In situ forming and reactive oxygen species-scavenging gelatin hydrogels for enhancing wound healing efficacy

Abstract

The overexpression of reactive oxygen species (ROS) contributes to the pathogenesis of numerous diseases such as atherosclerosis, myocardial infarction, cancer, and chronic inflammation. Therefore, the development of materials that can locally control the adverse effects resulting from excessive ROS generation is of great significance. In this study, the antioxidant gallic acid-conjugated gelatin (GGA) was introduced into gelatin-hydroxyphenyl propionic (GH) hydrogels to create an injectable hydrogel with enhanced free radical scavenging properties compared to pure GH hydrogels. The modified hydrogels were rapidly formed by an HRP-catalyzed cross-linking reaction with high mechanical strength and biodegradability. The resulting GH/GGA hydrogels effectively scavenged the hydroxyl radicals and DPPH radicals, and the scavenging capacity could be modulated by varying GGA concentrations. Moreover, in an in vitro H₂O₂-induced ROS microenvironment, GH/GGA hydrogels significantly suppressed the oxidative damage of human dermal fibroblast (hDFBs) and preserved their viability by reducing intracellular ROS production. More importantly, the ROS scavenging hydrogel efficiently accelerated the wound healing process with unexpected regenerative healing characteristics, shown by hair follicle formation; promoted neovascularization; and highly ordered the alignment of collagen fiber in a full-thickness skin defect model. Therefore, we expect that injectable GH/GGA hydrogels can serve as promising biomaterials for tissue regeneration applications, including wound treatment and other tissue repair related to ROS overexpression.

Statement of significance

Recently, many researchers have endeavored to develop injectable hydrogel matrices that can modulate the ROS level to normal physiological processes for the treatment of various diseases. Here, we designed an injectable gelatin hydrogel in which gallic acid, an antioxidant compound, was conjugated onto a gelatin polymer backbone. The hydrogels showed tunable properties and could scavenge the free radicals in a controllable manner. Because of the ROS scavenging properties, the hydrogels protected the cells from the oxidative damage of ROS microenvironment and effectively accelerated the wound healing process with high quality of healed skin. We believe that this injectable ROS scavenging hydrogel has great potential for wound treatment and tissue regeneration, where oxidative damage by ROS contributes to the pathogenesis.

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.