Pathological Study Of Diabetic Wound Healing With Mesenchymal Stem Cells Carried By Three-dimensional Hydrogel Scaffolds

ObjectiveThe growth of adipose-derived stem cells(ADSCs)on Gelatin Methacrylate(GelMA A)hydrogel scaffolds was studied.The ADSCs-loaded three-dimensional(3D)-printed GelMA A hydrogel scaffolds were implanted into the back wound skin of diabetic rats to observe the systemic histopathological changes during the wound healing,and to explore the mechanism of promoting diabetic wound healing,providing new ideas and theoretical support for the treatment of refractory wounds.MethodsThe primary ADSCs were cultured for 3rd-5th generation and then added to the surface of the scaffold.After 3 days,the growth of the cells on the scaffold was observed by scanning electron microscope.The diabetic rat model was established by intraperitoneal injection of Streptozocin(STZ),and the blood glucose was measured by tail blood collection.Forty-five diabetic rats were divided into three groups: control group,GelMA A scaffold group and ADSCs-loaded GelMA A scaffold group.The diabetic rats were killed on the 7th,14 th and 21 st day respectively.The wound healing of the three groups was observed by naked eye.Wound photos were taken and Image J was used to analyze the wound healing rate of the rats.Hematoxylin-eosin(H&E)staining was used to detect epithelialization and granulation tissue formation,as well as subcutaneous rebleeding after epithelial healing and calcification.Masson’s trichrome staining was used to detect collagen fibers and angiogenesis.The expression of Vascular endothelial growth factor growth factor(VEGF)and transforming growth factor β-1(TGF β-1)was detected by immunohistochemistry.Results(1)Scanning electron microscopy showed that ADSCs grew well in culture within the scaffold,mainly in a round or oval shape,with a few short protrusions.GelMA A hydrogel scaffold could promote the proliferation and adhesion of ADSCs.(2)Image J analysis showed that wound healing was faster in rats treated with ADSCs-loaded GelMA A scaffolds on day 14 than in control group and in the scaffold groups(P<0.001,P<0.01),the wound closure rate was higher in the ADSCs-GelMA A group at day 21 compared with the control and scaffold groups(P<0.01,P<0.05),suggesting that the use of ADSCs-loaded GelMA A scaffolds can promote wound contraction.(3)HE staining showed that on day 14,the length and thickness of the new epithelium in the ADSCs-GelMA A group were significantly increased compared with the control group(P<0.0001);at day 7,the thickness of granulation tissue in the ADSCs-GelMA A group was higher than that in the scaffold group and the control group(P<0.01),and at day14,the thickness of granulation tissue in the ADSCs-loaded scaffold group was significantly higher than that in the control group(P<0.001)and also higher than that in the scaffold group(P<0.01),which indicated that the ADSCs-loaded GelMA A scaffold could promote the differentiation and maturation of wound epidermis and the obvious proliferation of granulation tissue.(4)Masson staining showed that the deposition of collagen fibers in ADSCs-GelMA A group was significantly higher than that in control group at day 14 and 21(P<0.01);On day 7 after surgery,capillary density in ADSCs-loaded scaffold group was significantly higher than that in control group(P<0.0001),also higher than the scaffold group(P<0.05),indicating that ADSCs-loaded GelMA A scaffold can promote neovascularization as well as collagen synthesis and deposition in large amounts.(5)Immunohistochemical staining showed differences in VEGF immunohistochemical scores between the ADSCs-GelMA A group and the control group at day 7 and 14(P<0.001,P<0.05),the number of TGFβ-1 positive endothelial cells in the scaffold group was significantly higher than that in the control group(P<0.0001)and the scaffold group(P<0.001)on the 7th day,suggesting that GelMA A hydrogel scaffolds may promote the formation and maturation of endothelial capillary by promoting the expression of endothelial VEGF and TGFβ-1.(6)The results of HE staining showed that the scaffold may reduce calcium salt deposition during wound healing.On day 14,similar foreign body granulomatous reactions were observed in all groups.At this time,the number of multi-nucleated macrophages in the ADSCs-loaded GelMA A group was significantly higher than that in the control group(P<0.0001)and GelMA A group(P<0.001),suggesting that the ADSCs-loaded GelMA A scaffold promoted local chronic granulomatous inflammation of the wound,in which TGFβ-1 may play an important role.(7)On 21 day after surgery,almost all capillaries in the control group reappeared bleeding,and the bleeding in the scaffold group was significantly reduced,while that in the ADSCs-GelMA A group was even less,indicating that the scaffold and ADSCs could promote better wound healing by promoting the maturation of capillaries.ConclusionThe use of GelMA A hydrogel loaded with ADSCs contributed to faster and more mature wound healing in diabetic rats.It accelerates wound contraction,regenerative epidermis formation,promotes fibrogenesis,collagen deposition,and angiogenesis and maturation,and may promote wound healing through calcification and chronic granuloma formation.