Hydrogel comprising functional peptide adjustable in physical properties

Abstract

The present invention provides a hydrogel and a bioprinting method using same, the hydrogel comprising a modified functional peptide and a tyrosine-containing biodegradable polymer, wherein the modified functional peptide has tyrosine-containing linkers connected to both ends of a functional peptide, and spacers are independently interposed between the linkers and at least one of the two ends. In the present invention, the flexibility of a bioprinted structure can be adjusted by adjusting the spacer length of the hydrogel.

Description

Hydrogel containing functional peptides capable of controlling physical properties

[0001] The present invention relates to a hydrogel comprising a functional peptide, and specifically to a hydrogel comprising a functional peptide in which a tyrosine-containing linker is connected to both ends of the functional peptide.

[0002]

[0003] 3D bioprinting is a technology that enables the fabrication of three-dimensional tissues and organs by positioning cells at desired locations. When performing bioprinting using cells, printing only the cells can induce cell death. Therefore, bioprinting is carried out by encapsulating the cells in a material called bioink.

[0004] Bioinks must ensure high resolution for 3D bioprinting, including deposition ability, printability, and shape fidelity. Bioinks not only provide the high resolution essential for 3D bioprinting but also offer an environment that promotes cell growth, differentiation, and maturation; when cells are mixed into the bioink, the bioink provides the cells with a three-dimensional microenvironment. Since this three-dimensional microenvironment possesses a three-dimensional network structure similar to the extracellular matrix of our bodies, it can induce an enhanced effect on the cells' tissue regeneration capabilities.

[0005] Recently, the development of bioinks using decellularized extracellular matrix allows for tissue-specific regenerative performance because the extracellular matrix components are used as they are. Due to their high regenerative performance, they are being actively researched in the field of 3D bioprinting.

[0006] However, since decellularized extracellular matrix is ​​primarily produced from animal-derived tissues, characteristics vary by host, which can lead to differences between production batches, and there are limitations in immunogenicity and the potential for pathogen delivery due to differences between human and animal species.

[0007] Meanwhile, research is currently underway to mimic the extracellular matrix by adding peptides to bioinks. Peptides are the smallest units capable of performing protein functions, and research is being conducted to maximize regeneration efficiency by loading peptides that act as extracellular matrix or growth factors into bioinks.

[0008] Although peptide-loaded bioinks cannot replicate the complexity of the extracellular matrix found in decellularized extracellular matrix bioinks, they can ensure consistency of the material across production batches and control tissue regeneration ability depending on the functionality of the loaded peptides.

[0009] The invention of Korean Published Patent No. 10-2024-0105305 discloses a method for efficiently loading peptides into bioink without using a synthesis method. The invention discloses a method for loading functional substances, particularly functional peptides, into bioink by introducing a tyrosine-containing linker to the terminal of a functional peptide, adding this to a protein-containing bioink, and then irradiating it with light to cross-link the protein components forming the matrix while simultaneously chemically binding the functional peptide to the protein, thereby enabling the introduction of functional peptides into bioink in a simple manner.

[0010] In 2006, Engler et al. disclosed in Cell 126, 677 that cell differentiation can be regulated by the elasticity of the matrix. It is thought that if this concept is introduced to bioinks, it will be possible to diversify tissue regeneration targets by controlling the physical properties of the bioink.

[0011] Accordingly, the inventors discovered that when a hydrogel prepared by introducing a tyrosine-containing linker to both ends of a functional peptide and independently including a glycine residue as a spacer between the linker and the functional peptide is used as a bioink, the flexibility of a structure produced by 3D bioprinting can be controlled, and thus completed the present invention.

[0012] The present invention is a result obtained by carrying out the project number RS-2024-00423107 as the “Development of a Macro-scale Bionic Twin Model-based Therapeutic Evaluation Platform for Patients with Amyotrophic Lateral Sclerosis” and the project number RS-2024-00407234 as the “Development of Process and Material Technology for Implementing Customized Gradient Functions for Defective Areas by Imitating Continuous / Three-dimensional Gradients of Hard-Soft Tissue Interface Composition, Structure, and Environment with Precision (≤20μm)”.

[0013]

[0014] We aim to provide a new functional peptide loaded into a bioink that can control the flexibility of structures produced by 3D bioprinting.

[0015]

[0016] In one aspect, a hydrogel comprising a modified functional peptide and a tyrosine-containing biodegradable polymer is provided, wherein the modified functional peptide has tyrosine-containing linkers connected to both ends of the functional peptide, and a spacer is independently interposed between at least one of the ends and the linkers.

[0017] In another aspect, a step of forming a structure by bioprinting the hydrogel; and

[0018] A bioprinting method is provided, comprising the step of irradiating light onto the above structure.

[0019]

[0020] The hydrogel of the present invention can control the flexibility of a structure produced by bioprinting by adjusting the length of a spacer portion composed of glycine repeating units.

[0021]

[0022] Figure 1 is a schematic diagram illustrating a method of utilizing a functional peptide of a hydrogel as a crosslinking agent for a polymer.

[0023] Figure 2 is a graph showing the calculated water content of the hydrogel structures of the examples and comparative examples.

[0024] Figure 3 is a photograph of a three-dimensional structure obtained by bioprinting a hydrogel according to the present invention loaded with cells.

[0025] Figure 4 is a photograph of a muscle fiber obtained by culturing a three-dimensional structure obtained by bioprinting the hydrogels of Comparative Example 1 and Examples 1 to 3 loaded with cells.

[0026] Figure 5 is a graph showing the calculated fusion index of the muscle fibers obtained in Figure 4.

[0027] Figure 6 is a photograph of the structures of Comparative Example 1 and Example 2 implanted in the anterior tibialis muscle.

[0028] Figure 7a is a graph measuring the muscle contraction force of the anterior tibialis muscle implanted with the structures of Comparative Example 1 and Example 2.

[0029] Figure 7b is a graph measuring the percentage of muscle contraction force relative to normal muscle of the tibialis anterior muscle implanted with the structures of Comparative Example 1 and Example 2.

[0030] Figure 8 is a graph measuring the length of the anterior tibialis muscle implanted with the structures of Comparative Example 1 and Example 2.

[0031] Figure 9 is a photograph confirming whether blood vessels were formed in the defective area and the area where the structure was implanted.

[0032] Figure 10 is a graph of the area of ​​blood vessels created in the defective area and the area where the structure was implanted.

[0033] Figure 11 is a photograph confirming the generation of muscle fibers of the delivery cells in the area where the structure was implanted.

[0034] Figure 12 is a graph showing the number of huNu cells per MHC+ cell of the delivery cell at the site where the structure was implanted.

[0035] Figure 13 is a photograph confirming the presence of neuromuscular junctions in the area where the structure was implanted.

[0036] Figure 14a is an NMJ graph of the area where the structure was implanted.

[0037] Figure 14b is a graph of the NMJ recovery rate compared to normal tissue in the area where the structure was implanted.

[0038]

[0039] The present invention will be described in detail below.

[0040] In interpreting the specification, the term "includes" means that additional components may be included, and in interpreting the scope of rights, additional components should be interpreted as being excluded unless explicitly limited otherwise.

[0041] In the present invention, a hydrogel comprising a modified functional peptide having tyrosine-containing linkers connected to both ends of the functional peptide and a tyrosine-containing biodegradable polymer is provided.

[0042] In the hydrogel of the present invention, the biodegradable polymer forming the matrix after printing of the hydrogel may preferably be a protein, or tyrosine may be introduced as a non-protein-based biodegradable polymer.

[0043] The hydrogel of the present invention can be used as a bioink that serves as a material for bioprinting. The bioink contains a biocompatible material that mimics living cells and the extracellular matrix environment, thereby supporting cell adhesion, proliferation, and differentiation after printing. Bioinks are typically used in 3D printing, however, they require properties different from those of 3D printing materials such as metals, ceramics, and polymers. The bioink contains a biocompatible material that mimics living cells and the extracellular matrix environment, thereby supporting cell adhesion, proliferation, and differentiation after printing. Since cell differentiation can be controlled by the elasticity of the matrix, cell differentiation can be controlled by adjusting the elasticity or flexibility of the bioink.

[0044] The hydrogel of the present invention may include other additional components along with natural or non-natural polymer components that constitute the matrix, for example, the cell itself and / or functional materials necessary for cell growth or differentiation. The polymer is typically a natural or non-natural polymer capable of forming a hydrogel, preferably a protein, and specific examples include collagen, gelatin, and fibroin as a component of silk, and these exemplary polymers contain tyrosine amino acids within the protein, and various other proteins or polymers containing tyrosine in their sequences may also be used.

[0045] Meanwhile, the above polymer may be a non-protein biodegradable polymer such as alginate, hyaluronic acid, or chitosan, provided, however, that in this case, these polymers may have tyrosine amino acids chemically introduced. These tyrosine-functionalized biodegradable non-protein polymers may be functionalized through known methods.

[0046] The above protein may be a protein derived from nature or may be artificially produced by biotechnological methods. The above protein may be used in its natural sequence as is, and the scope of the present invention also includes cases where the sequence has been artificially partially modified or chemically partially modified.

[0047] The concentration of the biodegradable polymer in the hydrogel can be appropriately selected within the range of 0.1 to 10% (w / v). If the concentration of the biodegradable polymer is lower than the lower limit, it may be difficult to form the hydrogel, and if it is higher than the upper limit, the viscosity of the hydrogel may be excessive, making subsequent bioprinting difficult. As a specific example, if collagen is selected, it may be included within the range of 0.1 to 5%.

[0048] The above hydrogel is aqueous-based and may be prepared by including a protein for the matrix in a buffer solution for cell culture, and additionally adding cells and / or functional materials thereto.

[0049] The above functional substance is not limited but preferably includes a functional peptide. The sequence length of the functional peptide is not particularly limited, but typically may include 1 to 100 amino acids, 2 to 50 amino acids, 3 to 50 amino acids, or 5 to 50 amino acids.

[0050] The amino acids in functional peptides typically include amino acids derived from nature, and may also include artificially synthesized amino acids.

[0051] The above functional peptide is modified at both ends by a tyrosine-containing linker. The N-terminus or C-terminus of the functional peptide may be modified. The linker contains tyrosine residues and forms dityrosine bonds with the tyrosine of the biodegradable polymer forming the matrix upon light irradiation. The linker may contain 1 to 20, 1 to 10, 1 to 5, 2 to 10, or 2 to 5 tyrosine residues. If it contains 20 or more tyrosine residues, the modified linker may be too long compared to the length of the functional peptide, and thus the functional peptide may not be able to properly perform its function. For example, the linker may have a peptide sequence consisting solely of tyrosine residues.

[0052] The above functional peptide may be connected such that both ends are modified by tyrosine-containing linkers, and additionally, spacers are independently interposed between the linker and the ends of the functional peptide.

[0053] The spacer enables the functional peptide itself to fully exhibit its intended functionality. The spacer may, for example, include glycine residues, and may include 1 to 20, 1 to 10, 1 to 9, or 3 to 6 glycine residues. As the length of the glycine residues increases, the physical properties of the hydrogel may become more flexible. However, if 20 or more glycine residues are included, the flexibility of the hydrogel may become excessive, and its physical properties may be weakened. For example, the spacer may have a peptide sequence consisting solely of glycine residues.

[0054] The physical properties of the hydrogel can be controlled by adjusting the length of the spacers. For example, in an embodiment of the present invention, it was confirmed that the number of glycines, which are spacers, affects the muscle maturity of a structure bioprinted with a hydrogel.

[0055] The concentration of the functional peptide or modified functional peptide that can be loaded varies depending on the solubility of the peptide, and can be appropriately selected and added as needed depending on the purpose and the type of selected functional peptide or modified functional peptide.

[0056] Meanwhile, the hydrogel according to the present invention may include a photoinitiator and / or an oxidizing agent for forming dityrosine bonds.

[0057] Although not specifically limited as photoinitiators, specific examples include Rose Bengal, methylene blue, Eosin-Y, Ruthenium, and Riboflavin. When using a photoinitiator, specifically Riboflavin, it can be used at a concentration of 10 μM to 50 mM. If the concentration of Riboflavin is lower than the lower limit, dityrosine bonds may not form well. However, if it is higher than the upper limit, it does not cause a significant difference in crosslinking.

[0058] As an oxidizing agent, persulfates may be used as an example, and as specific examples, sodium persulfate or ammonium persulfate may be used. When selecting an oxidizing agent, specifically sodium persulfate, its concentration may be selected in the range of 2 mM to 50 mM. If the concentration of sodium persulfate is lower than the lower limit, oxidation may not proceed smoothly. However, even if it is higher than the upper limit, it does not cause a significant difference in the formation of the hydrogel.

[0059] Meanwhile, additional cells can be added to the hydrogel as needed. The types of cells can be added according to the purpose; for example, bone marrow-derived mesenchymal stem cells are 1 x 10⁶ 6 At a concentration of cells / ml, skeletal muscle cells are 1.5 x 10 7It can be added at a concentration of cells / ml. It is evident that the cell concentration can also be changed depending on the purpose.

[0060] In one aspect of the present invention, a cell regeneration structure is provided that helps regenerate cells by bioprinting the hydrogel.

[0061] In another aspect of the present invention,

[0062] A step of forming a structure by bioprinting the above hydrogel; and

[0063] A bioprinting method is provided, comprising the step of irradiating light onto the above structure.

[0064] The hydrogel according to the present invention is biodegraded by light irradiation, so that cross-linking between polymers and binding of modified functional peptides to the biodegradable polymers occur simultaneously.

[0065] A structure can be formed by curing the structure through the formation of a three-dimensional structure using a hydrogel according to the present invention via bioprinting or 3D bioprinting, and then irradiating the structure with light. The curing is achieved by crosslinking through the bonding between tyrosines present in the biodegradable polymer forming the matrix, namely, dityrosine bonds.

[0066] In addition, the irradiated light causes the formation of dityrosine bonds between the tyrosine in the added modified functional peptide and the tyrosine present in the biodegradable polymer.

[0067] In the light irradiation step, if cells are involved, it is preferable to use light in the visible light range to prevent cell death; specifically, light with a wavelength of 395 nm to 415 nm when riboflavin is used as a photoinitiator, or light with a wavelength of 520 nm to 540 nm when eosin Y is used as a photoinitiator. In addition, it is advisable to use appropriate wavelengths for each photoinitiator. The light intensity is 5 mW / cm². 2The above light intensity can be used. The light intensity can also be selected by appropriately adjusting it. In the embodiment of the present invention, 70 mw / cm² 2 used.

[0068] It is desirable to continue the light irradiation time until the hydrogel is cured, and this may vary depending on the polymer of the hydrogel. If the light irradiation time is too long, heat may be generated and cells may die; therefore, the curing time can be appropriately adjusted to achieve sufficient curing while preventing cell death.

[0069] In the embodiment of the present invention, 50 seconds were used.

[0070] When light irradiation occurs, the tyrosine in the collagen is oxidized, and at the same time, the tyrosine in the peptide is oxidized, and the tyrosines in the collagen chemically bond with each other to become dityrosine, which cross-links and hardens the hydrogel.

[0071] The chemical bonding between collagen and a functional peptide modified with tyrosine at both ends is as shown in Fig. 1.

[0072] Meanwhile, at the same time, the tyrosine of the peptide is also oxidized and meets the oxidized tyrosine of the collagen chain to become dityrosine, which enables the peptide to be loaded onto the collagen chain.

[0073] When forming a three-dimensional structure through bioprinting or 3D bioprinting, a functional peptide may be loaded first, and then additional functional peptides may be loaded after repeatedly contacting the structure with the functional peptide.

[0074]

[0075] The present invention will be explained below through specific embodiments. These embodiments are intended only to facilitate the implementation of the invention, and the scope of the invention should not be interpreted as being limited to these embodiments.

[0076]

[0077] <Preparation of Ingredients>

[0078] Functional peptides were commercially custom-made and used.

[0079] As the functional peptide used in Examples 1 to 3, the laminin peptide (having the IKVAV sequence) of Table 1 below was selected. Both ends of functional peptide C were modified with a spacer consisting of glycine residues, and then a linker consisting of tyrosine residues was modified at the ends of the spacers.

[0080] As Comparative Example 1, a functional peptide of SEQ ID NO. 4 without a spacer was selected, and as Comparative Example 2, a functional peptide of SEQ ID NO. 5 in which a linker exists on only one side was selected.

[0081]

[0082] Example 1 Sequence No. 1 YYYGGGIKVAVGGGYYY Example 2 Sequence No. 2 YYYGGGGGGIKVAVGGGGGGYYY Example 3 Sequence No. 3 YYYGGGGGGGGGIKVAVGGGGGGGGGYYY Comparative Example 1 Sequence No. 4 YYYIKVAVYYY Comparative Example 2 Sequence No. 5 IKVAVGGGYYY

[0083]

[0084] Example 1.

[0085] A hydrogel containing collagen as a matrix was prepared in the same manner as in Prior Art 1, and a functional peptide was loaded.

[0086] A 1% collagen hydrogel was prepared by dissolving porcine skin-derived atelocollagen (Collagen from porcine skin from Darim Tissen Co., Ltd.) in 0.5M acetic acid and then titrating the pH to 7 using 10N NaOH. Subsequently, a photoinitiator and an oxidizing agent were added to the hydrogel. A hydrogel solution was prepared by adding Riboflavin as a photoinitiator and sodium persulfate as an oxidizing agent at concentrations of 100μM and 10mM, respectively.

[0087] The functional peptide of SEQ ID NO. 1 prepared was added to the prepared hydrogel solution. Although the laminin peptide can be dissolved at a maximum concentration of 5 mg / ml, in the example, the peptide was added to the hydrogel at a concentration of 200 µg / ml to prepare a hydrogel containing the functional peptide.

[0088] Example 2.

[0089] A hydrogel containing a functional peptide was prepared in the same manner as in Example 1, except that the functional peptide of SEQ ID NO. 2 was used instead of SEQ ID NO. 1.

[0090] Example 3.

[0091] A hydrogel containing a functional peptide was prepared in the same manner as in Example 1, except that the functional peptide of SEQ ID NO. 3 was used instead of SEQ ID NO. 1.

[0092] Comparative Example 1.

[0093] A hydrogel containing a functional peptide was prepared in the same manner as in Example 1, except that the functional peptide of SEQ ID NO. 4 was used instead of SEQ ID NO. 1.

[0094] Comparative Example 2.

[0095] A hydrogel containing a functional peptide was prepared in the same manner as in Example 1, except that the functional peptide of SEQ ID NO. 5 was used instead of SEQ ID NO. 1.

[0096] Comparative Example 3.

[0097] A hydrogel was prepared in the same manner as in Example 1, except that no functional peptide was used.

[0098]

[0099] Experimental Example 1. Comparison of water content of hydrogels according to peptide loading

[0100] Hydrogels containing functional peptides of SEQ ID NOs 1 to 3 prepared in Examples 1 to 3, hydrogels containing functional peptides of SEQ ID NOs 4 and 5 as a comparative group (Comparative Examples 1 and 2), and a control hydrogel not containing functional peptides (Comparative Example 3) were prepared.

[0101] After dispensing 100 µl of each prepared hydrogel into a 24-well plate, irradiate with visible light at a wavelength of 405 nm at a light intensity of 70 mW / cm² 2 It was investigated for 50 seconds.

[0102]

[0103] The swelling ratio of each fabricated hydrogel structure was calculated using the above formula and is shown in Figure 2. W0 is the dry weight of the structure, and W1 is the weight of the structure after swelling in PBS.

[0104] Through Figure 2, it was confirmed that the water content increases as the length of the glycine increases. This is because the hydrogel cross-linked network has larger pores, and as the peptide length increases, this pore structure provides space to capture more water molecules, which increases the overall water absorption rate and consequently raises the water content (swelling ratio). An increase in water content can lead to more flexible physical properties.

[0105] On the other hand, Comparative Example 3, which did not include a peptide, Comparative Example 2, which included a linker at only one end (using IKVAVGGGYYY), and Comparative Example 1, which did not have a G (spacer), showed no significant difference.

[0106]

[0107] Experimental Example 2. Bioprinting using collagen hydrogel

[0108] After preparing the hydrogel as in Example 1, skeletal muscle cells 1.5 x 10 7It was added to the hydrogel at a concentration of cells / ml.

[0109] Peptide and cell-added collagen hydrogel at 405nm wavelength, 70mw / cm² 2 Muscle structures were printed using a photopolymerization-based 3D bioprinter equipped with light intensity. The thickness of the fabricated muscle structure layers was 100 µm, and the curing time for each layer was 50 seconds.

[0110] Bioprinting allows for the simultaneous fabrication of three-dimensional structures and peptide loading, as well as the loading of various cells and the fabrication of various tissue structures. By loading peptides with diverse biological activities, differentiation and growth of each tissue can be promoted.

[0111] Muscle structures were printed using the hydrogel of Example 1 with a bioprinter with structure sizes of 200m, 400m, and 800m, and this is shown in Fig. 3.

[0112]

[0113] Experimental Example 3. Confirmation of muscle fiber growth according to peptide length in bioprinted structures

[0114] After culturing muscle structures prepared with the hydrogels of Comparative Example 1 and Examples 1 to 3, which had a structure size of 800 m, for 14 days, a myosin heavy chain antibody (primary antibody) was treated, followed by treatment with a secondary fluorescent antibody to perform fluorescent immunostaining. Subsequently, cell nuclei were stained using DAPI (4′,6-diamidino-2-phenylindole), and the myosin heavy chains of each group were identified using a confocal fluorescence microscope and are shown in Figure 4.

[0115] Upon checking the formation of muscle fibers, high muscle fiber formation was observed when using the hydrogels of Examples 1 and 2. On the other hand, when using the hydrogel of Comparative Example 1, muscle fiber formation was inhibited due to its relatively rigid physical properties. When using the hydrogel of Example 3, although muscle fiber growth was observed, the structure was somewhat soft and appeared to have shrunk on day 14 of culture, indicating that muscle fiber formation was somewhat inhibited compared to other groups.

[0116] The Fusion index, an indicator used to evaluate the differentiation and maturation of muscle cells, was calculated according to the formula below and is shown in Figure 5. It was confirmed that the highest degree of muscle maturation was observed when the hydrogel of Example 2 was used.

[0117]

[0118]

[0119] Experimental Example 4. Confirmation of muscle function recovery following structural implantation

[0120] A 40% defect was introduced into the tibialis anterior muscle of 9-week-old Sprague-Dawley rats (SD rats), and the structures of Comparative Example 1 and Example 2 were implanted, as shown in Fig. 6.

[0121] After 4 weeks, to evaluate the physiological function of the muscle, the tendon of the tibialis anterior muscle was connected to a force transducer, and the muscle contraction force induced by applying electrical stimulation to the sciatic nerve was measured. The measurement results are shown in Fig. 7a for muscle contraction force and in Fig. 7b for the percentage of muscle contraction force relative to normal muscle. The muscle contraction force showed a recovery of more than 80% in the Comparative Example 1 and Example 2 groups compared to normal muscle. In particular, the structure of Example 2 was able to confirm a recovery of 88% of muscle function.

[0122] In addition, the muscle weight of each muscle was measured after 4 weeks and is shown in Fig. 8. Both Comparative Example 1 and Example 2 groups loaded with peptides showed a recovery of more than 80% compared to normal muscle weight.

[0123]

[0124] Experimental Example 5. Confirmation of angiogenesis following structure transplantation

[0125] 40% defect was introduced into the tibialis anterior muscle of 9-week-old Sprague-Dawley rats (SD rats), and the structures of Comparative Example 1 and Example 2 were implanted.

[0126] Vascular immunostaining was performed by treating the defective site and the site where the structure was transplanted with a CD31 antibody (primary antibody) and a secondary fluorescent antibody to confirm the formation of angiogenesis. Subsequently, CD31 positive areas were measured by analyzing images taken with a fluorescence microscope, and the results are shown in Figures 9 and 10. In Figure 9, CD31 represents blood vessels and vascular endothelial cells, while DAPI represents cell nuclei. High vascularization was observed in the peptide-loaded group, and particularly in Example 2, high vascularization was confirmed.

[0127]

[0128] Experimental Example 6. Confirmation of muscle fiber generation in delivery cells following structure transplantation

[0129] 40% defect was introduced into the tibialis anterior muscle of 9-week-old Sprague-Dawley rats (SD rats), and the structures of Comparative Example 1 and Example 2 were implanted.

[0130] To confirm the generation of muscle fibers by delivery cells at the site where the structure was implanted, measurements were taken using staining with HuNu antibodies (Anti-Human Nuclei Antibody) and are shown in Figs. 11 and 12. The 3D bioprinted muscle consists of 1.5 x 10 human skeletal muscle cells. 7 Since it was prepared in cells / ml, human nuclei (HuNu) staining was performed, and surrounding muscle staining was carried out. In Figure 11, HuNu represents the nuclei of human-derived cells, MHC represents muscle fibers, and DAPI represents the nuclei of all cells. As a result, human nuclei staining was confirmed in more than 60% of the newly regenerated muscle at the transplant site.

[0131]

[0132] Experimental Example 7. Confirmation of neuromuscular junction regeneration following structural implantation

[0133] 40% defect was introduced into the tibialis anterior muscle of 9-week-old Sprague-Dawley rats (SD rats), and the structures of Comparative Example 1 and Example 2 were implanted.

[0134] To confirm the presence of neuromuscular junctions in the site where the structure was implanted, muscle, acetylcholine receptors, and nerves were stained simultaneously. These results were measured using fluorescence immunostaining and confocal microscopy analysis and are shown in Fig. 13. Neuromuscular junctions (NMJs) were measured and are shown in Fig. 14a, while a graph showing the NMJ recovery rate relative to normal tissue in the site where the structure was implanted is shown in Fig. 14b. In Fig. 13, MHC represents muscle fibers, AChR represents acetylcholine receptors, Tuj1 represents nerve fibers, and DAPI represents the nucleus of all cells. As a result, high NMJ formation was confirmed in the peptide-loaded structure compared to the defective muscle, and high NMJ formation was confirmed particularly in Example 2.

Claims

1. A hydrogel comprising a modified functional peptide and a tyrosine-containing biodegradable polymer, wherein the modified functional peptide has tyrosine-containing linkers connected to both ends of the functional peptide, and a spacer is independently interposed between at least one of the ends and the linkers.

2. In Paragraph 1, The above hydrogel is characterized in that a functional peptide is chemically bonded to the biodegradable polymer by irradiating light after bioprinting.

3. In Paragraph 2, The above-mentioned bond is a dityrosine bond between the tyrosine of the linker and the tyrosine of the biodegradable polymer, in a hydrogel.

4. In Paragraph 1, The above tyrosine-containing biodegradable polymer is a hydrogel, which is a non-protein-based biodegradable polymer into which protein or tyrosine has been introduced.

5. In Paragraph 4, The above protein is a hydrogel selected from collagen, gelatin, and fibroin.

6. In Paragraph 4, The above-mentioned non-protein-based biodegradable polymer is a hydrogel selected from alginate, hyaluronic acid, and chitosan.

7. In Paragraph 1, The above tyrosine-containing linker is a hydrogel containing 1 to 20 tyrosine residues.

8. In Paragraph 1, The above spacer is a hydrogel comprising 1 to 20 glycine residues.

9. In Paragraph 1, A hydrogel comprising a tyrosine-containing linker composed of 1 to 20 tyrosine residues and a spacer composed of 1 to 20 glycine residues.

10. In Paragraph 1, The above hydrogel is a hydrogel that further contains cells.

11. In Paragraph 10, A hydrogel that aids in the regeneration of cells or tissues in the body by bioprinting the above hydrogel.

12. A step of forming a structure by bioprinting the hydrogel of claim 1; and A bioprinting method comprising the step of irradiating light onto the above structure.

13. In Paragraph 12, A bioprinting method characterized by irradiating light to form crosslinks between tyrosine-containing biodegradable polymers, while simultaneously a modified functional peptide crosslinks between the biodegradable polymers.

14. In Paragraph 13, A bioprinting method in which the above-mentioned crosslinking is achieved through dityrosine binding.

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