SLR - September 2009 - Jennifer Pappalardo
Reference:
Han, B., Woodell-May, J., Ponticiello, M., Yang, Z., and Nimni, M. (2009). The Effect of Thrombin Activation of Platelet-Rich Plasma on Demineralized Bone Matrix Osteoinductivity. TheJournal of Bone and Joint Surgery. 91:1459-1470.
Scientific Literature Review
Reviewed By: Jennifer Pappalardo, DPM
Residency Program: Saint Vincent Hospital/ Worcester Medical Center
Podiatric Relevance:
Demineralized bone matrix has been used in podiatric medicine as an adjunctive treatment to many surgical procedures due to the wide variety of osteoinductive proteins. In addition, platelet-rich plasma (PRP) has been noted to enhance the activity of demineralized bone matrix. Many commercially available formulations of PRP include activation with thrombin. Thrombin activation has been theorized to change the physical property of PRP and also decrease the osteoinductivity of demineralized bone matrix.
Methods:
PRP was prepared with the GPS II system (Biomet Biologics). The levels of TGF-β1, VEGF, and PDGF-BB in the PRP, platelet poor plasma, and whole blood supernatants were measured using commercially available ELISA kits. TGF-β was selected to be tested before and after thrombin activation. TGF-β activity was measured with mink lung cells transfected with plansminogen activator inhibitor-1 (PAI-1) promoter-luciferase (luc) reporter. Only active TGF-β in PRP will then express PAI-1 linked luciferase activity. Cell number was determined using Saos-2 cells derived from human osteosarcoma cells and rat bone marrow-derived stroma cells. Cells were plated with and without thrombin activated PRP. Cell numbers were then read using a microplate reader. Demineralized bone matrix biologic activity was measured in vitro utilizing C2C12 rat myoblast cell line production of alkaline phosphatase and in vivo following intramuscular implantation of 27 athymic rats. Three implantation types were studied: demineralized bone matrix plus inactivated PRP, demineralized bone matrix with thrombin activated PRP and demineralized bone matrix alone. Explantation underwent histological staining and the second half for alkaline phosphatase activity.
Results:
Growth factors noted in thrombin activated PRP, unactivated platelet poor plasma, and thrombin activated whole blood showed a four to sevenfold increases in growth factor concentration as compared to whole blood. Thrombin activated PRP significantly increased luciferase activity. However, with the increase in storage time, spontaneous activation and increased luciferase activity was noted in the previously inactivated PRP. When osteosarcoma cells (Saos-2) were tested for biologic potential with thrombin activated PRP and unactivated PRP, high doses of thrombin activated PRP decreased the cell number. These results that suggest thrombin may have an inhibitory effect on growth and cell viability. Lastly chondrogenesis and osteogenesis were tested in vivo. PRP activated with thrombin plus demineralized bone matrix increased alkaline phosphatase activity. Further testing resulted in more bone formation and bone marrow was seen in PRP non-activated with thrombin plus demineralized bone matrix.
Conclusions:
Demineralized bone matrix augmented with platelet rich plasma proves to be a successful adjunctive procedure for producing osteoinduction, however this study concludes that prior activation with thrombin should be discouraged.