Preparation method of stent for promoting collagenous fiber assembly and internal and external mineralization

Abstract

The invention provides a preparation method of a scaffold for promoting collagenous fiber assembly and internal and external mineralization. The preparation method comprises the following steps: A) adding glycine and potassium chloride into a compound solution formed by chelating non-collagen analogues with calcium ions to prepare an assembly solution; wherein the non-collagen analogue is one or two of polycarboxylic acid substances and glucuronic acid; b) adding a collagen solution into the assembly solution obtained in the step A) to obtain a mixed solution, and adjusting the pH value of the mixed solution to 7.0 + / -0.2; placing the mixed solution in an incubator at 37 + / -0.5 DEG C, incubating for 12-24 hours, and assembling collagen to obtain collagen fibers; c) placing the collagenous fibers prepared in the step B) in a collagenous fiber mineralizing solution for mineralization to obtain mineralized collagenous fibers, and washing, freezing and drying the mineralized collagenous fibers to obtain a mineralized collagenous fiber scaffold; according to the invention, the bone tissue generation process is simulated in a safer bionic manner, so that the collagenous fiber scaffold is assembled and mineralized more efficiently.

Description

Technical Field

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

[0002] Natural bone tissue is primarily composed of orderly arrays of hydroxycarbonate apatite crystals, type I collagen, and water. The hydroxycarbonate apatite crystals are arranged within and outside the collagen fibrils, forming a regular pattern of internal and external mineralization. Natural bone tissue possesses a complex structure, encompassing hierarchical gradients of nanocrystals and collagen fibrils across many length scales. This structure imparts natural bone with both the strength and toughness necessary to meet the functional needs of the organism.

[0003] The exceptional structure and properties of bone tissue enable it to play a vital role in human life. Trauma, infection, tumors, and other factors can damage bone tissue, leading to bone defects. Repairing and treating bone defects is crucial for restoring both physiological function and psychological well-being, and has long been a focus of intense medical attention. Autologous bone transplantation, using the patient's own bone tissue for transplantation, offers the best biocompatibility and lowest rejection response, effectively promoting bone healing and is considered the "gold standard" for bone repair. However, autologous bone transplantation can cause secondary trauma to the body and is limited in the amount of donor bone available. Tissue-engineered bone graft materials, with their excellent biocompatibility, biosafety, and biofunctionality, combined with degradability and the ability to induce osteogenesis, have garnered widespread attention. Bone implant materials must possess a comprehensive range of excellent mechanical properties, achieving 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, the design of biomimetic bone implant materials strives to mimic the biological structure of natural bone.

[0004] Many studies have mixed collagen and nanohydroxyapatite to create composite scaffolds for bone defect repair. These simple blends mimic only the composition of bone tissue, but not its structure and properties. Currently, numerous technologies attempt to mimic the ordered spatial hierarchy of collagen fibrils and hydroxyapatite found in natural bone. In the absence of cellular regulation, these technologies employ polyelectrolytes to mimic the function of osteogenesis-related proteins in vitro, precisely controlling the growth of hydroxyapatite crystals within and outside the collagen fibrils. This results in scaffolds that are highly biomimetic in composition, microstructure, and physiological function. A key component of these technologies is promoting the interaction between amorphous calcium phosphate precursors and collagen. This is typically achieved by activating the collagen surface and promoting collagen cross-linking to further enhance mineralization. However, current activation agents, primarily glutaraldehyde and EDC / NHS, are toxic and have poor biocompatibility. These agents differ significantly from the in vivo collagen activation process, posing potential biosafety risks. Therefore, finding a faster, safer, and more mineralized collagen assembly strategy is an urgent scientific challenge. 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 deficiencies or defects in the existing technology, simulate the bone tissue development process in a more bionic way, and thus achieve more efficient assembly and mineralization of collagen fiber scaffolds.

[0006] To achieve the above object, the technical solution of the present invention is achieved as follows: A method for preparing a scaffold that promotes collagen fiber assembly and internal and external mineralization comprises the following steps: A) adding glycine and potassium chloride to a complex solution formed by a non-collagen analogue chelating calcium ions to prepare an assembly solution; wherein the non-collagen analogue is one or both of a polycarboxylic acid substance and glucuronic acid; B) adding the collagen solution to the assembly solution obtained in step A) to obtain a mixed solution, adjusting the pH of the mixed solution to 7.0±0.2; placing the mixed solution in an incubator at 37±0.5°C for 12-24 hours to allow the collagen to assemble and obtain collagen fibers; C) placing the collagen fibers prepared in step B) in a collagen fiber mineralization solution for mineralization to obtain mineralized collagen fibers, washing the mineralized collagen fibers, freezing, and drying to obtain a mineralized collagen fiber scaffold.

[0007] Preferably, in step A), the preparation of the complex solution formed by the non-collagen analog chelating calcium ions comprises the following steps: dissolving calcium chloride in ultrapure water at a concentration of 0.05 to 0.2 M and dissolving it by magnetic stirring; adding a non-collagen analog and dissolving it, wherein the concentration of the non-collagen analog is 0.1 to 0.3 g / L, stirring for 10 to 30 minutes to obtain a complex solution formed by the non-collagen analog chelating calcium ions.

[0008] Preferably, in step A), the final concentration of glycine in the assembly solution is 30-60 mM; the final concentration of potassium chloride is 100-300 mM.

[0009] Preferably, in step B), a collagen solution having a concentration of 8-11 mg / mL is added to the assembly solution obtained in step A) at 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 with 0.5-3 M NaOH solution.

[0010] Preferably, in step C), 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 the non-collagen protein analog in the calcium ion solution to obtain solution A, wherein the concentration of the non-collagen protein 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; solution A and solution B are mixed in a volume ratio of 1:1-1:2 and stirred evenly to obtain a collagen fiber mineralization solution.

[0011] Preferably, 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. During the mineralization process, the collagen fiber mineralization solution is replaced every 1-3 days. Mineralization is carried out for 5-7 days to obtain mineralized collagen fibers.

[0012] Preferably, in step C), the obtained mineralized collagen fibers are washed with ultrapure water for 3 to 5 times and frozen at -80°C for 4 to 12 hours. Preferably, in step C), the drying is freeze-drying for 1 to 3 days at a temperature of -50°C.

[0013] Preferably, in step B), the collagen is rat tail tendon collagen.

[0014] Collagen can be commercially available or prepared by the following method: a) Fresh 8-week-old SD rat tails were disinfected, the skin removed, and the tail tendons extracted. The tails were soaked in sterile deionized water throughout the entire procedure to prevent collagen denaturation in the tail tendons.

[0015] b) The extracted rat tail tendon was placed in a Tris-HCl-NaCl solution (5 M, pH = 7.4) and immersed at 4°C for 12 h.

[0016] c) After rinsing with deionized water, place the rat tail tendon in an acetic acid solution (0.2-0.4 M). Stir continuously at room temperature for 2-3 days until the rat tail tendon is completely dissolved to obtain a collagen solution.

[0017] d) Place the collagen solution in a low-temperature high-speed centrifuge and centrifuge at 3000-4000 rpm / min for 10-20 minutes. Collect the supernatant in a sterile centrifuge tube and store at 4°C until use.

[0018] Preferably, in step A), the polycarboxylic acid substance is one or more of polyaspartic acid, polyglutamic acid, and polyacrylic acid.

[0019] The complex formed by non-collagen analogs chelating calcium ions participates in the assembly of collagen fibers. During the assembly process, the complex formed by non-collagen analogs chelating calcium ions is embedded in the gaps between collagen fibers, helping the collagen fibers to mineralize faster and to a higher degree. The collagen fibers are then placed in a mineralizing solution. The amorphous precursor (ACP) with liquid fluidity formed by the calcium and phosphate ions in the mineralizing solution enters the interior of the collagen fibers, gradually loses water, begins to solidify and crystallize, and forms thermodynamically more stable hydroxyapatite, completing the internal and external mineralization of the collagen fibers.

[0020] Compared with the prior art, the scaffold preparation method for promoting collagen fiber assembly and internal and external mineralization of the present invention has the following advantages: (1) Compared with the prior art, the collagen assembly environment in the present invention is closer to the environment containing calcium in the human body, and the assembly effect of the obtained collagen fibers is better.

[0021] (2) The complex formed by the non-collagen analogue chelated calcium ion used in the present invention to promote the assembly and mineralization of collagen fibers has good biocompatibility and is safer and has no toxic side effects compared to toxic cross-linking agents such as glutaraldehyde.

[0022] (3) The present invention simulates the bone tissue development process in a safer and more biomimetic manner, thereby achieving more efficient assembly and mineralization of collagen fiber scaffolds. This novel scaffold preparation method that promotes collagen fiber assembly and internal and external mineralization produces synthesized collagen fibers with 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 future repair of clinical bone tissue defects and has broader application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1This is a transmission electron microscopy result of the PAsp-Ca complex prepared in Example 1; the scale bar in A is 2 μm, and the scale bar in B is 200 nm.

[0024] Figure 2 Characterization diagrams of the collagen fibers prepared in Example 1 and Comparative Example 1; wherein A, B, and C are transmission electron microscopy results, atomic force microscopy surface morphology images, and atomic force microscopy Young's modulus images of the collagen fibers obtained in Comparative Example 1, respectively; D, E, and F are transmission electron microscopy results, atomic force microscopy surface morphology images, and atomic force microscopy Young's modulus images of the collagen fibers obtained in Example 1, respectively.

[0025] Figure 3 These are the transmission electron microscopy results of the mineralized collagen fibers obtained in Example 1 and Comparative Example 1; A and C are the transmission electron microscopy results of the mineralized collagen fibers obtained in Comparative Example 1 after mineralization for 3 d and 5 d, respectively; B and D are the transmission electron microscopy results of the mineralized collagen fibers obtained in Example 1 after mineralization for 3 d and 5 d, respectively.

[0026] Figure 4 Characterization diagram of the mineralized collagen fiber scaffold prepared in Example 1; wherein A is the result of field emission scanning electron microscopy 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 rats; 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 rats.

[0027] Figure 5 The diagram shows the results of osteogenic differentiation of SD rat bone marrow mesenchymal stem cells stained with alkaline phosphatase; A, B, and C are the results of 7 days of osteogenic differentiation induced by the blank cell culture control group, the collagen fiber scaffold of Comparative Example 2, and the mineralized collagen fiber scaffold obtained in Example 1; D, E, and F are the results of 14 days of osteogenic differentiation induced by the blank cell culture control group, the collagen fiber scaffold of Comparative Example 2, and the mineralized collagen fiber scaffold obtained in Example 1.

[0028] Figure 6 The figure shows the results of osteogenic differentiation of SD rat bone marrow mesenchymal stem cells stained with Alizarin red for 14 days; A, B, and C are the results of osteogenic differentiation induced by the blank cell culture control group, the collagen fiber scaffold of Comparative Example 2, and the mineralized collagen fiber scaffold of Example 1 for 14 days, respectively.

[0029] Figure 7 To establish the mandibular bone defect model in SD rats.

[0030] Figure 8The three-dimensional reconstruction comparison diagram of Micro-CT scanning results of mandibular bone defects in SD rats at 4 weeks; A, B, and C represent the results of the blank control group (no repair material is placed), the collagen fiber scaffold group of comparative example 2, and the mineralized collagen fiber scaffold group of example 1, respectively. DETAILED DESCRIPTION

[0031] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which this invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods described, unless otherwise specified, are conventional methods.

[0032] The present invention will be described in detail below with reference to the embodiments.

[0033] The concentration unit M mentioned in the embodiments of the present invention is mol / L; mM is 0.001 mol / L.

[0034] Example 1

[0035] A method for preparing a scaffold that promotes collagen fiber assembly and internal and external mineralization comprises the following steps: (1) Preparation of complex solution formed by non-collagen analogues chelating calcium ions Poly-L-aspartic acid (PAsp, molecular weight 5 kDa) was selected as the non-collagen analog. Calcium chloride dihydrate was dissolved in ultrapure water at a concentration of 0.13 M. After dissolution by magnetic stirring, PAsp was added and dissolved at a concentration of 0.1 g / L. The mixture was stirred for 0.5 h to form a PAsp-Ca complex solution, i.e., a complex solution formed by the non-collagen analog chelating calcium ions.

[0036] (2) Extraction and self-assembly of type I collagen After disinfecting the tail of a fresh 8-week-old SD rat, remove the skin and extract the tail tendon. The entire procedure was supplemented with sterile deionized water soaking to prevent collagen denaturation in the tail tendon. The extracted tail tendon was 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 tendon was placed in an acetic acid solution (0.2M). Stirring was continued at room temperature for 3 days until the tail tendon was completely dissolved to obtain a collagen solution. The collagen solution was placed in a low-temperature high-speed centrifuge and centrifuged at 3000 rpm / min for 10 minutes. After centrifugation, the supernatant was collected in a sterile centrifuge tube and stored at 4°C for later use.

[0037] Glycine and potassium chloride were added to the PAsp-Ca complex solution prepared in step (1) to make the final concentration of glycine in the solution 50 mM and the final concentration of potassium chloride 200 mM, thereby preparing an assembly solution.

[0038] A 10 mg / mL collagen solution was added to the assembly solution at a volume ratio of 4:1 to obtain a mixed solution, and the pH of the mixed solution was adjusted to 7.0 with a 1 M NaOH solution. The mixed solution was placed in an incubator at 37°C for 24 hours to allow collagen to assemble and obtain collagen fibers.

[0039] (3) Mineralization of collagen fibers and preparation of scaffolds Calcium chloride dihydrate was dissolved in deionized water to form a 3.34 mM calcium ion solution, and PAsp was then dissolved in the calcium ion solution to form a solution with a PAsp concentration of 240 μg / mL, 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, obtaining solution B. Solutions A and B were mixed in a 1:1 volume ratio and stirred to obtain a collagen fiber mineralization solution.

[0040] 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 an incubator at 37° C. The collagen fiber mineralization solution was replaced every 2 days. The mineralization was carried out for 7 days to obtain mineralized collagen fibers.

[0041] The mineralized collagen fibers were washed three times with ultrapure water, frozen at -80°C for 12 h, placed in a freeze dryer, and freeze-dried at -50°C for 2 days to obtain a mineralized collagen fiber scaffold.

[0042] (4) Sterilization of mineralized collagen fiber scaffolds The mineralized collagen fiber scaffold prepared in step (3) was placed under a 254 nm ultraviolet lamp and irradiated on both the front and back sides for 4 h for sterilization.

[0043] Comparative Example 1 This comparative example provides a method for preparing a mineralized collagen fiber scaffold without using a complex solution formed by chelating calcium ions with a non-collagen analog: (1) After disinfecting the tail of a fresh 8-week-old SD rat, remove the tail skin and extract the tail tendon. The entire operation is supplemented with sterile deionized water soaking to prevent collagen denaturation in the tail tendon. The extracted tail tendon is 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 tendon is placed in an acetic acid solution (0.2M). Stir continuously at room temperature for 3 days until the tail tendon is completely dissolved to obtain a collagen solution. Place the collagen solution in a low-temperature high-speed centrifuge and centrifuge at 3000rpm / min for 10 minutes. After centrifugation, collect the supernatant in a sterile centrifuge tube and store at 4°C for later use; (2) A 10 mg / mL collagen solution was added to an ultrapure aqueous solution containing 50 mM glycine and 200 mM potassium chloride at a volume ratio of 4:1. The pH of the mixture was adjusted to 7.0 with 1 M NaOH solution. The mixture was incubated at 37°C for 24 h to allow collagen to assemble and produce collagen fibers.

[0044] (3) The mineralization of collagen fibers and the preparation of the scaffold are the same as step (3) in Example 1.

[0045] (4) Sterilization of the mineralized collagen fiber scaffold is the same as step (4) in Example 1.

[0046] Comparative Example 2 This comparative example provides a method for preparing an unmineralized collagen fiber scaffold: (1) After disinfecting the tail of a fresh 8-week-old SD rat, remove the tail skin and extract the tail tendon. The entire operation was supplemented with sterile deionized water soaking to prevent collagen denaturation in the tail tendon. The extracted tail tendon was 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 tendon was placed in an acetic acid solution (0.2M). Stirring was continued at room temperature for 3 days until the tail tendon was completely dissolved to obtain a collagen solution. The collagen solution was placed in a low-temperature high-speed centrifuge and centrifuged at 3000 rpm / min for 10 minutes. After centrifugation, the supernatant was collected in a sterile centrifuge tube and stored at 4°C for later use.

[0047] (2) Calcium chloride dihydrate was dissolved in ultrapure water at a concentration of 0.13 M. After dissolution by magnetic stirring, PAsp was dissolved in the above solution at a concentration of 0.1 g / L. The mixture was stirred for 0.5 h to form a PAsp-Ca complex solution.

[0048] Glycine and potassium chloride were added to the PAsp-Ca complex solution to make the final concentration of glycine in the solution 50 mM and the final concentration of potassium chloride 200 mM, thereby forming an assembly solution.

[0049] A 10 mg / mL collagen solution was added to the assembly solution at a volume ratio of 4:1 to obtain a mixed solution. The pH of the mixed solution was adjusted to 7.0 with 1 M NaOH solution. The mixed solution was incubated at 37°C for 24 hours to allow collagen assembly and obtain collagen fibers.

[0050] The collagen fibers were washed three times with ultrapure water, frozen at -80°C for 12 h, placed in a freeze dryer, and freeze-dried at -50°C for 2 days to obtain a collagen fiber scaffold.

[0051] Sterilization of the stent The collagen fiber scaffold prepared in step (2) was placed under a 254 nm ultraviolet lamp and irradiated on both sides for 4 h for sterilization.

[0052] The scaffold materials prepared in Example 1, Comparative Example 1 and Comparative Example 2 were subjected to experiments related to characterizing their structure, performance and bone defect bone tissue regeneration.

[0053] 1. Characterization of PAsp-Ca complex: The PAsp-Ca complex solution prepared in Example 1 was dropped onto an ultra-thin carbon film copper mesh. After drying, the morphology of PAsp-Ca was characterized using a transmission electron microscope. Figure 1 As shown in A and B, the PAsp-Ca complex prepared in Example 1 is spherical.

[0054] 2. Characterization of the co-assembly effect of collagen and PAsp-Ca complex: The assembled collagen fibers in Example 1 and Comparative Example 1 were dropped onto an ultra-thin carbon film copper mesh and a mica sheet. 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 shown in Figure 2. Figure 2 As shown, the collagen fibers co-assembled with the PAsp-Ca complex had a larger diameter and a higher Young's modulus than the collagen fibers co-assembled without the PAsp-Ca complex.

[0055] 3. Characterization of the mineralization effect after the co-assembly of collagen and PAsp-Ca complex: The mineralized collagen fibers prepared in Example 1 and Comparative Example 1 were dropped onto an ultra-thin carbon film copper mesh and the morphology and mineralization degree of the mineralized collagen fibers were observed by transmission electron microscopy. Figure 3 As shown, the degree of mineralization in the collagen fibers obtained by co-assembly with the PAsp-Ca complex in Example 1 is higher than that in the collagen fibers of Comparative Example 1.

[0056] 4. Characterization of the mineralized collagen fiber scaffold prepared in Example 1: The sample was freeze-dried and then sprayed with gold. The surface micromorphology of the mineralized collagen fiber scaffold was observed by scanning electron microscopy. The results were as follows: Figure 4As shown in A; X-ray diffraction qualitatively determined that the inorganic minerals of the mineralized collagen fiber scaffold are mainly hydroxyapatite, which is similar to the content of natural bone. The results are as follows Figure 4 As shown in B; The content of organic and inorganic substances in the mineralized collagen fiber scaffold was analyzed by thermogravimetric analysis. Figure 4 As shown in Figure C, the inorganic content in the scaffold is similar to and exceeds that of natural bone.

[0057] 5. Cell experiments 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 / well in a blank cell culture control, a 24-well plate containing the collagen fiber scaffold of Comparative Example 2, and a mineralized collagen fiber scaffold of Example 1 for co-culture, with 3 replicates per group, and cultured until the cells grew to 80%. The culture medium was replaced with osteogenic induction medium (Biyuntian, China), and the culture medium was replaced every 3 days, and induced for 7 and 14 days respectively. The osteogenic induction medium was removed, and the cells were washed 3 times with PBS buffer, fixed with 4% paraformaldehyde for 10 to 15 minutes, and washed 3 times with ultrapure water. ALP staining reagent was added and incubated in the dark at 37°C for 30 minutes, the staining solution was removed, the cells were washed 2 to 3 times with ultrapure water, and observed under a microscope. The results are as follows Figure 5 As shown in , the mineralized collagen fiber scaffold prepared in Example 1 has the best osteoinduction effect.

[0058] Alizarin red staining: SD-BMSCs were seeded at a density of 20,000 / well in a blank cell culture control, a 24-well plate containing the collagen fiber scaffold of comparative example 2, and a mineralized collagen fiber scaffold of example 1 for co-culture, with 3 replicates per group, and cultured until the cells grew to 80%. The culture medium was replaced with osteogenic induction medium (Biyuntian, China), and the culture medium was replaced every 3 days for 14 days. The osteogenic induction medium was removed, and the cells were washed 3 times with PBS buffer, fixed with 4% paraformaldehyde for 10-15 minutes, and washed 3 times with PBS buffer. Alizarin red staining reagent was added and incubated at 37°C for 30 minutes, the staining solution was removed, and the cells were washed 2-3 times with PBS buffer. The calcium nodules were observed under a microscope, and the stained calcium nodules were orange-red. The results are as follows Figure 6 As shown in , the mineralized collagen fiber scaffold prepared in Example 1 had a higher content of stained calcium nodules than the other two groups, and had the best osteogenic induction effect.

[0059] 6. Animal Models Healthy SD male rats, 6 weeks old, weighing about 150-200g, were raised in an SPF environment. The 5mm extreme mandibular defect model was divided into 3 groups: a blank control group (no repair material was placed), a collagen fiber scaffold group of comparative example 2, and a mineralized collagen fiber scaffold group of Example 1. After preoperative weighing, the rats were anesthetized with a mass volume concentration of 2.5% Avertin, and intraperitoneally injected at a dose of 12.5μL / g body weight. After the rats were fully anesthetized, they were fixed on the mouse board. The skull and neck were prepared and fully disinfected with iodine, supplemented with local infiltration anesthesia. A transverse incision was made at the lower edge of the rat's mandible, and the skin and subcutaneous tissue were cut to the periosteum surface. The periosteum was bluntly separated using a periosteal stripper to fully expose the mandible. A circular defect with a diameter of about 5mm was solemnly prepared in the skull using a trephine drill, as shown in the figure. Figure 7 As shown, 0.9% saline was continuously used for cooling during the operation. Subsequently, residual bone debris was irrigated from the surgical area, and hemostasis was achieved with sterile gauze. The corresponding mineralized collagen fiber scaffold / collagen fiber scaffold was implanted, and the periosteum and skin were sutured sequentially with 5-0 silk sutures. Given the large wound surface, animals are prone to postoperative mortality. Therefore, they were placed on a warming pad before resuscitation, and their heart rate and respiration were closely monitored.

[0060] Image analysis: Rats were killed in batches 4 weeks after surgery, and intact mandibular specimens were removed and fixed in 4% paraformaldehyde solution. MicroCT scanning was performed, and three-dimensional reconstruction of the skull defect was performed. Figure 8 As shown in the figure, the bone defect grew from the periphery to the middle defect site. The amount of new 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.

[0061] 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 bracket), equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a scaffold that promotes collagen fiber assembly and internal and external mineralization, characterized by: The following steps are included: A) adding glycine and potassium chloride to a complex solution formed by a non-collagen analogue chelating calcium ions to prepare an assembly solution; wherein the non-collagen analogue is one or both of a polycarboxylic acid substance and glucuronic acid; B) adding the collagen solution to the assembly solution obtained in step A) to obtain a mixed solution, adjusting the pH of the mixed solution to 7.0±0.2; placing the mixed solution in an incubator at 37±0.5°C for 12-24 hours to allow the collagen to assemble and obtain collagen fibers; C) placing the collagen fibers prepared in step B) in a collagen fiber mineralization solution for mineralization to obtain mineralized collagen fibers, washing the mineralized collagen fibers, freezing, and drying to obtain a mineralized collagen fiber scaffold.

2. The method for preparing a scaffold that promotes 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 the non-collagen analog chelated calcium ions comprises the following steps: dissolving calcium chloride in ultrapure water at a concentration of 0.05 to 0.2 M and dissolving it by magnetic stirring; adding a non-collagen analog and dissolving it, wherein the concentration of the non-collagen analog is 0.1 to 0.3 g / L, stirring for 10 to 30 minutes to mix, and obtaining a complex solution formed by the non-collagen analog chelated calcium ions.

3. The method for preparing a scaffold for promoting 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; the final concentration of potassium chloride is 100-300 mM.

4. The method for preparing a scaffold for promoting collagen fiber assembly and internal and external mineralization according to claim 2, characterized in that: In step B), a collagen solution having a concentration of 8-11 mg / mL is added to the assembly solution obtained in step A) at 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 with 0.5-3 M NaOH solution.

5. The method for preparing a scaffold for promoting collagen fiber assembly and internal and external mineralization according to claim 1, characterized in that: In step C), 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 the non-collagen protein analog in the calcium ion solution to obtain solution A, wherein the concentration of the non-collagen protein 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; solution A and solution B are mixed in a volume ratio of 1:1-1:2 and stirred evenly to obtain a collagen fiber mineralization solution.

6. The method for preparing a scaffold for promoting collagen fiber assembly and internal and external mineralization according to claim 5, characterized in that: 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. During the mineralization process, the collagen fiber mineralization solution is replaced every 1-3 days. The mineralization is carried out for 5-7 days to obtain mineralized collagen fibers.

7. The method for preparing a scaffold for promoting collagen fiber assembly and internal and external mineralization according to claim 5, characterized in that: In step C), the obtained mineralized collagen fibers are washed 3 to 5 times with ultrapure water and frozen at -80°C for 4 to 12 hours.

8. The method for preparing a scaffold for promoting collagen fiber assembly and internal and external mineralization according to claim 5, characterized in that: In step C), the drying is freeze-drying, the freeze-drying temperature is -50°C, and the freeze-drying is performed for 1 to 3 days.

9. The method for preparing a scaffold for promoting collagen fiber assembly and internal and external mineralization according to claim 1, characterized in that: In step B), the collagen is rat tail tendon collagen.

10. The method for preparing a scaffold for promoting collagen fiber assembly and internal and external mineralization according to claim 1, characterized in that: In step A), the polycarboxylic acid substance is one or more of polyaspartic acid, polyglutamic acid, and polyacrylic acid.

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