A degradable zinc-based composite GBR membrane with a surface-loaded nanoelectrospun fiber coating and preparation and application thereof

Abstract

The present application relates to the technical field of biomedical materials, and particularly relates to a surface-loaded nanoelectrospun fiber coating degradable zinc-based composite GBR membrane and preparation and application thereof. The present application implants a nanofiber coating on the surface of zinc alloy and other zinc-based materials by using electrospinning technology, and obtains a surface-loaded nanoelectrospun fiber coating degradable zinc-based composite GBR membrane which can inhibit excessive release of zinc ions, improve the biocompatibility of zinc alloy and other materials, promote osteoblast differentiation and promote angiogenesis. The electrospun coating on the surface of the zinc-based composite GBR membrane serves as a physical barrier to block the invasion of epithelial cells and fibroblasts, and the fiber degradation releases drugs to promote angiogenesis; the porous zinc alloy and other materials in the intermediate layer not only provide mechanical support, but also release zinc ions through the degradation process to realize the multiple functions of antibiosis and bone formation promotion.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biomedical materials, and in particular to a degradable zinc-based composite GBR membrane loaded with a nanoelectrospun fiber coating on the surface and a preparation and application thereof. BACKGROUND

[0002] Guided bone regeneration (GBR) is the most common local bone augmentation technique. Existing guided bone regeneration membranes (such as collagen membranes, polytetrafluoroethylene membranes and titanium membranes) cannot simultaneously meet the clinical requirements of good biocompatibility, high mechanical strength, controllable degradation, good biological activity and easy operability.

[0003] In recent years, zinc (Zn) and its alloys have shown great potential as guided bone regeneration membranes in treating alveolar bone volume deficiency during dental implantation. Zinc-based materials have good mechanical properties, plasticity and certain antibacterial properties, and appropriate concentrations of Zn 2+ can promote bone matrix mineralization and inhibit inflammatory response, and are considered a promising GBR barrier membrane material. Compared with iron-based and magnesium-based materials, zinc has a more moderate degradation rate, and the released Zn 2+ or Zn-based corrosion products can be absorbed by the surrounding tissue and then excreted through the gastrointestinal tract or kidney filtration, and no harmful hydrogen gas is generated during the degradation process. However, the clinical use of zinc-based materials is still subject to many challenges, such as excessive early Zn 2+ release leading to insufficient cell biocompatibility and osteogenesis. Many current studies are trying to avoid these shortcomings, and common methods include alloying, surface modification and composite material synthesis. Among them, surface modification has been proven to effectively improve the surface properties of zinc-based materials, and the coating structure can effectively improve cell compatibility and improve biological activity.

[0004] Electrospinning is a technology for preparing nanofibers using high-voltage electric field. The nanofibers prepared by electrospinning usually have a diameter of tens of nanometers to several microns. After irregular deposition, a fibrous membrane in the form of non-woven fabric is formed, which has a very high surface area to volume ratio and high porosity, and the morphology and structure are similar to the fibrous structure of the natural extracellular matrix. Electrospinning also has good drug loading performance and slow release effect, and can be used for surface functionalization modification of zinc alloys. SUMMARY

[0005] In order to overcome the deficiencies and shortcomings of the prior art GBR membrane material, such as poor rigidity and the need for secondary surgery to remove the membrane, the primary purpose of the present application is to provide a preparation method of a degradable zinc-based composite GBR membrane with a surface loaded with nanoelectrospun fiber coating, which implants a nanofiber coating on the surface of a zinc alloy or other zinc-based material by using an electrospinning technology, so as to obtain a degradable zinc-based composite GBR membrane with a surface loaded with nanoelectrospun fiber coating, which can inhibit excessive release of zinc ions, improve the biocompatibility of zinc alloys and the like, promote differentiation of osteoblasts, and promote angiogenesis.

[0006] Another purpose of the present application is to provide the degradable zinc-based composite GBR membrane with a surface loaded with nanoelectrospun fiber coating prepared by the above preparation method, wherein the electrospun coating on the surface of the zinc-based composite GBR membrane acts as a physical barrier to block the invasion of epithelial cells and fibroblasts, and the porous zinc alloy or the like in the intermediate layer not only provides mechanical support, but also releases zinc ions through the degradation process to achieve the multiple functions of antibiosis and promotion of osteogenesis. This slow-release system with multiple synergistic effects not only fully utilizes the functions of various factors and drugs, but also provides a barrier effect through the electrospun nanofiber to provide an ideal microenvironment for the defect site and effectively promote the osteogenesis process.

[0007] Still another purpose of the present application is to provide the application of the above-mentioned degradable zinc-based composite GBR membrane with a surface loaded with nanoelectrospun fiber coating.

[0008] The present application is realized by the following means:

[0009] A preparation method of a degradable zinc-based composite GBR membrane with a surface loaded with nanoelectrospun fiber coating, comprising the following steps:

[0010] 1) Preparation of a porous zinc-based mesh:

[0011] A pure zinc or zinc alloy sheet is selected as a substrate, which is processed into a porous structure with a pore size of 0.05-2 mm and a pitch of 0.05-2 mm; then, the substrate is polished, cleaned and dried to obtain a porous zinc-based mesh;

[0012] 2) Preparation of a nanoelectrospun fiber coating:

[0013] ① The functional drug and the biomacromolecular material are dissolved in an organic solvent, or the biomacromolecular material is dissolved in an organic solvent alone to obtain a uniform spinning solution;

[0014] ② The spinning solution prepared in step 1) is loaded into a syringe, and the porous zinc-based web prepared in step 1) is fixed on the axle of a spinning machine as a receiving substrate to perform electrospinning, wherein the parameters of electrospinning are as follows: the pushing speed of the spinning solution is set to 0.5-3 mL / h, the voltage of the high-voltage power supply is set to 10-20 kV, the distance between the nozzle and the receiving device is 10-20 cm, the collection rotation speed is set to 100-300 r / min, and the single-side spinning time is 1-3 h; after one side of the porous zinc-based web is completed with spinning, the substrate is turned over to continue spinning on the other side according to the above parameters for 1-3 h to obtain a double-sided composite membrane;

[0015] ③ The double-sided composite membrane prepared in step 2) is further dried to obtain a degradable zinc-based composite GBR membrane with a nanometer electrospun fiber coating on the surface.

[0016] The zinc alloy in step 1) can be a Zn-Sr alloy (Sr content 0.1-5 wt%), a Zn-Mg alloy (Mg content 0.1-5 wt%), a Zn-Ag alloy (Ag content 0.1-5 wt%), a Zn-Cu alloy (Cu content 0.1-5 wt%), a Zn-Ca alloy (Ca content 0.1-5 wt%), Zn-Fe, Zn-Mn, Zn-Sn, and other zinc-based alloys.

[0017] The preparation method of the zinc alloy in step 1) can include conventional alloying processes such as casting, rolling, extrusion, and stretching, and heat treatment or cold working process can be used during alloying to adjust the mechanical properties and organizational structure of the alloy.

[0018] The thickness of the zinc or zinc alloy sheet selected in step 1) is 0.01-1 mm.

[0019] The processing method in step 1) is preferably by laser punching or plasma punching process.

[0020] The specific operation of polishing, cleaning, and drying in step 1) is preferably as follows:

[0021] The pure zinc or zinc alloy sheet with a porous structure is polished to 2000 mesh using silicon carbide sandpaper, and then sequentially cleaned in acetone and ethanol solutions for 5-15 min under ultrasonic, and finally dried at room temperature for standby.

[0022] The functional drug in step 2) 1) can be at least one of bone morphogenetic protein 2 (BMP-2), minocycline (Minocycline), deferoxamine (DFO), etc.

[0023] The biomacromolecule material in ① of step 2) can be one or two of gelatin (GEL), polycaprolactone (PCL), chitosan (CS), silk fibroin (SF), cellulose acetate (CA), etc. When being two components, the commonly used formulae include PCL:GEL, PCL:CS, PCL:SF, etc., and the common mass ratio is 1:1, 2:1 or other suitable ratios.

[0024] The amount of the biomacromolecule material in ① of step 2) is 0.1-3 g / 10 mL of the organic solvent.

[0025] The organic solvent in ① of step 2) includes hexafluoroisopropanol (HFIP), formic acid, acetic acid solution, or dichloromethane / N,N-dimethylformamide mixed solution, etc.

[0026] The loading amount of the functional drug in ① of step 2) is controlled to be 0.01-20wt%, i.e. the amount of the functional drug is 0.01-20wt% of the amount of the biomacromolecule material.

[0027] The specific operation of the dissolving in ① of step 2) is as follows:

[0028] Under magnetic stirring, the functional drug and the biomacromolecule material are added into the organic solvent for homogeneous dissolution until the solution is uniform; wherein the homogeneous dissolution temperature is 20-30℃, and the homogeneous dissolution time is 4-8 h.

[0029] The spinning solution in ② of step 2) is preferably loaded into a 10 mL syringe, a 20G needle is selected as the stainless steel needle, and the pushing is performed by an automatic injection pump, and the pushing speed is 0.5-3 mL / h.

[0030] The upper surface of the double-sided composite film in ② of step 2) preferably loads a polycaprolactone nanoelectrospun fiber coating loaded with minocycline, and the lower surface preferably loads a gelatin nanoelectrospun fiber coating loaded with bone morphogenetic protein 2.

[0031] The specific operation of the drying treatment in ③ of step 2) is preferably as follows:

[0032] The double-layer composite film prepared in step ② is subjected to drying treatment in a vacuum oven for 24-48 h, and the drying temperature is controlled to be 30-40℃, so as to remove the residual solvent.

[0033] A degradable zinc-based composite GBR film loaded with a nanoelectrospun fiber coating on the surface is prepared by the above preparation method.

[0034] The degradable zinc-based composite GBR film loaded with a nanoelectrospun fiber coating on the surface is used in the preparation of a treatment for dental implant products.

[0035] The present application has the following advantages and effects relative to the prior art:

[0036] (1) The present application implants a nanofiber coating on the surface of a zinc alloy or other zinc-based material using electrospinning technology, obtaining a degradable zinc-based composite GBR membrane with a surface-loaded nanoelectrospun fiber coating that can inhibit excessive release of zinc ions, improve the biocompatibility of zinc alloys, promote osteoblast differentiation, and promote angiogenesis.

[0037] (2) The surface of the degradable zinc-based composite GBR membrane provided by the present application has an electrospun coating that acts as a physical barrier to block the invasion of epithelial cells and fibroblasts, and the degradation of the fibers releases drugs to promote angiogenesis; the porous zinc alloy in the middle layer not only provides mechanical support but also releases zinc ions through the degradation process to achieve the multiple functions of antibiosis and bone formation promotion. This multi-factor synergistic release system not only fully utilizes the functions of various factors and drugs, but also provides a barrier effect through electrospun nanofibers, providing an ideal microenvironment for the defect site and effectively promoting the osteogenesis process.

[0038] (3) The zinc-based composite GBR membrane of the present application can be individually designed and customized by adjusting parameters such as drug loading type, release rate, membrane thickness, and morphology according to different clinical needs. This customizability allows the present application to adapt to various treatment scenarios, including different types of bone defects and other tissue engineering applications, with strong flexibility and adaptability.

[0039] (4) The preparation process of the present application is simple and low-cost. Since the preparation method of the electrospun coating is not limited by the shape of the substrate, it can be used to prepare coatings on substrates with complex shapes, thus providing a flexible solution for personalized and complex bone repair. The universality and efficiency of this process make the composite membrane of the present application highly competitive in practical applications. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 It is the actual object graph after the surface electrospun coating of the zinc alloy of Example 1 is modified.

[0041] Figure 2 It is the EDS point scanning and surface scanning graph of the zinc-strontium alloy prepared in Example 1.

[0042] Figure 3 It is the metallographic graph of the zinc-strontium alloy prepared in Example 1.

[0043] Figure 4 It is the surface SEM graph of the surface electrospun coating of the zinc alloy of Example 1 after modification.

[0044] Figure 5This is a cross-sectional SEM image of the zinc alloy surface after modification by electrospinning coating in Example 1.

[0045] Figure 6 The graph shows the diameter of the electrospun fibers of the composite GBR membrane prepared in Example 1.

[0046] Figure 7 The drug release curve of the composite GBR membrane prepared in Example 1 over 30 days.

[0047] Figure 8 The zinc ion release curve of the composite GBR membrane prepared in Example 1 after immersion in Hanks' solution for 30 days.

[0048] Figure 9 The image shows the experimental effect of the composite GBR membrane prepared in Example 1 on promoting angiogenesis.

[0049] Figure 10 The image shows the antibacterial zone of the composite GBR membrane prepared in Example 1.

[0050] Figure 11 The images show alkaline phosphatase staining and alizarin red staining of the composite GBR membrane prepared in Example 1 after osteogenic induction with a 50% concentration extract.

[0051] Figure 12 The image shows the effect of the composite GBR membrane prepared in Example 1 on guided bone regeneration in a rat mandibular defect model. Detailed Implementation

[0052] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0053] Example 1

[0054] This embodiment prepares an electrospun composite zinc alloy GBR film: a porous Zn-0.8Sr alloy mesh is prepared using laser cutting technology, and a polycaprolactone / gelatin (PCL / GEL) nano-electrospun fiber coating is constructed on the surface of the biodegradable Zn-0.8Sr alloy using electrospinning technology, which is then loaded with deferoxamine (DFO). The specific steps are as follows:

[0055] 1) Preparation of porous zinc-strontium alloy mesh: The Zn-0.8Sr alloy is prepared from high-purity Zn (99.99%) and Sr (99%) raw materials, wherein the mass percentage of Sr in the alloy is 0.8wt%. Figure 2 These are EDS point and area scan images of the alloy. Figure 3is its metallographic diagram. The Zn-0.8Sr alloy is cold-rolled into a sheet with a thickness of 0.2 mm according to a conventional method, and then a porous zinc alloy mesh with a pore diameter of 1.2 mm and a hole distance of 1.2 mm is prepared by using a laser punching technology. The porous zinc alloy mesh sample is mechanically polished to 2000 mesh by using silicon carbide sandpaper in stages, sequentially ultrasonically cleaned in acetone and ethanol for 10 min, and dried at room temperature for standby.

[0056] 2) Preparation of a nano-electrospun fiber coating:

[0057] ①Under the condition of magnetic stirring, 1 g of gelatin (Gel) and 1 g of polycaprolactone (PCL) and 0.65 mg of deferoxamine (DFO) are added to 10 mL of hexafluoroisopropanol solution, stirred and mixed at 30°C for 6 h until the solution is uniformly dissolved, and a homogeneous spinning solution is obtained.

[0058] ②The spinning solution prepared in step ① is loaded into a 10 mL syringe with a 20G stainless steel needle, and then the syringe is installed on the automatic injection pump of the spinning machine, the positive electrode of the high-voltage power supply is connected to the needle of the automatic injection pump, the negative electrode is connected to the electrospinning device, and the porous zinc alloy mesh prepared in step 1) is fixed on the wheel shaft of the spinning machine to receive the spinning, wherein the electrospinning parameters are: the high-voltage power supply voltage is set to 15 kV, the nozzle and the receiving distance are set to 15 cm, the pushing speed of the spinning solution is set to 2.0 mL / h, and the collection speed is set to 200 r / min, and the operation is performed for 2 h to obtain a Zn-0.8Sr alloy composite film covered with a PCL / GEL nano-electrospun fiber coating on one side. Turn over the single-sided composite film and continue to receive the spinning according to the same parameters for 2 h to obtain a double-sided Zn-0.8Sr alloy composite film.

[0059] ③The double-sided Zn-0.8Sr alloy composite film obtained in step ② is placed in a vacuum oven and dried for 24 h at a temperature of 30-40°C to remove the residual solvent on the nano-fibers, and a degradable Zn-0.8Sr alloy composite GBR film with a drug-loaded nano-electrospun fiber coating on the surface is obtained. Figure 1 Figure 4 is its surface SEM image, Figure 5 is its cross-sectional SEM image, and the electrospun nanofiber layer is observed to be formed on the surface of the zinc alloy, uniformly distributed, and without obvious fracture, with an average diameter of 987.2±188.3 nm. The cross-sectional SEM image shows that the electrospun fiber layer is uniformly distributed on the surface of the zinc alloy and closely connected with the zinc alloy, indicating that the electrospinning process is stable and controllable.

[0060] Effect implementation example 1

[0061] ​The Zn-0.8Sr alloy composite GBR membrane prepared in Example 1 was subjected to fiber diameter statistics, water contact angle test, tensile test, drug release test, corrosion degradation test, cytotoxicity test, antibacterial performance test, osteogenic induction experiment, angiogenic performance test, and animal model bone regeneration experiment, respectively. The specific test methods are as follows:

[0062] 1) Fiber diameter test: After gold plating of the sample for 60 seconds, scanning electron microscopy (SEM) was used for observation. 100 fibers were randomly selected in the electron microscope field of view for each sample, and the diameter of the fibers was measured and the average value was calculated using ImageJ software.

[0063] 2) Water contact angle test: Static contact angle method was used, 5 μL of deionized water was added, and the water contact angle on the surface of the sample was measured by a goniometer (WCA, SDC-200S) to evaluate the surface wettability. Each sample was measured 5 times at different positions and the average value was taken.

[0064] 3) Tensile test: Uniaxial tensile test was used, and the ASTM E8 / E8M-16(44) standard (thin film / sheet) was referred to. The sample width was 10 mm, the gauge length was 50 mm, and the thickness was measured according to the actual measurement. The crosshead speed was 10 mm / min, the tensile strength was the maximum load / initial cross-sectional area, and the elongation was the breaking elongation / initial gauge length x 100%. The yield strength was determined by the 0.2% offset method, and the load value was determined according to the 0.2% strain on the stress-strain curve, and the initial cross-sectional area of the sample was used.

[0065] 4) Drug release test: PBS solution (pH 7.4, 37°C) was used as the release medium, and the medium volume / sample surface area ratio was 1 mL / cm 2 . The sample was taken at regular intervals and an equal volume of new solution was added. The concentration of the drug was detected by high performance liquid chromatography (HPLC), using a C18 column (250 x 4.6 mm, 5 μm), the mobile phase was water and acetonitrile (containing 0.1% (volume percent) trifluoroacetic acid), the detection wavelength was 220 nm, and the external standard method was used for quantification. The drug release rate calculation method was the cumulative release drug amount / total drug loading amount.

[0066] 5) Immersion corrosion degradation test: According to the ASTM G31-12 standard, the sample surface area to medium volume ratio was 1 cm 2 / 20 mL, temperature 37±0.5℃, and the soaking medium was Hanks' balanced salt solution (NaCl 8.0 g / L, CaCl20.14 g / L, KCl 0.4 g / L, NaHCO30.35 g / L, glucose 1.0 g / L, MgCl2·6H2O 0.1 g / L, Na2HPO4·2H2O 0.06 g / L, MgSO4·7H2O 0.06 g / L, KH2PO40.06 g / L, pH 7.4). The sample was soaked in a 37℃ environment, and the zinc ion concentration was quantitatively analyzed by ICP-OES after soaking for 30 days. The initial thickness and the thickness after soaking of the sample were measured using a high-precision vernier caliper, the thickness change before and after the sample was soaked for 30 days was recorded, and the thickness difference was calculated. The corrosion rate was calculated by the following formula: corrosion rate = Δt / A x 12, unit: μm / year. Δt is the change in sample thickness (unit: mm), A is the surface area of the sample (unit: cm 2 ).

[0067] 6) Cytotoxicity test: Before the experiment, the test material was soaked in α-MEM (Gibco, USA) medium, and the material to medium ratio was 1.25 cm 2 / mL, incubated at 37℃ for 48 h, and the supernatant was collected as a 100% concentration of the extract, and stored at 4℃ for use; 1-fold and 2-fold dilutions of the medium were used to obtain 50% and 25% concentration extracts, respectively. Mouse calvarial osteogenic precursor cells (Mc3t3-e1, commercially available) were inoculated into a 96-well plate at a density of 5000 cells per well, and cell culture was performed in a 37℃, 5% CO2 incubator. After 1 day of culture, the medium was replaced with 100%, 50%, and 25% concentration extracts added with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution, and the old cell culture medium was replaced with fresh medium every 2 days. After 1, 3, and 5 days of culture, the original culture medium was replaced with 200 μL CCK-8 reagent, and then the culture was continued for 2 h. According to ISO10993-5, the optical density (OD) was measured at 450 nm using a microplate reader (Bio-Rad680, USA) to determine the cell viability.

[0068] 7) Hemangiogenic performance test: Before the experiment, the test material was soaked in DMEM medium (Gibco, USA), and the material to medium ratio was 1.25 cm 2 / mL, incubated at 37°C for 48 h, the supernatant was collected as 100% concentration extract and stored at 4°C for use; extracts were diluted 1-fold and 2-fold with culture medium to obtain 50% and 25% concentrations, respectively. Matrigel (Corning, 356234) was melted at 4°C and added to 96-well plates, 50 μL per well, and cured at 37°C for 30 min. Human umbilical vein endothelial cells (HUVECs, commercially available) were seeded on Matrigel at a density of 2 × 10⁶. 4 Cells / wells were cultured in a 50% concentration of extract for 4 h, with a pure titanium extract used as a control. Images were taken using an inverted fluorescence microscope. The number of junctions and total tube length of the vascular structures were analyzed using ImageJ software.

[0069] 8) Antibacterial performance test: According to ISO 20645 standard, the inhibition zone diameter (IZD) was used to assess the antibacterial activity of the sample against Staphylococcus aureus (ATCC 25923). 500 μL of diluted original bacterial suspension (1×10⁻⁶) was added to an agar plate. 6 The concentration of CFU / mL was measured, and the samples were then placed on agar plates, with three replicates per group. After 24 h of incubation, images of the inhibition zone were taken, and the diameter of the inhibition zone was calculated using calipers.

[0070] 9) Osteogenesis-inducing alkaline phosphatase staining experiment: 2×10 4 MC3T3-E1 cells (commercially available) at a density of / wells were seeded in 48-well plates. After 24 h, the original culture medium was removed, and 50% concentration of the extract (prepared as in step 6) was added. The control group received extract prepared with pure titanium. The culture medium was replaced every 2 days. After 21 days, each group was stained using the BCIP / NBT ALP kit (Beyotime, Shanghai, China). Images were observed and recorded using an inverted microscope. The images were then opened using ImageJ software, converted to 8-bit grayscale, and background impurities were removed. The threshold was adjusted to select ALP-positive areas, and the average stained area for each group was measured and calculated. Finally, the relative area ratio of the 50% concentration extract group was calculated based on the average stained area of ​​the control group.

[0071] 10) Osteogenesis-inducing Alizarin Red staining experiment: 2×10 4MC3T3-E1 cells (commercially available) with a pore density were seeded in a 48-well plate, and 24 h later, the original culture medium was removed, and 50% concentration of the extract (preparation method step 6) was added. The control group was added with the extract prepared from pure titanium, and the culture medium was updated every 2 days. After 21 days, 200 μL of alizarin red solution (OriCell, USA) was added to each well, and after 20 min of placement at room temperature, the dye solution was removed. The sample was observed using a body microscope and the photo was recorded. The same ImageJ operation process in step 9) was used to measure the average area of the calcified nodules (alizarin red staining positive area) in each group. Finally, the average staining area of the control group was taken as the reference to calculate the relative area ratio of the 50% concentration of the extract group.

[0072] 11) Animal model bone regeneration experiment: 8-week-old SD rats (commercially available) were intraperitoneally injected with 2% (mass percentage) pentobarbital at a dose of 50 mg / kg, and the left mandibular region and neck skin were prepared. The surgical area was disinfected with complex iodine, a sterile hole towel was laid, the mandibular angle was positioned, and a surgical knife was used to cut along the mandibular margin from the mandibular angle. The incision was about 2 cm. The skin and subcutaneous tissue were cut layer by layer, and the muscle tissue was bluntly separated to expose the mandibular body and mandibular angle. After the muscle layer and periosteum were bluntly separated, a circular mandibular defect with a depth of 2 mm and a diameter of 5 mm was created on the mandibular body using a bone trephine. The experimental materials in each group were gently placed in the defect, and the wound was sutured layer by layer. At 4 and 8 weeks after the operation, 3 rats were randomly sacrificed from each group, and the mandibular bone containing the defect area was removed and immersed in 4% (mass percentage) paraformaldehyde for 48 h. The implanted material was removed, and the local bone formation ability was evaluated by Micro-CT with parameters of 100 kV and 200 uA. After scanning, 3D reconstruction was performed using NRecon software, and the mandibular defect regeneration was observed.

[0073] It was detected that the average fiber diameter of the nanoelectrospun fiber coating of the Zn-0.8Sr alloy composite GBR membrane prepared in Example 1 was 0.987 μm (0.987 μm) Figure 6 , the water contact angle was 38.4°, the tensile strength of the zinc-based composite GBR membrane was 80 MPa, the yield strength was 52.0 MPa, and the elongation rate was 15.5%.

[0074] The drug release test results showed that the drug release rate of the nanoelectrospun fiber coating loaded with deferoxamine (DFO) within 30 days was 65.3% (65.3%) Figure 7 .

[0075] The immersion corrosion degradation test results showed that the zinc ion concentration released by the Zn-0.8Sr alloy composite GBR membrane in Hanks' solution was 0.61 μg / mL after immersion for 30 days, and the degradation rate was 25 μm / year (25 μm / year) Figure 8 .

[0076] Cytotoxicity experiment results show that the cell survival rate of mouse calvarial osteoblast precursor cells (Mc3t3-e1) cultured in Zn-0.8Sr alloy composite GBR membrane leaching liquor with concentrations of 100%, 50% and 25% for 5 days is 37.7%, 100.7% and 100.2% respectively, and the diluted leaching liquor shows good cell compatibility.

[0077] Angiogenesis test results show that the Huvec tube formation experiment shows that the Zn-0.8Sr alloy composite GBR membrane leaching liquor with a concentration of 50% has better angiogenesis effect than the control group (pure titanium leaching liquor) Figure 9 ).

[0078] Antibacterial performance test results show that the size of the bacterial inhibition zone of the sample after being cultured with Staphylococcus aureus (ATCC 25923) for 1 day is 4.3 mm, showing excellent antibacterial performance Figure 10 ).

[0079] Osteogenic induction experiment shows that after 21 days of osteogenic induction using Zn-0.8Sr alloy composite GBR membrane leaching liquor with a concentration of 50%, it shows good osteogenic differentiation and mineralization induction ability. Among them, the alkaline phosphatase activity staining positive area is increased by 8.71 times compared with the control group (pure titanium group), and the alizarin red staining positive area is increased by 7.52 times Figure 11 ).

[0080] Animal model bone regeneration experiment results show that compared with the control group (pure titanium group), the sample implanted into the mandibular bone defect model of SD rats for 4 weeks and 8 weeks shows excellent guided bone regeneration effect Figure 12 ).

[0081] Example 2

[0082] In this embodiment, an electrospun composite pure zinc GBR membrane is prepared: a porous zinc mesh is prepared by laser cutting technology, and a poly (caprolactone) / chitosan (PCL / CS) nanofiber coating is constructed on the surface of the zinc mesh by electrospinning technology. The specific steps are as follows:

[0083] 1) Preparation of porous zinc mesh: pure zinc is cold-rolled into a sheet with a thickness of 0.1 mm, and then a laser drilling technology is used to prepare a porous zinc mesh with a rhombic hole size of 1 mm×2 mm and a hole distance of 0.2 mm. The porous zinc mesh sample is mechanically polished to 2000 mesh using silicon carbide sandpaper, ultrasonically cleaned in acetone and ethanol for 10 min respectively, and dried at room temperature for standby.

[0084] 2) Preparation of nanofiber coating:

[0085] ①Under the condition of magnetic stirring, 0.2 g of chitosan (CS) and 0.2 g of polycaprolactone (PCL) were added into 10 mL of 90wt% acetic acid solution, and the mixture was stirred at 30℃ for 6 h until the solution was uniformly dissolved to obtain a homogeneous spinning solution.

[0086] ②The spinning solution prepared in step ① was loaded into a 10 mL syringe with a 20G stainless steel needle, and then the syringe was installed on the automatic injection pump of the spinning machine. The positive electrode of the high-voltage power supply was connected to the needle of the automatic injection pump, and the negative electrode was connected to the electrospinning device. The porous zinc mesh prepared in step 1) was fixed on the wheel shaft of the spinning machine to receive the spinning. The electrospinning parameters were as follows: the voltage of the high-voltage power supply was set to 18 kV, the distance between the nozzle and the receiving end was set to 10 cm, the pushing speed of the spinning solution was set to 1.0 mL / h, and the collection speed was set to 200 r / min. After running for 2 h, a pure zinc composite membrane covered with a PCL / GEL nanofiber coating on one side was obtained. The single-sided pure zinc composite membrane was turned over and continued to be collected according to the same parameters for 2 h to obtain a double-sided pure zinc composite membrane.

[0087] ③The double-sided composite pure zinc GBR membrane obtained in step ② was placed in a vacuum oven and dried for 24 h at a temperature of 30-40℃ to remove the residual solvent on the nanofiber, thereby obtaining a degradable zinc-based composite GBR membrane with a surface loaded with a nanofiber coating.

[0088] Effect Example 2

[0089] The specific detection method was the same as that in Effect Example 1.

[0090] It was detected that the average fiber diameter of the nanofiber coating of the zinc-based composite GBR membrane prepared in Example 2 was 0.331 μm, the water contact angle was 62.4°, the tensile strength of the composite membrane was 93.5 MPa, and the elongation rate was 16.1%.

[0091] The immersion corrosion degradation test results showed that the zinc ion concentration released by the zinc-based composite GBR membrane after immersion in Hanks' solution for 30 days was 0.45 μg / mL, and the degradation rate was 17 μm / year.

[0092] The cytotoxicity experiment results showed that the cell survival rates of mouse calvarial osteogenic precursor cells (Mc3t3-e1) cultured in zinc-based composite GBR membrane extracts with concentrations of 100%, 50% and 25% by volume fraction for 5 days were 12.3%, 110.2% and 102.4%, respectively. The diluted extract showed good cell compatibility.

[0093] The antibacterial performance test results show that the size of the bacterial inhibition ring of the sample after being cultured with Staphylococcus aureus (ATCC 25923) for 1 day is 3.9 mm.

[0094] The osteogenic induction experiment shows that the zinc-based composite GBR membrane membrane extract solution with a concentration of 50% exhibits good osteogenic differentiation and mineralization induction ability after 21 days of osteogenic induction. Among them, the alkaline phosphatase activity staining positive area is improved by 5.25 times compared with the pure titanium group, and the alizarin red staining positive area is improved by 4.61 times.

[0095] Example 3

[0096] In this embodiment, an electrospun composite Zn-1.2Mg alloy GBR membrane is prepared: a porous Zn-1.2Mg alloy mesh is prepared by laser cutting technology, and a poly (caprolactone) / silk fibroin (PCL / SF) nanoelectrospun fiber coating is constructed on the surface thereof by electrospinning technology, and osteogenic peptide (OP) is loaded. The specific steps are as follows:

[0097] 1) Preparation of porous Zn-1.2Mg alloy mesh: Zn-1.2Mg alloy is prepared from high-purity Zn (99.99%) and Mg (99%) raw materials, wherein the mass percentage of Mg in the alloy is 1.2%, and the Zn-1.2Mg alloy is cold-rolled into a thin sheet with a thickness of 0.25 mm according to the conventional method, and then a porous zinc alloy mesh with a pore diameter of 1.0 mm and a pore distance of 1.0 mm is prepared by laser punching technology. The porous zinc alloy mesh sample is mechanically polished to 2000 mesh by silicon carbide sandpaper, ultrasonically cleaned in acetone and ethanol for 10 min, respectively, and dried at room temperature for standby.

[0098] 2) Preparation of nanoelectrospun fiber coating:

[0099] ①Under the condition of magnetic stirring, 1.2 g of silk fibroin (SF) and 0.8 g of poly (caprolactone) (PCL) and 1.0 mg of osteogenic peptide (OP) are added to 10 mL of formic acid solution, and the mixture is stirred at 30°C for 5 h until the solution is uniformly dissolved, obtaining a homogeneous spinning solution.

[0100] ②Refer to Example 1 for electrospinning, wherein the electrospinning parameters are: the high-voltage power supply voltage is set to 16 kV, the nozzle and receiving distance is set to 12 cm, the spinning solution is set to 1.5 mL / h, and the collection speed is set to 200 r / min. After spinning on one side for 2 h, the other side is spun for 2 h, obtaining a double-sided Zn-1.2Mg alloy composite membrane.

[0101] ③ The double-sided Zn-1.2Mg alloy composite film obtained in step ② is placed in a vacuum oven at 35°C and dried for 24 h, with a drying temperature of 30-40°C, to remove the residual solvent on the nanofibers, to obtain a degradable Zn-1.2Mg alloy composite GBR film with a nanoelectrospun fiber coating loaded with drug-loaded nanoelectrospun fibers on the surface.

[0102] Effect Example 3

[0103] The specific detection method is the same as that in Effect Example 1.

[0104] It is detected that the average fiber diameter of the nanoelectrospun fiber coating of the Zn-1.2Mg alloy composite GBR film prepared in Example 3 is 0.752 μm, the water contact angle is 41.2°, the tensile strength of the Zn-1.2Mg alloy composite GBR film is 92.7 MPa, and the elongation rate is 14.8%.

[0105] The drug release test results show that the drug release rate of the nanoelectrospun fiber coating loaded with osteogenic peptide is 72.5% within 30 days.

[0106] The immersion corrosion degradation test results show that the Zn-1.2Mg alloy composite GBR film is immersed in Hanks' solution for 30 days, the zinc ion release concentration is 0.48 μg / mL, and the degradation rate is 21 μm / year.

[0107] The cytotoxicity experiment results show that the cell survival rates of mouse calvarial osteogenic precursor cells (Mc3t3-e1) cultured in Zn-1.2Mg alloy composite GBR film leaching liquor with concentrations of 100%, 50% and 25% for 5 days are 34.6%, 105.3% and 98.7%, respectively, and the diluted leaching liquor shows good cell compatibility.

[0108] The antibacterial performance test results show that the antibacterial circle diameter of the Zn-1.2Mg alloy composite GBR film sample prepared in Example 3 is 4.8 mm after being co-cultured with Staphylococcus aureus (ATCC 25923) for 1 day.

[0109] The osteogenic induction experiment shows that the Zn-1.2Mg alloy composite GBR film leaching liquor with a concentration of 50% shows good osteogenic differentiation and mineralization induction ability after being used for 21 days. Among them, the alkaline phosphatase activity staining positive area is increased by 4.92 times compared with the pure titanium group, and the alizarin red staining positive area is increased by 4.54 times.

[0110] Example 4

[0111] The present embodiment prepares a double-sided heterogeneous drug-loaded electrospun composite zinc alloy GBR membrane: using double-sided asynchronous electrospun technology, a poly-caprolactone (PCL) nanofiber coating loaded with minocycline is constructed on the upper surface (the side expected to face the gingival soft tissue) of the porous Zn-3Ag alloy mesh to achieve the functions of antibacterial, anti-inflammatory and barrier to soft tissue invasion; a gelatin (GEL) nanofiber coating loaded with bone morphogenetic protein 2 (BMP-2) is constructed on the lower surface (the side expected to face the bone defect area) to achieve the function of efficient promotion of osteogenic differentiation. The zinc-silver alloy itself can also provide antibacterial properties and support. The specific steps are as follows:

[0112] 1) Preparation of porous zinc-silver alloy mesh: Zn-3Ag alloy is prepared from high-purity Zn (99.99%) and Ag (99.99%) raw materials, wherein the mass percentage of Ag in the alloy is 3.0wt%. The Zn-3Ag alloy is cold-rolled into a thin sheet with a thickness of 0.2 mm according to the conventional method, and then a porous zinc alloy mesh with a pore size of 1.0 mm and a pore distance of 1.2 mm is prepared using laser drilling technology. The porous zinc alloy mesh sample is mechanically polished to 2000 mesh using silicon carbide sandpaper, sequentially ultrasonically cleaned in acetone and ethanol for 10 min, and dried at room temperature for standby use.

[0113] 2) Preparation of nanoelectrospun fiber coating 1 (lower surface osteogenic spinning layer, facing the bone surface):

[0114] ① Under the condition of magnetic stirring, 1.5 g of gelatin (Gel) is added to 10 mL of hexafluoroisopropanol (HFIP) solution, and stirred and mixed at 30°C for 4 h until completely dissolved; then 20 mg of bone morphogenetic protein 2 (BMP-2) is added and mixed gently by vortex to avoid protein inactivation caused by vigorous stirring, to obtain a homogeneous spinning solution 1 (BMP-2 / Gel solution).

[0115] ② The porous Zn-3Ag alloy mesh prepared in step 1) is fixed horizontally on the receiving device with the lower surface exposed upward; then the spinning solution 1 prepared in step ① is electrospun according to the method of embodiment 1, wherein the high-voltage power supply voltage is set to 15 kV, the distance between the nozzle and the receiving device is set to 12 cm, the pushing speed of the spinning solution is set to 1.0 mL / h, and the collection speed is set to 200 r / min. Run for 1.5 h to form a dense BMP-2-loaded gelatin nanoelectrospun fiber coating on the lower surface of the Zn-3Ag alloy mesh.

[0116] 3) Preparation of nanoelectrospun fiber coating 2 (upper surface antibacterial spinning layer, facing the soft tissue surface):

[0117] ①Under the condition of magnetic stirring, 2 g of polycaprolactone (PCL) was added into 10 mL of mixed solvent of dichloromethane (DCM) and N,N-dimethylformamide (DMF) (volume ratio 7:3) to be completely dissolved by magnetic stirring at 30 ℃ for 2 h; then 50 mg of minocycline was added, and magnetic stirring was continued at 30 ℃ for 2 h to obtain a homogeneous spinning solution 2 (minocycline-loaded PCL solution).

[0118] ②The porous Zn-3Ag alloy mesh was turned over, and the upper surface on which the lower coating had been prepared was faced upward. The spinning solution 2 (minocycline-loaded PCL solution) prepared in step ① was electrospun according to the method of Example 1, wherein the voltage of the high-voltage power supply was set to 18 kV, the distance between the nozzle and the receiving end was 15 cm, the pushing speed of the spinning solution was 1.5 mL / h, the collection rotation speed was set to 200 r / min, and the operation was performed for 2 h to form a more hydrophobic and denser drug-loaded PCL nanofiber membrane on the upper surface of the Zn-3Ag alloy mesh, which effectively blocked the migration of epithelial cells.

[0119] 4) Post-treatment: The finally obtained double-sided heterogeneous drug-loaded composite Zn-3Ag alloy membrane was placed in a vacuum oven for drying for 48 h at a drying temperature of 30 ℃ to completely remove all residual solvents, thereby obtaining a degradable Zn-3Ag alloy composite GBR membrane with a drug-loaded nanoelectrospun fiber coating on the surface.

[0120] Effect Example 4

[0121] The specific detection method was the same as that in Effect Example 1.

[0122] It was detected that the average fiber diameter on the upper surface of the nanoelectrospun fiber coating of the Zn-3Ag alloy composite GBR membrane prepared in Example 4 was 1.05 μm, and the water contact angle was 126.5°; the average fiber diameter on the lower surface was 0.82 μm, and the water contact angle was 29.8°. The overall tensile strength of the Zn-3Ag alloy composite GBR membrane was 108.1 MPa, and the elongation rate was 9.2%.

[0123] The drug release test results showed that the cumulative release rate of minocycline on the upper surface of the Zn-3Ag alloy composite GBR membrane prepared in Example 4 was 78.4% within 30 days, and the cumulative release rate of BMP-2 on the lower surface was 22.5% within 30 days.

[0124] The immersion corrosion degradation test results showed that the Zn-3Ag alloy composite GBR membrane prepared in Example 4 was immersed in Hanks' solution for 30 days, the zinc ion release concentration was 0.65 μg / mL, and the degradation rate was 28 μm / year.

[0125] The cytotoxicity experiment result shows that the cell survival rate of mouse skull osteogenic precursor cells (Mc3t3-e1) is 95.2% after being cultured in the Zn-3Ag alloy composite GBR membrane leaching liquor with a volume fraction of 50% for 5 days.

[0126] The antibacterial performance test result shows that the antibacterial ring diameter of the Zn-3Ag alloy composite GBR membrane sample prepared in Example 4 is 5.5 mm after being co-cultured with Staphylococcus aureus (ATCC 25923) for 24 h.

[0127] The osteogenic induction experiment shows that the Zn-3Ag alloy composite GBR membrane leaching liquor with a concentration of 50% exhibits good osteogenic differentiation and mineralization induction ability after being subjected to osteogenic induction for 21 days. The alkaline phosphatase activity staining positive area is increased by 7.21 times compared with the pure titanium group, and the alizarin red staining positive area is increased by 6.35 times.

[0128] Table 1: Mechanical property test results of the composite GBR membrane samples prepared in different examples

[0129] Sample name Tensile strength (MPa) Yield strength (MPa) Elongation (%) Example 1 80.0 52.0 15.5 Example 2 93.5 60.8 16.1 Example 3 92.7 63.0 14.8 Example 4 108.1 82.0 9.2

[0130] In summary, the present application uses the electrospinning technology to prepare a uniform distribution of nanofiber coating on the surface of zinc alloy and load drugs, which improves the biocompatibility of zinc alloy and the like, has good hydrophilicity and high surface area, is beneficial to cell adhesion and growth, and at the same time, the loading of drugs endows the zinc alloy with multiple functions, such as osteogenesis, angiogenesis, antibacterial function, and the like, and accelerates bone healing.

[0131] The embodiments of the present application are described above in combination with the drawings, and the embodiments in the present application and the features in the embodiments can be combined with each other without conflict, the present application is not limited to the above specific embodiments, the above specific embodiments are only illustrative, but not limited, and a person of ordinary skill in the art can make many forms under the inspiration of the present application without departing from the scope of the present application and the protection scope of the claims, which all belong to the protection of the present application.

Claims

1. A method for preparing a biodegradable zinc-based composite GBR film with a surface-loaded nano-electrospun fiber coating, characterized in that... It includes the following steps: 1) Preparation of porous zinc-based mesh: Pure zinc or zinc alloy sheets are selected as the substrate and processed into a porous structure with a pore size of 0.05–2 mm and a pore spacing of 0.05–2 mm; subsequently, they are polished, cleaned, and dried to obtain a porous zinc-based mesh. 2) Preparation of nano-electrospun fiber coating: ① Dissolve functional drugs and biomacromolecules in an organic solvent to obtain a homogeneous spinning solution; ② The spinning solution obtained in step ① is loaded into a syringe, and the porous zinc-based mesh obtained in step 1) is fixed on the wheel shaft of the spinning machine as a receiving substrate for electrospinning. The electrospinning parameters are as follows: the feed speed of the spinning solution is set to 0.5-3 mL / h, the voltage of the high-voltage power supply is set to 10-20 kV, the distance between the nozzle and the receiving device is 10-20 cm, the collection speed is set to 100-300 r / min, and the single-sided spinning time is 1-3 h. After one side of the porous zinc-based mesh is spun, the substrate is flipped over and spinning is continued on the other side according to the above parameters for 1-3 h to obtain a double-sided composite film. ③ The double-sided composite film obtained in step ② is further dried to obtain a biodegradable zinc-based composite GBR film with a surface-loaded nano-electrospun fiber coating. In step 2), the upper surface of the double-sided composite membrane is a coating of polycaprolactone nanoelectrospun fibers loaded with minocycline, and the lower surface is a coating of gelatin nanoelectrospun fibers loaded with bone morphogenetic protein 2.

2. The method for preparing a biodegradable zinc-based composite GBR film with a surface-loaded nano-electrospun fiber coating according to claim 1, characterized in that: The zinc alloy mentioned in step 1) is a Zn-Sr alloy, a Zn-Mg alloy, a Zn-Ag alloy, a Zn-Cu alloy, a Zn-Ca alloy, a Zn-Fe alloy, a Zn-Mn alloy, or a Zn-Sn alloy.

3. The method for preparing a biodegradable zinc-based composite GBR film with a surface-loaded nano-electrospun fiber coating according to claim 1, characterized in that: The thickness of the zinc or zinc alloy sheet selected in step 1) is 0.01 to 1 mm.

4. The method for preparing a biodegradable zinc-based composite GBR film with a surface-loaded nano-electrospun fiber coating according to claim 1, characterized in that: The specific dissolution operation described in step 2) ① is as follows: Functional drugs and biomolecular materials are added to an organic solvent for homogenization and dissolution under magnetic stirring until the solution is homogeneous; the homogenization and dissolution temperature is 20-30 °C and the homogenization and dissolution time is 4-8 h.

5. The method for preparing a biodegradable zinc-based composite GBR film with a surface-loaded nano-electrospun fiber coating according to claim 1, characterized in that: The specific operation of the drying process described in step 2) ③ is as follows: The double-layer composite film obtained in step ② is dried in a vacuum oven for 24–48 h at a temperature controlled at 30–40 °C.

6. The method for preparing a biodegradable zinc-based composite GBR film with a surface-loaded nano-electrospun fiber coating according to claim 1, characterized in that: The amount of biomacromolecule material used in step 2) ① is 0.1–3 g / 10 mL of organic solvent.

7. The method for preparing a biodegradable zinc-based composite GBR film with a surface-loaded nano-electrospun fiber coating according to claim 1, characterized in that: The organic solvent mentioned in step 2)① includes hexafluoroisopropanol, formic acid, acetic acid solution, or dichloromethane / N,N-dimethylformamide mixed solution.

8. The method for preparing a biodegradable zinc-based composite GBR film with a surface-loaded nano-electrospun fiber coating according to claim 1, characterized in that: The loading amount of the functional drug mentioned in step 2) ① is 0.01 to 20 wt%.

9. A biodegradable zinc-based composite GBR film with a surface-loaded nano-electrospun fiber coating, characterized in that... It is prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the biodegradable zinc-based composite GBR membrane with surface-loaded nano-electrospun fiber coating as described in claim 9 in the preparation of therapeutic dental implant products.

Images