Biomaterial composition comprising graphene oxide-comprising hydrogel and decm material for 3D (BIO)printing

Abstract

The invention relates to a composition for three-dimensional printing comprising a hydrogel comprising: a. graphene oxide, b.methacrylated derivatives of biopolymers, and c. radical polymerisation photoinitiator

Description

Biomaterial composition comprising graphene oxide-comprising hydrogel and dECM material for 3D (bio)printingField of the invention

[0001] The invention relates to a method for producing a composition of a biomaterial with unique characteristics comprising graphene oxide as a constituent. The method comprises both the method of obtaining a reproducible material with specific performance characteristics and the biomaterial composition that provides its significant utility in 3D bioprinting technology. The invention relates to both a graphene oxide-comprising hydrogel and a graphene oxide-based biomaterial enriched with a decellularized [exfra]cellular matrix dECM, with 3D bioprinting suitability, and improved mechanical properties. The invention is applicable in tissue engineering, 3D bioprinting, transplantology, regenerative medicine, and biomedical research.State of the art

[0002] In modem tissue engineering, great progress has been made in the development of hybrid scaffolds for cell cultures that replace the extracellular matrix. The design and production of functional tissue models using biomaterials is both the biggest challenge and limitation in the processing and clinical use of artificial constructs. Typically, the main component of artificial constructs is the ECM matrix, which consists of a complex network of structural and regulatory proteins arranged in a fibrous matrix that performs specific biological functions [I. Nishimura, R.L. Garrell, M. Hedrick, K. lida, S. Osher, B. Wu, Precursor tissue analogs as a tissue-engineering strategy, Tissue Eng. 9 (2003) 77-89, L.L. Nguyen, P.A. D’Amore, Cellular interactions in vascular growth and differentiation, Int. Rev. Cytol. 204, (2001 ), 1 -48], Designed scaffold that can replace the native ECM structure should maintain the three- dimensional cell structure and allow diffusion of nutrients, metabolites and soluble agents to ensure tissue regeneration [S. Heydarkhan-Hagvall, K. Schenke-Layland, A.P. Dhanasopon, F. Rofail, H. Smith, B.M. Wu, R. Shemin, R.E. Beygui, WR. MacLellan, Three-dimensional electrospun ECM-based hybrid scaffolds for cardiovascular tissue engineering, Biomaterials. 29 (2008), 2907-2914, J.P. Vacanti, R. Langer, Tissue engineering: the design and fabrication of living replacement devices for surgical reconstruction and transplantation, Lancet. 354 (1999) 32-34, R.M. Nerem, Tissue engineering: confronting the transplantation crisis, Proc. Inst. Meeh. Eng. Part H J. Eng. Med. 214 (2000) 95-99], In the design and production process of bioprintedscaffolds (with or without living cells), it is necessary to consider a number of features such as their structure, porosity, mechanical parameters, surface topography, stability, degradation, as well as biocompatibility.

[0003] The development of new biomaterials is of growing interest to many research groups. Among these, hydrogels such as alginate, gelatin, and hyaluronic acid have great potential in regenerative medicine and drug delivery systems. Hydrogels, as hydrophilic polymeric materials, are able to effectively retain water, which does not adversely affect their structural and physico-chemical properties. Due to their unique properties, these biomaterials are suitable for creating 3D scaffolds. Moreover, to modify their functional properties and improve their utility in tissue engineering, final formulations can be supplemented with other functional additives or polymer composites. The most common substances added to organic hydrogels are silica, hydroxyapatite, and gold and silver nanoparticles. Despite the many valuable advantages of using inorganic particles in the bioprinting process, a number of disadvantages have also been identified, including (i) an increase in shear stress during printing, which can inhibit cell proliferation and viability and affect cell functions; (ii) reduced diffusion of the cross-linking agents and (iii) poor material homogeneity that prevents the cross-linking process. One of the commonly used additives in modern tissue engineering is graphene oxide (GO), which is a carbon monolayer with a large surface area and numerous hydrophilic functional groups. These functional groups allow for a wide range of chemical modifications. The use of GO as an additive for biomaterials in biomedicine has not yet been tested and its impact on the immune system is not fully known. Regardless of the type of biomaterial, any new material introduced into the body / cells affects the immune system. Immune response is a complex process that aims to protect against foreign molecules, pathogens or other substances [S.P. Mukherjee, M. Bottini, B. Fadeel, Graphene and the immune system: a romance of many dimensions, Front. Immunol. 8 (2017) 673], The inclusion of a biomaterial into a living organism can trigger an immune response, as the immune system can identify this new material as foreign and potentially harmful. This can result in various effects, including inflammation at the site of implantation / incubation with the material or activation of different types of immune cells and cytokine production [M.J. Feito, M. Cicuendez, L. Casarrubios, R. Diez-Orejas, S. Fateixa, D. Silva, N. Barroca, P.A.A.P. Marques, M.T. Portoles, Effects of Graphene Oxide and Reduced Graphene Oxide Nanostructures on CD4+ Th2 Lymphocytes, Int. J. Mol. Sci. 23 (2022) 10625],Similarly, a GO-based biomaterial can have various biological consequences that depend on a wide range of factors, such as GO properties, concentration, additional modification of GO surface, exposure time, and individual cell characteristics [G. Peng, H.M. Sinkko, H. Alenius, N. Lozano, K. Kostarelos, L. Brautigam, B. Fadeel, Graphene oxide elicits microbiome-dependent type 2 immune responses via the aryl hydrocarbon receptor, Nat. Nanotechnol. 18 (2023) 42-48, X. Zhi, H. Fang, C. Bao, G. Shen, J. Zhang, K. Wang, S. Guo, T. Wan, D. Cui, The immunotoxicity of graphene oxides and the effect of PVP-coating, Biomaterials. 34 (2013) 5254-5261 ], It is therefore important to identify the biological utility (with particular emphasis on clinical applications) of new biomaterials and understand their impact on living organisms. To this end, it is essential to carry out tests (analyses) that allow to avoid and / or inhibit potential adverse immune reactions.

[0004] Document US11918703B2 refers to an extrudable photocrosslinkable hydrogel comprising a biochemically modified extracellular matrix (dECM) with an electroconductive nanomaterial embedded, photoinitiator, and solvent. The invention relates to a method for preparing electroconductive scaffolds using electroconductive extruded hydrogels for in situ defect-filling, conductive grafts, in situ or in vitro printed tissues or organs, adhesives for various tissues or bone adhesives. The document does not disclose the use of glycerol for hydrogel preparation and, unlike the present invention, does not use dECM in paste form. The use of hyaluronic acid and gelatin in non-methacrylated form is also a distinguishing feature.

[0005] Document US20240026182A1 discloses the composition and process for obtaining an ultra-strong, biocompatible, electroconductive, and stretchable hydrogel that comprises: step (a) of physical or chemical modification of natural polymers, e.g. preparation of silk nanofibres and double methacrylation of gelatin; step (b) of carboxylation of graphene oxide (GO); step (c) of carbodiimidation between methacrylated natural polymers from step (a) and carboxylated GO from (b); and step (d) of three-dimensional (3D) bioprinting from step (c) with / without silk nanofibres. These steps in this disclosure have been found to yield a biocompatible hydrogel with high mechanical strength for load-bearing soft tissues such as tendon and heart, as opposed to conventional hydrogels. The document does not disclose the use of hyaluronic acid, glycerol, and dECM for the preparation of bioinks.

[0006] Document EP3852821 B1 refers to a stable self-assembling matrix of a graphene oxide-protein composite comprising disordered protein (DP) and grapheneoxide (GO), wherein DP has the opposite charge to GO. Moreover, the graphene oxideprotein matrix is a 3D structure with the lumen defined by a membrane with an inner and outer surface. In addition, the above document discloses methods and kits for preparing such a graphene oxide-protein matrix as well as its use. The document does not disclose the use of ECM, LAP, hyaluronic acid, gelatin or glycerol for the production of bioink.

[0007] The American patent application US20230365775A1 discloses an electrically conductive hydrogel having a graphene network and a method of its manufacture, which comprises successively: (a) producing graphene-coated agarose microbeads (GAMs) having a positive or negative surface charge; (b) producing self-assembling granular hydrogel (GH) by mixing the graphene-coated agarose microbeads (GAMs) having a positive surface charge produced in (a) and the graphene-coated agarose microbeads (GAMs) having a negative surface charge produced in (a); and (c) performing thermal annealing of the granular hydrogel (GH) produced in step (b), wherein the graphene flakes may consist of graphene oxide. The electrically conductive hydrogel can be used especially in biomedical applications, such as scaffolds for tissue engineering, bioelectrodes, and biosensors. The document does not disclose the use of a photoinitiator (LAP), hyaluronic acid, or gelatin for hydrogel preparation.

[0008] Document W02023036970A3 is an international patent application that relates to a method of manufacturing a composite material consisting in the preparation of a suspension comprising crystalline nanochitin and carbon material selected from the group consisting of carbon nanotubes, carbon nanofibres, graphene, graphene oxide, and a mixture thereof. In another aspect, the invention relates to the use of a composite material as a bioink for 3D printing or as a substrate for cell growth. The document does not disclose the use of a photoinitiator (LAP), hyaluronic acid, or gelatin for the preparation of bioinks.

[0009] Document KR102422646B1 is a Korean patent disclosing a bioink composition comprising powdered ECM. In contrast to the present invention, in this invention, cells in the extracellular matrix remain alive. The bioink composition can further comprise a biocompatible polymer, e.g. gelatin, graphene, or hyaluronic acid. The document does not disclose the use of a photoinitiator or graphene oxide in the composition.

[0010] Document KR102412359B1 discloses a bioink composition for 3D printing based on graphene oxide and horseradish peroxidase (HRP) complex. In addition, the composition compriss hydroxyperoxide (H2O2), glucose oxidase (GOx), and glucose. In addition, KR102412359B1 discloses methods for producing a structure using the aforementioned materials. In the above composition, hyaluronic acid, glycerol, or LAP are not used.

[0011] Document WO2022252526A1 is an international patent application disclosing hydrogel material assembled from inorganic non-metallic nanoparticles to form a hydrogel network. Inorganic non-metallic nanoparticles can be graphene oxide. The document does not disclose the use of ECM, a photoinitiator (LAP), hyaluronic acid, or gelatin for hydrogel preparation.

[0012] Document PL240990B1 discloses a magnetic nanocomposite hydrogel for three-dimensional printing characterised in that it compriss a polymer component from the group of natural and synthetic polymers in the amount of 4-12% w / v, a viscosity modifier in the amount of 4-12% w / v, magnetic nanoparticles in the amount of 5-25% w / v, a stabiliser of magnetic nanoparticles in the amount of 0.1 -5% w / v, and a solvent in the amount of up to 100% w / v. The document also discloses a method of manufacturing a magnetic nanocomposite hydrogel for three-dimensional printing. The document does not disclose the use of graphene oxide as nanoparticles, nor does it use ECM, LAP, HAMA, or glycerol in the tested solution.

[0013] Document CN113651916A discloses a mineralised hydrogel and a method of its manufacture, preparation and use. The preparation of the hydrogel comprises the steps in which the hydrogel ink is provided, wherein the hydrogel ink comprises a polymeric monomer used to form the hydrogel, a high molecular polymer, a crosslinking agent, a photoinitiator, an inorganic filler, a mineralised enzyme subjected to immobilisation treatment, and deionised water; the hydrogel ink is transferred to a charging barrel of a 3D printer and ultraviolet irradiation is used during the forming process to obtain a cured formed structure; and soaking of the cured formed structure in an inorganic salt mineralising solution is carried out to obtain the mineralised hydrogel. The hydrogel ink disclosed in the above document is characterised by good printability, and the use of mineralised enzyme to induce the mineralisation is a green, eco-friendly, comfortable and fast method, the preparation of the mineralised hydrogel by 3D printing is carried out, and the prepared mineralised hydrogel has excellentmechanical properties and structural complexity. The document does not disclose the use of ECM for hydrogel production.

[0014] Document US20210108180A1 discloses hybrid hydrogels consisting of decellularized extracellular matrix (dECM) and biocompatible conductive nanomaterials. The hybrid hydrogels provide a tailored microenvironment for normal cell and tissue development. According to the above document, the mechanical and electrical properties of hydrogels can be modified. The hydrogels can be used in bioinks for high-throughput printing of tissues, and the engineered tissues generated using the hybrid hydrogels can be used to assess the biological activity of drug candidates. The document does not disclose the use of dECM in paste form. Gelatin, hyaluronic acid, LAP, and glycerol are not used in the preparation of the hydrogel.

[0015] The invention in patent CN112480746A discloses a 3D printing ink based on fish skin collagen, comprising a graphene oxide solution, a sodium alginate hydrogel substrate, a cross-linking agent and a dispersing agent. The content of components was as follows: 17-25 wt% of graphene oxide solution, 14-21 wt% of sodium alginate hydrogel medium, 2-6 wt% of cross-linking agent, 1 -2 wt% of dispersing agent, and the balance of water. 3D printing ink prepared with fish skin collagen as a cross-linking agent provide better biocompatibility; the viscosity of the ink and the mechanical properties of printed products are improved by the use of sodium alginate. A vortex mixer and centrifuge are used in the production process to improve the homogeneity as well as the quality of the ink. The document does not disclose the use of ECM, hyaluronic acid, and photoinitiator for hydrogel production.

[0016] Document KR102180865B1 discloses a composition of bioink that ensures both biocompatibility and electroconductivity and discloses its preparation method. The bioink compriss a large molecular polymer, obtained by polymerising silk fibroin (SF), a methacrylate compound, graphene oxide (GO) and a photoinitiator. The bioink disclosed in the document is characterised by high cytocompatibility. Therefore, it can be used as a medical material and for the production of various auxiliary materials for tissue engineering, including cells requiring electroconductivity, such as nervous cells or muscle cells. The composition described above does not compris ECM and glycerol.

[0017] Document CN103819656A refers to a graphene oxide / light-curable resin composite and a method for its production consisting in the preparation of a mixture of graphene oxide, an oligomer, and a photoinitiator. The graphene oxide / photo-curable resin composite compriss two phases of graphene oxide and photo-curable resin.Graphene oxide in an amount of 0.1 -1 wt% is evenly dispersed in the photocurable resin. The oligomers comprised in photo-curable resin are final acrylate resins or final methacrylate resins, unsaturated polyesters, polyene / thiol resins, epoxy resins, vinyl resins, or a mixture of polysiloxanes. According to the document described, the elongation at break and maximum bending strain of the unmodified photo-curable resin are improved to a certain extent and the impact strength of the coating is doubled, so that the part printed by the 3D printer has better mechanical properties. The document does not disclose the use of ECM, hyaluronic acid, and glycerol for bioink production.

[0018] Document CN114177356B refers to 3D printing and, in particular, to the preparation method and use of a light-curable 3D printing ink. The preparation method of a light-curable 3D printing ink is characterised by a content of 50-100 mg of graphene oxide powder, 15-20 g of acrylamide, 1 -5 g of polyethylene glycol (diol) diacrylate, and a photoinitiator (LAP). The 3D printing ink disclosed in this invention provide the possibility to customisable hydrogels. It can be used in transdermal drug delivery, skin cosmetology, cell cultures, and tissue engineering, and other fields. The document does not disclose the use of ECM, gelatin, hyaluronic acid, and glycerol for composition production.

[0019] Document CN115282326A discloses a method for 3D printing a functional hydrogel dressing that comprises the following steps: (1 ) preparing an ink comprising TO-CNF (tempo oxidised-cellulose nanofibres), GelMA, and a photoinitiator (LAP); (2) optimisation of printing parameters using the developed ink is carried out, the printable ink concentration and the printing parameters are then determined; (3) adding GelMA with dopamine to the printable ink; (4) adding PDA@rGO (dopamine hydrochloride + reduced graphene oxide) to the composite ink; (5) transferring the composite ink to a syringe, centrifugal defoaming, and pre-cooling in a refrigerator; (6) placing the syringe in an extrusion-type 3D printer for 3D printing; (7) performing UV cross-linking and Ca < 2+> complexation cross-linking on the printed hydrogel; and (8) performing a photothermal anti-bacterial treatment on the cross-linked wound dressing. The hydrogel prepared by this method has good photothermal, antibacterial, electrical conductivity, haemostasis, and biodegradability properties. Moreover, it keeps the wound moist and effectively promotes wound healing. The document does not disclose the use of ECM, hyaluronic acid, and glycerol for hydrogel production.

[0020] Document CN114681675A discloses a method for preparing a 3D printed hydrogel urethral stent comprising the following steps: S1 , preparing a SA / Gel / rGOhydrogel composite solution; S2, preparing a SA / Gel / rGO hydrogel scaffold using 3D printing; S3, placing the printed SA / Gel / rGO hydrogel stent in a low -temperature environment of -85°C to -80°C and freezing it for approx. 2-2.5 hours; S4, placing the frozen SA / Gel / rGO hydrogel stent in a vacuum freeze dryer, the freeze-drying time being 12 to 13 hours; S5, cross-linking the freeze-dried SA / Gel / rGO hydrogel scaffold and 5% CaCl2, the cross-linking time being 30-35 minutes, then, after cross-linking, washing the scaffold with sterilised water; S6, performing secondary freeze-drying of the purified SA / Gel / rGO hydrogel scaffold, then storing it in a vacuum comprise for later use. The SA / Gel / rGO nano-composite hydrogel is prepared by introducing a rGO solution into a mixed SA / Gel solution and is printed so that the swelling properties, pore size, and stretching properties of the hydrogel can be significantly improved. The document does not disclose the use of ECM, hyaluronic acid, and glycerol for hydrogel production.

[0021] Document CN108452375B discloses a method of preparing a 3D printed graphene oxide conductive hydrogel that comprises the following steps: (1 ) preparing a biomaterial composed of a polycaprolactone and gelatin composite, methacrylic acid modified gelatin, and graphene oxide and photoinitiator; (2) designing and (3) preparing 3D printing and printing. After curing by irradiation, a conducting hydrogel is obtained from graphene oxide. The conductive hydrogel is characterised by high porosity, good strength, elasticity, and good biocompatibility and conductivity. The document does not disclose the use of ECM, hyaluronic acid, glycerol, and LAP as a photoinitiator for hydrogel production.

[0022] The invention relates to a composition for three-dimensional printing comprising a hydrogel comprising: a)graphene oxide, b)methacrylated derivatives of biopolymers, and c)radical polymerisation photoinitiator

[0023] Preferably, the methacrylated derivatives of biopolymers are selected from the group consisting of methacrylated gelatin, methacrylated hyaluronic acid, and / or mixtures thereof

[0024] Preferably, the radical polymerisation photoinitiator is selected from the group consisting of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959, Sigma-Aldrich), diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO), phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide (BAPO), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (FAS), and / or mixtures thereof, particularly preferably, the radical polymerisation photoinitiator is lithium phenyl-2,4,6- trimethylbenzoylphosphinate.

[0025] Preferably, the composition for three-dimensional printing comprises a hydrogel comprising: a)graphene oxide, b)methacrylated gelatin and methacrylated hyaluronic acid, and c)lithium phenyl-2,4,6-trimethylbenzoylphosphinate photoinitiator.

[0026] Preferably, the composition comprises dECM (decellularized pancreatic extracellular matrix) derived from the decellularization of organs and digested with pepsin in an acidic environment.

[0027] Preferably, the composition comprises glycerol.

[0028] Preferably, the composition comprises methacrylated gelatin in the concentration range of 37.5-130 mg / ml; methacrylated hyaluronic acid in the concentration range of 0.56-6.5 mg / ml; graphene oxide in an aqueous suspension in the concentration range of 0.5-1 .0 mg / ml, and LAP photoinitiator in the concentration range of 0.1 -10 mg / ml, and glycerol in the concentration range of 50-100 mg / ml, preferably 89.8 mg / ml.

[0029] Preferably, dECM solution is derived from the decellularization of pancreas, liver, placenta, heart, and skin.

[0030] Preferably, the total dECM concentration is preferably in the range of 100-160 mg / ml and most preferably is 76.6 mg / ml. With a preferable dECM content, it is possible to obtain a rigid, stable printed fibre

[0031]

[0032] Another object of the invention is the use of the composition of the invention in a bioprinting process.

[0033] Preferably, in the use according to the invention, the printing temperature is in the range of 15-30°C, the pressure is from 5 to 75 kPa, and the printing speed is from 5 to 45 mm / s.

[0034] Yet another object of the invention is a method of three-dimensional printing using the composition of the invention.

[0035] The main objective of the invention is to develop a composition and a method of manufacturing a biomaterial comprising nGO, which is an agent that modifies andimproves its performance in the field of tissue engineering, regeneration and transplantation medicine, or biotechnology in general.

[0036] Moreover, during the studies, it was demonstrated that it is important to obtain a homogeneous material with unique performance properties including the following:

[0037] - optimal rheological properties: viscosity in the range of 35-1500 mPa s, storage modulus (G1) of 5-900 Pa, and loss modulus (G") of 2-15 Pa;

[0038] - optimal mechanical parameters of objects printed using the developed biomaterials: mechanical strength in the range of 300-600 kPa, elasticity in the range of 60-120 kPa;

[0039] - appropriate parameters characterising the suitability of the material for 3D printing technology while maintaining print parameters that are safe for the cellular material: temperature of 15-30°C, pressure of 5-75 kPa, print speed of 5-45 mm / s.

[0040] The invention also applies to the development of a composition and a method of obtaining a homogeneous biomaterial characterised by unique physico-chemical characteristics such as, e.g.: viscosity and complex modulus, mechanical strength and elasticity, printability, degradation and swelling, and biological characteristics, including biocompatibility and suitability for printing tissue models.

[0041] The test material is: a hydrogel comprising within its composition components such as:- methacrylated gelatin at a concentration of 37.5-130 mg / ml- methacrylated hyaluronic acid at 0.56-6.5 mg / ml- graphene oxide in an aqueous suspension, at a concentration of 0.5-1 .0 mg / ml- LAP photoinitiator at a concentration of 0.1 -10 mg / ml or a biomaterial comprising within its composition components such as: the hydrogel described in subsection A- a hydrogel material based on an acidic solution of dECM derived from the decellularization of organs, including pancreas, liver, placenta, heart, skin, and other organs and tissues, with pepsin, dECM-enriched with a final dECM concentration of 100-160 mg / ml, preferably obtaining in the biomaterial of 76.6 mg / ml, so that a rigid, stable printed fibre can be obtained- glycerol at a concentration of 50-100 mg / ml, the most preferable in the biomaterial being 89.8 mg / ml.2) The resulting standardised composition of the biomaterial with suitable application characteristics having the following advantages:- a desired in the context of the use of materials in the bioprinting technology viscosity value at the processing temperature in the range of 50-500 mPas;- a storage modulus value above the loss modulus value, indicating that elastic properties predominate over viscous properties;- good printability and resolution achieved under optimal printing parameters for biomaterials for cellular material-enriched biomaterials;- very good mechanical properties: obtaining a much higher elasticity (Young's modulus, conventional yield point) while maintaining a constant mechanical strength value;- biomaterial with moderate water absorption capacity, ensuring adequate hydration, support for cell growth, and long-term structural stability depending on the use, hydrogels have 6.5-8.5 times higher swelling ratio than corresponding biomaterials enriched with paste-like form of a dECM-based component;- biological properties allowing the use of tested materials in specialised applications such as tissue models, controlled drug delivery systems.Brief description of the drawing

[0042] Fig. 1 . shows a photograph of the hydrogel and the biomaterial comprising graphene oxide after cross-linking with a UV-VIS lamp (365 nm, 20 s, 13.0 mW / cm2).

[0043] Fig. 2. shows the relationship between the complex modulus and strain.

[0044] Fig. 3. shows the average viscosity measured for all material, both biomaterial and hydrogel, variants.

[0045] Fig. 4. shows optimal printing parameters.

[0046] Fig. 5. shows the results of the printed fibre-merging test: the relationship between both the dispersion rate of the material and printability coefficient and the size of the printed pore.

[0047] Fig. 6. shows the results of the printed fibre-collapse test on a platform.

[0048] Fig. 7. shows the results of the static compression test: 7A) compressive stress- sample / strain relationship for the tested materials: BREF, BGO1 , BGO2; 7B) mechanical parameters: I mechanical strength, II Young's modulus, III conventional yield point; 7C) photographs of test samples during the analysis.

[0049] Fig. 8. shows an evaluation of degradation of the materials.

[0050] Fig. 9. shows an evaluation of the swelling and absorbability of the materials.

[0051] Fig. 10 shows L929 cell lines after exposure to extracts obtained from individual biomaterials: HGO1 , HGO2, BGO1 , BGO2. The results are presented after incubation, at three time points (24, 48, 72 h). Pictures were taken using Olympus IX83 bright field (BF) microscope, 20x lens magnification.

[0052] Fig. 11. shows the effects of extracts obtained from HGO1 , HGO2, BGO1 , BGO2 biomaterials together with controls on L929 cell line. The results are presented at one time point (24 h). The guidelines laid down in ISO 10993-5:2009 (E) represent the continuous line representing the 70% viability limit.

[0053] Fig. 12 shows the effect of exposure of the biomaterials comprising GO on the viability and proliferation rate of L929 cell lines over time. Control: cells living under standard conditions. M. RLU: Relative Light Unit

[0054] Fig. 13. shows the effect of exposure of the biomaterials comprising graphene oxide on the proliferation rate of L929 cell lines over time.Embodiments of the inventionExample 1. Development of a composition of nGO-comprising homogeneous biomaterial and a method for obtaining thereof

[0054] The biomaterials were prepared as a composition of two main components:

[0055] a biomaterial in the form of a dECM-based hydrogel, supplemented with an additional amount of lyophilised decellularized pancreatic extracellular matrix (dECM), supplemented with an additional amount of dECM;

[0056] GO-based hydrogel consisting of a mixture of methacrylated gelatin, methacrylated hyaluronic acid, GO and glycerol.

[0057] According to an embodiment, dECM biomaterial was obtained as follows: dECM was digested in an acidic pepsin solution (1 mg / ml in 0.01 M HCI) and neutralised with 0.1 M NaOH. After the neutralisation step, an additional amount of lyophilised dECM was introduced in order to prepare the biomaterial. The GO-based hydrogel was prepared by mixing GelMa, HaMa, glycerol, LAP, and GO dissolved in PBS. To prepare the final composition of the nGO-comprising biomaterial, the GO- based hydrogel was mixed with the dECM biomaterial at a volume ratio of 1 :1 . Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP (1.85 mg / ml); used as the photoinitiator. The variant designations: HGO (HGO1 and HGO2), BREF, BGO (BGO1 and BGO2) of the invention refer to: GO-based hydrogels, reference biomaterials, and dECM-enriched biomaterials, respectively.

[0058] Table 1. Composition and concentrations of individual components of biomaterialsExample 2. Evaluation of physico-chemical properties of biomaterials

[0059] The physico-chemical properties, including the rheological properties, printability, mechanical properties, degradation, swelling, and absorbability, of the biomaterials of the invention were investigated.RHEOLOGY

[0060] A rheological analysis of hydrogels and biomaterials was performed using Anton Paar MCR 72 rheometer (Anton Paar, Austria) with a 50 mm diameter cone spindle- and-plate geometry system, was placed on the sample table, and the viscosity was tested at the shear rate of 100 s-1at the temperature of 25°C. The biomaterial storage modulus and loss modulus were tested under 1 -100% strain at the temperature of 14°C for hydrogels and at the temperature of 20°c for biomaterials. All the tests have been performed at a set zero-gap distance of 0.102 mm.

[0061] A biomaterial having a significant utility in the process of 3D bioprinting by the extrusion method should be characterised by appropriate values of rheological properties, such as a viscosity of not less than 30 mPas or a storage modulus (G1), which refers to the elastic properties and serves to evaluate the material as a elastic shape retention measure, of not less value than the loss modulus (G"), which represents the viscous part of the material or the amount of energy dispersed in the sample (LiXiaorui et al., “Biomaterial inks for extrusion-based 3D bioprinting: Property, classification, modification, and selection”, International Journal of Bioprinting, 2022 Dec 9;9(2):649). Research results showed that the biomaterials labelled BGO1 , BGO2, and BREF showed a higher storage modulus (G1) (higher energy storage capacity) than the loss modulus (G") over the range of oscillation amplitudes investigated (Fig.2), suggesting that they can be considered as elastic biomaterials. This feature is particularly important in the context of the use of such biomaterials in the bioprinting process, as greater elasticity of the biomaterials enables them to be extruded more easily. The storage modulus value obtained for the reference biomaterial (BREF, non- GO-BGO1 variant) is slightly lower than those determined for GO-supplemented biomaterials, which may indicate that the addition of a nanomaterial affects the elasticity of the biomaterial and enhances its solid-specific properties. Moreover, varying GelMa and HaMa concentrations (BGO1 and BGO2) in the final biomaterial composition did not bring about significant changes in the complex modulus component values. According to an embodiment, it was observed that the biomaterial as a composition of two components: the GO-based hydrogel and the dECM biomaterial, despite varying concentrations of GelMa and HaMa (BGO1 and BGO2) inthe resulting biomaterial, does not significantly change the complex modulus component values. (Fig. 2) Therefore, it can be concluded that the absence of a dECM- based component will significantly reduce the complex modulus component values (Fig. 2)

[0062] According to the invention, hydrogels have viscosity values 100 times lower than the corresponding biomaterials, while a comparison of individual HGO1 / BGO1 vs. HGO2 / BGO2 variants showed that the highest values were determined for HGO1 / BGO1 (Fig.3). This may be since HGO1 / BGO1 variants comprise lower concentrations of both HaMa and GelMa. The highest viscosity was determined for BGO1 (approx. 450 mPas) and the lowest for HGO2 (approx. 37 mPas). Therefore, it can be concluded that the developed biomaterial has good rheological characteristics, and the observed viscosity of the biomaterials may point in the direction of potential application in the bioprinting and its printability.PRINTABILITYFibre-merging test

[0063] The fibre-bonding test was performed according to a g-code generated model in which two layers were printed successively, one after the other, using a specific material. The prepared print follows the 0°-90° pattern, which provides 2D effect and increases the fibre distance (FD). The fibre distance was in the range of 1 -5 mm with increments of 1 mm. The printing speed, needle diameter, and printing distance used in the test were respectively: 20 mm / s, 21 G (0.609 mm) or 25G (0.437 mm), and 0.8 mm. During the test, the biomaterial was dosed at a temperature in the range of 24- 25°C and a pressure in the range of 30-75 kPa. (Fig. 4). Subseguently, the printed construct was cross-linked using an external UV-Vis 405 nm lamp for 15 s at 13 mW / cm2(Polbionica S. A, Poland). Images of the prints were taken with a camera immediately after production. The images were developed using Axio Vision software (Zeiss). Based on the results obtained, two parameters described by eguations were determined, namely the percentage diffusion rate (dispersion rate) (Dfr) and the printability (Pr). The diffusion rate in pores without dispersion of the material is 0 (i.e. At = Aa), and for ideal representation of the model the printabil ity is 1 .lW6SiP= —a

[0066] At, theoretical pore area,

[0067] Aa, actual pore area,

[0068] Pr, printability,

[0069] L, actual pore perimeter.2. Fibre-collapse test

[0070] According to an embodiment, the fibre-collapse test analysed the mid-span deflection of a suspended fibre. During the tests, a dedicated platform consisting of seven pillars was designed and printed. The individual pillars are 1 , 2, 3, 4, 5 and 6 mm apart. The dimensions of the two corner pillars are 5 x 10 x 6 mm3, while for the other five pillars they are 2 x 10 x 6 mm3. A single fibre of test material is deposited on the platform and a picture of the fibre was then taken. During the printing process, the temperature and pressure conditions were adjusted for the given biomaterial and printing was carried out with a rate of 20 mm / s using a 21 G (0.609 mm) needle. The collapse area coefficient (Cf), which is the percentage of the actual area after deflection of the suspended fibre relative to the theoretical area, was calculated using the formula:AcCf= - - 100% f ActAac, the actual area under the curve, Atc, the theoretical area under the curve, Cf, the collapse area factor.3. Smoothness and continuity of fibres

[0071] Fibre continuity during the printing of a 2-3 ml biocomponent according to the invention was determined using a zero-one system, where 0 indicates that the fibre is broken; and 1 that the fibre is continuous.

[0072] According to an embodiment, several fibres were printed using BREF, BGO1 , and BGO2 biomaterials while determining the optimum printing process parameters that allowed to produce a continuous fibre with satisfactory properties (Fig. 4). It can therefore be concluded that the use of GO-based biomaterials generates the need for a higher extrusion printing pressure compared to the reference sample (BREF). It was noted that for BGO1 and BGO2 biomaterials, the value of the pressure applied was approx. 20 and 30 kPa higher than the values determined for the reference biomaterial (BREF). It has been observed that when assessing the optimal printing parameters, not only the production method of 3D objects with the desired physico-chemical properties, but also of the impact of printing parameters on cell survival should be considered.

[0073] According to an embodiment, the printing temperature was also analysed as a function of the composition of the printed biomaterial, while comparing it with the reference biomaterial. During the studies, it was shown that the varying composition of the biomaterial does not affect the printing temperature (Fig.4). Therefore, in each of the presented variants it was possible to print a continuous fibre in the temperature range of 24 to 27°C.

[0074] Analyses of all biomaterials showed that as the pore size increases, the diffusion rate decreases and printability increases for pores larger than or equal to 4 mm2, as shown in Fig. 5. The lowest diffusion rate and the highest printabi lity for 4 mm2pores was shown for the BGO2 material.

[0075] The research showed that all materials enabled the printing of a stable and continuous fibre; however, a more preferable result was obtained using the BGO2 biomaterial, as shown in Fig. 6. For each biomaterial, a printability close to 0.8 was obtained, which makes it possible to infer a high suitability of the material for the bioprinting process.4. Mechanical properties

[0076] Mechanical compressive strength of the printed constructs was tested by a static compressive test using an apparatus consisting of: a computer with Axis FM software, Pronterface, an actuator and tripod control, a tripod with electric drive and control, an Axis FB50 actuator (maximum force 50 N) mounted on the tripod, a compression head.

[0077] According to an embodiment, cylindrical samples (d=10 mm and h=5 mm; 100% filling, cross-linked with an external UV-Vis lamp after each layer) were printed using a 3D printer (Cellink BIOX, Sweden). Each printed construct was subjected to an external force in the range of 0 to 0.05 N and to compression at a constant rate of 10 mm / min at room temperature, until 80% strain was achieved. The sampling points were collected every 0.025 s. After the measurement, a picture of the deformed sample was taken with a camera. Based on the results obtained, the mechanical strength of the samples was calculated as the maximum stress (force / printed sample surface area ratio) and the Young's modulus — as the slope of the stress / strain relationship straight line for the sample within the strain range 0.1 to 0.5. An important parameter is alsothe conventional elastic limit, i.e. the stress required to deform the samples by 10%. (Fig. 7).

[0078] The mechanical parameters of the 3D printed objects were determined using the static compression test. The elastic limit is the stress value required to deform 10% of the sample height, while the Young's modulus represents the stiffness of the material and is defined as the slope of the most linear part on the stress-strain curve, within the strain range of 0.1 -0.5. The results of the static compression test are shown in Fig 7. According to an embodiment, the external force applied to BGO1 and BGO2 biomaterials led to deformation of the 3D structure (Fig.7), while the GO-based biomaterials are characterised by higher values of Young's modulus and conventional yield point. Tested biomaterials were observed to have a lower crushing strength than the reference sample (BREF).5. Degradation

[0079] Degradation studies were performed in simulated body fluid (SBF) with and / or without an enzyme (0.1 mg / ml collagenase). Collagenase breaks down peptide bonds in collagen, which is the main component of the hydrogels and biomaterials tested. 300 mg of a biomaterial was placed in Petri dishes and then cross-linked using UV light (A=365 nm, 13 mW / cm2for 15 s). The samples were flooded with a suitable SBF solution and incubated at the temperature of 37°C for 21 days. At specified time points, sample weight loss or gain was monitored by fluid removal and lyophilisation. The time0 h consisted of lyophilised and weighed samples immediately after cross-linking. The test for each variant was carried out in 3 repetitions. The biodegradation ratio was calculated according to the following formula:w, dry mass of the sample for time 0 h, wt, dry mass of the sample after degradation time t,DEG, degradation ratio [%].

[0080] The results of the degradation ratio determination are shown in Fig.8. The degradation degree after 21 days ranged 50-90% for non-enzymatic degradation and 80-90% for enzymatic degradation. Biomaterials were observed to be more resistant to non-enzymatic degradation than hydrogels (BGO1 <HGO1 and BGO2<HGO2), while results of enzymatic degradation showed that both biomaterials and hydrogels had similar degradation ratios (HGO1 and BGO1 as well as HGO2 and BGO2). It cantherefore be concluded that the higher the cross-linked density of the biomaterials with GO, the more stable the 3D structure of the biomaterial, and thus the slower the degradation.6. Absorbability and swelling

[0081] The absorbability of hydrogels (HGO1 and HGO2) and biomaterials (BGO1 and BGO2) was determined by calculating water absorption. Hydrogels were cross-linked with UV light (A=365 nm, 13 mW / cm2for 15 s) and incubated in de-ionised water at the temperature of 37°C for 24, 48 and 72 hours. At the given time points water was collected and the sample weighed (ws). The test for each variant was carried out in 3 repetitions. The water absorption coefficient of the swollen gel was calculated as follows: ws— w0Water absorption = - 100% w0w0, the weight of the sample for time 0 h, ws, the weight of the sample after time t.

[0082] The swelling of hydrogels (HGO1 and HGO2) and biomaterials (BGO1 and BGO2) was determined by calculating the swelling ratio. The samples were crosslinked with UV light (A=365 nm, 13 mW / cm2for 15 s) then placed in an aqueous medium (de-ionised water) and stored at room temperature for 24 and 48 hours. After this time water was collected and the samples were lyophilised and weighed. At time 0 h the sample was lyophilised immediately after cross-linking. The test for each variant was carried out in 3 repetitions. The swelling ratio was determined as follows: SwellingMo, the dry mass of the sample after 0 h, Ms, the dry mass of the sample after time t.

[0083] From the results (Fig. 9), the hydrogels (HGO1 and HGO2) were observed to absorb significantly more water than the biomaterials (BGO1 and BGO2). The biomaterials were observed to absorb more water during the first 48 hours, and water absorption decreases afterwards as a consequence of biomaterial saturation. Analysisof water absorption per mg of sample (Fig. 9) showed that after 24 hours, the hydrogels (HGO1 and HGO2) had the lowest water absorption per mg of sample, with the trend reversing after 48 hours. In contrast, in the case of the biomaterials (BGO1 and BGO2), it was found that absorption was the highest after 24 hours and decreased over the following days. Thus, it can be concluded that dECM-comprising biomaterials easily saturate with water on the first day after being immersed in the liquid. The hydrogel swelling ratio was observed to be 6.5 (HGO2) and 8.5 (HGO1 ) times higher than that for biomaterials.

[0084] According to an embodiment, it can be concluded that the tested biomaterials have favourable rheological properties indicating that they can be used in 3D printing technology to print complex three-dimensional models, in particular in the development of functional tissue scaffold models. Due to the increased elasticity of printed constructs using graphene oxide-enriched materials, it is recommended that the tested materials be used in the development of tissue scaffolds requiring such mechanical properties.Example 3. Evaluation of biological properties of materialsEvaluation of cell viability in contact with graphene oxide (Go) -based biomaterial

[0085] The MTT test based on the reduction of yellow tetrazole salt (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide — MTT) to purple formazan crystals by metabolically active cells was used to assess cell viability.

[0086] Cell viability tests used: cell line: L929, mouse fibroblasts (ATCC®, cat no: CCL- 1TM) grown in DMEM cell medium with 10% FBS and 1 % antibiotic addition.

[0087] As shown in Table 1 , the biomaterial variants tested were used to test cell viability using the MTT test by placing the inserts in culture medium with 0.1 g crosslinked biomaterials per 1 ml of DMEM. The extracts were then incubated for 24 hours in a complete culture medium at the temperature of 37°C and in 5% CO2 environment. The extracts from under the inserts were then applied to the cells on the plates. The plates were incubated at time intervals of 24 h, 48 h, 72 h. The MTT solution was then removed and 100 pl of DMSO was applied. The plates were shaken until the crystals were fully dissolved. Then absorbance was then measured at 570 nm and 650 nm. The positive control was a culture incubated with 0.1 % Triton X-100 (Fig. 10).

[0088] Tested biomaterials with 1 % graphene oxide showed no cytotoxicity to the L929 cell line after 24-hour exposure, maintaining viability of > 70% (Fig. 11 ). Cell viability declines slightly over the following days of exposure. The composition of biomaterialscharacterising the sample: HG01 and HG02 showed no cytotoxicity even at 72 h of the test, as shown in Fig. 12. Over 70% — materials with the potential to be used in bioprinting and cell culture engineering.Evaluation of graphene oxide (GO) -based biomaterial exposure effect on L929 cell lines — quantitative lactate dehydrogenase (LDH) assay based on bioluminescence method

[0089] The LDH cytotoxicity assay allowed the cytotoxicity of biomaterials comprising graphene oxide to be estimated and cell proliferation to be assessed. For this purpose, according to an embodiment, an assay based on the reducing capacity of cell health indicators was also performed, using the reducing power of living cells to measure viability. The assay was performed by seeding L929 cells directly into HGO1 , HGO2, BGO1 , and BGO2 biomaterials incubated for 1 , 3 and 7 days. The control was cells grown under standard conditions.

[0090] The study found that during incubation of the cells on the biomaterial, the level of luminescence decreased, resulting in a decrease in the amount of LDH released (the luminescence signal is proportional to the LDH released).

[0091] Cells exposed to HGO1 on the first day were observed to have luminescence levels of 59,168 ± 7386. This value fell to 14,357 ± 594 (**p < 0.01 ) on day 1. On the other hand, for control cells, it was noted that LDH release was relatively constant, ranging from 17,000 to 20,000. It was observed that the HGO2 biomaterial showed a luminescence level comparable to control cells throughout the incubation period, averaging 15,000. The relative luminescence unit (RLU) values for BGO1 and BGO2 on the first day of incubation were 40,351 ± 3277 and 49,486 ± 6815, respectively. During the studies, it was observed that the relative luminescence unit values decreased over time until the last day of incubation after reaching values equivalent to those of control.

[0092] On the first day of the LDH assay, no statistically significant differences were observed between control cells in 2D culture and HGO2, between HGO1 and BGO2, and between BGO1 and BGO2. On the third day of the test, a statistical difference was shown by control cells in 2D culture and HGO2. On the 7th day of the experiment, statistically significant differences were observed only between control cells in 2D culture and BGO1 , BGO2, HGO1 , and HGO2. Moreover, the LDH assay on the first day showed no statistically significant differences between control cells in 2D culture and HGO2, between HGO1 and BGO2, and between BGO1 and BGO2. On the thirdday, there were no statistically significant differences between control cells in 2D culture and HG02, and on the seventh day, statistically significant differences were observed only for control cells in 2D culture and BGO1 , BGO2, HG01 , and HG02 (Fig. 12).Evaluation of L929 cell proliferation using Alamar Blue reagent

[0093] The L929 cell line proliferation evaluation study was conducted using Alamar Blue reagent, applied directly to the surface of the biomaterials (HGO1 , HGO2, BGO1 , BGO2) after 1 , 3 and 7 days. Cells were incubated for 24 hours with Alamar Blue reagent in the ratio of 1 : 10. After this time, 100 pl of the medium was transferred to black plates to reduce background and potential interferences from visible light. The absorbance of each sample was measured at 530 nm and 590 nm using a plate reader.

[0094] An analysis based on a test assessing the rate and viability of cell proliferation is presented in Fig. 13. It was observed that the level of fluorescence read was recorded at a similar level relative to relative fluorescence unit (RFU) control cells for HGO2 biomaterial approx. 19,000 at all three times RFU, for BGO1 biomaterial in the first RFU approx. 20,000 and on the third day of incubation, RFU approx. 20,000 for BGO1 , on the seventh day the fluorescence level drops significantly RFU approx. 9,000. Other HGO1 and BGO2 biomaterials showed fluorescence levels similar to control cells on the first (17,000 and 16,000, respectively) and third day of incubation (17,000 and 16,000, respectively). The fluorescence level decreased significantly on the 7th day of incubation for biocomponents: BGO1 , RFU approx. 9,000, and for BGO2, RFU approx. 1 ,000.

[0095] Statistically significant differences were identified at **p < 0.01 . The Alamar Blue test in all samples tested showed statistically significant differences at a given time point.

[0096] In the context of this invention, any trade name, trademark or product name shall be used solely to identify particular products or materials as they were known at the date of filing of this application, unless otherwise specified. This is for the purpose of describing the general characteristics or functions of products and should not limit the invention to specific commercial sources. The meaning or scope of trade names may evolve over time and their use herein should be understood in the context of their usual meaning at the time of filing.

Claims

Claims1 . A composition for three-dimensional printing comprising a hydrogel comprising: a) graphene oxide, b) methacrylated derivatives of biopolymers, and c) radical polymerisation photoinitiator2. The composition according to claim 1 , characterised in that the methacrylated derivatives of biopolymers are selected from the group consisting of methacrylated gelatin, methacrylated hyaluronic acid, and / or mixtures thereof3. The composition according to claim 1 or 2 characterised in that the radical polymerisation photoinitiator is selected from the group consisting of 2-hydroxy- 4'-(2-hydroxyethoxy)-2-methylpropiophenone, diphenyl(2,4,6- trimethylbenzoyl)phosphine oxide, phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and / or mixtures thereof, preferably lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

4. The composition for three-dimensional printing comprising a hydrogel according to claims 1 -3, characterised in that it comprises: a) graphene oxide, b) methacrylated gelatin and methacrylated hyaluronic acid, and c) lithium phenyl-2,4,6-trimethylbenzoylphosphinate photoinitiator.

5. The composition according to claims 1 -4, characterised in that it comprises dECM (decellularized extracellular matrix) derived from the decellularisation of organs and digested with pepsin in an acidic environment.

6. The composition according to claims 1 -5, characterised in that it comprises glycerol.

7. The composition according to claims 1 -6, characterised in that it comprises methacrylated gelatin in the concentration range of 37.5-130 mg / ml; methacrylated hyaluronic acid in the concentration range of 0.56-6.5 mg / ml; graphene oxide in an aqueous suspension in the concentration range of 0.5-1 .0 mg / ml, and LAP photoinitiator in the concentration range of 0.1 -10 mg / ml.

8. The composition according to claim 6, characterised in that it comprises glycerol at a concentration of 50-100 mg / ml, preferably 89.8 mg / ml.

9. The composition according to any one of claims 1 -8, characterised in that the dECM solution is derived from the decellularization of pancreas, liver, placenta, heart, and skin.

10. The composition according to any one of claims 1 -9, characterised in that the total dECM concentration is preferably in the range of 100-160 mg / ml, most preferably is 76.6 mg / ml.

11. The composition according to any one of claims 1 -10, characterised in that its viscosity is preferably in the range of 35-1500 mPa s, most preferably in the range of 50-500 mPa s.

12. The composition according to any one of claims 1 -11 , characterised in that the storage modulus (G1) is 5 to 900 Pa and the loss modulus (G") is 2 to 15 Pa.

13. Use of the composition according to claims 1 -12 in a bioprinting process.

14. The use according to claim 13, characterised in that the printing temperature is in the range of 15-30°C, the pressure is from 5 to 75 kPa, and the printing speed is from 5 to 45 mm / s.

15. A three-dimensional printing method using the composition according to claims 1 -12.

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