Comparative Study Of The Quality Evaluation Of Platelet-rich Plasma

Objective:Investigate the impact of different anticoagulants and coagulants with autologous platelet-rich plasma(PRP)in order to evaluate the quality of PRP standardization.Method:Bone marrow stem cells(BMSCs)were seeded into platelet-rich plasma with three various anticoagulants(EDTA,heparin sodium HS,and sodium citrate SC).Quality of PRP was evaluated and flow cytometric assay was used to detect the activity of the platelet(CD62p,PAC-1).BMSCs were also seeded into PRP with different coagulants(Thrombin,Collagen-I,ADP)as well as PRP un-activated(negative group)and L-DMEM complete culture without PRP(control group).various coagulants with PRP on proliferation,osteogenic differentiation of BMSCs were analyzed by Methyl thiazolyl tetrazolium assay(MTT),ALP staining,Von Kossa staining,Confocal microscopic observation,RT-PCR and Western Blot at the morphological,cellular and molecular levels.Results:Various anticoagulants(EDTA,heparin sodium HS,and sodium citrate SC)could affect the quality of PRP.EDTA group revealed the best quality and activity(CD62p,PAC-1).With various coagulants(Thrombin,Collagen-I and ADP)in the proliferation of BMSCs,the MTT assay showed that all groups promoted the proliferation of BMSCs increasing with time.On the seventh day of culture,the cell number of each PRP group was significantly higher than that in the control group(p<0.05),Collagen-I group increasing the most rapidly.The cumulative release of growth factors(TGF-?1,PDGF)at each time point(2h,24h,72h,120h)in PRP groups were higher than those in the negative and control group(P<0.05).Collagen-I was considered as the best PRP coagulant.With thrombin as a platelet coagulant,the release of growth factor of PRP was rapid and direct,while the release of growth factor in Collagen-I-activated PRP was sustained,slow and sufficient.And the total release of ADP-activated PRP growth factors was the lowest.The study demonstrated the similar outcome in osteogenic differentiation.In terms of gene expression,the PCR and western bolt results showed that the expression levels of OCN gene and RUNX2 protein in each PRP group were higher than that in the control group(p<0.05).Collagen-I group was expressed more than other groups.Conclusion:Various anticoagulants caused different degrees of lysis and spontaneous activation of platelets,which lead to different quality of PRP.Compared with HS and SC,EDTA could maintain the structural integrity of platelets,reduce their spontaneous activation,and increase the release of PRP growth factors for a longer period of time,thus ensuring the biomass of PRP.In addition,various coagulants also showed diverse results in the proliferation as well as osteogenic differentiation of BMSCs.Compared with Thrombin and ADP,Collagen-I may be a better choice.