| For orthodontic tooth movement, biochemical reaction surrounding the dental roots has been the focus of investigations. Orthodontic tooth movement results from the remodeling capacity of alveolar bone, the presence of osteoclasts at locations of bone remodeling is antecedent to orthodontic tooth movement, and a better understanding of osteoclasts differentiation at the compression sites could have a profound impact on the effective control of orthodontic tooth movement. Osteoprotegerin (OPG) is a novel secretory member of the tumor necrosis factor (TNF) receptor superfamily that negatively regulates osteoclastogenesis. The receptor activator of the NF-κB ligand (RANKL) is one of the key regulatory molecules in osteoclast formation and binds to OPG. There were few researches about the role of RANKL and OPG on bone remodeling during orthodontic tooth movement, so we performed in vivo and in vitro experiments to explore the role of OPG and RANKL on the induction of osteoclasts and bone remodeling at the compression site during tooth movement.Experiment in vivo72 adult male Wistar rats were randomized to either experimental group (n=60) or control group (n=12). In experimental group, an orthodontic appliance was devised to make the maxillary right first molar to move messily, 12 rats of control group were fixed with the same appliance, but no force was applied. 10 rats of experimental group were sacrificed at 1, 3, 5, 7, 10, and 14 days respectively, 2 rats of control group were also sacrificed at the same time. After the tissue samples were prepared, we examined RANKL and OPG protein localization and osteoclasts induction at the compression sites. Result show that TRAP-positive mononucleated and multinucleated cells were markedly induced at the compression side at 3, 5, and 7 days. Mononucleated osteoclast reached peak level at 3 days after tooth movement, while multinucleated cells reached peak level at 5 days. Immunohistochemical staining revealed that expression of RANKL and OPG protein was detected in osteoblasts, bone lining cells, fibroblasts and osteoclasts which mostly located in resorption lacunae. Compared with expression of RANKL and OPG protein in control group,RANKL protein was expressed strongly at the compressive site at 3, 5, and 7 days after experimental tooth movement, while OPG protein was expressed strongly at 5 and 7 days. The result of image analysis showed that there was a significant difference between control group and tooth movement group. In addition, RANKL expression increased in parallel with the changes in the numbers of osteoclasts. OPG protein was weekly expressed at early stage of tooth movement, and OPG expression became stronger after 5 days of tooth movement. At 7 days, OPG expression was still at a relatively higher level while RANKL expression and number of osteoclasts began to decrease.Experiment in vitroFurthermore, to clarify how RANKL and OPG produced at the compression site regulate osteoclastogenesis, we cultured M-CSF-dependent bone marrow macrophages (M-BMM) as preosteoclasts, in 15% FBS α-MEM supplemented with M-CSF, RANKL and/or OPG. After culture on coverslips and bovine cortical bone slices for 1, 3, 5, and 7 days, morphologic features, TRAP staining and bone absorptive lacunae were observed to evaluate osteoclast cells. The numbers of TRAP-positive cells were scored.The results show that when M-BMMs were further cultured with RANKL and M-CSF, TRAP-positive mononucleared and multinucleated cells were formed within 3 days,typical resorption lacunae were formed on bone slices. When M-BMMs were cultured with increasing concentrations of RANKL in the presence of M-CSF (50 ng/ml), TRAP-positive cells increased in a dose-dependent manner. In the absence of M-CSF, most of the M-BMMs died within 3 days,No TRAP-positive cells were formed. Adding OPG at a concentration of 50ng/ml or 25ng/ml to culture system, No TRAP-positive cells appeared, even in the presence of RANKL and M-CSF, at a concentration of 10ng/ml, only a few TRAP-positive cells wer...
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