Diabetic ulcers represent a significant and potentially life-threatening chronic complication of diabetes mellitus [1], [2], [3]. They are particularly prevalent in diabetic foot ulcers, which are notorious for their recalcitrance to healing and susceptibility to infection, often resulting in gangrene formation [4], [5]. This has emerged as the most prevalent cause of non-traumatic amputation in clinic [6], [7]. Numerous studies have revealed that the presence of excessive reactive oxygen species (ROS) in diabetic wounds is an important reason for their difficulty in healing [8]. In diabetic patients, high blood glucose levels lead to non-enzymatic glycosylation, and a large amount of ROS is released during this reaction [9], [10]. Excessive ROS in diabetic wounds not only induce the upregulation of pro-inflammatory factors, thereby eliciting a robust inflammatory response at the wound site and impeding the transition from the inflammatory phase to the proliferative and remodeling phases [8], [11], but also result in oxidative damage that affects endothelial cells, fibroblasts, and keratinocytes, which hampers vascular neogenesis, collagen deposition, and reepithelization processes, thus rendering healing of diabetic wounds challenging [12], [13], [14]. Meanwhile, the chronicity of open wounds and the hyperglycemic microenvironment make them highly susceptible to bacterial infections [15]. In severe instances, this can result in delayed wound healing and an elevated risk of amputation and mortality. Hence, it is imperative to eliminate ROS and prevent bacterial infection for effective healing of diabetic ulcer wounds [16].

Currently, antibiotics, antibacterial materials and photothermal antibacterial agents have been employed to inhibit bacterial infections for diabetic wound healing [17]. Non-steroidal anti-inflammatory drugs and artificial nano-enzymes have been utilized for the removal of ROS during wound healing [18], [19]. These therapeutic interventions have achieved certain efficacy, substantiating that bacterial inhibition or ROS scavenging can indeed accelerate the healing process of diabetic wounds. However, as research progresses, more refined criteria have been proposed for the eradication of bacteria and removal of ROS to enhance wound healing. Firstly, the skin hosts a diverse community of beneficial bacteria that play a crucial role in wound healing. It has been reported that some of these commensal bacteria can stimulate the immune system to produce anti-inflammatory cytokines, promoting wound healing [20]. However, traditional antibacterial methods, while effective at eliminating harmful pathogens, can also unintentionally harm the beneficial bacteria on skin surface, consequently hindering wound healing. Secondly, the ROS category encompasses a range of species, including superoxide anion (O2–•), hydroxyl radical (•OH), peroxynitrite anion (ONOO−), nitric oxide (NO•), hydrogen peroxide (H2O2) and others [21]. The aforementioned method for ROS scavenging can remove all ROS species nonspecifically, which may disrupt the body's oxidative/antioxidant equilibrium [22].

Indeed, low concentrations of O2–• and H2O2 serve as crucial regulatory signaling molecules involved in many signal transduction cascades, regulating vital biological processes such as apoptosis, cell proliferation and differentiation [23], [24]. NO• plays a crucial role as a neurotransmitter in vasodilation [25]. All of these processes are beneficial for wound repair. However, compared to other ROS, •OH and ONOO− exhibit stronger reactive activity, especially •OH, which is considered the most potentROS and can react with nucleic acids, lipids and proteins indiscriminately [26]. Both •OH and ONOO− exhibit high toxicity, resulting in tissue damage and impaired wound healing. Therefore, selectively inhibiting harmful bacteria and selectively scavenging •OH and ONOO− are novel requirements proposed following extensive research on diabetic ulcer treatment. However, achieving both these “selective” objectives simultaneously remains unreported, presenting a challenging yet pioneering endeavor.

Hydrogen (H2) gas, according to literature reports, has been documented to be able to selectively remove •OH and ONOO− [8], [22]. Since Ohsawa et al. discovered in 2007 that H2 can protect the brain from ischemia-reperfusion injury by neutralizing •OH [22], it has emerged as a cutting-edge therapeutic medical gas. H2 exhibits selective removal of these two highly toxic free radicals of •OH and ONOO−, thereby protecting cells and tissues from oxidative stress and inflammatory damage. It is widely acknowledged for its anti-inflammatory, antioxidant and anti-apoptotic effects [27].

In order to ensure their own survival, certain probiotics have the ability to produce antimicrobial substances, such as free fatty acids and bacteriocins, through metabolic processes. These substances effectively inhibit the growth of competing bacteria, thereby exerting selective bactericidal effects on harmful bacteria [28], [29], [30]. Recently, Lactobacillus has been incorporated into a hydrogel for the treatment of infected skin wounds [28]. The findings demonstrate that the Lactobacillus-loaded hydrogel successfully inhibits pathogenic bacteria such as Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Salmonella. Consequently, it exhibits an enhanced healing effect on mouse skin wounds infected with S. aureus.

Inspired by this, we have focused on a probiotic called Clostridium butyricum (C. butyricum), which can efficiently produce H2 in anaerobic environments and inhibit the growth of pathogenic bacteria, potentially achieving both of abovementioned “selective” objectives simultaneously. Therefore, C. butyricum holds great promise for diabetic ulcer wound healing. However, there are currently no reports available. This is mainly because as an anaerobic bacterium, C. butyricum cannot maintain physiological activity in the normoxic environment when applied externally on the skin, thus unable to exert its hydrogen production and antibacterial functions.

To address this issue, we propose the construction of a novel hydrogel, termed as anaerobic hydrogel, which provides an oxygen-free environment within its interior. This anaerobic hydrogel will serve as a carrier for C. butyricum to satisfy their anaerobic growth (abbreviated as C. butyricum@Anaerobic-Gel in Scheme 1). Vanillin (Van), a natural aldehyde compound extracted from vanilla beans, was selected as the oxygen-consuming substrate, taking advantage of its excellent biocompatibility, good solubility, light reaction color and high controllability in reactions [31], [32]. What is particularly noteworthy is that laccase and vanillin have been used to construct anaerobic hydrogels, serving as carriers for adipose-derived mesenchymal stem cells, to enable targeted delivery and retention of the stem cells within the organism [33]. In this study, Van is coupled with aldehyde-modified sodium alginate (ALG-CHO). The resulting Van-ALG-CHO is then mixed with a solution containing C. butyricum, laccase, and carboxymethyl chitosan (CMCS). Through the Schiff base reaction between aldehyde (–CHO) and amino (–NH2) groups, Van-ALG-CHO and CMCS are crosslinked to form the desired hydrogel. Consequently, both C. butyricum and laccase are encapsulated within this hydrogel system, achieving the construction of C. butyricum@Anaerobic-Gel successfully (Scheme 1A). In this system, Van undergoes an oxygen-consuming reaction with laccase to deplete dissolved oxygen in the hydrogel matrix effectively, thus creating an anaerobic microenvironment inside it (Scheme 1A). Even when applied topically on normoxic skin conditions, the internalized C. butyricum remains in an anaerobic state within the hydrogel, while maintaining its physiological activity for hydrogen production and antibacterial effects simultaneously. Therefore, C. butyricum@Anaerobic-Gel fulfills both requirements of “selective bacteria inhibition and selective ROS scavenging”, demonstrating promising potential for diabetic wound healing applications (Scheme 1B). The present study is anticipated to address the bottleneck issue associated with topical application of C. butyricum in normoxic environments on skin surfaces by establishing a suitable platform that sustains an anaerobic microenvironment. This not only paves the way for future utilization of C. butyricum in diabetic ulcer treatments, but also serves as a reference for the application of other anaerobic probiotics in normoxic environments.