Optimum temperature for extracellular matrix production by articular chondrocytes.

ABSTRACT

PURPOSE: The purpose of this study was to investigate the influence of temperature on the ability of the chondrocytes to produce extracellular matrix (ECM). MATERIALS AND METHODS: Articular chondrocytes were isolated from porcine knee joints. The chondrocytes were cultured at three different temperatures 32 °C, 37 °C, and 41 °C. The ability to produce ECM was assessed by gene expression analysis, histological, and biochemical evaluation in a pellet culture system. RESULTS: Wet weight of the pellets generated after 21 days, was significantly heavier when cultured at lower temperatures. Picrosirius red staining, employed to evaluate collagen production, was higher at lower temperatures, and safranin-O staining, used to evaluate sulphated glycosaminoglycan (GAG), was lower at 32 °C than at 37 °C and 41 °C. Collagen type IIA1 mRNA expression was markedly up-regulated at 41 °C. However, picrosirius red staining was inhibited at 41 °C. GAG and DNA content were measured by 1,9-dimethylmethylene blue (DMMB) assay and PicoGreen® assay, respectively. The GAG content per pellet was significantly low at 41 °C compared to that at 32 °C and 37 °C. The DNA content per pellet was larger at lower temperatures. The GAG content normalised with the DNA content per pellet was significantly lower at 32 °C compared to that at 37 °C and 41 °C. CONCLUSION: Our results suggest that a culture temperature of approximately 41 °C inhibits ECM production by decreasing DNA content and perhaps by collagen misfolding. Taken together, the optimum temperature for ECM production in articular chondrocytes may be between 32 °C and 37 °C.