W. Koevoet

Intrinsic differentiation potential of adolescent human tendon tissue: an in-vitro cell differentiation study

BackgroundTendinosis lesions show an increase of glycosaminoglycan amount, calcifications, and lipid accumulation. Therefore, altered cellular differentiation might play a role in the etiology of tendinosis. This study investigates whether adolescent human tendon tissue contains a population of cells with intrinsic differentiation potential.MethodsCells derived from adolescent non-degenerative hamstring tendons were characterized by immunohistochemistry and FACS-analysis. Cells were cultured for 21 days in osteogenic, adipogenic, and chondrogenic medium and phenotypical evaluation was carried out by immunohistochemical and qPCR analysis. The results were compared with the results of similar experiments on adult bone marrow-derived stromal cells (BMSCs).ResultsTendon-derived cells stained D7-FIB (fibroblast-marker) positive, but α-SMA (marker for smooth muscle cells and pericytes) negative. Tendon-derived cells were 99% negative for CD34 (endothelial cell marker), and 73% positive for CD105 (mesenchymal progenitor-cell marker). In adipogenic medium, intracellular lipid vacuoles were visible and tendon-derived fibroblasts showed upregulation of adipogenic markers FABP4 (fatty-acid binding protein 4) and PPARG (peroxisome proliferative activated receptor γ). In chondrogenic medium, some cells stained positive for collagen 2 and tendon-derived fibroblasts showed upregulation of collagen 2 and collagen 10. In osteogenic medium Von Kossa staining showed calcium deposition although osteogenic markers remained unaltered. Tendon-derived cells and BMCSs behaved largely comparable, although some distinct differences were present between the two cell populations.ConclusionThis study suggests that our population of explanted human tendon cells has an intrinsic differentiation potential. These results support the hypothesis that there might be a role for altered tendon-cell differentiation in the pathophysiology of tendinosis.

Effects of transforming growth factor‐β subtypes on in vitro cartilage production and mineralization of human bone marrow stromal‐derived mesenchymal stem cells

Human bone marrow stromal‐derived mesenchymal stem cells (hBMSCs) will differentiate into chondrocytes in response to defined chondrogenic medium containing transforming growth factor‐β (TGFβ). Results in the literature suggest that the three mammalian subtypes of TGFβ (TGFβ1, TGFβ2 and TGFβ3) provoke certain subtype‐specific activities. Therefore, the aim of our study was to investigate whether the TGFβ subtypes affect chondrogenic differentiation of in vitro cultured hBMSCs differently. HBMSC pellets were cultured for 5 weeks in chondrogenic media containing either 2.5, 10 or 25 ng/ml of TGFβ1, TGFβ2 or TGFβ3. All TGFβ subtypes showed a comparable dose‐response curve, with significantly less cartilage when 2.5 ng/ml was used and no differences between 10 and 25 ng/ml. Four donors with variable chondrogenic capacity were used to evaluate the effect of 10 ng/ml of either TGFβ subtype on cartilage formation. No significant TGFβ subtype‐dependent differences were observed in the total amount of collagen or glycosaminoglycans. Cells from a donor with low chondrogenic capacity performed equally badly with all TGFβ subtypes, while a good donor overall performed well. After addition of β‐glycerophosphate during the last 2 weeks of culture, the expression of hypertrophy markers was analysed and mineralization was demonstrated by alkaline phosphatase activity and alizarin red staining. No significant TGFβ subtype‐dependent differences were observed in expression collagen type X or VEGF secretion. Nevertheless, pellets cultured with TGFβ1 had significantly less mineralization than pellets cultured with TGFβ3. In conclusion, this study suggests that TGFβ subtypes do affect terminal differentiation of in vitro cultured hBMSCs differently. Copyright

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

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 chondrocytes in monolayer proliferated less and more slowly (two passages took 26.7 ± 2.1 days and were reached in 4.37 ± 0.30 population doublings) than nasoseptal chondrocytes (19.3 ± 2.5 days; 5.45 ± 0.20 population doublings). However, auricular chondrocytes produced larger pellets with more cartilage-like matrix than nasoseptal chondrocytes (2.2 ± 0.71 vs. 1.7 ± 0.13 mm in diameter after 35 days of culture). Although the matrix formed by auricular and nasoseptal chondrocytes contained collagen X, it did not mineralize in an in vitro model or after in vivo subcutaneous implantation. A DNA microarray study on expanded auricular and nasoseptal chondrocytes from the same donors revealed 1,090 differentially expressed genes. No difference was observed in the expression of known markers of chondrogenic capacity (e.g., collagen II, FGFR3, BMP2, and ALK1). The most striking differences were that the auricular chondrocytes had a higher expression of anabolic growth factors BMP5 and IGF1, while matrix-degrading enzymes MMP13 and ADAMTS5 were higher expressed in nasoseptal chondrocytes. This might offer a possible explanation for the observed higher matrix production by auricular chondrocytes. Moreover, chondrocytes isolated from auricular or nasoseptal cartilage had specific gene expression profiles even after expansion. These differently expressed genes were not restricted to known characterization of donor site subtype (e.g., elastic), but were also related to developmental processes.

Proteoglycan production is required in initial stages of new cartilage matrix formation but inhibits integrative cartilage repair

The optimal stimulus to repair or regenerate cartilage is not known. We therefore modulated collagen deposition, collagen crosslinking and GAG deposition simultaneously during cartilage matrix production and integrative repair, creating more insight into their role in cartilage repair processes. Insulin‐like growth factor 1 (IGF‐1; increases proteoglycan and collagen synthesis), β‐aminopropionitrile (BAPN; a reversible inhibitor of collagen crosslinking) and para‐nitrophenyl‐β‐D‐xyloside (PNPX; interferes with proteoglycan production) were used. Bovine articular chondrocytes were cultured in alginate beads for 3 weeks with or without IGF‐1, BAPN or PNPX alone and in all possible combinations, followed by 3 weeks in control medium. DNA content, GAG and collagen deposition and collagen crosslinks were determined. Cartilage constructs were cultured under the same conditions and histologically analysed for integration of two opposing cartilage matrices. In alginate cultures, inhibition of collagen crosslinking with BAPN, in combination with promotion of matrix synthesis using IGF1, was most beneficial for matrix deposition. Addition of PNPX was always detrimental for matrix deposition. For integration of opposing cartilage constructs, the combination of BAPN, IGF1 and temporary prevention of proteoglycan formation with PNPX was most beneficial. When a new matrix is produced, proteoglycans are important to retain collagen in the matrix. When two already formed cartilage matrices have to integrate, a temporary absence of proteoglycans and temporary inhibition of collagen crosslinking might be more beneficial in combination with stimulation of collagen production, e.g. by IGF1. Therefore, the choice of soluble factors to promote cartilage regeneration depends on the type of therapy that will be used. Copyright

Elevated Levels of Cartilage Oligomeric Matrix Protein during In Vitro Cartilage Matrix Generation Decrease Collagen Fibril Diameter

Cartilage oligomeric matrix protein (COMP) is a protein present in the cartilage matrix and is expressed more abundantly in osteoarthritis cartilage than in healthy cartilage. The present study was designed to investigate the effect of transforming growth factor β (TGFβ) on COMP deposition and the influence of COMP on collagen biochemistry in a long-term 3-dimensional culture. Bovine chondrocytes in alginate beads were cultured with or without 25 ng/mL TGFβ2 for 21 or 35 days. COMP was overexpressed in bovine chondrocytes using lentiviral transfection. COMP gene expression, COMP protein production, collagen and proteoglycan deposition, and collagen fibril thickness were determined. Addition of TGFβ2 resulted in more COMP mRNA and protein than the control condition without growth factors. Lentiviral transduction with COMP resulted in elevated gene expression of COMP and increased COMP levels in the alginate bead and culture medium compared to untransfected cells. Overexpression of COMP did not affect the deposition of collagen, collagen cross-linking, proteoglycan deposition, or the mechanical properties. Stimulating COMP production by either TGFβ2 or lentivirus resulted in collagen fibrils with a smaller diameter. Taken together, COMP deposition can be modulated in cartilage matrix production by the addition of growth factors or by overexpression of COMP. Inducing COMP protein expression resulted in collagen fibrils with a smaller diameter. Because it has been demonstrated that the collagen fibril diameter is associated with mechanical functioning of the matrix, modulating COMP levels should be taken into account in cartilage regeneration strategies.

221 COLLAGEN TYPE IX AND CARTILAGE OLIGOMERIC MATRIX PROTEIN INFLUENCE CARTILAGE MATRIX ASSEMBLY

concentrations of human recombinant Il-1b (5 ng/ml) and TNFa (20 or 40 ng/ml) and simultaneously with etanercept at 100mg/ml for 24 hours; gene expression of iNOS Il-6, and COX-2 and was assessed by the RT/PCR method using 18S rRNA as the housekeeping gene. Results: Etanercept determined a complete suppression of TNFainduced iNOS, Il-6, and COX-2 gene expression. This anti-TNFa drug also downregulated, with a dose-dependent effect, iNOS and COX-2 gene expression (−20% and −30% respectively, p< 0.05) after stimulation with Il-1b. Etanercept seemed to have no effects on Il-1b-induced Il-6 gene expression. Conclusions: To our knowledge, this is the first report of the effects of etanercept on iNOS, Il-6 and COX-2 gene expression, after inflammatory stimulation, on a human chondrocyte population. In summary, these data suggest that the beneficial effects of the treatment with etanecerpt in EOA could be further explained by the action of this biological drug at cartilage level.

365 STIMULATING CARTILAGE OLIGOMERIC MATRIX PROTEIN PRODUCTION IN CARTILAGE REGENERATION

Introduction: Cartilage Oligomeric Matrix Protein (COMP) is a protein present in the cartilage extracellular matrix. The function of COMP is not completely understood but it has been shown to bind to chondrocytes(1), collagen types I and II(2), and the non collagenous domains of collagen IX(3). COMP is also implicated in the interaction between chondrocytes and collagen as well as in collagen fibrillogenesis(4). COMP is expressed more abundantly, and more pericellularly in OA cartilage than in normal healthy cartilage(5). This changed distribution suggests a possible involvement in regeneration of the cartilage matrix(6). To obtain more insight in the role of COMP in cartilage regeneration, the present study was designed to investigate the effect of growth factors on COMP deposition and the influence of COMP on collagen deposition and mechanical properties. Materials and Methods: Articular chondrocytes, isolated from bovine MCP joints, were suspended in alginate beads (4 million cells/ml) and cultured in DMEM/F12 with 10% FCS and ascorbic acid with or without 25 ng/ml FGF2, TGFbeta2 or IGF-1. In addition, human COMP (Hs-COMP) was overexpressed in bovine chondrocytes using lentiviral transfection. COMP gene expression, COMP (ELISA and Western blot), proteoglycan (dimethylmethylene blue) and DNA (Hoechst) were measured. Total collagen content and collagen cross-links were analyzed by HPLC. All data are presented as mean ± sd. Results: Stimulation of primary bovine chondrocytes with TGFbeta2 resulted in significant increase of COMP gene expression. FGF2 lead to a significant decrease in COMP gene expression. IGF1 had no effect when compared to the control condition without growth factors. (Figure 1)

A36 MODULATION OF COLLAGEN NETWORK FORMATION TO IMPROVE FUNCTIONAL PROPERTIES IN CARTILAGE REGENERATION

Methods: 24 skeletally mature New Zealand White rabbits underwent anterior cruciate ligament transection. 6 additional sham operated animals were used as controls. 10 days after the surgery the transected animals received an intraarticular injection of CD-RAP encapsulated in liposomes or liposomes alone as vehicle control. Three different dose levels of CD-RAP were tested. Thereafter the animals were injected every 10 days until the end of the 9-week treatment period. The animals were analysed macroscopically, radiologically, and histologically. To score the radiological and histological data, the Kellgren and a modified Mankin score, respectively, were used. Results: CD-RAP treatment resulted in reduced osteophyte formation and reduced joint space narrowing. This effect was significant as shown by the radiological scoring There was a trend towards middle and high dose CD-RAP being more efficacious than the low dose group but this was not statistically significant. The histological evaluation by the modified Mankin score, which evaluated cartilage structure, cells, matrix (safranin-O), and tidemark integrity, confirmed the radiological results. The average scores for sham, vehicle, and treatments groups (low, mid and high dose CD-RAP) were 1.12±1.19, 7.28±2.19, 7.22±2.69, 4.08±0.8, 4.47±1.93. Both middle and high dose CD-RAP groups showed a significant improvement over the vehicle group. Looking at the individual parameters, CD-RAP appeared to impact structural integrity as well as matrix content. Conclusions: These data suggest that treatment with the cartilage specific protein CD-RAP may be a new therapeutic option for osteoarthritis patients. Next steps will include the long-term evaluation of liposome-encapsulated CD-RAP in a large animal model.