Effect of stretching on gene expression of beta1 integrin and focal adhesion kinase and on chondrogenesis through cell-extracellular matrix interactions

Differentiation of skeletal tissues, such as bone, ligament and cartilage, is regulated by complex interaction between genetic and epigenetic factors. In the present study, we attempted to elucidate the possible role of cell-extracellular matrix (ECM) adhesion on the inhibitory regulation in chondrogenesis responding to the tension force. The midpalatal suture cartilages in rats were expanded by orthopedic force. In situ hybridization for type I and II collagens, immunohistochemical analysis for fibronectin, alpha5 and beta1 integrins, paxillin, and vinculin, and cytochemical staining for actin were used to demonstrate the phenotypic change of chondrocytes. Immunohistochemical analysis for phosphorylation and nuclear translocation of extracellular signal-regulated kinase (ERK)-1/2 was performed. The role of the cell-ECM adhesion in the response of the chondroprogenitor cells to mechanical stress and the regulation of gene expression of focal adhesion kinase (FAK) and integrins were analyzed by using an in vitro system. A fibrous suture tissue replaced the midpalatal suture cartilage by the expansive force application for 14 days. The active osteoblasts that line the surface of bone matrix in the newly formed suture tissue strongly expressed the type I collagen gene, whereas they did not express the type II collagen gene. Although the numbers of precartilaginous cells expressing alpha5 and beta1 integrin increased, the immunoreactivity of alpha5 integrin in each cell was maintained at the same level throughout the experimental period. During the early response of midpalatal suture cartilage cells to expansive stimulation, formation of stress fibers, reorganization of focal adhesion contacts immunoreactive to a vinculin-specific antibody, and phosphorylation and nuclear translocation of ERK-1/2 were observed. In vitro experiments were in agreement with the results from the in vivo study, i.e. the inhibited expression of type II collagen and upregulation in integrin expression. The arginine-glycine-aspartic acid-containing peptide completely rescued chondrogenesis from tension-mediated inhibition. Thus, we conclude that stretching activates gene expression of beta1 integrin and FAK and inhibits chondrogenesis through cell-ECM interactions of chondroprogenitor cells.     

Beta1 integrins regulate chondrogenesis and rock signaling in adipose stem cells

β1 integrins play a controversial role during chondrogenesis. Since the maturation of chondrocytes relies on a signaling switch from cell-cell to cell-matrix interactions, we hypothesized that β1 integrins play a different role at the earlier (mainly cell-cell interaction) from the later stage (mainly cell-matrix interaction) of chondrogenesis. Our data showed: in plain medium, sox9, collagen X, and collagen II gene expressions of ASCs were induced by β1-integrin blockage at day 14. In chondrogenic medium, however, sox 9, sox6, and collagen II gene expression were induced at day 4 but inhibited at day 14. In addition, both β1-integrin blockage and TGF-β1 down-regulated Rock-1 and -2 gene expression and produced the round cells. We concluded that β1 integrins play a more important role at the later stages than earlier stages of chondrogenesis, and that the onset of chondrogenesis promoted by β1-integrin blockage might be through inhibiting Rock signaling.     

Chondrogenesis and hypertrophy in response to aggregate behaviors of human mesenchymal stem cells on a dendrimer-immobilized surface

Objectives

To investigate the behaviors of aggregates of human mesenchymal stem cells (hMSCs) on chondrogenesis and chondrocyte hypertrophy using spatiotemporal expression patterns of chondrogenic (type II collagen) and hypertrophic (type X collagen) markers during chondrogenesis.

Results

hMSCs were cultured on either a polystyrene surface or polyamidoamine dendrimer surface with a fifth generation (G5) dendron structure in chondrogenic medium and growth medium. At day 7, cell aggregates without stress fibers formed on the G5 surface and triggered differentiation of hMSCs toward the chondrogenic fate, as indicated by type II collagen being observed while type X collagen was undetectable. In contrast, immunostaining of hMSCs cultured on polystyrene, which exhibited abundant stress fibers and did not form aggregates, revealed no evidence of either type II and or type X collagen. At day 21, the morphological changes of the cell aggregates formed on the G5 surface were suppressed as a result of stress fiber formation. Type II collagen was observed throughout the aggregates whereas type X collagen was detected only at the basal side of the aggregates. Change of cell aggregate behaviors derived from G5 surface alone regulated chondrogenesis and hypotrophy, and this was enhanced by chondrogenic medium.

Conclusions

Incubation of hMSCs affects the expression of type II and X collagens via effects on cell aggregate behavior and stress fiber formation.

     

Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels: influence of collagen type II extracellular matrix on MSC chondrogenesis

Bone marrow mesenchymal stem cells (MSCs) are candidate cells for cartilage tissue engineering. This is due to their ability to undergo chondrogenic differentiation after extensive expansion in vitro and stimulation with various biomaterials in three-dimensional (3-D) systems. Collagen type II is one of the major components of the hyaline cartilage and plays a key role in maintaining chondrocyte function. This study aimed at analyzing the MSC chondrogenic response during culture in different types of extracellular matrix (ECM) with a focus on the influence of collagen type II on MSC chondrogenesis. Bovine MSCs were cultured in monolayer as well as in alginate and collagen type I and II hydrogels, in both serum free medium and medium supplemented with transforming growth factor (TGF) beta1. Chondrogenic differentiation was detected after 3 days of culture in 3-D hydrogels, by examining the presence of glycosaminoglycan and newly synthesized collagen type II in the ECM. Differentiation was most prominent in cells cultured in collagen type II hydrogel, and it increased in a time-dependent manner. The expression levels of the of chondrocyte specific genes: sox9, collagen type II, aggrecan, and COMP were measured by quantitative "Real Time" RT-PCR, and genes distribution in the hydrogel beads were localized by in situ hybridization. All genes were upregulated by the presence of collagen, particularly type II, in the ECM. Additionally, the chondrogenic influence of TGF beta1 on MSCs cultured in collagen-incorporated ECM was analyzed. TGF beta1 and dexamethasone treatment in the presence of collagen type II provided more favorable conditions for expression of the chondrogenic phenotype. In this study, we demonstrated that collagen type II alone has the potential to induce and maintain MSC chondrogenesis, and prior interaction with TGF beta1 to enhance the differentiation.     

Sustained Wnt protein expression in chondral constructs from mesenchymal stem cells

Wnt genes encode a number of secreted glycoproteins which are closely associated with the cell surface and the extracellular matrix. Recently, members of Wnt family have been implicated in regulating chondrocyte differentiation, but their roles in the chondrogenic process are not fully understood. To contribute to an understanding of the roles of Wnts during chondrogenesis, we have analysed the spatiotemporal expression patterns of Wnt using in vitro models for chondrogenesis of human bone marrow-derived mesenchymal stem cells (hMSCs). In chondrogenic aggregate culture system, RT-PCR analysis revealed expression of Wnt5a and Wnt4 during late chondrogenesis (days 10 and 15). Immunohistochemical analysis showed widespread distribution of Wnt5a and Wnt4 throughout the aggregates at this late phase of culture (days 14 and 21). In addition, in this aggregate culture system, immunohistochemical staining of Wnt4 and Wnt5a showed similar spatiotemporal expression patterns to that of type II collagen or type X collagen. To confirm the results obtained by immunostaining, the specificity of the anti-Wnt4 or anti-Wnt5a antibody was assessed by Western blot analysis. Of Wnt4 and Wnt5a, only Wnt5a was immunodetectable by Western blot analysis. Western blot analysis showed that Wnt5a was expressed as two different molecular weight forms of 40 and 44 kDa. Treatment with PNGase F, which removes N-linked oligosaccharides, revealed that the mass difference between these two forms could be accounted for by the N-glycosylation status of the protein. When hMSCs were seeded on a porous gelatin sponge, immunolocalization studies showed that type II collagen and type X collagen were detected particularly at the periphery at day 7 of culture. In contrast, Wnt4 and Wnt5a showed even distribution throughout the hMSC/gelatin sponge constructs. Their different spatial expression patterns suggest that Wnt4 and Wnt5a proteins are not functionally linked to type II collagen and type X collagen synthesis in in vitro chondrogenic models of hMSCs.     

bFGF influences human articular chondrocyte differentiation

BACKGROUND: The possible functional role of basic fibroblast growth factor (bFGF) in regulating the mitotic and metabolic activity of primary human articular chondrocytes was investigated. METHODS: [EF1]Chondrocytes were enzymatically isolated from femoral head cartilage, and were cultured in vitro in monolayer. bFGF-dependent cell proliferation, production of collagen type II and aggrecan were monitored 10 days after isolation. Furthermore, effect of bFGF on cell cycle, cell morphology, and mRNA expression of integrins and chondrogenic markers determined by real time PCR were analyzed. RESULTS: bFGF concentrations in supernatants of primary human articular chondrocytes peaked immediately after isolation and then declined. In a dose-dependent manner, bFGF enhanced cell amplification and viability. BFGF induced a decrease in the apoptotic cell population, while the number of proliferating cells remained unchanged. Supplementation of cell culture with bFGF reduced collagen type II mRNA by 49%, but increased expression of the integrin alpha(2) by 70%. bFGF did not significantly regulate the integrins alpha(1), alpha(5), alpha(10), alpha(v) and type I collagen. bFGF reduced the amount of collagen type II by 53%, which was correlated with diminished mRNA production. Monolayer cultured chondrocytes secreted significant amounts of aggrecan that decreased over time. Secretion of this cartilage-specific marker was further reduced by the addition of bFGF. DISCUSSION: These findings highlight the potential role of bFGF as an endogenous chondrocyte mediator that can enhance cell amplification and regulate cell differentiation.     

Hyaluronic acid and autologous synovial fluid induce chondrogenic differentiation of equine mesenchymal stem cells: a preliminary study

Mesenchymal stem cells (MSC) have the potential to differentiate into distinct mesenchymal tissues including cartilage, which suggest these cells as an attractive cell source for cartilage tissue engineering approaches. Our objective was to study the effects of TGF-beta1, hyaluronic acid and synovial fluid on chondrogenic differentiation of equine MSC. For that, bone marrow was aspirated from the tibia of one 18-month-old horse (Haflinger) and MSC were isolated using percoll-density centrifugation. To promote chondrogenesis, MSC were centrifuged to form a micromass and were cultured in a medium containing 10 ng/ml TGF-beta1 or 0.1mg/ml hyaluronic acid (Hylartil, Ostenil) or either 5%, 10% or 50% autologous synovial fluid as the chondrogenesis inducing factor. Differentiation along the chondrogenic lineage was documented by type II collagen and proteoglycan expression. MSC induced by TGF-beta1 alone showed the highest proteoglycan expression. Combining TGF-beta1 with hyaluronic acid could not increase the proteoglycan expression. Cultures stimulated by autologous synovial fluid (independent of concentration) and hyaluronic acid demonstrated a pronounced, but lower proteoglycan expression than cultures stimulated by TGF-beta1. The expression of cartilage-specific type II collagen was high and about the same in all stimulated cultures. In summary, hyaluronic acid and autologous synovial fluid induces chondrogenesis of equine mesenchymal stem cells, which encourage tissue engineering applications of MSC in chondral defects, as the natural environment in the joint is favorable for chondrogenic differentiation.     

Distribution of the collagen-binding integrin α10β1 during mouse development

We have previously identified and characterised the collagen type II-binding integrin subunit alpha10, which is a member of the beta1 family and is expressed by chondrocytes. In the present study, we examined the expression of the alpha10 integrin in various mouse tissues. Immunohistochemical analysis of alpha10 on cryosections from 3-day-old mice demonstrated that alpha10beta1 was present in the hyaline cartilage of joints, vertebral column, trachea and bronchi. In addition, alpha10 was found in the ossification groove of Ranvier, in the aortic and atrioventricular valves of the heart and in the fibrous tissue lining skeletal muscle and ligaments. Overall, the distribution was distinct from that of the collagen-binding integrins alpha1beta1 and alpha2beta1. We also found that alpha10beta1was the dominating collagen-binding integrin during cartilage development. Expression of alpha10 appeared at embryonic day 11.5 (E11.5) at the same time as chondrogenesis started as judged by collagen type II expression. At E13.5, alpha10 was present throughout the anlage as well as in the perichondrium and in mesenchyme just outside the perichondrium, where it localised with collagen type I. Four weeks after birth, alpha10 was prominent both at the articular surface and in the growth plate. In conclusion, we found that integrin alpha10beta1 was a major collagen-binding integrin during cartilage development and in mature hyaline cartilage. In addition, we found that alpha10beta1 was present in some fibrous tissues.     

Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: II. Stimulation by bone morphogenetic protein-2 requires modulation of N-cadherin expression and function

Bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-beta (TGF-beta) superfamily, is characterized by its ability to induce cartilage and bone formation. We have recently demonstrated that the multipotential, murine embryonic mesenchymal cell line, C3H10T1/2, when cultured at high density, is induced by BMP-2 or TGF-beta 1 to undergo chondrogenic differentiation. The high-cell-density requirement suggests that specific cell-cell interactions, such as those mediated by cell adhesion molecules, are important in the chondrogenic response. In view of our recent finding that N-cadherin, a Ca(2+)-dependent cell adhesion molecule, is functionally required in normal embryonic limb mesenchyme cellular condensation and chondrogenesis, we examine here whether N-cadherin is also involved in BMP-2 induction of chondrogenesis in C3H10T1/2 cells. BMP-2 stimulation of chondrogenesis in high-density micromass cultures of C3H10T1/2 cells was evidenced by Alcian blue staining, elevated [35S]sulfate incorporation, and expression of the cartilage matrix markers, collagen type II and cartilage proteoglycan link protein. With BMP-2 treatment, N-cadherin mRNA expression was stimulated 4-fold within 24 h, and by day 5, protein levels were stimulated 8-fold. An N-cadherin peptidomimic containing the His-Ala-Val sequence to abrogate homotypic N-cadherin interactions inhibited chondrogenesis in a concentration-dependent manner. To analyze the functional role of N-cadherin further, C3H10T1/2 cells were stably transfected with expression constructs of either full-length N-cadherin or a dominant negative, N-terminal deletion mutant of N-cadherin. Moderate (2-fold) overexpression of full-length N-cadherin augmented, whereas higher (4-fold) overexpression inhibited the BMP-2-chondrogenic effect. On the other hand, expression of the dominant negative N-cadherin mutant dramatically inhibited BMP-2 stimulated chondrogenesis. These data strongly suggest that upregulation of N-cadherin expression, at defined critical levels, is a candidate mechanistic component of BMP-2 stimulation of mesenchymal chondrogenesis.     

The effect of beta-xylosides on the chondrogenic differentiation of mesenchymal stem cells

Chondroitin/dermatan sulphate (CS/DS) sulphation motifs on cell and extracellular matrix proteoglycans (PGs) within stem/progenitor cell niches are involved in modulating cell phenotype during the development of many musculoskeletal connective tissues. Here, we investigate the importance of CS/DS chains and their motifs in the chondrogenic differentiation of bone marrow mesenchymal stem cells (bMSCs), using p-nitrophenyl xyloside (PNPX) as a competitive acceptor of CS/DS substitution on PGs. Comparison of cultures grown in control chondrogenic medium, with those grown in the presence of PNPX showed that PNPX delayed the onset of chondrogenesis, characterised by cell rounding and aggregation into spheroidal beads. PNPX reduced gene expression of SOX-9, aggrecan and collagen type II, and caused reduced levels of collagen type II protein. PNPX-treated cultures also showed delayed expression of a native CS/DS sulphation motif epitope recognised by antibody 6C3. This epitope appeared associated with a range of PGs, particularly biglycan, and its close association was lost after PNPX treatment. Overall our data show that perturbation of PG glycosylation with CS/DS GAGs using PNPX significantly delays the onset of chondrogenic differentiation of bMSCs, highlighting the importance of CS/DS during the initial stages of chondrogenesis. The delayed expression of the CS/DS sulphation motif recognised by 6C3 suggests that this motif, in particular, may have early involvement in chondrogenesis. The mechanism(s) by which CS/DS chains on PGs contribute to early chondrogenic events is unknown; however, they may be involved in morphogenetic signalling through the capture and cellular presentation of soluble bioactive molecules (e.g. growth factors).     

Analysis of collagen expression during chondrogenic induction of human bone marrow mesenchymal stem cells

Adult mesenchymal stem cells (MSCs) are currently being investigated as an alternative to chondrocytes for repairing cartilage defects. As several collagen types participate in the formation of cartilage-specific extracellular matrix, we have investigated their gene expression levels during MSC chondrogenic induction. Bone marrow MSCs were cultured in pellet in the presence of BMP-2 and TGF-β3 for 24 days. After addition of FGF-2, at the fourth passage during MSC expansion, there was an enhancing effect on specific cartilage gene expression when compared to that without FGF-2 at day 12 in pellet culture. A switch in expression from the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of the collagen type II gene was observed at day 24. A short-term addition of FGF-2 followed by a treatment with BMP-2/TGF-β3 appears sufficient to accelerate chondrogenesis with a particular effect on the main cartilage collagens.     

C-type natriuretic peptide regulation of limb mesenchymal chondrogenesis is accompanied by altered N-cadherin and collagen type X-related functions

AMDM, a form of osteochondrodysplasia, is due to the loss-of-function mutations in NPR-B gene. This study investigated the functional involvement of CNP-3, chick homolog of human CNP, and its receptor NPR-B in chondrogenesis utilizing the micromass culture of the chick limb mesenchymal cells. Results revealed CNP-3 and NPR-B expression in the chick limb bud making stage-specific peak levels first at Hamburger-Hamilton stage 23-24, and second at stage 30-31, corresponding to pre-chondrogenic mesenchymal condensation and initiation of chondrogenic maturation-hypertrophy in vivo, respectively. CNP-3 and NPR-B expression in vitro increased parallel to collagen type X expression, but not to that of collagen type II. Treatment of cultures with CNP significantly increased N-cadherin, and collagen type X expression, glycosaminoglycan synthesis and chondrogenesis. Collagen type II expression was not significantly affected. Thus, results implicated CNP-3/NPR-B signaling in pre-chondrogenic mesenchymal condensation, glycosaminoglycan synthesis and late differentiation of chondrocytes in the process of endochondral ossification.     

Expression of adhesion molecules and collagen on rat chondrocyte seeded into alginate and hyaluronate based 3D biosystems. Influence of mechanical stresses

Chondrocytes use mechanical signals, via interactions with their environment, to synthesize an extracellular matrix capable to withstanding high loads. Most chondrocyte-matrix interactions are mediated via transmembrane receptors such as integrins or non-integrins receptors (i.e. annexin V and CD44). The aim of this study was to analyze, by flow cytometry, the adhesion molecules (alpha5/beta1 integrins and CD44) on rat chondrocytes seeded into 3D biosystem made of alginate and hyaluronate. These biosystems were submitted to mechanical stress by knocking the biosystems between them for 48 hours. The expression of type I and type II collagen was also evaluated. The results of the current study showed that mechanical stress induced an increase of type II collagen production and weak variations of alpha5/beta1 receptors expression no matter what biosystems. Moreover, our results indicated that hyaluronan receptor CD44 expression depends on extracellular matrix modifications. Thus, these receptors were activated by signals resulted from cell environment variations (HA addition and modifications owing to mechanical stress). It suggested that this kind of receptor play a crucial role in extracellular matrix homeostasis. Finally, on day 24, no dedifferentiation of chondrocytes was noted either in biosystems or under mechanical stress. For all biosystems, the neosynthesized matrix contained an important level of collagen, which was type II, whatever biosystems. In conclusion, it appeared that the cells, under mechanical stress, maintained their phenotype. In addition, it seems that, on rat chondrocytes, alpha5/beta1 integrins did not act as the main mechanoreceptor (as described for human chondrocytes). In return, hyaluronan receptor CD44 seems to be in relation with matrix composition.     

Hydrolyzed fish collagen induced chondrogenic differentiation of equine adipose tissue-derived stromal cells

Adipose-derived stromal cells (ADSCs) are multipotent cells which, in the presence of appropriate stimuli, can differentiate into various lineages such as the osteogenic, adipogenic and chondrogenic. In this study, we investigated the effect of transforming growth factor beta 1 (TGF-β1) in comparison to hydrolyzed fish collagen in terms of the chondrogenic differentiation potential of ADSCs. ADSCs were isolated from subcutaneous fat of horses by liposuction. Chondrogenesis was investigated using a pellet culture system. The differentiation medium was either supplemented with TGF-β1 (5 ng/ml) or fish collagen (0.5 mg/ml) for a 3 week period. After the 3 weeks in vitro differentiation, RT-PCR and histological staining for proteoglycan synthesis and type II collagen were performed to evaluate the degree of chondrogenic differentiation and the formation of cartilaginous extracellular matrix (ECM). The differentiation of ADSCs induced by TGF-β1 showed a high expression of glycosaminoglycan (GAG). Histological analysis of cultures stimulated by hydrolyzed fish collagen demonstrated an even higher GAG expression than cultures stimulated under standard conditions by TGF-β1. The expression of cartilage-specific type II collagen and Sox9 was about the same in both stimulated cultures. In this study, chondrogenesis was as effectively induced by hydrolyzed fish collagen as it was successfully induced by TGF-β1. These findings demonstrated that hydrolyzed fish collagen alone has the potential to induce and maintain ADSCs-derived chondrogenesis. These results support the application of ADSCs in equine veterinary tissue engineering, especially for cartilage repair.     

Distribution of Fibronectin and Collagen during Mouse Limb and Palate Development

Indirect immunofluorescence has been used to study the distribution of fibronectin and collagen types I, II, and III in the developing primary and secondary palatal processes and forelimb buds of the Swiss Webster (NIH) mouse. In the palatal processes fibronectin and types I and III collagen are distributed throughout the mesenchyme. Fibronectin is present in the basement membrane, while types I and III collagen are localized in a linear, discontinuous fashion beneath the basement membrane. Fibronectin is not observed in the epithelium, including the presumptive fusion areas. In the forelimb bud these components show a similar distribution prior to chondrogenesis (early day 11). When chondrogenesis commences (late day 11 or early day 12) fibronectin and, to a lesser degree, types I and III collagen are apparently concentrated in the core mesenchyme, suggesting that fibronectin has a role in initiating chondrogenesis, perhaps by increasing cellular aggregation. Type II collagen is observed only in chondrogenic regions. The codistribution of fibronectin and types I and III collagen supports in vitro studies which indicate that cells use fibronectin to bind to collagen in the matrix. The developing chondrogenic regions appear to lose fibronectin gradually, concomitant with the appearance of type II collagen, suggesting that fibronectin is not involved in the maintenance of functional chondrocytes in their matrices.