Functionalized nerve conduit modified by smooth muscle extracellular matrix as well as preparation method and application of functionalized nerve conduit

Abstract

The invention provides a functional nerve conduit modified by a smooth muscle extracellular matrix as well as a preparation method and application of the functional nerve conduit, and belongs to the field of biological tissue materials. Smooth muscle cells are inoculated to the surface of a material, the functional nerve conduit is prepared through smooth muscle cell-derived extracellular matrix modification, the nerve conduit remarkably promotes dorsal root ganglion and PC12 axon regeneration, angiogenesis is promoted by inducing Schwann cell migration and VEGF secretion, a guiding bridge is provided for new axon, and the nerve conduit has the advantages that the nerve conduit has a good application prospect. The cut-off nerve is reconnected, the large-segment defective sciatic nerve is repaired, the function control and the muscle function after nerve regeneration are improved, and good application prospects are achieved.

Description

Technical Field

[0001] The invention belongs to the field of biological tissue materials, and in particular relates to a functionalized nerve conduit modified with a smooth muscle cell extracellular matrix, and a preparation method and application thereof. Background Art

[0002] Peripheral nerve injuries (PNIs) account for approximately 3-5% of trauma cases worldwide, often leading to muscle weakness, chronic pain, and loss of sensory and motor nerve function. For PNIs with severe defects, autologous nerve transplantation is still considered the gold standard in clinical practice. However, autologous nerve transplantation has problems such as limited donors, donor site complications, and mismatch with PNI size.

[0003] In recent years, advances in tissue engineering have brought great hope for the repair of PNIs and avoided the shortcomings of autologous transplantation. A variety of cell-loaded or acellular nerve guide conduits (NGCs) have been developed to replace autologous nerve transplantation. Among them, acellular NGCs are favored because of their low cost, low risk of infection, no ethical issues, and low immunogenicity. In addition to synthetic polymers, extracellular matrix (ECM) scaffolds obtained by decellularization of cultured cells or natural tissues are promising candidates for the manufacture of acellular NGCs, which show excellent biocompatibility and biodegradability. Although studies have shown that acellular neural tissue plays an important role in supporting axon guidance and nerve regeneration in PNIs. However, acellular neural tissue has the disadvantages of limited sources and poor mechanical properties. Other decellularized tissues, such as porcine small intestinal submucosa (SIS), porcine bladder basement membrane, and human amniotic membrane (HAM), have appropriate three-dimensional environments and mechanical support, but lack specific biochemical stimulation signals to promote the regeneration of damaged nerves.

[0004] In the repair process of peripheral nerve injury, angiogenesis is crucial for nerve fiber regeneration. Studies have found that in the early stages after nerve injury, angiogenesis is positively correlated with nerve fiber regeneration. Specifically, angiogenic factors such as VEGF promote the proliferation and migration of endothelial cells to form new blood vessels, thereby providing support for the survival and regeneration of neurons. In addition, angiogenesis can also guide the migration of Schwann cells, help axon regeneration and rebuild nerve connections.

[0005] Therefore, it is of great significance to develop an ideal NGCs that provides a favorable microenvironment and rapidly and successfully promotes the proliferation and migration of Schwann cells (SCs) as well as the regeneration of axons. Summary of the invention

[0006] The purpose of the present invention is to provide a functionalized nerve conduit modified with a smooth muscle cell extracellular matrix and a preparation method and use thereof.

[0007] The invention provides a composite material. The composite material is obtained by inoculating smooth muscle cells on the surface of a material, culturing, decellularizing and molding the resulting material.

[0008] Furthermore, the smooth muscle cells are derived from intestinal, uterine, stomach, bladder or vascular smooth muscle; the material is selected from natural materials or artificial synthetic materials; the inoculation density of the smooth muscle cells is 1×10 4 ~1×10 8 Pieces / cm 2 , preferably 1×10 5 ~1×10 7 Pieces / cm 2 .

[0009] Furthermore, the natural material is small intestinal submucosa, amniotic membrane or collagen, and the synthetic material is a synthetic polymer material, preferably polyurethane or polylactic acid. Furthermore, the small intestinal submucosa is the small intestinal submucosa of pigs, cattle, sheep or horses.

[0010] Furthermore, the preparation method of the small intestinal submucosal layer is as follows:

[0011] Take the small intestine, remove the serosal layer and the muscular layer, defat, decellularize, descale, freeze-dry, and sterilize to obtain;

[0012] The degreasing step is to soak the mixture of methanol and chloroform in a volume ratio of 1:1 for 10 to 14 hours, and then rinse with deionized water;

[0013] And / or, the decellularization is performed by enzymatic digestion, soaking in a 0.25% pancreatic enzyme solution overnight at 4°C, and then washing with physiological saline to remove the pancreatic enzyme;

[0014] And / or, the descaling is performed by soaking in a 0.5% sodium dodecyl sulfate solution for 4 to 6 hours and washing with deionized water;

[0015] And / or, the sterilization is performed using ethylene oxide.

[0016] The present invention also provides a method for preparing the composite material, the method comprising the following steps:

[0017] (1) inoculating smooth muscle cells on the surface of the material and culturing them to obtain a cell-material complex;

[0018] (2) decellularizing the cell-material complex to obtain a decellularized cell-material complex;

[0019] (3) Washing, shaping, and drying the decellularized cell-material complex to obtain the product.

[0020] Furthermore, in step (1), the culture time is 2-14 days, preferably 7 days.

[0021] Furthermore, in step (2), the decellularization treatment includes the following steps: repeatedly freezing and thawing the cell-material complex, treating it with a decellularization solution, and washing it.

[0022] Furthermore, in step (2), the freeze-thaw conditions are: first freezing at -100 to -60°C for 20 to 60 minutes, and then thawing at 30 to 45°C for 20 to 60 minutes;

[0023] The decellularization solution is 1% Triton X-100;

[0024] The treatment temperature is 30-45°C and the treatment time is 1-10 minutes;

[0025] The cleaning liquid is PBS.

[0026] Furthermore, in step (2), the freeze-thaw conditions are: first freezing at -80°C for 40 min, then thawing at 37°C for 40 min;

[0027] The decellularization solution is 1% Triton X-100;

[0028] The treatment temperature is 37°C and the treatment time is 5 minutes.

[0029] Furthermore, in step (2), the number of repeated freeze-thaw cycles is 2 times.

[0030] Furthermore, in step (3), the drying temperature is 30-45°C, preferably 37°C.

[0031] The invention also provides use of the composite material in preparing a nerve conduit.

[0032] Furthermore, the nerve conduit is a nerve conduit that promotes Schwann cell migration and / or promotes angiogenesis.

[0033] The present invention implants smooth muscle cells in a membrane material to prepare a smooth muscle cell extracellular matrix modified nerve conduit, which significantly promotes axon regeneration of dorsal root ganglia and PC12, significantly promotes Schwann cell migration, and promotes angiogenesis. The nerve conduit also has good biodegradability, repairs defective sciatic nerves, improves functional control and muscle function after nerve regeneration, and has good application prospects.

[0034] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0035] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Preparation and characterization of M-SIS membrane: (A) Scanning electron microscopy; (B) DAPI staining; (C) DNA content detection; (D) collagen content detection; (E) elastin content detection; (F) HGF content detection; (G) bFGF content detection; (H) TGF-b content detection.

[0037] Figure 2 The gross and microscopic morphology of the M-SIS catheter.

[0038] Figure 3 The effect of M-SIS on axon regeneration: (A) axon regeneration of dorsal root ganglion; (B) axon regeneration of undifferentiated PC12.

[0039] Figure 4 To evaluate the effect of M-SIS on Schwann cell migration and angiogenesis: (A) Scratch assay to detect the effect of M-SIS on Schwann cell migration and (B) the results; (C) Transwell assay to detect the effect of M-SIS on Schwann cell migration and (D) the results; (E) Images of M-SIS and SCs working together to promote angiogenesis and (F) the results; (G) M-SIS induces SCs to secrete VEGF.

[0040] Figure 5 To evaluate the effect of M-SIS catheter in the repair of sciatic nerve defects: (A) Gross image of the specimen taken 12 weeks after surgery; (B) HE staining; (C) TUJ1 immunofluorescence staining; (D) Transmission electron microscopy.

[0041] Figure 6 Neuroelectrophysiological and gait analysis 12 weeks after surgery: (A) electrophysiological detection images and (B) results; (C) footprint and (D) plantar pressure analysis.

[0042] Figure 7 This is the morphological analysis of the tibialis anterior muscle. (A) General view; (B) Muscle HE staining; (C) Ankle joint range of motion; (D) Muscle wet weight ratio; (E) Muscle bundle diameter statistics; (F) Ankle joint range of motion statistics. DETAILED DESCRIPTION

[0043] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0044] The "overnight" referred to in the present invention is 12±5h.

[0045] The preparation method of porcine small intestinal submucosa (SIS) is as follows:

[0046] 1) Separation of small intestinal submucosa: Cut the cleaned pig small intestine into sections of about 15 cm in length, scrape off the muscular layer and serosal layer of the small intestine, wash with saline and filter to obtain the small intestinal submucosa;

[0047] 2) Degreasing: Immerse the prepared small intestinal submucosal layer in a degreasing solution (a mixture of methanol and chloroform in a volume ratio of 1:1) for 12 hours, and then wash it repeatedly until there is no odor;

[0048] 3) Decellularization: Immerse the defatted small intestinal submucosal layer in a 0.25% pancreatic enzyme solution overnight at 4°C, rinse until there is no foam, immerse it in a 0.5% sodium dodecyl sulfate solution for 4-6 hours, and wash with deionized water and freeze-dry;

[0049] 4) Sterilization: sterilize with ethylene oxide.

[0050] Example 1. Preparation of SIS membrane modified with smooth muscle cell extracellular matrix

[0051] Press 1×10 5 Pieces / cm 2 Smooth muscle cells (SMCs) were inoculated on the porcine small intestinal submucosal layer (SIS) at a density of 1.5 and cultured at 37℃ for 7 days to obtain smooth muscle cell-SIS complex (SMCs-SIS). Then the smooth muscle cell-SIS complex was decellularized.

[0052] The specific steps of decellularization are as follows: the smooth muscle cell-SIS complex is frozen at -80°C for 40 min, then thawed at 37°C for 40 min, then washed in PBS 3 times, and the freeze / thaw cycle is repeated 2 times. The complex is then treated with preheated 1% Triton X-100 at 37°C for 5 min, and washed 6 times with PBS to obtain a smooth muscle cell-SIS membrane after decellularization (abbreviated as M-SIS membrane).

[0053] Example 2: Preparation of SIS catheter modified with smooth muscle cell extracellular matrix

[0054] The decellularized smooth muscle cell-SIS membrane obtained in Example 1 was wound along the hollow conduit in the same direction, dried at 37° C., and demolded to obtain an ECM-SIS conduit (abbreviated as M-SIS conduit). The pore size of the hollow conduit can be adjusted according to demand.

[0055] The following is a control sample preparation.

[0056] Comparative Example 1: Preparation of SIS catheter

[0057] Referring to the method of Example 1, the SIS prepared in Comparative Example 1 was wound along the hollow conduit in the same direction, dried at 37° C., and demolded to obtain the SIS conduit. The aperture of the hollow conduit can be adjusted according to requirements.

[0058] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0059] Experimental Example 1: Characterization of SIS membrane / catheter and M-SIS membrane / catheter

[0060] 1. Experimental methods

[0061] The attachment of extracellular matrix (ECM) on the surface of SIS, SMCs-SIS and M-SIS was observed by scanning electron microscopy;

[0062] The components of SMCs, collagen, elastin, HGF, bFGF, and TGF-β in SIS, SMCs-SIS, and M-SIS were detected by DAPI staining and DNA content;

[0063] The gross and microscopic morphologies of the SIS catheter and the M-SIS catheter were observed under a microscope.

[0064] 2. Experimental results

[0065] Compared with SIS, more extracellular matrix (ECM) attachment can be observed on the surface of M-SIS ( Figure 1 A). DAPI staining ( Figure 1 B) and DNA content detection ( Figure 1 C) showed that decellularization can effectively remove SMCs and retain most of the collagen ( Figure 1 D) and elastin ( Figure 1 In addition, SMCs-derived ECM coating modification increased the content of neurotrophic molecules, including HGF, bFGF, and TGF-β ( Figure 1 FH).

[0066] Depend on Figure 2 As can be seen in the figure, both SIS and M-SIS catheters are translucent catheters with dense and uniform walls.

[0067] Experimental Example 2: Effects of M-SIS membrane on axon regeneration, Schwann cell migration and angiogenesis

[0068] 1. Experimental methods

[0069] 1.1 Axon regeneration: Dorsal root ganglia and PC12 cells were inoculated on the surface of SIS and M-SIS, respectively, and the regeneration of axons was observed.

[0070] 1.2M-SIS promotes Schwann cell migration:

[0071] (1) Scratch assay: The scratch assay was used to detect SC migration. SIS and M-SIS were placed in DMEM medium containing 1% fetal bovine serum (surface area of ​​material: volume of medium = 1:6) and soaked at 37°C for 24 h to prepare conditioned medium CM. SCs were seeded in a 6-well plate. When the cells grew to 95% confluence, DMEM containing 1% FBS was replaced and cultured overnight. The next day, a 200 μL gun tip was used to scratch the SCs, and the SCs were cultured in CM containing 1% FBS for another 48 h. At the preset time points, the scratch healing status was imaged and analyzed using ImageJ software.

[0072] (2) Transwell experiment: To study the effect of M-SIS on SCs migration, SIS and M-SIS were placed in the lower chamber of a Transwell chamber with a pore size of 6.5 mm and 8 μm, respectively. 5 SCs were seeded in the upper chamber and cultured for 24 h. The cells were fixed with 4% PFA, stained with DAPI, and rinsed with DI water until the cells became clear. The number of cells that migrated to the lower surface of the chamber was observed under an optical microscope to evaluate the effect of M-SIS on inducing SCs migration.

[0073] 1.3 Promoting angiogenesis: Umbilical vein endothelial cells were inoculated on the surface of Matrigel matrix and cultured with the extract of SIS membrane and M-SIS membrane (i.e., tube formation culture system). The tube formation was observed and the angiogenesis-promoting effects of SIS and M-SIS were evaluated. The tube formation culture system was further co-cultured with Schwann cells using Transwell to evaluate the role of SCs and M-SIS in promoting angiogenesis.

[0074] 2. Experimental results

[0075] The results showed that M-SIS could significantly promote the dorsal root ganglion ( Figure 3 A) and PC12( Figure 3 B) Regeneration of nerve axons. M-SIS can significantly promote Schwann cell migration ( Figure 4 AD), and in the presence of Schwann cells, it can promote angiogenesis by inducing Schwann cells to secrete VEGF and work together with SCs ( Figure 4 EF).

[0076] Experimental Example 3: Effect of M-SIS catheter in repairing sciatic nerve defects in rats

[0077] 1. Experimental methods

[0078] To determine whether the M-SIS catheter promotes nerve regeneration in vivo, the SIS catheter and the M-SIS catheter were implanted to repair a 10 mm nerve defect in the rat sciatic nerve.

[0079] (1) To evaluate the effect of M-SIS catheter in the repair of sciatic nerve defects: The gross images of rats obtained 12 weeks after surgery were observed under a microscope, and the nerve defect repair was observed by HE staining, TUJ1 immunofluorescence staining, and transmission electron microscopy.

[0080] (2) Evaluation of the recovery of functional control after nerve regeneration: The recovery of functional control after nerve regeneration was evaluated through electrophysiological testing and gait analysis experiments. Electrophysiological evaluation uses the compound muscle action potential (CMAP) indicator to evaluate the recovery of nerve conduction pathways.

[0081] (3) Evaluation of the structure and function of the innervated muscles: The tibialis anterior (TA) muscle of the affected limb was obtained 12 weeks after surgery, and the structure and function of the innervated muscles were evaluated by observing the tibialis anterior muscle morphology, muscle wet weight ratio, muscle HE staining, muscle bundle diameter statistics, and ankle joint range of motion.

[0082] 2. Experimental results

[0083] (1) The role of M-SIS catheter in the repair of sciatic nerve defects

[0084] 12-week gross photograph ( Figure 5 A) shows that similar to the autologous nerve transplantation group, the nerve diameters in the SIS catheter group and the M-SIS catheter group were uniform, and the nerve catheters were completely biodegradable. Only obvious thin filamentous tissue was seen between the stumps of the nerves in the defect group. HE staining was used to evaluate the continuity of the nerve fiber structure, the biodegradability and biosafety of the catheter, and the scar formation in the repair area. No obvious inflammatory infiltration was observed at the implantation site in each group. Four weeks after surgery, obvious nerve bundle formation was observed in the M-SIS catheter ( Figure 5 B, red arrow), while almost no nerve fibers were seen in the SIS catheter. In the defect group, because both ends of the nerves were sutured and fixed to the marked positions on the fascia, there was no nerve tissue in the gap between the two ends of the nerves, only muscle tissue. 12 weeks after surgery, the regenerated nerve fibers in the autograft group and the M-SIS group were dense and orderly, with no obvious scar tissue formation, while the new tissues in the defect group and the SIS group were sparse and disordered ( Figure 5 B).

[0085] Axon regeneration TUJ1 immunofluorescence staining ( Figure 5 C) and transmission electron microscopy (TEM) were used to evaluate ( Figure 5D) The newly generated nerve fibers in the M-SIS group were more evenly and densely arranged along the long axis of the regenerated nerve, similar to the autologous transplantation group, but the newly generated nerve fibers in the defect group and the SIS group were sparsely distributed and wrapped by loose fibrous tissue, with insufficient axon regeneration and myelination.

[0086] (2) Restoration of nerve function after regeneration by M-SIS catheter

[0087] Compared with the defect group and SIS catheter group, the CMAP latency of rats in the M-SIS catheter group was shorter, the motor conduction velocity (MCV) was faster, and the CAMP amplitude was larger ( Figure 6 AB). To further evaluate motor function, we recorded the footprints of rats walking 12 weeks after implantation and show representative footprints from different groups ( Figure 6 C). The average contact area and maximum contact area of ​​the autologous transplantation group and the M-SIS catheter group were higher than those of the defect group and the SIS catheter group ( Figure 6 CD).

[0088] (3) M-SIS catheter affects the structure and function of innervated muscles

[0089] Compared with the autologous transplantation group and the M-SIS catheter group, the defect group and the SIS catheter group had significant muscle atrophy ( Figure 7 A), lower muscle wet weight ratio ( Figure 7 D). Muscle HE staining further shows the degree of muscle atrophy, which is manifested by a decrease in muscle fiber volume and an increase in loose connective tissue ( Figure 7 B, E). Due to long-term denervation of the injured limb, in addition to flexion contracture of the paw, all animals in the defect group and SIS catheter group had dorsiflexion contracture of the ankle joint, resulting in a significant decrease in ankle joint range of motion ( Figure 7 C, F).

[0090] The above results show that M-SIS membrane can significantly promote the regeneration of dorsal root ganglia and PC12 axons, significantly promote Schwann cell migration, and work together with SCs to promote angiogenesis. M-SIS catheter can effectively repair the defective sciatic nerve, improve the functional control of rat nerve regeneration, improve muscle atrophy, paw flexion contracture, dorsiflexion contracture and ankle joint range of motion, and can be completely biodegradable.

[0091] In summary, the present invention provides a functionalized nerve conduit modified with smooth muscle cell extracellular matrix, and a preparation method and use thereof. The present invention inoculates smooth muscle cells on the surface of the material, and prepares a functionalized nerve conduit through modification of the extracellular matrix derived from smooth muscle cells. The nerve conduit significantly promotes axon regeneration of dorsal root ganglia and PC12, and promotes angiogenesis by inducing Schwann cell migration and secretion of VEGF, providing a guiding "bridge" for new axons, reconnecting severed nerves, repairing large sections of defective sciatic nerves, and improving functional control and muscle function after nerve regeneration, and has good application prospects.

Claims

1. A composite material, characterized in that: The composite material is obtained by inoculating smooth muscle cells on the surface of the material, culturing, decellularizing and molding.

2. The composite material according to claim 1, characterized in that The smooth muscle cells are derived from intestinal, uterine, stomach, bladder or vascular smooth muscle; the material is selected from natural materials or artificial synthetic materials; the inoculation density of the smooth muscle cells is 1×10 4 ~1×10 8 Pieces / cm 2 , preferably 1×10 5 ~1×10 7 Pieces / cm 2 .

3. The composite material according to claim 2, characterized in that The natural material is small intestinal submucosa, amniotic membrane or collagen, and the artificial synthetic material is a synthetic polymer material, preferably polyurethane or polylactic acid.

4. A method for preparing the composite material according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: (1) inoculating smooth muscle cells on the surface of the material and culturing them to obtain a cell-material complex; (2) decellularizing the cell-material complex to obtain a decellularized cell-material complex; (3) Washing, shaping, and drying the decellularized cell-material complex to obtain the product.

5. The method according to claim 4, characterized in that In step (1), the culture time is 2-14 days, preferably 7 days.

6. The method according to claim 4, characterized in that In step (2), the decellularization treatment includes the following steps: repeatedly freezing and thawing the cell-material complex, treating it with a decellularization solution, and washing it.

7. The method according to claim 6, characterized in that In step (2), the freeze-thaw conditions are: first freezing at -100 to -60°C for 20 to 60 minutes, and then thawing at 30 to 45°C for 20 to 60 minutes; The decellularization solution is 1% Triton X-100; The treatment temperature is 30-45°C and the treatment time is 1-10 minutes; The cleaning liquid is PBS.

8. The method according to claim 7, characterized in that In step (2), the freeze-thaw conditions are: first freezing at -80°C for 40 min, then thawing at 37°C for 40 min; The decellularization solution is 1% Triton X-100; The treatment temperature is 37°C and the treatment time is 5 minutes.

9. Use of the composite material according to any one of claims 1 to 3 in the preparation of a nerve guide.

10. The use according to claim 9, characterized in that The nerve conduit is a nerve conduit that promotes Schwann cell migration and / or promotes angiogenesis.

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