CN108324991B - A sustained-release GDNF-SCs composite acellular neural scaffold - Google Patents

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

A sustained-release GDNF-SCs composite acellular neural scaffold

technical field

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

Background technique

The incidence of facial nerve injury caused by various reasons is high, which can cause facial paralysis, insufficiency of eyelid closure, facial muscle paralysis, atrophy, and eventually blindness and facial deformity. It seriously affects the facial appearance, function and the daily life and social activities of patients, reduces the quality of life, leads to severe inferiority complex, and even suicidal tendency, which brings great pressure to the family and society. In recent years, with the remarkable development of microneurosurgery and tissue engineering, domestic and foreign scholars have adopted methods such as autologous nerve transplantation and synthetic material stent transplantation. The functional repair of damaged facial nerve has been improved to some extent, but it is still not ideal. Therefore, there is an urgent need to find effective treatment methods to repair damaged facial nerve and improve its repair effect.

The repair of peripheral nerves is mainly based on direct anastomosis: one-stage end-to-end anastomosis, peroneal nerve insertion and transplantation, and two-stage facial-accessory or facial-hypoglossal nerve anastomosis. The repair techniques mostly use the principles of peripheral nerve repair: ① end-to-end anastomosis ② glue technology ③ laser welding technology. Although the restorations have improved with sophisticated microsurgical techniques, they are still not satisfactory. The effect of direct end-to-end anastomosis to repair facial nerve injury is limited simply by improving surgical skills. It is urgent to develop various nerve substitutes to bridge nerve defects and improve the repair effect of nerve injury.

At present, most of the peripheral nerve stent materials approved by the US Food and Drug Administration or European CE are nerve conduits made of degradable polymer materials. Most of them are just a hollow structure, mainly to improve the physical channel for nerve regeneration and form a relatively isolated closed environment to avoid the influence of surrounding tissues on nerve regeneration; but due to the lack of biomimetic three-dimensional structure, the biological characteristics are far from normal nerves. far, the induction of axon regeneration is limited.

There are still many problems in the nerve repair materials in the prior art:

1. The shape and structure of nerve repair materials are single, and most of them are tubular support stents or three-dimensional three-dimensional stents. Although it can guide the direction of nerve cell regeneration under certain conditions, it lacks a dynamic microenvironment scaffold for sustained action. It has been reported that the extracellular matrix is structurally composited with artificial multi-channel and multi-channel scaffolds, so that the longitudinal channels of the scaffold contain transverse channel connections and are loaded with different substrates. It can further promote the planted seed cells to form a neural network with information transmission function, and use it with spinal cord injury and other parts to connect the upper and lower nerve fibers at the injury site to play a role. However, the texture of the peripheral nerve is not as soft as that of the spinal cord, its physical shape and directional growth direction are fixed, and continuous microenvironmental nutritional support is required for damage repair. Therefore, a simple three-dimensional or optimized suture scaffold cannot meet the conditions for the self-growth of its nerve cells.

2. The space volume ratio of the neurotrophic factor embedded in the nerve repair material is not enough. It has been reported that neural scaffolds mostly use the three-dimensional structure of their materials to make neurotrophic factors adhere naturally to achieve a larger area coverage. However, the nerve repair process is a slow "crawling" process from proximal to distal. Neurotrophic factors that do not meet the physiological dose are easily exposed to tissue immune cells in the early stage of transplantation and are rejected and cleared. Fixed dynamic requirements.

3. Insufficient repair function of nerve repair materials: Due to the lack of sources of autologous nerve grafts and the need for secondary surgery, it is urgent to develop extracellular matrix scaffold materials that can replace autologous nerves. At present, it is believed that the ideal nerve graft should have the following characteristics: ① no immune antigenicity, good biocompatibility and cytocompatibility; ② physical properties (elasticity, strength, toughness, etc.) are close to the recipient tissue, and the degraded The product is non-toxic. ③ The axons of the regenerated nerve cells can grow into and reach the distal part through the graft; ④ The graft has a three-dimensional pore-like structure, which can meet the dynamic migration and enrichment pathways of glial cells, and provide a good microenvironment for cell growth ; ⑤ The graft can provide important neurotrophic factors and other substances necessary for axonal growth. At present, most of the peripheral nerve injury repair scaffolds can only provide environmental support for nerve fiber growth and quantitative neuronutrients at the same time. It cannot meet the needs of dynamic and orderly growth of nerve fibers, so it is impossible to efficiently and accurately connect the end-to-end nerve fibers, and the therapeutic effect is not ideal.

"Shenqiao", which was approved to be marketed in China in 2012, is a decellularized allogeneic nerve repair material obtained after natural nerve decellularization treatment. The higher molecular material nerve conduit has been greatly improved. It is mainly composed of extracellular matrix and retains the scaffolding structure of the nerve. After bridging the nerve stump, its three-dimensional structure and extracellular matrix provide a good physical and biological environment for the growth of regenerated nerves, which can guide and support the growth of regenerated nerve fibers from the proximal stump to the distal stump, and recover. Innervation of target organs. However, the scaffold itself does not contain glial cells and a good cell proliferation environment, and cannot provide a perfect tissue repair microenvironment.

In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

The structure of the existing tissue-engineered neural scaffold is mainly a simple extracellular matrix scaffold, and its three-dimensional structure can be combined with active seed cells to form a complex with biological activity. The scaffold transplantation combined with SCs to bridge the nerve defect can promote nerve repair to a certain extent, but the survival rate of the implanted cells is very low, and the repair effect is not ideal. The invention provides a barium chloride cross-linked alginate gel sustained-release microsphere transport system combined with a GDNF-SCs acellular nerve repair scaffold. Provides a microenvironment for neurotrophic factors to act. The repair material improves the survival rate of the implanted seed cells, sustains and maintains a stable concentration of neurotrophic factors in the damaged area, and can dynamically adjust the content of neurotrophic factors released by the seed cells according to the pathophysiological mechanism of the injured tissue, so that the damaged facial nerve can be adjusted. The repair effect is greatly improved.

In order to realize the above-mentioned purpose of the present invention, the following technical solutions are specially adopted:

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

The gel microspheres loaded with GDNF-SCs are filled in the pores of the decellularized neural scaffold;

The GDNF-SCs were constructed from Schwann cells overexpressing GDNF.

The scaffold complex achieves organic combination. The composite GDNF sustained-release microspheres were fused with decellularized neural scaffolds to construct a novel nerve graft complex. The sustained-release microspheres loaded with GDNF can grow by means of the three-dimensional structure of the neural scaffold, which not only reduces the changes in structure and function caused by the accumulation and extrusion between the microspheres, but also improves the proliferation activity of GDNF-SCs permeating the microspheres; The outer membrane structure of the scaffold itself keeps GDNF confined to the damaged area, realizing the controlled release of GDNF protein, allowing it to permeate the microspheres in a long-term, slow and stable manner, and maintain a stable protein concentration in the damaged local area; it limits the GDNF exuding from the microspheres. Migrate to surrounding undamaged tissues to avoid adverse reactions. The composite system organically combines the two major carriers of acellular nerve scaffold material and slow-release microspheres. The two complement each other and work together. It is an ideal nerve transplantation composite system for repairing damaged facial nerve materials. It solves the problem that gene therapy cannot control the amount of GDNF secretion in the past, and the implanted seed cells cannot be limited to the damaged area to play a targeted role, and the cells migrate to the non-injured area to play an unpredictable role.

Compared with the prior art, the beneficial effects of the present invention are:

(1) The internal structure of the present invention is gel slow-release microspheres, and its transport system is a transport system for encapsulating the transplanted seed cells or tissues into microspheres with a synthetic, porous and highly biocompatible matrix. Some innate characteristics of alginate make it the preferred matrix material for the construction of sustained-release microsphere systems: 1) Relatively stable aqueous matrix; 2) The technical process of microsphere preparation is simple, and the equipment environment requirements are not high; 3) Porous gel, small molecules The degree of dispersion is high, and the porosity can be controlled by a simple coating process; ④ It has good biodegradability under normal physiological conditions. The grid-shaped microporous gel microsphere of the invention has good stability and good biocompatibility. It can adjust the appropriate micropore size and permeability, so that the encapsulated GDNF can be slowly released, so that GDNF can slowly release the microspheres, regulate the survival of regenerated neurons and promote the regeneration of axons.

The sustained-release GDNF-SCs composite acellular neural scaffold provided by the present invention is a composite scaffold structure, the outer layer is a decellularized three-dimensional neural scaffold, which has good shaping and fiber spacing, and the inner layer is embedded with physical properties soft biological and cellular phase Barium chloride cross-linked gel slow-release microspheres with good capacity, which can be used for directional microscopic movement with the inflammatory response of damage repair after nerve repair and transplantation, so as to continuously and dynamically release neurotrophic substances in the microspheres, etc. After the ball is degraded, it is non-toxic and non-residual, and can be transformed and absorbed with tissue metabolism, and can provide the microenvironment required for nerve repair for a long time.

(2) Glial cell line-derived neurotrophic factor (GDNF) is the most specific dopaminergic neurotrophic factor discovered so far, which not only promotes the survival and maintenance of dopaminergic neurons and motor neurons Reconstruction of nerve synapses after injury is also a necessary neurotrophic factor for the development of sympathetic and parasympathetic neurons. It can also promote the survival and differentiation of various neurons, and has obvious protective effects on nerve damage caused by various reasons. Locally targeted transport of neurotrophic factors is the most effective mechanism for promoting the regeneration of injured nerves. The invention utilizes alginate slow-release microspheres combined with Schwann cells stably expressing GDNF, and by slowly releasing GDNF to avoid the harsh microenvironment at the early stage of injury and avoid severe immune rejection, greatly improving the bioavailability of GDNF and improving regeneration The unfavorable microenvironment in which the nerve is located.

(3) The sustained-release GDNF-SCs composite decellularized neural scaffold provided by the present invention conforms to the characteristics of an ideal nerve graft, and preserves the complete inner nerve fiber conduit structure to the greatest extent. The integrity of the internal structure of the decellularized neural scaffold, the presence of extracellular matrix and the sustained release of microspheres are of great significance for Schwann cell crawling, neurotrophic factor release and nerve fiber lengthening, and are the key factors for the successful repair of facial nerve injury. Shows good clinical application prospects.

Description of drawings

In order to illustrate the specific embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

Fig. 1 is the general shape of the rat sciatic nerve decellularized scaffold prepared in one example; A: normal nerve; B decellularized scaffold: it shows that the decellularized scaffold completely retains the outline of the sciatic nerve, and the cell components inside the nerve have been removed;

Figure 2 shows the comparison of toluidine blue staining of decellularized neural scaffolds prepared by different methods, 200×; A normal control group; B modified method decellularized group; C traditional Sondell method decellularized group; D Hudson method decellularized group; A group: The cross-section of the nerve fibers is round in different sizes, and the periphery of the myelin sheath is lightly stained with a grid-like structure, and structures such as extracellular matrix can be seen. Group B: no cells, axons and myelin sheath structures were found on the cross-section, but irregular circular cavities and wavy pore structures were found in the endoneurium. Group C: the observation results were similar to those of group B, but the nerves on the cross-section The arrangement of membrane tubes is disordered, and there is no neat gap. Group D: Part of the myelin structure is still preserved;

Figure 3 is the analysis of the components of the decellularized neural scaffolds; A normal neural laminin IHC staining 200×; B decellularized scaffolds laminin IF staining 200×; it shows that the stained decellularized scaffolds have removed cellular components, myelin sheaths, axons and other structures , the extracellular matrix laminin component is preserved;

Fig. 4 is barium chloride cross-linked gel sustained-release microspheres; A: general shape of gel microspheres B: general shape of GDNF-SCs gel microspheres;

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

Figure 6 shows the effect of GDNF on the activity of SCs; A: Apoptosis of normal SCs; B: Apoptosis of GDNF-SCs;

Figure 7 shows the GDNF-SCs in the proliferative state detected by the Edu method;

Figure 8 shows the CM-Dil probe tracking and labeling of GDNF-SCs, showing that GNDF is well expressed in the cytoplasm of gel microsphere cells;

Figure 9 is a decellularized neural scaffold composited with GDNF-SCs sustained-release microspheres; blue GDNF-SCs nucleus, green decellularized scaffold Laminin, 400×;

Figure 10 is the evaluation of the effectiveness of the graft in repairing nerve injury, IF staining at the 8W nerve regeneration end after transplantation, 200×; C: longitudinal section of nerve, red indicates nerve fibers, blue indicates DAPI; D: nerve cross-section, red indicates Nerve fibers, blue indicates DAPI, green indicates fusion scaffold, nerve fibers are arranged in an orderly and directional growth, and no neuroma is seen;

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

Detailed ways

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

The gel microspheres loaded with GDNF-SCs are filled in the pores of the decellularized neural scaffold;

The GDNF-SCs were constructed from Schwann cells overexpressing GDNF.

Preferably, in the above-mentioned sustained-release GDNF-SCs composite decellularized neural scaffold, the gel microspheres are barium chloride-crosslinked alginate gel sustained-release microspheres.

Preferably, the above-mentioned slow-release GDNF-SCs composite decellularized neural scaffold, the preparation method of the gel microspheres loaded with GDNF-SCs includes:

After the GDNF-SCs in the logarithmic growth phase are digested and washed, they are incubated with low-viscosity sodium alginate, and the mixed incubation solution is mixed with a barium chloride solution to adjust pH=7.0-7.8, more preferably pH = 7.2 to 7.6, most preferably pH = 7.4 for gel polymerization.

Preferably, in the above-mentioned sustained-release GDNF-SCs composite decellularized neural scaffold, in the solution system of mixed incubation and culture, the concentration of low-viscosity sodium alginate is 10g/L～30g/L, and the concentration of cells is 1 ～3×10 ⁵ /mL; the time of the mixed incubation is preferably 5min～15min;

More preferably, in the solution system of the mixed incubation and culture, the concentration of low-viscosity sodium alginate is 20 g/L, and the concentration of cells is 2×10 ⁵ /mL; the time of the mixed incubation and culture is preferably 10 min.

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

More preferably, the mixed incubation solution is added dropwise to the barium chloride solution for mixing; more preferably, the drop distance is 4cm～8cm; more preferably, the gelation time is 8min～12min , more preferably 10min.

Preferably, the above-mentioned sustained-release GDNF-SCs composite decellularized neural scaffold, the method further comprises:

The GDNF-SCs-loaded gel microspheres were cultured in HG-DMEM medium with 13%-17% FBS (more preferably 15% FBS) for 2-3 days.

Preferably, the above-mentioned sustained-release GDNF-SCs composite acellular neural scaffold, the preparation method of the acellular neural scaffold comprises:

1). The nerve fibers with the attachments on the epineurium removed are placed in a solution containing 120mmol/L～130mmol/L SB-10 for shaking and washing;

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

3). Shake and wash the nerve fibers in a 3%-5% sodium deoxycholate solution;

More preferably, the above-mentioned sustained-release GDNF-SCs composite acellular neural scaffold, the preparation method of the acellular neural scaffold comprises:

1). The nerve fibers with the attachments on the epineurium removed were placed in a solution containing 125mmol/L SB-10 for shaking and washing;

2). Shake and wash the nerve fibers in a solution containing 0.05mg/L Triton X-200 and 2.5mg/L SB-16;

3). The nerve fibers were shaken and washed in 4% sodium deoxycholate solution.

Preferably, in the above-mentioned sustained-release GDNF-SCs composite decellularized neural scaffold, in step 1), the oscillation cleaning time is 8h-16h;

In step 2), the time of shaking cleaning is 20h～28h;

In step 3), the time of shaking cleaning is 20h～28h;

The frequency of oscillation is 70 to 120 times/min.

Preferably, in the above-mentioned sustained-release GDNF-SCs composite decellularized neural scaffold, the nerve fibers are the sciatic nerve of a rat.

Preferably, the above-mentioned sustained-release GDNF-SCs composite acellular neural scaffold, the preparation method of the acellular neural scaffold further comprises:

4). Repeat steps 1) to 3) at least once.

Preferably, the above-mentioned sustained-release GDNF-SCs composite acellular neural scaffold, the construction method of the GDNF-SCs is a lentivirus-mediated GDNF transfection method.

The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

Example

1. Build the outer three-dimensional neural scaffold. Materials: Adult male SD rats, 250-300 g, were taken from bilateral 20 mm long sciatic nerves after anesthesia, the blood vessels and adipose tissue attached to the epineurium were removed, and placed in PBS for use. The sciatic nerve was placed in sterile distilled water for hypotonic shaking for 7 hours at a rate of 100 times/min, shaken overnight in 125 mmol/L SB-10 solution, and 100 mL aqueous solution containing 0.5 mg Triton X-200 and 25 mg SB-16. Shake for 24h, shake in 4% sodium deoxycholate solution for 24h; repeat the above steps once; finally rinse with distilled water and place in PBS4°C for later use. Among them, the general shape of the rat sciatic nerve decellularized scaffold is shown in Figure 1B.

2. HE staining and scanning electron microscope observation of nerve scaffolds were used to verify the structure and composition of scaffolds. One decellularized nerve was taken, trimmed to a length of 5 mm, fixed with 10% formaldehyde, embedded in conventional paraffin and sliced horizontally and longitudinally with a thickness of 5 μm, and stained with conventional HE. The structural changes of cells, axons, myelin sheath and nerve basement membrane were observed under light microscope. In addition, a 5mm long sciatic nerve was obtained by the same method, pre-fixed with 4% glutaraldehyde, post-fixed with 1% osmic acid, dried with acrylic acid, and coated with gold by vacuum spraying. Membrane and other structural changes.

There are two main methods for the preparation of decellularized nerves: heat treatment and chemical treatment. Heat treatment is the most common method described in the literature. The tissue is repeatedly freeze-thawed 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 duct to remove cellular debris, which will delay the process of nerve regeneration and damage the basement membrane. Several chemical treatments Not immunogenic. But these methods damage the extracellular matrix more than heat treatment. After studying the chemical decellularization reagents, Hudson et al. believed that more commonly used chemical reagents (Trtion-100, sodium deoxycholate) would damage the internal structure of decellularized nerve grafts and affect the regeneration of axons. The applicant of the present invention found that the effect of the simple Hudson method of acellular neural scaffold was not good, and on the basis of it, he developed an improved preparation method of the acellular neural scaffold, which was comprehensively applied (Trtion-200, SB-10, SB-16, and Sodium), and testing confirmed that it can better retain the integrity of the internal structure and the active components of the extracellular matrix while removing the cellular components, which is an ideal tissue engineering neural scaffold.

The toluidine blue staining results of the acellular neural scaffolds prepared by different methods are shown in Figure 2; the component analysis of the acellular neural scaffolds is shown in Figure 3.

3. Build barium chloride cross-linked gel slow-release microspheres. Preparation materials: sodium alginate (SIGMA); calcium chloride (SIGMA). Prepare a certain concentration of sodium alginate polymer solution, drop it into a certain concentration of barium chloride gel, the dripping tool is a 10mL syringe, the drip tip is a 9# needle, the drip distance is 8cm, and pH=7.4 is adjusted to mix it into a glue. When sodium alginate is dropped into the barium chloride gel, the surface of the droplet gels immediately to form microspheres, and a gel reaction occurs layer by layer inside the microspheres. The shrinkage of the translucent microspheres can be observed with the naked eye. Moisture is squeezed out into the external medium. The gel microspheres with certain mechanical properties, good permeability and good biocompatibility can be prepared.

4. Purification and culture of Schwann cells (SCs). The experimental SD rats were anesthetized with ketamine gluteus maximus injection (8 mg/kg), a 2cm incision was made on the back of the calf and the lateral malleolus, the sural nerve was bluntly separated, the proximal nerve was ligated, and the skin was sutured. 7 days later, the nerve 1 cm below the ligation site was aseptically removed, placed in sterile D-Hank solution at 4°C, the epineurium was removed under a microscope, and freshly prepared 0.125% trypsin and 0.15% collagenase mixture was added to the culture dish. After the nerves were minced, they were digested in a culture flask for 30 min; centrifuged at 12 cm and 1000 r/min for 5 min to discard the supernatant. Add DMEM medium containing 10% FBS, and place in a 37°C, 5% CO ₂ incubator for culture. After 24h of primary culture, 1×10mol/L cytarabine was added to inhibit fibroblasts. Change the medium every 2 days. When the primary cells were fused into sheets, fibroblasts were removed by trypsin rapid digestion and differential adhesion, and Schwann cells were repeatedly purified.

5. GDNF transfected Schwann cells. Purified Schwann cells in logarithmic growth phase were taken, digested with 0.25% trypsin, and plated in a 6 cm culture dish at a cell density of 5 × 10 cells/mL, and transfected when the cells reached 70% confluence. Add 3 mL of thawed virus solution to each petri dish with a final concentration of 2 g/mL polybrene; after incubating at 37°C for 3 h, discard the supernatant, add the virus solution again for transfection, and repeat the transfection 3 times. 48h after virus transfection, medium containing 0.5g/mL puromycin was added for screening, and stable cell lines were obtained in 3-7 days. The cultured cells were expanded for PCR and Western blot detection to verify the expression of GDNF.

6. Composite GDNF-SCs gel microspheres. The logarithmic growth of GDNF-SCs was taken, digested with 0.25% trypsin/0.02% EDTA for 5 min, and the rounding of SCs and the shrinkage of pseudopodia were observed under the microscope. Add 5 mL of 15% FBS DMEM, stop the digestion, centrifuge for 5 min, discard the supernatant, aspirate the remaining culture medium, rinse twice with 0.15 mmol/L sodium chloride solution, resuspend the cells in 15% FBS DMEM, and adjust the solubility to 1× 10 ⁶ /mL. Take 0.5mL of cell suspension and mix with 2mL of 25.0g/L low-viscosity sodium alginate solution, the concentration of cells is 2×10 ⁵ /mL. Incubate for 10 min, drop into a petri dish containing 20 mL of 0.6 mmol/L barium chloride solution at a height of 5 cm above the liquid surface, and the gel will become milky white or transparent pale yellow for 10 min. The balls clump together and facilitate rapid shaping. Aspirate the residual barium chloride solution, rinse twice with 20 mL of 0.15 mmol/L sodium chloride solution, add the microspheres to a 6-well cell culture plate, add 15-20 microspheres to each well, and add 3 mL of 15% FBS-HG-DMEM , submerged the gel microspheres. Culture in 37°C, 5% CO ₂ incubator, replace 15% FBS-HG-DMEM medium for 2-3 days.

7. Construction of GDNF-SCs gel microsphere acellular scaffold complex. After the nerve scaffold was sutured to the injured nerve stump, sterile cultured GDNF-SCs gel microspheres were uniformly injected into the pores of the three-dimensional scaffold. The fascia, muscle, and skin are sutured layer by layer. Provides a microenvironment for sustained release of neurotrophic factors for nerve repair at the injury site.

The schematic diagram of the sustained-release GDNF-SCs composite acellular neural scaffold is shown in Figure 11.

① The outer structure is a rat sciatic nerve acellular nerve scaffold, which is milky white in appearance, has good light transmission, and has fibrous pores. The whole is a three-dimensional three-dimensional network structure.

②The scaffold is embedded with barium chloride cross-linked gel slow-release microspheres, and the microspheres have a pore structure, which can meet the uniform infiltration of cell small molecule liquids such as culture medium;

③The pores of the gel microspheres have certain plasticity and ductility, which can accommodate the survival and proliferation of various seed cells, such as Schwann cells stably expressing GDNF neurotrophic factor.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand: It is still possible to modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements to some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present invention. range.

Claims (13)

1. A slow-release GDNF-SCs composite decellularized neural scaffold, comprising gel microspheres loaded with GDNF-SCs and acellular neural scaffolds; The gel microspheres loaded with GDNF-SCs are filled in the pores of the decellularized neural scaffold; The GDNF-SCs were constructed from Schwann cells overexpressing GDNF. 2 . The sustained-release GDNF-SCs composite acellular neural scaffold according to claim 1 , wherein the gel microspheres are barium chloride-crosslinked alginate gel sustained-release microspheres. 3 . 3. slow-release GDNF-SCs composite decellularized neural scaffold according to claim 2, is characterized in that, the preparation method of the gel microspheres that described is loaded with GDNF-SCs comprises: After the GDNF-SCs in the logarithmic growth phase were digested and washed, they were incubated with low-viscosity sodium alginate. change. 4. slow-release GDNF-SCs composite decellularized neural scaffold according to claim 3, is characterized in that, in the solution system of described mixing incubation culture, the concentration of low-viscosity sodium alginate is 10g/L～30g/L , the concentration of cells is 1-3×10 ⁵ /mL; the mixing incubation time is 5min-15min. 5. The slow-release GDNF-SCs composite decellularized neural scaffold according to claim 3, wherein the volume ratio of the solution after the mixed incubation culture and the barium chloride solution is 2.5:17～23; The concentration of barium chloride solution is 0.5 mmol/L ~ 0.7 mmol/L. 6. slow-release GDNF-SCs composite decellularized neural scaffold according to claim 3, is characterized in that, the solution after described mixing incubation is added dropwise in described barium chloride solution to mix; 8cm. 7. The slow-release GDNF-SCs composite acellular neural scaffold according to claim 3, wherein the time for gel polymerization is 8min～12min. 8. slow-release GDNF-SCs composite acellular neural scaffold according to claim 3, is characterized in that, described method also comprises: The gel microspheres loaded with GDNF-SCs were cultured in HG-DMEM medium with 13%-17% FBS for 2-3 days. 9. The slow-release GDNF-SCs composite acellular neural scaffold according to claim 1, wherein the preparation method of the acellular neural scaffold comprises: 1). The nerve fibers with the attachments on the epineurium removed were placed in a solution containing 120mmol/L~130mmol/L SB-10 for shaking and washing; 2). Shake and wash the nerve fibers in a solution containing 0.04mg/L~0.06mg/L Triton X-200 and 2.2mg/L~2.8 mg/LSB-16; 3). The nerve fibers were shaken and washed in a 3%-5% sodium deoxycholate solution. 10. The sustained-release GDNF-SCs composite decellularized neural scaffold according to claim 9, characterized in that, in step 1), the oscillation cleaning time is 8h~16h; In step 2), the oscillation cleaning time is 20h~28h; In step 3), the oscillation cleaning time is 20h~28h; The frequency of oscillation is 70~120 times/min. 11 . The sustained-release GDNF-SCs composite acellular nerve scaffold according to claim 9 , wherein the nerve fibers are sciatic nerves of rats. 12 . 12. The slow-release GDNF-SCs composite acellular neural scaffold according to any one of claims 9 to 11, wherein the preparation method of the acellular neural scaffold further comprises: 4). Repeat steps 1)~3) at least 1 time. 13. The sustained-release GDNF-SCs composite acellular neural scaffold according to claim 1, wherein the construction method of the GDNF-SCs is a lentivirus-mediated GDNF transfection method.

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