Collagen composite material for repairing joint injury as well as preparation method and application of collagen composite material

Abstract

The invention provides a collagen composite material for joint injury repair and a preparation method and application thereof.The collagen composite material comprises collagen and chitosan, the chitosan is water-soluble chitosan subjected to one or two or more combined treatment of carboxylation, hydroxylation, phosphatization and quaternization, and the chitosan is water-soluble chitosan subjected to one or two or more combined treatment of carboxylation, hydroxylation, phosphatization and quaternization. The weight ratio of the collagen to the water-soluble chitosan is (1: 15)-(1: 3). The composition and proportion of the collagen and the water-soluble chitosan are scientifically screened, the collagen composite material which is closer to natural synovial fluid in composition is formed, the water-soluble chitosan provides physical blocking inflammatory factors and a lubricating effect between cartilages, and the collagen has a pore structure, so that the cartilages have a good anti-inflammatory effect on the cartilages, and the cartilages have a good anti-inflammatory effect on the cartilages. Adhesion of cartilage cells is facilitated, nutrition is obtained, proliferation of the cartilage cells is promoted, and repair of damaged cartilage is achieved.

Description

Technical Field

[0001] The present application relates to the technical field of biomaterials, and in particular to a collagen composite material for repairing joint injuries, and a preparation method and application thereof. Background Art

[0002] Osteoarthritis is a chronic degenerative joint disease that mainly affects the elderly, often causing joint pain, stiffness, and dysfunction in patients; the knee joint is the most common site of osteoarthritis. Current treatment options usually include: oral and / or intra-articular injection of non-steroidal anti-inflammatory drugs, antipyretic analgesics, hyaluronic acid, sodium hyaluronate, etc.

[0003] CN114748499A discloses a medical chitosan for intraarticular injection and a preparation method thereof, and CN115154413A discloses a gel material for intraarticular injection and a preparation method thereof, both of which use chitosan (also known as chitosan in the field of medical materials) as the main material for injection. Its structure is similar to the glycosaminoglycan structure in the extracellular matrix, which can promote cell-to-cell adhesion and effectively provide a physical barrier between the cartilage structures at the joints. It can also be used as a supplement to the synovial fluid matrix to strengthen the lubrication between joints, slow down the progression of arthritis, and relieve joint pain. However, the effect of chitosan on joint repair and joint regeneration is not obvious.

[0004] Collagen is a protein that is abundant in human skin, bones, tendons, ligaments and other connective tissues. It has natural biocompatibility. Introducing collagen into joint cavity injection can help repair joint injuries. However, improper use of the two materials will make it difficult for the structural cross-linking effect between molecules to be fully exerted, and the effect on promoting the repair of chondrocytes is limited. Therefore, how to improve the synergistic effect of the two materials and prepare a joint injection of composite collagen and chitosan that is closer in composition to the natural joint fluid matrix and has a stronger repair effect is currently in urgent clinical need. Summary of the invention

[0005] To solve the above problems, the first aspect of the present application provides a collagen composite material for repairing joint injuries, wherein the collagen composite material includes collagen and chitosan, wherein the chitosan is water-soluble chitosan treated by a combination of one or more of carboxylation, hydroxylation, phosphate and quaternary ammonium salt, and the weight ratio of the collagen to the water-soluble chitosan is 1:15-1:3.

[0006] The present application uses a scientific ratio of water-soluble chitosan and collagen that have been treated with one or more of the following: carboxylation, hydroxylation, phosphate, and quaternary ammonium salts to form a collagen composite material that is closer in composition to natural joint fluid. The mucosal adhesion properties of water-soluble chitosan can self-drive the composite material to aggregate on the injured cartilage wound surface. Water-soluble chitosan provides physical barriers to inflammatory factors and lubrication between cartilages. Collagen has a porous structure that is more conducive to the adhesion of chondrocytes and the acquisition of nutrients, thereby promoting the proliferation of chondrocytes and achieving the repair of damaged cartilage. The synergistic effect of the two can promote the cartilage Cell proliferation and repair of damaged cartilage; and the introduced carboxyl and hydroxyl groups can also form hydrogen bonds between molecules, further improving the performance stability of the collagen composite scaffold material; in addition, the collagen molecular structure is stable, and the degradation rate in the body is slow, and the chondrocytes attached to it can also get more sufficient proliferation and repair time, further accelerating the repair of articular cartilage, slowing down the continuous damage of joints, and effectively preventing and treating traumatic or degenerative osteoarthritis, and has the advantages of good water solubility, good biocompatibility, fast onset, good repair effect, good stability, good re-dissolution stability, long shelf life, and easy storage.

[0007] In some embodiments, the deacetylation degree of the water-soluble chitosan is 70%-90%. Generally, the deacetylation degree of the water-soluble chitosan is positively correlated with the stability of its chemical properties. The deacetylation degree of the water-soluble chitosan in this application is limited to 70%-90% to ensure its solubility in water in practical applications and facilitate the purchase and qualitative analysis of raw materials.

[0008] In some embodiments, the weight average molecular weight of the water-soluble chitosan is 300 kDa-600 kDa. By limiting the weight average molecular weight of the water-soluble chitosan, the degree of polymerization of the water-soluble chitosan molecules is controlled to ensure its water solubility.

[0009] In some embodiments, the water-soluble chitosan is any one of methyl glycol chitosan, carboxymethyl chitosan, hydroxyethyl chitosan, hydroxypropyl chitosan, hydroxybutyl chitosan, trimethyl carboxymethyl chitosan, polyethylene glycol chitosan or hydroxypropyl trimethyl ammonium chloride chitosan quaternary ammonium salt, or a combination of two or more thereof. The water-soluble chitosan has good water solubility, which improves the biocompatibility of the collagen composite material with the natural joint fluid matrix.

[0010] In some embodiments, the collagen is a fibrous collagen, and the fibrous collagen comprises a folded type of any one of α1, α2 or α3 or a combination of two or more thereof. The use of a folded type of fibrous collagen comprising any one of α1, α2 or α3 or a combination of two or more thereof enhances the mechanical properties of collagen, slows down its degradation rate in the body, and provides a more stable matrix environment and sufficient nutrition for the growth of chondrocytes, thereby achieving the effect of lubrication and prevention and treatment of traumatic or degenerative osteoarthritis.

[0011] In some embodiments, the fibrous collagen is one or a combination of two or more of type I, type II, type III, type V, type IX, type XI and type XXVII collagen. Type I, type II, type III, type V, type IX, type XI and type XXVII collagen all contain unique hierarchical structures, and collagen molecules are assembled into long fibrils with high tensile strength in a parallel and staggered manner, which greatly enhances the mechanical strength of collagen, provides a sufficiently stable structural framework for the growth and repair of chondrocytes in the damaged cartilage wound, and promotes the repair of articular cartilage.

[0012] In some embodiments, the collagen is one or a combination of two or more animal collagens selected from bovine collagen, porcine collagen, fish collagen, sheep collagen, horse collagen, chicken collagen, duck collagen, goose collagen, mouse collagen, rabbit collagen and crocodile collagen, or recombinant human collagen, or a combination of the animal collagen and the recombinant human collagen. The collagen composite material is compatible with a variety of animal collagens, which is convenient for controlling costs.

[0013] In some embodiments, the animal collagen is collagen taken from the Achilles tendon, cartilage, cartilage, tail, tendon, ligament or skin of animals. By selecting collagen that is originally attached to the Achilles tendon, cartilage, cartilage, tail, tendon or ligament near the bones of the animal body, the biocompatibility of animal collagen and chondrocytes is guaranteed; supplemented by collagen extracted from the skin, the yield is high and the supply is sufficient.

[0014] In some embodiments, the fibrous collagen is a collagen modified by intracellular translation to contain specific groups and / or three-dimensional structures. By performing translation modification on collagen, the target groups are modified at fixed positions of collagen to enhance the interaction between collagen molecules, and / or, the three-dimensional structure within the molecule is specifically designed to enhance the structural stability of collagen in the body and the adhesion to chondrocytes, thereby providing a favorable matrix environment for chondrocyte healing.

[0015] In some embodiments, the collagen composite material is in the form of a lyophilized agent, and the collagen composite material further includes an auxiliary material consisting of one or a combination of two or more of a buffer salt, an osmotic pressure regulator, a lyophilization protectant, and a pH regulator. By adding auxiliary materials, a collagen composite material with similar physical and chemical properties to the natural joint fluid matrix is ​​formulated, further reducing the body's rejection reaction to the collagen composite material; the use of a lyophilized agent can reduce transportation costs, and the user can prepare a medicine for injection by adding an appropriate amount of water or saline according to the instructions.

[0016] The second aspect of the present invention provides a method for preparing the above-mentioned lyophilized agent, the preparation comprising the following steps:

[0017] S1. Weigh the required amount of collagen, water-soluble chitosan and excipients;

[0018] S2. adding a predetermined proportion of water to fully dissolve the collagen, the water-soluble chitosan and the excipients, adjusting the pH and osmotic pressure to obtain a solution;

[0019] S3. The solution is packaged, freeze-dried and sterilized by irradiation to obtain the collagen composite material in the form of a freeze-dried agent.

[0020] The preparation method is simple to operate and is convenient for automated production.

[0021] The third aspect of the present invention provides an application of any of the above-mentioned collagen composite materials in lubricating joints, anti-inflammation of joints, delaying cartilage aging and repairing cartilage. The collagen composite material scientifically mixes water-soluble chitosan and collagen to form a collagen composite material that is closer to natural joint fluid in composition. The mucosal adhesion property of water-soluble chitosan can self-drive the composite material to aggregate on the injured cartilage wound surface. Through the synergistic effect of water-soluble chitosan and collagen, water-soluble chitosan provides physical barrier to inflammatory factors and lubrication between cartilages. Collagen has a porous structure, which is more conducive to the adhesion of chondrocytes and obtaining nutrients, promotes chondrocyte proliferation and realizes the repair of damaged cartilage. In addition, the molecular structure of collagen is stable, and the chondrocytes attached thereto can get more sufficient proliferation and repair time, further accelerate the repair of articular cartilage, slow down the continuous damage of joints, effectively prevent and treat traumatic or degenerative osteoarthritis, and has the advantages of good water solubility, good biocompatibility, fast onset, good repair effect, good stability, good re-dissolution stability, long validity period, and easy storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the description of the embodiments of the present application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0023] Figure 1-Figure 5 Electron microscope scanning was performed on Examples 1, 4, 7 and 10 and Comparative Example 1 in the present application to obtain electron microscope photographs.

[0024] Figure 6 This is a bar chart comparing the results of D3 (the third day) and D7 (the seventh day) of the chondrocyte proliferation experiment in Examples 1-12 and Comparative Examples 1-4 of the present application. DETAILED DESCRIPTION

[0025] The technical solutions in this application will be described clearly and completely below in conjunction with specific implementation methods. Obviously, the implementation methods and embodiments described are only implementation methods and embodiments of a part of this specification, rather than all implementation methods and embodiments. Based on the implementation methods and embodiments in this specification, all other implementation methods and embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention. The terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions.

[0026] The term "and / or" herein is only a description of the association relationship of the associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the term "at least one" herein represents any combination of at least two of any one or more of a plurality of. For example, including at least one of A, B, and C can represent including any one or more elements selected from the set consisting of A, B, and C.

[0027] In addition, in order to better illustrate the present disclosure, numerous specific details are given in the following specific embodiments. It should be understood by those skilled in the art that the present disclosure can also be implemented without certain specific details. In some examples, methods and means well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present disclosure.

[0028] The first aspect of the present application provides a collagen composite material with joint repair efficacy, wherein the collagen composite material comprises collagen and chitosan, wherein the chitosan is a water-soluble chitosan that has been treated with one or a combination of two or more of carboxylation, hydroxylation, phosphate and quaternary ammonium salts, and the weight ratio of the collagen to the water-soluble chitosan is 1:15-1:3.

[0029] The present application adopts a scientific ratio of water-soluble chitosan and collagen to form a collagen composite material whose composition is closer to natural joint fluid, wherein the mucosal adhesion property of water-soluble chitosan can self-drive to guide the collagen composite material to aggregate to the injured cartilage wound surface, and then through the synergistic effect of collagen and water-soluble chitosan: water-soluble chitosan provides physical barrier to inflammatory factors and lubrication between cartilage, collagen has a porous structure, which is more conducive to the adhesion of chondrocytes and obtain nutrition, promotes chondrocyte proliferation and realizes the repair of damaged cartilage, and the molecular structure of collagen is stable, and the chondrocytes attached thereto can get more sufficient proliferation and repair time, further accelerate the repair of articular cartilage, slow down the continuous damage of joints, effectively prevent and treat traumatic or degenerative osteoarthritis, and has the advantages of good water solubility, good biocompatibility, fast onset, good repair effect, good stability, good re-dissolution stability, long validity period, and easy storage.

[0030] In the specific implementation process, the water-soluble chitosan is carboxylated or hydroxylated through an oxidation reaction. The introduced carboxyl and hydroxyl groups are hydrophilic and can form hydrogen bonds between water-soluble chitosan molecules, thereby improving the water solubility and enhancing the performance stability of its water-based solution; or the water-soluble chitosan is activated and then phosphated or quaternized. The quaternized water-soluble chitosan can be hydroxypropyltrimethylammonium chloride water-soluble chitosan quaternary ammonium salt; the phosphorylated water-soluble chitosan usually couples the phosphorylcholine group to the water-soluble chitosan skeleton.

[0031] In some embodiments, the weight ratio of collagen to water-soluble chitosan in the collagen composite material of the present application is 1:15-1:3. In the specific implementation process, collagen is selected from one or more animal collagens in bovine collagen, porcine collagen, fish collagen, sheep collagen, horse collagen, chicken collagen, duck collagen, goose collagen, mouse collagen, rabbit collagen and crocodile collagen, or recombinant human collagen, or a combination of animal collagen and recombinant human collagen. This collagen composite material can be selected from a variety of animal collagens and recombinant human collagens, which is convenient for cost control. Specifically, the source of animal collagen can also be limited, such as: collagen close to bones such as Achilles tendons, cartilage, cartilage, tail, tendons or ligaments attached to the animal body, to ensure the biocompatibility of animal collagen with chondrocytes; collagen extracted from the skin can also be selected, which has high yield, mature technology, sufficient supply and guaranteed yield.

[0032] In some embodiments, the water-soluble chitosan is enhanced in water solubility according to the degree of deacetylation, the degree of substitution of hydrophilic groups such as hydroxyl groups, the control of weight-average molecular weight, or the control of viscosity of the water-soluble chitosan, thereby ensuring the compatibility of the collagen composite material with the natural synovial fluid matrix, and at the same time increasing the content of the water-soluble chitosan in the collagen composite material to a certain extent, providing long-lasting lubrication and enhancing efficacy. Among them, the deacetylation degree can intuitively evaluate the water solubility of chitosan, and chitosan with a deacetylation degree of more than 70% can have good solubility and biocompatibility at the same time, and is more suitable for intervening in biological organisms for joint injury repair; and the substitution degree of hydrophilic groups such as hydroxyl groups is greater than or equal to 5%, which can effectively improve the water solubility of chitosan to exert a more lasting lubrication and inflammation relief effect; in addition, the viscosity of the water-soluble chitosan can also be controlled to 6mPa.s-12mPa.s to improve the biocompatibility of the present collagen composite material. The higher the viscosity of the water-soluble chitosan, the worse its fluidity. Limiting the viscosity of the water-soluble chitosan can ensure the compatibility of the present collagen composite material with the natural joint fluid matrix.

[0033] In some embodiments, the collagen is a fibrous collagen, and the fibrous collagen comprises any one of α1, α2 or α3 or a combination of two or more folded types. α1, α2 or α3 folded collagens all have a fibrous structure; in actual implementation, one or more combinations of existing type I, type II, type III, type V, type IX, type XI and type XXVII collagens can be used. All of the above collagens contain a unique hierarchical structure, and the collagen molecules are assembled into long fibrils with high tensile strength in a parallel and staggered manner, which can achieve the same enhanced mechanical properties of collagen, slow down the degradation rate of collagen in the body, and provide a sufficiently stable structural framework and sufficient nutrition for the growth and repair of chondrocytes in the damaged cartilage wound surface, thereby promoting the repair of articular cartilage and preventing and treating traumatic or degenerative osteoarthritis; among them, type II, type IX, and type XI collagens are cartilage matrix collagens, which are naturally close to the matrix environment at the body's joints and are more suitable for the treatment of articular cartilage repair.

[0034] In the specific implementation process, in order to further enhance the structural stability of collagen molecules, collagen containing specific groups or three-dimensional structures can also be modified by intracellular translation. The translation modification includes glycosylation modification of hydroxylysine in collagen, disulfide bond modification in propeptide extension, peptide segment interception or replacement, domain interception or replacement, and / or deletion or replacement of subunits, wherein peptide segment interception or replacement includes isomerization of peptide bond-proline bond and hydroxylation of lysine-proline residues. By modifying the target group at the fixed position of collagen to enhance the interaction force between collagen molecules, the three-dimensional structure of collagen molecules is designed in a targeted manner, and / or the advanced structural segments are intercepted. On the one hand, enhancing the interaction force between collagen molecules can improve the structural stability of collagen in the body; on the other hand, the three-dimensional stereo structure of collagen molecular structure can be more conducive to the attachment and growth of chondrocytes, and provide a favorable matrix environment for chondrocyte healing; on the third hand, the targeted interception of complex advanced structural segments can enhance the water solubility of collagen and improve the biocompatibility of collagen.

[0035] Among them, taking type I collagen as an example, glycosylation modification can regulate the biological activity of collagen, enhance its interaction with chondrocytes, improve biocompatibility, and promote chondrocyte adhesion and proliferation. Disulfide bond modification in propeptide extension can be used to connect two cysteine ​​residues in different peptide chains or the same peptide chain, thereby enhancing the three-dimensional structural stability of collagen and maintaining the helical structure and tensile resistance of collagen. Hydroxylation of lysine-proline residues can form hydrogen bonds between collagen molecules and enhance the structural stability of collagen in the body. The deletion or replacement of subunits aims to cut and delete part of the protein subunit composition on the collagen molecule, reduce the molecular weight, and enhance the water solubility of collagen. The isomerization of peptide-proline bonds usually refers to cis-forming the peptide bonds and using the cis configuration to stabilize specific structures of proteins, such as the polyproline type II helix. Furthermore, taking the construction of triple-helix collagen as an example, glycine is combined with any other two amino acids to form a Gly-xy active site, where x and y are usually selected from proline and hydroxyproline. The triple-helix structure of the collagen molecule can be formed, which slows down the degradation rate and maintains the continuous repair of chondrocytes.

[0036] In some embodiments, the collagen composite material is made into a lyophilized agent, and an auxiliary material including one or a combination of two or more of buffer salts, osmotic pressure regulators, lyophilization protectants, and pH regulators is added. By adding auxiliary materials consisting of one or more of buffer salts, osmotic pressure regulators, lyophilization protectants, and pH regulators, a collagen composite material with physical and chemical properties similar to those of natural joint fluid matrix is ​​formulated, further reducing the body's rejection reaction to the collagen composite material; in addition, the use of a lyophilized agent can reduce transportation costs, and the user can add an appropriate amount of water or saline according to the instructions to formulate a medicine for injection, or make it into a gel or solution, which are also common types of medicines. In the specific implementation process, taking the preparation of the collagen composite material into a freeze-dried preparation as an example, the content of the freeze-dried protective agent is 1%-25%, which can be selected from any one of sorbitol, mannitol, glycerol, dextran, sucrose, trehalose, glucose, lactose, maltose, dextran, tricaprylin, polyethylene glycol, and ethylene glycol or a combination thereof; the content of the buffer salt is 15%-60%, which can be selected from any one of sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium chloride, and calcium chloride or a combination thereof.

[0037] The second aspect of the present invention provides a method for preparing the above-mentioned collagen composite material lyophilized agent, and the preparation comprises the following steps: weighing the required amount of collagen, water-soluble chitosan and auxiliary materials; adding a predetermined proportion of water to fully dissolve the collagen, the water-soluble chitosan and the auxiliary materials, adjusting the pH and osmotic pressure to obtain a solution, packaging, lyophilizing, and irradiating to obtain.

[0038] The preparation method is simple to operate and is convenient for automated production.

[0039] In the specific implementation process, the dissolution of collagen and water-soluble chitosan in distilled water can be carried out by stirring, shearing, high-pressure homogenization, ultrasound or any combination thereof; wherein the stirring can be magnetic stirring, stirring at a stirring speed of 100rpm-3000rpm for 1h-6h, preferably stirring at 150rpm-2000rpm for 1h-4h. And taking the preparation of the present collagen composite material into a lyophilized agent as an example, after mixing, it should also include the steps of standing, filtering the clarified solution, and freeze-drying the clarified solution. wherein the sterilization method is filtration sterilization or irradiation sterilization, preferably any one of ultraviolet sterilization, infrared sterilization, and gamma ray; the standing time is 0.5h-2.5h, preferably 1h-2h.

[0040] The third aspect of the present invention provides an application of the above collagen composite material in lubricating joints, resisting joint inflammation, delaying cartilage aging and repairing cartilage. The present application forms a collagen composite material whose composition is closer to natural joint fluid by scientifically proportioning water-soluble chitosan and collagen. The mucosal adhesion property of water-soluble chitosan can self-drive to guide the collagen composite scaffold material to aggregate to the injured cartilage wound surface. Through the synergistic effect of water-soluble chitosan and collagen, water-soluble chitosan provides physical barrier to inflammatory factors and lubrication between cartilages. Collagen has a porous structure, which is more conducive to the adhesion of chondrocytes and acquisition of nutrients, promotes chondrocyte proliferation and realizes the repair of damaged cartilage. In addition, the molecular structure of collagen is stable, and the chondrocytes attached thereto can get more sufficient proliferation and repair time, further accelerate the repair of articular cartilage, slow down the continuous damage of joints, effectively prevent and treat traumatic or degenerative osteoarthritis, and has the advantages of good water solubility, good biocompatibility, fast onset, good repair effect, good stability, good re-dissolution stability, long shelf life, and easy storage.

[0041] The collagen composite material in the present application is used to prevent and treat any one of traumatic or degenerative osteoarthritis or its complications; specifically, joint repair or cartilage repair is selected from any one of or a combination of refractory wounds, knee osteoarthritis, synovitis, cartilage damage, and bursitis; the collagen composite material in the present application is used for any one of or a combination of people who need to maintain their bones and joints, people with degenerative diseases in the middle-aged and elderly, people with sports injuries, people who need to maintain their sports, and people who need to recover after surgery for bone and joint diseases.

[0042] During the specific implementation process, taking the lyophilized agent in this application as an example, the method of use is: dissolve the lyophilized agent in water for injection or physiological saline to prepare an injection solution with a concentration of 1 mg / mL-80 mg / mL, preferably 8 mg / mL-30 mg / mL, and inject it into the knee joint cavity.

[0043] The technical solution of the present application is described below in conjunction with Examples 1-15 and Comparative Examples 1-12:

[0044] Embodiment 1-3:

[0045] Example 1-3 provides a lyophilized preparation of a composite material of type I recombinant collagen and hydroxyethyl chitosan, the composition of which is:

[0046]

[0047]

[0048] The preparation of the lyophilized agent of Example 1-3 comprises the following steps:

[0049] According to the concentration of each component in the ingredient list, 15 mL of solution was prepared, three portions of 30 mg of type I recombinant collagen were accurately weighed in order, and 90 mg / 180 mg / 360 mg of hydroxyethyl chitosan, 150 mg / 300 mg / 600 mg of Na2HPO4, and 3.75 mg / 7.5 mg / 15 mg of trehalose were added respectively, and the formula of each example was fully dissolved in 15 mL of distilled water by magnetic stirring, the stirring speed was 200 rpm-600 rpm, the pH was adjusted to about 7.0, the osmotic pressure was adjusted to about 300 mOSM / L, and the solution was packaged, freeze-dried, and irradiated for sterilization.

[0050] Embodiment 4-6:

[0051] Example 4-6 provides a lyophilized preparation containing a collagen composite material, which is a combination of type I and type III recombinant collagen with hydroxyethyl chitosan and hydroxypropyl chitosan, and the composition thereof is:

[0052] Example 4 Example 5 Example 6 Components Concentration (mg / mL) Concentration (mg / mL) Concentration (mg / mL) Recombinant collagen type I 1.0 1.0 1.0 Recombinant collagen type III 1.0 1.0 1.0 Hydroxyethyl chitosan 3.0 6.0 12.0 Hydroxypropyl Chitosan 3.0 6.0 12.0 NaCl 1.67 1.74 1.81 <![CDATA[Na2HPO4]]> 4 8 16 Trehalose 0.25 0.5 1

[0053] The preparation of the lyophilized agent of Example 4-6 comprises the following steps:

[0054] According to the concentration of each component in the ingredient list, 15 mL of solution was prepared, and three portions of 15 mg type I recombinant collagen, three portions of 15 mg type III recombinant collagen, 45 mg / 90 mg / 180 mg hydroxyethyl chitosan, 45 mg / 90 mg / 180 mg hydroxypropyl chitosan, 25 mg / 26.1 mg / 27.12 mg NaCl, 60 mg / 120 mg / 240 mg Na2HPO4, and 3.75 mg / 7.5 mg / 15 mg trehalose were accurately weighed in order, and the formula of each embodiment was fully dissolved in 15 mL of pure water by magnetic stirring, the stirring speed was 200 rpm-600 rpm, the pH was adjusted to about 7.0, the osmotic pressure was adjusted to about 300 mOSM / L, and the solution was packaged, freeze-dried, and irradiated for sterilization.

[0055] Embodiment 7-9

[0056] Example 7-9 provides a lyophilized preparation of a composite material of type I recombinant collagen, hydroxyethyl chitosan and hydroxypropyl chitosan, the composition of which is:

[0057] Example 7 Example 8 Example 9 Components Concentration (mg / mL) Concentration (mg / mL) Concentration (mg / mL) Recombinant collagen type I 2.0 2.0 2.0 Hydroxyethyl chitosan 3.0 6.0 12.0 Hydroxypropyl Chitosan 3.0 6.0 12.0 NaCl 1.385 1.57 1.78 <![CDATA[Na2HPO4]]> 4 8 16 Trehalose 0.25 0.5 1.0

[0058] The preparation of the lyophilized agent of Example 7-9 comprises the following steps:

[0059] According to the concentration of each component in the ingredient list, 15 mL of solution was prepared, and three portions of 30 mg type I recombinant collagen, 45 mg / 90 mg / 180 mg hydroxyethyl chitosan, 45 mg / 90 mg / 180 mg hydroxypropyl chitosan, 20.8 mg / 23.6 mg / 26.7 mg NaCl, 60 mg / 120 mg / 240 mg Na2HPO4, and 3.75 mg / 7.5 mg / 15 mg trehalose were accurately weighed in order, and the formula of each embodiment was fully dissolved in 15 mL of pure water by magnetic stirring, the stirring speed was 200 rpm-600 rpm, the pH was adjusted to about 7.0, the osmotic pressure was adjusted to about 300 mOSM / L, and the solution was packaged, freeze-dried, and irradiated for sterilization.

[0060] Examples 10-12

[0061] Example 10-12 provides a lyophilized preparation of a composite material of type III recombinant collagen, hydroxyethyl chitosan and hydroxypropyl chitosan, the composition of which is:

[0062] Example 10 Embodiment 11 Example 12 Components Concentration (mg / mL) Concentration (mg / mL) Concentration (mg / mL) Recombinant collagen type Ⅲ 2.0 2.0 2.0 Hydroxyethyl chitosan 3.0 6.0 12.0 Hydroxypropyl Chitosan 3.0 6.0 12.0 NaCl 1.35 1.71 2.17 <![CDATA[Na2HPO4]]> 4 8 16 Trehalose 0.25 0.5 1.0

[0063] The preparation of the lyophilized agent of Example 10-12 comprises the following steps:

[0064] According to the concentration of each component in the ingredient list, 15 mL of solution was prepared, and three portions of 30 mg type III recombinant collagen, 45 mg / 90 mg / 180 mg hydroxyethyl chitosan, 45 mg / 90 mg / 180 mg hydroxypropyl chitosan, 20 mg / 25.7 mg / 32.55 mg NaCl, 60 mg / 120 mg / 240 mg Na2HPO4, and 3.75 mg / 7.5 mg / 15 mg trehalose were accurately weighed in order, and the formula of each embodiment was fully dissolved in 15 mL of pure water by magnetic stirring, the stirring speed was 200 rpm-600 rpm, the pH was adjusted to about 7.0, the osmotic pressure was adjusted to about 300 mOSM / L, and the solution was packaged, freeze-dried, and irradiated for sterilization.

[0065] Comparative Example 1:

[0066] Comparative Example 1 provides a lyophilized agent containing only two chitosans, which is composed of:

[0067] Components Concentration (mg / mL) Hydroxyethyl chitosan 6 Hydroxypropyl Chitosan 6 NaCl 1.06 <![CDATA[Na2HPO4]]> 8 Trehalose 0.5

[0068] The preparation of the lyophilized agent of Comparative Example 1 comprises the following steps:

[0069] According to the concentration of each component in the ingredient list, 15 mL of solution was prepared, 90 mg of hydroxyethyl chitosan, 90 mg of hydroxypropyl chitosan, 16 mg of NaCl, 120 mg of Na2HPO4, and 7.5 mg of trehalose were accurately weighed in order, and magnetic stirring was performed to fully dissolve them in 15 mL of pure water. The stirring speed was 200 rpm-600 rpm, the pH was adjusted to about 7.0, the osmotic pressure was adjusted to about 300 mOSM / L, and the solution was packaged, freeze-dried, and irradiated for sterilization.

[0070] Comparative Example 2:

[0071] Comparative Example 2 provides a lyophilized agent containing only medical chitosan, wherein the medical chitosan raw material used is commercially available medical chitosan, and the composition of the lyophilized agent is:

[0072] Components Concentration (mg / mL) Medical Chitosan 12 NaCl 1.06 <![CDATA[Na2HPO4]]> 8 Trehalose 0.5

[0073] The preparation of the lyophilized agent of Comparative Example 2 comprises the following steps:

[0074] According to the concentration of each component in the ingredient list, 15 mL of solution is prepared, 180 mg of medical chitosan, 16 mg of NaCl, 120 mg of Na2HPO4, and 7.5 mg of trehalose are accurately weighed in order, and magnetic stirring is used to fully dissolve them in 15 mL of pure water. The stirring speed is 200 rpm-600 rpm, the pH is adjusted to about 7.0, the osmotic pressure is adjusted to about 300 mOSM / L, and the solution is packaged, freeze-dried, and irradiated for sterilization.

[0075] Comparative Example 3

[0076] Comparative Example 3 provides a lyophilized agent containing only type I recombinant collagen, the composition of the lyophilized agent is:

[0077] Components Concentration (mg / mL) Recombinant collagen type Ⅰ 2 NaCl 0.25 <![CDATA[Na2HPO4]]> 2 Trehalose 0.125

[0078] The preparation of the lyophilized agent of Comparative Example 3 comprises the following steps:

[0079] According to the concentration of each component in the ingredient list, 15 mL of solution was prepared, 30 mg of type I recombinant collagen, 3.75 mg of NaCl, 30 mg of Na2HPO4, and 1.88 mg of trehalose were accurately weighed in order, and fully dissolved in 15 mL of pure water by magnetic stirring at a stirring speed of 200 rpm-600 rpm. The pH was adjusted to about 7.0, and the osmotic pressure was adjusted to about 300 mOSM / L. The solution was packaged, freeze-dried, and irradiated for sterilization.

[0080] Comparative Example 4

[0081] Comparative Example 4 provides a lyophilized agent containing only type III recombinant collagen, wherein the composition of the lyophilized agent is:

[0082] Components Concentration (mg / mL) Recombinant collagen type Ⅲ 2 NaCl 0.25 <![CDATA[Na2HPO4]]> 2 Trehalose 0.125

[0083] The preparation of the lyophilized agent of Comparative Example 4 comprises the following steps:

[0084] According to the concentration of each component in the ingredient list, 15 mL of solution was prepared, 30 mg of type III recombinant collagen, 3.75 mg of NaCl, 30 mg of Na2HPO4, and 1.88 mg of trehalose were accurately weighed in order, and fully dissolved in 15 mL of pure water by magnetic stirring at a stirring speed of 200 rpm-600 rpm. The pH was adjusted to about 7.0, and the osmotic pressure was adjusted to about 300 mOSM / L. The solution was packaged, freeze-dried, and irradiated for sterilization.

[0085] Test Example 1

[0086] The above-mentioned Examples 1, 4, 7 and 10 and Comparative Example 1 were subjected to electron microscope scanning, and the following results were obtained: Figure 1-5 The electron microscope photograph shown is by Figure 1-5 It can be seen that the lyophilized agent provided in the present application has a unique three-dimensional structure and therefore has higher mechanical properties, which helps to provide a more stable extracellular matrix environment for cartilage tissue.

[0087] Test Example 2

[0088] The above Examples 1-12 and Comparative Examples 1-4 were all applied to chondrocyte proliferation experiments:

[0089] 1. Experimental reagents

[0090] DMEM (Dulbecco's Modified Eagle Medium) basal medium and complete medium, mouse collagenase type IV (Solarbio, catalog number: C8140), PBS buffered saline, CCK-8 solution, 75% alcohol

[0091] 2. Mouse chondrocyte extraction and culture experiment

[0092] S1: 8-week-old C57 mice were killed by cervical dislocation and immersed in a beaker containing 75% alcohol for 5 min;

[0093] S2: The leg bones of mice were removed and placed in a culture dish containing 75% alcohol for 2 minutes, and then placed in a culture dish containing PBS. After that, the hyaline cartilage at the knee joint was separated in a 15 mL centrifuge tube using sterilized surgical scissors in a clean bench, and the cartilage was rinsed twice with an appropriate amount of PBS.

[0094] S3. Cut the washed cartilage into small pieces with sterilized surgical scissors, place them in a six-well plate, add 0.2% mouse collagenase type IV solution (10 mg type IV collagenase dissolved in 5 mL DMEM basal medium) until the cartilage pieces are just submerged, and incubate in a cell culture incubator for 1 hour. During this period, take out the six-well plate every 15 minutes or so and blow it several times with a pipette to allow the collagenase solution to fully contact the cartilage pieces, which is conducive to digestion into single cells;

[0095] S4. Add about 6 mL of DMEM complete medium to terminate digestion, pipette and filter with a 200-mesh filter, collect the filtrate in a 50 mL centrifuge tube, put it in a centrifuge, adjust the speed to 1000 rpm, and centrifuge for 5 min. Discard the supernatant, add 6 mL of DMEM complete medium to resuspend, put it in a centrifuge again, adjust the speed to 500 rpm, centrifuge for 5 min, collect the supernatant and culture it in a six-well plate at 2 mL / well, put it in a cell culture incubator and culture it for 24 h, and then change the medium;

[0096] S5. Collect the cartilage blocks remaining on the filter and the precipitate centrifuged in step S4, place them in a six-well plate, add the type IV collagenase solution again to immerse the cartilage blocks, and continue to incubate in the incubator for 2 hours. During this period, blow several times every 15 minutes or so to ensure that the cartilage blocks are fully in contact with the collagenase solution;

[0097] S6. Add 6 mL of DMEM complete medium to terminate digestion, filter again, collect the filtrate in a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 6 mL of DMEM complete medium to resuspend, put it in the centrifuge again, adjust the speed to 500 rpm, centrifuge for 5 min, collect the supernatant and culture it in a six-well plate at 2 mL / well, resuspend the remaining precipitate in 2 mL of DMEM complete medium, culture it in a six-well plate, put it in a cell culture flask and culture it for 24 h before changing the medium;

[0098] S7. Observe the cell growth under a microscope. When the cell growth area reaches 80%-90% of the bottom area of ​​the cell culture flask, inoculate the cells into a 96-well plate.

[0099] 3. Experimental study on the proliferation of chondrocytes by the freeze-dried agent of the present application

[0100] S1. The chondrocytes grown in the above six-well plate were plated in a 96-well plate, with about 5,000-10,000 cells per well, 100 ul of DMEM complete medium per well, and placed in a cell culture incubator overnight;

[0101] S2. The next day, add 90 μl of DMEM complete medium containing lyophilized agent to each well and incubate in the incubator for 48 hours. The preparation method of DMEM complete medium containing lyophilized agent is as follows:

[0102] Example 1-12: Take the lyophilized agents prepared in the above examples 1-12 respectively, add DMEM complete medium to dissolve, and prepare a solution to restore the content of each component in each example;

[0103] Comparative Examples 1-4: The lyophilized agents prepared in the above Comparative Examples 1-4 were respectively taken, and DMEM complete medium was added to dissolve to prepare solutions, and the contents of the components in each comparative example were restored;

[0104] S3. After the incubation is completed, add 10 μl of CCK-8 solution to each well and incubate in the incubator for about 4 h;

[0105] S4. Detect the absorbance at 450 nm using an enzyme reader.

[0106] Cell proliferation rate (%) = absorbance value of test group / absorbance value of blank group×100%, the results are shown in Table 1. The collagen composite material of the present invention can achieve the proliferation of chondrocytes.

[0107] Table 1

[0108]

[0109]

[0110] Summary: See Figure 6, from Examples 1-12, the cell proliferation rate is positively correlated with the addition ratio of collagen; from Example 1 and Example 7, it can be seen that, while keeping the ratio of the total amount of chitosan to collagen unchanged, the compounding of multiple chitosans can enhance the cell proliferation promoting effect of the composite material; by comparing Example 7 with Example 10, it can be seen that, while keeping the chitosan ratio unchanged, replacing type III collagen can achieve a better cell proliferation promoting effect, indicating that type III recombinant collagen has a better repair effect; by comparing Example 7 with Example 4, while keeping the ratio of the total amount of chitosan to the total amount of collagen unchanged, the compounding of multiple chitosans and multiple collagens can enhance the cell proliferation promoting effect of the composite material. The composite material in Example 4, which uses type I and type III recombinant collagen and hydroxyethyl chitosan and hydroxypropyl chitosan in a ratio of 1:1:3:3, has the best cell proliferation promoting effect, and the cell proliferation rate on the third day is as high as 123.61%, proving that its high efficacy can be maintained for more than three days, and the efficacy is long-lasting. And. Correspondingly, in Comparative Example 1 which only uses two types of chitosan and Comparative Example 2 which only uses medical chitosan, and also adds chitosan at the same concentration as in Example 2, it can be determined that there is no effect of promoting cell proliferation at all; and in Comparative Examples 3 and 4 which do not add chitosan at all and only use 2 mg / mL of collagen, the effect of promoting cell proliferation is also poor, indicating that the chitosan and collagen compound provided in Example 1 of the present application has better repair effect than using chitosan or collagen alone, which indirectly confirms the synergistic effect of collagen and chitosan in repairing damaged joints.

[0111] In summary, see Figure 5 At the microscopic level, the freeze-dried preparation using only one or more chitosans is flatter in structure and weaker in mechanical properties. It is difficult to act on the damaged joints for a long time and continue to maintain the structural framework that assists the growth and repair of chondrocytes on the damaged cartilage wound surface. Figure 6 This also explains why the cell proliferation promoting effect of comparative example 1-2 is close to zero; see Figure 1-Figure 4 The embodiment in which the corresponding collagen is added has an obvious layered structure. The collagen molecules and chitosan molecules are cross-linked and assembled into long fibrils with high tensile strength, which can provide enough stable growth and repair space and sufficient nutrition for the damaged wound surface, thereby promoting the repair of articular cartilage and preventing and treating traumatic or degenerative osteoarthritis.

[0112] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present implementation. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0113] Although examples of the present embodiment have been shown and described, those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present embodiment, and the scope of the present embodiment is defined by the claims and their equivalents.

Claims

1. A collagen composite material for repairing joint damage, characterized in that: The collagen composite scaffold material comprises collagen and chitosan, wherein the chitosan is water-soluble chitosan treated by one or more of carboxylation, hydroxylation, phosphate and quaternary ammonium salt treatments, and the weight ratio of the collagen to the water-soluble chitosan is 1:15-1:

3.

2. The collagen composite material for repairing joint damage according to claim 1, characterized in that: The deacetylation degree of the water-soluble chitosan is 70%-90%.

3. The collagen composite material for repairing joint damage according to claim 1, characterized in that: The weight average molecular weight of the water-soluble chitosan is 300 kDa-600 kDa.

4. The collagen composite material for repairing joint damage according to claim 1, characterized in that: The water-soluble chitosan is any one of methyl glycol chitosan, carboxymethyl chitosan, hydroxyethyl chitosan, hydroxypropyl chitosan, hydroxybutyl chitosan, trimethyl carboxymethyl chitosan, polyethylene glycol chitosan and hydroxypropyl trimethyl ammonium chloride chitosan quaternary ammonium salt, or a combination of two or more thereof.

5. The collagen composite material for repairing joint damage according to claim 1, characterized in that: The collagen is fibrous collagen, and the fibrous collagen includes a folded type of any one of α1, α2, or α3, or a combination of two or more thereof.

6. The collagen composite material for repairing joint damage according to claim 5, characterized in that: The fibrous collagen is one or a combination of two or more of type I, type II, type III, type V, type IX, type XI and type XXVII collagen.

7. The collagen composite material for repairing joint damage according to claim 5, characterized in that: The collagen is one or a combination of two or more animal collagens selected from bovine collagen, porcine collagen, fish collagen, sheep collagen, horse collagen, chicken collagen, duck collagen, goose collagen, rat collagen, rabbit collagen and crocodile collagen, or is recombinant human collagen, or is a combination of the animal collagen and the recombinant human collagen.

8. The collagen composite material for repairing joint damage according to claim 7, characterized in that: The animal collagen is collagen taken from the Achilles tendon, cartilage, cartilage, tail, tendon, ligament or skin of animals.

9. The collagen composite material for repairing joint damage according to claim 5, characterized in that: The fibrous collagen is a collagen modified by intracellular translation to contain specific groups and / or three-dimensional structures.

10. The collagen composite material for repairing joint damage according to any one of claims 1 to 9, characterized in that: The collagen composite material is in the form of a lyophilized agent, and the collagen composite material further comprises auxiliary materials consisting of one or a combination of two or more of a buffer salt, an osmotic pressure regulator, a lyophilization protective agent, and a pH regulator.

11. The method for preparing the collagen composite material for repairing joint damage according to claim 10, characterized in that: The steps include: S1. Weigh the required amount of the collagen, the water-soluble chitosan and the auxiliary materials; S2. adding a predetermined proportion of water to fully dissolve the collagen, the water-soluble chitosan and the excipients, adjusting the pH and osmotic pressure to obtain a solution; S3. The solution is packaged, freeze-dried and sterilized by irradiation to obtain the collagen composite material in the form of a freeze-dried agent.

12. Use of the collagen composite material for joint injury repair according to any one of claims 1 to 10 in lubricating joints, anti-inflammatory joints, delaying cartilage aging and cartilage repair.

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