Platelet Rich Plasma combined with an Electrospun Collagen Scaffold: in-vivo and in-vitro Wound Healing Effects

Abstract

Platelet Rich Plasma (PRP) is an autologous clinical treatment that has been demonstrated to expedite wound healing through the delivery of platelets at a minimum concentration that is three times that of whole blood. PRP is currently used in a variety of clinical applications, including but not limited to: orthodontics, osteogenic care, orthopedics, acute and chronic wounds and various cosmetic applications. Although the treatment has been investigated in many regards, there is a lack of exploration into means of activation through the use of electrospun (e-spun) collagen scaffold technology that allows for activation in situ, or directly in the wound. In this study, this combination therapy (PRP and e-spun collagen scaffolds) was created and investigated first using an in-vitro assay and then in an in-vivo murine full-thickness wound model. In the current study, a standardized double centrifuge protocol was utilized for the creation of PRP. Flow cytometry and manual counts were performed in order to quantify the degree of platelet concentration. Manual counts yielded a mean platelet count for whole blood and PRP 1 µL smears at 10.08 ± 3.09 platelets/μl and 878.76 ± 156.28829.7 platelets, respectively (p < 0.01). Flow cytometry reported a mean platelet count of 426,461 ± 47,394 for the processed PRP and a whole blood platelet count of 35,480 ± 6,463.75 (p < 0.01), supporting that the currently described method produces clinically defined PRP. Light and scanning electron microscopy were utilized to determine if electrospun collagen scaffolds were an activator of platelets. Analysis was performed using morphological features of the platelets as the activation measurement. Upon application to an electrospun scaffold, platelets presented with an activated state in which the discoid morphology was no longer present, but was replaced with extended pseudopods and blebs. The morphology of non-activated PRP remained discoid and smooth. The change in morphology was indicative of activation and validated that the scaffolds serve as an in-situ platelet activator. An in-vitro scratch assay was utilized as an outcome measure to test the potency and effectiveness of the resulting PRP. The scratch assay has been commonly used as a cellular migration assessment, or in the current study, as a high throughput means to observe in-vitro wound closure. Varying concentrations of PRP were created and applied to a scratched (wounded) fibroblast monolayer in order to determine its effects on the wound. A concentration of 0.25% PRP was determined to have the highest percent closure at hour 12 compared to the control that did not close until hour 16. An in-vivo murine full-thickness wound model was used to demonstrate the effectiveness of the PRP and collagen scaffold treatment in a biological model. Post wound creation (96 hours) the wound closure percentage of the PRP/electrospun collagen treatment was 100% compared to the control at 86.076 ± 11.2806% (p=0.01), supporting the use of a combination therapy over standardized care. There was...