The Effect of Thrombin Activation of Platelet-Rich Plasma on Demineralized Bone Matrix Osteoinductivity

Background: Demineralized bone matrix is an osteoinductive and osteoconductive material that is often used in orthopaedic surgery to induce bone formation. Autologous platelet-rich plasma, which contains proliferative and chemoattractant growth factors, has been used as a demineralized bone matrix adjuvant with mixed results. One variable during clinical use appears to be whether the platelet-rich plasma is activated with thrombin or is implanted in a liquid form with intact platelets. The objective of the present study was to determine if platelet-rich plasma can increase the osteoinductivity of demineralized bone matrix when used without thrombin activation. Methods: The bioactivity of the demineralized bone matrix was evaluated in vitro by determining alkaline phosphatase production by C2C12 myoblast cells. The effect of thrombin activation on platelet-rich plasma was studied in vitro by evaluating osteosarcoma and bone marrow stromal cells for cell number and transforming growth factor-beta 1 activation. Demineralized bone matrices supplemented with platelet-rich plasma, with or without thrombin activation, were implanted intramuscularly in athymic rats and were examined at fourteen, twenty-eight, and fifty-six days. Histological samples were analyzed for osteogenesis and chondrogenesis. Osteogenesis was further characterized on the basis of alkaline phosphatase activity. Results: In vitro, thrombin triggered an immediate release of growth factors from the platelet-rich plasma, and the platelet-rich plasma increased the number of both osteosarcoma and stromal cells in a dose-dependent manner. Thrombin activation of the platelet-rich plasma eliminated such stimulatory effects. In vivo, the platelet-rich plasma stimulated chondrogenesis on Day 14 and osteogenesis on Days 28 and 56, whereas thrombin-activated platelet-rich plasma acted as an inhibitor of such events. In addition, inflammatory cells were detected in demineralized bone matrix samples that were mixed with thrombin-activated platelet-rich plasma. These cells were not present in matrix mixed with platelet-rich plasma alone. Conclusions: Platelet-rich plasma significantly increased in vivo demineralized bone matrix osteoinductivity only when used without thrombin activation.