Smart Microneedles Regulate Reactive Oxygen Species and Deliver Matrix Metalloproteinases for Pathological Scar Treatment

Abstract

Excessive oxidative stress and abnormal collagen deposition are critical drivers of pathological scar (PS) formation. To investigate the differences between normal and keloid tissues, single-cell sequencing and histological staining were conducted on patient samples, revealing that heightened collagen proliferation and oxidative stress are central to PS. To modulate the oxidative microenvironment and remodel the extracellular matrix (ECM), a smart microneedle capable of regulating reactive oxygen species (ROS) and delivering matrix metalloproteinase (MMP) functions was developed. Composed of methacrylated alginate hydrogel and hollow manganese dioxide nanoparticles, this microneedle not only scavenges ROS but also promotes the expression of genes and proteins associated with antioxidant and anti-inflammatory responses. Furthermore, MMP release from the ROS-responsive hydrogel suppresses transforming growth factor signaling, degrades excessive collagen, and facilitates ECM remodeling. In vivo evaluations in rabbit and porcine PS models demonstrated that this smart microneedle reduces scar thickness and restores skin function, highlighting its promising clinical potential. Overall, this study provides a clinically relevant framework for material design and establishes a closed-loop therapeutic strategy that integrates pathological signal decoding with targeted intervention, offering a approach for PS treatment.

1. Introduction

Pathological scar (PS) represents an abnormal healing response following skin injury, characterized by excessive proliferation, fibrosis, and functional impairment, significantly affecting patients’ appearance and quality of life. Epidemiological studies reveal that PS affects 35% of individuals with surgical wounds and 70% of those with burn injuries. − The pathogenesis of PS is multifactorial, with oxidative stress and abnormal collagen deposition playing central roles in excessive scar tissue proliferation and hardening. Oxidative stress, through the production of reactive oxygen species (ROS) like hydrogen peroxide (H₂O₂), causes cellular damage and activates pro-fibrotic signaling pathways, promoting scar formation. Furthermore, abnormal collagen deposition leads to excessive extracellular matrix (ECM) accumulation and disrupted remodeling, exacerbating scar hardness and functional impairment. , Despite the availability of treatments such as pharmacotherapy, laser therapy, and surgical excision, − they are often associated with limited efficacy, frequent recurrence, and substantial side effects. Recent advances in therapeutic approaches, such as gene therapy and cell-based therapies, have shown promise in preclinical fibrotic models. For example, small interfering RNA (siRNA) targeting key pro-fibrotic factors like transforming growth factor-β (TGF-β) has been explored to downregulate collagen synthesis, while mesenchymal stem cell (MSC)-derived exosomes have demonstrated anti-inflammatory and regenerative effects in wound healing. , However, these therapies face challenges including off-target effects, immunogenicity, complex manufacturing processes, and suboptimal tissue delivery. Nanomedicine-based antifibrotic strategies, including polymeric nanoparticles or liposomes for drug delivery, have also been studied, though they are often limited by rapid clearance and lack of precise, controlled drug release. Consequently, regulating the pathological microenvironmentparticularly by modulating oxidative stress and remodeling the ECMwhile ensuring localized, controlled, and minimally invasive delivery, is essential for improving therapeutic outcomes in PS and overcoming the limitations of current treatments.

To address the oxidative microenvironment, hollow manganese dioxide nanoparticles (hMNP) have garnered significant interest due to their unique physicochemical properties. hMNP possess excellent catalytic activity, efficiently decomposing H₂O₂ and alleviating oxidative stress. , For instance, Chen et al. demonstrated that manganese dioxide (MnO₂), as an inorganic nanozyme, plays a pivotal role in scavenging ROS and supplying oxygen to the wound site. , offering substantial advantages in antioxidant therapy. Additionally, hMNP exhibit pH-responsive release properties, enabling drug release in acidic environments, such as those found in scar tissue, thus facilitating targeted drug delivery and spatiotemporal control of release. − These characteristics make hMNP promising candidates for targeted ROS elimination and controlled drug delivery in the treatment of PS.

ECM remodeling plays a critical role in the treatment of PS. Matrix metalloproteinases (MMPs) are enzymes capable of specifically degrading ECM components, and their selective breakdown of abnormal collagen deposits facilitates ECM remodeling in scar tissue. − In the later stages of PS, where collagen deposition is particularly pronounced, recent studies have shown that MMPs can effectively reduce fibrosis, enhance the local tissue microenvironment, and significantly improve the hardness and appearance of scars. As a result, MMP-based therapies have emerged as a promising strategy for PS treatment. , However, despite promising preclinical results, the therapeutic use of collagen-degrading proteases such as MMPs still faces major challenges, including low delivery efficiency, poor stability and short in vivo half-life, and the risk of off-target damage to healthy tissues. − Thus, developing an effective delivery system capable of efficiently targeting and releasing MMPs in scar tissue is essential for improving therapeutic outcomes.

Microneedle (MN) technology has gained attention as a promising method for transdermal drug delivery, particularly in PS treatment, due to its pain-free, efficient, and minimally invasive nature. , MNs can penetrate the outer skin layers and directly deliver drugs to target tissues, enhancing drug delivery efficiency and bioavailability. Among various MN systems, hydrogel-based MNs have garnered significant interest due to their excellent biocompatibility, sustained drug release capabilities, and potential for incorporating stimuli-responsive functions. − Recent studies have explored several hydrogel materials for MN fabrication, such as gelatin, hyaluronic acid, and polyethylene glycol, demonstrating their promise in controlled drug release and wound healing. However, existing hydrogel MNs still face limitations, including insufficient mechanical strength for reliable skin penetration, uncontrolled degradation rates, and compromised structural integrity during insertion and drug release. , In this context, sodium alginate has emerged as an advantageous material due to its biocompatibility, biodegradability, and unique gelling properties. Sodium alginate hydrogels are easily fabricated into MNs, providing a robust yet flexible structure that maintains integrity during both insertion and drug release. Methacrylated alginate hydrogel (AlgMA), in particular, exhibits excellent water absorption and moisturizing properties, offering an optimal moist environment for scar tissue, promoting skin healing, and reducing scar formation. AlgMA also features strong film-forming capabilities, mild gelation under physiological conditions, and the potential for ionically cross-linked networks, which can be fine-tuned to enhance mechanical strength and control degradation rates. Therefore, integrating AlgMA with MN systems creates a synergistic platform that combines the mechanical strength and controlled release capabilities of the hydrogel with the targeted delivery efficiency of MNs. This innovative system has significant potential to improve the clinical management of PS.

While individual studies have explored the use of hMNP, MMPs, and hydrogel-based MN systems, their integration into a single, mechanism-driven platform has not been realized. Our smart MN system combines ROS scavenging, MMP-mediated ECM remodeling, and MN penetration, enabling localized, spatiotemporally controlled dual-action therapy directly within scar tissue. This closed-loop approach addresses both the root cause and the consequences of fibrosis, overcoming the limitations of prior methods that rely on systemic drug delivery or single-function materials. By integrating these mechanisms into a unified platform, the system ensures precise therapeutic targeting, improved efficacy, and significant translational potential, as confirmed in both rabbit and porcine scar models. This provides a promising strategy for the clinical management of PS.

This study aims to develop a smart MN system that targets ROS and delivers MMPs for PS treatment through a dual strategy of “microenvironment regulation and ECM remodeling” (Scheme ). To differentiate between normal and keloid tissues, single-cell sequencing analysis was conducted, and surgical samples were collected from patients with PS for histological staining. Subsequently, the smart MN was fabricated using AlgMA and MMP-loaded hMNP. The morphology of the MN, the in vitro release behavior of MMPs, and the ROS scavenging ability of the AlgMA-MMP-loaded hMNP composite hydrogel were evaluated. Additionally, the effects of the composite hydrogel on the expression of antioxidant, inflammatory, and fibrosis-related genes and proteins in fibroblasts derived from scar tissues were assessed. For in vivo evaluation, a rabbit ear PS model was used to investigate the therapeutic effects of the smart MN. To further explore its clinical potential, a PS model using skin scalds in Bama miniature porcines was established to assess the therapeutic efficacy of the smart MN.

1. A Smart MN System to Promote PS Repair by a Dual Strategy of “Microenvironment Regulation and ECM Remodeling” .

^a The microneedle patch integrates a AlgMA hydrogel with MMP-loaded hMNP. Upon transdermal insertion, the MN enhances drug penetration and enable targeted delivery into scar tissue. The hMNPs could scavenge excess ROS, enhance antioxidant and inflammatory properties. Meanwhile, the ROS-responsive release of MMP from the composite hydrogel could down-regulate the expression of transforming growth factor (TGF-β1) and selectively degrade abnormally deposited collagen and promote ECM remodeling. This smart MNs also exhibit good clinical potential based on the good therapeutic effects on skin scars in Bama miniature porcines.

2. Results and Discussion

2.1. Single-Cell RNA Sequencing Analysis of Keloids and Normal Tissues

Single-cell sequencing enables the precise identification and resolution of distinct cell types at the individual level, revealing functional differences in tissue repair and pathological conditions. Given the complexity and high heterogeneity of cell types in keloid and normal skin tissues, this approach allows for the analysis of each cell type’s unique gene expression patterns. Moreover, it identifies specific pathological cell populations in keloids, such as hyperactive fibroblasts and macrophages, which may play pivotal roles in the hyperproliferation observed in keloids. Analyzing these pathological cells offers a deeper understanding of keloid pathogenesis and uncovers potential therapeutic targets to inhibit their activity. Bioinformatic analysis was employed to compare keloid and normal tissues. The UMAP algorithm was used to reduce the dimensionality of the data to a two-dimensional space, identifying and annotating a total of 17 distinct cell clusters (Figure A,B). Distinct cell types were identified based on canonical marker genes, including macrophages (IL-1β/Lyz), fibroblasts (Col1a2/Col1a1), endothelial cells (Pecam1/Vwf), mast cells (Tpsab1/Tpsab2), and keratinocytes (Krt1/Krt14) (Figure C). The UMAP plots also illustrated the clustered distribution of these cell types, such as endothelial cells, fibroblasts, macrophages, keratinocytes, and mast cells, in a two-dimensional space (Figure D,E). The expression of antioxidant-related genes, SOD1 and CAT, was compared between keloid and normal tissues using violin plots. These revealed a significant decrease in SOD1 expression in keloid tissues, with a smaller differential expression observed for CAT (Figure F). Dot plots further highlighted the expression levels and ratios of SOD1 and CAT, showing that both the expression level and ratio of SOD1 were higher in normal tissues compared to keloid tissues (Figure G). These findings suggest that keloid tissues are in a state of heightened oxidative stress, indicating that oxidative stress may play a pivotal role in keloid formation and progression. Overall, these results offer a perspective on keloid pathogenesis and lay the groundwork for developing antioxidant-based therapeutic strategies.

1.

Single-cell RNA sequencing analysis of keloidal and normal tissues. (A,B) UMAP clustering map of keloid and normal tissues, respectively. (C) Expression point map of cell types. (D,E) UMAP plot for cell type annotation of endothelial cells, fibroblasts, macrophages, keratinocytes and mast cells in keloid and normal tissues, respectively. (F) Vln plot of SOD1 and CAT expression in keloid and normal tissues. (G) Dot plot of SOD1 and CAT.

2.2. Histology Staining of Normal and Keloid Tissues in Human

To investigate the differences between normal and keloid tissues, surgical samples were collected from patients with PS, representing a range of genders and ages. These samples, obtained from clinical surgeries, were subjected to relevant histological staining. The cohort comprised four females and one male, aged between 18 and 38 years (Figure A), with scars localized to the jaw, forearm, ears, and chest. Pathological classifications included hyperplastic scars, keloids, and a hybrid of both (Figure B). The keloid boundaries are indicated by red dotted lines in the corresponding images. Patients 1 and 2 exhibited hyperplastic scars, patients 3 and 4 had keloids on their ears, and patient 5 presented a mixed keloid and hypertrophic scar on the anterior chest. Histological analysis, employing Hematoxylin–Eosin (H&E) and Masson’s trichrome staining, revealed marked thickening of the dermis in scar tissue compared to normal tissue. In the dermal layer of scar tissue, collagen fibers appeared disorganized, with areas of inflammatory cell infiltration (Figure C). Masson’s trichrome staining further confirmed excessive collagen proliferation and irregular fiber arrangement, reflecting an increase in collagen density within the scar tissue (Figure D). Immunohistochemical staining for CD31, a marker of angiogenesis, showed significantly elevated CD31 expression in scar tissues, suggesting that enhanced vascularization plays a key role in scar formation and progression (Figures E and S1A). Additionally, dihydroethidium (DHE) staining was conducted to assess ROS levels in the tissues, revealing a significant increase in ROS in scar tissue, which implicates oxidative stress as a critical factor in keloid pathogenesis (Figures F and S1B). These results suggest that excessive collagen proliferation, oxidative stress, and increased vascularization are central to keloid formation, providing potential therapeutic targets. These insights not only deepen our understanding of PS pathophysiology but also open avenues for developing therapeutic strategies.

2.

Histology staining of normal and keloid tissues in patients. (A) Basic information about the patients of the PS sample. (B) Photos of PS areas in different donors. (C) H&E and (D) Masson’s trichrome staining of NS tissue and PS tissue, respectively. (E) Immunohistochemical staining for CD31 in NS tissue and PS tissue. (F) DHE staining of NS tissue and PS tissue.

2.3. Characterizations of the hMNP, hMNP/AlgMA Composite Hydrogel and MNs

The hMNP was synthesized using a hard template method, wherein silica nanoparticles were served as a spherical core. Manganese dioxide was deposited on the surface of the template, followed by a chemically etching process to remove the silica template and obtain hMNP. Transmission electron microscopy (TEM) revealed that the particle diameter was approximately 200 nm and confirmed its hollow structure (Figure S2A). Energy dispersive X-ray spectroscopy (EDS) was employed for elemental composition analysis, demonstrating the distribution of oxygen (O, blue) and manganese (Mn, yellow), confirming the successful synthesis of hMNP (Figure A). Subsequently, we characterized the MNs substrate material, which is composed of AlgMA cross-linked via blue light irradiation. Scanning electron microscopy (SEM) images revealed that the pure AlgMA hydrogel exhibited a distinctly loose and porous structure. With the incorporation of hMNP, the pore structure of the hydrogel significantly changed, particularly at an hMNP concentration of 1%, where the pore structure of the hydrogel collapsed notably (Figure B). To fabricate the composite MNs, the precursor solution was introduced into a PDMS mold bearing microneedle array patterns, photo-cross-linked, removed from the mold, and subsequently dried. The MN patches exhibited a certain degree of flexibility, allowing them to adapt to the skin’s bending. After doping with hMNP, the needle tips displayed an opaque black color, indicating successful incorporation (Figure C). The photomacrograph and SEM images showed that the MNs were tapered and aligned in an orderly manner, with a height of approximately 500 μm and a base diameter of around 260 μm (Figures D and S2B). To enhance the mechanical strength, the composite microneedles underwent liquid nitrogen freezing treatment to become cryo-MNs. To assess the skin penetration ability of the MNs, mechanical compression testing was first performed, and it was found that each needle can withstand a force of approximately 0.5 N, which is above the critical force required to penetrate the skin (Figure E–G). This result was further validated through porcine skin penetration experiments, where H&E staining confirmed that excellent penetration performance was achieved by the microneedles. After penetration into the skin, no significant deformation or bending of the microneedles was observed (Figure H). Moreover, the degradation properties of MNs are critical for drug delivery applications. Before insertion, the MN tips remained intact; however, after 7 days of insertion, approximately 20% of the MNs had degraded, with around 50% degradation observed by day 14. By day 21, the tips had largely degraded, demonstrating the gradual degradation of the MNs over time (Figure G). This gradual degradation profile supports sustained drug delivery, allowing the MNs to release their therapeutic payload over an extended period. The degradation behavior is likely influenced by the incorporation of hMNP and its interaction with the alginate matrix, which may affect the dissolution rate and structural integrity of the MNs. In conclusion, the composite MNs combined robust mechanical properties, controlled degradability, and sustained release behavior, supporting their potential application in transdermal drug delivery for PS management.

3.

Characterizations of the hMNP, hMNP/AlgMA composite hydrogels and composite MNs. (A) TEM image and EDS elemental analysis of hMNP. (B) SEM images of composite hydrogels with different hMNP concentrations. (C) SEM images of the hMNP/AlgMA-0.5% composite MNs. (D) Photomacrograph of AlgMA MNs and hMNP/AlgMA-0.5% composite MNs. (E–G) Mechanical test of hMNP/AlgMA-0.5% composite MNs. (H) Penetration performance of MNs in porcine skin by H&E staining. (I) Subcutaneous degradation property of hMNP/AlgMA-0.5% composite MNs. Data are presented as mean ± SD (n = 3); *, p < 0.05, one-way ANOVA analysis.

2.4. Biocompatibility, Anti-Inflammatory and Antioxidant Properties of the Composite Hydrogels

To evaluate the biocompatibility of the composite hydrogel, human skin fibroblasts (HSFs) were seeded on its surface. Live/dead staining results showed high cell viability across all hydrogel formulations, except for the hMNP/AlgMA-1% hydrogel, which exhibited a significant number of dead cells (Figure A). Cytoskeleton staining further revealed that HSFs on AlgMA, hMNP/AlgMA-0.25%, and hMNP/AlgMA-0.5% hydrogels displayed excellent spread, suggesting strong adhesion and no adverse effects on cell growth. In contrast, HSFs on the hMNP/AlgMA-1% hydrogel exhibited noticeable wrinkling, suggesting cytotoxic effects at this concentration (Figure B). Additionally, CCK-8 assay results showed no significant differences in cell proliferation among AlgMA, hMNP/AlgMA-0.25%, and hMNP/AlgMA-0.5% hydrogels, while proliferation was markedly inhibited on the hMNP/AlgMA-1% hydrogel (Figure C). Consequently, the hMNP/AlgMA-0.5% composite hydrogel was selected for subsequent experimentation due to its optimal biocompatibility.

4.

(A) Live–dead staining and (B) cytoskeleton staining images of HSFs cultured on the composite hydrogels. (C) CCK-8 assay of HSFs cultured on the composite hydrogels. (D) In vitro release behavior of MMP1 from the composite hydrogels under different conditions. (E) ROS scavenging property of different hydrogels in fibroblasts by DCFH-DA staining. (F) Effect of the composite hydrogel on the expression of antioxidant, inflammatory and fibrosis-related proteins in fibroblasts derived from scar tissue. (G) Effect of composite hydrogel on the expression of antioxidant, inflammatory and fibrosis-related genes in fibroblasts derived from scar tissue. Data are presented as mean ± SD (n = 3); *, p < 0.05, **, p < 0.01, one-way ANOVA analysis.

PS tissue is intricately linked with oxidative stress, often characterized by excessive H₂O₂ production. To address this, hMNP-based pH-responsive drug delivery system enables targeted therapy at the scar site, modulates the oxidative microenvironment, and inhibits fibrosis progression. In this study, we investigated the drug release behavior of MMP1-loaded hMNP/AlgMA composite hydrogels in the presence of H₂O₂. The encapsulation efficiency of MMP1 in hMNP was 50.9 ± 2.6% (Figure S3). Upon incorporation into the hydrogel, drug release analysis revealed a significant acceleration in MMP1 release upon exposure to H₂O₂ (Figure D), highlighting the system’s ROS-responsive drug release behavior. Furthermore, pathological fibroblasts were isolated from human PS tissue and their identity was confirmed (Figure S4), then the effect of the hMNP/AlgMA composite hydrogel on ROS expression levels in these cells was evaluated. The results demonstrated a significant reduction in intracellular ROS levels following treatment with the composite hydrogel, indicating its effective antioxidant properties (Figures E and S5).

In addition to oxidative stress, chronic inflammation and fibrosis are key factors contributing to the complexity of treating PS. Under normal wound healing conditions, inflammation is typically a transient phenomenon that resolves in the early stages. However, in the context of PS formation, there is a persistent infiltration of inflammatory cells, such as macrophages and neutrophils, which leads to a sustained elevation of pro-inflammatory cytokines (e.g., IL-1β, IL-6, and TNF-α). This results in the establishment of a chronic inflammatory milieu that not only prolongs the tissue repair process but also contributes to the abnormal activation of fibroblasts. , Moreover, PS is characterized by an overactive wound repair process that bears mechanistic resemblance to organ fibrosis. In PS tissue, sustained activation of transforming growth factor-β1 (TGF-β1) signaling contributes to aberrant fibroblast proliferation and myofibroblast differentiation. Myofibroblasts are distinguished by high α-smooth muscle actin (α-SMA) expression, increased contractile activity, and excessive production of collagen, especially collagen types I and III, which collectively drive scar enlargement and fibrotic remodeling. − To further investigate the therapeutic potential of the composite hydrogel, we analyzed the expression levels of proteins and genes related to antioxidation, inflammation, and fibrosis in fibroblasts extracted from scar tissue. Using Western blot and qPCR, we found that treatment with AlgMA hydrogel alone had no significant effect on the expression levels of these markers. In contrast, the hMNP/AlgMA composite hydrogel significantly upregulated the expression of antioxidant-related proteins (CAT and SOD), as well as the anti-inflammatory cytokine (IL-10), while downregulating the expression of the pro-inflammatory cytokine (TNF-α) and the fibrosis-related protein (TGFβ1) (Figures F and S6). Similar trends were observed at the gene expression level (Figure G). These findings demonstrate that the hMNP/AlgMA composite hydrogel effectively exerts antioxidant, anti-inflammatory, and antifibrotic effects in vitro, highlighting its strong potential for PS treatment applications. To further explore the necessity of ROS regulation for effective ECM remodeling, we performed decoupling experiments using the following groups: Ctrl, hMNP/AlgMA, MMP-1/AlgMA, and MMP1@hMNP/AlgMA. We measured the expression of fibrosis-related proteins (COL1, α-SMA, CTGF, and TGF-β1). The results showed that while MMP-1 delivery alone exhibited some ECM remodeling effects, these effects were significantly enhanced when ROS was scavenged, demonstrating a positive synergistic effect between ROS regulation and MMP-1 activity (Figure S7). These findings demonstrate that the hMNP/AlgMA composite hydrogel holds significant promise as a therapeutic strategy for PS. Its ability to modulate the expression of key proteins and genes involved in oxidative stress, inflammation, and fibrosis positions it as a multifaceted approach to addressing the complex pathophysiology of PS.

2.5. In Vivo Efficacy of Composite MNs in Rabbit Ear PS Model

To evaluate the in vivo therapeutic efficacy of the composite MNs, a rabbit ear PS model was established and the animals were randomly assigned to five groups: saline group, PS group, AlgMA group, hMNP/AlgMA group, and MMP1@hMNP/AlgMA group (Figure A). To support the therapeutic mechanism and efficacy of our ROS-responsive microneedle system, several preliminary experiments were performed to ensure the effectiveness and safety of the composite microneedles. First, the enzymatic stability of MMP-1 during microfabrication, UV-cross-linking, and storage was assessed using a DQ-collagen fluorescence assay, and it was found that approximately 86.4% of the original MMP-1 activity was retained, indicating minimal loss during the microfabrication process (Figure S8A,B). In addition, the stability of MMP-1 during storage at 4 °C for 3 weeks was evaluated, and no significant decrease in enzymatic activity was observed, demonstrating that MMP-1 maintains its bioactivity under typical storage conditions. These findings provide direct evidence that MMP-1 functionality is preserved during microneedle fabrication and storage (Figure S8C,D).

5.

Evaluation of in vivo therapeutic effect of MNs on rabbit ear PS model. (A) Diagram of the establishment of PS model and evaluation of the therapeutic effect. (B) The photomacrograph of the PS model at different points in time. (C) The photomacrograph of scar areas at different time points after treatment with composite MNs. (D) DHE staining image under different interventions. (E) Immunofluorescence staining of SOD, CAT and IL-10, respectively. (F) Immunofluorescence staining of TNF-α.

To ensure long-term biosafety, H&E staining of major organs (heart, liver, spleen, lung, and kidney) was performed 4 weeks after microneedle implantation, and no detectable histopathological changes were observed, indicating that no overt organ toxicity occurred under the tested conditions. These findings provide preliminary evidence supporting the in vivo safety of the composite MNs system (Figure S9). Then, Scar formation was monitored at different time points postmodeling, and representative images of skin lesions were captured at various intervals post-treatment (Figure B,C). After 26 days of treatment, it was observed that the lesions in the MMP1@hMNP/AlgMA group were noticeably smoother than those in the PS group, with scar borders largely diminishing. Furthermore, the MMP1-free composite MNs exhibited limited therapeutic efficacy, as indicated by the reduced height and blurred borders of PS lesions. To assess the potential off-target effects of MMP-1 on normal dermis, peri-lesional skin adjacent to the treated scars was analyzed. The microneedle system incorporates a ROS-responsive release mechanism, ensuring that MMP-1 is primarily released within the high-ROS microenvironment of the scar tissue, where denatured collagen is enriched. In addition, MMP-1 exhibits higher specificity for denatured collagen compared to native collagen, further limiting activity in surrounding healthy tissue. To experimentally validate these design features, H&E and Masson’s trichrome staining (Figure S10) were performed on the peri-lesional skin. No significant collagen loss, structural disruption, atrophy, or ulceration was observed, indicating that the therapeutic activity of MMP-1 is largely confined to fibrotic tissue and that off-target ECM degradation is minimized. These findings indicate that composite MNs loaded with MMP1 significantly enhance the treatment outcomes for PS.

Given the crucial role of ROS and inflammation in scar formation, we further conducted DHE staining and immunofluorescence staining of the surgical site 7 days postimplantation to evaluate ROS levels and the expression of antioxidant and inflammation-related proteins. The fluorescence intensity in the MMP1@hMNP/AlgMA group was significantly reduced, confirming the effectiveness of MMP1-loaded composite MNs as ROS scavengers (Figures D and S11). Immunofluorescence staining further revealed that the expression levels of antioxidant-related protein (SOD and CAT) and anti-inflammation protein (IL10) were markedly reduced in the PS group. However, treatment with hMNP/AlgMA and MMP1@hMNP/AlgMA led to a significant increase in their expression levels, with the MMP1@hMNP/AlgMA group exhibiting the strongest fluorescence intensity and the largest stained area (Figures E, S12A–C). Additionally, the expression of the pro-inflammatory protein TNF-α was significantly elevated in the PS group, whereas its level was nearly restored to that of the NS group following treatment with MMP1@hMNP/AlgMA (Figures F and S12D). These findings suggest that MMP1-loaded composite MNs regulate the local wound microenvironment by scavenging ROS and exerting anti-inflammatory effects, ultimately inhibiting scar formation.

Additionally, histological assessments via H&E and Masson’s trichrome staining were performed to evaluate the changes in PS lesions following different treatments (Figure A,B). Before treatment, the PS group exhibited a dense fibroblasts and thickened collagen bundles in the dermis. However, treatment with MMP1@hMNP/AlgMA composite MNs, a significant reduction in the thickness of PS tissue was observed. Higher magnification images further revealed a marked decrease in collagen deposition and a more organized arrangement of fibroblasts, indicating an improved tissue structure. PS is an aberrant outcome of wound healing, with fibrosis at its pathological feature. It is characterized by abnormal fibroblast proliferation, excessive ECM deposition, and dysregulated tissue remodeling. , Among the regulators of fibrosis, TGF-β is particularly instrumental in PS formation. TGF-β1, via both Smad-dependent and -independent pathways, promotes ECM synthesis while inhibiting MMPs, ultimately leading to excessive collagen deposition. , In PS tissues, there is an accumulation of type I collagen and a reduced proportion of type III collagen, which results in increased tissue stiffness and a loss of the normal elasticity characteristic of healthy skin. Additionally, fibroblasts within PS differentiate into myofibroblasts, which enhances contractility and contributes to scar contracture. , Connective tissue growth factor (CTGF), a member of the CCN family, participates in embryonic development and multiple cellular processes, including proliferation, migration, differentiation, and ECM remodeling. It is also recognized as an important regulator of fibrosis, including hepatic, pulmonary, and renal fibrosis, as well as in PS formation, further driving fibrosis and tissue dysfunction. To further validate the therapeutic efficacy of MMP1@hMNP/AlgMA composite MNs, we performed immunofluorescence staining for key fibrosis-related proteins in PS tissues. The results revealed that the fluorescence intensity of α-SMA, CTGF, COL1, and TGF-β1 was significantly higher in PS tissues compared to control, indicating enhanced fibrosis. However, after treatment with hMNP/AlgMA and MMP1@hMNP/AlgMA composite MNs, the fluorescence intensity of these proteins was markedly reduced, with the most pronounced therapeutic effect observed in the MMP1@hMNP/AlgMA group (Figures C and S13). These findings underscore the capacity of MMP1@hMNP/AlgMA composite MNs to ameliorate fibrosis in PS by modulating the expression of key fibrosis-related proteins, thereby attenuating the fibrotic response and improving tissue repair quality.

6.

Histological staining of rabbit ear PS treated by different MNs. (A) H&E and (C) Masson’s trichrome staining. (C) Immunofluorescence staining of fibrosis-related proteins.

2.6. In Vivo Efficacy of Composite MNs in Porcine Skin PS Model

The MMP1@hMNP/AlgMA composite MNs demonstrated promising therapeutic effects in the rabbit ear PS model. To further assess their clinical potential, a PS model was established using skin scalds in 5- to 6 month-old Bama miniature porcines (Figure A). Porcine skin closely resembles human skin in terms of anatomical and physiological properties, including epidermal thickness, dermal structure, hair follicle distribution, collagen organization, and wound healing mechanisms. These similarities make the porcine model highly relevant for studying MN-based antiscarring strategies. Severe burns in porcine skin induce scar formation marked by persistent inflammation, excessive fibroblast proliferation, collagen accumulation, and abnormal angiogenesis, closely resembling human PS. Consequently, the porcine PS model offers a valuable platform for preclinical research with high clinical translational potential.

7.

In vivo evaluation of therapeutic effect on porcine skin PS model. (A) The photomacrograph of scar formed in porcine skin. (B) H&E and (C) Masson’s trichrome staining image under different interventions, respectively. (D) Immunohistochemical staining of fibrosis-associated proteins. (E) Immunohistochemical staining of inflammation-related proteins.

Consistent with our previous experiments, DHE staining was performed to assess ROS levels in porcine skin tissues before and after treatment with the MMP1@hMNP/AlgMA composite MNs. The results mirrored those observed in the rabbit ear PS model, showing significantly elevated ROS levels in the PS group, which were substantially reduced following treatment with the composite MNs (Figure S14). Additionally, H&E and Masson’s trichrome staining were conducted to examine changes in PS lesions before and after treatment. Prior to treatment, the PS group exhibited abundant fibroblasts and thick collagen bundles in the dermis. After treatment with composite MNs, the thickness of the PS tissue significantly decreased, with magnified images revealing a substantial reduction in collagen deposition and a more organized fibroblast arrangement (Figure B,C). To further confirm the therapeutic effects of composite MNs in the porcine PS model, immunohistochemical staining was performed to evaluate the expression of inflammation- and fibrosis-related proteins in the tissues. As expected, fibrosis-related proteins (α-SMA and TGF-β) and the pro-inflammatory protein (TNF-α) were significantly elevated in the PS group compared to the control group. However, after treatment with composite MNs, the expression levels of these proteins were downregulated, while anti-inflammatory proteins showed the opposite trend (Figures D and S15A,B). Specifically, IL-10 was significantly upregulated following treatment with composite MNs, highlighting the anti-inflammatory potential of the treatment (Figures E and S15C,D). The results demonstrate that MMP1@hMNP/AlgMA composite MNs exhibited superior efficacy in the rabbit ear PS model and similarly promising therapeutic effects in the porcine skin PS model. These findings highlight the potential of this approach for clinical application, offering a effective strategy for managing PS.

2.7. Future Perspectives and Remaining Challenges

The therapeutic landscape for pathological scars is evolving, with emerging modalities such as gene and cell-based therapies offering new avenues for intervention. Gene therapy, particularly using siRNA or antisense oligonucleotides to knock down pro-fibrotic genes like TGF-β1 or CTGF, has shown potential in preclinical studies by directly inhibiting the synthetic machinery of fibroblasts. However, its clinical translation is hampered by challenges related to efficient and safe delivery to target cells, the risk of off-target genomic effects, and the transient nature of gene silencing. Cell-based therapies, including the use of MSC or their derived exosomes, leverage paracrine signaling to modulate inflammation and promote regeneration. While promising, these approaches face hurdles such as cell viability post-transplantation, heterogeneity in cell products, scalability for manufacturing, and potential immunogenicity. Other nanomedicine-based antifibrotic strategies, such as drug-loaded polymeric or lipid nanoparticles, offer improved pharmacokinetics but often lack the ability to overcome the physical barrier of dense scar tissue and provide controlled, on-demand release at the pathological site.

In contrast, the smart MN system introduced here provides a distinct and synergistic approach, directly addressing the core pathological drivers identified in our initial patient sample analysis: oxidative stress and ECM dysregulation. By integrating a ROS-scavenging nanozyme (hMNP) with an ECM-remodeling enzyme (MMP-1) into a physically penetrating MN, this system offers localized, mechanism-based intervention. Unlike systemic gene or cell therapies, the MN platform provides a minimally invasive, targeted delivery method that bypasses the dense ECM barrier, ensuring that therapeutic agents are delivered precisely where needed. The ROS-responsive release mechanism adds an additional layer of spatiotemporal control, activating drug release specifically within the high-oxidative stress microenvironment of the scar, thereby minimizing off-target effects in surrounding healthy tissue. This stands in contrast to the passive diffusion or systemic distribution often seen with other nanomedicines. Moreover, this approach combines two therapeutic actions within a single platformreducing inflammation and oxidative stress while promoting ECM remodelingthus creating a closed-loop strategy that addresses both the root cause and the consequences of fibrosis.

Despite the promising results, several unresolved challenges and future research directions must be addressed to facilitate clinical translation.

First, while the stability and bioactivity of MMP-1 during MN fabrication and storage have been demonstrated, ensuring the long-term stability and activity of protein therapeutics in a commercially viable product remains a critical area for optimization. Future work could explore lyophilization techniques or the use of stabilizing excipients to enhance shelf life. Second, the long-term biosafety of the system, particularly concerning the fate of manganese ions (Mn²⁺) released from degraded hMNP, requires further rigorous evaluation. Although our preliminary data from major organs and serum biochemistry over an 8 week period showed no overt toxicity, comprehensive long-term biodistribution, accumulation, and clearance studies are needed. Future iterations may explore strategies to mitigate potential Mn²⁺ toxicity, such as designing more biodegradable nanoparticles or incorporating chelating agents. Third, the risk of off-target ECM degradation by MMP-1, if it were to diffuse to the surrounding healthy dermis, is a valid concern. This issue is mitigated in our design by two key features: (1) the ROS-responsive release mechanism ensures MMP-1 is primarily released within the high-ROS microenvironment of the scar, and (2) MMP-1 exhibits higher specificity for denatured collagen, which is more prevalent in the disorganized scar matrix. However, future studies should incorporate more sensitive assays to detect any subtle changes in the peri-lesional healthy skin to definitively rule out atrophy or ulceration. Finally, scaling up the manufacturing process of these composite MNs while maintaining batch-to-batch consistency, mechanical integrity, and drug loading efficiency remains a key translational hurdle. The use of biocompatible materials like alginate offers a strong foundation for scalable fabrication using established micromolding technologies.

In conclusion, while the smart MN system represents a significant advancement by providing a synergistic, mechanism-based treatment for PS, ongoing research should focus on decoupling the individual contributions of ROS scavenging and MMP delivery to establish causality. Additionally, further investigations will optimize material properties for improved mechanical penetration of hardened keloids and conduct comprehensive long-term safety and manufacturing studies to transition this promising technology from the laboratory to clinical application.

3. Conclusion

This study analyzed the cellular and environmental changes in normal and PS tissues from patients, identifying excessive collagen proliferation and oxidative stress as key factors in PS development. Building on this, this study further developed a dual-function MN system aimed at treating PS by improving the oxidative microenvironment and remodeling the ECM. This system effectively scavenged excess ROS and remodeled the ECM by selectively degrading excessive collagen deposits. The physical penetration of the MNs and the pH-responsive release of hMNP facilitated precise, spatiotemporally controlled therapy, ensuring efficient drug delivery directly to the scar tissue. Therapeutic efficacy was confirmed in both rabbit ear and porcine PS models, showing significant reductions in fibrosis-related markers, improved collagen organization, and enhanced skin regeneration. By simultaneously addressing oxidative stress and ECM remodeling, this strategy bridges the gap between anti-inflammatory and antifibrotic therapies, offering a minimally invasive, synergistic approach to PS management. Moreover, the sodium alginate-based MN system’s cost-effectiveness and scalable manufacturing potential, along with positive results in large animal models, position it as a promising solution for PS treatment. These findings highlight the clinical translation potential of our composite MNs, laying a foundation for future research in skin regeneration and wound healing.

4. Materials and Methods

4.1. scRNA Analysis

The GSE163973 data set, comprising three keloid and three normal skin samples, was retrieved from the Gene expression omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/). For quality control, a cutoff was applied: nFeature_RNA between 300 and 5500, mitochondrial content less than 10%, and red blood cell content under 3%. Following data normalization, feature selection, and dimensionality reduction, cells were classified into distinct clusters at the optimal resolution and visualized in the UMAP diagram. Cell types were annotated based on markers obtained from CellMarker 2.0 (http://117.50.127.228/CellMarker/CellMarkerBrowse.jsp). The expression levels of SOD1 and CAT were represented using violin and bubble plots. Cluster annotations: clusters 0, 6, and 10 corresponded to endothelial cells; clusters 1, 2, 4, 5, 11, 13, 14, 15, and 16 to fibroblasts; clusters 3, 7, and 8 to keratinocytes; cluster 9 to macrophages; and cluster 12 to mast cells.

4.2. Detection of Human Samples

4.2.1. Ethical Statement

Pathological scarring specimens were obtained from five keloidectomy patients (one male and four females). The study protocol was approved by the Ethics Committee of Linyi People’s Hospital (202401-H-027, YiLin, China) and informed consent was obtained from each donor.

4.2.2. Histological Analysis

After fixation in 4% paraformaldehyde at 4 °C for 48 h, the samples were dehydrated, embedded, and cut into 6 μm sections for H&E and Masson’s trichrome staining. For CD31 immunohistochemistry, sections were incubated with a primary antibody against CD31 (Abcam, ab182981, UK) and then with HRP-labeled goat antirabbit IgG (Beyotime, China). Signal development was achieved using DAB, and the nuclei were counterstained with hematoxylin.

4.2.3. DHE Staining

For DHE staining, tissues were fixed in 4% paraformaldehyde at 4 °C overnight, cryoprotected in sucrose, embedded in OCT, and sectioned into 6 μm slices (Beyotime, China), and fluorescence intensity was subsequently quantified using ImageJ software.

4.3. Preparation and Characterizations of the hMNP/AlgMA Composite MNs

4.3.1. Synthesis of hMNP

hMNP were synthesized according to previous protocols. Fourteen mL of ethanol (SCRC, China), 2 mL deionized water, and 500 μL of ammonia (SCRC, China) were mixed and stirred at 50 °C for 5 min, followed by the dropwise addition of 500 μL TEOS (Sigma-Aldrich, GER) at 45 °C. Stirring continued for 2 h after the addition. The solution was washed twice by centrifugation with ethanol and water, then dispersed in water.

The solid silica from the previous step was dispersed in 10 mL of water, while 600 mg of KMnO₄ (SCRC, China) was dissolved in 20 mL of water under sonication. The KMnO4 solution was added dropwise to the silica suspension, sonicated for 1 h, and then stirred overnight at room temperature.

The resulting material was dispersed in 20 mL of 2 M Na₂CO₃ (Aladdin, China) solution and stirred overnight at 60 °C, followed by washing with water four times to obtain the final hMNP.

4.3.2. Preparation of the Composite Hydrogels and MNs

The composite precursors were formulated by incorporating hMNP (2.5, 5, or 10 mg) into 1 mL of 10% (w/v) AlgMA containing 0.25% (w/v) LAP (EFL, China). These mixtures, designated as hMNP/AlgMA-0.25%, −0.5%, and −1%, respectively, were homogenized and cast into cylindrical molds. Finally, the gels were formed through UV-induced photo-cross-linking.

For fabrication of hydrogel-based microneedles (MNs), an appropriate volume of pregel solution was cast into a PDMS mold. The mold was then placed under negative pressure to remove trapped air and facilitate filling of the microneedle cavities. This procedure was repeated until the cavities were completely filled. The loaded molds were dried at 35 °C and subsequently cross-linked under UV light. To facilitate demolding, the PDMS molds were stored at −20 °C for 4 h before the MN arrays were peeled off. The obtained MNs were then kept at −80 °C for 4 h and finally immersed in liquid nitrogen for 1 h to generate cryo-MNs.

4.3.3. Morphology of the hMNP and Composite MNs

The hMNP were characterized using TEM (TECNAI G2 F20, FEI, USA) at an acceleration voltage of 120 kV. The surface morphology of the composite MNs was analyzed via SEM (Quanta 250, FEI, USA) to assess detailed structural characteristics.

4.3.4. Mechanical Properties of the MNs

The mechanical properties of the MNs were evaluated using uniaxial compression tests to assess structural integrity and skin penetration suitability. MN patches were prepared using 10% (w/v) AlgMA, with or without 0.5% (w/v) hMNP, and cross-linked via UV photopolymerization. To improve mechanical strength, selected patches underwent cryo-treatment. For compression testing, three MNs were fixed onto a rigid stainless-steel plate with double-sided adhesive tape, ensuring stable contact and alignment.

Compression testing was performed at room temperature using a universal testing machine (HengYi, China) fitted with a 20 N load cell. A flat probe was driven toward the MN tips at 0.5 mm/min until the preset displacement was reached. Both the applied force and displacement were continuously recorded. For each condition, at least n = 3 independent MNs were tested. The peak compressive force before structural deformation of the MNs was recorded as the critical failure load, and the results were expressed as mean ± SD.

Additionally, the mechanical properties of the composite MNs were further evaluated using porcine skin penetration tests and MN compression experiments, with validation through H&E staining of the skin tissues.

4.4. In Vitro Biocompatibility

HSFs were seeded onto different hMNP/AlgMA hydrogels in 24-well plates to evaluate cytocompatibility. After 2 days of culture, the samples were washed with PBS, fixed in 4% paraformaldehyde, and blocked with 3% BSA at 4 °C overnight. Following this, the HSFs were incubated with phalloidin and subsequently stained with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, China). Fluorescence images were captured using a fluorescence microscope (EVOS, USA). After confirming satisfactory cell spreading on the hydrogels, cell viability was assessed using a Calcein-AM/PI Double Staining Kit (Solarbio, Beijing, China), and fluorescence images were obtained.

HSFs were seeded into 24-well plates at a density of 5 × 10³ cells/well for proliferation analysis. After 12 h of attachment, composite hydrogels were introduced as upper chambers, and cell proliferation was determined by CCK-8 assay (NCM, China).

4.5. In Vitro Drug Loading and Release Tests

The loading efficiency of MMP1 was determined by quantifying the residual protein in the supernatant using an MMP1-ELISA kit (Solarbio, China). The calculation was performed according to the following formula

$$\mathrm{L}\mathrm{o}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{e}\mathrm{f}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{y}=(\mathrm{M}\mathrm{a}\mathrm{s}\mathrm{s}\mathrm{o}\mathrm{f}\mathrm{M}\mathrm{M}\mathrm{P}1\mathrm{u}\mathrm{s}\mathrm{e}\mathrm{d}-\mathrm{M}\mathrm{a}\mathrm{s}\mathrm{s}\mathrm{o}\mathrm{f}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{i}\mathrm{d}\mathrm{u}\mathrm{a}\mathrm{l}\mathrm{M}\mathrm{M}\mathrm{P}1)/(\mathrm{M}\mathrm{a}\mathrm{s}\mathrm{s}\mathrm{o}\mathrm{f}\mathrm{M}\mathrm{M}\mathrm{P}1\mathrm{u}\mathrm{s}\mathrm{e}\mathrm{d})\times 100\%$$

For the MMP1 release study, 1 mL of AlgMA hydrogel containing MMP1@hMNP was prepared for each sample (n = 3). The hydrogels were immersed in 2 mL of either PBS or PBS supplemented with 100 μM H₂O₂, and then incubated in a thermostatic shaker at 37 °C and 60 rpm. At predetermined time points (days 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 31), the supernatant was collected and replaced with fresh buffer. The amount of released MMP1 was quantified using an MMP1 ELISA kit.

4.6. DQ-Collagen Fluorescence Kinetics for MMP1 Stability

The stability of MMP1 encapsulated in hydrogel-based MNs was evaluated using DQ-Collagen I (Thermo Fisher, D12052, USA) as a fluorogenic substrate. MNs stored at 4 °C for 0, 1, 2, and 3 weeks were tested. Three MNs from each time point were incubated in 200 μL of reaction buffer containing 10 μg/mL DQ-Collagen I in a black 96-well plate at 37 °C.

Fluorescence intensity (Ex/Em 495/515 nm) was recorded every 5 min over 60 min using a microplate reader. Background fluorescence was subtracted, and kinetic curves were plotted as fluorescence versus time. Enzyme activity was quantified from the initial linear portion of the curve. The data are presented as mean ± standard deviation (n = 3 MNs per time point), allowing for the assessment of MMP1 stability during MN preparation and storage.

4.7. In Vivo Degradation Property of the MNs

To investigate the degradation properties of the composite MNs in vivo, the composite MNs were punctured through the skin, and morphological changes were observed at 0d, 7d, 14d, and 21d after extraction.

4.8. Antioxidant Properties of the Composite Hydrogels

4.8.1. Reactive Oxygen Species (ROS) Staining

For ROS detection, cells were seeded in 24-well plates at 1 × 10⁴ cells per well. After 12 h, AlgMA and hMNP/AlgMA-0.5% aqueous gels were cocultured with the cells. After 24 h, staining was performed using the DCFH-DA probe (Beyotime, China), and fluorescence images were obtained using a fluorescence microscope.

4.8.2. qPCR and Western Blot Analysis

To evaluate gene expression, cells (2 × 10⁵/well) were seeded onto AlgMA or hMNP/AlgMA-0.5% composite hydrogels, with those in 6-well plates as controls. Total RNA was isolated after 48 h and quantified via NanoDrop (ThermoFisher Scientific, USA). Subsequently, cDNA synthesis was performed using 5X All-in-One RT MasterMix (ABM, Canada), followed by qPCR using iTaq Universal SYBR Green Master Mix. Relative mRNA levels were determined using the 2^–ΔΔCt method, normalized to GAPDH.

Total proteins were isolated using RIPA lysis buffer supplemented with 1% PMSF and a protease/phosphatase inhibitor cocktail (Beyotime, China). Protein concentrations were subsequently quantified via the BCA assay (Beyotime, China). Proteins were denatured at 95 °C for 10 min, separated on 10% SDS-PAGE gels, and transferred to PVDF membranes. Membranes were blocked with 5% skimmed milk for 1 h, followed by overnight incubation at 4 °C with primary antibodies against CAT (CST, 12980, USA), SOD (Proteintech, 24127-1-AP, China), TNF-α (Proteintech, 80258-6-RR, China), TGFβ1 (Invitrogen, PA1-29032, USA), and IL-10 (Proteintech, 24127-1-AP, China). Immunoblot signals were captured via an Odyssey infrared imaging system and subsequently analyzed for optical density using ImageJ.

4.9. Animal Study

Experimental protocols were approved by the Animal Care and Use Committee of Linyi People’s Hospital (Nos. 202407-A-005 and 202411-A-009) and conducted in accordance with NIH guidelines. Fifteen healthy New Zealand white rabbits underwent a one-week acclimation period with ad libitum access to food and water prior to the study. After environmental adaptation, hypertrophic scar models were induced on the ventral side of both ears using a modified method based on Morris et al. The rabbits were individually housed with daily checks to monitor survival and prevent infection. To promote hypertrophic scar formation and facilitate observation of the healing process, wounds were left exposed to air with regular removal of surface exudate, without bandaging or medication. Scabs were removed at 2 weeks. By day 28, the rabbit ear wounds had completely healed, forming reddish, firm-textured hypertrophic scars raised above the surrounding skin.

The experimental groups were as follows: NS, PS, AlgMA, hMNP/AlgMA, and MMP1@hMNP/AlgMA. Therapeutic treatments were administered on day 30, and scar tissue was collected on day 37 for ROS level detection, as well as the expression of inflammation- and antioxidant-related proteins. Samples were collected on day 56 for histological analysis.

Additionally, the study was extended to validate the model in porcine skin scarring. Three adolescent wild-type Bama miniature porcine (female, 25–30 kg, 5–6 months old) were utilized. Preoperative preparation included a 12 h fast and hair removal from the dorsal region. The skin was then sterilized with Betadine solution and rinsed with 75% alcohol. Zoloftil (15 mg/kg) was administered intramuscularly 10 min before creating the skin lesions. Anesthesia was induced via intramuscular Zoletil (5 mg/kg; Virbac) and Rompun (2 mg/kg; Bayer), followed by maintenance with inhaled isoflurane (Abbott Laboratories). On the depilated dorsal surface of each pig, 20 symmetrical scald wounds (2 × 2 cm²) were generated using a YLS-5Q scald meter set at 120 °C for 20 s. The wounds were left exposed to air, and surface secretions were removed. The scabs were removed at 2 and 3 weeks, and the effect of modeling was confirmed after 5 weeks. Subsequently, treatment with the composite MNs was applied, and samples were collected for histological staining after 10 weeks.

4.10. Histological Analysis

Rabbits and porcines were sacrificed, and samples were collected at 37 days, 56 days, and 70 days postoperatively. H&E staining, Masson’s staining, and DHE staining were performed to assess tissue repair. At day 37, levels of oxidative stress (SOD, CAT) and inflammatory cytokines (IL-10, TNF-α) were assessed. Subsequently, fibrosis-related proteins (α-SMA, C-TGF, COL1, and TGF-β) were targeted for staining at day 56. Additionally, immunohistochemical staining for IL-10 (Proteintech), TNF-α (Proteintech), α-SMA (Proteintech, 67735-1-Ig, China), and TGF-β (Invitrogen) was performed on tissue sections collected at 70 days. Fluorescence intensity was subsequently quantified using ImageJ software.

4.11. Statistical Analysis

All statistical analyses were carried out using GraphPad Prism software. Results are presented as mean ± SD (n ≥ 3). Comparisons among groups were made using one-way ANOVA followed by Tukey’s multiple-comparison test, and significance was defined as p < 0.05.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.6c00171.

• Additional quantitative analysis of CD31 (human-derived PS tissue), DHE (human-, rabbit-, and porcine-derived PS tissue), and DCFH-DA (human-derived PS tissue) staining; TEM and SEM characterization of hMNP and composite MNs; MMP-1 standard curve, loading concentration, and enzymatic activity assay; immunofluorescence staining of human fibroblasts; qPCR analysis related to antioxidation and inflammation; Western blot analysis and quantification of fibrosis-related proteins; H&E and Masson’s trichrome staining of major organs; immunofluorescence analysis of SOD, CAT, IL-10, TNF-α, α-SMA, CTGF, COL1, and TGF-β (PDF)

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J.W., J.M. and Z.L. These authors contributed equally to this work. Jun Wu, Jinjin Ma, and Zexi Li contributed equally to this work. Jun Wu, Jinjin Ma, Zexi Li, Fengxuan Han, Bin Li, and Can Xiao designed the research; Jun Wu, Jinjin Ma, Zexi Li, Jiaying Li, Jie Hu, Fengyu Ma, and Yingkai Wu performed the experiments; Jun Wu, Jinjin Ma, and Zexi Li analyzed the data; Jun Wu, Jinjin Ma, Zexi Li, Fengxuan Han, Bin Li, and Can Xiao wrote the paper.

The authors declare no competing financial interest.