A method for complexing inorganic minerals and proteins

Abstract

The application provides a method for preparing inorganic mineral and protein composite materials, which comprises the following steps: injecting a uniformly mixed protein solution and an inorganic mineral solution into a shearing stress loading chamber, adding a dehydrating agent into a dialysis chamber, separating the shearing stress loading chamber and the dialysis chamber by a dialysis membrane, applying a shearing stress of 1-10 Pa, acting for 1-8 days, and reacting at a temperature of 10-50 DEG C; and the molecular weight cut-off range of the dialysis membrane is 3-50 kDa. The obtained composite material has excellent mechanical properties, including high strength and toughness, and exhibits good biocompatibility, can support cell adhesion, proliferation and differentiation, and thus effectively promotes bone tissue regeneration. The composite material prepared by the application has a wide application prospect in the fields of bone repair and other biomedical fields.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medical material preparation, and particularly relates to a method for compounding inorganic minerals and proteins, which is based on shear stress and dialysis concentration technology to prepare inorganic mineral and protein-based composite materials, and is suitable for the field of tissue engineering, such as the field of bone defect repair. BACKGROUND

[0002] With the rapid development of bone repair and tissue engineering field, developing biological medical materials with good mechanical properties and biocompatibility has become a research focus. Bone tissue has a complex hierarchical structure, and its regeneration and repair requires materials with high mechanical strength, excellent biocompatibility, and support for cell adhesion, proliferation and differentiation. Traditional bone repair materials have certain biological activity, but there are still deficiencies in mechanical properties, tissue integration and material processability. Therefore, it is particularly important to develop composite materials with excellent mechanical properties and biological functions.

[0003] In the application of bone repair and tissue engineering, proteins (such as collagen, serum protein, silk fibroin, fibronectin, etc.) have been widely used in the preparation of biological medical materials due to their excellent biocompatibility and ability to support cell growth. These proteins can form a three-dimensional fibrous structure similar to natural tissue in a high concentration state, which is crucial for simulating the mechanical properties and biological functions of bone tissue. However, due to the low concentration of protein solution and the uneven arrangement of fibers, the mechanical properties and structural stability are limited, making it difficult to meet the requirements of clinical application. Existing bone repair materials have deficiencies in mechanical properties, biocompatibility and tissue integration, and are difficult to meet multiple requirements such as high strength, toughness and cell support. SUMMARY

[0004] Therefore, the present application aims to provide a method for compounding inorganic minerals and proteins to overcome the deficiencies of the prior art. The present application is a method for preparing inorganic mineral and protein-based composite materials based on shear stress and dialysis concentration technology. It is particularly suitable for the field of biological medical materials, such as bone defect repair and tissue engineering applications. Through the method of the present application, the concentration of protein solution can be effectively improved, the order of fiber arrangement can be improved, and the compounding with inorganic minerals can be promoted, ultimately preparing a composite scaffold material with excellent mechanical properties and biocompatibility.

[0005] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0006] The application relates to a method for compounding inorganic minerals and proteins, which comprises the following steps: injecting a uniformly mixed protein solution and an inorganic mineral solution into a shearing stress loading chamber, adding a dehydrating agent into a dialysis chamber, separating the shearing stress loading chamber from the dialysis chamber through a dialysis membrane, applying shearing stress of 1-10 Pa, acting for 1-8 days, and reacting at a temperature of 10-50 DEG C; and the molecular weight cut-off range of the dialysis membrane is 3-50 kDa.

[0007] The shearing stress, acting time and reaction temperature can be adjusted, the shearing stress can improve the flowability of the proteins and orderly arrange the proteins along the shearing direction, so that a uniform three-dimensional fiber structure is formed.

[0008] The rotating speed of the rotating cone plate in the shearing stress loading chamber is calculated according to the following three formulas to apply quantitative shearing stress.

[0009]

[0010] Shearing rate (s -1 ); tau: shearing stress (Pa); eta: dynamic viscosity (Pa.s); theta: cone angle (radian); omega: angular velocity (rad / s);

[0011] Preferably, the initial concentration of the protein solution is 1-10 mg / mL; the concentration of the inorganic mineral solution is 1-100 mM; and the volume ratio of the protein solution to the inorganic mineral solution is 1:1-1:10.

[0012] Preferably, the inorganic mineral solution is a solution containing one or more than two of calcium ions, phosphate ions, strontium ions, iron ions, zinc ions, silver ions, magnesium ions, citrate, insoluble strontium salt and insoluble magnesium salt.

[0013] Preferably, the inorganic mineral is one or more than two of hydroxyapatite, calcium carbonate, calcium phosphate, amorphous calcium phosphate and amorphous strontium carbonate solution.

[0014] Preferably, the inorganic mineral solution contains an organic additive, and the organic additive is one or more than two of polyaspartic acid, sodium citrate, polyacrylic acid and carboxymethyl chitosan.

[0015] The functions of the organic additives include, but are not limited to: a) during the complexation of amorphous calcium phosphate, the organic additives stabilize the amorphous calcium phosphate and delay the crystallization process, so that the amorphous calcium phosphate can be more easily incorporated into the organic matrix (e.g. inside collagen fibers) and achieve better complexation; b) the organic additives provide additional functions, such as imparting antibacterial properties to the material by adding specific antibacterial materials (e.g. carboxymethyl chitosan). The synergistic effect of the above-mentioned inorganic minerals and organic additives makes the resulting composite material have better mineralization ability, biocompatibility and functionality in biomedical applications.

[0016] Preferably, the proteins are one or more of collagen, fibroin, keratin, elastin, fibrin, lysozyme, fibronectin, serum protein, lactoferrin, transferrin, insulin, myoglobin, hemoglobin, egg white albumin, β-lactoglobulin, α-lactalbumin, or amyloid-like proteins generated during phase transition of the above proteins.

[0017] Structural proteins: used to provide mechanical support and structural stability of the composite material, including collagen, fibroin, keratin, elastin and fibrin. Among them, collagen imparts good biocompatibility and mechanical strength to the material; fibroin and keratin can enhance the strength and toughness of the material; elastin is suitable for tissue repair materials that require flexibility; fibrin helps to gel and stabilize the structure of the material.

[0018] Functional proteins: used to enhance the biological activity, antibacterial properties and promote cell adhesion, growth and differentiation of the composite material, including lysozyme, fibronectin, serum protein (such as human serum albumin and bovine serum albumin), lactoferrin, transferrin, insulin, myoglobin, hemoglobin, egg white albumin, β-lactoglobulin and α-lactalbumin. Among them, lysozyme and lactoferrin have antibacterial effect; fibronectin promotes cell adhesion and proliferation; serum protein improves the biocompatibility of the material; transferrin and insulin promote cell differentiation and tissue healing; myoglobin and hemoglobin provide local oxygen transport support in repair materials with high oxygen demand; egg white albumin, β-lactoglobulin and α-lactalbumin provide nutritional support.

[0019] Amyloid-like proteins generated during phase transition of the above-mentioned proteins are also included in the protein solution to enhance the mechanical strength, biological activity and structural stability of the composite material.

[0020] Preferably, the dehydrating agent is one or more of polyethylene glycol solution, salt-containing solution, sodium polyacrylate, sodium alginate, sodium carboxymethyl cellulose or silica gel. The salt-containing solution is one or more of NaCl, KCl, Na2HPO4 and KH2PO4 solution.

[0021] Preferably, the concentration of the polyethylene glycol solution is 20-400 mg / mL, preferably 250-350 mg / mL; and the salt concentration of the salt solution is 3-30 g / L.

[0022] Preferably, the shear stress loading chamber is also added with a polysaccharide compound or a polyphenol compound; the concentration of the polysaccharide compound or the polyphenol compound added is 0-10 mg / mL; and the polysaccharide compound or the polyphenol compound is added when it is necessary to improve the biological activity, biocompatibility or mechanical property of the material; preferably, the polysaccharide compound is one or more than two of chondroitin sulfate and hyaluronic acid, and the polyphenol compound is procyanidine.

[0023] Preferably, the flow rate of the liquid in the shear stress chamber and the dialysis chamber is 1 mL / min-15 mL / min, so as to maintain the pressure balance on both sides of the dialysis membrane, remove the water and impurities in the solution, and achieve the concentration purpose.

[0024] Under the action of dialysis concentration and shear stress, a high-concentration inorganic mineral and protein-based composite material is collected. The obtained material not only has high concentration but also has uniform fiber structure arrangement, and has excellent mechanical properties and biocompatibility. The concentrated organic-inorganic composite material can be further processed according to the application needs, for example: the composite gel is converted into a dry scaffold material with a porous structure through freeze-drying technology; a dense scaffold is formed through mechanical compression, which is used for high-load bone repair applications; the freeze-dried material is prepared into a powder form, which is convenient for storage or use in different environments, or is used for mixed preparation of other composite materials.

[0025] The shear stress loading chamber in the above method is realized by a hydraulic shear loading device, the device has a rotating impeller or a piston structure capable of constantly applying shear stress, or the device is a cone and plate viscometer, a syringe pump or a flat plate flow chamber circulating device capable of providing constant direction fluid shear force.

[0026] The application also provides the application of the inorganic mineral and protein-based composite material prepared by the above-mentioned method of inorganic mineral and protein compounding in the preparation of bone repair or other biomedical products.

[0027] The shear thinning effect refers to the phenomenon that the viscosity of a solution decreases with the increase of the shear rate under the action of shear force. The application of appropriate fluid shear force can significantly improve the physical properties of a protein solution (such as a collagen solution). The shear thinning effect improves the flowability of the solution, making it more feasible to operate under high concentration conditions, which provides conditions for concentrating the protein. Under the action of shear stress, the protein not only can reduce the flow resistance, but also can realize more ordered arrangement along the shear direction, thereby forming a three-dimensional fiber matrix with uniform structure and excellent mechanical properties. Such a matrix not only can simulate the fiber arrangement in natural bone tissue, but also can improve the overall mechanical properties of the material.

[0028] In addition to shearing technology, dialysis concentration technology also plays an important role in concentrating protein solutions and preparing composite materials with inorganic minerals. By removing excess water and small molecule impurities, dialysis concentration technology can effectively increase protein concentration, thereby enhancing its binding to inorganic minerals (such as hydroxyapatite and calcium phosphate). The concentrated, high-concentration protein solution can tightly bind to inorganic minerals to form a high-strength composite material with excellent mechanical properties and bioactivity, capable of supporting cell growth and bone tissue regeneration.

[0029] The present invention combines shearing technology with dialysis concentration technology to improve the fluidity of protein solution and the orderliness of fiber arrangement by shear stress, and combines protein concentration with inorganic minerals by dialysis to prepare a composite scaffold material with excellent mechanical properties. These composite materials not only retain the hardness and compressive strength of inorganic minerals, but also have the flexibility and biocompatibility of biomatrix materials, which are very suitable for bone repair fields. By rationally controlling shear stress and concentration conditions, the composite material can have good fluidity and processing properties, while showing high strength and high toughness after final solidification, and is suitable for tissue engineering such as bone repair and other biomedical fields.

[0030] Compared with the prior art, the method of compounding inorganic minerals and proteins of the present invention has the following advantages:

[0031] By combining shear stress and dialysis concentration techniques, this invention not only effectively increases the concentration of the protein solution but also improves the arrangement of protein fibers and the uniformity of the material. This technology promotes the close bonding of protein and inorganic minerals, forming a high-strength composite material with excellent biological functions and mechanical properties, supporting cell adhesion, proliferation, and differentiation, thereby accelerating bone tissue regeneration.

[0032] Compared with existing technologies, this invention significantly increases the concentration and efficiency of protein-inorganic mineral complexation by combining shear stress and dialysis concentration. This allows for the formation of composite scaffold materials with excellent mechanical properties and biofunctionality at high concentrations. This material has broad applications in bone repair, tissue engineering, and other biomedical fields, providing a new method for the efficient preparation of high-performance biomedical materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0034] Figure 1 Schematic diagram of the preparation of inorganic mineral and protein-based composites based on shear stress and dialysis concentration technology;

[0035] Figure 2 Scanning electron microscope (SEM) images of concentrated mineralized collagen scaffolds and static non-concentrated mineralized collagen scaffolds prepared according to the method of Example 1;

[0036] Figure 3 Elemental distribution (EDS Mapping) images of mineralized collagen scaffolds prepared according to the method of Example 1;

[0037] Figure 4 Elemental analysis images of mineralized collagen scaffolds prepared according to the method of Example 1;

[0038] Figure 5 Thermogravimetric analysis comparison of concentrated mineralized collagen scaffolds and static non-concentrated mineralized collagen scaffolds prepared according to the method of Example 1;

[0039] Figure 6 Young's modulus comparison of concentrated mineralized collagen scaffolds and static non-concentrated mineralized collagen scaffolds prepared according to the method of Example 1;

[0040] Figure 7 Scanning electron microscope images of concentrated amorphous calcium phosphate and lysozyme composite materials prepared according to the method of Example 3;

[0041] Figure 8 Cell scanning electron microscope images of the surface of concentrated mineralized collagen scaffolds prepared according to the method of Example 1;

[0042] Figure 9 Scanning electron microscope of concentrated mineralized collagen scaffolds prepared according to the method of Example 4;

[0043] Figure 10 Scanning electron microscope of concentrated mineralized collagen scaffolds prepared according to the method of Example 5. DETAILED DESCRIPTION

[0044] 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 the present application belongs. The experimental reagents used in the following examples are all conventional biochemical reagents unless otherwise specified; and the experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0045] The present application will be described in detail below with reference to the examples.

[0046] Example 1: Preparation of high-concentration inorganic mineral and collagen composite materials based on shear stress and dialysis concentration

[0047] A 3 mg / mL acidic type I collagen solution was mixed with an amorphous calcium phosphate solution at a volume ratio of 1 : 1. The amorphous calcium phosphate solution had a polyaspartic acid concentration of 240 pg / mL, a calcium ion concentration of 1.67 mM, a phosphate ion concentration of 9.5 mM, a sodium chloride concentration of 150 mM, and a sodium citrate concentration of 10 mM. The mixed solution was placed in the upper shear stress loading chamber, the shear loading device was started, a shear stress of 1 Pa was applied, and the temperature was set to 37 °C.

[0048] A 300 mg / mL polyethylene glycol (PEG) solution was placed in the lower dialysis chamber as a dehydrating agent, and the dialysis membrane had a molecular weight cutoff range of 8000-14000 Da. Solutions on both sides of the dialysis membrane were continuously injected into the upper stress loading chamber and the lower dialysis chamber at a flow rate of 1 mL / min.

[0049] Under the action of dialysis and shear stress, after 5 days of treatment, the collagen-amorphous calcium phosphate composite material was gradually concentrated, and the collagen fibers were orderly arranged along the shear direction. After the treatment was completed, the composite material in the upper chamber was collected. The material exhibited high concentration, good fiber arrangement, and excellent mechanical properties, and was suitable for bone repair and tissue engineering applications.

[0050] Comparative Example 1: Preparation of inorganic mineral and collagen composite material under static conditions

[0051] A 3 mg / mL acidic type I collagen solution was mixed with an amorphous calcium phosphate solution at a volume ratio of 1 : 1. The amorphous calcium phosphate solution had a polyaspartic acid concentration of 240 pg / mL, a calcium ion concentration of 1.67 mM, a phosphate ion concentration of 9.5 mM, a sodium chloride concentration of 150 mM, and a sodium citrate concentration of 10 mM. The mixed solution was placed in a beaker, and the reaction temperature was set to 37 °C.

[0052] Under static conditions, after 5 days of treatment, scanning electron microscopy showed that the collagen fiber structure was relatively loose, and obvious pore regions could be seen under low magnification. High magnification images further showed that there were gaps between the fibers, and the three-dimensional network density was significantly lower than that of the shear concentrated sample, and the overall morphology was porous and discontinuous. The deposition of mineral substances such as calcium phosphate crystals on the surface of the collagen fibers was uneven, and the orientation was poor. Compared with the mineralized collagen scaffold prepared by the method of Example 1, the mechanical properties of the material were reduced.

[0053] Figure 2In the middle, scanning electron microscopy shows that the collagen fibers are significantly densified after shearing dialysis concentration, and there is no obvious pore or loose area between the fibers. Under low magnification, the whole structure is a uniform continuous network structure, and under high magnification, the fibers are tightly packed to form a high-density three-dimensional network. The fiber arrangement shows a high degree of order, and the longitudinal section observation shows that the collagen fibers are distributed in parallel layers, with clear layers and consistent orientation. Hydroxyapatite crystals are uniformly deposited on the surface and interstices of collagen fibers, forming a continuous mineralization layer. In the high-magnification SEM image, it can be seen that the nanoscale mineralized particles are tightly attached to the surface of the fibers, forming a typical "collagen-mineral" composite structure. The SEM results of the concentrated mineralized collagen show that the densification, ordering and efficient mineralization of the collagen structure are successfully achieved by the shearing dialysis process. Compared with the concentrated mineralized collagen with applied shear force, the sample without applied shear force is significantly inferior in density, arrangement order and mineralization uniformity.

[0054] Figure 3 In the middle, combined with energy spectrum analysis, calcium and phosphorus elements are uniformly distributed on the collagen fibers, further confirming the high mineralization degree and uniform distribution.

[0055] Figure 4 In the middle, the elemental analysis results explain that the calcium to phosphorus ratio of the mineralized collagen is 1.49, close to the calcium to phosphorus ratio of hydroxyapatite in natural bone;

[0056] Figure 5 In the middle, the thermogravimetric analysis results show that after 5 days of mineralization, the inorganic mineral residue weight of the concentrated mineralized collagen scaffold is 65.53%, which is significantly higher than that of the static group of 47.59%, indicating that the concentrated mineralized collagen scaffold prepared by the shearing dialysis method can significantly improve the deposition of inorganic minerals.

[0057] Figure 6 In the middle, the DMT modulus (representing Young's modulus) of the shear force group is 2.5 GPa on average, which is significantly higher than that of the static group of 1.2 GPa. Higher Young's modulus indicates that the stiffness of the mineralized collagen scaffold is significantly improved after shearing dialysis treatment, reflecting a more dense inorganic mineral-collagen composite structure. Figure 6 The comparison chart of Young's modulus also shows that the introduction of shear force can effectively improve the Young's modulus of the composite mineralized collagen scaffold.

[0058] Figure 8 In the middle, the SEM image shows that the cells have a large number of extended filiform pseudopodia and lamellar pseudopodia, and adhere well to the surface of the concentrated mineralized collagen scaffold. It shows that the high-strength composite material prepared based on this technology has excellent biological function and can support cell adhesion.

[0059] Example 2: Preparation of high-concentration inorganic mineral and hyaluronic acid and collagen protein composite material based on shearing stress and dialysis concentration

[0060] A 2 mg / mL solution of hyaluronic acid was mixed with a 3 mg / mL solution of acidic collagen at a volume ratio of 1 : 10, and then uniformly mixed with an amorphous strontium carbonate solution at a volume ratio of 1 : 2, the amorphous strontium carbonate solution having a concentration of 2 mg / mL of carboxymethyl chitosan, 10 mM of strontium ions, and 6 mM of carbonate ions. The mixed solution was placed in a shear stress loading chamber, a shear stress of 2 Pa was applied, and the temperature was set to 37 °C.

[0061] A 300 mg / mL solution of polyethylene glycol (PEG) was placed in the dialysis chamber as a dehydrating agent, and the molecular weight cut-off range of the dialysis membrane was 8000-14000 Da. The mixed solution and the PEG solution were injected into the shear stress chamber and the dialysis chamber at a flow rate of 1 mL / min, respectively.

[0062] After 5 days of treatment, the fibers of hyaluronic acid and collagen were ordered along the shear direction and were complexed with amorphous strontium carbonate. The complex material collected in the upper chamber exhibited excellent mechanical properties and fiber arrangement, and was suitable for bone repair and tissue engineering.

[0063] Example 3: Preparation of high-concentration amorphous calcium phosphate and lysozyme complex material based on shear stress and dialysis concentration

[0064] A 3 mg / mL solution of lysozyme was mixed with an amorphous calcium phosphate solution at a volume ratio of 1 : 1, the amorphous calcium phosphate solution having a concentration of 240 μg / mL of polyacrylic acid, 1.67 mM of calcium ions, 9.5 mM of phosphate ions, and 150 mM of sodium chloride. The mixed solution was placed in a shear stress loading chamber, a shear stress of 5 Pa was applied, and the temperature was set to 37 °C.

[0065] A 300 mg / mL solution of polyethylene glycol (PEG) was placed in the dialysis chamber as a dehydrating agent, and the molecular weight cut-off range of the dialysis membrane was 8000-14000 Da. The mixed solution and the PEG solution were injected into the shear stress chamber and the dialysis chamber at a flow rate of 1 mL / min, respectively.

[0066] After 5 days of treatment, lysozyme and amorphous calcium phosphate were gradually concentrated under shear stress. The complex material collected in the upper chamber had good mechanical properties and was suitable for bone repair and tissue engineering applications.

[0067] Figure 7In the present embodiment, the scanning electron microscope results show that after the shearing dialysis concentration treatment, the surface of the amorphous calcium phosphate and lysozyme composite material presents a continuous and dense composite network, and the amorphous calcium phosphate particles are uniformly embedded in the lysozyme matrix without obvious pores or cracks. This shows that the shearing force and the concentration effect promote the close interweaving of amorphous calcium phosphate and lysozyme, forming a hierarchical structure similar to natural biomineralization.

[0068] Example 4: Preparation of high-concentration inorganic mineral and collagen composite material based on shearing stress and dialysis concentration

[0069] An acidic type I collagen solution of 3 mg / mL was mixed with an amorphous calcium phosphate solution in a volume ratio of 1:1, and the concentration of polyaspartic acid in the amorphous calcium phosphate solution was 240 μg / mL, the concentration of calcium ions was 1.67 mM, the concentration of phosphate ions was 9.5 mM, the concentration of sodium chloride was 150 mM, and the concentration of sodium citrate was 10 mM. The mixed solution was placed in the upper shearing stress loading chamber, the shearing loading device was started, a shearing stress of 5 Pa was applied, and the temperature was set to 37°C.

[0070] A polyethylene glycol (PEG) solution with a concentration of 300 mg / mL was placed in the lower dialysis chamber as a dehydrating agent, and the molecular weight cutoff range of the dialysis membrane was 8000-14000 Da. The solutions on both sides of the dialysis membrane were continuously injected into the upper stress loading chamber and the lower dialysis chamber at a flow rate of 1 mL / min.

[0071] Under the action of dialysis and shearing stress, after 5 days of treatment, the collagen and amorphous calcium phosphate composite material was gradually concentrated, and the collagen fibers were orderly arranged along the shearing direction. After the treatment was completed, the composite material in the upper chamber was collected. The material exhibits high concentration, good fiber arrangement, and excellent mechanical properties, and is suitable for bone repair and tissue engineering applications.

[0072] Figure 9 In the present embodiment, the scanning electron microscope shows that after dialysis concentration under the treatment of 5 Pa shearing stress, the collagen fiber structure is significantly densified, and there are no obvious pores or loose areas between the fibers. The fiber arrangement exhibits high orderliness, and hydroxyapatite crystals are uniformly deposited on the surface and interstices of the collagen fibers, forming a continuous mineralized layer.

[0073] Example 5: Preparation of high-concentration inorganic mineral and collagen composite material based on shearing stress and dialysis concentration

[0074] A 3 mg / mL acidic type I collagen solution was mixed with an amorphous calcium phosphate solution at a volume ratio of 1:1, the amorphous calcium phosphate solution having a polyaspartic acid concentration of 240 μg / mL, a calcium ion concentration of 1.67 mM, a phosphate ion concentration of 9.5 mM, a sodium chloride concentration of 150 mM, and a sodium citrate concentration of 10 mM. The mixed solution was placed in the upper shear stress loading chamber, the shear loading device was started, a shear stress of 10 Pa was applied, and the temperature was set to 37°C.

[0075] A polyethylene glycol (PEG) solution having a concentration of 300 mg / mL was placed in the lower dialysis chamber as a dehydrating agent, and a dialysis membrane having a molecular weight cutoff range of 8000-14000 Da was used. The solutions on both sides of the dialysis membrane were continuously injected into the upper stress loading chamber and the lower dialysis chamber at a flow rate of 1 mL / min.

[0076] Under the action of dialysis and shear stress, the collagen-amorphous calcium phosphate composite material was gradually concentrated after 5 days of treatment, and the collagen fibers were orderly arranged along the shear direction. After the treatment was completed, the composite material in the upper chamber was collected. The material exhibited high concentration, good fiber arrangement, and excellent mechanical properties, and was suitable for bone repair and tissue engineering applications.

[0077] Figure 10 In the above, the scanning electron microscope showed that under the action of a shear stress of 10 Pa, after dialysis concentration, the collagen fiber structure was significantly densified, and there were no obvious pores or loose areas between the fibers. The fiber arrangement exhibited a high degree of order, and hydroxyapatite crystals were uniformly deposited on the surface and interstices of the collagen fibers, forming a continuous mineralization layer.

[0078] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for compounding inorganic minerals and proteins, characterized in that: The uniformly mixed protein solution and inorganic mineral solution are injected into a shear stress loading chamber, a dehydrating agent is added to a dialysis chamber, and the shear stress loading chamber and the dialysis chamber are separated by a dialysis membrane. The applied shear stress is 1-10 Pa, the action time is 5-8 days, the reaction temperature is 10-50°C, and the flow rate of the liquid in the shear stress chamber and the dialysis chamber is 1 mL / min-15 mL / min. The molecular weight cut-off range of the dialysis membrane is 3-14 kDa. The initial concentration of the protein solution is 1-10 mg / mL; the concentration of the inorganic mineral solution is 1-100 mM; and the volume ratio of the protein solution to the inorganic mineral solution is 1:1-1:

10. The inorganic mineral is one or more of hydroxyapatite, calcium carbonate, calcium phosphate, amorphous calcium phosphate, and amorphous strontium carbonate solution; the inorganic mineral solution contains an organic additive, and the organic additive is one or more of polyaspartic acid, sodium citrate, polyacrylic acid, and carboxymethyl chitosan; The protein is one or more of the amyloid-like proteins generated during the phase transition of collagen, fibroin, keratin, elastin, fibrin, lysozyme, fibronectin, serum albumin, lactoferrin, transferrin, insulin, myoglobin, hemoglobin, egg white albumin, β-lactoglobulin, α-lactalbumin, or the like; The dehydrating agent is a polyethylene glycol solution, and the concentration of the polyethylene glycol solution is 250-350 mg / mL.

2. The method for compounding inorganic minerals and proteins according to claim 1, characterized in that: A polysaccharide compound or a polyphenol compound is also added into the shear stress loading chamber; the concentration of the added polysaccharide compound or the polyphenol compound is 0-10 mg / mL.

3. The method for compounding inorganic minerals and proteins according to claim 1, characterized in that: The polysaccharide compound is one or more of chondroitin sulfate and hyaluronic acid, and the polyphenol compound is proanthocyanidin.

4. Use of the inorganic mineral and protein-based composite material prepared by the method for combining inorganic minerals and proteins according to any one of claims 1 to 3 in the preparation of bone repair or other biomedical products.

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