CN119455110A

CN119455110A - Preparation method of mineralized collagen scaffold material with bionic structure, mineralized collagen scaffold material and application thereof

Info

Publication number: CN119455110A
Application number: CN202411606848.9A
Authority: CN
Inventors: 郭泰林; 吴安航; 唐玲玲; 谭欢; 吴昭君; 王潇潇; 卢佳琛; 游静
Original assignee: Southwest Jiaotong University
Current assignee: Southwest Jiaotong University
Priority date: 2024-11-12
Filing date: 2024-11-12
Publication date: 2025-02-18

Abstract

The invention provides a preparation method of mineralized collagen scaffold material with a bionic structure, belonging to the field of biomedical engineering, comprising the steps of preparing an amorphous calcium phosphate solution stabilized by polyacrylic acid, 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, carrying out isoelectric focusing treatment on the collagen solution under constant voltage to form a compact membrane, stretching the compact membrane to generate 120% -200% deformation to obtain a collagen membrane with a bionic structure, soaking the collagen membrane in the amorphous calcium phosphate solution, and freeze-drying to obtain the norepinephrine-mineralized collagen scaffold material. The mineralized collagen scaffold material prepared by the method has excellent biocompatibility, osteoinductive property and enough mechanical strength, can effectively promote bone tissue defect repair, and has good application prospect in the field of bone tissue engineering. The invention also provides a mineralized collagen scaffold material with a bionic structure and application thereof.

Description

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

Technical Field

The invention belongs to the field of biomedical engineering, and in particular relates to a preparation method of a mineralized collagen scaffold material with a bionic structure, the mineralized collagen scaffold material and application thereof.

Background

Bone serves as the most important hard tissue in the human body and plays a supporting and protecting role. Bone tissue defects caused by sports, traffic accidents, diseases and the like are common clinical diseases, harm the health of patients and bring great pain to the patients. Currently, the main methods for bone tissue repair are implantation of autograft, allograft, xenograft or synthetic artificial bone. Autologous bone has dual functions of bone induction and bone conduction and has proved to be an effective means of bone repair clinically. However, its use is limited due to the lack of donor tissue and additional secondary defects. Meanwhile, the risk of pain or infection brought to patients by immune rejection or disease transmission caused by allogeneic bone transplantation and xenogeneic bone transplantation is high, and the occurrence of immune rejection reaction can be reduced by low-temperature treatment, but the mechanical strength and osteoinductive property of the bone graft can be correspondingly reduced. Therefore, the development of bone tissue defect repair materials with good performance is of great significance.

The ideal bone tissue repair material should have good bone conductivity, biocompatibility, absorbability and sufficient mechanical strength. Currently, commonly used bone repair materials include metallic materials, ceramic implants, and polymeric scaffold materials. The most clinically applied metal material is titanium alloy, titanium is nontoxic and harmless, corrosion resistance is good, and elastic modulus is close to human hard tissue. However, the titanium alloy surface is relatively smooth, resulting in poor bone-bonding properties. Ceramic implants are mainly calcium phosphate ceramics, including hydroxyapatite (hydroxyapat ite, HA), β -tricalcium phosphate, etc. have been widely studied in the field of bone repair, although similar to the inorganic composition in natural bone tissue, the poor fracture toughness and tensile strength of calcium phosphate ceramics have limited its application in the field of biomaterials. Polymeric scaffold materials are capable of complexing growth factors thereon to accelerate bone repair, but most polymers have weaker bone conduction capabilities.

Disclosure of Invention

In order to obtain the bone tissue defect repair material with good performance, the invention provides a preparation method of the mineralized collagen scaffold material with a bionic structure, and the mineralized collagen scaffold material prepared by the method has excellent biocompatibility, osteogenesis inducibility and enough mechanical strength, can effectively promote bone tissue defect repair, and has good application prospect in the field of bone tissue engineering.

The invention also provides a mineralized collagen scaffold material with a bionic structure and application thereof.

The invention is realized by the following technical scheme:

the invention provides a preparation method of a mineralized collagen scaffold material with a bionic structure, which comprises the following steps:

Preparing an amorphous calcium phosphate solution stabilized by polyacrylic acid, 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 constant voltage to form a compact membrane, and stretching the compact membrane to generate 120% -200% deformation to obtain a collagen membrane with a bionic structure;

And immersing the collagen membrane in the amorphous calcium phosphate solution, and then freeze-drying to obtain the norepinephrine-mineralized collagen scaffold material.

Further, 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 mL/L, and the concentration of phosphate ions is 0.015-0.06 mol/L.

Further, the preparation of the amorphous calcium phosphate solution stabilized by polyacrylic acid specifically comprises the following steps:

dissolving polyacrylic acid in water, and then adding CaCl ₂ to obtain a mixed solution;

And (3) dropwise adding a K ₂HPO₄ solution into the mixed solution, and then adding NaOH to adjust the pH to 9.5+/-0.2 to obtain an amorphous calcium phosphate solution stabilized by polyacrylic acid.

Further, in the collagen solution, the concentration of collagen is 0.5-1 mg/mL, and the mass ratio of the collagen to the norepinephrine is 100 (0.5-5).

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

Further, the isoelectric focusing treatment is performed on the collagen solution under a constant voltage to form a dense film, and then the dense film is stretched to generate 120% -200% deformation, so as to obtain the collagen film with a bionic structure, which specifically comprises the following steps:

And carrying out isoelectric focusing treatment on the collagen solution for 10-20 min under a constant voltage of 10-20V to form a compact film, and then stretching the compact film at a speed of 1-3 mm/min to generate 120-200% deformation, so as to obtain the collagen film with the bionic structure.

Further, the collagen membrane is soaked in the amorphous calcium phosphate solution and then is frozen and dried to obtain the norepinephrine-mineralized collagen scaffold material, which specifically comprises the following steps:

Immersing the collagen membrane in the amorphous calcium phosphate solution for 12+/-2 hours at 37+/-1 ℃, and then freeze-drying to obtain the norepinephrine-mineralized collagen scaffold material.

Based on the same inventive concept, the invention provides a mineralized collagen scaffold material with a bionic structure, which is prepared by the preparation method of the mineralized collagen scaffold material with the bionic structure.

Based on the same inventive concept, the invention provides an application of mineralized collagen scaffold material with a bionic structure in serving as or preparing a bone defect repair material.

Based on the same inventive concept, the invention provides a bone defect repair material, which comprises the mineralized collagen scaffold material with a bionic structure;

or the preparation raw materials of the bone defect repair material comprise the mineralized collagen scaffold material with a bionic structure.

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

1. The invention relates to a preparation method of mineralized collagen scaffold material with bionic structure, which comprises adding appropriate amount of Norepinephrine (NE) into collagen solution, preparing collagen film with bionic structure by isoelectric focusing method, immersing the collagen film in amorphous calcium phosphate solution stabilized by polyacrylic acid (PAA) for mineralization, freeze drying to obtain norepinephrine-mineralized collagen scaffold, the scaffold material constructed by the invention can be used for a bone defect repair system, can regulate and control the two-phase proportion and the mechanical strength of a micro scaffold according to different amorphous calcium phosphate solution concentrations, has higher bioactivity, bone tissue repair promoting capability and nerve repair promoting capability, and can be applied to biomedical fields such as a drug controlled release system, bone defect treatment and the like.

2. The invention relates to a preparation method of mineralized collagen scaffold material with bionic structure, which takes collagen as scaffold base material, and makes collagen fiber directional arrangement through isoelectric focusing method, the process adds norepinephrine to make it enter into material internal structure, then through immersing under 37 ℃ amorphous calcium phosphate solution, amorphous calcium phosphate solution is CaCl ₂ and K ₂HPO₄ solution stabilized by PAA, ca: P is 1.67, various functional groups on collagen surface are combined with calcium phosphate to form Hydroxyapatite (HA) mineral layer to enhance mechanical property and biological property of collagen scaffold, finally through freeze drying to obtain mineralized collagen scaffold loaded with norepinephrine with anisotropy, the structure is similar to natural hard bone, the method uses collagen protein and hydroxyapatite to simulate high arrangement of natural bone in vitro, anisotropic fine structure, so that the material obtains similar function and property to natural bone tissue, the addition of norepinephrine enhances bone regeneration capability from nerve growth promotion angle, the preparation process is easy to implement, the condition is mild, the production cycle is short, the bionic scaffold is innoxious, and provides great medical application in the field of the development of bionic scaffold with great theoretical and great basic value.

3. The mineralized collagen scaffold material with the bionic structure HAs the advantages that the mineralized collagen scaffold material HAs the high-degree arrangement and anisotropic structure, the mechanical property and the structural stability of the scaffold are enhanced, collagen HAs good biocompatibility and low antigenicity, HA formed by mineralization on the surface of the mineralized collagen scaffold material synergistically enhances the biological property of the material, the adhesion and growth of BMSC cells can be promoted, a favorable environment can be provided for the formation of new bone tissues, the mineralized HA HAs excellent biocompatibility and osteogenesis inducibility, the functions of promoting the growth of bones and blood vessels are achieved, the mineralized HA can be used for repairing hard bone parts, and meanwhile, hydroxyapatite HA serving as an inorganic mineral provides a certain degree of mechanical strength for the scaffold.

4. The mineralized collagen scaffold material with the bionic structure is prepared by mineralizing collagen at room temperature by using amorphous calcium phosphate after NE (NE) is added and isoelectric focusing is performed at 10-20V, the concentration of an amorphous calcium phosphate solution is changed, the mineralized inorganic matter content on the surface of the collagen can be regulated and controlled within a certain range, the mineralized collagen scaffold material can be used for repairing defects of hard bone tissues, has good biocompatibility, mechanical property and structural stability, and the scaffold after freeze drying has certain communicated pores, thereby being beneficial to the exchange of nutrient substances, the migration of cells and proliferation.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a scanning electron microscope image of norepinephrine-mineralized collagen scaffolds prepared in example 1.

FIG. 2 is a tensile test chart of norepinephrine-mineralized collagen scaffolds prepared in example 1.

FIG. 3 is the in vitro activity data of norepinephrine-mineralized collagen scaffolds prepared in example 1 on BMSC cells.

FIG. 4 is a CT image of the norepinephrine-mineralized collagen scaffolds prepared in example 1 acting in vivo for 4 weeks on repair of rat skull defects.

Detailed Description

The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.

Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, 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 belongs. In case of conflict, the present specification will control.

Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.

The whole idea of the invention is as follows:

bone tissue in an adult human accounts for about 15% of the total body weight of the human body, and is a motor organ with multiple structural layers and carrying a range of biomechanical loads. With the advent of nanoscale three-dimensional imaging techniques, researchers have proposed nine-level structures of bone, the first four-level structure belonging to a microstructure involving the arrangement of the main components of bone, mineralized collagen fibrils, and mineralized collagen fibers. The latter five-level structure belongs to the macroscopic structure, including the types of bones (woven bones, parallel fibrobones), tissue elements (lamellar bodies, bone units, fibreboard bones), cancellous and compact bones, etc. From the viewpoint of macrostructure, bone structure is mainly divided into compact bone mass located on the surface of bone with hard texture and strong compression resistance, and spongy/flaky bone trabeculae interwoven into bone mass located on the inside of bone. The compact bone consists of bone blocks and Hupeh tubes surrounding blood vessels, and is in a layered structure, and single sheets are formed by orderly arranging mineralized collagen fiber bundles. Cancellous bone exhibits a significant morphological characterization of porosity relative to cortical bone, producing more cellular components on its surface, which results in a cancellous bone with far less mechanical properties than cortical bone. From a microstructure point of view, a characteristic basic unit of bone is mineralized collagen fibrils, the main components of which are HA and type I collagen (TypeIco l l agen, co ii). It can be described by a mineral and collagen assembly model in which curved needle-like mineral particles merge laterally to form slightly distorted plates, forming a continuous fibrous cross phase at the surface and interstices of the collagen. The inventors believe that it is this highly aligned, anisotropic, biphasic fine structure design that gives bone tissue excellent tensile and toughness, as well as high stiffness and compressive strength.

Norepinephrine (NE) is a neurotransmitter synthesized and secreted primarily by postsympathetic neurons and adrenergic nerve endings in the brain, and is the main transmitter released by the latter, and is also a hormone synthesized and secreted by the adrenal medulla, but in a lesser amount. NE has been reported to exert a trophic effect on normal nerve cells by promoting release of glial cell derived neurotrophic factor (GDNF) from BMSCs, to have a bioactive factor that has a regulatory effect on damaged nerve repair functions, to maintain survival of sympathetic nerves and sensory nerves, to promote differentiation of nerve cells, and to determine the direction of axon extension. In skeletal development and regeneration, non-bone systems such as nervous system, vascular system, immune system, etc. play an indispensable role. Various types of nerves enter the bone and promote bone tissue regeneration by secreting various molecules and interacting with bone cell line cells (e.g., osteoblasts and bone marrow stromal cells) and other cells that colonize the bone microenvironment (e.g., osteoclasts and vascular endothelial cells). Despite the interdependent relationship between nerves and bones, most studies in the field of bone tissue engineering have ignored the role of the nervous system.

Based on the above, the invention provides a mineralized collagen scaffold material with a bionic structure, which can not only improve the mechanical property and stability of the collagen scaffold to meet the requirements in the bone defect treatment process, but also load neurotransmitters to realize the controlled release of trace drugs and the repair of nerves in the bone defect process so as to meet the requirements of clinical bone tissue repair.

The invention is inspired by natural bone, and by simulating the structure and the property of the natural bone tissue, the mechanical property and the biological property of the matrix material are improved by using a biomimetic mineralization strategy, and the bone tissue repair effect of the material is enhanced. 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. From the aspect of material structure, the invention prepares a highly arranged and anisotropic structure similar to natural bone by isoelectric focusing, and the directional arrangement ensures that the material has better mechanical property and structural stability, and meanwhile, the communicating gaps are beneficial to cell migration and proliferation. In addition, the nervous system has an indispensable role in the bone regeneration process, neurotransmitter NE is introduced into the bone regeneration material, and the osteoinductive capacity of the material is further improved from the perspective of nerve growth. The bionic material has good application prospect in bone tissue engineering.

The collagen protein is the most abundant protein, wherein the type I collagen protein accounts for 90% of all types of collagen proteins in the body, mainly exists in bones, bone tendons, corneas and ligaments, has excellent biocompatibility, low antigenicity and high swelling capacity, promotes cell attachment and growth, provides favorable environment for the formation of new bone tissues, has great effect on the mechanical stability and adjustability of the collagen tissues of mammals, and the norepinephrine belongs to catecholamine in chemical structure, is combined with the collagen through C-N bonds, promotes BMSC secretion of bioactive factors with regulation effect on damaged nerve repair functions along with the degradation of the material, maintains the survival of sympathetic nerves and sensory nerves, promotes the formation of bone repair from the aspect of nerve growth, and the amorphous calcium phosphate solution used in the subsequent mineralization step can also permeate into collagen gaps to promote the formation of collagen gaps and strengthen the mechanical property of a collagen bracket.

The invention also provides a preparation method of the mineralized collagen scaffold material with the bionic structure, and the mineralized collagen scaffold is mineralized by adopting the amorphous calcium phosphate solution, so that substances harmful to organisms are not generated, and the stability and mechanical property of the material are improved. The various functional groups on the surface of the collagen can combine with calcium ions in the solution to form sites, so that the formation of HA crystals is promoted. The amorphous calcium phosphate stabilized by PAA has the characteristics of flowing, positive charge and small size, so that minerals can permeate into collagen interstitial regions with negative charges, and collagen fiber interstitial mineralization is formed to further improve the mechanical properties of the material. The prepared norepinephrine-mineralized collagen scaffold material does not contain toxic chemical agents at all, and has high potential application value in the fields of biomineralization, bone tissue engineering and the like.

The preparation method takes collagen as a main raw material to prepare the scaffold material, has the advantages of simple operation steps, short period, mild condition, wide source, low toxicity risk of the material, good stability and degradability, adjustable mechanical property, capability of effectively promoting BMSC proliferation and nerve growth by adding norepinephrine into the material, and great application and popularization value.

Specifically, the invention relates to a preparation method of a mineralized collagen scaffold material with a bionic structure, which comprises the following steps:

s1, preparing an amorphous calcium phosphate solution stabilized by polyacrylic acid, wherein the molar ratio of Ca element to P element in the amorphous calcium phosphate solution is 1.67:1;

S2, preparing a collagen solution containing norepinephrine;

s3, carrying out isoelectric focusing treatment on the collagen solution under constant voltage to form a compact film, and then stretching the compact film to generate 120% -200% deformation to obtain the collagen film with a bionic structure;

s4, soaking the collagen membrane in the amorphous calcium phosphate solution, and then freeze-drying to obtain the norepinephrine-mineralized collagen scaffold material.

Wherein, 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 mL/L, and the concentration of phosphate ions is 0.015-0.06 mol/L.

In the invention, the molar ratio of Ca element to P element in the amorphous calcium phosphate solution is 1.67:1, and the PH is adjusted to 9.5+/-0.2, so that calcium phosphate salt can be promoted to crystallize to form hydroxyapatite. In the amorphous calcium phosphate solution, polyacrylic acid plays a role of attracting calcium ions and phosphorus ions through electrostatic force, so that calcium phosphorus crystallization is limited to a large extent, and amorphous calcium phosphate clusters are formed. The small size of the fluid, positively charged amorphous calcium phosphate clusters allows minerals to penetrate into the interstices of collagen.

The step S1 specifically comprises the following steps:

In the invention, the polyacrylic acid is firstly dissolved in water, and then CaCl ₂ and K ₂HPO₄ solution are sequentially added, so that the polyacrylic acid is ensured to be uniformly distributed in the solution, and the uniform combination of the polyacrylic acid with calcium ions and phosphorus ions is facilitated.

In the collagen solution, the concentration of collagen is 0.5-1 mg/mL, and the mass ratio of the collagen to the norepinephrine is 100 (0.5-5).

Preferably, the collagen comprises bovine collagen.

In the invention, the collagen is favorable for the formation of anisotropy by adopting the concentration range, the directional arrangement of collagen molecules is easily damaged by too high concentration, and the thickness of the collagen film is easily reduced by too low concentration, so that the operation of the subsequent steps is not favorable. The concentration range of norepinephrine is limited by the nature of norepinephrine, and too high concentration can produce negative effects, and too low concentration can not produce due effects.

The step S3 specifically comprises the following steps:

According to the invention, the voltage of the isoelectric focusing treatment is 10-20V, so that the anisotropic collagen film can be efficiently prepared under the condition of keeping the material structure, a large number of bubbles can be generated to influence the material structure due to the fact that the voltage is too high, and more power-on time is needed to influence the efficiency due to the fact that the voltage is too low.

In the invention, the compact film is stretched at a speed of 1-3 mm/min to generate 120-200% deformation, so that the directional arrangement structure of collagen molecules is further improved by stretching, and meanwhile, the anisotropic structure of the collagen molecules is still maintained after the collagen molecules form collagen bundles in the mineralization process.

The step S4 specifically comprises the following steps:

More specifically, the invention relates to a preparation method of mineralized collagen scaffold material with a bionic structure, which comprises the following steps:

(1) The method for preparing the amorphous calcium phosphate solution by adopting the polymer stabilization method comprises the steps of preparing anhydrous CaCl ₂、K₂HPO₄·3H₂ O, PAA serving as a raw material, weighing 0.04-0.08 g of PAA, dissolving in 40ml of RO water, stirring at room temperature for 15min, adding 0.222-0.888 g of anhydrous CaCl ₂, continuously stirring uniformly, weighing 0.04-0.08 g of PAA, dissolving in RO water, adding K ₂HPO₄ to prepare 40ml of K ₂HPO₄ solution with the concentration of 0.03-0.12M, slowly dripping the solution, continuously stirring at room temperature for 30min after dripping is finished, mixing uniformly, and finally adjusting the pH of the solution to be 1.67 by NaOH, wherein Ca is P, and the pH is adjusted to be 9.5+/-0.2.

(2) A preparation system of the mineralized collagen scaffold material with the bionic structure comprises the steps of firstly diluting 13mg/g of collagen into 0.5-1.0 mg/ml of collagen solution by adding RO water, adding 5-50 μl of 20mg/ml of norepinephrine solution, stirring for 30min, taking Ti sheets as anodes and graphite sheets as cathodes, applying constant voltage of 10-20V for 10-20 min to the collagen solution through an electrochemical workstation to obtain dense collagen sheets in directional arrangement, stretching the collagen sheets at a speed of 1-3 mm/min through a stretching texture analyzer to enable the collagen sheets to generate 120% -200% of stretching deformation, further improving the directional arrangement of collagen fibers, finally soaking the material in 0.025-0.1M of amorphous calcium phosphate solution at a constant temperature of 37+/-1 ℃ for 12+/-2 h, taking out the material, drying the material through a freeze dryer, and obtaining the mineralized collagen scaffold material with the norepinephrine-loaded bionic structure, and the mineralized collagen scaffold material is used for repairing bone defects.

The following describes a method for preparing a mineralized collagen scaffold material with a bionic structure, a mineralized collagen scaffold material and application thereof in detail by combining examples and experimental data.

Example 1

The preparation method of the mineralized collagen scaffold material with the bionic structure specifically comprises the following steps:

1) The preparation method of the amorphous calcium phosphate solution by adopting the polymer stabilization method comprises the steps of preparing anhydrous CaCl ₂、K₂HPO₄·3H₂ O, PAA serving as a raw material, weighing 0.05g of PAA, dissolving in 40ml of RO water, stirring at room temperature for 15min, adding 0.222g or 0.444g or 0.888g of anhydrous CaCl ₂, continuously stirring uniformly, weighing 0.05g of PAA, dissolving in RO water, adding K ₂HPO₄ to prepare 40ml of K ₂HPO₄ solution with the pH of 0.03M or 0.06M or 0.12M, slowly dripping the solution, continuously stirring at room temperature for 30min after dripping is completed, uniformly mixing, and finally adjusting the pH of the solution to be 1.67 by NaOH.

2) The preparation system of the norepinephrine-loaded biomimetic structure mineralized collagen scaffold is that 13mg/g bovine collagen (extracted from bovine achilles tendon-fresh bovine achilles tendon with aponeurosis, fat and muscle removed is cut into small pieces, soaked in 10% NaCl solution for about 24 hours, washed with deionized water for multiple times and naturally dried at normal temperature is used. 50g of achilles tendon is weighed and soaked in 0.5M acetic acid solution for 2 hours, pepsin is added according to the mass ratio of enzyme to achilles tendon of 1:50, and stirring and enzymolysis are carried out for 72 hours at 4 ℃. Filtering to remove solid block, freeze centrifuging at 4000rpm/min for 20min, collecting supernatant, salting out with 0.75M NaCl solution, dialyzing at 4deg.C for 3 days, freeze drying to determine collagen concentration), diluting with RO water to 20ml of 0.75mg/ml collagen solution, adding 20 μl of 20mg/ml norepinephrine solution, stirring for 30min, using Ti sheet as anode and graphite sheet as cathode, applying constant voltage 15V for 15min to the collagen solution via electrochemical workstation to obtain oriented compact collagen sheet, stretching with a stretching texture analyzer at 2mm/min to obtain 150% stretched deformation, further improving collagen fiber orientation, soaking in amorphous calcium phosphate solution with calcium ion concentration of 0.025M, 0.05M and 0.1M at 37deg.C for 12 hr, drying by freeze drying to obtain mineralized scaffold loaded with norepinephrine with anisotropy, and repairing bone defect.

Example 2

In this example, the stent material prepared in example 1 was subjected to performance testing, specifically as follows:

1. The cross sections of the collagen, the oriented collagen film and the prepared mineralized collagen scaffold sample in the norepinephrine-mineralized collagen scaffold preparation process of example 1 were cut along the oriented arrangement direction by a thin blade, fixed on a sample table through a double-sided conductive adhesive, subjected to 90s metal spraying treatment, and observed under an acceleration voltage of 3.5kV by using a Scanning Electron Microscope (SEM), and the result is shown in FIG. 1, EC represents the collagen film with a bionic structure of the invention, and ACP represents amorphous calcium phosphate. The preparation methods of the materials of EC+0.025MACP, EC+0.05MACP and EC+0.1MACP are as follows:

1) Step 1) as in example 1;

2) The procedure of example 1, step 2), was followed except that no norepinephrine solution was added to the collagen solution to obtain ec+0.025MACP, ec+0.05MACP, and ec+0.1MACP materials.

As shown in fig. 1, the amorphous and oriented collagen scaffolds present porous surfaces, while the other four plate layered scaffolds present long mineralized collagen fibers interlaced with each other by a plurality of. The amorphous collagen scaffold exhibits a disordered state, and the arrangement of collagen fibers is not directional and is randomly arranged. The collagen fibers in the collagen scaffold prepared by isoelectric focusing can be obviously provided with directionality, and compared with amorphous collagen, the basic D-band structure of the collagen can be more clearly seen. By comparing the shapes of the scaffold materials prepared by three mineralization concentrations, the mineral substances can be seen to cover the surface of the collagen fibers, and the situation that the complete anisotropic structure can be reserved under the condition of complete mineralization under the condition of mineralization of EC+0.05MACP, the mineralization of EC+0.025MACP is incomplete, and the shape of the collagen fibers is covered under the mineralization condition of EC+0.1MACP can be observed. Meanwhile, as can be observed by EC+0.05MACP+NE, the addition of NE does not change the surface morphology of the scaffold material.

2. Mineralized collagen scaffolds under different mineralization conditions were prepared into a sheet shape with a length of 2.5cm, a width of 2.5cm and a thickness of 1mm, and the sheet shape was fixed on a clamp of a universal mechanical testing machine, tested at a tensile rate of 2mm/min, and a stress-strain curve was drawn to characterize the tensile properties of the mineralized collagen scaffolds under different mineralization conditions, and the results are shown in FIG. 2.

As shown in fig. 2, the upper graph shows the experimental result of stretching along the direction of alignment (longitudinal direction), and the lower graph shows the experimental result of stretching perpendicular to the direction of alignment (transverse direction). It can be observed that the mineralization of different degrees obviously changes the mechanical properties of the material, the mechanical properties of the scaffold are improved along with the increase of the mineralization liquid concentration, the maximum tensile stress of the EC+0.1MACP material exceeds 2.5MPa, and the existence of hydroxyapatite crystals enhances the rigidity of the collagen fibers, but also reduces the elastic deformation range of the collagen fibers, so that the fracture toughness of the collagen fibers is reduced. The non-oriented collagen scaffold has the advantages that the longitudinal stretching rigidity and toughness of the non-mineralized bionic structure collagen scaffold are superior to those of the non-oriented collagen scaffold under the same condition, the transverse mechanical strength of the scaffold material is obviously weaker than that of the non-oriented collagen scaffold under the same condition, the transverse rigidity and toughness of the scaffold material are lower than those of the non-oriented collagen scaffold under the same group, and compared with the non-oriented collagen scaffold, the anisotropic scaffold has better longitudinal mechanical performance, but weaker transverse mechanical performance, so that the material has anisotropy.

3. Amorphous collagen, EC+0.05MACP and EC+0.05 MACPE material were co-cultured with BMSC cells, respectively, and on day 3, the medium was removed and 110. Mu.L of fresh medium containing cell counting reagent (Ce L L Count ing Kit-8, CCK-8) was added (wherein the ratio of CCK-8 assay to medium was 1:10). The relative cell activity was determined following the procedure described in the reagent specifications and the results are shown in FIG. 3.

As shown in fig. 3, it can be seen that after collagen mineralization, cells do not die in large amounts, demonstrating that the material has good biocompatibility. Comparing whether NE is added or not, it can be observed that the cell growth of the NE-containing material is better than the NE-free material, indicating that the addition of NE is beneficial for cell growth.

4. Rats at 8 weeks of age were anesthetized with 4% sodium pentobarbital (40 mg/kg) intraperitoneal injection. The rat head was peeled off and the surgical site was cleaned with iodophor. An incision is then made near the sagittal midline to expose the parietal bone. The cranium was removed and a 5mm defect was created on one side of the non-suture-related parietal bone using a trephine to avoid dural perforation. The surgical field was washed with normal saline and each defect was filled with a prepared mineralized collagen scaffold. After implantation, the skin is sutured. Rats were sacrificed 28 days post-surgery by intraperitoneal injection of excess sodium pentobarbital, and the skull was harvested and saved in 4% paraformaldehyde for Micro-Computed Tomography (μct) evaluation.

As shown in fig. 4, the skull of the rat to which the biomimetic mineralized collagen scaffold was added was observed to show a better repair effect, indicating that the implanted scaffold material had good in vivo biocompatibility. The new bone coverage of bone tissue generated by a rat implanted with the NE mineralized collagen scaffold is larger, on one hand, the mineralized collagen has a repairing effect on bone tissue defects and plays an important role in the bone formation process, and on the other hand, the NE can promote BMSC to release neurotrophic factors, promote nerve regeneration and promote new bone generation in cooperation with mineralized collagen.

Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims

1. A method for preparing a mineralized collagen scaffold material with a bionic structure, which is characterized by comprising the following steps:

Preparing a collagen solution containing norepinephrine;

2. The method for preparing the mineralized collagen scaffold material with the bionic structure according to claim 1, wherein the concentration of polyacrylic acid in the amorphous calcium phosphate solution 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.

3. The method for preparing a mineralized collagen scaffold material with a bionic structure according to claim 1 or 2, wherein the preparation of the amorphous calcium phosphate solution stabilized by polyacrylic acid specifically comprises the following steps:

4. The method for preparing the mineralized collagen scaffold material with the bionic structure according to claim 1, wherein the concentration of collagen in the collagen solution is 0.5-1 mg/mL, and the mass ratio of the collagen to the norepinephrine is 100 (0.5-5).

5. The method for preparing a mineralized collagen scaffold material with a bionic structure according to claim 1, wherein the collagen comprises any one of bovine collagen, porcine collagen and fish skin collagen.

6. The method for preparing a mineralized collagen scaffold material with a bionic structure according to claim 1, wherein the method comprises the steps of performing isoelectric focusing treatment on the collagen solution under constant voltage to form a dense membrane, and stretching the dense membrane to generate 120% -200% deformation to obtain the collagen membrane with the bionic structure, and specifically comprises the following steps:

7. The method for preparing a mineralized collagen scaffold material with a bionic structure according to claim 1, wherein the method comprises immersing the collagen membrane in the amorphous calcium phosphate solution, and freeze-drying to obtain the norepinephrine-mineralized collagen scaffold material, specifically comprising:

8. A mineralized collagen scaffold material with a biomimetic structure, characterized in that the mineralized collagen scaffold material is prepared by a preparation method of the mineralized collagen scaffold material with a biomimetic structure according to any one of claims 1 to 7.

9. Use of a mineralized collagen scaffold material with a biomimetic structure according to claim 8 as or in the preparation of a bone defect repair material.

10. A bone defect repair material, characterized in that the bone defect repair material comprises the mineralized collagen scaffold material with a bionic structure according to claim 8;

or the preparation raw material of the bone defect repair material comprises the mineralized collagen scaffold material with a bionic structure as claimed in claim 8.