Anti-oxidant anti-inflammatory and antibacterial tannin-crosslinked citrate-based mussel-inspired bioadhesives facilitate scarless wound healing

Abstract

The revolutionary role of tissue adhesives in wound closure, tissue sealing, and bleeding control necessitates the development of multifunctional materials capable of effective and scarless healing. In contrast to the use of traditionally utilized toxic oxidative crosslinking initiators (exemplified by sodium periodate and silver nitrate), herein, the natural polyphenolic compound tannic acid (TA) was used to achieve near instantaneous (<25s), hydrogen bond mediated gelation of citrate-based mussel-inspired bioadhesives combining anti-oxidant, anti-inflammatory, and antimicrobial activities (3A-TCMBAs). The resulting materials were self-healing and possessed low swelling ratios (<60%) as well as considerable mechanical strength (up to ∼1.0 MPa), elasticity (elongation ∼2700%), and adhesion (up to 40 kPa). The 3A-TCMBAs showed strong in vitro and in vivo anti-oxidant ability, favorable cytocompatibility and cell migration, as well as photothermal antimicrobial activity against both Staphylococcus aureus and Escherichia coli (>90% bacterial death upon near-infrared (NIR) irradiation). In vivo evaluation in both an infected full-thickness skin wound model and a rat skin incision model demonstrated that 3A-TCMBAs + NIR treatment could promote wound closure and collagen deposition and improve the collagen I/III ratio on wound sites while simultaneously inhibiting the expression of pro-inflammatory cytokines. Further, phased angiogenesis was observed via promotion in the early wound closure phases followed by inhibition and triggering of degradation & remodeling of the extracellular matrix (ECM) in the late stage (supported by phased CD31 (platelet endothelial cell adhesion molecule-1) PDGF (platelet-derived growth factor) and VEGF (vascular endothelial growth factor) expression as well as elevated matrix metalloprotein-9 (MMP-9) expression on day 21), resulting in scarless wound healing. The significant convergence of material and bioactive properties elucidated above warrant further exploration of 3A-TCMBAs as a significant, new class of bioadhesive.

Keywords: Anti-oxidant; Hydrogen bond crosslinking; Phased angiogenesis; Scarless wound healing; Tannic acid.

Graphical abstract

Scheme 1

Synthesis mechanism of 3A-TCMBAs and application for scarless wound healing.

Fig. 1

Characterizations of 3A-TCMBAs: (A) gel times, (B) sol contents, (C) swelling ratios, and (D) degradation rates. (*p < 0.05, **p < 0.01).

Fig. 2

Adhesive properties of 3A-TCMBAs: (A) photographs of macroscopic adhesion, (B) photographs of the adhesion ability to glass, rubber, iron, and plastic, (C) photographs of the adhesion to porcine skin under water flushing, (D) adhesive properties to various tissues, (E, F) schematic presentation of the lap shear test, the lap shear strengths to (G) porcine skin and (H) other substrates, (I) the macroscopic photographs of self-healing capacity, (J) rheological behavior with alternate strains switched from 1% to 100% for four cycles. (*p < 0.05, **p < 0.01).

Fig. 3

Mechanical characterizations of 3A-TCMBAs: (A) stress-strain curves, (B) tensile strengths, (C) Young's moduli, (D) elongations at break. (**p < 0.01).

Fig. 4

Photothermal and photothermal antibacterial properties: the curves of ΔT-NIR irradiation time for (A) 3A-TCMBAs with different TA contents at 0.85 W/cm² and (B) 3A-TC_15% under different NIR power densities; (C) temperature change of 3A-TC_15% over four NIR irradiation on/off cycles (0.5 W/cm²); (D) the representative thermal graphs of 3A-TC_15% upon 808 nm NIR irradiation (0.5 W/cm²) for 10 min; (E, F) contacting antibacterial activity of 3A-TCMBAs, (E) bacterial colonies of E. coli and S. aureus cocultured with PBS (1), iC-E-Ca²⁺ (2), 3A-TC_5% (3), 3A-TC_10% (4) and 3A-TC_15% (5) and treated without or with NIR; (F) representative images of colony forming units for E. coli and S. aureus suspensions on LB agar plates following 12 h incubation at 37 °C; relative bacterial viabilities of (G) E. coli and (H) S. aureus. (*p < 0.05, **p < 0.01).

Fig. 5

Anti-oxidant properties: (A) UV–vis spectra and (B) DPPH scavenging percentages of 3A-TCMBAs after incubating for 2 min; (C, D) dynamic DPPH scavenging of 3A-TC_15%; expression levels of (E) superoxide dismutase (SOD), (F) catalase (CAT) and (G) glutathione peroxidase (GPx) during the process of in vivo wound healing after being treated with control, 3A-TC_15% and 3A-TC_15%+NIR adhesive for 7, 14 and 21 days, respectively. (*p < 0.05, **p < 0.01).

Fig. 6

Cytotoxicity evaluation of 3A-TCMBAs: cytotoxicity against L929 cells after 24 h for (A) sol content and (B) degradation products, (C) representative Live/Dead images, (D) L929 cells migration images and (E) quantitative scratch closure rates, (F) representative images of the transwell migration assay of L929 cells and (G) quantitative migration cell OD values. (*p < 0.05, **p < 0.01).

Fig. 7

Wound closure results of the infected wounds: (A) Photothermal temperature change of SD rats for 3A-TC_15%, 3A-TC_15%+NIR and the control groups; (B) representative photographs of the wounds treated with physiological saline (control) and 3A-TC_15% (with and without NIR) on the 3rd, 7th, 14th and 21st day; (C) schematic diagram of wound closure; (D) quantitative statistical analysis of wounds closure. (**p < 0.01).

Fig. 8

Histological and inflammation-related immunohistochemical analysis: (A) H & E and (B) Masson's trichrome staining images of the infected skin wound after being treated with 3A-TCMBAs (with and without NIR), and physiological saline (control), (C) collagen densities, (D) epidermis thicknesses (day 21) and (E) quantitative hair follicle numbers (day 21); Immunohistochemistry staining images of (F) TNF-α, and (G) quantitative IL-1β, cell densities of (H) TNF-α positive cells and (I) IL-1β positive cells. (*p < 0.05, **p < 0.01).

Fig. 9

Angiogenesis, hair follicle and remodeling related immunohistochemical analysis: Immunohistochemical staining images and the corresponding dyeing scores of (A, B) CD31 (Red arrows represent new blood vessels), (C, D) PDGF, (E, F) VEGF at day 7 and 14; immunohistochemistry staining images and the corresponding dyeing scores after being treated of (G, H, I) of CD34 and MMP9 on day 21. (**p < 0.01).

Fig. 10

Wound closure assessment of skin incision: (A) representative images of untreated skin incisions and the skin incisions treated by surgical sutures, 3A-TC_15% (with and without NIR); (B) H & E staining images and (C) Masson's trichrome staining of the skin incisions on the 7th and 14th day; (D) photothermal temperature change images of SD rats for 3A-TC_15% and 3A-TC_15%+NIR groups.