Conductive nanofiber scaffold and preparation method and application thereof

Abstract

The application provides a conductive nanofiber scaffold and a preparation method and application thereof, the scaffold comprising a conductive nanofiber base formed by electrospinning of polyvinyl butyral and PEDOT:PSS, and a multifunctional bioactive peptide fixed on the surface of the base. The scaffold combined with exogenous microelectric stimulation can inhibit the mTORC1 pathway, induce M2 type polarization and metabolic reprogramming of macrophages, inhibit fibrosis, realize immune-metabolic-fibrosis multi-target synergistic regulation, and is used for treating diabetic bone defects.

Description

Technical Field

[0001] This invention relates to a conductive nanofiber scaffold, its preparation method and application, belonging to the field of bone repair technology. Background Technology

[0002] In diabetic patients, the long-term hyperglycemic state, accumulation of advanced glycation end products, and enhanced oxidative stress significantly reduce bone tissue regeneration capacity, prolonging bone defect healing time and even leading to delayed healing or nonunion. Studies have shown that diabetic bone defects involve persistent activation of M1 macrophages, high expression of inflammatory factors, and abnormal fibroblast activation, resulting in the formation of local fibrous scar tissue, which further hinders angiogenesis, osteogenic differentiation, and trabecular bone remodeling.

[0003] Currently, materials used for bone repair mainly include inorganic bone substitutes, polymer scaffolds, hydrogels, and bioactive factor delivery systems. However, most technologies only target a single aspect such as promoting bone growth, reducing inflammation, or improving mechanical support, making it difficult to simultaneously regulate inflammation, metabolism, and fibrosis abnormalities in diabetic bone defects. Conductive biomaterials and electrical stimulation have been used in recent years to promote bone regeneration, enhancing osteoblast activity, promoting angiogenesis, and regulating macrophage polarization. However, their synergistic regulation of immune metabolic reprogramming and fibrous scar formation in the diabetic pathological microenvironment remains insufficient.

[0004] Furthermore, many existing conductive scaffolds employ simple surface coatings of conductive components, resulting in issues such as unstable conductivity, insufficient bioactivity, and limited tissue integration. How to stably conduct microcurrents and provide continuous bioactive signals, while simultaneously achieving synergistic regulation of macrophages, fibroblasts, and related metabolic pathways, remains an unsolved technical challenge in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a conductive nanofiber scaffold, its preparation method and application, to solve the problems of chronic inflammation, metabolic disorders and fibrous scarring in the repair of diabetic bone defects.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a conductive nanofiber scaffold, characterized in that the conductive nanofiber scaffold comprises a conductive nanofiber substrate formed by electrospinning a composite of polyvinyl butyral and poly(3,4-ethylenedioxythiophene) and poly(styrene sulfonate), and a multifunctional bioactive peptide fixed on the surface of the conductive nanofiber substrate, wherein the structure of the multifunctional bioactive peptide is: [(DOPA)2-IEELEEELEER-(PEG4)-LYENRL-(DOPA)2], wherein DOPA is 3,4-dihydroxyphenylalanine and PEG4 is polyethylene glycol tetrakis.

[0008] In conjunction with the first aspect, the multifunctional bioactive peptide is further anchored to the surface of a conductive nanofiber substrate via terminal DOPA groups, wherein the conductive nanofiber substrate has an oriented structure.

[0009] Furthermore, the amount of the poly(3,4-ethylenedioxythiophene) and poly(styrene sulfonate) composite added is 1% to 5% of the mass of polyvinyl butyral.

[0010] Secondly, the present invention provides the application of a conductive nanofiber scaffold in the preparation of a medical device for use in conjunction with exogenous micro-electrical stimulation to treat diabetic bone defects.

[0011] In conjunction with the second aspect, furthermore, the voltage of the exogenous micro-electric stimulation is 20mV to 80mV, the stimulation time is 5 to 30 minutes each time, and the stimulation frequency is 1 to 2 times per day.

[0012] Furthermore, the diabetic bone defects include skull defects, jawbone defects, or long bone defects in a diabetic state, as well as bone defects with delayed fracture healing, nonunion, or accompanied by high inflammation or high fibrosis.

[0013] Thirdly, the present invention provides a method for preparing a conductive nanofiber scaffold, the method comprising the following steps: dissolving polyvinyl butyral in anhydrous ethanol, adding a composite of poly(3,4-ethylenedioxythiophene) and poly(styrene sulfonate), and mixing to obtain a spinning solution; preparing a conductive nanofiber substrate by electrospinning the spinning solution; immersing the conductive nanofiber substrate in a solution containing multifunctional bioactive peptides to react, thereby immobilizing the multifunctional bioactive peptides on the surface of the conductive nanofiber scaffold, and obtaining the conductive nanofiber scaffold after drying.

[0014] In conjunction with the third aspect, the electrospinning conditions are further as follows: voltage 10-20 kV, feed speed 0.5-2.0 mL / h, receiving distance 10-20 cm, and roller speed 300-800 rpm.

[0015] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0016] This invention provides a conductive nanofiber scaffold, its preparation method, and its application. The conductive nanofiber substrate is formed by electrospinning a composite of polyvinyl butyral and poly(3,4-ethylenedioxythiophene) and poly(styrene sulfonate). The conductive components are uniformly embedded inside the fiber, resulting in stable and long-lasting conductivity and effective transmission of micro-electrical stimulation. The ordered orientation of the electrospinning structure and the uniform conductive components inside the fiber mimic the structure of an electrical circuit wire, enabling efficient and oriented transmission of current.

[0017] The scaffold surface is fixed with a multifunctional bioactive peptide with the structure [(DOPA)2-IEELEEELEER-(PEG4)-LYENRL-(DOPA)2], which has both anti-fibrotic and osteopromoting functions.

[0018] This invention provides a conductive nanofiber scaffold that, when used in combination with exogenous micro-electric stimulation, promotes macrophage polarization towards the M2 type and induces their metabolism to shift from glycolysis to oxidative phosphorylation by inhibiting the mTORC1 signaling pathway. At the same time, it inhibits the expression of α-SMA and abnormal fibrosis in fibroblasts, thereby achieving multi-target synergistic regulation of the immune-metabolic-fibrotic microenvironment. Attached Figure Description

[0019] Figure 1 The figure shows the molecular structure and chromatogram of the synthesized IL peptide. In the figure, a is the molecular structure of the IL peptide, which is synthesized by solid-phase synthesis. The IL peptide is formed by the reaction of amino and carboxyl groups to form amide bonds. b is the detection result of the IL peptide by high performance liquid chromatography.

[0020] Figure 2 The figures show the physicochemical characterization of the conductive nanofiber scaffold; in the figures, a is the scanning electron microscope morphology of the scaffold, b is the energy dispersive spectroscopy (EDS) spectrum, and c is the atomic force microscope roughness analysis diagram.

[0021] Figure 3 The figures show the infrared spectrum of the conductive nanofiber scaffold and the drug release kinetics curve of the peptide. In the figures, a is the infrared spectrum of the conductive nanofiber scaffold and b is the drug release kinetics curve of the peptide.

[0022] Figure 4 The results show the conductivity of the conductive nanofiber scaffold.

[0023] Figure 5 The results of the cell compatibility experiment of bone marrow mesenchymal stem cells on conductive nanofiber scaffolds are shown; in the figure, a is the immunofluorescence image of live and dead cells, and b is the statistical graph of the percentage of live and dead cells.

[0024] Figure 6 The results of the cell compatibility experiment of bone marrow-derived macrophages with conductive nanofiber scaffolds are shown; in the figure, a is the immunofluorescence image of live and dead cells, and b is the statistical graph of the percentage of live and dead cells.

[0025] Figure 7 Figure 1 shows the experimental results of macrophage polarization regulated by conductive nanofiber scaffolds; in the figure, a is the immunofluorescence staining of macrophages with CD86, b is the quantitative fluorescence intensity of CD86, c is the immunofluorescence staining of macrophages with CD206, and d is the quantitative fluorescence intensity of CD206.

[0026] Figure 8 The image shows the results of macrophage extracellular acidification rate detection.

[0027] Figure 9 Figure 1 shows the in vitro experimental results of conductive nanofiber scaffold promoting osteogenic differentiation; in the figure, a is the alkaline phosphatase staining image, b is the alizarin red staining image, c is the quantitative analysis image of alkaline phosphatase activity, and d is the quantitative analysis image of absorbance.

[0028] Figure 10 The image shows the evaluation results of bone repair in a diabetic rat model of skull defects using conductive nanofiber scaffolds; in the figure, a is the Micro-CT three-dimensional reconstruction image, b is the quantitative analysis image of bone mineral density, and c is the quantitative analysis image of bone volume fraction.

[0029] Figure 11 The images show histological staining of conductive nanofiber scaffolds implanted in the skull defects of diabetic rats; in the images, a is HE staining and b is Masson staining.

[0030] Figure 12 The image shows the fibrosis assessment results after implantation of conductive nanofiber scaffolds into the skull defects of diabetic rats; in the figure, a is the Sirius red staining image; b is the α-SMA immunohistochemical staining image.

[0031] Figure 13 HE staining images of the heart, liver, spleen, lungs, and kidneys of diabetic rats 8 weeks after implantation of conductive nanofiber scaffolds into skull defects.

[0032] Figure 14 This is a diagram illustrating the mechanism of ternary regulation. In the diagram, a is a molecular feature data enrichment analysis diagram, b is a representative immunoblot diagram of PI3K / AKT signaling pathway regulation in bone marrow-derived macrophages under different treatment conditions, c is a representative immunoblot diagram of mTORC1 signaling pathway regulation in bone marrow-derived macrophages under different treatment conditions, and d is a representative immunoblot diagram of P-S6, S6, α-SMA, and KLF4 in fibroblasts under different treatment conditions. Detailed Implementation

[0033] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the embodiments of the present invention and the specific features in the embodiments are detailed descriptions of the technical solution of the present invention, rather than limitations thereof.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional experimental methods; unless otherwise specified, the reagents and materials used are commercially available.

[0035] Unless otherwise specified, the meanings of the abbreviations in this invention are as follows:

[0036] P represents a pure polyvinyl butyral (PVB) nanofiber scaffold; PP represents a conductive nanofiber scaffold formed by electrospinning a composite of polyvinyl butyral (PVB) and poly(3,4-ethylenedioxythiophene) and poly(styrene sulfonate) (PEDOT:PSS); IL peptide represents a multifunctional bioactive peptide with the structure [(DOPA)2-IEELEEELEER-(PEG4)-LYENRL-(DOPA)2]; PP@IL represents a conductive nanofiber scaffold functionalized with IL peptide; ES represents microelectrostimulation treatment; PP ES This indicates the PP stent combined with micro-electrical stimulation group; PP@IL ES This indicates the PP@IL stent combined with micro-electrical stimulation group.

[0037] The above abbreviations refer to the same technical terms in the specification, embodiments and drawings.

[0038] Example 1:

[0039] This embodiment is used to prepare a PVB / PEDOT:PSS conductive nanofiber scaffold PP. The specific steps are as follows:

[0040] Weigh out PVB powder and add it to anhydrous ethanol. Stir magnetically for 12 hours at room temperature to prepare a homogeneous PVB solution with a mass-volume concentration of 10% w / v.

[0041] Subsequently, 3% of the PVB mass was added to the PEDOT:PSS dispersion, and the mixture was shaken and stirred magnetically for 24 hours to ensure that the PEDOT:PSS was fully dispersed in the PVB spinning solution, thus obtaining a conductive electrospinning solution. Tests showed that when the PEDOT:PSS addition amount was within the range of 1% to 5% of the PVB mass, the scaffold could form a continuous fiber structure and maintain good conductivity.

[0042] The obtained spinning solution was loaded into a syringe, and electrospinning was performed under the following conditions: voltage 15 kV, feed rate 1.0 mL / h, receiving distance 15 cm, roller speed 500 rpm, ambient temperature 25±2℃, and relative humidity 40% to 50%. This yielded a PVB / PEDOT:PSS conductive nanofiber scaffold with a specific orientation structure, i.e., a PP scaffold. The obtained PP scaffold was vacuum dried for 24 hours to remove residual solvent and was then ready for use.

[0043] Example 2:

[0044] This embodiment is used to prepare a multifunctional bioactive peptide-modified conductive nanofiber scaffold PP@IL.

[0045] The IEELEEELEER and LYENRL peptides were synthesized via solid-phase synthesis. DOPA and PEG4 were then linked together via an amide bond formed by the reaction of amino and carboxyl groups to obtain a multifunctional bioactive peptide. This chemically synthesized peptide has the structure [(DOPA)2-IEELEEELEER-(PEG4)-LYENRL-(DOPA)2], specifically: (DOPA)-(DOPA)-Ile-Glu-Glu-Leu-Glu-Glu-Glu-Glu-Leu-Glu-Glu-Arg-(PEG4)-Leu-Tyr-Glu-Asn-Arg-Leu-(DOPA)-(DOPA). It achieves stable anchoring with the conductive nanofiber scaffold through the terminal double DOPA groups, exhibiting both anti-fibrotic and osteopromoting functions.

[0046] The IEELEEELEER fragment used in this embodiment is a biomimetic peptide sequence designed based on anti-fibrotic function. After being linked with the LYENRL fragment via PEG4, it synergistically achieves macrophage M2 polarization and fibroblast inhibition functions.

[0047] After purification by high-performance liquid chromatography, the purity of the IL peptide is not less than 95%. Figure 1 As shown in the figure, a is the molecular structure of IL peptide, and b is the high performance liquid chromatography detection report of IL peptide.

[0048] The IL peptide was dissolved in 10 mM Tris-HCl buffer to prepare a peptide fixation solution with a concentration of 1.0 mg / mL, and the pH was adjusted to 8.5.

[0049] The PP scaffold obtained in Example 1 was cut to the required size, sterilized by immersing in 75% ethanol for 30 minutes, and then washed three times with sterile PBS.

[0050] The sterilized PP scaffold was immersed in the above-mentioned IL peptide fixation solution and reacted with gentle shaking at 25°C in the dark for 24 hours.

[0051] During the reaction, the DOPA groups at both ends of the IL peptide adhere to the surface of PVB / PEDOT:PSS nanofibers through the catechol structure, while the functional structure of the peptide molecule is retained on the scaffold surface, thus forming a stable peptide functionalized modification layer.

[0052] After the reaction was completed, the scaffold was removed and washed three times each with PBS and deionized water to remove unbound free peptide molecules. Then it was vacuum dried at 37°C for 12 hours to obtain a multifunctional bioactive peptide-modified conductive nanofiber scaffold PP@IL.

[0053] When preparing the conductive nanofiber scaffold of the present invention, the concentration of polyvinyl butyral in anhydrous ethanol can be the electrospinning concentration conventional in the art, for example, a mass-volume concentration of 8% to 12%. The immobilization of the multifunctional bioactive peptides can be performed using Tris-HCl buffer, with a pH of 8.0 to 9.0, a peptide concentration of 0.5 to 2.0 mg / mL, a reaction temperature of 20°C to 30°C, and a reaction time of 12 to 24 hours.

[0054] The above parameter ranges can all achieve stable anchoring of peptides on the surface of conductive nanofiber substrates. The specific operating parameters given in Examples 1 and 2 of this invention are merely illustrative, and the scope of protection of the preparation method is not limited to these specific values.

[0055] Example 3:

[0056] This embodiment uses scanning electron microscopy, infrared spectroscopy, conductivity and peptide release kinetics to detect the scaffolds obtained in Embodiments 1 and 2.

[0057] like Figure 2 As shown in Figure a, scanning electron microscopy results revealed that the P-scaffold, PP-scaffold, and PP@IL-scaffold all exhibited continuous nanofiber structures with relatively regular fiber arrangement. The introduction of PEDOT:PSS and peptide modification slightly increased the fiber diameter, but did not disrupt the overall fiber structure of the scaffold. Figure 2 The energy spectrum shown in b and the atomic force microscopy roughness analysis diagram shown in c further confirm this result.

[0058] like Figure 3 The infrared spectrum shown in Figure a shows that, compared to the PP scaffold, the PP@IL scaffold exhibits a higher infrared spectrum at approximately 1650 cm⁻¹. -1 and 1540 cm -1 The presence of enhanced absorption peaks in amide I and amide II bands indicates that the IL peptide was successfully modified onto the surface of the conductive nanofibers.

[0059] Peptide drug release kinetic curves as follows Figure 3 As shown in Figure b, the peptide release rate was detected by high performance liquid chromatography, and the results showed that IL peptides on the surface of the PP@IL scaffold could be continuously released for about 14 days.

[0060] The four-probe method was used to detect the stent conductivity. The results are as follows: Figure 4 As shown, the conductivity of P in a pure PVB stent is less than 1.0 × 10⁻⁶. -8 The conductivity is S / cm, which is essentially non-conductive; the conductivity of the PP support is 1.32±0.21×10. -3 S / cm; The conductivity of the PP@IL stent is 9.84±1.07×10 -4 S / cm. The conductivity of the PP@IL scaffold remained at 8.71±0.95×10⁻⁶ after 7 days of PBS soaking. -4 The S / cm indicates that although IL peptide modification slightly reduced the conductivity, the scaffold still maintained stable conductivity, which can meet the needs of subsequent micro-electrical stimulation applications.

[0061] The micro-electrical stimulation application methods include in vitro cell experiments and in vivo animal experiments. In in vitro cell experiments, a scaffold seeded with cells is placed in a culture dish, and two parallel platinum electrodes are immersed in the culture medium. The electrodes are connected to a multifunctional electrical stimulator via wires. The applied voltage is 40 mV, each stimulation lasts 15 minutes, 1-2 times daily, and the stimulation waveform is a constant DC voltage. During stimulation, the cell culture incubator conditions are maintained at 37°C and 5% CO2.

[0062] In vivo animal experiments used a subcutaneously implanted micro-electrical stimulation device for micro-electrical stimulation. A small constant-voltage stimulation module was implanted subcutaneously in the back of diabetic rats, with positive and negative electrodes connected to the cortical bone on both sides of the skull defect area via insulated wires. The stimulation parameters were 40 mV, 15 minutes per session, 1-2 times daily, for 4 or 8 weeks. The sham-operated group received the same device but without electrical stimulation.

[0063] The exogenous micro-electric stimulation parameters of the present invention are not limited to 40 mV, 15 minutes, and once daily as described in the above embodiments. Those skilled in the art should understand that the conductive nanofiber scaffold of the present invention can produce a synergistic effect with electrical stimulation under conditions of 20–80 mV voltage range, 5–30 minutes per stimulation session, and 1–2 times daily.

[0064] Compare with Example 1:

[0065] This comparative example prepared a pure PVB nanofiber scaffold P without PEDOT:PSS. The scaffold was prepared according to the method of Example 1, but without the addition of PEDOT:PSS. Electrospinning was performed using only a 10% w / v PVB solution to obtain the pure PVB nanofiber scaffold P, which was used to evaluate the effect of the conductive component PEDOT:PSS on the conductivity and biological effects of the scaffold.

[0066] Compare with Example 2:

[0067] This control example prepared a peptide-free conductive nanofiber scaffold (PP). The PP scaffold was prepared according to the method of Example 1, but without the IL peptide modification described in Example 2. This control set was used to evaluate the effects of multifunctional bioactive peptide modification on macrophage polarization, BMSC osteogenic differentiation, and bone defect repair.

[0068] Compare with Example 3:

[0069] This comparative example prepared a conductive nanofiber scaffold combined with electrical stimulation to PP. ESPP scaffolds were prepared according to the method in Example 1, and combined with micro-electrical stimulation of 40 mV for 15 min / time in in vitro or in vivo experiments to obtain PP scaffolds. ES This group was used to evaluate the biological effects of conductive scaffolds combined with electrical stimulation but without peptide modification.

[0070] Compare with Example 4:

[0071] This control group prepared a PP@IL non-electrical stimulation group. The PP@IL scaffold was prepared according to the method in Example 2, but no micro-electrical stimulation was applied in in vitro and in vivo experiments. This group was used to evaluate the effect of multifunctional peptide modification itself on cell behavior and bone repair.

[0072] Example 4:

[0073] In this embodiment, cell compatibility experiments were conducted on conductive nanofiber scaffolds by seeding bone marrow mesenchymal stem cells and bone marrow-derived macrophages onto different scaffold surfaces for culture.

[0074] The results of the biocompatibility experiment of the conductive nanofiber scaffold with bone marrow mesenchymal stem cells are as follows: Figure 5 As shown, a is a live / dead staining immunofluorescence image, and b is a statistical graph of the percentage of live and dead cells. Figure 5 It can be seen that the bone marrow mesenchymal stem cells on the surface of each scaffold grew well and no obvious cell death was observed, indicating that the scaffold has good cell compatibility.

[0075] The results of the biocompatibility experiment of bone marrow-derived macrophages using conductive nanofiber scaffolds are as follows: Figure 6 As shown in the figure, a is the immunofluorescence image of live and dead cells, and b is the statistical graph of the percentage of live and dead cells. It can be seen that the macrophages derived from bone marrow also showed good survival status on the surface of each scaffold, which further confirmed the cell compatibility of the scaffold.

[0076] Example 5:

[0077] This embodiment investigates the effect of conductive nanofiber scaffolds combined with micro-electrical stimulation on macrophage polarization. Bone marrow-derived macrophages were seeded onto different scaffold surfaces and cultured in a high-glucose / LPS-induced inflammatory microenvironment. Experimental groups included the P group, PP group, and PP group. ES Group, PP@IL group and PP@IL ES Group. Among them, PP ES Group and PP@IL ES The group was given micro-electrical stimulation of 40 mV for 15 min / time.

[0078] like Figure 7 Immunofluorescence staining images of a and c in the middle and Figure 7 The fluorescence intensity quantification plots for groups b and d show that, compared with groups P and PP, PP... ESGroup and PP@IL ES CD86 expression was decreased and CD206 expression was increased in the group, including PP@IL. ES The changes were most significant in group PP@IL. This indicates that the PP@IL scaffold combined with microelectrostimulation can significantly inhibit M1 inflammatory polarization and promote the transformation of macrophages to the M2 repair phenotype.

[0079] Results of macrophage extracellular acidification rate detection are as follows Figure 8 As shown, according to Figure 8 It can be seen that PP@IL ES The glycolytic metabolism level of macrophages in the group was significantly reduced, suggesting that their metabolic mode has shifted towards oxidative phosphorylation.

[0080] Example 6:

[0081] This embodiment conducts an in vitro experiment to regulate macrophage polarization and promote osteogenic differentiation. Specifically, it investigates the effect of conductive nanofiber scaffolds combined with micro-electrical stimulation on osteogenic differentiation of bone marrow mesenchymal stem cells. Different groups of macrophage conditioned medium were collected to induce osteogenic differentiation of bone marrow mesenchymal stem cells.

[0082] contrast Figure 9 Image a shows the alkaline phosphatase staining pattern, and image c shows the quantitative analysis of alkaline phosphatase activity. It can be seen that PP@IL ES The alkaline phosphatase activity in the group was significantly higher than that in the PP group and the PP group. ES Group and PP@IL group. Comparison Figure 9 b is the alizarin red staining image, and d is the quantitative analysis image of absorbance at 420 nm, indicating that PP@IL ES The mineralized nodules in the group have the largest area.

[0083] The results of this embodiment indicate that the PP@IL scaffold combined with micro-electrical stimulation can further promote osteogenic differentiation of bone marrow mesenchymal stem cells by remodeling the macrophage immune microenvironment.

[0084] Example 7:

[0085] This embodiment conducts an in vivo experiment on the repair of diabetic cranial defects, specifically investigating the effect of conductive nanofiber scaffolds combined with micro-electrical stimulation on the repair of cranial defects in diabetic rats. A 5 mm cranial defect model was established in diabetic rats, and different scaffolds were implanted into the cranial defect area. Experimental groups included a model group, a P group, a PP group, and a PP... ES Group, PP@IL group and PP@IL ES Group. Micro-CT was used to evaluate bone defect repair at 4 and 8 weeks postoperatively.

[0086] The results are as follows Figure 10 The Micro-CT 3D reconstruction image shown in Figure a is compared with the model group, P group, PP group, and PP... ESCompared with the PP@IL group, the PP@IL group ES The newly formed bone tissue fills the bone defect area more completely, resulting in a superior in vivo bone regeneration and repair effect.

[0087] Figure 10 The quantitative analysis graphs of bone mineral density (b) and bone volume fraction (c) further confirm that PP@IL ES The group exhibits superior in vivo bone regeneration and repair effects.

[0088] Figure 11 This is a histological staining image of a conductive nanofiber scaffold implanted in vivo into a diabetic rat skull defect, with HE staining as shown. Figure 11 As shown in Figure a, PP@IL ES The defect area showed better continuity of newly formed bone tissue and less fibrous scar tissue; Masson staining showed... Figure 11 As shown in b, PP@IL ES The collagen deposits are denser and more maturely arranged.

[0089] The results of fibrosis assessment after in vivo implantation of conductive nanofiber scaffolds into diabetic rats with skull defects are as follows: Figure 12 As shown, according to Figure 12 As shown in image a, the Sirius red staining pattern, and image b, the α-SMA immunohistochemical staining pattern, indicate that PP@IL ES The group with the lowest degree of fibrosis indicates that the PP@IL scaffold combined with micro-electrical stimulation can promote new bone formation and bone tissue maturation.

[0090] Example 8:

[0091] This embodiment evaluates the in vivo biocompatibility of the conductive nanofiber scaffold. Major organs (heart, liver, spleen, lung, and kidney) from rats in each experimental group of Example 7 were collected and stained with hematoxylin and eosin (HE). The results are as follows: Figure 13 As shown, HE staining of the major organs revealed no obvious inflammatory infiltration, tissue necrosis, or structural damage, indicating that the PP@IL scaffold and its combined application with micro-electrical stimulation have good in vivo biocompatibility.

[0092] Example 9:

[0093] This embodiment explores the ternary regulatory mechanism of conductive nanofiber scaffold combined with micro-electrical stimulation by verifying single-cell transcriptomics and signaling pathways.

[0094] like Figure 14 The molecular feature data enrichment analysis plot shown in Figure a is PP@IL. ES The mTORC1 signaling pathway was significantly suppressed in the treatment group. According to... Figure 14 The immunoblot images of b and c show that PP@IL ESAfter treatment, the expression of proteins related to the PI3K / AKT / mTORC1 signaling pathway was downregulated in bone marrow-derived macrophages.

[0095] Figure 14 In the middle section (d), representative immunoblot images of P-S6, S6, α-SMA, and KLF4 in fibroblasts under different treatment conditions are shown. PP@IL ES After treatment, the expression of P-S6 and α-SMA (smooth muscle actin) in fibroblasts was downregulated, while the expression of KLF4 was upregulated. This indicates that the conductive nanofiber scaffold promotes the transition of macrophages from glycolysis to oxidative phosphorylation by inhibiting mTORC1, thereby maintaining the M2 repair phenotype, and inhibits pathological fibrosis by regulating mTORC1 / α-SMA.

[0096] As can be seen from the above embodiments and comparative examples, the present invention constructs an IL peptide-functionalized conductive nanofiber scaffold and combines it with micro-electrical stimulation to form PP@IL. ES This treatment system can synergistically regulate macrophage polarization, improve the local immune microenvironment, promote BMSC osteogenic differentiation, and significantly enhance the repair effect of diabetic bone defects in the diabetic inflammatory microenvironment. Compared with peptide-free scaffolds and non-electrical stimulation groups, the technical solution of this invention can more fully utilize the synergistic effect between conductive scaffolds, functional peptides, and micro-electrical stimulation, and has clear technical effects and application value.

[0097] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. For those skilled in the art, any equivalent substitutions, improvements, or modifications made to the material ratio, peptide concentration, stimulation parameters, scaffold shape, and application site without departing from the technical principles of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A conductive nanofiber scaffold, characterized in that, The conductive nanofiber scaffold comprises a conductive nanofiber substrate formed by electrospinning a composite of polyvinyl butyral and poly(3,4-ethylenedioxythiophene) and poly(styrene sulfonate), and a multifunctional bioactive peptide fixed on the surface of the conductive nanofiber substrate. The structure of the multifunctional bioactive peptide is: [(DOPA)2-IEELEEELEER-(PEG4)-LYENRL-(DOPA)2], wherein DOPA is 3,4-dihydroxyphenylalanine and PEG4 is polyethylene glycol tetrakis.

2. The bracket according to claim 1, characterized in that, The multifunctional bioactive peptide is anchored to the surface of a conductive nanofiber substrate via terminal double DOPA groups, and the conductive nanofiber substrate has an oriented structure.

3. The bracket according to claim 1, characterized in that, The amount of the poly(3,4-ethylenedioxythiophene) and poly(styrene sulfonate) composite added is 1% to 5% of the mass of polyvinyl butyral.

4. The application of the stent according to any one of claims 1 to 3 in the manufacture of medical devices, characterized in that, The medical device is used in conjunction with exogenous micro-electrical stimulation to treat diabetic bone defects.

5. The application according to claim 4, characterized in that, The voltage of the exogenous micro-electric stimulation is 20mV to 80mV, the stimulation time is 5 to 30 minutes each time, and the stimulation frequency is 1 to 2 times per day.

6. The application according to claim 4, characterized in that, The diabetic bone defects include skull defects, jaw defects, or long bone defects in a diabetic state, as well as bone defects with delayed fracture healing, nonunion, or accompanied by high inflammation or high fibrosis.

7. A method for preparing the stent according to claim 1, characterized in that, Includes the following steps: Polyvinyl butyral was dissolved in anhydrous ethanol, and a composite of poly(3,4-ethylenedioxythiophene) and poly(styrene sulfonate) was added to obtain a spinning solution. The spinning solution was then electrospun to obtain a conductive nanofiber substrate. The conductive nanofiber substrate was immersed in a solution containing multifunctional bioactive peptides to react, thereby immobilizing the multifunctional bioactive peptides on the surface of the conductive nanofiber scaffold. After drying, the conductive nanofiber scaffold was obtained.

8. The method according to claim 7, characterized in that, The electrospinning conditions are as follows: voltage 10–20 kV, feed speed 0.5–2.0 mL / h, receiving distance 10–20 cm, and roller speed 300–800 rpm.

Images