The Study Of The Correlation Between Phosphorus Metabolic Abnormalities And Alveolar Bone Loss Under The Conditions Of Simulated Weightlessness

Background The pathogenesis and preventive treatments of bone loss in weightlessness have been focusing issues for scholars all the time.However,these researches were mainly concentrated on long bones at present.To date,the morphological changes of alveolar bone in weightlessness have been rarely reported.Due to the supportive and protective functions to teeth,the abnormal process of metabolism,absorption and defect of alveolar bone led to many oral diseases.Unlike long bones,alveolar bone is non-weight bearing bone.The reaction of alveolar bone to a mechanical stimulus may be less affected than bone,but more affected by hormones and the calcium,phosphate concentration.Our former studies showed that there was bone loss after mouse tail-suspended for 4wk,and blood phosphorus levels in these mice were elevated.In recent years,experimental studies had identified that inorganic phosphorus,which was fundamental to hydroxyapatite crystallization,was an integral signalling molecule in the bone formation and mineralization.These studies have aroused more and more attention.So,is there any bone loss in alveolar bone under simulated weightlessness environment? Is it related to abnormal phosphorus metabolism induced by weightlessness? These are the questions we focused on our project.With the building of various osteocytic cell lines and mice models of osteocytic specific transgenosis,a variety of biological functions of osteocytes have been studied deeply,including the control of bone remodeling.Osteocytes are currently considered as a major source of molecules that regulate the activity of osteoclasts and osteoblasts,such as Sclerostin(SOST)and RANKL(Receptor Activator of NF-?B Ligand).Previous studies showed that disorder and dysfunction of RANK(Receptor Activator of NF-?B),RANKL and OPG(osteoprotegerin)led to various metabolic bone diseases.RANKL/OPG ratio was closely related to alveolar bone resorption.To better understand SOST-OPG-RANKL-RANK system may provide a new approach for the therapy of bone resorptive diseases.So,whether or not SOST-OPG-RANKL-RANK system involved in the process,if there is a correlation between phosphorus metabolic abnormalities and alveolar bone loss under the conditions of simulated weightlessness,can low phosphate diet prevent alveolar bone loss under simulated weightlessness? These were also the questions we focused on our project.Then,if the tail-suspended mouse were fed with low phosphate diet,alveolar bone loss,can it play a role in prevention of alveolar bone loss under simulated weightlessness? All these problems were the main purpose of the article.The project were divided into three parts.The first part confirmed the correlation between phosphorus metabolic abnormalities and alveolar bone loss under the conditions of simulated weightlessness in vivo.The second part confirmed the correlation between phosphorus metabolic abnormalities and alveolar bone loss under the conditions of simulated weightlessness in vitro.Meanwhile,this part discussed the role of SOST-OPG-RANKL-RANK system in the process of bone loss.The last part discussed the protective effect of low phosphate diet against the alveolar bone loss under simulated weightlessness.Part 1 The study of the correlation between phosphorus metabolic abnormalities and alveolar bone loss under the conditions of simulated weightlessness in vivoObjective To investigate the bone remodeling of alveolar bone under conditions of simulated weightlessness,and the correlation between phosphorus metabolic abnormalities and alveolar bone loss.Methods 1.Twelve rats were matched into two groups randomly,i.e.the control(CON)group and the tail-suspension(SUS)group.After 4-wk experiments,all rats were sacrificed and the alveolar bones in the mandibular molar region were fixed and collected as specimens.Micro-CT analysis was performed to calculate vertical and furcated bone loss in the mandibular molar region.The osteocytes and bone collagen fibers of two groups were observed by SEM.To determine the histomorphology of new bone formation in the alveolar bone,Ponceau S staining was performed.Meanwhile,TRAP staining and ALP staining were used for the alveolar bone slices to observe the activity of osteoclast and osteoblast respectively.Furthermore,apoptotic positive cells were found by TUNEL asssy.SOST m RNA expression was detected by the immunohistochemistry staining.Serum phosphorus level was measured by automatic biochemistry analyzer.2.The high-phosphorus animal models were set up by giving mouse high phosphate diet.Twelve mouse were matched into two groups randomly,i.e.the control(CON)group and the high phosphate(HPi)group.Micro-CT,SEM were also performed as in the high phosphate mice.Serum phosphorus level was measured by automatic biochemistry analyzer.Results 1.Experimental results of tail-suspension: Micro-CT showed significant bone loss in the interradicular furcation region of alveolar bone in the mandibular first molar in the SUS group as compared with control group.Images of SEM displayed a sharp change of osteocytes in alveolar bones from spindle to round in SUS group rats;collagen fibers cannot be gathered into orderly,regular shaped collagen fiber bundles.The histological analysis also revealed that the areas of new bones were significantly decreased in the SUS group compared to that in the control group.Analysis of the TRAP-stained sections showed that the number of TRAP positive cells were increased in the SUS group compared to that in the control group.ALP-stained sections showed that the number of ALP positive cells were decreased in the SUS group compared to that in the control group.TUNEL positive cells increased in SUS group.The immunohistochemistry staining showed SOST-positive osteocytes significantly increased in the SUS group.Elevated serum phosphorus level was found in SUS group as compared with control group.2.Experimental results of high-phosphate: Serum phosphorus levels were elevated in the HPi group as compared with control group,indicating the HPi modeling was successful.An analysis of 3D bone microarchitecture by micro-CT showed that the alveolar bone in HPi group had bone loss compared to the CON group.Images of SEM displayed a sharp change of osteocytes in alveolar bones from spindle to round in HPi group rats,and the collagen fibers gathered in disorder.Conclusions 1.The findings indicate that 4 wk of the tail-suspension can lead to a certain degree of bone loss in alveolar bone,and increase of serum phosphorus level.As well as in HPi fed mice.Which indicated that the correlation between phosphorus metabolic abnormalities and alveolar bone loss under the conditions of simulated weightlessness.2.Morphological and biological changes of osteocytes may be the cause of alveolar bone loss under the conditions of simulated weightlessness or high phosphorus environment.3.Due to the limitations of animal experiments,the results of this study require further verification of osteocytes culture in vitro.Part 2 The study of the correlation between phosphorus metabolic abnormalities and alveolar bone loss under the conditions of simulated weightlessness in vitroObjective To study the effect of simulated weightlessness and high phosphorus on the biological functions of osteocytes in vitro.And the possible role of SOST-OPG-RANKL-RANK system in it.Methods IDG-SW3 osteocytes were cultured in vitro.They were matched into the control group and MG group;the control group and HPi group.The morphological changes of osteocytes were observed under TEM(transmission electron microscope).The expression of SOST,RANKL and OPG m RNA were detected by real time-PCR(real-time reverse transcriptase Polymerase Chain Reaction).Results It was observed that the apoptotic features of osteocytes: nuclear pyrosis,nuclear fragmentation and the formation apoptotic body under the transmission electron microscope in both MG and HPi groups.Real time-PCR showed that the expression of SOST and RANKL were increased,OPG were decreased,RANKL/OPG ratio were elevated in these two groups.Conclusions 1.The morphologic changes of osteocytes,which showed apoptosis under the simulated weightlessness environment and the high-phosphate modeling.The results of in vitro studies further confirmed that the abnormal metabolism of phosphorus was associated with the alveolar bone loss under simulated weightlessness.2.Expression of SOST was increased,and RANKL/OPG ratio was elevated.These results suggested that SOST-OPG-RANKL-RANK system played an important role in it that osteocytes regulated the activity of osteoclasts and osteoblasts.Part 3 Study on the prevention of alveolar bone loss with low phosphate diet under simulated weightlessnessObjective To investigate whether low phosphate diet could prevent alveolar bone loss under simulated weightlessness.Methods The tail suspension method was used to simulate weightlessness,and low phosphate diet was used as the intervention measure.Eighteen mice were divided into three groups: the control(CON)group,tail-suspension(SUS)group and tail-suspension group with low phosphate diet(SUS-LP)group.Micro-CT analysis was performed to calculate vertical and furcated bone loss in mandibular first molars.To determine the histomorphology of new bone formation in the alveolar bone,Ponceau S staining were performed.Meanwhile,TRAP staining and ALP staining were used to observe the activity of osteoclast and osteoblast.Results Three dimensional reconstruction of Micro-CT showed that the number of trabecular bone was decreased,the trabecular space was relatively loose,and the three-dimensional network structure was worse in SUS group compared to that in the CON group.The number of trabecular bone was increased,the trabecular space was relatively tight,and the three-dimensional network structure was better in SUS-LP group compared to that in the SUS group.Ponceau S-stained sections showed that the areas of new bones were decreased in the SUS group compared to that in the CON group.Analysis of the TRAP-stained sections showed that the number of TRAP positive cells were increased in the SUS group compared to that in the CON group.ALP-stained sections showed that the number of ALP positive cells were decreased in the SUS group compared to that in the CON group.Ponceau S-stained sections showed that the areas of new bones were increased in the SUS-LP group compared to that in the SUS group.Analysis of the TRAP-stained sections showed that the number of TRAP positive cells were decreased in the SUS-LP group compared to that in the SUS group.ALP-stained sections showed that the number of ALP positive cells were increased in the SUS-LP group compared to that in the SUS group.Conclusions In this study,low phosphate diet can increase bone mass,reduce bone loss,and promote bone formation in alveolar bone,thus displaying obvious preventive effects.