Bioactive scaffolds that slowly release Apt19S to recruit endogenous stem cells and guide the regeneration of central nervous system axons and their applications

Abstract

The present invention discloses a bioactive scaffold for slowly releasing Apt19S to recruit endogenous stem cells and guide the straight regeneration of central nervous system axons and its application. The bioactive scaffold is composed of acellular optic nerve (DON) and aptamer Apt19S, and its preparation method comprises the following steps: 1. preparing a DON scaffold. 2. activating the carboxyl group on the surface of the DON scaffold. 3. The DON scaffold is combined with amino-modified Apt19S through an amidation reaction to obtain the DON‑Apt19S scaffold (abbreviated as DON‑A). This bioactive scaffold can continuously release Apt19S, recruit ALPL-positive endogenous neural stem cells and mesenchymal cells, guide the straight regeneration of central nervous system axons, and provide a microenvironment for neurogenesis and angiogenesis for in situ spinal cord repair. The bioactive scaffold has important transformation prospects and application value in the repair of central nervous system injuries.

Description

Technical Field

[0001] The present invention relates to the field of medical tissue engineering, and in particular to a bioactive scaffold DON-Apt19S (hereinafter referred to as DON-A) that utilizes decellularized optic nerve (DON) and DNA aptamer (Apt) 19S (Apt19S) to construct a sustained-release Apt19S to recruit endogenous stem cells and guide the direct regeneration of central nervous system axons. The scaffold can specifically bind to alkaline phosphatase (ALPL) receptors on the surface of stem cell membranes through Apt19S, thereby recruiting endogenous ALPL-positive neural stem cells and mesenchymal stem cells in the central nervous system, providing a central nervous system-specific extracellular matrix microenvironment support for the proliferation and differentiation of stem cells, and guiding the regeneration of central nervous system and blood vessels. Background Art

[0002] Traditionally, it has been held that a severely injured spinal cord cannot repair itself. Besides the presence of a hypoxic, ischemic, and inflammatory microenvironment that is unfavorable for regeneration, another important reason is the lack of endogenous stem cells, which prevents the replenishment of functional cells. The inflammatory factors produced during the acute phase of spinal cord injury act as a double-edged sword, leading to necrosis and apoptosis of nerve cells on the one hand, and activating endogenous stem cells on the other. This provides a possibility for mobilizing autologous stem cells to achieve self-repair of spinal cord injury (SCI): if autologous stem cells can be fully mobilized and induced to differentiate into functional cells to participate in tissue repair, the series of difficulties faced by cell transplantation strategies (such as immune rejection, difficulty in long-term survival, and insufficient functional integration) can be avoided, making it easier to translate into clinical applications.

[0003] The first two weeks after SCI are a critical period for neural stem cell activation and proliferation. However, these neural stem cells (NSCs) activated by inflammatory factors often face a fate of apoptosis or differentiation, contributing to scar formation, in the inflammatory microenvironment. Several slowly released chemokines have been shown to coordinate the migration of endogenous NSCs at the site of spinal cord injury, promoting endogenous repair after SCI. For example, collagen scaffolds containing a slow release of stromal cell-derived factor 1 (SDF-1) can recruit endogenous NSCs when transplanted into the injured spinal cord of rats. Furthermore, a microenvironment containing a slow release of cetuximab, IL-10, paclitaxel (PTX), and N-cadherin can promote the differentiation of endogenous NSCs into neurons, contributing to SCI repair. Studies have shown that biomaterials rich in neurotrophic factors or mesenchymal stem cell (MSC) exosomes can also promote the survival and further differentiation of endogenous NSCs that have migrated into the materials into neurons or oligodendrocytes, contributing to neural repair. Therefore, strategies to attract endogenous NSCs, prevent NSC apoptosis, and promote their differentiation into neurons play an important role in SCI repair. However, there are currently no reports on the simultaneous recruitment of endogenous NSCs and MSCs at the site of spinal cord injury. Furthermore, existing approaches primarily focus on improving the injury microenvironment to prevent endogenous stem cell apoptosis rather than specifically recruiting endogenous stem cells. Ischemia and hypoxia at the site of spinal cord injury, coupled with insufficient numbers of endogenous neural stem cells and uncontrollable differentiation, remain fundamental challenges hindering spinal cord self-repair.

[0004] DNA aptamer (Apt) 19S (Apt19S) is a small molecule nucleic acid drug discovered in recent years. As a ligand, it can bind to the ALPL receptor on the surface of stem cells, specifically recruiting stem cells with high ALPL expression (including embryonic stem cells, pluripotent stem cells, etc.). The proliferation and differentiation potential of these stem cells is often proportional to the expression of ALPL. In recent years, Apt19S has also been found to have a specific recruitment effect on MSCs and NSCs, and is used for targeted treatment of cartilage and neurodegenerative diseases. However, Apt19S is easily degraded when used for intravenous injection, so it is often used for local administration. Our previous studies suggest that DON scaffolds have good mechanical strength, can provide a central nervous system development microenvironment for NSCs, and have the potential to guide the regeneration of nerve axons.

[0005] In summary, we propose to utilize the characteristic of Apt19S that it can specifically bind to protein carboxyl groups after amino modification, and stably load it into the DON scaffold rich in protein carboxyl groups. We design a bioactive scaffold DON-A that can not only support the spinal cord neural regeneration channel but also stably and slowly release Apt19S. It can efficiently recruit and "hatch" ALPL-positive endogenous NSCs and MSCs at the spinal cord injury site, effectively achieving in situ neural and vascular regeneration of the damaged spinal cord and functional reconstruction of neural pathways. Summary of the Invention

[0006] The purpose of the present invention is to overcome the problem of insufficient endogenous neural stem cells after spinal cord injury and their difficulty in surviving in the injury microenvironment, and to develop a bioactive scaffold that slowly releases Apt19S to recruit endogenous stem cells and guide the direct regeneration of central nervous system axons. DON-A is used to be transplanted into the spinal cord injury site to promote in situ spinal cord nerve and blood vessel regeneration. The spatial microtopological structure of the DON scaffold and the abundant protein carboxyl groups on its surface are utilized to load amino-modified Apt19S through an amidation reaction to prepare the bioactive scaffold DON-A for slowly releasing Apt19S, thereby achieving sustained release of Apt19S, which then specifically binds to ALPL on the surface of the stem cell membrane, recruiting a large number of endogenous NSCs and MSCs into the DON scaffold, and utilizing the central nervous system extracellular matrix microenvironment provided by DON to achieve in situ spinal cord nerve and blood vessel regeneration.

[0007] One of the objectives of the present invention is achieved by the following technical solution:

[0008] A bioactive scaffold that slowly releases Apt19S to recruit endogenous stem cells and guide the direct regeneration of central nervous system axons is composed of a decellularized optic nerve (DON) scaffold and an aptamer Apt19S. Specifically, the bioactive scaffold includes the following preparation steps: 1) preparing a DON scaffold; 2) activating the carboxyl groups of the DON scaffold; and 3) preparing a DON-A bioactive scaffold: the carboxyl-activated DON scaffold and amino-modified Apt19S are co-incubated to obtain a DON-A bioactive scaffold that slowly releases Apt19S.

[0009] Furthermore, in step 1), the preparation step of the DON scaffold includes the following steps:

[0010] After removing adipose tissue from fresh porcine optic nerves, they were washed overnight at 4°C in distilled water containing penicillin-streptomycin and mycotoxin / amphotericin solution. Subsequently, Triton X-100, sodium deoxycholate, DNase, and RNase were used to remove cells from the optic nerve, preserving the extracellular matrix and its natural physical structure. The cell-free optic nerves were then immersed in sterile distilled water in a centrifuge tube and freeze-dried for 24 hours to obtain DON scaffolds.

[0011] Further preferably, in step 1), the preparation step of the DON scaffold comprises the following steps:

[0012] a) extracting a fresh optic nerve from an adult pig, removing fat tissue and nerve sheath from the nerve to obtain the first optic nerve;

[0013] b) The first optic nerve was placed in distilled water containing 1% penicillin-streptomycin, mycotoxins, and amphotericin at 4° C. for 24 hours to obtain the second optic nerve; wherein the mycotoxins were at a concentration of 100 μg / mL and the amphotericin was at a concentration of 2.5 μg / mL.

[0014] c) The second optic nerve was then immersed in a 3% Triton-X-100 solution and shaken on a shaker at 250 rpm for 12 hours to obtain the third optic nerve;

[0015] d) Subsequently, the third optic nerve was immersed in a 4% sodium deoxycholate aqueous solution and shaken at 250 rpm for 24 hours to obtain the fourth optic nerve;

[0016] e) The fourth optic nerve was then immersed in an aqueous solution of 50 U / ml DNase, 50 U / ml RNase, 10 mM MgCl2·6H2O, and 50 mM Tris, and shaken at 150 rpm at 37°C for 3 hours to decellularize the nerve, thereby obtaining the fifth optic nerve;

[0017] f) Then, the fifth optic nerve was immersed in sterile double-distilled water, placed in a centrifuge tube and freeze-dried for 24 hours to obtain the DON scaffold.

[0018] Furthermore, in step 2), the preparation step of activating the carboxyl group of the DON scaffold comprises the following steps:

[0019] The DON scaffold obtained in step 1) was placed in a 0.1 M morpholineethanesulfonic acid (MES, pH = 6) solution and incubated at room temperature for 30 minutes. Then, 80 mg of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and 60 mg of N-hydroxysuccinimide were added to the morpholineethanesulfonic acid solution and incubated for 20 minutes to activate the carboxyl groups on the DON surface.

[0020] Further preferably, the preparation step of activating the carboxyl group of the DON scaffold further comprises the following steps before the DON scaffold is placed in morpholineethanesulfonic acid:

[0021] The DON scaffold obtained in step 1) is made into a cylindrical DON scaffold with a diameter of 3 mm and a height of 2 mm.

[0022] Furthermore, in step 3), the preparation of the DON-A bioactive scaffold comprises the following steps:

[0023] The carboxyl-activated DON scaffold and the amino-modified Apt19S were incubated together, and a stable chemical bond was formed through an amidation reaction, thereby achieving stable loading and sustained release of Apt19S in the DON scaffold.

[0024] Further preferably, in step 3), the preparation step of the DON-A bioactive scaffold comprises the following steps:

[0025] The carboxyl-activated DON scaffold was placed in an amino-modified Apt19S solution and incubated for 12 hours. Through an amidation reaction, a stable chemical bond was formed to achieve stable loading and sustained release of Apt19S in the DON scaffold, thereby obtaining the Apt19S-carrying DON-A bioactive scaffold.

[0026] The application of the bioactive scaffold obtained by the above preparation method for slowly releasing Apt19S to recruit endogenous stem cells and guide the direct regeneration of central nervous system axons in spinal cord injury repair falls within the protection scope of the present invention.

[0027] Experiments have shown that the DON-A scaffold prepared by the present invention can confirm in vitro that Apt19S can specifically bind to NSCs and MSCs through ALPL. In vivo experiments have confirmed that DON-A transplanted into the injury site of rats with complete spinal cord transection can recruit a large number of endogenous stem cells, promote more NSCs to differentiate into neurons, and MSCs to differentiate into vascular endothelial cells, increase the potential of endogenous stem cells to differentiate into neurons and the ability to form blood vessels, guide the direct regeneration of central nervous system axons, and promote the recovery of motor function of paralyzed hind limbs.

[0028] The second object of the present invention is achieved by the following technical solution:

[0029] One application is the use of the above-mentioned bioactive scaffold for slowly releasing Apt19S to recruit endogenous stem cells and guide the straight regeneration of central nervous system axons in the repair of spinal cord injury.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The present invention utilizes the spatial microtopological structure of the DON scaffold and the abundant protein carboxyl groups on its surface to load amino-modified Apt19S to prepare the sustained-release Apt19S bioactive scaffold DON-A, thereby achieving sustained release of Apt19S, which then specifically binds to ALPL on the surface of the stem cell membrane, recruiting a large number of endogenous NSCs and MSCs into the DON scaffold, and utilizing the central nervous system extracellular matrix microenvironment provided by DON to achieve in situ spinal cord nerve and blood vessel regeneration.

[0032] 2. The DON-A bioactive scaffold of the present invention effectively recruits endogenous NSCs and MSCs by continuously releasing Apt19S, binds to their ALPL receptors, and promotes in situ nerve and blood vessel regeneration in the injured spinal cord, which has important application value in the repair of central nervous system such as spinal cord injury.

[0033] 3. In the present invention, DON retains its natural extracellular matrix components belonging to the central nervous system, and has the characteristics of guiding the adhesion and growth of axons and promoting the homing and developmental differentiation of cells derived from central nervous system tissue; DON retains its natural channels and spatial microtopological structures that allow nerve bundles to grow straight, and has the characteristics of guiding the straight regeneration of central nervous system axons. After transplantation to the spinal cord injury site, DON continuously releases Apt19S, which has the characteristics of specifically recognizing and binding to Apt19S and the alkaline phosphatase (ALPL) receptor on the surface of the stem cell membrane, recruiting endogenous neural stem cells and mesenchymal stem cell subpopulations that highly express ALPL receptors, and realizing the specific and efficient recruitment of dual stem cells. After transplantation to the spinal cord injury site, the recruited endogenous neural stem cells that highly express ALPL receptors can proliferate in the central nervous system extracellular matrix microenvironment provided by DON and then differentiate into neurons to participate in the repair of spinal nerve pathways or differentiate into oligodendrocytes to form new myelin. After transplantation into the injured spinal cord, the recruited endogenous mesenchymal stem cells, which highly express the ALPL receptor, can first proliferate and then differentiate into vascular endothelial cells in the extracellular matrix microenvironment of the central nervous system provided by DON, participating in angiogenesis or exerting paracrine effects to regulate the immune microenvironment and promote neural regeneration. After transplantation into the injured spinal cord, myofibroblasts and macrophages that lowly express ALPL are not recruited to the injured area by Apt19S, and thus do not aggravate inflammation and scar formation after spinal cord injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the construction of DON-A bioactive scaffold;

[0035] Figure 2 The figure shows the specific binding and sustained release of Apt19S to NSCs and MSCs in DON-A;

[0036] Figure 3 The figure shows the recruitment of NSCs and MSCs and cell differentiation by DON-A in vitro;

[0037] Figure 4 Figure 2 is the expression of ALPL in macrophages and vascular smooth muscle cells;

[0038] Figure 5 A diagram showing the recruitment of NSCs and the differentiation of NSCs into neurons and oligodendrocytes after DON-A was transplanted into the injury site.

[0039] Figure 6 This is a diagram showing the regeneration of blood vessels and nerves after DON-A was transplanted into the injury site.

[0040] Figure 7This figure shows the nerve fiber regeneration, myelination and motor function recovery after DON-A was transplanted into the injury site. DETAILED DESCRIPTION

[0041] Below, in conjunction with specific embodiments, the present invention is further described. It should be noted that, under the premise of no conflict, the various embodiments described below or the various technical features can be arbitrarily combined to form new embodiments. The present invention is further described below by specific examples, but the present invention is not limited to the following examples. In the following examples, all raw materials are purchased except for some synthetic materials.

[0042] In the following examples, unless otherwise specified, all methods are conventional.

[0043] 1. DON-A scaffold preparation

[0044] The biological material used to construct the bioactive scaffold of the present invention is decellularized porcine optic nerve, and the Apt19S used is synthesized by Shanghai Bioengineering.

[0045] Fresh optic nerves were isolated from adult pigs. In a centrifuge tube, the obtained optic nerves were washed overnight at 4°C in distilled water containing 1% penicillin-streptomycin and mycotoxin / amphotericin solution (mycotoxin was 100 μg / mL, amphotericin was 2.5 μg / mL). Subsequently, 3% Triton X-100, 4% sodium deoxycholate, 50 U / ml DNAse, 50 U / ml RNAse, 10 mM MgCl2·6H2O, 50 mM Tris, etc. were used to remove the cellular content of the nerves. The optic nerves were then immersed in sterile distilled water in a 1.5 ml centrifuge tube and freeze-dried for 24 hours. The central nervous system-derived DON was made into a cylindrical scaffold (3 mm in diameter, 2 mm in height), as shown in FIG. Figure 1 As shown in AB, the transverse (A) and longitudinal (B) structures of the cylindrical DON scaffold can be observed under a scanning electron microscope. The scaffold was immersed in 10 mL of morpholineethanesulfonic acid (MES, 0.1 M, pH = 6) for 30 minutes at room temperature. Then 80 mg of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and 60 mg of N-hydroxysuccinimide (NHS) were added to the MES solution to activate the carboxyl groups on the DON surface. After 20 minutes, the activation buffer was removed, and 2 nmol of amino-modified Apt19S or amino-modified FITC-labeled Apt19S was added to 1 ml of buffer and reacted in a reciprocating shaker at room temperature for 12 hours. Finally, the DON-A scaffold was washed 3 times with buffer to obtain the Apt19S-carrying DON bioactive scaffold, which was used within 24 hours. As Figure 1As shown in C, Apt19S binds to the DON scaffold by forming an amide bond and can specifically bind to NSCs and MSCs through ALPL.

[0046] 2. Functional testing of DON-A bioactive scaffold in repairing spinal cord injury

[0047] (1) In vitro biological activity assay of DON-A

[0048] (a) Characteristic detection of sustained-release Apt19S from DON-A scaffold.

[0049] Figure 2 G shows that the longitudinal section of the DON-A scaffold loaded with FITC-Apt19S emits green fluorescence under a fluorescence microscope, while the ordinary DON scaffold does not show green fluorescence ( Figure 2 H). A DON scaffold loaded with amino-modified FITC-labeled Apt19S or amino-free FITC-labeled Apt19S (for control) was immersed 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 removed 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. Figure 2 Figure 1 shows the release curve of amino-modified Apt19S from the DON scaffold. During the entire 30-day observation process, the Apt19S-loaded DON scaffold could continuously and stably release Apt19S into the solution.

[0050] (b) Detection of the specific binding of DON-A scaffold to ALPL-positive NSCs and its differentiation in vitro.

[0051] 3-5 day old SD rats were selected and their brains were removed by decapitation under sterile conditions. The brains were placed in cold D-Hank's solution and the hippocampus was isolated using an instrument under a dissecting microscope. NSCs were cultured using mechanical pipetting: the hippocampal tissue was first minced with ophthalmic scissors and then transferred into a centrifuge tube along with the D-Hank's solution. The cells were gently pipetted several times with a fine-tipped glass pipette until no obvious tissue clumps were visible to the naked eye. Pipetting should be done slowly and with moderate force to avoid the formation of bubbles. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, and the procedure was repeated once. The cell pellet was resuspended in NSC culture medium and the cell density was adjusted to approximately 1×10 5 / ml, transfer the cell suspension into a culture flask and culture in a 37°C, 5% CO2 incubator for suspension culture. When a large number of cell clones are observed to form, use a fine-tipped glass pipette to mechanically separate the NSC clones and pass them on the next day. After two weeks of passaging, take the second generation NSCs and perform nestin immunofluorescence cytochemistry identification by floating method. Figure 2 As shown in A, NSCs showed the morphology of nestin-positive neurospheres. Figure 2 B shows that after the neurospheres adhered to the wall, the nestin-positive cytoskeleton was wrapped by the ALPL-positive cell membrane, and ALPL had a clear cell membrane expression pattern. These nestin-positive cells were co-incubated with Apt19s with a green fluorescent group. Figure 2 C shows that nestin-positive neural stem cells can specifically bind to Apt19S, and Apt19S with a green fluorescent group (FITC) can be seen in the cell body and processes.

[0052] (c) Detection of the specific binding of DON-A scaffold to ALPL-positive MSCs and its differentiation in vitro.

[0053] 7-day-old SD suckling mice were selected. Under sterile conditions, the entire femur was taken and placed in a sterile DMEM culture medium containing 10% fetal bovine serum (FBS). The bone marrow in the bone marrow cavity was blown into the cell culture dish using a 1 ml syringe. Then the cells were blown to a uniform cell suspension and placed in a cell culture incubator. After 48 hours, the medium was changed to remove the non-adherent cells. When the cells were about 80% full, the cells were digested with 0.25% trypsin containing 0.02% EDTA and passaged at a ratio of 1:3. MSCs between the 3rd and 6th generations were taken for CD73 and CD90 immunofluorescence cytochemical identification. Figure 2 D indicates that the cell membrane of these MSCs highly expresses ALPL. Figure 2 EF indicated that MSCs can express CD73 and CD90 simultaneously and can specifically bind to Apt19s with FITC.

[0054] (d) Verification of the specific recruitment and differentiation support of NSCs and MSCs by the DON-A scaffold.

[0055] Suspension adsorption experiments were used to further verify the recruitment of NSCs and MSCs by Apt19S. First, neurospheres of green fluorescent protein (GFP) transgenic SD rats were digested with 0.125% pepsin for 10 minutes to obtain single-cell suspensions of NSCs. 5×10 4GFP-NSCs were added to each well of a 24-well plate. GFP-MSCs were digested with 0.25% pepsin for 3 minutes to obtain a single-cell suspension of MSCs. Then, 5×10 4 GFP-NSCs or 5 × 10 in 1 ml of DMEM medium containing 10% FBS 4 GFP-MSCs were seeded in each well of a 24-well plate, and DON-A scaffolds or DON slices were placed in each well and incubated for 12 hours. The experimental groups included a blank scaffold (DON) group, a DON-A group, and a DON-A group supplemented with ALPL blocking antibodies (DON-A+anti-ALPL) group. Figure 3 Figures AB show the attachment of GFP-NSCs and GFP-MSCs to the scaffolds in each group. NSCs and MSCs suspended in culture medium were significantly more attracted to the DON-A scaffold, with the number of cells attached exceeding three times that of the blank DON scaffold. Adding an ALPL-blocking antibody to the cell culture medium in the DON-A group significantly reduced the number of cells attached to the scaffold to levels consistent with the blank DON scaffold. This suggests that ALPL-positive NSCs and MSCs are more attracted to the DON-A scaffold through the specific binding of ALPL to Apt19S. Figure 3 CD shows the differentiation of NSCs and MSCs on blank and DON-A scaffolds. NSCs have the potential to differentiate into Map2-positive neurons, Olig2-positive oligodendrocytes, and GFAP-positive astrocytes, with no difference in the differentiation ratio between the two scaffolds. Figure 3 EF shows the differentiation ratio of NSCs and MSCs. MSCs implanted on DON-A scaffolds and MSCs implanted on DON scaffolds have the potential to be induced to differentiate into VWF-positive and CD31-positive vascular endothelial cells. There is no statistical difference in the differentiation ratio between the two groups. The above results suggest that DON-A scaffolds do not affect the normal differentiation of NSCs and MSCs. In addition, macrophages ( Figure 4 AC) and vascular smooth muscle cells ( Figure 4 DF) ALPL expression is extremely low, suggesting that after DON-A transplantation, macrophages and myofibroblasts with low ALPL expression will not be recruited to the injury site by Apt19S, nor will they aggravate inflammation and scar formation after spinal cord injury, indicating that DON-A transplantation is less associated with the risk of inflammation and fibrous scar formation.

[0056] (2) In vivo experiments

[0057] Experimental procedures: Female adult SD rats (weighing 220-250 g) were provided by the Experimental Animal Center of Sun Yat-sen University. After undergoing a 2 mm complete transverse spinal cord injury at the T10 level, the rats were randomly divided into three groups (n = 24 per group): (1) SCI group (no stent was implanted at the injury site), (2) DON group (a cylindrical DON stent with a diameter of 3 mm and a height of 2 mm was implanted at the injury site), and (3) DON-A group (a cylindrical DON-A stent with a diameter of 3 mm and a height of 2 mm was implanted at the injury site). To trace cells with proliferation and differentiation capabilities during the acute phase of SCI, EDU was injected into the peritoneal cavity of each group of rats starting on the third day of injury modeling and continued for 10 consecutive days.

[0058] (a) Proliferation and differentiation of endogenous stem cells in the spinal cord 4 weeks after DON-A transplantation

[0059] Figure 5 AC indicates the proliferation and differentiation of endogenous stem cells in the head, center, and tail of the injury / transplantation area of ​​the longitudinally sectioned spinal cord in each group were observed 4 weeks after surgery. In the DON-A group, more Tuj-positive cells were observed at the head and tail ends of the injury / transplantation area and inside the scaffold, and a large number of EDU-positive and Olig2-positive cells were distributed. Figure 5 D shows the statistical analysis results of the relative fluorescence density of Tuj in each group. Figure 5 EF shows the statistical analysis results of the number of EDU and Olig2-positive cells in each group. Figure 5 G shows the ratio of Tuj and EDU double-positive cells and Olig2 and EDU double-positive cells within the EDU-positive cells in each group. The results showed that the DON-A group had significantly more EDU-positive cells at both ends of the injury / transplantation area and within the scaffold than the other two groups. The DON-A group had significantly more Tuj expression at the head and center of the injury / transplantation area than the other two groups. However, there was no significant difference in Tuj expression between the DON-A and DON groups at the tail. The DON-A group had significantly more Olig2-positive cells at both ends and the center of the injury / transplantation area than the other two groups. A higher proportion of EDU-positive cells in the DON-A group differentiated into Tuj. In the transplanted junction area, a large number of EDU and Tuj double-positive cells were seen growing from the spinal cord tissue into the interior of the scaffold. Inside the scaffold, a large number of EDU / Tuj double-positive neurites were seen growing straight along the channels of the scaffold. Only a very small number of EDU-positive cells differentiated into Olig2, and most of them were distributed in the spinal cord tissue at both ends of the head and tail, suggesting that the recruited endogenous NSCs had a higher proportion of neuronal differentiation in the microenvironment created by DON.

[0060] (b) Differentiation of endogenous NSCs and MSCs and regeneration of blood vessels and nerves in the spinal cord 4 weeks after DON-A transplantation

[0061] Figure 6 AC shows the differentiation of spinal cord endogenous stem cells represented by EDU into astrocytes and vascular endothelial cells 4 weeks after surgery. Figure 6 EF shows the statistical analysis results of the relative fluorescence density of GFAP and VWF in each group. The DON-A group showed significantly higher GFAP expression in the spinal cord tissue at the cranial and caudal junctions than the DON and SCI groups. Furthermore, the DON-A group had significantly more VWF-positive vascular endothelial cells at both the cranial and caudal ends and in the center of the injury / transplantation area than the other two groups. Figure 6 G shows the ratio of GFAP / EDU- and VWF / EDU-positive cells. The DON-A group had significantly more GFAP / EDU- and VWF / EDU-positive cells than the SCI group. Dense and branched blood vessels formed by VWF / EDU-double-positive endothelial cells were observed at the graft junction in the DON-A group. These vessels regenerated along the channels of the DON scaffold into the interior of the scaffold, suggesting that the endogenous stem cells recruited in the DON-A group possessed robust angiogenic capacity. Figure 6 D shows that at both ends of the transplanted area in the DON-A group, the processes of GFAP / EDU double-positive astrocytes tended to grow parallel to the longitudinal axis of the spinal cord and grew alongside GAP43-positive neuronal processes. The results suggest that astrocytes differentiated from ALPL-positive NSCs recruited by Apt19S may be more similar to immature astrocytes during neurodevelopment, which not only facilitates the guidance of neural regeneration but also provides better nutritional support for newborn neurons. In addition, ALPL-positive NSCs recruited by Apt19S have stronger migration ability than astrocytes, and the newborn neurons differentiated from them have good axonal growth potential.

[0062] (c) Recovery of motor function and spinal nerve conduction pathways in rats after DON-A bioactive scaffold transplantation

[0063] The open field test was used every week after surgery to evaluate the motor function recovery of the rats' paralyzed hind limbs. The Basso, Beattie, and Bresnahan (BBB) ​​scoring system was used to quantitatively assess the ability to move volitionally and support body weight. Figure 7 F shows the changes in BBB scores of each group from 0 to 8 weeks. The joint movement of rats in the DON-A group began to be significantly higher than that of the other two groups at 4 weeks. At the time point of 4-8 weeks, the behavior showed a continuous upward trend. At 8 weeks, the recovery of motor function was significantly higher than that of the other two groups. Figure 7AC shows the histological evidence of nerve regeneration and nerve conduction pathways in rats 8 weeks after treatment. The results show that the NF-positive nerve fibers in the DON-A group were able to regenerate more significantly to the injured / transplanted area and the spinal cord tissue at both ends of the head and tail. These NF-positive nerve fibers were detected to be surrounded by MBP-positive myelin sheaths at both ends and showed no signs of myelin sheath degeneration; more NF-positive nerve fibers were observed to regenerate straightly inside the scaffold in the DON-A group than in the other two groups. Figure 7 DE showed that the relative fluorescence density of NF and MBP expression in the DON-A group was significantly higher than that in the other two groups.

[0064] In summary, the DON-A scaffold effectively recruits endogenous NSCs and MSCs by continuously releasing Apt19S, binds to their ALPL receptors, and quickly initiates repair in the "incubation" microenvironment provided by DON. DON-A transplantation can guide the direct regeneration of central nervous system axons, promote the in situ regeneration of nerves and blood vessels in the injured spinal cord, and thus promote the repair of neural conduction pathways. The invention and application of the bioactive scaffold of the present invention that slowly releases Apt19S to recruit endogenous stem cells and guide the direct regeneration of central nervous system axons in spinal cord injury repair challenges the traditional view that endogenous stem cells are insufficient to support self-repair after spinal cord injury, and has important clinical application and transformation prospects and important application value in central nervous system injury repair.

[0065] 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 bioactive scaffold that slowly releases Apt19S to recruit endogenous stem cells and guide the regeneration of central nervous system axons, characterized in that: The method comprises the following preparation steps: 1) Preparation of DON scaffolds; 2) Activate the carboxyl group of the DON scaffold; 3) Preparation of DON-A bioactive scaffold: The carboxyl-activated DON scaffold and the amino-modified Apt19S were co-incubated to obtain the Apt19S-released DON-A bioactive scaffold; In step 1), the preparation of the DON scaffold includes the following steps: Fresh porcine optic nerves were decellularized by removing adipose tissue and then washed overnight at 4°C in distilled water containing penicillin-streptomycin and mycotoxin-amphotericin solution. Subsequently, Triton X-100, sodium deoxycholate, DNase, and RNase were used to remove cells from the optic nerves, preserving the extracellular matrix and its natural physical structure. The decellularized optic nerves were then immersed in sterile distilled water in a centrifuge tube and freeze-dried for 24 hours to obtain DON scaffolds. In step 2), the preparation step of activating the carboxyl group of the DON scaffold includes the following steps: The DON scaffold obtained in step 1) was placed in a 0.1 M morpholineethanesulfonic acid solution and incubated at room temperature for 30 minutes. Then, 80 mg of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and 60 mg of N-hydroxysuccinimide were added to the morpholineethanesulfonic acid solution and incubated for 20 minutes to activate the carboxyl groups on the DON surface.

2. The bioactive scaffold for slowly releasing Apt19S to recruit endogenous stem cells and guide the regeneration of central nervous system axons according to claim 1, characterized in that: The preparation steps of the DON scaffold include the following steps: a) extracting a fresh optic nerve from an adult pig, removing fat tissue and nerve sheath from the nerve to obtain the first optic nerve; b) The second optic nerve was obtained by placing the first optic nerve in distilled water containing 1% penicillin-streptomycin and the mycotoxin-amphotericin at 100 µg / mL and 2.5 µg / mL at 4°C for 24 hours; c) The second optic nerve was then immersed in 3% Triton-X-100 solution and shaken at 250 rpm for 12 hours to obtain the third optic nerve; d) Subsequently, the third optic nerve was immersed in a 4% sodium deoxycholate aqueous solution and shaken at 250 rpm for 24 hours to obtain the fourth optic nerve; e) The fourth optic nerve was then immersed in an aqueous solution of 50 U / ml DNase, 50 U / ml RNase, 10 mM MgCl2·6H2O, and 50 mM Tris, and shaken at 150 rpm at 37°C for 3 hours to decellularize the nerve and obtain the fifth optic nerve; f) Then, the fifth optic nerve was immersed in sterile double-distilled water, placed in a centrifuge tube and freeze-dried for 24 hours to obtain the DON scaffold.

3. The bioactive scaffold for slowly releasing Apt19S to recruit endogenous stem cells and guide the regeneration of central nervous system axons according to claim 1, characterized in that: The preparation step of activating the carboxyl group of the DON scaffold further includes the following steps before the DON scaffold is placed in morpholineethanesulfonic acid: The DON scaffold obtained in step 1) is made into a cylindrical DON scaffold with a diameter of 3 mm and a height of 2 mm.

4. The bioactive scaffold for slowly releasing Apt19S to recruit endogenous stem cells and guide the regeneration of central nervous system axons according to claim 1, characterized in that: In step 3), the preparation of the DON-A bioactive scaffold includes the following steps: The carboxyl-activated DON scaffold and the amino-modified Apt19S were incubated together, and a stable chemical bond was formed through an amidation reaction, thereby achieving stable loading and sustained release of Apt19S in the DON scaffold.

5. The bioactive scaffold for slowly releasing Apt19S to recruit endogenous stem cells and guide the regeneration of central nervous system axons according to claim 4, characterized in that: In step 3), the preparation of the DON-A bioactive scaffold includes the following steps: The carboxyl-activated DON scaffold was placed in an amino-modified Apt19S solution and incubated for 12 hours. Through the amidation reaction, a stable chemical bond was formed, achieving stable loading and sustained release of Apt19S in the DON scaffold, and obtaining the DON-A bioactive scaffold loaded with Apt19S.

6. An application, characterized in that: Use of the bioactive scaffold for slowly releasing Apt19S as described in any one of claims 1 to 5 to recruit endogenous stem cells and guide the direct regeneration of central nervous system axons in the preparation of a product for repairing spinal cord injury.

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