| Objective: Supercritical large area bone defects are still a problem in clinical treatment,due to the existence of limitations of autograft and allograft bone,researchers committed to the development of artificial bone material,but in order to solve the adverse effect of artificial bone material,people pay attention to the new treatment of physical therapy combined with artificial bone material.Low-intensity pulsed ultrasound stimulation(LIPUS)has been recognized as a new physical therapy in recent years,which can promote bone healing and repair.Because the effect of LIPUS on bone healing and the effect of LIPUS on the exchange of material in three-dimensional porous material.In recent years,LIPUS is used as an auxiliary treatment in combination with artificial bone materials for bone defect repair.It provides a new idea for artificial bone repair of large bone defects.However,the study of the appropriate parameters(including strength,frequency,time,etc.)on the effect of LIPUS on the osteogenesis of three-dimensional porous materials is rarely reported.Studies have confirmed that the mechanism of LIPUS on osteoblasts is regulated by integrin alpha 5 beta 1/PI3K/Akt pathway,ROCK-Cot/Tpl2-MEK signaling pathway and m-TOR pathway.The mechanism of the above study is based on the two-dimensional space,and the mechanism based on the two-dimensional space cannot be completely used to explain the experimental results on the three-dimensional porous material.Therefore,it is necessary to explore the mechanism of the effect of LIPUS on the osteogenesis of three-dimensional porous materials.In recent years,studies have confirmed that a variety of miRNA can participate in the different process of osteoblasts.However,the relationship between miRNA and LIPUS on osteogenesis on porous materials has not been reported.So my research goals are as follows: 1?In vitro experiments of two frequencies LIPUS were carried out,which observed the effects of two different frequencies LIPUS on the proliferation and differentiation of osteoblast in vitro on porous scaffolds,screening optimal frequency.To provide data for LIPUS promote the three-dimensional porous material for repairing bone defect.2?Comparison of the effect of two frequencies LIPUS on porous material repairing rabbit mandibular bone defect through in vivo study,screening optimal frequency.To provide data for the optimal parameters of the study of LIPUS to three-dimensional scaffolds repair bone defect.3 ? Through detect the micro RNA expression profile responsive of the effect of LIPUS on osteoblast on three-dimensional porous material.It is a preliminary study on the changes of miRNA of osteoblasts on the three-dimensional porous material under the LIPUS stimulation.Molecular biological data were provided to explore the mechanism of LIPUS promoting osteogenesis on three-dimensional porous material.Methods: 1 ? The porous titanium alloy materials were randomly divided into high-frequency ultrasound group,low-frequency ultrasound group and control group.Mouse embryo osteoblast precursor cells(MC3T3-E1)were inoculated and cultured on the scaffolds.The adhesion status of osteoblasts on the scaffolds was evaluated by scanning electron microscopy(SEM).Cell counting kit-8(CCK-8)and flow cytometry PI staining were used to detect the effect of two frequencies LIPUS on the proliferation of osteoblasts.The effect of two frequencies LIPUS on the early and late differentiation of osteoblasts were determined by alkaline phosphatase(ALP)and osteocalcin(OCN)detection techniques.2?The porous titanium alloy scaffolds were implanted into the area of bone defect of rabbit mandible,then were randomly divided into high-frequency ultrasound group,low-frequency ultrasound group and control group,the X-ray examination of infection was examined after 3 weeks and 6 weeks.The new bone formation was observed in the different group by Micro-CT.Hard tissue sections were made,the active degree of new bone formation of samples was observed by fluorescence microscopy.The tissue sections were stained with toluidine blue,methylene blue and acid fuchsin staining and von Kossa silver staining,then to analyse new bone formation and bone mineralization in different group.3?The porous titanium alloy scaffolds and MC3T3-E1 cells were co-culture,then were randomly divided into ultrasound group and control group,after 7 days LIPUS stimulation,detected the differential expression of miRNA in osteoblast on three-dimensional culture.And the target genes of the differential expression of miRNA were predicted,the function clustering analysis of GO and KEGG Pathway were proceeded through bio-informatics analysis.Then the reliability of the sequencing results was validated by real-time PCR.Results: 1?SEM showed that MC3T3-E1 cells stretch well on porous titanium alloy materials,visible pseudopodia,cells connected by pseudopodia,boundaries fuzzy,and the cells grew into the scaffold voids,but the density and morphology of cells had no significant difference among the three groups.CCK-8 results revealed that the number of cells was increased with the culture time,but no significant difference among the three groups.After PI staining with 3 days,there was no statistical difference between them.The results of ALP displayed that ALP activity increased with the time increasing,ultrasound group had significant difference in 4,7,10 days compared of control group.There was no statistically significant difference between the two frequencies groups.OCN results showed that the content of OCN was increased with the increase of culture time.At 10,14 days,there were significant difference between ultrasound group and control group.There was no statistically significant difference between the two frequencies groups.2?X-ray images showed no signs of infection at all times,and the bone was tightly integrated with scaffolds.Micro-CT scan revealed that with the extension of time,the amount of new bone in the scaffolds gradually increased.At the same time point,the new bone formation in the ultrasound group was better than that in the control group,but there was no significant difference between the two ultrasonic group.Fluorescence microscopy demonstrated that with the time increasing,the fluorescence band width and fluorescence intensity decreased gradually,but the fluorescence distribution area expanded gradually.At the same time point,the fluorescence band width,fluorescence intensity and fluorescence distribution area of ultrasound group were better than those of the control group,but there was no significant difference between the two ultrasonic groups.Toluidine blue,methylene blue and acid fuchsin staining results revealed that the area of new bone formation increased with the time increasing,bone structure is more and more mature.At the same time point,the new bone formation area and bone maturity of the ultrasound group were superior to the control group,and there was no significant difference between the two ultrasonic groups.With the increase of time,the area and density of mineral deposition in von Kossa staining increased gradually.At the same time point,the amount of mineral deposits in the ultrasound group was better than those in the control group,but there was no significant difference between the two ultrasonic groups.3?LIPUS can down regulate the expression of miRNA in the osteoblasts on porous titanium alloy scaffolds,and there were 30 miRNAs.The target genes of down regulated miRNAs were predicted,and the target genes were analyzed by GO and KEGG.599 GO terms and 185 signal pathways were identified.Real-time PCR validated expression of 4 miRNAs,and the results were consistent with the sequencing results,indicating that the sequencing results were true and reliable.Conclusions: 1?Two frequencies LIPUS has no obvious effect on the proliferation of osteoblasts on porous titanium alloy scaffold,LIPUS can significantly promote cell differentiation,but there is no significant difference between the two frequencies groups.2?Two frequencies LPIUS can significantly promote the formation of new bone,but there is no significant difference between the two frequencies groups.3? LIPUS can down regulate the expression of miRNA in osteoblast on the porous titanium alloy scaffolds,thus provide valuable experimental data for the later verification of the functional mechanism of miRNA. |
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