| Objective Periodontitis is a common disease with the incidence of more than 80%. It will cause periodontium destruction, attachment loss, development of periodontal pocket, and loose tooth, which is the main reason for adult's tooth-loss. Peri-implantitis, quite similar to periodontitis in etiology,progression and therapy, has also been a leading cause of implant failure and a big challenge in the implant treatment. Nowadays, routine clinical treatment for periodontitis includes basic periodontal treatment and supplementary systematical or local administration of antibiotics. When a lot of osteoclasts are activated by bone-resorption stimulators mediators and alveolar bone resorption quickly develops, for example, rapid-progress periodontitis, it's hard to reverse the general trend of rapid alveolar absorption only with the physical and chemical therapy; In addition, at present there is no other ideal therapy available to promote the regeneration of periodontal or peri-implant tissue which have been damaged or absorbed. Therefore at the initial stage of the inflammation, we must give priority to inhibiting osteoclatogenesis, function, and the absorption of alveolar bone, which is crucial in the process of curing periodontitis or peri-implantitis and maintaining function of the tooth or implant. The newly-found OPG/RANK/RANKL system is regarded as the key regulative factor in the process of bone remodeling, and it contributes absolutely necessarily to the course of osteoclastogenesis and osteoclast activity. In recent years, numerous researches have been reported on making the best of OPG to prevent and cure bone resorption disease caused by osteoclast hyperfucntion. And it has been proved that OPG could take good effect in restraining bone resorption disease caused by osteoporosis, metastatic carcinoma of bone and otherwise. However, it's rare to see such researches about the application of OPG to the treatment of periodontitis or peri-implantitis, and until now there is no single report about the study of OPG gene therapy on alveolar bone absorption caused by experimental periodontitis. Through construction of recombinant pIasmid pcDNA3.1-hOPG and its transformation in vitro and in vivo, this study is undertaken to determine the effect of OPG gene therapy on alveolar bone resorption caused by experimental periodontitis in rats, and supply an experimental foundation for further clinical application of OPG gene therapy in the treatment of periodontitis and peri-implantitis.Methods (1) Based on the human OPG cDNA sequence, two pairs of specific primers were designed and constructed. Polymerase chain reaction (PCR) was employed to clone hOPG cDNA from plasmid MGC: 29565, then the product was ligated into the eukaryotic expression vector pcDNA3.1(-). The recombinant plasmid was first propagated in Escherichia coli DH5α, then extracted and purified. The product was confirmed with PCR and DNA sequence analysis. (2)Complexes were prepared using different recombinant plasmid (μg) to Lipofectamine 2000(μl) ratios of 1:2, 1:2.5, 1:3, then under the instruction of plasmid transfection, they were used to transfect C2Cl2 (mouse myoblast cell line) for optimization. The mRNA and protein expression of hOPG were detected with RT-PCR,Western blotting and immunohistochemistry, respectively. (3)Homogenized bone marrow cell preps isolated form 10～12-day-old rat pups were plated with cortical bone wafers (n=3 per group), and incubated with culture supernatants from C2Cl2 transfected 3 days later, in the presence of 10-7mol/l cholecalciferol and 10-8mol/l dexamethasone. After 10 days, SEM was taken to compare the difference of the area of pits on the wafer surface between experimental group and contrast group. (4)Thirty female healthy SD rats were randomly divided into three groups, groupâ… (n=10): 100μg physiological saline injected: groupâ…¡(n=10): 100μg pcDNA3.1(-) plasmid injected; groupâ…¢(n=10): 100μg pcDNA3.1-hOPG plasmid injected. One of the upper second molars of each animal was randomly tied by silk ligatures, provided with drinking water containing 10%glucose, and inoculated with mixed periodontopathic bacteria to create periodontitis, and the contralateral tooth was left unligated. Four weeks after ligature, all the animals were sacrificed and the specimens routinely processed. The distance from the alveolar bone crest to the cemento-enamel junction was measured for each mesial and distal surface of the upper second molars of each rat by radiograph and histologic measurement. Immunohistochemistry were used to observe the expression of OPG/RANKL in periodontal tissue and the number of osteoclasts was counted through TRAP staining. One-way analysis of variance (ANOVA) was used to intergroup analysis (α=0.05), multiple comparison was carried out by least significant difference (LSD) test, where ANOVA test showed significant differences (α=0.05). Paired t-test was used to intragroup comparison.Results (1) The recombinant pcDNA3.1-hOPG vector contained correct nucleotide sequence for human OPG cDNA fragment, which is identical with OPG sequence in GenBank. (2)Western blot result showed that hOPG protein was enhanced with plasmid to Lipofectamine 2000 ratio of 1:2, 1:2.5, however, the OPG expression decreased with the ratio of 1:3.(3) RT-PCR results revealed the mRNA in transfected C2Cl2 was increased by 5.79×103 times with the transfection ratio of 1:2.(4) Wafers SEM results showed that the average total pitted area of experimental group is much smaller than that of control group.(5) In experimental periodontitis models, compared with groupâ… and groupâ…¡, OPG IOD and OPG/RANKL in groupâ…¢were enhanced (P＜0.05),alveolar bone resorption and active osteoclasts decreased respectively(P＜0.05). Conclusions (1) A eukaryotic expression vector containing human OPG in pcDNA3.1(-) has been successfully constructed, which produced effective OPG ex and in vivo. (2) OPG overexpression mediated by recombinant plasmid ex and in vivo suppressed osteoclastogenesis and osteoclast activity, and helped to prevent alveolar bone height reduction caused by experimental periodontitis.
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