A smooth muscle extracellular matrix-modified functionalized nerve conduit and methods of making and uses thereof

Abstract

The application provides a smooth muscle cell extracellular matrix modified functionalized nerve conduit and a preparation method and use thereof, and belongs to the field of biological tissue materials. The application inoculates smooth muscle cells on the surface of a material, and obtains a functionalized nerve conduit through smooth muscle cell derived extracellular matrix modification. The nerve conduit significantly promotes the axon regeneration of dorsal root ganglion and PC12, and promotes angiogenesis by inducing Schwann cell migration and VEGF secretion, thereby providing a guiding "bridge" for the newly generated axon, reconnecting the cut nerves, repairing the large segment defect sciatic nerve, improving the functional innervation and muscle function after nerve regeneration, and having a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological tissue materials, specifically relating to a functionalized neural conduit modified with the extracellular matrix of smooth muscle cells, its preparation method, and its uses. Background Technology

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

[0003] In recent years, advances in tissue engineering have brought great hope for the repair of neural tube defects (PNIs) and avoided the drawbacks of autologous transplantation. Various cell-loaded or cell-free neural guiding conduits (NGCs) have been developed as alternatives to autologous nerve transplantation. Among them, cell-free NGCs are highly favored due to their low cost, low risk of infection, lack of ethical concerns, and low immunogenicity. Besides synthetic polymers, extracellular matrix (ECM) scaffolds obtained from decellularized cultured cells or natural tissues are promising candidates for manufacturing cell-free NGCs, exhibiting excellent biocompatibility and biodegradability. Although studies have demonstrated that decellularized neural tissue plays an important role in supporting axonal guidance and neural regeneration of PNIs, it suffers from limitations such as limited availability and poor mechanical properties. Other decellularized tissues, such as porcine submucosal layer (SIS), porcine bladder basement membrane, and human amnion (HAM), possess suitable three-dimensional environments and mechanical support, but lack specific biochemical stimuli that promote the regeneration of damaged nerves.

[0004] In the repair of peripheral nerve injuries, angiogenesis is crucial for nerve fiber regeneration. Studies have found a positive correlation between angiogenesis and nerve fiber regeneration in the early stages following nerve injury. Specifically, angiogenic factors such as VEGF promote the proliferation and migration of endothelial cells, forming new blood vessels, thereby supporting neuronal survival and regeneration. Furthermore, angiogenesis can guide the migration of Schwann cells, aiding in axonal regeneration and the reconstruction of neural connections.

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

[0006] The purpose of this invention is to provide a functionalized neural conduit modified with extracellular matrix of smooth muscle cells, its preparation method, and its uses.

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

[0008] Furthermore, the smooth muscle cells are derived from the smooth muscle of the intestine, uterus, stomach, bladder, or blood vessels; the material is selected from natural or synthetic materials; and the seeding density of the smooth muscle cells is 1 × 10⁻⁶. 4 ~1×10 8 pcs / cm 2 Preferably 1×10 5 ~1×10 7 pcs / cm 2 .

[0009] Further, the natural material is the submucosa of the small intestine, amnion, or collagen, and the synthetic material is a synthetic polymer material, preferably polyurethane or polylactic acid. Further, the submucosa of the small intestine is the submucosa of the small intestine of pigs, cattle, sheep, or horses.

[0010] Furthermore, the method for preparing the submucosa of the small intestine is as follows:

[0011] Take small intestine, remove the serosa and muscle layer, defatt, decellularize, remove dirt, freeze dry, and sterilize to obtain the product;

[0012] The degreasing process involves soaking in a mixed solution of methanol and chloroform at a volume ratio of 1:1 for 10–14 hours, followed by rinsing with deionized water.

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

[0014] And / or, the descaling process involves soaking in a 0.5% sodium dodecyl sulfate solution for 4–6 hours, followed by rinsing with deionized water;

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

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

[0017] (1) Smooth muscle cells were seeded onto the surface of the material and cultured to obtain a cell-material complex;

[0018] (2) The cell-material complex is decellularized to obtain the decellularized cell-material complex;

[0019] (3) Wash the decellularized cell-material complex, shape it, and dry it to obtain the final product.

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

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

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

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

[0024] The processing temperature is 30–45°C, and the time is 1–10 min;

[0025] The cleaning liquid is PBS.

[0026] Further, in step (2), the freeze-thaw conditions are: first freeze at -80℃ for 40 min, then thaw at 37℃ for 40 min;

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

[0028] The treatment was carried out at a temperature of 37°C for 5 minutes.

[0029] Furthermore, in step (2), the freeze-thaw cycle is repeated twice.

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

[0031] The present invention also provides the use of the above-mentioned composite material in the preparation of nerve conduits.

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

[0033] This invention involves seeding smooth muscle cells in a membrane material to prepare a smooth muscle extracellular matrix-modified nerve conduit. This nerve conduit significantly promotes axonal regeneration of the dorsal root ganglion and PC12, significantly promotes Schwann cell migration, and promotes angiogenesis. The nerve conduit also exhibits good biodegradability, repairs damaged sciatic nerves, and improves functional innervation and muscle function after nerve regeneration, demonstrating promising application prospects.

[0034] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0035] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0036] Figure 1 Preparation and characterization of M-SIS membranes: (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 macroscopic and microscopic morphology of the M-SIS catheter are shown.

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

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

[0040] Figure 5 Evaluation of the role of the M-SIS catheter in sciatic nerve defect repair: (A) Gross image taken 12 weeks postoperatively; (B) HE staining; (C) TUJ1 immunofluorescence staining; (D) Transmission electron microscopy.

[0041] Figure 6 Neurophysiological and gait analysis 12 weeks post-surgery: (A) Electrophysiological images and (B) Results; (C) Footprints and (D) Plantar pressure analysis.

[0042] Figure 7 Morphological analysis of the tibialis anterior muscle. (A) Gross 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 Implementation

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

[0044] In this invention, "overnight" refers to 12±5 hours.

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

[0046] 1) Separation of the submucosal layer of the small intestine: Cut the cleaned pig small intestine into sections of about 15cm in length, scrape off the muscle layer and serosa layer of the small intestine, wash with physiological saline and filter dry to obtain the submucosal layer of the small intestine.

[0047] 2) Degreasing: The prepared submucosal layer of the small intestine was immersed in a degreasing solution (a mixed solution of methanol and chloroform with a volume ratio of 1:1) for 12 hours, and then repeatedly washed until there was no odor.

[0048] 3) Decellularization: Immerse the defatted submucosal layer of small intestine in a 0.25% pancreatic enzyme solution overnight at 4°C. After rinsing until no foam remains, immerse it in a 0.5% sodium dodecyl sulfate solution for 4–6 hours, wash with deionized water, and freeze dry.

[0049] 4) Sterilization: Sterilize with ethylene oxide to obtain the product.

[0050] Example 1: Preparation of SIS membrane modified with extracellular matrix of smooth muscle cells

[0051] Press 1×10 5 pcs / cm 2 Smooth muscle cells (SMCs) were seeded onto the submucosa (SIS) of porcine small intestine at a specific density and cultured at 37°C for 7 days to obtain a smooth muscle cell-SIS complex (SMCs-SIS). The smooth muscle cell-SIS complex was then decellularized.

[0052] The specific steps for decellularization were as follows: the smooth muscle cell-SIS complex was frozen at -80°C for 40 min, then thawed at 37°C for 40 min, followed by washing three times in PBS, and the freeze / thaw cycle was repeated twice. The complex was then treated at 37°C with preheated 1% Triton X-100 for 5 min, and washed six times with PBS to obtain the decellularized smooth muscle cell-SIS membrane (abbreviated as M-SIS membrane).

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

[0054] The decellularized smooth muscle cell-SIS membrane prepared in Example 1 was wound in the same direction along a hollow conduit and dried at 37°C. After demolding, the ECM-SIS conduit (abbreviated as M-SIS conduit) was obtained. The pore size of the hollow conduit can be adjusted as needed.

[0055] The following describes the preparation of control samples using comparative examples.

[0056] Comparative Example 1: Preparation of SIS catheter

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

[0058] The following experimental examples demonstrate the beneficial effects of the present invention.

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

[0060] 1. Experimental Methods

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

[0062] The composition of SMCs, collagen, elastin, HGF, bFGF, and TGF-β in SIS, SMCs-SIS, and M-SIS was detected by DAPI staining and DNA content analysis.

[0063] The gross and microscopic morphology of SIS catheters and M-SIS catheters were observed under a microscope.

[0064] 2. Experimental Results

[0065] Compared to 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) indicates that decellularization can effectively remove SMCs while retaining most of the collagen ( Figure 1 D) and elastin ( Figure 1 ECM components such as E) and SMCs-derived ECM coatings. Furthermore, SMC-derived ECM coatings increase the content of neurotrophic molecules, including HGF, bFGF, and TGF-β. Figure 1 FH).

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

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

[0068] 1. Experimental Methods

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

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

[0071] (1) Scratch assay: The scratch assay was used to detect the migration of SCs. SIS and M-SIS were placed in DMEM medium containing 1% fetal bovine serum (material surface area: medium volume = 1:6) and immersed at 37°C for 24 h to prepare conditioned medium CM. SCs were seeded in 6-well plates. When the cells reached 95% confluence, the DMEM medium containing 1% FBS was replaced and the cells were cultured overnight. The next day, scratches were made with 200 μL of pipette tip, and 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 investigate the effect of M-SIS on SCs migration, SIS and M-SIS were placed in the lower chambers of Transwell chambers with pore sizes of 6.5 mm and 8 μm, respectively. 1×10⁻⁶ 5 Cells were seeded in the upper chamber and cultured for 24 h. Cells were fixed with 4% PFA, stained with DAPI, and washed with DI water until the cells were clear. The number of cells that migrated to the lower surface of the chamber was observed under a light microscope to evaluate the effect of M-SIS on SC migration.

[0073] 1.3 Promoting angiogenesis: Umbilical vein endothelial cells were seeded on the surface of Matrigel matrix and cultured with extracts of SIS membrane and M-SIS membrane (i.e., tube formation culture system). Tube formation was observed, and the angiogenesis-promoting effects of SIS and M-SIS were evaluated. Furthermore, the tube formation culture system was co-cultured with Schwann cells using Transwell to evaluate the combined angiogenesis-promoting effect of SCs and M-SIS.

[0074] 2. Experimental Results

[0075] The results showed that M-SIS could significantly promote dorsal root ganglia ( 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 induce Schwann cells to secrete VEGF, which, together with SCs, promotes angiogenesis. Figure 4 EF).

[0076] Experimental Example 3: The role of M-SIS conduit in the repair of 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 sciatic nerve of rats.

[0079] (1) Evaluation of the role of M-SIS catheter in sciatic nerve defect repair: The gross images of rats taken 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) Assessment of the recovery of functional innervation after nerve regeneration: The recovery of functional innervation after nerve regeneration was assessed through electrophysiological testing and gait analysis experiments. The compound muscle action potential (CMAP) index was used to evaluate the recovery of neural conduction pathways in the electrophysiological evaluation.

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

[0082] 2. Experimental Results

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

[0084] A partial photograph taken at 12 weeks ( Figure 5 A) shows that, similar to the autologous nerve transplantation group, the nerve diameters in both the SIS and M-SIS catheter groups were uniform, and the nerve catheters were completely biodegradable. Only fine filamentous tissue was visible between the nerve ends in the defect group. HE staining was used to assess the continuity of nerve fiber structure, the biodegradability and biosafety of the catheters, and scar formation in the repair area. No significant inflammatory infiltration was observed at the implantation sites in any group. Four weeks post-operation, significant nerve bundle formation was observed in the M-SIS catheter. Figure 5 B, red arrow), while almost no nerve fibers are visible in the SIS conduit. In the defect group, because both nerve ends were sutured and fixed to the marked positions on the fascia, no nerve tissue exists in the gap between the nerve ends, only muscle tissue. At 12 weeks post-operation, the regenerated nerve fibers in the autologous graft group and the M-SIS group were dense and orderly, with no obvious scar tissue formation, while the newly formed tissue in the defect group and the SIS group was sparse and disordered. Figure 5 B).

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

[0086] (2) Recovery of functional innervation after nerve regeneration by M-SIS conduit

[0087] Compared with the defect group and the SIS catheter group, the M-SIS catheter group rats had a shorter CMAP latency, faster motor conduction velocity (MCV), and larger CAMP amplitude. Figure 6 AB). To further assess motor function, we recorded footprints of rats walking 12 weeks post-implantation and present representative footprints from different groups (AB). Figure 6 C). The mean and maximum contact areas of the autologous transplantation group and the M-SIS catheter group were significantly higher than those of the defect group and the SIS catheter group. Figure 6 CD).

[0088] (3) M-SIS ducts affect the structure and function of nerve-innervated muscles.

[0089] Compared with the autologous transplantation group and the M-SIS catheter group, the defect group and the SIS catheter group showed significant muscle atrophy. Figure 7 A), with a lower muscle-to-wet weight ratio ( Figure 7 D). HE staining of the muscle further reveals the degree of muscle atrophy, manifested as reduced muscle fiber volume and increased loose connective tissue. Figure 7 B, E). Due to prolonged denervation of the injured limb, in addition to flexion contractures in the paw, all animals in the defect group and the SIS duct group exhibited dorsiflexion contractures of the ankle joint, resulting in a significant reduction in ankle joint range of motion. Figure 7 C, F).

[0090] The above results indicate that the M-SIS membrane can significantly promote the regeneration of dorsal root ganglia and PC12 nerve axons, significantly promote Schwann cell migration, and work synergistically with SCs to promote angiogenesis. The M-SIS catheter can effectively repair damaged sciatic nerves, improve the functional innervation of rats after nerve regeneration, improve muscle atrophy, paw flexion contractures, dorsiflexion contractures, and ankle joint range of motion, and is completely biodegradable.

[0091] In summary, this invention provides a functionalized nerve conduit modified with smooth muscle extracellular matrix, its preparation method, and its applications. This invention involves seeding smooth muscle cells onto the surface of a material and modifying it with a smooth muscle cell-derived extracellular matrix to prepare a functionalized nerve conduit. This nerve conduit significantly promotes axon regeneration in the dorsal root ganglion and PC12, and promotes angiogenesis by inducing Schwann cell migration and VEGF secretion, providing a guiding "bridge" for newly formed axons, reconnecting severed nerves, repairing large-segment defects of the sciatic nerve, and improving functional innervation and muscle function after nerve regeneration, demonstrating promising application prospects.

Claims

1. Use of a composite material for the preparation of a nerve conduit, characterized in that, The composite material is obtained by seeding smooth muscle cells on the surface of a material, culturing, decellularizing and molding.

2. Use according to claim 1, characterized in that, The smooth muscle cells are derived from intestinal, uterine, gastric, bladder or vascular smooth muscle; the material is selected from natural or synthetic materials; the smooth muscle cells are seeded at a density of 1 x 10 4 to 1 x 10 8 cells / cm 2 .

3. Use according to claim 2, characterized in that, The smooth muscle cells are seeded at a density of 1 x 10 5 1 x 10 7 cm 2 -2 cells / cm2.

4. Use 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.

5. Use according to claim 4, characterized in that, The artificial synthetic material is polyurethane or polylactic acid.

6. Use according to claim 1, characterized in that, The preparation method of the composite material comprises the following steps: (1) seeding smooth muscle cells on the surface of a material, culturing to obtain a cell-material composite; (2) decellularizing the cell-material composite to obtain a decellularized cell-material composite; (3) washing, molding and drying the decellularized cell-material composite.

7. Use according to claim 6, characterized in that, In step (1), the culturing time is 2-14 days.

8. Use according to claim 7, characterized in that, In step (1), the culturing time is 7 days.

9. Use according to claim 6, characterized in that, In step (2), the decellularization process comprises the following steps: repeatedly freezing and thawing the cell-material composite, then treating with a decellularization solution and washing.

10. Use according to claim 9, characterized in that, In step (2), the freezing and thawing conditions are: freezing at -100~-60℃ for 20~60 min, then thawing at 30~45℃ for 20~60 min; The decellularization solution is 1% Triton X-100; The treatment temperature is 30~45℃ and the treatment time is 1~10 min; The washing liquid is PBS.

11. Use according to claim 10, characterized in that, In step (2), the freezing and thawing conditions are: freezing at -80℃ for 40 min, then thawing at 37℃ for 40 min; The decellularization solution is 1% Triton X-100; The treatment temperature is 37℃ and the treatment time is 5 min.

12. Use according to claim 1, characterized in that, The nerve conduit is a nerve conduit for promoting Schwann cell migration and / or promoting angiogenesis.

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