Bone repair material as well as preparation method and application thereof

Abstract

The invention discloses a bone repair material as well as a preparation method and application thereof. The bone repair material is prepared from recombinant human collagen, recombinant human bone morphogenetic protein 2 and hydroxyapatite, wherein the ratio of the inorganic matter to the organic matter in the bone repair material is (64-66): (36-34). The bone repair material disclosed by the invention takes the recombinant human collagen as a core raw material, so that the components and the structure of the prepared material are ensured to be highly consistent with those of natural bone tissues; secondly, by adding the recombinant human bone morphogenetic protein 2, the osteogenic activity of the material is further enhanced. The finally obtained recombinant human collagen active bone repair material completely retains the triple helix structure of the recombinant human collagen, so that the recombinant human collagen active bone repair material has complete biological activity, and also has the outstanding advantages of good osteogenic activity, no disease and virus transmission risk and no immunogenicity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and in particular, the present application relates to a bone repair material and a preparation method and application thereof. BACKGROUND

[0002] Bone repair materials are mainly divided into natural bone repair materials and artificial bone repair materials, wherein the natural bone repair materials are divided into allogeneic bone, xenogeneic bone and decalcified bone matrix, and the artificial bone repair materials are divided into metal materials, inorganic non-metallic materials (biological ceramics, calcium sulfate bone cement, calcium phosphate bone cement, biological glass, etc.), high polymer materials, composite materials and tissue engineering materials, etc. Bone repair materials are currently mainly used in the fields of orthopedics, neurosurgery and oral medicine, and the orthopedic applications include spinal orthopedic surgery, trauma orthopedic surgery, joint orthopedic surgery, etc. The neurosurgical applications include skull base limited surgery, skull base elective surgery, saddle area elective surgery, bone flap decompression surgery and craniotomy surgery, etc. The applications in the field of oral medicine are mainly concentrated in dental defect, tooth loss and other dental implant surgery.

[0003] The substance filled between cells in bone tissue is called bone matrix, which includes two components of organic matter and inorganic matter. The organic matter accounts for about 35% of the dry weight of bone tissue, including a large amount of collagen fibers, i.e. bone collagen, which is synthesized by osteoblasts and accounts for 90% of the organic matter. The inorganic matter component, also known as bone salt or inorganic salt, accounts for about 65% of the dry weight of bone tissue, mainly including calcium, phosphorus and magnesium, etc., mainly existing in the form of hydroxyapatite (Ca 10 (PO4)6(OH)2, Hydroxyapatite crystal, HAp) crystals, which are distributed in the organic matter of the bone. In the main inorganic components of human bone tissue, the molar ratio of calcium to phosphorus (Ca / P ratio) is 1.67. The ratio of organic matter to inorganic matter in the bone matrix is different with age, and with the increase of age, the inorganic matter accounts for an increasing proportion, and the bone gradually becomes brittle and hard, and the elasticity and impact resistance continuously decrease.

[0004] Bone defect repair materials are one of the most clinically demanded biomedical materials. At present, most artificial bone repair materials are prepared by mineralization of animal collagen, however, animal-derived collagen has the risk of disease and virus transmission, and the heterogeneity of the material also leads to the risk of immunogenicity, in addition, the extraction method limits the production of a large amount of non-uniform collagen, thus greatly limiting the application of collagen in the field of biomaterials. SUMMARY

[0005] The present application aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, the present application proposes a bone repair material, which has consistent composition and structure with natural bone tissue, has the ability to directly bond with bone, does not interfere with the activity of bone cells on its surface and the physiological regeneration of surrounding bone tissue, and has a bone conduction promoting effect on the degradation and absorption process of bone tissue.

[0006] The present application is based on the following findings of the inventor: Collagen is the main component of animal connective tissue and also the most abundant and widely distributed functional protein in mammals, accounting for 25-30% of the total protein. As an important structural protein in the human body, collagen has a wide range of applications in tissue repair and medical and beauty filling fields. Traditional animal-derived collagen has the risk of disease and virus transmission and also has certain immunogenicity problems. Recombinant collagen overcomes the shortcomings and deficiencies of animal-derived collagen, but still faces a series of technical problems. For example, recombinant humanized collagen still needs to improve its mechanical properties and complete biological activity due to the lack of complete triple helix structure and proline hydroxylation modification. Recombinant human collagen perfectly solves the defects and deficiencies of recombinant humanized collagen, not only has the same amino acid sequence composition as natural human collagen, but also has the same full-length triple helix structure and proline hydroxylation modification as natural human collagen, has stable mechanical properties and complete biological activity, and more importantly, can be prepared on a large scale in vitro using a cell factory.

[0007] The unique structural characteristics, physicochemical properties, excellent biocompatibility, and biodegradability of collagen make it an ideal biomaterial, which has been widely used in difficult wound healing, peripheral skin care, dermal tissue filling, artificial dermal substitutes, bone or cartilage tissue repair, and hemostasis in surgical operations.

[0008] Recombinant human collagen is developed by simulating the production process of natural collagen in the human body, and has a full-length triple helix structure. It has the same hydroxyproline content and higher structure as natural human collagen, has strong stability, no immunogenicity, uniform structure, and other outstanding advantages. Compared with animal collagen, it overcomes the disadvantages of limited source, possible disease and virus transmission risk, etc. Compared with recombinant humanized collagen, it has complete biological activity, high mechanical strength, and excellent support performance in vivo application, and can completely replace animal collagen in the application of bone repair materials.

[0009] Bone morphogenetic protein (BMP) is initially found in the decalcified bone matrix and is considered to be a substance that can induce new bone formation. More than 20 BMPs have been found so far, which are named BMP-1, BMP-2, BMP-3, …, BMP-X respectively. Among them, BMP-2 is the strongest growth factor in all bone morphogenetic proteins for inducing bone formation. The full length of BMP-2 is composed of 396 amino acids, and the mature BMP-2 monomer is composed of 114 aa. It contains 7 cysteines, forms a homodimer through a pair of inter-chain disulfide bonds, and the remaining 6 cysteines in each monomer form 3 pairs of intra-chain disulfide bonds. Active BMP-2 exists in the form of a homodimer.

[0010] Therefore, in the first aspect of the present application, the present application provides a bone repair material. According to the embodiments of the present application, the bone repair material comprises: recombinant human collagen, recombinant human bone morphogenetic protein 2 (rhBMP-2) and hydroxyapatite; wherein the ratio of inorganic matter to organic matter in the bone repair material is (64-66):(36-34). The bone repair material of the present application takes recombinant human collagen as the core raw material, ensuring that the composition and structure of the prepared material are highly consistent with those of natural bone tissue. Secondly, by adding recombinant human bone morphogenetic protein 2, the osteogenic activity of the material is further enhanced. The final recombinant human collagen active bone repair material retains the triple helix structure of recombinant human collagen, thereby having its complete biological activity, and at the same time has the outstanding advantages of good osteogenic activity, no risk of disease and virus transmission, and no immunogenicity.

[0011] In the second aspect of the present application, the present application provides a method for preparing the bone repair material of the first aspect. According to the embodiments of the present application, the method comprises: mineralizing recombinant human collagen to obtain mineralized collagen; performing first mixing treatment on the mineralized collagen and recombinant human bone morphogenetic protein 2; and sequentially performing standing treatment and first centrifugal treatment on the first mixing treatment product to obtain the bone repair material. Thus, by simulating the composition and formation process of natural human bone, the present application uses de-terminal peptide recombinant human collagen, obtains mineralized recombinant human collagen through mineralization, and then adsorbs recombinant human bone morphogenetic protein 2 to obtain recombinant human collagen active bone repair material with osteogenic activity. The recombinant human collagen active bone repair material prepared by the method has an inorganic matter and organic matter composition consistent with that of natural human bone, can fully realize effective fusion with the healing site, and especially contains recombinant human bone morphogenetic protein 2 with osteogenic activity, greatly improving the induced osteogenic activity and osteogenic effect of the material.

[0012] In a third aspect, this application proposes a bone repair material. According to an embodiment of this application, the bone repair material is prepared using the method described in the second aspect. Therefore, the bone repair material prepared using the method described in this application not only has a composition and structure consistent with natural bone tissue and excellent biocompatibility, but also can directly form a chemical bond with bone tissue; simultaneously, the recombinant human bone morphogenetic protein 2 added to the material can exert a strong bone-inducing effect, effectively promoting bone tissue growth.

[0013] In a fourth aspect of this application, the application of the bone repair material described in any one of the first and third aspects or the bone repair material prepared by the method described in the second aspect is proposed, the application including at least one of the following: application in the preparation of artificial bone, biomaterials, and medical devices; application in the preparation of products for treating bone defects; and application in the preparation of products for promoting bone tissue regeneration.

[0014] Beneficial effects: 1. The bone repair material prepared in this application has the following advantages: (1) It has the same composition and structure as natural bone tissue, and has the characteristics of easy water absorption and convenient clinical application; (2) Biocompatibility: It can chemically bind directly to bone without preventing the normal activity of bone cells on its surface or interfering with the natural regeneration process of surrounding bone cells, and has conductivity for the decomposition and absorption of bone tissue. (3) Mechanical endurance: Based on the trabecular bone, it has a certain compressive strength and compressive modulus; (4) Biodegradability: It is replaced by the host bone within a certain period of time, does not affect the repair of bone tissue, and has no toxic side effects; (5) Bone induction: Through its own or added bone induction factors (recombinant human bone morphogenetic protein 2), it can stimulate or induce bone growth and directly induce mesenchymal cells to differentiate into osteoprogenitor cells and osteoblasts, thereby forming bone tissue; (6) Osteoconductivity: The mineralized collagen fiber scaffold has good osteoconductivity. Bone grows on the surface of the recipient area, and new bone tissue grows into the implant (bone tissue creeping replacement).

[0015] 2. This application uses telopeptide-removed recombinant human collagen in the preparation of bone repair materials. Its advantages are reflected in two aspects: First, the design of removing telopeptides can effectively avoid the immunogenicity that telopeptides may cause. Combined with the characteristics of recombinant human collagen itself, the final prepared active bone repair material is completely non-immunogenic. Second, the telopeptide-removed recombinant human collagen has a completely identical amino acid sequence to natural human collagen and has a full-length stable triple helix structure. The hydroxylation modification level is also consistent with that of natural human collagen. This fully guarantees the biological activity of the bone repair material in terms of structure and properties.

[0016] 3. The recombinant human bone morphogenetic protein 2 of this application is fused with a collagen-binding peptide at its N-terminus to enhance the binding ability of recombinant human bone morphogenetic protein 2 to collagen, improve the osteogenic properties of bone repair materials, accelerate the repair of bone damage, and at the same time reduce the tissue diffusion of recombinant human bone morphogenetic protein 2 factor and reduce its usage.

[0017] 4. The preparation method of this application employs physical processes such as collagen mineralization, adsorption of recombinant human bone morphogenetic protein 2 (RMP2), and freeze-drying. This method does not damage the amino acid sequence composition and higher-order structure (triple helix structure, hydroxylation modification) of collagen, thus fully ensuring the collagen biological activity and osteogenic activity of the recombinant human collagen active bone repair material. Direct adsorption of RMP2 after the mineralization process results in more uniform dispersion of RMP2. The recombinant human collagen active bone repair material can achieve stable release along with collagen metabolism, ensuring consistent osteogenic activity throughout the entire bone metabolism lifecycle.

[0018] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0019] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is an SDS-PAGE image of recombinant type I human collagen with telopeptide removal according to an embodiment of this application, wherein Lane 1 is non-reduced, Lane 2 is a marker, and Lane 3 is reduced. Figure 2 This is an SDS-PAGE image of recombinant type III human collagen with telopeptide removal according to an embodiment of this application, wherein Lane 1 is non-reduced, Lane 2 is a marker, and Lane 3 is reduced. Figure 3 This is an SDS-PAGE diagram of rhBMP-2 refolded solution according to an embodiment of this application, wherein Lane 1 is the reduced rhBMP-2 refolded solution, Lane 2 is the marker, and Lane 3 is the non-reduced rhBMP-2 refolded solution; Figure 4 The image shown is an SDS-PAGE diagram of the rhBMP-2 stock solution according to an embodiment of this application. Lane 1 is the marker, Lane 2 is the molecular sieve sample (non-reduced), and Lane 3 is the concentrated molecular sieve sample, i.e., the stock solution (non-reduced). Figure 5This is a photograph of the active bone repair material containing adsorbed rhBMP-2 recombinant type I human collagen according to an embodiment of this application. Figure 6 The Ca / P ratio of the adsorbed rhBMP-2 recombinant type I human collagen active bone repair material according to the embodiments of this application is; Figure 7 The Ca / P ratio of the adsorbed rhBMP-2 recombinant type III human collagen active bone repair material according to the embodiments of this application is; Figure 8 This is a microscopic image of the recombinant type I human collagen active bone repair material according to an embodiment of this application; Figure 9 This is a microscopic image of the recombinant type III human collagen active bone repair material according to an embodiment of this application; Figure 10 The image shown is an SDS-PAGE diagram of the rhBMP-2-1 stock solution according to an embodiment of this application, where Lane 1 is the marker and Lane 2 is the sample after molecular sieve concentration (non-reduction). Detailed Implementation

[0020] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0021] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0022] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0023] To facilitate understanding of this application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application pertains.

[0024] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0025] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0026] This application proposes a bone repair material, its preparation method, and its application, which will be described in detail below.

[0027] Bone repair materials In a first aspect, this application proposes a bone repair material. According to an embodiment of this application, the bone repair material comprises: recombinant human collagen, recombinant human bone morphogenetic protein 2, and hydroxyapatite; wherein the ratio of inorganic to organic matter in the bone repair material is (64-66):(36-34). The bone repair material of this application uses recombinant human collagen as the core raw material, ensuring that the composition and structure of the prepared material are highly consistent with natural bone tissue; secondly, by adding recombinant human bone morphogenetic protein 2, the osteogenic activity of the material is further enhanced. The resulting recombinant human collagen active bone repair material, because it completely retains the triple helix structure of recombinant human collagen, possesses its complete biological activity, while also exhibiting the outstanding advantages of good osteogenic activity, no risk of disease or viral transmission, and no immunogenicity.

[0028] In some embodiments of this application, the ratio of inorganic to organic matter in the bone repair material can be 64:36, 63.5:36.5, 65:35, 65.5:34.5, 66:34, or any range of the above combinations.

[0029] In some embodiments of this application, the recombinant human collagen has a triple helix structure.

[0030] In some embodiments of this application, the recombinant human collagen is telopeptide-removed recombinant human collagen. First, telopeptide removal removes the major antigenic epitopes of collagen, fundamentally blocking immunogenicity. Second, the protein maintains the same amino acid sequence, full-length triple helix conformation, and hydroxyproline modification level as natural human collagen, balancing "low immune risk" and "intact biological activity," providing a safe, efficient, and functionally complete collagen template for subsequent mineralization and rhBMP-2 loading.

[0031] In some embodiments of this application, the determinated recombinant human collagen is produced by enzymatic hydrolysis using a determinated peptidase. In some embodiments of this application, the determinated peptidase includes at least one of pepsin, trypsin, and Lys-c enzyme (lysine protease).

[0032] In some embodiments of this application, the recombinant human collagen includes at least one of recombinant type I human collagen and recombinant type III human collagen. According to embodiments of the present invention, the recombinant type I human collagen is telopeptide-free recombinant type I human collagen, and the recombinant type III human collagen is telopeptide-free recombinant type III human collagen.

[0033] In some embodiments of this application, the recombinant type I human collagen is composed of two alpha1 chains and one alpha2 chain, the amino acid sequence of the alpha1 chain is shown in SEQ ID NO:2, and the amino acid sequence of the alpha2 chain is shown in SEQ ID NO:3.

[0034] In some embodiments of this application, the recombinant type III human collagen is composed of three peptide chains with amino acid sequences shown in SEQ ID NO:4.

[0035] In some embodiments of this application, the amino acid sequence of recombinant human bone morphogenetic protein 2 is shown in SEQ ID NO:1. Therefore, using the recombinant human bone morphogenetic protein 2 of this application can significantly enhance its binding ability with recombinant human collagen, thereby enhancing the osteogenic activity of bone repair materials and accelerating the bone injury repair process; at the same time, it can also effectively reduce the diffusion of rhBMP-2 in tissues, reducing its actual usage.

[0036] In some embodiments of this application, the molar ratio of calcium to phosphorus in the bone repair material is 1.6-1.7. For example, it can be 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, etc., or it can be a range of any of the above values.

[0037] Methods for preparing bone repair materials In a second aspect of this application, a method for preparing the bone repair material described in the first aspect is proposed. According to an embodiment of this application, the method includes: mineralizing recombinant human collagen to obtain mineralized collagen; performing a first mixing treatment on the mineralized collagen and recombinant human bone morphogenetic protein 2; and sequentially subjecting the first mixing product to a static treatment and a first centrifugation treatment to obtain the bone repair material. Thus, this application, by simulating the composition and formation process of natural human bone, uses telopeptide-free recombinant human collagen, obtains mineralized recombinant human collagen through a mineralization process, and then adsorbs recombinant human bone morphogenetic protein 2 to obtain a recombinant human collagen active bone repair material with osteogenic activity. The recombinant human collagen active bone repair material prepared by this method has an inorganic and organic composition consistent with natural human bone, which can fully achieve effective fusion with the healing site. In particular, it contains recombinant human bone morphogenetic protein 2, which has osteogenic activity, greatly improving the material's osteogenic induction activity and osteogenic effect.

[0038] In some embodiments of this application, the recombinant human collagen includes at least one of recombinant type I human collagen and recombinant type III human collagen. According to embodiments of the present invention, the recombinant type I human collagen is telopeptide-free recombinant type I human collagen, and the recombinant type III human collagen is telopeptide-free recombinant type III human collagen.

[0039] In some embodiments of this application, the telopeptide-removed recombinant type I human collagen is prepared using the method described in patent publication CN119954936A, which includes: constructing genetically engineered bacteria expressing proline hydroxylase, full-length COL1A1 protein, and full-length COL1A2 protein; culturing the genetically engineered bacteria; and performing enzymatic digestion of the culture product using telopeptidase to obtain the recombinant type I human collagen; wherein the proline hydroxylase includes P4Ha2, P4Hb, and vP4H. It should be noted that the specific parameters used in this method have all been disclosed in patent CN119954936A. Therefore, the recombinant type I human collagen sequence of this application is completely identical to the full-length sequence of natural type I human collagen, the hydroxyproline content is essentially the same as that of natural type I human collagen, and it possesses a triple helix structure, exhibiting the complete biological activity of natural type I human collagen. Since it is entirely a human-derived sequence, it has no immunogenicity and poses no viral risk.

[0040] In some embodiments of this application, the telopeptide-removed recombinant type III human collagen is prepared using the method described in patent publication CN119874880A, comprising: constructing a genetically engineered bacterium expressing proline hydroxylase and full-length recombinant type III human collagen; culturing the genetically engineered bacterium; and performing enzymatic digestion of the culture product using telopeptidase to obtain the recombinant type III human collagen; wherein the proline hydroxylase includes P4Ha2, P4Hb, and vP4H. It should be noted that the specific parameters used in this method have all been disclosed in patent CN119874880A. Therefore, the recombinant type III human collagen sequence of this application is completely identical to the full-length sequence of natural type III human collagen, the hydroxyproline content is essentially the same as that of natural type III human collagen, and it possesses a triple helix structure, exhibiting the complete biological activity of natural type III human collagen. Since it is entirely a human-derived sequence, it has no immunogenicity and poses no viral risk.

[0041] In some embodiments of this application, the amount of the determinate peptidase added to the culture product is 100~500 U / L.

[0042] In some embodiments of this application, the determinate peptidase includes at least one of pepsin, trypsin, and Lys-c enzyme.

[0043] In some embodiments of this application, the temperature of the enzymatic digestion treatment is 2~10℃.

[0044] In some embodiments of this application, the enzyme digestion treatment time is 16 to 30 hours.

[0045] In some embodiments of this application, the genetically engineered bacteria are selected from Pichia pastoris strains. Exemplarily, Pichia pastoris strains include, but are not limited to, Pichia pastoris strain GS115, Pichia pastoris strain X33, Pichia pastoris strain GS190, and Pichia pastoris strain KM71. According to an optional embodiment of this application, the Pichia pastoris strain is selected from Pichia pastoris strain GS115.

[0046] In some embodiments of this application, the recombinant human collagen obtained using the preparation method disclosed in the patent is in solution form. According to embodiments of this application, the concentration of the recombinant human collagen solution is 3-5 mg / mL. For example, it can be 3.0 mg / mL, 3.2 mg / mL, 3.5 mg / mL, 3.7 mg / mL, 4 mg / mL, 4.2 mg / mL, 4.5 mg / mL, 4.7 mg / mL, 5 mg / mL, etc., or a range of any of the above values.

[0047] In some embodiments of this application, the mineralization process is carried out as follows: a calcium salt solution is added to the recombinant human collagen to obtain a first mixture; phosphoric acid is added to the first mixture to obtain a second mixture; the pH of the second mixture is adjusted to obtain a third mixture; the third mixture is then subjected to stirring, static sedimentation, and a second centrifugation treatment to obtain mineralized collagen. Thus, mineralization allows for the in-situ deposition of nano-hydroxyapatite between collagen fiber molecules, forming an inorganic / organic three-dimensional scaffold consistent with natural bone. This provides immediate mechanical support and endows the material with osteoconductivity and an osteogenic template, laying the microstructural foundation for subsequent loading of recombinant human bone morphogenetic protein 2 and crawling replacement of new bone.

[0048] In some embodiments of this application, the settling time is 60-96 hours. For example, it can be 60 hours, 65 hours, 70 hours, 72 hours, 80 hours, 84 hours, 90 hours, 96 hours, or any range of the above values.

[0049] In some embodiments of this application, the calcium ion concentration in the first mixture is 1-1.5 mM. For example, it can be 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, or any range of the above values.

[0050] In some embodiments of this application, the phosphate ion concentration in the second mixture is 0.5-1 mM. For example, it can be 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, or any range of the above values.

[0051] In some embodiments of this application, the pH value of the third mixture is 7.0-8.0. For example, it can be 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, etc., or it can be a range of any of the above values.

[0052] In some embodiments of this application, after the second centrifugation treatment, the precipitate product is the mineralized collagen.

[0053] In some embodiments of this application, the recombinant human bone morphogenetic protein 2 is obtained by expressing the sequence shown in SEQ ID NO:1 using an E. coli system, followed by inclusion body denaturation, renaturation, and purification. The recombinant human bone morphogenetic protein 2 exists in solution form. According to embodiments of this application, the concentration of the recombinant human bone morphogenetic protein 2 solution is 0.1-0.5 mg / mL. For example, it can be 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, etc., or it can be any range of the above values.

[0054] In some embodiments of this application, the mass ratio of the recombinant human bone morphogenetic protein 2 to the mineralized collagen is 1:100-1:1000. For example, it can be 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, etc., or it can be any range of the above values.

[0055] In some embodiments of this application, the first centrifugation process further includes: sequentially freeze-drying and sieving the product of the first centrifugation process to obtain the bone repair material.

[0056] In some embodiments of this application, the freeze-drying process includes:

[0057] That is, first pre-freezing (freezing control), then drying and desorption drying are performed sequentially.

[0058] In some embodiments of this application, the sieving process is performed using a 30-70 mesh sieve. For example, the sieve can be 30 mesh, 40 mesh, 50 mesh, 60 mesh, 70 mesh, etc.

[0059] Bone repair materials In a third aspect, this application proposes a bone repair material. According to an embodiment of this application, the bone repair material is prepared using the method described in the second aspect. Therefore, the bone repair material prepared using the method described in this application not only has a composition and structure consistent with natural bone tissue and excellent biocompatibility, but also can directly form a chemical bond with bone tissue; simultaneously, the recombinant human bone morphogenetic protein 2 added to the material can exert a strong bone-inducing effect, effectively promoting bone tissue growth.

[0060] Applications and methods In a fourth aspect of this application, the application of the bone repair material described in any one of the first and third aspects or the bone repair material prepared by the method described in the second aspect is proposed, the application including at least one of the following: application in the preparation of artificial bone, biomaterials, and medical devices; application in the preparation of products for treating bone defects; and application in the preparation of products for promoting bone tissue regeneration.

[0061] In a fifth aspect of this application, the present invention provides a method for repairing bone defects. According to an embodiment of this application, the method includes: mixing a bone repair material as described in any one of the first and third aspects, or a bone repair material prepared by the method described in the second aspect, with a pharmaceutically acceptable isotonic carrier, and then filling the mixture into the bone defect site.

[0062] In some embodiments of this application, the pharmaceutically acceptable isotonic carrier includes physiological saline, plasma, etc.

[0063] The sequences used in this application are shown in Table A.

[0064] Table A

[0065]

[0066]

[0067] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0068] Example 1: Preparation of recombinant human collagen active bone repair material 1. Preparation of telopeptide-removed recombinant type I human collagen and telopeptide-removed recombinant type III human collagen (1) Referring to the preparation method of recombinant type I human collagen described in Example 6 of the patent with publication number CN119954936A and patent name "Preparation method of recombinant type I human collagen", this application adjusts the key process parameters in the specific operation process to prepare telopeptide-free recombinant type I human collagen stock solution: pepsin is added to the supernatant of the culture product, and the amount added is controlled to be 100U / L; then enzymatic digestion is performed, and the enzymatic digestion temperature is set to 4℃ and the enzymatic digestion time is 24 hours.

[0069] The prepared telopeptide-free recombinant type I human collagen consists of three chains: two alpha1 chains (sequence shown in SEQ ID NO:2) and one alpha2 chain (sequence shown in SEQ ID NO:3). The electrophoresis diagram of the telopeptide-free recombinant type I human collagen is shown below. Figure 1 As shown, Lane 1 represents non-reduced collagen (without reducing agent, representing the total molecular weight of the three chains of telopeptide-reduced recombinant type I human collagen), Lane 2 represents the marker, and Lane 3 represents reduced collagen (reduction refers to the addition of a reducing agent during electrophoresis to break the inter-chain forces of the three chains of telopeptide-reduced recombinant type I human collagen, representing the molecular weight of a single chain). The results show that this method successfully prepared telopeptide-reduced recombinant type I human collagen. The 130 kDa band (reduced) represents the length of a single alpha chain, while the non-reduced band shows the length of the triple-helix collagen structure.

[0070] (2) Referring to the preparation method of recombinant type III human collagen described in Example 6 of the patent with publication number CN119874880A and patent name "Preparation method of recombinant type III human collagen", this application adjusts the key process parameters in the specific operation process to prepare telopeptide-free recombinant type III human collagen stock solution: pepsin is added to the supernatant of the culture product, and the amount added is controlled to be 100U / L; then enzymatic digestion is performed, and the digestion temperature is set to 4℃ and the digestion time is 24 hours.

[0071] The prepared recombinant type III human collagen with determinated peptides consists of three chains, the sequences of which are shown in SEQ ID NO:4. The electrophoresis diagram of the recombinant type III human collagen with determinated peptides is shown below. Figure 2 As shown, Lane 1 represents non-reduced collagen (without reducing agent, representing the total molecular weight of the three chains of telopeptide-reduced recombinant type III human collagen), Lane 2 represents the marker, and Lane 3 represents reduced collagen (reduction refers to the addition of a reducing agent during electrophoresis to break the inter-chain forces of the three chains of telopeptide-reduced recombinant type III human collagen, representing the molecular weight of a single chain). The results show that this method successfully prepared telopeptide-reduced recombinant type III human collagen. The 130 kDa band (reduced) represents the length of a single alpha chain, while the non-reduced band shows the length of the triple-helix collagen structure.

[0072] 2. Preparation of recombinant human bone morphogenetic protein 2 (rhBMP-2) To obtain recombinant human bone morphogenetic protein 2 (rhBMP-2) stock solution, rhBMP-2 with the sequence shown in SEQ ID NO:1 was expressed using E. coli. The process involved inclusion body denaturation, renaturation, and purification, as detailed below: 1) Inclusion body denaturation: Inclusion bodies were expressed in E. coli and mixed with denaturing buffer (containing 8M urea) at a ratio of 1:10 (w / w). The mixture was stirred at 500 rpm for 24 hours at room temperature of 25°C to obtain the denaturing solution. 2) Refolding treatment: The dilution refolding method was used. Equal volumes of dilution buffer were added to the denaturing solution twice, with an interval of 24 hours between the two additions. Refolding was carried out at room temperature. Timing started after the second addition of dilution buffer, and the solution was allowed to stand for 72 hours to obtain the rhBMP-2 refolding solution.

[0073] The dilution buffer consists of 10-20 mM Tris or Tris-HCl, 10-20 mM, 300-500 mM arginine, and 5-10 mM EDTA, with the pH adjusted to 5.0. 3) Purification and concentration: Hydrophobic chromatography purification: A BorgLon Phenyl FF column was used for elution with a 0-100% B gradient of 10 column volumes (CV) at a flow rate of 200-250 cm / H. Composition of the mobile phase in chromatography: A1:20mmol / LTris+1mol / L NaCl+0.5mol / L Urea, pH8.5 A2:20mmol / LTris+1mol / L NaCl+2mol / L Urea, pH8.5 B:20mmol / LTris+2mol / L Urea, pH8.5 After concentration by molecular sieve, recombinant human bone morphogenetic protein 2 (rhBMP-2) stock solution was obtained.

[0074] Gel electrophoresis (SDS-PAGE) was performed on the rhBMP-2 refolded solution and the rhBMP-2 stock solution. The electrophoretic image of the rhBMP-2 refolded solution is shown below. Figure 3 As shown, Lane 1 is the reduced rhBMP-2 renaturation solution, Lane 2 is the marker, and Lane 3 is the non-reduced rhBMP-2 renaturation solution; the electrophoresis diagram of the original rhBMP-2 solution is shown below. Figure 4 As shown, Lane 1 is the marker, Lane 2 is the molecular sieve sample (non-reduced), and Lane 3 is the concentrated molecular sieve sample, i.e., the original solution (non-reduced). The results show that a band is present at 25 kDa in the refolding solution, indicating that the target protein was successfully prepared. A band is present at 12 kDa in the reduced solution, indicating that the target protein was opened by the reducing agent and is the product after reduction at 25 kDa.

[0075] 3. Mineralized recombinant human collagen S1: Take an appropriate amount of the recombinant type I human collagen stock solution or recombinant type III human collagen stock solution prepared in step 1, place it in a beaker and start stirring. Slowly add 2.0M CaCl2 solution dropwise until the Ca in the solution reaches a certain concentration. 2+ Once the concentration reaches 1.2 mM, continue stirring for 30 minutes. S2: Slowly add 2.0M H3PO4 dropwise to the solution stirred in S1 until the final phosphate ion concentration in the solution reaches 0.72 mM, and continue stirring for 30 min; S3: In the solution stirred in S2, adjust the pH to 7.0-7.5 with 5.0M NaOH solution; continue stirring at 20℃ for 16h, and then let it stand to settle for 72h. S4: Centrifuge the solution after sedimentation in S3 at 6000 rpm for 30 min (Thermo ST 16R benchtop centrifuge), take the precipitate, and wash the precipitate 3 times with purified water. The volume of purified water added each time is the same as the total volume of sedimentation in S3. After fully resuspending the precipitate, the washing is completed. S5: Take the purified water resuspension from the third wash in S4 and centrifuge it again at 10,000 rpm for 30 min (Thermo ST 16R benchtop centrifuge) to obtain wet mineralized recombinant type I human collagen precipitate or wet mineralized recombinant type III human collagen precipitate.

[0076] 4. Mineralized recombinant human collagen adsorbs rhBMP-2 Prepare an aqueous solution of rhBMP-2 in physiological saline, with a concentration of 0.2 × 10⁻⁶. -2 At a concentration of mg / ml, wet-state mineralized recombinant human collagen precipitate was added to rhBMP-2 saline solution at a ratio of 1:3 (w / v). The mixture was stirred at 100 rpm for 10 min until homogeneous, and then allowed to stand at 2-8℃ for 6 h for adsorption. After adsorption, the mixture was centrifuged at 10000 rpm for 30 min (Thermo ST 16R benchtop centrifuge) to collect the precipitate, yielding either the mineralized recombinant human collagen precipitate or the mineralized recombinant human collagen precipitate adsorbed with rhBMP-2.

[0077] 5. Vacuum freeze-drying of mineralized recombinant human collagen with adsorbed rhBMP-2. The mineralized recombinant type I or type III human collagen precipitate with adsorbed rhBMP-2 was transferred into a freeze-drying tray, and the freeze-drying program was preset and started. The freeze-drying program is shown in Table 1. Table 1

[0078] 6. Screening of mineralized recombinant type I human collagen with adsorption of rhBMP-2 The mineralized recombinant type I human collagen lyophilized product with adsorbed rhBMP-2 obtained in step 5 is passed through a 50-mesh sieve, and the sieved particles are collected to obtain recombinant type I human collagen active bone repair material with adsorbed rhBMP-2 or recombinant type III human collagen active bone repair material with adsorbed rhBMP-2.

[0079] Among them, the sample image of the active bone repair material with adsorbed rhBMP-2 recombinant type I human collagen is shown in the figure. Figure 5 As shown.

[0080] Example 2: Detection of Recombinant Type I / III Human Collagen Active Bone Repair Material 1. Detection of the ratio of inorganic to organic matter Testing instruments: Kjeldahl nitrogen analyzer (K1160, Haineng Future Technology Group Co., Ltd.), moisture analyzer (V30S, METTLER TOLEDO). The inorganic and organic matter ratios of the recombinant type I human collagen active bone repair material (JHM08) prepared in Example 1 and the recombinant type III human collagen active bone repair material (JHM09) prepared in Example 1 were tested respectively. The test results are shown in Table 2. The results show that the recombinant human collagen active bone repair materials prepared by the method described in this application have an inorganic and organic matter composition that is relatively consistent with that of natural human bone. Among them, the recombinant type I human collagen active bone repair material is closer to the composition of natural bone.

[0081] Table 2

[0082] 2. Detection of the molar ratio of calcium to phosphorus (Ca / P) Testing instruments: Field emission scanning electron microscope (FE-SEM) (JSM-IT800, Jeol / Japan Electronics Corporation), EDS energy dispersive spectroscopy (spot analysis).

[0083] The Ca / P ratios of the recombinant type I human collagen active bone repair material (JHM08) prepared in Example 1 and the recombinant type III human collagen active bone repair material (JHM09) prepared in Example 1 were tested respectively. The test results are shown in Table 3. Figure 6 , Figure 7The results showed that the Ca / P molar ratio of the recombinant type I human collagen active bone repair material of this application reached 1.63, consistent with the natural human bone matrix, indicating that the mineral phase was high-purity hydroxyapatite crystals; the Ca / P ratio of the recombinant type III human collagen active bone repair material was only 1.23, suggesting that in addition to hydroxyapatite crystals, other phosphate crystals were present in the mineral phase. Microscopic observation of the hydroxyapatite crystals of JHM08 and JHM09 showed the following... Figure 8 , Figure 9 It can be seen that JHM08 is entirely composed of typical hydroxyapatite crystal structures, while JHM09, in addition to hydroxyapatite crystals, also shows other phosphate crystals.

[0084] Table 3

[0085] 3. Water absorption characteristics test The water absorption properties of the recombinant type I human collagen active bone repair material prepared in Example 1 were studied.

[0086] The weight ratio of recombinant type I human collagen active bone repair material to water is 1:0.4. When water is dropped into the recombinant type I human collagen active bone repair material, it is completely and evenly absorbed within 10 seconds. This demonstrates that the recombinant type I human collagen active bone repair material of this application has good water absorption characteristics, can quickly form a non-collapsed, malleable, moist mass, meeting the clinical requirements of "ready to use, no pre-wetting required, no stirring required," and simultaneously absorbs wound exudate, maintaining a local moist microenvironment.

[0087] Example 3: Optimization of Preparation Parameters The inventors discovered that different mineralization times affect the performance of the resulting recombinant type I human collagen active bone repair material during the preparation of mineralized recombinant type I human collagen. Therefore, the inventors used different mineralization times to prepare mineralized recombinant type I human collagen, thereby preparing different bone repair materials.

[0088] The preparation process of mineralized recombinant type I human collagen is the same as in Example 1, except that the settling time in step 3 of Example 1 is 24h, 48h, 60h, 72h, 84h, and 96h, respectively.

[0089] The mineralized recombinant type I human collagen was subjected to adsorption of rhBMP-2, the mineralized recombinant type I human collagen with adsorbed rhBMP-2 was vacuum freeze-dried, and the mineralized recombinant type I human collagen with adsorbed rhBMP-2 was screened, as in Example 1.

[0090] The inorganic-organic ratio and Ca / P ratio of the recombinant type I human collagen active bone repair material were tested according to the method described in Example 2. The experimental results are shown in Table 4. The results show that mineralization and static standing for ≥72 h is the critical point for the inorganic-organic ratio to reach the natural bone ratio of 65:35. Below 72 h, the material obtained is "calcium deficient", the Ca / P ratio decreases simultaneously, and the mechanical and degradation properties do not meet the standards.

[0091] Table 4

[0092] Comparative Example: Comparison of adsorption characteristics of different rhBMP-2 sequences binding (adsorbing) with mineralized collagen To verify whether the sequence of recombinant human bone morphogenetic protein 2 would affect the final bone repair material, the inventors used the same preparation method as in Example 1 to prepare recombinant human bone morphogenetic protein 2-1 (rhBMP-2-1) without collagen-binding peptide.

[0093] Recombinant human bone morphogenetic protein 2-1 without collagen-binding peptide has the sequence shown in SEQ ID NO:5. The original solution SDS-PAGE is as follows: Figure 10 In this context, Lane 1 is the marker, and Lane 2 is the concentrated sample after molecular sieve processing, i.e., the stock solution (non-reduced) of recombinant human bone morphogenetic protein 2-1 without collagen-binding peptides. Using the same methods as in Example 1—mineralized recombinant type I human collagen, adsorption of rhBMP-2 by mineralized recombinant type I human collagen, vacuum freeze-drying, and sieving—a recombinant type I human collagen active bone repair material adsorbing recombinant human bone morphogenetic protein 2-1 without collagen-binding peptides was prepared.

[0094] Recombinant human bone morphogenetic protein 2 (with or without collagen-binding peptide) and recombinant type I human collagen active bone repair material were dispersed in a solution containing 0.9% NaCl at a mass / volume (mg / ml) ratio of 1 / 100. The mixture was stirred at 25°C and 100 rpm for 60 min. After centrifugation, the supernatant was collected, and the BMP-2 content in the supernatant was determined, as shown in the table below.

[0095] As shown in the table above, the recombinant type I human collagen active bone repair material without collagen-binding peptide 2-1 showed a small amount of dissociation when dispersed in a solution containing 0.9% NaCl. However, the recombinant type I human collagen active bone repair material containing collagen-binding peptide 2 did not show dissociation. This indicates that the recombinant human bone morphogenetic protein 2 containing collagen-binding peptide has good binding properties with mineralized recombinant type I human collagen, laying a good foundation for in vivo application.

[0096] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0097] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A bone repair material, characterized in that, include: Recombinant human collagen, recombinant human bone morphogenetic protein 2, and hydroxyapatite; The ratio of inorganic to organic matter in the bone repair material is (64-66):(36-34).

2. The bone repair material according to claim 1, characterized in that, The recombinant human collagen is terminal-determined recombinant human collagen; Optionally, the recombinant human collagen has a triple helix structure; Optionally, the recombinant human collagen includes at least one of recombinant type I human collagen and recombinant type III human collagen.

3. The bone repair material according to claim 2, characterized in that, The recombinant type I human collagen is composed of two alpha1 chains and one alpha2 chain. The amino acid sequence of the alpha1 chain is shown in SEQ ID NO:2, and the amino acid sequence of the alpha2 chain is shown in SEQ ID NO:

3. Optionally, the recombinant type III human collagen is composed of three peptide chains with the amino acid sequence shown in SEQ ID NO:4; Optionally, the amino acid sequence of the recombinant human bone morphogenetic protein 2 is shown in SEQ ID NO:

1.

4. The bone repair material according to any one of claims 1-3, characterized in that, The molar ratio of calcium to phosphorus in the bone repair material is 1.6-1.

7.

5. A method for preparing the bone repair material according to any one of claims 1-4, characterized in that, include: Recombinant human collagen was mineralized to obtain mineralized collagen; The mineralized collagen was mixed with recombinant human bone morphogenetic protein 2 in a first mixing treatment. The first mixed product is subjected to a static treatment and a first centrifugation treatment in sequence to obtain the bone repair material.

6. The method according to claim 5, characterized in that, The mineralization process is carried out in the following manner: A calcium salt solution was added to the recombinant human collagen to obtain a first mixture; Phosphoric acid is added to the first mixture to obtain a second mixture; The second mixture was subjected to pH adjustment to obtain a third mixture; The third mixture was subjected to stirring, static sedimentation, and second centrifugation in sequence to obtain mineralized collagen.

7. The method according to claim 6, characterized in that, The settling time is 60-96 hours. Optionally, the calcium ion concentration in the first mixture is 1-1.5 mM; Optionally, the phosphate ion concentration in the second mixture is 0.5-1 mM; Optionally, the pH of the third mixture is 7.0-8.

0.

8. The method according to any one of claims 5-7, characterized in that, The first centrifugation process further includes: sequentially freeze-drying and sieving the product from the first centrifugation process to obtain the bone repair material.

9. A bone repair material, characterized in that, It is prepared by the method described in any one of claims 5-8.

10. The application of the bone repair material according to any one of claims 1-4, 9, or the bone repair material prepared by the method according to any one of claims 5-8, wherein the application includes at least one of the following: Application in the preparation of products for treating bone defects; Applications in the preparation of artificial bone, biomaterials, and medical devices; Application in the preparation of products that promote bone tissue regeneration.

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