MicroRNA signature associated with osteogenic lineage commitment

Cell-based approaches offer a potential therapeutic strategy for appropriate bone manufacturing. Capable of differentiating into multiple cell types especially osteoblasts spontaneously, unrestricted somatic stem cell (USSC) seems to be a suitable candidate. Recent studies have shown the involvement of microRNAs in several biological processes. miRNA microarray profiling was applied in order to identify the osteo-specific miRNA signature. Prior to this analysis, osteogenic commitment of osteoblasts was evaluated by measuring ALPase activity, biomineralization, specific staining and evaluation of some main osteogenic marker genes. To support our findings, various in silico explorations (for both putative targets and signaling pathways) and empirical analyses (miRNA transfections followed by qPCR of osteogenic indicators and ALPase activity measurement) were carried out. The function of GSK-3b inhibitor was also studied to investigate the role of WNT in osteogenesis. Transient modulation of multiple osteo-miRs (such as mir-199b, 1274a, 30b) with common targets (such as BMPR, TCFs, SMADs) as mediators of osteogenic pathways including cell-cell interactions, WNT and TGF-beta pathways, suggests a mechanism for rapid induction of the osteogenesis as an anti-miRNA therapy. The results of this research have identified the miRNA signature which regulates the osteogenesis mechanism in USSC. To conclude, our study reveals more details about the allocation of USSCs into osteogenic lineage through modulatory effect of miRNAs on targets and pathways required for creating a tissue-specific phenotype and may aid in future clinical interventions.     

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

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.     

Regulation of chondrogenesis by protein kinase C: Emerging new roles in calcium signalling

During chondrogenesis, complex intracellular signalling pathways regulate an intricate series of events including condensation of chondroprogenitor cells and nodule formation followed by chondrogenic differentiation. Reversible phosphorylation of key target proteins is of particular importance during this process. Among protein kinases known to be involved in these pathways, protein kinase C (PKC) subtypes play pivotal roles. However, the precise function of PKC isoenzymes during chondrogenesis and in mature articular chondrocytes is still largely unclear. In this review, we provide a historical overview of how the concept of PKC-mediated chondrogenesis has evolved, starting from the first discoveries of PKC isoform expression and activity. Signalling components upstream and downstream of PKC, leading to the stimulation of chondrogenic differentiation, are also discussed. Although it is evident that we are only at the beginning to understand what roles are assigned to PKC subtypes during chondrogenesis and how they are regulated, there are many yet unexplored aspects in this area. There is evidence that calcium signalling is a central regulator in differentiating chondroprogenitors; still, clear links between intracellular calcium signalling and prototypical calcium-dependent PKC subtypes such as PKCalpha have not been established. Exploiting putative connections and shedding more light on how exactly PKC signalling pathways influence cartilage formation should open new perspectives for a better understanding of healthy as well as pathological differentiation processes of chondrocytes, and may also lead to the development of novel therapeutic approaches.     

Upregulation of miR-23b Enhances the Autologous Therapeutic Potential for Degenerative Arthritis by Targeting PRKACB in Synovial Fluid-Derived Mesenchymal Stem Cells from Patients

The use of synovial fluid-derived mesenchymal stem cells (SFMSCs) obtained from patients with degenerative arthropathy may serve as an alternative therapeutic strategy in osteoarthritis (OA) and rheumatoid arthritis (RA). For treatment of OA and RA patients, autologous transplantation of differentiated MSCs has several beneficial effects for cartilage regeneration including immunomodulatory activity. In this study, we induced chondrogenic differentiation of SFMSCs by inhibiting protein kinase A (PKA) with a small molecule and microRNA (miRNA). Chondrogenic differentiation was confirmed by PCR and immunocytochemistry using probes specific for aggrecan, the major cartilaginous proteoglycan gene. Absorbance of alcian blue stain to detect chondrogenic differentiation was increased in H-89 and/or miRNA-23btransfected cells. Furthermore, expression of matrix metalloproteinase (MMP)-9 and MMP-2 was decreased in treated cells. Therefore, differentiation of SFMSCs into chondrocytes through inhibition of PKA signaling may be a therapeutic option for OA or RA patients.     

The Effect of Dexamethasone and Triiodothyronine on Terminal Differentiation of Primary Bovine Chondrocytes and Chondrogenically Differentiated Mesenchymal Stem Cells

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

Efficient differentiation of human iPSC‐derived mesenchymal stem cells to chondroprogenitor cells

Induced pluripotent stem cells (iPSC) hold tremendous potential for personalized cell‐based repair strategies to treat musculoskeletal disorders. To establish human iPSCs as a potential source of viable chondroprogenitors for articular cartilage repair, we assessed the in vitro chondrogenic potential of the pluripotent population versus an iPSC‐derived mesenchymal‐like progenitor population. We found the direct plating of undifferentiated iPSCs into high‐density micromass cultures in the presence of BMP‐2 promoted chondrogenic differentiation, however these conditions resulted in a mixed population of cells resembling the phenotype of articular cartilage, transient cartilage, and fibrocartilage. The progenitor cells derived from human iPSCs exhibited immunophenotypic features of mesenchymal stem cells (MSCs) and developed along multiple mesenchymal lineages, including osteoblasts, adipocytes, and chondrocytes in vitro. The data indicate the derivation of a mesenchymal stem cell population from human iPSCs is necessary to limit culture heterogeneity as well as chondrocyte maturation in the differentiated progeny. Moreover, as compared to pellet culture differentiation, BMP‐2 treatment of iPSC‐derived MSC‐like (iPSC–MSC) micromass cultures resulted in a phenotype more typical of articular chondrocytes, characterized by the enrichment of cartilage‐specific type II collagen (Col2a1), decreased expression of type I collagen (Col1a1) as well as lack of chondrocyte hypertrophy. These studies represent a first step toward identifying the most suitable iPSC progeny for developing cell‐based approaches to repair joint cartilage damage. J. Cell. Biochem. 114: 480–490, 2013. © 2012 Wiley Periodicals, Inc.     

Chondrogenic capability of osteoarthritic chondrocytes from the trapeziometacarpal and hip joints

Osteoarthritis is the most common degenerative disease of joints like the hip and the trapeziometacarpal joint (rhizarthrosis). In this in vitro study, we compared the chondrogenesis of chondrocytes derived from the trapezium and the femoral head cartilage of osteoarthritic patients to have a deeper insight on trapezium chondrocyte behavior as autologous cell source for the repair of cartilage lesions in rhizarthrosis. Chondrocytes collected from trapezium and femoral head articular cartilage were cultured in pellets and analyzed for chondrogenic differentiation, cell proliferation, glycosaminoglycan production, gene expression of chondrogenic and fibrous markers, histological and immunohistochemical analyses. Our results showed a higher cartilaginous matrix deposition and a lower fibrocartilaginous phenotype of the femoral chondrocytes with respect to the trapezium chondrocytes assessed by a higher absolute glycosaminoglycan and type II collagen production, thus demonstrating a superior chondrogenic potential of the femoral with respect to the trapezium chondrocytes. The differences in chondrogenic potential between trapezium and femoral head chondrocytes confirmed a lower regenerative capability in the trapezium than in the femoral head cartilage due to the different environment and loading acting on these joints that affects the metabolism of the resident cells. This could represent a limitation to apply the cell therapy for rhizoarthrosis.     

Regulation of cartilage formation and maturation by mitogen-activated protein kinase signaling

The majority of bones comprising the adult vertebrate skeleton are generated from hyaline cartilage templates that form during embryonic development. A process known as endochondral ossification is responsible for the conversion of these transient cartilage anlagen into mature, calcified bone. Endochondral ossification is a highly regulated, multistep cell specification program involving the initial differentiation of prechondrogenic mesenchymal cells into hyaline chondrocytes, terminal differentiation of hyaline chondrocytes into hypertrophic chondrocytes, and finally, apoptosis of hypertrophic chondrocytes followed by bone matrix deposition. Recently, extensive research has been carried out describing roles for the three major mitogen-activated protein kinase (MAPK) signaling pathways, the extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-jun N-terminal kinase (JNK) pathways, in the successive stages of chondrogenic differentiation. In this review, we survey this research examining the involvement of ERK1/2, p38, and JNK pathway signaling in all aspects of the chondrogenic differentiation program from embryonic through postnatal stages of development. In addition, we summarize evidence from in vitro studies examining MAPK function in immortalized chondrogenic cell lines and adult mesenchymal stem cells. We also provide suggestions for future studies that may help ameliorate existing confusion concerning the specific roles of MAPK signaling at different stages of chondrogenesis.     

Direct and progressive differentiation of human embryonic stem cells into the chondrogenic lineage

Treatment of common and debilitating degenerative cartilage diseases particularly osteoarthritis is a clinical challenge because of the limited capacity of the tissue for self‐repair. Because of their unlimited capacity for self‐renewal and ability to differentiate into multiple lineages, human embryonic stem cells (hESCs) are a potentially powerful tool for repair of cartilage defects. The primary objective of the present study was to develop culture systems and conditions that enable hESCs to directly and uniformly differentiate into the chondrogenic lineage without prior embryoid body (EB) formation, since the inherent cellular heterogeneity of EBs hinders obtaining homogeneous populations of chondrogenic cells that can be used for cartilage repair. To this end, we have subjected undifferentiated pluripotent hESCs to the high density micromass culture conditions we have extensively used to direct the differentiation of embryonic limb bud mesenchymal cells into chondrocytes. We report that micromass cultures of pluripotent hESCs undergo direct, rapid, progressive, and substantially uniform chondrogenic differentiation in the presence of BMP2 or a combination of BMP2 and TGF‐β1, signaling molecules that act in concert to regulate chondrogenesis in the developing limb. The gene expression profiles of hESC‐derived cultures harvested at various times during the progression of their differentiation has enabled us to identify cultures comprising cells in different phases of the chondrogenic lineage ranging from cultures just entering the lineage to well differentiated chondrocytes. Thus, we are poised to compare the abilities of hESC‐derived progenitors in different phases of the chondrogenic lineage for cartilage repair. J. Cell. Physiol. 224: 664–671, 2010. © 2010 Wiley‐Liss, Inc.     

Mammalian chondrocytes expanded in the presence of fibroblast growth factor 2 maintain the ability to differentiate and regenerate three-dimensional cartilaginous tissue

The differentiated phenotype of chondrocytes from hyaline cartilage is gradually lost during expansion in monolayers. Chondrocytes can reexpress their differentiated phenotype by transfer into an environment that prevents cell flattening, but serially passaged cells never completely recover their chondrogenic potential. We report that chondrocytes expanded (up to 2000-fold) in the presence of fibroblast growth factor 2 (FGF-2) dedifferentiated, but fully maintained their potential for redifferentiation in response to environmental changes. After seeding onto three-dimensional polymer scaffolds, chondrocytes expanded in the presence of FGF-2 formed cartilaginous tissue that was histologically and biochemically comparable to that obtained using primary chondrocytes, in contrast to chondrocytes expanded to the same degree but in the absence of FGF-2. The presence of FGF-2 inhibited the formation of thick F-actin structures, which otherwise formed during monolayer expansion, were maintained during tissue cultivation, and were associated with reduced ability of chondrocytes to reexpress their differentiated phenotype. This study provides evidence that FGF-2 maintains the chondrogenic potential during chondrocyte expansion in monolayers, possibly due to changes in the architecture of F-actin elements and allows more efficient utilization of harvested tissue for cartilage tissue engineering.     

Src kinase inhibition promotes the chondrocyte phenotype

Regulated differentiation of chondrocytes is essential for both normal skeletal development and maintenance of articular cartilage. The intracellular pathways that control these events are incompletely understood, and our ability to modulate the chondrocyte phenotype in vivo or in vitro is therefore limited. Here we examine the role played by one prominent group of intracellular signalling proteins, the Src family kinases, in regulating the chondrocyte phenotype. We show that the Src family kinase Lyn exhibits a dynamic expression pattern in the chondrogenic cell line ATDC5 and in a mixed population of embryonic mouse chondrocytes in high-density monolayer culture. Inhibition of Src kinase activity using the pharmacological compound PP2 (4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine) strongly reduced the number of primary mouse chondrocytes. In parallel, PP2 treatment increased the expression of both early markers (such as Sox9, collagen type II, aggrecan and xylosyltransferases) and late markers (collagen type X, Indian hedgehog and p57) markers of chondrocyte differentiation. Interestingly, PP2 repressed the expression of the Src family members Lyn, Frk and Hck. It also reversed morphological de-differentiation of chondrocytes in monolayer culture and induced rounding of chondrocytes, and reduced stress fibre formation and focal adhesion kinase phosphorylation. We conclude that the Src kinase inhibitor PP2 promotes chondrogenic gene expression and morphology in monolayer culture. Strategies to block Src activity might therefore be useful both in tissue engineering of cartilage and in the maintenance of the chondrocyte phenotype in diseases such as osteoarthritis.     

Chondrogenesis of human periosteum-derived progenitor cells in atelocollagen

Periosteum-derived progenitor cells (PDPCs) could be differentiated into cartilage using atelocollagen as a carrier and in the presence of transforming growth factor-β3 (TGF-β3). Chondrogenesis was verified by RT-PCR and Western blotting. Expression of the type II collagen mRNA was found from the differentiated PDPCs in atelocollagen 3 weeks after chondrogenic induction. The chondrogenic potential of the PDPCs was also verified by histochemical staining for type II collagen protein. Increased production of glycosaminoglycan shows that the PDPCs in atelocollagen could differentiate into chondrocytes under a chondrogenic environment. PDPCs can therefore be used as a cell source for cell-based therapies targeted toward the articular cartilage of the knee.     

Xeno-free chondrogenesis of bone marrow mesenchymal stromal cells: towards clinical-grade chondrocyte production

Current cell-based cartilage therapies relay on articular cartilage-derived autologous chondrocytes as a cell source, which possesses disadvantages, such as, donor site morbidity and dedifferentiation of chondrocytes during in vitro expansion. Due to these and other limitations, novel cell sources and production strategies are needed. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are a fascinating alternative, but they are not spontaneously capable of producing hyaline cartilage-like repair tissue in vivo. In vitro pre-differentiation of BM-MSCs could be used to produce chondrocytes for clinical applications. However, clinically compatible defined and xeno-free differentiation protocol is lacking. Hence, this study aimed to develop such chondrogenic differentiation medium for human BM-MSCs. We assessed the feasibility of the medium using three human BM-MSCs donors and validated the method by comparing BM-MSCs to three other cell types holding potential for articular cartilage repair. The effectiveness of the method was compared to conventional serum-free and commercially available chondrogenic differentiation media. The results show that the defined xeno-free differentiation medium is at least as efficient as conventionally used serum-free chondrogenic medium and performed significantly better on all cell types tested compared to the commercially available chondrogenic medium.     

Effects of oxygen and culture system on in vitro propagation and redifferentiation of osteoarthritic human articular chondrocytes

Regenerative medicine-based approaches for the repair of damaged cartilage rely on the ability to propagate cells while promoting their chondrogenic potential. Thus, conditions for cell expansion should be optimized through careful environmental control. Appropriate oxygen tension and cell expansion substrates and controllable bioreactor systems are probably critical for expansion and subsequent tissue formation during chondrogenic differentiation. We therefore evaluated the effects of oxygen and microcarrier culture on the expansion and subsequent differentiation of human osteoarthritic chondrocytes. Freshly isolated chondrocytes were expanded on tissue culture plastic or CultiSpher-G microcarriers under hypoxic or normoxic conditions (5% or 20% oxygen partial pressure, respectively) followed by cell phenotype analysis with flow cytometry. Cells were redifferentiated in micromass pellet cultures over 4 weeks, under either hypoxia or normoxia. Chondrocytes cultured on tissue culture plastic proliferated faster, expressed higher levels of cell surface markers CD44 and CD105 and demonstrated stronger staining for proteoglycans and collagen type II in pellet cultures compared with microcarrier-cultivated cells. Pellet wet weight, glycosaminoglycan content and expression of chondrogenic genes were significantly increased in cells differentiated under hypoxia. Hypoxia-inducible factor-3α mRNA was up-regulated in these cultures in response to low oxygen tension. These data confirm the beneficial influence of reduced oxygen on ex vivo chondrogenesis. However, hypoxia during cell expansion and microcarrier bioreactor culture does not enhance intrinsic chondrogenic potential. Further improvements in cell culture conditions are therefore required before chondrocytes from osteoarthritic and aged patients can become a useful cell source for cartilage regeneration.     

Ser/Thr-phosphoprotein phosphatases in chondrogenesis: neglected components of a two-player game

Protein phosphorylation plays a determining role in the regulation of chondrogenesis in vitro. While signalling pathways governed by protein kinases including PKA, PKC, and mitogen-activated protein kinases (MAPK) have been mapped in great details, published data relating to the specific role of phosphoprotein phosphatases (PPs) in differentiating chondroprogenitor cells or in mature chondrocytes is relatively sparse. This review discusses the known functions of Ser/Thr-specific PPs in the molecular signalling pathways of chondrogenesis. PPs are clearly equally important as protein kinases to counterbalance the effect of reversible protein phosphorylation. Of the main Ser/Thr PPs, some of the functions of PP1, PP2A and PP2B have been characterised in the context of chondrogenesis. While PP1 and PP2A appear to negatively regulate chondrogenic differentiation and maintenance of chondrocyte phenotype, calcineurin is an important stimulatory mediator during chondrogenesis but becomes inhibitory in mature chondrocytes. Furthermore, PPs are implicated to be mediators during the pathogenesis of osteoarthritis that makes them potential therapeutic targets to be exploited in the close future. Among the many yet unexplored targets of PPs, modulation of plasma membrane ion channel function and participation in mechanotransduction pathways are emerging novel aspects of signalling during chondrogenesis that should be further elucidated. Besides the regulation of cellular ion homeostasis, other potentially significant novel roles for PPs during the regulation of in vitro chondrogenesis are discussed.     

In vitro differentiation of bone and hypertrophic cartilage from periosteal-derived cells

Periosteal cells were enzymatically liberated from the tibiae of young chicks, introduced into cell culture, and allowed to reach confluence. The morphology of the cells gave the impression of a relatively homogeneous population of fibroblast-like cells. These cultured cells did not overtly express osteogenic or chondrogenic properties as judged by their morphology and the lack of reactivity with probes to phenotype-specific antigens of osteoblasts or chondrocytes. The cells were then replated at relatively high density and chronologically evaluated for the differentiation of bone and cartilage. These replated cells formed a multi-layer of fibroblast-like cells, the top portion of which eventually differentiated into bone tissue as evidenced by the presence of mineralization and immunocytochemical reactivity to bone Gla protein- and osteocyte-speciflc probes. Cells below this distinctive top layer differentiated into chondrocytes, which eventually further developed into hypertrophie chondrocytes as evidenced by their morphology and the presence of immunoreactive type X collagen in the matrix. Mineralization was also observed in the territorial matrix of these hypertrophie chondrocytes, when the culture was augmented with β-glycerophosphate. Periosteal-derived cells replated at a lower density as controls did not show signs of osteochondrogenic differentiation. These observations suggest that periosteal-derived cells of young chicks contain mesenchymal cells which possess the potential to undergo terminal differentiation into osteogenic or chondrogenic phenotypes depending on local environmental or positional cues.     

Esophageal cancer related gene 4 (ECRG4) is a marker of articular chondrocyte differentiation and cartilage destruction

With the aim of identifying novel genes regulating cartilage development and degeneration, we screened a cartilage-specific expressed sequence tag database. Esophageal cancer related gene 4 (ECRG4) was selected, based on the criteria of ‘chondrocyte-specific’ and ‘unknown function.’ ECRG4 expression was particularly abundant in chondrocytes and cartilage, compared to various other mouse tissues. ECRG4 is a secreted protein that undergoes cleavage after secretion. The protein is specifically expressed in chondrocytes in a manner dependent on differentiation status. The expression is very low in mesenchymal cells, and dramatically increased during chondrogenic differentiation. The ECRG4 level in differentiated chondrocytes is decreased during hypertrophic maturation, both in vitro and in vivo, and additionally in dedifferentiating chondrocytes induced by interleukin-1β or serial subculture, chondrocytes of human osteoarthritic cartilage and experimental mouse osteoarthritic cartilage. However, ectopic expression or exogenous ECRG4 treatment in a primary culture cell system does not affect chondrogenesis of mesenchymal cells, hypertrophic maturation of chondrocytes or dedifferentiation of differentiated chondrocytes. Additionally, cartilage development and organization of extracellular matrix are not affected in transgenic mice overexpressing ECRG4 in cartilage tissue. However, ectopic expression of ECRG4 reduced proliferation of primary culture chondrocytes. While the underlying mechanisms of ECRG4 expression and specific roles remain to be elucidated in more detail, our results support its function as a marker of differentiated articular chondrocytes and cartilage destruction.     

The Wnt antagonist Wif-1 interacts with CTGF and inhibits CTGF activity

Wnt inhibitory factor 1 (Wif-1) is a secreted antagonist of Wnt signalling. We recently demonstrated that this molecule is expressed predominantly in superficial layers of epiphyseal cartilage but also in bone and tendon. Moreover, we showed that Wif-1 is capable of binding to several cartilage-related Wnt ligands and interferes with Wnt3a-dependent Wnt signalling in chondrogenic cells. Here we provide evidence that the biological function of Wif-1 may not be confined to the modulation of Wnt signalling but appears to include the regulation of other signalling pathways. Thus, we show that Wif-1 physically binds to connective tissue growth factor (CTGF/CCN2) in vitro, predominantly by interaction with the C-terminal cysteine knot domain of CTGF. In vivo such an interaction appears also likely since the expression patterns of these two secreted proteins overlap in peripheral zones of epiphyseal cartilage. In chondrocytes CTGF has been shown to induce the expression of cartilage matrix genes such as aggrecan (Acan) and collagen2a1 (Col2a1). In this study we demonstrate that Wif-1 is capable to interfere with CTGF-dependent induction of Acan and Col2a1 gene expression in primary murine chondrocytes. Conversely, CTGF does not interfere with Wif-1-dependent inhibition of Wnt signalling. These results indicate that Wif-1 may be a multifunctional modulator of signalling pathways in the cartilage compartment.