A hydrogel composition for biomedical use

Abstract

The present disclosure relates to a hydrogel composition comprising: a hydrogel composition comprising: a first polymer comprising aldehyde groups; a second polymer comprising amine groups or amide groups; a crosslinking agent comprising amine groups; and at least one type of conductive nanoparticles.

Description

[0001] A hydrogel composition for biomedical use.

[0002] Technical field

[0003] The present disclosure relates to hydrogels for biomedical applications, methods of manufacturing such hydrogels, and uses of such hydrogels.

[0004] Background

[0005] Hydrogels are three-dimensional polymeric networks capable of retaining substantial amounts of water due to their hydrophilic nature. They are extensively utilized in various fields, including biomedical applications, tissue engineering, drug delivery, and wound healing, owing to their properties such as biocompatibility, tunable mechanical strength, and functional versatility.

[0006] In the current state of the art, hydrogels have been developed using a variety of natural and synthetic polymers, such as alginate, chitosan, poly(ethylene glycol), and polyacrylamide. These materials can be crosslinked through physical interactions, such as hydrogen bonding and ionic interactions, or through chemical means, such as covalent bonding. Hydrogels are also often functionalized with nanoparticles, drugs, or bioactive molecules to enhance their performance in specific applications.

[0007] Despite the advancements in hydrogel technology, several challenges persist. Traditional hydrogels often suffer from inadequate mechanical strength, limited self- healing capabilities, and suboptimal performance in dynamic environments. These limitations hinder their application in load-bearing tissues, long-term biomedical implants, and environments requiring rapid and efficient self-repair. Additionally, achieving a balance between biodegradability and mechanical integrity remains a critical challenge, as materials typically need to be robust enough to perform their intended function yet degrade appropriately within the biological environment to avoid long term adverse effects.

[0008] Another challenge arises from the crosslinking strategies typically employed. Physical or single-scale chemical crosslinking can provide either flexibility or strength, but rarely both. Many conventional systems lack dynamic or reversible bonds that would enable rapid network reformation after disruption. Consequently, most known hydrogels exhibit slow or incomplete recovery following damage, and their performance deteriorates under cyclic loading.

[0009] It is therefore an objective of the present disclosure to provide a hydrogel composition that addresses these limitations by offering enhanced mechanical strength, improved self-healing capabilities and biodegradability. The aim is to develop a hydrogel that can be effectively used in a wide range of biomedical applications, including load-bearing tissues, long-term implants, and dynamic environments, without compromising on performance or safety.

[0010] Summary

[0011] The present disclosure relates to an advanced hydrogel system that overcomes longstanding limitations of conventional hydrogel technologies. Hydrogels known in the art often exhibit inadequate mechanical robustness, limited self-healing behaviour, and reduced performance under dynamic or load-bearing conditions. Many such materials also face challenges in achieving a suitable balance between mechanical stability, degradability, and biocompatibility, restricting their use in long-term or regenerative biomedical applications.

[0012] The hydrogels provided according to the present disclosure are designed to combine structural integrity with dynamic adaptability. Through the integration of complementary crosslinking mechanisms and carefully tuned material interactions, the disclosed hydrogels form resilient, water-rich networks capable of withstanding mechanical stress while maintaining reversibility and biocompatibility. The compositions can be tailored for specific functions, offering improved mechanical strength, controlled degradation, and rapid self-repair after deformation.

[0013] As a result, the hydrogel systems described herein provide a versatile platform suitable for diverse biomedical and technological applications, including tissue repair, wound care, cell encapsulation, and bioelectronic interfaces. The invention thus addresses a central need in the art for hydrogel materials that combine high mechanical performance, biological compatibility, and responsiveness to dynamic physiological environments.

[0014] Therefore, in a first aspect, the present disclosure relates to a hydrogel composition comprising: • a first polymer comprising aldehyde groups;

[0015] • a second polymer comprising amine groups or amide groups;

[0016] • a crosslinking agent comprising amine groups; and

[0017] • at least one type of conductive nanoparticles.

[0018] The hydrogel of the present disclosure exhibits attractive properties such as biocompatibility and non-toxicity. It was surprisingly found that the hydrogel composition stimulates osseous repair. The hydrogel of the present disclosure has a high water content that provides cells and tissues with an optimal environment.

[0019] The hydrogel further has a self-healing capacity, which is very valuable in biomedical applications where the hydrogel typically has to function under mechanical stress. The rheological properties of the hydrogel composition result in a high elasticity hydrogel while still being mechanically stable. The conductive nature of the hydrogel opens up possibilities for applications also outside the biomedical field.

[0020] In a second aspect, the present disclosure relates to the use of the hydrogel as disclosed herein. The hydrogel use may be in a biomedical application.

[0021] However, the use of the present disclosure may alternatively be another area of application, such as tissue engineering, drug delivery, wound dressings and cell encapsulation. The mechanical and conductive properties in combination with the hydrogel being biocompatible and having a high water content make the hydrogel versatile for many different use cases.

[0022] In a further aspect, the present disclosure relates to a method of manufacturing the hydrogel comprising the steps of:

[0023] • preparing a first solution comprising a first polymer and a second polymer;

[0024] • preparing a second solution comprising at least one type of conductive nanoparticles and a crosslinking agent; and

[0025] • mixing the first solution with the second solution

[0026] In yet a further aspect the present disclosure relates to a A method of manufacturing a biocompatible hydrogel composition as disclosed herein, the method comprising:

[0027] • providing a first polymer comprising aldehyde groups; • providing a second polymer comprising amine groups or amide groups;

[0028] • providing at least one crosslinking agent distinct from the first and second polymers, the crosslinking agent comprising two or more amine and / or hydrazide functional groups;

[0029] • providing at least one type of electrically conductive nanoparticles; and

[0030] • combining the components under conditions effective to allow the formation of covalent imine and / or hydrazone linkages between the polymer chains, thereby producing a crosslinked hydrogel network in which the conductive nanoparticles are dispersed.

[0031] Detailed description

[0032] In the following embodiment and examples the hydrogel composition will be described in greater detail with reference to the accompanying drawings.

[0033] Definitions

[0034] A hydrogel as used herein refers to a three-dimensional polymeric network capable of retaining substantial amounts of water due to its hydrophilic nature, often exhibiting swelling ratios ranging from hundreds to thousands of times its dry weight. Many polymers are suitable for manufacturing hydrogels, including both natural polymers such as alginate, agarose, chitosan, gelatine, hyaluronic acid, and collagen, as well as synthetic polymers such as poly(ethylene glycol), poly(vinyl alcohol), poly(acrylic acid), poly(acrylamide), and poly(N-isopropylacrylamide). Common to all these polymers is their ability to absorb and retain significant amounts of water within their network. Their crosslinking ability, either physically through non-covalent interactions or chemically through covalent bonds, allows for the engineering of specific mechanical properties, ranging from soft and flexible to rigid and strong, depending on the crosslinking density and the nature of the polymer. Many hydrogels are also stimuli-responsive, meaning they can undergo physical or chemical changes in response to environmental factors like pH, temperature, ionic strength, or the presence of specific molecules.

[0035] A nanoparticle as used herein refers to a small particle that ranges between 1 to 100 nanometres in at least one dimension, exhibiting significantly different physical and chemical properties compared to their larger material counterparts. They can have various shapes, including flakes or sheets, spheres, tubes, fibres, rods, cubes, or prisms, and these shapes can influence their physical and chemical properties. Many nanoparticles are conductive, such as metallic nanoparticles (e.g., Au, Ag, Cu, Ni); carbon-based nanoparticles (e.g., Graphene and Graphene Oxide, carbon nanotubes, fullerenes); conductive polymer nanoparticles (e.g., polyaniline, polypyrrole, polythiophene); and metal oxide nanoparticles (e.g., ITO, TiO2, ZnO). Hence, many different types of nanoparticles can provide the hydrogel with its conductive properties; however, not all conductive nanoparticles provide suitable rheological properties.

[0036] A polymer, as used herein, refers to a large molecule composed of repeating structural units known as monomers. These monomers are covalently bonded to form long chains. Polymers can be either natural, such as alginate, agarose, chitosan, gelatine, hyaluronic acid, and collagen, or synthetic, such as poly(ethylene glycol), poly(vinyl alcohol), poly(acrylic acid), poly(acrylamide), and poly(N-isopropylacrylamide). Polymers are fundamental in the formation of hydrogels due to their intrinsic ability to absorb and retain significant amounts of water within their network, providing the necessary structural and functional properties for various applications.

[0037] As used herein, the term “crosslinking agent” refers to a chemical compound capable of connecting polymer chains together through covalent or ionic interactions, thereby forming or reinforcing a three-dimensional network. Crosslinking agents play a critical role in determining the mechanical properties, elasticity, and structural stability of the resulting hydrogel. A crosslinker can transform a polymer solution into a solid or semisolid gel by establishing linkages between individual polymer molecules, enhancing the gel’s integrity and resilience.

[0038] The crosslinking agent may comprise two or more reactive functional groups, such as amine or hydrazide moieties, that are capable of forming imine and / or hydrazone linkages with aldehyde groups present on the first polymer. Multiple crosslinking agents may be employed concurrently to fine-tune network architecture and mechanical performance, each providing distinct functional contributions such as flexibility, dynamic reversibility, or increased crosslink density.

[0039] The crosslinking agent(s) may be a non-polymeric, low-molecular-weight component, typically having a molecular weight below about 5 000 Da, preferably below about 2 000 Da, and more preferably below about 1 000 Da, as further defined herein. Representative examples include adipic acid dihydrazide, hydrazine hydrate, glycol hydrazide, and succinic dihydrazide.

[0040] As used herein, the term “low-molecular-weight crosslinking agent” refers to a non- polymeric compound having a molecular weight typically in the range of from 100 to 5 000 Da, preferably from 100 to 2 000 Da, and more preferably from 100 to 1 000 Da. In some embodiments, the molecular weight is less than 5 000 Da, such as less than 2 000 Da, or less than 1 000 Da. Such crosslinkers generally contain two or more reactive functional groups, such as amine or hydrazide moieties, capable of forming covalent imine and / or hydrazone linkages with the aldehyde-functionalised polymer. Examples include adipic acid dihydrazide (ADH), hydrazine hydrate, glycol hydrazide, succinic dihydrazide, and similar small-molecule bifunctional or multifunctional linkers.

[0041] Aminated as used herein, for example in describing aminated ADH, refers to a compound having at least one amine group.

[0042] Imine bonds, also known as Schiff bases, as used herein, refer to covalent bonds formed between an aldehyde or ketone and a primary amine. In the context of the present disclosure, imine bonds can be formed between the aldehyde groups of the first polymer and functional groups of the second polymer and / or of the crosslinking agent. The second polymer may contain amine or amide groups, with amine groups being involved in imine bond formation. These bonds are dynamic and can reversibly break and re-form, providing the hydrogel with self-healing properties and enhancing its overall durability and functionality.

[0043] Biocompatibility, as used herein, refers to the ability of a material to perform with an appropriate host response when applied in a specific biological environment. For the hydrogel described in this disclosure, biocompatibility means that the material is nontoxic and does not elicit an adverse reaction when used in biomedical applications. This property can be important for ensuring the hydrogel can be safely used in medical devices, implants, and other therapeutic applications without causing harm to the patient.

[0044] Self-healing, as used herein, refers to the hydrogel’s capability to repair itself after being damaged. This property is due to the dynamic nature of the imine bonds within the hydrogel network, which can reversibly break and re-form. This allows the hydrogel to regain its integrity and functionality after sustaining damage, making it particularly valuable for applications where mechanical stress and potential damage are common.

[0045] Mechanical strength, as used herein, refers to the ability of the hydrogel to withstand mechanical forces without breaking or deforming. This term encompasses properties such as tensile strength, compressive strength, and elasticity. A hydrogel with high mechanical strength is capable of maintaining its structure and functionality under various mechanical stresses, making it suitable for load-bearing applications and other uses that require durability.

[0046] Conductive properties, as used herein, refer to the hydrogel’s ability to conduct electricity. This is typically achieved by incorporating conductive nanoparticles, such as graphene oxide or metallic nanoparticles, into the hydrogel matrix. These conductive properties enable the hydrogel to be used in applications that require electrical conductivity, such as biosensors, electronic devices, and electroactive tissue scaffolds. The integration of conductive nanoparticles not only provides electrical functionality but can also enhance the mechanical properties of the hydrogel.

[0047] In a first aspect, the present disclosure relates to a hydrogel composition comprising a first polymer and a second polymer, wherein the first polymer and the second polymer can form one or more imine bonds between themselves.

[0048] The second polymer may have been aminated or amidated. Alternatively, or additionally, the second polymer may possess amine groups or hydrazide groups. The second polymer may be selected such that it can form imine bonds with the first polymer. However, as known to a skilled person, imine bonds may also be formed between different types of functional groups, and the scope of the present disclosure is not limited to the formation of imine bonds only between polymers comprising hydrazide groups.

[0049] In certain embodiments, the second polymer comprises functional groups that are reactive toward the aldehyde groups of the first polymer and / or toward the functional groups of the crosslinking agent, thereby contributing to the formation of the hydrogel network. Such reactive groups may include primary or secondary amines or hydrazide moieties capable of forming imine or hydrazone linkages. The second polymer may be a natural or synthetic macromolecule functionalised to contain such groups, for example poly-L-lysine, dopamine-containing polymers, silk methacrylate, gelatin, chitosan, or other amino-functionalised polysaccharides or polypeptides. In some embodiments, polymers containing amide linkages, such as silk or gelatin derivatives, also possess available amino side chains that participate in crosslinking reactions, while the amide backbone enhances the mechanical strength, flexibility, and biocompatibility of the hydrogel. The second polymer can thus participate chemically in network formation and simultaneously serve as a structural and bioactive component within the hydrogel.

[0050] The formation of the hydrogel network as described herein may proceed through various combinations of covalent and non-covalent interactions, depending on the choice of polymers and crosslinking agents. In some embodiments, the crosslinking involves only small-molecule linkers, while in others, both polymer-polymer and polymer-small-molecule linkages contribute to network formation. The present disclosure therefore encompasses single-scale and multiscale crosslinked systems, unless otherwise specified.

[0051] In certain embodiments, the hydrogel network exhibits a multiscale crosslinking architecture arising from the simultaneous presence of macromolecular polymers and small-molecule crosslinkers. The first polymer, functionalised with aldehyde groups, reacts with both the second polymer and the small-molecule crosslinking agent to form covalent imine and / or hydrazone linkages on the molecular scale. At the same time, the larger polymer chains of the first and second polymers become physically entangled and electrostatically associated with the conductive nanoparticles, which may further participate in electron or ion transport across the network, thereby imparting electrical functionality and enhancing stress dissipation through interfacial interactions. This hierarchical structure integrates short, dynamic covalent crosslinks with longer, flexible polymer bridges, resulting in a material that combines high mechanical strength, elasticity, and rapid self-healing.

[0052] The multiscale crosslinking mechanism distinguishes the present hydrogel compositions from conventional two-component systems, which rely solely on polymer-polymer or polymer-small-molecule reactions. In contrast, the combination of a macromolecular second polymer and a small-molecule hydrazide crosslinker provides complementary bonding dynamics: the small-molecule crosslinks rapidly reform after mechanical disruption, while the larger polymeric chains maintain network integrity. This synergistic architecture contributes to the observed improvements in mechanical modulus, toughness, and self-healing efficiency of the hydrogels described herein.

[0053] The coexistence of covalent polymer-polymer and polymer-small-molecule crosslinks defines a multiscale network architecture that is not disclosed in conventional two- component systems and underlies the improved mechanical strength and self-healing efficiency observed in the present hydrogels. This configuration is consistent with the experimental results reported in the Examples, which demonstrate enhanced mechanical performance, rapid recovery, and electrical conductivity. It will be appreciated that the relative contributions of these interactions can be adjusted by varying the component ratios and functionalisation levels, enabling the design of hydrogels with tunable properties for different biomedical and technological applications.

[0054] Furthermore, the hydrogel may comprise at least one type of conductive nanoparticles to enhance its properties. However, other embodiments of the present disclosure may not include conductive nanoparticles.

[0055] The first polymer may be selected from the group comprising oxidized alginate, methacrylated oxidized alginate, alginate, oxidized pectin, methacrylated oxidized pectin, and / or oxidized chitosan. These polymers are advantageous due to their biocompatibility, biodegradability, and ability to form hydrogels through crosslinking with suitable agents. Oxidized alginate and methacrylated oxidized alginate, for example, provide functional aldehyde groups necessary for forming imine bonds with amine or hydrazide groups.

[0056] The second polymer can be one or more of poly-L-lysine, 2-, 4-, or 8-arm PEG, dopamine, pectin, and / or silk methacrylate. These agents are chosen for their ability to form robust and stable imine bonds with a group present in the first polymer. Poly-L- lysine, for example, is a polypeptide that not only provides crosslinking functionality but also enhances cell adhesion due to its cationic nature. Aminated PEG and dopamine offer flexibility and additional functional sites for further chemical modifications, which can be tailored to specific applications.

[0057] The inclusion of at least one type of conductive nanoparticle, such as graphene oxide, introduces unique properties to the hydrogel composition. Graphene oxide is known for its electrical conductivity, mechanical strength, and large surface area. When incorporated into the hydrogel, it provides it with conductive properties, making the hydrogel suitable for applications requiring electrical conductivity, such as biosensors, stretchable and self-healing electronics, and electroactive tissue scaffolds. Additionally, graphene oxide can improve the mechanical properties of the hydrogel, enhancing its toughness and elasticity.

[0058] The ratio of the first polymer to the second polymer to the conductive nanoparticles can be adjusted to optimize the mechanical, electrical, and biological properties of the hydrogel. This flexibility allows the hydrogel to be tailored for specific uses, such as wound dressings, drug delivery systems, or tissue engineering scaffolds.

[0059] One advantage of this composition is its enhanced biocompatibility and biodegradability, which can be important for biomedical applications. The ability to form a stable, three-dimensional network through imine bonds provides structural integrity and mechanical strength, while the self-healing capability allows the hydrogel to repair itself after damage. The incorporation of conductive nanoparticles not only adds conductive properties but also reinforces the hydrogel, making it suitable for a wide range of advanced applications.

[0060] The listed polymers, substances, and conductive nanoparticles are merely examples and shall not be considered limiting.

[0061] In one embodiment of the present disclosure:

[0062] • the first polymer is selected from a group comprising oxidized alginate, methacrylated oxidized alginate, alginate, oxidized pectin, methacrylated oxidized pectin, and / or oxidized chitosan;

[0063] • the second polymer is selected from a group comprising poly-L-lysine, 2-, 4-, or 8-arm PEG, dopamine, pectin, and / or silk methacrylate; and

[0064] • the at least one type of conductive nanoparticles is graphene oxide. In one embodiment of the present disclosure, the hydrogel composition has a self- healing ability. This feature significantly enhances the functionality and longevity of the hydrogel in various applications. The self-healing property is particularly advantageous in biomedical and dynamic environments where the material may be subjected to mechanical stress and potential damage.

[0065] The self-healing capability can be attributed to dynamic imine bonds formed between the first polymer and the second polymer. For example, aldehyde groups of the first polymer and the amine or hydrazide groups of the second polymer. Imine bonds, also known as Schiff bases, have the inherent ability to reversibly break and reform, allowing the hydrogel network to repair itself after being damaged. This reversible bonding mechanism enables the hydrogel to regain its integrity and mechanical strength without the need for external intervention.

[0066] Self-healing can be achieved by carefully selecting the components of the composition. For example, the first and the second polymers can be selected to form stable yet dynamic imine bonds. The degree of self-healing can be tuned by adjusting the concentration and ratio of these components. For instance, increasing the concentration of the second polymer can enhance the density of imine bonds, thereby improving the self-healing efficiency.

[0067] Possible variations of this embodiment include the incorporation of additional functional additives that can further enhance the self-healing capability. For example, integrating at least one type of conductive nanoparticles or other reinforcing agents can improve the mechanical properties and provide additional sites for imine bond formation. This can result in a hydrogel that not only self-heals efficiently but also maintains high mechanical strength and stability.

[0068] The technical advantages of the self-healing hydrogel are many. In biomedical applications, the self-healing property can lead to more durable and reliable implants, tissue scaffolds, and wound dressings that maintain their functionality over extended periods. In dynamic environments, such as wearable electronics or soft robotics, the self-healing hydrogel can ensure continuous operation and longevity, reducing the need for frequent replacements and maintenance. The self-healing ability of the hydrogel allows for a wide variety of applications. The hydrogel can be prepared and cast / crosslinked into any shape, such as cast into a syringe. Then ruptured during implant / injection in order to self-heal into a new shape corresponding to the receiving cavity.

[0069] In one embodiment, the hydrogel composition includes an oxidized polysaccharide, such as oxidized alginate, as the first polymer; a polycationic amino-containing polymer, such as poly-L-lysine, as the second polymer; and conductive nanoparticles such as graphene oxide. Such combinations have been found to provide hydrogels exhibiting enhanced mechanical strength, self-healing behaviour, and electrical conductivity.

[0070] In one embodiment of the present disclosure, the crosslinking agent comprises two or more hydrazide functional groups (-NH-NH2) or derivatives thereof. Such hydrazide- based crosslinkers, for example adipic acid dihydrazide (ADH) or PEG-hydrazides, react with aldehyde groups of the first polymer to form hydrazone linkages. The resulting dynamic covalent bonds provide reversible crosslinking, which imparts self- healing behaviour, controlled degradation, and adjustable mechanical properties to the hydrogel network. This differs from amine-based imine crosslinking systems, as hydrazone bonds exhibit reversible dynamics under physiological conditions, enabling reformation of the network after mechanical disruption.

[0071] In one embodiment of the present disclosure, the first polymer comprises or consists of oxidized alginate, the second polymer comprises or consists of poly-L-lysine, and the at least one type of conductive nanoparticles is graphene oxide.

[0072] Thus, an example of a hydrogel composition comprises oxidized alginate as the first polymer, poly-L-lysine as the second polymer, and graphene oxide as the at least one type of conductive nanoparticle. This specific combination of components results in a hydrogel with enhanced properties suitable for a wide range of applications.

[0073] In one embodiment of the present disclosure, the crosslinking agent comprises one or more hydrazide groups (-NH-NH2), or derivatives thereof, such as wherein the crosslinking agent is capable of forming imine bonds with aldehyde groups. In one embodiment of the present disclosure, the crosslinking agent is adipic acid dihydrazide (ADH), hydrazine hydrate, ethylenediamine, triethylenetetramine, lysine, polyethyleneimine (PEI), glycol hydrazide, 2-, 4-, or 8-arm PEG-amine, and / or semicarbazide.

[0074] In one embodiment of the present disclosure, the first polymer comprises or consists of oxidized alginate, the second polymer comprises or consists of poly-L-lysine, the crosslinking agent is adipic acid dihydrazide and the at least one type of conductive nanoparticles is graphene oxide.

[0075] Thus, a hydrogel composition may comprise oxidized alginate as the first polymer, poly-L-lysine as the second polymer, adipic acid dihydrazide as the crosslinking agent, and graphene oxide as the conductive nanoparticle. This specific combination of components results in a hydrogel with enhanced properties suitable for a wide range of applications.

[0076] Oxidized alginate is an alginate derivative in which the hydroxyl groups of the polymer chain are oxidized to form aldehyde groups, offering several advantages in hydrogel applications. The modifications provide unique properties to the hydrogel, enhancing its functionality and utility in various fields, especially in biomedicine. The introduction of aldehyde groups through oxidation allows oxidized alginate to form covalent bonds with amine or hydrazide-containing compounds, such as proteins, peptides, or synthetic polymers. This ability to form Schiff base (imine) linkages provides a robust method for crosslinking. Oxidized alginate is biodegradable, and the degree of oxidation can be controlled to tune the degradation rate of the hydrogel. This feature is particularly useful in controlled release systems and temporary scaffolds in tissue engineering, where the scaffold needs to degrade at a rate matching tissue formation, allowing for the gradual transfer of load and space to the new tissue.

[0077] Poly-L-lysine (PLL) can enhance cell adhesion in hydrogels. Due to its cationic nature, PLL interacts with negatively charged cell membranes, facilitating cell attachment. This property makes PLL-coated hydrogels particularly useful in tissue engineering and regenerative medicine, where promoting cell adhesion and growth on scaffolds is critical. PLL also has inherent antimicrobial properties. Its positive charge can disrupt microbial cell membranes, leading to increased permeability and cell death. Incorporating PLL into hydrogels can thus impart antimicrobial activity, which is beneficial in wound dressings and other biomedical applications where infection control is important. PLL can be used in drug delivery applications by encapsulating and protecting drugs within a hydrogel matrix, particularly those that are negatively charged, through electrostatic interactions. PLL can also control the release rate of drugs by forming complexes that are responsive to environmental conditions like pH. Achieving a stable and homogenous distribution of PLL within the hydrogel matrix remains a technical challenge. One common strategy for hydrogel formation involves using crosslinking agents to link polymer chains together, creating a stable network. The choice of crosslinking agents and the nature of the bonds formed can be important in determining the hydrogel's mechanical properties, swelling behavior, and degradation rate. Covalent crosslinking typically results in robust and stable hydrogels, while physical crosslinking results in dynamic and reversible characteristics.

[0078] Graphene oxide (GO), a derivative of graphene with oxygen-containing groups, adds beneficial functions to hydrogels, such as mechanical strength, electrical conductivity, biocompatibility, and the ability to interact with various molecules. It can increase the tensile strength and elasticity of the hydrogel. GO'S two-dimensional structure improves the hydrogel's resistance to deformation and enhances its elasticity, making the hydrogel more durable and resilient. GO can also enhance the compressive strength of the hydrogel, making it suitable for load-bearing applications or use in environments where mechanical stability is important. GO provides the hydrogel with electrical conductivity, which is beneficial in applications such as in biosensors, bioelectronics, and for electrically stimulated tissue engineering, where electrical signals can influence cell behavior, as well as in electrochemical sensors and actuators. The conductive properties of GO-enhanced hydrogels make them suitable for detecting biological or chemical substances and for use in devices that respond to electrical stimuli. GO allows the hydrogel to absorb more water, which is useful in applications like wound dressings, where maintaining a moist environment is beneficial for healing. GO further provides a large surface area and functional groups that can be used to load and interact with various drugs and biomolecules. GO can also promote cell adhesion and cell proliferation. The integration of GO into hydrogels can be challenging, requiring effective methods to ensure uniform dispersion and stable incorporation into the polymer matrix. The dispersion of GO can be important for the mechanical stability and overall performance of the hydrogels. Good dispersion ensures uniform mechanical properties and conductivity. If the GO is not well dispersed it can lead to agglomeration, which results in inconsistent mechanical properties. There is a high tendency for the restacking of GO sheets due to their hydrophilic nature in the solution. PLL can act as a dispersing agent for GO through electrostatic interaction.

[0079] In one embodiment of the present disclosure, the hydrogel composition comprises oxidized alginate, poly-L-lysine, graphene oxide, and a crosslinking agent.

[0080] The hydrogel composition is biocompatible and can be implanted into a mammalian body, such as a human body.

[0081] The inclusion of a crosslinking agent can further enhance the mechanical properties and stability of the hydrogel. The crosslinking agent can form additional covalent or ionic bonds with the polymer chains, contributing to the creation of a more robust three- dimensional network. This additional crosslinking can improve the hydrogel's resilience and its ability to maintain structural integrity under various conditions.

[0082] The crosslinking agent may comprise at least one functional group selected from hydrazide groups (-NH-NH2), amine groups (-NH2), and derivatives thereof capable of forming imine bonds with aldehyde groups. These functional groups are particularly effective in reacting with the aldehyde groups present on oxidized alginate, forming stable imine bonds (Schiff bases). This further crosslinking not only enhances the mechanical strength of the hydrogel but also contributes to its self-healing properties. The dynamic nature of imine bonds allows them to reversibly break and re-form, enabling the hydrogel to self-repair after damage.

[0083] Crosslinkers can be important components in hydrogel systems as they are involved in establishing the three-dimensional network that defines a hydrogel's properties. They transform a liquid solution of polymer precursors into a gel state. The process involves the linking of polymer chains into a network that traps water and other molecules, leading to the formation of a semi-solid or solid material. Crosslinkers affect the elasticity and stiffness of the hydrogel. Higher crosslink density generally increases the stiffness and reduces the elasticity of the hydrogel. Crosslinkers enhance the toughness and tensile strength of the hydrogel, making it capable of withstanding mechanical stress and strain. The concentration of the crosslinking agent determines the extent of network formation and consequently modulates the hydrogel’s mechanical modulus and water retention. Higher crosslinker content typically yields stronger but less flexible gels, whereas lower concentrations enhance elasticity and self-healing ability.

[0084] In one embodiment of the present disclosure, the crosslinking agent is selected from the group consisting of adipic acid dihydrazide (ADH), hydrazine hydrate, ethylenediamine, triethylenetetramine, lysine, polyethyleneimine (PEI), glycol hydrazide, 2-, 4-, or 8-arm PEG-amine, and / or semicarbazide.

[0085] Each of these crosslinking agents offers unique advantages in terms of reactivity and the resulting hydrogel properties. For instance, adipic acid dihydrazide (ADH) is known for its ability to form strong covalent bonds with aldehyde groups, providing significant stability and robustness to the hydrogel network. Hydrazine hydrate and ethylenediamine can also form effective crosslinks, enhancing the overall mechanical properties of the hydrogel. Lysine, a naturally occurring amino acid, offers biocompatibility and can further enhance cell interactions with the hydrogel. Polyethyleneimine (PEI) and glycol hydrazide offer versatile reactivity due to their multiple amine groups, allowing for extensive crosslinking and improved structural integrity. Semicarbazide can form stable covalent bonds, contributing to the durability and functionality of the hydrogel.

[0086] By incorporating these crosslinking agents, the hydrogel composition can be tailored to achieve desired mechanical properties, stability, and self-healing capabilities, making it suitable for a wide range of biomedical applications.

[0087] However, many crosslinking agents have the ability to form imine bonds with aldehydes, and the list shall not be seen as limiting in any way.

[0088] In one embodiment of the present disclosure, the hydrogel composition comprises oxidized alginate, poly-L-lysine, graphene oxide, and adipic acid dihydrazide.

[0089] This specific combination of components provides a hydrogel with enhanced mechanical strength, biocompatibility, and self-healing properties. Oxidized alginate, also referred to herein as AIOx, as previously described, contains aldehyde groups that enable the formation of covalent bonds with amine-containing compounds. This makes it a good choice for creating a robust hydrogel matrix. The degree of oxidation can be controlled to adjust the degradation rate of the hydrogel, which is particularly useful for applications such as controlled drug release or temporary scaffolds in tissue engineering.

[0090] Poly-L-lysine (PLL) enhances cell adhesion due to its cationic nature, facilitating interactions with negatively charged cell membranes. This property can be important in tissue engineering and regenerative medicine, for the cell attachment and growth. PLL also has antimicrobial properties, adding another layer of functionality to the hydrogel, making it suitable for wound dressings and other applications requiring infection control. Additionally, PLL can form electrostatic interactions with drugs, controlling their release rate in drug delivery applications.

[0091] Graphene oxide (GO) provides the hydrogel with mechanical strength, electrical conductivity, and enhanced biocompatibility. Its two-dimensional structure improves the hydrogel's resistance to deformation and increases its elasticity. The conductive properties of GO make the hydrogel suitable for applications in biosensors, bioelectronics, and electrically stimulated tissue engineering. GO also offers a large surface area for drug loading and promotes cell adhesion and proliferation.

[0092] Adipic acid dihydrazide (ADH) is included as the crosslinking agent. ADH forms stable covalent bonds with the aldehyde groups on oxidized alginate, creating a strong and stable hydrogel network. This additional crosslinking improves the hydrogel's mechanical properties, making it more resilient and durable. ADH also contributes to the hydrogel's self-healing capabilities, as the dynamic imine bonds formed between ADH and oxidized alginate can reversibly break and re-form, allowing the hydrogel to repair itself after damage.

[0093] This specific combination of oxidized alginate, poly-L-lysine, graphene oxide, and adipic acid dihydrazide results in a hydrogel that is robust, adaptable, and versatile, suitable for a wide range of biomedical applications, including tissue engineering, wound healing, and drug delivery. In hydrogel formation, crosslinking agents link polymer chains together, forming a three-dimensional network structure. This network structure gives hydrogels their characteristic properties such as high water content and soft or elastic consistency. For examples wherein adipic acid dihydrazide (ADH) is used as a crosslinking agent, the hydrazide functional groups of ADH can react with functional groups, such as aldehyde groups, of the first polymer, such as oxidized alginate, through Schiff base reactions. This chemical reaction creates dynamic covalent bonds between polymer chains, effectively crosslinking them and stabilizing the hydrogel structure. The degree of crosslinking can be controlled by adjusting the concentration of ADH, which in turn affects the mechanical properties, swelling behavior, and degradation rate of the hydrogel.

[0094] ADH can be used to create hydrogels that are sensitive to pH changes, which is useful in drug delivery systems where the release of the drug can be controlled by the pH of the environment. The biocompatibility and the ability of ADH to form gels at body temperature makes it useful in the preparation of injectable hydrogels. Hydrogels cross-linked with ADH can be used as scaffolds in tissue engineering, as they provide a supportive matrix for cell growth and tissue regeneration. ADH-based hydrogels can be used in wound dressings due to their ability to maintain a moist environment, absorb exudate, and protect the wound from external contaminants. Since they are biodegradable, they are suitable for applications where the material is required to degrade and be absorbed by the body over time, such as in temporary implants or drug delivery systems.

[0095] In an AIOx - GO-PLL - ADH hydrogel, the oxidized alginate serves as a reactive matrix, GO-PLL provides mechanical reinforcement and functionality, and ADH facilitates crosslinking through imine bond formation. The dynamic nature of these imine bonds gives the hydrogel self-healing properties. The process begins with the grafting of GO-PLL onto the oxidized alginate. This step can allow for incorporating the unique properties of graphene oxide and PLL into the hydrogel, thereby enhancing the mechanical strength and introducing functional capabilities. The oxidized alginate can react with ADH through its aldehyde groups. The ADH molecules, possessing two hydrazide groups, form imine bonds with these aldehyde groups, resulting in a crosslinked network. One important feature of this hydrogel is its self-healing property, attributed to the dynamic nature of the imine bonds formed between the oxidized alginate and ADH. Imine bonds can reversibly break and re-form, enabling the hydrogel to self-repair after damage.

[0096] In one embodiment of the present disclosure, the hydrogel comprises a pore diameter of 20-70 pm. The pore size in hydrogels significantly affects their ability to absorb water, mechanical strength, and rheological behavior. Smaller pores typically result in higher viscosity, stiffness, and slower swelling, while larger pores lead to lower viscosity, softer mechanical properties, and quicker swelling and deswelling. These characteristics can be modified to match the specific needs of different applications. For instance, larger pores may be advantageous for applications requiring rapid fluid transport and cell migration, such as in tissue engineering and wound healing, whereas smaller pores might be preferred for controlled drug delivery systems.

[0097] In another embodiment of the present disclosure, the hydrogel exhibits a hydration ratio of 90-95%. A high hydration ratio can be important for biomedical applications as it ensures that the hydrogel can retain a substantial amount of water, closely mimicking the natural extracellular matrix. This high water content provides an optimal environment for cell growth and differentiation, making the hydrogel particularly suitable for applications such as wound healing, tissue engineering, and cell encapsulation. The ability to bind water also enhances the hydrogel's biocompatibility and functionality in various medical applications.

[0098] Further embodiments of the present disclosure reveal that the addition of at least one type of graphene oxide to the hydrogel composition can reduce the degradation rate of the hydrogel by up to 20%. This reduced degradation rate enhances the longevity and stability of the hydrogel in biological environments, making it suitable for long-term applications such as implants and sustained drug delivery systems. The incorporation of graphene oxide helps to reinforce the hydrogel matrix, thereby slowing down its breakdown and maintaining its structural integrity over extended periods.

[0099] Additionally, the hydrogel may have a viscosity of 100±20 Pa s at a shear rate of 1 / sec. Viscosity is a critical parameter that influences the handling and application of the hydrogel. A viscosity within this range ensures that the hydrogel is easy to handle and can be readily injected or molded into desired shapes. This property is particularly important for applications that require precise placement of the hydrogel, such as in minimally invasive surgical procedures or in the delivery of bioactive agents.

[0100] The hydrogel, in another embodiment, exhibits an ultimate compressive stress between 5-15 kPa. This range of compressive stress indicates that the hydrogel has sufficient mechanical strength to withstand external pressures without deforming or breaking. This property is critical for load-bearing applications and ensures the hydrogel's durability and reliability in dynamic environments. The ultimate compressive stress is a measure of the maximum stress that the hydrogel can withstand before failure, which can be important for applications such as cartilage repair or other orthopedic uses.

[0101] Moreover, the hydrogel may possess a compressive modulus of 15-60 kPa. The compressive modulus is a measure of the hydrogel's stiffness and its ability to resist deformation under compressive forces. This property is important for maintaining the structural integrity of the hydrogel in various biomedical applications, including tissue scaffolds and implants. A hydrogel with an appropriate compressive modulus can support tissue growth and provide a stable framework for cell attachment and proliferation.

[0102] The hydrogel, in another embodiment, has a toughness between 0.6-1.5 kJ / m3. Toughness is an indication of the hydrogel's ability to absorb energy and resist fracture. This property is particularly valuable in applications where the hydrogel may be subjected to mechanical stresses and strains, such as in joint or cartilage repair. The toughness of the hydrogel ensures that it can withstand impact and deformation, thereby providing long-term functionality in dynamic environments.

[0103] In another embodiment, the hydrogel has an ion conductivity of at least 3x10-3S / cm, for example as measured by electrochemical impedance spectroscopy (EIS) at 25 °C. The ion conductivity of the hydrogel can be an important property for applications in bioelectronics and electrically stimulated tissue engineering. The incorporation of conductive nanoparticles like graphene oxide contributes to this property, enabling the hydrogel to conduct electrical signals, which can influence cell behavior and improve the performance of bioelectronic devices. This conductive property is particularly beneficial for applications that require electrical interfacing with biological tissues, such as neural interfaces or cardiac tissue engineering. The ion conductive properties of the hydrogel can allow for electrical stimulation of cells to enhance cell proliferation, such as stimulation of human Mesenchymal Stem cells (hMSC). The conductive properties of the hydrogel further allow for electrical stimulation of cells to enhance, for example, osteogenesis.

[0104] In one embodiment of the present disclosure, the hydrogel composition includes a first polymer with a degree of oxidation between 10% and 70%, wherein the degree of oxidation refers to the proportion of hydroxyl groups on the polymer chain that are converted to aldehyde groups. This range of oxidation optimizes the balance between crosslinking density, mechanical strength, flexibility, and biodegradability. A higher degree of oxidation increases the number of reactive aldehyde groups, thereby promoting more extensive crosslinking with hydrazide or amine groups and enhancing the mechanical stiffness and stability of the hydrogel. Conversely, a lower degree of oxidation reduces crosslinking density, resulting in a more flexible and degradable network. Adjusting the oxidation level thus allows fine-tuning of the gelation rate, self- healing ability, and degradation behaviour, enabling the hydrogel to be tailored for specific biomedical applications — such as tissue engineering — where controlled degradation and mechanical compatibility are required.

[0105] In an embodiment of the present disclosure, the hydrogel composition comprises a second polymer at a concentration ranging from 0.1% to 2% w / v. The second polymer, which may contain amine or amide groups capable of forming imine bonds with the aldehyde groups on the oxidized polymer, plays a role in establishing the three- dimensional network of the hydrogel. The specified concentration range ensures sufficient crosslinking to achieve desirable mechanical properties, such as elasticity and strength, while maintaining the hydrogel’s biocompatibility and self-healing capabilities.

[0106] In a further embodiment of the present disclosure, the hydrogel composition includes conductive nanoparticles, such as graphene oxide, at a concentration between 0.05% and 1% w / v. The inclusion of conductive nanoparticles imparts electrical conductivity to the hydrogel, which is advantageous for applications such as biosensors, electroactive tissue engineering scaffolds, and drug delivery systems where electrical stimulation can enhance therapeutic outcomes. The specified concentration range is selected to ensure a uniform distribution of nanoparticles within the hydrogel matrix, providing consistent electrical properties while avoiding issues like nanoparticle aggregation, which could compromise the mechanical and functional characteristics of the hydrogel. Additionally, this range allows for sufficient conductivity to meet the requirements of various electronic and biomedical applications without adversely affecting the hydrogel’s biocompatibility and structural integrity.

[0107] The content of conductive nanoparticles directly affects electrical conductivity and mechanical reinforcement of the hydrogel. For example, increasing graphene oxide content from 0.2% to 0.5% (w / v) increased ionic conductivity from 3.8 x 10-3S cm-1to 4.7 x 10-3S cm-1, and modified compressive properties.

[0108] In a further aspect, the present disclosure relates to the use of the hydrogel disclosed herein for biomedical applications. The hydrogel described herein is particularly suited for a wide range of biomedical applications due to its biocompatibility, non-toxicity, and ability to provide an optimal environment for cell growth and tissue regeneration. The self-healing capacity and high mechanical strength make it ideal for use in load-bearing tissues, long-term implants, and environments requiring rapid self-repair. Additionally, the hydrogel's conductive properties open up possibilities for applications in bioelectronics, biosensors, and electrically stimulated tissue engineering, enhancing its versatility and functionality in the biomedical field.

[0109] In a further aspect, the present disclosure relates to the use of the hydrogel disclosed herein for additive manufacturing. The hydrogel's properties, including its high water content, self-healing ability, and mechanical stability, make it a suitable material for use in additive manufacturing techniques such as 3D printing. The conductive properties provided by the incorporation of graphene oxide further expand the potential applications, allowing for the fabrication of complex structures with integrated electronic functionalities. This can be particularly beneficial in the development of customized implants, tissue scaffolds, and other biomedical devices that require precise and intricate designs.

[0110] In a further aspect, the present disclosure relates to the use of the hydrogel disclosed herein for stimulating cell growth. The hydrogel provides an optimal environment for cell proliferation and differentiation due to its high water content, biocompatibility, and ability to support cell adhesion. The inclusion of poly-L-lysine enhances cell attachment, while the conductive properties of graphene oxide can influence cellular behavior through electrical stimulation. This makes the hydrogel particularly useful in tissue engineering, regenerative medicine, and other applications where promoting cell growth can be important. The hydrogel can be used as a scaffold for growing various types of cells, facilitating the development of functional tissues and organs for therapeutic purposes.

[0111] In a further aspect, the present disclosure relates to the use of the hydrogel disclosed herein to stimulate the osteogenic differentiation in human Mesenchymal Stem cells (hMSCs) encapsulated in said hydrogel. The hydrogel provides a conducive environment for the osteogenic differentiation of hMSCs, owing to its biocompatibility, mechanical stability, and ability to support cell adhesion and proliferation. The presence of graphene oxide enhances the conductive properties of the hydrogel, which can be used to electrically stimulate the cells, promoting osteogenesis and enhancing the development of bone tissue. This makes the hydrogel particularly suitable for applications in bone regeneration and tissue engineering, where effective stimulation of osteogenic differentiation is vital.

[0112] In a further aspect, the present disclosure relates to the use of the hydrogel disclosed herein for the encapsulation of cells. The hydrogel's high water content, biocompatibility, and mechanical properties make it an ideal matrix for encapsulating various cell types. The hydrogel provides a protective environment that supports cell viability and proliferation, making it suitable for applications in cell therapy, tissue engineering, and regenerative medicine. The ability to fine-tune the hydrogel's properties, such as pore size and crosslinking density, allows for the optimization of cell encapsulation conditions for different cell types and therapeutic purposes.

[0113] In a further aspect, the present disclosure relates to the use of the hydrogel disclosed herein for increasing cell viability. The hydrogel's biocompatible and non-toxic nature, along with its ability to support cell adhesion and proliferation, contributes to enhanced cell viability. The hydrogel matrix provides a hydrated environment that mimics the natural extracellular matrix, promoting better cell survival and function. This property is particularly advantageous in applications such as tissue engineering, where maintaining high cell viability can be important for successful tissue formation and integration.

[0114] In a further aspect, the present disclosure relates to the use of the hydrogel disclosed herein as a wound care adhesive. The hydrogel's adhesive properties, combined with its biocompatibility and ability to promote cell proliferation, make it suitable for use in wound care applications. The hydrogel can adhere to wound surfaces, providing a moist environment that supports healing while protecting the wound from external contaminants. The incorporation of antimicrobial agents, such as poly-L-lysine, further enhances the hydrogel's suitability for wound care by reducing the risk of infection.

[0115] In a further aspect, the present disclosure relates to the use of the hydrogel disclosed herein as a wound dressing. The hydrogel's high water content, biocompatibility, and mechanical properties make it an effective wound dressing material. It can maintain a moist environment, which is beneficial for wound healing, and its mechanical properties provide protection and support to the wound site. The hydrogel can be loaded with therapeutic agents, such as antibiotics or growth factors, to enhance the healing process. Its ability to conform to the wound shape and provide a barrier against external contaminants further contributes to its effectiveness as a wound dressing.

[0116] In a further aspect, the present disclosure relates to the use of the hydrogel disclosed herein to electrically stimulate cells or tissues. The incorporation of conductive nanoparticles, such as graphene oxide, imparts electrical conductivity to the hydrogel, enabling its use in applications that require electrical stimulation. Electrical stimulation can influence various cellular processes, such as proliferation, differentiation, and migration, making the hydrogel suitable for use in bioelectronic devices, biosensors, and electroactive tissue scaffolds. This property is particularly beneficial in tissue engineering applications, where electrical signals can enhance tissue regeneration and function.

[0117] In a further aspect, the present disclosure relates to a method to manufacture the hydrogel disclosed herein comprising the steps of: preparing a first solution comprising a first polymer comprising aldehyde groups and a second polymer comprising amine or amide groups; preparing a second solution comprising at least one type of conductive nanoparticles and a crosslinking agent; mixing the first and the second solution. The method involves preparing the polymers in a first solution and the crosslinking agent and nanoparticles in a second solution before combining them. This approach offers several advantages. Firstly, it allows for precise control over the crosslinking reaction. By keeping the components separate until the final mixing step, premature crosslinking is prevented, ensuring a more uniform and controlled gelation process. This results in a hydrogel with a more consistent and homogeneous structure.

[0118] Additionally, preparing separate solutions ensures that each component is fully dissolved or dispersed, improving the overall homogeneity of the final hydrogel. For example, in the first solution, the oxidized alginate is dissolved along with poly-L-lysine. This ensures that the first polymer and second polymer are fully solubilized and can interact effectively during the crosslinking process.

[0119] In the second solution, the conductive nanoparticle, such as graphene oxide, dissolved in a PLL solution is mixed with a crosslinking agent, such as adipic acid dihydrazide (ADH). This separate preparation allows for better dispersion of the conductive nanoparticles, enhancing the electrical conductivity and mechanical properties of the final hydrogel. Uniform dispersion of the nanoparticles is important for achieving consistent conductivity throughout the hydrogel.

[0120] Furthermore, the separate preparation allows for greater flexibility in optimizing the concentrations and properties of each solution independently. This flexibility enables the fine-tuning of the hydrogel's mechanical strength, porosity, and swelling behavior to meet specific application requirements. By adjusting the concentrations and ratios of the components in the separate solutions, the properties of the final hydrogel can be precisely controlled.

[0121] Overall, the method of preparing separate solutions for the polymers and the crosslinking agent and the nanoparticles, and then mixing them, offers significant advantages in terms of control, homogeneity, and optimization of the hydrogel's properties. This approach ensures a well-structured and functional hydrogel suitable for various biomedical and other applications. In one embodiment of the presently disclosed method, the first solution comprises oxidized alginate and poly-L-lysine; the second solution comprises graphene oxide and adipic acid dihydrazide; and the first and the second solution is mixed at a ratio in the range of 0.1 -3:0.1-3.

[0122] By mixing the first and second solutions in a specific ratio, such as at a ratio of 0.1- 3:0.1-3, the method allows for precise control over the final hydrogel properties. This ratio can be adjusted to tailor the hydrogel’s mechanical strength, porosity, and swelling behavior to specific application needs. This approach results in a homogeneous and functional hydrogel with enhanced performance suitable for a wide range of biomedical applications.

[0123] In one embodiment of the present disclosure, the first solution is prepared by dissolving 5-15% w / v oxidized alginate in 0.1 -0.5% poly-L-lysine; and the second solution is prepared by dissolving 2-8% w / v adipic acid dihydrazide (ADH) in 0.1 -0.5% poly-L- lysine solution comprising 0.1%-0.5% graphene oxide.

[0124] In one embodiment of the present disclosure, the first solution is prepared by dissolving 10% w / v oxidized alginate in 0.1% poly-L-lysine, and the second solution is prepared by dissolving 6% w / v adipic acid dihydrazide (ADH) in 0.1% poly-L-lysine solution comprising 0.1%-0.5% graphene oxide.

[0125] In one embodiment of the present disclosure, the 0.1% poly-L-lysine solution comprising 0.1 - 0.5% graphene oxide is prepared by dissolving graphene oxide in the poly-L-lysine solution, sonicating the solution for 30 min at room temperature, incubating the solution for 1 h at 37°C, and centrifugation at 12000 rpm for 5 min to remove excess poly-L-lysine. The graphene oxide can subsequently be dissolved in a 0.1% poly-L-lysine solution to achieve a 0.1%-0.5% graphene oxide concentration.

[0126] In one embodiment of the present disclosure, the first and a second solution are mixed in a 0.1 -3:1 ratio.

[0127] In one embodiment of the present disclosure, the first and a second solution are mixed in a 1 :0.1-3 ratio. In one embodiment of the present disclosure, the first and a second solution are mixed in a 1 :1 ratio.

[0128] Description of Drawings

[0129] FIG. 1 illustrates a Fourier-transform infrared spectroscopy (FTIR) spectra, showing a) Alginate and oxidized alginate (AIOx); b) GO, PLL and GO-PLL; and c) ADH and hydrogels with and without GO.

[0130] FIG. 2 illustrates X-ray Photoelectron Spectroscopy (XPS) spectra of hydrogels, showing a) Survey spectra; b) C1 s spectra; and c) N1s spectra for 0%GO-PLL, 0.2% GO-PLL, and 0.5%GO-PLL hydrogels.

[0131] FIG. 3 illustrates XPS spectra, showing a) Survey spectra and C1 s spectra of sodium alginate and AIOx; b) Survey spectra and C1 s spectra of GO and GO-PLL.

[0132] FIG. 4 illustrates the 1 H NMR spectra of Na-Alginate, AIOx formation, and after crosslinking of AIOx with ADH.

[0133] FIG. 5 illustrates Scanning Electron Microscopy (SEM) images of hydrogels, showing a) 0%GO-PLL, b) 0.2% GO-PLL, and c) 0.5%GO-PLL, d) the influence of GO on pore diameter calculated from the SEM images.

[0134] FIG. 6 illustrates a) the hydration ratio and b) the degradation behavior of 0% GO-PLL, 0.2% GO-PLL, and 0.5% GO-PLL hydrogels.

[0135] FIG. 7 illustrates a) Differential scanning calorimetry (DSC) of AIOx, AIOx-PLL, AIOx- GO-PLL 0.2%, and AIOx-GO-PLL 0.5%; and b) Thermogravimetric analysis (TGA) of the 0% GO-PLL, 0.2% GO-PLL, and 0.5% GO-PLL hydrogels.

[0136] FIG. 8 illustrates the rheological characteristics of hydrogels containing 0%GO-PLL, 0.20%GO-PLL, and 0.5% GO-PLL, showing a) Rheological analysis of the storage modulus (G1) of the hydrogel as a function of time, monitoring gelation time through changes in shear modulus; b) the viscosity of the hydrogel as a function of shear rate; c) the storage modulus (G1) and loss modulus (G") of the hydrogel as a function of oscillation strain; and d) the G' and G" of the hydrogel as a function of angular frequency.

[0137] FIG. 9 illustrates the mechanical characterization conducted 1 hour after preparation (solid bar) and after 24 hours (striped bar) of the hydrogel composites containing 0%GO-PLL, 0.2%GO-PLL, and 0.5% GO-PLL, showing a) the stress-strain curve; b) ultimate stress; c) compressive modulus; and d) toughness.

[0138] FIG. 10 illustrates the ionic conductivity of the hydrogel composites.

[0139] FIG. 11 illustrates compression studies before and after self-healing of the hydrogel samples, showing a) 0%GO-PLL, b) 0.2%GO-PLL, and c) 0.5% GO-PLL; d) the corresponding efficiency calculated from the compression strength; and e) the rheological self-healing analysis of 0.5% GO-PLL, illustrating G' and G" of the hydrogel during alternating step strain tests. The tests involve small strain (y = 0.1%) for 50 seconds followed by large strain (y = 100%) for 200 seconds, conducted at a constant angular frequency of 10 rad / s and a temperature of 37 °C.

[0140] FIG. 12 illustrates biocompatibility studies, showing a) Live / Dead staining after 1, 3, and 7 days of cell culture; b) quantification of cellular viability in respective hydrogels after 1 , 3, and 7 days of culture by flow cytometry; and c) Live / Dead staining, 3D visualization. Scale bar = 200 pm. Statistical significance: **p < 0.01.

[0141] FIG. 13 illustrates mineralization studies after 5 weeks, a) and b) showing XRD analysis of the hydrogels conducted after 5 weeks of culturing human Mesenchymal Stem Cells (hMSCs), hydroxyapatite peak encircled; c) SEM images of a hydrogel, taken after 5 weeks of hMSCs culture, revealing the presence of nanometer-sized hydroxyapatite granules and d) EDAX analysis performed on the hMSCs-laden hydrogels after 5 weeks of culture to quantify the amounts of Ca and P produced by the hMSCs.

[0142] FIG. 14 illustrates mineralization studies after 6 weeks, showing a) XRD analysis of the hydrogels conducted after 6 weeks of culturing hMSCs with 0%GO-PLL, 0.2%GO-PLL, or 0.5%GO-PLL, with hydroxyapatite peaks encircled; b) SEM images and c) EDAX analysis of 0.5% GO-PLL hydrogels without cells; d) SEM images and e) EDAX analysis of 0% GO-PLL hydrogels, taken after 6 weeks of hMSC culture, indicating the absence of hydroxyapatite formation. The SEM images do not show any hydroxyapatite particles, and the EDAX analysis does not detect the presence of calcium or phosphorus.

[0143] FIG. 15 illustrates osseous repair evaluation after 8 weeks of hydrogel implantation, showing a) Histomorphometric analysis of the percentage of repair among all hydrogels; b) the mature bone I immature bone (MB / IB ratio). Cross-sectional representative images stained with Verde Luz-orange G-acid fuchsin (VOF) of the defect site in the different experimental groups, illustrating c) and f) 0%Go-PLL; d) and g) 0.2%Go-PLL; e) and h) 0.2%GO-PLL. Same letters displayed in different histograms denote significant differences (p < 0.001) among these groups. CT: Connective tissue, DS: Defect site, MB: Mature bone, IB: immature bone. Scale bar = 500 pm. i-l) shows the in-vivo mineralization studies. (I) High magnification Images of the repaired tissue in Alox-GO (0.5%) group showing the ossification foci (OF) (j) with osteoprogenitorslike cells (arrows), the defect site (DS), immature bone tissues with low mineralization (IB) and mature mineralized bone (MB), (k) mature mineralized bone (surrounded by graphene fibers (arrowheads). (I) Hypertrophic cartilage (CA) is occupied by mineralized bone matrix (*).

[0144] FIG. 16 illustrates representative panoramic images in horizontal section showing in rat the reparative tissue response in the critical calvaria defect in the experimental groups a) AIOx-PLL (0% GO); b) AIOx-PLL (0.2% GO); and c) AIOx-PLL (0.5% GO).

[0145] Examples

[0146] Example 1. FTIR spectroscopic analysis

[0147] Fourier Transform Infrared (FTIR) spectroscopic analysis was conducted to confirm the oxidation of oxidized alginate (AIOx), the grafting of graphene oxide-poly-L-lysine (GO- PLL), and the crosslinking of the hydrogel with adipic acid dihydrazide (ADH). This was performed using a PerkinElmer Spectrum 100 FTIR spectrometer (USA), which was fitted with a diamond crystal for Attenuated Total Reflectance (ATR) measurements. The spectra for transmittance were recorded at a temperature of 25°C, spanning a spectral range from 4000 to 500 cm-1. This was achieved through 16 cumulative scans, each with a spectral resolution of 4 cm-1. In the characterization of sodium alginate and its oxidized form (AIOx) via Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR), distinct spectral features elucidate the structural transformations incurred through oxidation. For sodium alginate, the spectrum prominently exhibits a broad absorption peak centered at 3280 cm-1, attributable to the O-H stretching vibrations of hydroxyl groups, alongside characteristic peaks at approximately 1606 cm-1and 1409 cm-1, corresponding to the asymmetric and symmetric stretching vibrations of carboxylate ions (-COO-), respectively. The presence of a peak around 1018 cm-1, indicative of C-0 stretching, further confirms the polysaccharide structure. Upon oxidation, the alginate spectrum undergoes notable changes, especially the emergence of a new absorption peak around 1732 cm-1, which is characteristic of the aldehyde (-CHO) functional groups formed during the oxidation process. This peak directly signifies the successful introduction of aldehyde functionalities, a modification that can enhance AIOx's reactivity and crosslinking capability in hydrogel applications. Moreover, subtle shifts in the carboxylate peaks can also be observed, FIG. 1A, indicating alterations in the alginate's molecular environment post-oxidation.

[0148] In the investigation of the grafting process between graphene oxide (GO) and poly-L- lysine (PLL) via ATR-FTIR spectroscopy, distinct spectral signatures delineate the successful conjugation of these materials. Initially, the spectrum of GO is characterized by prominent peaks: a broad absorption around 3300 cm-1, attributed to O-H stretching vibrations from hydroxyl groups; a peak near 1733 cm-1, representing the C=O stretching of carboxylic groups; and additional peaks around 1626 cm-1and 1050 cm-1, corresponding to 0=0 stretching vibrations of the aromatic rings and C-0 stretching vibrations, respectively. Upon grafting with PLL, notable spectral modifications emerge, evidencing the interaction between GO and PLL. A peak shift in the amide I region (around 1650 cm-1) indicates the C=O stretching vibration from the PLL's amide groups to 1636 cm-1, suggesting the successful attachment of PLL to the GO surface. Similarly, the peak shifts in the amide II region (around 1540 cm-1) to 1540 cm-1, attributed to N-H bending and C-N stretching vibrations, further confirm the conjugation, FIG. 1 B.

[0149] In the comprehensive characterization of the hydrogel composition incorporating GO- PLL, oxidized alginate (AIOx), and ADH via ATR-FT-IR spectroscopy, significant spectral features provide insights into the intricate interactions and crosslinking within the hydrogel matrix. Upon adding ADH into the hydrogel formulation, further spectral shifts in the amide peaks become evident, attributed to the formation of imine bonds (- CH=N-), a direct indicator of the Schiff base reaction signifying the successful crosslinking within the hydrogel structure. These spectroscopic observations collectively validate the successful formulation of the GO-PLL, oxidized alginate (AIOx), and ADH hydrogel, demonstrating the effective crosslinking and interaction between components, FIG. 10.

[0150] Example 2. X-ray Photoelectron Spectroscopy (XPS) analysis

[0151] X-ray Photoelectron Spectroscopy (XPS) analysis was performed to investigate the surface composition and chemical state modifications during the formation of GO-PLL hydrogel, oxidation of oxidized alginate (AIOx), crosslinking of AIOx with adipic acid dihydrazide (ADH), and the final assembly of the GO-PLL hydrogel. XPS was carried out using XPS K-Alpha from Thermo Scientific with a monochromated Al Ka X-ray source with a photon energy of 1486.6 eV and ultrahigh vacuum (~8x10“6mbar). The results are shown in FIG. 2 and FIG. 3.

[0152] For the GO-PLL composite, the XPS spectra revealed significant changes in the surface chemistry of graphene oxide upon conjugation with poly-L-lysine (PLL). The C1 s region exhibited a decrease in the oxygen-containing functional groups, evidenced by the reduction in the intensity of the peaks associated with C-0 (epoxy and alkoxy groups) and C=O (carbonyl groups) functionalities, reflecting the interaction with PLL. Additionally, the introduction of PLL to GO was confirmed by the appearance of nitrogen signals in the N1s region, indicative of the amide linkages formed between GO and PLL, showcasing the successful grafting process, FIG. 2.

[0153] For AIOx, the XPS spectra highlighted the oxidation-induced changes in the alginate structure. The C1s region showed an increase in the intensity of the peak corresponding to carbonyl groups (C=O), primarily from the aldehyde functionalities introduced during the oxidation process. This was a clear indicator of the successful oxidation of alginate to AIOx, enhancing its reactivity for subsequent crosslinking reactions, FIG. 3A.

[0154] Upon crosslinking AIOx with ADH, the XPS spectra provided evidence of the chemical transformations indicative of successful crosslinking. In the N1s region, new peaks corresponding to imine bonds (C=N) were observed, confirming the formation of Schiff bases between the aldehyde groups of AIOx and the hydrazide groups of ADH, FIG. 3B.

[0155] Example 3. Nuclear Magnetic Resonance (NMR) spectroscopy

[0156] Nuclear Magnetic Resonance (NMR) spectroscopy was employed to elucidate the structural characteristics and verify the formation of oxidized alginate (AIOx) and its subsequent crosslinking with adipic acid dihydrazide (ADH), using a Bruker Ascend™ 400 MHz spectrometer. Analysis of the NMR data was facilitated through the application of MestReNova software, allowing for detailed interpretation of the spectra and identification of specific chemical shifts and patterns associated with the molecular structure of AIOx and the AIOx-ADH complex. For the preparation of NMR samples, solutions of AIOx and AIOx-ADH in D2O were used to minimize exchange with protons in the solvent. The NMR spectra were acquired under standardized conditions to ensure reproducibility and accuracy in the analysis.

[0157] 1 H NMR spectroscopic analysis of sodium alginate, AIOx, and AIOx-ADH are shown in FIG. 4. The proton signal ranging from 3.5 to 4.9 ppm in sodium alginate corresponds to the protons of G and M units. The formation of AIOx was confirmed by the two new signals corresponding to hemiacetal protons appearing at 5.3 and 5.6 ppm. After reaction with ADH, the signals corresponding to hemiacetal protons disappeared, indicating the Schiff base reaction between aldehyde groups present in the AIOx and amine groups present in the ADH molecules.

[0158] Example 4. Scanning Electron Microscopy (SEM) analysis

[0159] Scanning Electron Microscopy (SEM) analysis of the freeze-dried hydrogels was conducted using an FEI Quanta 200 ESEM FEG, set to an acceleration voltage of 10 kV and an emission current of 10 mA, to examine their porous structures. Prior to imaging, all samples were sputter-coated with a 10 nm layer of gold to ensure enhanced electrical conductivity and to achieve high-resolution images of the hydrogels' morphology. The SEM images of all hydrogels (AIOx-PLL, AIOx-O.2% GO- PLL, and 0.5% GO-PLL) revealed a well-defined porous structure. The pores appeared relatively uniform in size, and the average pore diameter, as calculated using Imaged software, was determined to be approximately 33.4 pm, 53.2 pm, and 64 pm for AIOx- PLL, AIOx-O.2% GO-PLL, and 0.5% GO-PLL, respectively, FIGs. 5A-5D. Example 5. Gel hydration ratio and degradation

[0160] The hydration ratio of the hydrogels was tested by measuring the weight immediately after preparation and their corresponding dry weight after freeze-drying. Remarkably, all hydrogel composites demonstrated a consistent hydration ratio, maintaining a high water content of 94-95%, FIG. 6A. Having 95% water content in the hydrogel is advantageous for biomedical applications because it closely mimics the natural extracellular matrix, promoting better cell viability and tissue integration. Additionally, the high water content ensures biocompatibility and provides a conducive environment for nutrient and waste exchange, which may be important for supporting cellular functions.

[0161] The degradation behavior of the AIOx-PLL, AIOx-O.2% GO-PLL, and AIOx-O.5% GO- PLL hydrogel composites was thoroughly investigated by comparing the wet weight and dry weight after freeze-drying over a period of 10 days to understand their performance in aqueous environments, FIG. 6B.

[0162] Regarding degradation, the hydrogels exhibited a controlled degradation rate, with all materials showing a reduction in mass between 10-20% during a 10-day period. This gradual degradation behavior is indicative of the hydrogels' stability and resilience over time, while also allowing for eventual breakdown, which can be important for biodegradable applications.

[0163] Example 6. Thermal characterization

[0164] The thermal behavior of the hydrogel composites was evaluated using a TA Instruments TGA Q500 Thermogravimetric Analyzer and a TA DSC Q200 Differential Scanning Calorimeter (DSC), both from TA Instruments, USA. The thermogravimetric analysis (TGA) was performed in a nitrogen atmosphere, gradually heating the sample from 30°C up to 900°C at a heating rate of 10°C per minute, with a nitrogen gas flow maintained at 60 mL per minute. This process allowed for the observation of mass changes over the temperature range, utilizing an ultra-sensitive scale to track these changes, as shown in FIGs. 7A-B. Figure 7A shows the DSC thermograms of AIOx, AIOx-PLL, and AIOx-GO-PLL composites containing 0.2% and 0.5% GO. The DSC curves indicate the thermal transitions of the different composites. The peaks observed in the range of approximately 150°C to 250°C suggest melting or phase transitions of the composite materials. The AIOx sample shows a prominent endothermic peak around 200°C, indicating a significant thermal event, likely related to the degradation or melting of the alginate oxide matrix. For the AIOx-PLL sample, the peak shifts slightly to higher temperatures compared to AIOx alone. The AIOx-GO-PLL composites with 0.2% and 0.5% GO display peaks that are further shifted compared to the AIOx-PLL sample. This indicates that GO influences the thermal stability and the phase behavior of the composites. Figure 7B displays the TGA curves, showing weight loss as a function of temperature for composites with different GO concentrations (0%, 0.2%, and 0.5%). The TGA curves represent the thermal degradation behavior of the composites. As temperature increases, the weight of the sample decreases due to the loss of material from thermal decomposition. The 0% GO sample shows a two-stage weight loss, the first starting around 100°C, likely due to moisture loss, and the second occurring between 200°C and 400°C, which is attributed to the degradation of the polymer backbone. The composites with 0.2% GO exhibit similar trends, but with slightly higher thermal stability as the temperature increases. The incorporation of GO helps to slow down the decomposition, as reflected by the delayed onset of the second stage of weight loss. However, a further increase of GO to 0.5% slightly lowers the degradation resistance, likely due to the agglomeration of GO particles, which reduces the effectiveness of the reinforcement and creates structural defects.

[0165] Example 7. Rheological characterization and viscosity

[0166] The rheological characteristics of the hydrogel samples containing 0%, 0.2%, and 0.5% graphene oxide - poly-L-lysine (GO-PLL) were evaluated using a Discovery Hybrid Rheometer HR-2 (TA Instrument, New Castle, United States) at a temperature of 25°C. This assessment involved measuring the loss modulus (G") and storage modulus (G') with the device's 40 mm parallel plate setup. To identify the hydrogel's linear viscoelastic region (LVR), tests varying the amplitude from 0.1 to 100 Pa at an angular frequency of 0.1 rad / s were conducted, as illustrated in FIG. 8C. Additionally, to examine the hydrogel's behavior under continuous shear, experiments were carried out at a controlled temperature of 25°C, applying shear rates from 0.1 to 100 s-1and maintaining a gap of 300 pm. These experiments were replicated three times to ensure accuracy, as shown in FIG. 8D.

[0167] Example 8. Mechanical characterization The evaluation of the hydrogel's mechanical properties was conducted on an Instron mechanical tester (Model 5967, UK), which was fitted with a 50 N load cell. Compressive tests were performed at a strain rate of 0.5 mm min-1 , with strain levels reaching up to approximately 40%. Cylindrical samples of the hydrogels, with dimensions of about 10 mm in diameter and 2 mm in height (N=6), were prepared and then incubated overnight in DPBS at a temperature of 37°C. Before conducting the mechanical tests, any excess moisture was removed from the samples with Kimwipes paper, and their dimensions were precisely measured using a digital Vernier caliper. The compressive modulus for each sample was calculated by analyzing the slope of the stress-strain curve within a strain range of 20 to 30%, as shown in FIGs. 9A-9D.

[0168] Example 9. Electrical characterization

[0169] Electrochemical impedance spectroscopy (EIS) was employed to evaluate the ionic conductivity of the hydrogels. The EIS measurements were conducted using a PalmSens instrument (USA) across a frequency spectrum ranging from 100 kHz to 10 Hz, with a signal amplitude of 10 mV. For these analyses, the hydrogels were placed between two stainless steel plates, creating a two-electrode configuration, and securely clamped to ensure proper contact for the EIS evaluation. The average ionic conductivities recorded were 1.9 x 10-3S / cm, 3.8 x 10-3S / cm, and 4.7 x 10-3S / cm for the AIOx-PLL, AIOx-O.2% GO-PLL, and AIOx-O.5% GO-PLL hydrogel composites, respectively, FIG. 10.

[0170] Example 10. Self-healing properties of the hydrogel

[0171] Leveraging the dynamic and reversible bonding mechanisms inherent to their composition, the AIOx-PLL hydrogel and AIOx-O.5% GO-PLL have demonstrated remarkable self-healing capabilities. Compressive strength analyses, depicted in FIGs. 11A-C, present the stress-strain relationships for AIOx-PLL, AIOx-O.2% GO-PLL, and AIOx-O.5% GO-PLL hydrogels, both pre- and post- a 24-hour healing period. The quantification of self-healing efficiency, inferred from the ultimate stress values of these compression tests, yielded impressive recovery rates: 85% for AIOx-PLL and AIOx- O.2% GO-PLL, and 89% for AIOx-O.5% GO-PLL after just 1 hour; and an enhancement to 95-100% for AIOx-PLL and AIOx-O.2% GO-PLL, with AIOx-O.5% GO-PLL achieving 92% after 24 hours. Cyclic rheological assessments further corroborate the self-healing effectiveness, FIG. 11 D. By examining the hydrogel's response to alternating low (0.1 %) and high (100%) strain rates, it was observed that AIOx-O.5% GO-PLL could undergo up to three full recovery cycles within 300 seconds each, as shown in FIG. 11E. This rheological behavior, particularly the ability to withstand and recover from extensive strain, emphasizes the hydrogel's structural integrity and resilience. Collectively, these findings underscore the potential of AIOx-GO-PLL hydrogels for applications demanding durable, self-repairing materials.

[0172] Example 11. Cell viability in the hydrogel

[0173] Human Mesenchymal Stem Cells (hMSCs) were cultured in DMEM (low glucose, GlutaMAX) supplemented with FBS 10% (v / v) and 1% (v / v) penicillin-streptomycin. Afterward, 50 pL of hMSC solution with a density of 6 x 1OA6 cells / mL was mixed with the hydrogel precursor and then deposited (10 pL) into each microwell array mounted on a glass slide. All hydrogel-encapsulated cell samples were cultured with either fresh hMSC growth media or differentiation media and incubated at 37 °C in a 5% CO2 incubator. The culture media was replaced every 2 days. The viability of cells encapsulated within the hydrogels over various culture durations (1 , 3, and 7 days) was evaluated using the LIVE / DEAD™ Viability / Cytotoxicity Kit from Thermo Fisher Scientific, USA, following the instructions provided by the manufacturer. The hydrogels were first rinsed twice with warm DPBS before being incubated in a staining solution containing 4 pM calcein-AM and 10 pM ethidium homodimer-1 , all dissolved in DPBS, for 15 minutes in a dark environment. After staining, the hydrogels were again rinsed with DPBS to wash away any unbound dye, and the stained cells were visualized using a Zeiss LSM 700 confocal laser scanning microscope. The captured images were processed with Imaged software, where they were separated into green and red fluorescence channels to distinguish between live and dead cells, respectively. The percentage of viable cells was determined by calculating the ratio of live cells to the total cell count within each image. For each time point and experimental condition, six samples were analyzed, with at least three images taken per sample to ensure a comprehensive assessment.

[0174] Live-dead staining quantification demonstrated that all hydrogel samples effectively promoted cell viability (Fig. 12 A-C). After 7 days of exposure, all samples showed a statistically significant increase in cell population. Additionally, the Live / Dead assay indicated a high percentage of live cells within a week of contact with the hydrogel samples. Example 12. Mineralization

[0175] The osteogenic differentiation potential of encapsulated human Mesenchymal Stem Cells (hMSC) was tested. After 5 and 6 weeks, the cell-laden hydrogel samples in differentiation media were washed three times with DPBS and three times with sterile water and then lyophilized for analysis by XRD, EDAX, and FTIR techniques. For osteogenic differentiation, the cells were cultured in DM EM (low glucose, GlutaMAX) containing 10% FBS, 1% (v / v) penicillin-streptomycin, 0.1 pm dexamethasone with 10 mM p-glycerophosphate, and 50 pg / mL ascorbic acid. The hMSCs were used at passage number 3.

[0176] From the XRD results (FIG. 13A and FIG. 13B), significant hydroxyapatite-related peaks associated with the (211) crystalline planes of hydroxyapatite were observed in the 0.5% GO-PLL samples in differentiation media (DM). However, all other samples (0.5% GO-PLL (GM and C), 0.2% GO-PLL (GM, DM, and C), 0 % GO-PLL (GM, DM, and C)) did not show any peaks associated with hydroxyapatite crystalline planes. To further investigate the mineral phase of the calcified matrix deposited by the hMSCs, hydrogels were analyzed via SEM, (FIG. 13C). Temporal mineralization led to the formation of particles resembling hydroxyapatite Ca5(PO4)3OH, the primary inorganic component of native bone. EDAX analysis confirmed the particles are primarily composed of calcium and phosphate (FIG. 13D). Similar to the XRD results, EDAX analysis of all other samples (0.5% GO-PLL (GM and C), 0.2% GO-PLL (GM, DM, and C), 0 % GO-PLL (GM, DM, and C)) did not show the presence of such granulate features.

[0177] XRD analysis was performed using a HUBER G670 X-ray powder diffractometer (Germany) employing the image plate detection method in the Guinier geometry. The analysis was performed in the 20 range of 10-80° at a step size of 0.005°, and the diffractometer was equipped with a secondary monochrome and Cu X-ray tube. The sealed tube X-ray generator was operated at 40 kV and 40 mA to provide Cu Ka1 radiations of wavelength 1.54056 A. The hydrogels were washed and lyophilized for 48 hours for SEM (FEI Quanta 200 ESEM FEG) imaging, operating at an accelerating voltage of 10 kV. All samples were sputter-coated with gold (10 nm) before SEM imaging. The EDAX measurements were performed for the same samples using an energy-dispersive X-ray spectrometer (EDX; Oxford Instruments 80 mm2X-Max silicon drift detector) connected to the SEM instrument. Example 13. Manufacturing of an Oxidized Alginate (AIOx) - Poly-L-Lysine (PLL) - Graphene Oxide (GO) - adipic acid dihydrazide (ADH) hydrogel

[0178] A first solution was prepared by dissolving 10% w / v AIOx in 200 pL of 0.1% PLL. A second solution was prepared by dissolving 12 mg of ADH (6%) in 200 pL of either 0.1% GO-PLL, 0.2% GO-PLL, or 0.5% GO-PLL, depending on the reguired GO concentration.

[0179] To prepare the GO-PLL for the second solution, 10 mg GO was dissolved in 10 mL 0.1% PLL. The mixture was sonicated at room temperature for 30 minutes, incubated at 37°C for 1 hour, and then centrifuged at 12,000 rpm for 5 minutes.

[0180] To form the hydrogel, the first and second solutions were mixed in a 1 :1 ratio, cast into syringes, and incubated at 37°C for about 30 minutes. Hydrogel crosslinking was achieved by mixing the two solutions, resulting in spontaneous crosslinking to form a solid hydrogel. Different ratios of the solutions were used to create hydrogels with varying compositions.

[0181] The present disclosure is not limited to the specific examples described. Any polymer bearing reactive aldehyde and amine (or hydrazide) groups capable of forming reversible covalent bonds can be used. The same crosslinking mechanism applies to other polysaccharides or synthetic polymers functionalised with such groups, and to crosslinking agents having two or more reactive amine and / or hydrazide functionalities, such as those exemplified herein. Likewise, various conductive nanoparticles may be employed provided they impart electrical conductivity without inhibiting the crosslinking reaction. Adjustments in concentrations, molecular weights, or reaction conditions to accommodate such variations are within the routine abilities of a person skilled in polymer chemistry. The examples merely illustrate the general principle of dynamic imine / hydrazone crosslinking underlying the invention.

[0182] Items

[0183] 1. A hydrogel composition comprising:

[0184] • a first polymer comprising aldehyde groups;

[0185] • a second polymer comprising amine groups or amide groups;

[0186] • a crosslinking agent comprising amine groups; and at least one type of conductive nanoparticles.

[0187] 2. The hydrogel composition according to any of the preceding items, wherein the first polymer is selected from a group comprising oxidized alginate, methacrylated oxidized alginate, alginate, oxidized pectin, methacrylated oxidized pectin, and / or oxidized chitosan.

[0188] 3. The hydrogel composition according to item 1, wherein the second polymer is selected from a group comprising poly-L-lysine, 2-, 4-, or 8-arm PEG, dopamine, pectin, and / or silk methacrylate.

[0189] 4. The hydrogel composition according to any of the preceding items, wherein the second polymer has been aminated, or amidated.

[0190] 5. The hydrogel composition according to any of the preceding items, wherein the crosslinking agent comprises one or more hydrazide groups (-NH-NH2), or derivatives thereof, such as wherein the crosslinking agent is capable of forming imine bonds with aldehyde groups.

[0191] 6. The hydrogel composition according to any one of the preceding items, wherein the crosslinking agent is adipic acid dihydrazide (ADH), hydrazine hydrate, ethylenediamine, triethylenetetramine, lysine, polyethyleneimine (PEI), glycol hydrazide, 2-. 4-, or 8-arm PEG-amine, and / or semicarbazide.

[0192] 7. The hydrogel composition according to any one of the preceding items, wherein the at least one type of conductive nanoparticles is selected from a group comprising carbon-based, nanoclay-based, silicate-based, calcium peroxide based, transition metal carbide based materials, zinc oxide tetrapods, and / or metal-ligand- frameworks.

[0193] 8. The hydrogel composition according to any one of the preceding items, wherein the at least one type of conductive nanoparticles is selected from a group comprising gold, silver, copper, nickel, graphene, graphene oxide, carbon nanotubes, fullerenes, polyaniline, polypyrrole, polythiophene, indium tin oxide (ITO), titanium dioxide (TiO2), zinc oxide (ZnO).

[0194] 9. The hydrogel composition according to any one of the preceding claims, wherein the first polymer can form an imine bond with the second polymer and / or the crosslinking agent.

[0195] 10. The hydrogel composition according to any one of the preceding items, having a self-healing ability.

[0196] 11 . The hydrogel composition according to any one of the preceding items, wherein the first polymer is oxidized alginate, the second polymer is poly-L-lysine, the crosslinking agent is adipic acid dihydrazide and the at least one type of conductive nanoparticles is graphene oxide.

[0197] 12. The hydrogel composition according to any one of the preceding items, wherein the degree of oxidation of the first polymer is between 30% and 70%.

[0198] 13. The hydrogel composition according to any one of the preceding items, wherein the concentration of the crosslinking agent is between 0.1% and 2% w / v.

[0199] 14. The hydrogel composition according to any one of the preceding items, wherein the concentration of the at least one type of conductive nanoparticles is between 0.05% and 1 % w / v.

[0200] 15. The hydrogel composition according to any one of the preceding items wherein the pore diameter is 20-70 pm.

[0201] 16. The hydrogel composition according to any one of the preceding items wherein the hydration ratio is 90-95%.

[0202] 17. The hydrogel composition according to any one of the preceding items wherein the added graphene oxide reduces the hydrogel degradation up to 20%. The hydrogel composition according to any one of the preceding items wherein the viscosity is 100+ / -20 Pa s at a shear rate of 1 / sec. The hydrogel composition according to any one of the preceding items wherein the ultimate compressive stress is between 5-15 kPa. The hydrogel composition according to any one of the preceding items wherein the compressive modulus is 15-60 kPa. The hydrogel composition according to any one of the preceding items wherein the toughness is between 0.6- 1.5 kJ / m3. The hydrogel composition according to any one of the preceding items wherein the ion conductivity is at least 3x1 O'3S / cm. Use of the hydrogel composition according to any one of the preceding items for biomedical applications. Use of the hydrogel according to any one of items 1-22 for additive manufacturing. Use of the hydrogel according to any one of items 1-22 for stimulating cell growth. Use of the hydrogel according to any one of items 1-22 for stimulating the osteogenic differentiation in human mesenchymal stem cells encapsulated in said hydrogel. Use of the hydrogel according to any one of items 1-22 for encapsulation of cells. Use of the hydrogel according to any one of items 1-22 for increasing cell viability. Use of the hydrogel according to any one of items 1-22 as a wound care adhesive. Use of the hydrogel according to any one of items 1-22Fejl! Henvisningskilde ikke fundet. as a wound dressing. 31. Use of the hydrogel according to any one of items 1-22 to electrically stimulate and monitor cells or tissues.

[0203] 32. A method of manufacturing the hydrogel of any one of items 1-22, the method comprising the steps of:

[0204] • preparing a first solution comprising a first polymer and a second polymer;

[0205] • preparing a second solution comprising at least one type of conductive nanoparticles and a crosslinking agent; and

[0206] • mixing the first solution with the second solution.

[0207] 33. The method of item 32, wherein:

[0208] • the first solution comprises oxidized alginate and poly-L-lysine;

[0209] • the second solution comprises graphene oxide and adipic acid dihydrazide; and

[0210] • the first and the second solutions are mixed at a ratio in the range of 0.1-3:0.1-3.

[0211] 34. The method of any one of items 32-33, wherein:

[0212] • the first solution is prepared by dissolving 5-15% w / v oxidized alginate in 0.1 -0.5% poly-L-lysine; and

[0213] • the second solution is prepared by dissolving 2-8% w / v adipic acid dihydrazide (ADH) in a graphene oxide poly-L-lysine solution comprising 0.1 -0.5% poly-L-lysine solution and 0.1%-0.5% graphene oxide.

[0214] 35. The method of any one of items 32-34, wherein:

[0215] • the first solution is prepared by dissolving 10% w / v oxidized alginate in 0.1% poly-L-lysine, and

[0216] • the second solution is prepared by dissolving 6% w / v adipic acid dihydrazide (ADH) in a graphene oxide poly-L-lysine solution comprising 0.1% poly-L-lysine solution and 0.1%-0.5% graphene oxide.

[0217] 36. The method according to items 32-35, wherein the graphene oxide poly-L-lysine solution is prepared by:

[0218] • dissolving graphene oxide in the poly-L-lysine solution; sonicating the solution for 30 min at room temperature; incubating the solution for 1 h at 37°C; centrifuging at 12000 rpm for 5 min to remove excess poly-L-lysine.

[0219] 37. The method according to any one of items 32-36, wherein the first and the second solution are mixed in a 0.1 -3:1 ratio.

[0220] 38. The method according to any one of items 32-37, wherein the first and the second solution are mixed in a 1:0.1-3 ratio.

[0221] 39. The method according to any one of items 32-38, wherein the first and the second solution are mixed in a 1 :1 ratio.

[0222] 40. The method according to any one of items 32-39, wherein the first and the second solution are mixed in a 1 :3 ratio, such as in a 1:2 ratio, such as in a 3:1 ratio, such as in a 2:1 ratio, such as in a 1 :1 ratio.

[0223] 41. A hydrogel produced by a method comprising the steps described in items 32-40.

Claims

44Claims1 . A biocompatible hydrogel composition comprising:• a first polymer comprising aldehyde groups;• a second polymer comprising amine groups or amide groups;• at least one crosslinking agent that is distinct from the first and second polymers and that comprises two or more amine and / or hydrazide functional groups, the crosslinking agent being capable of forming covalent imine and / or hydrazone linkages between polymer chains; and• at least one type of electrically conductive nanoparticles dispersed within the hydrogel.

2. The hydrogel composition according to any one of the preceding claims, wherein the first polymer is a polymer functionalised to comprise aldehyde groups selected from oxidized alginate, such as methacrylated oxidized alginate, oxidized pectin, such as methacrylated oxidized pectin, and / or oxidized chitosan.

3. The hydrogel composition according to any one of the preceding claims, wherein the second polymer is a polymer functionalised to comprise amine groups or amide groups including, but not limited to, poly-L-lysine, a dopamine-functionalised polymer, and / or silk methacrylate.

4. The hydrogel composition according to any one of the preceding claims, wherein the crosslinking agent forms imine and / or hydrazone linkages with aldehyde groups of the first polymer.

5. The hydrogel composition according to any one of the preceding claims, wherein the composition comprises a hydrogel network comprising: (i) covalent linkages between the first and second polymers; and (ii) covalent linkages between at least one of said polymers and a low-molecular-weight crosslinking agent, thereby forming a multiscale crosslinking architecture.

6. The hydrogel composition according to any one of the preceding claims, wherein the crosslinking agent comprises two or more hydrazide functional groups (-NH- NH2) capable of forming hydrazone linkages with the aldehyde groups of the first45 polymer.

7. The hydrogel composition according to any one of the preceding claims, wherein the crosslinking agent is• a hydrazide-based compound selected from adipic acid dihydrazide (ADH), glycol hydrazide, 2-, 4-, or 8-arm PEG-hydrazide, succinic dihydrazide, malonic dihydrazide, sebacic dihydrazide, poly(acryloyl hydrazide), or semicarbazide; or• an amine-based compound selected from hydrazine hydrate, lysine, polyethyleneimine (PEI), or triethylenetetramine.

8. The hydrogel composition according to any one of the preceding claims, wherein the at least one type of conductive nanoparticles is selected from the group consisting of carbon-based materials, transition metal carbide based materials, zinc oxide tetrapods, and / or metal-ligand-frameworks.

9. The hydrogel composition according to any one of the preceding claims, wherein the at least one type of conductive nanoparticles is selected from the group consisting of gold, silver, copper, nickel, graphene, graphene oxide, carbon nanotubes, fullerenes, polyaniline, polypyrrole, polythiophene, indium tin oxide (ITO), titanium dioxide (TiO2), and zinc oxide (ZnO).

10. The hydrogel composition according to any one of the preceding claims, wherein the first polymer can form an imine bond with the second polymer and / or the crosslinking agent.

11. The hydrogel composition according to any one of the preceding claims, wherein the hydrogel has a self-healing ability, such as a self-healing efficiency of at least 85% after 1 h, determined by recovery of ultimate compressive stress.

12. The hydrogel composition according to any one of the preceding claims, wherein the first polymer is oxidized alginate, the second polymer is poly-L-lysine, the crosslinking agent is adipic acid dihydrazide and the at least one type of conductive nanoparticles is graphene oxide.4613. The hydrogel composition according to any one of the preceding claims, wherein the degree of oxidation of the first polymer is between 30% and 70%.

14. The hydrogel composition according to any one of the preceding claims, wherein the concentration of the crosslinking agent is between 0.1% and 2% w / v.

15. The hydrogel composition according to any one of the preceding claims, wherein the concentration of the at least one type of conductive nanoparticles is between 0.05% and 1 % w / v.

16. The hydrogel composition according to any one of the preceding claims, wherein the pore diameter is 20-70 pm.

17. The hydrogel composition according to any one of the preceding claims, wherein the hydration ratio is 90-95%.

18. The hydrogel composition according to any one of the preceding claims, wherein incorporation of graphene oxide reduces hydrogel mass loss during degradation by up to 20 % relative to a corresponding hydrogel without graphene oxide.

19. The hydrogel composition according to any one of the preceding claims, wherein the viscosity is 100 ± 20 Pa s at a shear rate of 1 s-1.

20. The hydrogel composition according to any one of the preceding claims, wherein the ultimate compressive stress is between 5-15 kPa.

21. The hydrogel composition according to any one of the preceding claims, wherein the compressive modulus is 15-60 kPa.

22. The hydrogel composition according to any one of the preceding claims, wherein the toughness is between 0.6-1.5 kJ / m3.

23. The hydrogel composition according to any one of the preceding claims, wherein the ion conductivity is at least 3x1 O'3S cm-1, as measured by electrochemical impedance spectroscopy (EIS) at 25 °C.

24. Use of the hydrogel composition according to any one of the preceding claims, for biomedical applications.

25. Use of the hydrogel according to any one of claims 1-23 for additive manufacturing.

26. Use of the hydrogel according to any one of claims 1-23 for stimulating cell growth.

27. Use of the hydrogel according to any one of claims 1-23 for stimulating the osteogenic differentiation in human mesenchymal stem cells encapsulated in said hydrogel.

28. Use of the hydrogel according to any one of claims 1-23 for encapsulation of cells.

29. Use of the hydrogel according to any one of claims 1-23 for increasing cell viability.

30. Use of the hydrogel according to any one of claims 1-23 as a wound care adhesive.

31. Use of the hydrogel according to any one of claims 1-23 as a wound dressing.

32. Use of the hydrogel according to any one of claims 1-23 to electrically stimulate and monitor cells or tissues.

33. A method of manufacturing the hydrogel of any one of claims 1-23, the method comprising the steps of:• preparing a first solution comprising a first polymer and a second polymer;• preparing a second solution comprising at least one type of conductive nanoparticles and a crosslinking agent; and• mixing the first solution with the second solution.

34. The method of claim 33, wherein: the first solution comprises oxidized alginate and poly-L-lysine; the second solution comprises graphene oxide and adipic acid dihydrazide; andthe first and the second solutions are mixed at a ratio in the range of 0.1-3:0.1-3.

35. The method of any one of claims 33-34, wherein:• the first solution is prepared by dissolving 5-15% w / v oxidized alginate in 0.1 -0.5% poly-L-lysine; and• the second solution is prepared by dissolving 2-8% w / v adipic acid dihydrazide (ADH) in a graphene oxide poly-L-lysine solution comprising 0.1 -0.5% poly-L-lysine solution and 0.1%-0.5% graphene oxide.

36. The method of any one of claims 33-35, wherein:• the first solution is prepared by dissolving 10% w / v oxidized alginate in 0.1% poly-L-lysine, and• the second solution is prepared by dissolving 6% w / v adipic acid dihydrazide (ADH) in a graphene oxide poly-L-lysine solution comprising 0.1% poly-L-lysine solution and 0.1%-0.5% graphene oxide.

37. The method according to claims 33-36, wherein the graphene oxide poly-L-lysine solution is prepared by:• dissolving graphene oxide in the poly-L-lysine solution;• sonicating the solution for 30 min at room temperature;• incubating the solution for 1 h at 37°C;• centrifuging at 12000 rpm for 5 min to remove excess poly-L-lysine.

38. The method according to any one of claims 33-37, wherein the first and the second solution are mixed in a 0.1 -3:1 ratio.

39. The method according to any one of claims 33-38, wherein the first and the second solution are mixed in a 1:0.1-3 ratio.

40. The method according to any one of claims 33-39, wherein the first and the second solution are mixed in a 1 :1 ratio.4941. The method according to any one of claims 33-40, wherein the first and the second solution are mixed in a 1 :3 ratio, such as in a 1:2 ratio, such as in a 3:1 ratio, such as in a 2:1 ratio, such as in a 1 :1 ratio.

42. A hydrogel produced by a method comprising the steps described in claims 33-41.

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