Pre-crosslinking process, bio-ink composition containing pre-crosslinked methacrylate, and use thereof in bioprinting.

Abstract

A method for pre-crosslinking methacrylate, comprising the step of placing an aqueous solution containing methacrylate at a pH of 4 to 8, with a molecular weight of 1 kDa to 500,000 kDa, preferably 30 to 70 kDa, and having at least 0.5% polymerizable functional groups, at a concentration of 0.5 to 50% (w / v), and a photoinitiator at a concentration of 0.0125 to 2% (w / v), in a transparent container, wherein the mixture is subjected to a wavelength of 250 nm to 800 nm and an output of 1 to 1000 mW / cm². 2 The container is subjected to UV-VIS radiation for a period of no more than 1000 seconds, and rotated 180 degrees during half of the crosslinking time.

Description

[Technical Field]

[0001] This invention relates to a process for pre-crosslinking low-molecular-weight polymers by partial polymerization using UV-VIS radiation. This technique is applicable even to highly fluid hydrogels, which exhibit excessively liquid forms due to their mechanical properties and pose problems in bioprinting applications. This technique does not preclude the use of cells, i.e., bioprinting with biomaterials. [Background technology]

[0002] US20210046220A1 discloses a hydrogel composition comprising a crosslinked biodegradable polymer. The composition may optionally contain microgel cells crosslinked with a second agent. Paragraph

[0089] states that the resulting composition is subjected to approximately 20 mW / cm² to stabilize the resulting structure. 2 It states that UV irradiation was performed for 1 minute. US9045657B2 describes a viscoelastic ink for directly producing hydrogel structures, the ink comprising a long-chain polymer and a photopolymerizable moiety (a photopolymerizable monomer or a photopolymerizable group bonded to the long-chain polymer). The ink may further comprise a crosslinking agent, a photoinitiator, and water. Paragraph

[0125] states that the composition is exposed to approximately 100 mW / cm² of 320 nm light. 2 It is stated that it can be cured with UV irradiation for 1 minute at this intensity, and can be cured at an even lower intensity with 250nm light. EP3412728B1 discloses a microcapsule comprising a shell having a three-dimensional crosslinked structure including at least one bond selected from urethane bonds or urea bonds, and a core containing a polymerizable compound and a photopolymerization initiator. Paragraph

[0511] discloses that in the irradiation step, UV light irradiation for 0.01 seconds to 120 seconds, preferably 0.1 seconds to 90 seconds, is advantageous. WO2022109284A1 discloses a method for shrinking hydrogels. The method includes contacting a polyionic hydrogel with a polyionic solution containing an ionic polymer having the opposite net charge to the polyionic hydrogel for a time sufficient to reduce its volume. The document also describes hydrogel compositions produced using this method and methods for using these hydrogels in 3D printing. Paragraph

[0109] describes UV irradiation (approximately 10 mW / cm²). 2 It is disclosed that photocrosslinking was initiated by (360-480 nm, 40 seconds). US20220306877A1 describes a method for manufacturing structures for 3D printing of biocompatible structures, which includes manufacturing a photoreactive protein resin, curing the resin by irradiation with light of a selected wavelength to form a desired structure, thereby 3D printing a biocompatible structure. A suitable photoreactive protein resin can be prepared by combining an aqueous solution of acrylic or methacrylated globular protein with a photoreactive copolymer or photoinitiator. Structures printed from the photoreactive protein resin can be subjected to photocuring and drying to produce bioplastic structures. Paragraph

[0072] and Table 4 disclose photocuring times ranging from 2 to 18 seconds. WO2022233736A1 describes a radiation-curable composition comprising at least one water-soluble monofunctional unsaturated monomer, at least one water-soluble non-curable component, and at least one photoinitiator. Paragraph

[0093] states that the radiation-curable liquid composition can be cured by actinic light having sufficient energy to initiate polymerization or crosslinking reactions. Actinic radiation includes alpha rays, gamma rays, UV radiation, visible light, and electron beams, with UV radiation and electron beams, particularly UV radiation, being preferred. Paragraph

[0094] discloses that the irradiation time may be in the range of 0.5 to 10 seconds, preferably in the range of 0.6 to 6 seconds. US20170319746A1 discloses a composition comprising two materials crosslinked by a reaction induced by a common activator. According to paragraph

[0058] , in a preferred composition, the first crosslinking reaction is relatively fast, preferably on the order of about 0.1 to 10 seconds (e.g., about 1 second), and the second crosslinking reaction is relatively slow, preferably on the order of about 10 to 60 minutes (e.g., about 15 to 30 minutes). US20200255818A1 discloses a mixture for forming polymer-encapsulated cells. The mixture comprises a prepolymer, a photoinitiator, and cells. According to paragraph

[0136] , the mixture of cells, prepolymer, and photoinitiator can be cured by UV radiation to crosslink the prepolymer. In some cases, curing may involve UV radiation in the range of 300 nm to 450 nm for a time sufficient to crosslink the prepolymer to encapsulate the cells. In some cases, UV curing may take less than 30 seconds, less than 15 seconds, or less than 10 seconds. [Overview of the project] [Problems that the invention aims to solve]

[0003] The object of the present invention is to develop a bioprinting material preparation process that includes pre-crosslinking and, in particular, increases the viscosity of water-soluble low molecular weight polymer hydrogels. This process improves the mechanical properties of the hydrogel and affects its printability. 3D printing is impossible when the polymer solution concentration is low and the molecular weight is low. Pre-crosslinking by partial polymerization using UV-VIS light increases the viscosity of the material, thereby enabling printing with such pre-crosslinked (PCL) polymers. Another object of the present invention is to provide a bioprinting process using a bioink containing a pre-crosslinked polymer. [Means for solving the problem]

[0004] The present invention relates to a preliminary crosslinking process for methacrylate, comprising the following steps. A step of placing an aqueous solution containing a methacrylate with a pH of 4 to 8, a molecular weight of 1 kDa to 500,000 kDa, preferably 30 to 70 kDa, and containing at least 0.5% of functional groups to be polymerized, at a concentration of 0.5 to 50% (w / v), and a photoinitiator at a concentration of 0.0125 to 2% (w / v), into a transparent container, The aforementioned mixture has a wavelength of 250 nm to 800 nm and an output of 1 to 1000 mW / cm. 2 The UV-VIS irradiation is applied for a period not exceeding 1000 seconds. The container is rotated 180 degrees during half of the crosslinking time. Preferably, the methacrylate is selected from the group including GELMA, HAMA, ALGMA, and CHIMA. Preferably, the photoinitiator is selected from the group comprising lithium phenyl-2,4,6-trimethylbenzoylphosphine, 2-hydroxy-1-(4-(2-hydroxyethoxy)phenyl)-2-methylpropan-1-one and its derivatives, or derivatives of diazo compounds. Preferably, the aqueous solution is physiological saline, cell culture medium, or a weakly acidic solution. Preferably, the crosslinking is performed in a syringe with a cross-section of 1 to 100 nm. The present invention also relates to a bio-ink composition comprising pre-crosslinked methacrylate. Preferably, the pre-crosslinked methacrylate has a degree of conversion expressed as the content of converted methacrylate groups in the range of 0.1% to 99.9%, preferably in the range of 40% to 60%. Preferably, it contains pre-crosslinked methacrylate at a concentration of 0.5% to 50% (w / v) and uncrosslinked methacrylate at a concentration of 0.1% to 99.9% (w / v). Preferably, it is 5 × 10 per 1 mL of the bio-ink composition. 5 It further contains more than one L929 cell. The present invention further relates to compositions used in bioprinting processes. Preferably, the printing temperature is in the range of 5 to 50 °C, the pressure is in the range of 5 to 150 kPa, the printing speed is in the range of 1 to 100 mm / s, the needle diameter is in the range of 50 to 900 μm. After the printing is completed, UV-VIS light with a wavelength of 280 nm to 800 nm, preferably 405 nm, is output for 1 second to 360 seconds, preferably 30 seconds, at an output of 1 to 1000 mW / cm 2 , preferably 28.5 mW / cm 2 and irradiated. Preferably, the printing includes extrusion or volumetric printing.

[0005] The advantage of the present invention is that a bioink composition containing a methacrylate pre-crosslinked by the process according to the present invention enables printing of a material showing continuous dense fibers, enables printing with good / very good resolution, and can be additionally crosslinked after bioprinting. Compared with a solution without pre-crosslinking, the obtained material has significantly improved printability parameters.

Brief Description of the Drawings

[0006] The present invention is also represented in the following drawings. Figure 1 shows the scheme of the gcode file (template.gcode). Figure 2 shows a schematic diagram of the fiber deflection test platform. Figure 3 shows a microscopic photograph of the structure in the fiber bonding test. Figure 4 shows the percentage Dfr of the fiber diffusion rate (fiber bonding test). Figure 5 shows the printability Pr (fiber bonding test). Figure 6 shows a photograph of the structure in the fiber deflection test. Figure 7 shows the fiber crushing coefficient Cf (fiber deflection test). Figure 8 is a diagram showing the crosslinking kinetics of a selected hydrogel based on HAMA. Figure 9 shows the crosslinking kinetics of the hydrogel based on ALGMA. Figure 10 shows the crosslinking kinetics of the hydrogel based on GELMA. Figure 11 shows the growth degree of the cells subjected to printing in the tested biomaterials. Figure 12 shows the growth degree of the cells seeded on the tested biomaterials. Figure 13 shows the observation results after bright field (BF) transmission light and FDA / Pi staining (green / red) of the cells printed in the biomaterial 4% ALGMA + GELMA [3% (w / v) ALGMA (low viscosity): 1% (w / v) GELMA with 0.25% (w / v) LAP added]. Figure 14 shows the observation results after bright field (BF) transmission light and FDA / Pi staining (green / red) of the cells seeded on the biomaterial 2% HAMA + GELMA [1.5% (w / v) HAMA (130 - 300 kDa): 1% (w / v) GELMA with 0.25% (w / v) LAP added]. Figure 15 shows the observation results after bright field (BF) transmission light and FDA / Pi staining (green / red) of the cells seeded on the biomaterial 4% HAMA + GELMA [3% (w / v) HAMA (30 - 50 kDa): 1% (w / v) GELMA with 0.25% (w / v) LAP added]. Figure 16 shows the observation results after bright field (BF) transmission light and FDA / Pi staining (green / red) of the cells seeded on the biomaterial 4% ALGMA + GELMA [3% (w / v) ALGMA (low viscosity): 1% (w / v) GELMA with 0.25% (w / v) LAP added]. Figure 17 shows the observation results after bright field (BF) transmission light and FDA / Pi staining (green / red) of the cells seeded on the biomaterial 4% GELMA [4% (w / v) GELMA + 0.25% (w / v) LAP]. Figure 18 shows the observation results after bright field (BF) transmission light and FDA / Pi staining (green / red) of the cells seeded on the biomaterial 1.5% ALGMA + GELMA [0.75% (w / v) ALGMA (high viscosity): 1% (w / v) GELMA with 0.25% (w / v) LAP added]. Figure 19 is a schematic diagram showing the operating principle of the preliminary cross-linking technique by exemplifying a methacrylate derivative of a natural polymer. Figure 20 is a diagram showing the characteristics of the sample printout and includes the following photographs. 1: Smooth and dense fibers, a structure with good resolution, the structure can be lifted from the pan, is not brittle, and forms a dense and smooth drop. 2: Smooth and slightly lumpy fibers, a structure with very good resolution, the structure can be lifted from the pan, is not brittle, and forms a dense and smooth drop, but small lumps can be seen. 3: No difference in the fiber structure is observed compared with the case of performing preliminary cross-linking with the same parameters at a low printing temperature. Compact fibers, good extrudability. Figure 21 is a diagram showing the characteristics of the printout and includes the following photographs. 1: Very dense and slightly lumpy fibers. High-resolution printing. The 410 μm needle became clogged. The structure could be lifted from the pan and was not brittle. A very compact drop. 2: Smooth and slightly dense fibers, a structure with good resolution. The structure could be lifted from the pan and was not brittle. A compact and smooth drop. 3: Compact and slightly lumpy fibers. Good printing resolution. Clogging of the 410 μm needle. The structure could be lifted from the pan and was not brittle. A very compact drop. 4: Compact and slightly lumpy fibers. Very good printing resolution. Clogging of the 410 μm needle. The structure could be lifted from the pan and was not brittle. A very compact drop. Figure 22 shows the quality comparison of the extruded fibers. Figure 23 shows the disappearance of the methacrylate group signal during the cross-linking process at 2% HAMA, 0.25% LAP, 405 nm, P = 20 mW / cm 2 Figure 24 shows the disappearance of the methacrylate group signal during the cross-linking process at 2% HAMA, 0.25% LAP, 405 nm, P = 28.5 mW / cm 2 ​​Figure 25 shows 2% HAMA, 0.25% LAP, 365 nm, P=13 mW / cm². 2 This shows the disappearance of the methacrylate group signal during the crosslinking process. Figure 26 is a diagram illustrating the characteristics of the printout and includes the following photographs. 1: A smooth, highly fluid fiber. It is impossible to create structures with good resolution. 2: The fibers are somewhat fluid, but can produce structures with average / good resolution. Needle clogging does not occur. The drops exhibit some fluidity. The structures are flexible and can be removed from the pan. 3: Dense and slightly stretchable. Very good resolution structure. No needle clogging occurs. Compact and smooth drop. The structure is flexible and can be removed from the pan. Figure 27 is a diagram illustrating the characteristics of the printout and includes the following photographs. 1: The fibers are somewhat fluid, but can produce structures with average / good resolution. Needle clogging does not occur. The drops exhibit some fluidity. The structures are flexible and can be removed from the pan. 2: Dense, somewhat lumpy fibers, resulting in a very good resolution structure. No needle clogging occurs. The structure is flexible and can be removed from the pan. Compact and smooth drop. 3: Dense, somewhat lumpy fibers, resulting in a very good resolution structure. No needle clogging occurs. The structure is flexible and can be removed from the pan. Compact and smooth drop. [Modes for carrying out the invention]

[0007] The crosslinking level of the pre-crosslinked material is measured as the amount of conversion of methacrylate groups or other groups to be polymerized, and is in the range of 0.1% to 99.9%, preferably 40% to 60%. The crosslinking parameters should be selected so that the number of polymerizable groups does not fall below 0.5%, thereby enabling additional crosslinking after bioprinting and complete crosslinking of the printed material, as well as forming durable bonds in the structure. The entire process consists of three steps. 1. Prepare a material using pre-crosslinking to increase the viscosity of a methacrylate-based hydrogel. 2. Perform bioprinting using pre-crosslinked hydrogels. 3. To form durable bonds between fibers, the material is cross-linked / additionally cross-linked after printing (after each layer).

[0008] The crosslinking method according to the present invention can be applied to the following methacrylate solutions: GELMA (methacrylated gelatin), HAMA (methacrylated hyaluronic acid), ALGMA (methacrylated sodium alginate derivative), CHIMA (methacrylated chitosan), and other natural polymer derivatives, having a concentration in the range of 0.5% to 50%, and having a photoinitiator (selected from the group consisting of lithium phenyl-2,4,6-trimethylbenzoylphosphine, 2-hydroxy-1-(4-(2-hydroxyethoxy)phenyl)-2-methylpropan-1-one and its derivatives, or diazo compound derivatives, complexes and transition metal salts, peroxides and halogens, and other initiators) in the range of 0.0125% to 2%, and which, in their original form, cannot be used for high-resolution 3D printing. Materials that have been methacrylated (or functionalized with other substituents that enable crosslinking), namely gelatin, hyaluronic acid, alginic acid, chitosan, and other natural polymer derivatives having a molecular weight in the range of 1 kDa to 500,000 kDa, preferably 30 kDa to 70 kDa, and exhibiting a degree of substitution of 10 to 100% after functionalization with methacrylate, acrylate, styrene, or other substituents containing polymerizable double or triple bonds. This includes mixed polymerization between atoms of carbon, nitrogen, sulfur, and other elements. The process involves working with polymers that are completely soluble in an aqueous environment at concentrations of 0.1 to 100 mL, and includes physiological saline, cell culture media adapted to a specific cell system, or a weakly acidic solution with a pH of 4 to 8.

[0009] Pre-crosslinking requires preparing 0.1 mL to 10 mL of a suitable hydrogel solution in an open container or a container with appropriate UV-VIS light transmission, and subjecting it to pre-crosslinking at intervals of 1 second to 1000 seconds, i.e., for the time necessary to impart a printable form. The wavelength used for pre-crosslinking is in the range of 250 nm to 800 nm. The light output is 1 mW / cm². 2 ~1000mW / cm 2This is within the range. The preliminary crosslinking treatment is preferably carried out using a transparent cylindrical container equipped with a plunger, or another container that allows the thickness of the crosslinked layer to be controlled and the material to be transferred to a printer head with a cross-section of 1 mm to 100 mm.

[0010] Pre-crosslinking (PCL) materials can be used as a single material in bioinks, or as an additive to mixtures of other non-crosslinking materials at concentrations of 0.1% to 99.9%, or as pre-crosslinking materials at concentrations of 0.5% to 50%, accounting for 0.1% to 99.9% of the total blend. Pre-crosslinking (PCL) materials or their blends can be applied to biological tests using the reference cell line L929 in the initial stages of testing, or, in accordance with ISO 10993-5, to CCL1 (NCTC clone 929), CCL163 (Balb / 3T3 clone A31), CCL171 (MRC-5), CCL75 (WI-38), CCL81 (Vero), and CCL10 [BHK-21 (C-13) and V-79 379A], as well as other commercially available cell lines or cell lines isolated from human or animal tissues, provided that identical or similar MTT test results are obtained. In particular, it can be applied to endothelial cells, stem cells, pancreatic islet cells (α, β, δ), bone, muscle, or nerve cells, and in later stages of testing, specific cell lines can be used depending on the application. The number of cells in 1 mL of bioink is 5 × 10⁶. 5 The above is complete. Pre-crosslinking (PCL) material, its blend, and material with added cell lines can be homogenized by syringe mixing, a cell mixer specifically designed for biological applications, or other specialized equipment.

[0011] This technology encompasses extrusion and volumetric printing, as well as other printing techniques that require appropriate material viscosity to obtain uniform, dense fibers and maintain print resolution. Depending on the material used, the printing temperature can vary in the range of 5°C to 50°C. The printing pressure in extrusion printing is in the range of 5 to 150 kPa. The printing speed is in the range of 1 to 100 mm / s. The needle diameter is in the range of 50 to 900 μm. The printed model may have any dimensions expressed in mm, and may have a fill density of 5% to 100% to obtain an open workpiece printout.

[0012] I. Preparation of the solution 1) Solution GELMA4%(w / v)+LAP0.25%(w / v) A 10.0 mL solution of GELMA 4% (w / v) + LAP 0.25% (w / v) was prepared. This was lyophilized GELMA with a degree of substitution of 100%. Using a serum pipette, 10.0 mL of PBS × 1 was transferred to a 50 mL flask. LAP was weighed on a weighing vessel using an analytical balance, and then 25.0 ± 0.5 mg of LAP was transferred to the flask containing PBS × 1. The flask containing the solution was transferred to a thermoblock (30-50°C) and then stirred at 100-1000 rpm for 15-30 minutes (until the photoinitiator was dissolved). Then, the lyophilized GELMA was weighed and 400.0 ± 1.0 mg was added to the photoinitiator solution. The GELMA solution was placed in a thermoblock (30-50°C) and stirred at 100-1000 rpm until completely dissolved. After the GELMA was completely dissolved, the pH of the solution was checked and adjusted to a range of 7.2-7.4. The prepared solution was filtered through a 0.22-0.45 μm syringe filter. The filtered solution was transferred to a 50 mL flask wrapped in aluminum foil and stored in the refrigerator until use.

[0013] 2) Solution HAMA2%(w / v)+LAP0.25%(w / v) A 10.0 mL solution of HAMA 2% (w / v) + LAP 0.25% (w / v) was prepared. This solution contained hyaluronic acid with a molecular weight of 130-300 kDa and lyophilized HAMA with a degree of substitution of 25%. 2.0 mL of PBS×1 was transferred to a 15 mL flask. LAP was weighed on a weighing vessel using an analytical balance, and then 25.0 ± 0.5 mg was transferred to the flask containing PBS×1. The flask was wrapped in aluminum foil and placed in a thermoblock, and shaken at 100-1000 rpm and 30-50°C. After the photoinitiator had dissolved, the flask was placed in the refrigerator. 8.0 mL of PBS×1 was transferred to a 50 mL flask. Then, the lyophilized HAMA was weighed, and 200.0 ± 1.0 mg was transferred to the flask containing PBS×1. The HAMA solution was shaken in a thermoblock at 100-1000 rpm and 4-20°C for 0.5-4 hours. After the lyophilized material had dissolved, the cooled LAP solution was added, and the mixture was placed in the thermoblock for 10-30 minutes to mix the components. After the components were completely mixed, the pH of the solution was checked and adjusted to a range of 7.2-7.4. The prepared solution was filtered through a syringe filter with a diameter of 0.22–0.45 μm. The filtered solution was transferred to a 50 mL flask wrapped in aluminum foil and stored in the refrigerator until use.

[0014] 3) Solution HAMA4%(w / v)+LAP0.25%(w / v) A 10.0 mL solution of HAMA 4% (w / v) + LAP 0.25% (w / v) was prepared. This solution contained hyaluronic acid with a molecular weight of 30-50 kDa and lyophilized HAMA with a degree of substitution of 21%. 2.0 mL of PBS×1 was transferred to a 15 mL flask. LAP was weighed on a weighing vessel using an analytical balance, and then 25.0 ± 0.5 mg was transferred to the flask containing PBS×1. The flask was wrapped in aluminum foil and placed in a thermoblock, and shaken at 100-1000 rpm and 30-50°C. After the photoinitiator had dissolved, the flask was placed in the refrigerator. 8.0 mL of PBS×1 was transferred to a 50 mL flask. Then, the lyophilized HAMA was weighed, and 200.0 ± 1.0 mg was transferred to the flask containing PBS×1. The HAMA solution was shaken in a thermoblock at 100-1000 rpm and 4-20°C for 0.5-4 hours. After the lyophilized material had dissolved, the cooled LAP solution was added, and the mixture was placed in the thermoblock for 10-30 minutes to mix the components. After the components were completely mixed, the pH of the solution was checked and adjusted to a range of 7.2-7.4. The prepared solution was filtered through a syringe filter with a diameter of 0.22–0.45 μm. The filtered solution was transferred to a 50 mL flask wrapped in aluminum foil and stored in the refrigerator until use.

[0015] 4) Solution ALGMA1.5%(w / v)+LAP0.25%(w / v) A 10.0 mL solution of ALGMA 1.5% (w / v) + LAP 0.25% (w / v) was prepared. This is a highly viscous alginate, lyophilized ALGMA with a degree of substitution of 25%. Using a serum pipette, 10.0 mL of PBS × 1 was transferred to a 50 mL flask. Using an analytical balance, LAP was weighed on a weighing vessel, and then 25 ± 0.5 mg of LAP was transferred to the flask containing PBS × 1. The flask containing the solution was transferred to a thermoblock (20-30°C) and then stirred at 100-1000 rpm for 15-30 minutes (until the photoinitiator was dissolved). Subsequently, the lyophilized ALGMA was weighed, and 150.0 ± 1.0 mg was added to the photoinitiator solution. The ALGMA solution was placed in a thermoblock (20-30°C) and stirred at 100-1000 rpm for 1-24 hours until completely dissolved. After the ALGMA was completely dissolved, the pH of the solution was checked and adjusted to a range of 7.2-7.4. The prepared solution was filtered through a 0.22-0.45 μm syringe filter. The filtered solution was transferred to a 50 mL flask wrapped in aluminum foil and stored in the refrigerator until use.

[0016] 5) Solution ALGMA4%(w / v)+LAP0.25%(w / v) A 10.0 mL solution of ALGMA 4% (w / v) + LAP 0.25% (w / v) was prepared. This was a low-viscosity alginate, lyophilized ALGMA with a degree of substitution of 39%. Using a serum pipette, 10.0 mL of PBS × 1 was transferred to a 50 mL flask. Using an analytical balance, LAP was weighed on a weighing vessel, and then 25 ± 0.5 mg of LAP was transferred to the flask containing PBS × 1. The flask containing the solution was transferred to a thermoblock (20-30°C) and then stirred at 100-1000 rpm for 15-30 minutes (until the photoinitiator was dissolved). Subsequently, 400.0 ± 1.0 mg of lyophilized ALGMA was weighed and added to the photoinitiator solution. The ALGMA solution was placed in a thermoblock (20-30°C) and stirred at 100-1000 rpm for 1-24 hours until completely dissolved. After the ALGMA was completely dissolved, the pH of the solution was checked and adjusted to a range of 7.2-7.4. The prepared solution was filtered through a 0.22-0.45 μm syringe filter. The filtered solution was transferred to a 50 mL flask wrapped in aluminum foil and stored in the refrigerator until use.

[0017] 6) Preparation of Pre-Crosslinking (PreCL) Materials A needle was attached to a syringe to collect 1.0–3.0 mL of methacrylate solution, which was then sealed with a blue protective plug. The syringe containing the material was pre-crosslinked according to the parameters shown in Table 1. For this purpose, the syringe was placed under a Polbionic AUV-Vis lamp. The material was uniformly crosslinked by rotating the syringe 180° at half the crosslinking time. After crosslinking, the syringe was protected from light by wrapping it in aluminum foil. The protected syringe was allowed to stand for 2–5 minutes. Then, the pre-crosslinked solution was mixed between multiple syringes, and the plunger was pushed 20 times. The material thus prepared can be used directly for printing or for preparing blends for biological testing. [Table 1]

[0018] 7) Preparation of blends for biological testing Pre-crosslinking (PCL) material syringes were filled with appropriate amounts of GELMA (without pre-crosslinking) and L929 cell suspension (in the case of a variation where cells are suspended in biomaterial). The proportions used to prepare the material are shown in Table 2. The components were then mixed between multiple syringes, and the plunger was pushed 10-20 times until a homogeneous mixture was obtained. The material thus prepared was transferred to a cartridge and used for printing. For each layer, the structure was crosslinked according to a general pattern. [Table 2]

[0019] II. Printability Test Printability tests were conducted using a BioX CELLINK printer. The printing parameters for each material are shown in Table 3. The applied parameters resulted in uniform and dense fibers. The procedure for conducting the printability tests was based on the literature (Ahasan Habib, VenkatachalemSathish, Sankumallik, Bashirkhoda, 3D Printability of Alginate-CarboxymethyLCellulose Hydrogel, materials (Basel), 2018mar 20;11(3):454. doi:10.3390 / ma11030454). [Table 3]

[0020] 1) Fiber bonding test For the fiber bonding test, an appropriate g-code (template.gcode) was prepared. This assumes printing two consecutive layers using the test material without interlayer crosslinking with an external lamp. Printouts were performed using a BioX CELLINK printer. The printouts followed a 0°-90° pattern system, reflecting the 2D effect and increasing the inter-fiber distance. The inter-fiber distance was set in 1mm increments from 1 to 5mm. The printing speed, needle diameter, and extrusion width used in the test were 15mm / s, 25G (0.250mm), and 0.3mm, respectively. During the test, the material was supplied within the appropriate pressure and temperature range shown in Table 3. After each layer, the printouts were subjected to additional crosslinking with a Polbionica external UV-Vis lamp, irradiated with the following parameters: wavelength 405nm, time 30 seconds, power 28.5mW / cm². 2 Microscope images were taken after printing. The captured images were processed with ImageJ software. Based on the results obtained, two parameters defined by the following equations, namely the percentage of fiber diffusion rate Dfr (material propagation rate) and printability Pr, were calculated. The pore diffusion rate without material propagation is 0 (i.e., At=Aa), and the printability in the ideal model representation is 1.0.

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[0021] 2) Fiber deflection test The deflection of a suspended filament at the center of its span was analyzed to determine material collapse. A special platform was designed for the experiment, consisting of seven support columns spaced at known intervals of 1, 2, 3, 4, 5, and 6 mm. The dimensions of the five support columns within the structure were 2 × 10 × 6 mm. 3 The dimensions of the two support posts at both ends are 5 x 10 x 6 mm. 3 The following was done: A single filament of the test material was placed on the platform according to MR_test1.gcode, and a photograph of the printout was taken immediately afterward. The captured images were processed with ImageJ software. During this time, the temperature and pressure conditions were adjusted according to the test material, and the printout was performed at a speed of 15 mm / s using a 25G (0.250 mm) needle. Collapse area coefficient Cf is the ratio of the measured area after deflection of the suspended filament to the theoretical area.

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[0022] III. Biological Tests Biological tests were conducted using blends of pre-crosslinked (PreCL) materials with a low-concentration GELMA solution added. The printing conditions for each material are shown in Table 4. The applied parameters resulted in uniform and dense fibers. [Table 4] The biological tests included viability tests (FDA / Pi staining) and growth tests using Alamar Blue reagent at specified time points (0 hours, 24 hours, 72 hours, 7 days, and 14 days). This experiment focused on biological tests performed on structures seeded with L929 cells and structures containing L929 cells within the material. Two structures were printed out for each variant for the growth tests using Alamar Blue, and five structures were printed out for each variant for the viability tests using FDA / Pi. According to the gcode file 10x10x2x4_inf100.gcode, the dimensions of the structure printed for testing were 10×10×2mm, and the infill rate was 100%. The structure obtained from the pre-prepared solution was crosslinked layer by layer using a Polbionica UV-Vis lamp under the following conditions: wavelength 405nm, time 30 seconds, power 28.5mW / cm². 2 .

[0023] IV. Results 1) Fiber bonding test Three fiber bonding tests were performed for each material variant. Microscopic images of the resulting structures were then taken (see Figure 3). Based on the measurement results, the percentage fiber diffusion rate (Dfr) and printability (Pr) were calculated (the results are shown in the graphs in Figures 4-5). Conclusion: • All tested materials exhibited continuous, dense fibers, enabling good / very good resolution printouts. • It was not possible to print out 1 x 2 mm pores from any of the materials tested. • Printability exceeding 0.8 and a fiber diffusion rate of less than 40% were achieved in each material variant. • Diffusion rate decreases, and printability improves. This occurs as pore size increases. • Higher viscosity materials (1.5%(w / v)ALGMA and 2%(w / v)HAMA) exhibit better printability parameters compared to lower viscosity materials. These are also the materials that most closely resemble the ideal material (Dfr=0, Pr=1). Compared to solutions without pre-crosslinking, the resulting material exhibits significantly improved printability parameters.

[0024] 2) Fiber deflection test Three fiber deflection tests were performed on each material variant. Afterward, photographs of the resulting structures were taken (see Figure 6). Based on the measurement results, the fiber collapse coefficient (Cf) was calculated (the results are shown in the graph in Figure 7). Conclusion: • All tested materials exhibited continuous, dense fibers, enabling good / very good resolution printouts. • In each variant tested, fibers were printed out along the entire length of the platform, and no significant crushing of the fibers was observed. The fiber breakdown coefficient was less than 80%. • The fiber collapse coefficient showed similar values ​​regardless of the distance between consecutive support columns on the test platform. • Higher viscosity materials (1.5%(w / v)ALGMA and 2%(w / v)HAMA) showed better parameters in fiber deflection tests compared to lower viscosity materials. These materials also most closely resemble the ideal material (Cf=100). Compared to a solution without pre-crosslinking, the resulting material exhibits significantly improved printability parameters.

[0025] Measurement of crosslinking degree using NMP method 4%HAMA(30~50kDa, 405nm, 28.5mW / cm 2 ) and 2% HAMA (130~300kDa, 405nm, 28.5mW / cm²) 2 The crosslinking level was measured according to the material irradiation time. The results are shown in Figure 8. 4%ALGMA (low viscosity, 405nm, 28.5mW / cm 2 The crosslinking level was measured according to the material irradiation time. The results are shown in Figure 9. 3% GELMA (405nm, 28.5mW / cm²) 2 The crosslinking level was measured according to the material irradiation time. The results are shown in Figure 9.

[0026] Alamar Silver Exam A quantitative method for quantitatively evaluating the growth rate of cell lines. This reaction uses a staining agent containing a redox indicator (REDOX) that fluoresces and changes color in response to the chemical reduction of the culture medium accompanying cell proliferation. When living cells proliferate continuously, a reducing environment (fluorescent, red) is maintained, while when proliferation is inhibited, an oxidizing environment (non-fluorescent, blue) is maintained. This change can be detected by a fluorescence detector or absorbance.

[0027] Specification of biomaterials: • 1.5% (w / v) HAMA_WS (130~300kDa): 1% (w / v) GELMA with 0.25% (w / v) LAP added = 2% HAMA + GELMA • 3% (w / v) HAMA_WS (30~50kDa): 1% (w / v) GELMA with 0.25% (w / v) LAP added = 4% HAMA + GELMA • 3% (w / v) ALGMA_WS: 1% (w / v) GELMA with 0.25% (w / v) LAP added = 4% ALGMA + GELMA • 1.125% (w / v) ALGMA_WS: 1% (w / v) GELMA with 0.25% (w / v) LAP added = 1.5% ALGMA + GELMA ·4%(w / v)GELMA_WS+0.25%(w / v)LAP=4%GELMA Comments on Figure 11: The preliminary crosslinking process does not affect the viability of cells that are suspended in a hydrogel and subjected to 3D bioprinting by extrusion.

[0028] Evaluation of the viability of L929 cells printed on the tested material (see Figures 13-18): Both the cells used for printing and the seeded cells were grown in or on the biomaterial tested. Stepwise cell proliferation was observed from day 1 to day 7.

[0029] Pre-crosslinking to improve HAMA's printability procedure I. Preparation of the solution Solution HAMA0.5-5%+LAP0.0125%-2% I prepared 20 ml of HAMA solution. Using a pipette, 3 mL of PBS×1 was transferred to a 5 mL flask. Two portions of LAP were weighed on the weighing vessel of an analytical balance, and then appropriately transferred to flasks containing PBS×1 or other buffer suitable for the material. The flasks were wrapped in aluminum foil and placed in a thermoblock, and shaken at 800 rpm, 5-60°C. After the photoinitiator had dissolved, the flasks were placed in the refrigerator. Using a serum pipette, PBS×1 or other suitable buffer was transferred to a 50 mL flask by volume transfer. Then, two portions of lyophilized HAMA were weighed and transferred to flasks containing PBS×1 or other stabilizing buffer. The HAMA solution was shaken in a thermoblock at 100-1200 rpm, 10-50°C for 0.1-1 hours. After HAMA had completely dissolved in PBS×1, 3 mL and 2 mL of the dissolved photoinitiator solution were transferred to each flask. The HAMA solutions thus prepared were shaken for a further 10 minutes until the components were mixed.

[0030] Preparation of preliminary crosslinking materials Using a syringe with a needle, 0.1–10 mL of HAMA solution was collected and sealed with a blue protective plug. The syringe containing the material was pre-crosslinked according to the parameters shown in Table 5. For this purpose, the syringe was placed under a Polbionica UV-Vis lamp. The material was uniformly crosslinked by rotating the syringe 180° at half the crosslinking time. After crosslinking, the syringe was protected from light by wrapping it in aluminum foil. The protected syringe was placed on a thermoblock and allowed to stand for 2–5 minutes to stabilize the temperature to 10–30°C depending on the print variant. Subsequently, the pre-crosslinked solution was mixed in multiple syringes. The material thus prepared was transferred to a cartridge and used for printing. [Table 5] The material obtained according to the above procedure was pre-crosslinked for a total of 1 to 60 seconds at intervals of 1 to 15 seconds to obtain a printable form. The wavelengths used for pre-crosslinking included the wavelength range of 250 nm to 800 nm. The optical output at wavelengths of 365 nm and 405 nm was 1 mW / cm². 2 ~100mW / cm 2 It is within the range of [the specified range].

[0031] II. Printability Test: Preliminary Examination To evaluate the printability of the prepared materials, 20 × 20 mm structures (2 layers × 0.5 mm) were printed using two types of printing needles (580 μm and 410 μm). Printing parameters are shown in Table 6. After printing, the stability of the structures was evaluated to determine whether they could be lifted from the pan without damage. Furthermore, a drop of the pre-crosslinked solution was extruded for sensory evaluation. [Table 6] This technology encompasses extrusion and volumetric printing, as well as other printing techniques that require appropriate material viscosity to obtain uniform, dense fibers and maintain print resolution. Depending on the material used, the printing temperature can vary in the range of 5°C to 50°C. The printing pressure in extrusion printing ranges from 5 to 150 kPa. The printing speed ranges from 1 to 100 mm / second. The needle diameter ranges from 50 to 900 μm. The printed model may have any dimensions expressed in millimeters.

[0032] result Abstract printouts were prepared using each prepared variant of the pre-crosslinked HAMA solution. The extruded fibers were compact, allowing for printing at good / very good resolution. A detailed explanation, including photographs, is shown in Figures 20 and 21. Figure 20: 1: Smooth and dense fibers, a structure with good resolution, the structure can be lifted from the bread, not brittle, and forms a dense and smooth drop. 2: Smooth, slightly lumpy fibers, a structure with very good resolution, the structure can be lifted from the pan, not brittle, forming a dense and smooth drop, but small lumps can be seen. 3. No difference in fiber structure was observed compared to when pre-crosslinking was performed at a lower printing temperature with the same parameters. The fibers were dense and the extrudeability was good. Figure 21: 1: Very dense and somewhat lumpy fibers. High-resolution printing. Clogging occurred with a 410μm needle. The structure could be lifted from the pan and was not brittle. Very compact drop. 2: Smooth, slightly dense fibers, a structure with good resolution. The structure could be lifted from the pan and was not brittle. Compact and smooth drop. 3: Compact, somewhat lumpy fibers. Good print resolution. 410μm needle clogging. The structure could be lifted from the pan and was not brittle. Very compact drop. 4: Compact, somewhat lumpy fibers. Print resolution is very good. 410μm needle clogging. The structure could be lifted from the pan and was not brittle. Very compact drop. Furthermore, the quality of fibers extruded from pre-crosslinked material was compared with the quality of fibers extruded from uncrosslinked HAMA solution (see Figure 22).

[0033] Pre-crosslinking to improve the printability of ALGMA procedure I. Preparation of the solution Solution ALGMA0.5-10%+LAP Using a pipette, PBS x 1 was transferred to a 50 mL flask. Two LAP portions were weighed on the pan of an analytical balance and then appropriately transferred to the flask containing PBS x 1 based on volume. The flask was wrapped in aluminum foil and placed in a thermoblock, where it was shaken at a temperature of 5–50°C and a speed of 100–1200 rpm. After the photoinitiator had dissolved, the flask was placed in a refrigerator. Next, a portion of the ALGMA lyophilized material was weighed and transferred to a flask containing PBS x 1 or other stabilizing buffer. The ALGMA solution was shaken in a thermoblock at a speed of 100–1200 rpm and a temperature of 5–50°C for 0.1–4 hours. After the ALGMA had completely dissolved, the flask was wrapped in aluminum foil and stored in a refrigerator.

[0034] II. Preparation of preliminary crosslinking materials Six 2.0 ml portions of pre-crosslinked ALGMA were prepared according to the SOP "Pre-crosslinking of methacrylate solution". Two ml of the solution was drawn using a needle-equipped syringe and then sealed with a blue protective plug. The syringe containing the material was pre-crosslinked according to the parameters shown in Table 7. For this purpose, the syringe was placed under a Polbionic AUV-Vis lamp. The material was uniformly crosslinked by rotating the syringe 180° at half the crosslinking time. After crosslinking, the syringe was protected from light by wrapping it in aluminum foil. The protected syringe was left to stand for 2 to 5 minutes. Then, the pre-crosslinked solution was mixed between several syringes, and the plunger was pushed 10 times. The material thus prepared was transferred to a cartridge and used for printing. [Table 7]

[0035] III. Printability Test: Preliminary Examination To evaluate the printability of the prepared materials, 20 × 20 mm structures (2 layers × 0.5 mm) were printed using two types of printing needles (580 μm and 410 μm). Printing parameters are shown in Table 8. After printing, the stability of the structures was evaluated to determine whether they could be lifted from the pan without damage. Furthermore, a drop of the pre-crosslinked solution was extruded for sensory evaluation. [Table 8] The material obtained according to the above procedure was pre-crosslinked for a total of 1 to 60 seconds at intervals of 1 to 15 seconds to obtain a printable form. The wavelengths used for pre-crosslinking included the wavelength range of 250 nm to 800 nm. The optical output at wavelengths of 365 nm and 405 nm was 1 mW / cm². 2 ~100mW / cm 2 It is within the range of [the specified range].

[0036] result Printing was performed using each prepared variant of the pre-crosslinked ALGMA solution. Compared to the HAMA solution, the ALGMA solution requires a longer pre-crosslinking time to obtain sufficiently dense fibers that allow for printing models with good or very good resolution. Figures 26 and 27 show a detailed explanation, including images. Figure 26: 1: A smooth, highly fluid fiber. It is impossible to create structures with good resolution. 2: The fibers are somewhat fluid, but can produce structures with average / good resolution. Needle clogging does not occur. The drops exhibit some fluidity. The structures are flexible and can be removed from the pan. 3: Dense and slightly stretchable. Very good resolution structure. No needle clogging occurs. Compact and smooth drop. The structure is flexible and can be removed from the pan. Figure 27: 1: The fibers are somewhat fluid, but can produce structures with average / good resolution. Needle clogging does not occur. The drops exhibit some fluidity. The structures are flexible and can be removed from the pan. 2: Dense, somewhat lumpy fibers, resulting in a very good resolution structure. No needle clogging occurs. The structure is flexible and can be removed from the pan. Compact and smooth drop. 3: Dense, somewhat lumpy fibers, resulting in a very good resolution structure. No needle clogging occurs. The structure is flexible and can be removed from the pan. Compact and smooth drop. This technology encompasses extrusion and volumetric printing, as well as other printing techniques that require appropriate material viscosity to obtain uniform, dense fibers and maintain print resolution. Depending on the material used, the printing temperature can vary in the range of 5°C to 50°C. The printing pressure in extrusion printing ranges from 5 to 150 kPa. The printing speed ranges from 1 to 100 mm / second. The needle diameter ranges from 50 to 900 μm. The printed model may have any dimensions expressed in millimeters.

[0037] This technology involves the use of any material based on functionalized derivatives of naturally derived polymers that may be subject to partial and / or complete polymerization or crosslinking in the process of other chemical transformations carried out by the formation of covalent bonds. The factor initiating crosslinking is UV-Vis light acting directly on the material, or UV-Vis light initiating the decomposition of a photoinitiator. The polymer must exhibit significant or complete solubility in an aqueous environment in the concentration range of 0.1–40%. This technology can be applied to low-viscosity polymer solutions that make it impossible to produce prints with high resolution and fiber uniformity. Crosslinking causes an increase in the molecular weight of the polymer applied, and a subsequent change in the mechanical properties of the hydrogel, which manifest as an increase in material viscosity. Therefore, pre-crosslinking makes it possible to print the material at low concentration limits. A schematic diagram illustrating the entire process is shown in Figure 19.

[0038] The proposed preliminary crosslinking technique for the material should be fully measurable to determine the degree of crosslinking of the material, to the extent that it can be expressed as a percentage of the proportion of crosslinkable groups. The degree of crosslinking of the material should be in the range of 0.1% to 99% of the loss of crosslinkable functional groups.

Claims

1. A method for pre-crosslinking methacrylate, The method includes placing an aqueous solution containing a methacrylate with a pH of 4 to 8, a molecular weight of 1 kDa to 500,000 kDa, preferably 30 to 70 kDa, and containing at least 0.5% of the functional groups to be polymerized, at a concentration of 0.5 to 50% (w / v), and a photoinitiator at a concentration of 0.0125 to 2% (w / v), into a transparent container. The aforementioned mixture has a wavelength of 250 nm to 800 nm and an output of 1 to 1000 mW / cm. ２ The UV-VIS irradiation is applied for a period not exceeding 1000 seconds. The method involves rotating the container 180 degrees during half of the crosslinking time.

2. The method according to claim 1, characterized in that the methacrylate is selected from the group including GELMA, HAMA, ALGMA, and CHIMA.

3. The method according to claim 1, characterized in that the photoinitiator is selected from the group comprising lithium phenyl-2,4,6-trimethylbenzoylphosphine, 2-hydroxy-1-(4-(2-hydroxyethoxy)phenyl)-2-methylpropan-1-one and its derivatives, or derivatives of diazo compounds.

4. The method according to claim 1, characterized in that the aqueous solution is physiological saline, cell culture medium, or a weakly acidic solution.

5. The method according to any one of claims 1 to 4, characterized in that the crosslinking is performed in a syringe having a cross-section of 1 to 100 nm.

6. A bioink composition comprising a pre-crosslinked methacrylate obtained by the method of claims 1 to 5.

7. The composition according to claim 6, characterized in that the pre-crosslinked methacrylate has a degree of conversion expressed as the content of converted methacrylate groups in the range of 0.1% to 99.9%, preferably in the range of 40% to 60%.

8. The composition according to claim 6 or 7, characterized by containing pre-crosslinked methacrylate at a concentration of 0.5% to 50% (w / v) and uncrosslinked methacrylate at a concentration of 0.1% to 99.9% (w / v).

9. The above composition contains 5 × 10 per 1 mL of the bioink composition. ５ The composition according to claim 8, further comprising one or more L929 cells.

10. Use of the compositions according to claims 6 to 9 for bioprinting.

11. The printing temperature is in the range of 5 to 50°C, the pressure is in the range of 5 to 150 kPa, the printing speed is in the range of 1 to 100 mm / s, the needle diameter is in the range of 50 to 900 μm, and after the printing is completed, UV-VIS light with a wavelength of 280 nm to 800 nm, preferably 405 nm, is applied for 1 second to 360 seconds, preferably 30 seconds, at an output of 1 to 1000 mW / cm². ２ Preferably 28.5 mW / cm² ２ The use according to claim 10, characterized by irradiation with [a specific device / tool].

12. The use according to claim 10 or 11, characterized in that the printing includes extrusion or volumetric printing.

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