| Objective Due to local high sugar,diabetic wound is easy to induce bacterial infection,wound healing is difficult,and it can lead to gangrene,seriously,amputation and so on.In recent years,stem cell injection therapy has achieved some success in tissue and organ repair,but for patients with surface injury,stem cell injection therapy can cause low local cell retention rate and survival rate,and the repair effect is not good.In this study,4D chitosan-based thermosensitive hydrogel(4D-CTH)was prepared by using 4D bioprinting technology,which was coated with adipose-derived mesenchymal stem cell(ADSCs).pi RNA-hsa-32182 regulate the growth and migration,and evaluate the repair effect of combined treatment on diabetic wounds,aiming to provide a new treatment method for diabetic wound healing.Methods High-throughput sequencing technology was used to screen pi RNA-hsa-32182,which was differentially expressed in the skin samples of patients with diabetic foot ulcer and healthy people,and quantitative Real-Time PCR(q RT-PCR)verified the differential expression of pi RNA-hsa-32182 in human skin samples and human keratinocytes(Ha Ca T cells)treated with high glucose.pi RNA-hsa-32182 agomir and antagomir were designed and synthesized.The effects of knockdown and overexpression of pi RNA-hsa-32182 on cell growth and migration were verified by cell scratch assay under high glucose environment.The primary ADSCs of mice were extracted by enzyme digestion method,and the cell surface marker molecules were identified by flow cytometry.The multidirection differentiation ability of these stem cells was verified by the adipogenic and osteogenic differentiation experiments.4D-CTH with uniform pore size and adjustable shape was prepared by 4D bioprinting technology and co-cultured with ADSCs.The cell compatibility of 4D-CTH was observed by AM/PI staining and fluorescence scratch assay.Diabetic mice skin injury model was established and divided into PBS group,4D-CTH group,ADSCs group,pi RNA-hsa-32182 antagomir group and 4D-CTH+ADSCs+pi RNAhsa-32182 antagomir combined group.The wound healing effect was observed at 0,4,7and 14 days respectively.HE staining,Masson’s trichrome staining and Sirius red staining were used to evaluate the degree of epithelialization,collagen deposition and maturation at the wound site,and to evaluate the wound healing effect.The expressions of Ki67,CD31 and α-SMA at the healing site were detected by immunofluorescence assay and immunohistochemical assay,respectively,to explore the mechanism of promoting healing.Results(1)The expression of pi RNA-hsa-32182 was up-regulated in diabetic skin samples and high-glucose treated Ha Ca T cells(P<0.05),and treatment with pi RNA-hsa-32182 antagomir significantly promoted cell growth and migration(P<0.05).(2)The extracted ADSCs adhered to the wall and showed a typical long spindle shape.The results of cell surface markers identified by flow cytometry showed that CD44 and CD90 were positive,leukocyte marker CD45 was negative,macrophage marker CD11 b was negative,and homologous controls Ig G2 a and Ig G2 b were negative,which was consistent with the immunophenotype of adipose-derived mesenchymal stem cells.The results of adipogenic differentiation and oil red O showed the presence of red round lipid droplets in the cells,and the results of osteogenic differentiation and alizarin red staining showed the presence of red calcium nodules in the cells,suggesting that stem cells had the ability of multidirection differentiation.(3)Fluorescence scratch test showed that cell mobility in the4D-CTH group at 12 hours and 24 hours was 31.84% and 50.32%,and that in the control group was 26.96% and 46.15%,showing no statistical difference(P>0.05),indicating that4D-CTH vector did not affect cell migration and growth;AM/PI results showed that ADSCs and 4D-CTH co-cultured for 24,48 and 72 h,cells in 4D-CTH group adhered well,ADSCs showed a typical long spindle shape,and the cell activity was above 90%,showing no statistical difference compared with the control group(P>0.05),indicating that 4D-CTH has no toxicity and good cytocompatibility.(4)The results of animal experiments showed that compared with PBS,4D-CTH,ADSCs,pi RNA-hsa-32182 antagomir group,the wound area of the 4D-CTH +ADSCs +pi RNA-hsa-32182 antagomir combined group was significantly reduced(P<0.05).The results of HE staining,Masson’s trichrome staining and Sirius red staining showed that it increased epithelial thickness,collagen deposition and maturation.(5)The results of immunofluorescence and immunohistochemistry showed that the 4D-CTH+ADSCs+pi RNA antagomir combined group could significantly increase the expressions of Ki67,CD31 and α-SMA(P<0.05),which confirmed that the 4D-CTH +ADSCs + pi RNA-hsa-32182 antagomir combined group could accelerate the healing of diabetic wound mainly through promoting cell proliferation and migration,promoting angiogenesis.Conclusion 4D printed stem cell carrier combined with pi RNA-hsa-32182 group can significantly accelerate diabetic skin wound healing,which mainly plays a healing promoting effect by increasing epithelial thickness,collagen deposition and maturation,promoting cell proliferation and migration,and promoting angiogenesis. |
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