Three-dimensional printing composition comprising methacrylated chitosan

Abstract

The invention relates to a three-dimensional printing composition comprising a dECM hydrogel-based biomaterial, a hydrogel made of methacrylated PBS biopolymer derivatives with a radical polymerisation photoinitiator, a methacrylated chitosan solution in 1% acetic acid, neutralised to a final concentration of 3.1 mg / ml in the biomaterial, and / or a chitosan solution in 2% acetic acid, neutralised to a final concentration of 1.4 mg / ml in the biomaterial.

Description

Three-dimensional printing composition comprising methacrylated chitosanField of the Invention

[0001] The object of the present invention is a material comprising chitosan and its methacrylated equivalent of considerable usefulness in 3D tissue model bioprinting technology, tissue engineering, and regenerative medicine.Prior Art

[0002] Currently, bioprinting and biomaterials are intensively researched and developed. Technical solutions in these fields allow for 3D printing of tissue structures, and ultimately for treatment through regenerative medicine. These biomaterials play a key role in the creation of functional and structurally robust tissues. They are selected on the basis of their ability to integrate seamlessly with the 3D printing process, to maintain cellular structures, and to trigger the necessary biological responses in the body.

[0003] From prior art there are known bioink solutions comprising chitosan and its derivatives usable in the field of tissue engineering.

[0004] Document WO2022236125A1 discloses an invention involving reinforced hydrogel structures, methods of reinforcing hydrogel structures, and methods of treating ischemic disorders using the reinforced hydrogel structures. A composition comprising a three-dimensional (3D) hydrogel structure and a layer comprising a mesh immersed in a photocurable or photocured ink contacting the structure. One of the preferred embodiments indicates that the structure comprises a polymer comprising chitosan as one of a vast plurality of examples of polymer materials. The document does not contain detailed information related specifically to the use of chitosan or its methacrylated derivative. This solution requires the use of a mesh (optionally multilayered) made of a polymer, which may be polyglactin, polyglutamic acid, poly(lactic acid), PFTE, or polycaprolactone.

[0005] American application US11642849B2 discloses bioinks and a composition comprising a methacrylated gelatin (MAG) or methacrylated chitosan, as well as sucrose, a silicate-based nanoparticle material (e.g. laponite); and a pharmacologically acceptable solution. The composition is in the form of a viscous liquid (viscosity in a range of 30-900 mPas) and is intended for providing tissue repair in vivo, the contentsof the description clearly indicating that this solution concerns the repair and regeneration of bones.

[0006] Document IN415089B discloses a bioink composition comprising at least one silk fibroin, at least one biopolymer, an optional cross-linking agent, a cel I -attaching agent, a photoinitiator, and an additional agent. Biopolymers listed as usable in this bioink include gelatin, polyvinylpyrrolidone, methacrylated gelatin, methacrylated chitosan, polyethylene glycol) dimethacrylate, and carrageenan, whereas additionally listed are, e.g. hepatic extracellular matrix (hepatic ECM) and decellularized extracellular matrix (dECM).

[0007] The invention disclosed in patent WO2022260988A1 relates to a bioink composition for extrusion-based printing comprising an extracellular matrix (ECM) precursor, wherein it comprises an uncrosslinked polymer and “sacrificial” microparticles (meaning “consumed” during the bioprinting process). The microparticles have a melting temperature above the crosslinking temperature of the uncrosslinked polymer. Preferred embodiments indicate that these microparticles may comprise gelatin, chitosan, alginate, and / or gum arabic. The document also indicates that the uncrosslinked polymer can be selected from a group consisting of collagen, fibrinogen, reconstituted extracellular matrices, modified matrix-derived proteins (e.g. gelatin), modified glycosaminoglycans (e.g. hyaluronic acid, chondroitin sulphate), modified polysaccharides (e.g. alginate, dextran, chitosan), and functionalised polyethylene glycol.

[0008] Document KR102477439B1 relates to a bioink containing methacrylated carboxymethyl chitosan, a photoinitiator, and a polycation. This material can be cured by visible light, has excellent biocompatibility, and can induce differentiation of stem cells into osteoblasts. The carboxymethyl chitosan can have a degree of carboxylation of 60% or more. The indicated application of the bioink is in aiding osteoblast differentiation in bone defect reconstruction.

[0009] Document WO2022236266A1 relates to a bioink comprising an ionically crosslinkable polymer; comprising a plurality of ionically crosslinkable groups, wherein preferably it is a natural polymer selected from gelatin, collagen, hyaluronic acid, chitosan, alginate, cellulose, pectin, agarose, ulvan, silk, fibrin, and combinations thereof. The essence of this solution is the ability to crosslink the polymer without the use of cytotoxic photoinitiators or exposing the cells contained in the bioink to harmful effects of UV used for crosslinking. The concept of this solution is based on an ionic,preferably anionic crosslinking process, wherein the preferred crosslinkers are metal ions of the first and second group of the periodic table, and especially Ca2+Chitosan is listed as one among a number of equally useful natural polymers.

[0010] Among other things, document US11414556B2 relates to bioink comprising a pharmacologically acceptable aqueous carrier, approx. 1-20% w / v of a first covalently crosslinked methacrylated polymer — methacrylated gelatin, approx. 0.1-5% w / v of a second ionically crosslinked polymer — polysaccharide, and approx. 0.1-10% w / v of a nanosilicate, the polysaccharide preferably being carrageenan, alginate, or chitosan. According to the description, the bioink is used in 3D printing to provide a biodegradable and biocompatible 3D construct comprising a combination of a nanosilicate (e.g. laponite) and two different polymers, the polymeric chains forming a dual strengthening intertwined polymeric system.

[0011] Document KR1020220026523A discloses a bioink composition comprising a photocurable chitosan-based compound (preferably in an amount of 1 to 25% w / v) and a photoinitiator (preferably in an amount of 0.00001 to 0.05% w / v) and cured by irradiation with light with a wavelength of 400 to 700 nm. A particularly preferable embodiment indicates that the photocurable chitosan-based compound is prepared by substituting carboxymethyl chitosan with a (meth)acrylic group.

[0012] Document KR1020210007888A discloses a bioink composition for the preparation of cartilage, comprising hydrogel, mesenchymal stem cells, and a substance inducing the formation of cartilage. It is also indicated that the hydrogel is preferably selected from a group consisting of collagen, gelatin, hyaluronic acid, alginate, methyl cellulose, chitosan, chitin, synthetic peptides, and polyethylene glycol- based hydrogels. The substance inducing the formation of cartilage is a substance increasing the chondrocyte differentiation rate in the cartilage structure printed using the bioink composition.

[0013] American application US11918703B2 discloses a composition of an extrudable photocrosslinkable hydrogel comprising a biochemically modified extracellular matrix (ECM) with an electroconductive nanomaterial embedded; a photoinitiator and a solvent. An additional component of the hydrogel may be a natural polymer such as chitosan, gelatin, collagen, hyaluronic acid, laminin, fibrin, fibronectin, alginate, and mixtures thereof. Graphene, borophene, graphyne, silicene, germanene, stanine, and mixtures thereof are disclosed among preferable conductive nanomaterials.

[0014] Document W02022003203A1 discloses a method for obtaining individual fibres of biocompatible hydrogels with a predefined diameter, which uses a printing system comprising at least two co-axial nozzles, one central nozzle being surrounded by the other circumferential nozzle. In the disclosed method, a printable biocompatible hydrogel is fed into the first (central) nozzle, a non-toxic thermoreversible gelation polymer is fed into the other (circumferential) nozzle, and hydrogel fibres with a thermoreversible gelation polymer coating are prepared through the performance of simultaneous extrusion. The indicated biocompatible hydrogel to be used as the material extruded through the central nozzle is a polymer selected from: alginate, modified alginate comprising inserted cell attachment sites, gelatin, fibrinogen, hyaluronic acid, chitosan, polyethylene glycol diacrylate (PEGDA), collagen, nanocellulose, a decellularized extracellular matrix (ECM), protein matrices, ECM proteins, gelatin methacrylate (GelMA), alginate methacrylate (AlgMA), gellan gum, collagen methacrylate (ColMA), agarose, hyaluronic acid methacrylate (HAMA), laminin, xanthan gum or NiPAAM.

[0015] Document CN115998954B discloses the uses of salamander skin secretions in the preparation of a bioink for underwater printing. The bioink consists of: (a) at least one photo-curable biomaterial selected from GelMA, HAMA, ColMA, ChSMA, CSMA, DexMA, CMCSMA, AlgMA, PEGDA, PVAMA, SilMA, F127DA, HepMA; (b) salamander skin secretions; (c) a solvent; (d) a photoinitiator; (e) a thickener selected from a group comprising gelatin, hyaluronic acid and salts thereof, collagen, chondroitin sulphate, chitosan, dextran, carboxymethyl cellulose, alginic acid and salts thereof, polyethylene glycol, polyvinyl alcohol, silk fibroin, polyether F127, and heparin.

[0016] Document IN201911030419A discloses an invention related to a bioink in the form of a suspension for 3D bioprinting, comprising two different biopolymers in an aqueous medium, a crosslinking agent, and (optionally) viable cells. Specific properties of the suspension are indicated, including compressive strength within a range of 10- 12 kN / m2, swelling within a range of 120-160%, and 80% cell proliferation, the viscosity to shear rate ratio being 1 -10s1 . It is indicated that the first biopolymer can be selected from a group comprising agarose, gelatin, fibrin, cellulose, silk, gellan gum, hyaluronic acid (HA), agarose, chitosan, silk, decellularized extracellular matrix (dECM), polyethylene glycol (PEG), Pluronic, cellular aggregates, cellular spheroids, natural or synthetic molecules with nanomaterials such as nanoparticles, minerals, etc., whereas the second biopolymer can be a psyllium husk extract.

[0017] Document WO2022061254A1 discloses the composition of a photocrosslinkable agent comprising methacrylate-modified nanoparticles, wherein a single nanoparticle has a plurality of molecules attached to its surface, wherein at least a portion of these molecules comprise at least one nanoparticle surface attachment ligand and at least one terminal methacrylate ligand. The large number of various listed polymers from which the nanoparticles can be formed include, e.g. chitosan. Document WO2022061254A1 also discloses a crosslinkable bioink comprising a solvent, a plurality of methacrylate group-modified nanoparticles, and optionally a plurality of methacrylate group-modified macromolecules, among which methacrylated chitosan is listed along with others.

[0018] Document CN117138109A discloses a method for preparing a 3D printed hydrogel scaffold for cartilage repair, comprising: (a) providing methacrylated chitosan; (b) providing microspheres containing 2-([1 ,1 -biphenyl]-4-ylcarbamoyl)benzoic acid and a polylactic and glycolic acid copolymer; (c) preparing 3D printing bioink; and (d) preparing a hydrogel scaffold made of the bioink using 3D printing technology. The first step (a) specifically includes adding a methacrylate anhydride dropwise to a chitosan solution in acetic acid, stirring and reaction in a water bath at a temperature of 60°C for 3 to 6 hours, dialysis and freeze drying in order to produce methacrylated chitosan.

[0019] Document CN110194840A discloses a method for the preparation of chitosan hydrogel, which comprises dissolving methacrylated chitosan in a solution of a cell culture medium containing amino acids and / or protein molecules, heating the whole to obtain chitosan hydrogel, and compressing the hydrogel to obtain a material with the characteristics of a gel, in particular with the required elasticity. The methacrylated chitosan disclosed in this solution is advantageously obtained by dissolving the chitosan in an acidic solution until a concentration of 0.5-1 .5% w / v is obtained, followed by adding methacrylic anhydride at a ratio of 1 to 2:100 v / v and dialysing the resulting solution. The main indicated application of the chitosan hydrogel disclosed in the above document is 3D printing bioink, with no other chemicals used in the above preparation method, which results in excellent biocompatibility and high cell viability (over 95%) of such hydrogel (and the bioink containing it).The Essence of the Invention

[0020] The invention relates to a three-dimensional printing composition comprising a dECM hydrogel-based biomaterial, a hydrogel made of methacrylated PBS biopolymer derivatives with a radical polymerisation photoinitiator, a methacrylated chitosansolution in 1 % acetic acid, neutralised to a final concentration of 3.1 mg / ml in the biomaterial, and / or a chitosan solution in 2% acetic acid, neutralised to a final concentration of 1 .4 mg / ml in the biomaterial.

[0021] Preferably, the dECM hydrogel-based biomaterial contains dECM lyophilisate at a concentration of 10 to 100 mg / ml. The lyophilisate content of the dECM hydrogel affects the value of the storage modulus which is responsible for the elasticity of the material. The value of the storage modulus in such material is greater than the loss modulus, which in turn indicates the prevalence of elastic properties over viscous ones. Thus, it has a favourable effect on the performance characteristics of the biomaterial.

[0022] Preferably, the composition according to the invention comprises methacrylated gelatin in PBS at a concentration of 31 mg / ml in the final biomaterial composition, and methacrylated hyaluronic acid in PBS at a final concentration of 3.1 mg / ml in the biomaterial.

[0023] Preferably, the radical polymerisation photoinitiator is selected from a 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. More preferably, the composition according to the invention comprises lithium phenyl-2,4,6-trimethylbenzoylphosphinate at a concentration of 1.85 mg / ml as a radical polymerisation photoinitiator.

[0024] Preferably, the composition according to the invention comprises glycerol, preferably at a concentration of 89.0 mg / ml.

[0025] In a particularly preferable embodiment, the composition according to the invention comprises a dECM hydrogel-based biomaterial enriched with dECM lyophilisate with a final dECM concentration of 76.6 mg / ml, methacrylated gelatin hydrogel in PBS at a concentration of 31 mg / ml in the final biomaterial composition, and methacrylated hyaluronic acid with PBS at a final concentration of 3.1 mg / ml in the biomaterial, with lithium phenyl-2,4,6-trimethylbenzoylphosphinate as the photoinitiator at a final concentration of 1.85 mg / ml, methacrylated chitosan dissolved in 1 % acetic acid and neutralised to a final concentration of 3.1 mg / ml in the biomaterial, and / or chitosan dissolved in 2% acetic acid, neutralised to a final concentration of 1.4 mg / ml in the biomaterial.

[0026] Another object of the invention is a methacrylated chitosan preparation method comprising successive steps, in which: a 1 % (v / v) CH3COOH solution is placed in areflux condenser reaction vessel and heated up to a temperature of 50°C; in small portions, chitosan is added to the acetic acid solution so that a 1 % (m / v) solution is produced as a result, with simultaneous stirring of its contents at a rate of at least 1 ,000 rpm until the substrate is completely dissolved; the reaction mixture is sterilised by exposure to UV light for 15 minutes; methacrylic anhydride (MAA) is gradually added, followed by conducting a reaction under predetermined conditions for a time of 16 to 20 hours; a PBS solution is added in portions to the reaction mixture, until 5-time dilution of the reaction mixture is obtained, and it is neutralised with a 1 M NaHCO3solution until pH of about 7.0 is obtained; subsequently, the solution is dialysed for 5 days, with the water replaced twice a day — every 8 h on average; it is diluted with demineralised water and protected from excessive effects of light, and mixed at a rate of 400 rpm while heating up to 40°C; after completion of the dialysis process, the solution is thickened in a rotary evaporator at a temperature of 45°C, under a pressure of approximately 40-70 mbar; and the solution is thickened by cooling down to a temperature of -80°C, and freeze-dried at a temperature of 10°C, under a pressure of 0.10 mbar for 48 h

[0027] Preferably, in the method according to the invention, the chitosan is dissolved in the acetic acid solution while stirred at a rate of 1 ,000-1 ,200 rpm.

[0028] Preferably, in the method according to the invention, the methacrylic anhydride is added gradually for 3 hours, preferably at a rate of 5.9 ml / h.

[0029] Preferably, the composition according to the invention comprises methacrylated chitosan prepared by the method according to the invention.

[0030] Another object of the invention is the application of the composition according to the invention in a bioprinting process.

[0031] In a preferable embodiment according to the invention, the printing temperature ranges between 15 and 30°C, the pressure ranges between 5 and 75 kPa, and the printing speed ranges between 5 and 45 mm / s.

[0032] Yet another object of the invention is a three-dimensional printing method using the composition according to the invention.Brief Description of the Figures

[0033] Fig. 1. Shows1H NMR spectra collected for representative samples.

[0034] Fig. 2. Shows the results of rheological tests, where Fig. 2A is the dependence of the storage modulus on the temperature, and Fig. 2B is the dependence of thestorage modulus on the shear stress for BCH (biomaterial with chitosan) and BCM (biomaterial enriched with methacrylated chitosan).

[0035] Fig. 3. Shows the results of printability tests, where Fig. 3A shows the dependence of the diffusion rate and the printability on the pore size of a printed template, and Fig. 3B — the dependence of the fibre collapse rate on the distance between platform pillars of BCH (biomaterial with chitosan) and BCM (biomaterial enriched with methacrylated chitosan). Fig. 3C shows photographs of printed models.

[0036] Fig. 4. Shows the results of mechanical tests. Fig. 4A shows the stress-strain dependence of the test samples: BCH (biomaterial with chitosan), BCM (biomaterial enriched with methacrylated chitosan). Fig. 4B shows the mechanical parameters: mechanical strength and Young's modulus for BCH (biomaterial with chitosan) and for BCM (biomaterial enriched with methacrylated chitosan). Fig. 4C shows images of printed and damaged structures of BCH and BCM.

[0037] Fig. 5. Shows the dependence of the average mass of water per sample mass on time: Fig. 5A — in a water absorption test, and Fig. 5B — in a swelling test.

[0038] Fig. 6. Degree of degradation of tested materials over time for enzymatic — Fig. 6A and non-enzymatic — Fig. 6B degradation.

[0039] Fig. 7. Shows photomicrographs of cells seeded on the materials and embedded within the printed construct.

[0040] Fig. 8. Shows the results of the LDH test.

[0041] Fig. 9. Shows the results of a cell proliferation test using the Alamar Blue reagent. Fig. 9A. Cells cultured on the surface of biomaterials. Fig. 9B 3D bioprinted models.

[0042] Fig. 10. Shows a cytotoxicity assessment of the extracts of chitosan-enriched biomaterials for L929 (Fig. 10A) and RFP-HDFCs-Neo cells (Fig. 10B).

[0043] Chitosan is a derivative of chitin generated as a result of its partial deacetylation. It is an organic chemical compound from the polysaccharide group, with biofunctional properties, i.e. nontoxicity, biocompatibility, and degradability. Unmodified chitosan dissolves only in an environment with acidic pH. Modification of chitosan by methacrylation of amine groups according to the following reaction determines an increase in its solubility in an inert environment, for example PBS ( Phosphate Buffered Saline), and therefore affects its use as a bioink component.

[0044] It is possible to modify the performance characteristics of chitosan by using the methacrylation process. A methacrylation reaction can result in the creation of a product with varying degrees of substitution, which affects its physicochemical and mechanical properties and is essential for the use of this material in specific applicationsEmbodiments of the invention:Embodiment 1. Developing the composition and producing homogeneous biomaterial containing chitosan or its methacrylic equivalent.1 .Components of the biomaterials used in the tests:

[0045] According to the invention, the biomaterials were prepared as a composition of three components:

[0046] a dECM hydrogel-based biomaterial with a final dECM concentration of 76.6 mg / ml;

[0047] methacrylated gelatin hydrogel in PBS at a concentration of 31 mg / ml in the final biomaterial composition, and methacrylated hyaluronic acid with PBS at a final concentration of 3.1 mg / ml in the biomaterial, with LAP (lithium phenyl-2,4,6- trimethylbenzoylphosphinate) as the photoinitiator at a final concentration of 1.85 mg / ml;

[0048] methacrylated chitosan dissolved in 1 % acetic acid and neutralised to a final concentration of 3.1 mg / ml in the biomaterial, and / or chitosan dissolved in 2% acetic acid, neutralised to a final concentration of 1.4 mg / ml in the biomaterial.

[0049] The biomaterials were labelled as a chitosan biomaterial (BCH) and a biomaterial containing methacrylated chitosan (BCM). Both biomaterials were additionally enriched with glycerol up to a concentration of 89.0 mg / ml.2. Preparation of methacrylated chitosan (CH IMA)

[0050] In a preferable embodiment, the method comprises the following steps:

[0051] A 1 ,000 ml three-neck flask fitted with a stirring element was placed in a heating block on a magnetic stirrer. Selection of the size of the reaction flask depends on the volume of the resulting solution, the quantity of which must not exceed 60% of the flask volume. Illustrative selection of the reaction vessel is presented in Table 1 below.Table 1 . Selecting the proper volume of the reaction vessel.

[0052] Subsequently, using a measuring cylinder, 1 % of the CH3COOH volume was measured out in the 500 ml range and poured into the flask. The side necks of the flask should be secured with a rubber septum, with a thermocouple mounted in one of the lateral necks, so that the sensor would be submerged in the solution, but would not hinder the stirring. A reflux condenser was placed in the middle neck of the flask, and the flow of water started. The flask was protected against light by means of aluminium foil, and heating was activated to the preset temperature of 50°C.

[0053] In a further step, chitosan was weighed out using an analytical balance, and in a further step it was added in small portions to the flask with the acetic acid solution, so as to obtain a 1 % solution (mass / volume) as a result, with simultaneous stirring at a rate of no less than 1 ,000 rpm, preferably continuing the stirring at 1 ,000-1 ,200 rpm. The mixture was left continuously stirred for a day, until the substrate dissolved in its entirety.

[0054] After complete dissolution of the substrate, the pH of the resulting solution was measured and marked as pHi. The flask with the solution was then exposed to UV light for 15 minutes to sterilise the reaction mixture.

[0055] A 1 % (w / v) solution with pH1 = 4.02 was obtained.

[0056] Subsequently, 17.5 ml of methacrylic anhydride (MAA) were measured out into a syringe connected to a hose and needle, collected into a 20 ml syringe, and drippedinto the solution by means of a syringe pump at a fixed rate, so that the dosage time would be 3 hours, preferably at a rate of 5.9 ml / h.

[0057] The key parameter describing the efficiency of the methacrylation reaction is the degree of substitution DS, which is controlled by adjusting the quantity of methacrylic anhydride (MMA) used for the reaction. The dependence of the ratio of MMA used per 5 g of chitosan (deacetylation degree > 75%) on the target degree of substitution of the final product is shown below in Table 2.

[0058] Table 2. Adjusting the amount of MMA relative to DS.

[0059] Once all of the measured out MMA had been dripped in, the reaction proceeded under the preset conditions (T = 50°C, stirring rate 1 ,000-1 ,200 RPM) overnight.

[0060] Subsequently, PBS was added to the reaction mixture in portions, preferably a 2,000 ml solution, until obtaining 5-time dilution of the reaction mixture, and 1 ,000 ml of 1 M NaHCO3were neutralised to obtain pH « 7.00.

[0061] The solution was transferred into dialysis tubes (MWCO membrane = 12-14 kDa) and placed in 5-litre beakers filled with demineralised water. The beakers were protected with aluminium foil against excessive effects of light, placed on magnetic stirrers, and heated up to 40°C with simultaneous stirring at a rate of 400 rpm. The dialysis process was performed at a preset temperature for 5 days, with the water replaced twice a day (10 water replacements in total). After completion of the dialysis process, the solution was thickened to a final volume of about 1 ,000 ml in a rotary evaporator at a temperature of 45°C, under a pressure of approximately 40-70 mbar. The solution was frozen at a temperature of -80°C and freeze dried at a temperature of 10°C under a pressure of 0.10 mbar for 48 h to produce pure methacrylated chitosan (CHIMA).2. Measuring the degree of substitution of1H NMR

[0062] In order to measure the degree of substitution of1H NMR, a 2 mg sample of methacrylated chitosan (CH IMA) was dissolved in 600 pl of D2O with 0.00916 mmol of TMSP (a quantitative and chemical standard — 3-(trimethylsilyl)propionic acid). The samples were then placed in a 5 mm NMR tube (Agilent DirectDrive2 700 MHz). The temperature was set at 60°C. The samples were then placed in an NMR spectrometer (Agilent DirectDrive2700 MHz). After the temperature had stabilised, the samples were turned, the probe was tuned, the pulse was measured, and the heterogeneity of the magnetic field was corrected. Subsequently, the 1 H spectrum was measured (measurement parameters: number of scans 8, repetition time 15 s, and 45° pulse time 2.5 ps). The samples were then placed in an NMR spectrometer (Agilent DirectDrive2 700 MHz). After the temperature had stabilised, the samples were turned, the probe was tuned, the pulse was measured, and the heterogeneity of the magnetic field was corrected. Subsequently, the1H spectrum was measured (measurement parameters: number of scans 8, repetition time 15 s, and 45° pulse time 2.5 ps) (Fig. 1 ). The spectrum was analysed using the NMRGIue package in the Python software. After data import, exponential weighing was carried out (line extension: 2 Hz) along with Fourier transforms, baseline phasing and correction for the following regions: 5.85 ppm: 5.6 ppm, 3.3 ppm: 3.05 ppm, and 0.1 ppm: -0.1 ppm. Subsequently, peak integrals were counted in the 5.85 ppm: 5.65 ppm area (corresponding to the protons of the methacrylic groups), and peak integration was performed in the 3.3 ppm: 3.0 ppm area (the peak of the proton originating from the medium). The DSNMR value (degree of substitution) was calculated on the basis of these parameters using the following formula:

[0063] Example 2. Assessing the physicochemical properties of materials2.1. Rheology

[0064] The rheological properties of the dECM biomaterials prepared according to the invention with an addition of CH or CHIMA were evaluated using an MCR 72 rheometer (Anton Paar). For this purpose, dynamic viscosity was measured at a temperature of 20°C, at a shear rate of 100 / s, along with the storage modulus for a temperature of 20°C, for a constant frequency of 1 Hz and a variable amplitude of 0.1 -100%.

[0065] The values of the storage modulus G' for chitosan biomaterials (BCH) over the tested amplitude range fall within a range of 570.23-1462.4 Pa, with a loss modulusG" of 101 .97-461 .11 Pa. On the other hand, the values of the storage modulus G' for biomaterials containing methacrylated chitosan (BCM) over the tested amplitude range fall within a range of 90.71-306.77 Pa, and the loss modulus G" is 121.01-238.12 Pa. (Fig. 2A)

[0066] On the basis of the storage modulus test, it was observed that for all the samples tested, the storage modulus was higher than the loss modulus, indicating the prevalence of elastic over viscous properties. A wide range of linear elastic viscosity was observed for the BCH variant. Therefore, it can be concluded that the tested biomaterials are stable over the investigated oscillation amplitude range. The variants of biomaterials, in particular BCM, have shown a much lower value of the storage modulus compared to BCH, and for a shear strain value of about 0.7 the characteristics change, and the loss modulus is higher than the storage modulus, which means that the viscous properties are starting to be more significant than the elastic properties. (Fig. 1 B) The dynamic viscosity of the BCH biomaterial was 122.07 (± 45.18) mPas, whereas for BCM it was 398.42 (± 17.65) mPas. The value of dynamic viscosity of variants with an addition of the BCH biomaterial are about three times lower compared to variants of biomaterials with an addition of methacrylated chitosan. The performed studies have shown that the gelation temperature of all the variants of biomaterials falls within a temperature range of 20.5-20.7°C. (Fig. 2 A and B)2. 2. Printability

[0067] The printability of the biomaterials was assessed by the following tests: a combination test for fibres printed in the form of a template, a collapse test for a fibre printed on a 3D platform, and a continuity test for fibres printed using the tested biomaterial in a volume of 3 ml. a) Fibre combination test

[0068] According to an embodiment, the test involves the printing of two printed layers according to a designed model, one after the other, using the test material, without the use of crosslinking therebetween. The printing was performed using a BIO XTMprinter. The resulting 2D printing has a formula of 0-90°, and the distance between the fibres ranges from 1 to 5 mm and increments by 1 mm. It was printed using the following conditions: printing speed 20 mm / s, needle diameter 0.609 mm or 0.437 mm, and printing distance 0.8 mm. During the test, the test material was extruded within a suitable pressure and temperature range. The printing was crosslinked by means of an external UV-Vis lamp (POLBIONICA UV VIS) with a light wavelength of 405 nm,and for 15 s at 13 W / cm2. The images were processed using the Carl Zeiss Vision AxioVision Viewer 4.8 software. Two parameters described by equations were determined on the basis of the results, i.e. the percentage diffusion rate (Dfr) and the printability (Pr). The degree of diffusion in pores with no material propagation is 0 (i.e. At = Aa), and for perfect reproduction of the model, the printability is 1 .At — theoretical pore area,Aa— actual pore area,L — actual pore circumference. b) Fibre collapse test

[0069] According to an embodiment, this fibre test was analysed on the basis of midspan flexure of a suspended fibre. In the course of the performed tests, a special platform consisting of seven pillars spaced apart from each other by known distances of 1 , 2, 3, 4, 5, and 6 mm was designed and printed in the 3D technology. The dimensions of the five pillars inside the structure are 2 x 10 x 6 mm3, and the dimensions of the two outermost pillars are 5 x 10 x 6 mm3. The platform model is presented in Fig. 3C. A single fibre of the test material was deposited on the platform, and subsequently the fibre was photographed. During the printing process, the temperature and pressure conditions were adjusted to a given biomaterial, and printing proceeded at a speed of 20 mm / s using a 21 G needle (0.609 mm). The flexure area ratio (Ct), which is the percentage of the actual area after the flexure of the suspended fibre relative to a theoretical area, was calculated by the following formula:Aac— actual area under the curve,A tc— theoretical area under the curve,Cf — flexure area ratio.

[0070] Fig. 3 presents the dependence of the ratios, i.e. the material diffusion rate and the printability, on the pore size of a printed template, and of the collapse ratio of a fibre printed on the platform on the distance between the pillars. It was observed that the fibre collapse ratio decreases as the pore size increases, and therefore there is an increase in printability. The spreading rate of the material printed in the form of a template model in the fibre combination test for a pore size of 4 mm2is lower for the BCM material than for the BCH material, which means that the use of methacrylated chitosan as a biomaterial component can allow for the achievement of high model reproduction accuracy and high resolution. It can be concluded that the printability of both biomaterials is satisfactory, the parameter exceeding 0.8 for pores above 4 mm2. The fibre collapse ratio is more stable for BCM than for BCH. A stable fibre stretched over the platform pillars was obtained for distances exceeding 3 mm. Based on the results of the collapse test for a fibre printable on the platform, it was observed that the most stable fibre was obtained for the BCM material. For the BCH material, a smooth and continuous fibre was produced at a temperature of 24°C and an extrusion pressure of 60 kPa, whereas for the BCM material, the optimal printing parameters are a temperature of 25°C and a pressure of 55 kPa.Smoothness and continuity of fibres

[0071] Fibre continuity during printing with the use of 2-3 ml of the biomaterial according to the invention was determined using a 0 / 1 system, where 0 — the fibre is broken; 1 — the fibre is continuous. The performed studies have shown that for the BCH biomaterial, a smooth and continuous fibre was produced at a temperature of 24°C, and under an extrusion pressure of 60 kPa, whereas for the BCM material, the optimal printing parameters are a temperature of 25°C and a pressure of 55 kPa. All the tested materials exhibit continuity and smoothness of fibres.3. Mechanical tests

[0072] During the tests, the mechanical compressive strength of the biomaterials was tested by the static compression test. Cylindrical samples with the following dimensions: diameter (d) 10 mm and height (h) 5 mm, were designed and printed for the test using a BIO XTMprinter (with 100% filling and crosslinking by means of an external UV-Vis lamp after each layer). The samples were statically compressed at a constant rate of 10 mm / min. at room temperature until 80% deformation was achieved,and measurement points were collected every 0.025 s. The elastic limit is the value of stress required to deform 10% of the height of a sample, while Young's modulus corresponds to the stiffness of the material and is defined as the slope of the most rectilinear segment of the stress-strain curve, within a deformation range of 0.1 -0.5. The results of the static compression test are shown in Fig. 3. Another parameter to be evaluated was the conventional elastic limit, i.e. the stress required to deform a sample by 10%.

[0073] According to an embodiment, three cylindrical samples were printed for each biomaterial, and subsequently tested by a static compression test in order to determine the mechanical parameters of the printed object. On the basis of the results obtained, the average mechanical strength and average Young’s modulus were determined for the samples tested. (Fig. 4). Materials containing chitosan were observed to have lower values of mechanical parameters than materials containing methacrylated chitosan. Cylinders printed using the BCH material exhibited two times lower mechanical strength than the samples made from the BCM material, and furthermore, cylindrical samples made from chitosan after methacrylation have considerable flexibility. It can therefore be concluded that BCM will be found useful when printing structures subject to high stresses, i.e. tissue models with a vascular system, or pieces of cartilage or bone.4. Swelling, absorption capacity, and degradation

[0074] Swelling tests were performed on structures made of hydrogels and biomaterials containing various proportions of GelMa, HaMa, and chitosan or ChiMa, in order to determine the swelling ratio. The samples were crosslinked by UV light (A = 365 nm, 13 mW / cm2for 15 s), in an aqueous environment (deionised water), and stored at room temperature for 24 and 48 hours. After this time, the water was collected, and the samples were freeze dried and weighed. At the time of 0 h, a sample was freeze dried immediately after crosslinking. The test was performed in 3 repetitions for each variant. The swelling ratio was determined as followsw0— sample weight at the time of 0 h, ws— sample weight after the time t,Ot — swelling ratio

[0075] The absorption capacity of hydrogels and biomaterials containing various proportions of GelMa, HaMa, and chitosan or ChiMa, was determined by calculating water absorption. The samples were crosslinked by UV light (A = 365 nm, 13 mW / cm2for 15 s), subsequently placed in an aqueous environment (deionised water), and stored at room temperature for 24 and 48 hours. After this time, the water was collected, and the samples were freeze dried and weighed. At the time of 0 h, a sample was freeze dried immediately after crosslinking. The test was performed in 3 repetitions for each variant. The swelling ratio was determined as follows:w0— sample weight at the time of 0 h, ws— sample weight after the time t,Oa — swelling ratio.

[0076] The experiment was performed in 3 repetitions for each biomaterial.

[0077] When analysing water absorption at three time points, it was observed that the highest water absorption per mg of sample was recorded after 24 h, and in the consecutive days (48 and 72 h), the amount of water absorbed decreases, and the biomaterial becomes completely saturated with water (Fig. 5A). On the first day, the material containing methacrylated chitosan absorbed more water than the material containing chitosan, but this trend was reversed with each subsequent day. As for the swelling test (Fig. 5B), there were no statistical differences observed between variants of the biomaterial at a given time point. After freeze drying, the material containing methacrylated chitosan absorbed more water than the material enriched with chitosan, both at the time of 0 and after 24 h. It can therefore be concluded that the use of methacrylated chitosan in biomaterials results in an increased swelling ratio compared to biomaterials with chitosan.

[0078] Degradation tests were performed in a simulated body fluid (SBF) with and / or without the use of an enzyme (0.1 mg / ml of collagenase). Collagenase breaks down the peptide bonds in collagen, which is the main component of the tested hydrogelsand biomaterials. 300 mg of biomaterial were placed on Petri dishes, and subsequently crosslinked by UV light (A = 365 nm, 13 mW / cm2for 15 s). The samples were immersed in a suitable SBF solution, and subsequently incubated at a temperature of 37°C for a period of 21 days. At specified time points, a drop or increase in the mass of a sample was monitored by removing the fluid and freeze drying. The time of 0 h involved freeze dried and weighed samples immediately after crosslinking. The test was performed in 3 repetitions for each variant. The degree of biodegradation was calculated according to the following formula:w0— dry matter of the sample at the time of 0 h, wt — dry matter of the sample after the degradation time t,DEG — degradation rate [%].The degradation rates of the tested materials are similar (Fig. 6). Enzymatic degradation (Fig. 6A) after 21 days results in almost total degradation of the biomaterials, which is approximately 90% — 91 % for BCH, and 82% for BCM. On the other hand, non-enzymatic degradation (Fig. 6B) exhibited a degradation rate above 60%— 62% for BCH, and 66% for BCM.Example 3. Assessment of biological properties of materials — viability assessments of cells cultured on the surface of biomaterials and in printed 3D models

[0079] The analysed variants of biomaterials: BCH — biomaterial with chitosan, and BCM — biomaterial with methacrylated chitosan.Preparation and bioprinting of a cell-containing biomaterial

[0080] Two types of biomaterials were used in the tests: a printed material with cells seeded thereon, and a biomaterial with an addition of cells to hydrogel and printed. The concentration of the cells added to the biomaterials was 2 x 105cells / ml. The biomaterials were printed using a Bio X 3D bioprinter, a biomaterial being dosed via a 21 G needle using extrusion-based bioprinting at a speed of 20 mm / s at a temperature of 22°C, under a pressure of 25 kPa. The bioprinted construct was cultured at atemperature of 37°C and with 5% CO2in a culture medium dedicated to the tested cell line. Cell viability in the 3D model was assessed 24 hours after bioprinting. In addition, a positive control was prepared, consisting of cells in a 2D culture, cultured under standard conditions, i.e. at a temperature of 37°C and with 5% CO2.Preparation and seeding of cells on the surface of biomaterials

[0081] Cell viability was assessed as the result of interaction between cells and the surface of the biomaterial 24 hours after seeding. The cells were observed and analysed in terms of their coverage on each biomaterial. Before seeding the cells onto a biomaterial, a 24-well plate was coated with an approximately 1 mm thick layer of each tested biomaterial variant, and then crosslinked. The experiment used the RFP- HDFCs-Neo culture medium, with a density of 3 x 104cells / well. The cells seeded on the surface were cultured at a temperature of 37°C and with 5% CO2in a culture medium intended for the cell line. The positive control consisted of cells in a 2D culture, cultured under standard conditions, i.e. at a temperature of 37°C and with 5% CO2.1. Microscopic observations

[0082] Microscopic imaging of cells was performed using an inverted Olympus IX83 microscope. The photographs were taken with 2 4 x, and 10 * magnification in both the bright-field (BF) and fluorescent light mode, using tetramethylrhodamine filters (TRITC). Microscopic analyses were carried out 24 hours and 7 days after the introduction of cells onto the surfaces of the biomaterials and / or their 3D printing. The reference sample consisted of cells in a 2D culture, kept under standard conditions (37°C and 5% CO2). On the basis of the produced results, the presence of the cultured cells was observed both on the biomaterials (cell-seeded biomaterials) and after the printing process (3D bioprinted models). The lack of translucency in the biomaterial prevented the observation of individual cells in the printed 3D models in the TRITC light. For the BCM biomaterial (on the surface of which the cells were seeded) there was no observed cell growth 24 hours after seeding the cells. On the basis of the produced results, increased cell proliferation was observed on the BCM biomaterial after 7 days of culturing, along with an increased number of cells in the models printed using the bioink containing methacrylated chitosan (BCM). There was also an observed presence of cells in the bioprinted 3D models, in the biomaterials with both chitosan (BCH) and methacrylated chitosan (BCM) (Fig. 7).1. Assessing the cytotoxicity of biomaterials by means of the LDH test

[0083] The cytotoxicity of the BCH and BCM biomateriais towards the ceils of the RFP- HDFCs-Neo line was determined by the LDH~Glo test according to the manufacturer's instructions. The test was performed 24 hours after printing and seeding the cells on the biomaterial. The LDH test was conducted according to the manufacturer's protocol. The negative control consisted of cells cultivated under standard conditions, i.e. 37°C and 5% CO2, as well as cell-free biomaterials. The positive control consisted of cells and biomaterials with cells cultured under standard conditions, with an addition of 0.1 % Triton X-100.

[0084] According to an embodiment, cells cultured on biomaterials for the positive control of the BCH / BCM biomaterials exhibited RLU levels (Relative Luminescence Unit) comparable to the positive control of the 2D culture, amounting to: 14,906 ± 345.78, 15,720 ± 289.91 , and 14,948 ± 458.2, respectively. In the case of the negative control, where cells were cultured under standard conditions, the recorded RLU amounted to 9913.5 ± 0.71 . During the test, the luminescence levels were observed to be in line with the level observed for cells grown on the surface of the biomaterials: BCH, where RLU = 9052.75 ± 301.06, and BCM, where RLU = 9673.25 ± 120.73. Furthermore, in the medium collected from the printed models, it was observed that the RLU value of the positive control for cells suspended in BCH and BCM was comparable to the RLU value of the cell control in the 2D culture exposed to 0.1 % Triton X-100, for which the RLU levels were 21 ,782 ± 687.3, 22,123 ± 24.0, and 21 ,737.7 ± 412.3, respectively. The highest RLU level of 35,496.5 ± 7,989.46 was observed for BCH in a 3D~bioprintable model, whereas for a 3D-bioprintable model for BCM, the RLU level was considerably lower compared to the BCH model, amounting to 14,394 ± 1781 .6. The result for BCH was slightly higher than for the negative control (cells cultured under standard conditions), RLU = 12,407.7 ± 737.37. From the results of the LDH test, it can be concluded that no statistically significant differences were observed between the tested groups in the biomaterials with the cells seeded (Fig. 8A). On the other hand, an analysis of the results for models printed in the 3D technology demonstrated statistically significant differences between BCH and BCM at p < 0.0001 (Fig. 8B).3.3 Cell proliferation

[0085] The proliferation of RFP-HDFCs-Neo cells, both in the printed models and in those cultured directly on the surfaces of the biomaterials (BCH and BCM), was assessed on the basis of the Alamar Blue test performed in accordance with the manufacturer's protocol.

[0086] For each biomaterial variant with the cell lines according to the invention, measurements were made at two time points: 3 h and 24 h after seeding the cells on the surface of the biomaterial or printing the constructs with the cells. During the performed test, an increase in cell proliferation was observed within 24 hours after the beginning of the experiment in both types of biomaterials, both on the surface of the biomaterials (Fig. 9A, biomaterials seeded with cells) and the printed models with 3D cells (Fig. 9B, 3D printed models). In particular, increased cell proliferation was demonstrated within 24 hours after exposure by the cells cultured on the surface of the biomaterials (Fig. 9A). Cells cultured on the surface of the BCH biomaterial (after 3 h: RFU = 80,786.25 ± 79,944.9; after 24 h: RFU = 1 , 152, 168 ± 34,419.13) and the BCM biomaterial (after 3 h: RFU = 81 ,461.7 ± 9226.7: after 24 h: RFU = 1 ,215,878 ± 81 ,097.37) demonstrated a comparable increase in proliferation compared to the negative control consisting of cells cultured under standard conditions (after 3 h: RFU = 142,986 ± 18,705.8; after 24 h: RFU = 1 ,068,050 ± 193.8). The highest level of proliferation was demonstrated by cells cultured on the biomaterial enriched with methacrylated chitosan (Fig. 9A, BCM). In the case of the 3D printed models no significant increase in proliferation over time was observed compared to the negative control (after 3 h: RFU = 194,864 ± 16,631.8; after 24 h: RFU = 975,566 ± 52,365.9). In the case of BCH, following an increase in proliferation, the RFU values ranged from 17,495 ± 1623.9 to 154,587.5 ± 27,060.3, and in the case of BCM, the RFU values ranged from 12,970.7 ± 960.7 to RFU = 127,466.3 ± 13,370.6. In the course of the performed tests, it can be concluded that, in the case of biomaterials with seeded cells, there are statistically significant differences between all groups at p < 0.0001 ; after 24 hours, p < 0.0451 for BCH and BCM, whereas no differences were observed after 3 hours for BCH and BCM (Fig. 9A). As shown in Fig. 9B, significant differences at p < 0.0001 were observed for 3D bioprinted models in all of the tested groups.

Claims

Claims1. A three-dimensional printing composition comprising:— a dECM hydrogel-based biomaterial— a hydrogel made of methacrylated PBS biopolymer derivatives with a radical polymerisation photoinitiator— a methacrylated chitosan solution in 1 % acetic acid, neutralised to a final concentration of 3.1 mg / ml in the biomaterial, and / or a chitosan solution in 2% acetic acid, neutralised to a final concentration of 1 .4 mg / ml in the biomaterial.

2. The composition of claim 1 , characterised in that the dECM hydrogel contains dECM lyophilisate at a concentration of 10 to 100 mg / ml;3. The composition according to claim 1 or 2, characterised in that it comprises methacrylated gelatin in PBS at a concentration of 31 mg / ml in the final biomaterial composition, and methacrylated hyaluronic acid in PBS at a final concentration of 3.1 mg / ml in the biomaterial4. The composition according to claims 1-3, characterised in that the radical polymerisation photoinitiator is selected from a 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.

5. The composition according to claims 1-4, characterised in that it comprises lithium phenyl-2,4,6-trimethylbenzoylphosphinate at a concentration of 1.85 mg / ml;6. The composition according to claims 1-5, characterised in that it comprises glycerol, preferably at a concentration of 89.0 mg / ml.

7. The composition according to claims 1-6, characterised in that it comprises:— a dECM hydrogel-based biomaterial enriched with dECM lyophilisate with a final dECM concentration of 76.6 mg / ml;— methacrylated gelatin hydrogel in PBS at a concentration of 31 mg / ml in the final biomaterial composition, and methacrylated hyaluronic acid with PBS at a final concentration of 3.1 mg / ml in the biomaterial, with lithium phenyl-2,4,6-trimethylbenzoylphosphinate as the photoinitiator at a final concentration of 1 .85 mg / ml;— methacrylated chitosan dissolved in 1 % acetic acid and neutralised to a final concentration of 3.1 mg / ml in the biomaterial, and / or chitosan dissolved in 2% acetic acid, neutralised to a final concentration of 1 .4 mg / ml in the biomaterial.

8. A methacrylated chitosan preparation method, characterised in that it comprises successive steps, in which:— a 1 % (v / v) CHsCOOH solution is placed in a reflux condenser reaction vessel and heated up to a temperature of 50°C;— chitosan is added in small portions to the acetic acid solution so that a 1 % (m / v) solution is produced as a result, with simultaneous stirring of its contents at a rate of at least 1 ,000 rpm until the substrate is completely dissolved;— the reaction mixture is sterilised by exposure to UV light for 15 minutes;— methacrylic anhydride (MAA) is gradually added, followed by conducting a reaction under predetermined conditions for a time of 16 to 20 hours;— a PBS solution is added in portions to the reaction mixture, until 5-time dilution of the reaction mixture is obtained, and it is neutralised with a 1 M NaHCO3solution until pH of about 7.0 is obtained;— subsequently, the solution is dialysed for 5 days, with the water replaced twice a day — every 8 h on average;— it is diluted with demineralised water and protected from excessive effects of light, and mixed at a rate of 400 rpm while heating up to 40°C;— after completion of the dialysis process, the solution is thickened in a rotary evaporator at a temperature of 45°C, under a pressure of approximately 40-70 mbar; and— the solution is thickened by cooling down to a temperature of -80°C, and freeze- dried at a temperature of 10°C, under a pressure of 0.10 mbar for 48 h9. The method according to claim 8, characterised in that the chitosan is dissolved in the acetic acid solution while stirred at a rate of 1 ,000-1 ,200 rpm.

10. The method according to claims 8 or 9, characterised in that the methacrylic anhydride is added gradually for 3 hours, preferably at a rate of 5.9 ml / h.

11. The composition according to claims 1—7, characterised in that the methacrylated chitosan is prepared by the method according to claims 8-10.

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

13. Use according to claim 13, characterised in that the printing temperature ranges between 15 and 30°C, the pressure ranges between 5 and 75 kPa, and the printing speed ranges between 5 and 45 mm / s.

14. A three-dimensional printing method using the composition according to claims 1-7 and 11.

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