| Objective : The changes of periodontal tissue and bone density in the pressure side of the experimental teeth were studied by establishing animal models of periodontal disease and orthodontic tooth movement in rats.The expression of RANKL,OPG,TNF-?,COX-2 and the number of osteoclasts in periodontal tissues were observed to investigate the effect of lactoferrin on bone resorption and removable bone remodeling in orthodontic patients with periodontal disease.Methods:1.Twelve healthy male SD rats aged 8-10 weeks were randomly selected as an experimental group.Six rats were anesthetized with 10% chloral hydrate and injected with 50?l of saline containing 10g/L lipopolysaccharides(E-LPS)into the mesiobuccal root and buccal gingiva of the left maxillary first molar,once every 48 hours,four times.The remaining six rats served as a blank control group.The inflammation of gingiva and periodontal tissue of the first maxillary molar in the experimental group and the control group were observed,and all rats were sacrificed eight days later.The left maxillary tooth-periodontal tissue samples were taken and fixed with 4% polyformaldehyde.The alveolarbone resorption was observed by Micro-CT.The effect of preparation of periodontitis model was checked by histopathological section,hematoxylin-eosin(HE)staining and osteoclasts counting.2.Fifty-two healthy male SD rats aged 8-10 weeks were randomly divided into three groups: group A: blank control group(4 rats);group B: orthodontic tooth movement model group(24 rats);group C: orthodontic tooth movement model group(24 rats).Group B was further divided into group B1: LPS + orthodontic force + bovine lactoferrin(12 rats);group B2: LPS + orthodontic force + normal saline(12 rats);group C was further divided into group C1: orthodontic force + bovine lactoferrin(12 rats);C2 group: orthodontic force + normal saline(12 rats).According to the experimental periodontitis model,rats in group B were injected with 50?l saline containing 10g/L LPS at the same location,and once every 48 hours for four times.Rats in group C were injected with saline of the same volume at the same time.NiTi spring device was installed in each group after 4times injection,and the force was 20 g.From the second day after the tooth movement model was established,rats in groups B2 and C2 were given 0.9%sodium chloride solution(1ml/100g)daily.At the same time,according to the dosage of 1ml/100 g and the daily dose of bovine lactoferrin received by each rat,85mg/Kg,the bovine lactoferrin was dissolved in the corresponding volume of0.9% sodium chloride solution and the rats in group B1 and C1 were intragastrically administered.Six rats in B1,B2,C1 and C2 groups were sacrificed on the 5th and 14 th days after gastric perfusion,totaling 48 rats in group A.After a week of adaptive feeding,the rats in group A were sacrificed directly.The near-middle gingival crevicular fluid of the first maxillary molar was taken before the rats in each group were sacrificed.The lactoferrin content in the gingival crevicular fluid was measured quantitatively by ELISA.Bonemineral density(BMD)and trabecular bone were measured by Micro-CT.HE staining was used to observe the periodontal tissue changes and alveolar bone resorption on the pressure side of the experimental teeth.TRAP staining was used to observe the distribution and number of osteoclasts on the pressure side of the experimental teeth.Immunohistochemical staining was used to observe the expression of RANKL,OPG,TNF-? and COX-2 in the periodontal tissues of the pressure side of the experimental teeth.Image-pro-plus 6.0 image analysis system was used for semi-quantitative analysis.SPSS22.0 statistical software was used to analyze and process the experimental results.Results:1.The rat periodontitis model was successfully established.The alveolar bone resorption model of periodontitis in rats could be effectively prepared by local injection of E.coli strain LPS.2.The orthodontic tooth movement model induced by mechanical force in rats was successfully established.3.The expression of lactoferrin was detected in the gingival crevicular fluid of the proximal middle teeth of each group.The content of lactoferrin in gingival sulcus fluid of the lactoferrin group was significantly higher than that of the blank group(P <0.05),and the difference was not statistically significant(P >0.05).4.Micro-CT results showed that Tb.N(Trabecular Number,Tb.N)and Tb.Th(Trabecular Thickness,Tb.Th)in group B1 were higher than those in group B2 on the 5th and 14 th days,BS/BV(Bone Surface/Bone Volume,BS/BV)and Tb.Sp(Trabecular Separation/Spacing,Tb.Sp)were lower than those in group B2(P<0.05);there was no significant difference in Tb.N,Tb.Th,BS/BV and Tb.Sp between group C1 and group C2(P >0.05).5.HE staining showed that no osteoclasts and alveolar bone resorption were observed in group A;alveolar bone resorption occurred in pressure side in group B and group C;alveolar bone resorption was more serious in group B2 than in group B1,which was characterized by disordered arrangement of periodontal ligament fibers,discontinuity of alveolar bone surface,formation of osteoclasts and bone resorption lacunae,formation of inflammatory cells such as neutrophils and lymphocytes in connective tissue.There was no significant difference in infiltration between C1 group and C2 group.6.TRAP staining results showed that TRAP staining was negative in group A,and no obvious mature osteoclasts were observed.On day 5 and day 14,affected by lactoferrin,the number of mature osteoclasts in group B2 was significantly higher than that in group B1,and the difference was statistically significant(P<0.05).Mature osteoclasts were observed on the pressure side of C1 and C2 groups,and the difference was not statistically significant(P >0.05).7.Immunohistochemical results showed that RANKL and TNF-?expression levels in group B1 were significantly lower than those in group B2 at day 5 and day 14(P<0.05).The expression of OPG in group B1 was higher than that in group B2(P<0.05),and reached the peak on the 5th day.The expression levels of RANKL,TNF-?,COX-2 and OPG showed no significant difference between C1 and C2 groups on day 5 and day 14(P >0.05).Conclusion : Under orthodontic force,bovine lactoferrin may inhibit periodontal disease-related bone resorption by affecting LPS/TLR4/TNF-?pathway,but it does not affect the bone remodeling induced by mechanical force through COX-2/PGE2 pathway. |
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