmp4 and Bambi in this group. The inhibition of angiogenesis by Fumagillin (atrophy group decreased the formation of new blood vessels and led to a non-healing situation with diminished chondrogenesis. RT-PCR results showed an attempt towards overcoming the early perturbance by significant up regulation of the angiogenic regulators Vegfa, Angiopoietin 2 and Fgf1 at day 7 and a further continuous increase of Fgf1, -2 and Angiopoietin 2 over time. However µCT angiograms showed incomplete recovery after 42 days. Furthermore, lower expression values were detected for the Bmps at day 14 and 21. The Bmp antagonists Dan and Twsg1 tended to be higher expressed in the atrophy group at day 42. In conclusion, the investigated animal models are suitable models to mimic human fracture healing complications and can be used for longitudinal studies. Analyzing osteogenic and angiogenic signaling patterns, clear changes in expression were identified between these three healing models, revealing the importance of a coordinated interplay of different factors to allow successful bone healing.

  11. Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation

    Directory of Open Access Journals (Sweden)

    Adila A Hamid

    2012-01-01

    Full Text Available OBJECTIVES: Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction. MATERIALS AND METHODS: Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction. RESULTS: Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction. CONCLUSION: Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adiposederived stem cells was most prominent after one week of chondrogenic induction.

  12. Electrospun scaffolds for multiple tissues regeneration in vivo through topography dependent induction of lineage specific differentiation.

    Science.gov (United States)

    Yin, Zi; Chen, Xiao; Song, Hai-Xin; Hu, Jia-Jie; Tang, Qiao-Mei; Zhu, Ting; Shen, Wei-Liang; Chen, Jia-Lin; Liu, Huanhuan; Heng, Boon Chin; Ouyang, Hong-Wei

    2015-03-01

    Physical topographic cues from various substrata have been shown to exert profound effects on the growth and differentiation of stem cells due to their niche-mimicking features. However, the biological function of different topographic materials utilized as bio-scaffolds in vivo have not been rigorously characterized. This study investigated the divergent differentiation pathways of mesenchymal stem cells (MSCs) and neo-tissue formation trigged by aligned and randomly-oriented fibrous scaffolds, both in vitro and in vivo. The aligned group was observed to form more mature tendon-like tissue in the Achilles tendon injury model, as evidenced by histological scoring and collagen I immunohistochemical staining data. In contrast, the randomly-oriented group exhibited much chondrogenesis and subsequent bone tissue formation through ossification. Additionally, X-ray imaging and osteocalcin immunohistochemical staining also demonstrated that osteogenesis in vivo is driven by randomly oriented topography. Furthermore, MSCs on the aligned substrate exhibited tenocyte-like morphology and enhanced tenogenic differentiation compared to cells grown on randomly-oriented scaffold. qRT-PCR analysis of osteogenic marker genes and alkaline phosphatase (ALP) staining demonstrated that MSCs cultured on randomly-oriented fiber scaffolds displayed enhanced osteogenic differentiation compared with cells cultured on aligned fiber scaffolds. Finally, it was demonstrated that cytoskeletal tension release abrogated the divergent differentiation pathways on different substrate topography. Collectively, these findings illustrate the relationship between topographic cues of the scaffold and their inductive role in tissue regeneration; thus providing an insight into future development of smart functionalized bio-scaffold design and its application in tissue engineering.

  13. Aislamiento y caracterización de células “stem” mesenquimales de médula ósea humana según criterios de la Sociedad Internacional de Terapia Celular

    Directory of Open Access Journals (Sweden)

    Jose Alejandro Aristizabal-Castellanos

    2010-12-01

    Full Text Available Isolation and characterization of mesenchymal stem cells from human bone marrow according to the criteria of the InternationalSociety for Cellular Therapy. Bone marrow (BM is an important source for isolating mesenchymal stem cells (MSC useful inimmunomodulation and tissue regeneration therapies. Objective. To isolate and characterize mesenchymal stem cells obtained from BMmeeting the requirements of the International Society for Cellular Therapy. Materials and methods. BM samples were collected fromvolunteer donors attending the Orthopedics Service of the San Ignacio University Hospital (Bogotá, Colombia. Morphological characteristicswere evaluated by inverted microscopy and the immunophenotype was determined by flow cytometry. Protocols were developed foradipogenic, osteogenic and chondrogenic differentiation using the Oil Red O, alkaline phosphatase and safranin stains, respectively.Results. We collected 24 samples of BM from patients with total hip replacement (volume of BM sample: 5-45 ml. Cells with afibroblastoid morphology were isolated from 21 BM samples (isolation efficiency: 87.5%. No statistical significant differences were foundbetween the hematopoyetic antigens (CD34 and CD45, p>0.05 in the immunophenotypic evaluation (of MSC from BM; on the contrary,there were differences (p=0.006 between the hematopoyetic antigen CD45 and the mesenchymal antigens (CD13, CD44, CD73, CD90,CD105, HLA-I, and HLA-DR. Oil Red O stain revealed the presence of multilocular adipocytes, in the osteogenic induction we observedlocalized mineralization nodules, and chondrogenesis was positive as revealed by the safranin stain. Conclusion. MSC were satisfactorilyisolated from BM and characterized according to the international standards.

  14. Autologous chondrocyte transplantation for the treatment of articular cartilage defects in the knee joint. Techniques and results; Autologe Chondrozytentransplantation zur Behandlung von Knorpeldefekten des Kniegelenks. Techniken und Ergebnisse

    Energy Technology Data Exchange (ETDEWEB)

    Marlovits, S.; Kutscha-Lissberg, F.; Aldrian, S.; Resinger, C.; Singer, P.; Zeller, P.; Vecsei, V. [Universitaetsklinik fuer Unfallchirurgie, Medizinische Universitaet Wien (Austria)

    2004-08-01

    Currently the use of autologous chondrocytes as a cartilage-repair procedure for the repair of injured articular cartilage of the knee joint, is recommended. This review presents the technique of autologous chondrocyte transplantation (ACT) and their modifications as matrix-associated autologous chondrocyte transplantation (MACT). Beside the surgical procedure the experimental and clinical results are discussed. Furthermore the major complications and the indication guidelines are presented. Articular cartilage in adults has a poor ability to self-repair after a substantial injury. Surgical therapeutic efforts in treating cartilage defects have focused on bringing new cells capable of chondrogenesis into the lesions. With ACT good to excellent clinical results are seen in isolated posttraumatic lesions of the knee joint in the younger patient with the formation of hyalinelike repair tissue. The major complications are periosteal hypertrophy, delamination of the transplant, arthrofibrosis and transplant failure. The current limitations include osteoarthritic defects and higher patient age. With the right indication and operative technique ACT is an effective and save option for the treatment of large full thickness cartilage defect of the knee joint. (orig.) [German] Zur Behandlung umschriebener Defekte des artikulaeren Kniegelenkgelenkknorpels wird der Einsatz autologer Knorpelzellen zunehmend als neue biologische Methode empfohlen. Die Technik der autologen Chondrozytentransplantation (ACT) und deren Modifikationen als matrixassoziierte autologe Chondrozytentransplantation (MACT) werden dargestellt. Es erfolgt ein Ueberblick ueber die experimentellen und klinischen Ergebnisse mit der Darstellung der haeufigsten Komplikationen und den derzeit gueltigen Indikationsrichtlinien. Unter Verwendung qualitativ hochwertiger Zellen zeigen besonders posttraumatische Knorpeldefekte bei juengeren Patienten eine hohe Erfolgsquote mit der Ausbildung eines hyalinartigen

  15. Protein O-mannosylation is crucial for human mesencyhmal stem cells fate.

    Science.gov (United States)

    Ragni, E; Lommel, M; Moro, M; Crosti, M; Lavazza, C; Parazzi, V; Saredi, S; Strahl, S; Lazzari, L

    2016-01-01

    Human mesenchymal stem cells (MSC) are promising cell types in the field of regenerative medicine. Although many pathways have been dissected in the effort to better understand and characterize MSC potential, the impact of protein N- or O-glycosylation has been neglected. Deficient protein O-mannosylation is a pathomechanism underlying severe congenital muscular dystrophies (CMD) that start to develop at the embryonic developmental stage and progress in the adult, often in tissues where MSC exert their function. Here we show that O-mannosylation genes, many of which are putative or verified glycosyltransferases (GTs), are expressed in a similar pattern in MSC from adipose tissue, bone marrow, and umbilical cord blood and that their expression levels are retained constant during mesengenic differentiation. Inhibition of the first players of the enzymatic cascade, POMT1/2, resulted in complete abolishment of chondrogenesis and alterations of adipogenic and osteogenic potential together with a lethal effect during myogenic induction. Since to date, no therapy for CMD is available, we explored the possibility of using MSC extracellular vesicles (EVs) as molecular source of functional GTs mRNA. All MSC secrete POMT1 mRNA-containing EVs that are able to efficiently fuse with myoblasts which are among the most affected cells by CMD. Intriguingly, in a pomt1 patient myoblast line EVs were able to partially revert O-mannosylation deficiency and contribute to a morphology recovery. Altogether, these results emphasize the crucial role of protein O-mannosylation in stem cell fate and properties and open the possibility of using MSC vesicles as a novel therapeutic approach to CMD. PMID:26245304

  16. Resveratrol protects bone marrow mesenchymal stem cell derived chondrocytes cultured on chitosan-gelatin scaffolds from the inhibitory effect of interleukin-1β

    Institute of Scientific and Technical Information of China (English)

    Ming LEI; Shi-qing LIU; Yu-lan LIU

    2008-01-01

    Aim: To investigate the effects of resveratrol on interleukin-lbeta (IL-1β) induced catabolism in bone marrow mesenchymal stem cell (MSC) derived chon-drocytes cultured on chitosan-gelatin scaffolds (CGS). Methods: The chondro-genesis of alginate-encapsulated MSCs was evaluated by toluidine blue staining, RT-PCR, and immunostaing. MSC-derived chondrocyte morphology cultured on CGS was evaluated by a scanning electron microscope (SEM) and a laser confocal microscope (LCM). When these cells on CGS were pre-stimulated with IL-1β or co-treated with IL-1β and resveratrol in the absence and presence of the specific β1-integrin blocking antibody, collagen type Ⅱ, aggrecan, matrix metalloproteinase-13 (MMP-13) expression, and the translocation of nuclear factor kappaB (NF-κB) were analyzed by Western blot analysis. Results: Transforming growth factor beta 3 (TGF-β3) combined with insulin-like growth factor Ⅰ (IGF-Ⅰ) induced the cartilage-specific collagen type Ⅱ, aggrecan expression and extracellular matrix (ECM) accumulation at the end of a 3-week culture. CGS supported those differentiated chondrocytes' attachment, proliferation, migration, and ECM formation. When those cells cultured on CGS were stimulated with IL-1β alone, collagen type Ⅱ and aggrecan expression was inhibited. However, MMP-13 expression increased. Resveratrol reversed the catabolic effects by reducing the translocation of NF-κB. A specific β1-integrin blocking antibody abrogated the effects of resveratrol on IL-1β stimulated MSC-derived chondrocytes. Conclusion: These results indicated that resveratrol acta as a NF-κB inhibitor to protect MSC-derived chondrocytes on the CGS from the IL-1β catabolism and these effects are mediated by β1-integrin.

  17. Neocartilage formation from predifferentiated human adipose derived stem cells in vivo

    Institute of Scientific and Technical Information of China (English)

    Xiao-bing JIN; Yong-sheng SUN; Ke ZHANG; Jing WANG; Xiao-dong JU; Si-quan LOU

    2007-01-01

    Aim: To examine the chondrogenic potential of human adipose derived stem cells (hASC) induced by human transforming growth factor beta2 (hTGF beta2) in vitro, and to investigate if predifferentiated hASC can produce neocartilage in vivo. Methods: hASC were isolated from subcutaneous adipose tissue and cul-tured in pellets with the addition of hTGF beta2. Chondrogenic differentiation was assayed by RT-PCR, Western blotting, toluidine blue staining, and immuno-histochemistry staining for collagen type Ⅱ. For the in vivo study, intact induced cell pellets or the released cells embedded in alginate gel with different concentra-tions were implanted subcutaneously in nude mice. Specimens were harvested at different time points and carried with histological and immunohistochemistry ex-amination to evaluate the cartilage formation. Results: RT-PCR analysis revealed that hASC produced aggrecan and collagen type Ⅱ after 7 d of induction and continued throughout the culture period. This was also demonstrated by the Western blot analysis, positive staining of toluidine blue, and immunohistochem-istry for collagen type Ⅱ. After reseeding in the monolayer, the cells isolated from the pellets displayed a polygonal morphology compared with the primary spindle shape, hASC were released from the induced cell pellets when embedded in alginate gel (implanted cell concentration=5x106/mL or higher). They produced neocartilage after 12 weeks in vivo culture; however, intact induced cell pellets implanted subcutaneously rapidly lost their differentiated phenotype. Conclusion:Chondrogenesis of hASC in vitro can be induced by combining pellet culture and hTGF beta2 treatment. Predifferentiated hASC embedded in alginate gel have the ability of producing neocartilage in vivo.

  18. Characterization of the collagen component of cartilage repair tissue of the talus with quantitative MRI: comparison of T2 relaxation time measurements with a diffusion-weighted double-echo steady-state sequence (dwDESS)

    Energy Technology Data Exchange (ETDEWEB)

    Kretzschmar, M.; Hainc, N.; Studler, U. [University Hospital Basel, Department of Radiology, Basel (Switzerland); Bieri, O. [University Hospital Basel, Division of Radiological Physics, Basel (Switzerland); Miska, M. [University Hospital, Department of Orthopedics, Heidelberg (Germany); Wiewiorski, M.; Valderrabano, V. [University Hospital Basel, Department of Orthopedic Surgery, Basel (Switzerland)

    2015-04-01

    The purpose of this study was to characterize the collagen component of repair tissue (RT) of the talus after autologous matrix-induced chondrogenesis (AMIC) using quantitative T2 and diffusion-weighted imaging. Mean T2 values and diffusion coefficients of AMIC-RT and normal cartilage of the talus of 25 patients with posttraumatic osteochondral lesions and AMIC repair were compared in a cross-sectional design using partially spoiled steady-state free precession (pSSFP) for T2 quantification, and diffusion-weighted double-echo steady-state (dwDESS) for diffusion measurement. RT and cartilage were graded with modified Noyes and MOCART scores on morphological sequences. An association between follow-up interval and quantitative MRI measures was assessed using multivariate regression, after stratifying the cohort according to time interval between surgery and MRI. Mean T2 of the AMIC-RT and cartilage were 43.1 ms and 39.1 ms, respectively (p = 0.26). Mean diffusivity of the RT (1.76 μm{sup 2}/ms) was significantly higher compared to normal cartilage (1.46 μm{sup 2}/ms) (p = 0.0092). No correlation was found between morphological and quantitative parameters. RT diffusivity was lowest in the subgroup with follow-up >28 months (p = 0.027). Compared to T2-mapping, dwDESS demonstrated greater sensitivity in detecting differences in the collagen matrix between AMIC-RT and cartilage. Decreased diffusivity in patients with longer follow-up times may indicate an increased matrix organization of RT. (orig.)

  19. Embryonic development of Python sebae - II: Craniofacial microscopic anatomy, cell proliferation and apoptosis.

    Science.gov (United States)

    Buchtová, Marcela; Boughner, Julia C; Fu, Katherine; Diewert, Virginia M; Richman, Joy M

    2007-01-01

    This study explores the microscopic craniofacial morphogenesis of the oviparous African rock python (Python sebae) spanning the first two-thirds of the post-oviposition period. At the time of laying, the python embryo consists of largely undifferentiated mesenchyme and epithelium with the exception of the cranial base and trabeculae cranii, which are undergoing chondrogenesis. The facial prominences are well defined and are at a late stage, close to the time when lip fusion begins. Later (11-12d), specializations in the epithelia begin to differentiate (vomeronasal and olfactory epithelia, teeth). Dental development in snakes is different from that of mammals in several aspects including an extended dental lamina with the capacity to form 4 sets of generational teeth. In addition, the ophidian olfactory system is very different from the mammalian. There is a large vomeronasal organ, a nasal cavity proper and an extraconchal space. All of these areas are lined with a greatly expanded olfactory epithelium. Intramembranous bone differentiation is taking place at stage 3 with some bones already ossifying whereas most are only represented as mesenchymal condensations. In addition to routine histological staining, PCNA immunohistochemistry reveals relatively higher levels of proliferation in the extending dental laminae, in osseous mesenchymal condensations and in the olfactory epithelia. Areas undergoing apoptosis were noted in the enamel organs of the teeth and osseous mesenchymal condensations. We propose that localized apoptosis helps to divide a single condensation into multiple ossification centres and this is a mechanism whereby novel morphology can be selected in response to evolutionary pressures. Several additional differences in head morphology between snakes and other amniotes were noted including a palatal groove separating the inner and outer row of teeth in the upper jaw, a tracheal opening within the tongue and a pharyngeal adhesion that closes off the

  20. Imaging mesenchymal stem cells containing single wall nanotube nanoprobes in a 3D scaffold using photo-thermal optical coherence tomography

    Science.gov (United States)

    Connolly, Emma; Subhash, Hrebesh M.; Leahy, Martin; Rooney, Niall; Barry, Frank; Murphy, Mary; Barron, Valerie

    2014-02-01

    Despite the fact, that a range of clinically viable imaging modalities, such as magnetic resonance imaging (MRI), computed tomography (CT), photo emission tomography (PET), ultrasound and bioluminescence imaging are being optimised to track cells in vivo, many of these techniques are subject to limitations such as the levels of contrast agent required, toxic effects of radiotracers, photo attenuation of tissue and backscatter. With the advent of nanotechnology, nanoprobes are leading the charge to overcome these limitations. In particular, single wall nanotubes (SWNT) have been shown to be taken up by cells and as such are effective nanoprobes for cell imaging. Consequently, the main aim of this research is to employ mesenchymal stem cells (MSC) containing SWNT nanoprobes to image cell distribution in a 3D scaffold for cartilage repair. To this end, MSC were cultured in the presence of 32μg/ml SWNT in cell culture medium (αMEM, 10% FBS, 1% penicillin/streptomycin) for 24 hours. Upon confirmation of cell viability, the MSC containing SWNT were encapsulated in hyaluronic acid gels and loaded on polylactic acid polycaprolactone scaffolds. After 28 days in complete chondrogenic medium, with medium changes every 2 days, chondrogenesis was confirmed by the presence of glycosaminoglycan. Moreover, using photothermal optical coherence tomography (PT-OCT), the cells were seen to be distributed through the scaffold with high resolution. In summary, these data reveal that MSC containing SWNT nanoprobes in combination with PT-OCT offer an exciting opportunity for stem cell tracking in vitro for assessing seeding scaffolds and in vivo for determining biodistribution.

  1. A novel fibrin gel derived from hyaluronic acid-grafted fibrinogen

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Chiung L; Chen, Hui W; Wang, Tzu C; Wang, Yng J, E-mail: wang@ym.edu.tw [Institute of Biomedical Engineering, National Yang Ming University, No. 155, Sec. 2, Li-Nung St., Shih-Pai, Taipei, Taiwan 112 (China)

    2011-04-15

    Fibrinogen is a major plasma protein that forms a three-dimensional fibrin gel upon being activated by thrombin. In this study, we report the synthesis and potential applications of hybrid molecules composed of fibrinogen coupled to the reducing ends of short-chain hyaluronic acids (sHAs) by reductive amination. The grafting of sHAs to fibrinogen was verified by analyzing particle size, zeta potential and gel-electrophoretic mobility of the hybrid molecules. The sHA-fibrinogen hybrid molecules with graft ratios (sHA/fibrinogen) of up to 6.5 retained the ability to form gels in response to thrombin activation. The sHA-fibrin gels were transparent in appearance and exhibited high water content, which were characteristics distinct from those of gels formed by mixtures of sHAs and fibrinogen. The potential applications of the sHA-fibrin gels were evaluated. The sHA-fibrinogen gel with a graft ratio of 3.6 (S3.6F) was examined for its ability to encapsulate and support the differentiation of ATDC5 chondrocyte-like cells. Compared with the fibrinogen-formed gel, cells cultured in the S3.6F gel exhibited increased lacunae formation; moreover, the abundance of cartilaginous extracellular matrix molecules and the expression of chondrocyte marker genes, such as aggrecan, collagen II and Sox9, were also significantly increased. Our data suggest that the three-dimensional gel formed by the sHA-fibrinogen hybrid is a better support than the fibrin gel for chondrogenesis induction.

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

    Science.gov (United States)

    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.

  3. Creating an Animal Model of Tendinopathy by Inducing Chondrogenic Differentiation with Kartogenin.

    Directory of Open Access Journals (Sweden)

    Ting Yuan

    Full Text Available Previous animal studies have shown that long term rat treadmill running induces over-use tendinopathy, which manifests as proteoglycan accumulation and chondrocytes-like cells within the affected tendons. Creating this animal model of tendinopathy by long term treadmill running is however time-consuming, costly and may vary among animals. In this study, we used a new approach to develop an animal model of tendinopathy using kartogenin (KGN, a bio-compound that can stimulate endogenous stem/progenitor cells to differentiate into chondrocytes. KGN-beads were fabricated and implanted into rat Achilles tendons. Five weeks after implantation, chondrocytes and proteoglycan accumulation were found at the KGN implanted site. Vascularity as well as disorganization in collagen fibers were also present in the same site along with increased expression of the chondrocyte specific marker, collagen type II (Col. II. In vitro studies confirmed that KGN was released continuously from KGN-alginate in vivo beads and induced chondrogenic differentiation of tendon stem/progenitor cells (TSCs suggesting that chondrogenesis after KGN-bead implantation into the rat tendons is likely due to the aberrant differentiation of TSCs into chondrocytes. Taken together, our results showed that KGN-alginate beads can be used to create a rat model of tendinopathy, which, at least in part, reproduces the features of over-use tendinopathy model created by long term treadmill running. This model is mechanistic (stem cell differentiation, highly reproducible and precise in creating localized tendinopathic lesions. It is expected that this model will be useful to evaluate the effects of various topical treatments such as NSAIDs and platelet-rich plasma (PRP for the treatment of tendinopathy.

  4. Mouse limb skeletal growth and synovial joint development are coordinately enhanced by Kartogenin.

    Science.gov (United States)

    Decker, Rebekah S; Koyama, Eiki; Enomoto-Iwamoto, Motomi; Maye, Peter; Rowe, David; Zhu, Shoutian; Schultz, Peter G; Pacifici, Maurizio

    2014-11-15

    Limb development requires the coordinated growth of several tissues and structures including long bones, joints and tendons, but the underlying mechanisms are not wholly clear. Recently, we identified a small drug-like molecule - we named Kartogenin (KGN) - that greatly stimulates chondrogenesis in marrow-derived mesenchymal stem cells (MSCs) and enhances cartilage repair in mouse osteoarthritis (OA) models. To determine whether limb developmental processes are regulated by KGN, we tested its activity on committed preskeletal mesenchymal cells from mouse embryo limb buds and whole limb explants. KGN did stimulate cartilage nodule formation and more strikingly, boosted digit cartilaginous anlaga elongation, synovial joint formation and interzone compaction, tendon maturation as monitored by ScxGFP, and interdigit invagination. To identify mechanisms, we carried out gene expression analyses and found that several genes, including those encoding key signaling proteins, were up-regulated by KGN. Amongst highly up-regulated genes were those encoding hedgehog and TGFβ superfamily members, particularly TFGβ1. The former response was verified by increases in Gli1-LacZ activity and Gli1 mRNA expression. Exogenous TGFβ1 stimulated cartilage nodule formation to levels similar to KGN, and KGN and TGFβ1 both greatly enhanced expression of lubricin/Prg4 in articular superficial zone cells. KGN also strongly increased the cellular levels of phospho-Smads that mediate canonical TGFβ and BMP signaling. Thus, limb development is potently and harmoniously stimulated by KGN. The growth effects of KGN appear to result from its ability to boost several key signaling pathways and in particular TGFβ signaling, working in addition to and/or in concert with the filamin A/CBFβ/RUNX1 pathway we identified previously to orchestrate overall limb development. KGN may thus represent a very powerful tool not only for OA therapy, but also limb regeneration and tissue repair strategies. PMID

  5. Creating an Animal Model of Tendinopathy by Inducing Chondrogenic Differentiation with Kartogenin.

    Science.gov (United States)

    Yuan, Ting; Zhang, Jianying; Zhao, Guangyi; Zhou, Yiqin; Zhang, Chang-Qing; Wang, James H-C

    2016-01-01

    Previous animal studies have shown that long term rat treadmill running induces over-use tendinopathy, which manifests as proteoglycan accumulation and chondrocytes-like cells within the affected tendons. Creating this animal model of tendinopathy by long term treadmill running is however time-consuming, costly and may vary among animals. In this study, we used a new approach to develop an animal model of tendinopathy using kartogenin (KGN), a bio-compound that can stimulate endogenous stem/progenitor cells to differentiate into chondrocytes. KGN-beads were fabricated and implanted into rat Achilles tendons. Five weeks after implantation, chondrocytes and proteoglycan accumulation were found at the KGN implanted site. Vascularity as well as disorganization in collagen fibers were also present in the same site along with increased expression of the chondrocyte specific marker, collagen type II (Col. II). In vitro studies confirmed that KGN was released continuously from KGN-alginate in vivo beads and induced chondrogenic differentiation of tendon stem/progenitor cells (TSCs) suggesting that chondrogenesis after KGN-bead implantation into the rat tendons is likely due to the aberrant differentiation of TSCs into chondrocytes. Taken together, our results showed that KGN-alginate beads can be used to create a rat model of tendinopathy, which, at least in part, reproduces the features of over-use tendinopathy model created by long term treadmill running. This model is mechanistic (stem cell differentiation), highly reproducible and precise in creating localized tendinopathic lesions. It is expected that this model will be useful to evaluate the effects of various topical treatments such as NSAIDs and platelet-rich plasma (PRP) for the treatment of tendinopathy. PMID:26848746

  6. Targeted stimulation of retinoic acid receptor-γ mitigates the formation of heterotopic ossification in an established blast-related traumatic injury model.

    Science.gov (United States)

    Pavey, Gabriel J; Qureshi, Ammar T; Tomasino, Allison M; Honnold, Cary L; Bishop, Danett K; Agarwal, Shailesh; Loder, Shawn; Levi, Benjamin; Pacifici, Maurizio; Iwamoto, Masahiro; Potter, Benjamin K; Davis, Thomas A; Forsberg, Jonathan A

    2016-09-01

    Heterotopic ossification (HO) involves formation of endochondral bone at non-skeletal sites, is prevalent in severely wounded service members, and causes significant complications and delayed rehabilitation. As common prophylactic treatments such as anti-inflammatory drugs and irradiation cannot be used after multi-system combat trauma, there is an urgent need for new remedies. Previously, we showed that the retinoic acid receptor γ agonist Palovarotene inhibited subcutaneous and intramuscular HO in mice, but those models do not mimic complex combat injury. Thus, we tested Palovarotene in our validated rat trauma-induced HO model that involves blast-related limb injury, femoral fracture, quadriceps crush injury, amputation and infection with methicillin-resistant Staphylococcus aureus from combat wound infections. Palovarotene was given orally for 14days at 1mg/kg/day starting on post-operative day (POD) 1 or POD-5, and HO amount, wound dehiscence and related processes were monitored for up to 84days post injury. Compared to vehicle-control animals, Palovarotene significantly decreased HO by 50 to 60% regardless of when the treatment started and if infection was present. Histological analyses showed that Palovarotene reduced ectopic chondrogenesis, osteogenesis and angiogenesis forming at the injury site over time, while fibrotic tissue was often present in place of ectopic bone. Custom gene array data verified that while expression of key chondrogenic and osteogenic genes was decreased within soft tissues of residual limb in Palovarotene-treated rats, expression of cartilage catabolic genes was increased, including matrix metalloproteinase-9. Importantly, Palovarotene seemed to exert moderate inhibitory effects on wound healing, raising potential safety concerns related to dosing and timing. Our data show for the first time that Palovarotene significantly inhibits HO triggered by blast injury and associated complications, strongly indicating that it may prevent

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

    Directory of Open Access Journals (Sweden)

    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.

  8. Nell1-deficient mice have reduced expression of extracellular matrix proteins causing cranial and vertebral defects

    Energy Technology Data Exchange (ETDEWEB)

    Desai, Jayashree [University of Tennessee, Knoxville (UTK) & Oak Ridge National Laboratory (ORNL); Shannon, Mark E. [Applied Biosystems; Johnson, Mahlon D. [University of Tennessee Graduate School of Medicine; Ruff, David W. [Applied Biosystems; Hughes, Lori A [ORNL; Kerley, Marilyn K [ORNL; Carpenter, D A [ORNL; Johnson, Dabney K [ORNL; Rinchik, Eugene M. [University of Tennessee, Knoxville (UTK) & Oak Ridge National Laboratory (ORNL); Culiat, Cymbeline T [ORNL

    2006-01-01

    The mammalian Nell1 gene encodes a protein kinase C-b1 (PKC-b1) binding protein that belongs to a new class of cell-signaling molecules controlling cell growth and differentiation. Over-expression of Nell1 in the developing cranial sutures in both human and mouse induces craniosynostosis, the premature fusion of the growing cranial bone fronts. Here, we report the generation, positional cloning and characterization of Nell16R, a recessive, neonatal-lethal point mutation in the mouse Nell1 gene, induced by N-ethyl-N-nitrosourea. Nell16R has a T!A base change that converts a codon for cysteine into a premature stop codon [Cys(502)Ter], resulting in severe truncation of the predicted protein product and marked reduction in steady-state levels of the transcript. In addition to the expected alteration of cranial morphology, Nell16R mutants manifest skeletal defects in the vertebral column and ribcage, revealing a hitherto undefined role for Nell1 in signal transduction in endochondral ossification. Real-time quantitative reverse transcription-PCR assays of 219 genes showed an association between the loss of Nell1 function and reduced expression of genes for extracellular matrix (ECM) proteins critical for chondrogenesis and osteogenesis. Several affected genes are involved in the human cartilage disorder Ehlers-Danlos Syndrome and other disorders associated with spinal curvature anomalies. Nell16R mutant mice are a new tool for elucidating basic mechanisms in osteoblast and chrondrocyte differentiation in the developing skull and vertebral column and understanding how perturbations in the production of ECM proteins can lead to anomalies in these structures.

  9. Implication of NOD1 and NOD2 for the differentiation of multipotent mesenchymal stem cells derived from human umbilical cord blood.

    Directory of Open Access Journals (Sweden)

    Hyung-Sik Kim

    Full Text Available Toll-like receptors (TLRs and Nod-like receptors (NLRs are known to trigger an innate immune response against microbial infection. Although studies suggest that activation of TLRs modulate the function of mesenchymal stem cells (MSCs, little is known about the role of NLRs on the MSC function. In this study, we investigated whether NOD1 and NOD2 regulate the functions of human umbilical cord blood-derived MSCs (hUCB-MSCs. The genes of TLR2, TLR4, NOD1, and NOD2 were expressed in hUCB-MSCs. Stimulation with each agonist (Pam(3CSK(4 for TLR2, LPS for TLR4, Tri-DAP for NOD1, and MDP for NOD2 led to IL-8 production in hUCB-MSC, suggesting the expressed receptors are functional in hUCB-MSC. CCK-8 assay revealed that none of agonist influenced proliferation of hUCB-MSCs. We next examined whether TLR and NLR agonists affect osteogenic-, adipogenic-, and chondrogenic differentiation of hUCB-MSCs. Pam(3CSK(4 and Tri-DAP strongly enhanced osteogenic differentiation and ERK phosphorylation in hUCB-MSCs, and LPS and MDP also slightly did. Treatment of U0126 (MEK1/2 inhibitor restored osteogenic differentiation enhanced by Pam(3CSK(4. Tri-DAP and MDP inhibited adipogenic differentiation of hUCB-MSCs, but Pam(3CSK(4 and LPS did not. On chondrogenic differentiation, all TLR and NLR agonists could promote chondrogenesis of hUCB-MSCs with difference in the ability. Our findings suggest that NOD1 and NOD2 as well as TLRs are involved in regulating the differentiation of MSCs.

  10. Additive manufactured polymeric 3D scaffolds with tailored surface topography influence mesenchymal stromal cells activity.

    Science.gov (United States)

    Neves, Sara C; Mota, Carlos; Longoni, Alessia; Barrias, Cristina C; Granja, Pedro L; Moroni, Lorenzo

    2016-06-01

    Additive manufactured three-dimensional (3D) scaffolds with tailored surface topography constitute a clear advantage in tissue regeneration strategies to steer cell behavior. 3D fibrous scaffolds of poly(ethylene oxide terephthalate)/poly(butylene terephthalate) block copolymer presenting different fiber surface features were successfully fabricated by additive manufacturing combined with wet-spinning, in a single step, without any post-processing. The optimization of the processing parameters, mainly driven by different solvent/non-solvent combinations, led to four distinct scaffold types, with average surface roughness values ranging from 0.071 ± 0.012 μm to 1.950 ± 0.553 μm, average pore sizes in the x- and y-axis between 351.1 ± 33.6 μm and 396.1 ± 32.3 μm, in the z-axis between 36.5 ± 5.3 μm and 70.7 ± 8.8 μm, average fiber diameters between 69.4 ± 6.1 μm and 99.0 ± 9.4 μm, and porosity values ranging from 60.2 ± 0.8% to 71.7 ± 2.6%. Human mesenchymal stromal cells (hMSCs) cultured on these scaffolds adhered, proliferated, and produced endogenous extracellular matrix. The effect of surface roughness and topography on hMSCs differentiation was more evident for cells seeded at lower density, where the percentage of cells in direct contact with the surface was higher compared to more densely seeded scaffolds. Under osteogenic conditions, lower surface roughness values (0.227 ± 0.035 μm) had a synergistic effect on hMSCs behavior, while chondrogenesis was favored on rougher surfaces (1.950 ± 0.553 μm). PMID:27219645

  11. Chondroregulatory action of prolactin on proliferation and differentiation of mouse chondrogenic ATDC5 cells in 3-dimensional micromass cultures

    Energy Technology Data Exchange (ETDEWEB)

    Seriwatanachai, Dutmanee [Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok (Thailand); Krishnamra, Nateetip [Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok (Thailand); Department of Physiology, Faculty of Science, Mahidol University, Bangkok (Thailand); Charoenphandhu, Narattaphol, E-mail: naratt@narattsys.com [Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok (Thailand); Department of Physiology, Faculty of Science, Mahidol University, Bangkok (Thailand)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Mouse chondrogenic ATDC5 cells expressed PRL receptor mRNAs and proteins. Black-Right-Pointing-Pointer Low PRL concentration (10 ng/mL) increased chondrocyte viability and differentiation. Black-Right-Pointing-Pointer Higher PRL concentrations ( Greater-Than-Or-Slanted-Equal-To 100 ng/mL) decreased viability and increased apoptosis. -- Abstract: A recent investigation in lactating rats has provided evidence that the lactogenic hormone prolactin (PRL) increases endochondral bone growth and bone elongation, presumably by accelerating apoptosis of hypertrophic chondrocytes in the growth plate and/or subsequent chondrogenic matrix mineralization. Herein, we demonstrated the direct chondroregulatory action of PRL on proliferation, differentiation and apoptosis of chondrocytes in 3-dimensional micromass culture of mouse chondrogenic ATDC5 cell line. The results showed that ATDC5 cells expressed PRL receptor (PRLR) transcripts, and responded typically to PRL by downregulating PRLR expression. Exposure to a low PRL concentration of 10 ng/mL, comparable to the normal levels in male and non-pregnant female rats, increased chondrocyte viability, differentiation, proteoglycan accumulation, and mRNA expression of several chondrogenic differentiation markers, such as Sox9, ALP and Hspg2. In contrast, high PRL concentrations of Greater-Than-Or-Slanted-Equal-To 100 ng/mL, comparable to the levels in pregnancy or lactation, decreased chondrocyte viability by inducing apoptosis, with no effect on chondrogenic marker expression. It could be concluded that chondrocytes directly but differentially responded to non-pregnant and pregnant/lactating levels of PRL, thus suggesting the stimulatory effect of PRL on chondrogenesis in young growing individuals, and supporting the hypothesis of hypertrophic chondrocyte apoptosis in the growth plate of lactating rats.

  12. A microRNA signature associated with chondrogenic lineage commitment

    Indian Academy of Sciences (India)

    Behnaz Bakhshandeh; Masoud Soleimani; Seyed Hassan Paylakhi; Nasser Ghaemi

    2012-08-01

    Generating appropriate cartilage for clinical applications to heal skeletal tissue loss is a major health concern. In this regard, cell-based approaches offer a potential therapeutic strategy for cartilage repair, although little is known about the precise mechanism of chondrogenesis. Unrestricted somatic stem cell (USSC) is considered as a suitable candidate because of its potential for differentiating into multiple cell types. Recent studies show that microRNAs (miRNAs) are involved in several biological processes including development and differentiation. To identify the chondro-specific miRNA signature, miRNA patterns of USSCs and differentiated chondrocytes were investigated using microarrays and validation by qPCR. Prior to these analyses, chondrogenic commitment of differentiated USSCs was verified by immunocytochemistry, specific staining and evaluation of some main chondrogenic marker genes. Various in silico explorations (for both putative targets and signalling pathways) and empirical analyses (miRNA transfections followed by qPCR of some chondrogenic indicators) were carried out to support our results. Transient modulation of multiple chondro-miRs (such as mir-630, mir-624 and mir-376) with chondrocyte targets (such as TGFbR, MAP3K, collagens, SMADs and cadherins) as mediators of chondrogenic signalling pathways including cell–cell interactions, TGF-beta, and MAPK signalling suggests a mechanism for genetic induction of chondrogenic differentiation. In conclusion, this research reveals more details about the allocation of USSCs into the chondrocytes through identification of miRNA signature which modulates targets and pathways required for chondrogenic lineage and could provide guidelines for future clinical treatments and anti-miRNA therapies.

  13. Poly (l-lactide-co-caprolactone) scaffolds enhanced with poly (β-hydroxybutyrate-co-β-hydroxyvalerate) microspheres for cartilage regeneration

    International Nuclear Information System (INIS)

    Biodegradable polymers, either as porous scaffolds or microspheres, have been investigated broadly for cartilage tissue engineering. A combination of these two forms of materials could potentially maximize their benefits. In this study, porous poly (l-lactide-co-ε-caprolactone) (PLCL) scaffolds were integrated with poly (β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV) microspheres to enhance the mechanical properties of the scaffolds as well as to potentially regulate cell behavior through altering surface topography. PHBV microspheres fabricated with an emulsion solvent evaporation method were incorporated into PLCL scaffolds (0%, 20%, 40% and 50% W/W). Compressive modulus, surface topography and porosity of the composite scaffolds were evaluated, and in vitro and in vivo chondrogenesis within the chondrocyte-laden scaffolds was investigated by examining proliferation of chondrocytes and the deposition of glycosaminoglycan (GAG) and type II collagen. The results showed significant enhancement of the compressive modulus of the scaffolds incorporated with PHBV microspheres, while Young's modulus of the scaffolds with 50% PHBV incorporation was 3.3 times higher than PLCL scaffolds alone. The porosity of the composite scaffolds was kept constant for all levels of PHBV incorporation. Though the PLCL scaffolds incorporated with microspheres showed no significant effects on adhesion at 6 h as well as in vitro cartilage formation and proliferation of the chondrocytes at both 2 weeks and 4 weeks, total contents of GAG and type II collagen excreted increased significantly with time. The chondrocyte-laden scaffolds formed cartilage-like tissues at 4 and 8 weeks after implantation in nude mice, with increased staining density of type II collagen and GAG over time. In conclusion, incorporation of PHBV microspheres not only enhanced the compressive modulus of PLCL scaffolds, but could also serve as scaffolding structures for cartilaginous tissue formation. (paper)

  14. Skeletal dysplasia in perinatal lethal osteogenesis imperfecta. A complex disorder of endochondral and intramembranous ossification.

    Science.gov (United States)

    Marion, M J; Gannon, F H; Fallon, M D; Mennuti, M T; Lodato, R F; Kaplan, F S

    1993-08-01

    Osteogenesis imperfecta (OI) Type II is a rare heritable disorder of bone matrix that results in catastrophic congenital skeletal dysplasia. Two cases of OI Type II had symmetric rhizomelic skeletal dysplasia apparent on ultrasound at 16 and 20 weeks' gestation. Histologic and histochemical studies performed on skeletal tissue from fetal autopsies showed the following: (1) abnormal growth plate tissue characterized by failure of formation of primary bony spongiosa; (2) persistence of calcified cartilage bars in the diaphysis; (3) metaphyseal microfractures; (4) abundant cartilaginous fracture callus; (5) absence of bony callus; (6) failure of formation of intramembranous cortical diaphyseal bone; (7) angulation of long bones in portions of the metadiaphyses bordered by fracture callus; and (8) mechanical failure of the perichondral ring of LaCroix with a normal fibrous ossification groove of Ranvier. These findings suggest that skeletal dysplasia in OI Type II results from the action of muscular forces on a skeleton weakened by a complex disorder of endochondral and intramembranous ossification. The paucity of primary metaphyseal trabeculae and subperiosteal cortical bone leads to pathologic fractures of the immature fiber bone and an imperfect attempt at fracture repair. Angulation and shortening of long bones occurs between numerous sites of focal endochondral fracture callus. Mechanical failure of the fibrous perichondral ring leads to further collapse and shortening without obvious functional impairment of the fibrous ossification groove. Perinatal lethal OI provides insight into how a molecular disorder predominantly of Type I collagen metabolism results in pathology of numerous tissues, leading to severe skeletal dysplasia without primarily affecting chondrogenesis. PMID:8339500

  15. BMP-2 Is Involved in Scleral Remodeling in Myopia Development.

    Directory of Open Access Journals (Sweden)

    Honghui Li

    Full Text Available The development of myopia is associated with scleral remodeling, but it is unclear which factors regulate this process. This study investigated bone morphogenetic protein-2 (BMP-2 expression in the sclera of guinea pigs with lens-induced myopia (LIM and after recovery from myopia and evaluated the effect of BMP-2 on extracellular matrix (ECM synthesis in human scleral fibroblasts (HSFs cultured in vitro. Lens-induced myopia was brought about in two groups of guinea pigs (the lens-induced myopia and myopia recovery groups by placing -4.00 D lenses on the right eye for three weeks. The left eye served as a contralateral control. In the recovery group, the lenses were removed after one week. The refractive power and axial length of the eyes were measured, and the BMP-2 expression levels in the sclera were measured. After three weeks, the lens-induced eyes acquired relative myopia in both groups of guinea pigs. Immunostaining of the eyeballs revealed significantly decreased BMP-2 expression in the posterior sclera of the myopic eyes compared to the contralateral eyes. One week after lens removal, BMP-2 expression recovered, and no differences were observed between the experimental and contralateral eyes in the recovery group. HSFs were cultured with BMP-2 or transforming growth factor-β1 (TGF-β1. Type I and type III collagen synthesis was significantly up-regulated following BMP-2 treatment in culture after one and two weeks, but the ratio of type III to type I collagen mRNA was not increased. Biosynthesis of glycosaminoglycan (GAG and aggrecan was increased in HSFs treated with BMP-2. Some chondrogenesis-associated genes expression increased in HSFs treated with BMP-2. From this study, we concluded that BMP-2 is involved in scleral remodeling in the development and recovery of lens-induced myopia.

  16. BMP-2 Is Involved in Scleral Remodeling in Myopia Development

    Science.gov (United States)

    Li, Honghui; Cui, Dongmei; Zhao, Feng; Huo, Lijun; Hu, Jianmin; Zeng, Junwen

    2015-01-01

    The development of myopia is associated with scleral remodeling, but it is unclear which factors regulate this process. This study investigated bone morphogenetic protein-2 (BMP-2) expression in the sclera of guinea pigs with lens-induced myopia (LIM) and after recovery from myopia and evaluated the effect of BMP-2 on extracellular matrix (ECM) synthesis in human scleral fibroblasts (HSFs) cultured in vitro. Lens-induced myopia was brought about in two groups of guinea pigs (the lens-induced myopia and myopia recovery groups) by placing -4.00 D lenses on the right eye for three weeks. The left eye served as a contralateral control. In the recovery group, the lenses were removed after one week. The refractive power and axial length of the eyes were measured, and the BMP-2 expression levels in the sclera were measured. After three weeks, the lens-induced eyes acquired relative myopia in both groups of guinea pigs. Immunostaining of the eyeballs revealed significantly decreased BMP-2 expression in the posterior sclera of the myopic eyes compared to the contralateral eyes. One week after lens removal, BMP-2 expression recovered, and no differences were observed between the experimental and contralateral eyes in the recovery group. HSFs were cultured with BMP-2 or transforming growth factor-β1 (TGF-β1). Type I and type III collagen synthesis was significantly up-regulated following BMP-2 treatment in culture after one and two weeks, but the ratio of type III to type I collagen mRNA was not increased. Biosynthesis of glycosaminoglycan (GAG) and aggrecan was increased in HSFs treated with BMP-2. Some chondrogenesis-associated genes expression increased in HSFs treated with BMP-2. From this study, we concluded that BMP-2 is involved in scleral remodeling in the development and recovery of lens-induced myopia. PMID:25965995

  17. Isolation and characterization of multipotential mesenchymal cells from the mouse synovium.

    Directory of Open Access Journals (Sweden)

    Ippei Futami

    Full Text Available The human synovium contains mesenchymal stem cells (MSCs, which are multipotential non-hematopoietic progenitor cells that can differentiate into a variety of mesenchymal lineages and they may therefore be a candidate cell source for tissue repair. However, the molecular mechanisms by which this can occur are still largely unknown. Mouse primary cell culture enables us to investigate the molecular mechanisms underlying various phenomena because it allows for relatively easy gene manipulation, which is indispensable for the molecular analysis. However, mouse synovial mesenchymal cells (SMCs have not been established, although rabbit, cow, and rat SMCs are available, in addition to human MSCs. The aim of this study was to establish methods to harvest the synovium and to isolate and culture primary SMCs from mice. As the mouse SMCs were not able to be harvested and isolated using the same protocol for human, rat and rabbit SMCs, the protocol for humans was modified for SMCs from the Balb/c mouse knee joint. The mouse SMCs obtained showed superior proliferative potential, growth kinetics and colony formation compared to cells derived from muscle and bone marrow. They expressed PDGFRá and Sca-1 detected by flow cytometry, and showed an osteogenic, adipogenic and chondrogenic potential similar or superior to the cells derived from muscle and bone marrow by demonstrating in vitro osteogenesis, adipogenesis and chondrogenesis. In conclusion, we established a primary mouse synovial cell culture method. The cells derived from the mouse synovium demonstrated both the ability to proliferate and multipotentiality similar or superior to the cells derived from muscle and bone marrow.

  18. BMP and TGFbeta pathways in human central chondrosarcoma: enhanced endoglin and Smad 1 signaling in high grade tumors

    International Nuclear Information System (INIS)

    As major regulators of normal chondrogenesis, the bone morphogenic protein (BMP) and transforming growth factor β (TGFB) signaling pathways may be involved in the development and progression of central chondrosarcoma. In order to uncover their possible implication, the aim of this study was to perform a systematic quantitative study of the expression of BMPs, TGFBs and their receptors and to assess activity of the corresponding pathways in central chondrosarcoma. Gene expression analysis was performed by quantitative RT-PCR in 26 central chondrosarcoma and 6 healthy articular cartilage samples. Expression of endoglin and nuclear localization of phosphorylated Smad1/5/8 and Smad2 was assessed by immunohistochemical analysis. The expression of TGFB3 and of the activin receptor-like kinase ALK2 was found to be significantly higher in grade III compared to grade I chondrosarcoma. Nuclear phosphorylated Smad1/5/8 and Smad2 were found in all tumors analyzed and the activity of both signaling pathways was confirmed by functional reporter assays in 2 chondrosarcoma cell lines. Immunohistochemical analysis furthermore revealed that phosphorylated Smad1/5/8 and endoglin expression were significantly higher in high-grade compared to low-grade chondrosarcoma and correlated to each other. The BMP and TGFβ signaling pathways were found to be active in central chondrosarcoma cells. The correlation of Smad1/5/8 activity to endoglin expression suggests that, as described in other cell types, endoglin could enhance Smad1/5/8 signaling in high-grade chondrosarcoma cells. Endoglin expression coupled to Smad1/5/8 activation could thus represent a functionally important signaling axis for the progression of chondrosarcoma and a regulator of the undifferentiated phenotype of high-grade tumor cells

  19. Isolation of mesenchymal stem cells from equine umbilical cord blood

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    Thomsen Preben D

    2007-05-01

    Full Text Available Abstract Background There are no published studies on stem cells from equine cord blood although commercial storage of equine cord blood for future autologous stem cell transplantations is available. Mesenchymal stem cells (MSC have been isolated from fresh umbilical cord blood of humans collected non-invasively at the time of birth and from sheep cord blood collected invasively by a surgical intrauterine approach. Mesenchymal stem cells isolation percentage from frozen-thawed human cord blood is low and the future isolation percentage of MSCs from cryopreserved equine cord blood is therefore expectedly low. The hypothesis of this study was that equine MSCs could be isolated from fresh whole equine cord blood. Results Cord blood was collected from 7 foals immediately after foaling. The mononuclear cell fraction was isolated by Ficoll density centrifugation and cultured in a DMEM low glucose based media at 38.5°C in humidified atmosphere containing 5% CO2. In 4 out of 7 samples colonies with MSC morphology were observed. Cellular morphology varied between monolayers of elongated spindle-shaped cells to layered cell clusters of cuboidal cells with shorter cytoplasmic extensions. Positive Alizarin Red and von Kossa staining as well as significant calcium deposition and alkaline phosphatase activity confirmed osteogenesis. Histology and positive Safranin O staining of matrix glycosaminoglycans illustrated chondrogenesis. Oil Red O staining of lipid droplets confirmed adipogenesis. Conclusion We here report, for the first time, the isolation of mesenchymal-like stem cells from fresh equine cord blood and their differentiation into osteocytes, chondrocytes and adipocytes. This novel isolation of equine cord blood MSCs and their preliminary in vitro differentiation positions the horse as the ideal pre-clinical animal model for proof-of-principle studies of cord blood derived MSCs.

  20. Projection Stereolithographic Fabrication of Human Adipose Stem Cell-incorporated Biodegradable Scaffolds for Cartilage Tissue Engineering

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    Aaron X Sun

    2015-08-01

    Full Text Available Poor self-healing ability of cartilage necessitates the development of methods for cartilage regeneration. Scaffold construction with live stem cell incorporation and subsequent differentiation presents a promising route. Projection stereolithography (PSL offers high resolution and processing speed as well as the ability to fabricate scaffolds that precisely fit the anatomy of cartilage defects using medical imaging as the design template. We report here the use of a visible-light based PSL (VL-PSL system to encapsulate human adipose-derived stem cells (hASCs into a biodegradable polymer (poly-D,L-lactic acid/polyethylene glycol/ poly-D,L-lactic acid (PDLLA-PEG/hyaluronic acid (HA matrix to produce live cell constructs with customized architectures. After fabrication, hASCs showed high viability (84% and were uniformly distributed throughout the constructs, which possessed high mechanical property with a compressive modulus of 780 kPa. The hASC-seeded constructs were then cultured in Control or TGF-β3-containing chondrogenic medium for up to 28 days. In chondrogenic medium treated group (TGF-β3 group hASCs maintained 77% viability and expressed chondrogenic genes Sox9, collagen type II, and aggrecan at 11, 232, and 2.29 x 10(5 fold increases, respectively, compared to levels at day 0 in non-chondrogenic medium. The TGF-β3 group also produced a collagen type II and glycosaminoglycan (GAG-rich extracellular matrix, detected by immunohistochemistry, and Alcian blue and Safranin O staining suggesting robust chondrogenesis within the scaffold. Without chondroinductive addition (Control group, cell viability decreased with time (65% at 28 days and showed poor cartilage matrix deposition. After 28 days, mechanical strength of the TGF-β3 group remained high at 240 kPa. Thus, the PSL- and PLLA-PEG/HA based fabrication method using adult stem cells is a promising approach in producing mechanically competent engineered cartilage for joint cartilage

  1. Fibrodysplasia ossificans progressiva: a clue from the fly?

    Science.gov (United States)

    Kaplan, F S; Tabas, J A; Zasloff, M A

    1990-08-01

    Fibrodysplasia (myositis) ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by symmetrical congenital malformations of the blastemal anlage of the hands and feet and by progressive heterotopic chondrogenesis and ossification of the soft connective tissues. There is neither an established pathogenesis nor an effective treatment for this disabling disorder. We reevaluated the published data on the natural history of FOP and discovered an array of developmental gradients (characteristic patterns of disease expression) similar to developmental anomalies induced by pleiotropic mutations of the decapentaplegic (dpp) locus in Drosophila melanogaster. The protein encoded by the dpp locus is a member of the transforming growth factor-beta (TGF-beta) family of molecules. It shares 75% sequence homology with the c-terminal region of two recently cloned human bone morphogenetic proteins (BMP-2A, BMP-2B), also members of the TGF-beta family. The striking sequence identity across so large an evolutionary distance suggests that the BMP-2 genes in man and the dpp gene in the fly may be derived from a common ancestral gene. BMP is the only molecule discovered thus far that is capable of inducing endochondral ossification in vivo. Expression of endochondral bone formation is the basis for limb formation in embryogenesis, longitudinal bone growth in postnatal life, and local bone regeneration (fracture healing) following injury. We believe that FOP is a genetic disorder characterized by a disturbed developmental expression of this endochondral program and may represent a mutation resulting in a dominant gain of function. The developmental similarities between decapentaplegic in the fly and FOP in man suggest a useful model for the study of FOP. The use of such a model might be especially fruitful in suggesting a molecular basis for FOP. PMID:2117991

  2. The Experimental Studies of the Tissue Engineering Cartilage by Co-Culturing Microtia Chondrocytes and Adipose Tissue-Derived Stem Cells in Vivo%残耳软骨细胞与脂肪干细胞共培养体内构建软骨的实验研究

    Institute of Scientific and Technical Information of China (English)

    张洁; 蒋海越; 何乐仁; 赵延勇; 杨庆华; 韩娟; 宋宇鹏

    2011-01-01

    Objective To explore the feasibility of the chondrogenesis by co-culturing microtia chondrocytes and human adipose tissue-derived stem cells in vivo. Methods hADSCs and microtia chondrocytes were isolated in vitro. 24 nude mice were randomly divided into 4 groups: ①Exp group, injected with microtia chondrocytes and hADSCs by a mixing ratio of 1:1 and the cell concentration was 5.0×l07 cells/mL; ②Ctrl 1 group, injected with only microtia chondrocytes and the cell concentration was 5.0×107 cellshnL; ③Ctrl 2 group, injected with only hADSCs and the cell concentration was 5.0×l07 cells/mL;④Ctrl 3 group, injected with only microtia chondrocytes and the cell concentration was 2.5×107 cells/mL. 6 nude mice were injected each group at a dose of 0.2 mL. All samples were harvested 10 weeks after culturingin vivo. Gross observation, average wet weights, glycosaminoglycan (GAG) quantification, histology and immunohistochemisty were used to evaluate the chondrogenesis of all groups. Results In Exp, Ctrl 1, and Ctrl 3 group, all the specimens formed homogeneous cartilagelike tissue with typical histological structure at different extent. In Ctrl 2 group, the specimens formed fiber-like tissue.Average wet weight and GAG content of specimens in Exp group were more than 88% of Ctrl 1 group while they were less than 40% in Ctrl 3 group. Cartilage lacuna was detected by HE staining in Exp, Ctrl 1 and Ctrl 3 group at different extent,but not in Ctrl 2 group. Collagen type Ⅱ was detected by immunohistochemistry in Exp, Ctrl 1 and Ctrl 3 group at different extent, but not in Ctrl 2 group. Conclusion Microtia chondrocytes could promote chondrogenesis of ADSCs in vivo under the co-culturing system. Tissue engineering cartilage by co-culturing microtia chondrocytes and ADSCs in vivo is feasible.%目的 验证残耳软骨细胞与脂肪来源的间充质干细胞(Adipose derived stem cells,ADSCs)共培养,体内构建软骨的可行性.方法 分离培养同一先天性

  3. Immortalisation with hTERT Impacts on Sulphated Glycosaminoglycan Secretion and Immunophenotype in a Variable and Cell Specific Manner.

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    Tina P Dale

    Full Text Available Limited options for the treatment of cartilage damage have driven the development of tissue engineered or cell therapy alternatives reliant on ex vivo cell expansion. The study of chondrogenesis in primary cells is difficult due to progressive cellular aging and senescence. Immortalisation via the reintroduction of the catalytic component of telomerase, hTERT, could allow repeated, longitudinal studies to be performed while bypassing senescent phenotypes.Three human cell types: bone marrow-derived stromal cells (BMA13, embryonic stem cell-derived (1C6 and chondrocytes (OK3 were transduced with hTERT (BMA13H, 1C6H and OK3H and proliferation, surface marker expression and tri-lineage differentiation capacity determined. The sulphated glycosaminoglycan (sGAG content of the monolayer and spent media was quantified in maintenance media (MM and pro-chondrogenic media (PChM and normalised to DNA.hTERT expression was confirmed in transduced cells with proliferation enhancement in 1C6H and OK3H cells but not BMA13H. All cells were negative for leukocyte markers (CD19, CD34, CD45 and CD73 positive. CD14 was expressed at low levels on OK3 and OK3H and HLA-DR on BMA13 (84.8%. CD90 was high for BMA13 (84.9% and OK3 (97.3% and moderate for 1C6 (56.7%, expression was reduced in BMA13H (33.7% and 1C6H (1.6%. CD105 levels varied (BMA13 87.7%, 1C6 8.2%, OK3 43.3% and underwent reduction in OK3H (25.1%. 1C6 and BMA13 demonstrated osteogenic and adipogenic differentiation but mineralised matrix and lipid accumulation appeared reduced post hTERT transduction. Chondrogenic differentiation resulted in increased monolayer-associated sGAG in all primary cells and 1C6H (p<0.001, and BMA13H (p<0.05. In contrast OK3H demonstrated reduced monolayer-associated sGAG in PChM (p<0.001. Media-associated sGAG accounted for ≥55% (PChM-1C6 and ≥74% (MM-1C6H.In conclusion, hTERT transduction could, but did not always, prevent senescence and cell phenotype, including

  4. The effects of dynamic and three-dimensional environments on chondrogenic differentiation of bone marrow stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Youngmee; Kim, Sang-Heon; Kim, Soo Hyun [Biomaterials Research Center, Korea Institute of Science and Technology, PO Box 131, Cheonryang, Seoul, 130-650 (Korea, Republic of); Kim, Young Ha, E-mail: soohkim@kist.re.k [Department of Materials Science and Engineering, Gwangju Institute of Science and Technology, 261 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712 (Korea, Republic of)

    2009-10-15

    Articular cartilage is subjected to complex loading, which plays a major role in its growth, development and maintenance. Previously, we found that mechanical stimuli enhanced the development and function of engineered cartilage tissues in elastic mechano-active poly(lactide-co-caprolactone) (PLCL) scaffolds. In addition, it is well known that the three-dimensional spatial organization of cells and extracellular matrices in hydrogels is crucial to chondrogenesis. This study was conducted to enhance the chondrogenic differentiation of bone marrow stromal cells (BMSCs) in the hybrid scaffolds of fibrin gels and PLCL scaffolds in dynamic environments by compression. A highly elastic scaffold was fabricated from very elastic PLCL with 85% porosity and a 300-500{mu}m pore size using a gel-pressing method. A mixture of rabbit BMSCs and fibrin gels was then seeded onto the PLCL scaffolds and subjected to continuous compressive deformation of 5% strain at 0.1 Hz for 10 days in a chondrogenic medium containing 10 ng ml{sup -1} TGF-beta{sub 1}. The BMSCs-seeded scaffold constructs were then implanted subcutaneously into nude mice. As a control, the cell-PLCL scaffold constructs were cultured under dynamic conditions or the cell-PLCL/fibrin hybrid scaffold constructs and the cell-PLCL scaffold constructs were cultured under static conditions for 10 days in vitro. The results revealed that cells adhered onto the hybrid scaffolds of fibrin gels and PLCL scaffolds cultured under dynamic conditions. In addition, the accumulation of the extracellular matrix of cell-scaffold constructs, which was increased through mechanical stimulation, showed that chondrogenic differentiation was sustained and enhanced significantly in the stimulated hybrid scaffold constructs. Overall, the results of this study indicate that the proper periodic application of dynamic compression and the three-dimensional environments of the hybrid scaffolds composed of fibrin gels and elastic PLCL can encourage

  5. Cartilage-specific over-expression of CCN family member 2/connective tissue growth factor (CCN2/CTGF stimulates insulin-like growth factor expression and bone growth.

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    Nao Tomita

    Full Text Available Previously we showed that CCN family member 2/connective tissue growth factor (CCN2 promotes the proliferation, differentiation, and maturation of growth cartilage cells in vitro. To elucidate the specific role and molecular mechanism of CCN2 in cartilage development in vivo, in the present study we generated transgenic mice overexpressing CCN2 and analyzed them with respect to cartilage and bone development. Transgenic mice were generated expressing a ccn2/lacZ fusion gene in cartilage under the control of the 6 kb-Col2a1-enhancer/promoter. Changes in cartilage and bone development were analyzed histologically and immunohistologically and also by micro CT. Primary chondrocytes as well as limb bud mesenchymal cells were cultured and analyzed for changes in expression of cartilage-related genes, and non-transgenic chondrocytes were treated in culture with recombinant CCN2. Newborn transgenic mice showed extended length of their long bones, increased content of proteoglycans and collagen II accumulation. Micro-CT analysis of transgenic bones indicated increases in bone thickness and mineral density. Chondrocyte proliferation was enhanced in the transgenic cartilage. In in vitro short-term cultures of transgenic chondrocytes, the expression of col2a1, aggrecan and ccn2 genes was substantially enhanced; and in long-term cultures the expression levels of these genes were further enhanced. Also, in vitro chondrogenesis was strongly enhanced. IGF-I and IGF-II mRNA levels were elevated in transgenic chondrocytes, and treatment of non-transgenic chondrocytes with recombinant CCN2 stimulated the expression of these mRNA. The addition of CCN2 to non-transgenic chondrocytes induced the phosphorylation of IGFR, and ccn2-overexpressing chondrocytes showed enhanced phosphorylation of IGFR. Our data indicates that the observed effects of CCN2 may be mediated in part by CCN2-induced overexpression of IGF-I and IGF-II. These findings indicate that CCN2

  6. Effects of bound versus soluble pentosan polysulphate in PEG/HA-based hydrogels tailored for intervertebral disc regeneration.

    Science.gov (United States)

    Frith, Jessica E; Menzies, Donna J; Cameron, Andrew R; Ghosh, P; Whitehead, Darryl L; Gronthos, S; Zannettino, Andrew C W; Cooper-White, Justin J

    2014-01-01

    Previous reports in the literature investigating chondrogenesis in mesenchymal progenitor cell (MPC) cultures have confirmed the chondro-inductive potential of pentosan polysulphate (PPS), a highly sulphated semi-synthetic polysaccharide, when added as a soluble component to culture media under standard aggregate-assay conditions or to poly(ethylene glycol)/hyaluronic acid (PEG/HA)-based hydrogels, even in the absence of inductive factors (e.g. TGFβ). In this present study, we aimed to assess whether a 'bound' PPS would have greater activity and availability over a soluble PPS, as a media additive or when incorporated into PEG/HA-based hydrogels. We achieved this by covalently pre-binding the PPS to the HA component of the gel (forming a new molecule, HA-PPS). We firstly investigated the activity of HA-PPS compared to free PPS, when added as a soluble factor to culture media. Cell proliferation, as determined by CCK8 and EdU assay, was decreased in the presence of either bound or free PPS whilst chondrogenic differentiation, as determined by DMMB assay and histology, was enhanced. In all cases, the effect of the bound PPS (HA-PPS) was more potent than that of the unbound form. These results alone suggest wider applications for this new molecule, either as a culture supplement or as a coating for scaffolds targeted at chondrogenic differentiation or maturation. We then investigated the incorporation of HA-PPS into a PEG/HA-based hydrogel system, by simply substituting some of the HA for HA-PPS. Rheological testing confirmed that incorporation of either HA-PPS or PPS did not significantly affect gelation kinetics, final hydrogel modulus or degradation rate but had a small, but significant, effect on swelling. When encapsulated in the hydrogels, MPCs retained good viability and rapidly adopted a rounded morphology. Histological analysis of both GAG and collagen deposition after 21 days showed that the incorporation of the bound-PPS into the hydrogel resulted in

  7. Constitutive activation of IKK2/NF-κB impairs osteogenesis and skeletal development.

    Science.gov (United States)

    Swarnkar, Gaurav; Zhang, Kaihua; Mbalaviele, Gabriel; Long, Fanxin; Abu-Amer, Yousef

    2014-01-01

    Pathologic conditions impair bone homeostasis. The transcription factor NF-κB regulates bone homeostasis and is central to bone pathologies. Whereas contribution of NF-κB to heightened osteoclast activity is well-documented, the mechanisms underlying NF-κB impact on chondrocytes and osteoblasts are scarce. In this study, we examined the effect of constitutively active IKK2 (IKK2ca) on chondrogenic and osteogenic differentiation. We show that retroviral IKK2ca but not GFP, IKK2WT, or the inactive IKK2 forms IKK2KM and IKK2SSAA, strongly suppressed osteogenesis and chondrogenesis, in vitro. In order to explore the effect of constitutive NF-κB activation on bone formation in vivo, we activated this pathway in a conditional fashion. Specifically, we crossed the R26StopIKK2ca mice with mice carrying the Col2-cre in order to express IKK2ca in osteoblasts and chondrocytes. Both chondrocytes and osteoblasts derived from Col2Cre/IKK2ca expressed IKK2ca. Mice were born alive yet died shortly thereafter. Histologically, newborn Col2Cre+/RosaIKK2ca heterozygotes (Cre+IKK2ca_w/f (het)) and homozygotes (Cre+IKK2ca_f/f (KI)) showed smaller skeleton, deformed vertebrate and reduced or missing digit ossification. The width of neural arches, as well as ossification in vertebral bodies of Cre+IKK2ca_w/f and Cre+IKK2ca_f/f, was reduced or diminished. H&E staining of proximal tibia from new born pups revealed that Cre+IKK2ca_f/f displayed disorganized hypertrophic zones within the smaller epiphysis. Micro-CT analysis indicated that 4-wk old Cre+IKK2ca_w/f has abnormal trabecular bone in proximal tibia compared to WT littermates. Mechanistically, ex-vivo experiments showed that expression of differentiation markers in calvarial osteoblasts derived from newborn IKK2ca knock-in mice was diminished compared to WT-derived cells. In situ hybridization studies demonstrated that the hypertrophic chondrocyte marker type-X collagen, the pre-hypertrophic chondrocyte markers Indian hedgehog

  8. Chondrogenic differentiation of marrow clots after microfracture with BMSC-derived ECM scaffold in vitro.

    Science.gov (United States)

    Wei, Bo; Jin, Chengzhe; Xu, Yan; Du, Xiaotao; Yan, Chao; Tang, Cheng; Ansari, Mujtaba; Wang, Liming

    2014-10-01

    -dECM scaffold may favor the chondrogenesis of marrow clots following microfracture in vitro. In conclusion, these tissue engineering-like constructs could be potential candidates for cartilage repair. PMID:24665837

  9. Constitutive activation of IKK2/NF-κB impairs osteogenesis and skeletal development.

    Directory of Open Access Journals (Sweden)

    Gaurav Swarnkar

    Full Text Available Pathologic conditions impair bone homeostasis. The transcription factor NF-κB regulates bone homeostasis and is central to bone pathologies. Whereas contribution of NF-κB to heightened osteoclast activity is well-documented, the mechanisms underlying NF-κB impact on chondrocytes and osteoblasts are scarce. In this study, we examined the effect of constitutively active IKK2 (IKK2ca on chondrogenic and osteogenic differentiation. We show that retroviral IKK2ca but not GFP, IKK2WT, or the inactive IKK2 forms IKK2KM and IKK2SSAA, strongly suppressed osteogenesis and chondrogenesis, in vitro. In order to explore the effect of constitutive NF-κB activation on bone formation in vivo, we activated this pathway in a conditional fashion. Specifically, we crossed the R26StopIKK2ca mice with mice carrying the Col2-cre in order to express IKK2ca in osteoblasts and chondrocytes. Both chondrocytes and osteoblasts derived from Col2Cre/IKK2ca expressed IKK2ca. Mice were born alive yet died shortly thereafter. Histologically, newborn Col2Cre+/RosaIKK2ca heterozygotes (Cre+IKK2ca_w/f (het and homozygotes (Cre+IKK2ca_f/f (KI showed smaller skeleton, deformed vertebrate and reduced or missing digit ossification. The width of neural arches, as well as ossification in vertebral bodies of Cre+IKK2ca_w/f and Cre+IKK2ca_f/f, was reduced or diminished. H&E staining of proximal tibia from new born pups revealed that Cre+IKK2ca_f/f displayed disorganized hypertrophic zones within the smaller epiphysis. Micro-CT analysis indicated that 4-wk old Cre+IKK2ca_w/f has abnormal trabecular bone in proximal tibia compared to WT littermates. Mechanistically, ex-vivo experiments showed that expression of differentiation markers in calvarial osteoblasts derived from newborn IKK2ca knock-in mice was diminished compared to WT-derived cells. In situ hybridization studies demonstrated that the hypertrophic chondrocyte marker type-X collagen, the pre-hypertrophic chondrocyte markers

  10. Dmp1 Null Mice Develop a Unique Osteoarthritis-like Phenotype

    Science.gov (United States)

    Zhang, Qi; Lin, Shuxian; Liu, Ying; Yuan, Baozhi; Harris, Steph E; Feng, Jian Q.

    2016-01-01

    Patients with hypophosphatemia rickets (including DMP1 mutations) develop severe osteoarthritis (OA), although the mechanism is largely unknown. In this study, we first identified the expression of DMP1 in hypertrophic chondrocytes using immunohistochemistry (IHC) and X-gal analysis of Dmp1-knockout-lacZ-knockin heterozygous mice. Next, we characterized the OA-like phenotype in Dmp1 null mice from 7-week-old to one-year-old using multiple techniques, including X-ray, micro-CT, H&E staining, Goldner staining, scanning electronic microscopy, IHC assays, etc. We found a classical OA-like phenotype in Dmp1 null mice such as articular cartilage degradation, osteophyte formation, and subchondral osteosclerosis. These Dmp1 null mice also developed unique pathological changes, including a biphasic change in their articular cartilage from the initial expansion of hypertrophic chondrocytes at the age of 1-month to a quick diminished articular cartilage layer at the age of 3-months. Further, these null mice displayed severe enlarged knees and poorly formed bone with an expanded osteoid area. To address whether DMP1 plays a direct role in the articular cartilage, we deleted Dmp1 specifically in hypertrophic chondrocytes by crossing the Dmp1-loxP mice with Col X Cre mice. Interestingly, these conditional knockout mice didn't display notable defects in either the articular cartilage or the growth plate. Because of the hypophosphatemia remained in the entire life span of the Dmp1 null mice, we also investigated whether a high phosphate diet would improve the OA-like phenotype. A 8-week treatment of a high phosphate diet significantly rescued the OA-like defect in Dmp1 null mice, supporting the critical role of phosphate homeostasis in maintaining the healthy joint morphology and function. Taken together, this study demonstrates a unique OA-like phenotype in Dmp1 null mice, but a lack of the direct impact of DMP1 on chondrogenesis. Instead, the regulation of phosphate homeostasis

  11. Effects of bound versus soluble pentosan polysulphate in PEG/HA-based hydrogels tailored for intervertebral disc regeneration.

    Science.gov (United States)

    Frith, Jessica E; Menzies, Donna J; Cameron, Andrew R; Ghosh, P; Whitehead, Darryl L; Gronthos, S; Zannettino, Andrew C W; Cooper-White, Justin J

    2014-01-01

    Previous reports in the literature investigating chondrogenesis in mesenchymal progenitor cell (MPC) cultures have confirmed the chondro-inductive potential of pentosan polysulphate (PPS), a highly sulphated semi-synthetic polysaccharide, when added as a soluble component to culture media under standard aggregate-assay conditions or to poly(ethylene glycol)/hyaluronic acid (PEG/HA)-based hydrogels, even in the absence of inductive factors (e.g. TGFβ). In this present study, we aimed to assess whether a 'bound' PPS would have greater activity and availability over a soluble PPS, as a media additive or when incorporated into PEG/HA-based hydrogels. We achieved this by covalently pre-binding the PPS to the HA component of the gel (forming a new molecule, HA-PPS). We firstly investigated the activity of HA-PPS compared to free PPS, when added as a soluble factor to culture media. Cell proliferation, as determined by CCK8 and EdU assay, was decreased in the presence of either bound or free PPS whilst chondrogenic differentiation, as determined by DMMB assay and histology, was enhanced. In all cases, the effect of the bound PPS (HA-PPS) was more potent than that of the unbound form. These results alone suggest wider applications for this new molecule, either as a culture supplement or as a coating for scaffolds targeted at chondrogenic differentiation or maturation. We then investigated the incorporation of HA-PPS into a PEG/HA-based hydrogel system, by simply substituting some of the HA for HA-PPS. Rheological testing confirmed that incorporation of either HA-PPS or PPS did not significantly affect gelation kinetics, final hydrogel modulus or degradation rate but had a small, but significant, effect on swelling. When encapsulated in the hydrogels, MPCs retained good viability and rapidly adopted a rounded morphology. Histological analysis of both GAG and collagen deposition after 21 days showed that the incorporation of the bound-PPS into the hydrogel resulted in

  12. Expression of transmembrane carbonic anhydrases, CAIX and CAXII, in human development

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    Lerman Michael I

    2009-03-01

    Full Text Available Abstract Background Transmembrane CAIX and CAXII are members of the alpha carbonic anhydrase (CA family. They play a crucial role in differentiation, proliferation, and pH regulation. Expression of CAIX and CAXII proteins in tumor tissues is primarily induced by hypoxia and this is particularly true for CAIX, which is regulated by the transcription factor, hypoxia inducible factor-1 (HIF-1. Their distributions in normal adult human tissues are restricted to highly specialized cells that are not always hypoxic. The human fetus exists in a relatively hypoxic environment. We examined expression of CAIX, CAXII and HIF-1α in the developing human fetus and postnatal tissues to determine whether expression of CAIX and CAXII is exclusively regulated by HIF-1. Results The co-localization of CAIX and HIF-1α was limited to certain cell types in embryonic and early fetal tissues. Those cells comprised the primitive mesenchyma or involved chondrogenesis and skin development. Transient CAIX expression was limited to immature tissues of mesodermal origin and the skin and ependymal cells. The only tissues that persistently expressed CAIX protein were coelomic epithelium (mesothelium and its remnants, the epithelium of the stomach and biliary tree, glands and crypt cells of duodenum and small intestine, and the cells located at those sites previously identified as harboring adult stem cells in, for example, the skin and large intestine. In many instances co-localization of CAIX and HIF-1α was not evident. CAXII expression is restricted to cells involved in secretion and water absorption such as parietal cells of the stomach, acinar cells of the salivary glands and pancreas, epithelium of the large intestine, and renal tubules. Co-localization of CAXII with CAIX or HIF-1α was not observed. Conclusion The study has showed that: 1 HIF-1α and CAIX expression co- localized in many, but not all, of the embryonic and early fetal tissues; 2 There is no evidence of

  13. MR imaging features of gadofluorine-labeled matrix-associated stem cell implants in cartilage defects.

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    Hossein Nejadnik

    Full Text Available OBJECTIVES: The purpose of our study was to assess the chondrogenic potential and the MR signal effects of GadofluorineM-Cy labeled matrix associated stem cell implants (MASI in pig knee specimen. MATERIALS AND METHODS: Human mesenchymal stem cells (hMSCs were labeled with the micelle-based contrast agent GadofluorineM-Cy. Ferucarbotran-labeled hMSCs, non-labeled hMSCs and scaffold only served as controls. Chondrogenic differentiation was induced and gene expression and histologic evaluation were performed. The proportions of spindle-shaped vs. round cells of chondrogenic pellets were compared between experimental groups using the Fisher's exact test. Labeled and unlabeled hMSCs and chondrocytes in scaffolds were implanted into cartilage defects of porcine femoral condyles and underwent MR imaging with T1- and T2-weighted SE and GE sequences. Contrast-to-noise ratios (CNR between implants and adjacent cartilage were determined and analyzed for significant differences between different experimental groups using the Kruskal-Wallis test. Significance was assigned for p0.017. However, hMSC differentiation into chondrocytes was superior for unlabeled and GadofluorineM-Cy-labeled cells compared with Ferucarbotran-labeled cells, as evidenced by a significantly higher proportion of spindle cells in chondrogenic pellets (p<0.05. GadofluorineM-Cy-labeled hMSCs and chondrocytes showed a positive signal effect on T1-weighted images and a negative signal effect on T2-weighted images while Ferucarbotran-labeled cells provided a negative signal effect on all sequences. CNR data for both GadofluorineM-Cy-labeled and Ferucarbotran-labeled hMSCs were significantly different compared to unlabeled control cells on T1-weighted SE and T2*-weighted MR images (p<0.017. CONCLUSION: hMSCs can be labeled by simple incubation with GadofluorineM-Cy. The labeled cells provide significant MR signal effects and less impaired chondrogenesis compared to Ferucarbotran-labeled h

  14. Optimization and translation of MSC-based hyaluronic acid hydrogels for cartilage repair

    Science.gov (United States)

    Erickson, Isaac E.

    2011-12-01

    Traumatic injury and disease disrupt the ability of cartilage to carry joint stresses and, without an innate regenerative response, often lead to degenerative changes towards the premature development of osteoarthritis. Surgical interventions have yet to restore long-term mechanical function. Towards this end, tissue engineering has been explored for the de novo formation of engineered cartilage as a biologic approach to cartilage repair. Research utilizing autologous chondrocytes has been promising, but clinical limitations in their yield have motivated research into the potential of mesenchymal stem cells (MSCs) as an alternative cell source. MSCs are multipotent cells that can differentiate towards a chondrocyte phenotype in a number of biomaterials, but no combination has successfully recapitulated the native mechanical function of healthy articular cartilage. The broad objective of this thesis was to establish an MSC-based tissue engineering approach worthy of clinical translation. Hydrogels are a common class of biomaterial used for cartilage tissue engineering and our initial work demonstrated the potential of a photo-polymerizable hyaluronic acid (HA) hydrogel to promote MSC chondrogenesis and improved construct maturation by optimizing macromer and MSC seeding density. The beneficial effects of dynamic compressive loading, high MSC density, and continuous mixing (orbital shaker) resulted in equilibrium modulus values over 1 MPa, well in range of native tissue. While compressive properties are crucial, clinical translation also demands that constructs stably integrate within a defect. We utilized a push-out testing modality to assess the in vitro integration of HA constructs within artificial cartilage defects. We established the necessity for in vitro pre-maturation of constructs before repair to achieve greater integration strength and compressive properties in situ. Combining high MSC density and gentle mixing resulted in integration strength over 500 k

  15. Direct lentiviral-cyclooxygenase 2 application to the tendon-bone interface promotes osteointegration and enhances return of the pull-out tensile strength of the tendon graft in a rat model of biceps tenodesis.

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    Charles H Rundle

    Full Text Available This study sought to determine if direct application of the lentiviral (LV-cyclooxygenase 2 (COX2 vector to the tendon-bone interface would promote osteointegration of the tendon graft in a rat model of biceps tenodesis. The LV-COX2 gene transfer strategy was chosen for investigation because a similar COX2 gene transfer strategy promoted bony bridging of the fracture gap during bone repair, which involves similar histologic transitions that occur in osteointegration. Briefly, a 1.14-mm diameter tunnel was drilled in the mid-groove of the humerus of adult Fischer 344 rats. The LV-COX2 or βgal control vector was applied directly into the bone tunnel and onto the end of the tendon graft, which was then pulled into the bone tunnel. A poly-L-lactide pin was press-fitted into the tunnel as interference fixation. Animals were sacrificed at 3, 5, or 8 weeks for histology analysis of osteointegration. The LV-COX2 gene transfer strategy enhanced neo-chondrogenesis at the tendon-bone interface but with only marginal effect on de novo bone formation. The tendon-bone interface of the LV-COX2-treated tenodesis showed the well-defined tendon-to-fibrocartilage-to-bone histologic transitions that are indicative of osteointegration of the tendon graft. The LV-COX2 in vivo gene transfer strategy also significantly enhanced angiogenesis at the tendon-bone interface. To determine if the increased osteointegration was translated into an improved pull-out mechanical strength property, the pull-out tensile strength of the LV-COX2-treated tendon grafts was determined with a pull-out mechanical testing assay. The LV-COX2 strategy yielded a significant improvement in the return of the pull-out strength of the tendon graft after 8 weeks. In conclusion, the COX2-based in vivo gene transfer strategy enhanced angiogenesis, osteointegration and improved return of the pull-out strength of the tendon graft. Thus, this strategy has great potential to be developed into an

  16. Powerful bivariate genome-wide association analyses suggest the SOX6 gene influencing both obesity and osteoporosis phenotypes in males.

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    Yao-Zhong Liu

    Full Text Available BACKGROUND: Current genome-wide association studies (GWAS are normally implemented in a univariate framework and analyze different phenotypes in isolation. This univariate approach ignores the potential genetic correlation between important disease traits. Hence this approach is difficult to detect pleiotropic genes, which may exist for obesity and osteoporosis, two common diseases of major public health importance that are closely correlated genetically. PRINCIPAL FINDINGS: To identify such pleiotropic genes and the key mechanistic links between the two diseases, we here performed the first bivariate GWAS of obesity and osteoporosis. We searched for genes underlying co-variation of the obesity phenotype, body mass index (BMI, with the osteoporosis risk phenotype, hip bone mineral density (BMD, scanning approximately 380,000 SNPs in 1,000 unrelated homogeneous Caucasians, including 499 males and 501 females. We identified in the male subjects two SNPs in intron 1 of the SOX6 (SRY-box 6 gene, rs297325 and rs4756846, which were bivariately associated with both BMI and hip BMD, achieving p values of 6.82x10(-7 and 1.47x10(-6, respectively. The two SNPs ranked at the top in significance for bivariate association with BMI and hip BMD in the male subjects among all the approximately 380,000 SNPs examined genome-wide. The two SNPs were replicated in a Framingham Heart Study (FHS cohort containing 3,355 Caucasians (1,370 males and 1,985 females from 975 families. In the FHS male subjects, the two SNPs achieved p values of 0.03 and 0.02, respectively, for bivariate association with BMI and femoral neck BMD. Interestingly, SOX6 was previously found to be essential to both cartilage formation/chondrogenesis and obesity-related insulin resistance, suggesting the gene's dual role in both bone and fat. CONCLUSIONS: Our findings, together with the prior biological evidence, suggest the SOX6 gene's importance in co-regulation of obesity and osteoporosis.

  17. A balanced t(5;17 (p15;q22-23 in chondroblastoma: frequency of the re-arrangement and analysis of the candidate genes

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    Wijers-Koster Pauline

    2009-11-01

    originate from a distinct neoplastic clone, although the occurrence of two distinct clones is unlikely. Impairment of the CA10 gene might be pathogenetically relevant, as low expression was found in four cases. Diffuse expression of SRD5A1 and sex steroid signalling-related molecules confirms their role in neoplastic chondrogenesis.

  18. Hedgehog信号调控骨髓间充质干细胞成软骨细胞分化:调控方式及其串话机制尚待研究

    Institute of Scientific and Technical Information of China (English)

    刘宽; 吴兴

    2014-01-01

    背景:Hedgehog 信号通路在骨髓间充质干细胞成软骨细胞分化过程中发挥重要的调控作用,但具体的调控机制,以及与其他信号通路的串话机制仍需进一步的研究,是近来研究的热点。目的:介绍hedgehog信号在调控骨髓间充质干细胞成软骨分化过程中信号转导机制的研究现状与发展趋势,以及与其他信号通路的相互串话。方法:通过搜索CNKI,PubMed及Google Scholar等数据库,以“hedgehog,骨髓间充质干细胞,软骨形成,软骨细胞”和“hedgehog,IHH,SHH,bone marrow mesenchymal stem cel ,chondrogenesis, cartilage, chondrocyte”为检索词,查阅有关hedgehog信号与骨髓间充质干细胞分化相关的文献,最终共纳入36篇文献进行综述。结果与结论:骨髓间充质干细胞是目前公认的组织工程种子细胞来源,hedgehog信号通路是运动系统发育过程中重要的信号分子。Hedgehog信号蛋白IHH和SHH参与调控骨髓间充质干细胞的增殖与成软骨分化,以及软骨形成后的表型维持,且与其他信号通路发挥协同作用。然而,具体的调控方式以及与其他信号的串话机制,仍需进一步的研究,是这一领域未来研究的方向。

  19. Generation of spatially periodic patterns by a mechanical instability: a mechanical alternative to the Turing model.

    Science.gov (United States)

    Harris, A K; Stopak, D; Warner, P

    1984-04-01

    greater range of possible geometries than is possible using gradients of scalar properties. In cases (such as chondrogenesis) where cell differentiation is influenced by the local population density of cells and extracellular matrix, the physical patterns of force and distortion within this extracellular matrix should even be able to accomplish the spatial control of differentiation, usually attributed to diffusible 'morphogens'. PMID:6747520

  20. Macro- and micro-designed chitosan-alginate scaffold architecture by three-dimensional printing and directional freezing.

    Science.gov (United States)

    Reed, Stephanie; Lau, Grace; Delattre, Benjamin; Lopez, David Don; Tomsia, Antoni P; Wu, Benjamin M

    2016-03-01

    , while achieving porous zones that mimic articular cartilage zonal architecture. In future applications, precisely controlled micro- and macro-channels have the potential to assist immediate endogenous bone marrow uptake, stimulate chondrogenesis, and encourage vascularization of bone in an osteochondral scaffold. PMID:26741113

  1. Macro- and micro-designed chitosan-alginate scaffold architecture by three-dimensional printing and directional freezing.

    Science.gov (United States)

    Reed, Stephanie; Lau, Grace; Delattre, Benjamin; Lopez, David Don; Tomsia, Antoni P; Wu, Benjamin M

    2016-01-07

    , while achieving porous zones that mimic articular cartilage zonal architecture. In future applications, precisely controlled micro- and macro-channels have the potential to assist immediate endogenous bone marrow uptake, stimulate chondrogenesis, and encourage vascularization of bone in an osteochondral scaffold.

  2. Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo.

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    Yusuke Nakagawa

    Full Text Available Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1 whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2 whether aggregates of human MSCs promoted lubricin expression, and (3 whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats.For in vitro analysis, human bone marrow (BM MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages.In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4, which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in

  3. BMP-2 functions independently of SHH signaling and triggers cell condensation and apoptosis in regenerating axolotl limbs

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    Finnson Kenneth

    2010-02-01

    Full Text Available Abstract Background Axolotls have the unique ability, among vertebrates, to perfectly regenerate complex body parts, such as limbs, after amputation. In addition, axolotls pattern developing and regenerating autopods from the anterior to posterior axis instead of posterior to anterior like all tetrapods studied to date. Sonic hedgehog is important in establishing this anterior-posterior axis of limbs in all tetrapods including axolotls. Interestingly, its expression is conserved (to the posterior side of limb buds and blastemas in axolotl limbs as in other tetrapods. It has been suggested that BMP-2 may be the secondary mediator of sonic hedgehog, although there is mounting evidence to the contrary in mice. Since BMP-2 expression is on the anterior portion of developing and regenerating limbs prior to digit patterning, opposite to the expression of sonic hedgehog, we examined whether BMP-2 expression was dependent on sonic hedgehog signaling and whether it affects patterning of the autopod during regeneration. Results The expression of BMP-2 and SOX-9 in developing and regenerating axolotl limbs corresponded to the first digits forming in the anterior portion of the autopods. The inhibition of sonic hedgehog signaling with cyclopamine caused hypomorphic limbs (during development and regeneration but did not affect the expression of BMP-2 and SOX-9. Overexpression of BMP-2 in regenerating limbs caused a loss of digits. Overexpression of Noggin (BMP inhibitor in regenerating limbs also resulted in a loss of digits. Histological analysis indicated that the loss due to BMP-2 overexpression was the result of increased cell condensation and apoptosis while the loss caused by Noggin was due to a decrease in cell division. Conclusion The expression of BMP-2 and its target SOX-9 was independent of sonic hedgehog signaling in developing and regenerating limbs. Their expression correlated with chondrogenesis and the appearance of skeletal elements has

  4. Differentiation of cartilaginous anlage in entire embryonic mouse limbs cultured in a rotating bioreactor.

    Science.gov (United States)

    Duke, P.; Oakley, C.; Montufar-Solis, D.

    The embryonic mammalian limb is sensitive both in vivo and in vitro to changes in gravitational force. Hypergravity of centrifugation and microgravity of space decreased size of elements due to precocious or delayed chondrogenesis respectively. In recapitulating spaceflight experiments, premetatarsals were cultured in suspension in a low stress, low sheer rotating bioreactor, and found to be shorter than those cultured in standard culture dishes, and cartilage development was delayed. This study only measured length of the metatarsals, and did not account for possible changes in width and/or in form of the skeletal elements. Shorter cartilage elements in limbbuds cultured in the bioreactor may be due to the ability of the system to reproduce a more in vivo 3D shape than traditional organ cultures. Tissues subjected to traditional organ cultures become flattened by their own weight, attachment to the filter, and restrictions imposed by nutrient diffusion. The purpose of the current experiment was to determine if entire limb buds could be successfully cultured in the bioreactor, and to compare the effects on 3D shape with that of culturing in a culture dish system. Fore and hind limbs from E11-E13 ICR mouse embryos were placed either in the bioreactor, in Trowell culture, or fixed as controls. Limbbuds were cultured for six days, fixed, and processed either as whole mounts or embedded for histology. Qualitative analysis revealed that the Trowell culture specimens were flattened, while bioreactor culture specimens had a more in vivo-like 3D limb shape. Sections of limbbuds from both types of cultures had excellent cartilage differentiation, with apparently more cell maturation, and hypertrophy in the specimens cultured in the bioreactor. Morphometric quantitation of the cartilaginous elements for comparisons of the two culture systems was complicated due to some limb buds fusing together during culture. This problem was especially noticeable in the younger limbs, and

  5. Gene expression pattern of functional neuronal cells derived from human bone marrow mesenchymal stromal cells

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    Bron Dominique

    2008-04-01

    Full Text Available Abstract Background Neuronal tissue has limited potential to self-renew or repair after neurological diseases. Cellular therapies using stem cells are promising approaches for the treatment of neurological diseases. However, the clinical use of embryonic stem cells or foetal tissues is limited by ethical considerations and other scientific problems. Thus, bone marrow mesenchymal stomal cells (BM-MSC could represent an alternative source of stem cells for cell replacement therapies. Indeed, many studies have demonstrated that MSC can give rise to neuronal cells as well as many tissue-specific cell phenotypes. Methods BM-MSC were differentiated in neuron-like cells under specific induction (NPBM + cAMP + IBMX + NGF + Insulin. By day ten, differentiated cells presented an expression profile of real neurons. Functionality of these differentiated cells was evaluated by calcium influx through glutamate receptor AMPA3. Results Using microarray analysis, we compared gene expression profile of these different samples, before and after neurogenic differentiation. Among the 1943 genes differentially expressed, genes down-regulated are involved in osteogenesis, chondrogenesis, adipogenesis, myogenesis and extracellular matrix component (tuftelin, AGC1, FADS3, tropomyosin, fibronectin, ECM2, HAPLN1, vimentin. Interestingly, genes implicated in neurogenesis are increased. Most of them are involved in the synaptic transmission and long term potentialisation as cortactin, CASK, SYNCRIP, SYNTL4 and STX1. Other genes are involved in neurite outgrowth, early neuronal cell development, neuropeptide signaling/synthesis and neuronal receptor (FK506, ARHGAP6, CDKRAP2, PMCH, GFPT2, GRIA3, MCT6, BDNF, PENK, amphiregulin, neurofilament 3, Epha4, synaptotagmin. Using real time RT-PCR, we confirmed the expression of selected neuronal genes: NEGR1, GRIA3 (AMPA3, NEF3, PENK and Epha4. Functionality of these neuron-like cells was demonstrated by Ca2+ influx through glutamate

  6. Differential properties of human ACL and MCL stem cells may be responsible for their differential healing capacity

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    Fu Freddie H

    2011-06-01

    Full Text Available Abstract Background The human anterior cruciate ligament (hACL and medial collateral ligament (hMCL of the knee joint are frequently injured, especially in athletic settings. It has been known that, while injuries to the MCL typically heal with conservative treatment, ACL injuries usually do not heal. As adult stem cells repair injured tissues through proliferation and differentiation, we hypothesized that the hACL and hMCL contain stem cells exhibiting unique properties that could be responsible for the differential healing capacity of the two ligaments. Methods To test the above hypothesis, we derived ligament stem cells from normal hACL and hMCL samples from the same adult donors using tissue culture techniques and characterized their properties using immunocytochemistry, RT-PCR, and flow cytometry. Results We found that both hACL stem cells (hACL-SCs and hMCL stem cells (hMCL-SCs formed colonies in culture and expressed stem cell markers nucleostemin and stage-specific embryonic antigen-4 (SSEA-4. Moreover, both hACL-SCs and hMCL-SCs expressed CD surface markers for mesenchymal stem cells, including CD44 and CD90, but not those markers for vascular cells, CD31, CD34, CD45, and CD146. However, hACL-SCs differed from hMCL-SCs in that the size and number of hACL-SC colonies in culture were much smaller and grew more slowly than hMCL-SC colonies. Moreover, fewer hACL-SCs in cell colonies expressed stem cell markers STRO-1 and octamer-binding transcription factor-4 (Oct-4 than hMCL-SCs. Finally, hACL-SCs had less multi-differentiation potential than hMCL-SCs, evidenced by differing extents of adipogenesis, chondrogenesis, and osteogenesis in the respective induction media. Conclusions This study shows for the first time that hACL-SCs are intrinsically different from hMCL-SCs. We suggest that the differences in their properties contribute to the known disparity in healing capabilities between the two ligaments.

  7. Rabbit bone marrow mesenchymal stem cells seeding on electrospinning polycaprolactone/gelatin scaffold for cartilage repair%电纺聚己内酯-明胶纳米纤维膜复合兔骨髓间充质干细胞构建软骨组织工程支架

    Institute of Scientific and Technical Information of China (English)

    徐正良; 刘骥; 张长青

    2013-01-01

    Objective To construct an electrospinning polycaprolactone (PCD/gelatin scaffold seeded with rabbit bone marrow mesenchymal stem cells (BMSCs), and investigate its feasibility for cartilage tissue engineering. Methods PCL/gelatin (50 : 50) nanofiber membrane as scaffold was produced by electrospinning technique. And the third generation of rabbit BMSCs was harvested and seeded on the scaffolds to develop a stem cells/scaffolds complex, subsequently cultured for chondrogenesis. Meanwhile, the material was measured for the fiber diameter, pore size and porosity by scanning electron microscopy, tested for the mechanical properties, and examined for the proliferation, differentiation and biocompatibility of the cells with the scaffolds by in vivo implantation tests and in vitro cell experiments. Results The PCL/gelatin nanofiber membrane was of uniform diameter, high porosity and specific surface area, and appropriate mechanical properties. When cutured for 24 h, 48 h and 72 h on the scaffold, the number of the cells multiplied notablely, and the chondrogenetic process was highly enhanced The BMSCs growed and prolifered well, and was able to promote the stem cells differentiate into cartilage. In vivo tests showed that the material was non-toxic and little irritant to the tissue, hence great biocompatibility. Conclusions The electrospinning PCL/gelatin nanofibrous membrane has good mechanical properties, cell adherence and biocompatibility, thus, can be applied as the vehicle in the cartilaginous tissue engineering.%目的 探索构建电纺聚己内酯(PCL)-明胶纳米纤维膜复合体作为软骨组织工程支架的可行性.方法 采用静电纺丝技术制作PCL-明胶(50∶50)纳米纤维膜作为支架.取第三代兔骨髓间充质干细胞(BMSC),接种于上述支架,构建细胞-支架复合体并进行成软骨诱导培养.通过电镜分析、力学测试、体内植入试验和细胞实验等检测材料纤维直径、孔径和孔隙率、力学性能,

  8. 脂肪组织来源干细胞的分化潜能和应用%DIFFERENTIATION POTENTIAL AND APPLICATION OF STEM CELLS FROM ADIPOSE TISSUE

    Institute of Scientific and Technical Information of China (English)

    史琳丽; 杨向群

    2012-01-01

    目的 介绍脂肪组织来源的干细胞种类、分化潜能及在再生医学中的应用和优势. 方法 广泛查阅近年关于BMSCs、脂肪来源干细胞(adipose-derived stem cells,ADSCs)和去分化脂肪(dedifferentiated fat,DFAT)细胞的实验研究及临床研究文献,并进行整理、综合与分析. 结果 从脂肪基质成分可以分离得到ADSCs,ADSCs具有多向分化潜能,可分化为脂肪、骨、软骨、内皮、肌以及神经细胞等,并已较成功地应用于再生医学各领域.成熟脂肪细胞经过天花板培养法可以去分化为成纤维样细胞,即DFAT细胞,获得了多向分化潜能,也可以像ADSCs一样分化为脂肪、骨、软骨、内皮、肌以及神经细胞等.相比较目前常用的成体干细胞BMSCs,ADSCs、DFAT细胞来源广泛、取材更容易;而相对于ADSCs,DFAT细胞均一性高,增殖能力强. 结论 脂肪组织作为人体干细胞的重要来源,可能会给临床组织缺损的修复与再生带来新希望.%Objective To introduce types and differentiation potentials of stem cells from adipose tissue, and its applications on regenerative medicine and advantages. Methods The literature of original experimental study and clinical research about bone marrow mesenchymal stem cells (BMSCs), adipose-derived stem cells (ADSCs), and dedifferentiated fat (DFAT) cells was extensively reviewed and analyzed. Results ADSCs can be isolated from stromal vascular fraction. As ADSCs have multi-lineage potentials, such as adipogenesis, osteogenesis, chondrogenesis, angiogenesis, myogenesis, and neurogenesis, they have already been successfully used in regenerative medicine areas. Dramatically, mature fat cells can be dedifferentiated and changed into fibroblast-like cells, named DFAT cells, via ceiling culture method. DFAT cells also had the same multi-lineage potentials as ADSCs, differentiating into adipocytes, osteocytes, chondrocytes, endothelial cells, muscle cells, and nerve cells. Compared

  9. Effect of self-assembled peptide–mesenchymal stem cell complex on the progression of osteoarthritis in a rat model

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    Kim JE

    2014-05-01

    metalloproteinases 1, and matrix metalloproteinase 9 were diffusely stained in controls, whereas localized or minimal staining was observed in other groups. Modified Mankin scores were significantly lower in the SAP and SAP-MSC groups than in controls (P=0.001 and 0.013. Although not statistically significant, synovial inflammation scores were lower in the SAP (1.3±0.3 and SAP-MSC (1.3±0.2 groups than in controls (2.6±0.2. However, neither the cytokine level nor the behavioral score was significantly different between groups.Conclusion: Injection of SAP-MSC hydrogels showed evidence of chondroprotection, as measured by the histologic grading and decreased expression of biochemical markers of inflammation and apoptosis. It also lowered subchondral bone mineral density, which can be increased by OA. This suggests that the SAP-MSC complex may have clinical potential to inhibit OA progression.Keywords: self-assembled peptide, mesenchymal stem cell, osteoarthritis, apoptosis, chondrogenesis

  10. Flavonoid Compound Icariin Activates Hypoxia Inducible Factor-1α in Chondrocytes and Promotes Articular Cartilage Repair.

    Science.gov (United States)

    Wang, Pengzhen; Zhang, Fengjie; He, Qiling; Wang, Jianqi; Shiu, Hoi Ting; Shu, Yinglan; Tsang, Wing Pui; Liang, Shuang; Zhao, Kai; Wan, Chao

    2016-01-01

    Articular cartilage has poor capability for repair following trauma or degenerative pathology due to avascular property, low cell density and migratory ability. Discovery of novel therapeutic approaches for articular cartilage repair remains a significant clinical need. Hypoxia is a hallmark for cartilage development and pathology. Hypoxia inducible factor-1alpha (HIF-1α) has been identified as a key mediator for chondrocytes to response to fluctuations of oxygen availability during cartilage development or repair. This suggests that HIF-1α may serve as a target for modulating chondrocyte functions. In this study, using phenotypic cellular screen assays, we identify that Icariin, an active flavonoid component from Herba Epimedii, activates HIF-1α expression in chondrocytes. We performed systemic in vitro and in vivo analysis to determine the roles of Icariin in regulation of chondrogenesis. Our results show that Icariin significantly increases hypoxia responsive element luciferase reporter activity, which is accompanied by increased accumulation and nuclear translocation of HIF-1α in murine chondrocytes. The phenotype is associated with inhibiting PHD activity through interaction between Icariin and iron ions. The upregulation of HIF-1α mRNA levels in chondrocytes persists during chondrogenic differentiation for 7 and 14 days. Icariin (10-6 M) increases the proliferation of chondrocytes or chondroprogenitors examined by MTT, BrdU incorporation or colony formation assays. Icariin enhances chondrogenic marker expression in a micromass culture including Sox9, collagen type 2 (Col2α1) and aggrecan as determined by real-time PCR and promotes extracellular matrix (ECM) synthesis indicated by Alcian blue staining. ELISA assays show dramatically increased production of aggrecan and hydroxyproline in Icariin-treated cultures at day 14 of chondrogenic differentiation as compared with the controls. Meanwhile, the expression of chondrocyte catabolic marker genes

  11. Flavonoid Compound Icariin Activates Hypoxia Inducible Factor-1α in Chondrocytes and Promotes Articular Cartilage Repair.

    Directory of Open Access Journals (Sweden)

    Pengzhen Wang

    Full Text Available Articular cartilage has poor capability for repair following trauma or degenerative pathology due to avascular property, low cell density and migratory ability. Discovery of novel therapeutic approaches for articular cartilage repair remains a significant clinical need. Hypoxia is a hallmark for cartilage development and pathology. Hypoxia inducible factor-1alpha (HIF-1α has been identified as a key mediator for chondrocytes to response to fluctuations of oxygen availability during cartilage development or repair. This suggests that HIF-1α may serve as a target for modulating chondrocyte functions. In this study, using phenotypic cellular screen assays, we identify that Icariin, an active flavonoid component from Herba Epimedii, activates HIF-1α expression in chondrocytes. We performed systemic in vitro and in vivo analysis to determine the roles of Icariin in regulation of chondrogenesis. Our results show that Icariin significantly increases hypoxia responsive element luciferase reporter activity, which is accompanied by increased accumulation and nuclear translocation of HIF-1α in murine chondrocytes. The phenotype is associated with inhibiting PHD activity through interaction between Icariin and iron ions. The upregulation of HIF-1α mRNA levels in chondrocytes persists during chondrogenic differentiation for 7 and 14 days. Icariin (10-6 M increases the proliferation of chondrocytes or chondroprogenitors examined by MTT, BrdU incorporation or colony formation assays. Icariin enhances chondrogenic marker expression in a micromass culture including Sox9, collagen type 2 (Col2α1 and aggrecan as determined by real-time PCR and promotes extracellular matrix (ECM synthesis indicated by Alcian blue staining. ELISA assays show dramatically increased production of aggrecan and hydroxyproline in Icariin-treated cultures at day 14 of chondrogenic differentiation as compared with the controls. Meanwhile, the expression of chondrocyte catabolic

  12. Biodegradable chitosan scaffolds containing microspheres as carriers for controlled transforming growth factor-β1 delivery for cartilage tissue engineering

    Institute of Scientific and Technical Information of China (English)

    CAI Dao-zhang; ZENG Chun; QUAN Da-ping; BU Li-si; WANG Kun; LU Hua-ding; LI Xiao-feng

    2007-01-01

    Background Natural articular cartilage has a limited capacity for spontaneous regeneration. Controlled release of transforming growth factor-β1 (TGF-β1) to cartilage defects can enhance chondrogenesis. In this study, we assessed the feasibility of using biodegradable chitosan microspheres as carriers for controlled TGF-β1 delivery and the effect of released TGF-β1 on the chondrogenic potential of chondrocytes.Methods Chitosan scaffolds and chitosan microspheres loaded with TGF-β1 were prepared by the freeze-drying and the emulsion-crosslinking method respectively. In vitro drug release kinetics, as measured by enzyme-linked immunosorbent assay, was monitored for 7 days. Lysozyme degradation was performed for 4 weeks to detect in vitro degradability of the scaffolds and the microspheres. Rabbit chondrocytes were seeded on the scaffolds containing TGF-β1 microspheres and incubated in vitro for 3 weeks. Histological examination and type Ⅱ collagen immunohistochemical staining was performed to evaluate the effects of released TGF-β1 on cell adhesivity, proliferation and synthesis of the extracellular matrix.Results TGF-β1 was encapsulated into chitosan microspheres and the encapsulation efficiency of TGF-β1 was high (90.1%). During 4 weeks of incubation in lysozyme solution for in vitro degradation, the mass of both the scaffolds and the microspheres decreased continuously and significant morphological changes was noticed. From the release experiments, it was found that TGF-β1 could be released from the microspheres in a multiphasic fashion including an initial burst phase, a slow linear release phase and a plateau phase. The release amount of TGF-β1 was 37.4%, 50.7%,61.3%, and 63.5% for 1, 3, 5, and 7 days respectively. At 21 days after cultivation, type Ⅱ collagen immunohistochemical staining was performed. The mean percentage of positive cells for collagen type Ⅱ in control group (32.7%± 10.4%) was significantly lower than that in the controlled

  13. The expression of IGF-2 in tetrachlorodibenzodioxin-induced congenital skeleton malformation%IGF一2在四氯二苯并二恶英诱导的大鼠骨骼畸形中的表达

    Institute of Scientific and Technical Information of China (English)

    郭磊; 赵玉岩; 张世亮; 朱世博; 刘魁

    2008-01-01

    analysis.respectively.Results TCDD(15 μg/kg)induced single or coalescent developmental skeleton defect in fetal rat(33.3%),such as cross-foot,cleft spine,cleft palate,rumplesand so on,The chondrogenesis center of tarsal bones and quantity of chondrocyte were decreased.There were expanding rough endoplasmic reticulum,degradation of nuclear matrix,and disorder of mitochondria cristae in cell nucleus of chondrocyte.IGF-2 protein level in TCDD-treated cartilaginous'tissue was decreased significantly.In chondrocyte treated with TCDD(1 × 10-8 mol/L)in vitro,the expression of IGF-2 mRNA and protein were decreased 80%and 60%,respectively(P<0.05).Conelusions The low expression of IGF-2 in chondrocyte may be related to the TCDD-induced developmentalskeleton defect.

  14. Successful isolation, in vitro expansion and characterization of stem cells from Human Dental Pulp

    Directory of Open Access Journals (Sweden)

    Preethy SP

    2010-01-01

    acids (5 .Cell counting was done by Trypan Blue dye exclusion method and the cells were seeded in 6 well culture plates. The plates with cells were incubated at 37˚C with 5% CO2 for varying periods from 14 days-28 days. The cells were observed daily and media change was done every three days. RESULTS: Viable Dental Pulp tissue-cells were obtained after transportation of up to 48 hrs and the in vitro growth of cells was initially slow but colonies were identified from the 10th day onwards. The cells were harvested at different intervals of 14-28 days for each sample based on their growth and subjected to H & E staining .The H & E staining of the cultured cells of all the samples showed positive resultsCONCLUSION: We are able to transport extracted teeth and derive viable dental pulp tissue cells after enzymatic digestion and multiply them in culture after a maximum of 48 hrs after transportation. The cells could be grown in culture with a morphology resembling dental pulp stem cells while in culture expansion and in H&E studies. Further characterization of the cells is necessary to confirm their Stemness. References1.Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs in vitro and in vivo. Proc Natl Acad Sci U S A. 20002.Nosrat IV, Widenfalk J, Olson L, Nosrat CA. Dental pulp cells produce neurotrophic factors, interact with trigeminal neurons in vitro, and rescue motoneurons after spinal cord injury. Dev Biol. 2001 Oct 3.Iohara K, Zheng L, Ito M, Tomokiyo A, Matsushita K, Nakashima M. Side population cells isolated from porcine dental pulp tissue with self-renewal and multipotency for dentinogenesis, chondrogenesis, adipogenesis, and neurogenesis. Stem Cells. 2006 Nov4.Gandia C, Armiñan A, García-Verdugo JM, Lledó E, Ruiz A, Miñana MD, Sanchez-Torrijos J, Payá R, Mirabet V, Carbonell-Uberos F, Llop M, Montero JA, Sepúlveda P. Human dental pulp stem cells improve left ventricular function, induce angiogenesis, and reduce

  15. Porcine Fetal Bone Marrow Mesenchymal Stem Cells in Cartilage Tissue Engineering%胎猪BMSC体外构建软骨的实验研究

    Institute of Scientific and Technical Information of China (English)

    刘李娜; 何爱娟; 周广东; 曹卫刚

    2013-01-01

    Objective To investigate the optimal seed cell for cartilage engineering by comparing the chondrogenesis capability of porcine fetal bone marrow mesenchymal stem cells (fBMSCs) and adult porcine BMSCs (aBMSCs). Methods Fetal pigs with gestational age of 70 days were obtained by the uterine-incision delivery, and primary fBMSCs were isolated from the bone marrow. Primary aBMSCs were isolated from the bone marrow which was aspirated from iliac. Cell morphology of the two kinds of cells at passage 3 were observed after in vitro proliferation. The two kinds of cells at passages 3 were characterized by their osteogenic, adipogenic and chondrogenic differentiation potential. Then the fBMSCs and aBMSCs were separately seeded onto a polyglycolic acid/polylactic acid (PGA/PLA) scaffold with the concentration of 1 ×108 cells/mL. All specimens were harvested after 8 weeks' culture in vitro. Gross observation, glycosaminoglycan (GAG) quantification, total collagen quantification and histology were used to compare related characteristic differences of engineered cartilage formed by the two kinds of cells. Results fBMSCs had better proliferation and multiple differentiation capacity than aBMSCs. The two kinds of cells both formed mature cartilage after 8 weeks of culture in vitro, and the engineered cartilage of aBMSCs group had better appearance. The GAG content and total collagen content of the cartilage formed by fBMSCs were both higher than the cartilage formed by aBMSCs (P<0.01). Histology and immunohistochemistry demonstrated that the cartilage formed by fBMSCs have more compact tissue structure. The cartilage matrix staining of cartilage formed by fBMSCs were stronger than that of cartilage formed by aBMSCs. Conclusion The fBMSCs seems to be the optimal seed cells for cartilage tissue engineering.%目的比较胎猪骨髓间充质干细胞(Bone Marrow Mesenchymal Stem Cells,BMSCs)和成年猪BMSCs构建软骨能力的差异,寻找合适的同种异体组织工

  16. Effect of gap junction-mediated intercellular communication on growth differentiation factor 5-induced chondrogenic differentiation in vitro%缝隙连接细胞间通讯在生长分化因子5诱导软骨分化中的作用

    Institute of Scientific and Technical Information of China (English)

    郭珈宜; 崔宏勋; 郭马珑; 郭艳幸; 郭艳锦

    2014-01-01

    Objective To investigate the effect of gap junctional blocker oleamide on growth differentiation factor 5 (GDF-5) induced chondrogenic differentiation.Methods hunan mesenchymal stem cells (hMSCs) were isolated and cultured in vitro,and the cells from Passage 3 were used in this study.hMSCs were randomly grouped into three according to the kinds of induction medium (0,100 μg/L GDF-5,100 μg/L GDF-5 + 2.5 μmol/L oleamide).The proliferation of hMSCs was investigated by methyl thiazol tetrazolium (MTT) assay.The cells were resuspended and the pellets were constructed by centrifugation at a density of 5 × 109/L,and then continued to be kept for two weeks.Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect the expression of mRNA of type Ⅱ collagen,comexin 43 (Cx43)and aggrecan.Western blotting was performed to detect the expression of protein of type Ⅱ collagen,Cx43and aggrecan.Toluidine blue was performed to detect extracellular matrix staining.Results GDF-5 can promote the mRNA expression of type Ⅱ collagen,Cx43 and aggrecan (1.00 ± 0.03 vs.0.40 ± 0.12 ;1.00 ±0.07 vs.0.32 ±0.02; 1.00 ±0.02 vs.0.45 ±0.01,P <0.01),and the relative protein expression was also increased (1.50±0.11 vs.0.53 ±0.07; 1.70 ±0.14 vs.0.52 ±0.04; 1.00 ±0.12 vs.0.50±0.06,P<0.01).While oleamide could up-regulate the expression of mRNA of Cx43 (0.94 ±0.12 vs.0.45 ±0.01,P <0.01),but had no effect on type Ⅱ collagen and aggrecan mRNA expression.Oleamide could down-regulate the expression of protein of type Ⅱ collagen and aggrecan,but had no effect onCx43 protein expression(0.42 ± 0.04 vs.0.53 ±0.07; 0.38 ±0.06 vs.0.52 ±0.04,P<0.01).Toluidine blue staining showed that GDF-5 can promote the synthesis of aggrecan.Conclusion GDF-5 modulation of chondrogenesis involves gap junction-mediated intercellular communication.%目的 观察缝隙连接细胞间通讯(GJIC)在生长分化因子5(GDF-5)诱导人骨髓间充质干细胞(hMSCs)成