Adenosine triphosphate (ATP) has been implicated as an autocrine/paracrine signal in the mechanotransduction pathway of chondrocytes. In this study, human chondrocytes in a 3D agarose scaffold were cultured with exogenous ATP in varying doses to determine its effect on extracellular matrix synthesis by the cells. Further experiments determined basal ATP release, ATP degradation and expression of P2Y1 and P2Y2 purinoreceptors by the cultured cell constructs. Human chondrocytes were obtained by enzymatic digestion of cartilage samples obtained at the time of total joint arthroplasty. The chondrocytes were cultured in a 3D agarose scaffold using standard tissue culture techniques. Various concentrations of exogenous ATP were added to the cultures, along with the radioisotopes to assess matrix synthesis. The cultures were harvested after a 24 hr incubation and radioisotope incorporation was determined by scintillation counting to determine proteoglycan ([35S]-sulfate) and collagen ([3H]-proline) synthesis, respectively. DNA content was determined by the Hoescht 33258 binding assay, and the proteoglycan and collagen synthesis were normalized to DNA content. Basal ATP release and degradation of exogenous ATP were determined by luciferase assay and luminometry. Expression of P2Y1 and P2Y2 purinoreceptors were determined by flow cytometry.Purpose
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