Effects Of Dlx-5 And Msx-1 In The Pathogenic Mechanism Of Bisphosphonate Related Osteonecrosis Of The Jaw

Objective:1.Due to the different embryological origins of craniofacial skeletons and ilia,different gene expression patterns and bone metabolism have been found between the jaw bones and ilia.Distal-less homeobox genes and Msx homeogenes,particularly Dlx-5 and Msx-1,play major roles in bone metabolism.The study was conducted in rats with long-tern administration of zoledronic acid.The purpose of this study was to investigate the effects of zoledronate on craniofacial skeletons and ilia by detecting the changes in Dlx-5 and Msx-1 at both the protein and mRNA levels.2.We tend to develop a reliable animal model of Bisphosphonate-related osteonecrosis of the jaw(BRONJ)in rats.We tend to observe the clinical manifestation,imaging changes and histopathological features of BRONJ.The purpose was to investigate the effects of Dlx-5 and Msx-1 in the pathogenic mechanism of BRONJ.Methods:1.A total of 36 female Sprague-Dawley rats were randomly divided into three groups.The zoledronate group(ZOL,n=12),injected intraperitoneally with zoledronic acid for 12 weeks(0.2mg/kg,three times a week);the saline solution group(S,n=12),injected with saline solution for 12 weeks;the control group(C,n=12)without injection.Using immunohistochemistry,western blot and real time-polymerase chain reaction(RT-PCR),the levels of Dlx-5 and Msx-1 in craniofacial skeletons(including maxillae,mandibles and parietal bones)and ilium samples were examined.2.24 female SD rats were divided into two groups.The zoledronate group,injected intraperitoneally with zoledronic acid for 12 weeks(0.2 mg/kg,three times a week),and the control group,injected with saline solution for 12 weeks.The left first mandibular molars were extracted after 12 weeks.We observed the healing of tooth extraction.All of the animals were sacrificed eight weeks after teeth extraction.The left jaws of the rats were examined by X-ray and Micro-CT to explore the imaging changes.Hematoxylin-eosin(HE)staining and Masson staining were used to investigate the histopathological characteristics of mandibular soft and hard tissue.The BRONJ was diagnosed by gross observation,radiological examination and histopathlolgical examination.Through Western blot and RT-PCR,the expression level of Dlx-5 and Msx-1 were detected in the mandible of BRONJ samples and normal samples.Results:1.The expression of Dlx-5 at the protein and mRNA levels in the maxilla and mandible were increased in the ZOL group compared with the S and C groups(P<0.05).The expression levels of Msx-1 in the maxilla and mandible were decreased in the ZOL group(P<0.01).The expression levels of Dlx-5 and Msx-1 in the ilium were decreased in the ZOL group(P<0.05).The levels of Dlx-5 and Msx-1 expression in the parietal bone were similar among the three groups.The differences did not attain statistical significance(P>0.05).2.Eight weeks after teeth extraction,the clinical examination revealed incomplete mucosal healing and exposure of sequestrum in the ZOL-injected rats.Radiological examination and histopathlolgical analysis showed the presence of BRONJ.The results of Masson staining showed that collagen fibrils around the extraction were slender in the ZOL group.The results of Western blot and RT-PCR showed that the expression levels of Dlx-5 were increased(P<0.01)and the expression levels of Msx-1 were decreased in the zoledronate group(P<0.01).Conclusions:1.Site-specific differences in the influence of zoledronate on the craniofacial skeletons and ilia could be explained by differently altered tendencies in Msx-1 and Dlx-5 expression.Compared with the parietal bones and ilia,the jaw bones were the most susceptible to the application of zoledronate.2.Zoledronate intraperitoneal injection and teeth extraction can induce a stable BRONJ animal model in rats.Changes in the expression of Dlx-5 and Msx-1 can promote bone formation,further damage the balance of bone metabolism,leading local bone sclerosis,which play roles in the pathogenic mechanism of BRONJ.