| Orthodontic tooth movement is closely related to the remodeling of alveolar bone. In physiological conditions, bone remodeling process is controlled by a complex network of endocrine hormones and local cytokines. Generally, the regeneration of alveolar bone processes are as follows:growth factor-inducing bone marrow mesenchymal stem cells or periodontal ligament stem cells differentiate into osteoblast, which then synthesizes the mineralized bone matrix, and ultimately induces the formation of bone in a complex biological route. In the process of alveolar bone repair and regeneration, the proliferation and differentiation of osteoblast are influenced by various factors in vivo and in vitro. In order to satisfy the application requirement of clinical medicine and look for safety and steady, high-efficiency, low toxic and low cost new medicament, the regulation of the alveolar bone repair is increasingly becoming a hot spot in oral medicine research field. Since osteoblast is directly associated with the synthesis of mineralized bone matrix, the proliferation and activation of osteoblast using growth factors and other agents will be an attractive issue in the alveolar bone repair and regeneration.ODN (oligodeoxynucleotides, ODN) refers to the multi-nucleotide chains containing 50 or less nucleotide monomers. ODN is absorbed into cells through the form of receptor-mediated, and then perform a function in cells. Thio-modified phosphate could enhance the capability of ingesting ODN and resisting the digestion of nuclease, which is good for keeping stable in vivo and increasing the intracellular uptake rate. Previous studies have shown that ODN can use the classical mode of action to regulate the balance of bone formation and resorption, which demonstrated that ODN could play an important role in the process of osteoblast differentiation. In the previous study of our group,12 different sequences ODN were screened in and 2 ODNs could affect the bone marrow mesenchymal stem cell proliferation and osteogenic differentiation. Early preliminary results suggested that specific ODN sequences could promote bone marrow mesenchymal stem cell to differentiate into osteoblast, which provided a theoretical basis and experimental basis for further studies of ODNs in the regeneration of alveolar bone tissue.In this study, based on preliminary studies, we presented a scientific hypothesis that a specific sequence of ODN plays a regulatory role in the proliferation, maturation and activation of osteoblasts. The research was performed as follows:obtaining rat osteoblast cells and human osteoblast cells by cell culture in vitro; screening the ODNs with the ability to promote osteoblastic proliferation and activation by by methyl thiazolyl tetrazolium (MTT) and alkaline phosphatase (ALP) assays; investigating the effect of ODNs on the expression level of Sp7, runx-2, collagen-1 and RANKL at the transcription and translation levels by real time PCR and Western blotting analysis; observing the changes of histological structure of alveolar bone, bone mass and osteoclast count; elucidating the effect and mechanism of the sequence-specific oligonucleotides to the proliferation and activation of the osteoblast cells, which supplied the theoretical foundation for the research of regulation of ODN in the process of remodeling of periodontal tissue of the orthodontic tooth movement. In this study, the 11 ODNs were designed and synthesized by College of Molecular Biology in Jilin University. According to the characteristics of immune function, these ODNs were divided into three types:(1) immunostimulant, including BW001, FC001, BW006, FC002, YW001, FC004 and YW002; (2) immunosuppressant, including SAT05f, MT01 and FC003; (3) immunologic inertia, such as MS 19.The content of this research is divided into five parts:Experiment 1:The influence of various ODNs on rats osteoblast proliferation activity.Wistar rats were used as research subjects. Rat osteoblasts were cultured and identified by alizarin red staining. After determining the best concentration,11 different sequences of ODNs were added to the cells in the experimental groups, meanwhile PBS was added to the control groups. The ODNs, which had the effect on proliferation of osteoblastic cells, were selected by MTT colorimetry.Experiment2:The influence of various ODNs on rats osteoblast muturation activity.The third generation of the rat osteoblast cells were used, and cultivated in 96-well plates with 5 x 103 cells per well. The 11 ODNs mentioned in Experiment 1 were co-cultivated respectively with the rat osteoblast cells in 96-well plates for 72 hours, with equal volume of PBS solution as control. After that, the expression level of ALP of each group was detected by ALP assay kit. And then the best concentration of the effect on rats osteoblast muturation activity was determined.Experiment 3:The influence of ODN on bone remodeling of rat tooth movement model.The influence of ODN on periodontal tissue of tooth movement model of wistar rats was discussed through studies in vivo. The changes of alveolar bone in rats were evaluated morphologically. Then the distance of orthodontic tooth movement was measured. Meanwhile, the expression of osteoblasts/osteoclast-related factors (RANKL, OPG, Runx-2, Osterix and Collagen-â… ) under the stimulation of ODN was detected by immunohistochemical staining. Furthermore, the mechanism for the effect of ODN on periodontal remodeling of experimental rat tooth movement model was explored.Experiment 4:The influence of ODN on proliferation and activation of human osteoblast-like MG 63 cells.The human osteoblast-like cell line MG 63 cells were used as research objects, and the three generations of MG 63 cells were taken. The 11 ODNs were co-cultivated respectively with the cells of experimental groups. Meanwhile, equal volume of PBS solution was added to the control group. The ODNs, which had the effect on proliferation and activation of human osteoblast-like MG 63 cells, were selected by MTT colorimetry and ALP assay kit.Experiment 5:The study of mechanism for promotion of ODN on proliferation and activation of bone cells.The changes of cell cycle of MG 63 cells, which was induced by ODN MT01, were detected by flow cytometry. Then the difference of transcription and protein expression level of related factors (Sp7, Runx-2, Collagen-â… , RANKL and OPG) of MG 63 cells, which were induced by ODN in vitro, was tested quantitatively by Real-time PCR and Western blot respectively at the transcription and translation levels. At last, the mechanism for promotion of ODN on proliferation and activation of bone cells was discussed.Through the above 5 experiments, the results were as follows:Experiment 1After 24 h, NO.2,3,6,9 and 10 ODNs could promote the proliferation of osteoblasts (P <0.05). After 48 h, NO.1,6,9 and 10 ODNs could promote the proliferation of osteoblasts (P <0.05). After 72 h, NO.1,6,9 and 10 ODNs could promote the proliferation of osteoblasts (P O.05). After 96 h, NO.1,2,8 and 9 ODNs could promote the proliferation of osteoblasts (P <0.05), of which the effect of NO.9 ODN (ODN MT01) in the whole process had a significant difference (P<0.01).Experiment 2Compared to the PBS vehicle control group expression, initially selected five ODN (2, 3,8,9 and 10) could increase the expression of osteoblast ALP at different degrees (P<0.05). These ODNs were co-cultivated with rat osteoblasts for 72h as the concentration of 4.0,2.0, 1.0 and 0.5 mg.L-1 respectively, and then the ALP activity was detected. The results showed that the experimental groups and control group had no significant difference (P> 0.05) with the concentration of 4.0 and 0.5 mg.L-1, and NO.9,10 ODNs could promote the expression levels of osteoblast ALP significantly (P<0.05) with the concentration of 2.0 and 1.0 mg.L-1.Experiment 3The moving distance of first molar teeth of experimental groups was less than that of the control group (P<0.05); specific sequences ODN could influence the balance of bone formation and resorption during the remodeling of rat alveolar bone, and inhibit the movement of tooth. The expression of osteoblasts/osteoclast-related factors (RANKL, OPG, Runx-2, Osterix and Collagen-â… ) under the stimulation of ODN was detected by immunohistochemical staining. The results showed that the expression of OPG, Runx-2, Osterix and Collagen-â… of experimental groups was increased compared with control group, while the expression of RANKL was weakened.Experiment 4After 24 h, NO.2,3,5,9 ODNs could promote the proliferation of MG 63 cell (P <0.05); after 48h, NO.3,5,6,9,10 ODNs could promote the proliferation of MG 63 cell (P <0.05); after 72 h, NO.3,5,9,10 ODNs could promote the proliferation of MG 63 cell (P <0.05); after 96h, NO.6,8,9 ODNs could promote the proliferation of MG 63 cell (P<0.05). NO.3,5,6,9,10 ODNs were selected to co-cultivated with MG 63 cells, and alkaline phosphatase activity was detected. The results displayed that NO.9 ODN could increase the expression of ALP of MG 63 cells significantly (P<0.05)Experiment 5The results of flow cytometry assay showed that more cells at S phase were observed in group treated with ODN MT01 than control group. It demonstrated that ODN MT01 might affect the cell cycle of MG 63 cells, and promote the proliferation of MG 63 cells by increasing the proportion of cells at DNA synthesis phase. The mRNA expression levels of osteoblasts-related genes of MG 63 cells treated with ODN was determined by real time quantitative PCR. The results showed that the mRNA expressions of Runx-2, Sp7 and collagen-â… of experimental groups were higher than control group at different degrees. The protein expression level of osteoblasts-related factors of MG 63 cells treated with ODN was determined by western blot. The results displayed that compared with control group, the expression of Runx-2, Sp7, collagen-â… and OPG were increased at different degrees, while the expression of RANKL had a marked decrease. This suggested that a specific sequence ODN might increase OPG/RANKL ratio by upregulating the expression of OPG and downregulating the expression of RANKL at the same time, and promote the maturation and activation of osteoblasts through the OPG-RANK-RANKL pathway.According to the results above, we can draw the following conclusions:a specific sequence of the ODN (ODN MT01) could promote the proliferation, maturation and activation of osteoblast in vitro, and be able to regulate the remodeling of alveolar bone of rat tooth movement model in vivo. This effect could be achieved through the regulation of OPG-RANK-RANKL pathway, which increased the expression of OPG and inhibited the expression of RANKL. |
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