Bone repair material as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and relates to a bone repair material and a preparation method and application thereof. The preparation method comprises the following steps: mixing sodium hyaluronate gel and bone graft particles to prepare a mixture; performing heat treatment on the mixture under a second heat treatment condition, and removing the solvent to prepare the bone repair material, the second heat treatment conditions are as follows: (1) the heat treatment temperature is about 100-130 DEG C, and the heat treatment time is about 10-30 minutes; and (2) the mixture has a pH of about 3 to 5; the use amount of the sodium hyaluronate corresponding to every 100 g of the sodium hyaluronate gel is about 2 g to 6 g. The sliding performance of the bone repair material is effectively reduced, and the repair effect is excellent.

Description

Technical Field

[0001] This application belongs to the technical field of biomedical materials, and particularly relates to a bone repair material, a preparation method thereof, and an application thereof. Background Art

[0002] Diseases such as tumors, traumas, necrosis, and infections often lead to bone defects, and bone transplantation is usually required after the defects. The outstanding performance of autologous bone transplantation in aspects such as bone generation, osteoinduction, and osteoconduction is considered the "gold standard" for clinical treatment. However, this technique requires additional surgeries to obtain bone grafts, resulting in secondary traumas and potential donor site morbidity. In addition, the source of autologous bone is limited.

[0003] The three-dimensional structure of the human skeleton is mainly composed of inorganic mineral phases, water, and organic biomolecules. The organic biomolecules include collagen and non-collagen polysaccharides (GAGs). The inorganic minerals provide a basic scaffold, and the organic matrix provides elasticity. The properties of these two different compounds determine the optimal performance of bone materials. Based on the research of the human skeleton, some allogeneic bone grafts have been developed.

[0004] A single collagen component is added to bovine cancellous bone to form a plastic bone material to mimic the structure of human bone. For example, the commercially available product Bio-oss Collagen (Geistlich Biomaterials, Geistlich, Switzerland) is a combination of 90% bovine cancellous bone and 10% porcine collagen. This material can be applied to multiple clinical scenarios. In the maxillary sinus floor elevation surgery, the added collagen component can better protect the sinus floor mucosa from being punctured. However, this material introduces two different species sources, increasing the risk of immunogenic rejection.

[0005] WO2009131323A3 discloses a hyaluronic acid bone filling composite and a preparation method thereof. The material components include calcium phosphate-based bone materials (biphasic calcium phosphate or calcium nitrate tetrahydrate, including tricalcium phosphate and hydroxyapatite) and a hyaluronic acid derivative crosslinked with divinyl sulfone (DVS) or cystamine.

[0006] US8876532B2 discloses a viscous bone repair material, which includes porous absorbable particles (derived from inorganic bone minerals or natural bone minerals), P-15 polypeptide (type I collagen containing a fifteen-amino acid sequence), and an absorbable carrier (such as high molecular weight hyaluronic acid or hydroxypropyl cellulose). Among them, the bone particles account for 55% and the carrier material accounts for 45%. During clinical use, the bone material and the absorbable carrier are manually physically mixed, and the formed paste has certain plasticity.

[0007] However, the mechanical properties and bone repair performance of current bone repair materials still need to be improved. In view of this, the present application is specifically proposed. Summary of the Invention

[0008] One or more embodiments of the present application provide a bone repair material, a preparation method thereof, and an application, and the technical solution is as follows:

[0009] One or more embodiments of the present application provide a preparation method of a bone repair material, and the preparation method includes the following steps:

[0010] Mix sodium hyaluronate gel and bone graft particles to prepare a mixture;

[0011] Heat-treat the mixture under a second heat-treatment condition to remove the solvent and prepare a bone repair material;

[0012] The second heat-treatment condition includes:

[0013] (1) The temperature of the heat treatment is about 100°C to 130°C, and the heat treatment time is about 10 min to 30 min; and,

[0014] (2) The pH value of the mixture is about 3 to 5;

[0015] The dosage of sodium hyaluronate corresponding to every 100 g of the sodium hyaluronate gel is about 2 g to 6 g.

[0016] In some embodiments of the present application, the preparation steps of the sodium hyaluronate gel include: mixing sodium hyaluronate and a dispersion medium, and fully swelling to prepare a sodium hyaluronate gel.

[0017] In some embodiments of the present application, the dispersion medium includes one or more of PBS buffer solution and hydrochloric acid.

[0018] In some embodiments of the present application, the pH value of the dispersion medium is about 3 to 5.

[0019] In some embodiments of the present application, the intrinsic viscosity of the sodium hyaluronate is about 1.5 m 3 / kg to 3.9 m 3 / kg.

[0020] In some embodiments of the present application, the preparation steps of the bone graft particles include: cutting, crushing, degreasing, virus inactivation, deproteinization of the bone donor material, and heat treatment under a first heat-treatment condition to prepare the bone graft particles.

[0021] In some embodiments of the present application, the first heat-treatment condition includes: the temperature of the heat treatment is about 250°C to 550°C, and the heat treatment time is about 5 h to 8 h.

[0022] In some embodiments of the present application, the degreasing treatment is carried out in an organic reagent; optionally, the organic reagent includes one or more of isopropanol and ethanol; optionally, the mass ratio of the organic reagent to the cut and crushed bone donor material is (2.0 - 3.5):1.

[0023] In some embodiments of the present application, the virus inactivation treatment uses a protein denaturant; optionally, the protein denaturant includes an alkali; optionally, the protein denaturation includes a sodium hydroxide solution with a sodium hydroxide content of about 3.5 wt% - 4.5 wt%; optionally, the time of the virus inactivation treatment is about 0.5 h - 1.5 h.

[0024] In some embodiments of the present application, the conditions for the deproteinization treatment include: the deproteinizing reagent used includes one or more of a strong oxidizing reagent and a primary amine reagent;

[0025] Optionally, the temperature for the deproteinization treatment corresponding to the primary amine reagent is about 115°C - 125°C;

[0026] Optionally, the strong oxidizing reagent includes a hydrogen peroxide solution; further optionally, the hydrogen peroxide content in the hydrogen peroxide solution is 25 wt% - 35 wt%;

[0027] Optionally, the primary amine reagent includes an ethylenediamine solution; further optionally, the ethylenediamine content in the ethylenediamine solution is 80 wt% - 90 wt%;

[0028] Optionally, the mass ratio of the deproteinizing reagent to the bone donor material after virus inactivation treatment is (2.5 - 3.5):1.

[0029] In some embodiments of the present application, the species source of the bone donor material includes vertebrates;

[0030] Optionally, the vertebrates include cattle;

[0031] Optionally, the bone donor material includes limb bones, further optionally the femur, and even more preferably the cancellous bone of the femur.

[0032] In some embodiments of the present application, the mass ratio of the sodium hyaluronate gel to the bone graft particles is about (6 - 10):6.

[0033] In some embodiments of the present application, the mixture further includes a carbonate;

[0034] Optionally, the carbonate includes one or more of calcium carbonate and magnesium carbonate;

[0035] Optionally, the content of carbonate in the osteoinductive material is about 2 wt % to 6 wt %.

[0036] In some embodiments of the present application, the method of removing the solvent includes one or more of vacuum freeze drying and vacuum drying;

[0037] Optionally, the conditions for vacuum drying include: a drying temperature of about 60° C. to 90° C., and a drying time of about 2 h to 6 h.

[0038] In some embodiments of the present application, the content of sodium hyaluronate in the osteoinductive material is about 2 wt % to 8 wt %.

[0039] In some embodiments of the present application, the particle size of the bone graft particles ranges from about 0.25 mm to 2 mm.

[0040] In some embodiments of the present application, the bone repair material is in block, column or granular form.

[0041] One or more embodiments of the present application also provide a bone repair material, which is prepared by the preparation method.

[0042] One or more embodiments of the present application further provide a bone repair method, comprising using the bone repair material to repair a bone defect area of ​​a subject.

[0043] In some embodiments of the present application, the bone defect area occurs in the oral bone, skull bone or non-weight-bearing bone of the limbs.

[0044] In some embodiments of the present application, the bone repair material is directly filled into the bone defect area, or is filled into the bone defect area after absorbing water or blood.

[0045] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0047] Figure 1 This is a diagram of an instrument for detecting the carbonate content in bone repair materials;

[0048] Figure 2Schematic diagram of the placement of the bone repair material in the simulated oral dental socket (it can be directly placed in the dry state or placed after cutting with water / blood attached in vitro);

[0049] Figure 3 Observation by SEM scanning electron microscope;

[0050] Figure 4 Sliding property test results of the bone repair material provided in Example 1;

[0051] Figure 5 Sliding property test results of the bone repair material provided in Comparative Example 2;

[0052] Figure 6 Sliding property test results of the bone repair material provided in Comparative Example 3;

[0053] Figure 7 Sliding property test results of the bone repair material provided in Comparative Example 4;

[0054] Figure 8 Sliding property test results of the bone repair material provided in Comparative Example 6;

[0055] Figure 9 Micro-CT images of animal experiments on cranial bone defects with multiple bone repair materials. Detailed implementation manners

[0056] The present application will be further described in detail below in conjunction with the accompanying drawings, implementation manners and examples. It should be understood that these implementation manners and examples are only used to illustrate the present application and not to limit the scope of the present application. The purpose of providing these implementation manners and examples is to make the understanding of the disclosed content of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms and is not limited to the implementation manners and examples described herein. Those skilled in the art can make various changes or modifications without departing from the connotation of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given to provide a more thorough understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the specification of this application herein are only for the purpose of describing the implementation manners and examples and are not intended to limit this application.

[0058] Term

[0059] Unless otherwise stated or there is a contradiction, the terms or phrases used herein have the following meanings:

[0060] As used herein, the selection scope of the terms "and / or", "or / and", and "and / or" includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more than two related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, it includes combinations of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution connected by "logical AND").

[0061] In this application, the terms "multiple", "multiple types", "multiple times", "multiple elements", etc., unless otherwise specified, mean greater than or equal to 2 in quantity. For example, "one or more types" means one type or greater than or equal to two types.

[0062] As used herein, "its combinations", "any combinations thereof", "any combination manners thereof", etc. include all suitable combination manners of any two or more than two items in the listed items.

[0063] In this article, the "suitable" in "suitable combination manners", "suitable manners", "any suitable manners", etc. is subject to being able to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0064] In this article, "preferred", "better", "more preferable", "preferably" are only used to describe embodiments or examples with better effects. It should be understood that they do not constitute a limitation on the protection scope of this application.

[0065] In this application, "further", "furthermore", "especially", etc. are used for descriptive purposes, indicating differences in content, but should not be construed as a limitation on the protection scope of this application.

[0066] In this application, "optionally", "optional", "optional" mean that it can be there or not, that is, it refers to any one of two parallel solutions of "yes" or "no". If "optional" appears multiple times in a technical solution, unless otherwise specified and there are no contradictions or mutual restrictions, each "optional" is independent of each other.

[0067] In this application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only for descriptive purposes and cannot be construed as indicating or implying relative importance or quantity, nor can they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumerative description and should be understood not to constitute a closed limitation on quantity.

[0068] In this application, among the technical features described in an open-ended manner, there are included closed technical solutions composed of the listed features, as well as open technical solutions containing the listed features.

[0069] In this application, regarding numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical values are considered continuous within the above numerical intervals and include the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical range, as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, when t is an integer selected from 1 to 10, it means that t is any integer selected from the integer group composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this article should be understood to include any and all sub-ranges subsumed therein.

[0070] The temperature parameter in this application, unless otherwise specified, allows both constant temperature treatment and variation within a certain temperature range. It should be understood that the so-called constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.

[0071] In this application, %(w / w) and wt% both represent weight percentages, %(v / v) refers to volume percentages, and %(w / v) refers to mass-volume percentages.

[0072] All documents mentioned in this application are cited herein as references, as if each document was cited individually as a reference. Unless it conflicts with the purpose of this application and / or the technical solution, the cited documents involved in this application are cited in their entirety and for all purposes. When referring to cited documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When referring to cited documents in this application, examples and preferred methods of the relevant technical features cited can also be incorporated as references into this application, but only to the extent that this application can be implemented. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or be amended adaptively according to the description in this application.

[0073] In the first aspect of the embodiments of the present application, a method for preparing a bone repair material is provided, and the preparation method includes the following steps:

[0074] Mix sodium hyaluronate gel and bone graft particles to prepare a mixture;

[0075] Heat-treat the mixture under a second heat-treatment condition to remove the solvent and prepare a bone repair material;

[0076] The second heat-treatment condition includes:

[0077] (1) The temperature of the heat treatment is about 100°C to 130°C (such as 100°C, 101°C, 102°C, 103°C, 104°C, 105°C, 106°C, 107°C, 108°C, 109°C, 110°C, 111°C, 112°C, 113°C, 114°C, 115°C, 116°C, 117°C, 118°C, 119°C, 120°C, 121°C, 122°C, 123°C, 124°C, 125°C, 126°C, 127°C, 128°C, 129°C, 130°C), and the heat-treatment time is about 10 min to 30 min (such as 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min); and,

[0078] (2) The pH value of the mixture is about 3 to 5 (such as 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0);

[0079] The amount of sodium hyaluronate corresponding to every 100 g of the sodium hyaluronate gel is about 2 g to 6 g (for example, 2 g, 2.2 g, 2.4 g, 2.6 g, 2.8 g, 3 g, 3.2 g, 3.4 g, 3.6 g, 3.8 g, 4.0 g, 4.2 g, 4.4 g, 4.6 g, 4.8 g, 5.0 g, 5.2 g, 5.4 g, 5.6 g, 5.8 g, 6 g).

[0080] The bone repair material prepared in the embodiment of the present application is also a plastic oral bone induction material, which is assembled by a suitable sodium hyaluronate gel and bone graft particles. Among them, the bone graft particles are wrapped by a three-dimensional network gel of sodium hyaluronate and have a bionic structure highly similar to the natural bone tissue of the human body. In the preparation method provided by the embodiment of the present application, the mixture of the sodium hyaluronate gel and the bone graft particles is heat-treated under suitable conditions, so that the prepared material has excellent cohesiveness and reduces the slipperiness of sodium hyaluronate itself.

[0081] At present, there are various bone grafts available for clinical use to replace autologous bone grafts. Vertebrate bones, especially bovine bones, are considered to be the most similar to human bones and are widely used in many commercial products. Due to the risks of immunogenicity and virus contamination, some manufacturers use different methods of deproteinization, defatting, and heat treatment to prepare bone particles, such as Bio-Oss (Geistlich Biomaterials, Geistlich, Switzerland), osteograft (CeraMed Co., Denver, Co, USA), and Endobon (Merck Co., Darmstadt, Germany). However, most of these materials only retain the inorganic components by calcination and completely remove some bioactive components, so their bone formation and osteogenic induction functions are significantly reduced. And bone powder products are inconvenient for clinical use, such as in the anterior tooth area with sufficient blood supply and Type II-0, Type II-I, Type II-II alveolar bone defects. Especially for some specific indications and regional locations, their use is limited. For example, in the surgical process of maxillary sinus floor elevation to treat insufficient bone mass in the posterior maxilla, due to the relatively thin maxillary sinus mucoperiosteum itself, the rough outer surface of the bone material will cause complications such as perforation of the maxillary sinus mucoperiosteum during maxillary sinus elevation, leading to risks such as maxillary sinus infection, implant shedding, and treatment failure. The perforation rate reported in the clinical literature is about 60%. And the solution provided by the present application can overcome the above technical defects.

[0082] The material provided by the embodiment of the present application can be cut arbitrarily in vitro under water-attached and blood-attached conditions to match the defect size, or directly filled into the alveolar bone defect site in a dry block form for in-situ shaping (such as Figure 2), which is convenient for clinicians to operate and saves surgical time. In particular, it is more convenient to operate some complex bone defects or alveolar bone defects of Type II-0, Type II-I, and Type II-II, especially for the maxillary sinus floor lifting surgery, the sinus floor mucosa can be better protected. After implanting the alveolar bone defect, the ability of the treated sodium hyaluronate gel contained in the material to quickly absorb blood stabilizes the blood clot on the bioactive bone scaffold. When the pusher applies pushing pressure to the bone augmentation material implanted in the implantation socket, the blood quickly absorbed by the bone graft particles wrapped in sodium hyaluronate will be released, forming a hydraulic effect. This hydraulic effect pushes the bone augmentation material and the blood released by it toward the sinus floor and the surrounding direction, "tearing off" the sinus floor mucoperiosteum around the implantation socket from the sinus floor bone wall, and propping up the broken sinus floor residual bone plate and mucoperiosteum together, thereby forming a tent-like structure of a certain height, creating space for implanting bone augmentation materials and implants. The material of the present application has excellent bone formation and bone induction effects, and can still maintain the stability of the bioactive bone scaffold for the anterior tooth surgical area with large bleeding.

[0083] In addition, relative to the technical solution described in WO2009131323A3, the present application has good bone formation and osteoinduction functions. Moreover, it avoids introducing toxic chemical cross-linking agents, and tries to avoid affecting the biological behavior of the product. Compared with the technical solution described in US8876532B2, the material prepared by the present application does not need to be manually physically mixed with the bone material and the absorbable carrier to form a plastic paste during clinical use. It is easy to use and can effectively avoid the situation where the bone space contour is poorly maintained due to the sliding property of sodium hyaluronate itself. It is suitable for the anterior tooth surgical area with a large amount of bleeding and has a good use effect, avoiding the reduction of long-term bone quality and thus causing implant exposure, avoiding the necessity of secondary surgery, and reducing the economic burden on patients.

[0084] In some embodiments, the step of preparing the sodium hyaluronate gel comprises: mixing sodium hyaluronate and a dispersion medium, and fully swelling the mixture to prepare the sodium hyaluronate gel.

[0085] The embodiments of the present application do not particularly limit the type of the dispersion medium. For example, a sodium hyaluronate gel can be prepared using a dispersion medium with a pH value outside the range of about 3 to 5, and then the pH value of the sodium hyaluronate gel or the mixture of the sodium hyaluronate gel and the bone graft particles can be adjusted to about 3 to 5 before subsequent heat treatment; alternatively, a sodium hyaluronate gel can be directly prepared using a dispersion medium with a pH value of about 3 to 5 and mixed with the bone graft particles to make the pH value of the resulting mixture about 3 to 5, and then heat treatment can be directly carried out. In some embodiments, the dispersion medium includes one or more of PBS buffer solution and hydrochloric acid. In some embodiments, the pH value of the dispersion medium is about 3 to 5 (such as 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0).

[0086] In some embodiments, the intrinsic viscosity of the sodium hyaluronate is about 1.5 m 3 / kg - 3.9 m 3 / kg (such as 1.5 m 3 / kg, 1.6 m 3 / kg, 1.7 m 3 / kg, 1.8 m 3 / kg, 1.9 m 3 / kg, 2.0 m 3 / kg, 2.1 m 3 / kg, 2.2 m 3 / kg, 2.3 m 3 / kg, 2.4 m 3 / kg, 2.5 m 3 / kg, 2.6 m 3 / kg, 2.7 m 3 / kg, 2.8 m 3 / kg, 2.9 m 3 / kg, 3.0 m 3 / kg, 3.1 m 3 / kg, 3.2 m 3 / kg, 3.3 m 3 / kg, 3.4 m 3 / kg, 3.5 m 3 / kg, 3.6 m 3 / kg, 3.7 m 3 / kg, 3.8 m 3 / kg, 3.9 m 3 / kg).

[0087] In some embodiments, the preparation steps of the bone graft particles include: cutting and crushing the bone donor material, degreasing treatment, virus inactivation treatment, deproteinization treatment, and heat treatment under first heat treatment conditions to prepare the bone graft particles.

[0088] In some embodiments, the first heat treatment conditions include: the temperature of the heat treatment is about 250°C to 550°C (such as 250°C, 260°C, 270°C, 280°C, 290°C, 300°C, 310°C, 320°C, 330°C, 340°C, 350°C, 360°C, 370°C, 380°C, 390°C, 400°C, 410°C, 420°C, 430°C, 440°C, 450°C, 460°C, 470°C, 480°C, 490°C, 500°C, 510°C, 520°C, 530°C, 540°C, 550°C), and the time of the heat treatment is about 5h to 8h (such as 5h, 5.5h, 6h, 6.5h, 7h, 7.5h, 8h). The bone graft particles prepared under these non-calcination conditions are assembled into a bone repair material wrapped by a three-dimensional network gel of sodium hyaluronate, and the sliding property is effectively improved.

[0089] The embodiments of the present application do not particularly limit the method used for degreasing treatment. In some embodiments, the degreasing treatment is carried out in an organic reagent; optionally, the organic reagent includes one or more of isopropanol and ethanol; optionally, the mass ratio of the organic reagent to the cut and crushed bone donor material is (2.0 - 3.5):1, such as 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3.0:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1.

[0090] The embodiments of the present application do not particularly limit the method used for virus inactivation treatment. In some embodiments, the virus inactivation treatment uses a protein denaturant; optionally, the protein denaturant includes an alkali; optionally, the protein denaturant includes a sodium hydroxide solution with a sodium hydroxide content of about 3.5wt% - 4.5wt% (such as 3.5wt%, 3.6wt%, 3.7wt%, 3.8wt%, 3.9wt%, 4.0wt%, 4.1wt%, 4.2wt%, 4.3wt%, 4.4wt%, 4.5wt%); optionally, the time of the virus inactivation treatment is about 0.5h to 1.5h (such as 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1.0h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h).

[0091] The embodiments of the present application do not particularly limit the method adopted for the deproteinization treatment. In some embodiments, the conditions for the deproteinization treatment include: the deproteinization reagent used includes one or more of strong oxidation reagents and primary amine reagents. In one of the embodiments, the deproteinization treatment uses a strong oxidation reagent and a primary amine reagent. For example, the strong oxidation reagent and the primary amine reagent are used for treatment in sequence.

[0092] Optionally, the temperature for the primary amine treatment is about 115°C to 125°C (such as 115°C, 116°C, 117°C, 118°C, 119°C, 120°C, 121°C, 122°C, 123°C, 124°C, 125°C), and the number of treatment times is about 3 to 8 times (such as 3 times, 4 times, 5 times, 6 times, 7 times, 8 times); the duration of each treatment is about 6h to 12h (such as 6h, 7h, 8h, 9h, 10h, 11h, 12h).

[0093] Optionally, the strong oxidation reagent includes a hydrogen peroxide solution; further optionally, the content of hydrogen peroxide in the hydrogen peroxide solution is about 25wt% to 35wt% (such as 26wt%, 26wt%, 27wt%, 28wt%, 29wt%, 30wt%, 31wt%, 32wt%, 33wt%, 34wt%, 35wt%), and the treatment duration of the strong oxidation reagent is about 8h to 16h (such as 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h).

[0094] Optionally, the primary amine reagent includes an ethylenediamine solution; further optionally, the content of ethylenediamine in the ethylenediamine solution is about 80wt% to 90wt% (such as 80wt%, 81wt%, 82wt%, 83wt%, 84wt%, 85wt%, 86wt%, 87wt%, 88wt%, 89wt%, 90wt%); optionally, the mass ratio of the deproteinization reagent to the bone donor material after virus inactivation treatment is about (2.5 to 3.5):1 (such as 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3.0:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1).

[0095] The embodiments of the present application do not particularly limit the species source of the bone donor material, including but not limited to vertebrates (including but not limited to cattle). The embodiments of the present application do not particularly limit the site source of the bone donor material, including but not limited to limb bones, optionally the femur, and further optionally the cancellous bone of the femur.

[0096] In some embodiments, the mass ratio of the sodium hyaluronate gel to the bone graft particles is about (6 to 10):6, such as 6:6, 7:6, 8:6, 9:6, 10:6.

[0097] In some embodiments, the mixture further comprises a carbonate. The embodiments of the present application do not particularly limit the type of carbonate, including but not limited to calcium carbonate and magnesium carbonate. In some embodiments, the carbonate content in the bone repair material is about 2 wt% to 6 wt% (such as 2 wt%, 2.2 wt%, 2.4 wt%, 2.6 wt%, 2.8 wt%, 3 wt%, 3.2 wt%, 3.4 wt%, 3.6 wt%, 3.8 wt%, 4 wt%, 4.2 wt%, 4.4 wt%, 4.6 wt%, 4.8 wt%, 5 wt%, 5.2 wt%, 5.4 wt%, 5.6 wt%, 5.8 wt%, 6 wt%). The addition of the carbonate makes it more suitable for combination with the bone graft particles, enhancing the interaction and binding force between the hyaluronic acid and the bone particles. A layer of bone material in the form of carbonate is formed on the hydroxyapatite, which is more conducive to osteogenesis. At the same time, the surface of sodium hyaluronate contains a large number of hydroxyl groups, which can bind to cations (preferably Ca ions) to form a specific spatial structure, better promoting the stability of the whole system.

[0098] In some embodiments, the sodium hyaluronate content in the bone repair material is about 2 wt% to 8 wt% (such as 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%, 5.5 wt%, 6 wt%, 6.5 wt%, 7 wt%, 7.5 wt%, 8 wt%). This is in line with the set range of the sodium hyaluronate concentration, the mixing ratio of the gel and the bone particles. If the sodium hyaluronate content is too high, gel will overflow during the mixing process, and finally show stronger slipperiness. If the content is too low, the gel and the bone particles are difficult to be fully mixed, with poor viscosity and easy to disperse after rehydration.

[0099] The embodiments of the present application do not particularly limit the method for removing the solvent, including but not limited to vacuum freeze-drying and vacuum drying. Optionally, the conditions for vacuum drying include: the drying temperature is about 60°C to 90°C (such as 60°C, 63°C, 65°C, 70°C, 72°C, 75°C, 80°C, 82°C, 85°C, 90°C), and the drying time is about 2 h to 6 h (such as 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, 6 h).

[0100] In some embodiments, the particle size range of the bone graft particles is about 0.25 mm to 2 mm. For example, it is 0.25 mm to 0.5 mm, 0.5 mm to 1.0 mm, 1.0 mm to 1.5 mm, 1.5 mm to 2.0 mm, 0.25 mm to 1.0 mm, 1 mm to 2 mm.

[0101] In some embodiments, the pH value of the finished product after mixing and drying the sodium hyaluronate gel and the bone graft particles is about 6.5 to 9.0 (for example, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0).

[0102] The embodiments of the present application do not particularly limit the shape of the bone repair material. For example, it can be square, cylindrical or granular, etc.

[0103] In the second aspect of the embodiments of the present application, a bone repair material is provided, and the bone repair material is prepared by the preparation method.

[0104] In the third aspect of the embodiments of the present application, a bone repair method is provided, including repairing the bone defect area of a subject using the bone repair material.

[0105] In some embodiments, the bone defect area occurs in oral bone, cranial bone or bone in non - weight - bearing parts of the extremities.

[0106] In some embodiments, the bone repair material is directly filled into the bone defect area, or filled into the bone defect area after adsorbing water or blood, as Figure 2 shown.

[0107] The embodiments of the present application do not particularly limit the subject, which can be any animal that may have a bone defect and needs to be repaired. It can be a human or other non - human mammals. The term "mammal" in the present application mainly refers to warm - blooded vertebrate mammals, including but not limited to: cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (such as rats, mice), pigs, cows, sheep, horses, humans, etc. Primates are preferred, and humans are more preferred.

[0108] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions in the following examples, the guidance given in the present application is preferably referred to, and it can also be in accordance with the experimental manuals or conventional conditions in the art, or in accordance with the conditions recommended by the manufacturer, or refer to the experimental methods known in the art.

[0109] In the following specific embodiments, regarding the measurement parameters of raw material components, unless otherwise specified, there may be slight deviations within the weighing accuracy range. Regarding temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operation accuracy are allowed.

[0110] Example 1

[0111] This example provides a bone repair material and its preparation method, including the following:

[0112] 1. Preparation of sodium hyaluronate gel

[0113] 1.1 Prepare 0.2 mol / L sodium dihydrogen phosphate solution and 0.2 mol / L disodium hydrogen phosphate solution respectively, mix and stir evenly according to the mass ratio of sodium dihydrogen phosphate solution:disodium hydrogen phosphate solution = 93.5:6.5, and adjust the pH to 3.0 using hydrochloric acid.

[0114] 1.2 Add 4.5 g of injection-grade sodium hyaluronate dry powder with an intrinsic viscosity in the range of 2.3 m 3 / kg - 2.5 m 3 / kg to PBS buffer to make the total amount of the resulting mixture 100 g, stir to make it evenly dispersed, and let it stand and swell for 16 h to obtain sodium hyaluronate gel. The gel should be completely transparent without visible white particulate matter to the naked eye, and the concentration of the sodium hyaluronate gel is 4.5%.

[0115] 2. Preparation of bone particles

[0116] 2.1 Take the cancellous bone part of bovine femur, cut and crush it to form bone particles, and degrease it using a conventional organic reagent according to the mass ratio of organic reagent to bone particles of 3:1. The organic reagent is isopropanol.

[0117] 2.2 Soak the degreased bone particles obtained in 2.1 in 4 wt% sodium hydroxide for 1 h for virus inactivation.

[0118] 2.3 After virus inactivation, the bone particles are first placed in 30 wt% hydrogen peroxide solution (the mass ratio of hydrogen peroxide solution to bone particles is 3:1) for 10 h for deproteinization treatment, and then placed in a double-layer circulating reaction kettle for ethylenediamine deproteinization treatment at a temperature of 120 °C. The concentration of ethylenediamine in the ethylenediamine solution is 85 wt%, and the mass ratio of ethylenediamine solution to bone particles is 3:1. A total of 3 treatments are carried out, each for 8 h.

[0119] 2.4 After the deproteinization treatment, the bone particles are washed with purified water to remove reagent residues.

[0120] 2.5 After washing, the bone particles are placed in a vacuum oven for heat treatment. The heat treatment temperature is 400 °C, and the heat treatment time is 6 h.

[0121] 2.6 After heat treatment, the bone particles are screened, and the particle size range selected for screening this time is 0.5 mm to 1.0 mm.

[0122] 3. Preparation of bone repair material

[0123] 3.1 Mix and stir the sodium hyaluronate gel prepared in Step 1 and the bone particles prepared in Step 2 according to a mass ratio of 10:6, and add 0.01 g of calcium carbonate and mix.

[0124] 3.2 Heat-treat the mixture prepared in 3.1. The heat treatment temperature is 120 °C and the heat treatment time is 10 min.

[0125] 3.3 Place the product prepared in 3.2 in a mold for vacuum drying. The vacuum drying temperature is 80 °C and the vacuum drying time is 3 h.

[0126] 3.4 Package and sterilize the dried sample to form the final product.

[0127] For the bone repair material prepared in this example, the sodium hyaluronate content should be about 5% (w / w), and the carbonate content is 2 wt% to 3 wt%.

[0128] Example 2

[0129] This example is a variant of Example 1. The difference from Example 1 is only that:

[0130] In 3.2, the heat treatment temperature is 130 °C and the heat treatment time is 10 min.

[0131] Example 3

[0132] This example is a variant of Example 1. The difference from Example 1 is only that:

[0133] In 3.2, the heat treatment temperature is 120 °C and the heat treatment time is 20 min.

[0134] Example 4

[0135] This example is a variant of Example 1. The difference from Example 1 is only that:

[0136] In 3.2, the heat treatment temperature is 120 °C and the heat treatment time is 30 min.

[0137] Example 5

[0138] This example is a variant of Example 1. The difference from Example 1 is only that:

[0139] In the first step, sodium hyaluronate gel was prepared using hydrochloric acid solution with a pH value of 5.0 instead of PBS buffer solution.

[0140] Example 6

[0141] This example is a variant of Example 1. The differences from Example 1 are only as follows:

[0142] Under item 1.1, the pH value of the prepared PBS buffer solution was 4.

[0143] Under item 1.2, the dosage of sodium hyaluronate corresponding to every 100 g of sodium hyaluronate gel was 2 g.

[0144] Under item 2.1, the organic reagent was ethanol, and the mass ratio of the organic reagent to the bone particles was 2:1.

[0145] Under item 2.2, the degreased bone particles under item 2.1 were soaked in 3.5 wt% sodium hydroxide for 1.5 h for virus inactivation.

[0146] Under item 2.3, the deproteinization treatment was carried out in 80 wt% ethylenediamine reagent and 25 wt% hydrogen peroxide solution. The mass ratio of the reagent to the bone particles was 2.5:1, and the temperature for ethylenediamine deproteinization treatment was 115 °C.

[0147] Under item 2.5, the heat treatment temperature was 350 °C and the heat treatment time was 8 h;

[0148] Under item 2.6, the screening particle size range was 0.25 mm - 0.5 mm.

[0149] Under item 3.1, the mass ratio of sodium hyaluronate gel to bone particles was 6:6, and the addition amount of calcium carbonate was 0.02 g to ensure that the carbonate content in the finally obtained bone repair material was 3 wt% - 4 wt%.

[0150] Under item 3.2, the heat treatment temperature was 100 °C and the heat treatment time was 30 min.

[0151] Under item 3.3, the vacuum drying temperature was 60 °C and the time was 6 h.

[0152] Example 7

[0153] This example is a variant of Example 1. The differences from Example 1 are only as follows:

[0154] Under item 1.1, the pH value of the prepared PBS buffer solution was 5.

[0155] Under item 1.2, the concentration of sodium hyaluronate in the prepared sodium hyaluronate gel was 6 g.

[0156] Under item 2.1, the organic reagent is ethanol, and the mass ratio of the organic reagent to the bone particles is 3.5:1.

[0157] Under item 2.2, the degreased bone particles under item 2.1 are soaked in 4.5 wt% sodium hydroxide for 0.5 h for virus inactivation.

[0158] Under item 2.3, the deproteinization treatment is carried out in 90% ethylenediamine reagent and 35% hydrogen peroxide solution, the mass ratio of the reagent to the bone particles is 3.5:1, and the temperature for ethylenediamine deproteinization treatment is 125 °C.

[0159] Under item 2.5, the heat treatment temperature is 550 °C and the heat treatment time is 5 h.

[0160] Under item 2.6, the screening particle size range is 1 mm - 2 mm.

[0161] Under item 3.1, the mass ratio of sodium hyaluronate gel to bone particles is 10:6, and the addition amount of calcium carbonate is 0.03 g to ensure that the carbonate content in the finally obtained bone repair material is 4 wt% - 6 wt%.

[0162] Under item 3.2, the heat treatment temperature is 130 °C and the heat treatment time is 10 min.

[0163] Under item 3.3, the vacuum drying temperature is 90 °C and the time is 4 h.

[0164] Example 8

[0165] This application is a variant of Example 1, and the differences from Example 1 are only:

[0166] In the first step, hydrochloric acid solution with a pH value of 3.0 is used.

[0167] Example 9

[0168] This example is a variant of Example 1, and the differences from Example 1 are only:

[0169] Under item 3.1, magnesium carbonate is used instead of calcium carbonate.

[0170] Example 10

[0171] This example is a variant of Example 1, and the differences from Example 1 are only:

[0172] Under item 3.1, 0.04 g of calcium carbonate is added to make the content of carbonate ions in the prepared bone repair material 6.5 wt% - 8 wt%.

[0173] Example 11

[0174] This example is a variant of Example 1, and the only difference from Example 1 is that:

[0175] Under item 2.5, the heat treatment temperature is 200 °C and the heat treatment time is 10 h.

[0176] Example 12

[0177] This example is a variant of Example 1, and the only difference from Example 1 is that:

[0178] Under item 2.5, the heat treatment temperature is 575 °C and the heat treatment time is 4 h.

[0179] Example 13

[0180] This example is a variant of Example 1, and the only difference from Example 1 is that:

[0181] Calcium carbonate is not added under item 3.1.

[0182] Comparative Example 1

[0183] This comparative example is a comparative example of Example 1, and the only difference from Example 1 is that:

[0184] Under item 1.2, the dosage of sodium hyaluronate corresponding to every 100 g of sodium hyaluronate gel is 7 wt%.

[0185] Comparative Example 2

[0186] This comparative example is a comparative example of Example 1, and the only difference from Example 1 is that:

[0187] Under item 1.2, the dosage of sodium hyaluronate corresponding to every 100 g of sodium hyaluronate gel is 1 wt%.

[0188] Comparative Example 3

[0189] This comparative example is a comparative example of Example 1, and the only difference from Example 1 is that:

[0190] Under item 3.2, the heat treatment temperature is 140 °C and the heat treatment time is 10 min.

[0191] Comparative Example 4

[0192] This comparative example is a comparative example of Example 1, and the only difference from Example 1 is that:

[0193] Under item 3.2, the heat treatment temperature is 120 °C and the heat treatment time is 40 min.

[0194] Comparative Example 5

[0195] This comparative example is the comparative example of Example 8, and the difference from Example 1 is only that:

[0196] The heat treatment step under 3.2 is omitted.

[0197] Comparative Example 6

[0198] This comparative example is the comparative example of Example 8, and the difference from Example 1 is only that:

[0199] In the first step, a hydrochloric acid solution with a pH of 2.5 is used to prepare the hyaluronic acid gel.

[0200] Comparative Example 7

[0201] This comparative example is the comparative example of Example 1, and the difference from Example 1 is only that:

[0202] In the first step, a hydrochloric acid solution with a pH of 5.5 is used to prepare the sodium hyaluronate gel.

[0203] Comparative Example 8

[0204] This comparative example is the comparative example of Example 1, and the difference from Example 1 is only that:

[0205] Under 1.2, after preparing the sodium hyaluronate gel, the sodium hyaluronate gel is heat-treated at a temperature of 120 °C for a time of 10 min.

[0206] Under 3.2, no heat treatment is carried out.

[0207] Comparative Example 9

[0208] This comparative example is the comparative example of Example 1, and the difference from Example 1 is only that:

[0209] Under 3.2, the heat treatment temperature is 90 °C and the heat treatment time is 40 min.

[0210] Comparative Example 10

[0211] This comparative example is the comparative example of Example 1, and the difference from Example 1 is only that:

[0212] In the first step, a PBS buffer solution with a pH of 2.5 is used.

[0213] Comparative Example 11

[0214] This comparative example is the comparative example of Example 1, and the difference from Example 1 is only that:

[0215] In the first step, a PBS buffer solution with a pH of 5.5 is used.

[0216] During the experiment of preparing sodium hyaluronate gel, it was found that when the concentration exceeded 6 wt%, the fluidity of the sodium hyaluronate gel decreased, the viscoelasticity increased, and it was still relatively viscous after high-temperature treatment, which was not conducive to forming a coating layer on bone particles and had strong slipperiness. When the concentration of hyaluronic acid was lower than 2 wt%, the solution was dilute and lacked viscosity. Therefore, the concentration of sodium hyaluronate in the sodium hyaluronate gel was 2 wt% - 6 wt%.

[0217] In order to reduce the slipperiness of the sodium hyaluronate gel itself, it was heat-treated in an acidic environment. During the heat treatment of the sodium hyaluronate gel, it was found that in the same acidic environment, with the increase of the heating temperature and heating time, the slipperiness of the sodium hyaluronate decreased. When the heating temperature reached above 130 °C and the heating time exceeded 30 min, the sodium hyaluronate gel presented a dilute solution state, and the effect of coating bovine bone particles was poor. When the heating temperature was lower than 100 °C and the heating time was less than 10 min, the sodium hyaluronate gel presented a relatively viscous state and still had a certain degree of slipperiness. At the same heating temperature and heating time, with the continuous decrease of pH, when it was lower than 3, the sodium hyaluronate gel presented a dilute solution state, and the effect of coating bovine bone particles was poor. When the pH was higher than 5, the sodium hyaluronate gel was viscous and had strong slipperiness.

[0218] During the mixing experiment, it was found that when the mixing ratio of the sodium hyaluronate gel to bone particles exceeded 10:6, the osteogenic effect of the animal experiment decreased. During the oral bone defect repair process, in addition to having a stable blood clot, bone repair materials were also required to provide a stable scaffold for the cells to migrate, adhere and proliferate to generate new bone. Therefore, it was necessary to ensure that the amount of bone particles was not too small. When the proportion of bone particles increased, it would affect the mixing process with the gel, and the formed bone mass would be washed away in the case of excessive water absorption and blood absorption, which was not conducive to the stability of the bone repair material in the surgical area. Therefore, the ratio of the sodium hyaluronate gel to bone particles was (6 - 10):6.

[0219] Experimental Example 1, Detection of Carbonate Content

[0220] Hydrochloric acid standard titration solution (c = 0.1 mol / L): Pipette 9 mL of hydrochloric acid (ρ ≈ 1.19 g / mL), and dilute it to 1000 mL with water. Weigh accurately 0.20 g (accurate to 0.0001 g) of anhydrous sodium carbonate (>99.99%) which has been pre-ignited to constant weight at 270℃ - 300℃ and cooled to room temperature in a desiccator, place it in a 250 mL beaker, dissolve it with 50 mL of water, add 10 drops of bromocresol green-methyl red indicator solution, titrate with the prepared hydrochloric acid solution until the solution changes from green to dark red, boil for 2 min, cool and continue titrating until the solution is dark red. At the same time, conduct a blank control group (no sample is added in the blank control group, and other treatment methods are the same as those in the test group); every 1 mL of hydrochloric acid titration solution (0.1 mol / L) is equivalent to 5.30 mg of anhydrous sodium carbonate.

[0221] Sodium hydroxide standard titration solution (c = 0.1 mol / L): Weigh 110 g of sodium hydroxide, dissolve it in 100 mL of water free of carbon dioxide, mix well, transfer it into a polyethylene container, and close it for storage until the solution becomes clear. Pipette 5.4 mL of the supernatant, dilute it to 1000 mL with water free of carbon dioxide, and mix well. Weigh accurately 0.75 g (accurate to 0.0001 g) of potassium hydrogen phthalate (>99.99%) which has been dried to constant weight in an electric oven at 105℃ - 110℃ and cooled to room temperature, place it in a 250 mL beaker, dissolve it with 50 mL of water free of carbon dioxide. Add 2 drops of phenolphthalein solution (10 g / L), titrate with the prepared sodium hydroxide standard titration solution until it turns pink and remains pink for 30 s as the end point; every 1 mL of sodium hydroxide titration solution (0.1 mol / L) is equivalent to 20.42 mg of potassium hydrogen phthalate.

[0222] Barium chloride (BaCl2·2H2O) solution: 122 g / L.

[0223] Phosphoric acid: (1 + 1).

[0224] Hydrochloric acid: 1 mol / L.

[0225] Sodium hydroxide: 1 mol / L.

[0226] Methyl orange solution: 1 g / L.

[0227] Phenolphthalein solution: 0.25 g / L, prepared with 50% ethanol.

[0228] Sample preparation: Take an appropriate amount of the bone repair material samples provided by each example and comparative example, grind them finely, and dry them in a drying oven at 105℃ for 2 h, then cool to room temperature. Weigh accurately 0.5 g of the fine powder and place it in an acid extraction CO2 conical flask.

[0229] Inspection of device airtightness: As per Figure 1Connect the test device properly. Add an appropriate amount of water to each conical flask so that the mouth of the catheter is immersed in the aqueous solution. Seal the air inlet with a stopcock, close the piston of the dropping funnel, and gently heat the first conical flask. If bubbles appear at the mouth of the catheter in the last conical flask, stop heating. A liquid column will form in the catheter inserted under the liquid level of the last conical flask, indicating that the airtightness of the device is good.

[0230] Blank test: Conduct a blank control group simultaneously (no bone repair material sample is added to the blank control group, and other treatment methods are the same as those in the test group).

[0231] Sample determination:

[0232] Precisely weigh 0.5 g of the sample and transfer it to a conical flask for acid extraction of CO₂. After adding about 100 mL of water, cover the neck plug and introduce nitrogen that has passed through secondary gas washing for 10 minutes at a nitrogen rate of about 50 mL / min. The following entire process is carried out under nitrogen conditions.

[0233] Without cutting off the nitrogen, connect the secondary absorption device. Each absorption conical flask contains 10 mL of 1 mol / L sodium hydroxide solution, 10 mL of 122 g / L barium chloride solution, 1 mL of 0.25 g / L phenolphthalein solution, and 20 mL of water.

[0234] Add 50 mL of 50% phosphoric acid solution to the conical flask for acid extraction of CO₂ through the dropping funnel, close the piston of the dropping funnel, and react for 50 minutes.

[0235] Titration:

[0236] Remove the second absorption flask, remove and rinse the inner tube with water, combine the washing solution in the absorption flask, and titrate the solution in the absorption flask with about 1 mol / L hydrochloric acid until it is close to the end point.

[0237] Continue to titrate with the hydrochloric acid standard titration solution until the phenolphthalein just turns colorless.

[0238] Add a certain excess of the hydrochloric acid standard solution until the precipitate completely dissolves. Immerse the inner tube in this solution to dissolve all the attached barium carbonate. Add 3 drops of methyl orange solution and back-titrate the excess hydrochloric acid with the sodium hydroxide standard titration solution.

[0239] Operate in the same way to neutralize and titrate the solution in the first absorption flask.

[0240] Result calculation:

[0241] Calculate the content of carbonate radical according to the following formula, expressed as W CO3 2- (%).

[0242]

[0243] Where: v1: the volume of the hydrochloric acid standard titrant used to dissolve barium carbonate in the two absorption flasks, mL;

[0244] v2: the volume of the sodium hydroxide standard titrant consumed for back-titrating the excessive hydrochloric acid in the two absorption flasks, mL;

[0245] v3: the volume of the hydrochloric acid standard titrant used to dissolve barium carbonate in the two absorption flasks in the blank test, mL;

[0246] v4: the volume of the sodium hydroxide standard titrant consumed for back-titrating the excessive hydrochloric acid in the two absorption flasks, mL;

[0247] c1: the concentration of the hydrochloric acid titrant, mol / L;

[0248] c2: the concentration of the sodium hydroxide standard titrant, mol / L;

[0249] m: the mass of the sample, g;

[0250] 30: the molar mass of 1 / 2 carbonate, g / mol.

[0251] Table 1. Detection results of carbonate content

[0252] Experiment Number Corresponding Grouping Detection Result of Carbonate Content (%) 1 Example 1 2.28±0.06 2 Example 6 3.32±0.05 3 Example 7 4.90±0.04 4 Example 9 2.26±0.02 5 Example 10 6.75±0.09 6 Example 13 1.13±0.02 7 Comparative Example 1 2.34±0.05 8 Comparative Example 6 2.25±0.07 9 Comparative Example 7 2.24±0.06 10 Comparative Example 9 2.31±0.03

[0253] The detection results of calcium carbonate content show that after adding an appropriate amount of carbonate, a layer of bone material in the form of carbonate is formed on hydroxyapatite. The presence of carbonate plays a crucial role in the metabolic balance of inorganic minerals in animal and human bone tissues, is more conducive to osteogenesis. At the same time, the specific spatial structure formed after a large number of hydroxyl groups on the surface of sodium hyaluronate bind to cations also better promotes the stability of the whole system. With the increase in the proportion of added carbonate, the carbonate content in the product shows a corresponding upward trend, and different carbonates have no obvious influence on the detection of carbonate content; no carbonate was added in Example 13, and the carbonate content in the product is lower than the set value.

[0254] Experimental Example 2. pH value detection

[0255] The pH of the 0.05 mol / L potassium hydrogen phthalate solution is 4.01 at 25°C;

[0256] The pH of the mixed solution of 0.025 mol / L disodium hydrogen phosphate and 0.025 mol / L sodium dihydrogen phosphate is 6.86 at 25°C;

[0257] The pH of the 0.01 mol / L borax solution is 9.18 at 25°C;

[0258] Take one pack of each standard buffer, dissolve it in distilled water (purified water that has been freshly boiled and cooled), and make up the volume to 250 mL.

[0259] Take 0.5 g of the sample, weigh accurately, with the extraction medium / bio-bone repair material (v / w) being 12:1. The extraction medium is freshly boiled and cooled purified water. Extract at 37 ± 1°C for 72 ± 2 h, and wait for inspection.

[0260] Before determination, according to the regulations under each variety, select two standard buffer solutions with a pH difference of 3 units, so that the pH value of the test solution is between the two;

[0261] Use the first standard buffer solution closer to the pH value of the test solution to calibrate (locate) the instrument, so that the instrument indication is consistent with the listed value;

[0262] After the instrument is located, check the instrument indication with the second standard buffer solution. The error should not be greater than ±0.02 pH units. If it is greater than this deviation, carefully adjust the slope so that the indication is consistent with the listed value of the second standard buffer solution. Repeat the above positioning and slope adjustment operations until the difference between the instrument indication and the specified value of the standard buffer solution is not greater than ±0.02 pH units. Otherwise, check the instrument or replace the electrode and then calibrate it until it meets the requirements.

[0263] Measure the pH of the sample, measure it twice repeatedly and take the average value. The error between the two measurements does not exceed 0.1, and make a record;

[0264] Before each replacement of the standard buffer solution or the test solution, wash the electrode thoroughly with purified water, then suck the water on the electrode with filter paper, or it can also be washed with the replaced standard buffer solution or the test solution.

[0265] Table 2. pH value test results

[0266] Experiment Number Corresponding Grouping Detection Result of pH Value 1 Example 1 7.11±0.02 2 Example 2 7.11±0.04 3 Example 3 7.11±0.04 4 Example 4 7.13±0.03 5 Example 5 7.15±0.04 6 Example 6 7.13±0.02 7 Example 7 7.17±0.04 8 Example 8 7.16±0.03 9 Example 9 7.15±0.02 10 Example 10 7.13±0.05 11 Example 11 7.13±0.06 12 Example 12 7.16±0.01 13 Example 13 7.16±0.04 14 Comparative Example 1 7.14±0.06 15 Comparative Example 2 7.14±0.04 16 Comparative Example 3 7.11±0.03 17 Comparative Example 4 8.01±0.05 18 Comparative Example 5 9.46±0.04 19 Comparative Example 6 6.35±0.04 20 Comparative Example 7 7.13±0.05 21 Comparative Example 8 7.15±0.04 22 Comparative Example 9 7.13±0.03 23 Comparative Example 10 6.28±0.03 24 Comparative Example 11 7.12±0.04

[0267] The pH value test results show that preferably, when the heat treatment temperature of the bone particles does not exceed 550°C and the heat treatment time does not exceed 8 h, there is no obvious difference in the pH values of each example and the comparative example. The bone particles themselves are weakly alkaline. After being mixed with sodium hyaluronate gel prepared with PBS buffer solution or hydrochloric acid with a suitable pH value and dried, they can all maintain a pH close to that of human tissues. The solubility of calcium carbonate in water is extremely low, and it has no obvious effect on the pH value of the bone repair material.

[0268] Experimental Example 3. Detection of sodium hyaluronate content

[0269] Carbazole ethanol solution with a volume fraction of 0.125%: Weigh 0.125 g of carbazole and dissolve it in 100 mL of anhydrous ethanol;

[0270] Glucuronic acid (GA) standard solution: Weigh accurately about 0.1 g of glucuronic acid reference substance which has been dried to constant weight at 105 °C with phosphorus pentoxide as the desiccant under vacuum, place it in a 100 mL volumetric flask, dissolve it with water and dilute to the mark, shake well to obtain the stock solution. Accurately measure 5.0 mL of the stock solution, place it in a 100 mL volumetric flask, and make a solution containing 50 μg per 1 mL with water, shake well.

[0271] 0.025 mol / L sodium tetraborate sulfuric acid solution: Weigh 9.54 g of sodium tetraborate (Na2B4O7·10H2O), add it to 1 L of concentrated sulfuric acid, cover it. Shake it from time to time until sodium tetraborate is completely dissolved, and store it at room temperature.

[0272] Sample preparation: Take the bone repair material samples of each example and comparative example after being dried to constant weight at 105 °C, grind the samples into fine powder, accurately weigh 0.2 g and put it into a triangular flask, transfer about 70 mL of purified water to the triangular flask, extract it at 37 °C and 120 r / min for 24 hours, then transfer it to a volumetric flask and make up to 100 mL and shake well. Take 1 mL of the solution and place it in a test tube for testing. Prepare a series of glucuronic acid standard solutions according to the following table.

[0273] Table 3. Concentrations of glucuronic acid (GA) standard solution series

[0274] Test Tube Number 0 1 2 3 4 5 GA Standard Solution / mL 0 0.2 0.4 0.6 0.8 1.0 Distilled Water / mL 1.0 0.8 0.6 0.4 0.2 0 GA Content / (μg / mL) 0 20 40 60 80 100

[0275] Detection steps: Place each test tube of the standard solution series and the sample test tube together in an ice-water bath, slowly add 5 mL of 0.025 mol / L sodium tetraborate sulfuric acid (stored in a 4 °C refrigerator for at least 2 h before use) to each tube, shake well while adding. After adding, mix well and place it in a boiling water bath and boil for 15 min, then take it out and cool in an ice-water bath. Add 0.2 mL of carbazole ethanol solution to each test tube. After mixing well, place it in a boiling water bath and boil for 15 min, and cool to room temperature. Use test tube No. 0 as the control, and measure the absorbance of each standard tube and sample tube at 530 nm with a spectrophotometer.

[0276] Draw an absorbance-concentration curve with the standard tubes, and find the glucuronic acid content of the sample tube from the standard curve according to the absorbance of the sample tube. Calculate the concentration of sodium hyaluronate in the dilution solution: Subtract the absorbance of the blank solution from the absorbance of the dilution solution, and find the concentration of sodium hyaluronate in the dilution solution from the obtained standard curve.

[0277] Calculation formula:

[0278] W%=[(2.07*c) / (10000*m)]×100%, where

[0279] c: The glucuronic acid concentration obtained from the standard curve for the sample tube, g / mL;

[0280] m: mass of the weighed sample, g;

[0281] 2.07: conversion coefficient for converting glucuronic acid to sodium hyaluronate;

[0282] 10000: conversion coefficient for converting the mass of glucuronic acid in 100 mL of the sample solution from gg to g.

[0283] Table 4. Detection results of sodium hyaluronate content

[0284]

[0285]

[0286] Experimental Example 4. Sliding performance test

[0287] Bone repair material samples of each example and comparative example with the same specification were placed on a culture dish, and physiological saline was added dropwise until completely infiltrated. The state and sliding distance of the bone block were recorded at 1 min, 3 min, and 5 min respectively (the maximum sliding distance was 8 cm). Some examples and comparative examples are shown in Figure 4 - Figure 8 .

[0288] Table 5. Sliding performance test results

[0289]

[0290]

[0291] Note: " / " indicates that the bone repair material has dispersed; "8.0+" indicates that the bone repair material has slid to the maximum distance.

[0292] Experimental Example 5. SEM scanning electron microscopy microscopic morphology detection

[0293] After the bone repair material samples of each example and comparative example were sputter-coated with gold on an ion sputtering instrument (HITACHI E-1045), they were placed under a field emission scanning electron microscope (HITACHI SU8010) for scanning, and the acceleration voltage was 3.0 kV. The results are shown in Figure 3 .

[0294] The experimental results showed that the sodium hyaluronate gel on the surface of the bone particles in Comparative Example 1 agglomerated and was overall non-uniform; the sodium hyaluronate gel adhered less to the surface of the bone particles in Comparative Example 2. In Comparative Example 5, due to the lack of heat treatment, the binding degree between the bone particles and the hyaluronic acid gel was poor; similar to Comparative Example 5, in Comparative Example 8, due to the change in the operation sequence of heat treatment, the binding degree between the bone particles and the hyaluronic acid gel was also poor. However, in Example 1, the surface of the bone particles was uniformly coated with sodium hyaluronate.

[0295] Experimental Example 6. Efficacy test (animal experiment: rabbit cranial defect experiment)

[0296] An experimental New Zealand rabbit was anesthetized. After shaving the hair on the top of its head, it was placed on a dedicated operating table in the ventral position. It was disinfected with iodine and alcohol, and sterile drapes were laid. A 5-cm straight incision was made in the middle and slightly posterior position of the top of the head, cutting through the scalp to expose the coronal suture and sagittal suture of the skull. Modeling was carried out on both sides of the sagittal suture, behind the coronal suture and in front of the lambdoid suture. The skull was ground open with a high-speed turbine to form a circular bone window with a diameter of about 1.0 cm, and hemostasis was achieved with medical gauze. Different bone repair materials (the bone regeneration materials prepared in Example 1, Example 11, and Example 13 of the present invention) were placed at the skull defect site. Two bone defects, one on the left and one on the right, were created in each animal, and the wound was sutured after implanting the materials. Micro-CT scans were taken at 1 week, 2 weeks, 4 weeks, and 8 weeks after the operation to observe the repair of the skull defect. The experimental results are as Figure 9 shown. The early bone repair effect of Example 1 was excellent, and the bone defect could be basically repaired and the formation of new bone could be induced at 8 weeks.

[0297] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0298] The above-described embodiments only represent several implementation manners of the present application, which are convenient for understanding the technical solutions of the present application specifically and in detail, but should not be construed as a limitation on the scope of patent protection of the application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. In addition, it should be understood that after reading the above teachings of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning, or limited experiments based on the technical solutions provided by the present application are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application should be determined by the content of the appended claims, and the specification and drawings can be used to explain the content of the claims.

Claims

1. A method for preparing a bone repair material, characterized in that: The preparation method comprises the following steps: Mixing sodium hyaluronate gel and bone graft particles to prepare a mixture; heat-treating the mixture under a second heat-treatment condition to remove the solvent and prepare a bone repair material; The second heat treatment conditions include: (1) the heat treatment temperature is about 100° C. to 130° C., and the heat treatment time is about 10 min to 30 min; and, (2) the pH value of the mixture is about 3 to 5; The amount of sodium hyaluronate corresponding to every 100g of the sodium hyaluronate gel is about 2g to 6g.

2. The method for preparing a bone repair material according to claim 1, characterized in that: The preparation steps of the sodium hyaluronate gel include: mixing sodium hyaluronate and a dispersion medium, and fully swelling the mixture to prepare the sodium hyaluronate gel.

3. The method for preparing the bone repair material according to claim 2, characterized in that: The dispersion medium includes one or more of PBS buffer and hydrochloric acid.

4. The method for preparing a bone repair material according to claim 2, characterized in that: The pH value of the dispersion medium is about 3-5.

5. The method for preparing a bone repair material according to claim 2, characterized in that: The intrinsic viscosity of the sodium hyaluronate is about 1.5 μm 3 / kg~3.9m 3 / kg.

6. The method for preparing a bone repair material according to any one of claims 1 to 5, characterized in that: The steps of preparing the bone graft particles include: cutting and crushing the bone donor material, defatting, inactivating viruses, deproteinizing, and heat treating under a first heat treatment condition to prepare the bone graft particles.

7. The method for preparing a bone repair material according to claim 6, characterized in that: The first heat treatment conditions include: the heat treatment temperature is about 250° C. to 550° C., and the heat treatment time is about 5 h to 8 h.

8. The method for preparing a bone repair material according to claim 6, characterized in that: The degreasing treatment is carried out in an organic reagent; optionally, the organic reagent includes one or more of isopropanol and ethanol; optionally, the mass ratio of the organic reagent to the cut and crushed bone donor material is (2.0-3.5):

1.

9. The method for preparing a bone repair material according to claim 6, characterized in that: The virus inactivation treatment uses a protein denaturant; optionally, the protein denaturant includes an alkali; optionally, the protein denaturation includes a sodium hydroxide solution with a sodium hydroxide content of about 3.5wt% to 4.5wt%; optionally, the virus inactivation treatment time is about 0.5h to 1.5h.

10. The method for preparing a bone repair material according to claim 6, characterized in that: The conditions for deproteinization treatment include: the deproteinization reagent used includes one or more of a strong oxidizing agent and a primary amine reagent; Optionally, the deproteinization temperature corresponding to the primary amine reagent is about 115° C. to 125° C.; Optionally, the strong oxidizing agent comprises a hydrogen peroxide solution; further optionally, the content of hydrogen peroxide in the hydrogen peroxide solution is about 25 wt% to 35 wt%; Optionally, the primary amine reagent includes an ethylenediamine solution; further optionally, the content of ethylenediamine in the ethylenediamine solution is about 80wt% to 90wt%; Optionally, the mass ratio of the deproteinizing agent to the virus-inactivated bone donor material is about (2.5-3.5):

1.

11. The method for preparing a bone repair material according to claim 6, characterized in that: The species sources of the bone donor material include vertebrates; Optionally, the vertebrate comprises a bovine; Optionally, the bone donor material includes limb bones, further optionally femurs, and further femoral cancellous bones.

12. The method for preparing a bone repair material according to any one of claims 1 to 11, characterized in that: The mass ratio of the sodium hyaluronate gel to the bone graft particles is about (6-10):

6.

13. The method for preparing a bone repair material according to any one of claims 1 to 12, characterized in that: The mixture also includes a carbonate; Optionally, the carbonate includes one or more of calcium carbonate and magnesium carbonate; Optionally, the content of carbonate in the bone repair material is about 2 wt% to 6 wt%.

14. The method for preparing a bone repair material according to any one of claims 1 to 13, characterized in that: The method of removing the solvent includes one or more of vacuum freeze drying and vacuum drying; Optionally, the conditions for vacuum drying include: a drying temperature of about 60° C. to 90° C., and a drying time of about 2 h to 6 h.

15. The method for preparing a bone repair material according to any one of claims 1 to 14, characterized in that: The content of sodium hyaluronate in the bone graft particles is about 2 wt % to 8 wt %.

16. The method for preparing a bone repair material according to any one of claims 1 to 15, characterized in that: The particle size of the bone graft particles ranges from about 0.25 mm to about 2 mm.

17. The method for preparing a bone repair material according to any one of claims 1 to 16, characterized in that: The bone repair material is in block, column or granular form.

18. A bone repair material, characterized in that: The bone repair material is prepared by the preparation method according to any one of claims 1 to 17.

19. A bone repair method, characterized in that: The method comprises using the bone repair material according to claim 18 to repair a bone defect area of ​​a subject.

20. The bone repair method according to claim 19, characterized in that: The bone defect area occurs in the oral bones, skull bones or non-weight-bearing bones of the limbs.

21. The bone repair method according to any one of claims 19 to 20, characterized in that: The bone repair material is directly filled into the bone defect area, or is filled into the bone defect area after absorbing water or blood.

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