Classifications

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  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
  • A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
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  • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
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  • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
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  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/54—Biologically active materials, e.g. therapeutic substances
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  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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  • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
  • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
  • A61L2300/432—Inhibitors, antagonists
  • A61L2300/434—Inhibitors, antagonists of enzymes

Definitions

• STSGs play a critical role, given their low donor site morbidity, promotion of a conducive healing environment, and ability to cover relatively large areas using graft meshing techniques.
• skin grafts serve an important function in rapidly restoring the barrier function of the skin to prevent infection and reduce mortality, secondary contraction during graft healing results in hypertrophic scar (HTS) formation (8).
• HTS hypertrophic scar
• Healed skin grafts are also characterized by increased fragility, abnormal pigmentation, and poor texture, compared to unwounded skin (9).
• mice are loose skinned animals, healing predominantly through contraction with less than 10% of the scarring that occurs in humans (28, 29).
• scRNA-seq Single-cell RNA sequencing
• the present disclosure provides a method for treating a wound of a subject, the method comprising applying a skin graft to the wound in combination with a mechanotransduction blocker >0 to treat the wound of the subject.
• the present disclosure provides a method of reducing scar formation after applying a skin graft to a treatment site of a human subject, the method comprising applying the skin graft to the treatment site; delivering a focal adhesion kinase inhibitor to the skin graft; and reducing scar formation at the treatment site.
• FIG 1A-1E Development of translational porcine model using clinically relevant methods.
• A Schematic of full-thickness excisional wounds on the pig dorsum and harvested skin grafts from a donor site.
• B Staged photos of full thickness wounds made using an electric bovie.
• C Photos of harvested and meshed skin grafts (0.01 in) at a 1 :1.5 ratio.
• D Staged images showing the original unwounded skin, full thickness wound, skin graft secured with staples, and coverage with 3 layers of petrolatum gauze with a bolster dressing.
• E Photographic images of the graft at POD 0, 7, and 90.
• FIG 2A-2J Cellular subpopulations in scar resulting from STSG are characterized by increased mechanotransduction and inflammatory signaling.
• B Quantification of dermal thickness, aSMA + myofibroblasts, and collagen alignment using CurveAlign (109). Statistical comparison made using unpaired two-tailed t-tests ( * p ⁇ 0.05).
• FIG 3A-3G Disruption of mechanotransduction in large animals accelerates STSG healing, attenuates fibrotic scar formation, reduces contracture, and improves biomechanical properties.
• C Representative images tracking interstitial epithelialization, scar formation, and contracture over time.
• FIG 4A-4F FAKI-mediated inhibition of mechanotransduction on STSG of large animals reduces collagen and restores organization of collagen fiber networks.
• Trichrome staining shows delineations of superficial and deep scar.
• Scale bar 1mm.
• CT-Fire fiber length/width metrics
• Scale bar 10mm.
• D to F Quantification of the different collagen fiber network characteristics across the four groups in the deep dermis. Statistical comparisons made using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons tests ( * P ⁇ 0.05, * P ⁇ 0.01, ** P ⁇ 0.001). All data represent mean ⁇ SEM of biological replicates.
• FIG 5A-5L Mechanotransduction blockade causes an early (POD7) upregulation of anti-inflammatory pathways in myeloid cells.
• A Schematic of STSG and STSG+FAKI.
• C Porcine cells shown in a UMAP embedding colored by STSG or STSG+FAKI.
• D UMAP embedding of cells colored by cell type. Dashed line shows myeloid cells of interest.
• E Number of differentially expressed genes between STSG and STSG+FAKI by cell type.
• F Violin plots of fibrotic genes expressed by fibroblasts.
• G UMAP embedding of myeloid cells colored by STSG or STSG+FAKI.
• H UMAP embeddings of myeloid cells colored by cell type with RNA velocity streams overlaid.
• I Violin plots of fibrotic or anti-inflammatory genes expressed by monocyte lineage cells.
• J Violin plots of fibrotic or anti-inflammatory genes
• FIG 6A-6F Disruption of mechanotransduction shifts fibroblast transcriptional fates from pro-fibrotic to regenerative at late (POD90) time points.
• A Porcine cells were isolated from STSG treated with FAKI hydrogel (STSG+FAKI), control STSG, and unwounded skin. UMAP embedding of the fibroblasts colored by treatment group. RNA velocities shown as the main gene-averaged flow, visualized by velocity streamlines projected onto the UMAP embedding.
• B Six fibroblast lineages and terminal states as determined by CellRank (81).
• C Cells colored by latent time as computed by scVelo across all genes, quantifying overall differences in transcriptional dynamics between cells.
• Top Expression of group-defining genes and key pathways projected onto the UMAP embedding.
• Bottom Gene-specific RNA velocities. Dotted purple line represents estimated ‘steady-state’ ratio of unspliced:spliced mRNA. Positive velocities (higher abundance of unspliced mRNA than expected) indicate gene up-regulation.
• ANOVA analysis of variance
• FIG 8A-8I 3D organotypic scar system recapitulates two opposing trajectories of regeneration versus fibrosis in both human and porcine cells.
• B and C Immunofluorescent staining of top (B) fibrotic and (C) regenerative markers. Scale bar: 100pm.
• FIG 9A-9E Diverse cellular ecology observed in chronic porcine STSG and unwounded skin.
• A Cell-type defining genes to confirm our automated cell type annotations.
• B Representative proportions of each cell type between STSG and unwounded skin.
• E Additional feature plots of genes and enriched pathways in fibroblasts.
• FIG 10A-10C Hydrogel releases FAKI over time into the dermis.
• A Schematic of hydrogel delivery of FAKI to the STSG.
• ANOVA analysis of variance
• Tukey multiple comparisons tests
• All data represent mean ⁇ SEM of biological replicates.
• C Penetration of FAKI into the dermis over time.
• FIG 12A-12C Diverse cellular ecology was observed in early (POD7) STSG and
• FIG 13A-13F CellRankand scVelo analysis of late (POD90) fibroblasts.
• A Initial and (B) terminal states identified by CellRank.
• C Velocity vectors shown for each individual cell.
• D Velocity length indicates increased transcriptional magnitudes across all genes.
• E, F Gene- resolved velocities for (E) ENPP1 and (F) ACTA2. The dotted line represents the estimated ‘steady-state’ ratio of unspliced to spliced mRNA abundance.
• FIG 14A-14D Additional analysis of fibroblasts from late (POD90) STSG, STSG+FAKI, and unwounded skin.
• A Violin plots of cluster-defining differentially expressed genes. Box plots are overlaid to show the medians and interquartile ranges.
• B Fleatmap of the top differentially expressed genes by treatment group.
• C Select feature plots illustrating gene expression.
• D GeneTrail feature UMAP plots of key pathways that differentiate between the groups.
• FIG 15. Protein confirmation of scRNA-seq observations in human patient samples.
• FIG 16. Immunofluorescent staining of ocSMA in human collagen scaffolds. Scale bar: 100pm. Statistical comparisons made using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons tests ( ** P ⁇ 0.01). All data represent mean ⁇ SEM of biological replicates.
• FIG. Violin plots of in vitro porcine scRNA-seq data with box plots overlaid to show the medians and interquartile ranges.
• fibroblast refers to a cell responsible for synthesizing and organizing extracellular matrix.
• Two fibroblast lineages include Engrailed-1 lineage-negative fibroblasts (ENFs) and Engrailed-1 lineage-positive fibroblasts (EPFs).
• the EPF lineage includes all cells that express Engrailed-1 at any point during their development, and all progeny of those cells.
• 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 a part or interference with its blood supply. It can be a consequence of the normal healing response that leads to a scar, an abnormal reactive process or no known or understood cause.
• the term “scar” refers to a fibrous tissue that replaces normal tissue destroyed by injury or disease. Damage to the outer layer of skin (the epidermis) is healed by rebuilding the tissue, and in these instances, scarring is slight or absent. When the thick layer of tissue beneath the skin’s outer surface (i.e., the dermis) is damaged, however, rebuilding is more complicated. The body lays down collagen fibers (a protein which is naturally produced by the body) in a composition that is different from that found in uninjured skin, and this usually results in a noticeable scar.
• scar area refers to the area of normal tissue that is destroyed by injury or disease and replaced by fibrous tissue.
• Scars differ from normal skin in three key ways: (1) they are devoid of any dermal appendages (hair follicles, sweat glands, etc.); (2) their collagen structure is fundamentally different, with dense, parallel fibers rather than the “basketweave” pattern that lends normal skin its flexibility and strength; and (3) as a result of their inferior matrix structure, they are weaker than skin.
• scar-related gene refers to a nucleic acid encoding a protein that is activated in response to scarring as part of the normal wound healing process.
• scar-related gene product refers to the protein that is expressed in response to scarring as part of the normal wound healing process.
• Scar tissue consists mainly of disorganized collagenous extracellular matrix. This is produced by myofibroblasts, which differentiate from dermal fibroblasts in response to wounding, which causes a rise in the local concentration of Transforming Growth Factor-b, a secreted protein that exists in at least three isoforms called TGF-bI , TGF ⁇ 2 and TGF ⁇ 3 (referred to collectively as TGF-b).
• TGF-b is an important 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 in, e.g., PCT Application No. WO 2014/040074, the disclosure of which is incorporated herein by reference in its entirety.
• skin used herein in its conventional sense includes all surface tissues of the body and sub-surface structure thereat including, e.g., mucosal membranes and eye tissue as well as ordinary skin.
• the expression “skin” may include a wound zone itself. The re approximation of skin over the surface of a wound has long been a primary sign of the completion of a significant portion of wound healing. This reclosure of the defect restores the protective function of the skin, which includes protection from bacteria, toxins, and mechanical forces, as well as providing the barrier to retain essential body fluids.
• the epidermis which is composed of several layers beginning with the stratum corneum, is the outermost layer of the skin. The innermost skin layer is the deep dermis.
• the term “dermal appendages” includes hair follicles, sebaceous and sweat glands, fingernails, and toenails.
• the term “dermal location” refers to a region of a skin of a subject having any size and area.
• the dermal location may encompass a portion of skin of a subject such as, e.g., the scalp.
• the dermal location may include one or more layers of skin including, e.g., the epidermis and the dermis. In some cases, the dermal location includes a wound.
• wound includes any disruption and/or loss of normal tissue continuity in an internal or external body surface of a human or non-human animal body, e.g. resulting from a non-physiological process such as surgery or physical injury.
• wound or wound environment used herein refers to any skin lesion capable of triggering a healing process which may potentially lead to scarring, and includes wounds created by injury, wounds created by burning, wounds created by disease and wounds created by surgical procedures.
• the wound may be present on any external or internal body surface and may be penetrating or non-penetrating. The methods herein described may be beneficial in treating problematic wounds on the skin's surface.
• superficial and non-superficial wounds e.g. abrasions, lacerations
• wounds arising from thermal injuries e.g. burns and those arising from any cryo-based treatment
• any wound resulting from surgery include both superficial and non-superficial wounds, e.g. abrasions, lacerations, wounds arising from thermal injuries (e.g. burns and those arising from any cryo-based treatment), and any wound resulting from surgery.
• wound healing refers to a regenerative process with the induction of a temporal and spatial healing program, including, but not limited to, the processes of inflammation, granulation, neovascularization, migration of fibroblast, endothelial and epithelial cells, extracellular matrix deposition, re-epithealization, and remodeling.
• Hydrogel Hydrogels useful in the methods of the invention maintain viability of entrapped cells for a period of time sufficient to enhance wound healing. Hydrogels are known and used in the art for wound healing.
• hydrogels are, by weight, 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% water, with the remaining weight comprising a suitable polymer, e.g. pullulan and collagen, glycosaminoglycan, acrylate, 2-hydroxymethyl methacrylate and ethylenedimethacrylate copolymer, carboxymethylcellulose, chitosan, gelatin, etc., or other suitable hydrophilic polymers as known in the art.
• Hydrogels can swell extensively without changing their gelatinous structure and are available for use as amorphous (without shape) gels and in various types of application systems, e.g. flat sheet hydrogels and non-woven dressings impregnated with amorphous hydrogel solution.
• Flat sheet (film) hydrogel dressings have a stable cross-linked macrostructure and therefore retain their physical form as they absorb fluid.
• a cross-linked hydrogel film is fabricated using pullulan and collagen under conditions that provided for cross-linking and pore formation.
• Collagen is added to a mixture of pullulan, cross-linking agent and pore-forming agent (porogen), where the collagen is provided at a concentration of at least about 1 %, and not more than about 12.5% relative to the dry weight of the pullulan.
• Collagen may be provided at a concentration of about 1%, about 2.5%, about 5%, about 7.5%, about 10%, usually at a concentration of from about 2.5% to about 10%, and may be from about 4% to about 6% relative to the dry weight of the pullulan.
• the collagen is typically a fibrous collagen, e.g. Type I, II, III, etc.
• Cross-linking agents of interest include sodium trimetaphosphate (STMP) or a combination of or a combination of sodium trimetaphosphate and sodium tripolyphosphate (STMP/STPP).
• STMP sodium trimetaphosphate
• STMP/STPP sodium tripolyphosphate
• the cross-linking agent can be included in a wt/wt ratio relative to the pullulan of from about 5:1 to about 1:5, and 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. KCI.
• the porogen can be included in a wt/wt ratio relative to the pullulan of from about 5:1 to about 1:5, and 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.
• the suspension of collagen, pullulan, cross-linker and porogen, in the absence of cells, is poured and compressed to form sheets.
• Preferred thickness is at least about 1 mm and not more than about 5 mm, usually not more than about 3 mm, and may be from about 1 to 2.5 mm, e.g. about 1.25, 1.5, 1.75, 2 mm thick.
• Pores are formed in the hydrogel through rapid dessication of swollen hydrogels by phase inversion. Dehydration results in localized super-saturation 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 following KCI dissolution.
• the films may be stored in a dried state, and are readily rehydrated in any suitable aqueous medium.
• the aqueous nature of hydrogel substrates provides an ideal environment for cellular growth and sustainability.
• Mechanical features of the hydrogel include average pore size and scaffold porosity. Both variables vary with the concentration of collagen that is present in the hydrogel. For a hydrogel comprising 5% collagen, the average pore size will usually range from about 25 mhi to about 50 mhi, from about 30 mhi to about 40 mhi, and may be about 35 mhi. For a hydrogel comprising 10% collagen the average pore size will usually range from about 10 mhi to about 25 mhi, from about 12 mhi to about 18 mhi, and may be about 15 Dm. One of skill in the art will readily determine suitable hydrogels at other collagen concentrations.
• the scaffold porosity will usually range from about 50% to about 85%, and may range from about 70% to about 75%, and will decrease with increasing concentrations of collagen.
• Hydrogels lacking collagen do not display any birefringence with polarizing light microscopy, while the hydrogels comprising collagen are diffusely birefringent.
• Pullulan A polysaccharide produced by the fungus Aureobasidium pullulans. It is a linear homopolysaccharide consisting of alpha-(1-6) linked maltotriose units and exhibits water retention capabilities in a hydrogel state which makes it an ideal therapeutic vehicle for both cells and biomolecules. Additionally, pullulan contains multiple functional groups that permit cross-linking and delivery of genetic material and therapeutic cytokines. Furthermore, pullulan-based scaffolds have been shown to enhance both endothelial cell and smooth muscle cell behavior in vitro. Collagen.
• collagen refers to compositions in which at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more of the protein present is collagen in a triple helical configuration.
• Collagens are widely found in vertebrate species, and have 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, e.g. between mammalian species. Typical commercial animal sources include the bovine Achilles tendon, calfskin and the bones of cattle.
• the collagen used in the preparation of the oriented thin film is Type I, Type II, or Type III collagen, and is derived from any convenient source, e.g. bovine, porcine, etc., usually a mammalian source.
• Collagen has a triple-stranded rope-like coiled structure.
• the major collagen of skin, tendon, and bone is collagen I, containing 2 alpha-1 polypeptide chains and 1 alpha-2 chain.
• the collagen of cartilage contains only 1 type of polypeptide chain, alpha-1.
• the fetus also contains collagen of distinctive structure.
• the genes for types I, II, and III collagens, the interstitial collagens, exhibit an unusual and characteristic structure of a large number of relatively small exons (54 and 108 bp) at evolutionarily conserved positions along the length of the triple helical gly-X-Y portion. (COL25A1 ); XXVII (COL27A1); XXVIII (COL28A1).
• mammalian collagens e.g. bovine, porcine, equine, etc. collagen
• FAK Focal adhesion kinase
• FAK is a non-receptor cytoplasmic tyrosine kinase.
• FAK is one of the key mediators of skin mechanobiology and it is activated after cutaneous injury. Mechanical forces potentiate the activation of FAK through phosphorylation following injury of the skin.
• FAK contributes to cell signaling through its linking of mechanical stress from the ECM to the cytoplasmic cytoskeleton, activating inflammatory pathways.
• Fibroblasts are recruited to the wound by inflammatory signaling, where their secretion of profibrotic cytokines brings about increased collagen synthesis.
• Various pathologies that are associated with poor wound healing have been shown to have atypical levels of FAK. Mechanical force regulates pathologic scarring through inflammatory FAK-ERK-MCP1 pathways, and molecular strategies targeting focal adhesion kinase (FAK) can effectively uncouple mechanical force from fibrosis.
• Wound dressing films of the invention find use as a wound dressing, or artificial skin, by providing an improved substrate that minimizes scarring.
• An effective bioactive wound dressing can facilitate the repair of wounds that may require restoration of both the epidermis and dermis.
• a hydrogel thin film is placed onto, and accepted by, the debrided wound of the recipient and provide a means for the permanent re-establishment of the dermal and epidermal components of skin.
• the graft suppresses the formation of granulation tissue which causes scarring.
• Additional criteria for biologically active wound dressings include: rapid adherence to the wound soon after placement; proper vapor transmission to control evaporative fluid loss from the wound and to avoid the collection of exudate between the wound and the dressing material.
• Skin substitutes should act as barrier to microorganisms, limit the growth of microorganisms already present in the wound, be flexible, durable and resistant to tearing.
• the substitute should exhibit tissue compatibility, that is, it should not provoke inflammation or foreign body reaction in the wound which may lead to the formation of granulation tissue.
• An inner surface structure of a hydrogel thin film is provided that permits ingrowth of fibro-vascular tissue.
• An outer surface structure may be provided to minimize fluid transmission and promote epithelialization.
• Typical bioabsorbable materials for use in the fabrication of porous wound dressings, skin substitutes and the like include synthetic bioabsorbable polymers such as polylactic acid or polyglycolic acid, and also, biopolymers such as the structural proteins and polysaccharides.
• the finished dressing prior to cell seeding is packaged and preferably radiation sterilized.
• Such biologically active products can be used in many different applications that require the regeneration of dermal tissues, including the repair of injured skin and difficult-to-heal wounds, such as burn wounds, venous stasis ulcers, diabetic ulcers, etc.
• Split-thickness grafts are usually used; for these grafts, a thin layer of epidermis and some dermis are excised and placed on the recipient site. Such grafts are typically used for burns but may also be used to accelerate healing of small wounds. Because a significant amount of dermal elements remain at the donor site, the site eventually heals and can be harvested again.
• Full-thickness grafts are composed of epidermis and dermis and provide better appearance and function than split-thickness grafts. Flowever, because the donor site will not heal primarily, it must be a loose area of redundant skin (eg, abdominal or thoracic wall, sometimes scalp) so that the site can be sutured closed. Thus, full-thickness grafting is usually reserved for cosmetically sensitive areas (eg, face) or areas requiring a thicker, more protective skin layer (eg, hands). Because full-thickness grafts are thicker and more vascular, they do not have quite as high a survival rate as split-thickness grafts. Split-thickness skin grafts may be classified as thin (0.15-0.25mm) intermediate (0.3-0.4mm) or thick (0.5-0.6mm). Composite grafts. Composite skin grafts include two or more different types of tissues.
• composite skin grafts have cartilage with or without subcutaneous tissue and the overlying skin. Because they offer support and structure composite grafts are often used to repair full-thickness defects of the nasal ala and helical rim.
• autograft refers to a skin graft where the graft tissues is from the individual or subjects own body.
• graft refers to a skin graft where the graft tissues is from a different individual or subject other than the subject that is receiving treatment of a wound.
• xenograft refers to a skin graft where the graft tissue is from an individual that is a different species or is synthetic graft tissue relative to the subject that is receiving the graft tissue.
• a xenograft may be wherein a human is treated for a wound with a porcine skin graft.
• Skin graft methods are provided. Aspects of the methods include applying a skin graft to a wound in combination with a mechanotransduction blocker, such as a pharmacological mechanotransduction blocker, e.g., a focal adhesion kinase inhibitor. Also provided are pharmaceutical compositions and kits for use practicing methods of the invention.
• a mechanotransduction blocker such as a pharmacological mechanotransduction blocker, e.g., a focal adhesion kinase inhibitor.
• METHODS FOR TREATING A WOUND OF A SUBJECT As summarized above, methods are provided for treating a wound of a subject, the methods include applying a skin graft to the wound in combination with a mechanotransduction blocker to treat the wound of the subject.
• the wound may be any wound of a subject in need of treatment.
• Wounds that receive benefit from the methods described herein include, without limitation, partial- and full-thickness wounds, ulcers, including pressure ulcers, diabetic ulcers (e.g., diabetic foot ulcers), venous ulcers, lower leg ulcer, etc.; burns (second and third degree burns) including scalds, chemical burns, thermal burns such as flame burns and flash burns, ultraviolet burns, contact burns, radiation burns, electrical burns, etc.; gangrene; skin tears or lacerations, such as made by knives, etc.; a incisions such as made by knives, nails, sharp glass, razors, etc.; avuls; amputations; surgical wounds; failing or compromised skin/muscle grafts or flaps; bites; slash wounds, i.e., a wound where the length is greater than the depth; bruises; and the like, or a combination of one or more of the above.
• Subjects of the present disclosure may be any subject in need of treatment of a wound.
• the subject is a mammal.
• mammals that would benefit from the methods disclosed herein include, without limitation, canines; felines; equines; bovines; porcines; ovines; rodentia, such as mice or rats, etc. and primates, e.g., non-human primates, humans, etc.
• the mammal is a human.
• the methods of the present disclosure involve applying a skin graft to the wound.
• the skin graft may be any skin graft deemed useful in the treatment of the wound of the subject.
• Skin grafts that find use in the present disclosure include, without limitation, full-thickness grafts, partial- thickness grafts, composite grafts, etc.
• the skin graft may be an autograft, an allograft or a xenograft. In some embodiments, the skin graft is applied before application of the mechanotransduction blocker.
• Mechanotransduction blocker that find use in the present disclosure are any inhibitors that impair mechanotransduction signaling pathways.
• mechanotranduction blockers include, without limitation, integrin inhibitors, focal adhesion kinase (FAK) inhibitors, Talin inhibitors, Vinculin inhibitors, Paxillin inhibitors, Zyxin inhibitors, VASP inhibitors, p130 cas inhibitors, etc.
• the mechanotransduction inhibitor is a focal adhesion kinase (FAK) inhibitor.
• Non-limiting examples of FAK inhibitors include, without limitation, PF-56227, PF-573228, TAE226 (NVP-TAE226), BI-4464, GSK2256098, PF-431396, PND-1186 (VS-4718), Y15, Defactinib (VS-6063), Solanesol (Nonaisoprenol), etc.
• other types of inhibitors may be used.
• other types of FAK inhibitors include, without limitation, siRNA, anti-sense oligonucleotides (ASO). CRISPR-mediated knockout or knockdown of FAK, etc.
• the mechanotransduction blocker is a pharmacological mechanotransduction blocker.
• the mechanotransduction blockers of the present disclosure may be applied in any way deemed useful.
• the mechanotransduction blocker is applied systemically.
• the mechanotransduction blocker is applied locally at the site of the skin graft.
• the mechanotransduction blocker may be applied in a sustained release formulation.
• the sustained release formulation includes a gel formulation.
• the gel formulation includes a hydrogel.
• the hydrogel includes a carbohydrate based hydrogel, e.g., a biodegradable pullulan-based hydrogel. Pullulan-based hydrogels are known in the art and have been 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 herein specifically incorporated by reference.
• the methods disclosed herein provide a number of benefits to wound healing relative to other methods, i.e. a skin graft in the absence of a mechanotransduction blocker.
• the methods may promote the healing of the wound, reduce fibrosis, reduce contracture, mitigate scar formation, restore collagen architecture, or improve graft biomechanical properties.
• Embodiments of the methods disclosed herein reduce the amount of contracture occurring following the skin graft.
• Contracture is a measure of the change in scar area relative to the area of the skin graft. Contracture is the result of scar formation pulling on the edges of the skin surrounding the scar cause strain.
• the methods disclosed herein result in a range of reductions in contracture. For instance, contracture may 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 a 50% reduction in contracture relative to skin grafts in the absence of a mechanotransduction blocker.
• Embodiments of the methods disclosed herein promote wound healing following a skin graft.
• the promotion of wound healing is an increase in re-epithelialization.
• a range of increases in re-epithelialization may occur.
• re- epithelization may be increase 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 a 50% increase in re-epithelialization relative to skin grafts in the absence of a mechanotransduction blocker.
• Embodiments of the methods disclosed herein improve skin graft biomechanical properties.
• the improvement of skin graft biomechanical properties is a decrease in the firmness and an increase in elasticity of the skin graft as measured by the vertical deformation of the graft.
• a range of increases in deformation may occur.
• deformation may increase 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 a 50% increase in deformation of skin grafts relative to skin grafts in the absence of a mechanotransduction blocker.
• Embodiments of the methods disclosed herein restore collagen architecture.
• the restoration of collagen architecture is a decrease in the alignment and length of collagen fibers relative to skin grafts without a mechanotransduction blocker. Unwounded skin is generally characterized as having short and randomly aligned collagen.
• a restoration in collagen architecture is a decrease in the alignment of collagen fibers, a range of decreases in alignment of collagen fibers may occur.
• alignment of collagen fibers may decrease 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 a 50% decrease in alignment of collagen fibers of skin grafts relative to skin grafts in the absence of a mechanotransduction blocker.
• a restoration in collagen architecture is a decrease in the length of collagen fibers
• a range of decreases in length of collagen fibers may occur.
• length of collagen fibers may decrease by 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 a 40% decrease in length of collagen fibers of skin grafts relative to skin grafts in the absence of a mechanotransduction blocker.
• methods for reducing scar formation after applying a skin graft to a treatment site of a human subject, the methods including applying the skin graft to the treatment site; delivering a focal adhesion kinase inhibitor to the skin graft to reduce scar formation at the treatment site.
• the skin graft may be any skin graft deemed useful in the treatment of the wound of the subject.
• Skin grafts that find use in the present disclosure include, without limitation, full-thickness grafts, partial-thickness grafts, composite grafts, etc.
• the skin graft may be an autograft, an allograft or a xenograft.
• a treatment site of the present disclosure may be any site on the skin that is in need of treatment.
• Treatment sites that find use in the present disclose include, without limitation, hand, palm, lower arm, upper arm, under arm, chest, abdomen, shoulders, upper back, lower back, neck, face, scalp, pelvis, groin, upper leg, lower leg, feet, etc.
• FAK inhibitors that find use in the method disclosed herein are any FAK inhibitors that impair FAK based signaling.
• Non-limiting examples of FAK inhibitors include, without limitation, PF-56227, PF-573228, TAE226 (NVP-TAE226), BI-4464, GSK2256098, PF-431396, PND-1186 (VS-4718), Y15, Defactinib (VS-6063), Solanesol (Nonaisoprenol), etc.
• other types of inhibitors may be used.
• other types of FAK inhibitors include, without limitation, siRNA, anti-sense oligonucleotides (ASO). CRISPR- mediated knockout or knockdown of FAK, etc.
• FAK inhibitors of the present disclosure may be delivered in a number of different ways.
• the FAK inhibitor is delivered systemically.
• the FAK inhibitor is applied locally at the treatment site.
• the FAK inhibitor is applied in a sustained release formulation.
• the sustained release formulation includes a gel formulation.
• the gel formulation includes a hydrogel.
• the hydrogel includes a biodegradable pullulan-based hydrogel.
• the reduction in scar formation is reducing in the visual appearance of a scar.
• the reduction in scar formation is a reduction in the contracture that occurs during and after scar formation.
• the methods disclosed herein result in a range of reductions in contracture. For instance, contracture may 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 a 50% reduction in contracture relative to skin grafts in the absence of a mechanotransduction blocker.
• the mechantransduction blocker(s), such as described above may be administered in combination with other pharmaceutically active agents, including other agents that treat the underlying condition or a symptom of the condition, e.g., scarring.
• “In combination with” as used herein refers to uses where, for example, the first compound is administered during the entire course of administration of the second compound; where the first compound is administered for a period of time that is overlapping with the administration of the second compound, e.g.
• in combination can also refer to regimen involving administration of two or more compounds.
• “In combination with” as used herein also refers to administration of two or more compounds which may be administered in the same or different formulations, by the same of different routes, and in the same or different dosage form type.
• YAP inhibitors examples include, but are not limited to, YAP inhibitors.
• the YAP inhibitor is a small molecule agent that exhibits the desired activity, e.g., inhibiting YAP expression and/or activity.
• Naturally occurring or synthetic small molecule compounds of interest include numerous chemical classes, such as organic molecules, e.g., small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons.
• Candidate agents have functional groups for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
• the candidate agents may include cyclical 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 other ways, by employing the screening protocols.
• the YAP inhibitor is a photosensitizing agent.
• the YAP inhibitor is a benzoporphyrin derivative (BPD).
• BPD benzoporphyrin derivative
• the benzoporphyrin derivative may be any convenient benzoporphyrin derivative such as, e.g., 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. Application No. 2009/0304803, the disclosures of which are incorporated herein by reference in their entireties.
• the benzoporphyrin derivative is a photosensitizing agent.
• the YAP inhibitor is verteporfin (benzoporphyrin derivative monoacid ring A, BPD-MA; tradename: Visudyne®).
• aspects of the methods may include administering an effective amount of a mechanotransduction blocker in combination with a Piezo inhibitor.
• the Piezo inhibitor includes a Piezol and/or Piezo2 inhibitor.
• the Piezo inhibitor is a Piezol inhibitor.
• the Piezo inhibitor is a Piezo2 inhibitor.
• both a Piezol inhibitor and Piezo2 inhibitor are administered to a subject.
• the method consists essentially of administering a Piezo inhibitor.
• a “Piezo inhibitor” refers to a molecule that may inhibit Piezo protein function and signaling. In some cases, the Piezo inhibitor inhibits cellular mechanical signaling.
• 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 a Piezo protein with other signaling molecules. In certain embodiments, administering the Piezo inhibitor reduces mechanical activation of one or more cells, e.g., adipocytes, in a wound, wherein, e.g., the level of mechanical activation of the one or more cells, e.g., adipocytes, in a wound is reduced compared to a suitable control. Further details regarding Piezo inhibitors and methods of using the same are provided in United States Provisional Patent Application Serial No.
• combination therapy compounds may be administered by the same route of administration (e.g. intrapulmonary, oral, enteral, etc.) that the mechanotransduction blocker is administered.
• the compounds for use in combination therapy with the mechanotransduction blocker may be administered by a different route of administration.
• compositions are provided for practicing the methods disclosed herein.
• Pharmaceutical compositions comprise the mechanotransduction blocker of the present disclosure and a pharmaceutical acceptable excipient(s).
• a pharmaceutical acceptable excipient A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein.
• Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical 5 Dosage Forms and Drug Delivery Systems (1999) H.C.
• the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents, are readily available to the public.
• pharmaceutically acceptable auxiliary 0 substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
• the pharmaceutical compositions of the present disclosure comprise a mechanotransduction blocker.
• Mechanotransduction blockers that find use in the present disclosure are any blockers that impair mechanotransduction signaling pathways.
• Non-limiting 5 examples of mechanotranduction blockers include, without limitation, integrin inhibitors, focal adhesion kinase (FAK) inhibitors, Talin inhibitors, Vinculin inhibitors, Paxillin inhibitors, Zyxin inhibitors, VASP inhibitors, p130 cas inhibitors, etc.
• the mechanotransduction inhibitor is a focal adhesion kinase (FAK) inhibitor.
• Non-limiting examples of FAK inhibitors include, without limitation, PF-56227, PF-573228, TAE226 (NVP-TAE226), Bl- !O 4464, GSK2256098, PF-431396, PND-1186 (VS-4718), Y15, Defactinib (VS-6063), Solanesol (Nonaisoprenol), etc.
• other types of inhibitors include, without limitation, siRNA, anti- sense oligonucleotides (ASO). CRISPR-mediated knockout or knockdown of FAK, etc.
• the pharmaceutical composition includes a sustained release !5 formulation.
• Sustained release formulations of the present disclosure are any sustained release formulation that is capable of releasing the mechanotransduction blocker for a prolonged period of time.
• the sustained release formulation is capable of releasing the mechanotransduction blocker for a range of time.
• the sustained release formulation may release the mechanotransduction inhibitor for at least 12 hours, at least 24 hours, at least 36 hours, at least >0 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours or greater than
• the sustained release formulation of the present disclosure is capable of releasing the mechanotransduction blocker into a specified depth into the skin grafts.
• the depth into the skin graft is a measure of the distance from the stratum corneum to the farthest point into the tissue beneath the stratum corneum.
• the specified depth that the sustained release formulation releases into the skin graft 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 greater than 3 mm.
• the sustained release formulation contains a gel formulation.
• the gel formulation includes a hydrogel, e.g., a carbohydrate based hydrogel, a protein based hydrogel, etc.
• the hydrogel contains a biodegradable pullulan-based hydrogel. Pullulan-based hydrogels are known in the art and have been 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).
• the pharmaceutical composition is formulated in an aqueous buffer.
• Suitable aqueous buffers include, but are not limited to, acetate, succinate, citrate, and phosphate buffers varying in strengths from 5 mM to 100 mM.
• the aqueous buffer includes reagents that provide for an isotonic solution. Such reagents include, but are not limited to, sodium chloride; and sugars e.g., mannitol, dextrose, sucrose, and the like.
• the aqueous buffer further includes a non-ionic surfactant such as polysorbate 20 or 80.
• the pharmaceutical composition may further include a preservative.
• Suitable preservatives include, but are not limited to, a benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, and the like. In many cases, the formulation is stored at about 4°C. Pharmaceutical compositions may also be lyophilized, in which case they generally include cryoprotectants such as sucrose, trehalose, lactose, maltose, mannitol, and the like. Lyophilized formulations can be stored over extended periods of time, even at ambient temperatures.
• the mechanotransduction blocker is formulated with a second agent such as the combination therapies disclosed above in a pharmaceutically acceptable excipient(s).
• the subject pharmaceutical composition can be administered orally, subcutaneously, intramuscularly, parenterally, or other route, including, but not limited to, for example, oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal), intravesical or injection into an affected organ.
• Each of the active agents can be provided in a unit dose of from about 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, 1 mg, 5 mg, 10 mg, 50, mg, 100 mg, 250 mg, 500 mg, 750 mg or more.
• the pharmaceutical composition may be administered in a unit dosage form and may be prepared by any methods well known in the art. Such methods include combining the mechanotransduction blocker with a pharmaceutically acceptable carrier or diluent which constitutes one or more accessory ingredients.
• a pharmaceutically acceptable carrier is selected on the basis of the chosen route of administration and standard pharmaceutical practice. Each carrier must be "pharmaceutically acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. This carrier can be a solid or liquid and the type is generally chosen based on the type of administration being used.
• suitable solid carriers include lactose, sucrose, gelatin, agar and bulk powders.
• suitable liquid carriers include water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions, and solution and or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.
• Such liquid carriers may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.
• Preferred carriers are edible oils, for example, corn or canola oils. Polyethylene glycols, e.g. PEG, are also good carriers.
• KITS Any drug delivery device or system that provides for the dosing regimen of the instant disclosure can be used.
• a wide variety of delivery devices and systems are known to those skilled in the art.
• kits for practicing the methods described in the present disclosure may include the pharmaceutical composition described above as described above and a skin graft harvester.
• the pharmaceutical composition may be contained in a specific deliver device.
• Delivery devices include, without limitation, patches, gauze dressings, transparent film dressings, foam dressings, hydrocolloids dressings, alginate dressings, composite dressings, etc.
• the skin graft harvester of the present disclosure is any skin graft harvester capable of producing a split-thickness skin graft.
• Skin graft harvesters that find use in the present disclosure include, without limitation, a surgical knife, oscillating, Goulian knife, an air powered dermatome, an electric powered dermatome, etc.
• a subject kit can include any combination of components for performing the methods of the present disclosure.
• the components of a subject kit can be present as a mixture or can be separate entities. In some cases, 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-sold mixture such as a hydrogel. Components of a subject kit can be in the same or separate containers, in any combination.
• the subject kits may further include (in certain embodiments) instructions for practicing 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, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like.
• Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), flash drive, and the like, on which the information has been recorded.
• Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a remote site.
• Burns and other traumatic injuries represent a substantial 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.
• STSG autologous split-thickness skin grafting
• Acute mechanotransduction blockade upregulated myeloid CXCUO-mediated anti-inflammation with decreased CXCL14-mediated myeloid and fibroblast recruitment.
• mechanical signaling shifted fibroblasts toward pro-fibrotic differentiation fates, whereas disruption of mechanotransduction modulated mesenchymal fibroblast differentiation states to block those responses and instead drove fibroblasts toward pro-regenerative, adipogenic states similar to unwounded skin.
• 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.
• scRNA-seq data were analyzed using Seurat, a quantitative analysis package for clustering and embedding scRNA-seq data.
• Seurat a quantitative analysis package for clustering and embedding scRNA-seq data.
• the resultant skin was placed on a Skin Graft Carrier (Dermacarrier II, Zimmer 00770800010) and slowly fed through a Skin Graft Mesher (Zimmer 7701 ) to create a mesh graft of 1 :1.5 ratio ⁇ 41).
• the grafts were carefully spread out on the graft carrier and placed onto the exposed muscle fascia of the wound beds.
• Skin staples (Covidien 8886803712) were used to secure the graft to the wound bed (about 5 staples per edge).
• the grafts were covered with three layers of petrolatum gauze (Xeroform, Covidien SH84-433605) to prevent them from drying out and also to prevent bacterial infection.
• Bolster dressings were prepared by cutting 5.5cm x 5.5cm squares from VAC Granufoam (Small Dressing Kit, Acelity M8275065) and heavily secured on top of the gauze and STSG with additional skin staples. Finally, Telfa non-adherent dressings (Covidien 1961) and Tegaderm adherent dressings (3M 1624W) were used to cover the bolster sponge dressings. In order to prevent irritation and to minimize the animal's ability to impact the dressings, custom-designed polyester jackets tailored to the individual pigs were utilized (Lomir Biomedical Inc). Animals were given oral amoxicillin 10 mg/kg post-operatively twice a day for 5 days total.
• pigs were used, and each pig received 6 total STSG.
• STSG were randomly assigned to equally receive either FAKI hydrogels, blank hydrogels, or no hydrogel (standard dressings used for all STSG), with treatment conditions randomly assigned to different STSG across each pig dorsum.
• STSG were randomly assigned to equally receive either FAKI hydrogels or no hydrogel.
• treatment conditions were randomly assigned to different STSG across each pig dorsum to minimize any locational effects.
• Hydrogels were applied over the STSG before the standard dressing (petrolatum gauze + bolster sponge + Telfa + Tegaderm + custom jacket).
• Custom single cell RNA sequencing methods for porcine skin tissue identify cellular subpopulations that contribute to scarring
• STSGs did not restore normal skin appearance and instead exhibited permanent scar contracture with excessive fibrosis, hyperpigmentation, and raised hypertrophic scar (HTS) formation at postoperative day (POD) 90 (FIG. 2A).
• STSGs had a thickened dermis with more highly aligned collagen fibers and fewer dermal appendages, which are all classic signs of fibrotic healing.
• normal unwounded skin had a randomly aligned, “basket-weave” collagen architecture ( * P ⁇ 0.05) (FIG. 2, A and B).
• the STSG was also found to contain more pro-fibrotic myofibroblasts ( * P ⁇ 0.05) (FIG. 2, A and B).
• FIG. 2C Data for individual cells from both groups were subjected to blinded Louvain-based clustering and embedded into a two-dimensional UMAP (uniform manifold approximation and projection) space (45) (FIG. 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 annotations through the SingleR package (FIG. 2D) and verified with cell type-specific marker genes (FIG.9A).
• fibroblasts had over 400 differentially expressed genes (DEGs; average log fold change > 0.5 between STSG and skin), and myeloid and lymphoid cells had over 200 DEGs between STSG and skin (FIG. 2E; FIG. 9B).
• other cell types keratinocytes, endothelial cells, VSMCs
• keratinocytes, endothelial cells, VSMCs had 100 or fewer DEGs, indicating that they were not transcriptionally different between normal skin and skin grafts.
• Myeloid STSG cells were also enriched for fibrosis-driving genes, such as TNF (55), and demonstrated an upregulation of common mechanotransduction pathways such as the ERK1/ERK2 and EGF/EGFR pathways (56) (FIG.2, F and G). These pathways are downstream of FAK and suggest that these immune cells are mechanoresponsive even at later time points (57, 58).
• TNF fibrosis-driving genes
• F and G EGF/EGFR pathways
• myofibroblast-associated 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 STSGs had differentiated into more contractile myofibroblast phenotypes associated with increased collagen production and fibrosis, which was also supported by our immunofluorescent staining (59) (FIG.2, A, B, and I; FIG.9E).
• ACTA2 encoding smooth muscle alpha actin, aSMA
• RUNX1 runt-related transcription factor 1
• TAGLN transgelin
• COL11A1 collagen type XI alpha 1 chain
• STSG fibroblasts exhibited an enrichment of gene sets related to mechanotransduction and collagen production/organization driving scar formation, such as response to mechanical stimulus (GO-BP:0009612), focal adhesion (WP306), ECM assembly (GO-BP:0030198), YAP signaling (WP3967), response to TGFB signaling (WP560), and ossification (GO-BP:0001503) (FIG. 21).
• STSG fibroblasts were also characterized by a down- regulation of genes associated with adipogenesis (WP236), lipid transport (GO-BP:0006869), and regulation of endothelial cell migration (GO-BP:0010594), defined by regenerative, adipogenic markers such as APOE (apolipoprotein E), APOD (apolipoprotein D), CLEC3B (c-type lectin domain family 3 member B), and AGT (angiotensinogen) (60) (FIG.2J; FIG.9E). These pathways and genes suggested that STSG fibroblasts were shifted away from the more homeostatic, quiescent baseline observed in normal skin.
• WP236 adipogenesis
• lipid transport GO-BP:0006869
• GO-BP:0010594 regulation of endothelial cell migration
• APOE apolipoprotein E
• APOD apolipoprotein D
• CLEC3B c-type lectin domain family 3
• fibroblasts are generally regarded as the primary mediators of collagen deposition and scar contracture (26), recent studies have suggested a role for immune cells in regulating (and sustaining) fibrotic processes (61-64). Although there is some evidence that macrophages respond to mechanical cues in certain situations, these studies have yielded conflicting results, with literature suggesting that mechanical strain produces both pro- and anti inflammatory responses (57, 58). Overall, our transcriptomic data indicated that tissue that develops after STSG is different from unwounded skin and primarily characterized by increased mechanotransduction signaling in both inflammatory cells and fibroblasts, suggesting a shared common pathway in the context of STSG.
• VS-6062 using a biodegradable, biocompatible, and soft pullulan-based hydrogel optimized for sustained drug release during wound healing ⁇ 23
• FIG. 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, later generation small molecule FAKI that blocks tumor growth and has undergone Phase I trials against advanced solid tumors (ClinicalTrials.gov Identifier: NCT00666926) ⁇ 65).
• VS-6062 also has a strong selectivity for FAK relative to a wide range of other kinase targets ⁇ 66, 67), and we have previously characterized the release of this drug in our hydrogel ⁇ 23, 26).
• the hydrogel contains VS-6062 and is rehydrated in saline to convert it into a hydrogel dressing (FIG. 10A) that slowly releases the drug, which permeates through the STSG dermis over time (FIG. S2, B and C). Similar to other hydrogels, this hydrogel dressing provides coverage over a wound or STSG, preventing desiccation and facilitating moist wound healing (FIG. 3B).
• VAS Visual Analog Scale
• FIG. 4A Pharmacological blockade of mechanotransduction significantly promoted shorter and more randomly aligned collagen in the 5 deep dermis, similar to the typical basket weave-like collagen fiber network in unwounded skin ( * P ⁇ 0.05) (FIG. 4, B to E; FIG. 11, A and B). Furthermore, FAK inhibition decreased overall collagen deposition throughout both the superficial and deep dermis by decreasing fiber widths and overall architectural complexity ( ** p ⁇ 0.01, *** p ⁇ 0.001) (FIG. 4F; FIG. 11, B and C). Overall, FAKI-treated STSGs demonstrated dermal remodeling similar to that of unwounded skin !O throughout the entire thickness of the developing scar.
• Mechanotransduction blockade causes an acute upregulation of antiinflammatory pathways in myeloid cells
• fibroblasts only had about 50 DEGs, indicating that there were not strong differences in fibroblast gene expression at these early time points, despite the substantial differences observed later.
• fibroblasts exhibited similar expression of extracellular matrix markers such as COL1A1, COL3A1, FN1 (encodes for fibronectin), as well as inflammatory chemokines such as CXCL14 (69, 70) (FIG. 5F). These findings suggested that fibroblasts are not dramatically altered by mechanical signaling during the early stages of healing in this model.
• FIG. 51 myeloid CXCL10 expression (encodes for interferon gamma induced protein 10; IP-10) (FIG. 51), a secreted chemokine that inhibits fibroblast migration in response to pro-inflammatory markers (71). IP-10 therapy has previously been clinically used to reduce fibrosis (72-74). FAK inhibition also induced SOCS3 expression (FIG. 51), which is known to attenuate inflammatory IL6 expression (75).
• Myeloid cells which are involved in acute inflammation after soft tissue injury, have recently been identified as being mechanically sensitive in the context of a number of physiological processes in the body including proprioception, touch, balance, and hearing (3, 76, 77).
• mechanotransduction may affect myeloid transcriptional dynamics and alter healing potential (57, 58, 78), but the mechanisms remain incompletely understood (79).
• disruption of 5 mechanotransduction had a greater effect on myeloid cells than fibroblasts at early time points by reducing inflammatory recruitment and promoting CXCL10- mediated anti-inflammatory transcriptional profiles.
• RNA velocity analysis using scVelo and CellRank (FIG.6, A and B), which combines RNA velocity information with transcriptomic similarity to compute a global map of cellular fate potentials uncovering initial !O and terminal cell states (FIG. 13A) (80, 81).
• CellRank identified six transcriptionally distinct cell lineages (FIG.6B), which originated at the initial root state (labeled with * ).
• Fibroblasts from FAKI- treated STSGs exhibited transcriptional similarity to those from unwounded skin, primarily in lineages 1 and 2, whereas cells from normal STSGs shifted away from this [Skin & STSG+FAKI] cell state along the UMAP-1 axis into four more heterogenous lineages (3,4,5, and 6) (FIG. 6, A !5 and B; FIG. 13, A to C).
• Fibroblast lineages 3 and 4 showed a higher latent time score and velocity vector length (FIG. 6C; FIG. 13D) indicating more advanced differentiation states with a higher proportion of mature spliced RNA.
• lineages 1,2, 3, and 4 were selected for further analysis.
• Aggrecan is a chondrocyte marker, while the family of THBS genes encode for thrombospondins, ECM proteins that facilitate cell-matrix binding and are known to modulate mesenchymal chondrogenic and adipogenic differentiation states 85).
• Comparison of the top DEGs in STSGs also revealed upregulation of previously identified pro-fibrotic genes, such as SFRP2 and TGFB1 (FIG. 14, A to C) ⁇ 86, 87).
• SFF1P2 has been previously identified in healthy human skin scRNA-seq as a fibroblast subpopulation with high fibrogenic potential ⁇ 46-48), while transforming growth factor (TGF ⁇ 1 is a well-known promoter of fibrosis ⁇ 88).
• Apolipoproteins such as APOE and APOD, are key markers of lipid transport and lipid metabolism that are expressed in both lipid trafficking fibroblasts (lipofibroblasts) and adipocytes ⁇ 92, 93). Apolipoproteins have been found to attenuate inflammation, and lipofibroblasts have been found to be a fibroblast subpopulation that interacts with adipose tissue and can differentiate into adipocytes during normal tissue healing ⁇ 94). These lipofibroblasts also expressed PPARG and CFD (complement factor D, encoding for Adipsin), which also promote lipid accumulation and are critical transcription factors of adipogenesis (FIG.6E).
• PPARG and CFD complement factor D, encoding for Adipsin
• FAK inhibition in STSG also promoted adipogenic gene sets (adipogenesis, angiogenesis, epithelial cell migration) and stem cell markers ⁇ CD34 and NT5E) (FIG.6E; FIG. 14, B to D).
• the expression of these markers matched the expression seen in unwounded skin.
• thrombospondins have been found to drive hypertrophic scar formation in a T ⁇ Rb1 dependent manner (95), and T ⁇ Rb1 has been found to inhibit adipogenesis ⁇ 88).
• This down regulation of mechanotransduction, thrombospondins, and TGF was further supported by an upregulation of small leucine-rich proteoglycans (SLRPs) such as decorin ( DCN) (FIG. 6D, FIG. 14A), which helps control the fibrillogenesis of scar formation, is highly expressed in unwounded skin compared to fibrotic tissue, and inhibits TQRb1 to reduce HTS formation (87).
• SLRPs small leucine-rich prote
• FAK inhibition attenuated THBS4 and CXCL14 expression (FIG. 7, A and B), decreasing cellular recruitment and subsequent fibrotic matrix deposition.
• Disruption of mechanotransduction also initiated CD34 expression at late time points (FIG.7C), demonstrating 5 a shift of fibroblast transcriptional profiles toward more plastic, stem-like phenotypes.
• These stem like fibroblasts subsequently demonstrated increased adipocyte lipid trafficking apolipoprotein E (protein form of APOE) at both PODs 14 ( * P ⁇ 0.05) and 90 ( * P ⁇ 0.05) (FIG. 7D), indicating an increased differentiation toward lipofibroblast, regenerative phenotypes.
• strained fibroblasts were advanced in both latent time and velocity pseudotime, demonstrating large changes in transcription induced by mechanical strain (FIG. 8F).
• Strained I0 fibroblasts upregulated CXCL14, fibrotic thrombospondin ⁇ THBS2), and collagen (COL1A1) expression and FAK inhibition abrogated all of these pro-fibrotic responses and instead demonstrated a regenerative transcriptomic signature characterized by an upregulation of APOE and the anti-fibrotic genes EGR1 (early growth response 1) and PRDX1 (peroxiredoxin 1) (FIG. 8, H and I; FIG. 17), matching both our STSG (FIG. 5 and 6) and human findings (FIG. 8, A to C).
• fibroblast transcriptional states have not previously been investigated in the context of large animal fibrosis or as part of skin graft healing.
• human scRNA-seq several groups have previously characterized unwounded human skin ⁇ 46-48) and compared human keloid to human scar without unwounded skin (49).
• direct comparisons of fibrotic tissue to unwounded skin or to pharmacologically enriched regenerative populations with single cell resolution have been lacking.
• Thrombospondins have also been found to promote osteo/chondrogenesis and inhibit adipogenesis in mesenchymal cells ⁇ 105, 106).
• fibroblasts did not demonstrate large changes in transcriptional activity during the early stages of healing. Instead, at earlier time points, disruption of mechanotransduction primarily pushed myeloid-lineage cells toward CXCUO-mediated anti inflammatory and regenerative states. Whereas previous studies focused on mechanoresponsive fibroblasts as the “drivers” of fibrosis (25), secreting chemokines to promote inflammatory cell recruitment (25), our findings revealed a more complicated interplay between immune cell and fibroblast mechanotransduction signaling during the different stages of healing. Controlling mechanical signaling in myeloid cells at early time points modulated secretion of inflammatory signals that could directly influence fibroblast phenotypes.
• antibiotics were given prophylactically and post- operatively.
• Cefazolin 25 mg/kg IV was given 60 minutes prior to the initial insult and repeated at 12 hours after the end of the surgery.
• Oral amoxicillin 10 mg/kg was given twice a day for one week. Wounds were wrapped in the appropriate dressings. Control of pain was achieved by the administration of transdermal fentanyl 50 mcg/h 24 hours in advance of surgery.
• Hydromorphone (0.05 mg/kg) IM was used if the fentanyl patch came off or was not placed prior to surgery.
• Carprofen was used once post-operatively, followed by once every 24 hours for 1-2 days, then as needed based on pain assessment.
• the mixture was poured into silicon molds and then allowed to dry overnight in a sterile hood at room temperature. Dried films were washed with deionized water to remove non-crosslinked polymers, NaOH, and KCI. The pH of the wash solution was measured and continually washed until it reached a pH between 7.0 to 7.5. The swollen hydrogels were frozen at -80°C before lyophilization to produce dry (blank) patches.
• FAKI compound was obtained from Verastem Oncology (VS-6062) and Selleckchem (625249). We dissolved FAKI in acetone at 1 mg/mL; 1 mL of the solution was poured uniformly on the porous hydrogel and then allowed to evaporate the solvent in a sterile hood. Blank and FAKI-containing hydrogel patches were placed in individual plastic bags and sterilized using e- beam irradiation at a 20kGy irradiation dose.
• Cutometer assessment is one of the most common instruments to measure viscoelasticity in human patients ⁇ 111, 112), measuring the vertical deformation of the skin surface by applying negative pressure (suction) through a small circular diameter (8 mm probe). Deformation (suction) for two seconds followed by two seconds of relaxation (no suction) was applied three times and averaged. The firmness was measured as the amplitude at the end of the suction phase (R0 metric) and normalized against the values of STSG treated with blank hydrogels ⁇ 111). Increased deformation to a consistent negative pressure corresponds to less firm tissue, more similar to unwounded skin.
• Porcine specimens were harvested from the center of each wound at the end of the study. Human hypertrophic scar (HTS) and unwounded skin samples were obtained under the approved IRB (#54225). The tissue samples collected from the study would otherwise be discarded. No patient identifying information was retained with the samples. These specimens were immediately fixed in 4% paraformaldehyde, dehydrated, and cryo-embedded in optimal cutting temperature (OCT) compound for frozen sectioning on a microtome-cryostat.
• HTS Human hypertrophic scar
• IRB IRB
• Immunofluorescent 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) (Abeam, ab263898), Apolipoprotein E (APOE) (Abeam, ab52607), CD34 (Abeam, ab81289), and alpha smooth muscle actin (aSMA) (Abeam, ab5694).
• the amount of fluorescent area was quantified and normalized to the number of cells (individual DAPI nuclei) using a custom MATLAB image processing code written by the authors and previously published ⁇ 98). All histology and immunofluorescent images shown are representative images of multiple experiments. k. Single cell barcoding, library preparation, and single cell RNA sequencing
• Tissue solution was subjected to maximum speed vortex mixer (VWR) for 30 seconds before being placed in the oven, after 1 hour, and again after total of 2 hours to physically disrupt any tissue that had clumped together and maximize the tissue surface area exposed to enzymatic digestion at all times.
• VWR maximum speed vortex mixer
• the tissue solution was filtered through a 100 pm Nylon cell filter (Fisher-Scientific 08- 771-19) into a new conical tube, and 20ml_ of 10% FBS DMEM was added through the filter to quench the enzymatic reaction and release any cells trapped within the filter, maximizing downstream cell yield.
• Solutions were spun at 350 x gfor 5 min at 4°C in a centrifuge, supernatant was aspirated, and cells were then resuspended in 20mL 10% FBS DMEM and passed through a 70 pm Nylon cell filter.
• a 20ml_ solution of 10% FBS in PBS (FACS Buffer) was added through the filter to wash the remaining cells.
• This cellular suspension was then resuspended in a concentrated solution and submitted for 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 partitioning oil was loaded onto a single cell chip, processed on the Chromium Controller, and reverse transcription was performed at 53°C for 45min.
• cDNA was amplified for 12 cycles total (BioRad C1000 Touch thermocycler) with cDNA size selected using SpriSelect beads (Beckman Coulter, USA) and a 3:5 ratio of SpriSelect reagent volume to sample volume.
• cDNA was analyzed on an Agilent Bioanalyzer High Sensitivity DNA chip for qualitative control, fragmented for 5min at 32°C, followed by end repair and A-tailing at 65°C for 30min, and then double-sided size selected with SpriSelect. Sequencing adaptors were ligated to the cDNA at 20°C for 15min. cDNA was amplified using a sample-specific index oligo as primer, followed by another round of double-sided size selection. Final libraries were analyzed on an Agilent Bioanalyzer High Sensitivity DNA chip for qualitative control purposes. cDNA libraries were sequenced on a HiSeq 4000 lllumina platform aiming for 50,000 reads per cell.
• RNA velocity analysis was performed using the dynamical model of the scVelo package(SO).
• Partition-based graph abstraction (PAGA) was performed using the sc.tl.paga function in scVelo.
• FAK FAM-related non-kinase inhibits myofibroblast differentiation through differential MAPK activation in a FAK-dependent manner. J Biol Chem 283, 26839-26849 (2008).
• ⁇ 5 adhesion kinase links mechanical force to skin fibrosis via inflammatory signaling.
• RNA-seq Single-cell RNA-seq reveals fibroblast heterogeneity and increased mesenchymal fibroblasts in human fibrotic skin diseases. Nat Commun 12, 3709 (2021).
• Thrombospondin 1 a multifunctional protein implicated in the regulation of tumor growth.
• 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).
• a range includes each individual member.
• a group having 1-3 articles refers to groups having 1 , 2, or 3 articles.
• a group having 1-5 articles refers to groups having 1 , 2, 3, 4, or 5 articles, and so forth.

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