A scaffold-gel composite central nervous system repair material for specifically attracting and incubating endogenous neural stem cells and its application

Abstract

The present invention discloses a scaffold-gel composite central nervous system repair material for specifically attracting and incubating endogenous neural stem cells and its application. The composite central nervous system repair material is composed of a decellularized optic nerve scaffold, a decellularized spinal cord hydrogel, neurotrophic factors, and the aptamer Apt19S. The above-mentioned scaffold can continuously release Apt19S and neurotrophic factors, recruit a large number of endogenous stem cells, and provide a neurogenesis microenvironment and physical support performance support for in situ spinal cord repair. The composite central nervous system repair material can be transplanted into the spinal cord injury site to repair spinal cord injury, and can also be transplanted into the optic nerve or brain injury site to repair central nervous system injury.

Description

Technical Field

[0001] The present invention relates to the technical field of central nervous system repair materials, and in particular to a scaffold-gel composite central nervous system repair material for specifically attracting and incubating endogenous neural stem cells and applications thereof. Background Art

[0002] Spinal cord injury (SCI) disrupts neural pathway conduction, leading to loss of motor, sensory, and autonomic function below the injured segment. Although biological therapies hold promise for restoring disrupted neural pathways, this strategy faces several key challenges. Exogenous stem cell transplantation is considered a promising strategy for treating SCI; however, limitations, including source, ethical concerns, immune rejection, tumorigenicity, and low survival rates, have significantly hindered its clinical translation. In contrast, endogenous neural stem cells (NSCs) could offer an alternative to stem cell transplantation for SCI repair, offering the advantages of being non-tumorigenic and immune-rejective, as well as the potential for better integration with host neural circuitry. However, endogenous NSCs at the injury site are far from sufficient, resulting in limited neural repair during natural repair. Furthermore, a low rate of neuronal differentiation hinders their ability to perform repair functions. Therefore, a strategy that can both recruit endogenous NSCs and promote neuronal differentiation is urgently needed to replenish neurons lost after SCI and reconstruct neural pathways.

[0003] To date, bioactive molecules that can effectively attract endogenous NSCs have been scarce. Although some bioactive molecules have been shown to promote endogenous repair after SCI, these molecules primarily prevent endogenous NSC apoptosis by improving the immune microenvironment or providing a microenvironment rich in neurotrophic factors, rather than directly recruiting endogenous NSCs. Stromal cell-derived factor 1 (SDF-1), the most commonly used chemokine, has been shown to coordinate stem cell migration within the central nervous system; however, SDF-1 can also attract monocytes and exacerbate neuroinflammation. Furthermore, SDF-1 carries the potential risk of inducing neuropathic pain. Recently, an oligonucleotide aptamer drug called Apt19S has shown promise for further recruiting endogenous stem cells due to its ability to specifically target pluripotent stem cells. Compared to protein-based drugs, aptamers such as Apt19S offer advantages such as high affinity and selectivity for their targets, lack of immunogenicity and toxicity, and rapid clearance from the circulation. Recent studies have shown that Apt19S can specifically bind to bone marrow stem cells (BMSCs) and effectively recruit endogenous BMSCs for cartilage repair. Recently, Apt19S has also been shown to recruit neural stem cells and has been used to treat Parkinson's disease.

[0004] In addition to recruiting endogenous NSCs, a microenvironment for neural generation must be provided for NSCs recruited in situ. Our previous studies have demonstrated that neurotrophic factor (NT-3) is a neurotrophic factor that can promote neuronal differentiation and axonal regeneration, which can create a pro-regenerative microenvironment for endogenous repair after SCI. However, when conventional methods are used to achieve sustained release, NT-3 is susceptible to structural damage and loses its bioactivity. Decellularized tissue matrix hydrogel is a superior candidate material that helps to continuously deliver NT-3 in situ without losing bioactivity. Decellularized spinal cord matrix hydrogel (DSCM hydrogel) can self-assemble under mild conditions and degrade at an appropriate rate, making it an ideal carrier for encapsulating NT-3 without compromising its bioactivity, ensuring sustained release at the injury site. In addition, DSCM hydrogel is able to synergize with NT-3 through its strong neurogenic extracellular matrix to create a neurogenic microenvironment that mimics the microenvironment of the developing embryonic spinal cord.

[0005] However, in cases of complete transection or extensive tissue defects in SCI, DSCM hydrogels alone are prone to collapse and degradation, making it difficult to provide structural support for the spinal cord. In a previous study, we found that a decellularized optic nerve scaffold (DON) supported and bridged the injury site without collapse due to its superior mechanical strength; in addition, DONs mimicked the white matter of the spinal cord by guiding directional axonal regeneration with straight channels. The high porosity of the DON scaffold also enabled it to be filled with DSCM hydrogels.

[0006] In summary, we proposed the idea of ​​combining two acellular matrix scaffolds (DON and DSCM hydrogels) to simulate the central nervous system development microenvironment and provide sufficient mechanical strength and directional axon growth guidance, while loading Apt19S to attract endogenous stem cells and NT-3 to further provide conditions for neural development. We designed a biomimetic spinal cord scaffold that effectively recruits endogenous neural stem cells to the injury site and induces the formation of a large number of new neurons in situ. Summary of the Invention

[0007] To overcome the deficiency of endogenous neural stem cells at the site of spinal cord injury and the adverse effects of the injury microenvironment on neurogenesis, the present invention aims to develop a scaffold-gel composite central nervous system repair material for the specific attraction and incubation of endogenous neural stem cells. This scaffold can continuously release Apt19S and neurotrophic factors, recruiting a large number of endogenous stem cells and providing a neurogenesis microenvironment and physical support for in situ spinal cord repair.

[0008] One of the objectives of the present invention is achieved by the following technical solution:

[0009] A scaffold-gel composite central nervous system repair material for specifically attracting and incubating endogenous neural stem cells, comprising a decellularized optic nerve scaffold, a decellularized spinal cord hydrogel, a neurotrophic factor, and an aptamer Apt19S. The compounding requirements of the decellularized optic nerve scaffold, decellularized hydrogel, neurotrophic factor, and aptamer Apt19S are as follows:

[0010] A 2 mm long, 3 mm diameter decellularized optic nerve scaffold; 2 nmol of aptamer Apt19S; 20 μl of 10 mg / ml decellularized hydrogel; and 2.5 μg of neurotrophic factor.

[0011] Furthermore, the method for preparing the scaffold-gel composite central nervous system repair material for specifically attracting and incubating endogenous neural stem cells comprises the following preparation steps:

[0012] a) utilizing the abundant carboxyl groups in the decellularized optic nerve scaffold, amino-modified Apt19S is loaded onto the material through an amidation reaction, thereby achieving sustained release of Apt19S and obtaining an Apt19S-loaded decellularized optic nerve scaffold;

[0013] b) injecting the decellularized hydrogel added with neurotrophic factors into the Apt19S-loaded decellularized optic nerve obtained in step a) to obtain the composite central nervous system repair material.

[0014] Furthermore, in step a), the step of loading the Apt19S-loaded decellularized optic nerve scaffold comprises the following steps:

[0015] 1) Incubate the decellularized optic nerve scaffold in 2 ml of morpholineethanesulfonic acid (MES, 0.1 M, pH = 6) at room temperature for 30 minutes;

[0016] 2) Add 60 mg of carbodiimide and 80 mg of N-hydroxysuccinimide to the above solution and incubate at room temperature for another 20 minutes;

[0017] 3) The treated optic nerve scaffold was removed from the liquid, washed three times with D-hanks buffer, and then added with sterile water containing 2 nmol Apt19S. After reacting in the dark for 12 hours, an Apt19S-loaded decellularized optic nerve scaffold was obtained.

[0018] Furthermore, in step b), the step of injecting the decellularized spinal cord hydrogel containing the neurotrophic factor into the material obtained in step a) comprises the following steps:

[0019] 1) Use 1 M sodium hydroxide solution to adjust the pH of the decellularized material solution to about 7.4;

[0020] 2) At 4°C, add 10×DEME, the volume of which is 1 / 9 of the obtained solution volume;

[0021] 3) At 4°C, take 10 μl of the solution obtained in step 2 and add 10 μl of 1× DMEM solution containing 2.5 μg of neurotrophic factor;

[0022] 4) The solution obtained in step 3 is poured into the decellularized optic nerve scaffold loaded with Apt19S, and incubated at 37° C. for 20-30 minutes to form a hydrogel, thereby obtaining the scaffold-gel composite central nervous system repair material.

[0023] Further preferably, the method for preparing the acellular material component for preparing the scaffold-gel composite central nervous system repair material comprises the following preparation steps:

[0024] 1) Fresh porcine optic nerves were washed overnight at 4°C in distilled water containing penicillin-streptomycin and mycotoxin / amphotericin solution. The nerves were then decellularized using Triton X-100, sodium deoxycholate, DNase, and RNase. The optic nerves were then immersed in sterile distilled water in a 1.5 ml centrifuge tube and freeze-dried for 24 hours to obtain a decellularized optic nerve scaffold.

[0025] 2) Fresh porcine spinal cord was washed overnight at 4°C in distilled water containing penicillin-streptomycin and mycotoxin / amphotericin solution. The neural cellular content was then removed using Triton X-100, sodium deoxycholate, DNase, and RNase. The spinal cord was then immersed in sterile distilled water in a 1.5 ml centrifuge tube and freeze-dried for 24 hours to obtain acellular spinal cord powder.

[0026] 3) utilizing the abundant carboxyl groups on the decellularized optic nerve of the decellularized optic nerve scaffold obtained in step 1) to combine with the amino-modified Apt19S through an amidation reaction, thereby achieving the loading and sustained release of Apt19S and the decellularized optic nerve, thereby obtaining an optic nerve scaffold loaded with Apt19S;

[0027] 4) The decellularized spinal cord powder obtained in step 2) was digested in 0.01 M HCl containing pepsin at room temperature for 12 hours to obtain a pre-decellularized spinal cord hydrogel solution; the pH of the solution was adjusted to 7.4 using 1 M NaOH and isotonically balanced with 10× DMEM. The adjusted pre-hydrogel was mixed with NT-3 and injected into the Apt19S-loaded optic nerve scaffold obtained in step 3), followed by incubation at 37°C for 20-30 minutes to produce a scaffold / gel composite central nervous system repair material.

[0028] Further preferably, the neurotrophic factor is one or more of neurotrophin-3 (NT-3), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF) and insulin-like growth factor (IGF).

[0029] Further preferably, the decellularized hydrogel is one of decellularized spinal cord hydrogel, decellularized brain hydrogel, and decellularized optic nerve hydrogel.

[0030] The use of the above-mentioned Apt19S oligonucleotide drug in the preparation of biomaterials for repairing central nervous system damage also falls within the scope of protection of the present invention, and the use of the above-mentioned Apt19S oligonucleotide drug in the preparation of materials for promoting electrophysiological recovery of spinal cord transection sites also falls within the scope of protection of the present invention.

[0031] The second object of the present invention is achieved by the following technical solution:

[0032] One application is the use of the above-mentioned scaffold-gel composite central nervous system repair material for specifically attracting and incubating endogenous neural stem cells in repairing spinal cord injuries and central nervous system injuries.

[0033] Such as preparing materials that promote the repair of spinal cord injuries, preparing materials that promote electrophysiological recovery of spinal cord transection sites, etc.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. The composite central nervous system repair material of the present invention is a biomimetic spinal cord scaffold based on DSCM hydrogel and DON scaffold, which effectively meets the bioactive microenvironment and physical strength characteristics of the central nervous system required for in situ spinal cord repair. At the same time, the biomimetic spinal cord scaffold fully utilizes the specific binding between Apt19S and alkaline phosphatase (ALPL) receptors to recruit specific subtypes of NSCs with high ALPL expression, while providing a neurogenesis microenvironment for the recruited NSCs through the NT-3-rich DSCM hydrogel. ALPL-positive NSCs have strong proliferation capacity and a high rate of differentiation into neurons. In the current study, the biomimetic spinal cord scaffold reconstructs the neural pathways for brain-derived signal transmission by generating a large number of new neurons at the injury site, thereby forming an endogenous neuronal network. Our strategy replicates the process of spinal cord development, breaking the limitation of the poor self-repair ability of the central nervous system, and has great prospects in clinical translation and application. It is worth mentioning that our strategy of focusing on activating certain subtypes of endogenous NSCs rather than using allogeneic stem cells is also applicable to repairing other central nervous system injuries. In the future, this strategy can also be combined with brain-computer interface technology and epidural spinal cord stimulation to achieve higher levels of motor function reconstruction.

[0036] 2. The composite central nervous system repair material of the present invention utilizes the specific binding between Apt19S and alkaline phosphatase (ALPL) receptors to attract endogenous neural stem cells expressing ALPL, and utilizes gel to load neurotrophic factors to achieve sustained release of neurotrophic factors, thereby creating an "incubation" microenvironment for neurogenesis for endogenous neural stem cells. The composite central nervous system repair material can be transplanted into the site of spinal cord injury to repair spinal cord injury, or can be transplanted into the site of optic nerve or brain injury to repair central nervous system injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : Transwell cell migration assay results. A: Blank group. B: Decellularized optic nerve scaffold group. C: Apt19S-loaded decellularized optic nerve scaffold group. D: Statistical results of the number of migrated cells in each group.

[0038] Figure 2 : Results of NSC adsorption experiments. A: Decellularized optic nerve scaffold group. B: Apt19S-loaded decellularized optic nerve scaffold group. C: Statistical results of the number of adsorbed cells in the two groups.

[0039] Figure 3Schematic diagram and general image of the scaffold / gel composite central nervous system repair material. A: Construction process of the scaffold / gel composite central nervous system repair material. B: Top view of the scaffold / gel composite central nervous system repair material under a stereomicroscope. C: Side view of the scaffold / gel composite central nervous system repair material under a stereomicroscope. & denotes acellular optic nerve scaffold, * denotes acellular spinal cord hydrogel.

[0040] Figure 4 : Apt19S (A) and neurotrophin-3 (B) release curves;

[0041] Figure 5 : Biocompatibility testing. A: Live / dead cell assay results B: CCK-8 assay results;

[0042] Figure 6 :Differentiation detection of neural stem cells on scaffolds;

[0043] Figure 7 :Detection of the potential of neural stem cells to differentiate into neurons and form synaptic connections on scaffolds;

[0044] Figure 8 Motor function recovery 8 weeks after surgery. A. Open-field exercise test. B. Inclined grid climbing test. C. Statistical graph of BBB scores in each group from 0 to 8 weeks (n = 8, * indicates statistically significant P < 0.05 when compared with the SCI group at that time point, # indicates statistically significant P < 0.05 when compared with the DON group at that time point, and & indicates statistically significant P < 0.05 when compared with the A-DON group at that time point). D. Statistical graph of the number of foot grips in the inclined grid climbing test (n = 6, *P < 0.05).

[0045] Figure 9 :EdU tracer-recruited NSCs differentiation and endogenous neurogenesis. AD: Low-magnification immunofluorescence images showing the expression of Tuj-1 in SCI, DON, A-DON and A-DHN groups. A1-D3: High-magnification images showing Tuj-1 at the junction of the head / tail end of the injury / transplantation area and in the injury / transplantation area. + / EdU + cells (indicated by arrows) and GFAP + / EdU + cells (indicated by arrows);

[0046] Figure 10 :The maturation of newborn neurons and the statistical analysis of the number of newborn neurons in each group. A: Tuj-1 in A-DHN group + / EdU + Newly born neurons express Map2. B: EdU in the junction of the head and tail of the injury / transplantation area and in the injury / transplantation area +Cell number statistics. C: Tuj-1 in the injury / transplantation area + / EdU + Statistical graph of the number of newborn neurons. D: EdU in the injury / transplantation area + Tuj-1 cell differentiation + / EdU + Newborn neurons and GFAP + / EdU + Statistical diagram of the proportion of astrocytes. E: Tuj-1 in the rostral junction of the injury / transplantation area. + / EdU + Statistical graph of the number of newly born neurons. (F) Tuj-1 in the caudal junction of the injury / transplantation area + / EdU + Statistics of the number of newborn neurons. DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0048] The following is a detailed description of the main instruments, material preparation, experimental cells and reagents used in the present invention and specific examples:

[0049] 1. Main instruments

[0050] Clean bench (Suzhou Purification Electronic Equipment Factory); ordinary centrifuge (Kubota, Japan); constant temperature water bath (Beijing Medical Equipment Factory); 5% CO2 incubator (Queue, USA); freeze dryer (6L FREEZONE PLUS, Labconco, USA); stereo microscope (M205FA, Leica, Germany); inverted phase contrast microscope (Olympus, Japan); fluorescence microscope (Leica, Germany); scanning electron microscope (Philips, Netherlands); semi-thin microtome (RM2065, Leica, Germany); transmission electron microscope (Philips, Netherlands); laser confocal imaging system (Carl Zeiss, Germany); high-speed confocal microscope (Dragonfly, Oxford Instruments, UK); low-temperature oven (Shanghai Yuejin Medical Instrument Factory); high-temperature oven (Shanghai Yuejin Medical Instrument Factory); high-pressure sterilizer (Jiangyin Binjiang Medical Equipment Factory); constant temperature box microtome (Shandon, UK); ultrapure water analyzer (Molsheim, France); -80°C ultralow temperature freezer (Revco Tech, USA); microplate reader (Sunrise, TECAN, Austria).

[0051] 2. Material Preparation

[0052] The biomaterials used to construct the biomimetic spinal cord scaffold in this invention are decellularized porcine optic nerve and decellularized spinal cord hydrogel, prepared in-house by our laboratory. The Apt19S used was synthesized by Shanghai Bioengineering, and the neurotrophic factor NT-3 was purchased from Peptek Asia Ltd.

[0053] 1) Preparation of Decellularized Optic Nerves: Fresh optic nerves were isolated from adult pigs (approximately 2 years old, provided by Northwest Agriculture and Forestry University, China). The obtained optic nerves were washed overnight in ddH2O containing 1% penicillin-streptomycin and a mycotoxin / amphotericin solution at 4°C in a centrifuge tube. The nerves were then decellularized using 3% Triton X-100, 4% sodium deoxycholate, DNase, and RNase. The optic nerves were then immersed in sterile ddH2O in a 1.5 ml centrifuge tube and freeze-dried for 24 hours.

[0054] 2) Preparation of decellularized spinal cord hydrogel: Fresh spinal cord was isolated from adult pigs (approximately 2 years old, provided by Northwest Agriculture and Forestry University, China) and washed overnight at 4°C in ddH2O containing 1% penicillin-streptomycin and mycotoxin / amphotericin solution. The spinal cord was then immersed in a 3% Triton-X-100 + 1% double-antibody aqueous solution on a shaker (250 rpm) for 12 hours, followed by immersion in a 4% sodium deoxycholate aqueous solution on a shaker (250 rpm) for 24 hours, and then immersed in a DNase (100x) + RNase (100x) + MgCl2 + Tris 50 mmol aqueous solution on a 37°C constant temperature shaker (150 rpm) for 3 hours, with the solution changed once. Then wash with ddH2O three times, each time for 20 minutes. After washing, freeze-dry the decellularized spinal cord scaffold. After freeze-drying, cut the scaffold into small pieces. Take pepsin in a ratio of 1:10 to the decellularized material and add 0.01M hydrochloric acid. Stir at room temperature for 24 hours to prepare the decellularized spinal cord hydrogel.

[0055] 3). Apt19S synthetic sequence:

[0056] 1) Apt19S:

[0057] 5'-AGGTCAGATGAGGAGGGGGACTTAGGACTGGGTTTATGACCTATGCGTG-3'.

[0058] 2)Amino-modified Apt19S5'-NH2-(A)9-AGGTCAGATGAGGAGGGGGACTTAGGACTGGGTTTTATGACCTATGCGTG-3'

[0059] 3)Amino-modified FITC-Apt19S:5'-NH2-(A)9-AGGTCAGATGAGGAGGGGGACTTAGGACTGGGTTTATGACCTATGCGTG-FIT C-3'

[0060] 4) Preparation of scaffold / gel composite central nervous system repair materials:

[0061] 1) Incubate the decellularized optic nerve scaffold in 2 ml of morpholineethanesulfonic acid (MES, 0.1 M, pH = 6) at room temperature for 30 minutes.

[0062] 2) Add 60 mg of carbodiimide and 80 mg of N-hydroxysuccinimide to the above solution and incubate at room temperature for another 20 minutes.

[0063] 3) The treated optic nerve was removed from the liquid, washed three times with D-hanks buffer, and then added with sterile water containing 2 nmol Apt19S. After reacting in the dark for 12 hours, an Apt19S-loaded decellularized optic nerve scaffold was obtained.

[0064] 4) Use 1 M sodium hydroxide solution to adjust the pH of the decellularized spinal cord matrix hydrogel solution to about 7.4.

[0065] 5) At 4°C, add 10×DEME, the volume of which is 1 / 9 of the obtained solution volume.

[0066] 6) At 4°C, take 10 μl of the solution obtained in step 2 and add 10 μl of 1× DMEM solution containing 2.5 μg of neurotrophin-3.

[0067] 7) The solution obtained in step 6 was poured into the decellularized optic nerve scaffold loaded with Apt19S, and incubated at 37° C. for 20 minutes to form a hydrogel, thereby obtaining the scaffold / gel composite central nervous system repair material.

[0068] 3. Experimental cells and animals

[0069] Adult female SD rats (220-250 grams) and SD rat pups were provided by the Experimental Animal Center of Sun Yat-sen University. NSCs were isolated and cultured from their brain hippocampus to test the performance of the scaffold. SD rat pups were selected at 3 to 5 days old and were killed by overdose of anesthesia under sterile conditions. After decapitation, the brains were removed and placed in cold D-Hank's solution. The hippocampus was isolated using instruments under a dissecting microscope. NSCs were cultured using a mechanical pipette method: the hippocampal tissue was first cut into pieces with ophthalmic scissors, and then transferred into a centrifuge tube along with the D-Hank's solution. The tube was gently pipetted several times with a fine-tipped glass pipette until no obvious tissue blocks were visible to the naked eye. The pipette should be pipetted slowly and with moderate force to avoid bubbles. The tube was centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, and the operation was repeated once. The cell pellet was resuspended with NSCs culture medium, and the cell density was counted and adjusted to approximately 1×10 5 / ml, transfer this cell suspension into a culture flask and culture in a 37°C, 5% CO2 incubator for suspension culture. Once a large number of cell spheres are observed to form, mechanically dissociate the NSC spheres using a fine-tipped glass pipette the next day for passage. Two weeks after passage, NSCs from the second generation are collected for nestin immunofluorescence cytochemistry and floatation.

[0070] 4. Main Reagents

[0071] DMEM / F12 (Gibico), DMEM-LG (Gibico), premium fetal bovine serum (TBD), poly-lysine (Sigma), D-Hank's balanced solution (self-prepared), trypsin (Sigma), EDTA (Sangon), 0.0.1 mol / l PBS (Zhongshan Jinqiao), MTT (Ameresco), dimethyl sulfoxide (DMSO, Sangon), Hoechst 33342 (Sigma), DAPI (Sigma), goat serum (Zhongshan Jinqiao), CCK-8 kit (DOJINDO), chicken anti-rat NF antibody (Abcam, UK), mouse anti-rat SYP antibody (Sigma), mouse anti-rat SYP-647 antibody (Abcam), rabbit anti-rat PSD95 antibody (Abcam), mouse anti-rat Map2 antibody (Sigma), rabbit anti-rat GFAP antibody (Abcam), mouse anti Rat Nestin antibody (Abcam), rabbit anti-rat 5-HT antibody (Sigma), rabbit anti-rat VGluT1 antibody (Abcam), rabbit anti-rat Ki-67 antibody (Abcam), rabbit anti-rat Tuj-1 antibody (Abcam), rabbit anti-rat ALPL antibody (Abmart, China), mouse anti-rat Tuj-1 antibody (Sigma), rabbit anti-rat ALPL antibody (Elabscience, China), rabbit anti-rat β-actin antibody (Proteintech, USA), rabbit anti-rat GAPHD antibody (Sigma), Streptavidin Conjugate 555 (Invitrogen, USA), goat anti-chicken IgG Alexa fluor 488 (Abcam), goat anti-rabbit IgG Alexa fluor 488 (Abcam), goat anti-mouse IgG Alexa fluor 488 (Abcam), goat anti-rabbit IgG Alexa fluor 555 (Abcam), goat anti-mouse IgG Alexa fluor 555 (Abcam), goat anti-rabbit IgG Alexa fluor 647 (Abcam), goat anti-mouse IgG Alexa fluor 647 (Abcam), goat anti-mouse IgG HRP (Abcam), goat anti-rabbit IgG HRP (Abcam), NT-3 ELISA kit (Boster), goat anti-rabbit HRP (Jackson ImmunoResearch), protein quantification kit (Dingguo), cell lysate (Boster), protease inhibitor cocktail (Sigma), ECL luminescent substrate detection kit (Kangwei Century), Epon-812 (Ted Pella), Coomassie brilliant blue (Bio-rad), 30% polyacrylamide solution (Kangwei Century).

[0072] Specific operation techniques

[0073] Example 1: Verification of Apt19S recruitment of neural stem cells (NSCs)

[0074] 1) The steps for loading the decellularized optic nerve with Apt19S are as follows:

[0075] a. Incubate the decellularized optic nerve scaffold in 2 ml of morpholineethanesulfonic acid (MES, 0.1 M, pH = 6) at room temperature for 30 minutes.

[0076] b. Add 60 mg of carbodiimide and 80 mg of N-hydroxysuccinimide to the above solution and incubate at room temperature for another 20 minutes.

[0077] c. The treated optic nerve was removed from the liquid, washed three times with D-hanks buffer, and then added to sterile water containing 2 nmol Apt19S. After 12 hours of reaction in the dark, an Apt19S-loaded decellularized optic nerve scaffold (A-DON) was obtained.

[0078] 2) The ability of Apt19S to recruit NSCs was assessed using a transwell system (Costar, 24-well plate, 8 μm). Neurospheres were digested with 0.125% pepsin for 10 minutes to obtain a single cell suspension. Considering the suspended growth of NSCs, the membrane of the lower chamber of the transwell system was incubated with laminin for 30 minutes before cell seeding to improve cell attachment. 2×10 4 Cells were seeded into the upper chamber of the transwell system in 200 μL DMEM / F12 medium. 600 μL of DMEM / F12 medium supplemented with 1% fetal bovine serum (FBS, Gibco) was added to the lower chamber, either without any biomaterial, or with DON scaffolds or A-DON scaffolds (n=4). After incubation for 16 h, the chambers were washed 3 times with PBS and fixed with 4% paraformaldehyde for 20 minutes. After washing 3 more times, the filters were stained with crystal violet for 10 minutes. Three random 100x bright field images were captured for each chamber using a microscope (DM6B, Leica). The results are shown in Figure 5. Figure 1 As shown, compared with the DON and control groups, the A-DON group had significantly enhanced cell migration across the transwell chamber membrane, indicating that Apt19S was released from the A-DON scaffold to promote the migration of NSCs. Quantitative analysis also showed that the number of NSCs that migrated in the A-DON group was significantly higher than that in the DON and blank groups.

[0079] 3) NSC attachment experiment further verified the recruitment of NSCs by Apt19S. First, the neurospheres of green fluorescent protein (GFP) transgenic SD rats were digested with 0.125% pepsin for 10 minutes to obtain a single cell suspension. 5x10 4GFP-NSCs were added to each well of a 24-well plate. DON scaffolds with or without Apt19S were placed in each well and incubated for 12 hours. The scaffolds were then fixed with 4% paraformaldehyde for 20 minutes, and the attached cells were observed under a confocal microscope. Figure 3 As shown, after 24 hours of culture, the number of NSCs attached to the A-DON scaffold was significantly higher than that attached to the DON scaffold. Quantitative analysis showed that the number of NSCs attached to the A-DON scaffold was three times that of the DON scaffold.

[0080] Example 2: Preparation of scaffold / gel composite central nervous system repair material:

[0081] 1) Incubate the decellularized optic nerve scaffold in 2 ml of morpholineethanesulfonic acid (MES, 0.1 M, pH = 6) at room temperature for 30 minutes.

[0082] 2) Add 60 mg of carbodiimide and 80 mg of N-hydroxysuccinimide to the above solution and incubate at room temperature for another 20 minutes.

[0083] 3) The treated optic nerve was removed from the liquid, washed three times with D-hanks buffer, and then added with sterile water containing 2 nmol Apt19S. After reacting in the dark for 12 hours, an Apt19S-loaded decellularized optic nerve scaffold was obtained.

[0084] 4) Use 1 M sodium hydroxide solution to adjust the pH of the decellularized spinal cord matrix hydrogel solution to about 7.4.

[0085] 5) At 4°C, add 10×DEME, the volume of which is 1 / 9 of the obtained solution volume.

[0086] 6) At 4°C, take 10 μl of the solution obtained in step 2 and add 10 μl of 1× DMEM solution containing 2.5 μg of neurotrophin-3.

[0087] 7) The solution obtained in step 6 was poured into the decellularized optic nerve scaffold loaded with Apt19S, and incubated at 37° C. for 20 minutes to form a hydrogel, thereby obtaining the scaffold / gel composite central nervous system repair material.

[0088] Preparation process and appearance of preparation materials Figure 3 shown.

[0089] Example 3: Scaffold / gel composite central nervous system repair material with sustained release of Apt19S and neurotrophic factor-3

[0090] 1) A decellularized optic nerve scaffold loaded with amino-modified FITC-labeled Apt19S. 2) Apt19S loaded with amino-free FITC labeling (for control). 3) A scaffold / gel composite central nervous system repair material loaded with amino-modified FITC-labeled Apt19S. Immerse in a 24-well culture plate containing 1 ml of sterile buffer. The scaffold was then incubated at 37 ° C. At each predetermined time point (1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30 days), 1 ml of sterile buffer was taken out and an equal volume of new sterile buffer was added. FITC was used as a model guest molecule to monitor the release kinetics of Apt19S. The collected suspension was added to a 96-well culture plate and quantified at a wavelength of 525 nm using a multimode plate reader to measure the concentration of released Apt19S.

[0091] like Figure 4 As shown in A, the decellularized optic nerve scaffold loaded with amino-modified Apt19S released 14.99% of Apt19S in the first 3 days and 34.97% within 7 days, showing a sustained release pattern. In contrast, the decellularized optic nerve scaffold loaded with unamino-modified Apt19S showed a burst release pattern, with approximately 42.41% of the total Apt19S released within the first day. More importantly, within 30 days, the total release amount of the decellularized optic nerve scaffold loaded with amino-modified Apt19S (0.86 nmol) was 5.5 times greater than that of the decellularized optic nerve scaffold loaded with unamino-modified Apt19S (total release amount 0.13 nmol). The scaffold / gel composite central nervous system repair material showed a similar release pattern and release amount to the decellularized optic nerve scaffold loaded with amino-modified Apt19S in terms of Apt19S release, indicating that the sustained release of Apt19S can be achieved to continuously attract endogenous neural stem cells.

[0092] The in vitro release of neurotrophic factor-3 was carried out by immersing the scaffold / gel composite central nervous system repair material (200ng neurotrophic factor-3 per scaffold, n=3) in 1ml of DMEM / F12 containing 5% BSA in a 24-well plate. The solution was collected at each time point (1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30 days) and replaced with 1ml of new DMEM / F12 containing 5% BSA to protect the protein from degradation. The concentration of neurotrophic factor-3 was quantified using the NT-3 ELISA kit according to the manufacturer's instructions. The neurotrophic factor-3 release curve was drawn based on the ratio of the cumulative released neurotrophic factor-3 to the total amount of loaded neurotrophic factor-3 at each time point. As Figure 4Release curve B showed that on the first day, 38.6% of the total amount of neurotrophic factor-3 was released from the scaffold / gel composite central nervous system repair material in a burst release mode; then it showed a stable release form, with a total of 77.9% of the loaded neurotrophic factor-3 released slowly over a total period of 30 days.

[0093] Example 4: In vitro biocompatibility and functional testing of scaffold / gel composite central nervous system repair materials

[0094] To test the biocompatibility of various components of the scaffold / gel composite central nervous system repair material, and their effects on the regulation of NSC differentiation fate and the formation of synaptic potential, the in vitro experiments were divided into the following six groups: 1) Two-dimensional culture group (no material). 2) Decellularized optic nerve scaffold group (DON). 3) Decellularized optic nerve scaffold loaded with Apt19S (A-DON). 4) Decellularized optic nerve / decellularized spinal cord hydrogel composite scaffold (DH). 5) Decellularized optic nerve / decellularized hydrogel composite scaffold loaded with NT-3 (DHN). 6) Decellularized optic nerve / decellularized hydrogel composite scaffold loaded with Apt19S and NT-3 (i.e., the scaffold / gel composite central nervous system repair material constructed in this patent, A-DHN).

[0095] Experimental plan: Extracted NSCs were seeded onto scaffolds. Live / dead cell staining and CCK-8 cell viability assays were used to assess NSC survival on the biomimetic spinal cord scaffolds. Immunofluorescence staining and confocal microscopy were used to examine the expression of neuronal markers (Map2) and astrocyte markers (GFAP) to assess the differentiation direction of NSCs on the biomimetic spinal cord scaffolds. Immunofluorescence staining and western blotting were used to examine the expression of presynaptic membrane markers (SYP) and postsynaptic membrane markers (PSD95) to evaluate the potential of the biomimetic spinal cord scaffolds to promote synapse formation.

[0096] like Figure 5 As shown in A and 5B, there was no significant difference in the results between the groups in the CCK-8 and live / dead cell staining experiments, proving that the components of all materials had no effect on cell activity. Figure 6 As shown, we observed more Map2 positive cells in the DH group compared with the DON and A-DON groups (Map2 + ) neurons and fewer GFAP-positive (GFAP + ) astrocytes, proving that the decellularized spinal cord hydrogel can play a certain role in promoting neuronal formation. Compared with the DON, A-DON and DH groups, we found that the Map2 + Neurons account for the highest proportion, while GFAP +Astrocytes accounted for the lowest proportion, proving that NT-3 and decellularized spinal cord hydrogel in the constructed scaffold / gel composite central nervous system repair material synergistically promoted the differentiation of NSCs into neurons, while Apt19S had no effect on cell differentiation. Figure 7 As shown, Map2 + The neurons expressed a large amount of synaptic markers SYP and PSD95, while the other groups only expressed a small amount, proving that the scaffold / gel composite central nervous system repair material constructed by this patent has a strong effect of inducing neurons to form synaptic connections.

[0097] Example 5: In vivo scaffold / gel composite central nervous system repair material promotes motor function recovery and neurogenesis

[0098] To examine the in vivo effects of a scaffold / gel composite central nervous system repair material on motor function recovery after spinal cord injury and the chemotaxis and incubation of NSCs, four in vitro experiments were conducted: 1) spinal cord injury (SCI) group (no material); 2) DON group; 3) A-DON group; and 4) A-DHN group.

[0099] Experimental Plan: The Basso, Beattie, and Bresnahan (BBB) ​​score and grid climbing test were performed on the first day after surgery and weekly. For the BBB test, rats were allowed to crawl freely on an open, flat surface for five minutes. Hindlimb movement was observed and scored according to the BBB scoring criteria. Key observations included the frequency and range of motion of three joints (hip, knee, and ankle) in both or one hindlimb; movement posture; the ability of the hindlimbs to occasionally or consistently support the body; and the ability to occasionally or consistently coordinate forelimb and hindlimb movements. The grid climbing test involved a rat climbing grid stand constructed according to the method of Ramon-Cueto et al. Rats were placed on a 45° sloped grid and their hindlimb movements were observed for five minutes while climbing the grid. This performance focused on the animal's overall ability to climb the obstacle per unit time and the presence of coordinated forelimb and hindlimb movements. Additionally, starting on the third day after surgery, EdU (50 mg / kg body weight) was injected intraperitoneally every 24 hours for 10 consecutive days. Injected EdU was used to label cells with proliferative capacity and allow observation of the differentiation of endogenous NSCs. TM EdU-488 and BeyoClick TM EdU-555 kit was used for EdU immunofluorescence staining.

[0100] like Figure 8As shown in A, the spinal cord was completely transected 2 mm at the T10 level and removed, resulting in complete paralysis of the hind limbs. Subsequently, no material (SCI), DON, A-DON, and A-DHN scaffolds were transplanted into the injured area. During the 8-week observation period, the rats did not show signs of pain such as hind limb biting due to hyperesthesia and pain, obvious weight loss, aggression, and crying when touched. The open field movement test was used to evaluate the recovery of motor function within 8 weeks after surgery. Compared with the SCI group and the DON group, the A-DHN group and the A-DON group observed significant improvement in hind limb motor function at 8 weeks after SCI ( Figure 8 A, arrows indicate paralyzed hindlimbs). The inclined grid climbing test is an effective method for evaluating sensory-motor coordination between the hindlimbs and forelimbs and detecting the reorganization of descending motor pathways. Figure 3-7 As shown in Figure B, rats in the SCI and DON groups dragged their hindlimbs while climbing the inclined grid and used their forelimbs to climb. In contrast, rats in the A-DON and A-DHN groups could climb the grid voluntarily using their hindlimbs; however, rats in the A-DHN group showed larger hindlimb movements ( Figure 8 B).

[0101] The recovery of hindlimb motor function in each group was also quantitatively evaluated using the BBB score (n = 8, Figure 8 C). At week 8, the mean score of the A-DHN group was 7.625, indicating that the rats could touch the ground with their paws on their backs without bearing weight. In the A-DON group, the mean BBB score of the rats was 5.550, meaning that two joints of the hind limbs were widely movable and the third joint was slightly movable. At week 8, the mean BBB scores of the DON and SCI groups were 3.375 and 2.250, respectively. To more accurately display the results of the inclined grid climbing, we quantified the number of hind limb grasps in three rounds of grid climbing according to the method described in previous studies and found that the A-DHN group had the most foot grasps, followed by the A-DON group, while the SCI and DON groups had relatively few grasps ( Figure 8 D).

[0102] To evaluate the differentiation of NSCs recruited to the injury / transplantation area within 8 weeks after spinal cord injury, we injected EdU into rats from day 3 to day 14 after surgery to track proliferating cells. A large number of EdU cells were found in the transplantation area and the junction area between the rostral and caudal ends of the transplantation area in the A-DHN and A-DON groups. + cells, while less EdU was detected in these areas in the DON and SCI groups. + cell( Figure 9 AD). Tuj-1 + / EdU +The cells are newborn neurons differentiated from recruited NSCs and were found in the transplanted areas and the junction areas at the head and tail ends of the transplanted areas in the A-DON and A-DHN groups ( Figure 9 C1-D3). In the A-DNH group, Tuj-1 in the transplanted area + / EdU + Newborn neurons were more abundant and more mature than those in the A-DON group. + / EdU + Few new neurons ( Figure 9 A1-B3). In addition, we observed Map2 at the junction of the head end of the transplanted area in the A-DHN group. + / Tuj-1 + / EdU + of newborn neurons, which marks the maturity of newborn neurons ( Figure 10 A) Quantitative analysis showed that although EdU + The number of cells was only slightly higher than that in the A-DON group, but the Tuj-1 + / EdU + The number of newborn neurons was more than twice that of the A-DON group ( Figure 10 B, C). In contrast, EdU + Cells and Tuj-1 + / EdU + The number of newborn neurons was significantly lower than that in the A-DON and A-DHN groups ( Figure 10 B, C). It is noteworthy that in the A-DHN group, more than 50% of the transplanted area was covered with EdU. + Tuj-1 cells + / EdU + The proportion of newly born neurons decreased significantly to 19.2% in the A-DON group and further decreased to only about 10% in the DON and SCI groups ( Figure 3-10 D) In ​​the region at the head / tail of the injury / transplantation area, the EdU + The percentage of cells differentiating into newborn neurons was similar to the trend observed in the injury / transplantation area ( Figure 10 D,F).

[0103] For GFAP + / EdU + The number of astrocytes showed a completely opposite trend to that of newborn neurons. The highest number of GFAP was observed in the SCI group at the head / tail junction of the injury / transplantation area. + / EdU + astrocytes, while the number in the A-DHN group was the lowest ( Figure 10D, F). At the cranial / caudal junction of the injury / transplantation area, approximately 50% of EdU was expressed in the DON and SCI groups. + GFAP cells differentiate into + / EdU + Astrocytes, this proportion decreased slightly to 40% in the A-DON group and significantly decreased to approximately 25% in the A-DHN group ( Figure 10 D, F). However, GFAP was detected in the injury / transplantation area in all four groups. + / EdU + There are very few astrocytes ( Figure 10 C).

[0104] In summary, the scaffold / gel composite central nervous system repair material constructed by this patent can specifically attract endogenous neural stem cells, and through the loaded NT-3 and decellularized spinal cord hydrogel, synergistically promote the differentiation of recruited NSCs into neurons, and promote the formation of synaptic connections between neurons, ultimately forming an endogenous neural network.

[0105] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and replacements made by technicians in this field on the basis of the present invention fall within the scope of protection required by the present invention.

Claims

1. A scaffold-gel composite central nervous system repair material for specifically attracting and incubating endogenous neural stem cells, characterized in that: The invention is composed of a decellularized optic nerve scaffold, a decellularized spinal cord hydrogel, a neurotrophic factor and an aptamer Apt19S. The compounding requirements of the decellularized optic nerve scaffold, the decellularized spinal cord hydrogel, the neurotrophic factor and the aptamer Apt19S are as follows: A 2 mm long, 3 mm diameter acellular optic nerve scaffold; 2 nmol of aptamer Apt19S; 20 μl of 10 mg / ml acellular spinal cord hydrogel; and 2.5 μg of neurotrophic factor. The method for preparing the scaffold-gel composite central nervous system repair material for specifically attracting and incubating endogenous neural stem cells comprises the following preparation steps: a) Utilizing the abundant carboxyl groups within the decellularized optic nerve scaffold, amino-modified Apt19S was loaded onto the material via an amidation reaction, thereby achieving sustained release of Apt19S and obtaining an Apt19S-loaded decellularized optic nerve scaffold; b) injecting the decellularized spinal cord hydrogel added with neurotrophic factors into the decellularized optic nerve scaffold loaded with Apt19S obtained in step a) to obtain the composite central nervous system repair material.

2. The scaffold-gel composite central nervous system repair material for specifically attracting and incubating endogenous neural stem cells according to claim 1, characterized in that: The neurotrophic factor is one or more of neurotrophin-3, ciliary neurotrophic factor, glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, fibroblast growth factor and insulin-like growth factor.

3. An application, characterized in that: Use of the scaffold-gel composite central nervous system repair material for specifically attracting and incubating endogenous neural stem cells as described in any one of claims 1 to 2 in the preparation of products for repairing spinal cord injury and central nervous system injury.

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