| Background:Titanium and titanium alloys are the most common materials used for implant restoration in clinical practice.The research on surface modification of implants and related molecular mechanisms of implant osseointegration has always been a hot spot in this field.Osteoimmunity plays a vital role in the process of implant osseointegration,and autophagy also plays an important role in maintaining bone homeostasis.However,whether osteoimmunity affects osseointegration by affecting autophagy is still unclear.In recent years,exosomes and their contents have been shown to play an important role in cell-to-cell communication in the connection between the immune system and the skeletal system.At the same time,reports have pointed out that the release of exosomes and the activity of autophagy are a coordinated process,they are conducive to maintaining cell homeostasis.However,there are few studies on the effect of exosomes and autophagy in the osteoimmune microenvironment that affect osseointegration and related molecular mechanisms.Purposes:Smooth and micro-nano topography morphology titanium disks were fabricated for experiments in vitro.Exosomes were isolated from the supernatant of macrophage culture medium stimulated by different titanium disks.The relationship and the key roles among osteoimmunity,macrophage-derived exosome and autophagy in the process of osseointegration were studies.This study aimed to deeply analysis of the osteoimmune microenvironment formed by the micro-nano topography morphology titanium disks affects the mechanism of osseointegration through exosomes and autophagy,and to provide a theoretical reference for the research on the mechanism of improving the osseointegration of implants.Methods:1.Smooth titanium disks and micro-nano composite titanium disks were prepared to observe their surface morphology with scanning electron microscope,to detect surface roughness by using optical profiler,and to measure contact angle by using hanging drop method.2.RAW264.7 cells were cultured on the surface of smooth disks and micro-nano composite titanium disks.qRT-PCR was used to detect the expression of inflammatory factors in RAW264.7 cells.The supernatant of the culture medium was extracted,and the exosomes derived from RAW264.7 cells stimulated by different titanium disks were separated by ultracentrifugation.The shape of exosomes was examined with transmission electron microscope,the size of which were measured by nanoparticle tracking technology.Meanwhile,the expression of exosomes-related marker proteins was detected by western blot.3.In the one hand,to study the key role of autophagy,MC3T3-E1 cells were cultivated with or without autophagy inhibitors,and were divided into control group and RAW264.7 cells/MC3T3-E1 cells co-culture group.In another,to study the key role of exosomes,MC3T3-E1 cells were divided into control group,RAW264.7 cells/MC3T3-E1 cells co-culture group and RAW264.7 cell-derived exosomes/MC3T3-E1 cells co-culture group.The expression of autophagy-related markers and osteogenic markers in MC3T3-E1 cells were detected from gene and protein level.CCK-8 kits were performed to detect cell proliferation of MC3T3-E1 cells.Alizarin Red Staining and Semi-quantitative Analysis were performed to detect the osteogenic mineralization of MC3T3-E1 cells.Immunofluorescence staining was performed to detect autophagosomes of MC3T3-E1 cells.Results:1.The smooth titanium disk possessed a relatively flat surface without obvious scratches;the micro-nano composite titanium disk had a large number of continuous overlapping pits under the low power microscope.The pits were present on the surface of the first-order depression with a diameter of approximately 10-50 μm,and a secondary depression with a diameter of 2-8 μm was superimposed.Nanopores with a diameter of 50-200 nm could be observed under a high-power microscope.Compared with the smooth titanium disk,the contact angle of the micro-nano composite titanium disk is significantly reduced(p<0.05),and the average roughness is significantly increased(p<0.05).2.Compared with the smooth titanium disk,the micro-nano composite morphology titanium disk can stimulate the expression of anti-inflammatory related gene IL-10 and decrease the expression of pro-inflammatory related gene IL-1β in RAW264.7 cells.3.Compared with the control group,the co-culture group of RAW264.7 cells/MC3T3-El cells increased expression of osteoblast-related markers,and cell proliferation and mineralization were better.After autophagy is inhibited,the expression of osteoblast-related markers was decreased,as the same as cell proliferation and mineralization.4.After isolating by ultracentrifugation,transmission electron microscope was showed that extracellular vesicles’ circular double-layer membrane structure,and nanoparticle tracking analysis found that it is basically in the distribution range of 30-200 nm.Western blot detected the expression of exosomal surface proteins CD9,CD63,TSG101.5.After using M2 type macrophage-derived exosomes to stimulate MC3T3-E1 cells,contrast with the control group and the co-culture group of RAW264.7 cells/MC3T3-E1 cells,the co-culture group of RAW264.7 cell-derived exosomes/MC3T3-E1 cells increased expression of osteoblast-related markers,and cell proliferation and mineralization were better.Fluorescence staining results showed that the co-culture group of RAW264.7 cells/MC3T3-El cells and the co-culture group of RAW264.7 cell-derived exosomes/MC3T3-E1 cells can activate the autophagy level of MC3T3-E1 cellsConclusion:1.The micro-nano composite morphology can stimulate RAW264.7 cells to form an anti-inflammatory immune microenvironment,which is conducive to osteogenic differentiation and mineralization;2.Autophagy plays an important role in the anti-inflammatory osteoimmune microenvironment to promote osseointegration;3.Macrophage-derived exosomes play an important role in the anti-inflammatory osteoimmune microenvironment to promote osseointegration;4.Macrophage-derived exosomes may affect implant osseointegration by regulating the autophagy level of MC3T3-E1 cells. |
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