An intrinsically bioactive hydrogel with on-demand drug release behaviors for diabetic wound healing

Abstract

Prolonged, intense inflammation and excessive oxidative stress hinder diabetic wounds from healing normally, leading to disorders downstream including the postponement of re-epithelialization and extracellular matrix (ECM) formation. Herein, we report a hyaluronic acid (HA) and chitosan based hydrogel (OHA-CMC) with inherent antibacterial and hemostatic activities fabricated via Schiff base reaction. By encapsulating nanotechnologically-modified curcumin (CNP) and epidermal growth factor (EGF) into the hydrogel, OHA-CMC/CNP/EGF exhibited extraordinary antioxidant, anti-inflammatory, and migration-promoting effects in vitro. Meanwhile, OHA-CMC/CNP/EGF presented on-demand drug release in synchrony with the phases of the wound healing process. Specifically, curcumin was rapidly and constantly released to alleviate inflammation and oxidative stress in the early phase of wound healing, while a more gradual and sustained release of EGF supported late proliferation and ECM remodeling. In a diabetic full-thickness skin defect model, OHA-CMC/CNP/EGF dramatically improved wound healing with ideal re-epithelialization, granulation tissue formation, and skin appendage regeneration, highlighting the enormous therapeutic potential this biomaterial holds as a diabetic wound dressing.

Keywords: Anti-Inflammation; Antioxidant; Bioactive hydrogel; Diabetic wound healing; Drug delivery.

Graphical abstract

Fig. 1

Schematic representations of OHA-CMC/CNP/EGF hydrogel for diabetic wound healing. (A) OHA-CMC hydrogel was fabricated via reversible Schiff base reaction between OHA and CMC and presented excellent intrinsic antibacterial and hemostatic properties. (B) OHA-CMC/CNP/EGF released drugs in a stepwise manner to solve the key issues in different phases of diabetic wound healing.

Fig. 2

Characterizations of hydrogels. (A) FT-IR spectra of HA, OHA, chitosan, CMC, and representative hydrogel formed through the Schiff base reaction between OHA and CMC. (B) Rheological behavior of hydrogels. (C) ESR of hydrogels after immersing in PBS for 24 h. (D) Representative SEM images of hydrogels and drug-encapsulated hydrogels. Scale bar: 100 μm. (E) Pore size of hydrogels and drug-encapsulated hydrogels. (F, G) Degradation kinetics of hydrogels in PBS without (F) and with (G) 50 U/mL hyaluronidase.

Fig. 3

(A) Cytocompatibility of hydrogels indicated by live/dead staining. Scale bar: 200 μm. (B) Cell viability of NIH-3T3 following incubating with hydrogel extracts for 48 h. (C) Hemolytic ratio of hydrogels. (D, E) Antibacterial analysis of hydrogels. Digital photographs of S. aureus colonies (D) and bacterial viability (E) after incubating with hydrogels for 6 h. (F) Schematic diagram illustrating the assessment of the hemostatic performance for the hydrogel via a mouse liver bleeding model. (G) Representative photographs of bleeding livers following different treatments at predetermined time points. (H) Total mass of blood loss in 60s from punctured livers after different treatments.

Fig. 4

(A) Drug release profile of curcumin and EGF from OHA-CMC hydrogel. (B) UV–vis spectra of DPPH and DPPH treated with OHA-CMC/CNP containing different doses of curcumin. (C) DPPH scavenging percentage for the treatments with OHA-CMC/CNP containing different doses of curcumin. (D) Changes of DPPH scavenging percentage for free curcumin and OHA-CMC/CNP with the same dose of curcumin kept at room temperature and 37 °C for 10 days. *p < 0.05, **p < 0.01, and ****p < 0.0001 comparison between free curcumin and OHA-CMC/CNP at room temperature. ##p < 0.01, ###p < 0.001, and ####p < 0.0001 comparison between free curcumin and OHA-CMC/CNP at 37 °C.

Fig. 5

(A) The intracellular ROS level indicated by DCFH-DA probe following different treatments. Scale bar: 200 μm. (B) The quantitative analysis of intracellular ROS depletion with different treatments by flow cytometry. (C) The RT-PCR analysis of intracellular TNF-α, IL-1β, and IL-6 mRNA level after treatment with OHA-CMC containing different doses of curcumin. (D) Representative photographs of scratch wounds at 0 h, 12 h, and 24 h following the treatments with control, OHA-CMC, OHA-CMC/CNP, OHA-CMC/EGF, and OHA-CMC/CNP/EGF. Scale bar: 200 μm. (E) Quantification of the remaining scratch wound area percentage at 12 h and 24 h with different treatments.

Fig. 6

OHA-CMC/CNP/EGF hydrogel promoted wound healing in STZ-induced diabetic mice. (A) Schematic illustration of the operations on the mouse in an order of time. (B) Representative photographs of the diabetic wound at day 0, 5, 10, and 15 treated with control, OHA-CMC, OHA-CMC/CNP, OHA-CMC/EGF, and OHA-CMC/CNP/EGF groups. (C) Wound closure percentage at day 5, 10, and 15 after treatment with the five groups. (D) H&E staining images indicating the regenerated granulation tissue and epidermis in the wound region following different treatments for 10 and 15 days (white dashed line denotes the boundary of epidermis and dermis; black arrow denotes the granulation tissue). Scale bar: 500 μm. (E) Quantification of granulation tissue thickness following different treatments for 10 and 15 days. (F) Quantification of epidermal thickness following different treatments for 10 and 15 days.

Fig. 7

The effects of the hydrogels on diabetic wound healing evaluated on the histological level. (A) H&E staining images of the wound tissues treated with control, OHA-CMC, OHA-CMC/CNP, OHA-CMC/EGF, and OHA-CMC/CNP/EGF for 5, 10, and 15 days (blue dashed line denotes the inflammatory area; red arrow denotes the blood vessel; brown arrow denotes the hair follicle; white dashed line denotes the boundary of epidermis and dermis). Scale bar: 200 μm. (B) Masson's trichrome staining images of the wound tissues following different treatments for 10 and 15 days. Scale bar: 100 μm. (C) Quantitative analysis of regenerated blood vessels following different treatments for 10 and 15 days. The data was normalized against the control group at day 10 which was defined as 100%. (D) Quantification of regenerated hair follicles following different treatments for 15 days. The data was normalized against the control group which was defined as 100%. (E) Collagen deposition levels in the wound tissues following different treatments for 10 and 15 days. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

Mechanism examination of hydrogel-mediated acceleration in wound healing. (A) ROS level of the wound regions indicated by DHE staining following different treatments for 5 and 10 days. Scale bar: 100 μm. (B) Immunofluorescent staining of IL-6 performed on wound tissues from different groups after treating for 5 and 10 days. Scale bar: 200 μm. (C) Neovascularization evaluation through CD31 immunofluorescent staining for the wound tissues treated with different groups for 10 and 15 days. Scale bar: 50 μm. (D, E) Quantitative analysis of ROS (D) and IL-6 (E) labeled structures. The data was normalized against the control group at day 5 which was set as 100%. (F) Quantification of the percentage of CD31 positive cells following the treatments with different groups for 10 and 15 days.