Chondrocyte Cultivation in Hyaluronan-Tyramine Cross-Linked Hydrogel

The application of chondrocyte-seeded hydrogels, such as tyramine-substituted hyaluronan, is a highly promising strategy for damaged cartilage treatments. The rheological characteristics of hyaluronan-tyramine derivative scaffold were determined together with the hydrogel swelling, the water uptake, and the morphological characterization. The chondrocytes seeded in hyaluronan-tyramine hydrogel were viable and expressed markers typical for differentiated chondrocytes with no signs of hypertrophy or extensive expression of matrix metalloproteinases. The lyophilized unseeded hyaluronan-tyramine scaffolds were also implantated into trochlea osteochondral defects in rabbits. In conclusion, the hyaluronan-tyramine derivative was proven to be advisable material as a scaffold for cartilage reconstitution.     

Mechanical Forces Induce Changes in VEGF and VEGFR-1/sFlt-1 Expression in Human Chondrocytes

Expression of the pro-angiogenic vascular endothelial growth factor (VEGF) stimulates angiogenesis and correlates with the progression of osteoarthritis. Mechanical joint loading seems to contribute to this cartilage pathology. Cyclic equibiaxial strains of 1% to 16% for 12 h, respectively, induced expression of VEGF in human chondrocytes dose- and frequency-dependently. Stretch-mediated VEGF induction was more prominent in the human chondrocyte cell line C-28/I2 than in primary articular chondrocytes. Twelve hours of 8% stretch induced VEGF expression to 175% of unstrained controls for at least 24 h post stretching, in promoter reporter and enzyme-linked immunosorbent assay (ELISA) studies. High affinity soluble VEGF-receptor, sVEGFR-1/sFlt-1 was less stretch-inducible than its ligand, VEGF-A, in these cells. ELISA assays demonstrated, for the first time, a stretch-mediated suppression of sVEGFR-1 secretion 24 h after stretching. Overall, strained chondrocytes activate their VEGF expression, but in contrast, strain appears to suppress the secretion of the major VEGF decoy receptor (sVEGFR-1/sFlt-1). The latter may deplete a biologically relevant feedback regulation to inhibit destructive angiogenesis in articular cartilage. Our data suggest that mechanical stretch can induce morphological changes in human chondrocytes in vitro. More importantly, it induces disturbed VEGF signaling, providing a molecular mechanism for a stress-induced increase in angiogenesis in cartilage pathologies.     

Enhanced function of chondrocytes in a chitosan-based hydrogel to regenerate cartilage tissues by accelerating degradability of the hydrogel via a hydrolysable crosslinker

Chitosan-based hydrogels as scaffolds for culturing chondrocytes were prepared using linkers with and without hydrolysable poly(dl -lactide) (PLA) segments. The evaluation of the cultured chondrocytes in them indicated that the accelerated degradation of the hydrogel via hydrolysis of the PLA slightly promoted production of the sulfated glycosaminoglycan and drastically improved that of collagen from the encapsulated chondrocytes, which are the chondrospecific extracellular matrix components. Furthermore, the accelerated degradability significantly upregulated the gene expression for Collagen II production and downregulated that for Collagen I production of the encapsulated chondrocytes. Because major component of the articular cartilage tissue is Collagen II-rich hyaline cartilage, these results suggest the degradation of the scaffolds is an important parameter in cartilage tissue regeneration and the accelerated degradability may have benefits on promotion of cartilage tissue regeneration especially from the viewpoint of hyaline cartilage-like collagen production. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 137, 48893.     

Intra-Articular Administration of Cramp into Mouse Knee Joint Exacerbates Experimental Osteoarthritis Progression

Osteoarthritis (OA) is the most common type of arthritis and is associated with wear and tear, aging, and inflammation. Previous studies revealed that several antimicrobial peptides are up-regulated in the knee synovium of patients with OA or rheumatoid arthritis. Here, we investigated the functional effects of cathelicidin-related antimicrobial peptide (Cramp) on OA pathogenesis. We found that Cramp is highly induced by IL-1β via the NF-κB signaling pathway in mouse primary chondrocytes. Elevated Cramp was also detected in the cartilage and synovium of mice suffering from OA cartilage destruction. The treatment of chondrocytes with Cramp stimulated the expression of catabolic factors, and the knockdown of Cramp by small interfering RNA reduced chondrocyte catabolism mediated by IL-1β. Moreover, intra-articular injection of Cramp into mouse knee joints at a low dose accelerated traumatic OA progression. At high doses, Cramp affected meniscal ossification and tears, leading to cartilage degeneration. These findings demonstrate that Cramp is associated with OA pathophysiology.     

Role of Insulin-Transferrin-Selenium in Auricular Chondrocyte Proliferation and Engineered Cartilage Formation in Vitro

The goal of this study is to determine the effects of Insulin-Transferrin-Selenium (ITS) on proliferation of auricular chondrocytes and formation of engineered cartilage in vitro. Pig auricular monolayer chondrocytes and chondrocyte pellets were cultured in media containing 1% ITS at different concentrations of fetal bovine serum (FBS, 10%, 6%, 2%, 0%), or 10% FBS alone as a control for four weeks. Parameters including cell proliferation in monolayer, wet weight, collagen type I/II/X (Col I, II, X) and glycosaminoglycan (GAG) expression, GAG content of pellets and gene expression associated with cartilage formation/dedifferentiation (lost cartilage phenotype)/hypertrophy within the chondrocyte pellets were assessed. The results showed that chondrocytes proliferation rates increased when FBS concentrations increased (2%, 6%, 10% FBS) in ITS supplemented groups. In addition, 1% ITS plus 10% FBS significantly promoted cell proliferation than 10% FBS alone. No chondrocytes grew in ITS alone medium. 1% ITS plus 10% FBS enhanced cartilage formation in terms of size, wet weight, cartilage specific matrices, and homogeneity, compared to 10% FBS alone group. Furthermore, ITS prevented engineered cartilage from dedifferentiation (i.e., higher index of Col II/Col I mRNA expression and expression of aggrecan) and hypertrophy (i.e., lower mRNA expression of Col X and MMP13). In conclusion, our results indicated that ITS efficiently enhanced auricular chondrocytes proliferation, retained chondrogenic phenotypes, and promoted engineered cartilage formation when combined with FBS, which is potentially used as key supplementation in auricular chondrocytes and engineered cartilage culture.     

Anisotropic Chitosan Scaffolds Generated by Electrostatic Flocking Combined with Alginate Hydrogel Support Chondrogenic Differentiation

The replacement of damaged or degenerated articular cartilage tissue remains a challenge, as this non-vascularized tissue has a very limited self-healing capacity. Therefore, tissue engineering (TE) of cartilage is a promising treatment option. Although significant progress has been made in recent years, there is still a lack of scaffolds that ensure the formation of functional cartilage tissue while meeting the mechanical requirements for chondrogenic TE. In this article, we report the application of flock technology, a common process in the modern textile industry, to produce flock scaffolds made of chitosan (a biodegradable and biocompatible biopolymer) for chondrogenic TE. By combining an alginate hydrogel with a chitosan flock scaffold (CFS+ALG), a fiber-reinforced hydrogel with anisotropic properties was developed to support chondrogenic differentiation of embedded human chondrocytes. Pure alginate hydrogels (ALG) and pure chitosan flock scaffolds (CFS) were studied as controls. Morphology of primary human chondrocytes analyzed by cLSM and SEM showed a round, chondrogenic phenotype in CFS+ALG and ALG after 21 days of differentiation, whereas chondrocytes on CFS formed spheroids. The compressive strength of CFS+ALG was higher than the compressive strength of ALG and CFS alone. Chondrocytes embedded in CFS+ALG showed gene expression of chondrogenic markers (COL II, COMP, ACAN), the highest collagen II/I ratio, and production of the typical extracellular matrix such as sGAG and collagen II. The combination of alginate hydrogel with chitosan flock scaffolds resulted in a scaffold with anisotropic structure, good mechanical properties, elasticity, and porosity that supported chondrogenic differentiation of inserted human chondrocytes and expression of chondrogenic markers and typical extracellular matrix.     

The effect of BMP‐2 and VEGF loading of gelatin‐pectin‐BCP scaffolds to enhance osteoblast proliferation

A composite scaffold of gelatine (Gel)‐pectin (Pec)‐biphasic calcium phosphate (BCP) was successfully fabricated. Growth factors such as bone morphogenetic protein‐2 (BMP‐2) and vascular endothelial growth factor (VEGF) were loaded into the Gel‐Pec‐BCP hydrogel scaffolds by freeze‐drying. The surface morphology was investigated by scanning electron microscopy, and BCP dispersion in the hydrogel scaffolds was measured by energy dispersive and X‐ray diffraction spectroscopy. The results obtained from Fourier transform infrared spectroscopy and quantitative measurements showed successfully loading of BMP‐2 and VEGF into the Gel‐Pec‐BCP hydrogel scaffolds. In addition MC3T3‐E1 preosteoblasts were cultivated on the three types of scaffolds to investigate the effects of BMP‐2 and VEGF on cell viability and proliferation. The Gel‐Pec‐BCP scaffolds loaded with VEGF and BMP‐2 demonstrated more cell spreading and proliferation compared to those of the Gel‐Pec‐BCP scaffolds. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41241.     

Matrilin-3-Primed Adipose-Derived Mesenchymal Stromal Cell Spheroids Prevent Mesenchymal Stromal-Cell-Derived Chondrocyte Hypertrophy

Adipose-derived mesenchymal stromal cells (Ad-MSCs) are a promising tool for articular cartilage repair and regeneration. However, the terminal hypertrophic differentiation of Ad-MSC-derived cartilage is a critical barrier during hyaline cartilage regeneration. In this study, we investigated the role of matrilin-3 in preventing Ad-MSC-derived chondrocyte hypertrophy in vitro and in an osteoarthritis (OA) destabilization of the medial meniscus (DMM) model. Methacrylated hyaluron (MAHA) (1%) was used to encapsulate and make scaffolds containing Ad-MSCs and matrilin-3. Subsequently, the encapsulated cells in the scaffolds were differentiated in chondrogenic medium (TGF-β, 1–14 days) and thyroid hormone hypertrophic medium (T3, 15–28 days). The presence of matrilin-3 with Ad-MSCs in the MAHA scaffold significantly increased the chondrogenic marker and decreased the hypertrophy marker mRNA and protein expression. Furthermore, matrilin-3 significantly modified the expression of TGF-β2, BMP-2, and BMP-4. Next, we prepared the OA model and transplanted Ad-MSCs primed with matrilin-3, either as a single-cell suspension or in spheroid form. Safranin-O staining and the OA score suggested that the regenerated cartilage morphology in the matrilin-3-primed Ad-MSC spheroids was similar to the positive control. Furthermore, matrilin-3-primed Ad-MSC spheroids prevented subchondral bone sclerosis in the mouse model. Here, we show that matrilin-3 plays a major role in modulating Ad-MSCs’ therapeutic effect on cartilage regeneration and hypertrophy suppression.     

Integrated three‐dimensional fiber/hydrogel biphasic scaffolds for periodontal bone tissue engineering

Combining a tissue engineering scaffold made of a load‐bearing polymer with a hydrogel represents a powerful approach to enhancing the functionalities of the resulting biphasic construct, such as its mechanical properties or ability to support cellular colonization. This research activity was aimed at the development of biphasic scaffolds through the combination of an additively manufactured poly(?‐caprolactone) (PCL) fiber construct and a chitosan/poly(γ‐glutamic acid) polyelectrolyte complex hydrogel. By investigating a set of layered structures made of PCL or PCL/hydroxyapatite composite, biphasic scaffold prototypes with good integration of the two phases at the macroscale and microscale were developed. The biphasic constructs were able to absorb cell culture medium up to 10‐fold of their weight, and the combination of the two phases had a significant influence on compressive mechanical properties compared with hydrogel or PCL scaffold alone. In addition, due to the presence of chitosan in the hydrogel phase, biphasic scaffolds exerted a broad‐spectrum antibacterial activity. The developed biphasic systems appear well suited for application in periodontal bone regenerative approaches in which a biodegradable porous structure providing mechanical stability and a hydrogel phase functioning as absorbing depot of endogenous proteins are simultaneously required. © 2016 Society of Chemical Industry     

Silk Fiber-Reinforced Hyaluronic Acid-Based Hydrogel for Cartilage Tissue Engineering

A continuing challenge in cartilage tissue engineering for cartilage regeneration is the creation of a suitable synthetic microenvironment for chondrocytes and tissue regeneration. The aim of this study was to develop a highly tunable hybrid scaffold based on a silk fibroin matrix (SM) and a hyaluronic acid (HA) hydrogel. Human articular chondrocytes were embedded in a porous 3-dimensional SM, before infiltration with tyramine modified HA hydrogel. Scaffolds were cultured in chondropermissive medium with and without TGF-β1. Cell viability and cell distribution were assessed using CellTiter-Blue assay and Live/Dead staining. Chondrogenic marker expression was detected using qPCR. Biosynthesis of matrix compounds was analyzed by dimethylmethylene blue assay and immuno-histology. Differences in biomaterial stiffness and stress relaxation were characterized using a one-step unconfined compression test. Cell morphology was investigated by scanning electron microscopy. Hybrid scaffold revealed superior chondro-inductive and biomechanical properties compared to sole SM. The presence of HA and TGF-β1 increased chondrogenic marker gene expression and matrix deposition. Hybrid scaffolds offer cytocompatible and highly tunable properties as cell-carrier systems, as well as favorable biomechanical properties.     

The Regulatory Role of Signaling Crosstalk in Hypertrophy of MSCs and Human Articular Chondrocytes

Hypertrophic differentiation of chondrocytes is a main barrier in application of mesenchymal stem cells (MSCs) for cartilage repair. In addition, hypertrophy occurs occasionally in osteoarthritis (OA). Here we provide a comprehensive review on recent literature describing signal pathways in the hypertrophy of MSCs-derived in vitro differentiated chondrocytes and chondrocytes, with an emphasis on the crosstalk between these pathways. Insight into the exact regulation of hypertrophy by the signaling network is necessary for the efficient application of MSCs for articular cartilage repair and for developing novel strategies for curing OA. We focus on articles describing the role of the main signaling pathways in regulating chondrocyte hypertrophy-like changes. Most studies report hypertrophic differentiation in chondrogenesis of MSCs, in both human OA and experimental OA. Chondrocyte hypertrophy is not under the strict control of a single pathway but appears to be regulated by an intricately regulated network of multiple signaling pathways, such as WNT, Bone morphogenetic protein (BMP)/Transforming growth factor-β (TGFβ), Parathyroid hormone-related peptide (PTHrP), Indian hedgehog (IHH), Fibroblast growth factor (FGF), Insulin like growth factor (IGF) and Hypoxia-inducible factor (HIF). This comprehensive review describes how this intricate signaling network influences tissue-engineering applications of MSCs in articular cartilage (AC) repair, and improves understanding of the disease stages and cellular responses within an OA articular joint.     

Decellularized Cartilage Extracellular Matrix Incorporated Silk Fibroin Hybrid Scaffolds for Endochondral Ossification Mediated Bone Regeneration

Tissue engineering strategies promote bone regeneration for large bone defects by stimulating the osteogenesis route via intramembranous ossification in engineered grafts, which upon implantation are frequently constrained by insufficient integration and functional anastomosis of vasculature from the host tissue. In this study, we developed a hybrid biomaterial incorporating decellularized cartilage extracellular matrix (CD-ECM) as a template and silk fibroin (SF) as a carrier to assess the bone regeneration capacity of bone marrow-derived mesenchymal stem cells (hBMSC’s) via the endochondral ossification (ECO) route. hBMSC’s were primed two weeks for chondrogenesis, followed by six weeks for hypertrophy onto hybrid CD-ECM/SF or SF alone scaffolds and evaluated for the mineralized matrix formation in vitro. Calcium deposition biochemically determined increased significantly from 4-8 weeks in both SF and CD-ECM/SF constructs, and retention of sGAG’s were observed only in CD-ECM/SF constructs. SEM/EDX revealed calcium and phosphate crystal localization by hBMSC’s under all conditions. Compressive modulus reached a maximum of 40 KPa after eight weeks of hypertrophic induction. μCT scanning at eight weeks indicated a cloud of denser minerals in groups after hypertrophic induction in CD-ECM/SF constructs than SF constructs. Gene expression by RT-qPCR revealed that hBMSC’s expressed hypertrophic markers VEGF, COL10, RUNX2, but the absence of early hypertrophic marker ChM1 and later hypertrophic marker TSBS1 and the presence of osteogenic markers ALPL, IBSP, OSX under all conditions. Our data indicate a new method to prime hBMSC’S into the late hypertrophic stage in vitro in mechanically stable constructs for ECO-mediated bone tissue regeneration.     

A novel strategy for cartilage tissue engineering: Collagenase-loaded cryogel scaffolds in a sheep model

In this study, we developed a novel strategy, through which cartilage tissue pieces were placed in a sheep cartilage defect model and covered with a collagenase incorporated cryogel scaffold (in vivo cartilage tissue engineering, IVCTE group). While applying this strategy, the chondrocytes could be isolated inside the body and the treatment could be accomplished in one session. To compare our strategy, to another group, in which we used cultured cells and Chondro-gide, standard matrix-induced autologous chondrocyte implantation (MACI) was applied. Although the MACI applied group demonstrated better healing than IVCTE, the type II collagen synthesis was better in the IVCTE group compared to MACI applied group. Collagenase did not have detrimental effect on surrounding cartilage in IVCTE group. The preliminary results of the novel strategy applied group (IVCTE) were promising.     

Surface treatment with amino acids of porous collagen based scaffolds to improve cell adhesion and proliferation

The purpose of this study was to improve the biocompatibility of glutaraldehyde (GA) cross‐linked chitosan coated collagen scaffold for cartilage tissue regeneration. In order to prevent the potential toxicity of GA, we treated the designed scaffold with either glutamic acid or glycine. Amino acid treated scaffolds were characterized by scanning electron microscopy (SEM) techniques. Afterward, chondrocyte interaction with the composite scaffold was investigated assessing cell adhesion and proliferation using Hoechst staining and MTT cell proliferation assay, respectively. The SEM analyses of the scaffolds’ surface and cross‐section confirmed the adhesion of amino acids on the surface of the scaffolds. We also observed that scaffolds’ porosity was reduced due to the coverage of the pores by chitosan and amino acids, leading to low porosity. The use of amino acid improved the chondrocyte adhesion and proliferation inside the scaffolds’ pores when cells were cultured onto the chitosan‐coated collagen scaffolds. Overall, our in vitro results suggest the use of amino acid to improve the biocompatibility of natural polymer composite scaffold being crosslinked with glutaraldehyde. Such scaffold has improved mechanical properties; biocompatibility thus may be useful for tissue regeneration such as cartilage.

     

Study of Different Delivery Modes of Chondroitin Sulfate Using Microspheres and Cryogel Scaffold for Application in Cartilage Tissue Engineering

This study focuses on the development of an efficient delivery modes designed for chondroitin sulfate (CS) for application in cartilage tissue engineering. Novel three-dimensional (3-D) scaffold fabricated from natural polymers such as chitosan and gelatin blended with chondroitin sulfate (CGC) were synthesized using cryogelation technology. Other methods to deliver CS were also tried, which included incorporation into microparticles for sustained release and embedding the CS loaded microparticles in CG (chitosan-gelatin) cryogel scaffold. Novel CGC scaffolds were characterized by rheology, scanning electron microscopy (SEM), and mechanical assay. Scaffolds exhibited compression modulus of 50 KPa confirming the utility of these scaffolds for cartilage tissue engineering. Primary goat chondrocytes were used for the in vitro testing of all the delivery modes. So this study shows that CS microparticles when given freely with matrix (chitosan–gelatin) or embedded into scaffold has potential to enhance chondrocyte proliferation together with improved matrix production than in control without microspheres.     

Regeneration of Articular Cartilage in Lizard Knee from Resident Stem/Progenitor Cells

The epiphysis of femur and tibia in the lizard Podarcis muralis can extensively regenerate after injury. The process involves the articular cartilage and metaphyseal (growth) plate after damage. The secondary ossification center present between the articular cartilage and the growth plate is replaced by cartilaginous epiphyses after about one month of regeneration at high temperature. The present study analyzes the origin of the chondrogenic cells from putative stem cells located in the growing centers of the epiphyses. The study is carried out using immunocytochemistry for the detection of 5BrdU-labeled long retaining cells and for the localization of telomerase, an enzyme that indicates stemness. The observations show that putative stem cells retaining 5BrdU and positive for telomerase are present in the superficial articular cartilage and metaphyseal growth plate located in the epiphyses. This observation suggests that these areas represent stem cell niches lasting for most of the lifetime of lizards. In healthy long bones of adult lizards, the addition of new chondrocytes from the stem cells population in the articular cartilage and the metaphyseal growth plate likely allows for slow, continuous longitudinal growth. When the knee is injured in the adult lizard, new populations of chondrocytes actively producing chondroitin sulfate proteoglycan are derived from these stem cells to allow for the formation of completely new cartilaginous epiphyses, possibly anticipating the re-formation of secondary centers in later stages. The study suggests that in this lizard species, the regenerative ability of the epiphyses is a pre-adaptation to the regeneration of the articular cartilage.     

Compressive properties and creep resistance of a novel,porous, semidegradable poly(vinyl alcohol)/poly(lactic‐co‐glycolic acid) scaffold for articular cartilage repair

Tissue engineering for articular cartilage repair has shown success in ensuring the integration of neocartilage with surrounding natural tissue, but the rapid restoration of biomechanical functions remains a significant challenge. The poly(vinyl alcohol) (PVA) hydrogel is regarded as a potential articular cartilage replacement for its fair mechanical strength, whereas its lack of bioactivity limits its utility. To obtain a scaffold possessing expected bioactivity and initial mechanical properties, we herein report a novel salt‐leaching technique to fabricate a porous PVA hydrogel simultaneously embedded with poly(lactic‐co‐glycolic acid) (PLGA) microspheres. Through the investigation of environmental scanning electron microscopy, we found that the porous PVA/PLGA scaffold was successfully manufactured. The compression and creep properties were also comprehensively studied before and after cell culturing. The relationship between the compressive modulus and strain ratio of the porous PVA/PLGA scaffold showed significant nonlinear behavior. The elastic compressive modulus was influenced a little by the porogen content, whereas it went higher with a higher PLGA microsphere content. The cell‐cultured scaffolds presented higher compressive moduli than the initial ones. The creep resistance of the cell‐cultured scaffolds was much better than that of the initial ones. In all, this new scaffold is a promising material for articular cartilage repair. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40311.     

Role of iRhoms 1 and 2 in Endochondral Ossification

Growth of the axial and appendicular skeleton depends on endochondral ossification, which is controlled by tightly regulated cell–cell interactions in the developing growth plates. Previous studies have uncovered an important role of a disintegrin and metalloprotease 17 (ADAM17) in the normal development of the mineralized zone of hypertrophic chondrocytes during endochondral ossification. ADAM17 regulates EGF-receptor signaling by cleaving EGFR-ligands such as TGFα from their membrane-anchored precursor. The activity of ADAM17 is controlled by two regulatory binding partners, the inactive Rhomboids 1 and 2 (iRhom1, 2), raising questions about their role in endochondral ossification. To address this question, we generated mice lacking iRhom2 (iR2−/−) with floxed alleles of iRhom1 that were specifically deleted in chondrocytes by Col2a1-Cre (iR1∆Ch). The resulting iR2−/−iR1∆Ch mice had retarded bone growth compared to iR2−/− mice, caused by a significantly expanded zone of hypertrophic mineralizing chondrocytes in the growth plate. Primary iR2−/−iR1∆Ch chondrocytes had strongly reduced shedding of TGFα and other ADAM17-dependent EGFR-ligands. The enlarged zone of mineralized hypertrophic chondrocytes in iR2−/−iR1∆Ch mice closely resembled the abnormal growth plate in A17∆Ch mice and was similar to growth plates in Tgfα−/− mice or mice with EGFR mutations. These data support a model in which iRhom1 and 2 regulate bone growth by controlling the ADAM17/TGFα/EGFR signaling axis during endochondral ossification.     

COMP and TSP-4: Functional Roles in Articular Cartilage and Relevance in Osteoarthritis

Osteoarthritis (OA) is a slow-progressing joint disease, leading to the degradation and remodeling of the cartilage extracellular matrix (ECM). The usually quiescent chondrocytes become reactivated and accumulate in cell clusters, become hypertrophic, and intensively produce not only degrading enzymes, but also ECM proteins, like the cartilage oligomeric matrix protein (COMP) and thrombospondin-4 (TSP-4). To date, the functional roles of these newly synthesized proteins in articular cartilage are still elusive. Therefore, we analyzed the involvement of both proteins in OA specific processes in in vitro studies, using porcine chondrocytes, isolated from femoral condyles. The effect of COMP and TSP-4 on chondrocyte migration was investigated in transwell assays and their potential to modulate the chondrocyte phenotype, protein synthesis and matrix formation by immunofluorescence staining and immunoblot. Our results demonstrate that COMP could attract chondrocytes and may contribute to a repopulation of damaged cartilage areas, while TSP-4 did not affect this process. In contrast, both proteins similarly promoted the synthesis and matrix formation of collagen II, IX, XII and proteoglycans, but inhibited that of collagen I and X, resulting in a stabilized chondrocyte phenotype. These data suggest that COMP and TSP-4 activate mechanisms to protect and repair the ECM in articular cartilage.