KR102721585B1 - Method for Manufacturing collagen based shape memorable hydrogel and uses thereof - Google Patents

Abstract

The present invention relates to a method for producing a hydrogel having shape restoring ability, and uses thereof. The hydrogel produced by the F-gel production method of the present invention has high cell activity, shape fixing ability, and shape restoring ability. In addition, it was confirmed that the biocomposite of the present invention produced by a production method that combines the cryo-gel production method and the F-gel production method has cell activity and shape fixing ability superior to the cryo-gel, has a shape restoration speed superior to the F-gel, and has high shape restoring ability. In addition, it was experimentally confirmed that when a hydrogel containing a polymer, ceramic, or metallic material is produced by utilizing the F-gel and the biocomposite production method, all of them maintain shape memory characteristics, and that all structures have excellent shape restoring ability when the hydrogel undergoes the same post-processing process regardless of the production process (3D printing). Therefore, it is expected that the production of a three-dimensional structure having shape restoring ability is possible, and that non-invasive use is possible in which the deformed structure can be easily injected into the human body or other underwater spaces.

Description

Method for manufacturing collagen-based shape memorable hydrogel and uses thereof {Method for Manufacturing collagen-based shape memorable hydrogel and uses thereof}

The present invention relates to a method for producing a collagen-based hydrogel having shape restoring ability, its use, etc.

Hydrogels are cross-linked 3D polymer networks that contain large amounts of water. They can be used in a variety of applications, including tissue engineering, biosensors or biofunctionalizers, drug delivery, fabrication of organisms, and biophysical analysis, due to their ability to support cell culture and permeability, which facilitates efficient transport of materials. Recent research has focused on shape-memory hydrogels (SMH) with high bioactivity to expand the applications of hydrogels in precision medicine, such as self-tailored scaffolds that induce osteointegration, and minimally invasive surgery for tissue regeneration.

For biomedical applications, bioactive hydrogels generally require suitable physical properties; excellent bioactivity to provide cytophilic motifs to induce cell adhesion and proliferation; and controllable biodegradability. Therefore, natural biopolymers derived from mammals, such as collagen, gelatin, elastin, and decellularized extracellular matrix (dECM), are more promising than synthetic hydrogels, such as polyethylene glycol and Pluronic F-127.

Among protein-based hydrogels, collagen is the most abundant structural protein in organs and tissues such as skin, tendons, bones, cornea, and heart. Collagen monomers are right-handed triple helices (300 nm in length and 1.5 nm in diameter), and each chain has a characteristic repeating sequence (Gly-X-Y), where X and Y are mainly composed of proline (Pro) and hydroxyproline (Hyp), respectively. In addition, collagen monomers have a unique self-assembly property called 'collagen fibrillation' in physiological environments. In vivo, collagen can be synthesized and secreted by fibroblasts, and monomers can be assembled into collagen fibers or fibrillar tissues that form collagen tissue. Collagen fibrillation can be replicated in vitro by controlling pH, temperature, and ionic strength, thereby generating collagen gels. Collagen gels can be used for cell-mediated collagen fiber remodeling, organogenesis, and tissue engineering scaffolds to model the fibrous microenvironment of the extracellular matrix.

Macro/microporous collagen-based cryogels have been recently fabricated using phase separation of solvent (water) and solute (polymer, crosslinker) caused by the formation of ice crystals. Not only have they shown shape memory properties, they can be widely used in various applications including injectable scaffolds, cartilage regeneration, oxygen carriers, cell transplantation, drug delivery, and even immunotherapy.

As research on a method for manufacturing hydrogels of various shapes with shape-restoring ability using collagen as a main material is insufficient, the inventors of the present invention studied a method for manufacturing collagen-based hydrogels with shape-restoring ability and completed the present invention.

Republic of Korea Patent Publication No. 10-0692608

The present inventors confirmed that the hydrogel by the F-gel manufacturing method of the present invention has high cell activity, shape fixation ability, and shape restoring ability, and that a biocomposite manufactured by the fusion of the cryo-gel manufacturing method and the F-gel manufacturing method has cell activity and shape fixation ability superior to that of the cryo-gel, shape restoration speed superior to that of the F-gel, and high shape restoring ability. In addition, when a hydrogel containing a polymer, ceramic, and metallic material is manufactured using the F-gel and the biocomposite manufacturing method, it is experimentally confirmed that all of the shape memory characteristics are maintained, and that regardless of the manufacturing process (3D printing) of the hydrogel, if it undergoes the same post-processing process, it has excellent shape restoring ability in all structures, thereby completing the present invention.

Accordingly, the purpose of the present invention is to provide a method for preparing a starting material comprising collagen, comprising: (a) preparing a starting material comprising collagen;

(b) a step of adding PBS to the starting material and fiberizing it in an environment of 30 to 40°C; and

(c) It provides a method for producing a shape memory hydrogel, including a step of crosslinking the fiberized starting material using a crosslinking solution.

Another object of the present invention is to provide a method for producing a composition comprising: (a) preparing a starting material comprising collagen;

(b) a step of adding PBS to the starting material and fiberizing it in an environment of 30 to 40°C; and

(c) a shape memory hydrogel manufactured by a method including a step of crosslinking the fiberized starting material using a crosslinking solution,

The above hydrogel provides a shape memory hydrogel characterized by satisfying at least one of the following characteristics:

(i) increasing cell adhesion and proliferation effects; and

(ii) Increase in shape fixation ability.

Another object of the present invention is to provide a method for producing a method comprising: (a) preparing a starting material comprising collagen and a photocurable polymer;

(b) a step of freezing the starting material;

(c) a step of photo-curing the frozen starting material to perform primary cross-linking;

(d) a step of adding PBS to the first cross-linked starting material and fiberizing it in an environment of 30 to 40°C; and

(e) It provides a method for producing a shape memory hydrogel, including a step of secondarily cross-linking the fiberized starting material using a cross-linking solution.

In addition, the present invention comprises the steps of (a) preparing a starting material comprising collagen and a photocurable polymer;

(b) a step of freezing the starting material;

(c) a step of photo-curing the frozen starting material to perform primary cross-linking;

(d) a step of adding PBS to the first cross-linked starting material and fiberizing it in an environment of 30 to 40°C; and

(e) It provides a shape memory hydrogel manufactured by a method including a step of secondarily cross-linking the fiberized starting material using a cross-linking solution.

However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

In order to achieve the above-described purpose of the present invention, the present invention comprises the steps of: (a) preparing a starting material including collagen;

(b) a step of adding PBS to the starting material and fiberizing it in an environment of 30 to 40°C; and

(c) Provided is a method for producing a shape memory hydrogel, including a step of cross-linking the fiberized starting material using a cross-linking solution.

In addition, the present invention comprises the steps of (a) preparing a starting material including collagen;

(b) a step of adding PBS to the starting material and fiberizing it in an environment of 30 to 40°C; and

(c) a shape memory hydrogel manufactured by a method including a step of crosslinking the fiberized starting material using a crosslinking solution,

The above hydrogel provides a shape memory hydrogel characterized by satisfying at least one of the following characteristics:

(i) increasing cell adhesion and proliferation effects; and

(ii) Increase in shape fixation ability.

In addition, the present invention comprises the steps of (a) preparing a starting material comprising collagen and a photocurable polymer;

(b) a step of freezing the starting material;

(c) a step of photo-curing the frozen starting material to perform primary cross-linking;

(d) a step of adding PBS to the first cross-linked starting material and fiberizing it in an environment of 30 to 40°C; and

(e) Provided is a method for producing a shape memory hydrogel, including a step of secondarily cross-linking the fiberized starting material using a cross-linking solution.

In addition, the present invention comprises the steps of (a) preparing a starting material comprising collagen and a photocurable polymer;

(b) a step of freezing the starting material;

(c) a step of photo-curing the frozen starting material to perform primary cross-linking;

(d) a step of adding PBS to the first cross-linked starting material and fiberizing it in an environment of 30 to 40°C; and

(e) A shape memory hydrogel is provided, manufactured by a method including a step of secondarily cross-linking the fiberized starting material using a cross-linking solution.

In one embodiment of the present invention, the starting material may be characterized by further including, but not limited to, one or more selected from the group consisting of polymeric materials, ceramic materials, and metallic materials.

In another embodiment of the present invention, the cross-linking solution may be characterized by including at least one selected from the group consisting of EDC/NHS [(N-ethyl-N'-(3-(dimethylamino)propyl) carbodiimide/N-hydroxysuccinimide)], glutaraldehyde, tannic acid, genipin, formaldehyde, and LOX (lysyl oxidase), but is not limited thereto.

In another embodiment of the present invention, the hydrogel may be characterized by increasing the expression of one or more genes selected from the group consisting of talin (TLN), paxillin (PXN), vinculin (VCL), focal adhesion kinase (FAK), and RhoA kinase, but is not limited thereto. The gene may be characterized by expressing a cell-substrate adhesion-related protein in cultured cells when cells containing the gene are cultured, but is not limited thereto.

In another embodiment of the present invention, the photocurable polymer may be characterized as being methacrylate collagen, but is not limited thereto.

In another embodiment of the present invention, the starting material may be characterized in that it contains collagen and a photocurable polymer in a mass ratio (w/w %) of 0.1:3.9 to 2:2, but is not limited thereto.

In another embodiment of the present invention, the starting material of step (a) may be characterized by further including, but not limited to, one or more selected from the group consisting of polymeric materials, ceramic materials, and metallic materials.

In another embodiment of the present invention, the hydrogel may be characterized by satisfying one or more of the following characteristics, but is not limited thereto: (i) increasing cell adhesion and proliferation effect; and (ii) increasing shape restoration speed.

In another embodiment of the present invention, the hydrogel may be a shape memory hydrogel manufactured by a manufacturing method further including a 3D printing step between steps (a) and (b), but is not limited thereto.

The hydrogel prepared by the F-gel manufacturing method of the present invention has high cell activity, shape fixation ability, and shape restoring ability. In addition, it was confirmed that the biocomposite prepared by the manufacturing method that combines the cryo-gel manufacturing method and the F-gel manufacturing method has better cell activity and shape fixation ability than the cryo-gel, better shape restoration speed than the F-gel, and high shape restoring ability. In addition, it was experimentally confirmed that when hydrogels containing polymers, ceramics, and metallic materials were manufactured using the F-gel and the biocomposite manufacturing method, all of them maintained shape memory characteristics, and that all structures had excellent shape restoring ability when the hydrogel underwent the same post-processing process regardless of the manufacturing process (3D printing). Therefore, it is expected that the manufacturing of a three-dimensional structure with shape restoring ability is possible, and that the deformed structure can be easily injected into the human body or other underwater spaces for non-invasive use.

Figure 1 shows a method for manufacturing shape-restoring hydrogels using collagen-based materials, specifically, a Cryo-gel using a low-temperature process, an F-gel utilizing the fibrosis properties of collagen, and a Composite gel (C2CM2 and C1CM3) manufactured by combining two processes. In addition, shape-restoration test images of the three hydrogels are shown.
Figure 2 shows the results of evaluating the shape recovery characteristics according to the four manufactured hydrogels (Cryo-gel; F-gel; Composite gel (C2CM2 and C1CM3) according to the material ratio). Specifically, (A) the image of the shape-recovered structure through the structural shape and moisture resupply after applying 80% strain; (B) the ratio of moisture escaping from the structure after deformation and (C) the ratio of the deformed structure; (D) the ratio of moisture reabsorbed into the structure after shape recovery; and (E) the shape recovery ratio; (F) the time required for shape recovery; (G) the stress-strain curve according to the compression-recovery test of 20, 40, 60, and 80%; and (H) the stress-strain curve when 80% strain is applied and repeated 10 times.
Figure 3 shows (A) cell proliferation data evaluated using human adipose derived stem cells (hASCs); (B) image of cytoskeletal protein (F-actin) using immunofluorescence staining; (C) F-actin area; (D) aspect ratio of F-actin; (E) relative expression levels of cell surface adhesion protein-related genes (paxillin, talin, vinculin); and (F) data for evaluating the characteristics of the construct according to cell restitution ability, cell proliferation, and adhesion ability.
Figure 4 shows (A) a shape recovery test image of an F-gel-based composite structure containing a polymer material (heparin; HEP), a ceramic material (hydroxyapatite; HA), or a metallic material (iron oxide; Fe ₂ O ₃ ); (B) time data required for shape recovery; and (C) the recovery rate when a compression test was repeated five times.
Figure 5 shows (A) an optical image and (B) an SEM image of a 3D printed F-gel structure after the structure was fabricated using the F-gel fabrication technology and 3D printing technology; (C) an image of the shape restoration when the 3D printed F-gel structure was deformed, put into a syringe, and then released again; (D) an image of the shape deformation and restoration of the 3D printed F-gel structure; and (E) an optical image, SEM image, and shape deformation and restoration images of an F-gel containing a ceramic material (hydroxyapatite, HA) and a polymer material (heparin, Hep) fabricated using the 3D printing technology.
Figure 6 shows the characteristics of a biocomposite containing a polymer material (heparin; Hep) and a ceramic (hydroxyapatite; HA), specifically, (A) an optical image and SEM image of an alcyane-blue-dyed biocomposite-Hep hydrogel; (B) an optical image and SEM image of an alizarin-red-S-dyed biocomposite-HA hydrogel; and (C) shape restoring properties of the two structures.
Figure 7 shows (A) an optical image and an SEM image of a 3D printed biocomposite structure after fabrication of a structure using a biocomposite fabrication technology and a 3D printing technology; (B) an image of shape deformation and restoration of a 3D printed biocomposite structure; and (C) an image of shape restoration when a 3D printed structure is deformed, put into a syringe, and then released again.
Figure 8 shows a structure produced using cryo-gel production technology and 3D printing technology, and the deformation and shape restoration data of the structure.

The present inventors confirmed that the hydrogel manufactured by the F-gel manufacturing method has high cell activity, shape fixation ability, and shape restoring ability, and that the biocomposite manufactured by the fusion of the cryo-gel manufacturing method and the F-gel manufacturing method has cell activity and shape fixation ability superior to the cryo-gel, shape restoration speed superior to the F-gel, and high shape restoring ability. In addition, when manufacturing hydrogels containing polymers, ceramics, and metallic materials using the F-gel and the biocomposite manufacturing method, they all maintain shape memory characteristics, and the hydrogel has excellent shape restoring ability in all structures when going through the same post-processing process regardless of the manufacturing process (3D printing), thereby completing the present invention.

In the present invention, it was confirmed that F-gel has excellent shape restoration ability, performs hydration-dependent shape restoration, and exhibits efficient cell attachment and proliferation.

In another embodiment of the present invention, it was confirmed that the biocomposite (C2CM2 and C1CM3) had a faster shape recovery speed than F-gel and had cell attachment and proliferation capabilities as efficient as F-gel (see Example 1).

In another embodiment of the present invention, it was confirmed that F-gel and biocomposite (C2CM2 and C1CM3) manufactured by including polymeric materials, ceramic materials, or metallic materials not only had shape restoration characteristics similar to those of F-gel and biocomposite not including the above materials, but also had excellent shape restoration capabilities of 3D printed F-gel and biocomposite (see Examples 2 and 3).

The present invention is described in detail below.

The present invention comprises the steps of: (a) preparing a starting material comprising collagen;

(b) a step of adding PBS to the starting material and fiberizing it in an environment of 30 to 40°C; and

(c) Provided is a method for producing a shape memory hydrogel, including a step of cross-linking the fiberized starting material using a cross-linking solution.

In the present invention, “shape memorable hydrogel” means a hydrogel that remembers its initial shape and returns to its original shape from a deformed shape due to the inflow of fluid. The stimulus may be moisture, but is not limited thereto.

In the present invention, the starting material may further include at least one selected from the group consisting of a polymer material, a ceramic material, and a metallic material, but is not limited thereto. The polymer material may be a polymer that can chemically or physically bind to collagen, and in one embodiment of the present invention, may be heparin (6-[6-[6-[5-acetamido-4,6-dihydroxy-2-(sulfooxymethyl)oxan-3-yl]oxy-2-carboxy-4-hydroxy-5-sulfooxyoxan-3-yl]oxy-2-(hydroxymethyl)-5-(sulfoamino)-4-sulfooxyoxan-3-yl]oxy-3,4-dihydroxy-5-sulfooxyoxane-2-carboxylic acid), but is not limited thereto. In addition, the ceramic material may be an inorganic material that can be mixed and utilized with a collagen solution, and in one embodiment of the present invention may be hydroxyapatite (pentacalcium, hydroxide, triphosphate), but is not limited thereto. In addition, the metallic material may be a metallic material that can be mixed and utilized with a collagen solution, and in one embodiment of the present invention may be iron oxide (Fe ₂ O ₃ ), but is not limited thereto.

In the present invention, the cross-linking solution may be at least one selected from the group consisting of EDC/NHS [(N-ethyl-N'-(3-(dimethylamino)propyl) carbodiimide/N-hydroxysuccinimide)], glutaraldehyde, tannic acid, genipin, formaldehyde, and LOX (lysyl oxidase), but is not limited thereto.

The present invention comprises the steps of: (a) preparing a starting material comprising collagen;

(b) a step of adding PBS to the starting material and fiberizing it in an environment of 30 to 40°C; and

(c) a shape memory hydrogel manufactured by a method including a step of crosslinking the fiberized starting material using a crosslinking solution,

The above hydrogel provides a shape memory hydrogel characterized by satisfying at least one of the following characteristics:

(i) increasing cell adhesion and proliferation effects; and

(ii) Increase in shape fixation ability.

In the present invention, the step of (a) preparing a starting material including collagen;

(b) a step of adding PBS to the starting material to cause fiberization; and

(c) A shape memory hydrogel manufactured by a method including a step of crosslinking the fiberized starting material using a crosslinking solution may be referred to as an F-gel, but is not limited thereto.

In addition, in the present invention, a hydrogel manufactured by a manufacturing method including a step of cooling at a temperature of minus 10 degrees Celsius or lower and crosslinking using a crosslinking solution may be referred to as a cryogel, but is not limited thereto.

In the present invention, the hydrogel may increase the expression of one or more genes selected from the group consisting of talin (TLN), paxillin (PXN), vinculin (VCL), focal adhesion kinase (FAK), and RhoA kinase, but is not limited thereto.

In the present invention, the hydrogel may have a shape restoring ability that is restored by moisture, but is not limited thereto. In addition, the hydrogel may have increased cell activity and shape fixity ratio, but is not limited thereto. The shape fixity ratio is the ability to maintain a deformed shape.

The present invention comprises the steps of: (a) preparing a starting material comprising collagen and a photocurable polymer;

(b) a step of freezing the starting material;

(c) a step of photo-curing the frozen starting material to perform primary cross-linking;

(d) adding to the first cross-linked starting material and causing fiberization in an environment of 30 to 40°C; and

(e) Provided is a method for producing a shape memory hydrogel, including a step of secondarily cross-linking the fiberized starting material using a cross-linking solution.

In the present invention, the photocurable polymer may be, but is not limited to, methacrylate collagen, methacrylate gelatin, or methacrylate alginate.

In the present invention, the starting material may contain collagen and a photocurable polymer in a mass ratio (w/w %) of 0.1:3.9 to 2:2, 0.2:3.8 to 2:2, 0.3:3.7 to 2:2, 0.4:3.6 to 2:2, 0.5:3.5 to 2:2, 0.6:3.4 to 2:2, 0.7:3.3 to 2:2, 0.8:3.2 to 2:2, 0.9:3.1 to 2:2, 1:3 to 2:2, or 1.5:2.5 to 2:2, but is not limited thereto.

In the present invention, the photocurable polymer can be photocured by light having a wavelength of 200 to 400 nm, 250 to 400 nm, 300 to 400 nm, 310 to 400 nm, 320 to 400 nm, 330 to 400 nm, 340 to 400 nm, or 350 to 400 nm, but is not limited thereto.

In the present invention, the freezing of step (b) can be performed at a temperature below zero, such as -50 to 0°C, -40 to 0°C, -30 to 0°C, -50 to -5°C, -40 to -5°C, -30 to -5°C, -50 to -10°C, -40 to -10°C, or -30 to -10°C, but is not limited thereto. In step (b), all of the moisture can become ice crystals, but is not limited thereto.

In the present invention, the secondary crosslinking of step (e) may be performed at a temperature of 25 to 40°C, or 30 to 40°C, but is not limited thereto. In addition, the starting material of step (a) may further include at least one selected from the group consisting of a polymer material, a ceramic material, and a metallic material, but is not limited thereto. In addition, the crosslinking solution may include at least one selected from the group consisting of EDC/NHS [(N-ethyl-N'-(3-(dimethylamino)propyl) carbodiimide/N-hydroxysuccinimide)], glutaraldehyde, tannic acid, genipin, formaldehyde, and LOX (lysyl oxidase), but is not limited thereto.

In the present invention, a structure using the above manufacturing method may have shape restoring ability, but is not limited thereto.

In addition, the present invention provides a shape memory hydrogel manufactured by a method comprising the following steps:

(a) a step of preparing a starting material comprising collagen and a photocurable polymer;

(b) a step of freezing the starting material;

(c) a step of photo-curing the frozen starting material to perform primary cross-linking;

(d) a step of adding PBS to the first cross-linked starting material and fiberizing it in an environment of 30 to 40°C; and

(e) A step of secondarily cross-linking the above-mentioned fiberized starting material using a cross-linking solution.

In the present invention, the hydrogel manufactured by the above manufacturing method can be referred to as C1CM3 or C2CM2 using the mass ratio of collagen and photocurable polymer, but is not limited thereto.

In the present invention, the hydrogel may occupy an area of 50 to 100%, 50 to 80%, or 50 to 70% in a pentagonal diagram using five parameters of (1) shape recovery ratio (R _r ), (2) shape fixation ratio (R _f ), (3) shape recovery time, (4) cell proliferation, and (5) cell adhesion, but is not limited thereto.

In the present invention, the hydrogel may satisfy at least one of the following characteristics, but is not limited thereto: (i) increasing cell adhesion and proliferation effects; and (ii) increasing shape restoration speed. In addition, the hydrogel may increase the expression of one or more genes selected from the group consisting of talin (TLN; talin), paxillin (PXN; paxillin), and vinculin (VCL; vinculin), but is not limited thereto. The hydrogel may have shape restoration ability that is restored by moisture. In addition, the hydrogel may have increased cell activity ability or shape fixation ability, and may have an increased shape restoration speed compared to F-gel, but is not limited thereto.

In addition, the present invention may further include a step of 3D printing between steps (a) and (b), but is not limited thereto.

In the present invention, the 3D printing step may include, but is not limited to, a step of injecting the starting material into a printing solution barrel and printing. The temperature of the printing step may be, but is not limited to, 0°C or less, -50 to -10°C, -40 to -15°C, -35 to -25°C, or -20°C. In addition, the temperature of the barrel may be, but is not limited to, 0 to 10°C, 1 to 8°C, 2 to 6°C, or 4°C.

Hereinafter, preferred examples are presented to help understand the present invention. However, the following examples are provided only to help understand the present invention more easily, and the content of the present invention is not limited by the following examples.

[Experimental method]

1. Preparation and analysis of collagen solution

Type 1 collagen derived from porcine skin (MS Bio, Korea) was used. Collagen solutions (4 wt%, 8 wt%) were prepared by dissolving collagen powder in 0.1 M acetic acid (pH 4.0), and a 4 wt% neutralized collagen solution (pH 7.8) was prepared by mixing an 8 wt% collagen solution with 0.25 M NaHCO ₃ in a 1:1 ratio.

The rheological properties [storage modulus (G') and loss modulus (G'')] of acidic and neutralized collagen (4 wt%) were measured by a rotating cone and plate rheometer (Bohlin Gemini HR Nano, Malvern Instruments, U.K.; diameter 40 mm; cone angle 4°; gap 150 μm). Additionally, temperature sweeps (approximately 5 to 55 °C at 2 °C/min) were performed at a frequency of 1 Hz with a strain of 1%.

Circular dichroism spectroscopy (J-815, JASCO, Japan) was used to analyze the collagen α-helical structure. In addition, acidic collagen (pH 4.0) and neutralized collagen (pH 7.8) were prepared at a final concentration of 0.3 mg/mL. 350 μL sample was loaded into a quartz cuvette (1 mm path length; 110-QS, Hellma, USA), and the far-UV spectrum (wavelength range: ~200–260 nm) was measured. All ellipticity measurements were corrected by buffer spectra and normalized to obtain the mean molar residue ellipticity (MRE).

2. Preparation and characterization of collagen-based hydrogels

A 3D-printed cylindrical mold (material: ABS, diameter: 5 mm, height: 2.5 mm) was used for the fabrication of hydrogels. Acidic collagen (4 wt%, pH 4.0) and neutralized collagen (4 wt%, pH 7.8) were placed into the molds and cross-linked with 200 mM EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride)/50 mM NHS (N-hydroxy-succinimide) to obtain C-gel and uF-gel, respectively. For cryo-gel preparation, acidic collagen was frozen at -20 °C. Then, the frozen collagen was cross-linked by immersing it in 200 mM EDC/50 mM NHS dissolved in 75% EtOH at -20 °C for 24 h. After that, the cross-linked gel was placed in a refrigerator (4 °C) to melt ice crystals. To obtain fibrous F-gels, neutralized collagen was poured into a mold, immersed in phosphate-buffered saline (PBS), and incubated at 37°C for 24 h. EDC/NHS cross-linking was performed at 37°C for 4 h (100 mM EDC/25 mM NHS dissolved in 75% EtOH). All hydrogels were washed with triple-distilled water (3DW). The surface and internal structures of the samples were analyzed using a scanning electron microscope (SNE-3000M, SEC Inc., South Korea).

Quantification of free amine residues in hydrogels was performed using 2,2-dihydroxy-1,3-indanedione (ninhydrin assay) to analyze the degree of cross-linking (n = 6). Briefly, 200 mM citric acid with 0.16 w/v% tin II chloride was prepared in 3DW, and the pH of the solution was adjusted to 5.0 by adding 10 M NaOH. In addition, ninhydrin powder was dissolved in 2-ethoxyethanol (4 w/v%). These two solutions were mixed 1:1 to prepare the reaction solution. Blank (w/o collagen), 3 mg of collagen sponge, and 3 mg each of four types of frozen hydrogels (C-gel, Cryo-gel, uF-gel, and F-gel) were added to 1.5 mL microtubes, and 1 mL of the reaction solution was added. The samples were heated in a heating chamber at 100 °C for 15 min. After the reaction, the samples were cooled at 4°C for 5 minutes, and 250 μL of 50% (v/v) 2-propanol was added to each microtube. 200 μL of each sample was placed in a 9-well plate, and the optical density (OD) was measured at 570 nm using a microplate reader (EL800, BioTek, United States). The degree of crosslinking (%) was calculated using the following Equation 1.

[Formula 1]

Degree of crosslinking (%) = [1 - (OD _sample - OD _blank )/(OD _{collagen sponge} - OD _blank )] × 100

The porosity (%) of the fabricated hydrogel (n = 5) was calculated using the following equation 2. W _sample is the weight of the frozen sample, ρ _collagen is the density, and V _sample is the volume of the structure.

[Formula 2]

Porosity (%) = [1 - (W _sample /ρ _collagen ) / V _sample ] × 100

To evaluate the water leakage of hydrogels (n = 5), various compressive strains (ε = 20, 40, 60, and 80%) were applied to the hydrogels at a rate of 6 mm/min using a universal testing machine (Top-tech; Chemilab, South Korea). At each strain, the water squeezed out of the hydrogel was removed with a tissue. The leakage rate (%) was calculated by Equation 3 below. W _{hydrogel, initial} and W _{hydrogel, ε} represent the weights of the hydrogel before and after removing the discharged water, respectively.

[Formula 3]

Leakage rate (%) = [(W _{hydrogel, initial} - W _{hydrogel, ε} ) / W _{hydrogel, initial} ] × 100

The pore size distribution of the hydrogels was analyzed using a porosimeter (Autopore V 9620; Micromeritics Inc., United States). The normalized pore volume density was calculated using the pore diameter and cumulative volume data using Equation 4.

ΔV _i , Δr _i and V _max represent the increased pore volume, pore radius spacing and maximum accumulated pore volume, respectively.

[Formula 4]

Pore volume density = ΔV _i / (Δr _i × V _max )

To measure water absorption, cuboid (width: 5 mm, depth: 1 mm, height: 6 mm) Cryo-gel and F-gel were prepared in a mold according to the protocol described above. The samples were fixed with clips, and the lower surface (1 mm) was immersed in a rhodamine dye solution (Sigma-Aldrich). Optical images were taken at each time point, and the absorption distance was measured using FiJi software (National Institutes of Health (NIH), USA).

Shape fixity ratio (R _f ) and shape recovery ratio (R _r ) were calculated using Equations 5 and 6 below. ε _D and ε _R represent the strain after deformation [(h _i - h _D )/h _i ] and the strain after rehydration [(h _i - h _R )/h _i ], respectively, and ε _m is the strain.

[Formula 5]

R _f = ε _D /ε _m

[Formula 6]

R _r = (ε _m - ε _R )/ε _m

3. Mechanical properties of hydrogels

Cylindrical hydrogels (diameter: 5 mm, height: 2.5 mm, n = 4) were subjected to compression tests at a rate of 6 mm/min using a universal testing machine (UTM; Top-tech). The compressive modulus was calculated from the linear region of the stress-strain curve. Cyclic compression tests were performed using the UTM at a constant rate (6 mm/min), and the height recovery during these decompression tests was measured from video recordings and analyzed with FiJi software (NIH, USA).

4. Methacrylation of collagen

Photo-crosslinkable collagen was obtained by methacrylation of collagen. Briefly, a collagen solution of 3.75 mg/mL was prepared using stirred 0.5 M acetic acid. After the collagen was completely dissolved, the pH of the solution was adjusted to 8.0 by adding 10 M NaOH dropwise at 4 °C. Collagen methacrylate (Col-ma, methacrylated collagen) was obtained by adding methacrylic anhydrides (MAA; Sigma-Aldrich) to the collagen solution (7.5 μL of MAA per mL of collagen solution) for 3 days. After the reaction, Col-ma was precipitated with acetone and washed five times with 70% ethanol. The washed Col-ma was dialyzed against 3DW using dialysis tubing (3.5 kDa, Spectrum Chemical Mfg. Corp., United States) for 7 days. Afterwards, it was frozen (FD8505, IlshinBioBase, South Korea) and stored at 4℃ until use. The methacrylation rate of Col-ma was 64.8 ± 0.8%, which was measured by ninhydrin assay according to the protocol described above.

5. Manufacturing of Col-ma/collagen composite hydrogel

Collagen and Col-ma solutions were prepared with 0.1 M acetic acid (pH 4.0, 8 wt%). Each solution was neutralized by mixing 1:1 with 0.25 M NaHCO ₃ , and the final concentration was 4 wt% (pH 7.8). The photoinitiator [lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP; Sigma-Aldrich)] was dissolved in the neutralized Col-ma solution (3 mg of LAP per mL of Col-ma solution). Col-ma and collagen (Col) solutions were mixed at various ratios to prepare Col/Col-ma complex solutions.

For the fabrication of Col/Col-ma composite hydrogels, the precursor solution was placed into a cylindrical mold (diameter: 5 mm, height: 2.5 mm). Then, it was frozen at -20 °C for 4 h, and UV crosslinking (365 nm, 200 mW/cm ² for 120 s) was performed at -20 °C. After that, the hydrogel was immersed in PBS and incubated at 37 °C for 24 h. After incubation, the PBS was removed, and 100 mM EDC/25 mM NHS dissolved in 75% EtOH was added for secondary crosslinking (37 °C, 4 h). All the fabricated hydrogels were washed with triple-distilled water (3DW), and the surface morphology, mechanical properties, and shape recovery properties were analyzed according to the protocol described above.

6. In vitro testing

Human adipose-derived stem cells (hASC, PT-5006, Lonza, Basel, Switzerland) were cultured in DMEM low glucose (HyClone; USA) supplemented with 10% FBS (fetal bovine serum, Biowest, France) and 1% penicillin/streptomycin (PS, Thermific Fisher) at 37° _C and 5% CO2. Cells were seeded on various hydrogels (1.0 × ¹⁰³ cells/scaffolds), and the medium was changed every 2 days.

Cell proliferation (n = 5) was analyzed using the CCK-8 assay (Dojindo, Japan) according to the manufacturer's protocol. Briefly, the CCK-8 assay solution and cell culture medium were mixed at a ratio of 1:9, and 200 μL of the solution was injected into each sample at each cell culture time point. After incubation for 2 h at 37°C, the reaction mixture was pipetted into a 96-well plate, and the absorbance was measured at 450 nm using a microplate reader (EL800).

To visualize the morphology of hASC cultured in hydrogels, cells were fixed with 3.7% formaldehyde (Sigma-Aldrich). To permeabilize and block nonspecific antibody binding, hydrogels were treated with 0.1% Triton X-100 (Sigma-Aldrich) containing 2% bovine serum albumin (BSA, Sigma-Aldrich) for 2 h. Then, the samples were incubated overnight at 4°C with anti-vinculin primary antibody (5 μg/ml, Abcam, USA). After rinsing the samples twice with PBS, diamidino-2-phenylinodole (DAPI, 1:100 diluted in PBS, Invitrogen, USA), Alexa Fluor 594 phalloidin (1:100 diluted in PBS, Invitrogen), and Alexa Fluor 488-conjugated secondary antibodies (1:50 in PBS, Invitrogen) were used to visualize cell nuclei, F-actin, and vinculin, respectively, for 1 h. The stained cells were observed using a confocal microscope (LSM700, Carl Zeiss, Germany). In addition, the F-actin area and aspect ratio of the cells were measured using FiJi software.

Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) was performed to measure the relative gene expression levels of talin (TLN), paxillin (PXN), vinculin (VCL), focal adhesion kinase (FAK), and RhoA (ras homolog family member A). Total RNA from cultured hASCs was extracted by treatment with TRI reagent (Sigma-Aldrich), and the concentration of the isolated RNA was measured using a spectrophotometer (FLX800T; Biotek, USA). cDNA (complementary DNA) was synthesized using ReverTra Ace qPCR RT Master Mix (Toyobo Co., Ltd., Japan) according to the manufacturer's protocol. To perform quantitative RT-PCR, StepOnePlus Real-Time PCR System (Applied Biosystems, USA) and Thunderbird ^® SYBR ^® qPCR Mix (Toyobo, Japan) were used, and gene expression levels were normalized to the expression level of the housekeeping gene (beta-actin (ACTB)). Gene-specific primer sequences are shown in Table 1 below.

order Sequence number human ACTB Forward ctcgcctttgccgatcc 1 Reverse tctccatgtcgtcccagttc 2 human TLN Forward gccatcaagaaaaagctggt 3 Reverse ctgcagcgatggtctgtg 4 human PXN Forward tcgctgtcggatttcaagat 5 Reverse atgaaccctccctcgtcct 6 human VCL Forward cgattacgaacctgagctgc 7 Reverse tggtagcttcccgatgcaag 8 human FAK Forward gctcccttgcatcttccagt 9 Reverse attgcagcccttgtccgtta 10 human RhoA Forward gtccacggtctggtcttcag 11 Reverse cagccattgctcaggcaac 12

7. Manufacturing of composite hydrogels containing polymer, ceramic, or metallic materials

A Col 2%/Col-ma 2% solution containing a photoinitiator (LAP) was used to prepare C2CM2-Hep and C2CM2-HA composite hydrogels. For the preparation of C2CM2-Hep hydrogel, 0.04 mg of heparin was mixed with 1 mL of solution. In addition, for the preparation of C2CM2-HA hydrogel, 0.08 g of hydroxyapatite was mixed with 1 mL of solution. The solution was placed into a cylindrical mold and frozen at -20 °C for 4 h, followed by UV crosslinking (365 nm, 200 mW/cm ² for 120 s) at -20 °C. The hydrogels were immersed in PBS and then incubated at 37 °C for 24 h. Afterwards, PBS was removed, and 100 mM EDC/25 mM NHS dissolved in 75% EtOH was added for further cross-linking (37 °C, 4 h). All fabricated hydrogels were washed with 3DW, and the surface morphology and shape recovery characteristics were analyzed according to the protocol described above.

8. Fabrication of shape-restoring hydrogels using 3D printing

A three-axis printing system (DTR3-2210 T-SG; DASA Robot, Korea) equipped with a dispensing system (AD-3000C; Ugin-tech, Korea) coupled with a temperature control system was used. For the fabrication of 3D printed C2CM2 scaffolds (C2CM2 _print ), Col 2 wt%/Col-ma 2 wt% (pH 7.8, mixed with 3 mg/mL LAP) was injected into the printing solution barrel. The printing stage temperature was set at -20 °C, and the barrel temperature was 4 °C. The mesh structures were printed under fixed printing parameters (nozzle diameter: 250 μm, air pressure: 100 kPa, printing speed: 10 mm/s). The printed C2CM2 structures were cross-linked by UV irradiation (200 mW/cm ² for 120 s) at -20 °C, immersed in PBS, and incubated at 37 °C for 24 h. Afterwards, the structures were cross-linked with 100 mM EDC/25 mM NHS dissolved in 75% EtOH at 37 °C for 4 h, and all samples were washed with 3DW.

To fabricate F-gel, a neutralized Col 4 wt% solution was injected into the barrel, and a three-dimensional structure was fabricated using a dispenser and a 3D printer. The temperature of the barrel was set to 5 to 10°C, and the temperature of the printing stage was in the range of 30 to 40°C. After printing, the structure was cross-linked with EDC/NHS, and the structure was washed with 3DW.

To produce a cryo-gel, a 4 wt% collagen or 4 wt% photocurable collagen (Col-ma) solution dissolved in acid was injected into the barrel, and a three-dimensional structure was produced using a dispenser and a 3D printer. The produced structure was cross-linked in an EDC/NHS solution at -80°C, or, in the case of the Col-ma structure, photocured by irradiating it with UV.

9. Statistical Analysis

Data were expressed as mean ± standard deviation, and statistical analysis was performed using SPSS software (SPSS, Inc., USA). The t-test was used to compare two groups. For comparison of three or more groups, a single-factor analysis of variance (ANOVA) and Tukey's HSD post-hoc test were used, and p < 0.05 was considered statistically significant.

[Example]

Example 1. Biocomposite with shape restoration properties of cryo-gel and cell activity properties of F-gel

Geometrical changes due to deformation and rehydration of cryo-gel _col-ma , F-gel, and biocomposites (C1CM3 and C2CM2) were investigated. The results are shown in Fig. 2 (A) to (E).

As shown in Fig. 2(A) to (C), the moisture contents of C2CM2 and F-gel after compression were slightly lower than those of Cryo-gel _col-ma and C1CM3 (Cryo-gel _col-ma = 77.7 ± 2.4%; C1CM3 = 78.4 ± 2.6%; C2CM2 = 75.1 ± 4.4%, F-gel = 72.4 ± 2.0%). However, the shape fixation ratio increased as the Col weight ratio increased (R _{f, Cryo-gel} ^col-ma = 3.9 ± 3.4%; R _{f, C1CM3} = 38.5 ± 9.7%; R _{f, C2CM2} = 68.8 ± 7.7%, and R _{f, F-gel} = 94.8 ± 5.1%). Additionally, after rehydration, all hydrogels absorbed water to similar levels and restored their shapes (R _{r, Cryo-gelcol-ma} = 98.6 ± 6.6%, R _{r, C1CM3} = 97.4 ± 5.7%, R _{r, C2CM2} = 99.2 ± 4.0%, R _{r, F-gel} = 99.6 ± 1.5%).

The inventors of the present invention confirmed that F-gel has a slower recovery speed than Cryo-gel. Accordingly, the recovery speed of the biocomposite was analyzed. The results are shown in Fig. 2 (F).

As shown in (F) of Fig. 2, the biocomposites (C2CM2 and C1CM3) recovered their shape much faster than F-gel, and as fast as Cryo-gel (or Cryo-gel _col-ma ). This is a result of efficient water absorption through the relatively larger pores in the Col-ma region of the biocomposites, as shown in (H) of Fig. 2.

Additionally, the shape recovery properties of biocomposites (C1CM3 and C2CM2), cryo-gel _col-ma , and F-gel were evaluated for various compressive strains (ε = 20, 40, 60, and 80%) and numbers of compression-recovery cycles (1, 5, and 10). The results are shown in Fig. 2 (G) and (H).

As shown in (G) and (H) of Fig. 2, the biocomposite showed excellent shape recovery without hysteresis even when various cyclic compression conditions were applied. Therefore, it can be usefully used in injectable scaffolds or tissue engineering bioreactors that apply deformation forces to cartilage, muscle, or bone.

Cell proliferation of hASCs cultured on cryo-gel _col-ma , F-gel, and two biocomposites was determined using a CCK-8 assay. The results are shown in Fig. 3 (A).

As shown in Fig. 3(A), the number of cells surviving on day 7 in the F-gel and C2CM2 composites was much higher than that in the Cryo-gel _col-ma and C1CM3. This suggests that F-gel and C2CM2 provided a microenvironment with a nanofiber structure more similar to the ECM.

Additionally, DAPI/phalloidin images, and F-actin area and aspect ratio of cells cultured in each hydrogel for 1 day are shown in Figures 3 (B) to (D).

As shown in Figures 3 (B) to (D), the morphology of cells cultured in the C2CM2 biocomposite was much more similar to that of cells cultured in the F-gel.

In addition, the expression of local adhesion genes, talin (TLN), paxillin (PXN), and vinculin (VCL), was measured after 7 days of cell culture using RT-PCR. At this time, normalization was performed using Cryo-gel _col-ma , and the results are shown in Fig. 3 (E).

As shown in Fig. 3 (E), the gene expression level of cells cultured on F-gel was the highest, and the gene expression level of cells cultured on C2CM2 biocomposite was much higher than that of cells cultured on Cryo-gel _col-ma and C1CM3.

In summary, to compare the shape restoration properties and cellular activity of biocomposites, five parameters were used: (1) shape restoration ratio (R _r ), (2) shape fixation ratio (R _f ), (3) shape recovery time, (4) cell proliferation, and (5) cell adhesion. The shape restoration time was normalized to the minimum value, and the other parameters were normalized to their respective maximum values. The results are shown in Fig. 3 (F).

As shown in Fig. 3(F), the C2CM2 biocomposite occupied the largest area of the pentagonal diagram (Cryo-gel _col-ma = 21.7%, C1CM3 = 42.4%, C2CM2 = 68.8%, F-gel = 61.8%). This means that C2CM2 has better shape recovery properties than F-gel and supports greater cell activity than Cryo-gel and C1CM3.

Therefore, the present invention combines the advantages of Cryo-gel with a fast shape recovery rate and F-gel with high cell activity. After freezing at sub-zero temperatures, it is experimentally very difficult to additionally perform cross-linking. However, the present invention proposes a method for manufacturing a hydrogel with a fast shape recovery rate and high cell activity by additionally performing cross-linking after freezing, which is a significantly excellent invention.

Example 2. Application of F-gel-based composite structures

To utilize the F-gel biocomposite for various tissue engineering applications, heparin, hydroxyapatite (HA), and Fe ₂ O ₃ were used as additives in the biocomposite. Heparin is widely distributed in the extracellular matrix and has been widely used for the sustained release of growth factors because it can delay the release of negative charges through electrostatic interactions. In addition, HA is one of the main components of bone and is widely used for osteointegration and osteoconduction in bone tissue regeneration. Iron oxide (Fe ₂ O ₃ ) was used as one of the metallic materials.

Therefore, three different types of F-gel biocomposites were prepared: (1) heparin-conjugated F-gel biocomposites (F-GEL-HEP, 1 μg heparin/Col 1 mg), (2) HA-conjugated F-gel biocomposites (F-GEL-HA, HA 2 mg/Col 1 mg), and (3) iron oxide-conjugated F-gel biocomposites (F-GEL-Fe ₂ O _3, Fe ₂ O ₃ 0.1 mg/Col 1 mg). Then, shape recovery characteristics were analyzed. The results are shown in Fig. 4 (A) to (C).

As shown in (A) to (C) of Figure 4, compression and deformation were performed using a 500 g weight, and it was confirmed that the initial shape was restored after the compression was removed.

To fabricate a three-dimensional porous structure using the F-gel fabrication technology, a printed F-gel (PF-gel) scaffold was fabricated through 3D printing. A 16 × 16 × 2 mm ³ structure was fabricated using neutralized collagen (Fig. 5 (A)), and the SEM analysis result of the fabricated structure confirmed that the surface was composed of a fibrous structure (Fig. 5 (B)). In order to confirm whether this could be used as an injectable structure, an injection test was conducted through a nozzle, and it was confirmed that the deformed structure was restored to the initial design when water was injected (Fig. 5 (C)). In addition, in order to confirm the deformation recovery ability again, a 500 g weight was used to perform compression and deformation, and it was confirmed that the initial shape was restored when water was injected again (Fig. 5 (D)).

In addition, two different types of (1) heparin-conjugated F-gel biocomposites (PF-GEL-HEP, 1 μg heparin/Col 1 mg) and (2) HA-conjugated F-gel biocomposites (PF-GEL-HA, HA 2 mg/Col 1 mg) were prepared. Then, staining, SEM images, and shape recovery property analyses were performed. The results are shown in Fig. 5 (E).

As shown in Fig. 5(E), the alkane-blue-stained PF-GEL-HEP and the alizarin-red-S-stained PF-GEL-HA showed that heparin and HA were well distributed throughout the biocomposites. In addition, the SEM images showed the typical pore structure of the C2CM2 complex. In addition, shape recovery characteristics similar to those of the PF-GEL complex were observed in both biocomposites (PF-GEL-HEP, PF-GEL-HA).

Example 3. Application of C2CM2 biocomposite

To utilize the C2CM2 biocomposite for various tissue engineering applications, heparin and hydroxyapatite (HA) were used as dispersing agents in the biocomposite. Heparin is widely distributed in the extracellular matrix and has been widely used for the sustained release of growth factors because it can delay the release of negative charges through electrostatic interactions. In addition, HA is one of the main components of bone and is widely used for osteointegration and osteoconduction in bone tissue regeneration.

Therefore, two different types of (1) heparin-conjugated C2CM2 biocomposites (C2CM2-Hep, 1 μg heparin/Col 0.5 mg and Col-ma 0.5 mg) and (2) HA-conjugated C2CM2 biocomposites (C2C2-HA, HA 2 mg/Col 0.5 mg and Col-ma 0.5 mg) were prepared. Then, staining, SEM images, and shape recovery property analyses were performed. The results are shown in Fig. 6 (A) to (C).

As shown in (A) and (B) of Fig. 6, Alcian-blue-stained C2CM2-Hep and alizarin-red-S-stained C2CM2-HA showed that heparin and HA were well distributed throughout the biocomposites. In addition, SEM images showed the typical pore structure of the C2CM2 complex. In addition, as shown in (C) of Fig. 6, shape recovery characteristics similar to those of the C2CM2 complex were observed in both biocomposites (C2CM2-Hep, C2CM2-HA).

3D printing is another important application of C2CM2 biocomposites. Using a 3D printing system with a low-temperature printing step (-20°C) and cross-linking/collagen-fibrillation processes (UV cross-linking, fiber generation, and EDC/NHS cross-linking), a mesh structure (C2CM2 _print ) was fabricated and printed. The shape recovery characteristics of the mesh structure are shown in Fig. 7 (A) to (C).

As shown in Fig. 7 (A), the C2CM2 print meshwork (12 × 12 × 2 mm ³ ) displayed in the optical image and the SEM image are shown.

Additionally, an optical image showing the shape deformation and restoration of the C2CM2 _print is shown in Fig. 7b.

As shown in Fig. 7(B), the C2CM2 _print was deformed without cracks by dehydration and was well restored after reabsorbing water.

In addition, to verify the feasibility of C2CM2 _print as an injection scaffold, a printed rectangular mesh scaffold (10 × 10 × 1 mm ³ ) was deformed to produce a thin cylindrical structure, which was then placed into a 14 G nozzle (inner diameter: 1.54 mm). A shape restoration experiment was then performed. The results are shown in Fig. 7 (C).

As shown in Fig. 7(C), the scaffold was discharged from the nozzle using a water-filled syringe, and the printed biocomposite immediately restored its original shape. This result implies that the printed C2CM2 structure can be used as an injectable scaffold.

Example 4. Application of Cryo-gel

To utilize cryo-gel for various tissue engineering applications, a mesh structure (Pcryo-gel) was fabricated and printed using a 3D printing system. An optical image of the shape restoration of this mesh structure is shown in Fig. 8.

As shown in Fig. 8, Pcryo-gel was deformed without cracks by dehydration and was well restored after reabsorbing water.

In summary, the hydrogel produced by the F-gel production method of the present invention not only has high cell activity, shape fixation ability, and shape restoring ability, but also the biocomposite produced by the production method that fuses the cryo-gel production method and the F-gel production method of the present invention has cell activity and shape fixation ability superior to those of the cryo-gel, a shape restoration speed superior to that of the F-gel, and high shape restoring ability. As shown in Fig. 1, the F-gel utilizing the fibrosis properties of collagen; and the biocomposite produced by the production method that fuses the cryo-gel and the F-gel are restored to their initial shape even after 90% compressive strain, indicating that the present invention has remarkably excellent shape restoring ability.

The above description of the present invention is for illustrative purposes only, and those skilled in the art to which the present invention pertains will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

<110> Research and Business Foundation SUNGKYUNKWAN UNIVERSITY <120> Method for Manufacturing collagen based shape memorable hydrogel and uses its <130> MP21-252 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> human ACTB_F <400> 1 ctcgcctttg ccgatcc 17 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> human ACTB_R <400> 2 tctccatgtc gtcccagttc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> human TLN_F <400> 3 gccatcaaga aaaagctggt 20 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> human TLN_R <400> 4 ctgcagcgat ggtctgtg 18 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> human PXN_F <400> 5 tcgctgtcgg atttcaagat 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> human PXN_R <400> 6 atgaaccctc cctcgtcct 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> human VCL_F <400> 7 cgattacgaa cctgagctgc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> human VCL_R <400> 8 tggtagcttc ccgatgcaag 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> human FAK_F <400> 9 gctcccttgc atcttccagt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> human FAK_R <400> 10 attgcagccc ttgtccgtta 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> human RhoA_F <400> 11 gtccacggtc tggtcttcag 20 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> human RhoA_R <400> 12 cagccattgc tcaggcaac 19

Claims (14)

delete delete delete delete delete (a) a step of preparing a starting material comprising collagen and a photocurable polymer;
(b) a step of freezing the starting material;
(c) a step of photo-curing the frozen starting material to perform primary cross-linking;
(d) a step of adding PBS to the first cross-linked starting material and fiberizing it in an environment of 30 to 40°C; and
(e) a step of secondarily cross-linking the fiberized starting material using a cross-linking solution;
A method for producing a shape memory hydrogel, wherein the above starting materials include collagen and a photocurable polymer in a mass ratio (w/w %) of 1:3 to 2:2.
In Article 6,
A method for manufacturing a shape memory hydrogel, characterized in that the photocurable polymer is methacrylate collagen.
delete In Article 6,
A method for producing a shape memory hydrogel, characterized in that the starting material of the above step (a) further includes at least one selected from the group consisting of a polymer material, a ceramic material, and a metallic material.
In Article 6,
A method for producing a shape memory hydrogel, characterized in that the cross-linking solution comprises at least one selected from the group consisting of EDC/NHS [(N-ethyl-N'-(3-(dimethylamino)propyl) carbodiimide/N-hydroxysuccinimide)], glutaraldehyde, tannic acid, genipin, formaldehyde, and LOX (lysyl oxidase).
(a) a step of preparing a starting material comprising collagen and a photocurable polymer;
(b) a step of freezing the starting material;
(c) a step of photo-curing the frozen starting material to perform primary cross-linking;
(d) a step of adding PBS to the first cross-linked starting material and fiberizing it in an environment of 30 to 40°C; and
(e) is manufactured by a method including a step of secondarily cross-linking the fiberized starting material using a cross-linking solution,
The above starting material is a shape memory hydrogel containing collagen and a photocurable polymer in a mass ratio (w/w %) of 1:3 to 2:2.
delete In Article 11,
A shape memory hydrogel characterized in that the hydrogel increases the expression of one or more genes selected from the group consisting of talin (TLN; talin), paxillin (PXN; paxillin), and vinculin (VCL; vinculin).
In Article 11,
A shape memory hydrogel manufactured by a manufacturing method further comprising a 3D printing step between steps (a) and (b).