| Objective: The repair of critical size defect(CSD)has always been a hot issue in the medical field.Non-invasive physical interventions can enhance bone regeneration,The combined application of physical stimulation and bone defect repair materials help optimize the treatment of bone defects.Low-intensity pulsed ultrasound(LIPUS)is a non-invasive,pulsed mechanical wave,which could impose therapeutic and protective effects on fractures and bone defects.The US Food and Drug Administration approved LIPUS for the acceleration of fresh or delayed bone fracture healing and the treating of nonunion.Three-dimensional scaffold materials play an important role in bone tissue engineering for the repair of CSD.Among them,porous titanium alloy material has excellent biological compatibility,osteoconductivity and processing stability,through the regulation of its porosity,it can match the elastic modulus of bone tissue.However,the osteogenic effect and the depth of new bone growth are still insufficient in the repair of CSD with porous metal materials alone.The combined application of LIPUS and porous titanium alloy provide a new therapeutic strategy for the repair of CSD.Our previous experimental results showed that LIPUS could significantly promote the migration and osteogenic differentiation of osteoblasts in porous titanium alloy scaffolds as well as promote the growth of bone tissue and bone maturation in scaffolds.The molecular mechanism of LIPUS-promoting bone healing is very complex and has not yet been fully elucidated,while the molecular mechanism behind LIPUS-promoting osteogenesis in 3D scaffolds has not been reported,so further exploration is necessary.Micro RNAs(miRNAs)have emerged as a novel mechanism which can regulate the expression of mRNAs and proteins.They can silence their cognate target genes by inhibiting mRNA translation or degrading the mRNA molecules by binding to their 3’-untranslated regions(3’-UTR),thus regulate posttranscriptional gene expression.Alterations in miRNA expression are closely related to various bone diseases,more and more miRNAs have been found to be involved in the differentiation process of osteoblasts.At present,there are few studies on the role of miRNA about LIPUS promoting osteogenic differentiation and fracture healing,and there is no report on the effect of miRNA in LIPUS-promoted osteogenesis in porous materials.The previous high-throughput miRNA sequencing demonstrated that miR-1187 was significantly down-regulated under LIPUS irradiation,suggesting that miR-1187 may play a regulatory role in LIPUS-promoted osteoblast differentiation in porous titanium alloys.Bioinformatics prediction analysis results showed that BMP4 was the target gene of miR-1187.However,it is still unclear whether LIPUS can regulate osteogenesis of MC3T3-E1 in porous titanium alloy scaffolds through the miR-1187/BMP4 pathway.To conclude,this study intends to investigate whether LIPUS promotes osteoblast osteogenic differentiation and new bone formation in scaffold materials by regulating miR-1187 / BMP4 pathway in vivo and in vitro,and preliminarily explore the molecular biological mechanism of LIPUS on bone growth in three-dimensional scaffolds,so as to provide a theoretical basis for the combined application of LIPUS and threedimensional scaffolds to repair large-area bone defects.Methods:(1)The RT-qPCR was applied to the determine the expression level of miR-1187 with or without LIPUS irradiation.The RT-qPCR and Western Blot were applied to determine the mRNA and protein level of osteogenic markers including COLI,ALP,RUNX2;MC3T3-E1 cells were transfected with miR-1187 mimics and inhibitor,and RT-qPCR,Western Blot,ALP activity,ALP staining and Alizarin red staining were applied to determine the effect of miR-1187 on the osteogenic differentiation function of MC3T3-E1 cells;To observe the effect of LIPUS on osteogenic differentiation when miR-1187 mimics was transfected,RT-qPCR and Western Blot experiments were applied to determine the mRNA and protein expression levels of MC3T3-E1 in scaffolds.(2)To evaluate the effect of LIPUS on the expression of BMP4,the RT-qPCR and Western Blot were applied to determine the mRNA and protein level of BMP4.RT-qPCR,Western Blot and double luciferase experiments were applied to prove the targeting relationship between miR-1187 and BMP4.Using si RNA-BMP4 and p CDNA3.1-AMP-BMP4 plasmids to downregulation and overexpression of BMP4 respectively,and RT-qPCR,Western Blot,ALP activity,ALP staining and Alizarin red staining were applied to determine the effect of BMP4 on the osteogenic differentiation function of MC3T3-E1 cells.Rescue experiments were applied to demonstrate that LIPUS promoted the osteogenic differentiation of MC3T3-E1 in scaffolds through miR-1187/BMP4 axis,miR-1187 mimics was transfected into MC3T3-E1 with or without LIPUS loading,cotransfecting MC3T3-E1 wtih miR-1187 mimics and p CDNA3.1-AMP-BMP4 plasmids,RT-qPCR,Western Blot and ALP activity were applied to determine the mRNA and protein level of osteogenic marker.(3)Critical bone defect was prepared in the anterior region of the mandible of the rats,and porous titanium alloys with the same specifications as the bone defect were implanted.The rats were randomly divided into the Control group,the LIPUS group,the si-BMP4 group and the si-BMP4+LIPUS group.The same volume of si-BMP4 or PBS was injected into the bone defect area of each group by local injection.After 4 and 8 weeks,Micro-CT,toluidine blue staining and Von Kossa silver staining were used to observe the growth and maturation of bone tissue in the scaffold;RT-qPCR was applied to determine the mRNA level of BMP4 as well as other osteogenic marker including Col1,Alp,Runx2 and Ocn.Results:(1)LIPUS significantly down-regulated the expression of miR-1187 while promoting the osteogenic differentiation of MC3T3-E1 on porous titanium alloys(P<0.05).Transfection of miR-1187 mimics significantly down-regulated the mRNA and protein expression levels of COL-1,ALP,RUNX2(P<0.05),and significantly inhibited the activity of ALP and matrix mineralization(P<0.05).While transfection of miR-1187 inhibitor possessed the opposite effect,indicating that miR-1187 could inhibit the osteogenic differentiation of MC3T3-E1.LIPUS loading can rescue the inhibition of osteogenic differentiation of MC3T3-E1 cells on scaffolds caused by miR-1187 mimics transfection.(2)LIPUS loading significantly up-regulated the expression of BMP4 at mRNA and protein expression levels(P<0.05).The results of luciferase assay showed that the fluorescence value of pmir GLO-BMP4 3’UTR(WT)+ miR-1187 mimics group was significantly lower than that of other groups(P<0.05),suggesting that miR-1187 can directly bind to the predicted binding site on BMP4,while transfection of miR-1187 mimics and inhibitor can significantly down-regulate and upregulate the expression of BMP4,respectively(P<0.05),miR-1187 has a targeted binding regulatory relationship with BMP4.Silencing and overexpression of BMP4 can significantly down-regulate and up-regulate the mRNA and protein expressions levels of ALP,COL-1,RUNX2,OCN respectively(P<0.05).Co-transfection of p CDNA3.1-AMP-BMP4 plasmids or LIPUS loading could rescue the inhibition of osteogenic differentiation of MC3T3-E1 on scaffolds caused by transfection of miR-1187 mimics(P<0.05).(3)At 4 w and 8 w,Micro-CT and hard tissue section staining histological observation showed that the si-BMP4 group had the least new bone formation,and the control group and LIPUS+ si-BMP4 group had better new bone formation than the si-BMP4 group,while the LIPUS group was better than the Control group and the si-BMP4+LIPUS group(P<0.05).Von Kossa silver staining results showed that the si-BMP4 group had the lowest IOD value of mineralized sediments at each time point.The IOD value of the Control group and the LIPUS+ si-BMP4 group was better than that of the si-BMP4 group,while the LIPUS group has the largest IOD value(P < 0.05).RT-qPCR results revealed that LIPUS could up-regulate the expression level of Bmp4 as well as related osteogenic markers including Col-1、Alp、Runx2 and Ocn;And interfered the expression level of Bmp4 gene in the bone defect area of rats by si RNA technique.could inhibit the gene expression levels of Col-1、Alp、Runx2 and Ocn.While LIPUS loading could rescue the inhibition of Col-1、Alp、Runx2 and Ocn gene expression levels in the bone tissue caused by si-BMP4 to a certain extent.Conclusion:(1)In vitro,miR-1187 negatively regulates the osteogenic differentiation of MC3T3-E1,LIPUS can promote the osteogenic differentiation of osteoblasts by down-regulating the expression of miR-1187.(2)In vitro,BMP4 promote the osteogenic differentiation of MC3T3-E1,miR-1187 negatively regulates the osteogenic differentiation of MC3T3-E1 by target BMP4;LIPUS can promote the osteogenic differentiation of MC3T3-E1 in scaffolds by regulating miR-1187 / BMP4 axis.(3)In vivo,LIPUS can promote the formation and maturation of new bone by regulating the expression of BMP4 and up-regulating the expression of osteogenesis-related genes in the scaffold material in the rat mandibular bone defect area. |
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