Establishment Of A Rat Model Of Subcutaneous Ectopic Osteogenesis Promoted By Low-intensity Pulsed Ultrasound Stimulation

Objective:Our previous studies demonstrated that low-intensity pulsed ultrasound stimulation(LIPUS)could promote the migratory outgrowth and differentiation of MC3T3-E1 cells within porous titanium alloy scaffolds(Ti-6Al-4V)and promote osteogenesis.To elucidate its mechanism,it is essential to establish effective in vitro cell models and animal models.In this study,we created an animal model and screened the suitable implantation mode by implanting MC3T3-E1 cells with porous titanium alloy scaffolds material subcutaneously in rats,which lays the foundation for further study of the mechanism of LIPUS promoting osteogenesis in rat subcutaneous MC3T3-E1 cells and porous titanium alloy materials in vivo.Methods:(1)MC3T3-E1 cells were co-cultured with porous titanium alloy constructs,CCK8(cell counting kit-8,CCK-8)was used to analyze cell viability and choose the appropriate cell density.(2)DAPI staining was used to visualize the distribution of cells(the density of cells screened)growing on the material.(3)After implanting of the porous titanium alloy co-culture complex(co-culture group),simple material(simple material group),cell suspension and porous titanium alloy(suspension group)into the subcutaneous sac of rats,they were randomly divided into LIPUS and control groups,injected tetracycline at 6 w after intervention,sacrificed at 7 w,the samples were fixed embedded sections and stained.The results of tetracycline staining were observed under fluorescence microscope,and the calcium salt deposition was evaluated.The results of toluidine blue staining were observed under light microscope and the osteogenesis was evaluated.Results:(1)CCK8 results showed good cell viability within the composites(The screening density and volume were:1×10~6,5ul);(2)DAPI staining showed good cell growth and distribution on the materials;(3)The results of the experiments in vivo:fluorescence microscopy results showed calcium salt deposition inside the materials,and the LIPUS group had a higher fluorescence labeling rate than the control group(p<0.05),and in the LIPUS group,the co-culture group had a higher fluorescence labeling rate than the suspension group(P<0.05),the results of toluene blue staining showed that the LIPUS group had new bone formation,and the co-cultured group had a larger bone area than the suspension group(P<0.05).Conclusions:In this study,a rat model of subcutaneous heterotopic osteogenesis promoted by LIPUS was established.Under the limited conditions of this study,LIPUS could promote calcium salt deposition and subcutaneous heterotopic osteogenesis in rats,and the amount of osteogenesis was statistically different between the two implantation modes,and the co-cultured group had a better bone formation effect.