The Regulation And Mechanismof Recombinant Human Bone Morphogenetic Protein-2 And Human Periodontal Ligament Fibroblasts On The Formation Of Osteoclast Like Cells

The biologic foundation of orthodontic tooth's movement is dependent on the bone resorption and formation involved with human periodontium.Orthodontic force has a direct effect on periodontal ligament cells(PDLCs) that is a heterogenicitic cell mass,which some of them can differentiate into osteoblasts and cementoblasts. Furthermore,PDLCs can express osteoprotegerin(OPG) and receptor activator nuclear factor kappa B ligand(RANKL) to regulate the activity of ostoclast.The immediate contact of osteoblast/matrix cells and osteoclast precursors(OCP) plays an essential role in formation and differentiation of osteoclast(OC).Moreover,when some bone resorption stimulus had an effect on osteoblast/matrix cells,RANKL's binding with receptor activator of nuclear factor-κB receptor(RANK),which expressed on the surfaces of OCP and OC,lead to a cascade reaction to promote the OC differentiation,maturity and activation.Additionally,osteoblast/matrix cells secreted OPG,which was cloned and found to inhibit OC differentiation and activities by competing with the binding of RANKL to RANK.Bone morphogenetic protein-2(BMP-2),a member of the transforming growth factor-β(TGF-β) superfamily of polypeptides,which were originally identified by the ability to form ectopic bone and promote the regeneration of alveolar bone,cementum and periodontal ligament.Now,many abroad scholars found that BMP-2 had an effect on OC differentiation and activation involved with osteoblast/matrix cells through RANKL/OPG.However,the roles of BMP-2 and PDLCs in OC activation remain unknown.Orthodontic treatment induces site-specific bone resorption and formation by pressure and tension applied to PDLCs,meanwhile,BMP-2 has a double contribution on osteoblast and osteoclast,so to identify the role of BMP-2 in PDLCs will have a significant meaning about tooth movement,corrective efficiency,alveolar bone remolding and post-treatment stability.The aim of this study was to observe the effect of the human periodontal ligament fibroblasts(PDLFs) and rhBMP-2 on the formation of osteoclast like cells (OLC) in vitro by co-culture of PDLFs and peripheral blood mononuclear cells (PBMC) that were isolated from normal volunteers;to evaluate the expression of OPG and RANKL from PDLFs and preliminarily research the effect of rhBMP-2 on RANKL/OPG.The study was divided into two parts as follows:一:The effect of the rhBMP-2 and human PDLFs on OLC formationObjective:(1):To establish a direct co-culture model of PDLFs and PBMCs,and then survey the effect of PDLFs on OLC formation;(2):To observe the influention of rhBMP-2 on the number of tartrate-resistant acidic phosphatase TRAP(+) mono-nucleate cells and multi-nucleate cells(3):To investigate the effect and mechanism of rhBMP-2 and human PDLFs on the formation of OLC in vitro.Methods:Human PDLFs were cultured and identified in vitro;PBMC were isolated from normal healthy volunteers by Ficoll-Paque Plus lymph separating medium.Then these two type cells were co-cultured in the presence of dexamethasone and 1α, 25-(OH)₂D₃ for 18 days,setting up the groups of rhBMP(+) and rhBMP(-).The medium was changed every 3 days with half throughout the experiments.Then the numbers of tartrate-resistant acidic phosphatase(TRAP) positive multi-nucleate cells were recorded.Results:(1):The OLC formation was observed by the model of direct co-cultured PDLFs with PBMCs,which identified by TRAP staining and the pits on dentin slices;(2):The numbers of TRAP(+) multi-nucleate cells were increased by the medium with 100ng/m rhBMP-2(t=71.070,P＜0.001).There is a significant difference in the numbers of TRAP(+) mono-nucleate cells and TRAP(+) multi-nucleate cells between PDLFs-PBMCs co-cultured group and only PBMC cultured group(t=39.000,P＜0.001).Significantly more OLC were formed in the presence of rhBMP-2 and PDLFs(t=17.097,P＜0.001).(3):RhBMP-2 and PDLFs play an important role in the formation of OLC, simultaneously,the combination of rhBMP-2 and PDLFs significantly increased the number of OLC.Conclusion:(1):Significantly PBMCs can differentiate into TRAP(+) multi-nucleate OLC when co-cultured with PDLFs;(2):rhBMP-2-induced the formation of OLC involves the modulation of PDLFs. 二:The expression of OPG/RANKL from PDLFs and the effect of rhBMP-2 on OPG/RANKLObjective:(1):The purpose of this study was to evaluate the expression of OPG/RANKL from PDLFs in gene level and protein level;(2):To observe the effect of rhBMP-2 on OPG/RANKL protein,and then explore the mechanism of its effect on OLC formation;Methods:Human PDLFs were prepared by using tissue explant culture technique in vitro, and then detected RANKL protein expressed on PDLFs by immunocyto -chemistry. Meanwhile,the medium were collected every 3 days to detect OPG protein secreted from PDLFs with 100ng/ml rhBMP-2-induced by ELISA.Results:(1):The positive expression of RANKL protein was detected on cellular membrane and endochylema of PDLFs;also PDLFs can secrete OPG protein;(2):rhBMP-2 significantly reduced the quantity of OPG protein(F=45.433, P＜0.001).Conclusion:(1):Human PDLFs expressed OPG and RANKL in base condition;(2):rhBMP-2 had an effect on OPG,so we presume that the effect of rhBMP-2 on OC activation was due to OPG/RANKL pathway.