A hydrogel

Abstract

The invention relates to a hydrogel obtained from chia seed mucilage.

Description

[0001] A HYDROGEL

[0002] Technical Field

[0003] The invention relates to a hydrogel obtained from chia seed mucilage.

[0004] State of the Art

[0005] Hydrogel obtained by combining both synthetic and natural, as well as synthetic and natural polymers, can be used in tissue engineering applications. However, the hydrogel currently in use has several disadvantages. Typically, autograft, allograft, and xenograft methods can be used for tissue regeneration in tissue engineering applications.

[0006] Autograft is the method of transferring the tissue taken from an individual to a different place in the same individual. Autograft has several disadvantages, even though it is seen as the gold standard. The graft source is limited and can be painful for the patient since it is obtained from the patient. There is a risk of hematoma and infection (donor site morbidity') in the graft area, in addition to the need for surgery to remove the graft. It is an expensive graft method in addition to these.

[0007] Allograft is a tissue transplantation method between different individuals. Allografts can cause disease transport, bacterial infections, and immunogenic reactions in the grafted individual since they are obtained from different individuals. If they are sterilized, changes in their mechanical properties may occur.

[0008] Xenograft is a method that involves transferring tissue from one species to another. Grafts taken from animals can be transferred to humans with this method. However, there is a risk of disease transfer, immunogenic reactions in the donor, and rejection of the graft by the patient, as with allografts.

[0009] Hydrogel from natural and synthetic polymers can be used for the regeneration of tissues as an alternative to the currently used graft methods. However, hydrogels made from natural and synthetic polymers also have several disadvantages. Tissue regeneration may not be achieved in a healthy wav due to these disadvantages. Natural hydrogel has a weak mechanical force and may not always provide sufficient strength when implanted in a load-bearing area. For this, it is aimed at increasing strength by using it together with synthetic polymers. Microbial contamination may occur in hydrogels of natural polymer origin. Their determination is not high. For example, natural polymers such as collagen and gelatin can be removed from the body immediately by undergoing rapid degradation. In these cases, it is aimed at improving the durability of natural polymers through chemical interactions.

[0010] Synthetic polymers generally do not support cell adhesion, so cells cannot show activities such as adhesion, proliferation, and differentiation on synthetic polymers. Although they are necessary' for cell adhesion, chemical modifications can have toxic effects because they are the result of chemical processes.Hydrogels containing methacrylated polymers using the conventional or microwave-assisted methacrylation method are included in the known state of the art [1-6], The methaciylation process is based on the functionalization of polysaccharides with an OH group-Iinked polymer backbone or polypeptides with an NHz group-linked polymer backbone with methacrylate groups through covalent cross-links, as shown in Figure 1. Methacrylate groups can be provided using various chemicals (methacrylic anhydride, 2 aminoethyl methacrylate hydrochloride, glycidyl methacrylate, methacrylic acid, and aminoethyl methacrylate).

[0011] Patent application W02020254807A1 describes methacrylated collagen (collagen methacrylamide) using aminoethyl methacrylate. Patent application WO2021022382A1 describes gelatin (GEL-MA) methacrylated by the conventional method using glycidyl methacrylate or methacrylic anhydride. Patent application US2013017177A1 describes methacrylated gellan gum using glycidyl methacrylate.

[0012] Descriptions of the Figures

[0013] Figure 1 . Functionalization of polym er chains by methacrylation and obtaining crosslinking in the presence of a photoini tiator.

[0014] Figure 2. Methacrylation process flow chart

[0015] Figure 3. Chia mucilage and chiaMA group proton NMR analysis, a) Peaks of the vinyl group of the AEMA b) Peaks of the m ethyl group of the AEMA.

[0016] Figure 4. FTIR analysis of chia mucilage and ChiaMA groups.

[0017] Figure 5. Thermal behavior graphs of chia mucilage and ChiaMA groups, a) DTG graph b) TGA graph.

[0018] Figure 6.1. Cross-sectional and surface SEM images of chia mucilage and conventional ChiaMA hydrogels, (a, b) Chia mucilage, (c, d) conventional ChiaMA 2.5%, (e, f) conventional ChiaMA 5%. All magnifications are 25 Ox.

[0019] Figure 6.2. Cross-sectional and surface SEM images of 100 W, 250 W, and 500 W ChiaMA hydrogels synthesized by microwave-assisted methacrylation. (a, b) 100 W ChiaMA 2.5%, (c, d) 250 W chiaMA 2.5%, (e, f) 500 W chiaMA 2.5%. All magnifications are 25 Ox.

[0020] Figure 6.3. Cross-sectional and surface SEM images of 100 W, 250 W, and 500 W ChiaMA hydrogels synthesized by microwave-assisted methacrylation. (a, b) 100 W ChiaMA 5%, (c, d) 250 W ChiaMA 5%, (e, f) 500 W ChiaMA 5%. Ail magnifications are 250x.

[0021] Figure 7. XRD graph of chia mucilage and ChiaMA groups

[0022] Figure 8.1. Rheological analysis results of the hydrogels prepared using 2.5% ChiaMA. Frequency-dependent a) storage module, b) loss module; strain module c) storage module, d) loss module; temperature-dependent e) shear stress values.

[0023] Figure 8.2. Rheological analysis results of the hydrogels prepared using 5% ChiaMA. Frequency-dependent a) storage module, b) loss module; strain module c) storage module, d) loss module; temperature-dependent e) shear stress values.

[0024] Figure 9. Viscosity values of the solutions obtained by dissolving chia mucilage and ChiaMA. a) 2.5% concentration, b) 5% concentration.

[0025] A Brief Description of the Invention

[0026] Our invention includes the methacrylation of chia seed mucilage using both conventional and microwave -assisted methods. A polymer functionalization method called methacrylation was used for the development of hydrogels. The methacrylation process is based on the binding of the polysaccharides and / or polypeptides in the chia mucilage with the methacrylate groups in the AEMA using EDC / NHS chemistry, as shown in Figure 2. To make a hydrogel, these methacrylated precursors (polymers with a. functional group that can be cross-linked using light energy) quickly cross-link through free radical chain polymerization when a photoinitiator is present. Hydrogel developed with the invention can be used as bioink for printing in 3D bioprinter systems. In addition, it is generally' used in controlled drug release systems where hydrogels are used, in biosensors, in the treatment of cartilage and bone diseases, in the treatment of wounds and bums, in the cosmetic field, and in many other regenerative medicine fields.

[0027] The advantages of being well tolerated by the patient, not causing immunogenic reactions, being easily accessible in terms of source, and providing the functions of real tissue were obtained even though the disadvantages of graft methods were eliminated with the invention. The disadvantages of autografts having painful and limited resources are not in question in the system developed -with the invention. There is no risk of hematoma and infection. In addition, the rehabilitation period for the patient will be shorter since the operation required for graft removal will not be needed.

[0028] The risk of di sease transport in allograft and xenograft is eliminated thanks to the invention.

[0029] Tie invention exhibits a mechanically more stable structure compared to hydrogels of natural polymer origin. A strong web structure is formed thanks to the cross-linking of chia seed mucilage with methacrylation.

[0030] The mucilage of chia seeds, which is the main component of the inventi on, solves the issue of cells' inability to adhere to synthetic polymers. Tie content of chia seed mucilage is very' similar to the extracellular matrix structure (ECM) between the cells. Thus, it is expected that the cells will multiply, differentiate, and provide tissue regeneration by adhering to the hydrogen obtained with our invention.

[0031] Detailed Description of the Invention

[0032] The invention includes the methacrylation of chia seed mucilage (ChiaMA) using both conventional and microwave-assisted methods. Methacrylation can be performed using conventional and microwave-assisted methods. The reaction can be achieved by mixing the molecules m the reaction solution, such as solvent, reagent, and catalyst, by applying room temperature or conventional heating (e.g. oil bath) in the methacrylation process performed with the conventional method. It is the result of microwave energy's capacity to absorb molecules of a specific substance and transform electromagnetic energy into heat. Many molecules are stimulated rotationally by the easy absorption of microwave energy by dipole moment molecules. Tie rotational energy is converted into kinetic energy, and heating occurs when these stimulated molecules collide with other molecules. Conventional heating is a relatively slow and inefficient method. Therefore, a wide range of polymer synthesis and functionalization can be performed by using microwave energy as an alternative to standard conventional thermal heating.

[0033] Using ChiaMA, cross-linked hydrogel structures are obtained in the presence of a photoinitiator. Hydrogels with a viscoelastic structure can be used in tissue engineering applications. In addition, its printability as a bioink in 3D bioprinting systems was examined by performing rheological analyses.

[0034] Synthesis Method

[0035] First, chia seeds are added to distilled water to obtain chia mucilage, which is stirred in a magnetic stirrer at room temperature for 2-3 hours. Mucilage is released from chia seeds at tlie end of the specified time. Tie chia seed and mucilage mixture is transferred to the vacuum system in order to separate the chia mucilage from the seeds. Hie vacuum system consists of a funnel, a glass filtration bottle, a porous fabric placed on the funnel to separate the seeds from the mucilage, and a vacuum pump. Vacuum is applied after the seed and mucilage mixture is poured through the fabric (300 mbar). Thanks to the fabric, which has a smaller pore diameter than chia seeds, the vacuum pulls down the mucilage while the chia seeds remain on the fabric, allowing the seed and mucilage to be separated from each other, lire mucilage separated by vacuum is taken into falcon tubes and centrifuged between 5000-8000 rpm for 5 minutes since small seed pieces may be mixed in the mucilage. Seed fragments and excess water remaining on the surface after centrifugation are separated from the mucilage vrith the help of a pasteur pipette. The result is chia mucilage free from seeds. Then, chia mucilage is placed in the lyophilizer device and freeze-dried for about 1 week.

[0036] ChiaMA is synthesized as a result of the methacrylation of dried chia mucilage. Both conventional methacrylation and microwave-assisted methacrylation methods are used to obtain ChiaMA.

[0037] Conventional methacrylation:

[0038] The steps to synthesize ChiaMA using the conventional methacrylation method are as follows: First, dried chia mucilage (1 % w / v) in 10 mL phosphate buffered saline (PBS) (pH=7.4) is dissolved homogeneously at room temperature using a magnetic stirrer for about 1 hour. Then, N-(3~Dimethylammopropyl)-N’-ethyl carbodiimide hydrochloride (EDC) (-1.8% w / v) is added to the solution and mixed at room temperature for 15 minutes. Then, N- hydroxy succinimide (NHS) (-0.5% w / v) is added to the same solution and mixed at room temperature for 15 minutes. 80 mg of 2-aminoethyl methacrylate hydrochloride (AEMA) (0.8% w / v) is finally added to the solution containing EDC-NHS, and the reaction is terminated by mixing at room temperature for 1 day. The reaction is carried out in a dark environment by coating the reaction container in which the solution is located with aluminum foil. Then, the solution is taken into a 12-14 kDa dialysis membrane and dialyzed at 37°C for 3 days. The distilled water from the dialysis is replenished daily. The solution is dried in the lyophilizer system for 5 days after the dialysis. Tirus, conventional methacrylated chia mucilage is obtained.

[0039] Microwave-assisted methacrylation:

[0040] Tire amounts used in the dried chia mucilage, EDC, NHS, and AEMA conventional methacrylation method in 10 mL of PBS to synthesize ChiaMA by the microwave-assisted methacrylation methods were prepared in the same way. Then, the solution is placed in the microwave system. The microwave system is adjusted so that the maximum temperature of the solution is 50°C. Three different alternative powers (100 W, 250 W, and 500 W) are used in the microwave system.

[0041] For a 100 W microwave application, 100 W microwave energy is given to the solution for 30 seconds. Then, the solution is cooled on ice for about 3 minutes. The microwave application and cooling process are evaluated as one cycle. A total of 6 minutes (30 sec x 12) of microwave application is provided to the solution, and the reaction is completed upon performing the cycle 12 times.

[0042] For 250 W microwave application, 250 W microwave energy is given to the solution for 20 seconds. Then, the solution is cooled for about 2 minutes. The microwave energy and cooling cycle are performed 10 times. Then, 250 W microwave energy is applied for 30 seconds, and the solution is cooled, and this cycle is carried out 5 times. A total of approximately 6 minutes (20 sec x 10 + 30 sec x 5) of microwave application is performed.

[0043] For 500 W micro-wave application, 500 W microwave energy is given to the solution for 10 seconds. Then, it is cooled for about 2 minutes. The microwave process and cooling cycle are repeated 20 times. Then, the process is completed after 10 cycles by applying a microwave for 15 seconds and cooling. The total reaction is completed in approximately 6 minutes (10 sec x 20 -t- 15 sec x 10).

[0044] The reason why the durations are different between the groups is that higher temperatures are reached quickly with the increase in the power of the applied microwave. Therefore, the applied microwave time is inversely proportional to the increase in power. The solutions are taken into a 12-14 kDa dialysis membrane and dialyzed at 37°C for 3 days after the microwave application process is completed. The distilled water from the dialysis is replenished daily. The solution is dried in the lyophilizer system for 5 days after the completion of the dialysis, and the microwave-assisted methacrylated chia mucilage is obtained.

[0045] Hydrogel was obtained by cross-linking the dried ChiaMA obtained using conventional and microwave-assisted methacrylation methods. Tire photoinitiator lithium phenyl-2,4,6- trimethylbenzoyl phosphinate (LAP) was first dissolved in distilled water at room temperature using a magnetic stirrer to obtain the chiaMA hydrogels. Then, ChiaMA (2.5% and 5% w / v) was added to the solution and dissolved at room temperature using a magnetic stirrer. LAP was used at 3 different concentrations (0.3%, 0.5%, and 1% (w / v)) to see the effect of photoinitiator concentration on crosslinking. ChiaMA synthesized by both conventional and microwave-assisted methacrylation methods was used. Tire groups using different concentrations of LAP and ChiaMA are as follows:

[0046] UV light was applied to obtain hydrogel by cross-linking the prepared solutions. 'The solutions were first taken into a 24-well petri dish and applied for 30 seconds so that the LTV light was 3200 mW / cm2. Cross-linking was observed in all groups and hydrogel was obtained as a result of UV application.

[0047] The synthesis method can be summarized with the following process steps. Conventional methacrylation: i. Secretion of chia mucilage, separation of mucilage from seeds using a vacuum system and freeze-drying with a lyophilizer. ii. Methacrylation by conventional method and obtaining ChiaMA by preparing a solution containing chia mucilage, EDC, NHS and AEMA. iii. Freeze-drying of ChiaMA with dialysis and lyophilizer to remove unreacted chemicals from the environment.

[0048] Microwave-assisted methacrylation: i. Secretion of chia mucilage, separation of mucilage from seeds using a vacuum system and freeze-drying with a lyophilizer. ii. Preparation of the solution containing chia mucilage, EDC, NFIS and the AEMA solution. iii. Mixing and placing the solutions in the microwave system, obtaining ChiaMA by microwave-assisted methacrylation method with an alternative application of 100 W,

[0049] 250 W or 500 W. iv. Freeze-drying of ChiaMA with dialysis and lyophilizer to remove unreacted chemicals from the environment.

[0050] Characterization:

[0051] Characterization of ChiaMA synthesized by conventional and microwave-assisted methacrylation methods was performed. The analyses made for characterization are as follows:

[0052] 10 mg of dried methacrylated chia mucilage w'as dissolved in conventional ChiaMA , 100 W ChiaMA , 250 W ChiaMA and 500 W ChiaMA 600 pl deuterium oxide (D2O) in order to perform NMR analysis. Then, NMR analysis was performed at room temperature and the changes in the chemical structure of chia mucilage as a result of conventional and microwave- assisted methacrylation were shown. Figure 3 shows the proton NMR graph of chia mucilage and all ChiaMA groups. Vinyl hydrogens of the AEMA form distinctive peaks in the range of 5.20-6.30 ppm on the NMR chart and methyl hydrogens around 2 ppm. No peak formation was observed in the range of 5.20-6.30 ppm when the graph of chia mucilage was examined. Three small peaks were observed due to chia mucilage around 2 ppm. Two peaks occurred in the range of 5.20-6.30 ppm, while three small peaks formed in chia mucilage around 2 ppm began to combine in the conventional ChiaMA group when the NMR graph of the conventional ChiaMA group was examined. Two peaks occurred in the range of 5.20-6.30 ppm in the 100 W, 250 W and 500 W ChiaMA groups. Three small peaks in the chia mucilage at about 2 ppm were completely combined in the 100 W ChiaMA group and became a single peak, and it was observed that the density of this peak gradually increased in tlie 250 W and 500 W ChiaMA groups. The density of the peaks increased significantly with the increase of microwave energy in the 100 W, 250 W, 500 W ChiaMA groups while the two peak densities in the range of 5.2.0-6.30 ppm were the lowest in the conventional ChiaMA group.

[0053] The structure and functional groups of ChiaMA synthesized by chia mucilage, conventional and microwave-assisted methacrylation methods were determined by FTIR analysis, which enables the identification of chemical bonds in the molecular or compound structure of the substance as a result of the absorption of infrared light by the sample. The results of the FTIR analysis are given in Figure 4. The peaks seen in the 3700-3000 cm’1and 2980-2850 cm’1areas in all groups are found in all polysaccharides and belong to the -OH and -CH stress vibrations, respectively. In addition, the peak at 1033 cm’1generally belongs to the C-O-C stretch found in polysaccharides. The peak at 938 cm'1is due to the C-0 bonds in the carbohydrate groups of chia mucilage . It belongs to the amide 1 band, which is the most characteristic band for the large peak proteins in the area of 1700-1600 cm’1as can be seen in the graph of chia. However, it was observed that the amide 1 band formed three separate peaks in the conventional, 100 W, 250 W and 500 W ChiaMA graphs in tire range of these values. The new peak formed at 1652 cm’1corresponds to the stretching vibrations of the NH- CO bond as a result of the coupling reaction of chia mucilage and AEMA. Tire new peaks formed at 1708 cm’1and 1545 cm'1are due to the stretching vibration of the C=O and N-H bonds of the methacrylate group. The peak at 1157 cm’1was weakly formed on the chia graph, but the peak at 1 157 cm’1was strengthened as a result of the stretching vibration of the C-N bonds due to AEMA in the graphs of the methacrylated groups. The TGA method was used to determine the changes in the sample mass with time and temperature change. The TGA analysis of chia mucilage and ChiaMA hydrogels were performed at a heating rate of 10°C / min in the temperature range of 25-900°C. Both differential thermal therm ogravimetric (DTG) and thermogravimetric analysis (TGA) graphs of all groups are given in Figure 5. Chia mucilage gave maximum weight loss at 300°C, while ChiaMA groups gave maximum weight loss around 290°C and 345°C according to the DTG graph in Figure 5. a. The first weight loss occurred due to the evaporation of water up to approximately 95°C in all groups according to the TGA graph in Figure 5.b. Maximum (-10%) water loss up to 95°C was in chia mucilage, while less water loss occurred in ChiaMA groups. The least water loss (-3%) was in the conventional chia group. It was observed that all groups were stable up to 175 °C, and a large mass loss occurred in all groups between 175°C and 450°C. This loss is due to the fact that polysaccharide molecules become low molecular weight volatiles by depolarization. The highest loss (>90%) occurred in the conventional ChiaMA group, while the least loss (-60%) was observed in chia mucilage. The 100 W, 250 W and, 500 W ChiaMA groups gave less weight loss than the conventional chiaMA group.

[0054] Chia mucilage and all ChiaMA hydrogel groups were first dried with a lyophilizer, then cut in half, and made a 6 nm gold coating, and both section and surface images were taken on the SEM device to examine the surface morphologies.

[0055] There are cross-sectional and surface SEM images of chia mucilage and 2.5% and 5% conventional ChiaMA hydrogel groups in Figure 6.1. It is seen that chia mucilage has a porous structure (Figure 6.1. a and Figure 6. 1 ,b). The formation of a layer-by-layer structure was observed in the cross-sectional images of the hydrogels (Figure 6.1.c and Figure 6.1.e) as a result of conventional methacrylation. It is also seen that the surfaces of conventional ChiaMA hydrogels have a porous structure (Figure 6.1.d and Figure 6. 1 ,f).

[0056] Tire images of the 100 W, 250 W and 500 W ChiaMA hydrogels prepared at a concentration of 2.5% are given in Figure 6.2., and the images of the 100 W, 250 W and 500 W ChiaMA hydrogels prepared at a concentration of 5% are given in Figure 6.3. Layered formation is observed in the cross-sectional structures of all ChiaMA hydrogels obtained by using 100 W, 250 W and 500 W microwave energy. The formation of the porous structure was observed in the surface morphology of the ChiaMA hydrogels obtained by using 500 W micro wave energy while the surface m orphology of the ChiaMA hydrogels obtained by using 100 W and 250 W microwave energy has a flat structure. However, the porous structure formed on the surface was observed more intensively in the ChiaMA hydrogels prepared at a concentration of 2.5% than the ChiaMA hydrogels prepared at a concentration of 5%.

[0057] XRD is a powerful analysis that is used to resolve polysaccharide structures, providing information about the crystal Unity of the structure. The XRD analysis of chia mucilage and ChiaMA hydrogels was performed in the range of 5-60° at a scanning rate of 2° / min and the results are given in Figure 7. A wide peak was observed between -13-26 degrees for chia mucilage, conventional ChiaMA and 250 W ChiaMA. It has been stated that the structure of asymmetric, large peaks at 16.96, 20.04, 22 degrees is amorphous, and their crystallinity is low, thus expressing the semi-crystalline property according to the studies on plant-derived polysaccharides in the literature. It was stated in the studies that the semi-crystalline structure of the extracted polysaccharides -was caused by CO, CH and OH functional bonds. It is seen based on these data that ChiaMA, which is subjected to conventional and microwave-assisted methacrylation, has a semi-crystalline structure. In addition, a peak at an angle of -28 degrees was observed in the chia mucilage and 100 W, 250 W and 500 W ChiaMA groups. The intensity of this peak -was observed to be higher at chia mucilage, 250 W and 500 W, while it was lower at conventional and 100 W ChiaMA. It has been stated that chia mucilage is similar to the calcium carbonate structure of the peaks at 29.4, 31.4 and 39.4 degrees according to the XRD graph in the literature. The presence of magnesium, phosphorus and calcium in chia mucilage has been shown with these data. It can be found based on the studyin the literature that chia mucilage and all ChiaMA groups may have magnesium, phosphorus, and calcium content according to the peak value of -28 degrees.

[0058] The rheological properties of ChiaMA hydrogels have been determined using a rheometer. The LAP was first dissolved in distilled water at a concentration of 1% at room temperature to prepare the hydrogels. Then, ChiaMA was added to the solution and dissolved at room temperature. Cylindrical disc-shaped hydrogels with a thickness of 2 mm and a diameter of 16 mm were obtained by applying UV for 1 minute as 3200 mW / cm2upon obtaining the solution homogeneously. All hydrogel groups are given below: ® 500 W ChiaMA 5%

[0059] Rheological properties were determined by performing frequency-dependent, temperaturedependent, and strain-dependent dynamic analyses. Frequency-dependent dynamic analysis was performed in the range of 0.5-200 rad / s under 0.2% strain. Strain -dependent dynamic analysis was performed at a constant frequency of 1 Hz, in the strain range of 0-2%. Rheological analyses of ChiaMA hydrogels are shown in Figure 8.1. and Figure 8.2. The rheological analysis results of the ChiaMA hydrogels prepared at a concentration of 2.5% are given in Figure 8.1., and the tank and loss modulus values obtained when the varying frequency is applied in the range of 200 rad / s are given m Table 1. Ail ChiaMA hydrogels were found to maintain their structural integrity and exhibit elasticity in a stable structure in the frequency range of 0.5-200 rad / s according to the frequency-dependent dynamic analysis, it was observed that conventional ChiaMA hydrogel had higher elasticity than 100 W, 250 W and 500 W ChiaMA hydrogels at 2.5% concentration values. In addition, it was observed that the storage modulus (G1) increased more than the loss modulus (G") with frequency in all groups. According to this result (G’>G"), all hydrogel groups have viscoelastic properties. All ChiaMA hydrogels exhibited a stable structure and no deformation was observed as a result of strain as a result of dynamic analysis made dependent on strain by applying strain in the range of 0-2%. It was observed that 100 W and 500 W ChiaMA hydrogels had higher strength than conventional ChiaMA hydrogels compared to the storage module. All hydrogel groups had a decrease in shear stress up to ~37°C with tire increase m temperature according to the temperature-dependent dynamic analysis, but the shear stress became stable after ~37°C.

[0060] The rheological analysis results of the ChiaMA hydrogels prepared at a concentration of 5% are given in Figure 8.2., and the tank and loss module values obtained when the varying frequency is applied in the range of 200 rad / s are given in Table 2. It was observed that all hydrogel groups had elastic properties, and 100 W, 250 W and 500 W ChiaMA hydrogels had higher elasticity than conventional hydrogel by a large difference, if the changing frequency was applied in the range of 0-200 rad / s. The increase in the storage modulus G' with tire increase in frequency is higher than the increase in the loss modulus G". According to this result (G'>G"), all groups exhibit viscoelastic properties. All ChiaMA hydrogels exhibited a stable structure according to die strain-dependent dynamic analysis performed by deformation applied in the range of 0-2%. All of the hydrogels showed a stable structure with the shear stress remaining at a. constant value after ~37°C according to the temperature -dependent dynamic analysis. The viscosity values of the bioinks used in the 3D bioprinting systems provide information on the printability of the bioink. For this reason, biosolution was obtained by dissolving chia mucilage dried with a lyophilizer at room temperature in distilled water at 2.5% and 5% rates of chia mucilage methacrylated by conventional and microwave-assisted (100 W, 250 W, 500 W) methods as mentioned before. Hie viscosity was then determined in the rheometer device in the range of 15-45°C so that the temperature increased by 0.5°C every 30 seconds, thus examining the printability of the solutions of chia mucilage and ChiaMA hydrogels before cross-linking. The viscosity must be in the range of 30 mPa.s to 6 x 10 ' mPa.s in 3D extruder bioprinting systems. Chia mucilage has the highest viscosity value in both concentrations by itself considering the viscosity values according to Figure 9. a and Figure 9.b. The viscosity values of the ChiaMA groups prepared at a concentration of 2.5% vary between 300-1800 mPa.s and the viscosity values of the ChiaMA groups prepared at a concentration of 5% vary between 250-7000 mPa.s. The viscosity values of all groups at room temperature and body temperature, which are important parameters for 3D bioprinting, are given in Table 3 and Table 4. It was concluded that both concentrations were within the printable range for 3D extiuder bioprinting systems according to these values.

[0061] Chia, also known as Salvia hispanica L., is a plant belonging to the mint group with seeds that can vary7in black, beige, and brown colors in elliptical or oval shape, length between 1.8-2.5 mm and width between 1.5-2 mm. The chemical content of chia seeds is fairly rich. It contains approximately 42% carbohydrates, consisting of 16-26% protein, 30-34% fat, 34% nutritional fibers. It is also an excellent source of minerals such as calcium, phosphorus, magnesium, potassium, iron, and zinc. Chia seeds have a higher fatty acid content, 60% alpha linolenic, compared to other seed species. It also contains many fatty acids such as linoleic, oleic, palmitic acid, omega 3 and omega 6 acids. Chia seeds contain relatively high levels of protein compared to other cereals such as wheat, com, rice, oats. In addition, it has been confirmed as a result of the studies that there are various vitamins such as Bl, B2 and Niacin in chia seed. Chia seeds also have a rich source in terms of groups called phytochemicals with high biological activity, of plant origin. These are polyphenols such as gallic, caffeic, chlorogenic, cinnamic and ferulic acid and quercetin, kaempferol. Another benefit of chia seed is that it has a high fiber ratio. Chia seeds are capable of releasing mucilage by absorbing 27 times their own weight when they come into contact with water. Mucilage, also known as dietaiy fiber, located on the outside of the seed shell, constitutes 5 to 6% of the dry' weight of the seed. Mucilage fibers become visible, and these fibers are especially rich in polysaccharides (71.22%) when chia seeds come into contact with water and are completely hydrated. The secreted mucilage is an anionic heteropolysaccharide with a molecular weight between 800 and 2000 kDa. This structure, also known as xyloglucan, has a 1:2: 1 content of xylose, glucose, and methyl glucuronic acid. It can act as a soluble fiber due to its excellent water retention ability. This feature, which allows hydration, has an important role in the development of materials for biomedical applications. In addition, xyloglucan comes to the forefront with its properties such as being mutagenic and non-toxic, and not irritating in case of contact with blood. However, studies on tissue engineering with chia mucilage are fairly limited.

[0062] It is important that the hydrogels used in tissue engineering applications can imitate the extracellular matrix (ECM) structure. ECM is a very dynamic structure consisting of hundred s of proteins and carbohydrates, constantly renewing, and shaping itself, containing celldirecting, growth, migration, and differentiation factors. Hydrogels, on the other hand, have a favorable environment for cell transport with their hydrophilic structures, ability to encapsulate cells and bioactive molecules, and can imitate many features of the natural ECM for tissue regeneration. The natural structure and functions of the tissues are determined by the ECM layout of the tissue and this layout is important in terms of biosimilarity of materials.

[0063] Chia seeds were preferred to be similar to the ECM content with their rich content given above, thus supporting tissue regeneration by imitating the ECM structure. Synthesis of chia mucilage by methacrylation of ChiaMA is necessary for the formation of hydrogel structures through crosslinks. In addition, mechanical strength is added to the structure through the cross-links formed. Methacrylation of chia mucilage with the conventional method is completely new since methacrylation of chia mucilage has not been performed before in the literature. In addition, methacrylation of chia mucilage was performed for the first time with the microwave-assisted methacrylation method, which has advantages such as shorter reaction time and higher degree of methacrylation compared to the conventional method. Table 1 . Properties of 2.5% ChiaMA hydrogels at a frequency of 200 rad / s according to the frequency-dependent dynamic analysis. Table 2. Properties of 5% ChiaMA hydrogels at a frequency of 200 rad / s according to the frequency-dependent dynamic analysis.

[0064] Table 3. The viscosity values of the chia mucilage and ChiaMA solutions prepared at a concentration of 2.5% at 25°C and 37°C.

[0065] Table 4. Viscosity values of chia mucilage and ChiaMA solutions prepared at a concentration of 5% at 25 °C and 37°C. References:

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Claims

CLAIMS1. A hydrogel, characterized in comprising cross-linked, methacrylated chia seed mucilage derivative polysaccharides.

2. A hydrogel manufacturing method, characterized in comprising steps of: a. Secretion of chia mucilage, separation of mucilage from seeds and freeze- drying b. Methacrylation in a solution of N-(3-Dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride, N-hydroxy succinimide and 2-aminoethyl methacrylate hydrochloride c. Freezing with dialysis and lyophilizer to remove unreacted chemicals from the environment.

3. A method according to Claim 2, characterized in that the methacrylation process comprises steps of: i. Dissolving dried chia mucilage in buffer solution ii. Adding N-(3-Dimethylaminopropyl)-N' -ethyl carbodiimide hydrochloride and then N-hydroxy succinimide into the solution and mixing iii. Adding 2-aminoethyl methacrylate hydrochloride and mixing in a dark environment iv. Dialysis of the resulting solution v. Drying the solution with a lyophilizer at the end of the dialysis.

4. A method according to Claim 2, characterized in that the methacrylation process is microwave-assisted.

5. A method according to Claim 4, characterized in that the microwave-assisted methacrylation process comprises steps of: i. Dissolving dried chia mucilage in buffer solution ii. Adding N-(3-Dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride and then N-hydroxy succinimide into the solution and mixing iii. Adding 2-aminoethyl methacrylate hydrochloride dissolved in buffer solution to the mixture iv. Applying a microwave with a power of 100 W, 250 W or 500 W.

6. A hydrogel according to Claim 1, for use in tissue regeneration by mimicking the extracellular matrix structure.

7. A graft, characterized in that it comprises a hydrogel according to Claim 1 .

8. A bioink for use in 3D bioprinting systems, characterized in that it comprises a hydrogel according to Claim 1.

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