Compositions for mediating mechanotransduction disruption in skin graft procedures and use therein

Abstract

Skin grafting methods are provided. Method embodiments include applying a skin graft to a wound in combination with a mechanotransduction blocker, e.g., a pharmacological mechanotransduction blocker, e.g., a focal adhesion kinase inhibitor. Also provided are compositions and kits for use in practicing the methods of the invention.

Description

[Technical field]

[0001] Government Rights Recognition This invention was made with government support under NIH Grant No. DE026914 awarded by the National Institute of Health. The United States Government has certain rights in the invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS Pursuant to 35 U.S.C. §119(e), this application claims priority to the filing dates of U.S. Provisional Patent Application No. 63 / 227,811, filed July 30, 2021, and U.S. Provisional Patent Application No. 63 / 340,145, filed May 10, 2022, the disclosures of which are incorporated herein by reference. [Background technology]

[0003] In humans and other large mammals, injury typically results in scar formation, which is characterized by excessive fibrosis and loss of function (1-3). As the most superficial organ of the body, the skin is usually the first defense against external traumatic forces, making it particularly susceptible to injury and subsequent hypertrophic scar formation and contracture. Although skin injuries can have multiple etiologies, burns are among the most devastating and represent a major global public health burden (1, 4), especially in middle- and low-income countries (5). There are at least 40,000 hospitalized cases related to burn injuries annually, and each of the 128 U.S. burn centers receives an average of 200 hospitalizations per year for burns and burn-related skin diseases (6). Burn injuries limited to the skin and / or subcutaneous fat are usually reconstructed with full- or split-thickness skin grafts (STSGs) using autologous skin grafting (7).

[0004] In clinical practice, deep burns and other deep large surface area injuries rarely heal on their own. Instead, STSGs play a critical role given their low donor site morbidity, promotion of a beneficial healing environment, and ability to cover relatively large areas using graft mesh techniques. Although skin grafts perform an important function in rapidly restoring skin barrier function to prevent infection and reduce mortality, secondary contracture during graft healing leads to hypertrophic scar (HTS) formation (8). Healed skin grafts are also characterized by increased fragility, abnormal pigmentation, and poor texture compared to non-wounded skin (9). Revision rates after skin grafting have been reported to be as high as 20-30% (10), and younger patients often require multiple skin grafts as they grow, presenting additional challenges in donor site availability (11, 12). Over time, skin graft contractures may form and are commonly addressed with contracture release and new STSGs, potentially restarting the vicious cycle of contracture. Unfortunately, there are currently no FDA-approved pharmacological therapies available for patients with skin injuries to prevent debilitating post-skin graft fibrotic scar formation, contractures, and other functional complications (7, 13, 14).

[0005] Several recent studies have revealed how upregulation of mechanical signaling can drive the development of fibroproliferative scarring and fibrosis after injury in mice (15-21). Our group has identified the critical importance of mechanotransduction pathways in skin and shown that their inhibition can successfully attenuate scar formation and improve open wound healing (22-27). However, none of these studies have investigated in detail the primary signaling pathways that drive skin grafting or healing after skin grafting surgery. In addition, mice are animals with loose skin, which heal primarily via contractures and have less than 10% of the scarring that occurs in humans (28, 29). In contrast, humans and pigs are several orders of magnitude larger, are animals with taut skin, and heal via re-epithelialization over granulation tissue, resulting in much more scarring (27). Unfortunately, these differences impact the ability to transfer findings from mice to humans.

[0006] The molecular and cellular mechanisms underlying dermal remodeling and fibrosis following STSG in large animals remain incompletely understood ( 30 , 31 ). Single-cell RNA sequencing (scRNA-seq) technology has recently revolutionized how cells can be transcriptionally analyzed to elucidate disease pathophysiology ( 32 – 34 ). Summary of the Invention

[0007] The present disclosure provides a method for treating a wound in a subject, the method comprising applying a skin graft in combination with a mechanotransduction blocking agent to the wound to treat the wound in the subject.

[0008] In some cases, the present disclosure provides a method of reducing scar formation following application of a skin graft to a treatment site in a human subject, the method comprising applying a skin graft to the treatment site, delivering a focal adhesion kinase inhibitor to the skin graft, and reducing scar formation at the treatment site.

[0009] Also provided are pharmaceutical compositions and kits for practicing the subject methods. [Brief description of the drawings]

[0010] [Figure 1-1] Development of a technology transfer porcine model using clinically relevant methods. (A) Schematic of a full thickness excision wound on the dorsum of a pig and harvested skin graft from the donor site. (B) Staged photographs of a full thickness wound created using an electric bovie. (C) Photograph of harvested mesh skin graft (0.01 inches) at a 1:1.5 ratio. [Figure 1-2] (D) Staged images showing the native unwounded skin, full thickness wound, stapled skin graft, and coverage with three layers of petrolatum gauze and a bolster dressing. (E) Photographic images of the graft at 0, 7, and 90 days after surgery. [Figure 2-1] Cell subpopulations in STSG-derived scars are characterized by increased mechanotransduction and inflammatory signaling. (A) Left: Macroscopic photographs of non-wounded skin (top) and STSG (bottom) at day 90. Scale bar = 0.5 cm. Center: Trichrome staining (scale bar = 0.5 mm) and aSMA+ myofibroblast staining (scale bar = 100 μm). Right: Picrosirius red staining of collagen fibers (scale bar = 5 μm). (B) Quantification of dermal thickness, aSMA+ myofibroblasts, and collagen alignment using CurveAlign (109). Statistical comparisons made using unpaired two-tailed t-tests (*p<0.05). All data represent the mean ± SEM of biological replicates (n = 6 STSGs per condition). [Figure 2-2](C) Schematic showing porcine cells isolated from STSG and non-wounded skin tissues and processed for scRNA-seq. (D) UMAP embedding of all cells colored by cell type. (E) Number of differentially expressed genes for each cell type (average log fold change >0.5). (F) Gene feature plot and (G) Over-representation analysis (ORA) enriched pathways by Genetrail3 for myeloid cells. (H) UMAP embedding of fibroblasts. (I and J) Top feature and pathway plots for fibroblasts. [Figure 3-1] Disruption of mechanotransduction in large animals accelerates STSG healing, attenuates fibrotic scar formation, reduces contractures, and improves biomechanical properties. (A and B) Schematic showing large area (25 cm2) full thickness excision wounds with STSGs made on the lateral dorsal side (left and right) of red Duroc pigs. STSGs were treated with either standard bandage dressing, blank hydrogel (STSG+blank; STSG+B), or FAKI-releasing hydrogel (STSG+FAKI; STSG+F) (n=6 STSGs per condition). All wounds were assessed by macroscopic photography. (C) Representative images tracking stromal epithelialization, scar formation, and contracture over time. Scale bar=2.5 cm. [Figure 3-2] (D) Scar contracture over time was measured and quantified. (E) Re-epithelialization of STSGs at 7 days after surgery compared to non-wounded skin. (F) Visual analog scale (VAS) scoring assessed from digital photographs by three blinded plastic surgeons. (G) STSG hardness assessed by cutometer-induced deformation (n=6 STSGs per condition). Statistical comparisons made using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test (*P<0.05). All data represent the mean ± SEM of biological replicates. [Figure 4-1]FAKI-mediated inhibition of mechanotransduction in STSG of large animals reduces collagen and restores organization of the collagen fiber network. (A) Trichrome staining shows the borders of superficial and deep scars. Scale bar: 1 mm. (B) Picrosirius red staining of three scar groups (STSG, STSG+Blank, STSG+FAKI) at 90 days post-surgery was quantified and compared to non-wounded skin for alignment (CurveAlign) and fiber length / width metrics (CT-Fire) (98, 109, 110) (n=6 STSG per condition). Scale bar: 10 μm. [Figure 4-2] (C) Picrosirius red staining of the three scar groups (STSG, STSG+Blank, STSG+FAKI) at 90 days post-surgery was quantified and compared to non-wounded skin (n=6 STSG per condition) for alignment (CurveAlign) and fiber length / width metrics (CT-Fire) (98,109,110). Scale bar: 10 μm. (D-F) Quantification of distinct collagen fiber network features across the four groups in the deep dermis. Statistical comparisons made using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test (*P<0.05, *P<0.01, **P<0.001). All data represent the mean ± SEM of biological replicates. [Figure 5-1] Mechanotransduction blockade causes early (day 7 after surgery) upregulation of anti-inflammatory pathways in myeloid cells. (A) Schematic of STSG and STSG+FAKI. (B) Representative picture of early FAK inhibition in STSG. Scale bar = 1 cm. (C) Porcine cells shown in UMAP embeddings stained by STSG or STSG+FAKI. (D) UMAP embeddings of cells stained by cell type. Dashed lines indicate myeloid cells of interest. (E) Number of differentially expressed genes between STSG and STSG+FAKI by cell type. (F) Violin plot of fibrotic genes expressed by fibroblasts. [Figure 5-2](G) UMAP embedding of myeloid cells stained by STSG or STSG+FAKI. (H) UMAP embedding of myeloid cells stained by cell type and overlaid RNA velocity streams. (I) Violin plot of fibrotic or anti-inflammatory genes expressed by monocytic lineage cells. (J) ORA pathway plot by Genetrail3 for myeloid cells. [Figure 5-3] (K) Representative images of immunofluorescence staining of CXCL10 protein in porcine tissues over time. Scale bar = 200 μm. HM (high magnification) scale bar = 50 μm. (L) Quantification of F4 / 80 and CXCL10 protein in porcine tissues over time at 7 days post-surgery (n = 3 per condition), 14 days post-surgery (n = 3 per condition), and 90 days post-surgery (n = 6 per condition). Statistical comparisons performed using two-way analysis of variance (ANOVA) and Tukey's multiple comparison test (*P < 0.05, ***P < 0.001). All data represent the mean ± SEM of biological replicates. [Figure 6-1] Disruption of mechanotransduction shifts fibroblast transcriptional fates from profibrotic to regenerative at late (90 days post-surgery) time points. (A) Porcine cells were isolated from STSG treated with FAKI hydrogel (STSG+FAKI), control STSG, and non-wounded skin. UMAP embeddings of fibroblasts colored by treatment group. RNA velocities shown as main gene average flow visualized by velocity streamlines projected onto the UMAP embedding. (B) Six fibroblast lineages and terminal states determined by CellRank (81). (C) Cells colored by latency calculated across all genes by scVelo quantifying global differences in transcriptional dynamics between cells. (D) Heatmap showing smoothed gene expression of top genes with highest correlation to regenerative (left, lineages 1, 2) and fibrotic (right, lineages 3, 4) fate probability, sorted according to latency peak. Center: APOE and ACAN gene expression across six lineages. [Figure 6-2](E and F) Regenerative (left) vs. fibrotic (right) lineages analyzed at greater depth. Top: Expression of group-defining genes and key pathways projected onto UMAP embeddings. Bottom: Gene-specific RNA velocities. Purple dotted lines represent the estimated "steady-state" ratio of unspliced:spliced ​​mRNA. Positive velocities (higher than expected abundance of unspliced ​​mRNA) indicate gene upregulation. [Figure 7-1] Time course of STSG healing across regenerative and fibrotic lineages. Protein confirmation was performed using immunofluorescence staining of STSG and FAK-inhibited STSG porcine dermal tissue sections at 7 days post-surgery (n=3 per group), 14 days post-surgery (n=3 per group), and 90 days post-surgery (n=6 per group). (A) CXCL14, (B) THBS4, [Figure 7-2] Representative images for (C) APOE, and (D) CD34. Scale bar = 200 μm. HM (high magnification) scale bar = 50 μm. Statistical comparisons performed using two-way analysis of variance (ANOVA) and Tukey's multiple comparison test (*P<0.05, ***P<0.001). All data represent the mean ± SEM of biological replicates. [Figure 8-1] The 3D organotypic scar system recapitulates two opposing trajectories of regeneration versus fibrosis in both human and porcine cells. (A) Schematic: Fibroblasts were isolated from human patient samples, cultured, and seeded within 3D collagen scaffolds. Collagen scaffolds were subjected to either no strain (NS, black), strain (blue), or strain and FAKI (strain+FAKI, red). (n=3 per condition). Scale bar: 1 cm. (B and C) Immunofluorescence staining of (B) fibrotic and (C) regenerative markers in the top view. Scale bar: 100 μm. Statistical comparisons performed using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test (*p<0.05, **p<0.01, ****p<0.0001). All data represent the mean ± SEM of biological replicates. [Figure 8-2](D) Schematic: Porcine fibroblasts were tested. (E) UMAP embedding of fibroblasts with velocity embedding stream and colored by group. (*) indicates basal origin of differentiation. (F) UMAP embedding colored by latency. (G) Heatmap of top differentially expressed genes by group. (H and I) Regenerative (H) vs. fibrotic (I) lineages observed in our 3D system. Expression of group-defining genes projected onto UMAP embedding (top) or violin plot (bottom). [Figure 9-1] Diverse cell biology observed in chronic porcine STSG and non-wounded skin. (A) Cell type defining genes to confirm our automated cell type annotation. (B) Representative proportions of each cell type between STSG and non-wounded skin. (C) Heatmap of differentially expressed genes by cell type. [Figure 9-2] (D) Heatmap of differentially expressed genes by cell type. (E) Additional feature plots of genes and enriched pathways in fibroblasts. [Figure 10] Hydrogels release FAKI into the dermis over time. (A) Schematic of hydrogel delivery of FAKI into STSG. (B) FAKI hydrogels in dialysis membranes show steady release of FAKI over time. (n=2 for blank; n=3 for FAKI). Statistical comparisons performed using two-way analysis of variance (ANOVA) and Tukey's multiple comparison test (*P<0.05, **P<0.01, ***P<0.001). All data represent the mean ± SEM of biological replicates. (C) Penetration of FAKI into the dermis over time. [Figure 11-1] Fiber analysis was performed in both the deep and superficial dermis. Quantification of additional images and picrosirius red stained images was performed (n=6 per condition). Statistical comparisons were performed using analysis of variance (ANOVA) and Tukey's multiple comparison test (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Data represent the mean ± SEM of biological replicates. [Figure 11-2]Fiber analysis was performed in both the deep and superficial dermis. Quantification of additional images and picrosirius red stained images was performed (n=6 per condition). Statistical comparisons were performed using analysis of variance (ANOVA) and Tukey's multiple comparison test (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Data represent the mean ± SEM of biological replicates. [Figure 11-3] Fiber analysis was performed in both the deep and superficial dermis. Quantification of additional images and picrosirius red stained images was performed (n=6 per condition). Statistical comparisons were performed using analysis of variance (ANOVA) and Tukey's multiple comparison test (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Data represent the mean ± SEM of biological replicates. [Figure 12-1] Diverse cell biology was observed in early (day 7 after surgery) STSG and STSG+FAKI. (A) Cell type defining genes confirm our automated cell type annotation. (B) Violin plots of differentially expressed cluster-defining genes in (B) fibroblasts and (C) monocytes and macrophages. Box plots are overlaid to show median and interquartile range. [Figure 12-2] (C) Violin plots of differentially expressed cluster-defined genes in (B) fibroblasts and (C) monocytes and macrophages, with box plots overlaid to show median and interquartile range. [Figure 13] CellRank and scVelo analysis of late stage (day 90 post-surgery) fibroblasts. (A) Initial and (B) terminal states identified by CellRank. (C) Velocity vectors shown for each individual cell. (D) Velocity lengths indicate the increase in transcription magnitude across all genes. (E,F) Gene degradation rates for (E) ENPP1 and (F) ACTA2. Dotted lines represent the estimated "steady state" ratio of unspliced ​​vs. spliced ​​mRNA abundance. [Figure 14-1]Additional analysis of fibroblasts from late stage (90 days after surgery) STSG, STSG+FAKI, and non-wounded skin. (A) Violin plot of differentially expressed cluster-defined genes. Box plots are overlaid to show median and interquartile range. [Figure 14-2] (B) Heatmap of the top differentially expressed genes by treatment group. [Figure 14-3] (C) Selected feature plot showing gene expression. (D) UMAP plot with GeneTrail of major pathways distinguishing between groups. [Figure 15] Protein confirmation of scRNA-seq observations in human patient samples. Protein confirmation was performed using immunofluorescence staining of human hypertrophic scars (HTS) (n=6 samples) and non-wounded skin (n=3 samples) collected from patient samples. Staining for THBS4, which contributes to excessive scar formation, or APOE, which contributes to regenerative adipogenic dermal healing. Scale bar: 50 μm. Statistical comparisons were performed using unpaired two-tailed t-tests (*P<0.05, ****P<0.0001). All data represent the mean ± SEM of biological replicates. [Figure 16] Immunofluorescence staining of αSMA in human collagen scaffolds. Scale bar: 100 μm. Statistical comparisons were performed using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test (**P<0.01). All data represent the mean ± SEM of biological replicates. [Figure 17] Violin plot of in vitro porcine scRNA-seq data, with box plots overlaid to show median and interquartile range.

[0011] definition As used herein in its conventional sense, the term "fibroblast" refers to cells involved in the synthesis and organization of extracellular matrix. Two fibroblast lineages include Engrailed-1 lineage negative fibroblasts (ENF) and Engrailed-1 lineage positive fibroblasts (EPF). The EPF lineage includes all cells that express Engrailed-1 at any time during development, and all progeny of these cells.

[0012] As used herein in its conventional sense, the term "fibrosis" refers to the formation or development of excess fibrous connective tissue in an organ or tissue as a result of injury or inflammation of the site or interference with its blood supply. Fibrosis may be the result of scarring, an abnormal reactive process, or a normal healing response resulting in unknown or understood causes.

[0013] As used herein in its traditional sense, the term "scar" refers to fibrous tissue that replaces normal tissue destroyed by injury or disease. Damage to the outer layer of skin (epidermis) heals by remodeling tissue, and in these cases, scarring is minimal or absent. However, when a thick layer of tissue beneath the outer surface of the skin (i.e., the dermis) is damaged, the remodeling is more complex. The body provides collagen fibers (a protein naturally produced by the body) in a different composition than that found in uninjured skin, which usually results in significant scarring. After the wound heals, the scar continues to remodel as new collagen is formed, existing collagen is enzymatically remodeled, and blood vessels return to normal, which allows most scars to disappear and improve in appearance over a period of two years after injury. However, some visible scarring remains permanently, and hair follicles, as well as sweat and sebaceous glands, do not grow back. As used herein, the term "scar area" refers to the area of ​​normal tissue destroyed by injury or disease and replaced by fibrous tissue.

[0014] Scars differ from normal skin in three major ways: (1) they lack any dermal appendages (hair follicles, sweat glands, etc.); (2) their collagen structure is fundamentally different, having a high density of parallel fibers rather than the "basket weave" pattern that gives normal skin its flexibility and strength; and 3) as a result of their inferior matrix structure, they are weaker than skin.

[0015] As used herein, the term "scar-associated gene" refers to a nucleic acid encoding a protein which is activated in response to scarring as part of the normal wound healing process. As used herein, the term "scar-associated gene product" refers to a protein which is expressed in response to scarring as part of the normal wound healing process.

[0016] Scar tissue consists primarily of a disorganized collagen extracellular matrix. It is produced by myofibroblasts, which differentiate from dermal fibroblasts in response to wounding, causing an increase in the local concentration of transforming growth factor-β, a secreted protein that exists in at least three isoforms called TGF-βΙ, TGF-β2, and TGF-β3 (collectively referred to as TGF-β). TGF-β is a key cytokine associated with fibrosis in many tissue types (Beanes, S. et al, Expert Reviews in Molecular Medicine, vol. 5, no. 8, pp. 1-22 (2003)). Types of scars are further described, for example, in International Patent Application No. 2014 / 040074, the disclosure of which is incorporated herein by reference in its entirety.

[0017] The term "skin" as used herein in its conventional sense includes all surface tissues of the body and subsurface structures therein, including, for example, mucous membranes and ocular tissues, as well as normal skin. The term "skin" may include the wound zone itself. Reapproximation of the skin above the surface of the wound has long been the primary sign of the completion of a significant portion of wound healing. This reclosure of the defect restores the protective functions of the skin, which include protection from bacteria, toxins, and mechanical forces, as well as providing a barrier to retain essential body fluids. The epidermis, composed of several layers beginning with the stratum corneum, is the outermost layer of the skin. The innermost skin layer is the deep dermis.

[0018] As used herein in its conventional sense, the term "dermal appendages" includes hair follicles, sebum and sweat glands, fingernails, and toenails.

[0019] As used herein, the term "dermal location" refers to an area of ​​a subject's skin having any size and area. A dermal location can include a portion of a subject's skin, such as the scalp. A dermal location can include one or more skin layers, including, for example, the epidermis and dermis. In some cases, a dermal location includes a wound.

[0020] As used herein in its conventional sense, the term "wound" includes any disruption and / or loss of normal tissue continuity on the internal or external body surface of a human or non-human animal body resulting from a non-physiological process, such as, for example, surgery or physical injury. The phrase "wound" or "wound environment" as used herein refers to any skin lesion capable of inducing a healing process that can potentially result in scarring, including wounds created by injury, wounds created by burns, wounds created by disease, and wounds created by surgical procedures. Wounds may be present on any external or internal body surface and may be penetrating or non-penetrating. The methods described herein may be beneficial in treating problematic wounds on skin surfaces. Examples of wounds that may be treated by the methods of the present invention include both superficial and non-superficial wounds, such as abrasions, lacerations, wounds resulting from temperature injuries (e.g., those resulting from burns and any cryo-based treatments), and any wound resulting from surgery.

[0021] As used herein in its conventional sense, the term "wound healing" refers to a regenerative process involving the induction of a temporal and spatial healing program, including, but not limited to, the processes of inflammation, granulation, neovascularization, fibroblast, endothelial, and epithelial cell migration, extracellular matrix deposition, re-epithelialization, and remodeling.

[0022] Hydrogels. Hydrogels useful in the methods of the present invention maintain the viability of entrapped cells for a sufficient period of time to enhance wound healing. Hydrogels are known and used in the art for wound healing. Typically, hydrogels are up to about 50%, up to about 55%, up to about 60%, up to about 65%, up to about 70%, up to about 75%, up to about 80%, up to about 85%, up to about 90% by weight water, with the remaining weight comprising suitable polymers such as pullulan and collagen, glycosaminoglycans, acrylates, 2-hydroxymethylmethacrylate and ethylenedimethacrylate copolymers, carboxymethylcellulose, chitosan, gelatin, and the like, or other suitable hydrophilic polymers known in the art. Hydrogels can swell to a wide range without changing their gelatin structure, and are available for use as amorphous (shapeless) gels, as well as in various types of application systems, such as flat sheet hydrogels and nonwoven dressings impregnated with amorphous hydrogel solutions. Flat sheet (film) hydrogel dressings have a stable crosslinked macrostructure and therefore retain their physical form when they absorb fluid.

[0023] In some embodiments, crosslinked hydrogel films are produced using pullulan and collagen under conditions that provide crosslinking and pore formation. Collagen is added to a mixture of pullulan, crosslinking agent, and pore-forming agent (porogen), with collagen being provided at a concentration of at least about 1% and no more than about 12.5% ​​based on the dry weight of pullulan. Collagen may be provided at a concentration of about 1%, about 2.5%, about 5%, about 7.5%, about 10%, typically about 2.5% to about 10%, and may be about 4% to about 6% based on the dry weight of pullulan. Collagen is typically fibrous collagen, such as type I, type II, type III, etc. Crosslinkers of interest include sodium trimetaphosphate (STMP), or a combination or combinations of sodium trimetaphosphate and sodium tripolyphosphate (STMP / STPP). The crosslinker may be included in a weight / weight ratio of about 5:1 to about 1:5 relative to the pullulan, which may be about 4:1, 3:1, 2:1, 1.75:1, 1.5:1, 1.25:1, 1:1, 1:1.25, 1:1.5, 1:1.75, 2:1, 3:1, 4:1, etc. Porogens of interest for in-gel crystallization include any suitable salt, e.g., KCl. The porogens may be included in a weight / weight ratio of about 5:1 to about 1:5 relative to the pullulan, which may be about 4:1, 3:1, 2:1, 1.75:1, 1.5:1, 1.25:1, 1:1, 1:1.25, 1:1.5, 1:1.75, 2:1, 3:1, 4:1, etc. A suspension of collagen, pullulan, crosslinker, and porogen in the absence of cells is poured and compressed to form a sheet. The preferred thickness is at least about 1 mm and no more than about 5 mm, usually no more than about 3 mm, and can be about 1-2.5 mm, e.g., about 1.25, 1.5, 1.75, 2 mm thick. Pores are formed in the hydrogel via rapid drying of the swollen hydrogel by phase inversion. Dehydration results in localized supersaturation and crystallization of the porogen. Pullulan and collagen are forced to organize around the crystals in an interconnected network, which results in reticular scaffold formation after KCl dissolution.

[0024] The films can be stored dry and are easily rehydrated in any suitable aqueous medium. The aqueous nature of the hydrogel matrix provides an ideal environment for cell growth and sustainability.

[0025] Mechanical characteristics of hydrogels include average pore size and scaffold porosity. Both variables vary with the concentration of collagen present in the hydrogel. For hydrogels containing 5% collagen, the average pore size is usually in the range of about 25 μm to about 50 μm, about 30 μm to about 40 μm, and can be about 35 μm. For hydrogels containing 10% collagen, the average pore size is usually in the range of about 10 μm to about 25 μm, about 12 μm to about 18 μm, and can be about 15 μm. Suitable hydrogels with other collagen concentrations can be easily determined by those skilled in the art. Scaffold porosity is usually in the range of about 50% to about 85%, and can be in the range of about 70% to about 75%, and decreases with increasing concentration of collagen. Hydrogels lacking collagen do not show any birefringence under polarized optical microscopy, while hydrogels containing collagen have diffuse birefringence.

[0026] Pullulan. A polysaccharide produced by the fungus Aureobasidium pullulans. Pullulan is a linear homopolysaccharide consisting of alpha-(1-6) linked maltotriose units that exhibits water retention capacity in the hydrogel state, making it an ideal therapeutic vehicle for both cells and biomolecules. In addition, pullulan contains multiple functional groups that allow for cross-linking and delivery of genetic material and therapeutic cytokines. Furthermore, pullulan-based scaffolds have been shown to enhance both endothelial and smooth muscle cell behavior in vitro.

[0027] Collagen. As used herein, the term "collagen" refers to a composition in which at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% or more of the protein present is collagen in triple helical configuration. Collagen is found widely in vertebrate species and has been sequenced for many different species. Due to the high degree of sequence similarity between species, collagen from different species can be used for biomedical purposes, for example, between mammalian species. Typical commercial animal sources include bovine Achilles tendon, calfskin, and bovine bone. In some embodiments, the collagen used in preparing the oriented thin film is type I, type II, or type III collagen, and is derived from any convenient source, typically mammalian sources, such as bovine, porcine, etc.

[0028] Collagen has a triple-stranded rope-like coil structure. The predominant collagen in skin, tendons, and bones is collagen I, which contains two alpha 1 polypeptide chains and one alpha 2 chain. Cartilage collagen contains only one type of polypeptide chain, alpha 1. Fetal collagen also contains collagens with different structures. The genes for the interstitial collagens types I, II, and III show an unusual and characteristic structure, with multiple relatively small exons (54 and 108 bp) at evolutionarily conserved positions along the length of the triple-helical gly-XY segment.

[0029] The collagen types are I (COL1A1, COL1A2); II (COL2A1); III (COL3A1); IV (COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6); V (COL5A1, COL5A2, COL5A3); VI (COL6A1, COL6A2, COL6A3); VII (COL7A1); VIII (COL8A1, COL8A2); IX (COL9A1, COL9A2, COL9A3); X (COL10A1); XI (COL11A1, COL 11A2);XII(COL12A1);XIII(COL13A1);XIV(COL14A1);XV(COL15A1);XVI(COL16A1);XVII(COL17A1);XVIII(COL18A1);XIX(COL19A1);XX(COL20A1);XXI(COL21A1);XXII(COL22A1);XXIII(COL23A1);XXIV(COL24A1);XXV(COL25A1);XXVII(COL27A1);XXVIII(COL28A1). It will be appreciated by those skilled in the art that other collagens are equally suitable for the methods of the present invention, including mammalian collagens, e.g., bovine, porcine, equine, etc.

[0030] Focal adhesion kinase (FAK). FAK is a non-receptor cytoplasmic tyrosine kinase. FAK is one of the major mediators of skin mechanobiology and is activated after skin injury. Mechanical forces enhance the activation of FAK via phosphorylation after skin injury. FAK contributes to cell signaling through its linkage of mechanical stress from the ECM to the cytoplasmic cytoskeleton, which activates inflammatory pathways. Fibroblasts are recruited to wounds by inflammatory signaling, and their secretion of profibrotic cytokines leads to increased collagen synthesis. Various pathologies associated with poor wound healing have been shown to have atypical levels of FAK. Mechanical forces regulate pathological scarring via the inflammatory FAK-ERK-MCP1 pathway, and molecular strategies targeting focal adhesion kinase (FAK) can effectively uncouple mechanical forces from fibrosis.

[0031] Wound dressing. The film of the present invention finds use as a wound dressing or artificial skin by providing an improved matrix that minimizes scarring. An effective bioactive wound dressing can facilitate the repair of wounds that may require both epidermal and dermal restoration. For example, a hydrogel thin film can be placed on a recipient's debrided wound, thereby providing a means for the permanent re-establishment of the dermal and epidermal components of the skin. The graft inhibits the formation of granulation tissue that causes scarring.

[0032] Additional criteria for a biologically active wound dressing include rapid adhesion to the wound immediately after placement; adequate moisture permeability to control evaporative fluid loss from the wound and to avoid collection of exudate between the wound and the dressing material. The skin replacement should act as a barrier to microorganisms, limiting the growth of microorganisms already present in the wound, and be flexible, durable, and resistant to breakage. The replacement should be tissue compatible, i.e., should not induce inflammation or a foreign body reaction in the wound that could result in the formation of granulation tissue. An inner surface structure of the hydrogel thin film is provided that allows for the ingrowth of fibrovascular tissue. An outer surface structure may be provided to minimize fluid transfer and promote epithelialization.

[0033] Typical bioabsorbable materials for use in the manufacture of porous wound dressings and skin replacements and the like include synthetic bioabsorbable polymers such as polylactic acid or polyglycolic acid, and also biopolymers such as structural proteins and polysaccharides. The completed dressing, prior to cell seeding, is packaged and preferably radiation sterilized. Such biologically active products can be used in many different applications requiring regeneration of dermal tissue, including repair of injured skin and recalcitrant wounds, such as burn wounds, venous stasis ulcers, diabetic ulcers, and the like.

[0034] Split-thickness grafts. Split-thickness or partial-thickness skin grafts are commonly used, for which a thin layer of epidermis and some of the dermis are excised and placed onto a recipient site. Such grafts are typically used for burns, but can also be used to accelerate the healing of small wounds. A significant amount of dermal material remains at the donor site so that the site can eventually heal and be reharvested.

[0035] Full-thickness grafts. Full-thickness skin grafts are composed of epidermis and dermis and provide better appearance and function than split-thickness grafts. However, the donor site must be a loose area of ​​overlapping skin (e.g., abdomen or chest wall, often scalp) so that the donor site can be sutured closed, as it will not heal primarily. Thus, full-thickness grafts are usually reserved for cosmetically sensitive areas (e.g., face) or areas requiring a thicker, more protective layer of skin (e.g., hands). Full-thickness grafts do not have the same significantly higher survival rates as split-thickness grafts, because they are thicker and more vascular. Split-thickness skin grafts can be classified as thin (0.15-0.25 mm), medium (0.3-0.4 mm) or thick (0.5-0.6 mm).

[0036] Composite grafts. Composite skin grafts contain two or more different types of tissue. Most commonly, composite skin grafts have subcutaneous tissue and cartilage with or without overlying skin. Composite grafts are often used to repair full thickness defects of the nasal alar and helical rim because they provide support and structure.

[0037] As used herein, the term "autograft" refers to a skin graft where the graft tissue is from the individual's or subject's own body.

[0038] As used herein, the term "allograft" refers to a skin graft where the graft tissue is from a different individual or subject other than the subject receiving wound treatment.

[0039] As used herein, the term "xenograft" refers to a skin graft where the graft tissue is from an individual that is a different species to the subject receiving the graft tissue or is a synthetic graft tissue. For example, a xenograft may be when a human is treated for a wound with a pig skin graft. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Skin grafting methods are provided. Method embodiments include applying a skin graft to a wound in combination with a mechanotransduction blocker, e.g., a pharmacological mechanotransduction blocker, e.g., a focal adhesion kinase inhibitor. Also provided are pharmaceutical compositions and kits for use in practicing the methods of the invention.

[0041] Before the invention is described in more detail, it is to be understood that the invention is not limited to the particular embodiments described, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing only particular embodiments, and is not intended to be limiting, since the scope of the invention will be limited only by the appended claims.

[0042] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of this range, and any other stated or intervening value in this stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0043] A range is provided herein with the term "about" preceding the numerical value. The term "about" is used herein to provide literal support for the exact number it precedes, as well as a number that is close to or approximately the number it precedes. In determining whether a number is close to or approximately a specifically recited number, the number that is close to or approximately the unrecited number may be a number that, in the context provided, provides substantial equivalence to the specifically recited number.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but representative exemplary methods and materials are described below.

[0045] All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0046] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. It should be further noted that the claims may be construed to exclude any optional element. As such, this statement is intended to serve as a predicate to the use of such exclusive terminology, such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of claim elements.

[0047] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any method described may be carried out in the order of events recited or in any other order which is logically possible.

[0048] Although the apparatus and methods have been or will be described in functional descriptions for grammatical fluidity, it is expressly understood that the claims should not be construed as necessarily limited in any way by the syntax of "means" or "step" limitations unless expressly recited under 35 U.S.C. 112, but should be accorded the full scope of meaning and equivalents of the definitions provided by the claims under the doctrine of judicial equivalents, and that the claims, when expressly recited under 35 U.S.C. 112, should be accorded the full statutory equivalents under 35 U.S.C. 112.

[0049] Method for treating a wound in a subject As summarized above, there is provided a method for treating a wound in a subject, the method comprising applying a skin graft in combination with a mechanotransduction blocking agent to the wound to treat the wound in the subject. The wound may be any wound in a subject in need of treatment. Wounds receptive to benefit from the methods described herein include, but are not limited to, partial and full thickness wounds; ulcers, including pressure ulcers, diabetic ulcers (e.g., diabetic foot ulcers), venous ulcers, leg ulcers, and the like; burns (second and third degree burns), including burns, chemical burns, temperature burns such as flame burns and flash burns, ultraviolet burns, contact burns, radiation burns, electrical burns, and the like; gangrene; skin lacerations or lacerations, e.g., those made by knives, etc.; incisions, e.g., those made by knives, nails, sharp glass, razors, etc.; avulsions; amputations; surgical wounds; failed or compromised skin / muscle grafts or flaps; bite wounds; incisions, i.e., wounds in which the length is greater than the depth; and contusions, etc., or a combination of one or more of the above.

[0050] The subject of the present disclosure may be any subject in need of wound treatment. In some embodiments, the subject is a mammal. Non-limiting examples of mammals that may benefit from the methods disclosed herein include, but are not limited to, dogs, cats, horses, cows, pigs, sheep, rodents such as mice or rats, and primates, such as non-human primates, humans, etc. In a preferred embodiment, the mammal is a human.

[0051] The method of the present disclosure includes applying a skin graft to the wound. The skin graft can be any skin graft that is deemed useful in treating a wound in a subject. Skin grafts that find use in the present disclosure include, but are not limited to, full thickness grafts, partial thickness grafts, composite grafts, and the like. The skin graft can be an autograft, an allograft, or a xenograft. In some embodiments, the skin graft is applied prior to application of the mechanotransduction blocking agent.

[0052] In addition to applying the skin graft, the method also includes applying a mechanotransduction blocker. A mechanotransduction blocker that finds use in the present disclosure is any inhibitor that impairs the mechanotransduction signaling pathway. Non-limiting examples of mechanotransduction blockers include integrin inhibitors, focal adhesion kinase (FAK) inhibitors, talin inhibitors, vinculin inhibitors, paxillin inhibitors, zyxin inhibitors, VASP inhibitors, p130 cas In some embodiments, the mechanotransduction inhibitor is a focal adhesion kinase (FAK) inhibitor. Non-limiting examples of FAK inhibitors include, but are not limited to, PF-56227, PF-573228, TAE226 (NVP-TAE226), BI-4464, GSK2256098, PF-431396, PND-1186 (VS-4718), Y15, defactinib (VS-6063), solanesol (nonaisoprenol), and the like. In addition to the FAK inhibitors disclosed above, other types of inhibitors may be used. For example, other types of FAK inhibitors include, but are not limited to, siRNA, antisense oligonucleotides (ASO), CRISPR-mediated knockout or knockdown of FAK, and the like. In some embodiments, the mechanotransduction blocker is a pharmacological mechanotransduction blocker.

[0053] The mechanotransduction blocking agents of the present disclosure may be applied in any manner deemed useful. In some embodiments, the mechanotransduction blocking agents are applied systemically. In some embodiments, the mechanotransduction blocking agents are applied locally at the site of a skin graft. When the mechanotransduction blocking agents are applied locally, the mechanotransduction blocking agents may be applied in a sustained release formulation. In some embodiments, the sustained release formulation comprises a gel formulation. In some embodiments, the gel formulation comprises a hydrogel. In some embodiments, the hydrogel comprises a carbohydrate-based hydrogel, for example, a biodegradable pullulan-based hydrogel. Pullulan-based hydrogels are known in the art and are described in Wong et al. (Tissue Eng Part A. 2011 Mar;17(5-6):631-44) and Wong et al. (Macromol Biosci. 2011 Nov 10;11(11):1458-66), each of which is specifically incorporated herein by reference.

[0054] The methods disclosed herein provide several advantages to wound healing over other methods, i.e., skin grafts in the absence of mechanotransduction blockers, For example, the methods may promote wound healing, reduce fibrosis, reduce contracture, reduce scar formation, restore collagen architecture, or improve graft biomechanical properties.

[0055] Embodiments of the methods disclosed herein reduce the amount of contracture that occurs after skin grafting. Contracture is a measure of the change in scar area relative to the area of ​​the skin graft. Contracture causes distortion that is a result of scar formation pulling the edges of the skin around the scar. The methods disclosed herein result in a range of reductions in contracture. For example, contracture can be reduced by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or greater than 50% reduction in contracture relative to skin grafts in the absence of a mechanotransduction blocker.

[0056] Embodiments of the methods disclosed herein promote wound healing after skin grafting. In some embodiments, the promotion of wound healing is an increase in re-epithelialization. In embodiments where the promotion of wound healing results in an increase in re-epithelialization, a range of increases in re-epithelialization can occur. For example, re-epithelialization can be an increase of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more than 50% increase in re-epithelialization relative to skin grafts in the absence of a mechanotransduction blocker.

[0057] The embodiments of the methods disclosed herein improve skin graft biomechanical properties. In some embodiments, the improvement in skin graft biomechanical properties is a decrease in the stiffness and an increase in elasticity of the skin graft as measured by the vertical deformation of the graft. In embodiments where the improvement in skin graft biomechanical properties is a decrease in the stiffness and an increase in elasticity of the skin graft as measured by the vertical deformation of the graft, a range of increases in deformation can occur. For example, the deformation can be increased by an increase in the deformation of the skin graft of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more than 50% relative to the skin graft in the absence of the mechanotransduction blocker.

[0058] The embodiments of the methods disclosed herein restore collagen architecture. In some embodiments, the restoration of collagen architecture is a reduction in collagen fiber alignment and length relative to skin grafts without mechanotransduction blockers. Unwounded skin is generally characterized as having short, randomly aligned collagen. In embodiments where the restoration of collagen architecture is a reduction in collagen fiber alignment, a range of reductions in collagen fiber alignment can occur. For example, collagen fiber alignment can be reduced by a reduction in collagen fiber alignment of the skin graft of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or greater than 50% relative to skin grafts in the absence of mechanotransduction blockers. In embodiments where the restoration of collagen architecture is a reduction in collagen fiber length, a range of reductions in collagen fiber length can occur. For example, collagen fiber length can be reduced by a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 40%, or greater than 40% reduction in collagen fiber length of the skin graft relative to a skin graft in the absence of a mechanotransduction blocker.

[0059] Methods for reducing scarring As summarized above, there is provided a method for reducing scar formation following application of a skin graft to a treatment site in a human subject, the method comprising applying a skin graft to the treatment site and delivering a focal adhesion kinase inhibitor to the skin graft to reduce scar formation at the treatment site.

[0060] The skin graft may be any skin graft that is deemed useful in treating a wound in a subject. Skin grafts that find use in the present disclosure include, but are not limited to, full thickness grafts, partial thickness grafts, composite grafts, etc. The skin graft may be an autograft, an allograft, or a xenograft.

[0061] Treatment sites of the present disclosure can be any site on the skin in need of treatment. Treatment sites that find use in the present disclosure include, but are not limited to, the hands, palms, lower arms, upper arms, underarms, chest, abdomen, shoulders, upper back, lower back, neck, face, scalp, pelvis, groin, upper legs, lower legs, feet, etc.

[0062] The FAK inhibitors found to be used in the methods disclosed herein are any FAK inhibitors that impair FAK-based signal transduction.Non-limiting examples of FAK inhibitors include, but are not limited to, PF-56227, PF-573228, TAE226 (NVP-TAE226), BI-4464, GSK2256098, PF-431396, PND-1186 (VS-4718), Y15, defactinib (VS-6063), solanesol (nonaisoprenol), and the like.In addition to the FAK inhibitors disclosed above, other types of inhibitors can be used.For example, other types of FAK inhibitors include, but are not limited to, siRNA, antisense oligonucleotides (ASO), CRISPR-mediated knockout or knockdown of FAK, and the like.

[0063] The FAK inhibitors of the present disclosure can be delivered in several different ways. In some embodiments, the FAK inhibitors are delivered systemically. In some embodiments, the FAK inhibitors are applied locally at the treatment site. In some embodiments, the FAK inhibitors are applied in a sustained release formulation. In some embodiments, the sustained release formulation comprises a gel formulation. In some embodiments, the gel formulation comprises a hydrogel. In some embodiments, the hydrogel comprises a biodegradable pullulan-based hydrogel.

[0064] The reduction in scar formation may manifest itself in several different ways. In some embodiments, the reduction in scar formation is a reduction in the visual appearance of the scar. In some embodiments, the reduction in scar formation is a reduction in contracture that occurs during and after scar formation. The methods disclosed herein result in a range of reductions in contracture. For example, contracture may be reduced by a reduction in contracture of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or greater than 50% relative to skin grafts in the absence of a mechanotransduction blocker.

[0065] Combination therapy For use in the subject methods, mechanotransduction blockers such as those described above may be administered in combination with other pharma- ceutical active agents, including other agents that treat the underlying condition or symptoms of the condition, e.g., scarring. As used herein, "in combination with" refers to, for example, the use in which the first compound is administered during the entire course of administration of the second compound; the use in which the first compound is administered for a period that overlaps with the administration of the second compound, for example, the administration of the first compound begins before the administration of the second compound, and the administration of the first compound ends before the end of the administration of the second compound; the administration of the second compound begins before the administration of the first compound, and the administration of the second compound ends before the end of the administration of the first compound; the administration of the first compound begins before the start of the administration of the second compound, and the administration of the second compound ends before the end of the administration of the first compound; the administration of the second compound begins before the start of the administration of the first compound, and the administration of the first compound ends before the end of the administration of the second compound. Thus, "in combination" can also refer to a regimen that includes the administration of two or more compounds. As used herein, "in combination with" also refers to the administration of two or more compounds, which may be administered in the same or different formulations, may be administered by the same by different routes, and may be administered in the same or different dosage form types.

[0066] Examples of other agents for use in combination therapy in embodiments of the method of the present invention include, but are not limited to, YAP inhibitors. In some cases, the YAP inhibitor is a small molecule agent that exhibits a desired activity, e.g., a small molecule agent that inhibits YAP expression and / or activity. Naturally occurring or synthetic small molecule compounds of interest include numerous chemical classes, e.g., organic molecules, e.g., small organic compounds with molecular weights greater than 50 and less than about 2,500 daltons. Candidate agents include functional groups for structural interaction with proteins, particularly hydrogen bonding, typically including at least an amine, carbonyl, hydroxyl, or carboxyl group, and preferably including at least two of the functional chemical groups. Candidate agents may include cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules, including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof. Such molecules may be identified, among others, by using screening protocols.

[0067] In some cases, the YAP inhibitor is a photosensitizing drug. In some cases, the YAP inhibitor is a benzoporphyrin derivative (BPD). The benzoporphyrin derivative can be any convenient benzoporphyrin derivative, for example, those described in U.S. Patent No. 5,880,145, U.S. Patent No. 6,878,253, U.S. Patent No. 10,272,261, and U.S. Patent Application No. 2009 / 0304803, the disclosures of which are incorporated herein by reference in their entirety. In some cases, the benzoporphyrin derivative is a photosensitizing drug. In some cases, the YAP inhibitor is verteporfin (benzoporphyrin derivative monobasic acid ring A, BPD-MA, trade name: Visudyne®).

[0068] Further details regarding YAP inhibitors and methods of use thereof are provided in U.S. patent application Ser. No. 17 / 626,699, the disclosure of which is incorporated herein by reference.

[0069] In some cases, aspects of the method may include administering an effective amount of a mechanotransduction blocking agent in combination with a Piezo inhibitor. In some embodiments, the Piezo inhibitor includes a Piezo 1 and / or Piezo 2 inhibitor. In some cases, the Piezo inhibitor is a Piezo 1 inhibitor. In some cases, the Piezo inhibitor is a Piezo 2 inhibitor. In some cases, both a Piezo 1 inhibitor and a Piezo 2 inhibitor are administered to the subject. In some cases, the method consists essentially of administering a Piezo inhibitor. As used herein, "Piezo inhibitor" refers to a molecule that can inhibit Piezo protein function and signaling. In some cases, the Piezo inhibitor inhibits cell mechanical signaling. In some cases, the Piezo inhibitor reduces or inhibits Piezo protein expression (DNA or RNA expression) or activity (e.g., nuclear translocation). In some cases, the Piezo inhibitor reduces or inhibits the interaction of Piezo protein with other signaling molecules. In some embodiments, administering the Piezo inhibitor reduces the mechanical activation of one or more cells, e.g., adipocytes, in the wound, e.g., the level of mechanical activation of one or more cells, e.g., adipocytes, in the wound is reduced compared to a suitable control. Further details regarding Piezo inhibitors and methods of use thereof are provided in U.S. Provisional Patent Application No. 63 / 335,843, the disclosure of which is incorporated herein by reference.

[0070] In the context of combination therapy, the combination therapy compounds may be administered by the same route of administration that the mechanotransduction blocker is administered (e.g., pulmonary, oral, enteral, etc.) Alternatively, compounds for use in combination therapy with a mechanotransduction blocker may be administered by different routes of administration.

[0071] Pharmaceutical Compositions As summarized above, a pharmaceutical composition for carrying out the methods disclosed herein is provided, which comprises a mechanotransduction blocker of the present disclosure and a pharma- ceutical acceptable excipient.

[0072] A wide variety of pharma- ceutically acceptable excipients are known in the art and need not be discussed at length here. Pharmaceutically acceptable excipients are described, for example, in A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7th ed., “Pharmaceutical Dosage Forms and Drug Delivery Systems,” 1999, pp. 111-115, 1999; th ed., Lippincott, Williams, & Wilkins, and Handbook of Pharmaceutical Excipients (2000) AHKibbe et al., eds., 3 rd It has been thoroughly described in a variety of publications, including ed. Amer. Pharmaceutical Assoc.

[0073] Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers, or diluents, are readily available to the public. Moreover, pharma- ceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like, are readily available to the public.

[0074] The pharmaceutical compositions of the present disclosure include mechanotransduction blockers. Mechanotransduction blockers that find use in the present disclosure are any blockers that impair mechanotransduction signaling pathways. Non-limiting examples of mechanotransduction blockers include integrin inhibitors, focal adhesion kinase (FAK) inhibitors, talin inhibitors, vinculin inhibitors, paxillin inhibitors, zyxin inhibitors, VASP inhibitors, p130 casIn some embodiments, the mechanotransduction inhibitor is a focal adhesion kinase (FAK) inhibitor. Non-limiting examples of FAK inhibitors include, but are not limited to, PF-56227, PF-573228, TAE226 (NVP-TAE226), BI-4464, GSK2256098, PF-431396, PND-1186 (VS-4718), Y15, defactinib (VS-6063), solanesol (nonaisoprenol), and the like. In addition to the FAK inhibitors disclosed above, other types of inhibitors may be used. For example, other types of FAK inhibitors include, but are not limited to, siRNA, antisense oligonucleotides (ASO), CRISPR-mediated knockout or knockdown of FAK, and the like.

[0075] In some embodiments, the pharmaceutical composition comprises a sustained release formulation. The sustained release formulation of the present disclosure is any sustained release formulation that can release the mechanotransduction blocking agent for an extended period of time. The sustained release formulation can release the mechanotransduction blocking agent over a period of time. For example, the sustained release formulation can release the mechanotransduction inhibitor for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, or more than 96 hours.

[0076] The sustained release formulation of the present disclosure can release the mechanotransduction blocking agent to a specific depth into the skin graft. The depth into the skin graft is a measure of the distance from the stratum corneum to the furthest point into the tissue below the stratum corneum. In some embodiments, the specific depth into the skin graft that the sustained release formulation releases is at least 0.5 mm, at least 0.6 mm, at least 0.7 mm, at least 0.8 mm, at least 0.9 mm, at least 1 mm, at least 1.1 mm, at least 1.2 mm, at least 1.3 mm, at least 1.4 mm, at least 1.5 mm, at least 1.6 mm, at least 1.7 mm, at least 1.8 mm, at least 1.9 mm, at least 2 mm, at least 2.1 mm, at least 2.2 mm, at least 2.3 mm, at least 2.4 mm, at least 2.5 mm, at least 2.6 mm, at least 2.7 mm, at least 2.8 mm, at least 2.9 mm, at least 3 mm, or more than 3 mm.

[0077] In some embodiments, the sustained release formulation comprises a gel formulation. In some embodiments, the gel formulation comprises a hydrogel, such as a carbohydrate-based hydrogel, a protein-based hydrogel, etc. In some embodiments, the hydrogel comprises a biodegradable pullulan-based hydrogel. Pullulan-based hydrogels are known in the art and described in Wong et al. (Tissue Eng Part A. 2011 Mar;17(5-6):631-44) and Wong et al. (Macromol Biosci. 2011 Nov 10;11(11):1458-66).

[0078] In some embodiments, the pharmaceutical composition is formulated in an aqueous buffer. Suitable aqueous buffers include, but are not limited to, acetate, succinate, citrate, and phosphate buffers, ranging in strength from 5 mM to 100 mM. In some embodiments, the aqueous buffer includes a reagent that provides an isotonic solution. Such reagents include, but are not limited to, sodium chloride; and sugars, such as mannitol, dextrose, sucrose, and the like. In some embodiments, the aqueous buffer further includes a non-ionic surfactant, such as polysorbate 20 or 80. Optionally, the pharmaceutical composition may further include a preservative. Suitable preservatives include, but are not limited to, benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, and the like. In many cases, the formulation is stored at about 4° C. The pharmaceutical composition may also be lyophilized, in which case the pharmaceutical composition generally includes a cryoprotectant, such as sucrose, trehalose, lactose, maltose, and mannitol. The lyophilized formulation can be stored for extended periods of time, even at ambient temperatures.

[0079] In some embodiments, the mechanotransduction blocker is formulated in a pharma- ceutically acceptable excipient with a second agent, such as a combination therapy disclosed above.

[0080] The subject pharmaceutical compositions may be administered orally, subcutaneously, intramuscularly, parenterally, or by other routes including, but not limited to, oral, rectal, nasal, topical (including transdermal, aerosol, buccal, and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, and intradermal), intravesical, or by injection into the affected organ.

[0081] Each of the active agents may be provided in a unit dose of about 0.1 μg, 0.5 μg, 1 μg, 5 μg, 10 μg, 50 μg, 100 μg, 500 μg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 250 mg, 500 mg, or 750 mg or more.

[0082] The pharmaceutical compositions may be administered in unit dosage form and may be prepared by any method known in the art. Such methods include combining the mechanotransduction blocking agent with a pharma- ceutically acceptable carrier or diluent, which constitutes one or more accessory ingredients. The pharma- ceutically acceptable carrier is selected based on the chosen route of administration and standard pharmaceutical practice. Each carrier must be "pharma- ceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. The carrier may be solid or liquid, and the type is generally selected based on the type of administration being used.

[0083] Examples of suitable solid carriers include lactose, sucrose, gelatin, agar, and bulk powder. Examples of suitable liquid carriers include water, pharma- ceutically acceptable fats and oils, alcohols, or esters, emulsions, syrups, or elixirs, suspensions, solutions and / or suspensions, and solutions and / or suspensions that are reconstituted from non-effervescent granules, and effervescent preparations that are reconstituted from effervescent granules. Such liquid carriers may contain, for example, suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, thickeners, and melting agents. A preferred carrier is edible oil, such as corn oil or canola oil. Polyethylene glycol, such as PEG, is also a good carrier.

[0084] Any drug delivery device or system that provides the dosing regimen of the present disclosure may be used. A wide variety of delivery devices and systems are known to those of skill in the art.

[0085] kit Also, a kit for carrying out the method described in the present disclosure. Generally, the subject kit may include the above-mentioned pharmaceutical composition and a skin graft harvester as described above. The pharmaceutical composition may be contained in a specific delivery device. The delivery device includes, but is not limited to, a patch, a gauze dressing, a transparent film dressing, a foam dressing, a hydrocolloid dressing, an alginate dressing, a composite dressing, and the like.

[0086] The skin graft harvester of the present disclosure is any skin graft harvester capable of producing split thickness skin grafts. Skin graft harvesters that find use in the present disclosure include, but are not limited to, surgical knives, vibrating Goulian knives, pneumatic dermatomes, motorized dermatomes, and the like.

[0087] The subject kits can include any combination of components for carrying out the methods of the present disclosure. The components of the subject kits can be present as a mixture or can be separate entities. In some cases, the components are present as a lyophilized mixture. In some cases, the components are present as a liquid mixture. In some cases, the components are present as a semi-solid mixture, such as a hydrogel. The components of the subject kits, in any combination, can be in the same container or in separate containers.

[0088] The subject kits may further include (in some embodiments) instructions for carrying out the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, such as, for example, a sheet or sheets of paper on which the information is printed, in the kit packaging, in a package insert, etc. Yet another form in which these instructions may be present is as a computer readable medium having the information recorded thereon, such as, for example, a diskette, a compact disc (CD), a flash drive, etc. Yet another form in which these instructions may be present is a website address, which may be used via the Internet to access the information at the remote site.

[0089] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0090] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0091] General methods in molecular and cellular biochemistry are described in Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001), Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999), Protein Methods (Bollag et al., John Wiley & Sons 1996), Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999), Viral Vectors (Kaplift & Loewy eds., Academic Press 1995), Immunology Methods Manual (I. Lefkovits ed., Academic Press, 1997), and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons Reagents, cloning vectors, cells, and kits for the methods referred to or related to in this disclosure are available from commercial suppliers such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and repositories such as, for example, Addgene, Inc., American Type Culture Collection (ATCC).

[0092] I. Disrupting mechanotransduction reduces fibrosis and contracture in split-thickness skin grafts A. Summary Burns and other traumatic injuries represent a significant biomedical burden. The current standard of care for deep injuries is autologous split-thickness skin grafting (STSG), which frequently results in contractures, abnormal pigmentation, and loss of biomechanical function. Currently, there is no effective therapy that can prevent fibrosis and contracture after STSG. Here, we developed a clinically relevant porcine model of STSG and comprehensively characterized the porcine cell populations involved in healing at single-cell resolution. We identified upregulation of proinflammatory and mechanotransduction signaling pathways in standard split-thickness skin grafts. By blocking mechanotransduction using a small molecule focal adhesion kinase (FAK) inhibitor, we promoted healing, reduced contracture, attenuated scar formation, restored collagen architecture, and ultimately improved graft biomechanical properties. Acute mechanotransduction blockade upregulated myeloid CXCL10-mediated anti-inflammation with a decrease in CXCL14-mediated myeloid and fibroblast recruitment. At later time points, mechanosignaling shifted fibroblasts toward a profibrotic differentiation fate, whereas disruption of mechanotransduction orchestrated a mesenchymal fibroblast differentiation state to block these responses and instead drove fibroblasts toward a pro-regenerative adipogenic state similar to unwounded skin. We then confirmed these two distinct fibroblast transcriptional trajectories in human skin, human scar, and three-dimensional organotypic models of human skin. Taken together, pharmacological blockade of mechanotransduction significantly improved large animal healing after STSG by promoting both early anti-inflammatory and late regenerative transcriptional programs, resulting in healed tissue similar to unwounded skin. FAK inhibition is the current supplemental standard of care for traumatic and burn injuries.

[0093] B. Materials and Methods 1.Research design The overall goal of the study was to identify and therapeutically target molecular drivers of fibrosis in large organisms to improve healing outcomes after STSG. Each pig (n=5) received six STSGs (n=30 total STSG biological replicates). Treatment conditions were randomly assigned to different STSGs across each pig's dorsum. All assessments of various scar characteristics (wound contracture, VAS, re-epithelialization, and engraftment) were performed using images blinded to the observer. Analysis of collagen architecture was performed using quantitative computer algorithms CurveAlign and CT-FIRE in an unbiased manner. 10X genomics scRNA-seq data were captured and sequenced by the Stanford Functional Genomics Facility blinded to treatment group. scRNA-seq data were analyzed using Seurat, a quantitative analysis package for clustering and embedding of scRNA-seq data. For the in vitro experiments, (n=15) human collagen hydrogel biological replicates, (n=6) porcine hydrogel biological replicates were generated and each hydrogel was randomly assigned to a different treatment group. No data were omitted. These sample sizes were large enough to detect the effect of treatment across a range of variables. All animal work was performed in accordance with Stanford APLAC and AAALAC guidelines (APLAC protocols 31530 and 32962). Human tissue samples were collected under IRB #54225 from procedures where samples would otherwise be discarded. Patient identification information was not recorded for any of the samples.

[0094] 2.Development of a pig model for technology transfer in STSG We developed a novel porcine STSG model using surgical techniques commonly applied in the clinical treatment of burn wounds and other soft tissue defects. First, we performed a 25 cm 2A full-thickness excision wound of 100 mm was made on the back of an adult Red Duroc pig, leaving the underlying fascia intact. In the same surgical procedure, we harvested a thin STSG (0.01 in) from the same pig using a clinical grade electric dermatome (Zimmer Biomet). First, the unwounded skin of the donor site was lubricated (Surgilube) and the dermatome was passed over the skin at a controlled speed. The resulting skin was placed onto a Skin Graft Carrier (Dermacarrier II, Zimmer 00770800010) and slowly fed through a Skin Graft Mesher (Zimmer 7701) to create a 1:1.5 ratio mesh graft (41). The graft was carefully spread onto the graft carrier and placed onto the exposed fascia of the wound bed. Skin staples (Covidien 8886803712) were used to secure the graft to the wound bed (approximately 5 staples per edge). The graft was covered with three layers of petrolatum gauze (Xeroform, Covidien SH84-433605) to prevent the graft from drying out and to prevent bacterial infection. A bolster dressing was prepared by cutting VAC Granufoam (Small Dressing Kit, Acelity M8275065) into 5.5 cm x 5.5 cm squares and secured firmly over the gauze and STSG with additional skin staples. Finally, the bolster sponge dressing was covered with Telfa non-adhesive dressing (Covidien 1961) and Tegaderm adhesive dressing (3M 1624W). To prevent irritation and minimize the ability of the animal to affect the dressing, a custom designed polyester jacket fitted to each individual pig was used (Lomir Biomedical Inc.) Animals were given 10 mg / kg amoxicillin orally twice daily for a total of 5 days after surgery.

[0095] For the treatment experiments, (n=5) pigs were used, with each pig receiving a total of six STSGs. For each pig for the 90 day post-surgery experiment, the STSGs were randomly assigned to receive either FAKI hydrogel, blank hydrogel, or no hydrogel (standard dressing used for all STSGs), and treatment conditions were randomly assigned to different STSGs across each pig's dorsum. For each pig for the 7 day post-surgery and 14 day post-surgery experiments, the STSGs were randomly assigned to receive either FAKI hydrogel or no hydrogel, and treatment conditions were randomly assigned to different STSGs across each pig's dorsum to minimize any positional effects. Hydrogel was applied over the STSGs before the standard dressing (Vaseline gauze + bolster sponge + Telfa + Tegaderm + custom jacket). Sterile hydrogel was presoaked in sterile saline before application. The STSGs were left undisturbed for the first three days. The dressings and hydrogel were then changed every other day for the first 3 weeks after the initial injury until the third week. Each of these dressing changes was performed under sterile conditions. The skin staples were removed with a skin stapler remover (3M MMMSR3Z) and the wounds and grafts were gently cleaned with sterile saline. Photographs were taken at each dressing change and the hydrogel and bolster dressings were replaced with new dressings. The animals were sedated for a short period of time for each dressing change.

[0096] After 3 weeks, the dressings (and hydrogel) were changed twice a week until the third month. The STSGs were no longer bolster dressed, but instead, conventional dressings were used (Telfa+Tegaderm). Each STSG in the animals was biopsied at the end of the study for histological and molecular evaluation.

[0097] 3. Statistics Statistical analysis was performed in Prism8 (GraphPad, San Diego, California). When comparing two samples, t-tests were used. When comparing more than two samples, either one-way or two-way analysis of variance (ANOVA) and Tukey's multiple comparison test were used. Data are presented as mean ± SEM. P values ​​less than 0.05 were considered statistically significant.

[0098] C. Results 1. Porcine model of autologous split-thickness skin grafting We developed a clinically relevant porcine STSG model that uses standardized surgical techniques applied in the clinical treatment of burn wounds and other soft tissue defects. We chose to use red Duroc pigs because they closely recapitulate human skin physiology, skin wound healing kinetics, and scar formation after injury (29, 37) (Figure 1). Human and red Duroc skin have similar thickness and biomechanical properties, and unlike small animal models, both develop thick fibrotic scar tissue that becomes rigid and contracts over time, thereby healing deep dermal injuries (38).

[0099] To treat full-thickness injuries, such as severe burns, surgeons often resect necrotic injured tissue to expose the underlying fascia before applying the STSG (39, 40). To mimic this, we used a 25 cm 2Full-thickness excision wounds were made on the dorsum of adult Red Duroc pigs (Figure 1, A and B), leaving the underlying fascia intact. In the same surgical procedure, we harvested STSGs (0.01 inches) using a clinical-grade electric dermatome (Zimmer Biomet) and meshed the resulting grafts at a ratio of 1:1.5 (41) (Figure 1C). Clinically, STSGs (approximately 0.01 inches) are typically harvested to contain the epidermis and superficial dermis, allowing the donor site to re-epithelialize and heal (41, 42). These STSGs were then applied directly to the dorsal wound using petrolatum gauze, a bolster dressing, and skin staples to maximize engraftment (Figure 1D). Each animal also wore a custom-made compression jacket throughout the study. All STSGs underwent complete engraftment (incorporation). We initially observed the typical "mesh" appearance of the healing graft, characteristic of human STSG during the early healing phase, followed by significant graft and scar contracture, as well as hyperpigmentation and rough texture (Figure 1E). These observations were consistent with the time course of skin healing and contracture after STSG in humans (43).

[0100] 2. Custom single-cell RNA sequencing method for porcine skin tissue identifies cell subpopulations that contribute to scarring STSG did not restore normal skin appearance, instead showing permanent scar contracture, as well as excessive fibrosis, hyperpigmentation, and raised hypertrophic scar (HTS) formation at 90 days post-operative day (POD) (Figure 2A). Specifically, STSG had a thicker dermis, which had more highly aligned collagen fibers and fewer dermal appendages, all classic signs of fibrotic healing. In contrast, normal non-wounded skin had a randomly aligned "basket weave" collagen architecture (*P<0.05) (Figure 2, A and B). STSG was also found to contain more pro-fibrotic myofibroblasts (*P<0.05) (Figure 2, A and B).

[0101] To further explore the cellular and molecular changes that drove the differences between fibrotic STSG and non-wounded skin, we performed scRNA-seq using the 10X Genomics platform after complete healing (Figure 2C) (44, 45). Previous studies have performed scRNA-seq in non-wounded human skin (46-48) or compared human keloids to human scars (49), but no study has previously directly compared fibrotic tissue to non-wounded skin at single-cell resolution. To collect high-quality cells, we optimized our previously published method and harvested porcine tissue from both late-stage (day 90 after surgery) STSG scars and normal non-wounded skin (44).

[0102] We created a custom porcine transcriptome using the Sus Scrofa genome (Ensembl, v11.1) (50) generated according to the 10X protocol for non-model organisms (44), which we have made publicly available with instructions for implementation as part of a modified 10X CellRanger pipeline (see Data and Materials Availability statement). Briefly, all read fragments were aligned to this transcriptome using a standard base matching threshold (51), followed by single-cell demultiplexing and unique molecular identifier (UMI) batch correction (52), allowing us to generate a single-cell mRNA count matrix for each porcine sample. Data for individual cells from both groups were subjected to blinded Louvain-based clustering and embedded within a two-dimensional UMAP (Uniform Manifold Approximation and Projection) space (45) (Figure 2C). A total of 7,700 cells were captured and distinct populations of fibroblasts, myeloid cells, lymphoid cells, endothelial cells, vascular smooth muscle cells (VSMCs), and keratinocytes were identified using automated cell type annotation via the SingleR package (Figure 2D) and validated with cell type-specific marker genes (Figure 9A).

[0103] We observed that fibroblasts had more than 400 differentially expressed genes (DEGs; average log fold change >0.5 between STSG and skin), and myeloid and lymphoid cells had more than 200 DEGs between STSG and skin (Figure 2E, Figure 9B). In contrast, other cell types (keratinocytes, endothelial cells, VSMCs) had less than 100 DEGs, indicating that they were not transcriptionally different between normal skin and skin grafts (Figure 2E; Figure 9, C and D). These findings suggested a sustained increase in fibroproliferative and inflammatory cell activity in STSG at this late time point, corroborating our observation of an increased number of myofibroblasts in STSG compared to non-wounded skin (Figure 2, A and B).

[0104] 3. Fibrosis after STSG is associated with increased mechanotransduction signaling First, we investigated myeloid cells in our STSG and non-wounded skin scRNA-seq datasets. We investigated differential regulation of signaling pathways using Genetrail3, a pipeline for over-representation analysis (ORA) (53). Initially, we observed that STSG myeloid cells demonstrated an inflammatory phenotype characterized by upregulation of inflammatory markers such as CXCL8 and CD86, as well as enrichment for immune response activation signal transduction (GO-BP:0002758) and inflammatory response pathways (WP453) (54) (Figure 2, F and G). Myeloid STSG cells were also enriched for fibrosis-driving genes such as TNF (55) and demonstrated upregulation of common mechanotransduction pathways such as the ERK1 / ERK2 and EGF / EGFR pathways (56) (Figure 2, F and G). These pathways are downstream of FAK, suggesting that these immune cells are mechanoresponsive even at later time points ( 57 , 58 ).

[0105] Next, we compared the transcriptional profiles of fibroblasts in STSG and non-wounded skin after complete healing over 90 days (Figure 2H). These revealed a significant increase in the expression of myofibroblast-related and collagen-producing genes, such as ACTA2 (encoding smooth muscle alpha actin aSMA), RUNX1 (runt-related transcription factor 1), TAGLN (transgelin), and COL11A1 (collagen type XI alpha 1 chain), suggesting that fibroblasts in STSG had differentiated into a more contractile myofibroblast phenotype associated with increased collagen production and fibrosis, which was also supported by our immunofluorescence staining (59) (Figures 2, A, B, and I; Figure 9E). STSG fibroblasts showed enrichment for gene sets related to mechanotransduction and collagen production / organization driving scar formation, such as response to mechanical stimuli (GO-BP:0009612), focal adhesions (WP306), ECM assembly (GO-BP:0030198), YAP signaling (WP3967), response to TGFB signaling (WP560), and ossification (GO-BP:0001503) (Figure 2I). STSG fibroblasts were also characterized by downregulation of genes related to adipogenesis (WP236), lipid transport (GO-BP:0006869), and regulation of endothelial cell migration (GO-BP:0010594), defined by regenerative adipogenesis markers such as APOE (apolipoprotein E), APOD (apolipoprotein D), CLEC3B (c-type lectin domain family 3 member B), and AGT (angiotensinogen) (60) (Figure 2J, Figure S6E). These pathways and genes suggested that STSG fibroblasts had transitioned away from the more homeostatic quiescent baseline observed in normal skin.

[0106] Although fibroblasts are generally considered the primary mediators of collagen deposition and scar contracture (26), recent studies suggest a role for immune cells in regulating (and sustaining) the fibrotic process (61-64). There is some evidence that macrophages respond to mechanical cues in some circumstances, but these studies have yielded conflicting results, and the literature suggests that mechanical strain results in both pro- and anti-inflammatory responses (57, 58). Overall, our transcriptomic data showed that the tissue that develops after STSG, unlike non-wounded skin, is primarily characterized by increased mechanotransduction signaling in both inflammatory cells and fibroblasts, suggesting a shared common pathway in the context of STSG. Although the importance of mechanotransduction has been previously identified during open wound healing (23, 25-27), the contribution of mechanotransduction signaling after skin grafting remains to be investigated. To develop therapies to improve STSG outcome, we hypothesized that targeting mechanotransduction signaling might improve healing and reduce scar formation after STSG.

[0107] 4. Disrupting mechanotransduction attenuates fibrosis in STSG by reducing contracture, promoting engraftment, and improving biomechanical properties To inhibit mechanotransduction in STSG-treated porcine wounds, we delivered FAKI (VS-6062) using a biodegradable, biocompatible, soft pullulan-based hydrogel (23) optimized for sustained drug release during wound healing (Figure 3, A and B). FAK is a critical transducer of integrin-matrix forces to downstream intracellular pathways (26). VS-6062 (formerly Pfizer PF-00562271) is a potent, ATP-competitive, next-generation small molecule FAKI that blocks tumor growth and is undergoing phase I clinical trials for advanced solid tumors (ClinicalTrials.gov Identifier: NCT00666926) (65). VS-6062 also has strong selectivity for FAK over a broad range of other kinase targets (66, 67), and we have previously characterized the release of this drug in our hydrogels (23, 26). The hydrogel contains VS-6062, and rehydrating the hydrogel in saline converts it into a hydrogel dressing (Fig. 10A), which slowly releases the drug, which penetrates the STSG dermis over time (Fig. S2, B and C). Similar to other hydrogels, this hydrogel dressing covers the wound or STSG, prevents drying, and facilitates moist wound healing (Fig. 3B).

[0108] We measured scar surface area and found that FAK inhibition blocked scar contracture at early time points (7 days after surgery, P=0.09; 21 days after surgery, *P<0.05) (FIG. 3, C and D). In contrast, STSG and blank hydrogel-treated STSG had immediate scar contracture by 7 days after surgery, which continued to increase over time. At 28 days after surgery, untreated and blank hydrogel-treated STSG had contracted by more than 70%, whereas FAKI-treated STSG showed only 30% contracture (FIG. 3D). These observations were statistically significant and persisted across all time points (*P<0.05).

[0109] A panel of three blinded plastic surgeons quantified STSG re-epithelialization and scar appearance using a visual analog scale (VAS). The VAS is a scar scoring system commonly used by plastic surgeons to stratify scar severity and visually assess scar appearance (range 0-100, 0=unwounded skin, and 100=hypertrophic scar) (68). From these blinded scores, we found that FAKI hydrogel accelerated stromal re-epithelialization of STSG at 7 days after surgery (**P<0.01) (Figure 3E). FAKI hydrogel also significantly improved the fibrotic appearance of the STSG wound over time, demonstrating reduced scar formation (*P<0.05) (Figure 3F).

[0110] Using a tissue cutometer, a non-invasive clinical instrument that measures the viscoelastic properties of skin via deformation under negative pressure, we found that FAKI-treated STSG also had less hardness and stiffness than untreated STSG (*P<0.05) (Figure 3G), exhibiting biomechanical properties similar to non-wounded skin. Collectively, these data demonstrated that early intervention to disrupt cellular mechanotransduction pathways reduces a range of complications that typically occur after STSG, including contracture, stiffness, and scar appearance.

[0111] 5. Pharmacological blockade of mechanotransduction after STSG restores collagen architecture similar to that of non-wounded skin To confirm these gross anatomical findings, we used histological analysis to observe changes in tissue architecture following FAK inhibition (Figure 4A). Pharmacological blockade of mechanotransduction significantly promoted shorter, more randomly aligned collagen in the deep dermis, similar to the typical basket-weave-like collagen fiber network in non-wounded skin (*P<0.05) (Figure 4, B-E; Figure 11, A and B). Furthermore, FAK inhibition reduced overall collagen deposition across both the superficial and deep dermis by decreasing fiber width and overall architectural complexity (**p<0.01, ***p<0.001) (Figure 4F; Figure 11, B and C). Overall, FAKI-treated STSG demonstrated dermal remodeling similar to that of non-wounded skin across the entire thickness of the developing scar.

[0112] 6. Mechanotransduction blockade leads to acute upregulation of anti-inflammatory pathways in myeloid cells To understand the mechanisms driving these macroscopic and microscopic tissue changes, we investigated how disruption of mechanotransduction affects healing in cells (Figure 5A). Because untreated (control) STSG and blank hydrogel-treated STSG demonstrated no differences across a wide range of clinical scar measurements (Figures 3 and 4), we focused our scRNA-seq analysis on cells from untreated STSG and FAKI-treated STSG. First, we investigated the early stages of STSG incorporation in porcine tissue 7 days after STSG (Figure 5, A and B). Again, we captured a diverse cellular environment in both treated and untreated STSG (Figure 5, C and D; Figure 12), but during this early inflammatory phase of wound healing, monocytic lineage cells (macrophages, monocytes, dendritic cells) at the early time point had the most DEGs (>170) (Figure 5E), followed by neutrophils and lymphoid cells (~100 each) (all other cell types had less than 75). Unexpectedly, fibroblasts had only ~50 DEGs, indicating that while substantial differences in fibroblast gene expression were observed at later times, there were no strong differences in their expression at these early time points. For example, in both conditions, fibroblasts showed similar expression of extracellular matrix markers such as COL1A1, COL3A1, FN1 (encoding fibronectin), and inflammatory chemokines such as CXCL14 (69, 70) (Figure 5F). These findings suggested that fibroblasts are not dramatically modified by mechanical signaling during the early stages of healing in this model.

[0113] Instead, inhibiting mechanotransduction in myeloid cells (monocyte lineage and neutrophils) induced a variety of beneficial transcriptional transitions (Figure 5, G and H). For example, STSG monocytic lineage cells demonstrated increased expression of COL1A1, COL3A1, and FN1 that was abrogated with FAK inhibition (Figure 5I), suggesting that FAK inhibition reduced early ECM deposition from myeloid cells. These findings suggest a previously unexplored ability of myeloid cells to contribute to extracellular matrix formation.

[0114] Disruption of mechanotransduction also upregulated myeloid CXCL10 expression (encoding interferon gamma-inducible protein 10; IP-10) (Figure 5I). CXCL10 is a secreted chemokine that inhibits fibroblast migration in response to proinflammatory markers (71). IP-10 therapy has previously been used clinically to reduce fibrosis (72-74). FAK inhibition also induced SOCS3 expression (Figure 5I), which is known to attenuate proinflammatory IL6 expression (75). Using Genetrail3 to investigate differential signaling pathways (53), we observed that FAK-inhibited myeloid cells showed enrichment of gene sets related to the IL-10 anti-inflammatory pathway (WP4495), interferon alpha / beta signaling (WP1835), and classical antibody-mediated complement activation (GO-BP:0006958) (Figure 5J).

[0115] We then collected porcine STSG tissue over time (days 7, 14, and 90 after surgery) to map the time course of STSG healing at the protein level. Using immunofluorescence staining, we first investigated the presence of F4 / 80-positive macrophage populations in the tissue (Figure 5K). Untreated STSG contained significantly increased numbers of macrophages at early (day 7 after surgery, ***P<0.001) and late (day 90 after surgery, *P<0.05) time points, indicating a chronic proliferative inflammatory response, supporting our previous myeloid cell findings (Figure 2, C-G). STSG treated with FAKI significantly reduced the number of infiltrating inflammatory cells at both early (***P<0.001) and late (*P<0.05) time points (Figure 5, K and L). Among the remaining inflammatory cells, disruption of mechanotransduction significantly (*P<0.05) upregulated IP-10 (CXCL10) secretion 7 days after surgery ( Fig. 5, K and L ), indicating a time-dependent decrease in fibroblast recruitment into the wound and reduced downstream fibrosis.

[0116] Myeloid cells, involved in acute inflammation after soft tissue injury, have recently been identified as mechanosensitive in the context of several physiological processes in the body, including proprioception, touch, balance, and hearing (3, 76, 77). Within the context of wound healing, recent studies suggest that mechanotransduction may affect myeloid transcriptional dynamics and alter healing potential (57, 58, 78), although the mechanisms remain incompletely understood (79). Here, we observed that disruption of mechanotransduction had a greater effect on myeloid cells than fibroblasts at early time points by reducing inflammatory recruitment and promoting a CXCL10-mediated anti-inflammatory transcriptional profile. ECM-producing myeloid cells were recruited to STSGs in excess amounts, indicating a previously unappreciated importance of myeloid cell collagen production during skin graft integration. These findings demonstrated that mechanosensitive myeloid cells also respond to mechanotransduction blockade by promoting a pro-regenerative phenotype.

[0117] 7. Disruption of mechanotransduction shifts myofibroblast transcriptional state toward regenerative differentiation With these differences at early time points, we next sought to understand the role of mechanotransduction blockade at later time points. Because fibroblasts had the most genetic changes between STSG scars and normal skin at day 90 (Figure 2E), we specifically investigated the effect of FAK inhibition on fibroblast differentiation state. First, we performed RNA velocity analysis using scVelo and CellRank (Figure 6, A and B). RNA velocity analysis combines RNA velocity information with transcriptome similarity to calculate a global map of cell fate potential, which reveals early and terminal cell states (Figure 13A) (80, 81). CellRank identified six transcriptionally distinct cell lineages (Figure 6B), which arose in an early basal state (marked with *). Fibroblasts from FAKI-treated STSG showed transcriptional similarity with those from non-wounded skin, mainly in lineages 1 and 2, whereas cells from normal STSG shifted away from this [skin and STSG+FAKI] cell state along the UMAP-1 axis into four more heterogeneous lineages (3, 4, 5, and 6) (Figure 6, A and B; Figure 13, A-C). Fibroblast lineages 3 and 4 showed higher latency scores and velocity vector lengths (Figure 6C, Figure 13D), indicating a more advanced differentiation state and a higher proportion of spliced ​​mature RNA. Thus, lineages 1, 2, 3, and 4 were selected for further analysis.

[0118] Combining fate probability estimation with pseudo-temporal ordering along latency, we visualized gene expression along trajectories leading to terminal states. This revealed lineage drivers for regenerative lineages 1 and 2, and fibrotic lineages 3 and 4 (Figure 6, D-F; Figure 13, A and B). Along fibrotic states, we observed upregulation of fibrotic chondrogenic markers THBS2 (thrombospondin 2; along states 3 and 4), THBS4 (state 3), ACAN (aggrecan; state 3), ENPP1 (ectonucleotide pyrophosphatase 1), and myofibroblast differentiation marker ACTA2 (state 4) (82-84), suggesting that mechanical forces push a subset of fibroblasts toward a more fibrotic fate during scar formation (Figure 6, D-F; Figure 14, E and F). Aggrecan is a chondrocyte marker, and the family of THBS genes encodes thrombospondins, ECM proteins known to facilitate cell-matrix binding and regulate mesenchymal chondrogenic and adipogenic differentiation states (85). Comparison of the top DEGs in STSGs also revealed upregulation of previously identified profibrotic genes, such as SFRP2 and TGFB1 (Figure 14, A-C) (86, 87). SFRP2 was previously identified in healthy human skin scRNA-seq as a fibroblast subpopulation with high fibrogenic potential (46-48), and transforming growth factor (TGF) β1 is a well-known promoter of fibrosis (88). This was supported by enrichment for gene sets involved in contractile myofibroblast phenotype (actin filament organization; lineages 3, 4), chondrocyte differentiation (lineage 3), and ossification (lineages 3, 4) (Figure 6F, Figure 14D). STSG fibroblasts also upregulated the fibroblast-recruiting chemokine CXCL14 ( Fig. 6<em>F ; Fig. S14 , A–C), which has previously been found to stimulate fibroblast migration and proliferation while inhibiting regenerative differentiation ( 89 , 90 ).

[0119] In contrast, along regenerative lineages 1 and 2, FAKI-treated STSG and non-wounded skin demonstrated similar expression of genes, indicating repression of the profibrotic genes mentioned above, as well as upregulation of APOE (state 2), CLEC3B (state 2), CD34 (state 1), and PPARG (peroxisome proliferator-activated receptor gamma; state 1) unspliced ​​pre-mRNA, indicating induction of regenerative adipogenic transcription (91, 92) (Figure 6, D and E). Apolipoproteins such as APOE and APOD are key markers of lipid transport and lipid metabolism, and these markers are expressed in both lipid transporting fibroblasts (lipofibroblasts) and adipocytes (92, 93). Apolipoproteins have been found to attenuate inflammation, and lipofibroblasts are a fibroblast subpopulation that have been found to interact with adipose tissue and can differentiate into adipocytes during normal tissue healing (94). These lipofibroblasts also expressed PPARG and CFD (complement factor D, encoding adipsin), which also promote lipid accumulation and are critical transcription factors for adipogenesis (Figure 6E). FAK inhibition in STSG (lineages 1 and 2) also promoted adipogenic gene sets (adipogenesis, angiogenesis, epithelial cell migration) and stem cell markers (CD34 and NT5E) (Figure 6E; Figure S14, B-D). The expression of these markers was consistent with that found in non-wounded skin. In addition, thrombospondin has been found to drive hypertrophic scar formation in a TGFβ1-dependent manner (95), and TGFβ1 has been found to inhibit adipogenesis (88). This downregulation of mechanotransduction, thrombospondin, and TGF was further supported by the upregulation of small leucine-rich proteoglycans (SLRPs) such as decorin (DCN) (Figure 6D, Figure S14A). SLRPs help control the fibrillogenesis of scar formation, are highly expressed in non-wounded skin compared to fibrotic tissue, and inhibit TGFβ1, reducing HTS formation ( 87 ).

[0120] 8. Mechanotransduction coordinates the temporal evolution of regenerative fate We then collected porcine STSG tissue over a series of time points (days 7, 14, and 90 after surgery) to map the time course of fibroblast protein expression within healing STSG tissue. Using immunofluorescence staining, we found that untreated STSG demonstrated increased expression of the fibroblast-recruiting chemokine CXCL14 at early time points, followed by a significant increase in CXCL14 at later time points (day 90 after surgery; ***P<0.001) (Figure 7A). Furthermore, untreated cells expressed greater abundance of the chondrogenic matrix protein thrombospondin 4 (the protein form of THBS4) over the course of days 7 (***P<0.001), 14 (***P<0.001), and 90 (*P<0.05) after surgery (Figure 7B). Accordingly, FAK inhibition attenuated THBS4 and CXCL14 expression (Figure 7, A and B), decreasing cell recruitment and subsequent fibrotic matrix deposition. Disruption of mechanotransduction also initiated expression of CD34 at later time points (Figure 7C), demonstrating a shift in fibroblast transcriptional profile toward a more plastic stem-like phenotype. These stem-like fibroblasts then demonstrated an increase in the adipocyte lipid transporter apolipoprotein E (the protein form of APOE) at both 14 days (*P<0.05) and 90 days (*P<0.05) after surgery (Figure 7D), indicating increased differentiation toward a lipofibroblast regenerative phenotype.

[0121] To confirm the potential human relevance of these findings, we collected patient samples from clinical trials. We were unable to collect healed human STSG samples, but were able to collect human hypertrophic scars. We found that human hypertrophic scars demonstrated a significant increase in thrombospondin 4 (*P<0.05) compared to non-wounded skin, indicating upregulation of extracellular matrix protein deposition in highly fibrotic scar tissue. Furthermore, healthy human skin demonstrated a significant increase in apolipoprotein E (*P<0.05), suggesting the presence of healthy lipofibroblasts and adipocytes in the skin. These lipotransporting cells were not present in hypertrophic scars (Figure 15).

[0122] Overall, these data demonstrated that FAK inhibition after injury promotes a regenerative phenotype and shifts fibroblasts away from a fibrotic state. We identified a "regenerative axis" that leads FAK inhibition and non-wounded skin clusters, and a "fibrotic axis" that leads in the opposite direction and is characterized by untreated STSG fibroblasts in an environment with high mechanical stress. We next attempted to recapitulate the existence of these two trajectories in a precisely controlled mechanical environment in vitro.

[0123] 9. Precise Manipulation of Mechanical Force Modulates Fibroblast Transcriptional Signatures Our group and others have previously demonstrated that both porcine wounds and human skin are subjected to mechanical strains of approximately 5-20% (96, 97) and that physically unloading this mechanical tension with a physical dressing reduces fibrotic scar formation (38). To determine whether the increased mechanical strain experienced by cells within the healing STSG induces the same fibroblast transcriptional state observed in vivo, we used our previously published three-dimensional (3D) organotypic scar culture system, which allows for precise manipulation of strain (and therefore stress) without any other potential confounding factors (27, 98). Fibroblasts were first isolated from human skin and seeded into 3D collagen scaffolds (Figure 8A). These scaffolds were then subjected to either 10% strain (strain, S), 10% strain and FAK inhibition (strain+FAKI, S+FAKI), or 0% strain as a control (no strain, NS) (Figure 8, A-C). Because it was believed that healing tissues may not be subjected to maximum strain (due to pain), we used a 10% strain profile to represent the average wound strain environment.

[0124] In human cells, we observed that fibroblasts subjected to mechanical strain upregulated both fibroblast-recruited CXCL14 and fibrotic THBS4 expression (*P<0.05) compared to fibroblasts not subjected to strain (Figure 8B). Subsequent FAK inhibition of strained fibroblasts significantly decreased CXCL14 (*P<0.05), THBS4 (*P<0.05), and aSMA (**P<0.01) expression compared to strained fibroblasts (Figure 8B, Figure 16), and also significantly increased APOE (**P<0.01) and CD34 (****P<0.0001) expression (Figure 8C). These observations confirmed the beneficial transcriptional effect of FAKI on STSGs in vivo. Repeating these experiments using porcine cells and scRNA-seq, we then observed that fibroblasts subjected to strain demonstrated increased expression of collagens (COL1A1, COL3A1) and other ECM-related genes (FN1, ADAM12), as well as markers (including POSTN) that have been found to drive fibrosis (99) (Figure 8, D-G). Using RNA kinetics, we again identified two differentiation trajectories radiating from the implantation center (indicated with *) to either strain (fibrotic) or S+FAKI (regenerative) lineages (Figure 8, E and F), consistent with our in vivo analysis (Figure 6, A-C; Figure 13). Also similar to our observations in vivo (Figure 6C), fibroblasts subjected to strain progressed in both latency and kinetic pseudotime, demonstrating large changes in transcription induced by mechanical strain (Figure 8F). Fibroblasts subjected to strain upregulated CXCL14, fibrotic thrombospondin (THBS2), and collagen (COL1A1) expression, and FAK inhibition abrogated all of these profibrotic responses, instead demonstrating a regenerative transcriptomic signature characterized by upregulation of APOE and the antifibrotic genes EGR1 (early growth response 1) and PRDX1 (peroxiredoxin 1) (Figure 8, H and I; Figure 17), consistent with both our STSG (Figures 5 and 6) and human findings (Figure 8, A-C).Overall, we observed that physiological mechanical strain pushed both human and porcine fibroblasts toward the same transcriptional profile found in vivo, confirming that manipulating mechanical forces directly drives fibrotic and regenerative phenotypes in both large animals and humans.

[0125] Taken together, these findings characterize scarring in large animals and humans, demonstrating that mechanical forces promote fibroblasts to assume a distinct profibrotic program that can be prevented by inhibiting mechanotransduction and driven toward a regenerative commitment similar to that of non-wounded skin. Fibroblasts in healing STSG are subjected to the natural high mechanical stress in skin tissue, which then activates mechanotransduction signaling, leading to contractile chondrogenic differentiation and fibrotic collagen formation. In contrast, FAK inhibition pharmacologically protects fibroblasts from this high stress environment, preventing the fibrotic phenotype and promoting a more regenerative adipogenic phenotype.

[0126] 10. Discussion Skin grafting is the mainstay for the treatment of severe burns and traumatic injuries and has revolutionized the survival of burn patients (8). Unfortunately, skin grafts are not perfect substitutes for injured skin and can result in debilitating fibrohypertrophic scar formation and severe contractures, which may require multiple revision surgeries (8-12). In addition, there are currently no FDA-approved pharmacological therapies to prevent these complications and improve outcomes after grafting (7, 13, 14). To understand the mechanisms driving STSG healing, we developed a clinically relevant large animal model to characterize the cellular environment in STSG scars and non-wounded skin and found that STSG exhibited sustained elevations of fibroproliferative and inflammatory transcriptional programs driven by mechanically activated immune cells and myofibroblasts. Because mechanotransduction pathways were found to be upregulated, we determined that mechanotransduction was a suitable target to block excessive fibrosis and contracture following STSG.

[0127] Here, we demonstrate that sustained delivery of small molecules to modulate mechanotransduction via FAK inhibition can be used with STSG to reduce scar contracture, promote regenerative dermal remodeling, and improve biomechanical skin properties in a human-like porcine model. We created this sustained release form within a hydrogel commonly used in standard wound and skin graft treatments (100). This treatment is an effective therapy for human grafts, which can be incorporated within the current standard of care, providing a technology transfer opportunity to aid patients with traumatic and burn injuries.

[0128] To understand scar formation and healing, many groups, including ours, have comprehensively investigated the critical role of fibroblasts in a wide range of fibrotic disease states (23, 25, 26, 32, 33, 101, 102). However, fibroblast transcriptional status has not been previously investigated in the context of large animal fibrosis or as part of skin graft healing. In the context of human scRNA-seq, several groups have previously characterized non-wounded human skin (46-48) and compared human keloids to human scars without non-wounded skin (49). However, there is a lack of direct comparison of fibrotic tissues with non-wounded skin or pharmacologically enriched regenerating populations at single-cell resolution. In our studies, we found that both myeloid cells and fibroblasts during late-stage scar formation had the greatest transcriptional changes from cells in unwounded skin, and we identified that mechanosignaling in STSGs promotes differentiation into a heterogeneous profibrotic phenotype. By disrupting mechanotransduction, we demonstrated the ability to redirect the mesenchymal fate commitment of large animal and human fibroblasts away from their contractile myofibroblast phenotype toward an adipogenic regenerative differentiation fate, restoring the numbers of quiescent fibroblasts that produce small amounts of randomly organized collagen in unwounded skin.

[0129] The inventors have surprisingly determined that modulating mechanotransduction signaling can directly transition fibroblast differentiation between a relatively profibrotic fate and a relatively proregenerative fate in vivo. In various tissues, both mesenchymal progenitor cells (MPCs) and myofibroblasts, cell types previously thought to be fully differentiated, have been found to be plastic cell types that can be pushed toward either fibrosis, osteo / chondrogenesis, or adipogenesis to differentiate into adipocytes (33, 103). Several previous in vitro studies have also investigated how changes in mechanical stimuli affect actin cytoskeleton organization and MPC differentiation. For example, McBeath et al. found that increased cellular mechanical stress guided adherent MPCs toward osteogenesis and nonadherent round MPCs toward adipogenesis (104). Thrombospondin has also been found to promote osteo / chondrogenesis and inhibit adipogenesis in mesenchymal cells (105, 106). Here, we characterize these fibroblast fate on a 'fibrosis-regeneration axis', with mechanical signaling pushing and pulling cells along this axis.

[0130] Unexpectedly, fibroblasts did not demonstrate large changes in transcriptional activity during the early stages of healing. Instead, at early time points, disruption of mechanotransduction primarily pushed myeloid lineage cells toward a CXCL10-mediated anti-inflammatory and regenerative state. Previous studies have focused on mechanoresponsive fibroblasts (25) as “drivers” of fibrosis, secreting chemokines to promote the recruitment of inflammatory cells (26), but our findings reveal a more complex interplay between immune cells and fibroblast mechanotransduction signaling during different stages of healing. Controlling mechanosignaling in myeloid cells at early time points orchestrated the secretion of inflammatory signals that could directly affect fibroblast phenotype. Although several recent studies have suggested a role for immune cells in regulating and sustaining these fibrotic processes (61-63), our findings reveal that myeloid cells may, in fact, “drive” fibroblasts. Many studies investigating other fibrotic disease states, such as renal fibrosis, idiopathic pulmonary fibrosis, nonalcoholic steatohepatitis (NASH), or liver cirrhosis, have typically characterized the importance of each of these cell types in isolation. Based on our findings, future studies should explore the interactions between both cell types to improve therapeutic and technology transfer outcomes for a wide range of fibrotic disease states.

[0131] Taken together, our studies represent a comprehensive characterization of fibrosis in both human-scale models and human tissues, linking together the importance of tuning mechanical forces for tissue regeneration in both large organisms and humans. Pharmacological blockade of mechanotransduction can improve clinical outcomes by promoting early anti-inflammatory and late regenerative transcriptional programs to reduce fibrosis and improve quality of life. Reducing scar formation and promoting tissue regeneration in humans and other large organisms remains the "holy grail" of biomedical research (107), and our findings may have the potential to improve patient treatment for several fibrotic diseases.

[0132] 11. Supplementary Materials and Methods: A. Animal husbandry All animal studies were performed in accordance with the Administrative Panel on Laboratory Animal Care protocols approved by Stanford University (APLAC# 31530 and 32962). Female red Duroc pigs, 6-8 weeks of age, weighing approximately 16-20 kg at the time of surgery, were purchased from Pork Power Farms (Turlock, CA). All animals were acclimated for at least 1 week upon arrival. All animals were fed a laboratory swine grower diet and water ad libitum.

[0133] b. Preparing the animal for surgery Fasting of the animals was performed 12 hours prior to the surgical procedure. Anesthesia was administered in collaboration with personnel from the Stanford Veterinary Service Center (VSC). Initially, the animals were sedated with intramuscular administration of telazol. A peripheral intravenous line was established in the ear vein. To minimize pain, local anesthetic in the form of EMLA (2.5% lidocaine and 2.5% prilocaine) cream was applied over the vein prior to cannulation. During this time, 10 (mL / kg h) fluids were initiated. In accordance with institutional guidelines, general anesthesia was established with a single intramuscular dose of 6-8 mg / kg telazol as a preanesthetic agent, the animals were intubated with an endotracheal tube, and the animals were maintained on inhalation of 1.5-3% isoflurane throughout the procedure for maintenance of anesthesia. The animals were placed in a prone position and their backs were shaved. The skin was cleaned with Betadine solution and rinsed with alcohol to ensure the absence of skin pathogen contamination. The animals were draped in a sterile fashion to maintain a sterile field. Heat support and eye lubrication were also used. Each surgical procedure lasted approximately 2 hours after the animals were sedated.

[0134] Antibiotics were administered prophylactically and postoperatively to prevent surgical site infection. Cefazolin, 25 mg / kg, was administered intravenously 60 minutes prior to initial injury and repeated 12 hours after completion of surgery. Oral amoxicillin, 10 mg / kg, was administered twice daily for one week. Wounds were covered in appropriate dressings. Pain control was achieved by administration of transdermal fentanyl, 50 mcg / hour, 24 hours prior to surgery. Intramuscular hydromorphone (0.05 mg / kg) was used if the fentanyl patch had come off or was not in place prior to surgery. Carprofen was used once postoperatively, followed by once every 24 hours for 1-2 days, then as needed based on pain assessment.

[0135] c. Blank and FAKI-releasing pullulan-collagen hydrogel preparations We prepared blank and FAKI-releasing hydrogels as previously described (23) with some modifications. 1 g pullulan (TCI 9057-02-7, Tokyo) was mixed with 1 g trisodium trimetaphosphate (STMP) (Sigma Aldrich T5508) and 1 g potassium chloride (KCl) (Fisher Scientific 7447-40-7). The powder was mixed with deionized water to a total volume of 5 mL and mixed thoroughly, followed by 5 mL of a 10 mg / mL suspension of bovine collagen in 0.01 M hydrochloric acid (HCl). The resulting mixture was vortexed until homogenous, and then 0.65 mL of 1 N sodium hydroxide NaOH to initiate crosslinking was also added with gentle vortexing. The mixture was poured into a silicon mold and then dried overnight at room temperature in a sterile hood. The dried film was washed with deionized water to remove non-crosslinked polymer, NaOH, and KCl. The pH of the wash solution was measured and washing was continued until the wash solution reached a pH of 7.0 to 7.5. The swollen hydrogel was frozen at -80°C and then freeze-dried to produce a dry (blank) patch.

[0136] FAKI compounds were obtained from Verastem Oncology (VS-6062) and Selleckchem (625249). We dissolved FAKI in acetone at 1 mg / mL, poured 1 mL of the solution evenly onto the porous hydrogel, and then allowed the solvent to evaporate in a sterile hood. Blank and FAKI-containing hydrogel patches were placed in individual plastic pouches and sterilized using electron beam irradiation at a dose of 20 kGy.

[0137] d. Patch FAKI release study In the pullulan-collagen hydrogel used in our current study, FAKI molecules are physically encapsulated within the hydrogel pores. We performed an in vitro release study of FAKI from this scaffold and demonstrated that the controlled sustained release of FAKI lasted up to 72 hours. Briefly, we took three 2 blank hydrogels and three FAKI hydrogels and placed them in 15,000 or 25,000 MWCO dialysis membrane tubes (Thermo Fisher Scientific). We then added 0.5 mL of PBS to swell the hydrogels in the dialysis tubes, sealed the dialysis tubes on both sides, and then immersed the tubes in 10 mL of PBS in a 50 mL Falcon tube. We placed the tubes and shook them at 37°C, and transferred the samples into new tubes with fresh PBS at each of the following time points: 2 hours, 4 hours, 6 hours, 8 hours, 24 hours, and 96 hours. We then analyzed the FAKI content of the collected samples by LCMS (Applied Biosystems, API 4000 Q Trap) to calculate the FAKI content in each patch ( FIG. 10B ). To study the release on pig skin, we used FAKI patches (2 mg FAKI / 25 cm 2 ) was placed on pig skin for 12 and 24 hours to determine the time-dependent release. Upon 24 hours of treatment, FAKI was detected at a depth of 2 mm from the stratum corneum (FIG. 10C).

[0138] e. Scar contracture analysis The STSG was monitored photographically at each dressing change. Each image was taken with a ruler or standard measuring device to standardize the measurements. Scar area was tracked over time in ImageJ. Contracture was measured as the change in scar area and normalized to the area of ​​the STSG during the first dressing change. Two independent assessors measured the scar area and the resulting scar area was the average of the two measurements.

[0139] f. Visual scar and stromal epithelialization assessment Macroscopic photographs of each wound were taken at each dressing change. Quantification of scar metrics was performed on these macroscopic photographs by a panel of three blinded scar experts using a visual analog scale (VAS) for five components: vascularity, pigmentation, observer comfort (e.g., overall cosmesis), acceptability, and contour. A total score was calculated as a composite of all five scores. Lower scores indicate improved scar appearance. Blinded experts also assessed the amount of stromal epithelialization, defined as the amount of re-epithelialization between the interstitial spaces of the mesh in the graft over time. Scoring was also performed on non-wounded skin as an additional control.

[0140] g. Viscoelastic analysis of skin biomechanical properties A cutometer (Dual MPA 580, Courage+Khazaka Electronic) was used to assess the firmness and elasticity of healing tissue and non-wounded skin. Cutometer assessment is one of the most common instruments for measuring viscoelasticity in human patients (111, 112), which measures the vertical deformation of the skin surface by applying negative pressure (suction) through a small circular diameter (8 mm probe). A 2 s period of deformation (suction) followed by 2 s of relaxation (no suction) was applied three times and averaged. Stiffness was measured as the amplitude at the end of the suction phase (R0 metric) and normalized to the value of STSG treated with blank hydrogel (111). An increase in deformation to consistent negative pressure corresponds to tissue with less firmness, more similar to non-wounded skin.

[0141] h. Collection and cryosectioning of human and porcine tissue specimens Pig specimens were taken from the center of each wound at the end of the study. Human hypertrophic scar (HTS) and non-wounded skin samples were obtained under approved IRB (#54225). Tissue samples collected from the study were otherwise discarded. Patient identification information was not retained with the samples. The samples were immediately fixed in 4% paraformaldehyde, dehydrated, cryo-embedded in optimal cutting temperature (OCT) compound, and cryosectioned on a microtome cryostat.

[0142] i. Histological analysis of collagen architecture Masson's trichrome staining and picrosirius red staining were performed and images were taken with a Leica DM5000 B upright microscope. For picrosirius red, 40x magnification images were obtained using a polarized light microscope and analysis of fiber alignment was performed using CurveAlign (109) and MatFiber (98,110). The alignment intensity ranges from values ​​of 0 (completely random fiber alignment) to 1 (completely aligned fibers). Quantification of individual collagen fiber parameters (fiber length, width) was performed using CT-FIRE (http: / / loci.wisc.edu / software / ctfire) (109,113).

[0143] j. Immunofluorescence staining Immunofluorescence staining was performed using primary antibodies targeting CXCL10 (Thermo Fisher Scientific, PA5-46999), F4 / 80 (Thermo Fisher Scientific, MF48000), CXCL14 (Thermo Fisher Scientific, 10468-1-AP), thrombospondin 4 (THBS4) (Abcam, ab263898), apolipoprotein E (APOE) (Abcam, ab52607), CD34 (Abcam, ab81289), and alpha smooth muscle actin (aSMA) (Abcam, ab5694). The amount of fluorescent area was quantified and normalized to the number of cells (individual DAPI nuclei) using custom MATLAB image processing code written by the authors and published previously (98). All histology and immunofluorescence images shown are representative images of multiple experiments.

[0144] k. Single-cell barcoding, library preparation, and single-cell RNA sequencing The inventors have 2 100 pieces of tissue were obtained from the center of our STSG group or non-wounded skin. First, the tissue was cut into small 1 mm pieces using a scalpel. 2 The tissue was completely cut into pieces and then carefully minced with sharp scissors. To maximize cell capture for scRNA-seq, the tissue was very carefully cut to a precise tissue paste consistency. The dense fibrotic porcine extracellular matrix was completely degraded using increasing enzyme concentrations and a volume of 30 mL of Liberase (Sigma-Aldrich 5401127001) at 1 mg / mL in PBS. The cell digestion suspension was constantly stirred (rotated) for a total of 2 hours at 37°C. The tissue solution was subjected to a maximum speed vortex mixer (VWR) for 30 seconds, then placed in an oven for 1 hour, and subjected to the VWR again after a total of 2 hours to physically break up any tissue that had clumped together and maximize the tissue surface area exposed to the enzyme digestion at any given time.

[0145] The tissue solution was filtered through a 100 μm nylon cell filter (Fisher-Scientific 08-771-19) into a new conical tube, and 20 mL of DMEM with 10% FBS was added through the filter to quench the enzyme reaction, release any cells trapped in the filter, and maximize downstream cell yield. The solution was spun in a centrifuge at 350×g for 5 minutes at 4° C., the supernatant was aspirated, and the cells were then resuspended in 20 mL of DMEM with 10% FBS and passed through a 70 μm nylon cell filter. A 20 mL solution of 10% FBS in PBS (FACS buffer) was added through the filter to wash remaining cells.

[0146] The cell suspension was then resuspended in a concentration solution and subjected to droplet-based microfluidic single-cell RNA sequencing (scRNA-seq) at the Stanford Functional Genomics Facility (SFGF) using the 10x Chromium Single Cell platform (Single Cell 3' v3, 10x Genomics, USA). The cell suspension, reverse transcription master mix, and fractionated oil were loaded onto the single cell chip and processed in the Chromium Controller, and reverse transcription was performed at 53°C for 45 minutes. cDNA was amplified for a total of 12 cycles (BioRad C1000 Touch thermal cycler), and cDNA size selected using SpriSelect beads (Beckman Coulter, USA) and a 3:5 ratio of SpriSelect reagent volume to sample volume. cDNA was analyzed for quality control on an Agilent Bioanalyzer High Sensitivity DNA chip, fragmented for 5 min at 32°C, followed by end repair and A-tailing for 30 min at 65°C, and then double-sided size selected with SpriSelect. Sequencing adapters were ligated to the cDNA for 15 min at 20°C. cDNA was amplified using sample-specific index oligos as primers, followed by one more round of double-sided size selection. The final library was analyzed for quality control purposes on an Agilent Bioanalyzer High Sensitivity DNA chip. cDNA libraries were sequenced on a HiSeq 4000 Illumina platform with a target of 50,000 reads per cell.

[0147] l. Custom Pig Transcriptome We created a custom porcine transcriptome using the Sus Scrofa genome (Ensemble, v11.1), generated according to the 10X protocol for non-model organisms (44), which we have made publicly available with instructions for implementation as part of a modified 10X CellRanger pipeline (see Data Availability and Materials). Briefly, the porcine reference genome (Sus scrofa11.1, release 98) was obtained from the Ensembl web server (https: / / uswest.ensembl.org / Sus_scrofa / Info / Index). Fasta files for individual chromosomes were merged using batch concatenation in cshell. GTF annotations were filtered using the CellRanger (10X Genomics) "mkgtf" function using the following arguments: [Table 1]

[0148] These GTF and FASTQ files were then assembled using the CellRanger "mkref" command with default parameterization. All read fragments were aligned to the transcriptome using a standard base matching threshold (51) followed by cellular demultiplexing and UMI batch correction (52), allowing us to generate a single-cell mRNA count matrix for each pig sample.

[0149] m. Data processing, FASTQ generation, and read mapping Base calls were converted to reads using the Cell Ranger (10X Genomics; version 3.1) implementation of mkfastq and then aligned against our custom pig transcriptome using Cell Ranger's count function and SC3Pv3 chemistry with 5,000 expected cells per sample (51). Cell barcodes were filtered for high quality based on optimized thresholds of having at least 200 profiled unique transcripts, less than 10,000 total transcripts, and less than 10% of their transcriptome of mitochondrial origin (114).

[0150] n. Data normalization and generation of distinctive subpopulation markers Unique molecular identifiers (UMIs) from each cell barcode were retained for all downstream analyses. Raw UMI counts were normalized with a scale factor of 10,000 UMIs per cell and then natural log-transformed with a pseudocount of 1 using the R package Seurat (version 3.1.1) (52). Highly variable genes were identified and cells were scaled by regression to the fraction of mitochondrial transcripts. The aggregated data were then assessed across the first 15 principal components using uniform manifold approximation and projection (UMAP) analysis (115). Automated cell annotations were imputed to the ENCODE Blue database (116) using the SingleR toolkit (version 3.11). Because we used human annotations, cell types were confirmed by also examining cell-specific markers in our dataset. Differentially expressed genes were identified using a ROC test with Seurat's native FindMarkers function and a log fold change threshold of 0.5 to assign a predictive power to each gene.

[0151] o. Overrepresentation analysis using Genetrail3 Overrepresentation analysis (ORA) was performed for each cell using the 500 most expressed protein-coding genes and the gene sets Gene Ontology: Biological Processes (GO-BP) and WikiPathways (WP) using Genetrail 3 (53). P values ​​were adjusted using the Benjamini-Hochberg procedure, and gene sets were required to have between 2 and 1000 genes.

[0152] RNA kinetic analysis using p.scVelo RNA kinetics analysis was performed using the dynamic models in the scVelo package ( 80 ). Partition-based graph abstraction (PAGA) was performed using the sc.tl.paga function in scVelo.

[0153] To find genes with differentially regulated transcriptional dynamics compared to all other clusters, a Welch's t-test with overestimated variance as conservative was applied using the sc.tl.rank_velocity_genes function. Genes were ranked by their likelihood obtained from the dynamic model grouped by treatment.

[0154] q. CellRank Using CellRank, the dynamic process of fibroblast differentiation was revealed based on Markov state modeling of single-cell data. Initial and terminal cell states, as well as fibroblast fate probabilities, were calculated in CellRank based on dynamic RNA kinetic information provided by scVelo.

[0155] r. Fibroblast-assembled 3D collagen scaffold experiment We isolated and cultured dermal fibroblasts from both healthy human and porcine skin samples. Fibroblasts were isolated by mechanical and enzymatic digestion and cultured under standard conditions up to passage 3. Following our previously published protocol (98), we used primary fibroblasts to fabricate fibroblast-populated collagen hydrogels at a concentration of 200k cells / mL and 2mg / mL collagen (PureCol, Advanced Biomatrix 5005). Briefly, collagen scaffolds were fabricated in a cross shape with sponges in the arms and cultured on top of a layer (~0.5cm) of cured polydimethylsiloxane (PDMS; Sylgard 184 Silicone Elastomer Kit; Dow Corning) in a Petri dish. Pins were pushed through the sponges in the hydrogel cross arms to hold down the scaffolds during the initial preculture period (24h). We then subjected the scaffolds to either 0% strain (no strain) or 10% equibiaxial strain (either with or without FAKI) for an additional 48 hours. Untreated cells subjected to strain received 10 μL of DMSO in 20 mL of culture medium, and treated cells subjected to strain received 10 μL of 20 mM FAKI in DMSO in 20 mL of culture medium to achieve a final concentration of 10 μM FAKI.

[0156] We used sutures to paint nine titanium(IV) oxide dots (Sigma-Aldrich 248576) onto the surface of the central region of the gel to track and quantify the applied strain. We used a digital camera to image the markers before and after strain. Strain was applied by removing the pins, manually stretching the hydrogel cross arms, and pushing the pins through the arms again to hold the scaffold in the new stretched position. Photographs of the marker positions were used to calculate a single homogeneous deformation gradient tensor F, which provided a least-squares best-fit mapping of the nine marker positions from undeformed to deformed positions by solving the following overdetermined matrix equation: x=FX+p [1], where p is an optional vector that is included to account for translation between images. We converted the deformations into a strain tensor E using:

number

[0157] II. References: 1. Z. Zhu, J. Ding, EETredget, The molecular basis of hypertrophic scars. Burns Trauma 4,2 (2016). 2. RKVSethi, ED Kozin, PJ Fagenholz, DJ Lee, MGShrime, STGray, Epidemiological Survey of Head and Neck Injuries and Trauma in the United States. Otolaryngology-Head and Neck Surgery 151, 776-784 (2014). 3.TAWynn,TRRamalingam,Mechanisms of fibrosis:therapeutic translation for fibrotic disease.Nat Med 18,1028-1040(2012). 4.B.Berman,M.H.Viera,S.Amini,R.Huo,I.S.Jones,Prevention and Management of Hypertrophic Scars and Keloids After Burns in Children.Journal of Craniofacial Surgery 19,989-1006(2008). 5.C.Mock,M.Peck,M.Peden,E.Krug,W.H.Organization,A WHO plan for burn prevention and care.Geneva:World Health Organization 3,(2008). 6.L.Deeter,M.Seaton,G.J.Carrougher,K.McMullen,S.P.Mandell,D.Amtmann,N.S.Gibran,Hospital-acquired complications alter quality of life in adult burn survivors:report from a burn model system.Burns :journal of the International Society for Burn Injuries 45,42-47(2019). 7.A.W.Chua,Y.C.Khoo,B.K.Tan,K.C.Tan,C.L.Foo,S.J.Chong,Skin tissue engineering advances in severe burns:review and therapeutic applications.Burns Trauma 4,3(2016). 8.L.F.Rose,J.C.Wu,A.H.Carlsson,D.I.Tucker,K.P.Leung,R.K.Chan,Recipient wound bed characteristics affect scarring and skin graft contraction.Wound repair and regeneration :official publication of the Wound Healing Society[and]the European Tissue Repair Society 23,287-296(2015). 9.J.D.Villapalos,P.vol.2019. 10.R.Kandiyali,H.Thom,A.E.Young,R.Greenwood,N.J.Welton,Cost-effectiveness and value of information analysis of a low-friction environment following skin graft in patients with burn injury.Pilot Feasibility Stud 6,8(2020). 11.M.Singh,K.Nuutila,K.C.Collins,A.Huang,Evolution of skin grafting for treatment of burns:Reverdin pinch grafting to Tanner mesh grafting and beyond.Burns :journal of the International Society for Burn Injuries 43,1149-1154(2017). 12.T.Nuri,K.Ueda,Y.Fujimori,Ten-year Follow-up After Treating Extended Burn Scar Contracture with an Autologous Cultured Dermal Substitute.Plast Reconstr Surg Glob Open 6,e1782(2018). 13.K.Meier,L.B.Nanney,Emerging new drugs for scar reduction.Expert Opin Emerg Drugs 11,39-47(2006). 14.L.Block,A.Gosain,T.W.King,Emerging Therapies for Scar Prevention.Adv Wound Care(New Rochelle)4,607-614(2015). 15.D.Duscher,Z.N.Maan,V.W.Wong,R.C.Rennert,M.Januszyk,M.Rodrigues,M.Hu,A.J.Whitmore,A.J.Whittam,M.T.Longaker,G.C.Gurtner,Mechanotransduction and fibrosis.J Biomech 47,1997-2005(2014). 16.R.S.Gieni,M.J.Hendzel,Mechanotransduction from the ECM to the genome:are the pieces now in place? J Cell Biochem 104,1964-1987(2008). 17.N.Wang,J.D.Tytell,D.E.Ingber,Mechanotransduction at a distance:mechanically coupling the extracellular matrix with the nucleus.Nat Rev Mol Cell Biol 10,75-82(2009). 18.G.C.Gurtner,S.Werner,Y.Barrandon,M.T.Longaker,Wound repair and regeneration.Nature 453,314-321(2008). 19.S.Aarabi,K.A.Bhatt,Y.Shi,J.Paterno,E.I.Chang,S.A.Loh,J.W.Holmes,M.T.Longaker,H.Yee,G.C.Gurtner,Mechanical load initiates hypertrophic scar formation through decreased cellular apoptosis.FASEB J 21,3250-3261(2007). 20.A.Zehender,J.Huang,A.H.Gyorfi,A.E.Matei,T.Trinh-Minh,X.Xu,Y.N.Li,C.W.Chen,J.Lin,C.Dees,C.Beyer,K.Gelse,Z.Y.Zhang,C.Bergmann,A.Ramming,W.Birchmeier,O.Distler,G.Schett,J.H.W.Distler,The tyrosine phosphatase SHP2 controls TGFbeta-induced STAT3 signaling to regulate fibroblast activation and fibrosis.Nat Commun 9,3259(2018). 21.D.S.Foster,C.D.Marshall,G.S.Gulati,M.S.Chinta,A.Nguyen,A.Salhotra,R.E.Jones,A.Burcham,T.Lerbs,L.Cui,M.E.King,A.L.Titan,R.C.Ransom,A.Manjunath,M.S.Hu,C.P.Blackshear,S.Mascharak,A.L.Moore,J.A.Norton,C.J.Kin,A.A.Shelton,M.Januszyk,G.C.Gurtner,G.Wernig,M.T.Longaker,Elucidating the fundamental fibrotic processes driving abdominal adhesion formation.Nat Commun 11,4061(2020). 22.V.W.Wong,K.C.Rustad,S.Akaishi,M.Sorkin,J.P.Glotzbach,M.Januszyk,E.R.Nelson,K.Levi,J.Paterno,I.N.Vial,A.A.Kuang,M.T.Longaker,G.C.Gurtner,Focal adhesion kinase links mechanical force to skin fibrosis via inflammatory signaling.Nat Med 18,148-152(2011). 23.K.Ma,S.H.Kwon,J.Padmanabhan,D.Duscher,A.A.Trotsyuk,Y.Dong,M.Inayathullah,J.Rajadas,G.C.Gurtner,Controlled Delivery of a Focal Adhesion Kinase Inhibitor Results in Accelerated Wound Closure with Decreased Scar Formation.J Invest Dermatol 138,2452-2460(2018). 24.Q.Ding,C.L.Gladson,H.Wu,H.Hayasaka,M.A.Olman,Focal adhesion kinase(FAK)-related non-kinase inhibits myofibroblast differentiation through differential MAPK activation in a FAK-dependent manner.J Biol Chem 283,26839-26849(2008). 25.S.Mascharak,H.E.desJardins-Park,M.F.Davitt,M.Griffin,M.R.Borrelli,A.L.Moore,K.Chen,B.Duoto,M.Chinta,D.S.Foster,A.H.Shen,M.Januszyk,S.H.Kwon,G.Wernig,D.C.Wan,H.P.Lorenz,G.C.Gurtner,M.T.Longaker,Preventing Engrailed-1 activation in fibroblasts yields wound regeneration without scarring.Science 372,(2021). 26.V.W.Wong,K.C.Rustad,S.Akaishi,M.Sorkin,J.P.Glotzbach,M.Januszyk,E.R.Nelson,K.Levi,J.Paterno,I.N.Vial,A.A.Kuang,M.T.Longaker,G.C.Gurtner,Focal adhesion kinase links mechanical force to skin fibrosis via inflammatory signaling.Nature medicine 18,148-152(2011). 27.K.Chen,SHKwon,D.Henn,BAKuehlmann,R.Tevlin,CABonham,M.Griffin,AATrotsyuk,MRBorrelli,C.Noishiki,J.Padmanabhan,JABarrera,ZNMaan,T.Dohi,CJMay s,AHGreco,D.Sivaraj,JQLin,T.Fehlmann,AMMermin-Bunnell,S.Mittal,MShu,AIZamaleeva,A.Keller,J.Rajadas,MTLongaker,M.Januszyk,GCGurtner,Disrupting Biological sensors of force promote tissue regeneration in large organisms.Nat Commun 12,5256(2021). 28.RDGaliano,JtMichaels,M.Dobryansky,JPLevine,GCGurtner,A quantitative and reproducible murine model of excisional wound healing. J Anat 233,275-288(2018) The panniculus carnosus muscle: an evolutionary enigma at the intersection of distinct research fields. 30.C.P.Denton,P.A.Merkel,D.E.Furst,D.Khanna,P.Emery,V.M.Hsu,N.Silliman,J.Streisand,J.Powell,A.Akesson,J.Coppock,F.Hoogen,A.Herrick,M.D.Mayes,D.Veale,J.Haas,S.Ledbetter,J.H.Korn,C.M.Black,J.R.Seibold,Recombinant human anti-transforming growth factor beta1 antibody therapy in systemic sclerosis:a multicenter,randomized,placebo-controlled phase I / II trial of CAT-192.Arthritis and rheumatism 56,323-333(2007). 31.D.M.DeBruler,B.N.Blackstone,K.L.McFarland,M.E.Baumann,D.M.Supp,J.K.Bailey,H.M.Powell,Effect of skin graft thickness on scar development in a porcine burn model.Burns :journal of the International Society for Burn Injuries 44,917-930(2018). 32.C.F.Guerrero-Juarez,P.H.Dedhia,S.Jin,R.Ruiz-Vega,D.Ma,Y.Liu,K.Yamaga,O.Shestova,D.L.Gay,Z.Yang,K.Kessenbrock,Q.Nie,W.S.Pear,G.Cotsarelis,M.V.Plikus,Single-cell analysis reveals fibroblast heterogeneity and myeloid-derived adipocyte progenitors in murine skin wounds.Nature Communications 10,650(2019). 33. MVPlikus, CF Guerrero-Juarez, M. Ito, YRLi, PHDedhia, Y. Zheng, M. Shao, DLGay, R. Ramos, T.-C. Hsi, JWO h,X.Wang,A.Ramirez,SEKonopelski,A.Elzein,A.Wang,RJSuppositionchart,H.-L.Lee,CHLim,A.Nace,A.Gu o,E.Treffeisen,T.Andl,RNRamirez,R.Murad,S.Offermanns,D.Metzger,P.Chambon,ADWidgerow,T-L.Tuan,A.Mortazavi,RKGupta,BAHamilton,SEMillar,P.Seale,WSPear,MALazar,G.Cotsarelis,Regeneration of fat cells from myofibroblasts during wound healing.Science 355,748-752(2017). 34.A.Butler,P.Hoffman,P.Smibert,E.Papalexi,R.Satija,Integrating single-cell transcriptomic data across different conditions,technologies,and species.Nat Biotechnol 36,411-420(2018). Nat Commun 10,500(2019) 35. P. Ramos-Ibeas, F. Sang, Q. Zhu, WWCTang, S. Withey, D. Klisch, L. Wood, M. Loose, M. Surani, R. Alberio. 36.M.S.B.Raredon,T.S.Adams,Y.Suhail,J.C.Schupp,S.Poli,N.Neumark,K.L.Leiby,A.M.Greaney,Y.Yuan,C.Horien,G.Linderman,A.J.Engler,D.J.Boffa,Y.Kluger,I.O.Rosas,A.Levchenko,N.Kaminski,L.E.Niklason,Single-cell connectomic analysis of adult mammalian lungs.Sci Adv 5,eaaw3851(2019). 37.M.Januszyk,V.W.Wong,K.A.Bhatt,I.N.Vial,J.Paterno,M.T.Longaker,G.C.Gurtner,Mechanical offloading of incisional wounds is associated with transcriptional downregulation of inflammatory pathways in a large animal model.Organogenesis 10,186-193(2014). 38.G.C.Gurtner,R.H.Dauskardt,V.W.Wong,K.A.Bhatt,K.Wu,I.N.Vial,K.Padois,J.M.Korman,M.T.Longaker,Improving cutaneous scar formation by controlling the mechanical environment:large animal and phase I studies.Annals of surgery 254,217-225(2011). 39.J.L.Hunt,R.Sato,C.R.Baxter,Early tangential excision and immediate mesh autografting of deep dermal hand burns.Annals of surgery 189,147-151(1979). 40.D.N.Herndon,R.E.Barrow,R.L.Rutan,T.C.Rutan,M.H.Desai,S.Abston,A comparison of conservative versus early excision.Therapies in severely burned patients.Annals of surgery 209,547-552;discussion 552-543(1989). 41.D.C.Adams,M.L.Ramsey,Grafts in dermatologic surgery:review and update on full-and split-thickness skin grafts,free cartilage grafts,and composite grafts.Dermatol Surg 31,1055-1067(2005). 42.Y.Bian,C.Sun,X.Zhang,Y.Li,W.Li,X.Lv,J.Li,L.Jiang,J.Li,J.Feng,X.Y.Li,Wound-healing improvement by resurfacing split-thickness skin donor sites with thin split-thickness grafting.Burns :journal of the International Society for Burn Injuries 42,123-130(2016). 43.G.Y.Hur,D.K.Seo,J.W.Lee,Contracture of skin graft in human burns:effect of artificial dermis.Burns :journal of the International Society for Burn Injuries 40,1497-1503(2014). 44.M.Januszyk,K.Chen,D.Henn,D.S.Foster,M.R.Borrelli,C.A.Bonham,D.Sivaraj,D.Wagh,M.T.Longaker,D.C.Wan,G.C.Gurtner,Characterization of Diabetic and Non-Diabetic Foot Ulcers Using Single-Cell RNA-Sequencing.Micromachines(Basel)11,(2020). 45.A.Diaz-Papkovich,L.Anderson-Trocme,C.Ben-Eghan,S.Gravel,UMAP reveals cryptic population structure and phenotype heterogeneity in large genomic cohorts.PLoS Genet 15,e1008432(2019). 46.A.M.Ascension,S.Fuertes-Alvarez,O.Ibanez-Sole,A.Izeta,M.J.Arauzo-Bravo,Human Dermal Fibroblast Subpopulations Are Conserved across Single-Cell RNA Sequencing Studies.The Journal of investigative dermatology 141,1735-1744.e1735(2021). 47.V.Vorstandlechner,M.Laggner,P.Kalinina,W.Haslik,C.Radtke,L.Shaw,B.M.Lichtenberger,E.Tschachler,H.J.Ankersmit,M.Mildner,Deciphering the functional heterogeneity of skin fibroblasts using single-cell RNA sequencing.FASEB journal :official publication of the Federation of American Societies for Experimental Biology 34,3677-3692(2020). 48.H.He,H.Suryawanshi,P.Morozov,J.Gay-Mimbrera,E.Del Duca,H.J.Kim,N.Kameyama,Y.Estrada,E.Der,J.G.Krueger,J.Ruano,T.Tuschl,E.Guttman-Yassky,Single-cell transcriptome analysis of human skin identifies novel fibroblast subpopulation and enrichment of immune subsets in atopic dermatitis.J Allergy Clin Immunol 145,1615-1628(2020). 49.C.C.Deng,Y.F.Hu,D.H.Zhu,Q.Cheng,J.J.Gu,Q.L.Feng,L.X.Zhang,Y.P.Xu,D.Wang,Z.Rong,B.Yang,Single-cell RNA-seq reveals fibroblast heterogeneity and increased mesenchymal fibroblasts in human fibrotic skin diseases.Nat Commun 12,3709(2021). 50.D.R.Zerbino,P.Achuthan,W.Akanni,M.R.Amode,D.Barrell,J.Bhai,K.Billis,C.Cummins,A.Gall,C.G.Giron,Ensembl 2018.Nucleic acids research 46,D754-D761(2018). 51.A.Dobin,C.A.Davis,F.Schlesinger,J.Drenkow,C.Zaleski,S.Jha,P.Batut,M.Chaisson,T.R.Gingeras,STAR:ultrafast universal RNA-seq aligner.Bioinformatics 29,15-21(2013). 52.T.Stuart,A.Butler,P.Hoffman,C.Hafemeister,E.Papalexi,W.M.Mauck III,Y.Hao,M.Stoeckius,P.Smibert,R.Satija,Comprehensive Integration of Single-Cell Data.Cell,(2019). 53.N.Gerstner,T.Kehl,K.Lenhof,A.Muller,C.Mayer,L.Eckhart,N.L.Grammes,C.Diener,M.Hart,O.Hahn,J.Walter,T.Wyss-Coray,E.Meese,A.Keller,H.P.Lenhof,GeneTrail 3:advanced high-throughput enrichment analysis.Nucleic Acids Res 48,W515-w520(2020). 54.R.C.Russo,C.C.Garcia,M.M.Teixeira,F.A.Amaral,The CXCL8 / IL-8 chemokine family and its receptors in inflammatory diseases.Expert Rev Clin Immunol 10,593-619(2014). 55.N.Oikonomou,V.Harokopos,J.Zalevsky,C.Valavanis,A.Kotanidou,D.E.Szymkowski,G.Kollias,V.Aidinis,Soluble TNF mediates the transition from pulmonary inflammation to fibrosis.PLoS One 1,e108(2006). 56.J.J.Saucerman,P.M.Tan,K.S.Buchholz,A.D.McCulloch,J.H.Omens,Mechanical regulation of gene expression in cardiac myocytes and fibroblasts.Nature Reviews Cardiology 16,361-378(2019). 57.D.J.Tschumperlin,G.Ligresti,M.B.Hilscher,V.H.Shah,Mechanosensing and fibrosis.J Clin Invest 128,74-84(2018). 58.S.Adams,L.M.Wuescher,R.Worth,E.Yildirim-Ayan,Mechano-Immunomodulation:Mechanoresponsive Changes in Macrophage Activity and Polarization.Ann Biomed Eng 47,2213-2231(2019). 59.B.Aldeiri,U.Roostalu,A.Albertini,J.Wong,A.Morabito,G.Cossu,Transgelin-expressing myofibroblasts orchestrate ventral midline closure through TGFβ signalling.Development 144,3336-3348(2017). 60.J.Park,J.Park,J.Jeong,K.H.Cho,I.Choi,J.Kim,Identification of tetranectin as adipogenic serum protein.Biochem Biophys Res Commun 460,583-588(2015). 61.T.Satoh,K.Nakagawa,F.Sugihara,R.Kuwahara,M.Ashihara,F.Yamane,Y.Minowa,K.Fukushima,I.Ebina,Y.Yoshioka,A.Kumanogoh,S.Akira,Identification of an atypical monocyte and committed progenitor involved in fibrosis.Nature 541,96-101(2017). 62.P.M.Tang,D.J.Nikolic-Paterson,H.Y.Lan,Macrophages:versatile players in renal inflammation and fibrosis.Nat Rev Nephrol 15,144-158(2019). 63.X.M.Meng,D.J.Nikolic-Paterson,H.Y.Lan,Inflammatory processes in renal fibrosis.Nat Rev Nephrol 10,493-503(2014). 64.D.Henn,K.Chen,T.Fehlmann,A.A.Trotsyuk,D.Sivaraj,Z.N.Maan,C.A.Bonham,Jr.,J.A.Barrera,C.J.Mays,A.H.Greco,S.E.Moortgat Illouz,J.Q.Lin,S.R.Steele,D.S.Foster,J.Padmanabhan,A.Momeni,D.Nguyen,D.C.Wan,U.Kneser,M.Januszyk,A.Keller,M.T.Longaker,G.C.Gurtner,Xenogeneic skin transplantation promotes angiogenesis and tissue regeneration through activated Trem2(+) macrophages.Sci Adv 7,eabi4528(2021). 65.V.M.Golubovskaya,Targeting FAK in human cancer:from finding to first clinical trials.Front Biosci(Landmark Ed)19,687-706(2014). 66.F.J.Sulzmaier,C.Jean,D.D.Schlaepfer,FAK in cancer:mechanistic findings and clinical applications.Nat Rev Cancer 14,598-610(2014). 67.W.G.Roberts,E.Ung,P.Whalen,B.Cooper,C.Hulford,C.Autry,D.Richter,E.Emerson,J.Lin,J.Kath,K.Coleman,L.Yao,L.Martinez-Alsina,M.Lorenzen,M.Berliner,M.Luzzio,N.Patel,E.Schmitt,S.LaGreca,J.Jani,M.Wessel,E.Marr,M.Griffor,F.Vajdos,Antitumor activity and pharmacology of a selective focal adhesion kinase inhibitor,PF-562,271.Cancer Res 68,1935-1944(2008). 68.R.Fearmonti,J.Bond,D.Erdmann,H.Levinson,A review of scar scales and scar measuring devices.Eplasty 10,e43(2010). 69.P.Ortiz-Montero,A.Londono-Vallejo,J.P.Vernot,Senescence-associated IL-6 and IL-8 cytokines induce a self-and cross-reinforced senescence / inflammatory milieu strengthening tumorigenic capabilities in the MCF-7 breast cancer cell line.Cell Commun Signal 15,17(2017). 70.C.A.Feghali,T.M.Wright,Cytokines in acute and chronic inflammation.Front Biosci 2,d12-26(1997). 71.A.M.Tager,R.L.Kradin,P.LaCamera,S.D.Bercury,G.S.Campanella,C.P.Leary,V.Polosukhin,L.H.Zhao,H.Sakamoto,T.S.Blackwell,A.D.Luster,Inhibition of pulmonary fibrosis by the chemokine IP-10 / CXCL10.Am J Respir Cell Mol Biol 31,395-404(2004). 72.S.D.Oldroyd,G.L.Thomas,G.Gabbiani,A.M.El Nahas,Interferon-gamma inhibits experimental renal fibrosis.Kidney Int 56,2116-2127(1999). 73.G.Raghu,K.K.Brown,W.Z.Bradford,K.Starko,P.W.Noble,D.A.Schwartz,T.E.King,Jr.,A placebo-controlled trial of interferon gamma-1b in patients with idiopathic pulmonary fibrosis.N Engl J Med 350,125-133(2004). 74.T.Poynard,J.McHutchison,M.Manns,C.Trepo,K.Lindsay,Z.Goodman,M.H.Ling,J.Albrecht,Impact of pegylated interferon alfa-2b and ribavirin on liver fibrosis in patients with chronic hepatitis C.Gastroenterology 122,1303-1313(2002). 75.B.A.Croker,D.L.Krebs,J.G.Zhang,S.Wormald,T.A.Willson,E.G.Stanley,L.Robb,C.J.Greenhalgh,I.Forster,B.E.Clausen,N.A.Nicola,D.Metcalf,D.J.Hilton,A.W.Roberts,W.S.Alexander,SOCS3 negatively regulates IL-6 signaling in vivo.Nat Immunol 4,540-545(2003). 76.J.Wu,A.H.Lewis,J.Grandl,Touch,Tension,and Transduction-The Function and Regulation of Piezo Ion Channels.Trends Biochem Sci 42,57-71(2017). 77.J.Padmanabhan,M.J.Augelli,B.Cheung,E.R.Kinser,B.Cleary,P.Kumar,R.Wang,A.J.Sawyer,R.Li,U.D.Schwarz,J.Schroers,T.R.Kyriakides,Regulation of cell-cell fusion by nanotopography.Sci Rep 6,33277(2016). 78.K.Chen,D.Henn,D.Sivaraj,C.A.Bonham,M.Griffin,H.Choi Kussie,J.Padmanabhan,A.A.Trotsyuk,D.C.Wan,M.Januszyk,M.T.Longaker,G.C.Gurtner,Mechanical Strain Drives Myeloid Cell Differentiation Toward Pro-Inflammatory Subpopulations.Advances in wound care,(2021). 79.R.Agha,R.Ogawa,G.Pietramaggiori,D.P.Orgill,A review of the role of mechanical forces in cutaneous wound healing.J Surg Res 171,700-708(2011). 80.V.Bergen,M.Lange,S.Peidli,F.A.Wolf,F.J.Theis,Generalizing RNA velocity to transient cell states through dynamical modeling.Nat Biotechnol,(2020). 81.M.Lange,V.Bergen,M.Klein,M.Setty,B.Reuter,M.Bakhti,H.Lickert,M.Ansari,J.Schniering,H.B.Schiller,D.Pe’er,F.J.Theis,CellRank for directed single-cell fate mapping.bioRxiv,2020.2010.2019.345983(2020). 82.A.Jeschke,M.Bonitz,M.Simon,S.Peters,W.Baum,G.Schett,W.Ruether,A.Niemeier,T.Schinke,M.Amling,Deficiency of Thrombospondin-4 in Mice Does Not Affect Skeletal Growth or Bone Mass Acquisition,but Causes a Transient Reduction of Articular Cartilage Thickness.PLoS One 10,e0144272(2015). 83.S.A.Wong,D.P.Hu,J.Slocum,C.Lam,M.Nguyen,T.Miclau,R.S.Marcucio,C.S.Bahney,Chondrocyte-to-osteoblast transformation in mandibular fracture repair.J Orthop Res,(2020). 84.P.Smeriglio,F.C.Grandi,S.E.B.Taylor,A.Zalc,N.Bhutani,TET1 Directs Chondrogenic Differentiation by Regulating SOX9 Dependent Activation of Col2a1 and Acan In Vitro.JBMR Plus 4,e10383(2020). 85.B.Sid,H.Sartelet,G.Bellon,H.El Btaouri,G.Rath,N.Delorme,B.Haye,L.Martiny,Thrombospondin 1:a multifunctional protein implicated in the regulation of tumor growth.Crit Rev Oncol Hematol 49,245-258(2004). 86.M.Mastri,Z.Shah,K.Hsieh,X.Wang,B.Wooldridge,S.Martin,G.Suzuki,T.Lee,Secreted Frizzled-related protein 2 as a target in antifibrotic therapeutic intervention.Am J Physiol Cell Physiol 306,C531-539(2014). 87.D.Honardoust,M.Varkey,K.Hori,J.Ding,H.A.Shankowsky,E.E.Tredget,Small leucine-rich proteoglycans,decorin and fibromodulin,are reduced in postburn hypertrophic scar.Wound repair and regeneration :official publication of the Wound Healing Society [and] the European Tissue Repair Society 19,368-378(2011). 88.R.A.Ignotz,J.Massague,Type beta transforming growth factor controls the adipogenic differentiation of 3T3 fibroblasts.Proceedings of the National Academy of Sciences of the United States of America 82,8530-8534(1985). 89.M.Augsten,C.Hagglof,E.Olsson,C.Stolz,P.Tsagozis,T.Levchenko,M.J.Frederick,A.Borg,P.Micke,L.Egevad,A.Ostman,CXCL14 is an autocrine growth factor for fibroblasts and acts as a multi-modal stimulator of prostate tumor growth.Proceedings of the National Academy of Sciences of the United States of America 106,3414-3419(2009). 90.R.J.Waldemer-Streyer,A.Reyes-Ordonez,D.Kim,R.Zhang,N.Singh,J.Chen,Cxcl14 depletion accelerates skeletal myogenesis by promoting cell cycle withdrawal.NPJ Regen Med 2,16017-(2017). 91.N.J.Song,S.Kim,B.H.Jang,S.H.Chang,U.J.Yun,K.M.Park,H.Waki,D.Y.Li,P.Tontonoz,K.W.Park,Small Molecule-Induced Complement Factor D(Adipsin)Promotes Lipid Accumulation and Adipocyte Differentiation.PLoS One 11,e0162228(2016). 92.Z.H.Huang,C.A.Reardon,T.Mazzone,Endogenous ApoE expression modulates adipocyte triglyceride content and turnover.Diabetes 55,3394-3402(2006). 93.R.W.Mahley,T.L.Innerarity,S.C.Rall,Jr.,K.H.Weisgraber,Plasma lipoproteins:apolipoprotein structure and function.J Lipid Res 25,1277-1294(1984). 94.B.M.Varisco,N.Ambalavanan,J.A.Whitsett,J.S.Hagood,Thy-1 signals through PPARγ to promote lipofibroblast differentiation in the developing lung.Am J Respir Cell Mol Biol 46,765-772(2012). 95.W.Qian,N.Li,Q.Cao,J.Fan,Thrombospondin-4 critically controls transforming growth factor β1 induced hypertrophic scar formation.J Cell Physiol 234,731-739(2018). 96.V.W.Wong,K.Levi,S.Akaishi,G.Schultz,R.H.Dauskardt,Scar zones:region-specific differences in skin tension may determine incisional scar formation.Plastic and reconstructive surgery 129,1272-1276(2012). 97.R.Maiti,L.-C.Gerhardt,Z.S.Lee,R.A.Byers,D.Woods,J.A.Sanz-Herrera,S.E.Franklin,R.Lewis,S.J.Matcher,M.J.Carre,In vivo measurement of skin surface strain and sub-surface layer deformation induced by natural tissue stretching.Journal of the Mechanical Behavior of Biomedical Materials 62,556-569(2016). 98.K.Chen,A.Vigliotti,M.Bacca,R.M.McMeeking,V.S.Deshpande,J.W.Holmes,Role of boundary conditions in determining cell alignment in response to stretch.Proceedings of the National Academy of Sciences of the United States of America 115,986-991(2018). 99.J.Crawford,K.Nygard,B.S.Gan,D.B.O’Gorman,Periostin induces fibroblast proliferation and myofibroblast persistence in hypertrophic scarring.Exp Dermatol 24,120-126(2015). 100.D.Sivaraj,K.Chen,A.Chattopadhyay,D.Henn,W.Wu,C.Noishiki,N.J.Magbual,S.Mittal,A.M.Mermin-Bunnell,C.A.Bonham,A.A.Trotsyuk,J.A.Barrera,J.Padmanabhan,M.Januszyk,G.C.Gurtner,Hydrogel Scaffolds to Deliver Cell Therapies for Wound Healing.Front Bioeng Biotechnol 9,660145(2021). 101.T.Xie,Y.Wang,N.Deng,G.Huang,F.Taghavifar,Y.Geng,N.Liu,V.Kulur,C.Yao,P.Chen,Z.Liu,B.Stripp,J.Tang,J.Liang,P.W.Noble,D.Jiang,Single-Cell Deconvolution of Fibroblast Heterogeneity in Mouse Pulmonary Fibrosis.Cell Rep 22,3625-3640(2018). 102.S.Mahmoudi,E.Mancini,L.Xu,A.Moore,F.Jahanbani,K.Hebestreit,R.Srinivasan,X.Li,K.Devarajan,L.Prelot,C.E.Ang,Y.Shibuya,B.A.Benayoun,A.L.S.Chang,M.Wernig,J.Wysocka,M.T.Longaker,M.P.Snyder,A.Brunet,Heterogeneity in old fibroblasts is linked to variability in reprogramming and wound healing.Nature 574,553-558(2019). 103.K.Sudo,M.Kanno,K.Miharada,S.Ogawa,T.Hiroyama,K.Saijo,Y.Nakamura,Mesenchymal progenitors able to differentiate into osteogenic,chondrogenic,and / or adipogenic cells in vitro are present in most primary fibroblast-like cell populations.Stem Cells 25,1610-1617(2007). 104.R.McBeath,D.M.Pirone,C.M.Nelson,K.Bhadriraju,C.S.Chen,Cell shape,cytoskeletal tension,and RhoA regulate stem cell lineage commitment.Dev Cell 6,483-495(2004). 105.S.Y.Jeong,D.H.Kim,J.Ha,H.J.Jin,S.J.Kwon,J.W.Chang,S.J.Choi,W.Oh,Y.S.Yang,G.Kim,J.S.Kim,J.R.Yoon,D.H.Cho,H.B.Jeon,Thrombospondin-2 secreted by human umbilical cord blood-derived mesenchymal stem cells promotes chondrogenic differentiation.Stem Cells 31,2136-2148(2013). 106.H.S.Shitaye,S.P.Terkhorn,J.A.Combs,K.D.Hankenson,Thrombospondin-2 is an endogenous adipocyte inhibitor.Matrix Biol 29,549-556(2010). 107.P.Martin,Wound healing--aiming for perfect skin regeneration.Science 276,75-81(1997). 108.R.Edgar,M.Domrachev,A.E.Lash,Gene Expression Omnibus:NCBI gene expression and hybridization array data repository.Nucleic Acids Res 30,207-210(2002). 109.J.S.Bredfeldt,Y.Liu,C.A.Pehlke,M.W.Conklin,J.M.Szulczewski,D.R.Inman,P.J.Keely,R.D.Nowak,T.R.Mackie,K.W.Eliceiri,Computational segmentation of collagen fibers from second-harmonic generation images of breast cancer.J Biomed Opt 19,16007-16007(2014). 110.G.M.Fomovsky,J.W.Holmes,Evolution of scar structure,mechanics,and ventricular function after myocardial infarction in the rat.American journal of physiology.Heart and circulatory physiology 298,H221-228(2010). 111.H.S.Ryu,Y.H.Joo,S.O.Kim,K.C.Park,S.W.Youn,Influence of age and regional differences on skin elasticity as measured by the Cutometer.Skin research and technology :official journal of International Society for Bioengineering and the Skin(ISBS)[and]International Society for Digital Imaging of Skin(ISDIS)[and]International Society for Skin Imaging(ISSI)14,354-358(2008). 112.S.S.Fong,L.K.Hung,J.C.Cheng,The cutometer and ultrasonography in the assessment of postburn hypertrophic scar--a preliminary study.Burns :journal of the International Society for Burn Injuries 23 Suppl 1,S12-18(1997). 113.Y.Liu,A.Keikhosravi,G.S.Mehta,C.R.Drifka,K.W.Eliceiri,Methods for Quantifying Fibrillar Collagen Alignment.Methods Mol Biol 1627,429-451(2017). 114.D.Osorio,J.J.Cai,Systematic determination of the mitochondrial proportion in human and mice tissues for single-cell RNA sequencing data quality control.Bioinformatics,(2020). 115. E. Becht, L. McInnes, J. Healy, C.-A. Dutertre, IWKwok, LGNg, F. Ginhoux, EW Newell, Dimensionality reduction for visualizing single-cell data using UMAP.Nature biotechnology 37,38(2019). 116.RKAuerbach, B.Chen, AJButte,Relating genes to function:identifying enriched transcription factors using the ENCODE ChIP-Seq significance tool.Bioinformatics 29,1922-1924(2013). 117. RAHorn, VVSergeichuk, Congruences of a square matrix and its transpose. Linear Algebra and its Applications 389, 347-353 (2004).

[0158] In at least some of the above embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment as long as the substitution is technically feasible. It should be understood that those skilled in the art may make various other omissions, additions, and modifications to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter defined by the appended claims.

[0159] Those skilled in the art will understand that the terms used herein in general, and in the appended claims in particular (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including, but not limited to," etc.). It will be further understood by those skilled in the art that where a particular number of introduced claim recitations are intended, such intention will be expressly set forth in the claim, and in the absence of such recitation, no such intention exists. For example, as an aid to understanding, the appended claims below may include the use of the introductory phrases "at least one" and "one or more" to introduce the claim recitation. However, the use of such phrases should not be interpreted as meaning that the introduction of a claim recitation by the indefinite article "a" or "an" limits a particular claim that includes such introduced claim recitation to an embodiment that includes only one such recitation. The same claim includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" means "at least one" or "one or more"); the same applies to the use of definite articles used to introduce claim recitations. In addition, even if a specific number of recitations of an introduced claim are explicitly recited, one of ordinary skill in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations" without other modifiers means at least two recitations, or more than two recitations).Furthermore, when a convention similar to "at least one of A, B, and C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having only A, only B, only C, both A and B, both A and C, both B and C, and / or both A, B, and C, etc.). When a convention similar to "at least one of A, B, or C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, systems having only A, only B, only C, both A and B, both A and C, both B and C, and / or both A, B, and C, etc.). It will be further understood by those skilled in the art that virtually any disjunction and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0160] In addition, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure therefore also is described in terms of any individual members of the Markush group or any subgroups of members.

[0161] As will be understood by those skilled in the art, for all purposes, e.g., in terms of providing a written description, all ranges disclosed herein also encompass any possible subranges and combinations of those subranges. Any listed range can be readily recognized as fully indicating and allowing the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, a middle third, an upper third, etc. As will also be understood by those skilled in the art, all terms such as "up to," "at least," "greater than," "less than," etc. refer to ranges that are inclusive of the numbers recited and that can subsequently be broken down into subranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, etc.

[0162] Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0163] Thus, the foregoing is merely illustrative of the principles of the present invention. It will be appreciated that those skilled in the art will be able to devise various configurations that embody the principles of the present invention and are within the spirit and scope thereof, although not expressly described or shown herein. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present invention and the concepts contributed by the inventors to further the art, and are not to be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. In addition, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended to be made public, regardless of whether such disclosure is expressly recited in the claims.

[0164] Therefore, it is not intended that the scope of the present invention be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims. In the claims, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is expressly defined to be invoked to limit a claim only if the precise phrase "means for" or the precise phrase "step for" is recited at the beginning of the limitation to such claim, and if such precise phrase is not used in the limitation to the claim, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is not invoked.

Claims

1. A pharmaceutical composition comprising a pharmacological mechanotransduction blocker.

2. 10. The pharmaceutical composition of claim 1 for a method of treating a wound in a subject, the method comprising:

10. The method of claim 1, wherein said subject is treated with a pharmaceutical composition comprising applying a skin graft to said wound in combination with a mechanotransduction blocking agent.

3. 3. The pharmaceutical composition of claim 2, wherein the skin graft is a split-thickness skin graft.

4. The pharmaceutical composition of claim 2, wherein the wound is a deep injury wound.

5. The pharmaceutical composition of claim 4, wherein the deep injury wound is a burn wound.

6. The pharmaceutical composition of claim 4, wherein the deep injury wound is a traumatic wound.

7. 3. The pharmaceutical composition of claim 2, wherein the skin graft is applied to the wound prior to the mechanotransduction blocking agent.

8. 3. The pharmaceutical composition of claim 2, wherein the mechanotransduction blocker comprises a pharmacological mechanotransduction blocker.

9. 3. The pharmaceutical composition of claim 2, wherein the pharmacological mechanotransduction blocker comprises a focal adhesion kinase inhibitor.

10. 3. The pharmaceutical composition of claim 2, wherein the mechanotransduction blocking agent is administered to the wound in a sustained release formulation.

11. 11. The pharmaceutical composition of claim 10, wherein the sustained release formulation comprises a gel formulation.

12. The pharmaceutical composition of claim 11 , wherein the gel formulation comprises a hydrogel.

13. 13. The pharmaceutical composition of claim 12, wherein the hydrogel comprises a biodegradable pullulan-based hydrogel.

14. The pharmaceutical composition of claim 2 , wherein the method promotes healing of the wound.

15. 3. The pharmaceutical composition of claim 2, wherein the method reduces fibrosis.

16. 3. The pharmaceutical composition of claim 2, wherein the method reduces contracture.

17. 3. The pharmaceutical composition of claim 2, wherein the method reduces scar formation.

18. The pharmaceutical composition of claim 2 , wherein the method restores collagen architecture.

19. The pharmaceutical composition of claim 2 , wherein the method improves implant biomechanical properties.

20. The pharmaceutical composition of claim 2 , wherein the subject is a mammal.

21. 21. The pharmaceutical composition of claim 20, wherein the mammal is a human.

22. 10. The pharmaceutical composition of claim 1, wherein the pharmacological mechanotransduction blocker comprises a focal adhesion kinase inhibitor.

23. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition comprises a sustained release formulation.

24. 24. The pharmaceutical composition of claim 23, wherein the sustained release formulation comprises a gel formulation.

25. 25. The pharmaceutical composition of claim 24, wherein the gel formulation comprises a hydrogel.

26. 26. The pharmaceutical composition of claim 25, wherein the hydrogel comprises a biodegradable pullulan-based hydrogel.

27. The pharmaceutical composition of claim 1; a skin graft harvester; and 28. A pharmaceutical composition comprising a pharmacological mechanotransduction blocker for a method of reducing scar formation following application of a skin graft to a treatment site in a human subject, said method comprising: applying the skin graft to the treatment area; delivering a focal adhesion kinase inhibitor to the skin graft to reduce scar formation at the treatment site.

29. 29. The pharmaceutical composition of claim 28, wherein the skin graft is a split-thickness skin graft.

30. 30. The pharmaceutical composition of claim 28, wherein the delivering step comprises applying to the skin graft a biodegradable pullulan-based hydrogel containing the focal adhesion kinase inhibitor.

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