Bone repair material, preparation method therefor, and use thereof

Abstract

The present application belongs to the technical field of biomedical materials, and relates to a bone repair material, a preparation method therefor, and use thereof. The preparation method comprises the following steps: mixing a sodium hyaluronate gel and bone graft particles to prepare a mixture; and performing heat treatment on the mixture under a second heat treatment condition, removing the solvent, and preparing a bone repair material, wherein the second heat treatment condition comprises: (1) the temperature of the heat treatment is about 100 °C-130 °C, and the heat treatment time is about 10 min-30 min; and (2) the pH value of the mixture is about 3-5. The amount of sodium hyaluronate per 100 g of the sodium hyaluronate gel is about 2 g-6 g. The sliding performance of the bone repair material is effectively reduced, and the repair effect is excellent.

Description

Bone repair material, preparation method and application thereof TECHNICAL FIELD

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

[0002] Diseases such as tumors, trauma, necrosis and infection often lead to bone defects, and bone transplantation is usually required after the defects. Autologous bone transplantation is considered as the "gold standard" in clinical treatment due to its outstanding performance in osteogenesis, osteoinduction and osteoguiding. However, this technique requires additional surgery to obtain bone grafts, which leads to secondary trauma and potential morbidity in the donor area. In addition, the autologous bone source is limited.

[0003] The three-dimensional structure of human bone is mainly composed of inorganic mineral phase, water and organic biomolecules, including collagen and non-collagen polysaccharides (GAGs). The inorganic mineral provides the basic scaffold, and the organic matrix provides the elasticity, and the properties of the two different compounds determine the optimal performance of the bone material. According to the research on human bone, 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 simulate the structure of human bone, such as the product Bio-oss Collagen (Geistlich Biomaterials, Geistlich, Switzerland) on the market abroad, which is composed of 90% bovine cancellous bone and 10% porcine collagen. This material can be used in multiple clinical scenarios, and the increased collagen component can better protect the sinus mucosa from being pierced during maxillary sinus floor lifting surgery. However, this material introduces two different species sources, increasing the risk of immunogenic rejection.

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

[0006] US8876532B2 describes 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 fifteen amino acid sequences), and absorbable carriers (such as high molecular weight hyaluronic acid or hydroxypropyl cellulose). 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 to form a paste with certain plasticity.

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

[0008] SUMMARY

[0009] One or more embodiments of the present application provide a bone repair material and a preparation method and application thereof, and the technical solution is as follows:

[0010] One or more embodiments of the present application provide a preparation method of a bone repair material, and the preparation method comprises the following steps:

[0011] Mixing sodium hyaluronate gel and bone graft particles to prepare a mixture;

[0012] Heat treating the mixture under a second heat treatment condition to remove the solvent and prepare a bone repair material;

[0013] The second heat treatment condition comprises:

[0014] (1) the temperature of heat treatment is about 100-130℃, and the heat treatment time is about 10-30min; and

[0015] (2) the pH value of the mixture is about 3-5;

[0016] The amount of sodium hyaluronate corresponding to 100g of the sodium hyaluronate gel is about 2-6g.

[0017] In some embodiments of the present application, the preparation step of the sodium hyaluronate gel comprises mixing sodium hyaluronate and a dispersion medium, and swelling sufficiently to prepare a sodium hyaluronate gel.

[0018] In some embodiments of the present application, the dispersion medium comprises one or more of PBS buffer and hydrochloric acid.

[0019] In some embodiments of the present application, the pH value of the dispersion medium is about 3-5.

[0020] In some embodiments of the present application, the intrinsic viscosity of the sodium hyaluronate is about 1.5m 3 / kg-3.9m 3 / kg.

[0021] In some embodiments of the present application, the preparation step of the bone graft particles comprises cutting and crushing a bone donor material, defatting treatment, virus inactivation treatment, deproteinization treatment, and heat treatment under a first heat treatment condition to prepare the bone graft particles.

[0022] In some embodiments of the application, the first heat treatment condition comprises a temperature of about 250-550°C and a time of about 5-8 hours.

[0023] In some embodiments of the application, the defatting treatment is performed in an organic reagent; optionally, the organic reagent comprises one or more of isopropyl alcohol and ethanol; optionally, the mass ratio of the organic reagent to the cut and pulverized bone donor material is (2.0-3.5):1.

[0024] In some embodiments of the application, the viral inactivation treatment employs a protein denaturant; optionally, the protein denaturant comprises a base; optionally, the protein denaturant comprises a sodium hydroxide solution having a sodium hydroxide content of about 3.5-4.5 wt%; optionally, the viral inactivation treatment is performed for a time of about 0.5-1.5 hours.

[0025] In some embodiments of the application, the deproteinization treatment is performed under conditions comprising the use of a deproteinization reagent comprising one or more of a strong oxidizing reagent and a primary amine reagent.

[0026] Optionally, the primary amine reagent is used at a temperature of about 115-125°C.

[0027] Optionally, the strong oxidizing reagent comprises a hydrogen peroxide solution; further optionally, the hydrogen peroxide solution has a hydrogen peroxide content of 25-35 wt%.

[0028] Optionally, the primary amine reagent comprises an ethylenediamine solution; further optionally, the ethylenediamine solution has an ethylenediamine content of 80-90 wt%.

[0029] Optionally, the deproteinization reagent is used at a mass ratio of (2.5-3.5):1 to the viral inactivation treated bone donor material.

[0030] In some embodiments of the application, the bone donor material is derived from a vertebrate animal.

[0031] Optionally, the vertebrate animal is a bovine.

[0032] Optionally, the bone donor material comprises a limb bone, further optionally a femur, and more further optionally a femoral cancellous bone.

[0033] In some embodiments of the application, the mass ratio of the sodium hyaluronate gel to the bone graft particles is about (6-10):6.

[0034] In some embodiments of the application, the mixture further comprises a carbonate salt.

[0035] Optionally, the carbonate salt comprises one or more of calcium carbonate and magnesium carbonate.

[0036] Optionally, the content of carbonate in the osteoinductive material is about 2wt% to 6wt%.

[0037] In some embodiments of the present application, the method of removing solvent comprises one or more of vacuum freeze-drying and vacuum baking.

[0038] Optionally, the conditions of vacuum baking comprise a drying temperature of about 60°C to 90°C and a drying time of about 2h to 6h.

[0039] In some embodiments of the present application, the content of sodium hyaluronate in the osteoinductive material is about 2wt% to 8wt%.

[0040] In some embodiments of the present application, the particle size of the bone graft particles is about 0.25mm to 2mm.

[0041] In some embodiments of the present application, the bone repair material is in the form of a block, a cylinder or a particle.

[0042] One or more embodiments of the present application also provide a bone repair material prepared by the preparation method.

[0043] One or more embodiments of the present application further provide a bone repair method, comprising repairing a bone defect region of a subject using the bone repair material.

[0044] In some embodiments of the present application, the bone defect region occurs in an oral bone, a skull bone or a non-weight-bearing limb bone.

[0045] In some embodiments of the present application, the bone repair material is directly filled into the bone defect region or is filled into the bone defect region after absorbing water or blood.

[0046] The details of one or more embodiments of the present application are set forth in the accompanying description below, and other features, objects and advantages of the present application will be apparent from the description and the claims. BRIEF DESCRIPTION OF DRAWINGS

[0047] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the drawings needed in the description of the embodiments will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0048] Figure 1 is a diagram of the instrument device for detecting the carbonate content in bone repair materials;

[0049] Figure 2 is a diagram of bone repair materials placed in simulated oral cavity tooth socket (can be placed directly in dry state or placed after cutting with water / blood in vitro);

[0050] Figure 3 is a SEM scanning electron microscope observation;

[0051] Figure 4 is the sliding property test result of the bone repair material provided in Example 1;

[0052] Figure 5 is the sliding property test result of the bone repair material provided in Comparative Example 2;

[0053] Figure 6 is the sliding property test result of the bone repair material provided in Comparative Example 3;

[0054] Figure 7 is the sliding property test result of the bone repair material provided in Comparative Example 4;

[0055] Figure 8 is the sliding property test result of the bone repair material provided in Comparative Example 6;

[0056] Figure 9 is the Micro-CT image of the skull defect animal experiment of the bone repair materials. DETAILED DESCRIPTION

[0057] The present application will be further described below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to explain the present application and not intended to limit the scope of the present application, and the purpose of providing these embodiments and examples is to make the understanding of the present application more thorough and comprehensive. It should also be understood that the present application can be realized in many different forms and is not limited to the embodiments and examples described herein, and those skilled in the art can make various modifications or changes without departing from the spirit of the present application, and the equivalent forms obtained thereby also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the specification of the present application herein is only for the purpose of describing the embodiments and examples and is not intended to limit the present application.

[0059] Terminology

[0060] Unless otherwise stated or contradictory, the terms or phrases used herein have the following meanings:

[0061] The selection range of the terms "and / or", "or / and", "and / or" used in the present application includes any one of two or more relevant listed items, and also includes any and all combinations of the relevant listed items, which includes any two relevant listed items, any more relevant listed items, or all relevant listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in the present application, the 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 of A, B and A+B. For 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 (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination 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 (i.e. the technical solution connected by "logical and").

[0062] In the present application, "multiple", "various", "multiple times", "multiple" and the like refer to greater than or equal to 2 in number, unless otherwise specified. For example, "one or more" means one or greater than or equal to two.

[0063] As used herein, "combinations thereof", "any combination thereof", "any combination manner thereof" and the like include all suitable combination manners of any two or more of the listed items.

[0064] As used herein, "suitable combination manner", "suitable manner", "any suitable manner" and the like refer to the ability to implement the technical solutions of the present application, solve the technical problems of the present application, and achieve the intended technical effects of the present application.

[0065] As used herein, "preferably", "better", "better", "as appropriate" only describe the implementation manner or embodiment with better effect, and it should be understood that it does not constitute a limitation on the protection scope of the present application.

[0066] In the present application, "further", "more further", "in particular" and the like are used to describe the purpose, indicating the difference in content, but should not be understood as a limitation on the protection scope of the present application.

[0067] In the present application, "optionally", "optional", "optional" means optional, i.e. selected from two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified, and there is no contradictory relationship or mutual restriction, each "optional" is independent.

[0068] In the present application, the terms "first", "second", "third", "fourth" and the like in the "first aspect", "second aspect", "third aspect", "fourth aspect" and the like are only used 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 technical features indicated. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration and description, and should be understood as not constituting a closed limitation on the quantity.

[0069] In the present application, among the technical features described in an open manner, a closed technical solution consisting of the listed features is also included, as well as an open technical solution containing the listed features.

[0070] In the present application, with respect to a numerical interval (i.e. a numerical range), if no specific description is provided, the optional numerical distribution within the above-mentioned numerical interval is considered to be continuous, and includes the two numerical end points (i.e. the minimum value and the maximum value) of the numerical range, as well as every numerical value between the two numerical end points. If no specific description is provided, when a numerical interval only refers to integers within the numerical interval, including the two end point integers of the numerical range and every integer between the two end points, in this document, it is equivalent to directly listing every integer, for example, t is an integer selected from 1 to 10, which means that t is any integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe a feature or a characteristic, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this document should be understood to include any and all sub-ranges encompassed therein.

[0071] In the present application, the temperature parameter, if not specifically limited, allows both constant temperature treatment and variation within a certain temperature interval. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. It is allowed to fluctuate within the range of, for example, ±5°C, ±4°C, ±3°C, ±2°C, ±1°C.

[0072] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0073] All the documents mentioned in the present application are incorporated by reference into the present application as if each document was individually incorporated. The documents mentioned in the present application are incorporated by reference in their entirety, for all purposes, unless and except to the extent that such incorporation is inconsistent with the express intent and / or technical teachings of the present application. When the present application refers to the documents mentioned, the definitions of the relevant technical features, terms, names, phrases, etc. in the documents are also incorporated by reference. When the present application refers to the documents mentioned, the examples and preferred modes of the relevant technical features are also incorporated by reference into the present application, as far as the present application can be implemented. It should be understood that when the content of the references conflicts with the description in the present application, the present application is the priority or the description in the present application is amended adaptively.

[0074] In a first aspect, the present application provides a preparation method of a bone repair material, which comprises the following steps:

[0075] mixing the sodium hyaluronate gel and the bone graft particles to prepare a mixture;

[0076] heat treating the mixture under a second heat treatment condition to remove the solvent and prepare the bone repair material;

[0077] The second heat treatment condition comprises:

[0078] (1) the temperature of the heat treatment is about 100-130℃ (for example, 100℃, 101℃, 102℃, 103℃, 104℃, 105℃, 106℃, 107℃, 108℃, 109℃, 110℃, 111℃, 112℃, 113℃, 114℃, 115℃, 116℃, 117℃, 118℃, 119℃, 120℃, 121℃, 122℃, 123℃, 124℃, 125℃, 126℃, 127℃, 128℃, 129℃, 130℃), and the heat treatment time is about 10-30min (for example, 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min, 30min); and,

[0079] (2) the pH value of the mixture is about 3-5 (for example, 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);

[0080] The amount of sodium hyaluronate corresponding to each 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).

[0081] The bone repair material prepared by the embodiment of the present application is also a moldable oral bone induction material. The material is assembled by a suitable sodium hyaluronate gel and bone graft particles. The bone graft particles are wrapped by the three-dimensional network gel of sodium hyaluronate, and have a biomimetic structure similar to human natural bone tissue. In the preparation method provided by the embodiment of the present application, the mixture of sodium hyaluronate gel and bone graft particles is heat treated under suitable conditions, so that the prepared material has excellent cohesiveness and reduces the sliding property of sodium hyaluronate itself.

[0082] At present, various bone grafts can be used in clinical practice to replace autologous bone grafts. Vertebrates, especially bovine bones, are considered to be the most similar to human bones and are widely used in many commercial products. Due to the risk of immunogenicity and viral 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 inorganic components by using calcination process and completely remove some bioactive components, so their osteogenesis and osteoinduction functions are significantly reduced. Moreover, bone powder products are inconvenient to use in clinical applications, such as in the surgical area of the anterior teeth with sufficient blood supply and Type II-0, Type II-I, Type II-II alveolar bone defects. Especially for some specific indications and regional positions, the use is limited, such as the surgical process of maxillary sinus floor elevation to treat insufficient bone volume in the posterior maxillary teeth. Since the maxillary sinus mucosa itself is relatively thin, the rough surface of the bone material can cause complications such as perforation of the maxillary sinus mucosa during the maxillary sinus lifting process, causing maxillary sinus infection, implant shedding, treatment failure and other risks. The perforation rate reported in the clinical literature is as high as about 60%. The scheme provided by the present application can overcome the above technical defects.

[0083] The material provided by the embodiments of the present application can be cut at will under water or blood in vitro to match the size of the defect, or directly filled in the alveolar bone defect site in a dry block shape to be molded in situ (as shown in FIG. 2), which is convenient for the operation of the clinician and saves the operation time. In particular, it is more convenient for the operation of some complex bone defects or Type II-0, Type II-I, Type II-II alveolar bone defects, and especially for better protection of the sinus mucosa during the maxillary sinus floor lifting surgery. After the alveolar bone defect is implanted, the treated sodium hyaluronate gel contained in the material can quickly absorb the blood to stabilize the blood clots on the bioactive bone scaffold. When the pusher applies a pushing pressure to the bone augmentation material implanted in the implantation well, the blood absorbed by the sodium hyaluronate wrapped bone graft particles will be released, forming a hydraulic effect. This hydraulic effect will push the bone augmentation material and the released blood towards the sinus floor and the surrounding direction, tear the sinus mucosa and bone membrane from the sinus floor bone wall, support the broken residual sinus floor bone plate and mucosa together, and thus form a tent-like structure with a certain height, creating space for the implantation of bone augmentation material and implants. The material of the present application has excellent osteogenesis and osteoinduction effects, and can still maintain the stability of the bioactive bone scaffold in the surgical area of the front teeth with a large amount of bleeding.

[0084] In addition, compared with the technical solution described in WO2009131323A3, the osteogenesis and osteoinduction functions of the present application are good. Moreover, the introduction of toxic chemical cross-linking agents is avoided, and the biological behavior of the product is as far as possible. Compared with the technical solution described in US8876532B2, the material prepared in the present application does not need to manually mix the bone material with the absorbable carrier to form a plastic paste during clinical use, which is convenient to use, and can effectively avoid the situation that the bone space shape contour is maintained poorly due to the sliding property of sodium hyaluronate itself. It is suitable for the surgical area of the front teeth with a large amount of bleeding and has good use effect, avoids leading to a decrease in long-term bone quality and thus exposing the implant, avoids the necessity of secondary surgery, and reduces the economic burden of the patient.

[0085] In some embodiments, the preparation step of the sodium hyaluronate gel comprises: mixing sodium hyaluronate and a dispersion medium, swelling sufficiently, and preparing a sodium hyaluronate gel.

[0086] The embodiments of the present application do not particularly limit the type of dispersion medium, for example, a dispersion medium having a pH value outside the range of about 3-5 can be used to prepare the sodium hyaluronate gel, and the pH value of the sodium hyaluronate gel or the mixture of the sodium hyaluronate gel and the bone graft particles is adjusted to about 3-5 before subsequent heat treatment; or a dispersion medium having a pH value of about 3-5 can be directly used to prepare the sodium hyaluronate gel and mixed with the bone graft particle mixture to make the pH value of the obtained mixture about 3-5, and then directly heat treated. In some embodiments, the dispersion medium comprises one or more of PBS buffer and hydrochloric acid. In some embodiments, the pH value of the dispersion medium is about 3-5 (for example, 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).

[0087] In some embodiments, the sodium hyaluronate has a specific viscosity of about 1.5 m 3 / kg-3.9 m 3 / kg (for example, 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).

[0088] In some embodiments, the preparing step of the bone graft particles comprises: cutting and pulverizing the bone donor material, defatting treatment, virus inactivation treatment, deproteinization treatment, and heat treatment under first heat treatment conditions to prepare the bone graft particles.

[0089] In some embodiments, the first heat treatment conditions comprise: a temperature of the heat treatment is about 250-550℃ (for example, 250℃, 260℃, 270℃, 280℃, 290℃, 300℃, 310℃, 320℃, 330℃, 340℃, 350℃, 360℃, 370℃, 380℃, 390℃, 400℃, 410℃, 420℃, 430℃, 440℃, 450℃, 460℃, 470℃, 480℃, 490℃, 500℃, 510℃, 520℃, 530℃, 540℃, 550℃), and a time of the heat treatment is about 5-8h (for example, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h, 8h). The bone graft particles prepared under the non-calcination conditions are effectively improved in sliding property when assembled into the bone repair material wrapped by the sodium hyaluronate three-dimensional network gel.

[0090] The method for defatting treatment is not particularly limited in the embodiments, and in some embodiments, the defatting treatment is performed in an organic reagent; optionally, the organic reagent comprises one or more of isopropyl alcohol and ethanol; optionally, the mass ratio of the organic reagent to the cutting and pulverized bone donor material is (2.0-3.5):1, for example, 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.

[0091] The method for virus inactivation treatment is not particularly limited in the embodiments, and in some embodiments, the virus inactivation treatment uses a protein denaturant; optionally, the protein denaturant comprises an alkali; optionally, the protein denaturant comprises a sodium hydroxide solution with a sodium hydroxide content of about 3.5wt%-4.5wt% (for example, 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 for the virus inactivation treatment is about 0.5-1.5h (for example, 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1.0h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h).

[0092] The method for deproteinization is not particularly limited in the embodiments of the present application. In some embodiments, the conditions for deproteinization include that the deproteinization reagent used includes one or more of strong oxidizing reagents and primary amine reagents. In one embodiment, the deproteinization uses both strong oxidizing reagents and primary amine reagents. For example, the strong oxidizing reagent and the primary amine reagent are used in sequence.

[0093] Optionally, the temperature for primary amine treatment is about 115-125°C (for example, 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 times of treatment is about 3-8 times (for example, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times), and the duration of each treatment is about 6-12 hours (for example, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours).

[0094] Optionally, the strong oxidizing reagent includes hydrogen peroxide solution, and further optionally, the content of hydrogen peroxide in the hydrogen peroxide solution is about 25-35 wt% (for example, 26 wt%, 26 wt%, 27 wt%, 28 wt%, 29 wt%, 30 wt%, 31 wt%, 32 wt%, 33 wt%, 34 wt%, 35 wt%), and the duration of treatment with the strong oxidizing reagent is about 8-16 hours (for example, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours).

[0095] Optionally, the primary amine reagent includes ethylenediamine solution, and further optionally, the content of ethylenediamine in the ethylenediamine solution is about 80-90 wt% (for example, 80 wt%, 81 wt%, 82 wt%, 83 wt%, 84 wt%, 85 wt%, 86 wt%, 87 wt%, 88 wt%, 89 wt%, 90 wt%), and optionally, the mass ratio of the deproteinization reagent to the virus-inactivated bone donor material is about (2.5-3.5):1 (for example, 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).

[0096] The species source of the bone donor material is not particularly limited in the embodiments of the present application, including but not limited to vertebrate animals (including but not limited to bovine). The site source of the bone donor material is not particularly limited in the embodiments of the present application, including but not limited to limb bones, and optionally femur, and further optionally femur cancellous bone.

[0097] In some embodiments, the mass ratio of the sodium hyaluronate gel and the bone graft particles is about (6-10):6, for example, 6:6, 7:6, 8:6, 9:6, 10:6.

[0098] In some embodiments, the mixture further comprises a carbonate. The present application does not particularly limit the type of the carbonate, including but not limited to calcium carbonate, magnesium carbonate. In some embodiments, the content of carbonate in the bone repair material is about 2wt%-6wt% (for example, 2wt%, 2.2wt%, 2.4wt%, 2.6wt%, 2.8wt%, 3wt%, 3.2wt%, 3.4wt%, 3.6wt%, 3.8wt%, 4wt%, 4.2wt%, 4.4wt%, 4.6wt%, 4.8wt%, 5wt%, 5.2wt%, 5.4wt%, 5.6wt%, 5.8wt%, 6wt%). The addition of carbonate makes it more suitable for use in combination with bone graft particles, enhances the interaction and binding force between hyaluronic acid and bone particles. The formation of a layer of bone material with carbonate form on hydroxyapatite is more conducive to osteogenesis, while the surface of sodium hyaluronate contains a large number of hydroxyl groups, which can combine with cations (preferably Ca ions) to form a specific spatial structure, better promoting the stability of the whole system.

[0099] In some embodiments, the content of sodium hyaluronate in the bone repair material is about 2wt%-8wt% (for example, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%, 5.5wt%, 6wt%, 6.5wt%, 7wt%, 7.5wt%, 8wt%). It is consistent with the set range of sodium hyaluronate concentration, gel and bone particle mixing ratio. If the content of sodium hyaluronate is too high, the gel will overflow during the mixing process, and finally it will show stronger sliding property. If the content is too low, the gel and bone particles are difficult to mix fully, and the adhesion is poor, and it is easy to scatter after rehydration.

[0100] The present application does not particularly limit the way of removing the solvent, including but not limited to vacuum freeze-drying, vacuum drying. Optionally, the conditions of vacuum drying include: the drying temperature is about 60°C-90°C (for example, 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 2h-6h (for example, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h).

[0101] In some embodiments, the particle size of the bone graft particles ranges from about 0.25 mm to 2 mm. For example, from 0.25 mm to 0.5 mm, from 0.5 mm to 1.0 mm, from 1.0 mm to 1.5 mm, from 1.5 mm to 2.0 mm, from 0.25 mm to 1.0 mm, from 1 mm to 2 mm.

[0102] In some embodiments, the pH of the final product after mixing and drying of the sodium hyaluronate gel and the bone graft particles ranges from about 6.5 to 9.0 (for example, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0).

[0103] The shape of the bone repair material is not particularly limited in the embodiments of the present application, and can be, for example, a square block, a cylindrical shape, or a granular shape, etc.

[0104] In a second aspect of the embodiments of the present application, a bone repair material is provided, which is prepared by the preparation method.

[0105] In a third aspect of the embodiments of the present application, a bone repair method is provided, which comprises repairing a bone defect region of a subject using the bone repair material.

[0106] In some embodiments, the bone defect region occurs in the oral bone, the skull, or the non-weight-bearing part of the limbs.

[0107] In some embodiments, the bone repair material is directly filled into the bone defect region, or is filled into the bone defect region after absorbing water or blood, as shown in FIG. 2.

[0108] The subject is not particularly limited in the embodiments of the present application, and can be any animal that can 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, for example, cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (such as rats, mice), pigs, cows, sheep, horses, humans, etc., preferably primates, and more preferably humans.

[0109] The embodiments of the present application will be described in detail below with reference to the 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. The experimental methods in the following examples, if not specified, are preferred to refer to the guidance given in the present application, and can also be performed according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturers, or according to the known experimental methods in the art.

[0110] In the following detailed examples, the amount of the raw material components is measured with a certain degree of accuracy, and slight deviations within the range of the measurement accuracy are allowed unless otherwise specified. In the case of temperature and time parameters, acceptable deviations caused by the accuracy of the measuring instruments or the accuracy of the operation are allowed.

[0111] Example 1

[0112] The present example provides a bone repair material and a preparation method thereof, comprising the following contents:

[0113] 1. Preparation of sodium hyaluronate gel

[0114] 1.1 Prepare a 0.2 mol / L sodium dihydrogen phosphate solution and a 0.2 mol / L disodium hydrogen phosphate solution respectively, mix and stir uniformly 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.

[0115] 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 disperse uniformly, and let it stand and swell for 16 h to obtain a sodium hyaluronate gel, which should be completely transparent without visible white particulate matter. The concentration of the sodium hyaluronate gel is 4.5%.

[0116] 2. Preparation of bone particles

[0117] 2.1 Take the cancellous bone part of a bovine femur, cut and crush it to form bone particles, and use a conventional organic reagent to perform delipidization according to a mass ratio of organic reagent to bone particles of 3:1. The organic reagent is isopropyl alcohol.

[0118] 2.2 Soak the bone particles treated by delipidization in item 2.1 in 4 wt% sodium hydroxide for 1 h to perform viral inactivation.

[0119] 2.3 After viral inactivation, first place the bone particles in a 30 wt% hydrogen peroxide solution (mass ratio of hydrogen peroxide solution to bone particles is 3:1) for 10 h to perform deproteinization, and then place them in a double-layer circulating reactor for ethylenediamine deproteinization, with a temperature of 120°C, a concentration of ethylenediamine in the ethylenediamine solution of 85 wt%, and a mass ratio of ethylenediamine solution to bone particles of 3:1. Perform the deproteinization for a total of 3 times, 8 h each time.

[0120] 2.4 After deproteinization, wash the bone particles with purified water to remove reagent residues.

[0121] 2.5 Place the washed bone particles in a vacuum oven for heat treatment, with a heat treatment temperature of 400°C and a heat treatment time of 6 h.

[0122] 2.6 After the heat treatment, the bone particles are sieved, and the particle size range of 0.5mm-1.0mm is selected.

[0123] 3. Preparation of bone repair material

[0124] 3.1 The sodium hyaluronate gel prepared in step 1 and the bone particles prepared in step 2 are mixed and stirred according to the mass ratio of 10:6, and 0.01g of calcium carbonate is added and mixed.

[0125] 3.2 The mixture prepared in item 3.1 is subjected to heat treatment, and the heat treatment temperature is 120°C and the heat treatment time is 10min.

[0126] 3.3 The product prepared in item 3.2 is placed in a mold for vacuum drying, and the vacuum drying temperature is 80°C and the vacuum drying time is 3h.

[0127] 3.4 The sample after drying is packaged and sterilized to form the final product.

[0128] The bone repair material prepared in this example should have a sodium hyaluronate content of about 5%(w / w) and a carbonate content of 2wt%-3wt%.

[0129] Example 2

[0130] This example is a variation of Example 1, and the only difference from Example 1 is that:

[0131] In item 3.2, the heat treatment temperature is 130°C and the heat treatment time is 10min.

[0132] Example 3

[0133] This example is a variation of Example 1, and the only difference from Example 1 is that:

[0134] In item 3.2, the heat treatment temperature is 120°C and the heat treatment time is 20min.

[0135] Example 4

[0136] This example is a variation of Example 1, and the only difference from Example 1 is that:

[0137] In item 3.2, the heat treatment temperature is 120°C and the heat treatment time is 30min.

[0138] Example 5

[0139] This example is a variation of Example 1, and the only difference from Example 1 is that:

[0140] In the first step, the sodium hyaluronate gel was prepared using a hydrochloric acid solution with a pH value of 5.0 instead of a PBS buffer.

[0141] Example 6

[0142] This example is a variation of Example 1, and the only difference from Example 1 is that:

[0143] Under item 1.1, the pH value of the prepared PBS buffer is 4.

[0144] Under item 1.2, the amount of sodium hyaluronate used is 2g per 100g of sodium hyaluronate gel.

[0145] Under item 2.1, the organic reagent is ethanol, and the mass ratio of the organic reagent to the bone particles is 2:1.

[0146] Under item 2.2, the defatted bone particles under item 2.1 are soaked in 3.5wt% sodium hydroxide for 1.5h for virus inactivation.

[0147] Under item 2.3, the deproteinization treatment is performed using 80wt% ethylenediamine reagent and 25wt% hydrogen peroxide solution, and the mass ratio of the reagent to the bone particles is 2.5:1. The temperature for ethylenediamine deproteinization treatment is 115°C.

[0148] Under item 2.5, the heat treatment temperature is 350°C, and the heat treatment time is 8h.

[0149] Under item 2.6, the particle size range after sieving is 0.25mm-0.5mm.

[0150] Under item 3.1, the mass ratio of sodium hyaluronate gel to bone particles is 6:6, and the addition amount of calcium carbonate is 0.02g to ensure that the content of carbonate in the final obtained bone repair material is 3wt%-4wt%.

[0151] Under item 3.2, the temperature for heat treatment is 100°C, and the heat treatment time is 30min.

[0152] Under item 3.3, the temperature for vacuum drying is 60°C, and the time is 6h.

[0153] Example 7

[0154] This example is a variation of Example 1, and the only difference from Example 1 is that:

[0155] Under item 1.1, the pH value of the prepared PBS buffer is 5.

[0156] Under item 1.2, the concentration of sodium hyaluronate in the prepared sodium hyaluronate gel is 6g.

[0157] 2.1 under which the organic reagent is ethanol and the mass ratio of the organic reagent to the bone particles is 3.5:1.

[0158] 2.2 under which the bone particles subjected to the defatting treatment under 2.1 are subjected to viral inactivation by soaking in 4.5 wt% sodium hydroxide for 0.5 h.

[0159] 2.3 under which the deproteinization is performed using 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 of the ethylenediamine deproteinization is 125°C.

[0160] 2.5 under which the temperature of the heat treatment is 550°C and the time of the heat treatment is 5 h.

[0161] 2.6 under which the particle size range is 1 mm-2 mm.

[0162] 3.1 under which the mass ratio of the sodium hyaluronate gel to the bone particles is 10:6, and the amount of calcium carbonate added is 0.03 g to ensure that the content of carbonate ions in the final bone repair material is 4 wt%-6 wt%.

[0163] 3.2 under which the temperature of the heat treatment is 130°C and the time of the heat treatment is 10 min.

[0164] 3.3 under which the temperature of the vacuum drying is 90°C and the time of the vacuum drying is 4 h.

[0165] Example 8

[0166] This application is a variation of Example 1, and the only difference from Example 1 is that:

[0167] In Step 1, a hydrochloric acid solution with a pH value of 3.0 is used.

[0168] Example 9

[0169] This example is a variation of Example 1, and the only difference from Example 1 is that:

[0170] In 3.1, magnesium carbonate is used instead of calcium carbonate.

[0171] Example 10

[0172] This example is a variation of Example 1, and the only difference from Example 1 is that:

[0173] In 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%.

[0174] Example 11

[0175] This example is a variation of Example 1, and the only difference from Example 1 is that:

[0176] Under item 2.5, the temperature of the heat treatment is 200°C, and the time of the heat treatment is 10h.

[0177] Example 12

[0178] This example is a variation of Example 1, and the only difference from Example 1 is that:

[0179] Under item 2.5, the temperature of the heat treatment is 575°C, and the time of the heat treatment is 4h.

[0180] Example 13

[0181] This example is a variation of Example 1, and the only difference from Example 1 is that:

[0182] Under item 3.1, no calcium carbonate is added.

[0183] Comparative Example 1

[0184] This comparative example is a variation of Example 1, and the only difference from Example 1 is that:

[0185] Under item 1.2, the amount of sodium hyaluronate corresponding to 100g of sodium hyaluronate gel is 7wt%.

[0186] Comparative Example 2

[0187] This comparative example is a variation of Example 1, and the only difference from Example 1 is that:

[0188] Under item 1.2, the amount of sodium hyaluronate corresponding to 100g of sodium hyaluronate gel is 1wt%.

[0189] Comparative Example 3

[0190] This comparative example is a variation of Example 1, and the only difference from Example 1 is that:

[0191] Under item 3.2, the temperature of the heat treatment is 140°C, and the time of the heat treatment is 10min.

[0192] Comparative Example 4

[0193] This comparative example is a variation of Example 1, and the only difference from Example 1 is that:

[0194] Under item 3.2, the temperature of the heat treatment is 120°C, and the time of the heat treatment is 40min.

[0195] Comparative Example 5

[0196] This comparative example is a comparative example of Example 8, the only difference with respect to Example 1 being that:

[0197] The heat treatment step under 3.2 is omitted.

[0198] Comparative Example 6

[0199] This comparative example is a comparative example of Example 8, the only difference with respect to Example 1 being that:

[0200] In Step 1, a sodium hyaluronate gel was prepared using a hydrochloric acid solution having a pH of 2.5.

[0201] Comparative Example 7

[0202] This comparative example is a comparative example of Example 1, the only difference with respect to Example 1 being that:

[0203] In Step 1, a sodium hyaluronate gel was prepared using a hydrochloric acid solution having a pH of 5.5.

[0204] Comparative Example 8

[0205] This comparative example is a comparative example of Example 1, the only difference with respect to Example 1 being that:

[0206] After the sodium hyaluronate gel was prepared under 1.2, the sodium hyaluronate gel was heat treated, the heat treatment temperature being 120°C and the heat treatment time being 10 minutes.

[0207] No heat treatment was performed under 3.2.

[0208] Comparative Example 9

[0209] This comparative example is a comparative example of Example 1, the only difference with respect to Example 1 being that:

[0210] Under 3.2, the heat treatment temperature was 90°C and the heat treatment time was 40 minutes.

[0211] Comparative Example 10

[0212] This comparative example is a comparative example of Example 1, the only difference with respect to Example 1 being that:

[0213] In Step 1, a PBS buffer having a pH of 2.5 was used.

[0214] Comparative Example 11

[0215] This comparative example is a comparative example of Example 1, the only difference with respect to Example 1 being that:

[0216] In Step 1, a PBS buffer having a pH of 5.5 was used.

[0217] In the process of preparing sodium hyaluronate gel, it is found that when the concentration is more than 6wt%, the sodium hyaluronate gel has decreased flowability and increased viscoelasticity, and is still viscous after high temperature treatment, which is not convenient for forming a wrapping layer on the bone particles, and has strong sliding property. When the concentration of hyaluronic acid is less than 2wt%, the solution is too dilute and lacks viscosity. Therefore, the concentration of sodium hyaluronate in the sodium hyaluronate gel is 2wt%-6wt%.

[0218] In order to reduce the sliding property of sodium hyaluronate gel, it is subjected to heat treatment in an acidic environment. It is found that in the same acidic environment, with the increase of heating temperature and heating time, the sliding property of sodium hyaluronate decreases. When the heating temperature is higher than 130℃ and the heating time is more than 30min, the sodium hyaluronate gel is in a dilute solution state and has poor effect on wrapping bovine bone particles. When the heating temperature is lower than 100℃ and the heating time is less than 10min, the sodium hyaluronate gel is in a relatively viscous state and still has certain sliding property. Under the same heating temperature and heating time, with the continuous decrease of pH, when the pH is lower than 3, the sodium hyaluronate gel is in a dilute solution state and has poor effect on wrapping bovine bone particles. When the pH is higher than 5, the sodium hyaluronate gel is viscous and has strong sliding property.

[0219] In the mixing experiment, it is found that when the mixing ratio of sodium hyaluronate gel and bone particles is more than 10:6, the osteogenesis effect of animal experiment decreases. In the process of repairing oral bone defect, in addition to stable blood clots, bone repair materials also need to provide stable scaffolds for the migration, adhesion and proliferation of their own cells to generate new bone. Therefore, the amount of bone particles should not be too small. When the ratio of bone particles increases, it will affect the mixing process of the gel, and the formed bone mass will be dispersed in the solution under the condition of excessive water and blood, which is not conducive to the stability of the bone repair material in the surgical area. Therefore, the ratio of sodium hyaluronate gel and bone particles is (6-10):6.

[0220] Experimental Example 1, Carbonate Content Detection

[0221] Standard hydrochloric acid titrant (c = 0.1 mol / L): pipette 9 mL of hydrochloric acid (p approximately 1.19 g / mL) and dilute to 1000 mL with water. Precisely weigh 0.20 g (accurate to 0.0001 g), previously calcined to constant weight at 270-300°C with anhydrous sodium carbonate (> 99.99%) in a 250 mL beaker, add 50 mL of water to dissolve, 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, while a blank control group is prepared (the blank control group does not contain the sample, but is otherwise treated in the same way as the test group); each 1 mL of hydrochloric acid titrant (0.1 mol / L) corresponds to 5.30 mg of anhydrous sodium carbonate.

[0222] Standard sodium hydroxide titrant (c = 0.1 mol / L): weigh 110 g of sodium hydroxide and dissolve in 100 mL of water free of carbon dioxide, mix well, transfer to a polyethylene container and store closed until the solution is clear. Pipette 5.4 mL of the upper clear solution and dilute to 1000 mL with water free of carbon dioxide, mix well. Weigh 0.75 g (accurate to 0.0001 g) of potassium hydrogen phthalate (> 99.99%) dried to constant weight at 105-110°C in an electric oven and cooled to room temperature, place in a 250 mL beaker, add 50 mL of water free of carbon dioxide to dissolve. Add 2 drops of phenolphthalein solution (10 g / L) and titrate with the prepared sodium hydroxide standard titrant until pink, maintaining the color for 30 s without fading; each 1 mL of sodium hydroxide titrant (0.1 mol / L) corresponds to 20.42 mg of potassium hydrogen phthalate.

[0223] Barium chloride (BaCl2·2H2O) solution: 122 g / L.

[0224] Phosphoric acid: (1 + 1).

[0225] Hydrochloric acid: 1 mol / L.

[0226] Sodium hydroxide: 1 mol / L.

[0227] Methyl orange solution: 1 g / L.

[0228] Phenolphthalein solution: 0.25 g / L, prepared with 50% ethanol.

[0229] Sample preparation: take an appropriate amount of bone repair material sample provided in each example and comparative example, grind finely and dry in a 105°C desiccator for 2 h, cool to room temperature. Precisely weigh 0.5 g of fine powder and place in a conical flask for CO2 extraction.

[0230] Leak test: connect the test apparatus as shown in Fig. 1, add appropriate amount of water into each conical flask so that the catheter port is immersed in the water solution, use water clamp to seal the air inlet, close the piston of the dropping funnel, slightly heat the first conical flask, if bubbles appear at the catheter port of the last conical flask, stop heating, insert the catheter under the liquid surface of the last conical flask to generate a liquid column, and the air tightness of the apparatus is good.

[0231] Blank test: at the same time, a blank control group (no bone repair material sample is added in the blank control group, and other treatment modes are consistent with those of the test group) is prepared.

[0232] Sample determination:

[0233] Precisely weigh 0.5 g of the sample, transfer it to the acid-extracted CO2 conical flask, add about 100 mL of water, cover the neck plug, and pass nitrogen gas through the secondary gas washing for 10 min at a rate of about 50 mL / min. The whole process is carried out under nitrogen condition.

[0234] Without cutting off the nitrogen gas, connect the secondary absorption apparatus, and 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.

[0235] Add 50 mL of 50% phosphoric acid solution into the acid-extracted CO2 conical flask through the dropping funnel, close the piston of the dropping funnel, and react for 50 min.

[0236] Titration:

[0237] Remove the second absorption bottle, remove and rinse the inner tube with water, combine the washing liquid in the absorption bottle, and titrate the solution in the absorption bottle with about 1 mol / L hydrochloric acid to near the end point.

[0238] Continue titration with the hydrochloric acid standard titration solution until the phenolphthalein is just colorless.

[0239] Add a certain excess of the hydrochloric acid standard solution until the precipitate is completely dissolved, immerse the inner tube in the 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.

[0240] Neutralize and titrate the solution in the first absorption bottle in the same way.

[0241] Result calculation:

[0242] The content of carbonate is calculated according to the following formula, and W CO3 2- (%) is used to represent.

[0243] In the formula: v1: volume of hydrochloric acid standard titration solution used to dissolve barium carbonate in two absorption bottles, mL;

[0244] v2: volume of sodium hydroxide standard titration solution consumed for back titration of excess hydrochloric acid in two absorption bottles, mL;

[0245] v3: volume of hydrochloric acid standard titration solution used to dissolve barium carbonate in two absorption bottles for a blank test, mL;

[0246] v4: volume of sodium hydroxide standard titration solution consumed for back titration of excess hydrochloric acid in two absorption bottles, mL;

[0247] c1: concentration of hydrochloric acid titration solution, mol / L;

[0248] c2: concentration of sodium hydroxide standard titration solution, mol / L;

[0249] m: sample mass, g;

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

[0251] Table 1, Carbonate content detection results

[0252] The calcium carbonate content detection results show that after adding an appropriate amount of carbonate, a layer of bone material with carbonate form is formed on the hydroxyapatite, and the presence of carbonate is crucial for the metabolic balance of animal and human bone tissue inorganic minerals, which is more conducive to osteogenesis. At the same time, the specific spatial structure formed by the combination of a large number of hydroxyl groups on the surface of sodium hyaluronate and cations also better promotes the stability of the entire system. With the increase of the proportion of added carbonate, the carbonate content in the product shows an upward trend, and different carbonates have no significant effect on the detection of carbonate content; no carbonate is added in Example 13, and the carbonate content in the product is lower than the set value.

[0253] Experimental Example 2, pH value detection

[0254] 0.05 mol / L potassium hydrogen phthalate solution has a pH of 4.01 at 25°C;

[0255] 0.025 mol / L disodium hydrogen phosphate and 0.025 mol / L sodium dihydrogen phosphate mixed solution has a pH of 6.86 at 25°C;

[0256] 0.01 mol / L borax solution has a pH of 9.18 at 25°C;

[0257] Take one package of standard buffer solution, dissolve it in distilled water (newly boiled and cooled purified water), and dilute to 250 mL.

[0258] Take sample 0.5g, precision weighing, extraction medium / biological bone repair material (v / w) is 12:1, extraction medium is pure water boiled and cooled, 37±1℃ extraction 72±2h, to be detected.

[0259] Before determination, according to the provisions under each variety, select two standard buffer solutions with a difference of 3 units in pH value, so that the pH value of the test solution is between the two;

[0260] Take the first standard buffer solution close to the pH value of the test solution to calibrate the instrument (positioning), so that the instrument reading is consistent with the table value;

[0261] After the instrument is positioned, the second standard buffer solution is used to check the instrument reading, and the error should not be more than ±0.02 pH units. If it is greater than this deviation, the slope should be adjusted carefully to make the reading consistent with the table value of the second standard buffer solution. Repeat the positioning and slope adjustment operation until the instrument reading is within ±0.02 pH units of the specified value of the standard buffer solution. Otherwise, the instrument should be checked or replaced with a new electrode before calibration is performed again to meet the requirements.

[0262] Determine the pH of the sample, repeat the measurement twice and take the average value, the error of the two measurements should not exceed 0.1, and record it;

[0263] Before each change of standard buffer solution or test solution, the electrode should be washed thoroughly with purified water, and then the water on the electrode should be absorbed with filter paper. Alternatively, the electrode can be washed with the replaced standard buffer solution or test solution.

[0264] Table 2, pH value detection results

[0265] The pH value detection results show that when the bone particle heat treatment temperature is not more than 550℃ and the heat treatment time is not more than 8h, the pH values of the examples and the comparative examples have no significant difference. The bone particles themselves are weakly alkaline, and when mixed with hyaluronic acid sodium gel prepared with PBS buffer solution or hydrochloric acid and then dried, they can maintain a pH close to that of human tissue. The solubility of calcium carbonate in water is very low, and it has no significant effect on the pH value of the bone repair material.

[0266] Experimental example 3, hyaluronic acid sodium content detection

[0267] 0.125% volume fraction of carbazole ethanol solution: weigh 0.125g of carbazole and add 100mL of anhydrous ethanol to dissolve;

[0268] Glucuronic acid (GA) standard solution: about 0.1 g of glucuronic acid reference substance dried to constant weight at 105°C with phosphorus pentoxide as drying agent was precisely weighed into a 100 mL volumetric flask, dissolved and diluted to the mark with water, and shaken to homogeneity. 5.0 mL of the stock solution was precisely measured into a 100 mL volumetric flask, and water was added to make a solution containing 50 μg per 1 mL, and shaken to homogeneity.

[0269] 0.025 mol / L sodium tetraborate sulfuric acid solution: sodium tetraborate (Na2B4O7·10H2O) 9.54 g was weighed into 1 L of concentrated sulfuric acid, and covered. Shake at random times until the sodium tetraborate is completely dissolved, and store at room temperature.

[0270] Sample preparation: each example and comparative example bone repair material sample dried to constant weight at 105°C was ground into fine powder, and 0.2 g was precisely weighed into a triangular flask. About 70 mL of purified water was transferred into the triangular flask, and after 24 hours of extraction at 37°C and 120 r / min, it was transferred to a volumetric flask, diluted to 100 mL, and shaken to homogeneity. 1 mL of the solution was taken into a test tube, and measured. The glucuronic acid standard solution series was prepared according to the following table.

[0271] Table 3, glucuronic acid (GA) standard solution series concentration

[0272] Detection steps: the standard solution series test tubes and sample test tubes were placed in an ice water bath, and 5 mL of 0.025 mol / L sodium tetraborate sulfuric acid (stored in a 4°C refrigerator for at least 2 h before use) was slowly added to each tube while shaking. After addition, mix well and place in a boiling water bath for 15 min, then remove, and cool in an ice water bath. 0.2 mL of carbazole ethanol solution was added to each test tube. After thorough mixing, place in a boiling water bath for 15 min, and cool to room temperature. Use a 0 test tube as a control, and use a spectrophotometer to measure the absorbance of each standard tube and sample tube at 530 nm.

[0273] Draw the absorbance-concentration curve with the standard tube, and according to the absorbance of the sample tube, find the glucuronic acid content of the sample tube from the standard curve. Calculate the concentration of the diluted sodium hyaluronate solution: subtract the absorbance of the blank solution from the absorbance of the diluted solution, and find the concentration of sodium hyaluronate in the diluted solution from the standard curve.

[0274] Calculation formula:

[0275] W% = [(2.07 * c) / (10000 * m)] * 100%, wherein

[0276] c: the glucuronic acid concentration of the sample tube found from the table, g / mL;

[0277] m: the mass of the sample, g;

[0278] 2.07: Conversion factor of glucuronic acid into sodium hyaluronate;

[0279] 10000: Conversion factor of the mass of glucuronic acid in 100 mL sample solution in g into g.

[0280] Table 4: Results of sodium hyaluronate content detection

[0281] Experimental Example 4: Test of sliding property

[0282] The bone repair material samples of each example and comparative example with the same specification were placed on a culture dish, physiological saline was added dropwise until complete infiltration, and the state and sliding distance of the bone block were recorded at 1 min, 3 min and 5 min (the maximum sliding distance was 8 cm). Some examples and comparative examples are shown in FIGS. 4-8.

[0283] Table 5: Results of sliding property test

[0284] Note: " / " indicates that the bone repair material has been scattered; "8.0+" indicates that the bone repair material has slid to the maximum distance

[0285] Experimental Example 5: SEM scanning electron microscope micro-morphology detection

[0286] The bone repair material samples of each example and comparative example were sprayed with gold on an ion sputtering instrument (HITACHI E-1045) and then placed under a field emission scanning electron microscope (HITACHI SU8010) for scanning at an acceleration voltage of 3.0 kV. The results are shown in FIG. 3.

[0287] The experimental results show that in Comparative Example 1, the sodium hyaluronate gel on the surface of the bone particles is aggregated, and the whole is not uniform; in Comparative Example 2, the sodium hyaluronate gel on the surface of the bone particles is less adhered. In Comparative Example 5, since no heat treatment is performed, the combination degree of the bone particles and the hyaluronate gel is not good; similarly to Comparative Example 5, in Comparative Example 8, since the operation sequence of the heat treatment is changed, the combination degree of the bone particles and the hyaluronate gel is not good. In Example 1, the surface of the bone particles is uniformly wrapped with sodium hyaluronate.

[0288] Experimental Example 6: Effectiveness test (animal experiment: rabbit skull defect experiment)

[0289] The experimental New Zealand rabbits were anesthetized, the top head hair was shaved, and then placed on a special operating table in a prone position, and then disinfected with iodine alcohol and alcohol, and then a sterile drape was laid. A 5cm straight incision was made on the midline of the skull, the scalp was incised, and the coronal suture and sagittal suture of the skull were exposed. The modeling was performed on the posterior side of the coronal suture, the anterior side of the herringbone suture, and the two sides of the sagittal suture. The skull was ground with a high-speed turbine to form a circular bone window with a diameter of about 1.0cm, and medical gauze was used to stop bleeding. Different bone repair materials (the bone regeneration materials prepared in Example 1, Example 11, and Example 13 of the present application) were placed in the skull defects, and two bone defects were made on the left and right sides of each animal. After the material was implanted, the wound was sutured. Micro-CT photography was performed at 1 week, 2 weeks, 4 weeks, and 8 weeks after the operation to observe the repair of the skull defects. The experimental results are shown in FIG. 9. The early bone repair effect of Example 1 is excellent, and the bone defect can be basically repaired and new bone formation can be induced at 8 weeks.

[0290] Each of the technical features of the above-described embodiments and examples can be combined in any suitable manner. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments and examples are described, however, as long as the combinations of the technical features do not contradict each other, they should be considered within the scope of the present disclosure.

[0291] The above-described embodiments only express several embodiments of the present application, facilitate specific and detailed understanding of the technical solutions of the present application, but should not be understood as limiting the scope of patent protection. It should be noted that, for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. In addition, it should be understood that after reading the above description of the present application, those skilled in the art can make various modifications or improvements to the present application, and the equivalent forms also fall within the scope of the present application. It should also be understood that those skilled in the art can obtain technical solutions based on the technical solutions provided in the present application through logical analysis, reasoning or limited experiments, and these technical solutions are within the scope of the claims of the present application. Therefore, the scope of protection of the patent of the present application should be based on the contents of the appended claims, and the description and drawings can be used to explain the contents of the claims.

Claims

1. A method for preparing a bone repair material, characterized in that, The preparation method includes the following steps: A mixture was prepared by mixing sodium hyaluronate gel and bone graft particles; The mixture is heat-treated 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 approximately 100℃~130℃, and the heat treatment time is approximately 10min~30min; and, (2) The pH value of the mixture is approximately 3 to 5; The amount of sodium hyaluronate used per 100g of the sodium hyaluronate gel is approximately 2g to 6g.

2. The method for preparing the 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, allowing it to swell fully, and preparing 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 the bone repair material according to claim 2, characterized in that, The pH value of the dispersion medium is approximately 3 to 5.

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

6. The method for preparing the bone repair material according to any one of claims 1 to 5, characterized in that, The preparation steps of the bone graft particles include: cutting and crushing the bone donor material, degreasing, virus inactivation, deproteinization, and heat treatment under a first heat treatment condition to prepare the bone graft particles.

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

8. The method for preparing the bone repair material according to claim 6, characterized in that, The defatting process 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 pulverized bone donor material is (2.0-3.5):

1.

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

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

1.

11. The method for preparing the bone repair material according to claim 6, characterized in that, The species source of the bone donor material includes vertebrates; Optionally, the vertebrate includes cattle; Optionally, the bone donor material includes limb bones, more preferably the femur, and even more preferably cancellous femoral bone.

12. The method for preparing the 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 approximately (6-10):

6.

13. The method for preparing the bone repair material according to any one of claims 1 to 12, characterized in that, The mixture also includes carbonates; 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 the bone repair material according to any one of claims 1 to 13, characterized in that, Solvent removal methods include 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 hours to 6 hours.

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

16. The method for preparing the 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 approximately 0.25 mm to 2 mm.

17. The method for preparing the bone repair material according to any one of claims 1 to 16, characterized in that, The bone repair material is in the form of blocks, cylinders, or granules.

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, This includes repairing bone defect areas in the subject using the bone repair material as described in claim 18.

20. The bone repair method according to claim 19, characterized in that, The bone defect area occurs in the oral cavity bone, skull bone, or non-weight-bearing bone 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 it is filled into the bone defect area after absorbing water or blood.

Images