A method for preparing a scaffold that promotes collagen fiber assembly and internal and external mineralization

Abstract

The application provides a scaffold preparation method for promoting collagen fiber assembly and internal and external mineralization, and comprises the following steps: A) adding glycine and potassium chloride into a complex solution formed by non-collagen protein analogs chelating calcium ions to prepare an assembly solution; wherein the non-collagen protein analogs are one or both of polycarboxylic acid substances and glucuronic acid; B) adding a collagen protein solution into the assembly solution obtained in step A) to obtain a mixed solution, the pH of the mixed solution is adjusted to 7.0±0.2; the mixed solution is placed in a 37±0.5 DEG C incubator, and is incubated for 12-24 hours, so that the collagen protein is assembled, and collagen fibers are obtained; C) the collagen fibers prepared in step B) are placed in a collagen fiber mineralization solution to be mineralized, mineralized collagen fibers are obtained, and the mineralized collagen fibers are washed, frozen and dried to obtain a mineralized collagen fiber scaffold; the application simulates the bone tissue generation process in a safer and more bionic manner, so that the collagen fiber scaffold is more efficiently assembled and mineralized.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering scaffold technology, and in particular relates to a method for preparing a scaffold that promotes collagen fiber assembly and internal and external mineralization. Background Technology

[0002] Natural bone tissue is primarily composed of ordered hydroxyapatite carbonate crystals, type I collagen, and water. The hydroxyapatite carbonate crystals are arranged in an orderly fashion within and around the collagen fibers, forming regular internal and external mineralization. The structure of natural bone tissue is complex, containing nanocrystals and collagen fibers with hierarchical structural gradients spanning many length scales. This structure endows natural bone tissue with both good strength and toughness to meet the functional requirements of organisms.

[0003] The superior structure and properties of bone tissue enable it to play a vital role in human life activities. Trauma, infection, tumors, and other factors can damage bone tissue, leading to bone defects. The repair and treatment of bone defects is crucial for helping patients restore physiological function and mental well-being, and has always been a focus of close attention in the medical field. Autologous bone grafting, using the patient's own bone tissue, offers the best biocompatibility and lowest rejection rate, effectively promoting bone healing and is considered the "gold standard" for bone repair. However, autologous bone grafting can cause secondary trauma to the body and is limited by the amount of donor bone. Tissue-engineered bone graft materials have attracted widespread attention due to their good biocompatibility, biosafety, and biofunctionality, as well as their biodegradability and ability to induce osteogenic growth. Bone implant materials need to possess comprehensive and excellent mechanical properties, striving for a balance of strength, stiffness, and toughness with natural bone to withstand the load conditions in the body and effectively support the repair process of damaged bone. To achieve this goal, the design of biomimetic bone implant materials aims to mimic the biological structure of natural bone.

[0004] Many studies have combined collagen and nano-hydroxyapatite to create composite scaffolds for bone defect repair. However, these simple hybrid scaffolds only mimic the components of bone tissue, without mimicking its structure and properties. Currently, numerous technologies mimic the ordered spatial hierarchy of collagen fibers and hydroxyapatite in natural bone. In vitro, without cell regulation, polyelectrolytes are used to simulate the function of osteogenic proteins, precisely controlling the growth of hydroxyapatite crystals inside and outside collagen fibers. This results in scaffold materials that are highly biomimetic in composition, microstructure, and physiological function. A key aspect of these technologies is promoting the interaction between amorphous calcium phosphate precursors and collagen. This is generally achieved by activating the collagen surface and promoting collagen cross-linking to further advance mineralization. However, current activating agents are mainly glutaraldehyde and EDC / NHS, both toxic substances with poor biocompatibility and significant differences from the in vivo collagen activation process, posing potential biosafety risks. Therefore, finding a faster, safer, and more mineralized collagen biomimetic assembly and mineralization strategy is an urgent scientific problem to be solved. Summary of the Invention

[0005] In view of this, the present invention aims to propose a scaffold preparation method that promotes collagen fiber assembly and internal and external mineralization, so as to overcome the shortcomings or defects in the prior art, simulate the bone tissue formation process in a more biomimetic way, and thus achieve more efficient assembly and mineralization of collagen fiber scaffolds.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0007] A method for preparing a scaffold that promotes collagen fiber assembly and internal and external mineralization includes the following steps:

[0008] A) Glycine and potassium chloride are added to a solution of a complex formed by the chelation of calcium ions by a non-collagen analog to prepare an assembly solution; wherein the non-collagen analog is one or two of polycarboxylic acid substances and glucuronic acid.

[0009] B) Add collagen solution to the assembly solution obtained in step A) to obtain a mixture. Adjust the pH of the mixture to 7.0±0.2. Place the mixture in an incubator at 37±0.5℃ and incubate for 12~24h until collagen is assembled to obtain collagen fibers.

[0010] C) The collagen fibers prepared in step B) are placed in a collagen fiber mineralization solution for mineralization to obtain mineralized collagen fibers. The mineralized collagen fibers are then washed, frozen, and dried to obtain a mineralized collagen fiber scaffold.

[0011] Preferably, in step A), the preparation of the complex solution formed by the non-collagen analog chelating calcium ions includes the following steps: dissolving calcium chloride in ultrapure water at a concentration of 0.05~0.2M and stirring magnetically to dissolve it; adding a non-collagen analog and dissolving it, wherein the concentration of the non-collagen analog is 0.1~0.3g / L; stirring for 10~30min to mix evenly to obtain the complex solution formed by the non-collagen analog chelating calcium ions.

[0012] Preferably, in step A), the final concentration of glycine in the assembly solution is 30-60 mM; and the final concentration of potassium chloride is 100-300 mM.

[0013] Preferably, in step B), a collagen solution with a concentration of 8-11 mg / mL is added to the assembly solution obtained in step A) at a volume ratio of 5:1 to 3:1 to obtain a mixed solution, and the pH of the mixed solution is adjusted to 7.0±0.2 with 0.5-3M NaOH solution.

[0014] Preferably, in step C), the collagen fiber mineralization solution is prepared by dissolving calcium chloride in deionized water to form a calcium ion solution with a concentration of 2-4 mM, then dissolving a non-collagen analog in the calcium ion solution to obtain solution A, wherein the concentration of the non-collagen analog in solution A is 100-300 μg / mL; dissolving disodium hydrogen phosphate in a 200-400 mM NaCl aqueous solution to form a hydrogen phosphate ion solution with a concentration of 10-20 mM to obtain solution B; mixing solution A and solution B at a volume ratio of 1:1 to 1:2 and stirring until homogeneous to obtain the collagen fiber mineralization solution.

[0015] Preferably, in step C), the volume ratio of collagen fiber prepared in step B) to collagen fiber mineralization solution is 1:8 to 1:15. During the mineralization process, the collagen fiber mineralization solution is replaced every 1 to 3 days, and mineralization is carried out for 5 to 7 days to obtain mineralized collagen fiber.

[0016] Preferably, in step C), the obtained mineralized collagen fibers are washed with ultrapure water 3-5 times and frozen at -80°C for 4-12 hours. Preferably, in step C), the drying is freeze-drying for 1-3 days at a temperature of -50°C.

[0017] Preferably, in step B), the collagen is rat tail tendon collagen.

[0018] Collagen can be commercially available or prepared using the following methods:

[0019] a) After disinfecting the tails of fresh 8-week-old SD rats, the tail skin was removed and the tail tendons were extracted. The entire process was accompanied by soaking in sterile deionized water to prevent collagen degeneration in the tail tendons.

[0020] b) The extracted rat tail tendon was placed in Tris-HCl-NaCl solution (5M, pH=7.4) and soaked at 4°C for 12 hours.

[0021] c) After rinsing with deionized water, place the rat tail tendon in an acetic acid solution (0.2~0.4M). Stir continuously at room temperature for 2~3 days until the rat tail tendon is completely dissolved to obtain a collagen solution.

[0022] d) Place the collagen solution in a low-temperature high-speed centrifuge and centrifuge at 3000~4000 rpm / min for 10~20 min. After centrifugation, collect the supernatant in a sterile centrifuge tube and store it at 4℃ for later use.

[0023] Preferably, in step A), the polycarboxylic acid substance is one or more of polyaspartic acid, polyglutamic acid, and polyacrylic acid.

[0024] Non-collagen analogs chelate calcium ions to form complexes that participate in the assembly of collagen fibers. During assembly, these complexes are embedded in the interstices of collagen fibers, helping them to mineralize more quickly and to a greater extent. When the collagen fibers are then placed in a mineralization solution, the calcium and phosphorus ions in the solution form amorphous precursors (ACPs) with liquid flow properties, which enter the interior of the collagen fibers, gradually lose moisture, and begin to solidify and crystallize, forming thermodynamically more stable hydroxyapatite, thus completing the internal and external mineralization of the collagen fibers.

[0025] Compared with existing technologies, the scaffold preparation method for promoting collagen fiber assembly and internal and external mineralization described in this invention has the following advantages:

[0026] (1) Compared with the prior art, the collagen assembly environment in this invention is closer to the environment containing calcium in the human body, and the collagen fiber assembly effect is better.

[0027] (2) The complex formed by chelating calcium ions with non-collagen analogs used in this invention to promote the assembly and mineralization of collagen fibers has good biocompatibility and is safer and has no toxic side effects compared with toxic cross-linking agents such as glutaraldehyde.

[0028] (3) This invention simulates the bone tissue formation process in a safer and more biomimetic way, thereby achieving more efficient assembly and mineralization of collagen fiber scaffolds. The collagen fibers synthesized by this novel scaffold preparation method that promotes collagen fiber assembly and internal and external mineralization have larger diameters and higher mechanical properties. It has significant advantages in in vivo experiments (animal experiments) and in vitro experiments (osteogenic differentiation), providing new possibilities for the repair of bone tissue defects in future clinical practice and has a broader application prospect. Attached Figure Description

[0029] Figure 1The image shows the transmission electron microscopy (TEM) results of the PAsp-Ca complex prepared in Example 1; where the scale bar of A is 2 μm and the scale bar of B is 200 nm.

[0030] Figure 2 Characterization images of collagen fibers prepared in Example 1 and Comparative Example 1 are shown below; where A, B, and C are transmission electron microscopy (TEM) images, atomic force microscopy (AFM) surface morphology images, and AFM Young's modulus images of the collagen fibers obtained in Comparative Example 1, respectively; and D, E, and F are TEM images, AFM surface morphology images, and AFM Young's modulus images of the collagen fibers obtained in Example 1, respectively.

[0031] Figure 3 The images show transmission electron microscopy (TEM) results of the mineralized collagen fibers obtained in Example 1 and Comparative Example 1, respectively; where A and C are TEM results of the mineralized collagen fibers obtained in Comparative Example 1 after 3d and 5d of mineralization, respectively; and B and D are TEM results of the mineralized collagen fibers obtained in Example 1 after 3d and 5d of mineralization, respectively.

[0032] Figure 4 The images show the characterization of the mineralized collagen fiber scaffold prepared in Example 1; where A is the field emission scanning electron microscope result of the mineralized collagen fiber scaffold prepared in Example 1; B is the X-ray diffraction result of the mineralized collagen fiber scaffold prepared in Example 1 (PAsp-Ca co-assembled collagen mineralized for 7 days) and the mandibular bone block of SD rat; C is the thermogravimetric analysis result of the mineralized collagen fiber scaffold prepared in Example 1 (PAsp-Ca co-assembled collagen mineralized for 7 days) and the mandibular bone block of SD rat.

[0033] Figure 5 The image shows the osteogenic differentiation results of bone marrow mesenchymal stem cells from SD rats stained with alkaline phosphatase. A, B, and C represent the results of osteogenic differentiation induced for 7 days in the blank cell culture control group, the collagen fiber scaffold of Comparative Example 2, and the mineralized collagen fiber scaffold of Example 1, respectively. D, E, and F represent the results of osteogenic differentiation induced for 14 days in the blank cell culture control group, the collagen fiber scaffold of Comparative Example 2, and the mineralized collagen fiber scaffold of Example 1, respectively.

[0034] Figure 6 The image shows the results of osteogenic differentiation of bone marrow mesenchymal stem cells from SD rats stained with Alizarin Red after 14 days; where A, B, and C represent the results of osteogenic differentiation induced for 14 days by blank cell culture control group, collagen fiber scaffold of Comparative Example 2, and mineralized collagen fiber scaffold of Example 1, respectively.

[0035] Figure 7 To establish a mandibular bone defect model in SD rats.

[0036] Figure 8The images show the three-dimensional reconstruction comparison of Micro-CT scan results of bone defects in the mandible of SD rats at 4 weeks of age; where A, B, and C represent the results of the blank control group (without repair material), the collagen fiber scaffold group of Comparative Example 2, and the mineralized collagen fiber scaffold group of Example 1, respectively. Detailed Implementation

[0037] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0038] The present invention will be described in detail below with reference to the embodiments.

[0039] In the embodiments of the present invention, the concentration unit M is mol / L; mM is 0.001 mol / L.

[0040] Example 1

[0041] A method for preparing a scaffold that promotes collagen fiber assembly and internal and external mineralization includes the following steps:

[0042] (1) Preparation of a solution of a complex formed by the chelation of calcium ions by non-collagen analogues

[0043] Poly-L-aspartic acid (PAsp, molecular weight Mw: 5 kDa) was selected as a non-collagen analog. Calcium chloride dihydrate was dissolved in ultrapure water at a concentration of 0.13 M. After magnetic stirring, PAsp was added at a concentration of 0.1 g / L and dissolved. The mixture was stirred for 0.5 h to form a PAsp-Ca complex solution, which is a complex solution formed by the chelation of calcium ions by the non-collagen analog.

[0044] (2) Extraction and self-assembly of type I collagen

[0045] After disinfecting the tails of fresh 8-week-old SD rats, the tail skin was removed and the tail tendons were extracted. The entire procedure was performed with immersion in sterile deionized water to prevent collagen denaturation within the tendons. The extracted tail tendons were placed in a Tris-HCl-NaCl solution (5M, pH=7.4) and soaked at 4°C for 12 hours. After rinsing with deionized water, the tail tendons were placed in an acetic acid solution (0.2M). The mixture was stirred continuously at room temperature for 3 days until the tail tendons were completely dissolved, yielding a collagen solution. The collagen solution was centrifuged at 3000 rpm for 10 minutes using a low-temperature high-speed centrifuge. The supernatant was collected in sterile centrifuge tubes and stored at 4°C for later use.

[0046] Glycine and potassium chloride were added to the PAsp-Ca complex solution prepared in step (1) to make the final concentration of glycine 50mM and the final concentration of potassium chloride 200mM, thus preparing the assembly solution.

[0047] A 10 mg / mL collagen solution was added to the assembly solution at a volume ratio of 4:1 to obtain a mixture, and the pH of the mixture was adjusted to 7.0 with 1M NaOH solution. The mixture was placed in an incubator at 37°C and incubated for 24 hours to allow collagen to assemble and obtain collagen fibers.

[0048] (3) Mineralization of collagen fibers and preparation of scaffolds

[0049] Calcium chloride dihydrate was dissolved in deionized water to form a 3.34 mM calcium ion solution. PAsp was then dissolved in the calcium ion solution, resulting in a PAsp concentration of 240 μg / mL, thus obtaining solution A. Disodium hydrogen phosphate was dissolved in a 300 mM sodium chloride aqueous solution to form a 19 mM hydrogen phosphate ion solution, thus obtaining solution B. Solutions A and B were mixed at a volume ratio of 1:1 and stirred thoroughly to obtain a collagen fiber mineralization solution.

[0050] The collagen fibers obtained in step (2) were placed in a freshly prepared collagen fiber mineralization solution at a volume ratio of 1:10 and placed in a 37°C incubator. The collagen fiber mineralization solution was replaced every 2 days and the mineralization was carried out for 7 days to obtain mineralized collagen fibers.

[0051] The mineralized collagen fibers were washed three times with ultrapure water, frozen at -80℃ for 12 hours, and then placed in a freeze dryer and freeze-dried at -50℃ for 2 days to obtain a mineralized collagen fiber scaffold.

[0052] (4) Sterilization of mineralized collagen fiber scaffolds

[0053] The mineralized collagen fiber scaffold prepared in step (3) was placed under a 254nm ultraviolet lamp and sterilized by irradiating both sides for 4 hours.

[0054] Comparative Example 1

[0055] This comparative example provides a method for preparing a mineralized collagen fiber scaffold without using a complex solution formed by chelating calcium ions with non-collagen analogs:

[0056] (1) After disinfecting the tails of fresh 8-week-old SD rats, the tail skin was removed and the tail tendons were extracted. The entire process was performed with sterile deionized water immersion to prevent collagen denaturation within the tail tendons. The extracted tail tendons were placed in Tris-HCl-NaCl solution (5M, pH=7.4) and soaked at 4℃ for 12h. After rinsing with deionized water, the tail tendons were placed in acetic acid solution (0.2M). The mixture was stirred continuously at room temperature for 3 days until the tail tendons were completely dissolved to obtain a collagen solution. The collagen solution was centrifuged at 3000rpm / min for 10min in a low-temperature high-speed centrifuge. The supernatant was collected in sterile centrifuge tubes and stored at 4℃ for later use.

[0057] (2) A collagen solution with a concentration of 10 mg / mL was added to an ultrapure aqueous solution with a final concentration of 50 mM glycine and a final concentration of 200 mM potassium chloride at a volume ratio of 4:1, and the pH of the mixture was adjusted to 7.0 with 1 M NaOH solution. The mixture was placed in an incubator at 37°C and incubated for 24 h until collagen was assembled to obtain collagen fibers.

[0058] (3) The mineralization of collagen fibers and the preparation of the scaffold are the same as in step (3) of Example 1.

[0059] (4) The sterilization of the mineralized collagen fiber scaffold is the same as step (4) in Example 1.

[0060] Comparative Example 2

[0061] This comparative example provides a method for preparing unmineralized collagen fiber scaffolds:

[0062] (1) After disinfecting the tails of fresh 8-week-old SD rats, the tail skin was removed and the tail tendons were extracted. The entire process was performed with sterile deionized water immersion to prevent collagen denaturation within the tail tendons. The extracted tail tendons were placed in Tris-HCl-NaCl solution (5M, pH=7.4) and soaked at 4℃ for 12h. After rinsing with deionized water, the tail tendons were placed in acetic acid solution (0.2M). The mixture was stirred continuously at room temperature for 3 days until the tail tendons were completely dissolved to obtain a collagen solution. The collagen solution was centrifuged at 3000rpm / min for 10min in a low-temperature high-speed centrifuge. The supernatant was collected in sterile centrifuge tubes and stored at 4℃ for later use.

[0063] (2) Dissolve calcium chloride dihydrate in ultrapure water at a concentration of 0.13M. After dissolving by magnetic stirring, dissolve PAsp in the above solution at a concentration of 0.1g / L. Stir for 0.5h to form PAsp-Ca complex solution.

[0064] Glycine and potassium chloride were added to the PAsp-Ca complex solution to make the final concentration of glycine 50 mM and the final concentration of potassium chloride 200 mM, thus forming the assembly solution.

[0065] A 10 mg / mL collagen solution was added to the assembly solution at a volume ratio of 4:1 to obtain a mixture, and the pH of the mixture was adjusted to 7.0 with 1M NaOH solution. The mixture was placed in an incubator at 37°C and incubated for 24 hours to allow collagen to assemble and obtain collagen fibers.

[0066] Collagen fibers were washed three times with ultrapure water, frozen at -80℃ for 12 hours, and then placed in a freeze dryer and freeze-dried at -50℃ for 2 days to obtain a collagen fiber scaffold.

[0067] Sterilization of stents

[0068] The collagen fiber scaffold prepared in step (2) was placed under a 254nm ultraviolet lamp and sterilized by irradiating both the front and back sides for 4 hours.

[0069] The scaffold materials prepared in Example 1, Comparative Example 1, and Comparative Example 2 were characterized by experiments to assess their structure, properties, and bone tissue regeneration in cases of bone defects.

[0070] 1. Characterization of the PAsp-Ca complex: The PAsp-Ca complex solution prepared in Example 1 was dropped onto an ultrathin carbon film copper grid, dried, and the morphology of PAsp-Ca was characterized by transmission electron microscopy. The results are as follows: Figure 1 As shown in Figures A and B, the PAsp-Ca complex prepared in Example 1 is spherical.

[0071] 2. Characterization of the co-assembly effect of collagen and PAsp-Ca complex: The collagen fibers assembled in Example 1 and Comparative Example 1 were dropped onto an ultrathin carbon film copper grid and a mica sheet, respectively. The morphology of the assembled collagen fibers was observed by transmission electron microscopy, and the surface morphology and Young's modulus of the assembled collagen fibers were observed by atomic force microscopy. The results are as follows: Figure 2 As shown, collagen fibers co-assembled with the PAsp-Ca complex have a larger diameter and a higher Young's modulus than collagen fibers co-assembled without the PAsp-Ca complex.

[0072] 3. Characterization of the mineralization effect after co-assembly of collagen and PAsp-Ca complex: The mineralized collagen fibers prepared in Example 1 and Comparative Example 1 were dropped onto an ultrathin carbon film copper grid, and the morphology and degree of mineralization of the collagen fibers after mineralization were observed by transmission electron microscopy. The results are as follows: Figure 3 As shown, the degree of mineralization in the collagen fibers obtained by co-assembling with the PAsp-Ca complex in Example 1 is higher than that in the collagen fibers of Comparative Example 1.

[0073] 4. Characterization of the mineralized collagen fiber scaffold prepared in Example 1: After freeze-drying, the sample was sputter-coated with gold, and the surface microstructure of the mineralized collagen fiber scaffold was observed by scanning electron microscopy. The results are as follows: Figure 4As shown in Figure A; X-ray diffraction qualitative analysis determined that the inorganic minerals of the mineralized collagen fiber scaffold were mainly hydroxyapatite, with a content similar to that of natural bone, as shown in the results. Figure 4 As shown in Figure B; the content of organic and inorganic matter in the mineralized collagen fiber scaffold was analyzed by thermogravimetric analysis, and the results are as follows. Figure 4 As shown in Figure C, the inorganic content in the scaffold is similar to and exceeds that of natural bone.

[0074] 5. Cell experiments

[0075] Alkaline phosphatase staining: Sprague-Dawley (SD) rat bone marrow mesenchymal stem cells (SD-BMSCs) were extracted and cultured to the third generation. SD-BMSCs were seeded at a density of 20,000 per well in 24-well plates containing a blank cell culture control, a collagen fiber scaffold from Comparative Example 2, and a mineralized collagen fiber scaffold from Example 1, with three replicates per group, and cultured until the cells reached 80% cell growth. The culture medium was replaced with osteogenic induction medium (Beyotime, China), and the medium was changed every 3 days for induction at 7 and 14 days. The osteogenic induction medium was removed, and the cells were washed three times with PBS buffer, fixed with 4% paraformaldehyde for 10-15 minutes, and washed three times with ultrapure water. ALP staining reagent was added and incubated at 37°C in the dark for 30 minutes. The staining solution was removed, and the cells were washed 2-3 times with ultrapure water and observed under a microscope. The results are as follows: Figure 5 As shown, the mineralized collagen fiber scaffold prepared in Example 1 showed the best osteogenic induction effect.

[0076] Alizarin Red Staining: SD-BMSCs were seeded at a density of 20,000 per well in 24-well plates containing blank cell culture control, collagen fiber scaffolds (Comparative Example 2), and mineralized collagen fiber scaffolds (Example 1), with three replicates per group. Cells were cultured until 80% cellularity was achieved. The culture medium was then replaced with osteogenic induction medium (Beyotime, China), and the medium was changed every 3 days for 14 days of induction. The osteogenic induction medium was removed, and cells were washed three times with PBS buffer. Cells were fixed with 4% paraformaldehyde for 10-15 minutes and washed three times with PBS buffer. Alizarin Red staining reagent was added and incubated at 37°C for 30 minutes. The staining solution was removed, and cells were washed 2-3 times with PBS buffer. Calcium nodules were observed under a microscope; the stained calcium nodules appeared orange-red. Results are as follows: Figure 6 As shown, the mineralized collagen fiber scaffold prepared in Example 1 had a higher content of stained calcium nodules than the other two groups, and the best osteogenic induction effect.

[0077] 6. Animal models

[0078] Healthy male SD rats, 6 weeks old and weighing approximately 150-200g, were housed in an SPF-grade environment. A 5mm mandibular bone defect model was used, divided into three groups: a blank control group (no repair material implanted), a collagen fiber scaffold group (Comparative Example 2), and a mineralized collagen fiber scaffold group (Example 1). After preoperative weighing, rats were anesthetized with 2.5% aphthylamine (12.5 μL / g body weight) via intraperitoneal injection. Once fully anesthetized, the rats were fixed to a board. The cranial neck was prepared and thoroughly disinfected with iodine, followed by local infiltration anesthesia. A transverse incision was made along the lower edge of the mandible, cutting through the skin and subcutaneous tissue to the periosteum. The periosteum was bluntly dissected using a periosteal elevator to fully expose the mandible. A circular defect approximately 5mm in diameter was carefully prepared in the skull using a trephine drill. Figure 7 As shown, the animal was continuously cooled using 0.9% saline solution. Subsequently, residual bone fragments in the surgical area were rinsed, and hemostasis was achieved thoroughly with sterile gauze. A suitable mineralized collagen fiber scaffold / collagen fiber scaffold was implanted, and the periosteum and skin were sutured sequentially using 5-0 silk sutures. Given the large wound area and the high risk of postoperative death, the animal was placed on a warming mat before resuscitation, and its heart and respiration were closely monitored.

[0079] Imaging analysis: Rats were euthanized in batches 4 weeks post-surgery, and intact mandibular bone samples were dissected and fixed in 4% paraformaldehyde solution. MicroCT scans were performed, and three-dimensional reconstructions of the skull defects were performed. Results are as follows: Figure 8 As shown, bone defects grow from the periphery towards the center of the defect. The amount of newly formed bone in the collagen fiber scaffold group of Comparative Example 2 and the mineralized collagen fiber scaffold group of Example 1 was greater than that in the blank control group, and the repair effect was better.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications (including doping crystals in the support), equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a scaffold that promotes collagen fiber organization and internal and external mineralization, characterized by: The method comprises the following steps: A) adding glycine and potassium chloride into a complex solution formed by non-collagen protein analogues chelating calcium ions to prepare an assembly solution; wherein the non-collagen protein analogues are one or both of polycarboxylic acid substances and glucuronic acid; B) adding a collagen protein solution into the assembly solution obtained in step A) to obtain a mixed solution, and adjusting the pH of the mixed solution to 7.0±0.2; placing the mixed solution in a 37±0.5℃ incubator, and incubating for 12-24 hours to obtain collagen fibers after the collagen is assembled; C) mineralizing the collagen fibers prepared in step B) in a collagen fiber mineralization solution to obtain mineralized collagen fibers, and washing, freezing and drying the mineralized collagen fibers to obtain a mineralized collagen fiber scaffold; wherein the collagen fiber mineralization solution is prepared by the following method: dissolving calcium chloride in deionized water to form a calcium ion solution with a concentration of 2-4 mM, and then dissolving non-collagen protein analogues in the calcium ion solution to obtain solution A, wherein the concentration of the non-collagen protein analogues in solution A is 100-300 μg / mL; dissolving disodium hydrogen phosphate in a 200-400 mM NaCl aqueous solution to form a hydrogen phosphate ion solution with a concentration of 10-20 mM to obtain solution B; and mixing solution A and solution B in a volume ratio of 1:1-1:2, and stirring uniformly to obtain the collagen fiber mineralization solution.

2. The method for preparing a scaffold to promote collagen fiber assembly and internal and external mineralization according to claim 1, characterized in that: In step A), the preparation of the complex solution formed by non-collagen protein analogues chelating calcium ions comprises the following steps: dissolving calcium chloride in ultrapure water at a concentration of 0.05-0.2 M, and magnetically stirring to dissolve; adding non-collagen protein analogues and dissolving them, wherein the concentration of the non-collagen protein analogues is 0.1-0.3 g / L, and stirring for 10-30 minutes to mix uniformly to obtain the complex solution formed by non-collagen protein analogues chelating calcium ions.

3. The method for preparing a scaffold to promote collagen fiber assembly and internal and external mineralization according to claim 1, characterized in that: In step A), the final concentration of glycine in the assembly solution is 30-60 mM; and the final concentration of potassium chloride is 100-300 mM.

4. The method of claim 2, wherein the scaffold is prepared by the steps of: a) providing a scaffold; b) coating the scaffold with a collagen solution; c) drying the scaffold; d) coating the scaffold with a mineral solution; and e) drying the scaffold. In step B), the assembly solution obtained in step A) is added with a collagen protein solution with a concentration of 8-11 mg / mL in a volume ratio of 5:1-3:1 to obtain a mixed solution, and the pH of the mixed solution is adjusted to 7.0±0.2 by using a 0.5-3 M NaOH solution.

5. The method of claim 1, wherein the scaffold is prepared by the steps of: a) providing a scaffold; b) coating the scaffold with a collagen solution; c) drying the scaffold; d) coating the scaffold with a mineral solution; and e) drying the scaffold. In step C), the volume ratio of the collagen fibers prepared in step B) to the collagen fiber mineralization solution is 1:8-1:15, the collagen fiber mineralization solution is replaced every 1-3 days during the mineralization process, and the mineralization is performed for 5-7 days to obtain mineralized collagen fibers.

6. The method of claim 1, wherein the scaffold is prepared by the steps of: a) providing a collagen solution; b) providing a mineral solution; c) mixing the collagen solution and the mineral solution; d) providing a biocompatible polymer; e) mixing the collagen solution and the mineral solution with the biocompatible polymer; and f) forming the scaffold. In step C), the obtained mineralized collagen fibers are washed with ultrapure water for 3-5 times, and frozen at -80℃ for 4-12 hours.

7. The method of claim 1, wherein the scaffold is prepared by the steps of: a) providing a collagen solution; b) providing a mineral solution; c) mixing the collagen solution and the mineral solution; d) providing a biocompatible polymer; e) mixing the collagen solution and the mineral solution with the biocompatible polymer; and f) forming the scaffold. In step C), the drying is freeze-drying, and the freeze-drying temperature is -50℃, and the freeze-drying is performed for 1-3 days.

8. The method of claim 1, wherein the scaffold is prepared by the steps of: a) providing a collagen solution; b) providing a mineral solution; c) mixing the collagen solution and the mineral solution; d) providing a biocompatible polymer; e) mixing the collagen solution, the mineral solution, and the biocompatible polymer; and f) forming the scaffold. In step B), the collagen protein is rat tail tendon collagen protein.

9. The method of claim 1, wherein the scaffold is prepared by the steps of: a) providing a collagen solution; b) providing a mineral solution; c) mixing the collagen solution and the mineral solution; d) providing a biocompatible polymer; e) mixing the collagen solution and the mineral solution with the biocompatible polymer; and f) forming the scaffold. In step A), the polycarboxylic acid substances are one or more than two of polyaspartic acid, polyglutamic acid and polyacrylic acid.

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