Water-soluble, biocompatible, biodegradable, 3d-printable, and radiation-curable polymeric products used as bioink

Abstract

This invention provides water-soluble, biocompatible, biodegradable, 3D-printable, and radiation-curable polymeric bioinks for tissue engineering and a variety of other biomedical applications. Synthesized using tailor-designed polyurethane and polyurea oligomers with controlled molecular weights and compositions, these bioinks offer optimized viscosity profiles for 3D printing and enhanced mechanical properties and structural stability. They are applicable in tissue engineering, wound healing, drug discovery, and other biomedical fields.

Description

[0001] WATER-SOLUBLE, BIOCOMPATIBLE, BIODEGRADABLE, 3D-PRINTABLE, AND RADIATION-CURABLE POLYMERIC PRODUCTS USED AS BIOINK

[0002] Technical Field

[0003] The invention relates to water-soluble, biocompatible, biodegradable, 3D-printable, and radiation-curable polymeric products used as bioink.

[0004] Prior Art

[0005] Bioinks are natural or synthetic polymers used for producing artificial living tissues with three- dimensional (3D) printers and are preferred due to their rheological properties and ease of use. These inks contain living cells, which are dispersed in hydrogel materials to protect them from mechanical impacts during bioprinting and ensure their viability. The combination of cells and biopolymer hydrogels is defined as bioink. For bioink to be suitable for 3D printing, it must possess the required viscosity, be shapeable, and be biocompatible. Additionally, the bioink should maintain its shape after printing and be suitable for cross-linking methods. It has been observed that bioinks available in the current technology do not meet the desired combination of mechanical, physical, rheological and / or biological properties.

[0006] In the state of the art, there are very few bioinks that exhibit sufficient material properties. One of the most commonly used bioinks is gelatin methacrylate (GelMA), a hydrogel. GelMA is obtained by reacting gelatin, derived from the hydrolysis of collagen, a protein found abundantly in our body, with methacrylic anhydride. The purpose of this methacrylation process is to produce a biomaterial that can be cross-linked and maintain its structure after bioprinting. GelMA is currently considered the gold standard in bioprinting applications. The primary reasons for this are its ability to support cell viability due to its protein structure and its suitable viscosity for bioprinting. However, there are various limitations associated with the use of GelMA. For example, 3D-printed products obtained with GelMA do not possess the necessary mechanical properties for bioprinting active load-bearing tissues (e.g., skeletal muscle). Additionally, the products obtained through GelMA bioprinting have structural stability issues over the long term, making them unsuitable for tissue engineering. Therefore, there is a need for bioinks that support cell viability, have suitable rheological properties for 3D bioprinting, can maintain their structure over the long term after printing, and have appropriate mechanical properties (e.g., strength, elasticity).

[0007] In the prior art, patent application EP3233493B 1 relates to a new bioink, which is a biomaterial in the form of a water dispersion of cellulose nanofibrils and can be converted into desired 3D shapes using 3D bioprinting technology.

[0008] Patent application W02020 / 081982 relates to a bioink composed of gelatin methacrylate and collagen methacrylate, used for 3D printing of tissue structures.

[0009] Patent application TR 2023 / 014183, filed on 01 / 11 / 2023, is related to the preparation and use of poly(ethylene oxide) (PEO) based and maleamide terminated polyurethane, polyurea, polyamide, and polyester type bioinks.

[0010] In the prior art bioinks, including GelMA and other compositions, exhibit several disadvantages that limit their effectiveness in advanced tissue engineering. One major drawback is their inadequate mechanical properties, particularly in GelMA-based bioinks, which are insufficient for producing load-bearing tissues like skeletal muscle. Additionally, the products obtained from GelMA bioprinting suffer from long-term structural instability, making them unsuitable for applications requiring durable tissue constructs. Moreover, GelMA lacks the necessary strength and elasticity for the production of complex, active tissues, such as musculoskeletal tissues. While GelMA and other bioinks, like those using cellulose nanofibrils and gelatin- collagen methacrylate combinations, offer cross-linking capabilities, their overall rheological properties may not be fully optimized for all tissue types. These limitations, along with a restricted range of material options, necessitate the need for bioinks with enhanced mechanical, physical, and biocompatibility characteristics to support more diverse and complex bioprinting applications.

[0011] Summary of the Invention

[0012] The invention aims to develop synthetic, water-soluble, biocompatible, biodegradable and radiation-curable bioinks for 3D bioprinting, specifically for use in fabricating artificial tissues. These bioinks are based on tailor-designed PEO containing reactive functionally terminated polyurethane and polyurea oligomers which are compatible with biological systems. The focus is on creating synthetic bioinks that provide both structural support during the printing process and a favorable environment for cell growth. The invention aims to provide precise control over the molecular weight and viscosity of the bioinks by designing PEO containing polyurethane, polyurea, polyester, etc., type oligomers with precisely controlled reactive, acrylic terminal groups. By fine-tuning these parameters, the bioinks can be adapted to meet specific mechanical and printing needs for different tissue types, ensuring that the bioinks perform optimally under various 3D bioprinting conditions.

[0013] The invention aims to enable cross-linking of the bioinks through light-based methods (such as UV or visible light) or chemical agents, allowing the printed materials to form stable hydrogels. This ensures that the printed structures maintain their shape and mechanical integrity after printing, which is crucial for supporting cell viability and tissue development.

[0014] The invention further aims to provide bioinks with tunable mechanical properties, such as elasticity, strength, and long-term durability. By adjusting variables like the length of alkylene chains (R1 and R2 groups), the number of repeating units (n and x), and the crosslink density, the bioinks can be customized to suit specific applications, from soft tissues to load-bearing tissues like muscle, offering flexibility in bioprinting different tissue types.

[0015] In addition, the invention aims to ensure that the bioinks are biodegradable and degrade in a controlled manner over time. The rate of degradation can be adjusted based on the chemical composition, ensuring that the bioinks provide structural support during tissue regeneration and break down safely without causing toxicity once their function is complete.

[0016] The overall aim of the invention is to overcome the limitations of existing bioinks, such as GelMA, by offering synthetic bioinks with enhanced mechanical strength, flexibility, stability, and biocompatibility, making them suitable for complex tissue engineering applications, artificial organ production, wound healing, drug delivery systems, and other biomedical uses.

[0017] Drawings

[0018] Figure 1. FTIR spectrum of amino terminated polyurea oligomer

[0019] Figure 2. FTIR spectrum of isocyanate terminated polyurea oligomer

[0020] Figure 3. FTIR spectrum of hydroxyalkyl methacrylate terminated, tetrafunctional polyurea

[0021] Figure 4.1H-NMR spectrum of hydroxy alkyl methacrylate terminated, tetrafunctional polyurea Figure 5. FTIR spectrum of hydroxyalkyl methacrylate terminated tetrafunctional polyurea

[0022] Detailed Description

[0023] Within the scope of the invention, water-soluble polymeric products are developed that are based on polyethylene oxide) (PEO) and consist of oligomers and / or polymers with a wide variety of chemical structures, molecular weights, and physical properties, which are also biocompatible, biodegradable, and used as bioink.

[0024] The polymeric products used as bioink in the invention are prepared by using polyurethane and polyurea oligomers with amino (NH2), hydroxyl (OH), or isocyanate (N=C=O) terminal groups, whose chemical structures are specified below, as precursors to which acrylic reactive terminal groups are attached. Similarly, it is also possible to prepare bioinks based on hydroxyl (OH), amino (NH2) and carboxylic acid (COOH) terminated, PEO containing polyester and polyamide oligomers.

[0025] The chemical structure of amino terminated polyurea oligomers is shown below:

[0026] The chemical structure of hydroxyl terminated polyurethane oligomers is shown below:

[0027] The chemical structure of isocyanate terminated polyurea oligomers is shown below:

[0028] The chemical structure of isocyanate terminated polyurethane oligomers is shown below:

[0029] Wherein:

[0030] • R1 refers to an alkylene group (C2-C16) or an ether bond with a (C2-C16) structure,

[0031] • R2 refers to a linear or branched (C4-C12) alkylene, (C6-C16) cycloalkylene, or (C6- C16) phenylene group,

[0032] • n ranges from 4 to 200, preferably between 10 and 50,

[0033] • x ranges from 1 to 100, preferably between 2 and 50.

[0034] The synthesis methods of amino (NH2), hydroxyl (OH), and isocyanate (NCO) terminated reactive polyurea oligomers with the chemical structures mentioned above are provided below. It is also possible to synthesize polyurethane oligomers using hydroxyl terminated reactants and similar methods. Furthermore, same approach can be used in the preparation of polyester and polyamide-based oligomers and bioinks.

[0035] Synthesis of Amino Terminated Polyurea Oligomers

[0036] The reactions are carried out at room temperature in a 3 -neck, round -bottom Pyrex reactor equipped with a mechanical stirrer and a controlled dropping funnel. Based on the desired molecular weight, amine terminated poly (ethylene oxide) oligomer (H2N-PEO-NH2) is weighed into the reactor and dissolved in the reaction solvent to achieve a concentration of about 25-30% by weight. In a separate beaker, required amount of diisocyanate (OCN-R-NCO) is weighed and dissolved in the solvent to reach a similar concentration. Diisocyanate solution is then added drop wise into the reactor over approximately 15-20 minutes while stirring vigorously. After complete addition of the diisocyanate solution, amino terminated polyurea oligomer is obtained. Average molecular weight of the oligomer is controlled by the molar ratio of [H2N-PEO-NH2] to [OCN-R-NCO] used in the reaction.

[0037] Example 1. Amino terminated polyurea oligomer with a number average molecular weight of about 6000 g / mol is synthesized as follows. 6.45 g (10.00 mmol) of amino terminated PEO (Huntsman Chemical ED600, Mn=645 g / mol) was weighed into a 150 m , 3-neck, round- bottom Pyrex flask equipped with an overhead mechanical stirrer and a dropping funnel and dissolved in 20 mL of THF. 1.46 g (8.68 mmol) of hexamethylene diisocyanate (HDI) is weighed into a beaker, dissolved in 8 mL of THF, introduced into the addition funnel and added dropwise into the reactor at room temperature over 15-20 minutes. Completion of the reaction is confirmed by FTIR spectroscopy (Figure 1). Average molecular weight of the oligomer produced is determined by titration of the amino end groups with dilute hydrochloric acid.

[0038] Synthesis of Isocyanate Terminated Polyurea Oligomers

[0039] The reactions are conducted at room temperature in 3-neck, round-bottom Pyrex reactors equipped with mechanical stirring and a dropping funnel. Diisocyanate (OCN-R-NCO) is weighed according to the desired molecular weight and dissolved in pure reaction solvent (IPA or THF) to achieve a 25-30% concentration by weight and introduced into the reactor. In a separate container, amine terminated poly (ethylene oxide) (PEO) oligomer (H2N-PEO-NH2) is weighed and dissolved in IPA or THF to reach a concentration of 25-30% by weight. PEO solution is transferred into the dropping funnel and added dropwise into the reactor, at room temperature, over 15 minutes under vigorous stirring. Average molecular weight of the oligomer is controlled by the molar ratio of [OCN-R-NCO] to [H2N-PEO-NH2] used in the reaction.

[0040] Example 2. Procedure used in the synthesis of isocyanate terminated polyurea oligomer with a number average molecular weight of about 6000 g / mol is as follows. 1.92 g (11.41 mmol) of hexamethylene diisocyanate (HDI) is weighed into a 150 mL, 3-neck, round-bottom Pyrex flask equipped with an overhead mechanical stirrer and a dropping funnel and dissolved in 12 mL of THF. 6.71 g (10.40 mmol) amino terminated PEO (Huntsman Chemical ED600, Mn=645 g / mol) is weighed into a beaker, dissolved in 20 mL of THF, introduced into the addition funnel and added drop wise into the reactor at room temperature over 15-20 minutes. Completion of the reaction is confirmed by FTIR spectroscopy (Figure 2), where a strong isocyanate peak at 2274 cm1indicates the presence of NCO end groups. Average molecular weight of the oligomer is determined by reacting the isocyanate end-groups with an excess of standard dibutylamine solution in IPA, followed by the back titration of the remaining amine with dilute hydrochloric acid.

[0041] Acrylic or methacrylic terminated, free-radically crosslinkable bioinks produced in this invention are prepared by the reaction of amino (NH2), hydroxyl (OH), or isocyanate (NCO) terminal groups present on polyurethane and polyurea oligomers with hydroxyalkyl, aminoalkyl, isocyanatoalkyl, and glycidyl acrylates or methacrylates.

[0042] Synthesis of Hydroxy alkyl Methacrylate Terminated, Tetrafunctional Polyurea Based Bioinks

[0043] Reactions are conducted in a three-necked, round-bottom Pyrex reactor equipped with mechanical stirring, a thermometer, and a controlled dropping funnel. The required amount of glycidyl methacrylate (GMA) monomer is dissolved in THF at a concentration of approximately 50% by weight and transferred into the dropping funnel. GMA solution is added into the amino terminated polyurea oligomer solution in the reactor over 2-3 minutes, and the solution is heated to reflux. The completion of the reaction is confirmed by FTIR spectroscopy.

[0044] Example 3. 0.75 g (5.28 mmol) of glycidyl methacrylate is dissolved in 5 mL of THF and introduced into the reactor containing amino terminated polyurea oligomer synthesized in Example 1, together with 0.5 mL of methanol. The system is heated up to reflux for 8 hours. Product obtained is coagulated in hexane, filtered and dried at room temperature until constant weight. Formation of the product is confirmed by FTIR (Figure 3) and 1H-NMR (Figure 4) spectroscopy.

[0045] In 1H-NMR spectrum of hydroxy alkyl methacrylate-terminated, tetrafunctional polyurea two singlet peaks present at 5.58-6.14 ppm belong to the protons of the alkene (-C=CH2) group found in the glycidyl methacrylate group. The broad signal observed between 5.1-5.6 ppm is thought to belong to the -NH and OH protons. The multiplet signals found between 3.3-3.7 ppm are attributed to the protons in -OCH2. The sharp singlet signal observed at 1.9 ppm corresponds to the CH3 protons on the glycidyl methacrylate. The multiplet signals observed between 1.00-1.50 ppm indicate the protons belonging to alkyl CH2.

[0046] Synthesis of Acrylic Urethane Terminated Polyurea Based Bioinks

[0047] Acrylic urethane terminated polyurea based bioinks can be prepared by two different routes, which are either by the reaction of isocyanate-terminated polyurea oligomers with hydroxyalkyl acrylates or by the reaction of hydroxy terminated polyurea oligomers with isocyanatoalkly acrylates. In Example 4 given below detailed description of the synthesis of acrylic urethane-terminated polyurea based bioink is provided. Example 4. Reaction is carried out in a three-neck, round-bottom Pyrex reaction flask. 0.24 g (2.06 mmol) of 2-hydroxyethyl acrylate is dissolved in 5 mF of THF in a beaker, introduced into the addition funnel and added into the reactor containing the isocyanate terminated polyurea oligomer synthesized in Example 2. 50 ppm dibutyltin dilaurate in THF is added as catalyst and the reaction mixture is heated to reflux for 6 hours. Completion of the reaction is monitored by FTIR spectroscopy following the disappearance of strong isocyanate peak located at 2274 cm1and formation of strong acrylate carbonyl peak located at 1724 cm1(Figure 5). Product obtained is coagulated in hexane, filtered and dried at room temperature until constant weight. The yield was quantitative. Chemical structures of polyurea and polyurethane based bioinks invented, with difunctional or tetrafunctional reactive end groups, are shown below:

[0048] • Hydroxyalkyl acrylate and hydroxyalkyl methacrylate terminated, difunctional polyurea based bioinks:

[0049] • Hydroxyalkyl acrylate and hydroxyalkyl methacrylate terminated, tetrafunctional polyurea based bioinks:

[0050] • Hydroxyalkyl acrylate and hydroxyalkyl methacrylate terminated polyurethane based bioinks:

[0051] • Acrylamide and methacrylamide terminated poly urea based bioinks:

[0052] • Acrylic and methacrylic ester terminated polyurethane based bioinks:

[0053] • Acrylic and methacrylic urea- terminated polyureas based bioinks:

[0054] • Acrylic and methacrylic urethane-terminated polyurethane based bioinks:

[0055] • Acrylic and methacrylic urethane-terminated poly ureas based bioinks: Wherein;

[0056] • The group R1 represents an alkylene chain (C2-C16) or an ether linkage (C2-C16),

[0057] • The group R2 represents a linear or branched alkylene chain (C4-C12), a cycloalkylene group (C6-C16), or a phenylene group (C6-C16),

[0058] • The variable n ranges from 4 to 200, preferably between 10 and 50, • The variable x ranges from 1 to 100, preferably between 2 and 50,

[0059] • The variable m ranges from 2 to 6, preferably 2 or 3.

[0060] The methods used for synthesizing amino (NH2), hydroxyl (OH), and isocyanate (NCO) terminated reactive polyurea oligomers and acrylic and methacrylic terminated polymers have been described. Similar methods can be used to synthesize polyurethane based oligomers by employing hydroxyl terminated reactants. Furthermore, similar approaches can be used in the preparation of polyester and polyamide-based oligomers and bioinks. The polymeric products described in the invention are designed to be water-soluble, crosslinkable, and have tunable viscosity and mechanical properties, making them highly adaptable for various biomedical applications. Their water solubility ensures a safe application medium, while cross -linkability (triggered by light or chemical agents) allows the bioinks to solidify into stable structures that maintain shape and mechanical integrity after printing. The mechanical properties, such as elasticity, strength, and durability, can be customized by modifying the chemical structure, including the length of polymer chains and cross-linking density. This tunability makes the bioinks suitable for a wide range of tissue engineering applications, from soft tissues to load-bearing tissues, offering flexibility in meeting specific mechanical and biological requirements.

[0061] In one application, these polymeric products are used as bioinks for preparing scaffolds for tissue engineering applications. The water-soluble, cross-linkable, biocompatible, and biodegradable bioinks developed in this invention are suitable not only for tissue engineering applications but also for various fields, including chemistry, biology, biomedicine, artificial organ and tissue production, muscle and skeletal system injuries, tissue damage, burn treatments, tissue regeneration, controlled release systems, 3D cell culture applications, as well as in cosmetics and drug discovery.

Claims

CLAIMS1. Water-soluble, biocompatible, biodegradable, 3D-printable, and radiation curable synthetic polymeric products used as bioinks, comprising polyurea or polyurethane and at least one of the functional groups which are acrylate, methacrylate, acrylamide, methacrylamide, acrylic urea, methacrylic urea, acrylic urethane, methacrylic urethane, acrylic ester, methacrylic ester, or hydroxyalkyl, as shown below:wherein;- R1 is an alkylene group (C2-C16) or an ether bond with a structure (C2-C16),- R2 is a linear or branched (C4-C12) alkylene, (C6-C16) cycloalkylene, or (C6- C16) phenyl group, - n is between 4 and 200,- x is between 1 and 100, m is between 2 and 6.

2. The polymeric products according to claim 1, characterized in that n is a number between 10 and 50.

3. The polymeric products according to claim 1, characterized in that m is a number between 2 and 50.

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