Anisotropic nanofiber yarn composite scaffold as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and discloses an anisotropic nanofiber yarn composite scaffold as well as a preparation method and application thereof. The anisotropic nanofiber yarn composite scaffold is formed by compounding methylacryloyl modified gelatin and a nanofiber yarn scaffold prepared through electrostatic spinning. The anisotropic nanofiber yarn composite scaffold not only has more excellent mechanical properties, but also can provide a more suitable microenvironment for cell adhesion and growth, can significantly improve the remodeling efficiency of an extracellular matrix, and has a wide application prospect in the field of heart valve repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to an anisotropic nanofiber yarn composite scaffold, its preparation method, and its application. Background Technology

[0002] Valvular heart disease (VHD) is caused by hemodynamic changes due to abnormal valve structure or function. Its incidence is rising due to factors such as population aging, posing a serious threat to health. Currently, end-stage VHD can only be treated with valve replacement, but mechanical valves require lifelong anticoagulation, and bioprosthetic valves are prone to calcification and degeneration, both having significant limitations. Therefore, tissue-engineered heart valves (TEHVs) have become a research hotspot. They can mimic the structure and function of natural valves, combining the low thrombosis risk of bioprosthetic valves with the durability of mechanical valves. Their core advantage is their dynamic tissue regeneration capability, which can guide host cell proliferation and secretion of new extracellular matrix (ECM) during material degradation, achieving the transformation of artificial materials into human tissue and potentially overcoming the limitations of current treatments.

[0003] In existing technologies, the key to preparing TEHV is constructing a scaffold suitable for seed cell survival. Hydrogel scaffolds are ideal choices due to their mechanical properties being close to soft tissue, good biocompatibility, and ability to mimic the porous microenvironment of the ECM, supporting cell adhesion, proliferation, and differentiation. However, pure hydrogels suffer from insufficient mechanical properties, limited ability to promote chondrogenic differentiation, and weak inhibitory effect on osteogenic differentiation. Furthermore, their network structure is difficult to mimic the orientational structure of biological systems such as heart valves. Polylactic acid (PLA) has been widely used in tissue engineering due to its good processability, lack of cytotoxicity, and non-toxic degradation products. It can be prepared into anisotropic nanofibers through electrospinning. However, its bioactivity is insufficient, lacking cell adhesion signals, and requires synergistic modification with other materials. Methacrylamide-modified gelatin (GelMA), as a natural modified hydrogel, is mechanically tunable, biomimetic to the ECM, and has excellent photocrosslinking properties, making it widely used in soft tissue engineering. However, it lacks the anisotropy required for valves and may induce osteogenic differentiation of mesenchymal stem cells (MSCs) and ECM calcification. Although nanofiber membranes can be constructed into three-dimensional structures with nanoscale pores through electrospinning to provide an ECM-like microenvironment, their mechanical properties are limited by the planar structure, making it difficult to withstand physiological stress. Furthermore, their flexibility and weavability are insufficient, making it difficult to adapt to irregular defect sites. In addition, the inter-fiber connections are weak, and the structure is prone to collapse after long-term culture or implantation, affecting the stability of the scaffold and the continuity of tissue regeneration. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art by providing an anisotropic nanofiber yarn composite scaffold, its preparation method and application, overcoming the problems of loose structure, poor mechanical properties, insufficient stability and equipment limitations in the preparation of nanofiber yarns in the prior art.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for preparing anisotropic nanofiber yarn composite scaffold, comprising the following steps: S1. Preparing methacryloyl-modified gelatin; S2. The methacrylamide-modified gelatin and polylactic acid are added to hexafluoroisopropanol to obtain a spinning solution; the spinning solution is then subjected to electrospinning to obtain an anisotropic nanofiber membrane. S3. The anisotropic nanofiber membrane is curled and then thermally stretched to obtain oriented nanofiber yarn; hexafluoroisopropanol is coated on the oriented nanofiber yarn, and the oriented nanofiber yarn is pasted on both sides in the transverse direction to obtain a nanofiber yarn support. S4. Immerse the nanofiber yarn scaffold in a solution of methacryloyl-modified gelatin, wherein the methacryloyl-modified gelatin solution includes cell culture medium and photoinitiator; perform photocuring treatment to obtain anisotropic nanofiber yarn composite scaffold.

[0006] The anisotropic nanofiber yarn composite scaffold fabrication method of this invention produces a scaffold with regular orientation, excellent mechanical properties, and enhanced cell activity. It demonstrates significant application potential in the field of heart valve repair.

[0007] As a preferred embodiment of the preparation method of the anisotropic nanofiber yarn composite scaffold of the present invention, in step S1, the preparation method of the methacryloyl modified gelatin is as follows: gelatin is dissolved in sodium carbonate / sodium bicarbonate buffer solution, the pH is adjusted to alkaline, methacrylic anhydride is added after heating to react, the pH is adjusted to neutral to terminate the reaction, and the reaction product is dialyzed and freeze-dried to obtain methacryloyl modified gelatin.

[0008] More preferably, the concentration of the sodium carbonate / sodium bicarbonate buffer solution is 0.1M-0.5M; the concentration of the gelatin is 1wt%-20wt%; and the volume-to-mass ratio of the gelatin to methacrylic anhydride is 10g:(0.5-2)mL.

[0009] More preferably, the alkaline pH value is 8-10; the heating temperature is 40℃-60℃; the neutral pH value is 6-8; the dialysis time is 2-4 days; and the molecular weight cutoff is 3000-4000.

[0010] In a preferred embodiment of the preparation method of the anisotropic nanofiber yarn composite scaffold of the present invention, in step S2, the concentration of the spinning solution is 5wt%-15wt%; the mass ratio of polylactic acid to methacryloyl modified gelatin is 1:(0.5-1.5); the electrospinning parameters include: positive voltage of 10KV-15KV, negative voltage of 0KV-3KV, receiving distance of 10cm-20cm, syringe advance speed of 0.5mL / h-2mL / h, temperature of 20℃-25℃, relative humidity of 40%-60%, and collection rotation speed of 100rpm-2000rpm.

[0011] In a preferred embodiment of the preparation method of the anisotropic nanofiber yarn composite scaffold of the present invention, in step S3, the temperature of the thermal stretching is 40℃-60℃, the stretching rate is 20mm / min-40mm / min, and the stretching is to 1-2 times the original length.

[0012] In a preferred embodiment of the preparation method of the anisotropic nanofiber yarn composite scaffold of the present invention, in step S4, the concentration of the methacryloyl-modified gelatin in the solution is 5wt%-10wt%, and the concentration of the photoinitiator is 0.1wt%-0.5wt%; the photoinitiator is lithium phenyl(2,4,6-trimethylbenzoyl)phosphate; during the photocuring treatment, the initiation wavelength is 405nm, and the initiation time is 20s-60s; the cell culture medium is DEME-F12 medium.

[0013] In a preferred embodiment of the preparation method of the anisotropic nanofiber yarn composite scaffold of the present invention, in step S4, the solution of methacryloyl-modified gelatin further includes human adipose-derived mesenchymal stem cells; each mL of the methacryloyl-modified gelatin solution contains 10 0 -10 7 The cells mentioned.

[0014] In a second aspect, the present invention provides an anisotropic nanofiber yarn composite scaffold, which is prepared by the preparation method described in the first aspect.

[0015] The anisotropic nanofiber yarn composite scaffold of this invention exhibits multiple advantages through the ordered axial orientation of the fibers: its oriented structure can simulate the hierarchical characteristics of natural tissues such as tendons and nerves, providing contact guidance signals for cells to promote the orderly regeneration of functional tissues; the yarn structure formed by axial orientation significantly improves the axial tensile strength and fatigue resistance of the scaffold, matching the mechanical requirements of natural tissues and providing effective support; the yarn morphology endows the material with good flexibility and weavability, and a three-dimensional mesh scaffold can be constructed through weaving, fixing and other processes to adapt to different defect sites, and the pores between the yarns facilitate nutrient transport; at the same time, the stable bundle structure formed by the axial integration of fibers reduces fiber slippage and loosening, ensuring the structural integrity of the scaffold after long-term culture and implantation.

[0016] Thirdly, the present invention applies the anisotropic nanofiber yarn composite scaffold described in the second aspect to tissue-engineered heart valves.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The anisotropic nanofiber yarn composite scaffold of the present invention is constructed from a three-dimensional nanofiber yarn network. Its three-dimensional structure has better mechanical properties, and the multi-layered porous structure formed by three-dimensional fixation can provide more adhesion sites for cells. This structural advantage can promote the uniform colonization and spread of cells inside the scaffold, which is beneficial to enhancing the interaction between the material and the cells; (2) In the preparation method of the present invention, the preparation of oriented nanofiber yarn does not rely on special electrospinning equipment. Nanofiber yarn can be prepared by ordinary electrospinning equipment, which reduces the process threshold and has wide equipment adaptability. (3) The anisotropic nanofiber yarn composite scaffold of the present invention can construct a suitable microenvironment that can effectively induce cartilage differentiation and inhibit osteogenic differentiation. At the same time, the anisotropic structure can also promote the active secretion of matrix metalloproteinases by cells to regulate the extracellular matrix components, create favorable conditions for tissue self-repair, and significantly improve the remodeling efficiency of the extracellular matrix. It has broad application prospects in the field of heart valve repair. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the preparation method of anisotropic nanofiber yarn composite scaffold.

[0019] Figure 2 The images show SEM images of the scaffolds in Example 1 and Comparative Example 1, along with their corresponding orientations and diameter distributions. In the images, a represents the scaffold without thermal stretching of nanofiber yarn, and b represents the scaffold with thermal stretching of nanofiber yarn.

[0020] Figure 3 The figures show the mechanical properties of the scaffolds in Example 1 and Comparative Example 2; in the figures, a represents the fiber axial direction and b represents the fiber radial direction.

[0021] Figure 4 The live / dead staining and spreading rate of the cell-carrying scaffolds in Examples 2, 3, and 4 are shown.

[0022] Figure 5 The content of extracellular matrix components (glycosaminoglycans, hydroxyproline) in the cell-carrying scaffolds of Examples 2, 3, and 4. Detailed Implementation

[0023] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0024] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0025] Example 1: An anisotropic nanofiber yarn composite scaffold The preparation method of this anisotropic nanofiber yarn composite scaffold includes the following steps: (1) Preparation of methacryloyl-modified gelatin Weigh 0.318 g of sodium carbonate and 0.586 g of sodium bicarbonate, dissolve them in 100 mL of ultrapure water to obtain a 0.25 M sodium carbonate / sodium bicarbonate buffer solution; weigh 10 g of gelatin (Sigma, G9382 derived from cowhide, number average molecular weight (Mn) of 80000D) and add it to the above buffer solution, stir and dissolve at 50 °C to obtain a 10 wt% gelatin solution, and adjust the pH of the solution to 9 using 5 mol / L sodium hydroxide and 6 mol / L hydrochloric acid; add 1 mL of methacrylic anhydride directly under water bath heating and stirring at 50 °C and 500 rpm, and react for 1 h; adjust the pH to 7.4 to end the reaction, centrifuge the reaction product at 8000 rpm for 5 min to remove unreacted precipitate, then dialyze using a dialysis bag with a molecular weight cutoff of 3500 for 3 days, and freeze-dry to obtain methacryloyl modified gelatin.

[0026] (2) Preparation of anisotropic nanofiber membranes 0.6 g of polylactic acid (PLA, McLean, P890263, weight-average molecular weight (Mw) 110,000) and 0.4 g of the prepared methacryloyl-modified gelatin were weighed and dissolved together in 10 mL of hexafluoroisopropanol solution. The mixture was magnetically stirred until completely dissolved to obtain a spinning solution with a mass fraction of 10 wt%. The spinning solution was transferred to a 5 mL syringe, and a 20 cm diameter roller was used as the receiving device to collect the nanofibers. The parameters of the electrospinning device were set as follows: positive voltage 15 kV, negative voltage 3 kV, receiving distance 15 cm, needle size 20, syringe advance speed 0.8 mL / h, spinning environment temperature controlled at 25 °C, relative humidity maintained at 50%, roller collection speed 2000 rpm, and anisotropic nanofiber membrane was obtained after 8 minutes of spinning.

[0027] (3) Preparation of nanofiber yarn scaffold like Figure 1 As shown, the anisotropic nanofiber membrane was coiled and then thermally stretched at 60°C at a stretching rate of 30 mm / min along the fiber orientation direction until it reached 1.5 times its original length, resulting in oriented nanofiber yarn. The yarn was then neatly arranged and fixed in the same direction, and hexafluoroisopropanol was applied to the ends of the yarn to enhance adhesion. Subsequently, yarn of the same specification was pasted onto both sides transversely, and the yarn was cut into 1 cm × 1 cm pieces to obtain a nanofiber yarn scaffold.

[0028] The scaffold prepared in this way has a fully oriented feature: at the microscopic level, the fibers within a single yarn are highly oriented; at the macroscopic level, all yarns are also neatly arranged in a single direction. This feature can more effectively guide the oriented adhesion and migration of cells.

[0029] The orientation degree of the nanofiber scaffold is calculated using the Directionality plugin in the Fiji software. The orientation degree Ω is defined as the percentage of the area of ​​oriented fibers (i.e., the area of ​​nanofibers arranged between 80° and 100°) in the SEM image relative to the total area of ​​all fibers in the SEM image. In this embodiment, the orientation degree of the nanofiber yarn scaffold is Ω = 0.836.

[0030] (4) Preparation of anisotropic nanofiber yarn composite scaffold The prepared methacryloyl-modified gelatin was dissolved in DEME-F12 medium to prepare a gel precursor solution with a mass fraction of 7 wt%. The photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (EFL) was added to the solution, and its concentration was controlled at 0.25 wt%. After thorough mixing, the solution was filtered through a 0.22 μm sterile filter to obtain precursor solution A.

[0031] The prepared nanofiber yarn scaffold was sterilized by immersing it in 75% ethanol solution for 1 hour, followed by rinsing three times with sterile phosphate buffer. 0.3 mL of precursor solution A was added to a 12 mm diameter, 3 mm thick silicone mold, and the sterilized nanofiber yarn scaffold was then placed into the mold containing precursor solution A for composite bonding. Finally, it was photocured by irradiation with a 405 nm UV lamp for 30 seconds to obtain the anisotropic nanofiber yarn composite scaffold.

[0032] Example 2: A cell-carrying anisotropic nanofiber yarn composite scaffold The preparation method of this cell-carrying anisotropic nanofiber yarn composite scaffold includes the following steps: (1) Preparation of methacryloyl-modified gelatin Same as Example 1.

[0033] (2) Preparation of anisotropic nanofiber membranes Same as Example 1.

[0034] (3) Preparation of nanofiber yarn scaffold Same as Example 1.

[0035] (4) Preparation of anisotropic nanofiber yarn composite scaffold The prepared methacryloyl-modified gelatin was dissolved in DEME-F12 medium to prepare a gel precursor solution with a mass fraction of 7 wt%. The photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (EFL) was added to the solution, and its concentration was controlled at 0.25 wt%. After thorough mixing, the solution was filtered through a 0.22 μm sterile filter to obtain precursor solution A.

[0036] Press 10 6 The amount of cells / mL of precursor solution A was used to seed human adipose-derived mesenchymal stem cells (HADMSC, Procyon) into precursor solution A to obtain precursor solution B.

[0037] The nanofiber yarn scaffold prepared in Example 3 was sterilized by immersing it in 75% ethanol solution for 1 hour, followed by rinsing three times with sterile phosphate buffer. 0.3 mL of precursor solution B was added to a 12 mm diameter, 3 mm thick silicone mold, and the sterilized nanofiber yarn scaffold was then placed into the mold containing precursor solution B for composite formation. Finally, it was photocured by irradiation with a 405 nm UV lamp for 30 seconds to obtain the cell-carrying anisotropic nanofiber yarn composite scaffold.

[0038] Comparative Example 1: A Nanofiber Yarn Composite Scaffold The preparation method of this nanofiber yarn composite scaffold includes the following steps: (1) Preparation of methacryloyl-modified gelatin Same as Example 1.

[0039] (2) Preparation of anisotropic nanofiber membranes Same as Example 1.

[0040] (3) Preparation of nanofiber yarn scaffold The anisotropic nanofiber membrane was prepared and then curled to obtain oriented nanofiber yarns. The yarns were then arranged and fixed in the same direction, and hexafluoroisopropanol was applied to the ends of the yarns to enhance adhesion. Subsequently, yarns of the same specification were pasted on both sides in the transverse direction, and the yarns were cut into 1cm×1cm sizes to obtain nanofiber yarn supports.

[0041] The orientation degree of the nanofiber scaffold was calculated using the Directionality plugin in the Fiji software. The orientation degree Ω was defined as the percentage of the area of ​​oriented fibers (i.e., the area of ​​nanofibers arranged between 80° and 100°) in the SEM image relative to the total area of ​​all fibers in the SEM image. The orientation degree of the nanofiber yarn scaffold in this comparative example was Ω = 0.521.

[0042] (4) Preparation of nanofiber yarn composite scaffold The prepared methacryloyl-modified gelatin was dissolved in DEME-F12 medium to prepare a gel precursor solution with a mass fraction of 7 wt%. The photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (EFL) was added to the solution, and its concentration was controlled at 0.25 wt%. After thorough mixing, the solution was filtered through a 0.22 μm sterile filter to obtain precursor solution A.

[0043] The prepared nanofiber yarn scaffold was sterilized by immersing it in 75% ethanol solution for 1 hour, followed by rinsing three times with sterile phosphate buffer. 0.3 mL of precursor solution A was added to a 12 mm diameter, 3 mm thick silicone mold, and the sterilized nanofiber yarn scaffold was then placed into the mold containing precursor solution A for composite bonding. Finally, it was photocured by irradiation with a 405 nm UV lamp for 30 seconds to obtain the nanofiber yarn composite scaffold.

[0044] Comparative Example 2: A Nanofiber Composite Scaffold The preparation method of this nanofiber yarn composite scaffold includes the following steps: (1) Preparation of methacryloyl-modified gelatin Same as Example 1.

[0045] (2) Preparation of anisotropic nanofiber membranes Same as Example 1.

[0046] (3) Fabrication of nanofiber composite scaffolds The prepared methacryloyl-modified gelatin was dissolved in DEME-F12 medium to prepare a gel precursor solution with a mass fraction of 7 wt%. The photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (EFL) was added to the solution, and its concentration was controlled at 0.25 wt%. After thorough mixing, the solution was filtered through a 0.22 μm sterile filter to obtain precursor solution A.

[0047] The prepared anisotropic nanofiber membrane was cut into 1cm × 1cm pieces and then sterilized by immersion in 75% ethanol solution for 1 hour. It was then rinsed three times with sterile phosphate buffer. 0.3 mL of precursor solution A was added to a 12 mm diameter, 3 mm thick silicone mold. The sterilized, cut anisotropic nanofibers were then placed into the mold containing precursor solution A for composite formation. Finally, the mixture was photocured by irradiation with a 405 nm UV lamp for 30 seconds to obtain the nanofiber composite scaffold.

[0048] Comparative Example 3: A Cell-Loading Hydrogel Scaffold The preparation method of this cell-carrying hydrogel scaffold includes the following steps: (1) Preparation of methacryloyl-modified gelatin Same as Example 1.

[0049] (2) Preparation of cell-carrying hydrogel scaffolds The prepared methacryloyl-modified gelatin was dissolved in DEME-F12 medium to prepare a gel precursor solution with a mass fraction of 7 wt%. The photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (EFL) was added to the solution, and its concentration was controlled at 0.25 wt%. After thorough mixing, the solution was filtered through a 0.22 μm sterile filter to obtain precursor solution A.

[0050] Press 10 6 The amount of cells / mL of precursor solution A was used to seed human adipose-derived mesenchymal stem cells (HADMSC, Procyon) into precursor solution A to obtain precursor solution B.

[0051] Add 0.3 mL of precursor solution B into a silicone round mold with a diameter of 12 mm and a thickness of 3 mm, and finally irradiate with a 405 nm ultraviolet lamp for 30 s for photocuring to obtain a cell-carrying hydrogel scaffold.

[0052] Comparative Example 4: A cell-carrying nanofiber composite scaffold The preparation method of this nanofiber yarn composite scaffold includes the following steps: (1) Preparation of methacryloyl-modified gelatin Same as Example 1.

[0053] (2) Preparation of anisotropic nanofiber membranes Same as Example 1.

[0054] (3) Fabrication of nanofiber composite scaffolds The prepared methacryloyl-modified gelatin was dissolved in DEME-F12 medium to prepare a gel precursor solution with a mass fraction of 7 wt%. The photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (EFL) was added to the solution, and its concentration was controlled at 0.25 wt%. After thorough mixing, the solution was filtered through a 0.22 μm sterile filter to obtain precursor solution A.

[0055] Press 10 6 The amount of cells / mL of precursor solution A was used to seed human adipose-derived mesenchymal stem cells (HADMSC, Procyon) into precursor solution A to obtain precursor solution B.

[0056] The prepared anisotropic nanofiber membrane was cut into 1cm × 1cm pieces and then sterilized by immersion in 75% ethanol solution for 1 hour, followed by rinsing three times with sterile phosphate buffer. 0.3 mL of precursor solution B was added to a 12 mm diameter, 3 mm thick silica gel mold. The sterilized, cut anisotropic nanofibers were then placed into the mold containing precursor solution B for composite formation. Finally, the mixture was photocured by irradiation with a 405 nm UV lamp for 30 seconds to obtain the cell-carrying nanofiber composite scaffold.

[0057] Experimental example: (1) Micromorphological analysis was performed on the anisotropic nanofiber yarn composite scaffold of Example 1 and the nanofiber yarn composite scaffold of Comparative Example 1.

[0058] SEM images of anisotropic nanofiber yarn scaffolds before and after heat stretching treatment, and their corresponding orientation and diameter distribution, are shown below. Figure 2 As shown: After the hot stretching treatment, the macroscopic diameter of the nanofiber yarn scaffold in Example 1 was significantly reduced, with the average fiber diameter decreasing from 739 nm to 527 nm, and the fiber orientation degree increasing from 0.521 to 0.836. This indicates that the hot stretching treatment successfully improved the anisotropy of the hand-twisted yarn.

[0059] (2) The tensile properties of the stent are one of the key indicators for evaluating the application of heart valve stents in practice. They must meet the requirements of elastic modulus and deformation resistance that match the natural valve.

[0060] The anisotropic nanofiber yarn composite scaffold of Example 1 and the nanofiber composite scaffold of Comparative Example 2 were subjected to mechanical property testing, and the testing method was in accordance with the standard GB / T13022-1991.

[0061] Mechanical properties comparison Figure 3As shown, the comparison reveals that, in the axial direction (fiber orientation direction), the tensile properties of the stent of Example 1 are superior to those of the stent of Comparative Example 2. Its elastic modulus (27.55±4.58 MPa and 12.92±2.56 MPa) and elongation at break (169.0±36.01% and 37.0±7.81%) are 2.1 times and 4.6 times that of the stent of Comparative Example 2, respectively. The stent of Example 1 significantly strengthens the axial alignment of the fibers during spinning and hot stretching, forming a more continuous and dense oriented fiber structure. This exhibits the high stiffness and ductility required by heart valves, and can more effectively withstand axial loads and deformations during valve closure than the stent of Comparative Example 2. While the modulus of the stent of Example 1 (7.50±0.96 MPa) is lower than that in the axial direction (perpendicular to fiber orientation direction), it is still 4.8 times higher than that of the stent of Comparative Example 2 (1.30±0.45 MPa). During the coiling, fixing, and thermal stretching processes, the stent of Example 1 still retained a certain degree of mechanical support in the vertical direction. At the same time, although the elongation at break of 17.50±2.67% was slightly lower than that of the stent of Comparative Example 2, it was closer to the radial mechanical requirements of a natural valve (10%-20%).

[0062] In summary, the anisotropic nanofiber yarn composite scaffold of Example 1 exhibits superior tensile properties compared to the nanofiber composite scaffold of Comparative Example 2.

[0063] (3) Live / dead staining analysis was performed on the cell-carrying scaffolds prepared in Example 2, Comparative Example 3 and Comparative Example 4. The live / dead staining analysis was performed using the LIVE / DEAD cell viability / cytotoxicity assay kit (purchased from Thermo Fisher Scientific) to obtain focused live / dead images and spread rate data of the three cell-carrying scaffolds.

[0064] like Figure 4 As shown, in Comparative Example 3, although the cells in the hydrogel scaffold group showed a proliferative trend from day 7 to day 14 of culture, they maintained a highly rounded morphology (spreading rate of 8.6±5.5%). The cells grew randomly within the gel without a specific growth or spreading direction. In contrast, after introducing nanofibers into the scaffolds of Comparative Example 4 and Example 2, the cells showed partial spreading by day 7, and the spreading rates reached 27±6.8% and 62±4.3% respectively by day 14. Furthermore, the cells spread oriented along the fiber axis, indicating that the introduction of nanofibers effectively promoted and guided the directional spreading of cells. Simultaneously, the scaffold of Example 2 exhibited superior cell spreading induction ability compared to the scaffold of Comparative Example 4. The yarns in the scaffold of Example 2 exposed more cell adhesion sites, which was more conducive to cell adhesion and growth, and also effectively resisted cell contraction forces, thus helping to maintain the stability of the spreading morphology.

[0065] (4) Changes in the content of glycosaminoglycans and hydroxyproline in the extracellular matrix reflect the regulatory effect of the scaffold on the synthesis and remodeling of the extracellular matrix. The extracellular matrix content of the cell-loaded scaffolds prepared in Example 2, Comparative Example 3 and Comparative Example 4 was analyzed using a glycosaminoglycan kit (purchased from Nanjing Jiancheng) and a hydroxyproline assay kit (acid hydrolysis method) (purchased from Nanjing Jiancheng).

[0066] Experimental results are as follows Figure 5 As shown, glycosaminoglycan content exhibited a consistent increasing trend across all three scaffolds, with no statistically significant difference observed at days 7 and 14. This result indicates that regardless of whether nanofiber structures (membranes or yarns) are incorporated into the scaffold, stable glycosaminoglycan secretion by cells is supported, and the scaffold structure has no significant impact on glycosaminoglycan synthesis efficiency. It is speculated that this is because glycosaminoglycans, as a characteristic component of cartilage matrix, primarily depend on cellular basal metabolic activities for secretion, and all three scaffolds provide a suitable microenvironment for nutrient exchange, thus maintaining the stability of glycosaminoglycan synthesis.

[0067] Compared to glycosaminoglycans, the changes in hydroxyproline content differed. In Comparative Example 3, the hydrogel scaffold reached its peak hydroxyproline content at 7 days (68.68±1.18 ug / ml) and remained stable over 14 days (67.12±2.05 ug / ml), exhibiting a slower collagen metabolism rate. In contrast, the hydroxyproline content of the scaffolds in Comparative Example 4 and Example 2 decreased significantly from 7 to 14 days (from 65.94±0.87 to 53.39±3.16 ug / ml and from 68.68±1.18 to 45.18±3.42 ug / ml, respectively), with Example 2 showing a particularly high decrease of 34.2%. This result indicates that the introduction of nanofibers (especially oriented yarn structures) into the scaffold can significantly accelerate the metabolism and remodeling process of hydroxyproline. The scaffold of Example 2 has a higher fiber orientation (Ω = 0.836) and a three-dimensional network structure, which can enhance cell adhesion and spreading (spreading rate of 62±4.3%), upregulate the activity of matrix metalloproteinases (MMPs), promote the degradation of existing collagen by collagenase, and drive the synthesis and orderly deposition of new collagen. It shows a significant decrease in hydroxyproline content and exhibits more active matrix remodeling efficiency. This characteristic is highly matched with the dynamic extracellular matrix renewal requirements of natural heart valves.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. 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 be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing anisotropic nanofiber yarn composite scaffold, characterized in that, Includes the following steps: S1. Preparation of methacryloyl-modified gelatin; S2. The methacrylamide-modified gelatin and polylactic acid are added to hexafluoroisopropanol to obtain a spinning solution; the spinning solution is then subjected to electrospinning to obtain an anisotropic nanofiber membrane. S3. The anisotropic nanofiber membrane is rolled up and then thermally stretched to obtain oriented nanofiber yarn. Hexafluoroisopropanol is coated on the oriented nanofiber yarn, and the oriented nanofiber yarn is pasted on both the front and back sides in the transverse direction to obtain a nanofiber yarn support. S4. Immerse the nanofiber yarn scaffold in a solution of methacryloyl-modified gelatin, wherein the methacryloyl-modified gelatin solution includes cell culture medium and photoinitiator; perform photocuring treatment to obtain anisotropic nanofiber yarn composite scaffold.

2. The method for preparing the anisotropic nanofiber yarn composite scaffold according to claim 1, characterized in that, In step S1, the method for preparing the methacryloyl-modified gelatin is as follows: gelatin is dissolved in sodium carbonate / sodium bicarbonate buffer solution, the pH is adjusted to alkaline, methacrylic anhydride is added after heating, the pH is adjusted to neutral to terminate the reaction, and the reaction product is dialyzed and freeze-dried to obtain methacryloyl-modified gelatin.

3. The method for preparing the anisotropic nanofiber yarn composite scaffold according to claim 2, characterized in that, The sodium carbonate / sodium bicarbonate buffer solution has a concentration of 0.1M-0.5M; the gelatin concentration is 1wt%-20wt%; and the volume-to-mass ratio of gelatin to methacrylic anhydride is 10g:(0.5-2)mL.

4. The method for preparing the anisotropic nanofiber yarn composite scaffold according to claim 2, characterized in that, The alkaline pH value is 8-10; the heating temperature is 40℃-60℃; the neutral pH value is 6-8; the dialysis time is 2-4 days, and the molecular weight cutoff is 3000-4000.

5. The method for preparing the anisotropic nanofiber yarn composite scaffold according to claim 1, characterized in that, In step S2, the concentration of the spinning solution is 5wt%-15wt%; the mass ratio of polylactic acid to methacryloyl modified gelatin is 1:(0.5-1.5); the electrospinning parameters are set as follows: positive voltage 10KV-15KV, negative voltage 0KV-3KV, receiving distance 10cm-20cm, syringe advance speed 0.5mL / h-2mL / h, temperature 20℃-25℃, relative humidity 40%-60%, and collection rotation speed 100rpm-2000rpm.

6. The method for preparing the anisotropic nanofiber yarn composite scaffold according to claim 1, characterized in that, In step S3, the temperature of the hot stretching is 40℃-60℃, the stretching rate is 20mm / min-40mm / min, and the stretching is to 1-2 times the original length.

7. The method for preparing the anisotropic nanofiber yarn composite scaffold according to claim 1, characterized in that, In step S4, the concentration of the methacryloyl-modified gelatin in the solution is 5wt%-10wt%, and the concentration of the photoinitiator is 0.1wt%-0.5wt%. The photoinitiator is lithium phenyl(2,4,6-trimethylbenzoyl)phosphate. During the photocuring treatment, the initiation wavelength is 405nm, and the initiation time is 20s-60s. The cell culture medium is DEME-F12 medium.

8. The method for preparing the anisotropic nanofiber yarn composite scaffold according to claim 1, characterized in that, In step S4, the solution of methacryloyl-modified gelatin further includes human adipose-derived mesenchymal stem cells; each mL of the solution of methacryloyl-modified gelatin contains 10 0 -10 7 The cells mentioned.

9. An anisotropic nanofiber yarn composite scaffold, characterized in that, It is prepared by the preparation method according to any one of claims 1-8.

10. The application of the anisotropic nanofiber yarn composite scaffold according to claim 9 in tissue-engineered heart valves.

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