Effects of Mechanical Stress on Proliferation and Differentiation of Chondrocytes

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Mechanical strain is an important factor to regulate chondrocyte proliferation and differentiation. Response of chondrocyte to mechanical strain could be differentiation dependent. Chondrogenic ATDC5 cells differentiate into hypertrophic chondrocytes in response to insulin, and regulates type II, X collagen, aggrecan, ALP, and PTH/PTHrP receptor mRNA expression. In the present study, we examined whether ATDC5 cells respond to mechanical strain in differentiation specific manner.ATDC5 cells were cultured on plastic plates at 1×10^6 cell/plate in the medium of F12/DMEM containing 5% FBS and 10 μg/ml insulin. ATDC5 were stained with toluidine-blue to observe the morphologic change. We utilized an. in vitro cell loading system using four-point bending. The magnitude of strain was 4200 μstrain and the frequency was 0.5 Hz. To assess the effects of mechanical strain on ATDC5 cells, the expression of type II, X collagen, aggrecan, ALP, and PTH/PTHrP receptor mRNAs were determined using northern blot analysis. Six to 24 hours after beginning of loading on day 4 through day 28, total RNA was extracted and hybridized with various cDNA probe.ATDC5 cells initiated chondrogenic differentiation as reported before. Type II collagen mRNA expression was firstly observed on day 4, type X collagen and aggrecan on day 14. The effects of mechanical strain were not observed on day 4 or 7, but mechanical strain up-regulates type II, X collagen, and aggrecan mRNA in a time-dependent manner thereafter. However, there were no increase in ALP and PTH/PTHrP receptor expression.These results indicate that differentiated, but not undifferentiated ATDC5 cells up-regulates type II, X collagen, and aggrecan transcripts in response to mechanical strain.