Optimization Of Platelet-rich Plasma Through The Depletion Of Leukocyte Forpromoting Repair Of Bone Defects

OBJECTIVE: To evaluate the effects of L-PRP and P-PRP on bone repair and NF-?B pathway in vitro and in vivo,in order to identify the optimal PRP formulations for the treatment of bone defects.METHODS: Part ?: PCP was prepared with a single spin under different centrifugal conditions and P-PRP was prepared with different double-spin methods.Concentrations of blood cells,growth factors and pro-inflammatory cytokines,and platelet function were measured.Effects of different P-PRP formations on the proliferation,viablity and migration of HBMSC were evaluated.Part ?: L-PRP and P-PRP were prepared with WEGO PRP preparation system or the method developed in Part ?.Concentrations of leukocytes,platelets,growth factors and pro-inflammatory cytokines in L-PRP and P-PRP were measured.And correlation analysis was performed to evaluate the correlations between cellular composition and cytokine composition of PRP formulations.Part ?: HBMSC and EaHy926 were cultured with medium supplemented with 10% FBS,L-PRP or P-PRP,and then,the proliferation,viability,migration of HBMSC and EaHy926,tube formation of EaHy926,and osteogenic differentiation of HBMSC were evaluated.Part ?: HBMSC and EaHy926 were cultured with medium supplemented with 10% FBS,L-PRP or P-PRP,and then,NF-?B p65 expression in the nucleus,m RNA expression of COX-2 and i NOS,and PGE2 and NO production were evaluated.Part V: Calvarial defects in rats were treated with ?-TCP only,or L-PRP or P-PRP preprocessed ?-TCP.CAPE was delieved to inhibit NF-?B activation after L-PRP preprocessed ?-TCP implanted.The in vivo effects of L-PRP and P-PRP were assessed by histological and immunofluorescence examinations.RESULTS: Part ?: Centrifugation at 160×g for 10 min and 250×g for 15 min sucessively captured and concentrated platelets more efficiently with the preservation of platelet function and depletion of leukocytes and erythrocytes compared with other conditions.Moreover,the optimized P-PRP promoted HBMSC proliferation and migration significantly compared with P-PRP obtained from other conditions.Therefore,the method may the optimal double-spin method for P-PRP preparation.Part ?: While concentrations of platelets,PDGF-AB,TGF-?1 and VEGF were similar between L-PRP and P-PRP,concentrations of leukocytes,IL-1? and TNF-? were significantly higher in L-PRP.Further studies demonstrated that there were significantly positive correlations between platelet concentration and growth factors concentrations,and between leukocyte concentration and pro-inflammatory cytokines concentrations.Part ?: Compared with FBS,both L-PRP and P-PRP promoted the proliferation,viability,migration of HBMSC and EaHy926,tube formation of EaHy926,and osteogenic differentiation of HBMSC,with P-PRP showing greater effects.Part ?: L-PRP induced the nuclear translocation of NF-?B p65,upregulated m RNA expression of COX-2 and i NOS,and upregulated PGE2 and NO production of both HBMSC and EaHy926,while P-PRP did not.Part ?: Both L-PRP and P-PRP promoted healing process of rat calvarial defects,with P-PRP showing greater effects.Moreover,postoperative injections of CAPE enhanced significantly the positive effects of L-PRP on bone repair.CONCLUSIONS: Leukocytes in L-PRP may activate NF-?B pathway via the increased pro-inflammatory cytokines to induce the inferior effects on bone repair of L-PRP compared with P-PRP.Hence,P-PRP may be more suitable for the treatment of bone defects compared with L-PRP.