| BACKGROUNDBone is a dynamic tissue that continually undergoes a remodeling process to maintain normal structure and function.Bone remodeling is involved in multiple types of cells.These cells constitute the active regions where bone remodeling occurs and is known as basic multicellular units(BMU).Osteoclast and osteoblast are two main types of cells involved in bone remodeling in BMU.Osteoclasts are responsible for the resorption of aging bone,while osteoblasts are responsible for the formation of new bone.Under physiological conditions,the interaction between bone absorption and bone formation are relatively stable.Osteoblasts will make timely feedback to bone absorption stimulation,so that the new bone formed in each BMU accurately replaces the bone absorbed in these BMU.This precise coordination of bone resorption and bone formation in time and space mediated by osteoclasts and osteoblasts is defined as coupling.When the coupling balance is destroyed,bone structure and function will be abnormal,resulting in a series of bone diseases including osteoporosis,osteosclerosis or bone defect nonunion.For a long time,the precise process by which bone remodeling is coupled has been widely recognized under its increasing research.Nonetheless,the precisely coupling mechanism of bone resorption and bone formation remains unclear.The structure and function of bone has long been thought to be primarily regulated by hormones in the circulatory system.This view has been gradually replaced by new findings in the last decade of research.Currently,homeostasis of the bone system is thought to be regulated by local factors.In particular,the crosstalk between osteoclasts and osteoblasts determines local bone mass.Extracellular vesicles(EVs),as key mediators of intercellular communication,participate in the regulation of diversified biological processes.According to the size and biogenesis,EVs are mainly divided into small EVs(sEVs)with a diameter of less than 200 nm and large EVs(l EVs)with a diameter of more than 200 nm.During bone remodeling,active osteoclasts secrete large amounts of EVs,which carry bioactive molecules including proteins,nucleic acids,and lipids.These EVs are transferred to a variety of bone cells and form a complex and precise regulatory network of bone microenvironment,which is a crucial factor in maintaining bone homeostasis.Among them,the coupling effect of osteoclast-EVs on osteoblast and osteogenic differentiation has become the focus of research in this field.Coupling between osteoclasts and osteoblasts in bone remodeling is the key to maintaining bone homeostasis.Further exploration of EVs-mediated osteoclast-osteogenesis coupling mechanism will lay the foundation of bone remodeling regulatory network.At the same time,it will provide a new theoretical basis for clinical diagnosis and treatment of bone diseases characterized by coupling imbalance.In recent years,EVs has become a potential drug delivery carrier in clinical translational therapy due to its inspiring biocompatibility and bioinformation editability.Current studies have shown that EVs with specific biological characteristics and functions can be designed and produced using EVs bioengineering technology based on the understanding of EVs biogenesis,secretion,and internalization pathways.It will greatly improve the efficiency of EVs to perform biological functions.EVs derived from endothelial cells have been reported to specifically target bone tissue and inhibit pathological bone resorption through its anti-osteoclast differentiation properties,suggesting its potential for the treatment of osteoporosis.Nevertheless,there is little research that explored the effect of osteoclast-EVs on osteogenic differentiation potential of bone marrow mesenchymal stem cells(MSCs)and its effect on bone defect healing.Revealing the specific mechanism of osteoclast EVs on MSCs osteogenic differentiation can provide a theoretical basis for the development of bone repair materials using osteoclast-EVs as carriers delivering biological information,and is of key significance for the clinical treatment of bone defects.OBJECTIVEBased on the role of osteoclast secreted EVs on the MSCs osteogenic differentiation,our study aims to reveal the potential mechanism of osteoclast-EVs as a biological signal carrier for artificial bone repair materials in cranial defect repair.MATERIALS AND METHODS1.The mi RNA expression profile and biological characteristics of osteoclast-derived sEVs were investigated.(1)We established an osteoclast differentiation model in vitro.Osteoclast-derived sEVs were purified by ultracentrifugation,and the sEVs were characterized by transmission electron microscope(TEM),Nanoparticle tracking analysis(NTA)and Western blot;(2)Osteoclasts-sEVs at different differentiation stages were extracted,the mi RNA expression profile in the sEVs was exhibited by RNA-seq,and the specific distribution of mi RNA was statistically analyzed;(3)VENN and GSEA analysis were performed to compare mi RNA expression profile of sEVs and parental osteoclasts.GO analysis was performed to predicted biological functions of mi RNAs in sEVs.2.The internalization effect of MSCs on osteoclast-derived sEVs and the effect of osteoclast-derived sEVs on osteogenic differentiation and bone mineralization of MSCs were investigated in vitro.(1)After 24 hours co-incubation of sEVs and MSCs,the internalization effect of MSCs on sEVs and intracellular localization of sEVs were observed by confocal microscopy;(2)GW4869 was used for osteoclasts pretreatment,and MSCs were cultured in osteoclast conditioned medium.Cell samples were collected at 7 and 21 days after culture,respectively.q PCR and Western blot were performed to measure the expressions of osteogenic markers(Col1a1,Alp,Sp7 and Runx2)of MSCs and alizarin red staining was performed to detect the osteogenic mineralization of MSCs.(3)After 7 days co-incubation of sEVs and MSCs,q PCR and Western blot was performed to detect the expressions of osteogenic markers of MSCs.(4)After 21 days co-incubation of sEVs and MSCs,alizarin red staining was performed to detect the osteogenic mineralization of MSCs.3.The function of mi R-324 on osteogenic differentiation of MSCs in osteoclast-derived sEVs was investigated in vitro,and downstream targets of mi R-324 were screened and detected.(1)q PCR was performed to detect the expression of mi R-324 in osteoclast-derived sEVs,and transfection experiment was performed to overexpress or knock down the level of mi R-324 in osteoclast-derived sEVs.The effects of mi R-324 in sEVs on the expression of osteogenic markers,proliferation,and osteogenic mineralization in MSCs were assessed using co-incubation,CCK-8,q PCR,Western blot,and alizarin red staining.(2)Bioinformatics analysis predicted mi R-324 as a target of ARHGAP1.The targeting relationship between mi R-324 and downstream ARHGAP1 was detected using q PCR and luciferase reporter.(3)Adenovirus vector transfection was performed to overexpress or knock down the expression level of ARHGAP1 in MSCs.The effects of ARHGAP1 on the expression of osteogenic markers and osteogenic differentiation in MSCs were detected using q PCR and ALP staining.4.The specific molecular mechanism of osteoclast-derived sEVs transferred mi R-324 to promote osteogenic differentiation of MSCs were investigated in vitro and in vivo.(1)mi R-324 overexpressed sEVs were co-incubationd with MSCs,and the expression activity of ARHGAP1 was detected by luciferase reporter assay.(2)mi R-324 inhibitor was performed to eliminate the expression of mi R-324 in sEVs,and Annexin V was performed to block the internalization of sEVs in MSCs respectively.The expression of osteogenic markers,proliferation and osteogenic mineralization after upregulated the expression of ARHGAP1 in MSCs were measured using co-incubation,CCK-8,q PCR,Western blot and alizarine red staining.Meanwhile,the activation of Rho A/ROCK downstream signaling pathway of ARHGAP1 was detected by Western blot.(3)We established a mouse cranial defect model.Osteoclast-derived sEVs were used as active molecules to construct artificial bone repair materials on demineralized bone matrix(DBM)and implanted in the defect area.After 4weeks,micro-CT and histological staining were used for evaluating the effect of scaffold materials on bone remodeling and bone regeneration in the bone defect area.Immunohistochemical staining was used for evaluating the expression of ARHGAP1,Rho A,ROCK and p-MYPT1.RESULTS1.Bioinformatics analysis showed that the mi RNA expression profile of osteoclast-derived sEVs exhibited high overlap with the parental cells.Meanwhile,the contents of osteoclast-derived sEVs were closely involved in “small GTPase-mediated signaling pathway”,“positive regulation of osteogenic differentiation”,“bone development”and other biological processes.2.After 24 h co-incubation,there was obvious fluorescence co-localization between sEVs and MSCs;After 7 days co-incubation,sEVs significantly upregulated the expression of osteogenic markers in MSCs.After 21 days co-incubation,osteoclast-derived sEVs significantly promoted the formation of osteoblast mineralized crystals.After GW4869 pretreatment,the facilitation effect of osteoclastic conditioned medium on the expression of osteogenic markers and mineralization of MSCs was reversed.3.RNA seq analysis and low-throughput verification showed that mi R-324 was markedly enriched in osteoclast-derived sEVs.Knockdown mi R-324 in sEVs impaired the promoting effect of sEVs on the osteogenic differentiation of MSCs and interference of mi R-324 expression in MSCs prevents its differentiation into osteoblasts.The luciferase activity of ARHGAP1 binding site markedly decreased by mi R-324 in MSCs,while the mutation of 3’UTR binding site eliminated the response to mi R-324.A high expression ARHGAP1 in MSCs significantly inhibited the expression of osteogenic markers and mineralization.4.The luciferase activities of the 3’UTR of ARHGAP1 were suppressed by mi R-324;The sEVs treated with Annexin V or mi R-324 inhibitor both counteracted the promoting effect of sEVs on the expression of osteogenic markers in MSCs and the formation of mineralized crystals.Overexpressed ARHGAP1 in MSCs silenced the protein expression of Rho A and ROCK.Besides,overexpressed ARHGAP1 suppressed the expression of osteogenic markers in MSCs and osteogenic mineralization.sEV-mi R-324 derived from osteoclasts promoted the mineralization of the defect area and increased the activity of local osteoclasts and osteoblasts.Osteoclast-derived sEVs inhibited ARHGAP1 expression,while promoted Rho A and ROCK expression,and phosphorylated MYPT1.CONCLUSIONS1.Osteoclast-derived sEVs inherited the signature contents and biological characteristics from parental cells and may have biological functions in regulating osteogenic differentiation and bone formation.2.Osteoclast-derived sEVs were effectively internalized by MSCs and promoted osteogenic differentiation and mineralization of MSCs.3.mi R-321 enriched in osteoclast-derived sEVs was a key factor promoting osteogenic differentiation and mineralization of MSCs.ARHGAP1 in MSCs was a negative regulator of osteogenic differentiation.4.Osteoclast-derived sEVs regulated the osteogenic differentiation of MSCs via ARHGAP1/Rho A/ROCK/p-MYPT1 signaling,thus promoting bone regeneration and bone remodeling activities. |
PDF Full Text Request