Objective:Using silk fibroin(SF)and gelatin(GN)as matrix materials,Sr HPO4(Sr P)-loaded porous SG scaffolds were prepared by freeze-drying and cross-linking methods.The physical and chemical properties of the scaffolds were characterized,and the effects of the scaffolds on the osteogenic differentiation ability of mouse bone marrow mesenchymal stem cells(m BMSCs)and the osteoclastic differentiation ability of RAW264.7 cells were examined,laying a foundation for the animal experiment of osteoporotic bone defect repair in vivo and provide theoretical basis for its clinical application.Method:(1)Groups and physicochemical properties of scaffolds.pure SG scaffolds,SG scaffolds with 5,10 and 15 wt.%Sr P/SG were labeled as 5Sr P/SG,10Sr P/SG and 15Sr P/SG scaffolds,respectively.Fourier transform infrared spectroscopy(FTIR)was used to determine the structure of the scaffold.Scanning electron microscopy(SEM)was used to observe the morphology of the scaffold.Dynamic mechanical testing machine was used to measure the mechanical properties of the scaffold,and the swelling behavior and degradation behavior of the scaffold were detected by immersing the scaffold in phosphate buffer(PBS).(2)The effect of scaffolds on the osteogenic differentiation of m BMSCs..Cells on Sr P/SG scaffolds were stained by Live-dead cell staining kit,and the cell survival was observed by fluorescence microscope.The proliferation of m BMSCs cultured on Sr P/SG scaffolds for 1,3 and 7 days was quantified by CCK-8 reagent.Alkaline phosphatase(ALP)activity was quantitatively evaluated by normalizing ALP content relative to total protein content,and the expression of osteogenic related genes was measured by quantitative real-time PCR(q PCR).(3)The effect of scaffolds on the osteoclastic differentiation of RAW264.7 cells.The expression of genes related to osteoclastogenesis was detected by q PCR,the activity of tartrate resistant acid phosphatase(TRAP)was quantitatively detected by TRAP assay kit and TRAP staining kit was used to stain the TRAP.Result:(1)SEM images show that all scaffolds have porous structure,most of which are about 100μM;FTIR showed that the main molecular groups of the scaffolds were hydrogen oxygen bond(-OH),amide I,II and III.The stress-strain curves show that the compressive strength of 5Srp/SG and 10Sr P/SG scaffolds is almost the same as that of SG scaffolds,and the compressive strength of 15Sr P/SG scaffolds is significantly higher than that of SG scaffolds;When the amount of Sr P is 10 wt.%and 15 wt.%,the water absorption radio of scaffolds decreases,while with the increase of Sr P content,the swelling radio of scaffolds increases.The addition of Sr P increased the weight loss rate.After 28 days,the weight loss rate of 15Sr P/SG scaffolds was significantly higher than that of SG scaffolds.(2)The fluorescent images of live dead cell staining showed that m BMSCs had good activity on all scaffolds.At 7 and 14 days,the number of cells on all Sr P/SG groups was significantly higher than that on SG groupwhile the ALP activity of 15Sr P/SG group was also significantly higher than that on SG group.The q PCR results showed that compared with other groups,the expression of ALP,Runx2,Col-I and OC genes in 15Sr P/SG group was higher.(3)When compared with SG extracts,the osteoclast like cells in Sr P/SG extracts were less and the TRAP activity in Sr P/SG extracts was also remarkably lower.The expression of TRAP,MMP9 and CTK genes in Sr P/SG extracts were significantly lower than those in SG extract.Conclusion:The SG composite porous scaffolds loaded with strontium have been successfully prepared,which have suitable pore size,degradation performance,sustained release of Sr,good cell compatibility,can promote cell osteogenic differentiation and inhibit cell osteoclastic differentiation.It provides a theoretical basis for the next step of the animal osteoporotic bone defect repair experiment. |