Oxygen-releasing hydrogels

Abstract

Tissue oxygenation compositions comprising a hydrogel and an oxygen carrier compound are described. Tissue oxygenation precursor compositions and methods of using tissue oxygenation compositions for tissue oxygenation are also described.

Description

OXYGEN-RELEASING HYDROGELSCONTINUING APPLICATION DATA

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 678,110, filed August 1, 2024, the disclosure of which is incorporated by reference herein.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant Numbers R21EB025270, R21EB026175, R21EB030257, R21HL168656, R00CA201603,R01EB028143, R01HL153857, R01HL165176, R01HL166522, R01CA282451,R56EB034702 awarded by the National Institutes of Health and under Grant Numbers CBET-EBMS-1936105, CISE-IIS-2225698 awarded by the National Science Foundation. The Government has certain rights in this invention.BACKGROUND

[0003] Myocardial infarction (Ml) remains one of the leading causes of mortality worldwide, despite advances in medical and interventional treatment. A systematic review and metaanalysis from 2023 revealed that the global incidence of MI is 3.8% in individuals under 60 years old, rising to 9.5% in those over 60. Salari el al., BMC Cardiovasc Disord 23, 206 (2023). Available therapies do not adequately address all aspects of the myocardial injuryresponses, including oxidative stress, inflammatory responses, and apoptosis, which are known to aggravate damage to myocardial cells after infarction.

[0004] Low myocardial oxygen tension after MI, even following revascularization therapy (e.g., thrombolysis and percutaneous coronary intervention), has been hypothesized to be a major driver of post-MI cell death and ischemia-perfusion injury (IRI). Several oxygenreleasing materials have been developed in recent years with the goal of providing sustained oxygen-delivery to myocardial tissue. These materials include oxygen-releasing hydrogels based on perfluorocarbons (PFCs), calcium peroxide (CaO2), and magnesium peroxide (MgO2). Wang el al., J Mater Chem B 11, 7300-7320 (2023). In one example, an approach using hydroxyapatite-coated CaO2 microparticles was introduced, which extended oxygen- release from 3 to 10 days in gelatin hydrogels (Tomioka et al., Chemistry of Materials 35,5378-5391 (2023)), improving cell proliferation under hypoxic conditions. However, poly(D,L-lactic acid) and poly(lactic-co-glycolic acid) matrices with CaCh produced hydrogen peroxide, requiring catalase for cell proliferation. Hilde et al., Journal of Materials Science: Materials in Medicine 26, 1-4 (2015). Another study showed that hydrogen peroxide-containing, oxygen-releasing hydrogels could sustain oxygen-release for up to no more than 2 weeks. Hassan et al., Small 20, 2470244 (2024). Clinical application of these peroxide-based systems is limited by the fact that they all produce cytotoxic byproducts. In contrast, micro / nanobubble hydrogels offer biocompatibility and controlled oxygen-release, but can be unstable without the use of technically challenging encapsulation techniques. Recent improvements in encapsulation have extended oxygen-release (Chin et al., Biotechnol Prog 24, 358-366 (2008)), though maintaining a consistent, sustained release rate remains a challenge. Oh et al., Biomaterials 30, 757-762 (2009). While oxygen-loaded microspheres provide more controlled delivery, their efficacy is limited by short-term “burst” release of oxygen, which can increase the potential for oxidative damage. Maria M Coronel RG, Cherie L Stabler, Biomaterials 129, 139-151 (2017). These shortcomings underscore the need for non-toxic, stable materials that are technically straightforward to implement and can produce sustained oxygen-release at suitable amounts.SUMMARY

[0005] In one aspect, a tissue oxygenation composition comprising a hydrogel and an oxygen carrier compound is provided. In some embodiments, the composition further comprises hemoglobin. In further embodiments, the hemoglobin is provided within red blood cells. In additional embodiments, the oxygen carrier compound is emulsified perfluoropentane. In yet further embodiments, the hydrogel comprises gelatin methacryloyl (GelMA). In additional embodiments, the hydrogel comprises an adhesive group, such as tyramine.

[0006] In another aspect, a tissue oxygenation precursor composition comprising a hydrogel precursor and an oxygen carrier compound is provided. In some embodiments, the composition further comprises hemoglobin. In further embodiments, the hemoglobin is provided within red blood cells. In additional embodiments, the oxygen carrier compound is emulsified perfluoropentane. In yet further embodiments, the hydrogel comprises gelatin methacryloyl (GelMA). In additional embodiments, the hydrogel comprises an adhesive group, such as tyramine.

[0007] In another aspect, a method of tissue oxygenation comprising contacting a tissue with a tissue oxygenation composition comprising a hydrogel and an oxygen carrier compound is provided. In some embodiments, the tissue is cardiac tissue. In further embodiments, the tissue oxygenation composition provides sustained release of oxygen. In further embodiments, the tissue oxygenation composition further comprises hemoglobin. In yet further embodiments, the hydrogel comprises an adhesive group, such as tyramine. In some embodiments, the tissue is contacted by injection. In further embodiments, the tissue is contacted by application of the tissue oxygenation composition using a handheld 3D printer.

[0008] The inventors have provided a hydrogel-based oxygen-delivery system for tissue oxygenation and repair, such as myocardial protection after infarction. This platform combines gelatin methacryloyl-tyramine (GT) hydrogel with dodecafluoropentane (DDFP) and red blood cells (RBCs) to enable sustained oxygen-release. The formulation supports long-term oxygen-storage and controlled release, reducing oxidative stress under hypoxia. In a rat infarction model, epicardial application of the hydrogel significantly reduces scar burden and preserves cardiac functions. This work reveals a synergistic effect of DDFP and RBCs in modulating oxy gen-delivery, offering a promising strategy for treating ischemic injury.BRIEF DESCRIPTION OF THE FIGURES

[0009] The present invention may be more readily understood by reference to the following figures, wherein:

[0010] FIG. 1 provides a schematic representation of GT-synthesis and mechanism of Ru / SPS-induced photocrosslinking in GT under visible light.

[0011] FIG. 2 provides a schematic diagram showing the design of a handheld bioprinter.

[0012] FIG. 3 provides a schematic comparison of complete reperfusion and epicardial oxygen-releasing hydrogel functional reperfusion post-MI.

[0013] FIGS. 4A-4H provides graphs and images showing ex vivo oxygen-release of perfluoropentane-based particles, (a) Pre-emulsion solution of DDFP in 5% BSA solution.(b) Post-emulsion suspension of DDFP in 5% BSA solution, (c) Time-course dissolved oxygen concentrations from 2% DDFP at 4 °C. Data presented as mean ± s.d.; n = 3. (d) Time-course DDFP particle size distributions in different formulations. Data presented as mean ± s.d.; n = 3. (e) 7-day hemocompatibility assessment of DDFP / RBC mixtures. Data presented as mean ± s.d.; n = 6. Statistical analysis was performed using ANOVA, and no statistically significant differences were observed (p > 0.05). (f-h) Oxygen-release kinetics in zero-oxygen DPBS at (f) 4 °C, (g) 26 °C, and (h) 37 °C. Scale bars: 2 mm.

[0014] FIGS. 5A-5J provide graphs and images showing characterizations of GT composites, (a) Swelling behaviors of 10% GelMA versus 10% GT hydrogels. Data presented as mean ± s.d.; n = 3. Statistical analysis was performed using paired t-test, and no statistically significant differences were observed (p > 0.05). (b) Photograph of 10% GT lap-shear testing on an ex vivo porcine skin, (c) Lap-shear strength comparisons of 10% GT and 1% DDFP mixtures with varied RBC concentrations. Data presented as mean ± s.d.; n = 3. Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD post- hoc test. ** indicates p < 0.01, *** indicates p < 0.001. (d) Compressive stress comparisons of 10% GT and 1% DDFP mixtures with varied RBCs concentrations. Data presented as mean ± s.d.; n = 3. Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD post-hoc test. * indicates p < 0.05, ** indicates p < 0.01. (e) Apparent viscosity profile of 10% GT and 1% DDFP / 1% RBC mixtures at 26 °C. (f) Shear stress response of 10% GT and 1% DDFP / 1% RBC mixtures at 26°C across varied shear rates, (g) Strain-dependent moduli at 26 °C: oscillatory strain sweep of 10% GT and 1% DDFP / 1% RBC mixtures, (h) Temperature-responsive modulus of 10% GT and 1% DDFP / 1% RBC mixtures: oscillatory temperature sweep from 18 °C to 37 °C. (i) Complex viscosity versus temperature profiles of 10% GT and 1% DDFP / 1% RBC mixtures from 18 °C to 37 °C. (j) Temperature-dependence of moduli of 10% GT / 1% DDFP with varying RBC concentrations from 18 °C to 37 °C. Scale bar: 1 cm.

[0015] FIGS. 6A-6J provide graphs and images showing printability analyses of 10% GT for benchtop and handheld extrusion-bioprinting, (a-c) Optimizations of 10% GT extrusion parameters: filament diameter variation with (a) nozzle size, (b) printhead moving speed, and (c) extrusion pressure. Data presented as mean ± s.d.; n = 30. (d-f) Optimizations of 10% GT / 1% DDFP / 1% RBCs extrusion parameters: filament diameter variation with (d) nozzle size, (e) printhead moving speed, and (f) extrusion pressure. Data presented as mean+ s.d.; n = 30. (g) Planar benchtop extrusion bioprinting: precise patterning with 10% GT. (h) 3D benchtop extrusion bioprinting: complex architectures with 10% GT. (i) Handheld single-material extrusion bioprinting with 10% GT. (j) Handheld multi-material bioprinting with 10% GT. Scale bars: 2 mm.

[0016] FIGS. 7A-7T provide graphs and images showing dynamic oxy gen-release and biocompatibility assessments of bioprinted GT constructs with DDFP and RBCs. (a) Immediate surface smoothness of 10% GT / 1% DDFP post-extrusion (0 h). (b) Surface bubbling on 10% GT / 1% DDFP pattern indicating initiation of oxy gen-release (2 h). (c) Increased bubbling in 10% GT / 1% DDFP pattern showing continued oxygen-release (24 h). (d) Enhanced bubbling in 10% GT / 1% DDFP pattern denoting progressive oxygen-release (48 h). (e) Oxygen-release visualization at 37 °C in 10% GT / 1% DDFP from day 1 to day 19. (F-H) Oxygen dissolution-enhancement in zero-oxygen DPBS at (f) 4 °C, (g) 26 °C, and (h) 37 °C for GT with different DDFP / RBC blends, (i, j) PrestoBlue reduction percentages with 10% GT in (i) THP1 and (j) RAW 264.7 cell co-cultures. Data presented as mean ± s.d.; n = 6. Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD post-hoc test. # indicates a significant difference compared to the GelMA group (p < 0.05) on the respective day. (k-r) Pro-inflammatory Ml cytokine- and (s-t) antiinflammatory M2 cytokine-expressions in RAW 264.7 cells co-cultured with 10% GT and 1% DDFP / 1% RBCs. Data presented as mean ± s.d.; n = 3. Statistical analysis was performed using unpaired t-test. * indicates p < 0.05, ** indicates p < 0.01. Scale bars: 2 mm.

[0017] FIGS. 8A-8N provides graphs and images showing comprehensive analyses of neonatal rat cardiomyocytes under different conditions, (a) Live / dead staining at days 1, 7, 14, and 21. Scale bar: 200 pm. (b) Intracellular ATP levels at days 1, 7, 14, and 21. (c) cTnT levels quantified by ELISA at days 1, 7, 14, and 21. (d) Intracellular calcium levels detected by Fluo-4 staining at days 1 , 7, 14, and 21. (e) Representative TMRM-stained images showing mitochondrial membrane potentials in NG and AGRD groups at day 21; pseudocolor applied to highlight ATP levels; scale bar: 50 pm. (f) Mitochondrial membrane potential expressed as TMRM fluorescence intensity ratio (NG / AGRD) at days 1, 7, 14, and 21. (g) ROS levels detected by dihydroethidium staining; scale bar: 200 pm. (h) ROS fluorescence ratios (NG / AGRD) at days 1, 7, 14, and 21. (a-h) n = 6. (i-n) Relative geneexpression levels analyzed by qPCR at days 1, 7, 14, and 21 (n=3). Statistical analysis wasperformed using unpaired t-test. # indicates p < 0.0001 vs. NO. For (i-h), comparisons were made between experimental groups and NG. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. Data are presented as mean ± s.d. NO: Normoxic conditions, no GT coating applied. NG: Normoxic conditions, coated with 10% GT. A0: Anoxia conditions, no GT coating applied. AG: Anoxia conditions, coated with 10% GT. AGD: Anoxia conditions, coated with 10% GT and 1% DDFP. AGR: Anoxia conditions, coated with 10% GT and 1% RBCs. AGDR: Anoxia conditions, coated with 10% GT, 1% DDFP, and 1% RBCs.

[0018] FIGS. 9A-9N provide graphs and images showing assessments of cardiac functional recovery post-MI in vivo with in situ-applicated GT-based hydrogels, (a) Workflow diagram of animal experimental procedures, (b) Echocardiographic heart images pre-procedure (baseline), immediately post-MI, and at study termination, (c-g) Relative echocardiographic measurements of ventricular wall motion from baseline to study conclusion: (c) AAWT (%): percent change in anterior wall thickness, (d) A PWT (%): percent change in posterior wall thickness, (e) A LEDD (%): percent change in left ventricular end-diastolic diameter, (f) ALESD (%): percent change in left ventricular end-systolic diameter, (g) AFS(%): percent change in fractional shortening. Data presented as mean ± s.d.; Control group n=5, GT group n=7, and GT / DDFP / RBC group n=9. The percentage difference in the dimensions of the left ventricle between the pre-infarction baseline and the end-of-study was compared using one-way ANOVA. Tukey’s multiple comparisons test was used for post hoc pairwise comparisons. * indicates p < 0.05, ** indicates p < 0.01. (h-1) Assessments of left ventricular size, wall thickness, and contractility at 3 weeks post-experimental MI, featuring measurements of (h) anterior wall thickness (AWT), (i) posterior wall thickness (PWT), (j) left ventricular end-diastolic diameter (LVEDD), (k) left ventricular end-systolic diameter (LVESD), and (1) fractional shortening (FS) to track morphological and functional changes post-infarction. Data presented as mean + s.d.; Control group n=6, GT group n=7, and GT / DDFP / RBCs group n=9. Comparisons of echocardiograms at 3 weeks post-MI were performed using ANOVA, with pre-infarct baseline echocardiograms included as covariates. The Bonferroni multiple comparisons test was used for post hoc pairwise comparisons. * indicates p < 0.05. (m) Fractional shortening (FS) comparisons among control, 10% GT, and 10% GT / 1% DDFP / 1% RBCs groups across baseline, post-MI, and end of study. Data presented as mean + s.d.; control group: baseline n=6, post-MI n=7, and end of study n=6; 10% GT group: baseline n=8, post-MI n=8, and end of study n=7; 10%GT / 1% DDFP / 1% RBC groups: baseline n=9, post-MI n=9, and end of study n=9. Statistical analysis was performed using the Wilcoxon signed-rank test. * indicates p < 0.05. (n) Left ventricular end-systolic diameter (LVESD)-measurements across timepoints for control, 10% GT, and 10% GT I 1% DDFP / 1% RBCs groups. Data presented as mean ± s.d.; control group: baseline n=6, post-MI n=7, and end of study n=6; 10% GT group: baseline n=8, post-MI n=8, and end of study n=7; 10% GT / 1% DDFP / 1% RBC group: baseline n=9, post-MI n=9, and end of study n=9. Comparisons of echocardiographic measurements at different experimental stages (e.g., pre-procedural baseline, immediately after experimental myocardial infarction, and 3 weeks after myocardial infarction) were performed using the Wilcoxon signed-rank test. * indicates p < 0.05.

[0019] FIGS. 10A-10I provide graphs and images showing histomorphological assessments of cardiac tissues post-MI in vivo with in .v / m-ap licated GT-based hydrogels, (a) Representative photograph of blanched myocardial tissue at the time of experimental MI. (b) Epicardial application of oxygen-releasing hydrogel using a handheld bioprinter, (c) MI post-ligation in rat heart, (d) Uniform epicardial application of 10% GT / 1% DDFP / 1% RBC hydrogel post-infarction, (e) Restoration of myocardial coloration following therapeutic hydrogel application, (f) Representative images of Masson’s trichrome-stained, short-axis sections of the left ventricles taken from all 3 experimental cohorts, (g) Rat heart scar burden calculation by MIQuant application, (h) Comparative analyses of transmural left ventricular scar volumes in rat MI hearts across different treatments. Data presented as mean ± s.d.; control group n=7; 10% GT group: n=8; 10% GT / 1% DDFP / 1% RBC group: n=9. Left ventricular scar burden (measured with morphometric analysis) between study cohorts was compared using one-way ANOVA. Tukey’s multiple comparisons test was used for post hoc pairwise comparisons. * indicates p < 0.05, ** indicates p < 0.01. (i) Effect of different treatments on left ventricular scar midline lengths in rat MI hearts across different treatments. Data presented as mean ± s.d.; control group: n=7; 10% GT group: n=8; 10% GT / 1% DDFP / 1% RBC group: n=9. Left ventricular scar midline length (measured with morphometric analysis) between study cohorts was compared using oneway ANOVA. Tukey’s multiple comparisons test was used for post hoc pairwise comparisons. ** indicates p < 0.01, **** indicates p < 0.0001. Scale bar: 3 mm.DETAILED DESCRIPTION

[0020] The present invention provides tissue oxygenation compositions comprising a hydrogel and an oxygen carrier compound. Tissue oxygenation precursor compositions and methods of using tissue oxygenation compositions for tissue oxygenation are also provided.Definitions

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, the present specification, including definitions, will control.

[0022] The terminology as set forth herein is for description of the embodiments only and should not be construed as limiting the application as a whole. For example, as used in this specification and the appended claims, the singular forms "a", "an" and "the" can include plural referents unless the content clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicate otherwise. The word "or" means any one member of a particular list and also includes any combination of members of that list. Further, all units, prefixes, and symbols may be denoted in its SI accepted form. The conjunctive phrase “and / or” indicates that either or both of the items referred to can be present.

[0023] The phrase "consisting essentially of" means that the composition or method may include additional ingredients and / or steps, but only if the additional ingredients and / or steps do not materially alter the basic and novel characteristics of the claimed composition or method.

[0024] Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Throughout this disclosure, various aspects of this invention are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges, fractions, and individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specificallydisclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6, and decimals and fractions, for example, 1.2, 3.8, 11 / 2, and 43 / 4 This applies regardless of the breadth of the range.

[0025] As used herein, the term “about” means ±10% of the recited value.

[0026] A weight percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

[0027] As used herein, the term "organic group" is used to mean a hydrocarbon group that is classified as an aliphatic group, cyclic group, or combination of aliphatic and cyclic groups (e.g., alkaryl and aralkyl groups). In the context of the present invention, suitable organic groups for the compounds of this invention are those that do not interfere with the activity of the active compounds. In the context of the present invention, the term "aliphatic group" means a saturated or unsaturated linear or branched hydrocarbon group. This term is used to encompass alkyl, alkenyl, and alkynyl groups, for example.

[0028] As used herein, the terms "alkyl", "alkenyl", and the prefix "alk-" are inclusive of straight chain groups and branched chain groups. Unless otherwise specified, these groups contain from 1 to 20 carbon atoms, with alkenyl groups containing from 2 to 20 carbon atoms. In some embodiments, these groups have a total of at most 10 carbon atoms, at most 8 carbon atoms, at most 6 carbon atoms, or at most 4 carbon atoms. Alkyl groups including 4 or fewer carbon atoms can also be referred to as lower alkyl groups. Alkyl groups can also be referred to by the number of carbon atoms that they include (i.e., Ci - C4 alkyl groups are alky groups including 1-4 carbon atoms).

[0029] The term "aryl" as used herein includes carbocyclic aromatic rings or ring systems. Examples of aryl groups include phenyl, naphthyl, biphenyl, fluorenyl and indenyl. Aryl groups may be substituted or unsubstituted.

[0030] "Biocompatible" as used herein, refers to the capability of a material to be integrated into a biological system without harming or being rejected by the system. Examples of harm include inflammation, infection, fibrotic tissue formation, cell death, or thrombosis. Theterms "biocompatible" and "biocompatibility" when used herein are art-recognized and mean that the material is neither itself toxic to a subject, nor degrades (if it degrades) at a rate that produces byproducts at toxic concentrations, does not cause prolonged inflammation or irritation, or does not induce more than a basal immune reaction in the host.

[0031] Treat", "treating", and "treatment", etc., as used herein, refer to any action decreasing the level of ischemic injury in a subject or providing a benefit to a subject having a disease, including improvement in the condition through lessening or suppression of at least one symptom, delay in progression of the disease, etc. Treatment, as used herein, can also include increasing the oxygen level in a tissue in need thereof.

[0032] A “subject,” as used herein, can be any animal, and may also be referred to as the patient. Preferably the subject is a vertebrate animal, and more preferably the subject is a mammal, such as a research animal (e.g., a mouse or rat) or a domesticated farm animal (e.g., cow, horse, pig) or pet (e.g., dog, cat). In some embodiments, the subject is a human.Tissue Oxygenation Compositions

[0033] In one aspect, the invention provides a tissue oxygenation composition that includes a hydrogel and an oxygen carrier compound. The tissue oxygenation composition is capable of oxygenating tissue it is in proximity to or in contact with. The tissue oxygenation composition provides sustained oxygen delivery and can be used to reduce ischemic injury and promote tissue repair.

[0034] The tissue oxygenation composition primarily comprises one or more hydrogels. Hydrogels are water-rich polymers that can hold considerable amounts of water and are benign to embedded cells. Hydrogels are polymeric networks with hydrophilic chains crosslinked either covalently or physically (via intra- and intermolecular attractions). Hydrogels typically have high biocompatibility. Biocompatible hydrogels can be both natural hydrogels and synthetic hydrogels. Examples of natural hydrogels include but are not limited to acryloyl- and methacryloyl-modified hyaluronic acid, silk, collagen, gelatin, and alginate. Examples of synthetic hydrogels include but are not limited to acrylated or methacrylated polyethylene glycol and polyvinyl alcohol. In some embodiments, the hydrogel comprises gelatin methacryloyl (GelMA). In further embodiments, the hydrogel isa thermosensitive hydrogel, which changes from a gel to liquid when warmed to body temperature (about 37 °C).

[0035] Additional examples of polymers or co-polymers that are suitable for forming a hydrogel include polyacrylates, polymethacrylates, polyacrylamides, polymethacrylamides, polyvinylpyrrolidone and copolymers thereof. Other examples include polyethers, polyurethanes, and poly(ethylene glycol), functionalized by cross-linking groups or usable in combination with compatible crosslinking agents. In various embodiments, the polymer chains may be modified with reactive groups such as acrylates or methacrylates in order for the polymer chains to be photo-crosslinkable.

[0036] The composition consists primarily of water but also typically includes a significant amount of polymer by weight. In some embodiments, the hydrogel is present in a weight percent ranging from about 0.1% to about 20%, while in further embodiments, it is present in a weight percent ranging from about 5% to about 15%, while in yet further embodiments, it is present in a weight percent ranging from about 1% to about 10%, while in yet further embodiments it is present in a weight percent from about 5% to about 20%. Note that in some alternate embodiments, an oxygen carrier compound and hemoglobin source can be used even in the absence of any hydrogel.

[0037] The tissue oxygenation composition comprises an oxygen carrier compound. Oxygen carrier compounds are synthetic compounds designed to replace red blood cells in blood substitution. Examples of oxygen carrier compounds include magnesium peroxide, calcium peroxide, polymer particles encapsulating calcium peroxide, hydrogen peroxide, and perfluorocarbon-based compounds such as perfluorodecalin, perfluorooctylbromide, and dodecaperfluoropentane. Jagers et al., Pflugers Arch., 473(2): 139-150 (2021). In some embodiments, the oxygen carrier compound is perfluoropentane or emulsified perfluoropentane. The tissue oxygenation composition can include from 0.1 wt.% to 5.0 wt.% of the oxygen carrier compound, or from 0.5 wt.% to 2.0 wt.% of the oxygen carrier compound.

[0038] In some embodiments, the tissue oxygenation composition further comprises hemoglobin. While not intending to be bound by theory, it is believed that the hemoglobin acts as a buffer for the oxygen, helping to slow and sustain its release. The hemoglobin canbe included either alone or as part of a hemoglobin-based oxygen carrier. For example, in some embodiments the hemoglobin is provided within red blood cells, while in other embodiments the hemoglobin is provided as an erythrocruorin. See Kruczowska et al., J Mol Med (Berl), 101(8), 961-972 (2023).

[0039] In some embodiments, the hydrogel comprises an adhesive group. An adhesive group is a group that is attached (e.g., covalently) to the hydrogel to improve adhesion of the tissue oxygenation composition to tissue. Examples of adhesive groups include 3,4- dihydroxyphenylalanine (DOPA), transglutaminase, and tyramine. As an example, the synthesis of a GelMA hydrogel including a tyramine adhesive group is shown in Figure 1.

[0040] In some embodiments, the composition further comprises a therapeutic additive. The choice of therapeutic additive will vary depending on the particular application of the tissue oxygenation composition. Examples of therapeutic additives include a variety of therapeutic agents such as cardiovascular agents (e.g., propranolol, lisinopril, and amlodipine), antiinflammatory agents (e.g., indomethacin, naproxen, and celecoxib), reperfusion therapy drugs (e.g., alteplase, clopidogrel, and heparin), antibiotics (e.g., penicillin, cefalexin, and azithromycin), cell nutrients (e.g., proteins, peptides, amino acids, vitamins, carbohydrates, and minerals), and chemotherapeutic agents (e.g., doxorubicin, cisplatin, and methotrexate). The tissue oxygenation composition can comprise one or more additives in an amount of 0.1 wt % to about 5 wt % of the composition, based on total weight of the composition.

[0041] In some embodiments, the tissue oxygenation composition is provided as a 3D object for medical or pharmaceutical use, such as a tissue scaffold (e.g., an artificial transplant support), which may or may not include living cells. In some embodiments, the 3D object is a soft tissue construct (e.g., an artificial organ), which again may or may not include living cells. In some cases, the 3D object can be personalized for a specific subject by basing the 3D object on an image obtained from magnetic resonance imaging, computed tomography, or ultrasound. Examples of additional objects for medical or pharmaceutical use include implantable medical devices such as pacemakers and stents. A wide variety of tissue engineering applications for 3D-printed hydrogels are known to those skilled in the art. Advincula et al., MRS Commun., 11(5):539-553 (2021).

[0042] Three-dimensional (3D) printing (also known as additive manufacturing) is a method that takes information of the surface shape of a 3D object, models using computer-aided design / computer-aided manufacturing (CAD / CAM) software, and then slices that model into multiple layers, creating a stacked cross-sectional version of the 3D object. This information is then fed into a 3D printer capable of fabricating the 3D object using an additive manufacturing process for constructing 3D single or multi-layered structures disposed on a substrate.

[0043] In some embodiments, the 3D printed object is a tissue construct. In particular, the 3D printed object can be a soft tissue construct. Soft tissues connect and support other tissues and surround the organs in the body. They include muscles (e.g., the heart), fat, blood vessels, nerves, tendons, and tissues that surround the bones and joints. Examples of 3D soft tissue constructs include skin, musculoskeletal tissue, cardiac tissue, heart valve, liver, and neuronal tissue. The cells included in the tissue construct are preferably the type of cells normally found in the particular type of tissue, or precursor cells (e.g., stem cells) that will result in that particular type of tissue.

[0044] In some embodiments, the 3D object comprises one or more embedded viable cells and / or cell types. In some embodiments, the printed 3D article is a scaffold for depositing and / or growing cellular tissue. A scaffold for cellular growth can have any suitable three- dimensional shape or dimensions. As a non-limiting example, a scaffold can comprise a stack of alternating layers of strands comprising the cleavable and non-cleavable polymers. When the polymers contain cells, the cells may be substantially uniformly distributed throughout the polymer, or they may be suspended within a part of the polymer.

[0045] Standard cell culture techniques are typically used when handling the cells. In embodiments in which the 3D-printed object comprises cells, a portion of or the entire printed article can be placed under standard cell culture conditions (e.g., temperature, pressure, nutrient concentrations, etc.) in order for the cells to remain viable. In some embodiments, the 3D-printed object comprises from about 1 x 101to about 1 x 109viable cells, or from about 1 x 102to about 1 x 108viable cells, or from about 1 x 103to about 1 x 107viable cells, or from about 1 x 104to about 1 x 107viable cells, or from about 1 x 105to about 1 x 107viable cells (all being cells per milliliter).

[0046] Viable cells that can be included in a 3D-printed object include prokaryotic and eukaryotic cells. Non-limiting examples of eukaryotic cells include mammalian cells (e.g., stem cells, progenitor cells and differentiated cells). Stem cells have the ability to replicate through numerous population doublings (e.g., at least 60-80), in some cases essentially indefinitely, and also have the ability to differentiate into multiple cell types (e.g., pluripotent or multipotent). Other viable cells include immortalized cells that do not undergo normal replicative senescence, and can proliferate essentially indefinitely. Other living cells include embryonic stem cells, amniotic fluid stem cells, cartilage cells, bone cells, muscle cells, skin cells, pancreatic cells, kidney cells, nerve cells, liver cells, and the like. Viable cells are living cells.Tissue Oxygenation Precursor Compositions

[0047] In another aspect, the present invention provides a tissue oxygenation precursor composition, comprising a hydrogel precursor and an oxygen carrier compound. When used for 3D printing, hydrogel precursor compositions can be referred to as “inks” or “bioinks.”

[0048] A hydrogel precursor is a monomer or macromonomer that when cross-linked forms a hydrogel. Hydrogel precursors may comprise macromolecules including but not limited to modified polycaprolactone, gelatin, gelatin methacryloyl, alginate, alginate methacrylate, modified chitosan, chitosan methacrylate, glycol chitosan, glycol chitosan methacrylate, modified hyaluronic acid (HA), HA methacrylate, and other non-crosslinked natural or synthetic polymeric chains and the like. In various embodiments, the macromolecules may be modified to make the macromolecules crosslinkable. For example, covalent crosslinks may be formed using acrylates, methacrylates, or other types of conjugation chemistry. In various embodiments, the hydrogel precursor disclosed herein may comprise gelatin methacryloyl (GelMA) and / or alginate methacrylate (ALMA). In some embodiments, the hydrogel precursor comprises discrete gelatin methacryloyl (GelMA). In further embodiments, the tissue oxygenation precursor composition can include a crosslinking agent such as a photoinitiator.

[0049] The tissue oxygenation precursor composition can include a substantial amount of vinyl oligomers, which are stimulated form vinyl polymers upon heating by the thermalinitiator. A vinyl oligomer is polymer including a reactive vinyl (-CH=CH-) group, but with a more limited number of repeating units (e.g., 3-10) as compared with a regular polymer, or the polymer resulting from the 3D printing method described herein. The vinyl oligomers can be obtained from synthetic or natural sources. In some embodiments, the precursor composition includes both a synthetic and natural oligomer. Typically, the vinyl oligomers are acrylate oligomers, such as poly(ethylene glycol) diacrylate (PEGDA), or natural acrylates like gelatin methacryloyl (GelMA), or their mixture. These acrylates form the polymer backbone of the printed structure. Examples include PEGDA (average Mn = 700 Da) and GelMA, which enable a flexible range of mechanical properties for different applications.

[0050] The tissue oxygenation precursor can also include hemoglobin. In some embodiments, the hemoglobin is provided within red blood cells. In further embodiments, the oxygen carrier compound is emulsified perfluoropentane. In additional embodiments, the hydrogel precursor comprises an adhesive group, while in yet further embodiments the adhesive group comprises tyramine. Accordingly, in some embodiments, the hydrogel precursor is a GelMA-tyramine macromer. In yet further embodiments, the tissue oxygenation precursor composition comprises a therapeutic additive.Methods of Tissue Oxygenation

[0051] Another aspect of the present invention provides a method of tissue oxygenation. The method includes contacting a tissue with a tissue oxygenation composition. The tissue oxygenation composition includes a hydrogel and an oxygen carrier compound, and can comprise any of the tissue oxygenation or tissue oxygenation precursor compositions described herein.

[0052] “Contacting”, as used herein, refers to causing two items (e.g., the tissue and the tissue oxygenation composition) to become physically adjacent and in contact, or placing them in an environment where such contact will occur within a short timeframe. For example, contacting a tissue with the tissue oxygenation composition includes administering the composition to s subject at or near a site such that released oxygen will reach the intended site. In some embodiments, the step of contacting the site comprises surgicallyimplanting the composition, while in other embodiments the step of contacting the site comprises injection of the tissue oxygenation composition.

[0053] The tissue oxygenation composition or precursor composition used in the method can also include hemoglobin. In some embodiments, the hemoglobin is provided within red blood cells. In further embodiments, the oxygen carrier compound is emulsified perfluoropentane. In additional embodiments, the hydrogel comprises an adhesive group, while in yet further embodiments the adhesive group comprises tyramine. In yet further embodiments, the tissue oxygenation composition or precursor composition used in the method comprises a therapeutic additive.

[0054] Tissue oxygenation using a tissue oxygenation composition can be used to oxygenate a variety of different tissues and treat a variety of different diseases and conditions involving oxidative stress, such as ischemic injury, and in particular reperfusion injury. Reperfusion injury, also known as ischemia-reperfusion injury (IRI), is a complex pathological process that occurs when blood flow is restored to tissues after a period of ischemia (lack of blood supply and oxygen). Since treatment of reperfusion injury is an important application of the tissue oxygenation composition, tissue in which reperfusion injury is more likely to occur is a preferred type of tissue. Examples of tissues that are more susceptible to reperfusion injury include heart (i.e., cardiac), brain, kidney, liver, and lung tissue. However, reperfusion injury can also occur in other organs, where it is referred to as remote organ injury. Kalogeris et al., Compr Physiol., 7(1): 113-170 (2016).

[0055] In some embodiments, a method of tissue oxygenation can be used to treat or reduce the risk of ischemic injury (e.g., reperfusion injury) in a subject identified as being at increased risk of ischemic (e.g., reperfusion) injury. Risk factors for reperfusion include pre-existing conditions such as hypertension, hyperlipidemia, and diabetes, age and sex, as well as factors relating to the ischemia itself such as its duration and the severity of the blockage.

[0056] The method of tissue oxygenation can also be used to treat or reduce the risk of other diseases or disorders involving tissue oxygenation, or for conditions where tissue oxygenation provides other benefits. Examples of situations where tissue oxygenation can be beneficial include enhancing organ perfusion in organ transplantation, facilitating woundhealing or tissue regeneration, combating infection, pain management, and even improved athletic performance.

[0057] The tissue oxygenation composition typically provides sustained release of oxygen. Sustained release of oxygen is the gradual, relatively stable release of oxygen from the tissue oxygenation composition over time. In some embodiments, the tissue oxygenation composition provides relatively stable release of oxygen for at least a week, while in other embodiments the tissue oxygenation composition provides relatively stable release of oxygen for at least two weeks, at least three weeks, or at least a month.

[0058] The tissue oxygenation composition, or the tissue oxygenation precursor composition, can be contacted with the tissue in a variety of different ways. When a tissue oxygenation precursor composition is used, the hydrogel can be crosslinked in situ after being administered. In some embodiments, the tissue is contacted by injection of the tissue oxygenation composition (or precursor) at the site where tissue oxygenation is required. For example, for ischemia reperfusion prevention, the composition can be injected into or adjacent to cardiovascular tissue.

[0059] In some embodiments, the tissue oxygenation composition is formed into a 3D object intended for medical or pharmaceutical use, such as a tissue scaffold, which is then contacted with tissue. In further embodiments, the 3D object is prepared using 3D printing. Examples of 3D objects for medical or pharmaceutical use are described herein.

[0060] In other embodiments, the tissue is contacted by application of the tissue oxygenation composition using a handheld 3D printer. An example of a handheld bioprinter is shown in Figure 2, which shows the assembled device, the sections of the device (i.e., the electronic base, the motor base, the main base, and the front and rear syringe covers) as well as the interior components, such as the motor, potentiometer, and syringe. A handheld printer can be used to apply in the tissue oxygenation composition or tissue oxygenation precursor composition in essentially whatever shape and structure are desired at the site of contact.

[0061] The present invention is illustrated by the following example. It is to be understood that the particular example, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.EXAMPLEEpicardial Delivery of An Oxygen-Releasing Hydrogel Promotes Functional Recovery of the Heart after Myocardial Infarction

[0062] The oxygen-delivery system described in this study was designed to address the shortcomings in previously reported systems. Specifically, we developed a novel composite material consisting of gelatin methacryloyl functionalized with tyramine groups (GelMA- tyramine, or GT), perfluoropentane (DDFP) nanoemulsions, and red blood cells (RBCs). GT provides a biocompatible scaffold that adheres effectively to the tissue (Lim et al., Adv Healthc Mater 9, el901792 (2020)), enabling effective coverage of the ischemic myocardium. DDFP, as a highly efficient oxygen-carrier, offers exceptional oxygen solubility. Spiess BD, Shock 52, 7-12 (2019). Combined with GT, DDFP facilitates localized oxygen-delivery to the tissue. Addition of RBCs to the GT / DDFP mixture prolongs the duration of oxygen-release. This GT-based matrix provides sustained oxygen- release to myocardial tissue at suitable levels without the acute “burst” oxygen-delivery and associated oxidative stress observed with previously described systems. Smooth, sustained delivery of oxygen at reduced concentrations by the GT-based matrix has the potential to mitigate oxidative stress and to improve the safety of delivering oxygen to infarcted myocardium. Nguyen et al., Curr Opin Biotechnol 34, 225-231 (2015). As illustrated in Figure 3, this GT-based composite material can provide prolonged oxygen-delivery to infarcted heart tissue without evidence of toxicity, thus promoting the healing of myocardium following experimental MI.Results

[0063] DDFP-Preparation, Characterizations, and Measurements of Oxygen-Release

[0064] Pure DDFP is a hydrophobic substance that is not soluble in aqueous solution (Figure 4a). After low-flow oxygenation and 5 min of ultrasonication, a homogeneous DDFP emulsion was formed, characterized by its milky appearance (Figure 4b). The 2% emulsified DDFP 14 solution was stored at 4 °C and the dissolved oxygen concentration was monitored over 12 months. The initial concentration decreased substantially on day 1 due to the release of supersaturated oxygen, and then remained stable for the following 8 months, with further decreases observed in the 10thand 12thmonths. This result indicatedthat the 2% DDFP emulsion could stably store oxygen at 4 °C for up to 8 months, with gradual oxygen-release and maintenance of local oxygen levels above the typical level of dissolved oxygen in water (7-8 mg ml , Figure 4c). The particle size of the 2% DDFP emulsion stayed relatively stable during the first 8 months of storage at 4 °C. After the 8th month, the particle size increased notably (Figure 4d), suggesting a decrease in emulsion stability, consistent with observations on dissolved oxygen concentration. The 2% DDFP emulsion was mixed with RBCs to achieve final concentrations of 1% RBCs and 1% DDFP. The supernatant was collected after centrifugation, and the absorbance was measured at 540 nm. No statistically significant differences were observed between the absorbance values across different time points (0, 1, 3, 5, and 7 days; Figure 4e), indicating that the 2% DDFP and RBCs mixture did not induce hemolysis.

[0065] The oxy gen-release kinetics of 1% DDFP, 1% RBCs, or their mixture (1% DDFP + 1% RBCs) were assessed in deoxygenated Dulbecco’s phosphate-buffered saline (DPBS) at 4 °C (refrigeration temperature, Figure 4f), 26 °C (room temperature, Figure 4g), and 37 °C (body temperature, Figure 4h). The results revealed that the lower the temperature, the slower and lower the initial oxygen-release from DDFP. At body temperature, the restored activity of RBCs extended the total amount and duration of oxygen-release. At all temperatures, the mixture exhibited significantly longer oxygen-release duration compared to DDFP or RBCs used alone.

[0066] Synthesis and Properties of GT Hydrogels

[0067] The synthesis of GT involves reacting gelatin with methacrylic anhydride to produce GelMA (Yue et al., Biomaterials 73, 254-271 (2015)), followed by introducing tyramine groups using l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) / N- hydroxy succinimide (NHS) chemistry, forming GT. Lim et al., Adv Healthc Mater 9, el901792 (2020). This reaction occurs under mild conditions, making the resulting product suitable for biomedical applications. The GT macromolecules could be crosslinked using the tris-bipyridyl-ruthenium (II) hexahydrate (Ru) / sodium persulfate (SPS) photoinitiation system under visible light to produce a stable hydrogel network (Figure 1). The optimized concentrations of the photoinitiators were 0.5 mM of Ru and 5 mM of SPS, ensuring structural integrity and mechanical strength of the resulting hydrogel. The dual functionality of GT, determined by oligomeric methacryloyl and tyramine crosslinking, shows notableadvantages in stability and controlled release compared to conventional GelMA. As shown in Figure 5a, both GelMA and GT hydrogels exhibited similar swelling ratios. The average swelling ratio for GelMA was 104.1 ± 1.6%, while GT had a slightly higher average of 114.5 ± 7.1%. Statistical analyses revealed no significant differences between the two groups, indicating that the introduction of tyramine crosslinking did not notably affect the hydrogel’s water-absorption capacity.

[0068] Figure 5b displays a photographic record of the setup used for lap-shear tests on adhesion of 10% GT with an ex vivo model that utilizes pig skin. Lim el al., Adv Healthc Mater 9, el901792 (2020). In this study, the changes in shear strengths of GelMA and GT were evaluated after the addition of DDFP and varying concentrations of RBCs. The shear strength of GelMA hydrogel was 50.6 ± 6.3 kPa, which decreased to 40.3 ± 5.3 kPa after adding DDFP (p < 0.01), and further decreased to 28.9 ± 0.9 kPa with the addition of 1% RBCs, and to 12.8 + 2.3 kPa with 2.5% RBCs (p < 0.001). Figure 5c shows that the initial shear strength of GT was 270.9 ± 11.3 kPa, which dropped to 223.2 ± 5.1 kPa after adding DDFP (p < 0.01), and further decreased to 110.6 ± 11.2 kPa with 1% RBCs (p < 0.001). These results indicated that increasing RBC concentration reduced the adhesion properties of the hydrogel, particularly in terms of lap-shear strength, reflecting an inverse relationship between RBCs content and adhesion stability. Although higher RBC concentrations may limit use in environments with higher adhesive demands, GT with 1% DDFP and 1% RBCs maintained acceptable lap-shear strength for the applications described in this study.

[0069] Changes in compressive strengths of GelMA and GT hydrogels after the addition of DDFP and varying concentrations of RBCs were evaluated. The compressive strength of GelMA hydrogel was 87.1 ± 5.3 kPa, which slightly increased after the addition of 1% DDFP (89.3 ± 4.3 kPa), but significantly decreased to 60.7 ± 2.5 kPa with the addition of 1% RBCs, and further decreased to 33.6 ± 3.4 kPa with 2.5% RBCs (p < 0.01). The GT hydrogels exhibited a similar trend (Figure 5d), with an initial compressive strength of 101.2 ± 5.8 kPa, which decreased to 87.1 ± 5.7 kPa after the addition of 1% DDFP, and further dropped to 66.5 ± 1.9 kPa with 1% RBCs, and to 42.5 ± 2.5 kPa with 2.5% RBCs (p < 0.01). These results suggested that the inclusion of RBCs reduced the compressive performances of GelMA and GT hydrogels, especially at higher concentrations, leading to a reduction in mechanical properties. While higher concentrations of RBCs could limit its use in applications requiring high mechanical strengths, GT hydrogel containing 1% DDFP and1% RBCs maintained acceptable compressive strength for the applications intended in this study.

[0070] The rheological behaviors of GT and GT composites (including DDFP and RBCs) were systematically evaluated at 26 °C and 37 °C. As shown in Figure 5e, the addition of 1% DDFP to GT reduced the zero-shear viscosity from 2 Pa s to 0.6 Pa s, while the addition of 1% RBCs increased the zero-shear viscosity to 11 Pa s. The combination of 1% DDFP and 1% RBCs substantially increased viscosity across the entire shear-rate range, forming a shear-thinning bioink suitable for use at room temperature. At an extrusion shear rate of 50 s ', the shear stress remained below 10 Pa (Figure 5f), suggesting that the material is potentially suitable for bioprinting with RBCs. In addition, the presence of 1% DDFP consistently enhanced the viscosity, with RBCs serving as effective rheology-modifiers, especially at concentrations of 1-2%, where the zero-shear viscosity increased significantly from 0.6 Pa-s to 50 Pa- s. However, higher concentrations of RBCs (5%) disrupted the physical network of the GT hydrogel, leading to a decrease in viscosity and reduced printability.

[0071] Further, temperature-dependent studies (Figure 5h and 5i) revealed that the apparent viscosity of GT composites decreased significantly at 37 °C, attributed to the melting of GT, forming a low-viscosity Newtonian fluid (6 mPa-s). GT undergoes a conformational transition from triple helices to coils between 26 °C and 32 °C, with low viscosity values observed above 37 °C. The GT / 1% DDFP / 1% RBCs bioink exhibited the gel-like behavior at room temperature with a yield strain of approximately 1000%, making it suitable for extrusion bioprinting (Figure 5j). In contrast, the original 10% GT bioink and the 10% GT / 1% DDFP mixture behaved as sols, demonstrating poor printability. The presence of RBCs effectively adjusted the rheology, enhancing viscosity and shear-thinning behavior, although concentrations exceeding 2% disrupted the physical network of the GT hydrogel, reducing viscosity and printability. These findings were further validated by additional rheological comparisons at 26 °C and 37 °C. The addition of RBCs increased the apparent viscosity and shear stress at both temperatures, with the most pronounced effects observed at 1-2% of RBC concentrations; however, the disruption of the physical network of the GT hydrogel at 5% RBCs led to decreased viscosity and printability. Data in the flow tests at higher temperatures suggested that the effects of DDFP and RBCs on viscosity vary,necessitating further studies to optimize the rheological properties of GT / DDFP / RBC bioinks for use in high-temperature conditions.

[0072] Printability of GT / DDFP / RBC Composite Bioinks

[0073] The effects of varying concentrations of DDFP and RBCs on the printability and structural integrity of 10% GT were evaluated. Constructs containing 1% and 2% RBCs exhibited good structural fidelity, whereas those with 5% RBCs showed notable deformation and structural distortion. The addition of 1% DDFP alone had negligible adverse impact on printability, but the combination of 1% DDFP and 1% RBCs substantially improved detail-retention and structural stability. In addition, the 10% GT concentration was identified as the optimal printable formulation, as it demonstrated better structural integrity during the bioprinting process than GT at 5% and 15%.

[0074] The printability of 10% GT in combination with DDFP and RBCs was evaluated in detail, with the goal of optimizing both benchtop and handheld extrusion bioprinting applications. Relative impacts of multiple components of the bioprinting system (filament diameter, nozzle size, printhead moving speed, and extrusion pressure) on printing fidelity and extrusion performance were evaluated. The printability of 10% GT / 1% DDFP / 1% RBCs (Figure 6d-f) was similar to that of 10% GT alone (Figure 6a-c). The optimal extrusion conditions were determined to be a 0.58-mm nozzle, a printhead moving speed of 180 mm s’1, and an extrusion pressure of 241 .3 kPa. Under these conditions, the bioprinter was able to create high-fidelity planar patterns and complex 3D structures (Figure 6g and h), including hexagonal lattices and stacked constructs, with good structural fidelity and extrusion performance.

[0075] Initial in vitro printing experiments were conducted using a benchtop extrusion bioprinter. Insights gained from the use of the benchtop bioprinter were applied to the development of a handheld bioprinter to maximize translational potential. The handheld bioprinter was capable of precise plotting in the creation of sophisticated anatomical models and multi-material hierarchical structures, such as a DNA helix pattern (Figure 6i and j). These data demonstrated that the 10% GT / 1% DDFP / 1% RBCs combination was not only suitable for conventional benchtop-based extrusion bioprinting but also exhibited favorableversatility and adaptability for handheld bioprinting, making it well-suited for a wide range of biomedical applications.

[0076] Sustained Oxygen-Release and Biocompatibility of GT / DDFP / RBC Constructs

[0077] The sustained oxygen-release from the 10% GT constructs containing 1% DDFP was systematically analyzed to assess its effectiveness. After extrusion, the construct surface appeared smooth (Figure 7a). Within 2 h, bubbles started to form on the surface, indicating the initiation of oxygen-release (Figure 7b). The number of bubbles increased over the next 24 h (Figure 7c) and further increased by 48 h (Figure 7d), demonstrating gradual oxygen- release from the construct. Long-term oxygen-release at 37 °C showed extensive bubblegeneration and release over 19 days (Figure 7e). Additionally, detailed visualizations of oxygen-release dynamics at 4 °C and 26 “Cover the same 19-day period provided further insights. At 4 °C, the 10% GT construct containing 1% DDFP exhibited gradual and sustained oxygen-release from day 1 to day 19, with bubbles persisting throughout. Similarly, at 26 °C, the construct exhibited bubble-formation over time, indicating active oxygen-release. Images in Figures 7, demonstrate the presence of bubbles and the changes in the structural integrity of the constructs over time.

[0078] Compared to constructs using 1% DDFP or 1% RBCs alone, those containing both RBCs and DDFP consistently maintained higher levels of dissolved oxygen across all temperatures (Figure 7f-h). At 4 °C, the initial dissolved oxygen concentration for 1 % DDFP was 2.7 mg mL-1, which gradually decreased over 17 days. In contrast, the 1% DDFP and 1% RBCs mixture started at 2.2 mg mL'1and maintained oxygen levels above 1 mg mL1until day 21 (Figure 7f). At 26 °C, the initial concentration for 1% DDFP was 2.8 mg mL1, while the mixture started at 2.7 mg mL1, with the mixture maintaining higher oxygen levels over 20 days (Figure 7g). At 37 °C, the initial concentration for 1% DDFP was 2.9 mg mL-1, and the mixture began at 2.8 mg mL1, maintaining higher oxygen levels over 20 days (Figure 7h). These results indicate that physiological body temperature (37 °C) more effectively maintains higher oxygen levels over a longer period compared to refrigeration (4 °C) and room temperature (26 °C), These data also demonstrate the positive role of RBCs in extending oxy gen-deli very.

[0079] In GT constructs, the combination of 1% DDFP and 1% RBC capitalized on the oxygen-carrying capacity of RBCs and the oxygen-releasing properties of DDFP. The combination of DDFP and RBCs provided a more sustained oxygen-release profile than either component alone. RBCs contributed to the gradual release and retention of oxygen, effectively extending oxygen-delivery. The initial dissolved oxygen concentration for the 1% DDFP and 1% RBCs mixture was lower than that of the 1% DDFP alone. This should be attributed to the presence of RBCs, which naturally absorb oxygen and reduce the initial burst of oxygen-release. This synergistic effect between DDFP and RBCs is crucial for maintaining higher levels of dissolved oxygen, particularly at physiological temperatures, making the constructs more effective for potential biomedical applications that require prolonged oxygen-delivery.

[0080] To evaluate the biocompatibility and potential inflammatory responses of GT composite materials with DDFP and RBCs, co-culture experiments were conducted using THP-1 and RAW264.7 immune cells. Cell viability was measured using the PrestoBlue assay. These experiments demonstrated that the viability values of THP-1 and RAW264.7 cells in the GT / DDFP / RBC group were significantly higher than those in the GT-only group (Figure 5i and j). This observation suggested that the addition of DDFP and RBCs not only maintained but enhanced cell metabolic activities and survival. Further analyses of pro- inflammatory and anti-inflammatory cytokine expression levels were conducted using enzyme-linked immunosorbent assay (ELISA). Although the level of interleukin (IL)-ip was significantly elevated in the GT / DDFP / RBC group (p < 0.01), indicating the presence of an inflammatory response, the expression levels of other key pro-inflammatory cytokines (IL- la, tumor necrosis factor (TNF)-a, and IL-6) did not show significant changes (Figure 7k-r). Anti-inflammatory factors IL-4 and IL-10 revealed no significant differences between groups, with no statistically significant changes observed (Figure 7s-t). In conclusion, the GT constructs containing DDFP and RBCs were cytocompatible and maintained high viability of THP-1 and RAW264.7 cells while inducing only mild inflammatory responses. The results of these experiments suggested that the GT / DDFP / RBC composite could be suitable for in vivo applications requiring long-term oxygen-delivery, such as myocardial repair.

[0081] Protective Effects of GT Composite on Multiple Cell Types Under Anoxic andHypoxic Conditions and Cell Survival-Assessments with Different Hypoxia- Preconditioning Times

[0082] Using primary cardiomyocytes extracted from neonatal rat hearts within 3 days of birth, we evaluated the protective effects of the 10% GT / 1% DDFP / 1% RBC composite material under extremely low oxygen conditions (0.1%), simulating the anoxic environment of the MI zone. In cardiomyocytes, Live / Dead staining showed significant cell death in the uncoated anoxic group on day 1, while the GT / DDFP group and the GT / DDFP / RBC group maintained significantly higher survival rates, with the GT / DDFP / RBC group achieving the highest rate by day 21, comparable to normoxic controls without coating and normoxic controls with GT coating (Figure 8a). Metabolic activity measured by optical density (OD) and reduced lactate dehydrogenase (LDH) release confirmed the GT / DDFP / RBC group’s ability to maintain cell viability and membrane integrity under hypoxic conditions. The GT / DDFP / RBC group also preserved intracellular adenosine triphosphate (ATP) levels, which were severely depleted in the uncoated anoxic group but maintained close to normoxic levels in the GT / DDFP / RBC group by day 21 (Figure 8b).

[0083] The GT / DDFP / RBC group demonstrated further protective effects by maintaining functional markers such as cardiac troponin T (cTnT), as detected by ELISA, which was significantly preserved compared to the normoxic GT group (Figure 8c). The GT / DDFP / RBC group also maintained intracellular calcium homeostasis, mitigating calcium-overload observed in the uncoated anoxic group, and preserved mitochondrial membrane potential, which was severely disrupted in the uncoated anoxic group but restored in the GT / DDFP / RBC group to levels exceeding the normoxic GT-coated group by day 21 (Figure 8d-f). Oxidative stress-analyses revealed significantly reduced ROS levels in the GT / DDFP / RBC group compared to the uncoated anoxic group and the normoxic GT- coated group, particularly by day 21 , as demonstrated by ROS fluorescence and fluorescence ratio (Figures 8g and h).

[0084] Of note, qPCR results validated these findings, The GT / DDFP / RBC group demonstrated significantly preserved expression of cTnT and brain natriuretic peptide (BNP) compared to the uncoated anoxic group, with levels approaching or surpassing those of the normoxic GT group by day 21. Additionally, myosin heavy chain 7 (MYH7)-expression indicated enhanced cardiac remodeling, with the GT / DDFP / RBC group showing a notable increase compared to all the other groups. These observations highlight the protective role of the composite material in maintaining key markers of cardiac functions and remodeling, with the GT / DDFP / RBC group significantly upregulating stress-related genes such as hypoxia-inducible factor 1-alpha (HIFla), vascular endothelial growth factor A (VEGF-A), superoxide dismutase 2 (SOD2), and heme oxygenase 1 (Hmoxl) (Figure 6i- n). Additionally, the GT / DDFP / RBC group suppressed pro-apoptotic genes Bcl-2- associated X protein (BAX) and caspase-3 (Casp3) while upregulating anti-apoptotic B-cell lymphoma 2 (BCL-2), effectively reducing cell apoptosis.

[0085] Similarly, in skeletal myoblasts, fibroblasts, and vascular endothelial cells under hypoxic culture, Live / Dead staining and cell counting revealed similar trends, with significantly higher survival rates in the GT / DDFP / RBC group compared to the uncoated group. Validation experiments with C2C12 skeletal muscle cells were conducted due to challenges in quantifying cardiomyocytes caused by fusion during culture. C2C12 cells showed consistent responses as for cardiomyocytes. The protective effects of the GT composite on preconditioned C2C12 cells were subsequently assessed under hypoxic conditions (1% oxygen), simulating the peri-necrotic region surrounding ischemia, with varying preconditioning durations (0, 6, 18, 36 h). Survival rates for cells in the normoxic culture group and the GT / DDFP / RBC group were comparable for hypoxic preconditioning times of 0 and 6 h. Extension of the hypoxic preconditioning time to 18 and 36 h was associated with significant decrease in cell-survival in the normoxic culture group, while the GT / DDFP / RBC group was still able to maintain higher survival rates of the cells at all time points evaluated. This observation is consistent with the hypothesis that immediate normoxic treatment might trigger cell damage due to an unmitigated burst release of oxygen, whereas the presence of RBCs in the GT / DDFP / RBC group reduced the burst release of oxygen during the early treatment phase and prevented damage caused by excessive oxygen-release. Also in comparison, without RBCs (i.e., the GT / DDFP group), the cells showed inferior viability at later time points (14 and 21 days) under these conditions, attributed to the reduced oxygen-release duration than the GT / DDFP / RBC formulation. As a whole, these results demonstrate that the GT / DDFP / RBC coating (10% GT / 1% DDFP / 1% RBC) effectively enhanced survival, energy-metabolism, and functions across various cell types under anoxic and hypoxic conditions by maintaining oxygendelivery, preserving mitochondria] function, and reducing oxidative stress.

[0086] Echocardiographic Assessments of the Protective Effects of GT / DDFP / RBC Combination Therapy on Ventricular Scar Burden and Contractile Functions after Experimental MI

[0087] Experimental Mis were performed in rats (the procedural summary is displayed in Figure 9a). There were three study cohorts: one control group and two treatment groups. Rats in the control group underwent experimental MI with no subsequent treatment. Rats in one treatment group underwent epicardial application of bioprinted GT immediately after MI, whereas rats in the other treatment group underwent epicardial application of GT / DDFP / RBC composite immediately after MI.

[0088] Echocardiography was performed at three time points: immediately before experimental MI, immediately after experimental MI, and 3 weeks post-MI. Figure 9b displays typical M-mode echocardiographic images, showing normal anterior and posterior wall motion at baseline and loss of anterior wall motion with compensatory posterior wall hypercontractility noted immediately post-MI. Echocardiograms performed for all rats that survived three weeks after MI revealed differences between treated and untreated groups. Compared to the control and GT groups, the GT / DDFP / RBC group exhibited better preservation of anterior wall thickness (AWT) and posterior wall thickness (PWT) following MI (Figures 9c, 9d). The percentage changes in left ventricular end-diastolic diameter (LVEDD) and end-systolic diameter (LVESD) revealed that the extent of ventricular dilation was significantly smaller in the GT / DDFP / RBC treatment group compared to the control group and the GT treatment group (Figures 9e and f). Moreover, the percentage change in fractional shortening was significantly improved (Figure 9g and 1), demonstrating that the GT / DDFP / RBC treatment effectively preserved systolic function.

[0089] Further analyses of ventricular wall thickness and chamber size illustrated that the AWT / PWT in the GT / DDFP / RBC-treatment group at 3 weeks post-MI (Figures 7h and i) were significantly higher than those in the control group. Moreover, the LVEDD and LVESD (Figures 9j and k) were significantly smaller in the treatment group than those in the control group, further validating the structural protective effects of the therapy. Figure 9m demonstrates a significant improvement in fractional shortening in the GT / DDFP / RBC- treatment group. Post-MI increase in LVESD was markedly smaller in the GT / DDFP / RBC treatment groups, indicating that treatment reduced ventricular dilation after MI (Figure 9n).Collectively, these observations suggested that the GT / DDFP / RBC composite, but not GT alone, mitigates reduction in post-MI contractile dysfunction and chamber dilatation.

[0090] Histopathological Assessment of Cardiac Tissues Post-MI with GT / DDFP / RBC Combination Therapy

[0091] In vivo application of the GT-based hydrogels post-MI is depicted in Figure 10a- e, showing infarction-induction (Figure 10a), handheld bioprinting (Figure 10b), and the subsequent reoxygenation effect (Figure 8c-e). Histological analyses using Masson’s trichrome staining (Figure lOf) revealed substantial reduction in scar-formation in the GT / DDFP / RBC group compared to the control and GT groups. Quantitative analyses confirmed that the GT / DDFP / RBC group had significantly lower LV scar volume and midline length (Figure lOh and i, p < 0.01 and p < 0.0001, respectively) compared to the control and GT treatment groups, indicating enhanced myocardial protection and reduced adverse remodeling post-MI. These results demonstrate the superior efficacy of the oxygenreleasing GT / DDFP / RBC hydrogel in minimizing scar formation and promoting myocardial repair.Discussion

[0092] DDFP possesses several properties that make it an ideal oxygen-carrier. Its low boiling point (29.2 °C) allows DDFP to transition into a gaseous state at body temperature, while emulsification enables it to remain in the nanodispersed liquid form, ensuring controlled and sustained oxygen-release. Lundgren et al., Undersea Hyperb Med 31, 105- 106 (2004). As the temperature decreases, the oxygen solubility of DDFP increases. With a high oxygen solubility (80 mL of oxygen per 100 mL at 25 °C) and an oxygen-carrying capacity 100 times greater than other fluorocarbons (Johnson et al., Artificial Cells, Blood Substitutes, and Biotechnology 37, 156-162 (2009)), DDFP is highly efficient in oxygendelivery, releasing dissolved oxygen linearly according to the partial pressure of oxygen, thus preventing excessive release. Lambert et al., Nanomedicine (Lond) 14, 2697-2712 (2019). DDFP is excreted through the lungs without undergoing metabolism or accumulation, reducing the risks associated with long-term use. It also exhibits strong storage stability, with studies showing that DDFP can be stored at 4 °C for up to 8 months while maintaining a high dissolved oxygen concentration. DDFP emulsions have shownsignificant cardiac protection in myocardial infarction models, reducing infarct size and cardiac troponin I levels through an ATP-sensitive K+ channel-dependent mechanism. Strom et al., Cardiovasc Drugs Ther 28, 541-547 (2014). These characteristics make DDFP a reliable oxygen-carrier for myocardial protection and repair, offering effective oxygendelivery while minimizing potential toxicity risks, highlighting its great potential for medical applications.

[0093] RBCs, as natural oxygen-carriers, are essential for tissue oxygenation through two main mechanisms. First, they transport oxygen via hemoglobin, with its oxygen-affinity adjusted to optimize delivery. Jensen et al., J Exp Biol 212, 3387-3393 (2009). Second, RBCs act as oxygen- sensors, release vasodilators such as ATP and nitric oxide (NO) in response to hypoxia, thus enhancing local blood flow and matching oxygen-delivery with tissue demand. In this study, RBCs were incorporated into the GT hydrogel, leveraging their inherent oxygen-carrying capacity to enhance oxygen-delivery to ischemic myocardial tissue. The results of this study revealed that the combination of DDFP and RBCs exhibited a previously unappreciated synergistic effect. Use of RBCs, which can bind as well as release oxygen, served to prevent a “burst” of oxygen-release by DDFP. This served to both reduce the unwanted effects of excess oxygen-delivery (oxidative stress) and served to extend the duration of oxygen-delivery. Ellsworth et al., Med Sci Sports Exerc 36, 35-41 (2004). Additionally, RBCs have the potential to mitigate inflammatory responses by releasing NO, facilitating efferocytosis, and modulation of tissue inflammation through the scavenging of chemokines and nucleic acids. Anderson et al., J Immunol 201, 1343-1351 (2018). This study revealed that the 1% DDFP and 1% RBCs mixture maintained higher levels of dissolved oxygen across different temperatures, and RBCs effectively prolonged the oxygen-release duration, offering sustained and controlled oxygen-delivery.

[0094] In previous studies, various oxygen-release systems have exhibited limited ability to provide prolonged oxygen-delivery. Peroxide-based systems release oxygen rapidly but lack the ability to provide either prolonged or controlled release. Adding a hydroxyapatite coating to calcium peroxide microparticles can extend oxygen-release for up to 10 days, but without an inherent mechanism for modulation of oxygen-release. Tomioka et al., Chemistry of Materials 35, 5378-5391 (2023). Oxygen-carrying materials such as oxygen- encapsulated polymers or nanoparticles exhibit somewhat longer release times, ranging from several hours to a few days, but are limited by oxygen storage capacity and releasecontrol. A recent study introduced a self-oxygenating, tissue-adhesive, silk-based hydrogel that simultaneously released oxygen and stromal cell-derived factor- la for 14 days 7. The DDFP / RBC combination described in this study demonstrated a unique ability to sustain oxy gen-release for up to 21 days, significantly surpassing the capabilities of many of the previous systems without exerting any noticeable adverse side effects. This extended- release profile, along with controlled oxygen-delivery, make the DDFP / RBC combination better suited for biomedical applications requiring long-term oxygen-supply than previously described oxygen-delivery systems.

[0095] Several features of the GT hydrogel facilitate its use for epicardial bioprinting. At room temperature, the material is in a gel state, allowing for easy handling and application. This gel is sufficiently soft to be extruded through a handheld bioprinting tool. This ensures rapid and precise delivery to the target area of the heart (epicardial surface of an area of the ventricle affected by myocardial infarction). The hydrogel conforms completely to the epicardial surface of the heart, without clumping. At body temperature (37 °C), the GT hydrogel rapidly transitions into a viscous liquid state. In this study, the GT hydrogel liquid uniformly covered the target (infarcted) region of the epicardial surface of the heart without uncovered areas or clumping that might cause external compression of the heart. This viscosity made the GT hydrogel particularly useful for covering infarcts on the anterior wall and apex of the left ventricle. GT hydrogel has strong adhesive properties, ensuring stable attachment to the heart surface while providing continuous oxygen-release directly to the ischemic myocardial tissue. The GT hydrogel is photopolymerized in situ with visible- spectrum light, which catalyzes formation of methacryloyl and dityrosine bonds through Ru / SPS. The crosslinked GT hydrogel forms a solid scaffold that adheres to the epicardial surface of the heart. These features ensure ease of operation and specific application of the GT hydrogel to the targeted area of the heart.

[0096] Addition of DDFP and RBC to the GT hydrogel creates a system that allows for direct, controlled delivery of oxygen to ischemic / infarcted myocardium in a sustained manner. By gradually releasing oxygen, the GT / DDFP / RBC system reduces the oxidative stress and tissue damage that often accompany the high oxygen tension associated with rapid reperfusion. Sustained, local oxygen-delivery reduces apoptosis and necrosis of multiple cell types (cardiomyocytes, fibroblasts, endothelial cells) associated with myocardial ischemia and infarction and reduces the formation of scar. Jiaheng Liang et al.,Macromol Biosci 24, e2300302 (2024). Slow oxygen-release allows time for the opening of collateral circulation, producing sustained local blood flow and reduction of ischemic damage. Moreover, continuous oxygen-release can promote angiogenesis, thus facilitating functional reperfusion of myocardial tissue. This can reduce the negative impact of myocardial infarction on ventricular contractile function.

[0097] The results of this study demonstrate that the GT composite significantly promotes the viability and proliferation of various cell types under hypoxic conditions in vitro, including myocytes, fibroblasts, and endothelial cells. Burst release of oxygen observed for other delivery systems was not observed for the GT / DDFP / RBC composite. Compared to the control group and the group using GT alone, the GT / DDFP / RBC composite material, through gradual and controlled oxygen-release, significantly improved cell-survival and proliferation capacities of these cell types evaluated. In cardiomyocyte cultures, the GT / DDFP / RBC composite provided sustained oxy gen-release under hypoxic conditions, maintaining high cell viability and density over 21 days, similar to the levels observed in optimal oxygen environments. Previously published reports have indicated that normoxia (oxygen concentration 21%) is not the optimal growth environment for all cell types. Trenton L. Place FED, Adam J. Case, Free Radic Biol Med 113, 311-322 (2017). For instance, hepatocytes grow best at 13% oxygen, renal cells at 5%, and vascular endothelial cells at 13%. Moreover, reducing oxygen levels to 5% (with some studies suggesting a range of 2-9%) substantially enhances the functions of cardiac stem cells (Li et al., Cardiovasc Res 89, 157-165 (2011)) and cardiac mesenchymal cells. Bolli et al., Stem Cell Rev Rep 17, 900-910 (2021). The GT / DDFP / RBC system, by providing oxygen concentrations lower than normoxia, may mitigate the harmful effects of reactive oxygen species that have been associated with “burst” delivery of oxygen.

[0098] In vitro treatments of fibroblasts and endothelial cells with the GT / DDFP / RBC composite also resulted in enhanced cell viability and proliferation over time. The sustained release of oxygen at reduced concentrations mediated by GT / DDFP / RBC provides a stable oxygenating environment for these cells over time. The controlled oxygen-release strategy further demonstrates that the GT / DDFP / RBC system not only supports the repair of ischemic myocardium but could also be useful in clinical applications that call for enhancement of angiogenesis and tissue oxygenation.

[0099] The GT / DDFP / RBC hydrogel system has demonstrated significant potential in mitigating post-MI injury. Compared to the traditional full reperfusion strategies, this system effectively reduces oxidative stress and tissue-damage caused by rapid reoxygenation through controlled, gradual oxy gen-release. Soares et al., Int J Mol Sci 20(20), 5034 (2019). The stepwise release of oxygen by the hydrogel provides a gentler reoxygenation process for ischemic tissues, preventing myocardial damage from sudden blood flow-restoration and associated increase in oxidative stress. In vitro studies suggest that the GT / DDFP / RBC composite significantly improves the viability of cardiomyocytes under hypoxic conditions. The synergistic effect of DDFP and RBCs ensures more controlled and sustained oxygen-release, thus mitigating the risks of tissue-damage associated with reperfusion. The impact of the GT / DDFP / RBC composite was confirmed in vivo.

[0100] Rats that underwent epicardial application of the hydrogel system at the time of experimental MI exhibited marked improvements in cardiac contractile function, preservation of ventricular wall thickness in the infarcted territory, and reduced ventricular dilation. The GT / DDFP / RBC treatment group outperformed both the untreated and the GT- only treatment groups. It could be that the gradual rate of oxygen-release (with minimization of oxidative stress) was a key determinant of the reduction in left ventricular scar burden associated with GT / DDFP / RBC composite application in experimental MI in rats. It is also possible that reduction of oxidative stress is responsible, in part, for the improvement in post-MI recovery observed both in this study and in a prior study that describes the use of hypoxia-treatment to enhance the recovery of cardiac functions after experimental MI in a different species, i.e., mouse. Nakada et al., Nature 541, 222-227 (2017).Conclusion

[0101] The GT / DDFP / RBC hydrogel system offers a potential therapeutic strategy for myocardial protection and repair following MI. This study provides the first demonstration of the synergistic effect of DDFP and RBCs in sustained, controlled oxygen- release at suitably low levels. This study also demonstrates that GT hydrogel adheres strongly with the epicardial surface of the heart and that this adhesion is stable. In a rat model of experimental myocardial infarction, GT / DDFP / RBC effectively reduced theimpact of MI on left ventricular contractile function and scar burden. These findings suggest that the unique oxygen-releasing hydrogel system described in this study holds significant clinical translational potential. Limitations of the current work include utilization of a rat model of MI, which may not accurately represent all aspects of MI in humans and its responses to hypoxia. Future studies will be required to determine the extent to which reduction in the concentration of reactive oxygen species is responsible for the observed benefits of GT / DDFP / RBC on post-MI healing. In addition, further experiments will be required to determine if the impact of GT / DDFP / RBC on post-MI ventricular healing can be enhanced by the co-delivery of molecular and cellular therapeutics.Materials and Methods

[0102] Preparation of 2% w / v DDFP Emulsion

[0103] A 5% bovine serum albumin (BSA) (Sigma- Aldrich, Burlington, MA, USA) solution was prepared by dissolving 5% BSA in DPBS (Thermo Fisher Scientific, Waltham, MA, USA). DDFP was added to 10 mL of the BSA solution to prepare a 2% w / v DDFP solution (Figure 4a). Oxygen was bubbled into the mixture at a flow rate of 0.1 L min-1, and the mixture was ultrasonicated in an ice-water bath for 5 min to ensure cooling and stability, generating a stable 2% DDFP emulsion (Figure 4b).

[0104] Synthesis of GelMA and GT

[0105] The synthesis of GelMA followed standard procedures. Briefly, 10 g of porcine skin-derived gelatin was dissolved in 100 mL of DPBS at 50°C, and 5 mL of methacrylic anhydride (Sigma-Aldrich, Burlington, MA, USA) was added dropwise. The reaction proceeded at 50 °C for 2 h. The reaction was terminated by dilution with two volumes of DPBS, and the mixture was dialyzed using a 12-14-kDa molecular weightcutoff membrane at 40 °C for 7 days to remove unreacted methacrylic anhydride and byproducts, resulting in the final GelMA product. Yue et al., Biomaterials 73, 254-271 (2015).

[0106] GT was synthesized through the coupling reaction between tyramine groups and the carboxyl groups of GelMA. A 10% (w / v) GelMA solution was dissolved in 2-(N- morpholino) ethanesulfonic acid (MES) (Sigma-Aldrich, Burlington, MA, USA) buffer andreacted with 1.5 mM of EDC (Thermo Fisher Scientific, Waltham, MA, USA) and 0.75 mM of NHS (Thermo Fisher Scientific, Waltham, MA, USA) at 40 °C for 15 min. Then, 36.5 mM of tyramine was added, and the mixture was stirred at 40 °C for 24 h, followed by purification through dialysis. All polymer solutions were sterile filtered through a 0.22-pm filter and lyophilized into a white porous foam. GT was stored at 4 °C and reconstituted into a 10% (w / v) hydrogel before use.

[0107] Crosslinking of GelMA and GT

[0108] Lyophilized GelMA and GT were re-dissolved in PBS at 37 °C to a concentration of 10% w / v. A photoinitiator system containing 0.5 mM of Ru (Sigma- Aldrich, Burlington, MA, USA) and 5 mM of SPS (Sigma-Aldrich, Burlington, MA, USA) was then added to the solution. Crosslinking of the hydrogels was achieved by exposing the solution to visible light from an LED with wavelength of 400-700 nm at 160 mW cm'2, for 1 min.

[0109] In vitro Swelling of GT

[0110] To evaluate the swelling properties of GelMA and GT hydrogels, precise cylindrical hydrogel samples were prepared. Their initial dry weights were measured, and the hydrogels were incubated in DPBS at 37 °C for 48 h to reach a fully hydrated state. Afterward, the samples were freeze-dried to remove water, and the dry weights were measured again. The swelling ratio was calculated as the ratio of hydrated weight to dry weight, with three independent samples analyzed (n=3). The results of the swelling properties are shown in Figure 3 a.

[0111] Lap-Shear Strength and Compressive Stress Testing

[0112] To evaluate the lap shear strengths and compressive stresses of the hydrogels, lyophilized GelMA and GT were re-dissolved in DPBS at 37 °C to a final concentration of 10% w / v. The photoinitiator system containing Ru (0.5 mM) and SPS (5 mM) was then added, and the hydrogels were applied to the inner layer of porcine skin. Crosslinking was achieved by exposing the samples to visible light for 8 min (Figure 2b). After crosslinking, the samples were incubated in DPBS for 10 min before lap shear strength and compressive stress testing.

[0113] Lap shear strength testing was conducted using a universal testing machine (Instron 3342, Instron, Norwood, MA, USA). The samples included 10% GT, 10% GT / 1% DDFP, and mixtures with various concentrations of RBCs (Research Blood Components, LLC, Brighton, MA, USA). The samples were placed between parallel plates, and shear force was applied at a constant rate of 1 mm min-1until failure occurred. Lap shear strength was calculated by dividing the maximum force by the sample’s cross-sectional area.

[0114] Compressive stress testing was performed similarly using the same universal testing machine. The samples were compressed until failure, and compressive stress was calculated by dividing the maximum force by the sample’s cross-sectional area.

[0115] Rheological Properties Testing

[0116] The viscoelastic properties of 10% GT hydrogels were analyzed using a rotational rheometer equipped with a Peltier temperature control system to prevent sample slippage. Steady-state flow scans were performed at 26 °C and 37 °C, and shear stress at different shear rates was measured using a 60-mm parallel plate geometry to determine viscosity. Temperature sweeps were conducted from 18 °C to 37 °C, with an oscillation frequency of 1 Hz, to measure the storage modulus (G’) and loss modulus (G”). Additionally, an oscillatory strain sweep was performed at 26 °C to determine the linear viscoelastic region. After gel-formation, dynamic frequency sweeps were used to evaluate changes in storage and loss moduli, providing insights into the mechanical strength of the hydrogels.

[0117] Printability Evaluation of Hydrogels

[0118] To evaluate the printability of 10% GT and its composites, a 3D extrusion bioprinter (Allevi, Inc., Philadelphia, PA, USA) was used to optimize several printing parameters. Three nozzle diameters (0.41 mm, 0.58 mm, 0.84 mm), three printhead moving speeds (90 mm s'1, 180 mm s'1, 360 mm s'1), and three extrusion pressures (172.4 kPa, 241.3 kPa, 3,103 kPa) were assessed. Thirty measurements were taken for each condition (n=30), and filament structures were observed under a microscope (Ti-E, Nikon, Japan). Fiber diameters were analyzed using ImageJ software (version 1.53a, National Institutes of Health, Bethesda, MD, USA).

[0119] Oxy gen- Release Kinetics Measurements of 1% DDFP

[0120] To assess the oxy gen-release kinetics of different solutions in DPBS, 8 mL of DPBS was placed in a 50-mL centrifuge tube and treated with nitrogen gas for 5 min to create a zero-oxygen environment. An oxygen-sensor probe was then inserted to monitor oxygen levels. Subsequently, 2 mL of the test solution (1% DDFP emulsion, 1% RBCs solution, or a 1% DDFP and 1% RBCs mixture) was quickly added to the DPBS, and the centrifuge tube was sealed to prevent external oxygen from entering. The sample was gently agitated on a shaker, and the oxygen concentration in the DPBS was recorded every 5 min to observe the release kinetics (Figure 4f-h).

[0121] Oxy gen- Release Kinetics Measurements of 1% DDFP, 1% RBCs, and TheirMixtures in GT

[0122] To evaluate the oxygen-release behaviors of 1% DDFP, 1% RBCs, and their mixtures in a 10% GT hydrogel, 20% GT was mixed with 0.5-mM Ru and 5-mM SPS. Then, 2 mL of 1% DDFP, 1% RBCs, or their mixture was added, and the hydrogel was crosslinked under visible light for 5 min to form square hydrogel blocks. The hydrogel blocks were subsequently immersed in 8 mL of nitrogen-saturated DPBS, and the dissolved oxygen levels were monitored in real time using an oxygen sensor. The tubes were gently agitated on a shaker, and the changes in dissolved oxygen were recorded periodically to evaluate the release kinetics (Figure 7f-h).

[0123] Visualization of Qxy en-Release

[0124] To visualize the oxygen-release from the 10% GT and 1% DDFP mixture, an experiment was designed to print honeycomb structures, which were then immersed in DPBS at 37 °C, 26 °C, and 4 °C. The formation of oxygen bubbles was observed and photographed at predetermined intervals. After each observation, the samples were rinsed with fresh DPBS to remove residual bubbles, and DPBS was replaced. The experiment lasted for 19 days, documenting the duration and extent of oxy gen-release. Detailed results from day 1 to day 19 are shown in Figures 7e.

[0125] Cell Viability Assays

[0126] To evaluate the impact of different hydrogel compositions on cell viability, THP-1 and RAW264.7 cells (ATCC, Manassas, VA, USA) were co-cultured with synthesized GelMA, GT, and their composite materials for 7 days. The hydrogels were crosslinked into disk shapes as abovementioned, and the cells were seeded into the wells of 24-well plates at a density of 5xl06cells per well. Cell viability was assessed using PrestoBlue reagent (Thermo Fisher Scientific, Waltham, MA, USA) on days 1, 3, 5, and 7, and fluorescence was measured with a microplate reader at an excitation wavelength of 570 nm and an emission wavelength of 600 nm.

[0127] The percent reduction of PrestoBlue reagent, which indirectly reflects cell viability, was calculated using the following formulae:• Percent Reduction of PrestoBlueReagent=((RlxN2)-(R2xNl)(O2xAl)-(GlxA2))xl00• Percent Reduction of PrestoBlueReagent=((O2xA 1 )-(01 xA2)(Rl xN2)-(R2xN 1 ))x 100, where:• 01 and 02 represent the molar extinction coefficients of the oxidized form ofPrestoBlue reagent at 570 nm and 600 nm, respectively.• R1 and R2 represent the molar extinction coefficients of the reduced form ofPrestoBlue reagent at 570 nm and 600 nm, respectively.• Al is the absorbance of the test well at 570 nm.• A2 is the absorbance of the test well at 600 nm.• N1 is the absorbance of the media-only control well at 570 nm.• N2 is the absorbance of the media-only control well at 600 nm.• Extraction of Neonatal Rat Cardiomyocytes

[0128] The study protocol (2020N000128) was approved by the Brigham and Women’s Hospital Institutional Animal Care and Use Committee (IACUC) and adhered to the standards of the American Association for Laboratory Animal Care. Cardiomyocytes were isolated from the hearts of 1-3-day-old Sprague Dawley rats (Charles River Laboratories, Wilmington, MA, USA). The hearts were minced in ice-cold Hank’s balanced salt solution (HBSS) without Ca2+and Mg2+and digested four times with 0.1% collagenase type II at 37 °C for 15 min each. Dissociated cells were collected by centrifugation at 300x g for 5 min, resuspended in DMEM supplemented with 10% FBS, 10-mM 4-(2-hydroxyethyl)-! -piperazineethanesulfonic acid) (HEPES), 2-mM L-glutamine, and 1% penicillin- streptomycin. After 1 h of pre-plating to remove fibroblasts, non-adherent cardiomyocytes were plated on gelatin-coated dishes at 1.5 x 105 cells cm'2.

[0129] LDH- As sessment

[0130] LDH release was quantified using the Invitrogen CyQUANT LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific, Eugene, OR, USA). Mammalian cells were treated with test compounds in 96-well plates. Post-treatment, 50 pL of medium was transferred to a fresh plate, mixed with an equal volume of reaction mixture, and incubated for 30 min at room temperature in the dark. The reaction was terminated with 50 pL of the stop solution per well. Absorbance was measured at 490 nm, using 680 nm as a reference, on a microplate reader. Cytotoxicity was calculated based on LDH-release from treated versus lysed control cells.

[0131] ATP-Assessment

[0132] ATP levels were quantified using the Molecular Probes ATP Determination Kit (Molecular Probes, Eugene, OR, USA). This bioluminescence assay employs recombinant firefly luciferase and its substrate D-luciferin to measure ATP. In brief, samples were mixed with the reaction buffer, D-luciferin, firefly luciferase, and DTT as per the kit’s protocol. The reaction was carried out at 28 °C for 15 min, after which luminescence was measured using a luminometer set to detect emission at approximately 560 nm. ATP concentration was determined against a standard curve prepared with known ATP concentrations.

[0133] cT nT - Qu antification

[0134] cTnT levels in rats were determined using the TSZ ELISA Kit (TSZ ELISA, Waltham, MA, USA). Briefly, samples or standards were added to antihody-precoated wells followed by a biotinylated detection antibody and horseradish peroxidase (HRP)- streptavidin. After incubation and washing, the 3, 3 ’,5, 5 ’-tetramethylbenzidine (TMB) substrate was added, and the reaction was stopped with the stop solution. Absorbance was measured at 450 nm. The cTnT concentration was calculated from a standard curve according to the manufacturer’s protocol.

[0135] Calcium Imaging in Adherent Cells

[0136] Calcium flux in adherent cells was assessed using the Fluo-4 Direct™ Calcium Assay Kit (Invitrogen, Eugene, OR, USA). Cells were grown to near confluence in poly-D-Lysine-coated plates and treated with the assay reagent directly in the growth medium, eliminating the need for wash steps. Incubation was performed at 37 °C for 30 min, followed by 30 min at room temperature. Fluorescence was measured at excitation / emission wavelengths of 494 / 516 nm.

[0137] Mitochondrial Membrane Potential-Assay

[0138] Mitochondrial membrane potential was assessed using tetramethylrhodamine, methyl ester (TMRM) (Thermo Fisher Scientific, Carlsbad, CA, USA). Adherent cells were stained with TMRM by removing the growth medium, adding 100-nM TMRM staining solution, and incubating for 30 min at 37 °C. Cells were optionally washed with DPBS to reduce background fluorescence. TMRM fluorescence was measured at excitation / emission wavelengths of 550 / 580 nm using a tetramethylrhodamine isothiocyanate (TRITQ / red fluorescent protein (RFP) filter set.

[0139] Animal Model and Surgical Procedure

[0140] The in vivo study protocol (2020N000075) was approved by the Massachusetts General Hospital IACUC and adhered to the standards of the American Association for Laboratory Animal Care. The experiments were conducted on 63 Lewis rats (Charles River Laboratories, Wilmington, MA, USA), aged 12-15 weeks (Figure 9a). All rats underwent general anesthesia and were intubated. After a left thoracotomy, the pericardium was opened to expose the heart. A 6.0 Prolene suture was used to ligate the mid-left anterior descending (LAD) artery. The appearance of a blanched distal anterior ventricular wall confirmed the formation of MI. The chest was then sutured, and the rats were monitored continuously until full recovery from anesthesia.

[0141] Application of Oxygen-Releasing GT Hydrogel

[0142] Immediately after confirming successful LAD artery-ligation, the oxygenreleasing GT hydrogel was applied to the damaged ventricular region. A 20% w / v GTsolution was first prepared, followed by the addition of an equal volume of 2% DDFP solution and RBCs at 1% of the total solution volume. A Ru / SPS solution (0.5 / 5 mM) was then added while avoiding exposure to light. The mixture was loaded into a 1-mL syringe, and 100 pL of the solution was evenly applied to the anterior surface of the heart using a handheld bioprinter 36, customized according to a previously published study. Li et al., Small 19, e2205078 (2023). The oxygen-releasing GT hydrogel was crosslinked in situ by exposing the heart surface to visible light (160 mW cm'2, wavelength 400-700 nm) for 30 s, with the light source positioned approximately 5 cm from the heart.

[0143] Transthoracic Echocardiographic Assessments

[0144] All rats underwent M-mode and 2D transthoracic echocardiography (Philips IE33, Philips Healthcare, Andover, MA, USA) at three time points: i) before MI, ii) immediately after MI (while still under anesthesia), and iii) 3 weeks post-MI (at the end of the study). Echocardiography was performed under anesthesia, and only measurements with adequate image quality were recorded. Offline processing was conducted using the SyngoDynamics software package (Siemens, Malvern, PA, USA). Fractional shortening was calculated using the following formula:Fractional Shortening = [(Left Ventricular End-Diastolic Diameter - Left Ventricular End- Systolic Diameter) / Left Ventricular End-Diastolic Diameter] x 100The relative change in fractional shortening was calculated using the following formula: Relative Change in Fractional Shortening = [(Fractional Shortening at the End of Study / Baseline Fractional Shortening) I Baseline Fractional Shortening] x 100

[0145] Morphometric Analyses of Scar Burden Post-MI

[0146] Histopathological analyses of ventricular scar burden were performed on rats that survived 3 weeks after experimental MI. The rat hearts were fixed in 4% paraformaldehyde solution, embedded in paraffin blocks, sectioned along the short axis, and stained with Masson’s trichrome stain (Sigma-Aldrich, Burlington, MA, USA). Scar volume was calculated based on the area of myocardium affected by fibrosis. Morphometric analyses were performed using the MIQuant software (MATLAB, MS Windows 32-bit, open-source, available online), with results detailed in Figure lOf-h.

[0147] Statistical Analyses

[0148] To ensure data accuracy and reliability, comprehensive statistical methods were employed in this study. All data are presented as mean ± standard deviation (SD), and the sample size (n) for each group was recorded. Intraoperative and postoperative mortality rates between groups were compared using Fisher’s exact test. Comparisons of echocardiographic measurements across different experimental stages (e.g., baseline, immediately after MI, and 3 weeks post-MI) were assessed using the Wilcoxon signed-rank test. One-way analysis of variance (ANOVA) was used to evaluate changes in left ventricular systolic function before and after MI, and 3-week echocardiographic data were analyzed using ANOVA, with baseline data as the covariate. Comparisons of left ventricular scar burden were performed using ANOVA, followed by Turkey’s multiple comparison test for post hoc analysis. Repeated measures ANOVA was used to analyze within-group changes over time. Student’s t-test was used for between-group comparisons at different time points. All statistical analyses were conducted using SPSS 28 software (IBM, Chicago, IL, USA), and statistical significance was indicated by p-values.

[0149] The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

CLAIMSWhat is claimed is:

1. A tissue oxygenation composition, comprising a hydrogel and an oxygen carrier compound.

2. The tissue oxygenation composition of claim 1, wherein the composition further comprises hemoglobin.

3. The tissue oxygenation composition of claim 2, wherein the hemoglobin is provided within red blood cells.

4. The tissue oxygenation composition of claim 1, wherein the oxygen carrier compound is emulsified perfluoropentane.

5. The tissue oxygenation composition of claim 1, wherein the hydrogel comprises gelatin methacryloyl (GelMA).

6. The tissue oxygenation composition of claim 1, wherein the hydrogel comprises an adhesive group.

7. The tissue oxygenation composition of claim 6, wherein the adhesive group is tyramine.

8. A tissue oxygenation precursor composition, comprising a hydrogel precursor and an oxygen carrier compound.

9. The tissue oxygenation precursor composition of claim 8, wherein the composition further comprises hemoglobin.

10. The tissue oxygenation precursor composition of claim 9, wherein the hemoglobin is provided within red blood cells.

11. The tissue oxygenation precursor of claim 8, wherein the oxygen carrier compound is emulsified perfluoropentane.

12. The tissue oxygenation precursor composition of claim 8, wherein the hydrogel oligomer comprises discrete gelatin methacryloyl (GelMA).

13. The tissue oxygenation precursor composition of claim 8, wherein the hydrogel precursor comprises an adhesive group.

14. The tissue oxygenation precursor composition of claim 13, wherein the adhesive group comprises tyramine.

15. A method of tissue oxygenation, comprising contacting a tissue with a tissue oxygenation composition, comprising a hydrogel and an oxygen carrier compound.

16. The method of claim 15, wherein the tissue is cardiac tissue.

17. The method of claim 15, wherein the tissue oxygenation composition provides sustained release of oxygen.

18. The method of claim 15, wherein the tissue oxygenation composition further comprises hemoglobin.

19. The method of claim 15, wherein the hydrogel comprises an adhesive group.

20. The method of claim 15, wherein the tissue is contacted by injection.

21. The method of claim 15, wherein the tissue is contacted by application of the tissue oxygenation composition using a handheld 3D printer.

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