A Study On The Expression Of Osteoprotegerin During The Replacement Of Dog's Primary And Permanent Teeth

With the development of permanent tooth germs, primary dental roots begin to be absorbed, and finally primary teeth are replaced by permanent teeth. This process is called replacement of primary and permanent teeth, which is a complex and rigorous physiological process participated by multiple factors and multiple cells. At present, the main research direction are resorption of primary dental roots and development of permanent tooth germs. It has been proved that OC and OD are two important function cells involved in the absorption of deciduous tooth root and alveolar bone, they blazed a way for the erupting permanent tooth germs. However, these mechanisms are not fully clarified. So it is significant to study further OC for elucidating the mechanism of replacement of primary and permanent teeth.Osteoprotegerin is a newfound dissoluble tumor necrosis factor receptor in research area of bone metabolism for the past few years. It can not only inhibit OC formation and differentiation, but also increase bone density. It is a key factor to inhibit resorption of bone. In skeleton, majority OPG is secreted from osteoblast and marrow stroma cell. It has been proved that osteoblast can secrete OPG, which can interrupt signal passageway-RANK/RANKL of OC development by binding its ligand-OPGL, and inhibit osteoclast precursor cell's generation, differentiation, confluence and activation of mature osteoclast. Moreover, Hakeda et al found that OPG can inhibit directly bone-resorbing activity of mature OC by binding F-actin on the plasma membrane.Up to now, there has no literature on the expression of OPG in OC, and on the function of OPG in the process of replacement of primary and permanent teeth. Based on the fact that OC derives from hematopoietic cells and forms through mononuclear precursor fusing, and OPG express highly in monocyte, it is deduced that OPG may express in OC and OC can self-regulate and inhibit itself differentiation. In order to detect whether OPG is expressed in OC and OD during the succession of deciduou teeth, this investigation divided the process of the succession of deciduous teeth into three phases:stable phase of deciduous tooth root, absorptive phase of deciduous tooth root and permanent dentition phase.By IHC and ISH, the expression of OPG in OD, OC of deciduous tooth roots, alveolar bone and ameloblast, odontoblast of permanent tooth germ was detected at ptotein and mRNA level respectively and the role of OPG during the succession of deciduous teeth was analysed. Moreover, this investigation, referring to some documents on OC culture in vitro, cultured PBMC, separated from human perpheral blood cells with lymphocyte separating medium was cultured with cell medium mixed with 1,25(OH)2 D3,M-CSF and RANKL.By IHC and ISH, the expression of OPG in OLC, which appeared after 12 days of culture, was detected at protein and mRNA level respectively and then the role of OPG in OC formation and function was analysed. The main content and results are listed as below:Part1:Expression of OPG during the replacement of dogs' primary and permanent teeth1 Dogs' primary dentition roots stability stage: In this phase, the deciduous teeth roots were not absorbed biologically and alveolar absorption lacunae around deciduous tooth root were not found. The permanent teeth germs were developing and dentin and enamel matrix started to form.The alveolar around permanent teeth germs was in rebuilding phase and osteoclasts located in the alveolar absorbed lacunae. In the immunohistochemical and in situ hybridization experiments, OPG positive signals were seen in the multinucleated osteoclasts, osteoblasts. These results indicated that OPG secreted by OB negatively regulated the differentiation and function of OC by binding OPGL. Moreover OC probably synthetize and secrete OPG, which participate directly in the inhibition of defferentiation and maturation of OC by self-regulating, thus affect bone reconstruction. OPG positive signals were also seen in ameloblasts, odontoblasts of the permanent teeth germs. These results indicat that OPG takes part in the development of dogs' permanent tooth germs.2 Dogs' primary dentition root resorption stage: Dogs' deciduous tooth roots were undergoing absorption biologically and there were a lot of absorbed lacunae on the roots. The multinucleated osteoclasts were localized in the absorbed lacunae of the alveolar bone near the germs. Dentin and enamel of the permanent teeth germs went on forming and calcifying. In the immunohistoc- hemical experiment, the OPG-positive signals were observed on odontoclasts on absorpted root surface of primary tooth roots, the osteoclasts and osteoblasts in alveolar bone, and osteoclasts and osteoblasts dyed strongest, there was a significant difference(P<0.05). It indicated that the expression of OPG protein was enhanced in order to prevent alveolar bone and primary tooth roots absorbed excessively in the stage. In the in situ hybridization experiment, the OPG-positive signals were observed on osteoclasts, odontoclasts, osteoblasts, and osteoclasts dyed strongest(P<0.05). It indicated that osteoclasts enhance compensatively the excreting of OPG and inhibit differentiation and maturation of OC. The OPG-positive signals were observed as well on the ameloblasts and odontoblasts of the permanent teeth germs. It indicated that ameloblasts and odontobalsts expressed OPG, and were involved in the development of dogs' permanent teeth germs during the stages of the primary dentition roots resorption.3 Dogs' permanent teeth dentition stage: Dogs' deciduous teeth shed and permanent teeth erupted. Osteoclasts and osteoblasts were weak positive in im- munohistochemical and in situ hybridization experiment. Dying degree of these positive cells increased significantly(P<0.05). The results indicated osteoclasts' and osteoblasts' secreting low and the alveolar bone is in the weaker period of rebuilding.Part2:Expression of OPG in OLC cultured in vitroObserved by inverted microscope and HE staining, some cells enlarged after 6 days of the induced culture of human PBMC with three cytokines and multinucleate cells appeared after 12 days. Some of these multinucleate cells were very large, containing 3-7 nuclei. OLC cultured for 12 days were used for identification by the unique dying. The cells which were TRAP-positive and had bone absorptive lacuna by bone slice in one culture system were identified as OLC. OLC were positive in OPG immunohistochemical and in situ hybridization experiment. These positive results confirmed that OLC cultured in vitro express OPG and OPGmRNA.In brief, this investigation observed that OPG was expressed in OC,OD during the succession of deciduous teeth, and in OLC cultured in vitro for the first time. OPG probably negatively regulated the differentiation and function of OC and OD by indirect and direct role, controlling the alveolar bone and deciduous tooth absorption process and preventing rapid absorption.