Air printing of hydrogels

Abstract

There is provided a method of printing a hydrogel by extruding a gel forming system comprising microgel particles functionalized with a hydrogen-bonding supramolecular moiety to obtain extruded microgel particles. The extruded microgel particles form a hydrogel that substantially retains its shape in air.

Description

AIR PRINTING OF HYDROGELSCROSS-REFERENCE TO A RELATED APPLICATION

[0001] This disclosure claims priority from U.S. provisional application number 63 / 662,689 filed on June 21 , 2024, which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] This disclosure relates to the field of bioprinting hydrogels, particularly the bioprinting of hydrogels containing biological materials such as cells for applications such as cellular therapy.BACKGROUND OF THE ART

[0003] As pluripotent stem cell derived therapeutic cells for chronic endocrine disorders such as Type 1 Diabetes are moving into the clinic, the need for hydrogel-based delivery devices is becoming more apparent. Current clinical trials involve long-term systemic immune suppression to mitigate risk of allogeneic and auto-immune destruction of these transplanted allogeneic cells. In addition, the focus on genetically modified cells further militates for their encapsulation within retrievable and immuno-protective barrier devices.

[0004] While early delivery devices had focussed on semiporous polyolefin pouches, 3D- bioprinted hydrogel-only devices offer fewer opportunities for generation of foreign body responses, a larger effective interface area with the host metabolic system, and access to many 3D geometries, that together allow optimization of metabolic connection and oxygen supply to the transplanted cells.

[0005] Bioprinting is a method of three-dimensional (3D) printing of biocompatible materials such as hydrogels. Hydrogels often require to be printed into a printing bath in order to retain the shape of the print while the hydrogel gels. Hydrogel baths often introduce chemical components that are not biocompatible and require a separate removal step. When considering the incorporation of cells into 3D printed devices, reducing the time from printing to implantation is important in order to maximize cell viability. Therefore, improvements in the 3D printing of hydrogels are desired.SUMMARY

[0006] In one aspect, there is provided a method of printing a hydrogel, the method comprising: extruding a gel forming system comprising microgel particles functionalized with ahydrogen-bonding supramolecular moiety to obtain extruded microgel particles; wherein the extruded microgel particles form a hydrogel that substantially retains its shape in air.

[0007] In at least some embodiments, the extruding includes extruding into or through air onto a supporting surface. In at least some embodiments, the shape of the hydrogel is independent of the supporting surface.

[0008] In at least some embodiments, the gel forming system is extruded through a nozzle or needle.

[0009] In at least some embodiments, the gel forming system comprises microgel particles comprising a synthetic polymer functionalized with the hydrogen-bonding supramolecular moiety, optionally a first subset of microgel particles comprising a first synthetic polymer and a second subset of microgel particles comprising a second synthetic polymer, wherein one or both, preferably both, of the first synthetic polymer and the second synthetic polymer are functionalized with the hydrogen-bonding supramolecular moiety, preferably wherein the first synthetic polymer and the second synthetic polymer comprise cross-linking moieties.

[0010] In at least some embodiments, the gel forming system further comprises one or more gel forming polymers, optionally, wherein the one or more gel forming polymers are selected from alginate, hyaluronic acid, gelatin, elastin, cellulose, hydroxypropylcellulose, carboxymethylcellulose, methylcellulose, synthetic polymers and combinations thereof. In at least some embodiments, the one or more gel forming polymers and the microgel particles together have a total concentration of from 1 to 20 wt. % in the gel forming system. In at least some embodiments, the synthetic polymers are functionalized with moieties that crosslink with each other. In at least some embodiments, the synthetic polymers have a backbone that comprises monomeric units selected from 2-aminoethyl acrylate, 2-aminoethyl methacrylate, 2-aminoethyl acrylamide, 2-aminoethyl methacrylamide, 3-aminopropyl acrylate, 3-aminopropyl methacrylate, 4-aminobutyl acrylate, 4-aminobutyl methacrylate, 4-aminobutyl acrylamide, 4-aminobutyl methacrylamide, acrylic acid (AA), acrylamide, methacrylic acid (MAA), 2- hydroxypropylacrylamide (HPA), 2-hydroxypropylmethacrylamide (HPMA), 2- hydroxyethylacrylate (HEA), 2-hydroxyethylmethacrylate (HEMA), 2-(methacryloyloxy)ethyl 2- (trimethylammonio)ethyl phosphate (MPC), carboxy betaine methacrylate (CBM), carboxy betaine methacrylamide, N,N-dimethylaminoethylacrylate (DMAEA), N,N-dimethylaminoethyl methacrylate, 3-N,N-dimethylaminopropyl acrylamide, 3-N,N-dimethylaminopropylmethacrylamide, 3-N,N-dimethyliaminopropyl acrylate, 3-N,N-diaminopropyl methacrylate, 4- N,N-dimethylaminobutyl acrylamide, 4-N,N-dimethylaminobutyl methacrylamide, 1 ,3- bis(dimethylamino)propyl acrylate, 1 ,3-bis(dimethylamino)propyl methacrylate, 2-((2- (dimethylamino)ethyl)(methyl)amino)ethyl methacrylate, 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl acrylate, sodium acrylate, sodium methacrylate, sodium ethacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate, [2- (acryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 3-((3- methacrylamidopropyl)dimethylammonio)propane-1 -sulfonate, 2-(acryloyloxy)ethyl phosphate, 2-(methacryloyloxy)ethyl phosphate, (2-(methacryloyloxy)ethyl)phosphonate, (2- (acryloyloxy)ethyl)phosphonate, (2-acrylamidoethyl)phosphonate, (2- methacrylamidoethyl)phosphonate, 2-methacryloyloxyethyl phosphorylcholine, N- (carboxymethyl)-2-(acryloyloxy)-N,N-dimethylethan-1-aminium, N-(carboxymethyl)-2-(methacryloyloxy)-N,N-dimethylethan-1-aminium, N-(carboxymethyl)-3-methacrylamido-N,N- dimethylpropan-1-aminium, N-(carboxymethyl)-3-acrylamido-N,N-dimethylpropan-1-aminium, 2- (dimethylamino)ethyl acrylate, 2-(dimethylamino)ethyl methacrylate, / V-[3-(Dimethylamino)propyl] acrylamide, / V-[3-(dimethylamino)propyl] methacrylamide, / V-(3-Aminopropyl)acrylamide hydrochloride, / V-(3-aminopropyl)methacrylamide hydrochloride, vinyl sulfonic acid, vinyl phosphonic acid, 3-sulfopropyl methacrylate potassium salt, and 2- (methacryloyloxy)ethanesulfonic acid and combinations thereof. In at least some embodiments, the synthetic polymer is poly(methyl vinyl ether-alt-maleic anhydride) (PMM), poly-ornithine, poly- L-lysine (PLL), epsilon-polylysine, poly[APM-co-DMAEA-co-SBMA] terpolymer (PADS), or poly[3- aminopropylmethacrylamide-co-2-(dimethylamino)ethyl acrylate] (PAD). In at least some embodiments, the hydrogel is formed by two gelling reactions, the hydrogel is first partially gelled by the formation of hydrogen-bonds by the hydrogen-bonding supramolecular moiety, and then further gelled by the formation of crosslinks between the moieties of the synthetic polymers.

[0011] In at least some embodiments, the one or more gel forming polymers are the alginate. In at least some embodiments, the hydrogel is formed by two gelling reactions, the hydrogel is first partially gelled by the formation of hydrogen-bonds and then further gelled by the formation of ionic crosslinks to crosslink the alginate. In at least some embodiments, the one or more gel forming polymers are the alginate and the synthetic polymers. In at least some embodiments, the hydrogel is formed by three gelling reactions, the hydrogel is first partially gelled by the formationof hydrogen-bonds and then further gelled by the formation of ionic crosslinks to crosslink the alginate and the formation of crosslinks between the moieties of the synthetic polymers.

[0012] In at least some embodiments, the hydrogel is allowed to partially gel while the hydrogel is in contact with the air.

[0013] In at least some embodiments, the hydrogen-bonding supramolecular moiety is ureido-pyrimidinone (UPy).

[0014] In at least some embodiments, the gel forming system further comprises a saline such as HEPES buffered saline.

[0015] In at least some embodiments, the method further com prises coating the printed shape of the hydrogel with a further synthetic polymer. In at least some embodiments, the further synthetic polymer has a backbone that comprises monomers selected from 2-aminoethyl acrylate, 2-aminoethyl methacrylate, 2-aminoethyl acrylamide, 2-aminoethyl methacrylamide, 3- aminopropyl acrylate, 3-aminopropyl methacrylate, 4-aminobutyl acrylate, 4-aminobutyl methacrylate, 4-aminobutyl acrylamide, 4-aminobutyl methacrylamide, acrylic acid (AA), acrylamide, methacrylic acid (MAA), 2-hydroxypropylacrylamide (HPA), 2- hydroxypropylmethacrylamide (HPMA), 2-hydroxyethylacrylate (HEA), 2- hydroxyethylmethacrylate (HEMA), 2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethyl phosphate (MPC), carboxy betaine methacrylate (CBM), carboxybetaine methacrylamide, N,N- dimethylaminoethylacrylate (DMAEA), N,N-dimethylaminoethyl methacrylate, 3-N,N- dimethylaminopropyl acrylamide, 3-N,N-dimethylaminopropyl methacrylamide, 3-N,N- dimethyliaminopropyl acrylate, 3-N,N-diaminopropyl methacrylate, 4-N,N-dimethylaminobutyl acrylamide, 4-N,N-dimethylaminobutyl methacrylamide, 1 ,3-bis(dimethylamino)propyl acrylate, 1 ,3-bis(dimethylamino)propyl methacrylate, 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl methacrylate, 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl acrylate, sodium acrylate, sodium methacrylate, sodium ethacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate, [2- (acryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 3-((3- methacrylamidopropyl)dimethylammonio)propane-1 -sulfonate, 2-(acryloyloxy)ethyl phosphate, 2-(methacryloyloxy)ethyl phosphate, (2-(methacryloyloxy)ethyl)phosphonate, (2- (acryloyloxy)ethyl)phosphonate, (2-acrylamidoethyl)phosphonate, (2- methacrylamidoethyl)phosphonate, 2-methacryloyloxyethyl phosphorylcholine, N-(carboxymethyl)-2-(acryloyloxy)-N,N-dimethylethan-1-aminium, N-(carboxymethyl)-2-(methacryloyloxy)-N,N-dimethylethan-1-aminium, N-(carboxymethyl)-3-methacrylamido-N,N- dimethylpropan-1-aminium, N-(carboxymethyl)-3-acrylamido-N,N-dimethylpropan-1-aminium, 2- (dimethylamino)ethyl acrylate, 2-(dimethylamino)ethyl methacrylate, / V-[3-(Dimethylamino)propyl] acrylamide, / V-[3-(dimethylamino)propyl] methacrylamide, / V-(3-Aminopropyl)acrylamide hydrochloride, / V-(3-aminopropyl)methacrylamide hydrochloride, vinyl sulfonic acid, vinyl phosphonic acid, 3-sulfopropyl methacrylate potassium salt, 2-(methacryloyloxy)ethanesulfonic acid, and combinations thereof. In at least some embodiments, the further synthetic polymer is poly(methyl vinyl ether-alt-maleic anhydride) (PMM), poly-ornithine, poly-L-lysine (PLL), epsilon- polylysine, poly[APM-co-DMAEA-co-SBMA] terpolymer (PADS), or poly[3- aminopropylmethacrylamide-co-2-(dimethylamino)ethyl acrylate] (PAD).

[0016] Many further features and combinations thereof concerning the present improvements will appear to those skilled in the art following a reading of the instant disclosure.DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 is an image showing a hydrogel that was air printed across defined gaps in a support structure.

[0018] FIG. 2 is an image showing a patch that was air printed.

[0019] FIG. 3A is a microscopy image showing an air printed hydrogel patch.

[0020] FIG. 3B is a fluorescence microscopy image of Fig. 3A that was fluorescently tagged with rhodamine.

[0021] FIG. 4 is a fluorescence microscopy image showing the formation of a distinct coating (fluorescently labelled) on the printed structure.

[0022] FIG. 5A is a brightfield microscopy image of a doubly coated raft.

[0023] FIG. 5B is a fluorescence microscopy image of Fig. 5A showing the fluorescently labelled coating.

[0024] FIG. 5C is a combination of the images of Figs. 5A-5B.DETAILED DESCRIPTION

[0025] 3D bioprinting hydrogels has been used to encapsulate cells inside the hydrogels while printing a particular shape of the hydrogel. The printing of a specific shape can be tailored to a specific implantation area and the geometry can be adapted for a best fit. The size and geometry can also be modified on a case-by-case basis and adapted for each individual. The hydrogels encapsulate therapeutic cells and isolate them in vivo from the immune system. Achieving immuno-isolation of encapsulated cells remains a complex task, and requires generally (i) sufficient mechanical stiffness for reliable handling during implantation, (ii) sufficient diffusion across the hydrogel to enable metabolic connection of the encapsulated cells, yet with a molecular weight cut-off of around 150,000 Daltons, expressed as percent in vitro exclusion of IgG after 24 hrs of exposure to an external solution of IgG, (iii) the ability to physically exclude cellular and molecular immune components, and to prevent escape of the therapeutic cells, (iv) the ability to vascularize the outer and inner surfaces of the 3D printed device in order to ensure both good long-term oxygen supply, as well as rapid blood sugar sensing and correction such as in the case of insulin producing cells.

[0026] To achieve these properties, there is provided a gel forming system comprising microgel particles. The gel forming system can further comprise one or more gel formers selected from alginate, hyaluronic acid, gelatin, elastin, cellulose, hydroxypropylcellulose, carboxymethylcellulose, methylcellulose, or synthetic polymers, suitably other synthetic polymers as described herein. The synthetic polymers can be polymers that are functionalized to gel through covalent bonds (crosslinking). The microgels are functionalized with hydrogen-bonding moieties such as ureido-pyrimidinone (UPy) to support formation of supramolecular assemblies that lend temporary support to the printed structure. The microgels may further be modified to incorporate functional groups that can covalently crosslink with other synthetic polymers, or with other microgels, or both. An advantage of a covalently crosslinked network is that it provides more long-term stability in vivo. The microgels can also be modified to introduce additional functional groups onto the printed structure, including but not limited to passive immune evasive groups as well as active immune modification groups. Furthermore, the resulting hydrogels can accommodate a high cell loading capacity coupled with high total surface area / islet equivalent or other therapeutic cell, to maximize metabolic exchange while maintaining acceptable stand off stand-off distance between encapsulated cells and host tissue, suitably on average a 100 pm stand-off distance.

[0027] As evidenced by the Examples, it has been determined that the incorporation of self- complementary hydrogen-bonding moieties on microgels in a gel forming system allowed to print hydrogels of the gel forming system into air (i.e. in the absence of a liquid, gel or matrix bath) by taking advantage of their ability to form self-supporting supra-molecular assemblies. Printing in air is highly preferable to printing in a supporting bath system, as it can generate smoother surfaces, simplify the overall process, and eliminate the step of removing components from the support bath post-printing.

[0028] The microgels are microparticles of gelled polymers and the particles can have a size of between 1 and 1000 pm. The microparticles can be made of or comprise a synthetic polymer functionalized with the hydrogen-bonding supramolecular moiety. In some embodiments, a first subset of microgel particles comprising a first synthetic polymer and a second subset of microgel particles comprising a second synthetic polymer, wherein one or both, preferably both, of the first synthetic polymer and the second synthetic polymer are functionalized with the hydrogen-bonding supramolecular moiety, preferably the first synthetic polymer and the second synthetic polymer comprise cross-linking moieties. Although the example section used methacrylic anhydride microgels, any other suitable polymer (i.e. biocompatible) can be used for forming the functionalize microgels and the present disclosure is not limited to methacrylic anhydride microgels.

[0029] In preferred embodiments, the gel forming system comprises the microgels in a swelled form. For example, the gel forming system is provided in a buffer or buffer saline, in some embodiments, in a concentration of from 1 to 20 wt. %, from 1 to 15 wt. %, from 1 to 10 wt. %, from 2 to 20 wt. %, from 2 to 15 wt. %, from 2 to 10 wt. %, from 2 to 6 wt. %, from 5 to 15 wt. %, from 5 to 10 wt. %, from 5.5 to 8.5 wt. % or from 6 to 7.5 wt. %. The buffer is for example HEPES or HBS. The concentration ranges recited for the gel forming system would represent the total of microgels and additional optional gel formers as described herein. This enhances the shear thinning property and facility to print the gel forming system.

[0030] When taking alginate containing ink as an example, a traditional printing method would be to print the alginate containing ink into a gelling bath comprising calcium, that is thick, shearthinning and can maintain the shape of the printed alginate. By contrast, in the present printing method, the print can be made into a support bath that is a water-like calcium-containing liquid that crosslinks the alginate in place but does not hold a shape of its own nor does it have any effect on holding the shape of the printed structure. Alternatively, the incorporation of microgelsalso allows for printing directly through air onto a support in the absence of calcium, relying on the jamming interaction of the densely packed but swollen microgels suitably present at levels of 5 to 20 wt% in the printed material to maintain the shape of the extruded hydrogel device long enough for covalent crosslinking to take place. In some embodiments, the microgels are further functionalized with self-complementary hydrogen-bonding moieties such as ureido-pyrimidinone (UPy) that further support gel strength at rest after extrusion and allows reduction of the microgel loading to 1 to 10 and preferably 2 to 6 wt% in the extruded material (bioink), which can enhance metabolic connection of the encapsulated cells and reduce mechanical damage during extrusion.

[0031] One particular example of a self-complementary hydrogen-bonding supramolecular moiety is ureido-pyrimidinone (UPy) (1-(6-aminohexyl)-3-(6-methyl-4-oxo-1 ,4-dihydropyrimidin-2- yl)urea (UPy)). The UPy moiety can be used as a monomer unit to impart strength and processability to the hydrogels or polymers. The spontaneous nature of the noncovalent bond in the supramolecular materials enables the formation and functionalization under air which is a mild condition.

[0032] One role of the hydrogen-bonding supramolecular moiety is to enhance the interactions between microgels (e.g. adding UPy functionalization to generate hydrogen bonding between microgels that can improve their printability, particularly at lower microgel contents).

[0033] The hydrogen-bonding supramolecular moieties can be incorporated in the microgels in a concentration of 0.5 - 50 mol%, 0.5 - 40 mol%, 0.5 - 35 mol%, 0.5 - 30 mol%, 0.5 - 25 mol%, 0.5 - 20 mol%, 1 - 20 mol%, preferably 1 - 15 mol%.

[0034] It was found that in the absence of hydrogen-bonding supramolecular moieties, it was not possible to perform air printing unless the viscosity and solids loading of the hydrogel suspension is increased to a level that would make the thickness and porosity incompatible with cell transplantation and cell survival inside the hydrogels.

[0035] The hydrogel printing as described herein can be performed directly i.e. through air, without a supporting bath, onto a support surface (“air-printing”). Microgels, swollen hydrophilic substantially spherical particles, are incorporated into the printable materials at loadings where they form a jammed gel, such that the extruded hydrogel mixture is sufficiently stiff at rest (thixotropic) to hold its shape long enough without significant deformation for crosslinking of extruded gel formers to occur.

[0036] The microgels are suitably size-mono-disperse at 1 to 40, and preferably 3 to 10 micrometers in diameter. In some embodiments, they are formed by radical copolymerization of suitable monomers and crosslinkers under conditions that enable formation of self-stabilizing colloidal particles even in the absence of any added surfactant or steric stabilizer. These conditions include free radical polymerization initiated by radical initiators such as azobisisobutyronitrile (AIBN), in near-theta solvents such as mixtures of methylethylketone and heptane, with their ratios adjusted to provide colloidal stabilization through surface swelling of the growing particles, but to force desolvation of the particle core to enable formation of a multiplicity of monodisperse particles. Post-processing of the resulting microparticles into water-swellable substantially spherical microgel particles is used to introduce chemical groups that enable covalent crosslinking with the synthetic gel formers, as well as covalent crosslinking between microgel particles, as well as ureido-pyrimidinone (UPy) groups.

[0037] Jammed gels of such hydrogel particles are thixotropic, and can be used to facilitate direct printing of hydrogel structures. Common implementations involve either nanometer-range particles formed by emulsion-type processes, or much larger microgel particles formed by suspension processes.

[0038] The present process can involve single-digit micron-range microgel particles that are mono-disperse in size, free of any surfactants and stabilizer residues, and that can be modified to enable suitable properties. For example, incorporation of self-complementary hydrogen bonding groups such as UPy enable formation of jammed gels at solids loadings down to about 4 wt%, as opposed to about 10 wt% for microgels in the absence of hydrogen-bonding supramolecular moiety addition. The hydrogen-bonding between microgels increases the degree of shear-thinning, and reduces mechanical cell damage during printing. Decreasing the solids loading is further beneficial for encapsulation (co-printing) of cells and cell clusters as it increased access of immobilized cells to oxygen and nutrients by diffusion. In addition, post-functionalization of these microgels can enable incorporation of functional groups that facilitate covalent crosslinking with each other, and optionally with other added gel formers.

[0039] As explained above, the gel formers can comprise or be supplemented by synthetic polymers that form covalent bonding or ionic covalent bonding. The polymer backbone can also include synthetic polycations such as those described in US10407527 which is incorporated herein by reference (e.g. poly(aminopropylmethacrylamide) (poly(APM))), as well as polyampholyte polymers and polybetaines for example as described in US10369227 which isincorporated herein by reference. Examples of covalent bonding gel formers are described in US8815283, WO2023235976, WO2022120478 and WO2024059954 the contents of which are incorporated herein by reference in their entirety. The covalent bonding can be formed between thiol and vinyl groups such as acrylate groups. The thiol-ene polymer pair can enhance robustness in the gel former by forming a reinforcing network within the latter. This network is formed by a thiol - ene addition reaction that can be triggered during printing or in a subsequent step using a reducing agent such as tris (2-carboxyethyl) phosphine (TCEP) or tris(hydroxypropyl) phosphine (THPP) which liberate free thiol groups on the thiol functional polymer. Poly(methylvinylether-alt-maleic anhydride) (PMM) is one example of such functional polymer. In some embodiments, the thiol group is homocysteine thiolactone (HTL)), to form PMM-HTL and the vinyl group is cysteamine vinyl sulfone to form PMM-CVS. In some embodiments, the covalent crosslinking may be performed by click chemistry.

[0040] Examples of ionic bonding for dynamic bonding include using multivalent counter cations that bind anionic functional groups on polymer chains. In one embodiment, alginate can be lightly crosslinked with a low concentration of Ca2+counter cations. In another embodiment, alginate can form dynamic ionic crosslinks with counter cations with a lower binding affinity such as Mg2+, Sr2+, and Zn2+, in addition to using mixtures of these ions together and with Ca2+. The low concentration and weaker binding affinity allows for ionic crosslinks to form, break, and reform, leading to dynamic bonding. The concentration of these ions can be used to control the viscosity and shear thinning properties of the polymer mixture for extrusion.

[0041] Alternatives to poly(methyl vinyl ether-alt-maleic anhydride) (PMM) include polyornithine, poly-L-lysine (PLL), epsilon-polylysine, homopolymer of methacrylic anhydride, homopolymer of polyacrylic acid, homopolymer of polymethacrylic acid, or copolymers of acrylic acid and methacrylic acid, or copolymers of maleic anhydride with vinyl ethers selected from methyl vinyl ether, ethyl vinyl ether, propyl vinyl ether, butyl vinyl ether, hydroxy ethyl vinyl ether, and methoxy ethyl vinyl ether, copolymers of N-vinylpyrrolidone with maleic or itaconic or citraconic anhydrides.

[0042] In some embodiments, the synthetic polymer includes monomeric units selected from 2-aminoethyl acrylate, 2-aminoethyl methacrylate, 2-aminoethyl acrylamide, 2-aminoethyl methacrylamide, 3-aminopropyl acrylate, 3-aminopropyl methacrylate, 4-aminobutyl acrylate, 4- aminobutyl methacrylate, 4-aminobutyl acrylamide, 4-aminobutyl methacrylamide, acrylic acid (AA), acrylamide, methacrylic acid (MAA), 2-hydroxypropylacrylamide (HPA), 2-hydroxypropylmethacrylamide (HPMA), 2-hydroxyethylacrylate (HEA), 2- hydroxyethylmethacrylate (HEMA), 2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethyl phosphate (MPC), carboxy betaine methacrylate (CBM), carboxybetaine methacrylamide, N,N- dimethylaminoethylacrylate (DMAEA), N,N-dimethylaminoethyl methacrylate, 3-N,N- dimethylaminopropyl acrylamide, 3-N,N-dimethylaminopropyl methacrylamide, 3-N,N- dimethyliaminopropyl acrylate, 3-N,N-diaminopropyl methacrylate, 4-N,N-dimethylaminobutyl acrylamide, 4-N,N-dimethylaminobutyl methacrylamide, 1 ,3-bis(dimethylamino)propyl acrylate, 1 ,3-bis(dimethylamino)propyl methacrylate, 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl methacrylate, 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl acrylate, sodium acrylate, sodium methacrylate, sodium ethacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate, [2- (acryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 3-((3- methacrylamidopropyl)dimethylammonio)propane-1 -sulfonate, 2-(acryloyloxy)ethyl phosphate, 2-(methacryloyloxy)ethyl phosphate, (2-(methacryloyloxy)ethyl)phosphonate, (2- (acryloyloxy)ethyl)phosphonate, (2-acrylamidoethyl)phosphonate, (2- methacrylamidoethyl)phosphonate, 2-methacryloyloxyethyl phosphorylcholine, N- (carboxymethyl)-2-(acryloyloxy)-N,N-dimethylethan-1-aminium, N-(carboxymethyl)-2-(methacryloyloxy)-N,N-dimethylethan-1-aminium, N-(carboxymethyl)-3-methacrylamido-N,N- dimethylpropan-1-aminium, N-(carboxymethyl)-3-acrylamido-N,N-dimethylpropan-1-aminium, 2- (dimethylamino)ethyl acrylate, 2-(dimethylamino)ethyl methacrylate, / V-[3-(Dimethylamino)propyl] acrylamide, / \ / -[3-(dimethylamino)propyl] methacrylamide, / V-(3-Aminopropyl)acrylamide hydrochloride, / V-(3-aminopropyl)methacrylamide hydrochloride, vinyl sulfonic acid, vinyl phosphonic acid, 3-sulfopropyl methacrylate potassium salt, and 2- methacryloyloxy)ethanesulfonic acid, and combinations thereof.

[0043] Optionally, the printed hydrogels can be coated with polymers both to enhance molecular weight exclusion useful to exclude immune components, as well as to further enhance cohesion of the hydrogel device. Such coating polymers promote the exclusion of proteins that could otherwise harm transplanted cells or trigger a detrimental immune response, while still allowing sufficient diffusional transfer of oxygen, glucose, insulin, and metabolic waste products.

[0044] The coating polymers may be one of the above-described synthetic polymers. In some embodiments, the synthetic polymer for coating hydrogels is a poly[APM-co-DMAEA-co-SBMA] terpolymer (PADS) which is a type of PMM polymer. APM is 3-aminopropylmethacrylamide which contains a primary amine to react with a click chemistry group to functionalize the polymer.DMAEA is N,N-dimethylaminoethylacrylate and SBMA is [2-(methacryloyloxy)ethyl]dimethyl-(3- sulfopropyl)ammonium hydroxide. A key formulation is the 1 :1 :2 ternary copolymer, where 1 :1 :2 represents initial molar feed ratios of 1 :1 :2 of APM, DMAEA, and SBMA. The molar ratios can vary by up to ±50%, ±20%, ±10%, ±5% or ±3% each, and additional comonomers can be contemplated.

[0045] The three comonomers have significantly different reactivity ratios, which introduces drift during copolymerization - however, due to the controlled (‘living’) nature of this polymerization, this is translated into gradient copolymer chains where each chain has more SBMA at the initiator end, and more cationic comonomers at the other end. An advantage of the gradient distributions is that it facilitates electrostatic binding and covalent reaction of the cationrich end of the polymer to the anionic hydrogel, while leaving the SBMA-rich end to be tissuefacing. The amine of APM contributes reactivity as well as cationic density for coating onto anionic core hydrogels and / or for functionalization of the PADS. DMAEA (N,N-dimethylaminoethyl acrylate) has a tertiary amine in the pendant group which also contributes cationic charge density to assist with coating onto capsules. DMAEA has the additional benefit that it undergoes chargeshifting by hydrolysis with a half-life measured in hours to days. Finally, SBMA is [2- (methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, a zwitterionic monomer that further improves the antifouling properties of the coating by increasing the hydrophilicity of the materials.

[0046] A polymer derived of PADS that excludes SBMA, namely poly[3- aminopropylmethacrylamide-co-2-(dimethylamino)ethyl acrylate] (PAD), is also contemplated herein. PAD retains the advantage of APM and DMEAE as explained above and can be employed in the same manner as PADS.

[0047] The physical interactions, rather than chemical interactions, between the microgels in their jammed state (at high packing densities) afford the material with solid-like properties at low strain and liquid-like properties at high strain. These materials are thus inherently shear-thinning, which is a highly important property for the ability to facilely extrude the material through a syringe and for the material to rapidly recover the solid-like characteristics immediately after extrusion to retain the shape fidelity of the filament being extruded. As used herein the term or the concept of retaining a shape or substantially retaining a shape means that the macroscopic geometry or shape obtained by the output of the printer has substantially the same shape as the shape formed by the movement of the nozzle in a given plane or the overall shape coded in the printer.

[0048] The microgel particles can form jammed systems at concentrations of 1 - 20 wt% with respect to the total mass of the microgels and solvent, preferably 1-5 wt%.

[0049] Microgels can be functionalized with components to improve their cellular interactions (i.e. RGD peptide sequences), and to include functional groups to allow the microgels to covalently crosslink with each other and with optionally added reactive polymers after printing. Finally, alginate can also be functionalized with analogous groups that can interact with the jammed microgel systems.

[0050] In one embodiment, the material printed with these hydrogels is a high surface area device resembling as a hydrogel patch, formed by extrusion of a ca 200 - 1200, and preferably 400 - 800 micrometer thick continuous gel string into an overlapping pattern. Therapeutic cells are contained within the string segments. Partial fusion at the overlap points of string segments printed in such overlapping patterns and with moderate vertical offsets provide mechanical strength to the overall structure. The resulting devices can be printed in a variety of sizes and shapes, for example they can have 1 - 10 and preferably 3-5 printed layers, be of oval shape, and have dimensions of, e.g., 6 by 5 cm, 5 by 4 cm, or 6 by 4 cm, which is sufficient to encapsulate a therapeutic load of beta cells in one or multiple implantable hydrogel devices.

[0051] It should be noted that multiple materials can be co-printed in the hydrogel patches, including different cell types for therapeutic purposes. In the case of multiple cell types, they can be incorporated into a single material, separated into multiple materials, or some combination thereof.

[0052] Hydrogel patches may further include materials (either cell-containing or cell-free) that confer the overall device with increased resistance to deformation, including during handling and implantation. These include inner layers or perimeters printed using a hydrogel of higher solids loading, as well as dome-shaped devices.

[0053] For enhancement of strength, stiffening additives such as cellulose nanofibers that are chemically modified so as to covalently integrate with the other gel formers are optionally added. Additionally, these additives may be functionalized with components to enhance cell viability and function, or participate in covalent crosslinking with linear polymers or microgels, or induce more rapid vascularization (i.e. RGD peptide or gastrin functionalization).

[0054] Therapeutic islet cell clusters can be mixed with the gel formers, suitably at concentrations of 1 ,000 - 100,000, and preferably 10,000 to 50,000 lEQ / mL to form the hydrogel patches.

[0055] Although pancreatic islets are the main example contemplated herein, there are other stand-alone therapeutic cells that may be contemplated including mesenchymal stromal cells (MSC), hepatocytes or other endocrine cells. As well, supportive cells including MSCs, endothelial cells, vascular microvessels, fibroblasts, etc., can be added within or around the hydrogel devices to enhance the health and function of implanted islets, encourage vascular ingrowth into the implanted patches or both.

[0056] The overall design of the patch can have a filament size between 200 pm and 1200 pm, preferably between 300 pm - 700 pm, an infill density between 10% and 80%, preferably from 30%-70%. These parameters can be tuned to produce gaps within the structure that are roughly between 250 pm and 600 pm wide, for the purpose of enhancing natural vascularization. The infill pattern can be a rectilinear, hexagonal, or triangular grid. There may also be a perimeter on each layer made out of the same material (with the same cells) or a material to impart greater mechanical strength (with or without cells). The overall patch shape can be a rectangular prism, but, preferably, the edges are rounded. The layers themselves may also be tapered in the height of the patch as well.

[0057] Printing technologies offer multiple ways to improve the function of the printed patches. For example, multi-layer patches can be printed into shapes that facilitate rolling the patch along one axis, facilitating laparoscopic administration to the desired implantation site followed by unrolling. The hydrogel patches can be given dome-shaped structures by stepping the z- dimension upwards towards the interior of the hydrogel patch, effectively turning it into a flat dome that is much more resistant to folding deformations and other distortions. Some designs may facilitate implantation. For example, if printed with a 3,- 4- or, preferably, 6-axis printhead, the structure can have a curved, dome or bowl-like structure that may fit better into a specific intended implantation site, as well as further supporting the structure and limiting the chance of the patch deforming during handling, implantation or long-term in vivo implantations. Further, the patch can be designed and sized for implantation into specific animal models, including in the intraperitoneal cavity, within fascia outside of the peritoneal cavity, or subcutaneously.

[0058] It should be noted that the hydrogel patches may shrink up to approximately 25% during gelation, and the degree of shrinking needs to be taken into account within the design.

[0059] Coating polymers promote the exclusion of proteins that could otherwise harm transplanted cells or trigger a detrimental immune response, while still allowing sufficient diffusional transfer of oxygen, glucose, insulin, and metabolic waste products. These coating may also strengthen the mechanical characteristics of the hydrogel patches as a whole, mitigate immune recognition, and prevent cell escape.

[0060] The printed structures, along with therapeutic cells, may also include materials that could encourage vascular ingrowth. These could include:• Growth factors such as one or more of VEGF, PDGF, Ang, HGF, IGF, TNF-a, FGF-2, IL- 6, SCF, etc.,• Platelet-rich plasma components,• Immature vascular cells such as SC-derived endothelial cells, smooth muscle cells,• Microvessels isolated from adipose or pancreatic tissues,• Cell spheroids consisting of, or incorporating, endothelial cells, MSCs, fibroblasts in form of cell spheroids / organoids that are less than 1mm in diameter, preferably less than 400 pm.

[0061] These entities may be included into the printed hydrogel structure, may be coated onto the structure in a wash after printing, and / or be included in an additional hydrogel structure that encases the printed material.

[0062] The printed hydrogel patch, after all post-processing steps, can be placed in a mould slightly larger than the printed structure and the encompassing hydrogel can be subsequently formed around it in the mould.

[0063] The encompassing hydrogel may optionally contain (potentially recombinant human) collagen I, collagen III, collagen IV, laminin, fibrin, hyaluronic acid, etc., and combinations thereof.

[0064] This mould may contain an array of pins, to reserve space for improved post-implant penetration of vasculature into the hydrogel patch.EXAMPLE 1

[0065] Microgels comprised of methacrylic anhydride crosslinked with 7 mol% of bis(2- methacryloyl)oxyethyl disulfide were generated by precipitation polymerization and subsequently functionalized with 5 mol% ureidopyrimidinone (UPy). These microgels were then swollen in 4- (2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (HBS) at a concentration of 7.5 wt% and the resulting granular microgel matrix was shear thinning and able to be printed in air, without the use of a suspension bath system. The material itself, due to the self-complementary hydrogen-bonding interactions between two UPy-functional groups on differing microgel particles, supported its own weight when extruded across 5 mm, 10 mm, 20 mm and 25 mm gaps, as shown in Fig. 1.

[0066] Fig. 1 shows a printed filament solely comprised of 7.5 wt% UPy-functionalized microgels spanning across gaps of 5 mm, 10 mm, 20 mm, and 25 mm while supporting their own weight.

[0067] The UPy-functionalized microgels were combined with a mixture of alginate, and two additional synthetic gel formers: poly(methyl vinyl ether-alt-maleic anhydride (PMM) functionalized with cysteamine vinyl sulfone (CVS), and with homocysteine thiolactone (HTL)), to form PMM-CVS and PMM-HTL, respectively, to form a bioink system that can be printed in air and then be subsequently crosslinked in a calcium solution, while, at a slower rate, the synthetic gel formers generate their own interpenetrating network by forming covalent crosslinks within the microgel-laden, alginate gelled system.

[0068] This was done by dispersing 4.0 wt.% UPy-functionalized microgels with 2.5 wt.% MVG-AIg, 1 wt.% PMM-HTL, 1 wt.% PMM-CVS in HBS. The UPy-functionalization of the microgels is important because interactions between the self-complementary UPy groups generates hydrogen bonds that can stabilize the jammed microgels at lower concentrations, which is desirable to minimize the overall solids content of the system.

[0069] The bioink was extruded with a 20G nozzle with a flow rate of 2.0 pL / s and a tip speed of 5.0 mm / s onto a glass dish to form a 20 mm x 20 mm rectilinear hydrogel patch with 2 layers using a commercially available 3D printer. Immediately after printing, a 50 mM CaCl2 solution was poured onto the patch to rapidly crosslink the alginate and set the patch. The patch was left to cure for up to 20 (typically 5-10 mins) minutes to allow for the synthetic gel formers to crosslink and generate a dual interpenetrating network around the microgels.

[0070] Fig. 2 shows the resulting patch, which is opaque white due to phase separation upon CaCh addition. This can be adjusted through modification of the microgel functionality, using different concentrations of the respective microgels and polymers, or both.EXAMPLE 2Synthesis of MG-UPy / CVS Microgels

[0071] Methacrylic anhydride (MeAn) based microgels were synthesized as previously reported in WO2022120478A1. In a representative example, MeAn based particles (7.64 g, 0.0467 mol) were suspended in 300 mL of N,N-dimethylformamide (DMF) in a 500 mL round bottom flask equipped with a stir-bar. To the reaction mixture, triethylamine (4.72 g, 0.0467 mol) was added, followed by a drop-wise addition of UPy-hexylamine (0.623 g, 2.33 mmol) dissolved in 20 mL DMF. The reaction mixture was left overnight at room temperature and subsequently split equally into 2 fractions. To one half of the reaction mixture, triethylamine (0.432 g, 4.26 mol) was added, followed by the drop-wise addition of cysteamine vinyl sulfone hydrochloride (0.491 g, 2.12 mmol) dissolved in 2 mL of a 1 :1 mixture of dimethyl sulfoxide (DMSO) : DMF. The reaction mixture was left to stir at room temperature (22 °C) for 2 h and was subsequently washed 2x with a 2:1 mixture of DMF and diethyl ether, 1x with of 0.9 wt% NaCI, and 2x with of distilled water at approximately pH 5 by centrifugation and resuspension cycles. The microgels were further hydrolyzed in water at pH 11 for 20 minutes, and subsequently pH adjusted to pH 5 and lyophilized, resulting in MeAn particles functionalized with UPy and CVS (MG-UPy / CVS).Synthesis of MG-UPy / HTL Microgels

[0072] MeAn microgels functionalized with UPy and homocysteine thiolactone (HTL) were prepared similarly to the UPy / CVS microgels described above. Briefly, as described above to the CVS functionalized microgels, homocysteine thiolactone (0.326 g, 2.12 mmol) and triethylamine (0.432 g, 4.26 mmol) were added to the reaction mixture and left to stir for 2 h at room temperature. The microgels were purified as described above, resulting in UPy and HTL functionalized microgels (MG-UPy / HTL).Preparation of MG-UPy-CVS / HTL Microgels Solutions for Air-Printing

[0073] As a representative example, the following procedure was used to prepare a solution of microgels for air printing. To prepare a 6 wt.% suspension of total microgel, a 1 :1 wt. ratio of the CVS and HTL functionalized MG-UPy microgels (37.5 mg of each) was used. The lyophilizedmicrogels were resuspended in 1 mL of HEPES buffered saline (HBS), and the pH was adjusted to pH 7.4-7.8. The microgel suspension was loaded into a syringe and was used to air-print a 2- layer raft with 1 cm by 1 cm dimensions and a rectilinear pattern.Coating Air-Printed Hydrogel Patches

[0074] The air-printed MG raft was doubly coated with a layer-by-layer deposition approach of exposing the raft to a solution of a polycation, followed by washing steps and an exposure to a solution of polyanion, to demonstrate the ability to covalently crosslink the microgel structure with homocysteine thiolactone-polyamine-vinyl sulfone chemistry. As the air printed microgel structures are anionic and contain HTL and CVS groups, the MG LifeRaft was first coated with a polycation, PAD75-25k, which electrostatically binds to the structure and further causes ringopening of the homocysteine thiolactone groups of the HTL microgels by aminolysis, yielding free thiol groups which then react with the vinylsulfone groups of the CVS microgels in a thiolene manner. The polycation coated MG raft can then be coated with a polyanion, such as PHMS-60- 25k, to form a covalently crosslinked coating by reaction of residual amines of the polycation with HTL groups on the reactive polyanion, PHMS-60-25k.

[0075] As a representative example, the following procedure was used to form covalently crosslinked, coated MG hydrogel patch. An air printed 2-layer raft was transferred to a glassbottom 6-well plate containing 2 mL of HBS. The HBS was removed and 1.5 mL of a 0.5 wt.% solution of PAD75-25k was added to the raft for 6 min, followed by 2 washes with 1.5 mL of HBS. The raft was then exposed to 1.5 mL of 0.5 wt.% PHMS-60-25k (rhodamine labelled) in HBS for 6 min, followed by 2 washed with 1 .5 mL of HBS. The doubly coated MG raft was left overnight in HBS and imaged using a Nikon A1 confocal and Nikon Ti inverted microscopes. A stitched image of the overall microgel structure is shown in Figs. 3A-3B for the brightfield (3A) and TRITC (3B) channels. The TRITC channel shows the final coating polymer PHMS-60-25k (rhodamine labelled), which demonstrated the layer-by-layer coating of the MG raft was successful.

[0076] The doubly coated MG hydrogel patch was imaged on the confocal in the TRITC channel, and Fig. 4 shows that the polymer was PHMS-60-25k polymer had indeed formed a distinct coating layer on the surface of the structure.Chemical Stress Test for Covalent Crosslinking

[0077] To test for covalent crosslinking, the doubly coated MG hydrogel patch was treated with sodium citrate, followed by sodium hydroxide. Under these conditions, if the raft and coating polymers remain intact, this would suggest the presence of covalent cross-linkages via HTL / CVS / polyamine reactive groups.

[0078] As an example, a doubly coated MG raft was treated with 3 mL of 70 mM sodium citrate and 1 mL of 1 M sodium hydroxide. After about 30 min, and gentle agitation, the raft was on a Nikon A1 confocal, showing the brightfield (left), TRITC (middle), and merged brightfield / TRITC (right) channels in Figs. 5A-5C. These images show the microgel structure as well as the coating intact, which suggests covalently crosslinking of the air-printed, coated MG hydrogel patch.

Claims

WHAT IS CLAIMED IS:

1. A method of printing a hydrogel, the method comprising: extruding a gel forming system comprising microgel particles functionalized with a hydrogen-bonding supramolecular moiety to obtain extruded microgel particles; wherein the extruded microgel particles form a hydrogel that substantially retains its shape in air.

2. The method of claim 1 , wherein the extruding includes extruding into or through air onto a supporting surface.

3. The method of claim 2, wherein the shape of the hydrogel is independent of the supporting surface.

4. The method of any one of claims 1 to 3, wherein the gel forming system is extruded through a nozzle or needle.

5. The method of any one of claims 1 to 4, wherein the gel forming system comprises microgel particles comprising a synthetic polymer functionalized with the hydrogenbonding supramolecular moiety, optionally a first subset of microgel particles comprising a first synthetic polymer and a second subset of microgel particles comprising a second synthetic polymer, wherein one or both, preferably both, of the first synthetic polymer and the second synthetic polymer are functionalized with the hydrogen-bonding supramolecular moiety, preferably wherein the first synthetic polymer and the second synthetic polymer comprise cross-linking moieties.

6. The method of any one of claims 1 to 5, wherein the gel forming system further comprises one or more gel forming polymers, optionally, wherein the one or more gel forming polymers are selected from alginate, hyaluronic acid, gelatin, elastin, cellulose, hydroxypropylcellulose, carboxymethylcellulose, methylcellulose, synthetic polymers and combinations thereof.

7. The method of claim 6, wherein the one or more gel forming polymers and the microgel particles together have a total concentration of from 1 to 20 wt. % in the gel forming system.

8. The method of claim 6 or 7, wherein the synthetic polymers are functionalized with moieties that crosslink with each other.

9. The method of claim 8, wherein the hydrogel is formed by two gelling reactions, the hydrogel is first partially gelled by the formation of hydrogen-bonds by the hydrogenbonding supramolecular moiety, and then further gelled by the formation of crosslinks between the moieties of the synthetic polymers.

10. The method of claim 6 or 7, wherein the one or more gel forming polymers are the alginate.

11. The method of claim 10, wherein the hydrogel is formed by two gelling reactions, the hydrogel is first partially gelled by the formation of hydrogen-bonds and then further gelled by the formation of ionic crosslinks to crosslink the alginate.

12. The method of claim 6 or 7, wherein the one or more gel forming polymers are the alginate and the synthetic polymers.

13. The method of claim 12, wherein the hydrogel is formed by three gelling reactions, the hydrogel is first partially gelled by the formation of hydrogen-bonds and then further gelled by the formation of ionic crosslinks to crosslink the alginate and the formation of crosslinks between the moieties of the synthetic polymers.

14. The method of any one of claims 9, 11 or 13, wherein the hydrogel is allowed to partially gel while the hydrogel is in contact with the air.

15. The method of any one of claims 5 to 14, wherein the synthetic polymers have a backbone that comprises monomeric units selected from 2-aminoethyl acrylate, 2-aminoethyl methacrylate, 2-aminoethyl acrylamide, 2-aminoethyl methacrylamide, 3-aminopropyl acrylate, 3-aminopropyl methacrylate, 4-aminobutyl acrylate, 4-aminobutyl methacrylate, 4-aminobutyl acrylamide, 4-aminobutyl methacrylamide, acrylic acid (AA), acrylamide, methacrylic acid (MAA), 2-hydroxypropylacrylamide (HPA), 2- hydroxypropylmethacrylamide (HPMA), 2-hydroxyethylacrylate (HEA), 2- hydroxyethylmethacrylate (HEMA), 2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethyl phosphate (MPC), carboxy betaine methacrylate (CBM), carboxybetaine methacrylamide, N,N-dimethylaminoethylacrylate (DMAEA), N,N-dimethylaminoethyl methacrylate, 3-N,N- dimethylaminopropyl acrylamide, 3-N,N-dimethylaminopropyl methacrylamide, 3-N,N- dimethyliaminopropyl acrylate, 3-N,N-diaminopropyl methacrylate, 4-N,N-dimethylaminobutyl acrylamide, 4-N,N-dimethylaminobutyl methacrylamide, 1 ,3- bis(dimethylamino)propyl acrylate, 1 ,3-bis(dimethylamino)propyl methacrylate, 2-((2- (dimethylamino)ethyl)(methyl)amino)ethyl methacrylate, 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl acrylate, sodium acrylate, sodium methacrylate, sodium ethacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate, [2- (acryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 3-((3- methacrylamidopropyl)dimethylammonio)propane-1 -sulfonate, 2-(acryloyloxy)ethyl phosphate, 2-(methacryloyloxy)ethyl phosphate, (2-(methacryloyloxy)ethyl)phosphonate, (2-(acryloyloxy)ethyl)phosphonate, (2-acrylamidoethyl)phosphonate, (2- methacrylamidoethyl)phosphonate, 2-methacryloyloxyethyl phosphorylcholine, N- (carboxymethyl)-2-(acryloyloxy)-N,N-dimethylethan-1-aminium, N-(carboxymethyl)-2- (methacryloyloxy)-N,N-dimethylethan-1-aminium, N-(carboxymethyl)-3-methacrylamido- N,N-dimethylpropan-1-aminium, N-(carboxymethyl)-3-acrylamido-N,N-dimethylpropan-1- aminium, 2-(dimethylamino)ethyl acrylate, 2-(dimethylamino)ethyl methacrylate, / \ / -[3- (Dimethylamino)propyl] acrylamide, / V-[3-(dimethylamino)propyl] methacrylamide, / \ / -(3- Aminopropyl)acrylamide hydrochloride, / V-(3-aminopropyl)methacrylamide hydrochloride, vinyl sulfonic acid, vinyl phosphonic acid, 3-sulfopropyl methacrylate potassium salt, 2- (methacryloyloxy)ethanesulfonic acid, and combinations thereof.

16. The method of any one of claims 5 to 14, wherein the synthetic polymer is poly(methyl vinyl ether-alt-maleic anhydride) (PMM), poly-ornithine, poly-L-lysine (PLL), epsilon- polylysine, poly[APM-co-DMAEA-co-SBMA] terpolymer (PADS), or poly[3- aminopropylmethacrylamide-co-2-(dimethylamino)ethyl acrylate] (PAD).

17. The method of any one of claims 1 to 16, wherein the hydrogen-bonding supramolecular moiety is ureido-pyrimidinone (UPy).

18. The method of any one of claims 1 to 17, wherein the gel forming system further comprises a saline such as HEPES buffered saline.

19. The method of any one of claims 1 to 18, further comprising coating the printed shape of the hydrogel with a further synthetic polymer.

20. The method of claim 19, wherein the further synthetic polymer has a backbone that comprises monomers selected from 2-aminoethyl acrylate, 2-aminoethyl methacrylate, 2-aminoethyl acrylamide, 2-aminoethyl methacrylamide, 3-aminopropyl acrylate, 3- aminopropyl methacrylate, 4-aminobutyl acrylate, 4-aminobutyl methacrylate, 4- aminobutyl acrylamide, 4-aminobutyl methacrylamide, acrylic acid (AA), acrylamide, methacrylic acid (MAA), 2-hydroxypropylacrylamide (HPA), 2- hydroxypropylmethacrylamide (HPMA), 2-hydroxyethylacrylate (HEA), 2- hydroxyethylmethacrylate (HEMA), 2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethyl phosphate (MPC), carboxy betaine methacrylate (CBM), carboxybetaine methacrylamide, N,N-dimethylaminoethylacrylate (DMAEA), N,N-dimethylaminoethyl methacrylate, 3-N,N- dimethylaminopropyl acrylamide, 3-N,N-dimethylaminopropyl methacrylamide, 3-N,N- dimethyliaminopropyl acrylate, 3-N,N-diaminopropyl methacrylate, 4-N,N- dimethylaminobutyl acrylamide, 4-N,N-dimethylaminobutyl methacrylamide, 1 ,3- bis(dimethylamino)propyl acrylate, 1 ,3-bis(dimethylamino)propyl methacrylate, 2-((2- (dimethylamino)ethyl)(methyl)amino)ethyl methacrylate, 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl acrylate, sodium acrylate, sodium methacrylate, sodium ethacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate, [2- (acryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 3-((3- methacrylamidopropyl)dimethylammonio)propane-1 -sulfonate, 2-(acryloyloxy)ethyl phosphate, 2-(methacryloyloxy)ethyl phosphate, (2-(methacryloyloxy)ethyl)phosphonate, (2-(acryloyloxy)ethyl)phosphonate, (2-acrylamidoethyl)phosphonate, (2- methacrylamidoethyl)phosphonate, 2-methacryloyloxyethyl phosphorylcholine, N- (carboxymethyl)-2-(acryloyloxy)-N,N-dimethylethan-1-aminium, N-(carboxymethyl)-2- (methacryloyloxy)-N,N-dimethylethan-1-aminium, N-(carboxymethyl)-3-methacrylamido- N,N-dimethylpropan-1-aminium, N-(carboxymethyl)-3-acrylamido-N,N-dimethylpropan-1- aminium, 2-(dimethylamino)ethyl acrylate, 2-(dimethylamino)ethyl methacrylate, / \ / -[3- (Dimethylamino)propyl] acrylamide, / \ / -[3-(dimethylamino)propyl] methacrylamide, / \ / -(3- Aminopropyl)acrylamide hydrochloride, / V-(3-aminopropyl)methacrylamide hydrochloride, vinyl sulfonic acid, vinyl phosphonic acid, 3-sulfopropyl methacrylate potassium salt, and 2-(methacryloyloxy)ethanesulfonic acid, and combinations thereof.

21. The method of claim 19, wherein the further synthetic polymer is poly(methyl vinyl ether- alt-maleic anhydride) (PMM), poly-ornithine, poly-L-lysine (PLL), epsilon-polylysine, poly[APM-co-DMAEA-co-SBMA] terpolymer (PADS), or poly[3- aminopropylmethacrylamide-co-2-(dimethylamino)ethyl acrylate] (PAD).

Images