A three-layer composite hydrogel scaffold material and its preparation method and application

Abstract

The present invention is applicable to the technical field of biomedical materials, and provides a three-layer structure composite hydrogel scaffold material and its preparation method and application, the method being: silanized nano-scale hydroxyapatite powder is dispersed in a dioxane solution of polylactic acid, freeze-dried, and a porous polylactic acid / hydroxyapatite composite scaffold material is obtained; by an ultraviolet light grafting method, a precursor solution of polyethylene glycol diacrylate containing methacrylated hyaluronic acid is grafted to a porous polylactic acid / hydroxyapatite composite scaffold material to obtain a double-layer structure composite hydrogel scaffold material; by an ultraviolet light grafting method, a polyethylene glycol diacrylate precursor solution containing 2-methacryloyloxyethyl phosphorylcholine is grafted to a double-layer structure composite hydrogel scaffold material to obtain a three-layer structure composite hydrogel scaffold material. The present invention carries out biomimetic construction according to the structural characteristics of cartilage tissue, simulates articular cartilage structure, and is conducive to the repair and regeneration of articular cartilage.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a three-layer composite hydrogel scaffold material and a preparation method and application thereof. Background Art

[0002] The surface of the articular cartilage that connects to the bone in the joint, facing the joint cavity, is very smooth, facilitating movement between bones. Articular cartilage can evenly distribute forces, expanding the load-bearing surface. This not only maximizes the ability to withstand mechanical loads, but also protects the articular cartilage from damage. Articular cartilage is very smooth, not easily worn during joint movement, and allows for flexible movement. Articular cartilage can sustain lifelong movement without damage because of its excellent lubrication. In cases of synovial lesions, such as rheumatoid arthritis, synovial fluid secretion becomes abnormal, resulting in a loss of normal lubrication, affecting joint function and the nutrition of the articular cartilage. Articular cartilage is also elastic, capable of maximizing the absorption and buffering of stress. When articular cartilage is damaged, its force absorption capacity decreases, and joint damage and degeneration progressively worsen.

[0003] Current research on cartilage repair primarily focuses on synthetic articular cartilage replacements or biomaterials that can stimulate new tissue regeneration. Ideal cartilage replacements would mimic the structure, mechanical properties, and composition of cartilage. In recent years, polymer hydrogels have attracted increasing attention due to their potential as biomaterials for soft tissue repair and even regeneration. Therefore, narrowing the performance gap between polymer hydrogels and biological soft tissue, identifying polymer hydrogel materials that match the performance of biological soft tissue, and ultimately achieving the goal of replacing damaged biological soft tissue with hydrogel materials, remains a key issue in polymer hydrogel research. However, traditional polymer hydrogels exhibit several inherent performance shortcomings, and their widespread application as biomaterials requires addressing numerous challenges, including improving their mechanical strength, imparting cell affinity, reducing friction and wear, and ensuring their connection with natural tissue after implantation. Summary of the Invention

[0004] The purpose of the present invention is to provide a three-layer structure composite hydrogel scaffold material and its preparation method and application, aiming to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for preparing a three-layer composite hydrogel scaffold material comprises the following steps:

[0007] Step S1, uniformly dispersing silanized nano-hydroxyapatite powder in a dioxane solution of polylactic acid, and freeze-drying the mixture to obtain a porous polylactic acid / hydroxyapatite composite scaffold material;

[0008] Step S2: grafting a polyethylene glycol diacrylate precursor solution containing methacrylated hyaluronic acid onto a porous polylactic acid / hydroxyapatite composite scaffold material by an ultraviolet grafting method to obtain a double-layer composite hydrogel scaffold material;

[0009] Step S3: Grafting a precursor solution of polyethylene glycol diacrylate containing 2-methacryloyloxyethyl phosphorylcholine onto the double-layer composite hydrogel scaffold material by an ultraviolet grafting method to obtain a three-layer composite hydrogel scaffold material.

[0010] Furthermore, the specific operations of step S1 are:

[0011] The nano-hydroxyapatite powder is uniformly dispersed in an ethanol-water mixed solution containing a silane coupling agent KH-570, heated in a water bath, and then vacuum dried to obtain silanized nano-hydroxyapatite powder;

[0012] Polylactic acid and photoinitiator 2959 are dissolved in a dioxane solution in which silanized nano-hydroxyapatite powder is uniformly dispersed, and then freeze-dried to obtain a porous polylactic acid / hydroxyapatite composite scaffold material.

[0013] Furthermore, the volume fraction of the silane coupling agent KH-570 is 0.5% to 1.5%; the volume fraction of ethanol in the ethanol-water mixed solution is 80% to 90%; the concentration of the polylactic acid is 5 to 10 mg / ml; the concentration of the photoinitiator 2959 is 0.5 to 1%; and the concentration of the silanized nano-hydroxyapatite powder is 1 to 5 mg / ml.

[0014] Furthermore, the specific operations of step S2 are:

[0015] 4-Dimethylaminopyridine and tetrabutylammonium bromide were added to a hyaluronic acid solution and stirred. Under nitrogen protection, glycidyl methacrylate was added to react. After the reaction, sodium chloride and methanol solution were added for salting out. The white precipitate was centrifuged using a high-speed centrifuge, dissolved in deionized water for dialysis, and then freeze-dried to obtain methacrylated hyaluronic acid.

[0016] Methacrylated hyaluronic acid and photoinitiator 2959 were dissolved in an aqueous solution of polyethylene glycol diacrylate. The polyethylene glycol diacrylate precursor solution containing methacrylated hyaluronic acid was grafted onto a porous polylactic acid / hydroxyapatite composite scaffold material through an ultraviolet light grafting method to obtain a double-layer structured composite hydrogel scaffold material.

[0017] Furthermore, the concentration of the 4-dimethylaminopyridine is 0.005-0.01 mol / L; the concentration of the tetrabutylammonium bromide is 0.002-0.005 mol / L; the volume fraction of the glycidyl methacrylate is 5-10%; the concentration of the sodium chloride is 3-5%; the volume fraction of the methanol solution is 200-500%; the concentration of the methacrylated hyaluronic acid is 0-1%; the concentration of the photoinitiator 2959 is 0.5-1%; the concentration of the polyethylene glycol diacrylate is 7.5-15%; and the UV grafting time is 5-20 min.

[0018] Furthermore, the specific operations of step S3 are:

[0019] 2-Methacryloyloxyethyl phosphorylcholine and photoinitiator 2959 were dissolved in an aqueous solution of polyethylene glycol diacrylate. The polyethylene glycol diacrylate precursor solution containing 2-methacryloyloxyethyl phosphorylcholine was grafted onto a double-layer composite hydrogel scaffold material through an ultraviolet light grafting method to obtain a three-layer composite hydrogel scaffold material.

[0020] Furthermore, the concentration of the 2-methacryloyloxyethyl phosphorylcholine is 0-2%; the concentration of the photoinitiator 2959 is 0.5-1%; the concentration of the polyethylene glycol diacrylate is 7.5-15%; and the ultraviolet light grafting time is 5-20 minutes.

[0021] A three-layer composite hydrogel scaffold material is prepared by a preparation method of a three-layer composite hydrogel scaffold material.

[0022] Application of a three-layer composite hydrogel scaffold material in the preparation of articular cartilage material and / or articular cartilage repair material.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention uses biomimetic construction based on the structural characteristics of cartilage tissue. Cartilage tissue is roughly divided into a calcification zone, a growth zone, and a lubrication zone. The present invention simulates these three zones of cartilage tissue by constructing a three-layer structure. From bottom to top, each layer corresponds to a corresponding zone in turn.

[0025] (2) The silane coupling agent KH570 used in the present invention can improve the hydrophilicity of nano-hydroxyapatite, facilitating better bonding with hydrophobic polylactic acid. The porous polylactic acid / hydroxyapatite composite scaffold material prepared by freeze-drying technology has uniform and regular pore size and high mechanical strength.

[0026] (3) The polyethylene glycol diacrylate used in the present invention is a photosensitive material having the advantages of being non-toxic, non-sensitizing, biocompatible and biodegradable.

[0027] (4) The hyaluronic acid and phosphatidylcholine used in the present invention are both components of articular cartilage, which can better simulate the articular cartilage environment and restore the function of articular cartilage.

[0028] (5) The silanized nano-hydroxyapatite powder, methacrylated hyaluronic acid and 2-methacryloyloxyethyl phosphorylcholine prepared in the present invention are all polymer materials with double bonds, which can be cross-linked with polyethylene glycol diacrylate under the action of ultraviolet light grafting, so that the layers can be tightly bonded.

[0029] (6) The method adopted in the present invention has a simple process, low requirements on instruments, low cost and is easy to implement. It is a relatively excellent method for preparing a three-layer structure composite hydrogel scaffold material.

[0030] (7) The three-layer composite hydrogel scaffold material obtained by the present invention has excellent performance and reasonable structure, and can meet the requirements of most clinical applications such as articular cartilage implantation and articular cartilage repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Infrared spectra of nHA and KH570-nHA.

[0032] Figure 2 X-ray diffraction patterns of nHA and KH570-nHA.

[0033] Figure 3 Cross-sectional scanning electron micrographs (SEM) of porous PLA / HA composite scaffolds with varying KH570-nHA content and their porosity. (a) A porous PLA / HA composite scaffold with 10% KH570-nHA content; (b) A porous PLA / HA composite scaffold with 20% KH570-nHA content; (c) A porous PLA / HA composite scaffold with 30% KH570-nHA content; (d) A porous PLA / HA composite scaffold with 40% KH570-nHA content; (f) A porous PLA / HA composite scaffold with KH570-nHA content; and (e) The porosity of porous PLA / HA composite scaffolds with varying KH570-nHA content.

[0034] Figure 4 Figure 1 shows the compressive strength of porous polylactic acid / hydroxyapatite composite scaffolds with varying KH570-nHA contents. (a) Compressive strength curves of porous polylactic acid / hydroxyapatite composite scaffolds before and after nHA silanization; (b) Compressive strength curves of porous polylactic acid / hydroxyapatite composite scaffolds with varying KH570-nHA contents.

[0035] Figure 5 The infrared spectra and nuclear magnetic resonance 1H spectra of HA and GMAHA are shown in Figure 1. (a) is the infrared spectra of HA and GMAHA; (b) is the nuclear magnetic resonance 1H spectra of HA and GMAHA.

[0036] Figure 6 Schematic diagrams of cross-sectional scanning electron micrographs (SEM) of polyethylene glycol diacrylate composite hydrogel scaffolds containing different GMAHA contents. In the figure: (a) polyethylene glycol diacrylate composite hydrogel scaffold with 0% GMAHA content; (b) polyethylene glycol diacrylate composite hydrogel scaffold with 0.25% GMAHA content; (c) polyethylene glycol diacrylate composite hydrogel scaffold with 0.5% GMAHA content; and (d) polyethylene glycol diacrylate composite hydrogel scaffold with 0.75% GMAHA content.

[0037] Figure 7 Figure 1 shows the mechanical strength of polyethylene glycol diacrylate composite hydrogel scaffolds with different GMAHA contents. (a) Rheological characterization of polyethylene glycol diacrylate composite hydrogel scaffolds with different GMAHA contents; (b) Compressive strength curves of polyethylene glycol diacrylate composite hydrogel scaffolds with different GMAHA contents.

[0038] Figure 8 Schematic diagrams of cross-sectional scanning electron micrographs (SEM) of polyethylene glycol diacrylate composite hydrogel scaffolds with different MPC contents. (a) 0% MPC content polyethylene glycol diacrylate composite hydrogel scaffold; (b) 1% MPC content polyethylene glycol diacrylate composite hydrogel scaffold; (c) 1.5% MPC content polyethylene glycol diacrylate composite hydrogel scaffold; (d) 2% MPC content polyethylene glycol diacrylate composite hydrogel scaffold.

[0039] Figure 9 Figures showing the friction and wear test results of polyethylene glycol diacrylate composite hydrogel scaffolds with different MPC contents. (a) The friction and wear curves of polyethylene glycol diacrylate composite hydrogel scaffolds with different MPC contents; (b) The friction and wear values ​​of polyethylene glycol diacrylate composite hydrogel scaffolds with different MPC contents.

[0040] Figure 10 A physical image and a cross-sectional scanning electron microscopy (SEM) diagram of a three-layer composite hydrogel scaffold. (a) A physical image of a three-layer composite hydrogel scaffold; (b) A cross-sectional scanning electron microscopy (SEM) diagram of a three-layer composite hydrogel scaffold.

[0041] Figure 11 Fluorescence microscopic images of rBMSCs cell activity in the S, C, and L layers of the materials in Example 1. The S layer is a porous polylactic acid / hydroxyapatite composite scaffold; the C layer is a polyethylene glycol diacrylate composite hydrogel containing methacrylated hyaluronic acid; and the L layer is a polyethylene glycol diacrylate composite hydrogel containing 2-methacryloyloxyethyl phosphorylcholine.

[0042] Figure 12 The graph shows the results of an rBMSCs cell proliferation experiment using the S-layer, C-layer, L-layer, SC-layer, and SCL-layer materials in Example 1. The S-layer is a porous polylactic acid / hydroxyapatite composite scaffold material; the C-layer is a polyethylene glycol diacrylate composite hydrogel material containing methacrylated hyaluronic acid; the L-layer is a polyethylene glycol diacrylate composite hydrogel material containing 2-methacryloyloxyethyl phosphorylcholine; the SC-layer is a double-layer composite hydrogel scaffold material; and the SCL-layer is a triple-layer composite hydrogel scaffold material.

[0043] Figure 13 This is a laser confocal micrograph of rBMSCs cells in the SCL layer material in Example 1.

[0044] Figure 14 The following is an ALP-stained photograph of rBMSCs cells that have passed through the S-layer, C-layer, L-layer, SC-layer, and SCL-layer materials in Example 1. The S-layer is a porous polylactic acid / hydroxyapatite composite scaffold material; the C-layer is a polyethylene glycol diacrylate composite hydrogel material containing methacrylated hyaluronic acid; the L-layer is a polyethylene glycol diacrylate composite hydrogel material containing 2-methacryloyloxyethyl phosphorylcholine; the SC-layer is a double-layer composite hydrogel scaffold material; and the SCL-layer is a triple-layer composite hydrogel scaffold material. Implementation Method

[0045] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0046] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0047] An embodiment of the present invention provides a method for preparing a three-layer composite hydrogel scaffold material, comprising the following steps:

[0048] Step S1, uniformly dispersing silanized nano-hydroxyapatite powder in a dioxane solution of polylactic acid, and freeze-drying the mixture to obtain a porous polylactic acid / hydroxyapatite composite scaffold material;

[0049] Step S2: grafting a polyethylene glycol diacrylate precursor solution containing methacrylated hyaluronic acid onto a porous polylactic acid / hydroxyapatite composite scaffold material by an ultraviolet grafting method to obtain a double-layer composite hydrogel scaffold material;

[0050] Step S3: Grafting a precursor solution of polyethylene glycol diacrylate containing 2-methacryloyloxyethyl phosphorylcholine onto the double-layer composite hydrogel scaffold material by an ultraviolet grafting method to obtain a three-layer composite hydrogel scaffold material.

[0051] In an embodiment of the present invention, preferably, polylactic acid is mainly formed by dehydration condensation of lactic acid, lactic acid can be obtained by fermentation of natural products, and polylactic acid prepared from lactic acid can be degraded into small molecule products for natural circulation. Therefore, polylactic acid is an ideal green polymer material that meets the development concept of green environmental protection in the world today. Polylactic acid has excellent mechanical properties, physical properties, degradability, biocompatibility, bioresorbability and ductility. Hydroxyapatite is the main inorganic component of vertebrate bones and teeth, and the hydroxyapatite content in bones is 69wt%, which has excellent biocompatibility and biological activity. By compounding polylactic acid and hydroxyapatite, the toughness and rigidity of the composite material can be improved, the composite material bone bioactivity can be given, and the acidic products produced by the degradation of polylactic acid can be buffered, the degradation rate of polylactic acid can be slowed down, and the biocompatibility of the composite material with bone tissue can be enhanced.

[0052] Hyaluronic acid, an acidic mucopolysaccharide, is a major component of connective tissues such as the intercellular matrix, vitreous humor, and synovial fluid. It performs crucial physiological functions in the body, including water retention, maintaining the extracellular space, regulating osmotic pressure, lubricating, and promoting cell repair. Hyaluronic acid molecules contain numerous carboxyl and hydroxyl groups, forming intra- and intermolecular hydrogen bonds in aqueous solution. This gives it a potent water-retaining capacity, capable of binding more than 400 times its own volume of water. At high concentrations, its intermolecular interactions form a complex tertiary network structure, giving its aqueous solution remarkable viscoelasticity. As a major component of the intercellular matrix, hyaluronic acid directly regulates the exchange of electrolytes inside and outside cells, acting as a filter for physical and molecular information. Hyaluronic acid, with its unique physicochemical properties and physiological functions, has found widespread medical application.

[0053] 2-Methacryloyloxyethylphosphocholine is an amphiphilic molecule composed of a hydrophilic head and a hydrophobic tail. It is a type of phospholipid with a choline group inserted into the head. The phosphatidylcholine lipids in human articular cartilage have a zwitterionic structure, in which the positively charged (N+(CH3)3) group and the negatively charged (PO4-) group strongly adsorb water molecules to form a stable hydrated lubricating layer. This strongly bound water layer consistently separates the two friction surfaces, achieving an ultra-low coefficient of friction between the articular cartilage and providing lubrication.

[0054] As a preferred embodiment of the present invention, the specific operations of step S1 are:

[0055] The nano-hydroxyapatite powder is uniformly dispersed in an ethanol-water mixed solution containing a silane coupling agent KH-570, heated in a water bath, and then vacuum dried to obtain silanized nano-hydroxyapatite powder;

[0056] Polylactic acid and photoinitiator 2959 are dissolved in a dioxane solution in which silanized nano-hydroxyapatite powder is uniformly dispersed, and then freeze-dried to obtain a porous polylactic acid / hydroxyapatite composite scaffold material.

[0057] In an embodiment of the present invention, preferably, the specific steps include: uniformly dispersing the nano-hydroxyapatite powder in an ethanol-water mixed solution containing a silane coupling agent KH-570 that has been hydrolyzed at room temperature for 30 minutes, heating in a 65°C water bath for 2 hours and then heating to 80°C to evaporate the solvent, washing with ethanol and water five times in sequence, and vacuum drying in an 80°C oven to obtain silanized nano-hydroxyapatite powder.

[0058] Polylactic acid and photoinitiator 2959 were dissolved in a dioxane solution in which silanized nano-hydroxyapatite powder was evenly dispersed, and then freeze-dried to obtain a porous polylactic acid / hydroxyapatite composite scaffold material. The material was then washed three times with deionized water and anhydrous ethanol respectively.

[0059] As a preferred embodiment of the present invention, the volume fraction of the silane coupling agent KH-570 is 0.5% to 1.5%; the volume fraction of ethanol in the ethanol-water mixed solution is 80% to 90%; the concentration of the polylactic acid is 5 to 10 mg / ml; the concentration of the photoinitiator 2959 is 0.5 to 1%; and the concentration of the silanized nano-hydroxyapatite powder is 1 to 5 mg / ml.

[0060] As a preferred embodiment of the present invention, the specific operations of step S2 are:

[0061] 4-Dimethylaminopyridine and tetrabutylammonium bromide were added to a hyaluronic acid solution and stirred. Under nitrogen protection, glycidyl methacrylate was added to react. After the reaction, sodium chloride and methanol solution were added for salting out. The white precipitate was centrifuged using a high-speed centrifuge, dissolved in deionized water for dialysis, and then freeze-dried to obtain methacrylated hyaluronic acid.

[0062] Methacrylated hyaluronic acid and photoinitiator 2959 were dissolved in an aqueous solution of polyethylene glycol diacrylate. The polyethylene glycol diacrylate precursor solution containing methacrylated hyaluronic acid was grafted onto a porous polylactic acid / hydroxyapatite composite scaffold material through an ultraviolet light grafting method to obtain a double-layer structured composite hydrogel scaffold material.

[0063] In an embodiment of the present invention, preferably, the specific steps include: dissolving hyaluronic acid in deionized water, then adding 4-dimethylaminopyridine and tetrabutylammonium bromide to the hyaluronic acid solution and stirring, adding glycidyl methacrylate to react under nitrogen protection, adding sodium chloride and methanol solution for salting out after the reaction, separating the white precipitate using a high-speed centrifuge, and repeating the above operation three times, dissolving the white precipitate in deionized water and dialyzing it for one week, and then freeze-drying it to obtain methacrylated hyaluronic acid; dissolving methacrylated hyaluronic acid and photoinitiator 2959 in an aqueous solution of polyethylene glycol diacrylate, and grafting the polyethylene glycol diacrylate precursor solution containing methacrylated hyaluronic acid onto a porous polylactic acid / hydroxyapatite composite scaffold material through an ultraviolet grafting method to obtain a double-layer structure composite hydrogel scaffold material, and washing the surface with deionized water to remove uncross-linked residues.

[0064] As a preferred embodiment of the present invention, the concentration of the 4-dimethylaminopyridine is 0.005-0.01 mol / L; the concentration of the tetrabutylammonium bromide is 0.002-0.005 mol / L; the volume fraction of the glycidyl methacrylate is 5-10%; the concentration of the sodium chloride is 3-5%; the volume fraction of the methanol solution is 200-500%; the concentration of the methacrylated hyaluronic acid is 0-1%; the concentration of the photoinitiator 2959 is 0.5-1%; the concentration of the polyethylene glycol diacrylate is 7.5-15%; and the UV grafting time is 5-20 min.

[0065] As a preferred embodiment of the present invention, the specific operations of step S3 are:

[0066] 2-Methacryloyloxyethyl phosphorylcholine and photoinitiator 2959 were dissolved in an aqueous solution of polyethylene glycol diacrylate. The polyethylene glycol diacrylate precursor solution containing 2-methacryloyloxyethyl phosphorylcholine was grafted onto a double-layer composite hydrogel scaffold material through an ultraviolet light grafting method to obtain a three-layer composite hydrogel scaffold material.

[0067] In an embodiment of the present invention, preferably, the specific steps include: dissolving 2-methacryloyloxyethyl phosphorylcholine and photoinitiator 2959 in an aqueous solution of polyethylene glycol diacrylate, grafting the polyethylene glycol diacrylate precursor solution containing 2-methacryloyloxyethyl phosphorylcholine onto a double-layer composite hydrogel scaffold material by an ultraviolet grafting method to obtain a three-layer composite hydrogel scaffold material, and washing the surface with deionized water to remove uncrosslinked residues.

[0068] As a preferred embodiment of the present invention, the concentration of the 2-methacryloyloxyethyl phosphorylcholine is 0-2%; the concentration of the photoinitiator 2959 is 0.5-1%; the concentration of the polyethylene glycol diacrylate is 7.5-15%; and the UV grafting time is 5-20 min.

[0069] An embodiment of the present invention provides a three-layer structure composite hydrogel scaffold material prepared by a preparation method of a three-layer structure composite hydrogel scaffold material.

[0070] An embodiment of the present invention provides a use of a three-layer composite hydrogel scaffold material in the preparation of articular cartilage materials and / or articular cartilage repair materials. Example

[0071] This embodiment provides a method for preparing a three-layer composite hydrogel scaffold material, comprising the following steps:

[0072] Step 1: Dissolve polylactic acid and 0.5% photoinitiator 2959 in 5 ml of dioxane solution in which silanized nano-hydroxyapatite powder is evenly dispersed (the mass ratios of polylactic acid to silanized nano-hydroxyapatite powder are 9:1, 4:1, 7:3, 3:2, and 1:1, respectively). Then, freeze-dry the solution to obtain a porous polylactic acid / hydroxyapatite composite scaffold material (named S layer), which is then washed three times with deionized water and anhydrous ethanol.

[0073] The preparation method of the silanized nano-hydroxyapatite powder comprises the following steps:

[0074] Nano-hydroxyapatite powder (named nHA) was uniformly dispersed in an ethanol-water mixed solution (ethanol: water = 9:1) containing 1% silane coupling agent KH-570 that had been hydrolyzed at room temperature for 30 minutes. The mixture was heated in a 65°C water bath for 2 hours and then heated to 80°C to evaporate the solvent. The mixture was washed with ethanol and water five times in sequence and dried in a vacuum oven at 80°C to obtain silanized nano-hydroxyapatite powder (named KH570-nHA).

[0075] Step 2: Dissolve methacrylated hyaluronic acid (the concentrations of methacrylated hyaluronic acid are 0%, 0.25%, 0.5%, and 0.75%, respectively) and a photoinitiator 2959 with a concentration of 0.5% in a 12.5% ​​aqueous solution of polyethylene glycol diacrylate. Graft the polyethylene glycol diacrylate precursor solution containing methacrylated hyaluronic acid (named C layer) onto the porous polylactic acid / hydroxyapatite composite scaffold material through UV grafting. The UV grafting time is 10 minutes to obtain a double-layer composite hydrogel scaffold material (named SC layer), and wash the surface with deionized water to remove uncross-linked residues.

[0076] The method for preparing the methacrylated hyaluronic acid comprises the following steps:

[0077] (1) Dissolve 0.5 g of hyaluronic acid (named HA) in 50 ml of deionized water, then add 0.008 mol / L of 4-dimethylaminopyridine and 0.003 mol / L of tetrabutylammonium bromide into the hyaluronic acid solution and stir;

[0078] (2) Under nitrogen protection, add 4 ml of glycidyl methacrylate and react for 48 hours. After the reaction, add sodium chloride to a concentration of 5% and 150 ml of methanol solution for salting out. Use a high-speed centrifuge to separate the white precipitate. Repeat the above operation three times.

[0079] (3) The white precipitate was dissolved in 300 ml of deionized water and dialyzed for one week, followed by freeze-drying to obtain methacrylated hyaluronic acid (named GMAHA);

[0080] Step 3. Dissolve 2-methacryloyloxyethyl phosphorylcholine (named MPC, the concentrations of 2-methacryloyloxyethyl phosphorylcholine are 0%, 1%, 1.5%, and 2%, respectively) and a photoinitiator 2959 with a concentration of 0.5% in an aqueous solution of polyethylene glycol diacrylate with a concentration of 12.5%. Graft the polyethylene glycol diacrylate precursor solution containing 2-methacryloyloxyethyl phosphorylcholine (named L layer) onto a double-layer composite hydrogel scaffold material by ultraviolet light grafting. The ultraviolet light grafting time is 5 minutes to obtain a three-layer composite hydrogel scaffold material (named SCL layer), and wash the surface with deionized water to remove uncrosslinked residues.

[0081] The porous polylactic acid / hydroxyapatite composite scaffold material obtained above was subjected to performance testing.

[0082] Depend on Figure 1 It can be seen that KH570-nHA is at 2956cm -1 、2930cm -1 The CH stretching vibration absorption peaks of CH3 and CH2 appeared at 1719 cm -1 The absorption peak of C=O in C=C-COOR moves to the lower wave number direction due to conjugation with C=C; at 815cm -1 The Si-O-Si bending vibration absorption peak appeared at , indicating the successful preparation of silanized nano-hydroxyapatite.

[0083] Depend on Figure 2 It can be seen from the comparison that obvious Bragg diffraction peaks appear at 2θ values ​​of 25.9°, 31.77°, 32.19°, 32.9°, 34.04°, 46.7°, and 49.5°, which correspond to the characteristic diffraction peaks of n-HA appearing on the (002), (211), (112), (300), (202), (222), and (213) crystal planes, respectively. Figure 2 It can be observed that the positions and sharpness of the characteristic peaks of all energy spectra are similar. The test results show that the crystallinity of the modified nHA has not changed, proving that the silane coupling agent KH570 did not chemically react with nHA during the modification process, but was physically adsorbed on the surface of nHA.

[0084] Depend on Figure 3 It can be seen that the cross-section of the polylactic acid / hydroxyapatite composite scaffold material prepared in this example has a distinct porous structure. The pore size of the composite scaffold material is most uniform when the KH570-nHA content is 30%, but the overall porosity is roughly the same.

[0085] Depend on Figure 4It can be seen that nHA has little effect on the compressive strength of the porous polylactic acid / hydroxyapatite composite scaffold material before and after silanization. However, with the increase of KH570-nHA content, under the condition of 70% strain, the compressive strength of the porous polylactic acid / hydroxyapatite composite scaffold material first increases and then decreases, which may be related to the aggregation of KH570-nHA.

[0086] The methacrylated hyaluronic acid obtained above was subjected to performance testing. Figure 5 (a) It can be seen that the vibration absorption peak of -C=CC=O appears at 1644cm-1. Figure 5 (b) 1H-NMR (D2O, 343K): 5.6, 6.1, -CH=CH2, indicating that HA with side chains containing double bonds and free cross-linking was successfully obtained.

[0087] The performance of the polyethylene glycol diacrylate precursor solution containing methacrylated hyaluronic acid obtained above was tested. Figure 6 It can be seen that the polyethylene glycol diacrylate composite hydrogel scaffold material with different GMAHA contents has a porous structure. As the GMAHA content increases, the composite hydrogel scaffold material is more completely cross-linked and smaller pores begin to appear.

[0088] Depend on Figure 7 (a) It can be seen that after adding GMAHA, the storage modulus of the composite hydrogel scaffold material is significantly improved, indicating that the polyethylene glycol diacrylate composite hydrogel scaffold material with different GMAHA contents has good viscoelasticity; Figure 7 (b) It can be seen that after adding GMAHA, the compressive strength of the composite hydrogel scaffold material is significantly improved, it can have a larger deformation, and has good buffering performance.

[0089] The performance of the polyethylene glycol diacrylate precursor solution containing 2-methacryloyloxyethyl phosphorylcholine obtained above was tested. Figure 8 It can be seen that the polyethylene glycol diacrylate composite hydrogel scaffold material with different MPC contents has a porous structure. As the MPC content increases, the composite hydrogel scaffold material is more completely cross-linked and smaller pores begin to appear.

[0090] Depend on Figure 9 It can be seen that after adding MPC, the friction coefficient of the composite hydrogel scaffold material is significantly reduced, indicating that the polyethylene glycol diacrylate composite hydrogel scaffold material with different MPC contents has good surface lubrication properties. With the increase of MPC content, the friction coefficient gradually decreases and tends to be stable.

[0091] The three-layer composite hydrogel scaffold material obtained above was subjected to performance testing. Figure 10It can be seen that the three-layer composite hydrogel scaffold material layers are tightly bonded, not easy to scatter, and the overall pore size is uniform. Example

[0092] This embodiment provides a method for preparing a three-layer composite hydrogel scaffold material, comprising the following steps:

[0093] Step 1: dissolving polylactic acid and 1% photoinitiator 2959 in 5 ml of dioxane solution in which silanized nano-hydroxyapatite powder is uniformly dispersed. The remaining steps are the same as step 1 in Example 1.

[0094] Step 2: dissolving methacrylated hyaluronic acid (the concentrations of methacrylated hyaluronic acid are 0%, 0.25%, 0.5%, and 0.75%, respectively) and 1% photoinitiator 2959 in a 15% aqueous solution of polyethylene glycol diacrylate. The remaining steps are the same as step 2 in Example 1.

[0095] Step 3: Dissolve 2-methacryloyloxyethyl phosphorylcholine (MPC, with concentrations of 0%, 1%, 1.5%, and 2%, respectively) and 1% photoinitiator 2959 in a 15% aqueous solution of polyethylene glycol diacrylate. The remaining steps are the same as step 3 in Example 1. Example

[0096] This embodiment provides a method for preparing a three-layer composite hydrogel scaffold material, comprising the following steps:

[0097] Step 1: This step is the same as step 1 in Example 1;

[0098] Step 2: Dissolve methacrylated hyaluronic acid (the concentrations of methacrylated hyaluronic acid are 0%, 0.25%, 0.5%, and 0.75%, respectively) and 0.5% of photoinitiator 2959 in a 10% aqueous solution of polyethylene glycol diacrylate; the UV grafting time is 15 minutes; the remaining steps are the same as step 2 in Example 1;

[0099] Step 3: Dissolve 2-methacryloyloxyethyl phosphorylcholine (MPC, the concentrations of 2-methacryloyloxyethyl phosphorylcholine are 0%, 1%, 1.5%, and 2%, respectively) and 0.5% photoinitiator 2959 in a 10% aqueous solution of polyethylene glycol diacrylate; the UV grafting time is 10 minutes; and the remaining steps are the same as step 3 in Example 1. Example

[0100] The cytotoxicity of the three-layer composite hydrogel scaffold material in Example 1 was evaluated by in vitro culture of rat bone marrow mesenchymal stem cells (rBMSCs) using the Calcein-AM / PI Double Stain Kit (Yeasen, Shanghai, China). The specific procedure is as follows:

[0101] (1) Place the S layer, C layer and L layer materials in Example 1 in a 48-well culture plate with a drop density of 5×10 4 cell / mL cell suspension, add low-glucose complete medium for incubation, and place the cell culture plate in a cell culture incubator with 5% CO2 saturated humidity at 37°C;

[0102] (2) After culturing for 1 day and 4 days, the living cells were fluorescently stained and washed according to the kit instructions;

[0103] (3) After staining, observe and take pictures using a fluorescence microscope. The results are as follows: Figure 11 shown.

[0104] Depend on Figure 11 This indicates that the S layer, C layer and L layer materials in Example 1 are non-toxic to rBMSCs cells. Example

[0105] In vitro culture experiments using rat bone marrow mesenchymal stem cells (rBMSCs) were conducted to evaluate cell proliferation across the S, C, L, SC, and SCL layers described in Example 1. Cell proliferation on the surface of the materials was assessed using a Cell Counting Kit (CKK-8, Beyotime, Shanghai, China). The specific procedures are as follows:

[0106] (1) Place the S layer, C layer, L layer, SC layer and SCL layer materials in Example 1 in a 48-well culture plate with a drop density of 1×10 4 cell / mL cell suspension, add low-glucose complete medium, and culture the cell culture plate in a cell culture incubator with 5% CO2 saturated humidity at 37°C;

[0107] (2) After culturing the cells for 1, 4, and 7 days, the culture medium was aspirated and 500 μL of new culture medium containing 10% CKK-8 solution was added. The culture plate was placed in an incubator and cultured for 2 hours. 100 μL of culture medium was taken from each well and placed in a 96-well plate.

[0108] (3) Using an enzyme-labeled instrument (iMark, Bio-Rad, USA), measure the absorbance of each well at a wavelength of 450 nm. The results are as follows: Figure 12 shown.

[0109] Depend on Figure 12 It can be seen that the absorbance of the C layer material is the lowest, and the SCL layer material is significantly better than the SC layer material and the L layer material, but compared with the control group, the cell activity is higher than 80%, indicating that each layer of the three-layer composite hydrogel scaffold material has good biological proliferation characteristics. Example

[0110] In vitro culture experiments using rat bone marrow mesenchymal stem cells (rBMSCs) were conducted to evaluate cell adhesion and growth after culturing the SCL layer material described in Example 1. The cytoskeleton and nuclei were stained with rhodamine-labeled phalloidin and DAPI, respectively, and observed using a laser confocal microscope. The specific procedures are as follows:

[0111] (1) After rBMSCs were cultured on the SCL layer material described in Example 1 for 3 days, the samples were removed and gently rinsed with PBS three times, each time for 10 minutes;

[0112] (2) The samples were fixed with 4% paraformaldehyde at 4°C for 30 min and gently rinsed with PBS three times, each time for 10 min.

[0113] (3) Add 0.5% Triton-100, react for 20 minutes, and gently rinse with PBS three times, each time for 10 minutes;

[0114] (4) Rhodamine-labeled phalloidin (100 nM) and DAPI (5 μg / mL) were used to stain the cytoskeleton and cell nucleus, respectively;

[0115] (5) After staining, observe and take pictures using a laser confocal microscope, and process the pictures using imageJ software. The results are as follows: Figure 13 shown.

[0116] Depend on Figure 13 It can be seen that the rBMSCs pseudopodia on the SCL layer material in Example 1 are relatively long, indicating that this is beneficial for cell adhesion and growth. Example

[0117] Rat bone marrow mesenchymal stem cells (rBMSCs) were cultured in vitro to evaluate the differentiation of cells passing through the S-layer, C-layer, L-layer, SC-layer, and SCL-layer materials described in Example 1. BCIP / NBT was used to stain the S-layer, C-layer, L-layer, SC-layer, and SCL-layer materials described in Example 1. The specific procedure is as follows:

[0118] (1) The S layer, C layer, L layer, SC layer and SCL layer materials in Example 1 were placed in a 48-well culture plate, with a drop density of 5×10 4cell / mL cell suspension, add low-glucose complete medium;

[0119] (2) After rBMSCs were cultured on the S layer, C layer, L layer, SC layer, and SCL layer materials in Example 1 for 1 day, the low-glucose complete medium was replaced with osteogenic induction medium, and the culture medium was replaced every 2 days;

[0120] (3) After culturing rBMSCs for 7 and 14 days, the culture medium was discarded and the cells were rinsed with PBS three times;

[0121] (4) rBMSCs were fixed with 4% paraformaldehyde at 4°C for 30 min and then rinsed with PBS three times;

[0122] (5) Prepare BCIP / NBT staining working solution, add 500 μL of staining solution to each well, and incubate at room temperature overnight in the dark;

[0123] (6) After staining, discard the working solution, rinse with PBS three times, dry and take pictures. The results are as follows Figure 14 shown.

[0124] Depend on Figure 14 It can be seen that the surface of the L layer material has less purple, indicating that the rBMSCs differentiation is poor. The surface of the S layer material and the C layer material has more purple, indicating that it is more conducive to cell differentiation.

[0125] The above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention. These should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A method for preparing a three-layer composite hydrogel scaffold material, characterized in that: The following steps are involved: Step S1, uniformly dispersing silanized nano-hydroxyapatite powder in a dioxane solution of polylactic acid, and freeze-drying the mixture to obtain a porous polylactic acid / hydroxyapatite composite scaffold material; Step S2: grafting a polyethylene glycol diacrylate precursor solution containing methacrylated hyaluronic acid onto a porous polylactic acid / hydroxyapatite composite scaffold material by an ultraviolet grafting method to obtain a double-layer composite hydrogel scaffold material; Step S3: Grafting a precursor solution of polyethylene glycol diacrylate containing 2-methacryloyloxyethyl phosphorylcholine onto the double-layer composite hydrogel scaffold material by an ultraviolet grafting method to obtain a three-layer composite hydrogel scaffold material.

2. The method for preparing a three-layer composite hydrogel scaffold material according to claim 1, characterized in that: The specific operations of step S1 are: The nano-hydroxyapatite powder is uniformly dispersed in an ethanol-water mixed solution containing a silane coupling agent KH-570, heated in a water bath, and then vacuum dried to obtain silanized nano-hydroxyapatite powder; Polylactic acid and photoinitiator 2959 are dissolved in a dioxane solution in which silanized nano-hydroxyapatite powder is uniformly dispersed, and then freeze-dried to obtain a porous polylactic acid / hydroxyapatite composite scaffold material.

3. The method for preparing a three-layer composite hydrogel scaffold material according to claim 2, characterized in that: The volume fraction of the silane coupling agent KH-570 is 0.5% to 1.5%; the volume fraction of ethanol in the ethanol-water mixed solution is 80% to 90%; the concentration of the polylactic acid is 5 to 10 mg / ml; the concentration of the photoinitiator 2959 is 0.5 to 1%; and the concentration of the silanized nano-hydroxyapatite powder is 1 to 5 mg / ml.

4. The method for preparing a three-layer composite hydrogel scaffold material according to claim 1, characterized in that: The specific operations of step S2 are: 4-Dimethylaminopyridine and tetrabutylammonium bromide were added to a hyaluronic acid solution and stirred. Under nitrogen protection, glycidyl methacrylate was added to react. After the reaction, sodium chloride and methanol solution were added for salting out. The white precipitate was centrifuged using a high-speed centrifuge, dissolved in deionized water for dialysis, and then freeze-dried to obtain methacrylated hyaluronic acid. Methacrylated hyaluronic acid and photoinitiator 2959 were dissolved in an aqueous solution of polyethylene glycol diacrylate. The polyethylene glycol diacrylate precursor solution containing methacrylated hyaluronic acid was grafted onto a porous polylactic acid / hydroxyapatite composite scaffold material through an ultraviolet light grafting method to obtain a double-layer structured composite hydrogel scaffold material.

5. The method for preparing a three-layer composite hydrogel scaffold material according to claim 4, characterized in that: The concentration of the 4-dimethylaminopyridine is 0.005~0.01 mol / L; the concentration of the tetrabutylammonium bromide is 0.002~0.005 mol / L; the volume fraction of the glycidyl methacrylate is 5~10%; the concentration of the sodium chloride is 3~5%; the volume fraction of the methanol solution is 200~500%; the concentration of the methacrylated hyaluronic acid is 0~1%; the concentration of the photoinitiator 2959 is 0.5~1%; the concentration of the polyethylene glycol diacrylate is 7.5~15%; the UV grafting time is 5~20 min; wherein, the concentration of the methacrylated hyaluronic acid is not 0.

6. The method for preparing a three-layer composite hydrogel scaffold material according to claim 1, characterized in that: The specific operations of step S3 are: 2-Methacryloyloxyethyl phosphorylcholine and photoinitiator 2959 were dissolved in an aqueous solution of polyethylene glycol diacrylate. The polyethylene glycol diacrylate precursor solution containing 2-methacryloyloxyethyl phosphorylcholine was grafted onto a double-layer composite hydrogel scaffold material through an ultraviolet light grafting method to obtain a three-layer composite hydrogel scaffold material.

7. The method for preparing a three-layer composite hydrogel scaffold material according to claim 6, characterized in that: The concentration of the 2-methacryloyloxyethyl phosphorylcholine is 0-2%; the concentration of the photoinitiator 2959 is 0.5-1%; the concentration of the polyethylene glycol diacrylate is 7.5-15%; the ultraviolet light grafting time is 5-20 minutes; wherein the concentration of the 2-methacryloyloxyethyl phosphorylcholine is not 0.

8. A three-layer composite hydrogel scaffold material prepared according to the method for preparing a three-layer composite hydrogel scaffold material according to any one of claims 1 to 7.

9. Use of the three-layer composite hydrogel scaffold material according to claim 8 in the preparation of articular cartilage materials and / or articular cartilage repair materials.

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