| Periodontitis,which is characterized by periodontal pocket formation,attachment loss and alveolar bone resorption is a chronic,bacterial infectious and inflammatory disease.Bacteria and plaque are the major etiological factors,and the invasion of periodontal periodontal pathogens leads to the expressions of many proinflammatory mediators and the activation of inflammatory cascades.Those cytokines contribute to the periodontal inflammatory infiltration as well as the indirect damage to our system.A number of studies showed that inflammatory cytokines,such as TNF-a,interleukin-1β(IL-1β)and IL-6 can be detected in periodontal tissue,gingival crevicular fluid and even in peripheral blood.These inflammatory factors may affect the development of some systemic diseases,thus periodontitis was considered as a systematic chronic low-grade infection burden.Trauma,tumor excision and congenital deformity all contribute to jaw defect,thus,the repair of over critical size of bone defect has been a major challenge for oral and maxillofacial surgeons.Bone defect healing is a complex of osteogenetic and osteoclastic integration,which can be affected by age,nutrition and inflammation of our body.As an inflammatory disease,periodontitis can also result in high level of several inflammatory cytokines in periodontal pockets and peripheral blood,and among them TNF-a is the most important.However,whether there is an adverse influence of periodontitis on bone defect healing has not been reported yet.Therefore,in our study,we established a periodontitis model to investigate the influence of periodontitis on the mandibular bone regeneration in rats.Meanwhile,non-surgical treatment was given to animals with induced periodontitis and the effect of periodontal treatment on mandibular defect healing under periodontitis environment was evaluated.The purpose of our study was to provide a necessary theoretical support for the clinical mandibular bone defect treatment,and open up a new idea for mandibular defect healing and functional reconstruction of bone defect under a periodontitis mircroenvironment.MATERIALS AND METHODS1.Establishment of an experimental periodontitis model in rats.Experimental periodontitis model was established by ligaturing with orthodontic wire and silk suture plus local administration of 20 μl of lipopolysaccharide(LPS),and five rats were respectively sacrificed with an overdose anesthetic at 1,2,3 and 4 weeks.Maxillary specimens with teeth and periodontal tissues were collected.HE staining was conducted for morphological analysis.The distance between the cemento-enamel junction(CEJ)and the alveolar bone crest(ABC)of each root surface was estimated by measured separately using software Image J to valuate the bone resorption.The rat TNF-alpha ELISA kit was also used to detect the the serum concentration of TNF-a.2.The effect of experimental periodontitis on mandibular defect healing in rats.Another 108 male Wister rats were randomly assigned into 3 groups:1)control group,in which mandibular defects were established in rats with healthy periodontal tissue;2)periodontitis group,in which mandibular defects were established in rats with induced periodontitis;3)periodontitis plus rhTNFR:Fc group,in which the rats with induced periodontitis were treated with rhTNFR:Fc and then mandibular defects were established.Bone defects,4 ×2 × 1 mm in rectangle,were created over the mesial root of the left mandibular first molar by using a dental drill under continuous irrigation with saline.For rats in the periodontitis plus rhTNFR:Fc group,rhTNFR:Fc at a dose of 2.5 mg/kg was subcutaneously injected at neck every three days until the animals were sacrificed.Sterile saline were injected for substitution in other groups.At 1,2,4 and 8 weeks after the surgery,rats were sacrificed and the mandibles were isolated for further analysis.The expression of serum TNF-a was measured by rat TNF-alpha ELISA kit.Osteogenic differentiation markers such as ALP、Runx2、BMP2、OCN and osteocalstogenic differentiation marker NFATc1 in the mandibular defect were analysed by quantitative real-time polymerase chain reaction(PCR).Osteogenic differentiation markers such as Runx2 and OCN were examined by Western blot and immunohistochemistry in protein level,and new bone formation was histologically evaluated via HE staining.Tartrate-resistant acid phosphatase(TRAP)staining was also performed to determine the number of osteoclasts.3.The effect of non-surgical periodontal treatment on mandibular defect healing in periodontitis rats.Another 72 male Wister rats were collected to establish periodontitis model as described in part 1,then they were randomly assigned into 2 groups:periodontitis group and non-surgical periodontal treatment group.Mandibular defects were established in all experimental rats with induced periodontitis as described in part 2.For the periodontitis group,non intervention was give;while,for the periodontal treatment group,rats were treated with the removal of the orthodontic wire,periodontal scaling and irrigation.At 1,2,4 and 8 weeks after the surgery,rats were sacrificed and the mandibles were isolated for further analysis.The expression of serum TNF-α was measured by rat TNF-alpha ELISA kit.The gene expression of osteogenic differentiation markers ALP、Runx2、OCN and osteocalstogenic differentiation marker NFATc1 in healing tissue of mandibular defect was examined by Real-time PCR.Protein expressions of osteogenic differentiation markers such as Runx2 and OCN were examined by western blot and immunohistochemistry,and new bone formation was histologically evaluated.TRAP staining was also performed to determine the number of osteoclasts.Results1.Establishment of an experimental periodontitis model in rats.There were obvious epithelial cells erosion and alveolar bone loss presented in the first and second molars 2 weeks after the ligation plus LPS injection when compared to 1 week group.Direct measure of distance between CEJ and ABC showed that it was 1.159±0.085 mm in the first molar and 0.671±0.056 mm in the second molar 2 weeks after inducement,which was significantly higher than that at 1 week.Blood samples analysis indicated that there was a significant increase of TNF-a level in the serum after ligaturing at week 2(70.78±5.61 pg/ml)when compared with 1 week group.But there was no significant difference among the groups of 2,3 and 4 weeks for all detections.2.The effect of experimental periodontitis on mandibular defect healing in rats.An significantly increased TNF-a expression presented at 1,2,4 and 8 weeks in the periodontitis group compared with the control group,while there was a significantly decreased expression of TNF-a in the periodontitis plus rhTNFR:Fc group(at 1,2 and 4 weeks)when compared with the periodontitis group.For the HE staining analysis,the newly formed bone area was significantly lower in the periodontitis group(2,4 weeks);no significant difference of the newly formed bone area between the control group and the periodontits plus rhTNFR:Fc group was detected.Real-time PCR analysis showed that there were significantly lower expressions of osteogenesis-related such as ALP,BMP2(at 1,2 and 4 w),Runx2 and OCN(at 1,2,4 and 8 w)and significantly higher expression of NFATcl(at 1,2 and 4 w)in the periodontitis group.Meanwhile,there was no significant difference between the control group and the periodontitis plus rhTNFR:Fc group.Western blot analysis showed that the protein levels of Runx2 at 1,2 and 4 weeks and OCN at 2 and 4 weeks were significantly lower in the periodontitis group.Immunohistochemical staining also revealed that Runx2 positive cell with brown-stained nuclei at 1,2 and 4 weeks and OCN protein expression at 2 and 4 weeks were significantly decreased in the periodontitis group than in the control group.TRAP staining uncovered that there were more TRAP+ osteoclasts in the bone defect area at week 1 and 2 in the periodontitis group compared with the control group,whereas,there was no significant difference of TRAP+ osteoclast number between the control group and the periodontitis plus rhTNFR:Fc group.3.The effect of non-surgical periodontal treatment on mandibular defect healing in periodontitis ratsCompared with the periodontitis group,there was a significantly increased TNF-a expression presented at 1 week,while a significantly decreased expression was observed at 2,4 and 8 weeks in the treatment group.Real-time PCR analysis showed that there were significantly higher expressions of osteogenesis-related mRNA such as ALP、Runx2 and OCN and significantly lower expression of NFATc1 in the treatment group.Western blot analysis certificated that for Runx2 expression,there was lower expression at 1 week,higher expression at 2 and 4 weeks in the treatment group.When it comes to OCN expression,there was higher expression at 2 and 4 weeks in the treatment group.There was no significant difference between the two groups at 8 weeks.HE staining showed that the formation of new bone in each group increased with time.For the treatment group,there was more new bone formation at 2 and 4 weeks;and there were no significant difference among the two groups at 8 weeks.Immunohistochemical staining also revealed that for Runx2 expression,there was less Runx2+ cells at 1 week,more Runx2+ cells at 2 and 4 weeks in the treatment group;When it comes to OCN expression,there was higher expression at 2 and 4 weeks in the treatment group;there was no significant difference among the four groups at 8 weeks.TRAP staining uncovered that there were less TRAP+ osteoclasts in the treatment group at 2 weeks when compared with the periodontitis group.Whereas,there was no significant difference of TRAP+ osteoclast number between the two groups at 4 weeks.Conclusions1.Periodontitis was successfully established via orthodontic ligature ligaturing plus LPS injection around maxillary first molar for two weeks in rats.2.Periodontitis,characterized by high expression of serum inflammatory mediators,may restrain the mandibular bone healing via disturbing osteogenic and osteoclastic balance.3.Tumor necrosis factor-a(TNF-a)may act as a leverage in the procedure of mandibular defect healing under periodontal environment.4.Non-surgical periodontal treatment may delay the mandibular defect healing in short time,but as the periodontal condition improving,mandibular defect healing was promoted under this treatment. |
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