The Study Of Platelet-rich Fibrin And Platelet-rich Plasma For The Proliferation And Diferentiation Of ADSCs In Vitro

Background and Objectives:Platelet-rich plasma (PRP) is developed from autologous blood upon re-suspension of platelets. It contains many factors, such as transforming growth factor-β1(TGF-β1), platelet-derived growth factor AB (PDGF-AB),vascular endothelis growth factor (VEGF), insulin-like growth factor (IGF). Platelet-rich plasma is reported as the first generation of autogenous growth factors used clinically to stimulate bone regeneration. But its real efficacy is debated. Some studies showed that PRP had a limited effect in stimulating bone regeneration. It is suggested that PRP released growth factors quickly, just before the cell outgrowth from the surrounding tissue.And it is demonstrated that thrombin may have a toxic effect on the body cells.Platelet-rich fibrin is prepared naturally without addition of thrombin, and it is hypothesized that PRF has a natural fibrin framework and can protect growth factors from proteolysis.Unlike PRP, PRF does not dissolve quickly during the following hours after application.Due to the solid consistency of fibrin, PRF is slowly destroyed by remodeling, like a natural blood clot. he PRF contains high quantities of three main growth factors(TGF-β1、PDGF-AB and VEGF), and it sustains a very significant slow release of these key growth factors during at least1week.Furthermore, PRF is not only a platelet concentrate but also an immune node to stimulate defense mechanisms.It is even likely that the significant inflammatory regulation noted on surgical sites treated with PRF is the outcome of the effects from cytokines trapped in the fibrin network and released during the remodeling of this initial matrix.It is suggested that PRF would necessarily have very different effects from PRP. The purpose of our study was to compare the releasing of growth factors between Platelet-rich fibrin and Platelet-rich Plasma as well as their effect on the proliferation and differentiation of ADSCs in vitro.Materials and Methods:1. The comparison of Platelet-rich fibrin and Platelet-rich Plasma on the releasing of growth factorsTaking suction of the rabbit central ear artery blood, the PRF was prepared by one centrifugation method and the PRP was prepared by two centrifugation method. Five milliliters of fresha-modified Eagle Medium was added to PRF and PRP, and put them in a humidified atmosphere of5%CO2/95%air for incubating. The time points to collect exudates on days1,7,,14,21,28, and the levels of transforming growth factor-β1(TGF-β1) and platelet derived growth factor-AB(PDGF-AB) were quantified in PRF and PRP with Enzyme Linked Immunosorbent Assay.2. The comparison of the effect of Platelet-rich fibrin and Platelet-rich Plasma on theproliferation and differentiation of adipose-derived stem cells Excising rabbit inguinal fat tissue at aseptic condition and primary ADSCs were obtained by the method of enzyme digestion.The ADSCs were identified by the method of multi-directional differentiation and the experiments were carried out with cells from the third passages.The exudates of PRF and PRP were prepared for10%culture medium and used to cultue ADSCs.At the same time,set up positive group and negative group. Then,the cell proliferation were calculated by the method of blood cell countingin allgroups.At the same time, The exudates of PRF and PRP were prepared for10%osteogenic induction culture medium and used to cultue ADSCs.The next,the alkaline phosphatase reagent kit was adopted to determine the expression of alkaline phosphatase in all groups.Results:1. The comparison of Platelet-rich fibrin and Platelet-rich Plasma on the releasing of growth factorsPRF released the highest amount of TGF-β1at day14(P<0.01) and the highest amount of PDGF-AB at day7(P<0.05).PRP released the highest amounts of TGF-β1and PDGF-AB at the first day (P<0.01)2. The comparison of the effect of Platelet-rich fibrin and Platelet-rich Plasma on the proliferation and differentiation of adipose-derived stem cellsExudates of PRF collected at day14expressed the maximum proliferation and alkaline phosphatase(ALP) activity (P<0.05). Exudates of PRP collected at day1expressed the maximum proliferation and alkaline phosphatase(ALP) activity (P<0.05)Conclusions:1. Compare to PRP, PRF can gradually released growth factors.2. Compare to PRP, PRF expressed stronger and more durable effect on proliferation and alkaline phosphatase activity of ADSCs in vitro over time.