| Platelet Rich Plasma is a unique autologous clinical treatment modality. The creation process includes gathering whole blood, centrifugation, removal of the buffy coat and plasma layer, and re-centrifugation of the collected buffy coat and plasma to create a platelet rich solution called Platelet Rich Plasma (PRP). PRP has been demonstrated to expedite healing in clinical applications. PRP is currently used in a variety of clinical modalities including but not limited to: orthodontics, osteogenic care, orthopedics and cosmetics. What has yet to be thoroughly researched from a pre-clinical (non-clinical) setting is the use of PRP with combination therapies such as electrospun protein scaffolds as a means of initiating platelet activation. Collagen is a known platelet activator; furthermore, the use of a collagen protein scaffold may aid in delivering proteins that are synthesized in the later stages of the wound healing process. This combination treatment may lead to decreased wound healing time in clinical applications, assuming proper testing and pre-clinical studies are first completed.;The outcomes of this Ph.D. project occurred by completing an extensive literature review detailing platelets, wound healing, PRP and three research aims. Aim one encompassed the maximization, creation and activation of PRP using a scientifically accepted protocol, modified from Nagata et al. (2009) and Messora et. al. (2011). Once the various PRP solutions were synthesized using a standardized protocol, they were quantified and qualified using light microscopy (LM), scanning electron microscopy (SEM), ELISA testing, manual counting methods and flow cytometry.;Aim two of the project demonstrated the effectiveness of an optimized PRP in an in-vitro (bench-top) assay called a cell-migration assay or "scratch assay". The scratch assay is a way to measure cellular migration i.e. wound closure by creating a mock wound in a monolayer of epithelial cells. Initially, a coagulation assay was performed to first determine what percentages of PRP could be evaluated using the scratch assay. Once the percentages were determined, the mock wound with the PRP treatment was assessed for percent wound closure at the various pre-determined time-points.;Aim three involved the use of a murine in-vivo model testing PRP + collagen, collagen (stand-alone) and control sites on full-thickness wounds. The animals underwent a treatment period of 6 days allowing the various treatments to aid in the healing response. During this treatment period the animals were observed for behavioral recovery status and the wound sites were photographically assessed every 48 hours for percent wound closure. After the 6-day period the animals were euthanized and tissue was collected from the wound sites. The tissues were then analyzed for the resulting wound healing response. This included histological assessment techniques to identify re-epithelialization and epithelial thickness. |
