Tissue engineering vascular smooth muscle remodeling method based on conducting polymer and application

Abstract

The invention discloses a tissue engineering vascular smooth muscle remodeling method based on a conducting polymer and application thereof. The invention finds that the conductive polymer plays a role in promoting the migration of vascular smooth muscle cells and guiding the functional remodeling of the vascular smooth muscle cells. According to the method, a conducting polymer synthesized by biological enzyme is stably modified on a tissue engineering blood vessel in three modes of direct soaking, click chemical grafting or gelatin sponge stent loading. The conductive modified intravascular stent can reconstruct an electrophysiological microenvironment similar to a natural blood vessel, can significantly guide smooth muscle cells to orderly migrate and arrange along the circumferential direction of the blood vessel, and promotes the smooth muscle cells to be transformed to contraction phenotypes expressing markers such as alpha-SMA, SM22alpha, CNN1 and the like, so that a smooth muscle layer close to an autologous blood vessel is structurally and functionally reconstructed. According to the invention, the bottleneck of insufficient functional remodeling of the tissue engineering vascular smooth muscle layer in the prior art is broken through, and a new strategy is provided for regeneration treatment of cardiovascular diseases.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering and regenerative medicine technology, specifically relating to a method for remodeling tissue-engineered vascular smooth muscle based on conductive polymers and its applications. More specifically, it relates to the discovery that conductive polymers can effectively promote smooth muscle cell migration and functional remodeling, and their application in constructing functional tissue-engineered blood vessels. Background Technology

[0002] With socioeconomic development, increased life expectancy, and the high incidence of obesity, the incidence of various cardiovascular diseases is on the rise. In recent years, researchers have discovered that phenotypic changes in vascular smooth muscle cells (VSMCs) play a crucial regulatory role in the progression of various cardiovascular diseases. VSMCs, located in the vascular media, play a vital role in regulating vascular tone, blood pressure, and blood distribution under physiological conditions, and are a key factor in maintaining vascular homeostasis. Under traditional in vitro static culture conditions, VSMCs are highly susceptible to rapid transformation from a contractile to a synthetic form due to hypoxia, inflammatory factors, and material rigidity mismatch. This leads to the secretion of large amounts of collagen and matrix metalloproteinases, resulting in thickened neovascular walls, decreased compliance, and ultimately, clinical complications such as anastomotic stenosis and atherosclerosis. Currently, whether using autologous veins, decellularized matrix, or 3D-printed elastic scaffolds, it is difficult to induce high expression of contractile phenotypic markers such as α-SMA, SM22α, and calponin in smooth muscle cells (SMCs) within a 4–6 week remodeling cycle. Furthermore, the absence of electrophysiological signals prevents SMCs from forming synchronous calcium transients, further hindering the maturation of functional vascular media. Therefore, regulating the phenotypic transformation of vascular smooth muscle at the microenvironment level has become a critical challenge for vascular tissue engineering.

[0003] Conductive polymers, due to their excellent biocompatibility and conductivity, have shown broad application prospects in the biomedical field and have been extensively studied. However, current technologies mostly focus on the synthesis of conductive polymers, general improvement of biocompatibility, or their use as electrode materials, without revealing or anticipating their specificity and efficiency in guiding the orderly migration and arrangement of vascular smooth muscle cells and promoting their transformation to a contractile phenotype. In existing technologies, whether using autologous veins, decellularized matrix, or 3D-printed elastic scaffolds, it is difficult to effectively induce smooth muscle cells (SMCs) to regain high expression of contractile phenotype markers such as α-SMA, SM22α, and calponin within a 4-6 week remodeling cycle, and to form a functional smooth muscle layer.

[0004] Therefore, this invention aims to solve the above-mentioned technical problems by utilizing the in-situ electroactivity regulation capability of conductive polymers to achieve targeted induction and functional remodeling of SMC phenotype, and to provide a new strategy for vascular tissue engineering and regenerative therapy of cardiovascular and cerebrovascular diseases. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the aforementioned problems in existing technologies, this invention provides a method for remodeling tissue-engineered vascular smooth muscle based on conductive polymers. This invention aims to solve the technical challenges of disordered smooth muscle cell migration, loss of contractile phenotype, and insufficient functional remodeling in existing tissue-engineered vascular techniques.

[0007] (II) Technical Solution

[0008] To address the aforementioned technical problems, this invention is based on a novel discovery: that specific conductive polymers can create an electrophysiological microenvironment conducive to the directed migration and functional differentiation of smooth muscle cells. Based on this discovery, this invention provides a method for tissue-engineered vascular smooth muscle remodeling based on conductive polymers.

[0009] The core of the method lies in introducing a biocompatible conductive polymer into a tissue-engineered vascular system through a specific construction method. The electrophysiological microenvironment created by the conductive polymer is used to actively guide the migration behavior of smooth muscle cells and induce them to express contractile phenotype proteins, thereby achieving structural regeneration and functional reconstruction of the smooth muscle layer.

[0010] The "specific construction method" includes, but is not limited to, the following three:

[0011] 1. Direct modification method: The artificial blood vessel is directly immersed in a conductive polymer solution, and modification is achieved through physical adsorption.

[0012] 2. Click chemistry method: Conductive polymers are covalently grafted onto the surface of blood vessels through click chemistry reaction between DBCO-NHS and azide groups, resulting in a more stable bond.

[0013] 3. Scaffold loading method: Conductive polymers are loaded onto a biodegradable gelatin sponge scaffold with high porosity, and then an artificial blood vessel is inserted through it to form an "outer membrane" structure that can provide continuous electrical stimulation and structural support.

[0014] This invention provides a method for tissue-engineered vascular smooth muscle remodeling based on conductive polymers, comprising the following steps:

[0015] S1. Prepare conductive polymers with good biocompatibility and conductivity through enzymatic synthesis;

[0016] S2. Decellularize the blood vessels to form artificial tissue-engineered blood vessels from decellularized tissue.

[0017] S3. The conductive polymer from step S1 is modified onto the artificial tissue-engineered blood vessel from step S2 by means of physical adsorption, chemical grafting, or scaffold loading, thereby constructing a conductive vascular scaffold that can promote smooth muscle cell migration and functional remodeling.

[0018] The method described above, preferably, involves physical adsorption in step S3 as follows: the artificial tissue-engineered blood vessel is directly immersed in a conductive polymer solution and rinsed for 20-24 hours at 0-4°C and 50-100 rpm to allow the conductive polymer to adhere to the surface of the blood vessel, followed by rinsing with a maintenance solution to remove any unadhered portions.

[0019] The method described above, preferably, involves chemical grafting in step S3 as follows: first, reacting the artificial tissue-engineered blood vessel with a DBCO-NHS solution to modify its surface with DBCO groups; then, combining it with a conductive polymer containing azide groups (-N3) through a click chemical reaction to form a stable DBCO-conductive polymer vascular stent.

[0020] As described above, preferably, the scaffold loading in step S3 is as follows: a hollow cylindrical scaffold is made from gelatin sponge, and then a conductive polymer is loaded into the pores of the gelatin sponge through vacuum-atmospheric pressure cyclic adsorption to form a conductive gelatin sponge scaffold; finally, the artificial tissue-engineered blood vessel is passed through the hollow part of the conductive gelatin sponge scaffold to form a coaxial composite structure.

[0021] Furthermore, the concentration of the conductive polymer solution used is 0.01~10 mM.

[0022] Furthermore, the pore size of the gelatin sponge is 10~100 μm and the porosity is greater than 90%; the adsorption conditions are maintained at -0.08 MPa for 5 min, maintained at atmospheric pressure for 5 min, cyclicated 3 times, and then allowed to stand at 0~4℃ for 20~24 h for adsorption.

[0023] Further, the concentration of the DBCO-NHs solution is 0.01 mg / ml. Preferably, in step S1, the conductive polymer is synthesized by enzymatic reaction of a conductive monomer, a biological enzyme, and an oxidant; the conductive monomer is at least one of pyrrole, aniline, thiophene, and their derivatives; the biological enzyme is at least one of horseradish peroxidase, catalase, or glucose oxidase; and the oxidant is at least one of hydrogen peroxide, ferrous ions, or ferric ions. Specifically, the conductive polymer is prepared by dissolving the conductive monomer and biological enzyme in water, adding an oxidant, and then enzymatically reacting to obtain a conductive polymer solution; wherein the final concentration of the conductive monomer in water is 20-100 mM, the final concentration of the biological enzyme in water is 0.2 mg / mL, and the final concentration of the oxidant is 1-3 mM, and the reaction is carried out at 0-4 °C, in the dark, at 200-300 rpm for 20-24 h.

[0024] Further, the conductive monomer is at least one of pyrrole, pyrrole-3-carboxylic acid, 2,2'-dipyrrole, pyrrole-3-sulfonic acid, aniline, p-aminodiphenylamine, thiophene, 4-ethylenedioxythiophene, 3,4-ethyldioxypyrrole, pyrrole-thiophene copolymer and pyrrole-3,4-ethyldioxythiophene, or a comonomer composed of any two or more of the aforementioned monomers.

[0025] A conductive-modified tissue-engineered blood vessel prepared by the method described above.

[0026] The application of the conductive-modified tissue-engineered blood vessels prepared by the method described above in the preparation of artificial vascular grafts for the treatment of cardiovascular diseases.

[0027] Furthermore, the cardiovascular disease includes atherosclerosis, anastomotic stenosis, or vascular trauma.

[0028] (III) Beneficial Effects

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] 1. A novel function of conductive polymers was discovered: For the first time, the biological functions of conductive polymers in specifically promoting the directional migration of smooth muscle cells and inducing their contractile phenotype expression were systematically verified and applied, which would not have been easily foreseen by those skilled in the art.

[0031] 2. Functional remodeling is achieved: By reconstructing the electrophysiological microenvironment, the method of this invention can significantly guide the orderly regeneration of smooth muscle cells along the circumferential direction of blood vessels and promote their high expression of contraction markers such as α-SMA, SM22α, and CNN1, ultimately forming a smooth muscle layer that is close to the autologous blood vessels in both structure and functional characteristics.

[0032] 3. Provides multiple reliable implementation schemes: The three vascular stent construction methods provided by this invention are flexible and reliable, and the appropriate scheme can be selected according to different preclinical or clinical needs, all of which can effectively achieve functional remodeling of smooth muscle.

[0033] 4. Long-lasting and stable effects: Animal experiments show that the conductive vascular stent constructed by the method of this invention can maintain good vascular patency even 28 days to 3 months after implantation, and shows a mature and stable smooth muscle layer and an intact endothelial lumen. The long-term effect is significantly better than that of the control group. Attached Figure Description

[0034] Figure 1 Photographs of several conductive polymer materials prepared by bio-enzymatic polymerization;

[0035] Figure 2 A schematic diagram of the structure of decellularized tissue-engineered blood vessels (TEBV);

[0036] Figure 3 Photographs of tissue-engineered blood vessels after one day of direct immersion modification with PPy conductive polymer;

[0037] Figure 4 To identify tissue-engineered blood vessels modified with PANI conductive polymer via DBCO-NHS click chemistry and their SEM morphology.

[0038] Figure 5 A schematic diagram illustrating the process and structure of constructing tissue-engineered blood vessels by loading conductive polymers onto a gelatin sponge scaffold;

[0039] Figure 6 Ultrasound monitoring images of blood flow in rats 14 and 28 days after PPy conductive gelatin sponge vascular stent implantation.

[0040] Figure 7 HE and Masson staining results of PPy conductive gelatin sponge vascular stent implanted in vivo 28 days after implantation;

[0041] Figure 8 Immunofluorescence staining of α-SMA and CD31 for PPy conductive gelatin sponge vascular stents 28 days after in vivo reconstruction;

[0042] Figure 9 Immunofluorescence staining of CNN1, SM22α, and CD31 three months after implantation of PPy conductive gelatin sponge vascular stent;

[0043] Figure 10 The scratch test results of smooth muscle cells under the action of PPy conductive polymer showed that their migration ability was significantly enhanced. Detailed Implementation

[0044] This invention provides a conductive polymer synthesized by bio-enzymatic synthesis, which has the advantages of low toxicity, high biocompatibility and conductivity.

[0045] This invention relates to a method for modifying vascular scaffolds with conductive polymers to promote the targeted regeneration and functional remodeling of smooth muscle cells, and its application in constructing functional tissue-engineered blood vessels. The method comprises three specific modification preparation methods: 1. Directly immersing an artificial blood vessel or tissue-engineered vascular scaffold in a conductive polymer solution, allowing the conductive material to adhere uniformly to the scaffold surface through physical adsorption. 2. Molding gelatin sponge into a tubular hollow scaffold and loading it with conductive polymer material to form a conductive gelatin sponge scaffold, allowing the target artificial blood vessel or tissue-engineered blood vessel to penetrate the lumen of the conductive scaffold, achieving indirect modification of the conductive material. 3. Modifying the surface of the artificial blood vessel or tissue-engineered vascular scaffold with active groups containing dibenzocyclooctylene-succinimide ester (DBCO-NHS), introducing a conductive polymer material with surface-modified azide groups (-N3), and achieving a stable bond between the conductive material and the vascular scaffold through a highly efficient, bioorthogonal click chemistry reaction between DBCO and the azide groups.

[0046] This invention provides a bio-enzymatically polymerized conductive polymer, which is prepared from a conductive monomer, water, and a first polymerization triggering factor. The conductive monomer is at least one of pyrrole, pyrrole-3-carboxylic acid, 2,2'-dipyrrole, pyrrole-3-sulfonic acid, aniline, p-aminodiphenylamine, thiophene, 4-ethylenedioxythiophene, 3,4-ethyldioxypyrrole, pyrrolethiophene copolymer, and pyrrole-3,4-ethyldioxythiophene, or a comonomer composed of any two or more of the aforementioned monomers.

[0047] The first polymerization triggering factor includes a biological enzyme and an oxidant, wherein the biological enzyme is at least one of catalase (CAT), glucose oxidase and horseradish peroxidase (HRP).

[0048] Specifically, the present invention provides a method for preparing tissue-engineered vascular smooth muscle remodeling based on conductive polymers, which includes the following steps:

[0049] S1. To prepare an enzyme-synthesized conductive polymer with good biocompatibility and conductivity;

[0050] S2. Process the blood vessels to form tissue-engineered blood vessels;

[0051] S3. The conductive polymer from step S1 is added to the artificial tissue-engineered blood vessel formed in step S2 and directly immersed and repeatedly rinsed so that the conductive polymer from step S1 is basically adhered to the artificial tissue-engineered blood vessel in step S2. The neutral maintenance solution is used to gently rinse away the conductive polymer that has not adhered, thus forming a conductive artificial tissue-engineered blood vessel.

[0052] Alternatively, the artificial tissue-engineered blood vessel from step S2 can be immersed in DBCO-NHs solution at low temperature for 18-24 hours to allow DBCO-NHs to completely integrate with the artificial tissue engineering from step S2. The conductive polymer from step S2 can then be added to integrate with DBCO-NHs to form a DBCO-conductive polymer vascular stent.

[0053] Alternatively, a hollow cylinder can be made from gelatin sponge, and the conductive polymer from step S1 can be added and adsorbed for 18-24 hours to form a gelatin sponge conductive polymer scaffold. Artificial tissue-engineered blood vessels can then pass through the gelatin sponge conductive polymer scaffold to form a conductive gelatin sponge vascular scaffold.

[0054] The conductive polymer is prepared by dissolving a conductive monomer and a biological enzyme in water, adding an oxidant, and then performing an enzymatic reaction to obtain a conductive polymer solution. The final concentration of the conductive monomer in water is 20-100 mM, the final concentration of the biological enzyme in water is 0.2 mg / mL, and the final concentration of the oxidant is 1-3 mM. The reaction conditions are 0-4 °C, protected from light, and 200-300 rpm for 20-24 h. The conductive monomer is at least one of pyrrole, pyrrole-3-carboxylic acid, 2,2'-dipyrrole, pyrrole-3-sulfonic acid, aniline, p-aminodiphenylamine, thiophene, 4-ethylenedioxythiophene, 3,4-ethyldioxypyrrole, pyrrole-thiophene copolymer, and pyrrole-3,4-ethyldioxythiophene, or a comonomer composed of any two or more of the aforementioned monomers.

[0055] The biological enzyme is at least one of catalase (CAT), glucose oxidase, and horseradish peroxidase (HRP);

[0056] The oxidant is at least one of hydrogen peroxide, ferrous iron, and ferric iron.

[0057] Furthermore, after the reaction, the purified conductive polymer was obtained by dialysis using a dialysis bag with a molecular weight cutoff of 12-14 kDa.

[0058] To better explain and facilitate understanding of the present invention, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art. Unless otherwise specified, all reagents used in the examples are analytical grade or higher and can be commercially available products. For example, aniline monomers, pyrrole monomers, pyrrole carboxylic acid (pyrrole-3-carboxylic acid) monomers (PyCA), and 3,4-ethylenedioxythiophene monomers can be purchased from Aladdin, CAT enzymes can be purchased from Beyotime, and DBCO-NHS can be purchased from Sigma-Aldrich.

[0059] Example 1 Enzymatic Synthesis of Conductive Polymers

[0060] This embodiment provides several methods for preparing conductive polymers, which serve as the material basis for subsequent functional verification. The methods are as follows:

[0061] (1) A aniline monomer (ANI), pyrrole monomer (Py), pyrrole carboxylic acid (pyrrole-3-carboxylic acid) monomer (PyCA), and 3,4-ethylenedioxythiophene monomer (EDOT) were dissolved in water to obtain conductive monomer solutions of a certain concentration.

[0062] (2) Dissolve catalase (CAT) in water to obtain a CAT enzyme solution with a concentration of 2 mg / mL.

[0063] (3) Enzymatic reaction system: Under sterile conditions, add 45 mL of any conductive monomer and 5 mL of CAT enzyme solution at 2 mg / mL to make the final concentration of the conductive polymer monomer 50 mM and the final concentration of CAT enzyme 0.2 mg / mL.

[0064] (4) Using a micro-pump, add 2 mL of H2O2 solution (30 wt%, diluted 10 times with PBS) dropwise at a constant rate of 0.5 mL min⁻¹ to the above enzymatic reaction system, bringing the final H2O2 concentration to 2 mM. After the addition is complete, continue the reaction at 0–4 °C, in the dark, and at 300 rpm for 24 h. During the reaction, the color of the system gradually changes from colorless to dark green or black, eventually forming a conductive polymer solution.

[0065] (5) After the reaction was completed, the solution was transferred to a dialysis bag with a molecular weight cutoff of 12–14 kDa and dialyzed with deionized water for 24 h (changing the water every 8 h) to remove free monomers, oligomers, and salt ions. Each monomer yielded a conductive polymer corresponding to polyaniline (PANI), polypyrrole (PPy), polypyrrole-3-carboxylic acid (PPyCA), and poly(3,4-ethylenedioxythiophene) (PEDOT). Photos of the four products are attached. Figure 1 As shown.

[0066] Example 2: Verification of Direct Modification of Blood Vessels by Conductive Polymers and its Promotion of Orderly Smooth Muscle Regeneration

[0067] In this embodiment, the PPy conductive polymer was modified onto decellularized rat tissue-engineered blood vessels using a direct immersion method. The modified blood vessels were then subjected to in vitro cell culture or in vivo implantation experiments. The method is as follows:

[0068] Sterilized rat decellularized tissue-engineered blood vessels (TEBV) (its structural diagram is shown in Figure 1) were then used. Figure 2 The pretreated rat decellularized tissue-engineered blood vessels were placed in PBS, then blotted dry with sterile filter paper, and transferred to new sterile 50 mL centrifuge tubes for later use. The pretreated rat decellularized tissue-engineered blood vessels were then completely immersed in the PPy polymerization solution prepared in Example 1. The centrifuge tubes were sealed and placed in a constant temperature shaker at 4 °C and 60 rpm for 24 h. After the reaction, the blood vessels were removed and rinsed three times with 200 mL of PBS for 5 min each time, followed by rinsing once with 100 mL of sterile deionized water to remove residual salt ions. The final result was a soft, flexible PPy-modified tissue-engineered blood vessel coated with a black conductive polymer, as shown below. Figure 3 As shown.

[0069] Example 3: Stable Modification of Conductive Polymers Based on Click Chemistry and Effect Verification

[0070] In this embodiment, PANI conductive polymer is covalently grafted onto the surface of blood vessels using DBCO-NHS click chemistry, as follows:

[0071] 1 mg of diphenylcyclooctylene-active ester (DBCO-NHS) was dissolved in 1 mL of dimethyl sulfoxide (DMSO) to obtain a clear solution A (1 mg / mL). Solution A was diluted with ultrapure water to obtain a 1% (w / w) solution B, which was milky white (0.01 mg / mL) (i.e., 100 μL of solution A was added to 9.9 mL of water to obtain milky white solution B). Rat decellularized tissue-engineered blood vessels (TEBV) were immersed in solution B and shaken on a shaker at 100 rpm for about 4 hours. Then, they were transferred to the 100 μg / mL enzymatically polymerized PANI sample solution prepared in Example 1 and shaken on a shaker at 4 °C and 100 rpm for 24 hours. After the reaction, the samples were washed with PBS for 3 × 10 min to remove free DBCO-NHS and dried with nitrogen to obtain DBCO-PANI-TEBV (DBCO-PANI-TEBV is an abbreviation for tissue-engineered blood vessels of enzymatically polymerized PANI modified with DBCO-NHS). Figure 4 As shown in the figure, A is the SEM image of the control, namely the decellularized tissue-engineered blood vessel.

[0072] Scanning electron microscopy (SEM) sample preparation and observation were then performed: DBCO-PANI-TEBV was cut into 5 mm ring segments and immersed in 2.5% glutaraldehyde / PBS for 2 h at 4 ℃ for fixation. The samples were then subjected to a gradient of ethanol dehydration for 15 min each time, with ethanol concentrations increasing from 30% to 50% to 70% to 90% to 100%, with each gradient repeated twice. The samples were then transferred to HMDS for 15 min, removed, and placed on filter paper to evaporate at room temperature for 30 min until completely dry. The dried samples were then adhered to an aluminum sample stage with the cut surface facing upwards using carbon adhesive. Gold was deposited using an ion sputtering system for 60 s, and the morphology of the modified outer membrane of the lumen was observed using field emission scanning electron microscopy. Macroscopically, DBCO-PANI-TEBV appeared as a uniform yellow-green color and was flexible and bendable. SEM showed that the fibrous network structure remained intact on the surface of the outer membrane, with polyaniline (PANI) nanoparticles uniformly covering it. The PANI nanoparticles appeared as coral-like nanoprotrusions with a particle size of 50–100 nm (see details). Figure 4 (B)

[0073] Example 4: Construction and Functional Study of Conductive Polymer-Gelatin Foam Composite Scaffold

[0074] This embodiment constructs a conductive polymer-gelatin sponge composite scaffold and assembles it with rat tissue-engineered blood vessels into a coaxial structure, as follows:

[0075] 1) Weigh an appropriate amount of Type A gelatin, add deionized water, and prepare Type A gelatin (10% w / v, such as 10g gelatin dissolved in 100mL deionized water); place it on a 50℃ magnetic stirrer and stir continuously at 400~600rpm for 30 minutes to fully dissolve the gelatin.

[0076] 2) Pour the gelatin solution into the mold (mandrel diameter 1.8mm, outer mold diameter 2.5mm, length 10mm) using a syringe to form a hollow cylinder; place the mold containing the gelatin solution into a vacuum degassing machine with a vacuum degree of -0.1MPa and degas for 10 minutes.

[0077] 3) After degassing, immediately place the mold in liquid nitrogen and freeze it rapidly for 5 minutes.

[0078] 4) Quickly transfer the frozen mold to a -50℃ freeze dryer and dry for 24 hours to obtain hollow gelatin sponge cylinders.

[0079] The hollow gelatin sponge cylinder was completely immersed in the PPy solution prepared in Example 1 (e.g. Figure 5A) Room temperature vacuum-atmospheric pressure cycling (-0.08 MPa for 5 min, atmospheric pressure for 5 min, 3 cycles) promoted PPy entry into the pores. Afterwards, the mixture was allowed to stand at 4 ℃ for 24 h for adsorption. The mixture was then removed and gently rinsed three times with PBS to remove unadsorbed particles, yielding PPy-gelatin sponge conductive cylinders.

[0080] The obtained PPy-gelatin sponge conductive cylinders were subjected to scanning electron microscopy (SEM). The morphology showed that the gelatin sponge had a pore size of 100–200 μm, and the pore walls were uniformly coated with PPy nanoparticles. Figure 5 (B) The obtained PPy conductive gelatin sponge vascular scaffold was axially fitted onto the outer wall of the sterilized tissue-engineered vascular scaffold, and both ends were fixed with 8-0 polypropylene sutures to form a coaxial "vascular-conductive sponge" structure. Figure 5 As shown in C. Optional step to improve stability: Crosslink the assembly in 0.1% (w / v) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) solution at room temperature for 30 min, then wash three times with PBS.

[0081] Example 5: Verification of the promoting effect of conductive polymers on smooth muscle cell migration behavior

[0082] This embodiment uses PPy conductive gelatin sponge vascular stents transplanted into the carotid artery defect area of ​​SD rats via microsurgery to verify its feasibility in promoting smooth muscle regeneration.

[0083] Using gelatin sponge (without conductive materials) as the Ctrl, postoperative dynamic monitoring was performed on the PPy conductive gelatin sponge vascular stent transplanted according to Example 4. Vascular patency was assessed using high-frequency Doppler ultrasound contrast imaging, and the smooth muscle regeneration process of the transplanted blood vessels was analyzed using paraffin section HE staining and MASSON staining techniques. The study focused on verifying the vascular function status and vascular remodeling and regeneration effect at 14 days (acute phase) and 28 days (remodeling phase) postoperatively.

[0084] The test results of high-frequency Doppler ultrasound contrast imaging are as follows Figure 6 As shown. Doppler ultrasound quantitative hemodynamics ( Figure 6 The PPy transplanted segment of the vessel maintained structural patency (without stenosis or thrombosis) at 14 and 28 days post-operation.

[0085] Simultaneously, after tissue sectioning, HE staining was performed, and the HE results of vascular remodeling were as follows: Figure 7As shown, at 28 days, the PPy conductive gelatin sponge vascular stent had an intact lumen outline, a smooth intima, significantly increased media thickness, higher density of smooth muscle-like cells, and a more mature arrangement, with no obvious intima hyperplasia or rupture. MASSON staining showed that at 28 days, the PPy conductive gelatin sponge vascular stent area was dominated by light blue collagen, accompanied by a small amount of red muscle fibers. The collagen content was high and formed a ring-shaped dense collagen outer membrane, indicating a high degree of functional vascular remodeling maturity.

[0086] After tissue sectioning, immunofluorescence staining was performed using fluorescent dyes for labeling nuclear DAPI, platelet-endothelial cell adhesion molecule (CD31) (a marker of vascular endothelial cells), and α-smooth muscle actin (α-SMA) (a marker of smooth muscle cells / pericytes). Molecular evidence for vascular functional smooth muscle remodeling includes... Figure 8 As shown, at 28 days, the PPy conductive gelatin sponge vascular stent achieved functional vascular remodeling, possessing a tissue structure and cellular composition similar to that of natural arteries. The PPy conductive gelatin sponge vascular stent wall media exhibited continuous and dense α-SMA positive ring signals, with a thickness comparable to that of the autologous common carotid artery, indicating that the smooth muscle layer had regenerated and exhibited a mature contractile phenotype. A relatively complete and continuous CD31 positive endothelial layer formed on the luminal surface, suggesting rapid endothelialization and maintenance of the antithrombotic barrier function. Further immunofluorescence staining was performed 3 months after transplantation, as shown... Figure 9 The results showed that PPy conductive gelatin sponge vascular scaffolds significantly promoted and maintained the regeneration of mature smooth muscle layers (CNN⁺, SM22α⁺) three months after reconstruction, while achieving sustained and complete endothelialization (CD31⁺). In contrast, no effective smooth muscle reconstruction was observed in the control group, confirming the key advantages of PPy conductive scaffolds in long-term vascular remodeling. Among the markers, DAPI served as a nuclear marker, CD31 as a vascular endothelial cell marker, α-SMA as a vascular smooth muscle marker, Merged composite image, CNN1 as a marker of contractile vascular smooth muscle, and SM22α as a marker of contractile VSMCs. Contractile VSMCs express abundant contractile proteins (such as α-SMA, SM22α, CNN1, etc.), endowing engineered vessels with physiological vascular contraction capabilities, helping engineered vessels form a medial structure similar to natural vessels, and improving their mechanical strength and stability. Due to their contractile and secretory functions, differentiated VSMCs help maintain vascular compliance and antithrombotic capacity, reducing postoperative complications such as stenosis, dilation, or rupture. Contractile vascular smooth muscle cells, after being remodeled into engineered blood vessels, can significantly improve the contractile function, structural maturity, long-term patency, and biological stability of blood vessels, making them an indispensable key component for achieving functional tissue-engineered blood vessels.

[0087] Example 6: Verification of the promoting effect of conductive polymers on smooth muscle cell migration behavior

[0088] This embodiment aims to verify the effect of PPy conductive polymer prepared by enzymatic synthesis on the migration behavior of smooth muscle cells (SMCs). Figure 10 The method is as follows:

[0089] Human aortic smooth muscle cells (HASMC, purchased from procell) were seeded in 24-well plates at a density of 5 × 10⁶ cells / well. 4 Cells / well were cultured until confluence exceeded 90%. A uniform scratch was made in the center of the cell layer using a 200 μL pipette tip, and detached cells were removed by washing with PBS. Smooth muscle cell culture medium (purchased from ProCell), prepared according to Example 1 and diluted to contain 1 mmol of enzymatically synthesized PPy conductive polymer, was added to the experimental group, with PBS used as a control. Cells were cultured at 37°C and 5% CO2, and images of the scratched area were taken at 0, 24, 48, and 78 hours to observe scratch closure.

[0090] The results showed that the scratch width in the PPy experimental group was significantly reduced after 48 hours, and the cell migration rate calculated by area reached (68.3±5.2)%, significantly higher than that in the control group (42.1±4.7)% (p<0.01). These results indicate that the PPy conductive polymer effectively promotes the migration of smooth muscle cells. This experimental result directly confirms the core finding of this invention: conductive polymers can effectively promote the migration of smooth muscle cells, providing a cellular behavioral basis for guiding orderly cell regeneration in vivo.

[0091] This invention, based on the discovery of the novel function of conductive polymers in promoting smooth muscle cell migration and functional remodeling, has developed several effective methods for constructing tissue-engineered blood vessels. These methods can overcome the bottlenecks of existing technologies, achieving structural regeneration and functional maturation of the smooth muscle layer in engineered blood vessels, and have broad application prospects in the field of regenerative therapy for cardiovascular diseases.

[0092] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art can make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for tissue engineered vascular smooth muscle remodeling based on electrically conductive polymers, characterized by, The method comprises the following steps: S1, providing a conductive polymer synthesized by a biological enzymatic reaction; S2, providing a decellularized artificial tissue engineering blood vessel; S3, modifying the conductive polymer of step S1 to the artificial tissue engineering blood vessel of step S2 by physical adsorption, chemical grafting or scaffold loading, thereby constructing a conductive blood vessel scaffold capable of promoting smooth muscle cell migration and functional remodeling.

2. The method of claim 1, wherein, In step S3, the physical adsorption is: directly immersing the artificial tissue engineering blood vessel in a conductive polymer solution, flushing at 0-4°C and 50-100 rpm for 20-24 hours, so that the conductive polymer adheres to the surface of the blood vessel, and then washing with a maintenance solution to remove the unattached part.

3. The method of claim 1, wherein, In step S3, the chemical grafting is: first reacting the artificial tissue engineering blood vessel with a DBCO-NHS solution to modify the surface with a DBCO group; then combining it with a conductive polymer with an azide group through a click chemistry reaction to form a stable DBCO-conductive polymer blood vessel scaffold.

4. The method of claim 1, wherein, In step S3, the scaffold loading is: making a gelatin sponge into a hollow cylindrical scaffold, then loading the conductive polymer into the pores of the gelatin sponge through vacuum-normal pressure cycle adsorption to make a conductive gelatin sponge scaffold; finally passing the artificial tissue engineering blood vessel through the hollow part of the conductive gelatin sponge scaffold to form a coaxial composite structure.

5. The method of claim 2 or 4, wherein, The use concentration of the conductive polymer solution is 0.01-10 mM.

6. The method of claim 4, wherein, The pore size of the gelatin sponge is 10-100 μm, and the porosity is greater than 90%; the adsorption conditions are: maintaining at -0.08 MPa for 5 min, maintaining at normal pressure for 5 min, and after 3 cycles, adsorbing at 0-4°C for 20-24 h.

7. The method of claim 1, wherein, The conductive polymer is synthesized by a conductive monomer, a biological enzyme and an oxidizing agent; the conductive monomer is at least one of pyrrole, aniline, thiophene and its derivatives; the biological enzyme is at least one of horseradish peroxidase, catalase or glucose oxidase; and the oxidizing agent is at least one of hydrogen peroxide, divalent iron or trivalent iron ion.

8. A conductive modified tissue engineering blood vessel prepared by the method of any one of claims 1-7.

9. Use of the conductive modified tissue engineering blood vessel of claim 8 in the preparation of an artificial blood vessel graft for treating cardiovascular diseases.

10. Use according to claim 9, characterized in that, The cardiovascular diseases include atherosclerosis, anastomotic stenosis or vascular trauma.

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