An In Vitro Study Of Different Capacity Of Platelet-rich Plasma (PRP) With Different Concentrations For Proliferation,Osteogenic And Adipogenic Differentiation Of Human Bone Marrow Mesenchymal Stem Cells

Objective: Platelet-rich plasma (PRP) is a fraction with a high concentration of platelets isolated from autologous whole blood. The local release of multiple endogenous growth factors, with a natural percentage, plays an important role in wound healing and tissue regeneration, which supports PRP as an essential member in sports medicines and orthopedic surgery. Bone marrow-derived mesenchymal stem cells (BMSCs) are an attractive cell population in MSC family. By advantage of their high capacity for proliferation and differentiation, BMSCs have been recently presented as a promising candidate for extensive therapeutic possibilities in regenerative medicine. Although many studies have shown PRP were capable of regulating BMSCs proliferation and differentiation extensively, and exhibited a promoting prospect in regenerative medicine,the effects has not been elucidated in detail, and the optimum concentration of PRP is still unknown. Therefore, the objective of our study was to assess the effect of different concentrations of PRPs, and optimize PRP concentration for MSC proliferation and differentiation.Methods: The current study have shown the method to obtain PRP of different concentrations, and have explored the capacity of PRP for regulating BMSCs proliferation and differentiation with different concentrations (200×109/L,500×109/L,800×109/L,1000×109/L,1200×1O9/L,1500×109/L,1800×lO9/L,2000×109/L,2200×109/L,2500×1O9/L,up to 2700×109/L and 3000×109/L). Briefly, we characterized PRP with the antibodies of CD41a-PE and CD42b-FITC, used CCK-8 assay to test the influence of PRPs for MSC proliferation, evaluated osteogenic differentiation and adipogenic differentiation of MSC with the help of transmission electron microscopy, Oil Red O Staining, ALP staining, von Kossa, and real-time QPCR.Results: The curves of CCK-8 test shown that different concentrations of PRP significantly promoted MSC proliferation compared to the control group, and it was in a dose-dependent manner in the range from 200×109/L to 1800×109/L. However, it reached a platform when it was beyond 1800×109/L. The results of ALP staining and von Kossa staining documented that PRPs increased MSC differentiation in a dose-dependent manner,with a double to twelvefold increase of differentiation efficiency in the concentration ranging from 200×109/L to 1500×109/L compared with control, and the differentiation efficiency reached a peak at 1500×109/L. However, the differentiation efficiency decreased smoothly from 1500×109/L to 3000×109/L in a dose-dependent manner, and at the 3000×109/L point, the staining area was decreased and even similar to the control group. In the osteogenic induction medium, PRPs treatment can significantly enhance the osteogenic differentiation in a dose-depentent manner when PRP concentration is below 1500×109/L,which may result from a synergistic effect. Interestingly, beyond 1500×109/L, there was a steep fall, reaching four or six folds, and after 6 days, a higher concentration gave a dose-dependent staining area rise. The results of Oil Red O staining demonstrated that PRPs gradually amplified total and per-cell insulin-induced intra-cytoplasmic lipid accumulation in a dose-dependent manner, with a double to hundreds fold increase of differentiation efficiency in the concentration ranging from 200 x109/L to 3000x109/L compared with control. Below a concentration of 2000×109/L, lipid accumulation in test groups gradually increased with the increasing of concentration of PRP, while that greatly increased beyond 2000×109/L concentration.Conclusion: In this study, we investigated the effects of different concentration of PRPs on MSC proliferation and differentiation, and optimized the concentration for clinical applications. We found that the optimum concentration of PRP for promoting BMSCs proliferation was 1800×109/L in vitro, while it became 1500×109/L when it went to osteogenic differentiation. There was a dose-dependent manner for adipogenic differentiation beyond 2000×109/L. These results suggested that we can choose 1500×109/L, which is weak for adipogenic differentiation, as the optimum concentration for osteogenic differentiation-relative diseases, such as long bone defects, and choose 3000×109/L, which is weak for osteogenic differentiation conversely , to apply in plastic surgery.