Guiding dental pulp stem cell extracellular matrix nerve scaffold and preparation method thereof
Patent Information
- Application Number
- CN202511996641.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-27
- Publication Date
- 2026-03-17
Smart Images
Figure CN121668407A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomaterials technology, and in particular to a guided extracellular matrix neural scaffold for dental pulp stem cells and its preparation method. Background Technology
[0002] Peripheral nerve injury can lead to severe motor dysfunction, sensory deficits, and chronic complications, significantly reducing patients' quality of life. Despite progress in biomaterials development, numerous challenges remain in repairing long-segment nerve defects and regulating delicate muscle functions, including persistent pain and incomplete functional recovery. The transient nature of the Schwann cell repair phenotype is a key factor, limiting the effective time window for nerve repair. Premature fading of this phenotype is a major cause of neural regeneration failure in humans. Therefore, developing biomaterials that can stabilize and prolong the Schwann cell repair phenotype has become a key research focus in the field of neural tissue engineering.
[0003] The extracellular matrix (ECM) plays a crucial role in regulating cell fate. Through the interaction of bioactive ligands with cell surface receptors, the ECM can precisely regulate the differentiation, migration, and functional maturation of nerve cells. Therefore, biomimicry of the composition, mechanical properties, and spatial structure of the ECM is a research hotspot for regulating Schwann cell behavior and promoting nerve injury repair. Among numerous biomimetic ECM materials, fully-component decellularized matrix stands out due to its naturally occurring bioactive ligands and its regulatory effect on the microenvironment, achieving verifiable results in nerve injury repair. However, this material still faces the dual challenges of immune rejection and pathogen transmission risks, as well as the loss of structure and activity caused by the decellularization process. In contrast, decellularized matrix from in vitro expanded cells achieves pathogen-free production through simplified processing, better retains bioactive factors, thereby optimizing the regenerative microenvironment and supporting customized functions. Stem cell-derived ECM contains abundant and diverse regulatory proteins, which can mimic the natural stem cell microenvironment, providing multi-level and dynamic developmental signals, and has great application potential in regulating cell fate and tissue morphology. Dental pulp stem cells (DPSCs), derived from the neural crest, are homologous to Schwann cells and possess the potential to differentiate into them. Their secreted extracellular matrix (DPSC-ECM) not only exhibits excellent damage repair capabilities but also effectively regulates cell phenotype and promotes angiogenesis. These properties make DPSC-ECM an ideal candidate material for Schwann cell phenotype regulation and neural repair.
[0004] The effectiveness of nerve regeneration is closely related to the formation of an ordered nerve fiber structure. A lack of structural guidance leads to disordered extracellular matrix regeneration and low regeneration efficiency. Therefore, applying DPSC-ECM to nerve repair still requires structural remodeling and overcoming its insufficient mechanical properties. Summary of the Invention
[0005] This invention proposes a guided extracellular matrix neural scaffold for dental pulp stem cells and its preparation method, in order to solve the problems of existing DPSC-ECM lack of structure guidance leading to disordered extracellular matrix regeneration and low regeneration efficiency, and the fact that DPSC-ECM still needs to be remodeled when applied to nerve repair, resulting in insufficient mechanical properties.
[0006] In a first aspect, the present invention provides a method for preparing a guided extracellular matrix neural scaffold for dental pulp stem cells, the method comprising the following steps: Step S1: Culturing dental pulp stem cells and adding ascorbic acid to stimulate extracellular matrix secretion. After the culture is completed, decellularization is performed to obtain extracellular matrix-depleted DPSC-dECM derived from dental pulp stem cells. Step S2: Process DPSC-dECM into powder and dissolve it in a solvent to form a dECM suspension; dissolve polylactic acid-caprolactone copolymer (PLCL) in a solvent to form a PLCL solution; mix the dECM suspension and the PLCL solution to obtain an ECM / PLCL mixed solution. Step S3: Using electrospinning technology, firstly, using the ECM / PLCL mixed solution as the spinning solution, a DPSC-ECM membrane is spun on a collector and formed. The DPSC-ECM membrane is then rolled onto a mandrel to form the inner scaffold of the catheter. Next, using the PLCL solution as the spinning solution, a PLCL fiber layer is spun on the outer surface of the inner scaffold of the catheter to form a double-layer tubular scaffold precursor. Step S4: The double-layer tubular scaffold precursor is dried to obtain the DPSC-ECM bio-inducible neural conduit graft.
[0007] Furthermore, the specific method of step S1 includes: S1-1: Using primary human dental pulp stem cells (DPSCs) from the 2nd to 4th generation, when the DPSCs grow to 90% confluence in the culture dish, 50 μM ascorbate phosphate is added to the culture medium to continuously stimulate the cells to secrete extracellular matrix (ECM). S1-2: Under the above conditions, culture continuously for 14 days. If cell wrinkles and semi-transparent white membrane structures are found to appear at the edge of the culture dish, it indicates that ECM has been fully deposited and the culture can be terminated. S1-3: Use a cell scraper to physically scrape off the translucent white membrane structure formed in the culture dish, immerse the scraped membrane structure in a 0.5% Triton X-100 solution containing 20 mM ammonium hydroxide, and react at 37°C to remove cellular components; S1-4: After the decellularization reaction was completed, the membrane structure was thoroughly washed three times with phosphate-buffered saline (PBS) for 5 minutes each time to remove residual chemical reagents and cell debris, resulting in dental pulp stem cells (DPSC-dECM) without extracellular matrix. The DPSC-dECM was then placed in 1× PBS and frozen at -80°C for later use.
[0008] Furthermore, the preparation method of the dECM suspension in step S2 includes: (1) Homogenize the DPSC-dECM obtained in step S1 using a tissue homogenizer; (2) The homogenized sample was immediately frozen in liquid nitrogen and freeze-dried for 48 hours. The freeze-dried sample was then crushed into fine particles using an electric grinder, frozen again in liquid nitrogen, and freeze-dried for a second time for 48 hours. The resulting ECM powder was scraped and collected using a cell scraper. (3) Suspend it in hexafluoro-2-propanol HFIP and shake continuously at 4°C for 36 hours to fully dissolve it. Then dilute the solution with HFIP to prepare dECM suspensions with final concentrations of 35, 45, 55, 65 and 75 mg / mL. Before electrospinning, transfer the dECM suspension into a capped syringe and vortex to homogenize for 20 seconds.
[0009] Furthermore, the concentration of the dECM suspension is preferably 55 mg / mL.
[0010] Furthermore, the PLCL solution in step S2 is prepared by dissolving the PLCL polymer in HFIP and magnetically stirring at 4°C for 24 hours to obtain a 10% (w / v) PLCL solution.
[0011] Furthermore, the preparation method of the ECM / PLCL mixed solution in step S2 includes: (1) Mix 10% (w / v) PLCL solution with dECM suspension at a volume ratio of 1:9 and mix thoroughly under constant stirring; (2) Before electrospinning, transfer the ECM / PLCL mixed solution into a capped syringe and vortex to homogenize for 20 seconds.
[0012] Furthermore, the specific method of step S3 includes: S3-1: Preparation of the inner dECM electrospun film; (1) Load the dECM suspension into a 5 ml syringe, equip it with a 20 G metal needle, and spin it on a rotating drum collector; (2) The electrospinning parameters of the inner layer are: positive voltage 8 kV, negative voltage 3 kV, solution propulsion flow rate 0.2 mL / h, distance between needle tip and collector 12 cm, ambient temperature 20-25°C, needle height 34 cm, collector rotation speed 3000 rpm, and total spinning time 5 hours. (3) After electrospinning, peel one edge of the obtained film off the collector and fix it on a metal core rod with a diameter of 1 mm to ensure that the fibers are arranged parallel to the axis of the metal core rod; then rotate the metal core rod at a uniform speed so that the DPSC-ECM film evenly wraps its surface to form the inner support of the conduit. S3-2: Preparation of the outer PLCL reinforcement layer; (1) A 10% PLCL solution was used to deposit it outside the inner scaffold; (2) The electrospinning parameters of the outer layer are: positive voltage 10 kV, negative voltage 3 kV, solution propulsion flow rate 0.4 mL / h, distance between needle tip and collector 12 cm, ambient temperature 20-25°C, needle height 34 cm, collector rotation speed 70 rpm, and spinning time 0.5 hours. (3) After electrospinning is completed, a PLCL fiber layer is spun on the outer surface of the inner layer of the catheter to obtain a double-layer tubular stent precursor.
[0013] Secondly, the present invention also provides a guided extracellular matrix neural scaffold for dental pulp stem cells. The extracellular matrix neural scaffold is a double-layered tubular structure. The inner layer is formed by electrospinning a composite extracellular matrix of dental pulp stem cells and polylactic acid-caprolactone copolymer. The inner layer is composed of oriented fibers parallel to the duct axis. The outer layer covers the outer surface of the inner layer and is composed of randomly oriented fibers formed by electrospinning polylactic acid-caprolactone copolymer.
[0014] Compared with the prior art, the present invention has the following advantages: 1. Integration of Bioactivity and Physical Guidance: This invention uses DPSC-ECM / PLCL composite fibers with inner-layer orientation as the core. It not only fully preserves the rich bioactive components (such as laminin and collagen) in the extracellular matrix of dental pulp stem cells, providing Schwann cells with direct adhesion, proliferation, and phenotypic regulatory signals, but also effectively guides the directional migration and extension of Schwann cells and axons through its highly parallel nanofiber topology. This achieves a highly efficient synergy between "biochemical signals" and "physical contact guidance," solving the problem of insufficient guidance in traditional materials.
[0015] 2. Structural Biomimicry and Mechanical Performance Optimization: By constructing a double-layered tubular structure with an "inner-oriented / outer-random" design, the hierarchical organization of the natural nerve's "basal membrane-outer membrane" is precisely simulated. The inner-oriented structure provides guidance, while the outer random PLCL fiber layer provides excellent mechanical support and surgical operability. This gives the scaffold overall flexibility and compressive strength comparable to autologous nerves, avoiding the risk of post-implantation collapse and overcoming the poor mechanical properties of pure ECM materials.
[0016] 3. Controllable preparation process and good activity retention: The electrospinning process parameters (such as voltage, flow rate, and receiving rotation speed) used in this invention have been optimized to stably prepare inner membranes with uniform fiber diameter and high orientation. Crucially, this gentle processing method has been proven to maximize the retention of active proteins in DPSC-ECM, ensuring the biological function of the final product. The unique "secondary freeze-drying and grinding" pretreatment process also ensures the uniform dispersion and complete dissolution of ECM components in the spinning solution.
[0017] 4. Promoting the Construction of a Neural Regeneration Microenvironment: Experiments have shown that this scaffold can not only directly regulate the transformation of Schwann cells to a repair phenotype and activate their autophagy, but also synergize with Schwann cells to significantly promote angiogenesis, establishing a crucial nutrient delivery network for the early stages of neural regeneration. This multi-functional support for "cell phenotype regulation," "axonal guidance," and "angiogenesis" creates a more complete neural regeneration microenvironment.
[0018] 5. Significant repair effect, functional recovery close to that of autologous nerves: In a rat model of long-segment facial nerve defects, the nerve scaffold prepared in this invention showed repair effects similar to autologous nerve transplantation in terms of axonal regeneration density, myelin sheath thickness, nerve electrophysiological conduction function, and final motor function recovery. It is significantly better than pure synthetic material catheters, providing a highly promising new solution for the clinical repair of long-segment peripheral nerve defects.
[0019] Based on the implementation methods provided in the above aspects, this application can be further combined to provide more implementation methods. Attached Figure Description
[0020] The above and other objects, features, and advantages of exemplary embodiments of the present invention will become readily apparent upon reading the following detailed description with reference to the accompanying drawings. In the drawings, several embodiments of the invention are illustrated by way of example and not limitation, with the same or corresponding reference numerals denoteing the same or corresponding parts, wherein: Figure 1 The diagram illustrates the preparation process and main mechanism of action of DPSC-ECM bioguided neural grafts provided by this invention. Figure 2 The preparation and characterization diagram of DBNG provided by this invention; Figure 3 A biocompatibility evaluation diagram of DBNG provided by the present invention; Figure 4 This is a diagram illustrating the regulatory effect of DBNG on Schwann cells and axonal function provided by the present invention. Figure 5 The DBNG-induced angiogenesis diagram provided by this invention; Figure 6 This invention provides an image illustrating how DBNG promotes facial nerve injury repair in vivo. Figure 7 The rat facial nerve function recovery evaluation diagram provided by this invention; Figure 8 The image shows the effect of DBNG on facial nerve remyelination and functional recovery provided by this invention. Detailed Implementation
[0021] The exemplary embodiments disclosed in this application will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of this application are shown in the drawings, it should be understood that this application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of this application and to fully convey the scope of this application to those skilled in the art. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.
[0022] The effectiveness of nerve regeneration is closely related to the formation of an ordered nerve fiber structure; a lack of structural guidance leads to disordered extracellular matrix regeneration and low regeneration efficiency. Therefore, applying DPSC-ECM to nerve repair still requires structural remodeling and overcoming insufficient mechanical properties. Electrospinning technology can prepare fiber scaffolds with fiber diameters and porous mesh structures highly similar to those of natural ECM extracellular matrix fibers, which is an important means of reconstructing the three-dimensional biomimetic structure of the extracellular matrix. Poly(L-lactide-co-ε-caprolactone) (PLCL) has good biocompatibility, biodegradability, and mechanical properties, and has been commercially used in nerve repair products. Therefore, it has great potential in assisting DPSC-ECM reconstruction and maintaining its mechanical properties.
[0023] Based on the above needs, we developed a bioguided material integrating mechanical and biological signals. This material provides a microenvironment conducive to maintaining the Schwann cell phenotype by pre-constructing an early extracellular matrix structure. Using electrospinning technology, we processed DPSC-ECM into oriented structures and fabricated a tubular scaffold with an outer PLCL layer, ultimately forming a DPSC-ECM bioinstructive nerve graft (DBNG). In vitro experiments showed that this graft provides a scaffold for axial migration and extension of Schwann cells, while effectively enhancing cell secretion, adhesion, and autophagy. Furthermore, in terms of angiogenesis, DBNG exhibits a synergistic promoting effect with the Schwann cell repair phenotype. In the repair of long-segment facial nerve defects, DBNG effectively promotes early angiogenesis and maintains the Schwann cell repair phenotype, and significantly promotes axonal regeneration, ultimately achieving effective functional recovery. Mechanistic studies showed that DBNG activates the JAK / STAT signaling pathway, a key pathway essential for stabilizing the Schwann cell repair phenotype. These findings demonstrate that DBNG can both adapt to extracellular matrix remodeling after injury and support early angiogenesis and long-term Schwann cell repair phenotypes, highlighting its great potential in peripheral nerve regeneration therapy.
[0024] Combination Figures 1 to 8 As shown, an optional embodiment of the present invention provides a method for preparing a guided extracellular matrix neural scaffold for dental pulp stem cells, the specific steps of which include: 1. Materials used: The poly(L-lactic acid-co-ε-caprolactone) (PLCL, lactic acid:caprolactone = 50:50, molecular weight: 1.10 × 10⁻⁶) used in this study 5 The intrinsic viscosity (IV): 2.0-3.0 was purchased from Jinan Dagang Biotechnology Co., Ltd., China. Hexafluoro-2-propanol (HFIP, >99% purity), ascorbic acid, ammonium hydroxide, and Triton X-100 were purchased from Sigma-Aldrich, USA. Duchenne Modified Eagle Medium (DMEM), α-Minimum Essential Medium (α-MEM), penicillin / streptomycin, fetal bovine serum (FBS), and 0.25% trypsin-EDTA (containing phenol red) were all purchased from Gibco, USA.
[0025] 2. Preparation of DPSC-derived extracellular matrix; Extracellular matrix-depleted human dental pulp stem cells (DPSC-dECM) were prepared from primary human dental pulp stem cells (DPSCs) of passages 2 to 4 (P2-P4). Briefly, when cells reached approximately 90% confluence under optimal growth conditions, 50 μM ascorbate phosphate was added to the culture medium to stimulate extracellular matrix (ECM) secretion. After 14 days of continuous culture, culture was terminated when cell folds and membrane-like structures were observed at the edge of the culture dish. The translucent white membrane-like structure was carefully scraped and immersed in 0.5% Triton X-100 solution containing 20 mM ammonium hydroxide for decellularization at 37°C. Subsequently, it was washed three times with phosphate-buffered saline (PBS) for 5 minutes each time. The resulting DPSC-dECM was stored in 1×PBS and frozen at -80°C for later use.
[0026] 3. The decellularization efficiency was assessed by 4′,6-diamidinyl-2-phenylindole (DAPI) staining and residual DNA quantification. Specific procedures were performed according to the relevant kit instructions. DNA was extracted from DPSC cell slices and DPSC-dECM using a genomic DNA extraction kit from Takara (China), and the concentration was quantified using a Thermo Fisher Scientific (USA) NanoDrop 2000c microspectrophotometer.
[0027] 4. Preparation of electrospinning solution; (1) Preparation of dECM suspension; The obtained DPSC-dECM was homogenized using a tissue homogenizer (Bioprep-24, China). The homogenized sample was immediately flash-frozen in liquid nitrogen and freeze-dried for 48 hours. The freeze-dried sample was then pulverized into fine particles using an electric grinder (Siemens, Germany), flash-frozen again in liquid nitrogen, and freeze-dried a second time for 48 hours. The resulting ECM powder was carefully scraped and collected using a cell scraper and suspended in hexafluoro-2-propanol (HFIP), and continuously shaken at 4°C for 36 hours to ensure complete dissolution. This solution was then diluted with HFIP to prepare dECM suspensions with final concentrations of 35, 45, 55, 65, and 75 mg / mL. Before electrospinning, the dECM suspensions were transferred into capped syringes and homogenized by vortexing for 20 seconds.
[0028] (2) Preparation of PLCL solution: Dissolve PLCL polymer in HFIP and stir magnetically at 4°C for 24 hours to prepare a 10% (w / v) PLCL electrospinning solution.
[0029] (3) Preparation of ECM / PLCL mixed solution; A 10% (w / v) PLCL solution was mixed with a 55 mg / mL dECM suspension at a volume ratio of 1:9 and thoroughly mixed under constant stirring. Before electrospinning, the ECM / PLCL mixture was transferred into a capped syringe and vortexed for 20 seconds to homogenize.
[0030] 5. Preparation of DPSC-ECM bio-inducible neural conduit grafts (DBNG); A double-layered nerve conduit graft was prepared using electrospinning technology.
[0031] (1) Preparation of the inner layer (dECM electrospun film); The electrospinning solution was loaded into a 5 mL syringe equipped with a 20G metal needle, and spinning was performed on a rotating drum collector. The optimized electrospinning parameters for the inner layer were: positive voltage 8 kV, negative voltage 3 kV, solution flow rate 0.2 mL / h, needle tip-to-collector distance 12 cm, ambient temperature 20-25°C, needle height 34 cm, collector rotation speed 3000 rpm, and total spinning time 5 hours. After electrospinning, the resulting film was carefully peeled off one edge from the collector and fixed to a 1 mm diameter metal mandrel, ensuring the fibers were aligned parallel to the mandrel axis. The mandrel was then rotated at a constant speed to uniformly coat the DPSC-ECM membrane onto its surface, forming the inner scaffold of the conduit.
[0032] (2) Preparation of the outer layer (PLCL reinforcement layer); A 10% PLCL solution was deposited on the outer layer of the scaffold using modified electrospinning parameters. The outer layer parameters were adjusted as follows: positive voltage 10 kV, negative voltage 3 kV, flow rate increased to 0.4 mL / h (receiving distance maintained at 12 cm), ambient temperature 20-25°C, needle height 34 cm, collector rotation speed reduced to 70 rpm, and spinning time shortened to 0.5 hours. Finally, the resulting bilayer conduit structure was placed in a vacuum environment and freeze-dried overnight to obtain the DPSC-ECM bio-inducible neural conduit graft.
[0033] Inspired by the hierarchical structure and functional synergy mechanism of the peripheral nerve basement membrane, this study designed a DBNG neural repair scaffold with an inner bioactive guiding layer and an outer mechanical support layer. The complete fabrication process is as follows: Figure 1As shown, the inner layer of oriented DPSC-ECM nanofibers mimics the topology of the nerve basement membrane. The parallel fiber orientation can regulate Schwann cell migration and axonal growth direction through contact guidance. Simultaneously, the collagen, laminin, and other bioactive components retained in the DPSC-ECM provide signals for cell adhesion and proliferation. The outer layer of randomly arranged PLCL nanofibers mimics the mechanical protective function of the nerve epineurium. The excellent mechanical properties of PLCL improve the surgical operability of the scaffold, while its biodegradable nature avoids the risk of secondary surgical removal. The specific preparation process and characterization results are as follows: Figure 2 As shown in a, DPSCs were isolated from the patient's extracted permanent teeth, and after 14 days of ascorbic acid-induced culture, decellularization was performed to remove cellular components while retaining natural extracellular matrix components.
[0034] Studies have shown that at a concentration of 55 mg / mL, smooth and continuous oriented fibers with uniform diameter (903.54±117.48 nm) were successfully prepared, with no obvious microbead formation and high consistency in fiber orientation. When the concentration exceeds 65 mg / mL, the fiber diameter increases significantly, and excessive accumulation leads to uneven local fiber distribution. Therefore, 55 mg / mL was selected as the optimal spinning concentration.
[0035] The orientation of nanofibers is crucial for axially guiding Schwann cells and promoting the directional regeneration of neural bridges. This study employed a "DPSC-ECM / PLCL blend" strategy to synergistically optimize fiber orientation, mechanical properties, and bioactivity. Specific characterization results are as follows: The microstructures of pure PLCL fibers, DPSC-ECM / PLCL blend fibers, and pure DPSC-ECM fibers were observed using scanning electron microscopy (SEM). Figure 2 As shown in b, the results indicate that all three groups of fibers exhibit a uniform and continuous filamentous structure, without obvious agglomeration, breakage, or microbead formation, confirming that the blend system has good spinning compatibility. The morphology of the three materials after swelling in PBS was observed using atomic force microscopy (AFM), as shown below. Figure 2 As shown in b, the results indicate that all fibers maintained an intact filamentous structure without significant dissolution or collapse, confirming their good hydration stability. Quantitative analysis of fiber diameter showed that the average diameter of the DPSC-ECM / PLCL blend fibers was 394.79 ± 17.92 nm, which highly matches the reported optimal fiber diameter of 400 nm for promoting cell migration. This size provides sufficient adhesion area for SCs while reducing the problem of insufficient mechanical strength caused by excessively fine fibers. Further evaluation of orientation uniformity was conducted by measuring the fiber deflection angle (±90°). Figure 2As shown in Figure c: the pure PLCL group exhibits the best orientation due to its high polymer chain entanglement, with 88.05% of fibers neatly arranged within a ±20° range. The pure DPSC-ECM group, however, suffers from poor ECM spinning properties, with only 49.51% of ordered fibers within a ±20° range, resulting in dispersed orientation. In the DPSC-ECM / PLCL blend, the addition of PLCL enhances intermolecular interactions and reduces fiber breakage during spinning, increasing the proportion of ordered fibers within a ±20° range to 82.84%, significantly better than the pure ECM group. This demonstrates that PLCL can effectively regulate the orientation of ECM-based fibers, providing a structural basis for Schwann cell directed migration. The mechanical properties of individual fibers were measured using AFM nanoindentation technology. The results showed that the elastic modulus of the DPSC-ECM / PLCL blend fibers, within the range of 0-140 MPa, was significantly lower than that of pure PLCL fibers (180-250 MPa), falling within a suitable range for cell adhesion and migration. Basement membrane components in the extracellular matrix (such as type IV collagen, fibronectin, and laminin) are core functional targets regulating Schwann cell adhesion, polarization, and myelin formation; their expression and activity directly determine the bioregulatory efficacy of the scaffold. To verify the bioactivity of DPSC-ECM / PLCL blended fibers, immunofluorescence staining was used to label LA, FN, COL4, and COL1, such as... Figure 2 As shown in Figure j, the results indicated that all four proteins were positively expressed on the surface of the blended fibers and were evenly distributed, confirming that the electrospinning process did not destroy the bioactive components in the DPSC-ECM and preserved the key ligand signals for Schwann cell function regulation. The scaffold hydrophilicity directly affects cell adhesion efficiency and nutrient transport rate. The surface hydrophilicity of the three materials was assessed by water contact angle measurement, such as... Figure 2 As shown, the results indicate that the water contact angle of the pure DPSC-ECM scaffold is 10.48±0.86°, exhibiting a superhydrophilic state, which may be attributed to the abundance of hydrophilic groups in ECM. The water contact angle of the DPSC-ECM / PLCL blend scaffold is 13.80±0.62°, slightly higher than that of the pure ECM group, but still significantly lower than that of the pure PLCL scaffold (114.45±1.23°), confirming that the addition of PLCL did not significantly weaken the overall hydrophilicity of the scaffold.
[0036] The PLCL / DPSC-ECM oriented film was rolled into a tube as the inner layer, and then randomly arranged PLCL fibers were deposited on the outer layer by electrospinning to form a bilayer composite structure (DBNG). The physical appearance of the DBNG conduit and the pure PLCL nanofiber conduit are shown below. Figure 2As shown in f, DBNG exhibits good flexibility, with no obvious breakage or wrinkling during bending, indicating its excellent bending toughness. This allows it to adapt to the complex anatomical environment within the body, avoiding structural damage caused by mechanical brittleness during surgical implantation. Furthermore, intraoperative suture images are shown below. Figure 2 The g-scan showed that the diameter of the DBNG conduit perfectly matched that of the facial nerve, demonstrating the plasticity advantage of electrospinning technology in conduit size control, which can meet the size requirements of different nerve defect repairs. HE staining of the DBNG conduit cross-section was performed as follows: Figure 2 As shown in h, the results showed that the inner layer exhibited uniform layered eosinophilic staining without blue hematoxylin staining, further confirming the effectiveness of the decellularization treatment. The inner layer fibers were arranged in a tight and orderly manner, while the outer layer fibers were randomly distributed evenly, and the boundary between the two layers was clear.
[0037] The cross-section and surface morphology of the DBNG catheter were observed using scanning electron microscopy (SEM), such as... Figure 2 As shown in i, the results reveal a typical bilayer structure: the inner layer consists of PLCL / DPSC-ECM oriented fibers, arranged orderly along the duct axis, mimicking the bundle-like structure of natural nerves and providing directional guidance for axonal growth; the outer layer is formed by randomly distributed stacked PLCL fibers, creating micropores with a diameter of approximately 2.84 μm between the fibers. The overall porosity of the DBNG duct is 19.25 ± 0.78%. This porous structure ensures effective exchange of nutrients and metabolic waste while providing channels for cell migration, avoiding cell survival limitations caused by dense structures.
[0038] The mechanical properties of the DBNG catheter were evaluated using a universal testing machine, and the results showed that... Figure 2 As shown in m and n: the tensile strength of the inner PLCL / DPSC-ECM oriented membrane is 2.80±0.52 MPa, and the tensile strength of the outer random PLCL fiber layer is 4.70±2.08 MPa. The overall mechanical properties are comparable to those of autologous nerves, providing stable structural support during nerve regeneration and preventing catheter collapse due to muscle compression or tissue movement. Furthermore, cyclic compression tests showed that the stress-strain curve of the DBNG catheter did not deviate significantly under repeated compression loads, confirming its good mechanical stability and fatigue resistance. The DBNG catheter and pure PLCL catheter were immersed in PBS at 37°C, and their degradation behavior was monitored. No significant difference was observed in the degradation rate of the two scaffolds within 3 months. Moreover, throughout the degradation process, the pH value of the immersion solution for both materials remained between 7.2 and 7.4 without significant decrease, confirming that there was no significant accumulation of acidic substances in the degradation products, thus avoiding inflammatory reactions or cytotoxicity caused by local pH reduction. The ECM protein retention of the DBNG catheter during the 12-week degradation process was detected by the BCA method. Figure 2As shown in Figure 1, the results showed that the ECM protein content gradually decreased with degradation time, with the protein content in week 4 being significantly lower than that in week 2 (p<0.01), indicating that the ECM protein release rate was high in the early stage, which could quickly provide bioactive signals to the injury site; by week 12, the DBNG catheter could still retain a protein content of 147.42±5.72 μg / mg, confirming its ability to continuously release bioactive components, which can provide long-term support for axonal myelination in the later stage of nerve regeneration.
[0039] Figure 2 In the figure, Figure a shows a schematic diagram of the preparation of DPSC-ECM electrospun nanofibers; Figure b shows scanning electron microscope (SEM) images and pressure atomic force microscope (AFM) three-dimensional morphology images of PLCL, ECM / PLCL, and DPSC-ECM nanofibers, demonstrating the surface structure of the nanofibers after swelling; Figure c shows the histogram of fiber orientation angle frequency distribution; Figure d shows the elastic modulus measurement results of nanofibers with different compositions; Figure e shows the surface wettability assessment by contact angle measurement; Figure f shows the macroscopic morphology of DBNG, demonstrating its structural stability under bending conditions; Figure g shows an intraoperative photograph of DBNG; Figure h shows the HE staining results of DBNG; Figure i shows a cross-sectional image of scanning electron microscope (SEM) revealing a bilayer structure, with the inner layer of neatly arranged layered microstructures guiding axon growth, and the outer layer having interconnected pores (porosity: 19.25). ±0.78%) promotes nutrient exchange; Figure j shows immunofluorescence staining of key extracellular matrix components of the electrospun membrane; the in vitro degradation curve shows the mass retention rate (%) and pH value (n=3) in PBS within 12 weeks; Figure l shows the quantitative analysis of protein content changes during degradation; Figure m shows the tensile test: (left) schematic diagram of the tensile strength measurement device. (right) tensile stress-strain curves of PLCL catheter and DBNG. Figure n shows the compression test: (left) nerve growth cone (NGC) shows complete structural recovery after 20 compression cycles at 35% strain. (right) comparison of compression stress-strain curves of PLCL catheter and DBNG; Figure o shows the statistical comparison of mechanical properties of PLCL catheter and DBNG.
[0040] Biocompatibility is a core prerequisite for the application of tissue engineering materials in vivo. This study systematically evaluated the effects of DBNG on the survival, morphology, proliferation, and migration of Schwann cells through live / dead cell staining and CCK-8 proliferation assays, cytoskeleton staining, and migration assays, thus verifying its biosafety and cell compatibility. Figure 3a) Results: RSC96 cells were labeled using the Calcein-AM / PI double staining method. The survival status and morphological characteristics of cells after 3 days of culture on four scaffolds (blank control group, pure PLCL group, pure DPSC-ECM group, and DPSC-ECM / PLCL blend group) were observed using confocal laser scanning microscopy. Only dense green fluorescence signals were observed on the surfaces of all four scaffolds; no obvious red dead cell signals were detected, indicating that all scaffolds had no significant cytotoxicity and could support the stable survival of RSC96 cells. Quantitative analysis of the proportion of viable cells showed no statistically significant difference among the four groups (p>0.05), further confirming that the components of DBNG (DPSC-ECM and PLCL) and the preparation process (electrospinning, tubular forming) did not introduce toxic substances and would not induce cell apoptosis. To further verify the effect of DBNG on the proliferation capacity of RSC96 cells, the scaffold extract culture method was used, and cell proliferation was detected at 24h, 48h, and 72h using the CCK-8 assay. Normal culture medium without scaffold extract was used as a blank control. Figure 3 As shown in c. The results showed that the cell proliferation curves of all groups (including the blank control) showed a significant upward trend with the extension of culture time. Cell viability continued to increase within 72 hours. This result indicates that the addition of DPSC-ECM did not introduce inhibitory components that affect cell proliferation, and the mechanical support function of PLCL did not have a negative impact on cell metabolism. All three scaffolds had good proliferation compatibility.
[0041] Phalloid peptide staining Figure 3 As shown in Figure d, there were significant differences in the morphology of RSC96 cells on different scaffold surfaces: In the DPSC-ECM / PLCL blend group, cells exhibited a typical long spindle shape and were regularly arranged along the fiber axis. The angle between the long axis of the cells and the fiber direction was less than 15°, forming a continuous "cell chain" structure, which highly matched the morphology of the natural Bungarus bands. In contrast, in the pure DPSC-ECM group, cells were spindle-shaped, but the arrangement was still locally disordered, with some cells growing along non-axial directions. In the PLCL group and the blank control group, cells were round or short spindle-shaped, with no obvious directionality in their arrangement, and the intercellular connections were loose, failing to form a continuous cell layer. This demonstrates the ability of the DPSC-ECM / PLCL blend scaffold to synergistically regulate the axial alignment of Schwann cells through "topological structure + biological signal".
[0042] The effects of different scaffolds on the migration ability of RSC96 cells were evaluated using Transwell migration assays and scratch healing assays. Figure 3As shown in Figure j, the migration rate of the DPSC-ECM / PLCL group was 55.37 ± 6.92%, significantly higher than that of the PLCL group and similar to that of the pure DPSC-ECM group (p>0.05), indicating that the blended fibers can significantly enhance the migration ability of Schwann cells. The scratch healing experiment showed that after cells were cultured on the scaffold surface for 24 h, the migration distance of the DPSC-ECM / PLCL group was 312.27 ± 10.65 μm, significantly higher than that of the PLCL group (p<0.001) and the pure DPSC-ECM group (p<0.05), and the cells migrated directionally along the scratch direction, forming a continuous cell covering layer.
[0043] The transformation of Schwann cells to a repair phenotype and autophagy activation are core components of peripheral nerve regeneration, while the directional elongation of axons relies on the synergistic guidance of the "Schwann cell microenvironment + scaffold topology." This study analyzed the regulatory effects of scaffold materials on Schwann cells and neurons through co-culturing, and analyzed intercellular crosstalk through co-culturing Schwann cells and neurons, systematically revealing the mechanism by which DBNG regulates Schwann cell function and guides axonal regeneration. Figure 4 As shown in Figure a, this study constructed a Transwell co-culture system with RSC96+DBNG / PLCL in the upper chamber and PC12 in the lower chamber to simulate Schwann cell-axon interaction in vivo. Immunofluorescence staining showed that the fluorescence intensity of c-Jun in RSC96 cells of the DBNG group in the upper chamber was significantly higher than that in the PLCL group (p<0.01), and the cells exhibited a long spindle-shaped distribution, consistent with the morphology of the repair phenotype. Figure 4 As shown in b; the fluorescence intensity of neurofilament protein NF200 in PC12 cells of the lower chamber was significantly higher than that in the PLCL co-culture group and the blank control group (p<0.001). Figure 4 As shown in b. The supernatant of the Transwell co-culture system was extracted for ELISA detection as follows. Figure 4 The results showed that the levels of NGF and BDNF in the DBNG group were (434.28 ± 15.23) pg / mL and (134.41 ± 10.73) pg / mL, respectively, which were significantly higher than those in the Control group and the PLCL group (p<0.05). The level of VEGF secretion was (660.72 ± 20.00) pg / mL, which was significantly higher than that in the Control group (p<0.05). These results were consistent with the qRT-PCR results, indicating that DBNG can promote the secretion of functional factors by Schwann cells and provide nutritional support for axon regeneration.
[0044] The expression levels of transcriptional regulatory factors and cytokines in each experimental group were detected using quantitative real-time PCR (qRT-PCR). Figure 4As shown in f, compared with the blank control group and the PLCL group, the mRNA expression levels of c-Jun, SOX2, NGF, VEGF, TGF-β, and IL-6 in RSC96 cells of the DBNG group were significantly increased. Although the mRNA expression level of BNGF did not show a statistically significant difference compared with the PLCL group, it was still significantly higher than that of the blank control group.
[0045] Western blot analysis of autophagy-related markers revealed that, compared with the control group and PLCL group, the DBNG group showed significantly higher protein expression levels of Beclin1 (an autophagy initiation marker) and LC3B (an autophagosome formation marker) in RSC96 cells (p<0.05); and decreased protein expression of p62 (an autophagy substrate) (p<0.05). After treatment with the autophagy inhibitor chloroquine (CQ), the DBNG group maintained high Beclin1 expression and low p62 expression, with only a slight increase in LC3B due to inhibited autophagosome degradation. This confirms that DBNG's activation of autophagy does not depend on downstream autophagy pathways but directly initiates the autophagy pathway, further supporting Schwann cell repair phenotypic transformation. Western blot analysis of c-Jun protein expression in Schwann cells co-cultured with DBNG and PLCL showed that c-Jun expression in the DBNG group was significantly higher than that in the control group (p<0.05). P <0.01). The protein expression level of N-cadherin, a key molecule in cell adhesion, was increased compared with that in the PLCL group (p<0.001), confirming that DBNG can enhance Schwann cell adhesion.
[0046] The effects of four scaffolds (blank control, randomized PLCL, directed PLCL, randomized ECM / PLCL, and directed ECM / PLCL) on PC12 cell arrangement and axon length were compared as follows: Figure 4 As shown in j and 4k, PC12 cells in the blank control group were randomly distributed with no axonal orientation; while cells in all four scaffold groups were arranged along the fiber direction. The cell arrangement consistency in the A-ECM / PLCL group was significantly higher than that in the A-PLCL group, confirming that ECM components can enhance the topological guidance effect of fibers on cells. Furthermore, the average axon length of PC12 cells in the A-ECM / PLCL group was significantly longer than that in the A-PLCL group (p<0.01) and the random ECM / PLCL group (p<0.01). These results indicate that DBNG can not only create a favorable microenvironment by regulating Schwann cell phenotype and autophagy function, but also guide axonal directional extension through oriented fiber structure, providing a synergistic "cell-scaffold" solution for long-distance neural defect repair.
[0047] Angiogenesis is a core component of early repair of peripheral nerve injury. New blood vessels not only provide "nutrient channels" for nerve bridge formation and improve the local hypoxic microenvironment, but also prevent central nervous system necrosis in the injured area, laying a structural foundation for Schwann cell migration and axonal extension. This study systematically investigated the regulatory role of DBNG catheters in angiogenesis and the "material-cell" synergistic mechanism through in vitro cell experiments and in vivo chicken chorionic villus allantoic membrane experiments. Figure 5 As shown in Figure a, we prepared extracts of PLCL and DBNG materials, respectively, and co-cultured them with Schwann cells to obtain Schwann cell conditioned medium. First, we examined the effect of these materials on the migration ability of human umbilical vein endothelial cells (HUVECs). In Transwell experiments, although the mean number of migrating cells in the DBNG group was higher than that in the PLCL group, the difference between the two groups was not statistically significant, indicating that the growth factors released by the material extracts alone were insufficient to affect the migration behavior of vascular endothelial cells. When the materials were co-cultured with Schwann cells, the number of migrating cells increased significantly, possibly due to factors secreted by Schwann cells. The number of migrating cells in the DBNG-SCs group was significantly higher than that in the DBNG and PLCL-SCs groups, as shown in Figure a. Figure 5 As shown in b and 5c, the regulatory effect of DBNG on angiogenesis may stem from the synergistic effect of DPSC-ECM components and Schwann cells. Wound healing experiments also exhibited a similar trend. Figure 5 As shown in d and 5e. Furthermore, we used Matrigel medium to assess the tubular formation capacity of human umbilical vein endothelial cells (HUVECs), a model that simulates the morphological changes of endothelial cells during angiogenesis. Figure 5 As shown in f-5j, compared with the PLCL group, the DBNG group showed a significant increase in quantitative indicators such as the number and length of tubular formations, indicating that the DBNG extract contains bioactive substances that promote angiogenesis and help construct an early injury microenvironment. The number and length of blood vessels in the DBNG-SCs group were significantly higher than in all other groups, further confirming the synergistic pro-angiogenic effect of Schwann cells and DPSC-ECM components. Since the angiogenesis assay can only assess the released products, and to more reliably assess the pro-angiogenic potential of the material, we used a chicken chorionic allantoic membrane assay under artificial matrix confinement conditions. Figure 5 As shown in k, after 10 days of embryo culture, PLCL and DBNG catheters were placed on CAM and incubated for 4 days before harvesting. Analysis was performed by statistically analyzing the number and diameter of infiltrating vessels within the material, such as... Figure 5 The results showed that the number and diameter of vessels penetrating DBNG were significantly higher than those in PLCL (p<0.05). Figure 5As shown in m and 5n. This may be attributed to the abundance of angiogenesis-supporting bioactive factors in the inner layer of DBNG, DPSC-ECM. HE staining of ducts incubated on CAM revealed that although PLCL ducts have a porous structure, no endovascularity was observed; only a few vessels were visible at the hollow interface with CAM. In contrast, the inner layer of DBNG, with its bioactively oriented microlayer structure, not only showed cell infiltration but also the presence of microvessels, which is crucial for obtaining adequate blood supply in the early stages of neurogenesis. Figure 5 o was implemented.
[0048] To investigate the neural repair capacity of DBNG, this study established a rat facial nerve defect model with an 8 mm defect for in vivo experiments.
[0049] The experimental design and timeline are detailed in Figure 6a. During tissue collection, it was observed that all nerve grafts effectively bridged the facial nerve defect, restoring nerve continuity, and only slight adhesions were observed on the surface of each graft. Figure 6 b.
[0050] This study further analyzed the axonal regeneration and remyelination of facial nerve defects in rats at 8 and 12 weeks post-surgery using immunofluorescence staining, examining both transverse and longitudinal sections to verify the repair effect of DBNG. The results showed significant differences in the distribution of positive areas among the three scaffolds at each time point: at 8 weeks post-surgery, the area of the positive Tuj-1 regeneration mid-segment in the DBNG group (8.95 ± 5.02%) was significantly higher than that in the pure PLCL group, but not statistically different from the autologous nerve group (p>0.05). The area of the positive area in the distal regeneration segment (7.19 ± 0.59%) was significantly higher than that in the pure PLCL group, but still lower than that in the autologous nerve group (p<0.05). In the pure PLCL group, the Tuj-1 positive signal was scattered and punctate, without obvious continuous axonal bundle formation; S-100 positive cells were mostly clustered at the duct margins, failing to form a ring structure around the axon, and the positive area contained numerous blank gaps, suggesting incomplete myelination. At 12 weeks post-operation, the area of Tuj-1 positive region in the DBNG group further increased, significantly exceeding that in the pure PLCL group, with no statistically significant difference compared to the autologous nerve group in the mid-segment and distal segments (p>0.05). Analysis of the S100 positive area showed that the area of S100 positive region in the DBNG group was significantly higher than that in the pure PLCL group at all time points, with no statistically significant difference compared to the autologous nerve group. Longitudinal sections of the regenerated facial nerve were stained with Tuj-1 / S-100 immunofluorescence to observe the ability of nerve fibers to traverse the defect area. In both the sham surgery group and the autologous nerve group, numerous continuous Tuj-1 positive nerve bundles were observed traversing the defect area longitudinally, with the fiber direction consistent with the long axis of the nerve. The DBNG group showed a significantly higher number of highly oriented Tuj-1 positive nerve fibers traversing the defect area compared to the PLCL group (p<0.01), while the PLCL scaffold group only had a small number of scattered Tuj-1 positive fibers growing proximally in the defect area, without forming continuous traversing fiber bundles. The above results confirm that DBNG can form a functional "axon-myelin" complex structure in long facial nerve defects, and its distribution characteristics are highly similar to those of autologous nerves.
[0051] To assess the functional recovery of facial nerve injury in rats, this study evaluated beard movement ability at various postoperative repair stages. Results showed that after scaffold repair in each group, beard movement function on the experimental side of the rats with facial nerve defects gradually recovered. Specifically, the DBNG group and the autologous transplantation group showed similar cheek movement amplitudes at each time point, with no significant differences observed (p>0.05). Figure 7 As shown, DBNG effectively promoted the establishment of nerve reinnervation and achieved good facial muscle function recovery. To assess the recovery of conduction function in the newly formed nerves, compound muscle action potential (CMAP) recordings were performed on the buccal branch of the repaired facial nerve. Figure 7 As shown.
[0052] Statistical analysis of CMAP waveforms at 12 weeks post-surgery showed no statistically significant difference in CMAP amplitude between the DBNG group and the autologous nerve group. Figure 8 As shown in c, the amplitude was significantly higher than that of the PLCL group. The latency among the groups also showed a similar trend; the conduction latency of the DBNG group was close to that of the autoneuron, and both were significantly lower than those of the PLCL group (p<0.001). This indicates that the neural conduction function of the DBNG group was better restored. Figure 8 As shown in d. Muscle denervation leads to tissue degeneration and loss of function. Compared to the healthy side, the buccal nerve muscles on the injured side showed varying degrees of atrophy, accompanied by a decrease in the cross-sectional area of muscle fibers and collagen fiber accumulation. To assess muscle recovery, HE staining and Masson staining were performed on the muscle fibers in the facial nerve innervation area, as shown in d. Figure 8 As shown in e. The results showed that the DBNG group achieved good muscle recovery, with a muscle fiber area comparable to that of the autologous transplantation group, while the PLCL group showed significant muscle fiber atrophy ( Figure 8 f). Furthermore, regarding the area of myocollagen fibers, the DBNG group was similar to the autologous nerve group, both being significantly lower than the PLCL group (p<0.001). Figure 8 As shown in g. After toluidine blue staining, the density of myelinated nerve fibers was statistically analyzed. The density of myelinated nerve fibers in the DBNG group was 26979.10 ± 271.68 / mm². 2 Compared with the autologous nerve group, there was no statistically significant difference. Figure 8 As shown in i. To assess the remyelination of newly formed nerve fibers, this project performed transmission electron microscopy on the nerve tissue distal to the nerve scaffold, as shown in [example image]. Figure 8 As shown in h. The results showed that the mean diameter of myelinated axons in the DBNG group was significantly larger than that in the PLCL group (p<0.001), while there was no significant difference compared with the autologous transplantation group. Figure 8 As shown in j. Myelin thickness analysis showed that the myelin thickness in the DBNG group was significantly better than that in the PLCL group. Figure 8 As shown in k, the results were comparable to those of the autologous transplantation group. Both the DBNG group and the autologous nerve group showed similar repair effects in multiple evaluations, indicating that DBNG can provide the microenvironmental conditions required for efficient electrical conduction in facial nerve repair.
[0053] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. A method for preparing a guided extracellular matrix neural scaffold for dental pulp stem cells, characterized in that, The preparation method comprises the following steps: Step S1: Culturing dental pulp stem cells and adding ascorbic acid to stimulate extracellular matrix secretion, and performing decellularization treatment after the culture is completed to obtain a dental pulp stem cell-derived decellular extracellular matrix DPSC-dECM; Step S2: Processing the DPSC-dECM into a powder and dissolving it in a solvent to form a dECM suspension; dissolving polylactic-co-caprolactone PLCL in a solvent to form a PLCL solution; mixing the dECM suspension and the PLCL solution to obtain an ECM / PLCL mixed solution; Step S3: Using an electrospinning technology, first, using the ECM / PLCL mixed solution as a spinning solution to spin and form a DPSC-ECM membrane on a collector, and winding the DPSC-ECM membrane on a mandrel to form an inner layer scaffold of a conduit; and then using the PLCL solution as a spinning solution to spin and form a PLCL fiber layer on the outer surface of the inner layer scaffold of the conduit to obtain a double-layer tubular scaffold precursor; Step S4: Drying the double-layer tubular scaffold precursor to obtain a DPSC-ECM bioinduced nerve conduit graft.
2. The method for preparing a guided extracellular matrix neural scaffold for dental pulp stem cells according to claim 1, characterized in that, The specific method of step S1 comprises: S1-1: Using the 2nd to 4th generation of primary human dental pulp stem cells DPSCs, when the DPSCs grow to 90% confluence in a culture dish, adding 50 μM ascorbic acid phosphate to the culture medium to continuously stimulate the cells to secrete extracellular matrix ECM; S1-2: Under the above conditions, continuously culturing for 14 days, and if cell wrinkles and translucent white membrane-like structures are found to be curled at the edge of the culture dish, it indicates that the ECM has been fully deposited, and the culture is terminated; S1-3: Using a cell scraper to physically scrape off the translucent white membrane-like structure formed in the culture dish, and immersing the scraped membrane-like structure in a 0.5% Triton X-100 solution containing 20 mM ammonium hydroxide to react at 37°C to remove the cell components; S1-4: After the decellularization reaction is completed, the membrane-like structure is washed with phosphate buffered saline PBS three times, each for 5 minutes, to remove residual chemical reagents and cell fragments, to obtain a dental pulp stem cell-derived decellular extracellular matrix DPSC-dECM, which is stored in 1× PBS at -80°C for later use.
3. The method of claim 1, wherein the dental pulp stem cell-extracellular matrix nerve guide is prepared by the steps of: The preparation method of the dECM suspension in step S2 comprises: (1) Homogenizing the DPSC-dECM obtained in step S1 using a tissue homogenizer; (2) Immediately freeze the homogenized sample in liquid nitrogen and freeze dry for 48 hours, grind the freeze-dried sample into fine particles using an electric grinder, freeze again in liquid nitrogen, and perform a second round of freeze drying for 48 hours, and collect the obtained ECM powder using a cell scraper; (3) The dECM suspension was suspended in hexafluoro-2-propanol HFIP, and was continuously shaken at 4°C for 36 hours to fully dissolve, then the solution was diluted with HFIP to prepare dECM suspensions with final concentrations of 35, 45, 55, 65 and 75 mg / mL respectively, and the dECM suspension was moved into a syringe with a cap before electrospinning, and was homogenized by vortexing for 20 seconds.
4. The method of claim 3, wherein the method further comprises the step of: The concentration of the dECM suspension is preferably 55 mg / mL. 5. The method for preparing a guided extracellular matrix neural scaffold for dental pulp stem cells according to claim 1, characterized in that, The preparation method of the PLCL solution in step S2 is to dissolve the PLCL polymer in HFIP, and to magnetically stir at 4°C for 24 hours to prepare a 10% (w / v) PLCL solution.
6. The method of claim 1, wherein the method further comprises the step of: The preparation method of the ECM / PLCL mixed solution in step S2 includes: (1) The 10% (w / v) PLCL solution and the dECM suspension were mixed in a volume ratio of 1:9, and were fully mixed under constant stirring; (2) The ECM / PLCL mixed solution was moved into a syringe with a cap before electrospinning, and was homogenized by vortexing for 20 seconds.
7. The method of claim 1, wherein the method further comprises the step of: The specific method of step S3 includes: S3-1: Preparation of the inner layer dECM electrospun film; The dECM suspension was loaded into a 5-milliliter syringe equipped with a 20G metal needle, and was spun on a rotating drum collector; The electrospinning parameters of the inner layer were: positive voltage 8 kV, negative voltage 3 kV, solution propulsion flow rate 0.2 mL / h, needle tip to collector distance 12 cm, ambient temperature 20-25°C, needle height 34 cm, collector rotation speed 3000 rpm, and total spinning time 5 hours; After electrospinning was completed, the obtained film was peeled off from one side edge of the collector, and was fixed on a metal core rod with a diameter of 1 mm to ensure that the fibers were arranged in parallel along the axis of the metal core rod; then the metal core rod was rotated at a constant speed, so that the DPSC-ECM film uniformly wrapped the surface of the metal core rod to form the inner layer scaffold of the conduit; S3-2: Preparation of the outer layer PLCL reinforcing layer; The 10% PLCL solution was deposited outside the inner layer scaffold; The electrospinning parameters of the outer layer were: positive voltage 10 kV, negative voltage 3 kV, solution propulsion flow rate 0.4 mL / h, needle tip to collector distance 12 cm, ambient temperature 20-25°C, needle height 34 cm, collector rotation speed 70 rpm, and spinning time 0.5 hours; After electrospinning was completed, a PLCL fiber layer was spun on the outer surface of the inner layer scaffold of the conduit to obtain a double-layer tubular scaffold precursor.
8. A guided dental pulp stem cell extracellular matrix nerve scaffold prepared by the method of claims 1-7, wherein, The extracellular matrix nerve scaffold is a double-layer tubular structure, the inner layer is formed by electrospinning of a mixture of dental pulp stem cell decellularized extracellular matrix and polylactic-co-glycolic acid copolymer, and the inner layer is composed of oriented fibers parallel to the axis of the conduit; the outer layer covers the outer surface of the inner layer, and the outer layer is composed of randomly oriented fibers formed by electrospinning of polylactic-co-glycolic acid copolymer.