Natural protein-based biomaterials and methods of making and use thereof

Abstract

A biomaterial comprising a cross-linked natural protein, wherein the natural protein is an egg white protein, and methods of making and use thereof, and compositions thereof, as well as a method of preparing a biomaterial comprising the steps of: providing a natural protein; tethering a cross-linkable functional group to the natural protein to produce a modified natural protein; and cross-linking the cross-linkable functional group, thereby producing a biomaterial.

Description

NATURAL PROTEIN-BASED BIOMATERIALS AND METHODS OF MAKING AND USETHEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application No. 63 / 562,887, filed March 8, 2024, the disclosure of which is incorporated by reference herein in its entirety.BACKGROUND OF THE INVENTION

[0002] Engineering highly bioactive scaffolds that can accommodate cells and remodel in vivo is a fundamental goal in tissue engineering. Naturally derived hydrogels like gelatin, chitosan, and alginate have garnered significant interest due to their intrinsic biocompatibility and bioactivity. However, it is worth noting that they may not fully replicate the ECM or offer all the necessary nutrients for cells (Tagafoghi et al., 2020, Advanced Healthcare Materials, 9, 1901722; Raphael et al., 2017, Materials Letters, 190, 103; Yuk et al., 2016, Nature Materials, 15, 190; Briquez et al., 2016, Nature Reviews Materials, 1, 15006; Han et al., 2016, Nature Materials, 14, 477; Zhao et al., 2016, Advanced Healthcare Materials, 5, 108; Wang et al., 2016, Advanced Functional Materials, 29, 1806200; Liu et al., 2018, Biomaterials, 171, 83; Chan et al., 2018, Proceedings of the National Academy of Sciences, 115, 7503; Tavafoghi et al., 2021, Colloids Surf B Biointerfaces, 207, 111975; Tavafoghi et al., 2021, Biofabrication, 13, 042002).

[0003] Thus, there is a need in the art for compositions and methods for making and use of biocompatible hydrogels from natural proteins which can mimic the ECM. The present invention satisfies this unmet need.SUMMARY

[0004] The present invention provides, in part, a biomaterial comprising a cross-linked natural protein, wherein the natural protein is an egg white protein. In some embodiments, the present invention provides a comprising the biomaterial, wherein the biomaterial is present in water at a concentration of between 1% and 20%.

[0005] In some embodiments, the egg white protein is selected from the group consisting of ovalbumin, cystatin, avidin, ovomucoid, ovotransferrin, ovomucin, ferritin, lysozyme, andcombinations thereof. In some embodiments, the cross-linked natural protein comprises crosslinks between a cross-linkable functionality selected from the group consisting of olefin, acrylate, methacrylate, epoxide, vinyl, allyl, and combinations thereof.

[0006] In some embodiments, the cross-linked natural protein comprises cross-links between methacryloyl groups. In some embodiments, the cross-linked natural protein has a degree of cross-linking of at least 10%.

[0007] In some embodiments, the biomaterial is porous. In some embodiments, the biomaterial is a hydrogel having a swelling ratio between 2% and 7%.

[0008] In some embodiments, the biomaterial is biocompatible in vivo. In some embodiments, the biomaterial is biocompatible with cells selected from the group consisting of fibroblasts and endothelial cells.

[0009] The present invention further provides, in part, a method of endothelization comprising the step of exposing the biomaterial to endothelial cells, and a method of treating a subject in need thereof comprising the step of administering to the subject the biomaterial.

[0010] In some embodiments, the present invention provides a method of preparing a biomaterial comprising the steps of: providing a natural protein; tethering a cross-linkable functional group to the natural protein to produce a modified natural protein; and cross-linking the cross-linkable functional group, thereby producing a biomaterial.

[0011] In some embodiments, the natural protein is selected from the group consisting of ovalbumin, cystatin, avidin, ovomucoid, ovotransferrin, ovomucin, ferritin, and lysozyme. In some embodiments, the cross-linkable functional group is selected from the group consisting of olefin, acrylate, methacrylate, epoxide, vinyl, allyl, and carbonyl. In some embodiments, the cross-linkable functional group is a methacrylate. In some embodiments, the step of cross-linking the cross-linkable functional group comprises combining the modified natural protein with a photo-initiator; and exposing the modified natural protein to ultraviolet light. In some embodiments, the step of cross-linking the cross-linkable functional group comprises combining the modified natural protein with a photo-initiator; and exposing the modified natural protein to ultraviolet light for a duration of between 1 second and 30 seconds.

[0012] In some embodiments, the method further comprises the step of lyophilizing the biomaterial to produce a dried biomaterial. The present invention further provides a biomaterial synthesized using the method described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The foregoing purposes and features, as well as other purposes and features, will become apparent with reference to the description and accompanying figures below, which are included to provide an understanding of the invention and constitute a part of the specification, in which like numerals represent like elements, and in which:

[0014] Fig. 1, comprising Fig. 1A through Fig. IE, depicts synthesis, chemical characterization, and gelation kinetics of egg white methacryloyl (EWMA). Fig. 1A depicts a schematic illustrating the methacryloyl modification and crosslinking of EW with ultraviolet (UV) light to form EWMA hydrogels. Fig. IB depicts proton nuclear magnetic resonance (’H- NMR) spectra of pristine and methacryloyl-modified EW with a substitution degree of 40%. Solutions were prepared in D2O, and the measurements were performed at 4 °C. Peaks corresponding to methacrylate groups were shown in green (5-6 ppm) and pink (1.7-1.9 ppm). Amino groups were shown in blue (2.7-3 ppm). Fig. 1C depicts rheological characterization of hydrogel crosslinking kinetics, including GelMA 5% w / v and EWMA 5, 7.5, and 10% w / v and a schematic illustration of the experimental setup. Fig. ID depicts real-time monitoring of crosslinking kinetics and the variation of storage and loss modulus with UV irradiation time, i) GelMA 5%, ii) EWMA 5%, iii) EWMA 7.5%, and iv) EWMA 10%. Fig. IE depicts comparing the gelation time of 5% GelMA with varying concentrations of EWMA hydrogels. Asterisks show the results that are statistically significantly different with p-values less than 0.001 (***), and ns shows the non-significant differences.

[0015] Fig. 2, comprising Fig. 2A through Fig. 2E, depicts mechanical, physical, and morphological characterization of the EWMA hydrogels at different concentrations (5%, 7.5%, and 10% w / v) and GelMA hydrogel at 5% w / v. Fig. 2A depicts representative scanning electron microscope (SEM) images of hydrogels. Fig. 2B depicts compressive modulus for hydrogels after 2 hr incubation in DPBS. Fig. 2C depicts failure strength for hydrogels after 2 hr incubation in DPBS. Fig. 2D depicts the time-dependent swelling ratio of the hydrogels. Fig. 2E depicts time-dependent degradation of UV crosslinked hydrogels in DPBS with collagenase II (2.5 U / ml) at 37 °C. Mean values from a minimum of four replicates ± standard deviations are presented. Asterisks (*) indicate statistically significant differences (p < 0.05), while 'ns' denotes non-significant differences. 0.01 (**), and 0.0001(****).

[0016] Fig. 3, comprising Fig. 3A through Fig. 3F, depicts rheological characterization of crosslinked EWMA hydrogels at different concentrations (5%, 7.5%, and 10% w / v) and GelMA hydrogel at 5% w / v. Fig. 3A depicts storage modulus of the hydrogels versus shear strain. Fig. 3B depicts loss modulus of the hydrogels versus shear strain. Fig. 3C depicts storage modulus of the hydrogels versus angular frequency. Fig. 3D depicts loss modulus of the hydrogels versus angular frequency. Fig. 3E depicts storage modulus of the hydrogels at strain = 0.1% and angular frequency = 1 rad / s. Fig. 3F depicts loss modulus of the hydrogels at strain = 0.1% and angular frequency = 1 rad / s. Data represent the mean values of a minimum of five replicates ± standard deviations. Statistically significant differences are denoted by asterisks (** p < 0.01 and **** p < 0.0001), while 'ns' indicates non-significant differences.

[0017] Fig. 4, comprising Fig. 4A through Fig. 4M, depicts light-based additive manufacturing of EWMA constructs using Digital Light Processing (DLP) stereolithographic (SLA); Fig. 4A depicts an isometric view of the printed Y-shaped perfusable hydrogel construct, scale bar 4 mm. Fig. 4B depicts an isometric view of the printed Y-shaped perfusable hydrogel construct, scale bar 4 mm. Fig. 4C depicts a top view of the printed Y-shaped perfusable hydrogel construct, scale bar 4 mm. Fig. 4D depicts a close-up view of the bifurcation. Fig. 4E depicts a close-up visualization of the printed layers and the staircase effect observed in the channel walls. Fig. 4F depicts red and blue dye perfusion in the bifurcation and demonstration of construct perfusability. Fig. 4G depicts a 3D design of the Y-shaped bifurcation. Fig. 4H depicts a printed gyroid structure, showcasing 8 unit cells across with a 500 pm wall thickness, illustrates the capability of printing intricate structures using EWMA with different printing method (Scale bar: 4 mm). Fig. 41 depicts zoomed-in structure of gyroid print with 8-unit cells. Fig. 4J depicts optical microscopy image of gyroid structure showing one unit cell of gyroid 3D structure printed and the layering artifacts from the DLP printer. Fig. 4K depicts visualization of strut distance in EWMA-printed structure. Fig. 4L depicts strut distance and pore structure in EWMA-printed scaffold. Fig. 4M depicts representation of the 3D structure printed by DLP printer using EWMA as bioink.

[0018] Fig. 5, comprising Fig. 5A through Fig. 5C, depicts the cytocompatibility of EWMA hydrogels at different concentrations (5%, 7.5%, and 10% w / v) and GelMA hydrogel at 5% w / v with GFP-HUVECs in a 2D culture. Fig. 5 A depicts fluorescence images of hydrogels seeded with GFP-HUVECs on days 1 and 7 of culture. Fig. 5B depicts metabolic activity ofGFP-HUVECs seeded on the surface of the hydrogels on days 1 , 3, and 7 of culture. Fig. 5C depicts fluorescence images of intact and VE-cadherin / D API-stained GFP-HUVECs seeded on EWMA 7.5% on day 7 of culture, showing HUVECs remained functional on the surface of EWMA and expressed VE-cadherin to form adherents’ junctions between the endothelial cells (red: VE-cadherin, blue: nuclei, green: GFP).

[0019] Fig. 6, comprising Fig. 6A and Fig. 6B, depicts the vascularization and endothelialization of EWMA 7.5% w / v, GelMA 5% w / v, and Matrigel hydrogels using GFP- HUVECs seeded on the surface of the hydrogels. Fig. 6A depicts fluorescence images of the hydrogels seeded with GFP-HUVECs on days 1, 2, 3, and 4 of culture. Fig. 6B depicts cell spreading area on the hydrogel surface at different incubation times. Higher cell spreading on EWMA compared to GelMA and Matrigel shows higher endothelialization of the EWMA hydrogel. The EWMA and GelMA hydrogels were crosslinked with UV light at an intensity of 800 mW for 1 min. Matrigel was used in its as-received form. All samples were seeded with 7.5X103GFP-HUVECs and placed on a plate shaker in the incubator at 145 rpm and 37 °C. The data represent mean values from a minimum of four replicates ± their respective standard deviations. Asterisks (*) indicate statistically significant differences, with p-values denoted as follows: * p < 0.01, *** p < 0.001, **** p < 0.0001; 'ns' indicates non-significant differences.

[0020] Fig. 7, comprising Fig. 7A through Fig. 7C, depicts the cytocompatibility of the EWMA hydrogels at different concentrations (5%, 7.5%, and 10% w / v) and GelMA hydrogel at 5% w / v with C2C12 cells in a 3D culture. Fig. 7A depicts immunofluorescent stained images of C2C 12-laden hydrogels on day 7 of post-differentiation induction. Myosin heavy chain (MyHC) is stained in green; actin is stained with phalloidin in magenta, and nuclei are stained with DAP1 in blue. Fig. 7B depicts total myotube coverage percentages of differentiated C2C12 cells on day 7 post differentiation induction. Fig. 7C depicts average nuclear distribution in differentiated C2C12 cell-derived myotubes. The data represent mean values from a minimum of four replicates ± their respective standard deviations. Asterisks show results that are statistically significantly different with p-values less than 0.01 (**), 0.001 (***), and 0.0001(****), and ns show non-significant differences.

[0021] Fig. 8, comprising Fig. 8A through Fig. 8D, depicts a biocompatibility study of EWMA using mouse subcutaneous implant in vivo. Fig. 8A depicts cross-sectional histological images of H&E stained skin on day 7 after initial implantation (scale bar: 200 pm). Fig. 8Bdepicts cross-sectional histological images of H&E stained skin on day 30 after initial implantation (scale bar: 200 pm). Fig. 8C depicts immunohistochemical staining was conducted on the subcutaneous implants using F4 / 80 and CD80 markers, accompanied by nuclear staining. Widefield fluorescent images were captured seven days after subcutaneous implantation, with color-coding as follows: Red corresponds to F4 / 80-macrophages, green to CD80, and blue to DAPI. (Scale bar: 200 pm). Fig. 8D depicts H&E staining of heart, kidney, spleen, liver, and lung for the toxicity effect study (scale bar: 200 pm).DETAILED DESCRIPTION

[0022] It is to be understood that the figures and descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for the purpose of clarity, many other elements found in related systems and methods. Those of ordinary skill in the art may recognize that other elements and / or steps are desirable and / or required in implementing the present invention. However, because such elements and steps are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements and steps is not provided herein. The disclosure herein is directed to all such variations and modifications to such elements and methods known to those skilled in the art.

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.Definitions

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, each of the following terms has the meaning associated with it in this section.

[0025] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0026] ‘About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0027] As used herein, the term “subject” refers to a human or another mammal (e.g., primate, dog, cat, goat, horse, pig, mouse, rat, rabbit, and the like) that can have a disease, disorder, or condition; or be at risk for developing a disease, disorder, or condition; but may or may not have a disease, disorder, or condition or be at risk for developing a disease, disorder, or condition. In many embodiments of the present invention, the subject is a human being. In such embodiments, the subject is often referred to as an “individual” or a “patient.” The terms “individual” and “patient” do not denote a particular age.

[0028] As used here, “biocompatible” refers to any material, which, when implanted in a mammal, does not provoke an adverse response in the mammal. A biocompatible material, when introduced into an individual, is not toxic or injurious to that individual, nor does it induce immunological rejection of the material in the mammal.

[0029] The term “biodegradable” includes polymers, compositions and formulations, such as those described herein, that are intended to degrade during use. Biodegradable polymers typically differ from non-biodegradable polymers in that the former may be degraded during use. In one embodiment, such use involves in vivo use, such as in vivo therapy. In another embodiment, such use involves in vitro use. In general, biodegradation involves the degradation of a biodegradable polymer into its component subunits, or digestion, e g., by a biochemical process, of the polymer into smaller, non-polymeric subunits. Two types of biodegradation may generally be identified. For example, biodegradation may involve cleavage of bonds (whether covalent or otherwise) in the polymer backbone. In such biodegradation, monomers and oligomers typically result, and even more typically, such biodegradation occurs by cleavage of a bond connecting one or more of subunits of a polymer. Further, biodegradation may involve cleavage of a bond (whether covalent or otherwise) internal to side chain or that connects a side chain to the polymer backbone. For example, a therapeutic agent or other chemical moietyattached as a side chain to the polymer backbone may be released by biodegradation. In one embodiment, at least one type of biodegradation may occur during use of a polymer. As used herein, the term “biodegradation” encompasses all known types of biodegradation.

[0030] Ranges: throughout this disclosure, various aspects of the invention can be 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 subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges 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, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.Biomaterials

[0031] The present invention relates to, in part, biomaterials derived from natural proteins. In certain embodiments, the biomaterial of the present invention comprises a natural protein and a cross-link. Thus, in certain embodiments, the present invention provides biomaterials derived from natural proteins comprising a natural protein and a cross-link and compositions and methods of use thereof.

[0032] As used herein, the term "biomaterial" refers to a natural, synthetic, living, or non-living substance or material that may interact with biological systems and / or have a biological use. The term "biomaterial" is intended to encompass a material or substance that may have been engineered to take a form which, alone or as part of a complex system, may be used to direct, by control of interactions with components of living systems, the course of any therapeutic or diagnostic procedure. The term "biomaterial" is further intended to include a material that is biocompatible with a human or animal body. In certain embodiments, a biomaterial comprises a natural protein.

[0033] In certain embodiments, the present invention provides a biomaterial comprising a cross-linked natural protein, wherein the natural protein is an egg white protein. The cross-linked natural protein may be present in the biomaterial in any suitable amount based on the total weight of the biomaterial. For example, the cross-linked natural protein may be present in an amount between about 1% and about 50%, about 1% and about 45%, about 1% and about 40%,about 1% and about 35%, about 1% and about 30%, about 1% and about 25%, about 1% and about 20%, about 1% and about 15%, about 1% and about 10%, about 1% and about 5%, 5% and about 50%, about 5% and about 45%, about 5% and about 40%, about 5% and about 35%, about 5% and about 30%, about 5% and about 25%, about 5% and about 20%, about 5% and about 15%, about 5% and about 10%, 10% and about 50%, about 10% and about 45%, about 10% and about 40%, about 10% and about 35%, about 10% and about 30%, about 10% and about 25%, about 10% and about 20%, about 10% and about 15%, or at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 7.5%, 8%, 9%, or 10%.

[0034] In some embodiments, the present invention further provides a composition comprising the biomaterial described herein, wherein the biomaterial is present in an amount between about 1% and about 50%, about 1% and about 45%, about 1% and about 40%, about 1% and about 35%, about 1% and about 30%, about 1% and about 25%, about 1% and about 20%, about 1% and about 15%, about 1% and about 10%, about 1% and about 5%, 5% and about 50%, about 5% and about 45%, about 5% and about 40%, about 5% and about 35%, about 5% and about 30%, about 5% and about 25%, about 5% and about 20%, about 5% and about 15%, about 5% and about 10%, 10% and about 50%, about 10% and about 45%, about 10% and about 40%, about 10% and about 35%, about 10% and about 30%, about 10% and about 25%, about 10% and about 20%, about 10% and about 15%, or at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 7.5%, 8%, 9%, 10%, 20%, 30%, 40%, or 50%.

[0035] In some embodiments, the degree of cross-linking, or the percentage of natural protein chains which comprise a cross-link or a connection, is between about 1% and about 50%, about 5% and about 50%, about 10% and about 50%, about 15% and about 50%, about 20% and about 50%, about 25% and about 50%, about 30% and about 50%, about 35% and about 50%, about 40% and about 50%, about 45% and about 50%, or at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.

[0036] In some embodiments, the cross-linked natural protein comprises cross-links between a reactive group and can be a cross-linkable functionality selected from the group consisting of olefin, acrylate, methacrylate, epoxide, vinyl, allyl, and combinations thereof. In some embodiments, the cross-linkable functionality forms a covalent bond between two atoms when exposed to a stimulus including, but not limited to, light and heat. In some embodiments, the cross-linkable functionality forms a free-radical when exposed to a stimulus.

[0037] In certain embodiments, the natural protein is an egg white protein. In some embodiments, the egg white protein is selected from the group consisting of ovalbumin, cystatin, avidin, ovomucoid, ovotransferrin, ovomucin, ferritin, lysozyme, and combinations thereof. Thus, in some embodiments, the cross-linked natural protein comprises cross-links within a combination of various proteins and may comprise cross-links between the same protein, crosslinks between two different proteins, and combinations thereof.

[0038] In certain embodiments, the biomaterial is a hydrogel. In some embodiments, the biomaterial is porous. In some embodiments, the biomaterial has a porosity of between about 10% and about 90%, about 20% and about 90%, about 30% and about 90%, about 40% and about 90%, about 50% and about 90%, about 60% and about 90%, about 70% and about 90%, about 80% and about 90%, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the biomaterial has a mean pore size of between about 10 pm to about 300 pm, about 10 pm to about 250 pm, about 10 pm to about 200 pm, about 10 pm to about 150 pm, about 10 pm to about 100 pm, about 10 pm to about 50 pm, or at least about 10 pm, 20 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, 100 pm, 150 pm, 200 pm, 250 pm, or 300 pm.

[0039] In some embodiments, the biomaterial has a swelling ratio between about 1% and about 10%, about 2% and about 10%, about 3% and about 10%, about 4% and about 10%, about 5% and about 10%, about 6% and about 10%, about 7% and about 10%, about 8% and about 10%, about 9% and about 10%, 1% and about 9%, about 2% and about 9%, about 3% and about 9%, about 4% and about 9%, about 5% and about 9%, about 6% and about 9%, about 7% and about 9%, about 8% and about 9%, 1% and about 8%, about 2% and about 8%, about 3% and about 8%, about 4% and about 8%, about 5% and about 8%, about 6% and about 8%, about 7% and about 8%, 1% and about 7%, about 2% and about 7%, about 3% and about 7%, about 4% and about 7%, about 5% and about 7%, about 6% and about 7%, 1% and about 6%, about 2% and about 6%, about 3% and about 6%, about 4% and about 6%, about 5% and about 6%, about 2% and about 7%, about 3% and about 7%, about 4% and about 7%, about 5% and about 7%, about 6% and about 7%, or at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0040] In some embodiments, the biomaterial demonstrates enhanced stiffness. In some embodiments, the biomaterial demonstrates enhanced elasticity. In some embodiments, the biomaterial has a storage modulus higher than its loss modulus.

[0041] In some embodiments, the biomaterial has a storage modulus (G’) at 0.1% strain and angular frequency of 1 rad / s of between about 400 Pa and about 10000 Pa, about 400 Pa and about 9000 Pa, about 400 Pa and about 8000 Pa, about 400 Pa and about 7000 Pa, about 400 Pa and about 6000 Pa, about 400 Pa and about 5000 Pa, about 400 Pa and about 4000 Pa, about 400 Pa and about 3000 Pa, about 400 Pa and about 2000 Pa, about 400 Pa and about 1000 Pa, about 500 Pa and about 10000 Pa, about 500 Pa and about 9000 Pa, about 500 Pa and about 8000 Pa, about 500 Pa and about 7000 Pa, about 500 Pa and about 6000 Pa, about 500 Pa and about 5000 Pa, about 500 Pa and about 4000 Pa, about 500 Pa and about 3000 Pa, about 500 Pa and about 2000 Pa, about 500 Pa and about 1000 Pa, or at least about 400 Pa, 500 Pa, 600 Pa, 700 Pa, 800 Pa, 900 Pa, 1000 Pa, 2000 Pa, 3000 Pa, 4000 Pa, 5000 Pa, 6000 Pa, 7000 Pa, 8000 Pa, 9000 Pa, or 10000 Pa.

[0042] In some embodiments, the biomaterial has a loss modulus (G”) at 0.1% strain and angular frequency of 1 rad / s of between about 100 Pa and about 10000 Pa, about 100 Pa and about 9000 Pa, about 100 Pa and about 8000 Pa, about 100 Pa and about 7000 Pa, about 100 Pa and about 6000 Pa, about 100 Pa and about 5000 Pa, about 100 Pa and about 4000 Pa, about 100 Pa and about 3000 Pa, about 100 Pa and about 2000 Pa, about 100 Pa and about 1000 Pa, about 100 Pa and about 900 Pa, about 100 Pa and about 800 Pa, about 100 Pa and about 700 Pa, about100 Pa and about 600 Pa, about 100 Pa and about 500 Pa, about 100 Pa and about 400 Pa, about100 Pa and about 300 Pa, about 100 Pa and about 200 Pa, or at least about 100 Pa, 200 Pa, 300Pa, 400 Pa, 500 Pa, 600 Pa, 700 Pa, 800 Pa, 900 Pa, or 1000 Pa.

[0043] In certain embodiments, the biomaterial of the present invention are biocompatible. In some embodiments, the biomaterial of the present invention is biocompatible in vivo. In some embodiments, the biomaterial of the present invention is an antiviral biomaterial. In some embodiments, the biomaterial of the present invention is an antibacterial biomaterial. In some embodiments, the biomaterial is biocompatible with cells selected from the group consisting of fibroblasts and endothelial cells. Thus, in some embodiments, the present invention further provides a method of endothelization comprising the step of exposing the biomaterial as described herein to endothelial cells. In certain embodiments, the biomaterial demonstrates enhanced cytocompatibility and promotes unexpectedly improved endothelization of cells, including but not limited to, fibroblasts, endothelial cells, and human umbilical veinendothelial cells, and / or expression of protein molecules, including but not limited to vascular endothelial (VE)-cadherin.

[0044] The biomaterials described herein may be provided in various forms, including but not limited to, a gel, a sponge, a solution, a coating, a polymeric matrix, a bead, and combinations thereof. In certain embodiments, the biomaterial provided as a coating may be coated onto a surface or another synthetic material such as an implant, a graft, or a mesh.Methods of Making Biomaterials

[0045] The present invention further provides, in part, methods of making the biomaterials described herein. Thus, in certain embodiments, the present invention provides a method of preparing a biomaterial comprising the steps of: providing a natural protein; tethering a cross-linkable functional group to the natural protein to produce a modified natural protein; and cross-linking the cross-linkable functional group, thereby producing the biomaterial.

[0046] Exemplary natural proteins which may be employed in the present method include, but are not limited to, egg white proteins such as ovalbumin, cystatin, avidin, ovomucoid, ovotransferrin, ovomucin, ferritin, and lysozyme, and gelatin.

[0047] In certain embodiments, the step of tethering a cross-linkable functional group to the natural protein can be performed using any method known in the art of organic synthesis. As used herein, the term “tethering” describes the joining of a functional group to a protein of interest through a covalent bond or through a tethering group, such as through polyethylene glycol (PEG). In some embodiments, the step of tethering forms a covalent bond between any amino acid side chain of the natural protein and a reactive functionality. Exemplary reactive functionalities are described elsewhere herein and include cross-linkable moieties. In some embodiments, the step of tethering forms a covalent bond between an amino acid side chain and a cross-linkable functionality. In some embodiments, the amino acid side chain comprises a functional group selected from the group consisting of amino, carboxylic acid, hydroxyl, thiol, and combinations thereof. In some embodiments, the step of tethering forms a carbon-carbon bond, a carbon-oxygen bond, a carbon-sulfur bond, a sulfur-sulfur bond, a carbon-nitrogen bond, or any combination thereof. In some embodiments, the step of tethering forms a carbon-nitrogen bond between the natural protein and a reactive functionality. For example, the natural protein isreacted with methacrylic anhydride to form carbon-nitrogen bonds, thereby tethering methacryloyl groups to the natural protein to produce a modified natural protein.

[0048] In some embodiments, the step of tethering a cross-linkable functional group to the natural protein comprises mixing a natural protein with a cross-linking agent. The present method can employ any natural protein described herein. In some embodiments, the crosslinking agent is selected from the group consisting of anhydrides, acyl chlorides, peroxides, isocyanates, silanes, acrylamides, sulfides, and combinations thereof. In some embodiments, the cross-linking agent is added to the natural protein at a concentration of weight by volume of between about 5% and about 95%, about 10% and about 95%, about 15% and about 95%, about 20% and about 95%, about 25% and about 95%, about 30% and about 95%, about 35% and about 95%, about 40% and about 95%, about 45% and about 95%, about 50% and about 95%, about 55% and about 95%, about 60% and about 95%, about 65% and about 95%, about 70% and about 95%, about 75% and about 95%, about 80% and about 95%, about 85% and about 95%, about 90% and about 95%, or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0049] In some embodiments, the modified natural protein has a degree of substitution, or a percentage of side chains which have a cross-linkable functional group, of between about 5% and about 90%, about 10% and about 90%, about 15% and about 90%, about 20% and about 90%, about 25% and about 90%, about 30% and about 90%, about 35% and about 90%, about 40% and about 90%, about 45% and about 90%, about 50% and about 90%, about 55% and about 90%, about 60% and about 90%, about 65% and about 90%, about 70% and about 90%, about 75% and about 90%, about 80% and about 90%, about 85% and about 90%, between about 5% and about 80%, about 10% and about 80%, about 15% and about 80%, about 20% and about 80%, about 25% and about 80%, about 30% and about 80%, about 35% and about 80%, about 40% and about 80%, about 45% and about 80%, about 50% and about 80%, about 55% and about 80%, about 60% and about 80%, about 65% and about 80%, about 70% and about 80%, about 75% and about 80%, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0050] In certain embodiments, the step of cross-linking the cross-linkable functional group comprises combining the modified natural protein with a photo-initiator; and exposing the modified natural protein to light. In some embodiments, the light is ultraviolet light. In someembodiments, the light is any light which has a wavelength required to activate the photoinitiator, for example at a wavelength of at least about 100 nm, about 200 nm, 300 nm, 400 nm, 500 nm, or 600 nm. Exemplary photo-initiators which may be employed in the present method include, but are not limited to, type I photoinitiators or type II photoinitiators, trimethylbenzoyl diphenylphosphine oxide (TPO), camphorquinone, benzophenone, 1 -phenyl- 1,2-propanedi one, benzoin ethers, and a-hydroxyalkylphenones.

[0051] In some embodiments, the step of exposing the modified natural protein to light is performed for between about 1 second and about 30 seconds, about 5 seconds and about 30 seconds, 10 seconds and about 30 seconds, or at least about 1 second, 5 seconds, 30 seconds, or 1 minute. In some embodiments, the step of exposing the modified natural protein to light is performed until gelation. In some embodiments, the step of exposing the modified natural protein to light is performed until G’ surpasses G”.

[0052] In certain embodiments, the method described herein further comprises the step of drying or dehydrating the biomaterial to produce a dried biomaterial in forms such as a powder or film. In some embodiments, the method further comprises the step of lyophilizing the biomaterial to produce a dried biomaterial. Lyophilization of the biomaterial may comprise the steps of freezing the biomaterial, then evaporating residual liquid from the frozen biomaterial at a reduced pressure.Methods of Using Biomaterials

[0053] The present invention further provides, in part, methods of using the biomaterials described herein. In certain embodiments, the present invention provides a method of culturing cells comprising exposing the biomaterial described herein to a cell of interest, including but not limited to, fibroblasts and endothelial cells such as human umbilical vein endothelial cells (HUVECs), pulmonary artery endothelial cells, iliac artery endothelial cells, and blood brain barrier endothelial cells. In some embodiments, the present invention provides a method of endothelization comprising the step of exposing the biomaterial described herein to endothelial cells.

[0054] In certain embodiments, the present invention provides a method of treating a subject in need thereof comprising administering to the subject with a biomaterial describedherein. For example, in some embodiments, the biomaterial may be used to encapsulate a cell of interest and implanted into the subject.

[0055] In certain embodiments, the biomaterial of the present invention is uniquely suited for use as extracellular matrix (ECM) mimics and can comprise cell-binding and matrixmetalloproteinase-responsive motifs. In certain embodiments, the biomaterial of the present invention is uniquely suited in cell culturing, growth, and proliferation. In certain embodiments, the biomaterial of the present invention is uniquely suited to facilitate endothelization and can be applied in the engineering of biomedical grafts and implants.EMBODIMENTS

[0056] 1. A biomaterial comprising a cross-linked natural protein, wherein the natural protein is an egg white protein.

[0057] 2 A composition comprising the biomaterial of embodiment 1, wherein the biomaterial is present in water at a concentration of between 1% and 20%.

[0058] 3 The biomaterial of embodiments 1 or 2, wherein the egg white protein is selected from the group consisting of ovalbumin, cystatin, avidin, ovomucoid, ovotransferrin, ovomucin, ferritin, lysozyme, and combinations thereof.

[0059] 4 The biomaterial of any one of embodiments 1-3, wherein the cross-linked natural protein comprises cross-links between a cross-linkable functionality selected from the group consisting of olefin, acrylate, methacrylate, epoxide, vinyl, allyl, and combinations thereof.

[0060] 5. The biomaterial of any one of embodiments 1-4, wherein the cross-linked natural protein comprises cross-links between methacryloyl groups.

[0061] 6. The biomaterial of any one of embodiments 1-5, wherein the cross-linked natural protein has a degree of cross-linking of at least 10%.

[0062] 7 The biomaterial of any one of embodiments 1-6, wherein the biomaterial is porous.

[0063] 8 The biomaterial of any one of embodiments 1-7, wherein the biomaterial is a hydrogel having a swelling ratio between 2% and 7%.

[0064] 9. The biomaterial of any one of embodiments 1-8, wherein the biomaterial is biocompatible in vivo.

[0065] 10. The biomaterial of any one of embodiments 1-9, wherein the biomaterial is biocompatible with cells selected from the group consisting of fibroblasts and endothelial cells.

[0066] 11. A method of endothelization comprising the step of exposing the biomaterial of any one of embodiments 1-10 to endothelial cells.

[0067] 12. A method of treating a subject in need thereof comprising the step of administering to the subject the biomaterial of any one of embodiments 1 -11.

[0068] 13. A method of preparing a biomaterial comprising the steps ofproviding a natural protein; tethering a cross-linkable functional group to the natural protein to produce a modified natural protein; and cross-linking the cross-linkable functional group, thereby producing a biomaterial.

[0069] 14. The method of embodiment 13, wherein the natural protein is selected from the group consisting of ovalbumin, cystatin, avidin, ovomucoid, ovotransferrin, ovomucin, ferritin, and lysozyme.

[0070] 15. The method of embodiments 13 or 14, wherein the cross-linkable functional group is selected from the group consisting of olefin, acrylate, methacrylate, epoxide, vinyl, allyl, and carbonyl.

[0071] 16. The method of embodiments 13-15, wherein the cross-linkable functional group is a methacrylate.

[0072] 17. The method of any one of embodiments 13-16, wherein the step of crosslinking the cross-linkable functional group comprises combining the modified natural protein with a photo-initiator; and exposing the modified natural protein to ultraviolet light.

[0073] 18. The method of any one of embodiments, 13-17, wherein the step of crosslinking the cross-linkable functional group comprises combining the modified natural protein with a photo-initiator; and exposing the modified natural protein to ultraviolet light for a duration of between 1 second and 30 seconds.

[0074] 19. The method of any one of embodiments 13-18, further comprising the step of lyophilizing the biomaterial to produce a dried biomaterial.

[0075] 20. A biomaterial synthesized using the method of any one of embodiments13-19.EXPERIMENTAL EXAMPLES

[0076] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0077] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.

[0078] Egg white (EW), a natural biomaterial, holds great potential in tissue engineering applications as it contains proteins such as ovalbumin (54%), ovotransferrin (12%), ovomucoid (1 1%), ovomucin (3.5%), and lysozyme (3.5%), which are essential for supporting cell survival and functionality. Furthermore, EW has exhibited remarkable antiviral, antibacterial, and anticancer properties, rendering it a suitable choice for applications in tissue engineering (Jalili- Firoozinezhad et al., 2020, Materials Today, 40, 193; Abeyrathne et al., 2013, Poultry Science, 92, 3292). Compared to the commonly used natural proteins such as gelatin and collagen, EW provides a prevalent, accessible, and low-cost extracellular matrix (ECM) that can be used directly in its raw form, thus eliminating the need for a complicated purification process. Moreover, the non-mammalian source of EW reduces the risk of transmissible diseases, such as mad cow disease. It overcomes religious and cultural concerns associated with the use of mammalian-derived products (Yoon et al., 2016, PloS One, 11, e0163902).

[0079] EW has been studied for various applications, including wound healing (Guo et al., 2022, Biomaterials, 282, 121406), tissue engineering (Delkash et al., 2021, Journal of Functional Biomaterials, 12, 45), pharmaceuticals (Dong, 2021, Journal of Biomedical MaterialsResearch Part B: Applied Biomaterials, 106, 1045), and bioelectronics. EW-based hydrogels and cryogels exhibit functional properties such as stretchability, printability, and self-healing properties and the capability to incorporate conductive nanomaterials for fabricating electronic sensors and actuators (Chang et al., 2019, Journal of Materials Chemistry A, 7, 24626; Balaji et al., 2019, Biocatalysis and Agricultural Biotechnology, 17, 441). EW has been recently described as a substitute for ECMs to improve the angiogenesis and biocompatibility of bioscaffolds (Kaipparettu et al., 2008, Biotechniques, 45, 165). EW has also been introduced as an alternative to Matrigel, a complex protein matrix with high angiogenic properties for mammary gland cells' 3D organotypic culture. Despite high angiogenic properties, Matrigel lacks reproducibility as it is secreted by living cells (Mousseau et al., 2014, Laboratory Investigation, 94, 340; Khoo et al., 2011, Tissue Engineering. Part C, Methods, 17, 895; Benelli, 1999, The International Journal of Biological Markers, 14, 243). Moreover, the tumor cell source of Matrigel may limit its use in the human body (Gomillion, 2017, Comprehensive Biomaterials II, Elsevier, Oxford, 403). Like Matrigel, EW can induce the formation of prevascular networks in endothelial and smooth muscle cell co-cultures. The EW matrix supports the growth of various cell types from human, mouse, and rat origins, and it has demonstrated compatibility with the aortic ring assay, enabling the culture of vascular and tumor cells (Emamat et al., 2020, Clinical Nutrition ESPEN, 39, 15). HUVECs and rat aortic endothelial cells appeared to grow, migrate, and organize themselves in a vessel-like network within 48 hours in an EW matrix similar to that observed for the Matrigel. However, little is known about the potential of EW hydrogels for the endothelization of biomaterial surfaces (Nojima, 2018, NPG Asia Materials, 10, e460).

[0080] Endothelization of biomedical implants has been widely accepted as an ideal approach to improve biocompatibility and avoid implant rejection (Jana, 2019, Acta Biomaterialia, 99, 53). Furthermore, a highly integrated endothelial lining is needed on the lumen surface of artificial vascular grafts to improve their hemocompatibility and long-term potency (Ren et al., 2015, Chemical Society Reviews, 44, 5680); however, it is significantly challenging to form a confluent endothelium layer on biomaterial surfaces. Based on previous studies that demonstrated the high activity of endothelial cells in EW, one can hypothesize that EW can be used as a highly bioactive substrate to induce endothelization. Also, the high cell affinity of albumin, a significant protein in EW, can improve bioactivity and reduce thecomplexity of introducing cell-binding domains in EW (Rauvala, 1981, Journal of Cell Biology, 88, 149).

[0081] Three-dimensional (3D) bioprinting using photocrosslinkable hydrogels has gained considerable attention due to its versatility in various applications, including tissue engineering and drug delivery. Egg White (EW) is an organic biomaterial with excellent potential in tissue engineering. It offers abundant proteins, biocompatibility, bioactivity, tunable mechanical properties, and inherent antiviral and antibacterial characteristics. Here, a photocrosslinkable hydrogel derived from EW through methacryloyl modification has been developed, resulting in Egg White methacryloyl (EWMA). Upon exposure to UV light, synthesized EWMA becomes crosslinked, creating hydrogels with remarkable bioactivity. These hydrogels offer adjustable mechanical and physical properties compatible with most current bioprinters. The EWMA hydrogels closely resemble the native extracellular matrix (ECM) due to cell-binding and matrix metalloproteinase-responsive motifs inherent in EW. In addition, EWMA promotes cell growth and proliferation in 3D cultures. It facilitates endothelialization when investigated with human umbilical vein endothelial cells (HUVECs), making it an attractive replacement for engineering hemocompatible vascular grafts and biomedical implants. In summary, the EWMA matrix enables the biofabrication of various living constructs. This breakthrough enhances the development of physiologically relevant 3D in vitro models and opens many opportunities in regenerative medicine.

[0082] The present invention provides in part, a novel hydrogel. Hydrogels are of tremendous interest and have already made considerable impact in a variety of bioengineering and tissue engineering applications including their use as cell-laden bioinks for three- dimensional (3D) bioprinting. Hydrogels from natural sources that possess important amino acid motifs which are important for cell attachment, proliferation, and differentiation have been explored. Hydrogels made from collagen and its derivative gelatin have been developed but proved weak. The addition of methacryloyl led to the development of crosslinkable printable hydrogel. However, these gelatin-based hydrogels have their own limitations and associated concerns. Thus, experiments were carried out to develop a new crosslinkable hydrogel. Egg white (EW) has been a source of many functional proteins such as albumin, lysozyme, and ovomucin that enables multiple advantages since it contains several important functional proteins beside albumin which have metal binders and possess antiviral and antibacterial activities inaddition to tumor growth inhibiting properties. However, the complex isolation and purification process as well as the cost has hindered the extensive utilization of EW and its derivatives in multiple fields. Hence, the potential of egg white in tissue engineering and regenerative medicine remains to be explored.

[0083] The present invention relates to a new biomaterial and its processing method needed to convert EW into a photo-crosslinkable hydrogel which includes methacrylation of albumin to form EW methacryloyl (EWMA). Encouraging results and preliminary results of tests of cell cytocompatibility of the novel biomaterial have been obtained when cells are embedded and crosslinked in the EW hydrogel matrix. 3D EWMA hydrogels closely resemble essential properties of native extracellular matrix (ECM) due to the presence of cell-attaching and matrix metalloproteinase responsive peptide motifs, which allow cells to proliferate and spread in EWMA-based scaffolds. EDMA hydrogels are crosslinked when they are exposed to light irradiation forming hydrogels with tunable mechanical properties that match those of the native ECM. EWMA can also be microfabricated using different methodologies including micromolding, photomasking, bioprinting, self-assembly, and microfluidic techniques to general constructs with controlled architectures. Hybrid hydrogel systems can also be formed by mixing EWMA hydrogels with nanoparticles such as nanosilicate and graphene oxide and other polymers to form networks with desired combined properties and characteristics for specific biological applications.

[0084] The present invention can be specifically useful in using EWMA hydrogels in applications that include, but are not limited to, bone regeneration and engineering vascular tissues among others. Other applications of the hydrogel, besides tissue engineering, include fundamental single-single cell research, cell signaling, drug and gene delivery, and bio-sensing.

[0085] Blood vessels are made of albumin rich extracellular matrix, accordingly having an albumin rich biomaterial can enhance the biomimetic properties of lumen extracellular matrix. Hence, EWMA will lead to more cell adhesion and enhanced angiogenesis. On the other hand, there are several studies showing that albumin is an efficient coating for bone-related biomaterials associated with increasing seeding efficiency, cell proliferation, and calcium deposition in vitro and bone remodeling in vivo. Albumin facilitates new bone formation in nonunion and critical size defect models in rats. However, the use of albumin hydrogels in bone tissue engineering remains unknown. Thus, an albumin-based tissue matrix would be a usefulmatrix because it can play a structural -support role in cell and tissue engineering. Importantly, this unique characteristic of albumin can dramatically reduce the complexity of developing cell- scaffold-based medical products by avoiding complicated cell-scaffold interactions.

[0086] To evaluate the potential of the present novel biomaterial, important physical, mechanical, and biological properties are compared to the most prevalently used hydrogel (GelMA) in tissue engineering and regeneration. It was found that EWMA introduces lower degradation compared to GelMA which will be comparatively more durable for delivering therapies. Swelling ratio was half of the GelMA which will enable the material to be more mechanically stable for in vivo applications over time and changes in properties will not significantly affect the results. Cell compatibility was evaluated using live and dead assays and metabolic activity offering high cell compatibility compared to GelMA and two-dimensional culture for two cell types including human fibroblast and human umbilical ventricle endothelial cells (HUVECs).

[0087] Therefore, in this invention a novel photo-crosslinkable albumin rich hydrogel biomaterial and the method of its preparation and processing are disclosed. These include example applications in 3D bioprinting of living constructs and demonstration of in vitro cytocompatibility.

[0088] Here, EW hydrogels were developed that can be readily crosslinked through light irradiation, forming a highly bioactive ECM-mimetic microenvironment for biological compounds. In contrast to thermal or chemical crosslinking techniques, photocrosslinking will create hydrogels with tunable mechanical and physical properties that are easily microfabricated using various light-based methods, such as photomasking and stereolithography, to generate miniaturized structures. Also, photocrosslinking of EW eliminates the need for high temperatures conventionally involved in thermal crosslinking processes, thus providing opportunities for 3D culture and cell encapsulation within the hydrogels (Yue et al., 2015, Biomaterials, 73, 254). To prepare a photocrosslinkable hydrogel bioink, EW was modified with methacrylic groups to form an EWMA hydrogel that can be crosslinked with light in the presence of photo-initiators (Pls). Physical and mechanical characterizations were performed, including biodegradation, compression, and rheological analysis, to evaluate the strength and stability of the synthesized EWMA hydrogel under physiological conditions. Furthermore, the potential of EWMA hydrogels was assessed as a cell-laden ECM that can be used for bioprinting and tissueengineering applications. For this purpose, fibroblasts' cytocompatibility and metabolic activity encapsulated within EWMA hydrogels was evaluated. Also, the efficacy of EWMA hydrogels as an ECM to host endothelial cells and induce endothelization in a 2D culture of HUVECs was investigated.

[0089] The results and discussion of experiments of the instant invention are discussed herein.Chemical characterizations and crosslinking kinetics

[0090] EWMA was synthesized in Dulbecco’s Phosphate-Buffered Saline (DPBS) buffer using a one-pot method that yielded a white-yellowish foam (Fig. 1 A). During the methacryloyl modification of EW, the acidic by-product of the reaction (methacrylic acid) can drastically decrease the solution pH and inhibit the substitution reaction by protonating the free amino groups of lysine on EW (Sewald et al., 2018, Macromolecular Bioscience, 18, 1800168). Therefore, the pH of the solution was monitored and maintained in a basic range between 8 and 9 to favor the forward reaction by deactivating the acidic by-products. The methacryloyl modification of EW was verified by HNMR analysis (Fig. IB). The peak area for amino acid moi eties at 7.2 ppm was used to normalize the HNMR spectra since this area remained unchanged after methacryloyl substitution. HNMR spectra showed that the peak intensity for the methylene proton (NH2CH2CH2CH2CH2-) of free lysine at 2.9 ppm decreased drastically on EW after the reaction. On the other hand, the acrylic protons (CH2=C(CH3)CONH-) at 5.7-5.3 ppm and methyl protons (CH2=C(CH?)CO-) at 1.8 ppm were increased for EWMA, confirming the methacryloyl modification of EW. The degree of substitution (DS) of EWMA was calculated to be 40% by comparing the methylene proton intensity around 5= 2.9 ppm for the methacrylated and pristine EW.

[0091] The EWMA hydrogels were synthesized by mixing EWMA and Pls, followed by photocrosslinking with UV irradiation. A rheological test was carried out to investigate the gelation kinetics of EWMA under UV light. Fig. 1C, Fig. ID, and Fig. IE represent the crosslinking kinetics of the hydrogels. Upon irradiation with UV light, the crosslinking reaction started until the gelation point was reached (where G' surpassed G") within ~ 4-20 s. This point corresponds to a sharp increase in the storage modulus when enough free radicals are generated to drive the reaction. Overall, a shorter gelation time was observed for hydrogels containinghigher percentages of vinyl groups, accelerating the polymerization process. EWMA 5% exhibited a significantly longer gelation time than GelMA 5%, which can be explained by fewer vinyl groups on EWMA 5%. Additionally, the higher amount of vinyl groups on EWMA 10% resulted in a significantly shorter gelation time than EWMA 5%. With the increase in crosslinking time, the reaction rate became slower until the storage modulus reached a plateau at UV irradiation time ~ 1 min, which was used as a crosslinking time to prepare hydrogels for the rest of the characterizations.Mechanical and physical characterization of EWMA hydrogels

[0092] Scanning electron microscopy (SEM) characterizations (Fig. 2A) showed that the hydrogels had a porous structure, which is critical for cell growth and metabolism (Lien et al., 2009, Acta Biomaterialia, 5, 670). Based on SEM observations, the porosities of GelMA 5%, EWMA 5%, and EWMA 7.5% were found to be similar, whereas EWMA 10% exhibited a significantly lower level of porosity compared to these samples. Mechanical robustness is one of the critical factors to consider for the design of bio-scaffolds to support the dynamic motion of tissues, especially at load-bearing sites (Peppas, 2020, Biomaterials Science, Elsevier, 153). The mechanical characteristic of hydrogels was evaluated using compression testing. The compressive modulus for the EWMA hydrogels (Fig. 2B) increased with the increase of EWMA concentration due to the increase in crosslinking density on the samples. The compressive modulus for EWMA 5% (112±9.9 kPa) and 7.5% ( 136± 5.9 kPa) was comparable to that of the control, GelMA 5% (133+7.3 kPa); however, EWMA 10% (166+13.7 kPa) showed the highest compressive modulus. Similarly, the failure strength (Fig. 2C) of the EWMA hydrogels raised with the increase in EWMA concentration. Also, the failure strength for EWMA 7.5% (99±6.5 kPa) and 10% (178+5 kPa) was higher than that for GelMA 5%; however, EWMA 5% (29+2.9 kPa) showed a significantly lower strength.

[0093] While compressive strength is vital for designing mechanically sturdy scaffolds, it's equally important to consider factors such as porosity and stiffness. These elements can significantly impact nutrient transport to the enclosed cells and influence bioactivity (Mohaghegh et al., 2023, Acta Biomaterialia; Akbari et al., 2014, Advanced Functional Materials, 24, 4060). As discussed previously, EWMA 7.5% showed comparable stiffness and porosity to the GelMA 5%, while its mechanical strength was significantly higher. Therefore,among different concentrations of the EWMA hydrogel, EWMA 7.5% was chosen to study the endothelialization efficacy of the hydrogels.

[0094] The swelling of hydrogels post-implantation can lead to tissue compression and negatively affect their mechanical characteristics (Jiang et al., 2019, Biomaterials Science, 7, 1805; Spicer, 2020, Polymer Chemistry, 11, 184; Peppas et al., 2006, Advanced Materials, 18, 1345). Hydrogels with lower swelling ratios have shown higher mechanical robustness and a more stable ECM microenvironment to maintain the microstructural remodeling of tissues after implantation (Sun et al., 2017, Journal of Materials Chemistry B, 5, 8060). The swelling ratio of the hydrogels is shown in Fig. 2D. Overall, the EWMA hydrogels at all concentrations showed lower swelling ratios than GelMA hydrogels. The swelling ratios of the hydrogels incubated for 10 hr were 5.7, 3.9, and 4.4 for EWMA 5%, 7.5%, and 10%, respectively, which were significantly less than the swelling ratio of GelMA 5% (7.9). These findings suggest that EWMA offers a physically stable hydrogel, providing a dependable platform for studying cellular behaviors. It maintains its structural integrity without significant changes during the incubation and culture period (Brandl et al., 2007, Biomaterials, 28, 134; Nichol et al., 2010, Biomaterials, 31, 5536; Temenoff et al., 2003, Journal of Biomaterials Science, Polymer Edition, 14, 989). Besides, hydrogels should experience controlled degradation following implantation to facilitate tissue regeneration and in vivo remodeling. Nonetheless, a rapid degradation rate prior to complete tissue regeneration can lead to suboptimal mechanical performance (Sun et al., 2017, Journal of Materials Chemistry B, 5, 8060). GelMA 5% completely degraded in a collagenase type II solution only in 2 days, while the weight loss for EWMA hydrogels was less than 20% even after incubation for 10 days (Fig. 2E). Therefore, EWMA hydrogels with a moderately lower degradation rate than GelMA can provide a more robust hydrogel matrix for tissue regeneration in vivo (Chiu et al., 2013, PloS One, 8, e60728).Rheological characterization of EWMA hydrogels

[0095] The storage and loss moduli are presented against oscillatory shear strain and angular frequency for crosslinked EWMA and GelMA hydrogels (Fig. 3). It's worth noting that the storage modulus (G') consistently surpassed the loss modulus (G") across all samples, as depicted in Fig. 3 A and Fig. 3B, owing to their crosslinked nature (Massensini et al., 2015, Acta Biomaterialia, 27, 116; Pan et al., 2019, Gels, 4, 43; Leone et al., 2010, Journal of Materials Science: Materials in Medicine, 21, 2491). The storage and loss moduli offer valuable insightsinto the mechanical behavior and properties of viscoelastic materials. Typically measured as a function of angular frequency, these moduli's behavior at different frequencies provides crucial information about the material's response under varying deformation rates. Fig. 3C and Fig. 3D display the relationship between storage and loss moduli and angular frequency. GelMA 5% and EWMA 5% showed comparable storage modulus (at angular frequency=l rad s ' and strain=0.1%) as well as loss modulus; however, the moduli increased with the increase in EWMA concentration due to the increase in vinyl contents and crosslinking density on the hydrogels (Fig. 3E and Fig. 3F). To illustrate, the storage modulus of EWMA 10% reached a substantial 6570 Pa, which was notably 14 times higher than that of EWMA 5% at 466 Pa. Likewise, the loss modulus of EWMA 10% measured 1480 Pa, surpassing that of GelMA 5% at 64 Pa by a factor of 23.Cytocompatibility with fibroblasts

[0096] The use of synthetic hydrogels for tissue engineering applications has been limited by their poor bioactivity and biodegradation properties (Tsou et al., 2016, Bioactive Materials, 1, 39; ELSherbiny et al., 2013, Global Cardiology Science and Practice, 2013, 38; Deiber et al., 2009, Polymer, 50, 6065). EWMA hydrogels exhibit high bioactivity due to important proteins such as ovalbumin, cystatin, and avidin (Li et al., 2014, Scientific Reports, 4, 5600). Cystatin mimics the ECM composition to facilitate cell proliferation and growth. Ovalbumin is the main protein found in EW, playing a crucial role in angiogenesis and cell growth (Luo et al ., 2015, Journal of Biomaterials Applications, 29, 903). Avidin is a tetrameric glycoprotein and acts as an antibacterial agent in EW. Avidin also plays a key role in supporting 3D cell growth and proliferation due to containing the Arg-Tyr-Asp-Ser (RYDS) sequence, which mimics the function of Arg-Gly-Asp-Ser (RGDS) sequence in regulating cell adhesion and attachment. The cytocompatibility of the hydrogels was evaluated using HDFs in a 3D culture. The HDFs were encapsulated in the EWMA (5%, 7.5%, and 10% w / v) and GelMA (5% w / v) hydrogels, and the cell viability and proliferation were assessed using Calcein- AM / Ethidium Homodimer (Live / Dead) assay, PrestoBlue assay, and Phalloidin / DAPI staining. As is evident from the Live / Dead fluorescence images, the HDFs were successfully encapsulated within the hydrogels. Also, DAPI / Phalloidin fluorescence images showed a homogenous distribution of cells in all samples. However, the cell viability was significantly higher for the EWMA hydrogels compared to GelMA. For example, the cell viability was 99% for EWMA7.5% while it was 88% for GelMA 5%. The metabolic activity results also confirmed that HDFs grew well in all of the samples; however, the cell growth and proliferation for EWMA 5% and 7.5% was significantly higher than for the control, GelMA 5%, over the entire incubation period. The metabolic activity for EWMA 7.5% on days 1, 3, and 7 of culture was 4, 3, and 1.5 times higher than those for GelMA 5% w / v. However, with the increase of EWMA concentration to 10% w / v, the cell activity was reduced. This decreased viability can be attributed to the increase in stiffness and less nutrition transfer at higher polymer concentrations. Results showed that EWMA 7.5% and GelMA 5% had comparable stiffnesses and porosities (Fig. 3) needed for nutrient transfer and cell proliferation; therefore, a higher cell activity observed on EWMA 7.5% compared to GelMA 5% can be attributed to the EWMA hydrogel being intrinsically more bioactive than the GelMA hydrogel.3D printing of constructs using EWMA as a bioink

[0097] Fig. 4 represents the 3D printing endeavor using the Digital Light Processing (DLP) stereolithographic (SLA) technique for EWMA constructs. These images provide a comprehensive view of the printed Y-shaped perusable hydrogel construct from various isometric and top angles (Fig. 4A, Fig. 4B, Fig. 4C) with a detailed close-up of the bifurcation (Fig. 4D). Additionally, Fig 4E presents a close-up view that highlights the printed layers and the staircase effect observed in the channel walls. The successful perfusion of red and blue dye through the bifurcation illustrates the construct's perfusability and possibility of printing various structures using EWMA as a bioinks (Fig. 4F). The 3D design of the Y-shaped bifurcation is displayed in Fig. 4G, while Fig. 4H demonstrates a gyroid print with eight-unit cells across and a 500 pm wall thickness. Lastly, Fig. 41 and Fig. 4J offer a visual representation of the gyroid's thin members. This collectively highlights the precision and complexity achievable through light-based additive manufacturing in creating intricate and functional constructs using the SLA- optimized EWMA bioink formulation. The results suggest that EWMA holds promise for use in various printing applications and precision bio-printing methodologies, showcasing its versatility and potential.

[0098] Furthermore, an investigation into the printability of EWMA using an extruderbased 3D printer (BioX cell) was conducted. The findings clearly demonstrated the ease with which EWMA can be printed using this extruder printer. Moreover, the results indicated that, with EWMA, it is feasible to achieve multicolor printing, enabling the representation of two oreven more distinct cell types printing within a single construct. The process of printing EWMA utilizing the BIOX cell is visually detailed in the supplementary video. In conclusion, the findings underscore the potential of EWMA in a wide array of printing applications and bioprinting techniques, highlighting its versatility and promise.Endothelialization and endothelial cell activity

[0099] Matrigel has been widely used as a matrix for angiogenesis assays due to its ability to form 3D vascular networks. However, the angiogenic activity of Matrigel strongly depends on the composition of its growth factors, resulting in low-reproducibility assays due to variation in Matrigel composition (Kleinman, 2005, Seminars in Cancer Biology; Hughes et al., 2010, Proteomics, 10, 1886). Similar to Matrigel, the EW hydrogels have shown the ability to form vessel-like endothelial networks. Therefore, the methacryloyl modification of EW can provide a photocrosslinkable and naturally derived hydrogel with the ability to form a highly biocompatible and endothelial cell-friendly ECM for angiogenesis in tissue engineering and drug testing.

[0100] Fig. 5 shows the cytocompatibility of hydrogels seeded with endothelial cells (2D culture). Fluorescence images reveal successful adhesion and growth of HUVECs on the surface of the EW hydrogels (Fig. 5 A). EWMA 7.5% was thoroughly covered with a layer of HUVECs on day 7 of culture. Besides, the PrestoBlue™ assay (Fig. 5B) showed that the metabolic activity of HUVECs on day 7 of culture was significantly higher for EWMA hydrogels than for the GelMA hydrogel. The activity of endothelial cells on the surface of EWMA 7.5% was further confirmed by VE-cadherin / DAPI immunostaining of Green fluorescent protein- human umbilical vein endothelial cells (GFP-HUVECs) (Fig. 5C). VE-cadherin is an endothelial-specific adhesion molecule located at junctions between the endothelial cells. VE-cadherin plays a critical role in blood vessel formation and angiogenesis by regulating various cellular processes, such as cell proliferation and apoptosis, and modulating vascular endothelial growth factor (VEGF) functions (Vestweber, 2008, Arteriosclerosis, thrombosis, and vascular biology, 28, 223). The expression of VE-cadherin (red) on EWMA7.5% confirmed the formation of adherents junctions between endothelial cells on the surface of this sample.

[0101] The endothelialization of EWMA 7.5%, GelMA 5% and Matrigel was evaluated using GFP-HUVECs seeded on the surface of the hydrogels (Fig. 6). For the endothelialization assay, the samples were placed in the incubator while shaken on a plate shaker to trigger celldivision and growth (Stoker, 1974, Cell, 3, 207). Among different concentrations of EWMA hydrogels, EWMA 7.5% was chosen for the endothelialization assay due to its porosity and stiffness being comparable to those of the control sample, GelMA 5%. The cell spreading on day 4 of culture was 93% on EWMA 7.5%, while it was 60% and 22% on GelMA 5% and Matrigel, respectively. These results indicated higher endothelial cell activity on the EWMA hydrogel surface compared to other naturally derived hydrogels, such as GelMA and Matrigel, which can be attributed to the presence of cell-binding motifs and integrin receptors in EW. For example, the presence of cystatin in EW acts as a TGF-P receptor and regulates integrins, which are the main receptors for ECM. Rapid endothelialization of EWMA hydrogels can improve its biocompatibility and reduce the failure rate of vascular implants, demonstrating the high potential of EWMA as ECMs in vascular tissue engineering and angiogenesis testing.

[0102] Matrigel is an extract of proteins and biomolecules derived from mouse tumors, and its vascularization property is attributed to a carcinogenic nature and components rich in VEGF and ECM proteins, such as laminin, closely mimicking the complex environment of the basement membrane (Kubota et al., 1988, Journal of Cell Biology, 107, 1589; Mousa et al., 2017, Anti-Angiogenesis Strategies in Cancer Therapeutics, Academic Press, 21). In an earlier study by Mousseau et al., it was shown that similar to Matrigel, the “thermally” crosslinked EW can also support the formation of vascular networks. The vascularization of EW has been attributed to its micropatterns and functional proteins, such as cystatin and avidin. These proteins contain major integrin receptors and Arg-Gly-Asp (RGD)-mimi eking amino acid domains that can play a critical role in cell adhesion and organization. However, the methacrylation of EW resulted in vinyl groups occupying the cell binding sites on EW, thus altering the endothelial cell spreading pattern and vascularization of EWMA hydrogel. Furthermore, the photocrosslinking process may have led to variations in the stiffness of the EW gel compared to thermally crosslinked EW, potentially altering the local fiber structures and microenvironments essential for cell growth and the development of vascular networks. The critical role of matrix stiffness on the growth of endothelial cells and vascularization is also reported by other researchers (LaV alley, 2014, Advances in Regenerative Biology, 1, 25247; Crosby, 2019, Regenerative Biomaterials, 6, 61). Zhao et al. reported that the substrate stiffness can increase the migration and angiogenesis potential of HUVECs (Zhao et al., 2018, Journal of Cellular Physiology, 233, 3407). Substrate stiffness can also influence the morphology, cytoskeletal structure, and adhesionof endothelial cells. Cells generate large forces on the stiff substrates using actin-myosin complexes, which are part of the cytoskeleton, to form the mature focal adhesion. In contrast, the soft substrates cannot provide sufficient resistance to counterbalance large, cell-generated forces. Thus, cells cannot provide abundant stress fibers, adhere to them, and grow well on soft surfaces. Also, stiff substrates can support cell spreading, whereas soft substrates induce rounded cell morphologies. Enhancing tissue regeneration can be achieved by optimizing scaffold stiffness at the surface. Therefore, the stiffness of scaffolds is an important factor in tissue engineering, which can affect the differentiation, proliferation, and migration of cells on the surface of scaffolds and regulate angiogenesis and vascularization processes (Zhao et al., 2018, Journal of Cellular Physiology, 233, 3407).Cytocompatibility study of EWMA using myoblast cell line

[0103] The cytocompatibility of the hydrogels was evaluated using C2C12 cells in 3D culture (Fig. 7). The C2C12 cells were encapsulated in the EWMA (5%, 7.5%, and 10% w / v) and GelMA (5% w / v) hydrogels, and cell differentiation was assessed by immunofluorescent staining. As evident from the images (Fig. 7A), the C2C12 cells were successfully encapsulated within the hydrogels. Also, DAPI / Phalloidin fluorescence images showed a homogenous distribution of cells in all samples. However, the cells encapsulated in GelMA and EWMA showed different levels of differentiation after 7 days of culture. The myotube coverage area was comparable between GelMA 5% and EWMA 5% at 12.3% ± 1.9 and 11.5% ± 1.4, respectively. However, the cells encapsulated in EWMA 7.5% showed a significant increase in their myotube coverage area at 32.3% ± 3.2. In contrast, increasing EWMA concentration to 10% did not favor cell differentiation and showed the lowest myotube coverage area at only 4.8% ± 1.3 (Fig. 7B). The nuclei distribution per myotube quantification data (Fig. 7C) also confirmed that C2C12 cells fused more efficiently and formed larger myotubes in EWMA 7.5% compared to other hydrogels. While both GelMA 5% and EWMA 5% showed comparable levels of cell fusion, increasing EWMA concentration to 10% drastically limited the ability of the cells to fuse. This decreased cell fusion and differentiation can be attributed to the increase in stiffness and less nutrition transfer at higher polymer concentrations (Truong et al., 2019, Acta Biomaterialia, 94, 160; Stowers et al., 2015, Proceedings of the National Academy of Sciences, 112, 1953; Zigon- Branc et al., 2019, Tissue Engineering Part A, 25, 1369). Results showed that EWMA 7.5% and GelMA 5% had comparable stiffnesses and porosities (Fig. 2) needed for nutrient transfer andcell proliferation; therefore, the higher cell activity observed on EWMA 7.5% in comparison to GelMA can be attributed to the EWMA hydrogel being intrinsically more bioactive than the GelMA hydrogel.Assessing in vivo biocompatibility of EWMA

[0104] To assess the biocompatibility of EWMA hydrogels post-implantation, in vivo investigations were conducted involving the subcutaneous implantation of both GelMA and EWMA hydrogel scaffolds. Detailed morphological examination of the skin through H&E staining (Fig. 8A and Fig. 8B) revealed the absence of inflammation or toxicity associated with GelMA or EWMA hydrogels. Remarkably, after 30 days, enhanced vascularization was observed in the EWMA group compared to GelMA, evident by the presence of blood vessels (indicated by red color) in hematoxylin and Eosin (H&E) staining. In contrast, GelMA samples showed evidence of fibrosis, characterized by the influx of immune cells to the implant site over the same duration, further underscoring EWMA's enhanced potential to stimulate vascularization and cell proliferation.

[0105] For the subcutaneous implantation samples, immunohistochemical staining was conducted. F4 / 80 is a well-established marker used to identify mouse macrophage populations, particularly CD80 associated with pro-inflammatory subtypes (Najafabadi et al., 2021, Journal of Controlled Release, 337, 168; Zehtabi et al., 2023, Macromolecular Bioscience, 23, 2200333). Detection of the CD80 marker can reveal inflammation in the implanted region. Fig. 8C displays immunohistochemical staining of the full-thickness skin near the subcutaneous implantation site. F4 / 80 (red) and CD80 (green) markers were used in conjunction with a nuclear stain (blue). Interestingly, minimal CD80 pro-inflammatory marker staining was observed in all samples, indicating the absence of inflammation associated with EWMA implantation. In summary, the immunohistochemical analysis indicated no sign of inflammation or activation of the immune system, which indicated that EWMA can serve as promising photocrosslinkable hydrogel. Furthermore, evaluation of vital organs, including the heart, kidney, liver, and lungs, alongside skin tissues, demonstrated no significant indications of adverse immune responses or toxicity. These findings, as illustrated in Fig. 8D, emphasize the promising biocompatibility of EWMA hydrogels, positioning them as potential candidates for diverse biomedical applications, particularly in tissue engineering and regenerative medicine.

[0106] The experimental materials and methods are now described herein.Materials

[0107] The EW used in this work was extracted from commercially available eggs. The methacrylic anhydride (MA, purity 94%), dimethyl sulfoxide (DMSO, purity > 99%), Triton X- 100, and 2-hydroxy-4’-(2-hydroxyethyl)-2-methyl propiophenone (purity > 98%) were obtained from Sigma Aldrich. Growth factor reduced Matrigel was purchased from BD Biosciences, San Jose, CA, USA. Collagenase type II was obtained from Thermo Fisher Scientific. Dulbecco’s phosphate-buffered saline (DPBS), trypsin-ethylenediaminetetraacetic acid (EDTA), Dulbecco’s Modified Eagle Medium (DMEM), and penicillin / streptomycin (Pen / Strep) were purchased from Gibco, NY, USA. Fetal bovine serum (FBS) and bovine serum albumin (BSA) were supplied from Sigma-Aldrich. Endothelial cell culture system (EGM™-2 Endothelial Cell Growth Medium-2 BulletKit™) containing endothelial basal medium (EBM) and endothelial growth factors (EGFs) were obtained from Lonza, Basel, Switzerland. Vascular endothelial-cadherin (VE-cadherin, 7B4 or cadherin-5, GT 15250) antibody and AlphaBioCoat Solution (AC001) were obtained from Neuromics (a division of CA3 Biosciences, Inc). A highly cross-adsorbed Donkey anti-Goat IgG (H+L) secondary antibody was purchased from Biotium. PrestoBlue, Live / Dead cell viability / cytotoxicity kit, anti mouse CD80, anti mouse F4 / 80, and DAPI / phalloidin staining were manufactured by Invitrogen and purchased from Fisher Scientific, USA. Paraformaldehyde in 0.1 M phosphate buffer was obtained from Bioenno Tech, USA. All reagents were used without further purification.EWMA synthesis

[0108] EW was collected from fresh eggs and filtered two times through a mesh net (50- 100 pm) to remove impurities (the EW collected from a large batch of eggs to ensure the reproducibility and consistency of the product, n>100). To prepare a homogenized and clear EW solution, the EW was diluted with equal water weight and vigorously stirred for 1 hr at 4 °C. The solution was subsequently centrifuged at 5000 rpm for 20 min, and the supernatant was collected and freeze-dried (Free zone freeze dryer, Labconco, MO, USA) before the methacryloyl modification. EWMA was synthesized in DPBS using a one-pot synthesis method. Firstly, 4.5 g of EW was dissolved in 112.5 ml of DPBS (pH = 7.4) and mixed for 1 hr at 4 °C to obtain 4% w / v EW solution (Ball Jr., 1982, Poultry Science, 61, 1041). The solution was then centrifuged at 3500 rpm for 5 min, and the supernatant was used to synthesize EWMA. To prepare medium EWMA (substitution degree of -40%), 1.8 ml of MA was added to the reaction mixturedropwise, followed by continuous stirring overnight in an ice-covered container. The pH of the reaction solution was regularly monitored and kept between 8 and 9. The monitoring of pH was crucial for the methacryloyl modification reaction since the acidic by-product of the reaction (methacrylic acid) could decrease the pH of the solution, thus inhibiting the forward reaction by protonating free amino groups of EW. After the overnight reaction, the reaction mixture was centrifuged at 5000 rpm for 15 minutes to remove air bubbles. The solution was then dialyzed against deionized (DI) water using a dialysis membrane of 12-14 kDa at 4 °C to remove the unreacted MA and the by-products. Finally, the EWMA solution was lyophilized in the freeze- dryer and stored at 4°C for future use. EWMA molecules at 20% and 80% substitution degrees were also synthesized by adding 0.9 and 3.6 ml of MA, respectively. However, the 40% substitution degree was selected since the 20% degree did not provide adequate crosslinking densities, and the 80% degree performed the same as the 40% degree. In all experiments, GelMA (degree of substitution 63.8%, prepared as previously described (Tamayol et al., 2015, Advanced Healthcare Materials, 4, 2146) with a concentration of 5% w / v was used as the control sample. EWMA with the concentrations of 5% w / v, 7.5% w / v, and 10% w / v were named EWMA 5% EWMA 7.5%, EWMA 10%, respectively.Chemical characterization of EWMA

[0109] 1H NMR spectroscopy was used to determine the degree of methacrylation ofEWMA following the same method used for methacrylated gelatin (GelMA) (Sun et al., 2018, Polymers, 10, 1290). To prepare the samples for NMR analysis, EW and EWMA were dissolved in DMSO at room temperature at a concentration of 2% w / v. The primary amino groups of EW proteins, originating from the amino acid lysine, interact with methacryloyl groups. As a result, these amino groups are critical for calculating the substitution degree of EWMA. The peak area for primary amino groups around 8= 2.9 was measured for EW before and after methacloyl modication. The DS was calculated based on Equation 1 (Hoch et al., 2013, Journal of Materials ChemistryEWMA hydrogel preparation

[0110] EWMA pregel solutions at concentrations of 5, 7.5, and 10% w / v were prepared by dissolving 50, 75, and 100 mg of EWMA in DI water, respectively. Also, a widely used naturally derived biopolymer, GelMA, was prepared as a control at a concentration of 5% w / v.Subsequently, an ultraviolet (UV) PI, 2-hydroxy-4’-(2-hydroxyethyl)-2-methyl propiophenone (5 mgml '), was added to the pregel solutions. The hydrogels were formed by photocrosslinking the pregel solutions with 356 nm UV light at an intensity of 800 mW for 1 min (distance from the light source: 8 cm).Crosslinking kinetics of EWMA hydrogels

[0111] The crosslinking kinetics of the hydrogels were evaluated by real-time monitoring of variations in the storage (G1) and loss modulus (G") under UV light using an MCR 302 rheometer (Anton Paar, Graz, Austria). Rheological measurements in the oscillatory mode were performed on 100 pl of the prepolymers using a sandblasted measuring plate (PP08 / S, diameter= 8 mm). Oscillatory displacements with 1% strain and 1 Hz frequency were applied to the hydrogels, and UV irradiation was commenced after a 1 min stabilization period. The modulus versus UV irradiation time (~ 4 min) was recorded until an entire plateau was achieved. The gelation point was assigned to the time when the storage modulus surpassed the loss modulus for each measurement.Morphological characterizations

[0112] SEM analysis was carried out to analyze the physical structure and porosity of the hydrogels. The crosslinked hydrogels were lyophilized in a freeze-dryer. The lyophilized samples were then coated with iridium using an ion beam sputter deposition and etching system (IBS / e, South Bay Technology, CA, USA). SEM images were captured on a Supra 40 VP, Zeiss microscope, Germany, at an accelerating voltage of 2 kV.Swelling test

[0113] To evaluate the swelling ratio of EWMA hydrogels, disc-shaped hydrogel samples (diameter=8 mm, height=2 mm) were crosslinked, and samples were weighed (Wo). The discs were then immersed in DPBS and incubated at 37 °C. The hydrogel discs were removed from DPBS at different time points (0, 1, 2, 4, 6, 8, 10, 24, and 48 hr), the excess DPBS was removed using Kimwipes, and the samples were weighed again (Wt). The increase in the volume of the samples was directly related to the amount of DPBS absorbed, and the swelling ratio was calculated based on Equation 2 (Rastegar et al., 2021, Carbohydrate Polymers, 269, 118351). Each condition was tested with four replicates, and the results were subsequently averaged.Swelling ratio =Wt W° % (2)WQDegradation test

[0114] To evaluate the in vitro degradation of EWMA hydrogels, disc-shaped samples were prepared following the method explained in the previous section. The samples were then immersed in DPBS containing collagenase type II (2.5 U / ml) and incubated at 37 °C. At desired incubation times (1, 2, and 10 days), the samples were removed from DPBS, freeze-dried, and weighted. The weight loss percentages of the samples were calculated based on Equation 3, where Wi and Wf are the initial and final weights of samples after immersion in DPBS, respectively. Four replicates for each condition were conducted and then average results were calculated.Weight loss 100 (3)Compression test

[0115] To prepare samples for compression testing, 80 pl of EWMA pregel solutions were transferred into cylindrical poly(dimethylsiloxane) (PDMS) molds (height = 2 mm, diameter = 8 mm) and crosslinked them. Subsequently, the hydrogels were soaked in DPBS for 1 hour at room temperature prior to measurements. The compression properties of the samples were evaluated using an Instron 5542 machine (Norwood, MA, USA) with a strain rate of 1 mm / min. The compressive modulus was determined from the slope of the linear region of stressstrain curves, up to a strain of 0.1 mm / mm. The compressive strength was defined as the maximum stress at the point of failure. Four replicates for each condition were conducted and then average results were calculated.Rheological characterizations

[0116] To assess the rheological behavior of the hydrogels, disc-shaped samples were prepared using the same crosslinking method employed for compression testing. Afterward, these samples were allowed to equilibrate in DPBS for 1 hour at room temperature. The rheological properties of the hydrogels were examined using an MCR 302 rheometer equipped with a sandblasted measuring plate (PP08 / S) having a diameter of 8 mm. Oscillatory shear strain tests, ranging from 0.01% to 100%, and frequency sweeps covering an angular frequency range of 0.1 to 100 rad / s at a fixed strain of 0.1%, were conducted on the hydrogels at approximately 25 °C. Storage modulus (G') and loss modulus (G") were determined as functions of shear strain and angular frequency, with G and G" values reported at an angular frequency of 1 rad / s and a strain of 0.1%. Five replicates for each condition were conducted and the average values were subsequently reported.3D bioprinting using EW A

[0117] A digital micro-mirror (DMD) based light modulator was used in conjunction with a 385nm LED to produce an image with an effective pixel size of 27 pm at the focal plane. The print layer height for this SLA setup was 50 pm with an exposure time of lOs / layer and an intensity of 28 mW cm2with a 95%< uniformity cross the print plane. The dark time between layers was set to 0.5 s to eliminate dark polymerization induced pattern warping. The designed Y-shaped bifurcation had a wall thickness of 200 pm (~7 pixels across) and an internal diameter of 1.5 mm and was printed horizontally with layers parallel to the channel direction. This was especially challenging considering the low stiffness and high compliance of EWMA hydrogel. The PI Lithium phenyl (2,4,6-trimethylbenzoyl) (LAP) was used at a concentration of 1% w / v, and the photo-absorber tartrazine was used at a concentration of 0.3% w / v to achieve the optimal exposure profiles for the given optical configuration.

[0118] Furthermore, EWMA printing was conducted using the extruder perimeter of the Bio X printer. Briefly, freeze-dried EWMA was initially dissolved in DI water, achieving a concentration of 7.5%. Subsequently, this EWMA solution was mixed with water-based coloring agents, either green or pink, creating distinct colors for visualization. The prepared mixture was then loaded into a 3 ml syringe, which was subsequently affixed to the printer. The printing procedure was executed in accordance with the manufacturer's recommended settings, involving specific conditions such as a temperature of 17 °C, a pressure range of 90-100 pa, a printing speed of 9 mm S'2, and an adjusted bed temperature of 7 °C. These controlled parameters ensured the successful and precise printing of EWMA-based structures, offering versatility in color representation and material properties for potential applications in tissue engineering and beyond.The cytocompatibility of EWMA hydrogelsEndothelialization and cytocompatibility study of EWMA hydrogels using a 2D endothelial cell cultureFibroblast cell culture and encapsulation in the hydrogels

[0119] Cytocompatibility of the EWMA hydrogels was investigated using human dermal fibroblasts (HDFs) encapsulated in the hydrogels (3D culture). HDFs were obtained from American Type Culture Collection (ATCC, CRL-2522, Manassas, VA, USA). HDFs were cultured in a T175 Coming flask using DMEM supplemented with 10% FBS and 1% Pen / Strepin an incubator at 5% CO2 and 37 °C. At confluency of 80%, cells were enzymatically detached from the flask using trypsin-EDTA (0.25%) and were collected by centrifugation at 1200 rpm for 5 min. GelMA (5% w / v) and EWMA (5, 7.5, and 10% w / v) were dissolved in 100 pL of the PI solution (5 mg / mL), and HDFs at a concentration of 2x106 cells / mL were suspended in the pregel solutions. The mixtures were gently mixed and transferred to 24-well plates. The samples were then crosslinked using a 356 nm UV light at an intensity of 800 mW for 1 min (distance from the light source: 8 cm) and incubated in the incubator at 5% CO2 and 37 °C to be tested for fibroblast cell activity.Fibroblast cell viability

[0120] The well plates were removed from the incubator on day 7 of culture, and the cellladen hydrogels were washed three times with DPBS. 0.5 pL of calcein and 2 pL of ethidium homodimer were dissolved in 1 mL of DPBS as a stock solution, and 500 pL of the solution was added to each well. After incubation for 30 min at 37 °C, the dye solution was removed, and the samples were washed three times with DPBS. Finally, a fresh DPBS was added to the samples, and live (stained with calcein-AM in green) and dead (stained with ethidium homodimer in red) cells were visualized using an inverted fluorescence microscope (Zeiss Axio Observer 5, Walpole, MA). Image J 150. i software was used to count the live and dead cells. Cell viability was determined as the ratio of living cells to the total number of cells.Fibroblast metabolic activity

[0121] The metabolic activity of the cell-laden hydrogels was evaluated on days 1, 3, and 7 of culture using PrestoBlue assay. The PrestoBlue reagent was dissolved in the medium at a ratio of 1 :9, and 1 mL of the PrestoBlue solution was added to the samples, followed by incubation at 37 °C for 2 hr. As the cells reacted to the PrestoBlue reagent, the color of the media changed from violet to pink. Subsequently, 100 pL of media was transferred into the 96-well plates and the fluorescence intensity resulting from cell activity was measured at the excitation / emission wavelengths of 530 / 570 nm using a microplate reader (Synergy™ HTX Multi-Mode Microplate Reader, BioTek Winooski, Vermont, USA).DAP I and phalloidin staining

[0122] The cell-laden hydrogels were removed from the incubator on day 7 of culture and washed three times with DPBS. Samples were then treated with 4% v / v paraformaldehyde for 20 min at room temperature and washed again with DPBS. The samples were permeabilizedusing 0.2% v / v Triton X-100 for 20 min and washed three times with DPBS. 2% v / v BSA solution was added to the samples and incubated for 1 hr at room temperature. Subsequently, the samples were immersed in a staining solution containing 1 mb DPBS, 1 pL DAPI, and 4 pL phalloidin for 30 min at 37 °C and finally washed 3 times with DPBS. Actin and nuclei on the samples were stained with phalloidin and DAPI in red and blue, respectively, and visualized using the inverted fluorescence microscope.Endothelial cell culture and seeding on the hydrogels

[0123] Green fluorescent protein- human umbilical vein endothelial cells (GFP- HUVECs, Neuromics) were cultured in EBM supplemented with BulletKitEGFs and 1% v / v Pen / Strep in a 5% CO2 incubator at 37 °C. GFP-HUVECs were cultured in a T25 Corning cell culture flask pre-treated with AlphaBioCoat solution. At the confluence of -80%, cells were enzymatically detached from the flask using 0.25% trypsin-EDTA and collected by centrifugation at 1200 rpm for 5 min. Cells were resuspended in the media and counted using a hemocytometer (JuLI™ Br Live Cell Analyzer). Then, 100 pl of media containing 7.5 xlO5of GFP-HUVECs was transferred to the surface of samples.Endothelial cell activity and immunostaining

[0124] GFP-HUVECs seeded on the surface of the hydrogels were imaged over 7 days using a fluorescence microscope. PrestoBlue assay was performed following the method described previously to measure the metabolic activity of GFP-HUVEC cells seeded on the surface of the hydrogels on days 1, 3, and 7 of culture. VE-cadherin / DAPI immunostaining was performed to observe the endothelial cell-to-cell junctions on the surface of the EWMA 7.5% hydrogel following protocol (Cohen-Kaplan et al., 2020, Frontiers in Oncology, 10, 2). Briefly, the hydrogels were removed from the incubator on day 7 of culture, washed with DPBS, and fixed with 4% v / v paraformaldehyde for 20 min at room temperature. Paraformaldehyde was washed with DPBS, and samples were kept at 4 °C overnight. The cells were then permeabilized using 0.2% v / v Triton X-100 for 30 min and washed twice with DPBS. The samples were blocked with 2% v / v BSA for 30 min at room temperature, followed by treatment with 10 pgmL1VE-cadherin antibodies for 3 hr at 37 °C. The samples were then washed three times with DPBS and treated with 5 pgmL1Donkey anti-Goat IgG (H+L) secondary antibody in DPBS for 45 min at room temperature and washed again three times with DPBS. Finally, the samples were treated with 2 pgmL1DAPI solution in DPBS for 10 min at 37 °C and washedwith DPBS before microscopy analysis. Fluorescence microscopy was performed on the samples using an inverted fluorescence microscope to observe VE-cadherin (stained in red) at the endothelial cell junctions.Endothelialization and vascularization assay

[0125] To study the endothelization and vascularization of the hydrogels, 200 pl of EWMA 7.5% and GelMA 5% pregel solutions were transferred to a 24-well plate and crosslinked. 200 pl of as-received Matrigel was also transferred to the well plate, and all samples were sterilized under UV light for 30 min. GelMA and Matrigel are shown to have high compatibility with endothelial cells (Khayat et al., 2017, Journal of Dental Research, 96, 192; Ponce, 2009, Angiogenesis Protocols, Springer, 183), and both were used as control samples in this experiment. To seed the cells, GFP-HUVECs were suspended in the culture medium, and 100 pl of the suspension containing 7.5 *105of GFP-HUVECs was transferred to the surface of the samples. The sample plate was then placed on a plate shaker (Thermo Scientific) at 145 rpm and incubated in 5% CO2 at 37 °C for 1, 2, 3, and 4 days. Cells on the samples were imaged at different incubation times using the fluorescence microscope and Image J 150. i software was used to estimate the cell spreading area.Cytocompatibility of the hydrogels in the 3D structure

[0126] The cytocompatibility of the EWMA hydrogels was investigated using mouse myoblast cells (C2C12s) encapsulated in the hydrogels (3D culture). C2C12s were obtained from the American Type Culture Collection (ATCC, CRL-2522, Manassas, VA, USA). C2C12s were cultured in a T175 Corning flask using DMEM supplemented with 10% FBS and 1% Pen / Strep in an incubator at 5% CO2 and 37 °C. At a confluency of 80%, cells were enzymatically detached from the flask using trypsin-EDTA (0.25%) and collected by centrifugation at 1200 rpm for 5 min. GelMA (5% w / v) and EWMA (5, 7.5, and 10% w / v) were dissolved in 100 pl of the PI solution (5 mg mF1), and C2C12s at a concentration of 2x 106cells mF1were suspended in the pregel solution. The mixtures were gently mixed and loaded into 3 ml syringe and printed using Bio x cell bioprinter. The samples were then crosslinked using a 356 nm UV light at an intensity of 800 mW for 1 min (distance from the light source: 8 cm) and incubated in the incubator at 5% CO2 and 37 °C to be tested for cell activity.Cell viability

[0127] The well plates were removed from the incubator on day 7 of culture, and the cellladen hydrogels were washed three times with DPBS. 0.5 pl of calcein and 2 pl of ethidium homodimer were dissolved in 1 ml of DPBS as a stock solution, and 500 pl of the solution was added to each well. After incubation for 30 min at 37 °C, the dye solution was removed, and the samples were washed three times with DPBS. Finally, fresh DPBS was added to the samples, and live (stained with calcein-AM in green) and dead (stained with ethidium homodimer in red) cells were visualized using an inverted fluorescence microscope (Zeiss Axio Observer 5, Walpole, MA). Image J software version 1.50i was employed to enumerate both live and dead cells. Cell viability was subsequently calculated as the ratio of living cells to the total cell count Metabolic activity

[0128] The metabolic activity of the cell-laden hydrogels was evaluated on days 1, 3, and 7 of culture using the PrestoBlue assay. The PrestoBlue reagent was dissolved in the medium at a ratio of 1 :9, and 1 ml of the PrestoBlue solution was added to the samples, followed by incubation at 37 °C for 2 hr. As the cells reacted to the PrestoBlue reagent, the media color changed from violet to pink. Subsequently, 100 pl of media was transferred into the 96-well plates, and the fluorescence intensity resulting from cell activity was measured at excitation / emission wavelengths of 530 / 590 nm using a microplate reader (Synergy™ HTX Multi-Mode Microplate Reader, BioTek Winooski, Vermont, USA).DAPI, phalloidin, and myosin heavy chain staining

[0129] The cell-laden hydrogels were removed from the incubator on day 7 of culture and washed three times with DPBS. Samples were then treated with 4% v / v paraformaldehyde for 20 min at room temperature and washed with DPBS. The samples were permeabilized using 0.2% v / v Triton X-100 for 20 min and washed three times with DPBS. 2% v / v BSA solution was added to the samples and incubated for 1 hr at room temperature. Subsequently, the samples were immersed in a staining solution containing DPBS (1 ml), DAPI (1 pl), Myosin heavy chain (5 pl), and phalloidin (4 pl) for 30 min at 37 °C and then washed 3 times with DPBS. Actin and nuclei on the samples were stained with phalloidin and DAPI, respectively, and visualized using an inverted fluorescence microscope.Evaluation of in vivo biocompatibility and histological analysis

[0130] To evaluate biocompatibility and potential toxicity of the EWMA hydrogel, scaffold implantations were performed using two 8 mm incisions in the dorsal region of mice.All animal procedures were conducted under the supervision of the IACUC committee of the Lundquist Institute (#22747). The tested scaffold variations included: 1) control (no injury), 2) 5% GelMA, and 3) 7.5% EWMA. These scaffold samples, shaped as 5 mm diameter and 1 mm height discs, were implanted into dorsal sections of each mouse. Following implantation, the incisions were sutured, and strict adherence to the approved IACUC protocol ensured thorough monitoring of the animals.

[0131] After 7 and 30 days of scaffold implantation, mice were euthanized via CO2 inhalation. The dorsal skin and organs were carefully excised and immediately submerged in a PBS solution. Skin sections containing the scaffolds were dissected, fixed in 10% formalin for a minimum of 48 hours, and subsequently embedded in paraffin. The specimens were then sectioned and subjected to Hematoxylin and Eosin staining or immunofluorescence, following the manufacturer's recommended procedures.Statistical analyses

[0132] All results were presented as mean values ± standard deviations. GraphPad Prism software was used for the statistical comparison of data. The differences between the data were determined by one-way and two-way ANOVA analyses followed by Tukey’s multiple comparison test. Statistical significances were presented as * (p< 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001).

Claims

CLAIMSWhat is claimed is:

1. A biomaterial comprising a cross-linked natural protein, wherein the natural protein is an egg white protein.

2. A composition comprising the biomaterial of claim 1, wherein the biomaterial is present in water at a concentration of between 1% and 20%.

3. The biomaterial of claim 1, wherein the egg white protein is selected from the group consisting of ovalbumin, cystatin, avidin, ovomucoid, ovotransferrin, ovomucin, ferritin, lysozyme, and combinations thereof.

4. The biomaterial of claim 1, wherein the cross-linked natural protein comprises cross-links between a cross-linkable functionality selected from the group consisting of olefin, acrylate, methacrylate, epoxide, vinyl, allyl, and combinations thereof.

5. The biomaterial of claim 1, wherein the cross-linked natural protein comprises cross-links between methacryloyl groups.

6. The biomaterial of claim 1, wherein the cross-linked natural protein has a degree of cross-linking of at least 10%.

7. The biomaterial of claim 1, wherein the biomaterial is porous.

8. The biomaterial of claim 1, wherein the biomaterial is a hydrogel having a swelling ratio between 2% and 7%.

9. The biomaterial of claim 1, wherein the biomaterial is biocompatible in vivo.

10. The biomaterial of claim 1, wherein the biomaterial is biocompatible with cells selected from the group consisting of fibroblasts and endothelial cells.

11. A method of endothelization comprising the step of exposing the biomaterial of claim 1 to endothelial cells.

12. A method of treating a subject in need thereof comprising the step of administering to the subject the biomaterial of claim 1.

13. A method of preparing a biomaterial comprising the steps of: providing a natural protein, wherein the natural protein is an egg white protein; tethering a cross-linkable functional group to the natural protein to produce a modified natural protein; and cross-linking the cross-linkable functional group, thereby producing a biomaterial.

14. The method of claim 13, wherein the natural protein is selected from the group consisting of ovalbumin, cystatin, avidin, ovomucoid, ovotran sferrin, ovomucin, ferritin, and lysozyme.

15. The method of claim 13, wherein the cross-linkable functional group is selected from the group consisting of olefin, acrylate, methacrylate, epoxide, vinyl, allyl, and carbonyl.

16. The method of claim 13, wherein the cross-linkable functional group is a methacrylate.

17. The method of claim 13, wherein the step of cross-linking the cross-linkable functional group comprises combining the modified natural protein with a photo-initiator; and exposing the modified natural protein to ultraviolet light.

18. The method of claim 13, wherein the step of cross-linking the cross-linkable functional group comprises combining the modified natural protein with a photo-initiator; and exposing the modified natural protein to ultraviolet light for a duration of between 1 second and 30 seconds.

19. The method of claim 13, further comprising the step of lyophilizing the biomaterial to produce a dried biomaterial.

20. A biomaterial synthesized using the method of claim 13.

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