Bone regeneration multiple bionic scaffold material and preparation method thereof

Abstract

The invention discloses a bone regeneration multiple bionic scaffold material and a preparation method thereof, and relates to the technical field of bionic bones. According to the bone regeneration multi-bionic scaffold material and the preparation method thereof, a polylactic acid block copolymer is used as a raw material, a 3D printing technology is adopted to prepare a substrate scaffold meeting the bone defect morphology, and then the substrate scaffold is alternately soaked in a collagen mixed solution and a modified layered dihydroxide solution for multiple times to obtain the bone regeneration multi-bionic scaffold material. A bionic matrix layer composed of collagen / layered dihydroxide is formed on the surface of the base stent, and finally, the base stent with the bionic matrix layer attached to the surface is alternately soaked in dipotassium phosphate and calcium chloride solutions for multiple times in sequence; and performing in-situ mineralization on the substrate support with the bionic matrix layer attached on the surface to form calcium phosphate nanoparticles, and finally cleaning and drying to obtain the calcium phosphate nano-particles. The bone regeneration multi-bionic scaffold material prepared by the preparation method disclosed by the invention is relatively good in biocompatibility and relatively good in mechanical property.

Description

Technical Field

[0001] The invention relates to a bone regeneration multiple bionic scaffold material and a preparation method thereof. Background Art

[0002] In recent years, bone tissue engineering scaffold materials have shown broad application prospects in the repair and regeneration of defective bone tissue. Among them, bone regeneration multi-biomimetic scaffold materials have become one of the research hotspots because they can simulate the multi-level structure and complex microenvironment of natural bone tissue. However, the commonly used bone scaffold materials still have many limitations, especially the balance between mechanical properties and biological activity, which seriously restricts their widespread application in clinical practice.

[0003] Traditional bone scaffold materials mainly include inorganic materials such as hydroxyapatite (HA) and calcium phosphate ceramics, which have good biocompatibility and certain osteoconductivity, and can provide structural support for new bone formation. However, these materials generally have the problems of high brittleness and low compressive strength, which are difficult to meet the mechanical requirements of cancellous bone or even load-bearing bones. In addition, their degradation rate is usually difficult to match the growth rate of new bone tissue, which may cause structural collapse or long-term residue, affecting the effect of bone regeneration.

[0004] In order to improve the pore structure of scaffold materials to facilitate cell adhesion and nutrient transport, researchers have tried to use 3D printing technology to construct calcium phosphate scaffolds with precisely controllable porosity in recent years, such as porous scaffolds made from calcium phosphate derived from bovine bones. Although this method significantly optimizes the microstructure of the scaffold and improves the permeability and bone formation of cells, its fracture toughness is still limited by the inherent brittleness of inorganic components, and it is prone to fracture when subjected to dynamic loads, limiting its application in complex mechanical environments in vivo.

[0005] On the other hand, hydrogel materials are widely used to construct bionic bone scaffold systems due to their high water content, good cell compatibility and controllable physical and chemical properties. For example, gelatin methacryloyl (GelMA) hydrogel has been shown to have good cell adhesion and proliferation-promoting abilities. However, single-component hydrogels generally have low mechanical strength and are difficult to maintain long-term structural stability, especially in the physiological environment of the body, which is prone to premature degradation or deformation, thus hindering the stable reconstruction of bone tissue.

[0006] Therefore, how to develop a multi-biomimetic scaffold material for bone regeneration that has good mechanical properties, bioactivity and immunomodulatory functions has become a key issue that needs to be urgently addressed in the current field of bone tissue engineering. Summary of the invention

[0007] The purpose of the present invention is to provide a bone regeneration multiple bionic scaffold material and a preparation method thereof, so as to solve the technical problems mentioned in the above background technology.

[0008] The technical solution for achieving the purpose of the present invention is as follows:

[0009] In the first aspect, the present invention provides a bone regeneration multi-bionic scaffold material. The bone regeneration multi-bionic scaffold material is prepared by first using a polylactic acid block copolymer as a raw material and adopting 3D printing technology to obtain a base scaffold conforming to the morphology of bone defects, and then alternately impregnating the base scaffold in a collagen mixed solution and a modified layered double hydroxide solution for multiple times to form a bionic matrix layer composed of collagen, silk fibroin, and layered double hydroxide on the surface of the base scaffold. Finally, the base scaffold with the bionic matrix layer attached to its surface is alternately soaked in a dipotassium hydrogen phosphate solution and a calcium chloride solution for multiple times to in-situ mineralize calcium phosphate nanoparticles on the base scaffold with the bionic matrix layer attached to its surface, and then it is washed and dried to obtain.

[0010] Among them, collagen (COL), as an important component of natural bone, shows significant advantages in the field of bone tissue engineering; it has good biocompatibility, can coexist harmoniously with human tissues, and will not cause obvious immune rejection reactions; it has biodegradability, can be gradually decomposed as new bone grows, and the degradation rate is controllable; it has low immunogenicity, can reduce the attack and interference of the immune system; at the same time, COL plays a key role in the processes of cell adhesion, proliferation, and differentiation, can provide a suitable microenvironment for cells, and promote the interaction between cells and materials;

[0011] Layered double hydroxide (LDH) has been widely studied due to its unique layered structure and good drug loading and release properties; LDH is composed of a positively charged hydroxide layer and a negatively charged intermediate layer, and the common one is magnesium-aluminum bimetal; this structure endows LDH with excellent ion exchange and adsorption capabilities, and it can load a variety of bioactive substances, such as drugs, growth factors, etc., and achieve slow release, which has important application value in the drug delivery system. In the field of bone tissue engineering, LDH can not only be used as a drug carrier, but also participate in the construction of bionic scaffolds, and improve the physical and chemical properties and biological properties of the scaffolds by compounding with other materials, promote the adhesion, proliferation, and differentiation of bone cells, and enhance the bone repair effect;

[0012] 3D printing technology provides a new approach for the preparation of bone regeneration scaffolds; it can be customized according to the specific morphology of the bone defect of the patient. By precisely scanning and modeling the bone defect site of the patient, a scaffold highly matching the defect site is fabricated, greatly improving the accuracy and adaptability of the repair. Natural bone has an ordered hierarchical structure, including the shell-like dense bone on the surface layer and the cancellous bone in the core. This structure endows bone with good mechanical strength and adaptability; 3D printing technology can well simulate this ordered structure through modeling, achieve structural bionics, provide a support structure closer to the natural environment for bone regeneration, and is conducive to cell adhesion, proliferation, and differentiation;

[0013] In summary, the present invention forms a bionic matrix layer on a 3D-printed substrate scaffold by the layer-by-layer self-assembly process of a collagen mixed solution and a modified layered double hydroxide solution, and then uses the in-situ mineralization technology to in-situ mineralize calcium phosphate nanoparticles on the substrate scaffold with the bionic matrix layer attached to the surface, controllably constructing a collagen bionic interface assembly structure with bone immunomodulatory function, which can precisely regulate the chemical composition, topological morphology, drug release, and material degradation rate of the interface, achieve multi-dimensional synergistic promotion of bone regeneration, and effectively solve the deficiencies of existing bone regeneration materials in the coordination of immunomodulation and bone regeneration.

[0014] Further, the polylactic acid block copolymer is obtained by the reaction polymerization of L-lactide, citric acid, methoxypolyethylene glycol, and glycolic acid.

[0015] Further, the collagen mixed solution is obtained by mixing a collagen solution and a silk fibroin solution.

[0016] Further, the modified layered double hydroxide is obtained by intercalating glutathione into layered double hydroxide.

[0017] The second aspect of the present invention provides a preparation method of a bone regeneration multi-bionic scaffold material, including the following preparation steps:

[0018] (1) Using a polylactic acid block copolymer as a raw material, a substrate scaffold conforming to the bone defect morphology is fabricated by 3D printing technology;

[0019] (2) The substrate scaffold obtained in step (1) is alternately soaked in a collagen mixed solution and a modified layered double hydroxide solution for multiple times in sequence;

[0020] (3) The substrate scaffold treated in step (2) is alternately soaked in a dipotassium hydrogen phosphate solution and a calcium chloride solution for multiple times in sequence;

[0021] (4) The substrate scaffold treated in step (3) is washed and dried to obtain a bone regeneration multi-bionic scaffold material.

[0022] Furthermore, the porosity of the substrate stent printed by the 3D printing technology is 50% - 80%, and the pore diameter is 0.5 - 10 mm.

[0023] Furthermore, the preparation steps of the polylactic acid block copolymer are as follows: Under argon protection, first mix 2.1 - 2.4 parts by mass of L-lactide, 0.3 - 0.6 parts by mass of citric acid, 0.6 - 0.9 parts by mass of methoxypolyethylene glycol, 1.5 - 1.8 parts by mass of glycolic acid, 3.8 - 4.2 parts by mass of anhydrous toluene, and 0.8 - 1 part by mass of stannous octoate, react at 120 °C for 24 h, then cool to room temperature, add 6.6 - 6.65 parts by mass of dichloromethane and stir for 25 - 35 min, then precipitate and filter in an excess of anhydrous ether at -10 °C. The precipitate is added with 6.6 - 6.65 parts by mass of dichloromethane and stirred for 25 - 35 min, then precipitated and filtered in an excess of anhydrous ether at -10 °C. After repeating the above operation twice, dry in a vacuum oven at 60 °C to constant weight to obtain the polylactic acid block copolymer.

[0024] Furthermore, the collagen mixed solution is obtained by mixing a 1 mg / mL collagen solution and a silk fibroin solution with a mass fraction of 3 - 3.5 wt% according to a mass ratio of 6.5 - 7.5:2.5 - 3.5; the concentration of the modified layered double hydroxide solution is 1 mg / mL.

[0025] Furthermore, the preparation steps of the modified layered double hydroxide are as follows: Mix 60 mmol of sodium hydroxide and ultrapure water evenly to obtain solution A of 100 mL; dissolve 20 mmol of magnesium chloride hexahydrate, 10 mmol of aluminum chloride hexahydrate, and 15 mmol of glutathione in 100 mL of ultrapure water to obtain solution B; under the water bath condition of 40 °C, simultaneously drop solution A and solution B into 100 mL of ultrapure water at a rate of 3.5 - 4.5 mL / min, stir at 300 rpm during the dropping process, place the obtained precipitate in a water bath at 85 °C for constant temperature crystallization for 18 h, then centrifuge, wash 4 times with ultrapure water, place in a blast drying oven at 105 °C for constant temperature drying for 24 h, and then grind with an agate mortar and pass through a 200-mesh sieve to obtain the modified layered double hydroxide.

[0026] Furthermore, the soaking duration in step (2) is 10 min, and the alternate soaking is carried out 10 - 20 times; the soaking duration in step (2) is 30 min, and the alternate soaking is carried out 3 - 4 times.

[0027] Adopting the above technical solutions, the present invention has the following beneficial effects:

[0028] (1) For the bone regeneration multi - bionic scaffold material of the present invention, a collagen mixed solution and a modified layered double hydroxide solution are first used to form a bionic matrix layer on a 3D - printed substrate scaffold through a layer - by - layer self - assembly process. Then, an in - situ mineralization technique is employed to in - situ mineralize calcium phosphate nanoparticles on the substrate scaffold with the bionic matrix layer attached to its surface, controllably constructing a collagen bionic interface assembly structure with bone immunomodulatory function. This structure can precisely regulate the chemical composition, topological morphology, drug release, and material degradation rate of the interface, achieving multi - dimensional synergistic promotion of bone regeneration, effectively solving the deficiencies in the synergy of immune regulation and bone regeneration of existing bone regeneration materials. Moreover, the preparation process is relatively simple, has strong repeatability, and is suitable for large - scale production. By precisely controlling the parameters of each step, the stability of product quality can be ensured, providing reliable material support for clinical applications.

[0029] (2) The raw material polylactic acid block copolymer for 3D printing of the present invention is obtained by the reaction polymerization of L - lactide, citric acid, methoxypolyethylene glycol, and glycolic acid, forming a block copolymer with a hyperbranched structure centered on citric acid. The biocompatibility and biodegradability of each raw material of the polylactic acid block copolymer are relatively good. Cavities are formed in the polylactic acid block copolymer. The existence of the cavities can improve the bonding strength between the scaffold and bone tissue, increase the contact area between the scaffold and body fluid, accelerate the degradation of the scaffold, promote the dissolution of calcium and phosphate ions, and be beneficial to the mineralization of bone tissue. At the same time, the existence of the cavities is conducive to protein adsorption, the growth of fibrous tissue, unmineralized bone tissue, and vascularized tissue, promoting the penetration of nutrients and the excretion of metabolic wastes, and promoting the ingrowth of new bone tissue inside the bone repair scaffold, thus better realizing the integration of the bone repair scaffold and the host bone, and not affecting the mechanical properties of the bone regeneration multi - bionic scaffold material due to the existence of the cavities.

[0030] (3) The collagen mixed solution of the present invention is obtained by mixing a collagen solution and a silk fibroin solution; among them, the collagen material has good biocompatibility, biodegradability, bionics, and high versatility; however, pure collagen often has insufficient mechanical properties by itself, while silk fibroin is a natural polymer biomaterial with excellent mechanical properties, good biocompatibility, processability, and biodegradability. The addition of silk fibroin can improve the strength of collagen.

[0031] (4) The modified layered double hydroxide of the present invention is obtained by intercalating glutathione into layered double hydroxide. Glutathione is a tripeptide compound composed of glutamic acid, cysteine, and glycine, and is an important endogenous antioxidant in the human body, with functions such as scavenging free radicals, detoxifying, regulating the immune system, promoting cell metabolism and repair. In the bone regeneration scaffold, glutathione plays a role through multiple mechanisms: Firstly, glutathione neutralizes reactive oxygen species (ROS), reduces the damage of oxidative stress to bone cells, protects the activity of osteoblasts and mesenchymal stem cells, and thus promotes the stability of the bone tissue regeneration environment. Secondly, the anti-inflammatory property of glutathione can reduce the inflammatory response caused by the implanted material, and at the same time, by enhancing the activity of immune cells (such as promoting T cell differentiation), optimize the local microenvironment, reduce the risk of infection, and provide suitable conditions for bone regeneration. Thirdly, glutathione accelerates the energy supply and signal transduction of bone-related cells by activating sulfhydryl enzymes and regulating metabolic pathways (such as sugar and fat metabolism), and stimulates osteoblast differentiation and bone matrix synthesis. Fourthly, glutathione can bind to and remove toxic by-products (such as heavy metal ions) that may be released during the degradation of the bone scaffold material, reduce damage to surrounding tissues, and enhance the biosafety of the scaffold. Fifthly, intercalating glutathione into layered double hydroxide can effectively increase the layer spacing of layered double hydroxide, allowing collagen and silk fibroin to penetrate into the interlayer. Glutathione binds better to layered double hydroxide, and at the same time, during subsequent in-situ mineralization, layered double hydroxide better adsorbs phosphates, and then can more firmly in-situ mineralize calcium phosphate nanoparticles on the substrate scaffold with a biomimetic matrix layer on the surface, effectively enhancing the mechanical properties of the bone regeneration multiple biomimetic scaffold material. Description of the Drawings

[0032] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments in combination with the drawings, where

[0033] Figure 1 is the optical physical diagram of the bone regeneration multiple biomimetic scaffold material of the present invention.

[0034] Figure 2 is the SEM morphology characterization diagram of the bone regeneration multiple biomimetic scaffold material of the present invention. Detailed Embodiments

[0035] To better understand the above technical solution, the following will detail the above technical solution in combination with the drawings of the specification and specific embodiments.

[0036] Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention.

[0037] The following embodiments are only used to illustrate the technical solutions of the present invention more clearly, and cannot be used to limit the protection scope of the present invention.

[0038] The raw materials of the embodiments and comparative examples of the present invention are as follows:

[0039] Collagen solution: Add 35.21 g of collagen gel with an original collagen gel concentration of 1.42% to a glass flask, and then add 20 g of 1 mol / L sodium hydroxide and 500 ml of deionized water to the glass bottle to make a mixed solution; under the conditions of a stirring power of 800 rpm and room temperature, completely dissolve the collagen gel, and stir and react for 4 h to obtain a collagen solution with a concentration of 1 mg / mL.

[0040] Silk fibroin solution: Prepare a 0.5% aqueous sodium bicarbonate solution and boil it. Immerse the cocoon in the boiling aqueous sodium bicarbonate solution for 60 min to remove the sericin protein on its surface. After washing with deionized water and drying and sealing, obtain regenerated silk fibroin fiber; mix calcium chloride, ethanol, and water in a molar ratio of 1:2:8 to obtain a ternary solution; magnetically stir the regenerated silk fibroin fiber in the ternary solution at 72 °C, with a bath ratio of 1:10 and a time of 1 h to obtain a mixed solution; put the mixed solution into a dialysis bag with a molecular weight cut-off of 8000 D and dialyze it in flowing tap water for 48 h, then change to deionized water, change water once per hour, and continue for 48 h. After dialysis and filtration, obtain a silk fibroin solution with a mass fraction of 3 wt%.

[0041] (Example 1)

[0042] A preparation method of a bone regeneration multi-bionic scaffold material includes the following preparation steps:

[0043] (1) Using a polylactic acid block copolymer as a raw material, a base scaffold conforming to the bone defect morphology is prepared by 3D printing technology; the porosity of the base scaffold is 50% and the pore diameter is 1 mm;

[0044] (2) Immerse the base scaffold prepared in step (1) in the collagen mixed solution for 10 min, then take it out and dry it, and then immerse it in the modified layered double hydroxide solution for 10 min, and repeat the above immersion process 10 times; among them, the collagen mixed solution is obtained by mixing a 1 mg / mL collagen solution and a silk fibroin solution with a mass fraction of 3 wt% in a mass ratio of 6.5:3.5; the concentration of the modified layered double hydroxide solution is 1 mg / mL.

[0045] (3) Immerse the base scaffold treated in step (2) in a 60 mM potassium hydrogen phosphate solution for 30 min, then take it out and dry it, and then immerse it in a 100 mM calcium chloride solution for 30 min, and repeat the above immersion process 3 times;

[0046] (4) Wash and dry the base scaffold processed in step (3) to obtain a bone regeneration multi-bionic scaffold material.

[0047] The preparation steps of the polylactic acid block copolymer are as follows: Under argon protection, first mix 2.1 parts by mass of L-lactide, 0.3 parts by mass of citric acid, 0.6 parts by mass of methoxypolyethylene glycol, 1.5 parts by mass of glycolic acid, 3.8 parts by mass of anhydrous toluene, and 0.8 parts by mass of stannous octoate, react at 120 °C for 24 h, then cool to room temperature, add 6.6 parts by mass of dichloromethane and stir for 25 min, then precipitate and filter in an excess of anhydrous ether at -10 °C. The precipitate is continued to be added with 6.6 parts by mass of dichloromethane and stirred for 25 min, then precipitated and filtered in an excess of anhydrous ether at -10 °C. Repeat the above operation twice, and then dry to constant weight in a vacuum oven at 60 °C to obtain the polylactic acid block copolymer.

[0048] The preparation steps of the modified layered double hydroxide are as follows: Mix 60 mmol of sodium hydroxide and ultrapure water evenly to obtain solution A of 100 mL; dissolve 20 mmol of magnesium chloride hexahydrate, 10 mmol of aluminum chloride hexahydrate, and 15 mmol of glutathione in 100 mL of ultrapure water to obtain solution B; under the water bath condition of 40 °C, simultaneously drop solution A and solution B into 100 mL of ultrapure water at a rate of 3.5 - 4.5 mL / min, stir at 300 rpm during the dropping process, place the obtained precipitate in a water bath at 85 °C for constant temperature crystallization for 18 h, then centrifuge, wash 4 times with ultrapure water, place in a blast drying oven at 105 °C for constant temperature drying for 24 h, then grind with an agate mortar, and pass through a 200-mesh sieve to obtain the modified layered double hydroxide.

[0049] (Example 2)

[0050] A preparation method of a bone regeneration multi-bionic scaffold material, comprising the following preparation steps:

[0051] (1) Using the polylactic acid block copolymer as a raw material, prepare a base scaffold conforming to the bone defect morphology by 3D printing technology; the porosity of the base scaffold is 60% and the pore diameter is 0.5 mm;

[0052] (2) Immerse the base scaffold prepared in step (1) in a collagen mixed solution for 10 min first, then take it out and air dry, then place it in a modified layered double hydroxide solution for 10 min, and repeat the above immersion process 15 times; wherein, the collagen mixed solution is obtained by mixing a 1 mg / mL collagen solution and a 3 wt% silk fibroin solution in a mass ratio of 7:3; the concentration of the modified layered double hydroxide solution is 1 mg / mL.

[0053] (3) Immerse the base scaffold processed in step (2) in an 80 mM dipotassium hydrogen phosphate solution for 30 min, then take it out and let it dry. Then place it in a 100 mM calcium chloride solution and soak for 30 min. Repeat the above soaking process three times;

[0054] (4) Wash and dry the base scaffold processed in step (3) to obtain a bone regeneration multi-bionic scaffold material.

[0055] The preparation steps of the polylactic acid block copolymer are as follows: Under argon protection, first mix 2.2 parts by mass of L-lactide, 0.5 parts by mass of citric acid, 0.7 parts by mass of methoxypolyethylene glycol, 1.7 parts by mass of glycolic acid, 4 parts by mass of anhydrous toluene, and 0.9 parts by mass of stannous octoate, react at 120 °C for 24 h, then cool to room temperature, add 6.63 parts by mass of dichloromethane and stir for 30 min, then precipitate and filter in an excess of anhydrous ether at -10 °C. The precipitate is added with 6.63 parts by mass of dichloromethane and stirred for 30 min, then precipitated and filtered in an excess of anhydrous ether at -10 °C. Repeat the above operation twice, and then dry to constant weight in a vacuum oven at 60 °C to obtain the polylactic acid block copolymer.

[0056] The preparation steps of the modified layered double hydroxide are as follows: Mix 60 mmol of sodium hydroxide and ultrapure water evenly to obtain solution A of 100 mL; dissolve 20 mmol of magnesium chloride hexahydrate, 10 mmol of aluminum chloride hexahydrate, and 15 mmol of glutathione in 100 mL of ultrapure water to obtain solution B; under the water bath condition of 40 °C, simultaneously drop solution A and solution B into 100 mL of ultrapure water at a rate of 3.5 - 4.5 mL / min, stir at 300 rpm during the dropping process, place the obtained precipitate in a water bath at 85 °C for constant temperature crystallization for 18 h, then centrifuge, wash 4 times with ultrapure water, place in a blast drying oven at 105 °C for constant temperature drying for 24 h, then grind with an agate mortar, and pass through a 200-mesh sieve to obtain the modified layered double hydroxide.

[0057] (Example 3)

[0058] A preparation method of a bone regeneration multi-bionic scaffold material, comprising the following preparation steps:

[0059] (1) Using the polylactic acid block copolymer as a raw material, a base scaffold conforming to the bone defect morphology is prepared by 3D printing technology; the porosity of the base scaffold is 80% and the pore diameter is 0.8 mm;

[0060] (2) Immerse the substrate scaffold prepared in step (1) in the collagen mixed solution for 10 min, then take it out and let it dry, and then place it in the modified layered double hydroxide solution for 10 min. Repeat the above immersion process 20 times. Among them, the collagen mixed solution is obtained by mixing a 1 mg / mL collagen solution and a 3 wt% silk fibroin solution in a mass ratio of 7.5:2.5. The concentration of the modified layered double hydroxide solution is 1 mg / mL.

[0061] (3) Immerse the substrate scaffold treated in step (2) in a 100 mM dipotassium hydrogen phosphate solution for 30 min, then take it out and let it dry, and then place it in a 100 mM calcium chloride solution for 30 min. Repeat the above immersion process 3 times.

[0062] (4) Wash and dry the substrate scaffold treated in step (3) to obtain a bone regeneration multi-bionic scaffold material.

[0063] The preparation steps of the polylactic acid block copolymer are as follows: Under argon protection, first mix 2.4 parts by mass of L-lactide, 0.6 parts by mass of citric acid, 0.9 parts by mass of methoxypolyethylene glycol, 1.8 parts by mass of glycolic acid, 4.2 parts by mass of anhydrous toluene, and 1 part by mass of stannous octoate, react at 120 °C for 24 h, then cool to room temperature, add 6.65 parts by mass of dichloromethane and stir for 35 min, then precipitate and filter in excess anhydrous ether at -10 °C. The precipitate is continued to be added with 6.65 parts by mass of dichloromethane and stirred for 35 min, then precipitated and filtered in excess anhydrous ether at -10 °C. Repeat the above operation two more times, and then dry to constant weight in a vacuum oven at 60 °C to obtain the polylactic acid block copolymer.

[0064] The preparation steps of the modified layered double hydroxide are as follows: Mix 60 mmol of sodium hydroxide and ultrapure water evenly to obtain 100 mL of solution A; dissolve 20 mmol of magnesium chloride hexahydrate, 10 mmol of aluminum chloride hexahydrate, and 15 mmol of glutathione in 100 mL of ultrapure water to obtain solution B; under the water bath condition of 40 °C, simultaneously drip solution A and solution B into 100 mL of ultrapure water at a rate of 3.5 - 4.5 mL / min, stir at 300 rpm during the dripping process, place the obtained precipitate in a water bath at 85 °C for constant temperature crystallization for 18 h, then centrifuge, wash 4 times with ultrapure water, place it in a blast drying oven at 105 °C for constant temperature drying for 24 h, then grind with an agate mortar, and pass through a 200-mesh sieve to obtain the modified layered double hydroxide.

[0065] (Comparative Example 1)

[0066] The difference between Comparative Example 1 and Example 2 is only that in Comparative Example 1, the substrate scaffold was alternately immersed in a 1 mg / mL collagen solution and a modified layered double hydroxide solution, and the remaining steps and components were the same as those in Example 2.

[0067] (Comparative Example 2)

[0068] The difference between Comparative Example 2 and Example 2 is only that the substrate scaffold was alternately immersed in a collagen mixed solution and a double hydroxide solution, and the remaining steps and components were the same as those in Example 2.

[0069] Among them, the preparation steps of the double hydroxide are as follows: 200 mmol of sodium hydroxide and ultrapure water were mixed evenly to obtain 100 mL of solution A; 60 mmol of magnesium chloride hexahydrate and 30 mmol of aluminum chloride hexahydrate were dissolved in 100 mL of ultrapure water to obtain solution B; under the water bath condition of 40 °C, solution A and solution B were simultaneously dropped into 100 mL of ultrapure water at a rate of 3.5 - 4.5 mL / min, and stirred at 300 rpm during the dropping process. The obtained precipitate was placed in a water bath at 85 °C for constant temperature crystallization for 18 h, then centrifuged, washed 4 times with ultrapure water, placed in a blast drying oven at 105 °C for constant temperature drying for 24 h, then ground with an agate mortar, and passed through a 200-mesh sieve to obtain the modified layered double hydroxide.

[0070] (Comparative Example 3)

[0071] The difference between Comparative Example 3 and Example 2 is only that the raw material of the substrate scaffold was polylactic acid, and the remaining steps and components were the same as those in Example 2.

[0072] Effect Example

[0073] Mechanical properties: The compressive strength of the bone regeneration multi-bionic scaffold materials of the examples and comparative examples was tested using a universal mechanical testing machine; specimen size: Φ4.75 mm × 9.00 mm. Before testing, the two ends of the specimen were ground flat, and the loading speed was set to 0.5 mm / min (n = 5).

[0074] The following Table 1 shows the test results of the compressive strength of the bone regeneration multi-bionic scaffold materials of the examples and comparative examples:

[0075] Compressive strength (MPa) Compressive strength (MPa) Example 1 23.8 Comparative Example 1 22.7 Example 2 24.6 Comparative Example 2 15.2 Example 3 24.3 Comparative Example 3 10.4

[0076] It can be seen from Table 1 above that the bone regeneration multi-bionic scaffold materials prepared in Examples 1 - 3 have better compressive strength.

[0077] The difference between Comparative Example 1 and Example 2 is only that in Comparative Example 1, the substrate scaffold was alternately immersed in a 1 mg / mL collagen solution and a modified layered double hydroxide solution, rather than a collagen mixed solution obtained by mixing a collagen solution and a silk fibroin solution, and its compressive strength is lower than that of Example 2.

[0078] The difference between Comparative Example 2 and Example 2 is only that the base scaffold was alternately immersed in a collagen mixed solution and a dihydroxide solution, without using a modified layered dihydroxide solution, and its compressive strength is lower than that of Example 2.

[0079] The difference between Comparative Example 3 and Example 2 is only that the raw material of the base scaffold is polylactic acid instead of a polylactic acid block copolymer, and its compressive strength is lower than that of Example 2.

[0080] Osteogenic activity test: After co-culturing the bone regeneration multi-bionic scaffold materials of the examples and comparative examples with MG-63 cells for 14 days respectively, the culture medium in the well plate was aspirated, rinsed 3 times successively with PBS buffer and physiological saline, then 200 μl of cell lysate was added and placed at 4 °C for 30 min to fully lyse the cells. Then the cell lysate was collected and centrifuged at 12000 rmp for 15 min. 25 μL of the supernatant was taken out and added to 200 μL of the protein test working solution in a 96-well plate and incubated at 37 °C for 30 min. The absorbance value at 625 nm was detected with an enzyme-labeled instrument to obtain the total protein content in each group of cells. Next, according to the procedure of the alkaline phosphatase test kit, 50 μL of the supernatant was added to 100 μL of the working solution in a 96-well plate and incubated at 37 °C for 15 min, then 50 μl of the chromogenic solution was added, and the absorbance value at 420 nm was immediately detected. Finally, the relative activity of alkaline phosphatase is the ratio of the total amount of alkaline phosphatase to the total amount of protein.

[0081] The following Table 2 shows the test results of the alkaline phosphatase activity of the bone regeneration multi-bionic scaffold materials of the examples and comparative examples:

[0082]

[0083]

[0084] It can be seen from Table 1 above that the alkaline phosphatase activity of the bone regeneration multi-bionic scaffold materials prepared in Examples 1 to 3 is better, indicating that the bone regeneration multi-bionic scaffold materials prepared in Examples 1 to 3 all have a certain promoting effect on the early differentiation of osteoblasts and have a certain osteogenic activity.

[0085] The difference between Comparative Example 1 and Example 2 is only that in Comparative Example 1, the base scaffold was alternately immersed in a 1 mg / mL collagen solution and a modified layered dihydroxide solution, rather than a collagen mixed solution obtained by mixing a collagen solution and a silk fibroin solution, and its alkaline phosphatase activity is lower.

[0086] The difference between Comparative Example 2 and Example 2 is only that the base scaffold was alternately immersed in a collagen mixed solution and a dihydroxide solution, without using a modified layered dihydroxide solution, and its alkaline phosphatase activity is lower.

[0087] The difference between Comparative Example 3 and Example 2 is only that the raw material of the base stent is polylactic acid instead of polylactic acid block copolymer, and the alkaline phosphatase activity is lower.

[0088] The specific embodiments described above further elaborate on the objectives, technical solutions, and beneficial effects of the present invention. It should be understood that the above are only specific 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 principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A multiple biomimetic scaffold material for bone regeneration, characterized in that, The multi-bionic scaffold material for bone regeneration is prepared by first using a 3D printing technique with a poly(lactic acid) block copolymer as the raw material to obtain a base scaffold conforming to the bone defect morphology, then alternately impregnating the base scaffold multiple times in a collagen mixed solution and a modified layered double hydroxide solution to form a bionic matrix layer on the surface of the base scaffold, and finally alternately soaking the base scaffold with the bionic matrix layer attached thereto multiple times in a dipotassium hydrogen phosphate solution and a calcium chloride solution, followed by washing and drying.

2. The bone regeneration multi-bionic scaffold material according to claim 1, characterized in that The poly(lactic acid) block copolymer is obtained by the reaction polymerization of L-lactide, citric acid, methoxypolyethylene glycol, and glycolic acid.

3. The bone regeneration multi-bionic scaffold material according to claim 1, wherein, The collagen mixed solution is obtained by mixing a collagen solution and a silk fibroin solution.

4. The bone regeneration multi-bionic scaffold material according to claim 1, characterized in that, The modified layered double hydroxide is obtained by intercalating glutathione into layered double hydroxide.

5. A preparation method of a bone regeneration multi-bionic scaffold material, characterized in that, It includes the following preparation steps: (1) Using a 3D printing technique with a poly(lactic acid) block copolymer as the raw material to obtain a base scaffold conforming to the bone defect morphology; (2) Alternately soaking the base scaffold obtained in step (1) multiple times in a collagen mixed solution and a modified layered double hydroxide solution; (3) Alternately soaking the base scaffold treated in step (2) multiple times in a dipotassium hydrogen phosphate solution and a calcium chloride solution; (4) Washing and drying the base scaffold treated in step (3) to obtain the multi-bionic scaffold material for bone regeneration.

6. The bone regeneration multi-bionic scaffold material according to claim 5, characterized in that, The porosity of the base scaffold printed by the 3D printing technique is 50% - 80%, and the pore diameter is 0.5 - 10 mm.

7. The bone regeneration multi-bionic scaffold material according to claim 5, characterized in that The preparation steps of the poly(lactic acid) block copolymer are as follows: Under argon protection, first mix 2.1 - 2.4 parts by mass of L-lactide, 0.3 - 0.6 parts by mass of citric acid, 0.6 - 0.9 parts by mass of methoxypolyethylene glycol, 1.5 - 1.8 parts by mass of glycolic acid, 3.8 - 4.2 parts by mass of anhydrous toluene, and 0.8 - 1 part by mass of stannous octoate, react at 120 °C for 24 h, then cool to room temperature, add 6.6 - 6.65 parts by mass of dichloromethane and stir for 25 - 35 min, then precipitate and filter in an excess of anhydrous ether at -10 °C. The precipitate is continued to be added with 6.6 - 6.65 parts by mass of dichloromethane and stirred for 25 - 35 min, then precipitated and filtered in an excess of anhydrous ether at -10 °C. Continue to repeat the above operation twice, and then dry to a constant weight in a vacuum oven at 60 °C to obtain the poly(lactic acid) block copolymer.

8. The bone regeneration multi-bionic scaffold material according to claim 5, characterized in that, The collagen mixed solution is obtained by mixing a 1 mg / mL collagen solution and a 3 - 3.5 wt% silk fibroin solution in a mass ratio of 6.5 - 7.5:2.5 - 3.5; the concentration of the modified layered double hydroxide solution is 1 mg / mL.

9. The bone regeneration multi-bionic scaffold material according to claim 5, wherein The preparation steps of the modified layered double hydroxide are as follows: 60 mmol of sodium hydroxide and ultrapure water are mixed evenly to obtain 100 mL of solution A; 20 mmol of magnesium chloride hexahydrate, 10 mmol of aluminum chloride hexahydrate, and 15 mmol of glutathione are dissolved in 100 mL of ultrapure water to obtain solution B; under the water bath condition of 40 °C, solution A and solution B are simultaneously dropped into 100 mL of ultrapure water at a rate of 3.5 - 4.5 mL / min, and stirred at 300 rpm during the dropping process. The obtained precipitate is placed in a water bath at 85 °C for constant temperature crystallization for 18 h, then centrifuged, washed 4 times with ultrapure water, placed in a forced air drying oven at 105 °C for constant temperature drying for 24 h, then ground with an agate mortar, and passed through a 200-mesh sieve to obtain the modified layered double hydroxide.

10. The bone regeneration multi-bionic scaffold material according to claim 5, characterized in that, The soaking duration in step (2) is 10 min, with alternate soaking for 10 - 20 times; the soaking duration in step (2) is 30 min, with alternate soaking for 3 - 4 times.

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