The objective of our study was to compare the capacity of PRP with different concentrations for the proliferation and differentiation of BMSCs, then select the optimum concentration and put forward the suggestion for clinical application of PRP.
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 medience and orthopedic surgery. Bone marrow-derived mesenchymal stem cells(BMSCs) are an attractive cell population in MSC family.Considerating 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 capable of regulating BMSCs proliferation and differentiation extensively, and exhibited a promoting prospect in regenerative medicine, the effect has not been elucidated in detail ,and the optimum concentration of PRP is still unknown. Accordingly, the objective of our study was to assess the effect of different concentrations, and optimize PRP concentration for proliferation and differentiation.
This experiment shown the method to obtain PRP of different concentrations, and stuied 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, 1200×109/L, 1500×109/L, 1800×109/L, 2000×109/L, 2200×109/L, 2500×109/L, up to 2700×109/L and 3000×109/L). Briefly, we characterized PRP with the markers of CD41a-PE and CD42b-FITC,used CCK-8 to test the infuluence of PRP for proliferation, evaluated osteogenic differentiation, adipogenic differentiation in levels of morphology,cytology,and molecular biology with the help of transmission electron microscopy,Oil Red O Staining, ALP staining,von Kossa,and real-time QPCR, and obtained a detail dose-action curve.
CCK-8 test shown that different concentrations of PRP significantly promoted proliferation compared to 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. ALP staining and von Kossa staining documented that PRP increased BMSCs 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 pink 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 piont, the staining area was low and similar to serum control group. In the osteogenic-based culture medium, PRP treatment can significantly enhance the osteogenic differentiation in a dose-depentent manner 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-depentent staining area rise. Oil Red O Staining demenstrated that PRP gradually tamplified total and per-cell insulin-induced intracytoplasmic lipid accumulation in a dose-dependent manner, with a double to hundredsfold increase of differentiation efficiency in the concentration ranging from 200×109/L to 3000×109/L compared with control. Below 2000×109/L concentration, lipid accumulation in test groups gradually increased with the increasing of concentration of PRP, while that greatly increased beyond 2000×109/L concentration.
In this study, we investigated the effect of different PRP concentrations on BMSCs proliferation and differentiation, and optimized the priviledge 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-ralative diseases,such as long bone defects, and choose 3000×109/L,which is weak for osteogenic differentiation conversely , to apply in plastic surgery.