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Sample records for human cartilage explants

  1. Standardized butanol fraction of WIN-34B suppresses cartilage destruction via inhibited production of matrix metalloproteinase and inflammatory mediator in osteoarthritis human cartilage explants culture and chondrocytes

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    Huh Jeong-Eun

    2012-12-01

    Full Text Available Abstract Background WIN-34B is a novel Oriental medicine, which represents the n-butanol fraction prepared from dried flowers of Lonicera japonica Thunb and dried roots of Anemarrhena asphodeloides BUNGE. The component herb of WIN-34B is used for arthritis treatment in East Asian countries. The aim of this study was to determine the cartilage-protective effects and mechanisms of WIN-34B and its major phenolic compounds, chlorogenic acid and mangiferin, in osteoarthritis (OA human cartilage explants culture and chondrocytes. Methods The investigation focused on whether WIN-34B and its standard compounds protected cartilage in interleukin (IL-1β-stimulated cartilage explants culture and chondrocytes derived from OA patients. Also, the mechanisms of WIN-34B on matrix metalloproteinases (MMPs, tissue inhibitor of matrix metalloproteinases (TIMPs, inflammatory mediators, and mitogen-activated protein kinases (MAPKs pathways were assessed. Results WIN-34B was not cytotoxic to cultured cartilage explants or chondrocytes. WIN-34B dose-dependently inhibited the release of glycosaminoglycan and type II collagen, increased the mRNA expression of aggrecan and type II collagen, and recovered the intensity of proteoglycan and collagen by histological analysis in IL-1β-stimulated human cartilage explants culture. The cartilage protective effect of WIN-34B was similar to or better than that of chlorogenic acid and mangiferin. Compared to chlorogenic acid and mangiferin, WIN-34B displayed equal or greater decreases in the levels of MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, and markedly up-regulated TIMP-1 and TIMP-3. WIN-34B inhibited inflammatory mediators involved in cartilage destruction, such as prostaglandin E2, nitric oxide, tumor necrosis factor-alpha, and IL-1β. The phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK, and p38 was significantly reduced by WIN-34B treatment, while phosphorylation of JNK was only

  2. Standardized butanol fraction of WIN-34B suppresses cartilage destruction via inhibited production of matrix metalloproteinase and inflammatory mediator in osteoarthritis human cartilage explants culture and chondrocytes

    Science.gov (United States)

    2012-01-01

    Background WIN-34B is a novel Oriental medicine, which represents the n-butanol fraction prepared from dried flowers of Lonicera japonica Thunb and dried roots of Anemarrhena asphodeloides BUNGE. The component herb of WIN-34B is used for arthritis treatment in East Asian countries. The aim of this study was to determine the cartilage-protective effects and mechanisms of WIN-34B and its major phenolic compounds, chlorogenic acid and mangiferin, in osteoarthritis (OA) human cartilage explants culture and chondrocytes. Methods The investigation focused on whether WIN-34B and its standard compounds protected cartilage in interleukin (IL)-1β-stimulated cartilage explants culture and chondrocytes derived from OA patients. Also, the mechanisms of WIN-34B on matrix metalloproteinases (MMPs), tissue inhibitor of matrix metalloproteinases (TIMPs), inflammatory mediators, and mitogen-activated protein kinases (MAPKs) pathways were assessed. Results WIN-34B was not cytotoxic to cultured cartilage explants or chondrocytes. WIN-34B dose-dependently inhibited the release of glycosaminoglycan and type II collagen, increased the mRNA expression of aggrecan and type II collagen, and recovered the intensity of proteoglycan and collagen by histological analysis in IL-1β-stimulated human cartilage explants culture. The cartilage protective effect of WIN-34B was similar to or better than that of chlorogenic acid and mangiferin. Compared to chlorogenic acid and mangiferin, WIN-34B displayed equal or greater decreases in the levels of MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, and markedly up-regulated TIMP-1 and TIMP-3. WIN-34B inhibited inflammatory mediators involved in cartilage destruction, such as prostaglandin E2, nitric oxide, tumor necrosis factor-alpha, and IL-1β. The phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38 was significantly reduced by WIN-34B treatment, while phosphorylation of JNK was only inhibited by chlorogenic

  3. Explant cultures of human colon

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    Autrup, Herman; Barrett, L.A.; Jackson, F.E.

    1978-01-01

    Human colonic epithelium has been cultured as explants in a chemically defined medium for periods of 1 to 20 days. The viability of the explants was shown by the preservation of the ultrastructural features of the colonic epithelial cells and by active incorporation of radioactive precursors into...

  4. The metabolic dynamics of cartilage explants over a long-term culture period

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    E.K Moo

    2011-01-01

    Full Text Available INTRODUCTION: Although previous studies have been performed on cartilage explant cultures, the generalized dynamics of cartilage metabolism after extraction from the host are still poorly understood due to differences in the experimental setups across studies, which in turn prevent building a complete picture. METHODS: In this study, we investigated the response of cartilage to the trauma sustained during extraction and determined the time needed for the cartilage to stabilize. Explants were extracted aseptically from bovine metacarpal-phalangeal joints and cultured for up to 17 days. RESULTS: The cell viability, cell number, proteoglycan content, and collagen content of the harvested explants were analyzed at 0, 2, 10, and 17 days after explantation. A high percentage of the cartilage explants were found to be viable. The cell density initially increased significantly but stabilized after two days. The proteoglycan content decreased gradually over time, but it did not decrease to a significant level due to leakage through the distorted peripheral collagen network and into the bathing medium. The collagen content remained stable for most of the culture period until it dropped abruptly on day 17. CONCLUSION: Overall, the tested cartilage explants were sustainable over long-term culture. They were most stable from day 2 to day 10. The degradation of the collagen on day 17 did not reach diseased levels, but it indicated the potential of the cultures to develop into degenerated cartilage. These findings have implications for the application of cartilage explants in pathophysiological fields.

  5. The effects of ascorbic acid on cartilage metabolism in guinea pig articular cartilage explants.

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    Clark, Amy G; Rohrbaugh, Amy L; Otterness, Ivan; Kraus, Virginia B

    2002-03-01

    Ascorbic acid has been associated with the slowing of osteoarthritis progression in guinea pig and man. The goal of this study was to evaluate transcriptional and translational regulation of cartilage matrix components by ascorbic acid. Guinea pig articular cartilage explants were grown in the presence of L-ascorbic acid (L-Asc), D-isoascorbic acid (D-Asc), sodium L-ascorbate (Na L-Asc), sodium D-isoascorbate (Na D-Asc), or ascorbyl-2-phosphate (A2P) to isolate and analyze the acidic and nutrient effects of ascorbic acid. Transcription of type II collagen, prolyl 4-hydroxylase (alpha subunit), and aggrecan increased in response to the antiscorbutic forms of ascorbic acid (L-Asc, Na L-Asc, and A2P) and was stereospecific to the L-forms. Collagen and aggrecan synthesis also increased in response to the antiscorbutic forms but only in the absence of acidity. All ascorbic acid forms tended to increase oxidative damage over control. This was especially true for the non-nutrient D-forms and the high dose L-Asc. Finally, we investigated the ability of chondrocytes to express the newly described sodium-dependent vitamin C transporters (SVCTs). We identified transcripts for SVCT2 but not SVCT1 in guinea pig cartilage explants. This represents the first characterization of SVCTs in chondrocytes. This study confirms that ascorbic acid stimulates collagen synthesis and in addition modestly stimulates aggrecan synthesis. These effects are exerted at both transcriptional and post-transcriptional levels. The stereospecificity of these effects is consistent with chondrocyte expression of SVCT2, shown previously to transport L-Asc more efficiently than D-Asc. Therefore, this transporter may be the primary mechanism by which the L-forms of ascorbic acid enter the chondrocyte to control matrix gene activity.

  6. Hypotonic challenge modulates cell volumes differently in the superficial zone of intact articular cartilage and cartilage explant.

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    Turunen, Siru M; Lammi, Mikko J; Saarakkala, Simo; Koistinen, Arto; Korhonen, Rami K

    2012-05-01

    The objective of this study was to evaluate the effect of sample preparation on the biomechanical behaviour of chondrocytes. We compared the volumetric and dimensional changes of chondrocytes in the superficial zone (SZ) of intact articular cartilage and cartilage explant before and after a hypotonic challenge. Calcein-AM labelled SZ chondrocytes were imaged with confocal laser scanning microscopy through intact cartilage surfaces and through cut surfaces of cartilage explants. In order to clarify the effect of tissue composition on cell volume changes, Fourier Transform Infrared microspectroscopy was used for estimating the proteoglycan and collagen contents of the samples. In the isotonic medium (300 mOsm), there was a significant difference (p integrity of the mechanical environment of chondrocytes.

  7. Low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF) treatments affect degeneration of cultured articular cartilage explants

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    Tan, Lijun; Ren, Yijin; Kooten, van Theo G.; Grijpma, Dirk W.; Kuijer, Roel

    2015-01-01

    Purpose: Articular cartilage has some capacity for self-repair. Clinically used low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF) treatments were compared in their potency to prevent degeneration using an explant model of porcine cartilage. Methods: Explants of porcine

  8. The effect of protease inhibitors on the induction of osteoarthritis-related biomarkers in bovine full-depth cartilage explants

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    He, Yi; Zheng, Qinlong; Jiang, Mengmeng

    2015-01-01

    Objective The specific degradation of type II collagen and aggrecan by matrix metalloproteinase (MMP)-9, -13 and ADAMTS-4 and -5 (aggrecanase-1 and -2) in the cartilage matrix is a critical step in pathology of osteoarthritis (OA). The aims of this study were: i) To investigate the relative...... contribution of ADAMTS-4 and ADAMTS-5 to cartilage degradation upon catabolic stimulation; ii) To investigate the effect of regulating the activities of key enzymes by mean of broad-spectrum inhibitors. Methods Bovine full-depth cartilage explants stimulated with tumor necrosis factor alpha (TNF......I (ADAMTS-degraded aggrecan), AGNxII (MMP-degraded aggrecan), and CTX-II (MMP-derived type II collagen) were quantified in the explants-conditioned media. Results We found that: i) Active ADAMTS-4, MMP-9, -13 were released in the late stage of TNF-α/ OSM stimulation, whereas no significant active ADAMTS-5...

  9. Respiration rate in human pituitary tumor explants.

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    Anniko, M; Bagger-Sjöbäck, D; Hultborn, R

    1982-01-01

    Studies on the respiration rate of human pituitary tumor tissue have so far been lacking in the literature. This study presents the results from four adenomas causing acromegaly, all with different clinical degrees of the disease. Determination of oxygen uptake was performed in vitro with a spectrophotorespirometric system. Pieces of the tumors were explanted to an organ culture system with a high degree of stability. The secretion rate of growth hormone (GH) and prolactin (PRL) was determined. After 4-8 days in vitro, specimens were analyzed for respiration rate. This was approximately 1-1.5 microliters O2/h/micrograms dry weight. The activity of the pituitary tumor tissue was characterized by both the hormone secretion rate and the respiration rate. Particularly active foci were found to occur in the adenoma tissue. Depending on the individual tumor, the GH secretion rate was approximately 0.1-100 pmol/micrograms dry weight/h and PRL secretion rate approximately 0.4-18 micrograms/micrograms dry weight/h. The respiration rate--as is also the hormone secretion rate--is dependent on the time in vitro prior to analysis. The respiration rate in individual tumors is a parameter which does not reflect GH or PRL serum levels or clinical activity of the disease.

  10. Effects of compression on the loss of newly synthesized proteoglycans and proteins from cartilage explants

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    Sah, R.L.; Doong, J.Y.; Grodzinsky, A.J.; Plaas, A.H.; Sandy, J.D. (Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology Harvard-M.I.T., Cambridge (United States))

    1991-04-01

    The effects of mechanical compression of calf cartilage explants on the catabolism and loss into the medium of proteoglycans and proteins radiolabeled with (35S)sulfate and (3H)proline were examined. A single 2- or 12-h compression of 3-mm diameter cartilage disks from a thickness of 1.25 to 0.50 mm, or slow cyclic compression (2 h on/2 h off) from 1.25 mm to 1.00, 0.75, or 0.50 mm for 24 h led to transient alterations and/or sustained increases in loss of radiolabeled macromolecules. The effects of imposing or removing loads were consistent with several compression-induced physical mediators including fluid flow, diffusion, and matrix disruption. Cyclic compression induced convective fluid flow and enhanced the loss of 35S- and 3H-labeled macromolecules from tissue into medium. In contrast, prolonged static compression induced matrix consolidation and appeared to hinder the diffusional transport and loss of 35S- and 3H-labeled macromolecules. Since high amplitude cyclic compression led to a sustained increase in the rate of loss of 3H- and 35S-labeled macromolecules that was accompanied by an increase in the rate of loss of (3H)hydroxyproline residues and an increase in tissue hydration, such compression may have caused disruption of the collagen meshwork. The 35S-labeled proteoglycans lost during such cyclic compression were of smaller average size than those from controls, but contained a similarly low proportion (approximately 15%) that could form aggregates with excess hyaluronate and link protein. The size distribution and aggregability of the remaining tissue proteoglycans and 35S-labeled proteoglycans were not markedly affected. The loss of tissue proteoglycan paralleled the loss of 35S-labeled macromolecules.

  11. Suppression of MMP activity in bovine cartilage explants cultures has little if any effect on the release of aggrecanase-derived aggrecan fragments

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    Wang, Bijue; Chen, Pingping; Jensen, Anne-Christine Bay

    2009-01-01

    - and aggrecanase-derived fragments of aggrecan and type II collagen into the supernatant of bovine cartilage explants cultures using neo-epitope specific immunoassays, and to associate the release of these fragments with the activity of proteolytic enzymes using inhibitors. FINDINGS: Bovine cartilage explants were...... cultured in the presence or absence of the catabolic cytokines oncostatin M (OSM) and tumor necrosis factor alpha (TNFalpha). In parallel, explants were co-cultured with protease inhibitors such as GM6001, TIMP1, TIMP2 and TIMP3. Fragments released into the supernatant were determined using a range of neo......-epitope specific immunoassays; (1) sandwich (342)FFGVG-G2 ELISA, (2) competition NITEGE(373)ELISA (3) sandwich G1-NITEGE(373 )ELISA (4) competition (374)ARGSV ELISA, and (5) sandwich (374)ARGSV-G2 ELISA all detecting aggrecan fragments, and (6) sandwich CTX-II ELISA, detecting C-telopeptides of type II collagen...

  12. Electric field stimulation can increase protein synthesis in articular cartilage explants.

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    MacGinitie, L A; Gluzband, Y A; Grodzinsky, A J

    1994-03-01

    It has been hypothesized that the electric fields associated with the dynamic loading of cartilage may affect its growth, remodeling, and biosynthesis. While the application of exogenous fields has been shown to modulate cartilage biosynthesis, it is not known what range of field magnitudes and frequencies can alter biosynthesis and how they relate to the magnitudes and frequencies of endogenous fields. Such information is necessary to understand and identify mechanisms by which fields may act on cartilage metabolism. In this study, incorporation of 35S-methionine was used as a marker for electric field-induced changes in chondrocyte protein synthesis in disks of cartilage from the femoropatellar groove of 1 to 2-week-old calves. The cartilage was stimulated sinusoidally at 1, 10, 100, 10(3), and 10(4) Hz with current densities of 10-30 mA/cm2. Incorporation was assessed in control disks maintained in the absence of applied current at 37, 41, and 43 degrees C. The possibility that applied currents would induce synthesis of the same stress proteins that are caused by heating or other mechanisms was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examination of gel fluorographs. Total radiolabel incorporation in cartilage that had been stimulated relative to incorporation in the controls increased with current density magnitudes greater than 10 mA/cm2. The increase was greatest at 100 Hz and 1 kHz, and it depended on the position on the joint surface from which the cartilage samples were taken. Together, these results suggest that endogenous electric fields could affect cartilage biosynthesis. Stress proteins were not induced at any current density when the electrodes were electrically connected but chemically isolated from the media by agarose bridges. Stress proteins were observed for disks incubated at temperatures greater than 39 degrees C (no field) and when the stimulating platinum electrodes were in direct contact with the media

  13. Controlled release of C-type natriuretic peptide by microencapsulation dampens proinflammatory effects induced by IL-1β in cartilage explants.

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    Peake, Nick J; Pavlov, Anton M; D'Souza, Alveena; Pingguan-Murphy, Belinda; Sukhorukov, Gleb B; Hobbs, Adrian J; Chowdhury, Tina T

    2015-02-09

    C-type natriuretic peptide (CNP) exhibits potent anti-inflammatory effects in chondrocytes that have the potential to repair cartilage damage observed in osteoarthritis (OA). However, treatments for OA have been challenging due to poor targeting and delivery of therapeutics. The present study fabricated polyelectrolyte microcapsules loaded with CNP and examined whether the layer-by-layer (LbL) approach could have protective effects in cartilage explants treated with the pro-inflammatory cytokine, interleukin-1β (IL-1β). SEM showed uniform, 2 to 3 μm spherical microcapsules with morphological characteristic similar to templates loaded with or without CNP. The protein was localized around the external surface of the microcapsules with encapsulation efficiencies >82.9%. CNP release profiles were broadly similar following 9 days of culture. The presence of CNP microcapsules did not significantly affect cell viability (80%) with DNA values that remained stable throughout the culture conditions. Confocal imaging showed clustering of microcapsules in chondrocytes to natriuretic peptide receptor (Npr) 2 and 3. Treatment of cartilage explants with CNP microcapsules led to concentration-dependent inhibition of NO release in response to IL-1β and restoration of matrix synthesis. In summary, we demonstrate controlled delivery of CNP to dampen pro-inflammatory effects induced by IL-1β in cartilage explants. The LbL approach has the potential to promote cartilage repair in vivo.

  14. Effects of tenoxicam and aspirin on the metabolism of proteoglycans and hyaluronan in normal and osteoarthritic human articular cartilage.

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    Manicourt, D H; Druetz-Van Egeren, A; Haazen, L; Nagant de Deuxchaisnes, C

    1994-01-01

    1. As nonsteroidal anti-inflammatory drugs may impair the ability of the chondrocyte to repair its damaged extracellular matrix, we explored the changes in the metabolism of newly synthesized proteoglycan (PG) and hyaluronan (HA) molecules produced by tenoxicam and aspirin in human normal cartilage explants and in osteoarthritic (OA) cartilage from age-matched donors. 2. Explants were sampled from the medial femoral condyle and were classified by use of Mankin's histological-histochemical grading system. Cartilage specimens were normal in 10 subjects, exhibited moderate OA (MOA) in 10 and had severe OA (SOA) in 10. 3. Cartilage explants were pulsed with [3H]-glucosamine and chased in the absence and in the presence of either aspirin (190 micrograms ml-1) or tenoxicam (4-16 micrograms ml-1). After papain digestion, the labelled chondroitin sulphate ([3H]-PGs) and HA([3H]-HA) molecules present in the tissue and media were purified by anion-exchange chromatography. 4. In normal cartilage as well as in explants with MOA and SOA aspirin reduced more strongly PG and HA synthesis than the loss of [3H]-HA and [3H]-PGs. 5. In normal cartilage, tenoxicam did not affect PG metabolism whereas it reduced HA synthesis in a dose-dependent manner and did not change or even increased the net loss of [3H]-HA. In contrast, in OA cartilage, tenoxicam produced a stronger reduction in the loss of [3H]-PGs than in PG synthesis and this decrease occurred at lower concentrations in cartilage with SOA (4-8 micrograms ml-1) than in cartilage with MOA (8-16 micrograms ml-1).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7889262

  15. Glucosamine increases hyaluronic acid production in human osteoarthritic synovium explants

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    Uitterlinden EJ

    2008-09-01

    Full Text Available Abstract Background Glucosamine (GlcN used by patients with osteoarthritis was demonstrated to reduce pain, but the working mechanism is still not clear. Viscosupplementation with hyaluronic acid (HA is also described to reduce pain in osteoarthritis. The synthesis of HA requires GlcN as one of its main building blocks. We therefore hypothesized that addition of GlcN might increase HA production by synovium tissue. Methods Human osteoarthritic synovium explants were obtained at total knee surgery and pre-cultured for 1 day. The experimental conditions consisted of a 2 days continuation of the culture with addition of N-Acetyl-glucosamine (GlcN-Ac; 5 mM, glucosamine-hydrochloride (GlcN-HCl; 0.5 and 5 mM, glucose (Gluc; 0.5 and 5 mM. Hereafter HA production was measured in culture medium supernatant using an enzyme-linked binding protein assay. Real time RT-PCR was performed for hyaluronic acid synthase (HAS 1, 2 and 3 on RNA isolated from the explants. Results 0.5 mM and 5 mM GlcN-HCl significantly increased HA production compared to control (approximately 2 – 4-fold, whereas GlcN-Ac had no significant effect. Addition of 5 mM Gluc also increased HA production (approximately 2-fold, but 0.5 mM Gluc did not. Gene expression of the HA forming enzymes HAS 1, 2 and 3 was not altered by the addition of GlcN or Gluc. Conclusion Our data suggest that exogenous GlcN can increase HA production by synovium tissue and is more effective at lower concentrations than Gluc. This might indicate that GlcN exerts its potential analgesic properties through stimulation of synovial HA production.

  16. Synthesis of insulin-like growth factor binding protein 3 in vitro in human articular cartilage cultures.

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    Eviatar, Tamar; Kauffman, Hannah; Maroudas, Alice

    2003-02-01

    To quantify the rate of synthesis of insulin-like growth factor binding protein 3 (IGFBP-3) and insulin-like growth factor 1 (IGF-1) by in vitro cultures of normal and osteoarthritic (OA) human articular cartilage. Levels of IGF-1 and IGFBP-3 in media from in vitro cultures of human cartilage were determined by radioimmunoassay (RIA). IGFBPs were characterized by immunoblots and ligand blots. Ultrafiltration and RIA analysis of synovial fluid (SF) samples and washings of cartilage samples ex vivo were used to calculate partition coefficients and to estimate the amount of IGF-1 and IGFBP-3 in cartilage in vivo. OA cartilage synthesized 150 ng of IGFBP-3 per gm of cartilage per day, compared with 50 ng synthesized by normal cartilage. The surface zone of normal cartilage produced more IGFBP-3 than did the deep zone. Immunoblots and ligand blots confirmed the presence of IGFBP-3. IGFBP-3 synthesis was stimulated by exogenous IGF-1. No freshly synthesized IGF-1 was detected. The quantities of IGF-1 and IGFBP-3 present ex vivo were 11.3 and 78.7 ng/gm of cartilage in normal cartilage and 21.6 and 225.4 ng/gm in OA cartilage. The results show that while IGFBP-3 is synthesized in explant cultures, IGF-1 is not. The rate of IGFBP-3 synthesis is 3 times higher in OA than in normal cartilage. Both IGFBP-3 and IGF-1 penetrate into cartilage from SF in vivo. We estimate that the quantities of IGFBP-3 produced in culture by human cartilage are small compared with the amount supplied in the form of "small complexes" from the circulation. The high value of the partition coefficient of IGFBP-3 implies binding to the matrix.

  17. EXPLANTATION OF MESANGIAL CELL HILLOCKS - A METHOD FOR OBTAINING HUMAN MESANGIAL CELLS IN CULTURE

    NARCIS (Netherlands)

    MULLER, EW; KIM, Y; MICHAEL, AF; VERNIER, RL; VANDERHEM, GK; VANDERWOUDE, FJ

    1992-01-01

    A simple method is presented for selective cell culture of human mesangial cells using explanatation of mesangial cell hillocks. Glomeruli which had been incubated with collagenase were explanted on plastic tissue culture flasks. Three to 6 weeks after explantation, a rapidly growing multilayer of e

  18. EXPLANTATION OF MESANGIAL CELL HILLOCKS - A METHOD FOR OBTAINING HUMAN MESANGIAL CELLS IN CULTURE

    NARCIS (Netherlands)

    MULLER, EW; KIM, Y; MICHAEL, AF; VERNIER, RL; VANDERHEM, GK; VANDERWOUDE, FJ

    1992-01-01

    A simple method is presented for selective cell culture of human mesangial cells using explanatation of mesangial cell hillocks. Glomeruli which had been incubated with collagenase were explanted on plastic tissue culture flasks. Three to 6 weeks after explantation, a rapidly growing multilayer of e

  19. Thermogravimetry of irradiated human costal cartilage

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    Martinho Junior, Antonio C.; Machado, Luci D.B.; Dias, Djalma B.; Mathor, Monica B. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)]. E-mail: antonio_carlos_martinho@msn.com; lmachado@ipen.br; dbdias@ipen.br; mathor@ipen.br; Herson, Marisa R. [Universidade de Sao Paulo, SP (Brazil). Hospital das Clinicas. Banco de Tecidos do Instituto Central]. E-mail: marisah@vifm.org; Meumann, Nilton F.; Pasqualucci, Carlos Augusto G. [Universidade de Sao Paulo, SP (Brazil). Faculdade de Medicina. Servico de Verificacao de Obitos]. E-mail: svoc@usp.br

    2007-07-01

    Costal cartilage has been sterilized with gamma radiation using {sup 60}Co sources at two different doses, 25 kGy and 50 kGy, for storage in tissue banks. Samples of costal cartilage were deep-freezing as method of preservation. Thermogravimetry (Shimadzu TGA-50) was used to verify the water release of costal cartilage before and after irradiation. The TG tests were carried out at heating rate of 10 deg C/min from room temperature to 600 deg C under a flow rate of 50 mL/min of compressed air. Samples of costal cartilage were divided in 2 parts. One part of them was kept as reference material; the other part was irradiated. This procedure assures better homogeneity of the sample and reproducibility of the experimental results. The obtained data have shown that the TG curves have the same pattern, independently of the sample. Non-irradiated samples showed great variability of thermogravimetric curves among different donors and for the same donor. Further experimental work is being carried out on human cartilage preserved in glycerol in high concentration (> 98%) to compare with those deep freezing. (author)

  20. Collagen metabolism of human osteoarthritic articular cartilage as modulated by bovine collagen hydrolysates.

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    Saskia Schadow

    Full Text Available Destruction of articular cartilage is a characteristic feature of osteoarthritis (OA. Collagen hydrolysates are mixtures of collagen peptides and have gained huge public attention as nutriceuticals used for prophylaxis of OA. Here, we evaluated for the first time whether different bovine collagen hydrolysate preparations indeed modulate the metabolism of collagen and proteoglycans from human OA cartilage explants and determined the chemical composition of oligopeptides representing collagen fragments. Using biophysical techniques, like MALDI-TOF-MS, AFM, and NMR, the molecular weight distribution and aggregation behavior of collagen hydrolysates from bovine origin (CH-Alpha®, Peptan™ B 5000, Peptan™ B 2000 were determined. To investigate the metabolism of human femoral OA cartilage, explants were obtained during knee replacement surgery. Collagen synthesis of explants as modulated by 0-10 mg/ml collagen hydrolysates was determined using a novel dual radiolabeling procedure. Proteoglycans, NO, PGE(2, MMP-1, -3, -13, TIMP-1, collagen type II, and cell viability were determined in explant cultures. Groups of data were analyzed using ANOVA and the Friedman test (n = 5-12. The significance was set to p≤0.05. We found that collagen hydrolysates obtained from different sources varied with respect to the width of molecular weight distribution, average molecular weight, and aggregation behavior. None of the collagen hydrolysates tested stimulated the biosynthesis of collagen. Peptan™ B 5000 elevated NO and PGE(2 levels significantly but had no effect on collagen or proteoglycan loss. All collagen hydrolysates tested proved not to be cytotoxic. Together, our data demonstrate for the first time that various collagen hydrolysates differ with respect to their chemical composition of collagen fragments as well as by their pharmacological efficacy on human chondrocytes. Our study underscores the importance that each collagen hydrolysate

  1. Chondrogenic Priming at Reduced Cell Density Enhances Cartilage Adhesion of Equine Allogeneic MSCs - a Loading Sensitive Phenomenon in an Organ Culture Study with 180 Explants

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    Jan H. Spaas

    2015-09-01

    Full Text Available Background: Clinical results of regenerative treatments for osteoarthritis are becoming increasingly significant. However, several questions remain unanswered concerning mesenchymal stem cell (MSC adhesion and incorporation into cartilage. Methods: To this end, peripheral blood (PB MSCs were chondrogenically induced and/or stimulated with pulsed electromagnetic fields (PEMFs for a brief period of time just sufficient to prime differentiation. In an organ culture study, PKH26 labelled MSCs were added at two different cell densities (0.5 x106 vs 1.0 x106. In total, 180 explants of six horses (30 per horse were divided into five groups: no lesion (i, lesion alone (ii, lesion with naïve MSCs (iii, lesion with chondrogenically-induced MSCs (iv and lesion with chondrogenically-induced and PEMF-stimulated MSCs (v. Half of the explants were mechanically loaded and compared with the unloaded equivalents. Within each circumstance, six explants were histologically evaluated at different time points (day 1, 5 and 14. Results: COMP expression was selectively increased by chondrogenic induction (p = 0.0488. PEMF stimulation (1mT for 10 minutes further augmented COL II expression over induced values (p = 0.0405. On the other hand, MSC markers remained constant over time after induction, indicating a largely predifferentiated state. In the unloaded group, MSCs adhered to the surface in 92.6% of the explants and penetrated into 40.7% of the lesions. On the other hand, physiological loading significantly reduced surface adherence (1.9% and lesion filling (3.7% in all the different conditions (p Conclusion: The present study demonstrates that primed chondrogenic induction of MSCs at a lower cell density without loading results in significantly enhanced and homogenous MSC adhesion and incorporation into equine cartilage.

  2. Human neural progenitor cells promote photoreceptor survival in retinal explants.

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    Englund-Johansson, Ulrica; Mohlin, Camilla; Liljekvist-Soltic, Ingela; Ekström, Per; Johansson, Kjell

    2010-02-01

    Different types of progenitor and stem cells have been shown to provide neuroprotection in animal models of photoreceptor degeneration. The present study was conducted to investigate whether human neural progenitor cells (HNPCs) have neuroprotective properties on retinal explants models with calpain- and caspase-3-dependent photoreceptor cell death. In the first experiments, HNPCs in a feeder layer were co-cultured for 6 days either with postnatal rd1 mouse or normal rat retinas. Retinal histological sections were used to determine outer nuclear layer (ONL) thickness, and to detect the number of photoreceptors with labeling for calpain activity, cleaved caspase-3 and TUNEL. The ONL thickness of co-cultured rat and rd1 retinas was found to be almost 10% and 40% thicker, respectively, compared to controls. Cell counts of calpain activity, cleaved caspase-3 and TUNEL labeled photoreceptors in both models revealed a 30-50% decrease when co-cultured with HNPCs. The results represent significant increases of photoreceptor survival in the co-cultured retinas. In the second experiments, for an identification of putative survival factors, or a combination of them, a growth factor profile was performed on conditioned medium. The relative levels of various growth factors were analyzed by densitometric measurements of growth factor array membranes. Following growth factors were identified as most potential survival factors; granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GMCSF), insulin-like growth factor II (IGF-II), neurotrophic factor 3 (NT-3), placental growth factor (PIGF), transforming growth factors (TGF-beta1 and TGF-beta2) and vascular endothelial growth factor (VEGF-D). HNPCs protect both against calpain- and caspase-3-dependent photoreceptor cell death in the rd1 mouse and against caspase-3-dependent photoreceptor cell death in normal rat retinas in vitro. The protective effect is possibly achieved by a variety of

  3. Metabolic Response of Human Osteoarthritic Cartilage to Biochemically Characterized Collagen Hydrolysates

    Science.gov (United States)

    Schadow, Saskia; Simons, Viktor S.; Lochnit, Guenter; Kordelle, Jens; Gazova, Zuzana; Siebert, Hans-Christian; Steinmeyer, Juergen

    2017-01-01

    The most frequent disease of the locomotor system is osteoarthritis (OA), which, as a chronic joint disease, might benefit more from nutrition than acute illnesses. Collagen hydrolysates (CHs) are peptidic mixtures that are often used as nutraceuticals for OA. Three CHs were characterized biochemically and pharmacologically. Our biophysical (MALDI-TOF-MS, NMR, AFM) and fluorescence assays revealed marked differences between CHs of fish (Peptan® F 5000, Peptan® F 2000) and porcine (Mobiforte®) origin with respect to the total number of peptides and common peptides between them. Using a novel dual radiolabeling procedure, no CH modulated collagen biosynthesis in human knee cartilage explants. Peptan® F 2000 enhanced the activities of the aggrecanase ADMATS4 and ADMATS5 in vitro without loss of proteoglycan from cartilage explants; the opposite effect was observed with Mobiforte®. Interleukin (IL)-6, matrix metalloproteinase (MMP)-1, -3 and -13 levels were elevated in explants that were treated with Mobiforte® and Peptan® F 5000, but not with Peptan® F 2000. In conclusion, the heterogeneous peptide composition and disparate pharmacological effects between CHs suggest that the effect of a CH preparation cannot be extrapolated to other formulations. Thus, the declaration of a CH as a safe and effective nutraceutical requires a thorough examination of its pleiotropic effects. PMID:28117674

  4. Metabolic Response of Human Osteoarthritic Cartilage to Biochemically Characterized Collagen Hydrolysates

    Directory of Open Access Journals (Sweden)

    Saskia Schadow

    2017-01-01

    Full Text Available The most frequent disease of the locomotor system is osteoarthritis (OA, which, as a chronic joint disease, might benefit more from nutrition than acute illnesses. Collagen hydrolysates (CHs are peptidic mixtures that are often used as nutraceuticals for OA. Three CHs were characterized biochemically and pharmacologically. Our biophysical (MALDI-TOF-MS, NMR, AFM and fluorescence assays revealed marked differences between CHs of fish (Peptan® F 5000, Peptan® F 2000 and porcine (Mobiforte® origin with respect to the total number of peptides and common peptides between them. Using a novel dual radiolabeling procedure, no CH modulated collagen biosynthesis in human knee cartilage explants. Peptan® F 2000 enhanced the activities of the aggrecanase ADMATS4 and ADMATS5 in vitro without loss of proteoglycan from cartilage explants; the opposite effect was observed with Mobiforte®. Interleukin (IL-6, matrix metalloproteinase (MMP-1, -3 and -13 levels were elevated in explants that were treated with Mobiforte® and Peptan® F 5000, but not with Peptan® F 2000. In conclusion, the heterogeneous peptide composition and disparate pharmacological effects between CHs suggest that the effect of a CH preparation cannot be extrapolated to other formulations. Thus, the declaration of a CH as a safe and effective nutraceutical requires a thorough examination of its pleiotropic effects.

  5. Explant culture of human peripheral lung. I. Metabolism of benzo[alpha]pyrene

    DEFF Research Database (Denmark)

    Stoner, G.D.; Harris, C.C.; Autrup, Herman

    1978-01-01

    Human lung explants have been maintained in vitro for a period of 25 days. Autoradiographic studies indicated that the broncholar epithelial cells, type 2 alveolar epithelial cells, and stromal fibroblasts incorporated 3H-thymidine during the culture. After 7 to 10 days, type 2 cells were the pre...

  6. Culture of human adipose tissue explants leads to profound alteration of adipocyte gene expression.

    Science.gov (United States)

    Gesta, S; Lolmède, K; Daviaud, D; Berlan, M; Bouloumié, A; Lafontan, M; Valet, P; Saulnier-Blache, J S

    2003-03-01

    Primary culture of adipose tissue has often been used to investigate pharmacological and nutritional regulation of adipocyte gene expression. Possible alteration of adipocyte gene expression by primary culture on its own has not been explored in detail. In order to address this issue, explants were prepared from human subcutaneous adipose tissue recovered from plastic surgery and maintained for 0 to 48 h in DMEM supplemented with 10 % serum. At different time points, adipocytes were isolated from the explants by collagenase digestion, and mRNA expression and lipolysis were studied. Culture was associated with an accumulation of tumor necrosis factor-alpha (TNFalpha) in the culture medium, an increase in anaerobic glycolysis, and an increase in the basal lipolysis. In parallel, a rapid and dramatic decrease in the level of mRNA encoding for several adipocyte-specific proteins such as adipocyte lipid-binding protein, hormone-sensitive lipase, lipoprotein lipase, and peroxisome proliferation activating receptor-gamma2 was observed in isolated adipocytes. These downregulations were reminiscent of a dedifferentiation process. In parallel, primary culture was associated with an increase in adipocyte beta-actin, TNFalpha, glucose transporter-1 and hypoxia-induced factor-1alpha mRNAs. Treatment of explants with agents that increase cAMP (isobutylmethylxanthine and forskolin) prevented TNFalpha production and expression and culture-induced alterations of adipocyte gene expression. These data show that primary culture of human adipose tissue explants dramatically alters adipocyte gene expression.

  7. Characteristic of c-Kit+ progenitor cells in explanted human hearts

    OpenAIRE

    Matuszczak, Sybilla; Czapla, Justyna; Jarosz-Biej, Magdalena; Wiśniewska, Ewa; Cichoń, Tomasz; Smolarczyk, Ryszard; Kobusińska, Magdalena; Gajda, Karolina; Wilczek, Piotr; Śliwka, Joanna; Zembala, Michał; Zembala, Marian; Szala, Stanisław

    2014-01-01

    According to literature data, self-renewing, multipotent, and clonogenic cardiac c-Kit+ progenitor cells occur within human myocardium. The aim of this study was to isolate and characterize c-Kit+ progenitor cells from explanted human hearts. Experimental material was obtained from 19 adult and 7 pediatric patients. Successful isolation and culture was achieved for 95 samples (84.1 %) derived from five different regions of the heart: right and left ventricles, atrium, intraventricular septum,...

  8. Millicurrent stimulation of human articular chondrocytes cultivated in a collagen type-I gel and of human osteochondral explants

    Directory of Open Access Journals (Sweden)

    Silny Jiri

    2010-08-01

    Full Text Available Abstract Background Here we investigate the effect of millicurrent treatment on human chondrocytes cultivated in a collagen gel matrix and on human osteochondral explants. Methods Human chondrocytes from osteoarthritic knee joints were enzymatically released and transferred into a collagen type-I gel. Osteochondral explants and cell-seeded gel samples were cultivated in-vitro for three weeks. Samples of the verum groups were stimulated every two days by millicurrent treatment (3 mA, sinusoidal signal of 312 Hz amplitude modulated by two super-imposed signals of 0.28 Hz, while control samples remained unaffected. After recovery, collagen type-I, type-II, aggrecan, interleukin-1β, IL-6, TNFα and MMP13 were examined by immunohistochemistry and by real time PCR. Results With regard to the immunostainings 3 D gel samples and osteochondral explants did not show any differences between treatment and control group. The expression of all investigated genes of the 3 D gel samples was elevated following millicurrent treatment. While osteochondral explant gene expression of col-I, col-II and Il-1β was nearly unaffected, aggrecan gene expression was elevated. Following millicurrent treatment, IL-6, TNFα, and MMP13 gene expression decreased. In general, the standard deviations of the gene expression data were high, resulting in rarely significant results. Conclusions We conclude that millicurrent stimulation of human osteoarthritic chondrocytes cultivated in a 3 D collagen gel and of osteochondral explants directly influences cell metabolism.

  9. Biochemical effects on long-term frozen human costal cartilage

    Energy Technology Data Exchange (ETDEWEB)

    Santin, Stefany P.; Martinho Junior, Antonio C.; Yoshito, Daniele; Soares, Fernando A.N.; Mathor, Monica B., E-mail: mathor@ipen.b [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

    2011-07-01

    Currently, the progresses on treatment of musculoskeletal diseases with the evolving of artificial implants and the success of tissue transplantation between genetically different individuals have conducted to an increase in radiosterilization. Regarding to tissue transplantation, it is essential to have sterile tissue and many tissue banks use radiosterilization as an effective method to sterilize these tissues. However, high doses of ionizing radiation and the preservation method may induce structural modifications in the tissues, as degradation of structural scaffold, decreasing its mechanical properties. Particularly, cartilage have been preserved in high concentrations of glycerol or deep-frozen at -70 degree C for storage after radiosterilization. Therefore, it is important to study the modifications induced in cartilage by preservation methods and by radiosterilization to determine the appropriated parameters for high quality of human allografts. Costal cartilages were obtained from cadaveric donors and were frozen at -20 degree C for 2 years long in order to compare with previous studies for fresh, deep-frozen and glycerolised cartilages. The mechanical tests were carried out in a universal testing machine until sample failure. According our results, there is no significant statistical difference between stress at break of fresh, long-term - 20 degree C frozen cartilages and deep-frozen cartilage. This early result suggests, regarding to tensile property, that long-term - 20 degree C frozen cartilages corresponds to glycerolised costal cartilages irradiated with 25 kGy or deep-frozen cartilages irradiated with 25 and 50 kGy. Thus, this long-term frozen cartilages may be used for tissue banks, but more studies about effects of ionizing radiation are necessary. (author)

  10. The cytotoxicity of bupivacaine, ropivacaine, and mepivacaine on human chondrocytes and cartilage.

    Science.gov (United States)

    Breu, Anita; Rosenmeier, Katharina; Kujat, Richard; Angele, Peter; Zink, Wolfgang

    2013-08-01

    Intraarticular injections of local anesthetics are frequently used as part of multimodal pain regimens. However, recent data suggest that local anesthetics affect chondrocyte viability. In this study, we assessed the chondrotoxic effects of mepivacaine, ropivacaine, and bupivacaine. We hypothesized that specific cytotoxic potencies directly correlate with analgesic potencies, and that cytotoxic effects in intact cartilage are different than in osteoarthritic tissue. Human articular chondrocytes were exposed to equal and equipotent concentrations of bupivacaine, ropivacaine, and mepivacaine for 1 hour. Cell viability, apoptosis, and necrosis were determined at predefined time points using flow cytometry, live-dead staining, and caspase detection. Intact and osteoarthritic human cartilage explants were treated with equipotent concentrations of named drugs to determine cell viability applying fluorescence microscopy. Chondrotoxic effects increased from ropivacaine to mepivacaine to bupivacaine in a time-dependent and concentration-dependent manner. Compared with control, bupivacaine 0.5% decreased chondrocyte viability to 78% ± 9% (P = 0.0183) 1 hour and 16% ± 10% (P mepivacaine 2%, viable cells were scored 36% ± 6% (P mepivacaine exposure (P = 0.0059). Exposure to concentrations up to 0.25% of bupivacaine, 0.5% of ropivacaine, and 0.5% of mepivacaine did not reveal significant chondrotoxicity in flow cytometry. However, chondrotoxicity did not correlate with potency of local anesthetics. Immediate cell death was mainly due to necrosis followed by apoptosis. Cellular death rates were clearly higher in osteoarthritic compared with intact cartilage after bupivacaine, mepivacaine, and ropivacaine treatment in a decreasing order. Bupivacaine, ropivacaine, and mepivacaine are chondrotoxic in a time-dependent, concentration-dependent, and drug-dependent manner. Chondrotoxic and analgesic potencies do not directly correlate. Cellular death rates were higher in

  11. 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.

  12. 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.

  13. Explant culture: An advantageous method for isolation of mesenchymal stem cells from human tissues.

    Science.gov (United States)

    Hendijani, Fatemeh

    2017-04-01

    Mesenchymal stem cell (MSC) research progressively moves towards clinical phases. Accordingly, a wide range of different procedures were presented in the literature for MSC isolation from human tissues; however, there is not yet any close focus on the details to offer precise information for best method selection. Choosing a proper isolation method is a critical step in obtaining cells with optimal quality and yield in companion with clinical and economical considerations. In this concern, current review widely discusses advantages of omitting proteolysis step in isolation process and presence of tissue pieces in primary culture of MSCs, including removal of lytic stress on cells, reduction of in vivo to in vitro transition stress for migrated/isolated cells, reduction of price, processing time and labour, removal of viral contamination risk, and addition of supporting functions of extracellular matrix and released growth factors from tissue explant. In next sections, it provides an overall report of technical highlights and molecular events of explant culture method for isolation of MSCs from human tissues including adipose tissue, bone marrow, dental pulp, hair follicle, cornea, umbilical cord and placenta. Focusing on informative collection of molecular and methodological data about explant methods can make it easy for researchers to choose an optimal method for their experiments/clinical studies and also stimulate them to investigate and optimize more efficient procedures according to clinical and economical benefits. © 2017 John Wiley & Sons Ltd.

  14. Pistacia lentiscus fruit oil reduces oxidative stress in human skin explants caused by hydrogen peroxide.

    Science.gov (United States)

    Ben Khedir, S; Moalla, D; Jardak, N; Mzid, M; Sahnoun, Z; Rebai, T

    2016-10-01

    We investigated the efficacy of Pistacia lentiscus fruit oil (PLFO) for protecting human skin from damage due to oxidative stress. PLFO contains natural antioxidants including polyphenols, sterols and tocopherols. We compared the antioxidant potential of PLFO with extra virgin olive oil (EVOO). Explants of healthy adult human skin were grown in culture with either PLFO or EVOO before adding hydrogen peroxide (H2O2). We also used cultured skin explants to investigate the effects of PLFO on lipid oxidation and depletion of endogenous antioxidant defense enzymes including glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) one day after 2 h exposure to H2O2. We found that PLFO scavenged radicals and protected skin against oxidative injury. PLFO exhibited greater antioxidant and free radical scavenging activity than EVOO. Skin explants treated with PLFO inhibited H2O2 induced MDA formation by inhibition of lipid oxidation. In addition, the oil inhibited H2O2 induced depletion of antioxidant defense enzymes including GPx, SOD and CAT. We found that treatment with PLFO repaired skin damage owing to its antioxidant properties.

  15. Structures of benzo(a)pyrene-nucleic acid adducts formed in human and bovine bronchial explants

    DEFF Research Database (Denmark)

    1977-01-01

    obtained evidence that the same derivative is involved in the binding of BP to the DNA of human bronchial explants, although details of the specific isomer involved and of the structure of the adduct were not reported. We describe here studies on RNA and DNA adducts formed by human bronchial explants...... and provide evidence that the structures of the major adducts are similar to those formed in the analogous bovine system....

  16. Comparative digital cartilage histology for human and common osteoarthritis models

    Directory of Open Access Journals (Sweden)

    Pedersen DR

    2013-02-01

    Full Text Available Douglas R Pedersen, Jessica E Goetz, Gail L Kurriger, James A MartinDepartment of Orthopaedics and Rehabilitation, University of Iowa, Iowa City, IA, USAPurpose: This study addresses the species-specific and site-specific details of weight-bearing articular cartilage zone depths and chondrocyte distributions among humans and common osteoarthritis (OA animal models using contemporary digital imaging tools. Histological analysis is the gold-standard research tool for evaluating cartilage health, OA severity, and treatment efficacy. Historically, evaluations were made by expert analysts. However, state-of-the-art tools have been developed that allow for digitization of entire histological sections for computer-aided analysis. Large volumes of common digital cartilage metrics directly complement elucidation of trends in OA inducement and concomitant potential treatments.Materials and methods: Sixteen fresh human knees, 26 adult New Zealand rabbit stifles, and 104 bovine lateral plateaus were measured for four cartilage zones and the cell densities within each zone. Each knee was divided into four weight-bearing sites: the medial and lateral plateaus and femoral condyles.Results: One-way analysis of variance followed by pairwise multiple comparisons (Holm–Sidak method at a significance of 0.05 clearly confirmed the variability between cartilage depths at each site, between sites in the same species, and between weight-bearing articular cartilage definitions in different species.Conclusion: The present study clearly demonstrates multisite, multispecies differences in normal weight-bearing articular cartilage, which can be objectively quantified by a common digital histology imaging technique. The clear site-specific differences in normal cartilage must be taken into consideration when characterizing the pathoetiology of OA models. Together, these provide a path to consistently analyze the volume and variety of histologic slides necessarily generated

  17. Further assessment of neuropathology in retinal explants and neuroprotection by human neural progenitor cells

    Science.gov (United States)

    Mohlin, Camilla; Liljekvist-Soltic, Ingela; Johansson, Kjell

    2011-10-01

    Explanted rat retinas show progressive photoreceptor degeneration that appears to be caspase-12-dependent. Decrease in photoreceptor density eventually affects the inner retina, particularly in the bipolar cell population. Explantation and the induced photoreceptor degeneration are accompanied by activation of Müller and microglia cells. The goal of this study was to determine whether the presence of a feeder layer of human neural progenitor cells (hNPCs) could suppress the degenerative and reactive changes in the explants. Immunohistochemical analyses showed considerable sprouting of rod photoreceptor axon terminals into the inner retina and reduced densities of cone and rod bipolar cells. Both sprouting and bipolar cell degenerations were significantly lower in retinas cultured with feeder layer cells compared to cultured controls. A tendency toward reduced microglia activation in the retinal layers was also noted in the presence of feeder layer cells. These results indicate that hNPCs or factors produced by them can limit the loss of photoreceptors and secondary injuries in the inner retina. The latter may be a consequence of disrupted synaptic arrangement.

  18. Lubricin is expressed in chondrocytes derived from osteoarthritic cartilage encapsulated in poly (ethylene glycol) diacrylate scaffold.

    Science.gov (United States)

    Musumeci, G; Loreto, C; Carnazza, M L; Coppolino, F; Cardile, V; Leonardi, R

    2011-01-01

    Osteoarthritis (OA) is characterized by degenerative changes within joints that involved quantitative and/or qualitative alterations of cartilage and synovial fluid lubricin, a mucinous glycoprotein secreted by synovial fibroblasts and chondrocytes. Modern therapeutic methods, including tissue-engineering techniques, have been used to treat mechanical damage of the articular cartilage but to date there is no specific and effective treatment. This study aimed at investigating lubricin immunohistochemical expression in cartilage explant from normal and OA patients and in cartilage constructions formed by Poly (ethylene glycol) (PEG) based hydrogels (PEG-DA) encapsulated OA chondrocytes. The expression levels of lubricin were studied by immunohistochemistry: i) in tissue explanted from OA and normal human cartilage; ii) in chondrocytes encapsulated in hydrogel PEGDA from OA and normal human cartilage. Moreover, immunocytochemical and western blot analysis were performed in monolayer cells from OA and normal cartilage. The results showed an increased expression of lubricin in explanted tissue and in monolayer cells from normal cartilage, and a decreased expression of lubricin in OA cartilage. The chondrocytes from OA cartilage after 5 weeks of culture in hydrogels (PEGDA) showed an increased expression of lubricin compared with the control cartilage. The present study demonstrated that OA chondrocytes encapsulated in PEGDA, grown in the scaffold and were able to restore lubricin biosynthesis. Thus our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repairing cartilage lesions in patients with OA to reduce at least the progression of the disease.

  19. Glucocorticoids affect 24 h clock genes expression in human adipose tissue explant cultures.

    Directory of Open Access Journals (Sweden)

    Purificación Gómez-Abellán

    Full Text Available AIMS: to examine firstly whether CLOCK exhibits a circadian expression in human visceral (V and subcutaneous (S adipose tissue (AT in vitro as compared with BMAL1 and PER2, and secondly to investigate the possible effect of the glucocorticoid analogue dexamethasone (DEX on positive and negative clock genes expression. SUBJECTS AND METHODS: VAT and SAT biopsies were obtained from morbid obese women (body mass index ≥ 40 kg/m(2 (n = 6. In order to investigate rhythmic expression pattern of clock genes and the effect of DEX on CLOCK, PER2 and BMAL1 expression, control AT (without DEX and AT explants treated with DEX (2 hours were cultured during 24 h and gene expression was analyzed at the following times: 10:00 h, 14:00 h, 18:00 h, 22:00 h, 02:00 h and 06:00 h, using qRT-PCR. RESULTS: CLOCK, BMAL1 and PER2 expression exhibited circadian patterns in both VAT and SAT explants that were adjusted to a typical 24 h sinusoidal curve. PER2 expression (negative element was in antiphase with respect to CLOCK and in phase with BMAL1 expression (both positive elements in the SAT (situation not present in VAT. A marked effect of DEX exposure on both positive and negative clock genes expression patterns was observed. Indeed, DEX treatment modified the rhythmicity pattern towards altered patterns with a period lower than 24 hours in all genes and in both tissues. CONCLUSIONS: 24 h patterns in CLOCK and BMAL1 (positive clock elements and PER2 (negative element mRNA levels were observed in human adipose explants. These patterns were altered by dexamethasone exposure.

  20. The Relationship between MR Parameters and Biomechanical Quantities of Loaded Human Articular Cartilage in Osteoarthritis: An In-Vitro Study

    Science.gov (United States)

    Juráš, V.; Szomolányi, P.; Gäbler, S.; Frollo, I.; Trattnig, S.

    2009-01-01

    The aim of this study was to assess the changes in MRI parameters during applied load directly in MR scanner and correlate these changes with biomechanical parameters of human articular cartilage. Cartilage explants from patients who underwent total knee replacement were examined in the micro-imaging system in 3T scanner. Respective MRI parameters (T1 without- and T1 with contrast agent as a marker of proteoglycan content, T2 as a marker of collagen network anisotropy and ADC as a measure of diffusivity) were calculated in pre- and during compression state. Subsequently, these parameters were compared to the biomechanical properties of articular cartilage, instantaneous modulus (I), equilibrium modulus (Eq) and time of tissue relaxation (τ). Significant load-induced changes of T2 and ADC were recorded. High correlation between T1Gd and I (r = 0.6324), and between ADC and Eq (r = -0.4884) was found. Multi-parametric MRI may have great potential in analyzing static and dynamic biomechanical behavior of articular cartilage in early stages of osteoarthritis (OA).

  1. Boundary mode lubrication of articular cartilage by recombinant human lubricin.

    Science.gov (United States)

    Gleghorn, Jason P; Jones, Aled R C; Flannery, Carl R; Bonassar, Lawrence J

    2009-06-01

    Lubrication of cartilage involves a variety of physical and chemical factors, including lubricin, a synovial glycoprotein that has been shown to be a boundary lubricant. It is unclear how lubricin boundary lubricates a wide range of bearings from tissue to artificial surfaces, and if the mechanism is the same for both soluble and bound lubricin. In the current study, experiments were conducted to investigate the hypothesis that recombinant human lubricin (rh-lubricin) lubricates cartilage in a dose-dependent manner and that soluble and bound fractions of rh-lubricin both contribute to the lubrication process. An rh-lubricin dose response was observed with maximal lubrication achieved at concentrations of rh-lubricin greater than 50 microg/mL. A concentration-response variable-slope model was fit to the data, and indicated that rh-lubricin binding to cartilage was not first order. The pattern of decrease in equilibrium friction coefficient indicated that aggregation of rh-lubricin or steric arrangement may regulate boundary lubrication. rh-lubricin localized at the cartilage surface was found to lubricate a cartilage-glass interface in boundary mode, as did soluble rh-lubricin at high concentrations (150 microg/mL); however, the most effective lubrication occurred when both soluble and bound rh-lubricin were present at the interface. These findings point to two distinct mechanisms by which rh-lubricin lubricates, one mechanism involving lubricin bound to the tissue surface and the other involving lubricin in solution.

  2. Effects of Er:YAG laser irradiation on human cartilage

    Science.gov (United States)

    Glinkowski, Wojciech; Brzozowska, Malgorzata; Ciszek, Bogdan; Rowinski, Jan; Strek, Wieslaw

    1996-03-01

    Irradiation of the hyaline or fibrous cartilage excised from the body of a human cadaver with Er:YAG laser beam, single pulse with a dose of 1 J, produces a crater with a depth of approximately 500 micrometers and a diameter varying from 5 to 300 micrometers. Histological examination has revealed that the laser-made craters were surrounded by a thin rim (2-10 micrometer) of charred and coagulated tissue. No damage was observed in the cartilage surrounding the rim. The presence of sharp demarcation between the tissue areas ablated by laser energy and the undamaged areas argues for the potential usefulness of the Er:YAG laser in surgery of cartilages.

  3. 2-photon laser scanning microscopy on native human cartilage

    Science.gov (United States)

    Martini, Joerg; Toensing, Katja; Dickob, Michael; Anselmetti, Dario

    2005-08-01

    Native hyaline cartilage from a human knee joint was directly investigated with laser scanning microscopy via 2-photon autofluorescence excitation with no additional staining or labelling protocols in a nondestructive and sterile manner. Using a femtosecond, near-infrared (NIR) Ti:Sa laser for 2-photon excitation and a dedicated NIR long distance objective, autofluorescence imaging and measurements of the extracellular matrix (ECM) tissue with incorporated chondrocytes were possible with a penetration depth of up to 460 μm inside the sample. Via spectral autofluorescence separation these experiments allowed the discrimination of chondrocytes from the ECM and therefore an estimate of chondrocytic cell density within the cartilage tissue to approximately 0.2-2•107cm3. Furthermore, a comparison of the relative autofluorescence signals between nonarthritic and arthritic cartilage tissue exhibited distinct differences in tissue morphology. As these morphological findings are in keeping with the macroscopic diagnosis, our measurement has the potential of being used in future diagnostic applications.

  4. An animal explant model for the study of human cutaneous squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Daniel A Belkin

    Full Text Available We established a human tissue explant model to facilitate study of cutaneous squamous cell carcinoma. We accomplished this by implanting debulked SCC, from surgical discard, into nude rats. Human SCC remained viable and continued to proliferate for at least 4 weeks and showed evidence of neovascularization. At 4 weeks, SCC implants showed a trend toward increased PCNA positive cells compared to fresh SCC cells/mm(2 tissue supporting continued proliferation throughout engraftment. Von Willebrand's Factor (VWF positive cells were found within implants and likely represented rat vessel neovascularization. Human Langerhans' (Langerin+ cells, but no T cells (CD3+, CD8+, FoxP3+, macrophages (CD163, or NK cells (NKp46, were present in SCC implants at 4 weeks. These findings support the possibility that LCs fail to migrate from cutaneous SCC and thus contribute to lack of effective antitumor response. Our findings also provide a novel model system for further study of primary cutaneous SCC.

  5. Lubricin is expressed in chondrocytes derived from osteoarthritic cartilage encapsulated in poly(ethylene glycol diacrylate scaffold

    Directory of Open Access Journals (Sweden)

    G. Musumeci

    2011-09-01

    Full Text Available Osteoarthritis (OA is characterized by degenerative changes within joints that involved quantitative and/or qualitative alterations of cartilage and synovial fluid lubricin, a mucinous glycoprotein secreted by synovial fibroblasts and chondrocytes. Modern therapeutic methods, including tissue-engineering techniques, have been used to treat mechanical damage of the articular cartilage but to date there is no specific and effective treatment. This study aimed at investigating lubricin immunohistochemical expression in cartilage explant from normal and OA patients and in cartilage constructions formed by Poly (ethylene glycol (PEG based hydrogels (PEG-DA encapsulated OA chondrocytes. The expression levels of lubricin were studied by immunohistochemistry: i in tissue explanted from OA and normal human cartilage; ii in chondrocytes encapsulated in hydrogel PEGDA from OA and normal human cartilage. Moreover, immunocytochemical and western blot analysis were performed in monolayer cells from OA and normal cartilage. The results showed an increased expression of lubricin in explanted tissue and in monolayer cells from normal cartilage, and a decreased expression of lubricin in OA cartilage. The chondrocytes from OA cartilage after 5 weeks of culture in hydrogels (PEGDA showed an increased expression of lubricin compared with the control cartilage. The present study demonstrated that OA chondrocytes encapsulated in PEGDA, grown in the scaffold and were able to restore lubricin biosynthesis. Thus our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repairing cartilage lesions in patients with OA to reduce at least the progression of the disease.

  6. Special pattern of endochondral ossification in human laryngeal cartilages: X-ray and light-microscopic studies on thyroid cartilage.

    Science.gov (United States)

    Claassen, Horst; Schicht, Martin; Sel, Saadettin; Paulsen, Friedrich

    2014-04-01

    Endochondral ossification is a process that also occurs in the skeleton of the larynx. Differences in the ossification mechanism in comparison to growth plates are not understood until now. To get deeper insights into this process, human thyroid cartilage was investigated by the use of X-rays and a series of light-microscopic stainings. A statistical analysis of mineralization was done by scanning areas of mineralized cartilage and of ossification. We detected a special mode of endochondral ossification which differs from the processes in growth plates. Thyroid cartilage ossifies very slowly and in a gender-specific manner. Compared with age-matched women, bone formation in thyroid cartilage of men is significantly higher in the age group 41-60 years. Endochondral ossification is prepared by internal changes of extracellular matrix leading to areas of asbestoid fibers with ingrowing cartilage canals. In contrast to growth plates, bone is deposited on large areas of mineralized cartilage, which appear at the rims of cartilage canals. Furthermore, primary parallel fibered bone was observed which was deposited on woven bone. The predominant bone type is cancellous bone with trabeculae, whereas compact bone with Haversian systems was seldom found. Trabeculae contain a great number of reversal and arresting lines meaning that the former were often reconstructed and that bone formation was arrested and resumed again with advancing age. It is hypothesized that throughout life trabeculae of ossified thyroid cartilage undergo adaptation to different loads due to the use of voice.

  7. A comparative study of candidal invasion in rabbit tongue mucosal explants and reconstituted human oral epithelium.

    Science.gov (United States)

    Jayatilake, J A M S; Samaranayake, Y H; Samaranayake, L P

    2008-06-01

    The purpose of this study is to compare the light and scanning electron microscopic (SEM) features of tissue invasion by three Candida species (C. albicans, C. tropicalis, and C. dubliniensis) in two different tissue culture models: rabbit tongue mucosal explants (RTME) and reconstituted human oral epithelium (RHOE). Tongue mucosal biopsies of healthy New Zealand rabbits were maintained in explant culture using a transwell system. RHOE was obtained from Skinethic Laboratory (Nice, France). RTME and RHOE were inoculated with C. albicans, C. tropicalis, and C. dubliniensis separately and incubated at 37 degrees C, 5% CO(2), and 100% humidity up to 48 h. Light microscopic and SEM examinations of uninfected (controls) and infected tissues were performed at 24 and 48 h. C. albicans produced characteristic hallmarks of pathological tissue invasion in both tissue models over a period of 48 h. Hyphae penetrated through epithelial cells and intercellular gaps latter resembling thigmotropism. SEM showed cavitations on the epithelial cell surfaces particularly pronounced at sites of hyphal invasion. Some hyphae on RTME showed several clusters of blastospores attached in regular arrangements resembling "appareil sporifere". C. tropicalis and C. dubliniensis produced few hyphae mainly on RTME but they did not penetrate either model. Our findings indicate that multiple host-fungal interactions such as cavitations, thigmotropism, and morphogenesis take place during candidal tissue invasion. RTME described here appears to be useful in investigations of such pathogenic processes of Candida active at the epithelial front.

  8. Thermal neutron irradiation field design for boron neutron capture therapy of human explanted liver.

    Science.gov (United States)

    Bortolussi, S; Altieri, S

    2007-12-01

    The selective uptake of boron by tumors compared to that by healthy tissue makes boron neutron capture therapy (BNCT) an extremely advantageous technique for the treatment of tumors that affect a whole vital organ. An example is represented by colon adenocarcinoma metastases invading the liver, often resulting in a fatal outcome, even if surgical resection of the primary tumor is successful. BNCT can be performed by irradiating the explanted organ in a suitable neutron field. In the thermal column of the Triga Mark II reactor at Pavia University, a facility was created for this purpose and used for the irradiation of explanted human livers. The neutron field distribution inside the organ was studied both experimentally and by means of the Monte Carlo N-particle transport code (MCNP). The liver was modeled as a spherical segment in MCNP and a hepatic-equivalent solution was used as an experimental phantom. In the as-built facility, the ratio between maximum and minimum flux values inside the phantom ((phi(max)/phi(min)) was 3.8; this value can be lowered to 2.3 by rotating the liver during the irradiation. In this study, the authors proposed a new facility configuration to achieve a uniform thermal neutron flux distribution in the liver. They showed that a phi(max)/phi(min) ratio of 1.4 could be obtained without the need for organ rotation. Flux distributions and dose volume histograms were reported for different graphite configurations.

  9. Human Endogenous Retrovirus W Activity in Cartilage of Osteoarthritis Patients

    Directory of Open Access Journals (Sweden)

    Signy Bendiksen

    2014-01-01

    Full Text Available The etiology of viruses in osteoarthritis remains controversial because the prevalence of viral nucleic acid sequences in peripheral blood or synovial fluid from osteoarthritis patients and that in healthy control subjects are similar. Until now the presence of virus has not been analyzed in cartilage. We screened cartilage and chondrocytes from advanced and non-/early osteoarthritis patients for parvovirus B19, herpes simplex virus-1, Epstein Barr virus, cytomegalovirus, human herpes virus-6, hepatitis C virus, and human endogenous retroviruses transcripts. Endogenous retroviruses transcripts, but none of the other viruses, were detected in 15 out the 17 patients. Sequencing identified the virus as HERV-WE1 and E2. HERV-W activity was confirmed by high expression levels of syncytin, dsRNA, virus budding, and the presence of virus-like particles in all advanced osteoarthritis cartilages examined. Low levels of HERV-WE1, but not E2 envelope RNA, were observed in 3 out of 8 non-/early osteoarthritis patients, while only 3 out of 7 chondrocytes cultures displayed low levels of syncytin, and just one was positive for virus-like particles. This study demonstrates for the first time activation of HERV-W in cartilage of osteoarthritis patients; however, a causative role for HERV-W in development or deterioration of the disease remains to be proven.

  10. Human sclera maintains common characteristics with cartilage throughout evolution.

    Directory of Open Access Journals (Sweden)

    Yuko Seko

    Full Text Available BACKGROUND: The sclera maintains and protects the eye ball, which receives visual inputs. Although the sclera does not contribute significantly to visual perception, scleral diseases such as refractory scleritis, scleral perforation and pathological myopia are considered incurable or difficult to cure. The aim of this study is to identify characteristics of the human sclera as one of the connective tissues derived from the neural crest and mesoderm. METHODOLOGY/PRINCIPAL FINDINGS: We have demonstrated microarray data of cultured human infant scleral cells. Hierarchical clustering was performed to group scleral cells and other mesenchymal cells into subcategories. Hierarchical clustering analysis showed similarity between scleral cells and auricular cartilage-derived cells. Cultured micromasses of scleral cells exposed to TGF-betas and BMP2 produced an abundant matrix. The expression of cartilage-associated genes, such as Indian hedge hog, type X collagen, and MMP13, was up-regulated within 3 weeks in vitro. These results suggest that human 'sclera'-derived cells can be considered chondrocytes when cultured ex vivo. CONCLUSIONS/SIGNIFICANCE: Our present study shows a chondrogenic potential of human sclera. Interestingly, the sclera of certain vertebrates, such as birds and fish, is composed of hyaline cartilage. Although the human sclera is not a cartilaginous tissue, the human sclera maintains chondrogenic potential throughout evolution. In addition, our findings directly explain an enigma that the sclera and the joint cartilage are common targets of inflammatory cells in rheumatic arthritis. The present global gene expression database will contribute to the clarification of the pathogenesis of developmental diseases such as high myopia.

  11. Recapitulation of physiological spatiotemporal signals promotes in vitro formation of phenotypically stable human articular cartilage

    Science.gov (United States)

    Wei, Yiyong; Zhou, Bin; Bernhard, Jonathan; Robinson, Samuel; Burapachaisri, Aonnicha; Guo, X. Edward

    2017-01-01

    Standard isotropic culture fails to recapitulate the spatiotemporal gradients present during native development. Cartilage grown from human mesenchymal stem cells (hMSCs) is poorly organized and unstable in vivo. We report that human cartilage with physiologic organization and in vivo stability can be grown in vitro from self-assembling hMSCs by implementing spatiotemporal regulation during induction. Self-assembling hMSCs formed cartilage discs in Transwell inserts following isotropic chondrogenic induction with transforming growth factor β to set up a dual-compartment culture. Following a switch in the basal compartment to a hypertrophic regimen with thyroxine, the cartilage discs underwent progressive deep-zone hypertrophy and mineralization. Concurrent chondrogenic induction in the apical compartment enabled the maintenance of functional and hyaline cartilage. Cartilage homeostasis, chondrocyte maturation, and terminal differentiation markers were all up-regulated versus isotropic control groups. We assessed the in vivo stability of the cartilage formed under different induction regimens. Cartilage formed under spatiotemporal regulation in vitro resisted endochondral ossification, retained the expression of cartilage markers, and remained organized following s.c. implantation in immunocompromised mice. In contrast, the isotropic control groups underwent endochondral ossification. Cartilage formed from hMSCs remained stable and organized in vivo. Spatiotemporal regulation during induction in vitro recapitulated some aspects of native cartilage development, and potentiated the maturation of self-assembling hMSCs into stable and organized cartilage resembling the native articular cartilage. PMID:28228529

  12. Explanted diseased livers - a possible source of metabolic competent primary human hepatocytes.

    Science.gov (United States)

    Kleine, Moritz; Riemer, Marc; Krech, Till; DeTemple, Daphne; Jäger, Mark D; Lehner, Frank; Manns, Michael P; Klempnauer, Jürgen; Borlak, Jürgen; Bektas, Hueseyin; Vondran, Florian W R

    2014-01-01

    Being an integral part of basic, translational and clinical research, the demand for primary human hepatocytes (PHH) is continuously growing while the availability of tissue resection material for the isolation of metabolically competent PHH remains limited. To overcome current shortcomings, this study evaluated the use of explanted diseased organs from liver transplantation patients as a potential source of PHH. Therefore, PHH were isolated from resected surgical specimens (Rx-group; n = 60) and explanted diseased livers obtained from graft recipients with low labMELD-score (Ex-group; n = 5). Using established protocols PHH were subsequently cultured for a period of 7 days. The viability and metabolic competence of cultured PHH was assessed by the following parameters: morphology and cell count (CyQuant assay), albumin synthesis, urea production, AST-leakage, and phase I and II metabolism. Both groups were compared in terms of cell yield and metabolic function, and results were correlated with clinical parameters of tissue donors. Notably, cellular yields and viabilities were comparable between the Rx- and Ex-group and were 5.3±0.5 and 2.9±0.7×106 cells/g liver tissue with 84.3±1.3 and 76.0±8.6% viability, respectively. Moreover, PHH isolated from the Rx- or Ex-group did not differ in regards to loss of cell number in culture, albumin synthesis, urea production, AST-leakage, and phase I and II metabolism (measured by the 7-ethoxycoumarin-O-deethylase and uracil-5'-diphosphate-glucuronyltransferase activity). Likewise, basal transcript expressions of the CYP monooxygenases 1A1, 2C8 and 3A4 were comparable as was their induction when treated with a cocktail that consisted of 3-methylcholantren, rifampicin and phenobarbital, with increased expression of CYP 1A1 and 3A4 mRNA while transcript expression of CYP 2C8 was only marginally changed. In conclusion, the use of explanted diseased livers obtained from recipients with low labMELD-score might represent

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

    DEFF Research Database (Denmark)

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

    1999-01-01

    YKL-40, also called human cartilage glycoprotein-39, is a major secretory protein of human chondrocytes in cell culture. YKL-40 mRNA is expressed by cartilage from patients with rheumatoid arthritis, but is not detectable in normal human cartilage. The aim was to investigate the distribution of YKL......-40 in osteoarthritic (n=9) and macroscopically normal (n=5) human articular cartilage, collected from 12 pre-selected areas of the femoral head, to discover a potential role for YKL-40 in cartilage remodelling in osteoarthritis. Immunohistochemical analysis showed that YKL-40 staining was found...... staining for YKL-40 was in general low in normal cartilage. The present findings, together with previous observations, suggests that YKL-40 may be of importance in cartilage remodelling/degradation of osteoarthritic joints....

  14. Comparison of human nasal epithelial cells grown as explant outgrowth cultures or dissociated tissue cultures in vitro.

    Science.gov (United States)

    Jiao, Jian; Meng, Na; Wang, Hong; Zhang, Luo

    2013-12-01

    The purpose of this study was to compare cell growth characteristics, ciliated cell differentiation, and function of human nasal epithelial cells established as explant outgrowth cultures or dissociated tissue cultures. Human nasal mucosa of the uncinate process was obtained by endoscopy and epithelial cell cultures were established by explant outgrowth or dissociated tissue culture methods. Epithelial cell growth characteristics were observed by inverted phase contrast microscopy. Ciliated cell differentiation was detected by β-tubulin IVand ZO-1 immunocytochemistry. Basal and ATP-stimulated ciliary beat frequency (CBF) was measured using a highspeed digital microscopic imaging system. Both the explant and dissociated tissue cultures established as monolayers with tight junctions and differentiated cell composition, with both types of cultures comprising ciliated and non-ciliated epithelial cells. Fibroblasts were also frequently found in explant cultures but rarely seen in dissociated tissue cultures. In both culture systems, the highest ciliated cell density appeared at 7th-10th culture day and declined with time, with the lifespan of ciliated cells ranging from 14 to 21 days. Overall, 10% of the cells in explant cultures and 20% of the cells in the dissociated tissue cultures were ciliated. These two cultures demonstrated similar ciliary beat frequency values at baseline (7.78 ± 1.99 Hz and 7.91 ± 2.52 Hz, respectively) and reacted equivalently following stimulation with 100 μM ATP. The results of this study indicate that both the explant outgrowth and dissociated tissue culture techniques are suitable for growing well-differentiated nasal ciliated and non-ciliated cells, which have growth characteristics and ciliary activity similar to those of nasal epithelial cells in vivo.

  15. Effects of high hydrostatic pressure on bacterial growth on human ossicles explanted from cholesteatoma patients.

    Science.gov (United States)

    Dommerich, Steffen; Frickmann, Hagen; Ostwald, Jürgen; Lindner, Tobias; Zautner, Andreas Erich; Arndt, Kathleen; Pau, Hans Wilhelm; Podbielski, Andreas

    2012-01-01

    High hydrostatic pressure (HHP) treatment can eliminate cholesteatoma cells from explanted human ossicles prior to re-insertion. We analyzed the effects of HHP treatment on the microbial flora on ossicles and on the planktonic and biofilm states of selected isolates. Twenty-six ossicles were explanted from cholesteatoma patients. Five ossicles were directly analyzed for microbial growth without further treatment. Fifteen ossicles were cut into two pieces. One piece was exposed to HHP of 350 MPa for 10 minutes. Both the treated and untreated (control) pieces were then assessed semi-quantitatively. Three ossicles were cut into two pieces and exposed to identical pressure conditions with or without the addition of one of two different combinations of antibiotics to the medium. Differential effects of 10-minute in vitro exposure of planktonic and biofilm bacteria to pressures of 100 MPa, 250 MPa, 400 MPa and 540 MPa in isotonic and hypotonic media were analyzed using two patient isolates of Staphylococcus epidermidis and Neisseria subflava. Bacterial cell inactivation and biofilm destruction were assessed by colony counting and electron microscopy. A variety of microorganisms were isolated from the ossicles. Irrespective of the medium, HHP treatment at 350 MPa for 10 minutes led to satisfying but incomplete inactivation especially of gram-negative bacteria. The addition of antibiotics increased the efficacy of elimination. A comparison of HHP treatment of planktonic and biofilm cells showed that the effects of HPP were reduced by about one decadic logarithmic unit when HPP was applied to biofilms. High hydrostatic pressure conditions that are suitable to inactivate cholesteatoma cells fail to completely sterilize ossicles even if antibiotics are added. As a result of the reduced microbial load and the viability loss of surviving bacteria, however, there is a lower risk of re-infection after re-insertion.

  16. Working conditions of bipolar radiofrequency on human articular cartilage repair following thermal injury during arthroscopy

    Institute of Scientific and Technical Information of China (English)

    Huang Yuelong; Zhang Yujun; Ding Xiaoquan; Liu Songyang; Sun Tiezheng

    2014-01-01

    Background The thermal injury during bipolar radiofrequercy results in chondrocyte death that limits cartilage repair.The purpose was to determine the effects of various factors of bipolar radiofrequency on human articular cartilage after thermal injury,offering suitable working conditions for bipolar radiofrequency during arthroscopy.Methods Osteochondral explants from 28 patients undergoing total knee arthroplasty (TKA) in Department of Orthopaedic,Peking University Reople's Hospital from October 2013 to May 2014,were harvested and treated using bipolar radiofrequency in a light contact mode under the following conditions:various power setting of levels 2,4 and 6; different durations of 2 seconds,5 seconds and 10 seconds; irrigation with fluids of different temperatures of 4℃,22℃,and 37℃; two different bipolar radiofrequency probes ArthroCare TriStar 50 and Paragon T2.The percentage of cell death and depth of cell death were quantified with laser confocal microscopy.The content of proteoglycan elution at different temperatures was determined by spectrophotometer at 530 nm.Results Chondrocyte mortality during the treatment time of 2 seconds and power setting of level 2 was significantly lower than that with long duration or in higher level groups (time:P=0.001; power:P=0.001).The percentage of cell death after thermal injury was gradually reduced by increasing the temperature of the irrigation solutions (P=0.003),the depth of dead chondrocytes in the 37℃ solution group was significantly less than those in the 4℃ and 22℃ groups (P=0.001).The proteoglycan elution was also gradually reduced by increasing the temperature (P=0.004).Compared with the ArthroCare TriStar 50 group,the percentage of cell death in the Paragon T2 group was significantly decreased (P=0.046).Conclusions Thermal chondroplasty with bipolar radiofrequency resulted in defined margins of chondrocyte death under controlled conditions.The least cartilage damage during thermal chondroplasty

  17. Formation of Hyaline Cartilage Tissue by Passaged Human Osteoarthritic Chondrocytes.

    Science.gov (United States)

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

    2017-02-01

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

  18. Isolation, identification and differentiation of human embryonic cartilage stem cells.

    Science.gov (United States)

    Fu, Changhao; Yan, Zi; Xu, Hao; Zhang, Chen; Zhang, Qi; Wei, Anhui; Yang, Xi; Wang, Yi

    2015-07-01

    We isolated human embryonic cartilage stem cells (hECSCs), a novel stem cell population, from the articular cartilage of eight-week-old human embryos. These stem cells demonstrated a marker expression pattern and differentiation potential intermediate to those of human embryonic stem cells (hESCs) and human adult stem cells (hASCs). hECSCs expressed markers associated with both hESCs (OCT4, NANOG, SOX2, SSEA-3 and SSEA-4) and human adult stem cells (hASCs) (CD29, CD44, CD90, CD73 and CD10). These cells also differentiated into adipocytes, osteoblasts, chondrocytes, neurons and islet-like cells under specific inducing conditions. We identified N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) as an inducer of chondrogenic differentiation in hECSCs. Similar results using N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) were obtained for two other types of human embryonic tissue-derived stem cells, human embryonic hepatic stem cells (hEHSCs) and human embryonic amniotic fluid stem cells (hEASCs), both of which exhibited a marker expression pattern similar to that of hECSCs. The isolation of hECSCs and the discovery that N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) induces chondrogenic differentiation in different stem cell populations might aid the development of strategies in tissue engineering and cartilage repair.

  19. Fibrous cartilage of human menisci is less shock-absorbing and energy-dissipating than hyaline cartilage.

    Science.gov (United States)

    Gaugler, Mario; Wirz, Dieter; Ronken, Sarah; Hafner, Mirjam; Göpfert, Beat; Friederich, Niklaus F; Elke, Reinhard

    2015-04-01

    To test meniscal mechanical properties such as the dynamic modulus of elasticity E* and the loss angle δ at two loading frequencies ω at different locations of the menisci and compare it to E* and δ of hyaline cartilage in indentation mode with spherical indenters. On nine pairs of human menisci, the dynamic E*-modulus and loss angle δ (as a measure of the energy dissipation) were determined. The measurements were performed at two different strain rates (slow sinusoidal and fast single impact) to show the strain rate dependence of the material. The measurements were compared to previous similar measurements with the same equipment on human hyaline cartilage. The resultant E* at fast indentation (median 1.16 MPa) was significantly higher, and the loss angle was significantly lower (median 10.2°) compared to slow-loading mode's E* and δ (median 0.18 MPa and 16.9°, respectively). Further, significant differences for different locations are shown. On the medial meniscus, the anterior horn shows the highest resultant dynamic modulus. In dynamic measurements with a spherical indenter, the menisci are much softer and less energy-dissipating than hyaline cartilage. Further, the menisci are stiffer and less energy-dissipating in the middle, intermediate part compared to the meniscal base. In compression, the energy dissipation of meniscus cartilage plays a minor role compared to hyaline cartilage. At high impacts, energy dissipation is less than on low impacts, similar to cartilage.

  20. Effects of mechanical loading on human mesenchymal stem cells for cartilage tissue engineering.

    Science.gov (United States)

    Choi, Jane Ru; Yong, Kar Wey; Choi, Jean Yu

    2017-05-19

    Today, articular cartilage damage is a major health problem, affecting people of all ages. The existing conventional articular cartilage repair techniques, such as autologous chondrocyte implantation (ACI), microfracture, and mosaicplasty, have many shortcomings which negatively affect their clinical outcomes. Therefore, it is essential to develop an alternative and efficient articular repair technique that can address those shortcomings. Cartilage tissue engineering, which aims to create a tissue-engineered cartilage derived from human mesenchymal stem cells (MSCs), shows great promise for improving articular cartilage defect therapy. However, the use of tissue-engineered cartilage for the clinical therapy of articular cartilage defect still remains challenging. Despite the importance of mechanical loading to create a functional cartilage has been well demonstrated, the specific type of mechanical loading and its optimal loading regime is still under investigation. This review summarizes the most recent advances in the effects of mechanical loading on human MSCs. First, the existing conventional articular repair techniques and their shortcomings are highlighted. The important parameters for the evaluation of the tissue-engineered cartilage, including chondrogenic and hypertrophic differentiation of human MSCs are briefly discussed. The influence of mechanical loading on human MSCs is subsequently reviewed and the possible mechanotransduction signaling is highlighted. The development of non-hypertrophic chondrogenesis in response to the changing mechanical microenvironment will aid in the establishment of a tissue-engineered cartilage for efficient articular cartilage repair. © 2017 Wiley Periodicals, Inc.

  1. Engineering Cartilage

    Science.gov (United States)

    ... Research Matters NIH Research Matters March 3, 2014 Engineering Cartilage Artistic rendering of human stem cells on ... situations has been a major goal in tissue engineering. Cartilage contains water, collagen, proteoglycans, and chondrocytes. Collagens ...

  2. 308-nm excimer laser ablation of human cartilage

    Science.gov (United States)

    Prodoehl, John A.; Rhodes, Anthony L.; Meller, Menachem M.; Sherk, Henry H.

    1993-07-01

    The XeCl excimer laser was investigated as an ablating tool for human fibrocartilage and hyaline cartilage. Quantitative measurements were made of tissue ablation rates as a function of fluence in meniscal fibrocartilage and articular hyaline cartilage. A force of 1.47 Newtons was applied to an 800 micrometers fiber with the laser delivering a range of fluences (40 to 190 mj/mm2) firing at a frequency of 5 Hz. To assess the effect of repetition rate on ablation rate, a set of measurements was made at a constant fluence of 60 mj/mm2, with the repetition rate varying from 10 to 40 Hz. Histologic and morphometric analysis was performed using light microscopy. The results of these studies revealed that the ablation rate was directly proportional to fluence over the range tested. Fibrocartilage was ablated at a rate 2.56 times faster than hyaline cartilage at the maximum fluence tested. Repetition rate had no effect on the penetration per pulse. Adjacent tissue damage was noted to be minimal (10 - 70 micrometers ).

  3. Deferoxamine Suppresses Collagen Cleavage and Protease, Cytokine, and COL10A1 Expression and Upregulates AMPK and Krebs Cycle Genes in Human Osteoarthritic Cartilage

    Directory of Open Access Journals (Sweden)

    Elena V. Tchetina

    2016-01-01

    Full Text Available This study reports the effects of the iron chelator deferoxamine (DFO on collagen cleavage, inflammation, and chondrocyte hypertrophy in relation to energy metabolism-related gene expression in osteoarthritic (OA articular cartilage. Full-depth explants of human OA knee articular cartilage from arthroplasty were cultured with exogenous DFO (1–50 μM. Type II collagen cleavage and phospho-adenosine monophosphate-activated protein kinase (pAMPK concentrations were measured using ELISAs. Gene expression studies employed real-time PCR and included AMPK analyses in PBMCs. In OA explants collagen cleavage was frequently downregulated by 10–50 μM DFO. PCR analysis of 7 OA patient cartilages revealed that 10 μM DFO suppressed expression of MMP-1, MMP-13, IL-1β, and TNFα and a marker of chondrocyte hypertrophy, COL10A1. No changes were observed in the expression of glycolysis-related genes. In contrast, expressions of genes associated with the mitochondrial Krebs cycle (TCA, AMPK, HIF1α, and COL2A1 were upregulated. AMPK gene expression was reduced in OA cartilage and increased in PBMCs from the same patients compared to healthy controls. Our studies demonstrate that DFO is capable of suppressing excessive collagenase-mediated type II collagen cleavage in OA cartilage and reversing phenotypic changes. The concomitant upregulation of proanabolic TCA-related gene expressions points to a potential for availability of energy generating substrates required for matrix repair by end-stage OA chondrocytes. This might normally be prevented by high whole-body energy requirements indicated by elevated AMPK expression in PBMCs of OA patients.

  4. Deferoxamine Suppresses Collagen Cleavage and Protease, Cytokine, and COL10A1 Expression and Upregulates AMPK and Krebs Cycle Genes in Human Osteoarthritic Cartilage.

    Science.gov (United States)

    Tchetina, Elena V; Markova, Galina A; Poole, A Robin; Zukor, David J; Antoniou, John; Makarov, Sergey A; Kuzin, Aleksandr N

    2016-01-01

    This study reports the effects of the iron chelator deferoxamine (DFO) on collagen cleavage, inflammation, and chondrocyte hypertrophy in relation to energy metabolism-related gene expression in osteoarthritic (OA) articular cartilage. Full-depth explants of human OA knee articular cartilage from arthroplasty were cultured with exogenous DFO (1-50 μM). Type II collagen cleavage and phospho-adenosine monophosphate-activated protein kinase (pAMPK) concentrations were measured using ELISAs. Gene expression studies employed real-time PCR and included AMPK analyses in PBMCs. In OA explants collagen cleavage was frequently downregulated by 10-50 μM DFO. PCR analysis of 7 OA patient cartilages revealed that 10 μM DFO suppressed expression of MMP-1, MMP-13, IL-1β, and TNFα and a marker of chondrocyte hypertrophy, COL10A1. No changes were observed in the expression of glycolysis-related genes. In contrast, expressions of genes associated with the mitochondrial Krebs cycle (TCA), AMPK, HIF1α, and COL2A1 were upregulated. AMPK gene expression was reduced in OA cartilage and increased in PBMCs from the same patients compared to healthy controls. Our studies demonstrate that DFO is capable of suppressing excessive collagenase-mediated type II collagen cleavage in OA cartilage and reversing phenotypic changes. The concomitant upregulation of proanabolic TCA-related gene expressions points to a potential for availability of energy generating substrates required for matrix repair by end-stage OA chondrocytes. This might normally be prevented by high whole-body energy requirements indicated by elevated AMPK expression in PBMCs of OA patients.

  5. Activin A induces Langerhans cell differentiation in vitro and in human skin explants.

    Directory of Open Access Journals (Sweden)

    Tiziana Musso

    Full Text Available Langerhans cells (LC represent a well characterized subset of dendritic cells located in the epidermis of skin and mucosae. In vivo, they originate from resident and blood-borne precursors in the presence of keratinocyte-derived TGFbeta. In vitro, LC can be generated from monocytes in the presence of GM-CSF, IL-4 and TGFbeta. However, the signals that induce LC during an inflammatory reaction are not fully investigated. Here we report that Activin A, a TGFbeta family member induced by pro-inflammatory cytokines and involved in skin morphogenesis and wound healing, induces the differentiation of human monocytes into LC in the absence of TGFbeta. Activin A-induced LC are Langerin+, Birbeck granules+, E-cadherin+, CLA+ and CCR6+ and possess typical APC functions. In human skin explants, intradermal injection of Activin A increased the number of CD1a+ and Langerin+ cells in both the epidermis and dermis by promoting the differentiation of resident precursor cells. High levels of Activin A were present in the upper epidermal layers and in the dermis of Lichen Planus biopsies in association with a marked infiltration of CD1a+ and Langerin+ cells. This study reports that Activin A induces the differentiation of circulating CD14+ cells into LC. Since Activin A is abundantly produced during inflammatory conditions which are also characterized by increased numbers of LC, we propose that this cytokine represents a new pathway, alternative to TGFbeta, responsible for LC differentiation during inflammatory/autoimmune conditions.

  6. Enhanced functional integration of human photoreceptor precursors into human and rodent retina in an ex vivo retinal explant model system.

    Science.gov (United States)

    Yanai, Anat; Laver, Christopher R J; Gregory-Evans, Cheryl Y; Liu, Ran R; Gregory-Evans, Kevin

    2015-06-01

    Retinal disease is the major cause of irreversible blindness in developed countries. Transplantation of photoreceptor precursor cells (PPCs) derived from human embryonic stem cells (hESCs) is a promising and widely applicable approach for the treatment of these blinding conditions. Previously, it has been shown that after transplantation into the degenerating retina, the percentage of PPCs that undergo functional integration is low. The factors that inhibit PPC engraftment remain largely unknown, in part, because so many adverse factors could be at play during in vivo experiments. To advance our knowledge in overcoming potential adverse effects and optimize PPC transplantation, we have developed a novel ex vivo system. Harvested neural retina was placed directly on top of cultured retinal pigment epithelial (RPE) cells from a number of different sources. To mimic PPC transplantation into the subretinal space, hESC-derived PPCs were inserted between the retinal explant and underlying RPE. Explants cocultured with hESC-derived RPE maintained normal gross morphology and viability for up to 2 weeks, whereas the explants cultured on ARPE19 and RPE-J failed by 7 days. Furthermore, the proportion of PPCs expressing ribbon synapse-specific proteins BASSOON and RIBEYE was significantly higher when cocultured with hESC-derived RPE (20% and 10%, respectively), than when cocultured with ARPE19 (only 6% and 2%, respectively). In the presence of the synaptogenic factor thrombospondin-1 (TSP-1), the proportion of BASSOON-positive and RIBEYE-positive PPCs cocultured with hESC-derived RPE increased to ∼30% and 15%, respectively. These data demonstrate the utility of an ex vivo model system to define factors, such as TSP-1, which could influence integration efficiency in future in vivo experiments in models of retinal degeneration.

  7. Investigations of micron and submicron wear features of diseased human cartilage surfaces.

    Science.gov (United States)

    Peng, Zhongxiao; Baena, Juan C; Wang, Meiling

    2015-02-01

    Osteoarthritis is a common disease. However, its causes and morphological features of diseased cartilage surfaces are not well understood. The purposes of this research were (a) to develop quantitative surface characterization techniques to study human cartilages at a micron and submicron scale and (b) to investigate distinctive changes in the surface morphologies and biomechanical properties of the cartilages in different osteoarthritis grades. Diseased cartilage samples collected from osteoarthritis patients were prepared for image acquisition using two different techniques, that is, laser scanning microscopy at a micrometer scale and atomic force microscopy at a nanometer scale. Three-dimensional, digital images of human cartilages were processed and analyzed quantitatively. This study has demonstrated that high-quality three-dimensional images of human cartilage surfaces could be obtained in a hydrated condition using laser scanning microscopy and atomic force microscopy. Based on the numerical data extracted from improved image quality and quantity, it has been found that osteoarthritis evolution can be identified by specific surface features at the micrometer scale, and these features are amplitude and functional property related. At the submicron level, the spatial features of the surfaces were revealed to differ between early and advanced osteoarthritis grades. The effective indentation moduli of human cartilages effectively revealed the cartilage deterioration. The imaging acquisition and numerical analysis methods established allow quantitative studies of distinctive changes in cartilage surface characteristics and better understanding of the cartilage degradation process.

  8. Visible effects of rapamycin (sirolimus) on human skin explants in vitro.

    Science.gov (United States)

    Peramo, Antonio; Marcelo, Cynthia L

    2013-03-01

    In this manuscript, we report observations of the effects of rapamycin in an organotypic culture of human skin explants. The tissues were cultured for 5 days at the air-liquid interface or in submersed conditions with media with and without rapamycin at 2 nM concentration. Histological analysis of tissue sections indicated that rapamycin-treated samples maintained a better epidermal structure in the upper layers of the tissue than untreated samples, mostly evident when skin was cultured in submersed conditions. A significant decrease in the number of positive proliferative cells using the Ki67 antigen was observed when specimens were treated with rapamycin, in both air-liquid and submersed conditions but apoptosis differences between treated and untreated specimens, as seen by cleaved caspase-3 positive cells, were only observed in submersed specimens. Finally, a decrease and variability in the location in the expression of the differentiation marker involucrin and in E-cadherin were also evident in submersed samples. These results suggest that the development of topical applications containing rapamycin, instead of systemic delivery, may be a useful tool in the treatment of skin diseases that require reduction of proliferation and modulation or control of keratinocyte differentiation.

  9. Study on the Microstructure of Human Articular Cartilage/Bone Interface

    Institute of Scientific and Technical Information of China (English)

    Yaxiong Liu; Qin Lian; Jiankang He; Jinna Zhao; Zhongmin Jin; Dichen Li

    2011-01-01

    For improving the theory of gradient microstructure of cartilage/bone interface, human distal femurs were studied. Scanning Electron Microscope (SEM), histological sections and MicroCT were used to observe, measure and model the microstructure of cartilage/bone interface. The results showed that the cartilage/bone interface is in a hierarchical structure which is composed of four different tissue layers. The interlocking of hyaline cartilage and calcified cartilage and that of calcified cartilage and subchondral bone are in the manner of"protrusion-pore" with average diameter of 17.0 μm and 34.1 μm respectively. In addition, the cancellous bone under the cartilage is also formed by four layer hierarchical structure, and the adjacent layers are connected by bone trabecula in the shape of H, I and Y, forming a complex interwoven network structure. Finally, the simplified structure model of the cartilage/bone interface was proposed according to the natural articular cartilage/bone interface. The simplified model is a 4-layer gradient biomimetic structure, which corresponds to four different tissues of natural cartilage/bone interface. The results of this work would be beneficial to the design of bionic scaffold for the tissue engineering of articular cartilage/bone.

  10. The immunomodulatory effects of shark cartilage on the mouse and human immune system

    Directory of Open Access Journals (Sweden)

    ali Sheikhian

    2007-01-01

    Materials and methods: In an experimental study, the effects of different doses of shark cartilage on humoral (antibody titer immune response against sheep red blood cells (SRBC, were measured in mouse. In addition, we evaluated the modulatory effects of the shark cartilage on the natural killer (NK activity of the peritoneal cells of mouse against a tumor cell line called K562, according to the standard methods. The proliferative response of the human peripheral blood mononuclear cells was measured under the influence of shark cartilage. Results: Pure shark cartilage enhanced antibody response against SRBC in vivo. The hemagglutination titer which was 1/147 in the control group (injected with hen cartilage, increased to 1/1355 in the test group. The optimal dose was 100 mg/ml. both type of cartilage had blastogenic effect on peripheral blood mononuclear cells (the blastogenic index was 6.7 and 4.9 for impure shark cartilage and hen cartilage, respectively. NK activity was inhibited completely by pure shark cartilage (the amount of the killing activity of the effector peritoneal cells for the control and test groups against target cells was 25.9% and 5.5% respectively. Conclusion: Shark cartilage has a potent immunomodulatory effect on the specific immune mechanisms and some inhibitory effects on the innate immune mechanisms such as NC activity. Since the specific immunity has a more pivotal role against tumor formation, shark cartilage can be used as a cancer immunotherapeutic.

  11. Large, stratified, and mechanically functional human cartilage grown in vitro by mesenchymal condensation

    Science.gov (United States)

    Bhumiratana, Sarindr; Eton, Ryan E.; Oungoulian, Sevan R.; Wan, Leo Q.; Ateshian, Gerard A.; Vunjak-Novakovic, Gordana

    2014-01-01

    The efforts to grow mechanically functional cartilage from human mesenchymal stem cells have not been successful. We report that clinically sized pieces of human cartilage with physiologic stratification and biomechanics can be grown in vitro by recapitulating some aspects of the developmental process of mesenchymal condensation. By exposure to transforming growth factor-β, mesenchymal stem cells were induced to condense into cellular bodies, undergo chondrogenic differentiation, and form cartilagenous tissue, in a process designed to mimic mesenchymal condensation leading into chondrogenesis. We discovered that the condensed mesenchymal cell bodies (CMBs) formed in vitro set an outer boundary after 5 d of culture, as indicated by the expression of mesenchymal condensation genes and deposition of tenascin. Before setting of boundaries, the CMBs could be fused into homogenous cellular aggregates giving rise to well-differentiated and mechanically functional cartilage. We used the mesenchymal condensation and fusion of CMBs to grow centimeter-sized, anatomically shaped pieces of human articular cartilage over 5 wk of culture. For the first time to our knowledge biomechanical properties of cartilage derived from human mesenchymal cells were comparable to native cartilage, with the Young’s modulus of >800 kPa and equilibrium friction coeffcient of CMBs have capability to form mechanically strong cartilage–cartilage interface in an in vitro cartilage defect model. The CMBs, which acted as “lego-like” blocks of neocartilage, were capable of assembling into human cartilage with physiologic-like structure and mechanical properties. PMID:24778247

  12. Human serum provided additional values in growth factors supplemented medium for human chondrocytes monolayer expansion and engineered cartilage construction.

    Science.gov (United States)

    Chua, K H; Aminuddin, B S; Fuzina, N H; Ruszymah, B H I

    2004-05-01

    We have previously formulated an optimized human chondrocytes growth medium based on 2% fetal bovine serum supplementation. For clinical usage, the animal serum must be replaced by patient own serum. We investigated the effects of human serum concentration for human nasal septum chondrocytes monolayer culture and cartilage reconstruction. Human serum demonstrated a dose dependent manner in promoting chondrocytes growth and cartilage engineering.

  13. Replication of avian, human and swine influenza viruses in porcine respiratory explants and association with sialic acid distribution

    Directory of Open Access Journals (Sweden)

    Nauwynck Hans J

    2010-02-01

    Full Text Available Abstract Background Throughout the history of human influenza pandemics, pigs have been considered the most likely "mixing vessel" for reassortment between human and avian influenza viruses (AIVs. However, the replication efficiencies of influenza viruses from various hosts, as well as the expression of sialic acid (Sia receptor variants in the entire porcine respiratory tract have never been studied in detail. Therefore, we established porcine nasal, tracheal, bronchial and lung explants, which cover the entire porcine respiratory tract with maximal similarity to the in vivo situation. Subsequently, we assessed virus yields of three porcine, two human and six AIVs in these explants. Since our results on virus replication were in disagreement with the previously reported presence of putative avian virus receptors in the trachea, we additionally studied the distribution of sialic acid receptors by means of lectin histochemistry. Human (Siaα2-6Gal and avian virus receptors (Siaα2-3Gal were identified with Sambucus Nigra and Maackia amurensis lectins respectively. Results Compared to swine and human influenza viruses, replication of the AIVs was limited in all cultures but most strikingly in nasal and tracheal explants. Results of virus titrations were confirmed by quantification of infected cells using immunohistochemistry. By lectin histochemistry we found moderate to abundant expression of the human-like virus receptors in all explant systems but minimal binding of the lectins that identify avian-like receptors, especially in the nasal, tracheal and bronchial epithelium. Conclusions The species barrier that restricts the transmission of influenza viruses from one host to another remains preserved in our porcine respiratory explants. Therefore this system offers a valuable alternative to study virus and/or host properties required for adaptation or reassortment of influenza viruses. Our results indicate that, based on the expression of Sia

  14. Age variations in the properties of human tibial trabecular bone and cartilage

    DEFF Research Database (Denmark)

    Ding, Ming

    2000-01-01

    ) to investigate the age-related and osteoarthrosis-related changes in the mechanical properties of the human tibial cartilage-bone complex; and 3) to evaluate mutual associations among various properties. Normal specimens from human autopsy proximal tibiae were used for investigation of age variations...... in the properties of trabecular bone and the cartilage-bone complex, and osteoarthrotic specimens were used for the investigation of changes in the mechanical properties of the cartilage-bone complex induced by this disease process. The mechanical properties and physical/compositional properties of trabecular bone...... is parallel to the longitudinal loading axis of the tibia. The mechanical properties of the normal cartilage and bone vary with age and respond simultaneously to mechanical loading. Both cartilage and bone in early-stage OA are mechanically inferior to normal, and OA cartilage and bone have lost their unit...

  15. Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium

    DEFF Research Database (Denmark)

    Marthin, June Kehlet; Stevens, Elizabeth Munkebjerg; Larsen, Lars Allan

    2017-01-01

    BACKGROUND: Three-dimensional explant spheroid formation is an ex vivo technique previously used in studies of airway epithelial ion and water transport. Explanted cells and sheets of nasal epithelium form fully differentiated spheroids enclosing a partly fluid-filled lumen with the ciliated apical....... Nasal ciliary cells and sheets were removed on day 1 by nasal brush biopsy and analyzed with regard to ciliary beat pattern-and frequency using high-speed video imaging for standard reference values. Three-dimensional explant spheroid formation was initiated in the same individual on the same day...... was successful in 15 out of 18 (82%) sampled individuals. Thus, formation was successful in seven healthy controls and eight PCD patients, while unsuccessful in 3 with PCD due to infection. Median (range) number of days in culture before harvesting of spheroids was 4 (1-5) in healthy versus 2 (1-5) in PCD...

  16. Streamlined bioreactor-based production of human cartilage tissues.

    Science.gov (United States)

    Tonnarelli, B; Santoro, R; Adelaide Asnaghi, M; Wendt, D

    2016-05-27

    Engineered tissue grafts have been manufactured using methods based predominantly on traditional labour-intensive manual benchtop techniques. These methods impart significant regulatory and economic challenges, hindering the successful translation of engineered tissue products to the clinic. Alternatively, bioreactor-based production systems have the potential to overcome such limitations. In this work, we present an innovative manufacturing approach to engineer cartilage tissue within a single bioreactor system, starting from freshly isolated human primary chondrocytes, through the generation of cartilaginous tissue grafts. The limited number of primary chondrocytes that can be isolated from a small clinically-sized cartilage biopsy could be seeded and extensively expanded directly within a 3D scaffold in our perfusion bioreactor (5.4 ± 0.9 doublings in 2 weeks), bypassing conventional 2D expansion in flasks. Chondrocytes expanded in 3D scaffolds better maintained a chondrogenic phenotype than chondrocytes expanded on plastic flasks (collagen type II mRNA, 18-fold; Sox-9, 11-fold). After this "3D expansion" phase, bioreactor culture conditions were changed to subsequently support chondrogenic differentiation for two weeks. Engineered tissues based on 3D-expanded chondrocytes were more cartilaginous than tissues generated from chondrocytes previously expanded in flasks. We then demonstrated that this streamlined bioreactor-based process could be adapted to effectively generate up-scaled cartilage grafts in a size with clinical relevance (50 mm diameter). Streamlined and robust tissue engineering processes, as the one described here, may be key for the future manufacturing of grafts for clinical applications, as they facilitate the establishment of compact and closed bioreactor-based production systems, with minimal automation requirements, lower operating costs, and increased compliance to regulatory guidelines.

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

    Directory of Open Access Journals (Sweden)

    Rebecca Williams

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

  18. A novel method for the culture and polarized stimulation of human intestinal mucosa explants.

    Science.gov (United States)

    Tsilingiri, Katerina; Sonzogni, Angelica; Caprioli, Flavio; Rescigno, Maria

    2013-05-01

    Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food. Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out. To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina

  19. Mechanical properties of the normal human cartilage-bone complex in relation to age

    DEFF Research Database (Denmark)

    Ding, Ming; Dalstra, M; Linde, F

    1998-01-01

    OBJECTIVE: This study investigates the age-related variations in the mechanical properties of the normal human tibial cartilage-bone complex and the relationships between cartilage and bone. DESIGN: A novel technique was applied to assess the mechanical properties of the cartilage and bone by means...... normal donors aged 16-83 years were tested in compression. The deformation was measured simultaneously in bone and cartilage to obtain the mechanical properties of both tissues. RESULTS: The stiffnesses and elastic energies of both cartilage and bone showed an initial increase, with maxima at 40 years......, followed by a steady decline. The viscoelastic energy was maximal at younger ages (16-29 years), followed by a steady decline. The energy absorption capacity did not vary with age. Stiffnesses and elastic energies were correlated significantly between cartilage and bone. CONCLUSIONS: The present study...

  20. Construction of tissue-engineered cartilage using human placenta-derived stem cells

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Human placenta-derived stem cells (hPDSCs) were isolated by trypsinization and further induced into cartilage cells in vitro.The engineered cartilage was constructed by combining hPDSCs with collagen sponge and the cartilage formation was observed by implantation into nude mice.Results showed that hPDSCs featured mesenchymal stem cells and maintained proliferation in vitro for over 30 passages while remaining undifferentiated.All results indicated that hPDSCs have the potential to differentiate into functional cartilage cells in vitro when combined with collagen sponge,which provided experimental evidence for prospective clinical application.

  1. Human rheumatoid arthritis tissue production of IL-17A drives matrix and cartilage degradation: synergy with tumour necrosis factor-alpha, Oncostatin M and response to biologic therapies.

    LENUS (Irish Health Repository)

    Moran, Ellen M

    2009-01-01

    INTRODUCTION: The aim of this study was to examine IL-17A in patients, following anti-TNF-alpha therapy and the effect of IL-17A on matrix turnover and cartilage degradation. METHODS: IL-17A expression was examined by ELISA and immunohistology in the rheumatoid arthritis (RA) joints. RA whole synovial tissue explant (RA ST), primary synovial fibroblasts (RASFC), human cartilage and chondrocyte cultures were stimulated with IL-17A +\\/- TNF-alpha and Oncostatin M (OSM). Matrix metalloproteinase (MMP) and tissue inhibitor (TIMP-1) were assessed by ELISA and zymography. Cartilage proteoglycan release was assessed histologically by Safranin-O staining. Clinical parameters, IL-17A, MMP\\/TIMP were assessed in patients pre\\/post biologic therapy. RESULTS: IL-17A levels were higher in RA vs osteoarthritis (OA)\\/normal joints (P < 0.05). IL-17A up-regulated MMP-1, -2, -9, and -13 in RA ST, RASFC, cartilage and chondrocyte cultures (P < 0.05). In combination with TNF-alpha and OSM, IL-17A shifted the MMP:TIMP-1 ratio in favor of matrix degradation (all P < 0.05). Cartilage proteoglycan depletion in response to IL-17A was mild; however, in combination with TNF-alpha or OSM showed almost complete proteoglycan depletion. Serum IL-17A was detected in 28% of patients commencing biologic therapy. IL-17A negative patients demonstrated reductions post therapy in serum MMP1\\/TIMP4, MMP3\\/TIMP1 and MMP3\\/TIMP4 ratios and an increase in CS846 (all P < 0.05). No significant changes were observed in IL-17A positive patients. CONCLUSIONS: IL-17A is produced locally in the inflamed RA joint. IL-17A promotes matrix turnover and cartilage destruction, especially in the presence of other cytokines, mimicking the joint environment. IL-17A levels are modulated in vivo, following anti-TNF therapy, and may reflect changes in matrix turnover.

  2. Development and characterization of decellularized human nasoseptal cartilage matrix for use in tissue engineering.

    Science.gov (United States)

    Graham, M Elise; Gratzer, Paul F; Bezuhly, Michael; Hong, Paul

    2016-10-01

    Reconstruction of cartilage defects in the head and neck can require harvesting of autologous cartilage grafts, which can be associated with donor site morbidity. To overcome this limitation, tissue-engineering approaches may be used to generate cartilage grafts. The objective of this study was to decellularize and characterize human nasoseptal cartilage with the aim of generating a biological scaffold for cartilage tissue engineering. Laboratory study using nasoseptal cartilage. Remnant human nasoseptal cartilage specimens were collected and subjected to a novel decellularization treatment. The decellularization process involved several cycles of enzymatic detergent treatments. For characterization, decellularized and fresh (control) specimens underwent histological, biochemical, and mechanical analyses. Scanning electron microscopy and biocompatibility assay were also performed. The decellularization process had minimal effect on glycosaminoglycan content of the cartilage extracellular matrix. Deoxyribonucleic acid (DNA) analysis revealed the near-complete removal of genomic DNA from decellularized tissues. The effectiveness of the decellularization process was also confirmed on histological and scanning electron microscopic analyses. Mechanical testing results showed that the structural integrity of the decellularized tissue was maintained, and biocompatibility was confirmed. Overall, the current decellularization treatment resulted in significant reduction of genetic/cellular material with preservation of the underlying extracellular matrix structure. This decellularized material may serve as a potential scaffold for cartilage tissue engineering. N/A. Laryngoscope, 126:2226-2231, 2016. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

  3. Evaluation of taper joints with combined fatigue and crevice corrosion testing: Comparison to human explanted modular prostheses

    Energy Technology Data Exchange (ETDEWEB)

    Reclaru, L., E-mail: lucien.reclaru@pxgroup.com [PX Group S.A., Dep R and D Corrosion and Biocompatibility Group, Bd. des Eplatures 42, CH-2304 La Chaux-de-Fonds (Switzerland); Brooks, R.A. [Orthopaedic Research, Addenbrooke' s Hospital, University of Cambridge, Box 180 Hills Road, CB2 0QQ Cambridge (United Kingdom); Zuberbühler, M. [Smith and Nephew Orthopaedics AG, Schachenalle 29, 5001 Aarau (Switzerland); Eschler, P.-Y.; Constantin, F. [PX Group S.A., Dep R and D Corrosion and Biocompatibility Group, Bd. des Eplatures 42, CH-2304 La Chaux-de-Fonds (Switzerland); Tomoaia, G. [University of Medicine and Pharmacy Iuliu Hateganu of Cluj-Napoca, Dept. of Orthopaedics and Traumatology, Cluj-Napoca (Romania)

    2014-01-01

    The requirement for revision surgery of total joint replacements is increasing and modular joint replacement implants have been developed to provide adjustable prosthetic revision systems with improved intra-operative flexibility. An electrochemical study of the corrosion resistance of the interface between the distal and proximal modules of a modular prosthesis was performed in combination with a cyclic fatigue test. The complexity resides in the existence of interfaces between the distal part, the proximal part, and the dynamometric screw. A new technique for evaluating the resistance to cyclic dynamic corrosion with crevice stimulation was used and the method is presented. In addition, two components of the proximal module of explanted Ti6Al4V and Ti6Al7Nb prostheses were investigated by optical and electron microscopy. Our results reveal that: The electrolyte penetrates into the interface between the distal and proximal modules during cyclic dynamic fatigue tests, the distal module undergoes cracking and corrosion was generated at the interface between the two models; The comparison of the explanted proximal parts with the similar prostheses evaluated following cyclic dynamic crevice corrosion testing showed that there were significant similarities indicating that this method is suitable for evaluating materials used in the fabrication of modular prostheses. - Highlights: • Electrochemical crevice corrosion testing combined with fatigue test conducted on Ti6Al7Nb and Ti6Al4V modular prostheses • Cations released from integral prostheses • Comparison of human explanted modular prostheses with the similar prostheses evaluated in cyclic dynamic crevice corrosion.

  4. An investigation of donor and culture parameters which influence epithelial outgrowths from cultured human cadaveric limbal explants.

    Science.gov (United States)

    Baylis, Oliver; Rooney, Paul; Figueiredo, Francisco; Lako, Majlinda; Ahmad, Sajjad

    2013-05-01

    Limbal stem cell deficiency is a blinding disease which affects the cornea at the front of the eye. The definitive cure involves replacing the corneal epithelial (limbal) stem cells, for example by transplanting cultured limbal epithelial cells. One method of performing cultures is to grow a sheet of epithelial cells from a limbal explant on human amniotic membrane. The growth of limbal tissue can be variable. The aim of this study is to investigate how different donor and culture factors influence the ex vivo growth of cadaveric limbal explants. Limbal explant cultures were established from 10 different cadaveric organ cultured corneo-scleral discs. The growth rate and the time taken for growth to be established were determined. Statistical analysis was performed to assess correlation between these factors and donor variables including donor age, sex, time from donor death to enucleation, time from enucleation to organ culture storage and duration in organ culture. Growth curves consistently showed a lag phase followed by a steeper linear growth phase. Donor age, time between death and enucleation, and time between enucleation and organ culture were not correlated to the lag time or the growth rate. Time in organ culture had a significant correlation with the duration of lag time (P = 0.003), but no relationship with the linear growth rate. This study shows that an important factor correlating with growth variation is the duration of corneo-scleral tissue in organ culture. Interestingly, donor age was not correlated with limbal explant growth. Copyright © 2012 Wiley Periodicals, Inc.

  5. Mechanical and biochemical mapping of human auricular cartilage for reliable assessment of tissue-engineered constructs.

    Science.gov (United States)

    Nimeskern, Luc; Pleumeekers, Mieke M; Pawson, Duncan J; Koevoet, Wendy L M; Lehtoviita, Iina; Soyka, Michael B; Röösli, Christof; Holzmann, David; van Osch, Gerjo J V M; Müller, Ralph; Stok, Kathryn S

    2015-07-16

    It is key for successful auricular (AUR) cartilage tissue-engineering (TE) to ensure that the engineered cartilage mimics the mechanics of the native tissue. This study provides a spatial map of the mechanical and biochemical properties of human auricular cartilage, thus establishing a benchmark for the evaluation of functional competency in AUR cartilage TE. Stress-relaxation indentation (instantaneous modulus, Ein; maximum stress, σmax; equilibrium modulus, Eeq; relaxation half-life time, t1/2; thickness, h) and biochemical parameters (content of DNA; sulfated-glycosaminoglycan, sGAG; hydroxyproline, HYP; elastin, ELN) of fresh human AUR cartilage were evaluated. Samples were categorized into age groups and according to their harvesting region in the human auricle (for AUR cartilage only). AUR cartilage displayed significantly lower Ein, σmax, Eeq, sGAG content; and significantly higher t1/2, and DNA content than NAS cartilage. Large amounts of ELN were measured in AUR cartilage (>15% ELN content per sample wet mass). No effect of gender was observed for either auricular or nasoseptal samples. For auricular samples, significant differences between age groups for h, sGAG and HYP, and significant regional variations for Ein, σmax, Eeq, t1/2, h, DNA and sGAG were measured. However, only low correlations between mechanical and biochemical parameters were seen (Rbiochemical map of human auricular cartilage. Regional variations in mechanical and biochemical properties were demonstrated in the auricle. This finding highlights the importance of focusing future research on efforts to produce cartilage grafts with spatially tunable mechanics. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Postnatal expression in hyaline cartilage of constitutively active human collagenase-3 (MMP-13) induces osteoarthritis in mice

    National Research Council Canada - National Science Library

    Neuhold, L A; Killar, L; Zhao, W; Sung, M L; Warner, L; Kulik, J; Turner, J; Wu, W; Billinghurst, C; Meijers, T; Poole, A R; Babij, P; DeGennaro, L J

    2001-01-01

    ...). We have used tetracycline-regulated transcription in conjunction with a cartilage-specific promoter to target a constitutively active human MMP-13 to the hyaline cartilages and joints of transgenic mice...

  7. Human articular cartilage: in vitro correlation of MRI and histologic findings

    Energy Technology Data Exchange (ETDEWEB)

    Uhl, M.; Allmann, K.H.; Laubenberger, J.; Langer, M. [Department of Diagnostic Radiology, University Hospital of Freiburg (Germany); Ihling, C.; Tauer, U.; Adler, C.P. [Department of Pathology, University Hospital of Freiburg (Germany)

    1998-09-01

    The aim of our study was to correlate MRI with histologic findings in normal and degenerative cartilage. Twenty-two human knees derived from patients undergoing amputation were examined with 1.0- and 1.5-T MR imaging units. Firstly, we optimized two fat-suppressed 3D gradient-echo sequences. In this pilot study two knees were examined with fast imaging with steady precession (FISP) sequences and fast low-angle shot (FLASH, SPGR) sequence by varying the flip angles (40, 60, 90 ) and combining each flip angle with different echo time (7, 10 or 11, 20 ms). We chose the sequences with the best visual contrast between the cartilage layers and the best measured contrast-to-noise ratio between cartilage and bone marrow. Therefore, we used a 3D FLASH fat-saturated sequence (TR/TE/flip angle = 50/11 ms/40 ) and a 3D FISP fat-saturated sequence (TR/TE/flip angle = 40/10 ms/40 ) for cartilage imaging in 22 human knees. The images were obtained at various angles of the patellar cartilage in relation to the main magnetic field (0, 55, 90 ). The MR appearances were classified into five categories: normal, intracartilaginous signal changes, diffuse thinning (cartilage thickness < 3 mm), superficial erosions, and cartilage ulcers. After imaging, the knees were examined macroscopically and photographed. In addition, we performed histologic studies using light microscopy with several different stainings, polarization, and dark field microscopy as well as electron microscopy. The structural characteristics with the cartilage lesions were correlated with the MR findings. We identified a hyperintense superficial zone in the MR image which did not correlate to the histologically identifiable superficial zone. The second lamina was hypointense on MRI and correlated to the bulk of the radial zone. The third (or deep) cartilage lamina in the MR image seemed to represent the combination of the lowest portion of the radial zone and the calcified cartilage. The width of the hypointense second

  8. Endogenous HLA-DR-restricted presentation of the cartilage antigens human cartilage gp-39 and melanoma inhibitory activity in the inflamed rheumatoid joint

    NARCIS (Netherlands)

    van Lierop, M. J. C.; den Hoed, L.; Houbiers, J.; Vencovsky, J.; Ruzickova, S.; Krystufkova, O.; van Schaardenburg, M.; van den Hoogen, F.; Vandooren, B.; Baeten, D.; De Keyser, F.; Sonderstrup, G.; Bos, E.; Boots, A. M.

    2007-01-01

    Objective. The cartilage proteins melanoma inhibitory activity (MIA) and human cartilage gp-39 (HC gp-39) are candidate autoantigens in rheumatoid arthritis (RA). The present study was undertaken to investigate the endogenous HLA-DR4-restricted presentation of these self proteins, in order to seek i

  9. T cells fail to develop in the human skin-cell explants system; an inconvenient truth

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    Vanderlocht Joris

    2011-02-01

    Full Text Available Abstract Background Haplo-identical hematopoietic stem cell (HSC transplantation is very successful in eradicating haematological tumours, but the long post-transplant T-lymphopenic phase is responsible for high morbidity and mortality rates. Clark et al. have described a skin-explant system capable of producing host-tolerant donor-HSC derived T-cells. Because this T-cell production platform has the potential to replenish the T-cell levels following transplantation, we set out to validate the skin-explant system. Results Following the published procedures, while using the same commercial components, it was impossible to reproduce the skin-explant conditions required for HSC differentiation towards mature T-cells. The keratinocyte maturation procedure resulted in fragile cells with minimum expression of delta-like ligand (DLL. In most experiments the generated cells failed to adhere to carriers or were quickly outcompeted by fibroblasts. Consequently it was not possible to reproduce cell-culture conditions required for HSC differentiation into functional T-cells. Using cell-lines over-expressing DLL, we showed that the antibodies used by Clark et al. were unable to detect native DLL, but instead stained 7AAD+ cells. Therefore, it is unlikely that the observed T-lineage commitment from HSC is mediated by DLL expressed on keratinocytes. In addition, we did confirm expression of the Notch-ligand Jagged-1 by keratinocytes. Conclusions Currently, and unfortunately, it remains difficult to explain the development or growth of T-cells described by Clark et al., but for the fate of patients suffering from lymphopenia it is essential to both reproduce and understand how these co-cultures really "work". Fortunately, alternative procedures to speed-up T-cell reconstitution are being established and validated and may become available for patients in the near future.

  10. Cartilage integrity and proteoglycan turnover are comparable in canine experimentally induced and human joint degeneration

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    Femke Intema

    2010-10-01

    Full Text Available The value of experimental models of osteoarthritis (OA largely depends on the ability to translate observations to human OA. Surprisingly, direct comparison of characteristics of human and experimental OA is scarce. In the present study, cartilage integrity and matrix turnover in a canine model of joint degeneration were compared to human clinical OA. In 23 Beagle dogs, joint degeneration was induced in one knee, the contra-lateral knee served as a control. For comparison, human osteoarthritic and healthy knee cartilage were obtained at arthroplasty (n=14 and post-mortem (n=13. Cartilage was analyzed by histology and biochemistry. Values for cartilage integrity and proteoglycan (PG synthesis showed species specific differences; GAG content of healthy cartilage was 2-fold higher in canine cartilage and PG synthesis even 8-fold. However, the relative decrease in PG content between healthy and OA cartilage was similar for humans and canines (-17% vs. -15%, respectively, as was the histological damage (+7.0 vs. +6.1, respectively and the increase of PG synthesis (+100% vs. +70%, respectively. Remarkably, the percentage release of total and of newly formed PGs in human and canine controls was similar, as was the increase due to degeneration (+65% vs. +81% and +91% vs. +52%, respectively. Despite differences in control conditions, the observed changes in characteristics of cartilage integrity and matrix turnover are similar in a canine model of joint degeneration and human clinical OA. The canine Groove model shows that its characteristics reflect those of human OA which makes the model appropriate for studying human OA.

  11. Nd:YAG 1.44 laser ablation of human cartilage

    Science.gov (United States)

    Cummings, Robert S.; Prodoehl, John A.; Rhodes, Anthony L.; Black, Johnathan D.; Sherk, Henry H.

    1993-07-01

    This study determined the effectiveness of a Neodymium:YAG 1.44 micrometers wavelength laser on human cartilage. This wavelength is strongly absorbed by water. Cadaveric meniscal fibrocartilage and articular hyaline cartilage were harvested and placed in normal saline during the study. A 600 micrometers quartz fiber was applied perpendicularly to the tissues with a force of 0.098 N. Quantitative measurements were then made of the ablation rate as a function of fluence. The laser energy was delivered at a constant repetition rate of 5 Hz., 650 microsecond(s) pulsewidth, and energy levels ranging from 0.5 joules to 2.0 joules. Following the ablation of the tissue, the specimens were fixed in formalin for histologic evaluation. The results of the study indicate that the ablation rate is 0.03 mm/mj/mm2 for hyaline cartilage and fibrocartilage. Fibrocartilage was cut at approximately the same rate as hyaline cartilage. There was a threshold fluence projected to be 987 mj/mm2 for hyaline cartilage and fibrocartilage. Our results indicate that the pulsed Nd:YAG laser operating at 1.44 micrometers has a threshold fluence above which it will ablate human cartilage, and that its ablation rate is directly proportional to fluence over the range of parameters tested. Fibrocartilage and hyaline cartilage demonstrated similar threshold fluence and ablation rates which is related to the high water content of these tissues.

  12. A comparison of healthy human and swine articular cartilage dynamic indentation mechanics.

    Science.gov (United States)

    Ronken, S; Arnold, M P; Ardura García, H; Jeger, A; Daniels, A U; Wirz, D

    2012-05-01

    Articular cartilage is a multicomponent, poroviscoelastic tissue with nonlinear mechanical properties vital to its function. A consequent goal of repair or replacement of injured cartilage is to achieve mechanical properties in the repair tissue similar to healthy native cartilage. Since fresh healthy human articular cartilage (HC) is not readily available, we tested whether swine cartilage (SC) could serve as a suitable substitute for mechanical comparisons. To a first approximation, cartilage tissue and surgical substitutes can be evaluated mechanically as viscoelastic materials. Stiffness measurements (dynamic modulus, loss angle) are vital to function and are also a non-destructive means of evaluation. Since viscoelastic material stiffness is strongly strain rate dependent, stiffness was tested under different loading conditions related to function. Stiffness of healthy HC and SC specimens was determined and compared using two non-destructive, mm-scale indentation test modes: fast impact and slow sinusoidal deformation. Deformation resistance (dynamic modulus) and energy handling (loss angle) were determined. For equivalent anatomic locations, there was no difference in dynamic modulus. However, the HC loss angle was ~35% lower in fast impact and ~12% higher in slow sinusoidal mode. Differences seem attributable to age (young SC, older HC) but also to species anatomy and biology. Test mode-related differences in human-swine loss angle support use of multiple function-related test modes. Keeping loss angle differences in mind, swine specimens could serve as a standard of comparison for mechanical evaluation of e.g. engineered cartilage or synthetic repair materials.

  13. A spectroscopic approach to imaging and quantification of cartilage lesions in human knee joints

    Science.gov (United States)

    Johansson, A.; Sundqvist, T.; Kuiper, J.-H.; Öberg, P. Å.

    2011-03-01

    We have previously described a technology based on diffuse reflectance of broadband light for measuring joint articular cartilage thickness, utilizing that optical absorption is different in cartilage and subchondral bone. This study is the first evaluation of the technology in human material. We also investigated the prospects of cartilage lesion imaging, with the specific aim of arthroscopic integration. Cartilage thickness was studied ex vivo in a number of sites (n = 87) on human knee joint condyles, removed from nine patients during total knee replacement surgery. A reflectance spectrum was taken at each site and the cartilage thickness was estimated using the blue, green, red and near-infrared regions of the spectrum, respectively. Estimated values were compared with reference cartilage thickness values (taken after sample slicing) using an exponential model. Two-dimensional Monte Carlo simulations were performed in a theoretical analysis of the experimental results. The reference cartilage thickness of the investigated sites was 1.60 ± 1.30 mm (mean ± SD) in the range 0-4.2 mm. Highest correlation coefficients were seen for the calculations based on the near-infrared region after normalization to the red region (r = 0.86) and for the green region (r = 0.80).

  14. Hyaline cartilage formation and tumorigenesis of implanted tissues derived from human induced pluripotent stem cells.

    Science.gov (United States)

    Saito, Taku; Yano, Fumiko; Mori, Daisuke; Kawata, Manabu; Hoshi, Kazuto; Takato, Tsuyoshi; Masaki, Hideki; Otsu, Makoto; Eto, Koji; Nakauchi, Hiromitsu; Chung, Ung-il; Tanaka, Sakae

    2015-01-01

    Induced pluripotent stem cells (iPSCs) are a promising cell source for cartilage regenerative medicine. Meanwhile, the risk of tumorigenesis should be considered in the clinical application of human iPSCs (hiPSCs). Here, we report in vitro chondrogenic differentiation of hiPSCs and maturation of the differentiated hiPSCs through transplantation into mouse knee joints. Three hiPSC clones showed efficient chondrogenic differentiation using an established protocol for human embryonic stem cells. The differentiated hiPSCs formed hyaline cartilage tissues at 8 weeks after transplantation into the articular cartilage of NOD/SCID mouse knee joints. Although tumors were not observed during the 8 weeks after transplantation, an immature teratoma had developed in one mouse at 16 weeks. In conclusion, hiPSCs are a potent cell source for regeneration of hyaline articular cartilage. However, the risk of tumorigenesis should be managed for clinical application in the future.

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

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    Pingping Chen-An

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

  16. Anisotropy, inhomogeneity, and tension-compression nonlinearity of human glenohumeral cartilage in finite deformation.

    Science.gov (United States)

    Huang, Chun-Yuh; Stankiewicz, Anna; Ateshian, Gerard A; Mow, Van C

    2005-04-01

    The tensile and compressive properties of human glenohumeral cartilage were determined by testing 120 rectangular strips in uniaxial tension and 70 cylindrical plugs in confined compression, obtained from five human glenohumeral joints. Specimens were harvested from five regions across the articular surface of the humeral head and two regions on the glenoid. Tensile strips were obtained along two orientations, parallel and perpendicular to the split-line directions. Two serial slices through the thickness, corresponding to the superficial and middle zones of the cartilage layers, were prepared from each tensile strip and each compressive plug. The equilibrium tensile modulus and compressive aggregate modulus of cartilage were determined from the uniaxial tensile and confined compression tests, respectively. Significant differences in the tensile moduli were found with depth and orientation relative to the local split-line direction. Articular cartilage of the humeral head was significantly stiffer in tension than that of the glenoid. There were significant differences in the aggregate compressive moduli of articular cartilage between superficial and middle zones in the humeral head. Furthermore, tensile and compressive stress-strain responses exhibited nonlinearity under finite strain, while the tensile modulus differed by up to two orders of magnitude from the compressive aggregate modulus at 0% strain, demonstrating a high degree of tension-compression nonlinearity. The complexity of the mechanical properties of human glenohumeral cartilage was exposed in this study, showing anisotropy, inhomogeneity, and tension-compression nonlinearity within the same joint. The observed differences in the tensile properties of human glenohumeral cartilage suggest that the glenoid may be more susceptible to cartilage degeneration than the humeral head.

  17. Human Adipose-Derived Mesenchymal Progenitor Cells Engraft into Rabbit Articular Cartilage

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    Wen Wang

    2015-05-01

    Full Text Available Mesenchymal stem cells (MSCs are known to have the potential for articular cartilage regeneration, and are suggested for the treatment of osteoarthritis (OA. Here, we investigated whether intra-articular injection of xenogeneic human adipose-derived mesenchymal progenitor cells (haMPCs promoted articular cartilage repair in rabbit OA model and engrafted into rabbit articular cartilage. The haMPCs were cultured in vitro, and phenotypes and differentiation characteristics of cells were evaluated. OA was induced surgically by anterior cruciate ligament transection (ACLT and medical meniscectomy of knee joints. At six weeks following surgery, hyaluronic acid (HA or haMPCs was injected into the knee joints, the contralateral knee served as normal control. All animals were sacrificed at the 16th week post-surgery. Assessments were carried out by macroscopic examination, hematoxylin/eosin (HE and Safranin-O/Fast green stainings and immunohistochemistry. The data showed that haMPC treatment promoted cartilage repair. Signals of human mitochondrial can be directly detected in haMPC treated cartilage. The haMPCs expressed human leukocyte antigen I (HLA-I but not HLA-II-DR in vivo. These results suggest that intra-articular injection of haMPCs promotes regeneration of articular cartilage in rabbit OA model, and support the notion that MPCs are transplantable between HLA-incompatible individuals.

  18. Fourier transform infrared imaging of focal lesions in Human osteoarthritic cartilage

    Directory of Open Access Journals (Sweden)

    David-Vaudey E.

    2005-11-01

    Full Text Available Fourier Transform Infrared Imaging (FTIRI is a new method for quantitatively assessing the spatial-chemical composition of complex materials. This technique has been applied to examine the feasibility of measuring changes in the composition and distribution of collagen and proteoglycan macromolecules in human osteoarthritic cartilage. Human cartilage was acquired post-operatively from total joint replacement patients. Samples were taken at the site of a focal lesion, adjacent to the lesion, and from relatively healthy cartilage away from the lesion. Sections were prepared for FTIRI and histochemical grading. FTIRI spectral images were acquired for the superficial, intermediate, and deep layers for each sample. Euclidean distance mapping and quantitative partial least squares analysis (PLS were performed using reference spectra for type-II collagen and chondroitin 6-sulphate (CS6. FTIRI results were correlated to the histology-based Mankin scoring system. PLS analysis found relatively low relative concentrations of collagen (38 ± 10% and proteoglycan (22 ± 9% in osteoarthritic cartilage. Focal lesions were generally found to contain less CS6 compared to cartilage tissue adjacent to the lesion. Loss of proteoglycan content was well correlated to histological Mankin scores (r=0.69, p<0.0008. The evaluation of biological tissues with FTIRI can provide unique quantitative information on how disease can affect biochemical distribution and composition. This study has demonstrated that FTIRI is useful in quantitatively assessing pathology-related changes in the composition and distribution of primary macromolecular components of human osteoarthritic cartilage.

  19. Expression of NGF, Trka and p75 in human cartilage

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    A Gigante

    2009-06-01

    Full Text Available Nerve growth factor (NGF exerts its action through two types of receptor: high-affinity tyrosine kinase A receptor (trkA and low-affinity p75 receptor. NGF has a neurotrophic role in central and peripheral nervous system development, but there is also clear evidence of its involvement in the developing skeleton. The aim of the present immunohistochemical study was to investigate the expression and distribution of NGF, trkA, and p75 in normal cartilaginous tissues from adult subjects: articular and meniscal cartilage of the knee, cartilage from the epiglottis, and intervertebral disc tissue. Detection of NGF mRNA was also performed by in situ hybridization. Immunoreaction for NGF and the two receptors in articular chondrocytes, chondrocyte-like cells of meniscus and annulus fibrosus, and chondrocytes of the epiglottis demonstrated that they are all expressed in hyaline, fibrous and elastic cartilaginous tissues, suggesting that they could be involved in cartilage physio-pathology.

  20. Cartilage tissue engineering using pre-aggregated human articular chondrocytes

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    F Wolf

    2008-12-01

    Full Text Available In this study, we first aimed at determining whether human articular chondrocytes (HAC proliferate in aggregates in the presence of strong chondrocyte mitogens. We then investigated if the aggregated cells have an enhanced chondrogenic capacity as compared to cells cultured in monolayer. HAC from four donors were cultured in tissue culture dishes either untreated or coated with 1% agarose in the presence of TGFb-1, FGF-2 and PDGF-BB. Proliferation and stage of differentiation were assessed by measuring respectively DNA contents and type II collagen mRNA. Expanded cells were induced to differentiate in pellets or in Hyaff®-11 meshes and the formed tissues were analysed biochemically for glycosaminoglycans (GAG and DNA, and histologically by Safranin O staining. The amount of DNA in aggregate cultures increased significantly from day 2 to day 6 (by 3.2-fold, but did not further increase with additional culture time. Expression of type II collagen mRNA was about two orders of magnitude higher in aggregated HAC as compared to monolayer expanded cells. Pellets generated by aggregated HAC were generally more intensely stained for GAG than those generated by monolayer-expanded cells. Scaffolds seeded with aggregates accumulated more GAG (1.3-fold than scaffolds seeded with monolayer expanded HAC. In conclusion, this study showed that HAC culture in aggregates does not support a relevant degree of expansion. However, aggregation of expanded HAC prior to loading into a porous scaffold enhances the quality of the resulting tissues and could thus be introduced as an intermediate culture phase in the manufacture of engineered cartilage grafts.

  1. Explant culture: a simple, reproducible, efficient and economic technique for isolation of mesenchymal stromal cells from human adipose tissue and lipoaspirate.

    Science.gov (United States)

    Priya, Nancy; Sarcar, Shilpita; Majumdar, Anish Sen; SundarRaj, Swathi

    2014-09-01

    Adipose tissue has emerged as a preferred source of mesenchymal stem/stromal cells (MSC), due to its easy accessibility and high MSC content. The conventional method of isolation of adipose tissue-derived stromal cells (ASC) involves enzymatic digestion and centrifugation, which is a costly and time-consuming process. Mechanical stress during isolation, use of bacterial-derived products and potential contamination with endotoxins and xenoantigens are other disadvantages of this method. In this study, we propose explant culture as a simple and efficient process to isolate ASC from human adipose tissue. This technique can be used to reproducibly isolate ASC from fat tissue obtained by liposuction as well as surgical resection, and yields an enriched ASC population free from contaminating haematopoietic cells. We show that explanting adipose tissue results in a substantially higher yield of ASC at P0 per gram of initial fat tissue processed, as compared to that obtained by enzymatic digestion. We demonstrate that ASC isolated by explant culture are phenotypically and functionally equivalent to those obtained by enzymatic digestion. Further, the explant-derived ASC share the immune privileged status and immunosuppressive properties implicit to MSC, suggesting that they are competent to be tested and applied in allogeneic clinical settings. As explant culture is a simple, inexpensive and gentle method, it may be preferred over the enzymatic technique for obtaining adipose tissue-derived stem/stromal cells for tissue engineering and regenerative medicine, especially in cases of limited starting material.

  2. The Inhibitory Effect of Salmon Calcitonin on Tri-Iodothyronine Induction of Early Hypertrophy in Articular Cartilage

    OpenAIRE

    Pingping Chen-An; Kim Vietz Andreassen; Kim Henriksen; Yadong Li; Morten Asser Karsdal; Anne-Christine Bay-Jensen

    2012-01-01

    OBJECTIVE: Salmon calcitonin has chondroprotective effect both in vitro and in vivo, and is therefore being tested as a candidate drug for cartilage degenerative diseases. Recent studies have indicated that different chondrocyte phenotypes may express the calcitonin receptor (CTR) differentially. We tested for the presence of the CTR in chondrocytes from tri-iodothyronin (T3)-induced bovine articular cartilage explants. Moreover, investigated the effects of human and salmon calcitonin on the ...

  3. Time-of-flight secondary ion mass spectrometry-based molecular distribution distinguishing healthy and osteoarthritic human cartilage

    CERN Document Server

    Cillero-Pastor, Berta; Kiss, Andras; Blanco, Francisco J; Heeren, Ron M A

    2013-01-01

    Osteoarthritis (OA) is a pathology that ultimately causes joint destruction. The cartilage is one of the principal affected tissues. Alterations in the lipid mediators and an imbalance in the metabolism of cells that form the cartilage (chondrocytes) have been described as contributors to the OA development. In this study, we have studied the distribution of lipids and chemical elements in healthy and OA human cartilage. Time of flight-secondary ion mass spectrometry (TOF-SIMS) allows us to study the spatial distribution of molecules at a high resolution on a tissue section. TOF-SIMS revealed a specific peak profile that distinguishes healthy from OA cartilages. The spatial distribution of cholesterol-related peaks exhibited a remarkable difference between healthy and OA cartilages. A distinctive colocalization of cholesterol and other lipids in the superficial area of the cartilage was found. A higher intensity of oleic acid and other fatty acids in the OA cartilages exhibited a similar localization. On the ...

  4. Latent Transforming Growth Factor-beta1 Functionalised Electrospun Scaffolds Promote Human Cartilage Differentiation: Towards an Engineered Cartilage Construct

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    Erh-Hsuin Lim

    2013-11-01

    Full Text Available BackgroundTo overcome the potential drawbacks of a short half-life and dose-related adverse effects of using active transforming growth factor-beta 1 for cartilage engineering, a cell-mediated latent growth factor activation strategy was developed incorporating latent transforming growth factor-β1 (LTGF into an electrospun poly(L-lactide scaffold.MethodsThe electrospun scaffold was surface modified with NH3 plasma and biofunctionalised with LTGF to produce both random and orientated biofunctionalised electrospun scaffolds. Scaffold surface chemical analysis and growth factor bioavailability assays were performed. In vitro biocompatibility and human nasal chondrocyte gene expression with these biofunctionalised electrospun scaffold templates were assessed. In vivo chondrogenic activity and chondrocyte gene expression were evaluated in athymic rats.ResultsChemical analysis demonstrated that LTGF anchored to the scaffolds was available for enzymatic, chemical and cell activation. The biofunctionalised scaffolds were non-toxic. Gene expression suggested chondrocyte re-differentiation after 14 days in culture. By 6 weeks, the implanted biofunctionalised scaffolds had induced highly passaged chondrocytes to re-express Col2A1 and produce type II collagen.ConclusionsWe have demonstrated a proof of concept for cell-mediated activation of anchored growth factors using a novel biofunctionalised scaffold in cartilage engineering. This presents a platform for development of protein delivery systems and for tissue engineering.

  5. X-ray dark field imaging of human articular cartilage: Possible clinical application to orthopedic surgery

    Energy Technology Data Exchange (ETDEWEB)

    Kunisada, Toshiyuki [Department of Medical Materials for Musculoskeletal Reconstruction, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8558 (Japan); Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8558 (Japan)], E-mail: toshi-kunisada@umin.ac.jp; Shimao, Daisuke [Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, Ibaraki 300-2394 (Japan); Sugiyama, Hiroshi [Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Ibaraki 305-0801 (Japan); Takeda, Ken; Ozaki, Toshifumi [Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8558 (Japan); Ando, Masami [Research Institute for Science and Technology, Tokyo University of Science, Chiba 278-8510 (Japan)

    2008-12-15

    Despite its convenience and non-invasiveness on daily clinical use, standard X-ray radiography cannot show articular cartilage. We developed a novel type of X-ray dark field imaging (DFI), which forms images only by a refracted beam with very low background illumination. We examined a disarticulated distal femur and a shoulder joint with surrounding soft tissue and skin, both excised from a human cadaver at the BL20B2 synchrotron beamline at SPring-8. The field was 90 mm wide and 90 mm high. Articular cartilage of the disarticulated distal femur was obvious on DFI, but not on standard X-ray images. Furthermore, DFI allowed visualization in situ of articular cartilage of the shoulder while covered with soft tissue and skin. The gross appearance of the articular cartilage on the dissected section of the proximal humerus was identical to the cartilage shown on the DFI image. These results suggested that DFI could provide a clinically accurate method of assessing articular cartilage. Hence, DFI would be a useful imaging tool for diagnosing joint disease such as osteoarthritis.

  6. Analysis of human knee osteoarthritic cartilage using polarization sensitive second harmonic generation microscopy

    Science.gov (United States)

    Kumar, Rajesh; Grønhaug, Kirsten M.; Romijn, Elisabeth I.; Drogset, Jon O.; Lilledahl, Magnus B.

    2014-05-01

    Osteoarthritis is one of the most prevalent joint diseases in the world. Although the cause of osteoarthritis is not exactly clear, the disease results in a degradation of the quality of the articular cartilage including collagen and other extracellular matrix components. We have investigated alterations in the structure of collagen fibers in the cartilage tissue of the human knee using mulitphoton microscopy. Due to inherent high nonlinear susceptibility, ordered collagen fibers present in the cartilage tissue matrix produces strong second harmonic generation (SHG) signals. Significant morphological differences are found in different Osteoarthritic grades of cartilage by SHG microscopy. Based on the polarization analysis of the SHG signal, we find that a few locations of hyaline cartilage (mainly type II collagen) is being replaced by fibrocartilage (mainly type I cartilage), in agreement with earlier literature. To locate the different types and quantify the alteration in the structure of collagen fiber, we employ polarization-SHG microscopic analysis, also referred to as _-tensor imaging. The image analysis of p-SHG image obtained by excitation polarization measurements would represent different tissue constituents with different numerical values at pixel level resolution.

  7. Discovery of highly potent and selective small molecule ADAMTS-5 inhibitors that inhibit human cartilage degradation via encoded library technology (ELT).

    Science.gov (United States)

    Deng, Hongfeng; O'Keefe, Heather; Davie, Christopher P; Lind, Kenneth E; Acharya, Raksha A; Franklin, G Joseph; Larkin, Jonathan; Matico, Rosalie; Neeb, Michael; Thompson, Monique M; Lohr, Thomas; Gross, Jeffrey W; Centrella, Paolo A; O'Donovan, Gary K; Bedard, Katie L Sargent; van Vloten, Kurt; Mataruse, Sibongile; Skinner, Steven R; Belyanskaya, Svetlana L; Carpenter, Tiffany Y; Shearer, Todd W; Clark, Matthew A; Cuozzo, John W; Arico-Muendel, Christopher C; Morgan, Barry A

    2012-08-23

    The metalloprotease ADAMTS-5 is considered a potential target for the treatment of osteoarthritis. To identify selective inhibitors of ADAMTS-5, we employed encoded library technology (ELT), which enables affinity selection of small molecule binders from complex mixtures by DNA tagging. Selection of ADAMTS-5 against a four-billion member ELT library led to a novel inhibitor scaffold not containing a classical zinc-binding functionality. One exemplar, (R)-N-((1-(4-(but-3-en-1-ylamino)-6-(((2-(thiophen-2-yl)thiazol-4-yl)methyl)amino)-1,3,5-triazin-2-yl)pyrrolidin-2-yl)methyl)-4-propylbenzenesulfonamide (8), inhibited ADAMTS-5 with IC(50) = 30 nM, showing >50-fold selectivity against ADAMTS-4 and >1000-fold selectivity against ADAMTS-1, ADAMTS-13, MMP-13, and TACE. Extensive SAR studies showed that potency and physicochemical properties of the scaffold could be further improved. Furthermore, in a human osteoarthritis cartilage explant study, compounds 8 and 15f inhibited aggrecanase-mediated (374)ARGS neoepitope release from aggrecan and glycosaminoglycan in response to IL-1β/OSM stimulation. This study provides the first small molecule evidence for the critical role of ADAMTS-5 in human cartilage degradation.

  8. 3D Human cartilage surface characterization by optical coherence tomography

    Science.gov (United States)

    Brill, Nicolai; Riedel, Jörn; Schmitt, Robert; Tingart, Markus; Truhn, Daniel; Pufe, Thomas; Jahr, Holger; Nebelung, Sven

    2015-10-01

    Early diagnosis and treatment of cartilage degeneration is of high clinical interest. Loss of surface integrity is considered one of the earliest and most reliable signs of degeneration, but cannot currently be evaluated objectively. Optical Coherence Tomography (OCT) is an arthroscopically available light-based non-destructive real-time imaging technology that allows imaging at micrometre resolutions to millimetre depths. As OCT-based surface evaluation standards remain to be defined, the present study investigated the diagnostic potential of 3D surface profile parameters in the comprehensive evaluation of cartilage degeneration. To this end, 45 cartilage samples of different degenerative grades were obtained from total knee replacements (2 males, 10 females; mean age 63.8 years), cut to standard size and imaged using a spectral-domain OCT device (Thorlabs, Germany). 3D OCT datasets of 8  ×  8, 4  ×  4 and 1  ×  1 mm (width  ×  length) were obtained and pre-processed (image adjustments, morphological filtering). Subsequent automated surface identification algorithms were used to obtain the 3D primary profiles, which were then filtered and processed using established algorithms employing ISO standards. The 3D surface profile thus obtained was used to calculate a set of 21 3D surface profile parameters, i.e. height (e.g. Sa), functional (e.g. Sk), hybrid (e.g. Sdq) and segmentation-related parameters (e.g. Spd). Samples underwent reference histological assessment according to the Degenerative Joint Disease classification. Statistical analyses included calculation of Spearman’s rho and assessment of inter-group differences using the Kruskal Wallis test. Overall, the majority of 3D surface profile parameters revealed significant degeneration-dependent differences and correlations with the exception of severe end-stage degeneration and were of distinct diagnostic value in the assessment of surface integrity. None of the 3D

  9. The multicomponent phytopharmaceutical SKI306X inhibits in vitro cartilage degradation and the production of inflammatory mediators.

    NARCIS (Netherlands)

    Hartog, A.; Hougee, S.; Faber, J.; Sanders, A.; Zuurman, C.; Smit, H.F.; Kraan, P.M. van der; Hoijer, M.A.; Garssen, J.

    2008-01-01

    Clinical studies have demonstrated that SKI306X, a purified preparation of three medicinal plants, relieves joint pain and improves functionality in osteoarthritis patients. To study the biological action of SKI306X, bovine cartilage explants and human peripheral blood mononuclear cells (PBMC) were

  10. UV-independent induction of beta defensin 3 in neonatal human skin explants [v2; ref status: indexed, http://f1000r.es/53b

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    Erin Wolf Horrell

    2015-02-01

    Full Text Available In order to determine the effect of UV radiation on β-defensin 3 (BD3 expression in human skin, freshly-isolated UV-naïve skin was obtained from newborn male infants undergoing planned circumcision.  Skin explants sustained ex vivo dermis side down on RPMI media were exposed to 0.5 kJ/m2 UVB, and biopsies were taken from the explant through 72 hours after radiation.  mRNA expression was measured by qRTPCR and normalized to TATA-binding protein.  BD3 expression at each time point was compared with an untreated control taken at time 0 within each skin sample.  Extensive variability in both the timing and magnitude of BD3 induction across individuals was noted and was not predicted by skin pigment phenotype, suggesting that BD3 induction was not influenced by epidermal melanization.  However, a mock-irradiated time course demonstrated UV-independent BD3 mRNA increases across multiple donors which was not further augmented by treatment with UV radiation, suggesting that factors other than UV damage promoted increased BD3 expression in the skin explants.  We conclude that BD3 expression is induced in a UV-independent manner in human skin explants processed and maintained in standard culture conditions, and that neonatal skin explants are an inappropriate model with which to study the effects of UV on BD3 induction in whole human skin.

  11. UV-independent induction of beta defensin 3 in neonatal human skin explants [v1; ref status: indexed, http://f1000r.es/4s2

    Directory of Open Access Journals (Sweden)

    Erin Wolf Horrell

    2014-11-01

    Full Text Available In order to determine the effect of UV radiation on β-defensin 3 (BD3 expression in human skin, freshly-isolated UV-naïve skin was obtained from newborn male infants undergoing planned circumcision.  Skin explants sustained ex vivo dermis side down on RPMI media were exposed to 0.5 kJ/m2 UVB, and biopsies were taken from the explant through 72 hours after radiation.  mRNA expression was measured by qRTPCR and normalized to TATA-binding protein.  BD3 expression at each time point was compared with an untreated control taken at time 0 within each skin sample.  Extensive variability in both the timing and magnitude of BD3 induction across individuals was noted and was not predicted by skin pigment phenotype, suggesting that BD3 induction was not influenced by epidermal melanization.  However, a mock-irradiated time course demonstrated UV-independent BD3 mRNA increases across multiple donors which was not further augmented by treatment with UV radiation, suggesting that factors other than UV damage promoted increased BD3 expression in the skin explants.  We conclude that BD3 expression is induced in a UV-independent manner in human skin explants processed and maintained in standard culture conditions, and that neonatal skin explants are an inappropriate model with which to study the effects of UV on BD3 induction in whole human skin.

  12. Preservation of glial cytoarchitecture from ex vivo human tumor and non-tumor cerebral cortical explants: A human model to study neurological diseases.

    Science.gov (United States)

    Chaichana, Kaisorn L; Capilla-Gonzalez, Vivian; Gonzalez-Perez, Oscar; Pradilla, Gustavo; Han, James; Olivi, Alessandro; Brem, Henry; Garcia-Verdugo, Jose Manuel; Quiñones-Hinojosa, Alfredo

    2007-08-30

    For the human brain, in vitro models that accurately represent what occurs in vivo are lacking. Organotypic models may be the closest parallel to human brain tissue outside of a live patient. However, this model has been limited primarily to rodent-derived tissue. We present an organotypic model to maintain intraoperatively collected human tumor and non-tumor explants ex vivo for a prolonged period of time ( approximately 11 days) without any significant changes to the tissue cytoarchitecture as evidenced through immunohistochemistry and electron microscopy analyses. The ability to establish and reliably predict the cytoarchitectural changes that occur with time in an organotypic model of tumor and non-tumor human brain tissue has several potential applications including the study of cell migration on actual tissue matrix, drug toxicity on neural tissue and pharmacological treatment for brain cancers, among others.

  13. In situ measurements of human articular cartilage stiffness by means of a scanning force microscope

    Energy Technology Data Exchange (ETDEWEB)

    Imer, Raphael [Institute of Microtechnology, University of Neuchatel, Jaquet-Droz 1, 2007 Neuchatel (Switzerland); Akiyama, Terunobu [Institute of Microtechnology, University of Neuchatel, Jaquet-Droz 1, 2007 Neuchatel (Switzerland); Rooij, Nico F de [Institute of Microtechnology, University of Neuchatel, Jaquet-Droz 1, 2007 Neuchatel (Switzerland); Stolz, Martin [Maurice E. Mueller Institute, University of Basel, Klingelbergstr. 70, 4056 Basel (Switzerland); Aebi, Ueli [Maurice E. Mueller Institute, University of Basel, Klingelbergstr. 70, 4056 Basel (Switzerland); Kilger, Robert [Clinics for Orthopedic Surgery and Traumatology, Kantonsspital, 4101 Bruderholz (Switzerland); Friederich, Niklaus F [Clinics for Orthopedic Surgery and Traumatology, Kantonsspital, 4101 Bruderholz (Switzerland); Wirz, Dieter [Lab. for Orthopaedic Biomechanics, University of Basel, Klingelbergstr. 50-70, 4056 Basel (Switzerland); Daniels, A U [Lab. for Orthopaedic Biomechanics, University of Basel, Klingelbergstr. 50-70, 4056 Basel (Switzerland); Staufer, Urs [Institute of Microtechnology, University of Neuchatel, Jaquet-Droz 1, 2007 Neuchatel (Switzerland)

    2007-03-15

    Osteoarthritis is a painful and disabling progressive joint disease, characterized by degradation of articular cartilage. In order to study this disease at early stages, we have miniaturized and integrated a complete scanning force microscope into a standard arthroscopic device fitting through a standard orthopedic canula. This instrument will allow orthopedic surgeons to measure the mechanical properties of articular cartilage at the nanometer and micrometer scale in-vivo during a standard arthroscopy. An orthopedic surgeon assessed the handling of the instrument. First measurements of the elasticity-modulus of human cartilage were recorded in a cadaver knee non minimal invasive. Second, minimally invasive experiments were performed using arthroscopic instruments. Load-displacement curves were successfully recorded.

  14. Regeneration of human-ear-shaped cartilage by co-culturing human microtia chondrocytes with BMSCs.

    Science.gov (United States)

    Zhang, Lu; He, Aijuan; Yin, Zongqi; Yu, Zheyuan; Luo, Xusong; Liu, Wei; Zhang, Wenjie; Cao, Yilin; Liu, Yu; Zhou, Guangdong

    2014-06-01

    Previously, we had addressed the issues of shape control/maintenance of in vitro engineered human-ear-shaped cartilage. Thus, lack of applicable cell source had become a major concern that blocks clinical translation of this technology. Autologous microtia chondrocytes (MCs) and bone marrow stromal cells (BMSCs) were both promising chondrogenic cells that did not involve obvious donor site morbidity. However, limited cell availability of MCs and ectopic ossification of chondrogenically induced BMSCs in subcutaneous environment greatly restricted their applications in external ear reconstruction. The current study demonstrated that MCs possessed strong proliferation ability but accompanied with rapid loss of chondrogenic ability during passage, indicating a poor feasibility to engineer the entire ear using expanded MCs. Fortunately, the co-transplantation results of MCs and BMSCs (25% MCs and 75% BMSCs) demonstrated a strong chondroinductive ability of MCs to promote stable ectopic chondrogenesis of BMSCs in subcutaneous environment. Moreover, cell labeling demonstrated that BMSCs could transform into chondrocyte-like cells under the chondrogenic niche provided by co-cultured MCs. Most importantly, a human-ear-shaped cartilaginous tissue with delicate structure and proper elasticity was successfully constructed by seeding the mixed cells (MCs and BMSCs) into the pre-shaped biodegradable ear-scaffold followed by 12 weeks of subcutaneous implantation in nude mouse. These results may provide a promising strategy to construct stable ectopic cartilage with MCs and stem cells (BMSCs) for autologous external ear reconstruction.

  15. Human conchal cartilage and temporal fascia: an evidence-based roadmap from rhinoplasty to an in vivo study and beyond.

    Science.gov (United States)

    Cimpean, Anca Maria; Crăiniceanu, Zorin; Mihailovici, Dorina; Bratu, Tiberiu; Raica, Marius

    2014-01-01

    Conchal cartilage or cartilage/ temporal fascia composite grafting (DC-F) used for rhinoplasty is applied by plastic surgeons for reconstructive purposes. Previous studies on experimental models such as mice or rabbits have elucidated on the late events following grafting, with tissue specimens being harvested two months after implantation. Early microscopic and molecular events following DC-F grafting are completely unknown. We designed a chick embryo chorioallantoic membrane model for human grafts study, regarding the dynamic observation of graft survival and its mutual interrelation with the chick embryo chorioallantoic membrane microenvironment. The DC-F graft preserved its cartilage component in a normal state compared to cartilage graft-only because of protective factors provided by temporal fascia. Its strong adherence to the cartilage, lack of angiogenic factors and high content of collagen IV-derived fragments with anti-angiogenic effects make the temporal fascia a good protective tissue to prevent implanted cartilage degeneration. The cartilage graft produced high inflammation, stromal fibrosis and activated angiogenic cascade through VEGF-mediated pathways followed by cartilage degeneration. Also, high content of podoplanin from conchal cartilage chondrocytes exerted a major role in inflammation accompanying cartilage graft. The presently employed experimental model allowed us to characterize the early histological and molecular events triggered by temporal fascia, cartilage or composite graft DC-F implanted on chick embryo chorioallantoic membrane. Our microscopic and molecular observations may help explain some post-surgical complications generated after using cartilage alone as biomaterial for nasal augmentation, supporting the use of DC-F composite graft, with the aim to reduce unwanted post-surgical events. Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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

  17. Effect of Human Adipose Tissue Mesenchymal Stem Cells on the Regeneration of Ovine Articular Cartilage.

    Science.gov (United States)

    Zorzi, Alessandro R; Amstalden, Eliane M I; Plepis, Ana Maria G; Martins, Virginia C A; Ferretti, Mario; Antonioli, Eliane; Duarte, Adriana S S; Luzo, Angela C M; Miranda, João B

    2015-11-09

    Cell therapy is a promising approach to improve cartilage healing. Adipose tissue is an abundant and readily accessible cell source. Previous studies have demonstrated good cartilage repair results with adipose tissue mesenchymal stem cells in small animal experiments. This study aimed to examine these cells in a large animal model. Thirty knees of adult sheep were randomly allocated to three treatment groups: CELLS (scaffold seeded with human adipose tissue mesenchymal stem cells), SCAFFOLD (scaffold without cells), or EMPTY (untreated lesions). A partial thickness defect was created in the medial femoral condyle. After six months, the knees were examined according to an adaptation of the International Cartilage Repair Society (ICRS 1) score, in addition to a new Partial Thickness Model scale and the ICRS macroscopic score. All of the animals completed the follow-up period. The CELLS group presented with the highest ICRS 1 score (8.3 ± 3.1), followed by the SCAFFOLD group (5.6 ± 2.2) and the EMPTY group (5.2 ± 2.4) (p = 0.033). Other scores were not significantly different. These results suggest that human adipose tissue mesenchymal stem cells promoted satisfactory cartilage repair in the ovine model.

  18. Mechanism of Trypanosoma cruzi Placenta Invasion and Infection: The Use of Human Chorionic Villi Explants

    Directory of Open Access Journals (Sweden)

    Ricardo E. Fretes

    2012-01-01

    Full Text Available Congenital Chagas disease, a neglected tropical disease, endemic in Latin America, is associated with premature labor and miscarriage. During vertical transmission the parasite Trypanosoma cruzi (T. cruzi crosses the placental barrier. However, the exact mechanism of the placental infection remains unclear. We review the congenital transmission of T. cruzi, particularly the role of possible local placental factors that contribute to the vertical transmission of the parasite. Additionally, we analyze the different methods available for studying the congenital transmission of the parasite. In that context, the ex vivo infection with T. cruzi trypomastigotes of human placental chorionic villi constitutes an excellent tool for studying parasite infection strategies as well as possible local antiparasitic mechanisms.

  19. Differential response of the epithelium and interstitium in developing human fetal lung explants to hyperoxia.

    Science.gov (United States)

    Bustani, Porus; Hodge, Rachel; Tellabati, Ananth; Li, Juan; Pandya, Hitesh; Kotecha, Sailesh

    2006-03-01

    Hyperoxia is closely linked with the development of chronic lung disease of prematurity (CLD), but the exact mechanisms whereby hyperoxia alters the lung architecture in the developing lung remain largely unknown. We developed a fetal human lung organ culture model to investigate (a) the morphologic changes induced by hyperoxia and (b) whether hyperoxia resulted in differential cellular responses in the epithelium and interstitium. The effects of hyperoxia on lung morphometry were analyzed using computer-assisted image analysis. The lung architecture remained largely unchanged in normoxia lasting as long as 4 d. In contrast, hyperoxic culture of pseudoglandular fetal lungs resulted in significant dilatation of airways, thinning of the epithelium, and regression of the interstitium including the pulmonary vasculature. Although there were no significant differences in Ki67 between normoxic and hyperoxic lungs, activated caspase-3 was significantly increased in interstitial cells, but not epithelial cells, under hyperoxic conditions. These changes show that exposure of pseudoglandular lungs to hyperoxia modulates the lung architecture to resemble saccular lungs.

  20. Melanocortin agonists stimulate lipolysis in human adipose tissue explants but not in adipocytes.

    Science.gov (United States)

    Møller, Cathrine Laustrup; Pedersen, Steen B; Richelsen, Bjørn; Conde-Frieboes, Kilian W; Raun, Kirsten; Grove, Kevin L; Wulff, Birgitte Schjellerup

    2015-10-12

    The central melanocortin system is broadly involved in the regulation of mammalian nutrient utilization. However, the function of melanocortin receptors (MCRs) expressed directly in peripheral metabolic tissues is still unclear. The objective of this study was to investigate the lipolytic capacity of MC1-5R in differentiated adipocytes versus intact white adipose tissue. Non-selective MCR agonist α-MSH, MC5R-selective agonist PG-901 and MC4R-selective agonist LY2112688 significantly stimulated lipolysis in intact white adipose tissue, whereas stimulation of MCRs in differentiated adipocytes failed to do so. The lipolytic response of MC5R was decreased in intact human white adipose tissue when co-treating with β-adrenergic antagonist propranolol, suggesting that the effect may be dependent on neuronal innervation via noradrenalin release. When developing an anti-obesity therapeutic drug with selective MC4R/MC5R properties, effects on lipolysis in white adipose tissue may be physiologically relevant.

  1. Characterisation of a bystander effect induced in human tissue explant cultures by low LET radiation

    Energy Technology Data Exchange (ETDEWEB)

    Motersill, C.; O' Malley, K.; Seymour, C.B

    2002-07-01

    The existence of a bystander effect following both alpha and gamma irradiation of many cell lines is not now in dispute. The significance of this effect for cancer risk assessment and radiotherapy treatment planning requires demonstration of its relevance in vivo. The problem in demonstrating the existence of the effect in vivo is that other systemic effects may mask or confound the effect being investigated and it is practically impossible to attribute an effect in a particular cell to a signal produced in another irradiated cell. To approach this problem, an assay has been developed where fragments of human tissue can be irradiated ex vivo and the media harvested and added to unirradiated, clonogenic cells which have a well characterised and stable response to the bystander signal. The variation in the production of a signal from patient to patient can thus be assessed. The results of a study using tissue from over 100 patients attending Beaumont and St Vincent's Hospitals in Dublin for investigation of urological disorders including follow-up after treatment for transitional cell carcinoma (TCC) and resection of suspect prostatic lesions, are now available. Blood samples from the prostate group were also obtained. The results show that there is variation in the effect of the signal produced by irradiated tissue from different patients. This holds for bladder, prostate and blood. Gender, smoking status and the existence of a malignancy influence the expression of the signal by normal tissue. Male gender, smoking and a pre-existing malignancy all reduce the amount or effect of the signal produced into medium when the tissue is exposed. The effects of exposure to medium containing the signal are transmitted to distant progeny of the exposed cell population. The results may be important not only for understanding radiation risk mechanisms for protection but also for radiotherapy treatment planning where they may open new avenues for development of drugs for

  2. Tumorigenic risk of human induced pluripotent stem cell explants cultured on mouse SNL76/7 feeder cells

    Energy Technology Data Exchange (ETDEWEB)

    Kamada, Mizuna; Mitsui, Youji, E-mail: y-mitsui8310@hb.tp1.jp; Kumazaki, Tsutomu; Kawahara, Yuta; Matsuo, Taira; Takahashi, Tomoko, E-mail: t-takahashi@kph.bunri-u.ac.jp

    2014-10-24

    Highlights: • hiPS cell explants formed malignant tumors when SNL76/7 feeder cells were used. • Multi type tumors developed by interaction of SNL76/7 feeder cells with hiPS cells. • Tumorigenic risk occurs by co-culture of hiPS cells with SNL76/7 feeder cells. - Abstract: The potential for tumor formation from transplanted human induced pluripotent stem cell (hiPSC) derivatives represents a high risk in their application to regenerative medicine. We examined the genetic origin and characteristics of tumors, that were formed when 13 hiPSC lines, established by ourselves, and 201B7 hiPSC from Kyoto University were transplanted into severe combined immune-deficient (SCID) mice. Though teratomas formed in 58% of mice, five angiosarcomas, one malignant solitary fibrous tumor and one undifferentiated pleomorphic sarcoma formed in the remaining mice. Three malignant cell lines were established from the tumors, which were derived from mitomycin C (MMC)-treated SNL76/7 (MMC-SNL) feeder cells, as tumor development from fusion cells between MMC-SNL and hiPSCs was negative by genetic analysis. While parent SNL76/7 cells produced malignant tumors, neither MMC-SNL nor MMC-treated mouse embryo fibroblast (MEF) produced malignant tumors. When MMC-SNL feeder cells were co-cultured with hiPSCs, growing cell lines were generated, that expressed genes similar to the parent SNL76/7 cells. Thus, hiPSCs grown on MMC-SNL feeder cells have a high risk of generating feeder-derived malignant tumors. The possible mechanism(s) of growth restoration and the formation of multiple tumor types are discussed with respect of the interactions between MMC-SNL and hiPSC.

  3. Volumetric characterization of human patellar cartilage matrix on phase contrast x-ray computed tomography

    Science.gov (United States)

    Abidin, Anas Z.; Nagarajan, Mahesh B.; Checefsky, Walter A.; Coan, Paola; Diemoz, Paul C.; Hobbs, Susan K.; Huber, Markus B.; Wismüller, Axel

    2015-03-01

    Phase contrast X-ray computed tomography (PCI-CT) has recently emerged as a novel imaging technique that allows visualization of cartilage soft tissue, subsequent examination of chondrocyte patterns, and their correlation to osteoarthritis. Previous studies have shown that 2D texture features are effective at distinguishing between healthy and osteoarthritic regions of interest annotated in the radial zone of cartilage matrix on PCI-CT images. In this study, we further extend the texture analysis to 3D and investigate the ability of volumetric texture features at characterizing chondrocyte patterns in the cartilage matrix for purposes of classification. Here, we extracted volumetric texture features derived from Minkowski Functionals and gray-level co-occurrence matrices (GLCM) from 496 volumes of interest (VOI) annotated on PCI-CT images of human patellar cartilage specimens. The extracted features were then used in a machine-learning task involving support vector regression to classify ROIs as healthy or osteoarthritic. Classification performance was evaluated using the area under the receiver operating characteristic (ROC) curve (AUC). The best classification performance was observed with GLCM features correlation (AUC = 0.83 +/- 0.06) and homogeneity (AUC = 0.82 +/- 0.07), which significantly outperformed all Minkowski Functionals (p GLCM-derived statistical features can distinguish between healthy and osteoarthritic tissue with high accuracy.

  4. Human low density lipoprotein as a substrate for in vitro steroidogenesis assays with fathead minnow ovary explants

    Science.gov (United States)

    Gonad explant in vitro steroidogenesis assays are used as part of a multifaceted strategy to detect endocrine active chemicals capable of altering steroid hormone synthesis. An in vitro steroidogenesis assay used in our laboratory involves exposing fathead minnow (FHM) gonad exp...

  5. Human low density lipoprotein as a substrate for in vitro steroidogenesis assays with fathead minnow ovary explants

    Science.gov (United States)

    Gonad explant in vitro steroidogenesis assays are used as part of a multifaceted strategy to detect endocrine active chemicals capable of altering steroid hormone synthesis. An in vitro steroidogenesis assay used in our laboratory involves exposing fathead minnow (FHM) gonad exp...

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

    Science.gov (United States)

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

    2014-06-01

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

  7. Stimulation of proteoglycan synthesis by glucuronosyltransferase-I gene delivery: a strategy to promote cartilage repair.

    Science.gov (United States)

    Venkatesan, N; Barré, L; Benani, A; Netter, P; Magdalou, J; Fournel-Gigleux, S; Ouzzine, M

    2004-12-28

    Osteoarthritis is a degenerative joint disease characterized by a progressive loss of articular cartilage components, mainly proteoglycans (PGs), leading to destruction of the tissue. We investigate a therapeutic strategy based on stimulation of PG synthesis by gene transfer of the glycosaminoglycan (GAG)-synthesizing enzyme, beta1,3-glucuronosyltransferase-I (GlcAT-I) to promote cartilage repair. We previously reported that IL-1beta down-regulated the expression and activity of GlcAT-I in primary rat chondrocytes. Here, by using antisense oligonucleotides, we demonstrate that GlcAT-I inhibition impaired PG synthesis and deposition in articular cartilage explants, emphasizing the crucial role of this enzyme in PG anabolism. Thus, primary chondrocytes and cartilage explants were engineered by lipid-mediated gene delivery to efficiently overexpress a human GlcAT-I cDNA. Interestingly, GlcAT-I overexpression significantly enhanced GAG synthesis and deposition as evidenced by (35)S-sulfate incorporation, histology, estimation of GAG content, and fluorophore-assisted carbohydrate electrophoresis analysis. Metabolic labeling and Western blot analyses further suggested that GlcAT-I expression led to an increase in the abundance rather than in the length of GAG chains. Importantly, GlcAT-I delivery was able to overcome IL-1beta-induced PG depletion and maintain the anabolic activity of chondrocytes. Moreover, GlcAT-I also restored PG synthesis to a normal level in cartilage explants previously depleted from endogenous PGs by IL-1beta-treatment. In concert, our investigations strongly indicated that GlcAT-I was able to control and reverse articular cartilage defects in terms of PG anabolism and GAG content associated with IL-1beta. This study provides a basis for a gene therapy approach to promote cartilage repair in degenerative joint diseases.

  8. Fetal mesenchymal stromal cells differentiating towards chondrocytes acquire a gene expression profile resembling human growth plate cartilage.

    Directory of Open Access Journals (Sweden)

    Sandy A van Gool

    Full Text Available We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs differentiating towards chondrocytes as an alternative model for the human growth plate (GP. Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFβ3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development.

  9. Changes in the stiffness of the human tibial cartilage-bone complex in early-stage osteoarthrosis

    DEFF Research Database (Denmark)

    Ding, Ming; Dalstra, M; Linde, F

    1998-01-01

    Cylindrical human tibial cartilage-bone unit specimens were removed from 9 early-stage medial osteoarthrotic (OA) tibiae (mean age 74 years) and 10 normal age-matched tibiae (mean age 73 years). These specimens were divided into 4 groups: OA, lateral comparison, medial age-matched, and lateral ag...... and bone remained significant in the three control groups. Our findings suggest that both cartilage and bone in early-stage OA are mechanically inferior to normal, and that OA cartilage and bone have lost their unit function to mechanical loading....

  10. Expression of caspase-3 and -9 relevant to cartilage destruction and chondrocyte apoptosis in human osteoarthritic cartilage.

    Directory of Open Access Journals (Sweden)

    Matsuo M

    2001-12-01

    Full Text Available To clarify the involvement of the caspase family in the pathway of NO-induced chondrocyte apoptosis, osteoarthritis (OA cartilage obtained from 8 patients undergoing total hip arthroplasty were used for histopathological study. Cartilage samples taken from non-fibrillated areas of femoral head resected during surgery for femoral neck fracture were used for comparison. DNA fragmentation of chondrocytes was detected by the nick end-labeling (TUNEL method. Apoptosis was further confirmed by transmission electron microscopy. The distributions of nitrotyrosine (NT, caspase-3, and -9 were examined immunohistochemically. The populations of apoptotic as well as NT-, caspase-3-, and -9-positive cells were quantified by counting the number of cells in the superficial, middle, and deep layers, respectively. The TUNEL-positive cells were observed primarily in superficial proliferating chondrocytes, clustering chondrocytes, and deep-layer chondrocytes of OA cartilage. Few positive cells were seen in the proliferating chondrocytes in the middle layer. Positive reactions for caspase-3 and -9 were observed in chondrocytes in similar areas. Histological OA grade showed significant correlations with the mean populations of apoptotic chondrocytes (% apoptosis over the 3 areas. The populations of NT-positive cells (% NT over the same areas also showed significant correlation with OA grade. Positivity for caspase-3 closely correlated with the OA grade, % apoptosis and %NT. It was concluded that caspase-3 and -9 could play a role in NO-induced chondrocyte apoptosis in OA cartilage.

  11. Electromechanical Assessment of Human Knee Articular Cartilage with Compression-Induced Streaming Potentials.

    Science.gov (United States)

    Becher, Christoph; Ricklefs, Marcel; Willbold, Elmar; Hurschler, Christof; Abedian, Reza

    2016-01-01

    To assess the electromechanical properties of human knee articular cartilage with compression-induced streaming potentials for reliability among users and correlation with macroscopic and histological evaluation tools and sulfated glycosaminoglycan (sGAG) content. Streaming potentials are induced in cartilage in response to loading when mobile positive ions in the interstitial fluid temporarily move away from negatively charged proteoglycans. Streaming potential integrals (SPIs) were measured with an indentation probe on femoral condyles of 10 human knee specimens according to a standardized location scheme. Interobserver reliability was measured using an interclass correlation coefficient (ICC). The learning curves of 3 observers were evaluated by regression analysis. At each SPI measurement location the degradation level of the tissue was determined by means of the International Cartilage Repair Society (ICRS) score, Mankin score, and sGAG content. The computed ICC was 0.77 (0.70-0.83) indicating good to excellent linear agreement of SPI values among the 3 users. A significant positive linear correlation of the learning index values was observed for 2 of the 3 users. Statistically significant negative correlations between SPI and both ICRS and Mankin scores were observed (r = 0.502, P < 0.001, and r = 0.255, P = 0.02, respectively). No correlation was observed between SPI and sGAG content (r = 0.004, P = 0.973). SPI values may be used as a quantitative means of cartilage evaluation with sufficient reliability among users. Due to the significant learning curve, adequate training should be absolved before routine use of the technique.

  12. The normal human chondro-osseous junctional region: evidence for contact of uncalcified cartilage with subchondral bone and marrow spaces

    Directory of Open Access Journals (Sweden)

    Stoddart Robert W

    2006-06-01

    Full Text Available Abstract Background The chondro-osseous junctional region of diarthrodial joints is peculiarly complex and may be considered to consist of the deepest layer of non-calcified cartilage, the tidemark, the layer of calcified cartilage, a thin cement line (between the calcified cartilage and the subchondral bone and the subchondral bone. A detailed knowledge of the structure, function and pathophysiology of the normal chondro-osseous junction is essential for an understanding of the pathogenesis of osteoarthrosis. Methods Full thickness samples from human knee joints were processed and embedded in paraffin wax. One hundred serial sections (10 μm thick were taken from the chondro-osseous junctional region of a block from the medial tibial plateau of a normal joint. They were stained with haematoxylin and eosin and photographed. For a simple physical reconstruction images of each 10th sequential tissue section were printed and the areas of the photomicrographs containing the chondro-osseous junctional region were cut out and then overlaid so as to create a three-dimensional (3D model of this region. A 3D reconstruction was also made using computer modelling. Results Histochemical staining revealed some instances where prolongations of uncalcified cartilage, delineated by the tidemark, dipped into the calcified cartilage and, in places, abutted onto subchondral bone and marrow spaces. Small areas of uncalcified cartilage containing chondrocytes (virtual islands were seen, in two-dimensional (2D sections, to be apparently entombed in calcified matrix. The simple physical 3D reconstruction confirmed that these prolongations of uncalcified cartilage were continuous with the cartilage of zone IV and demonstrated that the virtual islands of uncalcified cartilage were cross-sections of these prolongations. The computer-generated 3D reconstructions clearly demonstrated that the uncalcified prolongations ran through the calcified cartilage to touch bone and

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

    Directory of Open Access Journals (Sweden)

    Haqqi Tariq M

    2011-08-01

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

  14. Phase II metabolism in human skin: skin explants show full coverage for glucuronidation, sulfation, N-acetylation, catechol methylation, and glutathione conjugation.

    Science.gov (United States)

    Manevski, Nenad; Swart, Piet; Balavenkatraman, Kamal Kumar; Bertschi, Barbara; Camenisch, Gian; Kretz, Olivier; Schiller, Hilmar; Walles, Markus; Ling, Barbara; Wettstein, Reto; Schaefer, Dirk J; Itin, Peter; Ashton-Chess, Joanna; Pognan, Francois; Wolf, Armin; Litherland, Karine

    2015-01-01

    Although skin is the largest organ of the human body, cutaneous drug metabolism is often overlooked, and existing experimental models are insufficiently validated. This proof-of-concept study investigated phase II biotransformation of 11 test substrates in fresh full-thickness human skin explants, a model containing all skin cell types. Results show that skin explants have significant capacity for glucuronidation, sulfation, N-acetylation, catechol methylation, and glutathione conjugation. Novel skin metabolites were identified, including acyl glucuronides of indomethacin and diclofenac, glucuronides of 17β-estradiol, N-acetylprocainamide, and methoxy derivatives of 4-nitrocatechol and 2,3-dihydroxynaphthalene. Measured activities for 10 μM substrate incubations spanned a 1000-fold: from the highest 4.758 pmol·mg skin(-1)·h(-1) for p-toluidine N-acetylation to the lowest 0.006 pmol·mg skin(-1)·h(-1) for 17β-estradiol 17-glucuronidation. Interindividual variability was 1.4- to 13.0-fold, the highest being 4-methylumbelliferone and diclofenac glucuronidation. Reaction rates were generally linear up to 4 hours, although 24-hour incubations enabled detection of metabolites in trace amounts. All reactions were unaffected by the inclusion of cosubstrates, and freezing of the fresh skin led to loss of glucuronidation activity. The predicted whole-skin intrinsic metabolic clearances were significantly lower compared with corresponding whole-liver intrinsic clearances, suggesting a relatively limited contribution of the skin to the body's total systemic phase II enzyme-mediated metabolic clearance. Nevertheless, the fresh full-thickness skin explants represent a suitable model to study cutaneous phase II metabolism not only in drug elimination but also in toxicity, as formation of acyl glucuronides and sulfate conjugates could play a role in skin adverse reactions.

  15. A superficial hyperechoic band in human articular cartilage on ultrasonography with histological correlation: preliminary observations

    Directory of Open Access Journals (Sweden)

    Tae Sun Han

    2015-04-01

    Full Text Available Purpose: To demonstrate the superficial hyperechoic band (SHEB in articular cartilage by using ultrasonography (US and to assess its correlation with histological images. Methods: In total, 47 regions of interest (ROIs were analyzed from six tibial osteochondral specimens (OCSs that were obtained after total knee arthroplasty. Ultrasonograms were obtained for each OCS. Then, matching histological sections from all specimens were obtained for comparison with the ultrasonograms. Two types of histological staining were used: Safranin-O stain (SO to identify glycosaminoglycans (GAG and Masson’s trichrome stain (MT to identify collagen. In step 1, two observers evaluated whether there was an SHEB in each ROI. In step 2, the two observers evaluated which histological staining method correlated better with the SHEB by using the ImageJ software. Results: In step 1 of the analysis, 20 out of 47 ROIs showed an SHEB (42.6%, kappa=0.579. Step 2 showed that the SHEB correlated significantly better with the topographical variation in stainability in SO staining, indicating the GAG distribution, than with MT staining, indicating the collagen distribution (P<0.05, kappa=0.722. Conclusion: The SHEB that is frequently seen in human articular cartilage on high-resolution US correlated better with variations in SO staining than with variations in MT staining. Thus, we suggest that a SHEB is predominantly related to changes in GAG. Identifying an SHEB by US is a promising method for assessing the thickness of articular cartilage or for monitoring early osteoarthritis.

  16. Analysis of the Mineral Composition of the Human Calcified Cartilage Zone

    Directory of Open Access Journals (Sweden)

    Ying Zhang, Fuyou Wang, Hongbo Tan, Guangxing Chen, Lin Guo, Liu Yang

    2012-01-01

    Full Text Available As the connecting tissue between the hyaline articular cartilage and the subchondral bone, calcified cartilage zone (CCZ plays a great role in the force transmission and materials diffusion. However, the questions that remain to be resolved are its mineral composition and organization. In this study, 40 healthy human knee specimens were harvested; first the CCZ was dissected and observed by Safranin O/fast green staining, then CCZ chemical characteristics were measured by using amino acid assay and X-ray diffraction. The percentage of dry weight of type II collagen as an organic compound of CCZ was 20.16% ± 0.96%, lower than that of the hyaline cartilage layer (61.39% ± 0.38%; the percentage of dry weight of hydroxyapatite as an inorganic compound was 65.09% ± 2.31%, less than that of subchondral bone (85.78% ± 3.42%. Our study provides the accurate data for the reconstruction of the CCZ in vitro and the elucidation of CCZ structure and function.

  17. Iron oxide labelling of human mesenchymal stem cells in collagen hydrogels for articular cartilage repair.

    Science.gov (United States)

    Heymer, Andrea; Haddad, Daniel; Weber, Meike; Gbureck, Uwe; Jakob, Peter M; Eulert, Jochen; Nöth, Ulrich

    2008-04-01

    For the development of new therapeutical cell-based strategies for articular cartilage repair, a reliable cell monitoring technique is required to track the cells in vivo non-invasively and repeatedly. We present a systematic and detailed study on the performance and biological impact of a simple and efficient labelling protocol for human mesenchymal stem cells (hMSCs). Commercially available very small superparamagnetic iron oxide particles (VSOPs) were used as magnetic resonance (MR) contrast agent. Iron uptake via endocytosis was confirmed histologically with prussian blue staining and quantified by mass spectrometry. Compared with unlabelled cells, VSOP-labelling did neither influence the viability nor the proliferation potential of hMSCs. Furthermore, iron incorporation did not affect hMSCs in undergoing adipogenic, osteogenic or chondrogenic differentiation, as demonstrated histologically and by gene expression analyses. The efficiency of the labelling protocol was assessed with high-resolution MR imaging at 11.7T. VSOP-labelled hMSCs were visualised in a collagen type I hydrogel, which is in clinical use for matrix-based articular cartilage repair. The presence of VSOP-labelled hMSCs was indicated by distinct hypointense spots in the MR images, as a result of iron specific loss of signal intensity. In summary, this labelling technique has great potential to visualise hMSCs and track their migration after transplantation for articular cartilage repair with MR imaging.

  18. Mechanical Stimulation Protocols of Human Derived Cells in Articular Cartilage Tissue Engineering - A Systematic Review.

    Science.gov (United States)

    Khozoee, Baktash; Mafi, Pouya; Mafi, Reza; Khan, Wasim S

    2017-01-01

    Mechanical stimulation is a key factor in articular cartilage generation and maintenance. Bioreactor systems have been designed and built in order to deliver specific types of mechanical stimulation. The focus has been twofold, applying a type of preconditioning in order to stimulate cell differentiation, and to simulate in vivo conditions in order to gain further insight into how cells respond to different stimulatory patterns. Due to the complex forces at work within joints, it is difficult to simulate mechanical conditions using a bioreactor. The aim of this review is to gain a deeper understanding of the complexities of mechanical stimulation protocols by comparing those employed in bioreactors in the context of tissue engineering for articular cartilage, and to consider their effects on cultured cells. Allied and Complementary Medicine 1985 to 2016, Ovid MEDLINE[R] 1946 to 2016, and Embase 1974 to 2016 were searched using key terms. Results were subject to inclusion and exclusion criteria, key findings summarised into a table and subsequently discussed. Based on this review it is overwhelmingly clear that mechanical stimulation leads to increased chondrogenic properties in the context of bioreactor articular cartilage tissue engineering using human cells. However, given the variability and lack of controlled factors between research articles, results are difficult to compare, and a standardised method of evaluating stimulation protocols proved challenging. With improved standardisation in mechanical stimulation protocol reporting, bioreactor design and building processes, along with a better understanding of joint behaviours, we hope to perform a meta-analysis on stimulation protocols and methods.

  19. Histological examinations of bone and cartilage in the axial skeleton of human triploidy fetuses.

    Science.gov (United States)

    Nolting, Dorrit; Hansen, Birgit Fischer; Keeling, Jean W; Kjaer, Inger

    2002-02-01

    The aim of this study was to examine histologically bone and cartilage in vertebral corpora of axial skeletons of eight human triploid fetuses, gestational ages 14-25 weeks, CRL 100-200 mm. The results were compared to earlier studies on vertebral development in trisomies 21, 18, 13, and to normal corpora development. After radiography in frontal and lateral projections, the vertebral column was sectioned into cervical, thoracic and lumbar segments, decalcified, dehydrated, and embedded in paraffin. The blocks were serially sectioned in the vertical plane and stained with Toluidine blue and Alcian blue/van Gieson. The radiographic characteristics of the vertebral corpora observed in frontal and lateral projection varied from small cleft vertebral corpora to fusions between the individual corpora. Histological examination of the vertebral corpora confirmed the abnormal pattern of ossification seen radiographically. As a new finding abnormal metachromasia of the ground substance was observed in the cartilage. Marked borderlines were registered in the cartilage between regions with differences in metachromasia. These borderlines were similar but more extensive than borderlines observed previously in trisomies 21, 18 and 13.

  20. Growth activity in human septal cartilage: age-dependent incorporation of labeled sulfate in different anatomic locations

    Energy Technology Data Exchange (ETDEWEB)

    Vetter, U.; Pirsig, W.; Heinze, E.

    1983-02-01

    Growth activity in different areas of human septal cartilage was measured by the in vitro incorporation of /sup 35/S-labeled NaSO/sub 4/ into chondroitin sulfate. Septal cartilage without perichondrium was obtained during rhinoplasty from 36 patients aged 6 to 35 years. It could be shown that the anterior free end of the septum displays high growth activity in all age groups. The supra-premaxillary area displayed its highest growth activity during prepuberty, showing thereafter a continuous decline during puberty and adulthood. A similar age-dependent pattern in growth activity was found in the caudal prolongation of the septal cartilage. No age-dependent variations could be detected in the posterior area of the septal cartilage.

  1. Effect of radiation on the growth of normal and malignant human oesophageal explant cultures pre-treated with bleomycin

    Energy Technology Data Exchange (ETDEWEB)

    Seymour, C.B.; Cusack, A.; Mothersill, C.; Hennessy, T.P.

    1988-05-01

    A method has been developed for testing the response of oesophageal explants from tumour and surrounding normal tissue in the same patient to chemotherapy and ..gamma..-radiation, singly and in combination. The test allows treatment combinations, time and order of administration of agents to the tissue to be accurately controlled. Cytotoxicity, determined by measuring the area of outgrowth from an explant 2 weeks after plating, is the most useful short-term end-point, although many other are possible. Results showing differential cytotoxicity of belomycin with and without radiation in squamous and adenocarcinoma of the oesophagus and surrounding normal tissue from the same patient indicate tumour cells are relatively resistant to radiation alone, low levels of belomycin with or without radiation preferentially spare tumour cells and high levels, in combination with any radiation dose tested, but not without radiation, spare normal cells and give a significantly high amount of relative tumour cell kill. Belomycin must be added to the cells just before or just after irradiation to obtain the normal-tissue sparing effect.

  2. Scaffold-free cartilage tissue engineering with a small population of human nasoseptal chondrocytes.

    Science.gov (United States)

    Chiu, Loraine L Y; To, William T H; Lee, John M; Waldman, Stephen D

    2017-03-01

    Cartilage tissue engineering is a promising approach to provide suitable materials for nasal reconstruction; however, it typically requires large numbers of cells. We have previously shown that a small number of chondrocytes cultivated within a continuous flow bioreactor can elicit substantial tissue growth, but translation to human chondrocytes is not trivial. Here, we aimed to demonstrate the application of the bioreactor to generate large-sized tissues from a small population of primary human nasoseptal chondrocytes. Experimental study. Chondrocytes were cultured in the bioreactor using different medium compositions, with varying amounts of serum and with or without growth factors. Resulting engineered tissues were analyzed for physical properties, biochemical composition, tissue microstructure, and protein localization. Bioreactor-cultivated constructs grown with serum and growth factors (basic fibroblast growth factor and transforming growth factor beta 2) had greater thickness, as well as DNA and glycosaminoglycan (GAG) contents, compared to low serum and no growth factor controls. These constructs also showed the most intense proteoglycan and collagen II staining. The combination of bioreactor conditions, serum, and growth factors allowed the generation of large, thick scaffold-free human cartilaginous tissues that resembled the native nasoseptal cartilage. There also may be implications for patient selection in future clinical applications of these engineered tissues because their GAG content decreased with donor age. NA. Laryngoscope, 127:E91-E99, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

  3. The acute effect of bipolar radiofrequency energy thermal chondroplasty on intrinsic biomechanical properties and thickness of chondromalacic human articular cartilage.

    Science.gov (United States)

    Dutcheshen, Nicholas; Maerz, Tristan; Rabban, Patrick; Haut, Roger C; Button, Keith D; Baker, Kevin C; Guettler, Joseph

    2012-08-01

    Radio frequency energy (RFE) thermal chondroplasty has been a widely-utilized method of cartilage debridement in the past. Little is known regarding its effect on tissue mechanics. This study investigated the acute biomechanical effects of bipolar RFE treatment on human chondromalacic cartilage. Articular cartilage specimens were extracted (n = 50) from femoral condyle samples of patients undergoing total knee arthroplasty. Chondromalacia was graded with the Outerbridge classification system. Tissue thicknesses were measured using a needle punch test. Specimens underwent pretreatment load-relaxation testing using a spherical indenter. Bipolar RFE treatment was applied for 45 s and the indentation protocol was repeated. Structural properties were derived from the force-time data. Mechanical properties were derived using a fibril-reinforced biphasic cartilage model. Statistics were performed using repeated measures ANOVA. Cartilage thickness decreased after RFE treatment from a mean of 2.61 mm to 2.20 mm in Grade II, II-III, and III specimens (P resistance to shear and tension could be compromised due to removal of the superficial layer and decreased fibril modulus, RFE treatment increases matrix modulus and decreases tissue permeability which may restore the load- bearing capacity of the cartilage.

  4. Cartilage tissue engineering of nasal septal chondrocyte-macroaggregates in human demineralized bone matrix.

    Science.gov (United States)

    Liese, Juliane; Marzahn, Ulrike; El Sayed, Karym; Pruss, Axel; Haisch, Andreas; Stoelzel, Katharina

    2013-06-01

    Tissue Engineering is an important method for generating cartilage tissue with isolated autologous cells and the support of biomaterials. In contrast to various gel-like biomaterials, human demineralized bone matrix (DBM) guarantees some biomechanical stability for an application in biomechanically loaded regions. The present study combined for the first time the method of seeding chondrocyte-macroaggregates in DBM for the purpose of cartilage tissue engineering. After isolating human nasal chondrocytes and creating a three-dimensional macroaggregate arrangement, the DBM was cultivated in vitro with the macroaggregates. The interaction of the cells within the DBM was analyzed with respect to cell differentiation and the inhibitory effects of chondrocyte proliferation. In contrast to chondrocyte-macroaggregates in the cell-DBM constructs, morphologically modified cells expressing type I collagen dominated. The redifferentiation of chondrocytes, characterized by the expression of type II collagen, was only found in low amounts in the cell-DBM constructs. Furthermore, caspase 3, a marker for apoptosis, was detected in the chondrocyte-DBM constructs. In another experimental setting, the vitality of chondrocytes as related to culture time and the amount of DBM was analyzed with the BrdU assay. Higher amounts of DBM tended to result in significantly higher proliferation rates of the cells within the first 48 h. After 96 h, the vitality decreased in a dose-dependent fashion. In conclusion, this study provides the proof of concept of chondrocyte-macroaggregates with DBM as an interesting method for the tissue engineering of cartilage. The as-yet insufficient redifferentiation of the chondrocytes and the sporadic initiation of apoptosis will require further investigations.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  6. Effects of radiofrequency energy on human chondromalacic cartilage: an assessment of insulation material properties.

    Science.gov (United States)

    Meyer, Marie L; Lu, Yan; Markel, Mark D

    2005-04-01

    The objective of this study was to establish guidelines for the selection of an insulation material used to surround the electrode of radiofrequency energy (RFE) probes used for thermal chondroplasty. These guidelines were established by identifying which insulation materials resulted in the least amount of chondrocyte death while smoothing the surface of chondromalacic cartilage. RFE causes electrolyte oscillation and molecular friction in the tissue to heat it and subsequently smooth the surface. Material properties investigated included the coefficient of thermal expansion (CTE), thermal conductivity (TC), and volume resistivity (VR). Fresh human chondromalacic cartilage samples of Outerbridge grades II and III were obtained from patients undergoing total knee arthroplasty. Stiffness measurements were taken pretreatment and posttreatment. RFE was applied to a 1-cm2 area for 15 s in a paintbrush treatment pattern. The insulation materials evaluated included Macor (decrease CTE, decrease TC, increase VR; in relation to CTE = 10 x 10(-6)/degrees C at 20 degrees C, TC = 3 W/mK, VR=1 x 10(14) ohm x cm), zirconia toughened alumina (ZTA) and 99.5% alumina (decrease CTE, increase TC, increase VR), aluminum nitride (decrease CTE, increase TC, decrease VR), Teflon (PTFE) (increase CTE, decrease TC, increase VR), partially stabilized zirconia (YTZP) (decrease CTE, decrease TC, decrease VR), and Ultem (increase CTE, decrease TC, decrease VR). There were no significant differences between pretreatment and posttreatment stiffness of the cartilage for any material investigated. Subjectively scored scanning electron microscopy (SEM) images revealed that the surfaces of all samples treated with RFE were relatively smooth with melted fronds. Prototype probes made with Macor, 99.5% alumina, and ZTA had TC or = 1 x 10(14) ohm x cm and resulted in a mean 37% less cell death than aluminum nitride or YTZP. There was no apparent relationship between CTE and the depth of chondrocyte

  7. Rotating three-dimensional dynamic culture of adult human bone marrow-derived cells for tissue engineering of hyaline cartilage.

    Science.gov (United States)

    Sakai, Shinsuke; Mishima, Hajime; Ishii, Tomoo; Akaogi, Hiroshi; Yoshioka, Tomokazu; Ohyabu, Yoshimi; Chang, Fei; Ochiai, Naoyuki; Uemura, Toshimasa

    2009-04-01

    The method of constructing cartilage tissue from bone marrow-derived cells in vitro is considered a valuable technique for hyaline cartilage regenerative medicine. Using a rotating wall vessel (RWV) bioreactor developed in a NASA space experiment, we attempted to efficiently construct hyaline cartilage tissue from human bone marrow-derived cells without using a scaffold. Bone marrow aspirates were obtained from the iliac crest of nine patients during orthopedic operation. After their proliferation in monolayer culture, the adherent cells were cultured in the RWV bioreactor with chondrogenic medium for 2 weeks. Cells from the same source were cultured in pellet culture as controls. Histological and immunohistological evaluations (collagen type I and II) and quantification of glycosaminoglycan were performed on formed tissues and compared. The engineered constructs obtained using the RWV bioreactor showed strong features of hyaline cartilage in terms of their morphology as determined by histological and immunohistological evaluations. The glycosaminoglycan contents per microg DNA of the tissues were 10.01 +/- 3.49 microg/microg DNA in the case of the RWV bioreactor and 6.27 +/- 3.41 microg/microg DNA in the case of the pellet culture, and their difference was significant. The RWV bioreactor could provide an excellent environment for three-dimensional cartilage tissue architecture that can promote the chondrogenic differentiation of adult human bone marrow-derived cells.

  8. Human articular chondrocytes express multiple gap junction proteins: differential expression of connexins in normal and osteoarthritic cartilage.

    Science.gov (United States)

    Mayan, Maria D; Carpintero-Fernandez, Paula; Gago-Fuentes, Raquel; Martinez-de-Ilarduya, Oskar; Wang, Hong-Zhang; Valiunas, Virginijus; Brink, Peter; Blanco, Francisco J

    2013-04-01

    Osteoarthritis (OA) is the most common joint disease and involves progressive degeneration of articular cartilage. The aim of this study was to investigate if chondrocytes from human articular cartilage express gap junction proteins called connexins (Cxs). We show that human chondrocytes in tissue express Cx43, Cx45, Cx32, and Cx46. We also find that primary chondrocytes from adults retain the capacity to form functional voltage-dependent gap junctions. Immunohistochemistry experiments in cartilage from OA patients revealed significantly elevated levels of Cx43 and Cx45 in the superficial zone and down through the next approximately 1000 μm of tissue. These zones corresponded with regions damaged in OA that also had high levels of proliferative cell nuclear antigen. An increased number of Cxs may help explain the increased proliferation of cells in clusters that finally lead to tissue homeostasis loss. Conversely, high levels of Cxs in OA cartilage reflect the increased number of adjacent cells in clusters that are able to interact directly by gap junctions as compared with hemichannels on single cells in normal cartilage. Our data provide strong evidence that OA patients have a loss of the usual ordered distribution of Cxs in the damaged zones and that the reductions in Cx43 levels are accompanied by the loss of correct Cx localization in the nondamaged areas. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  9. Phase contrast imaging X-ray computed tomography: quantitative characterization of human patellar cartilage matrix with topological and geometrical features

    Science.gov (United States)

    Nagarajan, Mahesh B.; Coan, Paola; Huber, Markus B.; Diemoz, Paul C.; Wismüller, Axel

    2014-03-01

    Current assessment of cartilage is primarily based on identification of indirect markers such as joint space narrowing and increased subchondral bone density on x-ray images. In this context, phase contrast CT imaging (PCI-CT) has recently emerged as a novel imaging technique that allows a direct examination of chondrocyte patterns and their correlation to osteoarthritis through visualization of cartilage soft tissue. This study investigates the use of topological and geometrical approaches for characterizing chondrocyte patterns in the radial zone of the knee cartilage matrix in the presence and absence of osteoarthritic damage. For this purpose, topological features derived from Minkowski Functionals and geometric features derived from the Scaling Index Method (SIM) were extracted from 842 regions of interest (ROI) annotated on PCI-CT images of healthy and osteoarthritic specimens of human patellar cartilage. The extracted features were then used in a machine learning task involving support vector regression to classify ROIs as healthy or osteoarthritic. Classification performance was evaluated using the area under the receiver operating characteristic (ROC) curve (AUC). The best classification performance was observed with high-dimensional geometrical feature vectors derived from SIM (0.95 ± 0.06) which outperformed all Minkowski Functionals (p analysis of chondrocyte patterns in human patellar cartilage matrix involving SIM-derived geometrical features can distinguish between healthy and osteoarthritic tissue with high accuracy.

  10. A low percentage of autologous serum can replace bovine serum to engineer human nasal cartilage

    Directory of Open Access Journals (Sweden)

    F Wolf

    2008-02-01

    Full Text Available For the generation of cell-based therapeutic products, it would be preferable to avoid the use of animal-derived components. Our study thus aimed at investigating the possibility to replace foetal bovine serum (FBS with autologous serum (AS for the engineering of cartilage grafts using expanded human nasal chondrocytes (HNC. HNC isolated from 7 donors were expanded in medium containing 10% FBS or AS at different concentrations (2%, 5% and 10% and cultured in pellets using serum-free medium or in Hyaff®-11 meshes using medium containing FBS or AS. Tissue forming capacity was assessed histologically (Safranin O, immunohistochemically (type II collagen and biochemically (glycosaminoglycans -GAG- and DNA. Differences among experimental groups were assessed by Mann Whitney tests. HNC expanded under the different serum conditions proliferated at comparable rates and generated cartilaginous pellets with similar histological appearance and amounts of GAG. Tissues generated by HNC from different donors cultured in Hyaff®-11 had variable quality, but the accumulated GAG amounts were comparable among the different serum conditions. Staining intensity for collagen type II was consistent with GAG deposition. Among the different serum conditions tested, the use of 2% AS resulted in the lowest variability in the GAG contents of generated tissues. In conclusion, a low percentage of AS can replace FBS both during the expansion and differentiation of HNC and reduce the variability in the quality of the resulting engineered cartilage tissues.

  11. Reference genes for normalization of gene expression studies in human osteoarthritic articular cartilage

    Directory of Open Access Journals (Sweden)

    Gomez-Reino Juan J

    2008-01-01

    Full Text Available Abstract Background Assessment of gene expression is an important component of osteoarthritis (OA research, greatly improved by the development of quantitative real-time PCR (qPCR. This technique requires normalization for precise results, yet no suitable reference genes have been identified in human articular cartilage. We have examined ten well-known reference genes to determine the most adequate for this application. Results Analyses of expression stability in cartilage from 10 patients with hip OA, 8 patients with knee OA and 10 controls without OA were done with classical statistical tests and the software programs geNorm and NormFinder. Results from the three methods of analysis were broadly concordant. Some of the commonly used reference genes, GAPDH, ACTB and 18S RNA, performed poorly in our analysis. In contrast, the rarely used TBP, RPL13A and B2M genes were the best. It was necessary to use together several of these three genes to obtain the best results. The specific combination depended, to some extent, on the type of samples being compared. Conclusion Our results provide a satisfactory set of previously unused reference genes for qPCR in hip and knee OA This confirms the need to evaluate the suitability of reference genes in every tissue and experimental situation before starting the quantitative assessment of gene expression by qPCR.

  12. Differences in Cartilage-Forming Capacity of Expanded Human Chondrocytes From Ear and Nose and Their Gene Expression Profiles

    NARCIS (Netherlands)

    Hellingman, C.A.; Verwiel, E.T.P.; Slagt, I.; Koevoet, W.; Poublon, R.M.L.; Nolst-Trenite, G.J.; de Jong, R.J.B.; Jahr, H.; van Osch, G.J.V.M.

    2011-01-01

    The aim of this study was to evaluate the potential of culture-expanded human auricular and nasoseptal chondrocytes as cell source for regeneration of stable cartilage and to analyze the differences in gene expression profile of expanded chondrocytes from these specific locations. Auricular chondroc

  13. Arthroscopic airbrush assisted cell implantation for cartilage repair in the knee: a controlled laboratory and human cadaveric study

    NARCIS (Netherlands)

    Windt, de T.S.; Vonk, L.A.; Buskermolen, J.K.; Visser, J.; Karperien, H.B.J.; Bleys, R.L.A.W.; Dhert, W.J.A.; Saris, D.B.F.

    2015-01-01

    Summary Objective The objective of this study was to investigate the feasibility of arthroscopic airbrush assisted cartilage repair. Methods An airbrush device (Baxter) was used to spray both human expanded osteoarthritic chondrocytes and choncrocytes with their pericellular matrix (chondrons) at 1

  14. Arthroscopic airbrush assisted cell implantation for cartilage repair in the knee : a controlled laboratory and human cadaveric study

    NARCIS (Netherlands)

    de Windt, T S; Vonk, L A; Buskermolen, J K; Visser, J.; Karperien, M; Bleys, R L A W; Dhert, W J A; Saris, D B F

    2015-01-01

    OBJECTIVE: The objective of this study was to investigate the feasibility of arthroscopic airbrush assisted cartilage repair. METHODS: An airbrush device (Baxter) was used to spray both human expanded osteoarthritic chondrocytes and choncrocytes with their pericellular matrix (chondrons) at 1 × 10(6

  15. Arthroscopic airbrush assisted cell implantation for cartilage repair in the knee : A controlled laboratory and human cadaveric study

    NARCIS (Netherlands)

    de Windt, T. S.; Vonk, L. A.; Buskermolen, J. K.; Visser, J.; Karperien, M.; Bleys, R. L A W; Dhert, W. J A; Saris, D. B F

    2015-01-01

    Objective: The objective of this study was to investigate the feasibility of arthroscopic airbrush assisted cartilage repair. Methods: An airbrush device (Baxter) was used to spray both human expanded osteoarthritic chondrocytes and choncrocytes with their pericellular matrix (chondrons) at 1×106cel

  16. OA cartilage derived chondrocytes encapsulated in poly(ethylene glycol) diacrylate (PEGDA) for the evaluation of cartilage restoration and apoptosis in an in vitro model.

    Science.gov (United States)

    Musumeci, G; Loreto, C; Carnazza, M L; Strehin, I; Elisseeff, J

    2011-10-01

    Osteoarthritis (OA) is characterized by cartilage attrition, subchondral bone remodeling, osteophyte formation and synovial inflammation. Perturbed homeostasis caused by inflammation, oxidative stress, mitochondrial dysfunction and proapoptotic/antiapoptotic dysregulation is known to impair chondrocyte survival in joint microenvironments and contribute to OA pathogenesis. However, the molecular mechanisms underlying the programmed cell death (apoptosis) of chondral cells are not yet well defined. The present study was conducted to evaluate apoptosis of chondrocytes from knee articular cartilage of patients with OA. The aim of this study was to investigate and compare the apoptosis through the expression of caspase-3 in tissue explants, in cells cultured in monolayer, and in cells encapsulated in a hydrogel (PEGDA) scaffold. Chondrocytes were also studied following cell isolation and encapsulation in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Specifically, articular cartilage specimens were assessed by histology (Hematoxlyn and Eosin) and histochemistry (Safranin-O and Alcian Blue). The effector of apoptosis caspase-3 was studied through immunohistochemistry, immunocytochemistry and immunofluorescence. DNA strand breaks were evaluated in freshly isolated chondrocytes from human OA cartilage using the TUNEL assay, and changes in nuclear morphology of apoptotic cells were detected by staining with Hoechst 33258. The results showed an increased expression of caspase-3 in tissue explants, in pre-confluent cells and after four passages in culture, and a decreased expression of caspase-3 comparable to control cartilage in cells encapsulated in hydrogels (PEGDA) after 5 weeks in culture. The freshly isolated chondrocytes were TUNEL positive. The chondrocytes after 5 weeks of culture in hydrogels (PEGDA) showed the formation of new hyaline cartilage with increased cell growth, cellular aggregations and extracellular matrix (ECM) production. This is of particular

  17. Radiocarbon dating reveals minimal collagen turnover in both healthy and osteoarthritic human cartilage

    DEFF Research Database (Denmark)

    Heinemeier, Katja M.; Schjerling, Peter; Heinemeier, Jan

    2016-01-01

    The poor regenerative capacity of articular cartilage presents a major clinical challenge and may relate to a limited turnover of the cartilage collagen matrix. However, the collagen turnover rate during life is not clear, and it is debated whether osteoarthritis (OA) can influence it. Using...... the carbon-14 ((14)C) bomb-pulse method, life-long replacement rates of collagen were measured in tibial plateau cartilage from 23 persons born between 1935 and1997 (15 and 8 persons with OA and healthy cartilage, respectively). The (14)C levels observed in cartilage collagen showed that, virtually......, no replacement of the collagen matrix happened after skeletal maturity and that neither OA nor tissue damage, per se, influenced collagen turnover. Regional differences in (14)C content across the joint surface showed that cartilage collagen located centrally on the joint surface is formed several years earlier...

  18. [Microdurimetric and biochemical study of human articular cartilage. Comparison of different joints].

    Science.gov (United States)

    Vignon, E; Arlot, M; Hartman, D; Noyer, D

    1980-12-01

    The micro-hardness and the density of fixed negative charges in cartilage of the shoulders, hips and knees of 6 subjects were studied. These two parameters were narrowly correlated. The resistance and proteoglycan concentration of the cartilage of the femoral head were greater than those of the knee and of the shoulder. They did not vary on each side. There is a significant correlation between the hardness of the cartilage of the femoral head and of the external femoral condyle. The histologically normal cartilage of the femoral head in arthrosis is at the lower limit of control values for hardness.

  19. MORPHOLOGY AND MORPHOMETRY OF ADULT HUMAN CRICOID CARTILAGE: A CADAVERIC STUDY IN NORTH INDIAN POPULATION

    Directory of Open Access Journals (Sweden)

    Rajan Kumar Singla

    2015-03-01

    Full Text Available Introduction: Out of different cartilages of larynx, cricoid is the strongest cartilage. It is the only cartilage which extends completely around the air passage. It is smaller but stronger and thicker than the thyroid cartilage. Though a lot of work has been done on thyroid cartilage it is not so for cricoid cartilage. This give us a impetus to design this study. Material and method: The material for present study comprised of 30 adult (M:F::25:5 apparently normal cadaveric larynges, obtained from the Anatomy Department of Govt. Medical College, Amritsar. Different morphometric diameters of the cricoid cartilage were measured with help of vernier caliper with least count 0.01 mm and these were noted on a predesigned proforma. All the data thus obtained was tabulated, analysed, scrutinized and compared with the earlier studies available in the literature. An attempt has been done to provide a base line data for this region. Result and Conclusion: Cricoid cartilage was oval in shape in all the specimens. Outer and inner transverse diameters and outer and inner anteroposterior diameters of cricoid cartilage were larger in males as compared to females. As we compare both diameters in males and females, outer transverse diameter was found to be larger than outer anteroposterior diameter, while inner anteroposterior diameter was larger than inner transverse diameter. Height and thickness of cricoid arch and lamina were observed to be larger in males as compared to females.

  20. In-vitro and in-vivo imaging of MMP activity in cartilage and joint injury

    Energy Technology Data Exchange (ETDEWEB)

    Fukui, Tomoaki; Tenborg, Elizabeth; Yik, Jasper H.N.; Haudenschild, Dominik R., E-mail: DRHaudenschild@ucdavis.edu

    2015-05-08

    Non-destructive detection of cartilage-degrading activities represents an advance in osteoarthritis (OA) research, with implications in studies of OA pathogenesis, progression, and intervention strategies. Matrix metalloproteinases (MMPs) are principal cartilage degrading enzymes that contribute to OA pathogenesis. MMPSense750 is an in-vivo fluorimetric imaging probe with the potential to continuously and non-invasively trace real-time MMP activities, but its use in OA-related research has not been reported. Our objective is to detect and characterize the early degradation activities shortly after cartilage or joint injury with MMPSense750. We determined the appropriate concentration, assay time, and linear range using various concentrations of recombinant MMPs as standards. We then quantified MMP activity from cartilage explants subjected to either mechanical injury or inflammatory cytokine treatment in-vitro. Finally, we performed in-vivo MMP imaging of a mouse model of post-traumatic OA. Our in-vitro results showed that the optimal assay time was highly dependent on the MMP enzyme. In cartilage explant culture media, mechanical impact or cytokine treatment increased MMP activity. Injured knees of mice showed significantly higher fluorescent signal than uninjured knees. We conclude that MMPSense750 detects human MMP activities and can be used for in-vitro study with cartilage, as well as in-vivo studies of knee injury, and can offering real-time insight into the degradative processes that occurring within the joint before structural changes become evident radiographically. - Highlights: • MMPSense750 is near-infrared fluorescent probe which can detect MMP activity. • MMPSense750 can detect human MMP-3, -9, and -13. • The reaction kinetics with MMPSense750 were different for the three MMPs. • MMPSense750 can visualized real time MMP activity in mouse injured knees. • MMPSense750 is convenient tool to evaluate real-time MMP activity non-invasively.

  1. In vitro and in vivo modulation of cartilage degradation by a standardized Centella asiatica fraction.

    NARCIS (Netherlands)

    Hartog, A.; Smit, H.F.; Kraan, P.M. van der; Hoijer, M.A.; Garssen, J.

    2009-01-01

    Osteoarthritis (OA) is a degenerative joint disease in which focal cartilage destruction is one of the primary features. The present study aims to evaluate the effect of a Centella asiatica fraction on in vitro and in vivo cartilage degradation. Bovine cartilage explants and bovine chondrocytes cult

  2. 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.

  3. Tensorial electrokinetics in articular cartilage.

    Science.gov (United States)

    Reynaud, Boris; Quinn, Thomas M

    2006-09-15

    Electrokinetic phenomena contribute to biomechanical functions of articular cartilage and underlie promising methods for early detection of osteoarthritic lesions. Although some transport properties, such as hydraulic permeability, are known to become anisotropic with compression, the direction-dependence of cartilage electrokinetic properties remains unknown. Electroosmosis experiments were therefore performed on adult bovine articular cartilage samples, whereby fluid flows were driven by electric currents in directions parallel and perpendicular to the articular surface of statically compressed explants. Magnitudes of electrokinetic coefficients decreased slightly with compression (from approximately -7.5 microL/As in the range of 0-20% compression to -6.0 microL/As in the 35-50% range) consistent with predictions of microstructure-based models of cartilage material properties. However, no significant dependence on direction of the electrokinetic coupling coefficient was detected, even for conditions where the hydraulic permeability tensor is known to be anisotropic. This contrast may also be interpreted using microstructure-based models, and provides insights into structure-function relationships in cartilage extracellular matrix and physical mediators of cell responses to tissue compression. Findings support the use of relatively simple isotropic modeling approaches for electrokinetic phenomena in cartilage and related materials, and indicate that measurement of electrokinetic properties may provide particularly robust means for clinical evaluation of cartilage matrix integrity.

  4. In vitro observation of cartilage-degeneration progression by Fourier-domain OCT

    Science.gov (United States)

    Marx, Ulrich; Schmitt, Robert; Nebelung, Sven; Tingart, Markus; Lüring, Christian; Rath, Björn

    2012-03-01

    Optical Coherence Tomography (OCT) as emerging clinical diagnostic imaging technology for dermatology and other semitransparent tissues has shown high potential in monitoring and evaluating the inner structure of articular cartilages. Since novel therapies for the limitation of cartilage degeneration in early stages of osteoarthritis are available, the early minimal invasive diagnosis of cartilage degradation is clinically essential for further treatment options. With the advancing performance and thus diagnostic opportunities of 3D-OCT devices, we carried out a systematic study by monitoring arthrotic alterations of porcine osteochondral explants that are mechanically induced under traumatic impaction. As for in-vitro tomographic imaging we utilized two OCT devices, a Thorlabs FD-OCT device with 92KHz A-scan rate and 1310nm as central wavelength and a self-developed FD-OCT device at 840nm central wavelength. This allows the comparison in image contrast and optical penetration of cartilage tissue between these two spectral bandwidths. Further we analyzed human biopsies of articular cartilages with various degrees of osteoarthritis. The 2D and 3D OCT tomograms are characterized qualitatively regarding the inner tissue structure and quantitatively regarding the tissue absorption parameters. Therefore, we are developing image processing algorithms for the automated monitoring of cartilage tissue. A scoring system for 3D-monitoring allows the characterization of the probe volume regarding the morphological structure and tissue compactness by processing the C - scan data.

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

    Science.gov (United States)

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

    2017-06-01

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

  6. Brief report: reconstruction of joint hyaline cartilage by autologous progenitor cells derived from ear elastic cartilage.

    Science.gov (United States)

    Mizuno, Mitsuru; Kobayashi, Shinji; Takebe, Takanori; Kan, Hiroomi; Yabuki, Yuichiro; Matsuzaki, Takahisa; Yoshikawa, Hiroshi Y; Nakabayashi, Seiichiro; Ik, Lee Jeong; Maegawa, Jiro; Taniguchi, Hideki

    2014-03-01

    In healthy joints, hyaline cartilage covering the joint surfaces of bones provides cushioning due to its unique mechanical properties. However, because of its limited regenerative capacity, age- and sports-related injuries to this tissue may lead to degenerative arthropathies, prompting researchers to investigate a variety of cell sources. We recently succeeded in isolating human cartilage progenitor cells from ear elastic cartilage. Human cartilage progenitor cells have high chondrogenic and proliferative potential to form elastic cartilage with long-term tissue maintenance. However, it is unknown whether ear-derived cartilage progenitor cells can be used to reconstruct hyaline cartilage, which has different mechanical and histological properties from elastic cartilage. In our efforts to develop foundational technologies for joint hyaline cartilage repair and reconstruction, we conducted this study to obtain an answer to this question. We created an experimental canine model of knee joint cartilage damage, transplanted ear-derived autologous cartilage progenitor cells. The reconstructed cartilage was rich in proteoglycans and showed unique histological characteristics similar to joint hyaline cartilage. In addition, mechanical properties of the reconstructed tissues were higher than those of ear cartilage and equal to those of joint hyaline cartilage. This study suggested that joint hyaline cartilage was reconstructed from ear-derived cartilage progenitor cells. It also demonstrated that ear-derived cartilage progenitor cells, which can be harvested by a minimally invasive method, would be useful for reconstructing joint hyaline cartilage in patients with degenerative arthropathies.

  7. A morphometric study of age-related changes in adult human epiglottis using quantitative digital analysis of cartilage calcification.

    Science.gov (United States)

    Kano, Mitsuhiro; Shimizu, Yoshinaka; Okayama, Keisuke; Igari, Toshirou; Kikuchi, Masayoshi

    2005-01-01

    The epiglottis plays an important role in deglutition in humans. The present study investigated age-related changes in the epiglottis using macroscopic and microscopic measurements. Epiglottic specimens from 281 Japanese adult cadavers (177 males, 104 females) were obtained. Specimens were divided into three groups according to age: group I: 20-39 years old (32 males, 26 females), group II: 50-69 years old (82 males, 36 females), and group III: 80-98 years old (63 males, 42 females). Width, height, and thickness were measured macroscopically. To evaluate the degree of calcium deposition, the calcium volume in digitalized von Kossa-stained sections was assessed using a quantitative analysis. An elemental analysis of the area detected with von Kossa staining was done using energy-dispersive X-ray fluorescence spectrometer (EDX). Measurements of the thickness and cell density in the superficial and deep layers of epiglottic cartilage were performed in horizontal histological sections. No significant differences in macroscopic width or height were found across the age groups in either sex. A series of three measurements in males was significantly larger than in females (p<0.05). The volume of the calcium deposit area was greater in males than in females (p<0.05) and was significantly increased in group III in males (p<0.05). The lower level of the epiglottic cartilage showed a greater calcium deposit area than the upper level. In the scanning image by line and surface analysis using EDX, the calcium deposit areas detected with von Kossa staining indicated a close association of calcium and phosphorus ions. The mean Ca/P molar ratio in the calcium deposit area was 1.32+/-0.12. Microscopic cartilage thickness increased significantly with age (p<0.05), and was greater in males than in females (p<0.05). Cartilage cell density in the superficial cartilage layer was higher than in the deep layer and was decreased in group III (p<0.05). Cartilage cell density was lower in

  8. Nanoparticles containing siRNA to silence CD4 and CCR5 reduce expression of these receptors and inhibit HIV-1 infection in human female reproductive tract tissue explants

    Directory of Open Access Journals (Sweden)

    Susan K. Eszterhas

    2011-09-01

    Full Text Available Human Immunodeficiency Virus-type 1 (HIV- 1 binds to CD4 and CCR5 receptors on target cells in the human female reproductive tract. We sought to determine whether reducing levels of messenger RNA (mRNA transcripts that encode these receptors in female reproductive tract cells could protect mucosal tissue explants from HIV- 1 infection. Explants prepared from the endometrium, endocervix, and ectocervix of hysterectomy tissues from HIV-1 sero-negative women were exposed to nanoparticles containing CD4- and CCR5-specific short-interfering RNA (siRNA sequences. Explants were then exposed two days later to HIV-1, and HIV-1 reverse transcripts were measured five days post-infection. Explants treated with nanoparticles containing CD4- and CCR5-specific siRNA showed reduced levels of CD4 and CCR5 transcripts, and significantly lower levels of HIV-1 reverse transcripts compared to those treated with an irrelevant siRNA. In female reproductive tract explants and in peripheral blood cell cultures, siRNA transfection induced the secretion of IFN-alpha (IFN-α, a potent antiviral cytokine. In female mice, murine-specific Cd4-siRNA nanoparticles instilled within the uterus significantly reduced murine Cd4 transcripts by day 3. Our findings demonstrate that siRNA nanoparticles reduce expression of HIV-1 infectivity receptors in human female reproductive tract tissues and also inhibit HIV-1 infection. Murine studies demonstrate that nanoparticles can penetrate the reproductive tract tissues in vivo and silence gene expression. The induction of IFN-α after siRNA transfection can potentially contribute to the antiviral effect. These findings support the therapeutic development of nanoparticles to deliver siRNA molecules to silence host cell receptors in the female reproductive tract as a novel microbicide to inhibit mucosal HIV-1 transmission.

  9. Application of stem-cell media to explant culture of human periosteum: An optimal approach for preparing osteogenic cell material

    Directory of Open Access Journals (Sweden)

    Kohya Uematsu

    2013-10-01

    Full Text Available As part of our clinical tests on bone regeneration using cultured periosteal sheets, here, we prepared cultured periosteal sheets in two types of stem-cell culture media, STK1 and STK3. Human periosteum was expanded either in 1% human serum–supplemented STK1 for 28 days, in 1% human serum–supplemented STK1 for 14 days followed by 1% human serum–supplemented STK3 for 14 days (1% human serum–supplemented STK1+3, or in 10% fetal bovine serum–supplemented Medium 199 for 28 days (control. Cultured periosteal sheet diameter and DNA content were significantly higher, and the multilayer structure was prominent in 1% human serum–supplemented STK1 and 1% human serum–supplemented STK1+3. The messenger RNA of osteoblastic markers was significantly upregulated in 1% human serum–supplemented STK1+3. Osteopontin-immunopositive staining and mineralization were evident across a wide area of the cultured periosteal sheet in 1% human serum–supplemented STK1+3. Subcutaneous implantation in nude mice following expansion in 1% human serum–supplemented STK1+3 produced the highest cultured periosteal sheet osteogenic activity. Expansion in 1% human serum–supplemented STK1+3 successfully induced cultured periosteal sheet growth while retaining osteogenic potential, and subsequent osteoblastic induction promoted the production of homogeneous cell material.

  10. Cartilage repair by human umbilical cord blood-derived mesenchymal stem cells with different hydrogels in a rat model.

    Science.gov (United States)

    Park, Yong-Beom; Song, Minjung; Lee, Choong-Hee; Kim, Jin-A; Ha, Chul-Won

    2015-11-01

    This study was carried out to assess the feasibility of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in articular cartilage repair and to further determine a suitable delivering hydrogel in a rat model. Critical sized full thickness cartilage defects were created. The hUCB-MSCs and three different hydrogel composites (hydrogel A; 4% hyaluronic acid/30% pluronic (1:1, v/v), hydrogel B; 4% hyaluronic acid, and hydrogel C; 4% hyaluronic acid/30% pluronic/chitosan (1:1:2, v/v)) were implanted into the experimental knee (right knee) and hydrogels without hUCB-MSCs were implanted into the control knee (left knee). Defects were evaluated after 8 weeks. The hUCB-MSCs with hydrogels composites resulted in a better repair as seen by gross and histological evaluation compared with hydrogels without hUCB-MSCs. Among the three different hydrogels, the 4% hyaluronic acid hydrogel composite (hydrogel B) showed the best result in cartilage repair as seen by the histological evaluation compared with the other hydrogel composites (hydrogel A and C). The results of this study suggest that hUCB-MSCs may be a promising cell source in combination with 4% hyaluronic acid hydrogels in the in vivo repair of cartilage defects.

  11. 3D Bioprinting Human Chondrocytes with Nanocellulose-Alginate Bioink for Cartilage Tissue Engineering Applications.

    Science.gov (United States)

    Markstedt, Kajsa; Mantas, Athanasios; Tournier, Ivan; Martínez Ávila, Héctor; Hägg, Daniel; Gatenholm, Paul

    2015-05-11

    The introduction of 3D bioprinting is expected to revolutionize the field of tissue engineering and regenerative medicine. The 3D bioprinter is able to dispense materials while moving in X, Y, and Z directions, which enables the engineering of complex structures from the bottom up. In this study, a bioink that combines the outstanding shear thinning properties of nanofibrillated cellulose (NFC) with the fast cross-linking ability of alginate was formulated for the 3D bioprinting of living soft tissue with cells. Printability was evaluated with concern to printer parameters and shape fidelity. The shear thinning behavior of the tested bioinks enabled printing of both 2D gridlike structures as well as 3D constructs. Furthermore, anatomically shaped cartilage structures, such as a human ear and sheep meniscus, were 3D printed using MRI and CT images as blueprints. Human chondrocytes bioprinted in the noncytotoxic, nanocellulose-based bioink exhibited a cell viability of 73% and 86% after 1 and 7 days of 3D culture, respectively. On the basis of these results, we can conclude that the nanocellulose-based bioink is a suitable hydrogel for 3D bioprinting with living cells. This study demonstrates the potential use of nanocellulose for 3D bioprinting of living tissues and organs.

  12. Paracrine Engineering of Human Explant-Derived Cardiac Stem Cells to Over-Express Stromal-Cell Derived Factor 1α Enhances Myocardial Repair.

    Science.gov (United States)

    Tilokee, Everad L; Latham, Nicholas; Jackson, Robyn; Mayfield, Audrey E; Ye, Bin; Mount, Seth; Lam, Buu-Khanh; Suuronen, Erik J; Ruel, Marc; Stewart, Duncan J; Davis, Darryl R

    2016-07-01

    First generation cardiac stem cell products provide indirect cardiac repair but variably produce key cardioprotective cytokines, such as stromal-cell derived factor 1α, which opens the prospect of maximizing up-front paracrine-mediated repair. The mesenchymal subpopulation within explant derived human cardiac stem cells underwent lentiviral mediated gene transfer of stromal-cell derived factor 1α. Unlike previous unsuccessful attempts to increase efficacy by boosting the paracrine signature of cardiac stem cells, cytokine profiling revealed that stromal-cell derived factor 1α over-expression prevented lv-mediated "loss of cytokines" through autocrine stimulation of CXCR4+ cardiac stem cells. Stromal-cell derived factor 1α enhanced angiogenesis and stem cell recruitment while priming cardiac stem cells to readily adopt a cardiac identity. As compared to injection with unmodified cardiac stem cells, transplant of stromal-cell derived factor 1α enhanced cells into immunodeficient mice improved myocardial function and angiogenesis while reducing scarring. Increases in myocardial stromal-cell derived factor 1α content paralleled reductions in myocyte apoptosis but did not influence long-term engraftment or the fate of transplanted cells. Transplantation of stromal-cell derived factor 1α transduced cardiac stem cells increased the generation of new myocytes, recruitment of bone marrow cells, new myocyte/vessel formation and the salvage of reversibly damaged myocardium to enhance cardiac repair after experimental infarction. Stem Cells 2016;34:1826-1835.

  13. A Simple Method to Isolate and Expand Human Umbilical Cord Derived Mesenchymal Stem Cells: Using Explant Method and Umbilical Cord Blood Serum.

    Science.gov (United States)

    Hassan, Ghmkin; Kasem, Issam; Soukkarieh, Chadi; Aljamali, Majd

    2017-08-31

    Mesenchymal stem cells (MSCs) are multipotent stem cells that can be isolated from umbilical cords and are therapeutically used because of their ability to differentiate into various types of cells, in addition to their immunosuppressive and anti-inflammatory properties. Fetal bovine serum (FBS), considered as the standard additive when isolating and culturing MSCs, has a major limitation related to its animal origin. Here, we employed a simple and economically efficient protocol to isolate MSCs from human umbilical cord tissues without using digestive enzymes and replacing FBS with umbilical cord blood serum (CBS). MSCs were isolated by culturing umbilical cord pieces in CBS or FBS supplemented media. Expansion and proliferation kinetics of cells isolated by explant method in the presence of either FBS or CBS were measured, with morphology and multi-differentiation potential of expanded cells characterized by flow cytometry, RT-PCR, and immunofluorescence. MSCs maintained morphology, immunophenotyping, multi-differentiation potential, and self-renewal ability, with better proliferation rates for cells cultured in CBS compared to FBS supplement media. We here present a simple, reliable and efficient method to isolate MSCs from umbilical cord tissues, where cells maintained proliferation, differentiation potential and immunophenotyping properties and could be efficiently expanded for clinical applications.

  14. First ex vivo study demonstrating that {sup 99m}Tc-NTP 15-5 radiotracer binds to human articular cartilage

    Energy Technology Data Exchange (ETDEWEB)

    Cachin, Florent; Culot, Damien [Jean Perrin Cancer Centre, Nuclear Medicine Department, Clermont-Ferrand (France); Universite d' Auvergne, UMR 990 INSERM, Clermont-Ferrand (France); Boisgard, Stephane [Gabriel Montpied University Hospital, Orthopaedic Surgery, Clermont-Ferrand (France); Universite d' Auvergne, UMR 990 INSERM, Clermont-Ferrand (France); Vidal, Aurelien; Auzeloux, Philippe; Madelmont, Jean-Claude; Chezal, Jean-Michel; Miot-Noirault, Elisabeth [Universite d' Auvergne, UMR 990 INSERM, Clermont-Ferrand (France); Filaire, Marc [Universite d' Auvergne, Anatomy Laboratory, Clermont-Ferrand (France); Askienazy, Serge [Cyclopharma Laboratoire, Saint-Beauzire (France)

    2011-11-15

    Preclinical data pointed to {sup 99m}Tc-NTP 15-5 as a good candidate for single photon emission computed tomography (SPECT) imaging of cartilaginous disease. We set out to investigate and quantify {sup 99m}Tc-NTP 15-5 ex vivo uptake by human articular cartilage relative to bone {sup 99m}Tc-hydroxymethylene diphosphonate (HMDP) radiotracer. Three osteoarthritic human tibial plateaux and four tibiofemoral joints were incubated with {sup 99m}Tc-NTP 15-5 and {sup 99m}Tc-HMDP for 2 h. Affinity of tracers for cartilage was determined by visual analysis of SPECT/CT acquisitions and measurement of cartilage to cortical bone uptake ratios. Cartilage to cortical bone uptake ratios were 3.90 {+-} 2.35 and 0.76 {+-} 0.24, respectively, for {sup 99m}Tc-NTP 15-5 and {sup 99m}Tc-HMDP radiotracers. Visual analysis of fused SPECT/CT slices showed selective, intense {sup 99m}Tc-NTP 15-5 accumulation in articular cartilage, whereas {sup 99m}Tc-HMDP binding was low. Interestingly, a cartilage defect visualized on CT was clearly associated with focal decreased uptake of {sup 99m}Tc-NTP 15-5. The tracer {sup 99m}Tc-NTP 15-5 is of major interest for human cartilage molecular imaging and could find clinical applications in osteoarthritis staging and monitoring. (orig.)

  15. Effects of the mesonephros and insulin-like growth factor I on chondrogenesis of limb explants.

    Science.gov (United States)

    Geduspan, J S; Solursh, M

    1993-04-01

    The mesonephros has been shown to have a growth-promoting influence in vivo on limb outgrowth. This influence has been studied in detail using an organ culture system. The results show that in the presence of the mesonephros limb explants formed larger cartilages than cultures without mesonephros. Furthermore, with mesonephros, morphology of the cartilages is comparable to that of skeletal elements in vivo while cartilages formed in cultures lacking mesonephros were amorphous. The mesonephric influence also promoted the formation of a well-organized extracellular matrix in the cartilage while cartilage in cultures without mesonephros formed an abnormal appearing matrix. Cartilage matrices in cultures with or without mesonephros were immunoreactive to type IX and type II collagens, cartilage proteoglycan PGH, and link protein although cultures lacking mesonephros had a very restricted distribution of type IX collagen immunoreactivity. Despite the different distribution of type IX collagen, long-form-type IX collagen transcripts appeared similar in both types of culture based on in situ hybridization. The mesonephric effect on limb explants could be partially duplicated by the addition of insulin-like growth factor I (IGF-I) to cultures without mesonephros. Furthermore, the mesonephric influence on cartilage growth and morphological differentiation could be blocked by the addition of a blocking antibody to IGF-I to cultures with mesonephros. The results support the hypothesis that IGF-I is one of the growth factors produced by the mesonephros which may play a role in early limb development and chondrogenesis.

  16. High seeding density of human chondrocytes in agarose produces tissue-engineered cartilage approaching native mechanical and biochemical properties.

    Science.gov (United States)

    Cigan, Alexander D; Roach, Brendan L; Nims, Robert J; Tan, Andrea R; Albro, Michael B; Stoker, Aaron M; Cook, James L; Vunjak-Novakovic, Gordana; Hung, Clark T; Ateshian, Gerard A

    2016-06-14

    Animal cells have served as highly controllable model systems for furthering cartilage tissue engineering practices in pursuit of treating osteoarthritis. Although successful strategies for animal cells must ultimately be adapted to human cells to be clinically relevant, human chondrocytes are rarely employed in such studies. In this study, we evaluated the applicability of culture techniques established for juvenile bovine and adult canine chondrocytes to human chondrocytes obtained from fresh or expired osteochondral allografts. Human chondrocytes were expanded and encapsulated in 2% agarose scaffolds measuring ∅3-4mm×2.3mm, with cell seeding densities ranging from 15 to 90×10(6)cells/mL. Subsets of constructs were subjected to transient or sustained TGF-β treatment, or provided channels to enhance nutrient transport. Human cartilaginous constructs physically resembled native human cartilage, and reached compressive Young's moduli of up to ~250kPa (corresponding to the low end of ranges reported for native knee cartilage), dynamic moduli of ~950kPa (0.01Hz), and contained 5.7% wet weight (%/ww) of glycosaminoglycans (≥ native levels) and 1.5%/ww collagen. We found that the initial seeding density had pronounced effects on tissue outcomes, with high cell seeding densities significantly increasing nearly all measured properties. Transient TGF-β treatment was ineffective for adult human cells, and tissue construct properties plateaued or declined beyond 28 days of culture. Finally, nutrient channels improved construct mechanical properties, presumably due to enhanced rates of mass transport. These results demonstrate that our previously established culture system can be successfully translated to human chondrocytes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Use of non-degenerate human osteochondral tissue and confocal laser scanning microscopy for the study of chondrocyte death at cartilage surgery

    Directory of Open Access Journals (Sweden)

    Huntley J. S.

    2005-02-01

    Full Text Available Although autologous osteochondral grafting has been widely applied in humans, most in vitro work has been on animal models. The aims of this study were to: (i elaborate a full thickness human femoral condylar model using discard material from knee arthroplasty resections, and (ii use this model to assess chondrocyte viability in response to surgical trauma. Homogeneous regions of human lateral femoral condyle bone-cartilage were procured from knee arthroplasty resections. These were graded prospectively, firstly by visual inspection, and then by confocal laser scanning microscopy (CLSM. Samples were subjected to tests of tissue hydration, including analysis of water content and swelling after excision from underlying bone. Surgical cuts were made in explants that were macroscopically and microscopically normal (i.e. Grade 0. Associated margins of death were assessed from both transverse and surface perspectives. Thirty-nine samples were obtained from anterior and distal femoral cuts (16 knees from 13 patients for (1 macroscopic grading, (2 microscopic analysis, (3 analyses of water content as cut and on re-equilibration after excision from bone. Thirteen were Grade 0 on macroscopic viewing - however one showed fibrillation on microscopy and was therefore reassigned Grade 1. Grade 0 tissue had a water content of 73.8±0.38%, in agreement with control values from the literature. Tissues of Grades 2 and 3 were significantly (P=0.03, and P=0.004 more hydrated (76.0±0.59%, 76.7±0.99% than Grade 0 tissue. Grade 0 tissue from the anterior cut did not swell significantly following excision from subchondral bone. However Grade 0 tissue from the distal cut showed a small but statistically significant (P=0.019 increase in water content (1.68±0.39% following excision. With increasing grade there was increased tendency to swell off the bone (P<0.0001. Transverse imaging showed that the Acufex MP surgical harvester caused a greater margin of cell death

  18. Allogeneic Mesenchymal Stem Cells Stimulate Cartilage Regeneration and Are Safe for Single-Stage Cartilage Repair in Humans upon Mixture with Recycled Autologous Chondrons

    NARCIS (Netherlands)

    de Windt, Tommy S; Vonk, Lucienne A; Slaper-Cortenbach, Ineke C M; van den Broek, Marcel P H; Nizak, Razmara; van Rijen, Mattie H P; de Weger, Roel A; Dhert, Wouter J A; Saris, Daniel B F

    Traditionally, mesenchymal stem cells (MSCs) isolated from adult bone marrow were described as being capable of differentiating to various lineages including cartilage. Despite increasing interest in these MSCs, concerns regarding their safety, in vivo behavior and clinical effectiveness have

  19. Growth plate regulation and osteochondroma formation: insights from tracing proteoglycans in zebrafish models and human cartilage.

    Science.gov (United States)

    de Andrea, Carlos E; Prins, Frans A; Wiweger, Malgorzata I; Hogendoorn, Pancras C W

    2011-06-01

    Proteoglycans are secreted into the extracellular matrix of virtually all cell types and function in several cellular processes. They consist of a core protein onto which glycosaminoglycans (e.g., heparan or chondroitin sulphates), are attached. Proteoglycans are important modulators of gradient formation and signal transduction. Impaired biosynthesis of heparan sulphate glycosaminoglycans causes osteochondroma, the most common bone tumour to occur during adolescence. Cytochemical staining with positively charged dyes (e.g., polyethyleneimine-PEI) allows, visualisation of proteoglycans and provides a detailed description of how proteoglycans are distributed throughout the cartilage matrix. PEI staining was studied by electron and reflection contrast microscopy in human growth plates, osteochondromas and five different proteoglycan-deficient zebrafish mutants displaying one of the following skeletal phenotypes: dackel (dak/ext2), lacking heparan sulphate and identified as a model for human multiple osteochondromas; hi307 (β3gat3), deficient for most glycosaminoglycans; pinscher (pic/slc35b2), presenting with defective sulphation of glycosaminoglycans; hi954 (uxs1), lacking most glycosaminoglycans; and knypek (kny/gpc4), missing the protein core of the glypican-4 proteoglycan. The panel of genetically well-characterized proteoglycan-deficient zebrafish mutants serves as a convincing and comprehensive study model to investigate proteoglycan distribution and the relation of this distribution to the model mutation status. They also provide insight into the distributions and gradients that can be expected in the human homologue. Human growth plate, wild-type zebrafish and fish mutants with mild proteoglycan defects (hi307 and kny) displayed proteoglycans distributed in a gradient throughout the matrix. Although the mutants pic and hi954, which had severely impaired proteoglycan biosynthesis, showed no PEI staining, dak mutants demonstrated reduced PEI staining and no

  20. MiniCD4 microbicide prevents HIV infection of human mucosal explants and vaginal transmission of SHIV(162P3 in cynomolgus macaques.

    Directory of Open Access Journals (Sweden)

    Nathalie Dereuddre-Bosquet

    Full Text Available In complement to an effective vaccine, development of potent anti-HIV microbicides remains an important priority. We have previously shown that the miniCD4 M48U1, a functional mimetic of sCD4 presented on a 27 amino-acid stable scaffold, inhibits a broad range of HIV-1 isolates at sub-nanomolar concentrations in cellular models. Here, we report that M48U1 inhibits efficiently HIV-1(Ba-L in human mucosal explants of cervical and colorectal tissues. In vivo efficacy of M48U1 was evaluated in nonhuman primate (NHP model of mucosal challenge with SHIV(162P3 after assessing pharmacokinetics and pharmacodynamics of a miniCD4 gel formulation in sexually matured female cynomolgus macaques. Among 12 females, half were treated with hydroxyethylcellulose-based gel (control, the other half received the same gel containing 3 mg/g of M48U1, one hour before vaginal route challenge with 10 AID(50 of SHIV(162P3. All control animals were infected with a peak plasma viral load of 10(5-10(6 viral RNA (vRNA copies per mL. In animals treated with miniCD4, 5 out of 6 were fully protected from acquisition of infection, as assessed by qRT-PCR for vRNA detection in plasma, qPCR for viral DNA detection in PBMC and lymph node cells. The only infected animal in this group had a delayed peak of viremia of one week. These results demonstrate that M48U1 miniCD4 acts in vivo as a potent entry inhibitor, which may be considered in microbicide developments.

  1. Imaging of irradiated human costal cartilage birefringence by PS-OCT

    Energy Technology Data Exchange (ETDEWEB)

    Martinho Junior, Antonio C.; Freitas, Anderson Z.; Santin, Stefany P.; Soares, Fernando A.N.; Mosca, Rodrigo C.; Bringel, Fabiana A.; Mathor, Monica B., E-mail: freitas.az@ipen.b, E-mail: rmosca@usp.b, E-mail: mathor@ipen.b [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

    2011-07-01

    Sterilization by ionizing radiation is a technique used for tissue banks around the world to avoid transmission of infectious diseases by human allografts. However, high doses of ionizing radiation may cause undesirable changes in tissue structure, decreasing its mechanical properties, for example. Optical Coherence Tomography (OCT) is a non destructive, non ionizing and real time method to investigate biological tissues without promote any change in tissue structure. Polarization Sensitive Optical Coherence Tomography (PS-OCT) is an OCT technique that combines polarimetry with low coherence reflectometry to provide depth resolved measurements from birefringent structures as collagen. Costal cartilages from 15 cadaveric donors were preserved in high concentration glycerol and each individual sample was divided in 6 fragments. One of them was kept as a control group and the others were irradiated with gamma radiation from a Co-60 source with doses of 15, 25, 50, 75 and 100 kGy. OCT and PS-OCT images of the same region of the samples were obtained from a device OCS 1300 SS (Thorlabs, USA) with a coupling polarization module PSOCT 1300 (Thorlabs, USA). According with our results, birefringence may be visualized in all test groups as well in the control group, suggesting that sterilization by ionizing radiation does not affect the collagen structure significantly to cause total loss of birefringence, even if high doses as 75 and 100 kGy are used. The next step of our work is to develop a new method to quantify the birefringence using the optical properties of the tissue. (author)

  2. Characterization of human primary chondrocytes of osteoarthritic cartilage at varying severity

    Institute of Scientific and Technical Information of China (English)

    YIN Jing; YANG Zheng; CAO Yong-ping; GE Zi-gang

    2011-01-01

    Background There is a difficulty in evaluating the in vivo functionality of individual chondrocytes,and there is much heterogeneity among cartilage affected by osteoarthritis (OA).In this study,in vitro cultured chondrocytes harvested from varying stages of degeneration were studied as a projective model to further understand the pathogenesis of osteoarthritis.Methods Cartilage of varying degeneration of end-stage OA was harvested,while cell yield and matrix glycosaminoglycan (GAG) content were measured.Cell morphology,proliferation,and gene expression of collagen type Ⅰ,Ⅱ,and Ⅹ,aggrecan,matrix metalloproteinase 13 (MMP-13),and ADAMTS5 of the acquired chondrocytes were measured during subsequent in vitro culture.Results Both the number of cells and the GAG content increased with increasing severity of OA.Cell spreading area increased and gradually showed spindle-like morphology during in vitro culture.Gene expression of collagen type Ⅱ,collagen type X as well as GAG decreased with severity of cartilage degeneration,while expression of collagen type Ⅰ increased.Expression of MMP-13 increased with severity of cartilage degeneration,while expression of ADAMTS-5 remained stable.Expression of collagen type Ⅱ,X,GAG,and MMP-13 substantially decreased with in vitro culture.Expression of collagen type Ⅰ increased with in vitro cultures,while expression of ADAMTS 5 remained stable.Conclusions Expression of functional genes such as collagen type Ⅱ and GAG decreased during severe degeneration of OA cartilage and in vitro dedifferentiation.Gene expression of collagen Ⅰ and MMP-13 increased with severity of cartilage degeneration.

  3. Conserving Cartilage In Microtia Repair: The Modular Component Assembly Approach To Rebuilding A Human Ear

    Science.gov (United States)

    Gandy, Jessica R.; Lemieux, Bryan; Foulad, Allen; Wong, Brian J.F.

    2016-01-01

    Objectives Current methods of microtia repair include carving an auricular framework from the costal synchondrosis. This requires considerable skill and may create a substantial donor site defect. Here, we present a modular component assembly (MCA) approach that minimizes the procedural difficulty and reduces the amount of cartilage to a single rib. Study Design Ex vivo study and survey Methods A single porcine rib was sectioned into multiple slices using a cartilage guillotine, cut into components outlined by 3D-printed templates, and assembled into an auricular scaffold. Electromechanical reshaping (EMR) was used to bend cartilage slices for creation of the helical rim. Chondrocyte viability was confirmed using confocal imaging. Ten surgeons reviewed the scaffold constructed with the MCA approach to evaluate aesthetics, relative stability, and clinical feasibility. Results An auricular framework with projection and curvature was fashioned from one rib. Surgeons found the MCA scaffold to meet minimal aesthetic and anatomic acceptability. When embedded under a covering, the region of the helix and anti-helix of the scaffold scored significantly higher on the assessment survey than that of an embedded alloplast implant (t-value=0.01). Otherwise, no difference was found between the embedded MCA and alloplast implants (t-value >0.05). EMR treated cartilage was found to be viable. Conclusion This study demonstrates that one rib can be used to create an aesthetic and durable framework for microtia repair. Precise assembly and the ability to obtain thin, uniform slices of cartilage were essential. This cartilage-sparing MCA approach may be an alternative to classic techniques. PMID:26720326

  4. Expression of BMP-receptor type 1A correlates with progress of osteoarthritis in human knee joints with focal cartilage lesions

    DEFF Research Database (Denmark)

    Schmal, Hagen; Pilz, Ingo H; Mehlhorn, Alexander T;

    2012-01-01

    was significantly higher in cartilage biopsies taken in type 3 lesions with intact subchondral layer compared with type 4 defects (P ...BACKGROUND AIMS: Bone morphogenetic protein-2 (BMP-2) and its receptor type 1A (BMPR-1A) play significant roles in cartilage metabolism. The aim of this study was to evaluate a possible correlation between intra-articular expression of these proteins and the degree of osteoarthritis (OA) in human...... knees. METHODS: Biopsies of synovia and debrided cartilage were taken in 15 patients undergoing autologous chondrocyte implantation. Expression of BMP-2 and BMPR-1A was evaluated semi-quantitatively by immunohistologic staining. These data were complemented by grading of cartilage lesions according...

  5. Age-related accumulation of Maillard reaction products in human articular cartilage collagen

    NARCIS (Netherlands)

    Verzijl, N.; Groot, J. de; Oldehinkel, E.; Bank, R.A.; Thorpe, S.R.; Baynes, J.W.; Bayliss, M.T.; Bijlsma, J.W.J.; Lafeber, F.P.J.G.; TeKoppele, J.M.

    2000-01-01

    Non-enzymic modification of tissue proteins by reducing sugars, the so-called Maillard reaction, is a prominent feature of aging. In articular cartilage, relatively high levels of the advanced glycation end product (AGE) pentosidine accumulate with age. Higher pentosidine levels have been associated

  6. Differential Gene Expression in Explanted Human Retinal Pigment Epithelial Cells 12-Hours Post-Exposure to 532 nm, 120 ps Pulsed Laser Light

    Science.gov (United States)

    2004-04-01

    RPE tissue donor was a 41 year old Caucasian, blue eyed, female that died of cancer. No ocular pathologies were noted. Explant preparation: Globes...be discussed below. However, other EST’s that were differentially regulated functions remain unknown thus remain fertile ground for future exploration...The induction of the chaperonin, as well as the induction of ubiquinol - cytochrome C reductase hinge protein [1.8] and its binding protein [2.7

  7. Quantitative in vivo CT arthrography of the human osteoarthritic knee to estimate cartilage sulphated glycosaminoglycan content : correlation with ex-vivo reference standards

    NARCIS (Netherlands)

    van Tiel, J; Siebelt, M; Reijman, M; Bos, P.K.; Waarsing, J H; Zuurmond, A-M; Nasserinejad, K; van Osch, G J V M; Verhaar, J A N; Krestin, G P; Weinans, H; Oei, E H G

    OBJECTIVE: Recently, computed tomography arthrography (CTa) was introduced as quantitative imaging biomarker to estimate cartilage sulphated glycosaminoglycan (sGAG) content in human cadaveric knees. Our aim was to assess the correlation between in vivo CTa in human osteoarthritis (OA) knees and ex

  8. Quantitative in vivo CT arthrography of the human osteoarthritic knee to estimate cartilage sulphated glycosaminoglycan content: correlation with ex-vivo reference standards

    NARCIS (Netherlands)

    Tiel, J. van; Siebelt, M.; Reijman, M.; Bos, P.K.; Waarsing, J.H.; Zuurmond, A.M.; Nasserinejad, K.; Osch, G.J.V.M. van; Verhaar, J.A.N.; Krestin, G.P.; Weinans, H.; Oei, E.H.G.

    2016-01-01

    Objective. Recently, computed tomography arthrography (CTa) was introduced as quantitative imaging biomarker to estimate cartilage sulphated glycosaminoglycan (sGAG) content in human cadaveric knees. Our aim was to assess the correlation between in vivo CTa in human osteoarthritis (OA) knees and ex

  9. Prefabrication of 3D cartilage contructs: towards a tissue engineered auricle--a model tested in rabbits.

    Directory of Open Access Journals (Sweden)

    Achim von Bomhard

    Full Text Available The reconstruction of an auricle for congenital deformity or following trauma remains one of the greatest challenges in reconstructive surgery. Tissue-engineered (TE three-dimensional (3D cartilage constructs have proven to be a promising option, but problems remain with regard to cell vitality in large cell constructs. The supply of nutrients and oxygen is limited because cultured cartilage is not vascular integrated due to missing perichondrium. The consequence is necrosis and thus a loss of form stability. The micro-surgical implantation of an arteriovenous loop represents a reliable technology for neovascularization, and thus vascular integration, of three-dimensional (3D cultivated cell constructs. Auricular cartilage biopsies were obtained from 15 rabbits and seeded in 3D scaffolds made from polycaprolactone-based polyurethane in the shape and size of a human auricle. These cartilage cell constructs were implanted subcutaneously into a skin flap (15 × 8 cm and neovascularized by means of vascular loops implanted micro-surgically. They were then totally enhanced as 3D tissue and freely re-implanted in-situ through microsurgery. Neovascularization in the prefabricated flap and cultured cartilage construct was analyzed by microangiography. After explantation, the specimens were examined by histological and immunohistochemical methods. Cultivated 3D cartilage cell constructs with implanted vascular pedicle promoted the formation of engineered cartilaginous tissue within the scaffold in vivo. The auricles contained cartilage-specific extracellular matrix (ECM components, such as GAGs and collagen even in the center oft the constructs. In contrast, in cultivated 3D cartilage cell constructs without vascular pedicle, ECM distribution was only detectable on the surface compared to constructs with vascular pedicle. We demonstrated, that the 3D flaps could be freely transplanted. On a microangiographic level it was evident that all the skin flaps

  10. Hypoxia inhibits hypertrophic differentiation and endochondral ossification in explanted tibiae.

    Directory of Open Access Journals (Sweden)

    Jeroen C H Leijten

    Full Text Available PURPOSE: Hypertrophic differentiation of growth plate chondrocytes induces angiogenesis which alleviates hypoxia normally present in cartilage. In the current study, we aim to determine whether alleviation of hypoxia is merely a downstream effect of hypertrophic differentiation as previously described or whether alleviation of hypoxia and consequent changes in oxygen tension mediated signaling events also plays an active role in regulating the hypertrophic differentiation process itself. MATERIALS AND METHODS: Fetal mouse tibiae (E17.5 explants were cultured up to 21 days under normoxic or hypoxic conditions (21% and 2.5% oxygen respectively. Tibiae were analyzed on growth kinetics, histology, gene expression and protein secretion. RESULTS: The oxygen level had a strong influence on the development of explanted fetal tibiae. Compared to hypoxia, normoxia increased the length of the tibiae, length of the hypertrophic zone, calcification of the cartilage and mRNA levels of hypertrophic differentiation-related genes e.g. MMP9, MMP13, RUNX2, COL10A1 and ALPL. Compared to normoxia, hypoxia increased the size of the cartilaginous epiphysis, length of the resting zone, calcification of the bone and mRNA levels of hyaline cartilage-related genes e.g. ACAN, COL2A1 and SOX9. Additionally, hypoxia enhanced the mRNA and protein expression of the secreted articular cartilage markers GREM1, FRZB and DKK1, which are able to inhibit hypertrophic differentiation. CONCLUSIONS: Collectively our data suggests that oxygen levels play an active role in the regulation of hypertrophic differentiation of hyaline chondrocytes. Normoxia stimulates hypertrophic differentiation evidenced by the expression of hypertrophic differentiation related genes. In contrast, hypoxia suppresses hypertrophic differentiation of chondrocytes, which might be at least partially explained by the induction of GREM1, FRZB and DKK1 expression.

  11. Biomaterial and Cell Based Cartilage Repair

    NARCIS (Netherlands)

    Zhao, X

    2015-01-01

    Injuries to human native cartilage tissue are particularly troublesome because cartilage has little ability to heal or regenerate itself. The reconstruction, repair, and regeneration of cartilage tissue continue to be one of the greatest clinical challenges, especially in orthopaedic and plastic sur

  12. Biomaterial and Cell Based Cartilage Repair

    NARCIS (Netherlands)

    Zhao, X

    2015-01-01

    Injuries to human native cartilage tissue are particularly troublesome because cartilage has little ability to heal or regenerate itself. The reconstruction, repair, and regeneration of cartilage tissue continue to be one of the greatest clinical challenges, especially in orthopaedic and plastic

  13. 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.

  14. Regeneration of Cartilage in Human Knee Osteoarthritis with Autologous Adipose Tissue-Derived Stem Cells and Autologous Extracellular Matrix

    Directory of Open Access Journals (Sweden)

    Jaewoo Pak

    2016-08-01

    Full Text Available This clinical case series demonstrates that percutaneous injections of autologous adipose tissue-derived stem cells (ADSCs and homogenized extracellular matrix (ECM in the form of adipose stromal vascular fraction (SVF, along with hyaluronic acid (HA and platelet-rich plasma (PRP activated by calcium chloride, could regenerate cartilage-like tissue in human knee osteoarthritis (OA patients. Autologous lipoaspirates were obtained from adipose tissue of the abdominal origin. Afterward, the lipoaspirates were minced to homogenize the ECM. These homogenized lipoaspirates were then mixed with collagenase and incubated. The resulting mixture of ADSCs and ECM in the form of SVF was injected, along with HA and PRP activated by calcium chloride, into knees of three Korean patients with OA. The same affected knees were reinjected weekly with additional PRP activated by calcium chloride for 3 weeks. Pretreatment and post-treatment magnetic resonance imaging (MRI data, functional rating index, range of motion (ROM, and pain score data were then analyzed. All patients' MRI data showed cartilage-like tissue regeneration. Along with MRI evidence, the measured physical therapy outcomes in terms of ROM, subjective pain, and functional status were all improved. This study demonstrates that percutaneous injection of ADSCs with ECM contained in autologous adipose SVF, in conjunction with HA and PRP activated by calcium chloride, is a safe and potentially effective minimally invasive therapy for OA of human knees.

  15. Human adult chondrocytes express hepatocyte growth factor (HGF) isoforms but not HgF: potential implication of osteoblasts on the presence of HGF in cartilage.

    Science.gov (United States)

    Guévremont, Melanie; Martel-Pelletier, Johanne; Massicotte, Frédéric; Tardif, Ginette; Pelletier, Jean-Pierre; Ranger, Pierre; Lajeunesse, Daniel; Reboul, Pascal

    2003-06-01

    HGF is increased in human OA cartilage, possibly from Ob's. RT-PCR shows HGF isoforms are differently regulated between chondrocytes and Ob. A paracrine cross-talk between subchondral bone and cartilage may occur during OA. Recently, hepatocyte growth factor (HGF) has been identified by immunohistochemistry in cartilage and more particularly in the deep zone of human osteoarthritic (OA) cartilage. By investigating HGF expression in cartilage, we found that chondrocytes did not express HGF; however, they expressed the two truncated isoforms, namely HGF/NK1 and HGF/NK2. Because the only other cells localized near the deep zone are osteoblasts from the subchondral bone plate, we hypothesized that they were expressing HGF. Indeed, we found that HGF was synthesized by osteoblasts from the subchondral bone plate. Moreover, OA osteoblasts produced five times more HGF than normal osteoblasts and almost no HGF/NK1, unlike normal osteoblasts. Because prostaglandin E2 (PGE2) and pro-inflammatory cytokines such as interleukin (IL)-1 and IL-6 are involved in OA progression, we investigated whether these factors impact HGF produced by normal osteoblasts. PGE2 was the only factor tested that was able to stimulate HGF synthesis. However, the addition of NS398, a selective inhibitor of cyclo-oxygenase-2 (COX-2) had no effect on HGF produced by OA osteoblasts. HGF/NK2 had a moderate stimulating effect on HGF production by normal osteoblasts, whereas osteocalcin was not modulated by either HGF or HGF/NK2. When investigating signaling routes that might be implicated in OA osteoblast-produced HGF, we found that protein kinase A was at least partially involved. In summary, this study raises the hypothesis that the HGF found in articular cartilage is produced by osteoblasts, diffuses into the cartilage, and may be implicated in the OA process.

  16. An RGD-restricted substrate interface is sufficient for the adhesion, growth and cartilage forming capacity of human chondrocytes

    Directory of Open Access Journals (Sweden)

    D Vonwil

    2010-11-01

    Full Text Available This study aimed at testing whether an RGD-restricted substrate interface is sufficient for adhesion and growth of human articular chondrocytes (HAC, and whether it enhances their post expansion chondrogenic capacity. HAC/substrate interaction was restricted to RGD by modifying tissue culture polystyrene (TCPS with a poly(ethylene glycol (PEG based copolymer system that renders the surface resistant to protein adsorption while at the same time presenting the bioactive RGD-containing peptide GCRGYGRGDSPG (RGD. As compared to TCPS, HAC cultured on RGD spread faster (1.9-fold, maintained higher type II collagen mRNA expression (4.9-fold and displayed a 19% lower spreading area. On RGD, HAC attachment efficiency (66±10% and proliferation rate (0.56±0.04 doublings/day, as well as type II collagen mRNA expression in the subsequent chondrogenic differentiation phase, were similar to those of cells cultured on TCPS. In contrast, cartilaginous matrix deposition by HAC expanded on RGD was slightly but consistently higher (15% higher glycosaminoglycan-to-DNA ratio. RDG (bioinactive peptide and PEG (no peptide ligand controls yielded drastically reduced attachment efficiency (lower than 11% and proliferation (lower than 0.20 doublings/day. Collectively, these data indicate that restriction of HAC interaction with a substrate through RGD peptides is sufficient to support their adhesion, growth and maintenance of cartilage forming capacity. The concept could thus be implemented in materials for cartilage repair, whereby in situ recruited/infiltrated chondroprogenitor cells would proliferate while maintaining their ability to differentiate and generate cartilage tissue.

  17. Articular cartilage repair with recombinant human type II collagen/polylactide scaffold in a preliminary porcine study.

    Science.gov (United States)

    Muhonen, Virpi; Salonius, Eve; Haaparanta, Anne-Marie; Järvinen, Elina; Paatela, Teemu; Meller, Anna; Hannula, Markus; Björkman, Mimmi; Pyhältö, Tuomo; Ellä, Ville; Vasara, Anna; Töyräs, Juha; Kellomäki, Minna; Kiviranta, Ilkka

    2016-05-01

    The purpose of this study was to investigate the potential of a novel recombinant human type II collagen/polylactide scaffold (rhCo-PLA) in the repair of full-thickness cartilage lesions with autologous chondrocyte implantation technique (ACI). The forming repair tissue was compared to spontaneous healing (spontaneous) and repair with a commercial porcine type I/III collagen membrane (pCo). Domestic pigs (4-month-old, n = 20) were randomized into three study groups and a circular full-thickness chondral lesion with a diameter of 8 mm was created in the right medial femoral condyle. After 3 weeks, the chondral lesions were repaired with either rhCo-PLA or pCo together with autologous chondrocytes, or the lesion was only debrided and left untreated for spontaneous repair. The repair tissue was evaluated 4 months after the second operation. Hyaline cartilage formed most frequently in the rhCo-PLA treatment group. Biomechanically, there was a trend that both treatment groups resulted in better repair tissue than spontaneous healing. Adverse subchondral bone reactions developed less frequently in the spontaneous group (40%) and the rhCo-PLA treated group (50%) than in the pCo control group (100%). However, no statistically significant differences were found between the groups. The novel rhCo-PLA biomaterial showed promising results in this proof-of-concept study, but further studies will be needed in order to determine its effectiveness in articular cartilage repair. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:745-753, 2016. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  18. Hyaline cartilage tissue is formed through the co-culture of passaged human chondrocytes and primary bovine chondrocytes.

    Science.gov (United States)

    Taylor, Drew W; Ahmed, Nazish; Hayes, Anthony J; Ferguson, Peter; Gross, Allan E; Caterson, Bruce; Kandel, Rita A

    2012-08-01

    To circumvent the problem of a sufficient number of cells for cartilage engineering, the authors previously developed a two-stage culture system to redifferentiate monolayer culture-expanded dedifferentiated human articular chondrocytes by co-culture with primary bovine chondrocytes (bP0). The aim of this study was to analyze the composition of the cartilage tissue formed in stage 1 and compare it with bP0 grown alone to determine the optimal length of the co-culture stage of the system. Biochemical data show that extracellular matrix accumulation was evident after 2 weeks of co-culture, which was 1 week behind the bP0 control culture. By 3 to 4 weeks, the amounts of accumulated proteoglycans and collagens were comparable. Expression of chondrogenic genes, Sox 9, aggrecan, and collagen type II, was also at similar levels by week 3 of culture. Immunohistochemical staining of both co-culture and control tissues showed accumulation of type II collagen, aggrecan, biglycan, decorin, and chondroitin sulfate in appropriate zonal distributions. These data indicate that co-cultured cells form cartilaginous tissue that starts to resemble that formed by bP0 after 3 weeks, suggesting that the optimal time to terminate the co-culture stage, isolate the now redifferentiated cells, and start stage 2 is just after 3 weeks.

  19. Articular cartilage defect detectability in human knees with MR-arthrography

    Energy Technology Data Exchange (ETDEWEB)

    Engel, A. [Orthopaedic Clinic, Univ. of Vienna (Austria); Kramer, J. [MR-Inst., Univ. of Vienna (Austria); Stiglbauer, R. [MR-Inst., Univ. of Vienna (Austria); Hajek, P.C. [MR-Inst., Univ. of Vienna (Austria); Imhof, H. [MR-Inst., Univ. of Vienna (Austria)

    1993-04-01

    One hundred and thirteen knee joints were examined, of which 48 showed damage of the hyaline cartilage in one or more locations. For the evaluation of the magnetic resonance (MR) arthrographic images we used the macroscopic staging according to Outerbridge, the defect staging according to Bauer, as well as a new MR-arthrographic staging. The results of the evaluation were compared with the surgical findings in 61 knee joints. This revealed a sensitivity of 86 %, a specificity of 100 % and accuracy of 90 %. All lesions that could not be classified on MR-arthrography were of stage-I chondromalacia. (orig.)

  20. Reliability of cut mark analysis in human costal cartilage: the effects of blade penetration angle and intra- and inter-individual differences.

    Science.gov (United States)

    Puentes, K; Cardoso, H F V

    2013-09-10

    Identification of tool class characteristics from cut marks in either bone or cartilage is a valuable source of data for the forensic scientist. Various animal models have been used in experimental studies for the analysis of individual and class characteristics. However, human tissue has seldom been used and it is likely to differ from that of non-humans in key aspects. This study wishes to assess how the knife's blade angle, and both intra- and inter-individual variation in cartilage samples affect the ability of costal cartilage to retain the original class characteristics of the knife, as measured microscopically by the distance between consecutive striations. The 120 cartilaginous samples used in this study originated from the ribcage of 6 male cadavers which were submitted to autopsy at the North Branch of the National Institute of Legal Medicine, in Portugal. Three different serrated knives were purchased from a large department store, and were used in the experimental cuts. Samples of costal cartilage from 2 individuals were assigned to each knife. Each individual provided 20 cartilage samples. Cartilage samples were manually cut using each of the three knives, following two motions: one straight up-and-down cutting motion and parallel and one perpendicular to the blade's teeth long axis forward cutting motion. Casts of the samples were made with Mikrosil(®). Image capture and processing were performed with an Olympus stereomicroscope and its software. The blade's penetration angle and inter-individual variation were shown to affect the identification of the tool class characteristics from the striation pattern observed in a kerf wall, although this seems to be related only to the degree of calcification of the costal cartilage. Intra-individual variation does not seem to significantly affect the identification of the tool class characteristics from the striation pattern observed in a kerf wall, for the same knife following the same motion. Although this

  1. Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo - Implications for Tissue Engineering and Clinical Applications.

    Science.gov (United States)

    Szabó, Dóra Júlia; Noer, Agate; Nagymihály, Richárd; Josifovska, Natasha; Andjelic, Sofija; Veréb, Zoltán; Facskó, Andrea; Moe, Morten C; Petrovski, Goran

    2015-01-01

    Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation.

  2. Shear loading of costal cartilage

    CERN Document Server

    Subit, Damien

    2014-01-01

    A series of tests were performed on a single post-mortem human subject at various length scales. First, tabletop tests were performed. Next, the ribs and intercostal muscles were tested with the view to characterize the load transfer between the ribs. Finally, the costal cartilage was tested under shear loading, as it plays an important in the transfer of the load between the ribs and the sternum. This paper reports the results of dynamic shear loading tests performed on three samples of costal cartilage harvested from a single post-mortem human subject, as well as the quantification of the effective Young's modulus estimated from the amount of cartilage calcification.

  3. 改良组织块酶消化法培养人龋损牙髓干细胞的实验研究%Culture of human caries dental pulp stem cells with combined explants method and enzymatic separation method

    Institute of Scientific and Technical Information of China (English)

    麻丹丹; 高杰; 吴补领

    2011-01-01

    AIM; To compare the successfulness and the growth of human caries dental pulp stem cells(hCDPSCs) cultured with three different methods. METHODS: Twenty-five normal and caries human third molars were collected, the dental pulp tissues were cultured by the tissue explant method, tissue-explan collagenase digestion method and the combination of explant method and enzymatic separation method, respectively. The adherence of the explants, the morphology and quantity of cells were observed under a phase-contrast microscope. Culture duration was recorded. The clones were identified by expression of Stro-1 and CD90 and the growth curve of normal DPSCs and CDPSCs was drawn. RESULTS; Human normal DPSCs and CDPSCs could be cultured by all the three methods. A large number of human normal DPSCs and CDPSCs were cultured by the com hined explant method and enzymatic separation method in a shorter time, and these cells exhibited more vitality and more different morphologies. The growth rate of CDPSCs was higher than that of normal DPSCs . CONCLISION: The improved combination of explant method and enzymatic separation method is an ideal method for the primary culture of hCDPSCs in vitro, it may provide a methodological foundation for studying the mechanism of the formation of the tertiary dentine when the tooth was damaged.%目的:比较3种方法培养人龋损牙髓干细胞的成功率和细胞生长状态,以探求人龋损牙髓干细胞的最佳培养方法.方法:取18~22岁成人新鲜正常和龋损离体第三磨牙各25个,采用组织块法、酶消化法、改良组织块酶消化法培养牙髓干细胞.通过倒置显微镜观察组织块的贴壁以及细胞的形态和数量,并记录培养所需时间;有限稀释法纯化牙髓干细胞,流式细胞仪检测正常和龋损牙髓干细胞表面标记物Stro-1、CD 90的表达情况,绘制正常和龋损牙髓干细胞生长曲线.结果:组织块法、酶消化法和改良组织块酶消化法均可以培养

  4. Cartilage Turnover Reflected by Metabolic Processing of Type II Collagen: A Novel Marker of Anabolic Function in Chondrocytes

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    Natasja Stæhr Gudmann

    2014-10-01

    Full Text Available The aim of this study was to enable measurement of cartilage formation by a novel biomarker of type II collagen formation. The competitive enzyme-linked immunosorbent assay (ELISA Pro-C2 was developed and characterized for assessment of the beta splice variant of type II procollagen (PIIBNP. This is expected to originate primarily from remodeling of hyaline cartilage. A mouse monoclonal antibody (Mab was raised in mouse, targeting specifically PIIBNP (QDVRQPG and used in development of the assay. The specificity, sensitivity, 4-parameter fit and stability of the assay were tested. Levels of PIIBNP were quantified in human serum (0.6–2.2 nM, human amniotic fluid (163–188 nM and sera from different animal species, e.g., fetal bovine serum (851–901 nM with general good linearity (100% (SD 7.6 recovery and good intra- and inter-assay variation (CV% < 10. Dose (0.1 to 100 ng/mL and time (7, 14 and 21 days dependent release of PIIBNP were evaluated in the conditioned medium from bovine cartilage explants (BEX and human cartilage explants (HEX upon stimulation with insulin-like growth factor (IGF-1, transforming growth factor (TGF-β1 and fibroblastic growth factor-2 (FGF-2. TGF-β1 and IGF-1 in concentrations of 10–100 ng/mL significantly (p < 0.05 induced release of PIIBNP in BEX compared to conditions without treatment (WO. In HEX, IGF-1 100 ng/mL was able to induce a significant increase of PIIBNP after one week compared to WO. FGF-2 did not induce a PIIBNP release in our models. To our knowledge this is the first assay, which is able to specifically evaluate PIIBNP excretion. The Pro-C2 assay seems to provide a promising and novel marker of type II collagen formation.

  5. Cartilage-selective genes identified in genome-scale analysis of non-cartilage and cartilage gene expression

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    Cohn Zachary A

    2007-06-01

    Full Text Available Abstract Background Cartilage plays a fundamental role in the development of the human skeleton. Early in embryogenesis, mesenchymal cells condense and differentiate into chondrocytes to shape the early skeleton. Subsequently, the cartilage anlagen differentiate to form the growth plates, which are responsible for linear bone growth, and the articular chondrocytes, which facilitate joint function. However, despite the multiplicity of roles of cartilage during human fetal life, surprisingly little is known about its transcriptome. To address this, a whole genome microarray expression profile was generated using RNA isolated from 18–22 week human distal femur fetal cartilage and compared with a database of control normal human tissues aggregated at UCLA, termed Celsius. Results 161 cartilage-selective genes were identified, defined as genes significantly expressed in cartilage with low expression and little variation across a panel of 34 non-cartilage tissues. Among these 161 genes were cartilage-specific genes such as cartilage collagen genes and 25 genes which have been associated with skeletal phenotypes in humans and/or mice. Many of the other cartilage-selective genes do not have established roles in cartilage or are novel, unannotated genes. Quantitative RT-PCR confirmed the unique pattern of gene expression observed by microarray analysis. Conclusion Defining the gene expression pattern for cartilage has identified new genes that may contribute to human skeletogenesis as well as provided further candidate genes for skeletal dysplasias. The data suggest that fetal cartilage is a complex and transcriptionally active tissue and demonstrate that the set of genes selectively expressed in the tissue has been greatly underestimated.

  6. Regeneration of human bones in hip osteonecrosis and human cartilage in knee osteoarthritis with autologous adipose-tissue-derived stem cells: a case series

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    Pak Jaewoo

    2011-07-01

    Full Text Available Abstract Introduction This is a series of clinical case reports demonstrating that a combination of percutaneously injected autologous adipose-tissue-derived stem cells, hyaluronic acid, platelet rich plasma and calcium chloride may be able to regenerate bones in human osteonecrosis, and with addition of a very low dose of dexamethasone, cartilage in human knee osteoarthritis. Case reports Stem cells were obtained from adipose tissue of abdominal origin by digesting lipoaspirate tissue with collagenase. These stem cells, along with hyaluronic acid, platelet rich plasma and calcium chloride, were injected into the right hip of a 29-year-old Korean woman and a 47-year-old Korean man. They both had a history of right hip osteonecrosis of the femoral head. For cartilage regeneration, a 70-year-old Korean woman and a 79-year-old Korean woman, both with a long history of knee pain due to osteoarthritis, were injected with stem cells along with hyaluronic acid, platelet rich plasma, calcium chloride and a nanogram dose of dexamethasone. Pre-treatment and post-treatment MRI scans, physical therapy, and pain score data were then analyzed. Conclusions The MRI data for all the patients in this series showed significant positive changes. Probable bone formation was clear in the patients with osteonecrosis, and cartilage regeneration in the patients with osteoarthritis. Along with MRI evidence, the measured physical therapy outcomes, subjective pain, and functional status all improved. Autologous mesenchymal stem cell injection, in conjunction with hyaluronic acid, platelet rich plasma and calcium chloride, is a promising minimally invasive therapy for osteonecrosis of femoral head and, with low-dose dexamethasone, for osteoarthritis of human knees.

  7. An in vitro comparative study of T2 and T2* mappings of human articular cartilage at 3-Tesla MRI using histology as the standard of reference

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Taehee; Park, Sunghoon [Ajou University School of Medicine, Department of Radiology, Suwon (Korea, Republic of); Ajou University Medical Center, Musculoskeletal Imaging Laboratory, Suwon (Korea, Republic of); Min, Byoung-Hyun [Ajou University School of Medicine, Department of Orthopaedic Surgery, Suwon (Korea, Republic of); Ajou University School of Medicine, Cartilage Regeneration Center, Suwon (Korea, Republic of); Yoon, Seung-Hyun [Ajou University School of Medicine, Cartilage Regeneration Center, Suwon (Korea, Republic of); Kim, Hakil [INHA University, School of Information and Communication Engineering, Incheon (Korea, Republic of); Lee, Hyun Young [Ajou University Medical Center, Regional Clinical Trial Center, Suwon (Korea, Republic of); Yonsei University College of Medicine, Department of Biostatistics, Seoul (Korea, Republic of); Kwack, Kyu-Sung [Ajou University School of Medicine, Department of Radiology, Suwon (Korea, Republic of); Ajou University Medical Center, Musculoskeletal Imaging Laboratory, Suwon (Korea, Republic of); Ajou University School of Medicine, Cartilage Regeneration Center, Suwon (Korea, Republic of)

    2014-07-15

    The aim of this study was to evaluate the correlations between T2 value, T2* value, and histological grades of degenerated human articular cartilage. T2 mapping and T2* mapping of nine tibial osteochondral specimens were obtained using a 3-T MRI after total knee arthroplasty. A total of 94 ROIs were analyzed. Histological grades were assessed using the David-Vaudey scale. Spearman's rho correlation analysis and Pearson's correlation analysis were performed. The mean relaxation values in T2 map with different histological grades (0, 1, 2) of the cartilage were 51.9 ± 9.2 ms, 55.8 ± 12.8 ms, and 59.6 ± 10.2 ms, respectively. The mean relaxation values in T2* map with different histological grades (0, 1, 2) of the cartilage were 20.3 ± 10.3 ms, 21.1 ± 12.4 ms, and 15.4 ± 8.5 ms, respectively. Spearman's rho correlation analysis confirmed a positive correlation between T2 value and histological grade (ρ = 0.313, p < 0.05). Pearson's correlation analysis revealed a significant negative correlation between T2 and T2* (r = -0.322, p < 0.05). Although T2* values showed a decreasing trend with an increase in cartilage degeneration, this correlation was not statistically significant in this study (ρ = -0.192, p = 0.129). T2 mapping was correlated with histological degeneration, and it may be a good biomarker for osteoarthritis in human articular cartilage. However, the strength of the correlation was weak (ρ = 0.313). Although T2* values showed a decreasing trend with an increase in cartilage degeneration, the correlation was not statistically significant. Therefore, T2 mapping may be more appropriate for the initial diagnosis of articular cartilage degeneration in the knee joint. Further studies on T2* mapping are needed to confirm its reliability and mechanism in cartilage degeneration. (orig.)

  8. Shark cartilage

    Science.gov (United States)

    ... sarcoma, that is more common in people with HIV infection. Shark cartilage is also used for arthritis, psoriasis, ... Neovastat) by mouth seems to increase survival in patients with advanced kidney cancer (renal cell carcinoma). This product has FDA “Orphan Drug ...

  9. Computer-aided diagnosis in phase contrast imaging X-ray computed tomography for quantitative characterization of ex vivo human patellar cartilage.

    Science.gov (United States)

    Nagarajan, Mahesh B; Coan, Paola; Huber, Markus B; Diemoz, Paul C; Glaser, Christian; Wismuller, Axel

    2013-10-01

    Visualization of ex vivo human patellar cartilage matrix through the phase contrast imaging X-ray computed tomography (PCI-CT) has been previously demonstrated. Such studies revealed osteoarthritis-induced changes to chondrocyte organization in the radial zone. This study investigates the application of texture analysis to characterizing such chondrocyte patterns in the presence and absence of osteoarthritic damage. Texture features derived from Minkowski functionals (MF) and gray-level co-occurrence matrices (GLCM) were extracted from 842 regions of interest (ROI) annotated on PCI-CT images of ex vivo human patellar cartilage specimens. These texture features were subsequently used in a machine learning task with support vector regression to classify ROIs as healthy or osteoarthritic; classification performance was evaluated using the area under the receiver operating characteristic curve (AUC). The best classification performance was observed with the MF features perimeter (AUC: 0.94 ±0.08 ) and "Euler characteristic" (AUC: 0.94 ±0.07 ), and GLCM-derived feature "Correlation" (AUC: 0.93 ±0.07). These results suggest that such texture features can provide a detailed characterization of the chondrocyte organization in the cartilage matrix, enabling classification of cartilage as healthy or osteoarthritic with high accuracy.

  10. Neonatal Desensitization Supports Long-Term Survival and Functional Integration of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells in Rat Joint Cartilage Without Immunosuppression

    Science.gov (United States)

    Zhang, Shufang; Jiang, Yang Zi; Zhang, Wei; Chen, Longkun; Tong, Tong; Liu, Wanlu; Mu, Qin; Liu, Hua; Ji, Junfeng; Ouyang, Hong Wei

    2013-01-01

    Immunological response hampers the investigation of human embryonic stem cells (hESCs) or their derivates for tissue regeneration in vivo. Immunosuppression is often used after surgery, but exhibits side effects of significant weight loss and allows only short-term observation. The purpose of this study was to investigate whether neonatal desensitization supports relative long-term survival of hESC-derived mesenchymal stem cells (hESC-MSCs) and promotes cartilage regeneration. hESC-MSCs were injected on the day of birth in rats. Six weeks after neonatal injection, a full-thickness cylindrical cartilage defect was created and transplanted with a hESC-MSC-seeded collagen bilayer scaffold (group d+s+c) or a collagen bilayer scaffold (group d+s). Rats without neonatal injection were transplanted with the hESC-MSC-seeded collagen bilayer scaffold to serve as controls (group s+c). Cartilage regeneration was evaluated by histological analysis, immunohistochemical staining, and biomechanical test. The role of hESC-MSCs in cartilage regeneration was analyzed by CD4 immunostaining, cell death detection, and visualization of human cells in regenerated tissues. hESC-MSCs expressed CD105, CD73, CD90, CD29, and CD44, but not CD45 and CD34, and possessed trilineage differentiation potential. Group d+s+c exhibited greater International Cartilage Repair Society (ICRS) scores than group d+s or group s+c. Abundant collagen type II and improved mechanical properties were detected in group d+s+c. There were less CD4+ inflammatory cell infiltration and cell death at week 1, and hESC-MSCs were found to survive as long as 8 weeks after transplantation in group d+s+c. Our study suggests that neonatal desensitization before transplantation may be an efficient way to develop a powerful tool for preclinical study of human cell-based therapies in animal models. PMID:22788986

  11. Integration of Stem Cell to Chondrocyte-Derived Cartilage Matrix in Healthy and Osteoarthritic States in the Presence of Hydroxyapatite Nanoparticles.

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    Rupak Dua

    Full Text Available We investigated the effectiveness of integrating tissue engineered cartilage derived from human bone marrow derived stem cells (HBMSCs to healthy as well as osteoarthritic cartilage mimics using hydroxyapatite (HA nanoparticles immersed within a hydrogel substrate. Healthy and diseased engineered cartilage from human chondrocytes (cultured in agar gels were integrated with human bone marrow stem cell (HBMSC-derived cartilaginous engineered matrix with and without HA, and evaluated after 28 days of growth. HBMSCs were seeded within photopolymerizable poly (ethylene glycol diacrylate (PEGDA hydrogels. In addition, we also conducted a preliminary in vivo evaluation of cartilage repair in rabbit knee chondral defects treated with subchondral bone microfracture and cell-free PEGDA with and without HA. Under in vitro conditions, the interfacial shear strength between tissue engineered cartilage derived from HBMSCs and osteoarthritic chondrocytes was significantly higher (p < 0.05 when HA nanoparticles were incorporated within the HBMSC culture system. Histological evidence confirmed a distinct spatial transition zone, rich in calcium phosphate deposits. Assessment of explanted rabbit knees by histology demonstrated that cellularity within the repair tissues that had filled the defects were of significantly higher number (p < 0.05 when HA was used. HA nanoparticles play an important role in treating chondral defects when osteoarthritis is a co-morbidity. We speculate that the calcified layer formation at the interface in the osteoarthritic environment in the presence of HA is likely to have attributed to higher interfacial strength found in vitro. From an in vivo standpoint, the presence of HA promoted cellularity in the tissues that subsequently filled the chondral defects. This higher presence of cells can be considered important in the context of accelerating long-term cartilage remodeling. We conclude that HA nanoparticles play an important role in

  12. Inhibition of oncostatin M in osteoarthritic synovial fluid enhances GAG production in osteoarthritic cartilage repair

    Directory of Open Access Journals (Sweden)

    M Beekhuizen

    2013-09-01

    Full Text Available Mediators in the synovial fluid are thought to play a major role in osteoarthritic cartilage turnover. The purpose of the current study was to investigate the role of oncostatin M (OSM in osteoarthritis (OA by evaluating the presence of the cytokine and its receptors in the OA joint and interfering with its activity in synovial fluid co-cultured with cartilage explants. OSM levels were increased in the synovial fluid of osteoarthritic patients compared to healthy donors. Immunohistochemistry confirmed the presence of both the leukaemia inhibitory factor (LIF and OSM receptors for OSM throughout the whole depth of osteoarthritic cartilage and synovial tissue, whereas in healthy cartilage their presence seemed more restricted to the superficial zone. Blocking OSM activity, using an activity inhibiting antibody, in 25 % osteoarthritic synovial fluid added to OA cartilage explant cultures increased glycosaminoglycan (GAG content from 18.6 mg/g to 24.3 mg/g (P < 0.03 and total production from 7.0 mg/g to 11.9 mg/g (P < 0.003. However, OSM exogenously added to cartilage explant cultures reflecting low and high concentrations in the synovial fluid (5 and 50 pg/mL did not affect cartilage matrix turnover, suggesting that factors present in the synovial fluid act in concert with OSM to inhibit GAG production. The current study indicates the potential to enhance cartilage repair in osteoarthritis by modulating the joint environment by interfering with OSM activity.

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

    Directory of Open Access Journals (Sweden)

    Jan-Philipp Stromps

    2014-01-01

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

  14. Enhanced human bone marrow mesenchymal stem cell functions in novel 3D cartilage scaffolds with hydrogen treated multi-walled carbon nanotubes.

    Science.gov (United States)

    Holmes, Benjamin; Castro, Nathan J; Li, Jian; Keidar, Michael; Zhang, Lijie Grace

    2013-09-13

    Cartilage tissue is a nanostructured tissue which is notoriously hard to regenerate due to its extremely poor inherent regenerative capacity and complex stratified architecture. Current treatment methods are highly invasive and may have many complications. Thus, the goal of this work is to use nanomaterials and nano/microfabrication methods to create novel biologically inspired tissue engineered cartilage scaffolds to facilitate human bone marrow mesenchymal stem cell (MSC) chondrogenesis. To this end we utilized electrospinning to design and fabricate a series of novel 3D biomimetic nanostructured scaffolds based on hydrogen (H2) treated multi-walled carbon nanotubes (MWCNTs) and biocompatible poly(L-lactic acid) (PLLA) polymers. Specifically, a series of electrospun fibrous PLLA scaffolds with controlled fiber dimension were fabricated in this study. In vitro MSC studies showed that stem cells prefer to attach in the scaffolds with smaller fiber diameter. More importantly, the MWCNT embedded scaffolds showed a drastic increase in mechanical strength and a compressive Young's modulus matching to natural cartilage. Furthermore, our MSC differentiation results demonstrated that incorporation of the H2 treated carbon nanotubes and poly-L-lysine coating can induce more chondrogenic differentiations of MSCs than controls. After two weeks of culture, PLLA scaffolds with H2 treated MWCNTs and poly-L-lysine can achieve the highest glycosaminoglycan synthesis, making them promising for further exploration for cartilage regeneration.

  15. Enhanced human bone marrow mesenchymal stem cell functions in novel 3D cartilage scaffolds with hydrogen treated multi-walled carbon nanotubes

    Science.gov (United States)

    Holmes, Benjamin; Castro, Nathan J.; Li, Jian; Keidar, Michael; Zhang, Lijie Grace

    2013-09-01

    Cartilage tissue is a nanostructured tissue which is notoriously hard to regenerate due to its extremely poor inherent regenerative capacity and complex stratified architecture. Current treatment methods are highly invasive and may have many complications. Thus, the goal of this work is to use nanomaterials and nano/microfabrication methods to create novel biologically inspired tissue engineered cartilage scaffolds to facilitate human bone marrow mesenchymal stem cell (MSC) chondrogenesis. To this end we utilized electrospinning to design and fabricate a series of novel 3D biomimetic nanostructured scaffolds based on hydrogen (H2) treated multi-walled carbon nanotubes (MWCNTs) and biocompatible poly(L-lactic acid) (PLLA) polymers. Specifically, a series of electrospun fibrous PLLA scaffolds with controlled fiber dimension were fabricated in this study. In vitro MSC studies showed that stem cells prefer to attach in the scaffolds with smaller fiber diameter. More importantly, the MWCNT embedded scaffolds showed a drastic increase in mechanical strength and a compressive Young’s modulus matching to natural cartilage. Furthermore, our MSC differentiation results demonstrated that incorporation of the H2 treated carbon nanotubes and poly-L-lysine coating can induce more chondrogenic differentiations of MSCs than controls. After two weeks of culture, PLLA scaffolds with H2 treated MWCNTs and poly-L-lysine can achieve the highest glycosaminoglycan synthesis, making them promising for further exploration for cartilage regeneration.

  16. Adeno-Associated Vector mediated gene transfer of Transforming Growth Factor-beta1 to normal and osteoarthritic human chondrocytes stimulates cartilage anabolism

    Directory of Open Access Journals (Sweden)

    Ulrich-Vinther M.

    2005-11-01

    Full Text Available The objective of the present study was to investigate whether cartilage anabolism in human primary osteoarthritic chondrocytes could be improved by adeno-associated virus (AAV vector-mediated gene transduction of transforming growth factor TGF-beta1 (TGF-beta1. A bi-cistronic AAV-TGF-beta1-IRES-eGFP (AAV-TGF-beta1 vector was generated and used for transduction of a normal human articular chondrocyte cell line (tsT/AC62 and primary human osteoarthritic articular chondrocytes harvested from 8 patients receiving total knee joint arthroplasty. Transduction efficiency was detected by fluorescent microscopy for gene expression of enhanced green fluorescent protein (eGFP. TGF-beta1 synthesis was determined by ELISA. To assess the influence of TGF-beta1 gene therapy on chondrocyte cartilage metabolism, mRNA expressions of type II collagen, aggrecan, and matrix metalloproteinase 3 (MMP-3 were determined by quantitative real-time PCR. AAV-TGF-beta1 transduction resulted in increased synthesis of TGF-beta1 in both osteoarthritic chondrocytes and the normal articular chondrocyte cell line. The expression levels of the transduced genes were correlated to "multiplicity of infection" (MOI and post-infectious time. In both osteoarthritic chondrocytes and the normal articular chondrocyte cell line, AAV-TGF-beta1 treatment increased mRNA expression of both type II collagen and aggrecan, but decreased MMP-3 mRNA expression. Osteoarthritic chondrocytes and the normal articular chondrocyte cell line could be transduced with equal efficiencies. In conclusion, it was demonstrated that AAV-TGF-beta1 gene transfer stimulates cartilage anabolism and decreases expression of enzymes responsible for cartilage degradation in human osteoarthritic chondrocytes. The results indicate that the AAV vector is an efficient mediator of growth factors to human articular chondrocytes, and that it might be useful in future chondrocyte gene therapy.

  17. Effect of antibiotics on in vitro and in vivo avian cartilage degradation.

    Science.gov (United States)

    Peters, T L; Fulton, R M; Roberson, K D; Orth, M W

    2002-01-01

    Antibiotics are used in the livestock industry not only to treat disease but also to promote growth and increase feed efficiency in less than ideal sanitary conditions. However, certain antibiotic families utilized in the poultry industry have recently been found to adversely affect bone formation and cartilage metabolism in dogs, rats, and humans. Therefore, the first objective of this study was to determine if certain antibiotics used in the poultry industry would inhibit in vitro cartilage degradation. The second objective was to determine if the antibiotics found to inhibit in vitro cartilage degradation also induced tibial dyschondroplasia in growing broilers. Ten antibiotics were studied by an avian explant culture system that is designed to completely degrade tibiae over 16 days. Lincomycin, tylosin tartrate, gentamicin, erythromycin, and neomycin sulfate did not inhibit degradation at any concentration tested. Doxycycline (200 microg/ml), oxytetracycline (200 microg/ml), enrofloxacin (200 and 400 microg/ml), ceftiofur (400 microg/ml), and salinomycin (10 microg/ml) prevented complete cartilage degradation for up to 30 days in culture. Thus, some of the antibiotics did inhibit cartilage degradation in developing bone. Day-old chicks were then administered the five antibiotics at 25%, 100%, or 400% above their recommended dose levels and raised until 21 days of age. Thiram, a fungicide known to induce experimental tibial dyschondroplasia (TD), was given at 20 ppm. Birds were then killed by cervical dislocation, and each proximal tibiotarsus was visually examined for TD lesions. The results showed that none of these antibiotics significantly induced TD in growing boilers at any concentration tested, whereas birds given 20 ppm thiram had a 92% incidence rate.

  18. MRI quantification of human spine cartilage endplate geometry: Comparison with age, degeneration, level, and disc geometry.

    Science.gov (United States)

    DeLucca, John F; Peloquin, John M; Smith, Lachlan J; Wright, Alexander C; Vresilovic, Edward J; Elliott, Dawn M

    2016-08-01

    Geometry is an important indicator of disc mechanical function and degeneration. While the geometry and associated degenerative changes in the nucleus pulposus and the annulus fibrosus are well-defined, the geometry of the cartilage endplate (CEP) and its relationship to disc degeneration are unknown. The objectives of this study were to quantify CEP geometry in three dimensions using an MRI FLASH imaging sequence and evaluate relationships between CEP geometry and age, degeneration, spinal level, and overall disc geometry. To do so, we assessed the MRI-based measurements for accuracy and repeatability. Next, we measured CEP geometry across a larger sample set and correlated CEP geometric parameters to age, disc degeneration, level, and disc geometry. The MRI-based measures resulted in thicknesses (0.3-1 mm) that are comparable to prior measurements of CEP thickness. CEP thickness was greatest at the anterior/posterior (A/P) margins and smallest in the center. The CEP A/P thickness, axial area, and lateral width decreased with age but were not related to disc degeneration. Age-related, but not degeneration-related, changes in geometry suggest that the CEP may not follow the progression of disc degeneration. Ultimately, if the CEP undergoes significant geometric changes with aging and if these can be related to low back pain, a clinically feasible translation of the FLASH MRI-based measurement of CEP geometry presented in this study may prove a useful diagnostic tool. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1410-1417, 2016.

  19. Optical properties of nasal septum cartilage

    Science.gov (United States)

    Bagratashvili, Nodar V.; Sviridov, Alexander P.; Sobol, Emil N.; Kitai, Moishe S.

    1998-05-01

    Optical parameters (scattering coefficient s, absorption coefficient k and scattering anisotropy coefficient g) of hyaline cartilage were studied for the first time. Optical properties of human and pig nasal septum cartilage, and of bovine ear cartilage were examined using a spectrophotometer with an integrating sphere, and an Optical Multi-Channel Analyser. We measured total transmission Tt, total reflection Rt, and on-axis transmission Ta for light propagating through cartilage sample, over the visible spectral range (14000 - 28000 cm-1). It is shown that transmission and reflection spectra of human, pig and bovine cartilage are rather similar. It allows us to conclude that the pig cartilage can be used for in-vivo studies instead of human cartilage. The data obtained were treated by means of the one-dimensional diffusion approximation solution of the optical transport equation. We have found scattering coefficient s, absorption coefficient k and scattering anisotropy coefficient g by the iterative comparison of measured and calculated Tt, Rt and Ta values for human and pig cartilage. We found, in particular, that for 500 nm irradiation s equals 37,6 plus or minus 3.5 cm-1, g equals 0,56 plus or minus 0.05, k approximately equals 0,5 plus or minus 0.3 cm-1. The above data were used in Monte Carlo simulation for spatial intensity profile of light scattered by a cartilage sample. The computed profile was very similar to the profile measured using an Optical Multi-Channel Analyzer (OMA).

  20. Incorporation of exudates of human platelet-rich fibrin gel in biodegradable fibrin scaffolds for tissue engineering of cartilage.

    Science.gov (United States)

    Chien, Chi-Sheng; Ho, Hsiu-O; Liang, Yu-Chih; Ko, Pai-Hung; Sheu, Ming-Thau; Chen, Chien-Ho

    2012-05-01

    The goal of this study was to assess the incorporation of exudates of human platelet-rich fibrin (hPRF) that is abundant in platelet cytokines and growth factors into biodegradable fibrin (FB) scaffolds as a regeneration matrix for promoting chondrocyte proliferation and re-differentiation. hPRF was obtained from human blood by centrifugation without an anticoagulant, and the exudate of hPRF was collected and mixed with bovine fibrinogen, and then thrombin was added to form the FB scaffold. Proliferation and differentiation of human primary chondrocytes and a human chondrosarcoma cell line, the SW-1353, embedded in the three-dimensional (3D) scaffolds and on the two-dimensional (2D) surface of the FB scaffolds so produced were evaluated in comparison with an agarose (AG) scaffold serving as the control. Results demonstrated that the amounts of these cytokines and growth factors in hPRF exudates were higher than those in the blood-derived products except for TGF-β1. Chondrocytes and SW1353 cells on the 2D and 3D FB scaffolds with the addition of the exudates of PRF exhibited more-available proliferation and differentiation than cells on 2D and 3D FB and AG scaffolds. It was concluded that FB scaffolds can provide an appropriate environment for chondrocyte proliferation and re-differentiation, and it could be improved by adding exudates of hPRF. These 3D scaffolds have great promise for cartilage tissue engineering. Copyright © 2012 Wiley Periodicals, Inc.

  1. Imaging of Osteoarthritic Human Articular Cartilage using Fourier Transform Infrared Microspectroscopy Combined with Multivariate and Univariate Analysis.

    Science.gov (United States)

    Oinas, J; Rieppo, L; Finnilä, M A J; Valkealahti, M; Lehenkari, P; Saarakkala, S

    2016-07-21

    The changes in chemical composition of human articular cartilage (AC) caused by osteoarthritis (OA) were investigated using Fourier transform infrared microspectroscopy (FTIR-MS). We demonstrate the sensitivity of FTIR-MS for monitoring compositional changes that occur with OA progression. Twenty-eight AC samples from tibial plateaus were imaged with FTIR-MS. Hyperspectral images of all samples were combined for K-means clustering. Partial least squares regression (PLSR) analysis was used to compare the spectra with the OARSI grade (histopathological grading of OA). Furthermore, the amide I and the carbohydrate regions were used to estimate collagen and proteoglycan contents, respectively. Spectral peak at 1338 cm(-1) was used to estimate the integrity of the collagen network. The layered structure of AC was revealed using the carbohydrate region for clustering. Statistically significant correlation was observed between the OARSI grade and the collagen integrity in the superficial (r = -0.55) and the deep (r = -0.41) zones. Furthermore, PLSR models predicted the OARSI grade from the superficial (r = 0.94) and the deep (r = 0.77) regions of the AC with high accuracy. Obtained results suggest that quantitative and qualitative changes occur in the AC composition during OA progression, and these can be monitored by the use of FTIR-MS.

  2. Nanoparticulate Mineralized Collagen Scaffolds and BMP-9 Induce a Long-Term Bone Cartilage Construct in Human Mesenchymal Stem Cells.

    Science.gov (United States)

    Ren, Xiaoyan; Weisgerber, Daniel W; Bischoff, David; Lewis, Michael S; Reid, Russell R; He, Tong-Chuan; Yamaguchi, Dean T; Miller, Timothy A; Harley, Brendan A C; Lee, Justine C

    2016-07-01

    Engineering the osteochondral junction requires fabrication of a microenvironment that supports both osteogenesis and chondrogenesis. Multiphasic scaffold strategies utilizing a combination of soluble factors and extracellular matrix components are ideally suited for such applications. In this work, the contribution of an osteogenic nanoparticulate mineralized glycosaminoglycan scaffold (MC-GAG) and a dually chondrogenic and osteogenic growth factor, BMP-9, in the differentiation of primary human mesenchymal stem cells (hMSCs) is evaluated. Although 2D cultures demonstrate alkaline phosphatase activity and mineralization of hMSCs induced by BMP-9, MC-GAG scaffolds do not demonstrate significant differences in the collagen I expression, osteopontin expression, or mineralization. Instead, BMP-9 increases expression of collagen II, Sox9, aggrecan (ACAN), and cartilage oligomeric protein. However, the hypertrophic chondrocyte marker, collagen X, is not elevated with BMP-9 treatment. In addition, histologic analyses demonstrate that while BMP-9 does not increase mineralization, BMP-9 treatment results in an increase of sulfated glycosaminoglycans. Thus, the combination of BMP-9 and MC-GAG stimulates chondrocytic and osteogenic differentiation of hMSCs.

  3. ARG098, a novel anti-human Fas antibody, suppresses synovial hyperplasia and prevents cartilage destruction in a severe combined immunodeficient-HuRAg mouse model

    Directory of Open Access Journals (Sweden)

    Matsubara Tsukasa

    2010-09-01

    Full Text Available Abstract Background The anti-human Fas/APO-1/CD95 (Fas mouse/human chimeric monoclonal IgM antibody ARG098 (ARG098 targets the human Fas molecule. The cytotoxic effects of ARG098 on cells isolated from RA patients, on normal cells in vitro, and on RA synovial tissue and cartilage in vivo using implanted rheumatoid tissues in an SCID mouse model (SCID-HuRAg were investigated to examine the potential of ARG098 as a therapy for RA. Methods ARG098 binding to each cell was analyzed by cytometry. The effects of ARG098 on several cells were assessed by a cell viability assay in vitro. Effects on the RA synovium, lymphocytes, and cartilage were assessed in vivo using the SCID-HuRAg mouse model. Results ARG098 bound to cell surface Fas molecules, and induced apoptosis in Fas-expressing RA synoviocytes and infiltrating lymphocytes in the RA synovium in a dose-dependent manner. However, ARG098 did not affect the cell viability of peripheral blood mononuclear cells of RA patients or normal chondrocytes. ARG098 also induced apoptosis in RA synoviocytes and infiltrating lymphocytes in the RA synovium in vivo. The destruction of cartilage due to synovial invasion was inhibited by ARG098 injection in the modified SCID-HuRAg mouse model. Conclusions ARG098 treatment suppressed RA synovial hyperplasia through the induction of apoptosis and prevented cartilage destruction in vivo. These results suggest that ARG098 might become a new therapy for RA.

  4. Lubrication of Articular Cartilage.

    Science.gov (United States)

    Jahn, Sabrina; Seror, Jasmine; Klein, Jacob

    2016-07-11

    The major synovial joints such as hips and knees are uniquely efficient tribological systems, able to articulate over a wide range of shear rates with a friction coefficient between the sliding cartilage surfaces as low as 0.001 up to pressures of more than 100 atm. No human-made material can match this. The means by which such surfaces maintain their very low friction has been intensively studied for decades and has been attributed to fluid-film and boundary lubrication. Here, we focus especially on the latter: the reduction of friction by molecular layers at the sliding cartilage surfaces. In particular, we discuss such lubrication in the light of very recent advances in our understanding of boundary effects in aqueous media based on the paradigms of hydration lubrication and of the synergism between different molecular components of the synovial joints (namely hyaluronan, lubricin, and phospholipids) in enabling this lubrication.

  5. β-Defensin-4 (HBD-4) is expressed in chondrocytes derived from normal and osteoarthritic cartilage encapsulated in PEGDA scaffold.

    Science.gov (United States)

    Musumeci, Giuseppe; Carnazza, Maria Luisa; Loreto, Corrado; Leonardi, Rosalia; Loreto, Carla

    2012-12-01

    Defensins are antibiotic peptides involved in host defense mechanisms, wound healing and tissue repair. Furthermore, they seem to play an important role in protection mechanisms in articular joints. The aim of this study was to investigate β-defensin-4 expression in chondrocytes taken from articular cartilage of knees of patients with osteoarthritis (OA) compared to normal cartilage, in vivo in explanted tissue, and in vitro in chondrocytes encapsulated in construct PEGDA hydrogels. The present investigation was conducted to try and elucidate the possible use of β-defensin-4 as a relevant marker for the eventual use of successive scaffold allografts, and to provide new insights for hydrogel PEGDA scaffold efficacy in re-differentiation or repair of OA chondrocytes in vitro. Articular cartilage specimens from OA cartilage and normal cartilage were assessed by histology, histochemistry, immunohistochemistry and Western blot analysis. The results showed strong β-defensin-4 immunoexpression in explanted tissue from OA cartilage and weak β-defensin-4 expression in control cartilage. The chondrocytes from OA cartilage after 4 weeks of culture in PEGDA hydrogels showed the formation of new hyaline cartilage and a decreased expression of β-defensin-4 immunostaining comparable to that of control cartilage. Our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repair of cartilage lesions in patients with OA using β-defensin-4 as a relevant marker. Copyright © 2012 Elsevier GmbH. All rights reserved.

  6. Enhancing FTIR imaging capabilities with two-dimensional correlation spectroscopy (2DCOS): A study of concentration gradients of collagen and proteoglycans in human patellar cartilage

    Science.gov (United States)

    Jiang, Eric Y.; Rieppo, Jarno

    2006-11-01

    This paper explores a new application of two-dimensional correlation spectroscopy (2DCOS) in FTIR spectroscopic imaging analysis of biological samples. A particular example demonstrated in this paper is the characterization of concentration gradients of collagen and proteoglycans in human patellar cartilage. A focal plane array detector-based FTIR imaging system has been proven to be an efficient tool to detect early collagen and proteoglycans degradation in developing osteoarthrosis through evaluating compositional changes of osteoarthritic cartilage along the depth. However, the closely overlapped bands of collagen and proteoglycans make normal spectral and spatial analysis difficult. With 2DCOS analysis of the imaging data, it is possible to enhance the spectral resolution and reveal distinctive compositional changes that are normally hidden with conventional approaches. The combined technique, FTIR imaging enhanced with 2DCOS, provides new possibilities to solve challenging problems in the analysis of complex biological systems.

  7. Quantitative analysis of the mRNA expression levels of BCL2 and BAX genes in human osteoarthritis and normal articular cartilage: An investigation into their differential expression.

    Science.gov (United States)

    Karaliotas, Georgios I; Mavridis, Konstantinos; Scorilas, Andreas; Babis, George C

    2015-09-01

    Osteoarthritis (OA) is primarily characterized by articular cartilage degeneration and chondrocyte loss. Although the role of apoptosis in cartilage pathobiology remains to be elucidated, the apoptotic B‑cell CLL/lymphoma 2 (BCL2) gene family is considered to be involved in OA. The purpose of the present study was to quantitatively analyze the mRNA expression profiles of the BCL2‑associated X protein (BAX) and BCL2 genes in human OA and in normal cartilage. Cartilage tissue samples were obtained from 78 patients undergoing total knee arthroplasty for OA (OA group) and orthopedic interventions for causes other than OA (control group). Total RNA was isolated from the cartilage tissue specimens and reverse transcribed into cDNA. A highly sensitive and specific reverse transcription quantitative polymerase chain reaction assay was developed for quantification of the mRNA levels of BAX and BCL2, using beta‑2 microglobulin as an endogenous control for normalization purposes. Gene expression analysis was performed using the comparative Ct (2(‑ΔΔCt)) method. The mRNA expression of BAX presented an increasing trend in the OA group compared with the control group, although without statistically significace (P=0.099). By contrast, the expression ratio of BCL2/BAX was found to be significantly decreased (2.76‑fold) in the OA group compared with the normal cartilage control group (P=0.022). A notable 4.6‑fold overexpression of median mRNA levels of BAX was also observed in patients with stage III OA compared with the control (P=0.034), while the BCL2/BAX ratio was markedly (2.5‑fold) decreased (P=0.024). A marked positive correlation was observed between the mRNA levels of BAX and BCL2 in the control group (r(s)=0.728; P<0.001), which was also present in the OA group, although to a lesser degree (r(s)=0.532; P<0.001). These results further implicate apoptosis in the pathogenesis of OA, through molecular mechanisms, which include the aberrant expression of the

  8. Unsupervised definition of the tibia-femoral joint regions of the human knee and its applications to cartilage analysis

    Science.gov (United States)

    Tamez-Peña, José G.; Barbu-McInnis, Monica; Totterman, Saara

    2006-03-01

    Abnormal MR findings including cartilage defects, cartilage denuded areas, osteophytes, and bone marrow edema (BME) are used in staging and evaluating the degree of osteoarthritis (OA) in the knee. The locations of the abnormal findings have been correlated to the degree of pain and stiffness of the joint in the same location. The definition of the anatomic region in MR images is not always an objective task, due to the lack of clear anatomical features. This uncertainty causes variance in the location of the abnormality between readers and time points. Therefore, it is important to have a reproducible system to define the anatomic regions. This works present a computerized approach to define the different anatomic knee regions. The approach is based on an algorithm that uses unique features of the femur and its spatial relation in the extended knee. The femur features are found from three dimensional segmentation maps of the knee. From the segmentation maps, the algorithm automatically divides the femur cartilage into five anatomic regions: trochlea, medial weight bearing area, lateral weight bearing area, posterior medial femoral condyle, and posterior lateral femoral condyle. Furthermore, the algorithm automatically labels the medial and lateral tibia cartilage. The unsupervised definition of the knee regions allows a reproducible way to evaluate regional OA changes. This works will present the application of this automated algorithm for the regional analysis of the cartilage tissue.

  9. Inhibition of oncostatin M in osteoarthritic synovial fluid enhances GAG production in osteoarthritic cartilage repair

    OpenAIRE

    Beekhuizen, M.; GJVM van Osch; AGJ Bot; MCL Hoekstra; DBF Saris; WJA Dhert; LB Creemers

    2013-01-01

    Mediators in the synovial fluid are thought to play a major role in osteoarthritic cartilage turnover. The purpose of the current study was to investigate the role of oncostatin M (OSM) in osteoarthritis (OA) by evaluating the presence of the cytokine and its receptors in the OA joint and interfering with its activity in synovial fluid co-cultured with cartilage explants. OSM levels were increased in the synovial fluid of osteoarthritic patients compared to healthy donors. Immunohistochemistr...

  10. The in vitro and in vivo capacity of culture-expanded human cells from several sources encapsulated in alginate to form cartilage

    Directory of Open Access Journals (Sweden)

    MM Pleumeekers

    2014-04-01

    Full Text Available Cartilage has limited self-regenerative capacity. Tissue engineering can offer promising solutions for reconstruction of missing or damaged cartilage. A major challenge herein is to define an appropriate cell source that is capable of generating a stable and functional matrix. This study evaluated the performance of culture-expanded human chondrocytes from ear (EC, nose (NC and articular joint (AC, as well as bone-marrow-derived and adipose-tissue-derived mesenchymal stem cells both in vitro and in vivo. All cells (≥ 3 donors per source were culture-expanded, encapsulated in alginate and cultured for 5 weeks. Subsequently, constructs were implanted subcutaneously for 8 additional weeks. Before and after implantation, glycosaminoglycan (GAG and collagen content were measured using biochemical assays. Mechanical properties were determined using stress-strain-indentation tests. Hypertrophic differentiation was evaluated with qRT-PCR and subsequent endochondral ossification with histology. ACs had higher chondrogenic potential in vitro than the other cell sources, as assessed by gene expression and GAG content (p < 0.001. However, after implantation, ACs did not further increase their matrix. In contrast, ECs and NCs continued producing matrix in vivo leading to higher GAG content (p < 0.001 and elastic modulus. For NC-constructs, matrix-deposition was associated with the elastic modulus (R2 = 0.477, p = 0.039. Although all cells – except ACs – expressed markers for hypertrophic differentiation in vitro, there was no bone formed in vivo. Our work shows that cartilage formation and functionality depends on the cell source used. ACs possess the highest chondrogenic capacity in vitro, while ECs and NCs are most potent in vivo, making them attractive cell sources for cartilage repair.

  11. Chondroitin sulphate and heparan sulphate sulphation motifs and their proteoglycans are involved in articular cartilage formation during human foetal knee joint development.

    Science.gov (United States)

    Melrose, James; Isaacs, Marc D; Smith, Susan M; Hughes, Clare E; Little, Christopher B; Caterson, Bruce; Hayes, Anthony J

    2012-09-01

    Novel sulphation motifs within the glycosaminoglycan chain structure of chondroitin sulphate (CS) containing proteoglycans (PGs) are associated with sites of growth, differentiation and repair in many biological systems and there is compelling evidence that they function as molecular recognition sites that are involved in the binding, sequestration or presentation of soluble signalling molecules (e.g. morphogens, growth factors and cytokines). Here, using monoclonal antibodies 3B3(-), 4C3 and 7D4, we examine the distribution of native CS sulphation motifs within the developing connective tissues of the human foetal knee joint, both during and after joint cavitation. We show that the CS motifs have broad, overlapping distributions within the differentiating connective tissues before the joint has fully cavitated; however, after cavitation, they all localise very specifically to the presumptive articular cartilage tissue. Comparisons with the labelling patterns of heparan sulphate (HS), HS-PGs (perlecan, syndecan-4 and glypican-6) and FGF-2, molecules with known signalling roles in development, indicate that these also become localised to the future articular cartilage tissue after joint cavitation. Furthermore, they display interesting, overlapping distributions with the CS motifs, reflective of early tissue zonation. The overlapping expression patterns of these molecules at this site suggests they are involved, or co-participate, in early morphogenetic events underlying articular cartilage formation; thus having potential clinical relevance to mechanisms involved in its repair/regeneration. We propose that these CS sulphation motifs are involved in modulating the signalling gradients responsible for the cellular behaviours (proliferation, differentiation, matrix turnover) that shape the zonal tissue architecture present in mature articular cartilage.

  12. Xylosyltransferase-I regulates glycosaminoglycan synthesis during the pathogenic process of human osteoarthritis.

    Directory of Open Access Journals (Sweden)

    Narayanan Venkatesan

    Full Text Available Loss of glycosaminoglycan (GAG chains of proteoglycans (PGs is an early event of osteoarthritis (OA resulting in cartilage degradation that has been previously demonstrated in both huma and experimental OA models. However, the mechanism of GAG loss and the role of xylosyltransferase-I (XT-I that initiates GAG biosynthesis onto PG molecules in the pathogenic process of human OA are unknown. In this study, we have characterized XT-I expression and activity together with GAG synthesis in human OA cartilage obtained from different regions of the same joint, defined as "normal", "late-stage" or adjacent to "late-stage". The results showed that GAG synthesis and content increased in cartilage from areas flanking OA lesions compared to cartilage from macroscopically "normal" unaffected regions, while decreased in "late-stage" OA cartilage lesions. This increase in anabolic state was associated with a marked upregulation of XT-I expression and activity in cartilage "next to lesion" while a decrease in the "late-stage" OA cartilage. Importantly, XT-I inhibition by shRNA or forced-expression with a pCMV-XT-I construct correlated with the modulation of GAG anabolism in human cartilage explants. The observation that XT-I gene expression was down-regulated by IL-1β and up-regulated by TGF-β1 indicates that these cytokines may play a role in regulating GAG content in human OA. Noteworthy, expression of IL-1β receptor (IL-1R1 was down-regulated whereas that of TGF-β1 was up-regulated in early OA cartilage. Theses observations may account for upregulation of XT-I and sustained GAG synthesis prior to the development of cartilage lesions during the pathogenic process of OA.

  13. Xylosyltransferase-I regulates glycosaminoglycan synthesis during the pathogenic process of human osteoarthritis.

    Science.gov (United States)

    Venkatesan, Narayanan; Barré, Lydia; Bourhim, Mustapha; Magdalou, Jacques; Mainard, Didier; Netter, Patrick; Fournel-Gigleux, Sylvie; Ouzzine, Mohamed

    2012-01-01

    Loss of glycosaminoglycan (GAG) chains of proteoglycans (PGs) is an early event of osteoarthritis (OA) resulting in cartilage degradation that has been previously demonstrated in both huma and experimental OA models. However, the mechanism of GAG loss and the role of xylosyltransferase-I (XT-I) that initiates GAG biosynthesis onto PG molecules in the pathogenic process of human OA are unknown. In this study, we have characterized XT-I expression and activity together with GAG synthesis in human OA cartilage obtained from different regions of the same joint, defined as "normal", "late-stage" or adjacent to "late-stage". The results showed that GAG synthesis and content increased in cartilage from areas flanking OA lesions compared to cartilage from macroscopically "normal" unaffected regions, while decreased in "late-stage" OA cartilage lesions. This increase in anabolic state was associated with a marked upregulation of XT-I expression and activity in cartilage "next to lesion" while a decrease in the "late-stage" OA cartilage. Importantly, XT-I inhibition by shRNA or forced-expression with a pCMV-XT-I construct correlated with the modulation of GAG anabolism in human cartilage explants. The observation that XT-I gene expression was down-regulated by IL-1β and up-regulated by TGF-β1 indicates that these cytokines may play a role in regulating GAG content in human OA. Noteworthy, expression of IL-1β receptor (IL-1R1) was down-regulated whereas that of TGF-β1 was up-regulated in early OA cartilage. Theses observations may account for upregulation of XT-I and sustained GAG synthesis prior to the development of cartilage lesions during the pathogenic process of OA.

  14. Assessment of chemical species of lead accumulated in tidemarks of human articular cartilage by X-ray absorption near-edge structure analysis

    Energy Technology Data Exchange (ETDEWEB)

    Meirer, Florian [Atominstitut, Vienna University of Technology, 1020 Wien (Austria); MiNALab, CMM-Irst, Fondazione Bruno Kessler, Via Sommarive 18, 38123 Trento (Italy); Pemmer, Bernhard, E-mail: bpemmer@ati.ac.at [Atominstitut, Vienna University of Technology, 1020 Wien (Austria); Pepponi, Giancarlo [MiNALab, CMM-Irst, Fondazione Bruno Kessler, Via Sommarive 18, 38123 Trento (Italy); Zoeger, Norbert; Wobrauschek, Peter [Atominstitut, Vienna University of Technology, 1020 Wien (Austria); Sprio, Simone; Tampieri, Anna [Istituto di Scienza e Tecnologia dei Materiali Ceramici CNR, Faenca (Italy); Goettlicher, Joerg; Steininger, Ralph; Mangold, Stefan [Institute for Synchrotron Radiation, Karlsruhe Institute of Technology, Campus South, 76344 Eggenstein-Leopoldshafen (Germany); Roschger, Paul [Ludwig Boltzmann Institute of Osteology, Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, 4th Medical Department, Hanusch Hospital, Vienna (Austria); Berzlanovich, Andrea [Department of Forensic Medicine, Medical University of Vienna, A-1090 Vienna (Austria); Hofstaetter, Jochen G. [Ludwig Boltzmann Institute of Osteology, Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, 4th Medical Department, Hanusch Hospital, Vienna (Austria); Department of Orthopaedics, Vienna General Hospital, Medical University of Vienna, A-1090 Vienna (Austria); Streli, Christina [Atominstitut, Vienna University of Technology, 1020 Wien (Austria)

    2011-03-01

    Lead is a toxic trace element that shows a highly specific accumulation in the transition zone between calcified and non-calcified articular cartilage, the so-called ‘tidemark’. Excellent agreement has been found between XANES spectra of synthetic Pb-doped carbonated hydroxyapatite and spectra obtained in the tidemark region and trabecular bone of normal human samples, confirming that in both tissues Pb is incorporated into the hydroxyapatite crystal structure of bone. During this study the µ-XANES set-up at the SUL-X beamline at ANKA was tested and has proven to be well suited for speciation of lead in human mineralized tissue samples. A highly specific accumulation of the toxic element lead was recently measured in the transition zone between non-calcified and calcified normal human articular cartilage. This transition zone, the so-called ‘tidemark’, is considered to be an active calcification front of great clinical importance. However, little is known about the mechanisms of accumulation and the chemical form of Pb in calcified cartilage and bone. Using spatially resolved X-ray absorption near-edge structure analysis (µ-XANES) at the Pb L{sub 3}-edge, the chemical state of Pb in the osteochondral region was investigated. The feasibility of the µ-XANES set-up at the SUL-X beamline (ANKA synchrotron light source) was tested and confirmed by comparing XANES spectra of bulk Pb-reference compounds recorded at both the XAS and the SUL-X beamline at ANKA. The µ-XANES set-up was then used to investigate the tidemark region of human bone (two patella samples and one femoral head sample). The spectra recorded at the tidemark and at the trabecular bone were found to be highly correlated with the spectra of synthetic Pb-doped carbonated hydroxyapatite, suggesting that in both of these very different tissues Pb is incorporated into the hydroxyapatite structure.

  15. Imaging of articular cartilage

    Directory of Open Access Journals (Sweden)

    Bhawan K Paunipagar

    2014-01-01

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

  16. 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

    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....... MLPCs obtained from BioE Inc (St. Paul, MN, USA) were expanded, and subsequently cultured in a standard micromass pellet system. Pellets were cultured for 21 days in control or chondrogenic induction medium under 5% or 21% oxygen tension. Chondrogenic potential was evaluated by histology (alcian blue......Nano (Aarhus University, Denmark). Micromass pellets cultured in induction medium were larger with a more dense and well-defined spherical structure. GAG production in induced pellets was shown by alcian blue and safranin O staining with most GAG observed centrally in 21%-, and peripherally in 5%-oxygen...

  17. Human Suprapatellar Fat Pad-Derived Mesenchymal Stem Cells Induce Chondrogenesis and Cartilage Repair in a Model of Severe Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Ignacio Muñoz-Criado

    2017-01-01

    Full Text Available Cartilage degeneration is associated with degenerative bone and joint processes in severe osteoarthritis (OA. Spontaneous cartilage regeneration is extremely limited. Often the treatment consists of a partial or complete joint implant. Adipose-derived stem cell (ASC transplantation has been shown to restore degenerated cartilage; however, regenerative differences of ASC would depend on the source of adipose tissue. The infra- and suprapatellar fat pads surrounding the knee offer a potential autologous source of ASC for patients after complete joint substitution. When infrapatellar- and suprapatellar-derived stromal vascular fractions (SVF were compared, a significantly higher CD105 (+ population was found in the suprapatellar fat. In addition, the suprapatellar SVF exhibited increased numbers of colony formation units and a higher population doubling in culture compared to the infrapatellar fraction. Both the suprapatellar- and infrapatellar-derived ASC were differentiated in vitro into mature adipocytes, osteocytes, and chondrocytes. However, the suprapatellar-derived ASC showed higher osteogenic and chondrogenic efficiency. Suprapatellar-derived ASC transplantation in a severe OA mouse model significantly diminished the OA-associated knee inflammation and cartilage degenerative grade, significantly increasing the production of glycosaminoglycan and inducing endogenous chondrogenesis in comparison with the control group. Overall, suprapatellar-derived ASC offer a potential autologous regenerative treatment for patients with multiple degenerative OA.

  18. A histomorphometric and scanning electron microscopy study of human condylar cartilage and bone tissue changes in relation to age

    DEFF Research Database (Denmark)

    Paulsen, Hans Ulrik; Thomsen, J.S.; Hougen, Hans Petter

    1999-01-01

    . EXPERIMENTAL SETTING AND DESIGN: A histomorphometric and scanning electron microscopic analysis of cartilage characteristics and bone remodelling activity. The Departments of Orthodontics and Cell Biology at Aarhus University, Denmark. An autopsy sample of condyles from 20 individuals, 18-31 years of age...

  19. Kinematic biomechanical assessment of human articular cartilage transplants in the knee using 3-T MRI: an in vivo reproducibility study

    Energy Technology Data Exchange (ETDEWEB)

    Juras, Vladimir; Szomolanyi, Pavol [Medical University of Vienna, Department of Radiodiagnostics, MR Centre of Excellence, Vienna (Austria); Slovak Academy of Sciences, Department of Imaging Methods, Institute of Measurement Science, Bratislava (Slovakia); Ludwig Boltzmann Institute for Clinical and Experimental Traumatology, Austrian Cluster for Tissue Regeneration, Vienna (Austria); Welsch, Goetz H.; Pinker, Katja; Trattnig, Siegfried [Medical University of Vienna, Department of Radiodiagnostics, MR Centre of Excellence, Vienna (Austria); Ludwig Boltzmann Institute for Clinical and Experimental Traumatology, Austrian Cluster for Tissue Regeneration, Vienna (Austria); Millington, Steven [Royal National Orthopaedic Hospital, London, Stanmore (United Kingdom); Mamisch, Tallal C. [Inselspital, Orthopedic Surgery Department, Berne (Switzerland)

    2009-05-15

    The aims of this study were to examine the clinical feasibility and reproducibility of kinematic MR imaging with respect to changes in T{sub 2} in the femoral condyle articular cartilage. We used a flexible knee coil, which allows acquisition of data in different positions from 40 flexion to full extension during MR examinations. The reproducibility of T{sub 2} measurements was evaluated for inter-rater and inter-individual variability and determined as a coefficient of variation (CV) for each volunteer and rater. Three different volunteers were measured twice and regions of interest (ROIs) were selected by three raters at different time points. To prove the clinical feasibility of this method, 20 subjects (10 patients and 10 age- and sex-matched volunteers) were enrolled in the study. Inter-rater variability ranged from 2 to 9 and from 2 to 10% in the deep and superficial zones, respectively. Mean inter-individual variability was 7% for both zones. Different T{sub 2} values were observed in the superficial cartilage zone of patients compared with volunteers. Since repair tissue showed a different behavior in the contact zone compared with healthy cartilage, a possible marker for improved evaluation of repair tissue quality after matrix-associated autologous chondrocyte transplantation (MACT) may be available and may allow biomechanical assessment of cartilage transplants. (orig.)

  20. Footprint-free human induced pluripotent stem cells from articular cartilage with redifferentiation capacity: a first step toward a clinical-grade cell source.

    Science.gov (United States)

    Boreström, Cecilia; Simonsson, Stina; Enochson, Lars; Bigdeli, Narmin; Brantsing, Camilla; Ellerström, Catharina; Hyllner, Johan; Lindahl, Anders

    2014-04-01

    Human induced pluripotent stem cells (iPSCs) are potential cell sources for regenerative medicine; however, clinical applications of iPSCs are restricted because of undesired genomic modifications associated with most reprogramming protocols. We show, for the first time, that chondrocytes from autologous chondrocyte implantation (ACI) donors can be efficiently reprogrammed into iPSCs using a nonintegrating method based on mRNA delivery, resulting in footprint-free iPSCs (no genome-sequence modifications), devoid of viral factors or remaining reprogramming molecules. The search for universal allogeneic cell sources for the ACI regenerative treatment has been difficult because making chondrocytes with high matrix-forming capacity from pluripotent human embryonic stem cells has proven challenging and human mesenchymal stem cells have a predisposition to form hypertrophic cartilage and bone. We show that chondrocyte-derived iPSCs can be redifferentiated in vitro into cartilage matrix-producing cells better than fibroblast-derived iPSCs and on par with the donor chondrocytes, suggesting the existence of a differentiation bias toward the somatic cell origin and making chondrocyte-derived iPSCs a promising candidate universal cell source for ACI. Whole-genome single nucleotide polymorphism array and karyotyping were used to verify the genomic integrity and stability of the established iPSC lines. Our results suggest that RNA-based technology eliminates the risk of genomic integrations or aberrations, an important step toward a clinical-grade cell source for regenerative medicine such as treatment of cartilage defects and osteoarthritis.

  1. The epidermal growth factor-like domain of the human cartilage large aggregating proteoglycan, aggrecan: increased serum concentration in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Møller, H J; Ingemann-Hansen, T; Poulsen, J H

    1994-01-01

    The large aggregating proteoglycan from human cartilage, aggrecan, has recently been shown to possess an immunologically detectable domain with close homology to epidermal growth factor (EGF), that is variably expressed by alternative mRNA splicing. Using a competitive ELISA we detected this domain...... in sera from both patients with RA and normal controls. The EGF-like domain could only be detected after digestion of sera with chondroitinase ABC, which demonstrates its proteoglycan origin. The concentration of the aggrecan EGF-like domain was considerably elevated in sera from patients with RA...

  2. A Simple Method for Establishing Adherent Ex Vivo Explant Cultures from Human Eye Pathologies for Use in Subsequent Calcium Imaging and Inflammatory Studies

    Directory of Open Access Journals (Sweden)

    Sofija Andjelic

    2014-01-01

    Full Text Available A novel, simple, and reproducible method for cultivating pathological tissues obtained from human eyes during surgery was developed using viscoelastic material as a tissue adherent to facilitate cell attachment and expansion and calcium imaging of cultured cells challenged by mechanical and acetylcholine (ACh stimulation as well as inflammatory studies. Anterior lens capsule-lens epithelial cells (aLC-LECs from cataract surgery and proliferative diabetic retinopathy (PDR fibrovascular epiretinal membranes (fvERMs from human eyes were used in the study. We hereby show calcium signaling in aLC-LECs by mechanical and acetylcholine (ACh stimulation and indicate presence of ACh receptors in these cells. Furthermore, an ex vivo study model was established for measuring the inflammatory response in fvERMs and aLC-LECs upon TNFα treatment.

  3. The effect of culture medium and carrier on explant culture of human limbal epithelium: A comparison of ultrastructure, keratin profile and gene expression.

    Science.gov (United States)

    Pathak, Meeta; Olstad, O K; Drolsum, Liv; Moe, Morten C; Smorodinova, Natalia; Kalasova, Sarka; Jirsova, Katerina; Nicolaissen, Bjørn; Noer, Agate

    2016-12-01

    Patients with limbal stem cell deficiency (LSCD) often experience pain and photophobia due to recurrent epithelial defects and chronic inflammation of the cornea. Successfully restoring a healthy corneal surface in these patients by transplantation of ex vivo expanded human limbal epithelial cells (LECs) may alleviate these symptoms and significantly improve their quality of life. The clinical outcome of transplantation is known to be influenced by the quality of transplanted cells. Presently, several different protocols for cultivation and transplantation of LECs are in use. However, no consensus on an optimal protocol exists. The aim of this study was to examine the effect of culture medium and carrier on the morphology, staining of selected keratins and global gene expression in ex vivo cultured LECs. Limbal biopsies from cadaveric donors were cultured for three weeks on human amniotic membrane (HAM) or on tissue culture coated plastic (PL) in either a complex medium (COM), containing recombinant growth factors, hormones, cholera toxin and fetal bovine serum, or in medium supplemented only with human serum (HS). The expanded LECs were examined by light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) for keratins K3, K7, K8, K12, K13, K14, K15 and K19, as well as microarray and qRT-PCR analysis. The cultured LECs exhibited similar morphology and keratin staining on LM, TEM and IHC examination, regardless of the culture condition. The epithelium was multilayered, with cuboidal basal cells and flattened superficial cells. Cells were attached to each other by desmosomes. Adhesion complexes were observed between basal cells and the underlying carrier in LECs cultured on HAM, but not in LECs cultured on PL. GeneChip Human Gene 2.0 ST microarray (Affymetrix) analysis revealed that 18,653 transcripts were ≥2 fold up or downregulated (p ≤ 0.05). Cells cultured in the same medium (COM or HS) showed more similarities in gene

  4. Chondrocalcin is internalized by chondrocytes and triggers cartilage destruction via an interleukin-1β-dependent pathway.

    Science.gov (United States)

    Bantsimba-Malanda, Claudie; Cottet, Justine; Netter, Patrick; Dumas, Dominique; Mainard, Didier; Magdalou, Jacques; Vincourt, Jean-Baptiste

    2013-01-01

    Chondrocalcin is among the most highly synthesized polypeptides in cartilage. This protein is released from its parent molecule, type II pro-collagen, after secretion by chondrocytes. A participation of extracellular, isolated chondrocalcin in mineralization was proposed more than 25 years ago, but never demonstrated. Here, exogenous chondrocalcin was found to trigger MMP13 secretion and cartilage destruction ex vivo in human cartilage explants and did so by modulating the expression of interleukin-1β in primary chondrocyte cultures in vitro. Chondrocalcin was found internalized by chondrocytes. Uptake was found mediated by a single 18-mer peptide of chondrocalcin, which does not exhibit homology to any known cell-penetrating peptide. The isolated peptide, when artificially linked as a tetramer, inhibited gene expression regulation by chondrocalcin, suggesting a functional link between uptake and gene expression regulation. At the same time, the tetrameric peptide potentiated chondrocalcin uptake by chondrocytes, suggesting a cooperative mechanism of entry. The corresponding peptide from type I pro-collagen supported identical cell-penetration, suggesting that this property may be conserved among C-propeptides of fibrillar pro-collagens. Structural modeling localized this peptide to the tips of procollagen C-propeptide trimers. Our findings shed light on unexpected function and mechanism of action of these highly expressed proteins from vertebrates.

  5. Hyaline cartilage degenerates after autologous osteochondral transplantation.

    Science.gov (United States)

    Tibesku, C O; Szuwart, T; Kleffner, T O; Schlegel, P M; Jahn, U R; Van Aken, H; Fuchs, S

    2004-11-01

    Autologous osteochondral grafting is a well-established clinical procedure to treat focal cartilage defects in patients, although basic research on this topic remains sparse. The aim of the current study was to evaluate (1) histological changes of transplanted hyaline cartilage of osteochondral grafts and (2) the tissue that connects the transplanted cartilage with the adjacent cartilage in a sheep model. Both knee joints of four sheep were opened surgically and osteochondral grafts were harvested and simultaneously transplanted to the contralateral femoral condyle. The animals were sacrificed after three months and the received knee joints were evaluated histologically. Histological evaluation showed a complete ingrowth of the osseous part of the osteochondral grafts. A healing or ingrowth at the level of the cartilage could not be observed. Histological evaluation of the transplanted grafts according to Mankin revealed significantly more and more severe signs of degeneration than the adjacent cartilage, such as cloning of chondrocytes and irregularities of the articular surface. We found no connecting tissue between the transplanted and the adjacent cartilage and histological signs of degeneration of the transplanted hyaline cartilage. In the light of these findings, long-term results of autologous osteochondral grafts in human beings have to be followed critically.

  6. Cartilage and soft tissue imaging using X-rays: propagation-based phase-contrast computed tomography of the human knee in comparison with clinical imaging techniques and histology.

    Science.gov (United States)

    Horng, Annie; Brun, Emmanuel; Mittone, Alberto; Gasilov, Sergei; Weber, Loriane; Geith, Tobias; Adam-Neumair, Silvia; Auweter, Sigrid D; Bravin, Alberto; Reiser, Maximilian F; Coan, Paola

    2014-09-01

    This study evaluates high-resolution tomographic x-ray phase-contrast imaging in whole human knee joints for the depiction of soft tissue with emphasis on hyaline cartilage. The method is compared with conventional computed tomography (CT), synchrotron radiation absorption-based CT, and magnetic resonance imaging (MRI). After approval of the institutional review board, 2 cadaveric human knees were examined at an synchrotron institution using a monochromatic x-ray beam of 60 keV, a detector with a 90-mm field of view, and a pixel size of 46 × 46 μm. Images of phase-contrast imaging CT were reconstructed with the filtered back projection algorithm and the equally sloped tomography method. Image quality and tissue contrast were evaluated and compared in all modalities and with histology. Phase-contrast imaging provides visualization of altered cartilage regions invisible in absorption CT with simultaneous high detail of the underlying bony abnormalities. The delineation of surface changes is similar to 3-T MRI using cartilage-dedicated sequences. Phase-contrast imaging CT presents soft tissue contrast surpassing that of conventional CT with a clear discrimination of ligamentous, muscular, neural, and vascular structures. In addition, phase-contrast imaging images show cartilage and meniscal calcifications that are not perceptible on conventional CT or on MRI. Phase-contrast imaging CT may facilitate a more complete evaluation of the human knee joint by providing concurrent comprehensive information about cartilage, the underlying subchondral bone, and their changes in osteoarthritic conditions.

  7. Biochemical markers of type II collagen breakdown and synthesis are positioned at specific sites in human osteoarthritic knee cartilage

    DEFF Research Database (Denmark)

    Jensen, Anne-Christine Bay; Levin Andersen, Thomas; Charni-Ben Tabassi, N;

    2007-01-01

    as for type IIA N propeptide (PIIANP), a biochemical marker reflecting synthesis of type IIA collagen. RESULTS: Helix-II and CTX-II were detected in areas where collagen damage was reported previously, most frequently around chondrocytes, but also frequently in regions not previously investigated......OBJECTIVE: To investigate whether type II collagen turnover markers used for osteoarthritis (OA) activity evaluation in body fluids can be detected at the level of specific histological features of OA cartilage tissue, as well as how they relate with each other at this level. METHODS: Adjacent...... sections were obtained from full-depth cartilage biopsies from 32 OA knees. Immunohistochemistry was performed for Helix-II and CTX-II, which are type II collagen fragments originating from the triple helix and the telopeptide region, respectively, and believed to reflect distinct breakdown events, as well...

  8. Moderate loading of the human osteoarthritic knee joint leads to lowering of intraarticular cartilage oligomeric matrix protein

    DEFF Research Database (Denmark)

    Helmark, Ida C; Petersen, Marie C H; Christensen, Helle E

    2012-01-01

    osteoarthritic joint determined by biochemical markers of cartilage turnover and inflammation in the synovial fluid (SF), serum and urine. Eleven subjects with OA of the knee(s), but with no other joint- or inflammatory disorders, volunteered for the study and had samples of blood, urine and synovial fluid drawn...... both at baseline and following 30-min one-legged knee-extension exercise. Workload: 60% of 1 RM (Repetition Maximum). Determination of cartilage oligomeric matrix protein (COMP), aggrecan, C-terminal collagen II peptide (CTX-II) and interleukin (IL)-6 were performed in synovial fluid (SF), serum...... and urine. A significant decrease was found in SF concentration of COMP following exercise, whereas aggrecan, CTX-II and IL-6 remained unchanged. No differences in any of the tested markers were found in serum and urine between baseline and post-exercise. Thirty minutes of mechanical loading of a single...

  9. Chondrogenic Differentiation Capacity of Human Umbilical Cord Mesenchymal Stem Cells with Platelet Rich Fibrin Scaffold in Cartilage Regeneration (In Vitro Study

    Directory of Open Access Journals (Sweden)

    Ni Putu Mira Sumarta

    2016-09-01

    Full Text Available Background: Human umbilical cord mesenchymal stem cell is a promising source of allogenous MSC with great chondrogenic differentiation capacity. Meanwhile, platelet rich fibrin (PRF is a natural fibrin matrix, rich in growth factors, forming a smooth and flexible fibrin network, supporting cytokines and cell migration, thus can be used as a scaffold that facilitate the differentiation of MSC. However, the differential capability of MSC cultured in PRF was still poorly understood. Method: We studied in vitro differentiation potential of MSC cultured in PRF by evaluating several markers such as FGF 18, Sox 9, type II collagen, aggrecan in 3 different culture medium. Result: The result showed that there was positive expression of FGF 18, Sox 9, type II collagen, aggrecan in all medium of in vitro culture. Conclusion: MSC cultured from human umbilical cord had the capacity of chondrogenic differentiation and able to produce cartilage extracellular matrix in vitro which means that hUCMSC is a potential allogeneic MSC for cartilage regeneration.

  10. Study of physical, chemical and structural effects caused by ionizing radiation and preservation on human costal cartilage; Estudo dos efeitos fisicos, quimicos e estruturais ocasionados pela radiacao ionizante e preservacao em cartilagem costal humana

    Energy Technology Data Exchange (ETDEWEB)

    Martinho Junior, Antonio Carlos

    2008-07-01

    Tissue Banks around the world have stored human cartilages obtained from cadaver donors for use in several kinds of reconstructive surgeries. To ensure that such tissues are not contaminated, they have been sterilized with ionizing radiation. However, high doses of gamma radiation may cause undesirable changes in the tissues, decreasing the mechanical properties of the grafts. In this work, we evaluate physical/chemical and structural changes in deep-frozen (-70 deg C) or high concentration of glycerol (> 98%) preserved costal cartilage, before and after sterilization by ionizing radiation at 3 different doses (15, 25 and 50 kGy). Samples of human costal cartilage were obtained from 20 cadaver donors ranging between 18 and 55 years old. A {sup 60}Co irradiator was used as irradiation source. Thermogravimetry (TG), Optical Coherence Tomography (OCT) and mechanical tension and compression tests were carried out to evaluate the changes in the cartilage. Regarding the thermogravimetric results, the obtained data has shown that the TG curves have the same pattern independently of the sample irradiated or not. On the other hand, non-irradiated samples showed great variability of thermogravimetric curves among different donors and for the same donor. Concerning the mechanical tests, when cartilages were irradiated with 15 kGy, their mechanical strength to tension was increased about 24%, in both deep-froze and preserved in glycerol samples. Samples deep-frozen, when irradiated with 25 and 50 kGy, presented a decrease of their mechanical behavior smaller than those preserved in high concentrations of glycerol and irradiated with the same dose. Therefore, deep-frozen cartilages can be sterilized with doses until 50 kGy and cartilages preserved in high concentrations of glycerol can be sterilized with doses until 25 kGy without significant changes in their bio-mechanical properties.(author)

  11. Differential expression of pathogenic genes of Entamoeba histolytica vs E. dispar in a model of infection using human liver tissue explants

    Science.gov (United States)

    González, Enrique; Magaña, Ulises; Morán, Patricia; Gudiño-Zayas, Marco; Partida, Oswaldo; Hernández, Eric; Rojas-Velázquez, Liliana; Maldonado, Héctor

    2017-01-01

    We sought to establish an ex vivo model for examining the interaction of E. histolytica with human tissue, using precision-cut liver slices (PCLS) from donated organs. E. histolytica- or E. dispar-infected PCLS were analyzed at different post-infection times (0, 1, 3, 24 and 48 h) to evaluate the relation between tissue damage and the expression of genes associated with three factors: a) parasite survival (peroxiredoxin, superoxide dismutase and 70 kDa heat shock protein), b) parasite virulence (EhGal/GalNAc lectin, amoebapore, cysteine proteases and calreticulin), and c) the host inflammatory response (various cytokines). Unlike E. dispar (non-pathogenic), E. histolytica produced some damage to the structure of hepatic parenchyma. Overall, greater expression of virulence genes existed in E. histolytica-infected versus E. dispar-infected tissue. Accordingly, there was an increased expression of EhGal/GalNAc lectin, Ehap-a and Ehcp-5, Ehcp-2, ehcp-1 genes with E. histolytica, and a decreased or lack of expression of Ehcp-2, and Ehap-a genes with E. dispar. E. histolytica-infected tissue also exhibited an elevated expression of genes linked to survival, principally peroxiredoxin, superoxide dismutase and Ehhsp-70. Moreover, E. histolytica-infected tissue showed an overexpression of some genes encoding for pro-inflammatory interleukins (ILs), such as il-8, ifn-γ and tnf-α. Contrarily, E. dispar-infected tissue displayed higher levels of il-10, the gene for the corresponding anti-inflammatory cytokine. Additionally, other genes were investigated that are important in the host-parasite relationship, including those encoding for the 20 kDa heat shock protein (HSP-20), the AIG-1 protein, and immune dominant variable surface antigen, as well as for proteins apparently involved in mechanisms for the protection of the trophozoites in different environments (e.g., thioredoxin-reductase, oxido-reductase, and 9 hypothetical proteins). Some of the hypothetical proteins

  12. Human Articular Cartilage Progenitor Cells Are Responsive to Mechanical Stimulation and Adenoviral-Mediated Overexpression of Bone-Morphogenetic Protein 2.

    Directory of Open Access Journals (Sweden)

    Alexander J Neumann

    Full Text Available Articular cartilage progenitor cells (ACPCs represent a new and potentially powerful alternative cell source to commonly used cell sources for cartilage repair, such as chondrocytes and bone-marrow derived mesenchymal stem cells (MSCs. This is particularly due to the apparent resistance of ACPCs to hypertrophy. The current study opted to investigate whether human ACPCs (hACPCs are responsive towards mechanical stimulation and/or adenoviral-mediated overexpression of bone morphogenetic protein 2 (BMP-2. hACPCs were cultured in fibrin-polyurethane composite scaffolds. Cells were cultured in a defined chondro-permissive medium, lacking exogenous growth factors. Constructs were cultured, for 7 or 28 days, under free-swelling conditions or with the application of complex mechanical stimulation, using a custom built bioreactor that is able to generate joint-like movements. Outcome parameters were quantification of BMP-2 and transforming growth factor beta 1 (TGF-β1 concentration within the cell culture medium, biochemical and gene expression analyses, histology and immunohistochemistry. The application of mechanical stimulation alone resulted in the initiation of chondrogenesis, demonstrating the cells are mechanoresponsive. This was evidenced by increased GAG production, lack of expression of hypertrophic markers and a promising gene expression profile (significant up-regulation of cartilaginous marker genes, specifically collagen type II, accompanied by no increase in the hypertrophic marker collagen type X or the osteogenic marker alkaline phosphatase. To further investigate the resistance of ACPCs to hypertrophy, overexpression of a factor associated with hypertrophic differentiation, BMP-2, was investigated. A novel, three-dimensional, transduction protocol was used to transduce cells with an adenovirus coding for BMP-2. Over-expression of BMP-2, independent of load, led to an increase in markers associated with hypertropy. Taken together ACPCs

  13. Induction of embryogenics calli in cupuassu explants

    OpenAIRE

    Maria das Graças Rodrigues Ferreira; Fernando Enrique Ninamango Cárdenas; Carlos Henrique Siqueira de Carvalho; Andréa Almeida Carneiro; Carlos Ferreira Damião Filho

    2004-01-01

    Objetivou-se a indução de calos embriogênicos em cupuaçuzeiro, em função do tipo de explante e meio de cultura. Foram testados como explantes, segmentos cotiledonares e eixos embrionários divididos em três partes: região da plúmula, radícula e hipocótilo. Os explantes foram cultivados em 2 diferentes meios de cultura: 1) MS suplementado com 2,4-D (1 mg L-1) e Cinetina (0,25 mg L-1); 2) MS acrescido de ANA (5 mg L-1) e Cinetina (0,25 mg L-1). Constatou-se que a região do hipocótilo foi a parte...

  14. PGE2 And Its Cognate EP Receptors Control Human Adult Articular Cartilage Homeostasis and Are Linked to the Pathophysiology of Osteoarthritis

    Science.gov (United States)

    Li, Xin; Ellman, Michael; Muddasani, Prasuna; Wang, James H-C; Cs-Szabo, Gabriella; van Wijnen, Andre J; Im, Hee-Jeong

    2009-01-01

    Objective To elucidate the pathophysiologic links between prostaglandin E2 (PGE2) and osteoarthritis by characterizing the catabolic effects of PGE2 and its unique receptors in human adult articular chondrocytes. Methods Human adult articular chondrocytes were cultured in monolayer or alginate beads with and without PGE2 and/or agonist, antagonist of EP receptors and cytokines. Cell survival, proliferation, and total proteoglycan synthesis and accumulation were measured in alginate beads. Chondrocyte-related gene expression and PI3k/Akt signaling were assessed by real-time PCR and western blotting, respectively, using a monolayer cell culture model. Results Stimulation of human articular chondrocytes with PGE2 through the EP2 receptor (i) suppresses proteoglycan accumulation and synthesis, (ii) suppresses aggrecan gene expression, (iii) does not appreciably affect expression of matrix-degrading enzymes; and (iv) decreases the collagen II:I ratio. EP2 and EP4 receptors are expressed at higher levels in knee compared to ankle cartilage, and in a grade-dependent fashion. PGE2 titration combined with IL-1 synergistically accelerates expression of pain-associated molecules such as inducible nitric oxide synthase (iNOS) and IL-6. Finally, stimulation with exogenous PGE2 or an EP2 agonist inhibits activation of Akt that is induced by insulin-like growth factor (IGF-1). Conclusion PGE2 exerts an anti-anabolic effect on human adult articular cartilage in vitro, and EP2/4 receptor antagonists may represent effective therapeutic agents for the treatment of osteoarthritis. PMID:19180509

  15. Platelet rich plasma (PRP) induces chondroprotection via increasing autophagy, anti-inflammatory markers, and decreasing apoptosis in human osteoarthritic cartilage.

    Science.gov (United States)

    Moussa, Mayssam; Lajeunesse, Daniel; Hilal, George; El Atat, Oula; Haykal, Gaby; Serhal, Rim; Chalhoub, Antonio; Khalil, Charbel; Alaaeddine, Nada

    2017-03-01

    Autophagy constitutes a defense mechanism to overcome aging and apoptosis in osteoarthritic cartilage. Several cytokines and transcription factors are linked to autophagy and play an important role in the degradative cascade in osteoarthritis (OA). Cell therapy such as platelet rich plasma (PRP) has recently emerged as a promising therapeutic tool for many diseases including OA. However, its mechanism of action on improving cartilage repair remains to be determined. The purpose of this study is to investigate the effect of PRP on osteoarthritic chondrocytes and to elucidate the mechanism by which PRP contributes to cartilage regeneration. Osteoarthritic chondrocytes were co-cultured with an increasing concentration of PRP obtained from healthy donors. The effect of PRP on the proliferation of chondrocytes was performed using cell counting and WST8 proliferation assays. Autophagy, apoptosis and intracellular level of IL-4, IL-10, and IL-13 were determined using flow cytometry analyses. Autophagy markers BECLIN and LC3II were also determined using quantitative polymerase chain reaction (qPCR). qPCR and ELISA were used to measure the expression of ADAMDTS-5, MMP3, MMP13, TIMP-1-2-3, aggregan, Collagen type 2, TGF-β, Cox-2, Il-6, FOXO1, FOXO3, and HIF-1 in tissues and co-cultured media. PRP increased significantly the proliferation of chondrocytes, decreased apoptosis and increased autophagy and its markers along with its regulators FOXO1, FOXO3 and HIF-1 in osteoarthritic chondrocytes. Furthermore, PRP caused a dose-dependent significant decrease in MMP3, MMP13, and ADAMTS-5, IL-6 and COX-2 while increasing TGF-β, aggregan, and collagen type 2, TIMPs and intracellular IL-4, IL-10, IL-13. These results suggest that PRP could be a potential therapeutic tool for the treatment of OA. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Proliferation and differentiation of adult human dental pulp cells cultured by tissue explant method%组织块法培养成体人牙髓细胞的增殖及分化状态水

    Institute of Scientific and Technical Information of China (English)

    姜新朋; 张颖丽; 黄洋; 郭世梁

    2009-01-01

    并保持低分化状态.%BACKGROUND: Human pulp tissue has been known to be less, and exhibit poor tolerance to enzymatic digestion and less adherent cells after step-by-step digestion of trypsin and collagenase, thereby often leading to a failure of passage. Only several kinds of dental pulp cells with poor activity can be obtained by the tissue explant-collagenase digestion. OBJECTIVE: To investigate human dental pulp cells cultured in vitro by tissue explant method. DESIGN, TIME AND SETTING: A cytological observation was performed at Heping Campus and School of Stomatology, Jilin University from 2005 to 2007. MATERIALS: Healthy young human teeth extracted for orthodontic correction or impaction. METHODS: Pulp tissue from the third molar teeth was collected, cut into small blocks with a size of 1.0 mm×1.0 mm×0.5 mm under the infiltration of small amount of Dulbecco's modified eagle's medium, and then transferred into a 6-well plate containing culture medium for incubation in a 5% CO2 and saturated humidity atmosphere at 37 ℃. During the process of incubation, pulp tissue was adjusted at a density of 3-6 blocks/well, with an equal spacing of 0.5 cm and the 6-well plate was kept inverted. Three hours later, the 6-well plate was turned over to make tissue blocks adhering to the plate wall. Culture was continued after addition of 2 mL of culture medium. Culture medium was renewed every 4-6 days. After 6-15 days, cells emigrated from the edge of tissue blocks and call outgrowth appeared around each tissue block. When cells closed to confluency, a digestion procedure of 2.0-3.0 minutes (0.25% trypsin and 0.02% ethylenadiamine tetraacetic acid) was followed by passage culture at a proportion of 1: (2-3) in 25 mL of culture flasks. Purified fibroblast-like cells were gradually obtained from primarily cultured cells by repeated digestion and passage. MAIN OUTCOME MEASURES: Cellular morphology was identified by immunohistochemistry; secreted dental pulp cells were determined using alkaline phosphatase activity

  17. Ex Vivo Produced Oral Mucosa Equivalent by Using the Direct Explant Cell Culture Technique

    Directory of Open Access Journals (Sweden)

    Kamile Öztürk

    2012-09-01

    Full Text Available Objective: The aim of this study is the histological and immunohistochemical evaluation of ex vivo produced oral mucosal equivalents using keratinocytes cultured by direct explant technique.Material and Methods: Oral mucosa tissue samples were obtained from the keratinized gingival tissues of 14 healthy human subjects. Human oral mucosa keratinocytes from an oral mucosa biopsy specimen were dissociated by the explant technique. Once a sufficient population of keratinocytes was reached, they were seeded onto the type IV collagen coated “AlloDerm” and taken for histological and immunohistochemical examinations at 11 days postseeding of the keratinocytes on the cadaveric human dermal matrix.Results: Histopathologically and immunohistochemically, 12 out of 14 successful ex vivo produced oral mucosa equivalents (EVPOME that consisted of a stratified epidermis on a dermal matrix have been developed with keratinocytes cultured by the explant technique.Conclusion: The technical handling involved in the direct explant method at the beginning of the process has fewer steps than the enzymatic method and use of the direct explant technique protocol for culturing of human oral mucosa keratinocyte may be more adequate for EVPOME production.

  18. Induction of tolerance with intranasal administration of human cartilage gp-39 in DBA/1 mice - Amelioration of clinical, histologic, and radiologic signs of type II collagen-induced arthritis

    NARCIS (Netherlands)

    Joosten, LAB; Coenen-de Roo, CJJ; Helsen, MMA; Lubberts, E; Boots, AMH; van den Berg, WB; Miltenburg, AMM

    2000-01-01

    Objective. Human cartilage glycoprotein 39 (HC gp-39) was recently identified as a candidate autoantigen in the pathogenesis of rheumatoid arthritis, In the present studies, we investigated the capacity of HC gp-39 to interfere in clinical disease induced by an unrelated autoantigen, type II collage

  19. Crosslinking by advanced glycation end products increases the stiffness of the collagen network in human articular cartilage: A possible mechanism through which age is a risk factor for osteoarthritis

    NARCIS (Netherlands)

    Verzijl, N.; Groot, J. de; Zaken, C.B.; Braun-Benjamin, O.; Maroudas, A.; Bank, R.A.; Mizrahi, J.; Schalkwijk, C.G.; Thorpe, S.R.; Baynes, J.W.; Bijlsma, J.W.J.; Lafeber, F.P.J.G.; TeKoppele, J.M.

    2002-01-01

    Objective. Age is an important risk factor for osteoarthritis (OA). During aging, nonenzymatic glycation results in the accumulation of advanced glycation end products (AGEs) in cartilage collagen. We studied the effect of AGE crosslinking on the stiffness of the collagen network in human articular

  20. Crosslinking by advanced glycation end products increases the stiffness of the collagen network in human articular cartilage: A possible mechanism through which age is a risk factor for osteoarthritis

    NARCIS (Netherlands)

    Verzijl, N.; Groot, J. de; Zaken, C.B.; Braun-Benjamin, O.; Maroudas, A.; Bank, R.A.; Mizrahi, J.; Schalkwijk, C.G.; Thorpe, S.R.; Baynes, J.W.; Bijlsma, J.W.J.; Lafeber, F.P.J.G.; TeKoppele, J.M.

    2002-01-01

    Objective. Age is an important risk factor for osteoarthritis (OA). During aging, nonenzymatic glycation results in the accumulation of advanced glycation end products (AGEs) in cartilage collagen. We studied the effect of AGE crosslinking on the stiffness of the collagen network in human articular

  1. Loading and Boundary Condition Influences in a Poroelastic Finite Element Model of Cartilage Stresses in a Triaxial Compression Bioreactor

    Science.gov (United States)

    Kallemeyn, Nicole A; Grosland, Nicole M; Pedersen, Doug R; Martin, James A; Brown, Thomas D

    2006-01-01

    Background: We developed a poroelastic finite element (FE) model of cartilage in dynamic triaxial compression to parametrically analyze the effects of loading and boundary conditions on a baseline model. Conventional mechanical tests on articular cartilage such as confined and unconfined compression, indentation, etc., do not fully allow for modulation of compression and shear at physiological levels whereas triaxial compression does. A Triaxial Compression Bioreactor, or TRIAX, has been developed to study chondrocyte responses to multi-axial stress conditions under cyclic loading. In the triaxial setting, however, a cartilage explant's physical testing environment departs from the ideal homogeneous stress state that would occur from strict linear superposition of the applied axial and transverse pressure. Method of Approach: An axisymmetric poroelastic FE model of a cartilage explant (4 mm diameter, 1.5 mm thick) in cyclic triaxial compression was created. Axial and transverse loads (2 MPa at 1 Hz.) were applied via a platen and containment sheath. Parameters of interest included the rise time and magnitude of the applied load, in addition to the containment sheath modulus and the friction coefficient at the cartilage/platen interfaces. Metrics of interest in addition to whole explant axial strain included axial (surface normal) stress, shear stress, pore pressure, and the fluid load carriage fraction within the explant. Results: Strain results were compared to experimental data from explants tested in the TRIAX under conditions similar to the baseline model. Explant biomechanics varied considerably over numbers of load cycles and parameter values. Cyclic loading caused an increase in accumulated strain for the various loading and boundary conditions. Conclusions: Unlike what would be expected from linear superposition of the homogeneous stresses from the applied axial and transverse pressure, we have shown that the stress state within the TRIAX is considerably

  2. Characterization of Myelomonocytoid Progenitor Cells with Mesenchymal Differentiation Potential Obtained by Outgrowth from Pancreas Explants

    Directory of Open Access Journals (Sweden)

    Marc-Estienne Roehrich

    2012-01-01

    Full Text Available Progenitor cells can be obtained by outgrowth from tissue explants during primary ex vivo tissue culture. We have isolated and characterized cells outgrown from neonatal mouse pancreatic explants. A relatively uniform population of cells showing a distinctive morphology emerged over time in culture. This population expressed monocyte/macrophage and hematopoietic markers (CD11b+ and CD45+, and some stromal-related markers (CD44+ and CD29+, but not mesenchymal stem cell (MSC-defining markers (CD90− and CD105− nor endothelial (CD31− or stem cell-associated markers (CD133− and stem cell antigen-1; Sca-1−. Cells could be maintained in culture as a plastic-adherent monolayer in culture medium (MesenCult MSC for more than 1 year. Cells spontaneously formed sphere clusters “pancreatospheres” which, however, were nonclonal. When cultured in appropriate media, cells differentiated into multiple mesenchymal lineages (fat, cartilage, and bone. Positive dithizone staining suggested that a subset of cells differentiated into insulin-producing cells. However, further studies are needed to characterize the endocrine potential of these cells. These findings indicate that a myelomonocytoid population from pancreatic explant outgrowths has mesenchymal differentiation potential. These results are in line with recent data onmonocyte-derivedmesenchymal progenitors (MOMPs.

  3. Direct Quantification of Solute Diffusivity in Agarose and Articular Cartilage Using Correlation Spectroscopy.

    Science.gov (United States)

    Shoga, Janty S; Graham, Brian T; Wang, Liyun; Price, Christopher

    2017-06-13

    Articular cartilage is an avascular tissue; diffusive transport is critical for its homeostasis. While numerous techniques have been used to quantify diffusivity within porous, hydrated tissues and tissue engineered constructs, these techniques have suffered from issues regarding invasiveness and spatial resolution. In the present study, we implemented and compared two separate correlation spectroscopy techniques, fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS), for the direct, and minimally-invasive quantification of fluorescent solute diffusion in agarose and articular cartilage. Specifically, we quantified the diffusional properties of fluorescein and Alexa Fluor 488-conjugated dextrans (3k and 10k) in aqueous solutions, agarose gels of varying concentration (i.e. 1, 3, 5%), and in different zones of juvenile bovine articular cartilage explants (i.e. superficial, middle, and deep). In agarose, properties of solute diffusion obtained via FCS and RICS were inversely related to molecule size, gel concentration, and applied strain. In cartilage, the diffusional properties of solutes were similarly dependent upon solute size, cartilage zone, and compressive strain; findings that agree with work utilizing other quantification techniques. In conclusion, this study established the utility of FCS and RICS as simple and minimally invasive techniques for quantifying microscale solute diffusivity within agarose constructs and articular cartilage explants.

  4. The chrondoprotective actions of a natural product are associated with the activation of IGF-1 production by human chondrocytes despite the presence of IL-1β

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    Bobrowski Paul

    2006-04-01

    Full Text Available Abstract Background Cartilage loss is a hallmark of arthritis and follows activation of catabolic processes concomitant with a disruption of anabolic pathways like insulin-like growth factor 1 (IGF-1. We hypothesized that two natural products of South American origin, would limit cartilage degradation by respectively suppressing catabolism and activating local IGF-1 anabolic pathways. One extract, derived from cat's claw (Uncaria guianensis, vincaria®, is a well-described inhibitor of NF-κB. The other extract, derived from the vegetable Lepidium meyenii (RNI 249, possessed an uncertain mechanism of action but with defined ethnomedical applications for fertility and vitality. Methods Human cartilage samples were procured from surgical specimens with consent, and were evaluated either as explants or as primary chondrocytes prepared after enzymatic digestion of cartilage matrix. Assessments included IGF-1 gene expression, IGF-1 production (ELISA, cartilage matrix degradation and nitric oxide (NO production, under basal conditions and in the presence of IL-1β. Results RNI 249 enhanced basal IGF-1 mRNA levels in human chondrocytes by 2.7 fold, an effect that was further enhanced to 3.8 fold by co-administration with vincaria. Enhanced basal IGF-1 production by RNI 249 alone and together with vincaria, was confirmed in both explants and in primary chondrocytes (P Conclusion The identification of agents that activate the autocrine production of IGF-1 in cartilage, even in the face of suppressive pro-inflammatory, catabolic cytokines like IL-1β, represents a novel therapeutic approach to cartilage biology. Chondroprotection associated with prevention of the catabolic events and the potential for sustained anabolic activity with this natural product suggests that it holds significant promise in the treatment of debilitating joint diseases.

  5. Study of ionizing radiation effects in human costal cartilage by thermogravimetry and optical coherence tomography; Estudo dos efeitos da radiacao ionizante em cartilagem costal humana por meio de termogravimetria e tomografia por coerencia optica

    Energy Technology Data Exchange (ETDEWEB)

    Martinho Junior, Antonio Carlos

    2012-07-01

    Tissue Banks around the world have stored human cartilages obtained from post mortem donors for use in several kinds of reconstructive surgeries. To ensure that such tissues are not contaminated, they have been sterilized with ionizing radiation. However, high doses of gamma radiation may cause undesirable changes in the tissues. In this work, we evaluated the possibility of use Optical Coherence Tomography (OCT) and Thermogravimetric Analysis (TGA) to identify possible structural modifications caused by both preservation methods of cartilage and gamma irradiation doses. Cartilages were obtained from cadaveric donors and were frozen at -70 deg C or preserved in glycerol. Irradiation was performed by {sup 60}Co source with doses of 15, 25 and 50 kGy. Our TGA results showed that glycerolized cartilages irradiated with different doses of radiation does not presented statistical differences when compared to the control group for the dehydration rate. However, the same was not observed for deep-frozen cartilages irradiated with 15 kGy. The results of OCT associated to total optical attenuation coefficient showed that doses of 15 kGy promote cross-link between collagen fibrils, corroborating the results obtained from TGA. Moreover, total optical attenuation coefficient values are proportional to stress at break of cartilages, what will be very useful in a near future to predict the quality of the allografts, without unnecessary loss of biological tissue, once OCT is a nondestructive technique. By PS-OCT images, we found that high doses of ionizing radiation does not promote sufficient impairments to promote complete loss of tissue birefringence. Thus, TGA and OCT are techniques that can be used for tissue banks to verify tissue quality before its transplant. (author)

  6. CALLUS INDUCTION FROM FLORAL EXPLANTS OF CUPUASSU

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    MARIA DAS GRAÇAS RODRIGUES FERREIRA

    2013-01-01

    Full Text Available There are few studies related to the in vitro cultivation of plants from theTheobroma genus and no effective micropropagation protocols for T.grandiflorum. The aim of this study was to evaluate the calli formation in cupuassu floral explants, targeting their organogenic or embryogenicdevelopment. Experiments were conducted in the Plant Tissue Culture Laboratory of EMBRAPA, Porto Velho, Rondônia, Brazil. Floral parts from unopened immature flower buds taken from seedless cupuassu trees were sterilized and employed as a source of explants. These explants were cultivated in Petri dishes in an induction medium consisting of MS salts and vitamins, supplemented with glycine(3 mg.L-1, lysine (0,4 mg.L-1, leucine (0,4 mg.L-1, arginine (0,4 mg.L-1, tryptophan (0,2 mg.L-1, 2,4-D (1 mg.L-1, kinetin (0,25 mg.L-1, coconut water (50 ml.L-1, sucrose (40 g.L-1, Gelrite (2,2 g.L-1 and pH adjusted to 5,8. Cultures were maintained in the dark for 3 weeks at 27°C and then subcultured for six weeks in medium without growth regulators supplemented with glycine (1 mg.L-1, lysine (0,2 mg.L-1, leucine (0,2 mg.L-1, arginine (0,2 mg.L-1, tryptophan (0,1 mg.L-1, coconut water (100 ml.L-1, sucrose (40 g.L-1, Gelrite (2,2 g.L-1 and pH 5,8. We used a completely randomized design with 10 replications of 5 explants per plate and four different explant sources: staminode, petal, ligule and ovary. As a result, we obtained a highercalli formation in theinduction medium when ovaries were used as source of explants. However, there was no development of somatic embryosor organogenic response in medium without growth regulators and further studies are being conducted.

  7. Cartilage Tissue Engineering via Avocado/Soybean Unsaponifible and Human Adipose Derived Stem Cells on Poly (lactide-co–glycolide /Hyaluronic acid composite scaffold

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    Zynolabedin Sharifian

    2016-09-01

    Full Text Available Background: Growth factors and chemical stimulants have key role in stem cell to chondrocyte differentiation in cartilage tissue engineering, but this agents have adverse effects on cells as well as they are expensive and they have short half time. Todays there is great interest in the application of herbal agent for treatment of diseases.Avocado/soybean unsaponifiable (ASU with herbal components has chondroprotective, anti-inflammatory and pro-anabolic effects that it causes stimulate of deposition of extracellular matrix in chondrocytes and relief of osteoarthritis. The aim of this study was an investigation of the chondrogenic effect of ASU in human adipose derived stem cells (hADSCs on PLGA/HA scaffold. Materials and Methods: The 3-D scaffold of Poly lactide-co –glycolide acid (PLGA prepared via solvent/casting leaching method and impregnated with hyaluronic acid to produce composite scaffold. The characterizations of the scaffold, such as surfaces morphology were observed by scanning electron microscopy (SEM and the degradation behaviour of the composite scaffold were evaluated. hADSCs seeded in PLGA/HA scaffold and cultured in chondrogenic media with and without ASU. The expression of chondrogenic related genes (Sox9, type II collagen, Aggrecan and hypertrophic marker (type X collagen were quantified by real time PCR and viability of cells in different groups were assessed by MTT. Results: Our results showed that the expression of genes related chondrogenesis markers Sox9 and type II collagen and aggrecan in differentiated cells in the presence of ASU were significantly increased compared with the control groups (P<0.05, on the other hand, type X collagen expression was not significantly increased. Conclusions: Our results indicated that ASU could be as an appropriate inducer for chondrogenesis of hADSCs and cartilage tissue engineering.

  8. The in vitro and in vivo capacity of culture-expanded human cells from several sources encapsulated in alginate to form cartilage

    NARCIS (Netherlands)

    M.M. Pleumeekers (Mieke); L. Nimeskern (Luc); J.L.M. Koevoet (Wendy); N. Kops (Nicole); R.M.L. Poublon (René); K.S. Stok (Kathryn); G.J.V.M. van Osch (Gerjo)

    2014-01-01

    textabstractAbstract Cartilage has limited self-regenerative capacity. Tissue engineering can offer promising solutions for reconstruction of missing or damaged cartilage. A major challenge herein is to define an appropriate cell source that is capable of generating a stable and functional matrix. T

  9. Effects of the re-innervation of organotypic skin explants on the epidermis.

    Science.gov (United States)

    Lebonvallet, Nicolas; Boulais, Nicholas; Le Gall, Christelle; Pereira, Ulysse; Gauché, Dominique; Gobin, Eric; Pers, Jacques-Olivier; Jeanmaire, Christine; Danoux, Louis; Pauly, Gilles; Misery, Laurent

    2012-02-01

    The nervous system takes part in skin homeostasis and interacts with skin cells. In in vitro organotypic skin models, these interactions are lost owing to the absence of nerve endings. We have developed an in vitro organotypic skin model based on a re-innervated human skin explant using primary sensory neurons from the dorsal root ganglia of rats. After 10 days of co-culture between skin explant and neurons, a dense network of nerve fibres was observed. The epidermis and dermis presented nerve fibres associated with cellular body from sensory neurons introduced in the co-culture. Epidermal thickness, cell density and quality of re-innervated skin explant were all higher when skin explants were re-innervated by sensory neurons at 10 days of culture. Proliferation of epidermal cell was not modified, but the apoptosis was significantly diminished. Hence, this innovative model of co-cultured skin explants and neurons allows better epidermal integrity and could be useful for studies concerning interactions between the skin and its peripheral nervous system.

  10. Human adipose stromal cells (ASC for the regeneration of injured cartilage display genetic stability after in vitro culture expansion.

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    Simona Neri

    Full Text Available Mesenchymal stromal cells are emerging as an extremely promising therapeutic agent for tissue regeneration due to their multi-potency, immune-modulation and secretome activities, but safety remains one of the main concerns, particularly when in vitro manipulation, such as cell expansion, is performed before clinical application. Indeed, it is well documented that in vitro expansion reduces replicative potential and some multi-potency and promotes cell senescence. Furthermore, during in vitro aging there is a decrease in DNA synthesis and repair efficiency thus leading to DNA damage accumulation and possibly inducing genomic instability. The European Research Project ADIPOA aims at validating an innovative cell-based therapy where autologous adipose stromal cells (ASCs are injected in the diseased articulation to activate regeneration of the cartilage. The primary objective of this paper was to assess the safety of cultured ASCs. The maintenance of genetic integrity was evaluated during in vitro culture by karyotype and microsatellite instability analysis. In addition, RT-PCR array-based evaluation of the expression of genes related to DNA damage signaling pathways was performed. Finally, the senescence and replicative potential of cultured cells was evaluated by telomere length and telomerase activity assessment, whereas anchorage-independent clone development was tested in vitro by soft agar growth. We found that cultured ASCs do not show genetic alterations and replicative senescence during the period of observation, nor anchorage-independent growth, supporting an argument for the safety of ASCs for clinical use.

  11. MRI of the cartilage

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    Imhof, H.; Noebauer-Huhmann, I.-M.; Krestan, C.; Gahleitner, A.; Marlovits, S.; Trattnig, S. [Department of Osteology, Universitaetklinik fuer Radiodiagnostik, AKH-Vienna, Waehringer Guertel 18-20, 1090 Vienna (Austria); Sulzbacher, I. [Universitaetsklinik fuer Pathologie Vienna, Waehringer Guertel 18-20, 1090 Vienna (Austria)

    2002-11-01

    With the introduction of fat-suppressed gradient-echo and fast spin-echo (FSE) sequences in clinical routine MR visualization of the hyaline articular cartilage is routinely possible in the larger joints. While 3D gradient-echo with fat suppression allows exact depiction of the thickness and surface of cartilage, FSE outlines the normal and abnormal internal structures of the hyaline cartilage; therefore, both sequences seem to be necessary in a standard MRI protocol for cartilage visualization. In diagnostically ambiguous cases, in which important therapeutic decisions are required, direct MR arthrography is the established imaging standard as an add-on procedure. Despite the social impact and prevalence, until recent years there was a paucity of knowledge about the pathogenesis of cartilage damage. With the introduction of high-resolution MRI with powerful surface coils and fat-suppression techniques, visualization of the articular cartilage is now routinely possible in many joints. After a short summary of the anatomy and physiology of the hyaline cartilage, the different MR imaging methods are discussed and recommended standards are suggested. (orig.)

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

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    Pedersen Christian

    2010-04-01

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

  13. Pharmacological influence of antirheumatic drugs on proteoglycans from interleukin-1 treated articular cartilage.

    Science.gov (United States)

    Steinmeyer, J; Daufeldt, S

    1997-06-01

    The purpose of this study was to examine whether drugs used in the treatment of arthritic disorders possess any inhibitory potential on the proteoglycanolytic activities of matrix metalloproteinases (MMPs), and to determine whether drugs which inhibit these enzymes also modulate the biosynthesis and release of proteoglycans (PGs) from interleukin-1-(IL-1) treated articular cartilage explants. The cartilage-bone marrow extract and the glycosaminoglycan-peptide complex (DAK-16) dose-dependently inhibited MMP proteoglycanases in vitro when tested at concentrations ranging from 0.5 to 55 mg/mL, displaying an IC50 value of 31.78 mg/mL and 10.64 mg/mL (1.9 x 10[-4] M) respectively. (R,S)-N-[2-[2-(hydroxyamino)-2-oxoethyl]-4-methyl-1-oxopentyl++ +]-L-leucyl-L-phenylalaninamide (U-24522) proved to be a potent inhibitor of MMP proteoglycanases (IC50 value 1.8 x 10[-9] M). None of the other tested drugs, such as possible chondroprotective drugs, nonsteroidal anti-inflammatory drugs (NSAIDs), disease modifying antirheumatic drugs (DMARDs), glucocorticoids and angiotensin-converting enzyme inhibitors tested at a concentration of 10(-4) M displayed any significant inhibition. Only U-24522, tested at a concentration ranging from 10(-4) to 10(-6) M, significantly inhibited the IL-1-induced augmentation of PG loss from cartilage explants into the nutrient media, whereas DAK-16 and the cartilage-bone marrow extract were ineffective. DAK-16 and the cartilage-bone marrow extract did not modulate the IL-1-mediated reduced biosynthesis and aggregability of PGs by the cartilage explants. The addition of 10(-5) M U-24522, however, partially maintained the aggregability of PGs ex vivo. In our experiments, both possible chondroprotective drugs as well as U-24522 demonstrated no cytotoxic effects on chondrocytes.

  14. Prostaglandin E2 role in inhibition of joint cartilage collagen destruction in patients with osteoarthritis

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    E V Chetina

    2009-01-01

    Full Text Available Prostaglandin E2 role in inhibition of articular cartilage collagen degradation in patients with osteoarthritis. Objective. To assess prostaglandin E2 (PGE2 role in inhibition of type II collagen digestion in explants of articular cartilage of pts with osteoarthritis (OA. Material and methods. Explants of articular cartilage of pts with OA were cultured with PGE2 1pg to 10 ng/ml. Type II collagen digestion was assessed with immuno-enzyme assay. Gene expression was evaluated with PCR in real time. Results. PGE2 10 pg/ml as well as transforming growth factor β2 (TGFβ2 suppressed type II collagen digestion in explants of articular cartilage of pts with OA. This concentration of PGE2 did not suppress proteoglycan (aggrecan degradation. Gene expression analysis in 5 OA pts showed that PGE2 10 pg/ml suppressed metallomonooxigenase (MMP-13, MMP-1 and marker of chondrocyte hypertrophy type X collagen (COL10A1 as well as proinflammatory cytokines interleukine (IL-1β and tumor necrosis factor (TNFα. Naproxen, nonselective cyclooxygenase(COX-2 and 1 inhibitor concentration from 5 to 30 mcg/ml blocked TGFβ2 induced collagen digestion inhibition proving that PGE2 mediate influence of this growth factor. Naproxen concentration 5 mcg/ml increased collagen degradation. Conclusion. The study showed that PGE2 is a chondroprotector because it is able to suppress selectively OA pts cartilage collagen degradation. Beside that cartilage chondrocyte hypertrophy in OA connected functionally with increased collagen digestion is also regulated by low concentrations of PGE2

  15. Elastic energy storage in human articular cartilage: estimation of the elastic modulus for type II collagen and changes associated with osteoarthritis.

    Science.gov (United States)

    Silver, Frederick H; Bradica, Gino; Tria, Alfred

    2002-03-01

    The viscoelastic mechanical properties of normal and osteoarthritic articular were analyzed based on data reported by Kempson [in: Adult Articular Cartilage (1973)] and Silver et al. (Connect. Tissue Res., 2001b). Results of the analysis of tensile elastic stress-strain curves suggest that the elastic modulus of cartilage from the superficial zone is approximately 7.0 GPa parallel and 2.21 GPa perpendicular to the cleavage line pattern. Collagen fibril lengths in the superficial zone were found to be approximately 1265 microm parallel and 668 microm perpendicular to the cleavage line direction. The values for the elastic modulus and fibril lengths decreased with increased extent of osteoarthritis. The elastic modulus of type II collagen parallel to the cleavage line pattern in the superficial zone approaches that of type I collagen in tendon, suggesting that elastic energy storage occurs in the superficial zone due to the tensile pre-tension that exists in this region. Decreases in the elastic modulus associated with osteoarthritis reflect decreased ability of cartilage to store elastic energy, which leads to cartilage fibrillation and fissure formation. We hypothesize that under normal physiological conditions, collagen fibrils in cartilage function to store elastic energy associated with weight bearing and locomotion. Enzymatic cleavage of cartilage proteoglycans and collagen observed in osteoarthritis may lead to fibrillation and fissure formation as a result of impaired energy storage capability of cartilage.

  16. Induction of advanced glycation end products and alterations of the tensile properties of articular cartilage

    NARCIS (Netherlands)

    Chen, A.C.; Temple, M.M.; Ng, D.M.; Verzijl, N.; Groot, J. de; TeKoppele, J.M.; Sah, R.L.

    2002-01-01

    Objective. To determine whether increasing advanced glycation end products (AGEs) in bovine articular cartilage to levels present in aged human cartilage modulates the tensile biomechanical properties of the tissue. Methods. Adult bovine articular cartilage samples were incubated in a buffer solutio

  17. The Immunosuppressant FTY720 (Fingolimod enhances Glycosaminoglycan depletion in articular cartilage

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    Stradner Martin H

    2011-12-01

    Full Text Available Abstract Background FTY720 (Fingolimod is a novel immunosuppressive drug investigated in clinical trials for organ transplantation and multiple sclerosis. It acts as a functional sphingosine-1-phosphate (S1P receptor antagonist, thereby inhibiting the egress of lymphocytes from secondary lymphoid organs. As S1P is able to prevent IL-1beta induced cartilage degradation, we examined the direct impact of FTY720 on cytokine induced cartilage destruction. Methods Bovine chondrocytes were treated with the bioactive phosphorylated form of FTY720 (FTY720-P in combination with IL-1beta or TNF-alpha. Expression of MMP-1,-3.-13, iNOS and ADAMTS-4,-5 and COX-2 was evaluated using quantitative real-time PCR and western blot. Glycosaminoglycan depletion from cartilage explants was determined using a 1,9-dimethylene blue assay and safranin O staining. Results FTY720-P significantly reduced IL-1beta and TNF-alpha induced expression of iNOS. In contrast FTY720-P increased MMP-3 and ADAMTS-5 mRNA expression. Furthermore depletion of glycosaminoglycan from cartilage explants by IL-1beta and TNF-alpha was significantly enhanced by FTY720-P in an MMP-3 dependent manner. Conclusions Our results suggest that FTY720 may enhance cartilage degradation in pro-inflammatory environment.

  18. Induction of advanced glycation end products and alterations of the tensile properties of articular cartilage

    OpenAIRE

    Chen, A C; Temple, M.M.; Ng, D.M.; Verzijl, N; de Groot, J.; TeKoppele, J.M.; Sah, R.L.

    2002-01-01

    Objective. To determine whether increasing advanced glycation end products (AGEs) in bovine articular cartilage to levels present in aged human cartilage modulates the tensile biomechanical properties of the tissue. Methods. Adult bovine articular cartilage samples were incubated in a buffer solution with ribose to induce the formation of AGEs or in a control solution. Portions of cartilage samples were assayed for biochemical indices of AGEs and tested to assess their tensile biomechanical p...

  19. From gristle to chondrocyte transplantation: treatment of cartilage injuries

    Science.gov (United States)

    Lindahl, Anders

    2015-01-01

    This review addresses the progress in cartilage repair technology over the decades with an emphasis on cartilage regeneration with cell therapy. The most abundant cartilage is the hyaline cartilage that covers the surface of our joints and, due to avascularity, this tissue is unable to repair itself. The cartilage degeneration seen in osteoarthritis causes patient suffering and is a huge burden to society. The surgical approach to cartilage repair was non-existing until the 1950s when new surgical techniques emerged. The use of cultured cells for cell therapy started as experimental studies in the 1970s that developed over the years to a clinical application in 1994 with the introduction of the autologous chondrocyte transplantation technique (ACT). The technology is now spread worldwide and has been further refined by combining arthroscopic techniques with cells cultured on matrix (MACI technology). The non-regenerating hypothesis of cartilage has been revisited and we are now able to demonstrate cell divisions and presence of stem-cell niches in the joint. Furthermore, cartilage derived from human embryonic stem cells and induced pluripotent stem cells could be the base for new broader cell treatments for cartilage injuries and the future technology base for prevention and cure of osteoarthritis. PMID:26416680

  20. The identification of CD163 expressing phagocytic chondrocytes in joint cartilage and its novel scavenger role in cartilage degradation.

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    Kai Jiao

    Full Text Available BACKGROUND: Cartilage degradation is a typical characteristic of arthritis. This study examined whether there was a subset of phagocytic chondrocytes that expressed the specific macrophage marker, CD163, and investigated their role in cartilage degradation. METHODS: Cartilage from the knee and temporomandibular joints of Sprague-Dawley rats was harvested. Cartilage degradation was experimentally-induced in rat temporomandibular joints, using published biomechanical dental methods. The expression levels of CD163 and inflammatory factors within cartilage, and the ability of CD163(+ chondrocytes to conduct phagocytosis were investigated. Cartilage from the knees of patients with osteoarthritis and normal cartilage from knee amputations was also investigated. RESULTS: In the experimentally-induced degrading cartilage from temporomandibular joints, phagocytes were capable of engulfing neighboring apoptotic and necrotic cells, and the levels of CD163, TNF-α and MMPs were all increased (P0.05. CD163(+ chondrocytes were found in the cartilage mid-zone of temporomandibular joints and knee from healthy, three-week old rats. Furthermore, an increased number of CD163(+ chondrocytes with enhanced phagocytic activity were present in Col-II(+ chondrocytes isolated from the degraded cartilage of temporomandibular joints in the eight-week experimental group compared with their age-matched controls. Increased number with enhanced phagocytic activity of CD163(+ chondrocytes were also found in isolated Col-II(+ chondrocytes stimulated with TNF-α (P<0.05. Mid-zone distribution of CD163(+ cells accompanied with increased expression of CD163 and TNF-α were further confirmed in the isolated Col-II(+ chondrocytes from the knee cartilage of human patients with osteoarthritis, in contrast to the controls (both P<0.05. CONCLUSIONS: An increased number of CD163(+ chondrocytes with enhanced phagocytic activity were discovered within degraded joint cartilage, indicating a

  1. The identification of CD163 expressing phagocytic chondrocytes in joint cartilage and its novel scavenger role in cartilage degradation.

    Science.gov (United States)

    Jiao, Kai; Zhang, Jing; Zhang, Mian; Wei, Yuying; Wu, Yaoping; Qiu, Zhong Ying; He, Jianjun; Cao, Yunxin; Hu, Jintao; Zhu, Han; Niu, Li-Na; Cao, Xu; Yang, Kun; Wang, Mei-Qing

    2013-01-01

    Cartilage degradation is a typical characteristic of arthritis. This study examined whether there was a subset of phagocytic chondrocytes that expressed the specific macrophage marker, CD163, and investigated their role in cartilage degradation. Cartilage from the knee and temporomandibular joints of Sprague-Dawley rats was harvested. Cartilage degradation was experimentally-induced in rat temporomandibular joints, using published biomechanical dental methods. The expression levels of CD163 and inflammatory factors within cartilage, and the ability of CD163(+) chondrocytes to conduct phagocytosis were investigated. Cartilage from the knees of patients with osteoarthritis and normal cartilage from knee amputations was also investigated. In the experimentally-induced degrading cartilage from temporomandibular joints, phagocytes were capable of engulfing neighboring apoptotic and necrotic cells, and the levels of CD163, TNF-α and MMPs were all increased (P0.05). CD163(+) chondrocytes were found in the cartilage mid-zone of temporomandibular joints and knee from healthy, three-week old rats. Furthermore, an increased number of CD163(+) chondrocytes with enhanced phagocytic activity were present in Col-II(+) chondrocytes isolated from the degraded cartilage of temporomandibular joints in the eight-week experimental group compared with their age-matched controls. Increased number with enhanced phagocytic activity of CD163(+) chondrocytes were also found in isolated Col-II(+) chondrocytes stimulated with TNF-α (PCD163(+) cells accompanied with increased expression of CD163 and TNF-α were further confirmed in the isolated Col-II(+) chondrocytes from the knee cartilage of human patients with osteoarthritis, in contrast to the controls (both PCD163(+) chondrocytes with enhanced phagocytic activity were discovered within degraded joint cartilage, indicating a role in eliminating degraded tissues. Targeting these cells provides a new strategy for the treatment of arthritis.

  2. Interleukin-29 Enhances Synovial Inflammation and Cartilage Degradation in Osteoarthritis.

    Science.gov (United States)

    Xu, Lingxiao; Peng, Qiuyue; Xuan, Wenhua; Feng, Xiaoke; Kong, Xiangqing; Zhang, Miaojia; Tan, Wenfeng; Xue, Meilang; Wang, Fang

    2016-01-01

    We have recently shown that IL-29 was an important proinflammatory cytokine in pathogenesis of rheumatoid arthritis (RA). Inflammation also contributes to the pathogenesis of osteoarthritis (OA). The aim of this study was to investigate the effect and mechanism of IL-29 on cytokine production and cartilage degradation in OA. The mRNA levels of IL-29 and its specific receptor IL-28Ra in peripheral blood mononuclear cells (PBMCs) were significantly increased in OA patients when compared to healthy controls (HC). In the serum, IL-29 protein levels were higher in OA patients than those in HC. Immunohistochemistry revealed that both IL-29 and IL-28Ra were dramatically elevated in OA synovium compared to HC; synovial fibroblasts (FLS) and macrophages were the main IL-29-producing cells in OA synovium. Furthermore, recombinant IL-29 augmented the mRNA expression of IL-1β, IL-6, IL-8, and matrix-metalloproteinase-3 (MMP-3) in OA FLS and increased cartilage degradation when ex vivo OA cartilage explant was coincubated with OA FLS. Finally, in OA FLS, IL-29 dominantly activated MAPK and nuclear factor-κB (NF-κB), but not Jak-STAT and AKT signaling pathway as examined by western blot. In conclusion, IL-29 stimulates inflammation and cartilage degradation by OA FLS, indicating that this cytokine is likely involved in the pathogenesis of OA.

  3. Potentialities of the {sup 99m}Tc-N.T.P. 15-5 for imaging of cartilage pathologies: situation of in vivo studies in animal and ex vivo on human specimens; Potentialites du 99mTc-NTP 15-5 pour l'imagerie des pathologies du cartilage: bilan des etudes in vivo chez l'animal et ex vivo sur specimens humains

    Energy Technology Data Exchange (ETDEWEB)

    Miot-Noirault, E.; Cachin, F.; Vidal, A.; Auzeloux, P.; Boisgard, S.; Moins, N.; Chezal, J.M. [Inserm, EA4231, UMR 990, 63 - Clermont-Ferrand (France); Gouin, F.; Redini, F. [Inserm, EA3822, UMR 957, 44 - Nantes (France); Askienazy, S. [Cyclopharma laboratoires, 63 - Saint-Beauzire (France)

    2010-07-01

    The strategy we develop is the use of quaternary ammonium function to vectorize a technetium macrocycle towards proteoglycans, in order to offer an innovative diagnostic strategy of degenerative and tumoral diseases of cartilage in nuclear medicine. The results got in relevant animal models demonstrate the importance of {sup 99}Tc-NTP15-5, (N-(triethylammonium)-3-propyl-[15]ane-N5 radiolabeled with {sup 99m}Tc ({sup 99m}Tc-NTP 15-5)), as cartilage radiotracer in nuclear medicine. The results obtained from human specimens encourage us to a clinical transfer. (N.C.)

  4. Enzymatic digestion of articular cartilage results in viscoelasticity changes that are consistent with polymer dynamics mechanisms

    Directory of Open Access Journals (Sweden)

    June Ronald K

    2009-11-01

    Full Text Available Abstract Background Cartilage degeneration via osteoarthritis affects millions of elderly people worldwide, yet the specific contributions of matrix biopolymers toward cartilage viscoelastic properties remain unknown despite 30 years of research. Polymer dynamics theory may enable such an understanding, and predicts that cartilage stress-relaxation will proceed faster when the average polymer length is shortened. Methods This study tested whether the predictions of polymer dynamics were consistent with changes in cartilage mechanics caused by enzymatic digestion of specific cartilage extracellular matrix molecules. Bovine calf cartilage explants were cultured overnight before being immersed in type IV collagenase, bacterial hyaluronidase, or control solutions. Stress-relaxation and cyclical loading tests were performed after 0, 1, and 2 days of incubation. Results Stress-relaxation proceeded faster following enzymatic digestion by collagenase and bacterial hyaluronidase after 1 day of incubation (both p ≤ 0.01. The storage and loss moduli at frequencies of 1 Hz and above were smaller after 1 day of digestion by collagenase and bacterial hyaluronidase (all p ≤ 0.02. Conclusion These results demonstrate that enzymatic digestion alters cartilage viscoelastic properties in a manner consistent with polymer dynamics mechanisms. Future studies may expand the use of polymer dynamics as a microstructural model for understanding the contributions of specific matrix molecules toward tissue-level viscoelastic properties.

  5. Small RNAs derived from lncRNA RNase MRP have gene-silencing activity relevant to human cartilage-hair hypoplasia.

    Science.gov (United States)

    Rogler, Leslie E; Kosmyna, Brian; Moskowitz, David; Bebawee, Remon; Rahimzadeh, Joseph; Kutchko, Katrina; Laederach, Alain; Notarangelo, Luigi D; Giliani, Silvia; Bouhassira, Eric; Frenette, Paul; Roy-Chowdhury, Jayanta; Rogler, Charles E

    2014-01-15

    Post-transcriptional processing of some long non-coding RNAs (lncRNAs) reveals that they are a source of miRNAs. We show that the 268-nt non-coding RNA component of mitochondrial RNA processing endoribonuclease, (RNase MRP), is the source of at least two short (∼20 nt) RNAs designated RMRP-S1 and RMRP-S2, which function as miRNAs. Point mutations in RNase MRP cause human cartilage-hair hypoplasia (CHH), and several disease-causing mutations map to RMRP-S1 and -S2. SHAPE chemical probing identified two alternative secondary structures altered by disease mutations. RMRP-S1 and -S2 are significantly reduced in two fibroblast cell lines and a B-cell line derived from CHH patients. Tests of gene regulatory activity of RMRP-S1 and -S2 identified over 900 genes that were significantly regulated, of which over 75% were down-regulated, and 90% contained target sites with seed complements of RMRP-S1 and -S2 predominantly in their 3' UTRs. Pathway analysis identified regulated genes that function in skeletal development, hair development and hematopoietic cell differentiation including PTCH2 and SOX4 among others, linked to major CHH phenotypes. Also, genes associated with alternative RNA splicing, cell proliferation and differentiation were highly targeted. Therefore, alterations RMRP-S1 and -S2, caused by point mutations in RMRP, are strongly implicated in the molecular mechanism of CHH.

  6. Physical mechanisms underlying the strain-rate-dependent mechanical behavior of kangaroo shoulder cartilage

    Science.gov (United States)

    Thibbotuwawa, Namal; Oloyede, Adekunle; Li, Tong; Singh, Sanjleena; Senadeera, Wijitha; Gu, YuanTong

    2015-09-01

    Due to anatomical and biomechanical similarities to human shoulder, kangaroo was chosen as a model to study shoulder cartilage. Comprehensive enzymatic degradation and indentation tests were applied on kangaroo shoulder cartilage to study mechanisms underlying its strain-rate-dependent mechanical behavior. We report that superficial collagen plays a more significant role than proteoglycans in facilitating strain-rate-dependent behavior of the kangaroo shoulder cartilage. By comparing the mechanical properties of degraded and normal cartilages, it was noted that proteoglycan and collagen degradation significantly compromised strain-rate-dependent mechanical behavior of the cartilage. Superficial collagen contributed equally to the tissue behavior at all strain-rates. This is different to the studies reported on knee cartilage and confirms the importance of superficial collagen on shoulder cartilage mechanical behavior. A porohyperelastic numerical model also indicated that collagen disruption would lead to faster damage of the shoulder cartilage than when proteoglycans are depleted.

  7. Making post-mortem implantable cardioverter defibrillator explantation safe

    OpenAIRE

    Räder, Sune B.E.W.; Zeijlemaker, Volkert; Pehrson, Steen; Svendsen, Jesper H

    2009-01-01

    Aims The aim of this study is to investigate whether protection with rubber or plastic gloves during post-mortem explantation of an implantable cardioverter defibrillator (ICD) offers enough protection for the explanting operator during a worst-case scenario (i.e. ICD shock). Methods and results We investigated the insulating properties of rubber and plastic gloves (double layer) within the first 60 min exposure (mimicking the maximum time of an explantation procedure) to saline (simulating t...

  8. Enzyme replacement in a human model of mucopolysaccharidosis IVA in vitro and its biodistribution in the cartilage of wild type mice.

    Directory of Open Access Journals (Sweden)

    Melita Dvorak-Ewell

    Full Text Available Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS, an enzyme that degrades keratan sulfate (KS. Currently no therapy for MPS IVA is available. We produced recombinant human (rhGALNS as a potential enzyme replacement therapy for MPS IVA. Chinese hamster ovary cells stably overexpressing GALNS and sulfatase modifying factor-1 were used to produce active ( approximately 2 U/mg and pure (>or=97% rhGALNS. The recombinant enzyme was phosphorylated and was dose-dependently taken up by mannose-6-phosphate receptor (K(uptake = 2.5 nM, thereby restoring enzyme activity in MPS IVA fibroblasts. In the absence of an animal model with a skeletal phenotype, we established chondrocytes isolated from two MPS IVA patients as a disease model in vitro. MPS IVA chondrocyte GALNS activity was not detectable and the cells exhibited KS storage up to 11-fold higher than unaffected chondrocytes. MPS IVA chondrocytes internalized rhGALNS into lysosomes, resulting in normalization of enzyme activity and decrease in KS storage. rhGALNS treatment also modulated gene expression, increasing expression of chondrogenic genes Collagen II, Collagen X, Aggrecan and Sox9 and decreasing abnormal expression of Collagen I. Intravenous administration of rhGALNS resulted in biodistribution throughout all layers of the heart valve and the entire thickness of the growth plate in wild-type mice. We show that enzyme replacement therapy with recombinant human GALNS results in clearance of keratan sulfate accumulation, and that such treatment ameliorates aberrant gene expression in human chondrocytes in vitro. Penetration of the therapeutic enzyme throughout poorly vascularized, but clinically relevant tissues, including growth plate cartilage and heart valve, as well as macrophages and hepatocytes in wild-type mouse, further supports development of rhGALNS as enzyme replacement therapy for

  9. Enzyme replacement in a human model of mucopolysaccharidosis IVA in vitro and its biodistribution in the cartilage of wild type mice.

    Science.gov (United States)

    Dvorak-Ewell, Melita; Wendt, Dan; Hague, Chuck; Christianson, Terri; Koppaka, Vish; Crippen, Danielle; Kakkis, Emil; Vellard, Michel

    2010-08-16

    Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), an enzyme that degrades keratan sulfate (KS). Currently no therapy for MPS IVA is available. We produced recombinant human (rh)GALNS as a potential enzyme replacement therapy for MPS IVA. Chinese hamster ovary cells stably overexpressing GALNS and sulfatase modifying factor-1 were used to produce active ( approximately 2 U/mg) and pure (>or=97%) rhGALNS. The recombinant enzyme was phosphorylated and was dose-dependently taken up by mannose-6-phosphate receptor (K(uptake) = 2.5 nM), thereby restoring enzyme activity in MPS IVA fibroblasts. In the absence of an animal model with a skeletal phenotype, we established chondrocytes isolated from two MPS IVA patients as a disease model in vitro. MPS IVA chondrocyte GALNS activity was not detectable and the cells exhibited KS storage up to 11-fold higher than unaffected chondrocytes. MPS IVA chondrocytes internalized rhGALNS into lysosomes, resulting in normalization of enzyme activity and decrease in KS storage. rhGALNS treatment also modulated gene expression, increasing expression of chondrogenic genes Collagen II, Collagen X, Aggrecan and Sox9 and decreasing abnormal expression of Collagen I. Intravenous administration of rhGALNS resulted in biodistribution throughout all layers of the heart valve and the entire thickness of the growth plate in wild-type mice. We show that enzyme replacement therapy with recombinant human GALNS results in clearance of keratan sulfate accumulation, and that such treatment ameliorates aberrant gene expression in human chondrocytes in vitro. Penetration of the therapeutic enzyme throughout poorly vascularized, but clinically relevant tissues, including growth plate cartilage and heart valve, as well as macrophages and hepatocytes in wild-type mouse, further supports development of rhGALNS as enzyme replacement therapy for MPS IVA.

  10. Fabrication of cartilage in predetermined shapes from human nasoseptal chondrocytes with tissue engineering method%鼻中隔软骨细胞组织工程法构建预定形态软骨

    Institute of Scientific and Technical Information of China (English)

    崔鹏程; 陈文弦; 罗家胜

    2001-01-01

    目的探讨利用人鼻中隔软骨细胞组织工程方法构建预定形态软骨的可能性。方法将人鼻中隔软骨细胞播种在聚乙醇酸(polyglycolic acid, PGA)无纺网支架材料上,制成片状和管状结构,埋入裸鼠体内,经4、6、8周后取材作大体及组织学观察。结果大体观察见裸鼠体内形成了预定的片状和管状软骨。组织学观察:6周时软骨细胞基本成熟, Masson 三色染色显示胶原形成,番红花-“O”染色证实其基质中存在糖氨多糖。对照组于6周时PGA纤维基本消失。结论人鼻中隔软骨细胞与PGA无纺网复合可在裸鼠体内形成预定形态软骨。%Objective To investigate the feasibility of fabricating a new cartilage in predetermined shapes with tissue engineering methods. Methods Human nasoseptal chondrocytes were seeded onto a nonwoven mesh of polyglycolic acid(PGA) to form a cell-PGA construct. The construction was then configured in sheet and tube shapes, and implanted subcutaneously into the dorsa of 11 athymic mice. The specimens were harvested 4,6,8 weeks after implantation and subjected to gross morphologic and histologic analysis. Results Gross observation showed that the predetermined sheet and tube shapes of new cartilage were formed. Histological observation demonstrated that new mature cartilages were formed in 6-week. A Masson's trichrome stain showed the interwining bands of collagen at the periphery of the cartilage. Staining of Safranin O evaluated that the new cartilage was bound of glycosaminoglycan. In the control group, the PGA of the specimens were completely absorbed at 6 weeks. Conclusion Human nasoseptal chondrocytes-PGA construct could develop into a new cartilage in predetermined shapes in athymic mice.

  11. Fetal Mesenchymal Stromal Cells Differentiating towards Chondrocytes Acquire a Gene Expression Profile Resembling Human Growth Plate Cartilage

    NARCIS (Netherlands)

    van Gool, S.A.; Emons, J.A.M.; Leijten, Jeroen Christianus Hermanus; Decker, E.; Sticht, C.; van Houwelingen, J.C.; Goeman, J.J.; Kleijburg, C.; Scherjon, S.; Gretz, N.; Wit, J.M.; Rappold, G.; Post, Janine Nicole; Karperien, Hermanus Bernardus Johannes

    2012-01-01

    Abstract We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether

  12. Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish

    Energy Technology Data Exchange (ETDEWEB)

    Chenais, Nathalie; Lareyre, Jean-Jacques; Le Bail, Pierre-Yves; Labbe, Catherine, E-mail: catherine.labbe@rennes.inra.fr

    2015-07-01

    The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression. Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation. - Highlights: • Recycled fin explants outgrow cells bearing stable mesenchymal traits. • Cell production and quality is enhanced in the recycled explant culture system. • Fresh fin primary culture is highly variable and loose epithelial traits over time.

  13. Production of immunoglobulins in gingival tissue explant cultures from juvenile periodontitis patients

    Energy Technology Data Exchange (ETDEWEB)

    Hall, E.R.; Falkler, W.A. Jr.; Suzuki, J.B. (Univ. of Maryland Dental School, Baltimore (USA))

    1990-10-01

    B lymphocytes and plasma cells are histologically observed in granulomatous periodontal tissues of juvenile periodontitis (JP) patients. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. An in vitro explant culture system was utilized to demonstrate the production of immunoglobulins by diseased JP tissues. Immunodiffusion studies using goat anti-human gamma, alpha, or mu chain serum revealed IgG to be the major immunoglobulin present in 92% of the day 1 supernatant fluids (SF) of the 47 JP gingival tissue explant cultures. IgA was present in 15% of the SF; however, no IgM was detected. Staph Protein A isolated 14C-labeled IgG from the SF, when allowed to react with goat anti-human gamma chain serum, formed lines of precipitation. Positive autoradiographs confirmed the biosynthesis of IgG by the explant cultures. The in vitro gingival tissue explant culture system described provides a useful model for the study of localized immunoglobulins produced by diseased tissues of JP patients.

  14. Prolonged in vitro precultivation alleviates post-implantation inflammation and promotes stable subcutaneous cartilage formation in a goat model.

    Science.gov (United States)

    Liu, Yi; Li, Dan; Yin, Zongqi; Luo, Xusong; Liu, Wei; Zhang, Wenjie; Zhang, Zhiyong; Cao, Yilin; Liu, Yu; Zhou, Guangdong

    2016-12-02

    Synthetic biodegradable scaffolds such as polylactic acid coated polyglycolic acid (PLA-PGA) are especially suitable for engineering shaped cartilage such as auricle, but they induce a serious inflammatory reaction particularly in the immunologically aggressive subcutaneous site, leading to resorption of the engineered autologous cartilage. Our previous study in a rabbit model has demonstrated 2 weeks of in vitro precultivation could significantly alleviate the post-implantation inflammation induced by PLA-PGA engineered cartilaginous grafts, but reproduction of this result failed in a preclinical goat model. The aims of the current study were to investigate whether prolonged in vitro precultivation could form a mature cartilaginous graft to resist the acute host response and promote stable subcutaneous cartilage formation in a preclinical goat model. Goat chondrocytes were seeded onto PLA-PGA scaffolds, in vitro precultivated for 2, 4, 8, and 12 weeks, and then implanted subcutaneously in autologous goats for 1 and 8 weeks. The in vitro engineered cartilage (vitro-EC) was examined histologically (hematoxylin and eosin, safranin-O, collagen II). The 1 week explants were examined histologically and stained for CD3, CD68, collagen I, and apoptosis. The 8 week explants were evaluated by histology, wet weight, volume, glycosaminoglycan (GAG) quantification and Young's modulus. With prolonged in vitro time, the quality of vitro-EC improved and the amount of scaffold residue decreased; more pronounced cartilage formation with fewer immune cells (CD3 and CD68 positive), apoptotic cells, and less collagen I expression were observed in explants that had been in vitro precultivated for a longer period. The subcutaneously regenerated neocartilage became more mature after prolonged implantation. These results suggested that prolonged in vitro precultivation allowed formation of a mature cartilaginous graft to resist the acute host response and promoted stable subcutaneous

  15. Anti-cartilage antibody.

    Science.gov (United States)

    Greenbury, C L; Skingle, J

    1979-08-01

    Antibody to cartilage has been demonstrated by indirect immunofluorescence on rat trachea in the serum of about 3% of 1126 patients with rheumatoid arthritis. Titres ranged from 1:20 to 1:640. The antibody was not found in 284 patients with primary or secondary osteoarthritis or in 1825 blood donors, nor, with the exception of two weak reactors, in 1314 paraplegic patients. In most cases the antibody appears to be specific for native type II collagen. Using this as an antigen in a haemagglutination test 94% of anti-cartilage sera were positive, whereas among 100 rheumatoid control sera there were only three weak positives. More than 80% of patients with antibody had some erosion of articular cartilage, but there was no correlation with age, sex, duration of disease, nor any recognisable clinical event or change.

  16. MORPHOMETRIC STUDY OF THYROID CARTILAGES IN WESTERN INDIA

    Directory of Open Access Journals (Sweden)

    Mohini M.Joshi

    2015-06-01

    Full Text Available Background: Morphometrical evaluation of the larynx has always been interesting for both morphologists and the physicians. A good understanding of the anatomy and the knowledge of variations in the laryngeal cartilages is important Objective: Objective of the present study was to collect exact and reliable morphometric data of thyroid cartilage in adult human larynx of regional population. Methods: The totals of 50 thyroid cartilage specimens were studied. The cartilages were preserved in 5% formalin. The measurements were taken with the help of Digital Vernier Caliper. The cartilages were weighed on Single pan electronic balance. For each of the parameters, the mean, standard deviation (S.D. and range was calculated. Results: Mean depth of superior thyroid notch was 9.7± 3.36 mm. Asymmetry between the length of superior horn of thyroid cartilages in left and right sides can be seen, but difference was not statistically significant (p>0.05. It is observed that inner thyroid angle varies from 55 to 1040 and outer thyroid angle varies from 53 to 990. In present study mean weight of thyroid cartilage was 6.70±1.55 grams. Conclusions: A fair amount of intersubject variability in the dimensions was observed. Bilateral asymmetry, though present in majority of specimens, was insignificant. Various dimensions of thyroid cartilages are smaller as compared to the western population.

  17. Curcumin reduces prostaglandin E2, matrix metalloproteinase-3 and proteoglycan release in the secretome of interleukin 1β-treated articular cartilage [v2; ref status: indexed, http://f1000r.es/1ks

    Directory of Open Access Journals (Sweden)

    Abigail L Clutterbuck

    2013-08-01

    Full Text Available Objective: Curcumin (diferuloylmethane is a phytochemical with potent anti-inflammatory and anti-oxidant properties, and has therapeutic potential for the treatment of a range of inflammatory diseases, including osteoarthritis (OA. The aim of this study was to determine whether non-toxic concentrations of curcumin can reduce interleukin-1beta (IL-1β-stimulated inflammation and catabolism in an explant model of cartilage inflammation. Methods: Articular cartilage explants and primary chondrocytes were obtained from equine metacarpophalangeal joints. Curcumin was added to monolayer cultured primary chondrocytes and cartilage explants in concentrations ranging from 3μM-100μM. Prostaglandin E2 (PGE2 and matrix metalloproteinase (MMP-3 release into the secretome of IL-1β-stimulated explants was measured using a competitive ELISA and western blotting respectively. Proteoglycan (PG release in the secretome was measured using the 1,9-dimethylmethylene blue (DMMB assay. Cytotoxicity was assessed with a live/dead assay in monolayer cultures after 24 hours, 48 hours and five days, and in explants after five days. Results: Curcumin induced chondrocyte death in primary cultures (50μM p<0.001 and 100μM p<0.001 after 24 hours. After 48 hours and five days, curcumin (≥25μM significantly increased cell death (p<0.001 both time points. In explants, curcumin toxicity was not observed at concentrations up to and including 25μM after five days. Curcumin (≥3μM significantly reduced IL-1β-stimulated PG (p<0.05 and PGE2 release (p<0.001 from explants, whilst curcumin (≥12μM significantly reduced MMP-3 release (p<0.01. Conclusion: Non-cytotoxic concentrations of curcumin exert anti-catabolic and anti-inflammatory effects in cartilage explants.

  18. Curcumin reduces prostaglandin E2, matrix metalloproteinase-3 and proteoglycan release in the secretome of interleukin 1β-treated articular cartilage [v1; ref status: indexed, http://f1000r.es/1cl

    Directory of Open Access Journals (Sweden)

    Abigail L Clutterbuck

    2013-07-01

    Full Text Available Objective: Curcumin (diferuloylmethane is a phytochemical with potent anti-inflammatory and anti-oxidant properties, and has therapeutic potential for the treatment of a range of inflammatory diseases, including osteoarthritis (OA. The aim of this study was to determine whether non-toxic concentrations of curcumin can reduce interleukin-1beta (IL-1β-stimulated inflammation and catabolism in an explant model of cartilage inflammation. Methods: Articular cartilage explants and primary chondrocytes were obtained from equine metacarpophalangeal joints. Curcumin was added to monolayer cultured primary chondrocytes and cartilage explants in concentrations ranging from 3μM-100μM. Prostaglandin E2 (PGE2 and matrix metalloproteinase (MMP-3 release into the secretome of IL-1β-stimulated explants was measured using a competitive ELISA and western blotting respectively. Proteoglycan (PG release in the secretome was measured using the 1,9-dimethylmethylene blue (DMMB assay. Cytotoxicity was assessed with a live/dead assay in monolayer cultures after 24 hours, 48 hours and five days, and in explants after five days. Results: Curcumin induced chondrocyte death in primary cultures (50μM p<0.001 and 100μM p<0.001 after 24 hours. After 48 hours and five days, curcumin (≥25μM significantly increased cell death (p<0.001 both time points. In explants, curcumin toxicity was not observed at concentrations up to and including 25μM after five days. Curcumin (≥3μM significantly reduced IL-1β-stimulated PG (p<0.05 and PGE2 release (p<0.001 from explants, whilst curcumin (≥12μM significantly reduced MMP-3 release (p<0.01. Conclusion: Non-cytotoxic concentrations of curcumin exert anti-catabolic and anti-inflammatory effects in cartilage explants.

  19. Principles of cartilage repair

    CERN Document Server

    Erggelet, Christoph; Mandelbaum, Bert R

    2008-01-01

    Cartilage defects affect patients of all age groups. Surgeons, teamdoctors, general practitioners and physiotherapists alike are expected to provide adequate care. Only individual treatment plans combining a well balanced choice of various options will be successful. Background knowledge, operative and non-operative therapies are described in concise chapters: Articular cartilage biology - Diagnostics - Surgical techniques - Symptomatic and alternative medications - Physiotherapy. Diagnostic findings and surgical procedures are generously illustrated by aquarelles and colour photographs. Recommendations for additional reading, description of important clinical scoring systems and a listing of analytic tools are added for further information.

  20. The junction between hyaline cartilage and engineered cartilage in rabbits.

    Science.gov (United States)

    Komura, Makoto; Komura, Hiroko; Otani, Yushi; Kanamori, Yutaka; Iwanaka, Tadashi; Hoshi, Kazuto; Tsuyoshi, Takato; Tabata, Yasuhiko

    2013-06-01

    Tracheoplasty using costal cartilage grafts to enlarge the tracheal lumen was performed to treat congenital tracheal stenosis. Fibrotic granulomatous tissue was observed at the edge of grafted costal cartilage. We investigated the junction between the native hyaline cartilage and the engineered cartilage plates that were generated by auricular chondrocytes for fabricating the airway. Controlled, prospecive study. In group 1, costal cartilage from New Zealand white rabbits was collected and implanted into a space created in the cervical trachea. In group 2, chondrocytes from auricular cartilages were seeded on absorbable scaffolds. These constructs were implanted in the subcutaneous space. Engineered cartilage plates were then implanted into the trachea after 3 weeks of implantation of the constructs. The grafts in group 1 and 2 were retrieved after 4 weeks. In group 1, histological studies of the junction between the native hyaline cartilage and the implanted costal cartilage demonstrated chondrogenic tissue in four anastomoses sides out of the 10 examined. In group 2, the junction between the native trachea and the engineered cartilage showed neocartilage tissue in nine anastomoses sides out of 10. Engineered cartilage may be beneficial for engineered airways, based on the findings of the junction between the native and engineered grafts. Copyright © 2012 The American Laryngological, Rhinological and Otological Society, Inc.

  1. Green fluorescent protein as marker in chondrocytes overexpressing human insulin-like growth factor-1 for repair of articular cartilage defects in rabbits

    Institute of Scientific and Technical Information of China (English)

    ZHANG Shao-kun; LIU Yi; SONG Zhi-ming; FU Chang-feng; XU Xin-xiang

    2007-01-01

    Objective:To label the primary articular chondrocytes overexpressing human insulin-like growth factor ( hIGF-1 ) with green fluorescent protein (GFP) for repair of articular cartilage defects in rabbits. Methods:GFP cDNA was inserted into pcDNA3.1-hIGF-1 to label the expression vector.The recombinant vector,pcGI,a mammalian expression vector with multiple cloning sites under two respective cytomegalovirus promoters/enhancers,was transfected into the primary articular chondrocytes with the help of lipofectamine.After the positive cell clones were selected by G418,G418-resistant chondrocytes were cultured in medium for 4 weeks.The stable expression of hIGF-1 in the articular chondrocytes was determined by in situ hybridization and immunocytochemical analysis and the GFP was confirmed under a fluorescence microscope. Methyl thiazolyl tetrazolium (MTT) and flow cytometer methods were employed to determine the effect of transfection on proliferation of chondrocytes. Gray value was used to analyze quantitatively the expression of type Ⅱ collagen. Results:The expression of hIGF-1 and GFP was confirmed in transfected chondrocytes by in situ hybridization, immunocytochemical analysis and fluorescence microscope observation. Green articular chondrocytes overexpressing hIGF-1 could expand and maintain their chondrogenic phenotypes for more than 4 weeks.After the transfection of IGF-1,the proliferation of chondrocytes was enhanced and the chondrocytes could effectively maintain the expression of type Ⅱ collagen. Conclusions:The hIGF-1 eukaryotic expression vector containing GFP marker gene has been successfully constructed.GFP,which can be visualized in real time and in situ, is stably expressed in articular chondrocytes overexpressing hIGF-1.The labeled articular chondrocytes overexpressing hIGF-1 can be applied in cell-mediated gene therapy as well as for other biomedical purposes of transgenic chondrocytes.

  2. Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish.

    Science.gov (United States)

    Chenais, Nathalie; Lareyre, Jean-Jacques; Le Bail, Pierre-Yves; Labbe, Catherine

    2015-07-01

    The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression. Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation.

  3. Cartilage differentiation in cephalopod molluscs.

    Science.gov (United States)

    Cole, Alison G; Hall, Brian K

    2009-01-01

    Amongst the various metazoan lineages that possess cartilage, tissues most closely resembling vertebrate hyaline cartilage in histological section are those of cephalopod molluscs. Although elements of the adult skeleton have been described, the development of these cartilages has not. Using serial histology of sequential developmental stages of the European cuttlefish, Sepia officinalis, we investigate these skeletal elements and offer the first description of the formation of any cellular invertebrate cartilage. Our data reveal that cuttlefish cartilage most often differentiates from uncondensed mesenchymal cells near the end of embryonic development, but that the earliest-forming cartilages differentiate from a cellular condensation which goes through a protocartilage stage in a manner typical of vertebrate primary cartilage formation. We further investigate the distribution and degree of differentiation of cartilages at the time of hatching in an additional four cephalopod species. We find that the timing of cartilage development varies between elements within a single species, as well as between species. We identify a tendency towards cartilage differentiation from uncondensed connective tissue in elements that form at the end of embryogenesis or after hatching. These data suggest a form of metaplasia from connective tissue is the ancestral mode of cartilage formation in this lineage.

  4. INJURED ARTICULAR CARTILAGE REPAIR

    Directory of Open Access Journals (Sweden)

    Ariana Barlič

    2008-02-01

    Surveys show that the most frequently used surgical methods are mosaicplasty and bonemarrow stimulation with microfracturing. The efficacy of the autologous chondrocyte implantationmethod should be superior to microfracturing on a long run. Especially when(regeneration of the hyaline cartilage instead of fibrous tissue (fibrocartilage is concerned.However, it has not been scientifically proved yet

  5. Ex vivo model exhibits protective effects of sesamin against destruction of cartilage induced with a combination of tumor necrosis factor-alpha and oncostatin M.

    Science.gov (United States)

    Khansai, Manatsanan; Boonmaleerat, Kanchanit; Pothacharoen, Peraphan; Phitak, Thanyaluck; Kongtawelert, Prachya

    2016-07-11

    Rheumatoid arthritis (RA) is an autoimmune disease associated with chronic inflammatory arthritis. TNF-α and OSM are pro-inflammatory cytokines that play a key role in RA progression. Thus, reducing the effects of both cytokines is practical in order to relieve the progression of the disease. This current study is interested in sesamin, an active compound in sesame seeds. Sesamin has been shown to be a chondroprotective agent in osteoarthritis models. Here, we have evaluated a porcine cartilage explant as a cartilage degradation model related to RA induced by TNF-α and/or OSM in order to investigate the effects of sesamin on TNF-α and OSM in the cartilage degradation model. A porcine cartilage explant was induced with a combination of TNF-α and OSM (test group) or IL-1β and OSM (control group) followed by a co-treatment of sesamin over a long-term period (35 days). After which, the tested explants were analyzed for indications of both the remaining and the degradation aspects using glycosaminoglycan and collagen as an indicator. The combination of TNF-α and OSM promoted cartilage degradation more than either TNF-α or OSM alone and was comparable with the combination of IL-1β and OSM. Sesamin could be offering protection against cartilage degradation by reducing GAGs and collagen turnover in the generated model. Sesamin might be a promising agent as an alternative treatment for RA patients. Furthermore, the generated model revealed itself to be an impressive test model for the analysis of phytochemical substances against the cartilage degradation model for RA. The model could be used to test for the prevention of cartilage degradation in other biological agents induced with TNF-α and OSM as well.

  6. Increased angiogenic response in aortic explants of collagen XVIII/endostatin-null mice.

    Science.gov (United States)

    Li, Qing; Olsen, Bjorn R

    2004-08-01

    Endostatin, a proteolytic fragment of basement membrane-associated collagen XVIII, has been shown to be a potent angiogenesis inhibitor both in vivo and in vitro when given at high concentrations. The precise molecular mechanisms by which it functions and whether or not it plays a role in physiological regulation of angiogenesis are not clear. In mice with targeted null alleles of Col18a1, there appears to be no major abnormality in vascular patterns or capillary density in most organs. Furthermore, the growth of experimental tumors is not increased. However, a detailed analysis of induced angiogenesis in these mice has not been performed. Therefore, we compared the angiogenic responses induced by in vitro culture of aortic explants from collagen XVIII/endostatin-null mice (ko) to wild-type (wt) littermates. We found a twofold increase in microvessel outgrowth in explants from ko mice, relative to wt explants. This increased angiogenesis was reduced to the wt level by the addition of low levels (0.1 microg/ml) of recombinant mouse or human endostatin during the culture period. To address cellular/molecular mechanisms underlying this difference in angiogenic response between ko and wt mice, we isolated endothelial cells from both strains and compared their biological behavior. Proliferation assays showed no difference between the two types of endothelial cells. In contrast, adhesion assays showed a striking difference in their ability to adhere to fibronectin suggesting that collagen XVIII/endostatin may regulate interactions between endothelial cells and underlying basement membrane-associated components, including fibronectin, such that in the absence of collagen XVIII/endostatin, endothelial cells are more adhesive to fibronectin. In the aortic explant assay, characterized by dynamic processes of microvessel elongation and regression, this may result in stabilization of newly formed vessels, reduced regression, and a net increase in microvessel outgrowth in

  7. Characterisation of a divergent progenitor cell sub-populations in human osteoarthritic cartilage: the role of telomere erosion and replicative senescence

    Science.gov (United States)

    Fellows, Christopher R.; Williams, Rebecca; Davies, Iwan R.; Gohil, Kajal; Baird, Duncan M.; Fairclough, John; Rooney, Paul; Archer, Charles W.; Khan, Ilyas M.

    2017-01-01

    In recent years it has become increasingly clear that articular cartilage harbours a viable pool of progenitor cells and interest has focussed on their role during development and disease. Analysis of progenitor numbers using fluorescence-activated sorting techniques has resulted in wide-ranging estimates, which may be the result of context-dependent expression of cell surface markers. We have used a colony-forming assay to reliably determine chondroprogenitor numbers in normal and osteoarthritic cartilage where we observed a 2-fold increase in diseased tissue (P  < 0.0001). Intriguingly, cell kinetic analysis of clonal isolates derived from single and multiple donors of osteoarthritic cartilage revealed the presence of a divergent progenitor subpopulation characterised by an early senescent phenotype. Divergent sub-populations displayed increased senescence-associated β–galactosidase activity, lower average telomere lengths but retained the capacity to undergo multi-lineage differentiation. Osteoarthritis is an age-related disease and cellular senescence is predicted to be a significant component of the pathological process. This study shows that although early senescence is an inherent property of a subset of activated progenitors, there is also a pool of progenitors with extended viability and regenerative potential residing within osteoarthritic cartilage. PMID:28150695

  8. Experimental articular cartilage repair in the Göttingen minipig

    DEFF Research Database (Denmark)

    Christensen, Bjørn Borsøe; Foldager, Casper Bindzus; Olesen, Morten Lykke;

    2015-01-01

    BACKGROUND: A gold standard treatment for articular cartilage injuries is yet to be found, and a cost-effective and predictable large animal model is needed to bridge the gap between in vitro studies and clinical studies. Ideally, the animal model should allow for testing of clinically relevant...... treatments and the biological response should be reproducible and comparable to humans. This allows for a reliable translation of results to clinical studies.This study aimed at verifying the Göttingen minipig as a pre-clinical model for articular cartilage repair by testing existing clinical cartilage...

  9. Tribology approach to the engineering and study of articular cartilage.

    Science.gov (United States)

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

    2004-01-01

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

  10. Making post-mortem implantable cardioverter defibrillator explantation safe

    DEFF Research Database (Denmark)

    Räder, Sune B E W; Zeijlemaker, Volkert; Pehrson, Steen;

    2009-01-01

    AIMS: The aim of this study is to investigate whether protection with rubber or plastic gloves during post-mortem explantation of an implantable cardioverter defibrillator (ICD) offers enough protection for the explanting operator during a worst-case scenario (i.e. ICD shock). METHODS AND RESULTS...... that the resting voltage over the operating person would not exceed 50 V. CONCLUSION: The use of intact medical gloves made of latex, neoprene, or plastic eliminates the potential electrical risk during explantation of an ICD. Two gloves on each hand offer sufficient protection. We will recommend the use...

  11. Tissue Culture Responses from Different Explants of Rice

    Institute of Scientific and Technical Information of China (English)

    WANG Xiu-hong; SHI Xiang-yuan; WU Xian-jun

    2005-01-01

    Different culture explants, including anther, young panicle, young embryo, and mature embryo, from 19 rice varieties were used for callus induction and green plantlet differentiation. The culture efficiency differed significantly among the four types of explants, and varied from genotype to genotype. Callus induction frequency presented significantly positive correlation each between anther and young panicle, anther and mature embryo, and young panicle and young embryo. Green plantlet differentiation showed no relationship between different types of explants. In addition, no relationship was found between callus induction frequency and green plantlet differentiation frequency.

  12. Absolute Quantification of Selected Proteins in the Human Osteoarthritic Secretome

    Directory of Open Access Journals (Sweden)

    Mandy J. Peffers

    2013-10-01

    Full Text Available Osteoarthritis (OA is characterized by a loss of extracellular matrix which is driven by catabolic cytokines. Proteomic analysis of the OA cartilage secretome enables the global study of secreted proteins. These are an important class of molecules with roles in numerous pathological mechanisms. Although cartilage studies have identified profiles of secreted proteins, quantitative proteomics techniques have been implemented that would enable further biological questions to be addressed. To overcome this limitation, we used the secretome from human OA cartilage explants stimulated with IL-1β and compared proteins released into the media using a label-free LC-MS/MS-based strategy. We employed QconCAT technology to quantify specific proteins using selected reaction monitoring. A total of 252 proteins were identified, nine were differentially expressed by IL-1 β stimulation. Selected protein candidates were quantified in absolute amounts using QconCAT. These findings confirmed a significant reduction in TIMP-1 in the secretome following IL-1β stimulation. Label-free and QconCAT analysis produced equivocal results indicating no effect of cytokine stimulation on aggrecan, cartilage oligomeric matrix protein, fibromodulin, matrix metalloproteinases 1 and 3 or plasminogen release. This study enabled comparative protein profiling and absolute quantification of proteins involved in molecular pathways pertinent to understanding the pathogenesis of OA.

  13. Advances in cartilage tissue engineering : in vitro

    NARCIS (Netherlands)

    E.W. Mandl (Erik)

    2004-01-01

    textabstractWithin the body three subtypes of cartilage can be distinguished: hyaline cartilage, elastic cartilage and fibrocartilage. Hyaline cartilage is the predominant subtype and is mainly located in articular joints and in less extent in the nasal septum and cricoid. Elastic cartilage can be

  14. Label-free characterization of articular cartilage in osteoarthritis model mice by Raman spectroscopy

    Science.gov (United States)

    Oshima, Yusuke; Akehi, Mayu; Kiyomatsu, Hiroshi; Miura, Hiromasa

    2017-02-01

    Osteoarthritis (OA) is very common joint disease in the aging population. Main symptom of OA is accompanied by degenerative changes of articular cartilage. Cartilage contains mostly type II collagen and proteoglycans, so it is difficult to access the quality and morphology of cartilage tissue in situ by conventional diagnostic tools (X-ray, MRI and echography) directly or indirectly. Raman spectroscopy is a label-free technique which enables to analyze molecular composition in degenerative cartilage. In this study, we generated an animal OA model surgically induced by knee joint instability, and the femurs were harvested at two weeks after the surgery. We performed Raman spectroscopic analysis for the articular cartilage of distal femurs in OA side and unaffected side in each mouse. In the result, there is no gross findings in the surface of the articular cartilage in OA. On the other hand, Raman spectral data of the articular cartilage showed drastic changes in comparison between OA and control side. The major finding of this study is that the relative intensity of phosphate band (960 cm-1) increases in the degenerative cartilage. This may be the result of exposure of subchondral bone due to thinning of the cartilage layer. In conclusion, Raman spectroscopic technique is sufficient to characterize articular cartilage in OA as a pilot study for Raman application in cartilage degeneration and regeneration using animal models and human subjects.

  15. Generating cartilage repair from pluripotent stem cells.

    Science.gov (United States)

    Cheng, Aixin; Hardingham, Timothy E; Kimber, Susan J

    2014-08-01

    The treatment of degeneration and injury of articular cartilage has been very challenging for scientists and surgeons. As an avascular and hypocellular tissue, cartilage has a very limited capacity for self-repair. Chondrocytes are the only cell type in cartilage, in which they are surrounded by the extracellular matrix that they secrete and assemble. Autologous chondrocyte implantation for cartilage defects has achieved good results, but the limited resources and complexity of the procedure have hindered wider application. Stem cells form an alternative to chondrocytes as a source of chondrogenic cells due to their ability to proliferate extensively while retaining the potential for differentiation. Adult stem cells such as mesenchymal stem cells have been differentiated into chondrocytes, but the limitations in their proliferative ability and the heterogeneous cell population hinder their adoption as a prime alternative source for generating chondrocytes. Human embryonic stem cells (hESCs) are attractive as candidates for cell replacement therapy because of their unlimited self-renewal and ability for differentiation into mesodermal derivatives as well as other lineages. In this review, we focus on current protocols for chondrogenic differentiation of ESCs, in particular the chemically defined culture system developed in our lab that could potentially be adapted for clinical application.

  16. PRP and Articular Cartilage: A Clinical Update

    Science.gov (United States)

    Rossi, Roberto; Castoldi, Filippo; Michielon, Gianni

    2015-01-01

    The convincing background of the recent studies, investigating the different potentials of platelet-rich plasma, offers the clinician an appealing alternative for the treatment of cartilage lesions and osteoarthritis. Recent evidences in literature have shown that PRP may be helpful both as an adjuvant for surgical treatment of cartilage defects and as a therapeutic tool by intra-articular injection in patients affected by osteoarthritis. In this review, the authors introduce the trophic and anti-inflammatory properties of PRP and the different products of the available platelet concentrates. Then, in a complex scenario made of a great number of clinical variables, they resume the current literature on the PRP applications in cartilage surgery as well as the use of intra-articular PRP injections for the conservative treatment of cartilage degenerative lesions and osteoarthritis in humans, available as both case series and comparative studies. The result of this review confirms the fascinating biological role of PRP, although many aspects yet remain to be clarified and the use of PRP in a clinical setting has to be considered still exploratory. PMID:26075244

  17. PRP and Articular Cartilage: A Clinical Update

    Directory of Open Access Journals (Sweden)

    Antonio Marmotti

    2015-01-01

    Full Text Available The convincing background of the recent studies, investigating the different potentials of platelet-rich plasma, offers the clinician an appealing alternative for the treatment of cartilage lesions and osteoarthritis. Recent evidences in literature have shown that PRP may be helpful both as an adjuvant for surgical treatment of cartilage defects and as a therapeutic tool by intra-articular injection in patients affected by osteoarthritis. In this review, the authors introduce the trophic and anti-inflammatory properties of PRP and the different products of the available platelet concentrates. Then, in a complex scenario made of a great number of clinical variables, they resume the current literature on the PRP applications in cartilage surgery as well as the use of intra-articular PRP injections for the conservative treatment of cartilage degenerative lesions and osteoarthritis in humans, available as both case series and comparative studies. The result of this review confirms the fascinating biological role of PRP, although many aspects yet remain to be clarified and the use of PRP in a clinical setting has to be considered still exploratory.

  18. Fluoroquinolone's effect on growth of human chondrocytes and chondrosarcomas. In vitro and in vivo correlation

    DEFF Research Database (Denmark)

    Multhaupt, H A; Alvarez, J C; Rafferty, P A

    2001-01-01

    Clinical and in vitro studies have demonstrated that fluoroquinolones are toxic to chondrocytes; however, the exact mechanism of fluoroquinolone arthropathy is unknown. We investigated the toxicity of ciprofloxacin on normal cartilage and on cartilaginous tumors. Normal human cartilage, enchondroma......, and chondrosarcoma explants were cultured either alone or with the addition of ciprofloxacin at 1, 10, or 20 mg/L of medium. Samples were collected up to twenty-one days after treatment and were processed for electron microscopy and conventional light microscopy. The specimens were characterized morphologically...... droplets, rough endoplasmic reticulum, and prominent Golgi apparatus; and a proteoglycan layer surrounding the cells. With prolonged ciprofloxacin treatment and with increased doses, there was an increase in dilated rough endoplasmic reticulum, the appearance of phagosomes, and disintegrated bundles...

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

    Directory of Open Access Journals (Sweden)

    Elena Vasil'evna Chetina

    2010-01-01

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

  20. An ovine tracheal explant culture model for allergic airway inflammation

    Directory of Open Access Journals (Sweden)

    Abeynaike Latasha

    2010-08-01

    Full Text Available Abstract Background The airway epithelium is thought to play an important role in the pathogenesis of asthmatic disease. However, much of our understanding of airway epithelial cell function in asthma has been derived from in vitro studies that may not accurately reflect the interactive cellular and molecular pathways active between different tissue constituents in vivo. Methods Using a sheep model of allergic asthma, tracheal explants from normal sheep and allergic sheep exposed to house dust mite (HDM allergen were established to investigate airway mucosal responses ex vivo. Explants were cultured for up to 48 h and tissues were stained to identify apoptotic cells, goblet cells, mast cells and eosinophils. The release of cytokines (IL-1α, IL-6 and TNF-α by cultured tracheal explants, was assessed by ELISA. Results The general morphology and epithelial structure of the tracheal explants was well maintained in culture although evidence of advanced apoptosis within the mucosal layer was noted after culture for 48 h. The number of alcian blue/PAS positive mucus-secreting cells within the epithelial layer was reduced in all cultured explants compared with pre-cultured (0 h explants, but the loss of staining was most evident in allergic tissues. Mast cell and eosinophil numbers were elevated in the allergic tracheal tissues compared to naïve controls, and in the allergic tissues there was a significant decline in mast cells after 24 h culture in the presence or absence of HDM allergen. IL-6 was released by allergic tracheal explants in culture but was undetected in cultured control explants. Conclusions Sheep tracheal explants maintain characteristics of the airway mucosa that may not be replicated when studying isolated cell populations in vitro. There were key differences identified in explants from allergic compared to control airways and in their responses in culture for 24 h. Importantly, this study establishes the potential for the

  1. Scaffolding Biomaterials for Cartilage Regeneration

    Directory of Open Access Journals (Sweden)

    Zhen Cao

    2014-01-01

    Full Text Available Completely repairing of damaged cartilage is a difficult procedure. In recent years, the use of tissue engineering approach in which scaffolds play a vital role to regenerate cartilage has become a new research field. Investigating the advances in biological cartilage scaffolds has been regarded as the main research direction and has great significance for the construction of artificial cartilage. Native biological materials and synthetic polymeric materials have their advantages and disadvantages. The disadvantages can be overcome through either physical modification or biochemical modification. Additionally, developing composite materials, biomimetic materials, and nanomaterials can make scaffolds acquire better biocompatibility and mechanical adaptability.

  2. Profiling the secretion of soluble mediators by end stage osteoarthritis synovial tissue explants reveals a reduced responsiveness to an inflammatory trigger.

    Directory of Open Access Journals (Sweden)

    Lobke M Gierman

    Full Text Available OBJECTIVE: Evidence is accumulating that synovial tissue plays an active role in osteoarthritis (OA, however, exact understanding of its contribution is lacking. In order to further elucidate its role in the OA process, we aimed to identify the secretion pattern of soluble mediators by synovial tissue and to assess its ability to initiate cartilage degeneration. METHODS: Synovial tissue explants (STEs obtained from donors without history of OA (n = 8 or from end stage OA patients (n = 16 were cultured alone or together with bovine cartilage explants in the absence or presence of IL-1α. The secretion of 48 soluble mediators was measured and the effect on glycosaminoglycan (GAG release and matrix metalloproteinase (MMP activity was determined. RESULTS: Normal and OA STEs secreted comparable levels of almost all measured soluble mediators. However, in the presence of IL-1α these mediators were less secreted by OA than by normal STEs of which 15 differed significantly (p<0.01. No effect of normal or OA STEs on GAG release from the cartilage explants was observed, and no differences in MMP activity between OA and normal STEs were detected. CONCLUSIONS: Unexpectedly, a comparable secretion profile of soluble mediators was found for OA and normal STEs while the reduced responsiveness of OA STEs to an inflammatory trigger indicates a different state of this tissue in OA patients. The effects could be the result of prolonged exposure to an inflammatory environment in OA development. Further understanding of the pro-inflammatory and inflammation resolving mechanisms during disease progression in synovial tissue may provide valuable targets for therapy in the future.

  3. Intraocular lens explantation or exchange: indications, postoperative interventions, and outcomes

    Directory of Open Access Journals (Sweden)

    Refik Oltulu

    2015-06-01

    Full Text Available ABSTRACT Purpose: To analyze the indications for explantation or exchange of intraocular lenses (IOLs, which were originally implanted for the correction of aphakia during cataract extraction. Methods: All cases that involved intraocular lens explantation or exchange in one institution between January 2008 and December 2014 were analyzed retrospectively. Results: In total, 93 eyes of 93 patients were analyzed. The median time interval between implantation and explantation of the anterior chamber intraocular lenses (AC IOL and posterior chamber intraocular lenses (PC IOL was 83.40 ± 83.14 months (range: 1-276 months and 55.14 ± 39.25 months (range: 1-168 months, respectively. Pseudophakic bullous keratopathy (17 eyes, 38.6% and persistent iritis (12 eyes, 27.8% in the AC IOL group and dislocation or decentration (30 eyes, 61.2% and incorrect IOL power (nine eyes, 18.4% in the PC IOL group were the most common indications for explantation of IOLs. The mean logMAR best corrected visual acuity (BCVA improved significantly from 1.30 preoperatively to 0.62 postoperatively in the PC IOL group (p<0.001 but did not improve significantly in the AC IOL group (p=0.186. Conclusions: The primary indication for IOL explantation or exchange was pseudophakic bullous keratopathy in the AC IOL group and was dislocation or decentration in the PC IOL group. PC IOL explantation or exchange is safe and improves visual acuity.

  4. Repair of Torn Avascular Meniscal Cartilage Using Undifferentiated Autologous Mesenchymal Stem Cells: From In Vitro Optimization to a First?in?Human Study

    OpenAIRE

    Whitehouse, Michael; Howells, Nicholas; Parry, Michael; Austin, Eric; Kafienah, Wael; Brady, Kyla; Goodship, Allen; Eldridge, Jonathan; Blom, Ashley; Hollander, Anthony

    2016-01-01

    Abstract Meniscal cartilage tears are common and predispose to osteoarthritis (OA). Most occur in the avascular portion of the meniscus where current repair techniques usually fail. We described previously the use of undifferentiated autologous mesenchymal stem cells (MSCs) seeded onto a collagen scaffold (MSC/collagen?scaffold) to integrate meniscal tissues in vitro. Our objective was to translate this method into a cell therapy for patients with torn meniscus, with the long?term goal of del...

  5. Superior results with continuous passive motion compared to active motion after periosteal transplantation. A retrospective study of human patella cartilage defect treatment.

    Science.gov (United States)

    Alfredson, H; Lorentzon, R

    1999-01-01

    Fifty-seven consecutive patients (33 men and 24 women), with a mean age of 32 years (range 16-53 years), who suffered from an isolated full-thickness cartilage defect of the patella and disabling knee pain of long duration, were treated by autologous periosteal transplantation to the cartilage defect. The first 38 consecutive patients (group A) were postoperatively treated with continuous passive motion (CPM), and the next 19 consecutive patients (group B) were treated with active motion for the first 5 days postoperatively. In both groups, the initial regimens were followed by active motion, slowly progressive strength training, and slowly progressive weight bearing. In group A, after a mean follow-up of 51 months (range 33-92 months), 29 patients (76%) were graded as excellent or good, 7 patients (19%) were graded as fair, and 2 patients (5%) were graded as poor. In group B, after a mean follow-up of 21 months (range 14-28 months), 10 patients (53%) were graded as excellent or good, 6 patients (32%) were graded as fair, and 3 patients (15%) were graded as poor. Altogether, nine of the fair or poor cases (50%) were diagnosed with chondromalacia of the patella. Our results, after performing autologous periosteal transplantation in patients with full-thickness cartilage defects of the patella and disabling knee pain, are good if CPM is used postoperatively. The clinical results using active motion postoperatively are not acceptable, especially not in patients with chondromalacia of the patella.

  6. 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

  7. Efficient Isolation of Mesenchymal Stem Cells from Human Bone Marrow by Direct Plating Method Combined with Modified Primary Explant Culture%直接铺种结合改良组织块培养法可有效分离人骨髓中的间充质干细胞

    Institute of Scientific and Technical Information of China (English)

    邢文; 庞爱明; 姚剑峰; 李园; 石慧; 盛梦瑶; 周圆; 赵迎旭; 许明江

    2013-01-01

    Human bone marrow is the major source of mesenchymal stem cells (MSC). It was reported that the standard density gradient centrifugation method was not efficient in isolating MSC and it may be caused by the existing of bone marrow particles. In previous studys, a lot of MSC were obtained by culturing bone marrow particles alone combined with standard method. However, it is time- and labor-consuming to obtain bone marrow particles by filtering and to isolate MNC by density gradient centrifugation. This study was purposed to explore the more simple and efficient method to isolate MSC from bone marrow. Seven normal bone marrow aspirates were collected and centrifugated. The bone marrow particles floated on surface layers were cultured by modified primary explant culture, whereas the bone marrow aspirates deposited were cultured by direct plating method, then the immun phenotype and differentiation capability of isolated cells were analyzed. The results showed that in 3 of 7 aspirates, bone marrow particles were floated on surface layers, whereas the other bone marrow cells and some particles were deposited after centrifugation. The MSC were reliably isolated from the floating layers or deposited aspirates by modified primary explant culture and direct plating method separately. After 3 passages the isolated MSC did not express CD45 and CD34, but expressed CD105 ,CD73, CD44,CD90,CD49e and they could differentiate into chondrocytes and adipocytes. It is concluded that normal human bone marrow MSC can be isolated simply and efficiently by direct plating method in combination with modified primary explant culture.%骨髓是间充质干细胞(MSC)的重要来源.研究显示,标准密度梯度离心法分离骨髓MSC的效率不高,骨髓小粒是造成该法低效的原因.通过组织块法分离骨髓小粒,再结合标准法,可从单份骨髓标本分离获得更多MSC,然而这种方法费时费力.本研究探求分离骨髓MSC更简单、更有效的方法.收集7

  8. Mechanical Compression of Articular Cartilage Induces Chondrocyte Proliferation and Inhibits Proteoglycan Synthesis by Activation of the Erk Pathway: Implications for Tissue Engineering and Regenerative Medicine

    Science.gov (United States)

    Ryan, James A.; Eisner, Eric A.; DuRaine, Grayson; You, Zongbing; Reddi, A. Hari

    2013-01-01

    Articular cartilage is recalcitrant to endogenous repair and regeneration and thus a focus of tissue engineering and regenerative medicine strategies. A pre-requisite for articular cartilage tissue engineering is an understanding of the signal transduction pathways involved in mechanical compression during trauma or disease. We sought to explore the role of the extracellular signal-regulated kinase 1/2 (ERK 1/2) pathway in chondrocyte proliferation and proteoglycan synthesis following acute mechanical compression. Bovine articular cartilage explants were cultured with and without the ERK 1/2 pathway inhibitor PD98059. Cartilage explants were statically loaded to 40% strain at a strain rate of 1−sec for 5 seconds. Control explants were cultured under similar conditions but were not loaded. There were four experimental groups: 1) no load without inhibitor 2) no load with the inhibitor PD98059, 3) loaded without the inhibitor, and 4) loaded with the inhibitor PD98059. Explants were cultured for varying durations, from 5 minutes to 5 days. Explants were then analyzed by biochemical and immunohistochemical methods. Mechanical compression induced phosphorylation of ERK 1/2, and this was attenuated with the ERK 1/2 pathway inhibitor PD98059 in a dose-dependent manner. Chondrocyte proliferation was increased by mechanical compression. This effect was blocked by the inhibitor of the ERK 1/2 pathway. Mechanical compression also led to a decrease in proteoglycan synthesis that was reversed with inhibitor PD98059. In conclusion, the ERK 1/2 pathway is involved in the proliferative and biosynthetic response of chondrocytes following acute static mechanical compression. PMID:19177463

  9. Linking Cellular and Mechanical Processes in Articular Cartilage Lesion Formation: A Mathematical Model.

    Science.gov (United States)

    Kapitanov, Georgi I; Wang, Xiayi; Ayati, Bruce P; Brouillette, Marc J; Martin, James A

    2016-01-01

    Post-traumatic osteoarthritis affects almost 20% of the adult US population. An injurious impact applies a significant amount of physical stress on articular cartilage and can initiate a cascade of biochemical reactions that can lead to the development of osteoarthritis. In our effort to understand the underlying biochemical mechanisms of this debilitating disease, we have constructed a multiscale mathematical model of the process with three components: cellular, chemical, and mechanical. The cellular component describes the different chondrocyte states according to the chemicals these cells release. The chemical component models the change in concentrations of those chemicals. The mechanical component contains a simulation of a blunt impact applied onto a cartilage explant and the resulting strains that initiate the biochemical processes. The scales are modeled through a system of partial-differential equations and solved numerically. The results of the model qualitatively capture the results of laboratory experiments of drop-tower impacts on cartilage explants. The model creates a framework for incorporating explicit mechanics, simulated by finite element analysis, into a theoretical biology framework. The effort is a step toward a complete virtual platform for modeling the development of post-traumatic osteoarthritis, which will be used to inform biomedical researchers on possible non-invasive strategies for mitigating the disease.

  10. New technique for preparing cartilage for intracordal injection: the freezing and grinding method.

    Science.gov (United States)

    Park, Young Min; Lee, Won Yong; Lim, Yun-Sung; Lee, Jin-Choon; Lee, Byung-Joo; Wang, Soo-Geun

    2014-07-01

    We developed a technique for preparing harvested cartilage that creates finer, more uniform pieces by freezing with liquid nitrogen and grinding with a mortar and pestle. Herein, we report the application of this new technique for intracordal cartilage injection. Experimental study. Human cartilage was obtained from surgical cases. In the standard method, harvested cartilage was prepared with scissors and a knife. In the experimental group, harvested cartilage was frozen with liquid nitrogen and ground with a mortar and pestle. It took an average of 60 minutes to manipulate cartilage using the standard technique, whereas it took an average of 10 minutes using the freezing and grinding method (P<0.001). The average size of cartilage flakes generated by the standard and new techniques were 727 and 48.6 μm, respectively. The cartilage flakes produced using scissors and a knife were able to pass through a 19-gauge needle, whereas those created using the freezing and grinding method were able to pass through a 24-gauge needle. Using the freezing and grinding method, cartilage was broken into fine, uniform pieces that could pass through a 24-gauge needle. This new technique will facilitate the production of commercial cartilage material for intracordal injection. Copyright © 2014 The Voice Foundation. All rights reserved.

  11. Histological Analysis of Failed Cartilage Repair after Marrow Stimulation for the Treatment of Large Cartilage Defect in Medial Compartmental Osteoarthritis of the Knee

    Directory of Open Access Journals (Sweden)

    Sakata,Kenichiro

    2013-02-01

    Full Text Available Bone marrow-stimulating techniques such as microfracture and subchondral drilling are valuable treatments for full-thickness cartilage defects. However, marrow stimulation-derived reparative tissues are not histologically well-documented in human osteoarthritis. We retrospectively investigated cartilage repairs after marrow stimulation for the treatment of large cartilage defects in osteoarthritic knees. Tissues were obtained from patients who underwent total knee arthroplasty (TKA after arthroscopic marrow stimulation in medial compartmental osteoarthritis. Clinical findings and cartilage repair were assessed. Sections of medial femoral condyles were histologically investigated by safranin O staining and anti-type II collagen antibody. Marrow stimulation decreased the knee pain in the short term. However, varus leg alignment gradually progressed, and TKA conversions were required. The grade of cartilage repair was not improved. Marrow stimulations resulted in insufficient cartilage regeneration on medial femoral condyles. Safranin O-stained proteoglycans and type II collagen were observed in the deep zone of marrow-stimulated holes. This study demonstrated that marrow stimulation resulted in failed cartilage repair for the treatment of large cartilage defects in osteoarthritic knees. Our results suggest that arthroscopic marrow stimulation might not improve clinical symptoms for the long term in patients suffering large osteoarthritic cartilage defects.

  12. [The anatomical structure similarity research on auricular cartilage and nasal alar cartilage].

    Science.gov (United States)

    Chen, Changyong; Fan, Fei; Li, Wenzhi; Li, Binbin; You, Jianjun; Wang, Huan

    2015-09-01

    There are many scaffold materials of repairing nasal alar cartilage defects. Auricuiar cartilage was used extensively in terms of its abundant tissues, good elasticity, little donor-site malformation, good plasticity etc. The authors dissected auricular cartilage and nasal alar cartilage, measured cartilage's morphous data and found some similar territories with nasal alar cartilage in the structure of auricular cartilage. An anatomical study was performed using 10 adult cadavers acquired through Plastic Surgery Hospital, Peking Union Medical College, Beijing, China. Seven male and three female cadav-ers were included in the study. Harvest 20 auricular cartilage specimens and 20 nasal alar cartilage specimens. Then, Computed Tomography Scan on the auricular cartilage and nasal alar cartilage were performed. The datas were imported into mimics and three-dimensional reconstructions of the auricular cartilage and nasal alar cartilage were carried on. Parts of the auricular cartilage, such as conchal fossa, tragus, intertragic notch, and cymba of auricular concha, curs of helix and curs of helix, triangular fossa, are ana-tomically similar to nasal alar cartilage. This study reports the anatomy of auricular cartilage and nasal alar cartilage, found some territories in the auricular cartilage, such as conchal fossa, tragus, intertragic notch, and cymba of auricular concha, curs of helix and curs of helix, triangular fossa, are anatomically similar to nasal alar cartilage. This research provides the anatomical basis that auricular cartilage was used to repair the nasal cartilage defect.

  13. Cartilage oligomeric matrix protein neoepitope in the synovial fluid of horses with acute lameness: A new biomarker for the early stages of osteoarthritis.

    Science.gov (United States)

    Skiöldebrand, E; Ekman, S; Mattsson Hultén, L; Svala, E; Björkman, K; Lindahl, A; Lundqvist, A; Önnerfjord, P; Sihlbom, C; Rüetschi, U

    2017-09-01

    Clinical tools to diagnose the early changes of osteoarthritis (OA) that occur in the articular cartilage are lacking. We sought to identify and quantify a novel cartilage oligomeric matrix protein (COMP) neoepitope in the synovial fluid from the joints of healthy horses and those with different stages of OA. In vitro quantitative proteomics and assay development with application in synovial fluids samples obtained from biobanks of well-characterised horses. Articular cartilage explants were incubated with or without interleukin-1β for 25 days. Media were analysed via quantitative proteomics. Synovial fluid was obtained from either normal joints (n = 15) or joints causing lameness (n = 17) or with structural OA lesions (n = 7) and analysed for concentrations of the COMP neoepitope using a custom-developed inhibition enzyme-linked immunosorbent assay (ELISA). Explants were immunostained with polyclonal antibodies against COMP and the COMP neoepitopes. Semitryptic COMP peptides were identified and quantified in cell culture media from cartilage explants. A rabbit polyclonal antibody was raised against the neoepitope of the N-terminal portion of one COMP fragment (sequence SGPTHEGVC). An inhibition ELISA was developed to quantify the COMP neoepitope in synovial fluid. The mean concentration of the COMP neoepitope significantly increased in the synovial fluid from the joints responsible for acute lameness compared with normal joints and the joints of chronically lame horses and in joints with chronic structural OA. Immunolabelling for the COMP neoepitope revealed a pericellular staining in the interleukin-1β-stimulated explants. The ELISA is based on polyclonal antisera rather than a monoclonal antibody. The increase in the COMP neoepitope in the synovial fluid from horses with acute lameness suggests that this neoepitope has the potential to be a unique candidate biomarker for the early molecular changes in articular cartilage associated with OA. © 2017 The Authors

  14. Isolation and characterization of mesenchymal stem cells derived from whole human umbilical cord by applying a direct explant technique%用组织贴壁法从整根脐带分离培养间充质干细胞及其生物学特性的检测

    Institute of Scientific and Technical Information of China (English)

    张颢; 张斌; 程梅; 陶艳玲; 扈江伟; 徐曼; 陈虎

    2012-01-01

    Objective To establish a technique for isolating mesenchymal stem cells from umbilical cord and their biological characteristics. Methods Mesenchymal stem cells (MSCs) were isolated from umbilical cord(UC) by applying a explant technique and explanding cultured in vitro. Growth curves were drawn, and The phenotypes and cell cycle were evaluated by flow cytometry. UC-MSCs were induced to differentiate into adipocytes, osteocytes and chondrocytes in special differentiation condition. Mutidifferentiation related genes and stem cell-related transcription factors, Nanog, Oct-4 Sox-2 were detected by TR-PCR. Results UC-MSCs were isolated using a explant technique , and characteristic of plastic adherence and fibroblast-like morphology, the adherent cells displayed an abundant presence of CD73, CD90, CD105 and absence of CD34, CD45, HLA-DR. Cell cycle showed that there werepercentages of stem cells as Go/G1 81. 56 % and S + G2 + M 18. 44% respectively. When cultured in differentiation media, they differentiated into adipocytes, osteocytes and chondrocytes. RT-PCR reactions confirmed that their mutidifferentiation related genes were positive. Moreover, stem cell-related transcription factors, Nanog, Oct-4 Sox-2 were positively expressed in UC-MSCs. Conclusions The explant technique is a feasible method to obtain MSCs from whole human umbilical cord, and UC can be considered as a novel and convenient source of adult MSCs displaying primitive pluripotent sten cells and high expansion potential.%目的 建立从整根脐带分离培养间充质干细胞(UC-MSCs)的技术,并对其生物学特性进行检测.方法 用组织贴壁法分离UC-MSCs,并通过传代进行纯化和扩增培养,绘制生长曲线,用流式细胞仪检测UC-MSCs表面抗原及细胞周期;在特定诱导体系中,检测UC-MSCs向脂肪、成骨及软骨分化的潜能;用RT-PCR检测多能干细胞标志多能干细胞标志Oct-4,Sox-2,NanogmRNA水平.结果 成功建立了UC-MSCs分离培养的

  15. Articular cartilage collagen: an irreplaceable framework?

    Directory of Open Access Journals (Sweden)

    D R Eyre

    2006-11-01

    Full Text Available Adult articular cartilage by dry weight is two-thirds collagen. The collagen has a unique molecular phenotype. The nascent type II collagen fibril is a heteropolymer, with collagen IX molecules covalently linked to the surface and collagen XI forming the filamentous template of the fibril as a whole. The functions of collagens IX and XI in the heteropolymer are far from clear but, evidently, they are critically important since mutations in COLIX and COLXI genes can result in chondrodysplasia syndromes. Here we review what is known of the collagen assembly and present new evidence that collagen type III becomes covalently added to the polymeric fabric of adult human articular cartilage, perhaps as part of a matrix repair or remodelling process.

  16. An amidated carboxymethylcellulose hydrogel for cartilage regeneration.

    Science.gov (United States)

    Leone, Gemma; Fini, Milena; Torricelli, Paola; Giardino, Roberto; Barbucci, Rolando

    2008-08-01

    An amidic derivative of carboxymethylcellulose was synthesized (CMCA). The new polysaccharide was obtained by converting a large percentage of carboxylic groups ( approximately 50%) of carboxymethylcellulose into amidic groups rendering the macromolecule quite similar to hyaluronan. Then, the polysaccharide (CMCA) was crosslinked. The behavior of CMCA hydrogel towards normal human articular chondrocytes (NHAC) was in vitro studied monitoring the cell proliferation and synthesis of extra cellular matrix (ECM) components and compared with a hyaluronan based hydrogel (Hyal). An extracellular matrix rich in cartilage-specific collagen and proteoglycans was secreted in the presence of hydrogels. The injectability of the new hydrogels was also analysed. An experimental in vivo model was realized to study the effect of CMCA and Hyal hydrogels in the treatment of surgically created partial thickness chondral defects in the rabbit knee. The preliminary results pointed out that CMCA hydrogel could be considered as a potential compound for cartilage regeneration.

  17. Altered Knee Joint Mechanics in Simple Compression Associated with Early Cartilage Degeneration

    Directory of Open Access Journals (Sweden)

    Y. Dabiri

    2013-01-01

    Full Text Available The progression of osteoarthritis can be accompanied by depth-dependent changes in the properties of articular cartilage. The objective of the present study was to determine the subsequent alteration in the fluid pressurization in the human knee using a three-dimensional computer model. Only a small compression in the femur-tibia direction was applied to avoid numerical difficulties. The material model for articular cartilages and menisci included fluid, fibrillar and nonfibrillar matrices as distinct constituents. The knee model consisted of distal femur, femoral cartilage, menisci, tibial cartilage, and proximal tibia. Cartilage degeneration was modeled in the high load-bearing region of the medial condyle of the femur with reduced fibrillar and nonfibrillar elastic properties and increased hydraulic permeability. Three case studies were implemented to simulate (1 the onset of cartilage degeneration from the superficial zone, (2 the progression of cartilage degeneration to the middle zone, and (3 the progression of cartilage degeneration to the deep zone. As compared with a normal knee of the same compression, reduced fluid pressurization was observed in the degenerated knee. Furthermore, faster reduction in fluid pressure was observed with the onset of cartilage degeneration in the superficial zone and progression to the middle zone, as compared to progression to the deep zone. On the other hand, cartilage degeneration in any zone would reduce the fluid pressure in all three zones. The shear strains at the cartilage-bone interface were increased when cartilage degeneration was eventually advanced to the deep zone. The present study revealed, at the joint level, altered fluid pressurization and strains with the depth-wise cartilage degeneration. The results also indicated redistribution of stresses within the tissue and relocation of the loading between the tissue matrix and fluid pressure. These results may only be qualitatively interesting

  18. A novel method for coral explant culture and micropropagation.

    Science.gov (United States)

    Vizel, Maya; Loya, Yossi; Downs, Craig A; Kramarsky-Winter, Esti

    2011-06-01

    We describe here a method for the micropropagation of coral that creates progeny from tissue explants derived from a single polyp or colonial corals. Coral tissue explants of various sizes (0.5-2.5 mm in diameter) were manually microdissected from the solitary coral Fungia granulosa. Explants could be maintained in an undeveloped state or induced to develop into polyps by manipulating environmental parameters such as light and temperature regimes, as well as substrate type. Fully developed polyps were able to be maintained for a long-term in a closed sea water system. Further, we demonstrate that mature explants are also amenable to this technique with the micropropagation of second-generation explants and their development into mature polyps. We thereby experimentally have established coral clonal lines that maintain their ability to differentiate without the need for chemical induction or genetic manipulation. The versatility of this method is also demonstrated through its application to two other coral species, the colonial corals Oculina patigonica and Favia favus.

  19. Mechanical overloading causes mitochondrial superoxide and SOD2 imbalance in chondrocytes resulting in cartilage degeneration.

    Science.gov (United States)

    Koike, Masato; Nojiri, Hidetoshi; Ozawa, Yusuke; Watanabe, Kenji; Muramatsu, Yuta; Kaneko, Haruka; Morikawa, Daichi; Kobayashi, Keiji; Saita, Yoshitomo; Sasho, Takahisa; Shirasawa, Takuji; Yokote, Koutaro; Kaneko, Kazuo; Shimizu, Takahiko

    2015-06-25

    Mechanical stress and aging are major risk factors of cartilage degeneration. Human studies have previously reported that oxidative damage increased, while SOD2 protein was reciprocally downregulated in osteoarthritic degenerated cartilage. However, it remains unclear whether mitochondrial superoxide imbalance in chondrocytes causes cartilage degeneration. We herein demonstrate that mechanical loading promoted mitochondrial superoxide generation and selective Sod2 downregulation in chondrocytes in vivo and that mitochondrial superoxide inducer also downregulated Sod2 expression in chondrocytes in vitro. A genetically manipulated model revealed that Sod2 deficiency in chondrocytes also resulted in mitochondrial superoxide overproduction and dysfunction, thus leading to cartilage degeneration. Intra-articular injection of a permeable antioxidant effectively suppressed the mechanical loading-induced mitochondrial superoxide generation and cartilage degeneration in mice. Our findings demonstrate that mitochondrial superoxide plays a pivotal role in the development and progression of osteoarthritis, and the mitochondrial superoxide balance may therefore be a promising target for the treatment of cartilage degeneration.

  20. 13C NMR relaxation studies on cartilage and cartilage components.

    Science.gov (United States)

    Naji, L; Kaufmann, J; Huster, D; Schiller, J; Arnold, K

    2000-08-07

    We have investigated the molecular motions of polysaccharides of bovine nasal and pig articular cartilage by measuring the 13C NMR relaxation times (T1 and T2). Both types of cartilage differ significantly towards their collagen/glycosaminoglycan ratio, leading to different NMR spectra. As chondroitin sulfate is the main constituent of cartilage, aqueous solutions of related poly- and monosaccharides (N-acetylglucosamine and glucuronic acid) were also investigated. Although there are only slight differences in T1 relaxation of the mono- and the polysaccharides, T2 decreases about one order of magnitude, when glucuronic acid or N-acetylglucosamine and chondroitin sulfate are compared. It is concluded that the ring carbons are motion-restricted primarily by the embedment in the rigid pyranose structure and, thus, additional limitations of mobility do not more show a major effect. Significant differences were observed between bovine nasal and pig articular cartilage, resulting in a considerable line-broadening and a lower signal to noise ratio in the spectra of pig articular cartilage. This is most likely caused by the higher collagen content of articular cartilage in comparison to the polysaccharide-rich bovine nasal cartilage.

  1. Quantitative imaging of excised osteoarthritic cartilage using spectral CT

    Energy Technology Data Exchange (ETDEWEB)

    Rajendran, Kishore; Bateman, Christopher J.; Younis, Raja Aamir; De Ruiter, Niels J.A.; Ramyar, Mohsen; Anderson, Nigel G. [University of Otago - Christchurch, Department of Radiology, Christchurch (New Zealand); Loebker, Caroline [University of Otago, Christchurch Regenerative Medicine and Tissue Engineering Group, Department of Orthopaedic Surgery and Musculoskeletal Medicine, Christchurch (New Zealand); University of Twente, Department of Developmental BioEngineering, Enschede (Netherlands); Schon, Benjamin S.; Hooper, Gary J.; Woodfield, Tim B.F. [University of Otago, Christchurch Regenerative Medicine and Tissue Engineering Group, Department of Orthopaedic Surgery and Musculoskeletal Medicine, Christchurch (New Zealand); Chernoglazov, Alex I. [University of Canterbury, Human Interface Technology Laboratory New Zealand, Christchurch (New Zealand); Butler, Anthony P.H. [University of Otago - Christchurch, Department of Radiology, Christchurch (New Zealand); European Organisation for Nuclear Research (CERN), Geneva (Switzerland); MARS Bioimaging, Christchurch (New Zealand)

    2017-01-15

    To quantify iodine uptake in articular cartilage as a marker of glycosaminoglycan (GAG) content using multi-energy spectral CT. We incubated a 25-mm strip of excised osteoarthritic human tibial plateau in 50 % ionic iodine contrast and imaged it using a small-animal spectral scanner with a cadmium telluride photon-processing detector to quantify the iodine through the thickness of the articular cartilage. We imaged both spectroscopic phantoms and osteoarthritic tibial plateau samples. The iodine distribution as an inverse marker of GAG content was presented in the form of 2D and 3D images after applying a basis material decomposition technique to separate iodine in cartilage from bone. We compared this result with a histological section stained for GAG. The iodine in cartilage could be distinguished from subchondral bone and quantified using multi-energy CT. The articular cartilage showed variation in iodine concentration throughout its thickness which appeared to be inversely related to GAG distribution observed in histological sections. Multi-energy CT can quantify ionic iodine contrast (as a marker of GAG content) within articular cartilage and distinguish it from bone by exploiting the energy-specific attenuation profiles of the associated materials. (orig.)

  2. Changes in articular cartilage in experimentally induced patellar subluxation

    Science.gov (United States)

    Ryu, J.; Saito, S.; Yamamoto, K.

    1997-01-01

    OBJECTIVES—Patellar subluxation was experimentally induced in young rabbits and the resulting cartilaginous changes were observed over a prolonged period of time to determine histological changes in the subluxated patellar cartilage.
METHODS—The tibial tuberosity in 12 week old rabbits was laterally displaced and fixed to the tibia with wire to induce lateral patellar subluxation. Pathological changes in patellar cartilage were examined for 120 weeks after surgery using computed tomography and stereoscopic microscopy.
RESULTS—Eight weeks after surgery, changes in articular cartilage consisting of horizontal splitting of the matrix were observed in the intermediate zone and were presumed to have been caused by shearing stress applied to the patellar cartilage. The cartilaginous changes caused by patellar subluxation progressed very little over the 120 weeks. Very few rabbits presented with osteoarthritic changes in the patellofemoral joint, most probably because the stress resulting from the malalignment of the patellofemoral joint was mild enough to permit recovery.
CONCLUSION—The mild, non-progressive pathological changes, in particular, basal degeneration, induced in this experiment in patellar cartilage were quite similar to the changes in articular cartilage seen in human chondromalacia patellae.

 PMID:9462171

  3. Serial explant culture provides novel insights into the potential location and phenotype of corneal endothelial progenitor cells.

    Science.gov (United States)

    Walshe, Jennifer; Harkin, Damien G

    2014-10-01

    The routine cultivation of human corneal endothelial cells, with the view to treating patients with endothelial dysfunction, remains a challenging task. While progress in this field has been buoyed by the proposed existence of progenitor cells for the corneal endothelium at the corneal limbus, strategies for exploiting this concept remain unclear. In the course of evaluating methods for growing corneal endothelial cells, we have noted a case where remarkable growth was achieved using a serial explant culture technique. Over the course of 7 months, a single explant of corneal endothelium, acquired from cadaveric human tissue, was sequentially seeded into 7 culture plates and on each occasion produced a confluent cell monolayer. Sample cultures were confirmed as endothelial in origin by positive staining for glypican-4. On each occasion, small cells, closest to the tissue explant, developed into a highly compact layer with an almost homogenous structure. This layer was resistant to removal with trypsin and produced continuous cell outgrowth during multiple culture periods. The small cells gave rise to larger cells with phase-bright cell boundaries and prominent immunostaining for both nestin and telomerase. Nestin and telomerase were also strongly expressed in small cells immediately adjacent to the wound site, following transfer of the explant to another culture plate. These findings are consistent with the theory that progenitor cells for the corneal endothelium reside within the limbus and provide new insights into expected expression patterns for nestin and telomerase within the differentiation pathway.

  4. Citrus tissue culture employing vegetative explants.

    Science.gov (United States)

    Chaturvedi, H C; Singh, S K; Sharma, A K; Agnihotri, S

    2001-11-01

    Citrus being a number one fruit of the world due to its high nutritional value, huge production of fruits and fruit products, the citrus industry may be considered a major fruit industry. Though citrus orchard area in India is comparable to USA, the produce is far less, while its export is nil. Biotechnology has played an outstanding role in boosting the citrus industry, e.g., in Spain, which is now the biggest exporter of citrus fruit with the application of micrografting. Amongst the fruit trees, perhaps the maximum tissue culture research has been done in citrus during the past four decades, however, the results of practical value are meagre. The shortfalls in citrus tissue culture research and some advancements made in this direction along with bright prospects are highlighted, restricting the review to vegetative explants only. Whilst utilization of nucellar embryogenesis is limited to rootstocks, the other aspects, like, regeneration and proliferation of shoot meristems measuring 200 microm in length--a global breakthrough--of two commercially important scion species, Citrus aurantifolia and C. sinensis and an important rootstock, C. limonia, improvement of micrografting technique, cloning of the same two scion species as well as some Indian rootstock species, employing nodal stem segments of mature trees, of immense practical value have been elaborated. A rare phenomenon of shift in the morphogenetic pattern of differentiation from shoot bud differentiation to embryoid formation occurred during the long-term culture of stem callus of C. grandis. Stem callus-regenerated plants of C. aurantifolia, C. sinensis and C. grandis showed variation in their ploidy levels and a somaclonal variant of C. sinensis, which produced seedless fruits was isolated. Tailoring of rooting in microshoots to a tap root-like system by changing the inorganic salt composition of the rooting medium, resulting in 100% transplant success, and germplasm preservation through normal growth

  5. Co-Expression and Co-Localization of Cartilage Glycoproteins CHI3L1 and Lubricin in Osteoarthritic Cartilage: Morphological, Immunohistochemical and Gene Expression Profiles.

    Science.gov (United States)

    Szychlinska, Marta Anna; Trovato, Francesca Maria; Di Rosa, Michelino; Malaguarnera, Lucia; Puzzo, Lidia; Leonardi, Rosy; Castrogiovanni, Paola; Musumeci, Giuseppe

    2016-01-01

    Osteoarthritis is the most common human arthritis characterized by degeneration of articular cartilage. Several studies reported that levels of human cartilage glycoprotein chitinase 3-like-1 (CHI3L1) are known as a potential marker for the activation of chondrocytes and the progression of Osteoarthritis (OA), whereas lubricin appears to be chondroprotective. The aim of this study was to investigate the co-expression and co-localization of CHI3L1 and lubricin in normal and osteoarthritic rat articular cartilage to correlate their modified expression to a specific grade of OA. Samples of normal and osteoarthritic rat articular cartilage were analyzed by the Kellgren-Lawrence OA severity scores, the Kraus' modified Mankin score and the Histopathology Osteoarthritis Research Society International (OARSI) system for histomorphometric evaluations, and through CHI3L1 and lubricin gene expression, immunohistochemistry and double immuno-staining analysis. The immunoexpression and the mRNA levels of lubricin increased in normal cartilage and decreased in OA cartilage (normal vs. OA, p < 0.01). By contrast, the immunoexpression and the mRNA levels of CHI3L1 increased in OA cartilage and decreased in normal cartilage (normal vs. OA, p < 0.01). Our findings are consistent with reports suggesting that these two glycoproteins are functionally associated with the development of OA and in particular with grade 2/3 of OA, suggesting that in the future they could be helpful to stage the severity and progression of the disease.

  6. Co-Expression and Co-Localization of Cartilage Glycoproteins CHI3L1 and Lubricin in Osteoarthritic Cartilage: Morphological, Immunohistochemical and Gene Expression Profiles

    Directory of Open Access Journals (Sweden)

    Marta Anna Szychlinska

    2016-03-01

    Full Text Available Osteoarthritis is the most common human arthritis characterized by degeneration of articular cartilage. Several studies reported that levels of human cartilage glycoprotein chitinase 3-like-1 (CHI3L1 are known as a potential marker for the activation of chondrocytes and the progression of Osteoarthritis (OA, whereas lubricin appears to be chondroprotective. The aim of this study was to investigate the co-expression and co-localization of CHI3L1 and lubricin in normal and osteoarthritic rat articular cartilage to correlate their modified expression to a specific grade of OA. Samples of normal and osteoarthritic rat articular cartilage were analyzed by the Kellgren–Lawrence OA severity scores, the Kraus’ modified Mankin score and the Histopathology Osteoarthritis Research Society International (OARSI system for histomorphometric evaluations, and through CHI3L1 and lubricin gene expression, immunohistochemistry and double immuno-staining analysis. The immunoexpression and the mRNA levels of lubricin increased in normal cartilage and decreased in OA cartilage (normal vs. OA, p < 0.01. By contrast, the immunoexpression and the mRNA levels of CHI3L1 increased in OA cartilage and decreased in normal cartilage (normal vs. OA, p < 0.01. Our findings are consistent with reports suggesting that these two glycoproteins are functionally associated with the development of OA and in particular with grade 2/3 of OA, suggesting that in the future they could be helpful to stage the severity and progression of the disease.

  7. The effect of adipose-derived stem cells on the survival of diced cartilage graft in rabbits

    Directory of Open Access Journals (Sweden)

    Mohammad reza Ebadi

    2016-10-01

    Full Text Available Backgrounds and Aims: The use of diced cartilage grafts in rhinoplasty in recent years, have been considered by most plastic surgeons. However, long-term resorption usually occurs. The aim of this study was to Examine the effects of adipose-derived stem cells on the viability of diced cartilage grafts. Materials and Methods: In this study, 10 New Zealand White male rabbits, weighing 2000-2500 g, approximately 12 to 16 weeks of age were used.Stem cells was harvested from inguinal adipose tissue of each rabbits. Grafts placed subcutaneously along the dorsal midline. Stem cells were injected in one side and the other side was control. The cartilage weights were recorded both before implantation and after explantation. Evaluation of living chondrocytes was conducted 12 weeks after implantation. Results: The mean difference of cartilage weights was varied between two groups (intervention and control sides; So that the average was significantly higher in stem cell side than that in the control side (p=0.021. The average number of live chondrocytes was significantly higher in the intervention side than the control side (p<0.001. Conclusions: These findings suggest that adipose-derived stem cells can maintain the viability of diced cartilage, although the exact mechanism remains to be defined. Because adipose-derived stem cells are autologous and easy to harvest, they may be useful for improving the long-term outcomes of diced cartilage grafting.

  8. Articular cartilage generation applying PEG-LA-DM/PEGDM copolymer hydrogels

    NARCIS (Netherlands)

    Zhao, Xing; Papadopoulos, Anestis; Ibusuki, Shinichi; Bichara, David A; Saris, Daniel B; Malda, Jos|info:eu-repo/dai/nl/412461099; Anseth, Kristi S; Gill, Thomas J; Randolph, Mark A

    2016-01-01

    BACKGROUND: Injuries to the human native cartilage tissue are particularly problematic because cartilage has little to no ability to heal or regenerate itself. Employing a tissue engineering strategy that combines suitable cell sources and biomimetic hydrogels could be a promising alternative to

  9. Articular cartilage generation applying PEG-LA-DM/PEGDM copolymer hydrogels

    NARCIS (Netherlands)

    Zhao, Xing; Papadopoulos, Anestis; Ibusuki, Shinichi; Bichara, David A.; Saris, Daniel B.; Malda, J; Anseth, Kristi S.; Gill, Thomas J.; Randolph, Mark A.

    2016-01-01

    Background: Injuries to the human native cartilage tissue are particularly problematic because cartilage has little to no ability to heal or regenerate itself. Employing a tissue engineering strategy that combines suitable cell sources and biomimetic hydrogels could be a promising alternative to

  10. Relative contribution of matrix metalloprotease and cysteine protease activities to cytokine-stimulated articular cartilage degradation

    DEFF Research Database (Denmark)

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

    2006-01-01

    explants were stimulated with oncostatin M (OSM) 10 ng/ml and tumor necrosis factor-alpha (TNF-alpha) 20 ng/ml in the presence or absence of the broad-spectrum MMP inhibitor GM6001 and the cysteine protease inhibitor, E64. Cartilage degradation was evaluated in the conditioned medium by glycosaminoglycans...... in vivo in CK null mice. CONCLUSION: Inhibition of MMP activity reduced both proteoglycan loss and type II collagen degradation. In contrast, inhibition of cysteine proteases resulted in an increase rather than a decrease in MMP derived fragments of collagen type II degradation, CTX-II, suggesting altered...

  11. Biotribology :articular cartilage friction, wear, and lubrication

    OpenAIRE

    Schroeder, Matthew O

    1995-01-01

    This study developed, explored, and refined techniques for the in vitro study of cartilage-on-cartilage friction, deformation, and wear. Preliminary results of in vitro cartilage-on- cartilage experiments with emphasis on wear and biochemistry are presented. Cartilage-bone specimens were obtained from the stifle joints of steers from a separate controlled study. The load, sliding speed, and traverse of the lower specimens were held constant as lubricant and test length were varied. Lubric...

  12. Correlated response of in vitro regeneration capacity from different source of explants inCucumis melo.

    Science.gov (United States)

    Molina, R V; Nuez, F

    1995-01-01

    The variation among and within different populations of the regeneration ability from leaf, cotyledon and hypocotyl explants has been studied. A control population and two lines selected by their regeneration capacity from leaf explants were used. Significant differences among the plants of the control population,for the organogenic response, were detected. The regeneration capacity varies depending on the type of explant. Selection in order to improve the regeneration frequency from leaf explants also raises the organogenic response in the other explant types. This result suggests the presence of a partial common genetic system controlling the regeneration frequency of the diverse types of explants.

  13. Hypoxia Inhibits Hypertrophic Differentiation and Endochondral Ossification in Explanted Tibiae

    NARCIS (Netherlands)

    Leijten, Jeroen Christianus Hermanus; Moreira Teixeira, Liliana; Landman, Ellie; van Blitterswijk, Clemens; Karperien, Hermanus Bernardus Johannes

    2012-01-01

    Purpose: Hypertrophic differentiation of growth plate chondrocytes induces angiogenesis which alleviates hypoxia normally present in cartilage. In the current study, we aim to determine whether alleviation of hypoxia is merely a downstream effect of hypertrophic differentiation as previously

  14. 3D Printing of Cytocompatible Water-Based Light-Cured Polyurethane with Hyaluronic Acid for Cartilage Tissue Engineering Applications

    Science.gov (United States)

    Shie, Ming-You; Chang, Wen-Ching; Wei, Li-Ju; Huang, Yu-Hsin; Chen, Chien-Han; Shih, Cheng-Ting; Chen, Yi-Wen; Shen, Yu-Fang

    2017-01-01

    Diseases in articular cartilages have affected millions of people globally. Although the biochemical and cellular composition of articular cartilages is relatively simple, there is a limitation in the self-repair ability of the cartilage. Therefore, developing strategies for cartilage repair is very important. Here, we report on a new liquid resin preparation process of water-based polyurethane based photosensitive materials with hyaluronic acid with application of the materials for 3D printed customized cartilage scaffolds. The scaffold has high cytocompatibility and is one that closely mimics the mechanical properties of articular cartilages. It is suitable for culturing human Wharton’s jelly mesenchymal stem cells (hWJMSCs) and the cells in this case showed an excellent chondrogenic differentiation capacity. We consider that the 3D printing hybrid scaffolds may have potential in customized tissue engineering and also facilitate the development of cartilage tissue engineering. PMID:28772498

  15. In Vitro Infection of Trypanosoma cruzi Causes Decrease in Glucose Transporter Protein-1 (GLUT1 Expression in Explants of Human Placental Villi Cultured under Normal and High Glucose Concentrations

    Directory of Open Access Journals (Sweden)

    Luciana Mezzano

    2012-01-01

    Full Text Available Trypanosoma cruzi, the etiologic Chagas' disease agent, induces changes in protein pattern of the human placenta syncytiotrophoblast. The glucose transporter protein-1 (GLUT1 is the primary isoform involved in transplacental glucose transport. We carried out in vitro assays to determine if T. cruzi infection would induce changes in placental GLUT1 protein expression under normal and high concentration of glucose. Using Western blot and immunohistological techniques, GLUT1 expression was determined in normal placental villi cultured under normal or high concentrations of glucose, with or without in vitro T. cruzi infection, for 24 and 48 hours. High glucose media or T. cruzi infection alone reduced GLUT1 expression. A yet more accentuated reduction was observed when infection and high glucose condition took place together. We inform, for the first time, that T. cruzi infection may induce reduction of GLUT1 expression under normal and high glucose concentrations, and this effect is synergic to high glucose concentrations.

  16. IN VITRO REGENERATION CASTOR (RICINUS COMMUNIS L.) USING COTYLEDON EXPLANTS

    Science.gov (United States)

    A novel plant regeneration protocol was established for castor (Ricinus communis L.), an important oilseed crop. Mature seed-derived cotyledon explants produced adventitious shoots when placed on Murashige and Skoog (MS) medium containing thidiazuron (TDZ). The rate of shoot regeneration was maximal...

  17. Simple, effective and economical explant-surface sterilization ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-19

    Oct 19, 2009 ... ISSN 1684–5315 © 2009 Academic Journals. Full Length Research ... highest reduction in bacterial and fungal contamination (0%) at time intervals between 20 - 45 min. The ... Key words: Explant, surface sterilization, cowpea, rice, sorghum, JIK® ... majority of commercial and scientific plant tissue culture.

  18. STRUCTURAL ANALYSIS OF ARTICULAR CARTILAGE OF THE HIP JOINT USING FINITE ELEMENT METHOD

    Directory of Open Access Journals (Sweden)

    Robert Karpiński

    2016-09-01

    Full Text Available The paper presents the results of a preliminary study on the structural analysis of the hip joint, taking into account changes in the mechanical properties of the articular cartilage of the joint. Studies have been made due to the need to determine the tension distribution occurring in the cartilage of the human hip. These distribution are the starting point for designing custom made human hip prosthesis. Basic anatomy, biomechanical analysis of the hip joint and articular cartilage are introduced. The mechanical analysis of the hip joint model is conducted. Final results of analysis are presented. Main conclusions of the study are: the capability of absorbing loads by articular cartilage of the hip joint is preliminary determined as decreasing with increasing degenerations of the cartilage and with age of a patient. Without further information on changes of cartilage’s mechanical parameters in time it is hard to determine the nature of relation between mentioned capability and these parameters.

  19. The Removal of Hydrogel Explants: An Analysis of 467 Consecutive Cases

    NARCIS (Netherlands)

    Crama, N.; Klevering, B.J.

    2016-01-01

    PURPOSE: To describe the complications associated with hydrogel explants and to describe the indications, surgical technique, and risks involved in the removal of a hydrogel explant. DESIGN: Single-center, retrospective interventional case series. PARTICIPANTS: Patients who underwent surgical remova

  20. A dual flow bioreactor with controlled mechanical stimulation for cartilage tissue engineering.

    Science.gov (United States)

    Spitters, Tim W G M; Leijten, Jeroen C H; Deus, Filipe D; Costa, Ines B F; van Apeldoorn, Aart A; van Blitterswijk, Clemens A; Karperien, Marcel

    2013-10-01

    In cartilage, tissue engineering bioreactors can create a controlled environment to study chondrocyte behavior under mechanical stimulation or produce chondrogenic grafts of clinically relevant size. Here we present a novel bioreactor that combines mechanical stimulation with a two compartment system through which nutrients can be supplied solely by diffusion from opposite sides of a tissue-engineered construct. This design is based on the hypothesis that creating gradients of nutrients, growth factors, and growth factor antagonists can aid in the generation of zonal tissue-engineered cartilage. Computational modeling predicted that the design facilitates the creation of a biologically relevant glucose gradient. This was confirmed by quantitative glucose measurements in cartilage explants. In this system, it is not only possible to create gradients of nutrients, but also of anabolic or catabolic factors. Therefore, the bioreactor design allows control over nutrient supply and mechanical stimulation useful for in vitro generation of cartilage constructs that can be used for the resurfacing of articulated joints or as a model for studying osteoarthritis disease progression.

  1. Leptin produced by joint white adipose tissue induces cartilage degradation via upregulation and activation of matrix metalloproteinases.

    Science.gov (United States)

    Hui, Wang; Litherland, Gary J; Elias, Martina S; Kitson, Gareth I; Cawston, Tim E; Rowan, Andrew D; Young, David A

    2012-03-01

    To investigate the effect of leptin on cartilage destruction. Collagen release was assessed in bovine cartilage explant cultures, while collagenolytic and gelatinolytic activities in culture supernatants were determined by bioassay and gelatin zymography. The expression of matrix metalloproteinases (MMP) was analysed by real-time RT-PCR. Signalling pathway activation was studied by immunoblotting. Leptin levels in cultured osteoarthritic joint infrapatellar fat pad or peri-enthesal deposit supernatants were measured by immunoassay. Leptin, either alone or in synergy with IL-1, significantly induced collagen release from bovine cartilage by upregulating collagenolytic and gelatinolytic activity. In chondrocytes, leptin induced MMP1 and MMP13 expression with a concomitant activation of STAT1, STAT3, STAT5, MAPK (JNK, Erk, p38), Akt and NF-κB signalling pathways. Selective inhibitor blockade of PI3K, p38, Erk and Akt pathways significantly reduced MMP1 and MMP13 expression in chondrocytes, and reduced cartilage collagen release induced by leptin or leptin plus IL-1. JNK inhibition had no effect on leptin-induced MMP13 expression or leptin plus IL-1-induced cartilage collagen release. Conditioned media from cultured white adipose tissue (WAT) from osteoarthritis knee joint fat pads contained leptin, induced cartilage collagen release and increased MMP1 and MMP13 expression in chondrocytes; the latter being partly blocked with an anti-leptin antibody. Leptin acts as a pro-inflammatory adipokine with a catabolic role on cartilage metabolism via the upregulation of proteolytic enzymes and acts synergistically with other pro-inflammatory stimuli. This suggests that the infrapatellar fat pad and other WAT in arthritic joints are local producers of leptin, which may contribute to the inflammatory and degenerative processes in cartilage catabolism, providing a mechanistic link between obesity and osteoarthritis.

  2. Safranin O reduces loss of glycosaminoglycans from bovine articular cartilage during histological specimen preparation.

    Science.gov (United States)

    Király, K; Lammi, M; Arokoski, J; Lapveteläinen, T; Tammi, M; Helminen, H; Kiviranta, I

    1996-02-01

    The ability of Safranin O, added to fixation and decalcification solutions, to prevent the escape of glycosaminoglycans (GAGs) from small cartilage tissue blocks during histological processing of cartilage has been studied. GAGs in the fixatives and decalcifying solutions used and those remaining in the 1 mm3 cubes of cartilage were assayed biochemically. The quantity of GAGs remaining in the cartilage cubes were determined from Safranin O-stained sections using videomicroscopy or microspectrophotometry. A quantity (10.6%) of GAGs were lost during a conventional 4% buffered formaldehyde fixation (48 h) and a subsequent decalcification in 10% EDTA (12 days) at 4 degrees C. Roughly one-quarter of the total GAG loss occurred during the 48 h fixation, and three-quarters during the 12 days of decalcification. Inclusion of 4% formaldehyde in the decalcification fluid decreased the loss of GAGs to 6.2%. The presence of 0.5% Safranin O in the fixative reduced this loss to 3.4%. When 0.5% Safranin O was included in the fixative and 4% formaldehyde in the decalcification solution, Safranin O staining of the histological sections increased on average by 13.5%. After fixation in the presence of 0.5% Safranin O, there was no difference in the staining intensities when decalcification was carried out in the presence of either Safranin O or formaldehyde, or both. It took 24 h for Safranin O to penetrate into the deep zone of articular cartilage, warranting a fixation period of at least this long. In conclusion, the addition of Safranin O to the fixative and either Safranin O or formaldehyde in the following decalcification fluid, markedly reduces the loss of GAGs from small articular cartilage explants during histological processing. However, for immunohistochemical studies, Safranin O cannot be included in the processing solutions, because it may interfere.

  3. Interaction of cochlear nucleus explants with semiconductor materials.

    Science.gov (United States)

    Mlynski, Robert; Volkenstein, Stefan; Hansen, Stefan; Brors, Dominik; Ebmeyer, Joerg; Dazert, Stefan

    2007-07-01

    Implantable hearing devices such as cochlear implants and auditory brainstem implants deliver auditory information through electrical stimulation of auditory neurons. The combination of microelectronic electrodes with auditory nerve cells may lead to further improvement of the hearing quality with these devices. Whereas several kinds of neurons are known to grow on semiconductor substrates, interactions of cochlear nucleus (CN) neurons with such materials have yet to be described. To investigate survival and growth behavior of CN neurons on different semiconductor materials. CN explants from postnatal day 10 Sprague-Dawley rats were cultured for 96 hours in Neurobasal medium on polished and unpolished silicon wafers (p-type Si [100] and p-type Si3N4[100]) as well as plastic surface. These surfaces had been coated with poly-L-lysine and laminin. Neuronal outgrowth was examined using image analysis software after immunohistologic staining for neurofilament. Neurite length and directional changes were quantified. Additionally, neurite morphology and adhesion to the semiconductor material was evaluated by scanning electron microscopy. Although proper adhesion of CN explants was seen, no neurite growth could be detected on unpolished silicon wafers (Si and Si3N4). Compared with the other test conditions, polished, laminin-coated Si3N4 wafers showed best biocompatibility regarding neurite length and number per explant. CN explants developed a mean of eight neurons with an average length of 236 mum in 96 hours of culture on these wafers. The results of this study demonstrate the general possibility of CN neuron growth in culture on semiconductors in vitro. The differences in neuron length and number per explant indicate that the growth of CN neurons is influenced by the semiconductor substrate as well as extracellular matrix proteins, with laminin-coated p-type Si3N4[100] being a preferable material for future hybrid experiments on auditory-neuron semiconductor chips.

  4. Absorção de macronutrientes por explantes de bananeira in vitro Macronutrient absorption by banana explants in vitro

    Directory of Open Access Journals (Sweden)

    Josefa Diva Nogueira Diniz

    1999-07-01

    Full Text Available Com o objetivo de estudar a absorção de macronutrientes (N, P, K, Ca, Mg e S em explantes de bananeira cv. Prata Anã, foram utilizados explantes de plantas estabelecidas in vitro, inoculados em meio básico de Murashige & Skoog (1962 contendo sacarose (30 g/L, e BAP (3,5 mg/L com sete tratamentos, representados pelos períodos de 0, 10, 20, 30, 40, 50 e 60 dias de cultivo e três repetições. As quantidades de macronutrientes totais absorvidas pelos explantes seguiram a ordem: K > N > Ca > ou = P > Mg @ S. O P foi o nutriente absorvido mais rapidamente pelos explantes, com 75% extraído do meio de cultivo nos primeiros 30 dias, cessando sua absorção aos 50 dias, restando ainda 9% no meio de cultivo. A absorção do S cessou também aos 50 dias, quando 66% deste nutriente ainda permanecia no meio de cultivo. Este resultado sugere haver uma relação, quanto à absorção, entre esses dois nutrientes. As maiores taxas de absorção de todos os nutrientes foram verificadas nos primeiros 20 dias. O rizoma, o pseudocaule e as folhas, se diferenciaram quanto à concentração e extração ou acúmulo de nutrientes.The absorption of the nutrients (N, P, K, Ca, Mg and S by banana (Musa sp. cv. Prata Anã explants on the basic medium of Murashige & Skoog (1962 supplemented with sucrose (30 g/L and BAP (3.5 mg/L were evaluated at 0, 10, 20, 30, 40, 50 and 60 days after inoculation. The seven treatments were arranged on a completely randomized design with three replicates. The sequence of nutrient absorption by the explants was K > N > Ca > or = P > Mg @ S. The P was the nutrient with the fastest absorption rate and at the 30th day the explants had already absorbed 75% of the P from the medium. The P absorption stopped by the 50th day. The S absorption stopped at the 50th day with 66% of it remaining in the medium. The results suggested a close relationship between these two nutrients. The highest rates of nutrient absorption were observed during the

  5. Morphologic differences observed by scanning electron microscopy according to the reason for pseudophakic IOL explantation

    DEFF Research Database (Denmark)

    Fernandez-Buenaga, Roberto; Alio, Jorge L.; Ramirez, Jose M.

    2015-01-01

    Purpose To compare variations in surface morphology, as studied by scanning electron microscopy (SEM), of explanted intraocular lenses (IOLs) concerning the cause leading to the explantation surgery. Methods In this prospective multicenter study, explanted IOLs were analyzed by SEM and energy-dis...

  6. Microvesicle-mediated release of soluble LH/hCG receptor (LHCGR from transfected cells and placenta explants

    Directory of Open Access Journals (Sweden)

    Randeva Harpal

    2011-05-01

    Full Text Available Abstract Placental hCG and pitutary LH transduce signals in target tissues through a common receptor (LHCGR. We demonstrate that recombinant LHCGR proteins which include the hormone-binding domain are secreted from transfected cells and that natural LHCGR is also secreted from human placental explants. LHCGR recombinant proteins representing varying lengths of the N-terminal extracellular domain were expressed in Chinese Hamster Ovary cells in suspension culture. Secretion was minimal up to 72h but by 96h 24-37% of the LHCGR had been released into the culture medium. The secreted proteins were folded and sensitive to glycosidases suggesting N-linked glycosylation. Secretion was independent of recombinant size and was mediated via structurally defined membrane vesicles (50-150nm. Similarly cultured human early pregnancy placental explants also released LHCGR via microvesicles. These studies provide the first experimental evidence of the possible mechanistic basis of the secretion of LHCGR.

  7. Endoscopic laser reshaping of rabbit tracheal cartilage: preliminary investigations

    Science.gov (United States)

    Tsang, Walter; Lam, Anthony; Protsenko, Dmitry; Wong, Brian J.

    2005-04-01

    Background: Tracheal cartilage deformities due to trauma, prolonged endotracheal intubation or infection are difficult to correct. Current treatment options such as dilation, laser ablation, stent placement, and segmental resection are only temporary or carry significant risks. The objectives of this project were to design and test a laser activated endotracheal stent system that can actively modify the geometry of tracheal cartilage, leading to permanent retention of a new and desirable tracheal geometry. Methods: Ex vivo rabbit tracheal cartilage (simulating human neonate trachea) were irradiated with an Er: Glass laser, (λ= 1.54um, 0.5W-2.5W, 1 sec to 5 sec). Shape change and gross thermal injury were assessed visually to determine the best laser power parameters for reshaping. A rigid endoscopic telescope and hollow bronchoscope were used to record endoscopic images. The stent was constructed from nitinol wire, shaped into a zigzag configuration. An ex vivo testing apparatus was also constructed. Results: The best laser power parameter to produce shape change was 1 W for 6-7 seconds. At this setting, there was significant shape change with only minimal thermal injury to the tracheal mucosa, as assessed by visual inspection. The bronchoscopy system functioned adequately during testing in the ex vivo testing apparatus. Conclusion: We have successfully designed instrumentation and created the capability to endoscopically reshape tracheal cartilage in an ex vivo rabbit model. The results obtained in ex vivo tracheal cartilage indicated that reshaping using Er: Glass laser can be accomplished.

  8. Does Radio Frequency Ablation (RFA) Epiphysiodesis Affect Joint Cartilage?

    DEFF Research Database (Denmark)

    Shiguetomi Medina, Juan Manuel; Abood, Ahmed Abdul-Hussein; Rahbek, Ole;

    Background: Epiphysiodesis made with RFA has resulted, in animal models, an effective procedure that disrupts the growth plate and induces LLD. This procedure involves an increase of temperature (>92°C) of the targeted region causing thermal damage. To our knowledge, no study that investigates...... the effect of this procedure in the adjacent joint articular cartilage has been reported Purpose / Aim of Study: Proof of concept that epiphysiodesis made with RFA is a safe procedure that disrupts the growth plate without damaging the adjacent joint articular cartilage Materials and Methods: RFA...... articular joint cartilage. This study resembles possible results of RFA epiphysiodesis on humans. Previous studies suggest that an 8 min ablation is enough to disrupt the growth plate. This study shows that RFA can be done safely in the growing physis even on triple-long procedures. It is important...

  9. Contrast Agent-Enhanced Computed Tomography of Articular Cartilage: Association with Tissue Composition and Properties

    Energy Technology Data Exchange (ETDEWEB)

    Silvast, T.S.; Jurvelin, J.S.; Aula, A.S.; Lammi, M.J.; Toeyraes, J. (Dept. of Clinical Neurophysiology, Kuopio Univ. Hospital, Kuopio (Finland))

    2009-01-15

    Background: Contrast agent-enhanced computed tomography may enable the noninvasive quantification of glycosaminoglycan (GAG) content of articular cartilage. It has been reported that penetration of the negatively charged contrast agent ioxaglate (Hexabrix) increases significantly after enzymatic degradation of GAGs. However, it is not known whether spontaneous degradation of articular cartilage can be quantitatively detected with this technique. Purpose: To investigate the diagnostic potential of contrast agent-enhanced cartilage tomography (CECT) in quantification of GAG concentration in normal and spontaneously degenerated articular cartilage by means of clinical peripheral quantitative computed tomography (pQCT). Material and Methods: In this in vitro study, normal and spontaneously degenerated adult bovine cartilage (n=32) was used. Bovine patellar cartilage samples were immersed in 21 mM contrast agent (Hexabrix) solution for 24 hours at room temperature. After immersion, the samples were scanned with a clinical pQCT instrument. From pQCT images, the contrast agent concentration in superficial as well as in full-thickness cartilage was calculated. Histological and functional integrity of the samples was quantified with histochemical and mechanical reference measurements extracted from our earlier study. Results: Full diffusion of contrast agent into the deep cartilage was found to take over 8 hours. As compared to normal cartilage, a significant increase (11%, P<0.05) in contrast agent concentration was seen in the superficial layer of spontaneously degenerated samples. Significant negative correlations were revealed between the contrast agent concentration and the superficial or full-thickness GAG content of tissue (|R|>0.5, P<0.01). Further, pQCT could be used to measure the thickness of patellar cartilage. Conclusion: The present results suggest that CECT can be used to diagnose proteoglycan depletion in spontaneously degenerated articular cartilage with a

  10. Transcriptomic signatures in cartilage ageing

    Science.gov (United States)

    2013-01-01

    Introduction Age is an important factor in the development of osteoarthritis. Microarray studies provide insight into cartilage aging but do not reveal the full transcriptomic phenotype of chondrocytes such as small noncoding RNAs, pseudogenes, and microRNAs. RNA-Seq is a powerful technique for the interrogation of large numbers of transcripts including nonprotein coding RNAs. The aim of the study was to characterise molecular mechanisms associated with age-related changes in gene signatures. Methods RNA for gene expression analysis using RNA-Seq and real-time PCR analysis was isolated from macroscopically normal cartilage of the metacarpophalangeal joints of eight horses; four young donors (4 years old) and four old donors (>15 years old). RNA sequence libraries were prepared following ribosomal RNA depletion and sequencing was undertaken using the Illumina HiSeq 2000 platform. Differentially expressed genes were defined using Benjamini-Hochberg false discovery rate correction with a generalised linear model likelihood ratio test (P ageing cartilage. Conclusion There was an age-related dysregulation of matrix, anabolic and catabolic cartilage factors. This study has increased our knowledge of transcriptional networks in cartilage ageing by providing a global view of the transcriptome. PMID:23971731

  11. Histological and molecular evaluation of patient-derived colorectal cancer explants.

    Directory of Open Access Journals (Sweden)

    Joshua M Uronis

    Full Text Available Mouse models have been developed to investigate colorectal cancer etiology and evaluate new anti-cancer therapies. While genetically engineered and carcinogen-induced mouse models have provided important information with regard to the mechanisms underlying the oncogenic process, tumor xenograft models remain the standard for the evaluation of new chemotherapy and targeted drug treatments for clinical use. However, it remains unclear to what extent explanted colorectal tumor tissues retain inherent pathological features over time. In this study, we have generated a panel of 27 patient-derived colorectal cancer explants (PDCCEs by direct transplantation of human colorectal cancer tissues into NOD-SCID mice. Using this panel, we performed a comparison of histology, gene expression and mutation status between PDCCEs and the original human tissues from which they were derived. Our findings demonstrate that PDCCEs maintain key histological features, basic gene expression patterns and KRAS/BRAF mutation status through multiple passages. Altogether, these findings suggest that PDCCEs maintain similarity to the patient tumor from which they are derived and may have the potential to serve as a reliable preclinical model that can be incorporated into future strategies to optimize individual therapy for patients with colorectal cancer.

  12. Glucosamine increases hyaluronic acid production in human osteoarthritic synovium explants

    NARCIS (Netherlands)

    E.J. Uitterlinden (Elian); C.F. Verkoelen; S.M. Bierma-Zeinstra (Sita); H. Jahr (Holger); H.H. Weinans (Harrie); J.A.N. Verhaar (Jan); G.J.V.M. van Osch (Gerjo); J.L.M. Koevoet (Wendy)

    2008-01-01

    textabstractBackground. Glucosamine (GlcN) used by patients with osteoarthritis was demonstrated to reduce pain, but the working mechanism is still not clear. Viscosupplementation with hyaluronic acid (HA) is also described to reduce pain in osteoarthritis. The synthesis of HA requires GlcN as one o

  13. Reactivation of HSV-1 following explant of tree shrew brain.

    Science.gov (United States)

    Li, Lihong; Li, Zhuoran; Li, Xin; Wang, Erlin; Lang, Fengchao; Xia, Yujie; Fraser, Nigel W; Gao, Feng; Zhou, Jumin

    2016-06-01

    Herpes Simplex Virus type I (HSV-1) latently infects peripheral nervous system (PNS) sensory neurons, and its reactivation leads to recurring cold sores. The reactivated HSV-1 can travel retrograde from the PNS into the central nervous system (CNS) and is known to be causative of Herpes Simplex viral encephalitis. HSV-1 infection in the PNS is well documented, but little is known on the fate of HSV-1 once it enters the CNS. In the murine model, HSV-1 genome persists in the CNS once infected through an ocular route. To gain more details of HSV-1 infection in the CNS, we characterized HSV-1 infection of the tree shrew (Tupaia belangeri chinensis) brain following ocular inoculation. Here, we report that HSV-1 enters the tree shrew brain following ocular inoculation and HSV-1 transcripts, ICP0, ICP4, and LAT can be detected at 5 days post-infection (p.i.), peaking at 10 days p.i. After 2 weeks, ICP4 and ICP0 transcripts are reduced to a basal level, but the LAT intron region continues to be expressed. Live virus could be recovered from the olfactory bulb and brain stem tissue. Viral proteins could be detected using anti-HSV-1 antibodies and anti-ICP4 antibody, during the acute stage but not beyond. In situ hybridization could detect LAT during acute infection in most brain regions and in olfactory bulb and brain stem tissue well beyond the acute stage. Using a homogenate from these tissues' post-acute infection, we did not recover live HSV-1 virus, supporting a latent infection, but using a modified explant cocultivation technique, we were able to recover reactivated virus from these tissues, suggesting that the HSV-1 virus latently infects the tree shrew CNS. Compared to mouse, the CNS acute infection of the tree shrew is delayed and the olfactory bulb contains most latent virus. During the acute stage, a portion of the infected tree shrews exhibit symptoms similar to human viral encephalitis. These findings, together with the fact that tree shrews are closely

  14. Quality of placental RNA: Effects of explant size and culture duration.

    Science.gov (United States)

    Brew, O; Nikolopoulou, E; Hughes, A; Christian, M; Lee, Y; Oduwole, O; Sullivan, M H F; Woodman, A

    2016-10-01

    We evaluated the impact of placental micro (≤50 mg) and macro (∼200 mg) explants, oxygen concentration and culture method on placental RNA quality after long-term culture. Our findings show that micro explants cultured at 8% oxygen have the best RNA quality and tissue structure. Macro explants were less viable after long-term culture. Macro explants and explants undergoing syncytial degeneration produced poor quality RNA and should be avoided. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Platelet-rich plasma plus human umbilical cord mesenchymal stem cells for cartilage repair%富血小板血浆及脐带间充质干细胞修复软骨损伤

    Institute of Scientific and Technical Information of China (English)

    徐静; 王黎明; 周立冬; 吴美; 崔辉; 赵璟; 曾独娟; 张仲文; 刘爱兵

    2014-01-01

    BACKGROUND:Chondrocytes co-cultured with bone marrow stromal stem cells on the scaffold of platelet-rich plasma are found to proliferate, and besides proliferative growth, bone marrow stromal cells exhibit a tendency of differentiating into chondrocytes. OBJECTIVE:To study the effect of platelet-rich plasma and human umbilical cord mesenchymal stem cells (hUCMSCs) on cartilage repair. METHODS:Forty healthy New Zealand white rabbits were selected to establish models of cartilage defects, and then randomly divided into normal saline group, platelet-rich plasma group, hUCMSCs group and combination group. Platelet-rich plasma was prepared by using double centrifugations to prepare passage 3 hUCMSCs. After modeling, intra-articular injection of normal saline (0.5 mL), 12.5%platelet-rich plasma (0.5 mL), 1×107 hUCMSCs (0.5 mL), 12.5%platelet-rich plasma+1×107 hUCMSCs (total y 0.5 mL) was done in corresponding groups, respectively. After 12 weeks of modeling, the injured cartilage was grossly observed, and hematoxylin-eosin staining was used to observe cartilage repair under light microscope;according to the O'Driscol histologic standard, histological examination was performed. RESULTS AND CONCLUSION:The repair effect in the normal saline group was significantly better that in the platelet-rich plasma group, hUCMSCs group, combination group (P<0.05), while the platelet-rich plasma group and combination group also exhibit better outcomes than the hUCMSCs group (P<0.05). These findings indicate that both platelet-rich plasma and hUCMSCs can promote cartilage repair;moreover, platelet-rich plasma with or without hUCMSCs is superior to hUCMSCs alone in the cartilage repair.%背景:研究者直接将富血小板血浆作为支架材料与骨髓基质干细胞、软骨细胞等复合后体外培养发现软骨细胞在富血小板血浆三维支架呈现增殖生长,骨髓基质干细胞在增殖的同时有向软骨细胞分化的倾向。目的:观察富血小板

  16. Shoot Regeneration from Leaf Explants of Withania somnifera (L. Dunal

    Directory of Open Access Journals (Sweden)

    Aruna Girish JOSHI

    2010-03-01

    Full Text Available Regeneration from leaf explants of Withania somnifera (L. for mass propagation was studied on Murashige and Skoog�s medium supplemented with Kinetin (Kn and 6-benzylaminopurine (BAP alone or in combination. Shoot buds were induced from the midrib on the abaxial side in presence of Kn and BAP (4 �M. These shoot buds developed into shoots on the same medium. Rooting of these shoots was achieved in 0.5 �M of IBA.

  17. Transcriptomic profiling of cartilage ageing

    Directory of Open Access Journals (Sweden)

    Mandy Jayne Peffers

    2014-12-01

    Full Text Available The musculoskeletal system is severely affected by the ageing process, with many tissues undergoing changes that lead to loss of function and frailty. Articular cartilage is susceptible to age related diseases, such as osteoarthritis. Applying RNA-Seq to young and old equine cartilage, we identified an over-representation of genes with reduced expression relating to extracellular matrix, degradative proteases, matrix synthetic enzymes, cytokines and growth factors in cartilage from older donors. Here we describe the contents and quality controls in detail for the gene expression and related results published by Peffers and colleagues in Arthritis Research and Therapy 2013 associated with the data uploaded to ArrayExpress (E-MTAB-1386.

  18. PHOTOCROSSLINKABLE HYDROGELS FOR CARTILAGE TISSUE ENGINEERING

    NARCIS (Netherlands)

    Levett, Peter Andrew

    2015-01-01

    For millions of people, damaged cartilage is a major source of pain and disability. As those people often discover upon seeking medical treatment, once damaged, cartilage is very difficult to repair. Finding better clinical therapies for damaged cartilage has generated a huge amount of research inte

  19. Foldable Lens Explantation and Exchange:The Reason and Solution

    Institute of Scientific and Technical Information of China (English)

    Danying Zheng; Zhenpin Zhang; Wenhui Yang; Weirong Chen

    2001-01-01

    Objective: To report the explantation and exchange of Hydrophilic Acrylic foldable intraocular lens (IOL) on 14 patients who had visual disturbances caused by the change of transparence on optic. Methods: Sixteen Hydrophilic Acrylic foldable intraocular lenses from 14 patients who presented with decreased visual acuity from 6 months to 1 year after normal phacoemulsification and IOL implantation associated with extensive transparent change on optic of the lens. The lenses were explanted with the bisection technique. All the eyes were reinserted with Acrysof foldable lenses. Results: Sixteen lenses were removed successfully and exchanged with the new lens in the capsule. The posterior capsular rupture and vitreous loss were found in the first two cases. One of them had the zonulysis due to the radial tear of the anterior capsule during the enlargement of the capsular opening. The anterior vitrectomy was performed with IOL fixed on the ciliary sulcus. The visual acuity of all the patients improved obviously without posterior complication. Conclusion: Foldable lens explantation with the bisection technique and exchange had a successful outcome with improvement of ocular condition. Eye science 2001; 17:54 ~56.

  20. Osthole Inhibits Proliferation and Induces Catabolism in Rat Chondrocytes and Cartilage Tissue

    Directory of Open Access Journals (Sweden)

    Guoqing Du

    2015-08-01

    Full Text Available Background/Aims: Cartilage destruction is thought to be the major mediator of osteoarthritis. Recent studies suggest that inhibition of subchrondral bone loss by anti-osteoporosis (OP drug can protect cartilige erosion. Osthole, as a promising agent for treating osteoporosis, may show potential in treating osteoarthritis. The purpose of this study was to investigate whether Osthole affects the proliferation and catabolism of rat chondrocytes, and the degeneration of cartilage explants. Methods: Rat chondrocytes were treated with Osthole (0 μM, 6.25 μM, 12.5 μM, and 25 μM with or without IL1-β (10ng/ml for 24 hours. The expression levels of type II collagen and MMP13 were detected by western Blot. Marker genes for chondrocytes (A-can and Sox9, matrix metalloproteinases (MMPs, aggrecanases (ADAMTS5 and genes implicated in extracellular matrix catabolism were evaluated by qPCR. Cell proliferation was assessed by measuring proliferating cell nuclear antigen (PCNA expression and fluorescence activated cell sorter. Wnt7b/β-catenin signaling was also investigated. Cartilage explants from two-week old SD rats were cultured with IL-1β, Osthole and Osthole plus IL-1β for four days and glycosaminoglycan (GAG synthesis was assessed with toluidine blue staining and Safranine O/Fast Green FCF staining, collagen type II expression was detected by immunofuorescence. Results: Osthole reduced expression of chondrocyte markers and increased expression of MMP13, ADAMTS5 and MMP9 in a dose-dependent manner. Catabolic gene expression levels were further improved by Osthole plus IL-1β. Osthole inhibited chondrocyte proliferation. GAG synthesis and type II collagen were decreased in both the IL-1β groups and the Osthole groups, and significantly reduced by Osthole plus IL-1β. Conclusions: Our data suggested that Osthole increases the catabolism of rat chondrocytes and cartilage explants, this effect might be mediated through inhibiting Wnt7b

  1. 35SULPHATE INCORPORATION ASSAY AS A NEW TOOL FOR MEASURING EARLY CARTILAGE DEGRADATION FOLLOWING BLOOD EXPOSURE IN VITRO AND IN VIVO IN F8 KO RATS

    DEFF Research Database (Denmark)

    Pulles, A. E.; Christensen, K. R.; Coeleveld, K.

    2017-01-01

    of radioactive 35Sulphate (35SO42-) in cartilage, is a sensitive method to detect early cellular changes in cartilage previously applied in human and larger animal models. Isolating cartilage of small animals can be challenging, so a technique to shave off rat’s cartilage was developed and the 35Sulphate...... with and approved by the Danish Animal Experiments Council, Ministry of Food, Agriculture and Fisheries, Denmark. Results: On average, a total of 1.6mg (0.8–3.1mg) cartilage per tibia could be obtained. The proteoglycan synthesis rate of healthy cartilage determined after four days of culturing in vitro...

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

    Directory of Open Access Journals (Sweden)

    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. Increased proliferation of explanted vascular smooth muscle cells: a marker presaging atherogenesis.

    Science.gov (United States)

    Absher, P M; Schneider, D J; Baldor, L C; Russell, J C; Sobel, B E

    1997-06-01

    The JCR:LA-cp homozygous cp/cp corpulent rat is genetically predisposed to develop atherosclerosis evident after 9 and 18 months of age in males and females and to manifest metabolic derangements resembling those seen in type II diabetes in humans (hyperinsulinemia, insulin resistance, hyperglycemia and hypertriglyceridemia). The present study was undertaken to determine whether vascular smooth muscle cells (SMCs) explanted from vessels destined to become atherosclerotic later in life exhibit intrinsic properties ex vivo that presage atherogenesis to provide a means for evaluating promptly intervention designed to modify it. SMCs were cultured from aortic explants of JCR:LA-cp corpulent (cp/cp) and lean control (+/+) rats of 4, 5, 6, and 9 months of age. Compared with SMCs from controls, SMCs from cp/cp rats exhibited increased proliferation, higher saturation density, increased augmentation of proliferation in response to selected mitogens and greater adherence to extracellular matrix proteins. The increased proliferative activity ex vivo anteceded by several months the development of atherosclerotic lesions in vivo. Thus, it is a promising marker in assessments of the efficacy of interventions designed to retard or prevent atherosclerosis.

  4. Strategies for Stratified Cartilage Bioprinting

    NARCIS (Netherlands)

    Schuurman, W.

    2012-01-01

    Multiple materials, cells and growth factors can be combined into one construct by the use of a state–of-the-art bioprinter. This technique may in the future make the fabrication of complete tissues or organs possible. In this thesis the feasibility of the bioprinting of cartilage and the difference

  5. Postnatal development of articular cartilage

    NARCIS (Netherlands)

    Turnhout, van M.C.

    2010-01-01

    Articular cartilage (AC) is the thin layer of tissue that covers the ends of the bones in the synovial joints in mammals. Functional adult AC has depth-dependent mechanical properties that are not yet present at birth. These depth-dependent mechanical properties in adult life are the result of a

  6. Postnatal development of articular cartilage

    NARCIS (Netherlands)

    Turnhout, van M.C.

    2010-01-01

    Articular cartilage (AC) is the thin layer of tissue that covers the ends of the bones in the synovial joints in mammals. Functional adult AC has depth-dependent mechanical properties that are not yet present at birth. These depth-dependent mechanical properties in adult life are the result of a dep

  7. Effect of shark cartilage on the cytotoxic activity of NK cells immune system

    Directory of Open Access Journals (Sweden)

    Afshar Bargahi

    2009-12-01

    Full Text Available Background: On the basis of traditional medicine Shark cartilage have been used in the treatment of cancer especially immune related cancers. Then, we hypotheses that shark cartilage contains immune stimulatory ingredients. Methods: The immune stimulatory effect of shark cartilage derived proteins on the cytotoxic activity of natural killer cells(NK cells from healthy human peripheral blood mononuclear cells (hPBMN was studied. Shark cartilage proteins were purified by ion-exchange chromatography and ultra filtration. The effect of each protein fraction on the modulation of cytotoxicity of NK cells, as effectors, against K562, as target cells, was assayed by enzymatic LDH test. Results: The results from cytotoxic assay of NK cells and SDS- Polyacrylamide gell electrophoresis of shark cartilage protein fractions indicated that AR10 fraction, containing proteins with molecular weight of about 14.5 KDa is the most active ingredients of shark cartilage. Conclusion: Shark cartilage contains a 14.5 KDa protein that modulates NK cells activity of human immune system.

  8. Estudo Morfológico do desenvolvimento da cartilagem quadrangular do nariz e implicações nas cirurgias septoplásticas Morphologic study of the development of the human nose quadrilateral cartilage and implications in the septoplastic surgery

    Directory of Open Access Journals (Sweden)

    Paulo S. G. Pereira

    2002-03-01

    Full Text Available Introdução: Este trabalho é um estudo morfológico do desenvolvimento da cartilagem quadrangular humana ao longo da vida. Para tal retiramos cartilagens quadrangulares em bloco com as demais cartilagens do nariz, utilizando a técnica de rinoplastia aberta, em cadáveres autopsiados no Serviço de Patologia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto - USP e no serviço de verificação de óbitos do interior, em Ribeirão Preto. Forma de estudo: Experimental. Métodos: As peças anatômicas foram cortadas em dois pontos dividindo-as em três partes, com relação ao eixo caudo-cefálico correspondendo à borda livre da cartilagem, terço caudal (posição 1, médio (posição 2 e cefálico (posição 3. Tem relação com a placa perpendicular do etmóide e volmer. As amostras foram fixadas em formol a 10% e incluídas em parafina. Foram examinados cortes histológicos de 7 µm de espessura e corados por hematoxilina e eosina (HE e Alcian Blue. Em cada posição, três diferentes locais, em relação ao eixo ântero-posterior do nariz: Terço superior (local 1, terço médio (local 2 e terço inferior (local 3 foram estudados sobre Microscopia Óptica, com métodos fotométricos, histológicos e morfométricos. Resultado: Mostra que o tecido cartilaginoso apresenta maior velocidade de crescimento até cinco anos de idade. A menor velocidade de crescimento ocorre a partir dos oito anos de idade. Não ocorreu diferença de atividade metabólica entre as diversas regiões estudadas. Conclusões: As intervenções cirúrgicas no septo cartilaginoso podem ser realizadas após os cinco anos de idade. A idade inicial mais adequada para as cirurgias do septo nasal ocorreria aos oito anos.Introduction: The present paper is a morphological study of human quadrilateral cartilage including 0 to 66 years old subjects. The quadrilateral cartilage was removed in block with the others nasal cartilages. The open rhinoplasty was the

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

    Science.gov (United States)

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

    2002-12-01

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

  10. The effect of adipose-derived stem cells on the increased survival of crushed cartilage graft in rabbits

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Ebadi

    2016-10-01

    Full Text Available Background: In recent years the use of diced cartilage grafts in reconstructive surgery particulary rhinoplasty have been considered by most plastic surgeons. However, long-term resorption usually occurs. Stem cells are a powerful tool for reconstructive surgery to rebuild and maintain tissue with reduced complications. Since the adipose tissue-derived stem cells (ADSCs can rebuild a wide variety of tissues such as skin, fat, bone and cartilage are used, this is a very good chance for cosmetic surgery. The aim of this study was to examine the effects of adipose-derived stem cells on the viability of diced cartilage grafts. Methods: This interventional study was performed on May 2014 in animal laboratory of Hazrat Fatima Hospital on 10 New Zealand white male rabbits, weighing 2000-2500 grams, approximately 12 to 16 weeks of age. Stem cells was harvested from inguinal adipose tissue of each rabbits. After completely removing the skin and perichondrium, cartilage became divided into two equal pieces using a scalpel. Then place the ear amputation was restored by nylon 4 zero. After weighing cartilages, on either side of the center line on the back of each rabbits, left and right, subcutaneous pocket created equal weight and each piece of cartilage was placed in an envelope. Stem cells were injected in one side and the other side was control. The cartilage weights were recorded both before implantation and after explantation. Evaluation of living chondrocytes was conducted 12 weeks after implantation. Results: The mean difference of cartilage weights was varied between two groups (intervention and control sides, So that the average was significantly higher in stem cell side than that in the control side (P= 0.021. The average number of live chondrocytes was significantly higher in the intervention side than the control side (P< 0.001. Conclusion: Despite the unclear mechanism, these results suggest that adipose-derived stem cells can maintain the

  11. Distribution of Basement Membrane Molecules, Laminin and Collagen Type IV, in Normal and Degenerated Cartilage Tissues

    Science.gov (United States)

    Toh, Wei Seong; Gomoll, Andreas H.; Olsen, Bjørn Reino; Spector, Myron

    2014-01-01

    Objective: The objective of the present study was to investigate the presence and distribution of 2 basement membrane (BM) molecules, laminin and collagen type IV, in healthy and degenerative cartilage tissues. Design: Normal and degenerated tissues were obtained from goats and humans, including articular knee cartilage, the intervertebral disc, and meniscus. Normal tissue was also obtained from patella-tibial enthesis in goats. Immunohistochemical analysis was performed using anti-laminin and anti–collagen type IV antibodies. Human and goat skin were used as positive controls. The percentage of cells displaying the pericellular presence of the protein was graded semiquantitatively. Results: When present, laminin and collagen type IV were exclusively found in the pericellular matrix, and in a discrete layer on the articulating surface of normal articular cartilage. In normal articular (hyaline) cartilage in the human and goat, the proteins were found co-localized pericellularly. In contrast, in human osteoarthritic articular cartilage, collagen type IV but not laminin was found in the pericellular region. Nonpathological fibrocartilaginous tissues from the goat, including the menisci and the enthesis, were also positive for both laminin and collagen type IV pericellularly. In degenerated fibrocartilage, including intervertebral disc, as in degenerated hyaline cartilage only collagen type IV was found pericellularly around chondrocytes but with less intense staining than in non-degenerated tissue. In calcified cartilage, some cells were positive for laminin but not type IV collagen. Conclusions: We report differences in expression of the BM molecules, laminin and collagen type IV, in normal and degenerative cartilaginous tissues from adult humans and goats. In degenerative tissues laminin is depleted from the pericellular matrix before collagen type IV. The findings may inform future studies of the processes underlying cartilage degeneration and the functional

  12. Investigation of polarization-sensitive optical coherence tomography towards the study of microstructure of articular cartilage

    Science.gov (United States)

    Kasaragod, Deepa; Lu, Zenghai; Le Maitre, Christine; Wilkinson, J. Mark; Matcher, Stephen

    2013-03-01

    This paper highlights the extended Jones matrix calculus based multi-angle study carried out to understand the depth dependent structural orientation of the collagen fibers in articular cartilage using polarization-sensitive optical coherence tomography (PS-OCT). A 3D lamellar model for the collagen fiber orientation, with a quadratic profile for the arching of the collagen fibers in transitional zone which points towards an ordered arrangement of fibers in that zone is the basis of the organization architecture of collagen fibers in articular cartilage. Experimental data for both ex-vivo bovine fetlock and human patellar cartilage samples are compared with theoretical predictions, with a good quantitative agreement for bovine and a reasonable qualitative agreement for human articular cartilage samples being obtained

  13. Usual interstitial pneumonia end-stage features from explants with radiologic and pathological correlations.

    Science.gov (United States)

    Rabeyrin, Maud; Thivolet, Françoise; Ferretti, Gilbert R; Chalabreysse, Lara; Jankowski, Adrien; Cottin, Vincent; Pison, Christophe; Cordier, Jean-François; Lantuejoul, Sylvie

    2015-08-01

    Idiopathic pulmonary fibrosis (IPF) is the most frequent and severe idiopathic interstitial pneumonia, with typical high-resolution computed tomography (HRCT) features and histologic pattern of usual interstitial pneumonia (UIP); its main differential diagnosis is fibrotic nonspecific interstitial pneumonia (F-NSIP). Usual interstitial pneumonia was mainly described from lung biopsies, and little is known on explants. Twenty-two UIP/IPF explants were analyzed histologically and compared with previous open lung biopsies (OLBs; n = 11) and HRCT (n = 19), when available. Temporospatial heterogeneity and subpleural and paraseptal fibrosis were similarly found in UIP/IPF explants and OLB (91%-95%). Fibroblastic foci were found in 82% of OLBs and 100% of explants, with a higher mean score in explants (P = .023). Honeycombing was present in 64% of OLBs and 95% of explants, with a higher mean score in explants (P = .005). Almost 60% of UIP/IPF explants showed NSIP areas and 41% peribronchiolar fibrosis; inflammation, bronchiolar metaplasia, and vascular changes were more frequent in UIP/IPF explants; and Desquamative Interstitial Pneumonia (DIP)-like areas were not common (18%-27%). Numerous large airspace enlargements with fibrosis were frequent in UIP/IPF explants (59%). On HRCT, honeycombing was observed in 95% of the cases and ground-glass opacities in 53%, correlating with NSIP areas or acute exacerbation at histology. Six patients had combined IPF and emphysema. Lesions were more severe in UIP/IPF explants, reflecting the worsening of the disease. Usual interstitial pneumonia/IPF explants more frequently presented with confounding lesions such as NSIP areas, peribronchiolar fibrosis, and airspace enlargements with fibrosis sometimes associated with emphysema.

  14. The influence of season collection of explants on micropropagation of peach rootstock GF-677

    Directory of Open Access Journals (Sweden)

    Elektra Spahiu

    2013-02-01

    Full Text Available The influence of season on the rate of multiplication on in vitro culture of peach rootstock GF- 677 was investigated on Murashige and Skoog (MS media, supplemented with GA3 0.1 mg/L and IAA 0.1mg/l. Benzyladenine (BAP at concentrations 1mg/l was used in the multiplication stage and 1mg/l IBA in the stage of rooting. Shoot-tip and nodal segment explants were collected from 5 years old rootstock GF-677 (Prunus persica x Prunus amygdalus in February 24th (from dormant shoots that have been sprouted in climatic room, March 22th, April 20th, May 18th and September 15th during the 2009 growing season and have been sterilized by sodium hypochlorite (NaOCl 10% for 20 min. The data on the effect of the season collection of the explants on number of shoots per explants, the mean shoot length and the percentage of rooted shoots were recorded six weeks after culture. In vitro performance of explants indicated a positive correlation between shoot proliferation and season collection The highest number of shoots per explants (3,5 was obtained on explants collected in March 22th (3,5, which was on a par with explants collected in February 24th (from shoots that have been sprouted in climatic room. Moreover, the highest shoot length was observed on explants collected on February and March (1,53cm and 1,505cm respectively. The percentage of rooted shoots from explants sampled on February was not markedly greater than those sampled on March. The number of shoots per explants, the shoot length and the percentage of rooted shoots on explants sampled in April, May and September were significantly lower than those sampled in February and March. The amount of chlorophyll a + b of the shoots coming from explants collected in March was markedly greater than those collected in February, April, May and September.

  15. Ontogeny of rat chondrocyte proliferation: studies in embryo, adult and osteoarthritic (OA) cartilage

    Institute of Scientific and Technical Information of China (English)

    Madaí A GóMEZ-CAMARILLO; Juan B.KOURI

    2005-01-01

    The aim of this work was to study the ontogeny of chondrocyte cell division using embryo, adult and osteoarthritic (OA) cartilage. We searched for mitosis phases and performed a comparative evaluation of mitotic index, basic fibroblast growth factor b (FGFb), transforming growth factor β1 (TGF-β1) receptors, cyclin dependent kinase (CDK1)and Cyclin-B expression in fetal, neonate, 3, 5, 8 weeks old rats and experimental OA. Our results showed that mitosis phases were observed in all normal cartilage studied, although, we found a decrease in mitotic index in relation to tissue development. No mitosis was detected in OA cartilage. We also found a statistical significant reduction in cell number in OA cartilage, compared with the normal tissue. Furthermore, FGFb and TGF-β1 receptors diminished in relation to tissue development, and were very scarce in experimental OA. Western blot assays showed CDK-1 expression in all cases, including human-OA cartilage. Similar results were observed for Cyclin-B, except for 8 weeks, when it was not expressed. Our results suggest that cell division seems to be scarce, if not absent within the OA cartilage studied.Nevertheless, the existence of factors essential for cell division leaves open the question concerning chondrocyte proliferation in OA cartilage, which is likely to be present in the early stages of the disease.

  16. Effect of peripheral nerve on the neurite growth from retinal explants in culture

    Institute of Scientific and Technical Information of China (English)

    LiuLi; SoKwokfai

    1990-01-01

    The effect of peripheral nerve (PN) on neurite outgrowth from retinal explants of adult hamsters was examined.Cultures of retinal explants,and co-cultures of retinal explants and PN were performed using chick retinal basement memebrane (BM) as substrate.The presence of PN increases the number and length of neurite outgrowth.In addition,a high proportion of neurites situated close to PN tend to grow towards it.Since there was no contact between retinal explants and PN,we suggest that PN might secete diffusible substances to attract the neurites to grow towards it.

  17. Micropropagation of Cyrtopodium paludicolum (Orchidaceae from root tip explants

    Directory of Open Access Journals (Sweden)

    Dayana Rotili Nunes Picolotto

    2017-06-01

    Full Text Available An efficient protocol for in vitro plant propagation of Cyrtopodium paludicolum has been developed using root tips dissected from well-developed seedlings. Root tips were cultured on Knudson medium supplemented with α-naphthaleneacetic acid (NAA, and/or thidiazuron (TDZ. TDZ did not induce protocorm-like bodies (PLBs in the NAA absence, indicating phytoregulators synergistic effect. Medium supplemented with 1.34 μM NAA and 2.27 μM TDZ resulted in better response on PBLs, and subsequent shoot differentiation (55.25 shoots per explant, and in better rooting number and root length responses, favoring acclimatization with 90% of survived plants. However, the medium supplemented with only NAA (1.34 μM resulted in 33.50 shoots per explant. Histological sections confirmed that only one PLB was induced per responsive root tip, and it showed numerous dispersed and extended meristemoids, or division centers that originated new PBLs. Additionally, this protocol could be an excellent model to study molecular aspects of root to shoot conversion.

  18. Microscopic and spectroscopic investigation of an explanted opacified intraocular lens

    Energy Technology Data Exchange (ETDEWEB)

    Simon, V., E-mail: viosimon@phys.ubbcluj.ro [Babeş-Bolyai University, Faculty of Physics and Interdisciplinary Research Institute on Bio-Nano-Sciences, 400084 Cluj-Napoca (Romania); Radu, T.; Vulpoi, A. [Babeş-Bolyai University, Faculty of Physics and Interdisciplinary Research Institute on Bio-Nano-Sciences, 400084 Cluj-Napoca (Romania); Rosca, C. [Optilens Clinic of Ophthalmology, 400604 Cluj-Napoca (Romania); Eniu, D. [Iuliu Haţieganu University of Medicine and Pharmacy, Department of Molecular Sciences, 400349 Cluj-Napoca (Romania)

    2015-01-15

    Highlights: • Changes on intraocular lens (IOL) surface after implantation. • Partial opacification of IOL central area. • Elemental composition on IOL surface prior to and after implantation. • First XPS depth profiling examination of the opacifying deposits. • Cell-mediated hydroxyapatite structuring. - Abstract: The investigated polymethylmethacrylate intraocular lens explanted an year after implantation presented a fine granularity consisting of ring-like grains of about 15 μm in diameter. In order to evidence the changes occurred on intraocular lens relative to morphology, elemental composition and atomic environments, microscopic and spectroscopic analyses were carried out using scanning electron microscopy (SEM), Fourier transform infrared (FTIR), energy-dispersive X-ray (EDS), and X-ray photoelectron (XPS) spectroscopies. The results revealed that the grains contain hydroxyapatite mineral phase. A protein layer covers the lens both in opacified and transparent zones. The amide II band is like in basal epithelial cells. The shape and size of the grains, and the XPS depth profiling results indicate the possibility of a cell-mediated process involving lens epithelial cells which fagocitated apoptotic epithelial cells, and in which the debris derived from cell necrosis were calcified. To the best of our knowledge, this is the first investigation on explanted intraocular lenses using XPS depth profiling in order to examine the inside of the opacifying deposits.

  19. Microscopic and spectroscopic investigation of an explanted opacified intraocular lens

    Science.gov (United States)

    Simon, V.; Radu, T.; Vulpoi, A.; Rosca, C.; Eniu, D.

    2015-01-01

    The investigated polymethylmethacrylate intraocular lens explanted an year after implantation presented a fine granularity consisting of ring-like grains of about 15 μm in diameter. In order to evidence the changes occurred on intraocular lens relative to morphology, elemental composition and atomic environments, microscopic and spectroscopic analyses were carried out using scanning electron microscopy (SEM), Fourier transform infrared (FTIR), energy-dispersive X-ray (EDS), and X-ray photoelectron (XPS) spectroscopies. The results revealed that the grains contain hydroxyapatite mineral phase. A protein layer covers the lens both in opacified and transparent zones. The amide II band is like in basal epithelial cells. The shape and size of the grains, and the XPS depth profiling results indicate the possibility of a cell-mediated process involving lens epithelial cells which fagocitated apoptotic epithelial cells, and in which the debris derived from cell necrosis were calcified. To the best of our knowledge, this is the first investigation on explanted intraocular lenses using XPS depth profiling in order to examine the inside of the opacifying deposits.

  20. Jellyfish collagen scaffolds for cartilage tissue engineering.

    Science.gov (United States)

    Hoyer, Birgit; Bernhardt, Anne; Lode, Anja; Heinemann, Sascha; Sewing, Judith; Klinger, Matthias; Notbohm, Holger; Gelinsky, Michael

    2014-02-01

    Porous scaffolds were engineered from refibrillized collagen of the jellyfish Rhopilema esculentum for potential application in cartilage regeneration. The influence of collagen concentration, salinity and temperature on fibril formation was evaluated by turbidity measurements and quantification of fibrillized collagen. The formation of collagen fibrils with a typical banding pattern was confirmed by atomic force microscopy and transmission electron microscopy analysis. Porous scaffolds from jellyfish collagen, refibrillized under optimized conditions, were fabricated by freeze-drying and subsequent chemical cross-linking. Scaffolds possessed an open porosity of 98.2%. The samples were stable under cyclic compression and displayed an elastic behavior. Cytotoxicity tests with human mesenchymal stem cells (hMSCs) did not reveal any cytotoxic effects of the material. Chondrogenic markers SOX9, collagen II and aggrecan were upregulated in direct cultures of hMSCs upon chondrogenic stimulation. The formation of typical extracellular matrix components was further confirmed by quantification of sulfated glycosaminoglycans.

  1. Mechanobiology and Cartilage Tissue Engineering

    Institute of Scientific and Technical Information of China (English)

    Céline; HUSELSTEIN; Natalia; de; ISLA; Sylvaine; MULLER; Jean-Franois; STOLTZ

    2005-01-01

    1 IntroductionThe cartilage is a hydrated connective tissue in joints that withstands and distributes mechanical forces. Chondrocytes utilize mechanical signals to maintain tissue homeostasis. They regulate their metabolic activity through complex biological and biophysical interactions with the extracellular matrix (ECM). Although some of the mechanisms of mechanotransduction are known today, there are certainly many others left unrevealed. Different topics of chondrocytes mechanobiology have led to the de...

  2. Dorsal Augmentation with Septal Cartilage

    OpenAIRE

    Murrell, George L.

    2008-01-01

    Deficiency of nasal dorsal projection may be inherent or acquired. Repair is most commonly performed with an onlay graft. When nasal septal cartilage is available, it is the author's preferred source for graft material. It is important to realize that dorsal augmentation is an operation performed for aesthetic not functional reasons. As such, patients understandably scrutinize their postoperative result, and attention to detail in all aspects of the surgery is critical in achieving a favorabl...

  3. Resident mesenchymal progenitors of articular cartilage.

    Science.gov (United States)

    Candela, Maria Elena; Yasuhara, Rika; Iwamoto, Masahiro; Enomoto-Iwamoto, Motomi

    2014-10-01

    Articular cartilage has poor capacity of self-renewal and repair. Insufficient number and activity of resident mesenchymal (connective tissue) progenitors is likely one of the underlying reasons. Chondroprogenitors reside not only in the superficial zone of articular cartilage but also in other zones of articular cartilage and in the neighboring tissues, including perichondrium (groove of Ranvier), synovium and fat pad. These cells may respond to injury and contribute to articular cartilage healing. In addition, marrow stromal cells can migrate through subchondral bone when articular cartilage is damaged. We should develop drugs and methods that correctly stimulate resident progenitors for improvement of repair and inhibition of degenerative changes in articular cartilage. Copyright © 2014. Published by Elsevier B.V.

  4. Diode laser (980nm) cartilage reshaping

    Science.gov (United States)

    El Kharbotly, A.; El Tayeb, T.; Mostafa, Y.; Hesham, I.

    2011-03-01

    Loss of facial or ear cartilage due to trauma or surgery is a major challenge to the otolaryngologists and plastic surgeons as the complicated geometric contours are difficult to be animated. Diode laser (980 nm) has been proven effective in reshaping and maintaining the new geometric shape achieved by laser. This study focused on determining the optimum laser parameters needed for cartilage reshaping with a controlled water cooling system. Harvested animal cartilages were angulated with different degrees and irradiated with different diode laser powers (980nm, 4x8mm spot size). The cartilage specimens were maintained in a deformation angle for two hours after irradiation then released for another two hours. They were serially measured and photographed. High-power Diode laser irradiation with water cooling is a cheep and effective method for reshaping the cartilage needed for reconstruction of difficult situations in otorhinolaryngologic surgery. Key words: cartilage,diode laser (980nm), reshaping.

  5. Harnessing Biomechanics to Develop Cartilage Regeneration Strategies

    OpenAIRE

    Athanasiou, KA; Responte, DJ; Brown, WE; Hu, JC

    2015-01-01

    Copyright © 2015 by ASME. As this review was prepared specifically for the American Society of Mechanical Engineers H.R. Lissner Medal, it primarily discusses work toward cartilage regeneration performed in Dr. Kyriacos A. Athanasiou's laboratory over the past 25 years. The prevalence and severity of degeneration of articular cartilage, a tissue whose main function is largely biomechanical, have motivated the development of cartilage tissue engineering approaches informed by biomechanics. Thi...

  6. Nanofibrous poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) scaffolds provide a functional microenvironment for cartilage repair.

    Science.gov (United States)

    Ching, Kuan Y; Andriotis, Orestis G; Li, Siwei; Basnett, Pooja; Su, Bo; Roy, Ipsita; Tare, Rahul S; Sengers, Bram G; Stolz, Martin

    2016-07-01

    Articular cartilage defects, when repaired ineffectively, often lead to further deterioration of the tissue, secondary osteoarthritis and, ultimately, joint replacement. Unfortunately, current surgical procedures are unable to restore normal cartilage function. Tissue engineering of cartilage provides promising strategies for the regeneration of damaged articular cartilage. As yet, there are still significant challenges that need to be overcome to match the long-term mechanical stability and durability of native cartilage. Using electrospinning of different blends of biodegradable poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate), we produced polymer scaffolds and optimised their structure, stiffness, degradation rates and biocompatibility. Scaffolds with a poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) ratio of 1:0.25 exhibit randomly oriented fibres that closely mimic the collagen fibrillar meshwork of native cartilage and match the stiffness of native articular cartilage. Degradation of the scaffolds into products that could be easily removed from the body was indicated by changes in fibre structure, loss of molecular weight and a decrease in scaffold stiffness after one and four months. Histological and immunohistochemical analysis after three weeks of culture with human articular chondrocytes revealed a hyaline-like cartilage matrix. The ability to fine tune the ultrastructure and mechanical properties using different blends of poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) allows to produce a cartilage repair kit for clinical use to reduce the risk of developing secondary osteoarthritis. We further suggest the development of a toolbox with tailor-made scaffolds for the repair of other tissues that require a 'guiding' structure to support the body's self-healing process.

  7. Imaging Bone–Cartilage Interactions in Osteoarthritis Using [18F]-NaF PET-MRI

    Directory of Open Access Journals (Sweden)

    Dragana Savic MSc

    2016-12-01

    Full Text Available Purpose: Simultaneous positron emission tomography–magnetic resonance imaging (PET-MRI is an emerging technology providing both anatomical and functional images without increasing the scan time. Compared to the traditional PET/computed tomography imaging, it also exposes the patient to significantly less radiation and provides better anatomical images as MRI provides superior soft tissue characterization. Using PET-MRI, we aim to study interactions between cartilage composition and bone function simultaneously, in knee osteoarthritis (OA. Procedures: In this article, bone turnover and remodeling was studied using [18F]-sodium fluoride (NaF PET data. Quantitative MR-derived T1ρ relaxation times characterized the biochemical cartilage degeneration. Sixteen participants with early signs of OA of the knee received intravenous injections of [18F]-NaF at the onset of PET-MR image acquisition. Regions of interest were identified, and kinetic analysis of dynamic PET data provided the rate of uptake (Ki and the normalized uptake (standardized uptake value of [18F]-NaF in the bone. Morphological MR images and quantitative voxel-based T1ρ maps of cartilage were obtained using an atlas-based registration technique to segment cartilage automatically. Voxel-by-voxel statistical parameter mapping was used to investigate the relationship between bone and cartilage. Results: Increases in cartilage T1ρ, indicating degenerative changes, were associated with increased turnover in the adjoining bone but reduced turnover in the nonadjoining compartments. Associations between pain and increased bone uptake were seen in the absence of morphological lesions in cartilage, but the relationship was reversed in the presence of incident cartilage lesions. Conclusion: This study shows significant cartilage and bone interactions in OA of the knee joint using simultaneous [18F]-NaF PET-MR, the first in human study. These observations highlight the complex biomechanical and

  8. Can Glucosamine Supplements Protect My Knee Cartilage from Osteoarthritis?

    Science.gov (United States)

    ... Can glucosamine supplements protect my knee cartilage from osteoarthritis? Answers from Brent A. Bauer, M.D. Study results on this question have ... build cartilage. The most common type of arthritis, osteoarthritis wears away the slick cartilage that covers the ...

  9. Organogênese direta de explantes cotiledonares e regeneração de plantas de mogango Direct organogenesis of cotyledon explants and plant regeneration of squash

    Directory of Open Access Journals (Sweden)

    André Luís Lopes da Silva

    2006-06-01

    Full Text Available Os objetivos foram induzir a organogênese direta de explantes cotiledonares de mogango e estudar a regeneração de plântulas completas a partir das brotações adventícias. Foram utilizados cotilédones como explantes, originados das plântulas de mogango com 20 dias após a semeadura. O meio basal utilizado foi o MS (MURASHIGE & SKOOG, 1962 suplementado com 30g L-1 de sacarose e 7g L-1 de agar. Foram testadas as concentrações de 6-benzilaminopurina (BAP de 0; 0,5; 1,0 e 2,0mg L-1. Explantes de ápices caulinares e segmentos nodais de brotações adventícias foram então cultivados em meio MS suplementado com 30g L-1 de sacarose e 7g L-1 de agar. Maiores concentrações de BAP no meio MS promoveram um aumento da percentagem de explantes cotiledonares com brotações adventícias e uma redução da percentagem de enraizamento. Explantes de segmentos nodais e ápices caulinares oriundos de brotações adventícias cresceram e enraizaram em meio MS sem reguladores de crescimento. Altas percentagens de enraizamento dependem do tamanho dos explantes utilizados.The objectives were to induce direct organogenesis of squash cotyledons and to study the regeneration of complete plantlets from adventitious shoot. Cotyledon explants of 20-day seedlings were cultured in MS (MURASHIGE & SKOOG, 1962 medium supplemented with 30g L-1 of sucrose and 7g L-1 of agar. The 6-benzilaminopurina (BAP concentrations of 0, 0.5, 1.0 and 2.0mg L-1 were tested. Apical and nodal explants from adventitious shoots were transferred to MS medium supplemented with 30g L-1 of sucrose and 7g L-1 of agar. Increasing BAP concentrations in the MS medium enhance the percentage of adventitious shoot and reduce the percentage of root organogenesis of squash cotyledon explants. Apical and nodal explants from adventitious shoot regenerated plantlets with roots in MS medium without growth regulators. High percentage of plantlet rooting depends upon the size of the explants.

  10. Cartilage collagen damage in hip osteoarthritis similar to that seen in knee osteoarthritis; a case–control study of relationship between collagen, glycosaminoglycan and cartilage swelling

    Directory of Open Access Journals (Sweden)

    Hosseininia Shahrzad

    2013-01-01

    Full Text Available Abstract Background It remains to be shown whether OA shares molecular similarities between different joints in humans. This study provides evidence for similarities in cartilage molecular damage in osteoarthritic (OA joints. Methods Articular cartilage from osteoarthritic hip joints were analysed and compared to non-OA controls regarding collagen, glycosaminoglycan and water content. Femoral heads from 16 osteoarthritic (OA and 20 reference patients were obtained from hip replacement surgery due to OA and femoral neck fracture, respectively. Cartilage histological changes were assessed by Mankin grading and denatured collagen type II immunostaining and cartilage was extracted by α-chymotrypsin. Hydroxyproline and Alcian blue binding assays were used to measure collagen and glycosaminoglycan (GAG content, respectively. Results Mankin and immunohistology scores were significantly higher in hip OA samples than in reference samples. Cartilage water content was 6% higher in OA samples than in references. 2.5 times more collagen was extracted from OA than from reference samples. There was a positive association between water content and percentage of extractable collagen pool (ECP in both groups. The amounts of collagen per wet and dry weights did not differ statistically between OA and reference cartilage. % Extractable collagen was not related to collagen per dry weight in either group. However when collagen was expressed by wet weight there was a negative correlation between % extractable and collagen in OA cartilage. The amount of GAG per wet weight was similar in both groups but the amount of GAG per dry weight was higher in OA samples compared to reference samples, which suggests a capacity for GAG biosynthesis in hip OA cartilage. Neither of the studied parameters was related to age in either group. Conclusions Increased collagen extractability and water content in human hip cartilage is associated with OA pathology and can be observed at

  11. {mu}-PIXE and SAXS studies at the bone-cartilage interface

    Energy Technology Data Exchange (ETDEWEB)

    Kaabar, W. [Department of Physics, University of Surrey, Guildford GU2 7XH (United Kingdom)], E-mail: w.kaabar@surrey.ac.uk; Gundogdu, O. [Umuttepe Campus, University of Kocaeli, 41380, Kocaeli (Turkey); Laklouk, A. [Food Science Department, Al-Fateh Unversity, Tripoli (Libyan Arab Jamahiriya); Bunk, O. [Swiss Light Source, Paul Scherrer Institute, 5232 Villigen (Switzerland); Pfeiffer, F. [Swiss Light Source, Paul Scherrer Institute, 5232 Villigen (Switzerland); Ecole Polytechnique Federale de Lausanne, 1015 Lausanne (Switzerland); Farquharson, M.J. [Department of Radiography, City University, London EC1V OHB (United Kingdom); Bradley, D.A. [Department of Physics, University of Surrey, Guildford GU2 7XH (United Kingdom)

    2010-04-15

    Micro Proton Induced X-ray Emission ({mu}-PIXE) analysis has been employed herein in investigating and quantifying the distribution of a number of essential elements in thin human diseased articular cartilage sections affected by osteoarthritis (OA). Various cations Ca, P and Zn have been reported to play an important role both in the normal growth and remodelling of articular cartilage and subchondral bone as well as in the degenerative and inflammatory processes associated with the disease; they act as co-factors of a class of enzymes known as metalloproteinases which are believed to be active during the initiation, progress and remodelling processes associated with osteoarthritis. Other important enzymes such as alkaline phosphatase are associated with cartilage mineralization. Synchrotron radiation X-ray fluorescence (SR-XRF) for mapping of elemental distributions in bone and cartilage has also been employed by the present group and others. In the current investigations using the cSAXS beamline at the Swiss light source, Small-Angle X-ray Scattering (SAXS) was carried out on decalcified human articular cartilage to explore the structural and organizational changes of collagen networks in diseased articular cartilage.

  12. Tamarind Seed (Tamarindus indica) Extract Ameliorates Adjuvant-Induced Arthritis via Regulating the Mediators of Cartilage/Bone Degeneration, Inflammation and Oxidative Stress

    OpenAIRE

    Mahalingam S Sundaram; Mahadevappa Hemshekhar; Santhosh, Martin S.; Manoj Paul; Kabburahalli Sunitha; Ram M. Thushara; NaveenKumar, Somanathapura K.; Shivanna Naveen; Sannaningaiah Devaraja; Rangappa, Kanchugarakoppal S.; Kempaiah Kemparaju; Girish, Kesturu S.

    2015-01-01

    Medicinal plants are employed in the treatment of human ailments from time immemorial. Several studies have validated the use of medicinal plant products in arthritis treatment. Arthritis is a joint disorder affecting subchondral bone and cartilage. Degradation of cartilage is principally mediated by enzymes like matrix metalloproteinases (MMPs), hyaluronidases (HAase), aggrecanases and exoglycosidases. These enzymes act upon collagen, hyaluronan and aggrecan of cartilage respectively, which ...

  13. Which cartilage is regenerated, hyaline cartilage or fibrocartilage? Non-invasive ultrasonic evaluation of tissue-engineered cartilage.

    Science.gov (United States)

    Hattori, K; Takakura, Y; Ohgushi, H; Habata, T; Uematsu, K; Takenaka, M; Ikeuchi, K

    2004-09-01

    To investigate ultrasonic evaluation methods for detecting whether the repair tissue is hyaline cartilage or fibrocartilage in new cartilage regeneration therapy. We examined four experimental rabbit models: a spontaneous repair model (group S), a large cartilage defect model (group L), a periosteal graft model (group P) and a tissue-engineered cartilage regeneration model (group T). From the resulting ultrasonic evaluation, we used %MM (the maximum magnitude of the measurement area divided by that of the intact cartilage) as a quantitative index of cartilage regeneration. The results of the ultrasonic evaluation were compared with the histological findings and histological score. The %MM values were 61.1 +/- 16.5% in group S, 29.8 +/- 15.1% in group L, 36.3 +/- 18.3% in group P and 76.5 +/- 18.7% in group T. The results showed a strong similarity to the histological scoring. The ultrasonic examination showed that all the hyaline-like cartilage in groups S and T had a high %MM (more than 60%). Therefore, we could define the borderline between the two types of regenerated cartilage by the %MM.

  14. In vitro morphogenic events in culture of Lotus corniculatus L. seedling root explants

    Directory of Open Access Journals (Sweden)

    Jan J. Rybczyński

    2011-01-01

    Full Text Available The experiments were carried out on Lotus corniculatus (L. seedling root explants of the cultivar varieties Skrzeszowicka, Caroll A10 and strain 175. Callus formation and shoot regeneration were the major explant response depended mainly on of the studied genotype and used plant growth regulators (PGRs. Primary cortex of proximal and distal end of explant was the most active tissue for callus proliferation. For shoot primordia differentiation deeper zones of cortex took a part. The process of meristematic centre initiation was not uniform and various level of shoot differentiation events were observed not earlier than 3 weeks of culture. Usually, the shoot primordia regeneration began on proximal rather than distal end of the explant. BAP rather than urea derivatives stimulated shoot proliferation in extended cultures. Increasing of BAP and TDZ concentrations brought about the explant polarity and expansion of the meristematic zones. The explant position in root did not have significant influence on the number of regenerated shoots. The cultures only had better bud formation by TDZ when compared to BAP. BAP stimulated bud formation and development of the shoots from them. Short term of TDZ treatment of explants stimulated meristem formation which developed into buds and shoots. CPPU stimulated callus proliferation and bud formation when explants pretreatment was prolonged from 12 to 36 hrs.

  15. Explant culture of rat colon: A model system for studying metabolism of chemical carcinogens

    DEFF Research Database (Denmark)

    Autrup, Herman; Stoner, G.D.; Jackson, F.

    1978-01-01

    An explant culture system has been developed for the long-term maintenance of colonic tissue from the rat. Explants of 1 cm2 in size were placed in tissue-culture dishes to which was added 2 ml of CMRL-1066 medium supplemented with glucose, hydrocortisone, beta-retinyl acetate, and either 2.5% bo...

  16. Articular Cartilage Changes in Maturing Athletes

    Science.gov (United States)

    Luria, Ayala; Chu, Constance R.

    2014-01-01

    Context: Articular cartilage has a unique functional architecture capable of providing a lifetime of pain-free joint motion. This tissue, however, undergoes substantial age-related physiologic, mechanical, biochemical, and functional changes that reduce its ability to overcome the effects of mechanical stress and injury. Many factors affect joint function in the maturing athlete—from chondrocyte survival and metabolism to structural composition and genetic/epigenetic factors governing cartilage and synovium. An evaluation of age-related changes for joint homeostasis and risk for osteoarthritis is important to the development of new strategies to rejuvenate aging joints. Objective: This review summarizes the current literature on the biochemical, cellular, and physiologic changes occurring in aging articular cartilage. Data Sources: PubMed (1969-2013) and published books in sports health, cartilage biology, and aging. Study Selection: Keywords included aging, athlete, articular cartilage, epigenetics, and functional performance with age. Study Design: Systematic review. Level of Evidence: Level 3. Data Extraction: To be included, research questions addressed the effect of age-related changes on performance, articular cartilage biology, molecular mechanism, and morphology. Results: The mature athlete faces challenges in maintaining cartilage health and joint function due to age-related changes to articular cartilage biology, morphology, and physiology. These changes include chondrocyte loss and a decline in metabolic response, alterations to matrix and synovial tissue composition, and dysregulation of reparative responses. Conclusion: Although physical decline has been regarded as a normal part of aging, many individuals maintain overall fitness and enjoy targeted improvement to their athletic capacity throughout life. Healthy articular cartilage and joints are needed to maintain athletic performance and general activities. Genetic and potentially reversible

  17. 骨形态发生蛋白与碱性成纤维细胞生长因子联合修复软骨缺损的效果评价%Effects of recombinant human bone morphogenetic protein combined with basic fibroblast growth factor on the repair of articular cartilage defects

    Institute of Scientific and Technical Information of China (English)

    朱国华; 蔡建平; 郭翠玲; 廖家新; 刘勇; 罗洪涛; 许国华; 胡红涛

    2012-01-01

    背景:多种细胞生长因子在骨软骨代谢过程中的协同作用越来越受到重视,但目前复合细胞生长因子修复软骨缺损报道较少,且修复效果尚无定论.目的:探讨骨形态发生蛋白和碱性成纤维细胞生长因子联合应用修复关节软骨缺损的效果.方法:24 只日本大耳白兔建立骨软骨缺损模型后随机等分为4 组,对照组缺损处仅填塞明胶海绵,其他3 组在对照组基础上,缺损处分别注射骨形态发生蛋白和碱性成纤维细胞生长因子、骨形态发生蛋白、碱性成纤维细胞生长因子.结果与结论:大体观察显示联合应用2 种细胞因子后,软骨缺损面基本修复但稍不平整,单独使用其中1 种细胞因子缺损面未完全修复,对照组无明显修复.联合应用2 种细胞因子缺损部位软骨细胞数多于其他3 组(P < 0.05),且Ⅱ型胶原免疫组化染色深于其他组.提示联合应用骨形态发生蛋白和碱性成纤维细胞生长因子可以促进关节软骨损伤的修复,疗效优于单独应用骨形态发生蛋白或碱性成纤维细胞生长因子.%BACKGROUND: The synergy of various cell growth factors attracts more and more attention in the course of cartilage metabolism.However, there are few reports of repairing cartilage defects with combined cell growth factors, and the effect remains unknown atpresent.OBJECTIVE: To study the repairing effect of recombinant human bone morphogenetic protein (rhBMP) combined with basicfibroblast growth factor (bFGF) on articular cartilage defects.METHODS: After the model of articular cartilage defects was made, 24 Japan big-eared white rabbits were randomly divided intofour groupsforintervention: rhBMP combined with bFGF (group A), single rhBMP (group B), single bFGF (group C), the fourthgroup was without injection and just filled with gelatin sponge (group D).RESULTS AND CONCLUSION: In general observation, articular cartilage defects were basically repaired but slightly

  18. [The effect of Solcoseryl on explant cultures of the hippocampus].

    Science.gov (United States)

    Lindner, G; Grosse, G; Goworek, K; Franz, C; Liebezeit, K

    1979-01-01

    Explants of hippocampus from fetal rats were cultivated in Maximow chambers in semisynthetic medium up to 12 days in vitro. The cultures were fixed Bouin, slided 15 micron, coloured with Klüver-Barrera and some morphological parameters were tested. 1. The nerve fiber index increased by influence of 1% Solcoseryl in relation to control cultures, which growed in minimal medium. An essential stimulation was observed by application of placentar serum and embryonal extract into the culture medium. 2. Die decrease of the number of neurons and glial cells per unit of area and a small decrease of the area of neuron nuclei was discussed in relation to the effect of the pharmacon Solcoseryl on O2- consumption. 3. Solcoseryl (firm Solco AG, Base) is an extract of calf blood. It can not substitute other tissue extracts.

  19. Environmental neurotoxins β-N-methylamino-l-alanine (BMAA) and mercury in shark cartilage dietary supplements.

    Science.gov (United States)

    Mondo, Kiyo; Broc Glover, W; Murch, Susan J; Liu, Guangliang; Cai, Yong; Davis, David A; Mash, Deborah C

    2014-08-01

    Shark cartilage products are marketed as dietary supplements with claimed health benefits for animal and human use. Shark fin and cartilage products sold as extracts, dry powders and in capsules are marketed based on traditional Chinese medicine claims that it nourishes the blood, enhances appetite, and energizes multiple internal organs. Shark cartilage contains a mixture of chondroitin and glucosamine, a popular nutritional supplement ingested to improve cartilage function. Sharks are long-lived apex predators, that bioaccumulate environmental marine toxins and methylmercury from dietary exposures. We recently reported detection of the cyanobacterial toxin β-N-methylamino-l-alanine (BMAA) in the fins of seven different species of sharks from South Florida coastal waters. Since BMAA has been linked to degenerative brain diseases, the consumption of shark products may pose a human risk for BMAA exposures. In this report, we tested sixteen commercial shark cartilage supplements for BMAA by high performance liquid chromatography (HPLC-FD) with fluorescence detection and ultra performance liquid chromatography/mass spectrometry/mass spectrometry (UPLC-MS/MS). Total mercury (Hg) levels were measured in the same shark cartilage products by cold vapor atomic fluorescence spectrometry (CVAFS). We report here that BMAA was detected in fifteen out of sixteen products with concentrations ranging from 86 to 265μg/g (dry weight). All of the shark fin products contained low concentrations of Hg. While Hg contamination is a known risk, the results of the present study demonstrate that shark cartilage products also may contain the neurotoxin BMAA. Although the neurotoxic potential of dietary exposure to BMAA is currently unknown, the results demonstrate that shark cartilage products may contain two environmental neurotoxins that have synergistic toxicities.

  20. Plant Regeneration of Sweet Potato via Somatic Embryogenesis from Different Explants

    Institute of Scientific and Technical Information of China (English)

    Ling ZHANG; Hongxuan XU; Baifu QIN; Zhihua LIA0; Min CHEN; Chunxian YANG; Yufan FU; Qitang ZHANG

    2012-01-01

    [Objective] This study aimed to regenerate plants of sweet potato (Ipomoea batatas) cultivar Xushu22 via somatic embryogenesis, using leaf and shoot apex as explants. [Method] The leaf and shoot apex of Xushu 22 were separately cultured on MSB medium and MSD medium. The induced embryogenic calluses were then cultured on MS medium. The regeneration frequency of leaf and shoot apex ex- plants were respectively calculated. [Result] The average frequency of leaf explants developing somatic callus was 95.69% compared to 30.56% in case of shoot apex explants. There were different types of morphogenic structures in the process of so- matic embryo development. Leaf explants gave a high regeneration frequency to 60.61%, while the regeneration frequency of shoot apices was 22%. In addition, no morphological variations were observed in the regeneration plants. [Conclusion] Leaf explant was better than shoot apices in plant regeneration of Xushu22 via somatic embryogenesis.

  1. Anatomical study of nasal cartilage in buffalo (Bubalus bubulus

    Directory of Open Access Journals (Sweden)

    Mahdi Yeganehzad

    2011-07-01

    Full Text Available This study used ten heads of adult buffalo taken from slaughterhouse. After transferring the samples to the anatomy hall, a split was carefully created on skin of muzzle and the skin was slowly separated from muscles and hypodermal connective tissue. Place of connection of cartilages to bone, cartilages to each other and shape of the cartilages were specified. In buffalo, nose apex has two nostrils fixed by bone and cartilage. After identifying and separating the cartilages, it was found that nasal cartilages in buffalo consisted of: 1 septum nasal located between two nostrils and reinforces it from inside. 2 dorso-lateral nasal cartilage constituting dorsal and lateral parts of the nostril. 3 ventro-lateral nasal cartilage constituting ventral and lateral parts of the nostril. 4 lateral accessory cartilage constituting lateral and ventral parts of the nostril. 5 medial accessory nasal cartilage located at Alar fold and connected to ventro-lateral nasal cartilage.

  2. Strategies for Zonal Cartilage Repair using Hydrogels

    NARCIS (Netherlands)

    Klein, Travis J.; Rizzi, Simone C.; Reichert, Johannes C.; Georgi, Nicole; Malda, Jos; Schuurman, Wouter; Crawford, Ross W.; Hutmacher, Dietmar W.

    2009-01-01

    Articular cartilage is a highly hydrated tissue with depth-dependent cellular and matrix properties that provide low-friction load bearing in joints. However, the structure and function are frequently lost and there is insufficient repair response to regenerate high-quality cartilage. Several hydrog

  3. Magnetic Resonance Imaging of Cartilage Repair

    Science.gov (United States)

    Trattnig, Siegfried; Winalski, Carl S.; Marlovits, Stephan; Jurvelin, Jukka S.; Welsch, Goetz H.; Potter, Hollis G.

    2011-01-01

    Articular cartilage lesions are a common pathology of the knee joint, and many patients may benefit from cartilage repair surgeries that offer the chance to avoid the development of osteoarthritis or delay its progression. Cartilage repair surgery, no matter the technique, requires a noninvasive, standardized, and high-quality longitudinal method to assess the structure of the repair tissue. This goal is best fulfilled by magnetic resonance imaging (MRI). The present article provides an overview of the current state of the art of MRI of cartilage repair. In the first 2 sections, preclinical and clinical MRI of cartilage repair tissue are described with a focus on morphological depiction of cartilage and the use of functional (biochemical) MR methodologies for the visualization of the ultrastructure of cartilage repair. In the third section, a short overview is provided on the regulatory issues of the United States Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) regarding MR follow-up studies of patients after cartilage repair surgeries. PMID:26069565

  4. Deformation of articular cartilage during static loading of a knee joint--experimental and finite element analysis.

    Science.gov (United States)

    Halonen, K S; Mononen, M E; Jurvelin, J S; Töyräs, J; Salo, J; Korhonen, R K

    2014-07-18

    Novel conical beam CT-scanners offer high resolution imaging of knee structures with i.a. contrast media, even under weight bearing. With this new technology, we aimed to determine cartilage strains and meniscal movement in a human knee at 0, 1, 5, and 30 min of standing and compare them to the subject-specific 3D finite element (FE) model. The FE model of the volunteer׳s knee, based on the geometry obtained from magnetic resonance images, was created to simulate the creep. The effects of collagen fibril network stiffness, nonfibrillar matrix modulus, permeability and fluid flow boundary conditions on the creep response in cartilage were investigated. In the experiment, 80% of the maximum strain in cartilage developed immediately, after which the cartilage continued to deform slowly until the 30 min time point. Cartilage strains and meniscus movement obtained from the FE model matched adequately with the experimentally measured values. Reducing the fibril network stiffness increased the mean strains substantially, while the creep rate was primarily influenced by an increase in the nonfibrillar matrix modulus. Changing the initial permeability and preventing fluid flow through noncontacting surfaces had a negligible effect on cartilage strains. The present results improve understanding of the mechanisms controlling articular cartilage strains and meniscal movements in a knee joint under physiological static loading. Ultimately a validated model could be used as a noninvasive diagnostic tool to locate cartilage areas at risk for degeneration.

  5. Abnormal Mechanical Loading Induces Cartilage Degeneration by Accelerating Meniscus Hypertrophy and Mineralization After ACL Injuries In Vivo.

    Science.gov (United States)

    Du, Guoqing; Zhan, Hongsheng; Ding, Daofang; Wang, Shaowei; Wei, Xiaochun; Wei, Fangyuan; Zhang, Jianzhong; Bilgen, Bahar; Reginato, Anthony M; Fleming, Braden C; Deng, Jin; Wei, Lei

    2016-03-01

    Although patients with an anterior cruciate ligament (ACL) injury have a high risk of developing posttraumatic osteoarthritis (PTOA), the role of meniscus hypertrophy and mineralization in PTOA after an ACL injury remains unknown. The purpose of this study was to determine if menisci respond to abnormal loading and if an ACL injury results in meniscus hypertrophy and calcification. The hypotheses were that (1) abnormal mechanical loading after an ACL injury induces meniscus hypertrophy and mineralization, which correlates to articular cartilage damage in vivo, and (2) abnormal mechanical loading on bovine meniscus explants induces the overexpression of hypertrophic and mineralization markers in vitro. Controlled laboratory study. In vivo guinea pig study (hypothesis 1): Three-month-old male Hartley guinea pigs (n = 9) underwent ACL transection (ACLT) on the right knee; the left knee served as the control. Calcification in the menisci was evaluated by calcein labeling 1 and 5 days before knee harvesting at 5.5 months. Cartilage and meniscus damage and mineralization were quantified by the Osteoarthritis Research Society International score and meniscus grade, respectively. Indian hedgehog (Ihh), matrix metalloproteinase-13 (MMP-13), collagen type X (Col X), progressive ankylosis homolog (ANKH), ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1), alkaline phosphatase (ALP), inorganic pyrophosphate (PPi), and inorganic phosphate (Pi) concentrations were evaluated by immunohistochemistry and enzyme-linked immunosorbent assay. In vitro bovine meniscus explant study (hypothesis 2): Bovine meniscus explants were subjected to 25% strain at 0.3 Hz for 1, 2, and 3 hours. Cell viability was determined using live/dead staining. The levels of mRNA expression and protein levels were measured using real-time quantitative reverse transcription polymerase chain reaction and Western blot after 24, 48, and 72 hours in culture. The conditioned medium was collected for sulfated

  6. Elemental and structural studies at the bone-cartilage interface

    Energy Technology Data Exchange (ETDEWEB)

    Kaabar, W., E-mail: w.kaabar@surrey.ac.uk [Department of Physics, University of Surrey, Guildford GU2 7XH (United Kingdom); Daar, E. [Department of Physics, University of Surrey, Guildford GU2 7XH (United Kingdom); Bunk, O. [Swiss Light Source, Paul Scherrer Institute, 5232 Villigen (Switzerland); Farquharson, M.J. [Department of Medical Physics and Applied Radiation Sciences, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4K1 (Canada); Laklouk, A. [Al-Fateh University, Tripoli (Libya); Bailey, M.; Jeynes, C. [Surrey Ion Beam Centre, University of Surrey, Guildford GU2 7XH (United Kingdom); Gundogdu, O. [Umuttepe Campus, University of Kocaeli, 41380 Kocaeli (Turkey); Bradley, D.A. [Department of Physics, University of Surrey, Guildford GU2 7XH (United Kingdom)

    2011-10-01

    Micro-Proton Induced X-ray Emission ({mu}-PIXE) and Proton Induced Gamma-ray Emission (PIGE) techniques were employed in the investigation of trace and essential elements distribution in normal and diseased human femoral head sections affected by osteoarthritis (OA). PIGE was exploited in the determination of elements of low atomic number z<15 such as Na and F whereas elements with z>15 viz Ca, Z, P and S were determined by PIXE. Accumulations of key elements in the bone and cartilage sections were observed, significant S and Na concentrations being found in the cartilage region particularly in normal tissues. Zn showed enhanced concentrations at the bone-cartilage interface. At a synchrotron facility, small angle X-ray scattering (SAXS) was utilized on a decalcified human femoral head section affected by OA, direct measurements being made of spatial alterations of collagen fibres. The SAXS results showed a slight decrease in the axial periodicity between normal collagen type I and that in diseased tissue in various sites, in contrast with the findings of others.

  7. Elemental and structural studies at the bone-cartilage interface

    Science.gov (United States)

    Bradley, D. A.; Kaabar, W.; Gundogdu, O.

    2012-02-01

    The techniques μProton-Induced X-and γ-ray Emission, μ-PIXE and μ-PIGE, were used to investigate trace and essential element distributions in sections of normal and osteoarthritic (OA) human femoral head. μ-PIGE yielded 2-D mappings of Na and F while Ca, Z, P and S were mapped by μ-PIXE. The concentration of chondroitin sulphate supporting functionality in healthy cartilage is significantly reduced in OA samples. Localised Zn points to osteoblastic/osteoclastic activity at the bone-cartilage interface. Small-angle X-ray scattering applied to decalcified OA-affected tissue showed spatial alterations of collagen fibres of decreased axial periodicity compared to normal collagen type I.

  8. 人脂肪基质干细胞复合藻酸钙体外构建工程软骨的实验研究%Construction of tissue engineered cartilage using human adipose derived stem cells and calcium alginate in vitro

    Institute of Scientific and Technical Information of China (English)

    赵大庆; 马钰; 李青; 韩宇; 邱建华

    2011-01-01

    Objective To study the feasibility of constructing tissue engineered canilage using human adipose derived stem cells (hADSC) and calcium alginate in vitro. Methods Human adult adipose tissues were obtained by liposuction procedures under local anesthesia, and then digested to isolate the hADSC. After cultured in the cartilage induction medium, the final concentration was adjusted to 5 × 106/mL. The cells were then mixed with 200 mmol/L calcium alginate gels to creale tissue engineered cartilage in a centrifuge tube. After 8 weeks of culture in citro, hematoxylin-eosin (HE) and Masson trichrome staining were performed to observe the construction of tissue engineered cartilage. Resujts Tissue engineered cartilaSe was constructed in vitro using hADSC in combination with calcium alginate gels after cultured in cartilage induction medium. The tissue engineered cartilage contained collagen and mucopolysaccharide. Conclusion It is demonstrated that hADSC could be used as a seed cell in cartilage tissue engineering and might provide a new way of repairing cartilage defect for clinical rehabilitation.%目的 观察人脂肪基质干细胞(hADSC)复合藻酸钙凝胶体外构建工程软骨的可行性.方法 取临床整形外科超声乳化的成人脂肪组织溶液,经消化、分离、培养得到hADSC,经成软骨诱导培养后与藻酸钠凝胶复合,使细胞终浓度为5×10/mL,然后滴入浓度200mmol/L CaCl溶液中,固化15min形成藻酸钙凝珠,将其在离心管中悬浮培养8周,行大体、苏木精-伊红(HE)染色、Masson's三色染色等组织学检测,观察组织工程软骨形成情况.结果 hADSC经成软骨诱导培养后复合藻酸钙凝胶可于体外较好地构建出组织工程软骨,形成软骨中有胶原及黏多糖等软骨特有基质成分.结论 hADSC可以作为软骨组织工程较理想的种子细胞,为软骨缺损的临床修复治疗提供了新的治疗方法.

  9. International Cartilage Repair Society (ICRS) Recommended Guidelines for Histological Endpoints for Cartilage Repair Studies in Animal Models and Clinical Trials.

    Science.gov (United States)

    Hoemann, Caroline; Kandel, Rita; Roberts, Sally; Saris, Daniel B F; Creemers, Laura; Mainil-Varlet, Pierre; Méthot, Stephane; Hollander, Anthony P; Buschmann, Michael D

    2011-04-01

    Cartilage repair strategies aim to resurface a lesion with osteochondral tissue resembling native cartilage, but a variety of repair tissues are usually observed. Histology is an important structural outcome that could serve as an interim measure of efficacy in randomized controlled clinical studies. The purpose of this article is to propose guidelines for standardized histoprocessing and unbiased evaluation of animal tissues and human biopsies. Methods were compiled from a literature review, and illustrative data were added. In animal models, treatments are usually administered to acute defects created in healthy tissues, and the entire joint can be analyzed at multiple postoperative time points. In human clinical therapy, treatments are applied to developed lesions, and biopsies are obtained, usually from a subset of patients, at a specific time point. In striving to standardize evaluation of structural endpoints in cartilage repair studies, 5 variables should be controlled: 1) location of biopsy/sample section, 2) timing of biopsy/sample recovery, 3) histoprocessing, 4) staining, and 5) blinded evaluation with a proper control group. Histological scores, quantitative histomorphometry of repair tissue thickness, percentage of tissue staining for collagens and glycosaminoglycan, polarized light microscopy for collagen fibril organization, and subchondral bone integration/structure are all relevant outcome measures that can be collected and used to assess the efficacy of novel therapeutics. Standardized histology methods could improve statistical analyses, help interpret and validate noninvasive imaging outcomes, and permit cross-comparison between studies. Currently, there are no suitable substitutes for histology in evaluating repair tissue quality and cartilaginous character.

  10. Cold Atmospheric Plasma Modified Electrospun Scaffolds with Embedded Microspheres for Improved Cartilage Regeneration.

    Directory of Open Access Journals (Sweden)

    Wei Zhu

    Full Text Available Articular cartilage is prone to degeneration and possesses extremely poor self-healing capacity due to inherent low cell density and the absence of a vasculature network. Tissue engineered cartilage scaffolds show promise for cartilage repair. However, there still remains a lack of ideal biomimetic tissue scaffolds which effectively stimulate cartilage regeneration with appropriate functional properties. Therefore, the objective of this study is to develop a novel biomimetic and bioactive electrospun cartilage substitute by integrating cold atmospheric plasma (CAP treatment with sustained growth factor delivery microspheres. Specifically, CAP was applied to a poly(ε-caprolactone electrospun scaffold with homogeneously distributed bioactive factors (transforming growth factor-β1 and bovine serum albumin loaded poly(lactic-co-glycolic acid microspheres. We have shown that CAP treatment renders electrospun scaffolds more hydrophilic thus facilitating vitronectin adsorption. More importantly, our results demonstrate, for the first time, CAP and microspheres can synergistically enhance stem cell growth as well as improve chondrogenic differentiation of human marrow-derived mesenchymal stem cells (such as increased glycosaminoglycan, type II collagen, and total collagen production. Furthermore, CAP can substantially enhance 3D cell infiltration (over two-fold increase in infiltration depth after 1 day of culture in the scaffolds. By integrating CAP, sustained bioactive factor loaded microspheres, and electrospinning, we have fabricated a promising bioactive scaffold for cartilage regeneration.

  11. Osteoarthritis-derived chondrocytes are a potential source of multipotent progenitor cells for cartilage tissue engineering.

    Science.gov (United States)

    Oda, Tomoyuki; Sakai, Tadahiro; Hiraiwa, Hideki; Hamada, Takashi; Ono, Yohei; Nakashima, Motoshige; Ishizuka, Shinya; Matsukawa, Tetsuya; Yamashita, Satoshi; Tsuchiya, Saho; Ishiguro, Naoki

    2016-10-21

    The natural healing capacity of damaged articular cartilage is poor, rendering joint surface injuries a prime target for regenerative medicine. While autologous chondrocyte or mesenchymal stem cell (MSC) implantation can be applied to repair cartilage defects in young patients, no appropriate long-lasting treatment alternative is available for elderly patients with osteoarthritis (OA). Multipotent progenitor cells are reported to present in adult human articular cartilage, with a preponderance in OA cartilage. These facts led us to hypothesize the possible use of osteoarthritis-derived chondrocytes as a cell source for cartilage tissue engineering. We therefore analyzed chondrocyte- and stem cell-related markers, cell growth rate, and multipotency in OA chondrocytes (OACs) and bone marrow-derived MSCs, along with normal articular chondrocytes (ACs) as a control. OACs demonstrated similar phenotype and proliferation rate to MSCs. Furthermore, OACs exhibited multilineage differentiation ability with a greater chondrogenic differentiation ability than MSCs, which was equivalent to ACs. We conclude that chondrogenic capacity is not significantly affected by OA, and OACs could be a potential source of multipotent progenitor cells for cartilage tissue engineering. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Growth plate cartilage shows different strain patterns in response to static versus dynamic mechanical modulation.

    Science.gov (United States)

    Kaviani, Rosa; Londono, Irene; Parent, Stefan; Moldovan, Florina; Villemure, Isabelle

    2016-08-01

    Longitudinal growth of long bones and vertebrae occurs in growth plate cartilage. This process is partly regulated by mechanical forces, which are one of the underlying reasons for progression of growth deformities such as idiopathic adolescent scoliosis and early-onset scoliosis. This concept of mechanical modulation of bone growth is also exploited in the development of fusionless treatments of these deformities. However, the optimal loading condition for the mechanical modulation of growth plate remains to be identified. The objective of this study was to evaluate the effects of in vitro static versus dynamic modulation and of dynamic loading parameters, such as frequency and amplitude, on the mechanical responses and histomorphology of growth plate explants. Growth plate explants from distal ulnae of 4-week-old swines were extracted and randomly distributed among six experimental groups: baseline ([Formula: see text]), control ([Formula: see text]), static ([Formula: see text]) and dynamic ([Formula: see text]). For static and dynamic groups, mechanical modulation was performed in vitro using an Indexed CartiGen bioreactor. A stress relaxation test combined with confocal microscopy and digital image correlation was used to characterize the mechanical responses of each explant in terms of peak stress, equilibrium stress, equilibrium modulus of elasticity and strain pattern. Histomorphometrical measurements were performed on toluidine blue tissue sections using a semi-automatic custom-developed MATLAB toolbox. Results suggest that compared to dynamic modulation, static modulation changes the strain pattern of the tissue and thus is more detrimental for tissue biomechanics, while the histomorphological parameters are not affected by mechanical modulation. Also, under dynamic modulation, changing the frequency or amplitude does not affect the biomechanical response of the tissue. Results of this study will be useful in finding optimal and non-damaging parameters

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

    Science.gov (United States)

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

    2014-05-01

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

  14. Knee cartilage extraction and bone-cartilage interface analysis from 3D MRI data sets

    Science.gov (United States)

    Tamez-Pena, Jose G.; Barbu-McInnis, Monica; Totterman, Saara

    2004-05-01

    This works presents a robust methodology for the analysis of the knee joint cartilage and the knee bone-cartilage interface from fused MRI sets. The proposed approach starts by fusing a set of two 3D MR images the knee. Although the proposed method is not pulse sequence dependent, the first sequence should be programmed to achieve good contrast between bone and cartilage. The recommended second pulse sequence is one that maximizes the contrast between cartilage and surrounding soft tissues. Once both pulse sequences are fused, the proposed bone-cartilage analysis is done in four major steps. First, an unsupervised segmentation algorithm is used to extract the femur, the tibia, and the patella. Second, a knowledge based feature extraction algorithm is used to extract the femoral, tibia and patellar cartilages. Third, a trained user corrects cartilage miss-classifications done by the automated extracted cartilage. Finally, the final segmentation is the revisited using an unsupervised MAP voxel relaxation algorithm. This final segmentation has the property that includes the extracted bone tissue as well as all the cartilage tissue. This is an improvement over previous approaches where only the cartilage was segmented. Furthermore, this approach yields very reproducible segmentation results in a set of scan-rescan experiments. When these segmentations were coupled with a partial volume compensated surface extraction algorithm the volume, area, thickness measurements shows precisions around 2.6%

  15. Hyaline cartilage cells outperform mandibular condylar cartilage cells in a TMJ fibrocartilage tissue engineering application.

    Science.gov (United States)

    Wang, L; Lazebnik, M; Detamore, M S

    2009-03-01

    To compare temporomandibular joint (TMJ) condylar cartilage cells in vitro to hyaline cartilage cells cultured in a three-dimensional (3D) environment for tissue engineering of mandibular condylar cartilage. Mandibular condylar cartilage and hyaline cartilage cells were harvested from pigs and cultured for 6 weeks in polyglycolic acid (PGA) scaffolds. Both types of cells were treated with glucosamine sulfate (0.4 mM), insulin-like growth factor-I (IGF-I) (100 ng/ml) and their combination. At weeks 0 and 6, cell number, glycosaminoglycan (GAG) and collagen content were determined, types I and II collagen were visualized by immunohistochemistry and GAGs were visualized by histology. Hyaline cartilage cells produced from half an order to a full order of magnitude more GAGs and collagen than mandibular condylar cartilage cells in 3D culture. IGF-I was a highly effective signal for biosynthesis with hyaline cartilage cells, while glucosamine sulfate decreased cell proliferation and biosynthesis with both types of cells. In vitro culture of TMJ condylar cartilage cells produced a fibrous tissue with predominantly type I collagen, while hyaline cartilage cells formed a fibrocartilage-like tissue with types I and II collagen. The combination of IGF and glucosamine had a synergistic effect on maintaining the phenotype of TMJ condylar cells to generate both types I and II collagen. Given the superior biosynthetic activity by hyaline cartilage cells and the practical surgical limitations of harvesting cells from the TMJ of a patient requiring TMJ reconstruction, cartilage cells from elsewhere in the body may be a potentially better alternative to cells harvested from the TMJ for TMJ tissue engineering. This finding may also apply to other fibrocartilages such as the intervertebral disc and knee meniscus in applications where a mature cartilage cell source is desired.

  16. An ex vivo porcine nasal mucosa explants model to study MRSA colonization.

    Directory of Open Access Journals (Sweden)

    Pawel Tulinski

    Full Text Available Staphylococcus aureus is an opportunistic pathogen able to colonize the upper respiratory tract and skin surfaces in mammals. Methicillin-resistant S. aureus ST398 is prevalent in pigs in Europe and North America. However, the mechanism of successful pig colonization by MRSA ST398 is poorly understood. To study MRSA colonization in pigs, an ex vivo model consisting of porcine nasal mucosa explants cultured at an air-liquid interface was evaluated. In cultured mucosa explants from the surfaces of the ventral turbinates and septum of the pig nose no changes in cell morphology and viability were observed up to 72 h. MRSA colonization on the explants was evaluated followed for three MRSA ST398 isolates for 180 minutes. The explants were incubated with 3×10(8 CFU/ml in PBS for 2 h to allow bacteria to adhere to the explants surface. Next the explants were washed and in the first 30 minutes post adhering time, a decline in the number of CFU was observed for all MRSA. Subsequently, the isolates showed either: bacterial growth, no growth, or a further reduction in bacterial numbers. The MRSA were either localized as clusters between the cilia or as single bacteria on the cilia surface. No morphological changes in the epithelium layer were observed during the incubation with MRSA. We conclude that porcine nasal mucosa explants are a valuable ex vivo model to unravel the interaction of MRSA with nasal tissue.

  17. Effect of vasoactive intestinal peptide on pulmonary surfactants phospholipid synthesis in lung explants

    Institute of Scientific and Technical Information of China (English)

    Lian LI; Zi-qiang LUO; Fu-wen ZHOU; Dan-dan FENG; Cha-xiang GUAN; Chang-qing ZHANG; Xiu-hong SUN

    2004-01-01

    AIM: To investigate the effect of vasoactive intestinal peptide (VIP) on pulmonary surfactants (PS) phospholipid synthesis in cultured lung explants. METHODS: Lung explants were cultured with serum-free medium, [methyl-3H]choline incorporation, total phospholipid, phosphatidylcholine, activity of choline-phosphate cytidylyltransferase (CCT) and CCTα mRNA level in lung explants were determined. RESULTS: (1) VIP (10-10-10-7 mol/L) for 16 h promoted [methyl-3H]choline incorporation in dose dependence and VIP (10-8 mol/L) for 2 h-16 h promoted [methylz3H]choline incorporation in time dependence. (2) VIP (10-8 mol/L) enhanced the contents of total phospholipidsand phosphatidylcholine in lung explants. (3) VIP (10-10-10-7 mol/L) elevated microsomal CCT activity of lung explants in dose dependence. (4) VIP (10-8 mol/L) increased expression of CCTα mRNA in lung explants and alveolar type Ⅱ cells (ATII). (5) [D-P-Cl-Phe(6)-Leu(17)]-VIP (10-6 mol/L), a VIP receptors antagonist, abolished the increase of [3H]choline incorporation, microsomal CCT activity and CCTα mRNA level induced by VIP (10-8 mol/L) in lung explants. CONCLUSION: VIP could enhance synthesis of phosphatidylcholine, the major component of pulmonary surfactants by enhancing microsomal CCT activity and CCTα mRNA level via VIP receptormediated pathway.

  18. Cartilage T2 assessment: differentiation of normal hyaline cartilage and reparative tissue after arthroscopic cartilage repair in equine subjects.

    Science.gov (United States)

    White, Lawrence M; Sussman, Marshall S; Hurtig, Mark; Probyn, Linda; Tomlinson, George; Kandel, Rita

    2006-11-01

    To prospectively assess T2 mapping characteristics of normal articular cartilage and of cartilage at sites of arthroscopic repair, including comparison with histologic results and collagen organization assessed at polarized light microscopy (PLM). Study protocol was compliant with the Canadian Council on Animal Care Guidelines and approved by the institutional animal care committee. Arthroscopic osteochondral autograft transplantation (OAT) and microfracture arthroplasty (MFx) were performed in knees of 10 equine subjects (seven female, three male; age range, 3-5 years). A site of arthroscopically normal cartilage was documented in each joint as a control site. Joints were harvested at 12 (n = 5) and 24 (n = 5) weeks postoperatively and were imaged at 1.5-T magnetic resonance (MR) with a 10-echo sagittal fast spin-echo acquisition. T2 maps of each site (21 OAT harvest, 10 MFx, 12 OAT plug, and 10 control sites) were calculated with linear least-squares curve fitting. Cartilage T2 maps were qualitatively graded as "organized" (normal transition of low-to-high T2 signal from deep to superficial cartilage zones) or "disorganized." Quantitative mean T2 values were calculated for deep, middle, and superficial cartilage at each location. Results were compared with histologic and PLM assessments by using kappa analysis. T2 maps were qualitatively graded as organized at 20 of 53 sites and as disorganized at 33 sites. Perfect agreement was seen between organized T2 and histologic findings of hyaline cartilage and between disorganized T2 and histologic findings of fibrous reparative tissue (kappa = 1.0). Strong agreement was seen between organized T2 and normal PLM findings and between disorganized T2 and abnormal PLM findings (kappa = .92). Quantitative assessment of the deep, middle, and superficial cartilage, respectively, showed mean T2 values of 53.3, 58.6, and 54.9 msec at reparative fibrous tissue sites and 40.7, 53.6, and 61.6 msec at hyaline cartilage sites. A

  19. Culturing Schwann Cells from Neonatal Rats by Improved Enzyme Digestion Combined with Explants-culture Method.

    Science.gov (United States)

    Liu, Di; Liang, Xiao-Chun; Zhang, Hong

    2016-08-01

    Objective To develop an improved method for culturing Schwann cells(SCs) by using both enzyme digestion and explants-culture approaches and compared with traditional explants-culture method and general hemi-explants-culture method. Methods Bilaterally sciatic nerves and brachial plexus nerves were dissected from 3 to 5-day-old neonatal SD rats and explants-culture method,general hemi-explants-culture method,and improved enzyme digestion combined with explants-culture method were adopted to culture SCs,respectively. SCs were digested and passaged after 7 days in culture and counted under the microscope. The purity of SCs was identified by S-100 immunofluorescence staining. Results The SCs of improved method group grew fastest and the total number of cells obtained was(1.85±0.13)×10(6);the SCs of the hemi-explants-culture method group grew slower than the improved method group and the total number of cells obtained was (1.10±0.10)×10(6);the SCs of the explants-culture method group grew slowest and the total number of cells obtained was (0.77±0.03)×10(6).The total number of cells obtained showed significant difference among the three groups(Pculture method group,and (74.50±4.23)% in the explants-culture method group(Pculture method can obtain sufficient amount of high-purity SCs in a short time and thus may be applied in further research on peripheral nerve regeneration.

  20. Genome-wide transcriptome and expression profile analysis of Phalaenopsis during explant browning.

    Directory of Open Access Journals (Sweden)

    Chuanjun Xu

    Full Text Available Explant browning presents a major problem for in vitro culture, and can lead to the death of the explant and failure of regeneration. Considerable work has examined the physiological mechanisms underlying Phalaenopsis leaf explant browning, but the molecular mechanisms of browning remain elusive. In this study, we used whole genome RNA sequencing to examine Phalaenopsis leaf explant browning at genome-wide level.We first used Illumina high-throughput technology to sequence the transcriptome of Phalaenopsis and then performed de novo transcriptome assembly. We assembled 79,434,350 clean reads into 31,708 isogenes and generated 26,565 annotated unigenes. We assigned Gene Ontology (GO terms, Kyoto Encyclopedia of Genes and Genomes (KEGG annotations, and potential Pfam domains to each transcript. Using the transcriptome data as a reference, we next analyzed the differential gene expression of explants cultured for 0, 3, and 6 d, respectively. We then identified differentially expressed genes (DEGs before and after Phalaenopsis explant browning. We also performed GO, KEGG functional enrichment and Pfam analysis of all DEGs. Finally, we selected 11 genes for quantitative real-time PCR (qPCR analysis to confirm the expression profile analysis.Here, we report the first comprehensive analysis of transcriptome and expression profiles during Phalaenopsis explant browning. Our results suggest that Phalaenopsis explant browning may be due in part to gene expression changes that affect the secondary metabolism, such as: phenylpropanoid pathway and flavonoid biosynthesis. Genes involved in photosynthesis and ATPase activity have been found to be changed at transcription level; these changes may perturb energy metabolism and thus lead to the decay of plant cells and tissues. This study provides comprehensive gene expression data for Phalaenopsis browning. Our data constitute an important resource for further functional studies to prevent explant browning.

  1. Genome-wide transcriptome and expression profile analysis of Phalaenopsis during explant browning.

    Science.gov (United States)

    Xu, Chuanjun; Zeng, Biyu; Huang, Junmei; Huang, Wen; Liu, Yumei

    2015-01-01

    Explant browning presents a major problem for in vitro culture, and can lead to the death of the explant and failure of regeneration. Considerable work has examined the physiological mechanisms underlying Phalaenopsis leaf explant browning, but the molecular mechanisms of browning remain elusive. In this study, we used whole genome RNA sequencing to examine Phalaenopsis leaf explant browning at genome-wide level. We first used Illumina high-throughput technology to sequence the transcriptome of Phalaenopsis and then performed de novo transcriptome assembly. We assembled 79,434,350 clean reads into 31,708 isogenes and generated 26,565 annotated unigenes. We assigned Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations, and potential Pfam domains to each transcript. Using the transcriptome data as a reference, we next analyzed the differential gene expression of explants cultured for 0, 3, and 6 d, respectively. We then identified differentially expressed genes (DEGs) before and after Phalaenopsis explant browning. We also performed GO, KEGG functional enrichment and Pfam analysis of all DEGs. Finally, we selected 11 genes for quantitative real-time PCR (qPCR) analysis to confirm the expression profile analysis. Here, we report the first comprehensive analysis of transcriptome and expression profiles during Phalaenopsis explant browning. Our results suggest that Phalaenopsis explant browning may be due in part to gene expression changes that affect the secondary metabolism, such as: phenylpropanoid pathway and flavonoid biosynthesis. Genes involved in photosynthesis and ATPase activity have been found to be changed at transcription level; these changes may perturb energy metabolism and thus lead to the decay of plant cells and tissues. This study provides comprehensive gene expression data for Phalaenopsis browning. Our data constitute an important resource for further functional studies to prevent explant browning.

  2. A cartilage-inspired lubrication system.

    Science.gov (United States)

    Greene, George W; Olszewska, Anna; Osterberg, Monika; Zhu, Haijin; Horn, Roger

    2014-01-14

    Articular cartilage is an example of a highly efficacious water-based, natural lubrication system that is optimized to provide low friction and wear protection at both low and high loads and sliding velocities. One of the secrets of cartilage's superior tribology comes from a unique, multimodal lubrication strategy consisting of both a fluid pressurization mediated lubrication mechanism and a boundary lubrication mechanism supported by surface bound macromolecules. Using a reconstituted network of highly interconnected cellulose fibers and simple modification through the immobilization of polyelectrolytes, we have recreated many of the mechanical and chemical properties of cartilage and the cartilage lubrication system to produce a purely synthetic material system that exhibits some of the same lubrication mechanisms, time dependent friction response, and high wear resistance as natural cartilage tissue. Friction and wear studies demonstrate how the properties of the cellulose fiber network can be used to control and optimize the lubrication and wear resistance of the material surfaces and highlight what key features of cartilage should be duplicated in order to produce a cartilage-mimetic lubrication system.

  3. Regulatory Challenges for Cartilage Repair Technologies.

    Science.gov (United States)

    McGowan, Kevin B; Stiegman, Glenn

    2013-01-01

    In the United States, few Food and Drug Administration (FDA)-approved options exist for the treatment of focal cartilage and osteochondral lesions. Developers of products for cartilage repair face many challenges to obtain marketing approval from the FDA. The objective of this review is to discuss the necessary steps for FDA application and approval for a new cartilage repair product. FDA Guidance Documents, FDA Panel Meetings, scientific organization recommendations, and clinicaltrials.gov were reviewed to demonstrate the current thinking of FDA and the scientific community on the regulatory process for cartilage repair therapies. Cartilage repair therapies can receive market approval from FDA as medical devices, drugs, or biologics, and the specific classification of product can affect the nonclinical, clinical, and regulatory strategy to bring the product to market. Recent FDA guidance gives an outline of the required elements to bring a cartilage repair product to market, although these standards are often very general. As a result, companies have to carefully craft their study patient population, comparator group, and clinical endpoint to best