Preparation method of methacrylated dECM

Abstract

A process for the methacrylation of dECM comprises placing a carbonate buffer at a concentration of 1 M into a reaction vessel and heating to a temperature of 50 DEG C, adding dECM to the carbonate buffer to obtain a dECM solution at a concentration of 4% (w / v), sterilizing the solution by irradiation with radiant ultraviolet rays for 15 minutes, adding methacrylic anhydride in an amount of 0.5 mL / 1 g of dECM and reacting at 50 DEG C for 1 hour, and then removing the dECM from the reaction vessel. Then adding a phosphate buffer salt solution to obtain a diluent which is 5 times of the mixture, putting the obtained solution into a dialysis tube, then putting the dialysis tube into deionized water, carrying out the stage at 40 DEG C for not more than 4 days, after the stage is completed, transferring the solution in the dialysis tube into an aluminum plate, and then standing at-80 DEG C for at least 3 hours, and then freeze-drying the frozen solution under the following conditions: the shelf temperature is 10 DEG C, the pressure is 0.1 mba, and the freeze-drying time is 48 hours. The invention also relates to the methacrylated dECM obtained by the above process, and to the use of the methacrylated dECM obtained by the above process in a bioprinting process.

Description

Technical Field

[0001] The subject of this invention is a method for preparing methacrylamide dECM, the methacrylamide dECM obtained by the method, and the use of the obtained methacrylamide dECM. Background Technology

[0002] There are known solutions in the prior art for the methacrylation of compounds subsequently used in bioprinting methods, including the methacrylation of decellularized extracellular matrix (dECM) components.

[0003] Document WO2019122351A1 discloses a combination of ECM (extracellular matrix) and cellulose nanofibers for 3D printing of human tissues. The disclosed method is characterized by extended cell lifespan and good printability. According to this disclosure, the ink used is named CELLINK®, and consists of dispersed nanofiberized cellulose with added cross-linking components.

[0004] Document WO2021014359A1 describes a method for preparing decellularized extracellular matrix in powder and liquid forms without detergent. This invention also relates to a method for preparing dECM-containing bio-ink for 3D printing. According to one embodiment, this document discloses a method for preparing bio-ink, which includes the following steps:

[0005] - Prepare a paste containing 5-50% (w / v) dECM powder and 1-10% (w / v) dECM solution;

[0006] - Incubate the paste at 7-10°C for at least 24 hours;

[0007] - Add 1.46-7.32% (w / v) methacrylamide gelatin, 0.15-1.10% (w / v) methacrylamide hyaluronic acid, 5-10% (w / v) glycerin and photoinitiator;

[0008] - Stir gently.

[0009] Document WO2022180565A1 discloses a bio-ink containing silk fibroin for 3D bioprinting and for supporting the production of hematopoietic models, platelet and blood cell production. According to embodiments, the bio-ink contains gelatin selected from the group consisting of, for example, methacrylamide gelatin or a derivative thereof.

[0010] Document WO2020081982A1 discloses a bio-ink that can be used for three-dimensional printing of structures. According to embodiments, the bio-ink composition may include methacrylamide gelatin and methacrylamide collagen.

[0011] Document IN201831038727A describes a composition of bio-ink, which, for example, consists of silk fibroin, methacrylamide gelatin, methacrylamide chitosan, dimethacrylamide polyethylene glycol, and decellularized extracellular matrix.

[0012] Document US9623051B2 discloses a method for preparing decellularized extracellular matrix compositions. These compositions can be used to coat culture media such as tissue culture media, osteogenic gels, and medical devices.

[0013] Document US20210324336A1 describes a bio-ink for printing three-dimensional structures, which includes methacrylamide hyaluronic acid and extracellular matrix components, and a biocompatible photoinitiator capable of crosslinking methacrylamide hyaluronic acid and ECM components to form a hydrogel.

[0014] Document US20210138114A1 discloses an extrudable photocrosslinked hydrogel composed of a biochemically modified extracellular matrix (ECM) embedded with conductive nanomaterials, a photoinitiator, and a solvent. This hydrogel is used to form in-situ or in vitro printed tissues or organs. The biochemical modification of the ECM involves enriching the matrix with methacrylamide functional groups, acrylamide functional groups, or mixtures thereof. The ECM is modified with methacrylic acid or methacrylic anhydride.

[0015] Document US20190106673A1 describes a bio-ink composition characterized by having elasticity similar to natural tissue. The disclosed composition comprises thiolized hyaluronic acid, methacrylamide collagen, and water.

[0016] Document WO2021250186A1 discloses a biodegradable bio-ink whose mechanical properties are related to structural stability and has surface-modifiable functional groups. The method includes the following steps: providing a (meth)acrylylated cross-linked biocompatible hydrogel carrier derived from the extracellular matrix; and reacting the carrier with a functionalized peptide to obtain a cross-linked biocompatible hydrogel.

[0017] The papers by Ranjithkumar Ravichandran et al. (“Functionalized type-I collagen as a hydrogel building block for bio-orthogonal tissue engineering applications”, 2016, Journal of Materials Chemistry. B, (4), 2, 318-326. http: / / dx.doi.org / 10.1039 / c5tb02035) and Mengxiang Zhu et al. (“Gelatinmethacryloyl and its hydrogels with an exceptional degree of controllability and batch-to-batch consistency”. Scientific Reports. 2019. https: / / doi.org / 10.1038 / s41598-019-42186-x) disclose the use of methacrylic anhydride for the methacrylylation of collagen and gelatin, respectively. Furthermore, in the second paper mentioned, the methacrylylation was carried out in a carbonate buffer solution. Summary of the Invention

[0018] The object of this invention is to produce methacrylamide dECM that can be used in therapeutic applications (e.g., regenerative medicine).

[0019] This invention relates to a dECM methacrylation method, which includes the following steps:

[0020] a) Place 1M carbonate buffer in a reaction vessel and heat to 50°C;

[0021] b) Add dECM to the carbonate buffer solution from step (a) to obtain a 4% (w / v) dECM solution;

[0022] c) Sterilize the solution obtained in step (b) by irradiating it with ultraviolet radiation for 15 minutes;

[0023] d) Add methacrylic anhydride at a rate of 0.5 mL / 1 g dECM;

[0024] e) After adding methacrylic anhydride, react at 50°C for 1 hour;

[0025] f) Add an aqueous phosphate buffer solution to obtain a 5-fold dilution of the mixture from step (e);

[0026] g) Place the solution from step (f) into a dialysis tube, and then place the dialysis tube into deionized water. This step should be carried out at 40°C for no more than 4 days.

[0027] h) After completing step (g), transfer the solution in the dialysis tube to an aluminum pan and place it at -80°C for at least 3 hours;

[0028] i) Freeze-dry the freeze solution in step (h) under the following conditions: shelf temperature 10°C, pressure 0.1 mbar, freeze-drying time 48 hours.

[0029] Preferably, in step (c), methacrylic anhydride is added using a syringe pump over a period of no more than 3 hours, or in portions at 15-minute intervals over a period of no more than 3 hours.

[0030] Preferably, in step (g), the deionized water is replaced at least 11 times.

[0031] Preferably, after completing step (g), the solution in the dialysis tube is concentrated to 25-35% of its initial volume using a rotary evaporator, and the volume of the concentrated solution is not less than 150 mL.

[0032] The present invention also relates to methacrylamide dECM obtained according to the above method, wherein the following equation is used at 60°C via 1 H NMR was used to determine the degree of substitution of the methacryloyl group:

[0033]

[0034] TMSP - Internal Standard, ranging from 75% to 135%.

[0035] In another aspect, the present invention relates to the use of methacrylamide dECM obtained by the method of the present invention in bioprinting methods.

[0036] Preferably, the bioprinting temperature is in the range of 10 to 35°C, the pressure is in the range of 5 to 50 kPa, the printing speed is 1 to 30 mm / s, the needle diameter is 100 to 900 μm, the crosslinking occurs at a wavelength of 365 nm to 405 nm, the time is 5 to 360 s, and the power is as high as 1 to 100 mW / cm. 2 The bio-ink contains 1% to 15% (w / v) of methacrylamide dECM and 0.1% to 0.5% (w / v) of LAP.

[0037] The advantage of this invention lies in the improved mechanical properties and stability of pancreatic ECM scaffolds. Pancreatic-derived methacrylamide-modified (dECM) serves as an advanced 3D framework with customized properties, particularly suitable for tissue engineering and 3D culture applications. This biomaterial is used to produce bioinks for 3D bioprinting, providing a biocompatible and bioactive environment that supports tissue-specific cell adhesion, migration, and regeneration. Its adaptability makes it a valuable tool for constructing scaffolds highly similar to the natural pancreatic tissue microenvironment, promoting cell proliferation and differentiation for therapeutic interventions. Thanks to the methacrylamide process, dECM achieves cross-linking capabilities without the need for additional cross-linking agents such as GELMA and HAMA, thus better mapping the natural cellular environment and reducing the cytotoxicity of the entire bioink. Attached Figure Description

[0038] The invention is illustrated in the accompanying drawings, wherein:

[0039] Figure 1 A schematic diagram of the reaction kit used in the dECM methacrylation process is shown.

[0040] Figure 2 A graph illustrating the degree of substitution measurement (DSNMR) is shown.

[0041] Figure 3 The ECMMA preparation scheme is shown.

[0042] Figure 4 The design of the model is shown, printed using a 3D printer.

[0043] Figure 5 A visualization of a thin-film bioprinting method is shown.

[0044] Figure 6 The cross-linked solution is shown.

[0045] Figure 7 The results of the crosslinking test are shown.

[0046] Figure 8 This diagram illustrates the functional analysis of pancreatic β cells in three concentrations of dECMMA bio-ink.

[0047] Figure 9 A graph illustrating cell viability relative to extract concentration is shown. Detailed Implementation

[0048] The extracellular matrix (ECM) is a complex protein structure produced by cells. It fills the spaces between cells. Its building blocks are mostly proteins and related polysaccharides. The molecules constituting this structure can be broadly classified into three categories: collagen, proteoglycans, and integrin-binding proteins, with their varying proportions determining their properties. The main component of the animal extracellular matrix is ​​collagen, and depending on the species, the chain structure and the content of individual amino acids vary. In the typical amino acid composition of collagen, glycine accounts for 33%, proline for 10%, 4-hydroxyproline for 10%, and 3-hydroxyproline (<0.5%), 5-hydroxylysine (1%), and lysine. The presence of numerous free amino groups allows for functionalization of this material through simple chemical transformations, such as the methacrylylation reaction, which involves replacing amino groups with methacrylic anhydride.

[0049]

[0050] Preparation of methacrylated dECM

[0051] 1. Place a 5-5000 mL three-necked flask equipped with a mixing element in the heating block of a magnetic stirrer. Figure 1 A schematic diagram of the reaction system is shown.

[0052] The choice of reaction flask size depends on the initial solution volume, which must not exceed 60% of the flask volume. Table 1 below provides an overview of reaction vessel selection.

[0053] Table 1. Selection of suitable reaction vessel volume.

[0054]

[0055] Then, measure 2.5 to 3000 mL of 1M carbonate buffer (CB) using a graduated cylinder, filter it through a 0.22 μm filter membrane, and pour it into a flask. Transfer any excess carbonate buffer to a glass bottle and irradiate with a UV lamp for 15 minutes.

[0056] 2. Seal the neck of the flask with a rubber diaphragm and install a thermocouple (pre-wiped with alcohol) in one of the side necks, ensuring the sensor is immersed in the solution without hindering mixing. Place an venting needle in the middle neck. Protect the flask from light by covering it with aluminum foil and heat it to the set temperature of 50°C.

[0057] 3. Next, weigh the ECM using a balance; its mass should be between 5 and 10,000 mg. Then, add small amounts of ECM in portions to a flask containing CB buffer to obtain a 4% (w / v) solution. When adding the substrate to the flask, remove the diaphragm with the vent needle and add it through the neck, taking care to prevent substrate precipitation on the grinding disc. If necessary, rinse with a small amount of demineralizing water, then reinstall the diaphragm with the vent needle. Stir the mixture continuously at 1000-1200 rpm until the substrate is completely dissolved.

[0058] 4. After the substrate is completely dissolved, measure the pH of the resulting solution and label it pH1. Then sterilize the reaction mixture by irradiating the flask containing the solution with ultraviolet light for 15 minutes. When measuring the pH of the solution using a pH meter, extreme care must be taken when inserting the electrode into the reaction mixture.

[0059] 5. Measure the methacrylic anhydride into a syringe fitted with a needle, following these steps:

[0060] a) Weigh the empty syringe (m1) with tube and needle;

[0061] b) Measure the target volume of methacrylic anhydride (MMA) into a syringe, ensuring there are no air bubbles in the system, and then weigh it again (m2);

[0062] c) After adding MMA, weigh the remixing metering system again to determine the accurate amount (m3) of MMA added to the reaction mixture.

[0063] A key parameter describing the efficiency of methacrylation is the degree of substitution (DS), which is controlled by adjusting the amount of methacrylic anhydride (MMA) used in the reaction. It is important to note that the degree of substitution is highly dependent on the characteristics of the substrate itself, specifically the collagen and fat content of the starting ECM. The relationship between the MMA ratio used per 1g of ECM and the target degree of substitution of the final product is shown below.

[0064] Table 2. Select the amount of MMA relative to DS.

[0065]

[0066] 6. Wrap the syringe containing MMA in aluminum foil and place it in the syringe pump. Insert the needle connected to the tubing into the diaphragm on the side neck of the flask, ensuring that the metered anhydride is added directly into the reaction mixture instead of flowing down the wall. Set an appropriate flow rate and continue metering until the solution in the syringe is completely expelled, constantly monitoring the mixing efficiency and the viscosity of the reaction mixture. If necessary, increase the mixer speed. The dropping rate of MMA should be adjusted according to its volume to ensure a total dropping time of 3 hours. For solutions without a syringe pump, MMA can be added in 15-minute intervals, ensuring a total metering time of 3 hours.

[0067] 7. After adding all the measured MMA, weigh the syringe again and calculate the exact amount of MMA added to the reaction (see item 5). Continue the reaction under the given conditions (T=50°C, stirring speed 1000-1200 RPM) for another hour.

[0068] 8. Next, measure the pH value (pH2) of the mixture after the reaction. If this treatment is not to continue on the same day, pour the demineralized water (depending on the flask's capacity) into the flask containing the mixture and place it in the refrigerator overnight. If the mixture needs to be stored for a longer period (2-3 days), freeze the flask containing the mixture at -20°C without adding water first.

[0069] 9. To determine the content of methacrylic acid, transfer 2-3 mL of the mixture into a Falcon apparatus, ensuring that any foam formed in the mixture does not affect the volume collected.

[0070] 10. Next, add the PBS×l solution in portions to the mixture until a 5-fold dilution of the reaction mixture is achieved (1:4 mixture / PBS×l).

[0071] 11. Pour the resulting solution into a pre-prepared dialysis tube following the procedure below:

[0072] a) Measure and cut dialysis tubing to a suitable length, chosen such that the post-reaction solution contained in one dialysis vessel does not exceed 1500 mL (considering space for tying the ends, approximately 80 cm of tubing holds approximately 500-600 mL of solution; one beaker holds 3 such tubing). Immerse the MWCO 1000 Da dialysis tubing in a sodium azide solution;

[0073] b) Place the tube into a beaker containing demineralized water and let it stand for 10 minutes. Then replace the water with fresh water, soak and let it stand for 10 minutes. Then replace the water with fresh water and let it stand for 10 minutes.

[0074] c) Gently remove the soaked tube from the beaker and tie it at one end. Place a funnel over its open end and pour in a small amount of water to check the tube's seal;

[0075] d) If no leak is found, drain the water from the tube, then quantitatively transfer the solution into the tube and rinse the reaction vessel with 3 × 10 mL of deionized water;

[0076] e) Connect the tubing and secure it with clamps on both sides, then place it in a 5L beaker sprayed with alcohol (pre-assembled with a "sun" type mixing element and a dialysis rack sprayed with alcohol).

[0077] f) Fill a beaker with deionized water (about 4L) and place it on a magnetic stirrer. Cover the entire beaker with aluminum foil to block out light.

[0078] 12. Dialysis is performed at 40°C (400 rpm) for 4 days, with water changes 3 times a day (usually at 8:00 AM, 12:00 PM, and 4:00 PM). The number of dialysis days may be adjusted due to possible time constraints. It is important that there are at least 11 transfers, for example, 4 water changes per day.

[0079] 13. After the dialysis process is complete, remove 2-3 mL of solution from the dialysis tube (note the foam, as it will affect the collection volume) to determine methacrylic acid. If a methacrylic acid signal is observed, continue dialysis (transfer 6 more times, procedure as in step 11). If no signal is observed, repeat the measurement in splitless mode. If the presence of methacrylic acid is confirmed, repeat dialysis; if no signal is observed, proceed to step 14.

[0080] 14. Quantitatively transfer the solution from the dialysis tube to a large beaker, then to 1000 mL round-bottom flasks, each containing approximately 600 mL of solution. Concentrate the solution on a rotary evaporator to approximately 25-35% of the initial volume (not less than 150 mL). Transfer the concentrated solution to a glass bottle and store it in a refrigerator or freezer. If the volume of the reaction mixture after dialysis is less than 1500 mL, the concentration step can be skipped, and step 15 can be performed.

[0081] The final concentration parameters are:

[0082] a) Bath temperature: 40°C

[0083] b) Pressure: 30 mbar

[0084] c) Rotation speed: 150 rpm

[0085] d) Cooler initial temperature: -8°C

[0086] The final parameters were achieved by gradually decreasing the pressure, starting at 140 mbar. At 90 mbar, the solution was evaporated for approximately 20 minutes (until no more foaming occurred), and then the pressure was gradually decreased again. Depressurizing too quickly would cause the solution to foam, and some of the contents of the flask would transfer directly to the receiver.

[0087] 15. After concentration, transfer approximately 150-180 mL of solution (using a smaller beaker) to an aluminum pan on a plastic stand (the pan should be pre-sprayed with alcohol and labeled with the sample name and pan number). Weigh the exact amount of solution added to each pan and label it m4.

[0088] 16. Place the dish containing the solution in a -80°C freezer for at least 3 hours. Transfer N frozen samples to a polystyrene foam box for freeze-drying. Freeze-drying parameters:

[0089] a. Shelf temperature: 10°C

[0090] b. Pressure: 0.100 mbar

[0091] c. Duration: 48 hours

[0092] 17. Transfer the obtained freeze-dried material to a pre-weighed plastic container (recorded as m5 on the side of the container), weigh the whole (container + product, recorded as m6 on the container), and subtract the two values ​​from each other to obtain the weight of the product (recorded as Δm on the container).

[0093] 18. Store containers containing materials at -20°C.

[0094] 19. Inspect the crosslinking and gelation of the resulting product. To do this, prepare a 10% (mass fraction) solution of the product in water, and then add LAP to achieve a concentration of 0.25% in the entire solution. Take 1 mL of the resulting solution, transfer it to a mold, and irradiate it with a wavelength of 365 nm or 405 nm (maximum power) for 1 minute.

[0095] 20. Determine the moisture content of the product obtained after freeze-drying. This is done using the following formula:

[0096] M% H2O = m6-m5 / m4-m5*100%

[0097] 21. After freeze-drying, weigh approximately 15 mg of the dried product, place it in a small vial with a rubber stopper, label it with the sample name and approximate amount of material, and place it in a freezer (-20°C).

[0098] Substitutability 1 H NMR measurements

[0099] In order to pass 1 To measure the degree of substitution using 1H NMR, 5 mg of sample was dissolved in 600 μl of D₂O containing 0.00916 mmol TMSP (quantitative standard – 3-(trimethylsilyl)propionic acid) and placed in a 5 mm NMR tube. The sample was then placed in an NMR spectrometer (Agilent DirectDrive2 700 MHz). The temperature was set to 60°C. After temperature stabilization, the sample was mixed, the probe was tuned, the pulse was measured, and magnetic field inhomogeneity was corrected. Next, the 1H spectrum was measured (measurement parameters: 8 scans, 15 s repetition time, 45° pulse time 2.5 µs). Spectral analysis was performed using the NMRGlue package in the Python environment. After importing the data, exponential weighting (line broadening: 2 Hz) was applied, and Fourier transform, phase adjustment, and baseline correction were performed for the 6.7 ppm region: 6.45 ppm, the 1.05 ppm region: 0.8 ppm, and the 0.1 ppm region: -0.1 ppm. Next, the integrated peak count in the 6.7 ppm region is 6.45 ppm (corresponding to protons in the double bond) and the integrated peak in the 1.05 ppm region is 0.8 ppm (corresponding to protons from the medium). Based on these parameters, the DS NMR value (degree of substitution) is calculated using the following formula:

[0100]

[0101] The calculation results are in Table 3 and Figure 2 The chart is shown.

[0102] Table 3. Degree of substitution calculated by NMR analysis.

[0103]

[0104] Hydrogel formulation: 10% ECMMA + 0.25% LAP

[0105] Using a pipette, quantitatively transfer 15 mL of PBS × 1 to a 50 mL dark glass vial. Using an analytical balance, weigh the LAP (photoinitiator – lithium phenyl-2,4,6-trimethylbenzoylphosphonate) into a weighing container, then quantitatively transfer 39.4 mg of LAP into the vial containing PBS × 1. Transfer the vial containing the solution to a water bath (30°C) and stir at 500 rpm for 15 minutes (until the photoinitiator dissolves). Then filter the LAP solution into a sterile 50 mL Falcon vial. Add 9.1 mL of the filtered LAP solution to another sterile 50 mL vial. Then, weigh the ECMMA lyophilized material (sterilized with a UV lamp beforehand) and add it to the photoinitiator solution in a volume of 1.01 g. Place the ECMMA solution in a water bath (30°C) until completely dissolved, mixing at a rate of 100-200 rpm.

[0106] Table 4. Reagents used in the preparation of hydrogels

[0107]

[0108] ECMMA preparation scheme as follows Figure 3 As shown.

[0109] Bioprinting of scaffolds

[0110] According to the program design ( Figure 4 A scaffold with dimensions of 10×10×3mm was printed. Table 5 contains detailed information about the model. The printing was performed on a CELLINK BioX 3D bioprinter. Each layer was cross-linked using an external UV-Vis lamp (Polbionica). The scaffold was printed using 10% ECMMA hydrogel. The scaffold was printed on a culture dish. Table 5 lists the detailed information about the model.

[0111] Table 5. Bioprinting Module Parameters

[0112]

[0113] During the printing process, select the continuous printing parameters based on the given bio-ink portion. The parameter ranges used are shown in Table 6. Figure 5 The completed printed photo is shown.

[0114] Table 6. Bracket Printing Parameters

[0115]

[0116] Weigh each slice, then transfer it to a petri dish and pour in sterile PBS containing antibiotics.

[0117] The crosslinking test solution was then prepared by adding the following substances sequentially to a 15 mL vial:

[0118] -750 mg ECMMA, 18.75 mg LAP and 6731.25 µl of PBS × 1 for a 10% solution;

[0119] -375 mg ECMMA, 18.75 mg LAP and 7106.25 µl of PBS were used for a 5% solution.

[0120] The solutions were placed in a hot block and stirred at 1000 rpm at 40°C until the samples were completely dissolved. Next, 1.5 mL of each solution was transferred to ten 4 mL glass vials and stirred at 405 nm, 28.5 mW / cm² at specified time points. 2 Cross-linking occurs. The cross-linking solution is as follows: Figure 6 As shown. The crosslinking test results are as follows. Figure 7 As shown.

[0121] The resulting bio-ink enabled sufficient diffusion of insulin and glucagon within the bioprinted structure. To confirm this, the function of pancreatic β-cells was analyzed in three concentrations of dECMMA bio-ink. Figure 8 The results confirm that dECMMA can be used for tissue engineering and regenerative medicine using pancreatic islet cells and other cell lines.

[0122] Cytotoxicity of ECMMA biomaterials was assessed using the indirect MTT assay.

[0123] The purpose of this procedure is to assess the metabolic activity of cells and examine how long the cytotoxicity of the test materials lasts: 10% ECMMA, 12.5% ​​ECMMA, 15% ECMMA, batch T18.

[0124] Cell viability and proliferation are related to the ability of enzymes present in living cells (mitochondrial dehydrogenases) to convert the water-soluble tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)) into water-insoluble purple formazan crystals. The supernatant needs to be aspirated from above the cells and formazan crystals before adding an organic solvent (e.g., isopropanol or dimethyl sulfoxide), which can introduce errors (such as accidentally removing some crystals) and reduce the sensitivity of the assay; therefore, caution must be exercised during the procedure.

[0125]

[0126] Preparation of extracts

[0127] The quality of the samples is shown in Table 7.

[0128] Table 7. Mass of samples used to prepare extracts

[0129]

[0130] The scaffold produced from ECMMA biomaterials was crushed and transferred into sterile 5 mL Eppendorf tubes. Culture medium was added to the biomaterials, and the sample was then placed in an incubator (37°C) for a specific time, depending on the experimental protocol.

[0131] Preparation of samples for MTT testing

[0132] The same procedure was used for each time period of 24, 48, and 72 hours. Extracts from the top of the biological material were collected into new Eppendorf tubes as 100% extracts. Each extract was also diluted 50%.

[0133] To prepare L929 cells for MTT assay, cells were seeded in 96-well plates for the collection of extracts. L929 cell lines were prepared for each extract as planned. A concentration of 10... 5 Cells at a density of 1 / mL were seeded into 100 μL of 96-well plates arranged in rows. PBS was added to the edge wells. The prepared plates were incubated at 37°C for 24 hours. Table 8 shows the cell viability.

[0134] Table 8. Survival rate of inoculated cells.

[0135]

[0136] Extracts collected from biological materials

[0137] The extract can be collected without centrifugation. The color of the culture medium remained unchanged at any time point.

[0138] Extracts of 100% and 50% ECMMA at concentrations of 10%, 12.5%, and 15% were applied to well plates. The results showed that the material was non-cytotoxic at the tested concentrations and therefore safe for use in bioprinting tissue models and biomimetic organs.

Claims

1. A method for dECM methacrylation, comprising the following steps: a) Place 1M carbonate buffer into the reaction vessel and heat to 50°C. b) Add dECM to the carbonate buffer solution from step (a) to obtain a 4% (w / v) dECM solution. c) Sterilize the solution obtained in step b) by irradiating it with ultraviolet radiation for 15 minutes. d) Add methacrylic anhydride at a rate of 0.5 mL / 1 g dECM. e) After adding methacrylic anhydride, react at 50°C for 1 hour. f) Add phosphate buffer solution to obtain a 5-fold dilution of the mixture from step e). g) Place the solution from step f) into a dialysis tube, then place the dialysis tube in deionized water. This step should be performed at 40°C for no more than 4 days. h) After completing step g), transfer the solution from the dialysis tube to an aluminum tray and then incubate at -80°C for at least 3 hours. i) Freeze-dry the freezing solution of step h) under the following conditions: shelf temperature 10°C, pressure 0.1 mbar, freeze-drying time 48 hours.

2. The method according to claim 1, characterized in that, In step c), the addition of methacrylic anhydride is performed using a syringe pump over a period not exceeding 3 hours, or in 15-minute intervals over a period not exceeding 3 hours.

3. The method according to claim 1, characterized in that, In step g), the deionized water is replaced at least 11 times.

4. The method according to claim 1, characterized in that, After completing step g), concentrate the solution in the dialysis tube to 25-35% of the initial volume using a rotary evaporator, and the volume of the concentrated solution shall not be less than 150 mL.

5. The methacrylamide-treated dECM obtained by the method according to claims 1 to 5.

6. The methacrylamide-modified dECM according to claim 6, characterized in that, Use the following equation to pass through at 60°C 1 HNMR was used to determine the degree of substitution of the methacryloyl group: TMSP - Internal Standard, ranging from 75% to 135%.

7. Use of the methacrylamide-modified dECM obtained by the method according to claims 1-5 in a bioprinting method.

8. The use according to claim 8, characterized in that, Bioprinting operates at temperatures ranging from 10 to 35°C, pressures from 5 to 50 kPa, printing speeds from 1 to 30 mm / s, needle diameters from 100 to 900 μm, crosslinking occurs at wavelengths from 365 nm to 405 nm, time ranges from 5 to 360 s, and power reaches 1 to 100 mW / cm². 2 The bio-ink contains 1% to 15% (w / v) of methacrylamide-modified dECM and 0.1% to 0.5% (w / v) of LAP.

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