Effects of platelet rich plasma and platelet lysate on proliferation of rat bone marrow mesenchymal stem cells: a comparative study

Abstract

Objective To compare the effects of platelet rich plasma(PRP)and platelet lysate(PL)on proliferation and cell cycle of cultured rat bone marrow stem cells(BMSCs).Methods BMSCs were obtained from twenty 4-week-old SD rats using the whole bone marrow isolation and cultivation method and were identified with flow cytometry.Blood samples were taken from the hearts of thirty 12-week-old SD rats and gradient centrifugation was used to prepare PRP,and PL was obtained after three times of centrifugation and repeated freezing and thawing.The third generation of BMSCs with good growth state were divided into seven groups according to different culture media:ordinary complete medium(A group),1% PRPconditioned medium(B group),1% PL-conditioned medium(C group),5% PRP-conditioned medium(D group),5% PLconditioned medium(E group),10% PRP-conditioned medium(F group),and 10% PL-conditioned medium(G group). The proliferation of BMSCs was assessed by MTT assay.The PCNA protein expression was assessed by immunofluorescence method.Cell cycle of BMSCs was tested by flow cytometry.Western blotting analysis was used to analyze CyclinD1and p27 Kip1protein expression of BMSCs.Results The proliferation of BMSCs was significantly promoted by 1%,5%,10% PRPand 1%,5%,10% PL-conditioned media after cultured for 24h,48h,and 72hin a time-and dose-dependent manner(P 0.05),with the effect of PRP and PL being similar at the same concentration(P0.05).Immunofluorescence assay showed that PRP and PL both significantly promoted PCNA protein expression in a dose-dependent manner(P0.01).Flow cytometry showed that different concentrations of PRP- and PL-conditioned media,compared with the ordinary complete medium,resulted in gradually reduced cells in G0/G1 phase,gradually increased cells in S,G2 /M phase,and significantly increased PI(P0.05),with the changes in a dose-dependent manner.However,the effects of the same concentration of PRP and PL on cell cycle were not significantly different(P0.05).Western blotting analysis showed that 5% PRP and 5% PL significantly up-regulated CyclinD1and down-regulated the expression of p27Kip1.Conclusion Different concentrations of PL and PRP can accelerate cell cycle progression of cultured rat BMSCs by up-regulation CyclinD1and inhibiting p27Kip1, promoting the proliferation of BMSCs in a time-and dose-dependent manner.PL and PRP at the same concentration have the same proliferation-promoting effect,indicating that PL may be used as an alternative of PRP to promote the repair of bone defects.