A collagen scaffold mimicking bone matrix in situ mineralization and a preparation method thereof

Abstract

The application provides a preparation method of a collagen in-situ mineralized scaffold simulating a bone matrix, S1: extraction of type I collagen fibers; S2: preparation of a biomimetic mineralization solution: carboxymethyl chitosan (CMC) is placed in deionized water, after stirring and dissolving, K2HPO4 is added, CaCl2.2H2O is added drop by drop, and is fully dissolved and mixed to form a carboxymethyl chitosan / amorphous calcium phosphate biomimetic mineralization solution (CMC / ACP); S3: preparation of a biomimetic collagen scaffold composed of a simulated mineralized bone matrix: the acid-recombined collagen extracted in step S1 is fully mixed with hyaluronic acid (HA) and is placed in a dialysis bag, an external PBS buffer is used to gradually increase the pH value, after self-assembly for a period of time, the CMC / ACP biomimetic mineralization solution prepared in step S2 is added in the dialysis bag and is fully mixed, after mineralization for a period of time, the content of the dialysis bag is centrifuged to remove supernatant, and is repeatedly washed with deionized water, finally, after centrifugation, the precipitate is frozen overnight, and the biomimetic scaffold is finally obtained through freeze-drying. The application provides the preparation method of the collagen in-situ mineralized scaffold simulating the bone matrix.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological hospital materials, and particularly relates to a collagen in-situ mineralization scaffold simulating a bone matrix and a preparation method thereof. BACKGROUND

[0002] Bone tissue defects caused by tumors, trauma, inflammation and other reasons are common and difficult problems in clinical practice. Due to the limitations of donor sources, bone immunogenicity, controllability of biodegradation and other aspects, autologous bone and artificial bone powder still cannot achieve the regeneration and repair of the defect area. In recent years, with the continuous deepening and expansion of tissue engineering research, biomimetic scaffolds have gradually become a promising new material, indicating a new way for the treatment of bone tissue-related diseases in clinical practice.

[0003] Natural bone tissue is mainly a composite structure formed by orderly arranging hydroxyapatite nanocrystals on the I collagen fiber network scaffold under the regulation of non-collagen protein and other extracellular matrix. In order to simulate the matrix composition of natural bone tissue in the defect area as much as possible and effectively reduce the immunogenicity caused by implantation, I collagen protein has become the most commonly used component in the construction of engineering scaffolds, showing good biocompatibility and providing certain environmental support for cell metabolic activity. However, such organic scaffolds represented by collagen have poor mechanical properties and limited stress bearing capacity. Therefore, inorganic components such as hydroxyapatite are often mixed into the organic scaffold by physical means to enhance its rigidity. However, this modification method cannot achieve uniform distribution of inorganic particles on the surface of the organic scaffold, and it also lacks the ability to mineralize the interior of the collagen fibers, and the pro-osteogenic effect on cells is relatively weak.

[0004] At present, the PILP (Polymer-Induced Liquid Precursor) system developed by simulating the stabilizing effect of non-collagen protein on the hydroxyapatite precursor in the mineralized tissue based on the mechanism of non-classical crystallization theory has been recognized as an effective means to realize the mineralization of the interior of the fibers, and has been introduced into the research and development of biomimetic mineralized scaffolds in recent years. However, with the deepening of research, the limitations of simply applying PILP have become more and more prominent, that is, it cannot achieve high-quality mineralization of collagen, so there is still a gap between this type of scaffold and the structural unit of real bone tissue.

[0005] Applying the mode of natural bone formation more and more close to the design and construction of scaffolds can more quickly and efficiently achieve bone defect repair and avoid secondary trauma to the host when the implant is removed. The simulation of the organic matrix of bone tissue mainly relies on the application of chemical cross-linking agents such as EDC / NNS and glutaraldehyde to integrate two or more natural / synthetic organic components. However, this simple cross-linking modification still has a large difference from the synergistic assembly mode initiated by the secretion of organic matrix by osteoblasts in the external environment during the process of bone tissue formation.

[0006] In the fully mineralized natural bone matrix, hydroxyapatite as the main inorganic component evenly covers the surface and inside of collagen fibers, and this special distribution mode endows the bone tissue with superior mechanical properties. However, in the existing research, physical means are often used to blend apatite with organic scaffolds to achieve the purpose of strengthening the load-bearing capacity of the latter. However, this improvement technology cannot achieve the uniform distribution of minerals on the surface of the scaffold, and cannot effectively play the original promoting effect of inorganic components on cell osteogenic differentiation. In addition, the most critical point limiting the application of this type of scaffold is that even if the material selected is a nanoscale inorganic crystal, it is also difficult to effectively penetrate into the collagen fiber gap to achieve internal mineralization. However, if the mineral is only deposited on the surface of the fiber, it will increase the brittleness of the scaffold while improving the rigidity of the scaffold, which is easy to cause stress concentration and break when bearing load, and cannot provide sufficient support in the bone defect area. Improving the internal mineralization of collagen fibers is an effective way to improve such defects.

[0007] In the process of batch production of scaffolds, collagen is often obtained from rat tendons, fish skin and bovine Achilles tendons. This collagen extracted by tissue digestion belongs to recombinant collagen, which has some differences with the structure of collagen in bone matrix, such as the loss of glycosaminoglycan in the collagen gap. Therefore, the self-mineralization ability of recombinant collagen scaffold is extremely limited, and even if the PILP system is used to improve the mineral deposition in the fiber gap, the internal mineralization effect is still not ideal, and the macroscopic mechanical behavior of the scaffold still has a gap with the ideal bone replacement material. SUMMARY

[0008] Therefore, the present application aims to provide a collagen in-situ mineralized scaffold simulating bone matrix and a preparation method thereof, which can avoid the potential defects of chemical cross-linking and further simulate the extracellular ring in bone tissue. The in-situ mineralized scaffold prepared by the technology can make up for the shortcomings of the existing scaffold technology to some extent and promote the new bone regeneration in the bone defect area.

[0009] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0010] The technical scheme of the collagen in-situ mineralized scaffold simulating bone matrix of the present application is as follows:

[0011] S1: Extraction of type I collagen fibers;

[0012] S2: Preparation of biomimetic mineralization solution: carboxymethyl chitosan (CMC) is placed in deionized water, and after being fully stirred and dissolved at room temperature, K2HPO4 is added. Then, CaCl2·2H2O is added dropwise, and is fully dissolved and mixed to form a carboxymethyl chitosan / amorphous calcium phosphate biomimetic mineralization solution (CMC / ACP);

[0013] S3: Preparation of the biomimetic collagen scaffold composed of simulated mineralized bone matrix: The acid reconstituted collagen extracted in step 1) is mixed with hyaluronic acid (HA) and then placed in a dialysis bag, and PBS buffer is placed outside the bag to gradually increase the pH value. After self-assembly for 2-3 days, the CMC / ACP biomimetic mineralization solution prepared in step 2) is added to the dialysis bag and mixed thoroughly. After mineralization for 5 days, the contents of the dialysis bag are centrifuged to remove the supernatant, and then repeatedly washed with deionized water. After the final centrifugation, the precipitate is frozen overnight at -20 to -40°C, and then freeze-dried to obtain the biomimetic scaffold.

[0014] In the present application, preferably, the extraction of type I collagen fibers in step S1 comprises the following steps:

[0015] A1: The tail of an 8-10 week old SD rat is soaked in a 75% ethanol solution for 20-30 minutes;

[0016] A2: The tail is removed and the tendon is separated and placed in a Tris-HCl-NaCl buffer solution and soaked at 4°C for 12-20 hours;

[0017] A3: The tendon is removed, washed with deionized water, and then placed in a 0.2-0.4 M acetic acid solution, and stirred at room temperature for 2-4 days to obtain an acetic acid solution of collagen;

[0018] A4: The acetic acid solution of collagen extracted in step S3 is centrifuged at 4°C at 3000 rpm / min for 30 minutes, and the lower tissue precipitate is discarded and stored at 4°C.

[0019] In the present application, preferably, in the CMC / ACP biomimetic mineralization solution of step 2), the CMC is 6-13 mg / mL, the K2HPO4 concentration is 1-6 mM, and the CaCl2·2H2O concentration is 2-10 mM, and the calcium to phosphorus molar ratio is controlled at 1:1 to 2:1.

[0020] In the present application, preferably, in step 3), the HA is 3 mg / mL, and the HA and the acid collagen solution are mixed in a dialysis bag with a molecular weight cut-off of 1000-14000 at a volume ratio of 1:10 (HA-Col), and a PBS buffer with a pH range of 7.2-7.4 is placed outside the bag. As the pH inside the bag gradually increases, the ion exchange balance between the inside and outside is achieved, and the HA-Col is simultaneously assembled.

[0021] In the present application, preferably, in step 3), the CMC / ACP solution with a pH of 7 is added to the dialysis bag at a volume ratio of 1:2 to 1:3 with respect to the HA-Col, thereby starting the mineralization process of the collagen.

[0022] The patent utilizes the driving force provided by pH buffering to realize the synergistic assembly of HA and acidic collagen solution, which is close to the actual process of bone matrix.

[0023] The patent optimizes the aggregation ability of the recombinant collagen to the cations in the surrounding environment by providing a large number of recognition sites of HA, further enhances the mineralization quality inside the fiber, and avoids the fracture of the scaffold due to stress concentration;

[0024] The biomimetic collagen scaffold prepared by the patent can further simulate the real mineralized matrix of bone tissue, establish a stable repair environment for the bone defect area, and play the functions of bone conduction and bone regeneration to promote new bone formation. The key point of the patent is the specific preparation method of the collagen in-situ mineralization scaffold, and the driving mode of the synergistic assembly of HA and collagen has not been reported in other researches.

[0025] Compared with the prior art, the collagen in-situ mineralization scaffold simulating bone matrix and the preparation method thereof have the following beneficial effects:

[0026] The patent is different from the traditional scaffold, which integrates other active ingredients after the collagen assembly is completed by using a crosslinking agent. The synergistic assembly of HA and collagen is realized by regulating pH, which simplifies the preparation process of the scaffold and avoids the batch difference caused by different crosslinking degrees. In addition, the intervention of HA can optimize the limited self-mineralization of the recombinant collagen, improve the mineralization quality, further improve the bone conduction and bone regeneration ability of the scaffold, and expand the application range of the scaffold. BRIEF DESCRIPTION OF DRAWINGS

[0027] The accompanying drawings, which form a part of the present application, are used to provide a further understanding of the present application, and the schematic embodiments of the present application and the description thereof are used to explain the present application, and do not constitute an improper limitation on the present application. In the drawings:

[0028] Figure 1 FTIR results of the HA molecule intervened in the initial and final stages of collagen assembly in embodiment 1 of the present application;

[0029] Figure 2 HA-collagen synergistic assembly intuitive diagram and TEM image of embodiment 1 of the present application;

[0030] Figure 3 TEM image of the influence of HA on collagen mineralization under CMC / ACP mineralization induction in embodiment 1 of the present application (Col group is a-c, HA-Col group is d-f, and the mineralization time points are 1d, 3d and 5d in turn);

[0031] Figure 4The images shown are macroscopic and microscopic SEM images of the stent in Comparative Example 1. (a, b are Col group, c, d are M-Col group, e, f are HA-Col group, g, h are M-HA-Col group; magnification is 500× in the left column and 5000× in the right column.)

[0032] Figure 5 The energy storage modulus and loss modulus of the biological scaffold (A is G', B is G”);

[0033] Figure 6 A) shows the adhesion of 3T3-E1 cells to the scaffold surface, and B) shows the cell proliferation under the CCK-8 assay.

[0034] Figure 7 Micro-CT images (A) and BV / TV values ​​(B) at different time points for a rat model with an 8mm ulnar diameter skull defect. Detailed Implementation

[0035] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0036] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0037] Example 1

[0038] The bone matrix simulated collagen in-situ mineralization scaffold of the present invention is prepared by the following steps:

[0039] (1) Extraction of type I collagen

[0040] Eight-week-old SD rat tails were immersed in 75% ethanol solution for 20 minutes. Subsequently, the tails were removed and the skin was exfoliated to detach the tail tendon. To avoid collagen denaturation, this procedure was performed under deionized water immersion conditions. The obtained tail tendon was then placed in Tris-HCl-NaCl buffer (0.05 mol / L, pH 7.4) and immersed at 4°C for 12 hours. After rinsing the tail tendon with deionized water to remove residual buffer, it was placed in 0.3M acetic acid solution and stirred continuously at room temperature for 3 days to obtain a collagen acetate solution. This solution was centrifuged for 30 minutes (4°C, 3000 rpm / min), and the supernatant was collected and stored at 4°C.

[0041] (2) Preparation of biomimetic mineralizing solution (CMC / ACP)

[0042] 30 mL of deionized water was added 400 mg of CMC (viscosity: 45, Mw: 13700, isoelectric point 3.6), and the mixture was stirred at room temperature until the powder was completely dissolved to obtain a CMC solution. Then, 41.76 mg of K2HPO4 was added to the CMC solution. After 58.8 mg of CaCl2-2H2O was dissolved in 10 mL of deionized water, the solution was added dropwise to the continuously stirred CMC-K2HPO4 solution to finally obtain a CMC / ACP biomimetic mineralization solution.

[0043] (3) Preparation of collagen in situ mineralization scaffold (M-HA-Col)

[0044] The dialysis bag was soaked in 75% ethanol solution for 15 minutes, washed with deionized water, and one end was tightly closed. 3 mg of hyaluronic acid (80-2000 kDa) was completely dissolved in 1 mL of deionized water, and 300 μL was added to 3 mL of collagen acetic acid solution and mixed. Then, the HA-Col mixture was placed in the dialysis bag and closed, and 15 mL of PBS buffer (pH 7.2-7.4) was added. After 2 days of synchronous assembly of the HA-Col solution, the PBS buffer was replaced daily. After 2 days, 6 mL of CMC / ACP solution prepared in (2) was added to the dialysis bag and mixed well, and then closed. The mineralization solution and the external buffer were replaced daily until the collagen was completely mineralized in 5 days. The mineralized HA-Col was taken out of the dialysis bag and centrifuged at 3000 rpm / min for 15 min at room temperature. The supernatant was discarded, and the precipitate was washed repeatedly with deionized water and centrifuged three times. The precipitate was placed in an environment of -20 to -40°C for 24 hours, and then freeze-dried to obtain a biomimetic collagen scaffold.

[0045] From Figure 1 It can be seen that compared with pure collagen, the cooperative assembly of HA and collagen did not cause a significant red shift of the characteristic peak band of collagen protein, including amide I, II and III bands, which proved that even if HA molecules intervene during the assembly of collagen, the latter can still maintain its unique conformation. However, compared with the simple mixing of HA with collagen, the cooperative assembly has a significant enhancement of the C=O stretching vibration frequency (wavenumber 1645 cm -1 ) of amide I band, which indicates that HA plays an important role in promoting the formation of helical conformation during the assembly of collagen. In addition, the C-OH stretching vibration peak of HA-Col at 1032 cm -1 and 1074 cm -1 is also weakened, which indicates that the hydroxyl group inside the HA molecule can form an ester bond with lysine or proline. As Figure 2 Compared with pure collagen (a), the HA-cooperative intervention (b) can effectively accelerate the assembly process of collagen and significantly increase the diameter of collagen fibers. As Figure 3, the mineralization degree of the collagen modified by HA (M-HA-Col) was obviously optimized, and the crystal axis orientation was parallel to the collagen fiber, which was similar to the collagen mineralization in the real bone matrix.

[0046] Comparative Example 1

[0047] The comparative example provides a preparation method of non-mineralized collagen scaffold (Col):

[0048] (1) The extraction step of type I collagen is the same as step (1) in Example 1.

[0049] (2) Preparation of non-mineralized collagen scaffold: 3 mL of the collagen acetic acid solution extracted in (1) was added to a dialysis bag, and PBS buffer was placed outside. After 2 days of collagen assembly, it was taken out from the dialysis bag and washed and centrifuged. The obtained precipitate was placed in an environment of -20 to -40℃ for 24 hours, and after freeze-drying, a non-mineralized collagen scaffold was obtained.

[0050] From Figure 4 , the non-mineralized HA-Col scaffold (e, f) also showed a porous morphology and the fiber bundle was obviously thicker, but the distribution degree of micrometer-sized pores was relatively lower than that of the Col group (a, b). However, after CMC / ACP mineralization, the M-HA-Col group (g, h) showed uniform pore distribution, and the number of micropores was obviously improved compared with before mineralization, and the fiber existed periodically rough swelling, indicating that the mineralization in the scaffold was good. The number of micropores in the M-Col group (c, d) decreased, and the collagen surface was covered with uneven inorganic sheet.

[0051] Comparative Example 2

[0052] The comparative example provides a preparation method of collagen biomimetic mineralized scaffold (M-Col):

[0053] (1) The extraction step of type I collagen is the same as step (1) in Example 1.

[0054] (2) Preparation of biomimetic mineralized pure collagen scaffold: 3 mL of the collagen acetic acid solution extracted in (1) was added to a dialysis bag, and PBS buffer was placed outside. After 2 days of collagen assembly, 6 mL of CMC / ACP solution prepared in step (2) of Example 1 was mixed with the pure collagen to start the collagen mineralization. After 5 days, the mineralized collagen was taken out and washed by centrifugation. The obtained precipitate was placed in an environment of -20 to -40℃ for 24 hours, and after freeze-drying, a mineralized pure collagen scaffold was obtained.

[0055] The M-HA-Col, Col and M-Col scaffolds obtained by Example 1, Comparative Example 1 and Comparative Example 2 were respectively evaluated for basic performance and bone tissue regeneration performance.

[0056] 1. M-HA-Col, Col and M-Col characterization

[0057] 1) Infrared Fourier spectroscopy (FTIR): To observe the combination form of HA and collagen under the condition of synchronous assembly, and to confirm the integration of the two in the assembly process.

[0058] Different samples were processed into powder texture. The wave number range of the infrared spectrometer was set to 400-4000 cm -1 , and the resolution was 16 cm -1 . According to the peak shift degree of the FTIR spectrum, it was determined whether there was a combination between HA and collagen.

[0059] 2) Transmission electron microscopy (TEM): To observe the effect of HA on the mineralization of collagen and to evaluate the incorporation of inorganic particles inside the collagen fibers.

[0060] The samples were dropped onto a copper mesh covered with a 400-mesh carbon support film, and TEM was used to observe the mineralization of MHC and MPC at different time points (1, 3, 5 days).

[0061] 3) Scanning electron microscopy (SEM): To observe the micro-morphology of the surface of each scaffold material.

[0062] Different scaffold materials were subjected to gold sputtering treatment, and SEM was used to observe their micro-morphology and compare the morphology differences between groups.

[0063] 4) Thermogravimetric analysis (TGA): To evaluate whether higher quality collagen mineralization can be achieved in the presence of HA and to measure the proportion of inorganic components inside the fibers.

[0064] 8-10 mg of M-HA-Col and M-Col material was weighed and calcined using an alumina crucible, with a temperature of 25-700°C under air flow, with a temperature rise of 10°C / min, and the total amount of inorganic residue was weighed to evaluate the mineralization effect of the scaffold.

[0065] As Figure 5 , the storage modulus and loss modulus of the scaffold were analyzed. For G' and G", the M-HA-Col group was the highest, indicating that it had strong deformation storage capacity and could quickly recover to its original shape after stress removal (reversible), and played a good rigid role. Correspondingly, the M-HA-Col group also had higher energy loss due to deformation (irreversible), and the viscous behavior was not significant. The Col group and the HA-Col group without mineralization showed obvious viscous behavior. The M-Col group was basically between the three.

[0066] 2. Comparison of mechanical properties of scaffold materials

[0067] 1) Dynamic mechanical analysis (DMA): HA-Col, M-HA-Col, Col and M-Col were prepared into samples with a size of 25x10x3mm. The rigidity and viscoelasticity of the scaffold were evaluated using a DMA analyzer in multi-frequency mode (frequency 1-20Hz, amplitude 20μm).

[0068] 2) Degradation rate and swelling rate: PBS buffer and DTT solution were used to evaluate the degradation rate and structural stability of different scaffold materials at different time periods.

[0069] Depend on Figure 6 As can be seen, the M-HA-Col scaffold exhibits good biocompatibility, and the cell proliferation rate is not significantly affected (B). Furthermore, compared to pure collagen scaffolds, the M-HA-Col scaffold provides better spatial support for cells, which is beneficial for their adhesion (A).

[0070] like Figure 7 In the blank control group, no significant wound healing was observed in the early stages, and only a very small amount of new bone formed after 12 weeks. In the Col group, new bone deposition led to a gradual reduction in the defect area, but the central area still lacked new tissue. In the M-HA-Col group, mineralized nodules were observed scattered in the center of the defect in the early healing stage, and new bone integration was good at the edges. By 12 weeks, the defect area was basically covered by regenerated tissue. Furthermore, based on the BV / TV results, the M-HA-Col scaffold induced more new bone tissue compared to the Col group.

[0071] 3. Cellular experiments (in vitro assessment of osteogenic properties)

[0072] 1) Mouse pre-osteoblasts (3T3-E1) were seeded onto a scaffold material to determine cell proliferation levels.

[0073] After the scaffolds were sterilized with Go60, 3T3-E1 cells were seeded onto different scaffolds, and the proliferation of cells at different time points (1, 3, 5, and 7 days) was assessed using the CCK-8 probe.

[0074] 2) Mouse pre-osteoblasts (3T3-E1) were seeded onto a scaffold material to observe the distribution of live and dead cells.

[0075] After 3T3-E1 cells were seeded onto the scaffold, the scaffold was stained with AO / EB at different time points (1, 3, 5, and 7 days), and the cell morphology and distribution were observed under a laser confocal microscope.

[0076] 3) Mouse bone marrow mesenchymal stem cells (BMSCs) were seeded onto a scaffold material to determine osteogenic differentiation levels.

[0077] BMSCs were seeded on scaffolds and collected at different time points (1, 3, 5, 7 days). RT-PCR was used to detect the expression of related osteogenic factors ALP, RUNX2, OCN, RANKL and OPG at the gene level.

[0078] 4. Animal experiments (in vivo evaluation of the pro-osteogenic performance)

[0079] 1) Establishment of a critical bone defect model in the skull of SD rats

[0080] Male rats aged 8 weeks and weighing 250-320 g were anesthetized, the head was prepared for skin and thoroughly disinfected, then incised and a through defect with a diameter of 8 mm was drilled using a trephine.

[0081] 2) Repair of skull defects in rats using different scaffold materials

[0082] Different biological scaffolds were placed in the defect area and the skull wound was sutured layer by layer.

[0083] 3) Evaluation of new bone formation in the bone defect area using micro-computed tomography (Micro-CT)

[0084] The rats were sacrificed at different time points (4 and 8 weeks), the calvaria was removed and fixed, and Micro-CT was used to evaluate the new bone formation in the defect area.

[0085] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a collagen scaffold that mimics in situ mineralization of bone matrix, characterized by: It comprises the following steps: S1: extraction of collagen type I fibers; S2: preparation of a biomimetic mineralization solution: carboxymethyl chitosan CMC is placed in deionized water, stirred and dissolved, then K2HPO4 is added, CaCl2·2H2O is added dropwise, and the mixture is fully dissolved to form a carboxymethyl chitosan / amorphous calcium phosphate CMC / ACP biomimetic mineralization solution; S3: preparation of a biomimetic collagen scaffold simulating the composition of a mineralized bone matrix: the acid-reconstituted collagen extracted in step S1 is fully mixed with hyaluronic acid HA, then placed in a dialysis bag, and PBS buffer is placed outside to gradually increase the pH value; after self-assembly for a period of time, the CMC / ACP biomimetic mineralization solution prepared in step S2 is added to the dialysis bag and fully mixed, and mineralized for a period of time; then the contents of the dialysis bag are centrifuged to remove the supernatant, and repeatedly washed with deionized water; finally, after centrifugation, the precipitate is frozen overnight, freeze-dried, and finally a biomimetic scaffold is obtained; In step S2, the carboxymethyl chitosan CMC is placed in deionized water, fully stirred and dissolved at room temperature, and then K2HPO4 is added; In step S3, the PBS buffer is placed outside to gradually increase the pH value, and the self-assembly time is 2-3 days.

2. A method of preparing a collagen-based bone matrix mimicking scaffold according to claim 1, characterized in that: The extraction of collagen type I fibers in step S1 comprises the following steps: A1: immerse the rat tail in an ethanol solution for a period of time; A2: remove the tail and separate the tendon, and immerse it in a Tris-HCl-NaCl buffer at low temperature; A3: remove the tendon, wash it with deionized water, and then immerse it in an acetic acid solution at room temperature with continuous stirring to obtain an acetic acid solution of collagen; A4: centrifuge the collagen acetic acid solution extracted in step A3, discard the lower tissue precipitate, and store it.

3. A method of preparing a collagen-based bone matrix mimicking scaffold according to claim 2, characterized in that: The rat tail in step A1 is a tail of an 8-10 week old SD rat; Step A1 is an ethanol solution with a mass fraction of 75%, and the immersion time is 20-30 minutes; The low-temperature immersion in step A2 is 12-20 hours in a 4°C environment; Step A3 is to remove the tendon, wash it with deionized water, and then immerse it in a 0.2-0.4 M acetic acid solution at room temperature with continuous stirring for 2-4 days to obtain an acetic acid solution of collagen; Step A4 is to centrifuge the collagen acetic acid solution extracted in step A3 at 4°C and 3000 rpm / min for 30 minutes, discard the lower tissue precipitate, and store it at 4°C.

4. The method for preparing a collagen in-situ mineralization scaffold simulating bone matrix according to claim 1, characterized in that: In step S3, the CMC / ACP biomimetic mineralization solution prepared in step S2 is added to the dialysis bag and fully mixed, and after mineralization for 5 days, the contents of the dialysis bag are centrifuged to remove the supernatant, and repeatedly washed with deionized water; finally, after centrifugation, the precipitate is frozen overnight at -20 to -40°C, and finally a biomimetic scaffold is obtained by freeze-drying.

5. The method for preparing a collagen in-situ mineralization scaffold simulating bone matrix according to claim 1, characterized in that: In the CMC / ACP biomimetic mineralization solution of step S2, the CMC is 6-13 mg / mL, the K2HPO4 concentration is 1-6 mM, and the CaCl2·2H2O concentration is 2-10 mM, with a calcium-phosphorus molar ratio of 1:1 to 2:

1.

6. The method for preparing a collagen in-situ mineralization scaffold simulating bone matrix according to claim 1, characterized in that: The HA in the step S3 is 3 mg / mL, the HA and the acidic collagen solution are mixed in a dialysis bag with a molecular weight cut-off of 1000-14000 at a volume ratio of 1:10 to obtain HA-Col, and a PBS buffer with a buffer range of 7.2-7.4 is used, and with the gradual increase of the pH in the bag, the ion exchange balance between the inside and the outside, and the synchronous assembly of the HA-Col.

7. The method for preparing a collagen in-situ mineralization scaffold simulating bone matrix according to claim 1, characterized in that: In the step S3, the CMC / ACP solution with a pH of 7 is added in the dialysis bag at a volume ratio of 1:2-1:3 with the HA-Col, so as to start the mineralization process of the collagen.

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