WO2025032337A1

WO2025032337A1 - Method for preparing a hydrogel

Info

Publication number: WO2025032337A1
Application number: PCT/GB2024/052096
Authority: WO
Inventors: Wenmiao Shu; Yifei Wan
Original assignee: University Of Strathclyde
Priority date: 2023-08-08
Filing date: 2024-08-08
Publication date: 2025-02-13
Also published as: GB202312148D0

Abstract

A method of manufacturing a hydrogel comprises: (i) providing a polymerizable or crosslinkable hydrogel material (101,102); (ii) freeze-drying (112) the hydrogel material (105); and (iii) polymerizing or crosslinking (113) the hydrogel material (120) in solid state.

Description

Method for preparing a hydrogel

Field of the Invention

The present invention relates to methods for preparing a hydrogels, to hydrogels produced by said methods, and to articles, such as medical articles, made from such hydrogels.

Surgical meshes are medical devices intended to be inserted in a patient’s body during surgery. A surgical mesh typically provides permanent or temporary support for a particular tissue area or organ of the patient. Exemplary applications include hernia repair and pelvic organ prolapse.

Materials currently used in mesh technology are generally categorised in two groups: absorbable materials or non-absorbable materials.

Absorbable materials are typically made of natural materials such as collagen, alginates, chitosan, or mesenchymal and porcine dermal matrix, but can also include synthetic materials such as polylactic acid (PLA), polyglycolic acid (PGL), or poly(lactic- co-glycolic acid) (PLGA). Absorbable materials typically show excellent integration into the body, but tend to suffer from rapid degradation, mechanical failure and/or poor scar tissue formation.

Non-absorbable materials are typically made of synthetic materials such as polypropylene (PP), polyethylene terephthalate (PET) polytetrafluoroethylene (PTFE) or expanded PTFE (ePTFE). Non-absorbable materials are typically affordable, and generally considered to represent the gold standard in relation to mesh durability and mechanical properties. However, they are more prone to triggering immune reaction and can cause adhesion, erosion, infection and may in some cases require mesh removal surgery.

Due to the disadvantages of non-absorbable materials, various attempts have been made to produce absorbable products with improved mechanical properties. However, current technologies tend to generate materials which still display inferior mechanical properties, or which are prohibitively expensive (for example, produced from decellularised animal or human scaffolds).

Therefore, there is a need in the art for low-cost mesh materials that exhibit good biocompatibility but also have improved mechanical properties.

A number of conventional techniques exist to produce hydrogel materials. Typically, hydrogels are made by a process that involves crosslinking a starting material such as gelatin provided within a liquid medium. This leads to hydrogels which are flexible but typically have low strength and fast degradation.

CN115636954A (YANG JIN et al) discloses a method of preparing a hyperelastic double-layer photo-thermal hydrogel which comprises preparing a composition comprising polyvinyl alcohol and cellulose fibres, and performing a series of steps including freezing, thawing, and crosslinking. No freeze-drying step is involved in this process.

CN110818921 B (Suzhou Institute Of Nano-Tech And Nano-Bionics) discloses a process comprising (1) mixing and reacting gellan gum and methacrylic anhydride to obtain double-bond modified gellan gum; (2) mixing and reacting glycidyl methacrylate and collagen to obtain double-bond modified collagen; and (3) mixing the double-bond modified gellan gum with a photoinitiator and the double-bond modified collagen, soaking in a divalent ion bath to obtain an ion-crosslinked hydrogel, and then performing a photoinitiation reaction to obtain the ultraviolet light secondary cured double-crosslinked hydrogel. Step (1) may comprise freeze-drying the double-bond modified gellan gum, and step (2) may comprise freeze-drying the double-bond modified collagen. However, such is dissolved in a buffer solution before crosslinking in step (3).

Other documents include preparing crosslinkable hydrogel materials, which can be freeze-dried to prepare a solid material (e.g. in powder form), followed by dissolving the solid material, and subsequent crosslinking, as for example in CN113244455B (Guangxi Medical University), CN114836047A (Zhejiang Sci-Tech University), CN114773549A (Zhejiang A & F University), CN113336973B (Sun Yat-Sen University), CN112851978B (Northwestern Polytechnical University), and CN109627462A (Univ Xian Technology).

US 9,028,857 B2 (Le visage et al) discloses a method for preparing a porous scaffold, which comprises preparing an alkaline solution of a polysaccharide, and crosslinking under alkaline condition with sodium trimetaphosphate during a sublimation step.

Acta Biomaterialia, vol. 6, no. 9, 2010, Autissier et al., "Fabrication of porous polysaccharide-based scaffolds using a combined freeze-drying/cross-linking process", p. 3640-3648, discloses the fabrication of porous polysaccharide-based scaffolds by crosslinking during a freeze drying process.

US 2020/0399431 A1 (Huang et al) discloses a method of preparing a covalently cross-linked hyaluronic acid aerogel, comprising promoting a chemical crosslinking through covalent bonds between the crosslinking agent and the hyaluronic acid by a freeze-drying technique to obtain a covalently cross-linked.

US 2014/0341836 A1 (Sawhney et al) discloses a method for making freeze dried hydrogel and structures therefrom, comprising combining precursor components to initiate crosslinking, allowing crosslinking to a desired level of complete crosslinking, and freeze-drying the partially crosslinked hydrogel.

It is an object of the invention to address and/or mitigate one or more problems associated with the prior art.

It is an object of the invention to produce low-cost mesh materials that exhibit good biocompatibility but also have good mechanical properties.

Summary

The present invention is based on the finding that it is possible to produce low- cost durable scaffold or hydrogel materials based on cross-linked or polymerised hydrogels that exhibit good biocompatibility but also have good mechanical properties.

According to a first aspect, there is provided a method of manufacturing a hydrogel, the method comprising:

(i) providing a polymerizable or crosslinkable hydrogel material;

(ii) freeze-drying the hydrogel material; and

(iii) polymerizing or crosslinking the hydrogel material in solid state.

Preferably, steps (i)-(iii) are performed sequentially.

The term “freeze-drying” herein refers to a conventional method also named lyophilisation or cryodesiccation. Freeze-drying involves dehydrating a material, e.g. the hydrogel material, by freezing the material below the solvent’s triple point, then reducing pressure to sublimate the solvent.

Typically, the solvent may comprise, may consist of, or may be water.

Without wishing to be bound by theory, it is believed that the polymerizable or crosslinkable hydrogel material, in its normal form, forms a hydrated, loose structure. By freeze-drying the polymerizable or crosslinkable hydrogel material, the solvent, e.g. water molecules, are removed from the hydrogel structure, which causes densification of the hydrogel structure.

Preferably, polymerizing or crosslinking the hydrogel material may be performed after freeze-drying. The inventors have found that by polymerizing or crosslinking the hydrogel material in solid state, i.e. after freeze-drying, the hydrogel may be “fixed” in its condensed structure. This results in a hydrogel with improved mechanical properties, e.g. increased strength. This is true even when the polymerized or cross-linked hydrogel material is subsequently rehydrated, as the polymerization or crosslinking of the hydrogel material in solid states ensures that it retains its denser structure even after rehydration. Thus, the present process provides a hydrogel structure with increased durability, strength and density. In contrast, it was observed that, if a hydrogel is polymerized or crosslinked before or during freeze-drying, the freeze-drying step typically causes the hydrogel structure to break or collapse, leading to poor mechanical properties upon rehydration.

Advantageously also, free-radical crosslinking or polymerisation is typically challenging in high water-content environments such as hydrogel compositions. Freeze- drying the hydrogel material first and performing the polymerizing or crosslinking in solid state may help promote free-radical crosslinking or polymerisation of the hydrogel material, particularly when using typically low water-soluble or non-water-soluble initiators.

The polymerizable or crosslinkable hydrogel material may comprise or may consist of a crosslinkable hydrogel. For example, the hydrogel may comprise a modified or functionalised hydrogel, such as an acrylated hydrogel, e.g. acrylated gelatin or acrylated collagen.

The polymerizable or crosslinkable hydrogel material may comprise or may consist of a polymerizable hydrogel. For example, the hydrogel may comprise an unsaturated monomer, e.g. a di- or poly-unsaturated monomer, for example a di- or polyacrylate monomer, such as polyethylene glycol diacrylate (PEGDA).

The polymerizable or crosslinkable hydrogel may comprise a hydrogel comprising a plurality of components.

The hydrogel may comprise or may form, e.g. upon crosslinking or polymerizing, an inter-penetrating network (IPN).

The hydrogel may comprise a non-polymerizable or non-crosslinkable first component and a polymerizable or crosslinkable second component.

The non-polymerizable or non-crosslinkable first component may comprise a natural polymer such as collagen, gelatin, alginate, or the like.

The polymerizable or crosslinkable second component may comprise an unsaturated component, e.g. a polymerizable component such as PEGDA. The hydrogel may comprise a plurality of polymerizable or crosslinkable components, e.g. a polymerizable or crosslinkable first component and a polymerizable or crosslinkable second component.

The polymerizable or crosslinkable first component may comprise modified or functionalised hydrogel, such as an acrylated hydrogel, e.g. acrylated gelatin or acrylated collagen.

The polymerizable or crosslinkable second component may comprise an unsaturated component, e.g. a polymerizable component such as PEGDA.

It will be appreciated that the particular hydrogel composition may depend on the desired application, e.g. as an implantable hernia repair scaffold, pelvic repair scaffold, or as a bone repair scaffold. For example, the composition may comprise a first component comprising a natural polymer such as collagen, gelatin, or alginate, in an amount of about 5-15 wt%, e.g., and a second component comprising or consisting of an unsaturated component, e.g. a polymerizable component such as PEGDA, in an amount of about 0.1-15 wt%, e.g. about 1-10 wt%.

Typically, the step of polymerizing or crosslinking the hydrogel material may comprise a free radical and/or addition reaction. It was surprisingly found that this type of reaction is better suited to a crosslinking and/or polymerisation reaction in solid state, i.e. after freeze-drying. In particular, free-radical crosslinking or polymerisation is typically challenging in high water-content environments such as hydrogel compositions. Freeze-drying the hydrogel material first and performing the polymerizing or crosslinking in solid state may help promote free-radical crosslinking or polymerisation of the hydrogel material, particularly when using typically low water-soluble or non-water-soluble initiators.

Step (iii) may comprise providing a stimulus to induce reaction in the hydrogel material.

Step (iii) may comprise irradiating the hydrogel material, e.g. exposing the hydrogel material to a radiation source such as UV, electron beam, gamma radiation, or light. Conveniently, step (iv) may comprise irradiating the hydrogel with UV radiation. In such instance, step (iii) may comprise photo-polymerizing or photo-crosslinking the hydrogel material. This may be preferred when the hydrogel is substantially transparent to the type of radiation intended to be used.

Step (iii) may comprise stimulating the hydrogel by heating and/or by increasing temperature. In such instance, step (iii) may comprise thermo-polymerizing or thermocrosslinking the hydrogel material. Step (iii) may comprise heating the hydrogel material. This may be preferred when the hydrogel is substantially not transparent to one or more types of radiation intended to be used such as UV light.

The method may comprise adding a crosslinking initiator or a polymerization initiator. Advantageously, the method may comprise (ia) adding a crosslinking initiator or a polymerization initiator before step (ii), typically between step (i) and step (ii). By such provision, the method may ensure that the initiator is present in the hydrogel structure upon freeze-drying. This may help ensure that polymerization or crosslinking is able to take place within the freeze-dried structure whilst in solid state.

The photoinitiator may comprise or may consist of lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), 2-Hydroxy-4'-(2-hydroxyethoxy)-2- methylpropiophenone (Irgacure® 2959), a ruthenium-based photoinitiator such as tris(bipyridine)ruthenium(ll), Eosin Y and/or its co-initiators TEOA and NVP; Camphorquinone (2,3-bornanedione) and/or riboflavin.

The thermal initiator may comprise or may consist of an azo compound such as 2,2'-Azobis(methylpropionitrile) (AIBN - Azobisisobutyronitrile), 4,4'-Azobis(cyanovaleric acid) (AVCA), 1 ,1 '-Azobis(cyclohexanecarbonitrile) (ABCN); benzoyl peroxide, di-tert- butyl peroxide, cumene hydroperoxide, and/or peracetic acid.

The method may comprise (iv) rehydrating the hydrogel. The method may comprise adding a solvent, e.g. water, to the hydrogel. Preferably, step (iv) may be carried out after step (iii).

According to a second aspect, there is provided a method of manufacturing a hydrogel, the method comprising, sequentially:

(i) providing a polymerizable or crosslinkable hydrogel material;

(ii) adding an initiator;

(iii) freeze-drying the hydrogel material; and

(iv) polymerizing or crosslinking the hydrogel material.

The method may comprise mixing the initiator and the polymerizable or crosslinkable hydrogel material, e.g. before step (iii).

The initiator may be dispersed within the polymerizable or crosslinkable hydrogel material.

Step (iv) may comprise providing a stimulus to induce reaction in the hydrogel material. Step (iv) may comprise irradiating the hydrogel material, e.g. exposing the hydrogel material to a radiation source such as UV, electron beam, gamma radiation, or light. Conveniently, step (iv) may comprise irradiating the hydrogel with UV radiation.

Step (iv) may comprise heating the hydrogel material.

Advantageously, adding a crosslinking or a polymerization initiator before step (ii), typically between step (i) and step (iii), may ensure that the initiator is present in the hydrogel structure upon freeze-drying. This may help ensure that polymerization or crosslinking is able to take place within the freeze-dried structure, e.g. whilst in solid state.

The method may comprise (v) rehydrating the hydrogel. The method may comprise adding a solvent, e.g. water, to the hydrogel.

According to a third aspect, there is provided a hydrogel structure obtained or obtainable by a method according to the first aspect or the second aspect.

The inventors have found that by polymerizing or crosslinking the hydrogel material in solid state and/or after freeze-drying, the hydrogel may be “fixed” in its condensed structure. This results in a hydrogel with improved mechanical properties, e.g. increased strength. This is true even when the polymerized or cross-linked hydrogel material is rehydrated, as the polymerization or crosslinking of the hydrogel material in solid states ensures that it retains its denser structure even after rehydration. Thus, the present process provides a hydrogel structure with increased durability, strength and density.

The hydrogel structure may comprise or may be a medical article, such as an implantable device, e.g. an implantable medical device. The hydrogel structure may comprise or may be an implantable scaffold or an implantable mesh.

According to a fourth aspect, there is provided an article, e.g. a medical article such as an implantable device, comprising a hydrogel structure according to the third aspect. The article may comprise or may be an implantable scaffold or an implantable mesh.

The article may comprise a plurality of layers.

One or more layers may comprise or may be formed of the hydrogel structure. One or more layers may comprise or may be formed of another material, e.g. a nonabsorbable and/or synthetic material such as polypropylene (PP), polyethylene terephthalate (PET) polytetrafluoroethylene (PTFE) or expanded PTFE (ePTFE). Advantageously, at least one, e.g. both, outer layer(s), may comprise or may be formed of the hydrogel structure. By such providing, the article may exhibit good biocompatibility properties, whist maintaining higher durability through one or more core layers.

The features described in relation to any aspect of the invention may equally apply to any other aspect and, merely for brevity, are not repeated. For example, features described in relation to products can apply in relation to methods, and vice versa.

Brief Description of Drawings

Embodiments of the invention are described with reference to the accompanying drawings, which show:

Figure 1 : a method of manufacturing a hydrogel according to a first embodiment;

Figure 2: a method of manufacturing a hydrogel according to a second embodiment;

Figure 3: Optical & SEM images of hydrogels according of two different embodiments;

Figure 4: Graph illustrating pore size measurements of hydrogels according to two different embodiments;

Figure 5: Graph showing the tensile strength of hydrogels according to different embodiments;

Figure 6: Graphs showing various mechanical properties of hydrogels according to different embodiments;

Figure 7: Graphs illustrating the influence of irradiation time on various mechanical properties of hydrogels according to different embodiments;

Figure 8: Microscopy images of biological live/dead cell staining with

15%Gelatin/5%PEGDA scaffold according to an embodiment

Figures 9 and 10: Graphs showing various mechanical properties of hydrogels according to alternative embodiments. Detailed Description

In the present disclosure, reference is made to a number of terms, which have the meanings provided below, unless a context indicates to the contrary. The nomenclature used herein for defining compounds, in particular the compounds according to the invention, is in general based on the rules of the IIIPAC organisation for chemical compounds, specifically the “IIIPAC Compendium of Chemical Terminology (Gold Book)”. For the avoidance of doubt, if a rule of the IIIPAC organisation is in conflict with a definition provided herein, the definition herein is to prevail. Furthermore, if a compound structure is in conflict with the name provided for the structure, the structure is to prevail.

The term “comprising” or variants thereof is to be understood herein to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The term “consisting” or variants thereof is to be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, and the exclusion of any other element, integer or step or group of elements, integers or steps.

The term “about” herein, when qualifying a number or value, is used to refer to values that lie within ± 5% of the value specified. For example, if a temperature is specified to be about 5 to about 13 °C, temperatures of 4.75 to 13.65 °C are included.

Reference to physical states of matter (such as liquid or solid) refer to the matter’s state at 25 °C and atmospheric pressure unless the context dictates otherwise.

Figure 1 illustrates a method of manufacturing a hydrogel according to a first embodiment illustrate an apparatus 105 for manufacturing a tubular biological structure according to a first embodiment.

In the first step 111 the method comprises mixing a hydrogel composition, which here consists of gelatin 101 and PEGDA 102, with a crosslinking initiator 103, which here is photoinitiator Irgacure® 2959, to generate a first mixture 105.

The first mixture 105 forms a hydrated, physically incorporated structure.

The second step 112 involves freeze-drying the material 105. By freeze-drying the hydrogel material 105, the solvent, here water, is removed from the hydrogel structure, which causes densification by reducing the pore size of the hydrogel structure to form freeze-dried hydrogel 115. The third step 113 involves polymerizing or crosslinking the hydrogel material in solid state, in this case by UV irradiation. In the present case, the PEGDA is polymerised forming an inter-penetrating network (IPN) of gelatin and complex network of PEGDA polymer.

The inventors have found that by polymerizing or crosslinking the hydrogel material 105 in solid state, i.e. after freeze-drying, the hydrogel may be “fixed” in its condensed structure. This results in a hydrogel 115 with improved mechanical properties, e.g. increased strength. This is true even when the polymerized or crosslinked hydrogel material is subsequently rehydrated, as the polymerization or crosslinking of the hydrogel material in solid states ensures that it retains its denser structure even after rehydration. Thus, the present process provides a hydrogel structure with increased durability, strength and density.

Figure 2 illustrates a method of manufacturing a hydrogel according to a second embodiment. The method 210 of Figure 2 is generally similar to the method 110 of the first embodiment, like parts denoted by like numerals, but incremented by ‘100’. However, in this embodiment, the method further comprises a fourth step 214 involving rehydrating the hydrogel 230 by adding a solvent, here water, to the hydrogel 230. This generates a rehydrated hydrogel 240, ready for use, e.g. implantation, which, although rehydrated, still retains its denser structure due to the present process, even after rehydration. Thus, the present process provides a hydrogel structure 240 with increased durability, strength and density.

Figure 3 shows optical microscopy images (3(a) and 3(c)) & SEM images (3(b) and 3(d)) of hydrogels according to two different embodiments. The hydrogel of Figs 3(a) and 3(b) was prepared using a mixture of 15wt% gelatin and 5wt% PEGDA, according to the method of Figure 1 (prior to rehydration). The hydrogel of Figs 3(c) and 3(d) was prepared using a mixture of 5wt% gelatin and 5wt% PEGDA, according to the method of Figure 1 (prior to rehydration).

Figure 4 shows a graph illustrating pore size measurements of the two hydrogels shown described in Figure 3.

As can be seen, both hydrogels have a low porosity, due to the removal of water during freeze-drying prior to crosslinking/polymerisation. The first hydrogel (made from a mixture of 15wt% gelatin and 5wt% PEGDA) had an average porosity of about 98pm ± 28 pm. The second hydrogel (made from a mixture of 5wt% gelatin and 5wt% PEGDA) had an average porosity of 263 pm ± 54 pm. Whilst the more concentrated mixture resulted in a lower pore size, it can be seen that both compositions led to an average pore size much lower than the typical pore size of a similar composition made using a conventional process.

Figures 5-7 generally relate to the investigation of mechanical properties of hydrogels made using the present methodology.

Figure 5 is a graph showing the tensile strength of hydrogels (in dry condition - i.e. without rehydration) made according to the present methodology, using various amounts and ratios of gelatin and PEGDA. These amounts ranged from 19wt% gelatin and 1wt% PEGDA to 5wt% gelatin and 15wt% PEGDA. It can be observed that all of these compositions led to high tensile strength (from about 7.23MPa to about 32MPa). All of these values are far higher than the tensile strength of corresponding compositions made using a conventional process. Typically, a conventional process involving crosslinking followed by freeze drying will lead to a hydrogel material that would disintegrate and tensile strength would not be easily measurable.

Figure 6 depicts graphs showing various mechanical properties ((a) and (b): tensile strength; (c) and (d): elongation at break) of hydrogels ((a) and (c): in dry condition; (b) and (d): after rehydration), made according to the present methodology, using various amounts and ratios of gelatin and PEGDA. These amounts and ratios were 15wt% gelatin and 5wt% PEGDA, 22.5wt% gelatin and 7.5wt% PEGDA, 10wt% gelatin and 10wt% PEGDA, and 15wt% gelatin and 15wt% PEGDA. It can be observed that all of these compositions led to high tensile strength, and high elongation at break. All of these values are far higher than the tensile strength and elongation at break of corresponding compositions made using a conventional process. Without freeze-drying, the UV cross-linked hybrid hydrogel of gelatin and PEGDA (conventional method) partially formed after immersion in 37°C. Conventional UV cross-linked hybrid hydrogels undergo freeze-drying and then rehydration, still resulting in structural integrity yet impractical to handle. Therefore, with or without freeze-drying, the conventional UV cross-linked hybrid hydrogel of gelatin and PEGDA exhibits a tensile strength of 0 Pa and/or tensile strength that would not be easily measurable.

Figure 7 shows graphs illustrating the influence of irradiation time on various mechanical properties ((a) and (b): tensile strength; (c) and (d): elongation at break) of hydrogels ((a) and (c): in dry condition; (b) and (d): after rehydration) of hydrogels made according to the present methodology, using 15wt% gelatin and 5wt% PEGDA;

It can be seen that, although all of the polymerised compositions exhibited superior properties, the mechanical properties of the compositions can be fine-tuned, e.g. based on the anticipated application, based on the exposure time to activating irradiation.

Other parameters allowing fine-tuning of these properties include, for example, pH.

Figure 8 shows microscopy images of a 15%Gelatin/ 5%PEGDA hydrogel scaffold hydrogels made according to the present methodology. (a)The gelatin/PEGDA mesh exhibits porous structure under bright field, (b) primary human umbilical vein endothelial cells (HLIVEC, green, FDA staining) were seeded into gelatin/PEGDA mesh for 7 days, (c) fibroblasts were proliferate followed with the porous structure of the mesh, and (d) no dead cells (red when dead, PI staining) observed. It can be seen that the biocompatibility of the present hydrogels is excellent, as evidenced by the cell staining shown in green (350) in Figures 8(b).

Comparison of crosslinking conditions

To test the different effects of free radical polymerisation conditions for materials with varied optical transparency, photoinitiators (Irgacure 2959) and thermal iniatiators (AIBN, and ACVA) were tested for a range of hydrogel materials, namely: gelatin/PEGDA, and gelatin/PEGDA with addition of HEMA (translucent), PVA (translucent), graphene (non-transparent) and HA (non-transparent).

Materials

The materials used in this study included gelatin from porcine skin (gel strength 300, Type A); Poly(ethylene glycol) diacrylate (PEGDA, average Mn 700); 2-Hydroxy-4'- (2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959, 98%); 2,2'-Azobis(2- methylpropionitrile) (AIBN, 98%); 4,4'-Azobis(4-cyanovaleric acid) (ACVA, >98.0% (T)); 2-Hydroxyethyl methacrylate (HEMA, >99%, contains <50 ppm monomethyl ether hydroquinone as inhibitor); Poly(vinyl alcohol) (PVA, Mw 89,000-98,000, 99+% hydrolysed); hydroxyapatite (HA, BioReagent, suitable for plant cell culture, powder). All materials above were sourced from Sigma-Aldrich. The graphene solution was sourced from Karbohm. Preparation of materials

Gelatin powder was dissolved in pure water at 50°C with a magnetic stirrer under 300 rpm for 20 mins. The additive (when used) was added to the gelatin solution for 30 minutes to keep the homogenous condition. Then, the initiator was mixed with PEGDA solution at room temperature and slowly dropped into the hydrogel solution for 5 mins. The hydrogel precursor was frozen at -20 °C for 24h and freeze-dried at 0.05mbar at - 80°C for 24h. After freeze-drying, the material is in dry condition. When a UV initiator (Irgacure® 2959) was used, the material was cured under ultraviolet light at 365 nm for 15 mins for each side. When a AIBN initiator was used, the material was heated to 60°C in an oven for 3h. When a ACVA initiator was used, the material was heated to 70°C in an oven for 3h. The materials can be stored in dry condition for further use.

Mechanical test

All materials for the mechanical properties test were immersed in phosphate- buffered saline (PBS) solution to rehydrate at 37°C overnight to achieve water equilibrium. Mechanical tests were conducted using a Mechanical Tester Model Mach-1 v500c (Biomomentum) equipped with a 100N load cell. For the tensile strength test, the material was cast into a mould to form a dumbbell shape with a cross-sectional area of 2mm x 2mm. The material was then subjected to tensile stress using two grippers at each end, at a velocity of 0.1mm/s, to determine the force required to break it. For the unconfined compression test, the material was cast into a mould to form a disc with a diameter of 12mm and a thickness of 2mm. The material was compressed with a ramp velocity of 0.1% of the thickness per second until it reached 10% strain. The compressive modulus was calculated from the linear portion of the stress-strain curve, specifically within the 50%-100% strain range.

All tests were performed in triplicate. For statistical analysis, a one-way ANOVA followed by a normality test was conducted. A p-value of less than 0.05 was considered statistically significant. All date is displayed in Prism 10. The results are shown in Table 1.

Table 1 : Effects of Various Initiators on various Hydrogel compositions After Rehydration and Mechanical Testing

G10_P10 WITH WITH WITH WITH

10 (W/W) % 5 (W/W) % 1 (W/W )% 10 (W/W) % HA HEMA PVA GRAPHENE

Elongation at modulus)

0 15±0 04 0 0 0

Elongation at modulus)

break “G10_P10” denotes the polymer composition material containing 10% Gelatin and 10% PEGDA.

When Irgacure® 2959 was used as initiator, it can be seen that, when an additive was used (HEMA, PVA, HA, or graphene), a crosslinked hydrogel was not formed after rehydration (resulting in no measurable mechanical properties), which means the crosslinking did not occur. It is believed that this is because UV light could not penetrate into the material scaffold to induce sufficient photo crosslinking reaction.

When a thermoinitiator was used (AIBN or AVCVA), it can be seen that these additives caused an improvement in the mechanical properties of the resulting hydrogel, demonstrating that crosslinking of the base hydrogel did occur. Alternative hydrogel compositions

The above examples were based on a hydrogel composition containing gelatin type A and PEGDA. As mentioned above, the principles of the present invention can be applied using alternative hydrogel material components, such as optionally modified gelatin, collagen or alginates, and other unsaturated polymerisable compounds.

The following examples describe using alternative gelatin derivatives, and in particular provide a comparison using:

(i) Gelatin (type A) + PEGDA (as above);

(ii) Gelatin (type B) + PEGDA; and

(iii) GELMA (methacrylated gelatin) + PEGDA.

Gelatin (type B) and methacrylated gelatin were also sourced from Sigma Aldrich.

As above, the hydrogel containing a UV initator (Irgacure® 2959) was freeze dried, then crosslinked by UV irradiation. As above, the base hydrogel material contained 10% of the gelatin compound and 10% of PEGDA.

The mechanical strength of the hybrid material was measured as described above.

The results are shown in Figures 9 and 10.

Figure 9 shows stress vs strain measured for each of the three types of crosslinked hydrogel, in a dry state.

Figure 10 shows elongation at break (Fig 10(a)), ultimate tensile strength (Fig 10(b)) and Young’s modulus (Fig 10(c)), for each of the three types of crosslinked hydrogel, in a dry state.

It can be seen from Figures 9 and 10 that the type of gelatin material and hydrogel mixture has an influence on the mechanical strength of the final material. However, in all cases, even when using a softer type of gelatin (type B), the resulting hydrogel structure has measurable mechanical properties, which were vastly improved over conventional hydrogels by carrying the present methodology.

It will be understood that the present embodiments are provided by way of example only, and that various modifications can be made to the present embodiments without departing from the scope of the invention.

Claims

CLAIMS:

1. A method of manufacturing a hydrogel, the method comprising, sequentially:

(i) providing a polymerizable or crosslinkable hydrogel material;

(ii) freeze-drying the hydrogel material; and

(iii) polymerizing or crosslinking the hydrogel material in solid state.

2. A method according to claim 1, wherein the polymerizable or crosslinkable hydrogel material comprises a crosslinkable hydrogel.

3. A method according to claim 1 , wherein the polymerizable or crosslinkable hydrogel material comprises a polymerizable hydrogel.

4. A method according to any preceding claim, wherein the hydrogel forms, upon crosslinking or polymerizing, an inter-penetrating network (IPN).

5. A method according to any preceding claim, wherein the polymerizable or crosslinkable hydrogel material comprises a first component comprising a natural polymer, and a second component comprising an unsaturated component.

6. A method according to claim 5, wherein the first component comprises collagen, gelatin, or alginate.

7. A method according to claim 5 or claim 6, wherein the amount of the first component in the polymerizable or crosslinkable hydrogel material is about 5-15 wt%.

8. A method according to any of claims 5 to 7, wherein the second component comprises or consists of PEGDA.

9. A method according to any of claims 5 to 8, wherein the amount of the second component in the polymerizable or crosslinkable hydrogel material is about 0.1-15 wt%.

10. A method according to any preceding claim, wherein step (iii) comprises irradiating the hydrogel material.

11. A method according to claim 10, comprising exposing the hydrogel material to UV radiation.

12. A method according to any one of claims 1 to 9, wherein step (iii) comprises heating the hydrogel material.

13. A method according to any preceding claim, wherein the method comprises:

(ia) adding a crosslinking initiator or a polymerization initiator before step (ii).

14. A method according to any preceding claim, the method comprising: (iv) rehydrating the hydrogel.

15. A method of manufacturing a hydrogel, the method comprising, sequentially:

(i) providing a polymerizable or crosslinkable hydrogel material;

(ii) adding an initiator;

(iii) freeze-drying the hydrogel material; and

(iv) polymerizing or crosslinking the hydrogel material.

16. A hydrogel structure obtained or obtainable by a method according to any of claims 1 to 15.

17. A medical article comprising a hydrogel structure according to claim 16.

18. A medical article according to claim 17, wherein the article is an implantable scaffold or an implantable mesh.