High-efficiency load osteogenic and angiogenic multi-drug wood-based elastic artificial periosteum and preparation method and application thereof

Abstract

This invention discloses a highly efficient wood-based elastic artificial periosteum loaded with multiple osteogenic and angiogenic drugs, its preparation method, and its applications. The artificial periosteum uses wood-derived elastic wood as a base layer, polydopamine as a bonding layer, and layered drug-loaded electrostatic self-assembly layers as a biofunctional layer. The base layer not only mimics the structural characteristics and mechanical anisotropy of the natural periosteum, but its elasticity also facilitates clinical implantation of the artificial periosteum. The bonding layer effectively connects the base layer and the biofunctional layer, while also imparting certain adhesive properties to the artificial periosteum. The biofunctional layer can efficiently load multiple drugs, achieving synergistic and long-lasting promotion of osteogenic and angiogenic biological functions during bone repair. The artificial periosteum prepared by this invention possesses structural and mechanical anisotropy, high porosity, high elasticity, and adhesive properties; moreover, its drug loading and mechanical properties are controllable, making it an ideal novel artificial periosteum material.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, and specifically relates to a wood-based elastic artificial bone membrane with high efficiency for loading multiple osteogenic and angiogenic drugs, its preparation method and application. Background Technology

[0002] Bone defects of critical size caused by trauma and tumor resection surgery remain a major challenge in orthopedics. In the past, there has been an overemphasis on the bone defect itself while neglecting the importance of the periosteum. The periosteum is a highly vascularized connective tissue membrane covering the surface of bone, delivering oxygen and nutrients to the bone and adjacent tissues through blood supply. An ideal artificial periosteum material should possess both structural and functional biomimetic properties. Structurally, the periosteum consists of an outer fibrous layer and an inner cambium. The outer fibrous layer is mainly composed of axially aligned collagen and high-density elastin, giving the periosteum anisotropy and elasticity. The inner cambium (also known as the osteogenic layer) adheres tightly to the bone and coordinates the osteogenic regeneration process by releasing cytokines.

[0003] Currently, publicly available artificial periosteum mainly includes decellularized periosteum scaffolds, electrospun membranes, growth factor-loaded micro / nanofiber membranes, and hydrogel membranes. Structurally, existing artificial periosteum often neglect the anisotropy of the natural periosteum in terms of structure and mechanics, as well as its certain flexibility and elasticity, and its adhesion is limited. For example, the national invention patent application No. 202210193993.3 discloses a method for preparing and applying a double-layer biomimetic artificial periosteum. Although this artificial periosteum mimics the double-layer structure of the natural periosteum and has certain biological functions, it neglects the anisotropy of the natural periosteum in terms of structure and mechanics, as well as the requirements for the adhesion of the artificial periosteum in practical applications.

[0004] Natural wood is abundant, primarily composed of neatly arranged fibers with high porosity and numerous tubular channels, crucial for the rapid transport of nutrients and water. Importantly, its naturally occurring anisotropic structure makes wood a "natural template" for mimicking the anisotropic collagen fiber layers in natural periosteum. However, wood's excessive strength and lack of flexibility can cause mechanical damage when used as an implant material. Research has shown that wood-based cellulose fiber materials, prepared by completely or partially removing lignin and hemicellulose from wood through alkaline treatment, not only retain the anisotropic mechanical and structural characteristics of wood but also improve its flexibility. In previous studies, we published an efficient antibacterial agent combination, a natural wood-based hydrogel periosteum material, and its application (application number 202210550879.1). This artificial periosteum, prepared by immersing a drug-loaded hydrogel into channels of completely de-lignin and hemicellulose-removed white wood, exhibits certain antibacterial and osteogenic effects. However, the completely de-lignin and hemicellulose-removed white wood lacks morphological stability in a humid environment, necessitating the introduction of hydrogels to impart stable morphology and good flexibility. However, the introduction of hydrogels occupies the unique channel structure of wood, and this type of periosteum design neglects vascularization and adhesion properties. Sufficient blood supply and adhesion properties are fundamental prerequisites and guarantees for the effective repair of damaged bone tissue in the clinical application of artificial periosteum.

[0005] Therefore, using wood to mimic the anisotropic fibrous structure of the periosteum is an effective method. However, how to design a type of wood-based artificial periosteum that can efficiently load osteogenic and angiogenic drugs, retain the unique channel structure of wood, and have the elasticity and adhesion properties required for clinical use has become an urgent problem to be solved. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a wood-based elastic artificial bone membrane that can efficiently load multiple osteogenic and angiogenic drugs.

[0007] Another objective of this invention is to provide a method for preparing the above-mentioned highly efficient wood-based elastic artificial bone membrane loaded with multiple osteogenic and angiogenic drugs.

[0008] Another object of the present invention is to provide the application of the above-mentioned highly efficient loaded osteogenic and angiogenic multidrug wood-based elastic artificial bone membrane.

[0009] The objective of this invention is achieved through the following technical solution: a highly efficient wood-based elastic artificial bone membrane loaded with multiple osteogenic and angiogenic drugs, comprising a base layer, an adhesive layer, and a biofunctional layer connected in sequence; wherein, the base layer is elastic wood, the adhesive layer is polydopamine, and the biofunctional layer is composed of a positively charged layer loaded with drugs or is formed by the self-assembly of a positively charged layer loaded with drugs and a negatively charged layer loaded with drugs.

[0010] The thickness of the elastic wood is 500–2000 μm, preferably 800–1500 μm.

[0011] The elastic wood is obtained by removing lignin and hemicellulose from natural wood under alkaline conditions; preferably, it is prepared through the following specific steps:

[0012] (1) Prepare an alkaline solution;

[0013] (2) Place the natural wood in an alkaline solution and impregnate it under vacuum to remove air bubbles from the wood pores. Then release the vacuum so that the mixed solution can fully penetrate the wood pores.

[0014] (3) The wood-containing solution obtained in step (2) is heated and reacted. After the reaction is completed, the wood obtained is soaked and washed with deionized water to remove residual chemical impurities. Finally, it is frozen and dried to obtain elastic wood.

[0015] The alkaline solution mentioned in step (1) is a mixed solution containing 3 mol / L NaOH and 0.5 mol / L Na2SO3.

[0016] The vacuum condition described in step (2) is a negative pressure with a relative vacuum degree of no more than -80 kPa.

[0017] The soaking time in step (2) is preferably 10 to 25 minutes.

[0018] The preferred time for releasing the vacuum in step (2) is 5 to 10 minutes.

[0019] The preferred conditions for the heating reaction in step (3) are 70-100°C for 0.5-9 hours; more preferably, 70-100°C for 3-9 hours.

[0020] The soaking time in step (3) is preferably 24 to 48 hours; the water should be changed and washed multiple times during this period.

[0021] The freezing conditions described in step (3) are preferably freezing at -20°C for 6 to 24 hours.

[0022] The drying method described in step (3) is preferably freeze drying.

[0023] The freeze-drying time is preferably 12 to 24 hours.

[0024] The natural wood mentioned includes any one of the following: modified or unmodified balsa wood, poplar, beech, linden, walnut, elm, and paulownia.

[0025] The thickness of the adhesive layer is 5–50 nm.

[0026] The thickness of the biological functional layer is 10–1000 nm.

[0027] The biofunctional layer is connected to the adhesive layer through a positively charged layer loaded with drugs.

[0028] The structure of the aforementioned biological functional layer is shown below: (ab) n -a m a represents a positively charged layer loaded with drug, and b represents a negatively charged layer loaded with drug; ab indicates that the positively charged layer loaded with drug and the negatively charged layer loaded with drug are connected, a m In (ab) n The outermost layer; n is an integer greater than or equal to 0, preferably 0 to 15; m is 0 or 1, and n and m cannot be 0 at the same time; when n is 0 and m is 1, the biofunctional layer is a positively charged layer loaded with drugs; when n is an integer greater than or equal to 1 and m is 0, the structure of the biofunctional layer is (ab). n The outermost layer (centered on the elastic wood) is a negatively charged layer loaded with drugs; when n is an integer greater than or equal to 1 and m is 1, the structure of the aforementioned biological functional layer is (ab). n (ab) n -1…-a, the outermost layer is a positively charged layer loaded with drugs.

[0029] The drug-loaded positively charged layer is obtained by encapsulating the drug with a positively charged natural polymer.

[0030] The positively charged natural polymer is preferably chitosan.

[0031] The drug in the positively charged layer loaded with the drug is preferably a negatively charged osteogenic drug; more preferably, it is pamidronate disodium.

[0032] The drug-loaded negatively charged layer is obtained by encapsulating the drug with a negatively charged natural polymer.

[0033] The negatively charged natural polymer is preferably at least one of hyaluronic acid salts, alginate, gelatin, and type I collagen.

[0034] The drug in the negatively charged drug-loaded layer is preferably a positively charged angiogenic drug; more preferably deferoxamine.

[0035] The preparation method of the above-mentioned wood-based elastic artificial periosteum with high-efficiency loading of osteogenic and angiogenic multiple drugs includes the following steps:

[0036] 1) Soak the elastic wood in a dopamine solution. After the dopamine forms a polydopamine layer on the surface of the elastic wood through an oxidative self-polymerization process, wash it to remove residual unreacted dopamine and impurities. Then freeze and dry to obtain elastic wood with a surface-modified polydopamine layer.

[0037] 2) Prepare a solution A1 with a pH value of 1 to 6 by preparing a positively charged natural polymer, and then add the drug to obtain solution A2; prepare a solution B1 with a pH value of 1 to 6 by preparing a negatively charged natural polymer, and then add the drug to obtain solution B2.

[0038] 3) The surface-modified dopamine elastic wood was immersed in solution A and then washed with deionized water to obtain elastic wood with a polydopamine layer and a positively charged layer loaded with drugs, which were sequentially modified from the inside to the outside.

[0039] 4) Immerse the elastic wood obtained in step 3) in solution B, and then wash it with deionized water to obtain elastic wood that is modified from the inside out with a polydopamine layer, a positively charged layer loaded with drugs, and a negatively charged layer loaded with drugs. Depending on the actual needs, the elastic wood finally obtained in step 3) or step 4) is a high-efficiency wood-based elastic artificial bone membrane loaded with multiple osteogenic and angiogenic drugs, or step 3) and step 4) are repeated several times to obtain a high-efficiency wood-based elastic artificial bone membrane loaded with multiple osteogenic and angiogenic drugs.

[0040] The above-mentioned method for preparing a wood-based elastic artificial periosteum with high-efficiency loading of multiple osteogenic and angiogenic drugs further includes the following steps:

[0041] 5) After the final layer is assembled, the resulting product is frozen and dried to produce a wood-based elastic artificial bone membrane that can efficiently load multiple osteogenic and angiogenic drugs.

[0042] The concentration of the dopamine solution mentioned in step 1) is preferably 0.2-3 g / L; more preferably 0.5-3 g / L.

[0043] The solvent in the dopamine solution described in step 1) is preferably an 8-12 mM Tris-HCl buffer solution with a pH of 8.0-9.0; more preferably, it is a 10 mM Tris-HCl buffer solution with a pH of 8.5.

[0044] The soaking time described in step 1) is preferably 1 to 30 hours; more preferably 6 to 24 hours.

[0045] The concentration of positively charged natural polymers in solution A1 described in step 2) is preferably 1 to 10 mg / mL; more preferably 1 to 9 mg / mL.

[0046] The solvent for solution A1 is preferably a 1-2% glacial acetic acid solution.

[0047] The drug concentration in solution A2 mentioned in step 2) is 0.01 to 1 mg / mL.

[0048] The zeta potential of solution A2 described in step 2) is +10 to +45 mV; more preferably +18 to +35 mV.

[0049] The concentration of negatively charged natural polymers in solution B1 described in step 2) is preferably 1 to 10 mg / mL.

[0050] The solvent for solution B1 is preferably deionized water or glacial acetic acid solution. If the negatively charged natural polymer is collagen, then glacial acetic acid solution is required for dissolution.

[0051] The concentration of the glacial acetic acid solution is preferably 1-2% by volume.

[0052] The drug concentration in solution B2 described in step 2) is 20–200 μg / mL; more preferably 20–180 μg / mL.

[0053] The zeta potential of solution B2 described in step 2) is -5 to -45 mV; more preferably -7 to -45 mV.

[0054] The soaking time described in steps 3) and 4) is preferably 15–60 minutes. Self-assembly occurs during the soaking process.

[0055] The washing process described in steps 3) and 4) is preferably performed 2 to 3 times.

[0056] The freezing temperature described in step 5) is preferably -20°C.

[0057] The drying method described in step 5) is preferably freeze drying.

[0058] The above-mentioned application of highly efficient loaded osteogenic and angiogenic multidrug wood-based elastic artificial bone membrane in the preparation of bone repair materials.

[0059] The present invention has the following advantages and effects compared with the prior art:

[0060] (1) This invention uses wood-derived elastic wood as the base material, which is green and environmentally friendly and has abundant sources; moreover, elastic wood has the characteristics of anisotropy, high porosity, high elasticity and biocompatibility.

[0061] (2) The present invention uses a layer-by-layer self-assembly method to encapsulate drugs. Under electrostatic interaction, natural polymers carrying different drugs self-assemble layer by layer to form a biological functional layer. This treatment method not only does not occupy the pore structure of wood, thus maintaining the structural characteristics of the wood substrate, but also controls the drug concentration based on the layer-by-layer self-assembly method, avoiding excessive concentration and drug toxicity.

[0062] (3) The artificial bone membrane prepared by the present invention has good adhesion properties. As an artificial bone membrane, it can fit well with damaged bone tissue and more effectively exert the ability of artificial bone membrane to promote bone healing.

[0063] (4) The artificial periosteum prepared by the present invention can release pamidronate disodium and deferoxamine for a long time, exerting a synergistic and long-lasting effect to promote osteogenic and angiogenic effects, and is an ideal new type of artificial periosteum material.

[0064] In summary, this invention utilizes a layer-by-layer self-assembly method to efficiently modify the surface of an elastic wood substrate with osteogenic and angiogenic drugs. This maintains the unique anisotropic channel structure of wood while efficiently loading drugs with different biological functions, achieving controlled sustained release during bone repair. The prepared artificial periosteum not only mimics the structure and mechanical anisotropy of the natural periosteum, exhibiting excellent elasticity and adhesion properties, but also possesses good biocompatibility and superior osteogenic and angiogenic characteristics, making it a promising candidate for use as an artificial periosteum material. Attached Figure Description

[0065] Figure 1 The images shown are photographs of the elastic wood specimen prepared in Example 1 of this invention; wherein, A is a conventional optical photograph, B is a scanning electron microscope (SEM) photograph in the R direction (perpendicular to the fiber growth direction), and C is a scanning electron microscope (SEM) photograph in the L direction (parallel to the fiber growth direction).

[0066] Figure 2 The graph shows the physical property test results of the artificial bone membrane prepared in Example 2 of this invention; where A is the solid surface potential and B is the LBL. 10- Scanning electron microscope images of the cross-sectional morphology.

[0067] Figure 3 This is a tensile stress-strain curve of the artificial bone membrane prepared in Example 3 of the present invention.

[0068] Figure 4 The image shows the detection results of the in vitro adhesion of the artificial bone membrane prepared in Example 4 of the present invention; where A is the overlap shear test result and B is the adhesion photograph.

[0069] Figure 5The image shows the effect of the artificial periosteum prepared in Example 5 of this invention on cell proliferation (A) and a laser confocal microscope (LSM) photograph of a single cell morphology (B).

[0070] Figure 6 The diagram shows the results of osteogenic performance analysis of the artificial periosteum prepared in Example 6 of this invention: where A is the quantitative analysis result of alkaline phosphatase (ALP) activity, and B is the quantitative analysis result of calcium nodules.

[0071] Figure 7 This is a photograph of cell migration on the artificial periosteum prepared in Example 7 of the present invention.

[0072] Figure 8 This is a graph showing the migration rate analysis results of cells on the artificial periosteum prepared in Example 7 of the present invention.

[0073] Figure 9 These are photographs of the chicken embryo allantoic membrane model before and after drug loading of the artificial periosteum prepared in Example 8 of this invention; where A is EW-P (without DFO loading), and B is the self-assembled two-layer artificial periosteum LBL. 2- (Contains DFO)

[0074] Figure 10 The artificial bone membrane prepared in Example 8 of this invention was compared with unloaded elastic wood (EW-P) and drug-loaded elastic wood (LBL) in the chicken embryo allantoic membrane experiment. 2- A quantitative analysis diagram of the number of blood vessels. Detailed Implementation

[0075] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, all raw materials and reagents in the present invention are commercially available conventional raw materials and reagents. The amounts of each component in the embodiments are expressed in parts by mass (g, mL).

[0076] Example 1: Preparation of a wood-based elastic artificial periosteum with high efficiency loading of multiple osteogenic and angiogenic drugs

[0077] (1) First, prepare a mixed solution containing 3M (M is an abbreviation for mol / L) NaOH and 0.5M Na2SO3. At room temperature, place a 1000μm thick piece of balsa wood into the solution and remove air bubbles from the wood by vacuum impregnation (a negative pressure with a relative vacuum degree not exceeding -80kPa). Maintain the vacuum environment for 10 minutes, and then release the vacuum for 5 minutes to facilitate the full penetration of the mixed solution into the wood pores. Transfer the wood and solution to a three-necked flask and heat to 70℃ for 9 hours. Remove the wood and soak it in deionized water for 24 hours to remove excess impurities. Freeze the wood at -20℃ for 12 hours, and then freeze-dry it for 12 hours to obtain elastic wood. Figure 1Image A shows a photograph of the prepared elastic wood, demonstrating that the chemically treated wood exhibits significantly higher flexibility compared to natural wood. The cross-section (R) and longitudinal section (L) of the elastic wood were observed using a scanning electron microscope. Figure 1 As shown in B and C, the results demonstrate that the chemically treated elastic wood not only maintains its anisotropic structure but also possesses high porosity.

[0078] (2) Prepare a 1.212 g / L Tris solution with tris(hydroxymethyl)aminomethane (Tris), add 1 mol / L HCl solution to it to obtain a Tris-HCl buffer solution with pH=8.5; then take 200 mL of Tris-HCl buffer solution in a beaker, add 0.2 g of dopamine powder and stir to dissolve to prepare a 1 g / L dopamine solution, then add the elastic wood from step (1) to the dopamine solution and stir at room temperature for 12 h to form a polydopamine layer on the surface of the material through an oxidative self-polymerization process. Then wash it several times with deionized water to remove residual unreacted dopamine and impurities. Then freeze it in a -20℃ freezer and freeze dry it in a freeze dryer to finally obtain elastic wood with surface modified polydopamine.

[0079] (3) Weigh 49.5 mL of deionized water and add 0.5 mL of glacial acetic acid to obtain a 1% glacial acetic acid solution. After stirring evenly, add 250 mg of chitosan to the solution to obtain a chitosan solution with a concentration of 5 mg / mL. Then, adjust the pH of the chitosan solution to about 6 with 1 mM NaOH solution to obtain solution A. Finally, add 3 mg of pamidronate disodium and stir to dissolve in solution A to obtain a polycationic solution with a drug concentration of 0.06 mg / mL and a zeta potential of about +18 mV.

[0080] (4) Weigh 50 mL of deionized water, add 50 mg of sodium hyaluronate, stir well to obtain a hyaluronic acid solution with a concentration of 1 mg / mL, and then adjust the pH of the hyaluronic acid solution to 6 with 1 mM HCl solution to obtain solution B. Add 1 mg of deferoxamine to solution B and store it in the dark to obtain a polyanionic solution with a drug concentration of 20 μg / mL. The zeta potential of the solution is approximately -44 mV.

[0081] (5) The polydopamine-modified elastic wood obtained in step (2) is first immersed in the polycationic solution in step (3) for 30 min, and then washed three times with deionized water. The first layer is assembled and named LBL. 1+ Next, LBL 1+ Immerse the product in the polyanionic solution from step (4) for 30 minutes, then wash away unstable components with deionized water to complete the self-assembled second layer, named LBL. 2-Repeat the above steps as needed to obtain high-efficiency load-bearing osteogenic and angiogenic multidrug wood-based elastic artificial bone membranes with different numbers of layers.

[0082] Example 2: Preparation of a wood-based elastic artificial periosteum with high efficiency loading of multiple osteogenic and angiogenic drugs

[0083] (1) First, prepare a mixed solution containing 3M (M is an abbreviation for mol / L) NaOH and 0.5M Na2SO3. At room temperature, place a 1500μm thick poplar wood piece into the solution and remove air bubbles from the wood by vacuum impregnation (a negative pressure with a relative vacuum degree not exceeding -80kPa). Maintain the vacuum environment for 12 minutes, then release the vacuum for 6 minutes to facilitate the full penetration of the mixed solution into the wood pores. Then transfer it to a three-necked flask and heat it to 80℃ for 7.5 hours. Finally, take out the wood and soak it in deionized water for 30 hours to remove excess impurities. Freeze the wood at -20℃ for 6 hours, and then freeze-dry it for 12 hours to obtain elastic wood.

[0084] (2) Prepare a 1.212 g / L Tris solution with tris(hydroxymethyl)aminomethane (Tris), add 1 mol / L HCl solution to obtain a Tris-HCl buffer solution with pH=8; then take 300 mL of Tris-HCl buffer solution in a beaker, add 0.6 g of dopamine powder and stir to dissolve to prepare a 2 g / L dopamine solution, then add the elastic wood from step (1) to the dopamine solution and stir at room temperature for 8 h to form a polydopamine layer on the surface of the material through an oxidative self-polymerization process. Then wash several times with deionized water to remove residual unreacted dopamine and impurities, then freeze in a -20℃ freezer and then dry with a freeze dryer to finally obtain elastic wood with surface modified polydopamine.

[0085] (3) Weigh 49.5 mL of deionized water and add 0.5 mL of glacial acetic acid to obtain a 1% glacial acetic acid solution. After stirring evenly, add 100 mg of chitosan to the solution to obtain a 2 mg / mL chitosan solution. Adjust the pH of the chitosan solution to about 5 with 1 mM NaOH solution to obtain solution A. Finally, add 0.5 mg of pamidronate disodium and stir to dissolve in solution A to obtain a polycationic solution with a drug concentration of 0.01 mg / mL. The zeta potential of the solution is about +26 mV.

[0086] (4) Weigh 50 mL of deionized water, add 100 mg of gelatin, stir well to obtain a gelatin solution with a concentration of 2 mg / mL, then adjust the pH of the gelatin solution to 5 with 1 mM HCl solution to obtain solution B, then add 2 mg of deferoxamine to solution B and store it in the dark to obtain a polyanionic solution with a deferoxamine concentration of 40 μg / mL and a zeta potential of approximately -45 mV.

[0087] (5) The polydopamine-modified elastic wood obtained in step (2) is first immersed in the polycationic solution of step (3) for 50 min, and then washed three times with deionized water. The first layer is assembled and named LBL. 1+ The surface potential of the solid is +24mV; then LBL is applied. 1+ Immerse the product in the polyanionic solution from step (4) for 50 minutes, then wash away any unstable components with deionized water. This completes the self-assembled second layer, which is named LBL. 2- The surface potential of the solid is -35mV.

[0088] Repeat the above steps to obtain high-efficiency load-bearing osteogenic and angiogenic multidrug wood-based elastic artificial bone membranes with different numbers of layers. Figure 2 In the figure, A represents the Zeta potential value of the solid surface of the material before and after modification. It can be seen that the charged properties of the solid surface changed after electrostatic self-assembly, which proves the feasibility of the electrostatic self-assembly method.

[0089] In addition, the cross-sectional morphology of the self-assembled 10th layer of artificial periosteum was observed by SEM. Figure 2 B in the figure shows the adhesive layer and the self-assembled layer on the wood substrate, which proves that the self-assembled parts were successfully modified to the elastic wood substrate layer.

[0090] Example 3: Preparation of a wood-based elastic artificial periosteum with high efficiency loading of multiple osteogenic and angiogenic drugs

[0091] (1) First, prepare a mixed solution containing 3M (M is an abbreviation for mol / L) NaOH and 0.5M Na2SO3. At room temperature, place a 2000μm thick piece of beech wood into the solution and remove air bubbles from the wood by vacuum impregnation (a negative pressure with a relative vacuum degree not exceeding -80kPa). Maintain the vacuum environment for 15 minutes, then release the vacuum for 10 minutes to facilitate the full penetration of the mixed solution into the wood pores. Transfer the wood and solution to a three-necked flask and heat to 90℃ for 5 hours. Remove the wood and soak it in deionized water for 30 hours to remove excess impurities. Freeze the wood at -20℃ for 12 hours, and then freeze-dry it for 16 hours to obtain elastic wood.

[0092] (2) Prepare a 1.212 g / L Tris solution with tris(hydroxymethyl)aminomethane (Tris), add 1 mol / L HCl solution to it to obtain a Tris-HCl buffer solution with pH=9; then take 200 mL of Tris-HCl buffer solution in a beaker, add 0.1 g of dopamine powder and stir to dissolve to prepare a 0.5 g / L dopamine solution, then add the elastic wood from step (1) to the dopamine solution and stir at room temperature for 24 h to form a polydopamine layer on the surface of the material through an oxidative self-polymerization process. Then wash it several times with deionized water to remove residual unreacted dopamine and impurities. Then freeze it in a -20℃ freezer and freeze dry it in a freeze dryer to finally obtain elastic wood with surface modified polydopamine.

[0093] (3) Weigh 98 mL of deionized water and add 2 mL of glacial acetic acid to obtain a 2% glacial acetic acid solution. After stirring evenly, add 400 mg of chitosan to the solution to obtain a chitosan solution with a concentration of 4 mg / mL. Then, adjust the pH of the chitosan solution to about 4 with 1 mM NaOH solution to obtain solution A. Finally, add 20 mg of pamidronate disodium and stir to dissolve it in solution A to obtain a polycationic solution with a drug concentration of 0.2 mg / mL and a solution zeta potential of about +29 mV.

[0094] (4) Weigh 100 mL of deionized water, add 900 mg of sodium alginate, stir well to obtain a sodium alginate solution with a concentration of 9 mg / mL, and then adjust the pH of the sodium alginate solution to 4 with 1 mM HCl solution to obtain solution B. Then add 5 mg of deferoxamine to solution B and store it in the dark to obtain a polyanionic solution with a deferoxamine concentration of 50 μg / mL and a zeta potential of approximately -31 mV.

[0095] (5) The polydopamine-modified elastic wood obtained in step (2) is first immersed in the polycationic solution of step (3) for 20 min, and then washed three times with deionized water. The first layer is assembled and named LBL. 1+ Next, LBL 1+ Immerse the product in the polyanionic solution from step (4) for 20 minutes, then wash away unstable components with deionized water to complete the self-assembled second layer, named LBL. 2- .

[0096] The products obtained in the above steps are: dopamine-modified elastic wood (named EW-P) and LBL. 1+ LBL 2- LBL 9+ LBL 10- The samples were divided into 10mm × 50mm rectangular samples for tensile property testing in the L direction (according to national standard GB / T 228.1-2021). Figure 3 The tensile stress-strain curves of different artificial periosteums are shown. The tensile modulus of the artificial periosteum can reach 400 MPa. The tensile strength of the artificial periosteum also increases with the increase of the number of self-assembled biofunctional layers, indicating that the layer-by-layer self-assembled biofunctional layers improve the mechanical strength of the elastic wood substrate, and the mechanical properties of the artificial periosteum can be controlled by changing the number of self-assembled layers.

[0097] Example 4: Preparation of a wood-based elastic artificial periosteum with high efficiency loading of multiple osteogenic and angiogenic drugs

[0098] (1) First, prepare a mixed solution containing 3M (M is an abbreviation for mol / L) NaOH and 0.5M Na2SO3. At room temperature, place a 1600μm thick elm wood into the solution and remove air bubbles from the wood by vacuum impregnation (a negative pressure with a relative vacuum degree not greater than -80kPa). Maintain the vacuum environment for 10 minutes, and then release the vacuum for 5 minutes to facilitate the full penetration of the mixed solution into the wood pores. Transfer the wood and solution to a three-necked flask and heat to 100℃ for 3 hours. Remove the wood and soak it in deionized water for 35 hours to remove excess impurities. Freeze the wood at -20℃ for 24 hours, and then freeze-dry it for 24 hours to obtain elastic wood.

[0099] (2) Prepare a 1.212 g / L Tris solution with Tris, add 1 mol / L HCl solution to it to obtain a Tris-HCl buffer solution with pH=8; then take 300 mL of Tris-HCl buffer solution in a beaker, add 0.3 g of dopamine powder and stir to dissolve to prepare a 1 g / L dopamine solution, then add the elastic wood from step (1) to the dopamine solution and stir at room temperature for 12 h to form a polydopamine layer on the surface of the material through an oxidative self-polymerization process. Then wash it several times with deionized water to remove residual unreacted dopamine and impurities. Then freeze it in a -20℃ freezer and dry it with a freeze dryer to finally obtain elastic wood with surface modified polydopamine.

[0100] (3) Weigh 98 mL of deionized water, add 2 mL of glacial acetic acid to obtain a 2% glacial acetic acid solution, then add 700 mg of chitosan to the solution to obtain a 7 mg / mL chitosan solution. Adjust the pH of the chitosan solution to approximately 4.5 with 1 mM NaOH solution to obtain solution A. Finally, add 50 mg of pamidronate disodium salt and stir to dissolve in solution A to obtain a polycationic solution with a pamidronate disodium salt concentration of 0.5 mg / mL and a zeta potential of approximately +35 mV.

[0101] (4) Weigh 99 mL of deionized water, add 1 mL of glacial acetic acid to obtain a 1% glacial acetic acid solution, then add 400 mg of type I collagen and stir well to obtain a type I collagen solution with a concentration of 4 mg / mL. Adjust the pH of the type I collagen solution to 4.5 with 1 mM NaOH solution to obtain solution B. Add 10 mg of deferoxamine to solution B and store it in the dark to obtain a polyanionic solution with a deferoxamine concentration of 100 μg / mL and a zeta potential of approximately -30 mV.

[0102] (5) The polydopamine-modified elastic wood obtained in step (2) is first immersed in the polycationic solution of step (3) for 30 min, and then washed three times with deionized water. The first layer is assembled and named LBL. 1+ Next, LBL 1+ Immerse the product in the polyanionic solution from step (4) for 30 minutes, then wash away unstable components with deionized water to complete the self-assembled second layer, named LBL. 2- Repeat the above steps as needed to obtain highly efficient, load-bearing, multi-drug-dependent, wood-based elastic artificial bone membranes with different numbers of layers.

[0103] The elastic wood (named EW), polydopamine-modified elastic wood (named EW-P), and LBL prepared in the above steps were compared. 10- Rectangles of 1mm × 10mm × 50mm were cut and their adhesion was compared using an overlap shear test (according to national standard GB / T 33334-2016). In short, a 10mm × 50mm piece of porcine skin membrane was adhered to a glass slide, and the adhesion test was performed using the other side of the porcine skin. Then, an artificial bone membrane was cut into 10mm × 50mm pieces and adhered to a glass slide. In the overlap shear test, a constant force was applied to compress the tissue to establish good contact. The two slides were then pulled parallel to each other towards the glass surface at a constant rate of 2mm / min to measure the shear tissue adhesion strength. The experimental results are as follows: Figure 4 As shown, compared to unmodified elastic wood, the adhesion force of the self-assembled artificial periosteum can reach 10 kPa, while that of elastic wood is only 2.5 kPa. Figure 4 (A) In addition, the artificial periosteum can fit well with the bone and will not fall off. Figure 4 (B) The adhesive properties of artificial periosteum may originate from the modification of the polydopamine layer, and also from the fact that the natural polymer layers after layer-by-layer self-assembly endow the artificial periosteum with a certain adhesive force.

[0104] Example 5: Preparation of a wood-based elastic artificial periosteum with high efficiency loading of multiple osteogenic and angiogenic drugs

[0105] (1) First, prepare a mixed solution containing 3M (M is an abbreviation for mol / L) NaOH and 0.5M Na2SO3. At room temperature, place a 500μm thick piece of linden wood into the solution and remove air bubbles from the wood by vacuum impregnation (a negative pressure with a relative vacuum degree not exceeding -80kPa). Maintain the vacuum environment for 15 minutes, then release the vacuum for 5 minutes to facilitate the full penetration of the mixed solution into the wood pores. Transfer the wood and solution to a three-necked flask and heat to 100℃ for 3 hours. Remove the wood and soak it in deionized water for 36 hours to remove excess impurities. Freeze the wood at -20℃ for 6 hours, and then freeze-dry it for 12 hours to obtain elastic wood.

[0106] (2) Prepare a 1.212 g / L Tris solution with tris(hydroxymethyl)aminomethane (Tris), add 1 mol / L HCl solution to it to obtain a Tris-HCl buffer solution with pH=8.5; then take 300 mL of Tris-HCl buffer solution in a beaker, add 0.9 g of dopamine powder and stir to dissolve to prepare a 3 g / L dopamine solution, then add the elastic wood from step (1) to the dopamine solution and stir at room temperature for 6 h to form a polydopamine layer on the surface of the material through an oxidative self-polymerization process. Then wash it several times with deionized water to remove residual unreacted dopamine and impurities. Then freeze it in a -20℃ freezer and dry it with a freeze dryer to finally obtain elastic wood with surface modified polydopamine.

[0107] (3) Weigh 148.5 mL of deionized water and add 1.5 mL of glacial acetic acid to obtain a 1% glacial acetic acid solution. After stirring evenly, add 1350 mg of chitosan to the solution to obtain a chitosan solution with a concentration of 9 mg / mL. Then adjust the pH of the chitosan solution to about 3.5 with 1 mM NaOH solution. Finally, add 100 mg of pamidronate disodium salt and stir to dissolve in solution A to obtain a polycationic solution with a pamidronate disodium salt concentration of 0.67 mg / mL and a solution zeta potential of about +23 mV.

[0108] (4) Weigh 150 mL of deionized water, add 750 mg of sodium hyaluronate, stir well to obtain a 5 mg / mL hyaluronic acid solution, then adjust the pH of the hyaluronic acid solution to 3.5 with 1 mM HCl solution to obtain solution B. Add 20 mg of deferoxamine to solution B and store it in the dark to obtain a polyanionic solution with a deferoxamine concentration of 133 μg / mL and a zeta potential of approximately -20 mV.

[0109] (5) The polydopamine-modified elastic wood obtained in step (2) is first immersed in the polycationic solution of step (3) for 15 min, and then washed three times with deionized water. The first layer is assembled and named LBL. 1+ Next, LBL1+ Immerse the product in the polyanionic solution from step (4) for 15 minutes, then wash away unstable components with deionized water to complete the self-assembled second layer, named LBL. 2- Repeat the above steps as needed to obtain high-efficiency load-bearing osteogenic and angiogenic multidrug wood-based elastic artificial bone membranes with different numbers of layers.

[0110] The elastic wood (named EW), polydopamine-modified elastic wood (named EW-P), and LBL prepared in the above steps were compared. 1+ LBL 2- LBL 9+ and LBL 10- Rectangular samples of 1mm × 10mm × 10mm were uniformly cut and sterilized under ultraviolet light before being used for cell culture. Mouse bone marrow mesenchymal stem cells (mBMSCs) (MUCMX-01001, Cyagen OriCell) were used as seed cells for cell compatibility evaluation in this experiment. mBMSCs were cultured at a density of 2 × 102 4 Cells were cultured at the specified cell density in 24-well plates (containing 89% DMEM, 10% FBS, and 1% penicillin-dextrose antibody), with three replicates per group. The proliferation capacity of mBMSCs at different time points on different material surfaces was then assessed using a CCK-8 assay kit. Figure 5 The results showed that the OD values ​​of mBMSC cells on all material surfaces increased over time. Compared with the proliferation data of unmodified elastic wood, the modified artificial bone membrane significantly promoted cell proliferation.

[0111] The adhesion and spreading behavior of cells on the artificial periosteum surface were also observed using laser confocal microscopy. After culturing mBMSCs on the material for 48 hours, the cytoskeleton and nucleus were stained with rhodamine-labeled phalloidin and DAPI staining solution, respectively. Cell morphology and spreading were then observed and photographed under a laser confocal microscope. The results are as follows: Figure 5 As shown in Figure B, the figure illustrates the cell spreading behavior on the material. It can be seen that cells exhibit distinct pseudopodia on the artificial periosteum, and the cell spreading demonstrates anisotropy. These results indicate that, compared to elastic wood, the artificial periosteum modified with layer-by-layer self-assembly drugs is more conducive to cell proliferation and adhesion.

[0112] Example 6: Preparation of a wood-based elastic artificial periosteum with high efficiency loading of multiple osteogenic and angiogenic drugs

[0113] (1) First, prepare a mixed solution containing 3M (M is an abbreviation for mol / L) NaOH and 0.5M Na2SO3. At room temperature, place a 1000μm thick walnut wood piece into the solution and remove air bubbles from the wood by vacuum impregnation (a negative pressure with a relative vacuum degree not exceeding -80kPa). Maintain the vacuum environment for 25 minutes, then release the vacuum for 7 minutes to facilitate the full penetration of the mixed solution into the wood pores. Transfer the wood and solution to a three-necked flask and heat to 85℃ for 6 hours. Remove the wood and soak it in deionized water for 40 hours to remove excess impurities. Freeze the wood at -20℃ for 16 hours, and then freeze-dry it for 20 hours to obtain elastic wood.

[0114] (2) Prepare a 1.212 g / L Tris solution with tris(hydroxymethyl)aminomethane (Tris), add 1 mol / L HCl solution to it to obtain a Tris-HCl buffer solution with pH=8.5; then take 200 mL of Tris-HCl buffer solution in a beaker, add 0.3 g of dopamine powder and stir to dissolve to prepare a 1.5 g / L dopamine solution, then add the elastic wood from step (1) to the dopamine solution and stir at room temperature for 10 h to form a polydopamine layer on the surface of the material through an oxidative self-polymerization process. Then wash it several times with deionized water to remove residual unreacted dopamine and impurities. Then freeze it in a -20℃ freezer and dry it with a freeze dryer to finally obtain elastic wood with surface modified polydopamine.

[0115] (3) Weigh 198 mL of deionized water and add 2 mL of glacial acetic acid to obtain a 1% glacial acetic acid solution. Add 400 mg of chitosan to the solution to obtain a 2 mg / mL chitosan solution. Adjust the pH of the chitosan solution to approximately 2.5 with 1 mM HCl solution to obtain solution A. Finally, add 40 mg of pamidronate disodium and stir to dissolve it in solution A to obtain a polycationic solution with a pamidronate disodium concentration of 0.2 mg / mL and a zeta potential of approximately +22 mV.

[0116] (4) Weigh 200 mL of deionized water and add 2000 mg of sodium alginate to obtain a sodium alginate solution with a concentration of 10 mg / mL. Then, adjust the pH of the sodium alginate solution to 2.5 with 1 mM HCl solution to obtain solution B. Add 30 mg of deferoxamine to solution B and store it in the dark to obtain a polyanionic solution with a deferoxamine concentration of 150 μg / mL and a zeta potential of approximately -7 mV.

[0117] (5) The polydopamine-modified elastic wood obtained in step (2) is first immersed in the polycationic solution of step (3) for 40 min, and then washed three times with deionized water. The first layer is assembled and named LBL. 1+ Next, LBL 1+Immerse the product in the polyanionic solution from step (4) for 40 minutes, then wash away unstable components with deionized water to complete the self-assembled second layer, named LBL. 2- Repeat the above steps as needed to obtain highly efficient, load-bearing, multi-drug-dependent, wood-based elastic artificial bone membranes with different numbers of layers.

[0118] mBMSCs at 2×10 4 / Pore density in elastic wood (named EW), polydopamine-modified elastic wood (named EW-P), LBL 1+ LBL 2- LBL 9+ and LBL 10- After culturing for 7 and 21 days, respectively, the alkaline phosphatase (ALP) secreted by cells in the early stage was quantitatively analyzed using the AKP assay kit and the BCA protein content assay kit. Calcium nodules formed in the late stage of cell formation were analyzed using 1% Alizarin Red staining solution and 10% CPC working solution to assess the material's ability to promote osteogenic differentiation of mBMSCs. Three replicates were set up for each group. Results are as follows: Figure 6 As shown in A and B, compared to EW and EW-P, the artificial periosteum after layer-by-layer self-assembly modification of the drug secreted ALP and calcium nodules had higher OD values, indicating the osteogenic effect of the artificial periosteum. Moreover, the osteogenic effect was more obvious with the increase of the number of drug self-assembly layers, because the increase of drug concentration within a certain range played an osteogenic role.

[0119] Example 7: Preparation of a wood-based elastic artificial periosteum with high efficiency loading of multiple osteogenic and angiogenic drugs

[0120] (1) First, prepare a mixed solution containing 3M (M is an abbreviation for mol / L) NaOH and 0.5M Na2SO3. At room temperature, place an 800μm thick elm wood into the solution and remove air bubbles from the wood by vacuum impregnation (a negative pressure with a relative vacuum degree not exceeding -80kPa). Maintain the vacuum environment for 15 minutes, then release the vacuum for 6 minutes to facilitate the full penetration of the mixed solution into the wood pores. Transfer the wood and solution to a three-necked flask and heat to 95℃ for 4 hours. Finally, remove the wood and soak it in deionized water for 25 hours to remove excess impurities. Freeze the wood at -20℃ for 24 hours, and then freeze-dry it for 14 hours to obtain elastic wood.

[0121] (2) Prepare a 1.212 g / L Tris solution with tris(hydroxymethyl)aminomethane (Tris), add 1 mol / L HCl solution to it to obtain a Tris-HCl buffer solution with pH=9; then take 400 mL of Tris-HCl buffer solution in a beaker, add 1 g of dopamine powder and stir to dissolve to prepare a 2.5 g / L dopamine solution, then add the elastic wood from step (1) to the dopamine solution and stir at room temperature for 7 h to form a polydopamine layer on the surface of the material through an oxidative self-polymerization process. Then wash it several times with deionized water to remove residual unreacted dopamine and impurities. Then freeze it in a -20℃ freezer and dry it with a freeze dryer to finally obtain elastic wood with surface modified polydopamine.

[0122] (3) Weigh 147.75 mL of deionized water and add 2.25 mL of glacial acetic acid to prepare a 1.5% glacial acetic acid solution. After stirring evenly, add 1200 mg of chitosan to the solution to obtain an 8 mg / mL chitosan solution. Then, adjust the pH of the chitosan solution to about 3 with 1 mM HCl solution to obtain solution A. Finally, add 150 mg of pamidronate disodium and stir to dissolve in solution A to obtain a polycationic solution with a drug concentration of 1 mg / mL and a zeta potential of about +19 mV.

[0123] (4) Weigh 150 mL of deionized water, add 750 mg of sodium alginate, stir well to obtain a sodium alginate solution with a concentration of 5 mg / mL, and then adjust the pH of the sodium alginate solution to 3 with 1 mM HCl solution to obtain solution B. Add 7.5 mg of deferoxamine to solution B and store it in the dark to obtain a polyanionic solution with a deferoxamine concentration of 50 μg / mL and a zeta potential of approximately -17 mV.

[0124] (5) The polydopamine-modified elastic wood obtained in step (2) is first immersed in the polycationic solution of step (3) for 25 min, and then washed three times with deionized water. The first layer is assembled and named LBL. 1+ Next, LBL 1+ Immerse the product in the polyanionic solution from step (4) for 25 minutes, then wash away unstable components with deionized water to complete the self-assembled second layer, named LBL. 2- Repeat the above steps as needed to obtain high-efficiency load-bearing osteogenic and angiogenic multidrug wood-based elastic artificial bone membranes with different numbers of layers.

[0125] To determine whether cell migration is affected by drug molecules, we used human umbilical vein endothelial cells (HUVECs, AW-CELLS-H0172, Anwei Biotechnology) at a concentration of 2 × 10⁻⁶. 4 / well density seeding was performed in 24-well plates and cultured until confluence. Scratches were constructed using pipette tips, with 3 replicates per group. Subsequently, elastic wood (designated EW) with dimensions of 1mm × 10mm × 10mm, polydopamine-modified elastic wood (designated EW-P), and LBL were used. 1+ and LBL 9+ The material group was agitated with 5 mL of culture medium (containing 89% DMEM, 10% FBS, and 1% penicillin) under sterile conditions for one day. The resulting conditioned medium was then added to the scratched wells of the plate and incubated for a period of time (1 mL of conditioned medium per well). Finally, the scratch healing process was observed using an inverted optical microscope after 12 hours of co-culturing with HUVECs. Migration photographs are shown below. Figure 7 As shown in the figure, HUVECs on the elastic wood did not migrate significantly after 12 hours, while HUVECs on the artificial periosteum modified by layer-by-layer self-assembly exhibited significant migration. Quantitative analysis of migration is shown in the figure. Figure 8 As shown, it is clear that the introduction of the drug can greatly improve the cell migration rate.

[0126] Example 8: Preparation of a wood-based elastic artificial periosteum with high efficiency loading of multiple osteogenic and angiogenic drugs

[0127] (1) First, prepare a mixed solution containing 3M (M is an abbreviation for mol / L) NaOH and 0.5M Na2SO3. Place a 1500μm thick piece of balsa wood into the solution at room temperature and remove air bubbles from the wood by vacuum impregnation (a negative pressure with a relative vacuum degree not exceeding -80kPa). Maintain the vacuum environment for 20 minutes, then release the vacuum for 10 minutes to facilitate the full penetration of the mixed solution into the wood pores. Transfer the wood and mixed solution to a three-necked flask and heat to 75℃ for 8 hours. Finally, remove the wood and soak it in deionized water for 36 hours to remove excess impurities. Freeze the wood at -20℃ for 8 hours, and then freeze-dry it for 14 hours to obtain elastic wood.

[0128] (2) Prepare a 1.212 g / L Tris solution with tris(hydroxymethyl)aminomethane (Tris), add 1 mol / L HCl solution to obtain a Tris-HCl buffer solution with pH=8.5; then take 400 mL of Tris-HCl buffer solution in a beaker, add 0.08 g of dopamine powder and stir to dissolve to prepare a 0.2 g / L dopamine solution, then add the elastic wood from step (1) to the dopamine solution and stir at room temperature for 30 h. A polydopamine layer is formed on the surface of the material through an oxidative self-polymerization process. Then wash several times with deionized water to remove residual unreacted dopamine and impurities. Then freeze in a -20℃ freezer and dry with a freeze dryer to finally obtain elastic wood with surface modified polydopamine.

[0129] (3) Weigh 247.5 mL of deionized water, add 2.5 mL of glacial acetic acid to obtain a 1% glacial acetic acid solution, then add 2250 mg of chitosan to the solution to obtain a 9 mg / mL chitosan solution. Adjust the pH of the chitosan solution to approximately 4 with 1 mM NaOH solution to obtain solution A. Finally, add 200 mg of pamidronate disodium salt and stir to dissolve in solution A to obtain a polycationic solution with a pamidronate disodium salt concentration of 0.8 mg / mL and a zeta potential of approximately +29 mV.

[0130] (4) Weigh 250 mL of deionized water, add 2250 mg of gelatin, stir well to obtain a gelatin solution with a concentration of 9 mg / mL, and then adjust the pH of the gelatin solution to 4 with 1 mM HCl solution to obtain solution B. Add 45 mg of deferoxamine to solution B and store it in the dark to obtain a polyanionic solution with a deferoxamine concentration of 180 μg / mL and a zeta potential of approximately -31 mV.

[0131] (5) The polydopamine-modified elastic wood obtained in step (2) is first immersed in the polycationic solution of step (3) for 25 min, and then washed three times with deionized water. The first layer is assembled and named LBL. 1+ Next, LBL 1+ Immerse the product in the polyanionic solution from step (4) for 25 minutes, then wash away unstable components with deionized water to complete the self-assembled second layer, named LBL. 2- Repeat the above steps as needed to obtain highly efficient, load-bearing, multi-drug-dependent, wood-based elastic artificial bone membranes with different numbers of layers.

[0132] To observe the angiogenesis effect of the material, we used a chicken embryo allantoic membrane model for verification. First, the chicken embryos were divided into a blank control group and an experimental group. After exposing the CAM (Cardiac Ambulatory Membrane), the material was cut into 1mm × 10mm × 10mm rectangles and placed in the central area of ​​the CAM with sparse blood vessels. The air cell ends were then sealed with transparent tape. After sealing, incubation continued for 24 hours and 48 hours, and angiogenesis was observed using a stereomicroscope. Figure 9 It can be seen that the two-layer artificial periosteum LBL carrying DFO self-assembled 2- (B) Compared to the drug-free EW-P(A), it exhibited more angiogenesis after 48 hours of incubation, and the blood vessels were radial. Figure 10 This is a quantitative analysis of the number of angiogenesis cells. After 48 hours of incubation, the artificial periosteum LBL... 2- The number of blood vessels in the drug-free EW-P was significantly higher than that in the drug-free EW-P, indicating that DFO has a significant angiogenesis-promoting effect.

[0133] Example 9: Preparation of a wood-based elastic artificial periosteum with high efficiency loading of multiple osteogenic and angiogenic drugs

[0134] (1) First, prepare a mixed solution containing 3M (M is an abbreviation for mol / L) NaOH and 0.5M Na2SO3. At room temperature, place a 900μm thick walnut wood piece into the solution and remove air bubbles from the wood by vacuum impregnation (a negative pressure with a relative vacuum degree not exceeding -80kPa). Maintain the vacuum environment for 16 minutes, then release the vacuum for 8 minutes to facilitate the full penetration of the mixed solution into the wood pores. Transfer the wood and mixed solution to a three-necked flask and heat to 95℃ for 4 hours. Finally, remove the wood and soak it in deionized water for 24 hours to remove excess impurities. Freeze the wood at -20℃ for 14 hours, and then freeze-dry it for 10 hours to obtain elastic wood.

[0135] (2) Prepare a 1.212 g / L Tris solution with tris(hydroxymethyl)aminomethane (Tris), add 1 mol / L HCl solution to it to obtain a Tris-HCl buffer solution with pH=8.5; then take 500 mL of Tris-HCl buffer solution in a beaker, add 1.5 g of dopamine powder and stir to dissolve to prepare a 3 g / L dopamine solution, then add the elastic wood from step (1) to the dopamine solution and stir at room temperature for 6 h to form a polydopamine layer on the surface of the material through an oxidative self-polymerization process. Then wash it several times with deionized water to remove residual unreacted dopamine and impurities. Then freeze it in a -20℃ freezer and freeze dry it in a freeze dryer to finally obtain elastic wood with surface modified polydopamine.

[0136] (3) Weigh 99 mL of deionized water and add 1 mL of glacial acetic acid to obtain a 1% glacial acetic acid solution. After stirring evenly, add 100 mg of chitosan to obtain a chitosan solution with a concentration of 1 mg / mL. Then, adjust the pH of the chitosan solution to about 5 with 1 mM NaOH solution to obtain solution A. Finally, add 5 mg of pamidronate disodium and stir to dissolve it in solution A to obtain a polycationic solution with a pamidronate disodium concentration of 0.05 mg / mL and a zeta potential of about +26 mV.

[0137] (4) Weigh 100 mL of deionized water, add 150 mg of sodium hyaluronate, stir well to obtain a hyaluronic acid solution with a concentration of 1.5 mg / mL, and then adjust the pH of the hyaluronic acid solution to 5 with 1 mM HCl solution to obtain solution B. Add 3 mg of deferoxamine to solution B and store it in the dark to obtain a polyanionic solution with a deferoxamine concentration of 30 μg / mL and a zeta potential of approximately -45 mV.

[0138] (5) The polydopamine-modified elastic wood obtained in step (2) is first immersed in the polycationic solution of step (3) for 60 min, and then washed three times with deionized water. The first layer is assembled and named LBL. 1+ Next, LBL 1+ Immerse the product in the polyanionic solution from step (4) for 60 minutes, then wash away unstable components with deionized water to complete the self-assembled second layer, named LBL. 2- Repeating the above steps, after alternating self-assembly in chitosan and hyaluronic acid solutions five times each, an artificial periosteum material with a self-assembled thickness of 350±10 nm was prepared and labeled as LBL. 10- The surface of the composite film has a negative potential.

[0139] Example 10: Preparation of a wood-based elastic artificial periosteum with high efficiency loading of multiple osteogenic and angiogenic drugs

[0140] (1) First, prepare a mixed solution containing 3M (M is an abbreviation for mol / L) NaOH and 0.5M Na2SO3. At room temperature, place a 1000μm thick piece of balsa wood into the solution and remove air bubbles from the wood by vacuum impregnation (a negative pressure with a relative vacuum degree not exceeding -80kPa). Maintain the vacuum environment for 10 minutes, then release the vacuum for 5 minutes to facilitate the full penetration of the mixed solution into the wood pores. Transfer the wood and solution to a three-necked flask and heat to 75℃ for 8 hours. Finally, remove the wood and soak it in deionized water for 24 hours to remove excess impurities. Freeze the wood at -20℃ for 16 hours, and then freeze-dry it for 24 hours to obtain elastic wood.

[0141] (2) Prepare a 1.212 g / L Tris solution with tris(hydroxymethyl)aminomethane (Tris), add 1 mol / L HCl solution to it to obtain a Tris-HCl buffer solution with pH=8.5; then take 500 mL of Tris-HCl buffer solution in a beaker, add 0.4 g of dopamine powder and stir to dissolve to prepare a 0.8 g / L dopamine solution, then add the elastic wood from step (1) to the dopamine solution and stir at room temperature for 13 h to form a polydopamine layer on the surface of the material through an oxidative self-polymerization process. Then wash it several times with deionized water to remove residual unreacted dopamine and impurities. Then freeze it in a -20℃ freezer and dry it with a freeze dryer to finally obtain elastic wood with surface modified polydopamine.

[0142] (3) Weigh 297 mL of deionized water and add 3 mL of glacial acetic acid to obtain a 1% glacial acetic acid solution. Then add 1500 mg of chitosan to the solution and dissolve it completely to obtain a 5 mg / mL chitosan solution. Next, adjust the pH of the chitosan solution to about 5.5 with 1 mM NaOH solution to obtain solution A. Finally, add 150 mg of pamidronate disodium salt and stir to dissolve it in solution A to obtain a polycationic solution with a pamidronate disodium salt concentration of 0.5 mg / mL and a zeta potential of about +19 mV.

[0143] (4) Weigh 300 mL of deionized water, add 1500 mg of gelatin, stir well to obtain a gelatin solution with a concentration of 5 mg / mL, and then adjust the pH of the gelatin solution to 5.5 with 1 mM HCl solution to obtain solution B. Add 30 mg of deferoxamine to solution B and store it in the dark to finally obtain a polyanionic solution with a deferoxamine concentration of 100 μg / mL and a zeta potential of approximately -40 mV.

[0144] (5) The polydopamine-modified elastic wood obtained in step (2) is first immersed in the polycationic solution of step (3) for 55 min, and then washed three times with deionized water. The first layer is assembled and named LBL. 1+ Next, LBL 1+ Immerse the product in the polyanionic solution from step (4) for 55 minutes, then wash away any unstable components with deionized water. This completes the self-assembled second layer, which is named LBL. 2- Repeat the above steps, alternating between self-assembly in chitosan and gelatin solutions 6 times each, and then immerse the material in chitosan solution for 55 minutes to prepare an artificial periosteum material with a self-assembled thickness of 400±10 nm, labeled as LBL. 13+ The surface of the composite film has a positive potential.

[0145] The above embodiments are the optimal implementation methods explored by the present invention, but the implementation methods of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A wood-based elastic artificial periosteum with high efficiency for loading multiple osteogenic and angiogenic drugs, characterized in that: It consists of a base layer, an adhesive layer, and a biofunctional layer connected in sequence; wherein, the base layer is elastic wood, the adhesive layer is polydopamine, and the biofunctional layer is formed by the self-assembly of a positively charged layer loaded with drug and a negatively charged layer loaded with drug. The aforementioned elastic wood is obtained by removing lignin and hemicellulose from natural wood under alkaline conditions; The drug in the positively charged drug-loaded layer is a negatively charged osteogenic drug; The drug in the negatively charged drug-loaded layer is a positively charged angiogenic drug.

2. The high-efficiency loaded osteogenic and angiogenic multidrug wood-based elastic artificial bone membrane according to claim 1, characterized in that: The thickness of the elastic wood is 500–2000 μm; The thickness of the adhesive layer is 5–50 nm; The thickness of the biological functional layer is 10–1000 nm.

3. The high-efficiency loading of multiple osteogenic and angiogenic drugs on a wood-based elastic artificial periosteum according to claim 1, characterized in that: The biofunctional layer is connected to the adhesive layer via a positively charged layer loaded with drug. The structure of the aforementioned biological functional layer is shown below: (ab) n -a m a represents a positively charged layer loaded with drug, and b represents a negatively charged layer loaded with drug; n is an integer greater than or equal to 0; m is 0 or 1, and n cannot be 0.

4. The high-efficiency loaded osteogenic and angiogenic multidrug wood-based elastic artificial periosteum according to claim 1, characterized in that: The elastic wood is prepared through the following specific steps: (1) Prepare an alkaline solution; (2) Place the natural wood in an alkaline solution and impregnate it under vacuum to remove air bubbles from the wood pores, then release the vacuum so that the mixed solution can fully penetrate the wood pores; (3) The wood-containing solution obtained in step (2) is heated and reacted. After the reaction is completed, the wood obtained is soaked and washed with deionized water to remove residual chemical impurities. Finally, it is frozen and dried to obtain elastic wood. The drug-loaded positively charged layer is obtained by encapsulating the drug with a positively charged natural polymer; The drug-loaded negatively charged layer is obtained by encapsulating the drug with a negatively charged natural polymer.

5. The high-efficiency loaded osteogenic and angiogenic multidrug wood-based elastic artificial bone membrane according to claim 4, characterized in that: The alkaline solution mentioned in step (1) is a mixed solution containing 3 mol / L NaOH and 0.5 mol / L Na2SO3; The vacuum condition mentioned in step (2) is a negative pressure with a relative vacuum degree not exceeding -80 kPa; The soaking time described in step (2) is 10 to 25 minutes; The time for releasing the vacuum as described in step (2) is 5 to 10 minutes; The heating reaction conditions described in step (3) are 70–100 °C for 0.5–9 h; The soaking time described in step (3) is 24–48 hours; the water should be changed and washed multiple times during this period. The freezing conditions described in step (3) are -20 °C for 6 to 24 hours; The drying method described in step (3) is freeze drying; The natural wood mentioned includes any one of modified or unmodified balsa wood, poplar, beech, linden, walnut, elm, and paulownia; The positively charged natural polymer mentioned is chitosan; The negatively charged natural polymer is at least one of hyaluronic acid, alginate, gelatin, and type I collagen.

6. The high-efficiency loading of multiple osteogenic and angiogenic drugs on a wood-based elastic artificial periosteum according to claim 1, characterized in that: The negatively charged osteogenic drug is pamidronate disodium; The positively charged angiogenic drug mentioned is deferoxamine.

7. The method for preparing the highly efficient wood-based elastic artificial periosteum loaded with multiple osteogenic and angiogenic drugs according to any one of claims 1 to 6, characterized in that... Includes the following steps: 1) The elastic wood is immersed in a dopamine solution. After the dopamine forms a polydopamine layer on the surface of the elastic wood through an oxidative self-polymerization process, it is washed to remove residual unreacted dopamine and impurities. Then it is frozen and dried to obtain elastic wood with a surface modified polydopamine layer. 2) Prepare a solution A1 with a pH value of 1 to 6 by preparing a positively charged natural polymer, and then add the drug to obtain solution A2; prepare a solution B1 with a pH value of 1 to 6 by preparing a negatively charged natural polymer, and then add the drug to obtain solution B2. 3) The surface-modified dopamine elastic wood was immersed in solution A2 and then washed with deionized water to obtain elastic wood with a polydopamine layer and a drug-loaded positively charged layer sequentially modified from the inside out. 4) Immerse the elastic wood obtained in step 3) in solution B2, and then wash it with deionized water to obtain elastic wood that is modified from the inside out with a polydopamine layer, a positively charged layer loaded with drugs, and a negatively charged layer loaded with drugs. Depending on the actual needs, the elastic wood finally obtained in step 3) or step 4) is a wood-based elastic artificial bone membrane with high efficiency loaded with multiple osteogenic and angiogenic drugs, or step 3) and step 4) are repeated several times to obtain a wood-based elastic artificial bone membrane with high efficiency loaded with multiple osteogenic and angiogenic drugs.

8. The method for preparing the highly efficient wood-based elastic artificial bone membrane loaded with multiple osteogenic and angiogenic drugs according to claim 7, characterized in that... It also includes the following steps: 5) After the final layer is assembled, the resulting product is frozen and dried to produce a wood-based elastic artificial bone membrane that can efficiently load multiple osteogenic and angiogenic drugs.

9. The method for preparing the highly efficient wood-based elastic artificial bone membrane loaded with multiple osteogenic and angiogenic drugs according to claim 7 or 8, characterized in that: The concentration of the dopamine solution mentioned in step 1) is 0.2–3 g / L; The solvent in the dopamine solution mentioned in step 1) is an 8-12 mM Tris-HCl buffer solution with a pH of 8.0-9.0; The soaking time described in step 1) is 1–30 h; The concentration of positively charged natural polymers in solution A1 described in step 2) is 1–10 mg / mL; The drug concentration in solution A2 mentioned in step 2) is 0.01–1 mg / mL; The Zeta potential of solution A2 mentioned in step 2) is +10 to +45 mV; The concentration of negatively charged natural polymers in solution B1 described in step 2) is 1–10 mg / mL; The drug concentration in solution B2 described in step 2) is 20–200 µg / mL; The zeta potential of solution B2 mentioned in step 2) is -5 to -45 mV; The soaking time described in steps 3) and 4) is 15–60 min; The number of times the washing is performed in steps 3) and 4) is 2 to 3.

10. The use of the highly efficient loaded osteogenic and angiogenic multidrug wood-based elastic artificial bone membrane according to any one of claims 1 to 6 in the preparation of bone repair materials.

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