Collagen composite material for joint injury repair and preparation method and application thereof

Abstract

The application provides a collagen composite material for joint injury repair and a preparation method and application thereof, wherein the collagen composite material comprises collagen and chitosan; the chitosan is water-soluble chitosan which is treated by one or more than two ways of carboxylation, hydroxylation, phosphatization and quaternary ammonium saltization; and the weight ratio of the collagen to the water-soluble chitosan is 1:15-1:3. The application adopts scientific screening of the composition and ratio of the collagen and the water-soluble chitosan, forms the collagen composite material which is more close to natural joint fluid in composition, provides physical blocking inflammatory factors and lubrication for the cartilage gap by the water-soluble chitosan, and the collagen has a porous structure, is more conducive to the adhesion and nutrition acquisition of chondrocytes, promotes chondrocyte proliferation and realizes the repair of damaged cartilage.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, in particular to a collagen composite material for joint injury repair and a preparation method and application thereof. BACKGROUND

[0002] Osteoarthritis is a chronic degenerative joint disease mainly affecting the middle-aged and elderly population, often causing joint pain, stiffness and dysfunction in patients. Among them, the knee joint is the most common site of osteoarthritis. The current treatment plan is usually 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 intra-articular injection and a preparation method thereof, and CN115154413A discloses a gel material for intra-articular 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 that of glycosaminoglycan in the extracellular matrix, which can promote cell-cell adhesion and effectively provide physical barriers between cartilage structures in the joint. It can also serve as a supplement to the synovial fluid matrix and enhance the lubrication between joints, slow down the progression of arthritis and relieve joint pain. However, chitosan has little effect on joint repair and regeneration.

[0004] Collagen is a protein that exists in the skin, bones, tendons, ligaments and other connective tissues of the human body, and has natural biocompatibility. Introducing collagen into intra-articular injection can help achieve joint injury repair. However, inappropriate use of two materials can make it difficult to fully utilize the intermolecular structure crosslinking effect, and the repair effect of chondrocytes is limited. Therefore, how to improve the synergistic effect of the two materials and prepare a composite collagen and chitosan joint injection that is more similar to the natural synovial fluid matrix and has stronger repair effect is currently in great demand in clinical practice. SUMMARY

[0005] To solve the above problems, the first aspect of the present application provides a collagen composite material for joint injury repair, which comprises collagen and chitosan, the chitosan being water-soluble chitosan treated by one or more than two ways of carboxylation, hydroxylation, phosphatization and quaternary ammonium saltization, and the weight ratio of the collagen to the water-soluble chitosan being 1:15-1:3.

[0006] The water-soluble chitosan treated by one or more than two combinations of carboxylation, hydroxylation, phosphatization and quaternary ammonium saltization is scientifically proportioned with collagen to form a collagen composite material which is more close to natural synovial fluid in composition. The mucous membrane adhesion property of the water-soluble chitosan can self-drivenly guide the collagen composite material to gather on the wound surface of the injured cartilage. The water-soluble chitosan provides physical barrier for inflammatory factors and lubrication between cartilages. The collagen has a porous structure, which is more conducive to the adhesion and nutrition acquisition of chondrocytes, promotes the proliferation of chondrocytes and realizes the repair of damaged cartilage. The synergistic effect of the water-soluble chitosan and the collagen can promote the proliferation of chondrocytes and realize the repair of damaged cartilage. 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 has good molecular structure stability and slow degradation rate in the body. The chondrocytes attached thereto can also have more sufficient proliferation and repair time, further accelerating the repair of joint cartilage, slowing down the continuous damage of the joint, effectively preventing and treating traumatic or degenerative osteoarthritis, and having the advantages of good water solubility, good biocompatibility, fast effect, good repair effect, good stability, good reconstitution stability, long effective period, easy to store and the like.

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

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

[0009] In some embodiments, the water-soluble chitosan is any one or more than two combinations 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. The above water-soluble chitosan has good water solubility, which improves the biocompatibility of the collagen composite material with the natural synovial fluid matrix.

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

[0011] In some embodiments, the fibrous collagen is one or more than two combinations 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 comprise a unique layered structure, and collagen molecules are assembled into long fibrils with high tensile strength in a parallel and staggered manner, greatly enhancing the mechanical strength of collagen, providing a sufficient stable growth repair structure framework for chondrocytes of damaged cartilage wounds, and promoting the repair of articular cartilage.

[0012] In some embodiments, the collagen is one or more than two combinations of bovine collagen, porcine collagen, fish collagen, sheep collagen, horse collagen, chicken collagen, duck collagen, goose collagen, mouse collagen, rabbit collagen and alligator collagen, or is recombinant human collagen, or is a combination of the animal collagen and the recombinant human collagen. The collagen composite material is compatible with various animal collagens, facilitating cost control.

[0013] In some embodiments, the animal collagen is collagen extracted from the Achilles tendon, cartilage, brittle bone, tail, tendon, ligament or skin of an animal. By selecting collagen from the Achilles tendon, cartilage, brittle bone, tail, tendon or ligament near the skeleton of an animal, the biocompatibility of the animal collagen with chondrocytes is ensured; supplemented with collagen extracted from the skin, the yield is high and the supply is sufficient.

[0014] In some embodiments, the fibrous collagen is collagen comprising specific groups and / or stereoscopic structures modified by intracellular translation. By modifying collagen through translation, the target group is modified at the designated position of the collagen to enhance the interaction force between collagen molecules, and / or the intramolecular three-dimensional structure is designed to improve the structural stability of collagen in the body and the adhesion of 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 comprises an excipient formed by one or more than two combinations of a buffer salt, an osmotic pressure regulator, a lyophilization protective agent, and a pH regulator. By adding the excipient, a collagen composite material similar in physicochemical properties to a natural joint fluid matrix is prepared, further reducing the body's rejection reaction to the collagen composite material; the use of a lyophilized agent can reduce transportation costs, and users can prepare a medicament for injection by adding an appropriate amount of water or physiological saline according to the instructions.

[0016] The second aspect of the present application provides a preparation method of the above-mentioned lyophilized agent, and the preparation comprises the following steps:

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

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

[0019] S3. performing a dispensing treatment, a lyophilization treatment, and an irradiation sterilization treatment on the solution, thereby obtaining the collagen composite material in the form of a lyophilized agent.

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

[0021] The third aspect of the present application provides the use of the collagen composite material in any of the above-mentioned aspects in lubricating joints, joint anti-inflammation, delaying cartilage aging, and cartilage repair. The collagen composite material is scientifically proportioned with water-soluble chitosan and collagen to form a collagen composite material that is more similar in composition to natural joint fluid. The mucous membrane adhesion property of the water-soluble chitosan can self-actively guide the composite material to gather on the wound surface of the injured cartilage. Through the synergistic effect of the water-soluble chitosan and the collagen, the water-soluble chitosan provides a physical barrier to inflammatory factors and lubrication between cartilages, and the collagen has a porous structure, which is more conducive to the adhesion and nutrition acquisition of chondrocytes, promotes the proliferation of chondrocytes, and realizes the repair of damaged cartilage. In addition, the collagen has good molecular structure stability, the chondrocytes attached thereto can have more sufficient proliferation and repair time, further accelerating the repair of joint cartilage, reducing the continuous damage of joints, effectively preventing and treating traumatic or degenerative osteoarthritis, and having the advantages of good water solubility, good biocompatibility, fast effect, good repair effect, good stability, good redissolution stability, long effective period, and easy storage. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the description of the embodiments of the present application. Obviously, the drawings in the following description only some of the embodiments of the present application, and for those skilled in the art, other drawings can be obtained without creative labor under the premise of these drawings.

[0023] Figures 1-5 The electron micrographs of the electron microscope scanning of the embodiments 1, 4, 7 and 10 in the present application and the comparative example 1 are shown.

[0024] Figure 6 The result comparison column chart of D3 (the third day) and D7 (the seventh day) of the cartilage cell proliferation experiment of the embodiments 1-12 and the comparative examples 1-4 in the present application is shown. DETAILED DESCRIPTION

[0025] The technical solutions in the present application will be described clearly and completely in the following combined with the specific embodiments. Obviously, the described embodiments and examples are only some of the embodiments and examples of the present specification, but not all the embodiments and examples. Based on the embodiments and examples in the present specification, all the other embodiments and examples obtained by those skilled in the art without creative labor are within the scope of the present application. The terms "include" and "have" and any variations thereof are intended to cover the non-exclusive inclusion.

[0026] The term "and / or" in the present document is only to describe the association relationship of the associated objects, which means that there can be three relationships, for example, A and / or B can mean that there are three cases of A alone, A and B together, and B alone. In addition, the term "at least one" in the present document means any one of the plurality or any combination of at least two of the plurality, for example, including at least one of A, B and C can mean 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, a large number of specific details are given in the following specific embodiments. Those skilled in the art should understand that the present disclosure can also be implemented without some specific details. In some examples, the methods and means well known to those skilled in the art are not described in detail, in order to highlight the main idea of the present disclosure.

[0028] The first aspect of the present application provides a collagen composite material with joint repair efficacy, the collagen composite material comprising collagen and chitosan, the chitosan being water-soluble chitosan treated by one or more than two combinations of carboxylation, hydroxylation, phosphatization and quaternary ammonium saltification, and the weight ratio of the collagen to the water-soluble chitosan being 1:15-1:3.

[0029] The present application adopts scientific proportioning of water-soluble chitosan and collagen to form a collagen composite material which is more close to natural joint fluid in composition, wherein the mucous membrane adhesion property of the water-soluble chitosan can self-drivenly guide the collagen composite material to gather to the wound surface of the injured cartilage, and then through the synergistic effect of the collagen and the water-soluble chitosan: the water-soluble chitosan provides physical barrier to inflammatory factors and lubrication between cartilages, the collagen has a porous structure, which is more conducive to the adhesion and nutrition acquisition of chondrocytes, promotes the proliferation of chondrocytes and realizes the repair of damaged cartilage, and the collagen has good molecular structure stability, and the chondrocytes attached thereto can obtain more sufficient proliferation and repair time, further accelerates the repair of joint cartilage, slows down the continuous damage of the joint, effectively prevents and treats traumatic or degenerative osteoarthritis, and has the advantages of good water solubility, good biocompatibility, fast effect, good repair effect, good stability, good reconstitution stability, long effective period, easy storage and the like.

[0030] In the specific implementation process, the water-soluble chitosan is carboxylated or hydroxylated through an oxidation reaction, the introduced carboxyl and hydroxyl groups have hydrophilicity, and can form hydrogen bonds between water-soluble chitosan molecules, thereby improving the water solubility and enhancing the performance stability of the water-based solution; or the water-soluble chitosan is activated and then phosphatized or quaternary ammonium saltified, the water-soluble chitosan quaternary ammonium salt after quaternary ammonium saltification can be hydroxypropyl trimethyl ammonium chloride water-soluble chitosan quaternary ammonium salt; the water-soluble chitosan after phosphatization is usually a water-soluble chitosan skeleton to which a phosphocholine group is coupled.

[0031] In some embodiments, the weight ratio of collagen to water-soluble chitosan in the collagen composite material in the present application is 1:15-1:3. In specific implementations, the collagen is selected from one of the following or a combination of two or more of the following: bovine collagen, porcine collagen, fish collagen, sheep collagen, horse collagen, chicken collagen, duck collagen, goose collagen, mouse collagen, rabbit collagen, and alligator collagen, or is recombinant human collagen, or is a combination of animal collagen and recombinant human collagen. The collagen composite material can be selected from a variety of animal collagens and recombinant human collagens, facilitating cost control. Specifically, the source site of the animal collagen can be limited, such as collagen from the Achilles tendon, cartilage, brittle bone, tail, tendon, or ligament of an animal, which is close to the skeleton, ensuring the biocompatibility of the animal collagen and chondrocytes; or the collagen extracted from the skin, which has high yield, mature technology, sufficient supply, and guaranteed yield.

[0032] In some embodiments, the water-soluble chitosan is controlled in terms of its degree of deacetylation, or the degree of substitution of hydrophilic groups such as hydroxyl groups, or the weight average molecular weight, or the viscosity of the water-soluble chitosan, to enhance the water-soluble chitosan, ensure the compatibility of the collagen composite material with the natural synovial fluid matrix, and to a certain extent, increase the content of water-soluble chitosan in the collagen composite material, provide long-lasting lubrication, and enhance the drug efficacy. The degree of deacetylation can be used to directly evaluate the water-soluble chitosan, and chitosan with a degree of deacetylation of more than 70% can have good solubility and biocompatibility, and is more suitable for joint damage repair in the intervention of biological organisms; the degree of substitution of hydrophilic groups such as hydroxyl groups is greater than or equal to 5%, which can effectively improve the water-soluble chitosan, and provide more long-lasting lubrication and inflammation relief effects; in addition, the viscosity of the water-soluble chitosan can be controlled to be 6 mPa.s-12 mPa.s to improve the biocompatibility of the collagen composite material. The higher the viscosity of the water-soluble chitosan, the worse the flowability, and limiting the viscosity of the water-soluble chitosan can ensure the compatibility of the collagen composite material with the natural synovial fluid matrix.

[0033] In some embodiments, the collagen is fibrous collagen comprising any one or more than two combinations of α1, α2 or α3 folding type. The α1, α2 or α3 folding type collagen all have fibrous structure; and in actual implementation, one or more than two combinations of the existing type I, type II, type III, type V, type IX, type XI and type XXVII collagen can be used, which all comprise unique layered structure, and collagen molecules are assembled into long fibrils with high tensile strength in parallel and staggered manner, which can achieve equivalent mechanical performance of enhanced collagen, delay the degradation rate of collagen in the body, provide sufficient stable growth repair structure framework and sufficient nutrition for chondrocytes in damaged cartilage wound, so as to promote the repair of articular cartilage and prevent and treat traumatic or degenerative osteoarthritis; wherein, the type II, type IX and type XI collagen is cartilage matrix collagen, which is naturally close to the matrix environment of the joint of the body, and is more suitable for the treatment scene 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 stereoscopic 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 subunit deletion or replacement, wherein the peptide segment interception or replacement includes isomerization of peptide bond-proline bond and hydroxylation of lysine-proline residue. By modifying the target group at the specific position of the collagen, the interaction force between the collagen molecules is enhanced, the three-dimensional structure of the collagen molecule is designed, and / or the high-order structure segment is intercepted. On the one hand, enhancing the interaction force between collagen molecules can improve the structural stability of collagen in the body; secondly, the three-dimensional structure of the collagen molecule structure can be more conducive to the adhesion and growth of chondrocytes, and provide a favorable matrix environment for chondrocyte healing; thirdly, targeted interception of complex high-order structure segments can enhance the water solubility of collagen and improve the biocompatibility of collagen.

[0035] Among them, taking type I collagen as an example, the glycosylation modification can regulate the biological activity of collagen, enhance the interaction with chondrocytes, improve the biocompatibility, promote the adhesion and proliferation of chondrocytes, the disulfide bond modification in the propeptide extension can be used to connect different peptide chains or two cysteine residues in the same peptide chain, enhance the stability of the three-dimensional structure of collagen, maintain the helical structure of collagen and tensile resistance; the hydroxylation of lysine-proline residues can form hydrogen bonds between collagen molecules, improve 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, and the isomerization of peptide bond-proline bond usually refers to the cis configuration of the peptide bond, and the cis configuration is used to stabilize the specific structure of the protein, such as the polyproline II type helix. Further, taking the collagen with a triple helix structure as an example, the Gly-x-y active site composed of glycine and any two kinds of amino acids is used, wherein x and y are usually proline and hydroxyproline, so that the triple helix structure of the collagen molecule can be formed, the degradation rate is slowed down, and the continuous repair of chondrocytes is maintained.

[0036] In some embodiments, the collagen composite material is prepared as a lyophilized agent, and the auxiliary materials including one or more than two combinations of buffer salts, osmotic pressure regulators, lyophilization protectants, and pH regulators are added. By adding one or more than two auxiliary materials of buffer salts, osmotic pressure regulators, lyophilization protectants, and pH regulators, the collagen composite material with similar physicochemical properties to the natural joint fluid matrix is prepared, and the rejection reaction of the body to the collagen composite material is further reduced. In addition, the use of lyophilized agents can reduce transportation costs, and users can prepare the injection agent by adding an appropriate amount of water or physiological saline according to the instructions, or prepare gel or solution, which are common types of pharmaceutical agents. In the specific implementation process, taking the collagen composite material as an example, the content of the lyophilization protectant is 1%-25%, which can be selected from any one or a combination of sorbitol, mannitol, glycerol, dextran, sucrose, trehalose, glucose, lactose, maltose, dextran, triolein, polyethylene glycol, and ethylene vinyl. The content of the buffer salt is 15%-60%, which can be selected from any one or a combination of sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium chloride, and calcium chloride.

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

[0038] The preparation method is simple in operation and convenient for automatic production.

[0039] In the specific implementation process, the dissolution of collagen and water-soluble chitosan in distilled water can adopt any one or a combination of stirring, shearing, high-pressure homogenization, and ultrasonic; wherein the stirring can be magnetic stirring, and the stirring speed is 100 rpm-3000 rpm, and the stirring time is 1 h-6 h, preferably 150 rpm-2000 rpm, and the stirring time is 1 h-4 h. Moreover, taking the collagen composite material into a freeze-dried agent as an example, after mixing, the steps of standing, filtering the clear solution, and freeze-drying the clear solution should also be included. The sterilization method is filtration sterilization or irradiation sterilization, preferably any one of ultraviolet sterilization, infrared sterilization, and gamma ray sterilization; the standing time is 0.5 h-2.5 h, preferably 1 h-2 h.

[0040] The third aspect of the present application provides a use of the above-mentioned collagen composite material in lubricating joints, joint anti-inflammatory, delaying cartilage aging, and cartilage repair. Through scientific proportioning of water-soluble chitosan and collagen, the collagen composite material which is more close to natural joint fluid in composition is formed. The mucous membrane adhesion property of water-soluble chitosan can self-drivenly guide the collagen composite scaffold material to gather 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, and collagen has a pore structure, which is more conducive to the adhesion and nutrition acquisition of chondrocytes, promotes the proliferation of chondrocytes and realizes the repair of damaged cartilage. Moreover, the collagen molecular structure has good stability, the chondrocytes attached thereto can obtain more sufficient proliferation and repair time, further accelerates the repair of joint cartilage, slows down the continuous damage of joints, effectively prevents and treats traumatic or degenerative osteoarthritis, and has the advantages of good water solubility, good biocompatibility, fast effect, good repair effect, good stability, good reconstitution stability, long effective period, easy to store, etc.

[0041] The collagen composite material in the present application is used for preventing and treating any one of traumatic or degenerative osteoarthritis or its complications; specifically, the joint repair or cartilage repair is selected from any one or a combination of refractory wounds, knee osteoarthritis, synovitis, cartilage injury, and bursitis; the collagen composite material in the present application is used for any one or a combination of bone joint maintenance population, middle-aged degenerative disease population, sports injury population, sports maintenance population, and postoperative rehabilitation population of bone joint disease.

[0042] In the specific implementation process, taking the freeze-dried agent in the present application as an example, the use method is as follows: the freeze-dried agent is dissolved in water for injection or physiological saline to prepare an injection solution with a concentration of 1 mg / mL-80 mg / mL, preferably a concentration of 8 mg / mL-30 mg / mL, and is injected into the knee joint cavity.

[0043] The technical solutions of the present application are described below in combination with Examples 1-15 and Comparative Examples 1-12:

[0044] Examples 1-3:

[0045] Examples 1-3 provide a lyophilized agent of a composite material of Type I recombinant collagen and hydroxyethyl chitosan, which consists of:

[0046]

[0047]

[0048] The preparation of the lyophilized agent of Examples 1-3 includes the following steps:

[0049] According to the concentration of each component in the ingredient table, 15 mL of solution is prepared, and three portions of 30 mg of Type I recombinant collagen are accurately weighed in sequence, 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 are added, respectively. Magnetic stirring is used to fully dissolve the formulations of each example in 15 mL of distilled water, with a stirring speed of 200 rpm-600 rpm, a pH adjustment of about 7.0, an osmotic pressure adjustment of about 300 mOSM / L, and then the solution is divided, lyophilized, and sterilized by irradiation.

[0050] Examples 4-6:

[0051] Examples 4-6 provide a lyophilized agent of a collagen-containing composite material of Type I and Type III recombinant collagen and hydroxyethyl chitosan and hydroxypropyl chitosan, which consists of:

[0052] Example 4 Example 5 Example 6 Component 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 Na2HPO4 4 8 16 Trehalose 0.25 0.5 1

[0053] The preparation of the lyophilized agent of Examples 4-6 includes the following steps:

[0054] According to the concentration of each component in the ingredient table, 15 mL of solution is prepared, and three portions of 15 mg of Type I recombinant collagen and three portions of 15 mg of Type III recombinant collagen are accurately weighed in sequence, and 45 mg / 90 mg / 180 mg of hydroxyethyl chitosan, 45 mg / 90 mg / 180 mg of hydroxypropyl chitosan, 25 mg / 26.1 mg / 27.12 mg of NaCl, 60 mg / 120 mg / 240 mg of Na2HPO4, and 3.75 mg / 7.5 mg / 15 mg of trehalose are added, respectively. Magnetic stirring is used to fully dissolve the formulations of each example in 15 mL of pure water, with a stirring speed of 200 rpm-600 rpm, a pH adjustment of about 7.0, an osmotic pressure adjustment of about 300 mOSM / L, and then the solution is divided, lyophilized, and sterilized by irradiation.

[0055] Examples 7-9

[0056] Examples 7-9 provide a lyophilized agent of a composite material of Type I recombinant collagen compounded with hydroxyethyl chitosan, hydroxypropyl chitosan, which consists of:

[0057] Example 7 Example 8 Example 9 Component 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 Na2HPO4 4 8 16 Trehalose 0.25 0.5 1.0

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

[0059] According to the concentration of each component in the ingredient table, 15 mL of solution is 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, 3.75 mg / 7.5 mg / 15 mg trehalose are accurately weighed in sequence, magnetic stirring is used to fully dissolve the formulations of each example 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 then it is divided, lyophilized, and irradiation sterilized.

[0060] Examples 10-12

[0061] Examples 10-12 provide a lyophilized agent of a composite material of Type III recombinant collagen compounded with hydroxyethyl chitosan, hydroxypropyl chitosan, which consists of:

[0062] Example 10 Example 11 Example 12 Component 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 Na2HPO4 4 8 16 Trehalose 0.25 0.5 1.0

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

[0064] According to the concentration of each component in the ingredient table, 15 mL of solution is 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, 3.75 mg / 7.5 mg / 15 mg trehalose are accurately weighed in sequence, magnetic stirring is used to fully dissolve the formulations of each example 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 then it is divided, lyophilized, and irradiation sterilized.

[0065] Comparative Example 1:

[0066] Comparative Example 1 provides a lyophilized agent containing only two kinds of chitosan, and the composition of the lyophilized agent is as follows:

[0067] Component Concentration (mg / mL) Hydroxyethyl chitosan 6 Hydroxypropyl chitosan 6 NaCl 1.06 Na2HPO4 8 Trehalose 0.5

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

[0069] According to the concentration of each component in the ingredient table, 15 mL of solution is prepared, and 90 mg of hydroxyethyl chitosan, 90 mg of hydroxypropyl chitosan, 16 mg of NaCl, 120 mg of Na2HPO4, 7.5 mg of trehalose are accurately weighed in sequence, and magnetically stirred to fully dissolve 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 then it is divided, lyophilized, and sterilized by irradiation.

[0070] Comparative Example 2:

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

[0072] Component Concentration (mg / mL) Medical chitosan 12 NaCl 1.06 Na2HPO4 8 Trehalose 0.5

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

[0074] According to the concentration of each component in the ingredient table, 15 mL of solution is prepared, and 180 mg of medical chitosan, 16 mg of NaCl, 120 mg of Na2HPO4, 7.5 mg of trehalose are accurately weighed in sequence, and magnetically stirred to fully dissolve 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 then it is divided, lyophilized, and sterilized by irradiation.

[0075] Comparative Example 3

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

[0077] Component Concentration (mg / mL) Recombinant collagen type I 2 NaCl 0.25 Na2HPO4 2 Trehalose 0.125

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

[0079] According to the concentration of each component in the ingredient table, 15 mL solution is prepared, 30 mg of type I recombinant collagen, 3.75 mg of NaCl, 30 mg of Na2HPO4, 1.88 mg of trehalose are accurately weighed in sequence, and they are fully dissolved in 15 mL of pure water by magnetic stirring, 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 then it is divided, freeze-dried, irradiation sterilization, and obtained.

[0080] Comparative Example 4

[0081] Comparative Example 4 provides a freeze-dried agent containing only type III recombinant collagen, and the composition of the freeze-dried agent is:

[0082] Component Concentration (mg / mL) Recombinant collagen type III 2 NaCl 0.25 Na2HPO4 2 Trehalose 0.125

[0083] The preparation of the freeze-dried agent of Comparative Example 4 includes the following steps:

[0084] According to the concentration of each component in the ingredient table, 15 mL solution is prepared, 30 mg of type I recombinant collagen, 3.75 mg of NaCl, 30 mg of Na2HPO4, 1.88 mg of trehalose are accurately weighed in sequence, and they are fully dissolved in 15 mL of pure water by magnetic stirring, 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 then it is divided, freeze-dried, irradiation sterilization, and obtained.

[0085] Test Example 1

[0086] The above Examples 1, 4, 7 and 10 and Comparative Example 1 are scanned by electron microscope, and the electron microscope photos as shown in Figures 1-5 can be obtained. Figures 1-5 It can be known that the freeze-dried agent provided by the present application has a unique three-dimensional structure, and therefore has high 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 are all applied to cartilage cell proliferation experiments:

[0089] 1. Experimental reagents

[0090] DMEM (Dulbecco's Modified Eagle Medium) basic medium and complete medium, mouse-derived type IV collagenase (Solarbio, item number: C8140), PBS buffer salt solution, CCK-8 solution, 75% alcohol

[0091] 2. Mouse chondrocyte extraction and culture experiment

[0092] S1: C57 mice aged 8 weeks were sacrificed by cervical dislocation and placed in a beaker containing 75% alcohol for 5 min;

[0093] S2: The leg bones of the mice were taken out, first placed in a culture dish containing 75% alcohol for 2 min, then placed in a culture dish containing PBS, and then the hyaline cartilage at the knee joint was separated in a sterile operating room with sterilized surgical scissors into a 15 mL centrifuge tube, and the cartilage was washed twice with an appropriate amount of PBS;

[0094] S3. The washed cartilage was cut into small pieces with sterilized surgical scissors and placed in a six-well plate. 0.2% mouse-derived type IV collagenase solution (10 mg type IV collagenase dissolved in 5 mL DMEM basic medium) was added to just immerse the cartilage pieces, and incubated in a cell culture incubator for 1 h. During this period, the six-well plate was taken out every 15 min or so and blown several times with a pipette to ensure that the collagenase solution and cartilage pieces were in full contact, facilitating digestion into single cells;

[0095] S4. About 6 mL of DMEM complete medium was added to terminate digestion, blown with a pipette, and filtered with a 200 μm filter screen. The filtrate was collected in a 50 mL centrifuge tube, placed in a centrifuge, and the speed was adjusted to 1000 rpm for 5 min. The supernatant was discarded, 6 mL of DMEM complete medium was added for resuspension, and then placed in a centrifuge with the speed adjusted to 500 rpm for 5 min. The supernatant was collected and cultured in a six-well plate at 2 mL / well, and placed in a cell culture incubator for 24 h before changing the medium;

[0096] S5. The cartilage pieces left on the filter screen and the precipitate from step S4 were collected and placed in a six-well plate. Type IV collagenase solution was added again to immerse the cartilage pieces, and the six-well plate was continued to be incubated in the incubator for 2 h, during which the six-well plate was blown several times every 15 min or so to ensure that the cartilage pieces and collagenase solution were in full contact;

[0097] S6. 6 mL of DMEM complete medium was added to terminate digestion, filtered again, and the filtrate was collected in a centrifuge tube, 1000 rpm, centrifuged for 5 min, the supernatant was discarded, 6 mL of DMEM complete medium was added for resuspension, and then placed in a centrifuge with the speed adjusted to 500 rpm for 5 min. The supernatant was collected and cultured in a six-well plate at 2 mL / well, and the remaining precipitate was also resuspended with 2 mL of DMEM complete medium and cultured in a six-well plate, and placed in a cell culture bottle for 24 h before changing the medium;

[0098] S7. The cell growth was observed under a microscope, and when the cell growth area reached 80%-90% of the area of the bottom of the cell culture bottle, it was inoculated in a 96-well plate.

[0099] 3. Proliferation test of the freeze-dried agent of the present application on chondrocytes

[0100] S1. The above six-hole plate full of cartilage cells was placed in a 96-well plate, about 5000-10000 cells per well, 100ul of DMEM complete medium per well, and placed in a cell incubator overnight;

[0101] S2. The next day, 90ul of DMEM complete medium containing the freeze-dried agent was added to each well, and the incubator was incubated for 48h. The preparation method of the DMEM complete medium containing the freeze-dried agent is as follows:

[0102] Examples 1-12: Take the freeze-dried agent prepared in the above examples 1-12 respectively, add DMEM complete medium to dissolve, configure into a solution, and reduce the content of each component in each example;

[0103] Comparative examples 1-4: Take the freeze-dried agent prepared in the above comparative examples 1-4 respectively, add DMEM complete medium to dissolve, configure into a solution, and reduce the content of each component in each comparative example;

[0104] S3. After incubation, 10ul of CCK-8 solution was added to each well, and the incubator was incubated for about 4h;

[0105] S4. The absorbance was detected at 450nm by using an enzyme marker.

[0106] Cell proliferation rate (%) = test group absorbance value / blank group absorbance value x 100%, the results are shown in Table 1. The collagen composite material of the application can realize the proliferation of cartilage cells.

[0107] Table 1

[0108]

[0109]

[0110] Summary: see Figure 6From Examples 1-12, the cell proliferation rate is positively correlated with the added proportion of collagen; from Example 1 and Example 7, it can be seen that, while keeping the ratio of total chitosan and collagen unchanged, the compounding of multiple chitosans can enhance the cell proliferation effect of the composite material; by comparing Example 7 and Example 10, it can be seen that, while keeping the chitosan ratio unchanged, the use of type III collagen can achieve better cell proliferation effect, indicating that the repair effect of type III recombinant collagen is better; by comparing Example 7 and Example 4, while keeping the ratio of total chitosan and total collagen unchanged, the compounding of multiple chitosans and multiple collagens can enhance the cell proliferation effect of the composite material. The cell proliferation effect of the composite material in Example 4, which is compounded by type I and type III recombinant collagens and hydroxyethyl chitosan and hydroxypropyl chitosan in a ratio of 1:1:3:3, is the best, and the cell proliferation rate on the third day is as high as 123.61%, which proves that its high drug efficacy can be maintained for more than three days, and the drug efficacy is good. And. Correspondingly, Comparative Example 1 which only uses two kinds of chitosans and Comparative Example 2 which only uses medical chitosan, both of which are added with chitosan at the same concentration as Example 2, can be determined to have no cell proliferation effect; Comparative Example 3 and Comparative Example 4 which do not add chitosan but only use 2 mg / mL of collagen also have poor cell proliferation effect, which indicates that the chitosan and collagen compounding scheme provided in Example 1 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, referring to Figure 5 At the micro level, the freeze-dried agent using only one or more chitosans has a more flat structure and weak mechanical properties, and it is difficult to long-acting on the damaged joint and continuously maintain the structure framework of assisting the growth and repair of chondrocytes on the damaged cartilage surface, while Figure 6 This also explains the reason why the cell proliferation effect of Comparative Examples 1-2 is close to zero; referring to Figures 1-4 Examples with added corresponding collagen have obvious layered structure, and collagen molecules and chitosan molecules are cross-linked and assembled into long fibrils with high tensile strength, which can provide sufficient stable growth and repair space and sufficient nutrition for the damaged wound, thereby achieving the effect of promoting the repair of articular cartilage and preventing and treating traumatic or degenerative osteoarthritis.

[0112] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "exemplary embodiment", "example", "specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present embodiment. In the present specification, the exemplary description of the above terms does not necessarily mean the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be appropriately combined in any one or more embodiments or examples.

[0113] Although the embodiments of the present embodiment have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made thereto 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 material includes collagen and chitosan, the collagen is a combination of type I recombinant collagen and type III recombinant collagen, the chitosan is water-soluble chitosan, and the water-soluble chitosan is a combination of hydroxyethyl chitosan and hydroxypropyl chitosan, 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 any one of claims 1 to 3, characterized in that: The collagen composite material is in the form of a lyophilized agent, and further comprises auxiliary materials 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.

5. The method for preparing the collagen composite material for repairing joint damage according to claim 4, characterized in that: The steps include: S1. Weigh the required amount of the collagen, the water-soluble chitosan and the excipients; S2. adding a predetermined ratio 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 subjected to subpackaging, freeze-drying and irradiation sterilization to obtain the collagen composite material in the form of a freeze-dried agent.

6. Use of the collagen composite material for repairing joint damage according to any one of claims 1 to 3 in a freeze-dried preparation for lubricating joints, resisting joint inflammation, delaying cartilage aging and repairing cartilage.

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