Methods and compositions for improving wound healing

Abstract

The present disclosure relates to compositions and methods for improving wound healing and tissue regeneration. More specifically, the present disclosure relates to compositions that direct an immune response within a wound to a regenerative response and methods of using such compounds. Such compositions and methods are particularly useful for altering immune responses caused by medical implants to avoid scarring and fibrosis at the site of implantation.

Description

[0001] Priority paragraph

[0002] This application claims the benefit of priority under 35 USC §119 to U.S. Provisional Application Serial No. 63 / 367,994, filed on July 8, 2022, and U.S. Provisional Application Serial No. 63 / 488,122, filed on March 2, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0004] This work was supported by the Division of Intramural Research of the National Institute of Biomedical Imaging and Bioengineering, part of the National Institutes of Health. The U.S. Government has certain rights in this invention. Background Art

[0005] The field of the present disclosure generally relates to wound healing and tissue engineering. The goal of tissue engineering is to replace the function of missing or damaged tissues and organs. This can be achieved by using biomaterial scaffolds (such as skin grafts) that integrate and help regenerate damaged or missing tissues and medical device implants made of synthetic materials to replace the function or beauty of the tissue (such as knee replacement) (see, for example, US2019 / 0060524, which is incorporated herein by reference in its entirety). However, at any time when a biomaterial or medical device is implanted in the human body, it changes the body's homeostasis and induces a series of immune responses that can actively lead to scaffold integration and tissue growth or produce immune-mediated pathologies, such as implant fibrosis or excessive inflammation and surrounding tissue damage. Therefore, understanding the mechanism by which our immune system interacts with engineering materials is necessary to produce next-generation medical devices that can work with the immune system.

[0006] The field of evaluating immune responses to engineered materials has developed over the past decade and has produced a large number of discoveries about how immune cells can regulate material acceptance or rejection and fibrosis. In conjunction with the fields of wound healing and developmental biology, several cells have been implicated in these processes. Specifically, type 2 immunity (including eosinophils, M2 macrophages and Th2 T cells and the signaling protein interleukin (IL)-4) has been described in the healing of various tissues (including liver, nerves, skin and muscle). Regulatory T cells and related cytokines (IL-10) have also been described in the healing process and have been used in material acceptance strategies in an attempt to inhibit excessive immune activation. In terms of pathology, Th17 T cells are associated with tissue fibrosis and autoimmunity, and are also associated with device fibrosis in mice and humans. The crosstalk between these immune cells (such as T cells, macrophages and fibroblasts) creates a complex cell signaling network in which multiple factors contribute to regeneration and acceptance or inflammation and fibrosis.

[0007] Although research to date has begun to elucidate the cellular processes involved in wound healing and fibrosis, the treatment success rate using existing methods remains inconsistent, and the treatment of wounds and avoidance of fibrosis remains a challenge. Therefore, there is a need for better and more specific treatments that allow tissue regeneration within wounds without the accompanying fibrosis. The present disclosure addresses this need. Summary of the invention

[0008] One aspect is a method comprising administering at a wound site a therapeutic composition that induces a pro-regenerative environment within the wound and / or within tissue surrounding the wound.

[0009] One aspect is a method of altering the immune response to a wound, the method comprising administering at the wound site a therapeutic composition that induces a pro-regenerative environment within the wound and / or within tissue surrounding the wound.

[0010] One aspect is a method of treating a wound in an individual, the method comprising administering at the wound site a therapeutic composition that induces a pro-regenerative environment within the wound and / or within tissue surrounding the wound.

[0011] One aspect is a method of treating an individual having a wound, the method comprising administering at the wound site a therapeutic composition that induces a pro-regenerative environment within the wound and / or within tissue surrounding the wound.

[0012] One aspect is a method of reducing or preventing fibrosis in a wound, the method comprising administering at the wound site a therapeutic composition that induces a pro-regenerative environment within the wound and / or within tissue surrounding the wound.

[0013] One aspect is a method of implanting a medical device in an individual, the method comprising: introducing the medical device into tissue within the individual, and administering a therapeutic composition at the site of implantation of the medical device, the therapeutic composition inducing a pro-regenerative environment within the wound and / or within the tissue surrounding the wound.

[0014] One aspect is a method of implanting a medical device in an individual, the method comprising introducing the medical device into tissue within the individual, wherein a therapeutic composition is applied to the medical device prior to implantation of the medical device into the individual, the therapeutic composition inducing a pro-regenerative environment within a wound and / or within tissue surrounding a wound.

[0015] In these aspects, the wound can be a burn, contusion, seroma, hematoma, laceration, avulsion, puncture, surgical wound, incision, ulcer, or a wound caused by a crush injury. In these aspects, administering the therapeutic composition can include introducing the therapeutic composition into the wound cavity and / or into the tissue surrounding the wound cavity, which can include applying (e.g., topically) the therapeutic composition to the skin surrounding the wound cavity.

[0016] In these aspects, therapeutic composition can include ointment, spray, lotion, gel, cream, foam, solution, suspension, emulsion, hydrogel or paste, which can include nanoparticles or microspheres. In these aspects, therapeutic composition can be formulated into quick release composition or slow release composition. In these aspects, therapeutic composition can release therapeutic agent in about 5 minutes, in about 10 minutes, in about 30 minutes, in about one hour, in about two hours, in about 12 hours, in about 24 hours, in about 48 hours, in about 5 days, in about one week, or in about one month or longer.

[0017] In these aspects, the therapeutic composition may include a therapeutic agent that induces a pro-regenerative environment. The therapeutic agent may direct the immune response away from a TH1 type response. The therapeutic agent may induce a TH2 driven immune environment. The therapeutic agent may increase the number of M2 macrophages in the wound and / or in the tissue surrounding the wound. The therapeutic agent may induce an influx of M2 macrophages in the wound and / or in the tissue surrounding the wound, and / or it may induce local proliferation of M2 macrophages in the wound and / or in the tissue surrounding the wound. The therapeutic agent may induce an increase in the number of classical dendritic cells (cDC1) in the wound and / or in the tissue surrounding the wound. The therapeutic agent may induce an influx of classical dendritic cells (cDC1) in the wound and / or in the tissue surrounding the wound, and / or it may induce local proliferation of cDC1 in the wound and / or in the tissue surrounding the wound. The cDC1 may be a cross presenting dendritic cell. The cDC1 may be an XCR1 + CD103 + Dendritic cells.

[0018] In these aspects, therapeutic agent can include peptide, protein, glycoprotein, lipoprotein, lipid, sugar, polysaccharide, glycosaminoglycan, nucleic acid molecule, organic molecule and combination thereof. In these aspects, therapeutic agent can include decellularized extracellular matrix (ECM) or component derived from decellularized extracellular matrix, and the component derived from decellularized extracellular matrix can include degradation product of ECM. In these aspects, therapeutic agent can include one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan, agrin and degradation product thereof. The therapeutic agent may comprise an extracellular matrix activator (matrikine) which may be selected from the group consisting of: metastatin, aff esten, angiostatin, tetrastatin, pentastatin, lamstatin, hexastatin, endostatin, restin 1, restin 2, restin 3, restin 4, endostatin, neostatin, anastellin, sibsttin, PEX, endor epellin, CUB1CUB2 domain, Ten / 2, Ten11 / 12 / 13, Ten14, kappa-elastin, the extracellular domain of syndecan-1, the extracellular domain of syndecan-2, the extracellular domain of syndecan-3, the extracellular domain of syndecan-4, elas tokine, laminin peptide A13, laminin peptide C16, laminin 332 (laminin 5), DGGRYY peptide, GHK tripeptide, VGVAPG peptide, PGP tripeptide, acetylated PGP tripeptide (AcPGP), tenascin-C (TNC), G3 domain of nidogen-1, tumor inhibitory factor, and any combination thereof. In certain aspects, the therapeutic agent may comprise a DAMP.

[0019] In some aspects, the medical device can be an implant, which can be selected from the group consisting of: breast implants, stents, ports, shunts, hip implants, knee implants, cochlear implants, hernias or other, surgical meshes, intraocular lens implants, pacemakers, metal / surgical screws, rods or pins, artificial discs, and spinal fusion hardware. In some aspects, the implant can include metals and metal alloys, plastic polymers, ceramics, hydrogels, and composite materials, which can include, but are not limited to, silicone, polyethylene, stainless steel, titanium, zirconium oxide, polyurethane foam, polylactic acid, amalgam, gold, alumina, silicates, chromium alloys, cobalt, and molybdenum.

[0020] One aspect is a therapeutic composition for treating a wound, wherein the therapeutic composition includes a therapeutic agent that induces a pro-regeneration environment in the wound and / or in the tissue around the wound. The therapeutic composition may include an ointment, spray, lotion, gel, cream, foam, solution, suspension, emulsion, hydrogel or paste, which may include nanoparticles or microspheres. The therapeutic composition may be formulated into a quick-release composition or a slow-release composition. The therapeutic composition may release the therapeutic agent in about 5 minutes, in about 10 minutes, in about 30 minutes, in about one hour, in about two hours, in about 12 hours, in about 24 hours, in about 48 hours, in about 5 days, in about one week, or in about one month or longer. In some aspects, the therapeutic composition may include a therapeutic agent that induces a pro-regeneration environment. The therapeutic agent may guide the immune response away from the TH1 type reaction. The therapeutic agent may induce an immune environment driven by TH2. The therapeutic agent may increase the number of M2 macrophages in the wound and / or in the tissue around the wound. The therapeutic agent may induce an influx of M2 macrophages within the wound and / or within the tissue surrounding the wound, and / or it may induce local proliferation of M2 macrophages within the wound and / or within the tissue surrounding the wound. The therapeutic agent may induce an increase in the number of classical dendritic cells (cDC1) within the wound and / or within the tissue surrounding the wound. The therapeutic agent may induce an influx of classical dendritic cells (cDC1) within the wound and / or within the tissue surrounding the wound, and / or it may induce local proliferation of cDC1 within the wound and / or within the tissue surrounding the wound. The cDC1 may be a cross-presenting dendritic cell. The cDC1 may be an XCR1 + CD103 +Dendritic cells. Therapeutic agents can include peptides, proteins, glycoproteins, lipoproteins, lipids, sugars, polysaccharides, glycosaminoglycans, nucleic acid molecules, organic molecules and combinations thereof. In some aspects, the therapeutic agent can include acellular extracellular matrix (ECM) or a component derived from the acellular extracellular matrix, and the component derived from the acellular extracellular matrix can include the degradation products of ECM. The therapeutic agent can include one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan, aggregated protein and its degradation products. The therapeutic agent may comprise an extracellular matrix activator selected from the group consisting of metastatin, affesten, angiostatin, tetrastatin, pentastatin, lamstatin, hexastatin, endostatin, dormant protein 1, dormant protein 2, dormant protein 3, dormant protein 4, endostatin, neostatin, anastellin, sibsttin, PEX, endorepellin, CUB1CUB2 domain, Ten / 2, Ten / 4. 11 / 12 / 13, Ten14, kappa-elastin, the extracellular domain of syndecan-1, the extracellular domain of syndecan-2, the extracellular domain of syndecan-3, the extracellular domain of syndecan-4, elastokine, laminin peptide A13, laminin peptide C16, laminin 332 (laminin 5), DGGRYY peptide, GHK tripeptide, VGVAPG peptide, PGP tripeptide, acetylated PGP tripeptide (AcPGP), tenascin-C (TNC), the G3 domain of nidogen-1, tumor inhibitor, and any combination thereof. The therapeutic agent may comprise a DAMP.

[0021] One aspect of the present disclosure is a method of recruiting NK cells to a wound or wound adjacent tissue, the method comprising administering a therapeutic agent or therapeutic composition of the present disclosure at the wound site.

[0022] One aspect of the present disclosure is a method of activating NK cells, the method comprising contacting the NK cells with a therapeutic agent of the present disclosure.

[0023] One aspect of the present disclosure is a method of inducing increased expression of Xcl1 in NK cells, the method comprising contacting the NK cells with a therapeutic agent of the present disclosure.

[0024] One aspect of the present disclosure is a kit comprising the therapeutic composition of the present disclosure. The kit may also include a needle, a syringe, a vial, an applicator, and instructions for using the therapeutic composition to treat a wound.

[0025] One aspect of the present disclosure is a therapeutic composition of the present disclosure when used to treat a wound, wherein the therapeutic composition induces a pro-regenerative environment in the wound and / or in the tissue surrounding the wound.

[0026] One aspect of the present disclosure is a therapeutic composition of the present disclosure when used to alter the immune response to a wound, wherein the therapeutic composition induces a pro-regenerative environment in the wound and / or in the tissue surrounding the wound.

[0027] One aspect of the present disclosure is a therapeutic composition of the present disclosure when used to reduce or prevent wound fibrosis, wherein the therapeutic composition induces a pro-regenerative environment in the wound and / or in the tissue surrounding the wound. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The patent or application file contains at least one drawing executed in color. Copies of the patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0029] Figure 1 Gating strategy for bone marrow panel is shown. Representative plots and gates of samples stained with 22-color bone marrow phenotyping panel. Example data from 7 days post injury.

[0030] Figures 2A-2C show immune cell infiltration into muscle lesions. Cells were counted on a hemocytometer before flow staining, demonstrating live immune cell counts. Figure 2A shows individual values ​​for each mouse; Figure 2B shows mean ± SEM. Figure 2C Shown is hematoxylin and eosin staining of muscle damage 7 days after injury.

[0031] Figures 3A-3D Pro-regenerative and pro-fibrotic materials are shown to recruit a variety of different innate immune cells. Prevalence of innate immune cells in biomaterial-treated muscle injuries. Figure 3A : t-SNE (left) for manually gated populations displayed 7 days after injury; Uniform Manifold Approximation and Projection (UMAP) (middle); FlowSOM (right) dimensionality reduction calculation. Macrophages = red, dendritic cells = blue, monocytes = green, eosinophils = light blue, basophils = black, neutrophils = orange, other immune cells = purple. Figure 3B : t-SNE (left), UMAP (middle) and FlowSOM (right) dimensionality reduction calculations displayed for injury treatment types. Control = black, ECM-tx = cyan, PE-tx = pink. Figure 3C : t-SNE (left), UMAP (center) and FlowSOM (right) dimensionality reduction calculations for computer-derived clusters (FlowSOM). Figure 3DIn silico phenotypic analysis of clusters is shown. Expression = scaled fluorescence intensity.

[0032] Figures 4A-4E Immune cell populations over time are shown as percentages of live CD45+ immune cells. Basophils: CD11b-CD200R3+, eosinophils: CD11b+Siglec-F+, neutrophils: CD11b+Ly6G+, total monocytes (CD11b+Ly6C+), classical monocytes Ly6Chi, CX3CR1lo, alternative monocytes: Ly6C+CX3CR1hi, non-DC antigen presenting cells (CD11b-CD11c-MHCII+), dendritic cells: CD11b-CD11c+MHCII+, total macrophages: CD11b+CD68 and / or F4 / 80+, macrophages: F4 / 80+, macrophages F4 / 80+CD68+, macrophages: CD68+. Control = black, ECM treated = cyan, PE treated = pink. Data are mean ± 95% confidence interval, n = 5.

[0033] Figure 5A and 5B Shown is the phenotypic analysis of MHCII+ antigen presenting cells in the wound microenvironment 7 days after injury. Figure 5A The percentage of MHCII+ cells identified as the listed immune cell types is shown. Figure 5B The proportion of each treatment group represented by different cell types is shown. SD = standard deviation. Black = neutrophils, pink = eosinophils, cyan = macrophages, purple = basophils, lavender = dendritic cells, blue = monocytes, gray = other immune cells. ANOVA with Tukey post hoc correction for multiple comparisons, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001.

[0034] Figures 6A-6C Phenotyping markers for macrophages and dendritic cells are shown. Fig. 6A -MHCII+ macrophages (F4 / 80+CD68+MHCII+). Figure 6B -MHCII- macrophages (F4 / 80+CD68+MHCII-). Figure 6C - Dendritic cells (CD11c+MHCII+). Black / grey = control lesions; cyan = ECM-treated lesions; pink = PE-treated lesions. n=5 Data are shown as individual points, bars are mean values.

[0035] Figures 7A-7H Cross-presenting dendritic cells are shown to be enriched by the pro-regenerative scaffold and peak at 7 days after injury. Fig. 7A Representative dot plots of CD45+CD11blo / -CD11c+MHCII+ dendritic cells 7 days after injury are shown. The four quadrants correspond to: Q1 = CD103-XCR1+, Q2 = CD103+XCR1+, Q3 = CD103+XCR1-, and Q4 = CD103-XCR1-. Figure 7B The proportions (%) of dendritic cells in the four quadrants at 3 days, 7 days, 21 days, and 42 days after injury are shown. Figure 7C Dendritic cell counts (in 10,000s) per quadrant at 3, 7, 21, and 42 days after injury are shown. Fig.7D tSNE is shown. Fig. 7E Shown is a UMAP of CD45+ immune cells 7 days post-injury for the three treatment groups, with the cDC1 population highlighted in red. Figure 7F The proportions of cDC1 populations from the three treatment groups are shown. Figure 7G Shown is the phenotypic analysis of 30 distinct immune cell clusters identified by the FlowSOM algorithm, normalized within each marker with min=0, max=100. Figure 7H FlowSOM scaled expression values ​​on different clusters, cluster 22 (cDC1) highlighted in red. Control = black, ECM treated = cyan, PE treated = pink. CD103+XCR1+cDC = red. Data are mean ± 95% confidence interval, n = 5 represents at least two independent experiments.

[0036] Figure 8 A-8D shows that ( Figure 8 A) 3 days after injury, ( Figure 8 B) 7 days after injury, ( Figure 8 C) 21 days after injury and ( Figure 8 D) Dendritic cell subsets 42 days after injury. ANOVA with Tukey post hoc correction for multiple comparisons, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001. Data are mean ± SEM, n = 5, representative of at least two independent experiments.

[0037] Figures 9A-9C CD103 and XCR1 expression on myeloid cells is shown. Fig.9A The mean fluorescence intensity of CD103 is shown. Fig. 9B Shown are the mean fluorescence intensities of XCR1 on dendritic cells (black), MHCII+ macrophages (blue), and MHCII- macrophages (yellow). Fig. 9CShown are F4 / 80- and F4 / 80+ fractions of XCR1+ cells purified (by MACS column) from ECM-treated VMLs 7 days after injury. Red = actin / phalloidin, blue = DAPI. N = 4–5, ANOVA with Tukey post hoc correction for multiple comparisons, * = P < 0.05, ** = P < 0.01, **** = P < 0.0001.

[0038] Fig.10 CD86 expression on cDC1 is shown. Representative FACS plots of CD11c+MHCII+ dendritic cells in the three treatment groups. Green = XCR1+CD103+cDC1; Purple = XCR1-CD103- dendritic cells.

[0039] Fig.11A and 11B The reproducibility of the findings across litters, backgrounds, and species is shown. XCR1+CD103+ cells are present in Fig.11A ) different nests and ( Fig. 11B ) species. Student's T-test with Tukey post hoc correction, ns = not significant. Rat data extracted from Adusei et al., Cells, Tissues, and Organs, 2022.

[0040] Fig.12 Shown are CD103+XCR1+ dendritic cells in the skin overlying muscle lesions. Proportion of CD11c+MHCII+ dendritic cells in ECM-treated mice 7 days after injury. Data are mean ± SD, n = 3-5. Student's t-test, p = 0.0003.

[0041] Fig.13A and 13B The phenotype of dendritic cells in local tissues, blood, and draining lymph nodes is shown. Fig.13A Representative FACS plots are shown, color scale = CD8a expression. Fig. 13B : Purple = local muscle tissue, green = peripheral blood, blue = inguinal lymph nodes (LN).

[0042] Figures 14A-14C Shown that treatment with locally injured profibrotic material enriched peripheral plasmacytoid DCs. (a) Innate-like cells in draining lymph nodes. (b) Innate-like cells in blood. (c) B220 / CD45R expression on dendritic cells in blood and LNs. * = P < 0.05; ** = P < 0.01, *** = P < 0.001. n = 5, ANOVA with Tukey post hoc.

[0043] Fig.15XCL-1 levels in peripheral blood 7 and 21 days after injury are shown. Y axis = pg / ml in plasma, X axis = weeks after injury. Black = control, cyan = ECM-tx, pink = PE-tx. Data are mean ± SEM, n = 5. Student's T test, ** = P < 0.01. No significant differences between treatment groups.

[0044] Figures 16A-16J It was shown that type 2 myeloid markers were enhanced on cross-presenting dendritic cells by pro-regenerative scaffold treatment and were dependent on adaptive immunity. Fig.16A The prevalence of CD11c in muscle tissue is shown as the proportion of CD45+ live immune cells. Fig. 16B CD103+XCR1+ dendritic cells are shown. Fig. 16C CD103+ / -XCR1+ dendritic cells are shown. Fig.16D CD103+XCR1+ / - dendritic cells are shown. Fig.16E The gating strategy for dendritic cells is shown. Fig.16F Representative FACS plots are shown. Blue = wild type C57BL / 6J mice, red = Rag1- / - B6 mice. Figure 16G Shown is the expression of CD301b on F4 / 80+ macrophages in wild-type (WT) and RAG-deficient mice (Rag1- / -). Fig.16H Shown is the expression of CD301b on cDC1 in WT and Rag1- / - mice. Fig.16I Shown is the expression of CD206 on F4 / 80+ macrophages in WT and Rag1- / - mice. Fig.16J CD206 expression on cDC1 in WT and Rag1- / - mice is shown. ANOVA with Tukey post hoc correction for multiple comparisons, *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001, n=5.

[0045] Fig.17 The lymphoid group gating strategy is shown. A representative sample from an inguinal lymph node 21 days after injury is shown.

[0046] Figures 18A-18F It was shown that T cell activation and the prevalence of HELIOS+iTregs were temporally modulated and differed between draining lymph nodes and peripheral blood. Figures 18A-18B Shown are the results of 7 days after injury ( Fig.18A ) and 21 days ( Fig.18B ) activated T cells in the draining (inguinal) lymph nodes (ILN) and peripheral blood. Figures 18C-18F Shown are the results of 7 days after injury ( Figures 18C-18D ) and 21 days after injury ( Figures 18E-18F ) The proportion of FoxP3+ and HELIOS+ iTreg in ILN (18C and 18E) and peripheral blood (18D and 18F). ANOVA with Tukey post hoc correction for multiple comparisons, *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001. n=5.

[0047] Fig.19 HELIOS+ regulatory CD8 over time is shown. Dark grey = 7 days post injury. Light grey = 21 days post injury. ANOVA with Kruskal-Wallis FDR post hoc.

[0048] Fig. 20A and 20B Shown are ST2-expressing B cells and pDCs in lymph nodes following injury.

[0049] Figures 21A-21E Shown is that trauma induces proliferation and activation of CD103+XCR1+ adaptive immune cells in draining lymph nodes. Fig.21A Representative dot plots of draining (inguinal) lymph nodes of uninjured mice and DLN of injured mice 21 days after injury. B cells = CD45 + CD11c-CD11b-CD3-B220 +, T cells = CD45 + B220-CD11c-TCRb + CD 3 + CD49b-CD4 + (CD4 + T cells) or CD8 + (CD8 + T cells), yd T cells = CD45 + B220-TCRb-CD49b-TCRyd +. Fig.21B The proportion of CD103+XCR 1+ adaptive immune cells is shown. Fig. 21C CD62L - The proportion of CD103+XCR 1+ adaptive immune cells. Fig.21D The proportion of CD103loXCR1- adaptive immune cells is shown. Fig.21E The proportion of CD62L-CD103loXCR1- adaptive immune cells is shown. Control = black, ECM = cyan, PE = pink. Data are mean ± 95% confidence interval, n = 3-5.

[0050] Fig. 22 Shows expansion of active CD103+XCR1+ adaptive immune cell population in draining lymph nodes after injury. Cells were isolated from inguinal lymph nodes. Upper panel = proportion of cells that are CD103+XCR1+, lower panel = proportion of CD103+XCR1+ adaptive immune cells that are CD62L- (active). Uninjured = grey Control lesion = black. N = 3 (unlesioned), N = 5 (control lesioned). All comparisons (unlesioned vs. control lesioned) were significantly different at P < 0.05, ANOVA with Tukey post hoc correction for multiple comparisons.

[0051] Fig.23 CD44 and CD62L expression on T cells is shown. Representative FACS plots show CD62L and CD44 expression on CD4+ T cells that are double positive (blue) or double negative (grey) for CD103 and XCR1.

[0052] Figures 24A-24C Shown is the expression of FoxP3+( Fig.24A ) and HELIOS+( Fig. 24B and 24C ) regulates CD103 and XCR1 expression on CD4 and CD8 T cells. Black = control lesions, cyan = ECM-treated, pink = PE-treated. Data are mean ± SEM, n = 5.

[0053] Fig.25 Comparison of CD103+XCR1+ adaptive immune cells in draining lymph nodes and peripheral blood at 7 and 21 days after injury is shown. Proportion of adaptive immune cells positive for both XCR1 and CD103 in draining (inguinal) lymph nodes (ILN) and peripheral blood. Data are mean ± SEM, n = 4-5

[0054] Figures 26A-26D Upregulation of Xcl1 and E-cadherin in muscle was shown to be associated with tissue damage and material implantation. Fig.26A Relative quantification of Xcl1 mRNA is shown as fold change relative to uninjured control (2 -ΔΔCt ). Fig.26B Shown are CD103 cells found at the material and injury interface, scale bar = 75 um. Fig.26C Quantification of E-cadherin expression at distal / uninjured muscle tissue, injury interface, material itself, and external overlaying capsule quantified by DAB is shown. N / A = not applicable for control injury or uninjured samples. Data are mean ± SEM, n = 3-5. Fig.26D Representative images of hematoxylin and eosin (H&E) and E-cadherin (DAB) staining at the injury interface are shown. Scale bar = 300 um.

[0055] Fig. 27 Cytokine and chemokine profiles of local muscle tissue 7 days after injury are shown. Shown as fold change relative to uninjured control, Control (Injury) = black, ECM = cyan, PE = pink. Representative of n = 5 blots.

[0056] Fig.28 Proposed mechanisms of tissue homeostasis and injury response through communication between CD103+XCR1+ innate and adaptive immune cells are shown.

[0057] Figures 29A-29C Tolerogenic natural killer cells are shown to be recruited to tissue damage and induced by DAMPs. Fig.29A : Gene expression in cells isolated from muscle injury shows that NK cells have high expression of the Tgfb1 gene. NK cells that secrete Tgfb1 are associated with a tolerogenic phenotype. Fig.29B : Shows that NK cells have >350-fold higher gene expression of Xcl1 (a chemokine that binds XCR1) compared to other adaptive immune cells in the microenvironment. Fig.29C Shown are Xcl1 expression levels in NK cells exposed to decellularized extracellular matrix (ECM) fragments or low molecular weight hyaluronic acid (LMW-HA) in vitro.

[0058] Fig.30 A potential mechanism for the recruitment of tolerogenic dendritic cells (DCs) into the lesion compartment is shown. DETAILED DESCRIPTION OF THE INVENTION

[0060] The present disclosure relates to methods and compositions for improving wound healing. More specifically, the present disclosure relates to compositions that guide the immune response in the wound to a pro-regenerative response and methods for using such compounds. The disclosed compositions and methods are particularly useful for changing the immune response caused by the response to the implant device, which typically causes scarring and fibrosis at the implant site. Therefore, the method of the present disclosure can generally be implemented by introducing the composition of the present disclosure at the wound site, which induces a pro-regenerative environment in the wound. In some aspects, the pro-regenerative environment comprises new dendritic cells as described herein.

[0061] Before further describing the present disclosure, it should be understood that the present disclosure is not limited to the specific embodiments described, as such may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, as the scope of the present disclosure will be limited only by the claims.

[0062] It must be noted that, as used herein and in the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly indicates otherwise. For example, a compound refers to one or more compound molecules. Thus, the terms "a / an", "one or more", and "at least one" may be used interchangeably. Similarly, the terms "comprising", "including", and "having" may be used interchangeably. It should also be noted that the claims may be drafted to exclude any optional elements. Therefore, this description is intended to serve as a priori basis for the use of such exclusive terms as "only", "only", etc., in connection with the statement of claim elements or the use of a "negative" limitation.

[0063] Publications disclosed herein are provided only because they are disclosed prior to the filing date of the present application. Anything herein should not be construed as admitting that the present disclosure is not entitled to be earlier than such publications by virtue of prior disclosure. In addition, the publication date provided may be different from the actual publication date, which may require independent confirmation. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials related to the publications cited.

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present disclosure pertains. Although any methods and materials similar or identical to the methods and materials described herein may also be used in the practice or testing of the present disclosure, preferred methods and materials are now described. Terms and phrases common to the various aspects disclosed herein are defined below.

[0065] Wound healing includes multiple growth factors and cytokines that regulate cell growth, cell differentiation and cell proliferation. Although wound healing is usually carried out in stages, these stages may overlap to some extent. After tissue damage, epithelial cells and / or endothelial cells release inflammatory mediators, which start antifibrinolysis-coagulation cascade reaction, which causes blood clot formation. Next is the inflammation and proliferation stage, in which leukocytes are recruited, then activated and induced to proliferate by chemokines and growth factors. Activated leukocytes secrete profibrotic cytokines, such as IL-13 and TGF-b. Stimulated epithelial cells, endothelial cells and myofibroblasts produce matrix metalloproteinases (MMPs) that destroy basement membranes, and recruit and activate other cytokines and chemokines of important components of tissue regeneration, neutrophils, macrophages, T cells, B cells and eosinophils. Activated macrophages and neutrophils remove tissue debris, dead cells and the organism of invasion. Soon after the initial inflammatory stage, myofibroblasts produce ECM components, and endothelial cells form new blood vessels. Myofibroblasts can be derived from local mesenchymal cells, raised from the bone marrow (in the bone marrow they are called fibroblasts), or they can be derived from epithelial-mesenchymal transition (EMT). In subsequent remodeling and maturation stages, activated myofibroblasts stimulate wound contraction. Collagen fibers also become more organized, blood vessels return to normal, scar tissue is eliminated, and epithelial and / or endothelial cells divide and migrate through the basal layer to regenerate epithelium or endothelium respectively, restoring damaged tissue to normal appearance. The above process generally describes the pro-regeneration response to wounds. However, in some cases, such as chronic wounds, the normal healing process is destroyed. Continuous inflammation, tissue necrosis and infection lead to chronic activation of myofibroblasts and excessive accumulation of ECM components, which promote the formation of permanent fibrotic scars. However, the inventors have found that by treating wounds with appropriate compositions, the immune response in the wound can be guided away from fibrotic reactions and turned to pro-regeneration reactions.

[0066] One aspect of the present disclosure is a method comprising administering at a wound site a therapeutic composition that induces a pro-regenerative environment within the wound and / or within tissue surrounding the wound.

[0067] The term "wound" refers to damage to the integrity of biological tissues, such as skin (including epidermis, dermis and hypodermis), mucous membranes and organ tissues (e.g., muscle, lung tissue, etc.). Wounds suitable for use with the methods of the present disclosure may be closed wounds, or they may be open wounds. Wounds of the present disclosure include, but are not limited to, burns, contusions, seromas, hematomas, lacerations, avulsions, punctures, surgical wounds (e.g., incisions), ulcers, and wounds caused by crushing injuries. Therefore, in one aspect, the wound is selected from the group consisting of burns, contusions, seromas, hematomas, lacerations, avulsions, punctures, surgical wounds (e.g., incisions), ulcers, or wounds caused by crushing injuries.

[0068] "Applying at the wound site", "applying to the wound" and the like means introducing the therapeutic composition into the cavity (e.g., incision) of the wound, and / or into the tissue surrounding the wound cavity. It is well known that although the most obvious sign of a wound (such as an incision) is the open cavity thus created, the tissue surrounding the cavity is also affected, and the infiltration and proliferation of immune cells in such tissue contribute to the healing of the wound. Therefore, in some aspects, the introduction of the therapeutic composition may include or exclude the tissue surrounding the wound cavity. In some aspects, the location of introduction may be determined based on the physical properties of the therapeutic composition. For example, if the composition is a cream or foam, it is best to apply the composition to the skin surface in the wound cavity. Alternatively, if the composition is a liquid, it may be applied to the skin in the wound cavity and / or it may be injected into the tissue in or around the wound cavity.

[0069] A "therapeutic composition" is a composition comprising a therapeutic agent that induces a pro-regenerative environment at and around the location where the therapeutic composition is administered to an individual. The therapeutic composition of the present disclosure may include any formulation suitable for delivering a therapeutic agent such that the therapeutic agent can induce a pro-regenerative environment. In some aspects, the therapeutic composition may be formulated as an ointment, spray, lotion, gel, cream, foam, solution, suspension, emulsion, hydrogel, or paste. In some aspects, the therapeutic composition may include liposomes, microspheres, or nanoparticles.

[0070] In certain aspects, the therapeutic composition can comprise at least one additional agent (eg, a pharmacologically acceptable excipient) such as a buffer, a stabilizer, a chelating agent, an antioxidant, a preservative, and any mixtures thereof.

[0071] In certain aspects, the therapeutic composition can be formulated as a slow-release composition. A slow-release composition is a composition that releases an active ingredient (e.g., a therapeutic agent) slowly over a period of time rather than all at once. Methods for preparing slow-release compositions are known to those skilled in the art.

[0072] "Therapeutic agent" refers to a molecule or combination of molecules that, when applied to an individual, induces a pro-regenerative response at the location where the therapeutic composition is applied to the individual and preferably adjacent to the location. The therapeutic agent can be any type of molecule that can induce a pro-regenerative environment. In some aspects, the therapeutic agent can include peptides, proteins, glycoproteins, lipoproteins, lipids, sugars, polysaccharides, glycosaminoglycans, nucleic acid molecules, organic molecules, or any combination thereof. Therefore, the therapeutic agent can include one or more than one type of molecule. The therapeutic agent of the present disclosure can be separated from biological materials (e.g., cells, organisms, etc.), it can be produced using recombinant DNA technology, it can be chemically synthesized, or it can be produced using a combination of such technologies. The term "separation" does not mean a specific degree of separation.

[0073] In some aspects, therapeutic agent can comprise decellularized extracellular matrix (ECM) or be derived from one or more components of decellularized extracellular matrix.ECM is the non-cellular part of tissue and organ, comprises the network of protein (for example, collagen, elastin, laminin) and other molecules (such as proteoglycan and polysaccharide).After damage, enzyme (particularly MMP) is activated, and the protein of these activated enzyme degradation ECM, such as collagen and elastin.These degradation products play the effect of limiting the migration of matrix cells and accelerating fibroblast proliferation among others.Therefore, in some aspects, therapeutic agent can comprise one or more components of ECM, or be derived from one or more components thereof.In some aspects, therapeutic agent can comprise one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan and aggregated protein.

[0074] The dynamic remodeling of ECM is required for wound healing. During remodeling, ECM undergoes a proteolytic process to release bioactive matrix fragments, which have been referred to as "extracellular matrix activators". Examples of extracellular matrix activators are disclosed in WO2022 / 055974, which is incorporated herein by reference in its entirety. Extracellular matrix activators have been shown to promote cell infiltration, progressive tissue damage or wound healing. Therefore, these signals represent important effector mechanisms of cell signaling and cell transport to target organs of ECM. In some aspects, the therapeutic agent may include one or more degradation products of ECM. In some aspects, the therapeutic agent includes extracellular matrix activators. In some aspects, the therapeutic agent may include one or more degradation products of one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycans and aggregated proteins. In certain aspects, the therapeutic agent comprises an extracellular matrix activator selected from the group consisting of metastatin, affesten, angiostatin, tetrastatin, pentastatin, lamstatin, hexastatin, endostatin, dormant protein 1, dormant protein 2, dormant protein 3, dormant protein 4, endostatin, neostatin, anastellin, sibsttin, PEX, endorepellin, CUB1CUB2 domain, Ten / 2, Ten11 / 12 / 13, Ten14, kappa-elastin, the extracellular domain of syndecan-1, the extracellular domain of syndecan-2, the extracellular domain of syndecan-3, the extracellular domain of syndecan-4, elastokine, laminin peptide A13, laminin peptide C16, laminin 332 (laminin 5), DGGRYY peptide, GHK tripeptide, VGVAPG peptide, PGP tripeptide, acetylated PGP tripeptide (AcPGP), tenascin-C (TNC), the G3 domain of nidogen-1, tumor inhibitory factor, and any combination thereof.

[0075] In certain aspects, the therapeutic agent may comprise one or more damage-associated molecular patterns (DAMPs). Damage-associated molecular patterns (DAMPs) are endogenous danger molecules that are released from damaged or dead cells and activate the innate immune system by, for example, interacting with pattern recognition receptors (PRRs). DAMPs can be derived from different sources, including, for example, extracellular proteins such as biglycan and tenascin C; intracellular proteins such as high-mobility group protein B1 (HMGB1), histones, S100 proteins, heat shock proteins (HSPs), and plasma proteins such as fibrinogen, Gc-globulin, and serum amyloid A (SAA). Examples of DAMPs include, but are not limited to, biglycan, decorin, versican, LMW hyaluronan, heparan sulfate, fibronectin including EDA domains, fibrinogen, tenascin C, uric acid, S100 proteins, heat shock proteins, adenosine triphosphate (ATP), F-actin, cyclophilin A, amyloid beta (Aβ), histones, HMGB1, HMGN1, IL-1α, IL-33, SAP130, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), mitochondrial DNA (mtDNA), mitochondrial transcription factor A (TFAM), formyl peptides, mitochondrial reactive oxygen species (mROS), calreticulin, defensins, calthelicidin (LL37), eosinophil-derived neurotoxins, granulysin, syndecan, and glypican.

[0076] In some aspects, the therapeutic agent may comprise a compound that induces the production or release of an ECM component, or one or more components therefrom, an ECM activator, or a DAMP.

[0077] As used herein, "pro-regeneration environment" refers to a combination of factors (e.g., immune cells, cytokines, etc.) in and around the wound, which guides the healing process away from fibrosis and promotes the regeneration of tissue in the wound, so that the damaged tissue returns to normal appearance. As discussed above, damage initiates a series of events, which triggers the remodeling of ECM and tissue and cell mobilization to the wound site to initiate host defense and tissue repair. After damage, some of the earliest cells to respond are polymorphonuclear cells, including neutrophils, eosinophils and basophils. Neutrophils are phagocytic cells that remove debris and recruit other cells, such as macrophages. Therefore, in the first few days after damage, a group of various immune cells are recruited to the injury site. In some aspects, therapeutic agents induce pro-regeneration environments, which may include an immune environment driven by Th2. The TH2 environment is characterized by the presence of eosinophils, basophils, mast cell degranulation and M2 macrophages, wherein the latter is related to wound healing and repair. In addition, the Th2 environment shows an increase in one or more cytokine levels, such as interleukin 4 (IL-4), IL-5, IL-10 and IL-13, which are important for inducing humoral immune responses. In some aspects, the Th2-driven environment can include a granulocyte compartment dominated by eosinophils at the site of injury. In some aspects, the therapeutic agent of the present disclosure can induce the influx of macrophages with an M2 phenotype. In some aspects, the therapeutic agent of the present disclosure can induce the local proliferation of M2 macrophages at the site of injury. In some aspects, the therapeutic agent of the present disclosure can induce macrophages to differentiate into M2 macrophages. In some aspects, the M2 macrophages in the Th2-driven environment can include high levels of CD206, CD301b and CD169. Such levels indicate local proliferation of tissue-resident cells. Therefore, the therapeutic agent of the present disclosure can induce the increase in the number of M2 macrophages with high levels of CD206, CD301b and CD169 in the wound.

[0078] The second most common antigen presenting cell (APC) found within the wound environment after injury is dendritic cells, such as CD11c + CD11b lo / neg Dendritic cells. The inventors have found that the therapeutic compositions of the present disclosure induce a pro-regenerative environment containing an increased number of classical dendritic cells (cDC1). cDC1 cells can initiate de novo T cell responses, as well as attract T cells, secrete cytokines and enhance local cytotoxic T cell function. cDC1 cells can also induce tolerance. In certain aspects, the therapeutic agents of the present disclosure can induce an increase in the number of classical dendritic cells (cDC1). Such cells can be cross-presenting dendritic cells, which can be XCR1 + CD103 +Dendritic cells. In certain aspects, the therapeutic agents of the present disclosure induce an influx of XCR1+CD103+cDC1. In certain aspects, the therapeutic agents of the present disclosure induce local proliferation of XCR1+CD103+cDC1. In certain aspects, the therapeutic agents of the present disclosure induce dendritic cells to differentiate into XCR1+CD103+cDC1.

[0079] In some aspects, the therapeutic agent can induce a therapeutic environment that can include an increase in NK cells. This increase may be due to an increase in the recruitment of NK cells to the therapeutic agent site, or it may be due to an increase in NK cell replication. In some aspects, such NK cells can be CD49b+TCRβ-. In some aspects, such NK cells can show an increase in Xcl1 gene expression, which can be induced by the therapeutic agent.

[0080] One aspect of the present disclosure is a method for changing an individual's immune response to a wound, the method comprising applying a therapeutic composition at the wound site, the therapeutic composition comprising a therapeutic agent that induces a pro-regeneration environment in the wound and / or in the tissue surrounding the wound, thereby changing the immune response to the wound. In some aspects, the wound may include burns, contusions, seromas, hematomas, lacerations, avulsions, punctures, surgical wounds, incisions, ulcers, and wounds caused by crushing injuries. In some aspects, the therapeutic composition may include ointments, sprays, lotions, gels, creams, foams, solutions, suspensions, emulsions, hydrogels, or pastes. In some aspects, the therapeutic composition may include liposomes, microspheres, or nanoparticles. In some aspects, the therapeutic agent may induce a pro-regeneration immune response in the wound or in the tissue adjacent to the wound. In some aspects, the pro-regeneration immune response may include a granulocyte compartment dominated by eosinophils. In some aspects, the therapeutic agent may induce an immune response driven by TH2. In some aspects, the therapeutic agent may induce M2 macrophages to flow into the wound. In some aspects, therapeutic agents can induce the local proliferation of M2 macrophages in wounds. In some aspects, therapeutic agents can induce macrophages to differentiate into M2 macrophages. In some aspects, the immune response driven by Th2 can include macrophages with high levels of CD206, CD301b and / or CD169. In some aspects, therapeutic agents can induce the local proliferation of tissue-resident macrophages with high levels of CD206, CD301b and / or CD169. In some aspects, therapeutic agents can cause the enrichment of cross-presenting dendritic cells in wounds or in tissues adjacent to wounds. In some aspects, therapeutic agents can induce the influx of cross-presenting dendritic cells. In some aspects, therapeutic agents can induce the local proliferation of cross-presenting dendritic cells. In some aspects, therapeutic agents can induce dendritic cells to differentiate into cross-presenting dendritic cells. In some aspects, cross-presenting dendritic cells can include cDC1 cells. In some aspects, cross-presenting dendritic cells can be XCR1+CD103+ dendritic cells. In some aspects, the cross-presenting dendritic cells can express moderate levels of CD86. In some aspects, the therapeutic agent can induce CD44 in the individual + CD26L - In some aspects, CD44 + CD26L - T cells may include CD4+ cells. In some aspects, CD44 + CD26L -T cells may include CD8+ cells. In some aspects, therapeutic agents may include one or more components of ECM. In some aspects, therapeutic agents may include one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan and aggregated protein. In some aspects, therapeutic agents may include one or more degradation products from one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan, aggregated protein and any combination thereof. In certain aspects, the therapeutic agent comprises an extracellular matrix activator selected from the group consisting of metastatin, affesten, angiostatin, tetrastatin, pentastatin, lamstatin, hexastatin, endostatin, dormant protein 1, dormant protein 2, dormant protein 3, dormant protein 4, endostatin, neostatin, anastellin, sibsttin, PEX, endorepellin, CUB1CUB2 domain, Ten / 2, Ten11 / 12 / 13, Ten14, kappa-elastin, the extracellular domain of syndecan-1, the extracellular domain of syndecan-2, the extracellular domain of syndecan-3, the extracellular domain of syndecan-4, elastokine, laminin peptide A13, laminin peptide C16, laminin 332 (laminin 5), DGGRYY peptide, GHK tripeptide, VGVAPG peptide, PGP tripeptide, acetylated PGP tripeptide (AcPGP), tenascin-C (TNC), the G3 domain of nidogen-1, tumor inhibitory factor, and any combination thereof. In certain aspects, the therapeutic agent may comprise one or more DAMPs.

[0081] One aspect of the present disclosure is a method for treating an individual with a wound, the method comprising applying a therapeutic composition at the wound site, the therapeutic composition comprising a therapeutic agent that induces a pro-regeneration environment in the wound and / or in the tissue surrounding the wound, thereby treating the individual. In some aspects, the wound may include burns, contusions, seromas, hematomas, lacerations, avulsions, punctures, surgical wounds, incisions, ulcers, and wounds caused by crushing injuries. In some aspects, the therapeutic composition may include ointments, sprays, lotions, gels, creams, foams, solutions, suspensions, emulsions, hydrogels, or pastes. In some aspects, the therapeutic composition may include liposomes, microspheres, or nanoparticles. In some aspects, the therapeutic agent may induce a pro-regeneration immune response in the wound or in the tissue adjacent to the wound. In some aspects, the pro-regeneration immune response may include a granulocyte compartment dominated by eosinophils. In some aspects, the therapeutic agent may induce an immune response driven by TH2. In some aspects, the therapeutic agent may induce M2 macrophages to flow into the wound. In some aspects, the therapeutic agent may induce local proliferation of M2 macrophages in the wound. In some aspects, therapeutic agents can induce macrophage differentiation into M2 macrophages. In some aspects, the immune response driven by Th2 can include macrophages with high levels of CD206, CD301b and / or CD169. In some aspects, therapeutic agents can induce local proliferation of tissue-resident macrophages with high levels of CD206, CD301b and / or CD169. In some aspects, therapeutic agents can cause the enrichment of NK cells in wounds or in tissues adjacent to wounds. This enrichment may be due to an increase in NK cell recruitment or an increase in local proliferation of NK cells. In some aspects, such NK cells can be CD49b+TCRβ-. In some aspects, such NK cells can show an increase in Xcl1 gene expression, which can be induced by therapeutic agents.

[0082] In some aspects, the therapeutic agent can cause an enrichment of cross-presenting dendritic cells in a wound or in tissue adjacent to a wound. In some aspects, the therapeutic agent can induce an influx of cross-presenting dendritic cells. In some aspects, the therapeutic agent can induce local proliferation of cross-presenting dendritic cells. In some aspects, the therapeutic agent can induce dendritic cells to differentiate into cross-presenting dendritic cells. In some aspects, the cross-presenting dendritic cells can include cDC1 cells. In some aspects, the cross-presenting dendritic cells can be XCR1+CD103+ dendritic cells. In some aspects, the cross-presenting dendritic cells can express moderate levels of CD86. In some aspects, the therapeutic agent can induce CD44 in an individual. + CD26L - In some aspects, CD44 + CD26L - T cells may include CD4+ cells. In some aspects, CD44+ CD26L - T cells may include CD8+ cells. In some aspects, the therapeutic agent may include one or more components of the ECM. In some aspects, the therapeutic agent may include one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan, aggregated protein and any combination thereof. In some aspects, the therapeutic agent may include one or more degradation products of one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan, aggregated protein and any combination thereof. In some aspects, the therapeutic agent includes an extracellular matrix activator selected from the group consisting of: metastatin, affesten, angiostatin, tetrastatin, pen tastatin, lamstatin, hexastatin, endotrophin, dormant protein 1, dormant protein 2, dormant protein 3, dormant protein 4, endostatin, neostatin, anastellin, sibsttin, PE X, endorepellin, CUB1CUB2 domain, Ten / 2, Ten11 / 12 / 13, Ten14, kappa-elastin, the extracellular domain of syndecan-1, the extracellular domain of syndecan-2, the extracellular domain of syndecan-3, the extracellular domain of syndecan-4, elastokine, laminin peptide A13, laminin peptide C16, laminin 332 (laminin 5), DGGRYY peptide, GHK tripeptide, VGVAPG peptide, PGP tripeptide, acetylated PGP tripeptide (AcPGP), tenascin-C (TNC), the G3 domain of nidogen-1, tumor inhibitory factor, and any combination thereof. In certain aspects, the therapeutic agent may comprise one or more DAMPs.

[0083] One aspect is a method for treating a wound of an individual, the method comprising applying a therapeutic composition at the wound site, the therapeutic composition comprising a therapeutic agent that induces a pro-regeneration environment in the wound and / or in the tissue surrounding the wound, thereby treating the wound. In some aspects, the wound may include burns, contusions, seromas, hematomas, lacerations, avulsions, stab wounds, surgical wounds, incisions, ulcers, and wounds caused by crushing injuries. In some aspects, the therapeutic composition may include ointments, sprays, lotions, gels, creams, foams, solutions, suspensions, emulsions, hydrogels, or pastes. In some aspects, the therapeutic composition may include liposomes, microspheres, or nanoparticles. In some aspects, the therapeutic agent may induce a pro-regeneration immune response in the wound or in the tissue adjacent to the wound. In some aspects, the pro-regeneration immune response may include a granulocyte compartment dominated by eosinophils. In some aspects, the therapeutic agent may induce an immune response driven by TH2. In some aspects, the therapeutic agent may induce M2 macrophages to flow into the wound. In some aspects, the therapeutic agent may induce local proliferation of M2 macrophages in the wound. In some aspects, therapeutic agents can induce macrophage differentiation into M2 macrophages. In some aspects, the immune response driven by Th2 can include macrophages with high levels of CD206, CD301b and / or CD169. In some aspects, therapeutic agents can induce local proliferation of tissue-resident macrophages with high levels of CD206, CD301b and / or CD169. In some aspects, therapeutic agents can cause the enrichment of NK cells in the wound or in the tissue adjacent to the wound. This enrichment may be due to an increase in NK cell recruitment or an increase in local proliferation of NK cells. In some aspects, such NK cells can be CD49b+TCRβ-. In some aspects, such NK cells can show an increase in Xcl1 gene expression, which can be induced by therapeutic agents. In some aspects, therapeutic agents can cause the enrichment of cross-presenting dendritic cells in the wound or in the tissue adjacent to the wound. In some aspects, therapeutic agents can induce the influx of cross-presenting dendritic cells. In some aspects, therapeutic agents can induce the local proliferation of cross-presenting dendritic cells. In some aspects, the therapeutic agent can induce dendritic cells to differentiate into cross-presenting dendritic cells. In some aspects, the cross-presenting dendritic cells can include cDC1 cells. In some aspects, the cross-presenting dendritic cells can be XCR1+CD103+ dendritic cells. In some aspects, the cross-presenting dendritic cells can express moderate levels of CD86. In some aspects, the therapeutic agent can induce CD44 in an individual. + CD26L - In some aspects, CD44 + CD26L - T cells may include CD4+ cells. In some aspects, CD44 + CD26L -T cells may include CD8+ cells. In some aspects, the therapeutic agent may include one or more components of the ECM. In some aspects, the therapeutic agent may include one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan and aggregated protein. In some aspects, the therapeutic agent may include one or more degradation products of one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan and aggregated protein. In some aspects, the therapeutic agent includes an extracellular matrix activator selected from the group consisting of: metastatin, affesten, angiostatin, tetrastatin, pentas tatin, lamstatin, hexastatin, endostatin, dormant protein 1, dormant protein 2, dormant protein 3, dormant protein 4, endostatin, neostatin, anastellin, sibsttin, PEX, endorepellin, CUB1CUB2 domain, Ten / 2, Ten11 / 12 / 13, Ten14, kappa-elastin, the extracellular domain of syndecan-1, the extracellular domain of syndecan-2, the extracellular domain of syndecan-3, the extracellular domain of syndecan-4, elastokine, laminin peptide A13, laminin peptide C16, laminin 332 (laminin 5), DGGRYY peptide, GHK tripeptide, VGVAPG peptide, PGP tripeptide, acetylated PGP tripeptide (AcPGP), tenascin-C (TNC), the G3 domain of nidogen-1, and tumor inhibitory protein. In certain aspects, the therapeutic agent may comprise one or more DA MPs.

[0084] One aspect of the present disclosure is a method for implanting a medical device in an individual, the method comprising: introducing the medical device into a tissue in the individual, and applying a therapeutic composition at the implanted medical device site, the therapeutic composition comprising a therapeutic agent that induces a pro-regeneration environment in the wound and / or in the tissue surrounding the wound. In some aspects, the wound may include burns, contusions, seromas, hematomas, lacerations, avulsions, punctures, surgical wounds, incisions, ulcers, and wounds caused by crushing injuries. In some aspects, the therapeutic composition may include an ointment, a spray, a lotion, a gel, a cream, a foam, a solution, a suspension, an emulsion, a hydrogel, or a paste. In some aspects, the therapeutic composition may include liposomes, microspheres, or nanoparticles. In some aspects, the therapeutic agent may induce a pro-regeneration immune response in the wound or in a tissue adjacent to the wound. In some aspects, the pro-regeneration immune response may include a granulocyte compartment dominated by eosinophils. In some aspects, the therapeutic agent may induce a TH2-driven immune response. In some aspects, the therapeutic agent may induce M2 macrophages to flow into the wound. In some aspects, the therapeutic agent can induce the local proliferation of M2 macrophages in the wound. In some aspects, the therapeutic agent can induce macrophage differentiation into M2 macrophages. In some aspects, the immune response driven by Th2 can include macrophages with high levels of CD206, CD301b and / or CD169. In some aspects, the therapeutic agent can induce the local proliferation of tissue-resident macrophages with high levels of CD206, CD301b and / or CD169. In some aspects, the therapeutic agent can cause the enrichment of NK cells in the wound or in the tissue adjacent to the wound. This enrichment may be due to an increase in NK cell recruitment or an increase in local proliferation of NK cells. In some aspects, such NK cells can be CD49b+TCRβ-. In some aspects, such NK cells can show the rise of Xcl1 gene expression, which can be induced by the therapeutic agent. In some aspects, the therapeutic agent can cause the enrichment of cross-presenting dendritic cells in the wound or in the tissue adjacent to the wound. In some aspects, the therapeutic agent can induce the influx of cross-presenting dendritic cells. In some aspects, the therapeutic agent can induce local proliferation of cross-presenting dendritic cells. In some aspects, the therapeutic agent can induce dendritic cells to differentiate into cross-presenting dendritic cells. In some aspects, the cross-presenting dendritic cells can include cDC1 cells. In some aspects, the cross-presenting dendritic cells can be XCR1+CD103+ dendritic cells. In some aspects, the cross-presenting dendritic cells can express moderate levels of CD86. In some aspects, the therapeutic agent can induce CD44 in an individual. + CD26L - In some aspects, CD44 + CD26L - T cells may include CD4+ cells. In some aspects, CD44+ CD26L - T cells may include CD8+ cells. In some aspects, therapeutic agents may include one or more components of ECM. In some aspects, therapeutic agents may include one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan, aggregated protein and any combination thereof. In some aspects, therapeutic agents may include one or more degradation products from one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan, aggregated protein and any combination thereof. In certain aspects, the therapeutic agent comprises an extracellular matrix activator selected from the group consisting of metastatin, affesten, angiostatin, tetrastatin, pentastatin, lamstatin, hexastatin, endostatin, dormant protein 1, dormant protein 2, dormant protein 3, dormant protein 4, endostatin, neostatin, anastellin, sibsttin, PEX, endorepellin, CUB1CUB2 domain, Ten / 2, Ten11 / 12 / 13, Ten14, kappa-elastin, the extracellular domain of syndecan-1, the extracellular domain of syndecan-2, the extracellular domain of syndecan-3, the extracellular domain of syndecan-4, elastokine, laminin peptide A13, laminin peptide C16, laminin 332 (laminin 5), DGGRYY peptide, GHK tripeptide, VGVAPG peptide, PGP tripeptide, acetylated PGP tripeptide (AcPGP), tenascin-C (TNC), the G3 domain of nidogen-1, tumor inhibitor, and any combination thereof. In certain aspects, the therapeutic agent may comprise one or more DAMPs. "Medical device" refers to any device implanted in an individual for the purpose of improving the health and / or function of an individual. An example of a medical device is an implant. Examples of implants include, but are not limited to, breast implants, stents, infusion ports, shunts, hip implants, knee implants, cochlear implants, hernia surgery mesh implants, intraocular lens implants, pacemakers, metal / surgical screws, metal / surgical rods, metal / surgical needles, artificial intervertebral discs, and spinal fusion cages. Such implants can be made using, for example, metals and metal alloys, plastic polymers, ceramics, hydrogels, and composite materials, which can include, but are not limited to, silicone, polyethylene, stainless steel, titanium, zirconium oxide, polyurethane foam, polylactic acid, amalgam, gold, alumina, silicates, chromium alloys, cobalt, and molybdenum.

[0085] One aspect is a method for reducing or preventing wound fibrosis, the method comprising applying a therapeutic composition at the wound site, the therapeutic composition comprising a therapeutic agent that induces a pro-regeneration environment in the wound and / or in the tissue around the wound, thereby reducing or preventing wound fibrosis. In some aspects, the wound may include burns, contusions, seromas, hematomas, lacerations, avulsions, stab wounds, surgical wounds, incisions, ulcers, and wounds caused by crushing injuries. In some aspects, the therapeutic composition may include ointments, sprays, lotions, gels, creams, foams, solutions, suspensions, emulsions, hydrogels, or pastes. In some aspects, the therapeutic composition may include liposomes, microspheres, or nanoparticles. In some aspects, the therapeutic agent may induce a pro-regeneration immune response in the wound or in the tissue adjacent to the wound. In some aspects, the pro-regeneration immune response may include a granulocyte compartment based on eosinophils. In some aspects, the therapeutic agent may induce an immune response driven by TH2. In some aspects, the therapeutic agent may induce M2 macrophages to flow into the wound. In some aspects, the therapeutic agent can induce the local proliferation of M2 macrophages in the wound. In some aspects, the therapeutic agent can induce macrophage differentiation into M2 macrophages. In some aspects, the immune response driven by Th2 can include macrophages with high levels of CD206, CD301b and / or CD169. In some aspects, the therapeutic agent can induce the local proliferation of tissue-resident macrophages with high levels of CD206, CD301b and / or CD169. In some aspects, the therapeutic agent can cause the enrichment of NK cells in the wound or in the tissue adjacent to the wound. This enrichment may be due to an increase in NK cell recruitment or an increase in local proliferation of NK cells. In some aspects, such NK cells can be CD49b+TCRβ-. In some aspects, such NK cells can show the rise of Xcl1 gene expression, which can be induced by the therapeutic agent. In some aspects, the therapeutic agent can cause the enrichment of cross-presenting dendritic cells in the wound or in the tissue adjacent to the wound. In some aspects, the therapeutic agent can induce the influx of cross-presenting dendritic cells. In some aspects, the therapeutic agent can induce local proliferation of cross-presenting dendritic cells. In some aspects, the therapeutic agent can induce dendritic cells to differentiate into cross-presenting dendritic cells. In some aspects, the cross-presenting dendritic cells can include cDC1 cells. In some aspects, the cross-presenting dendritic cells can be XCR1+CD103+ dendritic cells. In some aspects, the cross-presenting dendritic cells can express moderate levels of CD86. In some aspects, the therapeutic agent can induce CD44 in an individual. + CD26L - In some aspects, CD44 + CD26L - T cells may include CD4+ cells. In some aspects, CD44 + CD26L- T cells may include CD8+ cells. In some aspects, therapeutic agents may include one or more components of ECM. In some aspects, therapeutic agents may include one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan, aggregated protein and any combination thereof. In some aspects, therapeutic agents may include one or more degradation products from one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan, aggregated protein and any combination thereof. In certain aspects, the therapeutic agent comprises an extracellular matrix activator selected from the group consisting of metastatin, affesten, angiostatin, tetrastatin, pentastatin, lamstatin, hexastatin, endostatin, dormant protein 1, dormant protein 2, dormant protein 3, dormant protein 4, endostatin, neostatin, anastellin, sibsttin, PEX, endorepellin, CUB1CUB2 domain, Ten / 2, Ten11 / 12 / 13, Ten14, kappa-elastin, the extracellular domain of syndecan-1, the extracellular domain of syndecan-2, the extracellular domain of syndecan-3, the extracellular domain of syndecan-4, elastokine, laminin peptide A13, laminin peptide C16, laminin 332 (laminin 5), DGGRYY peptide, GHK tripeptide, VGVAPG peptide, PGP tripeptide, acetylated PGP tripeptide (AcPGP), tenascin-C (TNC), the G3 domain of nidogen-1, tumor inhibitor, and any combination thereof. In certain aspects, the therapeutic agent may comprise one or more DAMPs.

[0086] One aspect of the present disclosure is a therapeutic composition for treating a wound, the composition comprising a therapeutic agent that induces a pro-regeneration environment in the wound and / or in the tissue surrounding the wound. In some aspects, the wound may include burns, contusions, seromas, hematomas, lacerations, avulsions, punctures, surgical wounds, incisions, ulcers, and wounds caused by crushing injuries. In some aspects, the therapeutic composition may include ointments, sprays, lotions, gels, creams, foams, solutions, suspensions, emulsions, hydrogels, or pastes. In some aspects, the therapeutic composition may include liposomes, microspheres, or nanoparticles. In some aspects, the therapeutic agent may induce a pro-regeneration immune response in the wound or in the tissue adjacent to the wound. In some aspects, the pro-regeneration immune response may include a granulocyte compartment dominated by eosinophils. In some aspects, the therapeutic agent may induce an immune response driven by TH2. In some aspects, the therapeutic agent may induce M2 macrophages to flow into the wound. In some aspects, the therapeutic agent may induce local proliferation of M2 macrophages in the wound. In some aspects, therapeutic agents can induce macrophage differentiation into M2 macrophages. In some aspects, the immune response driven by Th2 can include macrophages with high levels of CD206, CD301b and / or CD169. In some aspects, therapeutic agents can induce local proliferation of tissue-resident macrophages with high levels of CD206, CD301b and / or CD169. In some aspects, therapeutic agents can cause the enrichment of NK cells in the wound or in the tissue adjacent to the wound. This enrichment may be due to an increase in NK cell recruitment or an increase in local proliferation of NK cells. In some aspects, such NK cells can be CD49b+TCRβ-. In some aspects, such NK cells can show an increase in Xcl1 gene expression, which can be induced by therapeutic agents. In some aspects, therapeutic agents can cause the enrichment of cross-presenting dendritic cells in the wound or in the tissue adjacent to the wound. In some aspects, therapeutic agents can induce the influx of cross-presenting dendritic cells. In some aspects, therapeutic agents can induce the local proliferation of cross-presenting dendritic cells. In some aspects, the therapeutic agent can induce dendritic cells to differentiate into cross-presenting dendritic cells. In some aspects, the cross-presenting dendritic cells can include cDC1 cells. In some aspects, the cross-presenting dendritic cells can be XCR1+CD103+ dendritic cells. In some aspects, the cross-presenting dendritic cells can express moderate levels of CD86. In some aspects, the therapeutic agent can induce CD44 in an individual. + CD26L - In some aspects, CD44 + CD26L - T cells may include CD4+ cells. In some aspects, CD44 + CD26L -T cells may include CD8+ cells. In some aspects, therapeutic agents may include one or more components of ECM. In some aspects, therapeutic agents may include one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan, aggregated protein and any combination thereof. In some aspects, therapeutic agents may include one or more degradation products from one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan, aggregated protein and any combination thereof. In certain aspects, the therapeutic agent comprises an extracellular matrix activator selected from the group consisting of metastatin, affesten, angiostatin, tetrastatin, pentastatin, lamstatin, hexastatin, endostatin, dormant protein 1, dormant protein 2, dormant protein 3, dormant protein 4, endostatin, neostatin, anastellin, sibsttin, PEX, endorepellin, CUB1CUB2 domain, Ten / 2, Ten11 / 12 / 13, Ten14, kappa-elastin, the extracellular domain of syndecan-1, the extracellular domain of syndecan-2, the extracellular domain of syndecan-3, the extracellular domain of syndecan-4, elastokine, laminin peptide A13, laminin peptide C16, laminin 332 (laminin 5), DGGRYY peptide, GHK tripeptide, VGVAPG peptide, PGP tripeptide, acetylated PGP tripeptide (AcPGP), tenascin-C (TNC), the G3 domain of nidogen-1, tumor inhibitor, and any combination thereof. In certain aspects, the therapeutic agent may comprise one or more DAMPs.

[0087] One aspect of the present disclosure is a method for recruiting NK cells to a wound or a wound-adjacent tissue, the method comprising applying a therapeutic composition at the wound site, the therapeutic composition comprising a therapeutic agent that induces a pro-regeneration environment in the wound and / or in the tissue surrounding the wound, thereby recruiting NK cells to the wound or the wound-adjacent tissue. The wound may include a burn, a contusion, a seroma, a hematoma, a laceration, an avulsion, a stab wound, a surgical wound, an incision, an ulcer, and a wound caused by a crush injury. In some aspects, the therapeutic composition may include an ointment, a spray, a lotion, a gel, a cream, a foam, a solution, a suspension, an emulsion, a hydrogel, or a paste. In some aspects, the therapeutic composition may include liposomes, microspheres, or nanoparticles. In some aspects, the therapeutic agent may induce an increase in the recruitment of NK cells to a wound or a wound-adjacent tissue. In some aspects, the therapeutic agent may induce an increase in the local proliferation of NK cells. In some aspects, the NK cell may be CD49b+TCRβ-. In some aspects, the NK cell may show an upregulation of Xcl1 gene expression, which may be induced by a therapeutic agent. In some aspects, the therapeutic agent may include one or more components of ECM. In some aspects, the therapeutic agent may include one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan, aggregated protein and any combination thereof. In some aspects, the therapeutic agent may include one or more degradation products of one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan, aggregated protein and any combination thereof.In certain aspects, the therapeutic agent comprises an extracellular matrix activator selected from the group consisting of metastatin, affesten, angiostatin, tetrastatin, pentastatin, lamstatin, hexastatin, endostatin, dormant protein 1, dormant protein 2, dormant protein 3, dormant protein 4, endostatin, neostatin, anastellin, sibsttin, PEX, endorepellin, CUB1CUB2 domain, Ten / 2, Ten11 / 12 / 13, Ten14, kappa-elastin, the extracellular domain of syndecan-1, the extracellular domain of syndecan-2, the extracellular domain of syndecan-3, the extracellular domain of syndecan-4, elastokine, laminin peptide A13, laminin peptide C16, laminin 332 (laminin 5), DGGRYY peptide, GHK tripeptide, VGVAPG peptide, PGP tripeptide, acetylated PGP tripeptide (AcPGP), tenascin-C (TNC), the G3 domain of nidogen-1, tumor inhibitor, and any combination thereof. In certain aspects, the therapeutic agent may comprise one or more DAMPs.

[0088] One aspect of the present disclosure is a method for activating NK cells, the method comprising contacting NK cells with a therapeutic agent of the present disclosure. In some aspects, the therapeutic agent causes the replication of NK cells. In some aspects, the therapeutic agent causes the migration of NK cells. In some aspects, the therapeutic agent increases the Xcl1 gene expression in NK cells. In some aspects, the secretion of the therapeutic agent-induced Xcl1 protein increases. In some aspects, contacting includes introducing the therapeutic agent into a wound or into a tissue adjacent to the wound. In some aspects, contacting includes introducing the therapeutic agent into an in vitro NK cell (e.g., tissue culture). In some aspects, the therapeutic agent may include one or more components of ECM. In some aspects, the therapeutic agent may include one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan, aggregated protein, and any combination thereof. In some aspects, the therapeutic agent may comprise one or more degradation products of one or more components selected from the group consisting of collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan, aggrecan, and any combination thereof. In some aspects, the therapeutic agent comprises an extracellular matrix activator selected from the group consisting of metastatin, affesten, angiostatin, tetrastatin, pentastatin, lamstatin, hexastatin, endostatin, dormant protein 1, dormant protein 2, dormant protein 3, dormant protein 4, endostatin, neostatin, anastellin, sibsttin, PEX, endorepellin, CUB1CUB2 domain, Ten / 2, Ten11 / 12 / 13, Ten14, kappa-elastin, the extracellular domain of syndecan-1, the extracellular domain of syndecan-2, the extracellular domain of syndecan-3, the extracellular domain of syndecan-4, elastokine, laminin peptide A13, laminin peptide C16, laminin 332 (laminin 5), DGGRYY peptide, GHK tripeptide, VGVAPG peptide, PGP tripeptide, acetylated PGP tripeptide (AcPGP), tenascin-C (TNC), the G3 domain of nidogen-1, tumor inhibitor, and any combination thereof. In certain aspects, the therapeutic agent may comprise one or more DAMPs.

[0089] One aspect of the present disclosure is a method for inducing an increase in Xcl1 expression in NK cells, the method comprising contacting NK cells with a therapeutic agent of the present disclosure. In some aspects, the therapeutic agent increases the Xcl1 gene expression in NK cells. In some aspects, the therapeutic agent induces an increase in the secretion of Xcl1 protein. In some aspects, contacting includes introducing the therapeutic agent into a wound or into a tissue adjacent to the wound. In some aspects, contacting includes introducing the therapeutic agent into an in vitro NK cell (e.g., a tissue culture). In some aspects, the therapeutic agent may include one or more components of ECM. In some aspects, the therapeutic agent may include one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan, aggregated protein, and any combination thereof. In some aspects, the therapeutic agent may include one or more degradation products from one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan, aggregated protein, and any combination thereof. In certain aspects, the therapeutic agent comprises an extracellular matrix activator selected from the group consisting of metastatin, affesten, angiostatin, tetrastatin, pentastatin, lamstatin, hexastatin, endostatin, dormant protein 1, dormant protein 2, dormant protein 3, dormant protein 4, endostatin, neostatin, anastellin, sibsttin, PEX, endorepellin, CUB1CUB2 domain, Ten / 2, Ten11 / 12 / 13, Ten14, kappa-elastin, the extracellular domain of syndecan-1, the extracellular domain of syndecan-2, the extracellular domain of syndecan-3, the extracellular domain of syndecan-4, elastokine, laminin peptide A13, laminin peptide C16, laminin 332 (laminin 5), DGGRYY peptide, GHK tripeptide, VGVAPG peptide, PGP tripeptide, acetylated PGP tripeptide (AcPGP), tenascin-C (TNC), the G3 domain of nidogen-1, tumor inhibitory factor, and any combination thereof. In certain aspects, the therapeutic agent may comprise one or more DAMPs.

[0090] A kit for treating a wound, the kit comprising at least a therapeutic composition, the therapeutic composition comprising a therapeutic agent that induces a pro-regeneration environment in the wound and / or in the tissue surrounding the wound. In some aspects, the wound may include burns, contusions, seromas, hematomas, lacerations, avulsions, punctures, surgical wounds, incisions, ulcers, and wounds caused by crushing injuries. In some aspects, the therapeutic composition may include ointments, sprays, lotions, gels, creams, foams, solutions, suspensions, emulsions, hydrogels, or pastes. In some aspects, the therapeutic composition may include liposomes, microspheres, or nanoparticles. In some aspects, the therapeutic agent may induce a pro-regeneration immune response in the wound or in the tissue adjacent to the wound. In some aspects, the pro-regeneration immune response may include a granulocyte compartment dominated by eosinophils. In some aspects, the therapeutic agent may induce a TH2-driven immune response. In some aspects, the therapeutic agent may induce M2 macrophages to flow into the wound. In some aspects, the therapeutic agent may induce local proliferation of M2 macrophages in the wound. In some aspects, therapeutic agents can induce macrophage differentiation into M2 macrophages. In some aspects, the immune response driven by Th2 can include macrophages with high levels of CD206, CD301b and / or CD169. In some aspects, therapeutic agents can induce local proliferation of tissue-resident macrophages with high levels of CD206, CD301b and / or CD169. In some aspects, therapeutic agents can cause the enrichment of NK cells in the wound or in the tissue adjacent to the wound. This enrichment may be due to an increase in NK cell recruitment or an increase in local proliferation of NK cells. In some aspects, such NK cells can be CD49b+TCRβ-. In some aspects, such NK cells can show an increase in Xcl1 gene expression, which can be induced by therapeutic agents. In some aspects, therapeutic agents can cause the enrichment of cross-presenting dendritic cells in the wound or in the tissue adjacent to the wound. In some aspects, therapeutic agents can induce the influx of cross-presenting dendritic cells. In some aspects, therapeutic agents can induce the local proliferation of cross-presenting dendritic cells. In some aspects, the therapeutic agent can induce dendritic cells to differentiate into cross-presenting dendritic cells. In some aspects, the cross-presenting dendritic cells can include cDC1 cells. In some aspects, the cross-presenting dendritic cells can be XCR1+CD103+ dendritic cells. In some aspects, the cross-presenting dendritic cells can express moderate levels of CD86. In some aspects, the therapeutic agent can induce CD44 in an individual. + CD26L - In some aspects, CD44 + CD26L - T cells may include CD4+ cells. In some aspects, CD44 + CD26L -T cells may include CD8+ cells. In some aspects, therapeutic agents may include one or more components of ECM. In some aspects, therapeutic agents may include one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan, aggregated protein and any combination thereof. In some aspects, therapeutic agents may include one or more degradation products from one or more components selected from the group consisting of: collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, basement membrane proteoglycan, aggregated protein and any combination thereof. In certain aspects, the therapeutic agent comprises an extracellular matrix activator selected from the group consisting of metastatin, affesten, angiostatin, tetrastatin, pentastatin, lamstatin, hexastatin, endostatin, dormant protein 1, dormant protein 2, dormant protein 3, dormant protein 4, endostatin, neostatin, anastellin, sibsttin, PEX, endorepellin, CUB1CUB2 domain, Ten / 2, Ten11 / 12 / 13, Ten14, kappa-elastin, the extracellular domain of syndecan-1, the extracellular domain of syndecan-2, the extracellular domain of syndecan-3, the extracellular domain of syndecan-4, elastokine, laminin peptide A13, laminin peptide C16, laminin 332 (laminin 5), DGGRYY peptide, GHK tripeptide, VGVAPG peptide, PGP tripeptide, acetylated PGP tripeptide (AcPGP), tenascin-C (TNC), G3 domain of nidogen-1, tumor inhibitor and any combination thereof. In certain aspects, the therapeutic agent may comprise one or more DAMPs. The kit of the present disclosure may comprise additional components, such as needles, syringes, vials, applicators, and instructions for treating wounds using the components of the kit.

[0091] This written description uses embodiments to disclose the present disclosure, including the best mode, and to enable any person skilled in the art to practice the present disclosure, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the present disclosure is defined by the claims, and may include other embodiments that occur to those skilled in the art. Such other embodiments are intended to fall within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if such other embodiments include equivalent structural elements with insubstantial differences from the literal language of the claims. Example

[0092] Materials and methods

[0093] Material preparation

[0094] The small intestine is derived from the American Yorkshire pigs (Wagner Meats) of 5 to 6 months of age. The submucosal layer (SIS) is mechanically separated by removing the muscular layer and then mechanically scraping the luminal layer. The resulting SIS is rinsed with distilled water and frozen at -80 ° C until decellularization. After thawing, SIS is cut into 1 inch fragments in a biological safety cabinet, then shaken vigorously in 4% ethanol (FisherScientific) and 0.1% peracetic acid (Sigma) or incubated on a stirrer for 30 minutes. The resulting decellularized ECM is neutralized by continuous washing with sterile 1xPBS and distilled water. The liquid is blotted with a sterile absorbent pad, and then the material is transferred to a 50ml conical tube and frozen at -80 ° C until lyophilized 48 hours. The dried material is sampled into a sterile cryogenic grinding container and ground into a fine powder. The resulting powder is hydrated with sterile saline to form a thick paste, which is then sampled into a slip-tip 1ml syringe for application to the wound site.

[0095] Polyethylene powder (PE) with a particle size of <150 μm was purchased from Goodfellow Cambridge Limited and soaked in distilled water, then rinsed with 70% ethanol and UV sterilized in ethanol for 30 minutes. PE was stored in 70% ethanol until use. The PE particles suspended in ethanol were transferred to Eppendorf tubes and dried overnight in a biosafety cabinet. Due to the hydrophobicity of PE, the sample could not be injected into a syringe and was applied directly to the wound as a powder.

[0096] Muscle volume reduction surgery

[0097] According to the method described previously, mice received bilateral volume reduction muscle trauma (1). In brief, the lower limbs of 6-8 week-old female C57BL / 6WT mice (Jackson laboratory) were shaved with an electric razor one day before surgery, and excess hair was removed by depilatory cream. On the second day, mice were anesthetized at a flow rate of 200cc / min under 4.0% isoflurane in oxygen in an anesthesia chamber, and received subcutaneous injection of buprenorphine for pain management. Then mice were maintained under 2.0% isoflurane during the procedure. The surgical site was disinfected three times with povidone iodine, followed by 70% isopropanol, and then a 1 cm incision was made in the fascia above the skin and quadriceps. Using surgical scissors, a 3-4 mm defect was created in the mid-abdomen section of the quadriceps by removing 1 / 3 of the quadriceps. After removing the muscle, a uniform amount (50ul) of polyethylene particles or porcine-derived ECM scaffolds were filled into the resulting tissue gap. The wound was then subsequently closed with 3-4 wound clips and the procedure was repeated on the contralateral leg. After surgery, the mice were placed under a heat lamp for 2-3 minutes to allow them to recover from anesthesia. The mice were then returned to their cages and maintained on a nutritious regular diet until the end of the study. This protocol was approved by the NIH Clinical Center Animal Care and Use Committee, and the animal protocol number is NIBIB 20-01.

[0098] Flow cytometry

[0099] After 3 days, 7 days, 21 days and 42 days after injury, mice were ethically killed and then injured muscles were harvested together with the scaffold. The dissected muscles were then finely diced and digested at 100 rpm on a shaker at 37 ° C with digestion medium (0.5 mg / mL Liberase TM (Sigma) and 0.2 mg / ml DNase I (Roche) in HEPES supplemented medium) for 45 minutes. The digested suspension was then filtered through a 70 μm cell strainer, washed with 1x PBS, and centrifuged at room temperature for 5 minutes at 350 g. The cell pellet was then immersed in 5mM EDTA in 1xPBS solution for 10 minutes to reduce cell aggregation. After incubation for 10 minutes, the cells were then washed with 1x PBS again and then centrifuged at 4 ° C for 5 minutes at 350 g. The pellet was then resuspended in 200 μL of a viability dye solution (Live / Dead Blue (Thermo Fisher) diluted 1:1000 in PBS) for 20 minutes on ice, followed by washing with wash buffer (1% BSA and 2 mM EDTA in 1x PBS). The cells were then stained with either myeloid or lymphoid group antibodies (Tables 1 and 2) and then incubated at 4°C for 30 minutes. After incubation, the cells were washed three times with wash buffer and analyzed on a Cytek Aurora.

[0100] Intracellular staining

[0101] After surface dyeing, follow the manufacturer's instructions by using True-Nuclear TM Cells were fixed and permeabilized using the Transcription Factor Kit (BioLegend) for intracellular staining with FoxP3 and HELIOS antibodies (Table 2). Briefly, after the final wash of surface staining, cells were resuspended in True-Nuclear TM 1x fixative concentrate and incubated at 4°C for 45 minutes. After incubation, cells were centrifuged at 400 x g for 10 minutes at 4°C and resuspended in True-Nuclear TM The cells were then washed once more with 1x Perm buffer and then incubated with HELIOS and FoxP3 antibody cocktail (in True-Nuclear TM 1x Perm buffer) and then incubated at 4°C for 45 minutes. TM The cells were washed twice with 1x Perm buffer. After the final wash, the cells were resuspended in wash buffer and analyzed on a flow cytometer.

[0102] Histopathology

[0103] The samples were fixed in 10% neutral buffered formalin for 48–72 hours and then transferred to 70% ethanol. The samples were then dehydrated using a graded ethanol step through 70%, 80%, 95%, and 100% ethanol, then cleared in xylene and embedded in paraffin. The quadriceps muscle group was then cut in a transverse manner to expose the center of injury and then mounted face down in a paraffin mold. Five (5) to 7 μm sections were then placed on charged slides and baked overnight at 56°C until dry. After rehydration, the samples were stained with hematoxylin and eosin (H&E) by incubating in Harris hematoxylin for 5 minutes, followed by washing with tap water and dipping twice in acid ethanol to destain, followed by rinsing with ethanol and dipping in eosin Y. The samples were then washed with 95% ethanol, dehydrated, coverslipped and mounted with Permount. Picosirius red (PSR) staining was performed according to the manufacturer's instructions, and after rehydration, samples were stained in PSR for 1 hour, then washed with water and dipped twice in acetic acid to destain, then dehydrated and mounted with Permount. Slides were imaged on an EVOS microscope (Thermo).

[0104] Immunohistochemistry

[0105] The samples were rehydrated and then incubated in citrate antigen retrieval buffer for 20 minutes, followed by slow cooling on the benchtop for 20 minutes. Endogenous peroxidase was quenched by incubation in 0.3% hydrogen peroxide in 1xPBS for 5 minutes. Elite ABC-HRP kit (rabbit, Vector Laboratories) was used to stain the samples. Briefly, after washing with 1xPBS, the samples were blocked in 2.5% normal goat serum for 1 hour. The samples were incubated in primary antibodies diluted in blocking buffer for 1 hour. Rabbit monoclonal anti-CD103 (AbCam) and anti-E-cadherin (AbCam) were diluted at 1:100. The slides were washed 3 times with 1xPBS and then incubated with biotinylated secondary antibodies for 30 minutes. The slides were washed 3 times with 1xPBS and then incubated in VECTASTAIN Elite ABC reagent for 30 minutes. The samples were washed 3 times with 1xPBS and then incubated in The slides were incubated in DAB EqV peroxidase (HRP) substrate (Vector Laboratories) for 1 min, 30 s (CD103) or 50 s (E-cadherin). The slides were washed with tap water and then counterstained in Harris hematoxylin (Sigma) for 5 min, then rinsed with tap water, destained in acid ethanol, dehydrated and mounted with Permount.

[0106] RNA isolation and RT-PCR

[0107] 7 days after injury, quadriceps muscle groups were dissected from mice and homogenized in 2ml 1xPBS at 5000rpm with a mechanical homogenizer for 30 seconds. Five hundred (500) microliters of the resulting homogenized mixture were transferred to an Eppendorf tube containing 500ul TRI reagent solution (SigmaAldrich). The samples were vortexed and then stored at -80°C until RNA isolation. After thawing, 200ul chloroform (Sigma Aldrich) was added to each sample and vortexed, then separated for 5 minutes at room temperature, followed by centrifugation at 8000xg and 4°C for 15 minutes. The aqueous phase was combined with an equal volume of 70% ethanol and vortexed. The resulting sample was passed through an RNeasy micropreparative spin column (Qiagen), then washed once with RW1 buffer and twice with RPE buffer according to the manufacturer's instructions. The membrane was dried and RNA was eluted into 30ul of RNase-free water. RNA concentration was determined by NanoDrop and quality control was performed to proceed with samples with A260 / A280>2. Samples were diluted to 100 ng / ul concentration and 11 ul was added to SuperScript Reverse Transcriptase IV reaction with random hexamers as primers (ThermoFisher Scientific) according to the manufacturer's instructions. Two (2) ul of the resulting cDNA was added along with 10 ul of TaqMan Fast Advanced Master mix, 7 ul of nuclease-free water and 1 ul of FAM-MGB primer / probe: Gusb, Xcl1 (Table 3).

[0108] Protein Isolation from Mouse Tissues and Cytokine Analysis

[0109] Muscle and lymph node samples were snap frozen in liquid nitrogen or ethanol dry ice slurry immediately after dissection and stored until processing. Frozen muscle samples were added to 2 ml of ice-cold 1xPBS containing protease inhibitors (ThermoFisher Scientific) and cut into pieces with a pair of scissors. The samples were homogenized on ice for 45 seconds at 5000–6000 rpm using a mechanical homogenizer. Subsequently, 2.5 ml of ice-cold 1xPBS containing protease inhibitors and 50 ul of 10% Triton-X100 were added, then vigorously mixed and placed on ice for 5 minutes, and then aliquoted, snap frozen in liquid nitrogen and stored until use. On the day of use, the samples were thawed and centrifuged at 10,000 x g for 10 minutes to precipitate the fragments. Protein concentration was determined by diluting the Pierce BCA protein assay kit (Thermo Scientific) with lysis buffer 1: 1. Two hundred (200) micrograms of protein were injected into the Proteome ProfilerTM array, mouse XL cytokine array kit, and assayed according to the manufacturer's instructions. Blots were imaged on a BioRad ChemiDoc with a 30 sec exposure.

[0110] ELISA

[0111] XCL-1 measurement in mouse plasma samples was performed by using mouse XCL-1 SimpleStep ELISA kit (Abcam). Determination was performed according to the manufacturer's guidelines. In brief, 50 μL of mouse plasma samples or protein lysates diluted 1:1 with blocking buffer were added to the appropriate wells of a pre-coated 96-well plate. The sample was then treated with 50 μL of antibody mixture and then incubated at room temperature for 1 hour. After incubation, the mixture in the well was aspirated and the wells were washed three times with wash buffer. After the final wash, 100 μL of TMB chromogenic solution was added to each well, and the plate was incubated for 10 minutes. After incubation, 100 μL of stop solution was added to each well, and OD was subsequently read at 450 nm.

[0112] Statistics and data analysis

[0113] Flow cytometry data were unmixed using SpectroFlo software (Cytek Biosciences) using designated single spectral controls (Supplementary Tables 1, 2). The resulting unmixed data were exported to .fcs and then analyzed on FlowJo (Supplementary Figs. 2, 16). Dimensionality reduction algorithms were funded through FlowJo plugins. t-Stochastic Neighbor Embedding (t-SNE) was run with the following parameters: learning configuration - opt-SNE, number of iterations - 2000, perplexity - 30, KNN algorithm - exact (high ground tree), gradient algorithm - Barnes-Hut. Uniform manifold projection was run with the following parameters: Euclidean nearest neighbors - 15, minimum distance - 0.5, number of components - 2. FlowSOM 3.0.18 was run with the following parameters: number of metaclusters - 30. Singleton live immune cells were clustered using all parameters except LIVE / DEAD Blue. Data generated by manual gating were analyzed in GraphPad Prism v9 and R 4.1.2. Immunohistochemistry for E-cadherin was quantified by automatic white balance of Fiji (ImageJ), followed by color deconvolution to separate the DAB channel. The target area was manually outlined, then measured and converted to optical density (OD) readings by extracting log (maximum intensity / mean intensity). Each replicate represents the quantification of sections from different animals. The resulting OD values ​​were plotted in GraphPad Prism v9 for data display and analysis. Chemiluminescent proteome analysis blots were quantified and normalized to background using Protein Array Tools 2.0.0.1 version via MatLab (R2022a version) by pixel intensity and then displayed in R 4.1.2 as fold changes relative to uninjured control muscle tissue. The technical replicates of the RT-PCR Ct values ​​were averaged after subtracting the housekeeping gene (Gusb), and the average ΔCt of the uninjured control was subtracted from all ΔCt values ​​before being converted to display fold changes of 2 (- ΔΔ Ct).

[0114] Statistical tests used are stated in the figure captions.

[0115] Example 1. Characterization of immune cell responses after injury

[0116] In order to evaluate the immune response to engineering materials in wounds and tissue regeneration, bilateral volume reduction muscle surgery was performed, followed by implantation of materials. After creating a 3 mm defect in the quadriceps muscle group, the resulting gap was backfilled with control (saline), hydrated decellularized extracellular matrix (ECM) powder or polyethylene powder (PE). ECM provides an example of a pro-regenerative material, and PE is an example of a pro-fibrotic material. A 22-color flow cytometer panel was used to interrogate the bone marrow immune response to the material response and the early stages of wound healing, including markers of granulocytes, macrophages, dendritic cells, and phenotypic analysis of their activation ( Figure 1 ). Material treatment increased cell infiltration, reaching a peak at 7 days after injury and maintaining half of the peak infiltration level to 42 days after injury. Saline-treated controls peaked cell infiltration early, 3 days after injury (Figures 2A-2C). Structurally, the ECM material degraded over time, but still existed 41 days after injury, as shown by Sirius red staining of collagen. Cells with granular cytoplasm and segmented nuclei associated with neutrophils were found in the corona around the PE particles, which were subsequently replaced by fibrotic capsules. Cell infiltration in the material area was correlated with the total cell count observed during flow cytometry analysis (Figures 2A-2C).

[0117] A. Implanted materials recruit various innate immune compartments, with fibrotic materials shifting granulocytes from a predominantly eosinophilic to a predominantly neutrophilic lineage

[0118] Flow cytometry revealed that a diverse set of innate immune cells were recruited to the injury microenvironment 7 days after injury, depending on material treatment and visualized by dimensionality reduction ( Figures 3A-3D Granulocytes (such as neutrophils, basophils, and eosinophils) as well as mature macrophages and immature monocyte-like myeloid cells, dendritic cells, and other immune cells (CD45+Lin-) were identified by manual gating ( Figure 3A Comparison of responses to the different materials revealed differences in the immune repertoire 7 days after injury ( Figure 3B Using FlowSOM (a self-organizing map (SOM) algorithm for generating clusters based on the expression of markers detected by flow cytometry), we identified distinct subsets of macrophages and dendritic cells and confirmed the presence of granulocyte- and monocyte-like cell populations ( Figure 3C-3D ).

[0119] As previously mentioned, a high prevalence of macrophages (F4 / 80+CD68+) was observed to persist throughout the injury recovery process ( Figures 4A-4E). In addition, neutrophils (Ly6G+) were observed to be preferentially recruited to profibrotic PE-treated muscle lesions. The pro-regenerative material produced a granulocyte compartment dominated by eosinophils, which were more autofluorescent than neutrophils, possibly due to granule formation or phagocytosis of extracellular matrix materials. CD200R3+ basophils were preferentially recruited to untreated control lesions and peaked between 7 and 21 days after injury, while eosinophils in ECM-treated lesions persisted from 7 to 42 days after injury, and neutrophils in PE-treated lesions peaked at 7 days after injury and slowly declined 42 days after injury, while a large proportion of total immune cells remained in the microenvironment. Both control and ECM-treated lesions recruited neutrophils early, but they were cleared 7 days after injury. Compared with other treatments, PE-treated lesions recruited higher levels of monocytes, with a preference for CX3CR1+ cells, which may represent the activation of a pathogenic type 2 immune response, which promotes fibrosis when neutrophilic inflammation begins to subside. Dendritic cells were present and persisted at low proportions (<4% of total CD45+ cells) throughout the time course of the response to injury and material implantation. Macrophages peaked early and began to decrease proportionally over time, shifting from CD11b+F4 / 80+CD68+ cells to a majority of CD11b+CD68+F4 / 80- cells at 42 days post-injury.

[0120] B. Enrichment of CD103+XCR1+ dendritic cells by pro-regenerative scaffolds

[0121] Since previous studies have demonstrated adaptive immunity during biomaterial integration and regeneration / fibrosis, the phenotype of antigen-presenting cells was further investigated. The majority of MHCII+ immune (CD45+) cells in the wound microenvironment are F4 / 80+ macrophages ( Figure 5A and 5B ). Macrophages in ECM-tx muscle lesions were found to have higher levels of CD206, CD301b, and CD169 expression, suggesting local proliferation of tissue-resident cells with type 2 polarization, a hallmark of Th2-driven inflammation ( Figures 6A-6C Although accounting for only 1%-2% of the total CD45+ immune cell infiltration, the second most common APC in the wound cavity is dendritic cells (CD11c+CD11b lo / neg ). - CD11c + MHCII hi Expression of XCR1, chemokine receptors, and CD103 on dendritic cells to identify dendritic cells capable of cross-presenting Figures 7A-7H , Figure 8 ). CD11b+ F4 / 80 + Macrophages also express low levels of CD103 and XCR1, but significantly lower than CD11c+CD11b lo Dendritic cells ( Figure 8 , Figures 9A-9C Type 1 classical dendritic cells (cDC1) were enriched by the pro-regenerative scaffolds, whereas the pro-fibrotic scaffolds primarily recruited double-negative cells in a pattern that persisted up to 42 days post-injury ( Fig. 7A and 7B cDC1s expressed moderate levels of the co-stimulatory molecule CD86, whereas double negative cells showed CD86 expression that may be associated with plasmacytoid DCs (pDCs). hi Bimodal distribution of dendritic cell subsets ( Fig.10 Neither population expressed CD8α in muscle tissue. Double-negative and XCR1-positive cells increased proportionally over time in all groups ( Figure 7B and 7C ), but XCR1 + CD103 + Classical dendritic cells peaked in proportion between 3 and 7 days after injury, and peaked in counts 7 days after injury ( Figure 7B and 7C During the entire injury recovery process, there were very few CD103 single-positive dendritic cells in the wound cavity ( Figure 7B and 7C ). When evaluating the cell populations by dimensionality reduction and hierarchical clustering algorithms, the FlowSOM algorithm identified this cell type as a distinct cluster and mapped it to an island using t-SNE and UMAP visualization ( Fig.7D and 7E When FlowSOM was applied to the three treatment groups within the same cluster population, more than 70% of the cells within the cDC1 cluster were derived from ECM-treated lesions, while only 15% and 6% were derived from PE-treated and control lesions, respectively ( Figure 7F-7H The identification of these cells was confirmed in muscle lesions of two litters of C57BL / 6 mice as well as Lewis rats, demonstrating that this is reproducible and applicable to multiple species within the murine family ( Figures 11A-11B ).

[0122] At 7 days post-injury, when CD103+XCR1+ DCs peaked in muscle tissue, these DCs were detected in the overlying skin incision sites of ECM-treated muscle injuries, but at a significantly lower proportion, likely because the skin wound was largely closed by this time ( Fig.12 ).

[0123] In addition to local muscle tissue, dendritic cells were detected in the blood and lymph nodes of injured mice. These DCs (CD11b - / lo CD11c + MHCII hi ) have different expressions of markers related to antigen cross-presentation ability (XCR1, CD103 and CD8a) according to their location ( Figures 13A-13B ). As mentioned, in muscle, cells express high levels of both XCR1 and CD103 and no CD8a. In lymph nodes, these cells express all three markers: XCR1, CD103, and CD8a, while in blood, most cells are XCR1 - CD103 - CD8a - Interestingly, a greater proportion of CD11c was present in the blood of PE-treated mice. + MHCII hi DCs, and the majority of these cells are B220 / CD45R + , suggesting a potential preferential recruitment of circulating pDCs for profibrotic material implants ( Figures 14A-14C In the lymph nodes, there were no significant differences in the proportion of dendritic cells, although PE-treated mice did have a higher proportion of TCRγδ+ T cells, consistent with previous literature on the role of these cells in fibrotic diseases ( Fig.14A Regarding chemokines that bind to XCR1, there were no significant differences in XCL-1 levels in the blood at 7 and 21 days after injury between treatment groups, although significantly higher amounts of XCL-1 were present in the blood in the early response to injury in all treatment groups ( Fig.15 ).

[0124] C. Phenotype and recruitment of CD103+XCR1+ dendritic cells depend on adaptive immune activity

[0125] Since dendritic cells are able to communicate directly with adaptive immune cells (such as T cells and B cells) through antigen presentation, the inverse relationship and role of adaptive immunity in the recruitment and activation of these cells within the scaffold microenvironment was investigated. - / - ) in the whole environment, the proportion of dendritic cells recruited was similar, tending to Rag1 - / - Less, but XCR1+CD103+cDC1 significantly less ( Figures 16A-16F ). When their recruitment was assessed based on the expression of XCR1 or CD103, loss of CD103 expression was the main reason for the loss of these cDC1s.

[0126] Like macrophages, DCs in wild-type mice had high levels of CD301b and CD206 ( Figure 16G and 16H ), CD103+DCs expressed the highest levels of CD206 ( Fig.16I and 16J ). Compared with wild-type mice, - / - CD301b and CD206 in mice ( Figures 16G-16J )'s ECM scaffold-mediated type 2 upregulation was lost.

[0127] Example 2. Examination of T cell involvement in material response

[0128] A. Enhanced early T cell activation and subsequent induction of regulatory CD8+ T cells by pro-regenerative scaffold treatment

[0129] A 19-color flow cytometry panel was developed to evaluate lymphocyte behavior in blood and draining lymph nodes ( Fig.17 ). At 7 days post-injury, the prevalence of CD44+CD62L-CD4 and CD8 T cells in the draining lymph nodes was increased in ECM-treated injuries compared to PE-treated injuries. Active CD4+ T cells in the blood were enriched by ECM treatment at 7 days post-injury, correlating with previous work showing that IL-4 expression peaks in the draining lymph nodes at this time. This correlated with a lower proportion of HELIOS+ regulatory CD4 and CD8 T cells in the draining lymph nodes at 7 days post-injury. In the peripheral blood, the prevalence of CD4+HELIOS+iTregs was slightly higher compared to control injuries, and was more prevalent at 21 days post-injury. All treatments trended towards a higher proportion of HELIOS+CD8 T cells at 21 days post-injury compared to 7 days post-injury, although this was not significant after correction for multiple comparisons ( Figures 18A-18F , Fig.19 ). In addition to these regulatory cells, we identified a higher proportion of ST2+ regulatory B cells in ECM-tx and ST2+ pDCs in ECM-tx and PE-tx injuries compared to controls. Whether these cells are functionally regulatory or pathogenic is uncertain. ( Fig. 20A and 20B ).

[0130] B. CD103+XCR1+ adaptive immune cells are induced by trauma and altered by material treatment

[0131] In addition to these regulatory T cells, a CD103+XCR1+ adaptive immune cell subset induced by trauma was also found ( Fig.21A , Fig. 22). This population includes B cells, CD4+T cells, CD8+T cells, and γδT cells, and increases over time ( Fig.21B Among B cells and CD4+ T cells, the majority of this population was CD62L- even in the absence of injury, and CD8+ and γδT cell activation increased over time, with CD8 peaking at 7 days post-injury and γδT cells increasing continuously until 21 days post-injury ( Fig. 21C In addition to the CD103+XCR1+ population, there is also a CD103+ population that is most prominent in CD8+ T cells and γδ T cells. lo XCR1-group ( Fig.21A and 21D Activation of these cells peaked 7 days after injury, as determined by loss of CD62L expression ( Fig.21E Interestingly, these cells in both the blood and draining lymph nodes were CD44 lo , possibly indicating antigen-independent activation, although more work is needed to test this hypothesis ( Fig.23 Since there are multiple active adaptive immune cells in sterile injury, these CD103 and XCR positive adaptive immune cells were evaluated to determine whether they behave in a regulatory manner. It was found that the proportion of CD103+XCR1+ HELIOS+iTregs increased over time, and increased more than FoxP3+HELIOS-Tregs ( Figures 24A-24C ). This pattern was true for both CD4+iTregs and CD8+iTregs.

[0132] When draining lymph nodes and peripheral blood were compared, a higher proportion of circulating CD103+XCR1+ adaptive immune cells was observed ( Fig.25 ). For all treatment groups, B cells had the highest proportion of CD103+XCR1+ cells at 21 days post-injury, followed by γδ T cells, except for ECM-treated mice which had a lower proportion of these cells in the circulation. Additionally, at 7 days post-injury, when CD103+XCR1+ dendritic cell recruitment peaked, a strong enrichment of CD103+XCR1+ T cells was observed in the wound cavity compared to the blood and draining lymph nodes, suggesting that there is a preferential recruitment of these cells to the wound cavity.

[0133] C. Upregulation of Xcl1, E-cadherin, and TGFβ-related signaling in the injury microenvironment as potential mediators of CD103+ cell recruitment

[0134] At 7 days post-injury, Xcl1 mRNA expression was significantly higher in ECM-treated muscle lesions compared to uninjured controls. Fig. 6AWithin the muscle region, CD103+ cells were readily present in ECM-treated lesions, with a variety of morphologies associated with both myeloid and lymphocytes. These cells were located at the lesion interface, within the implant, and around the capsule ( Fig.26B In some cases, these cells were found within immune infiltrate clusters, but were also scattered throughout the injury site. In addition, there was upregulation of E-cadherin (a ligand for integrin CD103) at the injury interface compared to more distal uninjured muscle tissue ( Fig.26C This was true for all treatment groups, although most pronounced with biomaterial implants—both ECM and PE induced this condition and it was highly prevalent in damaged muscle tissue as well as in areas of immune infiltration with fine hematoxylin and eosin ( Fig.26D ).

[0135] When the cytokine and chemokine milieu was evaluated by protein arrays, there were large differences in the immune signature depending on the presence of injury as well as the type of treatment. Material implantation was associated with an increase in CCL6, which is produced by neutrophils and macrophages that are enriched in these materials. Both PE and ECM treatment induced upregulation of myeloperoxidase (MPO), which is associated with reactive oxygen species (ROS) generation. Interestingly, injury upregulated endoglin, part of the TGFβ receptor, compared to uninjured controls, and TGFβ is a known inducer of CD103 expression ( Fig. 27 ).

[0136] Example 3. Examination of NK cells involved in material response

[0137] To further evaluate the immune response to biomaterials in injury, 3 mm defects in the quadriceps of mice were studied and three conditions were studied: no treatment (control), polyethylene (PE) was implanted into the injury site to induce a profibrotic response, or an extracellular matrix (ECM) was used to promote a pro-regenerative environment. Flow cytometry, ELISA, and RT-PCR were then used to evaluate the immune response at each wound site. Seven days after injury, NK cells (CD49b + TCRβ - ) increased (2.498% vs. 0.216% and 0.0734% ± 0.017, P < 0.0001). Gene expression analysis showed that these NK cells displayed high levels of Tgfb1 expression ( Fig.29A These NK cells are accompanied by XCR1, which is capable of antigen cross-presentation. + CD103 + In addition, ECM treatment induced an increase in XCL-1 levels in the injury microenvironment and peripheral blood ( Fig.29B ). Wild type and Rag1- / - There were no significant differences in XCL-1 concentrations between mice, suggesting that XCL-1 secretion is mediated by RAG1-independent cell types such as NK cells. This is consistent with XCR1 promoting regenerative behavior. + CD103 + The increase in dendritic cells is associated with Batgf3 - / - In the absence of these cells in mice, there were physical manifestations more distal to the injury site, such as necrotic muscle fibers and giant cells, indicating that the trauma spread beyond the initial injury.

[0138] NK cells were also exposed to decellularized extracellular matrix (ECM) fragments or low molecular weight hyaluronic acid (LMW-HA) in vitro and the levels of Xcl1 production were measured. Upregulation of Xcl1 was observed 24 hours after exposure, indicating that engagement of NK cells with damage-associated molecular patterns mediates Xcl1 secretion.

[0139] Comprehensive evaluation of these data, except for XCR1 + CD103 + Dendritic cells and CD49b + In addition to NK cells, XCL-1 is upregulated within the first week after injury. The presence of XCL-1 provides + CD103 + Possible mechanism of cell recruitment. - / - In mice, there was no significant difference in the concentration of XCL-1 compared to wild-type, indicating that RAG-independent recruitment is the source of XCL-1. NK cells were found in greater proportions at sites of ECM damage compared to controls and PE environments.

[0140] Results and Discussion

[0141] The results demonstrated that DCs capable of cross-presenting were induced by pro-regenerative materials in trauma. These were accompanied by M2 macrophages carrying MHCII and CD8+iTreg and ST2+ regulatory B cells in the periphery, as well as CD103+XCR1+ adaptive immune cells induced by trauma. Recruitment of DCs capable of cross-presenting and activation of CD103+XCR1+CD8 T cells peaked early in the response to injury and reached a maximum 7 days after injury. Upregulation of both Xcl1 gene expression in homogenized injured tissue and E-cadherin within the muscle injury site was detected, particularly at the interface of the injury and the material and in the capsule surrounding the material, which could be a mechanism for recruiting these CD103+ cells that could bind upregulated E-cadherin to migrate to the injury site. In addition, due to Rag1 - / - CD103 expression on dendritic cells of mice is low and is known to be expressed by T regSecreted TGFβ induced CD103 upregulation, suggesting a possible mechanism by which damage-associated TGFβ secretion by adaptive immune cells leads to CD103 upregulation in DCs.

[0142] These data suggest that the balance of antigen cross-presentation during injury recovery is regulated by engineered material implantation. In the context of materials that promote type 2 and regulatory immune responses (such as decellularized ECM scaffolds), this cross-presentation occurs in an environment that is more adapted to peripheral tolerance and antigen-specific wound repair. In the context of pro-inflammatory and pro-fibrotic materials (such as PE, which favor type 1 and type 17 immune responses), this may lead to autoimmune activation and the formation of autoreactive T cells and antibodies, leading to distal lesions and systemic immune dysregulation in some patients that have been reported.

[0143] The presence of CD103+XCR1+ innate and adaptive immune cells may represent a homeostatic regulation of the response to injury itself, which is amplified during trauma following reaction with cross-presenting dendritic cells ( Fig.28 and 30 ). Previous studies have shown that XCR1+ T cells can be induced by trogocytosis and communication with cross-presenting dendritic cells and are potential targets for cancer immunotherapy. Although the ECM scaffold introduces a new protein source of exogenous antigens, the control injuries as well as the PE-treated injuries were surgically induced sterile wounds that do not introduce non-self antigens, and therefore, these cells may react with self antigens, or in an antigen-independent manner. The cells and pathways that communicate with cross-presenting dendritic cells have not been previously described in the context of trauma and biomaterial implantation, and describe a new mechanism of immune response to injured and damaged self in traumatic injury.

Claims

1. A therapeutic composition for treating a wound in an individual, the therapeutic composition comprising a therapeutic agent that induces a pro-regenerative environment within the wound and / or within the tissue surrounding the wound, wherein the therapeutic agent comprises a decellularized extracellular matrix (ECM) or a component derived from a decellularized extracellular matrix.

2. The therapeutic composition of claim 1, wherein the component derived from the decellularized extracellular matrix comprises degradation products of the ECM.

3. The therapeutic composition of any one of claims 1 or 2, wherein the therapeutic agent comprises one or more components selected from the group consisting of collagen, laminin, fibronectin, elastin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, perlecan, aggregated proteins, and degradation products thereof.

4. The therapeutic composition of any one of claims 1-3, wherein the therapeutic agent comprises an extracellular matrix activin or a damage-associated molecular pattern (DAMP).

5. The therapeutic composition of claim 4, wherein the extracellular matrix activator comprises a member selected from the group consisting of metastatin, affesten, angiostatin, tetrastatin, pentastatin, lamstatin, hexastatin, endostatin, dormant protein 1, dormant protein 2, dormant protein 3, dormant protein 4, endostatin, neostatin, anastellin, sibsttin, PEX, endorepellin, CUB1CUB2 domain, Ten / 2, Ten11 / 12 / 13, Ten14, kappa-elastin, the extracellular domain of syndecan-1, the extracellular domain of syndecan-2, the extracellular domain of syndecan-3, the extracellular domain of syndecan-4, elastokine, laminin peptide A13, laminin peptide C16, laminin 332 (laminin 5), DGGRYY peptide, GHK tripeptide, VGVAPG peptide, PGP tripeptide, acetylated PGP tripeptide (AcPGP), tenascin-C (TNC), the G3 domain of nidogen-1, and tumor inhibitory protein.

6. The therapeutic composition of any one of claims 1-8, wherein the therapeutic agent induces local proliferation of M2 macrophages within the wound and / or within the tissue surrounding the wound.

7. The therapeutic composition of claims 1-9, wherein the therapeutic agent induces local proliferation and / or recruitment of classical dendritic cells (cDC1) within the wound and / or within the tissue surrounding the wound.

8. The therapeutic composition of claim 10, wherein the cDC1 is a cross-presenting dendritic cell.

9. The therapeutic composition of any one of claims 9 or 10, wherein the cDC1 is XCR1 + CD103 + Dendritic cells.

10. The therapeutic composition of any one of claims 1-12, wherein the therapeutic agent comprises a peptide, a protein, a glycoprotein, a lipoprotein, a lipid, a sugar, a polysaccharide, a glycosaminoglycan, a nucleic acid molecule, an organic molecule, or a combination thereof.

11. A therapeutic composition for recruiting NK cells to a wound or wound-adjacent tissue, the therapeutic composition comprising a therapeutic agent that induces a pro-regenerative environment in the wound and / or in the tissue surrounding the wound, wherein the therapeutic agent comprises a decellularized extracellular matrix (ECM) or a component derived from a decellularized extracellular matrix.

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