Application Of Platelet-Rich Plasma Of Different Concentrations In Bone Tissue Engineering

Objective To observe in vitro the effects of platelet-rich plasma ( PRP) of different concentrations on the proliferation, differentiation, and mRNA of autologous bone marrow stromal cells(MSCs), to evaluate the bone induction effect of PRP on MSCs, and to investigate the feasibility of bone tissue regeneration by tissue engineering with the obtained optimal concentration.Methods Two times of centrifugation was used to extract PRP from the venous blood of the dogs, and the blood count was performed. The serum rich in growth factors(SRGF) were activated and prepared with serum-free D-MEM/F-12 solution to get 1%, 5%, 10%, 20%, and 30% SRGF. Density gradient centrifugation was applied to extract and culture MSCs of the dogs and generated, which was observed and photographed every day. The effects of PRP of different concentrations(l%, 5%, 10%, 20%, and 30%) on MSCs proliferation were observed with MTT on days 1, 3, 5, 7, and 9. Enzyme kinetics was used to detect the effect of PRP of different concentrations on ALP activity on days 3, 6, 9, and 12. ALP staining was performed with improved Gomori staining test on PRP-induced MSCs. The formation of mineralized nodules was detected with Dah1 McGec-Russll's alizarin red stain. The contents of osteocalcin were observed on days 7, 14, 21, and 28. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the ALPmRNA and OCNmRNA levels in PRP and rhBMP-induced MSCs. Results Adherence of the MSCs after the primary culture began to occur at 24-48h, and the cells blended into a monolayer 12d later. Most of the cells assumed fibroblast fusiform cells. After the induction with PRP, the cells increased significantly, and gradually changed into square or polygon, and melted into monolayer 4-5d later and multilayer 8-12d later. The platelet count showed that the platelet concentration in PRP was 5.38 times that in the whole blood. MTT detection showed that various levels of proliferation occurred in all the PRP groups, especially in the 10% PRP group. Enzyme kinetics detection showed that the ALP activity increased in all the PRP groups, especially in the 10% PRP and rhBMP groups. For the PRP-induced MSCs, ALP staining was strong positive, calcified nodules increased, and the osteocalcin expression alsoincreased. The difference was significant. Semi-quantitative RT-PCR showed that both ALPmRNA and OCNmRNA levels of the MSCs increased after the induction of PRP of different concentrations, especially the 10% PRP and rhBMP groups. Conclusion PRP of different concentrations ( 1%, 5%, 10%, 20%, and 30% )can effectively make MSCs to differentiate to osteoblasts and promote the osteoanagenesis. The best concentration is 10% PRP, which can be used to repair bone defects. The study is a breakthrough in bone tissue engineering.