Fabrication of polymeric structures

Abstract

Provided herein is a method of forming a porous polymeric structure, the method comprising the steps of: providing an aqueous two-phase system (ATPS) comprising a first phase containing a first photo-curable macromer and a second phase containing a second photo-curable macromer; transmitting acoustic energy to the system to induce shear mixing of the phases, resulting in a monophasic system; removing the acoustic energy allowing de-mixing of the monophasic system; irradiating the de-mixed system with curing radiation to promote curing of the first and the second photo-curable macromer; and removing one of the cured macromers to obtain the porous polymeric structure.

Description

[0001] FABRICATION OF POLYMERIC STRUCTURES

[0002] FIELD OF THE INVENTION

[0003] The present disclosure relates to methods for the fabrication of polymeric structures, and specifically to method for the fabrication of porous polymeric structures, for example porous hydrogel structures.

[0004] BACKGROUND OF THE INVENTION

[0005] Porous polymeric structures find applications in a variety of fields. In bio-technological fields, pores within bio-compatible polymeric structures, such as porous hydrogel structures, are useful and necessary structures, for example to mimic blood vessels or to guide cell growth and development in micro-physiological environments.

[0006] A variety of microfluidic organ-on-chip models use microfabricated channels in biocompatible polymer, such as poly dimethylsiloxane (PDMS) or other bio-compatible thermoplastic materials to approximate blood vessels. These are most often rectangular in cross section and on the order of 100’s of microns tall and wide. In some cases, extrusion- or lightbased additive manufacturing techniques are used to directly fabricate hollow channels, for example to replicate blood vessels. In some instances, cells can be directly encapsulated within a polymeric scaffold material at the time of fabrication, but in many, cells are introduced into the channels following initial fabrication.

[0007] In this context, “aqueous two-phase systems” (also known as “water-in-water emulsions”) have emerged as a useful tool to create and control pore size and interconnectivity within a biocompatible polymer, for example within hydrogel structures. By carefully selecting appropriate polymer components and solution conditions, one can control the mechanism and kinetics by which these emulsions decompose into two separate phases (a process also referred to as “demixing”). One such mechanism, spinodal decomposition, can result in highly interconnected pore morphology. However, controlling the final pore size and interconnectivity has been a non-trivial task, which limits the applications of aqueous two-phase emulsions in in vitro models.

[0008] There remains therefore an opportunity to address limitations of existing procedures for the fabrication of porous polymeric structures.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention provides a method of forming a porous polymeric structure, the method comprising the steps of: providing an aqueous two-phase system (ATPS) comprising a first phase containing a first photo-curable macromer and a second phase containing a second photo-curable macromer, transmitting acoustic energy to the system to induce shear mixing of the phases, resulting in a monophasic system, removing the acoustic energy allowing de-mixing of the monophasic system, irradiating the de-mixed system with curing radiation to promote curing of the first and the second photo-curable macromer, and removing one of the cured macromers to obtain the porous polymeric structure.

[0011] The proposed method effectively exploits phase separation dynamics within aqueous two- phase systems to tailor the porous architecture of polymers generated from those systems. The transmission of acoustic energy to an aqueous two-phase system advantageously generates acoustically driven flow within the system that mechanically homogenizes the originating solutions of incompatible macromers before allowing those macromers to separate.

[0012] As the acoustic energy is removed, the homogeneous system tends to gradually de-mix into the originating solutions of incompatible macromers. Since the macromers are photocurable, exposure to curing radiation promotes cross-linking within each macromer, effectively freezing the system morphology into the de-mixing phase status at that time. Halting the phase separation at specified time points by irradiation with curing radiation affords formation of both distinct pore sizes and degrees of pore interconnectivity. By taking advantage of the transient phase separation mechanisms at play in the system, the method affords creation of both connected and disconnected pores. Advantageously, in case of bio-compatible polymers, this can be accomplished at physiological temperature, pH, and osmolarity.

[0013] In some embodiments, at least one of the first photo-curable macromer and the second photo- curable macromer is a hydrogel macromer. In those instances, by combining acoustic mixing and photocuring the method affords control over size, orientation, and interconnectivity of pores in hydrogels created from aqueous two-phase systems. When desired, the pores created remain consistent throughout the bulk of the hydrogel with potentially achievable length scales from units to hundreds of pm (e.g. 5-120 pm).

[0014] The method may be performed using an integrated 3D printing system which combines the required acoustic stimulation and irradiation with curing radiation.

[0015] Accordingly, in some embodiments the method comprises: introducing a print head having a cavity containing gas into the aqueous two-phase system to form a submerged gas-liquid interface between the gas and the aqueous two-phase system, wherein said gas-liquid interface is constrained to the print head and defines a printing surface, transmitting the acoustic energy to the submerged gas-liquid interface to induce the shear mixing of the phases, resulting in the monophasic system, removing the acoustic energy from the submerged gas-liquid interface, allowing the demixing of the monophasic system, and irradiating the de-mixed system with the curing radiation by projecting said curing radiation on the submerged gas-liquid interface with the print head, thereby promoting curing of the first and the second photo-curable macromer at the printing surface.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Embodiments of the invention will be now described with reference to the following nonlimiting drawings, in which:

[0018] Figure 1 shows a sample 3D reconstruction of a pore network inside a hydrogel of the kind described in the Examples, Figure 2 shows a schematic overview of the materials and fabrication process for aqueous two- phase emulsion hydrogels,

[0019] Figure 3 shows results of the confocal analysis of pore size for samples described in the Examples, in which the sample images were taken from the confocal Z-stacks at the time points tested,

[0020] Figure 4 shows a sample 2D image taken from the distance transform of the reconstructed pore network, in which a higher intensity corresponds to higher depth within the pore,

[0021] Figure 5 shows a plot of calculated pore diameter for the low and high DoF GelMA + PVA-T gels described in the Examples cured at various delay periods. The star indicates the manually calculated pore diameter based off the confocal Z-stacks, rather than the 3D reconstruction,

[0022] Figure 6 shows a plot of calculated pore diameter for the GelMA blends with PVA-Tyramine vs PVA in respect of samples described in the Examples,

[0023] Figure 7 shows a confocal microscopy image of a hydrogel of the kind described herein wherein concentric regions of the liquid pre-polymer solution were exposed to curing radiation at progressive longer delays following removal of the acoustic irradiation, and

[0024] Figure 8 shows a confocal microscopy image of a hydrogel of the kind described herein wherein rectangular regions of the liquid pre-polymer solution were exposed to curing radiation at progressive longer delays following removal of the acoustic irradiation.

[0025] DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention relates to a method of forming a porous polymeric structure.

[0027] By “porous polymeric structure” is meant a material made of polymer having voids within its volume. From the morphological standpoint, said voids may present any morphology conformal (i.e. having the same shape) to the corresponding phase morphology that form during de-mixing of the system. For example, said voids may present as closed individual voids separate from one another, or as a network or interconnected channels extending within the volume of the polymeric structure, or both.

[0028] The method of the invention comprises the provision of an aqueous two-phase system (ATPS). Conventionally, an aqueous two-phase system (ATPS), which may also be referred to as a “water-in-water emulsion”, is an all-aqueous system fabricated from two immiscible or partially miscible aqueous phases to create a multi-component system under certain conditions (for example temperature or pH). The immiscibility of the aqueous phases derives from the chemical incompatibility of constituents forming each aqueous phase. Said aqueous two-phase system may also be referred herein simply as “system”.

[0029] The aqueous two-phase system (ATPS) in the method of the invention comprises a first phase containing a first photo-curable macromer and a second phase containing a second photo- curable macromer. Formation of the required aqueous two-phase system in the method of the invention would therefore be understood as being inherently achieved by blending a first and a second photo-curable macromers which are incompatible.

[0030] The term “photo-curable macromer” is used herein to refer to an oligomer or a polymer having two or more functional groups that upon exposure to curing radiation promote cross-linking of the macromer. Accordingly, the photo-curable macromer may also be referred to as a photo- cross-linkable macromer. Said two or more functional groups will be understood to encompass functional groups that can directly cross-link upon irradiation, as well as groups that can crosslink upon irradiation in presence of a photo -initiator.

[0031] The aqueous two-phase system may be prepared according to any procedure known to the skilled person based on the photo-curable macromers intended for use.

[0032] For instance, the aqueous two-phase system may be prepared by dissolving the first photo- curable macromer in water within its range of critical concentrations to form a first aqueous solution, and the second photo-curable macromer in water within its range of critical concentration to form a separate second solution. The solutions may then be combined to obtain the aqueous two-phase system. Alternatively, the first (or second) photo-curable macromer may be added directly into the solution of the second (or first) photo-curable polymer within its range of critical concentrations.

[0033] Formation of aqueous two-phase systems may depend on a combination of thermodynamic and electrostatic factors, including the molecular weight of the macromers, the interfacial tension and relative concentration of the macromers, pH, temperature, and the presence of ions in solution. Given a selection of suitable macromers, a skilled person would be capable of devising suitable conditions for formation of the required aqueous two-phase system.

[0034] For instance, formation of the required aqueous two-phase system may be facilitated by dissolving a salt to the aqueous solution(s). The resulting alteration of the ionic environment within the aqueous solution may reduce the electrical potential between the two phases, increasing the effective interfacial tension and thus accentuating the chemical incompatibility of the macromers.

[0035] Accordingly, in some embodiments the aqueous two-phase system contains a dissolved salt.

[0036] The salt may be used in any amount conducive to provision of the required aqueous two-phase system. In some embodiments, the salt is used at physiological concentration.

[0037] Example of suitable salts in that regard include one or more of sodium chloride, potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, or sodium citrate.

[0038] Formation of the required aqueous two-phase system may also be facilitated by altering the pH of the aqueous solution(s) to achieve the required system criticality.

[0039] In some embodiments, the aqueous two-phase system is at a pH from 2 to 10.

[0040] In some embodiments, the aqueous two-phase system is at physiological pH. The term “physiological pH” as used herein refers to a pH value that is found in a normal, non-pathologic cell or subject. In some embodiments, physiological pH is between pH 5 - 8. In some embodiments, physiological pH is pH 7-7.5, for example, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, or pH 7.5. In some embodiments, physiological pH is pH 6.5-7.5. In some embodiments, physiological pH is pH 5, pH 5.5, pH 6, pH 6.5, pH 7, pH 7.5, or pH 8.

[0041] The possibility to perform the method of the invention using the aqueous two-phase system at physiological pH is particularly advantageous, especially in the context of biological applications. For instance, conventional aqueous two-phase systems may preclude the possibility of encapsulating cells directly within the material at the time of fabrication. In that regard, non-physiologic al pH would stress cells, resulting in either cell death or a shift in phenotype to disqualify them as representative of human physiology. This is an issue because introducing cells into micrometer-scale channels after fabrication is a cumbersome and error- prone process.

[0042] The amounts of the first photo-curable macromer and the second photo-curable macromer in the system may also be tailored to provide the required aqueous two-phase system.

[0043] In some embodiments, the first photo-curable macromer is provided in an aqueous solution at a concentration of at least 10 mg / ml, for example from 10 mg / ml to 300 mg / ml.

[0044] In some embodiments, the second photo-curable macromer is provided in an aqueous solution at a concentration of at least 1 mg / ml, for example from 1 mg / ml to 150 mg / ml.

[0045] The molecular weight of the macromers may also influence the formation of the required aqueous two-phase system. As the molecular weight of a macromer increases, so does the tendency to form the required aqueous two-phase system. As a result, macromers with increasingly high molecular weight may be used at corresponding increasingly lower amounts to achieve the required aqueous two-phase system, and vice versa. Formation of an aqueous two-phase system may also be influenced by the relative molecular weight of the macromers. In general, the higher the difference in molecular weight between the macromers, the stronger the tendency to phase separate.

[0046] Accordingly, in some embodiments at least one of the first and second macromer has a molecular weight of at least 30 kDa. In some embodiments at least one of the first and second macromer has a molecular weight of at least 80 kDa.

[0047] In some preferred embodiments, at least one of the first and second macromer has a molecular weight of from 85 kDa to 124 kDa. These instances are particularly advantageous for the provision of highly controllable aqueous two-phase systems.

[0048] Accordingly, in some embodiments the second photo-curable macromer has a molecular weight of at least 80 kDa.

[0049] In some embodiments, the second photo-curable macromer has a molecular weight of 85 kDa- 124 kDa.

[0050] The interfacial tension between the first and the second macromer may also influence the formation of the intended aqueous two-phase system. For instance, it was observed that macromers characterised by an interfacial tension in water of at least 70 mN / m (measured via the pendant drop method) can be particularly effective to provide highly controllable aqueous two-phase systems.

[0051] For example, the first and the second macromer may be characterised by an interfacial tension of at least 50 mN / m.

[0052] In some embodiments, the first and the second macromer may be characterised by an interfacial tension of at least 55 mN / m, at least 60 mN / m, at least 70 mN / m, or at least 80 mN / m.

[0053] Within the life sciences, there is a broad need to create more representative models of human blood vessels within in vitro models (also called organoid, organ-on-chip, or lab-on-chip models). The human vasculature is a multi-scale network of interconnected blood vessels, with diameters ranging from millimeters in arteries to micrometers in capillaries. Many current in vitro organoid models currently only include spheroids of one or a few tissue-specific cell types, and these are used to study, for example, how certain drugs will affect the growth of tumors. The problem is that these results will not be representative of actual physiology in many instances if the drug is not delivered via a blood vessel, because interactions between the cells in the spheroid / organoid and cells making up the blood vessel affect the phenotype of both. Further, in the context of regenerative medicine or tissue engineering, there is an unmet need of developing materials that either serve as scaffolds for regenerating bone or integrate with the patient’s blood vessels to restore blood flow to sites of injury.

[0054] In that context, the method of the invention is therefore particularly useful when performed using biologically relevant photo-curable macromers, for example when used to fabricate porous hydrogel structures. The method of the invention is particularly effective for high- viability tissue engineering, scalable manufacturing and rapid prototyping of biocompatible structures having porosity characteristics that can be tailored to the specific bio-application of interest.

[0055] Accordingly, in some embodiments, at least one of the first photo-curable macromer and the second photo-curable macromer is a hydrogel macromer.

[0056] In some embodiments, the first and the second photo-curable macromer are hydrogel macromers.

[0057] By “hydrogel macromer” is meant herein a compound that upon cross-linking forms a hydrogel. In this context, the term “hydrogel” means a cross-linked network of hydrophilic polymers (natural or synthetic) that can swell in water to capture many times their original mass without dissolution. In the context of the invention, hydrogels will therefore be taken to encompass those based on natural polymers and / or synthetic polymers. Accordingly, hydrogel macromers for use in the invention include macromolecules that comprise a hydrophilic or water-soluble region and one or more cross-linkable regions.

[0058] Hydrogel macromers may be made from a number of hydrophilic polymers. Examples in that regard include polyvinyl alcohols (PVA), polyethylene glycols (PEG), polyvinyl pyrrolidone (PVP), polyalkyl hydroxy acrylates and methacrylates (e.g. hydroxyethyl methacrylate (HEMA), hydroxybutyl methacrylate (HBMA), dimethylaminoethyl methacrylate (DMEMA)), polysaccharides (e.g. cellulose, dextran), polyacrylic acid, polyamino acids (e.g. polylysine, polyethyimine, PAMAM dendrimers), polyacrylamide (e.g. polydimethylacrylamid-co- HEMA, polydimethylacrylamid-co-HBMA, polydimethylacrylamid-co-DMEMA). Hydrogel macromers can be linear or can have a branched, hyperbranched, or dendritic structure.

[0059] In some embodiments, either or both the first and the second photo-curable macromer(s) is / are a hydrogel macromer which is a cross-linkable polysaccharide, such that upon exposure to curing radiation it provides for a polysaccharide hydrogel. Examples of polysaccharide hydrogels include hydrogels containing alginate, cellulose, and glycosaminoglycan.

[0060] In some embodiments, either or both the first and the second photo-curable macromer(s) is a hydrogel macromer comprising one or more of polyethylene glycol diacrylate (PEGDA), gelatin methacryloyl (GelMA), and hexanediol diacrylate (HDD A).

[0061] In some embodiments, the first photo-curable macromer is a hydrogel macromer comprising one or more of polyethylene glycol diacrylate (PEGDA), gelatin methacryloyl (GelMA), and hexanediol diacrylate (HDD A).

[0062] In some embodiments, the first photo-curable macromer is gelatin methacryloyl (GelMA).

[0063] In some embodiments, the second photo-curable macromer is selected from one or more of a polyvinyl alcohol (PVA), a polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), a polyalkyl hydroxy acrylate and methacrylate (e.g. hydroxyethyl methacrylate (HEMA), hydroxybutyl methacrylate (HBMA), dimethylaminoethyl methacrylate (DMEMA)), a polysaccharide (e.g. cellulose, dextran), a polyacrylic acid, a polyamino acid (e.g. polylysine, polyethyimine, PAMAM dendrimers), a polyacrylamide (e.g. polydimethylacrylamid-co- HEMA, polydimethylacrylamid-co-HBMA, polydimethylacrylamid-co-DMEMA), and a conjugate correspondent thereof.

[0064] In some embodiments, the second photo-curable macromer is one or more of polyvinyl alcohol (PVA) and tyramine-conjugated PVA (PVA-Tyramine).

[0065] In some embodiments, the first photo-curable macromer is gelatin methacryloyl (GelMA), and the second photo-curable macromer is one or more of polyvinyl alcohol (PVA) and tyramine- conjugated PVA (PVA-Tyramine). In some embodiments, the aqueous two-phase system contains viable cells suspended therein. For example, the aqueous two-phase system may contain living cells and cell nutrients. The use of those systems may afford the direct fabrication of polymeric structures, for example hydrogel structures, with integrated viable cells within their pores and which can afford cell reproduction, for example to form a shape- specific target tissue.

[0066] As discussed herein, photo-curable macromers for use in the invention comprise two or more cross-linkable groups that can promote cross-linking reactions under curing radiation of the kind described herein, either with or without the aid of a photo-initiator. Said two or more functional groups are therefore understood to encompass functional groups that can directly cross-link upon irradiation, as well as groups that can cross-link upon irradiation in presence of a photo-initiator.

[0067] Accordingly, in some embodiments the aqueous two-phase system comprises a photo-initiator. Photo-initiators can trigger cross-linking of the first and the second macromer upon exposure to the curing radiation. As they are known in the art, photo-initiators are compounds that upon radiation of light generate reactive species (e.g. by decomposition and / or activation of compounds present in the system) that activate polymerization of cross -linkable moieties.

[0068] Suitable examples of photo-initiators for use in the method of the invention include onium salts (e.g. iodonium and sulfonium salts), organimetallic salts (e.g. a metal salt with a non- nucleophilic counter anion, such as ferrocinium salts), pyridinium salts, abstraction type photoinitiators (e.g. benzophenone, xanthones, and quinones), cleavage-type photoinitiators (e.g. benzoin ethers, acetophenones, benzoyl oximes, and acylphosphines).

[0069] In some embodiments, the photo-initiator comprises Tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate.

[0070] In some embodiments, the photo-initiator comprises sodium persulfate. In some embodiments, the photo-initiator is a combination of Tris(2,2'- bipyridyl)dichlororuthenium(II) hexahydrate and sodium persulfate (also referred to as “Ru / SPS”).

[0071] The method of the invention comprises a step of transmitting acoustic energy to the two-phase aqueous system.

[0072] By the expression “acoustic energy” is meant energy that manifests as elastic waves propagating as a pressure variation through a transmission medium. In this context, acoustic energy in the method of the invention would be a form of energy resulting in the formation of elastic waves propagating as a pressure variation through the aqueous two-phase system, resulting shear mixing within the aqueous two-phase system.

[0073] Transmission of acoustic energy to the system induces shear mixing of the two aqueous phases, resulting in the formation of a homogeneous (i.e. monophasic) system. The transmission of acoustic energy to the system generates acoustic vibrations within the system, creating mechanical flow within the system. As a result, the aqueous system is mechanically sheared resulting in the two phases becoming miscible and homogenous, similar to if the solution was held at a temperature below its critical temperature.

[0074] The use of acoustic energy to homogenise the two phases is particularly advantageous, in that it affords control over phase separation and mixing dynamics independently of other conventional control parameters, such as temperature and pH. As a result, the method of the invention affords modulation of the phase dynamics of the aqueous two-phase system irrespective of temperature and pH, which can be selected to be suitable for a given application. This is particularly useful for biological applications, for example in the preparation of biologically compatible porous hydrogels, in that the method affords fabrication of hydrogel structures with tailored porosity morphology at physiological temperature and pH.

[0075] The acoustic energy may be used at any frequency conducive to homogenise the aqueous two- phase system into a homogeneous single phase.

[0076] In some instances, the frequency of the acoustic energy may be selected based on practical considerations in relation to the viscosity of the aqueous phases. For instance, lower frequencies may afford more effective mixing relative to higher frequencies as the viscosity of the aqueous solutions increases.

[0077] Typically, the acoustic energy would have a frequency of at least about 1 Hz. For example, the acoustic energy may have a frequency from about 1 Hz to about 200 Hz.

[0078] In some embodiments, the acoustic energy has a frequency of at least 10 Hz.

[0079] In some embodiments, the acoustic energy has a frequency of from 20 Hz to 25 Hz.

[0080] In some embodiments, the acoustic energy generates acoustic waves within the system having an amplitude, expressed in terms of pressure amplitudes, from O.lPa to IMPa, for example from O.lPa to 5kPa (as measured in the medium transmitting the waves).

[0081] In some embodiments, the acoustic energy generates acoustic waves within the system having a frequency from 1 Hz to 100 kHz, and an amplitude in terms of pressure range from 0.1 Pa to 5kPa.

[0082] Acoustic energy may be generated by any means known to the skilled person. For example, acoustic energy may be generated and transmitted to the system by an acoustic generator placed to emit acoustic energy that can travel to the system. Examples of suitable acoustic generators in that regard include electroacoustic devices such as a voice coil actuator, a piezoelectric actuator, a magneto- strictive actuator, and a capacitive transducer. The transducer may be arranged in the form of phased arrays or modified by acoustic holograms or acoustic metamaterials.

[0083] The method of the invention also provides a step of removing the acoustic energy, which allows de-mixing of the monophasic system.

[0084] Upon removal of the acoustically induced mixing, the mechanical shear of the system stops. At that point, the homogeneous system spontaneously and gradually reverts into a bi-phase configuration with the original two aqueous phases starting to separate. The transition from a homogeneous system into said bi-phase configuration is referred to herein as “de-mixing”.

[0085] During de-mixing, the system gradually evolves into a bi-phasic system in which one phase gets progressively enriched with the first photo-curable macromer and another phase gets progressively enriched with the second photo-curable macromer.

[0086] Thermodynamically driven dynamics of de-mixing are believed to dictate the evolution of the specific morphology of the separating phases. In that regard, de-mixing is believed to occur via several mechanisms. Without wanting to be limited by theory, it is postulated that two de- mixing mechanisms are relevant in the context of the present method, namely “spinodal decomposition” and “nucleation”.

[0087] During spinodal decomposition, one distinct phase begins to emerge simultaneously across all locations within the other phase. This is observed as the formation and enlargement of interconnected “channels” (also called “bi-continuous” channels) of one phase interspersed among the other phase. According to this mechanism, the system is believed to evolve into a co-continuous phase configuration, in which a phase containing the majority of one macromer exists as a three-dimensional network of interconnected channels within another phase containing the majority of the other macromer. A schematic representation of this configuration is shown in Figure 1.

[0088] During nucleation, spherical droplets of one phase first emerge at distinct locations within the other phase. Said spherical droplets then proceed to enlarge as molecules of one of the macromers migrate between the phases to accumulate preferentially inside those spherical droplets. According to this mechanism, the system is believed to evolve into a continuous matrix phase containing the majority of one macromer and a dispersed phase containing the majority of the other macromer. Example images of this configuration are shown in Figure 3 (control samples).

[0089] In the present method, it was found that the acoustically driven shear can homogenize the two aqueous phases such that, once the acoustic energy is removed, de-mixing starts via spinodal decomposition, followed by nucleation. That is, upon removal of the acoustic energy, the system begins to de-mix via spinodal decomposition, and then shifts at some point in time to instead de-mixing via nucleation. In the transition, the interconnected “channels” will pinch off from one another and form spherical droplets.

[0090] The method of the invention takes advantage of those dynamics, in that one can simply time the irradiation step, after removal of the acoustic energy, such that curing of the macromers effectively freezes the bi-phasic morphology of the system that exists at that point in time. Depending on the delay between the removal of the acoustic energy and the irradiation with curing radiation, one can therefore obtain cross-linked bi-phasic systems having a co- continuous bi-phase configuration, a continuous / dispersed bi-phase configuration, or a blend of the two.

[0091] Accordingly, in some embodiments de-mixing results in a co-continuous phase system, wherein one phase contains the majority of the first photo-curable macromer and the other phase contains the majority of the second photo-curable macromer. In other words, in those instances the irradiation with curing photo-radiation is performed while the system undergoes spinodal phase separation.

[0092] In said co-continuous system configuration, whether the majority of the first or of the second photo-curable macromer is contained in the phase that constitutes the network of interconnected channels may depend, for example, on the nature and relative amount of macromers. For instance, if the first macromer is used in higher amount than the second macromer, then the system may de-mix into a co-continuous system configuration in which the network of interconnected channels contains the majority of the second macromer, while the other (matrix) phase contains the majority of the first macromer (or vice versa, if the first macromer is used in lower amount than the second macromer).

[0093] For avoidance of doubt, reference made herein to the “majority” of a given macromer means above 50% of the total amount of said macromer within the system. For example, above 60%, above 75%, above 80%, or above 90% of the total amount of said macromer within the system.

[0094] In some embodiments, de-mixing results in formation of a continuous phase containing the majority of the first (or second) photo-curable macromer and a dispersed phase containing the majority of the second (or first) photo-curable macromer. In other words, in those instances the irradiation with the curing radiation is performed while the system undergoes nucleation phase separation. In those instances, whether the first or the second macromer will preferentially accumulate into the continuous or the dispersed phase will depend on the distribution of those macromers in the originating co-continuous configuration.

[0095] In some embodiments, de-mixing results in formation of a continuous phase containing the majority of the first photo-curable macromer and a dispersed phase containing the majority of the second photo-curable macromer. In other words, in those instances the irradiation with the curing radiation is performed while the system undergoes nucleation phase separation.

[0096] By way of example, if the first macromer is GelMA and the second macromer is PVA (or a modified PVA), and they are used according to GelMA / PVA (or GelMA / modified PVA) weight ratio above 1, upon removal of the acoustic energy the system may be expected to demix into an initial co-continuous configuration made of a network of interconnected channels containing the majority of PVA (or modified PVA) within a bulk phase containing the majority of GelMA. The system would then progressively de-mix into a continuous phase containing the majority of GelMA and a dispersed phase containing the majority of the PVA (or the modified PVA).

[0097] The relative amount of the first to the second macromer may be tailored to dictate how each macromer will distribute into the separating aqueous phases during de-mixing.

[0098] In some embodiments, the first macromer and the second macromer are used according to a weight ratio (i.e. weight of first macromer / weight of second macromer) of at least 1. For example, the first macromer and the second macromer are used according to a weight ratio of at least 1.5, at least 1.75, at least 2, at least 5, or at least 10. In some embodiments, the first macromer and the second macromer are used according to a weight ratio of from 1 to 10.

[0099] The method of the invention also comprises a step of irradiating the de-mixed system with curing radiation to promote curing of the photo-curable macromers. Any curing radiation that promotes curing of the photo-curable macromers may be used in the method of the invention. The choice of the specific curing radiation can depend on various factors, such as the type of macromers, desired curing speed, depth of cure, and compatibility with the curing apparatus. It should be noted that the following descriptions of curing radiations are provided as examples and not intended to be limiting.

[0100] In some embodiments, the curing radiation has a wavelength between about 190 nm and about 2000 nm. For example, the curing radiation may have a wavelength in the range between about 290 nm and about 600 nm, or between about 365 nm and about 405 nm.

[0101] In some embodiments, the curing radiation is ultraviolet (UV) radiation. UV radiation, typically within the range of 200 to 400 nm, can be generated using UV light-emitting diodes (LEDs), mercury vapor lamps, or other UV sources. This radiation can activate photo-initiators present in the system, if any, and / or activate cross-linkable moieties in the macromers, initiating a cross-linking reaction that leads to solidification of the macromers within the irradiated volume.

[0102] In some embodiments, the curing radiation is visible light radiation. Visible light, with wavelengths ranging from approximately 400 to 700 nm, can penetrate deeper into the system compared to UV radiation. Light sources such as high-intensity LED arrays or specialized lamps can emit the desired visible light spectrum. The photo-initiators and / or cross -linkable moieties present in the system absorb the visible light and undergo a similar chemical reaction as in the case of UV radiation, leading to curing the macromers.

[0103] In some embodiments, the curing radiation is infrared (IR) radiation. IR radiation, with wavelengths longer than those of visible light, ranging from approximately 700 nm to 1,100 nm, can effectively penetrate deeper into the system. This deeper penetration allows for curing thick or opaque layers that may hinder the passage of UV or visible light. IR sources, such as IR LEDs or IR lamps, can provide the necessary radiation to initiate the curing process.

[0104] In some embodiments, curing radiation has wavelength of 405 nm.

[0105] It should be appreciated that the examples of curing radiations mentioned herein are not exhaustive. Other types of radiation may also be suitable for use in the method of the invention, including for example X-ray radiation, electron beam radiation, laser radiation, or focused ion beam.

[0106] Furthermore, it is contemplated that combinations of different curing radiations may be used in specific embodiments. Sequential or simultaneous exposure to multiple wavelengths or spectral bands can be employed to optimize the curing process, enhance material properties, or achieve unique curing effects. The intensity, duration, and spatial modulation of the curing radiations can be adjusted to accommodate various liquid formulations, printing requirements, or specific design considerations.

[0107] For a given system composition, the curing radiation would be selected to deliver sufficient optical dose to promote local curing of the macromers. For example, the curing radiation may be selected and projected to deliver an optical dose of between about 0.1 mW / cm2and 1.6- IO10mW / cm2.

[0108] The curing radiation may be projected for any exposure time conducive to effective curing of the macromers. In some embodiments, the exposure time for a given projection is at least 0.1s, at least 0.5s, at least Is, at least 5 s, at least 10s, or at least 30s. In some embodiments, the exposure time for a given projection is from 0.1s to 60s.

[0109] A skilled person will appreciate that the energy density delivered to the system and the exposure time can be tailored to specific application-related requirements.

[0110] For instance, when the system contains a biological entity (e.g. a viable cell), the energy density delivered into the system and the exposure time would be tailored to ensure curing of the macromers while preserving cell viability. For example, the optical dose may be kept to the minimum level required to achieve curing, thereby maximising the preservation of cell viability

[0111] In some embodiments, the curing radiation is selected and projected to deliver an energy density of from about 0.1 mW / cm2to about 150 mW / cm2. Considering an exposure time from 0.1 to 10s, this would correspond to a total energy of 0.01 mJ / cm2to 1.5 J / cm2delivered to the system. In some embodiments, the curing radiation delivers an energy density of from about 10 mW / cm2and about 10,000 mW / cm2. Those instances are particularly suited for the formation of 3D objects made of a biologically relevant material.

[0112] For the case of non-biomedical materials, the energy delivered to the system can be higher. For example, for an exposure time of 0.1 to 10s, the energy delivered to the system can be from 0.01 mJ / cm2to 1.6- 108J / cm2.

[0113] The curing radiation may be projected by any means known to the skilled person, provided it reaches the macromers to promote curing.

[0114] Typically, curing radiation would be generated by a radiation source and directed to the system. Optical parameters of the emitted radiation such as direction, intensity, collimation, focus, wavelength, etc. may be controlled along the optical path of the radiation by optical components that would be known to the skilled person.

[0115] For instance, a projection system made of a radiation source, such as a digital light projector or a laser beam emitter, may be used in combination with optical components such as filters, lenses, mirrors, shutters, etc. to direct and control the emitted radiation delivered to the system while controlling projection parameters such as intensity, duration of the exposure, and focus of the projected radiation.

[0116] In some embodiments, the curing radiation is projected by means of one or more optical fibres.

[0117] The curing radiation may be projected along any direction conducive to promoting curing of the macromers. In some embodiments, the curing radiation is projected along a vertical axis.

[0118] Advantageously, the time and position at which the system is irradiated with curing radiation can be controlled to tailor the morphology characteristics of the separating phases, such as shape, size, orientation, and degree of interconnectedness of the interspersed phases, which will eventually result in the shape, size, orientation, and degree of interconnectedness of the porosity obtained by removal of the corresponding phase. As discussed herein, irradiating the system during de-mixing induces cross-linking of the macromers, effectively freezing the morphology configuration of the phases at that point in time. By irradiating the system with curing radiation at a moment when the system is undergoing spinodal decomposition, a phase of interconnected channels (which will eventually provide interconnected pores upon removal of that phase) can be consolidated within the structure. By waiting for a longer time, disconnected phase regions (which will eventually provide disconnected pores upon removal of that phase) can be consolidated by curing a system undergoing nucleation.

[0119] Across both time regimes, waiting for a longer time before curing results in formation of larger discontinuous phase regions, which eventually provide larger pores.

[0120] Also, since the mixing process involves flow within the solution, curing within short time spans after the removal of the acoustic energy results in pores that are aligned preferentially in the direction of flow that existed at that location.

[0121] In some embodiments, irradiation is performed while the system is undergoing spinodal separation.

[0122] In some embodiments, irradiation is performed while the system is undergoing nucleation separation.

[0123] The time at which the de-mixing mechanisms shifts from spinodal decomposition to nucleation may depended on the specific composition of the system. Typically, said transition time may be taken to occur at least 5 to 10 seconds after removal of the acoustic energy. For example, the de-mixing of the system may transition from spinodal decomposition to nucleation at least 5, at least 10, at least 20, at least 30, at least 45, or at least 60 seconds after removal of the acoustic energy.

[0124] Accordingly, in some embodiments the irradiation with curing photo-radiation begins at least 2 seconds after the acoustic energy is removed.

[0125] In some embodiments, the irradiation with curing photo-radiation begins at least 10 seconds after the acoustic energy is removed.

[0126] In some embodiments, the irradiation begins at least 30 seconds after the acoustic energy is removed.

[0127] In some embodiments, the irradiation begins immediately after the acoustic energy is removed (i.e. 0 seconds delay).

[0128] In some embodiments, the irradiation begins from 0 seconds, at least 1 second, at least 2 seconds, at least 3 seconds, at least 5 seconds, at least 10 seconds, at least 15 seconds, at least 20 seconds, at least 25 seconds, at least 30 seconds, or at least 45 seconds after the acoustic energy is removed.

[0129] It is also possible to irradiate different zones of the same system at different time delays for each zone, for example at sequentially increasing time delays, following removal of the acoustic energy. In those instances, irradiation will cure different zones of the same system at different degree of de-mixing. This makes it advantageously possible to eventually create zones having different pore sizes and architecture within the same material. Being able to create regions of differently sized pores is highly valuable, for example in situations where people want to create networks of blood vessels inside hydrogels. This means scientists can then use one structure to study interconnected blood vessels of different sizes, delivering much more biologically relevant results from their experiments.

[0130] Accordingly, in some embodiments the irradiation is performed on different zones of the system following removal of the acoustic energy, each zone being irradiated at sequentially increasing irradiation delay starting from when the acoustic energy is removed. The delay of each sequential delay may be any time delay disclosed herein. In some embodiments, said sequential delay is 0 seconds, 0.5 seconds, 2 seconds, and 10 seconds.

[0131] Those skilled in the art will appreciate that said different zones of the irradiated system may have any shape or dimension, depending on the intended application. For instance, said zones may be concentric circular zones, or adjacent rectangular zones. Examples in that regard are shown in Figures 7 and 8. The form of the exposed regions may be as complex as any shape as can be illuminated by the irradiation. The sequentially irradiated regions may overlap, be adjacent, or be disconnected with respect to one another.

[0132] The method of the invention also comprises a step of removing one of the cured macromers to obtain the porous polymeric structure.

[0133] Removal of one of the cured macromers will result in the removal of the phase containing the majority of that cured macromer. This will result in the formation of voids forming the porosity of the resulting polymeric structure, which structural integrity will be provided by the phase containing the majority of the other cured macromer, which is not removed. The macromer that is removed to create pores (i.e. voids) within the structure may also be referred herein as the “porogen”, and the corresponding phase containing the majority of that cured macromer may also be referred herein as the “porogen phase”.

[0134] Depending on how each macromer distributes within each phase during de-mixing, the removal would be of the macromer forming the phase intended to provide the required voids within the resulting polymeric structure.

[0135] For instance, if irradiation is performed during spinodal separation and the phase constituting the network of interconnected channels contains the majority of the cured second macromer, then the method would entail removal of the cured second macromer. This would result in a porous polymeric structure made of the majority of the cured first macromer, with pores shaped as a network of interconnected channels left by the removal of the cured second macromer.

[0136] Conversely, if irradiation is performed during spinodal separation and the phase constituting the network of interconnected channels contains the majority of the cured first macromer, then the method would entail removal of the cured first macromer. This would result in a porous polymeric structure made of the majority of the cured second macromer, with pores shaped as a network of interconnected channels left by the removal of the cured first macromer.

[0137] Similarly, if irradiation is performed during nucleation separation and the dispersed phase contains the majority of the cured second macromer, then the method would entail removal of the cured second macromer to generate voids corresponding to said dispersed phase. This would result in a porous polymeric structure made of the majority of the cured first macromer, with dispersed voids left by the removal of the cured second macromer.

[0138] Conversely, if irradiation is performed during nucleation separation and the dispersed phase contains the majority of the cured first macromer, then the method would entail removal of the cured first macromer to generate voids corresponding to said dispersed phase. This would result in a porous polymeric structure made of the majority of the cured second macromer, with dispersed voids left by the removal of the cured first macromer.

[0139] Accordingly, in some embodiments the method comprises a step of removing cured first macromer to obtain the porous polymeric structure. Removal of cured first macromer will result in the removal of the phase containing the majority of the cured first macromer. This will result in the formation of voids forming the porosity of the resulting polymeric structure, formed from the majority of the cured second macromer.

[0140] In some embodiments, the method comprises a step of removing cured second macromer to obtain the porous polymeric structure. Removal of cured second macromer will result in the removal of the phase containing the majority of the cured second macromer. This will result in the formation of voids forming the porosity of the resulting polymeric structure, formed from the majority of the cured first macromer.

[0141] By way of example, as discussed herein, if the first macromer is GelMA and the second macromer is PVA (or a modified PVA), and they are used according to GelMA / PVA (or GelMA / modified PVA) weight ratio above 1, upon removal of the acoustic energy the system may be expected de-mix into an initial co -continuous configuration made of a network of interconnected channels containing the majority of PVA (or modified PVA) within a bulk phase containing the majority of GelMA. If the system is irradiated at that stage to cure the macromers and lock that phase configuration, the method would then entail removal of the cured PVA (or modified PVA) to obtain a structure of cured GelMA hydrogel having pores in the form of a network of interconnected channels.

[0142] Alternatively, if the system is allowed to de-mix further into a continuous phase containing the majority of GelMA and a dispersed phase containing the majority of the PVA (or the modified PVA), the method would entail removal of the cured PVA (or modified PVA) to obtain a structure of cured GelMA hydrogel having pores in the form of disconnected voids.

[0143] Selective removal of one of the cured macromers may be achieved by any means known to the skilled person.

[0144] For instance, difference in bond nature between the cross-links of the cured first macromer and those of the cured second macromer may be exploited to selectively remove either the cured first or second macromer. For instance, cross-linking bonds characterising one cured macromer may be weaker than those characterising the other, making that macromer amenable to removal by washing. In some instances, cured macromer with the weaker cross-linking bonds may even spontaneously break over time.

[0145] In some embodiments, the first (or second) macromer is selected such that irradiation promotes formation of impermanent bonds. In those instances, the impermanent bonds may break up over time (for example, over the course of days) to leave behind the empty space within the structure as the first (or second) macromer are removed. Removal of the cured first (or second) macromer in those instances may also be facilitated by washing the structure. In some instances, the impermanent bonds may even spontaneously break over time, resulting in removal of the first (or second) cured macromer.

[0146] In some embodiments, the first (or second) macromer is selected such that the chemical bonds formed during curing remain stable under the conditions that the structures are kept at after fabrication, whereas those forming during curing of the second (or first) macromer do not.

[0147] It may also be said that in some embodiments, the first macromer and the second macromer are selected such that, for a given set of ambient conditions (e.g. physiological conditions), the cured first macromer is characterised by stronger intermolecular forces than those characterising the cured second macromer. As a result, the cured second macromer may be more amenable to removal (e.g. by washing or spontaneous mechanical breakage) than the cured first macromer. Stronger intermolecular forces may derive from the type, number, and / or strength of the specific chemical bonds that form during curing of the first macromer. In some embodiments, the first and the second macromer may be selected such that the chemical bonds formed during curing of the first macromer differ in nature from those that form during curing of the second macromer. As a result, one of the cured macromers (e.g. the cured second macromer) may be more amenable to removal (e.g. by chemical washing or spontaneous mechanical breakage) than the other cured macromer (e.g. the cured first macromer).

[0148] For instance, the first and second macromers may be selected to such that they promote different cross-linking chemistries under irradiation. In those instances, the cured first macromer may not be covalently bound to the cured second macromer. Accordingly, in some embodiments the cured first macromer is not covalently bound to the cured second macromer.

[0149] A degree of cross-linking between the first and second macromer may nevertheless be acceptable. In those instances, some degree of cross-linking between the first and the second macromer might be advantageous to reinforce the cured phase that is not removed. For example, in the case of hydrogels some additional degree of cross-linking derived from cross-linking between the first and second macromer may advantageously afford mechanical reinforcement to the overall hydrogel structure.

[0150] In those instances where there is some degree of cross-linking between the first and second macromer, removal of one of the two cured macromers may be achieved by selectively targeting the chemical structure of the cured macromer to remove.

[0151] For instance, the first and the second macromer may be selected such that one of the macromers (e.g. the second macromer) contains cleavable chemical bonds within its structure, so that the underlying polymer backbone forming upon curing could be broken up instead of the crosslinking groups. As a result, the corresponding cured macromer (e.g. the cured second macromer) may be more amenable to removal (e.g. by chemical washing or spontaneous mechanical breakage) than the cured first macromer.

[0152] In some embodiments, the first macromer may be selected to provide a higher degree of polymerisation relative to the second macromer, under the same curing conditions. As a result, the cured second macromer may be more amenable to removal (e.g. by chemical washing or spontaneous mechanical breakage) than the cured first macromer.

[0153] In some embodiments, the first macromer may be selected to provide a higher degree of crosslinking relative to the second macromer, under the same curing conditions. As a result, the cured second macromer may be more amenable to removal (e.g. by chemical washing or spontaneous mechanical breakage) than the cured first macromer.

[0154] For example, if the first macromer is GelMA and the second macromer is a tyramine modified PVA (PVA-Tyramine), the bonds that form during curing of GelMA are between methacrylamide and methacrylate groups, while the bonds that form during curing of PVA- Tyramine) are between the tyramine groups (also called dityrosine bonds). The dityrosine bonds will preferentially break up in the aqueous solvent (over those between methacrylamide and methacrylate groups in GelMA) as the resulting structure is incubated in water (e.g. under physiological conditions), while the bonds between the methacrylate / methacrylamide groups GelMA would not break.

[0155] Removal of one of the cured macromers may be achieved by any means known to the skilled person, based on the chemical nature of the cured macromer.

[0156] Removal may be passive removal. That is, the cured macromer (e.g. the cured second macromer) may spontaneously break up in time, for example as its bonds gradually dissolve in water with no need for external intervention.

[0157] Removal may also be active removal. That is, removal of the intended cured macromer (e.g. the cured second macromer) may be facilitated by adding an enzyme selected to target specific bonds of the cured macromer, or by adding an external stimulus (e.g. light or electricity) to break up target bonds.

[0158] In some embodiments, removal of the intended cured macromer is effected by washing the cured structure with a solvent. Said solvent may be water, or an aqueous solution. For example, removal may be effected by washing the cured structure with a buffered aqueous solution, such as Dulbecco's Phosphate-Buffered Saline (DPBS). This may be particularly useful when the cured structure is a hydrogel intended for biological applications.

[0159] The proposed method is therefore a straightforward procedure affording production of polymeric structures having customised pores in terms of morphology, distribution, and size. By acting on the delay between the removal of the acoustic energy and the irradiation with curing radiation, one can effectively tune the morphology, distribution, and size characteristics of pores within the final structure. For instance, by irradiating the system with curing radiation at a moment when the system is undergoing de-mixing via spinodal decomposition, interconnected pores can be created within the structure. By waiting for a longer time before irradiation, disconnected pores can be created by curing a system de-mixing under a nucleation regime. Across both time regimes, waiting for a longer time before curing results in larger pores. Since the mixing process necessarily involves flow within the solution, curing within short time spans after the cessation of mixing results in pores that are aligned preferentially in the direction of flow that existed at that curing location.

[0160] Terms such as “porosity” and correlated terms such as “pores” and “porous”, etc. refer herein to voids within the polymeric structure. As described herein, the method of the invention affords formation of voids (i.e. pores) within the polymeric structure that can have different shape, ranging for example from unconnected spheroidal voids (e.g. spherical bubbles within the polymer) to a network of interconnected channels having irregular shape (e.g. as represented in Figure 1). In that context, the notion of pore “size” will be understood in relation to a characteristic dimension of a given pore shape. For instance, the pore size of discrete spherical voids would refer to the diameter of the discrete spherical voids.

[0161] In the case of a network of interconnected channels, the pore size would refer to the diameter of a circle that is created by intersecting the channel with a plane that is normal to the axis of the channel. Here, the size of pores in the form of a network of interconnected channels was measured by first creating the 3D reconstruction of the porous network (Figure 1) and then performing a Distance Transform. This operation creates a 3D image filling in the “not- polymer” portion of the 3D reconstruction. In that 3D image, the pixel brightness / intensity represents the distance from that point to the nearest wall of the porous network. That value (because it typically occurs in the middle of the channel) was multiplied by 2 to get the “pore diameter” values, for example those listed in Figures 6 and 7.

[0162] The method of the invention can advantageously afford fabrication of polymeric structures having pores (i.e. internal voids) with size controllable within at least mesoporous (pore size 2 to 50 nm) and microporous (above 50) size regimes.

[0163] In some embodiments, the method affords fabrication of polymeric structures having pores with size of at least 10 pm. For example, the method may afford fabrication of polymeric structures having pores with size of at least 15 pm, at least 20 pm, at least 30 pm, at least 50 pm, at least 70 pm, at least 100 pm, at least 150 pm, or at least 200 pm.

[0164] In some embodiments, the method affords fabrication of polymeric structures having pores with size from 10 pm to 150 pm.

[0165] The method of the invention may be performed using an integrated system that allows for provision of the required acoustic energy and irradiation with curing radiation.

[0166] Accordingly, in some embodiments the method comprises: introducing a print head having a cavity containing gas into the aqueous two-phase system to form a submerged gas-liquid interface between the gas and the aqueous two-phase system, wherein said gas-liquid interface is constrained to the print head and defines a printing surface, transmitting the acoustic energy to the submerged gas-liquid interface to induce the shear mixing of the phases, resulting in the monophasic system, removing the acoustic energy from the submerged gas-liquid interface, allowing the demixing of the monophasic system, and irradiating the de-mixed system with the curing radiation by projecting said curing radiation on the submerged gas-liquid interface with the print head, thereby promoting curing of the first and the second photo-curable macromer at the printing surface. By "print head" is meant herein a component or assembly that can deliver curing radiation to the photo-curable macromers, such that the macromers cure locally where the radiation is delivered.

[0167] In some embodiments, the print head comprises an emitter of curing radiation. Said emitter may be any component that is suitable to emit curing radiation of the kind described herein. For example, the print head may comprise an optical transmitter for emitting radiation of the kind described herein.

[0168] In some embodiments, the print head transmits curing radiation which is emitted from a radiation source external to the print head.

[0169] Particularly when the print head transmits curing radiation emitted from a radiation source external to the print head, the print head would be transparent to the curing radiation at least along a main optical transmission axis used to project the curing radiation on the photo-curable macromers. For instance, the print head may be transparent to curing radiation projected along a vertical axis onto the photo-curable macromers.

[0170] In some embodiments, the print head comprises an optical conducting component. For example, the print head may include an optical fiber for projecting curing radiation onto the photo- curable macromers.

[0171] In some embodiments, the print head comprises a fibre optic to direct light, and an attachment designed to provide the required gas-liquid interface at the fibre tip.

[0172] The print head may be made of any material allowing for introduction of the print head into the system without compromising its structural integrity. For example, the print head may be made of a material that is chemically inert toward the photo-curable macromers. Suitable examples of print head materials for use in the invention include polymer materials, metals, and ceramic, for example glass.

[0173] In some embodiments, the print head is made of a polymer material. Examples of suitable polymer materials in that regard include polymers such as polyethylene, including low-density polyethylene (LDPE) and high-density polyethylene (HDPE), polypropylene (PP), polyvinyl chloride (PVC), polystyrene (PS), nylon, nylon 6, nylon 6,6, teflon (polytetrafluoroethylene), and thermoplastic polyurethanes (TPU). In some embodiments, said polymer material is a composite comprising a polymer of the kind described herein.

[0174] In some embodiments, the print head is made of metal. Examples of suitable metals in that regard include aluminium, stainless steel, and titanium.

[0175] In some embodiments, the print head is made of glass. For example, the print head may be entirely made of glass.

[0176] In some embodiments, the print head comprises one or more regions which articulate, for example in the form segments united by joints. This enables arbitrary positioning of the interface along a desired orientation, for example via the use of internal mirrors.

[0177] In some embodiments, the print head is capable to rotate about a centre axis. In those instances, the print head may be rotated to change its fluidic flow profile and, subsequently, the shape of the gas-liquid interface during printing.

[0178] The print head may have any dimension conducive to the print head functioning as intended. For example, the print head may have a largest dimension of 0.5 cm, 1 cm, 5 cm, 10 cm, or 30 cm.

[0179] The print head has a cavity containing gas, and the embodiment method comprises a step of introducing the print head into the aqueous two-phase system to form a submerged gas-liquid interface between the gas and the aqueous two-phase system.

[0180] By the expression "cavity containing gas" is meant an empty space provided within the print head which contains gas, for example air. The cavity containing gas is shaped and oriented such that when the print head is introduced into the aqueous two-phase system, the gas in the cavity prevents liquid from entering into the cavity, thereby providing the formation of a submerged gas-liquid interface. To ensure contact between the gas and the liquid, it will be appreciated that the cavity of the print head would present at least one opening that, once the print head is introduced into the aqueous two-phase system, permits contact between the gas and the aqueous two-phase system to ensure formation of the required gas-liquid interface.

[0181] By being "submerged", the gas-liquid interface is located below the surface level of the aqueous two-phase system.

[0182] The proposed principle is analogous to that of forcing an empty glass upside-down into a volume of water. As the upside-down glass is pushed into the water, the air pocket within the empty glass prevents water from entering inside the glass, effectively counteracting the hydrostatic pressure of water and providing a submerged air-water interface at the glass opening.

[0183] Formation of the gas-liquid interface may therefore be achieved by any cavity design that would be fit for that purpose.

[0184] In some embodiments, the print head comprises a cavity having one opening. In those instances, once the print head is introduced into the aqueous two-phase system the gas contained in the cavity induces formation of the required gas-liquid interface at said opening. For instance, the print head may be a hollow component introduced vertically into the aqueous two-phase system. The component is sealed at the top end with a radiation transparent window and left open at the bottom end, to define an internal cavity with an opening at the bottom of the print head. Gas in the cavity prevents liquid from entering into the cavity. The gas inside the cavity of the print head may be pressurised to form a convex gas-liquid interface protruding downward from the open cavity. An example in that regard is shown in the schematic of Figure 2.

[0185] In some configurations, the print head comprises a cavity having multiple openings. Those configurations afford the provision of multiple discrete gas-liquid interfaces, for example for simultaneous curing of different volumes of aqueous two-phase system. For example, the print head may have a squared cross-section and an internal cavity with a 3x3 array of openings, in turn defining a 3x3 array of submerged gas-liquid interfaces. In some embodiments, the print head comprises multiple cavities and multiple openings. For example, the print head may comprise multiple cavities, each having a corresponding opening. In use, these print heads advantageously afford provision of multiple gas-liquid interfaces, useful for simultaneous curing of different volumes of aqueous two-phase system.

[0186] In the method of the invention, the gas-liquid interface is constrained to the print head. Since the gas is contained in a cavity of the print head, the gas-liquid interface that forms when the print head is introduced into the aqueous two-phase system is cohesive with the print head. By the gas-liquid interface being "constrained" to the print head, relative movement between the interface and the print head is restricted such that the interface and the print head move together.

[0187] In some embodiments, the print head comprises an optical fibre for the transmission of curing radiation. The optical fibre may be for transmitting curing radiation form a radiation source external to the print head, or for transmitting curing radiation from a radiation source that is integral to the print head.

[0188] In some embodiments, the print head comprises a gas inlet for the introduction or extraction of gas into / from the cavity. In those instances, when the print head is introduced in the aqueous two-phase system, gas within the cavity can be pressurised or depressurised, resulting in a modification of the shape and extension of the gas-liquid interface.

[0189] In some embodiments, the print head has an internal cavity with an opening at a bottom side of the print head, such that when the print head is introduced into the aqueous two-phase system and said cavity is filled with pressurised gas, the gas-liquid interface that forms has a convex shape protruding downward from the opening. An example in that regard is shown in the schematic of Figure 2.

[0190] Said opening may have any dimension conducive to formation of the submerged gas-liquid interface as described herein. In some embodiments, said opening has a largest dimension of from 2 mm to 30 mm, from 2 mm to 15 mm, from 5 mm to 15 mm. In some embodiments, said opening has a maximum dimension of 10 mm, or 25 mm. The opening may have any shape conducive to formation of the submerged gas-liquid interface as described herein. In some embodiments, the opening has a circular shape, a squared shape, or a rectangular shape having a largest dimension of from 2 mm to 30 mm, from 2 mm to 15 mm, or from 5 mm to 15 mm, for example 10 mm, or 25 mm.

[0191] In some embodiment, the print head comprises an open cavity with a circular opening with a diameter of from 2 mm to 30 mm, from 2 mm to 15 mm, or from 5 mm to 15 mm, for example 10 mm or 25 mm.

[0192] The print head may have any shape conducive to the intended function.

[0193] In some embodiments, the print head has a tubular shape with an opening at an end and a window transparent to curing radiation at the opposite end. The tubular shape may have a circular or square cross-section.

[0194] The gas may be any gas that forms a gas-liquid interface when in contact with the aqueous two- phase system. For example, the gas may be air, or oxygen.

[0195] In some embodiments, the gas comprises oxygen.

[0196] In some embodiments, the gas comprises air.

[0197] In some embodiments, the submerged gas-liquid interface is provided by pressurising the gas within the cavity of the print head. The term "pressurising" will be understood to encompass the application of either positive or negative pressure to the gas, resulting in either an expansion or a contraction of the gas volume. This may be achieved by any means known to the skilled person.

[0198] In some embodiments, the print head comprises a gas inlet fluidly connected to the cavity of the print head. The gas inlet can be used to introduce gas into, and / or extract gas out of, the cavity of the print head. Since pressure of the gas in the cavity counters the hydrostatic pressure of the aqueous two-phase system on the gas-liquid interface, pressurising the gas within the cavity changes the shape and extension of the interface. For instance, a pressure increase of the gas would expand the gas to push against the interface, which would extend outward relative to the print head. Conversely, a pressure decrease would result in a contraction of the gas volume, with consequent retraction of the interface, for example within the print head boundaries.

[0199] Pressurising the gas within the cavity of the print head advantageously ensures that the shape of the gas-liquid interface can be retained during printing irrespective of the depth of the printing head within the aqueous two-phase system. In other words, the use of pressurising gas within the printing head creates and maintains a controlled surface tension at the gas-liquid interface at any aqueous two-phase system depth, helping to retain the shape of the interface during printing and printhead movement. This results in an enhancement of aqueous two-phase system influx rates compared to conventional ‘flat’ gas-liquid interfaces, which shape is determined and modulated by the depth dependent hydrostatic pressure of the aqueous two- phase system.

[0200] Gas used to pressurise the gas in the cavity of the print head may be the same or different than the gas contained in the cavity.

[0201] The gas-liquid interface may have any shape and extension that is conducive to equalising gas pressure within the cavity of the print head and the hydrostatic pressure of the aqueous two- phase system. Changes in the gas pressure within the print head can therefore afford dynamic deformation of the gas-liquid interface permitting the influx of fresh aqueous two-phase system at the interface, thus facilitating the generation of a continuous layer-less structure.

[0202] In some embodiments, the gas-liquid interface has a convex shape.

[0203] In some embodiments, the gas-liquid interface has a concave shape.

[0204] In some embodiments, the gas-liquid interface is substantially flat. In this context, convexity or concavity of the interface will be understood to be relative to the gas phase. That is, a convex interface curves / projects outwards from the gas phase, while a concave interface curves / projects inward into the gas phase.

[0205] By installing a camera collecting images of the gas-liquid interface it is possible to implement automatic control of the shape of the gas-liquid interface. In those instances, the method can be performed taking advantage of camera-based feedback to automatically modulate the shape and extension of the gas-liquid interface (for example by automatically adjusting pressure of the gas within the print head). Accordingly, in some embodiments the method comprises automatic modulation of the pressure induced by pressurising gas within the print head based on image feedback of the gas-liquid interface.

[0206] The embodiment method comprises a step of transmitting acoustic energy to the submerged gas-liquid interface.

[0207] It was observed that the gas-liquid interface can be susceptible to acoustic stimulation. By transmitting acoustic energy to the submerged gas-liquid interface it is therefore possible to impart rapid modulation of the shape of the interface according to the characteristics of the acoustic energy. Without wanting to be confirmed by theory, it is believed that the rapid modulation of the shape of the interface via acoustic excitation can advantageously promote the formation of capillary waves on the interface, significantly enhancing liquid influx around the interface, resulting in shear mixing (as shown for example in the schematic of Figure 2). The rate and distribution of material influx can itself be modulated by controlling parameters such as the characteristics of the acoustic energy (e.g. amplitude, frequency), the print head geometry, and / or the interface curvature.

[0208] Accordingly, in some embodiments the method comprises transmitting acoustic energy to the gas-liquid interface to induce an acoustic field on the interface in the form of Faraday waves. The application of said waves to the interface advantageously enhances liquid influx during printing, resulting in efficient shear mixing.

[0209] Transmission of acoustic energy to the submerged gas-liquid interface may be achieved by any means known to the skilled person. For instance, acoustic energy may be transmitted to the submerged gas-liquid interface by vibrating the print head.

[0210] Acoustic energy may also be transmitted on the submerged gas-liquid interface by introducing acoustic energy into the aqueous two-phase system, for example by generating acoustic energy within the liquid volume. In those instances, the acoustic energy may be made to travel through the aqueous two-phase system to the gas-liquid interface, affording the desired shape modulation of the submerged gas-liquid interface.

[0211] In some embodiments, acoustic energy may be introduced into the cavity of the print head. In those instances, the acoustic energy can travel through the cavity of the print head as pressure waves within the gas contained in the cavity, affording shape modulation of the submerged gas-liquid interface.

[0212] In these instances, acoustic energy may be generated by any means known to the skilled person. For example, acoustic energy may be transmitted to the gas-liquid interface by an acoustic generator placed to emit acoustic energy that can travel to the gas-liquid interface. The acoustic generator may be any device capable to transmit acoustic energy to the gas-liquid interface. Examples of suitable acoustic generators in that regard include electroacoustic devices such as a voice coil actuator, a piezoelectric actuator, a magneto-strictive actuator, and a capacitive transducer. The transducer may be arranged in the form of phased arrays or modified by acoustic holograms or acoustic metamaterials.

[0213] For instance, a suitable acoustic generator may be coupled to the cavity of the print head, such that emitted acoustic energy travel through the gas in the cavity to reach the gas-liquid interface. In other configurations, an acoustic generator may be submerged within the aqueous two-phase system, such that generated acoustic energy travels though the aqueous two-phase system to the gas-liquid interface. In further configurations, an acoustic wave generator may be coupled to the print head, such that emitted acoustic energy travels through the body of the print head to reach the submerged gas-liquid interface. Alternative configurations may be readily devised by a skilled person. It will be understood that the specific effects of acoustic stimulation on the gas-liquid interface may depend on the rheological characteristics of the aqueous two-phase system, in that the propagation dynamics of acoustic energy travelling through the aqueous two-phase system can vary depending on the density and viscosity of the aqueous two-phase system. A skilled person would be able to fine tune frequency and amplitude characteristics of the acoustic energy depending on the intended result.

[0214] Also, the specific effects of acoustic stimulation on the gas-liquid interface may depend on the specific geometry of the print head. In that regard, the choice of different geometries of print head can assist in creating customary shapes of the interface under acoustic stimulation, as well as different wave, and therefore mixing, patterns.

[0215] In the method of the invention, the gas-liquid interface defines a printing surface. By "printing surface" is meant herein a target area within the aqueous two-phase system where curing is promoted by the curing radiation.

[0216] Accordingly, in some embodiments projecting curing radiation through the submerged gasliquid interface comprises focusing the curing radiation on the gas-liquid interface, on the liquid side of the gas-liquid interface, or on the gas side of the gas-liquid interface.

[0217] In some embodiments, the curing radiation comprises cross-sectional images of a 3D object.

[0218] Cross-sectional images of the 3D object may be obtained from a digital representation of the target object, which may be acquired or generated by means known to the skilled person. Those means may include computer-aided design (CAD) software, three-dimensional scanning devices, or data obtained from other 3D digital modeling techniques. The digital representation would typically comprise a stacked sequence of the cross-sectional images that stacked together form a complete 3D optical representation of the target object.

[0219] By adopting a curing radiation comprising cross-sectional images of the 3D object, the method of the invention makes it possible to generate complete 3D target objects by translating the print head along a single directional axis, significantly simplifying the requirements of the printing equipment. At each subsequent translation, sequential cross-sections of the 3D object can be projected and therefore cured at the gas-liquid interface, affording layer-by-layer formation of the 3D object. This is a fundamental departure from conventional 3D printing systems based on single focus-spot printing (e.g. using single spot-focussed laser for spotcuring). In those conventional systems, the print head required for printing an entire 3D objects must be moveable relative to the system along multiple axial directions to effectively enable point-by-point curing of an entire 3D volume. Conventional printing systems in that regard may be characterised, for example, by having the print head (or the container of the liquid) mounted on a 3 to 6 DOF ("Degrees-Of-Freedom") robotic arm to enable movement of the focal spot across all points of a given target volume. In that regard, the method of the invention affords the adoption of significantly simplified printing systems.

[0220] In some embodiments, the cross-sectional images of the 3D object correspond to a 2D crosssection of the object. Those instances are useful for example when the gas-liquid interface is flat, defining a corresponding flat printing surface.

[0221] Accordingly, in some embodiments the curing radiation comprises cross-sectional images of the 3D object, said cross-sectional images representing flat cross-sections of the 3D object that are conform to a flat gas-liquid interface.

[0222] In some embodiments, the cross-sectional images of the 3D object correspond to a non-flat cross-section of the object. For instance, the cross-sectional image of the 3D object may correspond to a curved slice of the object, for example a convex or concave slice of the object. Those instances are useful for example when the gas-liquid interface is non-flat, defining a corresponding non-flat printing surface, such as a convex or concave gas-liquid interface defining a convex or concave printing surface, respectively.

[0223] Accordingly, in some embodiments the curing radiation comprises cross-sectional images of the 3D object, said cross-sectional images representing convex cross-sections of the 3D object that are conform to a convex gas-liquid interface.

[0224] In other embodiments, the curing radiation comprises cross-sectional images of the 3D object, said cross-sectional images representing concave cross- sections of the 3D object that are conform to a concave gas-liquid interface. In those instances where the gas-liquid interface (and the corresponding printing surface) is non-flat (e.g. curved, such as convex or concave), it is required to project corresponding nonflat cross-sections of the target 3D object to achieve correct spatial curing of the macromers in a three dimensional printing surface. That is, each projection should be confirm to the shape of the gas-liquid interface, which requires accounting for both in-plane and out-of-plane structures of the 3D object during the slicing process, as the interface spans three dimensions. Consequently, the discretization of the print geometry necessitates the adoption of a non-planar approach as opposed to planar layers. This may be achieved, for example by means of customised algorithms for the determination of non-flat cross sections of the target object.

[0225] Figure 2 shows a schematic overview of the materials and fabrication process for the aqueous two-phase emulsion hydrogels. A) Chemical structures of the two polymers used as well as a schematic drawing of the Dynamic Interface Printing print head. B) The gels were formed by first lowering the print head into the well containing the polymer blend, mixing the fluid using acoustic actuation, and then curing with 405nm light after a prescribed delay period.

[0226] The method can advantageously be applied to generate in vitro cell culture scaffolds. Patterning multiple pore sizes within a single layer of a larger structure would be valuable in the context of vascular biology, where models of blood vessels covering multiple length scales would provide novel insights into tissue function. Future work to integrate cells into the produced gels, whether during or after fabrication, could validate the utility of such an approach. This work also highlights the importance of selecting manufacturing methods that can leverage the inherent properties of the materials being used. We employed a novel biofabrication system to induce acoustic mixing in viscous fluids directly at the site of fabrication. To our knowledge, this is the first example in which the porous architecture of a hydrogel material was designed by halting the dynamic phase separation process. Going forward, additional materials, such as sono-inks, or patterning modalities, such as dielectrophoresis, could be included to enable more complex cellular microenvironments to be formed

[0227] The method of the present invention affords fabrication of porous polymeric structures, for example porous hydrogel structures, having pores in the form of interconnected channels in the microporous size regime at 37 °C and at physiological pH. Embodiments of the method will now be described in the following non-limiting Examples.

[0228] EXAMPLES

[0229] EXAMPLE 1

[0230] A series of aqueous two-phase systems containing GelMA, polyvinyl alcohol (PVA), and / or a tyramine-functionalized polyvinyl alcohol (PVA-T) was investigated, to evaluate their phase separation dynamics and fitness for purpose in designing 3D cell culture scaffolds.

[0231] Synthesis and of GelMA

[0232] Two batches of gelatin methacryloyl (GelMA) were synthesized. For both batches, 50g of gelatin (from porcine skin, Type A, 300 bloom, SigmaAldrich cat no. G2500) was dissolved in 0.25M carbonate-bicarbonate buffer (SigmaAldrich cat no. C3041) at a concentration of 20% (w / v) while heating the solution to 55 °C in a water bath and stirring at > 500RPM. Once the gelatin had dissolved (after approx. 2 hours), the pH was adjusted to 9.4 using IM NaOH.

[0233] Afterwards, methacrylic anhydride (SigmaAldrich cat no. 276685) was slowly added dropwise to the gelatin solution. For the GelMA batch with a high targeted degree of functionalization (DoF), 4.690ml (4.854g) methacrylic anhydride was used, while 1.585ml (1.640g) was used for the low DoF batch. Once the methacrylic anhydride was added, the reaction proceeded at 55°C for 1 hour. Afterwards, the reaction was halted by lowering the pH of the solution to 7.4 using 36% HC1. The solution was then diluted with two volumes of MilliQ H2O and purified via dialysis (12.4kDa MWCO tubing, SigmaAldrich cat no. D0655) for 1 week, swapping the dialysate water at least once a day. Following dialysis, the purified solution was frozen overnight and lyophilized for 1 week before being stored at -20°C until use.

[0234] To prepare fluorescent GelMA samples for imaging, Rhodamine B was conjugated onto the GelMA (GelMA-RB) using EDC / NHS chemistry. The same procedure was followed for both batches of GelMA. To start, 4.5g of GelMA was dissolved in 50mL of MES buffer (SigmaAldrich cat no. M3885) overnight at 37°C. On the same day, 450mg of Rhodamine B (SigmaAldrich cat no. R6626) was dissolved in 250mL of MES buffer in a separate round bottom flask under continuous stirring, which was then covered in aluminum foil to protect the sample from light. 297mg of l-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC, ThermoFisher cat no. 22980) along with 544mg of N-hydroxysuccinimide (NHS, SigmaAldrich cat no. 130672) was added to the Rhodamine B solution and allowed to react overnight at room temperature. The next day, the GelMA solution was added to the Rhodamine B solution and allowed to react for 24 hours at room temperature. The pH was then adjusted to 7.4 using IM NaOH to quench the reaction, and the contents were again dialyzed and lyophilized as above.

[0235] Two batches of GelMA were synthesized and referred to as either “low DoF” GelMA or “high DoF” GelMA throughout the subsequent experiments. The absolute degrees of functionalization of the two batches were 0.105 and 0.381 mmolmethacryioyi / gGeiMA, respectively. This corresponded to relative degrees of functionalization of 39% and 97%.z / 7 NMR Characterization of GelMA

[0236] The relative and absolute DOFs of GelMA were quantified using ’ H NMR (not shown). Samples of the synthesized GelMA as well as the unmodified gelatin were dissolved in D2O at 20 mg / ml concentrations. Nicotinamide (SigmaAldrich cat no. 72340) was dissolved in D2O at Img / ml for use as an internal standard. To prepare each NMR sample, 0.5ml of the GelMA or gelatin solutions were combined with 0.2ml of nicotinamide solution. Measurements were taken (64 scans, 4s relaxation delay) at 37°C using a 400 MHz JEOL spectrometer (JEOL Ltd., Tokyo, Japan).

[0237] Spectra were analyzed using the NMR plugin of MestreNova (Mestrelab Research, Santiago de Compostela, Spain) after being baseline corrected. To quantify the absolute DOF (expressed in terms of mrnolmethacryioyi / g), the integrals of the peaks corresponding to protons of both the methacrylate and methacrylamide groups (in the 5.6 to 5.8ppm range) were combined and normalized against the internal nicotinamide standard (peak at 8.95ppm). The absolute DOF was calculated according to the following equation: To quantify the relative DOF, (expressed in terms of % functionalization), the relative decrease in the lysine peak between the GelMA samples and unmodified gelatin was calculated. Here, the lysine peak (2.97 to 3.1ppm) in each sample was normalized against the peaks corresponding to aromatic groups on the protein (7.2 to 7.45ppm), and then the DOF was calculated as follows: 100

[0238] Synthesis of PVA-Tyramine

[0239] PVA-Tyramine was synthesized based off a two-step synthesis protocol. Briefly, polyvinyl alcohol (PVA, 87-89% hydrolyzed, MW 85-124kDa) was first modified to include a carboxyl group (PVA-COOH) for subsequent functionalization with tyramine (PVA-T). To add the carboxyl group, 10g of PVA was dissolved in 85ml DMSO (SigmaAldrich cat no. D2438) at 60°C under a nitrogen atmosphere while stirring. Then, 450mg succinic anhydride (SigmaAldrich cat. no. 239690) and 610ul triethylamine (SigmaAldrich cat. no. 90340) were added, and the reaction was allowed to proceed for 24h at 60°C.

[0240] The products were then precipitated in 500ml ethanol and vacuum filtered before being redissolved in 200ml boiling MilliQ water. The resulting solution was then purified via dialysis for 4 days, swapping the dialysate twice a day, before being freeze dried for an additional 4 days. Before proceeding to the tyramine functionalization, the degree of carboxyl group conjugation was measured using1H NMR. The measured conjugation was approx. 2.75%, and this value was used to calculate the required masses of EDC, NHS, and Tyramine HC1 in the subsequent reaction.

[0241] The PVA-COOH was then functionalized with tyramine using EDC / NHS chemistry. To start, 3.75g PVA-COOH was dissolved in 100ml of 50mM MES Buffer at 70°C under nitrogen atmosphere while stirring. The pH of the solution was adjusted to 4.7 using 36% HC1 and allowed to cool to room temperature before 1.335g NHS and 0.7157g EDC were added. The reaction was allowed to proceed for 45 minutes before the pH was raised to 5.4 using a IM sodium bicarbonate buffer. At this point, 800g Tyramine HC1 (SigmaAldrich cat. no. T2879) was added, and the reaction proceeded at room temperature for 24 hours. The products were then precipitated in IL acetone, redissolved in MilliQ water, and purified via salt- gradient dialysis. For this, consecutively lower concentrations of NaCl (150mM to OmM in 25mM steps) were prepared in the dialysate, swapping once per day. The dialysis proceeded for an additional 4 days afterwards, swapping the dialysate once per day. The purified products were then freeze dried and stored at -20°C until use.

[0242] Tyramine-functionalized polyvinyl alcohol was synthesized via a two-step reaction involving the addition of a carboxyl group followed by functionalization via EDC / NHS chemistry. Given the repeating structure of PVA, here the degree of substitution is reported as the percentage of repeating units functionalized with the given group.

[0243] 1H NMR Characterization of PVA-Tyramine

[0244] To analyze the degree of conjugation of the functional side groups, 20mg / ml solutions of PVA- T, PVA-COOH, and base PVA were prepared in D2O. An additional 20mg / ml sample of a low MW (18kDa) PVA-T was also prepared for comparison. TheXH NMR measurement settings matched those described above for the GelMA analysis. To calculate the degree of carboxylation, the peak at 2.4 to 2.65 ppm corresponding to the four contributing methylene protons of the carboxyl group was normalized against the peak at 3.4 to 4.2 ppm corresponding to the one methylene proton of the polyvinyl alcohol repeating unit backbone: 100

[0245] To calculate the degree of tyramine functionalization, a similar calculation was performed, this time using the peak at 6.7 to 7.2 ppm corresponding to the four contributing protons present in the tyramine aromatic group: . _ _ 100 EXAMPLE 2

[0246] Interfacial Tension Measurements

[0247] To quantify the shift in interfacial tension introduced by the functionalization reactions, samples of unmodified PVA, high MW PVA-T, low MW PVA-T, high DoF GelMA, and low DoF GelMA were measured via the pendant drop method using a DataPhysics OCA 25 tensiometer (DataPhysics Instruments GmbH, Germany). 30mg / ml solutions of the PVA samples and 50mg / ml solutions of the GelMA samples were prepared in MilliQ H2O, matching the concentrations used in later experiments. For each sample, three repeats were measured. The differences in mean interfacial tension were analyzed using one-way ANOVA in GraphPad Prism (GraphPad Software Boston, USA).

[0248] The interfacial tension of the low DoF and high DoF GelMA was 68 and 84 mN / m, respectively, indicating that the higher concentration of methacryloyl groups increased the interfacial tension. Modifying the high MW PVA with tyramine increased the interfacial tension from 57 mN / m to 73 mN / m. Despite differences in molecular weight, the low MW PVA-T showed a yet higher interfacial tension of 84 mN / m. Tyramine has been shown to form hydrogen bonds between the amine and hydroxyl groups of neighboring molecules. These results suggest that increased structuring of the PVA-T molecules was the main contribution to the higher interfacial tension.

[0249] Confocal Microscopy

[0250] All gels were imaged on a Nikon ARI confocal microscope (Nikon Corporation, Tokyo, Japan) using a 555nm excitation laser. The gels were imaged under swollen conditions using a 20x, 0.75NA objective. For each gel, three separate Z-stacks were acquired at random XY locations within the gel, at starting Z positions approx. 50pm above the glass coverslip bottom of the well plate. Depending on the size of the pores observed, the acquired Z-stacks were either 40pm thick with a 0.95 pm step size and a 1024xl024px field of view or 80pm thick with a 2 pm step size and a 2048x2048px field of view. Image Analysis

[0251] To analyze the pore structures observed within the gels, the confocal Z-stacks were processed using Imaris v 10.2.1 (Oxford Instruments pic, Abingdon, UK). Prior to being converted to the Imaris file format, the Z-stacks were pre-processed using ImageJ to normalize the histogram of each slice individually. Once imported to Imaris, the “pores” representing the PVA(-T) phase of the gels were segmented from the fluorescent GelMA-RB by using Imaris’ machinelearning based segmentation. To do this, portions of individual Z-stack slices were manually annotated to denote foreground and background. These manual annotations were made on sample images from gels representing each preprogrammed delay and then used to train the machine learning model.

[0252] The same model was then used across all Z-stacks to identify the pores as Surface objects (Figure 1), as defined within the software. These Surface objects were then used as the basis for a distance transformation algorithm which assigned a value to each pixel within the pores corresponding to how far that pixel was from the Surface border (Figure 4). This operation created a second channel within the dataset, and the maximum value of that channel (i.e. the deepest point within a pore for that Z-stack) was used to numerically compare pores between gels. These maximum values were doubled to serve as a measure of maximum pore diameter and then imported into GraphPad Prism for plotting and descriptive statistics. All analyses were conducted on a Dell Precision T8720 computer with a 16-core, 3.8 GHz Intel Xeon Gold processor, 256GB DDR4 RAM, and 24GB NVIDIA RTX A5000 GPU running Windows 10.

[0253] Overall, the image analysis procedure gave robust results. The porogen phase could be reliably segmented by the machine learning algorithm in Imaris (Figure 1). The maximum pore diameter was calculated based on the distance transform of the segmented porogen phase (Figure 4) and plotted against the delay period between acoustic mixing and photocuring (Figures 6 and 7). One exception was the gel made from the high DoF GelMA and high MW PVA-T when photocuring began 30s after mixing.

[0254] The apparently unintuitive decrease in pore diameter compared to the 10s delay was found to be caused by droplets of GelMA that had become encapsulated within deeper regions of the PVA-T phase. While these GelMA droplets were correctly identified as such, they introduced errors when calculating the distance transform of the larger structure. To account for this, the pore diameters for Z-stacks of the 30s delay gels were measured manually in ImageJ and marked separately on the plot with star symbols.

[0255] Spatial Tailoring of Pore Size using Dynamic Exposure

[0256] As a proof of concept for how this approach could be used to control pore size throughout the gel, additional gels were fabricated, this time using only the low DoF GelMA-RB combined with the high MW PVA-T. For these gels, both a 4 mm circle and a 4 mm x 4 mm rectangle were cured. Prior to curing, the prepolymer mixture was acoustically mixed as above. However, instead of one uniform exposure, the gel was broken up into four separate regions. The exposure in each region was programmed to begin at different times following the acoustic mixing (0, 0.5, 2, or 10s). Each region was exposed at approx. 140 mW / cm2and for Is, independently of the other regions. The gels were washed and incubated in DPBS as described above, then tiled confocal images were acquired to illustrate the change in pore size throughout the XY plane of the gels.

[0257] EXAMPLE 3

[0258] Fabrication of Porous Gels using Acoustic Mixing

[0259] Samples of the aqueous two-phase emulsion gels were prepared via acoustic mixing in a prototype biofabrication system (as shown in the schematic of Figure 2A). For all samples, the two-component prepolymer mixture comprised either the high or low DoF GelMA-RB combined with either unmodified high MW PVA, low MW PVA-T, or high MW PVA-T.

[0260] The respective concentrations of the two polymer components remained the same across all samples: 50mg / ml for GelMA-RB, and 30mg / ml for PVA(-T).

[0261] In all samples, the photoinitiator was a combination of ImM Tris(2,2'- bipyridyl)dichlororuthenium(II) hexahydrate (Ru, SigmaAldrich cat. no. 224758) and lOmM sodium persulfate (SPS, SigmaAldrich cat. no. S6172). All components were initially dissolved in DPBS prior to being combined. All concentrations listed are the final concentrations of the respective components. Stock solutions of 200mg / ml GelMA-RB, lOOmg / ml PVA(-T), lOmM Ru, and lOOmM SPS were used to create each mixture. 2ml of each mixture was prepared to manufacture the gels for that mixture.

[0262] To create the macromer mixtures, the GelMA and PVA were first combined, diluted with DPBS, and vortexed for 30s. The Ru and SPS were then added, and the sample was covered in aluminum foil to protect from light. The sample was then placed in a water bath at 37 °C to ensure the mixture did not thermally gel. Immediately prior to being loaded into the biofabrication system, the samples were vortexed for an additional 30s and repeatedly pipetted using a 1ml pipette tip 10 times. The entirety of the mixture was then pipetted into one well of a glass coverslip-bottom, methacrylate-treated 12-well plate (Cellink cat. no. D16110025295). The well plate was then loaded into the enclosure of the biofabrication system, whose internal temperature was maintained at 37°C.

[0263] To begin, the hollow cylindrical print head was first immersed in the macromer mixture to establish a trapped meniscus (as shown in the schematic of Figure 2B). This meniscus represents the interface at which the gels would be cured using 405 nm light. Prior to curing, however, an acoustic signal was played for 5s which caused the meniscus to vibrate. These vibrations drove fluid flow in the mixture to actively mix and homogenize the solution. After the 5s of acoustic mixing ceased, a preprogrammed delay elapsed before the photocuring began. For all gels, the exposure pattern was a 4 mm diameter circle. Once the photocuring ceased, the print head was removed from the prepolymer mixture, which was then transferred to the next well for the subsequent gel.

[0264] For each macromer mixture, the first gel manufactured was a control, meaning no acoustic mixing was performed: the print head was immersed in the solution, the meniscus was established, and photocuring commenced. The subsequent gels had preprogrammed delays ranging from 0s (i.e. no delay) to 30s. For prepolymer mixtures containing PVA-T, photocuring was performed at approx. 140mW / cm2for Is. For mixtures containing PVA, photocuring was performed at approx. 350mW / cm2for 5s. After each gel was cured, it was rinsed twice with 2ml of DPBS. After all the gels for a mixture had been manufactured, they were incubated overnight in DPBS at 37°C before being imaged. Across all solutions, acoustically vibrating the print interface could be used to mix the two phases and create a more homogenous solution compared to the control condition (Figure 3).

[0265] By choosing a ratio of 50 mg / ml GelMA to 30 mg / ml porogen (whether that porogen was PVA or PVA-T), the solution consistently decomposed into a system wherein the porogen created channels that were contained within a GelMA bulk phase. As the delay period between acoustic mixing and photocuring was increased, the porogen phase continued to grow. The pore sizes were highly consistent amongst all gels and all time points, suggesting that 5s of acoustic mixing was adequate in all cases.

[0266] The low molecular weight PVA-T did not separate sufficiently from the GelMA to create distinct pores over the time scales studied here. Typically, it is postulated that porogens of higher molecular weight require a lower critical concentration to undergo phase separation. The low MW PVA-T was excluded from subsequent analyses so that clearer conclusions could be drawn from comparing the modified and unmodified high MW PVA mixtures.

[0267] Both PVA and PVA-T formed a porogen phase inside the bulk GelMA phase. However, the substantially higher exposure intensities and times required to cure the GelMA + PVA gels preclude PVA as an effective choice for biofabrication of 3D cell culture scaffolds. This may be explained when considering two factors. Firstly, the Ru / SPS photoinitiator system catalyzes the crosslinking of both methacrylate groups and tyramine groups. Secondly, although the two phases are referred to as the GelMA “bulk” and PVA(-T) “porogen” phases, it is important to note that the two molecules never completely partition themselves between the two compartments, even at thermodynamic equilibrium. It is more accurate to consider the bulk phase to be a “high GelMA, low PVA(-T)” concentration phase and vice versa for the porogen phase. Evidence of this can be seen in the confocal images, where aggregates of Rhodamine B (chemically bound to GelMA, Figure 3) can still be seen within the porogen phase. Taken together, this suggests that PVA-T acted to structurally reinforce the gel on both a macroscopic scale (by forming a network in the porogen phase) and the microscopic scale (by crosslinking with tyramine groups present on the GelMA backbone). EXAMPLE 4

[0268] Dynamic Control of Pore Size and Morphology

[0269] Looking at Figure 5, pore size remains comparable between the GelMA + PVA-T mixtures across delay periods up to 10s, regardless of the degree of functionalization of the GelMA used. The PVA-T pore diameter starts at approx. 13pm for both GelMA batches, growing to 36pm for the low DoF GelMA system and 39pm for the high DoF GelMA system after 10s. After being allowed to separate for 30s, the two systems diverged slightly: 98pm for the low DoF GelMA and 117 pm for the high DoF GelMA.

[0270] However, although the pore size is quantitatively similar, the pore interconnectivity of the two GelMA + PVA-T systems appear qualitatively different. Comparing the slices from the Z- stacks in Figure 3, more disconnected pores are apparent in the high DoF GelMA system, especially for the 10s delay period and beyond. Compared to the control condition, both systems display a more interconnected morphology across all delay periods. We posit that shear homogenizes the two phases such that they begin to separate via spinodal decomposition. As the volume fractions of GelMA and PVA(-T) change over time, the system reaches a state where it begins to decompose via nucleation instead.

[0271] Whereas the GelMA degree of functionalization had only a minor effect on the generated pore sizes, functionalizing the PVA with tyramine had a significant effect on both the maximum pore diameter and the rate at which the pores grew (Figure 6). This can be explained by the degree of intermolecular interactions between the GelMA and PVA or PVA-T molecules. The higher degree of intermolecular interactions between tyramine groups on the PVA-T and GelMA polymer chains would act to improve the miscibility of the two phases31. This could also be thought of as lowering the interfacial tension between the GelMA and PVA-T phases, which has been shown to slow the rate of phase separation14.

[0272] Tailoring of Pore Size via Controlled Exposure Delays

[0273] Given the degree to which pore size could be controlled by exposure timing, we investigated whether subsequent exposures in a single layer could be used to tailor pore size. As a proof of concept, this was done for both a 3mm diameter circular gel made up of concentric rings and a 3mmx3mm square pattern made up of vertical bars. Both geometries were successfully fabricated, and both showed distinct differences in pore size in their respective arrangements. In both cases, the pores within the gel maintained an interconnected, bicontinuous morphology. These results indicate that the method presented here could be a valuable tool for tailoring the in vitro environment for 3D cell culture applications.

[0274] Controlling the timing of the delivery of curing radiation to the aqueous two-phase system made it possible to create different pore sizes within a single material.

[0275] Figure 7 shows a confocal microscopy image of a hydrogel of the kind described herein wherein concentric regions of the liquid pre-polymer solution were exposed to curing radiation at progressive longer delays. The numerical values for each concentric region indicate the delay of exposure time in seconds, starting from when acoustic mixing of the system is interrupted. In the Figure, the white outlines denote the regions where curing radiation exposure began 0 seconds after acoustic mixing of the system had ceased, where the curing radiation exposure began 0.5 seconds after acoustic mixing ceased, and the same for 2 second and 10 seconds. Where this "delay" was longer, the liquid aqueous two-phase system was allowed to undergo phase separation for a longer period of time before the curing radiation cured the material. Therefore, comparing the size of pores created in regions with different delay periods, the pores in regions with a longer delay period are larger than those in regions with a shorter delay period, as shown in the corresponding insets.

[0276] Figure 8 shows an alternative outcome of the same concept, in the context where the cured gel and the sequentially exposed regions are rectangular instead of circular. In the Figure, insets (1) and (2) show close-ups of corresponding porosity transition areas (1) and (2) from the main image. The insets show the boundary between regions irradiated at different delays, highlighting the corresponding difference in pore size (the pores being the darker regions of the images). The delay periods for different regions (0 seconds, 0.5 seconds 2 seconds, and 10 seconds) are denoted by the numbers below the bottom image.

[0277] The ability to create regions of multiple porosities within a single layer of polymer material is particularly relevant for layer-by-layer additive manufacturing techniques. By re-establishing a new layer of monophasic polymer solution above previously cured material, three- dimensional distributions of porosities can be created by curing additional layers. With this technique, material composites and metamaterial designs can be manufactured more quickly and at a higher degree of dimensional precision.

[0278] In general, there is significant interest in being able to tailor properties such as stiffness, topology, or chemical cues at length scales comparable to individual cells (10-150 pm). Doing so facilitates the curation of cellular responses such as migration, proliferation, and differentiation. However, many previous attempts to use biofabrication techniques to tailor hydrogels have been limited to length scales above 100pm by technical constraints such as the effective feature resolution of light-based photolithography techniques. Our approach allowed us to create interconnected porous channels at scales from approx. 5 to 120 pm. To our knowledge, this is also the first example where multiple porosities were patterned within a single material in a light-based additive manufacturing process. We therefore believe this method is a valuable tool for biofabrication. For example, many microfluidic organ-on-chip models consist of multiple cell-laden flow channels separated by a semi-permeable membrane. Being able to tailor the porosity (and therefore permeability) of these membranes both within and between devices would be useful for matching the permeability of the desired anatomical structures. Further investigations could validate the utility of this approach in specific organ- on-chip models.

[0279] EXAMPLE 5

[0280] When observing the pore morphology of the gels, the mixture of high DoF GelMA with PVA- T began to separate via nucleation at an earlier time point than low DoF GelMA with PVA-T (Figure 3). As our aim was to present a method by which pore architecture could be tailored within hydrogels, a quantitative analysis of the differences in polymer-polymer interactions is outside the scope of this work. However, by considering Flory-Huggins solution theory, a qualitative explanation can be given.

[0281] Since the composition and temperature of the two cases are identical, the Flory-Huggins interaction parameter, %, can be regarded to evaluate the difference in behavior. The Flory- Huggins parameter is a dimensionless quantity that characterizes the interactions of both polymer- solvent and polymer-polymer pairs:

[0282] Where z is the coordination number of the lattice being considered, kBis Boltzmann’s constant, T is temperature, and m12, and a>22are the pairwise interaction energies of the polymer species with each other and themselves, respectively. The composition (i.e., the volume fraction) and interaction parameter of a given polymer blend determines whether the system will be stable, unstable, or metastable. These states determine whether the solution will remain homogenously mixed (stable) or separate (i.e. demix) via spinodal decomposition (unstable) or nucleation (metastable).

[0283] If demixing is energetically favorable (corresponding to positive values of %), an initially homogenous mixture will separate until two phases exist whose compositions lie on the binodal curve, defined as the phase boundary between a one-phase and two-phase system. Importantly, our results show that both the high and low DoF GelMA mixtures initially undergo spinodal decomposition, meaning the system starts in an unstable state. As the composition of the two phases diverge, the system must first enter the metastable state before reaching equilibrium. The time at which the system transitions from spinodal decomposition to nucleation depends on the magnitude of % and therefore the relative magnitudes of the m12, and a>22terms.

[0284] Coming back to the high and low DoF GelMA + PVA-T mixtures, we believe that the higher concentration of methacryloyl groups would have led to a higher number of hydrogen bonds between GelMA chains. This is equivalent to saying the high DoF GelMA would have had a higher pairwise interaction energy with itself, reducing the % value for that system. This in turn led to the high DoF GelMA + PVA-T mixture transitioning to nucleation earlier than the low DoF GelMA + PVA-T. Of course, these speculations would need to be confirmed by more in- depth investigations into the thermodynamic properties of these polymer blends.

[0285] As used herein, the term “about”, in the context of numerical values, typically means + / -5% of the stated value, more typically + / -4% of the stated value, more typically + / -3% of the stated value, more typically, + / -2% of the stated value, even more typically + / -1% of the stated value, and even more typically + / -0.5% of the stated value.

[0286] Throughout this specification and the claims which follow, unless the context requires otherwise, the word ‘comprise’, and variations such as ‘comprises’ and ‘comprising’, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0287] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of forming a porous polymeric structure, the method comprising the steps of: providing an aqueous two-phase system (ATPS) comprising a first phase containing a first photo-curable macromer and a second phase containing a second photo-curable macromer, transmitting acoustic energy to the system to induce shear mixing of the phases, resulting in a monophasic system, removing the acoustic energy allowing de-mixing of the monophasic system, irradiating the de-mixed system with curing radiation to promote curing of the first and the second photo-curable macromer, and removing one of the cured macromers to obtain the porous polymeric structure.

2. The method of claim 1, comprising: introducing a print head having a cavity containing gas into the aqueous two-phase system to form a submerged gas-liquid interface between the gas and the aqueous two-phase system, wherein said gas-liquid interface is constrained to the print head and defines a printing surface, transmitting the acoustic energy to the submerged gas-liquid interface to induce the shear mixing of the phases, resulting in the monophasic system, removing the acoustic energy from the submerged gas-liquid interface, allowing the demixing of the monophasic system, and irradiating the de-mixed system with the curing radiation by projecting said curing radiation on the submerged gas-liquid interface with the print head, thereby promoting curing of the first and the second photo-curable macromer at the printing surface.

3. The method of claim 1 or 2, wherein said de-mixing results in a co-continuous system having a phase containing the majority of the first photo-curable macromer and another phase containing the majority of the second photo-curable macromer.

4. The method of any one of claims 1-3, wherein said de-mixing results in formation of a continuous phase containing the majority of the first photo-curable macromer and a dispersed phase containing the majority of the second photo-curable macromer.

5. The method of any one of claims 1-4, wherein the irradiation with curing radiation begins immediately after the acoustic energy is removed.

6. The method of any one of claims 1-5, wherein the irradiation with curing radiation begins at least 10 seconds after the acoustic energy is removed.

7. The method of any one of claims 1-6, wherein the irradiation with curing radiation begins 30 seconds after the acoustic energy is removed.

8. The method of any one of any one of claims 1-7, wherein the irradiation with curing radiation is performed on different zones of the de-mixed system, each zone being irradiated at sequentially increasing irradiation delay starting from when the acoustic energy is removed.

9. The method of any one of claims 1-8, wherein the acoustic energy has a frequency of at least 1 Hz.

10. The method of any one of claims 1-9, wherein the acoustic energy has a frequency of from 20 Hz to 25 Hz.

11. The method of any one of claims 1-10, wherein the curing radiation has a wavelength of 405 nm.

12. The method of any one of claims 1-11, wherein the first or second photo-curable macromer has a molecular weight of at least 30 kDa.

13. The method of any one of claims 1-12, wherein the first or second photo-curable macromer has a molecular weight of 85 kDa- 124 kDa.

14. The method of any one of claims 1-13, wherein the cured second macromer is removed to obtain the porous polymeric structure.

15. The method of any one of claims 1-14, wherein at least one of the first and second photo-curable macromer is a hydrogel macromer.

16. The method of any one of claims 1-15, wherein the first photo-curable macromer is a hydrogel macromer comprising one or more of polyethylene glycol diacrylate (PEGDA), gelatin methacryloyl (GelMA), and hexanediol diacrylate (HDD A).

17. The method of any one of claims 1-16, wherein the second photo-curable macromer is selected from one or more of a polyvinyl alcohol (PVA), a polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), a polyalkyl hydroxy acrylate and methacrylate, a polysaccharide, a polyacrylic acid, a polyamino acid, a polyacrylamide, and a conjugate correspondent thereof.

18. The method of any one of claims 1-17, wherein the ATPS comprises a photo-initiator selected from one or more of tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate, and sodium persulfate (SPS).

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