Rapamycin Influences Bone Remodeling In Inflammation

Background:As a common disease in oral clinic, chronic periodontitis not only impacts themastication and pronounciationfunctions of patients, but also leads to alveolar boneresorption, decreaing the chance for reservation of permanent teeth and the survivalof implantation. Therefore, an appropriate method to cure chronic periodontitis andpromote alveolar bone remodeling has become the concern for all dental researchers.Considering the crucial effect of immune factors in the development of periodontitis,researchers suggest applying immunosuppressive drugs to cure periodontitis. Inrecent years, as a frequently-applicated immunosuppressive drug, rapamycin hasgained huge concern because of its multiple functions amongphysiological activities.However, controversy still exists regarding to how rapamycin influences boneremodelling, a possible application in oral clinic is currently facing multiplechallenges. In our study, we aim to investigate how rapamycin influences boneremodelling through in vivo rat models and how rapamycin impactsosteoclastogenesis and osteogenesis via in vitro methods.Objective:To choose a appropriate concentration of rapamycin through in vitro assay andinvistigate how rapamycin of different concentrations influence osteoclastogenesisand osteogenesis in both normal and LPS-induced inflammatory environmentsand tospeculate its possible mechanism. Furthermore, to observe the influence ofrapamycin of different concentrations to bone remodelling in rat tooth-extractionsockets through in vivo study in order to provide a solid theoretical basis for afurther application in oral clinic and a more efficient and developed treatmenttherapy to cure chronic periodontitis. Methods:There are three parts in our study:1. Osteoclastogenesis assays: Mouse mononuclear/macrophage cell line Raw264.7cells were cultured in both normal and LPS-induced inflammatory conditions,and rapamycin of different concentrations（20ng/ml,200ng/ml）were added to thecells. After1,3,5days, total RNAs were extracted and the expression ofosteoclastogenesis-related genes were measured via RT-PCR to identify the impactof rapamycin to osteogenesis under normal and inflammatory conditions.Furthermore, Ly294002, a specific inhibitor of PI3Kwas added to speculate thepossible mechanism of how rapamycin affected osteoclastogenesis.2. Osteogenesis Study: Rat bone marrow mesenchymal stem cells (BMSCs)were extracted and nurtured in both normal and LPS-induced inflammatoryconditions and assays such as ALP, Alizarin Red Staining, RT-PCR were conductedto investigate how rapamycin influenced BMSCs osteogenesis.3. In vivo study: Left mandibular incisors were extracted in rats to conduct arat tooth socket model. Rats were divided into3groups randomly and PBS,20μgrapamycin,100μg rapamycin were injected into the socket individually. After2weeks,4weeks and8weeks, all animals were sacrificed and mandibules wereharvested. Residual alveolar bone height were measured to investigate howrapamycin of different concentrations influencedthe rate of alveolar boneremodelling in tooth extration sockets. Histology sections are conducted toinvestigate how rapamycin of different concentrations influenced bone formation intooth extration sockets.Results:1. Osteoclastogenesis studies indicated that rapamycin only could significantlypromote the expression of osteoclastogenesis-related genes, while inLPS-inducedinflammatory conditions, rapamycin could further significantly enhancethe osteoclastogenesis-enhancing bone resorption effect of inflammatory factors.After adding Ly294002, the expression of osteoclastogenesis-related genes were decreased obviously, indicating that rapamycin functions through mTOR/Aktpathway.2. Osteogenesis studies indicated that rapamycin alone could not enhance theexpression of ALP and osteogenesis-related genes or the formation ofcalcium nodule, while in LPS-inducedinflammatory environment, rapamycin couldsignificantly enhance the expression of ALP and osteogenesis-related genes or theformation of calcium nodule compared to the LPS-induced environment itself.3. In vivo study indicted low-concentration rapamycin had no significantinfluence to residual alveolar bone resorption within8weeks, while highconcentration rapamycin could obviously decrease the rate of alveolar boneresorption. Histology sections show rapamycin of both concentrations inhibited boneformation in rat tooth sockets.Conclusion:1. Rapamycin enhanced osteoclastogenesis, while in LPS-inducedinflammatory environment, osteoclastogenesis was further promoted by rapamycin.2. Rapamycin itself could not enhance osteogenesis, while inLPS-inducedinflammatoryenvironment, rapamycin enhanced osteogenesis.3. Rapamycin of a high concentration decreased the rate of resorption ofresidual alveolar bone, rapamycin of both high and low concentrations inhibitedbone formation in rat tooth sockets.