Full length articleIn situ forming and reactive oxygen species-scavenging gelatin hydrogels for enhancing wound healing efficacy
Graphical abstract
Introduction
Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical (OH●), and superoxide radical (O2●) are key signaling molecules that play important roles in both the normal metabolism and pathogenesis of living organisms [1]. At the physiological level, they function as redox messengers for cell proliferation, apoptosis, and homeostasis [2]. ROS are also essential in the immune system to attack and kill bacteria [3]. However, the overproduction of ROS can induce oxidative stress, which is related to numerous diseases including diabetes, myocardial infraction, atherosclerosis, rheumatoid arthritis, chronic inflammation, and cancer [4,5]. It has been documented that the excessive ROS production can impair cutaneous wound healing or regeneration of injured tissue by triggering deleterious processes such as necrosis, inflammation, and fibrotic scarring [6]. In addition, the overproduction of ROS also causes damage to biological molecules, including DNA, proteins, lipids, and carbohydrates. The ROS microenvironment also particularly contributes to the cell death, leading to the decreased efficacy of cellular implants in wound treatment therapy [7], [8], [9]. Therefore, designing biomaterials that can scavenge excess ROS at wound sites may offer effective treatment for the augmentation of wound repair and regeneration process [10,11].
One of the most plausible means to decrease the ROS-induced oxidative stress damage is the administration of small molecule ROS scavengers, including synthetic or natural compounds [4]. However, this approach has challenges such as the adverse effects caused by the nonspecific diffusion of ROS scavengers and the repeated, high dose administration due to the rapid clearance by the kidney [5,12]. Several targeted delivery systems using liposomes, polymeric micelles, or nanoparticles have been reported to incorporate and release ROS scavengers, which can prevent the severe adverse effects and improve the therapeutic efficacy in the desired tissues [6,13,14]. Among them, hydrogels have been widely used as the vehicles to locally deliver ROS scavengers in a sustained manner with controlled therapeutic dose to the targeted sites. In addition to serving as sustainable local reservoirs of ROS scavengers, the hydrogels also provide structural support to facilitate the tissue repair and regeneration. Furthermore, hydrogel dressing can maintain a moist wound microenvironment, which protects the wound from bacterial infection and absorbs large amount of exudates [15,16]. In particular, injectable hydrogels have attracted much attention due to their superior biocompatibility and easy encapsulation of therapeutic agents via minimally invasive procedures [17,18]. Recently, there is growing interest in developing injectable hydrogels as promising ROS-modulating materials for a wide range of biomedical applications such as tissue regeneration [19], [20], [21], wound healing [6,15,22,23], and cell-based therapies [8,24,25].
Horseradish peroxidase (HRP)-mediated reaction serves as an effective approach to induce the injectable hydrogels, because of its mild gelation condition, ease of handling, tunable gelation rate, and good crosslinking density [26], [27], [28]. We have recently developed a gelatin-hydroxylphenylpropionic acid (GH) polymer that can in situ covalently crosslink via HRP-mediated coupling of phenol moieties on polymer backbone [29], [30], [31]. In this study, gallic acid-conjugated gelatin (GGA) was introduced into GH hydrogels to create an injectable hydrogel with enhanced ROS scavenging properties compared to pure GH hydrogels (Fig. 1). Gallic acid (GA) belongs to a group of polyphenyl natural compounds that are widely found in the vegetal kingdom, such as nuts, grapes, honey, green tea, and pomegranate [32]. GA has gained significant attention because of numerous beneficial effects, including antioxidant, antibacterial, anti-inflammatory, and antitumorigenic activities. Given the antioxidant activity of GA, we hypothesized that the conjugation of GA onto gelatin chains would endow the ROS scavenging properties to hydrogels as well as reduce the adverse effects caused by burst leaching of small GA when incorporated in GH hydrogels. To test this hypothesis, we first prepared and characterized the physicochemical properties of injectable GH/GGA hydrogels in term of gelation time, mechanical strength, degradation rate, and ROS-scavenging capability. Next, the effect of hydrogels on intracellular ROS production and the survival of human dermal fibroblasts under hydrogen peroxide (H2O2)-induced ROS microenvironment were investigated. Finally, we demonstrated that the GH/GGA hydrogels promote the wound healing and repair in a full-thickness skin defect model through ROS-scavenging activities that modulate the oxidative stress of the wound microenvironment. We expect that our GH/GGA hydrogel system provides a promising ROS-scavenging carrier for wound treatment and tissue regeneration applications.
Section snippets
Materials
Gelatin (type A from porcine skin, >300 Bloom), 3-(4‑hydroxy-phenyl) propionic acid (HPA), gallic acid (GA), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). HRP (type VI, 250–330 U/mg solid), collagenase from Clostridium histolyticum (type II, 0.5–5.0 FALGPA U/mg solid), and H2O2 (30 wt% in H2O), Safranin O, 1′-diphenyl-2-picrylhydrazyl (DPPH), and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA)
Synthesis and characterization of GH and GGA polymers
The reaction of gelatin with HPA and GA occurred between the amino group of gelatin and the carboxylic group of HPA or GA to form the amide linkage, using the EDC/NHS-mediated coupling reaction. The synthesis of GH and GGA polymers is shown in Fig. 2a. The chemical structures of GH polymer was characterized as previously reported [29]. Here, we confirmed the structure of GGA polymer via 1H NMR spectra. Compared with gelatin, the 1H NMR spectrum of GGA shows new peaks at 7.1 (C–H) and 5.55 ppm
Conclusion
In this study, we developed a series of injectable gelatin hydrogels with ROS scavenging activity. By using HRP-catalyzed cross-linking reaction, the hydrogels were rapidly formed and highly cytocompatible. We demonstrated that the incorporation of GGA polymer increased the ROS scavenging effect against hydroxyl radicals and DPPH radicals compared to only GH hydrogels. In addition, the GH/GGA hydrogels can suppress intracellular ROS production and protect hDFBs from the damage of H2O2-induce
Acknowledgement
This research was supported by the National Research Foundation (NRF) and funded by the Korean government (MSIT) (NRF-2018R1A2B2004529)
Disclosures
The authors declare that they have no conflict of interest.
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These authors contributed equally to this work.