Enhanced photocurability of 3D printed hydrogel objects

Abstract

The present disclosure provides reinforced hydrogel structures, methods for reinforcing hydrogel structures, and methods for treating ischemic injuries using the reinforced hydrogel structures.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 185,305, filed May 6, 2021, the entire contents of which are incorporated by reference herein. [Background technology]

[0002] Three-dimensional (3D) hydrogel structures are seeing increasing use in a variety of biomedical applications, however, the mechanical properties of hydrogel constructs can often pose problems related to the durability of such constructs.

[0003] In the past, practitioners have altered mechanical properties by forming a "skin" on the surface of a hydrogel structure in a process that is tedious, time-consuming, and difficult to reproduce. In addition, the "skin" is sometimes fragile itself. For example, Raghavan et al. used a complex methodology that employs concentric molds to create hollow cylindrical structures. The delicate material is difficult to remove from the mold, and in a second step, the surface of the tube is treated with an agent that causes cross-linking of polymer chains within the gel structure. The method makes it difficult to control the depth of this treatment.

[0004] Others have used additives to induce chemical reactions throughout the hydrogel that change its mechanical properties. This has several different drawbacks. One is that the beneficial properties of the soft hydrogel scaffold that interacts with cells are lost when the material properties are changed in this way. A second drawback of this approach is that an additional material type must be added to the system (which may have a negative effect on the cell response). This more complex implant also takes longer to gain regulatory approval. For example, Gaharwar was able to increase the stiffness of the hydrogel by 10 times and its toughness by 20 times through a process of adding small amounts of spherical magnetic nanoparticles to a collagen-based hydrogel. Others, such as Fukao (J. Mater. Chem. B, 2020,8, 5184-5188), have toughened the hydrogel by adding ceramic particles. This approach is only effective in limited applications where the presence of the ceramic is not detrimental to tissue healing. Summary of the Invention

[0005] Thus, there is a need in the art to improve the mechanical properties of hydrogel structures in a manner that is repeatable, does not create overly complex devices, and / or does not dramatically alter the beneficial properties of soft hydrogel scaffolds.

[0006] Some embodiments of the present disclosure are directed to a method of reinforcing a three-dimensional (3D) hydrogel structure, comprising contacting the structure with a mesh immersed in an uncured, light-curable bio-ink, and irradiating the mesh immersed in the uncured, light-curable bio-ink, thereby adhering it to the 3D hydrogel structure.

[0007] Some embodiments of the present disclosure are directed to a composition comprising a three-dimensional (3D) hydrogel structure and a layer comprising a mesh immersed in a photocurable or photocurable ink, wherein the layer comprising a mesh immersed in a photocurable or photocurable ink is in contact with the structure.

[0008] Some embodiments of the present disclosure are directed to a method of treating an ischemic disease in a subject in need thereof, comprising implanting a reinforced structure or composition disclosed herein. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows an exemplary graph showing mechanical properties (burst pressure, suture retention, and compressibility) of a tissue engineered vascular graft (TEVG) compared to a saphenous vein (SV). Burst pressure: 2134 mmHg=284 kPa; suture retention strength: 1.92 N; and compressibility: 25.6% / 100 mmHg. See Pashneh-Tala et al., Tissue Engineering: Part B, Volume 22, pg. 68, Number 1, 2016. [Figure 2-1] Experimental setup for deploying burst tests on hydrogel tubing: Figure 2A: Formlabs pass through tubing with plugs and flares; Figure 2B: Luer lock standard fitting with cable ties. [Figure 2-2] Experimental setup for developing burst tests on hydrogel tubes. Figure 2C: Fixation of Formlabs reinforced with self-adhesive bandages, steri-strips, and adhesive / paper (stick and lip tubes). Figure 2D: Reinforcement with tube adhesive and filter paper. [Figure 2-3] Experimental setup for developing burst tests on hydrogel tubes (Figure 2E): Technical drawing of the construct shown in Figures 2A, 2B, and 2C (Figure 2F): Tube fixed to the bottom of the container to maintain a horizontal configuration and eliminate bending of the tube caused by tube buoyancy. [Figure 2-4]Experimental setup for developing burst tests on hydrogel tubes. Figure 2G: Tube setup for removing adhesive application. Figure 2H: Tube with reinforced attachment ends. Figure 2I: Tube with reinforced attachment ends (R3=inner diameter and R6=outer diameter). [Figure 3-1] Figures 3A-3D show the suture pull test apparatus as described in Example 8. Figure 3A: Fixture for mounting membrane for suture pull test clamped in a vice with a U-shaped port for placement of suture (Figure 3B) within the membrane inserted into the fixture. Figure 3C: Soaked reinforced hydrogel. Figure 3D: Fixture with sandpaper support for gripping and generating force when performing suture pull test. [Figure 3-2] FIG. 3E shows the suture pull test apparatus as described in Example 8. FIG. 3E: Suture type, 3 / 8 circle, was used in the suture pull test. [Figure 4-1] 4A-4B show the porcine arterial connections discussed in Example 5. Figure 4A: Purse string suture technique. Figure 4B: Connector connected to native vessel via purse string suture as shown in Figure 4B. [Figure 4-2] Figure 4C: Porcine artery connection as discussed in Example 5. Figure 4C: Mesh over tubing that is light cured. Figure 4D: Mesh placed around tubing and light cured. Figure 4E: Mesh placed over tubing and light cured. Figure 4F: Jagged top edge may cut through light cured ink. [Diagram 5] Figure 5 shows Vicryl™ (Somerville, NJ) implantable graft reinforcement of Example 4. Figure 5A: Tubular hydrogel reinforced with mesh and sutured to a Gore-tex™ (Flagstaff, AZ) smooth-walled vascular graft. Figure 5B: Vicryl woven mesh-reinforced tube (left panel) and expanded mesh (right panel). Figure 5C: Vicryl knitted mesh-reinforced tube (left panel) and expanded mesh (right panel). [Figure 6]Figure 1 shows the setup for mesh-reinforced tubular suture tensioning experiments of Example 3. The tube is placed in a vice with the suture passing through the mesh at the end of the tube. [Figure 7] FIG. 1 shows graphical data representing the force required to pull for seven different suture pulling experiments (reinforced tubes (lines 5-7) and naked unreinforced tubes (lines 1-3)) from Example 3. The Y-axis shows the force applied to the tube and the X-axis shows the displacement distance affected by the pull. [Figure 8] Figure 1 shows the results of the tensile force experiment of Example 3 for mesh-reinforced tubes and tubes without mesh. The reinforced tubular hydrogels show an 8-fold increase in suture pull force compared to the unmodified tubular hydrogels. [Figure 9] FIG. 1 shows the ePTFE angiograph experimental apparatus of Example 4, where the ends were brought close together, sewn together using a running suture, pulled and tied securely using a square knot, union sealed using cyanoacrylate adhesive, and water + India ink was pumped through the tubing with no leaks observed. [Figure 10-1] Figure 10 shows the rabbit PA (pulmonary artery) experimental setup of Example 6. Figure 10A: Mesh-reinforced tube implanted in a pig. Figure 10B: Tube connected in vivo to native rabbit tissue. [Figure 10-2] Figure 10C: A loop of 3D printed graft material (indicated by arrow) coupled to the rabbit's PA. [Figure 11] FIG. 10 shows a porcine PA defect experimental setup according to Example 10, showing a reinforced tube implanted in the pulmonary artery of a pig (see also FIG. 10A). [Figure 12]Schematic of PA defect correction. Figure 12A: Shows the isolated, clamped, and severed PA. Figure 12B shows a polyethylene spacer (middle longer tube) glued end-to-end to a 3D printed tube (arrows indicate overlay on 3D printed tube). Figure 12C: The animal is anesthetized, the pulmonary artery is severed, and the device shown in Figure 12B is sutured to each end of the severed PA, at each end. [Figure 13] FIG. 1 shows the experimental setup for continuous pumping experiments. [Figure 14] FIG. 1 illustrates the introduction of reinforced ends to enhance suture performance. Three hydrogel sheets printed with different energy levels are shown. The mesh network, as seen by visual inspection, was printed with a higher energy than the bulk of the sheet material. In each case, the bulk material was printed at 48 mJ / cm2, while the strands of the mesh are at a higher energy as labeled. The energy increase areas were controlled by using a gray scale to control exposure within the images. [Figure 15] Cuff or sleeve experimental setup according to Example 6. Fig. 15A: Arrow indicates interface between tubing and printed tube. Blunt tip needle was slid inside hydrogel tube. Fixed with cyanoacrylate glue. Fig. 15B: Both ends were directly connected to blunt tip needle, perfused with DPBS+India ink, submerged in 1x PBS, then pumped at 0.1 mL per stroke / ~25 strokes / min=2.5 mL / min and allowed to circulate overnight. [Figure 16] FIG. 3D printed hydrogel blood vessels. [Figure 17-1] 17A-B show experimental results of pulling force experiments on 17A (602 tubing sample), 17B (porcine carotid artery tissue alone). [Figure 17-2] FIG. 17 shows experimental results of pulling force experiments for 17C (602 N pulling force on tube and carotid artery). [Figure 18]Figure 13. Burst / cycling experiment: Sample connected to tubing for burst test B shows pressure increase over time until the system pressure reaches the maximum pressure of the experiment. No sample rupture occurred. [Figure 19] FIG. 19 shows the experimental setup for the tensile test according to Example 7, in which the sample (FIG. 19A) and the sample that was pulled to failure (FIG. 19B) were placed. [Figure 20-1] Figure 20A shows a typical 602n curve, and Figure 20B shows the tensile load of a porcine carotid artery. Figure 20B shows the tensile test. [Figure 20-2] Figure 20C shows an overlay of the two graphs from Figure 20A and Figure 20B. The overlay is important because it illustrates the magnitude of the difference between the hydrogel alone and the native tissue. [Figure 21] FIG. 15 shows a technical drawing (CAD drawing) of the device of FIG. 14. [Figure 22] FIG. 13 shows a padding pattern at the tube ends to increase suturing performance. [Diagram 23] FIG. 23A illustrates the use of the reinforcement process to repair cracks observed in a leaky conduit. FIG. 23B illustrates the reinforcement of a lobar scaffold hydrogel with a mesh. The procedure was performed as follows: 1. Identify the crack in the object. 2. Apply uncured ink via pipette onto the cracked surface 3. Apply light of a suitable wavelength using a flashlight for 2-4 seconds at a distance of approximately 3 inches from the surface 4. Retest the object and repeat the procedure if the crack persists. [Figure 24] FIG. 24A shows the uncured 602N ink, and FIG. 24B shows the cured ink. [Diagram 25] FIG. 1 illustrates 3D airway printing according to an embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present disclosure includes embodiments directed to methods of reinforcing a three-dimensional (3D) hydrogel structure. Additional embodiments include the reinforced three-dimensional (3D) hydrogel structure and a method of treating an ischemic disease in a subject in need thereof, the method comprising implanting the reinforced three-dimensional (3D) hydrogel structure.

[0011] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and so forth.

[0012] As used herein, "weight percent" (also expressed as "wt%") refers to the percentage (%) of one or more components relative to the total weight of the composition. Thus, a composition having a mass of 100 grams containing 10 grams of Compound A has a weight percent of 10% for Compound A. As used herein, weight percent is used synonymously with mass percent.

[0013] In the embodiments herein, various patterns of woven fabrics and their woven structures are described, which are defined in Bilisik et al., “3D Fabrics for Technical textile Applications” Submitted: March 15th 2015Reviewed: July 14th 2015Published: March 24th 2016, DOI: 10.5772 / 61224. These definitions of textile terminology are well known in the art: Knitted mesh is composed of a single fiber that is formed into interlocking loops to create a series of openings. Woven mesh is composed of multiple fibers that cross each other to create a series of openings. Felt, otherwise known as nonwoven, is a mat of individual fibers that are interlocked by tangling or the application of heat, chemicals, pressure, or a combination thereof. Bonded nonwoven is a felt that contains a second material that binds the intersections of the tangled fibers of the mat. Braided flat or tubular mesh is a construct with a circular cross section rather than flattened, created by interlacing three or more fibers. Velcro is a series of loops and hooks that, when pressed together, become entangled by capturing the hooks within the loops. Lace is a fabric with a woven fabric-like structure created by twisting, braiding, looping, interlacing, or a combination thereof.

[0014] All references cited herein are hereby incorporated by reference in their entirety.Definitions are provided to facilitate understanding of certain terms used throughout this specification.Unless otherwise defined, technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art.Any suitable material and / or method known to those skilled in the art can be used in carrying out the method described herein.

[0015] The terms "administering" or "implanting" as used herein include defining administration as well as the actual administration, and also includes physical administration by another person, e.g., a surgeon, to the subject being treated.

[0016] As used herein, "subject" or "patient" or "individual" refers to any subject, patient, or individual, and the terms are used interchangeably herein. As such, the terms "subject," "patient," and "individual" include mammals, particularly humans.

[0017] As used herein, the terms "comprising" or "comprises" are intended to mean that compositions and methods include the recited elements but do not exclude others. "Consisting essentially of," when used to define compositions and methods, shall mean excluding other elements that, when combined for the purposes described, have some essential significance to the combination. Thus, a composition consisting essentially of elements as defined herein does not exclude other materials or steps that do not significantly affect the basic and novel characteristics of the claimed invention. "Consisting of" shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transitional phrases are within the scope of the present invention. When an embodiment is defined by one of these terms (e.g., "comprising"), the disclosure should be understood to include alternative embodiments, such as "consisting essentially of" the embodiment, as well as "consisting of."

[0018] "Substantially" or "essentially" means nearly entirely or completely, e.g., 95%, 96%, 97%, 98%, 99%, or more of a given quantity.

[0019] The term "about" is understood by those skilled in the art and varies to some extent depending on the context in which the term is used. In cases where there are uses of the term that are not clear to those skilled in the art given the context in which the term is used, "about" means up to ±10% of the particular term. For example, in some embodiments, the term means ±5% of the particular term. A particular range is presented herein using a numerical value preceded by the term "about". The term "about" is used herein to provide literal support for the exact number that follows it, as well as a number that is close to or approximate to the number that follows the term. When determining whether a number is close to or approximate to a number specifically cited, the uncited close or approximate number may be a number that represents the substantial equivalent of the number specifically cited in the context in which it is presented.

[0020] When a range of numerical values ​​is presented, it is understood that every intermediate value between the upper and lower limits of that range (to one-tenth of the unit of the lower limit unless the context clearly dictates otherwise), and any other stated or intermediate numerical value within that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, but are also encompassed within the invention, subject to any specifically excluded limit in the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those limits are also included in the disclosure.

[0021] As used herein, the term "treatment" or "treating" refers to any treatment of a disease or condition, or related disorder, in a patient, including:

[0022] "Treatment" also includes inhibiting or preventing a disease or condition, i.e., arresting or suppressing the onset of clinical symptoms, such as neurological deficits resulting from cerebral ischemia, providing neuroprotection; and / or ameliorating a disease or condition, i.e., causing regression of clinical symptoms (e.g., increasing neurological performance or decreasing neurological deficits).

[0023] As used herein, the term "grayscaling" or "voxel printing" refers to the in situ processing of a mesh design into an object. Parameters can be changed and printed to vary the cure at specific 3D points in space, or voxels, within the resulting object. Each voxel can be printed with completely different parameters, so the object can be printed from the same material but with different properties.

[0024] This application is related to the following documents: (a) U.S. Provisional Patent Application No. 63 / 185293, entitled "USE OF FUNCTIONALIZED AND NON-FUNCTIONALIZED ECMS, ECM FRAGMENTS, PEPTIDES AND BIOACTIVE COMPONENTS TO CREATE CELL ADHESIVE 3D PRINTED OBJECTS," filed on May 6, 2021, and U.S. nonprovisional patent and / or PCT applications of the same title, filed on May 6, 2022; (b) U.S. Provisional Patent Application No. 63 / 185302, entitled "MODIFIED 3D-PRINTED OBJECTS AND THEIR USES," filed on May 6, 2021, and U.S. nonprovisional patent and / or PCT applications of the same title, filed on May 6, 2022; (c) U.S. Provisional Patent Application No. 63 / 185302, entitled "MODIFIED 3D-PRINTED OBJECTS AND THEIR USES," filed on May 6, 2021, and U.S. nonprovisional patent and / or PCT applications of the same title, filed on May 6, 2022; No. 63 / 185300, entitled "3D PRINTING HYDROGEL OBJECTS USING HYDROHILIC MONOMERS, HYDROPHOBIC MONOMERS, AND CROSSLINKERS," filed May 6, 2022, and U.S. nonprovisional patent and / or PCT applications of the same title; (d) U.S. Provisional Patent Application No. 63 / 185299, entitled "ADDITIVE MANUFACTURING OF HYDROGEL TUBE FOR BIOMEDICAL APPLICATIONS," filed May 6, 2021, and U.S. nonprovisional patent and / or PCT applications of the same title; (e) U.S. Provisional Patent Application No. 63 / 185299, entitled "ADDITIVE MANUFACTURING OF HYDROGEL TUBE FOR BIOMEDICAL APPLICATIONS," filed May 6, 2021, and U.S. nonprovisional patent and / or PCT applications of the same title; No. 63 / 185,298, entitled "METHOD FOR USE IN HYDROPHENOL APPLICATIONS," as well as each of the U.S. non-provisional patent and / or PCT applications of the same title, filed May 6, 2022, are incorporated by reference in their entirety.

[0025] Methods for enhancing three-dimensional (3D) hydrogel structures and compositions comprising same Certain embodiments of the present disclosure relate to a method of reinforcing a three-dimensional (3D) hydrogel structure, comprising contacting a reinforcing scaffold immersed in an uncured curable bio-ink with the structure, and irradiating the reinforcing scaffold immersed in the uncured curable bio-ink, thereby adhering it to the 3D hydrogel structure. In some embodiments, the curable bio-ink is photocurable, e.g., via UV irradiation at certain wavelengths.

[0026] In another aspect, the present disclosure provides a composition comprising a three-dimensional (3D) hydrogel structure and a layer comprising a mesh immersed in a photocurable or photocurable ink, wherein the layer comprising a mesh immersed in a photocurable or photocurable ink is in contact with the structure.

[0027] The 3D shape of the hydrogel structure is not particularly limited and may be in the shape of a tube, or may be substantially the same shape, size, and / or have the same relative dimensions as an organ or fragment of an organ.

[0028] In some embodiments, the 3D shape of the hydrogel structure is substantially the same shape, size, and / or has the same relative dimensions as an organ or organ fragment. In certain embodiments, the organ or organ fragment includes a blood vessel, trachea, bronchus, esophagus, ureter, renal tubule, bile duct, renal duct, bile duct, hepatic duct, nerve conduit, CSF shunt, lung, kidney, heart, liver, spleen, brain, gallbladder, stomach, pancreas, bladder, lymphatic vessel, skeletal bone, cartilage, skin, intestine, muscle, larynx, or pharynx. In further embodiments, the vascular shape includes a pulmonary artery, renal artery, coronary artery, peripheral artery, pulmonary vein, or renal vein. In certain embodiments, the structure includes a hemodialysis graft. Other embodiments include where the structure is substantially the shape of a lung lobe, lung, pulmonary airway tree, pulmonary vasculature, or combinations thereof. In some embodiments, the reinforcement includes maintaining gas flow or blood (liquid) flow through the structure when an external pressure is applied to the structure.

[0029] In some embodiments, the 3D shape of the hydrogel structure is in the shape of a tube. In certain embodiments, the structure comprises a hollow tube with a first end and a second end. In some embodiments, the structure comprises a first sub-tube and a second sub-tube, each having a first end, the structure comprises a first sub-tube and a second sub-tube that connect to each other at their first ends to form a tube joint, and a reinforcing scaffold contacts the joint. In some embodiments, the reinforcing scaffold contacts the first and / or second ends of the tube.

[0030] Certain embodiments include a distance between a first end and a second end of the tube defining a tube length, where the reinforcing scaffold is in contact with a partial length of the tube defined by a distance from the first end and / or the second end to a predetermined point on the tube that is X% of the tube length away from the first end and / or the second end in contact with the reinforcing scaffold, where X is selected from about 0.01% to about 0.1%, about 0.1% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, or about 40% to about 50%. In some embodiments, the partial length includes about 1 mm to about 2.5 mm, about 2.5 mm to about 5 mm, about 5 mm to about 7.5 mm, about 7.5 mm to about 1 cm, or about 1 cm to about 2.5 cm.

[0031] The reinforcing scaffold is not particularly limited. Exemplary reinforcing scaffolds of the present disclosure include knitted mesh, woven mesh, nonwoven constructs (e.g., felt), bonded nonwoven constructs, braided tubular fabrics, braided plain fabrics, lamina, perforated lamina, velcro, and lace (or other fabrics that do not unravel when cut). Additional materials include those described in Bilisik, K., et al, "3D fabrics for technical textile applications," which is incorporated herein by reference. In certain embodiments, the reinforcing scaffold is a mesh. In some embodiments, the reinforcing scaffold is substantially planar and includes a thickness of about 0.1 μm to about 2 mm. For example, embodiments of reinforcement scaffold thicknesses include about 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 15 μm, 20 μm, 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 500 μm, 750 μm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, and interior ranges therebetween.

[0032] Non-limiting examples of reinforcing scaffold materials of the present disclosure include polyglactin (Vicryl®), polyglycolic acid (PGA), polylactic acid (PLA), monofilament propylene (e.g., SoftMesh, Parietex-TET, TIGR, Marlex), Dacron, Teflon®, polytetraflourethylene, polycaprolactone mesh, PGA / PCL, PGA / PLA / PCL, and combinations thereof. In a preferred embodiment, a 95 / 5 PCL / PGA mesh is used. The material has a preferred degradation time, such as approximately 9-12 months. In some embodiments, the material is made from two or more types of fibers. For example, when the material is made from two or more types of fibers, at least one of the fibers can degrade more quickly, resulting in a reinforced scaffold that decreases in density over time, such as to enhance tissue ingrowth. An example is a composite that is a PLA mesh that incorporates fibers of PGA (woven or knitted or braided). Another example is a composite with PCL fibers that incorporate PGA fibers.

[0033] The placement of the reinforcement scaffold can be adjusted to improve the mechanical properties of some or all of the structure. In some embodiments, the reinforcement scaffold spirals around a partial length of the tube or envelops the tube on the inner and / or outer surface of the tube. In some embodiments, the reinforcement scaffold contacts both the inner and outer surfaces of the tube. In other embodiments, the reinforcement scaffold wraps from the inner surface of the tube to the outer surface of the tube at the first and / or second ends of the tube to surround the edge of the tube.

[0034] The present disclosure also includes reinforcing the structure at areas expected to be sutured, cracked, torn, weakened, mechanically stressed, thinned, infected, and / or diseased areas. In some embodiments, the reinforcement includes bending resistance and / or collapse prevention of the tube. In some embodiments, the reinforcement is at the suture site of the structure and increases the suture pull strength of the structure by about 2.5 times to about 5 times, about 5 times to about 7.5 times, about 7.5 times to about 10 times, about 10 times to about 15 times, or more than about 15 times, relative to the unreinforced suture site of the structure.

[0035] Additional embodiments include reinforcement at the site of the structure for connection to biological tissue or graft material.

[0036] The three-dimensional (3D) hydrogel structure is not particularly limited, and may be, for example, a composite structure consisting of one or more different polymerized monomers. The hydrogel material that may be used in the present invention may be known to those skilled in the art, as may the method of making it. For example, hydrogels as described in Calo et al., European polymer Journal Volume 65, April 2015, Pages 252-267 may be used. In some embodiments, the hydrogel structure comprises polymerized (meth)acrylate and / or (meth)acrylamide hydrogels. In some embodiments, the structure is selected from the group consisting of polymerized poly(ethylene glycol) di(meth)acrylate, polymerized poly(ethylene glycol) di(meth)acrylamide, polymerized poly(ethylene glycol) (meth)acrylate / (methacrylamide), poly(ethylene glycol)-block-poly(ε-caprolactone), polycaprolactone, polyvinyl alcohol, gelatin, methylcellulose, hydroxyethyl methylcellulose, hydroxypropyl methylcellulose, polyethylene oxide, polyacrylamide, polyacrylic acid, polymethacrylic acid, salts of polyacrylic acid, salts of polymethacrylic acid, poly(2-hydroxyethyl Poly(methacrylate), polylactic acid, polyglycolic acid, polyvinyl alcohol, polyanhydrides such as poly(methacrylic acid) anhydride, poly(acrylic acid) anhydride, polysebacic acid anhydride, collagen, poly(hyaluronic acid), hyaluronic acid-containing polymers and copolymers, polypeptides, dextran, dextran sulfate, chitosan, chitin, agarose gels, fibrin gels, soy-derived hydrogels, alginate-based hydrogels, polymers comprising poly(sodium alginate), hydroxypropyl acrylate (HPA), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and combinations thereof.In some embodiments, the Mw of the hydrogel polymer is about 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1000 Da, 1100 Da, 1200 Da, 1300 Da, 1400 Da, 1500 Da, 1600 Da, 1700 Da, 1800 Da, 1900 Da, a, 2000Da, 2100Da, 2200Da, 2300Da, 2400Da, 2500Da, 2600Da, 2700Da, 2800Da, 2900Da, 3000Da, 3100Da, 3200Da, 3300Da, 3400Da, 3500Da, 3600Da, 3700Da, 3800 570 0Da, 5800Da, 5900Da, 6000Da, 6100Da, 6200Da, 6300Da, 6400Da, 6500Da, 7000Da, 7500Da, 8000Da, 8500Da, 9000Da, 9500Da, 10000Da, 15000Da, or 20000Da.

[0037] In some embodiments, the hydrogel comprises a crosslinked polymer. In some embodiments, the polymer has a crosslinking rate of about 0% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100%, based on the percentage (%) of crosslinkable moieties in the polymer. The crosslinkable moieties may include, for example, (meth)acrylate groups.

[0038] The curable bioink is not particularly limited. In some embodiments, the bioink is identical or similar in composition to the monomers used in the three-dimensional (3D) hydrogel structure. In some embodiments, the curable bioink is a photocurable ink, for example, an ink that can be photocured in the UV spectrum in the range of 100-400 nm. Possible inks include photoinitiators and / or dyes that react to and absorb light in the range of 100-400 nm. Examples of photopolymerization initiators include benzophenone, phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide (BAPO), 2-hydroxy-2-methyl-1-phenyl-propan-1-one, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, 2,2'-azobis[2-methyl-n-(2-hydroxyethyl)propionamide], 2,2-dimethoxy-2-phenylacetophenone, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP), and ethyl(2,4,6-trimethylbenzoyl)phenylphosphinate.

[0039] In certain embodiments, the hydrogel comprises a 3D printed object. Those skilled in the art will understand printing methods known in the art, including, but not limited to, selective laser sintering (SLS), fused deposition modeling (FDM), 3D inkjet printing, digital light processing (DLP), and stereolithography. In fused deposition modeling (FDM), ink is deposited by an extrusion head following a tool path defined by a CAD file. The material is deposited in fine layers, as thin as 20 μm, and the part is built up bottom-up, one layer at a time. Some 3D printers based on fused deposition modeling are equipped with dual print nozzle heads that can extrude two different materials, one being the build material and the other being the support material, such as the struts. The support material can be washed with water.

[0040] 3D inkjet printing has been effectively optimized for speed, low cost, high resolution, and ease of use, making it suitable for visualization from the concept stage of engineering design through early stage functional testing. In the inkjet printing method, complex 3D objects are generated from ink compositions by a jetting step followed by UV / Vis light. The light-curable ink in the inkjet printing process can be jetted through several nozzles on a build platform with a pattern defined by a CAD file.

[0041] Among the 3D printing techniques, one that is effective is the Digital Light Process (DLP) method, or Stereolithography (SLA). In a 3D printer using the DLP or SLA method, an ink material is layered on a vat or spread on a sheet, and a predetermined area or surface of the ink is exposed to ultraviolet-visible (UV / Vis) light controlled by a digital micromirror device or a rotating mirror. In the DLP method, additional layers are repeatedly or successively deposited, and each layer is cured until the desired 3D article is formed. The SLA method differs from the DLP method in that the ink is solidified by an array of radiation beams. Other methods of 3D printing can be found in 3D Printing Techniques and Processes by Michael Degnan, Dec 2017, Cavendish Square Publishing, LLC, the disclosure of which is hereby incorporated by reference.

[0042] The compositions of the present disclosure may be packaged with instructions or inserts or included in a kit. Such instructions or inserts may address recommended storage conditions, such as time, temperature, and light, while considering the shelf life of the composition. Such kits may also include instructions regarding medical implantation into patients and follow-up care for patients. Such instructions or inserts may also address special advantages of the composition, such as ease of storage for preparations that may require use outside of the field, controlled hospital, clinic, or office conditions. In one embodiment, the instructions may include visual aids / pictorial and / or written instructions for the administrator, producer, or recipient of the composition.

[0043] In one embodiment, the kit may include one or more compositions as disclosed herein, where the composition may be enclosed in a first protective packaging, or a second protective packaging, or a third protective packaging to protect the physical integrity of the product. One or more of the first, second, or third protective packaging may include a foil pouch. The kit may further include instructions for use of the device. In one embodiment, the kit contains two or more devices.

[0044] Methods for Treating Ischemic Disease In one aspect, a method of treating an ischemic disease in a subject in need thereof is provided, comprising implanting a reinforced structure produced by the method described in any embodiment herein, comprising contacting a mesh immersed in an uncured light-curable bio-ink with a structure, and irradiating the mesh immersed in the uncured light-curable bio-ink, thereby adhering it to the 3D hydrogel structure.

[0045] Tortuous or tortuous arteries and veins are commonly observed in humans and animals. Mild tortuosity is asymptomatic, but severe tortuosity can cause ischemic attacks in distant organs. Clinical findings have linked tortuous arteries and veins with aging, atherosclerosis, hypertension, genetic defects, and diabetes. Han et al., J. Vasc. Res. 2012 May; 49(3): 185-197. The reinforced tubes of the present disclosure may be implanted into blood vessels that are tortuous or may begin to tortuosity to treat and / or prevent ischemic injury from occurring or progressing.

[0046] Ischemic disease can include cerebral or systemic ischemic disorder.In one embodiment, cerebral or systemic ischemic disorder is selected from microcirculatory disorder, intrapartum cerebral ischemia, cerebral ischemia during / after cardiac arrest or resuscitation, cerebral ischemia caused by problems during surgery, cerebral ischemia during carotid artery surgery, chronic cerebral ischemia caused by stenosis of the artery supplying blood to the brain, venous sinus thrombosis or cerebral vein thrombosis, cerebrovascular malformation, diabetic retinopathy, hypertension, hypercholesterolemia, myocardial infarction, heart failure, cardiac insufficiency, congestive cardiac insufficiency, myocarditis, pericarditis, myocarditis, coronary heart disease, angina pectoris, congenital heart disease, shock, limb ischemia, renal artery stenosis, diabetic retinopathy, thrombosis associated with malaria, artificial heart valve, anemia, hypersplenism syndrome, emphysema, pulmonary fibrosis, erectile dysfunction or pulmonary edema.

[0047] For example, the methods of the present disclosure may show an improvement in one or more characteristics of cerebral or generalized ischemic injury as measured by a medically accepted scale. The improvement may be, for example, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0048] Medically accepted measures or techniques for measuring improvement include, for example, cholesterol testing, high sensitivity C-reactive protein testing, lipoprotein(a), plasma ceramides, natriuretic peptides, low density lipoprotein cholesterol, high density lipoprotein cholesterol, triglycerides, electrocardiogram (EKG), Holter monitor, stress test, echocardiogram, positron emission tomography (PET), thallium scan, myocardial perfusion scan, implantable loop recorder, tilt table test, electrophysiology test, coronary angiography, magnetic resonance imaging, magnetic resonance angiography, cardiac CT scan, and event recorder.

[0049] Methods for surgically implanting synthetic or tissue engineered vascular grafts, including the reinforced tubes disclosed herein, are disclosed in the Examples and are known to those skilled in the art. Koobatian et al., J Vis Exp. 2015; (98): 52354. Methods known in the art for surgically implanting TEVGs can be modified by those skilled in the art as disclosed herein, i.e., to reinforce said vessels as described herein before suturing the TEVG to a native vessel or tissue.

[0050] In some embodiments, the subject is a human or an animal, hi some embodiments, the subject is selected from a human, horse, dog, cat, cow, pig, bird, or reptile.

[0051] The embodiments described herein are further illustrated, but in no way limited, by the following examples. EXAMPLES

[0052] [Example 1] How to create a reinforced pleura using mesh The purpose of this description is to document the effect on the pleura when applying mesh in the scaffold of the present invention. Ventilation experiments show the effectiveness of this process. To test this mesh, applicant used one of the prints in FIG. 25, but it leaked in the pleura during ventilation testing. Therefore, VKML90 / 10PLGA was applied around the scaffold and the mesh was covered with 602N. The 602N tube contains the following components as shown in Table 1.

[0053] [Table 1]

[0054] A handheld UV light with a wavelength of 385 (nm) was applied to all parts of the print for 1 minute until polymerization occurred at the surface. The coated pieces were then placed in a buffer for 1 hour before being assembled into a bioreactor and connected to a ventilator.

[0055] [Example 2] Preparation of 6 cm long tube-mesh ends for use in pigs This experiment established a procedure for connecting a reinforcing mesh to a hydrogel tube and curing it using light. The steps performed were as follows: 1. Cut out the tube. 2. Thoroughly wipe the application end with a Kimwipe to remove excess buffer and prevent a barrier layer from forming between the new preparation and the original object. 3. Immerse the end into the preparation or apply drops using a bulb pipette. 4. Apply the ends of the fabric and tack them in place using a flashlight. 5. Wrap around the area, continue soaking and allow to harden until set. 6. Attach the UV source to the ring stand. 7. With the lamp suspended from a ring stand, the tube was placed very close to the light source. 8. Tack onto small area of ​​mesh end. Rotate slowly while adding preparation as needed. 9. When the entire circumference of the tube is covered with mesh, use surgical scissors to trim off the excess. 10.Continue to coat the graft with drips of the 602N preparation and allow it to harden while rotating. It helps to place the tube on the plastic rod so that the fingers are not exposed to the UV light. From time to time, press the mesh onto the surface of the tube with tweezers to ensure a tight bond. 11. While bending the tube, check for any gaps in the bond at the interface with the mesh. If no gaps are observed and the mesh does not move when pulled slightly, place the tube in a bath of PBS to wash off any uncured material.

[0056] [Example 3] Suture tension assay Methods: Three short lengths of tubing were prepared for testing on an Instron instrument (Figure 6) for measuring suture tensile values. The cut tubing sections had mesh applied to one end in the same manner as the 6 cm tubing described in Example 2. The experiment compared tubing without mesh to reinforced mesh tubing. All were from the same batch, 10 cm in length.

[0057] The 3 mm bite was marked with ink, then the needle was passed through the target mark and the suture was extracted using fine tweezers. The suture was secured in the top grip of the device.

[0058] Results: In all reinforced tubes, the mesh area was torn away from the body of the tube. No cases were observed in which the suture was pulled away from the mesh itself. The non-reinforced tubes were easily severed by the suture and required approximately 8-10 times less force to pull up (Figure 8).

[0059] [Example 4] Hydrogel sutured without leaks The goal of this experiment was to suture a reinforced hydrogel tube to a PTFE graft tube, inject liquid into the joined tubes, and demonstrate the absence of leaks.

[0060] method: The reinforced tube was prepared as described in Example 2. The end of the PTFE graft tube and the mesh-reinforced end of the tube were sutured together as shown in Figure 5A.

[0061] result: Water containing red ink was injected through the graft as shown in Figure 9. No leaks were observed. The ends of the hydrogel tube were plugged and no leakage was observed at the interface.

[0062] [Example 5] Implantable graft suture anchor for use in porcine / pig The goal of this experiment was to create a more functional vascular graft for deployment in a porcine model. The ultimate goal was to develop a 602N vascular graft augmentation method to create printed parts suitable for suturing within an animal model.

[0063] Even after feedback from surgeons on the vascular graft tubes created, problems continued to arise when connecting the 602N tubes to the native vessels. Previously, hard plastic barbed connectors were connected to the ends of the graft (using cyanoacrylate) and purse string sutures (Figure 4A) were tied to the native vessels, constricting them around the connectors (Figure 4B). In addition to making the hydrogel tubes stiff and weakening the ends / making them unsuitable for long-term use, cyanoacrylate was observed to form clots within the connectors in rabbit models. A stronger interface needs to be developed.

[0064] By embedding surgical mesh anchors within the hydrogel, applicants eliminate the need for connectors entirely, allowing the graft to be sutured directly to the native vessel.

[0065] material and method: Flashlight: Sunlite 365nm UV flashlight (20mW / cm at center of glass) 2 The power was measured in microns (measured in microns), ink used: 602N. First, a 365nm UV flashlight was tested by curing 50um of 602N in a petri dish. The flashlight was held approximately 5cm away from the droplet and turned on for approximately 10 seconds (Figure 24A shows the uncured 602N ink, and Figure 24B shows the cured ink).

[0066] The first test sample was made using a sheet of 12 ply medical gauze. Strips of gauze were cut (1 x 5 cm) and wrapped around the ends of 602N grafts (5 mm ID, 1.5 mm wall thickness). A small bulb pet was used to dispense approximately 1 mL of uncured ink onto the top of the gauze and cured for approximately 30 seconds using a UV flashlight.

[0067] result: Initial results from the 12ply gauze anchor test were very promising. Loose threads caused the gauze anchor to shrink / fray in some areas before the ink could be cured. Despite an unattractive appearance, the gauze was able to distribute the stress of the sutures over the entire surface and allowed the graft to hold the sutures intact without fraying. The reinforced areas allowed penetration by the suture needle and showed no signs of fraying or crushing at moderate levels of tension.

[0068] The first reinforced graft was made using one layer of 12ply gauze (2 cm sections on each end). Testing with closed loop sutures was successful with no fracture / cracking of the graft. The reinforced gauze graft was able to withstand tension without tearing or breaking. Additional reinforced grafts were made using the same technique using Vicryl surgical mesh and sent to the surgical team for evaluation and feedback. The reinforcement was cured into a structure using approximately 2 mL of ink in a 1 mm thick layer and the ink was allowed to cure for approximately 40 seconds per side.

[0069] Additional reinforcement was tested by printing Formlab Elastic v1 Resin printed anchors and securing them to the ends using a 602N and UV flashlight. The anchors were designed to replace the plastic connectors and allow for purse string suture connections. Net / mesh ends were designed to secure the anchor to the graft once the ink cured, and grooved bands were designed to secure the purse string sutures and withstand compressive forces. After implementation, mixed results were seen, with the serrated edges (Figure 4D) causing the graft to be cut by the newly cured ink and detach from the graft (Figure 4F).

[0070] [Example 6] Pumping experiment For the assembly of the device in Figure 15, an Ismatec Reglo ICC from Cole Parmer (Barrington, IL) was used with Tygon tubing and luer locks. Results: After pumping overnight without leaks, pressure increased until the break point was reached. Breakage Occurrence: The tubing luer lock did not break away from the attachment interface even when pumping at 0.5 mL per stroke / approximately 80 strokes per minute = 40 mL / min. Final dimensions: 2 mm OD, 1.5 mm ID.

[0071] [Example 7] Pull test A single rectangular section of artery retrieved from the burst test fixture was pulled to fracture at the same rates as the dogbone samples. All data were converted to MPa as shown in Figures 20A-C.

[0072] [Example 8] Suture tension test method: a) Using the apparatus shown in Figures 3A-3D, before starting the test, select the grip surface to be used and press it against the grip. The surface has a bite distance channel of 5 mm. b) Membranes should be stored in Ca-free, Mg-free DPBS at 37° C. for a minimum of 2 hours before testing. c) Remove the membrane from the buffer and lightly blot the bottom edge against a Kimwipe. Align the top edge of the membrane with the top edge of the gripping fixture. • Ensure that the base layer is located on the bottom side as indicated by the notch in the membrane. • Ensure that the membrane is in contact with the sandpaper surface for grip. Tighten the grip until it feels tight around your fingers. Do not overtighten as this will pull the membrane up past the top of the grip face mount. d) Insert a new suture while maintaining contact between the needle and the bottom of the suture channel. Carefully pull the suture through the membrane by firmly grasping the needle from the exit side and gently withdrawing it. e) Align the suture ends and clamp them together on the piece of tape at right angles to the tape edge, then press the tape against one face of the upper grip. Center the suture edge on the upper grip face. f) Tighten the grip until it is securely closed. g) Use the jog feature to remove most of the slack from the length of the suture. Leave a small amount of bend in the suture. The final slack is removed using the preload feature within the method. h) Zero Displacement and Zero Load i) Start the test.

[0073] [Example 9] Pig pulmonary artery repaired using reinforced tube the purpose: To evaluate the ability of the artificial pulmonary artery to maintain physiological pressures induced by blood flow, an animal model is needed. The porcine model offers reproducibility in that it is similar in size to the human pulmonary system and is well known for its application in several translational research studies.

[0074] method: 1. Sedate the pig 30 min prior to surgery with an intramuscular injection of ketamine (20 mg / kg), acepromazine (1.1 mg / kg), and atropine (0.04 mg / kg). 2. The animal is placed on the operating table. An ECG probe, a rectal probe thermometer, and a pulse oximeter are placed on the animal to monitor heart rate, temperature, and saturation throughout the surgery. 3. Induce anesthesia with an intravenous slow bolus injection of propofol (0.14 mg / kg). 4. Start a propofol infusion at 1.4 mL / min at a dose of 0.4 mg / kg / min to maintain anesthesia throughout the procedure. Administer an intramuscular injection of meloxicam (0.4 mg / kg) for analgesia. Monitor the depth of anesthesia throughout the procedure (heart rate, oculo-ocular reflexes, jaw tone) and increase the dose of propofol if necessary. 5. Administer a continuous intravenous infusion of Ringer's solution throughout the entire surgery (50-60 drops / min). 6. The pig is placed in a prone position and lidocaine (10 mg / mL) spray is injected onto the arytenoid cartilages. 7. Five minutes after the lidocaine spray, intubate the pig using a 6.5 endotracheal tube and rapidly ventilate with 100% oxygen (tidal volume of 7 mL / kg and respiratory rate of 25-28 breaths / min) while using an Ohmeda 7800 ventilator. 8. Position the pig on its right side in preparation for thoracotomy. 9. Administer a second intramuscular injection of meloxicam (0.2 mg / kg) and a subcutaneous injection of bupivacaine (1 mg / kg) at the orifice site. 10. An approximately 15 cm incision is made through the right second intercostal space. 11. Perform a dissection maneuver to expose the pulmonary artery, taking special care to avoid the vagus nerve. 12. Use forceps to move the vena cava away to allow enough space for implanting the graft. 13. The pulmonary artery is clamped to stop blood passing through it. A partial incision is made on the pulmonary artery to introduce the artificial vascular connector. 14. The sample graft is inserted through the incision into the pulmonary artery and clamped in place with a suture. The clamp is removed from the artery to allow blood flow through the artificial pulmonary artery.

Claims

1. 1. A method for reinforcing a three-dimensional (3D) hydrogel structure, comprising: contacting a mesh immersed in an uncured, light-curable bio-ink with the structure; and irradiating the mesh immersed in the uncured, light-curable bio-ink, thereby bonding the mesh to the 3D hydrogel structure.

2. The method of claim 1 , wherein the structure comprises a hemodialysis graft.

3. The method of claim 1 , wherein the structure comprises a hollow tube having a first end and a second end.

4. The method of claim 1, wherein the mesh is substantially planar and has a thickness of about 0.1 μm to about 2 mm.

5. The method of claim 3 , wherein the mesh spirals around the hollow tube length or surrounds the hollow tube on an inner and / or outer surface of the tube.

6. The method of claim 3 , wherein the contacting step includes contacting the mesh with an interior and / or exterior surface of the hollow.

7. The method of claim 1 , wherein the contacting and irradiating steps are repeated two or more times to form two or more layers of the mesh.

8. 10. The method of claim 1, wherein the mesh comprises polyglactin (Vicryl®), polyglycolic acid (PGA), polylactic acid (PLA), monofilament propylene (SoftMesh, Parietex-TET, TIGR, or Marlex), Dacron, Teflon®, polytetrafluoroethylene, polycaprolactone (PCL), a PGA / PCL blend, a PGA / PLA / PCL blend, or a combination thereof.

9. The method of claim 1 , wherein the structure comprises a polymerized (meth)acrylate and / or (meth)acrylamide hydrogel.

10. The method of claim 1 , wherein the photocurable ink comprises a photoinitiator and / or a dye that reacts to and / or absorbs light having a wavelength of about 100 to about 400 nm.

11. The method of claim 1 , wherein the contacting step comprises printing both the structure and the mesh in contact with one another.

12. 10. The method of claim 1, wherein the reinforced structure has a burst pressure of 1,000 mmHg or greater and / or a suture retention strength of 1.5 N or greater.

13. 13. A method of treating an ischemic disease in a subject in need thereof, comprising the step of implanting a reinforced structure produced by the method of claim 1.

14. A composition comprising a three-dimensional (3D) hydrogel structure and a layer comprising a mesh immersed in a photocurable or photocurable ink, wherein the layer comprising the mesh immersed in the photocurable or photocurable ink is in contact with the structure.

15. The composition of claim 14 , wherein the ink is photocurable but not photocured.

16. The composition of claim 14 , wherein the ink is photocured.

17. The composition of claim 14 , wherein the structure comprises a hemodialysis graft.

18. The composition of claim 14 , wherein the structure comprises a hollow tube having a first end and a second end.

19. The composition of claim 14 , wherein the structure comprises a polymerized (meth)acrylate and / or (meth)acrylamide hydrogel.

20. The composition of claim 14, further comprising one or more types of cells.

Images