| PARTⅠPREPARATION,CHARACTERIZATION AND BIOSAFETY EVALUATION OF cRGD-NBsObjective:To prepare cyclic arginine-glycine-aspartic acid-modified nanoscale lipid bubbles（cRGD-NBs）and normal nanoscale lipid bubbles（NBs）,investigate their basic physicochemical properties and test their biosafety,and evaluate whether the former can actively target bone marrow mesenchymal stem cells（BMSCs）.Methods:Different types of phospholipid film-forming materials wrapped with perfluoropropane（C3F8）inert gas were used to prepare cRGD-NBs and NBs,and their size,morphology and distribution were observed under light microscopy.The morphology was further observed under transmission electron microscope（TEM）.When the materials were fabricated at 6 h,24 h and 48 h,their distribution under light microscopy was observed to assess the stability of the materials.The particle size and the surface potential of the materials were then examined by dynamic light scattering technology.The concentrations of cRGD-NBs and NBs were detected by hemocytometer.The chemical bonds of cRGD-NBs and NBs were analyzed by X-ray photoelectron spectroscopy（XPS）.cRGD-NBs or NBs were labeled by Dil labeling method and incubated with BMSCs for90 min,followed by laser confocal microscopy to observe the targeting ability of both materials.The cRGD-NBs or NBs were added to the BMSCs and co-incubated with the cells every day.After 1d,2d and 3d,the proliferation of each group of cells was detected by CCK-8 method to evaluate their biosafety.Results:After the preparation of cRGD-NBs and NBs,both nanobubbles showed regular morphology and good dispersion under light microscope;both of them appeared as round vesicles by TEM.The two types of nanobubbles were found to be stable within 6 h after fabrication when observed under light microscope at different time points.The average particle sizes of cRGD-NBs and NBs were subsequently detected to be577.5±102.2 nm and 441.8±69.44 nm,respectively;the surface potentials were-12.6±0.9 mV and-10.4±1.5 mV,respectively.Their concentration was about 1×109/m L as measured by hemocytometer.XPS analysis revealed that the atomic percentages of functional groups such as C=O/N-O（O1s spectrum）and C-N（N1s spectrum）on cRGD-NBs were significantly higher than those on NBs,which was consistent with the chemical structures of the raw materials used for their preparation.The Dil labeling method verified the good targeting ability of cRGD-NBs.Both cRGD-NBs and NBs showed no significant effect on cell proliferation after incubation with BMSCs for different times.Conclusions:cRGD-NBs and NBs were successfully prepared with uniform size and good dispersion,and both had good biosafety.The former showed good ability to target BMSCs.PART II LIPUS COMBINED WITH cRGD-NBs PROMOTES OSTEOGENIC DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLSObjective: To evaluate the biosafety of low intensity pulsed ultrasound（LIPUS）in combination with cRGD-NBs and to determine whether LIPUS/cRGD-NBs can enhance the pro-osteogenesis ability of LIPUS.Methods: After extracting mouse BMSCs,3-6 passages of cells were taken and divided into Control group and LIPUS group,and cultured in basal medium and osteogenic induction medium.Alkaline phosphatase（ALP）staining and ALP activity detection were performed in each group after treatment for corresponding days to evaluate the differences in early osteogenic differentiation,and western blot（WB）was used to detect the protein expressions of osteogenesis-related genes in each group.The cells were then divided into four groups（Control group,LIPUS group,LIPUS+NBs group,LIPUS+cRGD-NBs group）,all of which were cultured under osteogenic induction conditions.Live/dead cell staining was performed to detect the cell viability of each group after 3 days of treatment;flow cytometry（FCM）and EdU method were used to assess the apoptosis rate and the percentage of cell proliferation in each group after 3 days of treatment,respectively.ALP staining and WB detection were performed after treatment in each group for the corresponding days;the protein expression of runt-related transcription factor 2（RUNX2）,a marker gene related to osteogenic differentiation,was observed by immunofluorescence staining.Results: Under osteogenic culture conditions,the ALP-positive staining area,ALP activity and the protein expressions of osteogenesisrelated indicators such as osteopontin（OPN）,osteocalcin（OCN）,collagenⅠ（COLⅠ）and RUNX2 were significantly higher in the LIPUS group than in the Control group;there was no significant difference in osteogenesisrelated indexes between the two groups under normal culture conditions.After LIPUS combined with cRGD-NBs or NBs,the cells in each group remained viable（the proportion of dead cells was about 1%）,and there was no statistical difference in apoptosis rate and proliferation percentage.Compared with the LIPUS+NBs group,the LIPUS+cRGD-NBs group showed a significant increase in ALP staining and protein expressions of osteogenic differentiation-related genes（OPN,COLⅠ,RUNX2）and transient receptor potential melastatin 7（TRPM7）,while there was no statistical difference in the above indexes between the LIPUS+NBs group and the LIPUS group.Immunofluorescence detection of RUNX2 protein expression in each group showed the same trend as WB.Conclusions: LIPUS can promote the osteogenic differentiation of BMSCs under osteogenic induction conditions;LIPUS in combination with cRGD-NBs or NBs has good biosafety;LIPUS/cRGD-NBs can enhance the pro-osteogenesis ability of LIPUS.PART III ROLE OF CYTOSKELETAL MICROFILAMENTS IN THE PROMOTION OF OSTEOGENIC DIFFERENTIATION BY LIPUS/cRGD-NBsObjective: To observe the changes of cytoskeletal microfilaments after the effect of LIPUS combined with cRGD-NBs and the effect of interfering cytoskeletal microfilaments on the pro-osteogenesis ability of LIPUS/cRGD-NBs.Methods: BMSCs were divided into four groups（Control group,cRGD-NBs group,LIPUS group,LIPUS+cRGD-NBs group）,and microfilaments green fluorescent probe staining was performed at 0 h,0.5 h,2 h and 4 h after treatment in each group,and the morphology and fluorescence intensity of microfilaments in each group were observed under confocal microscope.The protein expressions of intracellular filamentous actin（F-actin）and globular actin（G-actin）were detected by WB.The cells were then divided into four groups: Control group,Dimethyl sulfoxide（DMSO）group,Jasplakinolide（JA）group,Cytochalasin D（Cyto D）group,and each group was treated with LIPUS/cRGD-NBs.The protein expressions of F-actin and G-actin were measured by WB;ALP staining was used to evaluate the differences in early osteogenic differentiation and WB was used to analyze the protein expressions of osteogenesis-related indicators（RUNX2,OPN,OCN）and TRPM7 in each group;immunofluorescence staining was used to observe RUNX2 protein expression in each group.Results: Compared with the Control group,the intracellular green dots were significantly increased and clustered around the nucleus at 2 h after LIPUS treatment,and the number of filamentous F-actin was significantly reduced;at 4 h after treatment,the number of filamentous Factin increased significantly and the fluorescence intensity increased,but there were still a few spots in the cells.The LIPUS+cRGD-NBs group showed enhanced fluorescence intensity and more obvious filamentous substances at all time points compared with the LIPUS group（especially at4 h）.By evaluating the F-actin/G-actin protein expression ratio at the above time points in each group,it was found that compared with the Control group,the ratio of the LIPUS group had no significant change at 0 h,decreased after 0.5 h,decreased more significantly after 2 h,and increased significantly after 4 h;the LIPUS+cRGD-NBs group exhibited elevated ratios at all time points compared with the LIPUS group（especially at 4 h）.After the action of cytoskeleton interfering drug JA（actin polymerizer）or Cyto D（actin depolymerizer）,the F-actin/G-actin ratio showed a significant increase and decrease,respectively,compared with the DMSO group;ALP-positive staining as well as protein expressions of osteogenesis-related indicators（RUNX2,OPN,OCN）and TRPM7 were significantly increased in the JA group,whereas the Cyto D group showed a significant decrease,all compared with the DMSO group.Immunofluorescence analysis of RUNX2 protein expression in each group revealed the same trend as WB.Conclusions: During the osteogenic differentiation of BMSCs promoted by LIPUS,microfilaments underwent transient depolymerization and rearrangement and then showed increased polymerization;in the process of pro-osteogenesis of LIPUS/cRGD-NBs,the microfilaments polymerization was further enhanced;and promoting the polymerization of microfilaments can enhance the pro-osteogenesis ability of LIPUS/cRGDNBs.PART IV ROLE OF TRPM7-MEDIATED INTRACELLULAR CALCIUM OSCILLATION AND COMBINED MICROFILAMENTS IN THE PROMOTION OF OSTEOGENIC DIFFERENTIATION BY LIPUS/cRGDNBsObjective: To explore whether the mechanosensitive ion channel TRPM7 plays a key role in the pro-osteogenesis by LIPUS/cRGD-NBs and whether TRPM7 is also associated with intracellular calcium signaling and actin cytoskeleton during this process.Methods: BMSCs were divided into four groups（Control group,cRGD-NBs group,LIPUS group,LIPUS+cRGD-NBs group）,and the protein expressions of osteogenesis-related indicators（COLⅠ,RUNX2,OPN,OCN）and TRPM7 in each group were detected by WB;TRPM7protein expression was observed in each group by immunofluorescence;the expression of TRPM7 in membrane and cytoplasmic proteins of each group was detected using membrane and cytoplasmic protein extraction kit.The cells were then divided into five groups（Control group,LIPUS group,LIPUS+cRGD-NBs group,LIPUS+cRGD-NBs+NC group,LIPUS+cRGDNBs+siTRPM7 group）,and ALP staining was performed to assess the early osteogenic ability and WB was used to analyze the protein expressions of osteogenesis-related indicators and TRPM7 in each group;immunofluorescence staining was performed to observe RUNX2 protein expression in each group.Calcium ion fluorescent probe Fluo-4 AM was used to stain cells to assess the effect of each group of treatments on the intracellular calcium ion concentration.The effects of knockdown of TRPM7 on cytoskeletal microfilaments morphology and the protein expressions of F-actin and G-actin were analyzed by fluorescent staining and WB;the effect of cytoskeleton interference drug JA or Cyto D on the fluorescence intensity of TRPM7 under LIPUS/cRGD-NBs treatment was observed by fluorescent staining.Results: After LIPUS treatment,the protein expressions of TRPM7 and osteogenesis-related indicators were significantly increased compared with those in the Control group,while those in the LIPUS+cRGD-NBs group were significantly higher than those in the LIPUS group;immunofluorescence analysis revealed that the fluorescence intensity of TRPM7 was significantly enhanced after LIPUS action,while the LIPUS+cRGD-NBs group showed stronger fluorescence;the expression of TRPM7 in membrane and cytoplasmic proteins of each group also showed the same trend as above.Compared with the LIPUS+cRGD-NBs+NC group,the ALP-positive staining area as well as protein expressions of osteogenesis-related indicators and TRPM7 were significantly reduced in the LIPUS+cRGD-NBs+siTRPM7 group;immunofluorescence detection of RUNX2 protein expression revealed the same trend as above.It was found by Fluo-4 AM that knockdown of TRPM7 significantly inhibited the increase of intracellular calcium ion concentration under the action of LIPUS/cRGD-NBs.Compared with the LIPUS+cRGD-NBs+NC group,the TRPM7 fluorescence expression in the LIPUS+cRGD-NBs+siTRPM7 group was significantly decreased,accompanied by a significant decrease in the number and fluorescence intensity of F-actin,and the ratio of Factin/G-actin decreased significantly.It was found by fluorescent staining that F-actin increased and thickened after the action of JA,and the fluorescence intensity of TRPM7 was significantly enhanced,but Cyto D showed the opposite results after action.Conclusions: The mechanosensitive ion channel TRPM7 may play a key regulatory role in the pro-osteogenic differentiation of LIPUS/cRGDNBs and mediate the increase of intracellular calcium ion concentration;in this process,TRPM7 and actin cytoskeleton may regulate each other.PART V THE EFFECT OF UP-REGULATION OF TGFβ1ON THE OSTEOGENIC DIFFERENTIATION EFFECT OF LIPUS/cRGD-NBsObjective: To clarify whether the expression of transforming growth factor beta1（TGFβ1）changes during the pro-osteogenic differentiation of LIPUS/cRGD-NBs,and to explore the effects of overexpression of TGFβ1on the osteogenic differentiation effect of LIPUS/cRGD-NBs and the osteogenesis induced by bone morphogenetic protein 9（BMP9）.Methods: BMSCs were divided into four groups（Control group,cRGD-NBs group,LIPUS group,LIPUS+cRGD-NBs group）,and TGFβ1protein expression was detected in each group by WB.C3H10T1/2 cells were infected with recombinant adenovirus encoding TGFβ1 and red fluorescent protein（RFP）to overexpress TGFβ1,and the positive cells were observed under fluorescence microscope at different titers of adenovirus to clarify the transfection efficiency of each group;real-time fluorescence quantitative polymerase chain reaction（RT-PCR）and enzyme-linked immunosorbent assay（ELISA）were used to detect the m RNA expression level of TGFβ1 and the concentration of TGFβ1 in the supernatant under different titer treatments to determine the appropriate transfection efficiency.The effect of overexpression of TGFβ1 on protein expressions of TGFβ1,TRPM7 and osteogenesis-related indicators under the action of LIPUS/cRGD-NBs was detected by WB;further analysis of whether overexpression of TGFβ1 affects BMP9-mediated protein levels of osteogenesis-related indicators and the TGFβ/Smad canonical osteogenic signaling pathway by using WB.Results: TGFβ1 protein expression was significantly increased after LIPUS treatment and further increased after LIPUS/cRGD-NBs treatment.The transfection efficiency of TGFβ1 recombinant adenovirus should be about 1% in follow-up experiments.After overexpression of TGFβ1,the protein levels of osteogenesis-related genes under the action of LIPUS/cRGD-NBs were significantly up-regulated,accompanied by a significant increase in the levels of TRPM7 and Smad2.After up-regulation of TGFβ1,the protein levels of BMP9-mediated osteogenic differentiationrelated genes and Smad2 were significantly increased,but there was no significant difference in Smad1,while Smad4 was significantly inhibited.Conclusions: The expression of TGFβ1 is up-regulated during LIPUS/cRGD-NBs promotion of osteogenic differentiation;overexpression of TGFβ1 may enhance the osteogenesis effect of LIPUS/cRGD-NBs;upregulation of TGFβ1 may synergize with the osteogenic differentiation effect induced by BMP9. |
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