Chondrocyte-conditioned Medium Research Articles

Conditioned Medium of Mesenchymal Stem Cells Prevents Degenerative Changes in Ankle Osteoarthritis in Rat Models

Category:Ankle; Ankle Arthritis; Basic Sciences/Biologics; TraumaIntroduction/Purpose:Osteoarthritis (OA) is the most common degenerative joint disorder, characterized mainly by progressive articular cartilage degradation, subchondral bone thickening, osteophyte formation, and synovial inflammation. About 80% of ankle OA is believed to be post-traumatic and affects the younger population leading to a higher risk of disabilities during life. Mesenchymal stem cells (MSCs) have shown promising effects in preventing the degenerative process of OA via paracrine effect and great differentiation potential. Recently, using chondrocyte conditioned medium (CCM) that contains all necessary secretomes was shown to enhance MSC differentiation and regeneration of the cartilage tissue. In this study, we aimed to assess the effects of intraarticular injection of MSCs plus CCM on the degenerative process of monoiodoacetate (MIA)-induced ankle OA in rats.Methods:Fifty male rats were randomly divided into 5 groups (n=10): Non-treated control group (C); the group treated with intra-articular Hyaluronic acid (HA). Hyaluronic acid has shown beneficial effects on the degenerative process of OA in different reports. The other treatment groups received intraarticular synovial derived MSCs (5′105), intraarticular CCM, and intraarticular MSCs combined with CCM (MSC+CCM), respectively. Induction of ankle OA was conducted via 1 mg MIA injection in the right tibiotarsal joint for two consecutive days. Three months after starting the treatments, radiological assessments of joint space width (Normal=0, reduced=1, abcent=2) and osteophytes of the tibia and talus (No osteophyte=0, small=1, moderate=2, severe=3) were performed. Ankle specimens were obtained for histopathological examination of the joint surface, matrix, cell distribution, cell viability, subchondral bone, and mineralization; lower scores indicated more severe damages. Scores for each assessment were recorded. Mann-Whitney U test was used for comparisons and p-value<0.05 was considered statistically significant.Results:Our findings showed significant differences between the controls and the treatment groups regarding all pathological scores including joint surface, matrix, cell distribution, cell viability, subchondral bone, and mineralization, that were higher and radiological scores including joint space width and osteophytes of the tibia and talus that were lower in the controls (P<0.05; Figure 1). Among the treatment groups, HA-treated rats showed significantly lower pathological and higher radiological scores compared to CCM, MSC, and MSC+CCM (P<0.05). Among CCM, MSC, and MSC+CCM treated groups, no significant difference was found regarding the pathological and radiological scores (P>0.05). However, although not statistically noticeable, the MSC+CCM group showed slightly better outcomes, both radiologically and histopathologically, compared to the MSC and CCM groups (Figure 1).Conclusion:Our results showed that the combination of CCM can have a similar effect on the degenerative changes in ankle OA. However, the combination of MSC+CCM showed better scores regarding pathological and radiological evaluation. Although further studies with a greater population seem to be needed to validate these outcomes, our study indicates the efficacy of CCM in reducing the degenerative rate of OA. CCM is also known as 'secret factors' since it is still a 'black box' in terms of identifying its content and effects.

Human chondrocyte-conditioned medium promotes chondrogenesis of bone marrow stem cells

Abstract Background Cell-based therapy for osteoarthritis requires culturing of good quality cells, especially with a chondrogenic lineage, for implantation. Objective To investigate the ability of chondrocyte-conditioned medium (CCM) to induced chondrogenesis. Methods Bone marrow mesenchymal stem cells (BMSCs) were subjected to chondrogenic induction using CCM and chondrocyte induction medium (CIM). The optimal condition for the collection of CCM was evaluated by quantifying the concentration of secreted proteins. The chondrogenic efficiency of BMSCs induced by CCM (iCCM) was evaluated using immunocytochemical analysis, Safranin-O staining, and gene expression. Results Protein quantification revealed that CCM obtained from cells at passage 3 at the 72 h collection point had the greatest amount of protein. Supplementation of CCM results in the aggregation of BMSCs; however, no clumping was visible as in iCIM. The expression of collagen type 2 was detected as early as day 7 for all groups except for non-induced BMSCs; however, the level of expression decreased with culture time. Similarly, all tested groups showed positive staining for Safranin-O as early as day 7. The induction of BMSCs by CCM caused the down-regulation of collagen type 1, along with the up-regulation of the collagen type 2, ACP and SOX9 genes. Conclusion The optimum CCM to induce BMSC into chondrocytes was collected at passage 3 after 72 h and was used in a 50:50 ratio of CCM to fresh medium.

Abstract 50: Chondrogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells by Co-Culture with Xenogeneic Swine Chondrocytes

PURPOSE: Cartilage has very limited ability for self repair and regeneration. Although autologous chondrocytes can be obtained from uninvolved areas of the joint to re-implant and repair lesions, the additional joint morbidity and costly in vitro expansion of primary chondrocytes limits widespread clinical application. Human bone marrow mesenchymal stem cells (hBMSCs) can be obtained by minimally invasive methods and could be a favorable source for cartilage regeneration. However, controlling the differentiation of BMSCs into the chondrocyte phenotype has hampered clinical implementation. The stimulation of human hBMSCs with xenogeneic chondrocytes has great potential for increasing the numbers of chondrogenic cells for cellular cartilage repair. The aim of this study was to determine the differentiation effects on human BMSCs and swine BMSc (sBMSc) by co-culture with swine chondrocytes or culture medium conditioned by chondrocytes. METHODS: Swine articular chondrocytes were isolated and grown in standard chondrocyte culture medium that was collected and used as chondrocyte-conditioned medium (CCM). hBMSCs and sBMSCs were expanded in normal bone marrow medium for 3 weeks and transferred to 6 well plates and Transwell plates for an additional one week. Then BMSCs cultured in 6-well plates were treated with CCM for 10 days. In the transwell cultures swine articular chondrocytes (sACs) were added in co-culture with the BMSCs for 10 days. Four different groups were performed: (1) hCCM, (2) hTranswell, (3) sCCM, (4) sTranswell. Real-time polymerase chain reaction was performed to evaluate the gene expression of collagen type II (COL2A1), and aggrecan (ACAN). RESULTS: After 10 days of culture, COL2A1 gene expression in hTranswell group was significantly increased 20-fold (p < 0.00001), compared with hCCM group, indicating that co-culture with chondrocytes had a greater effect on chondrogenic differentiation than the conditioned medium alone. ACAN expression in hTranswell group was significantly higher than hCCM group (p = 0.003), which further demonstrated that xenogeneic swine chondrocytes have a strong effect on chondrogenic differentiation of hBMSCs. ACAN gene expression in sTranswell group showed 30-fold greater than sCCM group (p = 0.00022), indicating this autologous co-culture model has potential to differentiate both hBMSC and sBMSC towards a chondrogenic phenotype. CONCLUSION: These results indicate that both xenogeneic co-culture models are capable differentiating hBMSC towards a chondrogenic phenotype. This approach could eliminate the need for autologous chondrocyte harvest by using a plentiful supply of xenogeneic chondrocytes to chondrodifferentiate a patient’s own MSCs for cartilage repair. Recent development of alpha1,3-galactosyltransferase gene-knockout (GalT-KO) swine may even provide a source of xenogeneic MSCs that can be used in humans without the consequence of immune rejection.

Enhanced osteogenic commitment of murine mesenchymal stem cells on graphene oxide substrate

BackgroundTissue engineering is an interdisciplinary field that attempts to restore or regenerate tissues and organs through biomimetic fabrication of scaffolds with specific functionality. In recent years, graphene oxide (GO) is considered as promising biomaterial due to its nontoxicity, high dispersity, and hydrophilic interaction, and these characteristics are key to stimulating the interactions between substrates and cells.MethodIn this study, GO substrates were fabricated via chemically immobilizing GO at 1.0 mg/ml on glass slides. Furthermore, we examined the osteogenic responses of murine mesenchymal-like stem cells, C3H10T1/2 cells, on GO substrates.ResultsC3H10T1/2 cells on GO substrates resulted in increased cell surface area, enhanced cellular adhesions, and instigated osteogenic differentiation. Furthermore, priming of C3H10T1/2 cells with chondrocyte-conditioned medium (CM) could further induce a synergistic effect of osteogenesis on GO substrates.ConclusionsAll of these data suggest that GO substrate along with CM is suitable for upregulating osteogenic responses of mesenchymal stem cells.

HIF-1-Dependent IL-6 Activation in Articular Chondrocytes Initiating Synovitis in Femoral Head Ischemic Osteonecrosis.

Ischemic osteonecrosis of the femoral head in children is associated with chronic hip synovitis and increased levels of the pro-inflammatory cytokine interleukin-6 (IL-6) in the synovial fluid due to unknown mechanisms. The purpose of this study was to investigate hypoxia-inducible factor-1 (HIF-1) activation as a molecular mechanism linking the induction of ischemic osteonecrosis to IL-6 production and the initiation of hip synovitis. Ischemic osteonecrosis was surgically induced in the right femoral head of 6 piglets. A histologic score, synovial fluid volume, and IL-6 level were used to assess hip synovitis. IL-6 immunostaining of articular cartilage and synovial tissue was performed as well. To study the role of HIF-1 in IL-6 activation, in vitro experiments using an HIF-1α activator (deferoxamine) and inhibitor (HIF-1 small interfering-RNA [siRNA]) were carried out. Synovial cell responses to hypoxic chondrocyte-conditioned media with and without an IL-6 receptor blocker (tocilizumab) were assessed on the basis of IL-1β and tumor necrosis factor-alpha (TNF-α) gene expressions and with a synovial cell-proliferation assay. Induction of ischemic osteonecrosis produced hip synovitis and increased IL-6 levels in the synovial fluid. Immunostaining and protein analysis demonstrated articular chondrocytes as a source of increased IL-6 production. When articular chondrocytes were cultured under hypoxic conditions, significantly increased HIF-1α and IL-6 expressions were observed. Under hypoxic culture conditions, IL-6 gene expression was significantly increased by HIF-1α activation using deferoxamine and inhibited by HIF-1α inhibition using HIF-1 siRNA. Synovial cells exposed to hypoxic chondrocyte-conditioned medium showed significant increases in IL-1β and TNF-α gene expressions and cell proliferation, which were inhibited by the IL-6 receptor blocker tocilizumab. Induction of ischemic osteonecrosis results in IL-6 production in the articular cartilage through an HIF-1-dependent pathway. IL-6 produced by hypoxic articular chondrocytes stimulates inflammatory cytokine responses in synovial cells, which were significantly decreased by tocilizumab. This study provides new insight into the inherent relationship between the induction of ischemia and the initiation of hip synovitis following ischemic osteonecrosis and suggests a potential therapeutic target in the treatment of the synovitis.

Chondrogenesis by bone marrow-derived mesenchymal stem cells grown in chondrocyte-conditioned medium for auricular reconstruction.

Bone marrow-derived mesenchymal stem cells (BMSCs) can be obtained by minimally invasive means and would be a favourable source for cell-based cartilage regeneration. However, controlling the differentiation of the BMSCs towards the desired chondrogenic pathway has been a challenge hampering their application. The major aim of the present study was to determine if conditioned medium collected from cultured auricular chondrocytes could promote chondrogenic differentiation of BMSCs. Auricular chondrocytes were isolated and grown in BMSC standard culture medium (SM) that was collected and used as chondrocyte-conditioned medium (CCM). The BMSCs were expanded in either CCM or SM for three passages. Cells were seeded onto fibrous collagen scaffolds and precultured for 2weeks with or without transforming growth factor-beta 3 (TGF-β3). After preculture, constructs were implanted subcutaneously in nude mice for 6 and 12weeks and evaluated with real-time polymerase chain reaction, histology, immunohistochemistry and biochemistry. Real-time polymerase chain reaction results showed upregulation of COL2A1 in the constructs cultured in CCM compared with those in SM. After 12weeks in vivo, abundant neocartilage formation was observed in the implants that had been cultured in CCM, with or without TGF-β3. In contrast, very little cartilage matrix formation was observed within the SM groups, regardless of the presence of TGF-β3. Osteogenesis was only observed in the SM group with TGF-β3. In conclusion, CCM even had a stronger influence on chondrogenesis than the supplementation of the standard culture medium with TGF-β3, without signs of endochondral ossification. Efficient chondrogenic differentiation of BMSCs could provide a promising alternative cell population for auricular regeneration. Copyright © 2016 John Wiley & Sons, Ltd.

Osteogenic priming of mesenchymal stem cells by chondrocyte-conditioned factors and mineralized matrix.

Transient cartilage and a mineralizing microenvironment play pivotal roles in mesenchymal cell ossification during bone formation. In order to recreate these microenvironmental cues, C3H10T1/2 murine mesenchymal stem cells (MSCs) were exposed to chondrocyte-conditioned medium (CM) and seeded onto three-dimensional mineralized scaffolds for bone regeneration. Expansion of C3H10T1/2 cells with CM resulted in enhanced expression levels of chondrogenic markers such as aggrecan, type II collagen, type X collagen, and Sox9, rather than of osteogenic genes. Interestingly, CM expansion led to reduced expression levels of osteogenic genes such as alkaline phosphatase (ALP), type I collagen, osteocalcin, and Runx2. However, CM-expanded C3H10T1/2 cells showed enhanced osteogenic differentiation as indicated by increased ALP and Alizarin Red S staining upon osteogenic factor exposure. In vivo, CM-expanded C3H10T1/2 mesenchymal cells were seeded onto mineralized scaffolds (fabricated with polydopamine and coated with simulated body fluids) and implanted into critical-sized calvarial-defect mouse models. After 8weeks of implantation, mouse skulls were collected, and bone tissue regeneration was evaluated by micro-computed tumography and Masson's trichrome staining. In accordance with the in vitro analysis, CM-expanded C3H10T1/2 cells gave enhanced bone mineral deposition. Thus, chondrocyte-conditioned factors and a mineralized microenvironment stimulate the bone formation of MSCs.

AB0074 Thrombospondin-1: a novel chondroitin sulfate target for osteoarthritis treatment

BackgroundRecent works by our group provide evidences of the usefulness of proteomics techniques for anti-osteoarthritis (OA) drug screening [1-3].ObjectivesThe aim of this study is to identify protein markers for monitoring...

Type IIB procollagen n-propeptide inhibits angigenesis in cartilage

Purpose: A fragment of type II procollagen N-propeptide, PIIBNP, contains the classic integrin binding sequence RGDRGD. This sequence is conserved across species, indicating potential biological function. We have previously shown that recombinant PIIBNP induces death of tumor cells that express integrin αvβ3 and αvβ5. Data from HUVEC cell tube formation assay, mouse aortic ring assay and mouse corneal assay showed that recombinant PIIBNP inhibits angiogenesis both in vitro and in vivo (1). PIIBNP is synthesized by chondrocytes liberated to matrix in cartilage. The goal of this study was to determine the role that PIIBNP plays in cartilage in keeping cartilage avascular and intact. Methods: Detection of PIIBNP from cartilage. Cartilage was isolated from limbs of P1 mice. Cartilage was smashed by crusher and PIIBNP was extracted with SDS loading buffer, resolved by SDS-PEGE, and detected by western blotting. Mouse chondrocyte isolation. Chondrocytes were obtained by enzymatic dissociation of ventral regions of rib cages from newborn mice. Chondrocytes were incubated with or without the presence of MMP inhibitor for 48 hours. The medium from chondrocytes culture were used as the conditioned medium. Cartilage-aorta co-culture: Bovine and mice bones were used as control in the cartilage-aorta co-culture experiments. Mouse cartilage or bone was placed into inserts of a 24-well plate and cocultured with aorta in 3D matrigel for 8 days. The microvessel outgrowth was visualized under a light microscope (NIKON ECLIPS E800) and images were digitally photographed using a Q capture Retiga 2000R camera, and quantified with an Image J software. Results: We have been able to detect PIIBNP in cartilage (Fig. 1A) and chondrocytes conditioned medium (Fig. 1B). In order to reasonably hypothesize an effect of PIIBNP in cartilage, we sought to determine weather cartilage inhibits angiogenesis in mouse aortic ring assays. The results from cartilage-aorta coculture experiments showed that microvessel outgrowth was seen only in control and in the presence of bone, but not in the presence of cartilage (Fig. 2A). Since we and other have detected PIIBNP in cartilage, it is possible that the PIIBNP in cartilage inhibited the microvessel outgrowth. To further confirm this, we performed aorta angiogenesis assay in the presence of chondrocyte conditioned medium and found that the chondrocyte conditioned medium inhibited 80 percent of the microvessel outgrowth as compared with the control (Fig B). MMT inhibitor GM6001 inhibits the production of PIIBNP and the cartilage treated with GM6001 did not inhibit microvessel outgrowth in mouse aortic ring assay. These data suggested that natural PIIBNP in cartilage inhibits angiogenesis. To further investigate the mechanism behind the antiangiogenesis of PIIBNP, we treated HUVEC cells in culture with recombinant proteins. Treatments with PIIBNP, but not GST or mPIIBNP, induced disappearance of VE-cadherin at cell-cell junctions (Fig 3A), suggesting that the engagement of PIIBNP on cell surface elicited a signaling events involved in the inhibition of angiogenesis by PIIBNP. This hypothesis is supported by the fact that the PIIBNP, but not GST or mPIIBNP, induced activation of the Src kinase (Fig 3B), consistent with previous report that integrin αvβ3 signaling induces Src activation and VE-cadherin internalization. Conclusions: We previously showed that recombinant PIIBNP induced tumor cell death via binding to integrin αvβ3 and αvβ5, and suppresses tumor growth in nude mice. The data presented here suggested that the natural occurring PIIBNP in cartilage most likely inhibits angiogenesis. We have detected the existence of PIIBNP from P1 chondrocyte conditioned medium which also inhibits angiogenesis. Since PIIBNP reaches the highest level during cartilage formation and it kills tumor cell, it is an excellent candidate for the molecular mechanism by which cartilage remains avascular and intact.

Chondrogenic Priming Adipose-Mesenchymal Stem Cells for Cartilage Tissue Regeneration

Chondrocytes lose their ability to produce cartilaginous matrix during multiplication in culture through repeated passages, resulting in inferior tissue phenotype. To overcome the limited amount of primary chondrocytes, we aimed to determine the optimal culture condition for in vitro/in vivo cartilage regeneration using human adipose-derived mesenchymal stem cells (AMSCs). To evaluate the effects exerted by the chondrocytic culture condition on AMSC, we utilized chondrocyte conditioned medium (CM) and/or co-culture methods to prime and differentiate AMSCs. We evaluated ultimate in vivo engineered cartilage with primed AMSCs with that of chondrocytes. To examine the link between conditioned factors and proliferation/differentiation, cell cycle progression of AMSCs were examined using 5-ethynyl-2'-deoxyuridine (EdU), and gene expression was monitored. We report that AMSCs can be stimulated to become chondrogenic cells when expanded with chondrocyte CM. Polymeric scaffolds co-seeded with CM- expanded AMSCs and primary chondrocytes resulted in in vivo cartilaginous tissues with similar biochemical content to constructs seeded with chondrocytes alone. These results indicate that chondrocyte CM consists of suitable morphogenetic factors that induce the chondrogenic priming of AMSCs for cartilage tissue engineering.

Osteoarthritic chondrocyte–secreted morphogens induce chondrogenic differentiation of human mesenchymal stem cells

The potential of stem cells to repair compromised cartilage tissue, such as in osteoarthritis (OA), depends strongly on how transplanted cells respond to factors secreted from the residing OA chondrocytes. This study was undertaken to determine the effect of morphogenetic signals from OA chondrocytes on chondrogenic differentiation of human mesenchymal stem cells (MSCs). The effect of OA chondrocyte-secreted morphogens on chondrogenic differentiation of human MSCs was evaluated using a coculture system involving both primary and passaged OA chondrocytes. The findings were compared against findings for human MSCs cultured in OA chondrocyte-conditioned medium. Gene expression analysis, biochemical assays, and immunofluorescence staining were used to characterize the chondrogenic differentiation of human MSCs. Mass spectrometry analysis was used to identify the soluble factors. Numerical analysis was carried out to model the concentration profile of soluble factors within the human MSC-laden hydrogels. The human MSCs cocultured with primary OA chondrocytes underwent chondrogenic differentiation even in the absence of growth factors; however, the same effect could not be mimicked using OA chondrocyte-conditioned medium or expanded cells. Additionally, the cocultured environment down-regulated hypertrophic differentiation of human MSCs. Mass spectrometry analysis demonstrated cell-cell communication and chondrocyte phenotype-dependent effects on cell-secreted morphogens. The experimental findings, along with the results of the numerical analysis, suggest a crucial role of soluble morphogens and their local concentrations in the differentiation pattern of human MSCs in a 3-dimensional environment. The concept of using a small number of chondrocytes to promote chondrogenic differentiation of human MSCs while preventing their hypertrophic differentiation could be of great importance in formulating effective stem cell-based cartilage repair.

Conditioned Media Enhance Osteogenic Differentiation on Poly(L-lactide-co-ε-caprolactone)/Hydroxyapatite Scaffolds and Chondrogenic Differentiation in Alginate

The biochemical factors that regulate cell proliferation and differentiation can provide a means of optimizing culture conditions to develop a tissue-engineered osteochondral construct. Thus, the objectives of this study were to determine the effects of chondrocyte conditioned medium (CM) on the osteogenic differentiation of mesenchymal stem cells (MSCs) cultured on poly(L-lactide-co-ε-caprolactone)/hydroxyapatite (PLA/PCL/HAP) scaffolds and to determine the effect of osteoblast CM on the chondrogenic differentiation of MSCs cultured in alginate. In addition, the biomaterial's effect on MSC differentiation was also investigated. MSCs were grown in two groups: (1) on porous PLA/PCL/HAP scaffolds in osteogenic differentiation medium or (2) encapsulated in alginate in chondrogenic differentiation medium. CM was taken from one group and administered to the 'opposite' group in volumetric concentrations of 25% or 50% at each medium change. The osteogenic group samples that were administered chondrocyte CM showed higher alkaline phosphatase activity than the controls that were not administered CM. Additionally, the cells that were given chondrocyte CM had higher osteocalcin and sialoprotein expression than the controls. Samples in the chondrogenic group that were administered osteoblast CM at a volumetric concentration of 50% produced more sGAG than the controls. The aggrecan and Sox9 expression was significantly higher in the samples given 50% CM as compared to the controls. The study also showed that culturing cells in alginate, without differentiation medium, can produce similar levels of differentiation as cells that were administered differentiation medium.

ADAM‐8 isolated from human osteoarthritic chondrocytes cleaves fibronectin at Ala271

Fibronectin fragments are thought to play a critical role in the initiation and progression of cartilage degradation in arthritis. In a recent study, fibronectin neoepitopes resulting from cleavage of intact fibronectin at the Ala(271)/Val(272) scissile bond, generating an approximately 30-kd fragment with the new C-terminus VRAA(271) and an approximately 50-85-kd fragment with the new N-terminus (272)VYQP, were identified in osteoarthritis (OA) cartilage. The present study was undertaken to isolate the enzymes responsible for this cleavage from human OA chondrocytes. Fibronectin-degrading activity in human OA chondrocyte-conditioned medium (OACCM) was purified using conventional chromatography. A fluorescent peptide was developed based on the fibronectin scissile bond (269)RAA downward arrowVal(272), and this peptide was used to track fibronectinase activity during purification. Western blotting with antibodies that detect the fibronectin neoepitopes VRAA(271) and (272)VYQP was used to confirm cleavage of intact fibronectin by the enzymatically active fractions. Mass spectrometry was used to identify the proteins found in the fibronectinase-enriched fractions, with further confirmation by Western blotting. In addition, a recombinant enzyme identified by mass spectrometry was tested by Western blotting and dimethylmethylene blue assay for its ability to produce fibronectin neoepitopes in OA cartilage. Purification of OACCM by chromatography resulted in isolation of a fibronectin-degrading enzyme, and mass spectrometry identified ADAM-8 as the fibronectinase present in these preparations. Furthermore, treatment of OA cartilage with recombinant human ADAM-8 promoted cartilage catabolism. The results of this study identify ADAM-8 as a fibronectinase in human OA chondrocytes. Because ADAM-8 is capable of producing the fibronectin neoepitopes VRAA(271) and (272)VYQP in human OA cartilage, this enzyme may be an important mediator of cartilage catabolism.

Regulation of In Vitro Vascular Calcification by BMP4, VEGF and Wnt3a

Vascular calcification is a common clinical complication of cardiovascular disease, diabetes and end-stage renal failure, associated with significant morbidity and mortality. In this study we demonstrate that factors secreted by the hypertrophic chondrocytes induce matrix mineralization and osteoblastic transformation in cultured mouse vascular smooth muscle cells (VSMCs). In addition, these factors render VSMCs responsive to BMP4 and Wnt3a ligands. Neither BMP-4 nor Wnt3a could induce mineralization in short-term (up to 8 days) cultures of primary mouse VSMCs. However, both ligands act synergistically with the chondrocyte-conditioned medium causing a further increase in VSMC calcification. Finally, we show that commitment of VSMCs towards the BMP-regulated mineralization can be induced by the chondrocyte-secreted bone anabolic factor VEGF. In addition, expression profiling suggests a novel role in vascular calcification for the matrix proteins previously known to regulate bone formation and mineralization (including MMP3, fibulin, 11betahydroxysteroid dehydrogenase 1 and retinoic acid receptor responder 2). The results of this study may contribute to further understanding of the cellular mechanisms responsible for vascular calcification and provide important information for the treatment of this pathology.

Morphogenetic signals from chondrocytes promote chondrogenic and osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are potentially useful cells for musculoskeletal tissue engineering. However, controlling MSC differentiation and tissue formation in vivo remains a challenge. There is a significant need for well-defined and efficient protocols for directing MSC behaviors in vivo. We hypothesize that morphogenetic signals from chondrocytes may regulate MSC differentiation. In micromass culture of MSCs, incubation with chondrocyte-conditioned medium (CCM) significantly enhanced the production of cartilage specific matrix including type II collagen. In addition, incubation of MSCs with conditioned medium supplemented with osteogenic factors induced more osteogenesis and accumulation of calcium and increased ALP activity. These findings reveal that chondrocyte-secreted factors promote chondrogenesis as well as osteogenesis of MSCs during in vitro micromass culture. Moreover, when MSCs expanded with chondrocyte-conditioned medium were encapsulated in hydrogels and subsequently implanted into athymic mice, basophilic extracellular matrix deposition characteristic of neocartilage was evident. These results indicate that articular chondrocytes produce suitable morphogenetic factors that induce the differentiation program of MSCs in vitro and in vivo.

The transglutaminase, Factor XIIIA, is present in articular chondrocytes

Objectives The transglutaminase (TGase) family includes seven different enzymes that catalyse a protein cross-linking reaction resulting in structural and functional alterations in substrate proteins. TGase activity is easily measureable in mature articular cartilage where it may contribute to CPPD deposition disease through its actions on growth factors, crystal components or extracellular matrix proteins. In contrast, low levels of TGase activity are found in chondrocytes from young animals. We previously demonstrated type II TGase protein in articular chondrocytes. Earlier work also suggested the presence of another form of TGase in chondrocytes. We sought to determine if articular chondrocytes contain the TGase, Factor XIIIA (FXIIIA).Methods Western blots with FXIIIA antibody were used to detect FXIIIA in young and old porcine articular chondrocytes and articular cartilage vesicles (ACVs). The presence of FXIIIA mRNA was confirmed by RT-PCR.Results Old chondrocyte conditioned medium, cytosol, and membrane fractions contained FXIIIA protein on Western blots, while less FXIIIA was detectable in cell fractions or media from young chondrocytes. ACVs also contained FXIIIA. FXIIIA mRNA was demonstrated by PCR in old and young chondrocytes.Conclusions FXIIIA is present in articular chondrocytes. FXIIIA levels correlate with TGase activity in chondrocytes. The presence of two forms of TGase in articular chondrocytes suggest an important function for this enzyme family in articular cartilage.

Interaction of strain and interleukin-1 in articular cartilage: effects on proteoglycan synthesis in chondrocytes

In temporomandibular joint disorders, the release of proinflammatory cytokines such as interleukin-1 (IL-1) initiates an inflammatory process disrupting cartilage homeostasis, ultimately leading to cartilage destruction. Additionally, mechanical stimuli affect articular chondrocyte metabolism. While articular chondrocytes generate nitric oxide (NO) in the presence of IL-1 proteoglycan synthesis is consecutively suppressed. The purpose of this study was to assess the effects of proinflammatory cytokines and mechanical strain in the form of cyclic tensile stretch on proteoglycan synthesis in chondrocytes, as compared to the NO competitive inhibitor L-N-monomethyl arginine (LMA), and to assess whether this effect is secondarily related to the activity of growth factors such as transforming growth factor beta (TGF- β). Lapine articular chondrocytes were exposed to one of four different treatment regimens: no cyclic tensile stretch, IL-1, cyclic tensile stretch, or IL-1 plus cyclic tensile stretch. NO production was determined as medium nitrite accumulation. TGF- β-bioactivity in chondrocyte conditioned medium was measured with the mink-lung epithelial cell bioassay. Proteoglycan synthesis was measured as the incorporation of 35-[S]-sodium sulfate into macromolecules separated from unincorporated label by gel filtration on PD-10 columns. In resting chondrocyte cultures, only baseline levels of NO were measured and the application of stretch for 24 h did not affect NO production. Addition of IL-1 provoked a large increase in NO synthesis which was abrogated in the presence of LMA. Application of stretch decreased the IL-1 induced NO synthesis, but did not modify the effect of LMA (being a competitive inhibitor of the inducible NO synthase) inhibiting IL-1 induced NO production. Glucosaminoglycan production was noted as proteoglycan synthesis showing almost no effect of cyclic stretch alone in comparison to the control condition, which correlates with the missing NO production in control and stretch conditions. Addition of IL-1 strongly inhibited proteoglycan synthesis, which was partly restored in the presence of LMA. However, cyclic stretch acted as a stronger restorer of proteoglycan synthesis in IL-1 treated conditions in the absence, and even more in the presence, of LMA. It was concluded that motion in the form of cyclic tensile stretch is a remarkable anti-inflammatory stimulus reversing the IL-1 induced suppression of proteoglycan synthesis in chondrocytes. These findings have therapeutic implications for the treatment of temporomandibular joint disorders, supporting early onset of postoperative and post-traumatic continuous passive motion therapy.

Vascular endothelial growth factor (VEGF) in cartilage neovascularization and chondrocyte differentiation: auto-paracrine role during endochondral bone formation.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induces endothelial cell migration and proliferation in culture and is strongly angiogenic in vivo. VEGF synthesis has been shown to occur in both normal and transformed cells. The receptors for the factor have been shown to be localized mainly in endothelial cells, however, the presence of VEGF synthesis and the VEGF receptor in cells other than endothelial cells has been demonstrated. Neoangiogenesis in cartilage growth plate plays a fundamental role in endochondral ossification. We have shown that, in an avian in vitro system for chondrocyte differentiation, VEGF was produced and localized in cell clusters totally resembling in vivo cartilage. The factor was synthesized by hypertrophic chondrocytes and was released into their conditioned medium, which is highly chemotactic for endothelial cells. Antibodies against VEGF inhibited endothelial cell migration induced by chondrocyte conditioned media. Similarly, endothelial cell migration was inhibited also by antibodies directed against the VEGF receptor 2/Flk1 (VEGFR2). In avian and mammalian embryo long bones, immediately before vascular invasion, VEGF was distinctly localized in growth plate hypertrophic chondrocytes. In contrast, VEGF was not observed in quiescent and proliferating chondrocytes earlier in development. VEGF receptor 2 colocalized with the factor both in hypertrophic cartilage in vivo and hypertrophic cartilage engineered in vitro, suggesting an autocrine loop in chondrocytes at the time of their maturation to hypertrophic cells and of cartilage erosion. Regardless of cell exposure to exogenous VEGF, VEGFR-2 phosphorylation was recognized in cultured hypertrophic chondrocytes, supporting the idea of an autocrine functional activation of signal transduction in this non-endothelial cell type as a consequence of the endogenous VEGF production. In summary we propose that VEGF is actively responsible for hypertrophic cartilage neovascularization through a paracrine release by chondrocytes, with invading endothelial cells as a target. Furthermore, VEGF receptor localization and signal transduction in chondrocytes strongly support the hypothesis of a VEGF autocrine activity also in morphogenesis and differentiation of a mesoderm derived cell.

Insulin-like growth factor binding protein-5 proteolytic activity in ovine articular chondrocyte culture

We have previously demonstrated that ovine articular chondrocytes synthesise and release insulin-like growth factor binding protein-5 (IGFBP-5) which subsequently undergoes proteolysis in the tissue culture medium. The IGFBP-5 proteolytic activity has now been characterised and its substrate specificity analysed using recombinant IGFBP-5 and purified chondrocyte-derived IGFBPs. Iodinated human recombinant IGFBP-5 was incubated with chondrocyte culture or conditioned medium in the presence or absence of various inhibitors. Serine protease inhibitors aprotinin and heparin effectively inhibited the breakdown of IGFBP-5. Furthermore, insulin-like growth factor-I (IGF-I) but not its structural analogues with reduced affinity for IGFBP-5, was also able to partially protect IGFBP-5 from degradation indicating that the association of IGF with the binding protein was required for the inhibition of the proteolytic activity. The inflammatory cytokine interleukin-1 did not have any effect on IGFBP-5 proteolysis. The proteolytic activity appears to be IGFBP-5-specific since the incubation of chondrocyte-derived IGFBPs with chondrocyte conditioned medium resulted in the loss of IGFBP-5 while the levels of the other two IGFBPs (IGFBP-2 and a 24 kDa IGFBP) remained unchanged. In conclusion, we show that IGFBP-5 is specifically cleaved by a serine protease released by primary cultures of ovine articular chondrocytes and also demonstrate the ability of IGF-I to inhibit the proteolytic activity both in cell culture and in cell-free conditions.

Articular chondrocytes produce factors that inhibit maturation of sternal chondrocytes in serum-free agarose cultures: a TGF-beta independent process.

Under normal conditions, articular chondrocytes persist throughout postnatal life, whereas "transient" chondrocytes, which constitute the bulk of prenatal and early postnatal cartilaginous skeleton, undergo maturation, hypertrophy, and replacement by bone cells. The mechanisms regulating the markedly different behavior and fate of articular and transient chondrocytes are largely unclear. In the present study, we asked whether articular chondrocytes possess dominant antimaturation properties which may subtend their ability to persist throughout life. Adult chicken articular chondrocytes and transient maturing chondrocytes from the core region of day 17, chick embryo cephalic sternum were cultured or cocultured in serum-free agarose conditions. When the sternal cells were grown by themselves, they quickly developed into hypertrophic type X collagen-synthesizing cells; however, when they were cocultured with as few as 10% articular chondrocytes or fed with articular chondrocyte-conditioned medium, their maturation was markedly impaired, as revealed by a sharp drop in type X collagen synthesis. A similar, albeit less potent, antimaturation activity characterized resting and proliferating immature chondrocytes isolated from other regions of embryonic sternum. Transforming growth factor-beta 2 (TGF-beta 2) was previously suggested to be an inhibitor of chondrocyte maturation. We found, however, that treatment with a neutralizing antiserum to TGF-beta did not counteract the inhibition of maturation in cocultures of articular and maturing core sternal chondrocytes. Indeed, articular chondrocytes produced and accumulated relatively low levels of TGF-beta in their culture medium, about 15 ng/ml/48 h, of which over 90% was latent; surprisingly, maturing sternal core chondrocytes accumulated over 10-fold more TGF-beta in the medium, about 150 ng/ml/48 h, of which over 20% was endogenously active. These results indicate that articular chondrocytes do possess dominant antimaturation properties which appear to be TGF-beta independent. The TGF-beta s may thus have a more prominent role in the terminal phases of chondrocyte maturation, as indicated by their abundance and greater activity in hypertrophic chondrocytes.