Composite skull repair hydrogel material with patterned adhesion-promoting surface and preparation method thereof

Abstract

The present invention discloses a composite skull repair hydrogel material with a patterned adhesion-promoting surface and a preparation method thereof. The preparation method comprises the following: 1) using a precipitation method to prepare mesoporous manganese dioxide (MMD), then combining a cyclic ultrasonic loading method to load osteogenic drugs in the pores of the MMD, and using an in-situ free radical polymerization method to coat a sustained-release polydopamine (PDA) coating on its surface to obtain MMD-O-PDA; 2) MMD-O-PDA is evenly mixed with an amino-containing UV-curable polymer solution, placed on a custom PDMS mold with a special patterned surface, covered with a transparent quartz sheet, and UV-cured and demolded; 3) in situ grafting of adhesion proteins on the surface of the composite hydrogel material with a cross-linking agent, and the hydrogel material is obtained after ultra-frequency dialysis. The material has a highly efficient cell adhesion-promoting effect, can quickly enrich osteoblasts in the defect site, and simultaneously has anti-inflammatory and full-cycle osteogenic effects, thereby efficiently promoting the repair of skull defect sites.

Description

Technical Field

[0001] The present invention relates to the field of biomedical material preparation, and in particular to a composite skull repair hydrogel material with a patterned adhesion-promoting surface and a preparation method thereof. Background Art

[0002] Skull defects have always been one of the difficult problems faced in clinical practice, and the incidence rate has been increasing year by year. Skull defects are common after trauma or decompressive craniectomy. Skull defects of critical size cannot heal on their own and need to be repaired with bone repair materials. Common skull repair materials in clinical practice are titanium alloy mesh or PEEK materials. The biocompatibility of titanium mesh is relatively general, the healing time of the defect edge is long, and the mechanical properties are far from those of autologous bone; PEEK material has better biocompatibility and mechanical properties, but the price is too expensive and most patients can hardly accept it. For this reason, it is urgent to design a biomaterial that can efficiently promote the repair of skull defects.

[0003] With the rapid development of tissue engineering, a variety of bioactive hydrogel materials that promote skull defect repair have emerged. However, common hydrogel materials have the problem of poor local cell recruitment energy in the defect and low cell adhesion efficiency, and their ability to inhibit local foreign body inflammatory response is extremely limited, resulting in low repair efficiency and even directly leading to repair failure. In addition, osteogenesis is a long-cycle process, and most drug-loaded hydrogels only play a role in promoting osteogenesis in the early stage and have almost no effect on the later callus reconstruction. To this end, the present invention designs a composite skull repair hydrogel material with a patterned adhesion-promoting surface. By coating the patterned surface with adhesion proteins, local cell adhesion is greatly promoted to solve the above problems. Summary of the Invention

[0004] The purpose of the present invention is to address the deficiencies of the existing technology and provide a composite skull repair hydrogel material with a patterned adhesion-promoting surface and a preparation method thereof. The method uses raw materials such as a UV-curable polymer with an amino group, MMD, a osteogenic drug, and an adhesion protein, and successfully prepares a composite skull repair hydrogel material with a patterned adhesion-promoting surface through a combination of processes such as precipitation method, ultrasonic loading method, in situ self-polymerization method, and chemical grafting method. The material can efficiently promote local cell adhesion, rapidly enrich osteoblasts in the defect site, and has both anti-inflammatory and full-cycle osteogenic effects, and can efficiently promote the repair of skull defect sites.

[0005] The preparation method of the hydrogel material comprises the following steps:

[0006] 1) Preparation of MMD and MMD-O-PDA: MMD was prepared using a precipitation method using potassium permanganate and hydrogen peroxide. After cleaning and dialysis, the osteogenic drug was loaded into the MMD pores using a cyclic ultrasonic loading method. After loading, the MMD surface was coated with a sustained-release polydopamine coating using an in situ free radical polymerization method to obtain MMD-O-PDA.

[0007] 2) Preparation of a composite hydrogel material: A UV-curable polymer with amino groups was dissolved in a dark and heated environment. After the temperature dropped to room temperature, MMD-O-PDA, MMD, and the UV-curable polymer solution with amino groups were premixed. The premixed solution was placed on a PDMS mold with a patterned surface previously coated with a release agent. The mold was then covered with a transparent quartz slide coated with a release agent on the contact surface and UV-cured. The composite hydrogel material was obtained after repeated freezing and demolding.

[0008] 3) Grafting Adhesive Proteins onto the Composite Hydrogel Surface: The hydrogel material is immersed in a crosslinker, then quickly removed and immediately immersed in an adhesive protein solution. Ultrapure water is used to remove any residual adhesive protein from the surface, and the above process is repeated 3-5 times. After grafting the adhesive protein, dialysis is performed to remove any residual crosslinker and irradiation is performed to sterilize the surface. This yields the desired composite skull repair hydrogel material with a patterned, adhesion-promoting surface.

[0009] Furthermore, in step 1), the preparation process of MMD and MMD-O-PDA is as follows: prepare a 0.05-0.2 mol / L KMnO4 aqueous solution, then slowly add 30% hydrogen peroxide dropwise, and use 1 mol / L KOH or NaOH to maintain the solution pH at a weak alkaline level (about 8-10); stop adding hydrogen peroxide after no obvious bubbles are generated, continue stirring at room temperature for at least 30 minutes, and let it stand for 2-8 hours; filter the product, wash it with deionized water 3-5 times, and then redisperse it in deionized water, stir and wash it for 24-48 hours, stop stirring and allow it to settle naturally; repeat the above filtration-washing-sedimentation steps for 1-2 weeks to obtain MMD.

[0010] Furthermore, in step 1), the prepared MMD is re-ground and placed in a PBS solution containing an osteogenic drug. Ultrasonic loading is applied to allow the drug to enter the micropores, and the precipitate is obtained by centrifugation and then freeze-dried, wherein the concentration of MMD is 0.1-0.5wt%; the osteogenic drug can be one or more of BMP-2, BMP-7, SGF, IGF, etc., with a concentration of 2-10mg / ml; the ultrasonic loading power is 80-120w, the loading time is 30-60min, and the loading temperature is 0-4°C; the above ultrasonic loading-centrifugal freeze-drying process is repeated 4-10 times. After the above-mentioned drug-loaded MMD is washed with PBS 3-5 times, it is placed in a Tris-HCl solution (pH=8.5) containing dopamine (DA) and reacted on a shaker at room temperature for 4-8h, wherein the concentration of DA is 4-8mg / ml; after the reaction, it is washed with PBS 3-5 times;

[0011] Furthermore, in step 2), the amino-containing UV-curable polymer may be a combination of one or both of GelMA and SilMA, with a concentration of 5-25%. If it is a combination, the mass ratio range is 3:1-1:3; the amount of MMD-O-PDA added is 0.1-0.2wt%, and the amount of blank MMD (i.e., the MMD material) added is 0.1-0.2wt%; the polymer solution contains a photoinitiator, and the photoinitiator is one or more of LAP, Irgacure 2100, and Darocur 1173, with a concentration of 0.1-0.25wt%;

[0012] Furthermore, the pattern on the surface of the PDMS mold is an array structure of micrometer-scale grooves or columns, wherein the width of the grooves is 1-5 μm, the depth of the grooves is 0.5-3 μm, and the spacing between the grooves is 400 nm-4 μm; the diameter of the columns is 4-12 μm, the height is 10-50 μm, and the spacing between the columns is 10-20 μm; the above-mentioned UV-curable polymer with amino groups is added dropwise to the upper part of the PDMS mold pattern, and the polymer solution is uniformly infiltrated into the micro-pattern surface by combining the ultrasonic diffusion process, and then Use a flat, sterile quartz sheet to gently cover the top, ultrasonic power 60-100W, ultrasonic time 5-10min; the release agent is one or both of glycerol and ethylene glycol; UV wavelength is 405nm, power 20-50w, time 5-10min; after curing, cyclic freeze demoulding is performed, place the mold in a -80℃ refrigerator for quick freezing for 3-5min, then take it out and let it thaw naturally. Repeat the freeze-thaw process 2-3 times, then gently remove the gel from the mold and soak it in sterile PBS solution for use;

[0013] Furthermore, in step 3), the crosslinking agent may be glutaraldehyde or genipin, with a concentration of 0.05-0.25wt%, and the immersion time of the composite hydrogel in the crosslinking agent is 3-5s; the adhesion protein may be one or more of Fibronectin, Laminin, ColⅢ, etc., with a concentration of 0.1-0.5wt%, and the immersion time is 1-2h; the dialysis is ultrahigh frequency dialysis, that is, using PBS solution, changing the solution every 15-30min, and dialysis for 3-6h.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] 1. This invention uses biodegradable hydrogels, MMD, osteogenic drugs, adhesive proteins, and other raw materials to successfully prepare a composite skull repair hydrogel material with a patterned adhesion-promoting surface through a combination of processes including template processing, precipitation, cyclic ultrasonic loading, in situ self-polymerization, surface grafting, and ultra-frequency dialysis. The selection of these raw materials and the combined processes are unique to this invention.

[0016] 2. This invention focuses on a full-cycle closed-loop repair strategy for skull defect repair. Initially, osteoblasts are recruited to provide a cellular foundation for defect repair, and the anti-inflammatory effect of manganese ions provides a favorable osteogenic microenvironment. Later, sustained release of osteogenic drugs provides excellent full-cycle support for osteogenesis, while the sustained release of manganese ions provides a long-lasting anti-inflammatory effect. Furthermore, the excellent biodegradability provides a spatial foundation for the subsequent attachment of new bone tissue. This full-cycle repair strategy is highly innovative.

[0017] 3. Cells are the basis of bone repair. In order to solve the problems of low cell migration rate and low adhesion efficiency of conventional hydrogel materials, the present invention innovatively designs a dual structure of gel micro-patterned surface and adhesion protein grafting. By increasing the proliferation activity and directional migration ability of osteoblasts through the micro-patterned surface, the cell components can fill the defect faster. At the same time, the introduction of surface adhesion proteins further greatly increases the cell adhesion efficiency and its biocompatibility, forming a synergistic effect with the micro-patterned surface, which greatly promotes the defect repair effect; 4. Taking into account the infection risk faced in the early stage of implantation, the present invention adds blank MMD, so that it can release a sufficient concentration of manganese ions in the early stage; manganese ions have anti-inflammatory and anti-tissue edema effects, as well as a weak osteogenic effect, which can significantly improve the success rate of implantation; at the same time, taking into account the conventional drug-loaded hydrogel There are problems such as uncontrollable drug release, low drug loading efficiency, and short drug half-life in gel. MMD-O-PDA is used as a nano drug-loading filler: MMD-O-PDA has both a polymer outer layer and a porous structure, forming a dual sustained-release effect, so that the osteogenic factors contained in it are released slowly in the initial stage and the action time is longer, which can play a role in the entire cycle of defect repair, matching the physiological osteogenesis process; and while MMD itself can slowly release manganese ions, the physical coating of the outer layer of hydrogel matrix acts as a third sustained-release effect, so that it can continuously release manganese ions throughout the osteogenesis cycle, playing a long-lasting anti-inflammatory role.

[0018] 5. The present invention achieves process innovation through the rational combination of various process technologies: a cyclic impregnation process is used to graft adhesive proteins onto the surface of amino-containing hydrogels, overcoming the problem of low protein adhesion strength in traditional adsorption processes; MMD has a nanoporous structure and high surface tension, which makes it difficult to achieve sufficient and uniform drug loading by conventional drug loading methods. The present invention cleverly solves this problem by using ultrasonic loading; the PDA coating formed by in situ self-polymerization and the porous structure of MMD form a dual sustained-release effect; the patterned PDMS template is combined with UV curing, which is simple to operate and avoids expensive 3D printing methods; rapid dialysis (i.e., ultra-frequency dialysis) removes residual cross-linkers, which not only quickly removes the cross-linkers but also retains the drug loaded in the MMD to the greatest extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 TEM images of MMD;

[0020] Figure 2 Schematic diagrams of several specific design examples of patterned molds for composite skull repair hydrogel materials with patterned adhesion-promoting surfaces;

[0021] Figure 3 Schematic diagram of the preparation process of composite skull repair hydrogel material with patterned adhesion-promoting surface;

[0022] Figure 4 Manganese ion release curve of the composite skull repair hydrogel material with a patterned adhesion-promoting surface (corresponding to Example 3). DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific examples. The preparation method of the composite skull repair hydrogel material with a patterned adhesion-promoting surface of the present invention is as follows: Figure 3 As shown, the mold involved is patterned into an array structure of micron-scale grooves or pillars, such as Figure 2 Schematic diagram of mold patterns used in some specific embodiments of the present invention. In other embodiments of the present invention, other micron-scale groove or column array structure patterns may also be used, which will not be described in detail here.

[0024] Example 1

[0025] 1) Preparation of MMD and drug-loaded sustained-release MMD: Prepare a 0.1 mol / L KMnO4 aqueous solution, then slowly add 30% hydrogen peroxide, while using 1 mol / L KOH to maintain the solution pH at a weak alkaline level (approximately 8-10); stop adding hydrogen peroxide after no obvious bubbles are generated, continue stirring at room temperature for 30 minutes, and let it stand for 4 hours; filter the product, rinse with deionized water three times, and then redisperse it in deionized water, stir and rinse for 24 hours, stop stirring and allow it to settle naturally; repeat the above filtration-washing-sedimentation steps for one week to produce MMD. The MMD was re-ground and placed in a PBS solution containing 5 mg / ml BMP-2 (containing 0.5% MMD). Ultrasonic loading was performed for 60 minutes to allow the drug to enter the micropores. The ultrasonic process was repeated five times, and the precipitate was obtained by centrifugation and freeze-dried. The obtained drug-loaded MMD was washed three times with PBS and then placed in a Tris-HCl solution (pH = 8.5) containing 4 mg / ml DA and reacted on a shaker at room temperature for 8 h. After the reaction, it was washed three times with PBS to obtain the drug-loaded sustained-release MMD material, namely MMD-O-PDA.

[0026] 2) Preparation of composite hydrogel material: GelMA (GelMA concentration 10% wt) was dissolved in PBS containing 0.25% LAP under light-protected conditions and heated at 60°C to obtain a GelMA solution. After the temperature was lowered to room temperature, drug-loaded sustained-release MMD and blank MMD (both concentrations in the premix were 0.1 wt%) were premixed with the GelMA solution. The premix was placed on a conventional PDMS mold (not micropatterned) previously coated with a trace amount of glycerol. The mold was then covered with a transparent quartz slide coated with a trace amount of glycerol and UV-cured. After repeated freezing and demolding, the composite hydrogel material was obtained.

[0027] 3) Grafting of adhesive proteins onto the surface of the composite hydrogel: The hydrogel material was immersed in a 0.1 wt% genipin solution, then quickly removed and immediately immersed in a 0.1 wt% fibronectin solution. After 1 hour, the residual adhesive proteins on the surface were removed using ultrapure water, and the above process was repeated 5 times. The hydrogel material was then dialyzed with PBS for 6 hours, with the solution changed every 30 minutes. Finally, the hydrogel material was irradiated and sterilized, and then packaged to obtain a composite skull repair hydrogel material.

[0028] The composite hydrogel material had a 28-day in vitro degradation rate of 75%. After cells were inoculated on the surface, complete cell adhesion was visible 12 hours later. After 7 days of culture, live-dead cell fluorescence staining showed that the proportion of live cells was 92%. It was used to repair a rat skull defect model, and 4 weeks after surgery, the degree of defect repair was general, and HE staining showed that the local inflammatory reaction was mild.

[0029] Example 2

[0030] 1) Preparation of MMD and drug-loaded sustained-release MMD: Prepare a 0.1 mol / L KMnO4 aqueous solution, then slowly add 30% hydrogen peroxide, while using 1 mol / L KOH to maintain the solution pH at a weak alkaline level (approximately 8-10); stop adding hydrogen peroxide after no obvious bubbles are generated, continue stirring at room temperature for 30 minutes, and let it stand for 4 hours; filter the product, rinse with deionized water three times, and then redisperse it in deionized water, stir and rinse for 24 hours, stop stirring and allow it to settle naturally; repeat the above filtration-washing-sedimentation steps for one week to produce MMD. The MMD was re-ground and placed in a PBS solution containing 5 mg / ml BMP-2 (containing 0.5% MMD). Ultrasonic loading was performed for 60 minutes to allow the drug to enter the micropores. The ultrasonic process was repeated five times, and the precipitate was obtained by centrifugation and freeze-dried. The drug-loaded MMD was washed three times with PBS and then placed in a Tris-HCl solution (pH = 8.5) containing 4 mg / ml DA and reacted on a shaker at room temperature for 8 h. After the reaction, the mixture was washed three times with PBS to obtain a drug-loaded sustained-release MMD material, namely MMD-O-PDA.

[0031] 2) Preparation of composite hydrogel material: GelMA (GelMA concentration: 10% wt) was dissolved in PBS containing 0.25% LAP under light-protected conditions and heated at 60°C to obtain a GelMA solution. After the temperature was lowered to room temperature, drug-loaded sustained-release MMD and blank MMD (both at a concentration of 0.1 wt% in the premix) were premixed with the GelMA solution. The premix was placed on a custom patterned PDMS mold (patterned with microgrooves, 5 μm in width, 3 μm in depth, and 4 μm in spacing) previously coated with a trace amount of glycerol. The mold was then covered with a transparent quartz slide coated with a trace amount of glycerol on the contact surface and UV-cured. After cyclic freezing and demolding, the composite hydrogel material was obtained.

[0032] 3) Grafting of adhesive proteins onto the surface of the composite hydrogel: The hydrogel material was immersed in a 0.1 wt% genipin solution, then quickly removed and immediately immersed in a 0.1 wt% fibronectin solution. After 1 hour, the residual adhesive proteins on the surface were removed with ultrapure water and the above process was repeated 5 times; then, the hydrogel material was dialyzed with PBS for 6 hours, with the solution changed every 30 minutes; finally, the hydrogel material was irradiated and sterilized, and then packaged to obtain the desired composite skull repair hydrogel material with a patterned adhesion-promoting surface.

[0033] Compared with Example 1, the surface micropatterning of the composite hydrogel was added. The degradation rate of the composite hydrogel material in vitro was 77% after 28 days. After cells were inoculated on the surface, complete cell adhesion was observed after 8 hours. After 7 days of culture, live and dead cell fluorescence staining showed that the proportion of live cells was 91%. It was used to repair a rat skull defect model, and the degree of defect repair was good 4 weeks after surgery. HE staining showed that the local inflammatory reaction was mild.

[0034] Example 3

[0035] 1) Preparation of MMD and drug-loaded sustained-release MMD: Prepare a 0.1 mol / L KMnO4 aqueous solution, then slowly add 30% hydrogen peroxide, while using 1 mol / L KOH to maintain the solution pH at a weak alkaline level (approximately 8-10); stop adding hydrogen peroxide after no obvious bubbles are generated, continue stirring at room temperature for 30 minutes, and let it stand for 4 hours; filter the product, rinse with deionized water three times, and then redisperse it in deionized water, stir and rinse for 24 hours, stop stirring and allow it to settle naturally; repeat the above filtration-washing-sedimentation steps for one week to produce MMD. The MMD was re-ground and placed in a PBS solution containing 5 mg / ml BMP-2 (containing 0.5% MMD). Ultrasonic loading was performed for 60 minutes to allow the drug to enter the micropores. The ultrasonic process was repeated five times, and the precipitate was obtained by centrifugation and freeze-dried. The drug-loaded MMD was washed three times with PBS and then placed in a Tris-HCl solution (pH = 8.5) containing 4 mg / ml DA and reacted on a shaker at room temperature for 8 h. After the reaction, the mixture was washed three times with PBS to obtain a drug-loaded sustained-release MMD material, namely MMD-O-PDA.

[0036] 2) Preparation of composite hydrogel material: GelMA (GelMA concentration: 10% wt) was dissolved in PBS containing 0.25% LAP under light-protected conditions and heated at 60°C to obtain a GelMA solution. After the temperature was lowered to room temperature, drug-loaded sustained-release MMD and blank MMD (both at a concentration of 0.1 wt% in the premix) were premixed with the GelMA solution. The premix was placed on a custom patterned PDMS mold (patterned in the shape of micropillars, with a diameter of 10 μm, a height of 50 μm, and a spacing of 20 μm between pillars) previously coated with a small amount of glycerol. The mold was then covered with a transparent quartz slide coated with a small amount of glycerol on the contact surface and UV-cured. After cyclic freezing and demolding, the composite hydrogel material was obtained.

[0037] 3) Grafting of adhesive proteins onto the surface of the composite hydrogel: The hydrogel material was immersed in a 0.1 wt% genipin solution, then quickly removed and immediately immersed in a 0.1 wt% fibronectin solution. After 1 hour, the residual adhesive proteins on the surface were removed with ultrapure water and the above process was repeated 5 times; then, the hydrogel material was dialyzed with PBS for 6 hours, with the solution changed every 30 minutes; finally, the hydrogel material was irradiated and sterilized, and then packaged to obtain the desired composite skull repair hydrogel material with a patterned adhesion-promoting surface.

[0038] Compared with Example 1, the surface micropatterning of the composite hydrogel was added, and the pattern was different from that of Example 2. The degradation rate of the composite hydrogel material was 74% in vitro after 28 days. After cells were inoculated on the surface, complete cell adhesion was observed within 5 hours. After culturing for 7 days, live and dead cell fluorescence staining showed that the proportion of live cells was 92%. It was used to repair a rat skull defect model, and the defect was repaired to a good extent 4 weeks after surgery. HE staining showed that the local inflammatory reaction was mild.

[0039] Example 4

[0040] 1) Preparation of MMD and drug-loaded sustained-release MMD: Prepare a 0.1 mol / L KMnO4 aqueous solution, then slowly add 30% hydrogen peroxide, while using 1 mol / L KOH to maintain the solution pH at a weak alkaline level (approximately 8-10); stop adding hydrogen peroxide after no obvious bubbles are generated, continue stirring at room temperature for 30 minutes, and let it stand for 4 hours; filter the product, rinse with deionized water three times, and then redisperse it in deionized water, stir and rinse for 24 hours, stop stirring and allow it to settle naturally; repeat the above filtration-washing-sedimentation steps for one week to produce MMD. The MMD was re-ground and placed in a PBS solution containing 5 mg / ml BMP-2 (containing 0.5% MMD). Ultrasonic loading was performed for 60 minutes to allow the drug to enter the micropores. The ultrasonic process was repeated five times, and the precipitate was obtained by centrifugation and freeze-dried. The drug-loaded MMD was washed three times with PBS and then placed in a Tris-HCl solution (pH = 8.5) containing 4 mg / ml DA and reacted on a shaker at room temperature for 8 h. After the reaction, the mixture was washed three times with PBS to obtain a drug-loaded sustained-release MMD material, namely MMD-O-PDA.

[0041] 2) Preparation of composite hydrogel material: GelMA (GelMA concentration: 10% wt) was dissolved in PBS containing 0.25% LAP under light-protected conditions and heated at 60°C to obtain a GelMA solution. After the temperature was lowered to room temperature, drug-loaded sustained-release MMD and blank MMD (both at a concentration of 0.1 wt%) were premixed with the GelMA solution. The premixed solution was placed on a custom patterned PDMS mold (patterned with microgrooves, 5 μm in width, 3 μm in depth, and 4 μm in spacing) previously coated with a trace amount of glycerol. The mold was then covered with a transparent quartz slide coated with a trace amount of glycerol and UV-cured. After cyclic freezing and demolding, the composite hydrogel material was obtained.

[0042] 3) Grafting of adhesive proteins onto the surface of the composite hydrogel: The hydrogel material was immersed in a 0.1 wt% genipin solution, then quickly removed and immediately immersed in a 0.2 wt% fibronectin solution. After 1 hour, the residual adhesive proteins on the surface were removed with ultrapure water and the above process was repeated 10 times. The hydrogel material was then dialyzed with PBS for 6 hours, with the solution changed every 30 minutes. Finally, the hydrogel material was irradiated and sterilized, and then packaged to obtain the desired composite skull repair hydrogel material with a patterned adhesion-promoting surface.

[0043] Compared with Example 2, the concentration of adhesion protein and the number of cyclic immersions were increased. The degradation rate of the composite hydrogel material in vitro was 75% after 28 days. After cells were inoculated on the surface, complete cell adhesion was observed 4 hours after culture. After 7 days of culture, live-dead cell fluorescence staining showed that the proportion of live cells was 96%. Due to the repair of the rat skull defect model, the degree of defect repair was good 4 weeks after surgery, and HE staining showed that the local inflammatory reaction was mild.

[0044] Example 5

[0045] 1) Preparation of MMD and drug-loaded sustained-release MMD: Prepare a 0.1 mol / L KMnO4 aqueous solution, then slowly add 30% hydrogen peroxide, while using 1 mol / L KOH to maintain the solution at a weakly alkaline pH (about 8-10); stop adding hydrogen peroxide after no obvious bubbles are generated, continue stirring at room temperature for 30 minutes, and let it stand for 4 hours; filter the product, rinse with deionized water three times, and then redisperse it in deionized water, stir and rinse for 24 hours, stop stirring and allow it to settle naturally; repeat the above steps for 1 week to produce MMD. The MMD was re-ground and placed in a PBS solution containing 5 mg / ml BMP-2 (containing 0.5% MMD). Ultrasonic loading was performed for 60 minutes to allow the drug to enter the micropores. After the ultrasonic process was repeated 5 times, the precipitate was obtained by centrifugation and freeze-dried. The drug-loaded MMD was washed three times with PBS and then placed in a Tris-HCl solution (pH = 8.5) containing 4 mg / ml DA and reacted on a shaker at room temperature for 8 h. After the reaction, the mixture was washed three times with PBS to obtain a drug-loaded sustained-release MMD material, namely MMD-O-PDA.

[0046] 2) Preparation of composite hydrogel material: GelMA (GelMA concentration: 10% wt) was dissolved in PBS containing 0.25% LAP under light-protected conditions and heated at 60°C to obtain a GelMA solution. After the temperature was lowered to room temperature, drug-loaded sustained-release MMD and blank MMD (both concentrations in the premix were 0.2 wt%) were premixed with the GelMA solution. The premix was placed on a custom patterned PDMS mold (patterned with microgrooves, 5 μm width, 3 μm depth, and 4 μm spacing) previously coated with a trace amount of glycerol. The mold was then covered with a transparent quartz slide coated with a trace amount of glycerol and UV-cured. After cyclic freezing and demolding, the composite hydrogel material was obtained.

[0047] 3) Grafting of adhesive proteins onto the surface of the composite hydrogel: The hydrogel material was immersed in a 0.1 wt% genipin solution, then quickly removed and immediately immersed in a 0.1 wt% fibronectin solution. After 1 hour, the residual adhesive proteins on the surface were removed with ultrapure water and the above process was repeated 5 times; then, the hydrogel material was dialyzed with PBS for 6 hours, with the solution changed every 30 minutes; finally, the hydrogel material was irradiated and sterilized, and then packaged to obtain the desired composite skull repair hydrogel material with a patterned adhesion-promoting surface.

[0048] Compared with Example 2, the dosage of MMD and MMD-O-PDA was increased. The degradation rate of the composite hydrogel material in vitro was 69% after 28 days. After cells were inoculated on the surface, complete cell adhesion was observed 8 hours after culture. After 7 days of culture, live-dead cell fluorescence staining showed that the proportion of live cells was 84%. Due to the repair of the rat skull defect model, the degree of defect repair was good 4 weeks after surgery, and HE staining showed that there was almost no local inflammatory reaction.

[0049] The foregoing description is merely a partial list of preferred embodiments of the present invention, intended only to facilitate understanding of the present invention and not to limit the present invention. It should be noted that variations and improvements are possible without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A method for preparing a composite skull repair hydrogel material with a patterned adhesion-promoting surface, characterized in that: The steps include: 1) Preparation of MMD and MMD-O-PDA: Mesoporous manganese dioxide (MMD) materials were prepared using a precipitation method using potassium permanganate and hydrogen peroxide. After washing and dialysis, the osteogenic drug was loaded into the MMD pores using a cyclic ultrasonic loading method. After loading, the MMD surface was coated with a slow-release polydopamine coating using in situ free radical polymerization to obtain MMD-O-PDA; 2) Preparation of composite hydrogel material: dissolving a UV-curable polymer with amino groups under light-proof and heating conditions, and after the temperature drops to room temperature, pre-mixing the MMD-O-PDA, the MMD material and the UV-curable polymer solution with amino groups, placing the pre-mixed liquid on a PDMS mold with a patterned surface previously coated with a release agent, covering the contact surface with a transparent quartz sheet coated with a release agent, and then UV curing, and cyclic freezing and demolding to obtain a composite hydrogel material; wherein, the pattern on the surface of the PDMS mold is an array structure of micron-scale grooves or columns, wherein the width of the grooves is 1-5um, the depth of the grooves is 0.5-3um, and the spacing between the grooves is 400nm-4um; the diameter of the columns is 4-12um, and the height is 100nm-150nm. The premixed liquid is added dropwise to the upper part of the PDMS mold pattern, and the premixed liquid is uniformly infiltrated into the micro-pattern surface by combining the ultrasonic diffusion process, and then a flat and sterile quartz sheet is used to gently cover the upper part of the PDMS mold, the ultrasonic power is 60-100W, and the ultrasonic time is 5-10min; the release agent is one or both of glycerol and ethylene glycol; the UV wavelength is 405nm, the power is 20-50W, and the time is 5-10min; after curing, the mold is cyclically frozen and demolded, and the mold is placed in a -80℃ refrigerator for quick freezing for 3-5min, then taken out and allowed to thaw naturally, and the freeze-thaw process is repeated 2-3 times, after which the gel is gently removed from the mold and soaked in a sterile PBS solution for use; 3) Grafting adhesive proteins onto the surface of the composite hydrogel: Immerse the composite hydrogel material in a crosslinker, then quickly remove it and immediately immerse it in an adhesive protein solution. After removal, use ultrapure water to remove residual adhesive proteins on the surface. Repeat the above process 3-5 times. After grafting the adhesive proteins, dialyze to remove residual crosslinkers and sterilize by irradiation to obtain the composite skull repair hydrogel material with a patterned adhesion-promoting surface.

2. The method for preparing the composite skull repair hydrogel material having a patterned adhesion-promoting surface according to claim 1, characterized in that: In step 1), a 0.05-0.2 mol / L KMnO4 aqueous solution is prepared, and then 30% hydrogen peroxide is slowly added dropwise while maintaining the solution pH at 8-10; the addition of hydrogen peroxide is stopped after no obvious bubbles are generated, and the mixture is stirred continuously at room temperature for at least 30 minutes and allowed to stand for 2-8 hours. The product is filtered, washed with deionized water 3-5 times, and then redispersed in deionized water, stirred and washed for 24-48 hours, and then allowed to settle naturally after stopping stirring. The above filtration-washing-sedimentation steps are repeated for 1-2 weeks to obtain the MMD material.

3. The method for preparing the composite skull repair hydrogel material having a patterned adhesion-promoting surface according to claim 1, characterized in that: In step 1), the prepared MMD material is re-ground and placed in a PBS solution containing an osteogenic drug. Ultrasonic loading is performed to allow the drug to enter the micropores of the MMD material. The precipitate is centrifuged and then freeze-dried to obtain the drug-loaded MMD. The MMD concentration in the solution is 0.1-0.5wt%. The osteogenic drug is one or more of BMP-2, BMP-7, SGF, and IGF, with a concentration of 2-10 mg / ml. The ultrasonic loading power is 80-120W, the loading time is 30-60 minutes, and the loading temperature is 0-4°C. The above ultrasonic loading-centrifugal freeze-drying process is repeated 4-10 times. The drug-loaded MMD is washed with PBS 3-5 times and then placed in a Tris-HCl solution containing dopamine (DA) at pH 8.

5. The reaction is carried out on a shaker at room temperature for 4-8 hours, wherein the DA concentration is 4-8 mg / ml. After the reaction is completed, the MMD is washed with PBS 3-5 times to obtain MMD-O-PDA.

4. The method for preparing the composite skull repair hydrogel material having a patterned adhesion-promoting surface according to claim 1, characterized in that: In step 2), the UV-curable polymer with an amino group is a combination of one or both of GelMA and SilMA, with a concentration of 5-25%. When the two components are combined, the mass ratio of the two components is 3:1-1:3; the amount of MMD-O-PDA added to the premix is ​​0.1-0.2wt%, and the amount of MMD material added is 0.1-0.2wt%; the photoinitiator is one or more of LAP, Irgacure 2100, and Darocur 1173, with a concentration of 0.1-0.25wt%.

5. The method for preparing a composite skull repair hydrogel material having a patterned adhesion-promoting surface according to claim 1, characterized in that: In step 3), the crosslinking agent is glutaraldehyde or genipin at a concentration of 0.05-0.25wt%, and the composite hydrogel is immersed in the crosslinking agent for 3-5s; the adhesion protein is one or more of Fibronectin, Laminin, and Col III at a concentration of 0.1-0.5wt%, and the immersion time is 1-2h; the dialysis uses PBS solution, and the solution is changed every 15-30 minutes, and the dialysis is performed for 3-6h.

6. A composite skull repair hydrogel material with a patterned adhesion-promoting surface, characterized in that: The method is prepared by the method according to any one of claims 1 to 5.

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