Collagen-based injectable and self-healing hydrogel with multifunction for regenerative repairment of infected wounds

Abstract

At present, the development trend of dressing materials is being multifunctional for convenient and long-term nursing care process of some complicated wounds. Here, basing on the theory of wound moist healing, an injectable and self-healing hydrogel comprising of collagen (COL), chitosan (CS) and oxidation modified Konjac glucomannan (OKGM), which acts as a macromolecular cross-linker to construct dynamic Schiff-base bonds was smartly designed. The strategy of introducing the silver nanoparticles (Ag NPs) into the COL-CS-OKGM hydrogel matrix achieved a markedly enhanced antibacterial activity derived from the synergistical effect between the Ag⁺ and the mild photothermal efficacy of Ag NPs, which also improved the local capillary blood circulation of the wound area to further facilitate wound healing process. The excellent syringeability and self-healing behaviors endowed the COL-CS-OKGM-Ag hydrogel with self-adapting ability for the wounds with irregular and large area needing frequent applying and changing without secondary injuries. In vitro and in vivo evaluations verified that so-designed COL-CS-OKGM-Ag hydrogel also with hemostatic performance is a promising multifunctional dressing for the treatments of infected wound with not only good biocompatibility and convenient use, but also with desired regenerative healing prognoses benefited from hydrogel moist environment and physiotherapy.

Keywords: collagen; hydrogel; injectable and self-healing; regenerative repair; wounds.

Graphical abstract

Figure 1.

TEM images of GA–Ag NPs under low power (a) and at high magnification (b). Size distribution of GA–Ag NPs (c). TGA (d), ultraviolet spectrophotometer (e) and XRD profile (f) of GA–Ag NPs. SEM images of CS-OKGM hydrogel (g), COL–CS–OKGM hydrogel (h) and COL–CS–OKGM–Ag hydrogel (i). Bar = 500 μm.

Figure 2.

FTIR spectra of (a) GA–Ag NPs and (b) hydrogels. (c) Strain amplitude sweep and (d) step–strain measurements of COL–CS–OKGM–Ag hydrogel. Viscosity and shear-thinning behavior of COL–CS–OKGM–Ag hydrogel (e). Photographs of hydrogel injection via 26G syringe needle (f) and (g) fusion of two separated hydrogels showing the easy self-healing behavior of COL–CS–OKGM–Ag hydrogel.

Figure 3.

(a) Thermal infrared images of hydrogels with different Ag NPs content under 808 nm NIR irradiation. (b) Temperature profiles of COL–CS–OKGM–Ag hydrogels with different silver concentrations during the extension of irradiation time. (c) Temperature variation of the COL–CS–OKGM–Ag hydrogel containing 200 μg/ml Ag NPs during three on/off laser irradiation cycles (808 nm 2 W/cm²). Photograph of differently treated hydrogels incubated at 37°C for 12 h against inhibitory bands of (d) E. coli and (e) S. aureus. (f) Photographs of E. coli and S. aureus treated by (I) PBS, (II) COL–CS–OKGM hydrogel, (III) COL–CS–OKGM–Ag hydrogel containing 200 μg/ml Ag, (IV) COL–CS–OKGM–Ag hydrogel containing 200 μg/ml Ag + NIR. (g) Statistical analyses for the bacterial numbers of E. coli and S. aureus (mean ± SD, n = 3, ***P < 0.001).

Figure 4.

(a) SEM images of E. coli and S. aureus and (b) CLSM images of S. aureus treated by (I) PBS, (II) COL–CS–OKGM hydrogel, (III) COL–CS–OKGM–Ag hydrogel containing 200 μg/ml Ag NPs, (IV) COL–CS–OKGM–Ag hydrogel containing 200 μg/ml Ag NPs + NIR. PI labeling dead bacteria, and SYTO-9 labeling live bacteria. (c) A schematic illustration for the mouse liver hemorrhage model treated by COL–CS–OKGM–Ag hydrogel. (d) Accumulated blood loss in 120 s for hepatic hemorrhage under different treatments (mean ± SD, n = 3, ***P < 0.001). (e) The hemostasis effect of COL–CS–OKGM–Ag hydrogel on damaged mouse liver within 120 s as positive control and untreated group as negative control.

Figure 5.

CLSM images of (a) HDF cells, (c) HUVEC and (e) NIH3T3 cells cultured on the surface of COL–CS–OKGM–Ag hydrogel containing from 0 to 200 μg/ml Ag NPs (bar = 200 μm, 50 μm). (b) Three-dimensional reconstruction of CLSM image of HDF cells embedded and cultured inside the hydrogel for 5 days. Cells were cultured on hydrogels with different silver content for 1, 3 and 5 days, the proliferation of (d) HUVECs and (f) NIH3T3 cells was detected by CCK-8. (g) Photographs of S. aureus infected wounds at predetermined time points for different treatment groups. (h) Temperature changes showed by thermal infrared images of mice irradiated by infrared laser and (i) percentage of wound area in vivo at 7 and 14 days.

Figure 6.

(a) H&E staining and Masson staining of the wound sites on 7th and 14th day. Scale bars = 200 μm. Arrow 1: scab skin; Arrow 2: blood vessels; Arrow 3: thickened epidermis; Arrow 4: collagen fiber; Arrow 5: epidermal tissue; Arrow 6: hair follicle; Arrow 7: fibroblasts (n = 3). (b) The epidermal thickness and (c) collagen deposition was measured on 14th day (mean ± SD, n = 3, **P < 0.01).

Figure 7.

(a) CD31 immunohistochemically staining on the 7th and 14th day of the wound treatment. Scale bars =100μm, black arrows: blood vessels. (b) Number of newly formed blood vessels from the immunohistochemical images on Day 14 (mean ± SD, n = 3, **P < 0.01). Note: I: blank; II: CS–OKGM–Ag hydrogel + NIR; III: COL–CS–OKGM–Ag hydrogel; IV: COL–CS–OKGM–Ag hydrogel + NIR.