Bionic nanofiber-hydrogel composite biological scaffold as well as preparation method and application thereof

Abstract

The invention discloses a bionic nanofiber-hydrogel composite biological scaffold as well as a preparation method and application thereof. The bionic nanofiber-hydrogel composite biological scaffold is a composite scaffold for simulating a gray-white matter partition structure of a spinal cord tissue, and comprises bionic nanofibers and a bionic hydrogel material, wherein the bionic nanofibers are arranged in an oriented manner and simulate a white matter ordered structure around the spinal cord, and the bionic hydrogel material is combined with the bionic nanofibers and simulates gray matter in the center of the spinal cord. According to the bionic nanofiber-hydrogel composite biological scaffold, the biological activity of loaded stem cells or adult cells can be improved, the problem that a large number of transplanted exogenous cells die due to unbalanced oxidative stress generated after tissue damage can be solved, and it is guaranteed that the transplanted cells reside in a damaged area and play a role; therefore, the spinal cord microenvironment is improved, proliferation and differentiation of endogenous neural stem cells are promoted, functional recovery after spinal cord injury is finally promoted, and great potential is achieved in spinal cord injury treatment.

Description

Technical Field

[0001] The present invention relates to a biological scaffold. Specifically, it relates to a bionic nanofiber-hydrogel composite biological scaffold and its preparation method, as well as its application in the preparation of products for treating spinal cord injury, belonging to the technical field of biomedical materials. Background Art

[0002] The spinal cord is an important part of the central nervous system, located within the spinal column, connecting the brain to the body's neural network. It is a core organ that maintains human movement, sensation, vital activities, and the ability to adapt to the environment. Its importance lies in its function of integrating and transmitting information, as well as its irreplaceable role in life support activities. Spinal cord injury (SCI) can lead to severe motor, sensory, and autonomic dysfunction, with consequences including quadriplegia or paraplegia, loss of sensation, bladder and bowel incontinence, sexual dysfunction, and chronic pain. SCI is caused by primary mechanical injury, amplified through a secondary injury cascade, resulting in the destruction and loss of neural and vascular units. Due to the limited regenerative ability of spinal cord neurons, the complex structure and function of the spinal cord, the complex pathological responses after injury, and the limitations of existing treatment methods, the regenerative repair of SCI remains one of the most challenging medical problems. To date, the treatment of spinal cord injury has mainly focused on immobilizing the spine to reduce secondary damage and improving living ability through rehabilitation training, but there is still a lack of effective means for neural function regeneration and repair [SILVANA, et al. Progress in Neurobiology, 2014, 114: 25-57].

[0003] Neurovascular development occurs in parallel between the nervous system and the vascular system in the early stage of embryogenesis. Only after the blood vessels in the perineural vascular plexus invade the central nervous system (CNS) during the development of the neural tube can the CNS continue to develop and enhance its functions. At the same time, the developing nervous system releases signals that cause the vascular buds to migrate in specific directions. During development, endothelial cells, pericytes, neurons, and astrocytes are closely associated. Their dynamic interactions support the ultimate formation of the complex blood-spinal cord barrier (BSCB) in the spinal cord. Its low permeability plays a very important role in protecting spinal cord tissue from interference by pathogens and the like. The signal pathways VEGF / VEGFR and the classical Wnt / β-catenin signal pathway have been shown to be closely related to vascular development and neural development. For example, the VEGF receptor sFlt-1 has been shown to be related to the migration of angioblasts, and the deletion of the Wnt7a / b gene also leads to difficulty in the entry of newly formed vascular buds into the neural tube to participate in neurovascular development [BAUTCH VL, et al. Cell Adhesion&Migration, 2009, 3(2): 199]. At the cellular level, this structure composed of endothelial cells and nerve cells is called the neurovascular unit (NVU). The NVU is a structural and functional complex composed of neurons, endothelial cells, glial cells, basement membrane, pericytes, and smooth muscle cells, and plays a crucial role in the interaction between neural activity and microcirculation. In addition to being responsible for the delivery of oxygen and nutrients to assist metabolism, it is also responsible for coupling intercellular signal transduction and maintaining the microenvironmental homeostasis of neural activity [RADU BM, et al. Mediators of Inflammation, 2013: 648268].

[0004] In the treatment of various neurological diseases, restoring the normal function of nerve cells is the ultimate goal, and this repair requires the co-reconstruction of nerves and blood vessels. After SCI, the BSCB is damaged, and its membrane permeability increases significantly, causing the spinal cord to be in a state of ion imbalance and infiltration of neurotoxic substances. A large number of inflammatory mediators and free radicals damage neuron function and exacerbate the occurrence and development of inflammation, resulting in secondary injury of the spinal cord [XU J, et al. Experimental Neurology, 2023, 359: 114273]. Due to the important influence of neurovascular interaction on maintaining neurogenesis, repair and neuron differentiation, a large number of studies have focused on restoring vascular reconstruction after SCI [ZHOU R, et al. Journal of Cerebral Blood Flow and Metabolism, 2023, 43: 1437]. The damage of NVU occurs before the degeneration of motor neurons, and repairing NVU can help functional recovery. Moreover, there is a close connection between angiogenesis and neurogenesis in spinal cord injury. Repairing blood vessels through angiogenic factors, genetic engineering, physical stimulation, cell transplantation and biomaterial implantation has a significant effect on the recovery of nerve function after SCI [YAO C, et al. Frontiers in Physiology, 2021, 12: 631500]. For example, in 2021, Ye et al. injected exogenous platelet-derived growth factor on the wound surface of SCI mice and found that it reduced the permeability of BSCB and protected NVU by promoting angiogenesis, reducing neuron apoptosis, inhibiting the excessive proliferation of astrocytes and promoting collagen synthesis [YE LX, et al. Neural Regeneration Research, 2021, 16(4): 765].

[0005] Endothelial cells, as an important component of the NVU, can secrete a variety of factors that promote nerve regeneration. The research by C. Leventhal et al. in 1999 showed that, compared with fibroblasts and astrocytes, when co-cultured with endothelial cells, explants from the subventricular zone of adult rats exhibited enhanced neuronal proliferation and neurite outgrowth [LEVENTHAL C, et al. Molecular and Cellular Neuroscience, 1999, 13(6): 450-464]; in 2004, Shen et al. co-cultured neural stem cells with vascularization-related cells such as endothelial cells and fibroblasts and found that endothelial cells enhanced the self-renewal ability of neural stem cells by stimulating the upregulation of Hes1 in the Notch signaling pathway, and its derived factors rather than endothelial cells themselves were good factors for promoting the differentiation of neural stem cells into neurons [SHEN Q, et al. Science, 2004, 304: 1338-1340]. Similarly in 2021, Papon Muangsanit et al. prepared a collagen gel containing human umbilical vein endothelial cells (Human Umbilical Vein Endothelilal Cells, HUVEC) for the treatment of transected sciatic nerves in rats and found that HUVEC formed aligned tubular structures in the tethered gel, which could promote the axonal growth of Schwann cells and was shown to have a significant ability to support the extension of neurons across the repair site [MUANGSANIT P, et al. Acta Biomaterialia 2021, 126: 224]. In 2023, Zhifeng You et al. for the first time compared the differences in the repair of spinal cord injury (SCI) between transplanted spinal cord microvascular endothelial cells (Spinal Cord Microvascular Endothelial Cells, SCMECs) and brain microvascular endothelial cells (Brain Microvascular Endothelial Cells, BMECs) and found that SCMECs were more capable of promoting neovascularization in the SCI injury area and showed better neuronal regeneration ability, promoting functional recovery after SCI [YOU Z, et al. Bioactive Materials, 2023, 29: 36-49].

[0006] To fill the cavity of the injured spinal cord and provide support for exogenous transplanted cells and endogenous nerve cells, biomaterials are often used to repair damaged nerve tissues and guide axonal growth. Among them, nanofiber scaffolds have the ability to well mimic the fascicular nerve structure and fibrous extracellular matrix. Research shows that neurites prefer aligned fibers rather than randomly arranged fibers. Nerve cells have more significant proliferation characteristics on aligned fibers, and disordered fibers also inhibit the migration and elongation of astrocytes [FACCENDINI A, et al. Pharmaceuticals, 2017, 10(3): 63]. Therefore, it plays an important role in the treatment of spinal cord injury and provides support for nerve regeneration and tissue repair [LIAO S, et al. Materials Today Bio, 2022, 17: 100454]. In 2023, Zhenni Chen et al. used decellularized spinal cord matrix (DSCM) as raw material to prepare aligned decellularized spinal cord fibers (A-DSCF) through electrospinning technology. A-DSCF not only retains various types of spinal cord extracellular matrix proteins to mimic natural spinal cord tissue, but also exhibits more prominent mechanical properties and enzyme stability than aligned collagen fibers, promotes axonal growth and synapse formation of neurons along the fiber bundle, and significantly promotes myelin formation in SCI and helps the recovery of motor function after loading neural progenitor cells (NPCs) [CHEN Z, et al. ACS Nano, 2023, 17(24): 25591-25613].

[0007] However, nanofiber scaffolds have defects in spinal cord injury applications. The pore size of nanofiber scaffolds is usually small, which is not conducive to the migration of cells into the interior of the scaffolds; and their relatively rigid mechanical strength affects nerve repair. Therefore, the nanofiber-hydrogel composite (NHC), which combines the mechanical properties of hydrogels and the characteristics of ordered nanofibers, is expected to become a new strategy for the effective treatment of spinal cord injury. The three-dimensional structure of the hydrogel and the arrangement structure of the fiber scaffold can provide support similar to the extracellular matrix of spinal cord tissue for nerve cells, promoting the adhesion, proliferation, and growth of nerve cells. Guide axons to grow in a specific direction, help neurons reconnect, and repair damaged neural networks. Fill the gap of spinal cord injury and provide a protective support environment, which helps to reduce secondary injury [LI J, JI Z, et al. Biology - Basel, 2022, 11(5): 781]; Secondly, hydrogels and nanofiber scaffolds can be used as carriers for loading cells and active factors. Loading them into the injury site can reduce the loss and off-target of cells and active factors, load neurotrophic factors (such as brain-derived neurotrophic factor, glial cell-derived neurotrophic factor) or other drugs, and slowly release these factors by controlling the degradation rate to promote the survival and growth of neurons. Deliver anti-inflammatory drugs, antioxidants, or anti-apoptotic factors to reduce the inflammatory response and oxidative stress after spinal cord injury and reduce secondary injury [SILVAD, et al. Advanced Healthcare Materials, 2023, 12(17): e2202803]. For example, in 2020, Xiaowei Li et al. treated SCI rats by injecting a composite material of hyaluronic acid hydrogel and PCL nanofibers into the injury center, which improved the inflammation in the injury microenvironment, enhanced angiogenesis, and provided a favorable growth environment for axon growth while restricting spinal cord collapse [LI X, et al. Biomaterials, 2020, 245: 119978]. Furthermore, in 2022, Haggerty et al. interacted PCL nanofibers with a hydrogel formed by mercapto-functionalized hyaluronic acid (HA-SH) and PEGDA to prepare an injectable NHC, and combined it with mesenchymal stromal cells (MSCs) for the treatment of spinal cord contusion. The results showed that the NHC scaffold could reduce the early inflammation in the contusion by promoting the polarization of macrophages in the injury site to the M2 phenotype and support the survival of axons and astrocytes after injury, fully demonstrating the protective effect of NHC combined with MSC transplantation on nerve tissue [HAGGERTY AE, et al. Cells, 2022, 11(7): 1137].

[0008] After SCI, vascular and neuronal injuries release free radicals and pro-inflammatory molecules, generating a large amount of reactive oxygen species (ROS), continuous infiltration and activation of inflammatory cells, which trigger subsequent secondary injuries. Therefore, improving the extracellular environment of the injured spinal cord is also an important regulatory factor in the treatment of SCI. Reactive oxygen species play an important role in spinal cord injury. Although a small amount of ROS is generated by the body's metabolism under physiological conditions, the body's antioxidant system can promptly remove it. After injury, local hypoxia and insufficient blood flow enhance the activity of the in vivo ROS-generating enzyme system, leading to the generation of a large amount of reactive oxygen species. ROS cause oxidative damage to cell membranes, proteins, and DNA, resulting in cell dysfunction and death [RAO S, et al. Journal of Nanobiotechnology, 2022, 20(1): 278]. Reactive oxygen species can activate the intracellular oxidative stress pathway, induce apoptosis of neurons and glial cells, and thus exacerbate spinal cord injury. They also promote the inflammatory response, activate microglia and macrophages, secrete more inflammatory factors, and form a vicious cycle. In addition, reactive oxygen species damage the blood-spinal cord barrier, causing plasma proteins and immune cells to infiltrate into the spinal cord, further aggravating the injury. ROS also stimulate the formation of glial scars, hinder nerve regeneration, and limit the recovery after spinal cord injury [XIA M, et al. Frontiers in Aging Neuroscience, 2022, 14: 905115]. Overall, reactive oxygen species exacerbate cell death, loss of nerve function, and have a negative impact on the repair process after spinal cord injury, becoming an important factor in secondary injury [WAKATSUKI S, et al. Experimental Neurology, 2022, 352: 114024]. Therefore, the regulation of ROS is an important condition for maintaining the activity of transplanted cells and protecting endogenous NSCs. When exogenous cells are transplanted into the injury area, the high oxidative stress microenvironment triggers cell apoptosis, greatly reducing the survival rate of transplanted cells and unable to produce the required therapeutic growth factors and cytokines, thus affecting the therapeutic effect. Therefore, regulating ROS in the injury area can also weaken its damage to exogenous transplanted cells and improve the effect of cell therapy [SAHUA, et al. ACS Applied Materials & Interfaces, 2021, 13(22): 25649-25662].

[0009] In summary, the selection of biological scaffolds in spinal cord injury treatment has always been a difficult problem to solve. For a single form of biological material, such as hydrogel, although it has good degradation performance, the balance between its mechanical properties and cell permeability has always been a major problem in applications. The composite material NHC of nanofibers combined with hydrogel is on the rise. Such materials have been proven to be well applied to soft tissues such as skin and muscle. However, the research on using NHC to promote vascular and nerve regeneration after spinal cord injury is not yet perfect. After SCI, the destruction of the BSCB leads to spinal cord ischemia and hypoxia, forming an oxidative stress injury microenvironment with excessive ROS accumulation, resulting in a large number of endogenous nerve cell apoptosis. Therefore, regulating the oxidative stress microenvironment and repairing the BSCB have also become important links in the treatment of spinal cord injury. At present, the treatment of spinal cord injury with NHC mainly utilizes its characteristics as a cell and drug carrier. There is no research on using NHC to regulate the injury microenvironment to protect transplanted cells and endogenous nerve cells and enhance the treatment effect. Summary of the Invention

[0010] The main object of the present invention is to provide a bionic nanofiber-hydrogel composite biological scaffold and its preparation method to overcome the deficiencies in the prior art.

[0011] Another object of the present invention is to provide the application of the bionic nanofiber-hydrogel composite biological scaffold.

[0012] To achieve the foregoing invention objects, the technical solutions adopted by the present invention include:

[0013] An embodiment of the present invention provides a bionic nanofiber-hydrogel composite biological scaffold. The composite biological scaffold is a composite biological scaffold that mimics the gray-white matter partition structure of the spinal cord tissue, including oriented bionic nanofibers that mimic the white matter ordered structure of the peripheral part of the spinal cord, and a bionic hydrogel material that combines with the bionic nanofibers and mimics the gray matter of the central part of the spinal cord.

[0014] An embodiment of the present invention also provides a preparation method of a bionic nanofiber-hydrogel composite biological scaffold, which includes:

[0015] Making the oriented bionic nanofibers that mimic the white matter ordered structure of the peripheral part of the spinal cord fully contact with the bionic hydrogel material that mimics the gray matter of the central part of the spinal cord, and photocrosslinking to obtain a bionic nanofiber-hydrogel composite biological scaffold.

[0016] An embodiment of the present invention also provides a bionic nanofiber-hydrogel composite biological scaffold prepared by the foregoing preparation method.

[0017] Furthermore, an embodiment of the present invention also provides the application of the foregoing bionic nanofiber-hydrogel composite biological scaffold in the preparation of a product with the function of treating spinal cord injury.

[0018] Correspondingly, an embodiment of the present invention further provides a product with the function of treating spinal cord injury, which includes: the aforementioned bionic nanofiber-hydrogel composite biological scaffold, and endothelial cells loaded on the composite biological scaffold.

[0019] Compared with the prior art, the beneficial effects of the present invention at least include:

[0020] The bionic nanofiber-hydrogel composite biological scaffold provided by the present invention simulates the gray-white matter partition structure of spinal cord tissue, can improve the biological activity of the loaded stem cells or somatic cells, and can solve the problem that a large number of transplanted exogenous cells die due to unbalanced oxidative stress generated after tissue injury, ensure that the transplanted cells reside in the injury area and form and function, thereby improving the spinal cord microenvironment, promoting the proliferation and differentiation of endogenous neural stem cells, and ultimately enhancing the recovery of function after spinal cord injury, and has great potential in the treatment of spinal cord injury. Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figures 1A - 1B It is a solution diagram for preparing the PPM nanofiber scaffold in Example 1 of the present invention;

[0023] Figures 1C - 1D It is a morphology diagram of the mGL-PBA hydrogel before and after photo-crosslinking in Example 1 of the present invention;

[0024] Figure 1E It is a synthesis route diagram of mGL-PBA in Example 1 of the present invention;

[0025] Figure 1F It is a schematic diagram of electrospinning to prepare the fiber membrane PPM in Example 1 of the present invention;

[0026] Figure 1G It is a gelation schematic diagram of the mGL-PBA hydrogel in Example 1 of the present invention;

[0027] Figures 1H - 1I It is a SEM microstructural diagram of the PPM nanofiber membrane prepared in Example 1 of the present invention;

[0028] Figures 1J - 1K It is a SEM microstructural diagram of the mGL-PBA hydrogel prepared in Example 1 of the present invention;

[0029] Figure 2A Macromorphology diagram of the bionic nanofiber-hydrogel composite biological scaffold PPMB prepared in Example 1 of the present invention;

[0030] Figure 2B Schematic diagram for the preparation of the bionic nanofiber-hydrogel composite biological scaffold PPMB in Example 1 of the present invention;

[0031] Figure 2C Bonding reaction diagram of PVA and mGL-PBA in Example 1 of the present invention;

[0032] Figure 2D SEM diagrams of the bionic PPM and PPMB prepared in Example 1 of the present invention;

[0033] Figure 3A Stress-strain curve diagrams of the bionic PPM and PPMB fiber membranes prepared in Example 1 of the present invention;

[0034] Figure 3B 、 Figure 3C Tensile and compression modulus test diagrams of the bionic PPM and PPMB fiber membranes prepared in Example 1 of the present invention respectively;

[0035] Figure 3D Fracture energy test diagrams of the bionic PPM and PPMB fiber membranes prepared in Example 1 of the present invention;

[0036] Figure 3E Deformation property diagrams of the bionic PPM and PPMB fiber membranes prepared in Example 1 of the present invention;

[0037] Figure 3F 、 Figure 3G Adhesion and extension property diagrams of the PPMB fiber membrane prepared in Example 1 of the present invention respectively;

[0038] Figure 4A XPS full spectrum diagrams of the PPM and PPMB prepared in Example 1 of the present invention;

[0039] Figure 4B C 1s fitting diagrams in the XPS full spectrum diagrams of the PPM and PPMB prepared in Example 1 of the present invention;

[0040] Figure 4C B 1s fitting diagrams in the XPS full spectrum diagrams of the PPM and PPMB prepared in Example 1 of the present invention;

[0041] Figure 4D O 1s fitting diagrams in the XPS full spectrum diagrams of the PPM and PPMB prepared in Example 1 of the present invention;

[0042] Figure 4EIt is the N 1s fitting diagram in the XPS full spectrum of PPM and PPMB prepared in Example 1 of the present invention;

[0043] Figure 5A It is the FTIR spectra of PPM, mGL-PBA and PPMB prepared in Example 1 of the present invention;

[0044] Figure 5B It is the schematic diagram of the water contact angle of PPM and PPMB prepared in Example 1 of the present invention;

[0045] Figure 6A It is the schematic diagram of the results of detecting the antioxidant capacity of PPM and PPMB by the ABTS method in Example 1 of the present invention;

[0046] Figure 6B It is the schematic diagram of the results of scavenging ROS by PPM, mGL-PBA and PPMB prepared in Example 1 of the present invention over time;

[0047] Figure 7A It is the detection result diagram of the biocompatibility of PPM in Example 1 of the present invention;

[0048] Figure 7B It is the detection result diagram of the biocompatibility of PPMB in Example 1 of the present invention;

[0049] Figure 8A It is the result diagram of the relative expression level of the PECAM-1 gene in hBEC cells grown on PPM and PPMB materials in Example 1 of the present invention;

[0050] Figure 8B It is the result diagram of the relative expression level of the EPHB4 gene in hBEC cells grown on PPM and PPMB materials in Example 1 of the present invention;

[0051] Figure 8C It is the result diagram of the relative expression level of the ETV2 gene in hBEC cells grown on PPM and PPMB materials in Example 1 of the present invention;

[0052] Figure 9 It is the schematic diagram of the ability of the PPM and PPMB scaffolds prepared in Example 1 of the present invention to protect cells under in vitro simulated oxidative stress microenvironment conditions;

[0053] Figure 10A It is the result diagram of the effect of PPMB-hBEC on the proliferation of NSCs in Example 1 of the present invention;

[0054] Figure 10B and Figure 10C It is the result diagram of the effect of PPMB-hBEC on the differentiation of NSCs in Example 1 of the present invention;

[0055] Figure 11 Schematic diagram of the animal experimental model after filling the damaged area with the PPMB scaffold loaded with hBECs in Example 1 of the present invention;

[0056] Figure 12 Schematic diagram of the construction of the complete transection spinal cord injury model of SD rats in Example 1 of the present invention;

[0057] Figure 13A Schematic diagram of CD144 immunofluorescence staining in Example 1 of the present invention;

[0058] Figure 13B Statistical chart of the percentage of CD144 fluorescence area in Example 1 of the present invention;

[0059] Figure 14A Schematic diagram of Nestin immunofluorescence staining at both ends and the injury center of the SCI injury in Example 1 of the present invention;

[0060] Figures 14B - 14D Result diagram of the relative quantitative analysis of Nestin fluorescence intensity in different regions in Example 1 of the present invention;

[0061] Figure 15A Schematic diagram of Tuj 1 immunofluorescence staining in Example 1 of the present invention;

[0062] Figure 15B Result diagram of the relative quantitative analysis of Nestin fluorescence intensity in Example 1 of the present invention;

[0063] Figure 16A Immunofluorescence staining picture showing the tissue oxidative stress level by DHE staining when the bionic nanofiber-hydrogel composite biological scaffold loaded with human central nerve cells regulates the oxidative stress microenvironment in Example 1 of the present invention;

[0064] Figure 16B Result diagram of the relative quantitative analysis of the average fluorescence intensity;

[0065] Figure 17A Result diagram of the relative expression level of the TGFββ gene when the bionic nanofiber-hydrogel composite biological scaffold loaded with endothelial cells regulates the inflammatory microenvironment in Example 1 of the present invention;

[0066] Figure 17B Result diagram of the relative expression level of the Arg1 gene;

[0067] Figure 17C Schematic diagram of the ratio of the expression levels of TNF-α and Argl genes;

[0068] Figure 18 Schematic diagram of evaluating the recovery of the hind limb motor function of rats after SCI using the BBB motor assessment scale in Example 1 of the present invention. Detailed implementation manners

[0069] To improve the disadvantages of the above technologies, the inventors of this case, through long-term research and a large number of practices, were able to propose the technical solution of the present invention, mainly to design a bionic reactive oxygen species-responsive nanofiber-hydrogel composite biological scaffold and combine it with endothelial cells for the treatment of spinal cord injury.

[0070] To facilitate the understanding of this application, the following will describe this application in more detail. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the understanding of the disclosure content of the present invention more thorough and comprehensive.

[0071] Specifically, as an aspect of the technical solution of the present invention, a bionic nanofiber-hydrogel composite biological scaffold involved therein is a composite biological scaffold that mimics the gray-white matter partition structure of the spinal cord tissue, specifically including oriented bionic nanofibers that mimic the white matter ordered structure around the spinal cord, and a bionic hydrogel material that combines with the bionic nanofibers and mimics the gray matter in the central part of the spinal cord.

[0072] In some embodiments, the amount of the bionic hydrogel material combined with 1.2 cm 2 of the bionic nanofibers is 10 - 30 μL, preferably 18 - 22 μL.

[0073] In some embodiments, the bionic nanofibers (hereinafter may be abbreviated as PPM) are prepared from methacrylated gelatin (mGL), polycaprolactone (PCL), and polyvinyl alcohol (PVA). The fiber scaffold prepared based on PCL has good mechanical properties, and the mixed use of PVA improves the affinity of cell seeding and adhesion.

[0074] In some other embodiments, the bionic nanofibers can be prepared from glycyrrhetinic acid, 3-aminophenylboronic acid, polyvinyl alcohol, and epigallocatechin gallate.

[0075] In some other embodiments, the bionic nanofibers can be prepared from polycaprolactone and polyvinyl alcohol.

[0076] In some other embodiments, the bionic nanofibers can be prepared from polycaprolactone, beeswax, and polyvinyl alcohol.

[0077] In some other embodiments, the bionic nanofibers can be prepared from fish collagen and polycaprolactone.

[0078] In some other embodiments, the bionic nanofibers can be prepared from citral, polycaprolactone, and polyvinyl alcohol.

[0079] In some other embodiments, the bionic nanofibers can be prepared from indomethacin, polycaprolactone, polyhexamethylene guanidine hydrochloride, and polyvinyl alcohol.

[0080] In some embodiments, the bionic hydrogel material (hereinafter may be simply referred to as mGL-PBA hydrogel) is prepared from methacrylated gelatin (mGL) and a compound containing a phenylboronic acid group. The phenylboronic acid groups grafted on the bionic hydrogel material can specifically couple with the polyvinyl alcohol component in the bionic nanofibers and interact to form phenylborate ester bonds.

[0081] Furthermore, the compound containing a phenylboronic acid group includes 3-aminomethylphenylboronic acid hydrochloride, but is not limited thereto.

[0082] In some other embodiments, the bionic hydrogel material can be prepared from alginate, phenylboric acid, and polyvinyl alcohol.

[0083] In some embodiments, the bionic nanofibers are prepared by a dual-nozzle electrospinning technique. The present invention uses the dual-nozzle electrospinning technique to prepare oriented PPM and combines it with mGL-PBA hydrogel for secondary forming into a cylindrical scaffold with a hydrogel in the center and ordered fibers and hydrogel on the periphery. Filling it in the damaged area can connect the two ends of the damaged spinal cord area, simulate the arrangement structure and gray and white matter structure of the natural spinal cord internal nerve bundles, thereby connecting the head and tail of the spinal cord injury and replacing the lost spinal cord tissue extracellular matrix.

[0084] Furthermore, the bionic nanofibers can be bionic nanofiber membranes.

[0085] Furthermore, the bionic nanofibers are nanoscale. Specifically, the diameter of the bionic nanofibers is 100 - 500 nm, and the porosity is 40 - 75%.

[0086] Compared with the existing simple physical mixing method, the bionic nanofiber-hydrogel composite biological scaffold provided by the present invention can strengthen the structural integrity through the interfacial bonding between the gel and fiber components and maintain sufficient mechanical properties and porosity to provide support for the growth and migration of nerve cells, endothelial cells, etc.

[0087] Even further, the phenylboronic acid (PBA) groups grafted on the bionic hydrogel material can specifically couple with the polyvinyl alcohol (PVA) component with a diol structure in the bionic nanofiber PPM and interact to form phenylborate ester bonds. The interaction between the two strengthens the connection between the hydrogel and the nanofibers; at the same time, it can also specifically react with ROS, effectively scavenging the ROS that breaks out after spinal cord injury, improving the damaged microenvironment, thereby reducing the damage to transplanted endothelial cells and endogenous cells and increasing the survival rate.

[0088] Furthermore, the pore size of the bionic hydrogel material is 10 - 200 μm.

[0089] In summary, the bionic nanofiber-hydrogel composite biological scaffold of the present invention has good mechanical properties and biocompatibility. The prepared cylindrical structure can macroscopically simulate natural spinal cord tissue, and its longitudinally arranged nanofibers can also simulate spinal nerve bundles, facilitating the migration of neurites along the fiber arrangement direction and promoting nerve regeneration. It can effectively maintain the growth and adhesion of endothelial cells and nerve cells, reduce the reactive oxygen species generated in the center of spinal cord injury, thereby reducing the death of nerve cells. It can also generate new blood vessels in the injury area, provide relevant active factors and oxygen for the differentiation of neural stem cells, and increase the probability of neural stem cells differentiating into neurons.

[0090] Furthermore, the material provided by the present invention relies on the cis-diol structure of PBA and PVA to form bonds, enabling the material to maintain integrity. Compared with simple nanofibers, it has sufficient porosity for cell migration; compared with a single hydrogel, it has sufficient mechanical properties to provide support for cells in the syringomyelia area and enables nerve cells to migrate along the nerve conduction direction, thus better promoting spinal cord injury repair. At the same time, the phenylborate ester bond PBAE generated by PBA and PVA can, like PBA, react specifically and rapidly with reactive oxygen species to further regulate the ROS environment in the injury area. At the same time, the PPMB scaffold can also up-regulate the expression of angiogenesis-related genes in endothelial cells and create a good environment for the survival of transplanted endothelial cells in the center of spinal cord injury, promoting the generation of new blood vessels and intervening in the proliferation and differentiation of neural stem cells, increasing the proportion of neurons, and promoting nerve regeneration.

[0091] As another aspect of the technical solution of the present invention, a preparation method of a bionic nanofiber-hydrogel composite biological scaffold includes: making the bionic nanofibers with an ordered structure and oriented arrangement simulating the white matter of the peripheral part of the spinal cord fully contact with the bionic hydrogel material simulating the gray matter of the central part of the spinal cord, and cross-linking by light to obtain the bionic nanofiber-hydrogel composite biological scaffold.

[0092] In some embodiments, the preparation method includes: reacting methacrylic anhydride gelatin, polycaprolactone, and polyvinyl alcohol to obtain oriented bionic nanofibers.

[0093] In other embodiments, the preparation method includes: reacting glycyrrhetinic acid, 3-aminophenylboronic acid, polyvinyl alcohol, and epigallocatechin gallate to obtain the bionic nanofiber-hydrogel composite biological scaffold.

[0094] In other embodiments, the preparation method includes: reacting polycaprolactone and polyvinyl alcohol to obtain oriented bionic nanofibers.

[0095] In some other embodiments, the preparation method includes: reacting beeswax, polycaprolactone and polyvinyl alcohol to obtain oriented bionic nanofibers.

[0096] In some other embodiments, the preparation method includes: reacting fish collagen and polycaprolactone to obtain oriented bionic nanofibers.

[0097] In some other embodiments, the preparation method includes: reacting citral, polycaprolactone and polyvinyl alcohol to obtain oriented bionic nanofibers.

[0098] In some other embodiments, the preparation method includes: reacting indomethacin, polycaprolactone, polyhexamethylene guanidine hydrochloride and polyvinyl alcohol to obtain oriented bionic nanofibers.

[0099] In some preferred embodiments, the preparation method includes: using a dual-nozzle electrospinning technique to obtain oriented bionic nanofibers.

[0100] In some more preferred embodiments, the preparation method specifically includes:

[0101] Mixing methacrylic anhydride gelatin, polycaprolactone with a first solvent to form a first spinning solution;

[0102] Mixing polyvinyl alcohol with a second solvent to form a second spinning solution;

[0103] Inputting the first spinning solution and the second spinning solution into a dual-nozzle electrospinning device respectively, and through electrospinning, obtaining oriented bionic nanofibers.

[0104] In some preferred embodiments, the mass ratio of methacrylic anhydride gelatin to polycaprolactone is 1:0.5 - 1:9, preferably 2:8 - 3:2.

[0105] In some preferred embodiments, the total concentration of methacrylic anhydride gelatin and polycaprolactone in the first spinning solution is 5 - 14 w / v%, preferably 6 - 10 w / v%.

[0106] Furthermore, the first solvent may include any one or a combination of two or more of 2,2,2-trifluoroethanol (TFE), ethanol, hexafluoroisopropanol, dichloromethane, dimethylformamide, etc., but is not limited thereto.

[0107] In some preferred embodiments, the concentration of polyvinyl alcohol in the second spinning solution is 5 - 10 w / v%, preferably 5 - 9 w / v%.

[0108] Furthermore, the second solvent may include water, preferably deionized water.

[0109] In some preferred embodiments, the preparation method specifically includes: using an injection pump to input the first spinning solution and the second spinning solution at a rate of 0.1-5 ml / h, preferably 0.5-2.5 ml / h.

[0110] In some preferred embodiments, the voltage used in the electrospinning is 5-60 kV, preferably 10-30 kV, the distance between the spinning needle and the receiver is 10-40 cm, preferably 10-20 cm, and the motor translation speed is 1-10 mm / s.

[0111] In some preferred embodiments, the preparation method further includes: using a roller receiver rotating at a speed of 1000-4500 rpm to collect the obtained bionic nanofibers with uniform thickness.

[0112] In some embodiments, the preparation method includes: reacting methacrylic anhydride gelatin with a compound containing a phenylboronic acid group to obtain a bionic hydrogel material.

[0113] In some other embodiments, the preparation method includes: reacting alginate with phenylboric acid first, and then with polyvinyl alcohol to obtain a bionic hydrogel material.

[0114] In some more preferred embodiments, the preparation method specifically includes: mixing methacrylic anhydride gelatin with water, stirring and dissolving it fully at 35-60 °C, adjusting the pH value to 5.5-7.5, preferably 5.5-6.5, and then adding a condensing agent and a compound containing a phenylboronic acid group to react to obtain the bionic hydrogel material.

[0115] In some embodiments, the compound containing a phenylboronic acid group may include 3-aminomethylphenylboronic acid hydrochloride, but is not limited thereto.

[0116] In some preferred embodiments, the condensing agent may include DMTMM, but is not limited thereto.

[0117] In some embodiments, the mass ratio of methacrylic anhydride gelatin, the condensing agent and the compound containing a phenylboronic acid group is 1:0.5:0.1-1:4:2, preferably 1:1:0.2-1:2:0.8.

[0118] Further, the preparation method further includes: dialyzing the hydrogel material with a 3500 Da dialysis bag for 3-3.5 days.

[0119] In some embodiments, the preparation method includes: curling the bionic nanofibers, dropping the bionic hydrogel material at the center, between layers and on the periphery, and crosslinking by light for 1-5 min, preferably 4-5 min, to obtain a bionic nanofiber-hydrogel composite biological scaffold.

[0120] In some other embodiments, the preparation method may further include: incorporating the bionic nanofibers into a bionic hydrogel material and subjecting it to photocrosslinking for 1 - 5 min, preferably 4 - 5 min, to obtain a bionic nanofiber-hydrogel composite biological scaffold.

[0121] In some more preferred embodiments, the present invention utilizes a dual-nozzle electrospinning technique. After blending PCL, PVA, and mGL, an oriented PPM nanofiber membrane with good mechanical properties and biocompatibility is prepared. The PPM membrane is curled to a suitable size, and mGL-PBA hydrogel is dropped between the layers and around the periphery. After photocrosslinking to form a gel, a PPMB bionic nanofiber gel composite scaffold is prepared. The PPMB composite scaffold mimics the macroscopic structure of natural spinal cord tissue and the arrangement structure of nerve bundles, facilitating the adhesion of nerve cells and the growth of axons.

[0122] As another aspect of the technical solution of the present invention, it also relates to a bionic nanofiber-hydrogel composite biological scaffold prepared by the aforementioned preparation method.

[0123] Correspondingly, as another aspect of the technical solution of the present invention, it also relates to the application of the bionic nanofiber-hydrogel composite biological scaffold in the preparation of a product with the function of treating spinal cord injury.

[0124] Furthermore, another aspect of the embodiments of the present invention also provides a product with the function of treating spinal cord injury, which includes: the aforementioned bionic nanofiber-hydrogel composite biological scaffold, and endothelial cells and stem cells loaded on the bionic nanofiber-hydrogel composite biological scaffold.

[0125] In some embodiments, the endothelial cells may be human central nervous endothelial cells. Of course, other all types of endothelial cells can also be used, such as at least one of endothelial cells derived from spinal cord tissue, endothelial cells derived from brain tissue, endothelial cells from umbilical vein blood, iPSC-induced endothelial cells, etc., but not limited thereto.

[0126] In some embodiments, the stem cells may include any one of mesenchymal stem cells, neural stem cells, etc., but not limited thereto.

[0127] Furthermore, the product has the function of promoting blood vessel and nerve regeneration in vivo.

[0128] Furthermore, the product has the function of regulating the differentiation of neural stem cells into neurons and promoting nerve regeneration in the injured spinal cord.

[0129] Furthermore, the product has the function of regulating the oxidative stress microenvironment.

[0130] Furthermore, the product has the function of regulating the spinal cord inflammatory microenvironment.

[0131] In summary, the present invention loads exogenous endothelial cells and stem cells through a bionic nanofiber-hydrogel composite biological scaffold and implants them into the injured spinal cord cavity, thereby regulating oxidative stress and the vascular microenvironment, reducing the damage caused by excessive ROS to endogenous nerve cells and transplanted endothelial cells, promoting angiogenesis and inducing nerve regeneration, and ultimately promoting the recovery of motor function after spinal cord injury.

[0132] The technical solution of the present invention will be further described in detail below in conjunction with several embodiments and the accompanying drawings. These embodiments are implemented on the premise of the technical solution of the invention, and detailed implementation manners and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0133] Unless otherwise specified, the various raw materials, reaction equipment, testing equipment, and testing methods used in the following embodiments are well-known in the art.

[0134] Example 1

[0135] 1. Synthesis and characterization of PPM and mGL-PBA

[0136] (1) Synthesis of PPM: A bionic nanofiber scaffold with nanoscale size was successfully prepared using the dual-nozzle electrospinning technique. 2,2,2-Trifluoroethanol (TFE) was used as the solvent, and methacrylated gelatin (GelMA) and polycaprolactone (PCL) were stirred overnight until completely dissolved, with GelMA / PCL = 2:8, so that the final polymer concentration was 8% (w / v). Deionized water was used as the solvent, and polyvinyl alcohol (PVA) was dissolved to 6% (w / v) at 90 °C. The obtained GelMA / PCL and PVA solutions were respectively placed in 5 ml plastic syringes equipped with 21-gauge stainless steel needles, which were connected to a high-voltage power supply. An injection pump was used to continuously supply the solution at a rate of 0.8 ml / h, and a voltage of 19 kV was applied to generate electrospinning. The distance between the needle tip and the receiver was 10 cm, and the translation motor was set at a speed of 10 mm / s. Finally, a nanofiber PPM with a uniform thickness was obtained on a roller receiver rotating at 2800 rpm. The tin foil with the fibers was placed in a vacuum drying oven and dried at 25 °C for 24 h to remove the residual solvent.

[0137] (2) Synthesis of mGL-PBA: 5 g of methacrylated gelatin (GelMA) was added to 1 L of ddH 2In O, after stirring at 37 °C until it was fully dissolved, the pH was adjusted to 6.5, and then 25 mM DMTMM was added as a condensing agent. After 30 min, 20 mM 3-aminomethylphenylboronic acid hydrochloride was added and reacted for 24 h. It was dialyzed with a 3500 Da dialysis bag for 3 days, and finally GelMA-PBA was obtained.

[0138] (3) Characterization of PPM and mGL-PBA:

[0139] Figure 1A and Figure 1B showed the solutions required for the preparation of PPM and mGL-PBA, and Figure 1C and Figure 1D showed the morphological diagrams of the chemical bonds before and after cross-linking during the synthesis of mGL-PBA and PPMB. Figure 1E was the synthesis route diagram of mGL-PBA. Figure 1F was the schematic diagram of preparing the fiber membrane PPM by electrospinning. Figure 1G was the schematic diagram of the gelation of mGL-PBA hydrogel. As Figures 1F - 1G shown, PPM and mGL-PBA were synthesized by double-nozzle electrospinning technology and photo-crosslinking, and their micro-morphologies were photographed by SEM. As Figures 1H - 1K shown, were the SEM images of PPM and mGL-PBA. From Figure 1H and Figure 1I it could be seen that the surface of the prepared PPM nanofiber membrane was smooth and the arrangement direction was basically the same; and from Figure 1J and Figure 1K it was observed that mGL-PBA presented a uniform pore structure after photo-gelation. After analysis, the diameter of the nanofibers was about 178.23 ± 29.08 nm, and the pore size of mGL-PBA was about 24.82 ± 2.05 μm. The above analysis showed that the prepared PPM nanofibers were nanoscale, and had a larger specific surface area and porosity compared with nanofibers of micron scale and above, which was more conducive to the transport of nutrients and was closer to the collagen fibers (20 - 200 nm) contained in natural ECM; at the same time, the mGL-PBA hydrogel provided sufficient space for cell adhesion and migration.

[0140] 2. Synthesis and Characterization of PPMB

[0141] As Figure 2A shown, was the macroscopic morphology diagram of the biomimetic nanofiber-hydrogel composite biocompatible scaffold PPMB. As Figures 2B - 2C shown, in order to simulate the structure of the natural spinal cord, the PPM nanofibers were wound around a syringe needle with a diameter of 0.7 mm, and mGL-PBA was dropped between the PPM nanofiber layers. After photo-crosslinking for 4 min, it was prepared into a PPMB with a length of 4 mm and a diameter of 2 mm. As Figure 2CAs shown, it is the bonding reaction diagram of PVA and mGL-PBA. mGL-PBA and PVA in PPM form dynamic borate ester bonds through esterification reaction, which are used to scavenge ROS in the damaged spinal cord microenvironment. At the same time, mGL in mGL-PBA further crosslinks with mGL in PPM to form a more compact pore structure. As Figure 2D shown, in order to observe the morphology of PPMB, PPM and PPMB were characterized by SEM. PPM and PPMB were respectively prepared into cylindrical structures with a length of 3 mm and a diameter of 2 mm, and their microscopic morphologies were analyzed by SEM. As Figure 2D can be seen, the prepared PPMB is composed of ordered nanofibers and hydrogel structures, which well simulates the macroscopic structure of natural spinal cord tissue and contains an arrangement similar to nerve bundles, facilitating cell adhesion and migration.

[0142] Subsequently, as Figures 3A - 3G shown, the mechanical properties of the prepared PPM and PPMB were tested. A universal material testing machine was used to perform tensile and compression tests on PPM and PPMB respectively. As Figure 3A shown, as shown by the stress-strain curve. As Figure 3B and Figure 3C shown, the compression modulus of PPM is 66.29 ± 8.21 MPa, the tensile modulus is 9.65 ± 3.00 MPa, and its fracture energy is calculated to be 44.93 KJ / m 2 . The compression modulus of PPMB is 78.79 ± 17.68 MPa, the tensile modulus is 3.63 ± 0.86 MPa, and its fracture energy is calculated to be 21.2544.93 KJ / m 2 (as Figure 3D shown). Figure 3E It is the deformation performance diagram of the bionic PPM and PPMB fiber membranes. Figure 3F 、 Figure 3G are respectively the adhesion and extension performance diagrams of the PPMB fiber membrane. It can be seen that compared with PPM, the mechanical properties of PPMB are more suitable for the adhesion and growth of endothelial cells and nerve cells.

[0143] Next, in order to verify the surface element composition of the synthetic materials, XPS was used to analyze PPM and PPMB, and the results are as Figures 4A - 4E shown. First, a full-spectrum analysis was carried out ( Figure 4A ). By comparison, it was found that the boron (B) element was detected on the surface of the PPMB material, while PPM only had C, N, and O elements. The fine spectrum of C 1s ( Figure 4B ) showed that PPMB showed the C-C, C-N, and C-O═C bonds of the PPM backbone. Secondly, by comparing the fine spectra of O 1s of PPM and PPMB ( Figure 4D) It was found that PPMB showed a peak at 531.55 eV, which was presumably the B-O bond in the mGL-PBA component; further analysis of the B 1s fine spectra of PPM and PPMB ( Figure 4C ) revealed that PPMB showed peaks at 190.55 eV and 189.60 eV, corresponding to B-O and B-C bonds respectively, indicating the formation of borate ester bonds between PVA and mGL-PBA; finally, observation of N 1s ( Figure 4E ) showed that due to the crosslinking of mGL in the two components, the N-H bonds in the PPMB material were significantly reduced, mainly being C-N-C bonds. Thus, the successful preparation of the PPMB material was evident.

[0144] Since the suspended hydroxyl groups of the PVA polymer in PPM reacted with mGL-PBA to form phenyl borate ester bonds. To prove the formation of phenyl borate ester bonds in the synthesis reaction, Fourier transform infrared spectra were collected using an FTIR spectrometer from 600 cm -1 to 4000 cm -1 , and the results are as Figure 5A shown. Due to the presence of the N-H stretching in mGL and the -OH stretching modes of PVA and PCL, peaks were present around 3270.1 cm -1 in each material. The stretching modes of C=C and C=O were present at 1640 - 1680 cm -1 and 1650 - 1690 cm -1 respectively. Peaks appeared at 1725 cm -1 in the PPM and PPMB materials, which was attributed to the characteristic peak of the -C=O bond in the PCL component. The peaks at 1450.2 cm -1 and 1334.4 cm -1 represented the stretching vibration absorption peaks of C-B and B-O bonds respectively. Compared with mGL-PBA and PPM, PPMB showed a peak at 1406.3 cm -1 , which was attributed to the stretching vibration of B-O-C, indicating the formation of dynamic borate ester bonds between the boric acid group on mGL-PBA and the cis-diol sites of PVA in PPM, and the successful composite of the PPMB material. Further, the water contact angles of the PPM and PPMB fiber membranes were detected, as Figure 5B shown. The water contact angles at 0 seconds and 10 seconds were selected for comparison. At 0 seconds, the water contact angles of PPM and PPMB were (99.43 ± 6.34)° and (80.93 ± 4.08)° respectively; at the 10th second, the water contact angles of PPM and PPMB were (74.04 ± 11.03)° and (60.00 ± 3.73)° respectively. It can be seen that the nanofiber membranes have good water absorption performance, and PPMB shows better hydrophilicity, with its contact angle lower than that of PPM, being more suitable for the adhesion of endothelial cells and conducive to the differentiation of neuron cells.

[0145] 3. Verification of the ROS-scavenging function of PPMB

[0146] After spinal cord injury, the body is in a state of oxidative stress, and a large amount of reactive oxygen species (ROS) accumulated in tissues cannot be scavenged by the antioxidant system, leading to apoptosis of cells and tissue damage in the injury area. Therefore, the mGL-PBA hydrogel material, mGL-PBA and PPMB materials are selected. Due to their PBA groups and phenylborate ester groups, they can react directly with H 2 O 2 to scavenge it, thus greatly improving the secondary spinal cord injury caused by excessive ROS. The ABTS kit was used to detect the ROS content in the solution, thereby reflecting the ability of the material to scavenge ROS. The prepared ABTS working solution contains abundant ABTS + and appears green. When the antioxidant acts, the generation of ABTS + is inhibited. Therefore, the absorbance of ABTS + is measured to judge the antioxidant capacity of the material.

[0147] The antioxidant capacities of PPM and PPMB were detected by the ABTS method. Specifically: 0.12 g of freeze-dried PPM and PPMB were weighed and immersed in 1 mM H 2 O 2 solution for 24 h. After sucking the supernatant of the reaction solution, it was mixed with the ABTS working solution, and the absorbance value was measured at 405 nm. The results are as Figure 6A shown. It can be seen that PPMB has a significant antioxidant effect compared with PPM. To further observe the optimal time for PPMB to scavenge ROS, 0.12 g of freeze-dried PPM and PPMB were weighed and reacted with 1 mM H 2 O 2 for 0, 12, 24, and 48 h respectively. At the corresponding time, the supernatant of the solution was sucked and mixed with the prepared ABTS working solution, and the absorbance value was measured at 405 nm. The results are as Figure 6B shown. Compared with PPM, PPMB is similar to mGL-PBA, maintaining its antioxidant effect continuously over time and maintaining a stable antioxidant effect at 48 h, proving that the PPMB material can fully play the function of scavenging ROS in the acute stage of spinal cord injury.

[0148] 4. Biocompatibility detection of PPM and PPMB

[0149] The CCK8 method was used to evaluate the cytotoxicity of PPM and PPMB, as shown in Figure 7A and Figure 7B . First, the conditioned medium prepared with PPM of different areas was interacted with human brain microvascular endothelial cells (hBEC) for 3 days ( Figure 7A), it can be seen that there is no significant difference in the survival of cells with the PPM material. Secondly, the conditioned medium of the prepared PPMB nanofiber membrane material was used to act on hBEC cells at different densities for 3 days ( Figure 7B ), and it was found that compared with the control group, the cells all showed good cell viability and there was no significant difference, indicating that the material has no obvious effect on cell viability, proving that the material has good biocompatibility and providing a theoretical basis for subsequent cell and in vivo experiments.

[0150] 5. Verification of the in vitro vascularization-promoting function of PPMB

[0151] By detecting the expression of angiogenesis-related genes in hBEC cells, it was detected whether the PPMB material has the potential to promote angiogenesis. Figure 8A . Figure 8B and Figure 8C show the effects of PPM and PPMB on hBEC angiogenesis in vitro. hBEC cells grown on PPM and PPMB materials were detected by RT-qPCR, and the expression of ETV2 ( Figure 8C ), EPHB4 ( Figure 8B ), and PECAM ( Figure 8A ) genes was upregulated.

[0152] Studies have shown that ETV2 is an important transcription factor for the development of endothelial and hematopoietic lineages, and overexpression of ETV2 can induce the positive differentiation of endothelial cells. The protein encoded by EPHB4 has been shown to interact with the EFN-B2 protein and plays an important role in vascular development in the nervous system. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is the CD31 antigen, which is expressed on the surface of endothelial cells, participates in the interaction between cells and between cells and the extracellular matrix, and the upregulation of PECAM-1 is also associated with vascular development. The above results indicate that the PPM and PPMB materials have a promoting effect on the angiogenesis of endothelial cells, and the effect of PPMB is better.

[0153] 6. Evaluation of the ability of PPMB to regulate the oxidative stress level of hBEC

[0154] To detect whether the nanofiber-hydrogel composite biocompatible scaffold can regulate ROS in the oxidative stress microenvironment after SCI to protect cell survival, the present invention added 1 mM H 2 O 2 to the culture medium to simulate a microenvironment with abnormal ROS, incubated with PPM and PPMB materials for 24 h, and observed the ROS content in cells after acting on hBEC. Figure 9 shows the ability of PPM and PPMB scaffolds to protect cells under in vitro simulated oxidative stress microenvironment conditions. Cells take up non-fluorescent H 2After being oxidized by intracellular ROS, DCFH-DA is converted into 2'-7'-dichlorofluorescein with green fluorescence, so it is used as a probe to detect intracellular ROS. Observation Figure 9 It can be seen that H 2 O 2 After the action of hBEC, a large amount of green fluorescence expression was presented, while the content of green fluorescence in cells after the action of PPMB was significantly reduced. The results showed that PPMB could play a role in scavenging excessive ROS, thereby regulating the oxidative stress microenvironment and reducing the damage caused by oxidative stress to cells.

[0155] 7. Effects of PPMB-hBEC on the proliferation and differentiation of NSCs

[0156] There are a large number of nerve cells in the spinal cord, and these mature nerve cells are differentiated from NSCs. Therefore, maintaining the number and activity of endogenous NSCs in the spinal cord is of great significance for nerve regeneration after SCI. Research has shown that the proliferation and maintenance of the stemness of endogenous NSCs in vivo are related to endothelial cells in the microenvironment, and in vitro, endothelial cells have the ability to help NSCs survive and proliferate and promote their directional differentiation into neurons. In order to test whether hBEC also has the ability to promote the proliferation and differentiation of NSCs into neurons, this experiment considered interacting hBEC with PPMB and intervening in NSCs to observe the effects on the proliferation and differentiation of NSCs( Figures 10A - 10C ). As Figure 10A shown, the results showed that the PPM and PPMB empty material groups could improve the survival rate of NSCs compared with the PBS group, suggesting that both PPM and PPMB materials could promote the proliferation of NSCs. In addition, when NSCs intervened after the interaction of PPM and PPMB with hBEC, their proliferation rate was as high as twice that of the untreated group, indicating that the presence of hBEC in vitro could greatly increase the proliferation rate of NSCs, and the PPM and PPMB materials would not have a significant impact on the effect of hBEC. Figure 10B And Figure 10C is the result diagram of the effect of PPMB-hBEC on the differentiation of NSCs.

[0157] 8. Animal experiments

[0158] Spinal cord injury models include incomplete SCI models and complete transection SCI models. The complete transection SCI model can more accurately simulate nerve and axon regeneration due to its completely separated spinal cord, and is considered the gold standard for research in the field of SCI research. As Figure 11 shown, it is a schematic diagram of an animal experiment model after filling the PPMB scaffold loaded with hBEC at the injury site. By simulating the complete transection SCI rat model and filling the PPMB scaffold loaded with hBEC at the injury site, the recovery situation after spinal cord injury was observed.

[0159] (1) Construction of a complete transection injury model of T9-T10 in SD rats

[0160] The location of spinal cord injury has an important impact on the repair and survival of animals. In this example, a 3-mm complete transection injury model was selected at the T9-T10 segment. As Figure 12 shown, the model was prepared through the steps of exposing the lamina, knocking out the lamina to expose the spinal cord, completely transecting the spinal cord, and filling with a biomaterial scaffold.

[0161] After treating the endothelial cells, they were resuspended in unphotocrosslinked mGL-PBA hydrogel and dropped onto the PPM membrane as shown above to prepare a biomimetic nanofiber gel composite scaffold PPMBC loaded with endothelial cells.

[0162] In this example, female SD rats weighing 190-210 g were used. The purchased rats were randomly grouped and set up with a complete transection injury group (SCI), a PPM nanofiber scaffold group (PPM), a PPM scaffold + mGL-PBA hydrogel group (PPMB), a PPM loaded with hBEC group (PPMC), and a PPM scaffold + mGL-PBA hydrogel loaded with hBEC group (PPMBC). The experiment was divided into an early experimental group and a long-term experimental group. In the early experimental group, samples were taken on the 10th day after treatment to analyze the gene and protein expression in the animals. In the long-term experimental group, BBB scores were taken during the treatment period, and footprint analysis was performed at the 8th week to observe the recovery of hindlimb motor function. Finally, samples were taken to detect the effects of the materials on various organs and to identify the gene and protein expression.

[0163] (2) Promotion of blood vessel and nerve regeneration in vivo by biomimetic nanoscaffolds loaded with endothelial cells

[0164] Ve-cadherin is a transmembrane adhesion protein specifically expressed on the surface of vascular endothelial cells. Blood vessels were stained with anti-Ve-cadherin antibody (CD144) to evaluate the blood vessel recovery after spinal cord injury. The results are as Figure 13A and Figure 13B shown. Figure 13A This is CD144 immunofluorescence staining. Blue represents Hochest33342, green represents CD144, scale bar: 100 μm. It can be seen that the expression level of CD144 in the injury center of the PPMC and PPMBC groups is higher than that in the PPM and PPMB groups. It can be seen that transplantation of the material loaded with cells hBEC is beneficial to the recovery of blood vessels in the injury center. Comparing PPMB (or PPMBC) and PPM (or PPMC), it can be seen that scavenging reactive oxygen species in the injury area can protect vascular endothelial cells and enable them to survive in the injury center. For further statistical analysis, Image J was used to quantitatively analyze the percentage of CD144 fluorescence area in the injury center, as Figure 13BAs shown, PPMBC showed the highest expression level of CD144 compared to other experimental groups.

[0165] Nestin can specifically label neural stem cells, such as Figure 14A and Figure 14B shown. Figure 14A Immunofluorescence staining of Nestin at both ends and the center of SCI injury. Blue represents Hochest 33342, green represents Nestin, red represents GFAP, scale bar: 100 μm. Figures 14B - 14D Relative quantitative analysis of Nestin fluorescence intensity in different regions. Immunofluorescence staining of endogenous neural stem cells at both ends and the center of the injury area with Nestin and quantitative analysis of the percentage of Nestin fluorescence area using Image J showed that the number of neural stem cells at both ends and the center in the PPMBC group was more than that in other experimental groups. And compared with the PPMC group, the survival of neural stem cells at both ends in the PPMB and PPMBC groups increased significantly. These phenomena indicate that due to the function of PPMB in scavenging reactive oxygen species, it can improve the oxidative stress microenvironment at both ends and the center of the injury, thus enabling endogenous neural stem cells to survive at both ends of the injury and migrate towards the injury center.

[0166] To further study the regulation of blood vessels and NSCs by PPMB-hBEC in rats, PPMB-hBEC was transplanted into the complete transection injury rat model. Tuj1 is a member of the tubulin family and is used to label early neurons. Therefore, immunofluorescence staining of the injured tissue with Tuj1 was performed ( Figure 15A ), Figure 15A in which blue represents Hochest 33342, green represents Tuj 1, white represents GFAP, scale bar: 100 μm. As the results showed, the expression level of Tuj1 in the PPMBC group was the highest, followed by the PPMC group. It can be seen that after scavenging reactive oxygen species by the action of PPMB, hBEC plays a role in regulating the differentiation of neural stem cells into neurons at the injury center, which helps to promote nerve regeneration in the injured spinal cord. Figure 15B Graph showing the results of relative quantitative analysis of Nestin fluorescence intensity.

[0167] (3) Regulation of oxidative stress microenvironment by biomimetic nanofiber-hydrogel composite biological scaffold

[0168] After spinal cord injury, the massive accumulation of ROS caused by hypoxia-ischemia results in oxidative damage to endogenous and exogenous transplanted cells, ultimately leading to cell death and having a negative impact on injury repair. To verify the scavenging effect of the biomimetic nanofiber-hydrogel composite biological scaffold on reactive oxygen species, immunofluorescence staining and quantitative analysis of the injured spinal cord tissue were performed using DHE probe ( Figure 16A and Figure 16B ),Figure 16A Immunofluorescence staining pictures for DHE staining to show the tissue oxidative stress level, Figure 16B which is a relative quantitative analysis chart of the average fluorescence intensity. The results show that both materials PPM and PPMB have the ability to scavenge reactive oxygen species. Due to the presence of PBA and PBAE groups, the antioxidant ability of PPMB far exceeds that of other groups. The enhanced reactive oxygen species in the transplanted cell group compared with the pure material group is considered to be due to the individual's immune rejection reaction after transplantation of hBEC. Antigen-presenting cells such as macrophages are further activated, and the released pro-inflammatory factors and chemokines attract immune cells to the injury area, thus causing an increase in ROS in the injury area.

[0169] (4) Role of bionic nanofiber-hydrogel composite biocompatible scaffolds loaded with endothelial cells in regulating the spinal cord inflammatory microenvironment

[0170] qPCR detection of inflammatory genes in the collected spinal cord tissues was performed, and the results are as Figures 17A - 17C shown. TGF-β can promote nerve regeneration by inhibiting inflammation and reducing apoptosis, and affect the transformation of macrophages from M1 type to M2 type. As Figure 17A shown, the expression of TGFβ gene in the PPMC and PPMBC groups showed an upward trend compared with other groups. Arg1 can hydrolyze L-arginine into urea and ornithine, thus reducing the production of nitric oxide (NO). Since NO is an important pro-inflammatory mediator, Arg1 plays an important anti-inflammatory role. And studies have shown that there is a close relationship between TGFβ and Arg1. TGFβ can significantly enhance the expression of Arg1 through pathways such as SMAD and MAPK. As Figure 17B can be seen, the Arg1 gene showed high expression in both the PPMC and PPMBC groups. Therefore, it is analyzed that the transplanted cell group exerts a good anti-inflammatory function by highly expressing Arg1 in vivo. And PPMB showed a higher Arg1 expression level compared with PPM, and PPMBC showed a higher Arg1 expression level compared with PPMC, indicating that PBA weakens the in vivo inflammatory response caused by ROS by scavenging ROS. TNF-α can activate M1 macrophages, thus promoting the inflammatory response and immune regulation. Taking the ratio of the expression levels of TNF-α and Arg1 ( Figure 17C ), it was found that the ratio in the transplanted cell group showed a lower phenomenon. Generally speaking, the inflammation in the PPMC and PPMBC groups in vivo was reduced.

[0171] (5) Behavioral evaluation

[0172] In this experiment, the BBB locomotor rating scale was used to evaluate the recovery of the hind limb motor function of rats after SCI, and the evaluation was carried out at the same time every week (as Figure 18As shown. According to the statistical results, starting from the 4th week, both the PPMC and PPMBC groups showed slight movements of two hind limb joints and extensive movements of one joint, which were significantly different from the slight movements of the three hind limb joints in the SCI group. This indicates that PPMC and PPMB have a promoting effect on the recovery of motor function after SCI.

[0173] In summary, the PPMB scaffold has good mechanical properties and appropriate surface hydrophilicity and hydrophobicity, providing good mechanical support for the adhesion of transplanted endothelial cells and endogenous nerve cells after spinal cord injury. Moreover, on the basis of providing mechanical support, the PPMB fiber gel composite scaffold increases the specific surface area of the material, providing a good three-dimensional network structure for cell survival.

[0174] Furthermore, the PPMB scaffold shows good antioxidant capacity both in vitro and after spinal cord injury, scavenging reactive oxygen species in the injury area and protecting transplanted cells and endogenous nerve cells. The PPMB scaffold can up-regulate the expression of angiogenesis-related factors, showing the potential to induce angiogenesis of endothelial cells.

[0175] Therefore, treating spinal cord injury with the PPMB scaffold composite endothelial cells can promote the formation of new blood vessels in the injury area. The PPMB scaffold composite endothelial cells can promote the differentiation of neural stem cells into neurons after spinal cord injury, having the ability to promote nerve regeneration. Thus, the treatment method of the PPMB scaffold composite endothelial cells shows a better effect of motor function recovery.

[0176] Example 2

[0177] The difference between this example and Example 1 is that the preparation method of the biomimetic hydrogel material includes:

[0178] (a) Preparation of alginate-phenylboronic acid (Alg-PBA) precursor:

[0179] 1. Dissolve 100 mg of alginate in 10 ml of deionized water, then add 23.5 mg of PBA and 37.5 mg of DMTMM, stir until completely dissolved, then adjust the pH of the solution to 6.5, and transfer it to a 6 - 8 kDa dialysis bag and dialyze with deionized water for 3 days.

[0180] (b) Preparation of Alg-PBA hydrogel:

[0181] 1. Dissolve Alg-PBA and PVA in sodium chloride respectively, and make the solution concentrations 2.5 wt% and 4 wt% respectively;

[0182] 2. Mix Alg-PBA and PVA at a volume ratio of 3:1 at room temperature to prepare an Alg-PBA-PVA dynamic hydrogel.

[0183] Example 3

[0184] In comparison with Example 1, this example is different in that the preparation method of the bionic nanofiber membrane includes:

[0185] (a) Preparation of GelMA:

[0186] 1. Dissolve 10 w / v% gelatin in DPBS and fully dissolve it at 50 °C;

[0187] 2. Add 0.8 ml of methacrylic anhydride to every 1 g of gelatin, stir and react for 2 h, then add an equal amount of DPBS to terminate the reaction. Use a 12 - 14 kDa dialysis membrane to dialyze in deionized water at 40 - 50 °C for 7 days, and freeze-dry to obtain a powder.

[0188] (b) Preparation of GelMA / PCL electrospun membrane:

[0189] 1. Dissolve GelMA and PCL in acetic acid and hexafluoroisopropanol respectively to obtain 150 and 100 mg / ml solutions;

[0190] 2. Mix an equal amount of GelMA and PCL under continuous stirring to form a 50:50 (v / v) polymer blend;

[0191] 3. Load the solution into a 27G needle, set the voltage at 18 kV and the flow rate at 2 ml / h at room temperature, and collect the nanofiber membrane on an aluminum foil 18 cm away from the needle;

[0192] 4. Treat the electrospun membrane in a vacuum drying oven for 2 days to remove residual solvents.

[0193] Example 4

[0194] (a) Preparation of phenylboronic acid-modified glycyrrhetinic acid nanofibers (GAPBA):

[0195] 1. Dissolve 3 g of glycyrrhetinic acid in 200 ml of a mixed solvent (DMF∶ddH 2 O = 2∶3), then add 0.825 g of 3-aminophenylboronic acid (APBA), and stir until completely dissolved;

[0196] 2. Add 1.164 g of DMTMM, continuously stir for 48 h, then transfer to a 3500 Da dialysis bag and dialyze with deionized water for 3 days, freeze-dry, and store at 2 - 8 °C;

[0197] (b) Preparation of hydrogel (PVA / EGCG):

[0198] 1. Dissolve PVA in PBS at 80 °C, and then slowly add EGCG (epigallocatechin gallate) in batches to obtain a PVA / EGCG mixed solution with a final concentration of 8% w / v PVA and 5 mg / ml EGCG.

[0199] (c) Preparation of hydrogel (PVA / EGCG):

[0200] 1. Dissolve GAPBA in PBS to prepare solutions with concentrations of 4% w / v, 6% w / v, and 8% w / v.

[0201] 2. Vortex and mix the solutions in 1 with the solution obtained in (b) in equal volumes.

[0202] Example 5

[0203] (a) Preparation of PCL emulsion:

[0204] 1. Dissolve PCL in toluene to prepare a solution with a concentration of 12.5 - 22 wt%.

[0205] (b) Preparation of PVA emulsion:

[0206] 1. Dissolve PVA in deionized water to prepare a solution with a concentration of 5 - 9 wt%.

[0207] (c) Preparation of PCL / PVA electrospinning:

[0208] 1. Drop the above PCL solution into the PVA solution, with a PCL:PVA volume ratio of 1:2.

[0209] 2. Ultrasonically treat the mixture in 1 for 20 min and continue stirring for 5 h.

[0210] 3. Place the mixture in a 2-ml syringe for electrospinning, set the flow rate to be constant at 0.5 ml / h, the voltage to 30 kV, and collect it on an aluminum foil 20 cm away from the needle.

[0211] 4. Wash with deionized water 5 times to remove PVA and store at room temperature.

[0212] Example 6

[0213] Preparation of PVA / PCL / beeswax nanofibers:

[0214] 1. Dissolve 10 g of PVA in 90 ml of water and stir at 90 °C for 4 h to obtain a 10 wt% PVA solution. Pour the PVA onto a glass plate to prepare a PVA film and dry it at 70 °C for 10 min.

[0215] 2. Dissolve 0 - 4 g of beeswax in 75 ml of chloroform, then add 10 g of PCL and 25 ml of ethanol to prepare a 10 w / v% mixture solution, and stir well for 4 h;

[0216] 3. Add the PCL / beeswax solution into a syringe, select a 21G needle, prepare electrospinning under the conditions of a flow rate of 2.8 ml / h and a voltage of 18 kV, and collect it on a PVA glass plate 15 cm away from the needle tip. Finally, dry it at 70 °C for 1 h to obtain a nanofiber membrane.

[0217] After testing, according to the preparation method of the electrospun nanofiber - hydrogel composite biocompatible scaffold in Example 1, partial process adjustments were made in Examples 2 - 6, but the microstructure and performance tests of the obtained composite biocompatible scaffolds were similar to those in Example 1.

[0218] Example 7

[0219] Compared with Example 1, the difference in this example is that the electrospinning solution consists of fish collagen and polycaprolactone, where the volume ratio of the collagen solution to the polycaprolactone solution is 1:9, the total concentration of collagen and polycaprolactone in the electrospinning solution is 8%, the solvent is replaced with acetic acid and hexafluoroisopropanol, the voltage used for electrospinning is 18 kV, the distance between the needle tip and the collector is 10 cm, and the flow rate of the injection pump is 0.3 ml / h.

[0220] Example 8

[0221] Compared with Example 1, the difference in this example is that the mass ratio of methacrylic anhydride - modified gelatin to polycaprolactone is 1:1, the voltage used for electrospinning is 15 kV, the distance between the needle tip and the collector is 15 cm, and the flow rate of the injection pump is 2 ml / h.

[0222] Example 9

[0223] Compared with Example 1, the difference in this example is that the nanofiber membrane consists of citral, polycaprolactone and polyvinyl alcohol, where the concentration of polyvinyl alcohol is 9% w / v, the electrospinning voltage is 20 kV, and the distance between the needle tip and the collector is 18 cm.

[0224] Example 10

[0225] Compared with Example 1, the difference in this example is that nanofibers are prepared by coaxial electrospinning technology, the electrospinning solution consists of a 14% w / v polycaprolactone and a 5% w / v polyvinyl alcohol solution, the electrospinning voltage is 60 kV, and the distance between the needle tip and the collector is 12 cm.

[0226] Example 11

[0227] This embodiment is different from Embodiment 1 in that: the first spinning solution component is indomethacin and polycaprolactone, the second spinning solution is polyhexamethylene guanidine hydrochloride and polyvinyl alcohol, wherein the concentration of polycaprolactone is 12% w / v, the concentration of polyvinyl alcohol is 10% w / v, and the first solvent is a mixed solution of dimethyl sulfoxide and 2,2,2-trifluoroethanol. The voltage used for the first spinning solution is 16 kV, the voltage used for the second spinning solution is 25 kV, and the injection pump flow rate is 0.6 ml / h.

[0228] In addition, the inventors of this case also referred to the foregoing embodiments and conducted tests with other raw materials, process operations, and process conditions described in this specification, and all obtained relatively ideal results.

[0229] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. A biomimetic nanofiber-hydrogel composite biological scaffold, characterized in that: The composite biological scaffold is a composite biological scaffold that simulates the gray-white matter partition structure of spinal cord tissue, including oriented bionic nanofibers that simulate the ordered structure of white matter around the spinal cord, and a bionic hydrogel material combined with the bionic nanofibers to simulate the gray matter in the central part of the spinal cord.

2. The biomimetic nanofiber-hydrogel composite biological scaffold according to claim 1, characterized in that: 1.2cm 2 The amount of the bionic hydrogel material bound to the bionic nanofiber is 10-30 μL, preferably 18-22 μL.

3. The biomimetic nanofiber-hydrogel composite biological scaffold according to claim 1, characterized in that: The bionic nanofiber is prepared from methacrylic anhydride gelatin, polycaprolactone and polyvinyl alcohol; or, from glycyrrhetinic acid, 3-aminophenylboronic acid, polyvinyl alcohol and epigallocatechin gallate; or, from polycaprolactone and polyvinyl alcohol; or, from polycaprolactone, beeswax and polyvinyl alcohol; or, from fish collagen and polycaprolactone; or, from citral, polycaprolactone and polyvinyl alcohol; Alternatively, it is prepared from indomethacin, polycaprolactone, polyhexamethyleneguanidine hydrochloride and polyvinyl alcohol; And / or, the biomimetic hydrogel material is prepared from methacrylic anhydride gelatin and a compound containing phenylboronic acid groups, or is prepared from alginate, phenylboronic acid and polyvinyl alcohol; Preferably, the phenylboronic acid groups grafted onto the biomimetic hydrogel material can be specifically coupled with the polyvinyl alcohol component in the biomimetic nanofibers and interact to form phenylboronic acid ester bonds.

4. The biomimetic nanofiber-hydrogel composite biological scaffold according to claim 3, characterized in that: The compounds containing phenylboronic acid groups include 3-aminomethylphenylboronic acid hydrochloride; And / or, the bionic nanofiber is prepared by double-nozzle electrospinning technology; preferably, the bionic nanofiber includes a bionic nanofiber membrane; And / or, the bionic nanofiber is nanoscale, preferably, the bionic nanofiber has a diameter of 100-500 nm and a porosity of 40-75%; And / or, the pore size of the bionic hydrogel material is 10-200 μm.

5. The method for preparing the biomimetic nanofiber-hydrogel composite biological scaffold according to any one of claims 1 to 4, characterized in that: include: The bionic nanofibers with an ordered structure and oriented arrangement simulating the white matter in the peripheral part of the spinal cord are fully contacted with the bionic hydrogel material simulating the gray matter in the central part of the spinal cord, and are cross-linked by light to obtain a bionic nanofiber-hydrogel composite biological scaffold.

6. The preparation method according to claim 5, characterized in that: include: Methacrylic anhydride gelatin, polycaprolactone and polyvinyl alcohol are reacted to prepare oriented bionic nanofibers; Alternatively, glycyrrhetinic acid, 3-aminophenylboronic acid, polyvinyl alcohol and epigallocatechin gallate are reacted to prepare a biomimetic nanofiber-hydrogel composite bioscaffold; alternatively, polycaprolactone and polyvinyl alcohol are reacted to prepare oriented biomimetic nanofibers; alternatively, beeswax, polycaprolactone and polyvinyl alcohol are reacted to prepare oriented biomimetic nanofibers; Alternatively, fish collagen and polycaprolactone are reacted to prepare oriented biomimetic nanofibers; Alternatively, citral, polycaprolactone and polyvinyl alcohol are reacted to obtain oriented bionic nanofibers; alternatively, indomethacin, polycaprolactone, polyhexamethylene guanidine hydrochloride and polyvinyl alcohol are reacted to obtain oriented bionic nanofibers.

7. The preparation method according to claim 6, characterized in that: include: The oriented bionic nanofibers were prepared by using double-nozzle electrospinning technology; Preferably, the preparation method comprises: Mixing methacrylic anhydride gelatin, polycaprolactone and a first solvent to form a first spinning solution; mixing polyvinyl alcohol with a second solvent to form a second spinning solution; The first spinning solution and the second spinning solution are respectively input into a double-nozzle electrospinning device to obtain oriented bionic nanofibers through electrospinning; Preferably, the mass ratio of methacrylic anhydride gelatin to polycaprolactone is 1:0.5-1:9; preferably, the total concentration of methacrylic anhydride gelatin and polycaprolactone in the first spinning solution is 5-14w / v%; preferably, the first solvent includes any one of 2,2,2-trifluoroethanol, ethanol, hexafluoroisopropanol, dichloromethane, and dimethylformamide, or a combination of two or more thereof; Preferably, the concentration of polyvinyl alcohol in the second spinning solution is 5-10 w / v%; preferably, the second solvent comprises water; Preferably, the preparation method comprises: using a syringe pump to input the first spinning solution and the second spinning solution at a rate of 0.1-5 ml / h; Preferably, the voltage used in the electrospinning is 5-60 kV, and the distance between the spinning needle and the receiver is 10-40 cm. Preferably, the preparation method further comprises: collecting the bionic nanofibers with uniform thickness using a drum receiver with a rotation speed of 1000-4500 rpm.

8. The preparation method according to claim 5, characterized in that: include: The biomimetic hydrogel material is prepared by reacting methacrylic anhydride gelatin with a compound containing phenylboronic acid groups; Alternatively, alginate is first reacted with phenylboronic acid and then with polyvinyl alcohol to obtain a biomimetic hydrogel material; Preferably, the preparation method comprises: mixing methacrylic anhydride gelatin with water, stirring at 35-60° C. to fully dissolve, and adjusting the pH value to 5.5-7.5, and then adding a condensing agent and a compound containing a phenylboronic acid group to react to obtain the hydrogel material; Particularly preferably, the compound containing a phenylboronic acid group includes 3-aminomethylphenylboronic acid hydrochloride; Particularly preferably, the condensing agent comprises DMTMM; Preferably, the mass ratio of the methacrylic anhydride gelatin, the condensing agent and the compound containing phenylboronic acid groups is 1:0.5:0.1-1:4:2; And / or, the preparation method comprises: curling the bionic nanofibers, and dripping bionic hydrogel materials in the center, between layers and at the periphery, and light-crosslinking for 1-5 minutes to obtain a bionic nanofiber-hydrogel composite biological scaffold; Alternatively, the preparation method comprises: mixing the bionic nanofiber into a bionic hydrogel material, light-crosslinking for 1-5 minutes, and preparing a bionic nanofiber-hydrogel composite biological scaffold.

9. Use of the bionic nanofiber-hydrogel composite biological scaffold according to any one of claims 1 to 4 in preparing a product having the function of treating spinal cord injury.

10. A product with the function of treating spinal cord injury, characterized in that: include: The biomimetic nanofiber-hydrogel composite bioscaffold according to any one of claims 1 to 4, and the endothelial cells and stem cells loaded on the biomimetic nanofiber-hydrogel composite bioscaffold; Preferably, the endothelial cells include any one or a combination of two or more of human central nervous system endothelial cells, spinal cord tissue-derived endothelial cells, brain tissue-derived endothelial cells, umbilical vein blood endothelial cells, and iPSC-induced endothelial cells; Preferably, the stem cells include any one of mesenchymal stem cells and neural stem cells; Preferably, the product has the function of promoting blood vessel and nerve regeneration in vivo; Preferably, the product has the function of regulating the differentiation of neural stem cells into neurons and promoting nerve regeneration in injured spinal cord; Preferably, the product has the function of regulating the oxidative stress microenvironment; Preferably, the product has the function of regulating the inflammatory microenvironment of the spinal cord.

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