Table_1_Mechanism of Exosomes Involved in Osteoimmunity Promoting Osseointegration Around Titanium Implants With Small-Scale Topography.XLSX

Exosomes are nanoscale extracellular vesicles. Several studies have shown that exosomes participate in intercellular communication and play a key role in osseointegration. However, it is unclear whether exosomes and their contents participate in the communication between the immune and skeletal systems in the process of osseointegration. In this study, we obtained smooth titanium disks by polishing and small-scale topography titanium disks by sandblasted large-grit acid-etched (SLA) technology combined with alkali thermal reaction. After stimulating mouse RAW264.7 cells with these two kinds of titanium disks, we co-cultured the MC3T3-E1 cells and the RAW264.7 cells, obtained and identified the exosomes derived from RAW264.7 cells, and studied the effect of the osteoimmune microenvironment and the exosomes on the osseointegration of mouse MC3T3-E1 cells. Cell counting kit-8 (CCK-8), real time quantitative PCR, western blotting, alizarin red staining, and quantitative and confocal fluorescence microscopy were used to study the effects of exosomes on MC3T3-E1 cells; RNA sequencing and correlation analysis were performed. We found that the osteoimmune microenvironment could promote the osseointegration of MC3T3-E1 cells. We successfully isolated exosomes and found that RAW264.7 cell-derived exosomes can promote osteogenic differentiation and mineralization of MC3T3-E1 cells. Through RNA sequencing and gene analysis, we found differentially expressed microRNAs that targeted the signal pathways that may be related, such as mTOR, AMPK, Wnt, etc., and thus provide a reference for the mechanism of osteoimmunue regulation of implant osseointegration. The study further elucidated the mechanism of implant osseointegration and provided new insights into the effect of exosomes on implant osseointegration, and provided reference for clinical improvement of implant osseointegration and implant success rate.

外泌体（exosomes）是一类纳米级细胞外囊泡。多项研究表明，外泌体参与细胞间通讯，并在骨整合过程中发挥关键作用。然而，在骨整合进程中，外泌体及其所载物质是否参与免疫系统与骨骼系统的信号通讯，目前仍不明确。本研究通过抛光工艺制备光滑钛盘，并采用喷砂大颗粒酸蚀（sandblasted large-grit acid-etched, SLA）联合碱热反应技术制备微尺度形貌钛盘。将两种钛盘分别刺激小鼠RAW264.7细胞后，共培养MC3T3-E1细胞与RAW264.7细胞，分离并鉴定RAW264.7细胞来源的外泌体，探究骨免疫微环境及外泌体对小鼠MC3T3-E1细胞骨整合的影响。本研究采用细胞计数试剂盒-8（CCK-8）、实时定量聚合酶链式反应（real time quantitative PCR）、蛋白质印迹法（western blotting）、茜素红染色（alizarin red staining）、定量分析及共聚焦荧光显微镜（confocal fluorescence microscopy）技术，探究外泌体对MC3T3-E1细胞的作用；同时开展RNA测序及相关性分析。研究结果显示，骨免疫微环境可促进MC3T3-E1细胞的骨整合。本研究成功分离出外泌体，且发现RAW264.7细胞来源的外泌体可促进MC3T3-E1细胞的成骨分化与矿化。通过RNA测序及基因分析，本研究筛选得到差异表达microRNA，其靶信号通路可能与mTOR、AMPK、Wnt等通路相关，为骨免疫调控种植体骨整合的机制研究提供参考。本研究进一步阐明了种植体骨整合的分子机制，为外泌体在种植体骨整合中的作用提供了全新的研究视角，并为临床提升种植体骨整合效果与种植成功率提供了重要参考依据。