BMP-2-containing composition as well as preparation method and application thereof

Abstract

The invention provides a composition containing BMP-2, the composition comprises BMP-2 active protein and a matrix material, the BMP-2 active protein is dispersed and distributed in the matrix material, the matrix material is a spongy material with a pore structure, and the weight ratio (w / w) of the matrix material to the BMP-2 active protein is (10-1000): 1. The composition prepared by the invention can quantitatively load BMP-2, realizes slow release of BMP-2, has certain mechanical strength, is degradable in material, and can be used for treating, preventing or improving orthopedic related diseases.

Description

A composition containing BMP-2 and its preparation method and use Technical Field

[0001] The present invention relates to the field of biomaterials, and in particular to a composition containing BMP-2, a preparation method thereof, and uses thereof. Background Art

[0002] Bone defects caused by trauma, tumor resection, and congenital diseases can lead to skeletal deformity and dysfunction, severely impacting patients' daily lives. Therefore, restoring the function and condition of the bone to its original state to the greatest extent possible is a goal that people pursue and explore. When treating small bone defects, the body's self-healing ability can be harnessed to induce the growth of new bone tissue at the defect site for repair. However, larger defects, such as those caused by congenital and acquired lesions such as trauma, tumors, and infection, can only be repaired through transplantation.

[0003] One of the key elements of an ideal bone transplant material is to fully simulate the microstructure of natural bone tissue and form an effective network structure within the bone scaffold material to ensure sufficient oxygen and nutrient exchange directly inside, further providing osteoconduction for the new bone. For example, gelatin, as a hydrolyzate of natural collagen, can act as a carrier of protein factors to load and transfer factors. It can also be used as a filler to improve the biological properties of the material. More importantly, the unique sol-gel properties of gelatin can be prepared into a gel material with good biocompatibility and interconnected pores through cross-linking. For example, bioactive glass is a bioactive material with osteoconductive and osteoinductive activities. It has the characteristics of forming chemical bonds with bone tissue and soft tissue, inducing osteoblast differentiation, inducing HA layer formation in a liquid environment, and adsorbing protein molecules in the surrounding environment. It has been widely used in bone tissue regeneration and repair research.

[0004] Another key element of an ideal bone graft material is the loading of osteoinductive factors that synergistically regulate bone regeneration and repair. The timely and appropriate supplementation of the required growth factors can induce the migration, recruitment, development, differentiation, and osteogenesis of adjacent cells (osteoinduction), effectively promoting bone defect repair. Among these, bone morphogenetic protein-2 (BMP-2) is the most potent and widely used bone growth factor known to date. It is also the only cell-active protein capable of initiating osteogenic differentiation and affecting the entire osteogenic differentiation process. BMP-2 promotes the synthesis of extracellular matrix components and cell proliferation, including chemotaxis of mesenchymal cells, which then differentiate into chondrocytes and, in turn, form new bone within the endochondral osteoblast nucleus, encompassing the bone marrow. The osteogenic effect of BMP-2 is dose-dependent; low doses of BMP-2 are less effective, while supraphysiological doses are less safe and may even cause a range of adverse reactions. Furthermore, BMP-2 is easily inactivated in body fluids, resulting in a rapid decrease in its therapeutic concentration and an inability to continuously stimulate target cells to fully exert its inductive activity.

[0005] Bone grafts containing BMP-2 have been previously disclosed, mostly using collagen sponges, hydroxyapatite, and calcium phosphate as carriers. For relevant literature, reference may be made to patents such as US6261586, CN01117292.4, CN201610498331.1, and CN201610392378.X. These patents disclose that existing BMP-2 bone grafts generally limit the diffusion of BMP-2 components through the molecular structure of the matrix material to ensure a stable concentration of BMP-2 around the host target cells. However, relying solely on physical action to achieve controlled release of BMP-2 generally requires a larger dose of BMP-2 to achieve a good osteogenesis effect. The preparation cost of such a bone graft containing BMP-2 is high, and due to the large amount of BMP-2 added, there may be safety issues.

[0006] In addition, as described in patent CN200510132792.9, loading of BMP-2 is achieved only by physically adsorbing the bioactive factor onto the matrix material. This adsorption method cannot achieve quantitative control and may lead to problems such as failure to achieve effective therapeutic effects in clinical applications.

[0007] Therefore, clinical medicine still needs to develop a medical product that can quantitatively load BMP-2 protein, achieve slow release of BMP-2, and have certain mechanical strength and biodegradable materials, so that it can be conveniently used to treat, prevent or improve orthopedic diseases. Summary of the Invention

[0008] To make the present invention easier to understand, some terms are first defined. Unless otherwise specified, the scientific and technical terms used herein shall have the meanings commonly understood by those of ordinary skill in the art.

[0009] Unless otherwise indicated, the practice of the present invention will employ conventional techniques in materials science, biochemistry, bioengineering, molecular biology, etc., which are all within the technical scope of the art and are fully explained in the technical literature and general textbooks in the art, such as biomedical polymer materials, Molecular Biology, biomaterials and tissue engineering, etc.

[0010] The BMP-2 active protein used in the present invention refers to bone morphogenetic protein-2 (BMP-2). BMP-2 is a member of the transforming growth factor B superfamily. It has the ability to induce undifferentiated mesenchymal stem cells to differentiate and proliferate into chondrocytes and osteoblasts, promote osteoblast differentiation and maturation, participate in bone and cartilage growth and development and their reconstruction process, and thus accelerate bone defect repair. Natural BMP-2 is synthesized in vivo as a precursor, and the signal peptide and propeptide are removed by proteolytic cleavage to obtain a mature peptide composed of 114 amino acid residues. The mature peptide correctly folds its conserved structure through 7 pairs of disulfide bonds, and the mature peptide homo- or heterodimers have biological activity. The BMP-2 active protein used in the present invention also includes BMP-2 mutants prepared by conservative sequence modifications such as amino acid substitutions, additions and / or deletions and still retaining the basic activity of BMP-2.

[0011] The "inorganic particles" used in the present invention are defined as granular materials made of inorganic substances alone or mixed with other substances, including but not limited to materials prepared by a certain process from raw materials such as phosphates, silicates, aluminates and / or raw materials such as calcides, oxides, nitrides, etc.

[0012] The term "calcium phosphate compound" as used herein is defined as a phosphate calcification, including but not limited to hydroxyapatite, tricalcium phosphate (TCP, Ca3(PO4)2), tetracalcium phosphate (Ca4(PO4)2O), dicalcium diphosphate (Ca2P2O7), calcium tripolyphosphate (Ca5(P3O 10 )2) and bioactive glass, etc.

[0013] The "bioactive glass" used in this invention is a type of amorphous silicate-based solid. For its definition, refer to the medical device industry standard YY0964-2014, "Bioglass and Glass-Ceramic Materials for Surgical Implants." Compared to traditional bone repair materials, bioactive glass (BG) is a bone repair material primarily composed of calcium oxide (CaO) and silicon dioxide (SiO2). During application, it releases inorganic ions such as silicon (Si), phosphorus (P), and calcium (Ca), resulting in excellent bioactivity. It also has advantages such as inducing osteoblast differentiation in bone tissue cells and promoting gene expression in osteoblasts, making it an excellent bone defect repair material. While the composition and structure of bioactive glass vary depending on the preparation process, its basic components are the SiO2-CaO-P2O5 ternary system.

[0014] The "freeze cross-linking" used in the present invention refers to a cross-linking reaction under low temperature (-20°C to 0°C) conditions, in which the amino groups in biological molecules such as proteins and short-chain polypeptides and the aldehyde groups in inorganic molecules undergo Schiff base reaction to form a tertiary structure.

[0015] The term "subject" or "patient" used in the present invention includes humans and non-human animals. Non-human animals include all vertebrates, for example mammals and non-mammals, such as non-human primates (monkeys, gorillas), sheep, dogs, pigs, rats, mice, cats, cows, horses and birds.

[0016] The present invention aims to provide a composition containing BMP-2 that can quantitatively load BMP-2 protein and achieve slow release, while also possessing sufficient mechanical strength and being biodegradable. This composition can provide or optimize the physiological microenvironment required for bone tissue regeneration and can be formulated as a pharmaceutical or medical device for convenient clinical use in the treatment, prevention, or improvement of orthopedic diseases.

[0017] The first aspect of the present invention provides a composition comprising BMP-2, comprising:

[0018] (1) BMP-2 active protein;

[0019] (2) a matrix material, wherein the matrix material comprises biomolecules and inorganic particles; wherein the biomolecules are selected from gelatin, collagen, elastin or a combination thereof; and the inorganic particles are selected from any one or a combination of the following calcium phosphate compounds: hydroxyapatite, monocalcium phosphate, dicalcium phosphate, tricalcium phosphate (TCP, Ca3(PO4)2), tetracalcium phosphate (Ca4(PO4)2O), dicalcium diphosphate (Ca2P2O7), calcium tripolyphosphate (Ca5(P3O 10 )2) or bioactive glass;

[0020] The BMP-2 active protein is diffusely distributed in a matrix material, the matrix material is a sponge-like material with a porous structure, and the weight ratio (w / w) of the matrix material to the BMP-2 active protein is 10-1000:1.

[0021] In another embodiment, the BMP-2 active protein in the composition of the present invention refers to a mature peptide with a full length of 114 amino acid residues (SEQ ID NO: 1), or a mature peptide with 115 amino acid residues with methionine added at the N-terminus (SEQ ID NO: 2), or an N-terminally truncated mature peptide (SEQ ID NO: 3), or mutants of the above three BMP-2 proteins. For the design of BMP-2 mutants (with 90% or higher homology), reference can be made to document CN201080011605.0. Conservative sequence modifications such as amino acid substitutions, additions and / or deletions will not significantly affect or change the biological activity of the BMP-2 protein. For example, the BMP-2 mutant is composed of an amino acid sequence of SEQ ID No: 1 comprising two or three amino acid substitutions, wherein the first and second amino acid substitutions are present at positions selected from S24 and N59, N59 and N102, or P36 and N59 of SEQ ID No: 1, or the three amino acid substitutions are present at positions S24, N59 and N102 of SEQ ID No: 1. The BMP-2 mutant with 90% or higher homology basically has the biological activity of the original BMP-2 protein, such as the S24E, N59K and N102YH mutations shown in SEQ ID NO: 4. In a preferred embodiment of the present invention, the BMP-2 active protein in the composition of the present invention refers to an N-terminally truncated BMP-2 mature peptide containing 108 amino acid residues, the sequence of which is shown in SEQ ID NO:3.

[0022] In another embodiment, the BMP-2 active protein in the composition of the present invention is extracted from bone tissue, chemically synthesized, or obtained using genetic recombination methods. The construction and expression of recombinant human bone morphogenetic protein-2 (BMP-2) mature peptide in prokaryotes, as well as the preparation, solubilization, and renaturation of inclusion bodies, are well documented. For details, see Xu Fang (CN01116754.8; CN200510050610.3), Liu Guoan et al. (CN200610049151.1), and Guo Wangming et al. (CN201010284844.5), all of which are incorporated herein by reference.

[0023] In another embodiment, in the composition described herein, the biomolecules in the matrix material are selected from gelatin, collagen, elastin, or a combination thereof. Gelatin is obtained by purifying collagen from animal skin, bones, cheeks, and ligaments through moderate hydrolysis (acid, alkaline, acid-base, or enzymatic methods), or is a mixture of these different gelatin products. Collagen is a family of secreted proteins containing at least 20 genetically distinct types. Collagen primarily provides structural support for the body, possessing a unique triple-helical structure consisting of three polypeptide chains (called α chains) and possessing specific biological functions. Elastin is a protein that maintains the elasticity of connective tissue, allowing many tissues in the body to regain their shape after stretching or contracting. In the human genome, elastin is encoded by the ELN gene.

[0024] In another embodiment, the collagen in the composition of the present invention is human collagen, animal-derived collagen, recombinant collagen or recombinant humanized collagen. The human body contains as many as 28 types of collagen, of which type I, type II, and type III collagen together account for 80% to 90% of the total collagen content. At present, the main types of collagen widely used in commercial scenarios include type I and type III. Animal-derived collagen is extracted and purified from animal tissues, completely retaining the triple helix structure and biological properties. It is the current main preparation technology and is widely used in medicine, cosmetics, food industry and other fields. Recombinant collagen protein is produced through recombinant DNA technology, by genetically manipulating and / or modifying the gene encoding the desired human collagen protein. The target gene is then introduced into appropriate host cells (such as bacteria, yeast, or other eukaryotic cells) using plasmids or viral vectors for expression and translation into collagen or collagen-like polypeptides. This is then extracted and purified. (For the definition of recombinant collagen, please refer to the medical device industry standard YY / T 1849-2022, "Recombinant Collagen.") Recombinant humanized collagen protein is a full-length or partial amino acid sequence fragment encoded by a gene encoding a specific type of human collagen, or a combination of functional human collagen fragments, produced using recombinant DNA technology. (For the definition of recombinant humanized collagen, please refer to the medical device industry standard YY / T 1888-2023, "Recombinant Humanized Collagen.") There are already documents that fully disclose recombinant collagen or recombinant humanized collagen and related preparation methods. For details, please refer to patents CN201880076990.3 (truncated), CN201811438582.6 (Jinbo), CN201110327873.X (Juzi), CN201110327865.5 (Jiangshan), CN201310033299.6 (Jiangsu Chuangjian) and related documents Biochem Biophys Res Commun. 2019; 508(4): 1018-23, ACS Biomater Sci Eng. 2020; 6(4): 1977-88 and Synthetic Biology 2023; 4(4): 808-823, all of which are incorporated herein by reference. In another embodiment, the biomolecule is preferably gelatin, recombinant collagen or recombinant humanized collagen. In another embodiment, exemplary recombinant collagen or recombinant humanized collagen sequences are shown in SEQ ID NO:5-SEQ ID NO:8.

[0025] In another embodiment, in the composition of the present invention, the inorganic particles in the matrix material are preferably selected from hydroxyapatite, tricalcium phosphate (TCP, Ca3(PO4)2), bioactive glass or a combination thereof.

[0026] In another embodiment, in the composition of the present invention, the inorganic particulate tricalcium phosphate in the matrix material is β-tricalcium phosphate (β-TCP).

[0027] In another embodiment, in the composition described herein, the inorganic particles in the matrix material are bioactive glass. Bioactive glass (BG) is a type of amorphous silicate-based solid that, when implanted in the body, can form bonds with both hard and soft tissues, but not necessarily adhesion. Furthermore, in an appropriate in vitro environment, such as when immersed in simulated body fluid (SBF) or Tris buffer (Tris buffer), it can form a layer of carbonated hydroxyapatite (CHAp) on the surface of the material. Bioglass with this property is considered bioactive glass (for a definition of bioactive glass, see the medical device industry standard YY0964-2014, "Bioglass and Glass-Ceramic Materials for Surgical Implants").

[0028] Bioactive glass varies in composition and structure depending on the preparation process, but its basic components are the SiO2-CaO-P2O5 ternary system. For example, AW-GC (Apatite-Wollastonite Glass-Ceramic), developed by Japanese scholars Horita et al., is a high-performance bioactive glass-ceramic material composed of apatite (A) and wollastonite (W) crystals. It is prepared through a specific composition design (e.g., the SiO2-CaO-MgO-P2O5 system) and heat treatment. For the types and preparation processes of bioactive glass, please refer to relevant patents CN201010248059.4, CN200710185030.4, CN200610124001.2, CN200610035111.1 and related literature such as Dent Mater, 2018.34(9): p.1323-1330, Mater Sci Eng C Mater Biol Appl. 2013; 33(7): 3592-600, Biomaterials. 2006; 27(11): 2414-25 and J Orthop Translat. 2022; 36: 120-31, etc.

[0029] In another preferred embodiment, the raw materials of the bioactive glass in the composition of the present invention have an effective component SiO2 mass fraction of ≥45%, a CaO mass fraction of ≥15%, and a P2O5 mass fraction of ≥3%.

[0030] In another preferred embodiment, the bioactive glass in the composition of the present invention is selected from 45S5 bioactive glass, 52S4.6 bioactive glass, S53P4 bioactive glass, AW-GC or a combination thereof.

[0031] In another embodiment, in the composition of the present invention, the inorganic particles in the matrix material have an average particle size of 10 to 150 μm.

[0032] In another embodiment, in the composition of the present invention, the inorganic particles in the matrix material have an average particle size of 30 to 120 μm.

[0033] In another embodiment, in the composition of the present invention, the inorganic particles in the matrix material have an average particle size of 50 to 100 μm.

[0034] In another embodiment, in the composition of the present invention, the inorganic particles in the matrix material have an average particle size of 70 to 90 μm.

[0035] In another embodiment, in the composition of the present invention, the weight ratio (w / w) of the biomolecules to the inorganic particles in the matrix material is 0.5 to 10:1.

[0036] In another embodiment, in the composition of the present invention, the weight ratio (w / w) of the biomolecules to the inorganic particles in the matrix material is 0.5 to 5:1.

[0037] In another embodiment, in the composition of the present invention, the weight ratio (w / w) of the biomolecules to the inorganic particles in the matrix material is 1 to 3:1.

[0038] In another embodiment, in the composition of the present invention, the weight ratio (w / w) of the biomolecules to the inorganic particles in the matrix material is 1 to 2:1.

[0039] In another embodiment, in the composition of the present invention, the weight ratio (w / w) of the biomolecules to the inorganic particles in the matrix material is 1 to 1.5:1.

[0040] In another embodiment, in the composition of the present invention, the biomolecules and inorganic particles in the matrix material form a sponge-like material with a porous structure through chemical bonding and physical adsorption. During the preparation process of mixing the biomolecules and inorganic particles, a crosslinking agent can be added to allow the amino groups in the biomolecules to react with the aldehyde groups in the crosslinking agent to form a Schiff base reaction, thereby forming a three-dimensional network structure through crosslinking. In another embodiment, the crosslinking agent is selected from formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, glyceraldehyde, vanillin, or glutaraldehyde, preferably formaldehyde or glutaraldehyde. The crosslinking agent can be added by any method, and the amount added is 0.1%-1% (w / w) of the weight of the biomolecules, preferably 0.2%-0.8% (w / w) of the weight of the biomolecules, and more preferably 0.5%-0.7% (w / w) of the weight of the biomolecules. For example, the crosslinking agent can be prepared as a separate solution and added to the solution of the biomolecules and inorganic material to undergo a chemical crosslinking reaction. After the reaction is complete and dried, the biomolecules and inorganic particles form a sponge-like material with a porous structure.

[0041] In another embodiment, in the composition of the present invention, the BMP-2 active protein is dispersed in the matrix material. The BMP-2 active protein is added to the matrix material in any manner during the chemical cross-linking reaction between the biomolecules and the inorganic material, and is uniformly mixed with the matrix material by stirring or other means, thereby effectively adsorbing the BMP-2 active protein into the three-dimensional network structure of the matrix material.

[0042] In another embodiment, in the composition of the present invention, the BMP-2 active protein is diffusely distributed in a matrix material, the matrix material is a sponge-like material with a porous structure, and the weight ratio (w / w) of the matrix material to the BMP-2 active protein is 10 to 500:1.

[0043] In another embodiment, in the composition of the present invention, the BMP-2 active protein is diffusely distributed in a matrix material, the matrix material is a sponge-like material with a porous structure, and the weight ratio (w / w) of the matrix material to the BMP-2 active protein is 50 to 500:1.

[0044] In another embodiment, in the composition of the present invention, the BMP-2 active protein is diffusely distributed in a matrix material, the matrix material is a sponge-like material with a porous structure, and the weight ratio (w / w) of the matrix material to the BMP-2 active protein is 90 to 200:1.

[0045] In another embodiment, in the composition of the present invention, due to the presence of residual crosslinker after the aldehyde groups of the crosslinker react with amino groups in biomolecules to form a Schiff base during the preparation process, the weight ratio of residual crosslinker to composition is less than 1 mg / g. That is, based on 1 part by weight of the composition, the residual crosslinker accounts for less than 0.1% (w / w). In another embodiment, in the composition of the present invention, the residual crosslinker accounts for less than 0.06% (w / w). In another embodiment, in the composition of the present invention, the residual crosslinker accounts for less than 0.03% (w / w).

[0046] In another embodiment, the composition of the present invention can be dried to form a sponge-like material having a porous structure by any known method, such as drying at room temperature and pressure, or freeze-drying using a freeze-drying apparatus. Drying, as used herein, means that the composition has a water content of less than 5%, more preferably less than 4%, 3%, 2%, 1%, 0.5% (w / w), or less. In another preferred embodiment, the composition of the present invention has a water content of 0.1% to 3% (w / w), calculated per part by weight of the composition.

[0047] In another embodiment, the composition of the present invention is a sponge-like material having a porous structure, which has a porosity of 70% or higher. The porosity detection method of the present invention can refer to the technical content disclosed in the document Bioact Mater. 2021; 6(10): 3396-410.

[0048] In another embodiment, the composition of the present invention is a sponge-like material having a porous structure with a porosity of 80% or more.

[0049] In another embodiment, the composition of the present invention is a sponge-like material having a porous structure with a porosity of 85% or more.

[0050] In another embodiment, the composition of the present invention is a sponge-like material having a porous structure with a porosity of 90% or more.

[0051] In another embodiment, the composition of the present invention has a compressive strength of 0.1 to 2.0 MPa and an elastic modulus of 3 to 20 MPa.

[0052] In another embodiment, the composition of the present invention has a compressive strength of 0.2 to 1.0 MPa and an elastic modulus of 6 to 16 MPa.

[0053] In another preferred embodiment, the present invention provides a composition comprising BMP-2, comprising:

[0054] (1) BMP-2 active protein, the sequence of which is shown in one of SEQ ID NO: 1 to SEQ ID NO: 4 or a mutant thereof;

[0055] (2) a matrix material, wherein the matrix material comprises biomolecules and inorganic particles, and the weight ratio (w / w) of the biomolecules to the inorganic particles is 0.5-10:1; wherein the biomolecule is gelatin or collagen, and the collagen is selected from human collagen, animal-derived collagen, recombinant collagen, or recombinant humanized collagen; and the inorganic particles are selected from any one or a combination of the following calcium-phosphorus compounds: hydroxyapatite, β-tricalcium phosphate, or bioactive glass;

[0056] The BMP-2 active protein is diffusely distributed in a matrix material, the matrix material is a sponge-like material with a porous structure, and the weight ratio (w / w) of the matrix material to the BMP-2 active protein is 10 to 1000:1.

[0057] In another preferred embodiment, the present invention provides a composition comprising BMP-2, comprising:

[0058] (1) BMP-2 active protein, the sequence of which is shown in SEQ ID NO: 3 or a mutant thereof;

[0059] (2) a matrix material, wherein the matrix material comprises biomolecules and inorganic particles, wherein the weight ratio (w / w) of the biomolecules to the inorganic particles is 1 to 3:1; wherein the biomolecule is gelatin or a recombinant humanized collagen having an amino acid sequence as shown in any one of SEQ ID NO:5 to SEQ ID NO:8; and the inorganic particles are selected from bioactive glass;

[0060] The BMP-2 active protein is diffusely distributed in the matrix material, which is a sponge-like material with a porous structure and a porosity of 70% or higher, and the weight ratio (w / w) of the matrix material to the BMP-2 active protein is 90 to 200:1.

[0061] The second aspect of the present invention provides a method A for preparing the above-mentioned composition containing BMP-2, comprising the following steps:

[0062] (1) adding inorganic particles to a biomolecule solution having a concentration of 3% to 15% (m / v) at a biomolecule to inorganic particle mass ratio (w / w) of 0.5 to 10:1; stirring uniformly, adding a crosslinking agent and stirring to react to form a suspension;

[0063] (2) Add BMP-2 active protein in an amount of (inorganic particles + biomolecules): BMP-2 active protein mass ratio of 10 to 1000:1 and stir evenly;

[0064] (3) injecting the stirred reaction system into a mold and placing it in a mold for molding, and freezing and crosslinking the generated gel for 24-120 hours;

[0065] (4) The gel is freeze-dried to obtain the composition containing BMP-2; wherein the BMP-2 active protein is diffusely distributed in the matrix material, and the matrix material is a sponge-like material with a porous structure.

[0066] In another embodiment, the present invention provides a method B for preparing the above-mentioned composition comprising BMP-2, comprising the following steps:

[0067] (1) adding inorganic particles to a biomolecule solution having a concentration of 3% to 15% (m / v) at a biomolecule:inorganic particle mass ratio (w / w) of 0.5 to 10:1 and stirring uniformly;

[0068] (2) adding BMP-2 active protein in an amount of (inorganic particles + biomolecules): BMP-2 active protein mass ratio of 10 to 1000:1, and then adding a crosslinking agent and stirring to react to form a suspension;

[0069] (3) injecting the stirred reaction system into a mold for forming, and refrigerating and cross-linking the generated gel for 24-120 hours;

[0070] (4) The gel is freeze-dried to obtain the composition containing BMP-2; wherein the BMP-2 active protein is diffusely distributed in the matrix material, and the matrix material is a sponge-like material with a porous structure.

[0071] In another embodiment, the inorganic particles, biomolecules, BMP-2 active protein, crosslinking agent, matrix material and their amounts in steps (1) to (4) of method A or B of the present invention are defined as described above.

[0072] In another embodiment, in step (1) of method A or B of the present invention, the biomolecule is selected from gelatin, collagen, elastin or a combination thereof; the inorganic particles are selected from any one or a combination of the following calcium phosphate compounds: hydroxyapatite, monocalcium phosphate, dicalcium phosphate, tricalcium phosphate (TCP, Ca3(PO4)2), tetracalcium phosphate (Ca4(PO4)2O), dicalcium diphosphate (Ca2P2O7), calcium tripolyphosphate (Ca5(P3O 10 )2) or bioactive glass.

[0073] In another embodiment, in step (1) of method A or B of the present invention, the inorganic particles are preferably added to the biomolecule solution having a concentration of 3% to 15% (m / v) in an amount such that the mass ratio (w / w) of the biomolecules to the inorganic particles is 0.5 to 5:1, 1 to 3:1, 1 to 2:1 or 1 to 1.5:1.

[0074] In another embodiment, in step (1) of method A or B of the present invention, the preferred inorganic particles are hydroxyapatite, β-tricalcium phosphate (β-TCP) or bioactive glass.

[0075] In another embodiment, in step (1) of method A or B of the present invention, the preferred inorganic particles are bioactive glass, the active ingredients of which are SiO2 by mass ≥ 45%, CaO by mass ≥ 15%, and P2O5 by mass ≥ 3%.

[0076] In another preferred embodiment, in step (1) of method A or B of the present invention, the preferred inorganic particles are bioactive glass selected from 45S5 bioactive glass, 52S4.6 bioactive glass, S53P4 bioactive glass, AW-GC or a combination thereof.

[0077] In another embodiment, in step (1) of method A or B of the present invention, the preferred biomolecule is gelatin.

[0078] In another embodiment, in step (1) of method A or B of the present invention, the preferred biomolecule is collagen, and the collagen is selected from human collagen, animal-derived collagen, recombinant collagen or recombinant humanized collagen.

[0079] In another embodiment, in step (1) of method A or B of the present invention, the preferred biomolecule is recombinant collagen or recombinant humanized collagen, the sequence of which is shown in any one of SEQ ID NO: 5 to SEQ ID NO: 8.

[0080] In another embodiment, in step (1) of method A or step (2) of method B of the present invention, the preferred cross-linking agent is glutaraldehyde, which is added in an amount of 0.1%-1.0% (w / w) of the mass of the biomolecule, preferably 0.2%-0.8%, and more preferably 0.5%-0.7% (w / w).

[0081] In another embodiment, in step (1) of method A of the present invention, the temperature of the cross-linking reaction is controlled to be 35°C to 50°C, more preferably 37°C to 45°C.

[0082] In another embodiment, in step (1) of method A of the present invention, the cross-linking reaction time is controlled to be 10 to 40 minutes.

[0083] In another embodiment, in step (1) of method A of the present invention, the cross-linking reaction time is controlled to be 10 to 30 minutes.

[0084] In another embodiment, in step (1) of method A of the present invention, the cross-linking reaction time is controlled to be 10 to 20 minutes.

[0085] In another embodiment, in step (2) of method A or B of the present invention, the BMP-2 active protein sequence is as shown in SEQ ID NO: 1 to SEQ ID NO: 4.

[0086] In another embodiment, in step (2) of method A or B of the present invention, BMP-2 active protein is added in an amount with a (inorganic particles + biological molecules): BMP-2 active protein mass ratio of 10-500:1, 50-500:1 or 90-200:1.

[0087] In another embodiment, in step (2) of method A of the present invention, the temperature of the BMP-2 active protein during stirring is controlled to be 35°C to 50°C, more preferably 37°C to 45°C.

[0088] In another embodiment, in step (2) of method A of the present invention, the stirring time after adding the BMP-2 active protein is controlled to be 10 to 40 minutes.

[0089] In another embodiment, in step (2) of method A of the present invention, the stirring time after adding the BMP-2 active protein is controlled to be 10 to 30 minutes.

[0090] In another embodiment, in step (2) of method A of the present invention, the stirring time after adding the BMP-2 active protein is controlled to be 10 to 20 minutes.

[0091] In another embodiment, in step (2) of method B of the present invention, the temperature of the cross-linking reaction is controlled to be 25°C to 55°C, preferably 35°C to 50°C, and more preferably 37°C to 45°C.

[0092] In another embodiment, in step (2) of method B of the present invention, the cross-linking reaction time is controlled to be 10 to 40 minutes.

[0093] In another embodiment, in step (2) of method B of the present invention, the cross-linking reaction time is controlled to be 10 to 30 minutes.

[0094] In another embodiment, in step (2) of method B of the present invention, the cross-linking reaction time is controlled to be 10 to 20 minutes.

[0095] In another embodiment, in step (3) of method A or B of the present invention, the molding temperature is 20°C to 35°C, more preferably 24°C to 30°C.

[0096] In another embodiment, in step (3) of method A or B of the present invention, the generated gel is frozen and cross-linked at -20°C to 0°C for 24-120 hours.

[0097] In another embodiment, in step (3) of method A or B of the present invention, the generated gel is frozen and cross-linked at -10°C to -2°C for 72-120 hours.

[0098] In another embodiment, in step (4) of method A or B of the present invention, the frozen cross-linked gel is freeze-dried at -50°C to 0°C to allow the water molecules in the matrix material to gradually sublime, thereby obtaining a composition of a sponge-like material with a porous structure containing BMP-2.

[0099] In another embodiment, in step (4) of method A or B of the present invention, the frozen cross-linked gel is freeze-dried at -45°C to -20°C to allow the water molecules in the matrix material to gradually sublime, thereby obtaining a composition containing BMP-2 and a sponge-like material with a porous structure. In another embodiment, the composition containing BMP-2 prepared according to method A or B of the present invention has a residual cross-linking agent content of less than 0.1% (w / w), preferably less than 0.06% (w / w), and more preferably 0.001% to 0.03% (w / w).

[0100] In another embodiment, the composition comprising BMP-2 prepared according to method A or B of the present invention has a water content of less than 5%, more preferably less than 4%, 3%, 2%, 1%, 0.5% (w / w), or less. In another preferred embodiment, the composition of the present invention has a water content of 0.1% to 3% (w / w), calculated per part by weight of the composition.

[0101] In another embodiment, the composition comprising BMP-2 prepared according to method A or B of the present invention is a sponge-like material having a porous structure with a porosity of 70%, 80%, 85%, 90%, 95% or more.

[0102] In another embodiment, the composition of the present invention has a compressive strength of 0.1 to 2.0 MPa and an elastic modulus of 3 to 20 MPa.

[0103] In another embodiment, the composition of the present invention has a compressive strength of 0.2 to 1.0 MPa and an elastic modulus of 6 to 16 MPa.

[0104] The third aspect of the present invention provides the medical use of the composition containing BMP-2. The present invention discloses the use of the composition containing BMP-2 in the preparation of a drug or medical device for treating, preventing or ameliorating orthopedic diseases.

[0105] As previously described, the composition containing BMP-2 prepared by the present invention comprises a matrix material comprising a porous, spongy material and a porous structure. The composition containing BMP-2 prepared by the present invention is capable of slow release of BMP-2 while maintaining BMP-2 protein activity. Furthermore, the matrix material possesses a certain mechanical strength and is degradable. This composition exhibits excellent biocompatibility and bioactivity, can provide or optimize the physiological microenvironment required for bone tissue regeneration, exhibits osteoconductive and osteoinductive properties, and can induce osteoprogenitor cell differentiation toward osteoblasts. It can be prepared as a pharmaceutical or medical device product for the treatment, prevention, or improvement of orthopedic diseases.

[0106] The composition containing BMP-2 prepared by the present invention can be used to treat, prevent or improve bone, cartilage or vertebrae related diseases, including filling and repairing bone defects, nonunion, delayed bone healing or nonunion, as well as spinal fusion, joint fusion and orthopedic bone graft repair. Bone-related conditions include femoral neck fractures, cervical spine fractures and wrist fractures, defects caused by cancer and injury, disease-related bone loss, such as bone loss after tooth extraction and bone loss related to periodontal disease, weakened bone quality, arthritis, osteolysis and other degenerative changes or healing of bone tissue, such as in the jaw, refractory bone wound healing, delayed bone healing, bone healing accompanied by bone resorption or the need for placement of metal or non-metal implants to stabilize or fix and reconstruct bone tissue; cartilage-related conditions include defects caused by cancer and injury, weakened cartilage quality, arthritis and perforation, degenerative changes of cartilage tissue, and refractory joint wound healing and the need for placement of metal or non-metal implants to stabilize, fix and reconstruct joints; vertebral-related conditions include spinal fractures / diseases or intervertebral disc displacement, fractures or degenerative changes of vertebral tissue, bone and other tissue defects, degeneration and degenerative changes caused by cancer, injury, systemic metabolism, infection or aging, or fixation and reconstructive treatment of vertebral tissue.

[0107] In a preferred embodiment, the present invention provides a structurally stable composition capable of effectively controlled-release BMP-2. In in vitro testing, the three-dimensional network structure formed by cross-linking the composition effectively delayed the material's in vitro degradation period to 8-16 days. BMP-2 binds to the matrix material through chemical bonding and physical adsorption, effectively delaying the release of the drug protein by 8-16 days.

[0108] In another preferred embodiment of the present invention, the composition containing BMP-2 prepared by the present invention has shown effectiveness in animal experiments. A composition containing bone morphogenetic protein 2 (BMP-2) provided by the present invention was implanted in a beagle dog femoral bone defect model. 4 weeks after surgery, the new bone (BV / TV, Tb.N) in the defect area was significantly higher than the normal bone marrow parameters, indicating that the bone content at this stage is high and it is an active period of new bone formation; 12 weeks after surgery, the new bone (BV / TV, Tb.Th, Tb.N) in the defect area was close to the normal bone marrow parameters, indicating that the new bone formation at this stage has reached a mature stage. The results of the study showed that the experimental group case using the composition containing bone morphogenetic protein 2 (BMP-2) not only achieved good new bone formation, but also the new bone formation trend was consistent with the expected release curve of the protein in the composition. Therefore, it is believed that the composition containing bone morphogenetic protein 2 (BMP-2) has a good effect in promoting bone healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] Figure 1: BMP-2 composition protein sustained release curve.

[0110] Figure 2: In vitro anti-disintegration test of BMP-2 composition.

[0111] Figure 3: Representative histopathological images. Animal 1003, Day 29, distal femoral defect, HE staining, objective magnification = 2×. Extensive new bone formation was observed in the defect area, its periphery, and the medullary cavity.

[0112] Figure 4: Representative histopathological images. Animal 1003, Day 29, distal femoral defect, HE staining, objective magnification = 2×. New bone formation and fibrosis are observed around the defect area.

[0113] Figure 5: Bone fracture scan results (1003 - 4 weeks post-operatively). Positions 1, 2, and 3 were selected based on the images: Position 1 is an image of the normal bone marrow cavity; Position 2 is outside the cortical bone, suggesting new bone callus; Position 3 is an image of the fractured bone, showing a higher density than Position 1. Based on the actual surgical situation, this suggests that the filler material has been mixed with the new bone.

[0114] Figure 6: Bone scan results (1002 - 12 weeks post-surgery). Positions 1, 2, and 3 were selected based on the image: Position 1 is the image of the normal bone marrow cavity; Position 2 is outside the bone cortex and is believed to be a newly formed callus; Position 3 is the newly formed bone, which is similar in morphology to Position 1. DETAILED DESCRIPTION

[0115] Example 1-1 Preparation of Composition 1

[0116] (1) Weigh 2.5 g of commercially available gelatin (Rousselot) and add it to a three-necked flask containing 50 mL of pure water. After mechanical stirring (260 rpm) in a 40°C water bath for 30 min, add 2 g of bioactive glass powder (BG component content: SiO2 mass fraction ≥ 45%, CaO mass fraction ≥ 15%, P2O5 mass fraction ≥ 3%, and bioactive glass powder with a particle size of < 90 μm accounts for no less than 90%) and stir for 15 min. Then, add 150 μL of 100 mg / ml glutaraldehyde solution dropwise to the system in small amounts and continue mechanical stirring for 15 min to obtain a cross-linked gelatin bioactive glass suspension.

[0117] (2) Then, add 9 mg of BMP-2 (SEQ ID NO: 3) protein powder and stir in a 40°C water bath for 15 min;

[0118] (3) Cool the flask to about 25°C, then pour the suspension into the mold. The size of the template hole is 1*1*1.5 (cm 3 ); Place the sample poured into the mold to form a transition gel, place it in a -4°C freeze-thaw cabinet, and take it out after frozen cross-linking for 4 days;

[0119] (4) When the freeze dryer is pre-cooled to -45°C, the sample taken out together with the mold is placed in the freeze drying chamber and freeze-dried for 3 days to obtain the sample. The water content is measured to be less than 3%.

[0120] Example 1-2 Preparation of Composition B1

[0121] (1) Weigh 5.0 g of commercially available gelatin (Shanghai Xintian Gelatin Co., Ltd.) and add it to a three-necked flask containing 57 mL of pure water. Let it stand at room temperature for 1-2 hours to swell. After mechanically stirring (280 rpm) in a 50°C water bath for 30 min, add 4 g of bioactive glass powder (BG content: SiO2 mass fraction ≥45%, CaO mass fraction ≥15%, P2O5 mass fraction ≥3%, and bioactive glass powder with a particle size of <90 μm accounts for no less than 90%) and stir for 15 min. Add 45 mg of BMP-2 (SEQ ID NO: 3) protein powder to the system and keep stirring in a 50°C water bath for 15 min.

[0122] (2) Weigh 78.9 mg of 50% glutaraldehyde and dilute it into 42 ml of water. Add the diluted glutaraldehyde to the system to obtain a cross-linked gelatin bioactive glass suspension;

[0123] (3) Then pour the suspension into a silicone mold with a mold size of 10*10*0.5 (cm 3 ); The silicone mold needs to be pre-cooled at 2-8°C in advance. The sample poured into the mold is placed on the freeze dryer plate, the temperature is set to 4°C, and refrigerated for cross-linking for 1 day before removal;

[0124] (4) Take out the silicone mold from the freeze dryer and demould the sample. The demoulded sample is cut into 1*1*0.5 (cm 3 ) block gel, placing the cut gel blocks on a tray;

[0125] (5) When the freeze dryer is pre-cooled to -45°C, the sample and the mold are placed in the freeze drying chamber and freeze-dried for 52 hours to obtain sample B1, whose water content is measured to be less than 5%.

[0126] Example 2 Preparation of other compositions

[0127] According to the preparation process described in Example 1-1, compositions were prepared according to different ratio parameters, as shown in the following table. Except for composition 3 in which the inorganic particles were hydroxyapatite, the inorganic particles of the other compositions were all bioactive glass (BG).

[0128] Multiple groups of compositions were prepared according to the preparation process described in Examples 1-2, compositions were prepared according to different ratio parameters, and performance tests were performed on the compositions.

[0129] The relative activity values ​​of the proteins in the composition are shown in the table below:

[0130] Example 3 Preparation of Composition 3 Containing Hydroxyapatite

[0131] (1) Weigh 2.5 g of commercially available gelatin (Rousselot) and add it to a three-necked flask containing 50 mL of pure water. Stir mechanically (260 rpm) in a 40°C water bath for 30 min, then add 1.5 g of hydroxyapatite (HA) powder and stir for 15 min.

[0132] (2) 150 μL of 100 mg / ml glutaraldehyde solution was added dropwise to the system in small amounts and several times, and mechanical stirring was continued for 15 min to obtain a cross-linked gelatin hydroxyapatite suspension;

[0133] (3) Then, 40.2 mg of BMP-2 (SEQ IN NO: 3) protein powder was added and stirred for 15 min;

[0134] (4) Cool the flask to about 25°C, then pour the suspension into the mold. The size of the template hole is 1*1*1.5 (cm 3 ); Place the sample poured into the mold in a freeze-thaw cabinet at -4°C after forming the transition gel, and remove it after 4 days of frozen cross-linking; Place the sample poured into the mold in a freeze-thaw cabinet at -4°C after 4 days of frozen cross-linking;

[0135] (5) When the freeze dryer is pre-cooled to -45°C, the sample is taken out together with the mold and placed in the freeze drying chamber. The sample is freeze-dried for 3 days and the water content is measured to be less than 5%.

[0136] Example 4 Curve detection of sustained release of BMP-2 protein in vitro in the composition

[0137] (1) Take one prepared sample and add it to a 50 mL centrifuge tube. Add 10 mL of 0.005 M PBS solution to each tube, and repeat for three samples. Place all the centrifuge tubes in a shaker at 37°C and 100 rpm.

[0138] (2) Sampling was performed at different times, the actual volume was recorded each time, and fresh 10 mL of 0.01 M PBS medium was added;

[0139] (3) Add an equal amount of 0.2 M HCl to the sampled liquid, vortex mix evenly, and then send the sample to detect the BMP-2 concentration;

[0140] (4) According to the release amount of BMP-2 at different times, a sustained-release curve of BMP-2 was drawn. The sustained-release curves of the protein of composition 1 and composition 2 are shown in FIG1 .

[0141] The results in FIG1 show that the gelatin / BG (1.25) protein of compositions 1 and 2 is sustained-released to 50% within one week, with a sustained-release period of 8-16 days and a cumulative release of 55-60%.

[0142] Example 5 Detection of BMP-2 activity in the composition

[0143] The relative in vitro activities of compositions 2, 5 and 6 were determined according to the following method.

[0144] C2C12 cells were passaged in DMEM medium containing 10% FBS 2-3 times per week. When passaged or plated for activity experiments, the cell culture medium was discarded, the cell surface was washed once with DPBS, and then 2-5 ml of 0.25% trypsin was added to digest at 37°C for 3-5 minutes. The cells were then resuspended in fresh medium. The cell suspension was plated at 100 μl / well in a 96-well plate at a cell density of 5×10 4cells / ml, and the edges of the cell plate were sealed with DMEM culture medium. After the cells in the 96-well plate adhered to the wall, the extract of the composition with different dilution concentrations (3000ng / ml as the starting point, 2-fold gradient dilution, a total of 10 gradients) and the control (rhBMP-2 original powder dissolved in 2% acetic acid solution) were added. After incubation in a 37°C, 5% CO2 incubator for 3 days, the cells were lysed and the ALP substrate (disodium p-nitrophenolphosphate) was added to detect the ALP content produced by the cells stimulated by the extract of the composition. The cell plate was placed on a microplate reader to measure the absorbance value at a main wavelength of 405nm and a secondary wavelength of 490nm. The 4-parameter fitting curves of the samples and standards were read, and the relative activity of the samples was calculated based on the Relative Potency%.

[0145] The relative activity values ​​of the proteins in the composition are shown in the table below.

[0146] Example 6 Composition Porosity Test

[0147] The porosity of compositions 1, 7, and 8 was measured using a density balance according to the Archimedean drainage method (reference Bioact Mater. 2021; 6(10): 3396-410). The specific steps are as follows:

[0148] (1) Weigh the freeze-dried sample and record it as W1;

[0149] (2) Place the sample in anhydrous ethanol, then vacuum until the sample is filled with anhydrous ethanol. Take out the sample, gently wipe off the excess anhydrous ethanol on the surface with saturated alcohol-wet gauze, and then weigh it, which is recorded as W2;

[0150] (3) Place the sample in a density balance (the density balance is filled with anhydrous ethanol) and weigh the sample, recording it as W3. Calculate according to the formula: Porosity = (W1-W) / (W1-W2).

[0151] The measured porosity data are shown in the table below:

[0152] Example 7 In vitro anti-collapse test of the composition

[0153] Composition 1 Anti-collapse Test: 0.5024g, 0.5103g, and 0.5089g of the freeze-dried samples were weighed and placed into 50ml centrifuge tubes, respectively. 10ml of PBS solution was added, and the tubes were shaken at 37°C and 100rpm for observation. The anti-collapse data for the samples are summarized in Figure 2 below. 50% of the samples collapsed after 5 days, and completely collapsed after approximately 10 days. This indicates that the three-dimensional network structure formed by cross-linking the composition effectively delays the in vitro degradation period of the material to 8-16 days.

[0154] Example 8 Composition Strength Determination

[0155] Using the preparation conditions of Composition 2, 18 samples were prepared for mechanical strength measurement.

[0156] The test was conducted using a microcomputer-controlled electronic universal material testing machine at a test speed of 2.0 mm / min, with 5% fracture (95% remaining) as the end point. The mechanical strength test data of the 18 samples are shown in the table below:

[0157] Example 9 Determination of glutaraldehyde residues in the composition

[0158] Two batches of samples of composition 2 were prepared for the detection of glutaraldehyde (GA) residues.

[0159] Extraction conditions reference standard:

[0160] 1. DB13 / T 5127.11-2019 Determination of toxic and hazardous substances in extracts of polymer materials for implantable medical devices Part 11: Glutaraldehyde migration by high performance liquid chromatography.

[0161] 2. GBT 16886.12-2005 Biological evaluation of medical devices Part 12: Sample preparation and reference samples.

[0162] Glutaraldehyde content detection method: refer to the "Chinese Pharmacopoeia" 2020, dilute glutaraldehyde solution [content determination].

[0163] Glutaraldehyde residue detection: refer to the "Chinese Pharmacopoeia" 2020, 3204 glutaraldehyde residue determination method.

[0164] The residual glutaraldehyde in Composition 2 was extracted using physiological saline at 0.2 g / mL in accordance with GB / T 16886.12, and the measured residual concentration was 13.0 ng / g, which meets the statutory standard (calculated according to the reference standard GB / T 16886.17-2005 "Biological Evaluation of Medical Devices Part 17: Establishment of Permissible Limits of Leachable Matter", the residual glutaraldehyde content should meet the requirement of <1 mg / g).

[0165] Example 10 Animal Experiment 1

[0166] The composition prepared in Example 1-1 was implanted into a bone defect in a beagle dog, and the osteogenesis level of the composition was evaluated. A wedge-shaped femoral bone defect was modeled on the left hind limb of the test animal. The defect site was 20 mm in length and 10-20 mm in depth (incomplete truncation, leaving one side of the cortical bone intact), and the composition 1 prepared in Example 1 was implanted. Blood was collected before surgery and at the end of the autopsy for hematological examination, serum biochemistry, and coagulation index testing; X-ray scans were performed on the left hind limb of the test animal before surgery, immediately after surgery, and at the end of the autopsy; two test animals (1002, 1003) were taken for Micro-CT scanning of the left bone defect site after autopsy to evaluate bone growth, and tissue morphometric analysis, material degradation release, and new bone formation were performed after the scan.

[0167] Pathological examination of the experimental group (animal 1003) four weeks after surgery revealed Goldner's trichrome staining and HE-stained whole-section scans of sections from the proximal, mid, and distal ends of the defect. Numerous spongy or pine-needle-like trabeculae were observed within the cortical bone and medullary cavity, indicating substantial new bone formation within the defect (Figures 3 and 4). Histomorphometric analysis revealed that the average new bone formation area across the defect at different sections accounted for 32.14% of the total defect area, demonstrating the material's excellent repair efficacy in this wedge-shaped defect model.

[0168] The table below shows bone volume fraction, trabecular thickness, trabecular number, and trabecular separation data at 4 and 12 weeks post-surgery. At 4 weeks post-surgery (Figure 5, Animal 1003), new bone formation (BV / TV, Tb.N) in the defect region was significantly higher than normal bone marrow parameters, indicating a period of active bone formation with high bone content. At 12 weeks post-surgery (Figure 6, Animal 1002), new bone formation (BV / TV, Tb.Th, Tb.N) in the defect region was similar to normal bone marrow parameters, making it difficult to distinguish between new bone and autologous bone on imaging, indicating that new bone formation had reached maturity at this stage.

[0169] Micro CT data

[0170] Example 11 Animal Experiment 2

[0171] Composition B2 prepared in Example 1-2 was subjected to a mouse muscle pouch osteogenesis induction test, the steps being as follows:

[0172] (1) Anesthetize mice: Use a disposable syringe to administer 2,2,2-tribromoethanol solution at a dose of 0.2 mL / 10 g intraperitoneally. Note: During injection, be sure to prevent the anesthetic from leaking through the needle.

[0173] (2) Place the anesthetized mouse prone on a surgical tray, trim the hair on the outside of the left thigh (note: do not trim the skin), disinfect the hair removal area with a 75% alcohol cotton ball, gently cut the skin with a sterile scalpel, and use curved hemostats to bluntly separate the skin and fascia for about 1 cm until the muscle bag is visible (note: prevent bleeding during blunt separation. If bleeding occurs, the mouse will be eliminated).

[0174] (3) Implant approximately 5 mg of material into the muscle bag of each mouse. Implant the divided n portions of material into n mice. Suture the skin and fascia once with a 1 / 2 circular needle (be careful to prevent bleeding). Finally, mark with picric acid and record.

[0175] (4) After 21 days of normal feeding, the mice were sacrificed, X-rayed, and dissected to remove the new bone in the implanted area, completely separating the non-bone portion. If the new bone could not be separated, the data were not included in the statistical sample. The mice were dried at room temperature for 1 hour and then weighed. The total weight of the induced new bone was calculated for each group.

[0176] (5)

[0177] Test results

[0178] The results showed that the osteogenic activity of composition B2 in mice was 4693 mg new bone / 400 mg composition.

Claims

1. A composition comprising BMP-2, comprising: (1) BMP-2 active protein; (2) a matrix material, wherein the matrix material comprises biomolecules and inorganic particles; wherein the biomolecules are selected from gelatin, collagen, elastin or a combination thereof; and the inorganic particles are selected from any one or a combination of the following calcium phosphate compounds: hydroxyapatite, monocalcium phosphate, dicalcium phosphate, tricalcium phosphate (TCP, Ca3(PO4)2), tetracalcium phosphate (Ca4(PO4)2O), dicalcium diphosphate (Ca2P2O7), calcium tripolyphosphate (Ca5(P3O 10 )2) or bioactive glass; The BMP-2 active protein is diffusely distributed in a matrix material, the matrix material is a sponge-like material with a porous structure, and the weight ratio (w / w) of the matrix material to the BMP-2 active protein is 10 to 1000:

1.

2. The composition according to claim 1, wherein: The BMP-2 active protein sequence in the composition is as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 4 or a mutant thereof.

3. The composition according to claim 2, characterized in that: The BMP-2 mutant has at least 90% of the biological activity of the BMP-2 protein through conservative sequence modification such as amino acid substitution, addition and / or deletion.

4. The composition according to claim 3, characterized in that: The BMP-2 mutation consists of an amino acid sequence of SEQ ID No: 1 comprising two or three amino acid substitutions, wherein the first and second amino acid substitutions are present at positions selected from S24 and N59, N59 and N102, or P36 and N59 of SEQ ID No: 1, or the three amino acid substitutions are present at positions S24, N59 and N102 of SEQ ID No:

1.

5. The composition according to claim 1, wherein: The BMP-2 active protein sequence in the composition is shown in SEQ ID NO:

3.

6. The composition according to claim 1, wherein: The collagen in the composition is human collagen, animal-derived collagen, recombinant collagen or recombinant humanized collagen.

7. The composition according to claim 1, wherein: The biological molecules in the composition are gelatin, recombinant collagen or recombinant humanized collagen.

8. The composition according to claim 7, characterized in that: The sequence of the recombinant collagen or recombinant humanized collagen in the composition is shown in any one of SEQ ID NO: 5 to SEQ ID NO:

8.

9. The composition according to claim 1, wherein: The inorganic particles in the matrix material of the composition are selected from hydroxyapatite, tricalcium phosphate (TCP, Ca3(PO4)2), bioactive glass or a combination thereof.

10. The composition according to claim 9, characterized in that: The inorganic tricalcium phosphate particles in the matrix material of the composition are β-tricalcium phosphate (β-TCP).

11. The composition according to claim 9, characterized in that: The inorganic particles in the matrix material of the composition are bioactive glass.

12. The composition according to claim 11, characterized in that: The raw materials of the bioactive glass in the composition contain effective components such as SiO2 with a mass fraction of ≥45%, CaO with a mass fraction of ≥15%, and P2O5 with a mass fraction of ≥3%.

13. The composition according to claim 11, characterized in that: The bioactive glass in the composition is selected from 45S5 bioactive glass, 52S4.6 bioactive glass, S53P4 bioactive glass, AW-GC or a combination thereof.

14. The composition according to claim 1, wherein: In the composition, the inorganic particles in the matrix material have an average particle size of 10 to 150 μm.

15. The composition according to claim 14, characterized in that: In the composition, the inorganic particles in the matrix material have an average particle size of 30 to 120 μm.

16. The composition according to claim 15, characterized in that: In the composition, the inorganic particles in the matrix material have an average particle size of 50 to 100 μm.

17. The composition according to claim 16, characterized in that: In the composition, the inorganic particles in the matrix material have an average particle size of 70 to 90 μm.

18. The composition according to claim 1, characterized in that: In the composition, the weight ratio (w / w) of the biomolecules and the inorganic particles in the matrix material is 0.5 to 10:

1.

19. The composition according to claim 18, characterized in that: In the composition, the weight ratio (w / w) of the biomolecules and the inorganic particles in the matrix material is 0.5 to 5:

1.

20. The composition according to claim 19, characterized in that: In the composition, the weight ratio (w / w) of the biomolecules and the inorganic particles in the matrix material is 1 to 3:

1.

21. The composition according to claim 20, characterized in that: In the composition, the weight ratio (w / w) of the biomolecules and the inorganic particles in the matrix material is 1 to 2:

1.

22. The composition according to claim 21, characterized in that: In the composition, the weight ratio (w / w) of the biomolecules and the inorganic particles in the matrix material is 1 to 1.5:

1.

23. The composition according to claim 1, wherein: In the composition, the weight ratio (w / w) of the matrix material to the BMP-2 active protein is 10 to 500:

1.

24. The composition according to claim 23, characterized in that: In the composition, the weight ratio (w / w) of the matrix material to the BMP-2 active protein is 50-500:

1.

25. The composition according to claim 24, characterized in that: In the composition, the weight ratio (w / w) of the matrix material to the BMP-2 active protein is 90-200:

1.

26. The composition according to claim 1, wherein: The composition further comprises a cross-linking agent, and the residual cross-linking agent accounts for less than 0.1% (w / w).

27. The composition according to claim 26, characterized in that: In the composition, the residual cross-linking agent accounts for less than 0.06% (w / w).

28. The composition according to claim 27, characterized in that: In the composition, the residual crosslinking agent accounts for less than 0.03% (w / w).

29. The composition according to any one of claims 26 to 28, characterized in that: The residual cross-linking agent is formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, glyceraldehyde, vanillin or glutaraldehyde.

30. The composition according to any one of claims 1 to 25, characterized in that: The composition has a water content of less than 5% (w / w).

31. The composition according to claim 30, characterized in that: The composition has a water content of 4%, 3%, 2%, 1%, 0.5% (w / w) or less.

32. The composition according to claim 31, characterized in that: The water content of the composition is 0.1% to 3% (w / w).

33. The composition according to any one of claims 1 to 25, characterized in that: The composition is a sponge-like material with a pore structure and a porosity of 70% or more.

34. The composition according to claim 33, characterized in that: The composition is a sponge-like material with a pore structure and a porosity of 80% or more.

35. The composition according to claim 34, characterized in that: The composition is a sponge-like material with a pore structure and a porosity of 85% or more.

36. The composition according to claim 35, characterized in that: The composition is a sponge-like material with a pore structure and a porosity of 90% or more.

37. The composition according to any one of claims 1 to 25, characterized in that: The composition has a compressive strength of 0.1 to 2.0 MPa and an elastic modulus of 3 to 20 MPa.

38. The composition according to any one of claims 1 to 25, characterized in that: The composition has a compressive strength of 0.2 to 1.0 MPa and an elastic modulus of 6 to 16 MPa.

39. A composition comprising BMP-2, comprising: (1) BMP-2 active protein, the sequence of which is shown in one of SEQ ID NO: 1 to SEQ ID NO: 4 or a mutant thereof; (2) a matrix material, wherein the matrix material comprises biomolecules and inorganic particles, and the weight ratio (w / w) of the biomolecules to the inorganic particles is 0.5 to 10:1; wherein the biomolecule is gelatin or collagen, and the collagen is selected from human collagen, animal-derived collagen, recombinant collagen, or recombinant humanized collagen; and the inorganic particles are selected from any one or a combination of the following calcium-phosphorus compounds: hydroxyapatite, β-tricalcium phosphate, or bioactive glass; The BMP-2 active protein is diffusely distributed in a matrix material, the matrix material is a sponge-like material with a porous structure, and the weight ratio (w / w) of the matrix material to the BMP-2 active protein is 10 to 1000:

1.

40. A composition comprising BMP-2, comprising: (1) BMP-2 active protein, the sequence of which is shown in SEQ ID NO: 3 or a mutant thereof; (2) a matrix material, wherein the matrix material comprises biomolecules and inorganic particles, wherein the weight ratio (w / w) of the biomolecules to the inorganic particles is 1 to 3:1; wherein the biomolecule is gelatin or a recombinant humanized collagen having an amino acid sequence as shown in any one of SEQ ID NO:5 to SEQ ID NO:8; and the inorganic particles are selected from bioactive glass; The BMP-2 active protein is diffusely distributed in the matrix material, which is a sponge-like material with a porous structure and a porosity of 70% or higher, and the weight ratio (w / w) of the matrix material to the BMP-2 active protein is 90 to 200:

1.

41. A method A for preparing the composition comprising BMP-2 according to claim 1, comprising the following steps: (1) adding inorganic particles to a biomolecule solution having a concentration of 3% to 15% (m / v) at a biomolecule to inorganic particle mass ratio (w / w) of 0.5 to 10:1; stirring uniformly, adding a crosslinking agent and stirring to react to form a suspension; (2) Add BMP-2 active protein in an amount of (inorganic particles + biomolecules): BMP-2 active protein mass ratio of 10 to 1000:1 and stir evenly; (3) injecting the stirred reaction system into a mold and placing it in a mold for molding, and freezing and crosslinking the generated gel for 24-120 hours; (4) freeze-drying the gel to obtain the composition containing BMP-2; wherein, The BMP-2 active protein is diffusely distributed in the matrix material, and the matrix material is a sponge-like material with a pore structure.

42. A method B for preparing the composition comprising BMP-2 according to claim 1, comprising the following steps: (1) adding inorganic particles to a biomolecule solution having a concentration of 3% to 15% (m / v) at a biomolecule:inorganic particle mass ratio (w / w) of 0.5 to 10:1 and stirring uniformly; (2) adding BMP-2 active protein in an amount of (inorganic particles + biomolecules): BMP-2 active protein mass ratio of 10 to 1000:1, and then adding a crosslinking agent and stirring to react to form a suspension; (3) injecting the stirred reaction system into a mold for forming, and refrigerating and cross-linking the generated gel for 24-120 hours; (4) freeze-drying the gel to obtain the composition containing BMP-2; wherein, The BMP-2 active protein is diffusely distributed in the matrix material, and the matrix material is a sponge-like material with a pore structure.

43. The method according to claim 41 or 42, characterized in that: In step (1) of the method, the biomolecule is selected from gelatin, collagen, elastin or a combination thereof; the inorganic particles are selected from any one or a combination of the following calcium phosphate compounds: hydroxyapatite, monocalcium phosphate, dicalcium phosphate, tricalcium phosphate (TCP, Ca3(PO4)2), tetracalcium phosphate (Ca4(PO4)2O), dicalcium diphosphate (Ca2P2O7), calcium tripolyphosphate (Ca5(P3O 10 )2) or bioactive glass.

44. The method according to claim 41 or 42, characterized in that: In step (1) of the method, inorganic particles are added to a biomolecule solution having a concentration of 3% to 15% (m / v) according to a biomolecule:inorganic particle mass ratio (w / w) of 0.5 to 5:1, 1 to 3:1, 1 to 2:1 or 1 to 1.5:

1.

45. The method according to claim 41 or 42, characterized in that: In step (1) of the method, the inorganic particles are hydroxyapatite, β-tricalcium phosphate (β-TCP) or bioactive glass.

46. ​​The method according to claim 45, wherein: In step (1) of the method, the inorganic particles are bioactive glass, and the mass fraction of the effective components SiO2 is ≥45%, the mass fraction of CaO is ≥15%, and the mass fraction of P2O5 is ≥3%.

47. The method according to claim 45, wherein: In step (1) of the method, the inorganic particles are bioactive glass selected from 45S5 bioactive glass, 52S4.6 bioactive glass, S53P4 bioactive glass, AW-GC or a combination thereof.

48. The method according to claim 41 or 42, characterized in that: In step (1) of the method, the biomolecule is collagen, which is selected from human collagen, animal-derived collagen, recombinant collagen or recombinant humanized collagen.

49. The method according to claim 41 or 42, characterized in that: In step (1) of the method, the biomolecule is gelatin, recombinant collagen or recombinant humanized collagen.

50. The method according to claim 49, wherein: In step (1) of the method, the biomolecule is recombinant collagen or recombinant humanized collagen, and its sequence is shown in any one of SEQ ID NO: 5 to SEQ ID NO:

8.

51. The method according to claim 41 or 42, wherein: In step (1) of method A or step (2) of method B, the cross-linking agent is selected from formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, glyceraldehyde, vanillin or glutaraldehyde.

52. The method according to claim 51, characterized in that: In step (1) of method A or step (2) of method B, the cross-linking agent is glutaraldehyde.

53. The method according to claim 51, wherein: In step (1) of method A or step (2) of method B, the cross-linking agent can be added in any manner, and the amount added is 0.1% to 1% (w / w) of the mass of the biomolecule.

54. The method according to claim 53, wherein: In step (1) of method A or step (2) of method B, the cross-linking agent can be added in any manner, and the amount added is 0.2% to 0.8% (w / w) of the mass of the biomolecule.

55. The method according to claim 54, characterized in that: In step (1) of method A or step (2) of method B, the cross-linking agent can be added in any manner, and the amount added is 0.5% to 0.7% (w / w) of the mass of the biomolecule.

56. The method according to claim 41, wherein: In step (1) of the method, the temperature of the cross-linking reaction is controlled to be 35°C to 50°C.

57. The method according to claim 56, characterized in that: In step (1) of the method, the temperature of the cross-linking reaction is controlled to be 37°C to 45°C.

58. The method of claim 41, wherein: In step (1) of the method, the cross-linking reaction time is controlled to be 10 to 40 minutes.

59. The method according to claim 58, wherein: In step (1) of the method, the cross-linking reaction time is controlled to be 10 to 30 minutes.

60. The method according to claim 59, wherein: In step (1) of the method, the cross-linking reaction time is controlled to be 10 to 20 minutes.

61. The method according to claim 42, wherein: In step (2) of the method, the temperature of the cross-linking reaction is controlled to be 25°C to 55°C.

62. The method according to claim 61, characterized in that: In step (2) of the method, the temperature of the cross-linking reaction is controlled to be 35°C to 50°C.

63. The method according to claim 62, wherein: In step (2) of the method, the temperature of the cross-linking reaction is controlled to be 37°C to 45°C.

64. The method of claim 42, wherein: In step (2) of the method, the cross-linking reaction time is controlled to be 10 to 40 minutes.

65. The method according to claim 64, characterized in that: In step (2) of the method, the cross-linking reaction time is controlled to be 10 to 30 minutes.

66. The method according to claim 65, characterized in that: In step (2) of the method, the cross-linking reaction time is controlled to be 10 to 20 minutes.

67. The method according to claim 41 or 42, characterized in that: In step (2) of the method, the BMP-2 active protein sequence is as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or a mutant thereof.

68. The method according to claim 67, wherein: In step (2) of the method, the BMP-2 mutant is modified by conservative sequence such as amino acid substitution, addition and / or deletion and has at least 90% of the biological activity of the BMP-2 protein.

69. The method according to claim 67, wherein: In step (2) of the method, the BMP-2 mutation consists of an amino acid sequence of SEQ ID No: 1 comprising two or three amino acid substitutions, wherein the first and second amino acid substitutions are present at positions selected from S24 and N59, N59 and N102, or P36 and N59 of SEQ ID No: 1, or the three amino acid substitutions are present at positions S24, N59 and N102 of SEQ ID No:

1.

70. The method of claim 67, wherein: In step (2) of the method, the BMP-2 active protein sequence is shown in any one of SEQ ID NO: 1 to SEQ ID NO:

4.

71. The method according to claim 41 or 42, characterized in that: In step (2) of the method, BMP-2 active protein is added in an amount with a (inorganic particles+biomolecules):BMP-2 active protein mass ratio of 10-500:1, 50-500:1 or 90-200:

1.

72. The method according to claim 41, wherein: In step (2) of the method, the temperature of the BMP-2 active protein during stirring is controlled to be 35° C. to 50° C.

73. The method according to claim 72, characterized in that: In step (2) of the method, the temperature of the BMP-2 active protein during stirring is controlled at 37° C. to 45° C.

74. The method of claim 41, wherein: In step (2) of the method, the stirring time after adding the BMP-2 active protein is controlled to be 10 to 40 minutes.

75. The method according to claim 74, characterized in that: In step (2) of the method, the stirring time after adding the BMP-2 active protein is controlled to be 10 to 30 minutes.

76. The method according to claim 75, characterized in that: In step (2) of the method, the stirring time after adding the BMP-2 active protein is controlled to be 10 to 20 minutes.

77. The method according to claim 41 or 42, characterized in that: In step (3) of the method, the molding temperature is 20°C to 35°C.

78. The method according to claim 77, characterized in that: In step (3) of the method, the placing and molding temperature is 24°C to 30°C.

79. The method according to claim 41 or 42, characterized in that: In step (3) of the method, the generated gel is frozen and cross-linked at -20°C to 0°C for 24-120 hours.

80. The method according to claim 79, wherein: In step (3) of the method, the generated gel is frozen and cross-linked at -10°C to -2°C for 72-120 hours.

81. The method according to claim 41 or 42, wherein: In step (4) of the method, the frozen cross-linked gel is freeze-dried at -50°C to 0°C to allow the water molecules in the matrix material to gradually sublime, thereby obtaining a composition of a sponge-like material with a porous structure containing BMP-2.

82. The method according to claim 81, characterized in that: In step (4) of the method, the frozen cross-linked gel is freeze-dried at -45°C to -20°C to allow the water molecules in the matrix material to gradually sublime, thereby obtaining a composition of a sponge-like material with a porous structure containing BMP-2.

83. The method according to claim 41 or 42, characterized in that: The composition containing BMP-2 prepared according to the method has a residual cross-linking agent content of less than 0.1% (w / w).

84. The method according to claim 83, wherein: The composition containing BMP-2 prepared according to the method has a residual cross-linking agent content of less than 0.06% (w / w).

85. The method according to claim 84, characterized in that: The composition containing BMP-2 prepared according to the method has a residual cross-linking agent content of 0.001% to 0.03% (w / w).

86. The method according to claim 41 or 42, wherein: The composition containing BMP-2 prepared according to the method has a water content of less than 5%.

87. The method according to claim 86, characterized in that: The composition containing BMP-2 prepared according to the method has a water content of less than 4%, 3%, 2%, 1%, 0.5% (w / w) or lower.

88. The method according to claim 87, wherein: The composition containing BMP-2 prepared according to the method has a water content of 0.1% to 3% (w / w).

89. The method according to claim 41 or 42, characterized in that: The composition containing BMP-2 prepared according to the method is a sponge-like material with a porous structure and a porosity of 70%, 80%, 85%, 90%, 95% or higher.

90. The method according to claim 41 or 42, characterized in that: The composition containing BMP-2 prepared according to the method has a compressive strength of 0.1 to 2.0 MPa and an elastic modulus of 3 to 20 MPa.

91. The method according to claim 90, characterized in that: The composition containing BMP-2 prepared according to the method has a compressive strength of 0.2 to 1.0 MPa and an elastic modulus of 6 to 16 MPa.

92. Use of the composition according to any one of claims 1 to 40 or the composition comprising BMP-2 prepared according to the method according to any one of claims 41 to 91 in the preparation of a drug or medical device for treating, preventing or ameliorating orthopedic diseases.

93. The use according to claim 92, characterized in that: The composition comprising BMP-2 can be used to treat, prevent or improve bone, cartilage or vertebrae related diseases.

94. The use according to claim 93, characterized in that: The applications include filling and repairing bone defects, bone nonunion, delayed bone union or nonunion, as well as spinal fusion, joint fusion and orthopedic bone grafting repair.

95. The use according to claim 93, characterized in that: The bone-related conditions include femoral neck fractures, cervical spine fractures and wrist fractures, defects caused by cancer and injury, disease-related bone loss, such as bone loss after tooth extraction and bone loss related to periodontal disease, weakened bone quality, arthritis, osteolysis and other degenerative changes or healing of bone tissue, such as in the jaw, refractory bone wound healing, delayed bone healing, bone healing accompanied by bone resorption or the need to place metal and non-metal implants to stabilize or fix and reconstruct bone tissue; cartilage-related conditions include defects caused by cancer and injury, weakened cartilage quality, arthritis and perforation, degenerative changes of cartilage tissue, and refractory joint wound healing and the need to place metal or non-metal implants to stabilize, fix and reconstruct joints; vertebral-related conditions include spinal fractures / diseases or intervertebral disc displacement, fractures or degenerative changes of vertebral tissue, bone and other tissue defects, degeneration and degenerative changes caused by cancer, injury, systemic metabolism, infection or aging, or fixation and reconstructive treatment of vertebral tissue.

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