The Application Of Human Platelet Lysate On Proliferation And Osteogenic Differentiation Of Human Dental Pulp Mesenchymal Cells In Vitro

Objective Periodontitis is mainly manifested as the destruction of periodontal tissue and it is the main reason of the tooth losing.Recent years,with the establishment of the concept of regenerative medicine and development of tissue engineering technology,autologous replantation of stem cells for the treatment of periodontal tissue defects has emerged.However,the components of fetal bovine serum(FBS)which used in vitro culture of the stem cells are complex.Risks such as allogeneic immune responses and the spread of potential diseases may arise in future clinical applications.Human platelet lysate(HPL)is a growth factor enrichment.A large amount of growth factors stored in the platelet alpha particles.This experiment explored the effect of HPL on the proliferation and osteogenic differentiation of human Dental Pulp Mesenchymal Cells(h DPSCs)in vitro.Provide a basis for HPL to replace of FBS.Methods Primary culture of the healthy pulp tissue was performed with 5%,10%,20%HPL and 10% FBS respectively.After the cells climbed out,morphology and growth state of the cells were observed by inverted fluorescence microscopy and scanning electron microscopy(SEM).Then the survival rate of the cells was calculated.Flow cytometry was used to detect the expression of mesenchymal stem cell-specific surface antigens CD34,CD45,CD44,CD90,CD105 and Stro-1.The cell proliferation was detected by the cell proliferation and toxicity test kit(cck-8)at 1d,2d,3d to 7d.Trypan blue staining was used to detect the viability of the cells.Real-time fluorescent polymerase chain reaction(Q-PCR)was used to detect the expression of osteogenic genes alkaline phosphatase(ALP),bone morphogenetic protein-2(BMP-2),osteocalcin(OCN)and runt-related transcription factor 2(RUNX2).Alizarin red staining was used to detect bone mineralization ability in vitro.All experimental datas were analyzed by/SPSS 22.0 statistical software.Results Fibroblast-like cells climbed out of the tissue blocks in each experimental and control groups.The inverted fluorescence microscope and SEM showed similar morphology of the cells,which were long fusiform and occasionally polygonal.Flow cytometry results showed that CD34(-),CD45(-),CD44(+),CD90(+),CD105(+)and Stro-1(+),which accorded with the phenotypic characteristics of human mesenchymal-derived cells.Cck-8 results showed that the proliferation rate of h DPSCs in 10% HPL was faster than that in 10% FBS group,and the 5% HPL group was similar to that in 10% FBS group at 4d(P<0.05).Alizarin red staining showed that the osteogenesis ability of the 10% HPL group was better than 10% FBS group.The results of Q-PCR showed that the expression of osteogenic genes BMP2,ALP,OCN and RUNX2 in the 10% HPL group were better than those in the 10% FBS group,and the difference was statistically significant(P<0.05).Conclusions Appropriate concentration of HPL can promote the proliferation and osteogenic differentiation of DPSCs effectively.Under certain conditions,HPL can be used in place of FBS for cell expansion.It provides a theoretical basis for the clinical safety of HPL for DPSCs culture.