Biomaterials and methods for producing biomaterials

Abstract

This invention provides a biomaterial composed of biopolymers that exhibits excellent flexibility and strength, as well as a method for producing such a biomaterial. [Solution] The gel is mainly composed of a collagen-hyaluronic acid complex in which hyaluronic acid is intertwined between collagen fibers.

Description

Technical Field

[0001] The present invention relates to a biomaterial composed of a biopolymer and a method for producing the biomaterial.

Background Art

[0002] Collagen gel known as a biomaterial is not limited to a culture substrate for culturing functional cells such as liver cells, but is also used as a medical material such as an artificial blood vessel and an artificial skin due to its high biocompatibility. Since ordinary collagen gel is very soft and difficult to handle, it is widely practiced to increase its strength using a crosslinking agent.

[0003] However, when trying to increase the strength of collagen gel by adding a crosslinking agent to collagen extracted alone, the surface becomes hard and the internal structure becomes soft, resulting in a very brittle material. Therefore, the collagen gel of Patent Document 1 constructs the structure of the collagen gel by neutralizing an acidic collagen aqueous solution in which a single collagen is dispersed in an acidic aqueous solution before treatment with a crosslinking agent, and reacting the crosslinking agent while containing moisture in the collagen gel. Since the crosslinking remains only between the collagen fibers while maintaining the distance between the collagen fibers, crosslinking can be performed while maintaining the internal structure of the collagen gel, and the vibration durability and strength are enhanced.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] The collagen gel described in Patent Document 1 has increased strength compared to conventionally crosslinked collagen gels because it is crosslinked while maintaining its internal structure. However, it has the problem of lacking flexibility and strength for use in applications such as bioadhesives and wound dressings where even greater mechanical strength is required. Hybrid materials combining collagen, a biopolymer, and chemically synthesized synthetic polymers are known as biomaterials with high mechanical strength. However, even if the synthetic polymer itself is harmless, the possibility of adverse effects due to accumulation in the body cannot be ignored. Therefore, there is a need for biomaterials composed of biopolymers that have excellent flexibility and strength.

[0006] This invention was made in view of these problems, and aims to provide a biomaterial composed of biopolymers that is excellent in flexibility and strength, as well as a method for producing such a biomaterial. [Means for solving the problem]

[0007] To solve the aforementioned problems, a biomaterial according to one aspect of the present invention is: It is characterized by being a gel whose main component is a collagen-hyaluronic acid complex, in which hyaluronic acid is intertwined between collagen fibers. According to these characteristics, the main component is a collagen-hyaluronic acid complex that maintains the composite structure of collagen and hyaluronic acid constructed in the body. Because the collagen constituting this collagen-hyaluronic acid complex is intertwined and gelled, a biomaterial composed of biopolymers with excellent flexibility and strength can be obtained.

[0008] The collagen-hyaluronic acid complex is characterized by a content of 4-6%. This characteristic allows for a balanced enhancement of both the flexibility and strength of biomaterials.

[0009] The aforementioned collagen-hyaluronic acid complex is characterized by a composition ratio of collagen:hyaluronic acid = 8-12:0.8-1.2. This characteristic allows for a balanced enhancement of both the flexibility and strength of biomaterials.

[0010] The aforementioned gel is characterized by being neutralized. This characteristic allows for increased strength of biomaterials.

[0011] A method for producing biomaterials according to another aspect of the present invention is: A shredding process that cuts a material containing a collagen-hyaluronic acid complex, A degreasing step is performed to degrease the shredded material obtained in the above shredding step, The degreased material obtained in the degreasing step is immersed in an acidic solution and then crushed in a crushing step, An extraction step is performed in which the crushed liquid obtained by the crushing step is filtered to extract the collagen-hyaluronic acid complex, A freeze-drying step is performed to freeze-dry the extract obtained in the extraction step, A dispersion step is performed to uniformly disperse the freeze-dried extract obtained in the freeze-drying step by heating and stirring it in an acidic solvent at 40-70°C. A cooling step is performed to gel the dispersion obtained by the above dispersion step by cooling the dispersion liquid, It is characterized by having the following features. According to these characteristics, a collagen-hyaluronic acid complex is extracted from the raw material while maintaining the composite structure of collagen and hyaluronic acid that is constructed in vivo, without enzymatic or high-temperature treatment. The freeze-dried extract of this collagen-hyaluronic acid complex is then heated and stirred in an acidic solvent at 40-70°C to uniformly disperse it, and the dispersion is cooled to gel, thereby obtaining a biomaterial composed of biopolymers with excellent flexibility and strength.

[0012] The method is characterized by immersing the gel obtained by the aforementioned cooling step in a buffer solution with a pH of 7.4. According to this characteristic, the neutralization of the gel can increase the strength of the biomaterial.

[0013] The freeze-dried residue obtained by freeze-drying the residue obtained by the extraction step is enzymatically treated with a proteolytic enzyme and heated and stirred to be uniformly dispersed. According to this feature, a biomaterial excellent in adhesiveness can be obtained from the residue obtained after extracting the collagen-hyaluronic acid complex from the material.

[0014] The material is characterized by being a chicken crest. According to this feature, the collagen-hyaluronic acid complex can be extracted with a high yield and at a low cost, and a biomaterial having a good balance between flexibility and strength can be obtained.

Brief Description of the Drawings

[0015] [Figure 1] It is an image diagram showing the structure of the collagen-hyaluronic acid complex in the biomaterial of the present invention. [Figure 2] It is a flowchart showing a method for producing a biomaterial in an embodiment of the present invention. [Figure 3] It is a graph showing the weights of the freeze-dried extracts, residues, etc. obtained in each step of the method for producing a biomaterial in the embodiment. [Figure 4] (a) is a graph showing the collagen content estimated by colorimetric quantitative analysis for extracts 1 and 2 obtained in the extraction step of the method for producing a biomaterial in the embodiment, (b) is a graph showing the hyaluronic acid content in the same way, and (c) is a graph showing the weight loss when the temperature is raised from 0°C to 500°C in the same way. [Figure 5] (a) is a photographic view showing the state before a high-strength biomaterial is compressed in a compression test, and (b) is a photographic view showing the state before a high-strength biomaterial is pulled in a tensile test. [Figure 6](a) is a graph showing the compression test results (stress-strain curves) of biomaterials (2.5%, 5%, 7.5% acidic chicken comb gel) prepared by gelling a dispersion uniformly dispersed at 60°C and collagen gel (5% collagen gel) as a comparative example, and (b) is a graph showing the compression test results of a biomaterial (5% acidic chicken comb gel) prepared by gelling a dispersion uniformly dispersed at 100°C. [Figure 7] (a) is a graph showing the compression test results of the biomaterial (5% acidic chicken comb gel) in Fig. 6(a) and the biomaterial (5% pH 7.4 chicken comb gel) neutralized with a phosphate buffer at pH 7.4, and (b) is a graph showing the tensile test results (stress-strain curve) of the 5% pH 7.4 chicken comb gel. [Figure 8] (a) and (b) are photographic views showing the degree of elongation of the biomaterial (5% pH 7.4 chicken comb gel) of the example under tension. [Figure 9] (a) is a photographic view showing film gels obtained by evaporating moisture from the biomaterial (1.5% acidic chicken comb gel) of the example at 60°C and 25°C, (b) is a photographic view showing the swelling behavior when these film gels are immersed in a phosphate buffer at pH 7.4, and (c) is a graph showing the weight change when these film gels are immersed in a phosphate buffer at pH 7.4. [Figure 10] It is a photographic view showing the process of producing a film material from a uniform colloidal solution obtained by treating chicken comb residue with a proteolytic enzyme.

Mode for Carrying Out the Invention

[0016] Hereinafter, the biomaterial of the embodiment according to the present invention and the method for producing the biomaterial will be described in detail. However, the present invention can be implemented in many different forms and is not limited to the examples of the embodiments and examples shown below.

[0017] (Biomaterial) The biomaterial of the present invention (hereinafter sometimes referred to as "this biomaterial") is a gel mainly composed of a collagen-hyaluronic acid complex (see Figure 1), which is a biopolymer in which hyaluronic acid is intertwined between collagen fibers, and therefore exhibits excellent flexibility and strength against compression and tension.

[0018] The term "main component" refers to the component that is present in the highest amount, excluding water (H2O), in the composition of this biomaterial.

[0019] In this biomaterial, flexibility tends to improve as the content of the collagen-hyaluronic acid complex, the main component, decreases, in other words, as the gel concentration of the biomaterial decreases, while strength tends to improve as the content increases. However, from the viewpoint of improving flexibility and strength in a good balance, the content is 1-10%, preferably 4-6%, and more preferably 4.5-5.5%. In addition, this biomaterial consists almost entirely of water other than the collagen-hyaluronic acid complex, with a water content of 90% or more.

[0020] Furthermore, the composition ratio of the collagen-hyaluronic acid complex contained in this biomaterial is collagen:hyaluronic acid = 8-12:0.8-1.2. In addition, from the viewpoint of enhancing the flexibility and strength of this biomaterial in a balanced manner, the composition of the collagen-hyaluronic acid complex is preferably 60-80% collagen, 3-10% hyaluronic acid, and 10-37% other components. Other components include water, proteoglycans, etc.

[0021] As will be explained in more detail later, the collagen-hyaluronic acid complex, the main component of this biomaterial, retains its triple helix structure, consisting of three polypeptide chains, without gelatinization, because no enzymatic or heat treatment is performed during the extraction process from animal-derived materials. Furthermore, the collagen-hyaluronic acid complex is extracted and gelled using an acidic solution with a pH of 2 to 3.8, which suppresses the dissolution or precipitation of hyaluronic acid. As a result, the collagen-hyaluronic acid complex, the main component of this biomaterial, retains the state in which hyaluronic acid is intertwined between collagen fibers, that is, the composite structure of collagen and hyaluronic acid molecules constructed in living organisms.

[0022] Furthermore, any part of an animal containing the collagen-hyaluronic acid complex can be used as the raw material for this biomaterial. For example, in chickens, the comb, wingtips, and feet contain a large amount of the collagen-hyaluronic acid complex, and by using the comb, which is discarded in large quantities, as the raw material, the aforementioned collagen-hyaluronic acid complex can be extracted in high yield and at low cost. In addition, the comb is particularly preferred over the wingtips and feet because the composition ratio of the collagen-hyaluronic acid complex contained in the comb is collagen:hyaluronic acid = 8~12:0.8~1.2.

[0023] Furthermore, as will be explained in more detail later, this biomaterial is expected to be improved in terms of flexibility and strength against compression and tension because, by heating and stirring the collagen-hyaluronic acid complex in an acidic solvent at 40-70°C, more preferably 60-70°C, and uniformly dispersing it, at least a portion of the triple helix structure of the collagen constituting the collagen-hyaluronic acid complex unravels in the dispersion. As the dispersion cools, the polypeptide chains come together again and partially form a helical structure with neighboring polypeptide chains, and the collagen-hyaluronic acid complexes, whose composite structure of collagen and hyaluronic acid molecules is substantially maintained as it is constructed in the body, become entangled, and the entire dispersion gels.

[0024] Furthermore, when this biomaterial is formed, for example, as a cylindrical acidic gel with a diameter of 1.5 mm and a height of 9 mm at a gel concentration of 5%, it possesses high flexibility that prevents fracture even at a compressive strain of approximately 70%, and high strength that prevents fracture even under compressive stress greater than approximately 0.03 MPa.

[0025] Furthermore, this biomaterial is not limited to a block shape; it may be formed into any shape, such as a strip or film.

[0026] Furthermore, the strength of this biomaterial can be improved by neutralizing it, for example, by immersing it in a phosphate buffer solution with a pH of 7.4. In other words, even if the gel concentration of this biomaterial is lower than 5%, neutralization can increase its strength so that it does not break even under compressive stresses greater than, for example, approximately 0.03 MPa. Note that the buffer solution is not limited to pH 7.4; any buffer solution that can neutralize this biomaterial with a pH of 6 to 8 may also be used.

[0027] Furthermore, despite being a gel, this biomaterial exhibits only about 5% volume change due to swelling, making it resistant to volume fluctuations.

[0028] Furthermore, this biomaterial can be converted into a so-called xerogel by evaporating the water at room temperature (1-30°C), and this xerogel can be returned to a gel with excellent flexibility and strength by immersing it in a pH 7.4 phosphate buffer. Thus, this biomaterial can be stored for a long period at room temperature and can be easily converted back into a gel for use simply by immersing it in a pH 7.4 phosphate buffer.

[0029] (Methods for preparing biomaterials) Next, the method for producing the biomaterial (hereinafter sometimes referred to as "this production method") will be explained in detail using Figure 2. As shown in Figure 2, this production method comprises a shredding step S1 for shredding a material containing the collagen-hyaluronic acid complex, a degreasing step S2 for degreasing the shredded material obtained in the shredding step S1, a crushing step S3 for immersing the degreasing material obtained in the degreasing step S2 in an acidic solution and then crushing it, an extraction step S4 for filtering the crushed liquid obtained in the crushing step S3 to extract the collagen-hyaluronic acid complex, a freeze-drying step S5 for freeze-drying the extract obtained in the extraction step S4, a dispersion step S6 for heating and stirring the freeze-dried extract obtained in the freeze-drying step S5 in an acidic solvent at 40-70°C to uniformly disperse it, and a cooling step S7 for cooling the dispersion obtained in the dispersion step S6 to gel it.

[0030] Thus, in this production method, unlike conventional collagen or hyaluronic acid extraction methods, the collagen-hyaluronic acid complex is extracted from the material without enzymatic treatment or high-temperature treatment. As a result, the collagen-hyaluronic acid complex retains the composite structure of collagen and hyaluronic acid molecules that is constructed in the body.

[0031] Furthermore, in the crushing step S3, crushing the material using an acidic solution with a pH of 2 to 3.8 suppresses the dissolution or precipitation of hyaluronic acid from the collagen-hyaluronic acid complex.

[0032] Furthermore, in extraction step S4, the collagen-hyaluronic acid complex is extracted using an acidic solution with a pH of 2 to 3.8, thereby suppressing the dissolution or precipitation of hyaluronic acid from the collagen-hyaluronic acid complex.

[0033] Furthermore, in the dispersion step S6, uniformly dispersing the freeze-dried extract using an acidic solution with a pH of 2 to 3.8 suppresses the dissolution or precipitation of hyaluronic acid from the collagen-hyaluronic acid complex.

[0034] Furthermore, in the dispersion step S6, by heating and stirring at 40-70°C until a uniform dispersion is achieved, the thermal denaturation of collagen can be controlled so that the triple helix structure of collagen constituting the collagen-hyaluronic acid complex does not completely unravel in the dispersion.

[0035] Furthermore, it is presumed that this manufacturing method, by neutralizing the gel obtained in the cooling step S7 by immersing it in a pH 7.4 buffer solution, promotes collagen fibrillation and increases the structural regularity of the collagen in the gel, thereby increasing the strength of this biomaterial.

[0036] Embodiments for carrying out the biomaterial and method for producing the biomaterial according to the present invention will be described below based on examples. [Examples]

[0037] In this embodiment, a collagen-hyaluronic acid complex is extracted from chicken combs as a raw material, and a gel is prepared as a biomaterial.

[0038] (Extraction of collagen-hyaluronic acid complex) First, in the shredding process S1 (see Figure 2), the chicken comb was shredded into small pieces of about 1-2 cm in size using scissors or the like, to obtain chicken comb pieces as shredding material.

[0039] Next, in degreasing step S2 (see Figure 2), 200 ml of acetone was added to the chicken comb pieces and stirred for 3 hours, and the acetone portion in which the lipids had dissolved was filtered and recovered. Then, another 200 ml of acetone was added to the residue from the first filtration and stirred for 3 hours, and the acetone portion in which the lipids had dissolved was filtered and recovered. Next, 200 ml of acetone was added to the residue from the second filtration and stirred for 24 hours, and the acetone portion was filtered and recovered. As a result, defatted chicken comb pieces and acetone extract of chicken combs, which were sufficiently degreased materials, were obtained.

[0040] Next, in crushing step S3 (see Figure 2), 200 ml of 0.5 M acetic acid was added to the defatted chicken comb pieces and gently stirred for 24 hours. The acetic acid portion was then filtered and recovered. This yielded the chicken comb immersion liquid. The residue was then crushed using a Waring Blender (Osaka Chemical Co., Ltd., model 7011HB) until no further changes were observed. Next, 200 ml of 0.5 M acetic acid was added to this crushed chicken comb material, and it was crushed again using the Waring Blender. This yielded the crushed chicken comb liquid.

[0041] Next, in extraction step S4 (see Figure 2), 200 ml of 0.5 M acetic acid was added to the crushed chicken comb liquid and stirred for 24 hours, and the acetic acid portion was filtered (pressed). This yielded chicken comb extract 1. Furthermore, 200 ml of 0.5 M acetic acid was added to the residue after extraction of extract 1 and stirred for 24 hours, and the acetic acid portion was filtered (pressed). This yielded chicken comb extract 2.

[0042] Next, in freeze-drying step S5 (see Figure 2), extracts 1 and 2 of the chicken comb were freeze-dried to obtain freeze-dried extract 1 from extract 1 and freeze-dried extract 2 from extract 2.

[0043] In addition to the extracts 1 and 2 mentioned above, Figure 3 shows a comparison of the weights of the freeze-dried products obtained by freeze-drying the acetone extract obtained in the degreasing step S2, the immersion liquid obtained in the crushing step S3, and the residue after extracting extracts 1 and 2 in the extraction step S4.

[0044] As shown in Figure 3, 3.1 ± 1.5 g of freeze-dried extract 1 and 0.5 ± 0.4 g of freeze-dried extract 2 were obtained from 101.44 ± 7.11 g of chicken comb. This confirms that the collagen-hyaluronic acid complex can be extracted from chicken comb with a high yield of approximately 3%.

[0045] Furthermore, IR (infrared absorption spectroscopy) analysis was performed on freeze-dried extracts 1 and 2. 1 ¹H NMR (proton nuclear magnetic resonance) analysis revealed that the main component of freeze-dried extracts 1 and 2 is protein, and it was inferred that hyaluronic acid is complexed with this protein.

[0046] Next, the results of colorimetric quantitative analysis of chicken comb extracts 1 and 2 using either a hydroxyproline assay or carbazole sulfate are shown in Figure 4. As shown in Figure 4(a), the collagen content of extract 1 estimated by the hydroxyproline assay was approximately 74%, and the collagen content of extract 2 was approximately 69%.

[0047] Furthermore, as shown in Figure 4(b), it was confirmed that the hyaluronic acid content of extract 1, estimated by the carbazole sulfuric acid method, was approximately 6.5%, and the hyaluronic acid content of extract 2 was approximately 6.8%.

[0048] Furthermore, as shown in Figure 4(c), when extracts 1 and 2 were heated, both suffered a weight loss of approximately 10% at 100°C, confirming that the water content of extracts 1 and 2 is approximately 10%.

[0049] From steps S1 to S4 of this preparation method and the resulting analysis, it was confirmed that extracts 1 and 2 contain collagen-hyaluronic acid complexes that retain the complex structure of collagen and hyaluronic acid molecules constructed in vivo. Furthermore, although the water retained by hyaluronic acid is removed when extracts 1 and 2 are freeze-dried while retaining the complex structure of the collagen-hyaluronic acid complex, the complex structure of the collagen-hyaluronic acid complex is largely retained in the freeze-dried extracts 1 and 2.

[0050] (Gel preparation) Next, the freeze-dried extracts 1 and 2 obtained through the five steps (S1 to S5) described above—shredding, degreasing, crushing, extraction, and freeze-drying—are used to prepare the gel, which is the biomaterial of the present invention.

[0051] First, in dispersion step S6 (see Figure 2), a 5% aqueous acetic acid solution was added to the freeze-dried extract and heated and stirred at approximately 60°C until a homogeneous dispersion was obtained. In this example, homogeneous dispersions with freeze-dried extract concentrations of 2.5 wt%, 5.0 wt%, and 7.5 wt% were prepared.

[0052] Next, in the cooling step S7 (see Figure 2), each homogeneous dispersion was poured into a cylindrical mold with a diameter of 1.5 mm and cooled at room temperature (25°C). As a result, each homogeneous dispersion gelled, and chicken comb gel as a biomaterial was obtained. Hereinafter, a chicken comb gel with a gel concentration of 2.5% prepared from a homogeneous dispersion with a concentration of 2.5 wt% of the freeze-dried extract will be referred to as 2.5% acidic chicken comb gel, a chicken comb gel with a gel concentration of 5% prepared from a homogeneous dispersion with a concentration of 5 wt% of the freeze-dried extract will be referred to as 5% acidic chicken comb gel, and a chicken comb gel with a gel concentration of 7.5% prepared from a homogeneous dispersion with a concentration of 7.5 wt% of the freeze-dried extract will be referred to as 7.5% acidic chicken comb gel.

[0053] As a comparative example, a collagen gel was prepared by adding a 5% aqueous acetic acid solution to a freeze-dried product of commercially available collagen aqueous solution (Nitta Gelatin Co., Ltd., Cellmatrix Type I-A), heating and stirring at approximately 60°C until a homogeneous dispersion was obtained, and then cooling this homogeneous dispersion to room temperature (25°C). In this example, the collagen gel is a 5% collagen gel prepared from a homogeneous dispersion of freeze-dried collagen with a concentration of 5.0 wt%, and is therefore referred to as a 5% collagen gel.

[0054] (Compression test) Next, a compression test (see Figure 5(a)) was conducted on the aforementioned 2.5-7.5% acidic chicken comb gel (3 samples) and 5% collagen gel (1 sample) at a rate of 10 mm / min. The results are shown in Figure 6. As shown in Figure 6(a), the comparative example, the 5% collagen gel, fractures at a compressive strain of approximately 70% and a compressive stress of approximately 0.03 MPa. In the stress-strain curves showing the compression test results in Figure 6, etc., the inflection points or breaks in the graph indicate gel fracture.

[0055] As shown in Figure 6(a), the 2.5% acidic chicken comb gel exhibited high flexibility, with one of the three samples not fracturing even at a compressive strain of over 70% (specifically 74%). However, two of the three samples fractured at compressive stresses of 0.02 MPa or less, confirming that it tends to have lower strength compared to the 5% collagen gel.

[0056] All three samples of 5% acidic chicken comb gel demonstrated high flexibility, remaining unbroken even at compressive strain rates of approximately 70% or higher (specifically 72%, 80%, and 84%), while also possessing high strength, remaining unbroken even at compressive stresses of 0.04 MPa or higher (maximum approximately 0.08 MPa). In other words, 5% acidic chicken comb gel was found to possess high flexibility comparable to 5% collagen gel, while also exhibiting high strength.

[0057] While the 7.5% acidic chicken comb gel showed a tendency towards lower flexibility compared to the 5% collagen gel, with all three samples fracturing at compressive strain rates of approximately 70% or less (specifically 62%, 63%, and 68%), all samples demonstrated high strength, remaining unfractured even under compressive stresses of 0.06 MPa or higher (maximum approximately 0.08 MPa).

[0058] As shown in Figure 6(b), the 5% acidic cockscomb gel prepared from a homogeneous dispersion heated and stirred at 100°C in dispersion step S6 fractured under a compressive stress of 0.01 MPa or less in all samples. This confirms that the strength of the 5% acidic cockscomb gel prepared from a homogeneous dispersion heated and stirred at approximately 60°C in dispersion step S6 was significantly lower.

[0059] (Neutralization of the gel) Next, a new 5% acidic chicken comb gel, which demonstrated high flexibility and strength in the compression test described above, was prepared. This gel was then immersed in pH 7.4 phosphate buffer and allowed to stand at 5°C for 24 hours to neutralize it, resulting in 5% pH 7.4 chicken comb gel (3 samples). The results of the compression test performed on the 5% pH 7.4 chicken comb gel are shown in Figure 7(a). Figure 7(a) also shows a comparison with the 5% acidic chicken comb gel (see Figure 6(a)).

[0060] As shown in Figure 7(a), the 5% pH 7.4 cockscomb gel fractured at approximately 65% ​​compressive strain, indicating a tendency towards lower flexibility compared to the 5% collagen gel. However, all samples demonstrated high strength, with no fractures up to a compressive stress of approximately 0.08 MPa. In other words, it was confirmed that the strength of the 5% pH 7.4 cockscomb gel improved through neutralization.

[0061] Furthermore, the 5% pH 7.4 cockscomb gel exhibited consistent flexibility and strength in all samples after neutralization, confirming that the gel quality is stable.

[0062] Furthermore, the 5% pH 7.4 chicken comb gel had a swelling degree of 1.05, which could be determined from the weight before and after neutralization. In other words, although the biomaterial is a gel, the volume change due to swelling was only about 5%, confirming that it has the property of being resistant to volume changes.

[0063] (Tensile test) Next, tensile tests were performed on 5% pH 7.4 chicken comb gel (3 samples) and 5% acidic chicken comb gel (3 samples) at a rate of 10 mm / min (see Figure 5(b)). The results are shown in Figure 7(b).

[0064] As shown in Figure 7(b), the 5% acidic cockscomb gel fractured at a tensile stress of approximately 0.003 MPa and a tensile strain of approximately 50%, while the 5% pH 7.4 cockscomb gel fractured at a tensile stress of approximately 0.006 MPa and a tensile strain of approximately 55-60%. This confirms that neutralization improves both flexibility and strength in tensile strength.

[0065] Furthermore, as shown in Figure 8, when both ends of a strip-shaped 5% pH 7.4 cockscomb gel are held and pulled in the longitudinal direction, it stretches by approximately 50%, confirming that the 5% pH 7.4 cockscomb gel has high flexibility.

[0066] (Film version) Next, the film formation of the cockscomb gel will be explained using Figure 9. As shown in Figure 9(a), 300 mg of lyophilized extract was added to 20 ml of distilled water and 300 μl of 0.1 M acetic acid to create an acidic solvent. This solution, with a concentration of approximately 1.5 wt% of the lyophilized extract, was heated and stirred at approximately 65% ​​for 1 hour. 10 ml of this homogeneous dispersion was then placed into PTFE petri dishes. Films were prepared by evaporating the water while maintaining the solution state at 60°C, and by evaporating the water while the gel was formed at 25°C.

[0067] Next, these films were immersed in a pH 7.4 phosphate buffer solution, and their swelling behavior was observed (see Figure 9(b)). The weight changes of these films are shown in Figure 9(c). As shown in Figure 9(b), the film gels whose water was evaporated at 60°C swelled horizontally, and after 30 minutes, the gel began to partially disintegrate. However, the film gels whose water was evaporated at 25°C showed almost no weight change even after 24 hours, starting from a state where the water content was approximately 10% after 4 hours (see Figure 9(c)). Furthermore, the film gels whose water was evaporated at 25°C did not swell much horizontally.

[0068] As explained above, it was confirmed that a biomaterial (chicken comb gel) composed of biopolymers with excellent flexibility and strength against compression and tension can be obtained by extracting a collagen-hyaluronic acid complex from chicken combs without enzyme treatment or high-temperature treatment, while maintaining the composite structure of collagen and hyaluronic acid that is constructed in vivo, and then freezing the freeze-dried extract of this collagen-hyaluronic acid complex, heating and stirring the dispersion in an acidic solvent at 40-70°C to uniformly disperse it, and then cooling the dispersion to gel it.

[0069] Furthermore, it was confirmed that using chicken comb as a material for biomaterials allows for the extraction of collagen-hyaluronic acid complexes in high yield and at low cost, while also yielding biomaterials with a good balance of flexibility and strength.

[0070] Furthermore, by using a freeze-dried extract obtained by freeze-drying the collagen-hyaluronic acid complex, it becomes easier to adjust the gel concentration in the chicken comb gel.

[0071] Furthermore, since flexibility and strength can be adjusted by controlling the content of the collagen-hyaluronic acid complex (a biopolymer) and neutralizing the gel, without adding additives such as fillers or crosslinking agents, it is easy to handle as a biomaterial.

[0072] (Preparation of biomaterials using chicken comb residue) In the gel preparation method described above, the weight of the freeze-dried residue obtained by freeze-drying the chicken comb residue after extracting the collagen-hyaluronic acid complex from the chicken comb as extracts 1 and 2 is more than double the weight of the freeze-dried extract obtained by freeze-drying extracts 1 and 2. Therefore, we will investigate the potential use of the chicken comb residue.

[0073] Furthermore, component analysis of the chicken comb residue before freeze-drying revealed that it contained 23.4% hyaluronic acid (carbazole sulfate method), 28.5% collagen (hydroxyproline assay), 41.1% total protein (BCA method), and 26.1% moisture. In other words, the chicken comb residue is mainly composed of collagen peptides and hyaluronic acid.

[0074] This section describes a method for preparing biomaterials using chicken comb residue. Note that the steps from extracting the collagen-hyaluronic acid complex from the chicken comb as extracts 1 and 2 to obtaining the chicken comb residue are the same as those described in the gel preparation method, and therefore a detailed explanation is omitted.

[0075] As shown in Figure 10, first, 1.0 g of freeze-dried residue obtained by freeze-drying chicken comb residue was added to 100 ml of pH 7.0 phosphate buffer, and then 0.1 g of papain (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), a plant-derived proteolytic enzyme, was added and the mixture was heated and stirred overnight at 45°C to obtain a homogeneous colloidal solution.

[0076] Next, 20 ml of a homogeneous colloidal solution of the residue was added to a 50 mm diameter Teflon® petri dish, and after degassing, it was allowed to stand at 60°C to form a film, thereby obtaining a film material.

[0077] As shown in the right-hand figure of Figure 10, the film material obtained from the homogeneous colloidal solution adheres strongly to Teflon, which has very low surface tension, confirming that it has excellent adhesive properties.

[0078] Thus, it was confirmed that the homogeneous colloidal solution of the residue treated with proteolytic enzymes, mainly composed of collagen peptides and hyaluronic acid, can be utilized as a biomaterial for applications such as bioadhesives. In other words, a biomaterial with excellent adhesive properties can be obtained from the residue after extracting the collagen-hyaluronic acid complex to produce comb gel from chicken combs, thus enabling the efficient use of materials without waste.

[0079] Although embodiments of the present invention have been described above with reference to the drawings, the specific configurations are not limited to these embodiments, and any changes or additions that do not depart from the spirit of the present invention are also included.

[0080] For example, in the above embodiment, a chicken comb was used as the material for the biomaterial, but the material is not limited to this, and any animal species or part can be freely selected as long as it has animal tissue from which a collagen-hyaluronic acid complex can be extracted.

[0081] Furthermore, in the above embodiment, a freeze-dried extract obtained by freeze-drying the collagen-hyaluronic acid complex extract was used to prepare the gel, but the invention is not limited to this, and the extract itself may be used to prepare the gel. [Industrial applicability]

[0082] The present invention has industrial applicability as a biomaterial and method for producing biomaterials, which can be used as medical materials such as bioadhesives, wound dressings, scaffolding materials, and drug carriers, and consists of a gel composed of biopolymers that is excellent in flexibility and strength. Furthermore, the present invention allows for the inexpensive production of high-value-added biomaterials by extracting collagen-hyaluronic acid complexes in high yield from chicken combs, which are mostly discarded, and gelling them using the properties of collagen. In addition, the xerogel obtained by evaporating water from the biomaterial gel of the present invention at room temperature has excellent structural stability and can be stored for a long time as it returns to a gel when water is added. Moreover, the biomaterial of the present invention is easy to handle as a biomaterial and has a wide range of applications because its flexibility and strength can be adjusted by adjusting the collagen-hyaluronic acid complex content (gel concentration) and neutralizing the gel without adding any additives.

Claims

1. A biomaterial characterized by being a gel whose main component is a collagen-hyaluronic acid complex in which hyaluronic acid is intertwined between collagen fibers.

2. The biomaterial according to claim 1, characterized in that the collagen-hyaluronic acid complex content is 4 to 6%.

3. The biomaterial according to claim 1, characterized in that the composition ratio of the collagen-hyaluronic acid complex is collagen:hyaluronic acid = 8 to 12:0.8 to 1.

2.

4. The biomaterial according to any one of claims 1 to 3, characterized in that the gel is neutralized.

5. A shredding process that cuts a material containing a collagen-hyaluronic acid complex, A degreasing step is performed to degrease the shredded material obtained in the above shredding step, The degreased material obtained in the degreasing step is immersed in an acidic solution and then crushed in a crushing step, An extraction step is performed in which the crushed liquid obtained by the crushing step is filtered to extract the collagen-hyaluronic acid complex, A freeze-drying step is performed to freeze-dry the extract obtained in the extraction step, A dispersion step is performed to uniformly disperse the freeze-dried extract obtained in the freeze-drying step by heating and stirring it in an acidic solvent at 40 to 70°C. A cooling step is performed to gel the dispersion obtained by the above dispersion step by cooling the dispersion liquid, A method for producing a biomaterial, characterized by comprising the following features.

6. The method for producing a biomaterial according to claim 5, characterized in that the gel obtained by the cooling step is immersed in a buffer solution with a pH of 7.

4.

7. The method for producing a biomaterial according to claim 5, characterized in that the freeze-dried residue obtained by freeze-drying the residue obtained by the extraction step is enzymatically treated with a proteolytic enzyme and heated and stirred to uniformly disperse it.

8. The method for producing a biomaterial according to any one of claims 5 to 7, characterized in that the material is a chicken comb.

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