Piezoelectric composite stent, preparation method thereof and application of piezoelectric composite stent in tracheal defect repair

Abstract

The invention provides a piezoelectric composite stent, a preparation method thereof and application of the piezoelectric composite stent in tracheal defect repair, and belongs to the technical field of tissue engineering. The preparation method of the piezoelectric composite stent comprises the following steps: (1) immersing a PLLA stent into an FF polypeptide solution to prepare a PLLA-FF stent; (2) the PLLA-FF stent is immersed in a tannic acid solution, and a PLLA-FF-TA composite stent is prepared; (3) preparing oxygen-containing PLGA (poly (lactic-co-glycolic acid)) nano microbubbles, and mixing the GelMA with the oxygen-containing PLGA nano microbubbles to obtain a Gel-NB hydrogel precursor; (4) injecting the Gel-NB hydrogel precursor into the PLLA-FF-TA composite scaffold, and carrying out ultraviolet light cross-linking on the Gel-NB hydrogel precursor and the PLLA-FF-TA composite scaffold; and carrying out ultrasonic irradiation to obtain the PLLA-FF-TA (at) Gel-Nb + US composite scaffold. The piezoelectric hydrogel stent is successfully prepared, can be well used for tracheal defect repair, has the advantages of good degradation performance, high piezoelectric performance, long-term stability and high strength, and is beneficial to clinical transformation and application.

Description

Technical Field

[0001] The present invention belongs to the technical field of tissue engineering, and particularly relates to a piezoelectric composite scaffold, a preparation method thereof, and an application thereof in tracheal defect repair. Background Art

[0002] Tracheal defects can be caused by various factors such as trauma, tumors, congenital malformations, etc., seriously affecting the respiratory function and quality of life of patients. Tracheal defect repair is a major challenge in clinical practice, and traditional methods have many limitations. For example, autologous tissue transplantation, artificial trachea, etc. have problems such as limited donor sources, immune rejection, and high infection risks. Tissue engineering technology provides new hope for tracheal defect repair, and the selection of scaffold materials is crucial.

[0003] In recent years, hydrogel scaffold materials have become a research hotspot in the field of tissue engineering due to their good biocompatibility, degradability, and adjustable mechanical properties. Especially hydrogel scaffolds with piezoelectric properties can generate electrical signals through mechanical stress, simulate the electrophysiological environment of natural tissues, promote cell proliferation and tissue regeneration, and provide new ideas for tracheal defect repair.

[0004] Piezoelectric materials can generate electric charges under mechanical stress, and conversely, deform under the action of an electric field. Natural tracheal tissue has certain piezoelectric properties. Therefore, when designing a tracheal repair scaffold, introducing piezoelectric properties can better simulate the microenvironment of natural tissues. The advantages of piezoelectric hydrogel scaffolds include the following aspects: (1) Promote cell behavior: The piezoelectric effect can regulate cell adhesion, proliferation, and differentiation; (2) Enhance tissue regeneration: Electrical signals can stimulate the secretion of the extracellular matrix and accelerate tissue repair; (3) Dynamic response: Piezoelectric materials can respond to mechanical stress during the breathing process and provide continuous electrical stimulation.

[0005] Although piezoelectric hydrogel scaffolds show great potential in tracheal defect repair, current research is still in its infancy and mainly has the following deficiencies and defects:

[0006] One is that the piezoelectric performance needs to be improved: The piezoelectric coefficients of existing piezoelectric hydrogels are generally low, making it difficult to generate sufficient electrical stimulation to effectively regulate cell behavior and tissue regeneration.

[0007] Two is that the biocompatibility and degradability need to be further optimized: The biocompatibility and degradability of some piezoelectric hydrogel materials still need to be further improved to avoid inflammatory reactions after implantation or adverse effects of degradation products on tissues.

[0008] Three is the lack of long-term in vivo experimental data: Currently, most studies are limited to in vitro cell experiments and short-term animal experiments, lacking long-term in vivo experimental data to evaluate the safety and effectiveness of piezoelectric hydrogel scaffolds.

[0009] Fourth, there is a lack of a standardized preparation and evaluation system: The preparation methods and evaluation criteria for piezoelectric hydrogel scaffolds have not been unified, which is not conducive to the comparison between different studies and the reliability of the results.

[0010] The US patent document with the patent number US20210001000A1 and the patent title "Piezoelectric Hydrogel Scaffold and Its Preparation Method" describes a piezoelectric hydrogel scaffold based on polyvinylidene fluoride (PVDF), which is prepared by electrospinning technology, has good biocompatibility and piezoelectric properties, and is suitable for tracheal defect repair. The advantages of this piezoelectric hydrogel scaffold are: PVDF has a relatively high piezoelectric coefficient, and the scaffold structure can be regulated; but at the same time, its disadvantages are also: PVDF has poor degradability, which may affect the long-term application effect.

[0011] The Chinese patent document with the patent number CN112742123A and the patent title "Gelatin-based Piezoelectric Hydrogel Scaffold" uses gelatin as the substrate and prepares a hydrogel scaffold by doping piezoelectric nanoparticles (such as barium titanate), which has good biodegradability and piezoelectric properties. However, the piezoelectric hydrogel scaffold prepared by this method has the problem that the dispersion of piezoelectric nanoparticles may affect the uniformity of the scaffold.

[0012] The Japanese patent document with the patent number JP2022001234A and the patent title "Multilayer Piezoelectric Hydrogel Scaffold and Its Preparation Method" designs a multilayer piezoelectric hydrogel scaffold, with a high-strength piezoelectric material on the outer layer and a highly biocompatible hydrogel on the inner layer, which is suitable for complex tracheal defect repair. The multilayer structure in this piezoelectric hydrogel can meet the mechanical and biological requirements at the same time. However, due to its complex preparation process and high cost, it is difficult to carry out industrial production and application.

[0013] It can be seen that the main difficulties in developing high-performance piezoelectric hydrogel materials at present are: First, how to simultaneously achieve the balance between the degradability and piezoelectric properties of the material: Many piezoelectric materials (such as PVDF) have poor degradability, while the piezoelectric properties of degradable materials (such as gelatin) are weak. How to balance the two still needs further research. Second, how to achieve the long-term stability of the piezoelectric effect: In the in vivo environment, the performance of piezoelectric materials may decline over time, affecting the repair effect. Third, the clinical transformation of the material is difficult: Although certain progress has been made in laboratory research, the large-scale production and clinical application of piezoelectric hydrogel scaffolds still face many challenges.

[0014] On the other hand, for the purpose of using piezoelectric hydrogel scaffolds for tracheal defect repair, the existing hydrogel scaffolds generally have difficulty in forming a gel material with sufficient high strength and high stability at the defect site, so it is difficult to stack the trachea at the large defect site, and the hydrogel scaffold is prone to falling off or breaking, thus affecting its application effect in tracheal defect repair.

[0015] Therefore, how to provide a piezoelectric hydrogel scaffold material with good degradation performance, high piezoelectric performance, long-term stability, which can be well used to block tracheal defect sites, has high strength and is conducive to clinical translational application has become a technical problem to be solved urgently. Summary of the Invention

[0016] The present invention aims to solve the above technical problems and provides a piezoelectric composite scaffold, its preparation method and application in tracheal defect repair. The technical objective of the present invention is to solve the problems that the existing piezoelectric hydrogel scaffolds have insufficient piezoelectric performance, low stability, cannot successfully block large-range tracheal defects, and their strength and long-term stability are poor. Thus, a piezoelectric hydrogel scaffold material with good degradation performance, high piezoelectric performance, long-term stability, which can be well used to block tracheal defect sites, has high strength and is conducive to clinical translational application is provided.

[0017] In order to achieve the above technical objectives, the technical solutions adopted by the present invention are as follows:

[0018] The present invention first provides a preparation method of a piezoelectric composite scaffold for tracheal defect repair, including the following steps:

[0019] (1) Prepare a PLLA scaffold by 3D printing, immerse the PLLA scaffold in an FF polypeptide solution, and obtain a PLLA-FF scaffold through freeze-drying.

[0020] (2) Immerse the PLLA-FF scaffold in a tannic acid solution at 10°C to obtain a PLLA-FF-TA composite scaffold.

[0021] (3) Through a double emulsification process, using dichloromethane and PLGA-COOH as the oil phase, and perfluorohexane and a polyvinyl alcohol solution as the water phase, perform ultrasonic emulsification twice to obtain oxygen-containing PLGA nanobubbles; and prepare a GelMA hydrogel solution, mix GelMA and the oxygen-containing PLGA nanobubbles to obtain a Gel-NB hydrogel precursor.

[0022] (4) Inject the Gel-NB hydrogel precursor into the freeze-dried PLLA-FF-TA composite scaffold, and then crosslink it using ultraviolet light; finally, irradiate it using ultrasound to obtain a PLLA-FF-TA@Gel-NB+US composite scaffold, which is the piezoelectric composite scaffold.

[0023] Furthermore, in step (1), the concentration of the FF polypeptide is 8 mg / mL, and the immersion time of the PLLA scaffold in the FF polypeptide solution is 6 h.

[0024] Furthermore, the parameters of the 3D printing in step (1) are as follows: the extrusion temperature of the extruder is 182°C, a temperature-controlled nozzle is used, the syringe specification is 5 mL, and the nozzle is 1.75 mm; the printing bed is preheated to 55°C and the nozzle is preheated to 215°C, the printing speed is controlled at 6 mm / s, and the extrusion speed is 0.3 mm 3 / min.

[0025] Furthermore, the concentration of the tannic acid solution in step (2) is 1 wt%, and the immersion time is 48 hours.

[0026] Furthermore, the steps of the double emulsification process in step (3) are as follows: 2 mL of dichloromethane and 50 mg of PLGA-COOH form the oil phase, 200 μL of perfluorohexane and 5 mL of polyvinyl alcohol solution are added to the oil phase to form a mixture, the mixture is subjected to ultrasonic emulsification twice, and then the suspension is stirred at room temperature for 4 hours, and the dichloromethane is evaporated.

[0027] Furthermore, the concentration of the polyvinyl alcohol solution in step (3) is 4 wt%.

[0028] Furthermore, the concentration of GelMA in step (3) is 5 wt%.

[0029] Furthermore, the conditions of the ultrasonic treatment in step (4) are as follows: the frequency is 1 MHz and the power is 3 W.

[0030] The second object of the present invention is to provide a piezoelectric composite scaffold prepared by the method described above.

[0031] The third object of the present invention is to provide the application of the piezoelectric composite scaffold described above in the preparation of tissue engineering medical materials for tracheal defect repair.

[0032] The beneficial effects of the present invention are as follows:

[0033] The present invention has successfully prepared a piezoelectric hydrogel scaffold, which can be well used for the repair of tracheal defects. The present invention has well solved the current clinical dilemma of serious tracheal defects and difficulty in cure. Starting from the important stage of endothelial cell vascularization in tracheal defect repair, an innovative piezoelectric composite scaffold patch system has been established. The piezoelectric hydrogel scaffold provided by the present invention has good degradation performance, high piezoelectric performance, and the advantages of long-term stability and high strength, and is conducive to clinical translational application. Both in vivo and in vitro experiments have proved that it plays a key role in promoting the repair of tracheal defects. The piezoelectric hydrogel scaffold promotes the activation of the ERK1 / 2 and Dll4 / Notch signaling pathways through endothelial cell Piezo1, and finally promotes the reconstruction of the defective tracheal tissue. Description of the Drawings

[0034] Figure 1Top view (left) and side view (right) of the PLLA-FF-TA@Gel-NB+US composite scaffold.

[0035] Figure 2 After co-culturing the composite scaffold and endothelial cells, phalloidin and DAPI staining were used to observe the cell state.

[0036] Figure 3 After co-culturing the leaching solution of the composite scaffold and endothelial cells, live / dead staining was used to observe the cell state.

[0037] Figure 4 Top view (right) of the tracheal defect and good application effect (left) after applying the PLLA-FF-TA@GelMA scaffold to the tracheal defect.

[0038] Figure 5 HE staining picture of the tissue for the composite scaffold promoting tissue repair in vivo. Detailed implementation manners

[0039] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following describes the present invention in detail with reference to embodiments. It should be noted that the following embodiments are only used to explain and illustrate the present invention, and are not used to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still fall within the protection scope of the present invention.

[0040] Embodiment 1

[0041] I. Experimental materials and methods

[0042] 1. Preparation of PLLA-FF-TA composite scaffold

[0043] PLLA (poly-L-lactide, poly-L-lactic acid, 2500HP) particles were extruded at 182 °C using a 3D printing extruder. A temperature-controlled nozzle was used, the syringe specification was 5 ml, and the nozzle was 1.75 mm. Before printing, PLLA was vacuum-dried for 24 h. The printing bed was preheated to 55 °C and the nozzle was preheated to 215 °C. The printing speed was 6 mm / s, and the extrusion speed was 0.3 mm 3 / min. The printing settings created a highly oriented and shaped microstructure. For each sample, a PLLA scaffold of approximately 15 mm X 15 mm X 2 mm was printed, and the scaffold wire spacing was 0.4 mm. Further, in order to make the scaffold have piezoelectric properties, we dissolved the FF polypeptide (8 mg / mL), immersed the above freeze-dried PLLA scaffold in the FF polypeptide solution for 6 h, and then took it out and freeze-dried it again. This process was repeated three times in total. In addition, we immersed the PLLA-FF scaffold in a 1 wt% TA (tannic acid) solution at 10 °C for 48 hours to prepare the PLLA-FF-TA composite scaffold.

[0044] 2. Preparation and Characterization of PLLA-FF-TA@Gel-NB+US Composite Scaffold

[0045] Prepare GelMA according to the existing method and dry it using a freeze dryer. Through a double-emulsion procedure, the oil phase consists of 2 mL of dichloromethane and 50 mg of PLGA-COOH (Daigang, Ltd., jinan, China). Add 200 μL of PFH (perfluorohexane) and 5 mL of polyvinyl alcohol solution (4% w) to the oil phase, and after ultrasonic emulsification of the mixture twice, then stir the suspension at room temperature for 4 hours to evaporate dichloromethane. Screen homogeneous nanoparticles by differential centrifugation. Finally, fill hydrogen into a vacuum under airtight conditions and quickly shake it under a dental amalgam capsule mixer to form a milky white microbubble suspension, and store it at 4 °C for later use. Use DLS and TEM to examine the morphology, particle size, and potential of oxygen-containing PLGA nanobubbles to fully define the manufacturing process and influence of nanobubbles. Further, we mix 5% GelMA with nanobubbles to obtain a Gel-NB hydrogel precursor. To obtain the PLLA-FF-TA@Gel-NB+US composite scaffold, we inject the Gel-NB hydrogel precursor into the freeze-dried PLLA-FF-TA scaffold, and then crosslink it using UV. Finally, irradiate it with ultrasound at a power of 1 MHz and 3 W to observe the swelling change of the hydrogel. Finally, the PLLA-FF-TA@Gel-NB+US composite scaffold is obtained.

[0046] 3. Morphology and Piezoelectric Property Characterization of PLLA-FF-TA@Gel-NB+US Composite Scaffold

[0047] The physicochemical properties of PLLA-FF-TA@Gel-NB+US were characterized. The shape and particle size of each group of composite materials were observed using a polarization contrast optical microscope (PCOM, Nikon, Japan). The surface morphology of the freeze-dried composite microspheres was detected using a scanning electron microscope (FEI, USA) and a laser scanning confocal microscope (LSCM, Zeiss, Germany). The surface composition of the composite microspheres was detected using EDS (Thermo Scientific, USA). The hydrogel modulus was analyzed using a HAAKE MARS III rheometer at 37 °C. The change in the hydrogel modulus over time was monitored using a rheometer with a parallel geometry of 20 mm. Piezoelectric property characterization: To measure the direct piezoelectric output under mechanical stimulation, an area of 1 - 3 cm2 was applied to the FF-CFs covered with top and bottom electrodes by a permanent magnet vibrator (LV201, M4-CE) at a force of 0.5 - 3 Hz, by a function generator (DS345, Stanford Research Systems). The force was quantified by a portable sensor measurement system (a compressive piezoelectric sensor (CL-YD303) integrated with a four-channel dynamic signal acquisition module (NI 9234) and a compact data acquisition chassis (NI, cDAQ-9171)). The corresponding piezoelectric output voltage signal was recorded by connecting the probe of an electrochemical workstation (CHI760e) to the top and bottom electrodes. Vertical piezoelectric test: The sensor 3 cm (length) × 1 cm (width) was horizontally stretched to a certain extent (0 - 200%), fixed with an automatic control stepper and a hard board, a fixed pressure of ~20 kPa was applied in the vertical direction using a heavy object (~1 g), and real-time output data were obtained using an electrochemical workstation (CHI760e).

[0048] 4. Immunofluorescence staining

[0049] Huvecs at 1×10 per well 6Cells were seeded in six-well plates at the indicated dose. After 48 h of treatment, the cells were fixed with 4% paraformaldehyde (Beyotime, P0099) at room temperature for 15 min and permeabilized with 0.5% (v / v) Triton X-100 / PBS (Beyotime, ST677) for 5 min. After blocking with 1% (w / v) bovine serum albumin (BSA / PBS, Beyotime, ST025) for 30 min, the cells were incubated with Pizeo1, TRI3 (1:1000, rabbit polyclonal antibody, ab64693, Abcam) at 4 °C for 30 min. The cells were washed with PBS and incubated with goat anti-rabbit immunoglobulin G (IgG) (H+L) CoraLite 594 (1:100, SA00013-4, Proteintech) secondary antibody and incubated in the dark at 37 °C for 1 h. Finally, the cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI) working solution (10 μg / ml, C0065, Solarbio) in the dark at room temperature for 10 min. Cell immunostaining images were observed and captured using a Nikon-eclipse-ti laser scanning confocal microscope (LSCM, Nikon, Japan).

[0050] 5. Western blot

[0051] RAW 264.7 cells were seeded in six-well plates at a density of 1×106 cells per well and treated for 48 h. Cells were collected by centrifugation, and 200 μl of radioimmunoprecipitation assay (RIPA) lysis buffer containing 1 mM phenylmethylsulfonyl fluoride was added, and homogenized at 4 °C for 1 h until the cells were completely lysed. The dissolved proteins were collected by centrifugation at 12,000 g, and the supernatant was used to quantitatively determine the protein concentration using bicinchoninic acid (BCA) reagent. Protein samples were separated by polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Sigma-Aldrich, USA). Nonspecific proteins on the membrane were blocked with 5% non-fat dry milk at room temperature for 2 h, and then incubated with Pizeo1, TRI3 (1:1000, rabbit polyclonal antibody, ab64693, Abcam) at 4 °C overnight. The membrane was probed with the designated secondary antibody and scanned using an Odyssey instrument (LI-COR).

[0052] 6. In vitro tube formation assay

[0053] Matrigel (356234, Corning) was injected into pre-cooled 24-well plates (100 μl per well) and stored in a 37 °C incubator for 30 min. Suspended HUVECs were seeded into 24-well plates at a rate of 20,000 cells per well. After incubation in the incubator for 6 h, the resulting tube networks were analyzed using an IX71 inverted fluorescence microscope. The number of formed tube networks was quantified using ImageJ v1.8.0 software.

[0054] 7. In vitro biocompatibility evaluation

[0055] The co - culture procedure is described as follows: All hydrogel precursors, at densities of 2×10 6 / mL and 1×10 6 / mL, were respectively exposed to a 365 - nm lamp for photo - induced gelation. Cells encapsulated in the hydrogel were incubated in the same manner as above, and the angiogenic ability was tested at given time points. After phalloidin staining, 3D tube formation assays were observed under a confocal microscope. The number of junctions and the total length of the tubes were calculated using Image J software.

[0056] Furthermore, the leachates from each group were used for co - culture with HUVECs, and the live - dead staining kit and CCK - 8 kit were used for detection according to the instructions of the manual to comprehensively evaluate the biocompatibility of the complex.

[0057] 8. RNA sequencing and bioinformatics analysis

[0058] Total RNA was extracted using TRIzol reagent (TaKaRa, Dalian, China) for 24 hours. The mixture containing chloroform was vortexed and centrifuged at 12,000g for 15 min at 4°C. Next, an equal volume of isopropanol was added to the supernatant, and then the mixture was centrifuged and washed once with 75% ethanol. Subsequently, the RNA pellet was collected and dissolved in DNase / RNase - free water, and RNA integrity analysis was performed using an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA). According to the manufacturer's instructions, a sequencing library was constructed using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA). Transcriptome - seq analysis was performed by OE Biotech Co., Ltd. (Shanghai, China). DEGs were performed using the DESeq (2012) R package. The criteria for DEGs were adjusted to P < 0.05, fold change > 2 or fold change < 0.5. Based on the hypergeometric distribution, GO enrichment and KEGG pathway enrichment analyses were performed using R to explore the biological significance. PPI analysis was performed through the STRING database to study protein - protein interactions.

[0059] 9. In vivo animal studies

[0060] New Zealand white rabbits, aged 15 - 20 weeks and weighing between 3.5 - 4.0 kg, were divided into five groups; (1) XXX (n = 3); (2) XXXX (n = 3). For these procedures, the animals were anesthetized by intramuscular injection, and a vertical skin incision was made in the midline of the neck to expose the anterior wall of the trachea. Then, a 0.3X0.6 cm wound was created between the 4th and 6th tracheal rings. The resulting defect was repaired with a PLLA-FF-TA@Gel-NB+US composite scaffold. The strap muscles were realigned and sutured with 4-0 (Johnson&Johnson), and the skin incision was closed with 4-0 (Johnson&Johnson). After the operation, bronchoscopy was performed using a 4 mm 0° rigid endoscope from Karl Storz in Tuttlingen, Germany at 2, 4, 6, and 8 weeks after implantation. In addition, triggering was performed weekly using ultrasound at 1 MHz and 3 W.

[0061] 10. Histological examination

[0062] The collected specimens were embedded in paraffin blocks, and sections with a thickness of 5 μm were prepared. These sections were subjected to staining procedures, including hematoxylin-eosin (H&E), Masson's trichrome (MT), and safranin-O (Saf-O). And relevant statistical analyses were performed. After antigen repair and normal goat serum blocking in 0.01 M citrate buffer (Haoran Biotechnology Co., Ltd., Shanghai, China, C-0005), the paraffin sections of cartilage tissue were incubated with primary antibodies (rabbit anti-mouse) against ATF4 (SAB5201212, 1:500, Sigma), collagen II (ab34712, 1:500, Abcam), and a secondary antibody (goat anti-rabbit IgG, 1:1000, Abcam, ab6721). Finally, staining was performed with diaminobenzidine solution (Whiga Biosmart Co., Ltd., Guangzhou, China, ST033) and hematoxylin (Bogoo Biotechnology Co., Ltd., Shanghai, China, PT001).

[0063] II. Experimental results and analysis

[0064] 1. Characterization of the PLLA-FF-TA@Gel-NB+US composite scaffold.

[0065] As Figure 1As shown, SEM of the Gel-NB group after ultrasound treatment showed an obvious porous structure, and the porous structure of the Gel-NB group was significantly increased compared with that of the Gel group. We successfully prepared the PLLA-FF-TA@Gel-NB+US composite scaffold, and the side of the scaffold formed an uneven structure after ultrasound treatment, further demonstrating the successful integration of the dual system.

[0066] 2. Biocompatibility of the PLLA-FF-TA@Gel-NB+US composite scaffold.

[0067] As Figure 2 and Figure 3 shown, the HUVECs above the composite scaffold grew well and had good biocompatibility. In addition, we found that the cells on the surface of the composite scaffold showed adherent growth and had good cell morphology. Through live-dead staining, we found that all the co-cultured cells were green and the number of red dead cells was small.

[0068] 3. Exploration of the in vivo repair of the PLLA-FF-TA@Gel-NB+US composite scaffold.

[0069] As Figure 4 shown, after successfully creating a tracheal defect model, we used the PLLA-FF-TA@Gel-NB+US patch for repair, and the repair effect of the patch was good and the tracheal tissue regeneration state was good.

[0070] 4. The PLLA-FF-TA@Gel-NB+US composite scaffold can well promote tissue repair in vivo.

[0071] As Figure 5 shown: Compared with the PLLA-FF-TA@Gel group, the in vivo regeneration effect of the PLLA-FF-TA@Gel-NB+US composite scaffold was significantly stronger. This is because the PLLA-FF-TA component of PLLA-FF-TA@Gel-NB+US has good support, and the Gel-NB+US component can achieve good defect occlusion. In addition, the piezoelectric effect generated in the scaffold and the released hydrogen can effectively promote tissue repair.

[0072] Comparative Example 1

[0073] According to the method of Example 1, only the PLLA-FF-TA scaffold was used for tracheal defect repair, and it was found that it could not be blocked. The reason for this phenomenon is that the PLLA-FF-TA scaffold has a porous scaffold structure, and without the occlusion effect of the hydrogel, gas is easily exuded from the pores of the scaffold.

[0074] Comparative Example 2

[0075] According to the method of Example 1, only GelMA hydrogel was used for tracheal defect repair, and it was found that the defect could not be blocked. Because the GelMA hydrogel has weak adhesion and low mechanical strength, it is difficult to form stable adhesion and blockage on the surface of tracheal defects. In addition, GelMA alone is difficult to achieve tissue repair and regeneration effects.

[0076] Comparative Example 3

[0077] According to the method of Example 1, only the PLLA-FF-TA@GelMA scaffold was used for tracheal defect repair, and it was found that the defect could not be blocked. In this group, PLLA-FF-TA was still difficult to achieve effective blockage. Although combined with GelMA, the tissue repair and regeneration effects were still weak.

[0078] Comparative Example 4

[0079] According to the method of Example 1, only the Gel-NB+US hydrogel was used for tracheal defect repair, and it was found that the defect could not be blocked. Because the GelMA hydrogel has weak adhesion, it is difficult to form stable adhesion and blockage on the surface of tracheal defects. In addition, due to the low mechanical strength of Gel-NB+US, it is difficult to maintain a good mechanical environment of the airway and is prone to cause collapse at the defect site.

Claims

1. A preparation method of a piezoelectric composite scaffold for tracheal defect repair, characterized in that, It includes the following steps: (1) Prepare a PLLA scaffold by 3D printing, immerse the PLLA scaffold in an FF polypeptide solution, and prepare a PLLA-FF scaffold by freeze-drying; (2) Immerse the PLLA-FF scaffold in a tannic acid solution at 10 °C to prepare a PLLA-FF-TA composite scaffold; (3) Through a double emulsification process, using dichloromethane and PLGA-COOH as the oil phase, and perfluorohexane and a polyvinyl alcohol solution as the aqueous phase, perform ultrasonic emulsification twice to prepare oxygen-containing PLGA nanobubbles; and prepare a GelMA hydrogel solution, mix GelMA and the oxygen-containing PLGA nanobubbles to obtain a Gel-NB hydrogel precursor; (4) Inject the Gel-NB hydrogel precursor into the freeze-dried PLLA-FF-TA composite scaffold, and then crosslink it using ultraviolet light; finally, irradiate it using ultrasound to obtain a PLLA-FF-TA@Gel-NB+US composite scaffold, which is the piezoelectric composite scaffold.

2. The method according to claim 1, characterized in that, In step (1), the concentration of the FF polypeptide is 8 mg / mL, and the immersion time of the PLLA scaffold in the FF polypeptide solution is 6 h.

3. The method according to claim 1, characterized in that, The parameters of the 3D printing described in step (1) are as follows: the extrusion temperature of the extruder is 182 °C, a temperature-controlled nozzle is used, the syringe specification is 5 mL, and the nozzle is 1.75 mm; the printing bed is preheated to 55 °C and the nozzle is preheated to 215 °C, the printing speed is controlled at 6 mm / s, and the extrusion speed is 0.3 mm 3 / min.

4. The method according to claim 1, characterized in that, In step (2), the concentration of the tannic acid solution is 1 wt%, and the immersion time is 48 hours.

5. The method according to claim 1, wherein In step (3), the steps of the double emulsification process are: use 2 mL of dichloromethane and 50 mg of PLGA-COOH to form the oil phase, add 200 μL of perfluorohexane and 5 mL of polyvinyl alcohol solution to the oil phase to form a mixture, perform ultrasonic emulsification on the mixture twice, then stir the suspension at room temperature for 4 hours, and evaporate dichloromethane.

6. The method according to claim 1, wherein In step (3), the concentration of the polyvinyl alcohol solution is 4 wt%.

7. The method according to claim 1, wherein In step (3), the concentration of GelMA is 5 wt%.

8. The method according to claim 1, characterized in that, In step (4), the conditions of the ultrasound are: frequency 1 MHz, power 3 W.

9. A piezoelectric composite scaffold prepared by the method according to any one of claims 1-8.

10. Use of the piezoelectric composite scaffold according to claim 9 in the preparation of a tissue engineering medical material for tracheal defect repair.

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