Macrophage-containing hydrogel for chronic wounds

Abstract

The disclosure relates, in one aspect, to compositions comprising a hydrogel matrix and a plurality of macrophages at least partially encapsulated therein. In certain embodiments, the compositions of the disclosure are useful for treating or ameliorating a wound of a subject in need thereof. In certain embodiments, the sound is a diabetic sound (e.g., diabetic foot ulcer).

Description

[0001] TITLE

[0002] Macrophage-Containing Hydrogel for Chronic Wounds

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under R01 HL 130037 and R21 AR083080 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0005] CROSS-REFERENCE TO RELATED APPLICATIONS

[0006] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 645,559, filed May 10, 2024, which is incorporated herein by reference in its entirety.

[0007] BACKGROUND

[0008] Regenerative medicine seeks to harness the body’s natural healing abilities, which invariably requires control over the immune system. Smaller injuries are capable of regeneration via highly coordinated actions by immune cells to facilitate inflammation, resolution, and repair. In particular, macrophages are upstream regulators of several critical supporting cells like fibroblasts, endothelial cells, and satellite cells in the case of muscle, making macrophages key targets for various cell therapies. Macrophages rapidly shift phenotypes in response to local microenvironmental cues, including mechanical, chemical, and biological stimuli towards a range of phenotypes that are generalized as pro-inflammatory or pro-regenerative.

[0009] Macrophages within wounds must transition from a pro-inflammatory phenotype (also known as Ml) to a reparative phenotype (also known as M2) in order for successful healing to occur. These phenotypes are themselves quite diverse and comprise multiple subtypes. Ml -like macrophages are critical for the initiation of wound healing, especially through secretion of cellrecruiting chemokines and pro-angiogenic growth factors. M2-like macrophages are critical for stabilization of wound healing and resolution of inflammation, especially through secretion of anti-inflammatory factors and growth factors that stabilize growing blood vessels and promote proliferation of fibroblasts. Macrophages in chronic wounds, including diabetic foot ulcers, venous leg ulcers, pressure ulcers, and wounds in elderly or otherwise immunocompromised patients, are defective in both recruitment and response to external stimuli. There is a need for technologies that can replace the functions of dysfunctional macrophages in a temporally controlled way. This disclosure addresses this need through the transplantation of phenotypically controlled macrophages via immunomodulatory biomaterials.

[0010] Previous work has successfully demonstrated the ability to influence a non-polarized macrophage towards the pro-regenerative phenotype ex vivo, which is critical for successful tissue repair. However, simply transplanting pro-regenerative macrophages is insufficient because these macrophages can be cleared within hours to various other organs and cannot survive the harsh inflammatory environment they are transplanted into. Additionally, maintaining polarized macrophages with the desired phenotype requires sustained exposure to the appropriate stimuli. Macrophages that are pre-polarized and transplanted into these wounds and injuries are subject to the myriad of cytokines and growth factors within the area and evolve with time.

[0011] Overall, cell therapy with pro-regenerative macrophages remains as a promising therapeutic approach but is limited by two main challenges. First, macrophages are highly plastic cells that rapidly shift phenotype in response to microenvironmental cues. Therefore, a strategy is needed to sustain their pro-regenerative phenotype in situ following administration, to prevent them from re-polarizing in response to pro-inflammatory cues at the site of injury. Second, transplanted macrophages are rapidly cleared or cannot survive the harsh inflammatory environment of a wound. Therefore, engineered macrophages will require a delivery system that can retain them within the site of injury to facilitate their regenerative potential while they support cell-cell interactions and secrete regenerative factors and cytokines.

[0012] Biomaterials are crucial for macrophage cell therapy because they can support the survival, retention, and function of therapeutic regenerative macrophages within sites of injury. Realizing this potential, however, requires a deeper understanding of how specific biomaterial properties influence macrophage behavior, both in vitro and in vivo, particularly in the context of dysfunctional tissue repair. However, selecting, designing, and optimizing a biomaterial requires careful consideration to ensure control over transplanted macrophages while avoiding adverse immune responses, all while providing the appropriate cues to guide macrophage behavior for optimal healing outcomes. To complicate the matter, assays and metrics to understand a biomaterial’s impact on a macrophage cell therapy has not been well reported or explored within the published literature.

[0013] There is thus a need in the art for biomaterials which can support the survival, retention, and function of therapeutic regenerative macrophages within sites of injury and methods of use thereof. The disclosure addresses this unmet need.

[0014] BRIEF SUMMARY

[0015] In one aspect, the invention provides a composition comprising:

[0016] (a) a hydrogel matrix, and

[0017] (b) a plurality of macrophages, wherein the plurality of macrophages is at least partially encapsulated in the hydrogel matrix.

[0018] In various embodiments, the hydrogel matrix is biocompatible.

[0019] In various embodiments, the hydrogel matrix comprises a crosslinked polypeptide, crosslinked polysaccharide, and / or crosslinked synthetic polymer.

[0020] In various embodiments, wherein at least one of the following applies:

[0021] (a) the crosslinked polypeptide comprises collagen (e.g., gelatin);

[0022] (b) the crosslinked polysaccharide comprises hyaluronic acid; and

[0023] (c) the crosslinked synthetic polymer comprises a polyol (e.g., polyethylene glycol).

[0024] In various embodiments, the crosslinked polypeptide, crosslinked polysaccharide, or crosslinked synthetic polymer is crosslinked by a mechanism selected from the group consisting of covalent crosslinking (e.g., photo-crosslinking and enzymatic crosslinking) and ionic crosslinking (e.g., Ca2+).

[0025] In various embodiments, the covalent crosslinking comprises photo-crosslinking of a polypeptide, polysaccharide, or synthetic polymer which is at least partially functionalized with an acryloyl moiety (e.g., acryloyl or methacryloyl) or mal eimide moiety.

[0026] In various embodiments, the hydrogel matrix has a concentration of the crosslinked polypeptide, polysaccharide, or synthetic polymer of about 0.1% (w / v) to about 10% (w / v).

[0027] In various embodiments, the hydrogel matrix comprises a crosslinked gelatin methacryloyl (GelMA).

[0028] In various embodiments, the GelMA comprises methacrylate-functionalized gelatin, wherein the methacrylate and gelatin have a ratio of about 25 pmol methacrylate / g gelatin to about 100 nmol methacrylate / g gelatin, optionally wherein the methacrylate and gelatin have a ratio of about 66.7 pmol methacrylate / g gelatin.

[0029] In various embodiments, the plurality of macrophages comprise primary macrophages.

[0030] In various embodiments, the primary macrophages comprise or consist essentially of bone marrow derived macrophages (BMDMs).

[0031] In various embodiments, the plurality of macrophages comprise allogenic macrophages.

[0032] In various embodiments, at least a portion of the plurality of macrophages have been polarized with interleukin-4 (IL-4).

[0033] In various embodiments, the plurality of macrophages comprise macrophages which have not been polarized with interleukin-4 (IL -4).

[0034] In various embodiments, at least a portion of the plurality of macrophages exhibit an Ml phenotype for a period of about 4 days following at least partial encapsulation in the hydrogel matrix.

[0035] In various embodiments, the Ml phenotype is characterized by a pro-inflammatory phenotype, optionally wherein the pro-inflammatory phenotype is characterized by increased expression, production, and / or secretion of CD38, CD86, PDL1, and / or CXCR4, as compared to an unactivated macrophage.

[0036] In various embodiments, at least a portion of the plurality of macrophages exhibit an M2 phenotype after about 7 days following at least partial encapsulation in the hydrogel matrix.

[0037] In various embodiments, the M2 phenotype is characterized by a reparative phenotype, optionally wherein reparative phenotype is characterized by increased expression, production, and / or secretion of cytokines Argl, CD163, CD301b, and / or CD206, as compared to an unactivated macrophage.

[0038] In various embodiments, the plurality of macrophages are substantially retained within the hydrogel matrix after a period of about 7 to about 21 days.

[0039] In various embodiments, the composition has a concentration of macrophages of about 1.0 x 106macrophages / mL to about 3.0 x 107macrophages / mL, optionally wherein the composition has a concentration of about 2.5 x 107macrophages / mL.

[0040] In various embodiments, the composition further comprises at least one additional therapeutic agent. In various embodiments, the therapeutic agent is an antimicrobial agent.

[0041] In various embodiments, the invention provides a method for treating or ameliorating a wound of a subject in need thereof, the method comprising applying the composition to the wound.

[0042] In various embodiments, the wound is a chronic wound.

[0043] In various embodiments, the wound is a diabetic wound.

[0044] In various embodiments, the diabetic wound is diabetic foot ulcer.

[0045] In various embodiments, the composition is applied to the wound for a period of about 3 days to about 23 days.

[0046] BRIEF DESCRIPTION OF THE FIGURES

[0047] The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present application.

[0048] FIGs. 1 A-1D: Encapsulation within GelMA hydrogels causes macrophages to transition from pro-inflammatory to reparative in vitro. FIG. 1A: Murine macrophages were encapsulated within GelMA hydrogels or seeded within porous gelatin scaffolds and compared over 7 days in vitro to macrophages cultured on non-tissue culture-treated polystyrene well plates. FIG. IB: Macrophage viability for days 1, 3, and 7 using an amine-reactive fluorescent dye that permeates cells with compromised membranes and flow cytometry. FIGs. 1C-1D: Mean fluorescent intensity (MFI) from flow cytometry analysis of inflammatory phenotype markers (CD38, CD86, PDL1, and CXCR4; FIG. 1C), the fibrotic marker CD9 and reparative phenotype markers (ARG1, CD163, CD301b, and CD206) (FIG. ID). Two-way ANOVA, Tukey’s multiple comparison test. n=6, Bars show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001.

[0049] FIGs. 2A-2C: GelMA-encapsulated macrophages secrete high levels of inflammatory cytokines and cell-recruiting chemokines. FIG. 2A: cytokines, chemokines, and growth factors secreted by macrophages. FIGs. 2B-2C: Top 8 greatest differences between groups for IL6, TNFa, RANTES (CCL5), MDC (CCL22), TARC (CCL17) and KC (CXCL1) (FIG. 2B) and IL10, MIP-2 (CXCL2), IL20, IFNg, IL11, and MCP-1 (CCL2) (FIG. 2C). Two-way ANOVA, Tukey’s multiple comparison test. n=6, Bars show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0 ooi, ****p < 0.0001. FIGs. 3A-3C: Parallel in vitro and in vivo analysis of macrophage phenotype reveals dramatic increase of reparative phenotype marker expression for macrophage-loaded scaffolds. FIG. 3 A: Schematic of parallel in vitro and in vivo experimental setup. FIG. 3B: Recovery of live, GFP+ macrophages represented as a percentage of spiked blank controls in which macrophages were combined with biomaterials just prior to digestion for flow cytometry to calculate a recovery. FIG. 3C: Mean Fluorescence Intensity (MFI) of recovered macrophage phenotype expression using flow cytometry of inflammatory phenotype markers (CD38, PDL1, and CXCR4) and reparative phenotype markers (CD9, ARG1, CD163, CD301b, and CD206) over days 1 and 7. All groups loaded with 1E6, IL-4 polarized macrophages. One-way ANOVA, Sidak’s multiple comparison test. n=6, Bars show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 between analyzed groups. »p < 0.05, »»p < 0.01, “‘p < 0.001, ••••p < 0.0001 between the in vitro vs. in vivo comparisons of the same biomaterial carrier.

[0050] FIGs. 4A-4B: Tissue sectioning and staining of treatment groups at day 3 confirms presence of transplanted GFP+ macrophages retained within the site of injury. FIG. 4A: 4x magnification of representative F4 / 80 (macrophages), GFP (delivered macrophages), and CD31 (endothelial cells) for non-injured / untreated (naive), untreated, GelMA-only, Porous scaffold- only (Scaffold), GelMA w / encapsulated GFP macrophages (GelMA + Mac), and Porous scaffold w / encapsulated GFP macrophages (Scaff + Mac). Row 4 shows 32x magnification of representative regions at the interface of the injury and biomaterial. FIGs. 4B: Quantitation and analysis of PEC AM-1 positive staining surrounding within 200 um of the injury and biomaterial interface.

[0051] FIGs. 5A-5B: Host macrophage phenotype and immune cell recruitment is primarily driven by the biomaterial carrier by day 7. FIG. 5A: Mean Fluorescence Intensity (MFI) of recovered macrophage phenotype expression using flow cytometry of inflammatory phenotype markers (CD38, PDL1, and CXCR4) and reparative phenotype markers (CD9, ARG1, CD163, CD301b, and CD206) over days 1 and 7. All groups loaded with 1E6, IL-4 polarized macrophages. Two-way ANOVA, Tukey’s multiple comparison test. n=6, Bars show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 between analyzed groups. “U” indicates statistical significance compared to the untreated control. FIG. 5B: Percentage of the initial cell population that was positive for each marker representing a specific immune cell. Two-way ANOVA, Tukey’s multiple comparison test. n=6, Bars show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 between analyzed groups. “U” indicates statistical significance compared to the untreated control.

[0052] FIGs. 6A-6C: Exemplary experimental protocols and timelines taken to setup simultaneous sample processing for flow cytometry (FIGs. 6A-6B) and imaging studies (FIG. 6C) described herein.

[0053] FIGs. 7A-7B: GelMA-encapsulated macrophages secrete high levels of inflammatory cytokines and cell-recruiting chemokines; differences between groups for IL4, VEGF, IFNB-1, MCP5, GSCF, IL17, and IP10 (FIG. 7A) and MCSF, MIG (CXCL9), MIPa, MIPb, IL16, MIP3a, and MIP3b (FIG. 7B).

[0054] FIG. 8: Images depicting H&E staining of quadricep muscle tissue samples obtained from mice in treatment and control groups (z.e., untreated, GelMA only, Gelatin Scaffold (e.g. Surgifoam™), GelMA + Mac, and + Mac).

[0055] FIG. 9: Quantification of PECAM-1 / CD31 expression across treatment groups. Bar graph depicts the number of red-positive pixels (indicative of PECAM-1 / CD31 signal) within the total image for each group (i.e., naive, untreated, GelMA only, Scaffold, Scaffold + Macs, and GelMa + Macs). Data reflect mean values ± standard deviation from total image quantification.

[0056] FIGs. 10A: Bar graphs depicting percentage of the initial cell population that was positive for each marker representing a specific immune cell (i.e., CD9, CD38, Argl, CD163, PDL1, CXCR4, CD301b, and CD206. FIG. 10B: Bar graphs depicting mean fluorescence intensity (MFI) of recovered host immune cell distribution of B Cells, T Cells Resident Macrophages, Classical Monocytes, NK Cells, Neutrophils, Non-Classical Monocytes, and Dendritic Cells using flow cytometry immune cell markers over days 1 and 7.

[0057] FIG. 11 : Schematic showing that biomaterial selection is crucial for taking advantage of a desired regenerative effect. Overview schematic of biomaterial porosity on regenerative effect such that the macroporous scaffold would potentially best support interactions with host immune cells because of the larger pores that could facilitate cell-cell interactions, whereas the nanoporous hydrogel would improve retention of transplanted macrophages and serve as a secretome factory that could influence the host environment.

[0058] FIGs. 12A-12I: Day 1 marker expressions utilizing exemplary GelMA formulations of the disclosure for CD9 (FIG. 12A), CD38 (FIG. 12B), CD86 (FIG. 12C), PDL1 (FIG. 12D), CXCR4 (FIG. 12E), ARG1 (FIG. 12F), CD 163 (FIG. 12G), CD301b (FIG. 12H), and CD206 (FIG. 121).

[0059] FIGs. 13A-13I: Day 3 marker expressions utilizing exemplary GelMA formulations of the disclosure for CD9 (FIG. 13A), CD38 (FIG. 13B), CD86 (FIG. 13C), PDL1 (FIG. 13D), CXCR4 (FIG. 13E), ARG1 (FIG. 13F), CD 163 (FIG. 13G), CD301b (FIG. 13H), and CD206 (FIG. 131).

[0060] FIGs. 14A-14I: Day 7 marker expressions utilizing exemplary GelMA formulations of the disclosure for CD9 (FIG. 14A), CD38 (FIG. 14B), CD86 (FIG. 14C), PDL1 (FIG. 14D), CXCR4 (FIG. 14E), ARG1 (FIG. 14F), CD163 (FIG. 14G), CD301b (FIG. 14H), and CD206 (FIG. 141).

[0061] FIG. 15: Viability of macrophages using Live / Dead Aqua with certain exemplary GelMA formulations of the disclosure.

[0062] DETAILED DESCRIPTION OF THE INVENTION

[0063] Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0064] Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement "about X to Y" has the same meaning as "about X to about Y," unless indicated otherwise. Likewise, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z," unless indicated otherwise.

[0065] In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B " In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

[0066] In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0067] Description

[0068] Diabetic foot ulcers occur in up to 34% of patients living with type 2 diabetes worldwide, or 26 million people annually, leading to over 82,000 lower limb amputations annually in the United States and a 5-year mortality rate of up to 74%. The biological mechanisms behind impaired wound healing are complex, but dysfunctional macrophage recruitment and activation have been identified as primary factors.

[0069] Macrophages are innate immune cells that play a central role orchestrating tissue regeneration following injury or disease. Macrophage cell therapy holds great potential in regenerative medicine but is limited by two main challenges: (1) retention of transplanted cells within the site of injury, and (2) sustained control over macrophage phenotype. Biomaterial carriers hold potential to address these challenges, but it is still poorly understood how they can influence macrophage behavior. To address these questions, survival and phenotype changes in transplanted regenerative macrophages in response to nanoporous gelatin methacryloyl (GelMA) hydrogels and macroporous gelatin scaffolds were investigated in vitro and in vivo in a murine volumetric muscle loss (VML) quadricep model as a preclinical model of impaired tissue repair. The host immune cell response to the biomaterials with or without encapsulated macrophages was also analyzed and changes in resident macrophage phenotype were evaluated. Macrophages encapsulated in GelMA hydrogels exhibited a pro-inflammatory phenotype that transitioned to reparative over one week in vitro, while porous scaffolds had more neutral effects.

[0070] Thus, in one aspect, the compositions of the disclosure are useful for treating or ameliorating a wound of a subject in need thereof, including diabetic foot ulcers.

[0071] Definitions

[0072] The term "about" as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

[0073] The terms “antimicrobial agent” and “antibiotic agent” are used interchangeably herein to refer to naturally occurring, semisynthetic, or fully synthetic agents which inhibit the growth of microbes (i.e., bacteria, fungi, viruses, parasites and microbial spores) thereby preventing their development and microbial or pathogenic action. An antibiotic agent can be selected from the group consisting of small organic or inorganic molecules; saccharides; oligosaccharides; polysaccharides; biological macromolecules, e.g., peptides, proteins, and peptide analogs and derivatives; peptidomimetics; antibodies and antigen binding fragments thereof; nucleic acids; nucleic acid analogs and derivatives; glycogens or other sugars; immunogens; antigens; an extract made from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combinations thereof. As used herein, the term “antibiotic agent” is intended to embrace antibacterial agents or antimicrobial agents, antifungal agents, antiprotozoal agents, antiviral agents and mixtures thereof.

[0074] Non-limiting, exemplary antibiotic agents include, but are not limited to, acrosoxacin, amifioxacin, amikacin, amoxycillin, ampicillin, aspoxicillin, azidocillin, azithromycin, aztreonam, balofloxacin, biapenem, brodimoprim, cefaclor, cefadroxil, cefatrizine, cefcapene, cefdinir, cefetamet, ceftmetazole, cefoxitin, cefprozil, cefroxadine, ceftarolin, ceftazidime, ceftibuten, ceftobiprole, cefuroxime, cephalexin, cephalonium, cephaloridine, cephamandole, cephazolin, cephradine, chlorquinaldol, chlortetracycline, ciclacillin, cinoxacin, ciprofloxacin, clarithromycin, clavulanic acid, clindamycin, clofazimine, cloxacillin, colistin, danofloxacin, dapsone, daptomycin, demeclocycline, dicloxacillin, difloxacin, doripenem, doxycycline, enoxacin, enrofloxacin, erythromycin, fleroxacin, flomoxef, flucioxacillin, flumequine, fosfomycin, gentamycin, isoniazid, imipenem, kanamycin, levofloxacin, linezolid, mandelic acid, mecillinam, meropenem, metronidazole, minocycline, moxalactam, mupirocin, nadifloxacin, nalidixic acid, netilmycin, netromycin, nifuirtoinol, nitrofurantoin, nitroxoline, norfloxacin, ofloxacin, oxytetracycline, panipenem, pefloxacin, phenoxymethylpenicillin, pipemidic acid, piromidic acid, pivampicillin, pivmecillinam, prulifloxacin, rufloxacin, sparfloxacin, sulbactam, sulfabenzamide, sulfacytine, sulfametopyrazine, sulphacetamide, sulphadiazine, sulphadimidine, sulphamethizole, sulphamethoxazole, sulphanilamide, sulphasomidine, sulphathiazole, teicoplanin, temafioxacin, tetracycline, tetroxoprim, tigecyclin, tinidazole, tobramycin, tosufloxacin, trimethoprim, vancomycin, and pharmaceutically acceptable salts or esters thereof.

[0075] The term “biocompatible” as used herein refers to a material that is substantially nontoxic in the in vivo environment of its intended use, and that is not substantially rejected by the subject’s physiological system.

[0076] The term “crosslink” as used herein refers to a bond or chain of atoms attached between and linking two different polymer chains.

[0077] The term “encapsulated” as used herein refers to a state in which one or more cells (e.g., macrophages), proteins, nucleic acids, small molecules, or other biologically or chemically active substances, are physically confined, enclosed, or embedded within a surrounding matrix, shell, membrane, or structure (e.g., a hydrogel). In certain embodiments, the term “encapsulated” indicates that the cells, proteins, nucleic acids, small molecules or the like are substantially retained within the encapsulating material under physiological or intended use conditions. As used herein, “encapsulated” does not require complete isolation of the agent from the external environment, and may include partial diffusion from the encapsulating structure. In certain embodiments, the partial diffusion results in the escape of no more than about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about 25% of the encapsulated material (e.g., macrophages) over a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or about 23 days.

[0078] The term “hydrogel” as used herein refers to a crosslinked material which is not water- soluble and can contains at least 10% by weight of water within its polymer matrix when fully hydrated.

[0079] The term “macrophage” as used herein refers to a type of white blood cell derived from monocytes that resides within tissues and functions primarily in the detection, phagocytosis, and destruction of pathogens and apoptotic cells. As used herein, the term encompasses various macrophage subtypes, including but not limited to, classically activated, alternatively activated, tissue-resident, bone marrow-derived, primary, autologous, and allogeneic macrophages, as well as macrophages polarized in vitro or ex vivo using cytokines or other stimuli.

[0080] The term “polarization” as used herein in the context of macrophages, refers to a process whereby macrophages adopt different functional programs in response to signals in their microenvironment. In certain embodiments, macrophage polarization results in a change of a macrophage from an Ml phenotype (proinflammatory) to an M2 phenotype (reparative). In certain embodiments, the Ml phenotype (proinflammatory) is characterized by increased expression, production, and / or secretion of CD38, CD86, PDL1, and / or CXCR4, as compared to an unactivated macrophage. In certain embodiments, the M2 phenotype (reparative) is characterized by increased expression, production, and / or secretion of cytokines Argl, CD163, CD301b, and / or CD206, as compared to an unactivated macrophage.

[0081] The terms “patient,” “subject,” or “individual” are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In a non-limiting embodiment, the patient, subject or individual is a human.

[0082] The term “polymer” may include, according to some embodiments, any molecule comprising repeating structural units connected to each other, typically, by covalent chemical bonds. The term “polymer” may include, according to some embodiments, a homopolymer (which is a polymer derived from one monomer species), a copolymer (which is a polymer derived from two (or more) monomeric species) or a combination thereof. A polymer, as referred to herein, may include a mixture of polymers. A polymer, as referred to herein, may include linear and / branched polymers which consist of a single main chain with one or more polymeric side chains.

[0083] The term “polyol” as used herein refers to an organic compound comprising two or more hydroxyl functional groups, which may be linear, branched, or cyclic, and may comprise aliphatic, aromatic, or heterocyclic structural elements. The term encompasses both small molecule and polymeric polyols, including but not limited to sugar alcohols (e.g, erythritol, sorbitol, xylitol), polyether polyols (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG), and copolymers thereof), and polyester polyols. In non-limiting embodiments, the term “polyol” also includes functionalized polyols, such as derivatives bearing one or more additional reactive or functional groups (e.g., carboxyl, amine, thiol, or acrylate), and includes functionalized PEGs (e.g., methoxy -PEG, PEG-diacrylate, PEG-diamine) and other functionalized polyalkylene glycols. Polyols may vary in molecular weight and may be mono-, di-, or multi-functional depending on the number of hydroxyl groups or other reactive termini present.

[0084] The term “polypeptide” as used herein refers to a polymer of amino acids residues of at least eight or more amino acids bonded via covalent peptide bonds. The polypeptide can be linear or branched and may comprise naturally occurring and / or amino acid analogs. In nonlimiting embodiments, the term polypeptide includes protein-derived polymers like collagen and gelatin.

[0085] The term “polysaccharide” as used herein refers to polymeric carbohydrate structures, formed of repeating units (e.g., mono- or di-saccharides) joined together by glycosidic bonds. These structures may be linear, but may also contain various degrees of branching. The polysaccharide may be a homo- or heteropolysaccharide. The polysaccharide may contain noncarbohydrate units. Examples of polysaccharides include, but are not limited to, hyaluronic acid, amylose, amylopectin, pectins, glycogen, agar, alginate, carrageenans, chitin, beta-glucans, dextrins, carboxymethylcellulose, carboxy ethyl cellulose, hydroxypropylcellulose, methylcellulose and combinations thereof. One exemplary combination of the afore-mentioned polysaccharides is starch which consists of amylopectin and amylose. Usually polysaccharides comprise more than 100 monosaccharide or disaccharide units, such as 200 to 25,000. The molecular weight of the polysaccharides can be greater than 20,000, for example 20,000 to 1,000,000 g / mol or even higher.

[0086] The terms “treating” and “ameliorating” as used herein refer to the various steps involved in wound healing. Wound treatment is not limited to skin treatment, but also includes tissue repair of other types of wounds as described above. Further, treatment of a wound may comprise reduction and / or amelioration of pain and / or infections associated with the wound.

[0087] The term “wound” as used herein refers to bums, ulcers (e.g., diabetic ulcers, vascular deficiencies, swelling and burn-derived ulcers) exfoliated skin, or other skin problems (e.g., allergies), scratches, cuts, abrasions, and damage to tissues or skin caused by surgical procedures (e.g., those caused by minimally invasive surgery, laparoscopic surgery, robotic surgery, incision biopsy, general surgery and cosmetic surgery), inter alia. Wounds can range from superficial (e.g., affecting only the epidermis) to deeper trauma (e.g., lesions affecting a layer of tissue under the skin or epidermis). The wound may be of any length or shape, for example, in certain embodiments, the wound may be straight, jagged or curved.

[0088] The term “wound dressing” applies to all compositions and / or materials, including gel compositions, fabrics, textiles, or any combination thereof, which is directly placed upon a wound for protection of the wound against infection and mechanical damage and for absorbing exudate and debris from the wound.

[0089] Compositions

[0090] In one aspect, the disclosure provides a composition comprising a hydrogel matrix and a plurality of macrophages. In certain embodiments, the plurality of macrophages is at least partially encapsulated and / or immobilized in the hydrogel matrix.

[0091] In certain embodiments, the hydrogel matrix is biocompatible.

[0092] In certain embodiments, the hydrogel matrix comprises a crosslinked polypeptide. In certain embodiments, the hydrogel matrix comprises a crosslinked polysaccharide. In certain embodiments, the hydrogel matrix comprises a crosslinked synthetic polymer. In certain embodiments, the crosslinked polypeptide comprises collagen. In certain embodiments, the polypeptide comprises gelatin. In certain embodiments, the gelatin is Type A gelatin. In certain embodiments, the gelatin is Type B gelatin. In certain embodiments, the crosslinked polysaccharide comprises hyaluronic acid. In certain embodiments, the crosslinked synthetic polymer comprises a polyol. In certain embodiments, the polyol comprises polyethylene glycol.

[0093] In certain embodiments, the crosslinked polypeptide is crosslinked by covalent crosslinking. In certain embodiments, the crosslinked polypeptide is crosslinked by ionic crosslinking. In certain embodiments, the crosslinked polysaccharide is crosslinked by covalent crosslinking. In certain embodiments, the crosslinked polysaccharide is crosslinked by ionic crosslinking. In certain embodiments, the crosslinked synthetic polymer is crosslinked by covalent crosslinking. In certain embodiments, the crosslinked synthetic polymer is crosslinked by ionic crosslinking.

[0094] In certain embodiments, the covalent crosslinking comprises photo-crosslinking. In certain embodiments, the covalent crosslinking comprises enzymatic crosslinking. In certain embodiments, the covalent crosslinking comprises photo-crosslinking of a polypeptide, polysaccharide, or synthetic polymer which is at least partially functionalized with an acryloyl e.g., acryloyl or methacryloyl) moiety. In certain embodiments, the covalent crosslinking comprises photo-crosslinking of a polypeptide, polysaccharide, or synthetic polymer which is at least partially functionalized with a mal eimide moiety. In certain embodiments, the crosslinking comprises ionic crosslinking. In certain embodiments, the ionic crosslinking comprises use of Ca2+as a crosslinker.

[0095] In certain embodiments, photo-crosslinking comprises contacting the polypeptide, polysaccharide, or synthetic polymer and the acryloyl (e.g., acryloyl or methacryloyl) moiety in the presence of a photoinitiator and UV light. In certain embodiments, photo-crosslinking comprises contacting the polypeptide, polysaccharide, or synthetic polymer and the methacryloyl moiety in the presence of a photoinitiator and UV light. In certain embodiments, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

[0096] In certain embodiments, the hydrogel matrix has a concentration of the crosslinked polypeptide of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,

[0097] 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,

[0098] 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,

[0099] 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3,

[0100] 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10.0 (w / v%).

[0101] In certain embodiments, the hydrogel matrix has a concentration of the crosslinked polysaccharide of about 0.1, 0.2, 0.3, 0.4, 0.5, 0 6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,

[0102] 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0,

[0103] 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,

[0104] 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10.0 (w / v%).

[0105] In certain embodiments, the hydrogel matrix has a concentration of the crosslinked synthetic polymer of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,

[0106] 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0,

[0107] 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,

[0108] 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10.0 (w / v%). In certain embodiments, the hydrogel matrix comprises a crosslinked gelatin methacryloyl (GelMA).

[0109] In certain embodiments, the GelMA comprises methacrylate-functionalized gelatin, wherein the methacrylate and gelatin have a ratio of about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,

[0110] 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,

[0111] 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,

[0112] 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100 pmol / g gelatin.

[0113] In certain embodiments, the methacrylate and gelatin have a ratio of about 66.7 pmol / g gelatin (z.e., pmol methacrylate or methacryloyl to grams gelatin).

[0114] In certain embodiments, the plurality of macrophages comprise primary macrophages. In certain embodiments, the plurality of macrophages consist essentially of primary macrophages. In certain embodiments, the plurality of macrophages consist of primary macrophages. In certain embodiments, the primary macrophages is a bone marrow derived macrophage (BMDM).

[0115] In certain embodiments, the plurality of macrophages comprise allogenic macrophages. In certain embodiments, the plurality of macrophages consist essentially of allogenic macrophages. In certain embodiments, the plurality of macrophages consist of allogenic macrophages.

[0116] In certain embodiments, at least a portion of the plurality of macrophages have been polarized with interleukin-4 (IL-4). In certain embodiments, polarization with IL-4 comprises administration of a lipid nanoparticle (LNP) comprising IL-4-encoding mRNA or DNA.

[0117] In certain embodiments, the plurality of macrophages comprise macrophages which have not been polarized with interleukin-4 (IL-4). In certain embodiments, the plurality of macrophages consist essentially of macrophages which have not been polarized with interleukin- 4 (IL-4). In certain embodiments, the plurality of macrophages consist of macrophages which have not been polarized with interleukin-4 (IL-4).

[0118] In certain embodiments, at least a portion of the plurality of macrophages exhibit an Ml phenotype for a period of about 7 days following at least partial encapsulation in the hydrogel matrix. In certain embodiments, at least a portion of the plurality of macrophages exhibit an Ml phenotype for a period of 5, 6, 7, 8, or 9 days following at least partial encapsulation in the hydrogel matrix.

[0119] In certain embodiments, the Ml phenotype is characterized by a pro-inflammatory phenotype. In certain embodiments, the pro-inflammatory phenotype is characterized by increased expression, production, and / or secretion of CD38, CD86, PDL1, and / or CXCR4, as compared to an unactivated macrophage.

[0120] In certain embodiments, at least a portion of the plurality of macrophages exhibit an M2 phenotype after about 7 days following at least partial encapsulation in the hydrogel matrix. In certain embodiments, the M2 phenotype is characterized by a reparative phenotype. In certain embodiments, the reparative phenotype is characterized by increased expression, production, and / or secretion of cytokines Argl, CD163, CD301b, and CD206, as compared to an unactivated macrophage.

[0121] In certain embodiments, at least a portion of the plurality of macrophages exhibit an Ml phenotype for a period of about 3 days following at least partial encapsulation in the hydrogel matrix. In certain embodiments, at least a portion of the plurality of macrophages exhibit an Ml phenotype for a period of 1, 2, 3, 4, 5, 6, 7, 8, or 9 days following at least partial encapsulation in the hydrogel matrix.

[0122] In certain embodiments, the Ml phenotype is characterized by a pro-inflammatory phenotype. In certain embodiments, the pro-inflammatory phenotype is characterized by increased expression, production, and / or secretion of CD38, CD86, PDL1, and CXCR4, as compared to an unactivated macrophage. In certain embodiments, the Ml phenotype is characterized by increased secretion of pro-inflammatory cytokines like tumor necrosis factor alpha (TNFa), interleukin- 1 -beta (IL1B), interleukin-6 (IL6). In certain embodiments, the Ml phenotype is characterized by increased secretion of cell-recruiting chemokines like CCL1, CCL2, CCL5, CCL8, CXCL10, CXCL11, CXCL12, CXCL2, CXCL1, CCL17, and CCL22. In certain embodiments, the Ml phenotype is characterized by increased secretion of growth factors like vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF).

[0123] In certain embodiments, at least a portion of the plurality of macrophages exhibit a pro- inflammatory phenotype for a period of about 7 days following at least partial encapsulation in the hydrogel matrix, and subsequently exhibit a reparative phenotype after about 7 days following at least partial encapsulation in the hydrogel matrix.

[0124] In certain embodiments, at least a portion of the plurality of macrophages exhibit an M2 phenotype after about 7 days following at least partial encapsulation in the hydrogel matrix. In certain embodiments, the M2 phenotype is characterized by a reparative phenotype. In certain embodiments, the reparative phenotype is characterized by increased expression, production, and / or secretion of cytokines Argl, CD 163, CD301b, and CD206, as compared to an unactivated macrophage. In certain embodiments, the reparative phenotype is characterized by increased expression, production, and / or secretion of growth factors that typically act at later stages of wound healing, including platelet derived growth factor (PDGF), tissue inhibitor of metalloprotease 3 (TIMP3), CCL17, CCL18, and CCL22.

[0125] In certain embodiments, the plurality of macrophages shifts in phenotype over time from a population that is predominantly pro-inflammatory to a population that is predominantly reparative. In certain embodiments, this phenotype shift occurs over a period of about 7 days. In certain embodiments, the plurality of macrophages shift in phenotype within the hydrogel matrix after a period of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 days.

[0126] In certain embodiments, the plurality of macrophages are substantially retained within the hydrogel matrix after a period of about 3 to about 23 days. In certain embodiments, the plurality of macrophages are substantially retained within the hydrogel matrix after a period of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 days.

[0127] In one aspect, the disclosure provides a method for treating or ameliorating a wound of a subject in need thereof. In certain embodiments, the method comprises applying the composition of the disclosure to the wound.

[0128] In one aspect, the macrophages are unable to migrate out of the hydrogel, and the macrophages are shielded from immune cells originating from within the wound, thus allowing the safe transplantation of allogeneic macrophages. FIG. 4A (GFP staining at day 3 in vivo) shows that transplanted macrophages do not exit the hydrogel. Accordingly, in various embodiments, allogeneic macrophages are protected from immune response from the host.

[0129] In certain embodiments, the composition has a concentration of macrophages of less than about 1.0 x 106, 2.0 x 106, 3.0 x 106, 4.0 x 106, 5.0 x 106, 6.0 x 106, 7.0 x 106, 8.0 x 106, 9.0 x 106, 1.0 x 107, 2.0 x 107, or 3.0 x 107macrophages / mL. In certain embodiments, the composition has a concentration of macrophages of about 1.0 x 106, 2.0 x 106, 3.0 x 106, 4.0 x 106, 5.0 x 106, 6.0 x 106, 7.0 x 106, 8.0 x 106, 9.0 x 106, 1.0 x 107, 2.0 x 107, or 3.0 x 107macrophages / mL. In certain embodiments, the composition has a concentration of macrophages of greater than about 1.0 x 106, 2.0 x 106, 3.0 x 106, 4.0 x 106, 5.0 x 106, 6.0 x 106, 7.0 x 106, 8.0 x 106, 9.0 x 106, 1.0 x 107, 2.0 x 107, or 3.0 x 107macrophages / mL. In certain embodiments, the composition has a concentration of about 2.5 x 107macrophages / mL.

[0130] In certain embodiments, the composition further comprises at least one additional therapeutic agent. In certain embodiments, the therapeutic agent is an antimicrobial agent.

[0131] In certain embodiments, the disclosure provides a composition comprising a biocompatible hydrogel matrix and a plurality of macrophages at least partially encapsulated and / or immobilized in the hydrogel matrix, wherein the biocompatible hydrogel matrix comprises crosslinked gelatin methacryloyl (GelMA); wherein at least a portion of the plurality of macrophages exhibit a pro-inflammatory phenotype for a period of about 7 days following at least partial encapsulation in the hydrogel matrix; and wherein at least a portion of the plurality of macrophages subsequently exhibit a reparative phenotype after about 7 days following at least partial encapsulation in the hydrogel matrix.

[0132] Methods

[0133] In one aspect, the disclosure provides a method for treating or ameliorating a wound of a subject in need thereof. In certain embodiments, the method comprises applying the composition of the disclosure to the wound.

[0134] In certain embodiments, the wound is a chronic wound. In certain embodiments, the wound is a diabetic wound. In certain embodiments, the diabetic wound is diabetic foot ulcer.

[0135] In certain embodiments, the composition is applied to the wound for a period of about 7 days to about 21 days. In certain embodiments, the composition is applied to the wound for a period ofless than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 days. In certain embodiments, the composition is applied to the wound for a period of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 days. In certain embodiments, the composition is applied to the wound for a period of greater than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 days.

[0136] EXAMPLES

[0137] Various embodiments of the present application can be better understood by reference to the following Examples which are offered by way of illustration. The scope of the present application is not limited to the Examples given herein. Materials and Methods

[0138] GFP+ Bone marrow derived macrophage (BMDM) cell culture

[0139] GFP+ BMDM macrophages were used as the delivery cargo to distinguish them from the host macrophages within the site of injury at the time of analysis. To culture the GFP+ macrophages, the femur and tibia were removed from male C57BL / 6-Tg(UBCGFP) 30 Scha / J mice, 8-12 weeks of age, for bone marrow harvest / extraction. The heads of the bone shafts were clipped using surgical scissors and their contents were flushed using a 27 gauge syringe of sterile RPMI 1640 (GIBCO CAT: 11875-093). Once the marrow was harvested from all leg bones, the tube was centrifuged at 500 G’s for 5 minutes, and resuspended in complete media (RPMI 1640 + 10% Fetal Bovine Serum + 1% Penicillin Streptomycin) with 25 ng / mL of murine MCSF. Cells were cultured in non-tissue culture treated T75 flasks (Thermo Scientific, CAT: 12-566-85) and kept in a sterile incubator. Media was changed on day 3 of culture. On day 5 of culture, cells were polarized using 40 ng / mL of IL-4 (Peprotech, CAT: 214-14) for two days. On day 7, macrophages were gently detached using 6 mL of TryplE buffer (Thermo Fisher, CAT: 12604021), and a cell scraper. At this point, macrophages are fully prepared for in vitro or in vivo experiments and were immediately loaded onto a plate control, 5% w / v nanoporous GelMA with 0.5% LAP hydrogels crosslinked using 365 nm ultraviolet light, and 4 mm macroporous scaffolds.

[0140] Preparation for in vitro studies

[0141] For in vitro studies, all macrophage cultures on day 7 were loaded into their respective biomaterial carrier or control and plated onto a 6-well plate (VMW, CAT: 351146) in 5 mL of complete media with 25 ng / mL of murine MCSF. Media was collected and changed daily until the final timepoint. Experiments were setup such that BMDM’s were harvested and polarized fresh for each timepoint and biomaterial devices and groups were prepared so that all of the in vitro sacrifice timepoints would convene on the same day as further detailed and illustrated in FIGs. 6A-6C. All in vitro samples were digested in 1 mL of 3 mg / mL collagenase type II in RPMI 1640 for 20 minutes at 37 °C on an incubator shaker. Once digested, all well contents were carefully transferred into a labeled 1.5 mL, V-bottom Eppendorf tube and centrifuged at 500G for 5 min. The supernatant was aspirated and samples were reconstituted in 200 pL of FACS (IX PBS + ) split and analyzed for cell count and viability using the Trypan Blue colorimetric assay on a cell countess, and phenotype marker expression using flow cytometry.

[0142] Porous scaffold preparation

[0143] Biomaterial carriers were geometrically-matched to ensure they fill the injury identically. Porous scaffold sheets of 8cm x 12.5cm x 0.2cm (W x L x H) were punched using a 4mm biopsy and left overnight in complete media, which comprises of RPMI 1640 + 10% Fetal Bovine Serum (FBS) (VWR, CAT: 45000-734) + 1% Penicillin-Streptomycin (Thermo Fisher, CAT: 15070063) + 25 ng / mL of M-CSF (Peprotech, CAT: 315-02). After 12 hours, GFP+ macrophages were loaded onto pre-soaked porous scaffold scaffolds in 10 pL of 1 x 10 macrophages and left at 37 °C for 2 hours prior to either application in vitro or implantation in vivo. For in vitro studies, all porous scaffolds were cultured in 5 mL of complete media + 25 ng / mL MCSF.

[0144] GelMA synthesis

[0145] Gelatin Methacryloyl was synthesized from gelatin type A, from porcine skin, 300g Bloom (Sigma- Aldrich, G1890-500G). First, 6 g of gelatin was dissolved in 30 mL of 0.1 M carbonate buffer, pH= 9.0 (3.8 g ofNaHCCh and 477.1 mg of Na2COa in 500 mL of distilled water) and heated at 60 °C for 4 hrs. Next, 10% v / w of methacrylic anhydride (Sigma Aldrich, 276685-500 mL) was added to the 6 g of gelatin solution. For this study, 6 g of gelatin was dissolved in 30 mL of carbonate buffer and added 600 pL of methacrylic anhydride was added. Reactions were shaken at 37 °C for 24 hours. The next day, the newly modified GelMA was cooled to room temperature and then carefully transferred into dialysis bags (MWCO: 12-14 kDa) (Repligen Corp, Part#: S432706) and dialyzed in distilled water at 40 °C for one week. The distilled water was changed daily. After one week of dialysis, the contents of the dialysis bags were carefully transferred into an empty 50 mL conical tube, frozen, and lyophilized for one week. All GelMA batches were chemically characterized using NMR and their mechanics reported using a TA Instrument, Discovery rheometer. For all in vitro and in vivo studies, 5% w / v of the GelMA was used by resuspending in RPMI 1640 + 10% Fetal Bovine Serum + 1% Penn / Strep + 25 ng / mL of MCSF. After counting macrophages, they were split into 1.5 mL tubes such that the number of cells was 1E6. They were centrifuged at 500 G’s for 5 min, aspirated, and resuspended in 40 pL of the GelMA mixture. After plated, they were crosslinked under UV for 1 min and then cultured in 5 mL of complete media. Animal Surgeries

[0146] All in vivo studies were conducted using six male C57BL / 6 mice per group and timepoint. Each mouse was first sedated in an isoflurane chamber and then lightly fixed on a surgical pad under a nosecone. They were then administered 0.05 mg / kg of buprenorphine, and then the hip / leg were prepared using a topical Nair product. After dry wiping with a kimwipe, each exposed leg was wiped with alternating iodine and ethanol wipes to sterilize the skin. Next, a 1-1.5 cm incision was made right above the knee to fit a 4 mm biopsy punch. Once the quadricep muscle body was injured using a 4 mm biopsy punch and the muscle removed to form the void, sterile cotton tipped swabs gently soaked up any fluid and blood. The in vivo controls for the study comprised of an un-injured, or naive, group and an injured, but untreated group. Experimental groups comprised GelMA, GelMA + Macrophages, Scaffold, and Scaffold + Macrophages. For the nanoporous GelMA hydrogel, the hydrogel treatment was crosslinked in situ using a 365 UV light source fixed in the operating room. Animals were sutured using #6 sterile sutures and observed daily until sacrificed for analysis.

[0147] Tissue Preparation

[0148] After transplanting each treatment for the allotted treatment period, animals were sacrificed, and the leg was exposed to operate on. The quadriceps muscle group was carefully excised by slicing the patellar tendon using a scalpel and cutting the insertion points along the pelvic crest. Once the entire quadricep group was removed, it was minced using scissors and then digested in 3 mL of 3 mg / mL collagenase type II (Worthington, CAT: LS004176) in RPMI 1640 for 20 minutes at 37 °C.

[0149] Flow Cytometry

[0150] All samples were plated at approximately 1 million cells per well, where appropriate Fluorescence Minus One (FMOs) and Unstained controls were created by mixing cells from all samples. Staining dilutions, catalog numbers and vendors are as such: Cell samples were first stained with 200 pL Live / Dead for 30 minutes at 4 °C. Next, samples were stained with 50 pL FcR block for 10 minutes at 4 °C. Samples then received 50 pL of extracellular marker staining for 15 minutes at 4 °C. They were washed and fixed using BD fixation and Permeation buffer for 20 minutes at 4 °C. Samples were washed twice with the appropriate washing buffer and stained for intracellular markers for 30 minutes at room temperature. Finally, cells were washed and resuspended in FACS buffer at 4 °C until analysis. All samples were analyzed using a BD Fortessa. Sample analysis was conducted using FlowJo.

[0151] Histology and Immunohistochemistry (IHC)

[0152] Histopathological and IHC assays were performed by the Translational Pathology Shared Resources at Sidney Kimmel Cancer Center, Thomas Jefferson University. First, the mouse quadriceps muscles tissue samples were harvested and fixed in 10% neutral formal buffer (NFB) fixative at room temperature for 48 hours. Next, we removed approximately 0.3 cm of muscle and tissue distal from the injury / center of the muscle body on both sides. Working with approximately 0.6 cm of muscle such that the injury was the center, the muscle was cut in 4 um thick slices and placed on slides and stained for Masson’s Trichrome and H&E. Antibodies and epitope retrieval methods for PECAM-1 / CD31 (Santa Cruz, Cat: SC-1506-R, 1 / 200 dilution) was conducted using Heat Induced Epitope Retrieval (HIER) and Uni-TRIEVE (INNOVEX, CAT: NB325-500) at 70 °C for 1 hour.

[0153] To support the challenge of recovering the encapsulated, GFP+ therapeutic macrophages from the biomaterial delivery systems, a subsect of animals were allocated for tissue sectioning, staining, and imaging at day 3. For this study, the quadriceps was carefully excised, fixed, and sectioned along the sagittal plane. The samples were sectioned so that adjacent slices would be stained with H&E, anti-GFP, anti-F4 / 80, and anti-PECAM-1.

[0154] PECAM-1 / CD31 Quantification and Analysis

[0155] To quantify the PECAM-1 presence within the images, slices of interest were first identified and saved as high-resolution .tiff images. These images were then called into a MATLAB R2023b code and function which would allow the user to select a freeform region of interest (ROI). Next, it extracted the RGB channels from the entire image and creates a threshold for the detection of “red” (threshold=32). A mask is created and applied to the ROI. Finally, the code tabulates all positive “red” pixels, as well as, total pixels within the ROI and outputs the pixel counts. The merged ROI + mask + original image is saved and output. For further information on the code, please see the supplemental for the commented functions. Statistical Analysis

[0156] GraphPad Prism vlO was used for all statistics and data visualization. All data was reported as mean values + / - standard deviation. Outliers were detected and removed using ROUT method. Normality and distribution of data was verified using the Kolmogorov- Smirnov test. Figures will denote statistical test specifics. Overall, we analyzed our data utilizing one-way ANOVA, two-way ANOVA, non-parametric tests, or multiple t-tests.

[0157] Example 1: Macrophage encapsulation within GelMA hydrogels promotes a phenotype transition from pro-inflammatory to reparative in vitro

[0158] To evaluate the effects of the biomaterial carrier on macrophage retention and phenotype, murine bone marrow derived macrophages (BMDMs) were pre-polarized to a reparative phenotype with interleukin-4 (IL-4), encapsulated within GelMA hydrogels or seeded within porous gelatin scaffolds, and characterized over 7 days in culture in comparison to standard culture on non-tissue culture-treated polystyrene (FIG. 1 A). While viability was high for both carriers after 1 day, by day 7 viability within GelMA hydrogels had dropped to 60% (FIG. IB). Both biomaterial carriers lost a significant amount of cells compared to their initial loading, with the hydrogel having less than 8% of the initial cells loaded remaining by day 7 (FIG. 10A).

[0159] Flow cytometry showed that encapsulation within the hydrogels caused macrophages to take on a strong initial pro-inflammatory phenotype characterized by high expression of CD38, CD86, PDL1, and CXCR4. By day 7, however, expression of these pro-inflammatory markers decreased, and instead hydrogel-encapsulated macrophages expressed higher levels of reparative phenotype markers Argl, CD163, CD301b, and CD206, compared to porous scaffolds and well plate controls. Expression of CD9, a fibrotic marker, was lower in hydrogel-encapsulated macrophages and almost completely subsided by day 7. In contrast, porous scaffolds had more subtle effects on macrophage phenotype, causing increased expression of CD9, slight but significant increases in expression of PDL1, Argl, and CD163, and lower expression of CD301b and CD206, compared to the well plate controls and depending on the time point. These initial results show that GelMA caused a pro-inflammatory -to-reparative phenotype switch in macrophages, concomitant with greater cell death, compared to porous scaffolds and to well plate controls (FIGs. 1C-1D). Example 2: GelMA-encapsulated macrophages secrete high levels of inflammatory cytokines and cell-recruiting chemokines.

[0160] Next, the effects of the biomaterial carriers on secretion of 44 cytokines, chemokines, and growth factors that are involved in inflammation and tissue regeneration were investigated. Of the 44, 18 were not detected. Hierarchical clustering of the remaining 26 showed that macrophages encapsulated in hydrogel at day 1 secreted high levels of pro-inflammatory cytokines and chemokines compared to the control and scaffold-loaded macrophages (FIG. 2A).

[0161] Despite the high viability of macrophages recovered from the scaffold at day 7, the scaffold did not yield elevated secretions of any analyte at any timepoint compared to the control. However, the hydrogel dramatically increased the release of several analytes within the first day. In addition, despite low numbers of viable macrophages at day 7 compared to scaffolds and well plate controls, levels of IFNg, IL20, MCP-1, IL20, IFNB-1, IL16, IL17 and MIP-3b remained significantly higher for hydrogel-encapsulated macrophages.

[0162] Example 3: Retention of transplanted macrophages is dramatically improved by both porous carriers in a 4 mm murine volumetric muscle loss model

[0163] After demonstrating phenotypic influence using the hydrogel and relative inertness of the scaffold, a murine VML model was used with a 4 mm biopsy punch to transplant the macrophage treatment groups with and without their biomaterial carrier within the site of injury. To understand the environmental impact on macrophage phenotype, a parallel in vitro and in vivo experimental setup was designed (FIG. 3A). All experimental groups and timepoints were set up such that they were all sacrificed, processed, and analyzed on the same day with details outlined in FIGs. 6A-6C. The macrophages recovered from their biomaterial carriers from the VML injury correlated similarly to the in vitro findings (FIG. 3B). Treatment groups containing GFP+ macrophages were gated for cells / singlets / GFP+ / Live and were adjusted to a spiked blank to calculate a final percent live recovery.

[0164] Example 4: Porous scaffold carriers had significantly increased reparative marker expression when challenged in vivo

[0165] FIG. 3C illustrates the MFI marker expression comparing the environmental effects between n vitro to in vivo samples, as well as how the biomaterial carrier affects the phenotype marker expression. At day 1, the trends were largely the same between the scaffold and hydrogel when comparing their behavior in vitro and in vivo without significant shifts. When comparing the in vitro carrier trends to the in vivo carrier trends, CD38, PDL1, and CD301b had the same trend with little to no change. CD9, CXCR4, and CD206 marker expression trends were the most dramatic where the respective marker expression for the scaffolds were significantly higher than the hydrogel in vitro and then shifted towards no differences in vivo. Inversely, the carriers had no effect in vitro but in vivo saw an increased expression of Argl in scaffold-loaded macrophages compared to hydrogel-encapsulated. CD 163 also had no carrier effects in vitro but in vivo saw increased marker expression in hydrogel-encapsulated macrophages.

[0166] By day 7, all GelMA in vivo samples were recovered with too few encapsulated GFP macrophages to analyze, and so all in vivo conclusions were limited to the porous scaffold- loaded macrophages. At day 7, the scaffold carriers effects on CD9, CD38, CXCR4, and CD301b saw no differences between the in vitro and in vivo experiments. More interestingly, the in vivo scaffold carrier increased PDL1 proinflammatory marker expression and dramatically increased the expression of reparative Argl, CD163, and CD206.

[0167] Example 5: Macrophage-encapsulated GelMA carrier suggests increased CD31+ angiogenesis at the interface of the site of injury by day 3

[0168] FIG. 4A captures representative sections from all controls and treatment groups. The top row of representative images shows the distribution of F4 / 80+ stained macrophages within the tissue slices. The highest concentration of these macrophages can be found at the site of injury. To distinguish between host and transplanted macrophages, the second row represents the GFP+ stained, transplanted macrophages. The scaffold+mac and GelMA+mac groups had increased presence of GFP+ macrophages compared to their respective carrier controls, which helps support that transplanted macrophages were retained within the site of injury even by day 3. The transplanted macrophages within the scaffolds are dispersed primarily around the edges of the scaffold, whereas the transplanted macrophages within the GelMA+mac group were localized on the edge in direct contact with the host tissue. Additionally, there were fewer GFP macrophages within the GelMA carrier, which is supported by the low viability results.

[0169] Adjacent slices were stained with PEC AM- 1 and analyzed to quantify the amount of positively stained PECAM-1 pixels within a given region-of-interest at the interface of the biomaterial and injury. The GelMA+mac treatment yielded the highest amount of PECAM-1 staining compared to the uninjured, naive control. Although quantitative differences amongst carriers with or without transplanted macrophages were not found, the PECAM-1 images for the carrier-only had decreased PECAM-1+ cells at the interface (FIG. 4B).

[0170] Example 6: Interactions with endogenous immune cells is primarily driven by the biomaterial carrier where both carriers influenced host macrophage phenotype

[0171] Compared to the untreated group, both carriers dramatically decreased the host macrophage phenotype marker expression (FIG. 5A). The scaffolds most notably decreased CD9, CD38, and CD206 expression by day 7 where the GelMA hydrogel more dramatically decreased these same markers including PDL1. Surprisingly, the presence of transplanted macrophages had little influence on host macrophages to increase reparative markers with slight statistical differences in CXCR4 and Argl between carriers.

[0172] The trend where the presence of transplanting macrophages having little to no effect on the host immune cell continued when investigating the host immune cell demographics. Besides neutrophils and dendritic cells, differences were driven by the biomaterial carrier itself. At day 1, the scaffold recruited more neutrophils to the site of injury whereas the scaffold+macs treatment dampened the recruitment of neutrophils to normal injury levels. Inversely, dendritic cells were heavily recruited by the scaffold+macs group, but also the GelMA-only treatment. Both the scaffold and GelMA+macs group maintained similar dendritic cell levels as the untreated control.

[0173] GelMA with or without encapsulated macrophages dramatically increased the recruitment of both classical and non-classical monocytes while decreasing B-Cell recruitment by day 7 compared to the scaffold treatment groups with or without loaded macrophages. Although GelMA reduced the B-Cell recruitment by a difference of a few percentages compared to the other groups, monocyte recruitment was nearly doubled by the GelMA treatment while maintaining levels of T cells and NK cells compared to the untreated control by day 7.

[0174] Example 7: Exemplary GelMA hydrogel formulation studies

[0175] Macrophages were harvested and cultured from C57 / BL6 mice prior to encapsulation within their respective hydrogel formulation groups. Hydrogels were synthesized by modifying porcine gelatin with anhydrous methacrylic anhydride (MA) with appropriate ratios (Table 1). Once the macrophages and hydrogels were prepared, each sample was resuspended in the respective formulation of the aqueous gel with 1,000,000 IL-4 polarized macrophages. Gel+Cell mixtures were plated into a 24-well plate at 40 pL, crosslinked for 60 s using a 365 nm UV lightsource, and then cultured in 2 mL of RPMI + 10% FBS + 1% PS + 25 ng / mL murine MCSF. Culture media was changed daily. The procedure was repeated for preparing the “day 3” and “day 1” timepoint plates so that all plates could be sacrificed / digested / stained on the same day.

[0176] Table 1. Exemplary GelMA formulation ratios for synthesis

[0177] Table 2 outlines the experimental groups that were prepared and studied for this experiment. After the groups were prepared and ready to be digested, they were mechanically “pushed” off of the surface of the plate using a cell scraper and digested with 2 mL of 3 mg / mL Collagenase Type II for 20 min at 37 °C, 50 RPM. They were transferred to a 2 mL, V-bottom microcentrifuge tube and centrifuged at 500 G for 5 min. Supernatant was removed and the sample was resuspended in 200 pL of FACS buffer.

[0178] Table 2. Formulations for experimental group (n=6) All 200 pL of the sample was transferred to a 96-well, U-bottom plate and centrifuged at 500 G, 5 min and underwent staining for markers and colors as listed in Table 3.

[0179] Table 3. Markers and colors for flow cytometer stain

[0180] All statistics were performed using a one-way ANOVA and compared to the “plate only” group for significance. For simplification, CD9, CD86, CD38, and PDL1 markers were used to describe a pro-inflammatory macrophage phenotype, whereas, Argl, CD 163, CD206, and CD301b were used to describe a pro-reparative macrophage phenotype. CXCR4 marker expression was used to capture a phenotype transition from the inflammatory towards the reparative phenotype and is often associated with cell proliferation.

[0181] At day 1 (FIGs. 12A-12I), slight significance was observed between all formulations and the plate only macrophage group. However, a dramatic elevations of CD86, PDL1 , and CXCR4 was observed within the first day, which suggests that the GelMA vehicle promotes a pro- inflammatory environment. When observing the reparative markers, a slight significance was observed, but nearly no significant difference within CD301b marker expression. At day 3 (FIGs. 13A-13I), CD9 greatly decreased its marker expression within all formulation groups, where each weight percent group demonstrates a slight trend of increased CD9 expression with respect to theoretical degree-of-methacrylation. However, CD86, PDL1 , and CXCR4 still maintain high expression, but reparative markers Argl and CD301b begin to increase expression as well.

[0182] Without wishing to be bound by any theory, the data suggest that the encapsulated macrophages are beginning to take on a reparative phenotype while still presenting a population that is pro- inflammatory. At day 7 (FIGs. 14A-14I), although significant expression of pro-inflammatory markers continued to be observed, it was also observed that all formulations presented increased reparative marker expression of the encapsulated macrophages. All formulations demonstrated a decrease in viability over time, with the 5% w / v% formulations having an initial viability of less than 80% within the first day (FIG. 15).

[0183] Enumerated Embodiments

[0184] The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance: Embodiment 1. A composition comprising:

[0185] (a) a hydrogel matrix, and

[0186] (b) a plurality of macrophages, wherein the plurality of macrophages is at least partially encapsulated in the hydrogel matrix.

[0187] Embodiment 2. The composition of Embodiment 1, wherein the hydrogel matrix is biocompatible.

[0188] Embodiment 3. The composition of Embodiment 1 or 2, wherein the hydrogel matrix comprises a crosslinked polypeptide, crosslinked polysaccharide, and / or crosslinked synthetic polymer.

[0189] Embodiment 4. The composition of Embodiment 3, wherein at least one of the following applies:

[0190] (a) the crosslinked polypeptide comprises collagen {e.g., gelatin);

[0191] (b) the crosslinked polysaccharide comprises hyaluronic acid; and

[0192] (c) the crosslinked synthetic polymer comprises a polyol {e.g., polyethylene glycol). Embodiment 5. The composition of Embodiment 3 or 4, wherein the crosslinked polypeptide, crosslinked polysaccharide, or crosslinked synthetic polymer is crosslinked by a mechanism selected from the group consisting of covalent crosslinking e.g., photo-crosslinking and enzymatic crosslinking) and ionic crosslinking (e.g., Ca2+).

[0193] Embodiment 6. The composition of Embodiment 5, wherein the covalent crosslinking comprises photo-crosslinking of a polypeptide, polysaccharide, or synthetic polymer which is at least partially functionalized with an acryloyl moiety (e.g., acryloyl or methacryloyl) or maleimide moiety.

[0194] Embodiment 7. The composition of any one of Embodiments 1-6, wherein the hydrogel matrix has a concentration of the crosslinked polypeptide, polysaccharide, or synthetic polymer of about 0.1% (w / v) to about 10% (w / v).

[0195] Embodiment 8. The composition of any one of Embodiments 1-7, wherein the hydrogel matrix comprises a crosslinked gelatin methacryloyl (GelMA).

[0196] Embodiment 9. The composition of Embodiment 8, wherein the GelMA comprises methacrylate-functionalized gelatin, wherein the methacrylate and gelatin have a ratio of about 25 pmol methacrylate / g gelatin to about 100 pmol methacrylate / g gelatin, optionally wherein the methacrylate and gelatin have a ratio of about 66.7 pmol methacrylate / g gelatin.

[0197] Embodiment 10. The composition of any one of Embodiments 1-9, wherein the plurality of macrophages comprise primary macrophages.

[0198] Embodiment 11. The composition of Embodiments 10, wherein the primary macrophages comprise or consist essentially of bone marrow derived macrophages (BMDMs).

[0199] Embodiment 12. The composition of any one of Embodiments 1-11, wherein the plurality of macrophages comprise allogenic macrophages.

[0200] Embodiment 13. The composition of any one of Embodiments 1-12, wherein at least a portion of the plurality of macrophages have been polarized with interleukin-4 (IL-4).

[0201] Embodiment 14. The composition of any one of Embodiments 1-12, wherein the plurality of macrophages comprise macrophages which have not been polarized with interleukin-4 (IL-4). Embodiment 15. The composition of any one of Embodiments 1-14, wherein at least a portion of the plurality of macrophages exhibit an Ml phenotype for a period of about 4 days following at least partial encapsulation in the hydrogel matrix.

[0202] Embodiment 16. The composition of Embodiment 15, wherein the Ml phenotype is characterized by a pro-inflammatory phenotype, optionally wherein the pro-inflammatory phenotype is characterized by increased expression, production, and / or secretion of CD38, CD86, PDL1, and / or CXCR4, as compared to an unactivated macrophage. Embodiment 17. The composition of any one of Embodiments 1-16, wherein at least a portion of the plurality of macrophages exhibit an M2 phenotype after about 7 days following at least partial encapsulation in the hydrogel matrix.

[0203] Embodiment 18. The composition of Embodiment 17, wherein the M2 phenotype is characterized by a reparative phenotype, optionally wherein reparative phenotype is characterized by increased expression, production, and / or secretion of cytokines Argl, CD163, CD301b, and / or CD206, as compared to an unactivated macrophage.

[0204] Embodiment 19. The composition of any one of Embodiments 1-18, wherein the plurality of macrophages are substantially retained within the hydrogel matrix after a period of about 7 to about 21 days.

[0205] Embodiment 20. The composition of any one of Embodiments 1-19, wherein the composition has a concentration of macrophages of about 1.0 x 106macrophages / mL to about 3.0 x 107macrophages / mL, optionally wherein the composition has a concentration of about 2.5 x 107macrophages / mL.

[0206] Embodiment 21. The composition of any one of Embodiments 1-20, wherein the composition further comprises at least one additional therapeutic agent.

[0207] Embodiment 22. The composition of Embodiment 21, wherein the therapeutic agent is an antimicrobial agent.

[0208] Embodiment 23. A method for treating or ameliorating a wound of a subject in need thereof, the method comprising applying the composition of any one of Embodiments 1-22 to the wound.

[0209] Embodiment 24. The method of Embodiment 23, wherein the wound is a chronic wound.

[0210] Embodiment 25. The method of Embodiment 23 or 24, wherein the wound is a diabetic wound.

[0211] Embodiment 26. The method of Embodiment 25, wherein the diabetic wound is diabetic foot ulcer.

[0212] Embodiment 27. The method of any one of Embodiments 23-26, wherein the composition is applied to the wound for a period of about 3 days to about 23 days.

[0213] The terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the present application. Thus, it should be understood that although the present application describes specific embodiments and optional features, modification and variation of the compositions, methods, and concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of embodiments of the present application.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising:(a) a hydrogel matrix, and(b) a plurality of macrophages, wherein the plurality of macrophages is at least partially encapsulated in the hydrogel matrix.

2. The composition of claim 1, wherein the hydrogel matrix is biocompatible.

3. The composition of claim 1 or 2, wherein the hydrogel matrix comprises a crosslinked polypeptide, crosslinked polysaccharide, and / or crosslinked synthetic polymer.

4. The composition of claim 3, wherein at least one of the following applies:(a) the crosslinked polypeptide comprises collagen (e.g., gelatin);(b) the crosslinked polysaccharide comprises hyaluronic acid; and(c) the crosslinked synthetic polymer comprises a polyol (e.g., polyethylene glycol).

5. The composition of claim 3 or 4, wherein the crosslinked polypeptide, crosslinked polysaccharide, or crosslinked synthetic polymer is crosslinked by a mechanism selected from the group consisting of covalent crosslinking (e.g., photo-crosslinking and enzymatic crosslinking) and ionic crosslinking (e.g., Ca2+).

6. The composition of claim 5, wherein the covalent crosslinking comprises photocrosslinking of a polypeptide, polysaccharide, or synthetic polymer which is at least partially functionalized with an acryloyl moiety (e.g., acryloyl or methacryloyl) or mal eimide moiety.

7. The composition of any one of claims 1-6, wherein the hydrogel matrix has a concentration of the crosslinked polypeptide, polysaccharide, or synthetic polymer of about 0.1% (w / v) to about 10% (w / v).

8. The composition of any one of claims 1-7, wherein the hydrogel matrix comprises a crosslinked gelatin methacryloyl (GelMA).

9. The composition of claim 8, wherein the GelMA comprises methacrylate-functionalized gelatin, wherein the methacrylate and gelatin have a ratio of about 25 pmol methacrylate / g gelatin to about 100 pmol methacrylate / g gelatin, optionally wherein the methacrylate and gelatin have a ratio of about 66.7 pmol methacrylate / g gelatin.

10. The composition of any one of claims 1-9, wherein the plurality of macrophages comprise primary macrophages.

11. The composition of claim 10, wherein the primary macrophages comprise or consist essentially of bone marrow derived macrophages (BMDMs).

12. The composition of any one of claims 1-11, wherein the plurality of macrophages comprise allogenic macrophages.

13. The composition of any one of claims 1-12, wherein at least a portion of the plurality of macrophages have been polarized with interleukin-4 (IL-4).

14. The composition of any one of claims 1-12, wherein the plurality of macrophages comprise macrophages which have not been polarized with interleukin-4 (IL-4).

15. The composition of any one of claims 1-14, wherein at least a portion of the plurality of macrophages exhibit an Ml phenotype for a period of about 4 days following at least partial encapsulation in the hydrogel matrix.

16. The composition of claim 15, wherein the Ml phenotype is characterized by a pro- inflammatory phenotype, optionally wherein the pro-inflammatory phenotype is characterized by increased expression, production, and / or secretion of CD38, CD86, PDL1, and / or CXCR4, as compared to an unactivated macrophage.

17. The composition of any one of claims 1-16, wherein at least a portion of the plurality of macrophages exhibit an M2 phenotype after about 7 days following at least partial encapsulation in the hydrogel matrix.

18. The composition of claim 17, wherein the M2 phenotype is characterized by a reparative phenotype, optionally wherein reparative phenotype is characterized by increased expression, production, and / or secretion of cytokines Argl, CD163, CD301b, and / or CD206, as compared to an unactivated macrophage.

19. The composition of any one of claims 1-18, wherein the plurality of macrophages are substantially retained within the hydrogel matrix after a period of about 7 to about 21 days.

20. The composition of any one of claims 1-19, wherein the composition has a concentration of macrophages of about 1.0 x 106macrophages / mL to about 3.0 x 107macrophage s / mL, optionally wherein the composition has a concentration of about 2.5 x 107macrophages / mL.

21. The composition of any one of claims 1-20, wherein the composition further comprises at least one additional therapeutic agent.

22. The composition of claim 21, wherein the therapeutic agent is an antimicrobial agent.

23. A method for treating or ameliorating a wound of a subject in need thereof, the method comprising applying the composition of any one of claims 1-22 to the wound.

24. The method of claim 23, wherein the wound is a chronic wound.

25. The method of claim 23 or 24, wherein the wound is a diabetic wound.

26. The method of claim 25, wherein the diabetic wound is diabetic foot ulcer.

27. The method of any one of claims 23-26, wherein the composition is applied to the wound for a period of about 3 days to about 23 days.

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