Objective To determine the cellular and matrix responses to experimental wounding of articular cartilage. Methods Immature and mature bovine articular cartilage was used as an in vitro model system to study the cellular responses to cartilage wounding. Explant cultures were wounded centrally with a trephine and maintained for up to 10 days. TUNEL labeling together with ultrastructural analyses were used to assess the nature of the observed cell death. In vitro labeling with ³H-thymidine was used to detect cell proliferation, and 2 antibodies (COL2-3/4M and BC-13) were used to detect changes in matrix turnover. Results Cell death was observed as a response to wounding and was considered to be a combination of necrosis and apoptosis. In immature tissue, cell death was more pronounced, particularly in the articular surface region. Within the area of cell death, many cells that did not die subsequently underwent proliferation. The collagenous network showed evidence of denaturation in the area of the wound, but “aggrecanase” activity was not detected. Conclusion There are 2 contrasting, but related, responses to cartilage wounding—apoptosis and proliferation. In order to improve cartilage repair, future studies need to elucidate the regulatory mechanisms that determine these responses.

Tissue engineering offers new strategies for developing treatments for the repair and regeneration of damaged and diseased tissues. These treatments, using living cells, will exploit new developments in understanding the principles in cell biology that control and direct cell function. Arthritic diseases that affect so many people and have a major impact on the quality of life provide an important target for tissue engineering. Initial approaches are in cartilage repair; in our own programme we are elucidating the signals required by chondrocytes to promote new matrix assembly. These principles will extend to other tissues of the musculoskeletal system, including the repair of bone, ligament and tendon.

Chondrocytes form and maintain the extracellular matrix of cartilage. The cells can be isolated from cartilage for applications such as tissue engineering, but their expansion in monolayer culture causes a progressive loss of chondrogenic phenotype. In this work, we have investigated the isolation of human articular chondrocytes from osteoarthritic (OA) cartilage at joint replacement, their expansion in monolayer culture, and their transduction with adenoviral, retroviral, and lentiviral vectors, using the gene encoding green fluorescent protein as a marker gene. The addition of growth factors (transforming growth factor β₁, fibroblast growth factor 2, and platelet-derived growth factor BB) during cell culture was found to greatly increase cell proliferation and thereby to selectively enhance the efficiency of transduction with retrovirus. With adenoviral and lentiviral vectors the transduction efficiency achieved was 95 and 85%, respectively. Using growth factor-supplemented medium with a retroviral vector, efficiency in excess of 80% was achieved. The expression was stable for several months with both retrovirus and lentivirus when analyzed by fluorescence-activated cell-sorting flow analysis and immunoblotting. Transduction with SOX9 was investigated as a method to reinitiate cartilage matrix gene expression in passaged human OA chondrocytes. Endogenous collagen II expression (both mRNA and protein) was increased in monolayer culture using both adenoviral and retroviral vectors. Furthermore, collagen II gene expression in chondrocytes retrovirally transduced with SOX9 was stimulated by alginate bead culture, whereas in control chondrocytes it was not. These results demonstrated methods for rapid expansion and highly efficient transduction of human OA chondrocytes and the potential for the recovery of key features of chondrocyte phenotype by transduction with SOX9 .

Human articular chondrocytes rapidly lose their phenotype in monolayer culture. Recently we have shown that overexpression of the transcription factor SOX9 greatly enhanced re-expression of the phenotype in three-dimensional aggregate cultures. Here we show that endogenous SOX9 mRNA can be rapidly up-regulated in subcultured human articular chondrocytes if grown in alginate, in monolayer with cytochalasin D, or with specific inhibition of the RhoA effector kinases ROCK1 and -2, which all prevent actin stress fiber formation. Disruption of actin stress fibers using any of these redifferentiation stimuli also supported the superinduction of SOX9 by cycloheximide. The superinduction was blocked by inhibitors of the p38 MAPK signaling pathway and involved the stabilization of SOX9 mRNA. Furthermore stimulation of chondrocyte p38 MAPK activity with interleukin-1beta resulted in increased levels of SOX9 mRNA, and this was again dependent on the absence of actin stress fibers in the cells. In this study of chondrocyte redifferentiation we have provided further evidence of the early involvement of SOX9 and have discovered a novel post-transcriptional regulatory mechanism activated by p38 MAPK, which stabilized SOX9 mRNA.