The Effects Of Platelet-rich Plasma Prepared By Different Methods On Cell Proliferation And Osteogenic Differentiation Of Adipose-derived Stem Cells

Objective Platelet-Rich Plasma is autologous whole blood platelet concentrates obtained by centrifugal method.It will release a variety of cytokines after activation,such as platelet-derived growth factor,transforming growth factor-?,insulin-like growth factor and epidermal growth factor,vascular endothelial growth factor,epithelial cell growth factor,and so on.These cytokines have some positive effects on cell proliferation,differentiation,morphogenesis,migration,angiogenesis and bone regeneration.The activated PRP contains fibrin,fibronectin and other protein and can be used as a natural scaffold materials for tissue engineering to create a favorable local environment for tissue regeneration.At present,there are many reports on the preparation of PRP,and it is found that the different preparation methods will affect the concentration of platelet and the growth factor in the final PRP,subsequently influencing its effectiveness.Adipose-derived stem cells(ADSCs)has been recently isolated from adipose tissue with multipotential differentiation potential.It can differentiate into osteoblasts,chondrocytes,adipocytes,smooth muscle cells and myocardial cells under some certain conditions.Autologous ADSCs is an excellent seed cell resource for tissue engineering used for the healing and regeneration of tissues and organs due to its accessibility and rapid amplification characteristics in vitro.Although previous studies have confirmed that PRP can play a vital role in a variety of cells,few of them study on ADSCs.This study used three different methods to prepare PRP and evaluated the differences in platelet concentration of the products as well as the growth factor release.The ADSCs were co-cultured with different PRPs and the effects of PRP on ADSCs proliferation and osteogenic differentiation were tested to explore the possibilities of PRP+ADSCs composite material for tissue engineering technology before clinical application.Methods 1.In this study we adopted the traditional twice centrifugation method,thrice-centrifugation method and a modified Tubex method to prepare PRP and evaluated the recovery rates of platelet in PRP using blood platelets count method.2.The rabbit ADSCs were obtained using an enzymatic digestion method and characterized by flow cytometry analysis.Its osteogenic differentiation and adipogenic differentiation properties were evaluated by Alizarin red staining and oil red O staining.3.The aforementioned PRPs were activated and added into ?-MEM at a mass ratio of 5%.After co-cultured with ADSCs,their effects on cell viability were evaluated by CCK-8 cell viability kit at different time points.4.The 5% PRP prepared by different methods were used to culture ADSCs.After cultured for different times,their effects on osteogenesis differentiation ability of ADSCs were evaluated by alizarin red staining,alkaline phosphatase staining and quantitation,and Quantitative PCR.Results1.The results from thrombocytometry showed that compared with twice centrifugation method and thrice-centrifugation method,the PRP prepared by Tubex had a higher recovery rates of platelet and the concentrations of growth factors were also significantly higher than the other two methods.2.The cells were isolated from rabbit adipose tissue by enzyme digestion method.The results from flow cytometry analysis,alizarin red staining and oil red O staining showed that the characters of the obtained cells were consistent with these reported in literatures,the cells can be defined as ADSCs.3.After 21-days cultivation,the formation of mineralized nodules were observed in all PRP-treated groups,and the alkaline phosphatase activities of ADSCs were significantly higher than the untreated cells.The results of real-time quantitative PCR showed that the expressions of osteogenic differentiation marker genes were significantly higher in PRP-treated cells than those induced by traditional mineralized solution.By comparing the effects of three kinds of PRP on osteogenic differentiation of ADSCs,we found that the PRP prepared by Tubex method could promote the osteogenic differentiation of adipose derived stem cells better than that of the other two methods.Conclusion Based on these above results,we found that the content of platelets and growth factors in PRP prepared by Tubex were higher than those prepared by twice centrifugation method and thrice-centrifugation method,as well as its effect on cell proliferation and osteogenic differentiation of ADSCs.The preparation of PRP by the Tubex closure device is simple and can improve the recovery rate of platelet.The abundant cytokines in PRP are conducive to the proliferation and differentiation of adipose derived stem cells.