CN108324991B

CN108324991B - Slow-release GDNF-SCs composite acellular nerve scaffold

Info

Publication number: CN108324991B
Application number: CN201810469180.6A
Authority: CN
Inventors: 王辉; 宁昕杰; 李张昱; 巴越洋; 陆新华; 罗骏成; 梁家骥; 高群
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-05-16
Filing date: 2018-05-16
Publication date: 2020-10-30
Anticipated expiration: 2038-05-16
Also published as: CN108324991A

Abstract

The invention relates to the field of biomedicine, in particular to a slow-release GDNF-SCs composite acellular nerve scaffold, which comprises gel microspheres loaded with GDNF-SCs and an acellular nerve scaffold; the gel microspheres loaded with GDNF-SCs are filled in the pores of the acellular nerve scaffold; the GDNF-SCs are constructed by Schwann cells over-expressing GDNF.

Description

Slow-release GDNF-SCs composite acellular nerve scaffold

Technical Field

The invention relates to the field of biomedicine, in particular to a slow-release GDNF-SCs composite acellular nerve scaffold.

Background

The incidence of facial nerve injury caused by various reasons is high, and facial paralysis, eyelid insufficiency, facial paralysis and atrophy are caused after the injury, and finally blindness and facial deformity are caused. Seriously affecting the beauty and functions of the face and the daily life and social activities of the patient, reducing the living quality, causing the serious self-inferior psychology of the patient and even suicide tendency, and bringing great pressure to families and society. With the remarkable development of microneurosurgery and tissue engineering in recent years, scholars at home and abroad adopt methods such as autologous nerve transplantation, synthetic material stent transplantation and the like, and the function repair of injured facial nerves is improved to a certain extent but still is not ideal. Therefore, there is an urgent need to find a treatment method for effectively repairing damaged facial nerves, which improves the repairing effect thereof.

Repair of peripheral nerves is dominated by direct anastomosis: first stage end-end anastomosis, fibular nerve insertion transplantation, second stage facial-auxiliary or facial-sublingual nerve anastomosis. The repair technology mostly adopts the repair principle of peripheral nerves: the method comprises the steps of end-to-end anastomosis, adhesive bonding technology, laser welding technology and the like. Despite the improvement in repair efficiency achieved by sophisticated microsurgical techniques, it is still unsatisfactory. The effect of repairing facial nerve injury by directly end-to-end anastomosis method simply by improving surgical operation skill is limited, and various nerve substitutes are urgently needed to be developed to bridge nerve defect and improve the repairing effect of nerve injury.

At present, the peripheral nerve scaffold material approved by the food and drug administration in the United states or the CE in Europe to be marketed is mostly a nerve conduit prepared from degradable high polymer materials. Most of the devices are only of a hollow structure, and mainly improve a physical channel for nerve regeneration to form a relatively isolated closed environment so as to avoid the influence of peripheral tissues on nerve regeneration; however, due to the lack of biomimetic three-dimensional structure, the biological properties are far from those of normal nerves, and the function of inducing axon regeneration is limited.

The nerve repair material in the prior art has a plurality of problems:

1. the nerve repair material has a single morphological structure and is mostly a tubular support stent or a three-dimensional stent. Although the dynamic microenvironment stent can directionally guide the regeneration direction of nerve cells under certain conditions, the dynamic microenvironment stent with continuous action is lacked. It has been reported that extracellular matrix is used to structurally compound an artificial multi-channel, multi-channel scaffold, so that the longitudinal channels of the scaffold contain transverse pore channel connections and are loaded with different substrates. Can further promote the planted seed cells to form a neural network with an information transmission function, and can be applied to parts such as spinal cord injury and the like to connect the upper and lower nerve fibers of the injury part to play a role. However, the texture of peripheral nerves is not as soft as that of spinal cord, the physical form and the directional growth direction of the peripheral nerves are fixed, and a microenvironment nutrition support with continuous action is needed during injury repair, so that a simple three-dimensional or optimized suture scaffold cannot meet the condition of nerve cell self-growth.

2. The spatial volume ratio of neurotrophic factors embedded in the nerve repair material is not sufficient. It has been reported that the three-dimensional structure of the material of the nerve scaffold is mostly used for naturally pasting the neurotrophic factors so as to achieve the effect of covering a larger area. However, the nerve repair process is a slow "creeping" process from the near end to the far end, the neurotrophic factors which do not conform to the physiological dose are easily exposed to tissue immune cells at the initial stage of transplantation to reject and remove, and the uniform coating can not meet the dynamic requirements of nerve repair to the maximum extent.

3. The nerve repair material has insufficient repair function: due to the defects of insufficient source of the autologous nerve graft, secondary operation and the like, the extracellular matrix scaffold material capable of replacing the autologous nerve is urgently required to be developed. It is presently believed that an ideal nerve graft should have the following characteristics: no immunological antigenicity, good biocompatibility and cell compatibility; ② the physical characteristics (elasticity, strength, toughness and the like) are close to the receptor tissue, and the degraded product has no toxicity. The regenerated nerve cell axon can grow into and pass through the transplant to reach a far-end part; the transplant has a three-dimensional pore-like structure, can meet the dynamic migration and enrichment pathway of glial cells, and provides a microenvironment for good cell growth; the transplant can provide important neurotrophic factors necessary for the growth of axon. At present, most of the peripheral nerve injury repair scaffolds can only provide environmental support for nerve fiber growth and quantitative neurotrophic substances at the same time. The requirement of dynamic and ordered growth of nerve fibers cannot be met, so that the end-to-end nerve fibers cannot be butted efficiently and accurately, and the treatment effect is not ideal.

The Shenqiao approved for marketing in 2012 is a decellularized allogeneic nerve repairing material obtained after natural nerve decellularization treatment, and a nerve conduit made of a higher molecular material is greatly improved, mainly consists of an extracellular matrix, and retains a neural scaffold structure. After bridging the nerve broken end, the three-dimensional structure and the extracellular matrix of the nerve broken end can provide good physical and biological environment for the growth of the regenerated nerve, guide and support the growth of the regenerated nerve fiber from the near broken end to the far broken end, and restore the innervation of the target organ. However, the scaffold itself does not contain glial cells and has a good cell proliferation environment, and a complete tissue repair microenvironment cannot be provided yet.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The existing tissue engineering nerve scaffold material structure is mainly a pure extracellular matrix scaffold, and the three-dimensional structure of the material can be combined with active seed cells to form a complex with biological activity. The scaffold transplanted composite SCs bridge nerve defects to promote nerve repair to a certain extent, but the survival rate of implanted cells is low, and the repair effect is not ideal. The invention provides a barium chloride cross-linked alginate gel slow-release microsphere transport system composite GDNF-SCs acellular nerve repair scaffold, which is embedded with a medium capable of guiding seed cells to slowly release on the basis of a three-dimensional scaffold to provide a microenvironment for the action of neurotrophic factors. The repairing material improves the survival rate of implanted seed cells, sustains and maintains the concentration of the neurotrophic factors with locally stable damage, and can dynamically adjust the content of the neurotrophic factors released by the seed cells according to the pathophysiological mechanism of damaged tissues, so that the repairing effect of damaged facial nerves is greatly improved.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the invention relates to a slow-release GDNF-SCs composite acellular nerve scaffold, which comprises gel microspheres loaded with GDNF-SCs and an acellular nerve scaffold;

the gel microspheres loaded with GDNF-SCs are filled in the pores of the acellular nerve scaffold;

the GDNF-SCs are constructed by Schwann cells over-expressing GDNF.

The scaffold complex achieves organic combination. The composite GDNF sustained-release microspheres are fused with the acellular nerve scaffold to construct a novel nerve transplantation complex. The GDNF-loaded sustained-release microspheres can grow by means of the three-dimensional structure of the nerve scaffold, thereby not only reducing the change of the structure and the function caused by stacking and extruding among the microspheres, but also improving the proliferation activity of GDNF-SCs permeating the microspheres; the outer membrane structure of the bracket limits GDNF to the damaged area, thereby realizing the controlled release of GDNF protein, ensuring the GDNF protein to slowly and stably permeate out of microspheres for a long time and maintaining the stable protein concentration of the damaged part; limiting the migration of the GDNF seeping out of the microspheres to the surrounding undamaged tissues and avoiding the occurrence of adverse reactions. The composite system organically combines the acellular nerve scaffold material and the slow release microsphere, and the acellular nerve scaffold material and the slow release microsphere are complementary to each other and play a role together, so that the composite system is an ideal nerve transplantation composite system for repairing damaged facial nerve materials. Solves the problems that the prior gene therapy can not control the GDNF secretion amount, the implanted seed cells can not be limited to the damaged area to play the role in a targeted way, and the cells migrate to the non-damaged area to play the unpredictable role.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention has an internal structure of gel sustained-release microspheres, and the transportation system is a transportation system which wraps transplanted seed cells or tissues into microspheres by using a synthetic, porous and high-biocompatibility matrix. Alginate has some inherent properties which make it the first matrix material for the construction of slow release microsphere systems: firstly, a relatively stable aqueous phase matrix; secondly, the technical process for preparing the microspheres is simple and has low requirements on equipment environment; the porous gel has high small molecule dispersion degree, and the porosity can be controlled by a simple coating process; and fourthly, the biodegradable polyester has good biodegradability under normal physiological conditions. The latticed microporous gel microspheres have good stability and good biocompatibility. The size and permeability of the micropores can be adjusted to ensure that the coated GDNF can be slowly released, the GDNF can be slowly released to microspheres, the survival of the regenerated neurons can be adjusted, and the regeneration of axons can be promoted.

The slow-release GDNF-SCs composite acellular nerve scaffold provided by the invention is of a composite scaffold structure, the outer layer is an acellular three-dimensional nerve scaffold and has good shaping and fiber spacing, the inner layer is embedded with barium chloride cross-linked gel slow-release microspheres with good physical property, soft biology and cell compatibility, the barium chloride cross-linked gel slow-release microspheres can directionally and microscopically move along with the inflammatory reaction of injury repair after being used for nerve repair transplantation so as to continuously and dynamically release nerve nutrient substances and the like in the microspheres, the gel microspheres are non-toxic and residue-free after being degraded and can be transformed and absorbed along with tissue metabolism, and a microenvironment required for nerve repair can be provided for a.

(2) Glial cell line-derived neurotrophic factor (GDNF) is a dopaminergic neurotrophic factor found at present with the strongest specificity, which not only promotes the survival of dopaminergic neurons and motor neurons and the reconstruction of nerve synapses after injury, but also is a neurotrophic factor necessary for the development of sympathetic and parasympathetic neurons, can promote the survival and differentiation of various neurons, and has obvious protective effect on nerve injury caused by various reasons. Local targeted delivery of neurotrophic factors is the most effective mechanism for promoting regeneration of damaged nerves. The invention utilizes alginate slow-release microspheres to combine with Schwann cells which stably express GDNF, avoids the severe microenvironment at the initial stage of injury and avoids serious immunological rejection by slowly releasing GDNF, greatly improves the bioavailability of GDNF and improves the unfavorable microenvironment of regenerated nerves.

(3) The slow-release GDNF-SCs composite acellular nerve scaffold provided by the invention conforms to various characteristics of an ideal nerve transplant body, and retains a complete fiber channel structure inside a nerve to the maximum extent. The integrity of the internal structure of the acellular nerve scaffold, the existence of extracellular matrix and the slow release effect of the microspheres have important significance on the Schwann cell crawling, the release of neurotrophic factors and the extension of nerve fibers, are key factors for successfully repairing facial nerve injury, and show good clinical application prospects.

Drawings

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

FIG. 1 is a schematic diagram of a rat sciatic nerve decellularized scaffold prepared by an example; a: normal nerves; b, acellular scaffold removal: the acellular scaffold is shown to completely retain the contour of the sciatic nerve, and the cell components in the nerve are removed;

FIG. 2 shows the toluidine blue staining comparison of decellularized nerve scaffold prepared by different methods, 200X; a normal control group; b, cell removal group by improved method; c traditional Sondell method decellularized group; d Hudson method decellularized group; group A: the cross section of nerve fiber is round with different sizes, the periphery of myelin sheath is lightly stained to be in a grid structure, and extracellular matrix and other structures can be seen. Group B: no cell, axon and myelin sheath structure were found on the cross section, irregular circular cavities and wave-like pore structures were formed in the endoneurium, group C: the observation was similar to group B, but the alignment of the endoneurial tubes was disturbed in the transverse plane, and no regular gaps were observed. Group D: part of myelin sheath structure is remained;

FIG. 3 is an analysis of the composition of a decellularized neural scaffold; a normal neural laminin IHC staining 200 ×; b decellularized scaffold laminin IF staining 200 ×; the staining of the decellularized scaffold is shown to remove cell components, myelin sheath, axon and other structures, and the extracellular matrix laminin components are preserved;

FIG. 4 is a barium chloride cross-linked gel sustained release microsphere; a: gel microsphere gross morphology B: GDNF-SCs gel microsphere general shape;

FIG. 5 shows the results of protein expression of GDNF in SCs and GDNF-SCs;

FIG. 6 is the effect of GDNF on SCs activity; normal SCs apoptotic conditions; b GDNF-SCs apoptosis;

FIG. 7 is a graph of Edu testing for GDNF-SCs in proliferative states;

FIG. 8 is a CM-Dil probe tracer labeled GDNF-SCs showing that GNDF is well expressed in the cytoplasm of the gel microsphere cells;

FIG. 9 is a acellular nerve scaffold of composite GDNF-SCs sustained release microspheres; blue GDNF-SCs cell nucleus, green decellularized scaffold Laminin, 400X;

FIG. 10 is an assessment of effectiveness of the grafts in repairing nerve damage, after implantation with IF staining of 8W nerve regeneration ends, 200 ×; and (C) diagram: the longitudinal section of the nerve is shown as nerve fiber in red, and DAPI in blue; and (D) diagram: nerve cross section, red nerve fiber, blue DAPI and green fusion scaffold, the nerve fiber is arranged and grows orderly and directionally, and no neuroma is generated;

FIG. 11 is a schematic diagram of the structure of a sustained-release GDNF-SCs composite acellular nerve scaffold.

Detailed Description

the GDNF-SCs are constructed by Schwann cells over-expressing GDNF.

Preferably, the sustained-release GDNF-SCs composite acellular nerve scaffold is a barium chloride crosslinked alginate gel sustained-release microsphere.

Preferably, the method for preparing the gel microspheres loaded with GDNF-SCs comprises the following steps:

GDNF-SCs in the logarithmic phase are digested, washed, mixed with low-viscosity sodium alginate, incubated and cultured, the solution after the incubation and culture is mixed with a barium chloride solution, and gel polymerization is performed by adjusting the pH to 7.0 to 7.8, more preferably 7.2 to 7.6, and most preferably 7.4.

Preferably, in the solution system of the mixed incubation culture, the concentration of the low-viscosity sodium alginate is 10g/L to 30g/L, and the concentration of the cells is 1 to 3 x 10⁵Per mL; the time of the mixed incubation culture is preferably 5min to 15 min;

more preferably, in the mixed incubation culture solution system, the concentration of the low viscosity sodium alginate is 20g/L, and the concentration of the cells is 2X 10⁵Per mL; the time of the mixed incubation culture is preferably 10 min.

Preferably, the volume ratio of the solution after the mixed incubation culture to the barium chloride solution is 2.5: 17-23, preferably 2.5: 20; the concentration of the barium chloride solution is 0.5 mmol/L-0.7 mmol/L, preferably 0.6 mmol/L;

more preferably, the solution after the mixed incubation culture is added dropwise to the barium chloride solution to mix; more preferably, the drop distance is 4 cm-8 cm; more preferably, the gel polymerization time is 8 to 12min, and still more preferably 10 min.

Preferably, the sustained release GDNF-SCs composite decellularized nerve scaffold as described above, the method further comprising:

and (2) culturing the gel microspheres loaded with the GDNF-SCs in HG-DMEM culture medium of 13-17% FBS (more preferably 15% FBS) for 2-3 days.

Preferably, the sustained-release GDNF-SCs composite acellular nerve scaffold as described above is prepared by a method comprising:

1) placing the nerve fiber with the attachments on the nerve adventitia removed in SB-10 solution with the concentration of 120 mmol/L-130 mmol/L for oscillation and cleaning;

2) oscillating and cleaning the nerve fiber in a solution containing 0.04-0.06 Triton X-200 and 2.2-2.8 mg/LSB-16;

3) oscillating and cleaning the nerve fibers in 3% -5% sodium deoxycholate solution;

more preferably, the sustained-release GDNF-SCs composite acellular nerve scaffold comprises the following steps:

1) the nerve fibers with the attachments on the nerve adventitia removed are placed in a solution containing 125mmol/L SB-10 for shaking and cleaning;

2) washing the nerve fiber in a solution containing 0.05mg/L Triton X-200 and 2.5mg/L SB-16 by shaking;

3) the nerve fibers are washed in a 4% sodium deoxycholate solution by shaking.

Preferably, in the step 1), the time for shaking and cleaning is 8-16 h;

in the step 2), the time of oscillation cleaning is 20-28 h;

in the step 3), the time of oscillation cleaning is 20-28 h;

the oscillation frequency is 70-120 times/min.

Preferably, the sustained release GDNF-SCs composite acellular nerve scaffold as described above, wherein the nerve fiber is sciatic nerve of rat.

Preferably, the sustained-release GDNF-SCs composite acellular nerve scaffold as described above, the preparation method of the acellular nerve scaffold further comprises:

4) repeating steps 1) to 3) at least 1 time.

Preferably, the sustained-release GDNF-SCs composite acellular nerve scaffold is constructed by a lentivirus-mediated GDNF transfection method.

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Examples

1. And constructing an outer three-dimensional nerve scaffold. Adult male SD rats (250-300 g) are selected as the material, sciatic nerves with the length of 20mm on both sides are taken after anesthesia, blood vessels and adipose tissues attached to the adventitia are removed, and the sciatic nerves are placed in PBS for later use. Placing sciatic nerve in sterile distilled water, performing hypotonic concussion treatment for 7h at a speed of 100 times/min, shaking in 125mmol/L SB-10 solution overnight, shaking in 100mL aqueous solution containing 0.5mg Triton X-200 and 25mg SB-16 for 24h, and shaking in 4% sodium deoxycholate solution for 24 h; repeating the steps for 1 time; finally, the mixture is washed by distilled water and placed in PBS4 ℃ for standby. Wherein, the general shape of the rat sciatic nerve acellular scaffold is shown in figure 1B.

2. And (4) carrying out staining and scanning electron microscope observation on the neural scaffold HE to verify the structure and the components of the scaffold. Taking 1 nerve after decellularization, trimming to 5mm long, fixing with 10% formaldehyde, embedding 5 μm thick transverse and longitudinal sections with conventional paraffin, and performing conventional HE staining. Structural changes of cells, axons, myelin sheaths, nerve basement membranes and the like are observed under a light microscope. Taking 5mm long sciatic nerve by the same method, fixing 4% glutaraldehyde, fixing 1% osmic acid, drying with acetone, vacuum spraying gold film, and observing structural changes of nerve cross section cell, axon, myelin sheath, nerve basement membrane, etc. by field emission scanning electron microscope.

The preparation method of the acellular nerve mainly comprises two methods of heat treatment and chemical treatment. Heat treatment is the most common method described in the literature. The tissue is subjected to repeated freeze thawing to kill the cells and remove the immunogenicity of the tissue, but the remaining components of the cells are not extracted. In the early stages of nerve transplantation, Schwann cells and macrophages invade the basement membrane tract to remove cellular debris, which delays the nerve regeneration process and destroys the basement membrane, and several chemical treatments exist to render the graft non-immunogenic by removing cellular debris. However, these methods can damage the extracellular matrix and are more damaging than thermal treatments. The study of chemical decellularization reagents by Hudson et al suggests that the more commonly used chemical reagent (Trtion-100, sodium deoxycholate) has a greater disruption to the internal structure of the decellularized nerve graft and affects axonal regeneration. The applicant of the invention finds that the effect of the pure Hudson method acellular nerve scaffold is poor, and on the basis, an improved preparation method and comprehensive application (Trtion-200, SB-10, SB-16 and sodium deoxycholate) of the acellular nerve scaffold are developed by self, and detection proves that the integrity of an internal structure and active components of an extracellular matrix can be well kept while cell components are removed, so that the pure Hudson method acellular nerve scaffold is an ideal tissue engineering nerve scaffold.

The toluidine blue staining results of the decellularized nerve scaffold prepared by different methods are shown in figure 2; the analysis of the acellular nerve scaffold components is shown in FIG. 3.

3. Building the barium chloride cross-linked gel slow-release microspheres. Preparing materials: sodium alginate (SIGMA); calcium chloride (SIGMA). Preparing a sodium alginate high-molecular solution with a certain concentration, dripping into a barium chloride gel with a certain concentration, wherein the dripping tool is a 10mL syringe, the dripper is a 9# needle head, the dripping distance is 8cm, and the pH value is adjusted to 7.4 to mix the sodium alginate high-molecular solution into the gel. After sodium alginate is dripped into the barium chloride gel, the surface of the liquid drop is immediately gelled to form microspheres, the interior of the microspheres undergoes gel reaction layer by layer, the contraction of the semitransparent microspheres can be observed by naked eyes, and water in the microspheres is extruded to enter an external medium. The gel microsphere with certain mechanical property, good permeability and biocompatibility can be prepared.

4. Purifying and culturing Schwann Cells (SCs). Experimental SD rats are anesthetized by ketamine injection (8mg/kg) to the gluteus maximus and 2c is performed right behind the lower leg and on the lateral malleolusm long incision, blunt separation of sural nerve, ligation of proximal nerve segment, and suturing of skin. 7D, taking 1cm of nerves below the ligation part under aseptic condition, placing in 4 ℃ sterile D-Hank solution, removing the nerve adventitia under a microscope, adding a freshly prepared mixed solution of 0.125% trypsin and 0.15% collagenase into a culture dish, shearing the nerves, and digesting in a culture bottle for 30 min; centrifuging at 1000r/min for 5min at a centrifugation radius of 12cm, and discarding the supernatant. Adding 10% FBS-containing DMEM culture solution, and standing at 37 deg.C and 5% CO₂Culturing in an incubator. After 24h of primary culture, 1X 10mol/L cytarabine was added to inhibit fibroblasts. The solution was changed 1 time every 2 days. When the primary cells are fused into a sheet, removing fibroblasts by using a trypsin rapid digestion method and a differential adherence method, and repeatedly purifying the Schwann cells.

5. GDNF transfects Schwann cells. Purified Schwann cells in the logarithmic growth phase are taken, digested by 0.25% trypsin, and then spread in a 6cm culture dish at a cell density of 5X 10/mL, and transfected when the cells grow to 70% confluence. 3mL of unfrozen virus liquid is added into each culture dish, and polybrene with the final concentration of 2g/mL is added; after incubation for 3h at 37 ℃, the supernatant was discarded, and the viral fluid was added again for transfection, and the transfection was repeated 3 times. And (3) after the virus is transfected for 48 hours, adding a culture medium containing 0.5g/mL puromycin for screening, and obtaining a stable cell strain after 3-7 days. The cells were amplified and cultured for PCR, Westernblot assay to verify GDNF expression.

6. Composite GDNF-SCs gel microspheres. Taking GDNF-SCs with logarithmic growth, digesting with 0.25% pancreatin/0.02% EDTA for 5min, observing SCs under a mirror to become round and shrink pseudopodically, flicking the culture dish to ensure that most cells are detached. Adding 15% FBS DMEM5mL, stopping digestion, centrifuging for 5min, discarding supernatant, completely sucking residual culture solution, repeatedly washing with 0.15mmol/L sodium chloride solution twice, re-suspending cells with 15% FBS DMEM, and adjusting solubility to 1 × 10⁶and/mL. Mixing 0.5mL cell suspension with 2mL 25.0g/L low viscosity sodium alginate solution with cell concentration of 2 × 10⁵and/mL. Culturing in incubator for 10min, dripping 20mL of 0.6mmol/L barium chloride solution into culture dish at height of 5cm, gelatinizing for 10min to obtain milky white or transparent light yellow, and shaking the solution to avoid agglomeration of microsphere in the culture dish for fast formation. Sucking out residual barium chloride solution20mL0.15mmol/L sodium chloride solution is washed for 2 times, microspheres are added into a 6-well cell culture plate, 15-20 microspheres are added into each well, and 3mL of L5% FBS-HG-DMEM is added to submerge the gel microspheres. 37 ℃ and 5% CO₂Culturing in an incubator, and replacing 15% FBS-HG-DMEM culture solution for 2-3 days.

7. And (3) constructing a GDNF-SCs gel microsphere acellular scaffold complex. After the nerve scaffold is sutured to the damaged nerve end, the sterile-cultured GDNF-SCs gel microspheres are evenly injected into the pores of the three-dimensional scaffold. The fascia, the muscle and the skin are sutured layer by layer. Provides a microenvironment for slowly releasing the neurotrophic factors for repairing the nerves at the injury.

A schematic structure diagram of the sustained-release GDNF-SCs composite acellular nerve scaffold is shown in FIG. 11.

Firstly, the outer layer structure is a rat sciatic nerve acellular nerve scaffold, the appearance is milk white, the light transmittance is good, the outer layer structure is provided with fiber pores, the whole body is a three-dimensional net structure, and the details are shown in a general specimen of the nerve scaffold in fig. 2;

secondly, barium chloride cross-linked gel slow-release microspheres are embedded in the bracket, and the microspheres have pore structures, so that the uniform infiltration of small cell molecular liquid such as culture medium can be met;

and pores of the gel microspheres have certain plasticity and ductility, and can accommodate survival and proliferation of various seed cells, such as Schwann cells for stably expressing GDNF neurotrophic factors.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A sustained-release GDNF-SCs composite acellular nerve scaffold comprises gel microspheres loaded with GDNF-SCs and an acellular nerve scaffold;

the GDNF-SCs are constructed by Schwann cells over-expressing GDNF.

2. The sustained-release GDNF-SCs composite acellular nerve scaffold of claim 1, wherein the gel microspheres are barium chloride crosslinked alginate gel sustained-release microspheres.

3. The sustained-release GDNF-SCs composite acellular nerve scaffold of claim 2, wherein the preparation method of the GDNF-SCs-loaded gel microspheres comprises the following steps:

digesting and cleaning GDNF-SCs in logarithmic phase, mixing with low-viscosity sodium alginate, incubating and culturing, mixing the solution after incubation and incubation culture with a barium chloride solution, and adjusting pH = 7.2-7.6 for gel polymerization.

4. The sustained-release GDNF-SCs composite acellular nerve scaffold according to claim 3, wherein in the mixed incubation culture solution system, the concentration of low-viscosity sodium alginate is 10-30 g/L, and the concentration of cells is 1-3 x 10⁵Per mL; the time of mixed incubation culture is 5-15 min.

5. The sustained-release GDNF-SCs composite acellular nerve scaffold of claim 3, wherein the volume ratio of the solution after the mixed incubation culture to the barium chloride solution is 2.5: 17-23; the concentration of the barium chloride solution is 0.5 mmol/L-0.7 mmol/L.

6. The sustained-release GDNF-SCs composite acellular nerve scaffold according to claim 3, wherein the solution after the mixed incubation culture is added dropwise to the barium chloride solution for mixing; the drop distance is 4 cm-8 cm.

7. The sustained-release GDNF-SCs composite acellular nerve scaffold according to claim 3, wherein the gel polymerization time is 8-12 min.

8. The sustained-release GDNF-SCs composite decellularized nerve scaffold of claim 3, wherein said method further comprises:

and culturing the gel microspheres loaded with the GDNF-SCs in an HG-DMEM culture medium containing 13% -17% FBS for 2-3 days.

9. The sustained-release GDNF-SCs composite acellular nerve scaffold of claim 1, wherein the preparation method of the acellular nerve scaffold comprises the following steps:

1) placing the nerve fibers with the attachments on the nerve adventitia removed in a SB-10 solution containing 120 mmol/L-130 mmol/L for oscillation cleaning;

2) oscillating and cleaning the nerve fibers in a solution containing 0.04-0.06 mg/L Triton X-200 and 2.2-2.8 mg/L LSB-16;

3) oscillating and cleaning the nerve fibers in 3% -5% sodium deoxycholate solution.

10. The sustained-release GDNF-SCs composite acellular nerve scaffold as claimed in claim 9, wherein in the step 1), the time for shaking and cleaning is 8-16 h;

in the step 2), the oscillation cleaning time is 20-28 h;

in the step 3), the oscillation cleaning time is 20-28 h;

the oscillation frequency is 70-120 times/min.

11. The sustained-release GDNF-SCs composite decellularized nerve scaffold of claim 9, wherein the nerve fiber is a sciatic nerve of a rat.

12. The sustained-release GDNF-SCs composite acellular nerve scaffold according to any one of claims 9 to 11, wherein the preparation method of the acellular nerve scaffold further comprises the following steps:

4) repeating steps 1) -3) at least 1 time.

13. The sustained-release GDNF-SCs composite acellular nerve scaffold of claim 1, wherein the GDNF-SCs is constructed by lentivirus-mediated GDNF transfection.