Collagen fibrils with highly bone-mimicking mineralization of inner fibrils and their preparation method

Abstract

The present invention discloses a type I collagen fibril with highly bone-mimicking mineralization and a preparation method thereof. The preparation method is to dissolve collagen in an acid solution to prepare a collagen acid solution, and then successively add a calcium salt solution and a phosphate solution dropwise into the collagen acid solution for thorough mixing, with continuous stirring and a certain temperature condition throughout the process. Then, under the condition of maintaining stirring, the pH is adjusted, and then the stirring is stopped and the mixture is allowed to stand. The precipitated part is separated, washed, and dried to obtain a type I collagen fibril with highly bone-mimicking mineralization. This preparation method realizes uniform, continuous, and sufficient mineralization both inside and on the outer surface of the collagen fibril, achieving a high degree of biomimicry of the basic unit of natural bone tissue, namely its secondary microstructure (bone-mineralized collagen fibril). The collagen fibril prepared by this preparation method can provide a simulated three-dimensional scaffold microenvironment for osteoblasts and supply calcium ions and phosphate ions to promote bone regeneration and repair.

Description

Technical Field

[0001] The present invention belongs to the technical field of collagen fibril preparation processes, and relates to a collagen fibril with highly bone-mimicking mineralization of inner fibrils and a preparation method thereof. The collagen fibril with highly bone-mimicking mineralization of inner fibrils can be used as a regenerative repair material for hard tissue defects such as bone and teeth. Background Art

[0002] In China, there are about 6 million bone defect patients caused by traffic accidents, trauma, natural disasters, diseases, etc. every year, which makes there is a great demand for bone repair materials used in clinical bone defect treatment. In the field of bone defect treatment, the repair materials used include autologous bone, allogeneic bone and artificial bone. Autologous bone, as the gold standard of clinical treatment methods, has advantages such as good osteoinductivity and fast osteogenesis speed, but has defects such as a single acquisition channel, secondary injury to patients caused by material collection, extended operation time and limited bone mass; allogeneic bone also has excellent osteoconductivity and osteoinductivity, and the source is wider than that of autologous bone, but allogeneic bone has the risks of immune rejection and disease transmission.

[0003] After a long-term development, artificial bone materials have evolved from the initial inert fillers to bioactive bone-induced regenerative repair materials, from the initial single-component to multi-component composite materials, and from industrial materials to bionic materials. In the 1990s, people used tissue engineering methods to construct bioactive tissues or organs in vitro for the repair of human defect sites. Bone tissue engineering includes scaffold materials, seed cells and cell growth factors. As the key core, scaffold materials can be divided into metal materials, polymer materials, inorganic materials and organic-inorganic composite materials. Based on the respective advantages and disadvantages of metal materials, polymer materials and inorganic materials, composite materials composed of organic components and inorganic components complement each other in terms of material structure and performance, having both the strength and bioactivity of inorganic components and the toughness and excellent biocompatibility of organic components.

[0004] Natural bone tissue is an organic-inorganic composite material. The inorganic components are mainly hydroxyapatite, carbonate apatite, etc., accounting for about 65% of the total mass of bone tissue; the organic components are mainly type I collagen, accounting for about 34% of the total weight of bone tissue. Natural bone tissue is roughly divided into seven levels of structure from macroscopic to microscopic: bone tissue, cancellous bone and compact bone, osteon, mineralized collagen fiber layers arranged parallel and staggered, mineralized collagen fiber bundles, mineralized collagen fibrils and nano-hydroxyapatite and collagen fibrils.

[0005] Mineralized collagen fibrils are the secondary structure of bone tissue and the basic building blocks of bone tissue. For decades, many scholars have conducted extensive research on how to prepare mineralized collagen fibrils in vitro. Initially, it was just biomimicry in terms of composition, that is, simply mixing collagen fibrils with hydroxyapatite. However, this kind of mixture does not have the uniform and orderly combination between collagen fibrils and nano-hydroxyapatite in natural bone tissue. Later, some researchers soaked collagen sponges in simulated body fluid to achieve the nucleation and growth of hydroxyapatite nanocrystals on the outer surface of collagen fibrils (Cui FZ, Li Y, Ge J. Self-assembly of mineralized collagen composites. Mater Sci Eng R Rep. 2007, 57(1–6):1–27; Xia Z, Yu X, Jiang X, Brody HD, Rowe DW, Wei M. Fabrication and characterization of biomimetic collagen–apatite scaffolds with tunable structures for bone tissue engineering. Acta Biomaterialia. 2013, 9:7308-7319; Wang Y, Hua Y, Zhang Q, Yang J, Li H, Li Y, Cao M, Cai Q, Yang X, Zhang X, Li C. Using biomimetically mineralized collagen membranes with different surface stiffness to guide regeneration of bone defects. Journal of Tissue Engineering and Regenerative Medicine. 2018, 12:1545-1555.). This method can, to a certain extent, mimic the structure and composition of the basic unit of natural bone tissue, but it cannot achieve uniform and sufficient mineralization inside the collagen fibrils and is not true biomimetic mineralization. Summary of the Invention

[0006] To solve the problems in the above-mentioned existing technologies, the present invention provides a collagen fibril with highly bone-mimicking mineralization in the inner fibrils and its preparation method. This preparation method realizes uniform, continuous, and sufficient mineralization on both the inner and outer surfaces of the collagen fibrils, achieving a high degree of biomimicry of the basic unit of natural bone tissue, namely its secondary microstructure (bone-mineralized collagen fibrils). The collagen fibril with highly bone-mimicking mineralization in the inner fibrils prepared by this preparation method can provide a simulated three-dimensional scaffold microenvironment for osteoblasts and supply calcium ions and phosphate ions to promote bone regeneration and repair.

[0007] To achieve the above-mentioned objectives, the present invention is realized by a technical solution composed of the following technical measures.

[0008] A preparation method of a collagen fibril with highly bone-mimicking mineralization in the inner fibrils mainly includes the following steps:

[0009] (1) Dissolve collagen in an acid solution to prepare a collagen acid solution with a collagen concentration of 0.1 - 3.0 mg / mL. Add a calcium salt solution dropwise to the collagen acid solution and mix well to obtain a collagen-calcium ion composite solution. Then add a phosphate solution dropwise to the collagen-calcium ion composite solution and mix well to obtain an intermediate solution;

[0010] Among them, the acid solution is any one of phosphoric acid solution, malic acid solution, and citric acid solution, and the pH value is 2 - 3;

[0011] In the collagen-calcium ion composite solution, the content of calcium ions is 0.05 - 0.12 mol calcium ions per gram of collagen; in the intermediate solution, the molar ratio of calcium ions to phosphate ions is (1 - 2):1;

[0012] The calcium salt solution is added dropwise to the collagen solution at a dropping rate of 40 - 120 drops per minute;

[0013] The phosphate solution is added dropwise to the collagen-calcium ion composite solution at a dropping rate of 40 - 120 drops per minute;

[0014] During the whole process of step (1), maintain the temperatures of the collagen solution, the collagen-calcium ion composite solution, and the intermediate solution at 18 - 30 °C respectively, and continuously stir throughout the process at a stirring rate of 500 - 1200 r / min;

[0015] (2) Under the condition of maintaining stirring, adjust the pH of the intermediate solution obtained in step (1) to 7 - 9.5 by dropwise adding a strong base solution. Precipitation appears in the intermediate solution and it turns into a suspension. Stop stirring and let it stand for at least 36 h. Separate the precipitated part, wash it, and dry it to prepare a collagen fibril with highly bone-mimicking mineralization in the inner fibrils;

[0016] In step (2), the temperatures of the intermediate solution and the suspension are maintained at 18 - 30 °C respectively.

[0017] In this article, the collagen is animal-derived collagen. Generally, those skilled in the art can select a suitable collagen source according to the actual application. In one technical solution, the collagen used is type I collagen extracted from pigskin, cowhide, fish skin, or bovine Achilles tendon.

[0018] In this article, the calcium salt solution in step (1) is a calcium salt solution prepared by dissolving a calcium salt in a conventional aqueous solvent, and the selection of the calcium salt can refer to the calcium salts commonly used in the preparation of mineralized collagen fibrils in the art.

[0019] In one technical solution, the molar concentration of the calcium salt solution in step (1) is 0.05 - 2.5 mol / L, and it is prepared by dissolving any one of calcium chloride and calcium nitrate in deionized water.

[0020] In one technical solution, the calcium salt solution in step (1) is added dropwise to the collagen solution at a dropping rate of 50 - 100 drops per minute.

[0021] In this article, the phosphate solution in step (1) is a phosphate solution prepared by dissolving a phosphate in a conventional aqueous solvent, and the selection of the phosphate can refer to the phosphates commonly used in the preparation of mineralized collagen fibrils in the art.

[0022] In one technical solution, the molar concentration of the phosphate solution in step (1) is 0.05 - 2.5 mol / L, and it is prepared by dissolving any one of disodium hydrogen phosphate, sodium dihydrogen phosphate, diammonium hydrogen phosphate, diammonium dihydrogen phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate in deionized water.

[0023] In one technical solution, the phosphate solution in step (1) is added dropwise to the collagen-calcium ion complex solution at a dropping rate of 50 - 100 drops per minute.

[0024] In this article, the pH of the intermediate solution obtained in step (1) is adjusted to 7 - 9.5 by dropwise adding a strong base solution, where the strong base solution is a strong base solution commonly used in the chemical industry to adjust the pH value of a solution, such as sodium hydroxide solution, potassium hydroxide solution, etc.

[0025] The key point of the present invention lies in that, on the basis of the existing preparation process of mineralized collagen fibrils, it is innovatively discovered that by further selecting and changing the process conditions therein, uniform, continuous and sufficient mineralization can be achieved both inside and on the outer surface of the collagen fibrils, that is, the mineralization of the collagen fibrils reaches intrafibrillar mineralization. Since the basic unit of natural bone tissue is mineralized collagen fibrils, that is, the secondary structure of bone, the mineralization mode of this secondary structure is intrafibrillar mineralization. Therefore, the collagen fibrils prepared by the technology of the present invention achieve a high degree of biomimicry of the secondary structure of bone tissue. Through electron microscopy observation and comparison, it can be fully demonstrated that the interior of the collagen fibrils is combined with uniform, continuous and sufficient nanoapatite crystals, the crystals are in the shape of long flakes, and are arranged along the long axis direction of the collagen fibrils, forming a biomimetic structure.

[0026] It should be emphasized that through exploratory comparative experiments, it is found that only when the temperature condition of its preparation process, the stirring rate and the dropping rate in step (1) are simultaneously satisfied, can the above-mentioned collagen fibrils with highly intrafibrillar bone-mimicking mineralization be successfully prepared. In contrast, although other researchers in this field adopted a relatively similar process preparation flow when preparing collagen fibrils, it can be clearly found through the electron microscopy photos published currently that none of them achieved uniform, continuous and sufficient mineralization inside the collagen fibrils, and they could not prepare truly biomimetic-structured mineralized collagen fibrils. During the repeated exploratory experiments of the present invention, it was surprisingly found that by further selecting and changing the process conditions, a qualitative change effect was caused, and in fact, collagen fibrils with highly intrafibrillar bone-mimicking mineralization were prepared.

[0027] In addition, through comparative experiments, it was also found that the specific selection of the acid solution when preparing the collagen acid solution would also significantly affect the internal mineralization of the collagen fibrils.

[0028] During the further exploration process, the inventors of the present invention also found that the collagen fibrils with highly intrafibrillar bone-mimicking mineralization can be further combined with bone formation-promoting drugs without affecting the uniform distribution of the above-mentioned nano-mineral crystals.

[0029] Based on this, in a more preferred technical solution, the bone formation-promoting drug is dissolved in a water solvent to prepare a bone formation-promoting drug solution, and the bone formation-promoting drug solution is added dropwise to the intermediate solution obtained in step (1) as an intermediate solution containing a bone-promoting drug. This intermediate solution containing a bone-promoting drug can directly replace the intermediate solution obtained in step (1) to participate in the subsequent preparation steps;

[0030] Among them, the mass ratio of the bone formation-promoting drug to collagen is 1:(1 - 5);

[0031] During the whole process, the temperature of the intermediate solution is maintained at 18 - 30 °C, the dropping rate is 40 - 120 drops per minute, and continuous stirring is carried out throughout the process, with the stirring rate being 500 - 1200 r / min.

[0032] In the above - mentioned preferred technical solution, the bone - forming promoting drug selection includes chemical molecular drugs with bone - forming promoting effects that have been disclosed in the prior art, such as bone morphogenetic protein - 2 (BMP - 2), transforming growth factor - β2 (TGF - β2), vascular endothelial growth factor (VEGF), etc., and can also include extracts of traditional bone - tonifying Chinese medicines, such as extracts of icariin, resveratrol, curcumin, berberine, astragalus, naringin, etc.

[0033] The collagen fibrils with highly bone - mineralized endo - fibrils prepared by the present invention can provide a simulated three - dimensional scaffold microenvironment for osteoblasts, and provide calcium ions and phosphate ions to promote bone regeneration and repair, such as being applied to bio - regenerative repair materials, dental medical filling materials, regenerative filling materials for dental alveolar defects, etc.

[0034] To better illustrate the above applications, on the other hand, the present invention also provides a method for preparing a plasticizable preparation based on the above - mentioned collagen fibrils with highly bone - mineralized endo - fibrils, which mainly includes the following steps:

[0035] (Ⅰ) Dissolve the polysaccharide binder in purified water to prepare a binder solution, where the volume of purified water is 50 - 200 times the mass of the polysaccharide binder;

[0036] (Ⅱ) Add collagen to the binder solution obtained in step (Ⅰ), and carry out a swelling treatment with stirring for 10 - 50 h to obtain a collagen mixture;

[0037] (Ⅲ) Add the collagen fibrils with highly bone - mineralized endo - fibrils and nano - calcium phosphate salts to the collagen mixture obtained in step (Ⅱ), and continuously stir and disperse for 24 - 72 h to obtain a pre - preparation;

[0038] Among them, the content of collagen in the pre - preparation is 5 - 30 wt% of the dry weight of the pre - preparation, the content of the collagen fibrils with highly bone - mineralized endo - fibrils is 20 - 80 wt% of the dry weight of the pre - preparation, and the content of calcium phosphate salts is 10 - 30 wt% of the dry weight of the pre - preparation;

[0039] (Ⅳ) Vacuum - treat the pre - preparation obtained in step (Ⅲ) to remove air bubbles, and then add it to a mold and carry out freeze - drying treatment to prepare a freeze - dried sponge - like plasticizable preparation.

[0040] In this article, the polysaccharide binder in step (Ⅰ) is a conventional binder added in bio - regenerative repair materials, and the selection of the polysaccharide binder can refer to the polysaccharide binders commonly used in regenerative repair materials in this field.

[0041] In one of the technical solutions, the polysaccharide binder described in step (Ⅰ) is selected from one or more of sodium alginate, chitosan, hyaluronic acid, modified starch, cellulose, konjac glucomannan, and sodium carboxymethyl cellulose.

[0042] In this article, the nano calcium phosphate salt described in step (Ⅲ) is a conventional nano-scale inorganic filler added in the biological regeneration and repair material, and the selection of the nano calcium phosphate salt can refer to the calcium phosphate salts commonly used in the regeneration and repair materials in this field.

[0043] In one of the technical solutions, the nano calcium phosphate salt described in step (Ⅲ) is selected from one or more of nano-hydroxyapatite, nano-α-tricalcium phosphate, nano-β-tricalcium phosphate, nano-tetracalcium phosphate, nano-octacalcium phosphate, and nano-amorphous calcium phosphate.

[0044] In this article, operations such as dissolution, mixing, stirring, and drying all follow the conventional principles in chemical engineering processes, and those skilled in the art can perform specific operations according to common general knowledge.

[0045] The present invention has the following beneficial effects:

[0046] (1) The internally fibrillar highly bone-mimetic mineralized collagen fibril and its preparation method provided by the present invention achieve uniform, continuous, and sufficient mineralization both inside and on the outer surface of the collagen fibril, reaching a high degree of biomimesis of the basic unit of natural bone tissue, namely its secondary microstructure (bone-mineralized collagen fibril). The internally fibrillar highly bone-mimetic mineralized collagen fibril prepared by this preparation method can provide a simulated three-dimensional scaffold microenvironment for osteoblasts and provide calcium ions and phosphate ions to promote bone regeneration and repair.

[0047] (2) The internally fibrillar highly bone-mimetic mineralized collagen fibril and its preparation method provided by the present invention, in one of the technical solutions / embodiments, are prepared into a shapeable preparation that can be arbitrarily shaped by compounding a composite collagen fibril containing a bone formation-promoting drug with pure collagen, nano-hydroxyapatite, and a small amount of polysaccharide. On the one hand, it avoids introducing other external components to reduce the potential biological safety risk; on the other hand, the loaded bone formation-promoting drug can be slowly released to promote the proliferation and differentiation of osteoblast-related cells at the bone defect site, further enhancing the ability of promoting regeneration and repair.

[0048] (3) The plasticizable preparation obtained from the intrafibrillar highly bone-mimetic mineralized collagen fibrils provided by the present invention is infiltrated and rehydrated with normal saline or PBS buffer in a volume ratio of 1:1 and kneaded evenly to obtain a dough-like mineralized collagen material that can be shaped arbitrarily. Then, the mineralized collagen material is placed in a syringe, and continuous strip materials can be extruded from the syringe, and the extruded materials are continuous and do not break, greatly expanding its application range and convenience of use. Description of the Drawings

[0049] Figure 1 Photographs of the specimens prepared in Example 1 and Example 2 of the present invention. In the figure, a is the powder of intrafibrillar highly bone-mimetic mineralized collagen fibrils prepared in Example 1, and b is the powder of composite collagen fibrils containing astragalus polysaccharide prepared in Example 2.

[0050] Figure 2 Photograph of the freeze-dried sponge-like plasticizable preparation prepared in Application Example 1 of the present invention.

[0051] Figure 3 Infrared spectra of the specimens prepared in Example 1 and Example 2 of the present invention. In the figure, the black line "biomimetic mineralized collagen fibrils" is the intrafibrillar highly bone-mimetic mineralized collagen fibrils prepared in Example 1, and the red line "biomimetic mineralized collagen-astragalus polysaccharide composite fibrils" is the composite collagen fibrils containing astragalus polysaccharide prepared in Example 2; it can be seen that the characteristic absorption peaks of the specimen in Example 2 are at 3378, 2927, 1626, 1417, 1050 cm -1 nearby. Most of the absorption peaks in the specimen of Example 2 coincide with the peaks existing in the specimen of Example 1. The characteristic peaks at 2927 and 1416 cm -1 belong to the characteristic absorption peaks of polysaccharides, and the presence of astragalus polysaccharide can be identified.

[0052] Figure 4 XRD spectra of the specimens prepared in Example 1 and Example 2 of the present invention. In the figure, the black line "biomimetic mineralized collagen fibrils" is the intrafibrillar highly bone-mimetic mineralized collagen fibrils prepared in Example 1, and the red line "biomimetic mineralized collagen-astragalus polysaccharide composite fibrils" is the composite collagen fibrils containing astragalus polysaccharide prepared in Example 2; compared with the hydroxyapatite standard colorimetric card, the (002), (211), (310), (222), (213) characteristic peaks of hydroxyapatite can be obtained from the spectra, confirming that the calcium phosphate minerals of the specimens in Example 1 and Example 2 are hydroxyapatite crystals. At the same time, it shows that the introduction of astragalus polysaccharide does not affect the formation of hydroxyapatite nanocrystals.

[0053] Figure 5This is an electron microscope photograph of the specimens prepared in Example 1 and Example 2 of the present invention. In the figure, a is the microscopic morphology of the intrafibrillar highly bone-mimicking mineralized collagen fibrils prepared in Example 1, and b is the microscopic morphology of the composite collagen fibrils containing astragalus polysaccharide prepared in Example 2. It can be seen that the surfaces of the collagen fibrils in Example 1 and Example 2 are both covered with uniform and sufficient minerals, and it is further verified that the introduction of astragalus polysaccharide has no effect on the uniform mineralization inside the collagen fibrils.

[0054] Figure 6 This is an energy spectrum analysis diagram of the specimens prepared in Example 1 and Example 2 of the present invention. In the figure, the left figure is the intrafibrillar highly bone-mimicking mineralized collagen fibrils prepared in Example 1, and the right figure is the composite collagen fibrils containing astragalus polysaccharide prepared in Example 2. It is known from the energy spectrum analysis that the calcium-phosphorus ratio of the specimen in Example 1 is 1.53 ± 0.016, and the calcium-phosphorus ratio of the specimen in Example 2 is 1.52 ± 0.086, which is very close to the calcium-phosphorus ratio of calcium-deficient hydroxyapatite in natural bone tissue.

[0055] Figure 7 This is a transmission electron microscope photograph of the specimens prepared in Example 1 and Example 2 of the present invention. In the figure, a is the transmission microscopic morphology of the intrafibrillar highly bone-mimicking mineralized collagen fibrils prepared in Example 1, and b is the transmission microscopic morphology of the composite collagen fibrils containing astragalus polysaccharide prepared in Example 2. It can be seen that uniformly and sufficiently nano-mineral crystals are combined inside the collagen fibrils in Example 1, the crystal particles are in the shape of long flakes and are arranged orderly along the axial direction of the collagen fibrils. The introduction of astragalus polysaccharide in Example 2 does not affect the combination of collagen and nano-hydroxyapatite.

[0056] Figure 8 This is a transmission electron microscope photograph of the comparative specimen prepared in Comparative Example 1 of the present invention. It can be clearly seen that in Comparative Example 1, the nano-calcium-phosphorus mineral crystals aggregate at both ends of the collagen fibrils in the field of view, and the nano-calcium-phosphorus mineral crystals in the middle of the fibrils are very sparse, which is a typical heterogeneous mineralization, and the size of the nano-calcium-phosphorus mineral crystals is relatively large.

[0057] Figure 9 This is a transmission electron microscope photograph of the comparative specimen prepared in Comparative Example 2 of the present invention. It can be clearly seen that the nano-calcium-phosphorus mineral crystals combined on the collagen fibrils in Comparative Example 2 are sparse, and uniform and continuous mineralization inside the collagen fibrils is not achieved.

[0058] Figure 10 This is a transmission electron microscope photograph of the comparative specimen prepared in Comparative Example 3 of the present invention. It can be clearly seen that the nano-calcium-phosphorus mineral crystals combined on the collagen fibrils in Comparative Example 3 are sparse and have the characteristics of irregular orientation.

[0059] Figure 11Schematic diagram of the plasticity of the shapeable preparation obtained in Application Example 1 of the present invention. After infiltrating and rehydrating the shapeable preparation obtained in Application Example 1 with physiological saline in a volume ratio of 1:1 and kneading evenly, it can be shaped arbitrarily, indicating that the preparation has excellent arbitrary plasticity; when it is extruded in a syringe, it can maintain a continuous strip shape. Detailed implementation mode

[0060] To further understand the present invention, the preferred implementation schemes of the present invention are described below in conjunction with the embodiments. However, it should be understood that these descriptions are only for further explaining the features and advantages of the present invention, rather than limiting the claims of the invention. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technical solution of the present invention. Although it is believed that those of ordinary skill in the art are fully aware of the following terms, the following definitions are still stated to help explain the subject matter disclosed by the present invention.

[0061] A method for preparing a collagen fibril with highly bone-like mineralization of endogenous fibrils mainly includes the following steps:

[0062] (1) Dissolve collagen in an acid solution to prepare a collagen acid solution, where the collagen concentration is 0.1 - 3.0 mg / mL. Add the calcium salt solution dropwise to the collagen acid solution and mix well to obtain a collagen-calcium ion composite solution. Then add the phosphate solution dropwise to the collagen-calcium ion composite solution and mix well to obtain an intermediate solution;

[0063] Among them, the acid solution is any one of phosphoric acid solution, malic acid solution, and citric acid solution, and the pH value is 2 - 3;

[0064] In the collagen-calcium ion composite solution, the content of calcium ions is 0.05 - 0.12 mol calcium ions per gram of collagen; in the intermediate solution, the molar ratio of calcium ions to phosphate ions is (1 - 2):1;

[0065] The calcium salt solution is added dropwise to the collagen solution at a dropping rate of 40 - 120 drops per minute;

[0066] The phosphate solution is added dropwise to the collagen-calcium ion composite solution at a dropping rate of 40 - 120 drops per minute;

[0067] During the whole process of step (1), the temperatures of the collagen solution, the collagen-calcium ion composite solution, and the intermediate solution are maintained at 18-30 °C respectively, and continuous stirring is carried out throughout the process at a stirring rate of 500-1200 r / min;

[0068] (2) Under the condition of maintaining stirring, the pH of the intermediate solution obtained in step (1) is adjusted to 7-9.5 by dropping a strong base solution. A precipitate appears in the intermediate solution and turns into a suspension. Stop stirring and let it stand for at least 36 h. Separate the precipitated part, wash and dry it to prepare collagen fibrils with highly bone-mimicking mineralization of inner fibrils;

[0069] In step (2), the temperatures of the intermediate solution and the suspension are maintained at 18-30 °C respectively.

[0070] In this article, the collagen is animal-derived collagen. Generally, those skilled in the art can select a suitable collagen source according to actual applications. In one embodiment, the collagen is type I collagen extracted from pigskin, cowhide, fish skin, or bovine tendon.

[0071] In one embodiment, the collagen concentration in step (1) is 0.1-3.0 mg / mL, such as 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2.0 mg / mL, 2.1 mg / mL, 2.2 mg / mL, 2.3 mg / mL, 2.4 mg / mL, 2.5 mg / mL, 2.6 mg / mL, 2.7 mg / mL, 2.8 mg / mL, 2.9 mg / mL, 3.0 mg / mL or any range or point value between them.

[0072] In this article, the calcium salt solution in step (1) is a calcium salt solution prepared by dissolving a calcium salt in a conventional aqueous solvent, and the selection of the calcium salt can refer to the calcium salts commonly used in the preparation of mineralized collagen fibrils in the art.

[0073] In one embodiment, the molar concentration of the calcium salt solution in step (1) is 0.05 - 2.5 mol / L, such as 0.05 mol / L, 0.10 mol / L, 0.15 mol / L, 0.20 mol / L, 0.25 mol / L, 0.30 mol / L, 0.4 mol / L, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L, 1.0 mol / L, 1.1 mol / L, 1.2 mol / L, 1.3 mol / L, 1.4 mol / L, 1.5 mol / L, 1.6 mol / L, 1.7 mol / L, 1.8 mol / L, 1.9 mol / L, 2.0 mol / L, 2.1 mol / L, 2.2 mol / L, 2.3 mol / L, 2.4 mol / L, 2.45 mol / L, 2.5 mol / L or any range or point value therebetween; the calcium salt solution is prepared by dissolving any one of calcium chloride and calcium nitrate in deionized water.

[0074] In one embodiment, the calcium salt solution in step (1) is added dropwise to the collagen solution at a dropping rate of 40 - 120 drops / minute, such as 40 drops / minute, 50 drops / minute, 60 drops / minute, 70 drops / minute, 80 drops / minute, 90 drops / minute, 100 drops / minute, 110 drops / minute, 120 drops / minute or any range or point value therebetween.

[0075] In one embodiment, the calcium salt solution in step (1) is added dropwise to the collagen solution at a dropping rate of 50 - 100 drops / minute.

[0076] In this article, the phosphate solution in step (1) is a phosphate solution prepared by dissolving phosphate in a conventional aqueous solvent, and the selection of the phosphate can refer to the phosphates commonly used in the preparation of mineralized collagen fibrils in the art.

[0077] In one embodiment, the molar concentration of the phosphate solution in step (1) is 0.05 - 2.5 mol / L, such as 0.05 mol / L, 0.10 mol / L, 0.15 mol / L, 0.20 mol / L, 0.25 mol / L, 0.30 mol / L, 0.4 mol / L, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L, 1.0 mol / L, 1.1 mol / L, 1.2 mol / L, 1.3 mol / L, 1.4 mol / L, 1.5 mol / L, 1.6 mol / L, 1.7 mol / L, 1.8 mol / L, 1.9 mol / L, 2.0 mol / L, 2.1 mol / L, 2.2 mol / L, 2.3 mol / L, 2.4 mol / L, 2.45 mol / L, 2.5 mol / L or any range or point value therebetween; the phosphate solution is prepared by dissolving any one of disodium hydrogen phosphate, sodium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate in deionized water.

[0078] In one embodiment, in step (1), the phosphate solution is added dropwise to the collagen-calcium ion composite solution, and the dropping rate is 40 - 120 drops / minute, such as 40 drops / minute, 50 drops / minute, 60 drops / minute, 70 drops / minute, 80 drops / minute, 90 drops / minute, 100 drops / minute, 110 drops / minute, 120 drops / minute or any range or point value therebetween.

[0079] In one embodiment, in step (1), the phosphate solution is added dropwise to the collagen-calcium ion composite solution, and the dropping rate is 50 - 100 drops / minute.

[0080] In one embodiment, the acid solution in step (1) is any one of phosphoric acid solution, malic acid solution, and citric acid solution, and the pH value is 2 - 3, such as 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3 or any range or point value therebetween.

[0081] In one embodiment, during the whole process in step (1), the temperatures of the collagen solution, the collagen-calcium ion composite solution, and the intermediate solution are maintained at 18 - 30 °C, such as 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C or any range or point value therebetween.

[0082] In one of the embodiments, the whole process in step (1) is accompanied by continuous stirring, and the stirring rate is 500 - 1200 r / min, such as 500 r / min, 550 r / min, 600 r / min, 650 r / min, 700 r / min, 750 r / min, 800 r / min, 850 r / min, 900 r / min, 950 r / min, 1000 r / min, 1050 r / min, 1100 r / min, 1150 r / min, 1200 r / min or any range or point value therebetween.

[0083] In one of the embodiments, the temperatures of the intermediate solution and the suspension in step (2) are maintained at 18 - 30 °C, such as 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C or any range or point value therebetween.

[0084] In this article, the pH of the intermediate solution obtained in step (1) is adjusted to 7 - 9.5 by dropping a strong base solution, where the strong base solution is a strong base solution commonly used in the chemical industry to adjust the pH value of a solution, such as sodium hydroxide solution, potassium hydroxide solution, etc.

[0085] The key point of the present invention is that, based on the existing preparation process of mineralized collagen fibrils, it is innovatively discovered that further selection and change of the process conditions can achieve uniform, continuous and sufficient mineralization on both the internal and external surfaces of the collagen fibrils, that is, the mineralization of the collagen fibrils reaches intrafibrillar mineralization. Since the basic unit of natural bone tissue is mineralized collagen fibrils, that is, the secondary structure of bone, the mineralization mode of this secondary structure is intrafibrillar mineralization. Therefore, the collagen fibrils prepared by the technology of the present invention achieve a high degree of biomimicry of the secondary structure of bone tissue. Through electron microscopy observation and comparison, it can be fully proved that the interior of the collagen fibrils binds with uniform, continuous and sufficient nanoapatite crystals, the crystals are in the shape of long flakes, and are oriented along the long axis direction of the collagen fibrils, forming a biomimetic structure.

[0086] It should be emphasized that through exploratory comparative experiments, it is found that only when the temperature condition of its preparation process, the stirring rate and the dropping rate in step (1) are simultaneously satisfied, can the above-mentioned collagen fibrils with highly bone-mimicking mineralization of inner fibrils be successfully prepared. In contrast, although other researchers in this field adopted relatively similar technological preparation processes when preparing collagen fibrils, it can be clearly observed from the electron microscope photos in the currently published articles that they have not achieved uniform, continuous and sufficient mineralization of the interior of the collagen fibrils, and cannot prepare truly biomimetic-structured mineralized collagen fibrils. During the repeated exploratory experiments of the present invention, it was surprisingly found that qualitative changes were caused by further selection and change of process conditions, and in fact, collagen fibrils with highly bone-mimicking mineralization of inner fibrils were prepared.

[0087] In addition, through comparative experiments, it was also found that the specific selection of the acid solution when preparing the collagen acid solution will also significantly affect the internal mineralization of the collagen fibrils.

[0088] During further exploration, the inventors of the present invention also found that the collagen fibrils with highly bone-mimicking mineralization of inner fibrils can further bind to bone formation-promoting drugs without affecting the uniform distribution of the above-mentioned nano-mineral crystals.

[0089] Based on this, in a more preferred embodiment, the bone formation-promoting drug is dissolved in a water solvent to prepare a bone formation-promoting drug solution, and the bone formation-promoting drug solution is added dropwise to the intermediate solution obtained in step (1) as an intermediate solution containing a bone-promoting drug. This intermediate solution containing a bone-promoting drug can directly replace the intermediate solution obtained in step (1) to participate in the subsequent preparation steps;

[0090] Among them, the mass ratio of the bone formation-promoting drug to collagen is 1:(1-5), such as 1:1, 1:2, 1:3, 1:4, 1:5 or any range or point value between them;

[0091] During the whole process, the temperature of the intermediate solution is maintained at 18-30 °C, the dropping rate is 40-120 drops / minute, and continuous stirring is carried out throughout the process, and the stirring rate is 500-1200 r / min.

[0092] In the above-mentioned preferred embodiment, the bone formation-promoting drugs include chemical molecular drugs that have been disclosed in the prior art and have the effect of promoting bone formation, such as bone morphogenetic protein-2 (BMP-2), transforming growth factor-β2 (TGF-β2), vascular endothelial growth factor (VEGF), etc., and can also include extracts of traditional bone-benefiting Chinese medicines, such as extracts of icariin, resveratrol, curcumin, berberine, astragalus, naringin, etc.

[0093] The collagen fibrils with highly bone-mimicking mineralization prepared by the present invention can provide an imitative three-dimensional scaffold microenvironment for osteoblasts, and provide calcium ions and phosphate ions to promote bone regeneration and repair. For example, they can be applied to bio-regenerative repair materials, medical filling materials for dentistry, and regenerative filling materials for dental alveolar defects, etc.

[0094] To better illustrate the above applications, on the other hand, the present invention also provides a method for preparing a moldable preparation based on the above-mentioned collagen fibrils with highly bone-mimicking mineralization, which mainly includes the following steps:

[0095] (Ⅰ) Dissolve the polysaccharide binder in purified water to prepare a binder solution, where the volume of the purified water is 50 - 200 times the mass of the polysaccharide binder;

[0096] (Ⅱ) Add collagen to the binder solution obtained in step (Ⅰ), and perform a swelling treatment with stirring for 10 - 50 h to obtain a collagen mixture;

[0097] (Ⅲ) Add the collagen fibrils with highly bone-mimicking mineralization and nano calcium phosphate salts to the collagen mixture obtained in step (Ⅱ), and continuously stir and disperse for 24 - 72 h to obtain a pre-preparation;

[0098] Among them, the content of collagen in the pre-preparation is 5 - 30 wt% of the dry weight of the pre-preparation, the content of the collagen fibrils with highly bone-mimicking mineralization is 20 - 80 wt% of the dry weight of the pre-preparation, and the content of calcium phosphate salts is 10 - 30 wt% of the dry weight of the pre-preparation;

[0099] (Ⅳ) Subject the pre-preparation obtained in step (Ⅲ) to vacuum treatment to remove air bubbles, and then add it to a mold and perform freeze-drying treatment to prepare a freeze-dried sponge-like moldable preparation.

[0100] In this article, the polysaccharide binder described in step (Ⅰ) is a conventional binder added in bio-regenerative repair materials, and the selection of the polysaccharide binder can refer to the polysaccharide binders commonly used in regenerative repair materials in the art.

[0101] In one implementation, the polysaccharide binder described in step (Ⅰ) includes one or more of sodium alginate, chitosan, hyaluronic acid, modified starch, cellulose, konjac glucomannan, and sodium carboxymethylcellulose.

[0102] In this article, the nano calcium phosphate salts described in step (Ⅲ) are conventional nano-scale inorganic fillers added in bio-regenerative repair materials, and the selection of the nano calcium phosphate salts can refer to the calcium phosphate salts commonly used in regenerative repair materials in the art.

[0103] In one of the embodiments, the nano calcium phosphate salt in step (III) is selected from one or more of nano-hydroxyapatite, nano-α-tricalcium phosphate, nano-β-tricalcium phosphate, nano-tetracalcium phosphate, nano-octacalcium phosphate, and nano-amorphous calcium phosphate.

[0104] In this text, operations such as dissolution, mixing, stirring, drying, etc. all follow the conventional principles in chemical engineering processes, and those skilled in the art can perform specific operations according to common knowledge.

[0105] The present application will be further explained in detail below with reference to the embodiments. However, those skilled in the art should understand that these embodiments are provided for illustrative purposes only and are not intended to limit the present application.

[0106] Example

[0107] The implementation scheme of the present application will be described in detail below in combination with the embodiments. However, those skilled in the art will understand that the following embodiments are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. For those not specified in the embodiments, the operations are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase. The present application should not be construed as being limited to the specific embodiments described.

[0108] 1. Raw materials

[0109] Highly purified collagen (type I) extracted from calfskin.

[0110] CaCl2 and Na2HPO4 powders were purchased from Chengdu Kelong Chemical Co., Ltd.

[0111] Astragalus polysaccharide powder was purchased from Shanghai Aladdin Reagent Co., Ltd.

[0112] 2. Test methods

[0113] The morphology of the sample fiber clusters was observed and analyzed using a (S-4800, Hitachi, Japan) type scanning electron microscope; the sample was fixed on a conductive adhesive for artificial sputtering coating. The main element distribution analysis was carried out using energy dispersive spectroscopy;

[0114] A small amount of finely ground sample was completely dispersed in absolute ethanol to form a suspension. The suspension was absorbed with a capillary and then dropped on a copper grid for drying. Then, the copper grid carrying the sample was placed under a transmission electron microscope (Tecnai G2 F20S-TWIN, FEI, USA) to analyze the microscopic structural relationship between the nanoapatite and collagen fibrils on the sample.

[0115] Example 1

[0116] Example 1 A method for preparing collagen fibrils with highly bone-mimicking mineralization of internal fibrils mainly includes the following steps:

[0117] (1) Dissolve 0.45 g of collagen in 500 ml of citric acid solution to prepare a collagen acid solution with a pH of 2.5. Add 25 ml of a calcium chloride solution with a concentration of 1.0 mol / L dropwise to the collagen acid solution and mix well to obtain a collagen-calcium ion composite solution. Then add 25 ml of a disodium hydrogen phosphate solution with a concentration of 0.6 mol / L dropwise to the collagen-calcium ion composite solution and mix well to obtain an intermediate solution.

[0118] The calcium chloride solution is added dropwise to the collagen solution at a dropping rate of 70 drops per minute.

[0119] The disodium hydrogen phosphate solution is added dropwise to the collagen-calcium ion composite solution at a dropping rate of 70 drops per minute.

[0120] During the whole process of step (1), maintain the temperature of the collagen solution, the collagen-calcium ion composite solution, and the intermediate solution at 25 °C, and continuously stir throughout the process at a stirring rate of 700 r / min.

[0121] (2) Under the condition of continuous stirring, adjust the pH of the intermediate solution obtained in step (1) to 8.5 by dropping sodium hydroxide solution. Precipitation appears in the intermediate solution and it turns into a suspension. Stop stirring and let it stand for 72 h. Separate the precipitated part, wash it, and dry it to prepare collagen fibrils with highly bone-mimicking mineralization of internal fibrils, which are used as test samples.

[0122] In step (2), maintain the temperature of the intermediate solution and the suspension at 25 °C.

[0123] Use the prepared collagen fibrils with highly bone-mimicking mineralization of internal fibrils as samples for testing, and the test results are as Figures 1 - 7 shown.

[0124] Example 2

[0125] Example 2 A method for preparing composite collagen fibrils containing astragalus polysaccharide and with highly bone-mimicking mineralization of internal fibrils mainly includes the following steps:

[0126] (1) Dissolve 0.45 g of collagen in 500 ml of citric acid solution to prepare a collagen acid solution with a pH of 2.5. Add 25 ml of a calcium chloride solution with a concentration of 1.0 mol / L dropwise to the collagen acid solution and mix well to obtain a collagen-calcium ion composite solution. Then add 25 ml of a disodium hydrogen phosphate solution with a concentration of 0.6 mol / L dropwise to the collagen-calcium ion composite solution and mix well to obtain an intermediate solution.

[0127] Add 10 ml of astragalus polysaccharide solution with a concentration of 10 mg / ml dropwise to the intermediate solution and mix well to obtain an intermediate solution containing astragalus polysaccharide;

[0128] The calcium chloride solution is added dropwise to the collagen solution at a dropping rate of 80 drops per minute;

[0129] The disodium hydrogen phosphate solution is added dropwise to the collagen-calcium ion composite solution at a dropping rate of 80 drops per minute;

[0130] The astragalus polysaccharide solution is added dropwise to the intermediate solution at a dropping rate of 80 drops per minute;

[0131] During the whole process of step (1), maintain the temperatures of the collagen solution, collagen-calcium ion composite solution, intermediate solution, and intermediate solution containing astragalus polysaccharide at 25°C, and continuously stir throughout the process at a stirring rate of 800 r / min;

[0132] (2) Under the condition of maintaining stirring, adjust the pH of the intermediate solution containing astragalus polysaccharide obtained in step (1) to 8 by dropping sodium hydroxide solution. A precipitate appears in the intermediate solution and turns into a suspension. Stop stirring and let it stand for 72 h. Separate the precipitated part, wash and dry it to prepare a composite collagen fibril containing astragalus polysaccharide, which is used as a test sample;

[0133] In step (2), maintain the temperatures of the intermediate solution containing astragalus polysaccharide and the suspension at 25°C.

[0134] Use the prepared composite collagen fibril containing astragalus polysaccharide as a sample for testing. The test results are as Figures 1 - 7 shown.

[0135] Comparative Example 1

[0136] Comparative Example 1 is prepared according to the preparation method of Example 1. However, during the whole process of step (1), maintain the temperatures of the collagen solution, collagen-calcium ion composite solution, and intermediate solution at 37°C, which is the conventional active temperature of type I collagen. Finally, prepare collagen fibrils, which are used as test comparison samples.

[0137] Use the prepared collagen fibrils as comparison samples for testing. The test results are as Figure 8 shown.

[0138] Comparative Example 2

[0139] Comparative Example 2 is prepared according to the preparation method of Example 1. However, during the continuous stirring in the whole process of step (1), set the stirring rate to 1500 r / min. Finally, prepare collagen fibrils, which are used as test comparison samples.

[0140] The prepared collagen fibrils were used as comparative specimens for testing, and the test results are as Figure 9 shown.

[0141] Comparative Example 3

[0142] Comparative Example 3 was prepared according to the preparation method of Example 1, but during the whole process of step (1), continuous stirring was carried out, and the stirring rate was set at 300 r / min. Finally, collagen fibrils were prepared and used as test comparative specimens.

[0143] The prepared collagen fibrils were used as comparative specimens for testing, and the test results are as Figure 10 shown.

[0144] Comparative Example 4

[0145] Comparative Example 4 was prepared according to the preparation method of Example 1, but in step (1), the dropping rates of the calcium chloride solution and the disodium hydrogen phosphate solution were 20 drops / minute. Finally, collagen fibrils were prepared and used as test comparative specimens.

[0146] The prepared collagen fibrils were used as comparative specimens for testing, and the test results were similar to those of Comparative Example 1.

[0147] Comparative Example 5

[0148] Comparative Example 5 was prepared according to the preparation method of Example 1, but in step (1), the dropping rates of the calcium chloride solution and the disodium hydrogen phosphate solution were 150 drops / minute. Finally, collagen fibrils were prepared and used as test comparative specimens.

[0149] The prepared collagen fibrils were used as comparative specimens for testing, and the test results were similar to those of Comparative Example 1.

[0150] Application Example 1

[0151] A method for preparing a plasticizable preparation using the composite collagen fibrils containing astragalus polysaccharide prepared in Example 2 as a raw material mainly includes the following steps:

[0152] (I) Dissolve 0.21 g of sodium carboxymethylcellulose in 30 ml of purified water to prepare an adhesive solution;

[0153] (II) Add 0.42 g of collagen to the adhesive solution obtained in step (I), and carry out a swelling treatment with stirring for 50 h to obtain a collagen mixture;

[0154] (III) Add 1.0 g of the composite collagen fibrils containing astragalus polysaccharide prepared in Example 2 and 0.44 g of nano-hydroxyapatite to the collagen mixture obtained in step (II), and continuously stir and disperse for 48 h to obtain a pre-preparation;

[0155] (Ⅳ) The pre - preparation obtained in step (Ⅲ) is vacuum - treated to discharge air bubbles, and then added to a mold and subjected to freeze - drying treatment to prepare a freeze - dried sponge - like moldable preparation.

[0156] As Figure 11 shown, the moldability of the prepared freeze - dried sponge - like moldable preparation was tested.

[0157] The above - mentioned embodiments are the preferred embodiments of the present invention. However, the embodiments of the present invention are not limited by the above - mentioned embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for preparing collagen fibrils with highly bone-mimicking mineralization of inner fibrils, characterized in that It mainly includes the following steps: (1) Dissolve collagen in an acid solution to prepare a collagen acid solution with a collagen concentration of 0.1 - 3.0 mg / mL. Add the calcium salt solution dropwise to the collagen acid solution and mix well to obtain a collagen-calcium ion composite solution. Then add the phosphate solution dropwise to the collagen-calcium ion composite solution and mix well to obtain an intermediate solution; Among them, the acid solution is any one of phosphoric acid solution, malic acid solution, and citric acid solution, and the pH value is 2 - 3; In the collagen-calcium ion composite solution, the content of calcium ions is 0.05 - 0.12 mol calcium ions per gram of collagen; in the intermediate solution, the molar ratio of calcium ions to phosphate ions is (1 - 2):1; The calcium salt solution is added dropwise to the collagen solution at a dropping rate of 40 - 120 drops per minute; The phosphate solution is added dropwise to the collagen-calcium ion composite solution at a dropping rate of 40 - 120 drops per minute; During the whole process of step (1), maintain the temperatures of the collagen solution, the collagen-calcium ion composite solution, and the intermediate solution at 18 - 30 °C respectively, and continuously stir throughout the process with a stirring rate of 500 - 1200 r / min; (2) Under the condition of maintaining stirring, adjust the pH of the intermediate solution obtained in step (1) to 7 - 9.5 by dropwise adding a strong base solution. Precipitation appears in the intermediate solution and it turns into a suspension. Stop stirring and let it stand for at least 36 h. Separate the precipitated part, wash it, and dry it to prepare collagen fibrils with highly bone-mimicking mineralization of endofibrils; In step (2), maintain the temperatures of the intermediate solution and the suspension at 18 - 30 °C respectively.

2. The preparation method according to claim 1, characterized in that: The collagen used is type I collagen extracted from pigskin, cowhide, fish skin, or bovine tendon.

3. The preparation method according to claim 1, characterized in that: The molar concentration of the calcium salt solution in step (1) is 0.05 - 2.5 mol / L, which is prepared by dissolving any one of calcium chloride and calcium nitrate in deionized water.

4. The preparation method according to claim 1, characterized in that: The molar concentration of the phosphate solution in step (1) is 0.05 - 2.5 mol / L, which is prepared by dissolving any one of disodium hydrogen phosphate, sodium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate in deionized water.

5. The preparation method according to claim 1, characterized in that: Dissolve the bone formation promoting drug in a water solvent to prepare a bone formation promoting drug solution. Add the bone formation promoting drug solution dropwise to the intermediate solution obtained in step (1) to obtain an intermediate solution containing the bone promoting drug. This intermediate solution containing the bone promoting drug can directly replace the intermediate solution obtained in step (1) to participate in the subsequent preparation steps; Among them, the mass ratio of the bone formation promoting drug to collagen is 1:(1 - 5); During the whole process, maintain the temperature of the intermediate solution at 18 - 30 °C, the dropping rate at 40 - 120 drops per minute, and continuously stir throughout the process with a stirring rate of 500 - 1200 r / min.

6. The preparation method according to claim 5, characterized in that: The bone formation promoting drug selected includes at least one of bone morphogenetic protein-2, transforming growth factor-β2, and vascular endothelial growth factor.

7. The preparation method according to claim 5, wherein: The bone formation-promoting drug selection includes at least one extract of icariin, resveratrol, curcumin, berberine, astragalus, and naringin.

8. An inner fibril highly osteomimetic mineralized collagen fibril prepared by the preparation method of the inner fibril highly osteomimetic mineralized collagen fibril according to claim 1.

9. The application of the collagen fibril according to claim 8 in the preparation of a biological regeneration repair material and a medical filling material.

10. A method for preparing a plasticizable preparation from the intrafibrillar highly bone-mimicking mineralized collagen fibrils according to claim 8, characterized in that It mainly includes the following steps: (Ⅰ) Dissolve the polysaccharide binder in purified water to prepare a binder solution, where the volume of the purified water is 50 to 200 times the mass of the polysaccharide binder; (Ⅱ) Add collagen to the binder solution obtained in step (Ⅰ), and perform a swelling treatment with stirring for 10 to 50 h to obtain a collagen mixture; (Ⅲ) Add the inner fibril highly osteomimetic mineralized collagen fibril and nano calcium phosphate to the collagen mixture obtained in step (Ⅱ), and continuously stir and disperse for 24 to 72 h to obtain a pre-preparation; Among them, the content of collagen in the pre-preparation is 5 to 30 wt% of the dry weight of the pre-preparation, the content of the inner fibril highly osteomimetic mineralized collagen fibril is 20 to 80 wt% of the dry weight of the pre-preparation, and the content of calcium phosphate is 10 to 30 wt% of the dry weight of the pre-preparation; (Ⅳ) Vacuum-treat the pre-preparation obtained in step (Ⅲ) to remove air bubbles, and then add it to a mold and perform freeze-drying treatment to obtain a freeze-dried sponge-like moldable preparation.

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