A nanofilament-based in-vitro spinal cord model and its preparation method and application

Abstract

The application belongs to the technical field of biomedical materials, and particularly relates to an in-vitro spinal cord-like model based on nanofilament yarns and a preparation method and application thereof. The in-vitro spinal cord-like model based on nanofilament yarns is designed and prepared, and has the characteristics of precise anatomical positioning, reasonable cell spatial distribution and the like. In the application, nanofilament yarns are used as inner cores, and nanofiber tubes are used as outer sheaths. On the one hand, the nanofilament yarns that play the role of nerve-like conducting bundles guide the oriented arrangement of NSPCs and promote the directional differentiation of the NSPCs. On the other hand, the nanofiber tubes play the role of artificial dura mater, prevent epidural adhesion and provide a stable intraluminal microenvironment for nerve regeneration. Finally, the nanofilament yarns carry model drugs to realize batch screening of the drugs in the spinal cord-like model.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to an in vitro spinal cord-like model based on nanofilaments as well as a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an acknowledgement or any form of suggestion that this information forms prior art already known to those of ordinary skill in the art.

[0003] Spinal cord injury (SCI) is a common neurological injury disease, which often leads to loss of sensory and motor function below the injury segment and dysfunction of bladder and bowel, seriously affecting the quality of life and life expectancy of patients. However, due to the limited nerve regeneration ability of the spinal cord and the inhibitory microenvironment of SCI, the current clinical treatment means is limited. With the development of nanotechnology and neurobiology, neural tissue engineering (NTE) has become the most promising treatment mode for SCI, which provides a guided biomimetic microenvironment to regulate the differentiation fate of stem cells, thereby promoting the repair and regeneration of injured tissues. However, the natural spinal cord shows a heterogeneous structure, including various cell types and directional distribution along the neural transmission bundle. Therefore, it is of great significance to construct an in vitro model that can simulate the cell kinetic behavior and stereoscopic spatial distribution of the central nervous system (CNS) for the treatment of neurological diseases such as SCI.

[0004] Organoids are an important means to construct in vitro biomimetic models. However, the organoid biomimetic models constructed based on the random self-assembly process of stem cells often have poor controllability, general repeatability and low spatial specificity. Recently, the differentiation pattern of organoids guided by NTE and other means has attracted widespread attention. For example, by changing the microstructure of the tissue scaffold through 3D bioprinting and other means, the tissue morphology and development pattern of organoids are adjusted. However, the printing resolution of microns cannot provide anisotropic nanophysical cues, which affects the interaction between cells and scaffolds, and is not conducive to the differentiation and development of the biomimetic model.

[0005] The introduction of traditional textile technology into electrospinning provides a new solution for constructing a new generation of spinal cord models. Nanoyarn (NY) is the main configuration unit for constructing a bio-textile scaffold and is the core element affecting the biological performance of the model. Its nanoscale three-dimensional structure has the advantages of high specific surface area, high porosity, and ECM-like morphology, which can guide the directional arrangement of stem cells, promote their differentiation and maturation. There are various ways to process electrospun NY at present, such as using a double nozzle to spray nanofibers with different charges to bundle the yarn by using a conjugate electrospinning device. However, the above method still has many defects: a. The orientation degree and crystallinity of the nanofibers in the yarn are low, which is not suitable for highly oriented nerve tissue. In order to improve the fiber orientation, the existing post-processing method will damage the structure or biological activity of the drug molecules loaded in the yarn, which seriously limits the application range of the drug; b. The uniformity and mechanical properties of the yarn are poor, which cannot meet the needs of traditional textile processing technology; c. The textile process is difficult to control, and the continuity and yield are low. Therefore, it still has technical challenges to combine the preparation process of new NY, the bionic structure of the spinal cord, and the delivery of active drugs to construct an in vitro spinal cord model.

[0006] In addition, stem cell selection is also important for model application. Neural progenitor and stem cells (NSPCs) are a class of cells with self-renewal ability and multi-differentiation potential. However, due to the existence of oxidative and inflammatory microenvironment in the acute phase of SCI, the therapeutic effect of directly injecting NSPCs into the SCI defect area is poor, and most cells are difficult to colonize and survive. At the same time, most of the transplanted NSPCs differentiate into astrocytes rather than neurons, which will exacerbate the formation of neural scar and is not conducive to the repair of spinal cord injury; on the other hand, there is a continuous neuronal phenotype transformation after injury: from excitatory neurons to inhibitory neurons, which is more detrimental to the recovery of neural function. Therefore, how to construct a multi-cell lineage spinal cord bionic scaffold with precise spatial distribution and heterogeneous communication network has become a problem to be solved in the construction of NTE model. SUMMARY

[0007] In order to solve the problems in the prior art, the purpose of the present application is to provide an in vitro spinal cord model based on nanoyarn and a preparation method and application thereof.

[0008] In order to achieve the above-mentioned purpose, the present application is realized by the following technical solutions:

[0009] In a first aspect, the present application provides a preparation method of an in vitro spinal cord model based on nanoyarn, comprising the following steps:

[0010] S1, the spinning material, the medicine is stirred and dissolved in the solvent to form a spinning solution, the solution is rotated into uniaxial arranged nanofibers using a rotating cylinder as a directional electrospinning device of a nanofiber collector, the nanofibers formed on the drum collector are twisted to form a roving, and the roving is drawn using a stretching device to obtain a nanofiber yarn;

[0011] S2, the spinning material is stirred and dissolved in the solvent to form a spinning solution, the solution is rotated into uniaxial arranged nanofibers using a rotating cylinder as a directional electrospinning device of a nanofiber collector, the nanofibers on the collector are cut into 2D film pieces along the longitudinal direction of the collector, the 2D film pieces are wound into a hollow tube, and the nanofiber yarn is filled into the hollow tube to obtain a spinal cord-like scaffold;

[0012] S3, the stem cells are planted in the spinal cord-like scaffold to obtain an in vitro spinal cord-like model based on a nanofiber yarn.

[0013] In a second aspect, the present application provides an in vitro spinal cord-like model based on a nanofiber yarn, which is obtained by the preparation method of the in vitro spinal cord-like model based on a nanofiber yarn.

[0014] In a third aspect, the present application provides application of the in vitro spinal cord-like model based on a nanofiber yarn in the field of biomimetic models.

[0015] The beneficial effects of the one or more technical solutions of the present application are as follows:

[0016] 1. Due to the randomness of the self-assembly process of stem cells, the conventional spinal cord-like model has general repeatability, limited biological functions, and is difficult to evolve into highly oriented nerve transmission bundles; the present application uses NY as a biomimetic transmission bundle and a nanofiber tube as an artificial dura mater to construct a new spinal cord-like model. The model has a biomimetic topological structure similar to ECM, the fibers are arranged in the same direction and have uniform diameters, the average diameter of the nanofibers is 500 nm, and the average diameter of the NY formed by the nanofibers is 200-300 mu m, which is conducive to the directional migration and infiltration of NSPCs.

[0017] 2. In the biocompatibility experiment, the proportion of living cells on the NY is more than 95%, which is consistent with that of a two-dimensional culture dish, indicating that the in vitro spinal cord-like model based on a nanofiber yarn of the present application has negligible negative impact on the survival ability of cells and good biocompatibility.

[0018] 3. NY can provide a guiding regenerative microenvironment, which is conducive to ensuring the long-term survival and growth of NSPCs. In addition, the anisotropic fiber structure of ECM-like can promote its differentiation into neurons and oligodendrocytes, and the axons are significantly elongated, arranged in the same direction as the nanofibers. It is worth mentioning that when cultured in 3D NYs, most neurons differentiate into excitatory subtypes, laying the foundation for the reconstruction of neural function. In addition, in the NY spinal cord model, the inventors observed complex synaptic networks and neurotransmitter responsiveness, indicating that the differentiated neurons have strong neural function.

[0019] 4. The present application can carry bioactive drugs by optimizing the preparation process of NY, which widens the drug selection space of the spinal cord model. BRIEF DESCRIPTION OF DRAWINGS

[0020] The drawings accompanying the specification of this application form a part thereof, serve to provide further understanding of the application, and together with the description, explain the application. The specific embodiments of the application and its description are used to explain the application without being an improper limitation of the application.

[0021] Figure 1 A is a schematic diagram of the preparation of the spinal cord model; B is a scanning electron micrograph of the spinal cord scaffold of Example 1, scale: 50um; C is a local enlarged view of the scanning electron micrograph of the spinal cord scaffold of Example 1, scale: 5um; D is a scanning electron micrograph of the spinal cord scaffold of Example 2, scale: 50um; E is a local enlarged view of the scanning electron micrograph of the spinal cord scaffold of Example 2, scale: 5um; F is a scanning electron micrograph of the spinal cord scaffold of Example 3, scale: 50um; G is a local enlarged view of the scanning electron micrograph of the spinal cord scaffold of Example 3, scale: 5um; H is a digital photo of the spinal cord scaffold; I is a statistical diagram of the diameters of the nanofilaments in each group; J is a statistical diagram of the diameters of the spinal cord scaffolds; K is a mechanical curve of the nanofilaments in each group; L is the Young's modulus of the nanofilaments in each group; M is the breaking strain rate of the nanofilaments in each group; N is the ultimate strength of the nanofilaments in each group; O is the sustained-release curve of the loaded curcumin; P is the Fourier infrared spectrum of the nanofilaments in each group; Q is the X-ray diffraction spectrum of the nanofilaments in each group;

[0022] Figure 2A is the live and dead staining of NSPCs after 1 day culture on normal 2D culture dish (scale bar: 50um), the right side is a local enlarged view (scale bar: 10um); B is the live and dead staining of NSPCs after 1 day culture on nanoscrolls of Example 1 (scale bar: 50um), the right side is a local enlarged view (scale bar: 10um); C is the live and dead staining of NSPCs after 1 day culture on nanoscrolls of Example 2 (scale bar: 50um), the right side is a local enlarged view (scale bar: 10um); D is the live and dead staining of NSPCs after 3 days culture on normal 2D culture dish (scale bar: 50um), the right side is a local enlarged view (scale bar: 10um); E is the live and dead staining of NSPCs after 3 days culture on nanoscrolls of Example 1 (scale bar: 50um), the right side is a local enlarged view (scale bar: 10um); F is the live and dead staining of NSPCs after 3 days culture on nanoscrolls of Example 2 (scale bar: 50um), the right side is a local enlarged view (scale bar: 10um); G is the 3D reconstruction of NSPCs on nanoscrolls of Example 1; H is the 3D reconstruction of NSPCs on nanoscrolls of Example 2; I is the cell viability of NSPCs cultured for 1, 3, 7 days in different groups; J is the immunofluorescence staining of Nestin of NSPCs after 1 day culture on normal 2D culture dish, to observe the arrangement direction of cells; K is the immunofluorescence staining of Nestin of NSPCs after 1 day culture on nanoscrolls of Example 1, to observe the arrangement direction of cells; L is the quantitative analysis of the cell arrangement direction of NSPCs on normal 2D culture dish and nanoscrolls of Example 1; M is the cell survival rate of NSPCs in different groups;

[0023] Figure 3A is the immunofluorescence staining of Ki67, SOX2, DAPI (scale: 50 um) of NSPC after 7 days of culture on ordinary 2D culture dish, to observe the cell stemness; B is the immunofluorescence staining of Ki67, SOX2, DAPI (scale: 50 um) of NSPC after 7 days of culture on the nanofilament of Example 1, to observe the cell stemness; C is the immunofluorescence staining of Ki67, SOX2, DAPI (scale: 50 um) of NSPC after 7 days of culture on the nanofilament of Example 2, to observe the cell stemness; D is the immunofluorescence staining of MAP2, GFAP, DAPI (scale: 50 um) of NSPC after 7 days of culture on ordinary 2D culture dish, to observe the differentiation direction of the cells; E is the immunofluorescence staining of MAP2, GFAP, DAPI (scale: 50 um) of NSPC after 7 days of culture on the nanofilament of Example 1, to observe the differentiation direction of the cells; F is the immunofluorescence staining of MAP2, GFAP, DAPI (scale: 50 um) of NSPC after 7 days of culture on the nanofilament of Example 2, to observe the differentiation direction of the cells; G is the immunofluorescence staining of NF200, vGlut2, DAPI (scale: 50 um) of NSPC after 7 days of culture on ordinary 2D culture dish, to observe the proportion of excitatory neurons; H is the immunofluorescence staining of NF200, vGlut2, DAPI (scale: 50 um) of NSPC after 7 days of culture on the nanofilament of Example 1, to observe the proportion of excitatory neurons; I is the immunofluorescence staining of NF200, vGlut2, DAPI (scale: 50 um) of NSPC after 7 days of culture on the nanofilament of Example 2, to observe the proportion of excitatory neurons; J is the immunofluorescence staining of Tuj-1 (scale: 50 um) of NSPC neurospheres after 7 days of culture on the surface of the nanofilament of Example 1, to observe the length of axon extension; K is the immunofluorescence staining of Tuj-1 (scale: 50 um) of NSPC neurospheres after 7 days of culture on the surface of the nanofilament of Example 2, to observe the length of axon extension; L is the quantitative analysis of the length of axon extension of the nanofilament of Example 1 and the nanofilament of Example 2; M is the quantitative analysis of the complexity of axon of the nanofilament of Example 1 and the nanofilament of Example 2; N is the proportion of Ki67 + cells after 7 days of culture of NSPC in each group; O is the proportion of SOX2 + cells after 7 days of culture of NSPC in each group; P is the fluorescence intensity quantitative diagram of MAP2 after 7 days of culture of NSPC in each group; Q is the fluorescence intensity quantitative diagram of GFAP after 7 days of culture of NSPC in each group; R is the fluorescence intensity quantitative diagram of NF200 after 7 days of culture of NSPC in each group; S is the fluorescence intensity quantitative diagram of vGlut2 after 7 days of culture of NSPC in each group. DETAILED DESCRIPTION

[0024] A preparation method of an in-vitro spinal cord model based on nanofilament yarn according to a first exemplary embodiment of the present application comprises the following steps:

[0025] S1, stirring and dissolving a spinning material and a drug in a solvent to form a spinning solution, using a rotating cylinder as a directional electrospinning device of a nanofiber collector to spin the solution into uniaxially arranged nanofibers, twisting the nanofibers formed on the drum collector to form a primary yarn, and using a stretching device to stretch the primary yarn to obtain a nanofilament yarn;

[0026] S2, stirring and dissolving a spinning material in a solvent to form a spinning solution, using a rotating cylinder as a directional electrospinning device of a nanofiber collector to spin the solution into uniaxially arranged nanofibers, cutting the nanofibers on the collector into a 2D film along the longitudinal direction of the collector, winding the 2D film into a hollow tube, and filling the nanofilament yarn into the hollow tube to obtain a spinal cord-like scaffold;

[0027] S3, planting stem cells in the spinal cord-like scaffold to obtain an in-vitro spinal cord model based on nanofilament yarn.

[0028] In one or more embodiments of the embodiment, the spinning material in step S1 is silk fibroin and polycaprolactone, and the mass ratio of polycaprolactone to silk fibroin is 3-5:1; the solvent in step S1 is hexafluoroisopropanol, and the mass concentration of the spinning material is 5-15%; and the drug in step S1 is curcumin, and the mass of the drug is 0-5% of the mass of the spinning material.

[0029] In one or more embodiments of the embodiment, the applied voltage for electrospinning in step S1 is 8-16 kV, the spinning distance is 3-100 cm, the feeding rate of the spinning solution is 0.1-5 mL / h, the spinning time is 1-60 min, and the rotating speed of the drum collector is 800-3000 r / min.

[0030] In one or more embodiments of the embodiment, the drawing ratio of the drawing in step S1 is 5-100%.

[0031] In one or more embodiments of the embodiment, the spinning material in step S2 is polycaprolactone, and the solvent is hexafluoroisopropanol, and the mass concentration of the spinning material is 5-15%.

[0032] In one or more embodiments of the embodiment, the applied voltage for electrospinning in step S2 is 8-16 kV, the spinning distance is 3-100 cm, the feeding rate of the spinning solution is 0.1-5 mL / h, the spinning time is 10-360 min, and the rotating speed of the drum collector is 800-3000 r / min.

[0033] In one or more embodiments of this embodiment, the diameter of the hollow tube in step S2 is 1-30 mm.

[0034] In one or more embodiments of this embodiment, the stem cells in step S3 are any one or a combination of several of neural stem / progenitor cells, pluripotent induced stem cells, mesenchymal stem cells, oligodendrocyte precursor cells, olfactory ensheathing cells, Schwann cells, and downstream cells differentiated from the above-mentioned cells.

[0035] In a second typical embodiment of the present application, a nanofilament-based in vitro spinal cord-like model is obtained by the preparation method of the nanofilament-based in vitro spinal cord-like model described above.

[0036] In a third typical embodiment of the present application, the application of the nanofilament-based in vitro spinal cord-like model described above in the field of biomimetic models.

[0037] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with specific examples and comparative examples.

[0038] Example 1

[0039] Polycaprolactone and silk fibroin were dissolved in a hexafluoroisopropanol solvent at a mass ratio of 4:1, and the total mass concentration of polycaprolactone and silk fibroin was 10%, to obtain a spinning solution. The prepared solution was loaded into a syringe with a needle. A rotating cylinder was used as a directional electrospinning device for the nanofiber collector to spin the solution into uniaxially arranged nanofibers. The applied voltage, spinning distance, and solution feeding rate were set to 12 kV, 16 cm, and 0.8 mL / h, respectively. The rotating speed of the drum collector was 1500 r / min. After electrospinning for 8 min, the electrospun nanofibers formed on the drum collector were twisted to form a roving. Then, the roving was stretched at a draft ratio of 20% using a stretching device to obtain a nanofilament.

[0040] Polycaprolactone was dissolved in a hexafluoroisopropanol solvent to prepare a uniform spinning solution with a polymer mass concentration of 10%. The prepared solution was loaded into a syringe with a needle. A rotating cylinder was used as a directional electrospinning device for the nanofiber collector to spin the solution into uniaxially arranged nanofibers. The applied voltage, spinning distance, and solution feeding rate were set to 12 kV, 16 cm, and 0.8 mL / h, respectively. The rotating speed of the drum collector was 1500 r / min. After electrospinning for 90 min, the nanofibers on the collector were cut into 2D membrane pieces along the longitudinal direction of the collector. Subsequently, the 2D nanofiber membrane was wound into a hollow tube with a diameter of 3 mm. The nanofilament was filled into the hollow tube to obtain a spinal cord-like scaffold.

[0041] NSPCs were extracted from Sprague-Dawley (SD) rats. First, the cortex of E13-15 day fetal rats was extracted, then enzymolysis was performed using ACCUTASE for 5 min at 37℃, and a single cell suspension was obtained by passing through a 40-um cell strainer. The cell suspension was dispersed in NSPC proliferation medium (DMEM / F12 + B27 + EGF + FGF + penicillin-streptomycin), and incubated at 37℃ in a humidified atmosphere of 5% CO2. The cells were half-changed every 2 days, and the NSPCs were subcultured once a week. The NSPCs subcultured to the 4th generation were enzymolysed into a single cell suspension using ACCUTASE. About 1 x 10 5 -1 x 10 7 / mL cells were seeded in the sterilized NY inside the spino-cord-like scaffold along the fiber arrangement direction of NY, and the system was cultured in NeuroCult™ differentiation medium (Stemcells, Canada), half-changed every 2 days, for 7 days, to obtain a nanowire-based in vitro spino-cord-like model.

[0042] Example 2

[0043] Polycaprolactone and silk fibroin were dissolved in a hexafluoroisopropanol solvent at a mass ratio of 4:1, and the total mass concentration of polycaprolactone and silk fibroin was 10%. Curcumin was added in an amount of 1% of the total mass of polycaprolactone and silk fibroin to obtain a spinning solution. The prepared solution was loaded into a syringe with a needle. A rotating cylinder was used as a directional electrospinning device for the nanofiber collector to spin the solution into uniaxially arranged nanofibers. The applied voltage, spinning distance, and solution feeding rate were set to 12 kV, 16 cm, and 0.8 mL / h, respectively. The rotating speed of the drum collector was 1500 r / min. After electrospinning for 8 min, the electrospun nanofibers formed on the drum collector were twisted to form a roving. Then, the roving was stretched at a draft rate of 20% using a stretching device to obtain a nanowire.

[0044] Polycaprolactone was dissolved in a hexafluoroisopropanol solvent to prepare a uniform spinning solution with a polymer mass concentration of 10%. The prepared solution was loaded into a syringe with a needle. A rotating cylinder was used as a directional electrospinning device for the nanofiber collector to spin the solution into uniaxially arranged nanofibers. The applied voltage, spinning distance, and solution feeding rate were set to 12 kV, 16 cm, and 0.8 mL / h, respectively. The rotating speed of the drum collector was 1500 r / min. After electrospinning for 90 min, the nanofibers on the collector were cut into 2D membrane pieces along the longitudinal direction of the collector. Subsequently, the 2D nanofiber membrane was wound into a hollow tube with a diameter of 3 mm. The nanowire was filled into the hollow tube to obtain a spino-cord-like scaffold.

[0045] NSPCs were extracted from Sprague-Dawley (SD) rats. First, the cortex of E13-15 day fetal rats was extracted, then enzymolysis was performed using ACCUTASE for 5 min at 37°C, and a single cell suspension was obtained by passing through a 40-um cell strainer. The cell suspension was dispersed in NSPC proliferation medium (DMEM / F12 + B27 + EGF + FGF + penicillin-streptomycin), and incubated at 37°C in a humidified atmosphere of 5% CO2. The cells were half-volume exchanged every 2 days, and the NSPCs were subcultured once a week. The NSPCs subcultured to the 4th generation were enzymolysed into a single cell suspension using ACCUTASE. About 1 x 10 5 -1 x 10 7 / mL cells were seeded in the sterilized NY inside the spino-cord-like scaffold along the fiber arrangement direction of NY, and the system was cultured in NeuroCult™ differentiation medium (Stemcells, Canada), half-volume exchanged every 2 days, and cultured for 7 days to obtain a nanoyarn-based in vitro spino-cord-like model.

[0046] Example 3

[0047] Polycaprolactone and silk fibroin were dissolved in a hexafluoroisopropanol solvent at a mass ratio of 4:1, and the total mass concentration of polycaprolactone and silk fibroin was 10%. Curcumin was added in an amount of 5% of the total mass of polycaprolactone and silk fibroin to obtain a spinning solution. The prepared solution was loaded into a syringe with a needle. A rotating cylinder was used as a directional electrospinning device for the nanofiber collector to spin the solution into uniaxially arranged nanofibers. The applied voltage, spinning distance, and solution feeding rate were set to 12 kV, 16 cm, and 0.8 mL / h, respectively. The rotating speed of the drum collector was 1500 r / min. After electrospinning for 8 min, the electrospun nanofibers formed on the drum collector were twisted to form a roving. Then, the roving was stretched at a draft rate of 20% using a stretching device to obtain a nanoyarn.

[0048] Polycaprolactone was dissolved in a hexafluoroisopropanol solvent to prepare a uniform spinning solution with a polymer mass concentration of 10%. The prepared solution was loaded into a syringe with a needle. A rotating cylinder was used as a directional electrospinning device for the nanofiber collector to spin the solution into uniaxially arranged nanofibers. The applied voltage, spinning distance, and solution feeding rate were set to 12 kV, 16 cm, and 0.8 mL / h, respectively. The rotating speed of the drum collector was 1500 r / min. After electrospinning for 90 min, the nanofibers on the collector were cut into 2D membrane pieces along the longitudinal direction of the collector. Subsequently, the 2D nanofiber membrane was wound into a hollow tube with a diameter of 3 mm. The nanoyarn was filled into the hollow tube to obtain a spino-cord-like scaffold.

[0049] NSPCs were extracted from Sprague-Dawley (SD) rats. First, the cerebral cortex of E13-15 day fetal rats was extracted, then enzymolyzed with ACCUTASE for 5 min at 37 °C, and a single cell suspension was obtained by passing through a 40-um cell strainer. The cell suspension was dispersed in NSPC proliferation medium (DMEM / F12 + B27 + EGF + FGF + penicillin-streptomycin), and incubated at 37 °C in a humidified atmosphere of 5% CO2. The cells were half-volume exchanged every 2 days, and the NSPCs were subcultured once a week. The NSPCs subcultured to the 4th generation were enzymolyzed into a single cell suspension with ACCUTASE. About 1 x 10 5 -1 x 10 7 mL cells were seeded in the sterilized NY inside the spinal cord-like scaffold along the fiber arrangement direction of NY, and the system was cultured in NeuroCult™ differentiation medium (Stemcells, Canada), half-volume exchanged every 2 days, for 7 days to obtain a nanoyarn-based in vitro spinal cord-like model.

[0050] Experimental Example 1

[0051] As shown in Figure 1 B-G, scanning electron microscopy showed that the spinal cord-like scaffolds of Examples 1-3 all had smooth surfaces and oriented morphologies, with average yarn diameters of 281.4 ± 5.2, 239.9 ± 4.4, and 217.2 ± 2.1 μm Figure 1 I), respectively, and average internal nanofiber diameters of 610.5 ± 74.9 nm, 541.9 ± 52.1 nm, and 483.7 ± 47.2 nm Figure 1 J), respectively. Digital photos of the spinal cord-like scaffolds are shown in Figure 1 H.

[0052] The tensile strength of the spinal cord-like scaffolds of Examples 1-3 was tested to evaluate their mechanical properties. All samples showed similar stress-strain curves, as shown in Figure 1 K. As shown in Figure 1 L-N, compared with Example 1, Examples 2 and 3 showed higher Young’s modulus and ultimate strength, which was mainly due to the decrease in nanoyarn diameter caused by curcumin loading.

[0053] The chemical groups of the spinal cord-like scaffolds of Examples 1-3 were characterized by Fourier transform infrared spectroscopy. As shown in Figure 1 P, for Example 1, the peak at 2949 cm -1 was attributed to the stretching vibration of -CH2, while the peak at 1471 cm -1 corresponded to its bending vibration. The peak at 1722 cm -1 belonged to the C=O stretching vibration. The peak at 1296 cm -1The peaks at 1240, 1166, and 1049 cm⁻¹ are attributed to the stretching vibrations of the CO / CC groups. -1 The nearby peaks correspond to the tensile vibrations of CO. All characteristic peaks originating from the polycaprolactone polymer were detected. (At 1645 cm⁻¹) -1 and 1538cm -1 The peak centered on the amide region is responsible for amide regions I and II, both of which are characteristic peaks of the β-sheet of silk fibroin. Therefore, no new chemical groups are generated during the fabrication of the spinal cord scaffold. Figure 1 As shown in Q, X-ray diffraction (XRD) analysis revealed that the spinal cord-like scaffolds of Examples 1-3 all exhibited two sets of diffraction peaks located at approximately 21.3° and 23.6°, corresponding to the (110) and (200) crystal planes, respectively. In contrast, lower peaks were detected in Examples 2 and 3, indicating that the addition of curcumin reduced crystallinity to some extent.

[0054] Plot the release curve of curcumin, such as... Figure 1 As shown in Figure O, the potential of the spinal cord-like scaffold in drug screening and delivery was evaluated. The spinal cord-like scaffold exhibited a two-stage release pattern, with a burst release of some degree of drug within 24 hours (7.06% and 2.53% in Examples 2 and 3, respectively), followed by a slow linear release pattern over 49 days (10.96% and 9.68% in Examples 2 and 3, respectively). Since the sustained release of curcumin provides long-term neuroprotection and allows for neurogenesis, the curcumin-loaded spinal cord-like scaffold has the therapeutic potential to promote the repair of injured spinal cords; furthermore, the above results also demonstrate the potential of the spinal cord-like scaffold as a drug screening platform.

[0055] Experimental Example 2

[0056] PC12 type neurons were seeded onto ordinary two-dimensional tissue culture plates (control group), nanoyarns of Example 1, and nanoyarns of Example 2, as follows: Figure 2 As shown in AF and 2M, live cells constituted the majority of the cell population (>95%), and almost no dead cells were observed after 1 to 3 days of culture, confirming the biocompatibility of the nanoyarn (F(2,6) = 0.091, P = 0.9118). Key features of live cells on the nanoyarn surface were reconstructed using an ultra-high resolution 3D imaging system, such as... Figure 2 As shown in G and 2H, cells are uniformly distributed on NY, capable of sensing aligned nanostructures and reshaping their morphology parallel to the direction of the nanofibers. After 1 day of culture, as... Figure 2 As shown in JL, NSPCs exhibit a highly oriented arrangement on nanofibers, whereas they are randomly oriented on ordinary 2D culture dishes. The Cell Counting Kit-8 (CCK-8) was used to study the proliferation ability of NSPCs on nanofibers, such as...Figure 2 As shown in Figure 1, the results showed that the proliferation rate of cells on Example 1 and 2 was significantly reduced (F(4, 18) = 11.35, P < 0.0001) during the 7-day culture, which might be related to the reduction of NSPCs stemness and promotion of differentiation by nanoyarns.

[0057] Experimental Example 3

[0058] After culturing NSPCs on normal two-dimensional culture dish (control group), nanoyarns of Example 1 and nanoyarns of Example 2 for 7 days, the expression of cell proliferation marker Ki67 and cell stemness marker SOX2 were examined, as shown in Figures 2A-C. Figure 3 The results showed that nanoyarns significantly reduced the proportion of Ki67 + proliferating cells (from 58.64% ± 4.26% to 9.25% ± 3.40%) (F(2, 12) = 331.1, P < 0.0001) and SOX2 + stem cells (from 15.09% ± 3.18% to 1.05% ± 0.93%) (F(1, 12) = 91.95, P < 0.00001) (Figures 2D-F, P, Q). Figure 3 As shown in Figures 2D-F, P, Q, the expression level of microtubule-associated protein MAP2, which specifically locates in mature neuron dendrites, was increased in Example 1 and Example 2 groups (F(2, 6) = 183.1, P < 0.0001), while the astrocyte marker GFAP was significantly reduced (F(1, 6) = 19.47, P < 0.001), which indicated that nanoyarns induced NSPCs to differentiate into neuron differentiation rather than astrocytes. Figure 3 vGlut2 (marker of excitatory neurons with glutamatergic phenotype) and NF200 (component of mature neuron axon cytoskeleton) immunofluorescence staining showed that nanoyarns significantly increased the density of NF200 + neurons, while vGlut2 + vesicles were hardly detected in normal culture dish (Figures 2G-I, R, S). Figure 3 As shown in Figures 2J, K, NSPCs neurospheres were cultured on nanoyarns of Example 1 and nanoyarns of Example 2 for 7 days, and the growth of neurite and branching complexity of neurons were evaluated. As shown in Figures 2L, M, Sholl quantitative analysis showed that NY and Cur significantly increased the length and number of axons. In summary, nanoyarns and curcumin promoted NSPCs to differentiate into excitatory neurons and induced their maturation. Figure 3 Figure 3

[0059] ​​The above merely provides the preferred embodiments of the present application, and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the principles and technical scope of the present application shall fall into the scope of the present application.

Claims

1. A method for preparing a nanofilament-based in vitro spinal cord model, characterized by, The method comprises the following steps: S1, stirring and dissolving a spinning material and a drug in a solvent to form a spinning solution, using a rotating cylinder as a directional electrospinning device of a nanofiber collector to rotate the spinning solution into uniaxially arranged nanofibers, twisting the nanofibers formed on the drum collector to form an initial yarn, and using a stretching device to stretch the initial yarn to obtain a nanofiber yarn; S2, stirring and dissolving a spinning material in a solvent to form a spinning solution, using a rotating cylinder as a directional electrospinning device of a nanofiber collector to rotate the spinning solution into uniaxially arranged nanofibers, cutting the nanofibers on the collector into 2D film pieces along the longitudinal direction of the collector, winding the 2D film pieces into a hollow tube, and filling the nanofiber yarn into the hollow tube to obtain a spinal cord-like scaffold; S3, planting stem cells in the sterilized nanofiber yarn inside the spinal cord-like scaffold along the fiber arrangement direction of the nanofiber yarn, and culturing the obtained system in a NeuroCultTM differentiation medium, performing half-volume liquid exchange every 2 days, and continuously culturing for 7 days to obtain an in-vitro spinal cord-like model based on a nanofiber yarn. In step S1, the spinning material is silk fibroin and polycaprolactone, and the mass ratio of polycaprolactone to silk fibroin is 3-5:1; in step S1, the solvent is hexafluoroisopropanol, and the mass concentration of the spinning material is 5-15%; in step S1, the drug is curcumin, and the mass of the drug is 1-5% of the mass of the spinning material. In step S2, the spinning material is polycaprolactone, and the solvent is hexafluoroisopropanol; the mass concentration of the spinning material is 5-15%. In step S3, the stem cells are neural stem / progenitor cells.

2. The method for preparing a nanofilament-based in vitro spinal cord-like model according to claim 1, wherein, In step S1, the applied voltage of the directional electrospinning device is 8-16 kV, the spinning distance is 3-100 cm, the feeding rate of the spinning solution is 0.1-5 mL / h, the spinning time is 1-60 min, and the rotating speed of the drum collector is 800-3000 r / min.

3. The method for preparing an in vitro spinal cord-like model based on nanoyarn as described in claim 1, characterized in that, In step S1, the draft rate of the draft is 5-100%.

4. The method for preparing an in vitro spinal cord-like model based on nanoyarn as described in claim 1, characterized in that, In step S2, the applied voltage of the directional electrospinning device is 8-16 kV, the spinning distance is 3-100 cm, the feeding rate of the spinning solution is 0.1-5 mL / h, the spinning time is 10-360 min, and the rotating speed of the drum collector is 800-3000 r / min.

5. The method for preparing an in vitro spinal cord-like model based on nanoyarn as described in claim 1, characterized in that, In step S2, the diameter of the hollow tube is 1-30 mm.

6. An in vitro spinal cord-like model based on nanofilaments, characterized in that, The in-vitro spinal cord-like model based on a nanofiber yarn is obtained by the preparation method of any one of claims 1-5.

7. The in-vitro spinal cord-like model based on a nanofiber yarn of claim 6 in the field of biomimetic models.

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