Inorganic mineral and protein compounding method

Abstract

The invention provides a method for compounding an inorganic mineral and a protein matrix, which comprises the following steps: injecting a protein solution and an inorganic mineral solution which are uniformly mixed into a shear stress loading chamber, adding a dehydrating agent into a dialysis chamber, separating the shear stress loading chamber from the dialysis chamber through a dialysis membrane, applying shear stress of 1-10Pa, and acting for 1-8 days, the reaction temperature is 10-50 DEG C; the molecular weight interception range of the dialysis membrane is 3-50 kDa. The obtained composite material has excellent mechanical properties including high strength and toughness, shows good biocompatibility, and can support adhesion, proliferation and differentiation of cells, thereby effectively promoting bone tissue regeneration. The composite material prepared by the invention has wide application prospects in bone repair and other biomedical fields.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of biomedical materials, and in particular relates to a method for compounding inorganic minerals and proteins. This method is based on shear stress and dialysis concentration techniques to prepare inorganic mineral and protein-based composite materials, and is applicable to the field of tissue engineering, such as bone defect repair. Background Art

[0002] With the rapid development of the fields of bone repair and tissue engineering, the development of biomedical materials with good mechanical properties and biocompatibility has become the research focus. Bone tissue has a complex hierarchical structure, and its regeneration and repair require materials with high mechanical strength, excellent biocompatibility, and the ability to support cell adhesion, proliferation, and differentiation. Although traditional bone repair materials have certain biological activities, they still have deficiencies in mechanical properties, tissue integration, and material processability. Therefore, it is particularly important to develop composite materials that simultaneously possess excellent mechanical properties and biological functions.

[0003] In bone repair and tissue engineering applications, proteins (such as collagen, serum protein, silk fibroin, fibronectin, etc.) have been widely used in the preparation of biomedical materials due to their excellent biocompatibility and ability to support cell growth. At high concentrations, these proteins can form three-dimensional fiber structures similar to natural tissues, which is crucial for mimicking the mechanical properties and biological functions of bone tissue. However, due to the usually low concentration of protein solutions and uneven fiber arrangement, their mechanical properties and structural stability are limited, making it difficult to meet the requirements of clinical applications. Existing bone repair materials have deficiencies in mechanical properties, biocompatibility, and tissue integration, and it is difficult to simultaneously meet multiple requirements such as high strength, toughness, and cell support. Summary of the Invention

[0004] In view of this, the present invention aims to propose a method for compounding inorganic minerals and proteins to overcome the deficiencies of the prior art. The present invention is a method for preparing inorganic mineral and protein-based composite materials based on shear stress and dialysis concentration techniques. It is particularly applicable to the field of biomedical materials, such as bone defect repair and tissue engineering applications. Through the method of the present invention, the concentration of protein solutions can be effectively increased, the arrangement orderliness of fibers can be improved, and their compounding with inorganic minerals can be promoted, ultimately preparing composite scaffold materials with excellent mechanical properties and biocompatibility.

[0005] To achieve the above object, the technical solution of the present invention is realized as follows:

[0006] A method for compounding inorganic minerals and proteins, wherein a uniformly mixed protein solution and an inorganic mineral solution are injected into a shear stress loading chamber, a dehydrating agent is added to a dialysis chamber, the shear stress loading chamber and the dialysis chamber are separated by a dialysis membrane, the applied shear stress is 1 to 10 Pa, the action time is 1 day to 8 days, and the reaction temperature is 10 to 50 °C; the molecular weight cut-off range of the dialysis membrane is 3 to 50 kDa;

[0007] The magnitude of the shear stress, the action time, and the reaction temperature are adjustable. The shear stress can improve the fluidity of the protein and arrange it orderly along the shear direction, thereby forming a uniform three-dimensional fiber structure.

[0008] The rotation speed of the rotating cone plate in the shear stress loading chamber is calculated according to the following three formulas to apply a quantitative shear stress:

[0009]

[0010] Shear rate (s -1 ); τ: Shear stress (Pa); η: Dynamic viscosity (Pa·s); θ: Cone angle (radian); Ω: Angular velocity (rad / s);

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

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

[0013] Preferably, the inorganic mineral is one or more 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 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 composite process of amorphous calcium phosphate, it plays a stabilizing role, delays the process of its crystal phase transformation, maintains it in a liquid metastable state, making it easier to enter the organic matrix (such as inside collagen fibers) and achieve better composite; b) It provides additional functions, such as endowing the material with antibacterial properties by adding specific organic matrices (such as antibacterial materials like carboxymethyl chitosan). With the synergistic effect of the above inorganic minerals and organic additives, the resulting composite material has better mineralization ability, biocompatibility and functionality in biomedical applications.

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

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

[0018] Functional proteins: Used to enhance the bioactivity, antibacterial properties of the composite material and promote cell adhesion, growth and differentiation, including lysozyme, fibronectin, serum proteins (such as human serum albumin and bovine serum albumin), lactoferrin, transferrin, insulin, myoglobin, hemoglobin, ovalbumin, β-lactoglobulin and α-lactalbumin. Among them, lysozyme and lactoferrin have antibacterial effects; fibronectin promotes cell attachment and proliferation; serum proteins improve the biocompatibility of the material; transferrin and insulin promote cell differentiation and tissue healing; myoglobin and hemoglobin provide local oxygen delivery support in repair materials with high oxygen demand; ovalbumin, β-lactoglobulin and α-lactalbumin provide nutritional support.

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

[0020] Preferably, the dehydrating agent is one or more of polyethylene glycol solution, salt solution, sodium polyacrylate, sodium alginate, sodium carboxymethyl cellulose or silica gel. The salt solution is NaCl, KCl, Na 2 HPO 4 and KH 2 PO4 One or more in the solution.

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

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

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

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

[0025] In the above method, the shear stress loading chamber is realized by a hydraulic shear loading device, which has a rotating impeller or piston structure capable of constantly applying shear stress, or the device is a cone - plate viscometer, an injection pump or a flat - plate flow chamber circulation device capable of providing a constant - direction fluid shear force.

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

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

[0028] In addition to shear technology, dialysis concentration technology also plays an important role in the concentration of protein solutions and the preparation of their composites with inorganic minerals. Dialysis concentration technology can effectively increase the concentration of proteins by removing excess water and small-molecule impurities, thereby enhancing their binding to inorganic minerals (such as hydroxyapatite, calcium phosphate, etc.). The concentrated high-concentration protein solution can be closely combined with inorganic minerals to form a composite material with high strength. This composite material has excellent mechanical properties and biological activity and can support cell growth and bone tissue regeneration.

[0029] The present invention combines shear technology and dialysis concentration technology. By shear stress, it improves the fluidity of the protein solution and the orderliness of fiber arrangement, and by dialysis, it concentrates the combination of proteins and inorganic minerals 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 biological matrix materials, and are very suitable for the field of bone repair. By reasonably controlling the shear stress and concentration conditions, the composite materials can have good fluidity and processing properties, and at the same time show high strength and high toughness after final curing, and are applicable to tissue engineering such as bone repair and other biomedical fields.

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

[0031] By combining shear stress and dialysis concentration technology, the present invention not only effectively increases the concentration of protein solutions but also improves the arrangement of protein fibers and the uniformity of materials. This technology can promote the close combination of proteins and inorganic minerals to form a composite material with high strength, which has excellent biological functions and mechanical properties and can support cell adhesion, proliferation, and differentiation, thus accelerating bone tissue regeneration.

[0032] Compared with the prior art, the present invention combines shear stress and dialysis concentration, significantly improving the concentration and composite efficiency of proteins and inorganic minerals, and forming a composite scaffold material with excellent mechanical properties and biological functions under high-concentration conditions. This material can be widely applied in bone repair, tissue engineering, and other biomedical fields, providing a new method for efficiently preparing high-performance biomedical materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0034] Figure 1 Schematic diagram for preparing an inorganic mineral and protein-based composite material based on shear stress and dialysis concentration techniques;

[0035] Figure 2 Scanning electron microscope (SEM) images of a concentrated mineralized collagen scaffold and a static non-concentrated mineralized collagen scaffold prepared based on the method of Example 1;

[0036] Figure 3 Element distribution (EDS Mapping) image of a mineralized collagen scaffold prepared based on the method of Example 1;

[0037] Figure 4 Element analysis image of a mineralized collagen scaffold prepared based on the method of Example 1;

[0038] Figure 5 Thermogravimetric analysis comparison graph of a concentrated mineralized collagen scaffold and a static non-concentrated mineralized collagen scaffold prepared based on the method of Example 1;

[0039] Figure 6 Young's modulus comparison graph of a concentrated mineralized collagen scaffold and a static non-concentrated mineralized collagen scaffold prepared based on the method of Example 1;

[0040] Figure 7 Scanning electron microscope image of a concentrated amorphous calcium phosphate and lysozyme composite material prepared based on the method of Example 3;

[0041] Figure 8 Cell scanning electron microscope image of the surface of a concentrated mineralized collagen scaffold prepared based on the method of Example 1;

[0042] Figure 9 Scanning electron microscope of a concentrated mineralized collagen scaffold prepared based on the method of Example 4;

[0043] Figure 10 Scanning electron microscope of a concentrated mineralized collagen scaffold prepared based on the method of Example 5. Detailed implementation mode

[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 invention belongs. The test reagents used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified.

[0045] The present invention will be described in detail below in conjunction with examples.

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

[0047] Mix an acidic type I collagen solution at 3 mg / mL with an amorphous calcium phosphate solution at a volume ratio of 1:1. The concentration of polyaspartic acid in the amorphous calcium phosphate solution is 240 μg / mL, the calcium ion concentration is 1.67 mM, the phosphate ion concentration is 9.5 mM, the sodium chloride concentration is 150 mM, and the sodium citrate concentration is 10 mM. Place the mixed solution in the shear stress loading chamber above, start the shear loading device, apply a shear stress of 1 Pa, and set the temperature to 37 °C.

[0048] Place a polyethylene glycol (PEG) solution at 300 mg / mL as a dehydrating agent in the dialysis chamber below. The molecular weight cut-off range of the dialysis membrane is 8000 - 14000 Da. The solutions on both sides of the dialysis membrane are continuously injected into the stress loading chamber above and the dialysis chamber below at a flow rate of 1 mL / min respectively.

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

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

[0051] Mix an acidic type I collagen solution at 3 mg / mL with an amorphous calcium phosphate solution at a volume ratio of 1:1. The concentration of polyaspartic acid in the amorphous calcium phosphate solution is 240 μg / mL, the calcium ion concentration is 1.67 mM, the phosphate ion concentration is 9.5 mM, the sodium chloride concentration is 150 mM, and the sodium citrate concentration is 10 mM. Place the mixed solution in a beaker and set the reaction temperature 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 were visible under low magnification. High-magnification images further showed gaps between the fibers, and the three-dimensional network density was significantly lower than that of the shear-concentrated samples, presenting a porous and discontinuous morphology overall. The deposition of mineral substances (such as calcium phosphate crystals) on the surface of collagen fibers was uneven and had poor orientation. Compared with the mineralized collagen scaffolds prepared by the method of Example 1, the mechanical properties of the material were reduced.

[0053] Figure 2 In [reference], scanning electron microscopy showed that after shear dialysis concentration, the collagen fiber structure was significantly densified, and there were no obvious pores or loose regions between the fibers. Under low magnification, a uniform continuous network structure was visible as a whole, and high-magnification microscopy further showed that the fibers were closely packed, forming a three-dimensional network with high density. The fiber arrangement showed a high degree of order. Longitudinal section observation showed that the collagen fibers were distributed in parallel layers, with clear layers and consistent orientation. Hydroxyapatite crystals were uniformly deposited on the surface and in the gaps of collagen fibers, forming a continuous mineralized layer. In high-magnification SEM images, nanoscale mineral particles were seen to be closely attached to the fiber surface, forming a typical "collagen-mineral" composite structure. The SEM results of the concentrated mineralized collagen showed that the densification, ordering, and efficient mineralization of the collagen structure were successfully achieved through the shear dialysis process. Compared with the concentrated mineralized collagen with shear force applied, the samples without shear force applied were significantly inferior in terms of densification, arrangement order, and mineralization uniformity.

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

[0055] Figure 4 In [reference], the elemental analysis results showed that the calcium-phosphorus ratio of the mineralized collagen was 1.49, close to the calcium-phosphorus ratio of hydroxyapatite in natural bone;

[0056] Figure 5 In [reference], the thermogravimetric analysis results showed that after 5 days of mineralization, the residual weight of inorganic minerals in the concentrated mineralized collagen scaffold was 65.53%, significantly higher than 47.59% of the static group, indicating that the concentrated mineralized collagen scaffold prepared by the shear dialysis method could significantly improve the deposition of inorganic minerals.

[0057] Figure 6 In [reference], the average DMT modulus (representing Young's modulus) of the shear force group was 2.5 GPa, significantly higher than 1.2 GPa of the static group. The higher Young's modulus indicated that the stiffness of the mineralized collagen scaffold was significantly improved after shear dialysis treatment, reflecting a denser inorganic mineral-collagen composite structure. Figure 6 The comparison graph of Young's modulus also showed that the introduction of shear force could effectively increase the Young's modulus of the composite mineralized collagen scaffold.

[0058] Figure 8 Among them, SEM images showed that the cells had a large number of extended filopodia and lamellipodia and adhered well to the surface of the concentrated mineralized collagen scaffold, indicating that the high-strength composite material prepared based on this technology had excellent biological functions and could support cell adhesion.

[0059] Example 2: Preparation of a composite material of high-concentration inorganic minerals, hyaluronic acid and collagen based on shear stress and dialysis concentration

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

[0061] A polyethylene glycol (PEG) solution at a concentration of 300 mg / mL 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 respectively injected into the shear stress chamber and the dialysis chamber at a flow rate of 1 mL / min.

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

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

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

[0065] A polyethylene glycol (PEG) solution at a concentration of 300 mg / mL 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 respectively injected into the shear stress chamber and the dialysis chamber at a rate of 1 mL / min.

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

[0067] Figure 7 In [reference], the scanning electron microscope results showed that after the shear dialysis concentration treatment, the surface of the composite material of amorphous calcium phosphate and lysozyme presented a continuous and dense composite network. The amorphous calcium phosphate particles were evenly embedded in the lysozyme matrix, and there were no obvious pores or cracks. This indicated that the shear force and the concentration effect promoted the close intertwining of amorphous calcium phosphate and lysozyme, forming a hierarchical structure similar to natural biomineralization.

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

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

[0070] A 300 mg / mL polyethylene glycol (PEG) solution was placed in the lower dialysis chamber as a dehydrating agent, and the molecular weight cut-off 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 respectively.

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

[0072] Figure 9 In [reference], the scanning electron microscope showed that under the treatment of 5 Pa shear stress, 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 showed a high degree of order, and hydroxyapatite crystals were evenly deposited on the surface and in the gaps of the collagen fibers, forming a continuous mineralized layer.

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

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

[0075] Place a polyethylene glycol (PEG) solution at a concentration of 300 mg / mL as a dehydrating agent in the dialysis chamber below. The molecular weight cut-off range of the dialysis membrane is 8000 - 14000 Da. The solutions on both sides of the dialysis membrane are continuously injected into the stress loading chamber above and the dialysis chamber below at a flow rate of 1 mL / min respectively.

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

[0077] Figure 10 Among them, the scanning electron microscope shows that under the treatment of a shear stress of 10 Pa, after dialysis concentration, the collagen fiber structure is significantly densified, and there are no obvious pores or loose areas between the fibers. The fiber arrangement shows a high degree of order, and hydroxyapatite crystals are uniformly deposited on the surface and in the gaps of the collagen fibers, forming a continuous mineralized layer.

[0078] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

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 the shear stress loading chamber, and the dehydrating agent is added into the dialysis chamber. The shear stress loading chamber and the dialysis chamber are separated by a dialysis membrane. The applied shear stress is 1 to 10 Pa, the action time is 1 to 8 days, and the reaction temperature is 10 to 50°C; the molecular weight cutoff range of the dialysis membrane is 3 to 50 kDa.

2. The method for compounding inorganic minerals and proteins according to claim 1, characterized in that: 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.

3. The method for compounding inorganic minerals and proteins according to claim 1, characterized in that: The inorganic mineral solution is a solution containing one or more of calcium ions, phosphate ions, strontium ions, iron ions, zinc ions, silver ions, magnesium ions, citrate, insoluble strontium salts, and insoluble magnesium salts.

4. The method for compounding inorganic minerals and proteins according to claim 1, characterized in that: The inorganic mineral is one or more of hydroxyapatite, calcium carbonate, calcium phosphate, amorphous calcium phosphate, and amorphous strontium carbonate solution; preferably, 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.

5. The method for compounding inorganic minerals and proteins according to claim 1, characterized in that: The protein is 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 the phase transition of the above proteins.

6. The method for compounding inorganic minerals and proteins according to claim 1, characterized in that: The dehydrating agent is one or more of polyethylene glycol solution, saline solution, sodium polyacrylate, sodium alginate, sodium carboxymethyl cellulose or silica gel; the saline solution is one or more of NaCl, KCl, Na2HPO4 and KH2PO4 solution.

7. The method for compounding inorganic minerals and proteins according to claim 6, characterized in that: The concentration of the polyethylene glycol solution is 20 to 400 mg / mL, preferably 250 to 350 mg / mL; the salt concentration of the saline solution is 3 to 30 g / L.

8. 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 to the shear stress loading chamber; the concentration of the added polysaccharide compound or the polyphenol compound is 0 to 10 mg / mL; preferably, the polysaccharide compound is one or more of chondroitin sulfate and hyaluronic acid, and the polyphenol compound is proanthocyanidin.

9. The method for compounding inorganic minerals and proteins according to claim 1, characterized in that: The flow rate of the liquid in the shear stress chamber and the dialysis chamber is 1 mL / min to 15 mL / min.

10. Use of the inorganic mineral and protein-based composite material prepared by the method for combining inorganic minerals and proteins as described in any one of claims 1 to 9 in the preparation of bone repair or other biomedical products.

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