Abstract
Achieving rapid and scar-free wound repair is a key goal in the field of regenerative medicine. Herein, a dynamically Schiff base-crosslinked hydrogel (F/R gel) with phase-adaptive regulating functions is constructed to integratedly promote rapid re-epithelization with suppressed scars on chronic infected wounds. Specifically, the gel effectively eliminates multidrug-resistant bacterial biofilm at infection stage via antimicrobial activity of ε-polylysine firstly dissociated from hydrogel matrix in infectious microenvironment, and interrupts the severe oxidative stress-inflammation cycle at wound site by the released ceria nanozyme, thus stimulating a pro-regenerative environment to ensure tissue repair. Subsequently, fibroblast growth factor/c-Jun siRNA co-loaded microcapsules gradually disintegrate to release drugs, facilitating neoangiogenesis and cell proliferation but simultaneously blocking c-Jun overexpression for fibrotic scar suppression. Notably, the F/R gel facilitates normal-like skin regeneration with no perceptible scars formed on infected male mouse wound and female rabbit ear wound models. Our work offers a promising regenerative strategy emphasizing immunomodulatory and fibroblast subtype modulation for scarless wound repair.
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Introduction
Chronic intractable wounds have been a major challenge in global healthcare system, causing huge social and economic burdens. As pathological wounds that cannot spontaneously heal in an orderly and timely manner, chronic wounds have an extremely complex pathophysiology, and are commonly seen in patients with diabetes, drug-resistant bacterial infection and immunodeficiency1,2. The incidence has risen significantly with aging of global population and increasing diabetes prevalence3,4. According to World Health Organization (WHO) and other relevant data, chronic wounds affect approximately 50 million people annually in China5, and the number has been increasing in recent years, especially for diabetic foot ulcers and pressure ulcers6. Unfortunately, achieving effective healing of chronic wounds is particularly difficult compared to acute wounds, which generally follow a predictable sequence of inflammation, proliferation, and remodeling phases. Featured with occurrence of multiple healing phases in an unpredictable and nonlinear manner, chronic wounds often suffer from a much complicated and protracted healing trajectory7. Meanwhile, chronic wounds are often associated with diabetes, vascular hindrances or pressure injuries, and in most cases tend to persistently stagnate in severe infection-inflammation stage, resulting in delayed wound closure6,8. In case of wounds infected with drug-resistant bacteria, for example, antibiotics are primarily used clinically to treat infections. However, commonly used antibiotics are difficult to inhibit drug-resistant bacteria, and at the same time, bacterial biofilm at wound sites further increases drug resistance and aggravates inflammatory response9,10. Moreover, the worsening of inflammatory response in turn increases accumulation of noxious reactive oxygen species (ROS) at wound sites, forming a vicious ROS-inflammation cycle that heavily hinders tissue regeneration11,12,13. In addition, the lack of new blood vessel formation results in an inadequate blood supply with limited oxygen and nutrient delivery, which also delays wound repair14,15. Up to now, various wound dressings have been developed for chronic wound managements, among which promoting transition from inflammation to proliferation phase is the most-proposed and studied therapeutic target for healing16,17,18,19. Notwithstanding these advanced wound dressings meet the needs of antimicrobial activity, maintaining redox balance, and promoting proliferation to some extent, most of them may ignore how to rationally orchestrate essential facets of the different healing process to achieve scarless wound repair.
Scar formation after wound closure is a complex biological process involving multiple cell types, signaling molecules and biochemical pathways20. Although some common wounds can basically heal naturally with almost normal skin appearance, it is very difficult for adult human wounds (particularly chronic wounds) to restore full-functional skins, which seriously affects healing quality8,21. Imperfect repair eventually leads to formation of semi- or non-functional scars at initial sites, marked by malformed tissue bulges and critical absence of skin appendages (like hair follicles and glands)22,23. Severe bacterial infection and inflammation stress are associated with scar formation to a certain extent, but underlying molecular mechanism is far more complex and has not been thoroughly studied at present24,25,26. In addition, delay in any healing stage has a knock-on effect in subsequent stages, ultimately leading to persistent scarring during long remodeling phase27,28. Currently, as a classical regulatory pathway for fibrosis, transforming growth factor beta (TGF-β) has been adopted as a promising therapeutic target for anti-scarring wound repair29. Recently, Wu et al. developed a photo-crosslinking hydrogel dressing that facilitated scarless wound repair through pulsatile release of TGF-β inhibitors30. This innovative dressing significantly enhanced skin wound closure and effectively suppressed scar formation in both murine skin wound model and preclinical large animal wound model. In another recent pioneering study, Gu et al. designed a core-shell structured microneedle patch loaded with verteporfin drug as photo-controlled programmed platform to facilitate scarless skin repair by modulating immune microenvironment and blocking Engrailed-1 (En1) activation28. However, studies on achieving scar-free repair of chronic wounds are very scarce at present, and the known reports are still limited to traditional targets. TGF-β, for example, has pleiotropic effects for wound healing, in which it promotes wound closure by regulating cell migration, proliferation, and extracellular matrix (ECM) deposition in early stages, whereas it causes fibrosis and scar formation in later stages30,31. Therefore, wound dressings targeting TGF-β should be carefully administered, otherwise normal healing process may be interfered. Besides, for ideal scarless wound repair, treatments should avoid intervention after scar formation, but rather during healing process to reduce hypertrophic scarring risk32. As a major heterodimeric transcription factor AP-1 component, c-Jun gene is a key driver of multiple organ tissue scarring on humans and mice33,34,35, and importantly has been reported to be overexpressed in fibroblasts which mediate formation of pathological fibrotic scars after wound closure36. During healing process, c-Jun overexpression affects fibroblast subpopulations at different dermal sites and triggers excessive ECM production for skin fibrosis36,37,38, revealing a potential regulatory target for achieving scarless wound healing.
In this work, we report a healing phase-adaptive regulating hydrogel (F/R gel) with hierarchically delivering performance for programmed modulation of chronic infected wounds (Fig. 1a). The gel with chronological release functions was fabricated by instant Schiff base-crosslinking reaction between cationic polypeptide (ε-polylysine, εPL) and aldehyde hyaluronic acid (HA-CHO), accompanied by encapsulating εPL-modified oxygen-deficient nanoceria (εPL-CeOv nanozyme) and basic fibroblast growth factor/c-Jun siRNA co-loaded PLGA microcapsules (F/R MCs). This hydrogel exhibited remarkable adhesive and self-healing properties, adaptable to irregular wound coverage. Drug-resistant bacteria are highly prone to forming weakly acidic biofilms on wound surfaces, which pose a significant obstacle to wound healing. The Schiff base bond in hydrogel matrix is very sensitive to cleavage under acidic condition, leading to fast hydrogel degradation. During the early stages of wound healing, low-molecular εPL and ultrasmall εPL-CeOv rapidly released, exerting their anti-biofilm and ROS-scavenging activities. The large specific surface area and abundant oxygen vacancies on εPL-CeOv surface allowed cerium ions to interconvert between Ce3+ and Ce4+, which laid the foundational molecular mechanism for its enzymatic activity such as superoxide dismutase and catalase. Combined with the inherent antimicrobial property of εPL, the εPL-CeOv could further modulate local inflammation via alleviating oxidative stress of wound tissues, which was critical for promoting smooth transition of inflammation to proliferation. The F/R MCs, which were uniformly distributed in the hydrogel and relatively larger in size, gradually released as the hydrogel degraded, and this process was accompanied by the release of bFGF as well as c-Jun siRNA. Specifically, in the proliferative phase, bFGF accelerated wound healing by promoting cell proliferation and angiogenesis, while c-Jun siRNA inhibited c-Jun overexpression and regulate the ratio of fibroblast subtypes, leading to scar-free skin regeneration. Therefore, the immunomodulatory and fibroblast subtypes regulation by F/R gel’s phase-adaptive functions promoted anti-bacterial infection, angiogenesis, opportune ECM deposition, and dermal fibrosis suppression. Upon this design, ultrafast wound healing with much reduced scar formation was verified on both infected mouse wound and rabbit ear hyperplastic scar wound models.
a Illustration of F/R gel preparation for programmed regulation on chronic infected wounds. b TEM image, c HRTEM image and the corresponding lattice analysis of CeOv nanoparticles. d SEM image of F/R MCs. e Representative CLSM image of F/R MCs. FITC-labeled bFGF and FAM-labeled siRNA (green) were loaded in Nile red-labeled PLGA microcapsules (red). f Fluorescence staining of c-Jun expression (green) in 3T3 cells. Three independent experiments were performed, and representative results are shown in b-f.
Results
Preparation and characterization
Cationic antimicrobial εPL with abundant amide groups along polypeptide chains was reacted with HA-CHO polymer via reversible Schiff base reaction to form F/R gel’s network, in which crystalline CeOv nanozyme and F/R MCs were uniformly embedded (Fig. 1a). HA-CHO was successfully synthesized by oxidating HA with sodium periodate (Supplementary Fig. 1). Uniform CeOv nanozyme (2.8 ± 0.8 nm) was synthesized via thermal decomposition and surface hydrophilically modified with εPL to endow homogeneous dispersion in aqueous solutions (Supplementary Fig. 2–8), which showed no obvious cytotoxicity to cells (Supplementary Fig. 9). Furthermore, F/R MCs loaded with bFGF and c-Jun siRNA were prepared by classical W/O/W double emulsion technology, and smooth-faced microspheres of ~ 6.23 ± 2.70 µm were shown via scanning electron microscopy (SEM) (Fig. 1d). Using fluorescent labeling method, we demonstrated that bFGF and siRNA drugs were successfully loaded into the microcapsules (Fig. 1e). Degradation tests revealed a slow-rate disintegration of the microcapsules in physiological solutions (Supplementary Fig. 10), and the loaded drugs (bFGF and c-Jun siRNA) displayed sustained release profiles from F/R MCs (Fig. 2k). Importantly, c-Jun gene silencing efficiency of F/R MCs on fibroblasts was verified by immunofluorescence staining, which showed remarkably reduced expression after incubation (Fig. 1f).
a Schematic illustration of the gel preparation. b SEM image and elemental distribution in F/R gel. c Representative time sweep rheological plots of F/R gel. d G′ and G′′ on strain sweep. e Rheological variation of F/R gel when alternate step strain switched from 1% to 800%. Representative photographs of the gel’s (f) self-healing process, (g) adhesion, and (h) stretchable property. i Representative pictures (illustration was created with BioRender.com) and (j) tensile strain test using porcine skin. k In vitro release properties of F/R gel’s components in neutral PBS (pH = 7.4) and acidic PBS (pH = 4.5) (n = 3 independent samples, and data are presented as mean ± SD). l Graphical representation of the F/R gel’s dynamically phase-adaptive regulating functions for scarless wound healing process (created with BioRender.com). Three independent experiments were performed, and representative results are shown in b, f–g.
After simply mixing two component solutions, aldehyde group in HA-CHO and amino group in εPL chain (free εPL or εPL-CeOv) would undergo a rapid Schiff base reaction to form F/R gel within seconds (Fig. 2a). Abundant porous microstructure was observed (Supplementary Fig. 11), and Ce element was uniformly distributed in hydrogel network (Fig. 2b). Rheological results revealed that stable three-dimensional gel was formed in about 3.0 seconds (Fig. 2c), with the Young’s modulus at ~ 6.69 kPa comparable to that of the blank Gel (Supplementary Fig. 12). By adjusting shear strain values at 0%-1000% (1 Hz), both F/R gel’s storage modulus (G’) and loss modulus (G”) kept decreasing with increase of shear stress, while an inflection point for G” > G appeared at about 770%, indicating shear thinning behavior (Fig. 2d, Supplementary Fig. 13). When the strain reduced to 1%, the gel recovered to elastic dominant state (G” <G’), and this process was reversible with multiple cycles, revealing good injectability and self-healing properties (Fig. 2e, f). As a kind of ready-to-use hydrogels, F/R gel could be conveniently applied via mixing for instant use when needed. All the free components of F/R gel possessed excellent stability after long-term storage (Supplementary Fig. 14, 15). Besides, excellent adhesion performance was also demonstrated by steady adhering to both the experimenter’s skin (The procedure was carried out with participants’ awareness and authorization.) whether the finger joint was cyclically straight or bent (Fig. 2g), and the various types of fresh wounds created on mice (Supplementary Fig. 16). Also, the gel showed good tensile property (Fig. 2h–j). Moreover, the loading capacity of F/R gel for εPL-CeOv and F/R MCs had important influences on gel’s network morphology and mechanical property, which revealed that an increased loading amount significantly reduced the gel’s pore structure, viscosity, and swelling properties (Supplementary Fig. 17, 18).
As the hydrogel network was mainly formed by Schiff base reaction and electrostatic interaction, F/R gel could undergo dissociation firstly triggered by weakly acidic microenvironment, and at this point, the low-molecular εPL or ultrasmall εPL-CeOv released immediately to exert antibacterial and immunomodulatory functions. Due to much larger size and slower degradation characteristic of PLGA microcapsules, disintegration of F/R MCs was somewhat delayed than εPL or εPL-CeOv release, and hence the loaded bFGF/siRNA could be liberated for facilitating regeneration cascades. To validate this delivery strategy, release profiles of εPL-CeOv, bFGF, and c-Jun siRNA over time were evaluated. As shown in Fig. 2k, when incubated in neutral PBS, εPL-CeOv released first from the gel, in which ~ 91.13% was detected to release into external solutions at day 6. With gel degradation, the release of bFGF and siRNA drugs became gradually detectable. By day 7, the cumulative release amount of bFGF was close to 85%, while the siRNA drug reached ~ 74.48%. Moreover, acidic microenvironment (pH = 4.5) triggered faster drug release rates especially for εPL-CeOv, which was consistent with the acid sensitivity of Schiff base bond. In addition, the accelerated breaking of Schiff-base hydrogel network in weakly acidic environment was further demonstrated by the in vitro hydrogel degradation experiments (Supplementary Fig. 19), which was more favorable for the release of εPL-CeOv and F/R MCs from F/R gel. We also compared the drug release rates of free F/R MCs and F/R gel, and found that the encapsulation of MCs in hydrogel matrix significantly delayed the release of bFGF and siRNA drugs, regardless of the solution’s pH values (Supplementary Fig. 20).
Therefore, the gel system had the property of hierarchical drug delivery, in which 1) antibacterial εPL-containing components (free εPL, εPL-CeOv) released first to fight against bacterial infections, 2) closely followed by ROS and inflammation clearance regulated through CeOv nanozyme, and then 3) pro-proliferative bFGF and c-Jun-inhibiting siRNA started to facilitate neoangiogenesis, cell proliferation, and simultaneously modulate fibroblast subtypes. This strategy offers the possibility of precise drug delivery on demand according to different wound healing stages, and holds great potentials for achieving rapid anti-fibrotic wound healing (Fig. 2l).
Antibacterial and anti-biofilm activities
For wounds infected by drug-resistant bacteria, effective inhibition of infection is very prerequisite to promote subsequent tissue repair. Antimicrobial effects of F/R gels were investigated using Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and methicillin-resistant Staphylococcus aureus (MRSA) as model strains. Ciprofloxacin (CIP), a clinically broad-spectrum antibiotic, was chosen as a positive control. We first evaluated the F/R gel’s ability to kill planktonic bacteria by colony counting and live/dead bacterial staining assays (Supplementary Fig. 21). The colony number significantly reduced after treated with εPL-CeOv, blank Gel, or F/R gel, which was comparable to CIP (Fig. 3a, b). It should be noted that the blank Gel’s antibacterial property was derived from εPL, one of hydrogel matrix components. Importantly, F/R gel inhibited the growth of both Gram-negative and Gram-positive bacteria, with inhibition rate on both E. coli and S. aureus reaching ~ 89%. The elimination rate on MRSA was even closer to 95%. Live/dead bacterial staining further confirmed the gel’s ability to instantly trigger bacterial death (Fig. 3a, b). In addition, bio-SEM images showed destructive changes on bacteria morphology, including distorted bacterial shapes, surface wrinkles and even ruptures (Fig. 3c).
a Representative images of bacteria colonies and live/dead staining assay after different treatments as indicated. b Statistical analysis of the colony counts and live/dead bacteria (n = 3 independent samples). c SEM images of the bacteria before and after F/R gel treatment (red arrows indicate rupture changes in cell wall). d Experimental illustration of biofilm destruction effect by F/R gel (created with BioRender.com). e Crystalline violet staining and live/dead bacteria staining images of mature biofilms after various treatments. f Biofilm evaluation values (including average thickness and biomass) of mature biofilms (n = 3 independent samples). g Green fluorescence intensity versus biofilm depth quantified from CLSM planes in e. Three independent experiments were performed and representative results are shown in a, c, e. Data are presented as mean ± SD and statistical significance was analyzed via one-way ANOVA with Tukey’s multiple comparison test. P value: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Bacterial biofilms remain intractable in wound healing due to the dense physical structure. Extracellular polymers of biofilms are rich in polysaccharides and nucleic acids, which contribute to inherent antibiotic resistance. Disruption and inhibition of biofilms is crucial for chronic wound managements, especially for those infected with drug-resistant bacteria. The F/R gel’s efficacy to inhibit biofilms formation and dissociate mature biofilms was examined (Fig. 3d, Supplementary Fig. 22a). Using crystal violet staining, we found that F/R gel inhibited E. coli, S. aureus, and MRSA biofilms by ~ 95.08%, ~ 87.10%, and ~ 87.89%, respectively, which dramatically exceeded the effectiveness of CIP (Supplementary Fig. 22b, c). Meanwhile, the gel could also effectively destroy mature biofilms with destruction rates of ~ 82.53% for E. coli, ~ 81.38% for S. aureus, and ~ 71.35% for MRSA, respectively (Fig. 3e, Supplementary Fig. 23). The strong biofilm separation ability was further verified by live/dead staining (Fig. 3e). After comprehensively analyzing the evaluation parameters including biofilm thickness, biomass, roughness, and green fluorescence intensity as a function of biofilm depth (Fig. 3f, g, Supplementary Fig. 24), we concluded that F/R gel presented a stronger ability of destroying biofilms than CIP. Besides, there was no significant difference between the biofilm destruction efficiency of F/R gel and blank Gel. This suggested that the hydrogel matrix components, i.e., εPL and εPL-CeOv, played a crucial role in dissociating biofilms, whereas the addition of F/R MCs did not impair its antimicrobial activity.
Antioxidant and anti-inflammatory activities
ROS leads to intense oxidative stress and cellular damage, thus slowing down wound healing process. Scavenging excessive ROS and maintaining ROS homeostasis are essential for normal wound healing. The main component that scavenges ROS is εPL-CeOv, which can accept or transfer electrons through the rapid redox cycling between Ce3+/Ce4+ oxidation states (Fig. 4a). The scavenging efficiencies of εPL-CeOv on hydrogen peroxide (H2O2), hydroxyl radicals (•OH), superoxide anion radicals (•O2−), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals were all concentration-dependent (Supplementary Fig. 25). Meanwhile, εPL-CeOv also possessed CAT-mimetic activity, which could rapidly catalyze decomposition of H2O2 (Supplementary Fig. 26). Besides, there was no significant decrease in the removal efficiency after five cycles, indicating that εPL-CeOv had excellent ROS scavenging stability (Supplementary Fig. 27). Blank Gel and F/R gel almost kept the same scavenging efficiency as pure εPL-CeOv did, while F/R MCs showed little scavenging activity (Fig. 4b–f). Therefore, cross-linking of εPL-CeOv with HA-CHO did not interfere its activity, and uniform nanozyme distribution in gel matrix ensured potent antioxidant capacity.
a Diagram for ROS scavenging ability of F/R gel. Scavenging tests of F/R gel on (b) ABTS free radical, (c) DPPH free radical, (d) hydrogen peroxide, (e) hydroxyl free radical, and (f) superoxide anion (n = 3 independent samples, and data are presented as mean ± SD). g Particle model for the oxygen-deficient CeOv nanozyme and its unit cell configurations. h Simulated electron density mapping images of CeO2 (upper panel) and CeOv (bottom panel). Red dashed circle indicates surface oxygen vacancy. i Top view for the optimized structures of ABTS+•, DPPH•, H2O2, •OH, and •O2- free radicals adsorbed on CeOv (111) facets. The corresponding Ebinding values are indicated. j Reaction energy profiles for the simulated SOD-like and CAT-like catalytic processes on CeOv (111) facets. The optimized structures of key intermediate steps are shown as insets.
To gain a deep insight into the underlying catalytic mechanisms of CeOv nanozyme at the atomic level, density functional theory (DFT) calculation was performed. Ultrasmall CeOv synthesized via thermal decomposition were characterized by abundant oxygen vacancies on surface (Fig. 4g), accompanied with Ce3+/Ce4+ redox couple for catalytic activity39,40,41. Primarily, the calculated charge density diagrams (Fig. 4h) demonstrated that the presence of oxygen vacancy led to distinct electron redistribution and reduction of adjacent Ce, which changed Ce valence state. According to Bader charge analysis calculation, Ce valence of CeOv reduced by ~ 0.47 e− when compared with the perfect CeO2 surface. Besides, the projected density of states (PDOS) showed that the Ce 4 f states shifted to a much lower energy position, revealing the occurrence of Ce4+→Ce3+ reduction on CeOv surface (Supplementary Fig. 28). We simulated the optimized structures of various ROS adsorbed on CeOv (111) facet and calculated the corresponding binding energies (Ebinding). Compared with CeO2 (Supplementary Fig. 29-30), CeOv surface showed stronger adsorption to these five free radicals, in which the Ebinding values were -3.87 eV for ABTS+•, -3.97 eV for DPPH•, -4.83 eV for H2O2, -6.46 eV for •OH, and -7.46 eV for •O2−, respectively (Fig. 4i). The significantly increased adsorption energy enhanced catalytic activity of CeOv on these free radicals. Taking two important enzymatic catalysis (SOD and CAT-mimicking) as examples, key intermediates and reaction pathways of •O2− disproportionation or H2O2 decomposition are depicted in Fig. 4j. Notably, as a Brønsted base, •O2− can be easily converted into HO2• in aqueous solution via the following reversible reaction42:
Initially, HO2• radicals can easily interact with CeOv surface by combining with oxygen vacancy around two Ce3+ in the lattice with a Gibbs free energy (ΔG) of -2.36 eV. An electron from Ce3+ transfers to an oxygen atom and generates an electronegative oxygen atom, accompanied by the transition of Ce3+ to Ce4+ state. The intermediates in oxygen vacancy further combine with protons to form H2O2, thus mimicking SOD activity40. For CAT-mimicking process, adsorption of H2O2 on CeOv surface causes Ce4+→Ce3+ valence transition, accompanied with generation of protons and oxygen molecules at the same time. The reduced Ce3+ reacts with another H2O2 molecule by transferring protons to form H2O, and the CAT-mimicking catalytic cycle is restored upon final reoxidation of Ce3+ to Ce4+ in this process42.
Excessive ROS accumulation causes intense oxidative stress and inflammatory response in wound tissues11,43, and at the same time, immoderate secretion of inflammatory factors mainly by M1-type macrophages in turn induces more ROS generation, which further exacerbates local inflammation and tissue damage, that is to say, persistent inflammation-excessive ROS cycle results in aberrant wound healing44,45. We verified at cellular level if F/R gel had ability to interrupt the inflammatory ROS cycle, i.e., to scavenge excess intracellular ROS, to protect cells from ROS damage, as well as to inhibit macrophage polarization towards proinflammatory M1 type (Fig. 5a). Oxidative stress cell models of 3T3 and HUVEC were established by tert-Butyl hydroperoxide (tBHP) stimulation. Compared to tBHP group, intracellular ROS intensity of F/R gel group was reduced more than 6-fold in 3T3 and 2-fold in HUVEC, respectively (Fig. 5b, c). Moreover, fluorescence images clearly showed more intense ROS signal when treated with tBHP, while negligible ROS level was detected in both 3T3 and HUVEC after F/R gel treatment (Fig. 5d, e, Supplementary Fig. 31). Hydroxyphenyl fluorescein (HPF) staining results further confirmed intracellular ROS scavenging effect of F/R gel (Fig. 5d, e), and as a consequence, a significant cell viability recovery was observed (Fig. 5d, Supplementary Fig. 31, 32, shown in the calcein/PI staining). Meanwhile, we observed a significant reduction in the number of EdU-positive cells in the tBHP-stimulated group, while F/R gel’s treatment elicited an enhanced cell proliferation (Supplementary Fig. 33). No significant difference of EdU fluorescence intensity was found between the F MCs and F/R MCs groups, indicating the proliferative effect of bFGF drug loaded in MCs. In F/R gel group, the presence of εPL-CeOv also scavenged cellular oxidative stress, and thus provided an optimal microenvironment for cell proliferation. Additionally, F/R gel could activate endothelial cells and subsequently promote endothelial tube formation under oxidative stress environment (Supplementary Fig. 34).
a Experimental illustration of cell experiments (created with BioRender.com). b Flow cytometry of DCFH-DA fluorescence on 3T3 and HUVEC cells. c Statistical data of intracellular DCFH-DA fluorescent intensity (n = 3 independent samples). d Fluorescence images of DCFH-DA probe (green), HPF probe (green), and live/dead cell staining assay (Calcein, green and PI, red) on 3T3 cells. e The corresponding statistical analysis (n = 3 independent samples). f Flow cytometry of CD86 marker on macrophages and g the corresponding statistical data (n = 3 independent samples). h Microscopic images of red blood cells and i hemolysis rates upon various treatments (n = 3 independent samples). j Experimental illustration of subcutaneous implantation test (created with BioRender.com). k H&E staining images of skin tissues after F/R gel implantation for 28 days. l Blood routine and biochemical indexes (n = 3 biologically independent samples). Three independent experiments were performed and representative results are shown in b, d, f, h, i, k. Data are presented as mean ± SD and statistical significance was analyzed via one-way ANOVA with Tukey’s multiple comparison test. P value: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
During the early stage of chronic wound healing, macrophages tend to polarize into M1 phenotype, a proinflammatory state that releases various inflammatory cytokines such as TNF-α, IL-1β, IL-6 and free radicals. Persistent M1-type macrophage infiltration exacerbates local inflammation and leads to delayed wound repair. Therefore, inhibiting excessive M1 macrophage polarization could reduce inflammatory response and effectively shorten wound healing time. Under lipopolysaccharide (LPS) stimulation, macrophages tended to polarize into M1 phenotype as revealed by the significantly increased fluorescence intensity of CD86 (M1-type marker) (Fig. 5f). In contrast, CD86 positive rate of F/R gel-incubated macrophages was only ~ 2.56%, and reduced almost 6.7-fold when compared with LPS group (Fig. 5g). Two dominant factors of F/R gel contributed to the suppressed macrophage activation: 1) the released bFGF could regulate expression of macrophage surface receptors46,47 and proinflammatory cytokines by inhibiting the activation of relevant signaling pathways, such as NF-κB48; 2) the antioxidant εPL-CeOv could scavenge intracellular noxious ROS and further inhibited M1 macrophage polarization.
Biocompatibility assessment
Good biocompatibility of wound dressings is a key prerequisite for applications. We first tested F/R gel’s biocompatibility by determining hemocompatibility. Deionized water (Positive group) was used as a hypotonic solution to cause hemolysis on erythrocytes, and no intact cells were seen under the microscope (Fig. 5h). As a result, the supernatant after centrifugation turned red, and the hemolysis rate approached 100%. After F/R gel incubation, intact erythrocytes could be seen under a microscope. The supernatant in F/R gel group was cleared and transparent with a hemolysis rate close to 0%, comparable to Negative group (Fig. 5i). Moreover, after co-cultured with three different cell types (C2C12, 3T3 and HUVEC) for 24, 48, and 72 h respectively, F/R gel did not cause any damages on cell survival, and all the cell viability values were comparable to or even slightly higher than Control group (Supplementary Fig. 35–37). Meanwhile, we further subcutaneously transplanted F/R gel into mouse skins for in vivo biocompatibility test (Fig. 5j). After 30 days, hematoxylin-eosin (H&E) staining results showed no discernible inflammatory response or tissue aberration in mouse skins and major organs (Fig. 5k, Supplementary Fig. 38, 39). In addition, all the tested blood routine and biochemistry indexes fell well within normal value ranges (Fig. 5l). By observing the retention effect at wound site by small animal live imaging technology, we found that the hydrogel encapsulation provided prolonged retention time in the wounds, while the administered dose during treatment was not sufficient to cause obvious accumulation in major organs (such as heart, liver, spleen, lung, and kidney) (Supplementary Fig. 40, 41).
F/R gel facilitates rapid healing of MRSA-infected chronic wounds on mice
To investigate whether F/R gel could accelerate chronic infected wound healing in vivo, two full-thickness excisional wounds (round, 6.0 mm) were created on the back of C57BL/6 mice and infected with MRSA, followed by treatments with PBS (Control), Beifuji gel (a commercial wound gel), F/R MCs, Gel, and F/R gel, respectively. Figure 6a briefly depicts the modeling procedure, dressing administration, and healing monitoring timeline. Digital photographs clearly displayed a much faster wound closure rate in F/R gel group (Fig. 6b and Supplementary Fig. 42), as determined by the smallest wound areas measured on day 4, 7, and 14 (Fig. 6c). Specially, when F/R gel was applied for only 4 days, the nonhealed would area decreased to ~ 63.25%, and swiftly reached ~ 32.56% on day 7. In contrast, all other groups showed obviously delayed healing speed, for instance, the nonhealed area in Control group was still as high as ~ 89.51% on day 4 and ~ 84.22% on day 7. Notably, wounds in F/R gel group were almost completely healed and closest to normal skin appearance on day 14. On the whole, F/R gel accelerated wound closure time by approximately 6 to 7 days, representing a reduction of nearly one-third in total healing duration. Detailed analysis on the wound healing rates clearly revealed that F/R gel treatment elicited a potent pro-healing effect in the early healing stage (Day 0 → 7), but showed a moderative healing rate in the later stage for avoiding over-proliferation (Supplementary Fig. 43). Meanwhile, we observed that Control, Beifuji, and F/R MCs groups exhibited signs of severe infection (with formation of biofilms) and inflammation on day 4 and 7, while Gel and F/R gel groups showed insignificant infection. During treatment, wound exudates were collected for colony agar plate coating assay, and F/R gel group had the lowest colony counts of bacteria (Fig. 6d, e). Bacteria colony gradually decreased as time prolonged, and there were almost no bacteria in F/R gel group on day 7. Combined with these data, we could generally determine that the wounds in F/R gel group had smoothly transitioned into proliferative phase on day 4-7, however, the wounds in other groups were still in infection or inflammatory phase during this period.
a Experimental process of the whole animal experiments on mice (created with BioRender.com). b Optical photos and simulated graphs of wound closure changes from day 0 to day 14. c Variation curve of wound areas within 14 days (n = 3 biologically independent samples). d Colony counting assay using wound exudates on day 2–7 (n = 3 independent samples) and (e) representative images. f Immunofluorescent staining of DHE (red), CD86 (green), CD206 (red), CD31 (red), VEGF (red), Ki67 (green) and DAPI (blue), H&E staining, and Masson staining images of wound tissues during wound healing. Red arrows mark the newborn hair follicles. g Relative mRNA expressions of CD86, iNOS, IL-1β, TNF-α, CD206, Arg1, IL-10, and Ki67 (n = 3 biologically independent samples). Three independent experiments were performed and representative results are shown in b, e, f. Data are presented as mean ± SD and statistical significance was analyzed via two-way ANOVA with Tukey’s multiple comparison test. P value: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
To systematically assess healing quality, tissue samples were collected on day 4, 7, and 14, respectively, followed by immunofluorescent and histopathological observation. Intense ROS (dihydroethidium, DHE) and CD86 fluorescence were found in Control, Beifuji, and F/R MCs groups, while slight or negligible signals were observed in Gel and F/R gel groups on day 4 (Fig. 6f). For M2-type macrophage (CD206) staining, the trend was reversed. Statistical data (Supplementary Fig. 44) and quantitative real-time PCR (qRT-PCR) experiments (Fig. 6g) verified the results. Because F/R gel treatment suppressed bacterial infection, reduced excessive ROS and inhibited proinflammatory factor secretion, redox homeostasis at wound site was restored to normal, thus the wound inflammation gradually subsided and progressed to proliferative stage. H&E staining results on day 7 revealed severe inflammatory cell infiltration in the Control group, while F/R gel group showed insignificant inflammation (Fig. 6f, Supplementary Fig. 45). Proliferative phase is a critical stage in wound healing process involving fibroblast proliferation, neovascularization, and extracellular matrix deposition. On day 7, higher CD31 and VEGF expressions in F/R gel group were observed at the wound site, suggesting more neovascularization after treatment (Fig. 6f, Supplementary Fig. 46). Moreover, F/R gel group showed the highest Ki67 expression (Fig. 6f, Supplementary Fig. 47), and qRT-PCR assay detected a nearly 4-fold increase of Ki67 mRNA level compared with Control group (Fig. 6g), revealing vigorous cell proliferation probably attributed to bFGF release, thereby promoting skin tissue regeneration. As wounds continued to heal, Ki67 expression gradually decreased on day 14 in F/R gel group (Supplementary Fig. 48) to avoid over-proliferation and transition into remodeling phase, while other groups were still in proliferating at this time. Importantly, an intriguing finding was the formation of newly formed hair follicles and sebaceous glands in the regenerated skins of F/R gel group on day 14, which was hardly observed in any of other groups (Fig. 6f). Besides, Masson’s trichrome staining images indicated more mature and well-organized collagen deposition in F/R gel group (Fig. 6f).
On day 14, we conducted a comprehensive evaluation of neovascularization in the wound tissues. The newly formed blood vessels beneath the mouse skins were first monitored (Supplementary Fig. 49a). Similar to normal skin, the F/R gel-treated wounds exhibited a denser and more regular vascular network. This observation was consistent with the results from a laser speckle contrast imaging (LSCI) system, which assessed the blood perfusion levels in the wounds. Collectively, these data clearly demonstrated a pronounced pro-angiogenic effect with higher local perfusion in the wounds after F/R gel treatment (Supplementary Fig. 49b).
F/R gel inhibits scar formation of MRSA-infected chronic wounds on mice
Macroscopic appearance and staining results showed visible accelerated wound closure and regeneration of newly formed epidermis across all the wound sites in F/R gel group on day 14 (Supplementary Fig. 50), highlighting the gel’s crucial role in regulating skin regeneration. Interestingly, F/R gel group exhibited distinct features with flatter wound surfaces and continuous/coherent epidermis-like normal skins, revealing its robust potential in preventing scar formation (Fig. 6b). We performed a comprehensive assessment on the regenerated skins by appearance inspection and histopathological evaluation, focusing on parameters including scar area, scar pigmentation area, epidermis thickness, collagen fraction, and skin appendages. After complete wound closure, we noticed that the new skins in the F/R gel group had better elasticity, more uniform epidermal color, less pigmentation, and fewer tissue depression. On day 28, the newborn epidermis color was nearly the same as surrounding tissues with no significant chromatic aberration, while Control, Beifuji, F/R MCs, and Gel groups presented little changes during day 21-28 monitoring period (Fig. 7a and Supplementary Fig. 51). Approximately 59.88% of the newborn skin in Control group arose scar-like tissues on day 21, and this value increased to ~ 64.28% on day 28. For F/R gel group, the scar area was only ~ 17.51% on day 21, and sharply decreased close to 0% (Fig. 7b). For scar pigmentation assessment, statistical scoring results showed a more similar color appearance to the surrounding skins in F/R gel group (higher L value, but lower A value). Epidermal thickness and skin appendage formation were further analyzed by H&E staining (Fig. 7c, d). The epidermal thickness in F/R gel group was closer to normal skin (~ 20.54 μm vs. ~ 20.57 μm); however, the value in the Control group increased 6 times thicker. Regeneration of a thinner epidermis is often advantageous for wound healing in preventing thick scab or keloid formation, which otherwise interferes normal skin functions. More encouragingly, mature skin appendages were also observed in F/R gel group, and statistical numbers restored to the levels comparable to normal tissues (Fig. 7c, d). CK19 was used as hair follicle cell marker for immunofluorescence staining, and the results further confirmed formation of hair follicles (Fig. 7d and Supplementary Fig. 51). In addition, nerve bundle (β3-Tublin positive) regeneration was also observed in the regenerated tissues of F/R gel group (Fig. 7d and Supplementary Fig. 51), revealing its potential activation effect on mature neurons into repair status to induce neural reconstruction. Therefore, organized formation of newly generated appendages and nerve fibers underscored the superior wound-healing quality of F/R gel.
a Optical photos and simulated graphs of scar area changes from day 21 to 28. b Scar changing curve and color analysis of scar tissues post-treatments (a higher L value represents a whiter color, and a higher A value represents a redder color) (n = 3 biologically independent samples). c Epidermal thickness (n = 6 biologically independent samples) and count of follicles (n = 3 biologically independent samples) analyzed by H&E staining on day 28. d H&E staining, Masson’s staining, and immunofluorescent staining of COL I (red), COL III (green), TGF-β (red), c-Jun (red), CK19 (green), β3-Tublin (red) and DAPI (blue) images of wound tissues on day 21 to 28. e Collagen volume ratio, fractal dimension value, and lacunarity value based on the tissue slice analysis (n = 3 biologically independent samples). f Relative mRNA expressions of COL I, COL III, and c-Jun on day 28 (n = 3 biologically independent samples). Three independent experiments were performed and representative results are shown in a, d. Data are presented as mean ± SD and statistical significance was analyzed via two-way ANOVA with Tukey’s multiple comparison test. P value: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
In order to observe the distribution and arrangement of collagen deposition, Masson trichrome staining was performed. F/R gel group showed a more mature collagen deposition with orderly and simpler arrangement than other groups (Fig. 7d). Statistical analysis further indicated well-defined collagen fiber alignments represented by lower collagen volume fraction values (CFV) (Fig. 7e). Collagen volume ratio of normal skin was analyzed at about 20%, while Control group had a ratio of about 78.83%, which implied abnormal collagen deposition and severe tissue fibrosis at wound site. Whereas F/R gel group had a collagen volume ratio of ~ 23.90%, which was close to normal skin tissue. Meanwhile, higher lacunarity but lower fractal dimension values were observed in F/R gel group, revealing porous microstructures and uniform collagen alignment. Type I collagen in skins mainly functions as maintaining tissue structure and connection, while type III collagen is associated with elasticity49. The expression of collagen I in F/R gel group was significantly reduced, whereas the trend was totally reversed for collagen III expression (Fig. 7d–f, Supplementary Fig. 52), signifying its capability to prevent fibrotic scar formation. In addition, the c-Jun overexpression was significantly mitigated due to siRNA delivery, and a decrease of TGF-β expression was also observed in tissues, implying the close association of c-Jun blocking and TGF-β signal inhibiting for fibrotic scar suppression. It should be noted that the uninfluenced c-Jun expression in F/R MCs group might be due to the rapid loss of free F/R MCs in healing process, highlighting the necessity of gel system for synergistically promoting scarless wound healing. To further elaborate the specific role of c-Jun siRNA in F/R gel for scar reduction, we supplemented another batch of animal experiments in which the F gel (gel containing only bFGF-loaded microspheres) group was added (Supplementary Fig. 42 and 49). The results clearly revealed that F gel treatment only accelerated wound closure rate but hardly inhibited scarring tissues, hence highlighting the importance of F/R gel’s components for anti-scar wound healing.
Fluorescence-activated cell sorting (FACS) and transcriptome sequencing analysis
It is demonstrated that differential expression of c-Jun and downstream genes such as TGF-β is closely related to fibroblast differentiation50. c-Jun overexpression promotes profibrotic myofibroblast phenotype, and drives dermal fibrosis through excess ECM deposition by specific fibroblast lineages36. We hypothesized that dual delivery of bFGF and c-Jun siRNA could stimulate fibroblast proliferation but simultaneously modulate distinct fibroblast subpopulations to realize anti-fibrotic wound healing. To verify this speculation, we performed FACS experiments to analyze the subtype proportions of fibroblasts in traumatic tissues on day 7 (Fig. 8a). The proportion of reticular fibroblasts in F/R gel group was significantly lower than all the control groups, while the variation tendency for lipofibroblasts was quite the other way (Fig. 8b, Supplementary Fig. 53, 54). Excessive reticular fibroblasts led to immoderate collagen deposition and unfavorable matrix remodeling, which in turn caused fibrosis. However, through modulating effect of F/R gel in healing process, a different fibroblast subtype proportion with less reticular fibroblasts but more lipofibroblasts was observed, which contributed to scarless repair (Fig. 8c). The α-smooth muscle actin (α-SMA, profibrotic myofibroblast phenotype) expression levels in F/R gel group kept very moderate and stable during the whole healing process (Fig. 8d, e), while in sharp contrast, persistent increased α-SMA expressions were observed in other groups, signifying excessive ECM proliferation and development of dermal fibrotic scars. Although changes of stem cell antigen-1 (Sca-1, adipose fibroblast phenotype) expression were not as pronounced as α-SMA, statistical analysis showed that F/R gel group had higher levels of Sca-1 expression at all time points (Fig. 8d, e).
a Schematic process of FACS experiment on wound tissues (created with BioRender.com). b Fluorescence-activated cell sorting results of fibroblast subtypes in wound tissues (n = 3 biologically independent samples). c Schematic diagram of fibroblast subtype regulated by c-Jun silencing strategy (created with BioRender.com). d Immunofluorescent staining images of α-SMA (green) and Sca-1 (red) markers in wound tissues on day 7, 14, and 28. e Mean fluorescent intensity of α-SMA and Sca-1 signals (n = 3 biologically independent samples). f Heatmap of Pearson’s correlation coefficient matrix for tested samples. g The corresponding volcano plots analyzed on day 14 between F/R gel and Control groups. h Heatmaps of the screened differentially expressed genes involved in healing process. i KEGG enrichment and (j) GO enrichment analysis of the differentially expressed genes. Three independent experiments were performed and representative results are shown in b, d. Data are presented as mean ± SD and statistical significance was analyzed via two-way ANOVA with Tukey’s multiple comparison test. P value: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
To further explore potential mechanism by which F/R gel promoted scarless wound healing, transcriptome analysis was performed on tissues harvested on day 14. For all the biological replicates, square of Pearson’s correlation coefficient (R2) was greater than 0.92, indicating good reliability of tissue samples (Fig. 8f). Volcano plot analysis showed 318 differentially expressed genes in F/R gel group, of which 175 genes were significantly up-regulated and 143 genes down-regulated (Fig. 8g). Among them, we particularly noted that Eln is an encoded elastin that is involved in formation and maintenance of elastic fibers that provide elasticity to tissues and organs51. Eln was significantly upregulated in the F/R gel group (Fig. 8h), which might contribute to the regeneration of more elastic skin tissues at wound sites. In addition, genes associated with the promotion of vascular regeneration (Myl7) and muscle regeneration (Myog), as well as hair follicle development and appendage regeneration (Sp6, Gsdma3, and Elf5), were significantly up-regulated in F/R gel group. Importantly, the expressions of genes associated with fibrotic scar formation, such as Ugt8a, Il-11, Gcgr, and Tgfbr3l, were significantly downregulated. Meanwhile, the overexpression of c-Jun in the F/R gel group was also downregulated due to the delivery of siRNA. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes revealed an enrichment of metabolic pathways, mainly involving IL-17, NF-κB, JAK-STAT, HIF-1, and TGF-β pathways, all of which play important roles in inflammation, immune regulation, cell proliferation, and tissue fibrosis during wound healing process (Fig. 8i). Gene Ontology (GO) analysis showed that the differentially expressed genes were mainly involved in biological processes such as cellular response to TGF-β stimulation (Fig. 8j).
F/R gel promotes scarless healing of rabbit ear wounds
We then evaluated the anti-scar healing effect on rabbit ear wound model, which usually cannot heal by contraction, and results in reproducible formation of hypertrophic scars by delayed re-epithelialization52. Slightly different from reported modeling process, here we established the rabbit ear hyperplasia wound model by removing full-thickness skins (round, 8 mm) and implementing infection with MRSA, hence bacteria-infected wounds were obtained to evaluate F/R gel’s role in promoting scarless healing (Fig. 9a). By carefully monitoring the whole wound healing and scar proliferation process, we were surprised to find that F/R gel not only improved wound healing efficiency, but also tremendously prevented occurrence of proliferative scarring tissues (Fig. 9b). On day 7, the wounds in F/R gel group began to close at an accelerated rate, and the wound healing efficiency reached ~ 87.66% on day 14, which was significantly superior to other groups (Fig. 9c). Meanwhile, severe red proliferative scarring tissues appeared after complete closure in Control group on day 21, and hypertrophic scars did not fade by day 28. The average red scarring area of the Control group was determined to be ~ 28.3 mm2, which was about 56.33% of initial wound size. In contrast, the regenerated tissues in F/R group gradually recovered to be the same as surrounding uninjured skins, including flat epidermis, uniform color appearance, and similar touch elasticity. Scar proliferation in each group was evaluated by ultrasonic imaging, H&E staining, Masson trichrome staining, and calculation of four indexes: scar evaluation index (SEI), scar thickness, scar bulge angle, and scar pigmentation (Fig. 9d–f). Ultrasonography revealed that serious tissue bulges at wound sites were more pronounced in Control, Beifuji, F/R MCs, and Gel groups, while F/R gel group showed flatter and closer tissue thickness to normal skins (Fig. 9f). H&E images displayed the same variation trend, and also showed that the epidermis was significantly thickened in Control group, however, epidermis thickness in F/R gel group was similar to normal tissue. SEI, scar thickness, and scar bulge angle values of F/R gel group were all the smallest ones among the tested groups (Fig. 9e). Besides, there was no significant difference on the regenerated skin color between the F/R gel group and normal epidermis, but Control group showed distinct scar pigmentation scores. Masson staining results showed obvious hypertrophic features of proliferated collagen fibers with coarse shapes, disorganized arrangements, and swirling structures in Control, Beifuji, F/R MCs, and Gel groups (Fig. 9f). On the contrary, the F/R gel’s treatment contributed to a significantly reduced cellularity and collagen fiber deposition, which was comparable to normal skin. In view of the fact that a more consistent collagen arrangement trend in remodeling phase results in increased skin strength and usually signifies better healing quality23,53, we further visualized and quantified the distribution of collagen fiber orientation. Similar to collagen fiber orientation of normal skins, the collagen arrangement in F/R gel group was relatively concentrated and orderly, while other groups all showed scattered distribution (Fig. 9g). Together, these results strongly suggested that our dynamically phase-adaptive regulating gel could effectively accelerate wound closure while mitigate skin fibrotic scarring on preclinical rabbit ear infected wound model.
a Experimental illustration for modeling process of rabbit ear wounds infected with MRSA and the following animal experiments (created with BioRender.com). b Optical photos, simulated graphs, and c statistical data of wound healing changes from day 0 to 28 (n = 3 biologically independent samples). d Schematic diagram of the evaluation indicators related to scarring effect on rabbit ears. e Statistical data of SEI, scar thickness, scar bulge angle and color measurement (n = 3 biologically independent samples). f Ultrasonography (red dotted lines indicate hyperplastic scarring area), H&E staining, and Masson’s staining images of the rabbit ear wound tissues. g Analysis of collagen orientation distribution based on Masson’s staining images in f. Three independent experiments were performed and representative results are shown in b, f, g. Data are presented as mean ± SD and statistical significance was analyzed via two-way ANOVA with Tukey’s multiple comparison test. P value: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Discussion
Effective clinical treatment options for chronic wounds are very scarce at present, especially for those caused by severe drug-resistant bacterial infections, and preventing scar formation in the healing skin is even more difficult. The mechanisms of scarring are complex, involving multiple cell types, signaling molecules, and biochemical pathways. While some common wounds can heal spontaneously with a normal skin appearance, chronic wounds are difficult to restore fully functional skin. Moreover, scar treatment after wound healing is usually limited in therapeutic effectiveness and proneness to recurrence. Therefore, to achieve rapid wound healing without scarring, two key issues should be addressed: 1) accelerate the wound healing process to ensure repair speed, and 2) inhibit profibrotic pathway and prevent scar formation to ensure repair quality. If these two challenges are tackled separately, the treatment process would require the elaborated use of different drugs at various wound healing stages, which inevitably complicates the approach and hardly achieves ideal curative effect. Currently available dressings typically focus on correcting factors associated with scar formation, including modulating microenvironment28,54, minimizing mechanical stimuli20,55,56,57,58, balancing healing stages59,60,61, and modulating traditional profibrotic targets30,62,63. However, there are few suitable therapeutic approaches that simultaneously accelerate wound closure rate and inhibit fibrotic scarring by dynamically modulating regeneration cascades and influencing proportion of fibroblast subtypes during wound management (Supplementary Table 5).
We have developed a dynamically phase-adaptive hydrogel platform (F/R gel) with injectable, self-healing, and adhesion properties that can be easily adjusted in injection dose to suit wound size and area. The systematic in vivo and ex vivo experiments revealed that in early stages of wound healing, the wound microenvironment influenced by bacterial biofilms accelerated the degradation of hydrogel matrix. The controlled release of low-molecular εPL and ultrasmall εPL-CeOv performed antimicrobial effect by inhibiting bacteria survival and biofilm formation. Suppressed biofilm growth and dissociated mature biofilms of Gram-positive, as well as Gram-negative bacteria with higher efficiency were observed when compared to conventional antibiotics. Meanwhile, the released εPL-CeOv nanozyme exerted its ROS scavenging activity to regulate local immune microenvironment by neutralizing excess inflammatory cytokines and inhibiting macrophage polarization toward a pro-inflammatory phenotype, thereby helping the wound rapidly pass through inflammatory phase. Previous studies have highlighted the critical importance of smooth transition from the inflammatory to the proliferative phase for successful would healing, and a failure in this transition can lead to prolonged inflammation as well as impaired re-epithelialization, ultimately resulting in intractable nonhealing wounds62,63. As shown in our results, the untreated mice in control group exhibited much increased inflammation and delayed epithelialization. Taking advantage of the larger size and slower degradation characteristic of F/R MCs, the loaded bFGF and siRNA drugs gradually released from the disintegration of MCs. bFGF accelerated wound healing rate by promoting cell proliferation and angiogenesis in the proliferative phase. Meanwhile, c-Jun siRNA regulated fibroblast subsets (reduced the number of reticular fibroblasts while increased lipofibroblasts) by inhibiting c-Jun overexpression, thereby preventing excessive fibrogenesis and reducing scar formation. Moreover, by testing on a rabbit ear wound model that is more closely similar to human beings28,64, we found that the hydrogel significantly facilitated wound closure while effectively mitigated formation of hypertrophic scars, further confirming its anti-scar healing effect.
In conclusion, our work proposed a promising strategy to manage difficult-to-heal chronic wounds through programmed modulation on healing process realized by graded delivery of functional active components. We demonstrated that rational design of hydrogel dressing could be well adaptive to wound healing phases, and finally achieved scarless skin regeneration via regulating the immune microenvironment as well as the regenerated fibroblast subtypes. Therefore, the developed hydrogel platform offers an innovational methodology for scarless repair of refractory infection wounds, and has great potentials for future wound managements.
Methods
Cell lines and animals
All the animal experiments were conducted in compliance with the Chinese National Standard and under the protocols that were approved by the Laboratory Anima Ethics Committee of Oujiang Laboratory [No. OJLAB22121616] and the Institutional Animal Care and Use Committee, Wenzhou Institute, University of Chinese Academy of Sciences [No. WIUCAS23080801]. NIH-3T3 cell line (3T3 cell), human umbilical vein endothelial cells line (HUVEC) and C2C12 mouse myoblasts (C2C12) were purchased from Procell Life Science & Technology Co., Ltd. These cell lines were cultured in DMEM supplemented with 1% streptomycin/penicillin and 10% fetal bovine serum (FBS, Gibco) in a cell incubator (37 °C, humidified atmosphere of 5% CO2). All materials used in the cell experiments were sterilized by UV irradiation sterilization, and the gel preparation device was sterilized with 75% ethanol before use. C57BL/6 mice (male, 8-10 weeks, 20-24 g) and New Zealand white rabbits (female, 3-4 months, 2-2.5 kg) were sourced from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. and Zhejiang Provincial Laboratory Animal Center, respectively.
Synthesis & characterization of εPL-CeOv and F/R MCs
Synthesis of CeOv nanocrystals: 1.0 mmol of Ce(NO3)3 · 6H2O, 3.0 mmol of oleylamine, and 5.0 g ODE were mixed at room temperature, and then fully dissolved at 80 °C for 30 min. After that, the reaction system was heated to 260 °C for 2 h under N2 condition. The product was obtained by centrifugation and purified with ethanol. Finally, the prepared dark brown precipitate was dispersed in cyclohexane for later use.
Modification of CeOv nanocrystals with εPL: CeOv nanocrystals dispersed in cyclohexane were transferred to a syringe as the organic phase. εPL powder dissolved in 10 mL of deionized water was as the aqueous phase. The organic phase was slowly injected into the aqueous phase with ultrasonication by an ultrasonic cell-crushing instrument. Once the injection was completed, the organic cyclohexane was completely volatilized under magnetic stirring to form an aqueous solution. After centrifuged at 2500 x g for 10 min to remove free εPL, the final εPL-CeOv precipitation was freeze-dried using a lyophilizer (Labconco™, ThermoScientific).
Synthesis of F/R MCs: PLGA microcapsules loaded with bFGF and c-Jun siRNA (i.e., F/R MCs) were prepared by using the traditional W/O/W double emulsion method. Firstly, 200 μg of bFGF and 2.5 μg of c-Jun siRNA were dissolved in deionized water, while 10 mg PLGA power was dissolved in dichloromethane. Next, the PLGA organic solution was mixed with bFGF/siRNA aqueous solution, and then immediately emulsified by homogenization at 9000 x g for 1 min. After that, the prepared single emulsion was further emulsified by a mechanical stirrer at 100 x g in 1% w/v PVA solution. Finally, the above emulsion was added into 200 mL PVA solution (0.1%, w/v), followed by continued stirring for 4 h. The obtained F/R MCs were filtered, washed with water, and freeze-dried overnight.
The TEM, HRTEM and SAED micrographs were taken by FEI Talos F200X at 200 kV. The size distribution was obtained by counting >500 nanocrystalline particles using ImageJ. XPS analysis was collected using a Thermo Scientific Escalab 250Xi instrument. The εPL content of εPL-CeOv was determined by thermogravimetric analysis (TGA, Mettler, USA) at a heating rate of 5 °C/min in air. The absorption property, chemical and crystal structure of εPL-CeOv were characterized by UV-visible spectrometer (UV-vis, Evolution 350, ThermoScientific), Fourier transformation infrared spectrometer (FTIR, Nicolet Nexus is50, ThermoScientific) and X-ray diffraction analysis (XRD, D8 ADVANCE, BRUKER). The angle of XRD was set from 10° to 90° with a rate of 6 °/min at 40 kV and 40 mA using a Cu Kα radiation source (λ = 0.15406 nm).
Synthesis and characterization of the F/R gel
Hyaluronic acid was oxidized by NaIO4 treatment to obtain aldehyde hyaluronic acid (HA-CHO). Briefly, 1% (w/v) hyaluronic acid aqueous solution was first prepared. Then, NaIO4 solution (0.2 mg/mL) was added dropwise to the solution under stirring, and the reaction was kept in the dark at room temperature for 4 h. Subsequently, the reaction was terminated by the addition of ethylene glycol for a duration of 1 h. The solution was dialyzed (MW cutoff at ~ 3500 Da) against water for 3 days. Finally, the HA-CHO product was lyophilized for further use. The oxidation degree of HA-CHO was quantified by calculating the number of aldehyde groups using the hydroxylamine hydrochloride titration method. FTIR spectrometer and proton nuclear magnetic resonance (1H NMR, Bruker AVANCE III 600 M, Germany) analysis used to identify the aldehyde groups in HA-CHO polymer. The solvent used in NMR analysis was deuterated water.
Solution A was prepared by dispersing εPL-CeOv nanocrystals (5 mg) into εPL aqueous solutions (1 mL, 25 mg/mL), while solution B was by dissolving HA-CHO powder (50 mg) into deionized water (1 mL). Schiff-based hydrogel (blank gel) was fabricated immediately by fully mixing equal volume of Solution A and B. For the F/R gel preparation, the F/R MCs (1.0 mg/mL) were firstly dispersed in solution A, and then mixed with solution B in the same way as above. The final concentrations of bFGF and siRNA in F/R gel were calculated as 23 ng/mL and 0.25 ng/mL, respectively. The SEM micrographs (EMAX mics2, HORIBA) were taken to observe the hydrogel morphology.
Measurements of ROS scavenging activity
To assess the ABTS radical scavenging activity, we employed the Total Antioxidant Capacity Assay Kit with the rapid ABTS method (Beyotime, Shanghai). In brief, ABTS radicals were produced by incubating a 7 mM ABTS stock solution with 2.45 mM potassium persulfate in darkness for 16 hours. Then, the ABTS radical solution was diluted by PBS to reach a proper absorbance at 734 nm. Subsequently, 1 mL of varying concentrations of the εPL-CeOv solution or the F/R gel was combined with 2 mL of ABTS solution and kept in the dark at room temperature for 10 minutes. The absorbance at 734 nm was recorded by microplate reader.
DPPH radical scavenging activity was measured by the 2,2-Diphenyl-1-picrylhydrazyl (DPPH). DPPH solution (0.2 mg/mL) was prepared using methanol as the solvent. εPL-CeOv solution (1 mL) or F/R gel was mixed with 1 mL DPPH, shaken, and incubated in the dark for 10 min at room temperature. The absorbance at 517 nm was recorded using a microplate reader.
The H2O2 concentration was monitored at 240 nm by UV-Vis spectrophotometer to assess H2O2 scavenging ability. εPL-CeOv solution (1 mL) or F/R gel was mixed with 2 mL of 10 mM H2O2, shaken, and incubated in the dark for 10 min at room temperature. The absorbance at 240 nm was then measured.
Hydroxyl Free Radical assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to evaluate •OH scavenging activity. Different concentrations of the εPL-CeOv solution (0.2 mL) or the F/R gel were mixed with the working solution, and placed in the dark for 1 min at 37 °C. To halt the reaction, the chromogenic agent was promptly added, and the mixture was left to react for 20 minutes at room temperature. The absorbance at 550 nm was recorded by a microplate reader.
Superoxide anion assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to evaluate •O2− scavenging activity. Different concentrations of the εPL-CeOv solution (0.05 mL) or the F/R gel were mixed with the working solution, incubated in the dark at 37 °C for 1 min, and then treated with a chromogenic agent. The absorbance at 550 nm was recorded by a microplate reader after 20 min at room temperature.
Intracellular ROS scavenging study
Two different cell lines were cultivated and used for the experiments: 3T3 cells and HUVEC were seeded into 6-, 12-, and 24-well plates severally at the density of 5×105 cells/well, 2×105 cells/well, and 1×105 cells/well. After 24 h incubation, different samples (F/R MCs, Gel and F/R gel) were added into the wells and incubated for another 24 h. Then, the cells were treated with 100 μM tBHP at 37 °C for 4 h. The cells seeded in 24-well plates were incubated with cell counting kit-8 to detect the cell viability. The cells seeded in 12-well plates were gently rinsed thrice with serum-free medium, followed by the addition of 10 μM DCFH-DA solution for 30 min’s incubation at 37 °C in the dark. Afterwards, the cells were washed with serum-free medium thrice to remove unloaded DCFH-DA probe, and then imaged by laser confocal microscope. Meanwhile, the HPF probe staining and the live/dead cell assay kit were also performed to detect the intracellular ROS level and cell viability changes. In addition, we performed cellular EdU staining according to the kit instructions. The cells seeded in 6-well plates were incubated with the DCFH-DA probe in the same way above, and then performed by flow cytometry analysis to quantify intracellular ROS levels separately.
In vitro regulation of macrophage polarization effect
RAW 264.7 cells (2×105) were cultured in a 6-well plate and incubated at 37 °C for 24 h. Then, the cells were treated with different groups (F/R MCs, Gel and F/R gel) for 24 h, and 250 ng/mL lipopolysaccharide (LPS) was added into for another 24 h. After centrifuged at 150 g for 3 min at 4 °C, the cells were resuspended in 500 μL of flow cytometry buffer containing APC anti-mouse CD86 antibody. The regulation of macrophage polarization effect was analyzed by flow cytometry.
Anti-scar healing effect of the F/R gel on MRSA-infected mouse wound model
To establish the infected wound model on mice, a round full-thickness wound (diameter ~ 6 mm) was created on the dorsal region of C57BL/6 mice (male, 8-10 weeks), followed by inoculation with 20 μl of MRSA bacteria (1×108 CFU/ml) solution on the wound area. The mice were randomly grouped and treated with PBS (100 μL), Beifuji gel (100 μL), F/R MCs (0.5 mg/mL, 100 μL), Gel (100 μL), and F/R gel (100 μL), respectively. Wound changes were carefully monitored during animal experiment.
On day 2, 4 and 7, the exudates at the wound site were gently dipped with sterilized cotton swabs, immediately diluted with LB culture medium, and coated on agar plates. After incubated at 37 °C for 12 h, representative images of bacterial colonies were taken for counting. Furthermore, the wound tissues after different treatments were collected at preselected time points and stored in 4% paraformaldehyde for H&E staining, Masson Trichrome staining and immunofluorescence staining (including DHE, CD86, CD206, CD68, CD31, VEGF, Ki67, CK10, COL I, COL III, TGF-β, c-Jun, CK19, β3-tubulin), respectively. The H&E staining and Masson staining slices were recorded using a virtual slide microscope (Olympus VS200). Immunofluorescence staining slices were photographed by laser confocal microscope. Information on antibodies used for immunofluorescence staining was listed in Table S1.
According to the manufacturer’s instructions, total mRNA was extracted from wound tissues using Trizol reagent, and then reverse-transcribed into cDNA using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, NanJing). qRT-PCR was performed using Taq Pro Universal SYBR qPCR Master Mix (Vazyme, NanJing) on a LightCycler® 96 Instrument (Roche, Switzerland). Levels of iNOS, CD86, TNF-α, IL-1β, IL-10, Arg1, CD206, Ki67, COL I, COL III, c-Jun and GAPDH transcripts were examined. The data were analyzed using the 2−ΔΔCt method, and the expression was normalized to that of GAPDH. The experiments were independently performed three times. The mRNA primer sequences used are listed in Table S2.
Mitigation of hyperplastic scar formation by the F/R gel on MRSA-infected rabbit ear wound model
Adult female New Zealand white rabbits (female, 3–4 months, 2-2.5 kg) were used to establish the hyperplastic scar wound model with MRSA infection. The rabbits were anaesthetized by isoflurane, and then each ear received six wounds using an 8 mm biopsy punch. The perichondria in the wounds were completely removed using a scalpel, and then 20 μl of MRSA bacteria (1×108 CFU/ml) solution was inoculated into the wound area. Then the wounds were treated at days 0, 4, 7, 14, 21 with PBS (100 μL), Beifuji gel (100 μL), F/R MCs (0.5 mg/mL, 100 μL), Gel (100 μL), and F/R gel (100 μL), respectively. The wounds were photographed during the treatments and the scars were collected on day 30 for H&E staining and Masson Trichrome staining.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
All data supporting the conclusions in the paper are present in the paper and/or the Supplementary Information. Data underlying Figs. 1–9 and Supplementary Figs. 1–54 are deposited in Figshare (https://doi.org/10.6084/m9.figshare.28138757). All data underlying this study are available from the corresponding author upon request.
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Acknowledgements
We thank the Scientific Research Center of Wenzhou Medical University for kind consultation and instrument availability that supported this work. This research was supported by National Natural Science Foundation of China under Grant No. 82202466 (Z.L.L.) and No. 82172428 (J.X.), Zhejiang Provincial Natural Science Foundation of China under Grant No. LZ24H150001 (Z.L.L.), Ningbo Yongjiang Talent Introduction Programme (Z.L.L.), Wenzhou City Major Scientific and Technological Innovation Project under Grant No. ZY2020023 (J.X.), and the Start-up Funding of Wenzhou Medical University under Grant No. 89223001 (Z.L.L.).
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F.Z. and Z.Z.L. conceived and designed the experiments. F.Z., H.J.Z., S.F.W., M.Y.G., K.Y.D., X.Y.C., and Z.L. performed the experiments. Y.L., Q.Q.H., A.Y. D., S.H.D., J.W., K.Q.S., and Z.M.C. provided technical and material support. F.Z. and Z.Z.L. analyzed the data and drafted the manuscript. Z.Z.L. and J.X. reviewed and edited the manuscript. All authors have read and approved the article.
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Zhang, F., Zhang, H., Wang, S. et al. A dynamically phase-adaptive regulating hydrogel promotes ultrafast anti-fibrotic wound healing. Nat Commun 16, 3738 (2025). https://doi.org/10.1038/s41467-025-58987-w
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DOI: https://doi.org/10.1038/s41467-025-58987-w
