Characterization of the Proliferating Layer Chondrocytes of Growth Plate for Cartilage Regeneration

Abstract

Cell-based therapy is an ideal way of repairing defective cartilage. At present, the articular chondrocytes (ACs) is the cell source for cartilage repair. Problematically, in serial culture, the dedifferentiation of ACs occurs, which may result in graft failure. In the present study, we examined chondrogenic capacity and physical characteristics of proliferating layer chondrocytes (PLCs) from the growth plate cartilage and evaluated its regenerative activity for cartilage repair. We found that PLCs preserved more chondrogenic phenotypes, such as polygonal appearances, whereas ACs appeared fibroblast-like after seventh passage. Profoundly, the ACs expressed higher apoptosis-related proteins, such as cleaved caspase-9 and cleaved caspase-3, than PLCs. Also, the PLCs have higher proliferation rate than ACs, and the cell doubling time is 20.9 h for PLCs and 29.5 h for ACs. Using flow cytometry, we demonstrated that 26.6% of PLCs entered the S-phase after 16 h serum re-addition to starved cells compared with 13.3% of ACs. Otherwise, col2a1, aggrecan, sox5, sox6, and sox9 mRNAs were significantly increased in PLCs compared with ACs; in contrast, the col1a and col10a1 mRNA expression level in PLCs was lower than that in ACs. The glycosaminoglycan content in PLCs was higher than that in ACs by the direct 1,9-dimethylmethylene blue assay. Histological and immunohistochemical evaluations have demonstrated that significantly more chondrogenic extracellular matrix was detected in the PLCs group compared with the ACs group after implantation in nude mice. Taken together, our data indicate the PLCs preserved much more chondrogenic phenotypes than ACs in vitro and in vivo. These results might imply that PLCs act as the better cell source for reapairing and regenerating growth plate and articular cartilage.

Impact Statement

In recent years, cell-based therapy is a promising strategy for repairing defect cartilage. However, in vitro expansion of articular chondrocytes (ACs) for collecting enough cell numbers eventually develops cell de-differentiation. In the present study, we choose the proliferative layer chondroctytes (PLCs) of growth plate as new candidate. The novel findings include (1) the higher proliferation potential of PLCs in comparison with the ACs, (2) PLCs produced more GAG than ACs, (3) the increased in GAG matrix production, (4) and lower senescence in PLCs. From these results, we found PLCs might be suitable as cell source for cartilage regeneration.