Living microecological hydrogels for wound healing

Abstract

Chronic hard-to-heal wounds draw great attention worldwide, as their treatments are limited by infections and hypoxia. Inspired by the natural oxygen production capacity of algae and the competitive advantage of beneficial bacteria over other microbes, we presented a living microecological hydrogel (LMH) with functionalized Chlorella and Bacillus subtilis encapsulation to realize continuous oxygen delivery and anti-infections for promoting chronic wound healing. As the hydrogel consisted of thermosensitive Pluronic F-127 and wet-adhesive polydopamine, the LMH could keep liquid at a low temperature while quickly solidifying and tightly adhering to the wound bed. It was demonstrated that by optimizing the proportion of the encapsulated microorganism, the Chlorella could continuously produce oxygen to relieve hypoxia and support the proliferation of B. subtilis, while B. subtilis could eliminate the colonized pathogenic bacteria. Thus, the LMH substantially promoted the healing of infected diabetic wounds. These features make the LMH valuable for practical clinical applications.

Fig. 1.. Schematic illustrations of application of the LMH for hard-to-heal wounds.

LMH was constructed by simultaneously using oxygen-produced Chlorella and antimicrobial beneficial B. subtilis into a hydrogel. The microecological hydrogel could fill the wound bed. The Chlorella in the microecological hydrogel could produce oxygen to enhance survival of the B. subtilis and relieve hypoxia, while the B. subtilis could control infections through releasing antimicrobial agents. The LMH could heal the infected diabetic wound using an antibacterial and by relieving hypoxia.

Fig. 2.. Preparation and characterization of the LMH.

(A) Schematic illustrations of the composition of the LMH. (B) LMH maintained liquid at room temperature of 25°C and gelled at body temperature of 37°C. (C) Hydrogel loaded with simple Chlorella or both Chlorella and B. subtilis. (D) Gelation time of different concentrations of simple Pluronic hydrogel at 37°C. (E) Gelation time of simple 18% Pluronic hydrogel, 18% Pluronic hydrogel mixed with PDA, 18% Pluronic hydrogel mixed with PDA and algae, and LMH. Scale bar, 5 μm (C). n = 3 per group (D and E).

Fig. 3.. Proliferation and dissolved-oxygen released capabilities of the LMH.

(A) Schematic illustration and statistical analysis of F-127–PDA hydrogel for proliferation of Chlorella and B. subtilis. (B) Proliferation rate of the Chlorella in the hydrogel with 0.1, 0.25, 0.5, and 1% PDA added. (C) Proliferation rate of the B. subtilis in the hydrogel with 0.1, 0.25, 0.5, and 1% PDA added. (D) Proliferation rates of Chlorella under different kinds of lights with or without hydrogel encapsulation. (E) The amount of dissolved oxygen released from Chlorella under different kinds of lights with or without hydrogel encapsulation. The influence of concentrations of B. subtilis on the proliferation of Chlorella without (F) or within (G) hydrogel. The influence of concentrations of Chlorella on the proliferation of B. subtilis without (H) or within (I) hydrogel. n = 3 per group (B to I).

Fig. 4.. Antibacterial and anti-hypoxia capabilities of the LMH.

(A) Image of the inhibition zone formed between B. subtilis and S. aureus. (B) Inhibition zone formed by (i) LBH loaded with simple B. subtilis and (ii) LMH loaded with both B. subtilis and Chlorella. (C) Representative photographs of L929 fibroblasts that suffered from hypoxia in different treatments. (D) Representative photographs of the tube formation of HUVECs that suffered from hypoxia in different treatments. (E) Statistical analysis of the cell viability of L929 fibroblasts that suffered from hypoxia in different treatments. (F) Statistical analysis of the tube formation of HUVECs that suffered from hypoxia in different treatments. Scale bars, 200 μm (C and D). n = 6 per group (E and F). **P < 0.01.

Fig. 5.. The LMH accelerated the wound healing process of the infected diabetic wounds.

(A) Representative images of full-thickness skin defect wounds treated by PBS, hydrogel, LAH, LBH, and LMH. (B) Schematic illustrations of the healing process of the wound bed. (C) The regenerated granulation tissues were stained by hematoxylin and eosin (H&E) staining after 9 days. (D) Quantification of the wound area healed by different treatments. (E) Quantification of granulation tissue thickness in different groups. Scale bars, 5 mm (A) and 1 mm (C). n = 6 per group. *P < 0.05 and **P < 0.01.

Fig. 6.. The mechanism of LMH promoting chronic wound repair.

(A) The CD31-positive blood vessel endothelial cells were stained as red. (B) Immunohistochemical staining of HIF-1α in granulation tissues in different groups. (C) Masson’s trichrome staining of granulation tissues in different treatments. (D) Double immunofluorescence staining of α-SMA and vimentin of granulation tissues in different treatments. (E to G) Quantification of (E) blood vessel number, (F) HIF-1α densities, and (G) collagen deposition. Scale bars, 100 μm (A to D). n = 6 per group. *P < 0.05 and **P < 0.01.