Abstract
Purpose
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.
Method
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.
Results
Cartilage was obtained and cultured from 22 patients. We identified responders (16/22) and non-responders (6/22) to ATP stimulation. Patients demographics, co-morbidities and medications were reviewed and no correlating characteristics were identified. The average increase in [3H]-proline incorporation was 242% the control (range 115%–388%, p<0.02) and the average increase in [35S]-sulfate incorporation was 238% (range 124%–711%, p<0.02). The expression of P2Y1 and P2Y2 receptors varied widely between individuals, with a range of 11–76% expression and of 3–67% expression for P2Y1 and P2Y2 receptors, respectively. Almost all cells expressing P2Y2 receptors also expressed P2Y1 receptors, and 4/8 patients also had significant cell populations expressing P2Y1 but not P2Y2 receptors (range of 4–17% of cells). Of the 8 patients studied, only 1 patient had measurable ATP within the culture media. ATP degradation within the culture media was measured, with the measured ATP half-life and elimination rate constants were determined. The ATP elimination rate constant values showed good correlation to P2Y1 receptor expression (R=0.99).
Conclusion
P2Y1 and P2Y2 receptors were expressed on a significant proportion of chondrocytes from patients with osteoarthritis and there was a significant correlation of the expression of these receptors to the ATP elimination rate constants. The addition of exogenous ATP increased both the proteoglycan and collagen synthesis of the developing cartilage constructs in a subset of patients and appears to be a promising technique to improve extracellular matrix production in these constructs.
