Morphologically plastic bionic mineralized composite bone filling material, its preparation method and application

Abstract

The present invention provides a morphologically plastic biomimetic mineralized composite bone filling material, its preparation method and application, belonging to the technical field of the preparation of bone filling materials. The method of the present invention uses decalcified cancellous bone as a carrier, and in-situ mineralization occurs in a mineralization solution to obtain a bone filling material. At the same time, the bone filling material is fully mixed with a thermosensitive hydrogel derived from decalcified cancellous bone to form a composite bone filling material, which is pre-filled in a syringe. The decalcified cancellous bone after in-situ mineralization has the pore structure of natural bone and nano-hydroxyapatite, and at the same time can be biodegradable, biocompatible and osteoinductive, which is more conducive to the growth and proliferation of blood vessels and cells. In addition, using a decalcified cancellous bone hydrogel with a natural bone-like porous three-dimensional structure as a carrier to achieve injectability, not only can the in-situ mineralized bone filling material be easily directly filled into the tooth extraction socket, but also due to the unique thermosensitivity of the hydrogel, it can self-crosslink under the action of body temperature, making the material stably exist and not easily fall off.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of bone filling materials, and particularly relates to a bionic mineralized composite bone filling material with a plastic morphology, a preparation method thereof, and an application thereof. Background Art

[0002] During dental implantation, after tooth extraction, the alveolar ridge undergoes progressive and irreversible resorption, often causing problems such as bone loss and alveolar ridge flattening, which pose difficulties for denture restoration or implant placement, and seriously affect the aesthetics of patients at the same time. A large number of clinical studies have shown that using bone filling materials for extraction socket preservation can effectively reduce the resorption of the alveolar ridge, provide sufficient bone mass and good bone quality for later implant restoration, which is also the most commonly used treatment method at present. From the perspective of materials, its products are mainly divided into four categories: ① natural biological-derived materials, such as decalcified bone matrix (allogeneic bone, xenogeneic bone), chitosan, alginate, etc.; ② synthetic inorganic materials, such as hydroxyapatite (HA), tricalcium phosphate (TCP), bioactive glass; ③ synthetic organic materials, such as polylactic acid and its copolymers; ④ synthetic composite materials, such as organic-inorganic composites such as collagen-TCP, etc.

[0003] Generally speaking, an ideal bone filling material needs to have both: ① osteoconductivity, that is, the supporting role of the "soil", which is conducive to the growth of capillaries and osteocytes into the material; ② osteoinductivity, that is, the nutritional role of the "nutrient", which can promote the differentiation of mesenchymal cells into osteoblasts. Although decalcified bone matrix has been widely proven to have both definite osteoinductive and osteoconductive effects, it is still quite difficult to use in actual clinical practice: ① On the one hand, the decalcified bone matrix particles are relatively loose, that is, they cannot be accurately filled in the narrow extraction socket, nor can they stay stably at the extraction socket, and are easily washed away by blood or flushing operations; ② If preformed in advance, when the sample is too large, it cannot be implanted into the extraction socket, and when the sample is too small, the resulting dead space will cause alveolar bone defect; ③ Lack of calcium and phosphorus required for bone formation, and the osteogenic speed is relatively slow.

[0004] Some studies have begun to focus on how to treat cancellous bone in a relatively mild way, so that it has no immunogenicity and can retain osteoinductivity, and can in-situ mineralize a hydroxyapatite coating that is relatively consistent with the human bone structure components, making the degradation and calcium and phosphorus release of the bone filling material controllable. Existing products are composed of composite materials such as bone morphogenetic protein-2 and hydroxyapatite, etc., but they are expensive, and their release rate is difficult to control, and it is easy to cause risks of over-repair or incomplete repair.

[0005] For example, Chinese Patent Document CN116712613A provides a bone filling material, which is prepared by crosslinking between raw material A (hyaluronic acid or chitosan) and raw material A, and covalently crosslinking between collagen and collagen; hydroxyapatite is deposited on collagen by physical deposition to obtain the bone filling material. It has the same material composition, microscopic and macroscopic structure as natural periosteum, and has good mechanical properties and biodegradation characteristics. However, due to simple physical mixing, uniform distribution of inorganic and organic phases cannot be achieved.

[0006] Chinese Patent Document CN105169482A provides a dental bone substitute material, which is prepared by a chemical treatment and calcination process to prepare a porous scaffold of fetal bovine cancellous bone, and a calcium phosphate mineralized crystal is formed on the surface treatment. It has a shorter in vivo metabolism time than bone substitute materials from other animal sources and better meets the needs of clinical bone grafting. However, due to using calcined bone as a carrier, it lacks the organic components of natural bone tissue and has no osteoinductivity.

[0007] Chinese Patent Document CN1106861C provides a nano-phase calcium phosphate / collagen / polylactic acid bone composite porous material, which involves a method for in-situ mineralized collagen, and its composition is similar to human bone. However, its carrier is purified collagen, which has no osteoinductivity and it is difficult to arbitrarily control the degree of mineralization.

[0008] Chinese Patent Document CN109276761B provides a bone repair material containing rh-BMP2 and phosphate, which uses chitosan as a sustained-release carrier to achieve a continuous and effective working concentration of BMP-2. However, it lacks the collagen scaffold structure of bone tissue, cannot reproduce in-situ biomimetic mineralization, and the release rate of inorganic components is uncontrollable.

[0009] In addition, Chinese Patent Document CN 109381745 B provides a collagen coral hydroxyapatite composite bone filling scaffold material, which can not only effectively avoid the risk of disease transmission in natural tissue extraction, but also has a natural pore structure that synthetic hydroxyapatite does not have, and solves the disadvantage that coral hydroxyapatite particles are not easy to shape. However, this bone filling scaffold material lacks the organic components of natural bone tissue, has no osteoinductivity, and its pre-shaping is difficult to adapt to complex bone defect wounds.

[0010] Chinese Patent Document CN 114366854 B provides a silicone rhinoplasty material with a composite decalcified bone matrix, but this material lacks the inorganic components of natural bone tissue, and only relies on silicone to play a supporting role and has no new bone formation effect.

[0011] Therefore, on the one hand, how to solve the lack of osteoinductivity in existing bone filling materials, so that the bone filling materials can retain sufficient active substances and have high osteoinductivity; on the other hand, it can also achieve better mineralization effect and more rapid repair of bone defect sites; at the same time, it is necessary to solve the problems such as the lack of calcium and phosphorus required for bone formation in common decalcified cancellous bone products, poor plasticity of bone filling materials, and great difficulty in surgical operation, which have become technical problems to be solved urgently. Summary of the Invention

[0012] The present invention is to solve the above technical problems, and thus provides a morphologically plastic biomimetic mineralized composite bone filling material, its preparation method and application. The technical object of the present invention is to provide a bone filling material with high decalcification efficiency, high content of active ingredients, in-situ mineralization, good mineralization effect, and at the same time having morphological plasticity, injectability and convenient operation, so as to solve the problems of low content of active ingredients, lack of osteoinductivity, poor mineralization effect, poor plasticity and great operation difficulty existing in existing bone filling materials.

[0013] In order to achieve the above technical object, the technical scheme adopted by the present invention is as follows:

[0014] The present invention first provides a preparation method of a morphologically plastic biomimetic mineralized composite bone filling material, comprising the following steps:

[0015] (I) Preparation of decalcified cancellous bone

[0016] (1) Crush cancellous bone into bone powder with a particle size of 0.3 - 0.8 mm, and then add it to an EDTA solution according to a solid-liquid ratio of 1:5 - 1:30 w / v, and oscillate at room temperature for decalcification for 4 - 16 hours;

[0017] (2) Add the cancellous bone treated in step (1) to a peracetic acid solution according to a solid-liquid ratio of 1:4 w / v, and oscillate at room temperature for 2 hours;

[0018] (3) Add the cancellous bone treated in step (2) to isopropanol according to a solid-liquid ratio of 1:5 - 1:10 w / v, and oscillate at room temperature for defatting for 24 hours;

[0019] (4) Add the cancellous bone treated in step (3) to a 1 - 4 wt% sodium deoxycholate solution according to a solid-liquid ratio of 1:5 - 1:10 w / v, and oscillate at room temperature for decellularization for 8 - 24 hours to prepare decalcified cancellous bone;

[0020] (II) Preparation of mineralized decalcified cancellous bone

[0021] (5) Add the decalcified cancellous bone obtained in step (4) to a 2 - 3 wt% sodium trimetaphosphate solution according to a solid-liquid ratio of 1:5 - 1:10 w / v, and oscillate at room temperature for 1 hour for phosphorylation treatment;

[0022] (6) Immerse the decalcified cancellous bone after phosphorylation treatment into the mineralization solution at a material-liquid ratio of 1:200 - 1:500 w / v, and carry out biomimetic mineralization by oscillation at 37°C for 1 - 5 days. The composition of the mineralization solution is: sodium chloride 100 - 160 mM, dipotassium hydrogen phosphate 2 - 4 mM, magnesium chloride 3 - 5 mM, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 35 - 40 mM, sodium bicarbonate 15 - 22 mM, calcium chloride 6 - 9 mM, and the pH value is 6.5 - 7.0, to obtain mineralized decalcified cancellous bone;

[0023] (III) Preparation of thermosensitive cancellous bone hydrogel

[0024] (7) Take the decalcified cancellous bone obtained in step (4), and add it to a 0.1 wt% porcine pepsin solution at a material-liquid ratio of 1:100 w / v, and stir at 4°C for 24 - 48 hours;

[0025] (8) Add an alkali to the product obtained in step (7) to adjust the pH to 7.2 - 7.4, then add PBS buffer solution to adjust the ionic strength of the solution to 0.5 - 1.5×PBS, and then add 1×PBS to adjust the concentration of the hydrogel solution to 0.4 - 0.8 wt%;

[0026] (IV) Preparation of biomimetic mineralized composite bone filling material

[0027] (9) Take the mineralized decalcified cancellous bone obtained in step (6), and add it to the hydrogel solution obtained in step (8) at a material-liquid ratio of 1 - 3:1 w / v, and stir at 4°C for 24 hours to obtain the biomimetic mineralized composite bone filling material.

[0028] The above preparation method provided by the present invention first prepares a mineralized decalcified cancellous bone material with a structure, composition and microscopic morphology similar to that of autologous bone through biomimetic mineralization technology. Through phosphorylation treatment, the nucleation sites of the decalcified cancellous bone are increased, which shortens the total mineralization time on the one hand and makes the mineralization process more stable on the other hand. Simulating natural bone mineralization, an inorganic salt coating is formed in situ on the decalcified cancellous bone, which has a better effect of promoting new bone formation and guiding bone regeneration. Through the phosphorylation and mineralization treatment of the present invention, the problems of low content of active ingredients and long degradation period of existing bone filling materials are well solved. This method first mineralizes nanoscale hydroxyapatite on a natural decalcified cancellous bone scaffold, removes immunogenicity, well retains active ingredients, and can regulate cell signal transduction, so that it can be self-regulated by the body to release calcium and phosphorus and efficiently form bone.

[0029] The bone filling material prepared by the method of the present invention is obtained by taking cancellous bone from animal femurs, performing pretreatment, decalcification, virus inactivation, degreasing, decellularization, and biomimetic mineralization. Based on the natural bone porous three-dimensional collagen scaffold, it is mineralized into a material mainly composed of nanoscale hydroxyapatite, and well retains the osteoinductivity. It can not only provide a three-dimensional scaffold structure for regenerated tissue cells, but also has the functions of regulating the behavior of regenerated cells (including cell morphology, adhesion, proliferation, migration, differentiation, and apoptosis), and has good biocompatibility and biodegradability. In addition, its preparation process flow is simple, with low cost and short time, and the mineralization degree of the final product can be regulated.

[0030] On this basis, the present invention further composites the above mineralized decalcified cancellous bone with a thermosensitive cancellous bone hydrogel to prepare a morphologically plastic biomimetic mineralized composite bone filling material. The composite material uses decalcified cancellous bone as a carrier, undergoes in-situ mineralization in a mineralization solution to obtain a bone filling material, and at the same time fully mixes the bone filling material with a thermosensitive hydrogel derived from decalcified cancellous bone to form a composite bone filling material, making it have morphologically plasticity and injectability, and its operation method is simple. This method solves the problems that common decalcified cancellous bone products lack calcium and phosphorus required for bone formation, have poor plasticity, and are difficult in surgical operation.

[0031] The decalcified cancellous bone after in-situ mineralization of the present invention has the pore structure of natural bone and nanoscale hydroxyapatite, and at the same time is biodegradable, has biocompatibility and osteoinductivity, which is more conducive to the growth and proliferation of blood vessels and cells, and has the advantage of faster repair speed in clinical practice.

[0032] The present invention creatively uses a collagen scaffold with a natural bone porous three-dimensional structure as an organic component, and maintains the natural porosity of cancellous bone, and the pore size is suitable for the growth of bone tissue and blood vessel tissue. Therefore, it can not only provide a three-dimensional scaffold structure for regenerated tissue cells, but also contains rich non-collagenous other signal macromolecules and osteoinductive protein components, making the bone filling material have excellent osteogenic performance. The surface nanoscale hydroxyapatite coating releases calcium, phosphorus and active substances with the body's self-regulation. After inducing osteogenesis at the defect site, the exposed collagen scaffold can be degraded relatively quickly and provide the required substances for the osteogenic site. Therefore, the bone filling material also has excellent degradability that can be adjusted according to human needs.

[0033] Furthermore, the concentration of the EDTA solution in step (1) is 10 wt%; as a preferred scheme, the pH value of the EDTA solution is 7.4.

[0034] Furthermore, the concentration of the peracetic acid solution in step (2) is 0.3 wt%.

[0035] Furthermore, the pH of the sodium deoxycholate solution in step (4) is 8.

[0036] Furthermore, the pH of the sodium trimetaphosphate solution in step (5) is 10.

[0037] Furthermore, in step (6) during the oscillating biomineralization process, the mineralization solution is changed every 24 hours.

[0038] Furthermore, in steps (1) to (6), the cancellous bone after each step of treatment is washed. The steps of the washing are as follows: The treated cancellous bone is poured into purified water at a material-liquid ratio of 1:10 - 1:20 w / v, and oscillated and washed at room temperature for 1 hour, and the liquid is changed every 5 minutes.

[0039] Furthermore, the pH of the porcine pepsin solution in step (7) is 2 - 3.

[0040] Furthermore, the operations of adjusting the pH and ionic strength in step (8) are as follows: 0.1 M sodium hydroxide is added dropwise to the product obtained in step (7) under stirring at 4°C until the pH range is 7.2 - 7.4, and then 10×PBS buffer solution is slowly added to the product to adjust the ionic strength of the solution to 0.5 - 1.5×PBS.

[0041] The beneficial effects of the present invention are as follows:

[0042] (1) In the composite bone filling material prepared by the method of the present invention, the proportion of the organic phase is 15% - 25 wt%, and the composition of the inorganic phase is 35% - 55 wt% (the remaining components are water), and its composition and structure are similar to those of natural bone;

[0043] (2) The composite bone filling material prepared by the present invention contains abundant osteogenic-related active factors such as bone morphogenetic protein BMP-2 and has excellent osteoinductivity;

[0044] (3) The composite bone filling material of the present invention removes immunogenicity, well retains active ingredients, and can regulate cell signal transduction, enabling it to be self-regulated by the body to release calcium and phosphorus and efficiently form bone;

[0045] (4) The composite bone filling material of the present invention has excellent plasticity and injectability;

[0046] (5) The composite bone filling material of the present invention can be used for the rapid repair of oral and maxillofacial bone defects and can be biodegradable within 3 months, which is coordinated with the bone formation time. Description of the Drawings

[0047] Figure 1 It is a physical picture of the mineralized cancellous bone filling material sample prepared in the embodiment of the present invention.

[0048] Figure 2 Scanning electron microscope images of the mineralized cancellous bone filling material prepared in the embodiment of the present invention (left: before mineralization; right: after mineralization).

[0049] Figure 3 The enlarged view of the inorganic phase of the mineralized cancellous bone filling material prepared in the embodiment of the present invention. Figure 4 XRD results of the inorganic phase of the mineralized cancellous bone filling material prepared in the embodiment of the present invention (the phase composition of the surface coating is hydroxyapatite).

[0050] Figure 5 The rabbit mandible after 8 weeks of modeling (left: Bio-Oss; right: sample of the embodiment). Figure 6 X-ray photograph of the rabbit mandible after 8 weeks of modeling (left: Bio-Oss; right: sample of the embodiment).

[0051] Figure 7 The rabbit mandible after 12 weeks of modeling (left: Bio-Oss; right: sample of the embodiment). Figure 8 Self-assembly process of the thermosensitive cancellous bone hydrogel at 37°C (left: room temperature state; right: after self-assembly at 37°C).

[0052] Figure 9 The effect of the concentration of the thermosensitive cancellous bone hydrogel on the self-assembly crosslinking time of the material.

[0053] Figure 10 Self-assembly crosslinking of the thermosensitive cancellous bone hydrogel with different ionic strengths.

[0054] Figure 11 Microstructure of the thermosensitive cancellous bone hydrogel.

[0055] Figure 12 Calcium ion release of the morphologically plastic bionic mineralized composite bone filling material (●: decalcified cancellous bone; ■: sample of the embodiment).

[0056] Figure 13 Phosphate ion release of the morphologically plastic bionic mineralized composite bone filling material (●: decalcified cancellous bone; ■: sample of the embodiment).

[0057] Figure 14 The stable retention of the two groups of products immediately after implantation (left: sample of the embodiment; right: decalcified cancellous bone).

[0058] Figure 15 X-ray photograph results after 12 weeks of modeling (left: sample of the embodiment; right: decalcified cancellous bone). Detailed implementation manners

[0059] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention will be specifically described below in conjunction with embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention and are not used to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above-mentioned inventive content still fall within the protection scope of the present invention.

[0060] Example 1

[0061] This example provides a preparation method for a morphologically plastic bionic mineralized composite bone filling material. The specific preparation steps are as follows:

[0062] (1) Preparation of decalcified cancellous bone

[0063] 1.1 Pretreatment: Take the whole femur, use a bone cutting machine to cut the cancellous bone parts at both ends of the femur, and discard the cortical bone part in the middle. Then use the bone cutting machine to completely remove the cortical bone on the outer circle of the bone block, leaving only the cancellous bone part. Finally, use the bone cutting machine to cut the cancellous bone into bone grains. Take the bone grains and crush them, pour them into purified water at a material-liquid ratio of 1:10 (w / v), oscillate and wash at room temperature for 1 hour, change the liquid every 5 minutes, and sieve to obtain bone powder with a particle size of 0.3 mm.

[0064] 1.2 Decalcification: Take the sample prepared in step 1.1, add 10 wt% EDTA solution (pH~7.4) at a material-liquid ratio of 1:5 (w / v), and decalcify by oscillation at room temperature for 4 hours. After completion, pour it into purified water at a liquid ratio of 1:10 (w / v), oscillate and wash at room temperature for 1 hour, and change the liquid every 5 minutes.

[0065] 1.3 Virus inactivation: Take the sample prepared in step 1.2, add 0.3 wt% peracetic acid solution at a material-liquid ratio of 1:4 (w / v), and oscillate at room temperature for 2 hours. After completion, pour it into purified water at a material-liquid ratio of 1:10 (w / v), oscillate and wash at room temperature for 1 hour, and change the liquid every 5 minutes.

[0066] 1.4 Degreasing: Take the sample prepared in step 1.3, add isopropanol at a material-liquid ratio of 1:5 (w / v), and degrease by oscillation at room temperature for 24 hours, changing the liquid every 8 hours. After completion, pour it into purified water at a material-liquid ratio of 1:10 (w / v), oscillate and wash at room temperature for 1 hour, and change the liquid every 5 minutes.

[0067] 1.5 Decellularization: Take the sample prepared in step 1.4, add 1 wt% sodium deoxycholate solution (pH~8) at a material-liquid ratio of 1:5 (w / v), and decellularize by oscillation at room temperature for 8 hours. After completion, pour it into normal saline at a material-liquid ratio of 1:10 (w / v), oscillate and wash at room temperature for 30 minutes, changing the liquid every 5 minutes. Then pour it into purified water at a material-liquid ratio of 1:10 (w / v), oscillate and wash at room temperature for 30 minutes, changing the liquid every 5 minutes, to obtain decalcified cancellous bone.

[0068] (2) Preparation of Mineralized Demineralized Spongy Bone

[0069] 2.1 Phosphorylation treatment: Take the demineralized spongy bone prepared by method (1), add 2 wt% sodium trimetaphosphate (pH ~ 10) according to the solid-liquid ratio of 1:5 (w / v), and oscillate at room temperature for 1 hour. After that, pour into purified water according to the solid-liquid ratio of 1:10 (w / v), and oscillate and wash at room temperature for 1 hour, changing the liquid every 5 minutes.

[0070] 2.2 Preparation of biomimetic mineralization solution: Weigh sodium chloride, dipotassium hydrogen phosphate, and magnesium chloride in sequence and add them into purified water. After complete dissolution, continue to add appropriate amounts of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and sodium bicarbonate to maintain the pH of the system, and then add calcium chloride and stir until the solution is clear. The final concentrations of the solution are 100 mM sodium chloride, 2 mM dipotassium hydrogen phosphate, 3 mM magnesium chloride, 35 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 15 mM sodium bicarbonate, 6 mM calcium chloride, and the pH value is 6.5.

[0071] 2.3 Biomimetic mineralization: Take the sample after phosphorylation in step 2.1, add the mineralization solution prepared in step 2.2 according to the solid-liquid ratio of 1:200, and carry out biomimetic mineralization by oscillating at 37°C, changing the liquid every 24 hours. After that, pour into purified water according to the solid-liquid ratio of 1:10 (w / v), and oscillate and wash at room temperature for 1 hour, changing the liquid every 5 minutes.

[0072] (3) Preparation of Thermosensitive Spongy Bone Hydrogel

[0073] 3.1 Digestion: Take demineralized spongy bone, add 0.1 wt% porcine pepsin solution (pH 2 - 3) according to the solid-liquid ratio of 1:100 (w / v), and stir at 4°C for 24 - 48 hours until it becomes semi-transparent liquid without obvious visible particles to the naked eye.

[0074] 3.2 Neutralization: Take the sample in step 3.1, continue to stir at 4°C, and add 0.1 M sodium hydroxide dropwise until the pH range is 7.2 - 7.4.

[0075] 3.3 Adjusting ionic strength: Take the sample in step 3.2, continue to stir at 4°C, slowly add 10× phosphate buffer solution (PBS) to adjust the ionic strength of the solution to 0.5 - 1.5× PBS, and then add 1× PBS to adjust the hydrogel concentration to 0.4 - 0.8 wt%.

[0076] (4) Preparation of Composite Bone Filling Material

[0077] Take the sample in step 2.3, add the sample in step 3.3 according to the solid-liquid ratio of 1 - 3:1 (w / v), stir at 4°C for 24 hours, and fill it into a prefilled syringe to obtain an injectable composite bone filling material.

[0078] Example 2

[0079] Referring to the method of Example 1, the differences are as follows:

[0080] In step 1.1, bone meal with a particle size range of 0.4 - 0.6 mm was taken at a material - liquid ratio of 1:12 (w / v).

[0081] In step 1.2, a 10 wt% EDTA solution was added at a material - liquid ratio of 1:10 (w / v), and decalcification was carried out by shaking at room temperature for 10 hours.

[0082] In step 1.4, isopropanol was added at a material - liquid ratio of 1:8 (w / v).

[0083] In step 1.5, a 2 wt% sodium deoxycholate solution was added at a material - liquid ratio of 1:8 (w / v), and decellularization was carried out by shaking at room temperature for 12 hours; it was poured into normal saline at a material - liquid ratio of 1:15 (w / v).

[0084] In step 2.1, 2.5 wt% sodium trimetaphosphate was added at a material - liquid ratio of 1:8 (w / v).

[0085] In step 2.2, the final concentrations of the mineralization solution were 120 mM sodium chloride, 3 mM dipotassium hydrogen phosphate, 4 mM magnesium chloride, 38 mM 4 - (2 - hydroxyethyl)piperazine - 1 - ethanesulfonic acid, 18 mM sodium bicarbonate, 8 mM calcium chloride, and the pH value was 6.8.

[0086] In step 2.3, the mineralization solution was added at a material - liquid ratio of 1:300 (w / v).

[0087] When cleaning the treated cancellous bone in all the above steps, purified water was poured in at a material - liquid ratio of 1:20 (w / v), and it was shaken and cleaned at room temperature for 1 hour, with the liquid changed every 5 minutes.

[0088] In step 3.1, it was stirred at 4°C for 48 hours until it became semi - transparent liquid without obvious visible particles to the naked eye.

[0089] In step 3.3, the concentration of the hydrogel solution was adjusted to 0.8 wt%.

[0090] In step (iv), the addition was carried out according to a material - liquid ratio of 3:1 (w / v).

[0091] Example 3

[0092] Referring to the method of Example 1, the differences are as follows:

[0093] In step 1.1, bone meal with a particle size of 0.8 mm was taken at a material - liquid ratio of 1:18 (w / v).

[0094] In step 1.2, a 10 wt% EDTA solution was added at a material - liquid ratio of 1:30 (w / v), and decalcification was carried out by shaking at room temperature for 16 hours.

[0095] In Step 1.4, isopropanol is added at a material-liquid ratio of 1:10 (w / v).

[0096] In Step 1.5, a 4 wt% sodium deoxycholate solution is added at a material-liquid ratio of 1:10 (w / v), and the cells are removed by shaking at room temperature for 24 hours; then it is poured into normal saline at a material-liquid ratio of 1:20 (w / v).

[0097] In Step 2.1, 3 wt% sodium trimetaphosphate is added at a material-liquid ratio of 1:10 (w / v).

[0098] In Step 2.2, the final concentrations of the mineralization solution are 160 mM sodium chloride, 4 mM dipotassium hydrogen phosphate, 5 mM magnesium chloride, 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 22 mM sodium bicarbonate, 9 mM calcium chloride, and the pH value is 7.0.

[0099] In Step 2.3, the mineralization solution is added at a material-liquid ratio of 1:500 (w / v).

[0100] In all the above steps, when washing the treated cancellous bone, purified water is poured in at a material-liquid ratio of 1:20 (w / v), shaken at room temperature for 1 hour, and the liquid is changed every 5 minutes.

[0101] In Step 3.1, stir at 4 °C for 36 hours until it becomes semi-transparent liquid without obvious visible particles to the naked eye.

[0102] In Step 3.3, the concentration of the hydrogel solution is adjusted to 0.6 wt%.

[0103] In Step (Four), add according to a material-liquid ratio of 2:1 (w / v).

[0104] Performance test:

[0105] After testing, the composite bone filling materials prepared by the above methods in Examples 1-3 of the present invention have the following characteristics:

[0106] (1) In the composite bone filling material, the proportion of the organic phase is 15%-25 wt%, and the proportion of the inorganic phase is 35%-55 wt% (the remaining components are water);

[0107] (2) The composite bone filling material contains abundant osteogenic-related active factors such as bone morphogenetic protein BMP-2 and has high osteogenic inductivity;

[0108] (3) The inorganic phase of the composite bone filling material is cluster-shaped or spherical hydroxyapatite with a diameter of about 1 μm. Each cluster-shaped hydroxyapatite is composed of nanoscale flaky hydroxyapatite, having a larger specific surface area and higher bioavailability;

[0109] (4) The composite bone filling material has injectability, good plasticity, and convenient operation.

[0110] The composite bone filling material described in (5) can be used for the rapid repair of oral and maxillofacial bone defects and can be biodegraded within 3 months, which is coordinated with the bone formation time.

[0111] The schematic diagram of the mineralized decalcified cancellous bone filling material product prepared in step (two) of the present invention is as Figure 1 shown.

[0112] Experimental Example 1: Comparing the effects of different pretreatment methods on the decalcification efficiency of cancellous bone

[0113] According to the preparation method of Example 1, decalcification was carried out with a 10 wt% EDTA solution. Samples were taken at the time points shown in the following table. After washing with purified water, the calcium content was measured according to the method of "GB / T 9695.13-2009 Determination of calcium content in meat and meat products". The results are shown in Table 1 below:

[0114] Table 1 Effects of pretreatment methods on the decalcification efficiency (calcium content %) of cancellous bone

[0115]

[0116] As can be seen from Table 1, cancellous bone with different particle sizes has a corresponding impact on the decalcification efficiency. In particular, the pretreatment method of preparing into bone powder used in the present invention enables the decalcification time of cancellous bone to be only 4 h, greatly reducing the time cost and the risk of microbial contamination.

[0117] Experimental Example 2: Comparing the effects of different decalcification methods on the bioactivity of bone filling materials.

[0118] Traditional decalcification methods are mostly treatment with 0.6 M hydrochloric acid for 24 - 72 h, which has a greater impact on the active components of cancellous bone. Referring to the preparation method of Example 1, cancellous bone powder samples under different decalcification conditions were prepared according to the conditions in Table 2. Each group of samples was extracted with a 4 M guanidine hydrochloride / Tris-HCl solution, and the guanidine hydrochloride was removed by dialysis to make its concentration lower than 0.06 M. The content of BMP-2 in the samples was quantitatively detected using an enzyme-linked immunosorbent assay (ELISA) kit; the denaturation temperature of the samples was quantitatively detected using differential scanning calorimetry (DSC). The results are shown in Table 2 below:

[0119] Table 2 Effects of decalcification methods on the bioactivity and structure of cancellous bone

[0120]

[0121]

[0122] As shown in Table 2, with the dissolution of calcium and phosphate salts during the initial decalcification phase, BMP-2 is released, leading to an increase in its measured content. However, after complete calcium removal, BMP-2 content gradually decreases with increasing EDTA treatment time. However, even after just one hour of hydrochloric acid decalcification, BMP-2 content has already significantly decreased. This demonstrates that the present invention's decalcified cancellous bone is gentle and short-term, effectively retaining the BMPs necessary for bone formation. Furthermore, the material's denaturation temperature gradually decreases with increasing treatment time, indicating that longer treatment times lead to more severe structural damage in the material. This further demonstrates that the short treatment time of the present decalcification process is beneficial for maintaining the material's biological activity and structure.

[0123] Experimental Example 3: Effects of different mineralization processes on the formation of inorganic phase.

[0124] Traditional mineralization involves immersion in simulated body fluid (SBF), but collagen lacks mineralization nucleation sites, resulting in a long and unstable mineralization process. Referring to the preparation method of Example 1, decalcified cancellous bone was treated using the different mineralization processes listed in Table 3. Samples were taken at the time points indicated in the table below and rinsed with purified water. The inorganic phase content was determined according to the residue on ignition method in 0841 of the General Rules of Part IV of the Chinese Pharmacopoeia (2020 Edition), as shown in Table 3. Simultaneously, a visual inspection of the mineralization of each replicate sample from the three groups of samples after three days of mineralization was performed, as shown in Table 4.

[0125] Table 3 Effect of mineralization mode on cancellous bone mineralization degree (inorganic phase content%)

[0126]

[0127]

[0128] Table 4. Mineralization of parallel samples in each mineralization method group after 3 days (×: not mineralized; √: mineralized)

[0129]

[0130] As shown in Tables 3 and 4, traditional SBF immersion is not suitable for decalcified bone materials and has extremely low mineralization efficiency. When simply immersing in the patented mineralizing solution, some samples can be successfully mineralized to form a hydroxyapatite coating, but some samples cannot be mineralized, resulting in a low mineralization success rate and unstable process. However, using a combined treatment method of phosphorylation + immersion in the patented mineralizing solution, all samples can be successfully mineralized. At the same time, the mineralization treatment is stable and efficient, meeting the needs of industrial production.

[0131] Experimental Example 4

[0132] The mineralized decalcified cancellous bone material prepared in step (2) of the embodiments of the present invention was subjected to performance testing. Taking the sample prepared in step (2) of Example 1 as an example (the samples prepared in the remaining examples are not very different), scanning electron microscopy (SEM) showed that the surface of the decalcified cancellous bone before mineralization was relatively smooth and lacked an inorganic phase coating. After mineralization, a nanoscale inorganic phase coating was formed on the surface of the decalcified cancellous bone, showing a cluster shape ( Figure 2 ), with a diameter of about 1 μm. After magnification, it can be seen that a single cluster-shaped hydroxyapatite is composed of multiple nanoscale sheet-like hydroxyapatites ( Figure 3 ), having a larger specific surface area and higher bioavailability. The X-ray diffraction analysis results showed that the component of the inorganic phase coating was hydroxyapatite ( Figure 4 ).

[0133] Experimental Example 5

[0134] A rabbit mandibular bone defect model with a size of diameter 10 mm * depth 4 mm was selected. Taking Bio-Oss as a control, after 8 weeks of operation, when the samples were taken, it was found that both this product (taking the mineralized decalcified cancellous bone sample obtained in step (2) of Example 1 as an example, and the samples of the remaining examples were similar) and the control Bio-Oss had material residues ( Figure 5 ). The X-ray photograph showed that the edges of the modeling areas in both groups were blurred, new bone formation was obvious, and they were connected into pieces, with no obvious difference ( Figure 6 ), indicating that this product has good osteogenic performance. After 12 weeks of operation, when the samples were taken, it was found that the control still had obvious material residues, while this product had no residues, indicating that this product has good biodegradability ( Figure 7 ).

[0135] Experimental Example 6

[0136] (1) Self-assembly process of thermosensitive cancellous bone hydrogel at 37°C

[0137] The thermosensitive cancellous bone hydrogel was prepared according to step (3) of Example 1, and its self-assembly process was observed. The results are as Figure 8 shown. It can be seen that at room temperature, the cancellous bone hydrogel presented a viscous liquid state and could flow down along the wall when inverted ( Figure 8 left in the figure). After being placed at 37°C, self-assembly cross-linking could occur, changing from a liquid state to a solid state and stably existing ( Figure 8 right in the figure). (2) Influence of the concentration of thermosensitive cancellous bone hydrogel on the self-assembly cross-linking of the material

[0138] According to the method of Example 1, thermosensitive cancellous bone hydrogels with different concentrations were prepared. 1 mL was taken and placed in a 1.5 mL centrifuge tube, and incubated in a 37°C water bath for 5 minutes. After the incubation, the centrifuge tube was inverted to observe whether it flowed down along the wall. The results are shown in Table 5 below:

[0139] Table 5 Influence of the concentration of thermosensitive cancellous bone hydrogel on the self-assembly cross-linking of the material.

[0140] Gel concentration (%) Room temperature property Incubated at 37°C for 5 min 1.0 Viscous liquid Can self-assemble, cannot flow down along the wall, can exist stably 0.8 Relatively viscous liquid Can self-assemble, cannot flow down along the wall, can exist stably 0.4 Relatively viscous liquid Can self-assemble, cannot flow down along the wall, can exist stably 0.2 Non-viscous liquid Can self-assemble, cannot flow down along the wall, relatively fragile 0.1 Non-viscous liquid Cannot self-assemble

[0141] As can be seen from Table 5, when the concentration of the thermosensitive cancellous bone hydrogel is lower than 0.4 wt%, it cannot stably exist after self-assembly. However, when the concentration is higher than 0.8 wt%, the viscosity is too high, which is not conducive to sufficient mixing with the mineralized decalcified cancellous bone. Therefore, the hydrogel with a concentration of 0.4% - 0.8 wt% described in the present invention can have the characteristics of sufficient mixing and stable existence.

[0142] (3) Influence of the concentration of the thermosensitive cancellous bone hydrogel on the self-assembly cross-linking time of the material

[0143] Prepare hydrogels with different concentrations according to the method of Example 1, add them into a 96-well plate (100 μL / well), and place them in a microplate reader preheated to 37°C. Measure the absorbance at 405 nm every 1 minute for 15 minutes. Calculate the normalized absorbance NA (A is the absorbance at a given time, A0 is the initial absorbance, and A max is the maximum absorbance), and plot the curve.

[0144]

[0145] According to the fitting curve, finally obtain the semi-gel time (t 1 / 2 ), gel rate (S), and lag time (t flag ). The results are shown in Table 6 and Figure 9 :

[0146] ① The semi-gel time (t 1 / 2 ) is defined as the time when the absorbance reaches 50%;

[0147] ② The gel rate (S) is defined as the maximum slope of the linear region of the gel curve;

[0148] ③ The lag time (t flag ) is defined as the intercept of the linear region of the gel curve when the absorbance is 0%.

[0149] Table 6 Influence of the concentration of the thermosensitive cancellous bone hydrogel on the self-assembly cross-linking time of the material.

[0150] Gel concentration (%) <![CDATA[Half gel time (t 1 / 2 )]]> Gelation rate (S) <![CDATA[Lag time (t flag )]]> 0.8 2.21 0.37 1.15 0.4 3.89 0.30 2.49 0.2 5.69 0.21 3.86

[0151] As can be seen from Table 6 and Figure 9 it can be known that the higher the concentration of the thermosensitive cancellous bone hydrogel, the shorter the self-assembly cross-linking time. The hydrogel with a concentration of 0.4% - 0.8 wt% described in the present invention can complete self-assembly cross-linking within 5 minutes, which is convenient for clinical use and has strong operability.

[0152] (4) Influence of the ionic strength of the thermosensitive cancellous bone hydrogel on the self-assembly cross-linking of the material

[0153] Prepare hydrogels with different ionic strengths according to the method of Example 1. Take 1 mL and place it in a 2 mL centrifuge tube. Incubate it in a 37 °C water bath for 5 minutes. After incubation, invert the centrifuge tube and observe whether it flows down along the wall. The results are as follows Figure 10 :

[0154] It can be Figure 10 seen that within the range of ionic strength from 0.5 to 1.5×PBS, self-assembly cross-linking can occur within 5 minutes, and it exists stably without flowing down along the wall. Considering that the ionic strength under physiological conditions of the human body is about 1×PBS, the thermosensitive cancellous bone hydrogel described in the present invention can stably self-assemble and cross-link under physiological conditions of ionic strength.

[0155] (5) Scanning electron microscopy (SEM) shows that the thermosensitive cancellous bone hydrogel presents a three-dimensional network scaffold structure( Figure 11 ), which is beneficial for the ingrowth of osteoblasts and capillaries.

[0156] (6) Ion release of the morphologically plastic biomimetic mineralized composite bone filling material

[0157] Place the decalcified cancellous bone and the composite bone filling material described in the present invention in physiological saline, incubate them with shaking at 37 °C, take the supernatant every day, and use an electrochemical method to detect the calcium ions( Figure 12 ) and phosphate ions( Figure 13 ) contents in the supernatant.

[0158] (7) Select a Beagle dog alveolar bone defect model with a size of approximately 8 mm in width × 10 mm in length × 10 mm in depth, and use decalcified cancellous bone as a control. Just implant it. This product can stay smoothly and stably in the extraction socket without being washed away by blood, while the control will spill during the implantation process, and there is no need to use an oral repair membrane in combination( Figure 14 ). X-ray photography after 12 weeks of operation shows that obvious high-density shadow particles of the filling material can be seen in the defect areas of both groups, the defect boundary lines disappear, the formation of new bone increases significantly, there is a bone marrow cavity formation, and the osteogenic effect of this product is significantly better than that of the control( Figure 15 ), indicating that this product has good osteogenic performance.

Claims

1. A preparation method of a morphologically plastic bionic mineralized composite bone filling material, characterized in that, It includes the following steps: (1) Preparation of decalcified cancellous bone (1) Crush the cancellous bone into bone powder with a particle size of 0.3 - 0.8 mm, and then add it to an EDTA solution according to a solid-liquid ratio of 1:5 - 1:30 w / v, and perform decalcification by shaking at room temperature for 4 - 16 hours; (2) Add the cancellous bone treated in step (1) to a peracetic acid solution according to a solid-liquid ratio of 1:4 w / v, and shake at room temperature for 2 hours; (3) Add the cancellous bone treated in step (2) to isopropanol according to a solid-liquid ratio of 1:5 - 1:10 w / v, and shake at room temperature for 24 hours to degrease; (4) Add the cancellous bone treated in step (3) to a 1 - 4 wt% sodium deoxycholate solution according to a solid-liquid ratio of 1:5 - 1:10 w / v, and shake at room temperature for 8 - 24 hours to decellularize, thus obtaining decalcified cancellous bone; (2) Preparation of mineralized decalcified cancellous bone (5) Add the decalcified cancellous bone obtained in step (4) to a 2 - 3 wt% sodium trimetaphosphate solution according to a solid-liquid ratio of 1:5 - 1:10 w / v. The pH of the sodium trimetaphosphate solution is 10, and shake at room temperature for 1 hour for phosphorylation treatment; (6) Immerse the phosphorylated decalcified cancellous bone in a mineralization solution according to a solid-liquid ratio of 1:200 - 1:500 w / v, and perform biomimetic mineralization by shaking at 37°C for 1 - 5 days. The composition of the mineralization solution is: sodium chloride 100 - 160 mM, dipotassium hydrogen phosphate 2 - 4 mM, magnesium chloride 3 - 5 mM, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 35 - 40 mM, sodium bicarbonate 15 - 22 mM, calcium chloride 6 - 9 mM, and the pH value is 6.5 - 7.0, thus obtaining mineralized decalcified cancellous bone; (3) Preparation of thermosensitive cancellous bone hydrogel (7) Take the decalcified cancellous bone obtained in step (4), and add it to a 0.1 wt% porcine pepsin solution according to a solid-liquid ratio of 1:100 w / v. The pH of the porcine pepsin solution is 2 - 3, and stir at 4°C for 24 - 48 hours; (8) Adjust the pH and ionic strength of the product obtained in step (7). Adjust the pH to 7.2 - 7.4, then slowly add 10× PBS buffer solution to adjust the ionic strength to 0.5 - 1.5× PBS, and then add 1× PBS to adjust the concentration of the hydrogel solution to 0.4 - 0.8 wt%; (4) Preparation of biomimetic mineralized composite bone filling material (9) Take the mineralized decalcified cancellous bone obtained in step (6), and add it to the hydrogel solution obtained in step (8) according to a solid-liquid ratio of 1 - 3:1 w / v, and stir at 4°C for 24 hours to obtain the biomimetic mineralized composite bone filling material.

2. The preparation method according to claim 1, wherein The concentration of the EDTA solution in step (1) is 10 wt%.

3. The preparation method according to claim 1, characterized in that, The pH value of the EDTA solution in step (1) is 7.

4.

4. The preparation method according to claim 1, characterized in that, The concentration of the peracetic acid solution in step (2) is 0.3 wt%.

5. The preparation method according to claim 1, characterized in that, The pH of the sodium deoxycholate solution in step (4) is 8.

6. The preparation method according to claim 1, characterized in that, In step (6) during the process of biomimetic mineralization by shaking, the mineralization solution is changed every 24 hours.

7. The preparation method according to any one of claims 1-6, characterized in that, In steps (1) to (6), the cancellous bone after each step of treatment is washed. The washing steps are as follows: The treated cancellous bone is poured into purified water at a solid-liquid ratio of 1:10 to 1:20 w / v, and oscillated and washed at room temperature for 1 hour, with the liquid changed every 5 minutes.

8. The preparation method according to claim 1, wherein, The operation of adjusting the pH in step (8) is as follows: 0.1 M sodium hydroxide is added dropwise to the product obtained in step (7) under stirring at 4°C until the pH ranges from 7.2 to 7.

4.

9. The biomimetic mineralized composite bone filling material obtained by the preparation method according to any one of claims 1-8, characterized in that, The proportion of the organic phase in the biomimetic mineralized composite bone filling material is 15-25 wt%, and the proportion of the inorganic phase is 35-55 wt%.

10. Use of the biomimetic mineralized composite bone filling material prepared by the method according to any one of claims 1-8 or the biomimetic mineralized composite bone filling material according to claim 9 in the preparation of an oral and maxillofacial bone defect filling material.

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