مرضیه رنجبر محمدی

Introduction and objectives: Diabetic ulcers are among the most common complications of diabetes mellitus which involves approximately 10-25% of diabetics. In this study, we have designed, characterized, and evaluated the effects of scaffolds composed of electrospun nanofibers and decellularized amnion membrane hydrogel containing cobalt- and copper-doped bioactive glasses to improve diabetic wound healing in male Wistar rats. Materials and methods: In the present study, extracted silk fibroin (SF) from the silk cocoon and isolated collagen (Col) from the rat tail tendon along with polyvinyl alcohol (PVA) and chitosan (CS) (SF-Col/PVA-CS) were used to fabricate electrospun nanofibers, in which vitamin D3 and cerium oxide nanoparticles (CeNPs) were utilized as additives. The prepared nanofibers were characterized by SEM, FTIR, Tensile test, contact angle, swelling, weight loss, release profile of vitamin D3 and CeNPs MTT, scratch assay (cell migration), cell adhesion, and blood compatibility. Cobalt and copper-doped mesoporous borate bioactive glasses (MBGs) were made by sol-gel method and characterized by SEM, MTT, blood compatibility, cell adhesion, scratch assay, chorioallantoic membrane (CAM assay), and antibacterial activity. The human amniotic membrane (HAM) was decellularized both physically and chemically. The efficiency of decellularization was assessed by DNA extraction and quantification, DAPI staining, analysis of hydroxyproline and collagen content, as well as tissue staining. After that, 20% MBGs was added to decellularized HAM (dHAM) hydrogel and characterized using FTIR, swelling, weight loss, protein release profile, cobalt, and copper ions release, gelling time, rheological investigation, MTT, and blood compatibility. Finally, a diabetic ulcer model was created on type 2 diabetic male Wistar rats in four experimental groups including (1) diabetic ulcer without treatment; (2) diabetic ulcer treated with dHAM hydrogel; (3) diabetic ulcer treated with dHAM hydrogel containing cobalt and copper-doped MBG, and (4) diabetic ulcer treated by dHAM hydrogel containing cobalt and copper-doped MBG and SF-Col-D/PVA-CS-CeNP scaffold. Eventually, macroscopic, microscopic, and molecular evaluations were conducted after 3, 7, 14, and 21 days using H&E, Masson's trichrome, immunohistochemistry for collagen type I, and the expression levels of inflammatory genes. Results: SF-Col-D/PVA-CS-CeNP nanofibers illustrated a mean diameter of 621.4 ± 506.1 nm, a contact angle of 68º after 6 seconds, swelling ratio of 379.5 ± 6.7%, weight loss of 35.7 ± 2.8% after 28 days immersion in PBS, a release level of 72.38±2.4 μg/ml for vitamin D3 after 96 hours and 0.65±0.02 mg/l for CeNP after 21 days. The scaffolds showed good cell and blood compatibility and the fibroblastic cells grew well on the scaffolds. MBGs significantly increased cell migration and angiogenesis up to 31% and 45% when compared to the control group, respectively, and excellent antibacterial properties as well (P<0.05). The achieved data showed that the HAM was successfully decellularized while preserving its integrity and structure. The release of cobalt and copper from dHAM hydrogel was 2.54 ± 0.02 and 3.83 ± 0.02 mg/L after 21 days, respectively. Based on macroscopic and microscopic evaluation of wounds, it was found that macroscopic wound area percentage was accelerated after 3 days (81.8±1 % in Group 4 (G4), 7 days (66.63±9% and 55±8% in G3 and G4, respectively), and 14 days (less than 37% in all treated groups). Moreover, reepithelization was increased after 7 days post-treatment in G3 (46.5±5%) and G4 (47.4±3%). Furthermore, the gene expression profile of wounds revealed that the expression levels of IL-6 and TNF-a were decreased after 3 days in both G3 and G4, and TNF-a was reduced after 7 days in G4 when compared to the diabetic control group (p<0.05). Conclusion: The results of the present study showed that the nanofibrous scaffolds alongside dHAM hydrogel containing Co and Cu-doped BGs can play an effective therapeutic role in improving and accelerating wound healing in diabetic ulcers. Key words: Diabetic ulcer, Electrospun nanofibers, Mesoporous bioactive glass, Decellurized amniotic memebran, hydrogel