Self-standing hydrogels based on poly(vinyl alcohol) and carbon nanostructures

Abstract

It relates to a porous self-standing homogeneous hydrogel comprising poly(vinyl alcohol) and carbon nanostructures, wherein: the carbon nanostructures are present in an amount from 7 to 85% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures and the pores have an average particle size equal to or higher than 8 µm. It also relates to processes for the preparation of these hydrogels including phase inversion and cross-linking methods, and to their use in nerve regeneration.

Description

[0001] Self-standing hydrogels based on poly(vinyl alcohol) and carbon nanostructures

[0002] This application claims the priority of the European Patent Application EP23382781.5 filed on 27.07.2023.

[0003] Technical Field

[0004] The present invention relates to 3D self-standing scaffolds based on poly(vinyl alcohol) (PVA) and carbon nanostructures with the ability to stabilize a large amount of carbon nanostructures within the polymeric matrix. These materials may be used as implants for electroactive cells and tissues, in particular as neural interface implants, for example for detecting, transmitting, or monitoring electrical signals to the brain, or for treating CNS damage in a subject in need thereof.

[0005] Background Art

[0006] After a person reaches maturity and adulthood, highly specific cells namely neurocytes, cannot proliferate in the same manner as in the neonatal or pre-neonatal phase. Therefore, upon a neural injury, for instance in the spinal cord, patients suffer paraplegia or tetraplegia, losing motor and sensory functions. Since neural cells are highly specific and differentiated cells which does not undergo mitosis like epithelial cells for example, these cells cannot regenerate on their own and soon lose function drastically. This process is further exacerbated by the formation of scar tissue as the cells attempt to repair themselves, leaving the patient either partially or completely injured for life. In the case of injuries to the peripheral nervous system (PNS), most prominently to the sciatic nerve, some degree of regeneration is seen with less severe traumas, due to the less complex structure of the nerve compared to the CNS. Hence, little hope of regaining proper function is left. In a study following 380 patients monitored over 24 years, healing was found in only 36% of cases.

[0007] Efforts are directed towards neuronal regeneration due to the increase of injuries around the world where spinal cord injuries alone amount to about 500,000 cases every year, in addition to the impact of nerve traumas on the patients’ lives decreasing their life expectancy between 2 to 5 times. The spinal cord consists of grey matter (motor / sensory neurons, interneurons, and unmyelinated neuroglia) encapsulated within the white matter (myelinated sensory and motor neurons). The spinal cord is highly organized into 4 tracts transmitting either sensory or motor signals towards or from the upper CNS to the rest of the body. On the other hand, the sciatic nerves are the longest and widest nerve fibers in the human body, responsible for supplying sensory and motor functions to the lower limbs. They stem from the spinal nerves between lumbar segment 4 through sacral segment 3 and exit the pelvis through the greater sciatic foramen progressing downwards to the biceps femoris muscle and to the rest of the leg and foot.

[0008] The pathophysiology of the CNS is similar to that of the PNS in the formation of scar tissue notably during the acute phase, demyelination, inflammation and ion imbalance occur followed by excessive proliferation of astrocytes and fibroblasts infiltration with further demyelination forming a glial scar. Finally, the cystic cavity formed prohibit migration of pro-regenerative cells, hence, permanent damage become inevitable in the case of the CNS, whereas severe complications are seen with injuries to the PNS.

[0009] Any injury to the nerve section or subsequently to the spinal cord can result in loss of the nerve functions. Additionally, the formation of neuromas at the lesion site within 4 months, if unoccupied by new axons, prevents regeneration of the nerve similar to the spinal cord injury (SCI), with current treatments being insufficient to regain proper former function.

[0010] Current efforts to develop advanced treatments for various neuropathies split into different approaches such as neuro-robotics, biological approach (stem cell therapy etc...) and synthetic materials / nanomedicine, it is noteworthy that the latter is the least studied approach, additionally, most efforts focus on the brain, given its complexity and the efforts it requires.

[0011] The current most promising biological practice is nerve grafting. Autografts and isografts from one side are considered the ‘gold standard’ for nerve regeneration. However, recent studies show that with injuries exceeding 2 cm, axonal growth is reduced. Additionally, long grafts had elevated senescence markers expression.

[0012] On the other hand, material and polymer science converged to give birth to advanced polymeric-based materials and scaffolds for tissue engineering and regeneration, many of which, are still considered underperforming or like the case of chitosan-based conduits, do not find their way past clinical studies.

[0013] Efforts are still directed towards achieving nerve regeneration via synthetic materials.

[0014] Nonetheless, the search for the ideal material is far from over as many of the developed materials fall short on mimicking nerve tissue properties.

[0015] As to mimicking the neuronal tissue, electrically conductive scaffolds became evident as a better alternative. Such materials are achieved by conductive additives including conductive polymers as PEDOT and Polypyrrole (Ppy), and carbon nanomaterials, such as graphene and carbon nanotubes (CNT). Soon after, CNT-based 3D scaffolds were increasingly being reported. However, concerns about cytotoxicity and biocompatibility, among others, hinder their progress into clinical trials. With all the scientific research insurgence that these CNT-based materials have seen, not only diminished those fears but proved their superiority as conductive additives, promoting neuronal maturation, proliferation, and synapses signaling. Additionally, CNTs show superior performance over other conductive additives.

[0016] Hydrogels containing PVA and CNTs have been described in the prior art (Hongwei et al, Separation and purification technology 2021 , vol. 264, 118459) in the context of a completely different use, which is wastewater treatment.

[0017] Therefore, there is still the need to provide new materials which overcome the problems of the prior art and may be used as neural interface implants.

[0018] Summary of Invention

[0019] The inventors have developed new 3D-biocompatible porous materials based on poly(vinyl alcohol) (PVA) and carbon nanostructures with the ability of mimicking the neuronal tissue. Thus, these materials may be used as implants for reconnecting and / or regenerating purposes, for example for detecting, transmitting, or monitoring electrical signals to the brain, or for treating CNS damage in a subject in need thereof for example through nerve regeneration by promoting neuronal growth.

[0020] The 3D-biocompatible materials based on poly(vinyl alcohol) (PVA) and carbon nanostructures form a homogeneous polymeric matrix which has the ability to stabilize within the matrix a large amount of carbon nanostructures (up to 75% in weight with respect to the sum of the carbon nanostructures and the PVA) unlike most of the materials of the prior art. This is a great advantage since the presence of carbon nanostructures, such as CNTs, was found to be fundamental for cell growth and enhancing neuronal signaling.

[0021] As illustrated in the examples below, the scaffolds of the invention showed increased cell attachment and axonal growth compared to controls in cell culture studies. Furthermore, in in vivo experiments where the sciatic nerve of mice (Mus musculus) was exposed by an incision in the gluteal minimus region and the nerve was cut and wrapped with the scaffold of the invention, it was found that mice regained mobility after 10 days post-surgery which shows that there is no toxic effect on the organism, and by the 3 weeks mark sacrifice, mice could use their healthy limbs and started walking (presenting a limp in the damaged leg as expected), and hair regrowth was observed in the area affected by the surgical process, which was seen as a positive sign.

[0022] Additionally, all mice survived the excision surgery experiment and the compression injury surgery experiment, and mice with CNT scaffold did not show an inflammatory reaction, or signs of pain or distress, unlike control mice, which did present some inflammatory reaction. Immunostaining of the extracted mouse sciatic nerve at week 3 with CNT scaffold, showed a high signal of neurofilament marker (which marks the neurofilaments in axons), which shows promising results / proof for regeneration.

[0023] Further, the scaffolds of the invention encompass different types of materials depending on its preparation process, including hydrogels in the form of films, designated herein as PIMs or PIM hydrogels, or cross-linked hydrogels, designated herein as PBCLs or PBCLC hydrogels. The first ones are obtained by phase inversion methods and the second ones by crosslinking as detailed in the examples below.

[0024] The materials of the present invention can also advantageously be molded into or onto different devices in order to obtain materials with different geometries. This is especially advantageous for applications in which a specific geometry is needed. More specifically, to obtain nerve-like geometries, such as mimicking the shape and size of the sciatic nerve or the spinal cord. Additionally, it can be used for personalized treatment to the shape and size of the neural lesion adaptable to each patient. The ability to mold the materials would also allow various approaches to neural regeneration, such as targeting lesions in the form of nerve guidance conduit (NGC), or implantable stent (as bulk material), among others.

[0025] For example, the hydrogels can be molded into hollow or filled cylinder-like structures by utilizing a hollow capillary tube (with no limitation to the width or diameter of the capillary) as illustrated in the examples. This results in formed hydrogel around the outerwalls of the capillary which can be used as Nerve Guidance Conduit NGC in in-vivo studies, and additionally to a filled thread-like hydrogel in the inside of the capillary forming a structure similar in shape and dimensions of the nerve.

[0026] The above processes are simple and scalable, which makes the materials of the invention interesting from an industrial point of view. Therefore, a first aspect of the invention relates to a porous self-standing homogeneous hydrogel, in particular suitable for use as an implant, which comprises poly(vinyl alcohol) and carbon nanostructures, wherein: a) the carbon nanostructures are present in an amount from 7 to 85% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; and b) the pores have an average size equal to or higher than 8 pm. The average pore size is particularly measured by means of Scanning Electron Microscopy (SEM) in dry state.

[0027] A second aspect of the invention relates to a device comprising the porous self-standing homogeneous hydrogel of the first aspect.

[0028] A third aspect of the invention relates to a process for the preparation of the porous selfstanding homogeneous hydrogel as defined herein, particularly a PIM hydrogel, which comprises the steps of: a1) providing an aqueous solution comprising poly(vinyl alcohol); b1) providing an aqueous dispersion comprising carbon nanostructures; c1) mixing the solution of step a1) and the dispersion of step b1) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures, wherein the amount of carbon nanostructures in the obtained dispersion is from 7 to 85% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; d1) spreading an amount of the dispersion of step c1) onto a support; e1) immersing the support with the dispersion of step d1) into a non-solvent to induce phase inversion and obtain a supported gel; f1) removing the supported gel from the non-solvent; g1) if desired, repeating steps d1), e1) and f1) one or more times until obtaining the desired gel thickness, provided that when step d1) is repeated the dispersion of step c1) is spread onto the gel on the support obtained in the previous step; hi) immersing the supported gel obtained in the previous step into an aqueous solution to obtain a supported hydrogel; and i1) removing the supported hydrogel from the aqueous solution, and separating the hydrogel from the support to obtain a self-standing hydrogel, or alternatively, iT) separating the hydrogel from the support to obtain a self-standing hydrogel, and removing the self-standing hydrogel from the aqueous solution. A fourth aspect of the invention relates to a process for the preparation of the porous selfstanding homogeneous hydrogel as defined herein, particularly a PIM hydrogel, which comprises the steps of: a2) providing an aqueous solution comprising poly(vinyl alcohol); b2) providing an aqueous dispersion comprising carbon nanostructures; c2) mixing the solution of step a2) and the dispersion of step b2) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures, wherein the amount of carbon nanostructures in the obtained dispersion is from 7 to 85% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; d2) adding a porogenic agent to the dispersion of step c2) to obtain a mouldable mass; e2) moulding the mouldable mass obtained in d2) into a moulded mass; f2) soaking the moulded mass obtained in step e2) in a non-solvent to induce phase inversion and obtain a self-standing gel; g2) removing the self-standing gel from the non-solvent; h2) immersing the self-standing gel obtained in step g2) into an aqueous solution to obtain a self-standing hydrogel; i2) heating the aqueous solution of step h2) to a temperature equal to or higher than 35 °C to remove the porogenic agent and obtain a porous self-standing hydrogel; and j2) removing the self-standing hydrogel from the aqueous solution.

[0029] A fifth aspect of the invention relates to a process for the preparation of the porous selfstanding homogeneous hydrogel as defined herein, particularly a PBCLC hydrogel, which comprises the steps of: a3) providing an aqueous solution comprising poly(vinyl alcohol); b3) providing an aqueous dispersion comprising carbon nanostructures; c3) mixing the solution of step a3) and the dispersion of step b3) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures, wherein the amount of carbon nanostructures in the obtained dispersion is from 7 to 85% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; and d3) adding a crosslinking agent and heating at a temperature from 80 to 120 °C; e3) cooling to a temperature equal to or lower than 60 °C to obtain a porous self-standing hydrogel.

[0030] A sixth aspect of the present invention relates to a neural implant made of the porous selfstanding homogeneous hydrogel as defined herein.

[0031] A seventh aspect of the present invention relates to the use of the neural implant as defined herein for detecting, transmitting, or monitoring electrical signals to the brain. An eighth aspect of the present invention relates to the neural implant as defined herein for use in treating CNS damage in a subject in need thereof.

[0032] Brief Description of Drawings

[0033] FIG. 1 shows SEM images of PIM materials of the surface part (S) and the part in contact with the glass slide (labelled “side 2”, S2) at different magnifications (x300, x1300, x3000).

[0034] FIG. 2 shows SEM images of PBCLC materials of the surface part at different magnifications (x300, x1300, x3000).

[0035] Detailed description of the invention

[0036] All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific terms as used in the present application are as set forth below and are intended to apply uniformly throughout the specification and claims unless an otherwise expressly set out definition provides a broader definition.

[0037] For the purposes of the invention, any ranges given include both the lower and the upper endpoints of the range. Ranges given, such as temperatures, times, sizes, and the like, should be considered approximate, unless specifically stated.

[0038] The term “about” or “around” or “approximately” as used herein refers to a range of values ± 10% of a specified value. For example, the expression “about 10” or “around 10” includes ± 10% of 10, i.e., from 9 to 11.

[0039] Poly(vinyl alcohol) (PVA) is a synthetic polymer of formula [CH2CH(OH)]nthat belongs to the class of polyvinyl esters, which are derived from polyvinyl acetate. PVA is a water- soluble polymer and is commonly used for its unique properties, including its biocompatibility, film-forming ability, and adhesion to a wide range of surfaces.

[0040] The term “scaffold” as used herein refers to a biocompatible material that provides a surface suitable for adherence and proliferation of cells and optionally further provides mechanical stability. The term “biocompatible” as used herein refers to a material which does not elicit a significant foreign body response, such as for example, an immune or an inflammatory response. The term “hydrogel”, as used herein, refers to a type of gel (soft material) that is composed of a polymeric three-dimensional network, capable of retaining a large amount of water or other aqueous solutions. The term hydrogel is derived from the fact that these gels are formed by hydrophilic polymers that are capable of absorbing and retaining water, giving them a gel-like consistency.

[0041] The term "self-standing" hydrogel as used herein refers to a hydrogel which is capable of standing or staying in an erect mode and substantially retains its original shape in the absence of external forces.

[0042] The term “homogeneous” as used herein refers to a material in which the components forming the material are distributed uniformly and regularly, i.e., the components are not segregated into different areas or sections of the material.

[0043] The term "aerogel" as used herein refers to the gel resulting from the removal of the solvent, typically an aqueous solvent such as water from the interior of the hydrogel, e.g., an aerogel may be obtained when the hydrogel is freeze-dried.

[0044] The terms “molecular weight”, “average molecular weight” and “Mw” have the same meaning and are used herein interchangeably. The molecular weight is calculated by the following equation:

[0045] . J. rsrr where Nj is the number of molecules of molecular mass Mj. The mass average molecular mass can be determined by light scattering, size exclusion chromatography (SEC), and sedimentation velocity.

[0046] The term “swelling by volume” (Sv%) as used herein refers to the increase in volume due to the accumulation of fluid in the materials. It can be determined by the following equation: wherein Vsis the volume of the swollen gel in water (in mm3), and Vd is the volume of the dry gel (in mm3).

[0047] The term “swelling by weight or swelling mass” (Sw%) as used herein refers to the increase in mass due to the accumulation of fluid in the material. It can be determined by the following equation:

[0048] Ws — Wd Sw% = — —— — x 100 Wd wherein Wsis the weight of the swollen gel in water (in mg), and Wd is the weight of the dry gel (in mg).

[0049] Ws, Wd can be measured as shown in the examples, and Vs, Vd, can be calculated on the basis of height and thickness measurements as shown in the examples.

[0050] The term "pore", as used herein, refer to any small opening or interstice. The term “porous” as used herein refers to the property of a material having pores, i.e., voids or open spaces. The term “porosity” as used herein refers to the measure of the void spaces within a material, typically expressed as a percentage of the total volume or surface area of the material. It describes the extent to which a material contains pores, voids, or open spaces that are not filled with solid material. The porosity of the hydrogels of the invention can be calculated from the following equation:

[0051] Ws — Wd

[0052] %p = x 100 p H2O x Vs wherein Ws, Wd, and Vsare as defined above, and p is the density of water (in mg / mm3).

[0053] The “pore size” or “average pore size” as used herein, refers to the average diameter of the cross-sectional dimension of the pores. By “average” is meant the arithmetic mean calculated from various measurements, for example by taking the measurements from 20 to 100 pores from 3 different areas of the hydrogel. It can be measured by means of Scanning Electron Microscopy (SEM) or Transmission Electron Microscopy (TEM), particularly Scanning Electron Microscopy (SEM) in dry state. Thus, the term pore size measured “in dry state” refers to the measurement of the pore size where the hydrogel has been lyophilized (i.e. , freeze dried) to preserve the pore structure as much as possible. If desired, the measurement of the average pore size can be corrected taking into account a correction factor (swelling). In such case, the pore size may be calculated by the following equation: pore size (pm) = [dried scaffold pore size] ■ (1+ (Sv% / 100)) wherein the dried scaffold pore size is the pore size in dry state of a dried sample (lyophilized or frozen), e.g. measured from a SEM or TEM image as mentioned above and shown in the examples, and Sv% is the swelling by volume as defined above. The term "porogenic agent" also designated as “porogen” refers to any compound which has the ability to form pores within a structure or material, i.e., the material based on PVA and carbon nanostructures. Any reagent having the suitable size, e.g., micrometric size, can be used.

[0054] The term "mouldable" as used herein refers to a material which can be formed into a shaped body. The term "moulded" or “molded” as used herein are used interchangeably and refer to a material which has already been formed into a desired shaped body.

[0055] The term “support” as used herein refers to a material that has at least a hard and smooth surface. A suitable support to prepare some of the hydrogels of the invention is a nontreated glass surface.

[0056] The term “implant”' as used herein refers to a material inserted or grafted surgically into the body, either temporarily or permanently, to detect, transmit, or monitor signals or improve, assist or maintain a function.

[0057] The term “non-solvent” refers to a material, which does not substantially dissolve a second or reference material, i.e., the material comprising PVA and carbon nanostructures.

[0058] The expression “obtainable by” is used herein for defining a material (e.g., the PIM hydrogel or the PBCLC hydrogel) by its preparation process and refers to the material that can be obtained through the preparation process disclosed herein. For the purposes of the invention, the expressions “obtainable”, “obtained” and similar equivalent expressions are used interchangeably and, in any case, the expression “obtainable” encompasses the expression “obtained”.

[0059] The term “phase inversion” as used herein refers to a process in which the properties or characteristics of a substance or system change as it transitions from one phase to another. Herein it refers to the change from a liquid phase to a solid phase. This process can lead to changes in various properties such as porosity, permeability, mechanical strength of the material, structure, and morphology. It is widely used in industrial applications i.e., films, membrane, coatings, foams, and other materials. The term “crosslinking agent” refers to a substance that is used to create chemical bonds between polymer chains, resulting in the formation of a three-dimensional network structure.

[0060] The term “thermosensitive” as used herein refers to the property of the hydrogels to exhibit a phase a reversible transition from solid to liquid / dispersion when heated.

[0061] As mentioned above, the first aspect of the present invention relates to a porous selfstanding homogeneous hydrogel comprising poly(vinyl alcohol) and carbon nanostructures, wherein: a) the carbon nanostructures are present in an amount from 7 to 85% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; and b) the pores have an average size equal to or higher than 8 pm.

[0062] It also forms part of the present invention a porous self-standing homogeneous hydrogel consisting of poly(vinyl alcohol) and carbon nanostructures, wherein: a) the carbon nanostructures are present in an amount from 7 to 85% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; and b) the pores have an average size equal to or higher than 8 pm.

[0063] According to one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the porous self-standing homogeneous hydrogel is biocompatible.

[0064] For the purposes of the invention, there is no particular limitation on the type of poly(vinyl alcohol) that can be used. According to one embodiment, the poly(vinyl alcohol) has an average molecular weight from 8 to 200 kDa, more particularly from 140 to 190 kDa.

[0065] In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the poly(vinyl alcohol) is present in an amount from 5 to 10% by weight with respect to the volume of the aqueous solution, particularly water, more particularly, in an amount about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by weight with respect to the volume of the aqueous solution, particularly water.

[0066] With regard to the nanostructures, different materials can be used. In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the carbon nanostructures are selected from carbon nanotubes (CNTs), carbon nanohorns, carbon nanoonions, nanodiamonds, fullerenes, graphene, graphene dots, carbon nanofibers, graphitized carbon nanofibers, and the corresponding functionalized derivatives from all these above-mentioned materials.

[0067] The term “carbon nanotube”, also referred to herein simply as CNT, refers to a hollow material which consists of graphene sheets rolled into cylindrical shapes, wherein graphene layers form one or more concentric cylinders along the tube axis.

[0068] Carbon nanostructures may be prepared by methods well-known in the art, for example by chemical vapor deposition (CVD) from hydrocarbons. They are also commercially available. CNTs include single-walled nanotubes (SWNT) and multi-walled carbon nanotubes (MWCNTs).

[0069] The diameter and the length of carbon nanostructures, such as CNTs, can be measured by methods well-known in the art such as transmission electron microscopy (TEM). Typically, average internal and external diameter values and average length values are given. The term “average diameter” refers to the average value taken from at least three different diameter measurements. The term “average length” refers to the average value taken from at least three different length measurements.

[0070] In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the carbon nanostructures are different from graphene containing structures.

[0071] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the carbon nanostructures are pristine carbon nanostructures. The term “pristine carbon nanostructures” as used herein refers to carbon nanostructures, e.g., CNTs, that have no observable surface modifications and have not been functionalized, modified, or chemically reacted with other elements such as oxygen.

[0072] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the carbon nanostructures are carbon nanotubes (CNTs), more particularly, MWCNTs. In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the carbon nanostructures, particularly MWCNTs, have an internal average diameter from 5 to 10 nm.

[0073] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the carbon nanostructures, particularly MWCNTs, have an external average diameter from 20 to 30 nm.

[0074] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the carbon nanostructures, particularly MWCNTs, have a length equal to or lower than 2 pm, more particularly from 0.5 to 2 pm.

[0075] In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the carbon nanostructures, particularly MWCNTs, have i) an internal average diameter from 5 to 10 nm; ii) an external average diameter from 20 to 30 nm; and iii) a length equal to or lower than 2 pm, more particularly from 0.5 to 2 pm.

[0076] In the porous self-standing homogeneous hydrogels of the first aspect, the carbon nanostructures are present in an amount from 7 to 85% by weight, more particularly from 7 to 75% by weight, with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures.

[0077] According to one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the carbon nanostructures are present in an amount from 15 to 85% by weight, more particularly from 18 to 75% by weight, even more particularly from 18 to 70% by weight, with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures.

[0078] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the carbon nanostructures are present in an amount about 15%, about 18%, about 20%, about 25%, about 30%, about 33%, about 35%, about 40%, about 44%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures. More particularly, the self-standing homogeneous hydrogels comprising carbon nanostructures in an amount from 15 to 85% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures are PIM hydrogels, obtainable by phase inversion methods.

[0079] As mentioned above, the self-standing homogeneous hydrogels of the present invention can be molded onto or into devices and materials with different geometries can be obtained without any limitation, such as e.g., threadlike structure, cylindrical geometry, or irregular shape to fit a lesion site.

[0080] A second aspect of the invention relates to a device comprising the porous self-standing homogeneous hydrogel of the first aspect.

[0081] In one embodiment of the second aspect, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the porous self-standing homogeneous hydrogel is a PIM hydrogel.

[0082] In another embodiment of the second aspect, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the porous self-standing homogeneous hydrogel is a PBCL hydrogel.

[0083] In another embodiment of the second aspect, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the invention relates to a device comprising the self-standing homogeneous hydrogel which has cylindrical geometry. More particularly, the device is a hollow or filled tube-like structure such as a capillary. In such embodiment, the self-standing homogeneous hydrogel, in particular a PIM hydrogel, is placed outerwalls of the capillary and in the inside of the capillary. This has the advantage that a structure similar in shape and dimensions of a nerve is formed.

[0084] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the carbon nanostructures are present in an amount from 7 to 18% by weight, more particularly from 8 to 12% by weight, with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures.

[0085] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the carbon nanostructures are present in an amount about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, or about 18% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures. More particularly, the self-standing homogeneous hydrogels comprising carbon nanostructures in an amount from 7 to 18% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures are PBCLC hydrogels, obtainable by using a crosslinking agent.

[0086] The porous self-standing homogeneous hydrogel of the first aspect of the invention comprises pores of an average size equal to or higher than 8 pm, particularly measured by means of Scanning Electron Microscopy (SEM) in dry state. In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the pores have an average size in a range from 8 to 250 pm, more particularly, from 10 to 200 pm, even more particularly, from 10 to 100 pm, and even more particularly, from 10 to 50 pm. More particularly, the pores have an average size about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, or about 20 pm.

[0087] In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the pore size is measured by the following equation: pore size (pm) = [dried scaffold pore size] ■ (1+ (Sv% / 100)) wherein: the dried scaffold pore size is the pore size of the hydrogel measured by Scanning Electron Microscopy (SEM) after placing the hydrogel at -20 °C for 24 h, then at -80 °C and lyophilizing it until the gel does not contain any water and the weight is constant (i.e. , when the difference between two consecutive weightings is equal to or lower than 0.50 mg per g of substance taken), and

[0088] Sv% is the swelling by volume calculated according to the following equation: wherein:

[0089] Vsis the volume of the swollen gel in water (in mg) measured after immersing the hydrogel in water for at least 72 h and removing the excess water, and

[0090] Vd is the volume of the dry gel (in mg) measured after placing the swollen hydrogel at -20 °C for 24 h, then at -80 °C and lyophilizing it until the gel does not contain any water and the weight is constant (i.e. , when the difference between two consecutive weightings is equal to or lower than 0.50 mg per g of substance taken).

[0091] More particularly, the pore size may be determined by: a) providing a disc of the porous self-standing homogeneous hydrogel; b) placing the disc at -20 °C for 24 h, then at -80 °C and lyophilizing it until the gel does not contain any water and the weight is constant (i.e., when the difference between two consecutive weightings is equal to or lower than 0.50 mg per g of substance taken); c) measuring the dried scaffold pore size by Scanning Electron Microscopy (SEM); and d) calculating the pore size by the following equation: pore size (pm) = [dried scaffold pore size] ■ (1+ (Sv% / 100)). wherein the dried scaffold pore size is obtained in step c) and Sv% is the swelling by volume. In particular, the swelling by volume (Sv%) can be determined by: a) providing a disc of the porous self-standing homogeneous hydrogel; b) immersing the disc in water for at least 72 h and removing the excess water; c) weighing the disc (swelling weight, Ws, in mg), and measuring its thickness (swelling height, hs), and diameter (swelling diameter, ds, in mm) using a digital micrometer; d) calculating the swelling volume (Vs, in mm3) according to the equation Vs=TT rS2 hs, wherein rs is the radius (1 / 2 ds, in mm); e) placing the disc at -20 °C for 24 h, then at -80 °C and lyophilizing it until the gel does not contain any water and the weight is constant (i.e., when the difference between two consecutive weightings is equal to or lower than 0.50 mg per g of substance taken); and f) weighing the disc (dry weight, Wd, in mg), and measuring its thickness (dry height, hd), and diameter (dry diameter, d , in mm) using a digital micrometer; g) calculating the dry volume (Vd, in mm3) according to the equation Vd=TT rd2- hd, wherein rd is the radius (1 / 2-dd, in mm); h) calculating the swelling by volume Sv%) according to the following equation:

[0092] According to another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the self-standing hydrogel as defined above wherein the pores are randomly distributed. For the purposes of the invention, the term “randomly distributed” refers to the fact that the pores have not been actively aligned and do not follow any designed pattern of orientation to each other. There is no particular limitation in the percentage of porosity of the porous self-standing homogeneous hydrogel of the invention. In a embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the self-standing hydrogel as defined herein wherein the porosity of the material ranges from 65 to 83%. The porosity of the self-standing hydrogel is measured as described below in the examples.

[0093] In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the porosity is calculated by the following equation:

[0094] Ws — Wd

[0095] %p = x 100 p H2O x Vs wherein:

[0096] Wsis the weight of the swollen gel in water (in mg) measured after immersing the hydrogel in water for at least 72 h and removing the excess water,

[0097] Wd is the weight of the dry gel (in mg) measured after placing the swollen gel at -20 °C for 24 h, then at -80 °C and lyophilizing it until the gel does not contain any water and the weight is constant (i.e. , when the difference between two consecutive weightings is equal to or lower than 0.50 mg per g of substance taken), p is the density of water (in mg / mm3), and

[0098] Vsis the volume of the swollen gel in water (in mm3) measured after immersing it in water for at least 72 h and removing the excess water.

[0099] In particular, Ws, Wd, and Vscan be determined by: a) providing a disc of the porous self-standing homogeneous hydrogel; b) immersing the disc in water for at least 72 h and removing the excess water; c) weighing the disc (swelling weight, Ws, in mg), and measuring its thickness (swelling height, hs), and diameter (swelling diameter, ds, in mm) using a digital micrometer; d) calculating the swelling volume (Vs, in mm3) according to the equation Vs=TT rS2 hs, wherein rs is the radius (1 / 2 ds, in mm); e) placing the disc at -20 °C for 24 h, then at -80 °C and lyophilizing it until the gel does not contain any water and the weight is constant (i.e., when the difference between two consecutive weightings is equal to or lower than 0.50 mg per g of substance taken); and f) weighing the disc (dry weight, Wd, in mg).

[0100] The porous self-standing hydrogels may have different thickness values. For the purposes of the invention, the term thickness refers to the measure of the extent or distance between two opposite surfaces of the material in a direction perpendicular to those surfaces. It is a dimension that describes how thick or thin an object or material is in a given direction.

[0101] Thus, in an embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the selfstanding hydrogel as defined above wherein the thickness of the material is equal to or higher than 100 pm, more particularly equal to or higher than 200 pm, even more particularly equal to or higher than 500 pm. The thickness of the self-standing hydrogel is measured by an accurate digital micrometer.

[0102] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the self-standing hydrogel is in the form of a film, more particularly having a thickness in a range from 10 to 500 pm. More particularly, the self-standing homogeneous hydrogels of this embodiment are PIM hydrogels, obtainable by phase inversion methods.

[0103] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the self-standing homogeneous hydrogel as defined herein is crosslinked. More particularly, the selfstanding homogeneous hydrogels of this embodiment are PBCLC hydrogels, obtainable by using a crosslinking agent.

[0104] The self-standing homogeneous hydrogels of the invention may be coated to provide better cell attachment. According to one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the hydrogel further comprises a coating, particularly a coating for improving cell attachment. There is no particular limitation in the coatings that can be used as for this purpose for the material of the present invention. For example, the coating may comprise hyaluronic acid (HA), Poly-dopamine (PDA), poly-D-Lysine (PDL), extracellular matrix (ECM) proteins, fibronectin, collagen IV, laminins, vitronectin, or mixtures thereof. The material may also be coated with Matrigel®, Geltrex™ Matrix (gelatinous protein mixtures secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells) or

[0105] In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the hydrogel of the invention further comprises a coating, particularly selected from Poly-dopamine (PDA), poly-D-Lysine (PDL), and mixtures thereof, more particularly the coating comprises PDL. The present invention also relates to porous self-standing homogeneous hydrogels designated as PIM hydrogels which are obtainable by phase inversion methods.

[0106] In particular, the present invention also relates in a third aspect to a process for the preparation of the porous self-standing homogeneous hydrogel as defined herein, in particular a PIM hydrogel, which comprises the steps of: a1) providing an aqueous solution comprising poly(vinyl alcohol); b1) providing an aqueous dispersion comprising carbon nanostructures; c1) mixing the solution of step a1) and the dispersion of step b1) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures, wherein the amount of carbon nanostructures in the obtained dispersion is from 7 to 85% by weight, particularly from 15 to 85% by weight, more particularly from 18 to 70% by weight, with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; d1) spreading an amount of the dispersion of step c1) onto a support; e1) immersing the support with the dispersion of step d1) into a non-solvent to induce phase inversion and obtain a supported gel; f1) removing the supported gel from the non-solvent; g1) if desired, repeating steps d1), e1) and f1) one or more times until obtaining the desired gel thickness, provided that when step d1) is repeated the dispersion of step c1) is spread onto the gel on the support obtained in the previous step; hi) immersing the supported gel obtained in the previous step into an aqueous solution to obtain a supported hydrogel; and i1) removing the supported hydrogel from the aqueous solution, and separating the hydrogel from the support to obtain a self-standing hydrogel, or alternatively, iT) separating the hydrogel from the support to obtain a self-standing hydrogel, and removing the self-standing hydrogel from the aqueous solution.

[0107] More particularly, it also forms part of the invention a porous self-standing homogeneous hydrogel as defined herein, in particular a PIM hydrogel, more particularly a porous selfstanding homogeneous hydrogel comprising poly(vinyl alcohol) and carbon nanostructures wherein: a) the carbon nanostructures are present in an amount from 15 to 85% by weight, more particularly from 18 to 70% by weight, with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; and b) the pores have an average size equal to or higher than 8 pm; which is obtainable by a process which comprises: a1) providing an aqueous solution comprising poly(vinyl alcohol); b1) providing an aqueous dispersion comprising carbon nanostructures; c1) mixing the solution of step a1) and the dispersion of step b1) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures, wherein the amount of carbon nanostructures in the obtained dispersion is from 7 to 85% by weight, particularly from 15 to 85% by weight, more particularly from 18 to 70% by weight, with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; d1) spreading an amount of the dispersion of step c1) onto a support; e1) immersing the support with the dispersion of step d1) into a non-solvent to induce phase inversion and obtain a supported gel; f1) removing the supported gel from the non-solvent; g1) if desired, repeating steps d1), e1) and f1) one or more times until obtaining the desired gel thickness, provided that when step d1) is repeated the dispersion of step c1) is spread onto the gel on the support obtained in the previous step; hi) immersing the supported gel obtained in the previous step into an aqueous solution to obtain a supported hydrogel; and i1) removing the supported hydrogel from the aqueous solution and separating the hydrogel from the support to obtain a self-standing hydrogel, or alternatively, iT) separating the hydrogel from the support to obtain a self-standing hydrogel and removing the self-standing hydrogel from the aqueous solution.

[0108] In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the process further comprises a step after step j1) of functionalizing or coating the self-standing hydrogel with one or more agents to promote cell attachment.

[0109] Step c1) comprises mixing the solution of step a1) and the dispersion of step b1) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures. In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step c1) is carried out at a temperature from 80 to 100 °C, particularly from 90 to 98 °C.

[0110] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step c1) is carried out for a period of time from 0.5 to 3 hours, particularly from 1 to 2 hours.

[0111] Step d1) comprises spreading the dispersion of step c1) onto a support. In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step d1) comprises spreading the dispersion of step c1) in an amount from 0.5 to 3 mL, particularly from 1 to 2 mL, onto the support.

[0112] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the support used in step d1) is non-treated glass.

[0113] Step e1) involves a phase inversion method in which a non-solvent is used. As a result of this step a gel on the support is obtained, i.e. , a supported gel. In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the non-solvent is a polar or apolar organic solvent, more particularly water miscible. More particularly, the non-solvent is acetone, dimethylformamide, acetonitrile, glycerol, or combinations thereof.

[0114] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step e1) is carried out at a temperature from 15 to 30 °C, particularly from 20 to 25 °C.

[0115] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step e1) is carried out for a period of time from 2 to 72 hours, particularly from 16 to 24 hours.

[0116] Step f1) comprises removing the supported gel from the non-solvent. In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, steps d 1 ) , e1) and f1) are repeated until the thickness of the gel is equal to or higher than 500 pm.

[0117] Step hi) comprises immersing the gel supported onto the support obtained in the previous step into an aqueous solution, particularly water, to obtain a supported hydrogel. In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step hi) is carried out at a temperature from 20 to 50 °C, particularly from 30 to 45 °C.

[0118] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step hi) is carried out for a period of time from 2 to 48 hours, particularly from 16 to 24 hours.

[0119] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the self-standing hydrogel is obtained by removing the supported hydrogel from the aqueous solution, particularly water, and then separating the hydrogel from the support (e.g., by peeling of the hydrogel) (step i1).

[0120] Alternatively, in another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the self-standing hydrogel is obtained by separating the hydrogel from the support (e.g., by peeling of the hydrogel) in the aqueous solution, particularly water, to obtain a selfstanding hydrogel and then removing it from the aqueous solution, particularly water (step iT).

[0121] As mentioned above, the self-standing homogeneous hydrogels of the present invention may be molded into or onto devices such as hollow tube-like structures or nerve-like structures by utilizing a hollow capillary tube. These materials with specific geometry can be prepared by using the process above comprising steps a1)-c1) and after step c1) molding the mixture into a device of interest having the desired geometry by dipping the device into the PVA / CNT mixture, particularly warm, followed by immersion in a nonsolvent to induce phase inversion.

[0122] There is no limitation on the devices that may be used for molding the self-standing homogeneous hydrogels into a particular geometry or shape. Thus, such devices include any rigid and solvent resistant materials such as glass, stainless steel, silicone, and the like.

[0123] Thus, in one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the invention relates to a process for the preparation of a device comprising the porous selfstanding homogeneous hydrogel, in particular a PIM hydrogel, as defined in the second aspect which comprises the steps of: a1) providing an aqueous solution comprising poly(vinyl alcohol); b1) providing an aqueous dispersion comprising carbon nanostructures; c1) mixing the solution of step a1) and the dispersion of step b1) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures, wherein the amount of carbon nanostructures in the obtained dispersion is from 7 to 85% by weight, particularly from 15 to 85% by weight, more particularly from 18 to 70% by weight, with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; dT) dipping a device into the mixture of step c1) e1 ’) immersing the device with the dispersion of step cT) into a non-solvent to induce phase inversion; fT) removing the device comprising the gel from the non-solvent; hT) immersing the device comprising the gel obtained in the previous step into an aqueous solution to obtain a device comprising the hydrogel; and iT) removing the device comprising the hydrogel from the aqueous solution.

[0124] More particularly, it also forms part of the invention a device comprising the porous selfstanding homogeneous hydrogel, in particular a PIM hydrogel, more particularly wherein the porous self-standing homogeneous hydrogel comprises poly(vinyl alcohol) and carbon nanostructures wherein: a) the carbon nanostructures are present in an amount from 15 to 85% by weight, more particularly from 18 to 70% by weight, with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; and b) the pores have an average size equal to or higher than 8 pm; which is obtainable by a process which comprises: a1) providing an aqueous solution comprising poly(vinyl alcohol); b1) providing an aqueous dispersion comprising carbon nanostructures; c1) mixing the solution of step a1) and the dispersion of step b1) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures, wherein the amount of carbon nanostructures in the obtained dispersion is from 7 to 85% by weight, particularly from 15 to 85% by weight, more particularly from 18 to 70% by weight, with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; d1 ’) dipping a device into the mixture of step c1) eT) immersing the device with the dispersion of step cT) into a non-solvent to induce phase inversion; fT) removing the device comprising the gel from the non-solvent; hT) immersing the device comprising the gel obtained in the previous step into an aqueous solution to obtain a device comprising the hydrogel; and iT) removing the device comprising the hydrogel from the aqueous solution.

[0125] In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the device is a hollow capillary tube.

[0126] The invention also relates in a fourth aspect to a process for the preparation of the porous self-standing homogeneous hydrogel as defined herein, in particular a PIM hydrogel, which comprises the steps of: a2) providing an aqueous solution comprising poly(vinyl alcohol); b2) providing an aqueous dispersion comprising carbon nanostructures; c2) mixing the solution of step a2) and the dispersion of step b2) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures, wherein the amount of carbon nanostructures in the obtained dispersion is from 7 to 85%, particularly from 15 to 85% by weight, more particularly from 18 to 70%, by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; d2) adding a porogenic agent to the dispersion of step c2) to obtain a mouldable mass; e2) moulding the mouldable mass obtained in d2) into a moulded mass; f2) soaking the moulded mass obtained in step e2) in a non-solvent to induce phase inversion and obtain a self-standing gel; g2) removing the self-standing gel from the non-solvent; h2) immersing the self-standing gel obtained in step g2) into an aqueous solution to obtain a self-standing hydrogel; i2) heating the aqueous solution of step h2) to a temperature equal to or higher than 35 °C to remove the porogenic agent and obtain a porous self-standing hydrogel; and j2) removing the self-standing hydrogel from the aqueous solution.

[0127] More particularly, it also forms part of the invention a porous self-standing homogeneous hydrogel as defined herein, in particular a PIM hydrogel, more particularly a porous selfstanding homogeneous hydrogel comprising poly(vinyl alcohol) and carbon nanostructures wherein: a) the carbon nanostructures are present in an amount from 15 to 85% by weight, more particularly from 18 to 70% by weight, with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; and b) the pores have an average size equal to or higher than 8 pm; which is obtainable by a process which comprises: a2) providing an aqueous solution comprising poly(vinyl alcohol); b2) providing an aqueous dispersion comprising carbon nanostructures; c2) mixing the solution of step a2) and the dispersion of step b2) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures, wherein the amount of carbon nanostructures in the obtained dispersion is from 7 to 85% by weight, particularly from 15 to 85% by weight, more particularly from 18 to 70%, by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; d2) adding a porogenic agent to the dispersion of step c2) to obtain a mouldable mass; e2) moulding the mouldable mass obtained in d2) into a moulded mass; f2) soaking the moulded mass obtained in step e2) in a non-solvent to induce phase inversion and obtain a self-standing gel; g2) removing the self-standing gel from the non-solvent; h2) immersing the self-standing gel obtained in step g2) into an aqueous solution to obtain a self-standing hydrogel; i2) heating the aqueous solution of step h2) to a temperature equal to or higher than 35 °C to remove the porogenic agent and obtain a porous self-standing hydrogel; and j2) removing the self-standing hydrogel from the aqueous solution.

[0128] Step c2) comprises mixing the solution of step a2) and the dispersion of step b2) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures. In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step c2) is carried out at a temperature from 80 to 100 °C, particularly from 90 to 98 °C.

[0129] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step c2) is carried out for a period of time from 0.5 to 3 hours, particularly from 1 to 2 hours.

[0130] Step d2) comprising adding a porogenic agent to the dispersion of step c) to obtain a mouldable solid may be carried out by using any suitable porogenic agent. Porogenic agents are non-soluble in water at room temperature and typically have a micrometric particle size.

[0131] Non limiting examples of porogenic agents that can be used include gelatin, polystyrene, or Tetrabutylammonium hexafluorophosphate (TBAPF6). In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the porogenic agent is gelatin.

[0132] By using the appropriate porogenic agent the total porosity and the pore size in the hydrogel of the invention can be tailored and controlled. Generally speaking, the higher the average particle size of the porogenic agent, the higher the pore size in the hydrogel, and the higher the amount of porogenic agent that is added, the higher the porosity in the hydrogel.

[0133] In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the porogenic agent has an average particle size from 500 nm to 800 pm, more particularly in the range from 20 to 500 pm, more particularly from 100 to 250 pm. In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the porogenic agent is added in an amount from 10 to 300 mg, particularly from 50 to 260 mg, with respect to each 1mL of PVA / CNT dispersion.

[0134] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step d2) is carried out at a temperature from 15 to 30 °C, particularly from 20 to 25 °C.

[0135] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step d2) is carried out for a period of time from 0.5 to 2 minutes, particularly from 0.5 to 1 minute.

[0136] Step e2) comprising the moulding the mouldable mass obtained in d2) into a moulded mass may be carried out by using a syringe or a mold template.

[0137] Step f2) involves a phase inversion method in which the moulded mass obtained in step e2) is soaked in a non-solvent is used. As a result, a self-standing gel is obtained.

[0138] In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the non-solvent is an a polar or apolar organic solvent, more particularly water miscible. More particularly, the non-solvent is acetone, dimethylformamide, acetonitrile, glycerol, or combinations thereof.

[0139] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step f2) is carried out at a temperature from 15 to 30 °C, particularly from 20 to 25 °C.

[0140] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step f2) is carried out for a period of time from 5 to 72 hours, particularly from 16 to 24 hours.

[0141] Step g2) comprises removing the self-standing gel from the non-solvent.

[0142] Step h2) comprises immersing the self-standing gel obtained in step g2) into an aqueous solution, particularly water, to obtain a hydrogel. In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step h2) is carried out at a temperature from 15 to 50 °C, particularly from 40 to 45 °C. Generally speaking, the higher the temperature, the less time will be needed to obtain a porous self-standing hydrogel.

[0143] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step h2) is carried out for a period of time from 24 to 96 hours, particularly from 72 to 96 hours.

[0144] Step i2) comprises heating to a temperature equal to or higher than 35 °C to remove the porogenic agent and obtain a porous self-standing hydrogel. Finally, step j2) comprises removing the self-standing hydrogel from the aqueous solution.

[0145] The invention also relates in a fifth aspect to a process for the preparation of the porous self-standing homogeneous hydrogel as defined herein, in particular a PBCLC hydrogel, more particularly a porous self-standing homogeneous hydrogel comprising poly(vinyl alcohol) and carbon nanostructures wherein: a) the carbon nanostructures are present in an amount from 7 to 18% by weight, more particularly from 8 to 12% by weight, with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; and b) the pores have an average size equal to or higher than 8 pm; which comprises the steps of: a3) providing an aqueous solution comprising poly(vinyl alcohol); b3) providing an aqueous dispersion comprising carbon nanostructures; c3) mixing the solution of step a3) and the dispersion of step b3) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures, wherein the amount of carbon nanostructures in the obtained dispersion is from 7 to 85% by weight, particularly from 7 to 18% by weight, more particularly from 8 to 12%, by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; and d3) adding a crosslinking agent and heating at a temperature from 80 to 120 °C; e3) cooling to a temperature equal to or lower than 60 °C to obtain a porous self-standing hydrogel.

[0146] The present invention also relates to porous self-standing homogeneous hydrogels designated as PBCLC hydrogels which are obtainable by using a crosslinking agent.

[0147] It also forms part of the invention a porous self-standing homogeneous hydrogel as defined herein, in particular a PBCLC hydrogel, which is obtainable by a process which comprises: a3) providing an aqueous solution comprising poly(vinyl alcohol); b3) providing an aqueous dispersion comprising carbon nanostructures; c3) mixing the solution of step a3) and the dispersion of step b3) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures, wherein the amount of carbon nanostructures in the obtained dispersion is from 7 to 85% by weight, particularly from 7 to 18% by weight, more particularly from 8 to 12% by weight, with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; and d3) adding a crosslinking agent and heating at a temperature from 80 to 120 °C; e3) cooling to a temperature equal to or lower than 60 °C to obtain a porous self-standing hydrogel.

[0148] Step c3) comprises mixing the solution of step a3) and the dispersion of step b3) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures. In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step c3) is carried out at a temperature from 80 to 100 °C, particularly from 90 to 98 °C.

[0149] In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the amount of the aqueous solution, particularly water present in the dispersion of step c3) is such that it is absorbed in the porous self-standing hydrogel obtained in step e3).

[0150] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step c3) is carried out at a temperature from 80 to 100 °C, particularly from 90 to 98 °C.

[0151] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step c3) is carried out for a period of time from 0.5 to 3 hours, particularly from 1 to 2 hours.

[0152] Step d3) comprising adding a crosslinking agent and heating at a temperature from 80 to 120 °C may be carried out by any suitable cross-linking agent, including phenolic acids or aldehydes. In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the crosslinking agent is 4-amino benzoic acid (4ABA). To the knowledge of the inventors 4ABA has never been used before for this purpose. This molecule has the advantage that is biocompatible and does not induce toxicity. Step e3) cooling to a temperature equal to or lower than 60 °C to obtain a porous selfstanding hydrogel. In one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step e3) is carried out at a temperature equal to or lower than 50 °C, equal to or lower than 40 °C, equal to or lower than 30 °C, equal to or lower than 20 °C, equal to or lower than 10 °C, or equal to or lower than 5 °C. In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step e3) is carried out by cooling to a temperature at about room temperature (20-25 °C) or a temperature equal to or lower than 4 °C. Generally speaking, the lower the temperature, the less time will be needed to obtain a porous self-standing hydrogel.

[0153] When carrying out step e3) the formed self-standing hydrogel takes the shape of the recipient. Thus, the self-standing hydrogel may be molded by performing step e3) in a mold of a desired shape.

[0154] The obtained porous self-standing hydrogel obtainable by using a crosslinking agent is thermosensitive, which means that it can be molded into any desired shape by heating it. Hence, the self-standing hydrogel may be later molded in another desired shape by heating the self-standing hydrogel obtained in step e3) at a temperature ranging from 65 to 100°C, molding the hydrogel and cooling to a temperature equal to or lower than 60 °C, particularly at a temperature at about room temperature (20-25 °C) or a temperature equal to or lower than 4 °C.

[0155] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the crosslinking agent is added in an amount from 0.5 to 2.5 % w / v, particularly from 1 to 1.5 % w / v, with respect to the aqueous solution, particularly water.

[0156] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step d3) is carried out at a temperature from 80 to 120 °C, particularly from 100 to 120 °C.

[0157] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, step d3) is carried out for a period of time from 1 to 6 hours, particularly from 3 to 4 hours.

[0158] The self-standing hydrogels of the invention may be stored under wet or dry conditions. According to one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the selfstanding hydrogel is kept immersed in an aqueous solution, such as water or Phosphate buffered saline (PBS) buffer solution, until use. To keep the hydrogel in water has the advantage that the biological properties are maintained. In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, a bacteriostatic preservative such as sodium azide may be optionally added to the aqueous solution.

[0159] Alternatively, according to another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the water of the self-standing hydrogel is removed such to obtain an aerogel. More particularly, the aerogel is obtained by freeze-drying the self-standing hydrogel under appropriate conditions. To store the gel as a dried material has the advantage that the biological properties are maintained. When the material is to be used, the aerogel may be immersed in an aqueous solution, such as water or Phosphate buffered saline (PBS) buffer solution, to recover the self-standing hydrogel.

[0160] Thus, it also forms part of the invention a porous self-standing homogeneous aerogel comprising poly(vinyl alcohol) and carbon nanostructures, wherein: a) the carbon nanostructures are present in an amount from 7 to 85% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; and b) the pores have an average size equal to or higher than 8 pm.

[0161] All embodiments of the porous self-standing homogeneous hydrogel as defined herein also apply to the porous self-standing homogeneous aerogel.

[0162] In a sixth aspect, the present invention relates to a neural implant made of the porous selfstanding homogeneous hydrogel as defined herein. In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the neural implant is an electrically conductive implant.

[0163] The neural implant may be used in signaling, for detecting, transmitting, or monitoring electrical signals to the brain. It may also be used in the treatment of CNS damage in a subject in need thereof. The expression “CNS damage” refers to partial or total loss of CNS function, including loss of sensory or of motor function, or both depending on the precise nature of the injury.

[0164] Thus, the invention also relates to the use of the neural implant as defined herein for use in treating CNS damage in a subject in need thereof. This aspect may also be formulated as a method for the treatment of CNS damage in a subject in need thereof which comprises applying a neural implant to the subject. It also forms part of the invention the porous self-standing homogeneous hydrogel as defined herein for the manufacture of the neural implant as defined herein for the treatment of CNS damage in a subject in need thereof.

[0165] According to one embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the CNS damage is associated to a condition or disorder selected from acute trauma, contusion, stroke, spinal cord injury, surgery, spinal cord cancer, and CNS chronic disease.

[0166] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the treatment comprises regenerating neural cells in or around a site of CNS damage, neural reconnection, or alternatively at least partially restoring sensory or motor function.

[0167] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the CNS chronic disease is selected from the group consisting of multiple sclerosis, Amyotrophic Lateral Sclerosis (ALS), Huntington's Disease, and Alzheimer's disease.

[0168] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below throughout all the description, the treatment comprises nerve regeneration, more particularly wherein the nerve regeneration is carried out by promoting neuronal growth.

[0169] Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word “comprise” encompasses the case of “consisting of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

[0170] Examples

[0171] 1. Materials

[0172] Polyvinyl Alcohol (MW 146000 - 186000, 99+% hydrolyzed) CAS 9002-89-5 and 4-Amino benzoic acid (PABA, 4ABA, Vitamin H1) CAS 150-13-0 were bought from Sigma Aldrich.

[0173] Multi-wall Carbon Nanotubes (MWCNT) (Outer Diameter: 20-30 nm, Length: 0.5 - 2 pm, Purity: >98+5, CAS 99685-96-8) were obtained from Nanostructured & Amorphous Materials, Inc. - USA and used without further treatment. LDH CytoTox 96® NonRadioactive Cytotoxicity Assay was bought from Promega. Phosphate buffered saline (PBS) buffer solution (1X) and Dulbecco’s Modified Eagle Medium (DMEM) were obtained from Gibco, Invitrogen, CA. Gelatin powder from porcine skin (CAS 9000-70-8, solubility 50mg / ml) was bought from Sigma Aldrich.

[0174] 2. Characterization methods

[0175] 2.1 Thermogravimetry Analysis (TGA)

[0176] TGA analysis was carried out under nitrogen. As CNT is mainly stable up to 600 °C, a temperature where the PVA has already been degraded, the amount of PVA degraded and the amount of CNT remaining was calculated at 500 °C, taking into account that the PVA at 500 °C has a residue of 14% (4ABA degrades below 230 °C).

[0177] 2.2 Conductivity and sheet resistance

[0178] Conductivity and sheet resistance were measured using a 4 point-probe (4PP) system. PIM gels were cut into 8 mm diameter disks and placed on a glass slide for measurements a minimum of 3 replicas of each gel were measured and reported. In the case of PBCLC hydrogels were soaked in water or PBS.

[0179] 2.3 Swelling a) PIM or PBCLC discs of 8 mm in diameter were immersed in Mill iQ water after synthesis for at least 48 h (for PIMs) or 72 h (for PBCLCs) to reach equilibrium. b) The excess water was removed using absorbing paper. Then, the discs were weighed (swelling weight, Ws, in mg), and their thickness (swelling height, hs), and diameter (swelling diameter, ds, in mm) of the discs were measured using an accurate digital micrometer. The swelling volume (Vs, in mm3) was calculated according to the equation Vs=TT rs2hs, wherein rsis the radius (1 / 2 ds, in mm). c) The scaffolds were placed at -20 °C for 24 h, then at -80 °C overnight and then lyophilized using an Alpha 2-4 LSCpIus part no. 102142 at a condenser temperature between -80 °C and -92 °C with a vacuum of 0.16 Pa. When the weight was constant (i.e., when the difference between two consecutive weightings is equal to or lower than 0.50 mg per g of substance taken), the weight (dry weight, Wd, in mg), thickness (dry height, hd), and diameter (dry diameter, d , in mm) of the discs were measured using an accurate digital micrometer. The dry volume (Vd, in mm3) was calculated according to the equation Vd=TT rd2hd, wherein rd is the radius (1 / 2-dd, in mm). d) The swelling behavior (either swelling by weight or swelling mass Sw%, or swelling by volume Sv%) was then calculated according to the following equations:

[0180] Ws — Wd Vs - Vd

[0181] Sw% = - - — x 100 Sv% = - - — x 100

[0182] Wd Vd

[0183] 2.4 Porosity

[0184] The porosity was calculated using the following protocol: a) PIM or PBCLC discs of 8 mm were first placed at -20 °C for 24 h, then at -80 °C overnight and lyophilized using an Alpha 2-4 LSCpIus part no. 102142 at a condenser temperature between -80°C and -92°C with a vacuum of 00.16 Pa. When the weight was constant (i.e. , when the difference between two consecutive weightings is equal to or lower than 0.50 mg per g of substance taken), the discs were weighed (dry weight, Wd, in mg) was measured. b) The discs were soaked in water for 24 h to reach equilibrium. The excess water was removed using absorbing paper. Then, the discs were weighed (swelling weight, Ws, in mg), and their thickness (swelling height, hs), and diameter (swelling diameter, ds, in mm) of the discs were measured using an accurate digital micrometer. The swelling volume (Vs, in mm3) was calculated according to the equation Vs=TT rs2hs, wherein rsis the radius

[0185] (1 / 2 ds, in mm). c) The porosity (p) was then calculated using the following equation:

[0186] Ws — Wd

[0187] %p = x 100 p H2O x Vs wherein Ws, Wd, and Vsare as defined above, and p is the density of water (in mg / mm3).

[0188] 2.5 Pore size (measured by Scanning Electron Microscopy, SEM)

[0189] For measuring the pore size, PIM or PBCLC scaffolds were placed at -20 °C for 24 h, then at -80 °C overnight, and finally, lyophilized. Then, the pore size was measured from SEM imaging (dried scaffold pore size, in mm). Since after lyophilization, the volume of the gels decreased noticeably, a correction factor based on the swelling by volume (1+ (Sv% / 100)) was applied to calculate the pore size. Thus, the pore size was calculated by the following equation: pore size (pm) = [dried scaffold pore size] ■ (1+ (Sv% / 100)). 2.6 Young’s modulus

[0190] Compression tests were performed using a universal testing machine where a strainstress curve is plotted. Tensile modulus is calculated taking the slope of the first exponential increase of the Strain-Stress.

[0191] 3. Fabrication of PVA / CNT 3D scaffolds and Nerve Guidance Conduits using Phase Inversion Method (PIM)

[0192] 20% of PVA solution was prepared by dissolving 500 mg of PVA in 2.5 mL of MilliQ water (Millipore, USA) and incubating at 80 °C for at least 30 minutes. A specified amount of MWCNT (from 20% - 75% w / w with reference to PVA weight) was dispersed in 2.5 mL of water with sonication in a bath for 20 minutes and mixed into the PVA Solution to dilute it to a final concentration of 10% (w / v). The dispersion was kept at near boiling point until PVA was completely solubilized (at least 3 hours).

[0193] Following the above procedure, the following dispersions shown in table 1 were obtained (the proportion (w / w) of CNTs and PVA are with respect to the sum CNTs+PVA):

[0194] Table 1

[0195] Dispersion Mass CNT (in .. . Mass PVA (in .. . .. .

[0196] . . r- , % CNT (w / w)e' % PVA (w / w) % PVA (w / v) label 5mL MilliQ) 5mL MilliQ)

[0197] 75%CNT 1500 mg 75% 500 mg 25% 10%

[0198] 70%CNT 1200 mg 70% 500 mg 30% 10%

[0199] 60%CNT 750 mg 60% 500 mg 40% 10%

[0200] 50%CNT 500 mg 50% 500 mg 50% 10%

[0201] 40%CNT 340 mg 40% 500 mg 60% 10%

[0202] 33%CNT 220 mg 33% 500 mg 67% 10%

[0203] 20%CNT 130 mg 20% 500 mg 80% 10%

[0204] 3.1 Layering Method

[0205] On a clean glass slide, around 1.5 mL of the PVA / CNT dispersion as prepared above was smeared with the help of another clean glass slide. The glass slide was submerged in a Pyrex® Circular Glass Dish containing acetone, making sure it was completely horizontal to avoid a non-homogeneous surface for 10-20 minutes. Afterwards, the glass slide was removed from the acetone and air dried for 30 seconds. For the second layer, 1.5 mL of the PVA / CNT dispersion as mentioned above was added onto the first layer and smeared homogeneously. Then, it was submerged in acetone contained in a Pyrex® Circular Glass Dish making sure it was completely horizontal. It was left overnight. Once the phase inversion was completed, the glass slide was removed from the acetone and then, it was submerged in a glass dish containing ultrapure water at a temperature between 25-37 °C for 2 hours. Then, the porous dry gel film formed onto the glass was peeled gently and left for (at least) another 3 hours. After this procedure a hydrogel with a thickness of ca. 500 pm was obtained.

[0206] Following the above procedure, the following materials shown in table 2 (and designated as PIMs) were obtained:

[0207] Table 2

[0208] Material PIMcontrol PIM20 PIM33 PIM44 PIM50 PIM60 PIM70PIM75 o%18.2%27 5%34. "I % 48.4% 54% 62.1%

[0209] % CNT (w / w)a 69%aCalculated amount of CNT and PVA at 500 °C From TGA plot, considering the 14% of PVA residue, machine error ± 5%.

[0210] Several parameters were measured for PIM20, PIM50, and PIM75:

[0211] Table 3

[0212] .. . . Conductivity Young’s PoreD.dSwelling Swelling aena4PP (S / m)bModulus KPa sizec orosiV volumedmassd

[0213] PIM20 0.75 25417±1265% 110%450%

[0214] PIM50 1.06152

[0215] 23,03 PIM75 1.35 6623 3±21 63.8 19.79% 270.3% pmbCalculated from 4-point-probecCalculated from SEM images by the equation defined above including the correction factordCalculated from swelling analyses in water

[0216] 3.2 Dispersion and controlled porosity method

[0217] A PVA / CNT dispersion was prepared as mentioned above and was added into a 15 mL disposable plastic container. Meanwhile, a porogen (gelatin) was sieved through two precise sieves placed on top of each other. The top sieve allowed particles over 250 pm to pass through. The bottom sieve allowed particles under 100 pm to pass through. Thus, leaving in the middle all of the particles in a range from 100 to 250 pm. Afterwards, 260 mg of the porogenic material previously sieved to have a particle size in the range 100- 250 pm was mixed with 1 mL of the PVA / CNT dispersion until a homogeneous and mouldable mass (paste-like material) was formed. The paste-like material was fed to a syringe with its end cut. Then, to mould the mouldable material into specific cylinder shape, the blunt end of the syringe was placed on a flat surface and the material was pushed against the surface firmly, resulting in a disk-like shape of around 0.5 cm thickness and 2 cm diameter (which can be changed as desired). The moulded material was submerged in a glass dish containing acetone. The material underwent phase inversion for 24 hours. Then, the obtained moulded, self-standing scaffold was rehydrated in ultrapure water for over 5 hours at a temperature of 35-40 °C and the gelatin was removed.

[0218] Following the above procedure, the following materials shown in table 3 (and designated as PIMs) were obtained:

[0219] Table 4

[0220] Material % CNT (w / w)aConductivity (mS / m)bPores'ze<:

[0221] GPPIMcntrl 0 - 225.57± 79.88 pm

[0222] GPPIM50 50 14 - 9000 232.47± 39.32 pmaCalculated amount of CNT and PVA at 500 °C From TGA plot, considering the 14% of PVA residue, machine error ± 5%.bCalculated from 4-point-probe.cCalculated from SEM images by the equation defined above.

[0223] 3.3 Optimization for in vitro studies

[0224] To support growth of cells on PIMs, the scaffolds were coated with various agents to promote cell attachment. For this purpose, 0.1 mg / mL aqueous solution of Poly-D-Lysine (PDL) was used. Scaffolds PIM control, PIM50, and PIM75 were coated with PDL according to the following procedure. The scaffolds were placed at -20 °C for 24 h, then at -80 °C overnight, and finally, lyophilized as described in section 2.3. Each scaffold was incubated in a 2 - 3 mL PDL, depending on size, for 48 hours at 60 °C followed by another lyophilization step (as explained above) prior to cell studies in the presence of excess PDL on the surface (this increased the content of PDL which will in turn maximise cellular attachment and will allow cells to infiltrate).

[0225] 3.4. Preparation of a nerve-like structure

[0226] To obtain a hollow like structure, or nerve-like structure, a glass capillary tube was used. Firstly, a mixture of PVA / CNT was prepared as described above, while the mixture was still warm, the glass capillary tube was dipped in the mixture for few seconds allowing the mixture to travel on the inside of the capillary walls, and coating the outside walls, the excess mixture was evened out by gently passing the capillary over a flat surface until a homogenous thickness was obtained. The as-prepared molded mixture was directly dipped in acetone (non-solvent) and eventually was left overnight to undergo phase inversion, the capillary with the formed scaffold on the surface is hydrated with water for at least 5 hours at 37 °C - 45 °C. thus obtaining, a thread-like structure in the inner part of the glass capillary tube, and a hollow tubular structure on the outside walls. 4. Fabrication of dynamically crosslinked PVA / CNT 3D Hydrogels (PBCLC) PVA / CNT dispersions were prepared by dissolving specified amounts of PVA and MWCNT in milliQ water until complete dissolution of PVA (at 95 °C for 1 - 2 hours). An aqueous solution of ABA was added to the PVA / CNT dispersion to reach a final concentration of 1.5% w / v of 4ABA, and the resulting dispersion was mixed under reflux and left under reflux for 3 hours with occasional vortexing (it is not necessary to dissolve PVA under reflux but as soon as the 4ABA was added the mixture was set under reflux). The mixture was cooled down and left at 4 °C for an average time of 72 hours for 5 mL of PBCLC hydrogels. Longer incubation periods are generally necessary to achieve complete crosslinking when larger amounts of gel are prepared or higher concentrations of CNT used.

[0227] Following the above procedure, the following materials, designated as PBCLC, shown in table 3 were obtained:

[0228] Table 5

[0229] Expen- _ . .. x . .. ConduCtlV _ ... u / Pllina

[0230] .. . . . mental % .. . Young’s .dr>eSwelling swelling

[0231] Material ity by 4PP .. . . ' ;lhModu usrc Pore size0Porosity 36. „ macc

[0232] CNT vo ume6masse

[0233] . , .aa (S / m) (w / w)b

[0234] PBCLC10 8% 2.5 57.8 kPa 10±8 pim 73% 28%600%

[0235] PBCLC7.5 10.8% 4.2 14.7 kPa 11 ±7 pim 66.5% 20%750%

[0236] PBCLC5 10% 2 7.7 kPa 12±8 pirn 83% 38% 1100%

[0237] 40 698±55 Knno /

[0238] PBCLC10.50 56.5% 0.736 1166 KPa „„„ 60% 5%

[0239] 913 pirn500%aCalculated amount of CNT and PVA at 500°C From TGA plot, considering the 14% of PVA residue.bCalculated from 4-point-probe (section 2.2)cCalculated from compression tests (section 2.8)dCalculated from SEM images (section 2.7)eCalculated from swelling analyses (section 2.6)

[0240] 4.1 Optimization for in-vitro studies

[0241] To modulate the pH and neutralize the gels PBCLC5, PBCLC7.5, and PBCLC10, an optimization procedure for in vitro studies was developed which involved soaking PBCLC overnight in a non-solvent. The gels were thus soaked overnight in an alkaline solution (10 mM NaOH) at room temperature. The gels were then lyophilized and coated with PDL as explained in section 3.2.

[0242] 5. Methods and Methodology for in-vitro cell studies

[0243] 5.1 Cell studies

[0244] Below, the results obtained using PVA / CNT with different CNT percentages (PIM60, PIM70, PIM75) are reported, showing the tendency of cells to form longer axons and better proliferation in the presence of higher MWCNT concentration (seeding density (SD) = 2 million cells / cm2, 6 mm scaffolds as discs, I Bl DI 8 pwell flask I P96, non-tissue cultured).

[0245] 5.1 .1 .Cell seeding protocol to study the effect of CNT on cell attachment and proliferation (using PIM hydrogels, non coated):

[0246] PIM60, PIM70, PIM75 scaffolds were prepared as described in the experimental section

[0247] 3.1 layering method. The scaffolds were cut to 1 cm x 1 cm and placed in an I Bl DI 8-well flask (non-tissue-culture treated). Scaffolds were treated by freezing them at -20 °C for 24 h, followed by freezing at -80 °C overnight and then lyophilizing. After which, all PIM scaffolds were UV-sterilized for 30 minutes each side before cell culture. SH-SY5Y cells were cultured to a confluency of around 80% to 90% and harvested by trypsinization followed by a washing step in PBS before using them in cell culture studies with the hydrogels. 50 to 70 pL of a 2 million cell / cm2suspension was drop casted carefully on top of the scaffolds and incubated at 37 °C for 20 minutes to maximize cell-scaffold interaction. Lastly, 350 pL of cell culture medium was added to the sides of the well taking great care not to disturb the cells on the scaffolds. After 24 hours of incubation, the scaffolds were moved to another well (preferably non-treated) and topped with fresh cell culture medium and incubated for 3 days at 37 °C, 5% CO2. The cells on the scaffolds were then fixed using 4% Paraformaldehyde (PFA) for 2 hours then carefully washed 3 times in PBS, taking great care not to disturb any cells on top of or inside the scaffolds.

[0248] 5.1.2. Cell seeding protocol to study the effect of various coatings (using PIM50)

[0249] 3 replicates of PIM50 hydrogel were placed in separated wells of I Bl DI 8 wells non-treated (glass bottom) flasks and were UV-sterilized for 20 min. I Bl DI plates were covered before removing them out of the hood. All I Bl DI plates with gels inside were placed at -20 °C overnight, then at -80 °C for at least 5-6 hours or overnight, and then lyophilized. Then the gels were coated either with PDL, PDA or a mixture PDL:PDA (50:50).

[0250] To perform the coating with PDL or PDA the gels inside the I BI DI were coated with PDL (0.1 mg / mL) by adding 1 mL of PDL or PDA over the dried scaffolds (1 reps) inside each vial, placing them at 37 °C for 48 hours. Then 2 mg / mL of dopamine in Tris-HCL buffer pH 8.5 was prepared. The above mixture (before dopamine started polymerizing) was guickly aspirated using a sterile syringe. A filter (0.2 pm) was placed over the syringe head and 1 mL of the mixture in the vial containing the scaffold (for each rep) was emptied and placed at 37 °C for 48 hours.

[0251] PDA: PDL coating was prepared as mentioned above. To perform the coating with

[0252] PDA: PDL, the scaffolds were coated at 37°C (exactly like the above-mentioned PDA coating). After 24 hours each scaffold was removed and placed in 1 mL of sterile PDL 0.1 mg / mL. The scaffolds were left in PDL solution for another 24 hours at 37 °C, to obtain a total of 48 hours of coating. The gels were removed and placed again in a sterile IBIDI in a well. The gels were lyophilized by freezing at -20 °C for 24 h followed by freezing at -80 °C overnight then lyophilizing.

[0253] 5.1.3. Cell seeding protocol to study the Effect of CNT using PIM hydrogels (coated with PDL)

[0254] PIM hydrogels were synthesized and coated as explained in section 5.1.2 with either 50% or 75% w / w of CNT. The seeding protocol was the same for all experiments following the method described in 5.1.1.

[0255] 5.1.4. Cell seeding protocol to study the effect of porosity and pore size with PBCLC and PIM hydrogels.

[0256] For PIM hydrogels, a mixture of PVA / CNT was prepared and for each 1 mL of this mixture, gelatine powder already sieved to particle size between 100 - 250 urn, was mixed in as stated in step a2 to j2 of the fourth aspect. The obtained controlled porosity PIM hydrogels were then treated with PDL as explained in section 5.1.2. the PIM gels with controlled porosity are herein referred to GPPIMXX where XX refers to the percentage of CNT by weight. The seeding protocol followed is described in section 5.1.1

[0257] For PBCLC hydrogels, a mixture of PVA / CNT crosslinked with 4ABA as described in step a3 to e3 of the fifth aspect described within this text, gelatine powder already sieved to particle size between 100 - 250 urn, was mixed in as stated in step a2 to j2 of the fourth aspect. The obtained controlled porosity PBCLC hydrogels were then optimized for in vitro cells as described in section 4.1. The PBCLC hydrogels with controlled porosity are herein referred to GPPBCLCXX where XX refers to the percentage of PVA by weight. The seeding protocol was the same for all experiments following the method described in 5.1.1.

[0258] Immunostaining for all in vitro protocols:

[0259] Fixed cells were first incubated in a blocking solution (5% Bovine serum albumin, 0.5% Triton x-100, 0.02% Sodium Azide) for 2 h. To visualize filamentous Actin, which is the protein that allows as to visualize cell wall and morphology, ActinGreen™ 488 ReadyProbes™ Reagent was used according to the manufacturer protocol and incubated overnight at 4 °C, along the incubation with ActinGreen™ , in specific cases, Bill tubulin, marker for neuronal progenitors, was checked for by incubating the samples with anti- beta-lll tubulin monoclonal antibody conjugated to Alexa Fluor 647 (1 pg mL-1 dilution, Abeam) in blocking solution as diluent. The scaffolds were then washed 3 times with PBS before adding DAPI (4',6-diamidino-2-phenylindole dihydrochloride - CAS. 28718-90-3) staining to stain the DNA of the nucleus. Lastly, the scaffolds were washed again 3 times with PBS to remove the excess DAPI.

[0260] 6. Results:

[0261] 6.1. Studying and modulating various factors of PBCLC and PIM hydrogels

[0262] All in vitro experiments were carried out using neuroblastoma cells, SH-SY5Y cells are commonly employed as models due to their rapid growth on various surfaces, offering insights into the behavior of mature neurons. In contrast, utilizing other neuronal cell lines, such as primary mature neurons, would be inappropriate for studying proliferation, given that adult neurons cease mitosis upon reaching differentiation. The advantage of employing neuroblastoma cells lies in the ability to assess their maturity and differentiation by examining factors like morphology, p-lll tubulin, cell processes length, and viability. Conseguently, data derived from studies using neuroblastoma cells can be extrapolated to mature neurons in a more cost-effective and time-efficient manner. Neuroblastoma cells are essentially cancer cells, cancer is known for the development of hard tissue associated with the uncontrolled overgrowth of a tumor. This hypothesis can further be extended to all cell types; hence, for each tissue targeted, cell growth will be highest and closest to the native tissue when grown on a material with a preset young’s modulus.

[0263] 6.1.1 Studying the effect of CNT using PIM hydrogel (non coated)

[0264] MWCNT (PIM60, PIM70, PIM75) were shown to facilitate the attachment of cells and axonal growth. For PIM-control, prepared following the layering method explained in section 3.1 above, no cell attachment was observed. PIMcontrol was used as a reference point. However, as CNT% increased the CNT-Cell interface promoted such attachment and proliferation. Additionally, at CNT% over 60%, larger migration of cells into the PIM scaffolds was observed. This resulted in the obstruction of the fluorescence of actin green by the nanotubes which proves that cells have penetrated the gels.

[0265] 6.1.2 Studying the effect of various coatings to enhance cellular attachment using PIM50 SH-SY5Y neuroblastoma cells with PDL coating showed longest axonal-like growth and highest cell attachment whereas the use of PDA resulted in shorter axonal-like growth and distorted round morphology. PDL supported the growth of neuroblastoma cells compared to the 2D control. Additionally, PDA was noticed to have higher cytotoxicity according to the preliminary results. It is worth mentioning that both sides of the scaffolds were examined with consistent results supporting the positive effect of PDL on cellular attachment were large tissue-like aggregations are formed possessing thin and long axonlike (up to 90 pm) growths as opposed to PDA coating. On the other hand, mixing a coating of PDA and PDL resulted in more cellular attachment. However cells in various parts of the scaffold showed a rounded morphology, and few axon-like growths were seen. All Optical Density (OD) results were background corrected to mitigate the effect of serum on LDH assay readings.

[0266] 6.1.3 Studying the Effect of CNT using PIM hydrogels (coated with PDL)

[0267] PIM hydrogels with a content of 60% w / w and above without any further coating cells started to attach and spread. Cellular attachment within 10 days without coating revealed the necessity to increase onset interaction between CNTs and cells in order to harness the effects of CNT. It was observed that after 10 days of incubation, as CNT content increased, cell coverage increased, nterestingly, PIMcntrl (0% CNT) could not support cell attachment with or without coating and therefore, removed from analysis, instead, PIM50 vs PIM75 were used to study such effect. Moreover, cell processes was seen to reach 200 .m in length in both materials, this is due to the increased amount of CNT in both. It is worth mentioning, that due to tissue-like formation of cell aggregates and the presence of great amount of CNT, oversaturation of f-actin fluorescence could not be avoided, especially with the CNT effect in guenching and blocking fluorescence.

[0268] 7. In vivo studies

[0269] The in vivo experiments were done in triplicates by excision (sciatic nerve fully cut) (3x controls (denoted by cntrlMX where x refers to order of sacrifice from 1 to 3, 1 being sacrificed at week 3, 2 being sacrificed at week 6 and 3 being sacrificed at week 9), 3x control scaffolds (PIM-control, no CNT) denoted by PIMcntrlMX where x refers to order of sacrifice from 1 to 3, 1 being sacrificed at week 3, 2 being sacrificed at week 6 and 3 being sacrificed at week 9), 3x scaffolds with 50% CNT (PIM50), denoted by PIM50MX where x refers to order of sacrifice from 1 to 3, 1 being sacrificed at week 3, 2 being sacrificed at week 6 and 3 being sacrificed at week 9),). The mice were sacrificed at 3, 6, 9 weeks intervals. PIM50 was used for these experiments whereby the sciatic nerve was exposed by an incision along the gluteus minimus region, cutting the nerve around > 5 mm cut, and wrapping the nerve with the scaffold in a hollow-tube like roll.

[0270] It is worth mentioning that the post traumatic process involves the following: After 1 hour of injury, calpains cleave neurofilaments and die back starts. Conduction through the severed nerve is still mediated after 24 hours, however, degeneration occurs in the proximal stump till the first healthy node of Ranvier, daughter axons are normally pruned but those which survive will start elongation, this gives rise to bifurcations in the regenerated nerve tissue. In the distal stump, wallarian degeneration happens within the 1 st week. It is worth mentioning that Schwann cells during the 1 st week after injury clear up cell remnants via phagocytosis which allows healthy regeneration. On the other hand, in rodents, regeneration happens for nerve gaps <5mm within 2 - 3 months spontaneously, however, for nerve gaps >5mm (5mm - 10mm in rats) spontaneous regeneration is halted or severely delayed.

[0271] Mice survived in all groups (Control, PIM-control, PIM50) and in both the excision experiment and the compression injury experiment. Control and PIM-control mice had a visible inflammatory reaction, which PIM50 did not present.

[0272] In the group treated with PIM50, all mice survived throughout the whole duration of the experiment and regained mobility after 10 days post-surgery, on the 3 weeks mark sacrifice, the mice started walking but with a limp (dependence on the healthy leg), hair regrowth was seen and evaluated as a positive sign. Additionally, no signs of pain or distress were seen after the 3 weeks mark nor throughout the 9 weeks.

[0273] Moreover, by the end of the experiment period (9 weeks) the mice could support weight using the operated leg and the limp noticeably reduced to near normal; however, the mice upon lifting their rear limbs preferentially support their weight on the non-operated leg, this could be due to muscle atrophy seen after the surgery or the absence of full nerve regeneration.

[0274] MRI after 3 weeks post-operation showed a noticeably thicker sciatic nerve above lesion site compared to the non-operated side. Upon performing autopsy to harvest surrounding muscles and inspect the regeneration process through histological studies it was observed that the scaffold was surrounded and stabilized by a fibrotic / connective tissue (collagenous in nature confirmed by Sirius red staining that stains collagen), which allowed it to only be in contact with the sciatic nerve and by that avoiding any interaction with other tissues such as muscle tissue. This fibrotic tissue seen, shows the protective effect of the scaffolds in prohibiting the infiltration of fibroblasts (which are responsible for creating the extracellular matrix) and microglia to the injury site and causing a glial scar that would prohibit the regeneration of the sciatic nerve. Additionally, tubular-like structures were formed most probably expected to be due to angiogenesis. The sciatic nerve was also seen fully attached to the scaffold which suggests a positive interaction between the two (meaning that the nerve was growing through the scaffold).

[0275] After 3 weeks, subject denoted by PIM50-M1, showed positive signal of NF-M (neurofilament marker to immunolabel axons in neuronal progenitors) in the NGC canal but showed negative signal of S100 (marker to stain microglial cells including myelinating Schwann cells) suggesting that more time is needed for myelinating Schwann cells to invade the lesion site, nonetheless, a strong aggrupation of S100 signal on the outer edge of the NGC suggest either Schwann cells myelinating bifurcations that were guided on the outer side of the NGC or merely Schwann cells trying to invade the lesion site.

[0276] At week 6, cells showing strong NF-M signal suggested the recruitment of neuronal progenitors into the scaffold or even elongated daughter nerves that grew within the scaffold itself and not the canal, Schwann cells, as evident by S100 and identified through cell morphology, were seen wrapping the NGC. This showed that the cells recognize the scaffold as self and further assert the positive NF signal.

[0277] At week 9, both NF and S100 signal was seen inside the canal in the distal part as well, positive for regeneration. The whole regeneration process generally compared to other NGC in Literature showed faster regeneration kinetics only at 50% of CNT in the final NGC.

[0278] Citation List

[0279] Jian Hongwei et al: "A Janus porous carbon nanotubes / poly (vinyl alcohol) composite evaporator far efficient solar-driven interfacial water evaporation", Separation and purification technology 2021, vol. 264, 118459

[0280] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

[0281] Clause 1. A porous self-standing homogeneous hydrogel, comprising poly(vinyl alcohol) and carbon nanostructures, wherein: a) the carbon nanostructures are present in an amount from 7 to 85% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; and b) the pores have an average size equal to or higher than 8 pm, particularly wherein the average pore size is measured by means of Scanning Electron Microscopy (SEM) in wet dry state, more particularly wherein the average pore size is measured by means of Scanning Electron Microscopy (SEM) and calculated taking into account the swelling volume as defined herein.

[0282] Clause 2. The hydrogel according to clause 1, wherein the carbon nanostructures are selected from the group consisting of carbon nanotubes, heteroatom-doped carbon nanotubes, carbon nanofibers, graphitized carbon nanofibers, and heteroatom-doped carbon nanofibers.

[0283] Clause 3. The hydrogel according to any of the clauses 1-2, wherein the poly(vinyl alcohol) is present in an amount from 5-10% by weight with respect to the volume of the aqueous solution.

[0284] Clause 4. The hydrogel according to any of the clauses 1-3, wherein the porosity of the material ranges from 65 to 83%, particularly wherein the porosity is measured the swelling volume as defined herein.

[0285] Clause 5. The hydrogel according to any of the clauses 1-4, wherein the pores are randomly distributed.

[0286] Clause 6. The hydrogel according to any of the clauses 1-5, wherein the thickness of the material is equal to or higher than 100 pm.

[0287] Clause 7. The hydrogel according to any of the clauses 1-6, wherein the carbon nanostructures are present in an amount from 15 to 85% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures.

[0288] Clause 8. The hydrogel according to any of the clauses 1-6, wherein the carbon nanostructures are present in an amount from 7 to 18% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures.

[0289] Clause 9. The hydrogel according to any of the clauses 1-6, which is cross-linked.

[0290] Clause 10. A process for the preparation of the hydrogel as defined in any of the clauses 1 to 6, which comprises the steps of: a1) providing an aqueous solution comprising poly(vinyl alcohol); b1) providing an aqueous dispersion comprising carbon nanostructures; c1) mixing the solution of step a1) and the dispersion of step b1) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures, wherein the amount of carbon nanostructures in the obtained dispersion is from 7 to 85% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; d1) spreading an amount of the dispersion of step c1) onto a support; e1) immersing the support with the dispersion of step d1) into a non-solvent to induce phase inversion and obtain a supported gel; f1) removing the supported gel from the non-solvent; g1) if desired, repeating steps d1), e1) and f1) one or more times until obtaining the desired gel thickness, provided that when step d1) is repeated the dispersion of step c1) is spread onto the gel on the support obtained in the previous step; hi) immersing the supported gel obtained in the previous step into an aqueous solution to obtain a supported hydrogel; and

[0291] 11) removing the supported hydrogel from the aqueous solution and separating the hydrogel from the support to obtain a self-standing hydrogel, or alternatively, iT) separating the hydrogel from the support to obtain a self-standing hydrogel and removing the self-standing hydrogel from the aqueous solution.

[0292] Clause 11. A process for the preparation of the hydrogel as defined in any of the clauses 1 to 6 which comprises the steps of: a2) providing an aqueous solution comprising poly(vinyl alcohol); b2) providing an aqueous dispersion comprising carbon nanostructures; c2) mixing the solution of step a2) and the dispersion of step b2) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures, wherein the amount of carbon nanostructures in the obtained dispersion is from 7 to 85% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; d2) adding a porogenic agent to the dispersion of step c2) to obtain a mouldable mass; e2) moulding the mouldable mass obtained in d2) into a moulded mass; f2) soaking the moulded mass obtained in step e2) in a non-solvent to induce phase inversion and obtain a self-standing gel; g2) removing the self-standing gel from the non-solvent; h2) immersing the self-standing gel obtained in step g2) into an aqueous solution to obtain a self-standing hydrogel;

[0293] 12) heating the aqueous solution of step h2) to a temperature equal to or higher than 35 °C to remove the porogenic agent and obtain a porous self-standing hydrogel; and j2) removing the self-standing hydrogel from the aqueous solution.

[0294] Clause 12. A process for the preparation of the hydrogel as defined in any of the clauses 1-6, which comprises the steps of: a3) providing an aqueous solution comprising poly(vinyl alcohol); b3) providing an aqueous dispersion comprising carbon nanostructures; c3) mixing the solution of step a3) and the dispersion of step b3) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures, wherein the amount of carbon nanostructures in the obtained dispersion is from 7 to 85% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; and d3) adding a crosslinking agent and heating at a temperature from 80 to 120 °C; e3) cooling to a temperature equal to or lower than 60 °C to obtain a porous self-standing hydrogel. Clause 13. An implant for electroactive cells and tissues, in particular a neural implant, made of the porous self-standing homogeneous hydrogel as defined in any of the clauses 1-9.

[0295] Clause 14. An implant for electroactive cells and tissues, in particular a neural implant, as defined in clause 13 for use in detecting, transmitting or monitoring electrical signals to the brain.

[0296] Clause 15. The neural implant as defined in clause 13, for use in treating CNS damage in a subject in need thereof.

Claims

Claims1. A porous self-standing homogeneous hydrogel suitable for use as an implant, which comprises poly(vinyl alcohol) and carbon nanostructures, wherein: a) the carbon nanostructures are present in an amount from 7 to 85% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; and b) the pores have an average size equal to or higher than 8 pm, wherein the average pore size is measured by means of Scanning Electron Microscopy (SEM) in dry state.

2. The hydrogel according to claim 1, wherein the carbon nanostructures are selected from the group consisting of carbon nanotubes, heteroatom-doped carbon nanotubes, carbon nanofibers, graphitized carbon nanofibers, and heteroatom-doped carbon nanofibers.

3. The hydrogel according to any of the claims 1-2, wherein the poly(vinyl alcohol) is present in an amount from 5-10% by weight with respect to the volume of the aqueous solution.

4. The hydrogel according to any of the claims 1-3, wherein the pores are randomly distributed.

5. The hydrogel according to any of the claims 1-4, wherein the thickness of the material is equal to or higher than 100 pm.

6. The hydrogel according to any of the claims 1-5, wherein the carbon nanostructures are present in an amount from 15 to 85% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures.

7. The hydrogel according to any of the claims 1-5, wherein the carbon nanostructures are present in an amount from 7 to 18% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures.

8. The hydrogel according to any of the claims 1-5, which is cross-linked.

9. A device comprising the porous self-standing homogeneous hydrogel as defined in any of the claims 1-8.

10. A process for the preparation of the hydrogel as defined in any of the claims 1 to 6, which comprises the steps of: a1) providing an aqueous solution comprising poly(vinyl alcohol); b1) providing an aqueous dispersion comprising carbon nanostructures; c1) mixing the solution of step a1) and the dispersion of step b1) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures, wherein the amount of carbon nanostructures in the obtained dispersion is from 7 to 85% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; d1) spreading an amount of the dispersion of step c1) onto a support; e1) immersing the support with the dispersion of step d1) into a non-solvent to induce phase inversion and obtain a supported gel; f1) removing the supported gel from the non-solvent; g1) if desired, repeating steps d1), e1) and f1) one or more times until obtaining the desired gel thickness, provided that when step d1) is repeated the dispersion of step c1) is spread onto the gel on the support obtained in the previous step; hi) immersing the supported gel obtained in the previous step into an aqueous solution to obtain a supported hydrogel; and i1) removing the supported hydrogel from the aqueous solution and separating the hydrogel from the support to obtain a self-standing hydrogel, or alternatively, iT) separating the hydrogel from the support to obtain a self-standing hydrogel and removing the self-standing hydrogel from the aqueous solution.

11. A process for the preparation of the hydrogel as defined in any of the claims 1 to 6 which comprises the steps of: a2) providing an aqueous solution comprising poly(vinyl alcohol); b2) providing an aqueous dispersion comprising carbon nanostructures; c2) mixing the solution of step a2) and the dispersion of step b2) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures, wherein the amount of carbon nanostructures in the obtained dispersion is from 7 to 85% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; d2) adding a porogenic agent to the dispersion of step c2) to obtain a mouldable mass; e2) moulding the mouldable mass obtained in d2) into a moulded mass; f2) soaking the moulded mass obtained in step e2) in a non-solvent to induce phase inversion and obtain a self-standing gel; g2) removing the self-standing gel from the non-solvent; h2) immersing the self-standing gel obtained in step g2) into an aqueous solution to obtain a self-standing hydrogel;i2) heating the aqueous solution of step h2) to a temperature equal to or higher than 35 °C to remove the porogenic agent and obtain a porous self-standing hydrogel; and j2) removing the self-standing hydrogel from the aqueous solution.

12. A process for the preparation of the hydrogel as defined in any of the claims 1-6, which comprises the steps of: a3) providing an aqueous solution comprising poly(vinyl alcohol); b3) providing an aqueous dispersion comprising carbon nanostructures; c3) mixing the solution of step a3) and the dispersion of step b3) to obtain a dispersion comprising poly(vinyl alcohol) and carbon nanostructures, wherein the amount of carbon nanostructures in the obtained dispersion is from 7 to 85% by weight with respect to the sum of the weights of the poly(vinyl alcohol) and of the carbon nanostructures; and d3) adding a crosslinking agent and heating at a temperature from 80 to 120 °C; e3) cooling to a temperature equal to or lower than 60 °C to obtain a porous self-standing hydrogel.

13. An implant for electroactive cells and tissues made of the porous self-standing homogeneous hydrogel as defined in any of the claims 1-8 or the device as defined in claim 10.

14. An implant for electroactive cells and tissues as defined in claim 13 for use in detecting, transmitting or monitoring electrical signals to the brain.

15. The implant as defined in claim 13, for use in treating CNS damage in a subject in need thereof.

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