Preparation method of mineralized collagen scaffold material with bionic structure, mineralized collagen scaffold material and application thereof

Abstract

The application provides a preparation method of a mineralized collagen scaffold material with a biomimetic structure, and belongs to the field of biomedical engineering.The preparation method comprises the following steps: preparing a polyacrylic acid-stabilized amorphous calcium phosphate solution, wherein the molar ratio of Ca element to P element in the amorphous calcium phosphate solution is 1.67:1; preparing a collagen solution containing norepinephrine; performing isoelectric focusing treatment on the collagen solution under a constant voltage to form a dense film, then stretching the dense film to produce a deformation of 120% to 200%, and obtaining a collagen film with a biomimetic structure; soaking the collagen film in the amorphous calcium phosphate solution, and then performing freeze-drying to obtain a norepinephrine-mineralized collagen scaffold material.The mineralized collagen scaffold material prepared by the method has excellent biocompatibility, osteogenic induction and sufficient mechanical strength, can effectively promote bone tissue defect repair, and has a good application prospect in the field of bone tissue engineering.The application further provides a mineralized collagen scaffold material with a biomimetic structure and an application thereof.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering, and specifically relates to a method for preparing a mineralized collagen scaffold material with a biomimetic structure, the mineralized collagen scaffold material, and its applications. Background Technology

[0002] Bone, as the most abundant hard tissue in the human body, plays a vital role in support and protection. Bone defects caused by sports, traffic accidents, and diseases are common clinical conditions, endangering patients' health and causing significant suffering. Currently, the main methods for bone repair are implantation of autologous bone grafts, allogeneic bone grafts, xenogeneic bone grafts, or synthetic artificial bone. Autologous bone, with its dual functions of osteoinduction and osteoconduction, has been clinically proven to be an effective means of bone repair. However, its application is limited due to the lack of donor tissue and additional secondary defects. Furthermore, allogeneic and xenogeneic bone grafts pose a high risk of pain or infection due to immune rejection or disease transmission. While cryogenic treatment can reduce the occurrence of immune rejection, its mechanical strength and osteoinduction properties will correspondingly decrease. Therefore, developing high-performance bone defect repair materials is of great significance.

[0003] Ideal bone repair materials should possess good osteoconductivity, biocompatibility, absorbability, and sufficient mechanical strength. Currently, commonly used bone repair materials include metallic materials, ceramic implants, and polymer scaffold materials. Titanium alloys are the most widely used metallic material clinically. Titanium is non-toxic, harmless, has good corrosion resistance, and its elastic modulus is close to that of human hard tissue. However, the relatively smooth surface of titanium alloys leads to poor bone integration. Ceramic implants are mainly calcium phosphate ceramics, including hydroxyapatite (HA) and β-tricalcium phosphate, which have been extensively studied in the field of bone repair. Although their inorganic composition is similar to that of natural bone tissue, the poor fracture toughness and tensile strength of calcium phosphate ceramics limit their application in the field of biomaterials. Polymer scaffold materials can be inlaid with growth factors to accelerate bone repair, but most polymers have weak osteoconductivity. Summary of the Invention

[0004] To obtain bone tissue defect repair materials with good performance, this invention provides a method for preparing a mineralized collagen scaffold material with a biomimetic structure. The mineralized collagen scaffold material prepared by this method has excellent biocompatibility, osteogenic induction and sufficient mechanical strength, which can effectively promote the repair of bone tissue defects and has good application prospects in the field of bone tissue engineering.

[0005] The present invention also provides a mineralized collagen scaffold material with a biomimetic structure and its application.

[0006] This invention is achieved through the following technical solution:

[0007] This invention provides a method for preparing a mineralized collagen scaffold material with a biomimetic structure, the method comprising:

[0008] Prepare an amorphous calcium phosphate solution stabilized by polyacrylic acid, wherein the molar ratio of Ca to P in the amorphous calcium phosphate solution is 1.67:1;

[0009] Prepare a collagen solution containing norepinephrine;

[0010] The collagen solution is subjected to isoelectric focusing treatment under constant voltage to form a dense film, and then the dense film is stretched to produce a deformation of 120% to 200% to obtain a collagen film with a biomimetic structure.

[0011] The collagen membrane was immersed in the amorphous calcium phosphate solution and then freeze-dried to obtain norepinephrine-mineralized collagen scaffold material.

[0012] Furthermore, in the amorphous calcium phosphate solution, the concentration of polyacrylic acid is 1-2 mg / mL, the concentration of calcium ions is 0.025-0.1 mol / L, and the concentration of phosphate ions is 0.015-0.06 mol / L.

[0013] Furthermore, the preparation of the amorphous calcium phosphate solution stabilized by polyacrylic acid specifically includes:

[0014] Polyacrylic acid was dissolved in water, and then CaCl2 was added to obtain a mixture.

[0015] K2HPO4 solution was added dropwise to the mixture, and then NaOH was added to adjust the pH to 9.5±0.2 to obtain an amorphous calcium phosphate solution stabilized by polyacrylic acid.

[0016] Furthermore, in the collagen solution, the concentration of collagen is 0.5–1 mg / mL, and the mass ratio of collagen to norepinephrine is 100:(0.5–5).

[0017] Preferably, the collagen includes any one of bovine collagen, porcine collagen, and fish skin collagen.

[0018] Furthermore, the process of performing isoelectric focusing treatment on the collagen solution under a constant voltage to form a dense film, followed by stretching the dense film to produce a deformation of 120% to 200%, to obtain a collagen film with a biomimetic structure, specifically includes:

[0019] The collagen solution is subjected to isoelectric focusing treatment at a constant voltage of 10-20V for 10-20 minutes to form a dense film. The dense film is then stretched at a speed of 1-3mm / min to produce a deformation of 120%-200%, thereby obtaining a collagen film with a biomimetic structure.

[0020] Furthermore, the step of immersing the collagen membrane in the amorphous calcium phosphate solution and then freeze-drying it to obtain a norepinephrine-mineralized collagen scaffold material specifically includes:

[0021] The collagen membrane was immersed in the amorphous calcium phosphate solution at 37±1℃ for 12±2h, and then freeze-dried to obtain norepinephrine-mineralized collagen scaffold material.

[0022] Based on the same inventive concept, the present invention provides a mineralized collagen scaffold material with a biomimetic structure, wherein the mineralized collagen scaffold material is prepared by the above-mentioned method for preparing a mineralized collagen scaffold material with a biomimetic structure.

[0023] Based on the same inventive concept, this invention provides the application of a mineralized collagen scaffold material with a biomimetic structure as or in the preparation of bone defect repair materials.

[0024] Based on the same inventive concept, the present invention provides a bone defect repair material, wherein the bone defect repair material comprises the above-mentioned mineralized collagen scaffold material with a biomimetic structure;

[0025] Alternatively, the raw materials for preparing the bone defect repair material may include one of the above-mentioned mineralized collagen scaffold materials with a biomimetic structure.

[0026] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0027] 1. This invention discloses a method for preparing a biomimetic mineralized collagen scaffold material. The method involves adding an appropriate amount of norepinephrine (NE) to a collagen solution, preparing a collagen membrane with a biomimetic structure using isoelectric focusing, then immersing the collagen membrane in an amorphous calcium phosphate solution stabilized by polyacrylic acid (PAA) for mineralization, and finally freeze-drying to obtain a norepinephrine-mineralized collagen scaffold. The scaffold material constructed by this invention can be used in bone defect repair systems. The phase ratio and mechanical strength of the micro-scaffold can be adjusted according to different concentrations of amorphous calcium phosphate solution. It exhibits high bioactivity, bone tissue repair promotion ability, and nerve repair promotion ability, and can be applied in biomedical fields such as drug controlled-release systems and bone defect treatment.

[0028] 2. This invention discloses a method for preparing a biomimetic mineralized collagen scaffold material. The method uses collagen as the scaffold substrate, and collagen fibers are oriented using isoelectric focusing. Norepinephrine is added during this process to allow it to penetrate the internal structure of the material. The material is then soaked in an amorphous calcium phosphate solution at 37°C. The amorphous calcium phosphate solution is a PAA-stabilized CaCl2 and K2HPO4 solution with a Ca:P ratio of 1.67. Multiple functional groups on the collagen surface combine with calcium phosphate salts to form a hydroxyapatite (HA) mineral layer, enhancing the mechanical and biological properties of the collagen scaffold. Finally, the material is freeze-dried to obtain a norepinephrine-loaded scaffold. Norepinephrine provides anisotropic mineralized collagen scaffolds with structures similar to natural bone. This method, starting from the perspective of material structure, uses collagen and hydroxyapatite to simulate the highly arranged and anisotropic fine structure of natural bone in vitro, enabling the material to acquire functions and properties similar to natural bone tissue. The addition of norepinephrine enhances the material's bone regeneration ability from the perspective of promoting nerve growth. It can be applied to bone tissue engineering. The preparation process is simple, easy to implement, has mild conditions, and a short production cycle. It is non-toxic and harmless to organisms, providing rich theoretical guidance and technical foundation for the design and development of biomimetic scaffolds, and has great application value in the biomedical field.

[0029] 3. This invention provides a mineralized collagen scaffold material with a biomimetic structure. This material has a highly arranged, anisotropic structure, which enhances the mechanical properties and structural stability of the scaffold. The collagen has good biocompatibility and low antigenicity. The HA formed by surface mineralization synergistically enhances the biological properties of the material, promotes the attachment and growth of bone marrow mesenchymal stem cells (BMSCs), and provides a favorable environment for the formation of new bone tissue. Furthermore, the mineralized HA has excellent biocompatibility and osteogenic induction, and has the function of promoting osteogenic and angiogenesis, which can be used for the repair of hard bone. At the same time, hydroxyapatite HA, as an inorganic mineral, provides a certain degree of mechanical strength to the scaffold.

[0030] 4. This invention provides a biomimetic mineralized collagen scaffold material. This material is obtained by adding NE to collagen at room temperature, followed by isoelectric focusing at 10-20V and mineralization with amorphous calcium phosphate. Changing the concentration of the amorphous calcium phosphate solution can regulate the content of inorganic substances mineralized on the collagen surface within a certain range. It can be used for the repair of bone tissue defects in hard bone, and has good biocompatibility, mechanical properties and structural stability. After freeze-drying, the scaffold has certain interconnected pores, which is conducive to the exchange of nutrients, cell migration and proliferation. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 The image shows a scanning electron microscope (SEM) image of the norepinephrine-mineralized collagen scaffold prepared in Example 1.

[0033] Figure 2 The image shows a tensile test of the norepinephrine-mineralized collagen scaffold prepared in Example 1.

[0034] Figure 3 Data on the in vitro activity of the norepinephrine-mineralized collagen scaffold prepared in Example 1 on BMSC cells.

[0035] Figure 4 The image shows a CT scan of the norepinephrine-mineralized collagen scaffold prepared in Example 1 after 4 weeks of in vivo treatment of skull defects in rats. Detailed Implementation

[0036] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0037] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0038] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0039] The overall concept of this invention is as follows:

[0040] Bone tissue accounts for approximately 15% of the total body weight in an adult human body. It is a multi-layered structural organ responsible for movement and bearing a range of biomechanical loads. With the advent of nanoscale three-dimensional imaging technology, researchers have proposed a nine-level structure of bone. The first four levels belong to the microstructure, involving the main components of bone, mineralized collagen fibrils, and their arrangement. The latter five levels belong to the macrostructure, including bone type (woven bone, parallel fibrous bone), tissue elements (lamellar bodies, osteoid units, fibrolamellar bone), cancellous bone, and compact bone. From a macroscopic structural perspective, bone structure is mainly divided into compact bone, which is hard and highly resistant to pressure, located on the bone surface, and cancellous bone, located on the inner side of the bone, composed of numerous spongy / sheet-like trabeculae. Compact bone consists of bone fragments and Haversian canals surrounding blood vessels, exhibiting a layered structure, with individual sheets composed of orderly arranged bundles of mineralized collagen fibers. Compared to compact bone, cancellous bone exhibits a significantly more porous morphology, producing more cellular components on its surface, resulting in significantly inferior mechanical properties compared to compact bone. From a microscopic structural perspective, a characteristic basic unit of bone is mineralized collagen fibrils, primarily composed of HA and Type I collagen (Collagen I). This can be described using a mineral and collagen assembly model: curved, needle-like mineral particles laterally merge to form slightly twisted plates, creating a continuous fibrous cross-phase on the surface and in the spaces between collagen fibers. The inventors believe that it is precisely this highly aligned, anisotropic, biphasic, and intricate structural design that gives bone tissue its excellent tensile strength and toughness, as well as high stiffness and compressive strength.

[0041] Norepinephrine (NE) is both a neurotransmitter, primarily synthesized and secreted by postganglionic sympathetic neurons and adrenergic nerve endings in the brain, and a hormone, synthesized and secreted by the adrenal medulla, but in smaller quantities. It has been reported that NE can promote the release of glial cell-derived neurotrophic factor (GDNF) from bone medullary stem cells (BMSCs), thus acting as a bioactive factor that nourishes normal nerve cells, regulates the repair function of damaged nerves, maintains the survival of sympathetic and sensory nerves, promotes nerve cell differentiation, and determines the direction of axonal extension. In bone development and regeneration, non-bone systems such as the nervous, vascular, and immune systems play indispensable roles. Various types of nerves enter the bone and promote bone tissue regeneration by secreting various molecules and interacting with bone cell lineages (such as osteoblasts and bone marrow stromal stem cells) and other cells colonizing the bone microenvironment (such as osteoclasts and vascular endothelial cells). Despite the interdependent relationship between nerves and bone, most research in bone tissue engineering has neglected the role of the nervous system.

[0042] Based on this, the present invention proposes a mineralized collagen scaffold material with a biomimetic structure. This material can improve the mechanical properties and stability of the collagen scaffold to meet the needs of bone defect treatment, and can also load neurotransmitters to achieve controlled release of micro-drugs and nerve repair during bone defect treatment, thereby meeting the needs of clinical bone tissue repair.

[0043] Inspired by natural bone, this invention enhances the mechanical and biological properties of the matrix material through a biomimetic mineralization strategy, mimicking the structure and properties of natural bone tissue, thereby improving the material's bone tissue repair effect. Collagen and hydroxyapatite are the most abundant organic and inorganic components in natural bone, respectively, and are often used as biomimetic composite materials in tissue engineering due to their excellent biocompatibility and biodegradability. This invention, from a material structure perspective, uses isoelectric focusing to prepare a highly aligned, anisotropic structure similar to natural bone. This directional alignment gives the material better mechanical properties and structural stability, while its interconnected voids facilitate cell migration and proliferation. Furthermore, the nervous system plays an indispensable role in bone regeneration; introducing the neurotransmitter NE into the bone regeneration material further enhances the material's osteogenic induction capacity from the perspective of nerve growth. This biomimetic material has excellent application prospects in bone tissue engineering.

[0044] This invention uses collagen (bovine Achilles tendon extract) as raw material. Collagen is the most abundant protein, with type I collagen accounting for 90% of all types of collagen in the body. It is mainly found in bones, tendons, cornea, and ligaments, and has excellent biocompatibility, low antigenicity, high swelling capacity, and the ability to promote cell adhesion and growth, as well as provide a favorable environment for the formation of new bone tissue. It also plays a significant role in the mechanical stability and tunability of mammalian collagen tissue. Norepinephrine, chemically classified as a catecholamine, binds to collagen through CN bonds. As the material degrades, it promotes the secretion of bioactive factors by BMSCs that regulate the repair function of damaged nerves, maintains the survival of sympathetic and sensory nerves, and promotes bone repair from the perspective of nerve growth. The amorphous calcium phosphate solution used in the subsequent mineralization step can also penetrate into the collagen interstitial space, promoting the formation of interstitial space mineralization and enhancing the mechanical properties of the collagen scaffold.

[0045] This invention also proposes a method for preparing a biomimetic mineralized collagen scaffold material. The method uses an amorphous calcium phosphate solution to mineralize the collagen scaffold, which does not produce substances harmful to organisms and improves the material's stability and mechanical properties. Various functional groups on the collagen surface can bind with calcium ions in the solution to form sites, promoting the formation of HA crystals. The amorphous calcium phosphate stabilized by PAA is fluid, positively charged, and small in size, allowing minerals to penetrate into the negatively charged intercellular spaces of the collagen fibers, forming intercellular mineralization that further enhances the material's mechanical properties. The prepared norepinephrine-mineralized collagen scaffold material is completely free of toxic chemicals and has high potential application value in fields such as biomineralization and bone tissue engineering.

[0046] This invention uses collagen as the main raw material to prepare scaffold materials. The operation steps are simple, the cycle is short, the conditions are mild, and the source is widely available. The material has low toxicity risk, good stability and degradability, and adjustable mechanical properties. When norepinephrine is added to the material, it can effectively promote BMSC proliferation and nerve growth, which has great application and promotion value.

[0047] Specifically, the present invention provides a method for preparing a mineralized collagen scaffold material with a biomimetic structure, comprising:

[0048] S1. Prepare an amorphous calcium phosphate solution stabilized by polyacrylic acid, wherein the molar ratio of Ca to P in the amorphous calcium phosphate solution is 1.67:1;

[0049] S2. Prepare a collagen solution containing norepinephrine;

[0050] S3. The collagen solution is subjected to isoelectric focusing treatment under constant voltage to form a dense film, and then the dense film is stretched to produce a deformation of 120% to 200% to obtain a collagen film with a biomimetic structure.

[0051] S4. The collagen membrane is immersed in the amorphous calcium phosphate solution and then freeze-dried to obtain norepinephrine-mineralized collagen scaffold material.

[0052] In the amorphous calcium phosphate solution, the concentration of polyacrylic acid is 1-2 mg / mL, the concentration of calcium ions is 0.025-0.1 mol / L, and the concentration of phosphate ions is 0.015-0.06 mol / L.

[0053] In this invention, the molar ratio of Ca to P in the amorphous calcium phosphate solution is 1.67:1, and the pH is adjusted to 9.5 ± 0.2 to promote the crystallization of calcium phosphate into hydroxyapatite. In the amorphous calcium phosphate solution, polyacrylic acid attracts calcium and phosphorus ions through electrostatic forces, significantly restricting calcium and phosphorus crystallization and forming amorphous calcium phosphate clusters. These fluid, small-sized, and positively charged amorphous calcium phosphate clusters allow the minerals to penetrate into the interstitial spaces of collagen.

[0054] Step S1 specifically includes:

[0055] Polyacrylic acid was dissolved in water, and then CaCl2 was added to obtain a mixture.

[0056] K2HPO4 solution was added dropwise to the mixture, and then NaOH was added to adjust the pH to 9.5±0.2 to obtain an amorphous calcium phosphate solution stabilized by polyacrylic acid.

[0057] In this invention, the advantage of first dissolving polyacrylic acid in water and then adding CaCl2 and K2HPO4 solutions in sequence is that it ensures that the polyacrylic acid is evenly distributed in the solution, which is beneficial for its uniform binding with calcium and phosphorus ions.

[0058] The collagen solution contains collagen at a concentration of 0.5–1 mg / mL, and the mass ratio of collagen to norepinephrine is 100:(0.5–5).

[0059] Preferably, the collagen includes bovine collagen.

[0060] In this invention, using the above-mentioned concentration range for collagen is beneficial for the formation of anisotropy. Too high a concentration can easily disrupt the directional arrangement of collagen molecules, while too low a concentration can lead to a thinner collagen membrane, which is detrimental to subsequent steps. Similarly, using the above-mentioned concentration range for norepinephrine is limited by the properties of norepinephrine itself; too high a concentration will produce negative effects, while too low a concentration will fail to achieve the desired effect.

[0061] Step S3 specifically includes:

[0062] The collagen solution is subjected to isoelectric focusing treatment at a constant voltage of 10-20V for 10-20 minutes to form a dense film. The dense film is then stretched at a speed of 1-3mm / min to produce a deformation of 120%-200%, thereby obtaining a collagen film with a biomimetic structure.

[0063] In this invention, the voltage of the isoelectric focusing treatment is 10-20V, which can efficiently prepare anisotropic collagen membranes while maintaining the material structure. Too high a voltage will cause a large number of bubbles to be generated, affecting the material structure, while too low a voltage will require more energizing time, affecting efficiency.

[0064] In this invention, the purpose of stretching the dense membrane at a speed of 1 to 3 mm / min to produce a deformation of 120% to 200% is to further improve the directional arrangement structure of collagen molecules through stretching, while ensuring that the anisotropic structure is maintained after the collagen molecules form collagen bundles during the mineralization process.

[0065] Step S4 specifically includes:

[0066] The collagen membrane was immersed in the amorphous calcium phosphate solution at 37±1℃ for 12±2h, and then freeze-dried to obtain norepinephrine-mineralized collagen scaffold material.

[0067] More specifically, the present invention provides a method for preparing a mineralized collagen scaffold material with a biomimetic structure, comprising:

[0068] (1) An amorphous calcium phosphate solution was prepared using a polymer-stabilized method. The specific method is as follows: Prepare raw materials: anhydrous CaCl2, K2HPO4·3H2O, and PAA. Weigh 0.04-0.08g of PAA and dissolve it in 40ml of RO water. Stir at room temperature for 15min and then add 0.222-0.888g of anhydrous CaCl2 and continue stirring until homogeneous. Weigh 0.04-0.08g of PAA and dissolve it in RO water. Add K2HPO4 to prepare 40ml of 0.03-0.12M K2HPO4 solution and slowly add it dropwise to the above solution. After the addition is complete, continue stirring at room temperature for 30min to mix it evenly. The final solution Ca:P ratio is 1.67. Adjust the pH to 9.5±0.2 with NaOH.

[0069] (2) The fabrication system of the biomimetic mineralized collagen scaffold material is as follows: First, 13 mg / g of collagen is diluted with RO water to a collagen solution of 0.5-1.0 mg / ml, and 5-50 μl of 20 mg / ml norepinephrine solution is added and stirred for 30 min. Ti sheet is used as the anode and graphite sheet is used as the negative electrode. A constant voltage of 10-20 V is applied to the above collagen solution for 10-20 min through an electrochemical workstation to obtain a densely arranged collagen sheet with orientation. The collagen sheet is stretched at a speed of 1-3 mm / min using a stretching texture apparatus to produce a tensile deformation of 120%-200%, which further improves the orientation of collagen fibers. Finally, the material is soaked in 0.025-0.1 M amorphous calcium phosphate solution at a constant temperature of 37±1℃ for 12±2 h. After drying the material in a freeze dryer, a biomimetic mineralized collagen scaffold material loaded with norepinephrine is obtained for the repair of bone defects.

[0070] The following will provide a detailed description of the preparation method, the mineralized collagen scaffold material with a biomimetic structure, and its application, in conjunction with embodiments and experimental data.

[0071] Example 1

[0072] This embodiment describes a method for preparing a mineralized collagen scaffold material with a biomimetic structure, specifically including:

[0073] 1) An amorphous calcium phosphate solution was prepared using a polymer-stabilized method, as follows: Anhydrous CaCl2, K2HPO4·3H2O, and PAA were prepared. 0.05 g of PAA was weighed and dissolved in 40 ml of RO water. After stirring at room temperature for 15 min, 0.222 g, 0.444 g, or 0.888 g of anhydrous CaCl2 was added and stirring continued until homogeneous. 0.05 g of PAA was weighed and dissolved in RO water. K2HPO4 was added to prepare 40 ml of a 0.03 M, 0.06 M, or 0.12 M K2HPO4 solution, which was then slowly added dropwise to the above solution. After the addition was complete, stirring was continued at room temperature for 30 min to ensure homogeneity. The final solution Ca:P ratio was 1.67. The pH was adjusted to 9.5 using NaOH.

[0074] 2) The fabrication system of the biomimetic mineralized collagen scaffold loaded with norepinephrine is as follows: First, 13 mg / g bovine collagen (extracted from bovine Achilles tendon - fresh bovine Achilles tendon, after removing the tendon membrane, fat and muscle, was cut into small pieces, soaked in 10% NaCl solution for about 24 hours, then washed several times with deionized water and air-dried at room temperature. 50 g of Achilles tendon was weighed and soaked in 0.5 M acetic acid solution for 2 hours. Pepsin was added at an enzyme-to-achilles tendon mass ratio of 1:50, and the mixture was stirred at 4℃ for 72 hours for enzymatic hydrolysis. The solid block was removed by filtration, and the filtrate was centrifuged at 4000 rpm / min for 20 min. The supernatant was collected and salted out with 0.75 M NaCl solution. Then, it was dialyzed at 4℃ for 3 days. Finally, the collagen concentration was determined using a freeze dryer) was diluted with RO water to a total of 20 ml to a collagen solution of 0.75 mg / ml. 20 μl of the solution was added. A 20 mg / ml norepinephrine solution was stirred for 30 min. Using a Ti sheet as the anode and a graphite sheet as the negative electrode, a constant voltage of 15 V was applied to the collagen solution for 15 min using an electrochemical workstation to obtain a densely packed collagen sheet with directional alignment. The collagen sheet was stretched at a speed of 2 mm / min using a stretching texture apparatus to produce a 150% stretching deformation, further improving the directional alignment of collagen fibers. Finally, the material was immersed in amorphous calcium phosphate solutions with calcium ion concentrations of 0.025 M, 0.05 M, and 0.1 M at a constant temperature of 37 °C for 12 h. After drying the material in a freeze dryer, an anisotropic mineralized collagen scaffold loaded with norepinephrine was obtained for the repair of bone defects.

[0075] Example 2

[0076] This embodiment tests the performance of the scaffold material prepared in Example 1, as detailed below:

[0077] 1. The collagen, oriented collagen membrane, and prepared mineralized collagen scaffold samples from the preparation process of the norepinephrine-mineralized collagen scaffold in Example 1 were cut into cross-sections along the oriented alignment direction using a thin blade. These sections were then fixed to the sample stage with double-sided conductive adhesive and subjected to 90s gold sputtering. The cross-sectional micromorphology was observed using a scanning electron microscope (SEM) at an accelerating voltage of 3.5 kV. The results are as follows: Figure 1 , Figure 1 In this invention, EC represents the collagen membrane with a biomimetic structure, and ACP represents amorphous calcium phosphate. The preparation methods for the EC+0.025MACP, EC+0.05MACP, and EC+0.1MACP materials are as follows:

[0078] 1) Same as step 1) in Example 1;

[0079] 2) No norepinephrine solution was added to the collagen solution, and the remaining operations were the same as step 2) in Example 1 to obtain EC+0.025MACP, EC+0.05MACP and EC+0.1MACP materials.

[0080] like Figure 1 As shown, amorphous collagen scaffolds and oriented collagen scaffolds exhibit porous surfaces, while the other four types of lamellar scaffolds consist of multiple, interwoven long mineralized collagen fibers. Amorphous collagen scaffolds exhibit a disordered state, with collagen fibers arranged randomly without directionality. In contrast, collagen fibers in scaffolds prepared by isoelectric focusing show a clear directionality, revealing the basic D-band structure of collagen more clearly compared to amorphous collagen. Comparing the morphologies of scaffold materials prepared at three mineralization concentrations reveals that minerals cover the collagen fiber surface. It can be observed that under EC+0.05MACP mineralization conditions, the anisotropic structure is preserved even with complete mineralization; EC+0.025MACP mineralization is incomplete; and under EC+0.1MACP mineralization conditions, the morphology of collagen fibers is obscured. Furthermore, using EC+0.05MACP+NE, it can be observed that the addition of NE does not alter the surface morphology of the scaffold material.

[0081] 2. Mineralized collagen scaffolds under different mineralization conditions were prepared into sheets 2.5 cm long, 2.5 cm wide, and 1 mm thick. These sheets were fixed in the fixtures of a universal testing machine and tested at a tensile rate of 2 mm / min. Stress-strain curves were plotted to characterize the tensile properties of the mineralized collagen scaffolds under different mineralization conditions. The results are as follows: Figure 2 .

[0082] like Figure 2 As shown, the upper figure shows the experimental results of tensile testing along the oriented alignment direction (longitudinal direction), and the lower figure shows the experimental results of tensile testing perpendicular to the oriented alignment direction (transverse direction). It can be observed that different degrees of mineralization significantly alter the mechanical properties of the material. The mechanical properties of the scaffold increase with increasing mineralization solution concentration. The maximum tensile stress of the EC+0.1MACP material exceeds 2.5 MPa. The presence of hydroxyapatite crystals enhances the stiffness of the collagen fibers but also reduces their elastic deformation range, thereby decreasing their fracture toughness. Due to the influence of the collagen fiber alignment direction, the unmineralized biomimetic collagen scaffold exhibits better stiffness and toughness in longitudinal tension than the non-oriented collagen scaffold under the same conditions. Under the same conditions, the scaffold material's mechanical strength in the transverse direction is significantly weaker than in the longitudinal direction, and both its stiffness and toughness in the transverse direction are lower than those in the longitudinal direction of the same group. Compared to the non-oriented collagen scaffold, the anisotropic scaffold has better mechanical properties in the longitudinal direction but weaker mechanical properties in the transverse direction, demonstrating the material's anisotropy.

[0083] 3. Amorphous collagen, EC+0.05MACP, and EC+0.05MACP+NE materials were co-cultured with BMSC cells. On day 3, the culture medium was removed, and 110 μL of fresh culture medium containing Cell Counting Kit-8 (CCK-8) was added (the ratio of CCK-8 detection solution to culture medium was 1:10). Relative cell viability was measured according to the reagent instructions. The results are as follows: Figure 3 .

[0084] like Figure 3 As shown, after collagen mineralization, no large-scale cell death occurred, proving that the material has good biocompatibility. Comparing materials with and without added NE, it can be observed that the cell growth of the material with NE is better than that of the material without NE, indicating that the addition of NE is beneficial to cell growth.

[0085] 4. Eight-week-old rats were anesthetized by intraperitoneal injection of 4% sodium pentobarbital (40 mg / kg). The rat's head was skinned and the surgical site was cleaned with povidone-iodine. An incision was then made near the midsagittal line to expose the parietal bone. The periosteum was removed, and a 5 mm defect was created on one side of the un-sutured parietal bone using a trephine drill to avoid dural perforation. The surgical area was cleansed with saline, and each defect was filled with a prepared mineralized collagen scaffold. After implantation, the skin was sutured. Twenty-eight days post-surgery, the rats were euthanized by intraperitoneal injection of an overdose of sodium pentobarbital, and the skull was harvested and preserved in 4% paraformaldehyde for micro-computed tomography (μCT) evaluation.

[0086] like Figure 4As shown, the skulls of rats implanted with biomimetic mineralized collagen scaffolds exhibited better repair effects, indicating that the implanted scaffold material has good in vivo biocompatibility. Rats implanted with NE-loaded mineralized collagen scaffolds showed greater new bone coverage, which can be attributed to two factors: firstly, mineralized collagen plays a crucial role in repairing bone defects and in bone formation; secondly, it may be due to NE promoting the release of neurotrophic factors from BMSCs, thus promoting nerve regeneration and synergistically promoting new bone formation with mineralized collagen.

[0087] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0088] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0089] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing a mineralized collagen scaffold material with a biomimetic structure, characterized in that, The preparation method includes: Prepare an amorphous calcium phosphate solution stabilized by polyacrylic acid, wherein the molar ratio of Ca to P in the amorphous calcium phosphate solution is 1.67:1; Prepare a collagen solution containing norepinephrine; The collagen solution is subjected to isoelectric focusing treatment under constant voltage to form a dense film, and then the dense film is stretched to produce a deformation of 120% to 200% to obtain a collagen film with a biomimetic structure. The collagen membrane was immersed in the amorphous calcium phosphate solution and then freeze-dried to obtain norepinephrine-mineralized collagen scaffold material. In the amorphous calcium phosphate solution, the concentration of polyacrylic acid is 1-2 mg / mL, the concentration of calcium ions is 0.025-0.1 mol / L, and the concentration of phosphate ions is 0.015-0.06 mol / L. The preparation of the amorphous calcium phosphate solution stabilized by polyacrylic acid specifically includes: Polyacrylic acid was dissolved in water, and then CaCl2 was added to obtain a mixture. K2HPO4 solution was added dropwise to the mixture, and then NaOH was added to adjust the pH to 9.5±0.2 to obtain an amorphous calcium phosphate solution stabilized by polyacrylic acid. The collagen in the collagen solution is bovine collagen.

2. The method for preparing a mineralized collagen scaffold material with a biomimetic structure according to claim 1, characterized in that, The collagen solution contains collagen at a concentration of 0.5–1 mg / mL, and the mass ratio of collagen to norepinephrine is 100:(0.5–5).

3. The method for preparing a mineralized collagen scaffold material with a biomimetic structure according to claim 1, characterized in that, The process of isoelectric focusing treatment of the collagen solution under constant voltage to form a dense film, followed by stretching of the dense film to produce a deformation of 120% to 200%, to obtain a collagen film with a biomimetic structure, specifically includes: The collagen solution is subjected to isoelectric focusing treatment at a constant voltage of 10-20V for 10-20 minutes to form a dense film. The dense film is then stretched at a speed of 1-3mm / min to produce a deformation of 120%-200%, thereby obtaining a collagen film with a biomimetic structure.

4. The method for preparing a mineralized collagen scaffold material with a biomimetic structure according to claim 1, characterized in that, The process involves immersing the collagen membrane in the amorphous calcium phosphate solution, followed by freeze-drying to obtain a norepinephrine-mineralized collagen scaffold material, specifically comprising: The collagen membrane was immersed in the amorphous calcium phosphate solution at 37±1℃ for 12±2h, and then freeze-dried to obtain norepinephrine-mineralized collagen scaffold material.

5. A mineralized collagen scaffold material with a biomimetic structure, characterized in that, The mineralized collagen scaffold material is prepared by any one of the preparation methods of a mineralized collagen scaffold material with a biomimetic structure according to any one of claims 1-4.

6. The application of the biomimetic mineralized collagen scaffold material as described in claim 5 in the use of or preparation of bone defect repair materials.

7. A bone defect repair material, characterized in that, The bone defect repair material includes a mineralized collagen scaffold material with a biomimetic structure as described in claim 5; Alternatively, the raw materials for preparing the bone defect repair material may include a mineralized collagen scaffold material with a biomimetic structure as described in claim 5.

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