Development of a new extraction method for platelet-rich plasma and partial purification of platelet-derived growth factor and transforming growth factor beta

Abstract

Platelet-rich plasma (PRP) is the cell free plasma, which has an enriched concentration of platelets and clotting factors with the ability to enhance the natural healing process. PRP is often used by physicians in an office setting to accelerate the healing of a variety of sports related injuries, chronic wounds and enhance skin rejuvenation. PRP mimics the wound healing cascade by enhancing the recruitment, proliferation and differentiation of cells involved in tissue regeneration. Although PRP is used to enhance healing, the efficacy thereof is debated as no clear-cut set of parameters is available that device manufacturers and protocols should follow. The lack of uniformity in the PRP preparation methods results in differing PRP volume, platelet contents and unavoidably platelet-derived growth factors. Therefore, the aim of this study was to develop a simple and rapid method for preparing autologous PRP in an office setting using a tabletop centrifuge for point-of-care use. The simplified preparation procedure involved a single centrifugation step of 18 ml of whole blood, which sufficiently enriched the platelet content in the PRP fraction. As activated platelets express and release growth factors and cytokines that mediate the different phases of the wound healing cascade, the extracted PRP fraction was activated with an ethanol, calcium chloride (CaCl2) and platelet poor plasma (PPP) preparation in glass containers, without the collection of additional blood as required in some protocols. The activated PRP formed a fibrin clot, trapping the degranulating platelets and its released growth factors. The concentration of TGF- 1 obtained from the fibrin clot was 45.49 ± 3.80 ng/ml, in range with the available literature. During the in vitro studies, the extracted PRP by the developed method was able to significantly induce cell proliferation in a dose dependent manner. Cells enumerated with the crystal violet assay indicated that the cells treaded with 5% or 10% PRP significantly increased the percentage of viable cells to 165-176% and 156-158%, when compared to the positive controls. Cells enumerated with the MTT-assay indicated that the cells treaded with 5% or 10% PRP increased the percentage of viable cells to 79-91% and 87-105% which is comparable to that of the positive control. Data from the cellular proliferation assays indicate that sufficient plateletderived growth factors had been obtained with the preparation procedure. Furthermore, data from the in vivo studies indicated that the extracted PRP was able to augment soft tissue regeneration and bone formation. Treatment with the activated PRP resulted in symptom reduction and accelerated healing of various injuries. The simplified preparation and the use of the provided study product packaged in a kit developed during this study will enable physicians to easily obtain autologous PRP, in an office setting for point-of-care use, with the ability to induce tissue regeneration.