Effects of mechanical strain on ANK, ENPP1 and TGF-β1 expression in rat endplate chondrocytes in�vitro

Abstract

We investigated the effects of mechanical strain on the progressive ankylosis (ANK) gene and extracellular nucleotide phosphatase/phosphodiesterase (ENPP)1 mRNA expression and TGF-β1 protein expression in rat endplate chondrocytes in vitro. Endplate chondrocytes were isolated and cultured in vitro. Following identification with toluidine blue and immunocytochemical staining, chondrocytes were subjected to 10% elongation with various frequencies (0.5, 1, 1.5 and 2 Hz) using a Flexercell Tension Plus system at various intervals (3, 6, 12, 24, 36 and 48 h). As a control, cells that had been cultured statically on the same type of plate but were not subjected to stretch were also observed. Real-time reverse transcription-polymerase chain reaction and the enzyme-linked immunosorbent assay were used to study the effects of mechanical strain on ANK and ENPP1 mRNA expression and TGF-β1 concentration in the supernatant, respectively. Following treatment, the shape of the chondrocytes displayed a significant change from the original polygon to a typical spindle cell morphology; and the arrangement of the cells exhibited a change from a haphazard arrangement to an alignment with a certain direction. In the 0.5 Hz, 24-h group, the ANK gene expression was significantly increased compared to the control group (P<0.05); whereas in the other groups, the ANK and ENPP1 expression levels were reduced. With the increased frequencies in the 24-h group, the ANK gene expression gradually reduced. Changes in the expression of ANK and ENPP1 followed similar trends. TGF-β1 in the supernatant increased gradually in each frequency group, with a clear increase in the 0.5 Hz group. We conclude that various frequencies of mechanical strain can affect the expression of ANK, ENPP1 and endogenous TGF-β1 in endplate chondrocytes. Our results indicate that 0.5 Hz, 24 h may be the optimal stimulation condition to prevent calcification occurrence and to maintain the function of endplate chondrocytes.