4:48 Platelet rich plasma stimulates stem cell chemotaxis, proliferation and potentiates osteogenic differentiation

Abstract

Purpose of study: Platelets are known to perform multiple functions during injury and tissue repair. As a repository of multiple growth factors such as PDGF, EGF, VEGF, and TGF-β, degranulation of platelets at wound sites serves to initiate or enhance the healing cascade. Armed with this knowledge, clinicians have used platelet rich plasma (PRP) in conjunction with bone graft materials to enhance osseous repair. As mesenchymal stem cells (MSDs) are known to be an essential component of the bone repair process, we sought to characterize their response to PRP. Because we have previously reported the profound mitogenic effect of PRP on MSCs, the purpose of this study was to extend our understanding of PRP's effect on MSCs during the chemotactic and cell differentiation phases of healing.Methods used: PRP was isolated from fresh human blood using a commercially available point-of-care system, designed to obtain PRP from a small amount of blood. Samples of the starting material and PRP were analyzed to determine the absolute concentrations of platelets. PRP, platelet poor plasma (PPP) and unfractionated blood were clotted with thrombin/CaCl2. PRP and PPP releasates were diluted in serum-free DMEM to create working stocks. Human NSCs (hMSCs) were isolated and culture expanded from bone marrow, as described previously. The ability of concentrated platelet releasate to stimulate the chemotactic migration of hMSCs was measured using a modified Boyden chamber. Cells were allowed to migrate for 4 hours, then stained and counted. The ability of PRP to support proliferation of hMSCs without loss of their osteogenic potential was demonstrated by first expanding hMSCs in media supplemented to 10% with PRP, or PPP, releasate. After 5 to 7 days in the test media, hMSCs were grown in media containing osteoinductive supplements (OS). Morphologic changes, alkaline phosphatase activity and matrix mineralization were used as differentiation markers.of findings: The efficiency of platelet recovery was approximately 70%, thus creating an approximately five-fold increase in platelet-derived growth factors. Exposure of hMSCs to PRP releasate caused a significant chemotactic effect in a dose-dependent manner. Control cultures with PPP or serum-free media exhibited significantly lower chemotactic rates. Human MSCs exposed to OS media after growth in PRP-supplemented media continued to proliferate more rapidly than the controls, and also displayed greater alkaline phosphatase activity and mineral deposition on a per cell basis, thus indicating an enhancement of differentiated function.Relationship between findings and existing knowledge: These studies demonstrate a direct effect of PRP releasate on purified human MSCs, which play a pivotal role in the process of musculoskeletal tissue repair. In view of the data presented, and clinical reports of enhanced bone healing, we suggest that local application of PRP causes migration of hMSCs to the wound site, followed by their substantial replication to form a repair blastema. As the bioactive factors diffuse away from the fibrin scaffold, now densely populated by hMSCs, the cells cease dividing and are primed to respond to the residual PRP releasate and endogenous inductive cues that stimulate differentiation.Overall significance of findings: The demonstration that autologous PRP is a source of concentrated growth factors that possesses chemotactic, mitogenic and osteopromotive activity lends further credence to its therapeutic role in clinical orthopedics.Disclosures: Device or drug: Symphony Platelet Concentrate System. Status: Approved.Conflict of interest: Stephen Haynesworth, grant research support; Stephen Haynesworth, consultant; Sudha Kadiyala, other support; Sudha Kadiyala, stockholder; Scott Bruder, employee; Scott Bruder, stockholder, JNJ, DePuy AcroMed.