Versatile dopamine-functionalized hyaluronic acid-recombinant human collagen hydrogel promoting diabetic wound healing via inflammation control and vascularization tissue regeneration

Abstract

The management of chronic wounds in diabetes remains challenging due to the complexity of impaired wound healing, delayed healing, susceptibility to infection, and elevated risk of reopening, highlighting the need for effective chronic wound management with innovative approaches such as multifunctional hydrogels. Here, we have produced HA-DA@rhCol hydrogels consisting of dopamine-modified hyaluronic acid and recombinant human collagen type-III (rhCol) by oxidative coupling of the catechol group using the H₂O₂/HRP catalytic system. The post-reactive hydrogel has a good porous structure, swelling rate, reasonable degradation, rheological and mechanical properties, and the catechol group and dopamine impart to the hydrogel tissue adhesiveness, antioxidant capacity, and excellent photothermal effects leading to superior in vitro antimicrobial activity. In addition, the ability of rhCol to confer hydrogels to promote angiogenesis and wound repair has also been investigated. Cytotoxicity and hemolysis tests demonstrated the good biocompatibility of the hydrogel. Wound closure, collagen deposition and immunohistochemical examination confirmed the ability of the hydrogel to promote diabetic wound healing. In summary, the adhesive hemostatic antioxidative hydrogel with rhCol to promote wound healing in diabetic rat is an excellent chronic wound dressing.

Keywords: Antibacterial; Antioxidant and anti-inflammation; Diabetic wound; Hyaluronic acid hydrogel; Recombinant human collagen.

Graphical abstract

Fig. 1

Schematic illustration of HA-DA@rhCol hydrogel preparation and application for diabetic wound care in rat.

Fig. 2

Characterization of HA-DA@rhCol hydrogels. A) Full infrared spectrum and B) UV–Vis spectra of HA, HA-DA and Dopamine. C) Gelation time of HA-DA@rhCol with different HA-DA concentrations. D) SEM images of hydrogels, a. 0.5% HA-DA@rhCol; b. 1.0% HA-DA@rhCol; c. 1.5% HA-DA@rhCol; d. 2.0% HA-DA@rhCol. E) Swelling rate and F) Water retention and G) Degradation rate of hydrogels. (n = 3). Error bars indicated means ± SD.

Fig. 3

Rheological and mechanical characterizations, adhesion and in vivo hemostatic performance of HA-DA@rhCol hydrogels. A) Shear rheology and B) Stress-strain profiles of these hydrogels by compression. C) Stress-strain profiles of these hydrogels by tensile tests. D) Photographs of adhesive strength test, E) Adhesive strength of different hydrogels, n = 3. F) Hemostatic performance of hydrogel HA-DA@rhCol. G) Photographs of hydrogels sticking to fingers. H) Photographs of hydrogels glued to different organs. Error bars indicated means ± SD. Statistical significances were analyzed using t-test. *P < 0.05, **p < 0.01, ***p < 0.001 compared with control group.

Fig. 4

Cell compatibility of HA-DA@rhCol hydrogels. A) Hemolysis rate of HA-DA@rhCol hydrogel. B) pictures of Hemolysis. C) Dead/live staining of a. Control, b. 1.5% HA, c. 1.5% HA-DA, d. 1.5% HA-DA@rhCol. D) Cell viability detected by MTT of NIH3T3 co-cultured with the hydrogels. Error bars indicated means ± SD. Statistical significances were analyzed using t-test. *P < 0.05 compared with control group.

Fig. 5

Antioxidant efficiency and inflammation control of hydrogels. A) The images of DPPH scavenging of all groups for 10 s and 1 min and B) DPPH scavenging capabilities by 1.5% HA, 1.5% HA-DA and 1.5% HA-DA@rhCol, n = 3. C) and D) Flowcytometry of DCFH-DA labeled Raw264.7 cells in fluorescein isothiocyanate FITC-A channel in different group, n = 3. E) Intracellular ROS-scavenging performance of 1.5% HA, 1.5% HA-DA and 1.5% HA-DA@rhCol. F) Fluorescence images of RAW 264.7 cells after treatment with LPS (control) or LPS + 1.5% HA, 1.5% HA-DA and 1.5% HA-DA@rhCol. CD86: M1 macrophages. Error bars indicated means ± SD. Statistical significances were analyzed using t-test. *P < 0.05, ***p < 0.001 compared with control group.

Fig. 6

Photothermal effect and antibacterial activity of hydrogels. A) Real-time infrared thermal images of 1.5% HA, 1.5% HA-DA and 1.5% HA-DA@rhCol, and B) photothermal curves of 1.5% HA-DA@rhCol under laser power intensity, n = 3. C) Photothermal curves of 1.5% HA-DA@rhCol under 1 W/cm⁻² in four on/off cycles, n = 3. D) Colony formation and SEM photograph (bar = 1.5 μm) of S. aureus and E. coli. Bacteria were cocultured with various concentrations of the Fe/PDA@GOx@HA, irradiated with or without an 808 nm laser at 1 W cm⁻² for 30 min.

Fig. 7

Cell migration and angiogenesis of hydrogels. A) Scratch test of HaCaT cells and B) Tube formation of HUVECs co-cultured with 1.5% HA, 1.5% HA-DA and 1.5% HA-DA@rhCol. C) and D) Quantitative analysis of cell migration and tube formation, n = 3. Statistical significances were analyzed using t-test. *P < 0.05, **p < 0.01, ***p < 0.001 compared with control group.

Fig. 8

Treatment efficiency of the chronic wound by 1.5% HA-DA@rhCol in diabetic rat. A) Representative photographs of the diabetic chronic wound treated with different dressing. (B) Simulation plots of wound closure. C) H&E staining of the wound indicated the healing condition at 11 days. D) Masson's staining and E) Sirius red staining of the wound sections at day 7 and day 14. F) The quantitative analysis of wound closure, G) wound length in H&E staining and H) collagen volume fraction in Masson's staining. n = 3. Statistical significances were analyzed using t-test and one-way ANOVA. *P < 0.05, **p < 0.01, ***p < 0.001 compared with control group.

Fig. 9

Modulation of inflammation microenvironment and regeneration in diabetic wound. A) Immunofluorescence images of marker F4/80 (Green) and CD206 (Red) in wound tissue, and nuclei were stained with DAPI (blue). B) Immunohistochemistry staining of F4/80 and CD31 on the wound sections at day 11. C) Quantitative analysis of CD206 fluorescence intensity/F4/80 fluorescence intensity ratio. D) and E) Quantitative analysis of F4/80 and CD31 positive area at day 11, respectively. Statistical significances were analyzed using one-way ANOVA. *P < 0.05, **p < 0.01, ns, p > 0.05 compared with control group.

Fig. 10

Mechanistic analysis of wound healing with HA-DA@rhCol treatment. A) Volcano plot of transcriptomic analysis of differentially expressed genes (n = 3 biologically independent samples). B) Heatmap analysis of differentially expressed genes. C) and D) GO analysis of differentially up-regulated and down-regulated expressed genes. E) and F) Enriched KEGG pathways of HA-DA@rhCol versus Control. The size of the dots indicates the number of genes associated with indicated KEGG terms.