A dopamine-methacrylated hyaluronic acid hydrogel as an effective carrier for stem cells in skin regeneration therapy

Abstract

Adipose-derived stem cells (ADSCs) show potential in skin regeneration research. A previous study reported the failure of full-thickness skin self-repair in an injury area exceeding 4 cm in diameter. Stem cell therapies have shown promise in accelerating skin regeneration; however, the low survival rate of transplanted cells due to the lack of protection during and after transplantation leads to low efficacy. Hence, effective biomaterials for the delivery and retention of ADSCs are urgently needed for skin regeneration purposes. Here, we covalently crosslinked hyaluronic acid with methacrylic anhydride and then covalently crosslinked the product with dopamine to engineer dopamine-methacrylated hyaluronic acid (DA-MeHA). Our experiments suggested that the DA-MeHA hydrogel firmly adhered to the skin wound defect and promoted cell proliferation in vitro and skin defect regeneration in vivo. Mechanistic analyses revealed that the beneficial effect of the DA-MeHA hydrogel combined with ADSCs on skin defect repair may be closely related to the Notch signaling pathway. The ADSCs from the DA-MeHA hydrogel secrete high levels of growth factors and are thus highly efficacious for promoting skin wound healing. This DA-MeHA hydrogel may be used as an effective potential carrier for stem cells as it enhances the efficacy of ADSCs in skin regeneration.

Fig. 1. Assessment of the formation of spheroids composed of ADSCs and cell viability.

A IF staining showed that the spheroids composed of ADSCs had good morphology and the cells migrated from the spheroid, suggesting that the ADSCs maintained good proliferation and migration abilities after spheroid formation. B Images of different cross-sections of the spheroids revealed that the cells were evenly distributed.

Fig. 2. DA-MeHA synthesis, characteristics and biocompatibility.

A The DA-MeHA synthesis process. B ¹H NMR spectrum of DA-MeHA. C TGA thermograms of HA, MeHA, DA-MeHA and their blends with a temperature increase rate of 20 °C/min. (a) gray line—HA, (b) red line—MeHA, and (c) blue line—DA-MeHA. D SEM images of HA, MeHA and DA-MeHA. HA formed crystal-like structures while MeHA and DA-MeHA formed similar porous leaf-like structures. E, F CCK-8 assays and live/dead staining showed that the DA-MeHA hydrogel provides a biocompatible 3D culture environment for encapsulating ADSCs in vitro (*p < 0.05 and **p < 0.01).

Fig. 3. The DA-MeHA hydrogel encapsulating spheroids composed of ADSCs accelerated the regeneration of skin defects in mice.

A Images captured on the 7th and 14th days after surgery showed that the skin defect healed remarkably faster after treatment with DA-MeHA hydrogel-encapsulated spheroids composed of ADSCs than the other groups. B Wound healing rates. The wound healing rate of the hydrogel+ADSCs spheroid group was significantly higher than that of the skin defect group and hydrogel group. C HE staining showed that the structure and morphology of the regenerated epidermis and dermis in the hydrogel + ADSCs spheroid group were closest to those of the control group, followed by the hydrogel group. Abbreviations: e epidermis, p papillary dermis, and r reticular dermis. Arrowheads indicate sebaceous glands and asterisks indicate hair bulbs (*p < 0.05 and **p < 0.01).

Fig. 4. DA-MeHA hydrogels encapsulating suspended ADSCs accelerated skin regeneration in mice better than injection of ADSCs alone.

A Images captured on the 7th and 14th days after surgery showed that wounds treated with DA-MeHA hydrogel-encapsulated suspensions of ADSCs healed remarkably faster than those in the other groups. B Wound healing rates. The wound healing rate in the hydrogel+ADSCs group was significantly higher than that in the skin defect, hydrogel, and ADSCs groups. C HE staining showed that the regenerated epidermal and dermal structures and their morphologies in the hydrogel + ADSCs group were closest to those of the control group. The epidermal papillary layer formed gradually in the center of the wound in only the hydrogel+ADSCs group (C-k), and newborn intradermal hair follicles and enhanced neovascularization were gradually observed (C-l). Abbreviations: e epidermis, p papillary dermis, and r reticular dermis. Arrowheads indicate sebaceous glands, asterisks indicate hair bulbs, and triangles indicate areas of neovascularization (*p < 0.05 and **p < 0.01).

Fig. 5. DA-MeHA hydrogel-encapsulated ADSCs promoted epidermal regeneration and dermal collagen formation and accelerated neovascularization.

A Cytokeratin 14 (CK14) is a surface marker of the epidermis the is used to evaluate skin defect re-epithelialization. IF staining showed higher CK14 expression in the hydrogel+ADSCs group than in the other groups. B Considering the migration of fibroblasts to the wound area, the collagen I level was measured to evaluate the therapeutic effects of the different treatments consisting of the DA-MeHA hydrogel and ADSCs. IF staining showed higher collagen I expression in the hydrogel+ADSCs group than in the other groups. C Images of IF staining from the hydrogel + ADSCs group showed that CK14 and collagen I were coexpressed in the skin. D CD31 is a surface marker of vascular endothelial cells. IF staining showed higher CD31 expression in the hydrogel+ADSCs group than in the other groups. E α-SMA is a specific marker of myofibroblasts (MFB) that is used to evaluate skin defect re-epithelialization. IF staining revealed higher α-SMA expression in the hydrogel+ADSCs group than in the other groups. F IF staining of the hydrogel + ADSCs group showed that α-SMA and CD31 were coexpressed in the regenerated skin, indicating that the ADSCs embedded in the DA-MeHA hydrogel retained a good ability to promote angiogenesis, and the effect was better than that of injection of ADSCs.

Fig. 6. The DA-MeHA hydrogel regulated Notch signaling.

A–E DA-MeHA hydrogel-encapsulated spheroids or suspensions of ADSCs upregulated the expression of Notch1 and Notch2 while downregulating the expression of Notch3, Jagged1 and Jagged2. F No significant difference in Hes1 expression was observed among the groups. n = 4 samples/group (*p < 0.05 and **p < 0.01).

Fig. 7. DA-MeHA hydrogel-encapsulated spheroids composed of ADSCs downregulated inflammatory responses.

A DA-MeHA hydrogel-loaded spheroids composed of ADSCs downregulated the expression of TNF-α. B DA-MeHA hydrogel-loaded spheroids composed of ADSCs downregulated the expression of HMGB1. n = 4 samples/group (*p < 0.05 and **p < 0.01).