CN116139336A - Multichannel biological scaffold and preparation method and application thereof - Google Patents

Abstract

The invention discloses a multichannel biological stent and a preparation method and application thereof. The multichannel biological scaffold comprises a plurality of core-shell structure fibers, wherein the core-shell structure fibers have a core-shell structure, and the core-shell structure fibers comprise core-structure fibers and shell structure fibers; the surface of the core structure fiber and the surface of the shell structure fiber are provided with topological structures, the topological structures comprise any one of a longitudinal groove microstructure, a porous microstructure and a smooth structure, and a communicated pore structure is arranged between the core structure fiber and the shell structure fiber in the core-shell structure fiber. The multichannel biological scaffold prepared by the invention provides a matrix environment similar to the spinal cord tissue environment, so that nerve cell directional migration and neuron axon directional regeneration extension are guided, and the loaded drug molecules and/or active factors are slowly released in the spinal cord injury area, thereby being beneficial to recovery of damaged nerve functions.

Description

Multichannel biological scaffold and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological materials, and particularly relates to a multichannel biological stent, and a preparation method and application thereof.

Background

The technical proposal of nerve tissue engineering is that the nerve scaffold material is ideal. Poor butt joint and gaps of two sections of ends after the spinal cord is transected easily produce glial cell 'scar', and the regeneration of spinal cord nerve fibers is blocked. Filling biological scaffold materials can prevent formation of glial scar, and studies show that: in an in vivo spinal cord injury model, a fiber scaffold with high arrangement can realize more robust directional axon regeneration than a fiber scaffold with random orientation, guide neuron axon extension, enable newly formed tissues to be close to original tissue structures, and facilitate establishment of functional connection, which is the basis of functional recovery. The pure cell suspension or the pure induction factors and growth factors cannot be guaranteed to reach the preset position so as to play a preset role, and the ideal bracket material can be used as a carrier of the cells and the factors so as to construct a good microenvironment for the cells and the factors. Curcumin as a traditional Chinese medicinal material has anti-inflammatory and antioxidant effects, and has neuroprotective effects, including inhibition of neuroinflammatory reactions; reducing formation of glial scar by down-regulating overexpression of glial fibrillary acidic protein; reducing local nerve tissue free radical release and lipid peroxidation, thereby protecting spinal cord tissue from oxidative stress; reducing neuronal apoptosis to improve spinal microenvironment. However, most of the current methods for treating spinal cord injury by using curcumin are daily intraperitoneal injection after constructing a spinal cord injury model, but few methods for treating spinal cord full-transection models and controlled release of specific damaged tissue parts of curcumin are available.

Disclosure of Invention

The invention mainly aims to provide a multichannel biological stent, and a preparation method and application thereof, so as to overcome the defects of the prior art.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention comprises the following steps:

the embodiment of the invention provides a multichannel biological bracket, which comprises a plurality of core-shell structure fibers, wherein the core-shell structure fibers are provided with a core-shell structure, and the core-shell structure fibers comprise core-structure fibers and shell structure fibers; the surface of the core structure fiber and the surface of the shell structure fiber are provided with topological structures, the topological structures comprise any one of a longitudinal groove microstructure, a porous microstructure and a smooth structure, a communicated pore structure is arranged between the core structure fiber and the shell structure fiber in the core-shell structure fiber, and the surfaces of the core structure fiber and the shell structure fiber are independently loaded with drug molecules and/or active factors;

wherein the groove width of the longitudinal groove microstructure on the surface of the nuclear structure fiber is 1-30 mu m, and the groove height is 50-100 mu m; the groove width of the longitudinal groove microstructure on the surface of the shell structure fiber is 1-50 mu m, and the groove height is 20-30 mu m.

The embodiment of the invention also provides a preparation method of the multichannel biological scaffold, which comprises the following steps: the preparation method comprises the steps of extruding natural polymer material-drug molecules and/or active factor gel by using a coaxial dispensing needle head with at least 2 channels by using a wet spinning preparation technology, and then carrying out crosslinking solidification, rotary collection and freeze drying treatment to obtain the multichannel biological scaffold. The embodiment of the invention also provides a functional product for repairing spinal cord injury, which comprises the multichannel biological scaffold.

Compared with the prior art, the invention has the beneficial effects that: the multichannel biological scaffold prepared by the invention provides well-defined spatial guidance and highly ordered microstructure morphology, and provides a matrix environment similar to the spinal cord tissue environment, so that directional migration of nerve cells is guided, directional regeneration and extension of nerve cell axons are promoted, and the loaded curcumin is slowly released in a spinal cord injury area, thereby being beneficial to differentiation of nerve stem cells to nerve cells, reducing generation of glial scars and being beneficial to recovery of damaged nerve functions to a certain extent.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings may be obtained according to the drawings without inventive effort to those skilled in the art.

FIG. 1 is a schematic diagram of the construction of a multichannel biological stent in an exemplary embodiment of the invention;

FIG. 2A is a diagram of a coaxial dispensing needle in an embodiment of the present invention, wherein the inner dispensing needle/outer dispensing needle specifications from left to right are 22G/14G, 26G/16G,25G/18G, respectively;

FIGS. 2B-2C are pictures of wet fibers collected on a resin support using a special spin collection process apparatus according to one embodiment of the present invention;

FIG. 3A is a photograph showing the collection of wet core structural fibers by a resin scaffold in accordance with one embodiment of the present invention;

FIG. 3B is a graph showing collection of wet core structural fibers loaded with curcumin through a resin scaffold in accordance with one embodiment of the present invention;

FIG. 3C is a photograph of dried core structural fibers collected by a resin scaffold in accordance with one embodiment of the present invention;

FIG. 3D is SEM pictures of different concentrations (4% and 6%) of core structural fibers, including longitudinal microstructures and transverse microstructures, in an embodiment of the invention;

FIG. 4A is a diagram of a multi-channel biological stent with core-shell structured fibers loaded with different fluorochromes, respectively, according to an embodiment of the present invention;

FIG. 4B is a scanning electron microscope image of the surface topography of a multichannel biological stent in an embodiment of the invention;

FIG. 4C is a laser confocal scanning fluorescence microscope image of a multichannel biological stent according to an embodiment of the invention, wherein core-shell structure fibers are respectively loaded with different fluorescent dyes;

FIGS. 4D-4F are SEM pictures of cross-sections of a multichannel biological stent in an embodiment of the invention;

FIG. 5A is a standard graph of curcumin according to an embodiment of the present invention;

FIG. 5B is a graph showing the cumulative release rate of curcumin loaded into a slow release medium by a core fiber and a shell fiber, respectively, according to an embodiment of the present invention;

FIG. 6A is an immunofluorescent-stained photograph of SD rat-derived neural stem cells cultured for 5d on a multichannel bioscaffold in accordance with an embodiment of the invention;

FIG. 6B is an immunofluorescent-stained photograph of a multi-channel bioscaffold of a nuclear structural fiber-loaded curcumin and having an ordered longitudinal groove microstructure on the surface transplanted to a full-transection spinal cord injury site 14d in a rat in accordance with an embodiment of the invention;

FIG. 6C is a photograph of 8d immunofluorescent staining of human neural precursor cells and astrocytes cultured on a multichannel biological stent according to one embodiment of the present invention;

FIG. 6D is an SEM image of human neural precursor cells and astrocytes cultured on a multichannel biological scaffold for 8D according to one embodiment of the present invention;

FIG. 7 is an immunofluorescent dye photograph of a multichannel bioscaffold of comparative example 1, in which the core structural fibers of the present invention were not loaded with curcumin and the surface was free of ordered longitudinal groove microstructures, transplanted to a rat full-transection spinal cord injury site 14 d.

Detailed Description

In view of the defects of the prior art, the inventor of the present invention has provided a technical scheme of the present invention through long-term research and a great deal of practice, and mainly utilizes a wet spinning preparation technology, a special rotation collection technology and a freeze drying method to obtain a multichannel biological scaffold with a longitudinal groove microstructure, wherein the multichannel biological scaffold comprises core fibers and shell fibers in core-shell structure fibers, and is respectively loaded with drugs and/or bioactive factors with different effects and multichannel biological scaffold-loaded cells. The invention respectively loads the drugs and/or factors with a controlled release period on the nuclear fiber and the shell fiber according to the requirements to achieve different slow release rates so as to achieve corresponding treatment effects, and more importantly, the multichannel biological scaffold not only can promote the linear growth of nerve cells and the elongation of cytoskeleton, but also has the order and connectivity, can provide a limited space for the growth of cells, is beneficial to the exchange of nutrient substances among cells and promotes the transplantation of cells of the scaffold, so that the multichannel biological scaffold for simultaneously constructing cells, drugs and/or factors which grow in alignment is a biomedical material with huge application potential.

The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Specifically, as one aspect of the technical scheme of the invention, the multichannel biological scaffold comprises a plurality of core-shell structure fibers, wherein the core-shell structure fibers are provided with a core-shell structure, and the core-shell structure fibers comprise core-structure fibers and shell structure fibers; the surfaces of the core structure fiber and the shell structure fiber are provided with topological structures, the topological structures are longitudinal groove microstructures (ordered longitudinal groove microstructures), a communicated pore structure is arranged between the core structure fiber and the shell structure fiber in the core-shell structure fiber, and the surfaces of the core structure fiber and the shell structure fiber are independently loaded with drug molecules and/or active factors;

wherein the groove width of the longitudinal groove microstructure on the surface of the nuclear structure fiber is 1-30 mu m, and the groove height is 50-100 mu m; the groove width of the longitudinal groove microstructure on the surface of the shell structure fiber is 1-50 mu m, and the groove height is 20-30 mu m.

In some preferred embodiments, the topology has a size of 1 to 100 μm.

In some preferred embodiments, the core structural fibers and the shell structural fibers in the multichannel biological scaffold have a communicated pore structure therebetween, and the pores have a cross-sectional diameter of 30-100 μm.

In some preferred embodiments, the individual fibers (core-shell structured fibers) in the multichannel biological scaffold have a cross-sectional diameter of 300 to 800 μm.

In some preferred embodiments, at least one layer of intermediate shell structure fibers is disposed between the core structure fibers and the shell structure fibers.

In some preferred embodiments, the sources of the core structural fiber, the shell structural fiber and the middle shell structural fiber are all natural polymer materials, and the natural polymer materials comprise one or more than two of collagen, silk fibroin, hyaluronic acid, chitosan, alginic acid, chondroitin sulfate, carboxymethyl starch, polylysine, polyglutamic acid, carboxymethyl glucose, elastin and heparin, and are not limited to the above.

Further, the natural polymer material comprises collagen.

In some preferred embodiments, the drug molecule comprises any one or a combination of two or more of curcumin, paclitaxel, baicalein, ganglioside, methylprednisolone, minocycline, scopolamine, and the like, and is not limited thereto.

Further, the drug molecule comprises curcumin.

In some preferred embodiments, the active factor includes any one or a combination of two or more of brain-derived neurotrophic factor (BDNF), neurotrophic factor III (NT-3), and neurotrophic factor IV/V (NT-4/5), and is not limited thereto.

In some preferred embodiments, the surface of the multichannel biological scaffold is loaded with cells.

In some preferred embodiments, the multi-channel biological scaffold is a collagen fiber multi-channel biological scaffold, the collagen fiber multi-channel biological scaffold has a core-shell structure, and the neural stem cells are promoted to differentiate into neurons by loading curcumin on the core structure, so that the multi-channel biological scaffold which is favorable for regeneration and functional repair of spinal cord injury nerves is constructed.

Further, the surfaces of the core structure fiber and the shell structure fiber in the collagen fiber multichannel biological scaffold are provided with ordered longitudinal groove microstructures, wherein the groove width of the surface of the shell structure fiber is 10-50 μm, the groove height is 20-30 μm, the groove width of the surface of the core structure fiber is 10-30 μm, and the groove height is 50-100 μm.

Further, the core structure fiber and the shell structure fiber in the core-shell structure fiber are provided with a communicated pore structure, and the diameter of the cross section of the pore is 30-100 mu m.

Further, the cross-sectional diameter of the individual fibers in the collagen fiber multichannel biological scaffold is 300-800 μm.

Further, the collagen fiber multichannel biological scaffold comprises one or more drugs and/or active factors which are uniformly distributed in the interior and the surface of the collagen fiber scaffold in a physical blending mode (physical adsorption).

The multichannel biological scaffold disclosed by the invention has the advantages that the secondary structure is formed by the self multichannel surface grooves, so that the specific surface area of the whole fiber and the scaffold is increased, and the interaction between cells and the biological scaffold is tighter. Specifically, the groove microstructure can indirectly promote cell proliferation by promoting cell adhesion; meanwhile, the increased specific surface area of the groove microstructure provides more available surface for cell proliferation; in addition, the grooves with specific dimensions have capillary action, so that the fluid conveying performance of the stent can be remarkably improved, and the delivery efficiency of nutrient substances and oxygen in the multichannel stent can be improved, thereby achieving the effect of promoting cell proliferation. Different division work functions, particularly supporting and wrapping functions of the shell structure, are achieved by adjusting process parameters (such as raw material concentration, fiber diameter and groove size) of the core-shell structure in the multiple channels, so that mechanical supporting functions are provided, channel penetration continuity is maintained, protection functions are provided for cells carried by the core structure, and loss caused by scouring of tissue fluid is avoided. The core structure is used for loading the drugs and factors which need to be slowly released, and the slow release effect is better than that of the shell structure; the continuous performance of the penetration of the channel is maintained, so that oxygen, nutrient substance delivery and metabolic waste removal can be more efficiently realized, and cells can migrate to the inside of the bracket rapidly.

Another aspect of the embodiments of the present invention further provides a method for preparing the multichannel biological scaffold, which includes: the preparation method comprises the steps of extruding natural polymer material-drug molecules and/or active factor gel by using a coaxial dispensing needle head with at least 2 channels by using a wet spinning preparation technology, and then carrying out crosslinking solidification, rotary collection and freeze drying treatment to obtain the multichannel biological scaffold.

Further, in extrusion molding, the pushing pump speed is 0.001-90 mL/h, the coaxial dispensing needle head is at least 2 channels, and the specification of the dispensing needle head is 34-14G.

Further, the push pump rate in extrusion molding is 0.1 to 90mL/h.

In some preferred embodiments, the schematic construction of the multichannel biological scaffold is shown in fig. 1, wherein drug a and drug B may be the same or different drug molecules or active factors.

In some preferred embodiments, the coaxial dispensing needles include any of a combination of at least one inner dispensing needle and one outer dispensing needle, a combination of two identical inner dispensing needles and one outer dispensing needle, a combination of two different inner dispensing needles and one outer dispensing needle, a combination of at least one inner dispensing needle and an outer dispensing needle that is capable of encapsulating all inner dispensing needles.

In some preferred embodiments, the means of crosslinking curing comprises chemical crosslinking and/or physical crosslinking.

In some preferred embodiments, the crosslinking cure is at a temperature of 10 to 30 ℃ for a time of 10 to 24 hours.

In some preferred embodiments, the cross-linking curing solution used for the cross-linking curing comprises 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide.

Further, the mass ratio of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to the N-hydroxysuccinimide is 2:1-5:1.

Further, the pH value of the crosslinking curing solution is 7.5-9.

In some preferred embodiments, the spin collection employs spin collection devices with spin rates of 0.1-10 mm/s, with spin directions Xiang Wei being forward and/or reverse.

In some more specific embodiments, the method of preparing a multichannel biological scaffold comprises:

providing two different collagen solutions, namely a first collagen solution and a second collagen solution; then adding curcumin into the first collagen solution and the second collagen solution respectively, and stirring and centrifuging to obtain first collagen-curcumin gel and second collagen-curcumin gel;

transferring the first collagen-curcumin gel and the second collagen-curcumin gel into 2 syringes respectively, mounting coaxial dispensing needles at the positions of the needles of the syringes, mounting inner dispensing needles corresponding to the syringes filled with the first collagen-curcumin gel, mounting outer dispensing needles corresponding to the syringes filled with the second collagen-curcumin gel, and then fixing the syringes on an extrusion push pump respectively;

mixing sodium bicarbonate solution with 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to form a cross-linked curing solution; and the syringe is placed below the coaxial dispensing needle of the syringe, and the height of the push pump is adjusted, so that the top end of the coaxial dispensing needle is just immersed into the crosslinking curing solution.

And turning on the push pump extrusion switch to start extruding the multichannel biological scaffold, enabling the core structure and the shell structure to come out of the needle head simultaneously, standing at room temperature for reaction, and then washing, rotary collection and vacuum freeze drying to obtain the multichannel biological scaffold.

Further, the concentration of the collagen solution is 20 mg/mL-70 mg/mL.

Further, the mass ratio of the curcumin to the collagen is 0.1% -5%.

Another aspect of embodiments of the present invention also provides a functional product for repairing spinal cord injury, comprising the multichannel biological scaffold described above.

The multi-channel biological scaffold with the patterned surface of the core-shell structure fiber is prepared by controlling the preparation process and the collection parameters, so that the longitudinal grooves with different intervals and depths can be prepared, the morphology and arrangement of nerve cells can be adjusted, the tubular space order can be maintained, and the directional migration capability and the nutrition diffusion permeability of cells can be promoted. In addition, the inner core structure is loaded with curcumin, the crosslinking density of the outer shell structure is improved, and the shell structure has stronger mechanical supporting force than the core structure, so that the core structure is prevented from being washed by tissue fluid, the slow release of the loaded medicine at the damaged part is ensured, and adverse effects of the damaged microenvironment on internal migration cells are avoided.

The (ordered) longitudinal groove microstructure of the multichannel biological scaffold can be applied to nerve cell perception and can regulate and control ordered migration of the nerve cells: the anisotropic mechanical stimulus given to the cells by the longitudinal groove structure is an intrinsic mechanism of mechanical transduction of the cells, namely, the anisotropic mechanical stimulus is perceived through an adhesion part, and the morphological characteristics of the cells are regulated from a gene level, and the cells extend along the groove direction to adapt to the biological scaffold structure or morphology. The multichannel biological scaffold with the special surface topological structure is designed, so that the purpose of repairing spinal cord injury is achieved. To obtain a special process, namely a longitudinal groove microstructure, firstly the centrifugation step (speed, time and temperature) in the process is critical to ensure that bubbles are effectively removed, and secondly, proper raw material parameters and proper extrusion molding speed are selected to ensure that the material has 'flow orientation' under the action of shear stress and 'stretching orientation' under the action of stretching, so that the material is realized.

The technical scheme of the present invention is further described in detail below with reference to several preferred embodiments and the accompanying drawings, and the embodiments are implemented on the premise of the technical scheme of the present invention, and detailed implementation manners and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

The experimental materials used in the examples described below, unless otherwise specified, were all commercially available from conventional biochemicals.

Example 1: construction of multichannel biological scaffold

1. Dissolution of raw materials

1g of collagen material is dissolved in 1% acetic acid solution to prepare two solutions with the solution concentration of 40mg/mL and 60mg/mL respectively, and the two solutions are kept stand for 4 hours at room temperature, curcumin with the mass of 0.5% of that of the collagen (the curcumin is dissolved by dimethyl sulfoxide) is respectively added into the two solutions, and the two solutions are stirred uniformly and centrifuged at 2000rpm for 10 minutes at room temperature to remove bubbles, so that the collagen-curcumin gel is obtained.

2. Extrusion molding

Transferring the gel into 2 10mL syringes respectively, mounting coaxial dispensing needles at the positions of the needles of the syringes (refer to figure 2A), mounting inner dispensing needles corresponding to the syringes filled with 40mg/mL (4%) of collagen solution-curcumin gel, mounting outer dispensing needles corresponding to the syringes filled with 60mg/mL (6%) of collagen solution-curcumin gel, and fixing the syringes on an extrusion push pump respectively. The specification of the push pump is 10cc, the volume is 10mL, and the push pump speeds corresponding to the inner and outer dispensing needles are 2mL/h and 15mL/h respectively. Fig. 2A shows the inner and outer dispensing needles in different size combinations, which can be adjusted to control the diameter and pore size of the core and shell structures according to the intended use of the stent, and further, the number of core-shell layers can be increased to increase the number of multi-layer channels as drug and cell carriers.

3. Crosslinking curing

(1) 1L of a 0.1M sodium bicarbonate solution was prepared. 1.5g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 500mg of N-hydroxysuccinimide were added in a mass ratio of=3:1, and the mixture was dissolved with stirring to prepare a solution.

(2) Pouring the prepared solution into an open container, placing the solution below the coaxial dispensing needle of the injector in the step 2, and adjusting the height of the push pump to enable the top end of the coaxial dispensing needle to be just immersed into the solution.

(3) And (2) turning on a push pump extrusion switch in the step (2) to start extrusion of the multichannel biological scaffold, so that the core structure and the shell structure come out of the needle at the same time.

(4) After completion of the reception, the reaction was allowed to stand at room temperature for 24 hours.

4. Washing treatment

After 24h, the multichannel bioscaffold (which may be forceps or a strainer) was gently collected, transferred to a new vessel, added with pure water, gently stirred for washing 5 times, 10min each.

5. Spin collection

The multichannel biological scaffold in step 4 was collected on a resin support frame (see fig. 3A and 3B) using an adjustable automatic rotation collection device (see fig. 2B-2C), set to a rotation rate of 1mm/s. Figures 3A and 3B show that the fibers have better mechanical properties and uniform fiber diameters when carrying the drug or not.

6. Vacuum freeze drying

(1) The medical low-temperature preservation box at-80 ℃ which is needed by freeze-drying is opened in advance, so that the temperature is reduced to the needed temperature.

(2) And (5) placing the materials in the step (5) into a medical low-temperature preservation box at the temperature of-80 ℃ for prefreezing for 1h.

(3) The vacuum freeze dryer was turned on and pre-frozen (pre-heating time typically 20min, depending on the specific instrument requirements) to a vacuum of 0.1mbar.

(4) After pre-freezing the bundles, taking the materials out of a medical low-temperature preservation box at the temperature of-80 ℃, putting the materials into a vacuum freeze dryer plate layer one by one, starting a starting switch, and sublimating and drying for 12 hours to obtain the multichannel biological stent (see figures 3C and 4A). Fig. 4A shows that the core-shell structure of the multichannel biological scaffold has unity, and the drug curcumin is continuously distributed in the core-structure fiber, ensuring uniform distribution of the drug.

Example 2: characterization of multichannel biological scaffolds

SEM observation of multichannel biological scaffold morphology and pore size

And (3) spraying metal on the multichannel biological scaffold in a dry state, observing under a scanning electron microscope, and photographing. The surface morphology (see fig. 3D and 4B) and cross section (see fig. 3D, 4e and 4F) were observed, and 3 fields of view were taken, respectively, and the stent diameter range and cross section pore size and range were counted. The result of FIG. 4B shows that the total diameter is 450-500 μm from the surface morphology of the bracket, and the surface of the bracket core-shell structure is provided with an ordered groove structure; from the cross-sectional views 4D and 4E, it can be seen that the core structure fiber diameter is 200 μm to 350 μm and the pore size between the core-shell structures is 100 μm to 200. Mu.m. The groove width of the longitudinal groove microstructure on the surface of the fiber with the nuclear structure is 20+/-5 mu m, and the groove height is 70+/-10 mu m; the groove width of the longitudinal groove microstructure of the shell structure fiber surface is 30+/-5 mu m, and the groove height is 25+/-2 mu m.

Note that: FIG. 3D is an SEM image of the core structure fiber of different concentrations (4%, 40mg/mL collagen solution-curcumin gel preparation core structure and 6%:60mg/mL collagen solution-curcumin gel preparation shell structure) in an embodiment of the invention, including a longitudinal microstructure and a transverse microstructure.

2. Fluorescent dye marked multichannel stent for verifying connectivity

The multichannel biological scaffold with the core structure and the shell structure marked by blue and green fluorescence respectively is prepared, crosslinked and solidified, washed, freeze-dried, re-soaked in PBS buffer solution, pore connectivity between the core structure and the shell structure is observed through a confocal laser scanning fluorescence microscope, and a photographic layer is scanned (see figure 4C). The results in fig. 4C show that voids between the core-shell structures remain in the wet state, with a pore size of 50-150 μm and connectivity.

3. In-vitro slow-release experiment for loading curcumin in core-shell structure

Precisely weighing 2.5mg of curcumin in a 25mL volumetric flask, and preparing a storage liquid by ethanol to a constant volume. Then, the stock solutions with different volumes are respectively sucked into a 5mL volumetric flask, ethanol is used for metering to a scale, the standard solutions with the concentration of 0.02 mug/mL, 0.1 mug/mL, 0.5 mug/mL, 1 mug/mL, 2 mug/mL, 4 mug/mL, 6 mug/mL, 8 mug/mL and 10 mug/mL are accurately prepared, the wavelength 423nm of an absorption peak is determined through spectral scanning, the absorbance of the standard solution is measured by using the wavelength, the linear regression is carried out on the curcumin concentration (C) by absorbance (A), a regression equation is obtained, and a standard curve is drawn (refer to figure 5A).

Placing the scaffold with core-shell structures respectively loaded with curcumin into a slow release medium: the cumulative drug release rate was calculated by sampling 2mL of PBS (pH 7.4) solution containing Tween 80 (0.5%) at 1, 2, 4, 8, 24, 32, 48, 72, 96 hours, and supplementing the same with Wen Dengliang slow release medium, measuring absorbance at 423nm of the sample with PBS solution containing 0.5% Tween 80 as a blank, and calculating the concentration according to the regression equation (see FIG. 5B). The results of fig. 5B show that the short term core structural fibers release significantly less curcumin for the load than the shell structural fibers.

Example 3: in vitro culture of neural stem cells by multichannel biological scaffold

1. Isolated culture of primary Neural Stem Cells (NSCs) and seeding onto scaffolds

Separating brain end of SD milk mice within 24h of birth, shearing, adding pancreatin substitute, digesting and blowing for several times in a 37 ℃ incubator for 3min, 5min and 5min respectively, adding equal amount of culture medium to stop digestion, filtering tissue caking with 400 mesh nylon membrane, centrifuging the filtrate: 350g,5min, discarding the supernatant, transferring the cells into a culture flask for balling culture, changing the liquid for the 4 th day, digesting the neurospheres with pancreatin substitutes for 15min for the 7 th day, and dissipating the neurospheres into single cells. The cell suspension was seeded onto the sterilized scaffolds and the fluid was changed every 2 d.

2. Immunofluorescence staining observation of influence of groove microstructure on bracket surface on NSCs adhesion distribution

The multichannel biological scaffold carrying NSCs is fixed for 30min at room temperature by 4% paraformaldehyde, and is soaked in PBS for 3 times; 0.5% TritonX-100 membrane permeation at room temperature for 5min, PBS 3 times; blocking for 1h at room temperature with 5% BSA solution; adding anti-Nestin, GFAP primary antibody, incubating overnight at 4 ℃, and washing 3 times with PBS; adding secondary antibodies of corresponding species and DAPI, incubating for 2h at room temperature, and washing 3 times with PBS; and observing and photographing through a confocal laser scanning fluorescence microscope. The results show that the multichannel biological scaffold is beneficial to adhesion of neural stem cells and obviously shows linear distribution along with the length extension of the fiber grooves of the shell structure (see fig. 6A and 6C), and demonstrate that the multichannel biological scaffold can give anisotropic mechanical stimulation to attached cells through a highly ordered surface groove structure, so that the cell perceives the anisotropic mechanical stimulation to adjust the morphological characteristics of the cell, and the cell perceives the anisotropic mechanical stimulation to extend along the groove direction to adapt to the morphology of a matrix. Green fluorescence represents the marker Nestin of neural stem cells, red fluorescence represents the marker GFAP of astrocytes, and blue represents DAPI-stained nuclei.

3. Multichannel biological stent morphology for supporting NSCs

The multichannel bioscaffold bearing NSCs was fixed with 4% paraformaldehyde for 30min at room temperature, washed 3 times with pbs, dehydrated with gradient ethanol (25%, 50%, 75%, 85%, 95%, 100%) for 10min each, and then freeze-dried and SEM-characterized after gold spraying (see fig. 6D). Fig. 6D shows that NSCs mainly adhere to the rough fiber grooves of the shell structure and exhibit a directional stretching state consistent with the extending direction of the grooves, which is consistent with the immunofluorescent staining result (see fig. 6A and 6C), and shows that the ordered groove structure (see fig. 3D and 4B) distributed on the surface of the prepared biological multichannel scaffold is favorable for directional migration of nerve cells.

Example 4: repair of rat spinal cord full-transection injury by multi-channel biological scaffold

1. Construction of rat spinal cord full-transection injury model

SPF-class female SD rats, weighing 200 g-220 g, were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg), were subjected to dehairing in the prone position, were subjected to lumbar dorsal dehairing, were sterilized, were cut at the T8-T10 position, were cut along the spinous processes in bilateral muscles, were pried open with a needle holder and were subjected to removal of the lamina at the T9 position, and were exposed to spinal cord. The micro needle holder clamps the spinal cord, a knife is cut at the front end and the rear end immediately by micro scissors, a section of spinal cord tissue with the length of about 4mm is removed, and the gelatin sponge is immediately compacted to stop bleeding. After the blood is stopped, the stent material is implanted into the damaged part.

2. Immunofluorescent staining to detect expression of neurons and astrocytes

After 14d of surgery, anesthetized rats were heart perfused with 4% paraformaldehyde, spinal cords containing the injured sites were removed, 4% paraformaldehyde was fixed overnight at 4 ℃, then 20% and 30% sucrose solutions were replaced respectively overnight at 4 ℃ for dehydration, finally, OCT embedding was performed, and frozen sections were performed. Immersing the slices in PBS for 3 times, each time for 5min; after PBS is dried, the region of tissue to be dyed is surrounded by an immunohistochemical pen; blocking with ready-to-use goat serum at room temperature for 1h, and removing serum; adding anti-Tuj-1, GFAP primary antibody, incubating overnight at 4 ℃, and washing 3 times with PBS; adding secondary antibodies of corresponding species, incubating for 2 hours at room temperature in a dark place, and washing with PBS for 3 times; air-dried, and sealed with DAPI-containing tablet for 15min, and photographed by confocal laser scanning fluorescence microscope (see FIG. 6B). Fig. 6B shows that the multi-channel biological scaffold with curcumin loaded on the core structure is beneficial for the differentiation of neural stem cells to neurons, which does not appear on the shell structure, but rather shows astrocytes distributed on the shell structure fiber, and the whole is observed, the core-shell structure shows a larger number of cells recruited, and the core structure fiber shows a circle of cells conforming to the appearance outline of the core-shell structure, which indicates that the multi-channel biological scaffold has connectivity between the core-shell structures and allows endogenous neurons to migrate to the pores of the core-shell structure, red fluorescence represents a marker Tuj-1 of neurons, green fluorescence represents a marker GFAP of astrocytes, and blue color represents DAPI-stained nuclei.

Comparative example 1

The preparation method of the multichannel biological scaffold is the same as that of the embodiment 1, and is different in that the surfaces of the core structure fiber and the shell structure fiber of the prepared multichannel biological scaffold are not provided with longitudinal groove microstructures;

the multichannel biological scaffold of the comparative example is used for repairing rat spinal cord full-transversal injury, and is characterized by photographing through laser confocal scanning fluorescence microscopy, wherein fig. 7 shows that a simple nuclear structure, namely, a multichannel biological scaffold without curcumin is not observed to differentiate into neurons, a nuclear shell structure is observed to be less in number, the whole nuclear shell structure is observed to be more recruited, the missing longitudinal groove microstructure also causes disorder of a circle of cells of the appearance outline of the nuclear shell structure fiber, the condition that the longitudinal groove microstructure on the surface of the nuclear shell structure in the multichannel biological scaffold is a necessary condition for promoting ordered orientation migration of the neurons is shown, red fluorescence represents a marker Tuj-1 of the neurons, green fluorescence represents a marker GFAP of astrocytes, and blue fluorescence represents a cell nucleus dyed by DAPI.

In addition, the inventors have conducted experiments with other materials, process operations, and process conditions as described in this specification with reference to the foregoing examples, and have all obtained desirable results.

It should be understood that the technical solution of the present invention is not limited to the above specific embodiments, and all technical modifications made according to the technical solution of the present invention without departing from the spirit of the present invention and the scope of the claims are within the scope of the present invention.

Claims (10)

1. The multichannel biological bracket is characterized by comprising a plurality of core-shell structure fibers, wherein the core-shell structure fibers have a core-shell structure, and the core-shell structure fibers comprise core-structure fibers and shell structure fibers; the surfaces of the core structure fiber and the shell structure fiber are provided with topological structures, the topological structures are longitudinal groove microstructures, a communicated pore structure is arranged between the core structure fiber and the shell structure fiber in the core-shell structure fiber, and the surfaces of the core structure fiber and the shell structure fiber are independently loaded with drug molecules and/or active factors;

wherein the groove width of the longitudinal groove microstructure on the surface of the nuclear structure fiber is 1-30 mu m, and the groove height is 50-100 mu m; the groove width of the longitudinal groove microstructure on the surface of the shell structure fiber is 1-50 mu m, and the groove height is 20-30 mu m.

2. The multi-channel biological stent of claim 1, wherein: the size of the topological structure is 1-100 mu m;

and/or the cross-sectional diameter of the pores between the core structural fibers and the shell structural fibers in the multichannel biological scaffold is 30-100 μm;

and/or the cross-sectional diameter of the core-shell structure fiber in the multichannel biological scaffold is 300-800 mu m.

3. The multi-channel biological stent of claim 1, wherein: at least one layer of middle shell structure fiber is arranged between the core structure fiber and the shell structure fiber.

4. A multi-channel biological stent according to claim 3, wherein: the sources of the core structure fiber, the shell structure fiber and the middle shell structure fiber are all natural polymer materials, and the natural polymer materials comprise one or more than two of collagen, silk fibroin, hyaluronic acid, chitosan, alginic acid, chondroitin sulfate, carboxymethyl starch, polylysine, polyglutamic acid, carboxymethyl glucose, elastin and heparin; preferably, the natural polymer material includes collagen.

5. The multi-channel biological stent of claim 1, wherein: the medicine molecules comprise any one or more than two of curcumin, taxol, baicalein, ganglioside, methylprednisolone, minocycline and scopolamine; preferably, the drug molecule comprises curcumin;

and/or the active factors comprise any one or more than two of brain-derived neurotrophic factors, neurotrophic factors III and neurotrophic factors IV/V;

and/or, the surface of the multichannel biological scaffold is loaded with cells.

6. A method of preparing a multi-channel biological stent according to any one of claims 1 to 5, comprising: the preparation method comprises the steps of extruding natural polymer material-drug molecules and/or active factor gel by using a coaxial dispensing needle head with at least 2 channels by using a wet spinning preparation technology, and then carrying out crosslinking solidification, rotary collection and freeze drying treatment to obtain the multichannel biological scaffold.

7. The method of manufacturing according to claim 6, wherein: the coaxial dispensing needle comprises any one of at least one combination of an inner dispensing needle and an outer dispensing needle, two identical combinations of the inner dispensing needle and the outer dispensing needle, two different combinations of the inner dispensing needle and the outer dispensing needle, at least one combination of the inner dispensing needle and the outer dispensing needle which can wrap all the inner dispensing needles;

and/or the push pump speed is 0.001-90 mL/h in extrusion molding; preferably 0.1 to 90mL/h.

8. The method of manufacturing according to claim 6, wherein: the cross-linking and curing mode comprises chemical cross-linking and/or physical cross-linking; and/or the temperature of the crosslinking and curing is 10-30 ℃ and the time is 10-24 hours;

and/or the cross-linking curing solution adopted by the cross-linking curing comprises any one of mixed solution of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, 1-2.5 wt% glutaraldehyde solution and 1-10 mmol/L genipin; preferably, the mass ratio of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to the N-hydroxysuccinimide in the mixed solution of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and the N-hydroxysuccinimide is 2:1-5:1; preferably, the pH of the crosslinking curing solution is 7.5 to 9.

9. The method of manufacturing according to claim 6, wherein: the rotation rate of the rotation collecting device adopted by the rotation collecting is 0.1-10 mm/s, and the rotation direction Xiang Wei is forward and/or reverse.

10. A functional product for repairing spinal cord injury, characterized by comprising the multichannel biological scaffold according to any one of claims 1-5.

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