Exploration On Protective Effects Of Young Rat Serum-derived Exosomes On Bone Quality In Senior Ovariectomized Rats

ObjectiveAs an essential mediator of intercellular communication,exosomes are involved in physiological and pathological processes widely and show great potential in the diagnostic and therapeutic fields.The contents carried by exosomes change at different ages,with consequent changes in the biological effects produced.The scientific question of interest in this study is whether young serum-derived exosomes(SDEs)exert protective effects on bone quality in osteoporosis by selectively enriching certain bioactive substances.We characterized the changes in bone quality in senior ovariectomized(OVX)rats,observed the effects of SDEs from young rats on osteogenic differentiation of bone mesenchymal stem cells(BMSCs)in senior OVX rats after fatigue loading,and then observed the effects of SDEs from young rats on bone microdamage and bone microstructure in OVX rats.The above studies investigated the protective effects and possible mechanisms of SDEs from young rats on bone quality.Taken together,we aim to explore the potential of SDEs from young rats in the prevention and treatment of osteoporosis in seniors.In addition,to clarify whether SDEs from humans have similar effects,the effects of SDEs from young females on BMSCs from senior postmenopausal females were initially assessed to provide a basis for the subsequent research of the functioning target.Methods1.Characterization of bone quality in senior OVX rats after fatigue loading.7-month-old SD female rats were ovariectomized and raised until22-month-old.Measured vertebral bone mineral density(BMD)of rats by Dual-energy X-ray absorptiometry(DXA)before OVX(pre-OVX at seven months of age),three weeks after OVX(post-OVX at seven months of age),and at 22-month-of age(post-OVX at 22 months of age).Perform fatigue loading test of the tibias in vivo at 22 months of age(the loading group)and rats without fatigue loading test(the non-loading group)as controls.Microdamage parameters of the tibias were analyzed:cortical bone area,cortical bone area ratio,average microcrack length(Cr.Le),microcrack density(Cr.Dn),and microcrack surface density(Cr.S.Dn).Analyze cortical bone microstructural parameters of the tibias by micro-computed tomography(μ-CT): volume bone mineral density(v BMD),cortical bone thickness(Cor.Th),and total porosity(percent)[Po(tot)].Examine the biomechanical properties of the tibias by three-point bending test: maximum bending strength,maximum load value,stiffness constant K,and elastic modulus.Extracted BMSCs from the tibias and identified BMSCs by flow cytometry.Under osteogenic induction,detect the level of osteogenic differentiation of BMSCs by alizarin red staining,RT-q PCR,and Western Blot.2.Detection of the effects of SDEs from young rats on the osteogenic differentiation capacity of BMSCs in senior OVX rats after fatigue loading.SDEs were extracted from 1-month-old SD female rats and identified by transmission electron microscopy,detection of marker proteins by Western Blot,and particle size analysis.Observe the uptake of SDEs by BMSCs through laser confocal microscopy.Under osteogenic induction,treated BMSCs of senior OVX rats after fatigue loading with SDEs.Alizarin red staining,RT-q PCR,and Western Blot were used to detect the osteogenic differentiation level of BMSCs.RT-q PCR verified the higher abundance of micro RNAs(miRNAs)in SDEs and used bioinformatics tools to predict target genes.Osteogenic differentiation and expression of target genes in BMSCs overexpressing miRNA and in BMSCs intervened with SDEs that had knocked down miRNA were analyzed separately.Then,analyzed Pathway signaling of BMSCs overexpressing miRNAs by RNA-seq technology.3.Detection of the effects of SDEs from young rats on bone microdamage and microstructure of the tibias in OVX rats after fatigue loading.Di R-labeled SDEs were injected into the tail vein or tibial marrow cavity and traced by fluorescence imaging.Raise 7-month-old OVX rats to 10-month-old.Make double bone labeling in vivo by intraperitoneal injection of calcein and tetracycline.Their tibias was subjected to fatigue loading in vivo—injected SDEs of young rats into the tibial marrow cavity.After three weeks,stained the tibias with basic fuchsin to analyze the bone microdamage parameters and mineral apposition rate(MAR)between the two groups.Detect cortical microstructural parameters of the tibias: bone volume/total volume(BV/TV),v BMD,Cor.Th,Po(tot)byμ-CT.Detect bone microstructural parameters of distal and proximal cancellous bone: v BMD,BV/TV,trabecular number(Tb.N),Connectivity Density(Conn.Dn),trabecular thickness(Tb.Th),bone structure model index,and SMI and bone trabecular separation(Tb.Sp)by μ-CT.4.Assessment of the effects of SDEs from young females on the osteogenic differentiation of BMSCs in senior postmenopausal females.SDEs of young and senior females were extracted and identified.Under osteogenic induction,BMSCs of senior postmenopausal females were treated with SDEs of young or senior females,detected their osteogenic differentiation by alizarin red staining,RT-q PCR,and Western Blot.Results1.Bone quality of senior OVX rats changed after fatigue loading.The decline in vertebral BMD of rats became slower after the rapid decline in BMD after OVX.BMD was significantly lower in 7-month-old post-OVX rats than in 7-month-old pre-OVX rats(p < 0.05),and BMD was significantly reduced in 22-month-old post-OVX rats compared to pre-OVX 7-month-old rats(p < 0.01),and compared to 7-month-old post-OVX rats,BMD was not significantly reduced in 22-month-old post-OVX rats(p > 0.05).Characterization of bone quality of the tibias in senior OVX rats after fatigue loading.(1)Microcracks ranging from tens to hundreds of micrometers in length were visible in hard tissue slices of the tibias in both fatigue loading and non-loading groups of rats.There was no significant difference in percentage of bone cortical area in the tibias.Microdamage parameters of the tibias: Cr.Le,Cr.Dn and Cr.S.Dn were higher in the loading group than in the non-loading group(p <0.001).(2)Microstructural parameters of the tibias: volume bone mineral density(v BMD),cortical bone thickness(Cor.Th),and total porosity(percent)[Po(tot)] of the tibias in the loading group were not statistically different from those in the non-loading group were not statistically different(p > 0.05).(3)Biomechanical properties of the tibias: maximum bending strength,maximum load value,constant stiffness K,and elastic modulus of the tibias in the loading group were not significantly different compared to the non-loading group(p > 0.05).(4)The purity of BMSCs from tibias of senior OVX rats met experimental requirements.Under osteogenic induction,compared with the non-loading group,BMSCs in the loading group showed reduced calcium nodule formation,and reduced expression of m RNAs for the osteogenic genes ALP,OCN,Collagen I,and Runx2.At the protein level,the expression of Collagen I and Runx2 decreased.2.SDEs of young rats promoted the osteogenic differentiation of BMSCs in senior OVX rats after fatigue loading.SDEs of young rats are disc-like structures of approximately 30-150 nm in diameter and identified marker proteins TSG 101 and CD9,and CD81.Laser confocal microscopy observed the uptake of SDEs by BMSCs.Under osteogenesis induction,BMSCs of senior OVX rats after fatigue loading treated with SDEs showed increased calcium nodule formation,and m RNA expression levels of osteogenic genes OCN,Collagen I,and Runx2,increased expression of Collagen I and Runx2 at the protein level.miRNA-19b-3p was more abundant in SDEs of young rats.Overexpression of miRNA-19b-3p resulted in increased expression of the osteogenic genes ALP,Collagen I,and Runx2 in BMSCs at the m RNA level,increased expression of Collagen I at the protein level,decreased expression of PTEN,and darker staining of ALP.Expression of the osteogenic genes ALP,Collagen I,and Runx2 at the m RNA level and Collagen I at the protein level was reduced(p < 0.01),and PTEN expression was increased in BMSCs after treatment of SDEs knocked out of miRNA-19b-3p.RNA-seq analysis speculated PI3K/Akt as the possible signaling pathway.3.SDEs of young rats improved bone microdamage and microstructure of the tibias in OVX rats after fatigue loading.At 48 h of tail vein injection of Di R-labeled SDEs of young rats,found no significant concentrations in live and isolated bone tissue of rats.Injected SDEs into the bone marrow cavity of the tibias and found significant concentrations at the location of the tibias.(1)Microdamage parameters of the tibias in OVX rats: the microfracture surface density(Cr.S.Dn)reduced in the SDEs injection group(p < 0.01),and the cortical bone area,bone cortical area ratio,Cr.Le,and Cr.Dn was not different from those in the PBS injection group(p > 0.05).(2)MAR was increased in the SDEs injection group(p < 0.01).(3)Cortical bone microstructural parameters at the load-bearing site of the tibias: no significant differences in tibial v BMD,BV/TV,Cor.Th and Po(tot)(p > 0.05).(4)Proximal cancellous bone microstructural parameters: v BMD,BV/TV,trabecular number(Tb.N),trabecular junction density(Conn.Dn)in the SDEs injection group increased,and trabecular separation(Tb.Sp)decreased.The differences were not statistically significant(p > 0.05).(5)Distal cancellous bone microstructural parameters: v BMD,BV/TV,Tb.N,and Conn.Dn increased in the SDEs injection group,with statistically significant differences in v BMD,BV/TV,and Tb.N(p < 0.05).Trabecular separation(Tb.Sp)of the tibias in SDEs injection group decreased but with no significant differences.4.SDEs of young females promote the osteogenic differentiation of BMSCs in senior postmenopausal females.Observe the uptake of SDEs of young females by BMSCs through Laser confocal microscopy.Treatment with SDEs of young females resulted in increased calcium nodule formation and higher m RNA and protein expression levels of the osteogenic genes Collagen I and Runx2 in BMSCs than treated with SDEs of senior females and PBS.At the m RNA and protein level,the expression of Runx2 in BMSCs treated with SDEs of senior females was not significantly different from that treated with PBS.Conclusion1.Senior OVX rats produced bone microdamage after fatigue loading of the tibias in vivo,with unaltered bone microstructure and biomechanical properties and reduced osteogenic differentiation capacity of BMSCs.2.SDEs of young rats could improve the osteogenic differentiation of BMSCs after fatigue loading of the tibias in senior OVX rats,and their effects were partially mediated by miRNA-19b-3p,with PTEN as a possible target protein and PI3K/Akt as a possible signaling pathway.3.SDEs of young rats can reduce bone microdamage,promote new bone formation,improve bone microstructure,and protect the bone quality of the tibias in OVX rats after fatigue loading.4.SDEs of young females promote osteogenic differentiation of BMSCs in senior postmenopausal females.