Injectable and biofunctionalized fibrin hydrogels co-embedded with stem cells induce hair follicle genesis

Abstract

Fibrin-based hydrogels have been widely used in various tissue engineering because of their biocompatibility, biodegradability, tunable mechanical characteristics and nanofibrous structural properties. However, their ability to support stem cells for hair follicle neogenesis is unclear. In this study, we investigated the effect of fibrin hydrogels in supporting skin-derived precursors (SKPs) in hair follicle neogenesis. Our results showed that SKPs in fibrin hydrogels with high cell viability and proliferation, the stemness of SKPs could be maintained, and the expression of hair induction signature genes such as akp2 and nestin was enhanced. Moreover, hair follicle reconstruction experiments showed de novo hair genesis in mice and the hairs persisted for a long time without teratoma formation. More importantly, the blood vessels and sebaceous glands were also regenerated. Our study demonstrated that fibrin hydrogels are promising in hair follicle regeneration and have potential application in clinical settings for alopecia and wound healing.

Keywords: alopecia; fibrin hydrogels; fibrinogen; hair follicle neogenesis; skin-derived precursors.

Graphical abstract

Figure 1.

Characterization of fibrin hydrogels. (A) The schematic structure of fibrinogen and the fibrin hydrogel formation process. (B and C) Fibrinogen solution in different concentration and fibrin hydrogel in different concentration. (D and E) SEM images of different concentration fibrin hydrogels. Scale bar: 300 μm (D), 100 μm (E). (F) Swelling capacity of different concentration fibrin hydrogels. (G) The viscosity of 80 mg/mL fibrinogen with a change in the temperature.

Figure 2.

Evaluation of SKPs compatibility. (A) Morphology of SKPs in fibrin hydrogels. (B) Live and dead staining of SKPs in fibrin hydrogels cultured for 1 day and 4 days, in which live cells are visualized with green and dead cells appear red. Scale bar: 100 μm. (C) The viability of SKPs before and after cultured in fibrin hydrogel for 3 days. (D) Cell proliferation of SKPs cultured in fibrin hydrogels for 1 day, 4 days and 7 days.

Figure 3.

Cytological analysis of SKPs in fibrin hydrogels (SKP-F). (A and B) Representative immunofluorescence images of BMP6, nestin and fibronectin expression of SKPs in fibrin hydrogels. Scale bar: 100 µm. (C) AP staining images of SKPs and SKPs cultured in fibrin hydrogels. Scale bar: 100 µm. (D and E) Real-time PCR analysis of SKPs in fibrin hydrogels for 3 days for their expression of stemness genes and HF induction-associated genes.

Figure 4.

Fibrin hydrogels support stem cell for de novo hair genesis. (A) Representative images of hair growth observed 4 weeks after transplantation (N = 3). (B) HE staining of hair genesis tissue shows the hair follicle, epidermis and dermis regenerated. Scale bar: 100 µm. (C and D) Representative IF images of K1 and K14 in regenerated tissue of fibrin hydrogels and Matrigel. Scale bar: 100 µm. (E) The numbers of hair shafts per wound in each wound and each group (N ≥ 3).

Figure 5.

Appendages regenerated after the transplantation of stem cells and fibrin hydrogels. (A and B) Representative images of IF staining of biotin which showed the regeneration of sebaceous gland in regenerated tissue of fibrin hydrogels and matrigel. Scale bar: 100 µm. (C and D) Representative images of IF staining of CD31, which showed the regeneration of blood vessels in regenerated tissue of fibrin hydrogels and matrigel. Scale bar: 100 µm.

Figure 6.

Biocompatibility evaluation. (A) The morphological images of mice after transplantation of 6 months. (B and C) Representative images of the hair genesis tissue after transplantation of 4 weeks and 6 months. Scale bar: 2 mm. (D) HE staining images of regenerated tissue after transplantation for 6 months. Scale bar: 100 µm.