A hybrid hydrogel encapsulating human umbilical cord mesenchymal stem cells enhances diabetic wound healing

Abstract

Background: Diabetic wound is a severe complication of diabetes. Stem cell is considered as a promising therapy for diabetic skin wounds. Hydrogel can supply niche for cells adhesion and survival to improve the efficacy of stem cell therapy, but the development of hydrogel with suitable properties remains a great challenge. Thus, our study was conducted to combine an optimized hydrogel with stem cell to improve complex diabetic wound treatment.

Methods: This study constructed a hydrogel with low toxicity and adjustable mechanical properties from gelatin methacrylate (GelMA) and chitosan-catechol (Chi-C), and encapsulated human umbilical cord-mesenchymal stem cells (hUMSCs) to repair full-thickness diabetic wound.

Results: We explored the relationship between mechanical stiffness and cell proliferation and differentiation potency, and found 10% GelMA hydrogel with an optimal stiffness improved hUMSCs adhesion, proliferation, and differentiation potency maintenance in vitro. Assistant with optimized hydrogel encapsulating hUMSCs, diabetic wound healing process was greatly accelerated, including accelerated wound closure, inhibited secretion of inflammatory factors TNF-α and IL-1β, promoted vascular regeneration and collagen deposition after treatment of hUMSCs.

Conclusions: The optimized hydrogel encapsulating hUMSCs improved diabetic wound healing, and has a broad implication for the treatment of diabetic complication. Diabetic wound is a severe complication of diabetes. Stem cell is considered as a promising therapy for diabetic skin wounds. Hydrogel can supply niche for cells adhesion and survival to improve the efficacy of stem cell therapy. This study constructed a hydrogel with low toxicity and adjustable mechanical properties from gelatin methacrylate (GelMA) and chitosan-catechol (Chi-C), and encapsulated human umbilical cord-mesenchymal stem cells (hUMSCs) to repair full-thickness diabetic wound. Hydrogel of 10% GelMA with an optimal stiffness improved hUMSCs adhesion, proliferation, and differentiation potency maintenance in vitro. Assistant with optimized hydrogel encapsulating hUMSCs, diabetic wound healing process was greatly accelerated, including accelerated wound closure, inhibited secretion of inflammatory factors TNF-α and IL-1β, promoted vascular regeneration and collagen deposition after treatment of hUMSCs. The study supplies an alternative treatment for diabetic complication. Hydrogel-hUMSCs combined treatment accelerates wound closure in diabetic mice. A. Representative images of wounds during 21-day in vivo experiments. B. Quantification of wound closure rate (%) over 21-day period. C. HE staining of wounds at days 7, 14 and 21. The bar corresponds to 200 μm.

Diabetic wound is a severe complication of diabetes. Stem cell is considered as a promising therapy for diabetic skin wounds. Hydrogel can supply niche for cells adhesion and survival to improve the efficacy of stem cell therapy. This study constructed a hydrogel with low toxicity and adjustable mechanical properties from gelatin methacrylate (GelMA) and chitosan-catechol (Chi-C), and encapsulated human umbilical cord-mesenchymal stem cells (hUMSCs) to repair full-thickness diabetic wound. Hydrogel of 10% GelMA with an optimal stiffness improved hUMSCs adhesion, proliferation, and differentiation potency maintenance in vitro. Assistant with optimized hydrogel encapsulating hUMSCs, diabetic wound healing process was greatly accelerated, including accelerated wound closure, inhibited secretion of inflammatory factors TNF-α and IL-1β, promoted vascular regeneration and collagen deposition after treatment of hUMSCs. The study supplies an alternative treatment for diabetic complication. Hydrogel-hUMSCs combined treatment accelerates wound closure in diabetic mice. A. Representative images of wounds during 21-day in vivo experiments. B. Quantification of wound closure rate (%) over 21-day period. C. HE staining of wounds at days 7, 14 and 21. The bar corresponds to 200 μm.

Fig. 1

Characterization of the hydrogel with different GelMA concentration (6%, 10%, 14%). A ¹H NMR spectrum of GelMA and Chi-C. B Time sweep sequence and C frequency sweep sequence of hydrogel. D Static compression test of hydrogel. Cyclic compression test of the hydrogel. E In vitro degradation behavior. G–I Morphology of lyophilized hydrogel with GelMA concentration of 6%, 10% and 14%

Fig. 2

Immunophenotypical analysis of hUMSCs. A HUMSCs were labeled with specific antibodies for the indicated MSCs (upper panels) or hematopoietic (bottom panels) markers and analyzed by flow cytometry. B Evaluation of hUMSCs proliferation at 1st, 2nd, 3rd day

Fig. 3

Biocompatibility of hydrogels. A CCk-8 assay of hUMSCs culture in leach liquor of three hydrogels or medium as control for 1, 3 and 7 days. B Cellular morphology of hUMSCs culture on hydrogels or tissue culture plate in inducing media for 14 days

Fig. 4

Differentiation of hUMSCs on hydrogels. Staining images of hUMSCs culture on hydrogels or tissue culture plate in inducing media (osteoblastic, chondroblast and adipose differentiation) for 21 days

Fig. 5

Hydrogel-hUMSCs combined treatment accelerates wound closure in diabetic mice. A Representative images of wounds during 21-day in vivo experiments. B Quantification of wound closure rate (%) over 21-day period. C HE staining of wounds at days 7, 14 and 21. The bar corresponds to 200 μm

Fig. 6

Immune factors expression of wounds for day 7. Expression of TNF-α at the wound sites of rats in the control, the hydrogel and hUMSCs alone or combined group at 7 days after the operation, as revealed by IHC. The bar corresponds to 500 μm

Fig. 7

Hydrogel-hUMSCs combined treatment promotes angiogenesis and re-epithelialization. Expression of CD31, α-SMA and VEGF at the wound sites of rats in the control, the hydrogel and hUMSCs alone or combined group 14 days after the operation, detected by immunofluorescence or IHC. High magnification corresponds to boxed area in the low-magnification images

Fig. 8

Masson’s trichrome staining of wound sections at day 7 or 14 post-wounding for four conditions