A nanohybrid fiber and a preparation method and application thereof

Abstract

The application discloses a kind of nanometer hybrid fiber and its preparation method and application, belong to bone repair material field.Nanometer hybrid fiber is made of composite polyester spinning;Composite polyester is made of 1,4-butanediol, zinc oxide, succinic acid and tetrabutyl titanate;According to mole ratio, 1,4-butanediol: succinic acid=(1.05~1.2):1;Zinc oxide addition is 1,4-butanediol and succinic acid total mass 0.5%~1.5%;Tetrabutyl titanate addition is succinic acid mole amount 0.1%~0.2%.The application provides a kind of nanometer hybrid fiber with good three-dimensional network entanglement, also provides a kind of biomimetic periosteum with porous network structure, provides nanometer hybrid fiber and biomimetic periosteum have excellent mechanical properties, antibacterial, biocompatibility and other functions;Biomimetic periosteum also has antioxidant, promotes osteogenesis ability;Provided preparation method is simple, can effectively solve the agglomeration problem of nanometer filler, realizes the uniform dispersion of each component.

Description

Technical Field

[0001] This invention relates to the field of bone repair materials technology, and in particular to a nano-hybrid fiber, its preparation method, and its application. Background Technology

[0002] The skeletal system forms the basic framework of the human body, and bone tissue is the fundamental building block of this system. It is a complex and metabolically active organ, providing mechanical support and protecting internal organs. As the body's largest calcium and phosphorus reserve, it plays a crucial role in regulating calcium and phosphorus balance, maintaining nerve conduction, and facilitating muscle contraction. However, borderline bone defects caused by exogenous trauma, pathological factors, and iatrogenic surgery have become a serious challenge for clinical orthopedics.

[0003] Currently, clinically available transplant materials include autologous bone, allogeneic bone, and artificial bone. Autologous bone transplantation is considered the "gold standard" for bone defect repair; however, the limited availability of autologous bone cannot meet the needs of large-area defects, and it also carries risks such as secondary damage to the donor site and infection. While allogeneic bone transplantation addresses the issues of donor site defects and scarcity to some extent, it presents ethical concerns, rejection reactions, and risks of disease transmission. Therefore, artificial bone repair materials, with their wide availability, good biocompatibility, and adjustable mechanical properties, have become a research hotspot in clinical bone transplantation materials.

[0004] Currently, artificial bone repair materials are mainly classified into three categories: ceramic-based, metal-based, and polymer-based. Among them, polymer-based artificial bone materials have broad application prospects in bone repair materials due to their good biocompatibility and processing performance. However, polymer matrices have several drawbacks, including a lack of bioactive signals, poor cell affinity, the generation of acidic products during degradation (especially PLA and PLGA), which may induce aseptic inflammation and lead to premature loss of scaffold mechanical properties, and insufficient osteoinductive activity, making it difficult to meet the clinical needs of bone regeneration and repair. Summary of the Invention

[0005] The purpose of this invention is to provide a nano-hybrid fiber, its preparation method, and its application, in order to solve the problems of lack of bioactive signals, poor cell affinity, and poor bone-inducing activity in artificial bone repair materials, and to provide a new material system for the clinical translation of functional bone repair materials.

[0006] To achieve the above objectives, the present invention provides a nano-hybrid fiber, wherein the nano-hybrid fiber is spun from a composite polyester; the composite polyester is prepared from 1,4-butanediol, ZnO powder, succinic acid, and tetrabutyl titanate; the molar ratio of 1,4-butanediol to succinic acid is (1.05~1.2):1; the amount of zinc oxide added is 0.5%~1.5% of the total mass of 1,4-butanediol and succinic acid; and the amount of tetrabutyl titanate added is 0.1%~0.2% of the molar mass of succinic acid.

[0007] A method for preparing the nano-hybrid fiber as described above includes the following steps: S1. Mix 1,4-butanediol and ZnO powder and sonicate. After sonication, continue stirring for a period of time to obtain a suspension. S2. Add the suspension obtained in S1 and succinic acid to a four-necked flask and react until the water output reaches more than 90% of the theoretical value. Then, heat to 200-250℃ and maintain at a vacuum of 500-1000Pa for 0.5-1h. Then, reduce the vacuum to below 50Pa and continue the reaction for 2-3h. Purge with protective gas until the system temperature drops to 140-160℃ and discharge. After cooling, hot press to obtain composite polyester. S3. Dissolve the composite polyester obtained in S2 in hexafluoroisopropanol to obtain an electrospinning solution, and electrospin to obtain nano-hybrid fibers.

[0008] Preferably, in step S1, the ultrasonic treatment is performed at 30-50 kHz and 10-20 W for 30-40 minutes, and the stirring is performed at 300-500 rpm for more than 12 hours.

[0009] Preferably, in S2, the four-necked flask is connected to a mechanical stirrer, a condenser, an inert gas protection device, and a glass stopper, respectively. The reaction conditions after connecting the four-necked flask are heating, stirring speed of 350-450 r / min, protective atmosphere, and heating from 130°C to 180°C.

[0010] Preferably, the hot pressing in S2 is carried out at 130-150℃ and 0.5-1MPa; the concentration of composite polyester in the electrospinning solution in S3 is 100-150mg / mL; the electrospinning parameters are: voltage 15-20kV, feed speed 1.0-1.2mL / h, needle movement distance 10-15cm, distance between needle and receiving roller 10-15cm, rotation speed of receiving roller 200-300r / min, ambient temperature 30-40℃, and ambient humidity 30-40%; after spinning in S3, the obtained fiber / fiber membrane is dried in an oven at 37℃ for no less than 24h.

[0011] The application of nano-hybrid fibers in bone repair, as described above.

[0012] A double-layered biomimetic periosteum, made from the aforementioned nano-hybrid fibers, is prepared as follows: (1) After dissolving poly-L-lactic acid in hexafluoroisopropanol, curcumin was added and dissolved to obtain an electrospinning solution. Electrospinning was then performed to obtain a PLLA-Cur fiber layer. (2) Dissolve the above-mentioned composite polyester in hexafluoroisopropanol to obtain an electrospinning solution, and spin it on the PLLA-Cur fiber layer obtained in (1) to obtain a double-layer biomimetic endothelial membrane.

[0013] Preferably, the mass-to-volume ratio of poly-L-lactic acid:hexafluoroisopropanol:curcumin in the electrospinning solution prepared in (1) is 10g:0.1L:1g; the electrospinning parameters are: voltage 15-20kV, feed speed 1.0-1.2mL / h, nozzle moving distance 10-15cm, distance between nozzle and receiving roller 10-15cm, rotation speed of receiving roller 2400r / min, ambient temperature 30-40℃, and ambient humidity 30-40%.

[0014] Preferably, the concentration of nano-hybrid fibers in the electrospinning solution prepared in (2) is 100-150 mg / mL; the electrospinning parameters are: voltage 15-20 kV, feed speed 1.0-1.2 mL / h, nozzle moving distance 10-15 cm, distance between nozzle and receiving roller 10-15 cm, rotation speed of receiving roller 300 r / min, ambient temperature 30-40 ℃, and ambient humidity 30-40%.

[0015] The application of the double-layered biomimetic periosteum in bone repair, as described above.

[0016] Therefore, the present invention provides a nano-hybrid fiber, its preparation method, and its application, the specific technical effects of which are as follows: (1) This invention provides a nano-hybrid fiber with uniform fiber membrane diameter, good three-dimensional network entanglement, and the ability to obtain fibers of different diameters by adjusting the component ratio; it also has multiple functions such as excellent mechanical properties, good surface hydrophilicity, antibacterial properties, biocompatibility and low hemolysis rate; (2) The present invention also provides a double-layer biomimetic periosteum, which has a continuous porous network structure and has excellent strength and toughness, good surface hydrophilicity, sustained release effect, anti-inflammatory, antioxidant, antibacterial, biocompatibility, bone differentiation and mineralization promotion and other functions, and the hemolysis rate meets international standards; it provides a new material system for the clinical transformation of functional bone repair materials; (3) The preparation method of nano-hybrid fibers and double-layer biomimetic dermal membrane provided by the present invention is simple and easy to operate. Different materials with different performance advantages can be obtained by adjusting the component ratio, which is suitable for industrial production. It can effectively solve the problems of agglomeration and interfacial compatibility of nanofillers, so that the components in the material are uniformly dispersed and stably combined.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum in Example 1 of the present invention; Figure 2 These are SEM images of the PBS-ZnO nanohybrid fiber membrane in Example 2 of this invention; where (a) is PBS; (b) is PBS-ZnO-0.5; (c) is PBS-ZnO-1.0; (d) is PBS-ZnO-1.5; (e) is the diameter distribution of PBS fibers; (f) is the diameter distribution of PBS-ZnO-0.5 fibers; (g) is the diameter distribution of PBS-ZnO-1.0 fibers; and (h) is the diameter distribution of PBS-ZnO-1.5 fibers. Figure 3 These are the mechanical property measurement results in Example 2 of the present invention; where (a) is the stress-strain curve; (b) is the tensile strength and elongation at break; and (c) is the Young's modulus. Figure 4 This refers to the contact angle measurement results in Example 2 of the present invention; Figure 5 This is the antibacterial effect of different nano-hybrid fiber membranes in the in vitro antibacterial performance test of Example 2 of the present invention; wherein (a) is the colony co-cultured with different nano-hybrid fiber membranes; (b) is the effect of different nano-hybrid fiber membranes on the antibacterial performance of the present invention. E. coli (c) is the antibacterial rate; S.aureus The antibacterial rate; Figure 6 These are SEM images of the bacterial morphology observation portion in Embodiment 2 of the present invention; Figure 7 These are the results of fluorescent staining for bacterial liveness and death in Example 2 of this invention; where (a) is a fluorescent micrograph; and (b) is a result of... E. coli (c) is the antibacterial rate; S.aureus The antibacterial rate; Figure 8 These are the cell compatibility test results in Example 2 of the present invention; where (a) is cell live / dead fluorescence staining; (b) is the CCK-8 absorbance value at different culture time points; and (c) is the cell viability calculated based on the CCK-8 results. Figure 9 This refers to the hemolysis experiment results in Example 2 of the present invention; Figure 10 This is a photograph of the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum prepared in Example 3 of this invention; Figure 11 This refers to the FTIR test results in Embodiment 3 of the present invention; Figure 12 These are SEM images of the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum in Example 3 of this invention; wherein (a) is PBS fiber; (b) is PBS-ZnO-1.0 fiber; (c) is PLLA fiber; (d) is PLLA-Cur fiber; (e) is the diameter distribution map of PBS fiber; (f) is the diameter distribution map of PBS-ZnO-1.0 fiber; (g) is the diameter distribution map of PLLA fiber; and (h) is the diameter distribution map of PLLA-Cur fiber. Figure 13 These are the mechanical property measurement results in Example 3 of the present invention; where (a) is the stress-strain curve; and (b) is the tensile strength and elongation at break. Figure 14 This refers to the contact angle test results in Embodiment 3 of the present invention; Figure 15 This is an analysis of the in vitro release capacity of curcumin in Example 3 of the present invention; wherein (a) is the UV-Vis absorption spectrum of Cur in 1% Tween-80 / PBS medium; (b) is the linear standard curve of Cur; (c) is the UV-Vis absorption spectrum of Cur released from PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum as a function of time; and (d) is the cumulative release curve of Cur from PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum. Figure 16 These are the results of the in vitro antioxidant experiment in Example 3 of this invention; Figure 17 This refers to the intracellular reactive oxygen species scavenging experiment in Example 3 of this invention; Figure 18 These are the in vitro antibacterial performance test results in Example 3 of the present invention; where (a) is a photograph of a culture dish; and (b) is the statistical result of the antibacterial rate. Figure 19These are the cell compatibility experiment results in Example 3 of the present invention; wherein (a) is the fluorescence staining result of cell live / dead staining; (b) is the CCK-8 absorbance value at different culture time points; and (c) is the cell viability calculated based on the CCK-8 results. Figure 20 This refers to the hemolysis experiment results in Example 3 of the present invention; Figure 21 These are the results of the in vitro osteogenic differentiation evaluation test in Example 3 of the present invention; where (a) is the ALP staining result; and (b) is the quantitative analysis result. Figure 22 These are the results of the alizarin red staining experiment in Example 3 of the present invention; where (a) is the ARS staining result; and (b) is the quantitative analysis result. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] The instruments, equipment, reagents and materials used in the embodiments were all obtained through commercial means; the methods and steps not described in detail in the embodiments are all conventional techniques in the art.

[0023] Example 1 The specific steps for preparing PBS-ZnO composite polyester are as follows: S11. Add 20g of 1,4-butanediol (1,4-BDO) and different masses of ZnO powder (0.5, 1.0 and 1.5 wt% of the total mass of SA and 1,4-BDO) to a 50mL beaker, sonicate at a frequency of 40kHz and a power of 15W for 30min, and then stir at 300rpm for 12h to obtain a uniformly dispersed ZnO / BDO suspension.

[0024] S12. Add the ZnO / BDO suspension obtained in step S11 and 23.6 g of succinic acid (SA) (alcohol-acid molar ratio 1:1.1) to a 250 mL four-necked flask, and add 0.12 g of tetrabutyl titanate (TBT) (equivalent to 0.15 mol% of SA by mass). Add a mechanical stirrer, a condenser, an argon gas protection device, and a glass stopper. Then place the flask in an oil bath preheated to 130 °C, turn on the mechanical stirrer (350 r / min) and argon gas protection (250 mL / min), raise the temperature to 180 °C, and maintain the reaction temperature until the water output reaches more than 90% of the theoretical value (approximately 3 h).

[0025] S13. Heat to 230℃ while maintaining a vacuum of 1000Pa in the flask for 0.5h. Then maintain the vacuum below 50Pa and continue the reaction for 2.5h. Introduce argon gas and wait for the system temperature to drop to 150℃ before discharging to obtain PBS-ZnO composite polyester.

[0026] The PBS-ZnO composite polyester was hot-pressed at 135℃ and 0.5 MPa. The composite polyesters with ZnO additions of 0, 0.5, 1.0 and 1.5 wt% were named PBS, PBS-ZnO-0.5 wt%, PBS-ZnO-1.0 wt%, and PBS-ZnO-1.5 wt%, respectively.

[0027] The prepared PBS-ZnO composite polyester was subjected to 1H NMR spectroscopy (NMR spectroscopy). 1 H NMR) test, with specific parameters as follows: The chemical structure of the PBS-ZnO-1.0 wt% hot-pressed film was characterized using an Avance 600 MHz nuclear magnetic resonance spectrometer. 5 mg of polyester was dissolved in 0.5 mL of deuterated chloroform (CDCl3) for testing, with the solvent peak (δ = 7.26 ppm) of CDCl3 chemical shift used as a reference.

[0028] The results are as follows Figure 1 As shown, the peak at δ=1.70ppm corresponds to the methylene group (-CH2-) in the butanediol unit, the peak at δ=2.62ppm corresponds to the methylene group (-CH2COO-) adjacent to the carbonyl group in the succinic acid unit, and the peak at δ=4.11ppm corresponds to the methylene group (-OCH2-) adjacent to the ester oxygen group in butanediol. The integral area ratio of 0.95:0.99:1.06 is basically consistent with the theoretical value of 1:1:1. The absence of residual monomer peaks confirms the successful synthesis of the PBS-ZnO composite polyester. Compared with the pure PBS spectrum in the literature, no significant chemical shift changes were observed in its characteristic peaks, indicating that no strong chemical bonding occurred between the ZnO and PBS chains.

[0029] Example 2 The specific steps for preparing PBS-ZnO nanofibers are as follows: 1.5 g of the PBS-ZnO composite polyester prepared in Example 1 was dissolved in 10 mL of hexafluoroisopropanol (HFIP), and then magnetically stirred at room temperature (500 r / min, 12 h) to completely dissolve the PBS-ZnO, yielding an electrospinning solution with a concentration of 150 mg / mL. The fiber membrane was prepared using the following electrospinning process: voltage 18 kV, feed speed 1 mL / h, needle movement distance 10 cm, needle-receiving roller distance 15 cm, receiving roller rotation speed 300 r / min, ambient temperature 30 °C, and ambient humidity 40%.

[0030] The collected fiber membranes were dried in an oven at 37°C for 24 hours to remove residual solvent. Fiber membranes with ZnO loadings of 0 wt%, 0.5 wt%, 1 wt%, and 1.5 wt% were named PBS, PBS-ZnO-0.5, PBS-ZnO-1.0, and PBS-ZnO-1.5, respectively.

[0031] The prepared PBS-ZnO nanofiber membrane was characterized as follows: (1) Surface structure morphology.

[0032] The morphology of the PBS-ZnO hybrid fiber membrane was characterized using a SU8010 scanning electron microscope. The test voltage was 3 kV and the gold sputtering time was 120 s. The diameter of at least 50 fibers was randomly measured using ImageJ software, and their distribution was statistically analyzed.

[0033] The results are as follows Figure 2 As shown, all PBS-ZnO hybrid fiber membranes formed a continuous and randomly oriented porous network structure, which is beneficial for cell migration and nutrient transport. Statistical results of the average diameter of PBS-ZnO fibers with different ZnO loadings show that the introduction of ZnO altered the fiber diameter and surface morphology, exhibiting a clear concentration dependence. The diameter of the PBS-ZnO-0.5 fiber membrane (707.4±312.6 nm) was increased compared to PBS (562.9±175.1 nm). As the ZnO content continued to increase, the polyester viscosity began to decrease, leading to a decrease in the spinning solution viscosity and a corresponding decrease in fiber diameter, especially for the PBS-ZnO-1.5 fiber, where the diameter decreased to 274.5±129.9 nm.

[0034] from Figure 2(d) A beaded structure can be observed on the fiber surface. This may be due to the aggregation of ZnO nanoparticles at high content, which disrupts the continuity of the polymer solution; at the same time, ZnO may increase the conductivity of the solution and enhance the charge density carried by the jet, causing the jet to undergo more severe stretching in the electric field, thereby producing finer fibers.

[0035] (2) Mechanical performance testing.

[0036] The mechanical properties of the nanofiber hybrid membrane were measured using a CTM-2050 universal testing machine. The thickness of the fiber membrane was measured using a thickness gauge at a tensile rate of 10 mm / min, and the sample size was 1 cm × 5 cm. Each sample was measured three times, and the average value was taken.

[0037] The results are as follows Figure 3 As shown, when the ZnO addition amount is 0.5wt%, the tensile strength of the fiber membrane is 2.74±0.08MPa, which is 15.5% higher than that of pure PBS (2.36±0.05MPa), the elongation at break is 90.57±2.52%, and the Young's modulus is 18.84±0.72MPa. This may be because the intrinsic viscosity of the PBS-ZnO-0.5wt% composite polyester is the highest (1.07dL / g), which is beneficial to the formation of chain entanglement and rheological properties in the spinning solution, thereby producing a fiber membrane with relatively uniform fiber diameter and good three-dimensional network entanglement.

[0038] As the ZnO content continued to increase to 1.0 wt%, the tensile strength of the fiber membrane decreased slightly to 2.58 ± 0.04 MPa, but the Young's modulus reached its highest value of 24.23 ± 0.39 MPa, an increase of approximately 49.2% compared to pure PBS. This indicates that at this content, the reinforcing effect of ZnO nanoparticles as rigid fillers was most significant, effectively improving the material's resistance to deformation by restricting molecular chain movement. When the ZnO content further increased to 1.5 wt%, the tensile strength decreased to 2.07 ± 0.04 MPa, and the Young's modulus also decreased to 21.81 ± 1.16 MPa. This may be because the intrinsic viscosity of the composite polyester decreased (0.70 dL / g), leading to a decrease in spinning jet stability and making it prone to structural defects such as uneven fiber diameter and beading (compared to...). Figure 2 (The morphological observation results are consistent), which reduces the overall load-bearing capacity of the fiber network, resulting in a decrease in mechanical properties.

[0039] Meanwhile, with the increase of ZnO content, the elongation at break of the fiber membrane gradually decreased (from 105.20±3.05% in PBS to 70.37±4.02%). This indicates that while the introduction of ZnO strengthens the PBS matrix, it also leads to a decrease in its ductility. This phenomenon may be due to the restriction of polymer molecular chain movement by nanoparticles and the increase in polymer crystallinity.

[0040] (3) Contact angle test.

[0041] The contact angle of the nanofiber hybrid membrane was measured using a Theta-Flex contact angle meter. The sample size was 1 cm × 3 cm, and it was attached to the glass sample stage with double-sided tape. The measurement was performed using the seated drop method. Three measurements were taken for each sample group, and the average value was calculated.

[0042] The results are as follows Figure 4 As shown, the surface of the pure PBS electrospun membrane exhibits hydrophobic properties, with a water contact angle of 128.9 ± 0.9°. With the introduction of ZnO nanoparticles, the contact angle of the composite fiber membrane showed a significant decreasing trend. When the ZnO addition amount was 0.5 wt%, 1 wt%, and 1.5 wt%, the contact angles were 118.4 ± 0.9°, 117.7 ± 0.8°, and 113.1 ± 0.6°, respectively. The results indicate that ZnO loading improves the surface hydrophilicity of the PBS electrospun membrane.

[0043] (4) In vitro antibacterial performance test.

[0044] ① Plating method: The plating method was used to study the effect of PBS-ZnO nanofiber membranes on Escherichia coli (E. coli). E. coli ) and Staphylococcus aureus ( S.aureus The in vitro antibacterial activity of PBS and PBS-ZnO nanofiber membranes (1.0 cm × 1.0 cm) was evaluated. Before testing, PBS and PBS-ZnO nanofiber membranes (1.0 cm × 1.0 cm) were sterilized under ultraviolet light in a clean bench for 30 min. Then, 100 μL of the membrane was diluted with 10... 2 The bacterial culture was added to 24-well plates and co-cultured with the fiber membrane. The co-cultured culture was then removed and subjected to 10... 3 After dilution, 100 μL of the bacterial suspension was evenly spread onto fresh LB agar plates, three times per group. The plates were incubated at 37°C for 18 hours. A control group was prepared by adding only an equal volume of bacterial suspension. After bacterial count, the antibacterial rate was calculated according to Formula III: (Formula III) In the formula, R is the antibacterial rate (%), A is the total number of bacteria in the control group, and B is the total number of bacteria in the experimental group.

[0045] Pure PBS E. coli and S.aureus The PBS-ZnO-0.5 solution showed almost no antibacterial effect, with bactericidal rates of 3.6% and 7.4%, respectively. However, the introduction of ZnO significantly improved its antibacterial properties. PBS-ZnO-0.5 achieved bactericidal rates exceeding 82% for both bacteria, and when the ZnO concentration was 1.5 wt%, a bactericidal rate of 99.9% was achieved for both bacteria. Figure 5 The introduction of antibacterial properties can effectively prevent early bacterial infection after implantation surgery, creating a favorable microenvironment for subsequent bone cell adhesion and growth.

[0046] ② Bacterial morphology was observed using SEM, and the results are as follows: Figure 6 As shown, bacteria co-cultured with PBS maintained their morphology, normal size, and intact cell surface structure. In contrast, bacteria co-cultured with PBS-ZnO-1.0 nanohybrid fiber membranes exhibited significant changes, with their cell membranes noticeably shrunken and even ruptured. This phenomenon may be due to the PBS-ZnO fiber membrane releasing positively charged Zn... 2+ and E. coli and S.aureus Negatively charged cell membranes undergo electrostatic interactions, resulting in the adsorption of substances onto the membrane. This adsorption increases cell membrane permeability, leading to the leakage of intracellular substances. Simultaneously, Zn enters the cell... 2+ It interferes with the protein metabolism of bacteria, ultimately leading to the death of the bacteria.

[0047] ③ Bacterial live / dead fluorescence staining: After centrifuging the bacterial cultures co-cultured with different groups of samples for 5 hours, the supernatant was discarded and the samples were washed thoroughly with PBS. Staining was performed using a bacterial live / dead staining kit, followed by incubation in the dark for 20 minutes. The stain was then added to a glass slide. After standing, the slides were observed and photographed using an inverted fluorescence microscope.

[0048] The results are as follows Figure 7 As shown, a significant shift from green fluorescence (live bacteria) to red fluorescence (dead bacteria) was observed with increasing ZnO content. Quantitative analysis further confirmed this ZnO concentration-dependent antibacterial trend. E. coli The antibacterial rate increased from 7.3±0.3% when the ZnO content was 0wt% to 99.5±0.5% when the ZnO content was 1.5wt%; similarly, for S.aureus The antibacterial rate increased from 4.8±1.0% to 99.1±0.5%. This result is consistent with the conclusions obtained by the plating method, jointly demonstrating that the PBS-ZnO nanofiber membrane possesses good antibacterial properties. Combined analysis of colony counting, live / dead fluorescence staining, and SEM showed that the PBS-ZnO nanofiber membrane exhibited good, concentration-dependent antibacterial properties against both Gram-negative and Gram-positive bacteria.

[0049] (5) Cell compatibility test. The complete culture medium used in the experiment was: 89% α-DMEM + 10% FBS + 1% P / S.

[0050] ① The cells used were embryonic mouse cranial osteoblast precursors (MC3T3-E1). The cells were removed from an ultra-low temperature freezer at -80℃ and rapidly thawed in a 37℃ water bath. They were then transferred to centrifuge tubes containing 3 times the volume of complete culture medium. After centrifuging the mixture (1000 rpm, 3 min), the supernatant was discarded, and complete culture medium was added. The mixture was then pipetted and transferred to 10 mL culture dishes and cultured in a cell culture incubator at 37℃ and 5% CO2.

[0051] ② After sterilizing with ultraviolet light in the clean bench for 30 min, place the sample (1cm×1cm) into a 24-well plate. Soak the sample in 75% (v / v) ethanol 3 times and irradiate it under ultraviolet light for 30 min each time. Then, soak the sample in PBS solution 3 times for 30 min each time. After washing with PBS 3 times, add complete culture medium and place it in a cell culture incubator for later use.

[0052] ③ After the cells in the culture dish from step ① have grown to cover the entire culture dish, digest them with trypsin and add complete culture medium to prepare a cell suspension. Use 5 × 10⁻⁶ cells / day. 3 Cells were seeded at a density of [number] cells / well on fibrous membranes in 24-well plates and cultured at 37°C and 5% CO2 for 1, 2, and 3 days, respectively. Cell proliferation and virulence were assessed using a CCK-8 assay kit. The specific method was as follows: After co-culturing with the fibrous membrane for 1, 2, and 3 days, the 24-well plates were removed, the original complete culture medium was discarded, and 10 μL of CCK-8 staining agent and 190 μL of DMEM were added to each well. The plates were then returned to a cell culture incubator and incubated in the dark for 1 hour. The solution was then transferred to 96-well plates, and the absorbance at 450 nm was measured using a microplate reader. Three samples were used for each group, and the average value was taken.

[0053] Cell viability / death assay kits were used to detect cell viability. After discarding the old complete culture medium from cell plates cultured for 1, 2, and 3 days, 200 μL of staining solution was added to each well (the volume of staining solution added was 0.08% of the complete culture medium, with 2 μL of each of the two staining solutions), and the plates were incubated at 37°C in the dark for 30 min. The cell state on the surface of the material was observed using a fluorescence microscope.

[0054] The results are as follows Figure 8 As shown in (a), after co-culturing with all groups (control group, PBS group and PBS-ZnO-1.0 group) for 3 days, MC3T3-E1 cells showed high density and good morphology of green fluorescence (live cells), with only sporadic red fluorescence (dead cells). This indicates that both PBS and PBS-ZnO-1.0 fiber membranes have good biocompatibility.

[0055] Subsequently, cell viability was further quantitatively analyzed using a CCK-8 assay, and the results were as follows: Figure 8As shown in (b) and (c): On day 1 of culture, cell viability in all groups was close to 100%, indicating that cell adhesion and activity were not affected by the material. On days 2 and 3, cell viability in both the PBS group and the PBS-ZnO-1.0 group remained above 98%, with no statistically significant difference compared to the control group. These quantitative results are consistent with fluorescence microscopy observations, jointly confirming that both PBS and the nanohybrid fiber membrane loaded with 1.0 wt% ZnO have good cell compatibility and no cytotoxicity.

[0056] (6) Hemolysis test.

[0057] Physiological saline was used as a negative control, and deionized water as a positive control. Fresh anticoagulated rabbit blood was diluted with physiological saline to a 2.5% (v / v) red blood cell suspension. A 1 cm × 1 cm fibrin membrane was mixed with 2 mL of the suspension and incubated at 37°C for 1 h. Subsequently, the fibrin membrane was removed, and the mixture was centrifuged (2000 rpm, 10 min). The absorbance of the supernatant was measured at 545 nm using an ELISA reader. The hemolysis rate was calculated according to Formula IV. (Form IV) Among them OD S OD N and OD P The absorbance values ​​are for the sample group, negative control group, and positive control group, respectively.

[0058] The results are as follows Figure 9 As shown, the hemolysis rates of the pure PBS membrane and the PBS-ZnO-1.0 nanofiber membrane were 4.09±0.77% and 3.80±0.07%, respectively, which were not significantly different from the negative control value of 3.85±0.19%. According to the international standard (ISO 10993-4 / ASTM F756), a hemolysis rate of less than 5% is considered to meet the biosafety requirements. The hemolysis rates of both the pure PBS membrane and the PBS-ZnO-1.0 nanofiber membrane were below the 5% threshold, demonstrating their good blood compatibility.

[0059] Pure PBS electrospun membranes inherit the good biocompatibility of PBS and do not produce obvious hemolytic effects when in direct contact with red blood cells. Therefore, the introduction of ZnO not only endows the hybrid fiber membrane with antibacterial and cell migration-promoting functions, but also retains good blood compatibility, achieving a synergy between functionality and safety.

[0060] Example 3 The specific steps for preparing the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum are as follows: Preparation of S31 and PLLA-Cur fiber layers.

[0061] 1.0 g of poly-L-lactic acid (PLLA) was dissolved in 10 mL of hexafluoroisopropanol (HFIP) and magnetically stirred at room temperature (500 r / min, 12 h) until completely dissolved. 0.1 g of curcumin (Cur) was added to prepare an electrospinning solution. Electrospinning was performed using the following process: voltage 18 kV, feed speed 1 mL / h, nozzle moving distance 15 cm, distance between nozzle and receiving roller 15 cm, receiving roller rotation speed 2400 r / min, ambient temperature 30 °C, and ambient humidity 40%.

[0062] S32. Prepare PBS-ZnO / PLLA-Cur bilayer biomimetic bone membrane.

[0063] 1.5 g of PBS-ZnO-1.0 wt% (composite polyester) was added to 10 mL of HFIP solution, and then magnetically stirred at room temperature (500 r / min, 12 h) until completely dissolved to obtain an electrospinning solution with a concentration of 150 mg / mL. Electrospinning was then performed on the surface of the PLLA-Cur fiber layer obtained in step S31, using the same process as step S31, except that the receiving roller speed was adjusted to 300 r / min.

[0064] Using a PBS / PLLA fiber membrane as a control, the preparation method was as follows: 1.0 g of PLLA was dissolved in 10 mL of HFIP and magnetically stirred at room temperature (500 r / min, 12 h) until completely dissolved to prepare an electrospinning solution. The PLLA electrospinning membrane was obtained using the same process as in step S31. 1.5 g of PBS polyester was added to 10 mL of HFIP solution and then magnetically stirred at room temperature (500 r / min, 12 h) until completely dissolved to obtain an electrospinning solution with a concentration of 150 mg / mL. The PBS / PLLA fiber membrane was obtained by electrospinning on the PLLA electrospinning membrane using the same process as in step S32. A photograph of the prepared PBS-ZnO / PLLA-Cur bilayer biomimetic bone membrane (experimental group) is shown below. Figure 10 As shown.

[0065] Comparative Example 1 The specific steps for preparing PLLA fiber membranes are as follows: Dissolve 1.0 g PLLA in 10 mL HFIP and stir magnetically at room temperature (500 r / min, 12 h) until completely dissolved to obtain an electrospinning solution. Electrospinning can then be performed using the process described in step S31 of Example 3.

[0066] Comparative Example 2 The specific steps for preparing PLLA-Cur fiber membranes are as follows: Dissolve 1.0 g PLLA in 10 mL HFIP and stir magnetically at room temperature (500 r / min, 12 h) until completely dissolved. Add 0.1 g curcumin (Cur) to prepare an electrospinning solution and perform electrospinning using the process in step S31 of Example 3.

[0067] The physicochemical properties of the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum prepared in Example 3, and the fibrous membranes prepared in Comparative Examples 1 and 2, were characterized as follows: (1) FTIR testing was performed. The chemical structures of PLLA fiber membranes and PLLA-Cur fiber membranes were analyzed by FTIR. The results are as follows: Figure 11 As shown, both membranes are at 2997 cm⁻¹ -1 and 2947cm -1 The peak at 1747 cm⁻¹ shows the CH stretching vibration of methyl (-CH₃) and methylene (-CH₂-). -1 A strong absorption peak for the ester carbonyl group (C=O) appears at 1174 cm⁻¹, and reaches 1174 cm⁻¹. -1 and 1045cm -1 Stretching vibration peaks of CO in the ester bond were observed, which are typical characteristics of PLLA.

[0068] Furthermore, in PLLA-Cur electrospun membranes, at 1633 cm⁻¹ -1 and 1515cm -1 Two distinct characteristic peaks not present in PLLA were observed: the stretching vibration of the ketone carbonyl group (C=O) in Cur, and the coupled vibration of C=O and C=C in its aromatic ring skeleton. This result indicates that Cur has been successfully loaded into electrospun fibers.

[0069] (2) The prepared PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum, such as Figure 10 As shown in the figure, the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum is yellow, confirming the successful loading of Cur; at the same time, the bilayer membrane exhibits good overall flexibility. The morphology of the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum and the control group fibrous membrane were observed and statistically analyzed using SEM, and the results are as follows: Figure 12 (a)-(d) All samples formed a continuous, uniform, and bead-free fibrous structure. The average diameters of the PBS and PBS-ZnO-1.0 fiber membranes were 572.7±150.1 nm and 614.9±164.9 nm, respectively; the diameter of the PLLA fibers was 1117.1±202.1 nm, while the diameter of the PLLA-Cur fibers was slightly smaller, at 1029.5±147.8 nm. Figure 12(e)-(h)), which may be due to the addition of curcumin changing the polarity or surface tension of the spinning solution, thereby affecting the stretching and solidification process of the jet in the electric field.

[0070] (3) Mechanical property tests were conducted using the same method as in Example 2. The results are as follows: Figure 13 As shown, the control group membranes exhibited distinctly different mechanical behaviors: the PBS-ZnO-1.0 fiber membrane had an elongation at break of 79.5±1.9%, demonstrating soft tissue-like toughness, but with lower tensile strength (2.57±0.04 MPa); the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum, on the other hand, had higher tensile strength (22.82±1.44 MPa) and an elongation at break of approximately 49.1±5.7%. Notably, the mechanical properties of the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum fell between the two, with a tensile strength of 7.34±0.14 MPa and an elongation at break of 73.8±1.2%, indicating that the bilayer membrane achieved a synergistic improvement in both strength and toughness.

[0071] (4) Conduct contact angle testing, using the same method as in Example 2. The results are as follows: Figure 14 As shown, the contact angle of the PLLA layer on the outer side of the control group's fibrous membrane was 134.9±0.1°, and the contact angle of the PBS layer on the inner side was 129.6±0.8°. In contrast, the hydrophilicity of the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum was improved, with the contact angle of the inner side (PBS-ZnO-1.0) decreasing to 117.9±0.4°, and the contact angle of the outer side (PLLA-Cur layer) decreasing to 120.1±0.4°. The improved hydrophilicity of the inner PBS-ZnO layer facilitates early cell adhesion, nutrient penetration, and integration with surrounding soft tissues. The relatively hydrophobic PLLA-Cur layer on the outer side mimics the barrier and protective function of the fibrous layer, helping to maintain fibrous structural stability, regulate the sustained-release behavior of curcumin, and may regulate its interaction with surrounding soft tissues (such as the periosteum and muscle), preventing excessive adhesion.

[0072] (5) Curcumin simulated in vitro release experiment.

[0073] A PBS solution containing 1% (w / v) Tween-80 (pH 7.4) was used as the release medium. A series of Cur standard solutions with concentrations (2.5, 5, 7.5, 10, and 15 μg / mL) were prepared. The absorbance was measured at the maximum absorption wavelength of 425 nm using a UV-Vis spectrophotometer, and a standard curve was plotted to obtain the linear equation Y = aX + b.

[0074] A 1cm × 1cm PBS-ZnO / PLLA-Cur bilayer biomimetic bone membrane was placed in a centrifuge tube, and 1.5 mL of the prepared release medium was added. Subsequently, the membrane was taken out at 1, 3, 6, 12, 24, 36, 48, 72, 96, 120, 144 and 168 h, respectively, and the absorbance of the corresponding release medium was measured. The cumulative release concentration at each time point was calculated according to the standard curve.

[0075] The results are as follows Figure 15 As shown, curcumin release exhibits a kinetic pattern of rapid initial release followed by slow, sustained release. Within the first 12 hours, Curcumin is released rapidly, with the cumulative release rate increasing rapidly from 28.3% at 1 hour to 56.6% at 24 hours. This is likely due to the rapid diffusion of Curcumin loaded on or near the surface of the outer PLLA-Cur fibers upon contact with the release medium. In the middle and later stages (12-120 hours), the release tends to level off, with the final cumulative release rate reaching 66.9%. This release kinetic is attributed to the large specific surface area and good pore connectivity of the electrospun fibers, as well as the excellent sustained-release properties of PLLA as a carrier. In the early stages of bone defect healing, reactive oxygen species (ROS) are generated in large quantities along with the inflammatory response. Excessive oxidative stress can hinder osteogenic differentiation and exacerbate inflammation. The PBS-ZnO / PLLA-Cur bilayer biomimetic bone membrane prepared in this invention, through rapid initial release of Curcumin, can promptly scavenge free radicals, exerting a synergistic anti-inflammatory and antioxidant effect, creating favorable conditions for bone regeneration.

[0076] (6) In vitro antioxidant experiment.

[0077] Accurately weigh 3.94 mg of DPPH reagent and dissolve it in 100 mL of anhydrous ethanol to obtain a 0.1 mM DPPH ethanol solution, which should be stored in the dark. Immerse a 1 cm × 1 cm double-layer electrospun membrane in 3 mL of DPPH ethanol solution and react at room temperature in the dark. Remove the electrospun membrane at 2, 4, 8, 12, 20, and 30 mins after immersion, and measure the absorbance of the solution at 517 nm using a UV-Vis microplate reader; calculate the DPPH scavenging efficiency (SA%) using formula V. (Formula V) Where A B and A S The absorbance values ​​are represented by 0.1 mM DPPH solution and DPPH solution after soaking the sample for different times.

[0078] The results are as follows Figure 16As shown, the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum exhibits highly efficient and sustained antioxidant properties, achieving a DPPH scavenging rate of 79.5±0.9% within 30 min. The kinetic process shows a rapid initial growth followed by a slower growth: the scavenging rate reaches 41±1.5% within the first 2 min, indicating rapid release of surface-loaded Cur; from 4 to 20 min, the scavenging rate rapidly increases from 59.5±1.1% to 77.9±0.9%, reflecting the continuous release of Cur from within the fibers and its participation in free radical scavenging; subsequently, the rate of increase in scavenging efficiency slows down and eventually approaches saturation. These results confirm that Cur can maintain excellent antioxidant activity within the electrospun fiber matrix, and the sustained-release characteristics based on the fiber structure enable the material to continuously eliminate free radicals, thus providing an important functional basis for effectively regulating local oxidative stress levels and promoting osteogenic repair in the early stages of bone repair.

[0079] (7) Intracellular reactive oxygen species scavenging experiment.

[0080] The intracellular ROS scavenging capacity of MC3T3-E1 cells with a double membrane was detected using a reactive oxygen species (ROS) assay kit. The specific steps are as follows: MC3T3-E1 cells were loaded at 2 × 10⁶ cells per well. 4 MC3T3-E1 cells were seeded at a density of 1000 mcg / mL in 24-well plates and cultured for 24 hours to allow for complete adherence. The original FBS-containing complete medium was then discarded and replaced with FBS-free complete medium containing Rosup (1 μg / mL) to stimulate ROS production for 2 hours. Next, 1 cm × 1 cm fiber membranes were placed in the wells and co-incubated with the cells for 6 hours. Untreated cells served as a positive control, and cells treated with Rosup alone served as a negative control. After washing twice with PBS, the cells were stained with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) at 37°C in the dark for 20 minutes. Finally, the samples were incubated with 4',6-diamino-2'-phenylindole (DAPI) staining solution at 37°C for 5 minutes and observed using a fluorescence microscope.

[0081] The results are as follows Figure 17 As shown, the fluorescence intensity of cells treated with the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum was significantly reduced, indicating that this material can effectively scavenge ROS induced by Rosup and exhibits significant intracellular antioxidant activity. In contrast, the control group fibrous membrane did not show obvious ROS scavenging ability. These results confirm at the cellular level that the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum possesses excellent antioxidant function and can effectively alleviate cell damage caused by oxidative stress, providing direct evidence for its role in regulating the oxidative microenvironment during bone repair.

[0082] (8) In vitro antibacterial performance test. The method is the same as (4) in Example 2, and the results are as follows: Figure 18 As shown, the control group fiber membrane... E. coli and S.aureus The antibacterial rates of all groups were below 5.5%, while the experimental group... E. coli and S.aureus The antibacterial rate of all of them reached over 98%.

[0083] (9) Cell compatibility test. The method was the same as (5) in Example 2, and the results were as follows. Figure 19 As shown, after 3 days of culture, cells in both the control group's fibrous membrane and the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum proliferated well, exhibiting typical spindle-shaped and polygonal morphologies. Green fluorescence (live cells) dominated the field of view, with only a very small amount of red fluorescence (dead cells). The live / dead staining results clearly indicated that the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum did not adversely affect cell survival. Subsequently, the CCK-8 assay was used to quantitatively evaluate cell proliferation activity. On day 1 of culture, the cell viability of the control group's fibrous membrane and the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum were 99.4% and 97.2% of the control group (no sample added, only an equal volume of culture medium added), respectively, indicating good cell adhesion to the material surface in the initial stage. As the culture time was extended to day 3, the cell viability of both groups further increased, and there was no statistically significant difference compared to the control group. The combined results of live / dead staining and CCK-8 assays indicate that the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum did not produce toxicity to MC3T3-E1 cells, and the cells were able to adhere well to the material surface, spread out, and proliferate continuously.

[0084] (10) Hemolysis test. The method is the same as (6) in Example 2, and the results are as follows. Figure 20 As shown, the hemolysis rates of the control group's fibrous membrane and the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum were 4.10±0.07% and 4.57±0.28%, respectively, which were not significantly different from the negative control group (physiological saline, 4.22±0.33%). According to international standards, the hemolysis rates of both were below the safety threshold of 5%, indicating that they do not possess hemolytic activity. The experimental results demonstrate that the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum has reliable blood safety.

[0085] This demonstrates that the present invention, through electrospinning and multilayer structure design, endows the material with bioactivity without compromising its essential blood safety as a bone repair implant, providing crucial safety evidence for its further application in blood-rich bone defect environments.

[0086] (11) Evaluation of in vitro osteogenic differentiation of PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum.

[0087] Rat bone marrow mesenchymal stem cells (BMSCs) were used in osteogenic differentiation induction medium (a-mem medium + 10% FBS + 1% penicillin-streptomycin + 100 μM dexamethasone + 50 μM vitamin C + 10 μM β-glycerophosphate sodium), and the cell resuscitation steps were the same as in Example 2 (5).

[0088] Sample sterilization: After sterilizing with ultraviolet light in a clean bench for 30 minutes, place the sample (1cm×1cm) into a 24-well plate, soak it in PBS solution and irradiate it under ultraviolet light for 1 hour; after washing, add complete culture medium and place it in a cell culture incubator for later use.

[0089] The results are as follows Figure 21 As shown, compared with the control group fibrous membrane, the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum exhibits a darker and denser purplish-black area, indicating that the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum has good osteogenic differentiation ability.

[0090] (12) Alkaline phosphatase (ALP) staining experiment.

[0091] Alkaline phosphatase (ALP) is an early marker of osteogenic differentiation. This experiment used the BCIP / NBT alkaline phosphatase colorimetric kit to detect ALP expression levels. Quantitative analysis results of ALP staining intensity (…) Figure 21 The staining results were consistent with those in (11), further confirming that the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum can more effectively promote early osteogenic differentiation of BMSCs.

[0092] (13) Alizarin Red staining experiment.

[0093] Calcium nodules are products of extracellular matrix mineralization during osteogenic differentiation and maturation, and are one of the hallmarks of osteogenic differentiation. Alizarin Red dye can specifically bind to calcium ions to form an orange-red complex, thus enabling the staining and observation of calcium nodules. The specific steps are as follows: First, the cell-fiber membrane complexes cultured for 14 days after osteogenic induction differentiation were fixed. Then, 500 μL of alizarin red staining solution was added to each well and incubated at room temperature for 10 min. The samples were washed three times with PBS to remove unbound dye. Finally, the staining effect of calcium nodules was observed under an optical microscope.

[0094] The results are as follows Figure 22As shown, compared with the control group fibrous membrane, the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum exhibited a deeper and more extensive orange-red positive area, indicating a significant increase in calcium nodule deposition. Quantitative analysis further confirmed that the ARS staining intensity on the PBS-ZnO / PLLA-Cur bilayer biomimetic periosteum was significantly higher than that on the control group fibrous membrane, indicating that it effectively promotes late osteogenic differentiation and mineralization of BMSCs.

[0095] Therefore, this invention provides a nano-hybrid fiber with uniform diameter and well-entangled three-dimensional network; it also provides a bilayer biomimetic periosteum with a continuous porous network structure. Both the provided nano-hybrid fiber and bilayer biomimetic periosteum possess multiple functions, including excellent strength and toughness, good surface hydrophilicity, sustained-release effect, anti-inflammatory, antioxidant, antibacterial, biocompatibility, and ability to promote bone differentiation and mineralization, and the hemolysis rate meets international standards. This provides a new material system for the clinical translation of functional bone repair materials. The provided preparation method is simple and easy to operate, and materials with different performance advantages can be obtained by adjusting the component ratio, making it suitable for industrial production. It can effectively solve the problems of nanofiller aggregation and interfacial compatibility, enabling uniform dispersion and stable bonding of each component in the material.

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A nano-hybrid fiber, characterized in that: The nano-hybrid fiber is spun from a composite polyester; the composite polyester is prepared from 1,4-butanediol, ZnO powder, succinic acid, and tetrabutyl titanate; the molar ratio of 1,4-butanediol to succinic acid is (1.05~1.2):1; the amount of zinc oxide added is 0.5%~1.5% of the total mass of 1,4-butanediol and succinic acid; the amount of tetrabutyl titanate added is 0.1%~0.2% of the molar mass of succinic acid.

2. A method for preparing nano-hybrid fibers as described in claim 1, characterized in that, The steps are as follows: S1. Mix 1,4-butanediol and ZnO powder and sonicate. After sonication, continue stirring for a period of time to obtain a suspension. S2. Add the suspension obtained in S1 and succinic acid to a four-necked flask and react until the water output reaches more than 90% of the theoretical value. Then, heat to 200-250℃ and maintain at a vacuum of 500-1000Pa for 0.5-1h. Then, reduce the vacuum to below 50Pa and continue the reaction for 2-3h. Purge with protective gas until the system temperature drops to 140-160℃ and discharge. After cooling, hot press to obtain composite polyester. S3. Dissolve the composite polyester obtained in S2 in hexafluoroisopropanol to obtain an electrospinning solution, and electrospin to obtain nano-hybrid fibers.

3. The method for preparing a nano-hybrid fiber according to claim 2, characterized in that: In S1, the ultrasonic treatment is performed at 30-50 kHz and 10-20 W for 30-40 minutes, and the stirring is performed at 300-500 rpm for more than 12 hours.

4. The method for preparing a nano-hybrid fiber according to claim 2, characterized in that: In S2, the four-necked flask is connected to a mechanical stirrer, a condenser, an inert gas protection device, and a glass stopper. The reaction conditions after connecting the four-necked flask are heating, stirring speed of 350-450 r / min, protective atmosphere, and heating from 130°C to 180°C.

5. The method for preparing a nano-hybrid fiber according to claim 2, characterized in that, In step S2, hot pressing is performed at 130-150℃ and 0.5-1MPa. In step S3, the concentration of composite polyester in the electrospinning solution is 100-150mg / mL. The electrospinning parameters are: voltage 15-20kV, feed speed 1.0-1.2mL / h, needle movement distance 10-15cm, distance between needle and receiving roller 10-15cm, rotation speed of receiving roller 200-300r / min, ambient temperature 30-40℃, and ambient humidity 30-40%. After spinning in step S3, the obtained fiber / fiber membrane is dried in an oven at 37℃ for no less than 24h.

6. The application of the nano-hybrid fibers according to claim 1 in bone repair.

7. A double-layered biomimetic periosteum, characterized in that, Made from the nano-hybrid fiber described in claim 1, the preparation steps are as follows: (1) After dissolving poly-L-lactic acid in hexafluoroisopropanol, curcumin was added and dissolved to obtain an electrospinning solution. Electrospinning was then performed to obtain a PLLA-Cur fiber layer. (2) Dissolve the composite polyester of claim 1 in hexafluoroisopropanol to obtain an electrospinning solution, and spin it on the PLLA-Cur fiber layer obtained in (1) to obtain a double-layer biomimetic endothelial membrane.

8. The double-layered biomimetic periosteum according to claim 7, characterized in that, The mass-to-volume ratio of poly-L-lactic acid:hexafluoroisopropanol:curcumin in the electrospinning solution prepared in (1) is 10g:0.1L:1g; the electrospinning parameters are: voltage 15-20kV, feed speed 1.0-1.2mL / h, nozzle moving distance 10-15cm, distance between nozzle and receiving roller 10-15cm, rotation speed of receiving roller 2400r / min, ambient temperature 30-40℃, and ambient humidity 30-40%.

9. The double-layered biomimetic periosteum according to claim 7, characterized in that, The concentration of nano-hybrid fibers in the electrospinning solution prepared in (2) is 100-150 mg / mL; the electrospinning parameters are: voltage 15-20 kV, feed speed 1.0-1.2 mL / h, nozzle moving distance 10-15 cm, distance between nozzle and receiving roller 10-15 cm, rotation speed of receiving roller 300 r / min, ambient temperature 30-40 ℃, and ambient humidity 30-40%.

10. The application of the double-layer biomimetic periosteum as described in claim 7 in bone repair.

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