A self-hygroscopic, rapidly self-gelling polysaccharide-based sponge with robust wet adhesion for non-compressible hemorrhage control and infected wounds healing

Abstract

Uncontrollable non-compressible hemorrhage and traumatic infection have been major causes of mortality and disability in both civilian and military populations. A dressing designed for point-of-care control of non-compressible hemorrhage and prevention of traumatic infections represents an urgent medical need. Here, a novel self-gelling sponge OHN@ε-pL is developed, integrating N-succinimidyl ester oxidized hyaluronic acid (OHN) and ε-poly-L-lysine (ε-pL). Upon application to the wound site, the sponge can rapidly absorb interfacial fluids and undergo a phase transition from sponge to gel. The transformed gel facilitates robust tissue adhesion and achieves synergistic hemostasis by enriching coagulation factors within the sponge phase and providing a barrier effect in the gel phase. The in vitro and in vivo studies revealed that the optimized OHN@ε-pL₃ sponge possesses self-gelling capability, tissue adhesion, enhanced coagulation ability, and exhibits excellent biocompatibility and antibacterial efficacy. In hemostasis, OHN@ε-pL₃ sponges exhibited reduced blood loss and decreased hemostatic time compared to commercial hemostatic agents, as demonstrated in rat liver, femoral vein, and tail truncation bleeding models. Furthermore, the OHN@ε-pL₃ sponge exhibited superior performance in accelerating wound closure and healing of S. aureus-infected wounds. Collectively, OHN@ε-pL sponges represent a promising candidate for medical dressings, specifically for managing uncontrollable non-compressible hemorrhage and traumatic infections.

Graphical abstract

A self-gelling sponge (OHN@ε-pL₃) developed by compounding N-succinimidyl easter oxidized hyaluronic acid (OHN) with ε-poly-L-lysine (ε-pL) can self-gel upon absorbing blood and then seal the wound, thereby exerting hemostatic and pro-coagulation performance. The superior antibacterial properties of the OHN@ε-pL₃ gel also help to alleviate wound inflammation, promote collagen deposition, and enhance angiogenesis, thereby accelerating the healing of infected wounds.

Highlights

• •A self-hygroscopic, fast-gelling polysaccharide sponge with strong wet adhesion has been developed.
• •The sponge transforms into an adhesive gel when it contacts blood or water-based fluids.
• •The sponge enhances coagulation and antibacterial processes, improving hemostasis and aiding infected wound healing.

1. Introduction

Uncontrolled bleeding from severe injuries in accidents, conflicts, and natural disasters causes over two million deaths worldwide annually [1,2]. In combat trauma, 90 % of fatal injuries involve non-compressible bleeding, which also accounts for 30 %–40 % of civilian deaths from severe bleeding [3]. Most trauma deaths occur within the first hour, known as the “Golden Hour”, making effective hemostasis vital for patient survival [4]. Since the body's coagulation system inability to promptly halt severe bleeding, using hemostatic materials for fast and effective on-site intervention is crucial to save lives and reduce complications from excessive bleeding [1,5].

Currently, the main types of hemostatic materials include powders, gauzes, hydrogels, sponges [[6], [7], [8]]. Sponges and gauzes are generally inadequate for addressing irregular wounds, as they lack shape adaptability. The hemostatic powder, when applied to the wound, can rapidly absorb blood and concentrate clotting factors to promote hemostasis. However, commercial hemostatic powders, including zeolite-based QuickClot, chitosan-based Celox, etc., generally exhibit weak mechanical strength, poor tissue adhesiveness, and low biocompatibility [1,9]. Furthermore, they generally fail to form stable blood clots or even dissolve in the bloodstream, hence struggling to stop arterial or venous bleeding [6,7,10]. By contrast, adhesive hydrogels such as fibrin glue, Coseal, and albumin-based sealants are commonly employed in clinical settings as adjunctive hemostatic agents [2,11,12]. They serve as physical barriers to prevent wound from bleeding. However, most commercial bio-glue adhesives exhibit limitations, such as low biocompatibility, slow adhesion, poor mechanical properties, and weak adhesive strength [13]. Blood or body fluids can weaken hydrogel's adhesion to tissue, lowering its hemostatic efficacy [2,7]. Adhesive hydrogels with strong wet adhesion are mainly used for hemostasis in hypotensive bleeding models [14,15], but are ineffective for deeper wounds or arterial/venous hemostasis in large animals [12,16]. Certain adhesive hydrogels, such as fibrin glue, are utilized in the form of two-component in-situ gelling systems but face challenges in storage, transportation, and usage [17]. Additionally, some hydrogels require photo-curing, extra coupling agents, or enzyme-mediated coagulation, raising further biosafety concerns [12,18,19]. Consequently, there is a pressing necessity to develop novel hemostatic materials capable of absorbing blood, establishing wet adhesion with tissues to physically arrest bleeding, and matching irregular shapes or deep wounds to facilitate rapid hemostasis, as well as ease of storage, transportation and usage [20,21].

Recently, research on hemostatic materials with wet adhesion properties has garnered significant attention. These materials are characterized by their capacity to remove interfacial water from tissue surface, enabling rapid cross-linking with the tissue and serving as a barrier to promote hemostasis. X. H. Zhao et al. developed a dry double-sided tape (DST) capable of removing moisture from the tissue surface and forming a stable and robust adhesion with tissue though rapid cross-linking with surface groups [21]. Moreover, studies have focused on developing functionalized polymer into powders that, upon contact with blood and/or tissue fluids, transformed into adhesive hydrogels in situ, effectively sealing bleeding wounds [1,6,10]. However, these wet-adhesive hemostatic materials only partially address the weak adhesion problem at wet tissue surface but are still imperfect. For instance, DST is constrained in its effectiveness for irregular deep wounds and is inadequate for managing non-compressible hemorrhage. And self-gelling hemostatic powders are susceptible to dislodgment by high-pressure blood flow, thereby compromising their effectiveness. Additionally, these powders pose challenges in controlling dosage accurately and removing residual material from the wound [1].

Given the limitations of existing hemostatic materials and the requirements for managing complex bleeding scenarios, herein, we have developed a polysaccharide-based hemostatic sponge characterized by self-hygroscopicity, self-gelling, and wet-adhesion properties. The hemostatic sponge is primarily composed of OHN and ε-pL. It is characterized by the integration of the active fluid-absorbing properties of sponges, the sealing properties of adhesive gels, and the activation of the coagulation system, making it potentially ideal for various bleeding scenarios.

Oxidized hyaluronic acid (OHA) is derived from hyaluronic acid (HA) and features highly reactive aldehyde groups [22]. HA is a crucial part of the extracellular matrix in mammalian tissues [23]. It regulates cell behaviors [24], aids in tissue repair [25], and enhances platelet migration and fibrin clot formation during hemostasis [26]. HA has multiple reactive groups and can be oxidized to form a double aldehyde group, enabling it to react with amines in a Schiff base reaction [24], thus expanding its applications [27]. Recently, OHA has become popular for hydrogel construction [28,29]. ε-Poly-L-lysine (ε-pL) is a natural antibacterial peptide extracted from the fermentation broth of Streptomycesalbulus, with a polymerization degree of 25–35. ε-pL is a cationic polymer with potent antibacterial properties against both Gram-negative and Gram-positive bacteria [30,31]. It kills bacteria by disrupting their cell membranes and binding to ribosomes, interrupting material, energy, and information flow [32]. Therefore, this amino-containing molecule is commonly used to create durable antibacterial hydrogels [31].

The OHN is prepared by introducing N-succinimidyl (NHS) ester groups into the carboxyl groups of OHA. The synthesis of OHN@ε-pL sponges leverages the distinct solubility properties of its polymers: the polysaccharide polymer (OHN) is exclusively soluble in the aqueous phase, whereas ε-pL exhibits solubility in both aqueous and organic solvents (e.g., methanol). Consequently, we smartly compounded OHN sponge with ε-pL in the organic phase to create the OHN@ε-pL composite sponge (Scheme 1a). Upon contact with water-based fluids such as blood or tissue fluid, the OHN@ε-pL sponge undergoes a rapid dissolution of its components, which then react to form a gel, accompanying a phase transition from sponge to gel (Scheme 1b). The sponge combines the high liquid absorption of porous materials with the barrier properties of gels, making it highly effective for hemostasis and preventing wound infection (Scheme 1c).

Scheme 1.

Schematic illustration of the preparation and application of self-gelling OHN@ε-pL sponge for hemostasis and infected wound healing. (a) and (b) Schematic illustration of the preparation and self-gelling mechanism of OHN@ε-pL sponge. (c) Application of the OHN@ε-pL sponge in hemostasis and infected wound healing.

Herein, we systematically investigated the physicochemical properties of the multifunctional OHN@ε-pL sponge, focusing on its self-hygroscopicity, self-gelatinization, and wet adhesion. Additionally, its hemostatic performance was assessed in various hemorrhagic models, including rat liver, femoral vein, tail truncation, and pericardial bleeding, as well as its healing effect on infected wounds. The test results demonstrated that the OHN@ε-pL sponge exhibited robust self-gelling and wet adhesion capabilities, along with excellent biocompatibility and antibacterial properties. The OHN@ε-pL₃ sponge exhibited superior hemostatic efficacy across various bleeding scenarios compared to commercial hemostatic agents (e.g., gauze, fibrin glue, gelatin sponge), significantly enhancing RBC and platelet adhesion and clotting. Moreover, the OHN@ε-pL₃ sponge demonstrated accelerated wound closure and healing in S. aureus-infected skin wounds.

2. Results and discussion

2.1. Preparation and characterization of OHN@ε-pL sponge, and its phase transition from sponge to gel

The polysaccharide-based sponge was synthesized by compounding OHN with ε-pL. The preparation processes for the OHN@ε-pL sponge are detailed in Fig. 1a. Initially, OHA was prepared by oxidizing HA with NaIO₄, achieving a degree of oxidation quantified at 37.6 ± 4.4 %, as determined by the hydroxylamine hydrochloride method. Subsequently, the N-succinimidyl ester group was grafted onto the carboxyl group of OHA to obtain OHN. The OHN was then impregnated into a methanol solution of ε-pL and subjected to vacuum drying to obtain OHN@ε-pL sponges with ε-pL evenly distributed on the surface, which was verified through Scanning Electron Microscope (SEM) analysis and immunofluorescence staining (Fig. 1k and S1). The morphology of each of the OHA, OHN, and OHN@ε-pL sponges were shown in Fig. 1b. Upon contact with an aqueous solution, the OHN and ε-pL components of the OHN@ε-pL sponges rapidly dissolved, exposing respective reactive groups (e.g., -NH₂, -CHO, -NHS ester). This triggered the occurrence of Schiff base and amide reactions, causing the phase transition from sponge to gel. Nevertheless, the OHN sponge without compounding ε-pL failed to gel upon exposure to water (Fig. 1c and d). In conjunction with the structural characterization of the OHN@ε-pL sponge and its gel scaffold using FTIR and ¹HNMR spectroscopy (Figs. S2 and S3), it was confirmed that Schiff base and amide reactions occurred during the phase transition from sponge to gel. Fig. 1e illustrates the viscosity of OHN and OHN@ε-pL sponges in PBS over the same time period. The viscosity test results indicated that the static viscosity of the OHN sponge solution is 473.47 mPa s, much less than that of the OHN@ε-pL sponge post self-gelling (1.48 × 10⁷ mPa s). Furthermore, the viscosity of OHN@ε-pL gel was much higher than the minimum standard viscosity threshold for hydrogel (1 × 10⁴ mPa s) [33], which further indicated that a cross-linking reaction occurred inside the OHN@ε-pL sponge during dissolution, and ultimately formed a gel rather than a solution. In the test, OHA and OHA@ε-pL sponges were served as controls, and yielded conclusions similar to those of the OHN and OHN@ε-pL sponge, indicating that both OHA and OHN sponges, when compounded with ε-pL, can undergo a gelation transition during the dissolution process. The difference between OHA@ε-pL and OHA@ε-pL gels was mainly manifested in the magnitude of their viscosity, potentially attributable to variations in their cross-linking reactions. Apparently, the static viscosity value of the OHA@ε-pL gel (1.42 × 10⁷ mPa s) was observed to be lower than that of the OHN@ε-pL gel. This discrepancy can be attributed to the primary cross-linking mechanism in the OHA@ε-pL sponge gelling process being the Schiff base reaction. In contrast, the OHN@ε-pL gelling sponge underwent both amide and Schiff base reactions, resulting in a denser cross-linking network and consequently higher viscosity.

Fig. 1.

(a) Synthetic route for the OHA and schematic illustration of the preparation of OHN@ε-pL sponge. (b) Macro-morphologic views of OHA, OHN and OHN@ε-pL sponges. (c) Schematic illustration of the morphological changes of OHN and OHN@ε-pL sponges after exposure to aqueous solution. (d) Morphology changes images of OHN and OHN@ε-pL sponges after exposure to aqueous solution. (e) Viscosity curves of polysaccharide-based sponge compounded with ε-pL (OHA@ε-pL, OHN@ε-pL) and without ε-pL (OHA, OHN) after dissolution. (f) Rheological behavior of gels in amplitude scanning mode. (g) Quantitative statistics of storage modulus (G′) of the gels in (f) above (n = 6). (h) Liquid absorption ratios of sponges composed of OHN (5 wt%) and ε-pL (1 wt%, 2 wt%, 3 wt%, 5 wt%, or 7 wt%) (n = 3). (i) Rheological behaviors of gels OHA@ε-pL₃ and OHN@ε-pL₃ cycled four time at 1min intervals with alternating strain between 1 % and 300 % s. (j) Images of self-healing behavior of OHN@ε-pL₃ gel; the divided gel pieces healed into a single gel after 10 min of contact at room temperature. (k) SEM images of OHN@ε-pL sponges, Magnification 100 × , Scale bar = 200 μm; detailed magnified image of ε-pL uniformly distributed on the surface of OHN scaffold, Magnification 5000 × , Scale bar = 5 μm. (l) Degradation behavior of the gel scaffolds in vitro. (m) Self-gelatinization and adhesion process of OHN@ε-pL sponge in contact with blood. (n) Liquid absorption ratios of OHN@ε-pL₃ sponge and commercial hemostatic products in water, PBS and blood (n = 3). (o) Self-expanding ratios of OHN@ε-pL₃ sponge versus commercial hemostatic sponges (n = 3) (∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001; ns: not significant).

The rheological behavior of OHN@ε-pL sponges after gelling, composed of OHN (5 wt%) and varying concentrations of ε-pLx (x = 1 wt%, 3 wt%, and 5 wt%), were investigated in an amplitude sweep mode. Their storage modulus (G′) and loss modulus (G″) exhibited an upward trend with the increase of ε-pL concentration (Fig. 1f and g). These observations indicated that higher ε-pL concentrations result in a denser cross-linking network and a more stable gel structure, thus leading to an elevated G′ value. Specifically, the G’ value of the OHN@ε-pL₁ gel was 2867 ± 39.99 Pa, significantly higher than that of the OHA@ε-pL₁ gel (1490 ± 26.06 Pa) (Fig. 1g). The difference between the two gel groups was likely due to the OHA@ε-pL₁ sponge forming a single cross-link network via Schiff base reaction, whereas the OHN@ε-pL₁ sponge formed a more stable, multiple cross-link network through both amide and Schiff base reactions. In the test, it was determined that the maximum solubility of ε-pL in methanol is 3 wt%, and higher solubility requires adding an aqueous solvent. However, water also dissolves the OHN sponge, causing premature cross-linking and altering its morphology and properties.

Fig. S4 illustrated the morphology of OHN@ε-pLx (x = 3 wt% or 5 wt%) sponges. From a macro perspective, the OHN@ε-pL₃ sponge, composed with OHN sponge and 3 wt% of ε-pL dissolved in methanol appeared loose, porous and soft performance, whereas OHN@ε-pL₅ sponge composed with OHN sponge and 5 wt% of ε-pL dissolved in methanol-water solution exhibited a collapsed structure. Moreover, the OHN@ε-pL₅ sponge felt stiffer, potentially hindering fluid absorption and wound shape adaptation. Additionally, ε-pL is a complex hydrochloride that lowers pH at high concentrations, impacting the OHN@ε-pL gelation reaction and gel stability. Thus, a 3 wt% dosage of ε-pL is more suitable for compounding with the OHN sponge.

The precursor concentration affects the composite's diameter, porosity, and liquid absorption [1]. We prepared a series of OHNy@ε-pL₃ (y = 1 wt%, 2 wt%, 3 wt%, 5 wt% or 7 wt%) sponges by compounding 3 wt% of ε-pL with varying OHN concentrations. The SEM observation and pore size data for each group were shown in Fig. S5. SEM images showed the sponge had an interconnected porous honeycomb structure, aiding rapid liquid absorption and permeation. Statistical plots indicated that increasing OHN concentration from 1 wt% to 7 wt% reduced the average pore size from 129.0 ± 27.47 μm to 67.92 ± 7.42 μm. As the concentration increased, the average pore size decreased, while the liquid absorption ratio in PBS rose from 3781 ± 347.4 % to 5359 ± 92.39 % between 1 wt% and 5 wt% OHN (Fig. 1h). Actually, increasing the OHN concentration to 7 wt% reduced the sponge's average liquid absorption ratio, likely due to smaller pore sizes affecting liquid transfer. Hence, the liquid absorption capacity of the OHN@ε-pL sponge is dependent on OHN concentration. An optimal OHN concentration improves liquid absorption and wet adhesion. Therefore, sponges with 5 wt% OHN were chosen for further studies.

2.2. Characterization of the gel properties

The phase transition of OHN@ε-pL sponges to gels is predicated on a dynamic Schiff base network formed by the interaction of activated amino and aldehyde groups, as well as a cross-linking network established through covalent amide bonds between N-succinimidyl ester and amino groups. The incorporation of a dynamic Schiff base network enables the hydrogel with exceptional self-healing properties and plasticity [34,35]. The self-healing capability of OHA@ε-pL and OHN@ε-pL gel was tested with a rheometer in alternating strain mode. Fig. 1i showed the gel structure remained intact (G’ > G″) at 1%strain, but was disrupted (G’ < G″) at 300 % strain. As the oscillatory strain alternated between 1 % and 300 %, the gel structure alternated between reconstruction and destruction, showing self-healing behavior of the two gels. Interestingly, after the first cycle, both G′ and G″ in the OHN@ε-pL gels did not return to their initial values, with G′ decreasing from ∼18832 Pa to ∼8353.7 Pa and G″ from ∼510.5 Pa to ∼483.4 Pa. This might be because the irreversible amide bonds formed by N-succinimidyl ester and amino groups in the gel likely break under high strain (300 %), leaving only the dynamic Schiff base network after the first cycle. Conversely, the OHA@ε-pL gel's dynamic Schiff base network enables both G′ and G″ to revert to their initial values in every cycle.

A macroscopic healing test was also conducted to assess the gel's self-healing performance. As shown in Fig. 1j, the two-colored gels merged seamlessly within 10 min and supported their own weight and tensile force without separating. This indicated the gel's excellent self-healing ability due to dynamically crosslinked Schiff base networks. However, the mechanical properties of the gel after self-healing appeared to have changed when compared to the intact gel. Specifically, the self-healed gel exhibited reduced tensile strength and stretchable displacement (Fig. S6), which was consistent with results observed in rheological tests under alternating strain modes. The degradation behavior of the gel scaffold can reflect the extent of the sponge's reaction during the self-gelling process. Fig. 1l showed the degradation curves of gel scaffolds formed by OHN5@ε-pLx (x = 1 wt%, 3 wt%, or 5 wt%) sponges in PBS (pH 7.4). The mass residues of OHN@ε-pL₁, OHN@ε-pL₃, and OHN@ε-pL₅ gel scaffolds decreased to 41.67 ± 0.65 %, 50.77 ± 1.94 %, and 56.17 ± 0.42 % of their initial mass, respectively, within 10 h. The degradation rate plummeted after 10 h and stabilized after 50 h, likely due to the release of unreacted components into the solution. Further gel scaffold degradation occurred through amide bond hydrolysis. Whereafter, the degradation slowed as stable imide bond cross-links formed at pH 7.4, with increased crosslink density further reducing the rate (Fig. 1l). We also assessed the swelling and degradation properties of OHN@ε-pL₃ in its gelled state. As depicted in Fig. S7, OHN@ε-pL₃ was in a water-absorbing and swelling phase during the first 5 h after immersion in PBS. Afterward, the gel mass decreased, indicating degradation due to the solvent and oscillation. By 72 h, the gel mass reduced to its initial size, demonstrating stability in a pH 7.4 solvent for that period. Self-gelling sponges gel upon water contact. Movie S1 showed OHN@ε-pL₃ sponges transitioning phases when exposed to water. Tests indicated that OHN sponges compounded with ε-pL quickly absorbed water and swelled in 30 s. Concurrently, OHN and ε-pL dissolved and swiftly cross-linked into a gel. Unlike OHN sponges, which rapidly dissolve in water, OHN@ε-pL₃ sponges retained their shape after fully absorbing water and could be lifted with tweezers without damage, as shown in Movie S1. Crucially, this process occurred within 30 s, specifically 25.33 ± 5.03 s. Subsequently, the swollen gel then shrank in volume (Fig. S8). Overall, upon contact with aqueous phase, the self-gelling sponge experienced a process of water absorption and swelling, followed by self-gelling and volume shrinkage. To further assess whether the self-gelling sponge maintains its capability in blood, water was replaced with anticoagulated whole blood for testing. Fig. 1m displayed the self-gelling process of the OHN@ε-pL sponge when in contact with blood. As shown Movie S2, the sponge rapidly absorbed blood within 3 s without external pressure. After 5 min, a noticeable shrinkage in the gel's volume could be observed, likely due to the expulsion of excess liquid from the gel as internal cross-linked network forms, indicating the formation of a stabilized gel. Once a stable gel formed, the sponge demonstrated adhesive performance (Fig. 1m and Movie S2).

Further, we compared the liquid absorption ratios of OHN@ε-pL sponge with commercial hemostatic products (e.g., gauze, Yunnan Baiyao powder, and gelatin sponge) in water, PBS, and blood (Fig. 1n). The OHN@ε-pL₃ sponge demonstrated average liquid absorption ratios of 4725 ± 60.31 % for water, 5525 ± 85.63 % for PBS, and 5216 ± 76.2 % for blood, all significantly higher than commercial hemostatic products. Additionally, the OHN@ε-pL sponge demonstrated superior liquid absorption and self-expansion capabilities due to its loose and porous structure. It achieved an expansion ratio of 350.2 ± 15.85 %, significantly higher than the 133.0 ± 9.14 % of commercial hemostatic sponges (Fig. 1o). Unlike commercial sponges that passively absorb liquid, the OHN@ε-pL sponge can actively absorb and rapidly expand due to its excellent hydrophilic properties. Thus, when applied to deep wounds, the OHN@ε-pL sponge can exert superior physical pressure on the tissue wall and function like an “Expandable Hemostatic Sponge” to stop bleeding [36].

Given the self-gelling and self-expanding properties of the OHN@ε-pL sponge, it was not difficult to deduce that the OHN@ε-pL sponge initially self-expands upon contact with liquid. Once fully saturated, reactive groups in OHN and ε-pL trigger gelation, crosslinking, volume shrinkage, and water discharge. These properties confirmed the OHN@ε-pL sponge's self-hygroscopicity, rapid self-gelling, and effectiveness for deep, non-compressible wound hemostasis.

2.3. Adhesion, sealing and burst pressure properties of the OHN@ε-pL sponge after self-gelling

The gel hemostat should be able to cure rapidly in situ, adhere firmly to wound tissue, and possess sufficient mechanical strength and flexibility to handle diverse wound conditions [3,7]. It should provide strong adhesion to wet tissue, facilitating sealing moist and soft wounds [37]. Here, we studied the self-gelling and adhesion of OHN@ε-pL sponges between wet tissues (Fig. 2a). After placing a sponge between a PMMA plate with dSIS adhered to it and a wet pigskin for 5 min, and gently lifting the plate, the pigskin was also lifted, indicating that the OHN@ε-pL₃ sponge had been transformed into an adhesive gel and adhered to the tissue. Fig. 2b showed the morphology of the OHN@ε-pL₃ sponge after liquid absorption to fully gelled. When twiddled with fingers or adhered to the finger joints and kept the joint mobilization, the gel structure remained intact, showing excellent adhesiveness and deformability. Wound are commonly irregular shapes and endure stress from various orientations. To evaluate the shape adaptability and mechanical flexibility of the OHN@ε-pL₃ gel, it was applied to pigskin and subjected to stretching, extruding, and twisting to observe any detachment.

Fig. 2.

Self-gelling of OHN@ε-pL sponge, as well as adhesion and shape adaptability after gelation. (a) Photographs of OHN@ε-pL₃ sponge undergoing self-gelling and wet-adhesion processes between wet tissues. (b) Photographs of the morphology of OHN@ε-pL₃ sponge after self-gelling and the adhesion effect between fingers and at joints. (c) Photographs showing the adhesion capacity and mechanical compliance of OHN@ε-pL₃ sponge after self-gelling. (d) Water rinse-resistant photographs of OHN@ε-pL₃ gel after adhesion to the surface of pigskin, and representative SEM image of the adhesion interface between the gel and pigskin. (e) Sealing of a water-leaking porcine casing with OHN@ε-pL₃ gel. (f) The home-designed device for measuring burst pressure and (g) Schematic illustration of the burst pressure test. (h) Quantitative statistics of burst pressure for fibrin glue, OHA@ε-pL₃ and OHN@ε-pL₃ (n = 4). The blue area indicates normal systolic pressure of the human body (90–140 mmHg). (i) Schematic illustration of the self-gelling and adhesion mechanisms of the sponge between wet tissues, in three stages: i) active absorption interface water, ii) dissolution of the OHN and ε-pL components, and iii) groups reaction between OHN, ε-pL and tissues, leading to gel formation and adhesion. (∗∗∗∗p < 0.0001; ns: not significant).

As shown in Fig. 2c, the gel adapted well to the deformation of the pigskin without shedding or losing integrity. Its adhesion stability to tissue was then tested by rinsing with water (Movie S3). Fig. 2d demonstrated that after the OHN@ε-pL₃ sponge fully gelatinized on pigskin, rinsing with water did not detach the gel. SEM images revealed no distinct boundary between the gel and pigskin, suggesting the adhesion resulted from a chemical reaction rather than simple physical adsorption. Notably, the firm adhesion of the gel to the tissue effectively resisted blood pressure and exerted hemostatic effect through physical closure. Fig. 2e showed that the gel, once firmly adhered to the dSIS, could hold over 50 g of water without leaking. The OHN@ε-pL₃ gel's maximum burst strength measured by a home-designed device was 144.9 ± 7.24 mmHg, significantly higher than fibrin glue (92.60 ± 7.99 mmHg), OHA@ε-pL₃ (83.68 ± 8.37 mmHg), and normal systolic blood pressure (90–140 mmHg) in normal subjects (Fig. 2f–h). The mechanism underlying the wet tissue adhesion of the OHN@ε-pL₃ gel appears to be primarily attributed to the covalent bonding that arises from the reaction between the reactive aldehyde and N-succinimidyl ester groups within the gel and the amino groups present on the tissue surface. In addition to covalent bonding, hydrogen bonding, electrostatic interactions, and hydrophobic interactions likely play significant roles in establishing a robust network within the gel matrix and at its interface with tissue or other substrates, thus enhancing the adhesion strength (Fig. 2i).

2.4. Adhesive performance and adhesive strength

An ideal bio-adhesive promotes bio-integration and enhances tissue regeneration under pathological conditions [7,38]. The strong adhesive properties of the bio-adhesive ensure its stable attachment to the target tissue [34]. The adhesion capabilities of the OHN@ε-pL₃ gel on wet tissues were evaluated. Adhesion tests revealed that the OHN@ε-pL₃ gel adhered immediately upon contact with wet fresh organs (heart, liver, spleen, lungs, and kidneys) and was able to support its weight (Fig. 3a). More interestingly, the gel can also firmly stick to various surfaces such as glass, plastic, latex, wood, and steel, and can support over 35.1g of weight (Fig. 3b). The adhesive behavior of the gel can be ascribed to its numerous hydroxyl, amino, and carboxyl groups that create extensive hydrogen and covalent bonds at its interface with tissues or substrates, ensuring strong adhesion.

Fig. 3.

Adhesive performance and adhesive strength of the OHN@ε-pL₃ gel. (a) Photographs of OHN@ε-pL gel adhered to rat fresh organs or tissues. (b) Photographs of OHN@ε-pL gel adhered to substrates. (c) Schematic illustration of the Lap-shear test. (d) Representative time-stress curves of the Lap-shear test, F_max = 55.07 N. (e) Lap-shear strength of fibrin glue and OHN@ε-pL₃ gel (n = 3). (f) Schematic illustration of the Tensile adhesion test. (g) Representative time-stress curves of the Tensile adhesion test, F_max= 57.88 N. (h) Tensile strength of fibrin glue and OHN@ε-pL₃ gel (n = 3). (i) Schematic illustration of the T-peel test. (j) Representative time-stress curves of the T-peel test, F_plateau = 37.46 N. (k) Interfacial toughness of fibrin glue and OHN@ε-pL₃ gel (n = 3). (∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001).

Given the kinetic properties of tissues and organs, both shear and tensile stresses at the wound site possess the potential to induce wound rupture [39]. The OHN@ε-pL₃ gels were subjected to Lap-shear, Tensile and T-peel tests to assess their adhesion properties between two wet tissues, following the ASTM standards [40]. Fibrin glue was chosen as the control. The Lap-shear test revealed that OHN@ε-pL₃ gel had a shear strength of 142.9 ± 6.44 kPa, significantly higher than fibrin glue's 10.72 ± 1.01 kPa (Fig. 3c–e). In tensile test, OHN@ε-pL₃ gels exhibited an anti-adhesive tensile strength of 108.2 ± 19.20 kPa, which is 5.4 times higher than the 20.45 ± 12.78 kPa of fibrin glue (Fig. 3f–h). Meanwhile, it exceeded the maximum adhesive strength reported for most adhesive gels (Fig. S9). Similarly, in T-peel test, the interfacial toughness of OHN@ε-pL₃ gel (2437 ± 840.4J·m⁻²) was 7.5 times higher than that of the fibrin glue (325.0 ± 71.06 J m⁻²) (Fig. 3i–k). Thus, the OHN@ε-pL₃ sponge demonstrated superior mechanical properties compared to clinical fibrin glue post-gelation, suggesting its potential as a sponge-gel two-phase dressing with promising clinical application prospects.

2.5. Biocompatibility of the OHN@ε-pL sponge

The biocompatibility of hemostatic materials constitutes a fundamental prerequisite for their prospective clinical implementation [1]. Therefore, the biocompatibility of OHN@ε-pL sponges was investigated. The cytocompatibility of OHN@ε-pLx (x = 0.5 wt%, 1 wt%, 3 wt%, 5 wt%) sponge extracts in NIH-3T3 cells were assessed using the CCK-8 assay. The results indicated that all sponge extracts, except ONH@ε-pL₅ group, exhibited cell survival rates above 80 %, suggesting they were not significantly cytotoxicity (Fig. 4a) [32]. Similarly, the cell proliferation rate of the OHN@ε-pLx sponge extracts on NIH-3T3 cells was also assessed. The results indicated that on day 3, the proliferation rates of the OHN@ε-pL₁ and OHN@ε-pL₃ groups were significantly higher than the control group, while the OHN@ε-pL₅ group exhibited a lower proliferation rate than the control group. On day 5, cell proliferation showed a similar trend as that on day3, with the OHN@ε-pL₁ group exhibiting the highest proliferation, followed by the OHN@ε-pL₃ group (Fig. 4a). NIH-3T3 cells cultured in the extracts for 24 h were subjected to live/dead staining. Analysis of the fluorescence intensity from Calcein AM and PI staining revealed that all groups exhibited strong green fluorescence and weak or negligible red fluorescence, indicating high cell viability and favorable cytocompatibility of the extracts (Fig. 4b). In addition, the cytocompatibility of the extracts in HSF cells was also evaluated at the same method. Fig. 4c showed that the survival rate of HSF cells exceeded 80 % across all extract groups, as generally decreased with increasing ε-pL concentration. Overall, HSF cells proliferation in the extracts differed slightly from that of NIH-3T3 cells, most notably in the OHN@ε-pL₅ group. Similarly, live/dead staining of HSF cells revealed that most cells in all groups were alive (Fig. 4d) [32]. The cytocompatibility evaluation of OHN@ε-pLx (x = 0.5 wt%, 1 wt%, 3 wt%, 5 wt%) sponges showed that ε-pL concentration influenced cell viability, proliferation, and apoptosis similarly in fibroblasts from different sources. The alterations in pH and charge effects resulting from ε-pL solubilization are likely the principal factors contributing to cellular physiological activity [41]. Assays indicated that increasing ε-pL concentration lowered pH and enhanced positive charge effects on cell membranes. A 3 wt% ε-pL concentration is optimal, as it does not significantly disrupt normal cell functions, demonstrating the good cytocompatibility of the OHN@ε-pL₃ sponge.

Fig. 4.

Biocompatibility of the OHN@ε-pL sponges. (a) Cell viability of the NIH-3T3 cells after incubation with the OHN@ε-pLx (x = 0.5 wt%, 1 wt%, 3 wt% or 5 wt%) gel extracts for 24 h (n = 5), and proliferation of NIH-3T3 cells on days 1, 3 and 5. (b) Representative Live/Dead staining images of NIH-3T3 cells incubated with the gel extracts for 24 h (n = 3). (c) Cell viability of the HSF cells after incubation with the gel extracts for 24 h (n = 5), and proliferation of HSF cells incubated with the extracts on days 1, 3 and 5. (d) Representative Live/Dead staining images of HSF cells incubated with the gel extracts for 24 h (n = 3). (e) Hemolysis ratios of groups OHN@ε-pLx (x = 3 wt%, 5 wt%) (n = 3). The hemolysis rate at the red line is 5 %. Inset: Photograph of hemolysis test. (f) and (g) Representative H&E images of OHN and OHN@ε-pL₃ sponges and their surrounding tissues on days 3 and 7 (n = 3). The asterisks, triangles, and dashed lines represent the implanted gel, inflammatory cells, and the dividing line between normal tissue and the gel, respectively. “Tis.” and “Mat.” are abbr. for “tissue” and “material”, respectively. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, ns: not significant).

Hemocompatibility of OHN@ε-pL sponges was evaluated using hemolysis test as previously conducted [42]. As shown in Fig. S10, after addition of 5 % (v/v) erythrocyte stock solution to the TritonX-100 solution, the solution turned progressively clear and transparent, while in the PBS group, the RBCs remained evenly dispersed and intact. In OHN@ε-pL₃ and OHN@ε-pL₅ sponge groups, the RBCs were seen adhering and aggregating on their surfaces. After incubation at 37 °C for 1 h, uneven blood clots appeared on the gel surface in the OHN@ε-pL₃ and OHN@ε-pL₅ groups, suggesting no significant hemolysis in the sponge groups. The supernatant from each group was aspirated after centrifugation. As shown in Fig. 4e inset, all sponge group supernatants were clear and colorless, similar to the PBS group. The hemolysis rates for the OHN@ε-pL₃ and OHN@ε-pL₅ groups were 3.65 ± 0.46 % and 4.67 ± 0.35 %, respectively, both below 5 %, indicating good hemocompatibility for the OHN@ε-pLx (x = 3 wt%, 5 wt%) sponges (Fig. 4e).

The degradation and histocompatibility of the sponges in vivo were further evaluated by implanting them subcutaneously on the back of rats. As shown in Figs. S11a and S11b, on postoperative days 3 and 7, the skin at the implantation site was peeled off to examine the degradation of the subcutaneous sponge. No notable edema, congestion, or tissue necrosis around the implant were observed during the experimental (Fig. S11b). H&E staining revealed a significant acute inflammatory response at the material and tissue interface by day 3, with inflammatory cells present. After 7 days of implantation, inflammation and the number of inflammatory cells at the material and tissue interface significantly decreased. H&E staining also showed a fibrous capsule forming around the implanted material. By day 3, the size of the fibrous capsule had shrunk, likely due to the sponge implanted transformation into a gel, leading to the capsule to flatten. By day 7, the OHN sponge had largely degraded, forming a cavity in the capsule. Conversely, the OHN@ε-pL₃ sponge absorbed tissue fluid during implantation, transformed into a gel, and slowed down the degradation rate, thereby maintaining the filling of fibrous capsule (Fig. 4f and g). The results of H&E staining were consistent with the macroscopic morphology observed in subcutaneous OHN and OHN@ε-pL₃ degradation (Fig. S11b). Furthermore, no significant abnormalities were observed in the H&E staining of major organs in rats with subcutaneous implants after 7 days compared to the control group (Fig. S12). These results indicated that the OHN@ε-pL₃ sponge is biocompatible and degrades at an appropriate rate in vivo.

The exceptional biocompatibility of the OHN@ε-pL₃ sponge is ascribed to its specific composition. The OHN constituting the OHN@ε-pL₃ sponge is derived from HA, a physiologically active substance that aids in tissue repair, cyto-protection, and immune cell growth [43]. Another component, ε-pL, is a natural, non-toxic, and biocompatible product, which can reduce cytotoxicity and immune response [44]. Therefore, the developed OHN@ε-pL₃ sponge demonstrates excellent biocompatibility and holds significant potential as a versatile biomaterial for a wide range of applications.

2.6. In vivo hemostatic performance of the OHN@ε-pL sponge

Advanced hemostatic materials should be able to handle various bleeding scenarios. In this study, a standardized rat liver crucial incision, rat tail truncation, rat femoral vein incision, and rat pericardium incision bleeding model were used to evaluate the hemostatic efficacy of OHN@ε-pL sponges. Initially, the sealing hemostatic performance of the OHN@ε-pL₃ sponge was evaluated in vivo using a rat model of non-compressible liver bleeding. As depicted in Fig. 5a, a 10 mm cross incision was made in the rat liver, and hemostasis was performed using an OHN@ε-pL₃ sponge, with commercial gauze, gelatin sponge, and fibrin glue serving as controls (Movie S4). Fig. 5b showed that the blank group had the largest blood stains at 30, 60, and 120 s, indicating severe bleeding. The commercial gauze, gelatin sponge, and fibrin glue groups had progressively smaller stains, with the OHN@ε-pL₃ sponge group exhibiting the smallest stains. The blank group experienced the greatest blood loss (2702 ± 235.70 mg) and the longest hemostatic time (338.5 ± 55.94 s). Apparently, blood loss and hemostasis time were lower in OHN@ε-pL₃ sponge, commercial gauze, gelatin sponge, and fibrin glue groups compared to the blank group, whereas the OHN@ε-pL₃ sponge showed the greatest reduction, outperforming both the blank and commercial hemostatic agents, but was not significantly different from the fibrin glue group (Fig. 5c and d). The size of the blood stains observed on the filter paper after hemostasis was consistent with the quantitative measurements of blood loss. Likewise, the hemostatic effect of OHN@ε-pL₃ sponges in the rat tail-truncated bleeding model was comparable to that seen in hepatic hemostasis (Fig. S13a, Movie S5). Briefly, the OHN@ε-pL₃ sponge reduced blood loss by ∼89.38 % and shortened hemostasis time by ∼68.21 % compared to the control group. Similarly, in comparison to other commercial hemostatic agents, the OHN@ε-pL₃ sponge demonstrated substantial reductions in blood loss and a significant decrease in hemostasis time. The size of the blood stains on the filter paper corresponded to the quantified blood loss (Figs. S13b–S13d).

Fig. 5.

In vivo hemostatic efficacy of the OHN@ε-pL₃ in rat models. (a) Schematic illustration of the liver crucial incision bleeding model and hemostatic process. (b) Representative images of the bleeding and hemostatic process. (c) Summary of hemostatic time and (d) blood loss for different samples in the rat liver bleeding model (n = 4). (e) Schematic illustration of femoral vein bleeding model and hemostatic process in rats. (f) Representative images of the bleeding and hemostatic process. (g) Cumulative hemostatic time and (h) blood loss in the rat femoral vein bleeding model (n = 4). (i) Schematic illustration of pericardial bleeding model and hemostatic process in rats. The red dashed line represents the incision line in the epicardial surface, and the yellow dashed line represents the histological section incision line. (j) Photograph of an OHN@ε-pL₃ sponge patch on a beating rat heart in vivo. (k) Representative H&E images of the histological section from the pericardial bleeding model. (l) The inserted blue box area presents the bleeding site in contact with the OHN@ε-pL₃ sponge, and (m) the inserted green boxed area presents the sponge, gel, and tissue sections. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns: not significant).

Vascular injury generally leads to high-pressure blood flow and potentially fatal hemorrhage [1]. Thus, the hemostatic capacity of OHN@ε-pL₃ sponges was further challenged in a rat femoral vein bleeding model by incising the femoral vein in the hindlimbs to induce bleeding (Fig. 5e–Movie S6). In the control group, removing the hemostatic forceps from both ends of the vessel led to continuous bleeding for the first 120 s, with a blood loss of 5158 ± 376.60 mg, and uncontrolled bleeding lasting up to 355.0 ± 24.40 s. In contrast, the OHN@ε-pL₃ sponge rapidly absorbed blood, and formed an adhesive gel to seal the wound, resulting in an average blood loss of 453.5 ± 302.20 mg and a reduced bleeding time to 106.5 ± 36.20 s. Both blood loss and hemostatic time were significantly reduced in the OHN@ε-pL₃ group compared to the control group, with no secondary bleeding observed during the 10 min observation period. In terms of the size of blood stains resulting from blood loss across the different groups, the control group exhibited the largest blood stains, followed sequentially by the commercial gauze, gelatin hemostatic sponge and fibrin glue groups, Notably, the OHN@ε-pL₃ sponge group demonstrated the smallest blood stains (Fig. 5f). Based on the statistical analysis of blood loss and hemostatic time, the three commercial hemostatic agents showed significantly lower blood loss and hemostatic time compared to the control group, but higher than the OHN@ε-pL₃ sponge group (Fig. 5g and h). And the blood stain sizes on the filter paper corroborated the blood loss statistics (Fig. 5f and g). The OHN@ε-pL₃ sponge demonstrated strong hemostatic performance in a femoral vein bleeding model, indicating its promising potential for effectively sealing acute hemorrhage from vascular rupture. Furthermore, to assess the adhesion of OHN@ε-pL₃ sponges to wet, dynamic tissue in vivo, a 5 mm bleeding incision was made in rat pericardium, and the hemostatic effect of the sponge on the epicardial surface was evaluated (Fig. 5i–Movie S7). Fig. 5j showed that the cylindrical OHN@ε-pL₃ sponge adhered firmly to the epicardial surface of a beating heart after being pressed for 5 s, allowing for complete absorption of the tissue interface fluid. Subsequently, the sponge established a firm adhesion to the epicardial surface, effectively preventing hemorrhage. The sponge adhering to the epicardial surface can even resist forceps pulling. Simultaneously, once the self-gelling sponge adhered stably to the tissue surface, it can also be removed with increased tensile force, essential for changing dressings or preventing secondary injury and infection [13,45]. Pathologic sections were performed in the vertical direction extending the incision. Fig. 5k showed the H&E staining of the sponge attached to the epicardium. Enlarged views of the hemorrhage site and its surrounding tissues (Fig. 5l and m) revealed that OHN@ε-pL₃ sponges at the hemorrhage site were filled with RBCs, and blood clotting formed in the epicardial defect, with some gelatinized sponge between the defect and the outer sponge (Fig. 5l). Similarly, as can be seen in Fig. 5m, the part attached to the epicardial surface was in the gel phase, while above the gel was the untransformed sponge phase. These observations further demonstrated that during hemostasis, the OHN@ε-pL₃ sponge first quickly absorbed blood or interfacial water, then transformed into a gel that adhered to tissue and formed a physical barrier to stop bleeding.

Based on the hemostatic effects observed across various bleeding models, the OHN@ε-pL₃ sponge exhibited superior hemostatic capacity compared to the currently available commercial hemostatic agents. The OHN@ε-pL₃ sponge's porous structure enables quick blood absorption and attracts coagulation factors to aid clotting. Its self-hygroscopic properties convert interfacial fluids into solvents, which dissolve the sponges and expose their reactive groups like -NH₂ and -NHS esters. The reactive groups interact with each other or with tissue and blood components to form a gel or adhere, creating a strong barrier to stop bleeding. Additionally, the positively charged ε-pL in the sponge can attract blood cells, platelets, and plasma fibronectin, promoting blood clotting via electrostatic interactions [46]. Thus, the synergy of multiple mechanisms of the OHN@ε-pL₃ sponge greatly enhances its hemostatic effectiveness, making it suitable for various bleeding scenarios.

2.7. In vitro blood-clotting activity and hemostatic mechanism of OHN@ε-pL sponge

Activation of coagulation factors is crucial for initiating the endogenous coagulation pathway to exert hemostasis [5]. Here, the activation effect of the OHN@ε-pL₃ sponge on coagulation factors was performed through in vitro coagulation assays. Fig. 6a illustrated the coagulation mechanism of the OHN@ε-pL₃ sponge in a rat liver bleeding model. SEM detection of the liver hemorrhage site revealed that the OHN@ε-pL₃ gel adhered tightly to the liver, with dense RBCs present within the gel, likely attracted by the gel's self-hygroscopic properties before its transition (Fig. 6b). A high-magnification SEM image of the gel's interior revealed a dense distribution of RBCs, with their surfaces covered by a network of fibrin filaments from activated coagulation pathways (Fig. 6c) [10,19]. It's reported that the adhesion and aggregation of RBCs and platelets, along with platelet activation, are essential for blood clotting and hemostasis [1,47]. To better understand the coagulation mechanism, RBCs and platelets adhesion assays were conducted. Initially, SEM was employed to characterize the adhesion morphology of RBCs and platelets on the material (Fig. 6d). As can be seen, a large number of RBCs aggregated on the gel surfaces, and there was no significant difference in RBCs density among these groups. Interestingly, RBCs in the OHN@ε-pL₃ group were surrounded by numerous filamentous fibrins, aligning with in vivo observations (Fig. 6c). Conversely, the groups showed significant differences in platelet surface distribution. The OHN@ε-pL₃ group showed increased platelet aggregation and notable shape changes, transforming from irregular discs to shuttle or polygonal forms, with their surfaces covered in crisscrossed fibrin, compared to the gauze, gelatin sponge, and fibrin glue groups.

Fig. 6.

In vitro coagulation property of the OHN@ε-pL₃ sponge. (a) Schematic illustration of coagulation mechanism of OHN@ε-pL₃ sponge for in vivo liver hemostatic applications. (b) SEM image of the liver (orange pseudo-color section), and OHN@ε-pL₃ gel (brown section) adhering to the liver bleeding site. The red pseudo-color section presents the RBCs inside the gel, Magnification 200 × , Scale bar = 30 μm, and (c) High-magnification SEM images of the internal view of OHN@ε-pL₃ gel after liver hemostasis. The blue arrow indicates the fibrin formed during clotting. Magnification 2000 × , Scale bar = 5 μm. (d) Representative SEM images of RBCs (red pseudo-color) and platelets (orange pseudo-color) adhering to gauze, gelatin sponge, fibrin glue, and OHN@ε-pL₃ sponge samples. Magnification 2000 × , Scale bar = 5 μm. (e) Schematic illustration of the pro-coagulant mechanism of the OHN@ε-pL₃ sponge. (f) Schematic illustration of coagulation induced by addition of hemostatic agents to anticoagulated whole blood. (g) Representative images of time-dependent blood clotting behavior. The green dashed box represents the time clotting occurred. (h) Quantitative statistics of clotting time (n = 3). (i) BCI values at different time points. (j) and (k) Adhesion rates of RBCs and platelets (n = 3). (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns: not significant).

The principal functions of platelets are to clot and stop bleeding, as well as to repair ruptured blood vessels. Meanwhile, prothrombin in plasma converts to thrombin, which then transforms fibrinogen into fibrin, an elongated fibrous protein. The fibrin fibers combine with RBCs to form a blood clot, achieving hemostasis [48]. SEM observations showed that OHN@ε-pL₃ sponges activated platelets and produced fibrin, which could form a “fibrous wall” with RBCs to block the wound. This indicated that OHN@ε-pL₃ sponges activated the coagulation pathway more effectively than other groups. Additionally, ε-pL is highly cationic, enabling it to bind to negatively charged cell surfaces and promote cell adhesion through charge interactions [44]. In summary, the hemostatic performance of the OHN@ε-pL₃ sponge might rely on its porous structure, electrostatic effects, and interaction with cell membranes during its transition from sponge to gel, which helps recruit RBCs and platelets and activate coagulation pathway, exerting hemostatic effects (Fig. 6e).

Furthermore, the clotting capacity of OHN@ε-pL₃ sponges was validated in vitro by detecting the clotting time after co-culture with whole blood (Fig. 6f–h). Coagulation assays indicated a clotting time of about 7 min in the blank group, aligning with previous reports [1,10]. Incubating anticoagulated blood with gauze, gelatin sponge, and fibrin glue resulted in clotting times of about 5, 4, and 3 min, respectively, all significantly faster than the blank group, showing varying procoagulant effects. The OHN@ε-pL₃ sponge notably activated platelets, shortening the coagulation time to 2 min. The stable clotting times after co-incubation with whole blood were 461.7 ± 25.17 s for the blank, 308.3 ± 12.58 s for gauze, 271.7 ± 16.80 s for gelatin sponge, 214.7 ± 11.72 s for fibrin glue, and 156.3 ± 18.72 s for the OHN@ε-pL₃ sponge, aligning with observed coagulation phenomena (Fig. 6g and h). The blood clotting index (BCI) was measured to assess the procoagulant properties of the OHN@ε-pL₃ sponge; a lower BCI indicates superior clotting ability [1,3]. It was observed that the BCI value of the OHN@ε-pL₃ sponge group was significantly lower than that of other groups before 6 min (Fig. 6i), demonstrating its superior and effective clotting ability among all hemostatic agents. In the quantitative test, the OHN@ε-pL₃ sponge group exhibited the highest RBCs and platelets adhesion, surpassing fibrin glue, gelatin sponge, gauze, and the blank control group (Fig. 6j and k). Interestingly, the statistics of adherent RBCs and platelets in the adhesion test appeared to be inconsistent with macro-observations, that is because their adhesion to the OHN@ε-pL₃ sponge is predicated on a coagulation reaction, rather than deposition or physical adsorption (Fig. 6d). In contrast, in the gauze, gelatin sponge, and fibrin glue groups, insufficient coagulation led to differential RBCs or platelets adhesion, possibly due to differing experimental procedures. Further, we evaluated the hemostatic mechanism of the hemostatic sponge by examining four key coagulation parameters (APTT, PT, Fib and TT). Research showed that coagulation can be divided into endogenous and exogenous pathways, depending on the initiation process and the prothrombin activators involved [13,49]. The activated partial thromboplastin time (APTT) primarily reflects the condition of the endogenous coagulation system, while the prothrombin time (PT) pertains to the exogenous system. The results of the hemostatic sponge's coagulation tests were depicted in Fig. S14. Compared to the control group, the shortened APTT indicated that the hemostatic sponge enhanced coagulation factor activity. Increased fibrinogen levels (Fib) and decreased thrombin time (TT) suggested that the sponge accelerated hemostasis by boosting coagulation factor activity, increasing fibrinogen content, and expediting the conversion of fibrinogen to fibrin. Conversely, a decrease in PT signified a relatively poor performance in activating the exogenous coagulation pathway, showing that the hemostatic sponge mainly promoted hemostasis via the endogenous pathway.

In summary, studies on in vitro coagulation activity and hemostatic mechanisms of OHN@ε-pL₃ sponges have demonstrated that their rapid hemostatic efficacy might be attributed to the synergistic effects of rapid blood absorption, self-gelling properties, and promoting the activation of endogenous coagulation pathways.

2.8. Wound closure capacity of OHN@ε-pL sponge

The prompt and efficient closure of damaged skin serves not only to safeguard the internal environment but also to establish a moist physiological milieu conducive to wound healing [32,50]. To investigate the wound closure efficiency of the OHN@ε-pL₃ sponge, a full-thickness skin incision was constructed on the backs of rats (Fig. 7a) to assess its adhesion and wound closure capabilities in vivo. Immediately after the incision of the skin, surgical sutures or commercial tissue glue, likely Beixiu® fibrin glue and Histoacryl® cyanoacrylate glue, were applied as controls. When OHN@ε-pL₃ sponge was applied to the incision, it quickly absorbed the blood and formed an adhesive gel that adhered to the wound (Fig. 7b). After 3 days, the incisions in the blank, surgical suture and fibrin glue groups showed no dehiscence and were crusted, while those in the cyanoacrylate group were significantly dehiscent. Upon applying the OHN@ε-pL₃ sponge to the incision, it rapidly transformed into a gel and firmly sealed the wound. By day 5 after treatment, the scabs in the blank control group had partially detached, and the incisions exhibited partial dehiscent due to tension in the dorsal skin. Likewise, the incisions in the cyanoacrylate group remained dehiscent, while those in the OHN@ε-pL₃ sponge group were well-bridged by day 5, with scabs shedding and fully healed by day 7. In contrast, the incisions in the blank, surgical suture, and fibrin glue groups had shrunk by day 7 but were not fully healed, with scabs still present. Meanwhile, the cyanoacrylate group showed smaller incisions, but dehiscence persisted, likely due to undegraded adhesive residue obstructing incision closure and tissue healing. By day 10, the incisions treated with OHN@ε-pL₃ sponges fully healed without visible scars. Yet, the surgical suture group had fully healed wounds with visible scars, whereas the blank, fibrin glue, and cyanoacrylate groups had incompletely healed wounds (Fig. 7b and c). Observations showed that sutures and commercial adhesives performed similar effects to previous reports [38,51], while OHN@ε-pL₃ sponges exhibited superior tissue adhesion and wound healing performance.

Fig. 7.

In vivo adhesion and wound closure performance. (a) Schematic illustration of adhesive glue application in rats full-thickness skin incision model. (b) Representative photographs of rat dorsal incision skin treated with sutures, fibrin glue, cyanoacrylate glue, and OHN@ε-pL₃ sponge (n = 4). (c) Schematic of the dynamic wound healing process on days 0, 3, 5, 7, and 10. (d) and (e) H&E staining and Masson staining of the wound sites on postoperative day 7. The orange dotted lines indicate the boundary of the unhealed incisional wound. Inset: local magnification of H&E staining or Matson staining. Scale bars = 5 mm (images in b), 500 μm (images in d, e), 100 μm (inset in d, e).

Histologic analysis of the skin at the incision sites was performed with H&E and Masson staining on day 7 after surgery. H&E staining revealed complete regeneration of the OHN@ε-pL₃ sponge-treated epidermis, with visible hair follicles and sebaceous glands near the incisions (Fig. 7d). However, significant unhealed dermal gaps were observed in the control, suture, fibrin glue, and especially the cyanoacrylate groups, with the cyanoacrylate group showing widest gaps. This is likely due to cyanoacrylate's poor degradability and cytotoxicity, which hinder wound healing [38]. Quantification analysis of the incision dermal gap on day 7 revealed that the dermal gaps in the OHN@ε-pL₃ sponge-treated group were significantly smaller compared to other four groups. Additionally, significant inflammatory cells were observed in the dermis of the fibrin glue and cyanoacrylate groups (Fig. S15). Masson staining performed on day 7 revealed that collagen deposition within the OHN@ε-pL₃ sponge groups was dense, well-organized, and closely resembled the structure of normal skin (Fig. 7e). These findings indicate that the OHN@ε-pL₃ sponge positively impacted wound closure and facilitated the wound healing process.

2.9. Wound healing effect of OHN@ε-pL sponge on infected skin wound

Severe wound contamination from war or civilian trauma can cause infections that may lead to serious consequences even death [20]. The antibacterial activity of the OHN@ε-pL₃ sponge against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was evaluated in vitro. In vitro antibacterial assays were conducted by incubating OHN@ε-pL₃ and OHN@ε-pL₅ sponges with bacteria for 6 h. The antibacterial effects were assessed using agar plates and live/dead staining, with PBS as the negative control and ampicillin (AMP) as the positive control. Fig. 8a and b revealed the antibacterial effect. In the PBS group, colony counts were 691.3 ± 40.80 × 10² for E. coli and 848.3 ± 26.41 × 10² for S. aureus. In contrast, significantly fewer colonies were observed in the AMP and OHN@ε-pLx (x = 3 wt% and 5 wt%) sponge treatment groups, with 256.0 ± 55.38 × 10², 35.67 ± 4.93 × 10² and 34.67 ± 2.52 × 10² colonies for E. coil, respectively, and 123.3 ± 7.57 × 10², 116.0 ± 21.63 × 10² and 68.0 ± 6.08 × 10² colonies for S. aureus, respectively, compared to the negative control group. Additionally, the antibacterial activity of OHN@ε-pL₃ sponges was further tested using live/dead bacterial staining kits. Fluorescent staining revealed strong green fluorescence in live E. coli and S. aureus in the control group, while those treated with the self-gelatinized OHN@ε-pL₃ sponge showed mainly red fluorescence, indicating disrupted bacterial cell membranes (Fig. 8c) [32,52]. The live bacteria's fluorescence intensity was consistent with the observed staining (Fig. 8d). The antibacterial efficacy of the OHN@ε-pL₃ sponge, as determined by both the agar plate method and live/dead staining, demonstrated consistency, thereby confirming the sponge's superior in vitro antibacterial effectiveness. Based on prior studies, we attribute the inhibitory performance of OHN@ε-pL₃ sponge to the positively charged ε-pL, which interacts with negatively charged bacterial membranes. This interaction disrupts the bacterial membranes by altering their surface zeta potential, thereby inhibiting bacterial activity. Additionally, ε-pL inhibits bacterial growth by disrupting the transmembrane pH gradient, increasing cell membrane permeability, inhibiting protein synthesis, degrading proteins, or binding to DNA to block transcription (Fig. 8e) [32,53].

Fig. 8.

In-vitro/-vivo antibacterial and wound healing performance of the OHN@ε-pL sponge. (a) Representative images of E. coli and S. aureus colonies on agar plates and (b) Quantitative counting of the colonies number (n = 3). (c) Live/dead fluorescence staining images of E. coli and S. aureus (Green, live bacteria; red, dead bacteria. Scale bar = 25 μm). (d) Quantification of the relative fluorescence intensity of living bacteria (n = 5). (e) Schematic illustration of the antibacterial mechanisms of OHN@ε-pL sponge. (f) Schematic illustration of S. aureus-infected mouse full-thickness skin wounds model treated with OHN@ε-pL₃ sponge. (g) Representative images of the wounds treated with SilverCare® Ag⁺ gel, OHN, and OHN@ε-pL₃ sponge on days 0, 3, 7, 10, and 14, and (h) Corresponding dynamic wound healing process. (i) Bacteria collected from the wounds on days 3 after treatments, and cultured on agar plates for colonies number counting. (j) H&E staining and (k) Masson staining images of the wound tissue on days 7 and 14. Scale bar = 2000 μm (Original magnification, 10×), 200 μm (Original magnification, 80×). (l) Representative images of CD31 immunostaining on days 7 and 14. (m) Wound healing rates at days 3, 7, 10, and 14 after treatment (n = 5). (n) Counting of colonies in wounds on day 3 (n = 3). (o) The granulation tissue thickness in wound tissue on days 3 and 7 (n = 5). (p) Collagen deposition ratio in wound tissue on days 7 and 14 (n = 5). (q) Quantification of the number of blood vessels in the wound tissue on days 7 and 14 (n = 5). (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns: not significant).

To further investigate the in vivo antibacterial and wound healing effects of the OHN@ε-pL₃ sponge, a mouse model of dorsal full-thickness skin wound infection with S. aureus was established in a P2 biosafety lab. 24 h after inoculation of S. aureus on the wounds, treatment with SilverCare® Ag⁺ gel, OHN, or OHN@ε-pL₃ sponge was administered. Wound sizes were monitored and recorded at specific intervals, and samples were collected (Fig. 8f). By the 2nd day post-inoculation with S. aureus (recorded as day 0), a white bacterial biofilm covered all wound surfaces, confirming the successful establishment of the infection model (Fig. 8g). Biofilms obstruct drug penetration and absorption; therefore, clinical practices and current research often involve the removal of biofilms or the use of microneedle drug delivery systems to enhance drug absorption and efficacy. For instance, P. Makvandi et al. developed a skin tissue model and validated its advantages in microneedle drug delivery system research, as well as reviewed the progress of existing antimicrobial microneedle drug delivery systems [54,55]. In the current study, biofilms were meticulously excised using tweezers prior to the application of the dressing, thereby facilitating direct contact between the self-gelling sponge and the subcutaneous tissue. On day 3, after removing the intervention materials, noticeable bacterial biofilm still remained in the control and OHN sponge-treated groups, while the Ag⁺ gel and OHN@ε-pL₃ sponge-treated groups showed no visible biofilm. Fresh red granulation tissue appeared on wounds treated with OHN@ε-pL₃ sponge, with no significant difference in healing rates among the groups by day 3 (Fig. 8m). Flushing fluid from each wound on day 3 was taken for plate coating, and colonies were counted (Fig. 8i). Notably, the colony counts for the Ag⁺ gel and OHN@ε-pL₃ sponge groups were significantly lower (220.7 ± 45.46 × 10³ and 258.0 ± 30.12 × 10³, respectively) compared to the control and OHN sponge groups (2464 ± 277.8 × 10³ and 974.0 ± 67.29 × 10³, respectively) on day 3 (Fig. 8n). On days 7 and 10, the OHN@ε-pL₃ sponge-treated group had significantly smaller wound areas and higher healing rates at 30.5 ± 11.96 % and 78.18 ± 2.88 %, respectively, compared to the other groups (Fig. 8m). Furthermore, by day 10, wounds in the OHN@ε-pL₃ sponge groups displayed evident epithelialization and were almost healed. The Ag ⁺ gel group exhibited a faster wound healing rate compared to the control and OHN sponge groups, likely because the antibacterial properties of both Ag⁺ gel and OHN@ε-pL₃ sponge effectively alleviated wound infection and inflammation, thereby accelerating wound healing. The in vivo antibacterial performances were essentially comparable to the in vitro results (Fig. 8i and n). By day 14, the wounds in the OHN@ε-pL₃ sponge group were almost completely healed, with a healing rate of 98.65 ± 0.38 %, significantly outperforming the other groups. Also, the wound traces over time reflected that the OHN@ε-pL₃ sponge accelerated healing process of infected wounds more effectively than the control, Ag⁺ gel, and OHN sponge groups (Fig. 8h).

Subsequently, histological analysis of the infected wound tissue was performed using H&E and Masson staining. H&E staining revealed that on day 7, the control group exhibited the largest wound area, with significant inflammatory cell infiltration. Conversely, the OHN@ε-pL₃ sponge group showed the smallest wound area and fewer inflammatory cells in the granulation tissue (Fig. 8j and Inset). By day 7, the wound treated with the OHN@ε-pL₃ sponge exhibited a significantly greater thickness of granulation tissue compared to the blank and OHN sponge groups, with similar growth trends observed on day 3 (Fig. 8o). Pathologic analysis indicated that the OHN@ε-pL₃ sponge has modulated the wound's immune microenvironment, thereby promoting wound healing and tissue reconstruction. By day14, macroscopic view and H&E staining of the wounds demonstrated a reduction in wound size in all groups. Notably, the OHN@ε-pL₃ sponge group exhibited no significant inflammatory cells in H&E staining, with mature granulation tissue and minimal skin appendage formation. Collagen deposition plays a vital role in wound healing process, significantly influencing the skin's mechanical strength and serving as a key indicator of scar formation [3,56]. Masson staining revealed greater collagen deposition in wounds treated with Ag⁺ gel and OHN@ε-pL₃ sponge by day 7 (Fig. 8k and Inset, and 8p). In the blank control group, collagen fibers were loosely arranged, whereas in the OHN@ε-pL₃ sponge-treated group, they were densely and orderly arranged (Fig. 8k and Inset). Moreover, collagen deposition in the OHN@ε-pL₃ sponge group was also higher compared to other groups, resembling normal skin (Fig. 8p). The Masson staining revealed that the OHN@ε-pL₃ sponge promoted collagen deposition more effectively during wound healing.

Vascular dysfunction constitutes a primary factor contributing to impaired wound healing [57]. To assess the impact of OHN@ε-pL₃ sponge on wound healing angiogenesis, CD31 expression was analyzed using immunofluorescence staining. Infected wounds treated with OHN@ε-pL₃ sponges exhibited significantly higher CD31 expression on day 7 compared to the control and OHN sponge groups, suggesting enhanced angiogenesis at the wound site (Fig. 8l and q). By day 14, the OHN@ε-pL₃ sponge group showed fewer blood vessels than on day 7, suggesting the wound is shifting from the proliferative phase to the remodeling phase, based on the pattern of angiogenesis during wound healing [38,58]. Interestingly, the Ag⁺ gel group showed a similar blood vessel density trend to the OHN@ε-pL₃ sponge group at days 7 and 14, suggesting that preventing wound infection accelerates angiogenesis and healing. In summary, OHN@ε-pL₃ sponge accelerated the healing process of infected wounds in full-thickness skin wounds in mouse by promoting granulation tissue regeneration, angiogenesis, and collagen deposition.

IL-6 and TNF-α are versatile cytokines essential for regulating inflammation [32]. Therefore, IL-6 and TNF-α are used to indicate the inflammatory response in wound healing. Fig. 9a–c indicated that on days 3 and 7, the blank control group showed the highest levels of IL-6 and TNF-α in granulation tissues, while the OHN@ε-pL₃ group exhibited the lowest levels, followed by the Ag⁺ gel group. Interleukin-10 (IL-10) is a crucial anti-inflammatory cytokine that suppresses immune cell activation and cytokine production [59,60]. IL-10 expression on days 3 and 7 was higher in the Ag⁺ gel and OHN@ε-pL₃ sponge groups compared to the blank control and OHN sponge groups (Figs. S16a and S16b). These findings further confirmed that preventing wound infections can alleviate excessive inflammation, which can otherwise cause hypertrophic scars or chronic non-healing wounds [61]. Immunohistochemical analysis and quantification of IL-6, TNF-α, and IL-10 in wound tissues revealed that both the Ag⁺ gel and OHN@ε-pL₃ sponge reduced inflammation in infected wounds, with the OHN@ε-pL₃ sponge showing the best performance. VEGF plays an important role in the inflammatory and proliferative stages, promoting vasodilation, and the migration and proliferation of keratinocytes and endothelial cells [62]. VEGF staining was performed on the wound granulation tissues obtained on days 3 and 7 to assess the progress of wound healing. The staining results showed that the OHN@ε-pL₃ sponge group exhibited increasingly stronger VEGF expression from day 3 to day 7, significantly surpassing that of the other groups (Fig. 9a and d). The high VEGF expression levels may result from the OHN@ε-pL₃ sponge's antibacterial properties, which inhibited excessive inflammation and encouraged tissue cell growth and migration via immunomodulation [3,63]. Furthermore, ELISA kits were also used to evaluate the levels of IL-6, TNF-α, IL-10, and VEGF in granulation tissue on day 7. The results aligned with immunohistochemistry findings (Fig. 9e), showing that the OHN@ε-pL₃ sponge group had the lowest IL-6 and TNF-α levels, and the highest IL-10 and VEGF levels among all groups. These results indicated that the OHN@ε-pL₃ sponge can effectively inhibit inflammation in S. aureus-infected wounds and accelerate healing process by inhibiting inflammatory factor overexpression.

Fig. 9.

Immunohistochemical staining of wound granulation tissue on days 3 and 7. (a), (b), (e) and (d) Representative images and quantification of IL-6, TNF-α, and VEGF staining in the granulation tissue, Magnification 800×, Scale bar = 50 μm. The blue arrow indicates IL-6, TNF-α, and VEGF-positive cells, respectively. (e) ELISA quantification of IL-6, TNF-α, VEGF, and IL-10 factors in granulation tissue. (f) Schematic illustration of OHN@ε-pL sponges exerting hemostasis, anti-inflammation, and promoting proliferation and reconstruction in injured tissues. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns: not significant).

These promising experimental results revealed that the easy-to-prepare OHN@ε-pL₃ sponges possesses suitable biocompatibility and is capable of absorbing blood and transforming it to a gel, simultaneously enriching coagulation factors. The formed OHN@ε-pL₃ gel adheres firmly to the skin, serving as a barrier to prevent bleeding and protect the wound from infection. The physical sealing effect of the OHN@ε-pL₃ gel, combined with the activation of the coagulation pathway, can effectively prevent wound bleeding. Additionally, the antibacterial properties of the OHN@ε-pL₃ gel inhibit excessive inflammation in infected wounds and promotes angiogenesis and skin tissue reconstruction. In summary, OHN@ε-pL₃ sponge dressing effectively regulates various wound phases — hemostatic, inflammatory, proliferative, and remodeling — by quickly halting bleeding, reducing inflammation, enhancing collagen deposition and angiogenesis, thus accelerating epithelialization and skin healing (Fig. 9f). These findings underscore the substantial potential of the OHN@ε-pL₃ sponge for clinical applications.

However, the application study of the OHN@ε-pL₃ sponge in this research has some limitations. Specifically, we have only assessed the short-term effects of the OHN@ε-pL₃ sponge on hemostasis and wound healing in small animal models, without evaluating its effectiveness in larger animal models or its potential long-term impact on tissue healing and regeneration. In future studies, we will further investigate the sponge's hemostatic and tissue healing effects in large animal models, as well as its long-term impact, to provide evidence for the necessity of scaling up production and the feasibility of potential clinical applications.

3. Conclusion

In this study, we engineered a polysaccharide-based sponge (OHN@ε-pL₃) exhibiting self-hygroscopic properties, rapid self-gelling capability, and strong wet adhesion. This was achieved by integrating an N-succinimidyl ester oxidized hyaluronic acid (OHN) sponge with ε-poly-L-lysine (ε-pL) in a methanol solution. The sponge demonstrated the capacity to swiftly absorb water from blood or tissue interfaces, which serves as a solvent for OHN and ε-pL to dissolve and expose its reactive groups, thus enabling the phase transition from sponge to gel as these groups undergo chemical reactions. This phase transition eliminates tissue interfacial water, thereby facilitating interactions between the gel and the tissue interface to achieve wet adhesion. The cationic ε-pL in the gel promotes the aggregation and adhesion of RBCs and platelets, activating intrinsic coagulation pathways. The self-gelling and wet adhesion properties of the OHN@ε-pL₃ sponges synergize with intrinsic coagulation pathway activation, significantly improving hemostatic function, as demonstrated by their superior performance in models of rat liver, truncated tail, femoral vein, and cardiac bleeding. Furthermore, OHN@ε-pL₃ sponges possess excellent biocompatibility and antibacterial properties. In a full-thickness skin defect model in mouse infected with S. aureus, the OHN@ε-pL₃ gel exhibited superior antibacterial activity, and significantly attenuated inflammation in infected wounds and enhanced collagen deposition, angiogenesis, and re-epithelialization, accelerating wound healing process. In conclusion, our study demonstrated that the OHN@ε-pL₃ sponge represents an optimal candidate for hemostatic dressings in management of clinical acute and/or non-compressible hemorrhages, preventing infection and accelerating wound healing.

4. Experimental section

Preparation of the OHA、OHN and OHN@ε-pL sponges: Oxidized hyaluronic acid (OHA) and N-succinimidyl ester oxidized hyaluronic acid (OHN) were first prepared, with detailed synthesis procedures available in Supporting Information. OHA and OHN solutions of varying concentrations were prepared, poured into the molds, and freeze-dried with a lyophilizer (FD-1A-50, Shanghai Shun system Instrument Manufacturing Co., Ltd., China) to produce OHA and OHN sponges with different mass percentages. ε-pL was dissolved in methanol to create solutions with concentrations of 0.5 wt%, 1 wt%, and 3 wt%. The solvent utilized for the 5 wt% ε-pL solution comprised a mixture of methanol and water in a 9:1 vol ratio. OHA or OHN sponges were soaked in ε-pL methanol solution for 5 min with gentle shaking, then vacuum dried using a vacuum dryer (DZF6020, Shanghai Shanzhi Instrument Co., Ltd., China) to create OHA@ε-pL or OHN@ε-pL sponges. The OHN@ε-pL sponge's chemical structure was analyzed using 1H NMR, FTIR spectroscopy, and SEM, as detailed in the Supporting Information.

Rheological Properties Study: The viscosity and rheological properties of the sponge's solutions or gels were measured with a rotational rheometer (MCR302, Anton Paar, Austria). Further details are in the Supporting Information.

Characterization of OHN@ε-pL sponges and the properties after self-gelling: To investigate the physicochemical properties of OHN@ε-pL sponges and their self-gelling process, the following analyses were performed by referring to previously reported methods [38]: self-healing behavior test, in vitro degradation tests, liquid absorption ratio test, self-expanding test, tissue adhesion test, burst pressure test, and adhesion strength test. Details are provided in the Supporting Information.

Cytocompatibility Evaluation: The cytocompatibility evaluation involved assessing cytotoxicity, cell proliferation, and live/dead cell staining. The effects of OHN@ε-pL sponges on NIH-3T3 and HSF cells were evaluated using CCK-8 and live/dead assays, with details in the Supporting Information.

Hemocompatibility and In Vivo Biocompatibility Evaluation: The hemolytic activity assay followed a previously described protocol [1,42]. The biocompatibility of OHN@ε-pL sponges was tested by implanting them subcutaneously in the backs of SD rats (6–8 weeks old, 200–250 g, male). Tissues together with the implant were collected at specific intervals post-implantation for H&E analysis. All animal procedures were reviewed and approved by the Laboratory animal care and ethics committee of Guangdong Laidi Biomedical Research Institute Co., LTD (Ethics number: 2023005-2). Details are described in the Supporting Information.

In Vivo Hemostatic Test in Rat Model: The hemostatic effect of OHN@ε-pL sponges was evaluated by constructing bleeding models of liver, femoral vein, cardiac, and tail truncation in rats using SD rats (8–10 weeks old, 250–300 g, male). Commercial gauze, gelatin hemostatic sponge, and fibrin glue served as controls. Rats were anesthetized with a 1.5 wt% sodium pentobarbital (100 g mL⁻¹) intraperitoneal injection during surgery. The details are shown in the Supporting Information.

In Vitro Blood Clotting, RBCs/Platelets Adhesion and Aggregation Evaluation of the OHN@ε-pL sponge: Whole blood clotting, RBCs adhesion, and platelet aggregation tests were conducted to study the clotting of the OHN@ε-pL sponge. The methodology was adapted from previous reports [1]. Detailed procedures are given in the Supporting Information.

Skin Incision Adhesion Assessment: The in vivo tissue adhesion and wound closure ability of OHN@ε-pL₃ sponges were tested on a rat skin incision model, using sutures, fibrin glue, and cyanoacrylate as positive controls, and saline as a negative control [38]. Detailed procedures are in the Supporting Information.

In Vitro Antibacterial Tests: The antibacterial activity of OHN@ε-pL sponges was evaluated against E. coli (ATCC25922) and S. aureus (ATCC6538). Detailed procedures are provided in the Supporting Information.

Wound Healing Effect of OHN@ε-pL Sponge on S. aureus - Infected Skin Wound: The therapeutic effect of OHN@ε-pL sponge on S. aureus-infected skin wounds was investigated referencing to the previously reported procedures [3,64]. Skin tissues were collected on days 3, 7, and 14 for histopathological analysis, including H&E, Masson, immunofluorescence, and immunohistochemical staining, to evaluate the healing effects of OHN@ε-pL sponges on infected wounds. ELISA kits (Beijing Dakewe Biotech Co., Ltd., China) were used to detect IL-6, TNF-α, IL-10, and VEGF levels in granulation tissues. Details are provided in the Supporting Information.

All data were expressed as mean ± S.D. unless otherwise specified, and statistical analyses were performed in GraphPad software (V8.0.1, GraphPad Software Inc.). Two groups with normally distributed data were compared by student's t-test. Data from three or more groups were compared by one-way ANOVA with the Tukey post hoc test. P-values were marked in the figure using asterisks indicating ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (ns = not significant).

CRediT authorship contribution statement

Xiong-Xin Lei: Writing – original draft, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Meng-Long Liu: Writing – review & editing, Resources, Project administration, Funding acquisition, Data curation. Chao-Feng Lu: Methodology, Formal analysis, Data curation. Li-Li Han: Methodology, Investigation, Data curation. Jie-Zhi Jia: Methodology, Formal analysis, Data curation. Zheng Li: Investigation, Data curation. Na Xu: Methodology, Data curation. Jiang-Feng Li: Investigation, Data curation. Xuan-Jian Fu: Methodology, Data curation. Ya-Bin Jin: Formal analysis, Data curation. Ri-Kuan Tong: Methodology, Data curation. Yun-Long Yu: Writing – review & editing, Supervision, Methodology, Formal analysis, Data curation. Gao-Xing Luo: Validation, Software, Resources, Funding acquisition. Yang Chen: Writing – review & editing, Validation, Funding acquisition, Data curation, Conceptualization.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

All animal procedures were reviewed and approved by the Laboratory animal care and ethics committee of Guangdong Laidi Biomedical Research Institute Co., LTD (Ethics number: 2023005-2).

Declaration of interest statement

There are no conflicts of interest to declare.

Appendix A. Supplementary data

The following are the Supplementary data to this article: