Nanometer wool protein / hydroxyapatite composite biomimetic bone material and preparation process thereof
The preparation of nano-wool protein/hydroxyapatite composite material has solved the problem of insufficient rigidity and toughness of bone repair materials, and realized the preparation of efficient bone tissue engineering materials with good biocompatibility and osteoconductivity.
Patent Information
- Application Number
- CN202310332130.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-03-31
- Publication Date
- 2026-01-23
- Estimated Expiration
- 2043-03-31
AI Technical Summary
Existing bone repair materials are insufficient to meet clinical needs in terms of rigidity and toughness. Traditional materials have problems such as high mechanical strength requiring secondary surgery, limited toughness of inorganic materials, and degradation rate of polymer materials not matching the growth rate of new bone.
Using a nano-wool protein/hydroxyapatite composite material, hydroxyapatite crystals are formed at the active sites of the protein through an in-situ biomimetic mineralization process using the wool protein complex as a template, generating a nano-cluster aggregate material that combines an inorganic-organic network structure.
It improves the compressive strength and toughness of biomimetic bone materials, has good biocompatibility and osteoconductivity, is suitable as a bone tissue engineering material, is easy to mass-produce and is environmentally friendly.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biomimetic bone composite materials, and aims to provide a biomimetic material for artificial bone tissue engineering applied to the repair of bone wound surfaces and its preparation process. Background Technology
[0002] The main approaches to bone injury repair are autologous and allogeneic bone grafting, but further development is limited by the availability of materials. Traditional metals, ceramics, or synthetic polymers and their composites often increase trauma and cause a series of problems such as immune rejection and infection, hindering their widespread clinical application. The availability of suitable bone replacement materials has always been a pursuit of the medical field and patients. The demand for biomaterials such as artificial bone has spurred rapid development in the technological development and production research of related materials, and also necessitates the support of new technologies and products.
[0003] However, the main bone repair materials used clinically are still classified into several categories, including metallic materials, inorganic non-metallic materials, and tissue engineering materials such as polymers. While metallic materials have high mechanical strength, they cannot degrade in vivo, requiring secondary surgery and causing trauma. Inorganic non-metallic materials have good osteoconductivity but limited toughness. Polymers are relatively easy to control in terms of biomechanical properties, but their degradation rate is difficult to match with the rate of new bone growth. Hydroxyapatite, the main inorganic component of human bone, possesses good biocompatibility and osteoconductivity. Composite materials prepared with hydroxyapatite also have biomimetic structures, thus being widely used as a major component in the preparation of artificial bone materials. However, hydroxyapatite (HA) itself is highly brittle and cannot be used alone as a biomaterial. By mimicking the structure and function of natural bone tissue, materials with bone tissue-like properties manufactured using existing technologies have achieved a certain degree of biosimilarity. Researchers have also attempted to use collagen, silk fibroin, and other proteins as the main organic components in artificial bone to compensate for some deficiencies. However, they still struggle to meet more stringent requirements in terms of rigidity and toughness. Based on theoretical analysis and calculations, this invention creatively utilizes wool protein complex extracted from wool as active sites for phosphorus and calcium sources. Through an in-situ biomimetic mineralization process, it achieves the preparation and application of an organic / inorganic composite nanomaterial with a network structure. The preparation process of this nano-wool protein / hydroxyapatite composite biomimetic bone material is simple and reliable, significantly improving stiffness and toughness, while also meeting the requirements for compressive strength and porosity of bone tissue engineering materials. Summary of the Invention
[0004] To improve the rigidity and toughness of bone repair materials, this invention provides a preparation process and material of a nano-wool protein / hydroxyapatite composite biomimetic bone material. Using a wool protein complex as a template and calcium and phosphate ions as ion sources, hydroxyapatite crystals are formed at the protein active sites through an in-situ mineralization process under the action of appropriate regulators. During the formation of these hydroxyapatite crystals, the hydroxyapatite crystals coat the surface of the wool protein molecular clusters, generating a nano-cluster aggregate material. The preparation steps of the nano-wool protein / hydroxyapatite composite biomimetic bone material of this invention include:
[0005] I. Preparation of Wool Protein Solution
[0006] Clean, pulverized wool was added to a NaHCO3 solution to remove surface impurities. The wool was then washed with distilled water until neutral, and the water was replaced with ethanol before vacuum drying to obtain wool raw material. The wool raw material was then immersed in a NaOH:CO(NH2)2 aqueous solution at a ratio of 1:1, heated and stirred, cooled, centrifuged to remove precipitate, and the supernatant was filtered through a membrane to obtain a wool protein solution.
[0007] II. Preparation of Mineralizing Solution
[0008] Take a certain amount of the wool protein solution from step one and mix it with the calcium ion solution. After stirring thoroughly, add a surfactant, pH adjuster, and performance regulator to obtain the precursor solution.
[0009] III. In-situ biomimetic mineralization process
[0010] Prepare a separate aqueous solution of phosphate ions at the appropriate concentration. After adding a pH adjuster to adjust the pH to 10-11, slowly add a certain amount of the aqueous solution of phosphate ions, stirring to ensure uniform dispersion. Allow it to stand for a certain period of time to carry out the in-situ mineralization reaction. After the reaction is complete, let it stand for a period of time, discard the supernatant, filter, and dry the filtered solids to obtain the nano-wool protein / hydroxyapatite composite biomimetic bone material. Detailed Implementation Plan
[0011] The technical solution of the present invention is not limited to the specific implementation schemes listed below, but also includes any combination of specific implementation schemes.
[0012] Specific Implementation Plan 1
[0013] I. Preparation of Wool Protein Solution
[0014] Clean, pulverized wool was added to a NaHCO3 solution to remove surface impurities. The wool was then washed with distilled water until neutral, and the water was replaced with ethanol before vacuum drying to obtain wool raw material. The wool raw material was then immersed in a NaOH:CO(NH2)2 aqueous solution at a ratio of 1:1, heated and stirred, cooled, centrifuged to remove precipitate, and the supernatant was filtered through a membrane to obtain a wool protein solution.
[0015] II. Preparation of Mineralizing Solution
[0016] A certain amount of the wool protein solution from step one is mixed with a calcium ion solution. After thorough stirring, a surfactant, a pH adjuster, and a performance regulator are added to obtain the precursor solution.
[0017] III. In-situ biomimetic mineralization process
[0018] Prepare a separate aqueous solution of phosphate ions at the appropriate concentration. Add a pH adjuster to the mineralization solution from step two to bring the pH to 10-11. Then, slowly add a certain amount of the aqueous solution of phosphate ions, stirring to ensure uniform dispersion. Continue adding the pH adjuster to maintain the pH of the reaction system at 10-11. Allow the mixture to stand for a certain period of time to carry out the in-situ mineralization reaction. After the reaction is complete, let it stand for a period of time, discard the supernatant, filter, and dry the filtered solids to obtain the nano-wool protein / hydroxyapatite composite biomimetic bone material.
[0019] Preferably, in step one, the raw wool is mechanically sheared to a length of 0.5-2 cm, washed with hot water, added to a NaHCO3 solution to remove surface impurities, taken out and washed with distilled water until neutral, and then vacuum dried after replacing the water with ethanol to obtain the raw wool.
[0020] Preferably, the specific steps for replacing water with ethanol in step one are as follows: prepare ethanol with a concentration of 80%–95%, add the washed wool to the ethanol, stir at 200–300 rpm for 3–6 hours at 30–50°C, filter, and then dry the wool in a vacuum oven at 50–80°C to obtain ethanol-treated wool. The vacuum oven drying conditions are: pressure 0.1 MPa, temperature 30–50°C, and time 2–3 hours.
[0021] Preferably, the specific centrifugation step in step one is as follows: the obtained wool solution is centrifuged at a rate of 4000 rpm for 5 to 10 minutes.
[0022] Preferably, the treatment method in step one is as follows: add a pH adjuster to the wool protein complex solution, adjust the pH of the solution to 7-8, and then add it to a dialysis bag for dialysis.
[0023] Preferably, in step one, the molecular weight cutoff of the filter membrane is 8000-14000, the dialysis time is 48-72 hours, and the distilled water is replaced every 12 hours.
[0024] Preferably, the calcium ion solution in step two is calcium nitrate tetrahydrate Ca(NO3)2·4H2O solution.
[0025] Preferably, the surfactant in step two is Gemini dodecylamine surfactant. The preparation method of the Gemini dodecylamine surfactant-modified wool protein complex solution is as follows: prepare a 0.03 mol / L Gemini dodecylamine surfactant solution, add a certain amount of wool protein complex solution, and magnetically stir at 200-300 rpm in a 70°C oil bath for 10 min, then cool to 25°C for later use.
[0026] Preferably, the pH adjuster in step two is 10% phosphoric acid and 25% ammonia.
[0027] Preferably, the performance modifiers added to the reaction system are cross-linked polyamides and chitosan / zinc oxide particles. A performance modifier is a substance added to the original material to give it different properties.
[0028] Preferably, the first performance regulator selected in step two is cross-linked polyamide. The preparation method of the cross-linked polyamide is as follows: 10-20% of the total weight of the target composite material is weighed as polyamide, dissolved in methanol, a cross-linking agent is added, the mixture is heated and stirred, the solution is rotary evaporated, and then vacuum dried to obtain a cross-linked polyamide gel; the stirring conditions are: pressure 0.1 MPa, temperature 50-60℃, stirring speed 500-600 rpm, and stirring time 10-12 h; the vacuum drying conditions are: pressure 0.8-1 MPa, temperature 50-60℃, and drying time 5-6 h; the specific cross-linking agent is epichlorohydrin; wherein the molar ratio of epichlorohydrin to polyamide is (0.5-1):(3-5).
[0029] Preferably, the second performance regulator selected in step two is chitosan / zinc oxide particles. The preparation method of the chitosan / zinc oxide particles is as follows: chitosan and zinc acetate are dissolved in a 1% acetic acid solution, heated and stirred, and sodium hydroxide is added until the solution pH = 10-11. After vacuum drying, chitosan / zinc oxide particles are obtained. The stirring conditions are: pressure 0.1 MPa, temperature 50-60℃, stirring speed 500-600 rpm, and stirring time 4-5 h. The vacuum drying conditions are: pressure 0.8-1 MPa, temperature 50-60℃, and drying time 5-6 h; wherein the molar ratio of chitosan to zinc acetate is (0.5-1):(0.5-1).
[0030] Preferably, the phosphate ion solution used in step three is an ammonium dihydrogen phosphate solution.
[0031] Preferably, the phosphate ions added in step three should meet the requirement that the calcium-to-phosphorus ratio is 1.67.
[0032] Preferably, the conditions for static settling in step three are: pressure of 0.1 MPa, temperature of 25°C, and time of 24–72 h.
[0033] Preferably, the drying conditions of the vacuum oven in step three are: pressure of 0.1 MPa, temperature of 70°C, and drying time of 5 to 6 hours.
[0034] Preferably, in step three, distilled water is continuously added during the filtration process for washing until the pH of the filtrate is 7-8.
[0035] Specific Example Scheme Two: This scheme differs from Specific Example Scheme One in that the method for removing surface impurities from the wool in step one is to treat the wool with a hydrogen peroxide pretreatment solution. Everything else is the same as Specific Example Scheme One.
[0036] The preparation method of the hydrogen peroxide pretreatment solution is as follows: Take 20-40% hydrogen peroxide solution and mix it evenly with 5g / L trisodium phosphate solution, add it in a ratio of wool to pretreatment solution bath of 1:50, stir magnetically at 200-300 rpm for 40-60 minutes at 30-50℃, take it out and wash it with water, and put the wool in a vacuum oven to dry at 50-80℃ to obtain the hydrogen peroxide-treated wool.
[0037] Specific Example Scheme 3: This implementation scheme differs from Specific Example Scheme 1 in that the drying method in step four is freeze-drying at -80℃ for 2-3 days. Everything else is the same as in Specific Example Scheme 1.
[0038] Beneficial effects of this invention:
[0039] This invention discloses a nano-wool protein / hydroxyapatite composite biomimetic bone material and its preparation method. This material imparts high toughness to hydroxyapatite without affecting the rigidity of the biomimetic bone material. This invention employs a network structure biomimetic bone ceramic material combining inorganic and organic matter, possessing a composition similar to bone. The wool keratin in the wool protein composite exhibits good biocompatibility and adsorption properties, beneficial for wound healing, bone regeneration, hemostasis, and peripheral nerve repair. The nano-clusters of hydroxyapatite, in addition to being a major inorganic component of bone, possess excellent osteoconductivity, and the rigidity is increased after the nano-hydroxyapatite particles are stacked. This invention uses a wool protein complex as an organic material, which is added to a solution of calcium ions and phosphate ions. At room temperature, it forms an organic-inorganic composite material with inorganic nano-hydroxyapatite. The helical wool protein can increase the compressive strength of hydroxyapatite. The good adsorption properties of wool protein allow nano-hydroxyapatite to be adsorbed on the surface of the protein macromolecules and fill the triple helical space of the wool protein. The triple helical structure plays a supporting role in the interior, thereby enhancing the compressive strength of the nano-wool protein / hydroxyapatite composite biomimetic bone material.
[0040] This invention employs a simple and environmentally friendly in-situ composite process involving the addition of wool protein solution during hydroxyapatite formation. Compared to traditional casting methods, this process facilitates large-scale production, promotes in-situ generation of the reinforcing phase without pollution, produces materials with good interfacial bonding, and is easily moldable with a template. The resulting biomimetic bone material exhibits excellent thermodynamic stability and can also be used as a raw material for 3D printing. This invention provides a new approach to the preparation of biomimetic bone materials, offering a novel solution to address the issues of poor rigidity, toughness, and biocompatibility in traditional biomimetic bone materials. Attached Figure Description
[0041] Figure 1 , Figure 2 Scanning electron microscope image of the nano-wool protein / hydroxyapatite composite biomimetic bone material prepared in Example 1;
[0042] Figure 3 , Figure 4 Scanning electron microscope image of the nano-wool protein / hydroxyapatite composite biomimetic bone material prepared in Example 3;
[0043] Figure 5 Comparison of compressive strength of the nano-wool protein / hydroxyapatite composite biomimetic bone materials prepared in Examples 1 and 3.
[0044] Figure 6 Comparison of porosity of nano-wool protein / hydroxyapatite composite biomimetic bone materials prepared in Examples 4 and 5.
[0045] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.
[0046] Example 1
[0047] (I) Wool pretreatment and wool protein refining
[0048] Clean, pulverized wool was added to a 0.2–0.5 mol / L NaHCO3 solution to remove surface impurities. The solution was then rinsed with distilled water until neutral, and 80% ethanol was prepared. After replacing the water with ethanol, the wool was vacuum-dried at 70°C to obtain the wool raw material. 2.4 g of NaOH and 3.6 g of CO(NH2)2 were weighed and mixed with 100 ml of distilled water to prepare a 1:1 molar ratio of NaOH solution and CO(NH2)2 solution. The wool raw material was immersed in these solutions, and the supernatant was dialyzed through a filter membrane to obtain a wool protein solution.
[0049] (II) Preparation and Composition of Mineralizing Solution
[0050] Weigh 10g Ca(NO3)2·4H2O and mix it with 200ml distilled water. Then mix it with 100ml of the wool protein complex solution from step two. Stir magnetically at a rate of 100-300 rpm for 20-30 minutes to obtain the precursor solution.
[0051] (III) In-situ mineralized nano-wool protein / hydroxyapatite composite biomimetic bone material
[0052] Weigh 2.92g of NH4H2PO4 and mix it with 200ml of distilled water. Add ammonia and phosphoric acid to the mineralization solution from step two to adjust the pH to 10-11. Then, slowly add the prepared phosphate ion aqueous solution and stir to disperse it evenly. Continue to add pH adjuster to maintain the pH of the reaction system at 10-11. Stir magnetically for 3 hours, and allow it to settle for 48-72 hours under a pressure of 0.1MPa and a temperature of 25℃ for in-situ mineralization. After filtration, dry the filtered solids at a pressure of 0.1MPa and a temperature of 70℃ for 5-6 hours to obtain the nano-wool protein / hydroxyapatite composite biomimetic bone material.
[0053] (IV) Scanning electron microscope images of the nano-wool protein / hydroxyapatite composite biomimetic bone material obtained in this embodiment are shown below. Figure 1 and Figure 2 As shown, hydroxyapatite molecules adhere to the surface of wool protein molecules, thereby enhancing the mechanical properties and toughness of the ceramic material. Figure 5 As shown, the compressive strength of the nano-wool protein / hydroxyapatite composite biomimetic bone material is 40-75 MPa, which is consistent with the basic strength of human bone.
[0054] Example 2
[0055] (I) Wool pretreatment and wool protein refining
[0056] Clean, pulverized wool was added to a 0.2–0.5 mol / L NaHCO3 solution to remove surface impurities. The solution was then rinsed with distilled water until neutral, and 80% ethanol was prepared. After replacing the water with ethanol, the wool was vacuum-dried at 70°C to obtain the wool raw material. 2.4 g of NaOH and 3.6 g of CO(NH2)2 were weighed and mixed with 100 ml of distilled water to prepare a 1:1 molar ratio of NaOH solution and CO(NH2)2 solution. The wool raw material was immersed in these solutions, and the supernatant was dialyzed through a filter membrane to obtain a wool protein solution.
[0057] (II) Preparation and Composition of Mineralizing Solution
[0058] Weigh 20g Ca(NO3)2·4H2O and mix it with 200ml distilled water. Then mix it with 125ml of the wool protein complex solution from step two. Stir magnetically at a speed of 100-300 rpm for 20-30 minutes to obtain the precursor solution.
[0059] (III) In-situ mineralized nano-wool protein / hydroxyapatite composite biomimetic bone material
[0060] Weigh 5.84g of NH4H2PO4 and mix it with 200ml of distilled water. Add ammonia and phosphoric acid to the mineralization solution from step two to adjust the pH to 10-11. Then, slowly add the prepared phosphate ion aqueous solution and stir to disperse it evenly. Continue to add pH adjuster to maintain the pH of the reaction system at 10-11. Stir magnetically for 3 hours, and allow it to settle for 48-72 hours under a pressure of 0.1MPa and a temperature of 25℃ for in-situ mineralization. After filtration, dry the filtered solids at a pressure of 0.1MPa and a temperature of 70℃ for 5-6 hours to obtain the nano-wool protein / hydroxyapatite composite biomimetic bone material.
[0061] Example 3
[0062] (I) Wool pretreatment and wool protein refining
[0063] Clean, pulverized wool was added to a 0.2–0.5 mol / L NaHCO3 solution to remove surface impurities. The solution was then rinsed with distilled water until neutral, and 80% ethanol was prepared. After replacing the water with ethanol, the wool was vacuum-dried at 70°C to obtain the wool raw material. 2.4 g of NaOH and 3.6 g of CO(NH2)2 were weighed and mixed with 100 ml of distilled water to prepare a 1:1 molar ratio of NaOH solution and CO(NH2)2 solution. The wool raw material was immersed in these solutions, and the supernatant was dialyzed through a filter membrane to obtain a wool protein solution.
[0064] (II) Preparation and Composition of Mineralizing Solution
[0065] Weigh 10g Ca(NO3)2·4H2O and mix it with 200ml distilled water. Then mix it with 125ml of the wool protein complex solution from step two. Stir the mixture magnetically at a rate of 100-300 rpm for 20-30 minutes. Mix this 100ml solution with a 0.03mol / L Gemini dodecylamine surfactant solution. Stir the mixture magnetically at 200-300 rpm for 30 minutes in an oil bath at 70℃ under a pressure of 0.1MPa. Then cool the mixture to 25℃ to obtain the precursor solution.
[0066] (III) In-situ mineralized nano-wool protein / hydroxyapatite composite biomimetic bone material
[0067] Weigh 2.92g of NH4H2PO4 and mix it with 200ml of distilled water. Add ammonia and phosphoric acid to the mineralization solution from step two to adjust the pH to 10-11. Then, slowly add the prepared phosphate ion aqueous solution and stir to disperse it evenly. Continue to add pH adjuster to maintain the pH of the reaction system at 10-11. Stir magnetically for 3 hours, and allow it to settle for 48-72 hours under a pressure of 0.1MPa and a temperature of 25℃ for in-situ mineralization. After filtration, dry the filtered solids at a pressure of 0.1MPa and a temperature of 70℃ for 5-6 hours to obtain the nano-wool protein / hydroxyapatite composite biomimetic bone material.
[0068] (IV) Scanning electron microscope images of the nano-wool protein / hydroxyapatite composite biomimetic bone material prepared in this embodiment are shown below. Figure 3 and Figure 4 As shown, the adhesion between hydroxyapatite molecules and wool protein molecules is stronger, and nano-hydroxyapatite is attached to the surface of wool protein in a helical structure.
[0069] The compressive strength of the two wool protein / hydroxyapatite nanoceramic materials in Example 1 and Example 3 was compared through a compressive strength test. Different amounts of wool protein were added for each material, and the comparison results are as follows: Figure 5 As shown, the compressive strength of the wool protein / hydroxyapatite nanoceramic material treated with Gemini dodecylamine surfactant is higher. However, when the wool protein content increases to 16%, the compressive strength decreases. This is because the high content of wool protein complex leads to the agglomeration of nano-hydroxyapatite, which changes the structure of the material and reduces its compressive strength.
[0070] Example 4
[0071] (I) Wool pretreatment and wool protein refining
[0072] Clean, pulverized wool was added to a 0.2–0.5 mol / L NaHCO3 solution to remove surface impurities. The solution was then rinsed with distilled water until neutral, and 80% ethanol was prepared. After replacing the water with ethanol, the wool was vacuum-dried at 70°C to obtain the wool raw material. 2.4 g of NaOH and 3.6 g of CO(NH2)2 were weighed and mixed with 100 ml of distilled water to prepare a 1:1 molar ratio of NaOH solution and CO(NH2)2 solution. The wool raw material was immersed in these solutions, and the supernatant was dialyzed through a filter membrane to obtain a wool protein solution.
[0073] (II) Preparation and Composition of Mineralizing Solution
[0074] Weigh 10g Ca(NO3)2·4H2O and mix it with 200ml distilled water. Then mix it with 125ml of the wool protein complex solution from step two and stir magnetically at a speed of 100-300 rpm for 20-30 minutes to obtain the precursor solution.
[0075] (III) Preparation of Crosslinked Polyamides
[0076] Weigh 1g of polyamide, dissolve it in 50ml of methanol, add 0.2ml of epichlorohydrin, heat and stir, evaporate the solution by rotary evaporation, and dry under vacuum to obtain cross-linked polyamide gel; the stirring conditions are: pressure 0.1MPa, temperature 50-60℃, stirring speed 500-600 rpm, stirring time 10-12h to obtain cross-linked polyamide solution; the vacuum drying conditions are: pressure 0.8-1MPa, temperature 50-60℃, drying time 5-6h; wherein the molar ratio of epichlorohydrin to polyamide is (0.5-1):(3-5).
[0077] (IV) In-situ mineralized nano-wool protein / hydroxyapatite / crosslinked polyamide composite biomimetic bone material
[0078] Weigh 2.92g of NH4H2PO4 and mix it with 200ml of distilled water. Add ammonia and phosphoric acid to the mineralization solution from step two to adjust the pH to 10-11. Slowly add the prepared phosphate ion aqueous solution and the prepared cross-linked polyamide. Stir to disperse evenly. Continue to add pH adjuster to maintain the pH of the reaction system at 10-11. Stir magnetically for 3 hours. Allow to settle at 0.1MPa and 25℃ for 48-72 hours for in-situ mineralization. After filtration, dry the filtered solids at 0.1MPa and 70℃ for 5-6 hours to obtain the nano-wool protein / hydroxyapatite composite biomimetic bone material.
[0079] (V) The contact angle experiment showed that the contact angle was greater than 90°, which proved the hydrophilicity of the composite biomimetic bone material and that it is beneficial to cell growth.
[0080] Example 5
[0081] (I) Wool pretreatment and wool protein refining
[0082] Clean, pulverized wool was added to a 0.2–0.5 mol / L NaHCO3 solution to remove surface impurities. The solution was then rinsed with distilled water until neutral, and 80% ethanol was prepared. After replacing the water with ethanol, the wool was vacuum-dried at 70°C to obtain the wool raw material. 2.4 g of NaOH and 3.6 g of CO(NH2)2 were weighed and mixed with 100 ml of distilled water to prepare a 1:1 molar ratio of NaOH solution and CO(NH2)2 solution. The wool raw material was immersed in these solutions, and the supernatant was dialyzed through a filter membrane to obtain a wool protein solution.
[0083] (II) Preparation and Composition of Mineralizing Solution
[0084] Weigh 10g Ca(NO3)2·4H2O and mix it with 200ml distilled water. Then mix it with 125ml of the wool protein complex solution from step two and stir magnetically at a speed of 100-300 rpm for 20-30 minutes to obtain the precursor solution.
[0085] (III) Preparation of chitosan / zinc oxide particles
[0086] Chitosan and zinc acetate were dissolved in a 1% acetic acid solution, heated and stirred, and sodium hydroxide was added until the solution pH was 10–11. After vacuum drying, chitosan / zinc oxide particles were obtained. The stirring conditions were: pressure 0.1 MPa, temperature 50–60℃, stirring speed 500–600 rpm, and stirring time 4–5 h. The vacuum drying conditions were: pressure 0.8–1 MPa, temperature 50–60℃, and drying time 5–6 h; wherein the molar ratio of chitosan to zinc acetate was (0.5–1):(0.5–1).
[0087] (IV) In-situ mineralized nano-wool protein / hydroxyapatite / chitosan / zinc oxide composite biomimetic bone material
[0088] Weigh 2.92g of NH4H2PO4 and mix with 200ml of distilled water. Add ammonia and phosphoric acid to the mineralization solution from step two to adjust the pH to 10-11. Slowly add the prepared phosphate ion aqueous solution and the prepared cross-linked polyamide. Stir to disperse evenly. Continue to add pH adjuster to maintain the pH of the reaction system at 10-11. Stir magnetically for 3 hours. Allow to settle at 0.1MPa and 25℃ for 48-72 hours for in-situ mineralization. After filtration, dry the filtered solids at 0.1MPa and 70℃ for 5-6 hours to obtain the nano-wool protein / hydroxyapatite / chitosan / zinc oxide composite biomimetic bone material.
[0089] (V) Porosity determination of the composite materials prepared in Examples 4 and 5: Weigh several portions of the composite material, each portion being a 5mm × 5mm cylindrical composite material, and record the initial mass of each portion as m1; place them in anhydrous ethanol, seal them, and apply negative pressure until no bubbles are generated on the material surface and then stop applying negative pressure, at which point the mass of each portion is recorded as m2; place the material in a 10ml graduated cylinder and add anhydrous ethanol to the 10ml mark, and record the amount of ethanol added as V0. Wherein, P is the porosity, and ρ is the density of anhydrous ethanol. Process the data according to formula (1) and plot. Figure 6 .
[0090] P=(m2-m1) / ρ(10-V0) (1)
[0091] The porosity comparison diagrams for Examples 4 and 5 are shown below. Figure 6 As shown in the figure, the porosity of the composite material with added cross-linked polyamide is generally higher than that with added chitosan / zinc oxide particles. The porosity determines the number of pores in the material. The more porous nano-wool protein / hydroxyapatite / cross-linked polyamide composite bone-like material is conducive to the circulation of blood and nutrients, and also to the growth of nerves in the bone-like material.
Claims
1. A method for preparing a nano-wool protein / hydroxyapatite composite biomimetic bone material, comprising the following steps: (I) Preparation of wool protein solution Clean, crushed wool is added to NaHCO3 solution to remove surface impurities. It is then washed with distilled water to neutralize the solution. After replacing the water with ethanol, the wool is vacuum dried to obtain wool raw material. The wool raw material is then immersed in an aqueous solution of NaOH:CO(NH2)2 = 1:1, heated and stirred. After cooling, it is centrifuged to remove the precipitate. The clear liquid is then filtered through a membrane to obtain a wool protein solution. (II) Preparation of Mineralizing Solution Take a certain amount of the wool protein solution from step one and mix it with the calcium ion solution. After stirring thoroughly, add a certain amount of Gemini dodecylamine surfactant solution. Stir thoroughly with magnetic force in an oil bath at 70°C, then cool to 25°C. Subsequently, add a pH adjuster and a performance regulator to obtain the precursor solution. (III) In-situ biomimetic mineralization process Prepare a phosphate ion aqueous solution of appropriate concentration. Add a pH adjuster to the mineralization solution in step two to make the pH 10-11. Slowly add a certain amount of phosphate ion aqueous solution, stir to disperse it evenly, and let it stand for a certain period of time to carry out the in-situ mineralization reaction. After the reaction is completed, let it stand for a period of time and discard the supernatant. After filtration, dry the filtered solids to obtain the nano-wool protein / hydroxyapatite composite biomimetic bone material.
2. The preparation method of the nano-wool protein / hydroxyapatite composite biomimetic bone material according to claim 1, characterized in that... Step 1: The raw wool is mechanically sheared to a length of 0.5-2cm.
3. The preparation method of the nano-wool protein / hydroxyapatite composite biomimetic bone material according to claim 1 or 2, characterized in that... The vacuum drying method in step one is as follows: the vacuum drying pressure is 0.1 MPa, the temperature is 40℃, and the time is 10~12h.
4. The preparation method of the nano-wool protein / hydroxyapatite composite biomimetic bone material according to claim 1, characterized in that... The specific steps for replacing water with ethanol in step one are as follows: Prepare ethanol with a concentration of 80% to 95%, add the washed wool to the ethanol, stir at 200 to 300 rpm for 3 to 6 hours in an environment of 30 to 50°C, filter, and then place the wool in a vacuum oven at 50 to 80°C to dry, thus obtaining wool treated with ethanol. The vacuum oven drying conditions are: pressure of 0.1 MPa, temperature of 30 to 50°C, and time of 2 to 3 hours.
5. The preparation method of the nano-wool protein / hydroxyapatite composite biomimetic bone material according to claim 1, characterized in that... The treatment method for the wool protein complex solution in step one is as follows: add a pH adjuster to the wool protein complex solution, adjust the pH of the solution to 7~8, and then add a filter membrane for dialysis.
6. The method for preparing the nano-wool protein / hydroxyapatite composite biomimetic bone material according to claim 1, characterized in that... The first performance regulator selected in step two is cross-linked polyamide. The preparation method of the cross-linked polyamide is as follows: according to the weight percentage, 10-20% of the total weight of the target composite material of polyamide is weighed, dissolved in methanol, a cross-linking agent is added, heated and stirred, the solution is rotary evaporated, and vacuum dried to obtain cross-linked polyamide gel; the stirring conditions are: pressure of 0.1 MPa, temperature of 50-60℃, stirring speed of 500-600 rpm, and stirring time of 10-12 h; the vacuum drying conditions are: pressure of 0.8-1 MPa, temperature of 50-60℃, and drying time of 5-6 h; the specific cross-linking agent is epichlorohydrin; wherein the molar ratio of epichlorohydrin to polyamide is (0.5-1):(3-5).
7. The method for preparing the nano-wool protein / hydroxyapatite composite biomimetic bone material according to claim 1, characterized in that... The second performance regulator selected in step two is chitosan / zinc oxide particles. The preparation method of the chitosan / zinc oxide particles is as follows: chitosan and zinc acetate are dissolved in a 1% acetic acid solution, heated and stirred, and sodium hydroxide is added until the solution pH=10~11. After vacuum drying, chitosan / zinc oxide particles are obtained. The stirring conditions are: pressure 0.1MPa, temperature 50~60℃, stirring speed 500~600 rpm, stirring time 4~5h; the vacuum drying conditions are: pressure 0.8~1MPa, temperature 50~60℃, drying time 5~6h; wherein the molar ratio of chitosan to zinc acetate is (0.5~1):(0.5~1).
8. In the preparation method of the nano-wool protein / hydroxyapatite composite biomimetic bone material according to claim 1, the static settling method in step three is: settling for 24-72 hours under a pressure of 0.1 MPa and a temperature of 25°C.
9. The preparation method of the nano-wool protein / hydroxyapatite composite biomimetic bone material according to claim 1, wherein the drying method in step three is: pressure of 0.1 MPa, temperature of 70℃, and drying time of 5-6 h.