Articular Cartilage Fragmentation Improves Chondrocyte Migration by Upregulating Membrane Type 1 Matrix Metalloprotease

Objective This study was undertaken to elucidate the mechanism of improved chondrocyte migration after juvenile articular cartilage fragmentation. Design In vitro organ culture with rabbit cartilage fragments and cell culture with rabbit chondrocytes were performed. In part A, minced juvenile cartilage fragments (~0.5 × 0.5 × 0.5 mm) from rabbits, planted in gelatin sponge and fibrin glue, were cultured for 2, 4, or 6 weeks in vitro and compared with the cartilage chunks (~4 × 4 × 1 mm) and membrane type 1 matrix metalloprotease (MT1-MMP) inhibitor groups. Chondrocyte outgrowth was evaluated on histology and confocal laser scanning microscopy. MT1-MMP expression was compared between the cartilage fragment group and the cartilage chunks group. In part B, articular chondrocytes were harvested from juvenile rabbits, MT1-MMP was transfected into the cells, and cell migration was evaluated using the Transwell and wound healing tests. Results The histology and confocal microscopy results revealed that cell accumulation occurred at the edge of cartilage fragments, and outgrowth was better in the cartilage fragment group than those in the cartilage chunks group. Similar results were observed for MT1-MMP expression. After MT1-MMP inhibition, cells did not accumulate at the edge of the cartilage fragments, and chondrocyte outgrowth did not occur. Furthermore, overexpression of MT1-MMP enhanced the migration of articular chondrocytes. Conclusions Juvenile articular cartilage fragmentation improved chondrocyte migration by upregulating MT1-MMP.

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.

Mesenchymal stem cell-derived exosomal microRNA-136-5p inhibits chondrocyte degeneration in traumatic osteoarthritis by targeting ELF3

Background Emerging evidence suggests that microRNAs (miRs) are associated with the progression of osteoarthritis (OA). In this study, the role of exosomal miR-136-5p derived from mesenchymal stem cells (MSCs) in OA progression is investigated and the potential therapeutic mechanism explored. Methods Bone marrow mesenchymal stem cells (BMMSCs) and their exosomes were isolated from patients and identified. The endocytosis of chondrocytes and the effects of exosome miR-136-5p on cartilage degradation were observed and examined by immunofluorescence and cartilage staining. Then, the targeting relationship between miR-136-5p and E74-like factor 3 (ELF3) was analyzed by dual-luciferase report assay. Based on gain- or loss-of-function experiments, the effects of exosomes and exosomal miR-136-5p on chondrocyte migration were examined by EdU and Transwell assay. Finally, a mouse model of post-traumatic OA was developed to evaluate effects of miR-136-5p on chondrocyte degeneration in vivo. Results In the clinical samples of traumatic OA cartilage tissues, we detected increased ELF3 expression, and reduced miR-136-5p expression was determined. The BMMSC-derived exosomes showed an enriched level of miR-136-5p, which could be internalized by chondrocytes. The migration of chondrocyte was promoted by miR-136-5p, while collagen II, aggrecan, and SOX9 expression was increased and MMP-13 expression was reduced. miR-136-5p was verified to target ELF3 and could downregulate its expression. Moreover, the expression of ELF3 was reduced in chondrocytes after internalization of exosomes. In the mouse model of post-traumatic OA, exosomal miR-136-5p was found to reduce the degeneration of cartilage extracellular matrix. Conclusion These data provide evidence that BMMSC-derived exosomal miR-136-5p could promote chondrocyte migration in vitro and inhibit cartilage degeneration in vivo, thereby inhibiting OA pathology, which highlighted the transfer of exosomal miR-136-5p as a promising therapeutic strategy for patients with OA.

Chondrocytes From Device-Minced Articular Cartilage Show Potent Outgrowth Into Fibrin and Collagen Hydrogels

Background: Transplantation of autologous minced cartilage is an established procedure to repair chondral lesions. It relies on the migration of chondrocytes out of cartilage particles into a biomaterial. So far, there is no efficient way to finely mince cartilage. No consensus exists on the nature of the biomaterial to be used to promote chondrocyte migration. Purpose/Hypothesis: This study aimed to investigate the potential clinical use of a custom-made mincing device as well as a possible alternative biomaterial to fibrin glue. The device was tested for its effect on chondrocyte viability and on subsequent chondrocyte migration into either a fibrin or a collagen gel. We hypothesized that device mincing would allow finer cutting and consequently more cell migration and that the gelation mechanism of the collagen biomaterial, which uses the clotting of platelet-rich plasma, would enhance matrix production by outgrown chondrocytes. Study Design: Controlled laboratory study. Methods: Cartilage from 12 patients undergoing knee arthroplasty was taken from the femoral condyles and subsequently either hand minced or device minced. The viability and the degree of outgrowth were quantified with live/dead assay on the generated cartilage particles and on the gels in which these particles were embedded, respectively. Matrix deposition in the biomaterials by the outgrown cells was investigated with histology. Results: The device allowed rapid mincing of the cartilage and produced significantly smaller pieces than hand mincing. The initial chondrocyte viability in cartilage particles dropped by 25% with device mincing as compared with no mincing. However, the viability in hand-minced, device-minced, and unminced samples was no longer different after 7 and 28 days in culture. Outgrowth scores were similar among the 3 groups. Fibrin and collagen biomaterials equally supported chondrocyte outgrowth and survival, but neither promoted matrix deposition after in vitro culture. Conclusion: The outgrowth potential, the viability after 28 days in culture, and the matrix deposition were not different between the mincing techniques and the tested biomaterials, yet device mincing is faster and results in significantly smaller cartilage particles. Clinical Relevance: Device mincing could become the standard method to mince cartilage for second-generation cartilage repair techniques.

Cartilage Grafting

Arthritis remains a widespread and yet unsolved therapeutic dilemma. Cartilage grafting has proven to be difficult and satisfactory results are often elusive. There are several inherent difficulties. These include both chondrocyte migration and the lack of sufficient uptake of nutrients to allow for graft survival. With autografts, there is also the paucity of symptom-free donor sites. Accordingly, multiple alternative therapies for cartilage regeneration and/or substitution have been developed over time. In this article, the authors shall discuss the options for the treatment of damaged cartilage with a focus on the cartilage grafting techniques.

Repair of an Osteochondral Defect With Minced Cartilage Embedded in Atelocollagen Gel: A Rabbit Model

Background: Autologous chondrocyte implantation (ACI) is often performed for large cartilage defects. Because this technique has several disadvantages, including the need for second-stage surgery, cartilage repair using minced cartilage has been suggested. However, this technique could be improved using 3-dimensional scaffolds. Purpose: To examine the ability of chondrocyte migration and proliferation from minced cartilage in atelocollagen gel in vitro and evaluate the repairable potential of minced cartilage embedded in atelocollagen gel covered with a periosteal flap in a rabbit model. Study Design: Controlled laboratory study. Methods: Minced cartilage or isolated chondrocytes from rabbits were embedded in atelocollagen gel and cultured for 3 weeks. Chondrocyte proliferation and matrix production were evaluated in vitro. An osteochondral defect at the trochlear groove was created in 56 rabbits, which were divided into 4 groups. The defect was left empty (defect group), filled with allogenic minced cartilage (minced cartilage group), filled with isolated allogenic chondrocytes embedded in atelocollagen gel (ACI group), or filled with atelocollagen gel (atelocollagen with periosteal flap group). At 4, 12, and 24 weeks after surgery, repair of the defect was evaluated in these 4 groups. Results: In vitro, the number of chondrocytes and abundant matrix on the surface of the gel significantly increased in the minced cartilage group compared with the ACI group ( P < .05). In vivo, the minced cartilage and ACI groups showed good cartilage repair compared with the empty defect and atelocollagen/periosteal flap groups ( P < .05); there was no significant difference in the Pineda score between the minced cartilage and ACI groups. Conclusion: Minced cartilage in atelocollagen gel had good chondrocyte migration and proliferation abilities in vitro, and osteochondral defects were well repaired by implanting minced cartilage embedded in the atelocollagen gel in vivo. Implantation of minced cartilage embedded in atelocollagen gel showed good cartilage repair equivalent to ACI. Clinical Relevance: Implantation of minced cartilage embedded in atelocollagen gel as a 1-step procedure has outcomes similar to those of ACI but is cheaper and more convenient than ACI.