Preparation method and application of neurotized tissue engineering blood vessel

Abstract

The invention relates to a preparation method and application of a neural tissue engineering blood vessel. The preparation method comprises the following steps: preparing neural stem cell / neural precursor cell neural spheres; preparing a gelatin sponge into a hollow cylinder, adding neural spheres, and incubating to prepare a neural gelatin sponge; and penetrating the decellularized artificial blood vessel or other tissue engineering blood vessels into the neurogelatin sponge for incubation and culture to obtain the neurotized tissue engineering blood vessel. The vascular graft with a nerve secretion function is constructed by optimizing preparation and dynamic culture parameters of neural spheres and combining a cooperative processing technology of a gelatin sponge stent and an acellular vascular matrix, and is used for repairing coronary artery, carotid artery and peripheral vascular defects; the method has the advantages that rapid re-endothelialization, innervation and anti-inflammatory microenvironment formation of transplanted blood vessels can be promoted, repair and fusion of the blood vessels can be accelerated, a new neurotization scheme is provided for tissue engineering blood vessels, and a new method is provided for solving the difficult problems of reconstruction and long-term stability of transplanted blood vessels clinically.

Description

Technical Field

[0001] This invention relates to a neuroengineered tissue vessel, its preparation method and application, belonging to the field of tissue engineered vessel construction and regenerative medicine technology. Background Technology

[0002] In the fields of vascular repair, vascular transplantation, and cardiovascular disease treatment, tissue-engineered blood vessels (TEBVs) have attracted much attention due to their potential clinical application value. Especially in the replacement therapy of small-diameter vessels, such as those less than 6 mm in diameter, TEBVs show great research and application potential. Currently, research on TEBVs mainly focuses on structural design and surface functional modification, such as improving their functionality and biocompatibility through biomimetic strategies and grafting of active ingredients. Although biomimetic structural designs, such as biomimetic layered fiber arrangements, and surface functional modifications, such as heparin coating for anticoagulation, have improved short-term graft patency rates, clinical translation is still limited by postoperative complications such as thrombosis, anastomotic intimal hyperplasia, and vascular calcification. Studies have shown that these complications are closely related to the lack of neuroregulatory function in grafts—the nervous system dynamically regulates vasomotor activity and endothelial homeostasis by releasing neurotransmitters such as acetylcholine and norepinephrine, and existing TEBV technologies struggle to achieve synergistic neurovascular regeneration.

[0003] In recent years, research on artificial tissue-engineered vascular scaffolds has attempted to induce neural regeneration of the adventitia of tissue-engineered vessels by introducing nerve growth factors, but the following bottlenecks still exist: 1) Lagging neural regeneration: Traditional tissue-engineered vascular scaffolds lack an adventitia and cannot simulate the activity and paracrine functions of nerve cells, such as the secretion of VEGF and BDNF, resulting in neural regeneration lagging behind vascular reconstruction; 2) Scaffold-host interface mismatch: Commercially available synthetic scaffolds such as PLGA and PCL, due to uneven pore size and excessive mechanical strength, limit nerve cell infiltration and host tissue integration; 3) Single function of decellularized matrix: Existing decellularized vessels only serve as kinetic supports, and their residual ECM components, such as collagen IV and laminin, do not form functional coupling with the neurally active scaffold. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] To address the aforementioned problems in the prior art, this invention provides a method for preparing neurally engineered blood vessels. This method utilizes neural stem cells / neural progenitor cells (NSCs / NPCs) to assemble neurospheres, which are then attached to a biodegradable three-dimensional gelatin sponge scaffold. This allows NSCs / NPCs to be rapidly transferred onto the tissue-engineered blood vessels, maintaining their activity and paracrine function, promoting the directional extension of nerve axons along the adventitia of blood vessels, and achieving synchronous regeneration of nerves and blood vessels. The biodegradable gelatin sponge's degradation cycle matches the vascular regeneration and reconstruction process, promoting vascular repair and long-term patency after transplantation, thus overcoming the bottleneck in the clinical application of small-diameter vascular grafts.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0008] A method for preparing neuroengineered blood vessels, comprising the following steps:

[0009] S1. Neurospheres formed by inducing and culturing neural stem cells or neural progenitor cells (NSCs or NPCs) to differentiate and culture.

[0010] S2. Make the gelatin sponge into a hollow cylinder that can be attached to the surface of blood vessels; add the culture medium used for the cells in step S1 and incubate overnight to allow the gelatin sponge to completely absorb the culture medium and form a pre-wet gelatin scaffold.

[0011] S3. Add the neurospheres formed in step S1 to the pre-wet gelatin scaffold prepared in step S2 and incubate them to make a neural three-dimensional gelatin sponge scaffold with attached neural stem cells or neural progenitor cells (NSCs or NPCs).

[0012] S4. Decellularize the blood vessels to form artificial tissue-engineered blood vessels from decellularized tissue.

[0013] S5. Insert the artificial tissue-engineered blood vessel processed in step S4 into the hollow cavity of the neural three-dimensional gelatin sponge scaffold prepared in step S3, and incubate and culture it to achieve coupling of the nerve and blood vessel interface, thereby obtaining the neural tissue-engineered blood vessel.

[0014] In the preparation method described above, preferably, the neural stem cells are obtained by induction of embryonic stem cells (ESCs), amniotic fluid stem cells, adult neural stem cells, or induced pluripotent stem cells (iPSCs); the neural progenitor cells are obtained by direct transdifferentiation of fetal neural tissue, umbilical cord blood or umbilical cord tissue, or somatic cells.

[0015] The diameter of the neurosphere is 100–200 μm.

[0016] The preparation method described above, preferably, involves the formation of the neurospheres using any one of the following methods: suspension culture without substrate, centrifugation-assisted spheroidization, or hydrogel encapsulation.

[0017] The suspension substrate-free culture method: 5×10 5 ~1×10 6 Single-cell suspensions of NSCs or NPCs (cells / mL) were statically cultured in ultra-low adsorption culture plates.

[0018] The centrifugation-assisted pelleting method includes the use of ultra-low adhesion 96-well plates or Stemcell AggreWell. TM 800, aliquot the single-cell suspension of NSCs or NPCs into ultra-low adhesion 96-well plates, with 2000–10000 cells / well; or 4.5 × 10⁻⁶ cells / well. 5 ~1.5×10 7 Single-cell suspensions of NSCs / NPCs were placed in StemcellAggreWell. TM Centrifuge at 300–500 × g for 3–10 minutes in a microwell at 800°C to induce cell aggregation at the bottom of the well, and then incubate statically.

[0019] The hydrogel encapsulation method includes encapsulating NSCs or NPCs in a natural matrix or hydrogel, dropping the encapsulated material into a culture plate, gelling it, adding culture medium, and allowing it to stand for incubation; wherein the natural matrix is ​​Matrigel or collagen, and the hydrogel is PEG or sodium alginate.

[0020] Furthermore, the gelation temperature was 37°C, and the culture conditions were carried out at 37°C and 5% CO2.

[0021] Preferably, the NSCs or NPCs can secrete soluble factors that inhibit inflammatory immune responses or cell growth, and the cell growth factors they secrete include neurotrophic factors and vascular endothelial growth factor (VEGF).

[0022] Furthermore, the nutrient factors include at least two of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and glial cell-derived neurotrophic factor (GDNF).

[0023] In the preparation method described above, preferably, in step S2, the pore size of the gelatin sponge is 100-300 μm and the porosity is >90%; the matching error between the hollow diameter of the hollow cylinder and the outer diameter of the blood vessel is ≤5%.

[0024] In the preparation method described above, preferably, in step S2, the hollow cylinder is prepared by pouring a gelatin aqueous solution into a blood vessel mold, followed by vacuuming, freezing, and drying.

[0025] Furthermore, the mass concentration of the gelatin aqueous solution is 7-15%, and the drying temperature is -20℃ to -50℃ for 10-24 hours.

[0026] In the preparation method described above, preferably, in step S4, the decellularization treatment involves alternating perfusion treatment with an SDS aqueous solution and PBS at a mass concentration of 0.075–2% for 2–3 times; wherein each treatment with the SDS aqueous solution lasts for 2–4 hours; and the PBS perfusion wash is performed 2–4 times, 10 minutes per wash.

[0027] Then, soak in a mixed enzyme solution containing 0.5–2% DNase and 0.05–0.2% RNase for 2–4 hours, wash with PBS 2–4 times, and finally sterilize.

[0028] Furthermore, sterilization is preferably performed by gamma ray irradiation, with an irradiation dose of 11–25 kGy.

[0029] In the preparation method described above, preferably, in step S5, the incubation culture is carried out using Neurobasal medium containing BDNF (brain-derived neurotrophic factor) and GDNF (glial cell-derived neurotrophic factor) at a final concentration of 20 ng / mL and B27 at a volume ratio of 2%, and the medium is dynamically rotated for 7–14 days.

[0030] The application of the neuralized tissue-engineered blood vessels obtained by the preparation method described above in the preparation of vascular repair grafts.

[0031] (III) Beneficial Effects

[0032] The beneficial effects of this invention are:

[0033] The neuralized tissue-engineered blood vessel provided by this invention, through the synergistic design of neural stem cells / neural progenitor cells (NSC / NPC)-gelatin sponge scaffold co-assembly, solves the problem of delayed neural regeneration in the practical application of neuralized blood vessels and avoids poor tissue function recovery that may be caused by slow neural regeneration.

[0034] The method for preparing neuroengineered blood vessels provided by this invention, through the preparation of a decellularized vascular matrix, establishes a functional coupling interface between the decellularized vascular matrix and a neuroengineered gelatin sponge scaffold, overcoming the long-standing technical bottleneck of synchronizing the sequential regeneration of nerves and blood vessels. In tissue engineering and regenerative medicine applications, the synchronous regeneration of nerves and blood vessels is crucial for the complete restoration of tissue function. This invention provides synchronous sequential regeneration of nerves and blood vessels, enabling them to regenerate synchronously according to physiological needs during tissue repair. This avoids situations where one side regenerates too quickly or too slowly, leading to tissue dysfunction or incomplete defect repair. It brings a new solution to the repair of complex tissue defects in the fields of tissue engineering and regenerative medicine, expanding the indications for this technology in clinical applications and potentially bringing better treatment outcomes and prognoses to many patients.

[0035] The method for preparing neuroengineered blood vessels provided by this invention provides a gelatin sponge scaffold that can form a good interface integration with the host tissue, providing a stable matrix environment for the growth and repair of neural tissue. Its pore size and mechanical properties match the host interface, which can solve the problem of interface integration.

[0036] The gelatin sponge scaffold and decellularized vascular matrix provided by this invention possess excellent properties and are highly promising functional biomaterials for neural reconstruction. In the field of cardiovascular transplantation and repair devices, this material can provide conditions for rapid reendothelialization of small-diameter vessels while achieving good nerve innervation, thereby significantly reducing the risk of serious complications such as postoperative thrombosis and intimal hyperplasia, and improving the success rate of vascular transplantation surgery and the long-term survival rate of patients. Furthermore, in complex tissue defect repair scenarios such as peripheral nerve injury repair scaffolds, where synergistic regeneration of nerves and blood vessels is required, this material can also leverage its unique advantages, providing new ideas and methods for the treatment of related diseases. It is expected to be widely promoted in future clinical applications, meeting the clinical needs of repairing various complex tissue defects, and possesses enormous market potential and application value. Attached Figure Description

[0037] Figure 1 To construct nerve cell spheres using a substrate-free suspension culture method;

[0038] Figure 2 Stemcell AggreWell 800 aggregates into neurospheres;

[0039] Figure 3 Samples for the preparation of hollow gelatin sponge scaffolds;

[0040] Figure 4 The pore size and porosity of the gelatin sponge;

[0041] Figure 5For the construction of neuralized three-dimensional gelatin scaffolds;

[0042] Figure 6 The results show the phenotypic and ultrastructural identification of cells after differentiation into neurospheres;

[0043] Figure 7 For the construction of neurally engineered blood vessels and their transplantation into rat carotid arteries;

[0044] Figure 8 For the temporal assessment of vascular patency and nerve regeneration after neuroengineered tissue vascular transplantation;

[0045] Figure 9 This study aims to provide a temporal assessment of vascular patency and nerve regeneration after existing artificial blood vessel transplantation. Detailed Implementation

[0046] This invention prepares a neuralized artificial blood vessel scaffold by selecting neural stem cells / neural progenitor cells (NSCs / NPCs), biodegradable gelatin sponge, and decellularized matrix artificial blood vessels. Specifically, by optimizing the induction of NSCs / NPCs and the preparation of neural cell spheres, combined with the application of a three-dimensional gelatin sponge scaffold, a three-dimensional microenvironment adapted to the adhesion and paracrine function of neural stem cells (NSCs / NPCs) is constructed. Combined with dynamic rotation incubation technology of decellularized blood vessels, uniform distribution of neural cells at the host interface is achieved, ultimately promoting rapid neural innervation of the transplanted blood vessel and the formation of an anti-inflammatory microenvironment. In cases of vascular injury and transplantation needs, the pores of the three-dimensional scaffold can be used to encapsulate NSCs / NPCs, forming a three-dimensional neural stem / progenitor cell scaffold. The neural stem / progenitor cells can secrete various growth factors and cytokines, exerting a remote influence on the regeneration of artificial blood vessel tissue after transplantation; they also differentiate into glial cells and neurons, attaching to the outer side of the blood vessel, promoting angiogenesis, accelerating vascular repair and fusion, providing a new neuralization approach for tissue-engineered blood vessels, and offering a new method to solve the clinical challenges of in vivo remodeling and long-term stability of transplanted blood vessels.

[0047] The present invention provides a method for preparing neuroengineered blood vessels, comprising the following steps:

[0048] S1. Induced differentiation of neural stem cells / neural progenitor cells (NSCs / NPCs) and cultured to collect the formed neurospheres (100-200 μm);

[0049] S2. Under aseptic conditions, a hollow cylinder that can be attached to the surface of an artificial blood vessel is made using gelatin sponge. The culture medium used for the cells in step S1 is added and incubated overnight to allow the gelatin sponge to completely absorb the culture medium and form a pre-wet gelatin scaffold.

[0050] S3. Add the neurospheres formed in step S1 to the pre-wet gelatin scaffold prepared in step S2 and incubate overnight to prepare a neural three-dimensional gelatin sponge scaffold with attached neural stem cells / neural progenitor cells (NSCs / NPCs).

[0051] S4. Decellularize the blood vessels to form artificial tissue-engineered blood vessels from decellularized tissue.

[0052] S5. Combine the neural three-dimensional gelatin scaffold prepared in step S3 with the artificial tissue-engineered blood vessel processed in step S4, and perform dynamic rotation incubation culture for 7-14 days to achieve nerve-blood vessel interface coupling and obtain neural tissue-engineered blood vessels.

[0053] In practical applications, the neuralized artificial blood vessel incubated in step S5 is transplanted to the site of vascular injury or the site requiring vascular repair. The transplantation sites include, but are not limited to, the sites of coronary artery, carotid artery, or peripheral vascular injury.

[0054] Furthermore, in step S1, taking the induction of differentiation of iPSCs or mesenchymal stem cells (MSCs) into NPCs or NSCs as an example:

[0055] iPSCs were induced and cultured for 5–7 days using the SMADi inhibition method (medium containing 10 μM SB431542 + 0.1 μM LDN193189) to obtain neuroectodermal precursor cells, which were then cultured in a neural induction medium (medium containing 20 ng / mL LEGF and 20 ng / mL bFGF) for 10–14 days to obtain NPCs.

[0056] Embryonic stem cells (ESCs) were induced and cultured for 5–7 days using the SMADi inhibition method (medium containing 10 μM SB431542 and 0.1 μM DN193189) to obtain neuroectodermal precursor cells, which were then induced and cultured for 10–14 days using neural induction medium (medium containing 20 ng / mL EGF + 20 ng / mL bFGF) to obtain NSCs.

[0057] The induced NSCs / NPCs will be 2000 to 1×10 5 Cells were cultured in 96-well low-adhesion U-shaped plates or 4.5 × 10⁻⁶ cells per well. 6 Up to 1.5×10 7 Single-cell suspensions of NSCs / NPCs were placed in Stemcell AggreWell. TMCentrifuge at 300–500 × g for 5 minutes in a microwell to induce cell aggregation at the bottom of the well, then allow to stand and culture to form neurospheres. Collect the formed neurospheres (100–200 μm in diameter) and suspend them in Neurobasal medium containing 2% B27 for later use.

[0058] The NSCs or NPCs described in this invention preferably secrete soluble factors that inhibit inflammatory immune responses or cell growth, and the secreted cell growth factors include neurotrophic factors and vascular endothelial growth factor (VEGF). Further, the trophic factors include at least two of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and glial cell-derived neurotrophic factor (GDNF).

[0059] Decellularization treatment was performed using the carotid artery of a rat as an example: The carotid artery (1.5-2.5 mm in diameter) of an adult SD rat was perfused sequentially with 0.075% SDS and retained for 4 h, then perfused with PBS and retained for 10 min. This process was repeated three times with alternating SDS and PBS, followed by DNase / RNase (4 h) and then PBS (10 min) perfusion and flushing three times.

[0060] After rinsing with PBS, the cells were sterilized by gamma ray (11–25 kGy) to obtain decellularized vascular matrix (DNA residue <50 ng / mg).

[0061] To better explain and facilitate understanding of the present invention, a detailed description of the invention is provided below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, all reagents used in the embodiments are of analytical grade or higher and can be commercially available products.

[0062] Example 1

[0063] This embodiment examines the use of a suspension substrate-free culture method and StemcellAggreWell. TMThe method used to achieve the effect of 800 cells aggregating into neurospheres was as follows: Neural progenitor cells (NPCs) differentiated from human induced pluripotent stem cells (iPSCs, available from the Chinese Academy of Sciences Stem Cell Bank) were used as material. NPCs were digested into single-cell suspensions using Accutase (available from Stemcell). Specifically, iPSCs were induced using the SMADi inhibition method in a medium containing 10 μM MSB431542 and 0.1 μM LDN193189 for 5-7 days. Subsequently, they were cultured in neural induction medium (containing 20 ng / mL EGF and 20 ng / mL bFGF) for 10-14 days to obtain NPCs. The obtained NPCs were then digested into single-cell suspensions using Accutase and counted.

[0064] I. Suspension substrate-free culture method: Use NPC amplification medium (available from Stemcell), directly at 5×10⁻⁶. 5 ~1×10 6 NPC single-cell suspensions of cells / mL were seeded and cultured in ultra-low adsorption 6-well plates; after 1 day of culture on a horizontal shaker at 70 rpm at 37°C and 5% CO2, nerve cell spheroids formed; and the results were observed under an optical microscope. Figure 1 In the substrate-free suspension culture method, aggregated cell clusters with a diameter of 50–300 μm appeared after 24 hours. Figure 1 The display shape is regular and the edges are clear.

[0065] II. (Containing 5×10) 6 A suspension of NPC single cells was placed in StemcellAggreWell TM In microwells containing 800 cells per well, the cells were centrifuged at 300g for 5 minutes to induce cell aggregation at the bottom of the well. After incubation at 37°C and 5% CO2 for 1 day, nerve cell spheroids were formed. The results were observed under a light microscope. Figure 2 As shown. AggreWell TM Within 24 hours, 800 samples successfully formed uniform spherical structures of nerve cells, with a diameter of 100–200 μm, a single sphere rate of >90% within the micropores, and regular morphology with clear edges (see [link]). Figure 2 ).

[0066] Both of the above technical solutions can form neurospheres with a size of 100–200 μm. AggreWell TM The 800 method uses micropores to physically confine and force cells to aggregate uniformly, enabling the efficient preparation of neurospheres with controllable size, high activity, and stem stability. Traditional matrix-free suspension culture methods can be improved by adjusting cell density and using methods such as horizontal rotating shakers to reduce costs and adapt to large-scale applications.

[0067] Example 2

[0068] This embodiment takes the preparation of a hollow gelatin sponge scaffold suitable for rat carotid artery repair as an example, and the method is as follows:

[0069] 1) Weigh an appropriate amount of Type A gelatin, add deionized water, and prepare Type A gelatin (10% w / v, such as 10g gelatin dissolved in 100mL deionized water); place it on a 50℃ magnetic stirrer and stir continuously at 400-600rpm for 30 minutes to fully dissolve the gelatin.

[0070] 2) Pour the gelatin solution into the mold (mandrel diameter 1.8mm, outer mold diameter 2.5mm, length 10mm) using a syringe to form a hollow cylinder; place the mold containing the gelatin solution into a vacuum degassing machine with a vacuum degree of -0.1MPa and degas for 10 minutes.

[0071] 3) After degassing, immediately place the mold in liquid nitrogen and freeze it rapidly for 5 minutes.

[0072] 4) Quickly transfer the frozen mold to a -50℃ freeze dryer and dry for 24 hours to obtain a hollow cylindrical gelatin sponge support (e.g. Figure 3 ).

[0073] In specific applications, sterilization should be ensured under aseptic conditions, which can be achieved through irradiation sterilization.

[0074] After the scaffold was demolded, the cross-section was morphologically analyzed using an optical microscope and ImageJ software. The results are as follows: Figure 3 As shown, the hollow stent has an inner diameter of (1.8±0.2) mm and an outer diameter of (2.5±0.3) mm, which matches well with the size of the rat carotid artery. The gelatin sponge stent exhibits a uniformly interconnected porous structure inside, such as... Figure 4 The pore size distribution is approximately 100–300 μm, and through image analysis, the porosity calculated as (95.2 ± 1.5)% (total pore area / total image area × 100%) meets the high porosity requirement.

[0075] Example 3

[0076] The neurospheres and hollow gelatin sponge scaffolds prepared in Examples 1 and 2 were seeded to construct a three-dimensional gelatin scaffold with nerves. The specific steps are as follows:

[0077] 1) Add the hollow cylindrical gelatin sponge scaffold to NPC amplification medium (500μL~1mL) and incubate overnight to allow the gelatin sponge to completely absorb the medium and form a pre-wetted scaffold.

[0078] 2) Construction of the three-dimensional gelatin scaffold for nerves: The neurosphere suspension prepared in Example 1 was injected into the pre-wetted scaffold at an inoculation density of 1×10⁻⁶. 4 ~5×10 4 pcs / cm 3 The process involves supplementing 500 μL to 1 mL of Neurobasal medium containing a final concentration of 10 ng / mL EGF (recombinant human epidermal growth factor) and 10 ng / mL bFGF (recombinant human epidermal growth factor) and 2% B27 (v / v), and incubating at 37°C and 5% CO2 for 24 h to form a neurogelatin sponge scaffold.

[0079] The growth of neurospheres was observed under a fluorescence microscope and a bright field microscope, such as... Figure 5 Figure A shows a light micrograph of a neurosphere implanted in a gelatin sponge scaffold. The figure shows that the size of the neurosphere matches the pore size of the collagen sponge scaffold, and the neurosphere can grow in the collagen scaffold. Figure 5 B represents the growth of GFP (green fluorescent protein) transfected neural progenitor cell spheres in a collagen sponge scaffold. GFP transfected neural progenitor cell spheres are used to indicate the survival, differentiation, migration, and connection with the host of transplanted neural stem cells. Figure 5 The C-value shows that the neural progenitor cells of Example 1 were labeled using flow cytometry. The markers used were PAX6 and SOX1, which represent the ratio of iPSCs to neural progenitor cells / neural stem cells. It can be seen that about 74.3% of the cells showed PAX6 positivity.

[0080] Example 4

[0081] In this embodiment, NPCs neurospheres prepared according to Example 1 were cultured in a three-dimensional gelatin scaffold (i.e., the neurogelatin sponge scaffold in Example 3) and induced to differentiate. Differentiation induction involved changing the differentiation medium (containing final concentrations of BDNF (20 ng / mL), GDNF (20 ng / mL), and Neurobasal medium at a volume ratio of 2% B27) and maintaining culture for 7–14 days. To comprehensively evaluate the phenotype and ultrastructure of differentiated cells within the scaffold, after 14 days of culture, the cell-scaffold complex was fixed with paraformaldehyde / glutaraldehyde, and identified by immunofluorescence staining and by preparing ultrathin sections for transmission electron microscopy (TEM) examination.

[0082] Immunofluorescence staining results as follows Figure 6CLSM imaging clearly showed that, after induction, neural stem cells derived from NPCs successfully differentiated into neural lineages. Numerous NF200 (a marker of mature axons) positive (NF200+) cells were observed, with strong fluorescence signals in their cell bodies and extended synapses (axons and dendrites), clearly confirming the presence of mature neurons. Simultaneously, significant MBP-positive (MBP+) signals were detected, clearly indicating the successful differentiation and presence of glial cells with myelin-forming capabilities. Transmission electron microscopy, as shown... Figure 6 The B result, obtained through high-resolution TEM imaging, revealed the intricate ultrastructure between differentiated cells. In longitudinal sections of neuronal axons, the formation of myelin sheath structures was clearly observed. These myelin sheaths exhibited a highly organized multi-layered concentric lamellar structure. This structure was formed by the tight, regular spiral winding of the glial cell membrane around the axon. Between adjacent membrane layers, alternating electron-dense major-dense lines and electron-transparent intraperiod lines were visible—a typical ultrastructural feature of mature, physiologically functional myelin sheaths.

[0083] Example 5

[0084] This embodiment uses neural progenitor cell spheres (NPCs spheres) prepared in Example 1 and a neurogelatin sponge scaffold developed in Example 2. Through three-dimensional co-culture technology, the NPCs spheres are directionally induced to differentiate into a neuralized tissue layer. This layer is then functionally assembled with a decellularized vascular scaffold sterilized by gamma ray irradiation to construct a biomimetic neuralized tissue-engineered blood vessel. Figure 7 ).

[0085] The specific operation is as follows: 1) Take the neurosphere suspension from Example 1 (density 1~5×10) 4 pcs / cm 3 The porous gelatin sponge scaffold prepared in Example 2 was injected into the pre-wetted state, and 500 μL to 1 mL of Neurobasal medium containing 10 ng / mL EGF + 10 ng / mL bFGF in 2% B27 was added. The scaffold was incubated at 37°C and 5% CO2 for 24 h to form a neurogelatin sponge scaffold.

[0086] 2) Take carotid arteries (1.5-2.5 mm in diameter) from adult SD rats and perfuse them sequentially. Use 0.075% SDS solution and retain for 4 hours, then discard the SDS. Perfuse with PBS and retain for 10 minutes, then discard the PBS. Continue alternating between SDS and PBS for a total of 3 times. Soak in a mixed enzyme solution of DNase (1%) / RNase (0.1%) for 4 hours, then soak in PBS for 10 minutes, and rinse thoroughly 3 times using a horizontal circular shaker at 150 rpm. After rinsing with PBS, sterilize with gamma rays (11-25 kGy) to obtain decellularized vascular matrix.

[0087] After the above operations, routine HE and Masson staining were used to check for any cell residue. The preparation of the mixed enzyme solution of DNase (1%) / RNase (0.1%) was as follows:

[0088] 1) Preparation of RNase A stock solution (10 mg / mL): Dissolve 100 mg RNase A in 10 mL of sterile 10 mM Tris-HCl (pH 7.5); boil for 10 minutes, slowly cool to room temperature, aliquot and store at -20 °C.

[0089] 2) Preparation of DNase I stock solution (10 mg / mL): Dissolve 100 mg of DNase I in 10 mL of Ca2+ solution. + / Mg2 + Buffer solution (10 mM Tris-HCl and 1 mM CaCl2); filter sterilize (0.22 μm), aliquot and store at -20°C (avoid repeated freeze-thaw cycles).

[0090] 3) Mixed working solution (1% DNase / 0.1% RNase): Take 1 mL of DNase I stock solution (10 mg / mL) and add 0.1 mL of RNase A stock solution (10 mg / mL). Add 8.9 mL of buffer (PBS or Tris-HCl), mix well, and filter through a 0.22 μm filter. Final concentration: DNase I 1% (10 mg / mL), RNase A 0.1% (1 mg / mL).

[0091] 3) Insert the decellularized vascular matrix obtained above into the hollow cavity of the neurogelatin sponge scaffold obtained in step 1) (the gap between the gelatin sponge and the decellularized blood vessel is ≤0.1 mm); place it in a dynamic rotating culture system (rotation speed 10 rpm), and incubate it in Neurobasal medium containing BDNF with a final concentration of 20 ng / mL + GDNF with 20 ng / mL and 2% B27 for 7-14 days to achieve neuro-vascular interface coupling and obtain neuroengineered blood vessels.

[0092] The neuroengineered blood vessel was transplanted into the carotid artery defect area of ​​SD rats via microsurgery to verify its feasibility in promoting synchronous neurovascular regeneration.

[0093] The neurochemical culture results of the neurogelatin sponge scaffold showed that after 7 days of differentiation culture following inoculation with NPCs balls, the neurogelatin sponge scaffold exhibited a homogeneous cell infiltration state (e.g., Figure 7 (A). Macroscopically, the scaffold transformed from a dry, white, porous structure to a translucent gel, indicating enhanced extracellular matrix secretion and scaffold hydration. Nerve bundle-like structures formed by the migration of NPCs (non-cellular matrix) spheres were clearly observed at the edges, extending and penetrating the scaffold pores. Characterization of the pretreatment of decellularized vascular scaffolds: Vascular scaffolds decellularized with 0.5% SDS aqueous solution combined with 11 kGy γ-ray irradiation for sterilization (e.g., scaffold A). Figure 7 (B) Maintains the spiral collagen fiber bundle structure of the natural artery. Digital photographs show it to be a milky white, translucent white tubular shape with no surface fissures. Finally, this neuroengineered blood vessel was transplanted, and the results showed that the neuroengineered blood vessel (decellularized blood vessel wrapped with a neurogelatin sponge layer scaffold) successfully bridged an 8mm defect in the rat carotid artery. Figure 7 C).

[0094] Example 6

[0095] Postoperative dynamic monitoring was performed on the composite neurogenic vascular stent transplanted into the rat common carotid artery in Example 5. Vascular patency was assessed using high-frequency Doppler ultrasound contrast imaging, and the integration and regeneration process of transplanted nerve cells in the adventitia was analyzed using frozen section immunofluorescence staining. The focus was on verifying vascular function and nerve ingrowth efficiency at 14 days (acute phase) and 28 days (remodeling phase) post-transplantation, providing crucial evidence for the synchronous neurovascular regeneration mechanism.

[0096] Test results are as follows Figure 8 As shown. Doppler ultrasound quantitative hemodynamics ( Figure 8 A) The transplanted vessel segment maintained structural patency (no stenosis or thrombosis) at both 14 and 28 days post-surgery. Molecular evidence for directional nerve ingrowth is as follows: Figure 8 B, GFP + Nerve fibers (green fluorescence) showed sequential growth. 14 days post-surgery: GFP + The fibers are scattered in a dotted pattern, migrating along the collagen basement membrane towards the adventitia of blood vessels. 28 days post-surgery: GFP + The fibers form bundle-like nerve networks that deeply infiltrate the outer membrane collagen fibers. Transplanted NPCs can differentiate into myelinated nerve fibers and complete structural integration.

[0097] Comparative Example 1

[0098] The decellularized matrix vessels of the SD rat carotid artery, which were not neurogenic, obtained in Example 5 were implanted using steps 1) and 2) of Example 5. Step 3) involved inserting the decellularized vascular matrix into the hollow cavity of the neurogelatin sponge scaffold without subsequent culture. These were then directly transplanted into the rat common carotid artery as a control. Postoperative dynamic monitoring was performed, and vascular patency was assessed using high-frequency Doppler ultrasound contrast imaging. Frozen section immunofluorescence staining was used to analyze the integration and regeneration process of transplanted nerve cells in the adventitia. The focus was on verifying vascular function and nerve ingrowth efficiency at 14 days (acute phase) and 28 days (remodeling phase) post-transplantation.

[0099] Test results are as follows Figure 9 As shown. Doppler ultrasound quantitative hemodynamics ( Figure 9 In the A) transplanted segment, decreased patency (partial stenosis or thrombosis) was observed at both 14 and 28 days post-surgery. Molecular immunofluorescence identification of nerve ingrowth was as follows: Figure 9 B, PGP9.5 + Nerve fiber (a marker of a nerve fiber). 14 days post-surgery: PGP 9.5 + No significant positive signal was observed in the fibers. 28 days post-surgery: Scattered PGP 9.5 was visible. + Fiber impregnation.

[0100] The results show that the neuroengineered blood vessels prepared in this invention exhibit scattered migration 14 days post-procedure: GFP + Fiber migration along the collagen basement membrane towards the adventitia indicates that the transplanted NPC has survived and adapted to the damaged microenvironment, initiating axonal extension; guided by vascular-secreted chemokines (such as SDF-1) or basement membrane components (laminin), it exhibits directional migration. At 28 days post-surgery, fascicular networks and myelination indicate GFP. + The fibers form an orderly bundle structure, deeply infiltrating the outer membrane collagen, suggesting that NPCs differentiate into mature neurons / Schwan cells, promoting fiber aggregation into bundles through the secretion of neural adhesion molecules (such as L1CAM); and structurally integrating with host tissue. Myelination: confirmed that differentiated Schwan cells or oligodendrocytes (central model) complete functional repair, supporting electrical signal transmission. This indicates that nerve trunk / precursor cells can secrete various growth factors and cytokines, exerting a remote influence on the regeneration of artificial blood vessel tissue after transplantation; and can differentiate into glial cells and neurons, attaching to the outer side of blood vessels, promoting angiogenesis, and accelerating vascular repair and fusion.

[0101] The existing decellularized artificial vascular stent showed a negative result 14 days post-surgery, suggesting that early (acute) nerve fibers may exhibit degenerative changes due to surgical trauma: distal degradation after axonal breakage, and temporary disappearance of PGP9.5 expression. Inflammation suppression: the local inflammatory microenvironment (such as macrophage infiltration and pro-inflammatory factors) may inhibit nerve regeneration. Scattered positive results at 28 days post-surgery indicate that nerve fiber regeneration has begun, but a complete network has not yet formed. This may be in the following stages: axonal budding: newly formed nerve fibers extend from the injury edge towards the target area; early regeneration stage: the regeneration rate is slow (approximately 1-3 mm / day), and only some fibers may reach the target area at 28 days. In other words, nerve fibers show significant degeneration or functional silencing in the short term (14 days), and although there are signs of regeneration at 28 days, the regeneration efficiency is low.

[0102] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art can make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing neuroengineered blood vessels, characterized in that, It includes the following steps: S1. Neural spheres formed by inducing and culturing neural stem cells or neural progenitor cells to differentiate and culture. S2. Make the gelatin sponge into a hollow cylinder that can be attached to the surface of blood vessels; add the culture medium used for the cells in step S1 and incubate overnight to allow the gelatin sponge to completely absorb the culture medium and form a pre-wet gelatin scaffold. S3. Add the neural spheres formed in step S1 to the pre-wet gelatin scaffold prepared in step S2 and incubate them to form a neural three-dimensional gelatin sponge scaffold with attached neural stem cells or neural progenitor cells. S4. Decellularize the blood vessels to form artificial tissue-engineered blood vessels from decellularized tissue. S5. Insert the artificial tissue-engineered blood vessel processed in step S4 into the hollow cavity of the neural three-dimensional gelatin sponge scaffold prepared in step S3, and incubate and culture it to achieve coupling of the nerve and blood vessel interface, thereby obtaining the neural tissue-engineered blood vessel.

2. The preparation method according to claim 1, characterized in that, The neural stem cells are derived from embryonic stem cells, amniotic fluid stem cells, adult neural stem cells, or induced pluripotent stem cells; the neural progenitor cells are derived from fetal neural tissue, umbilical cord blood, or umbilical cord tissue, or directly transdifferentiated from somatic cells. The diameter of the neurosphere is 100–200 μm.

3. The preparation method according to claim 1, characterized in that, The neurospheres are formed using any one of the following methods: suspension culture without substrate, centrifugation-assisted sphere formation, or hydrogel encapsulation.

4. The preparation method according to claim 3, characterized in that, The suspension substrate-free culture method: 5×10 5 ~1×10 6 A single-cell suspension of neural stem cells or neural progenitor cells per cell / mL was statically cultured in an ultra-low adsorption culture plate. The centrifugation-assisted pelleting method includes the use of ultra-low adhesion 96-well plates or Stemcell AggreWell. TM 800, dispense single-cell suspensions of NSCs or NPCs into ultra-low adhesion 96-well plates, with 2000-10000 cells per well; Or 4.5×10 5 ~1.5×10 7 A single-cell suspension of neural stem cells or neural progenitor cells was placed in StemcellAggreWell TM Centrifuge at 300–500 × g for 3–10 minutes in a microwell at 800°C to induce cell aggregation at the bottom of the well, and then incubate statically. The hydrogel encapsulation method involves encapsulating neural stem cells or neural progenitor cells in a natural matrix or hydrogel, dropping the encapsulated material into a culture plate, gelling it, adding culture medium, and then allowing it to be cultured statically. The natural matrix is ​​Matrigel or collagen, and the hydrogel is PEG or sodium alginate.

5. The preparation method according to claim 1, characterized in that, In step S2, the pore size of the gelatin sponge is 100-300 μm, and the porosity is >90%; the matching error between the hollow diameter of the hollow cylinder and the outer diameter of the blood vessel is ≤5%.

6. The preparation method according to claim 1, characterized in that, In step S2, the hollow cylinder is prepared by pouring a gelatin aqueous solution into a blood vessel mold, followed by vacuuming, freezing, and drying.

7. The preparation method according to claim 1, characterized in that, In step S4, the decellularization process involves alternating perfusion treatment with 0.075–2% SDS aqueous solution and PBS 2–3 times; each SDS aqueous solution treatment lasts for 2–4 hours; and the PBS perfusion wash is performed 2–4 times, 10 minutes per wash. Then, soak in a mixed enzyme solution containing 0.5–2% DNase and 0.05–0.2% RNase for 2–4 hours, wash with PBS 2–4 times, and finally sterilize.

8. The preparation method according to claim 7, characterized in that, The sterilization is performed by gamma ray irradiation sterilization, with an irradiation dose of 11–25 kGy.

9. The preparation method according to claim 1, characterized in that, In step S5, the incubation culture is carried out using Neurobasal medium containing BDNF and GDNF at a final concentration of 20 ng / mL and B27 at a volume ratio of 2%, and is dynamically rotated for 7 to 14 days.

10. The use of the neuralized tissue-engineered blood vessels obtained by the preparation method according to any one of claims 1-9 in the preparation of vascular repair grafts.

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