Collagen hydrogel for cartilage repair and preparation method thereof

Abstract

The invention provides collagen hydrogel for cartilage repair and a preparation method thereof, and belongs to the technical field of hydrogel. Comprising the following steps: (1) carrying out pretreatment, impurity removal, enzymolysis and purification on pigskin to obtain a collagen solution; (2) mixing the collagen solution and the chitosan solution, then adding tannic acid, uniformly stirring, and carrying out in-situ crosslinking to form gel, so as to obtain primarily physically crosslinked collagen / chitosan double-network gel; and (3) soaking the collagen / chitosan double-network gel subjected to primary physical crosslinking into a sodium carboxymethyl cellulose solution, and carrying out network crosslinking, so as to obtain the collagen hydrogel for cartilage repair. The collagen hydrogel for cartilage repair is prepared through a simple two-step physical cross-linking method, the collagen hydrogel is endowed with antibacterial performance on the basis of remarkably improving the mechanical property of the collagen gel, meanwhile, a chemical cross-linking agent does not need to be introduced, and the excellent biocompatibility of collagen is reserved; a new strategy is provided for a preparation method of a material in the field of bone repair.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogels, and in particular to a collagen hydrogel for cartilage repair and a preparation method thereof. Background Art

[0002] Collagen is the most abundant structural protein in the human body. It can form molecular scaffolds and provide mechanical strength for tissues and organs. At the same time, collagen can bind to a variety of cell surface receptors, regulate cell activities, and play a vital role in tissue regeneration. In addition, collagen has good biocompatibility, biodegradability, absorbability, suitable porosity, and beneficial cell growth and metabolic interconnection structure. These characteristics make collagen gel scaffolds have great application prospects in the fields of bone repair, wound dressings, and drug sustained-release carriers. However, the defects of collagen are its weak rigidity and fast degradation rate, which limit the application of collagen scaffolds.

[0003] At present, the mechanical properties of collagen scaffolds are mainly enhanced by introducing various chemical crosslinking agents such as glutaraldehyde, genipin, carbodiimide, etc. or by physical crosslinking such as UV, freeze drying, or by combining collagen with other organic and inorganic materials. However, these methods still have certain limitations. For example, although the collagen scaffold formed by chemical crosslinking has the advantages of relatively stable structure and properties, high mechanical strength, and strong resistance to degradation in vivo, the chemical crosslinking agents or initiators introduced during the preparation process may remain in the scaffold, reducing the biocompatibility of the collagen scaffold. Conventional physical crosslinking or composite collagen scaffolds have limited improvement in mechanical properties and can be used as wound dressings in the skin field, but still do not meet the requirements of the bone repair field.

[0004] In addition, bone defect-associated infection (OAI) can place a heavy burden on bone repair. When various composite materials are implanted during bone defect treatment, they are easily infected by bacteria, which invade the wound through injured soft tissue and proliferate rapidly, leading to symptoms such as osteitis or osteonecrosis. Bacterial infection during bone material implantation can also reduce the bone induction capacity of the site, thereby slowing down new bone formation. In order to improve the safety and effectiveness of bone repair, it is essential that collagen scaffolds have antibacterial properties.

[0005] Therefore, under the premise of ensuring that the excellent biological properties of collagen are not affected, how to significantly improve its mechanical properties and at the same time give it antibacterial properties is an important issue that needs to be solved in the application and development of collagen scaffolds in the field of bone repair. Summary of the invention

[0006] The purpose of the present invention is to propose a collagen hydrogel for cartilage repair and a preparation method thereof. In view of the shortcomings of the prior art, the present invention prepares a high-strength antibacterial double-physical cross-linked collagen-based multi-network hydrogel for cartilage repair through a simple two-step physical cross-linking method, which gives the collagen gel antibacterial properties on the basis of significantly improving its mechanical properties. At the same time, the excellent biocompatibility of collagen is retained without the introduction of chemical cross-linking agents, providing a new strategy for the preparation of materials in the field of bone repair.

[0007] The technical solution of the present invention is achieved in this way:

[0008] The present invention provides a method for preparing a collagen hydrogel for cartilage repair, comprising the following steps:

[0009] (1) Preparation of collagen solution: pre-treating, removing impurities, enzymolysis and purification of pig skin to obtain collagen solution;

[0010] (2) Preparation of collagen / chitosan double network hydrogel: The collagen solution and the chitosan solution were mixed, and then tannic acid was added and stirred evenly, and in situ cross-linked to form a gel, thereby obtaining a preliminary physically cross-linked collagen / chitosan double network gel;

[0011] (3) Preparation of collagen hydrogel for cartilage repair: The collagen / chitosan double network gel that has been initially physically cross-linked is immersed in a sodium carboxymethyl cellulose solution to perform network cross-linking to obtain a collagen hydrogel for cartilage repair.

[0012] As a further improvement of the present invention, the specific method of the pig skin pretreatment in step (1) is as follows: take the completely thawed pig skin raw material, first use an oil remover to remove part of the fat layer of the pig skin, then use a skin slicer to remove the epidermis and subcutaneous fat layer of the pig skin, leaving only the dermis; finally, use a pelletizer to cut the sheet pig dermis into small particles.

[0013] As a further improvement of the present invention, the specific method of removing impurities in step (1) is as follows: according to a solid-liquid ratio of 1:4-1:10 g / mL, the pig dermal particles are soaked in a 5-15wt% sodium chloride solution for 20-24 hours, and then the salt-treated pig dermal particles are soaked in a 1-3wt% NaOH solution for 4-8 hours.

[0014] As a further improvement of the present invention, the specific method of enzymatic hydrolysis in step (1) is as follows: first, soak the treated pigskin particles in a 2-4wt% acetic acid solution for 4-16 hours, then add pepsin at a mass ratio of pepsin: pigskin of 1:30-60, and treat at 10°C-25°C for 32-48 hours to obtain a collagenase hydrolysate.

[0015] As a further improvement of the present invention, the specific method of purification described in step (1) is as follows: first, the collagenase hydrolysate is pre-filtered using a deep membrane bag, and then 2-4wt% acetic acid solution is continuously added to the collagenase hydrolysate as a replacement fluid, and the hydrolysate is subjected to acid purification through a hollow fiber, and the change in conductivity is detected as the acid purification end point. When the conductivity of the collagen solution reaches 1-1.2ms / cm, it is the acid purification end point. Finally, water for injection is continuously added to the collagen solution as a replacement fluid, and the hydrolysate is subjected to water purification through a hollow fiber. When the pH reaches 4.8-6.0, it is the water purification end point. Stop adding purified water, and use a hollow fiber to concentrate the collagen solution to 5mg / mL-15mg / mL to obtain a collagen solution.

[0016] As a further improvement of the present invention, the molecular weight of the collagen in step (1) is as high as 360-380 kDa.

[0017] As a further improvement of the present invention, the specific steps in step (2) are as follows:

[0018] Water-soluble chitosan is dissolved in PBS buffer with a pH of 7-7.6, and the collagen solution and the water-soluble chitosan solution are mixed in a volume ratio of 1:1-9:1, and then the tannic acid solution is added in a volume ratio of 15-25:1 and mixed to obtain a pre-gel solution with a pH of 7.0-7.4 and an osmotic pressure in the range of 270-360. The pre-gel solution is poured into a mold and allowed to stand at 30-35°C for 0.5-1h to prepare a preliminary physically cross-linked collagen / chitosan double network gel.

[0019] As a further improvement of the present invention, the mass percentage concentration of collagen in the collagen solution in step (2) is 0.5-1.5wt%, the mass percentage concentration of water-soluble chitosan in the water-soluble chitosan solution is 0.5-2wt%, and the mass percentage concentration of tannic acid in the tannic acid solution is 20-30wt%.

[0020] As a further improvement of the present invention, the degree of substitution of the sodium carboxymethyl cellulose in step (3) should be 0.7-1.0, the molecular weight should be 200-500 KDa, and the compressive elastic modulus of the obtained collagen hydrogel for cartilage repair should be 2-5 MPa.

[0021] The first aspect of the present invention provides a high-strength antibacterial dual-physical cross-linked collagen-based multi-network hydrogel material for cartilage repair. It is composed of natural polymer materials such as collagen (COL), chitosan (CS), physical cross-linking agent tannic acid (TA) and sodium carboxymethyl cellulose (CMC), and has excellent biocompatibility. The hydrogel improves its mechanical properties through a two-step simple physical cross-linking method, without the introduction of other chemical cross-linking agents. The mechanical properties of the gel can reach the range required for cartilage repair scaffolds (compression modulus 0.5-5MPa), and can be used for repairing cartilage defects in the human body. The chitosan and tannic acid components in the gel serve as natural antibacterial materials, which improve the mechanical properties of the gel while giving it good antibacterial properties. When used for cartilage defects, no antibiotics are required to achieve antibacterial efficacy.

[0022] The collagen used is animal-derived collagen extracted from pig skin by enzymatic hydrolysis. The immunogenicity of collagen is removed by enzymatic telopeptide removal and membrane purification. The obtained collagen has a molecular weight of up to 360-380kda and has a complete triple helix structure. Membrane purification technology is used to remove impurities and the acetic acid solution introduced during the purification process to obtain a high-concentration neutral collagen solution.

[0023] The chitosan used is commercial water-soluble chitosan purchased. The solubility of chitosan modified by carboxymethylation in neutral and alkaline solutions is significantly enhanced. The tannic acid and sodium carboxymethyl cellulose used are both commercial reagents purchased. Among them, sodium carboxymethyl cellulose is preferably CMC with high degree of substitution and molecular weight, the degree of substitution should be 0.7-1.0, and the molecular weight should be 100-500KDa.

[0024] The second aspect of the present invention provides a method for preparing the above-mentioned high-strength antibacterial dual-physical cross-linked collagen-based multi-network hydrogel material for cartilage repair, comprising the following steps:

[0025] 1) Preparation of collagen solution

[0026] The preparation of collagen solution mainly includes four steps: pig skin pretreatment, impurity removal, enzymatic hydrolysis and purification.

[0027] The first step is to take the completely thawed pig skin raw material and pre-process it to obtain pig skin particles, which is specifically divided into three steps: degreasing, peeling, and pelletizing. First, use a degreasing machine to remove part of the fat layer of the pig skin to reduce the thickness of the pig skin and facilitate subsequent operations; then use a peeling machine to remove the epidermis and subcutaneous fat layer of the pig skin, leaving only the dermis; finally, use a pelletizer to cut the sheet pig dermis into small particles.

[0028] The second step is to treat the pig dermis particles obtained in the first step with high concentration salt solution and alkaline solution respectively to remove impurities in the pig dermis. The specific method is to soak the pig dermis particles in 5-15wt% sodium chloride solution for 24 hours according to the solid-liquid ratio of 1:4-1:10g / mL to remove salt-soluble impurities in the pig dermis. Then soak the salt-treated pig dermis particles in 1-3wt% NaOH solution for 4-8 hours to remove fat in the pig skin.

[0029] The third step is to perform enzymatic hydrolysis and extraction on the pig dermis particles obtained in the second step. First, soak the treated pig skin particles in 2-4wt% acetic acid solution for 4-16 hours to unfold the collagen fibers for subsequent collagen extraction; then add pepsin at a mass ratio of 1:50 (pepsin: pig skin), treat at 10℃-25℃ for 32-48 hours, and obtain collagenase hydrolyzate.

[0030] The fourth step is to purify the collagenase hydrolysate obtained in the third step. First, the collagenase hydrolysate is pre-filtered with a deep membrane bag to remove residual pigskin particles and impurities. Then, 2-4wt% acetic acid solution is continuously added to the collagenase hydrolysate as a replacement fluid, and the collagenase hydrolysate is subjected to acid purification by a hollow fiber, and the change in conductivity is detected as the acid purification end point, and the conductivity of the collagen solution reaches 1.1ms / cm. It is the acid purification end point. Finally, water for injection is continuously added to the collagen solution as a replacement fluid, and the collagenase hydrolysate is subjected to water purification by a hollow fiber, and the change in pH is detected as the water purification end point. When the pH reaches 4.8-6.0, it is the water purification end point, and the addition of purified water is stopped, and the collagen solution is concentrated to 5mg / mL-15mg / mL by a hollow fiber to obtain a collagen solution.

[0031] 2) Preparation of collagen / chitosan double network hydrogel

[0032] The collagen solution and chitosan solution prepared in 1) are mixed, and then tannic acid is added and stirred evenly. The intermolecular interaction between tannic acid, collagen and chitosan is utilized to form a gel by in situ crosslinking, thereby obtaining a preliminary physically crosslinked collagen / chitosan double network gel.

[0033] Tannic acid (TA) is a natural polyphenol antimicrobial small molecule with antioxidant and antibacterial properties. Its large number of phenolic groups can provide a variety of interaction sites, including hydrogen bonds, ionic bonds, coordination bonds and hydrophobic interactions, and can be used as a physical crosslinker for hydrogels. Collagen is the main component of the extracellular matrix and has good biocompatibility and degradability. Chitosan is a natural cationic polymer with broad-spectrum antimicrobial activity and tissue adhesion. By first mixing the collagen solution and the chitosan solution to form an interpenetrating network pregel solution of collagen / chitosan, and then introducing tannic acid into the pregel solution, after simple stirring and mixing, abundant hydrogen bonds are established between the tannic acid hydroxyl group as a hydrogen donor and the collagen carboxyl and amine groups as hydrogen acceptors, as well as between the chitosan amino groups, and a hydrogel can be formed very quickly.

[0034] In the process of preparing the preliminary physically cross-linked collagen / chitosan double network gel, the mechanical properties and antibacterial properties of the gel are optimized by optimizing the ratio of collagen and chitosan. The preferred mass percentage concentration of collagen is 0.5-1.5wt%, the mass percentage concentration of chitosan is 0.5-2wt%, and the mass percentage concentration of tannic acid is 20-30wt%. The specific method is: dissolve water-soluble chitosan in PBS buffer with a pH of 7-7.6, mix the high-concentration collagen solution and the water-soluble chitosan solution in a volume ratio of 1:1-9:1, and then add the tannic acid solution in a volume ratio of 20:1 to mix well to obtain a pre-gel solution with a pH of 7.0-7.4 and an osmotic pressure in the range of 270-360. Pour the pre-gel solution into a mold and let it stand at 30-35°C for 0.5-1h to prepare a preliminary physically cross-linked collagen / chitosan double network gel.

[0035] 3) Preparation of collagen hydrogel for cartilage repair

[0036] Prepare a sodium carboxymethyl cellulose (CMC) solution, soak the preliminary physically cross-linked collagen / chitosan double network gel prepared in step 2) in the CMC solution, and use the Donnan equilibrium between the collagen / chitosan gel network and the CMC solution to change the ratio of the gel network molecular chain size to the gel network size, the molar charge ratio and the swelling degree, thereby regulating and enhancing the interaction between collagen and chitosan molecules. In the process of enhancing and rearranging the interaction between the molecular chains, the active functional groups on the polymer molecular chains have more opportunities to react with each other, forming new chemical bonds or stronger physical interactions, thereby resulting in network cross-linking to obtain a collagen hydrogel for cartilage repair.

[0037] CMC is a negatively charged polyelectrolyte material, which can balance the compression of hydrogels with polyelectrolyte gels through the Donnan effect. When collagen / chitosan hydrogel is immersed in CMC solution, the Na in the CMC solution is + Ions can diffuse into the gel to a certain extent, but macromolecular CMC cannot freely enter. At the same time, some ions (H + OH - ) may also diffuse to a certain extent. In this way, a difference in ion concentration is formed between the CMC solution inside and outside the gel, and Donnan equilibrium begins to be established. According to the Donnan equilibrium principle, this ion concentration difference will generate osmotic pressure. The difference in osmotic pressure between the external polymer solution and the solution inside the cross-linked polymer network drives water out of the polymer network, causing the gel network to shrink. With the change in the migration trend of water and the distribution of ions, the movement of collagen and chitosan molecular chains is affected. On the one hand, the reduction in water makes the distance between the molecular chains relatively closer, enhancing the electrostatic interactions and hydrogen bonds that originally existed between them. On the other hand, changes in ion concentration may affect the charge state and conformation of the molecular chains, further promoting rearrangement and aggregation between the molecular chains. In this process, the active functional groups on the molecular chains have more opportunities to react with each other, forming new chemical bonds or stronger physical interactions, thereby leading to network cross-linking.

[0038] In the process of establishing the second physical cross-linked network, the mechanical properties of COL / CS / CMC multiple network hydrogels were optimized by exploring the effects of CMC solutions with different degrees of substitution and different molecular weights on the properties of collagen / chitosan gels. CMC with higher degrees of substitution and molecular weight has a better effect on compressing collagen / chitosan hydrogels than CMC with lower degrees of substitution and molecular weight, and the obtained double physically cross-linked COL / CS / CMC multiple network hydrogels have a denser network structure and better mechanical properties. Preferably, when the CMC, degree of substitution should be in the range of 0.7-1.0 and the molecular weight is in the range of 200-500KDa, the compressive elastic modulus of the obtained double physically cross-linked COL / CS / CMC multiple network hydrogel can reach 2-5MPa.

[0039] The present invention further protects a collagen hydrogel for cartilage repair prepared by the above preparation method.

[0040] The present invention has the following beneficial effects:

[0041] 1. The present invention prepares a high-strength antibacterial double-physical cross-linked COL / CS / CMC multi-network hydrogel by a simple two-step physical cross-linking method. On the basis of significantly improving the mechanical properties of collagen gel, it is endowed with antibacterial properties, and at the same time, the good biocompatibility of collagen is retained without the introduction of chemical cross-linking agents. The hydrogel has the characteristics of inducing human bone marrow mesenchymal stem cells (hBMSCs) to differentiate into chondrocytes, has a certain mechanical strength and antibacterial properties, and is suitable for the field of cartilage repair.

[0042] 2. The double physically cross-linked COL / CS / CMC multi-network hydrogel prepared by the present invention is composed of natural polymer materials such as collagen (COL), chitosan (CS), physical cross-linking agent tannic acid (TA) and sodium carboxymethyl cellulose (CMC) and has excellent biocompatibility.

[0043] 3. The hydrogel preparation method of the present invention creatively utilizes the intermolecular interactions between tannic acid and collagen and chitosan, as well as the Donnan equilibrium between the collagen / chitosan gel network and the CMC solution, and improves the mechanical properties through a two-step simple physical cross-linking method without introducing other chemical cross-linking agents. The mechanical properties of the gel can reach the range required for cartilage repair scaffolds (compression modulus 0.5-5MPa).

[0044] 4. The chitosan and tannic acid components in the gel serve as natural antibacterial materials. They improve the mechanical properties of the gel while giving it good antibacterial properties. When used for cartilage defects, they can achieve antibacterial effects without the use of additional antibiotics.

[0045] 5. The double physically cross-linked COL / CS / CMC multi-network hydrogel prepared by the present invention has a compression modulus of up to 5MPa, is non-cytotoxic, has good biocompatibility, has the ability to promote cell migration and induce cell differentiation, and has a simple preparation method and has broad application prospects in the field of cartilage repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0047] Figure 1 In the figure, (a) is the SEM image of the initial physically cross-linked collagen / chitosan double network gel, and (b) is the SEM image of the collagen hydrogel for cartilage repair.

[0048] Figure 2(a) is the electrophoresis diagram of the collagen solution, (b) is the circular dichroism spectrum of the collagen solution, and (c) is the micro-differential scanning thermal spectrum of the collagen solution.

[0049] Figure 3 This is a picture of BMSCs cell migration. DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] Embodiment 1:

[0052] (1) Preparation of collagen solution

[0053] Take the completely thawed pig skin raw material, use a degreasing machine to remove part of the fat layer of the pig skin, then use a skin slicer to remove the epidermis and subcutaneous fat layer to only keep the dermis, and finally use a pelletizer to cut the sliced ​​pig dermis into small particles.

[0054] The obtained porcine dermis particles were immersed in a 10 wt % sodium chloride solution at a solid-liquid ratio of 1:4 g / mL for 24 hours to remove salt-soluble impurities, and then the salt-treated porcine dermis particles were immersed in a 3 wt % NaOH solution for 6 hours to remove fat.

[0055] The treated pigskin particles were soaked in 3 wt % acetic acid solution for 16 h to expand the collagen fibers, and then pepsin was added at a mass ratio of 1:50 (pepsin: pigskin), and treated at 25° C. for 48 h to obtain a collagenase hydrolysate.

[0056] The collagen hydrolysate was pre-filtered with a deep membrane bag, and 3wt% acetic acid solution was continuously added to the collagen hydrolysate as a replacement fluid for acid purification. The acid purification was completed when the conductivity of the collagen solution reached 1.1ms / cm. Finally, water for injection was continuously added to the collagen solution as a replacement fluid for water purification. When the pH reached 4.8, the addition of purified water was stopped, and the collagen solution was concentrated to 15mg / mL with a hollow fiber.

[0057] (2) Preparation of collagen / chitosan double network hydrogel

[0058] Water-soluble chitosan was dissolved in PBS buffer at pH 7.4, and a collagen solution with a mass percentage concentration of 1.5wt% (15mg / mL) and a water-soluble chitosan solution with a mass percentage concentration of 2wt% were mixed at a volume ratio of 1:1, and then a tannic acid solution with a mass percentage concentration of 25wt% was added at a volume ratio of 20:1 to mix well to obtain a pre-gel solution with a pH of 7.2 and an osmotic pressure of 320. The pre-gel solution was then poured into a mold and allowed to stand at 35°C for 1h to prepare a preliminary physically cross-linked collagen / chitosan double network gel.

[0059] (3) Preparation of collagen hydrogel for cartilage repair

[0060] A CMC solution with a degree of substitution of 0.7 and a molecular weight of 400KDa-500KDa was prepared, and the prepared preliminary physically cross-linked collagen / chitosan double network gel was immersed in the CMC solution. As the immersion time increased, the gel volume gradually decreased, and the gel size no longer changed significantly after 3 hours.

[0061] Embodiment 2:

[0062] (1) Preparation of collagen solution

[0063] Take the completely thawed pig skin raw material, use a degreasing machine to remove part of the fat layer of the pig skin, then use a skin slicer to remove the epidermis and subcutaneous fat layer to only keep the dermis, and finally use a pelletizer to cut the sliced ​​pig dermis into small particles.

[0064] The obtained porcine dermis particles were immersed in a 10 wt % sodium chloride solution at a solid-liquid ratio of 1:8 g / mL for 16 hours to remove salt-soluble impurities, and then the salt-treated porcine dermis particles were immersed in a 2 wt % NaOH solution for 6 hours to remove fat.

[0065] The treated pigskin particles were soaked in 3 wt % acetic acid solution for 16 h to expand the collagen fibers, and then pepsin was added at a mass ratio of 1:50 (pepsin: pigskin), and treated at 20° C. for 48 h to obtain a collagenase hydrolysate.

[0066] The collagen hydrolysate was pre-filtered with a deep membrane bag, and 3wt% acetic acid solution was continuously added to the collagen hydrolysate as a replacement fluid for acid purification. The acid purification was completed when the conductivity of the collagen solution reached 1.1ms / cm. Finally, water for injection was continuously added to the collagen solution as a replacement fluid for water purification. When the pH reached 6.0, the addition of purified water was stopped, and the collagen solution was concentrated to 15mg / mL with a hollow fiber.

[0067] (2) Preparation of collagen / chitosan double network hydrogel

[0068] Water-soluble chitosan was dissolved in PBS buffer at pH 7.4, and a collagen solution with a mass percentage concentration of 1.5wt% (15mg / mL) and a water-soluble chitosan solution with a mass percentage concentration of 2wt% were mixed at a volume ratio of 5:1, and then a tannic acid solution with a mass percentage concentration of 25wt% was added at a volume ratio of 20:1 to mix well, thereby obtaining a pre-gel solution with a pH of 7.4 and an osmotic pressure of 280. The pre-gel solution was then poured into a mold and allowed to stand at 35°C for 1h to prepare a preliminary physically cross-linked collagen / chitosan double network gel.

[0069] (3) Preparation of collagen hydrogel for cartilage repair

[0070] Prepare a CMC solution with a degree of substitution of 0.7 and a molecular weight of 400KDa-500KDa, and immerse the prepared preliminary physically cross-linked collagen / chitosan double network gel in the CMC solution. As the immersion time increases, the gel volume gradually decreases, and the gel size no longer changes significantly after 3 hours.

[0071] Embodiment 3:

[0072] (1) Preparation of collagen solution

[0073] Take the completely thawed pig skin raw material, use a degreasing machine to remove part of the fat layer of the pig skin, then use a skin slicer to remove the epidermis and subcutaneous fat layer to only keep the dermis, and finally use a pelletizer to cut the sliced ​​pig dermis into small particles.

[0074] The obtained porcine dermis particles were immersed in a 10% sodium chloride solution at a solid-liquid ratio of 1:8 g / mL for 16 hours to remove salt-soluble impurities, and then the salt-treated porcine dermis particles were immersed in a 2wt% NaOH solution for 6 hours to remove fat.

[0075] The treated pigskin particles were soaked in 3 wt % acetic acid solution for 16 h to expand the collagen fibers, and then pepsin was added at a ratio of 1:50 (pepsin: pigskin) and treated at 20° C. for 48 h to obtain a collagenase hydrolysate.

[0076] The collagen hydrolysate was pre-filtered with a deep membrane bag, and 3wt% acetic acid solution was continuously added to the collagen hydrolysate as a replacement fluid for acid purification. The acid purification was completed when the conductivity of the collagen solution reached 1.1ms / cm. Finally, water for injection was continuously added to the collagen solution as a replacement fluid for water purification. When the pH reached 4.8, the addition of purified water was stopped, and the collagen solution was concentrated to 15mg / mL with a hollow fiber.

[0077] (2) Preparation of collagen / chitosan double network hydrogel

[0078] Water-soluble chitosan was dissolved in PBS buffer at pH 7.4, and a collagen solution with a mass percentage concentration of 1.5wt% (15mg / mL) and a water-soluble chitosan solution with a mass percentage concentration of 2wt% were mixed at a volume ratio of 5:1, and then a tannic acid solution with a mass percentage concentration of 25wt% was added at a volume ratio of 20:1 to mix well, to obtain a pre-gel solution with a pH of 6.7 and an osmotic pressure of 280. The pre-gel solution was then poured into a mold and allowed to stand at 35°C for 1h to prepare a preliminary physically cross-linked collagen / chitosan double network gel.

[0079] (3) Preparation of collagen hydrogel for cartilage repair

[0080] Prepare a CMC solution with a degree of substitution of 1.0 and a molecular weight of 400KDa-500KDa, and immerse the prepared preliminary physically cross-linked collagen / chitosan double network gel in the CMC solution. As the immersion time increases, the gel volume gradually decreases, and the gel size no longer changes significantly after 2 hours.

[0081] Embodiment 4:

[0082] (1) Preparation of collagen solution

[0083] Take the completely thawed pig skin raw material, use a degreasing machine to remove part of the fat layer of the pig skin, then use a skin slicer to remove the epidermis and subcutaneous fat layer to only keep the dermis, and finally use a pelletizer to cut the sliced ​​pig dermis into small particles.

[0084] The obtained porcine dermis particles were immersed in a 10wt% sodium chloride solution at a solid-liquid ratio of 1:8g / mL for 16 hours to remove salt-soluble impurities, and then the salt-treated porcine dermis particles were immersed in a 2wt% NaOH solution for 6 hours to remove fat.

[0085] The treated pigskin particles were soaked in 3 wt % acetic acid solution for 16 h to expand the collagen fibers, and then pepsin was added at a mass ratio of 1:50 (pepsin: pigskin), and treated at 25° C. for 48 h to obtain a collagenase hydrolysate.

[0086] The collagen hydrolysate was pre-filtered with a deep membrane bag, and 3wt% acetic acid solution was continuously added to the collagen hydrolysate as a replacement fluid for acid purification. The acid purification was completed when the conductivity of the collagen solution reached 1.1ms / cm. Finally, water for injection was continuously added to the collagen solution as a replacement fluid for water purification. When the pH reached 4.8, the addition of purified water was stopped, and the collagen solution was concentrated to 15mg / mL with a hollow fiber.

[0087] (2) Preparation of collagen / chitosan double network hydrogel

[0088] Water-soluble chitosan was dissolved in PBS buffer at pH 7.4, and a collagen solution with a mass percentage concentration of 1.5wt% (15mg / mL) and a water-soluble chitosan solution with a mass percentage concentration of 2wt% were mixed at a volume ratio of 1:1, and then a 25% tannic acid solution with a mass percentage concentration was added at a volume ratio of 20:1 to mix well to obtain a pre-gel solution with a pH of 7.2 and an osmotic pressure of 320. The pre-gel solution was then poured into a mold and allowed to stand at 35°C for 1h to prepare a preliminary physically cross-linked collagen / chitosan double network gel.

[0089] (3) Preparation of collagen hydrogel for cartilage repair

[0090] A CMC solution with a degree of substitution of 1.0 and a molecular weight of 400KDa-500KDa was prepared, and the prepared preliminary physically cross-linked collagen / chitosan double network gel was immersed in the CMC solution. As the immersion time increased, the gel volume gradually decreased, and the gel size no longer changed significantly after 2 hours.

[0091] Comparative Example 1:

[0092] The difference compared with Example 4 is that the mixing volume ratio of the collagen solution to the chitosan solution is 9:1, and the other steps are the same. A pre-gel solution with a pH of 5.8 and an osmotic pressure of 220 is obtained. The prepared preliminary physically cross-linked collagen / chitosan double network gel is immersed in the CMC solution for 2 hours to reach an equilibrium state.

[0093] Comparative Example 2:

[0094] The difference compared with Example 4 is that the mixing volume ratio of the collagen solution to the chitosan solution is 1:9, and the other steps are the same. A pre-gel solution with a pH of 7.4 and an osmotic pressure of 355 is obtained. The prepared preliminary physically cross-linked collagen / chitosan double network gel is immersed in the CMC solution for 2 hours to reach an equilibrium state.

[0095] Comparative Example 3:

[0096] The difference compared with Example 4 is that the molecular weight of the CMC solution used for immersion is 100KDa-200KDa, and the other steps are the same. A pre-gel solution with a pH of 5.8 and an osmotic pressure of 280 is obtained. The prepared preliminary physically cross-linked collagen / chitosan double network gel is immersed in the CMC solution for 2 hours to reach an equilibrium state.

[0097] Comparative Example 4:

[0098] The difference compared with Example 4 is that the concentration of the collagen solution is 5 mg / mL, and the other steps are the same. A pre-gel solution with a pH of 5.8 and an osmotic pressure of 280 is obtained. The prepared preliminary physically cross-linked collagen / chitosan double network gel is immersed in the CMC solution for 2 hours to reach an equilibrium state.

[0099] Test Example 1 Performance Evaluation

[0100] Mechanical properties of collagen hydrogel for cartilage repair

[0101] The gel samples of the above examples and comparative examples were prepared into cylinders with a diameter of 1 cm and a height of 1 cm, and the samples were subjected to compression performance tests using a universal testing machine at a test rate of 5 mm / min. Each group of samples had 5 samples, and the average value was taken as the test result. The test results are shown in Table 1.

[0102] Table 1

[0103]

[0104] From Table 1, it can be concluded that adjusting the ratio of collagen and chitosan, the degree of substitution and molecular weight of CMC can regulate the mechanical properties of the prepared gel. Comparative Example 1, Example 4 and Comparative Example 1 show that the mechanical properties of the gel prepared by CMC with high degree of substitution and large molecular weight are higher, which may be related to the high molecular weight and high degree of substitution CMC, which can produce greater steric hindrance and charge density effects in the system, thereby producing a stronger Donnan effect, forming a denser network result, and improving the mechanical properties of the gel. Comparative Examples 3, 4 and Comparative Examples 1, 2, and 3 show that when the amount of collagen or chitosan added is too large, the mechanical properties of the gel will decrease, which may be related to the characteristics of the two materials. When the amount of chitosan added is too much, the rigid chitosan gel network dominates, and the gel is fragile as a whole when subjected to stress, resulting in a decrease in the compressive elastic modulus. When the amount of collagen added is too much, the flexible polysaccharide gel network dominates, and the gel lacks toughness, resulting in a decrease in the compressive elastic modulus. When the ratio of collagen to chitosan is appropriate, the synergistic effect of the rigid network and the flexible network can be fully exerted, so that the mechanical properties of the gel can reach the best. For example, the elastic modulus of the gel in Example 4 can reach 5.08±0.15 MPa.

[0105] Test Example 2: Morphology and structure of hydrogel

[0106] Figure 1 a and Figure 1 b are SEM images of COL / CS double network hydrogel and COL / CS / CMC multiple network hydrogel. The test method includes: freeze-drying the hydrogel, cutting it into samples with a thickness of about 2 mm, adhering it to the sample stage with conductive glue, spraying gold, and observing its microscopic morphology through a scanning electron microscope (SEM). Figure 1The results showed that both COL / CS double network hydrogel and COL / CS / CMC multiple network hydrogel had uniform three-dimensional network structure, and the network structure of COL / CS / CMC hydrogel was denser than that of COL / CS double network hydrogel, which indicated that COL / CS double network hydrogel immersed in CMC solution would further undergo physical crosslinking. Compared with COL / CS double network hydrogel, the mechanical properties of COL / CS / CMC multiple network hydrogel were significantly improved, and its compression modulus increased from 374kDa to 5.08MPa. This result further proved that the Donnan equilibrium effect would cause the gel to undergo physical crosslinking.

[0107] Test Example 3 Characterization of collagen activity

[0108] Figure 2 a is the gel electrophoresis diagram of the collagen solution in Example 4. The results show that the molecular weight of the collagen extracted by this method is consistent with the molecular weight of the standard product and can reach 365-375 kda, and there are no other impurity protein bands. Figure 2 b and Figure 2 c are the circular dichroism spectrum and micro differential scanning calorimetry spectrum of the collagen solution of Example 4, respectively. Figure 1 b It can be seen that the collagen solution has a negative peak near 190nm and a positive peak near 220nm; it has a characteristic thermal absorption peak at 42°C. The above results indicate that the extracted collagen has a complete triple helix structure and is biologically active.

[0109] Test Example 4 In vitro cytotoxicity test

[0110] The hydrogel of Example 4 was extracted with DMEM complete culture medium at a ratio of 0.2 g / mL and extracted at 37°C for 72 h to obtain a hydrogel extract. Then, hBMSCs were cultured at a concentration of 1×10 4 Cells were seeded in 96-well plates at a density of 1.5 × 10 / well and incubated at 37°C and 5% CO 2 The cells were cultured in an incubator for 24 hours, then the old culture medium was sucked out, and the hydrogel extract was added to a 96-well plate at 100 μl / well to obtain an experimental group, and DMEM complete culture medium was used as a control group. The experimental group and the control group were cultured for 48 hours, and then taken out, and MTT solution was added at 50 μL / well. After further culture for 3 hours, the supernatant was sucked out and DMSO was added at 150 μL / well. The OD value of each well was measured at 490 nm using an enzyme marker. The cytotoxicity test results showed that the cell survival rate treated with the gel extract prepared in Example 4 was not significantly different from that of the blank control group, and the survival rate was above 90%, indicating that the gel was non-cytotoxic and had good biocompatibility.

[0111] Test Example 5: Ability of hydrogel to promote cell migration

[0112] BMSCs were seeded in DMEM / F12 medium (containing 10% FBS and 100 U / mL double antibody) and cultured in an incubator at 37°C and 5% CO2 until the logarithmic growth phase. Then BMSCs were seeded into the culture medium (cell seeding concentration 1×10 6 cells / mL), and after culturing for 24 hours, the central area of ​​cell growth was scratched with a micropipette tip or other hard object to remove the cells in the central part. After removing the scratched cells in the sample group, the pre-gel solution of Example 4 was added, and the blank group was added with a serum-free culture medium. Then, the cells were cultured for the time set in the experiment (different time points were allowed), the cell culture plate was taken out, and the surrounding cells were observed under a microscope to see whether they migrated to the central scratched area, and photos were taken. The results are shown in FIG. Figure 3 As shown, it was shown that the hydrogel prepared in Example 4 had a significantly improved ability to promote the migration of BMSCs.

[0113] Test Example 6 Ability to induce cell differentiation

[0114] BMSCs were inoculated in DMEM / F12 culture medium (containing 10% FBS and 100 U / mL double antibody) and incubated at 37°C with 5% CO 2 The BMSCs were then inoculated into the gel of Example 4 (cell inoculation concentration 1×10 5 Cells / mL), after culturing for 24 hours, the culture medium was replaced with chondrogenic differentiation medium, and the control group was not inoculated with gel. The medium was changed every two days, and after culturing for 14 days, ColII analysis was performed on the cells co-cultured with the hydrogel for 14 days to detect the chondrogenic differentiation ability. Compared with the control group, the ColII content in the sample group increased, indicating that the hydrogel of the present invention prepared in Example 4 has the ability to promote the differentiation of hBMSCs into chondrocytes and can be used to treat cartilage defects.

[0115] Test Example 7 Antibacterial Properties of Hydrogel

[0116] Table 2 shows the antibacterial properties of the cartilage repair hydrogel of Example 4. The test method is to use Staphylococcus aureus and Escherichia coli as Gram-positive bacteria and Gram-negative bacteria to test the antibacterial activity of the hydrogel, specifically including: activating the bacteria with liquid culture medium to obtain a bacterial suspension, diluting it with physiological saline to 1×10 5 cfm / mL, the hydrogel of Example 4 was added to the diluted bacterial suspension as the experimental group, and incubated on a shaker at 37°C and 120 rpm for 24 h. The bacterial suspension without hydrogel was used as the control group. The hydrogel concentration in the experimental group was 0.2 g / mL. After culturing for 24 h, the colony concentration of the control group was diluted to 1×10 3cfm, and dilute the experimental group with the same dilution multiple, respectively aspirate 100 μL and apply it on the solid culture medium plate, place it in a 37℃ incubator for 24 h, then count the live bacteria, determine the number of live bacteria, and calculate the bactericidal rate.

[0117] Table 2 Antibacterial test results

[0118]

[0119] The results show that the bactericidal rate of the hydrogel of the present invention against Escherichia coli is 88.9%, and the bactericidal rate of Staphylococcus aureus is 91.2%. The hydrogel of the present invention has a significantly improved antibacterial effect. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a collagen hydrogel for cartilage repair, characterized in that: The following steps are involved: (1) Preparation of collagen solution: pre-treating, removing impurities, enzymolysis and purification of pig skin to obtain collagen solution; (2) Preparation of collagen / chitosan double network hydrogel: The collagen solution and the chitosan solution were mixed, and then tannic acid was added and stirred evenly, and in situ cross-linked to form a gel, thereby obtaining a preliminary physically cross-linked collagen / chitosan double network gel; (3) Preparation of collagen hydrogel for cartilage repair: The collagen / chitosan double network gel that has been initially physically cross-linked is immersed in a sodium carboxymethyl cellulose solution to perform network cross-linking to obtain a collagen hydrogel for cartilage repair.

2. The preparation method according to claim 1, characterized in that: The specific method of the pig skin pretreatment in step (1) is as follows: take the completely thawed pig skin raw material, first use an oil remover to remove part of the fat layer of the pig skin, then use a skin slicer to remove the epidermis and subcutaneous fat layer of the pig skin, leaving only the dermis; finally, use a pelletizer to cut the sheet pig dermis into small particles.

3. The preparation method according to claim 1, characterized in that: The specific method of removing impurities in step (1) is as follows: according to the solid-liquid ratio of 1:4-1:10 g / mL, the pig dermal particles are soaked in a 5-15wt% sodium chloride solution for 20-24 hours, and then the salt-treated pig dermal particles are soaked in a 1-3wt% NaOH solution for 4-8 hours.

4. The preparation method according to claim 1, characterized in that: The specific method of enzymatic hydrolysis in step (1) is as follows: first, soak the treated pigskin particles in 2-4wt% acetic acid solution for 4-16 hours, then add pepsin according to the mass ratio of pepsin: pigskin of 1:30-60, and treat at 10℃-25℃ for 32-48h to obtain collagenase hydrolyzate.

5. The preparation method according to claim 1, characterized in that: The specific method of purification described in step (1) is as follows: first, the collagenase hydrolysate is pre-filtered with a deep membrane bag, and then 2-4wt% acetic acid solution is continuously added to the collagenase hydrolysate as a replacement fluid, and the hydrolysate is acid-purified by a hollow fiber, and the change in conductivity is detected as the acid purification end point. When the conductivity of the collagen solution reaches 1-1.2ms / cm, it is the acid purification end point. Finally, water for injection is continuously added to the collagen solution as a replacement fluid, and the hydrolysate is water-purified by a hollow fiber. When the pH reaches 4.8-6.0, it is the water purification end point, and the addition of purified water is stopped. The collagen solution is concentrated to 5mg / mL-15mg / mL using a hollow fiber to obtain a collagen solution.

6. The preparation method according to claim 1, characterized in that: The molecular weight of the collagen described in step (1) is as high as 360-380 kDa.

7. The preparation method according to claim 1, characterized in that: The specific steps in step (2) are as follows: Water-soluble chitosan is dissolved in PBS buffer with a pH of 7-7.6, and the collagen solution and the water-soluble chitosan solution are mixed in a volume ratio of 1:1-9:1, and then the tannic acid solution is added in a volume ratio of 15-25:1 and mixed to obtain a pre-gel solution with a pH of 7.0-7.4 and an osmotic pressure in the range of 270-360. The pre-gel solution is poured into a mold and allowed to stand at 30-35°C for 0.5-1h to prepare a preliminary physically cross-linked collagen / chitosan double network gel.

8. The preparation method according to claim 1, characterized in that: The mass percentage concentration of collagen in the collagen solution in step (2) is 0.5-1.5wt%, the mass percentage concentration of water-soluble chitosan in the water-soluble chitosan solution is 0.5-2wt%, and the mass percentage concentration of tannic acid in the tannic acid solution is 20-30wt%.

9. The preparation method according to claim 1, characterized in that: The degree of substitution of the sodium carboxymethyl cellulose in step (3) should be 0.7-1.0, the molecular weight should be 200-500 KDa, and the compressive elastic modulus of the obtained collagen hydrogel for cartilage repair should be 2-5 MPa.

10. A collagen hydrogel for cartilage repair prepared by the preparation method according to any one of claims 1 to 9.

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