| Background and ObjectiveThe development of dental implantation techniques has contributed to promote the application of dental implantation, which has become one of the important methods for the restoration of partially and totally edentulous patients. Compared with traditional methods, the restorable effect of dental implantation is better in the function and aestheics. However, an important factor which confines the application of dental implant is a insufficient volume of the bone at recipient areas. To solve this problem,we carry out many bone incremental technologies, such as bone condensing,bone splitting,maxillary sinus floor lifting,bone grafting and guided bone regeneration(GBR), but these methods have some limitations.In 2000, Choukroun's platelet-rich fibrin(Choukroun's PRF)that is known as the second generation platelet-concentrate was first developed by Choukroun in Frence.It has the advantages of PRP and has strong osteogenic ability. The preparation of Chouhoun's PRF is simple;it completely derived from the autologous blood without any biological agents, avoiding the ethical controversy and blood cross-infection risk. Chouhoun's PRF is used as a fibrin membrane or gel enriched with platelets and growth factors. Various growth factors play a synergistic role in promoting bone and soft tissue repair. The current researches of Chouhoun's PRF are still at a preliminary stage, and still needs a lot of experimental and clinical researches to analyze and evaluate the mechanism of stimulation osteogenesis and clinical effect. In this study, we research on the bone regeneration ability of Choukroun's PRF and observe the effect of Choukroun's PRF on bone marrow mesenchymal stem cells and human osteoblast-like cells(MG-63) in order to discuss the effect of Choukroun's PRF on the early role of osteogenic processes and provid the experimental basis for clinical application.MethodsThe bone marrow was taken from the tibial tuberosity of four rabbits.The BMSCs were separated and eultivated by the density gradient centrifugation and the passage bolting of the adherence cells at extraorgan. The growth character of the BMSCs was observed. The autologous blood of animals was collected and centrifugated to preparate Choukroun's PRF. Four groups were randomly divided: laser group 1 (L1):cells were carried in standard conditions with a PRFmembrane; control group1(C1): cells were carried in standard conditions;laser group 2 (L2): cells were carried in differentiation conditions with a PRF membrane;control group2(C2) cells were carried in differentiation conditions.The MTT values were detected at all time points in the laser group 1 and the control group 1; the positive expression of collagen typeâ… were detected at all time points in the laser group 2 and the control group 2.MG-63 was seeded into H-DMEM with 10% fetal bovine serum. The blood of human was collected and centrifugated to preparate Choukroun's PRF.Two groups were randomly divided: laser group3(L3) and control group3(C3). In the laser group 3, the MG-63 cells were interfused with Choukroun's PRF .The MTT values and the positive expression of APL,collagen typeâ… ,OPG and RANKL were detected at all time points . Results1.BMSCs morphology:After the primary culture,cells were inoculated with different sizes of the round. They began their attachment after 24-36 hours of primary culture.36-48 hours later, the attached cells began to increase, and the cells were spindle or polygonal. 4 days later,cells began to proliferate.And most of the cells were triangular, polygonal and spindle while some spindle cells that had larger nuclei extended multiple short processes. After 6-8 days, most of the cells arranged as fishes or spiral,when cells aggregated and cell-conjunetions were close.10-12 days later, with cell colony inereasing,the cells fused into flake and paved the flask's bottom. After digestion, most of the subeultured cells were round and quickly adhered. Cells increased rapidly and changed to a polygonal or triangle shape.The morphology of subeultured cells had no significant difference with primary cells.About 7 days, the cells fused into single deck.2.The effect of Choukroun's PRF on proliferation activity of BMSCs:MTT results showed that: on the 1st day and 3rd day,there was no significant difference between the absorbance A of L1 group and that of C1 group (P> 0.05);in the 5th day and 7th day,there was significant difference between the absorbance A of the L1 group and that of C1 group (P <0.05).3.The effect of Choukroun's PRF on the expression of coll I in BMSCs :On the 3rd and 7th days afer adding Choukroun's PRF into conditioned medium, coll I were brown and distributed in the cells cytoplasm. With immunohistochemical method, the expression of coll I in L2 group were higher than that in C2 group. (P <0.05). 4.The effect of Choukroun's PRF on proliferation activity of MG-63 cells:MTT results showed that: on the 1st day,3rd day,5th day and 7th day,the absorbance A of L3 group were higher than that of C3 group (P <0.05).5. The effect of Choukroun's PRF on ALP activity of MG-63 cells:On the 3rd and 7th days adding Choukroun's PRF into standard medium, ALP were brown and distributed in the cells cytoplasm. With immunohistochemical method, the expression of ALP in L2 group were higher than that in C2 group. (P <0.05).6. The effect of Choukroun's PRF on the expression of coll I,OPG and RANKL in MG-63 cells:On the 3rd and 7th days adding Choukroun's PRF into standard medium, coll I,OPG and RANKL were brown and distributed in the cells cytoplasm. With immunohistochemical method, the expression of coll I,OPG and RANKL in L2 group were higher than that in C2 group. (P <0.05).But on 7th day ,the RANKL/OPG ratio was lower than that on 3rd day (P <0.05).ConclusionIn the experimental group,we compared the effect of Choukroun's PRF on bone marrow mesenchymal stem cell and human osteoblast-like cells (MG-63) proliferation and differentiation by adding the Choukroun's PRF in the medium and the blank control.,and the conclusions as follows:1.In the early phase of proliferation,Choukroun's PRF can promote proliferation of bone marrow mesenchymal stem cells;2.In the early phase of differentiation,Choukroun's PRF can promote bone marrow mesenchymal stem cells to differentiate into bone cells, involved in the early bone repair;3.In the early phase of proliferation,Choukroun's PRF can promote proliferation of human osteoblast-like MG-63 cells;4.In the early phase of differentiation,Choukroun's PRF can promote human osteoblast-like MG-63 cells to secrete bone-related matrix , and promote early bone formation;5.In the early phase of differentiation,Choukroun's PRF can promote human osteoblast-like MG-63 cells to expresse OPG and RANKL, decreasing the RANKL / OPG ratio, and inhibit the activation of osteoclasts and promote bone tissue repair.
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