Wharton's jelly mesenchymal stem cells embedded in PF-127 hydrogel plus sodium ascorbyl phosphate combination promote diabetic wound healing in type 2 diabetic rat

Abstract

Background: Diabetic cutaneous ulcers (DCU) are a complication of diabetes with diabetic foot ulcers being the most common, and the wounds are difficult to heal, increasing the risk of bacterial infection. Cell-based therapy utilizing mesenchymal stem cells (MSCs) is currently being investigated as a therapeutic avenue for both chronic diabetic ulcers and severe burns. Wharton's jelly mesenchymal stem cell (WJMSC) with PF-127 hydrogel and sodium ascorbyl phosphate (SAP) improved skin wound healing in mice. Whether this combination strategy is helpful to diabetic ulcers wound healing remains to be explored.

Methods: Firstly, the WJMSCs embedded in PF-127 and SAP combination were transplanted onto excisional cutaneous wound bed in type 2 diabetic Sprague Dawley (SD) rats. Two weeks after transplantation, the skin tissue was collected for histological and immunohistochemical analysis. Further, overexpressing-EGFP WJMSCs were performed to investigate cell engraftment in the diabetic cutaneous ulcer. The apoptosis of WJMSCs which encapsulated with combination of PF-127 and SAP was detected by TUNEL fluorescence assay and RT-PCR in vitro. And the mitochondrial damage induced by oxidative stress assessed by MitoTracker and CMH2DCFDA fluorescence assay.

Results: In diabetic cutaneous wound rat model, PF-127 plus SAP-encapsulated WJMSCs transplantation promoted diabetic wound healing, indicating improving dermis regeneration and collagen deposition. In diabetic wound healing, less pro-inflammatory M1 macrophages, more anti-inflammatory M2 tissue-healing macrophages, and neovascularization were observed in PF-127 + SAP + WJMSCs group compared with other groups. SAP supplementation alleviated the apoptosis ratio of WJMSCs embedded in the PF-127 in vitro and promoted cell survival in vivo.

Conclusion: PF-127 plus SAP combination facilitates WJMSCs-mediated diabetic wound healing in rat through promoting cell survival, the macrophage transformation, and angiogenesis. Our findings may potentially provide a helpful therapeutic strategy for patients with diabetic cutaneous ulcer.

Keywords: Diabetic wound healing; PF-127 hydrogel; Rat; Sodium ascorbyl phosphate; Wharton’s jelly mesenchymal stem cells.

Fig. 1

PF-127 plus SAP combination facilitates WJMSC-mediated diabetic wound healing. a Schematic of in vivo wound-healing assay after WJMSCs transplantation in STZ-induced diabetic SD rats. b Representative images of wounded skin were showed at 0, 3, 7, 10, 14 days post-transplantation. c The percentage of residual wound area of each group was analyzed at day 7 after transplantation. Error bars represent mean ± SEM; n = 8 independent experiments. Significance was determined using one-way ANOVA. **p < 0.01, ***p < 0.001. d The percentage of residual wound area of each group was analyzed at day 14 after transplantation. Error bars represent mean ± SEM; n = 8 independent experiments. Significance was determined using one-way ANOVA. **p < 0.01, ***p < 0.001

Fig. 2

Histology analysis of the dermis regeneration and collagen deposition. a Hematoxylin and eosin staining in diabetic wound bed and surrounding normal tissues were analyzed at day 14 after transplantation. Scar bar: 500 μm. b The quantitative data of the dermis thickness in each group were showed at day 14 after transplantation. Error bars represent mean ± SEM; n = 8 independent experiments. Significance was determined using one-way ANOVA. ***p < 0.001. c The quantitative data of the number of newborn hair follicles in each group at day 14 post-transplantation per field. Error bars represent mean ± SEM; n = 8 independent experiments. Significance was determined using one-way ANOVA. ***p < 0.001. d The quantitative data of the scar width in each group were showed at day 14 post-transplantation. Error bars represent mean ± SEM; n = 8 independent experiments. Significance was determined using one-way ANOVA. ***p < 0.001. e Masson’s trichrome staining of diabetic wound bed and surrounding normal tissues was showed at day 14 after transplantation. Scar bar: 500 μm. N, normal skin tissue, shown on both sides of the black imaginary line; H, healed skin tissue, shown between black and red imaginary lines; W, wound bed, unhealed skin tissue, shown between the red imaginary lines

Fig. 3

Immunohistochemistry identifies the macrophage transformation, cell proliferation, and neovascularization. a Immunohistochemical staining with anti-CD86, CD163, Ki-67, and CD31 antibodies on diabetic wound in each group was performed and analyzed at 14 days post-transplantation. Scar bar: 50 μm. b, c The quantitative data of the total number of CD86-positive M1 macrophages (b) and CD163-positive M2 macrophages (c) per field were analyzed. Error bars represent mean ± SEM; n = 3 independent experiments. Significance was determined using one-way ANOVA. *p < 0.05, ***p < 0.001. d The quantitative data of the percentage of Ki-67-positive proliferating cells per field were analyzed. Error bars represent mean ± SEM; n = 3 independent experiments. Significance was determined using one-way ANOVA. *p < 0.05, ***p < 0.001. e The quantitative data of the total number of CD31-positive newly formed blood vessels were analyzed. Error bars represent mean ± SEM; n = 3 independent experiments. Significance was determined using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

PF-127 plus SAP combination promotes engraftment of WJMSCs in diabetic wound. a–c Bright-field and fluorescence microscopy of WJMSCs stably expressing Flag-tagged EGFP was performed (A-B) and either western blotted to confirm EGFP expression with the indicated antibodies. The tubulin was shown as internal control. d Schematic of OE-EGFP WJMSCs migration by fluorescence assay at 24 h and 72 h after transplantation in ulcer wound. e, f At 24 h after transplantation, EGFP-overexpressing WJMSCs in different groups were detected (E) and the number of EGFP-positive WJMSCs was analyzed quantitatively in each group (F). Error bars represent mean ± SEM; n = 3 independent experiments. Significance was determined using one-way ANOVA. ***p < 0.001. Scale bar: 50 μm. Enlarge scale bar: 25 μm. g, h At 72 h after transplantation, EGFP-overexpressing WJMSCs in different groups were detected (g) and the number of EGFP-positive WJMSCs was analyzed quantitatively in each group (H). Error bars represent mean ± SEM; n = 3 independent experiments. Significance was determined using one-way ANOVA. ***p < 0.001. Scale bar: 50 μm. Enlarge scale bar: 25 μm

Fig. 5

SAP supplementation alleviated the apoptosis of WJMSCs encapsulated with PF-127. a The cell apoptosis was detected by TUNEL immunofluorescence in different groups. TUNEL, green, represents apoptotic cell; DAPI, blue, represents cell nuclei. Scale bar, 100 μm. b The quantitative data of the percentage of TUNEL-positive cell per field were analyzed. Error bars represent mean ± SEM; n = 3 independent experiments. Significance was determined using one-way ANOVA. *p < 0.05, **p < 0.01. c The mRNA level of proapoptotic and antiapoptotic gene was measured by qRT-PCR in different groups. Error bars represent mean ± SEM; n = 3 independent experiments. Significance was determined using one-way ANOVA. *p < 0.05, **p < 0.01. d The protein level of proapoptotic and antiapoptotic gene was measured by western blot in different groups. e The quantitative data of protein level of different genes in different groups. Error bars represent mean ± SEM; n = 3 independent experiments. Significance was determined using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. f CMH2DCFAD staining was analyzed in different groups, CMH2DCFAD: green, represents the ROS fluorescence intensity; Hoechst, blue, represents cell nuclei. Scale bar: 20 μm. g The quantitative data of the percentage of CMH2DCFAD-positive cell per field were analyzed. Error bars represent mean ± SEM; n = 3 independent experiments. Significance was determined using one-way ANOVA. ***p < 0.001. h Mitochondrial location and distribution patterns were detected in different groups. Scale bar, 20 μm. Enlarge scale bar, 10 μm

Fig. 6

Working model of WJMSC + PF127 + SAP transplantation in diabetic rats. Transplantation of WJMSCs encapsulated with PF-127 and SAP can improve the diabetic wound healing and accelerate the macrophage transformation, cell proliferation, and neovascularization in the diabetic rats