A bio-instructive, bioactive in situ polymerizable wound matrix promotes scar-free burn wound repair

Summary

Early initiation of wound regeneration and healing is the primary determinant of survival for burn patients. While superficial burns do not require secondary interventions and undergo spontaneous healing, full-thickness burns exceed the intrinsic capacity of body to induce skin regeneration. Herein, we developed a wound matrix system, referred to as PEGScarX that provides critical bioactive and bio-instructive cues to modulate cell fate decisions for burn wound healing. Our results indicate that PEGScarX promotes faster wound healing kinetics and reduces scar formation in burn wounds. PEGScarX induces fibroblast and epidermal stem cell repopulation, inhibits transdifferentiation of fibroblasts to myofibroblasts, and induces a pro-regenerative immune cell niche. These findings unravel an opportunity to reduce morbidity and mortality in burn patients due to inadequate wound regeneration and reduce the occurrence of pathological scaring.

Graphical abstract

Highlights

• •PEGScarX provides bioactive and bio-instructive cues to modulate cell fate
• •PEGScarX minimizes scar formation in burn wounds
• •Fibroblast-to-myofibroblast transition is inhibited
• •snRNA-seq reveals a higher proportion of fibroblasts and epidermal stem cells

Cell biology; Biomaterials; Polymers

Introduction

Burns are a global health problem leading to significant morbidity and mortality in those who succumb to these injuries. According to the American Burn Association, every year over 450,000 serious burn injuries occur in the United States that require medical treatment accounting for $1.5 billion in treatment costs and additional $5 billion in costs associated with lost work in a single year (https://ameriburn.org/national-burn-awareness-week-2020/ABANBAW). While the World Health Organization (WHO) estimates that 11 million burn injuries of all types occur annually worldwide, 180,000 of which are fatal (https://www.who.int/news-room/fact-sheets/detail/burnsWBFS). Thus, burn injuries continue to be one of the leading causes of death even in those who survive the primary insult. This is a direct consequence of the loss of structural and functional integrity of the skin which increases the risk of bacterial contamination and translocation, exposing the patient to persistent infectious and inflammatory mediators as long as the wound remains open leading to a sustained systemic inflammatory response syndrome. Additive to this catastrophic cascade of pathophysiology is the loss of fluids, electrolytes, and proteins which increase the metabolic demands of the body, leading to hyperinflammation and hypermetabolism, both of which further increase the risk of sepsis, multi-organ failure (MOF), and death. To this end, recent analyses have shown that sepsis associated MOF is the most common cause of death.^1,2,3 Thus, early initiation of wound regeneration and healing, as well as preventing wound infections are primary determinants of survival for trauma and burn patients.

While superficial burns, characterized by the presence of viable dermis, do not require secondary interventions and undergo spontaneous healing, full-thickness burns exceed the intrinsic capacity of body to induce skin regeneration and eventually result in either non-healing wounds or less-than-ideal epithelial scars and wound contractures with reduced functionality. Thus, bioengineered matrices mimicking properties of the natural extracellular matrix (ECM) offer significant therapeutic potential and the ability to temporally control skin regeneration and wound healing. The ECM of the skin tissue constantly adapts to changing intrinsic and extrinsic biological and mechanical forces and is responsible for the transduction of these forces into cells to alter the wound healing process and regulate the susceptibility to either non-healing wounds or excessive scar formation. This fundamental understanding has led to the development of precisely tailored wound matrices with bioinstructive and bioactive properties that coordinate tissue formation and regeneration by delivering an initial signal to the cells (either endogenous or exogenous cells) to initiate their programmed functions². Cells in turn respond by initiating receptor-ligand interactions that trigger signaling cascades to regulate gene expression and integrate multiple external cues into a single programmed response.⁴ In addition to ligand-initiated molecular signaling, cells also interpret matrix stiffness by mechanotransduction. Mechanical forces not only act on cells but they also reorganize the matrix to dictate tissue orientation⁵ and provide feedback across multiple time and length scales of the wound healing cascade. Consequently, collectively these cell-cell and cell-matrix interactions govern cell migration, proliferation, and differentiation to influence epithelialization, fibrosis, angiogenesis, wound contraction, and remodeling.

Creating a conducive environment for cell survival and function in complex wound microenvironments is challenging. To this end, several innovative hydrogels and wound patches have been developed exploiting the electrical, mechanical, or biological activation of cells or a combination thereof. Hydrogels with electrical activation have gained popularity during the last few decades.⁶ A self-adaptive hydrogel with crucial features for wound repair was synthesized through the combination of tannic acid, human-like collagen, polyvinyl alcohol, and borax and used in combination with electrical stimulation therapy to facilitate intercellular interaction and increase cell migration and angiogenesis.⁷ Wearable piezoelectric nanogenerator (PENG) have been developed that produce a direct current pulse combined with an electro responsive hydrogel that included a phosphatase and tensin homolog (PTEN) inhibitor (BPV@PCP). The PENG devices transform movements into electrical signals, leading to the concurrent charging of both the wound and the BPV@PCP.⁸ Supercapacitors and sodium hyaluronate-based hydrogels are used to create self-powered electrically stimulated wound dressings.⁹ Likewise, mechanical stimulation of hydrogel characteristics affects mechanosignaling pathways, allowing spatiotemporal control over biological processes, including cell proliferation and tissue regeneration.⁶ Yufeng Shou developed a mechano-responsive hydrogel for therapeutic MSC direct production. They created a 3D magnetic hydrogel containing magnetic particles embedded in gelatin hydrogel, called gelatin methacryloyl/poly (ethylene glycol) diacrylate-magnetic particle hydrogel (GPM). Dynamic mechanical stimulation (DMS) significantly improved matrix and integrin β1 interactions, enhancing MSC proliferation and distribution.¹⁰ Using N-dimethyl bisacrylamide (NIPAM) and glutaraldehyde (GTA) crosslinked hyaluronic acid (HA), a hydrogel having interpenetrating polymer networks (IPN) was developed.¹¹ This hydrogel is temperature responsive and tissue adhesive, making it an effective wound dressing to accelerate wound healing. Yixiao Dong et al. developed a hyaluronic acid hydrogel scaffold to deliver stem cells and enhance wound healing outcomes. The hydrogel was composed of hyperbranched diacrylate poly (ethylene glycol) (HB-PEGDA), thiol-functionalized hyaluronic acid (HA-SH), and a peptide composed of arginine, glycine, and aspartic acid (RGD peptide). This PEG-HA-RGD hydrogel was mixed with adipose derived stem cells (ADSCs). The hydrogel was then tested on the second-degree burns on mice models. The authors reported that the hydrogel-mediated delivery of adipose stem cells (ASCs) significantly enhanced burn wound healing outcomes by increasing cell survival, reducing scar formation, and promoting neovascularization.¹² Multifunctional and smart hydrogels provide numerous wound healing stimuli simultaneously. An enhanced smart hydrogel for wound therapy was created in a study by Bai et al. to wirelessly monitor the condition of wounds in real-time. This device leverages a novel organohydrogel triboelectric nanogenerator developed from natural skin, thereby rendering it bioresorbable, devoid of battery demands, and capable of wireless use.¹³

These hydrogel-based treatments may improve wound healing, reduce scar formation, physical discomfort, and functional limitations especially in burns, surgical incisions, and challenging-to-heal wounds, where excessive scarring can cause long-term impairment and low self-confidence. State-of-the-art hydrogels have the potential not only to enhance patients’ quality of life but also to contribute significantly to the efficiency and effectiveness of healthcare systems.

Thus, here in, we demonstrate the development, preclinical efficacy, and mechanism of action of an in situ polymerizable, bio-instructive, and bioactive wound matrix, referred to as PEGScarX. We engineered PEGScarX as a modular wound matrix system composed of components which when combined, allow in situ polymerization on to burn wounds and modulate the dynamic wound healing cascade. Hence, the goal of our matrix design is to achieve a wound matrix that will impact the current standard of care by being conducive to endogenous cell infiltration, adhesion, and proliferation to promote granulation tissue formation and re-epithelialization; provide a barrier to pathogens and thereby prevent bacterial biofilm formation; biocompatible for biotherapeutic and/or cell delivery with tunable mechanical properties; and be available in minutes and in large quantities to allow coverage of large and severe wounds.

Results

Synthesis and rheological performance of PEGScarX

Ideal wound regeneration of excisional full-thickness burn wounds requires an optimal microenvironment that can prevent fluid loss and can provide a scaffold for cellular infiltration, proliferation, and matrix deposition followed by matrix remodeling to achieve skin regeneration similar to uninjured skin. Therefore, we aimed to create a wound matrix that can seal the wound upon application and has properties mimicking the natural dermal ECM. We achieved this by covalently incorporating a matrix metalloprotease degradable peptide between acrylate PEG side chains. A 10% w/v solution of this PEG macromer was combined in equal ratios with thiol-HA, thiol-gelatin, and tropoelastin which resulted in instantaneous in situ polymerization in less than 1 min to form the PEGScarX wound matrix when deposited on excisional burn wounds.

Rheological analyses were conducted in a similar scheme where the precursor solutions were immediately deposited onto the sample plate. Our results demonstrate that PEGScarX loses its solid behavior and yields after 20–30% strain material demonstrating compliant behavior (Figures 1A and 1B). Collagenase-mediated degradation of PEGScarX was confirmed by incubating 1 cm² matrices in 10 mg/mL collagenase with a degradation rate of 41.66 ± 16.99 mg/h (Figure 1C). We further analyzed swelling kinetics of the PEGScarX in saline solution and observed a 15.4 ± 2.06-fold increase in weight compared to dry weight (Figure 1D).

Figure 1.

Material and biological characterization of PEGScarX

(A and B) Storage modulus (G′) and loss modulus (G″) of PEGScarX. After 20–30% strain material loses its solid behavior and yields.

(C) Quantification of degradation in exogenous collagenase.

(D) Swelling kinetics in PBS.

(E) A 1 cm² PEGScarX sheet immersed in cell culture media with embedded MSCs. Scale bar = 1 cm.

(F) Bright field micrographs of cell spreading on day 5. Scale bar = 100 μm.

(G and H) Cell viability of MSCs embedded in PEGScarX demonstrate no hypoxic centers or cell death in the geometrical center. Scale bar = 200 μm.

(I–K) Immunofluorescence micrographs of (I) cell spreading using actin staining and ECM deposition (J) collagen I and III and (K) Laminin deposition. Scale bar = 50 μm.

(L) Cell viability of MSCs post-harvest from PEGScarX using exogenous collagenase. Data are represented as mean ± SEM. Scale bar = 50 μm.

PEGScarX is cytocompatible and promotes cell spreading and deposition of ECM proteins

To assess cytocompatibility, we embedded mesenchymal stem cells (MSCs) from discarded burn tissue in 1 cm² PEGScarX matrices at 0.5 × 10⁶ cells/cm² (Figure 1E). Bright-field micrographs revealed spindle shaped MSCs embedded within the matrix (Figure 1F). Cell viability was assessed on day 3 post-encapsulation and no hypoxic centers of cell death were observed in 3D z stacks projections of immunofluorescence micrographs (Figures 1G and 1H). Cells were allowed to proliferate and spread for further 21 days, and cell adhesion and spreading was analyzed by actin filament immunofluorescence staining demonstrating the characteristic spindle-shape morphology and spreading of MSCs (Figure 1I). Further, we observed that PEGScarX matrices were conducive to deposition of matrix proteins such as collagen family and laminin family by the embedded MSCs as evidenced by positive immunofluorescent staining for collagen I and III and laminin (alpha-helical domains I and II) (Figures 1J–1L).

PEGScarX modulates cytokine expression and growth factor expression in cells

Biophysical cues from the three-dimensional matrix are transmitted via cell-matrix interactions to influence intracellular signaling cascades impacting the cellular secretions of cytokines and growth factors. We elucidated the effect of PEGScarX on cytokine, chemokine, growth factor, and immunomodulatory proteins secretion from embedded MSCs and compared to monolayer MSCs (ML). No statistically significant differences were observed in secretion of pro-inflammatory cytokines interleukin (IL)-1α, tumor necrosis factor alpha (TNF-α), IL-6, or bioactive subunit of IL-12 (p70) which corroborates that PEGScarX does not induce a cytotoxic or proinflammatory response (Figure 2A). Concentrations were ML: 9.07 ± 4.07 pg/mL vs. PEGScarX: 3.12 ± 0.84 pg/mL, ML: 5.64 ± 2.46 pg/mL vs. PEGScarX: 3.41 ± 1.17 pg/mL, ML: 140.7 ± 33.72 pg/mL vs PEGScarX: 216.9 ± 58.0 pg/mL, and ML: 1.83 ± 0.71 pg/mL vs PEGScarX: 1.27 ± 0.08 pg/mL, for IL-1 α, TNF-α, IL-6, and IL-12 (p70), respectively. Chemokine secreted concentrations (Figure 2B) were also similar between cell cultured in monolayers or embedded in PEGScarX corresponding to ML: 1179 ± 462.7 pg/mL vs. PEGScarX: 227.4 ± 126.8 pg/mL, ML: 22.78 ± 2.72 pg/mL vs. PEGScarX: 7.92 ± 7.92 pg/mL, ML: 27.32 ± 4.29 pg/mL vs. PEGScarX: 23.04 ± 1.89 pg/mL and ML 95.51 ± 48.15 vs. PEGScarX: 33.06 ± 12.61 pg/mL for MCP-1 (monocyte chemoattractant protein 1), MIG, MIP-1α (macrophage iflammatory protein 1-alpha), and IL-8, respectively. Among immunomodulators (Figure 2C), IL-2 and IL-7 concentration were similar between the groups corresponding to ML: 0.59 ± 0.28 pg/mL vs. PEGScarX: 0.5 ± 0.02 pg/mL, ML: 4.62 ± 0.82 pg/mL vs. PEGScarX: 2.44 ± 0.32 pg/mL, respectively, while IL-15, and IL-22 were secreted at significantly lower level from cells embedded in PEGScarX compared to monolayer cultured cells with concentrations of ML: 4.64 ± 1.08 pg/mL vs. PEGScarX: 0.82 ± 0.42 pg/mL and ML: 16.48 ± 7.63 pg/mL vs. PEGScarX: 0 ± 0 pg/mL, respectively. Finally, no differences were observed in growth factor secretion (Figure 2D) for macrophage colony stimulating factor (M-CSF) (ML: 855.8 ± 105.9 pg/mL vs. PEGScarX: 339.8 ± 216.6 pg/mL) and vascular endothelial growth factor (VEGF) (ML: 188.9 ± 55.72 pg/mL vs. PEGScarX: 112.8 ± 12.66 pg/mL), platelet derived growth factor (PDGF) was only secreted from cells embedded in PEGScarX at 115.5 ± 10.04 pg/mL, while fibroblast growth factor (FGF) concentrations were significantly higher in PEGScarX at 23.74 ± 10.26 pg/mL compared to 6.123 ± 1.48 pg/mL in monolayer controls.

Figure 2.

Analysis of factor secretion and cell-mediated degradation

(A–D) Milliplex analysis of (A) pro-inflammatory cytokines, (B) chemokines, (C) immunomodulatory factors, and (D) growth factor secretion of PEGScarX-embedded MSCs.

(E) Contraction and degradation assay of PEGScarX with and without normal human dermal fibroblasts.

(F) Quantification of PEGScarX area does not demonstrate any contraction (reduction in area) with or without cells in vitro, (inset) fibroblast containing PEGScarX degraded over time due to cell-secreted MMPs and cell viability does demonstrate any cell death (7 days) and began to attach on the wells as PEGScarX degraded (14 days) while continuing to proliferate and spread. Scale bar, 1 cm. Data are represented as mean ± SEM. ∗: p < 0.05 using t test.

Cell-mediated matrix contraction and degradation of PEGScarX with embedded human dermal fibroblasts

Fibroblasts develop geometric and mechanical interactions via cell adhesions that occur three-dimensionally through attachment sites with the surrounding ECM. These cellular forces and the state of cellular mechanical loading regulate matrix contraction that govern wound contraction. Loading wound matrices with fibroblasts in vitro is a widely acceptable model to study wound contraction. Matrices that resist deformation develop mechanical tension in the cells leading to matrix contraction. To assess whether PEGScarX limits such mechanical loading on cells and thereby prevent contraction, we embedded normal human dermal fibroblasts at 1 × 10⁶/cm² in PEGScarX. PEGScarX did not demonstrate any contraction with or without fibroblasts in vitro (Figure 2E). PEGScarX area at 48 h was 0.67 ± 0.13 cm² and 0.85 ± 0.06 cm² without and with cells, respectively (Figure 2F). PEGScarX degraded over time due to cell-secreted MMPs and demonstrated increase in area due to breakdown of crosslinks. PEGScarX embedded with cells were placed in tissue culture well plates and allowed to culture. Cell-mediated degradation of PEGScarX did not lead to cell death of the embedded cells. As the matrix degraded, cells began to attach to the underlying tissue culture plastic and continued to proliferate and maintain their spindle-shaped morphology (Figure 2E-inset).

PEGScarX promotes faster wound healing kinetics in vivo with superior wound histological features

We next tested PEGScarX in a porcine thermal injury model where PEGScarX was deposited on to full thickness excisional burn wounds on day 2 post-burn for instantaneous in situ polymerization. Polymerization on the wounds was achieved within ∼1–2 min. Photographs of the wounds were taken at each dressing change and healing kinetics were analyzed. The wounds were allowed to heal for ∼40 days at which time tissue was collected. Our results demonstrated that PEGScarX promotes faster healing kinetics and superior wound histological features compared to commercial dermal regenerative matrix (DRM). Deposition of granulation tissue was scored on a scale from 0 to 5 (Figure 3A). By day 4 post-burn, PEGScarX treated wounds demonstrated deposition of coarse granulation tissue while granulation tissue was absent in no treatment controls and DRM wounds. By day 18 post-burn, PEGScarX wounds had granulation tissue evolved from coarse to mix to a fine structure signifying that PEGScarX provides a framework for cell migration, proliferation, and angiogenesis much earlier than control and DRM. By day 13, % epithelialized area were 51.26 ± 13.41%, 13.71 ± 27.32%, and 66.09 ± 5.60% for control, DRM, and PEGScarX treated wounds (Figure 3B). We further analyzed wound histomorphological features in wound tissues collected on day 40 post-burn at which times most of the wounds have re-epithelialized. All wounds represented distinct keratinized epithelia, epidermal, and dermal layers (Figure 3C). Fibrosis was observed in control and DRM wounds representing that these wounds were still in the remodeling phase compared to PEGScarX wounds. Further, PEGScarX demonstrated significantly higher number of rete ridges comparable to uninjured skin (Figure 3D). Dermal collagen morphology exhibited more mature collagen fibers with basket-weave like pattern (Figure 3E) with higher percent of type I collagen (Figure 3E, inset) and higher collagen density (Figure 3F) in PEGScarX wounds.

Figure 3.

PEGScarX promotes collagen deposition and wound re-epithelialization

(A) Granulation tissue scores in excisional burn wounds.

(B) Quantification of wound re-epithelialization.

(C) H&E wound histology. Scale bar = 102 μm.

(D) Quantification of epidermal rete ridges.

(E) Masson’s trichrome staining for collagen density. Scale bar = 102 μm.

(F) Quantification of collagen density.

Data are represented as mean ± SEM.

∗: p < 0.05 using ANOVA with least significant difference (LSD) post-hoc analysis.

PEGScarX reduces scar formation

Concurrently with epidermal and dermal regeneration, reconstitution of the skin barrier after a burn wound requires a delicate balance between contraction by myofibroblasts and re-epithelialization which ultimately determines the extent of scar formation and the functional quality of the repaired wound. To assess this, we evaluated the healed wounds for vascularity, pigmentation, pliability, and height and computed a cumulative score also known as the Vancouver Scar Score. Scar scores were significantly lower in PEGScarX treated wounds (5.84 ± 1.03) compared to control (7.98 ± 0.78) and DRM treatment (9.84 ± 0.89) (Figures 4A and 4B). This was further confirmed by α-SMA immunohistochemical staining (Figure 4C) representing significantly lower contraction in PEGScarX wounds (3.96 ± 2.66), while α-SMA intensities were 5.90 ± 4.65 and 8.74 ± 5.99 for control and DRM wounds, respectively (Figure 4D). Further, anti-scarring mediator transforming growth factor β3 (TGFβ3) levels were significantly higher in PEGScarX wounds (1.63 ± 0.79) compared to 1.28 ± 0.78 and 0.69 ± 0.23 in control and DRM, respectively (Figure 4E). Gene expression of wound remodeling proteases and structural proteins further demonstrated differential expression in PEGScarX wounds (Figures 4F and 4G).

Figure 4.

PEGScarX treatment mitigates wound scar formation

(A) Gross appearance of wound scar on day 40 post-burn

(B) Vancouver Scar Scale scores for assessment of wound scar.

(C–E) (C) Immunohistochemical staining; scale bar = 100 μm, and (D) quantification of α-SMA expressing myofibroblasts in wound histological sections on day 40 post-burn. Western blot analysis of (E) TGF-β3 in wounds on day 40 post-burn.

(F and G) Gene expression analysis of wound remodeling mediators.

Data are represented as mean ± SEM.

∗: p < 0.05 using ANOVA with least significant difference (LSD) post-hoc analysis. +: p < 0.05 between DRM and PEGScarX using ANOVA with least significant sifference (LSD) post-hoc analysis.

PEGScarX promotes neovascularization and reduces wound inflammation

Neovascularization of the wounds was confirmed by immunohistochemical stains for CD-31 (Figure 5A). Similar results were seen for blood vessel density (Figure 5B) and VEGF with levels significantly higher in PEGScarX wounds corresponding to fold changes of 1.39 ± 0.79, 0.99 ± 0.49, and 0.82 ± 0.24 in PEGScarX, controls and DRM wounds, respectively (Figure 5C). We further assessed wound inflammation and observed lowest expression of immune cell marker CD11b in PEGScarX treated wounds (Figures 5D and 5E). These results corroborated with gene expression analysis which demonstrated that PEGScarX treated wounds had significantly increased expression of neoangiogenesis markers SDF-1α and VEGF, and IL-6, a key modulator of inflammatory and reparative processes (Figure 5F).

Figure 5.

PEGScarX promotes neovascularization and reduces wound inflammation

(A) Immunohistochemical analysis of neovascularization using anti-CD31 to stain for blood vessels. Scale bar = 100 μm.

(B) Quantification of number of blood vessels.

(C) Western blot analysis of VEGF in wound sections demonstrated significantly higher levels in PEGScarX.

(D) Immunohistochemical analysis of wound inflammation using anti-CD11b. Scale bar = 100 μm.

(E) Quantification of CD11b+ cells demonstrate significantly reduced inflammation in PEGScarX compared to controls.

(F) Gene expression analysis of inflammation and neoangiogenesis factors. Data are represented as mean ± SEM.

∗: p < 0.05 using ANOVA with least significant difference (LSD) post-hoc analysis.

snRNA-seq analysis of PEGScarX treated full thickness burn wounds

We collected biopsy tissue from full thickness burn wounds treated with PEGScarX, DRM, and no treatment controls on post-burn day 14 and submitted the tissue to single-nucleus RNA sequencing (snRNA-seq) using the 10X Genomics platform (v2 chemistry). We examined the similarities and differences among the constituent skin cells across the high-dimensional gene expression space by performing t-SNE dimensionality reduction and identified 22 clusters with distinct expression profiles through Louvain clustering (Figure 6A). Employing known markers with the most representative expressed genes, we were able to identify 10 main cell phenotypes present in the healing wound on post-burn day 14 (Figure 6B). Clusters # 1, 10, 17, 5, 9, and 13 comprised fibroblasts based on the expression of DCN, COL1A2, PDGFRA, and COL3A1, while epidermal stem cells (ITGA6 and CD34) were identified in clusters # 3, 8, and 22. Skin immune cells including macrophages, Langerhans cells, dendritic cells and B cells were identified in 7 clusters (cluster # 4, 6, 7, 11, 15, 16, and 19) based on representative marker expression of macrophages (CD68, AIF1, CTSS, and LYZ), langerhans cells (CD86), dendritic cells (FCER1g), and B cells (MS4A1 and CD79a). Endothelial cells enriched in PECAM1, PROX1, and LYVE1, and vascular cells expressing RGS5 and PDGFRB, constituted clusters # 2, 18, 20, and clusters # 12, respectively. Next, we determined the percent proportion of the classified cell types within each treatment group (Figures 6C and 6D) and observed that the highest proportion of fibroblasts (46.04%), epidermal stem cells (18.34%), and vascular cells (6.75%) were detected in PEGScarX treated wounds, and immune cells (69.12%) and keratinocytes (0.97%) represented the highest population pool in DRM treated wounds while endothelial cells (23.26%) were highest in control wounds. To determine the range of biological diversity within each treatment group (Figure 7A), we conducted differential expression analysis (Figures 7B and 7C) followed by gene ontology analysis for most significant genes (false discovery rate [FDR]<0.05 and fold change of >2 and <-2) and examined cell phenotypes across signaling pathways to explore variability in the mechanisms of wound regeneration by our various treatments. Inflammation mediated by chemokine and cytokine signaling pathway and VEGF signaling pathway were significantly underrepresented in PEGScarX wounds at post-burn day 14 compared to control wounds (Figure 7D). Between PEGScarX and DRM wounds, integrin signaling pathway, TGF-beta signaling pathway, Wnt signaling pathway, angiogenesis, endothelin signaling pathway, and cadherin signaling pathway were significantly overrepresented (Figure 7E).

Figure 6.

snRNA sequencing analysis

(A) t-SNE plot depicting single-nuclei transcriptomes from whole porcine skin from d14 post-burn. Each dot represents a single cell (n = 21,726). Average expression of cell type markers were projected on the t-SNE plot to identify all cell populations.

(B) t-SNE plot with cell-type classification of graph-based clusters.

(C) Percent proportion of each cell-type within each treatment group.

(D) Average expression of each cell type within each treatment group.

Figure 7.

Graph-based clustering and cell-type classification

(A) t-SNE graph-based clustering plots of each treatment with cell-type classification.

(B and C) Differential gene expression analysis of across all cell types represented by volcano plots comparing (B) PEGScarX vs. Control wounds and (C) PEGScarX vs. DRM treated wounds.

(D and E) Pathway enrichment analysis of differentially expressed genes across all cell type.

p < 0.05, FDR < 0.05, Log₂FC < -2 and >2.

PEGScarX induces fibroblast and epidermal stem cell repopulation and function

Fibroblasts represented the highest proportion in graph-based clustering analysis of the snRNA-seq data (Figure 8A), we therefore wanted to understand the specific function of the subpopulations within the fibroblast clusters relevant to skin wound healing. In particular, fibroblasts can be classified based on their specific dermal locations with papillary fibroblasts expressing COL13A1 and COL18A1 located close to epidermal-dermal junction (Figure 8B), reticular fibroblasts expressing MGP or MFAP5 located deeper in the dermis within a very dense network of matrix fibers, and fibroblast subpopulations with mesenchymal progenitor expression, referred herein as mesenchymal fibroblasts (Figure 8C). From our differential gene expression analysis (Figures 8D and 8E), no significant differences were observed in gene marker expression of papillary and reticular fibroblasts between PEGScarX and control wounds. However, COL18A1 (log₂FC = 2.09, p=1 2.77E-07) and MFAP5 (log₂FC = 2.72, p=1.29E-50) differential gene expressions were significant between PEGScarX and DRM treated wounds. Further, studies have shown that fibroblast subpopulations expressing mesenchymal progenitor and differentiation-associated genes POSTN and COL11A1 are implicated in overexpression of collagens in keloid and skin fibrosis.¹⁴ Interestingly, POSTN was significantly downregulated in PEGScarX wounds. POSTN, which encodes periostin, plays an important role in keloid formation and increases collagen expression in keloid. Specifically, POSTN was downregulated in PEGScarX compared to DRM and controls at a fold change of log₂FC = −2.69 (p=3.61E-41) and log₂FC = −3.12 (p=1.07E-46), respectively. While COL11A1 in PEGScarX demonstrated fold changes of log₂FC = −4.27 (p=2.00E-39) vs. DRM and log₂FC = −3.97 (p=6.15E-98) vs. control.

Figure 8.

Graph-based clustering analysis of fibroblasts

(A–C) Transcriptional signatures of (A) fibroblasts, (B) papillary fibroblasts, and (C) mesenchymal fibroblasts.

(D and E) Differential expression analysis for fibroblast clusters only represented by volcano plots.

(F) Graph-based clustering analysis depicting transcriptional signatures of epidermal stem cells.

(G and H) Differential gene expression analysis of stem cell clusters.

In addition to fibroblasts, graph based clustering analysis also revealed a higher proportion of epidermal stem cells in PEGScarX wounds (Figures 6D and 8F). Within the differentially expressed genes in the epidermal stem cells cluster (Figures 8G and 8H), HES1 was strongly expressed in PEGScarX wounds compared to both control and DRM wounds.

PEGScarX does not affect fibroblast activation but inhibits transition to myofibroblasts

We further explored differential gene expression analysis for fibroblasts clusters to understand the transcriptional status of fibroblasts and heterogeneity in its subpopulations after each treatment. As expected acta2 that is used as a marker for α-SMA myofibroblast formation was downregulated at log₂FC = − 4.09 (p= 9.49E-64) in PEGScarX wounds compared to controls and at log₂FC = − 4.25 (p= 2.55E-49) compared to DRM wounds. Fibroblast activation markers such as fibroblast specific protein 1 (FSP1/S100A4) was downregulated in PEGScarX compared to control at fold change of log₂FC = −6.72 (p= 2.61E-11) and compared to DRM wounds at fold change of log₂FC = − 5.33 (p=8.18E-16). Interestingly, expression of other fibroblast markers, such as fibroblast activation protein (FAP), platelet-derived growth factor receptor-α and -β (PDGFRA and PDGFRB), and endoglin (ENG) remained unchanged.

PEGScarX promotes a pro-regenerative immune cell niche

The proportion of immune cells was highest in the DRM treatment group based on the signature gene expression; albeit we conducted subcluster analysis to further understand the functional profile of the immune cells between the treatment groups (Figures 9A–9C). We observed increased expression of C1Qb and SLPI in PEGScarX wounds. Among cytokines and chemokines, CCL2 and CCR5 were highly expressed while MRC1/CD206 was only significant in PEGScarX compared to DRM.

Figure 9.

Graph-based clustering analysis of immune cells

Transcriptional signatures of (A) macrophages, (B) dendritic and Langerhans cells, and (C) T cells.

Discussion

Biophysical and mechanical properties of ECM scaffolds provide critical cues that maintain tissue homeostasis and modulate downstream signaling cascades to control cell fate decisions for tissue regeneration.^4,5,6 Herein, we developed a wound matrix system consisting of an acryl-PEG derivative with conjugated cell-degradable peptide moieties that initiates a Michael-type click reaction between the acrylates and thiol groups of hyaluronic acid (HA) and gelatin to form covalent crosslinks with embedded tropoelastin. With this modular arrangement, each component of the matrix functions to mimic the natural ECM components required to support intrinsic wound regeneration. More specifically, HA forms an assembly with the initial fibrin matrix and interacts with cell surface receptors to induce cell-cell adhesions and cell-substrate adhesions to allow epidermal cells to migrate from the peripheral wound edges into the wound bed and stimulate the wound inflammatory response to induce chemotaxis. The infiltrating cells begin to secrete hyaluronidase which degrades HA and macrophages sense this signal to begin phagocytosis and wound debridement. HA does not support cell adhesion; hydrophobic and polycationic gelatin permits cell adhesion and proliferation of the infiltrating cells and allows the cells to achieve their characteristic morphologies to promote their factor secretions and function.^15,16 In addition to serving as a scaffold for cell attachment, gelatin absorbs wound exudate, acts as a hemostatic agent to initiate the wound healing mechanism, and provides a porous scaffold to stimulate the migration of cells to the injury site. It further enhances the formation of new tissues by providing structural and mechanical strength at the wound site. Finally, during active remodeling, the degradation products of HA stimulate wound vascularization. Tropoelastin in addition to its role in promoting cell attachment and migration, mimics prenatal development to induce elastin fiber synthesis affording stretch, resilience, and durability to the healing wound thereby limiting scar formation and contractures. Ultimately, the decreasing gradients of HA initiate cell and tissue differentiation. We observed that PEGScarX wounds demonstrated a >2 log₂FC in CD44 expression in fibroblasts which is the receptor for HA internalization by cells. This suggests that PEGScarX utilizes CD44 to control exaggerated inflammatory response and induces fibroblast migration from peripheral wound edges into the wounded area.¹⁷ CD44 has been demonstrated to play roles in regulating the immune microenvironment and fibrosis.¹⁸ Specifically, the CD44 receptor is believed to play a key role in stromal tissue and immune cells responses to HA by mediating the expression of pro-inflammatory cytokine IL-6 and chemokines IL-8, CXCL1, CXCL2, CXCL6, and CCL8 genes in dermal fibroblasts.¹⁹ In addition to being a receptor for HA and regulating local HA homeostasis,²⁰ CD44 also mediates a multitude of cascades including promoting stem cell enrichment,²¹ maintaining cell stemness, inhibiting apoptosis, and promoting cell survival after injury by binding osteopontin.²² To this end, CD44 regulates keratinocyte proliferation in response to extracellular stimuli,²⁰ and in fibroblasts, CD44 induces genes that repair damaged epithelium and prevent fibrosis.²³ Nevertheless, the role of CD44 needs to be further explored in future studies.

Further, our in vivo results in a preclinical thermal injury model indicated confirmation of in vitro PEGScarX characteristics and that in vivo integration of PEGScarX supports epidermal cell migration, proliferation, and matrix deposition by cells. This is critical because the necessary excision of necrotic burn tissue in full-thickness burn injury devoids the wound site of endogenous cells and relies on cell infiltration from the peripheral wounds edges to heal the wound. If not healed timely, it can lead to slow non-healing wounds, infection, pain, and hypertrophic scarring diminishing the patients’ quality of life.²⁴ Interestingly, PEGScarX wounds demonstrated early and improved granulation deposition, superior wound histomorphological features, and reduced scar formation. Scar outcomes are a result of an interplay of wound angiogenesis, fibroblast-to-myofibroblast transition, expression of TGF-β, deposition and subsequent remodeling of ECM, and alignment of collagen fibers. In our results, we observed lower expression of α-SMA in PEGScarX treated wounds compared to non-treated controls. This is indicative of a reduction of fibrosis and attenuation of contraction. These results were further corroborated by snRNA-seq analysis and markers that have been known to promote skin fibrosis.^14,25

It is worthy to note that wound biopsies were taken on post-burn day 14 for snRNA-seq analysis; this time point in the wound healing cascade is crucial because it overlaps the transition from inflammatory phase and granulation tissue deposition to the re-epithelialization phase. Interestingly, re-epithelialization requires migration, and proliferation of epidermal stem cells (ESCs) to re-establish an intact keratinocyte layer. HES1 was strongly expressed in PEGScarX wounds compared to both control and DRM wounds. It has been demonstrated that WNT signaling and Notch signaling affect the proliferation of ESCs, the differentiation and migration of keratinocytes, and follicle regeneration by targeting HES1, which ultimately lead to enhanced wound healing.²⁶

Intuitively, PEGScarX wounds represented an enrichment of papillary and reticular fibroblasts, and reduced differentiation of mesenchymal fibroblasts toward a phenotype that increases wound fibrosis. These findings unravel alternative opportunities to develop methods such as small molecule inhibitors to target mesenchymal fibroblast and reduce fibrosis in wounds and keloids.¹⁴

Studies have shown that ECM signatures drive the gene expression in macrophages to affect their activation status and therefore role in wound healing.²⁷ Interestingly, we observed that the immune cell clusters were significantly enriched in genes that contribute to wound healing by influencing macrophages to induce reepithelialization, revascularization and matrix transformation. Specifically, matrisome-associated genes of the complement cascade C1Qb were strongly expressed in PEGScarX. Further, we also observed increased expression in PEGScarX wounds of secretory leukocyte protease inhibitor (SLPI), a serine protease inhibitor expressed by macrophages, required for normal cutaneous wound healing and inhibiting TGF-β activation and elastase activity.²⁸ Among cytokines and chemokines, CCL2 and CCR5 were highly expressed implicating a role in macrophage recruitment, re-epithelialization, and neovascularization. MRC1/CD206 was only significant in PEGScarX compared to DRM signifying the switch toward alternatively activated macrophages in these wounds.

Wound healing is a multifactorial response that requires timely and coordinated efforts from all epidermal cells, their growth factor and cytokine/chemokine secretions, migration, and phenotypic differentiation. These cascades are particularly challenging in burn wounds due to the systemic pathophysiological manifestations of hypermetabolism and hyperinflammation that impacts wound healing. Pathological wound healing may be either non-healing wounds or pathological scarring from “over”-healed wounds. Hypertrophic scars and keloids are the two subtypes of pathological scarring that occur after thermal injury, therapies are needed that can avert this during wound healing predominantly by preventing a prolonged acute inflammatory phase, increased collagen deposition, and myofibroblast expression.^29,30

To this end, modified natural and synthetic biomaterials such as acrylated gelatin, collagen, and PEG require exposure to ultraviolet or visible light and addition of toxic photo-initiators to form hydrogel matrices. Here, we demonstrate an instantaneous, in-situ polymerizable modular matrix generated from thiol-Michael addition click reaction for biomatrix delivery to burn wounds without the need of a light source or specialized equipment. We present a wound healing matrix design using materials engineering approaches from regenerative medicine to inculcate characteristics that are growth inductive to cells from the surrounding uninjured skin, is cellularly degradable with tailored substrate stiffness, provides a scaffold for endogenous cellular infiltration, promote granulation tissue formation, is conducive to cell proliferation and cell matrix deposition, and includes biofactors that support anti-scarring.

Limitations of the study

In this study, traditional molecular biology techniques were not very robust in capturing dynamic changes during the wound healing process due to the vociferous interaction between cells in the healing wounds. Therefore, we resorted to conduct snRNA-seq analysis at day 14 because we observed that day 14 is the most optimal time point in capturing differential changes in wounds and pig wound healing in our model has overlapping signals from inflammatory and proliferation phases of wound healing at day 14. In future studies, conducting time-series experiments will be essential to analyze the dynamic changes in the healing wounds and to reveal the role of PEGScarX at different stages of wound healing using robust techniques, such as snRNA-seq and spatial transcriptomics, etc.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Marc G. Jeschke (marc.jeschke@hhsc.ca).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •Data reported in this paper will be shared by the lead contact upon reasonable request.
• •This paper does not report original code.
• •Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

This work was supported by National Research Council Canada Center for Research and Applications in Fluidic Technologies fellowship. The authors would like to acknowledge the animal care staff and committee at Sunnybrook Research Institute.

Author contributions

A.A. designed and performed the experiments, analyzed the data, and wrote the manuscript; M.E., Y.C., F.C., B.C., G.R., S.H., and A.M. performed the experiments and edited the manuscript; M.G.J. designed the experimental outlines and edited the manuscript.

Declaration of interests

All authors declare they have no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-Laminin antibody	abcam	ab11575; RRID: AB_298179
Anti-Collagen I + Collagen III antibody	abcam	ab34710; RRID: AB_731684
Anti CD11b	abcam	ab133357; RRID: AB_2650514
Anti-VEGFA antibody	abcam	ab46154; RRID: AB_2212642
Anti a-SMA	abcam	ab7817; RRID: AB_262054
Anti CD31	abcam	ab28364; RRID: AB_726362
Chemicals, peptides, and recombinant proteins
Fetal Bovine Serum	VWR	Cat#89510–194
DMEM	Thermofisher	Cat#MT10014CV
Critical commercial assays
LIVE/DEAD™ Viability/Cytotoxicity Kit, for mammalian cells	thermofisher scientific	L3224
F-actin phalloidin	thermofisher scientific	A12379
human cytokine/chemokine milliplex	thermofisher scientific	HCYTMAG-60K-PX29
Experimental models: Organisms/strains
Yorkshire pig	Caughell Farms	N/A
Software and algorithms
ImageJ	NIH	https://ImageJ.nih.gov/
Prism9	GraphPad	https://www.graphpad.com/
Partek Flow software	illumina	https://www.illumina.com/

Experimental model and study participant details

Porcine and housing conditions

All procedures involving animals were performed in compliance with the Animals for Research Act of Ontario and the Guidelines of the Canadian Council on Animal Care (CACC). This study was approved by the Sunnybrook Research Institute Animal Care Committee. Female Yorkshire pigs weighing 34-40 kg were purchased from Caughell Farms. Pigs were maintained at room temperature (22-23C) in individual cages. Pigs were acclimated for 1–2 weeks prior to study commencement.

Method details

Pig thermal injury model

Under anesthesia (ketamine at 0.2mg/kg sc. combined with Atropine 0.5 – 1.0 mg depending on the heart rate and isoflurane 5%/l/O2 intubation) and analgesia (Buprenorphine 0.05mg/kg subcutaneous (sc.), 5 × 5 cm dorsal full-thickness burns were created using a heated aluminum device (200°C) for 20 seconds under a constant force of 4N measured using a digital force gauge (Mark-10 Corporation). A 5 cm interspace was maintained between adjacent wounds. The wounds were covered with paraffin gauze and wet to dry gauze dressings that were kept in place by an adhesive bandage, and an elastic stocking porcine suit. Burned skin was removed after 48 hours by inflicting full thickness excisional wounds (post-excision day 0).

Wound analysis and imaging

Wounds were photographed at each dressing changed for gross appearance and wound area. Wound area was measured using NIH ImageJ software. Wound closure was calculated as percent area of original wound. The wound closure rate was calculated using the following equation: % Wound closure = [(Day 0 wound area − Day X wound area)/Day 0 wound area] × 100.

Wound gross examination

Gross wound examination will be conducted by taking photographs of the wounds post-injury until sacrifice. Granulation tissue was scored by a plastic surgeon by looking at actual animal wounds. The photographs were used to score for Vancouver scar scale by scoring for vascularity, pigmentation, pliability, and height. Evaluation for scarring was done by external blinded burn surgeons.

Histochemical and immunohistochemical analysis

Wound tissue was explanted at sacrifice and trimmed, followed by fixation in 10% formalin for 48 h at room temperature, then stored in 70% ethanol at 4°C until serial sectioning (5 μm thick) at SRI Histology Core. Histological sections were stained with hematoxylin and eosin (H & E) for morphological analysis and Masson’s Trichrome for collagen density. Immunohistochemical analysis was conducted to analyze wound contraction (alpha smooth muscle actin; α-SMA), neovascularization (CD 31) and wound inflammation (CD 11b). Stained slides were imaged under Leica light microscope (LEICADM 2000 LED) and analyzed with the evaluator blinded to treatment/control conditions. Histological images were analyzed on ImageJ software by thresholding RGB images and converted to gray scale to determine collagen density, % area positive for a-SMA, no. of blood vessels and no. of inflammatory cells.

RNA isolation and RT-qPCR

Total RNA was isolated from pig tissue samples using RNAzol RT. Reverse transcription of normalized RNA was then performed to obtain cDNA using a high-capacity cDNA reverse transcriptase kit. RT-qPCR was performed using the Step One Plus Real-Time PCR System (Applied Biosciences) and gene expression was calculated using 2^−ΔΔCt method expressed as a fold change, using GAPDH as housekeeping control.

Western blotting

Pig skin wound tissue was homogenized in RIPA lysis buffer (50mM Tris-Cl, pH 7.4; 150mM NaCl; 1% NP-40; 0.25% Na-doxycholate; 1mM PMSF) supplemented with phosphatase and protease inhibitors and centrifuged (10 min, 5,000 g, 4C). The infranatant was collected and a bicinchoninic assay (BCA) was conducted according to the manufacturer’s protocol (ThermoFisher Scientific). Samples were then prepped using a cocktail of RIPA buffer and 2X Laemmli sample buffer to achieve equal concentrations. Protein lysates were loaded into acrylamide gels, transferred onto a nitrocellulose membrane, blocked for 1h, then incubated overnight at 4C with the appropriate primary antibody. The following day, membranes were washed with TBS-T and incubated with the corresponding secondary antibody for 1h. Chemiluminescent signals were detected using and densitometry was quantified using ImageStudio Lite. (Li-COR Biosciences). Proteins were normalized to loading control (vinculin for TGFb3 and GAPDH for VEGF).

Covalent modification of PEG

Covalent modification of acrylated PEG was conducted to include cell-degradable peptides. A collagenase-sensitive peptide was covalently incorporated in amine-reactive PEG.¹⁰ Briefly, the peptide was dissolved in PBS with pH adjusted to 8.0 and reacted with 3.4 kDa acryl-PEG-SVA at 4°C overnight. The resulting product was dialyzed, lyophilized, and reconstituted in sterile water to achieve a 10% (w/v) solution (S1). S1 was combined with solution S2 composed of 1 mg/mL thiol-modified hyaluronic acid, 1 mg/mL thiol-modified gelatin and tropoelastin. S1 and S2 were combined in 1:1 ratio to yield the wound matrix PEGScarX.

PEGScarX mechanical properties

Assessment of PEGScarX rheological properties and swelling/degradation kinetics were conducted. Rheological properties of PEGScarX were studied using TA instruments HR-3 Discovery Hybrid Rheometer. Degradation kinetics were determined by swelling the 1 cm² PEGScarX for 24 h in PBS at 37°C and then incubated with 1 mg/mL collagenase at 25°C. The change in wet weight was measured over time. For swelling kinetics, 1 cm² PEGScarX was lyophilized to achieve the dry weight. Lyophilized PEGScarX was swelled in PBS at 25°C and change in weight was measured over time.

PEGScarX biocompatibility

For biocompatibility studies, mesenchymal stem cells derived from burn tissue expanded in DMEM/F-12 media (Gibco) supplemented with 10% FBS and 2% of Antibiotic/Antimycotic inside a humidified incubator at 37°C with 5% CO (https://ameriburn.org/national-burn-awareness-week-2020/ABANBAW). Once a confluency of ∼80 to 90% was achieved, the cells was trypsinized and 1 × 10⁶ MSCs were embedded in PEGScarX to assess cell viability, adhesion and spreading.

Milliplex analysis

Milliplex analysis of cytokines, chemokines and growth factors secretion was conducted. MSCs were isolated, expanded and embedded in PEGScarX. The cell culture supernatant was collected at 48 h and cytokines, chemokines, growth factors, and immunomodulatory proteins were detected using the HCYTOMAG-60 K MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay (EMD Millipore Corporation, Germany).

Contraction assay

PEGScarX was prepared as described above and the contractile properties of the PEGScarX were assessed. Contraction assays were conducted with or without normal human dermal fibroblasts in vitro to measure any reduction in area over time. Images were taken with a ruler placed next to PEGScarX. The images were then traced on NIH FIJI software to measure the area.

Single nuclei RNA sequencing analysis

Single nuclei RNA sequencing analysis on explanted pig skin tissue was conducted according to 10X Genomics protocols. Library preps are all done according to 3′ v3.1. The sequencing was done on a NovaSeq 6000 instrument according to 10X guidelines. Sequencing data was analyzed on Partek Inc. (2020). Partek® Flow® (Version 10.0). Low-quality barcodes or with high mitochondrial genome were excluded. Qualified cells with detected gene number between 200 and 5,000 were selected for analysis. After quality control, counts were filtered and normalized. Normalized counts were submitted to principal component analysis and cells were clustered by graph-based clustering and visualized by t-SNE. Individual cell types were submitted to ANOVA followed by pathway enrichment analysis.

Quantification and statistical analysis

Statistical analyses were performed on Prism v9 (GraphPad). All data is reported as mean ± standard error of the mean (SEM). Statistical significance between treatments was determined by conducting a one-way analysis of variance (ANOVA) followed by Fisher’s Least Significant Difference (LSD) post-hoc analysis. p < 0.05 was considered significant.

Published: April 17, 2025