| Objective: The repair of large area or large segment bone defect has always been a hot issue in the medical field.Physical factors stimulating bone growth and repair has become one of the methods of bone defect treatment.Low intensity pulsed ultrasound(LIPUS)is a non-invasive physical therapy that propagates in the tissue in the mechanical form of high-frequency sound pressure wave.It has been approved by the U.S.Food and Drug Administration and can be used in clinical application to treat fresh fractures,delayed fracture healing and nonunion.Three-dimensional scaffold material plays a very important role in bone tissue engineering for the repair of large-area bone defects.It not only plays a supporting role and maintains the shape of the original tissue,but also plays a template role,providing a place for cells to live,grow,differentiate and proliferate,so as to guide the regeneration of damaged tissue and control the structure of regenerated tissue.Porous Ti6Al4 V is a kind of titanium alloy scaffold material with high strength and toughness.Its porous structure can provide space for the growth of bone tissue and provide a conduction channel for LIPUS.Through the regulation of its porosity,it can match the elastic modulus of bone tissue,and its corresponding bone conduction can promote the repair of bone tissue.Therefore,the combined application of LIPUS and Ti6Al4 V scaffolds has become a new idea for the repair of large-area bone defects.Cell migration is a physiological process of normal growth and development.The migration of osteoblasts and bone marrow mesenchymal stem cells is so important for the healing of fractures and bone defects.LIPUS can regulate the aggregation,migration and differentiation of osteoblasts,affect the secretion of related cytokines,and promote the healing of bone defects from different pathways.Our previous experimental results showed that LIPUS could promote the migration level of osteoblasts in porous titanium alloy scaffolds,and promote the growth of bone tissue and bone maturation in scaffolds.The results of full transcriptome sequencing analysis of osteoblasts on porous Ti6Al4 V scaffolds demonstrated that mmu-mi R-30c-1-3p was significantly down regulated under LIPUS irradiation.It was suggested that mi R-30c-1-3p may play a role in LIPUS promoting osteoblast migration and bone tissue growth on scaffolds.In order to better understand the function of mi R-30c-1-3p,bioinformatics prediction analysis results showed that RHOB was the target gene of mir-30c-1-3p and STAT3 was the transcriptional regulator of mi R-30c-1-3p.Studies showed that the high expression of RHOB was related to osteoblast migration,while STAT3 was an important regulator of cell migration and osteogenesis.Preliminary results also showed that RHOB was highly expressed,and STAT3 was lowly expressed under the irradiation of LIPUS.Therefore,we speculate that under the irradiation of LIPUS,the down-regulated transcription factor STAT3 inhibits the transcription level of mi R-30c-1-3p,antagonizes the function of its negative regulation target gene RHOB,increases the expression of RHOB,and then promotes the migration of osteoblasts and the growth depth of bone tissue on titanium alloy porous materials.In conclusion,this study intends to prove that LIPUS promotes osteoblast migration level and bone tissue growth depth in scaffold materials by regulating STAT/mi R-30c-1-3p / RHOB 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 three-dimensional scaffolds to repair large-area bone defects.Methods:1.Osteoblasts were inoculated on Ti6Al4 V scaffolds and randomly divided into ultrasound group and sham irradiated control group.RT-q PCR,Western blot and ELISA were used to evaluate the effects of LIPUS on the expression of migration related indexes SDF-1,TGFβ and CXCR4.The effects of LIPUS on the migration of osteoblasts on scaffolds were evaluated wound healing assays,Transwell assays and laser confocal microscope.2.RT-q PCR and Western blot were used to evaluate the effects of LIPUS on the expression of STAT3,mi R-30c-1-3p,RHOB of the STAT3 / mir-30c-1-3p / RHOB axis.STAT3,mi R-30c-1-3p and RHOB were transfected respectively,and TR-q PCR,Western blot and ELISA were used to evaluate the effects of STAT3,mi R-30c-1-3p and RHOB on the expression of downstream migration related indexes SDF-1,TGFβ and CXCR4.The effects of transfection of STAT3,mi R-30c-1-3p and RHOB on the migration of osteoblasts on scaffolds were evaluated by wound healing assays,Transwell assays and laser confocal microscope.RT-q PCR,Western blot and double luciferase experiments were used to prove the targeting relationship between STAT3 and mi R-30c-1-3p,and mi R-30c-1-3p and RHOB.Recovery experiments were applied to demonstrate that LIPUS promoted the migration of osteoblasts into scaffolds through STAT3 /mi R-30c-1-3p / RHOB axis.3.Ti6Al4 V scaffolds were implanted into the anterior teeth of the mandible of rats and randomly divided into LIPUS group and sham irradiation group.Mi R-30c-1-3p agomir and phosphate buffer solution were injected locally regularly,respectively.Hard tissue section staining was used to observe the bone growth and bone mature in the scaffold material.The RNA of bone tissue of new formation was extracted respectively,and the expression levels of Stat3,mi R-30c-1-3p,Rhob and downstream migration related genes Sdf-1,Tgfβ and Cxcr4 in bone tissue in scaffold were detected by RT-q PCR,so as to verify that the effects of LIPUS and mi R-30c-1-3p on gene expression levels of Rhob,Sdf-1,Tgfβ and Cxcr4 in cells and bone tissue growth and maturation in scaffold material in vivo.Results:1.The results of RT-q PCR,Western blot and ELISA showed that LIPUS could promote the expression of genes and proteins of SDF-1,TGFβ and CXCR4.The results of wound healing assays,Transwell assays and laser confocal microscope showed that LIPUS could promote the migration of osteoblasts in the scaffold.2.The results of RT-q PCR and Western blot showed that LIPUS promoted the expression of of RHOB and inhibit the expression of STAT3 and mi R-30c-1-3p.The results of RT-q PCR,Western blot,ELISA,wound healing assays,Transwell assays and laser confocal microscope showed that mi R-30c-1-3p mimics and RHOB si RNA inhibited the migration of osteoblasts in the scaffold and decreased the expression level of SDF-1,TGFβ and CXCR4,while STAT3 si RNA promoted the migration of osteoblasts in the scaffold and increased the expression level of SDF-1,TGFβ and CXCR4.The results of RT-q PCR,Western blot and double luciferase showed that STAT3 was a potential transcription factor of mi R-30c-1-3p,and mi R-30c-1-3p had a targeted binding regulatory relationship with RHOB.After osteoblast transfection,STAT3 /mi R-30c-1-3p / RHOB axis and the expression levels of downstream genes and proteins were restored by LIPUS and restorative experimens.3.Histological observation results showed that LIPUS promoted the formation of bone tissue and bone maturity in Ti6Al4 V scaffold in rat mandibular,while mi R-30c-1-3p agomir inhibited the formation of bone tissue and bone maturity,and LIPUS could reverse the inhibitory effect of mi R-30c-1-3p agomir on bone formation and bone maturity to a certain extent.The RT-q PCR results showed that LIPUS promoted the gene expression level of Rhob and inhibit the gene expression levels of Stat3 and mi R-30c-1-3p of STAT3 / mir-30c-1-3p / RHOB axis in new bone tissue in scaffolds.LIPUS promoted the gene expression levels of Sdf-1,Tgfβ and Cxcr4 in bone tissue of scaffold material,while mi R-30c-1-3p agomir inhibited the gene expression levels of Rhob,Sdf-1,Tgfβ and Cxcr4 in bone tissue.LIPUS restored the inhibitory effect of mi R-30c-1-3p agomir on the gene expression levels of Rhob,Sdf-1,Tgfβ and Cxcr4 in bone tissue to a certain extent.Conclusion:1.LIPUS can promote the migration of osteoblasts in scaffold materials and the expression level of SDF-1,TGFβ and CXCR4.2.In vitro,LIPUS can promote the migration of osteoblasts in scaffolds and the expression level of SDF-1,TGFβ and CXCR4 by regulating STAT3 / mi R-30c-1-3p /RHOB axis.3.In vivo,LIPUS may promote the bone growth and bone mature in scaffolds and gene expression levels of migration related indicators in anterior mandibular area of rat by down regulating the expression level of mi R-30c-1-3p. |
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