Effect And Mechanism Of Culture Media Derived From Antler Reserve Mesenchyme Cells On Alveolar Bone Regeneration

Periodontitis is an inflammatory disease,which initiated by dysbiosis of the local microbial community.It results in destruction of tooth-supporting tissue by overactivating host immune response.In addition,periodontitis is not only the most common reason of tooth loss in the world,but also increasing the risk of a variety of systemic diseases,which brings great psychological and economic burden to patients.Therefore,effective treatments of periodontitis are essential to maintain human health.The ideal outcome of periodontal treatment is to control infection,rebuild periodontal tissue structure and restore periodontal tissue function.However,due to the complex structure of periodontal tissue,it is difficult to achieve functional periodontal regeneration by traditional treatment methods.With the development of tissue engineering,transplantation of exogenous mesenchymal stem cells（MSCs）for periodontal tissue regeneration has become one of the hot fields.Studies have shown that exogenous MSCs can effectively promote the regeneration and repair of periodontal supporting tissue.However,because of the shortcomings of high cost and low survival rate in using of MSCs,indirect treatment based on MSCs has attracted increasing attention.A large amount of evidence shows that MSCs could promote tissue regeneration by secreting a series of bioactive factors,which are widely defined as culture media（CM）.Culture media derived from mesenchymal stem cells（MSC-CM）can significantly drive biological processes such as metabolism,defense response and tissue regeneration.Compared with stem cell-based therapies,the preparation of CM is more economical and the evaluation of safety and efficacy is simpler.Studies have shown that the components and concentrations of cytokines and growth factors contained in MSC-CM from different cell sources are different.Therefore,it plays a key role to find an ideal MSCs in improving the efficacy of MSC-CM.Generally speaking,mammals cannot regenerate the complex tissues and organs,but the periodic regeneration of velvet antler is an exception.Velvet antler is known as the"pearl in the crown"of modern regenerative medicine.It is the only mammalian bone organ that can be completely regenerated so far.Antler stem cells are the key to initiating antler regeneration,which can achieve antler regeneration through continuously self-renew and differentiation.As an important factor driving the rapid growth of velvet antler,reserve mesenchyme cells（RMCs）have attracted more and more attention.RMCs are located in the outermost layer of the proliferation zone of the antler growth center,which have great proliferation potential and can maintain the amazing growth rate of antler（up to 2 cm/d）.Another study showed that under identical culture conditions,RMCs proliferated faster（8.6–11.7-fold increase in cell numbers）and exhibited increased osteogenic differentiation（17.4-fold increase in calcium mineralization）compared to human mesenchymal stem cells（h MSCs）.The rapid growth of velvet antler provides a rare system for the study of tissue regeneration.In this system,the rapid proliferation of cells is not only accurately regulated,but also not cancerous.This may be related to the fact that antler stem cells have the properties of MSCs and partial attributes of embryonic stem cells（ESCs）.Therefore,the rapidly growing velvet antler provides a valuable model for understanding how certain properties of ESCs are retained in postnatal tissues.The periodic regeneration of velvet antler not only provides a valuable opportunity for humans to understand how mammalian organs could complete regenerate,but also provides a potential inexhaustible source of RMCs,which are more accessible than other types of mammalian stem cells.Therefore,it has great potential of RMCs to applicate in tissue regeneration.However,as heterologous stem cells,there are still a lot of limitations in the clinical application of RMCs.Therefore,the research on the culture media derived from RMCs（RMC-CM）are increasing.In recent years,RMC-CM has been successively applied to promote regenerative healing of skin wounds,alleviate liver fibrosis and alleviate osteoarthritis,providing a theoretical basis for regenerative medicine and bone remodeling.RMC-CM is expected to play a role in the field of periodontal tissue regeneration due to its angiogenic,immunomodulatory and chemotactic abilities.Bone homeostasis is maintained by the balance between bone formation by osteoblasts and bone resorption by osteoclasts.In inflammation,lipopolysaccharide（LPS）of bacterial can break this balance and cause pathological bone resorption.Inhibiting the differentiation of osteoclasts under inflammatory conditions and reducing bone resorption are also important steps in bone regeneration.In this study,we explored the effect and mechanism of RMC-CM on alveolar bone regeneration from the two aspects of osteogenesis and osteoclasis,which has not been reported.Firstly,we identified the main components and functions of RMC-CM.In Chapter2,we proved that RMC-CM was different from（human bone marrow mesenchymal stem cells culture media,h BMSC-CM）in terms of composition and function by liquid chromatography-mass spectrometry（LC-MS/MS）analysis.The development and application of RMC-CM will provide a new therapeutic strategy for periodontal tissue regeneration.As h BMSC-CM has been widely used in periodontal tissue regeneration and its efficacy has been certified,it is selected as the control.Secondly,we evaluated the ability of RMC-CM on regulating osteoblastic differentiation.In Chapter 3,we established an alveolar bone defect model by surgery,and evaluated the effect of RMC-CM on alveolar bone regeneration with no inflammation through Micro-CT,histological staining and other methods.We also clarify SERPINE1 and LILRB3 as the target of RMC-CM in promoting alveolar bone regeneration by combining the results of transcriptome sequencing analysis with the expression of osteoblast related genes of rat bone marrow mesenchymal stem cells（r BMSCs）under osteogenic conditions.Thirdly,in the process of periodontitis,inflammation related factors lead to more bone resorption from osteoclasts than bone formation,which is the key mechanism of alveolar bone defect.Therefore,we evaluated the ability of RMC-CM on regulating osteoclast differentiation under inflammatory conditions.In Chapter 4,we established an animal experimental periodontitis model and evaluated the regeneration effect of RMC-CM on alveolar bone regeneration under inflammation through Micro-CT,histology staining and immunology analysis.We confirmed that RMC-CM achieved the therapeutic effect of inhibiting periodontal inflammation and promoting alveolar bone regeneration by promoting M2 type polarization of macrophage and inhibiting osteoclast differentiation.Finally,we sort out the above results.First,RMC-CM can inhibit osteoclast differentiation.Secondly,RMC-CM can cause the down regulation of Lilrb3transcription.Third,Lilrb3 can be enriched in the related pathways of osteoclast differentiation,and by searching the KEGG signal pathway database,it was found that leukocyte immunoglobulin like receptor（LILR）can activate PLCγ-IP3-Ca²⁺signaling pathway.Based on the above results,we speculated that RMC-CM may inhibit the PLCγ-IP3-Ca²⁺signal pathway by causing the down-regulation of LILRB3 transcription,thereby inhibit osteoclast differentiation and promote alveolar bone regeneration.In Chapter 5,we induced osteoclast differentiation of macrophages.Aiming at the key points of PLCγ-IP3-Ca²⁺signaling pathway,we detected the expression of PLCG2,NFATc1,CTSK and TRAP under the induction of RMC-CM,and preliminarily clarified that RMC-CM can achieve alveolar bone regeneration by regulating PLCγ-IP3-Ca²⁺signaling pathway.In conclusion,our study clarified that RMC-CM could promote the regeneration of alveolar bone caused by non-inflammatory factors and inflammatory factors.However,as RMC-CM is a mixture of a variety of cytokines and growth factors,whether the effect of RMC-CM on alveolar bone regeneration is the result of the synergistic effect of a variety of factors or the independent effect of some of them is still unknown.It needs to be further discussed in the follow-up study.