Coaxial electrostatic spinning stent coupled with electret electrical stimulation and atorvastatin, preparation of coaxial electrostatic spinning stent and application of coaxial electrostatic spinning stent in bone defect vascularized bone regeneration

Abstract

The invention discloses a coaxial electrostatic spinning stent coupled with electret electrical stimulation and an angiogenic drug, which utilizes the characteristic of sequential degradation (slow degradation of a core layer and relatively fast degradation of a shell layer) of a core-shell structure of a coaxial electrostatic spinning technology, and takes a PCL material which is slowly degraded as a matrix of the stent, thereby being beneficial to maintaining a space necessary for bone regeneration. AVT with a good angiogenesis promoting effect is loaded in a shell layer, the hydrophilicity of the PCL stent is improved, meanwhile, early-stage ordered release of the shell layer AVT is utilized to promote early-stage angiogenesis, SiO2 is loaded in a coaxial electrostatic spinning core layer, the influence of the surrounding body fluid environment is avoided, and electret electrical stimulation for promoting osteogenesis is continuously provided. The hydrophilic stent with the'electricity and medicine 'integrated core-shell fiber structure prepared by the invention is good in overall hydrophilicity and slow in degradation, continuous electrical stimulation promotes osteogenesis, early released AVT promotes early vascularization, and ultimately, skull critical bone defect vascularization bone regeneration is promoted.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and relates to a coaxial electrospun scaffold coupled with electret electric stimulation and atorvastatin, preparation and application in vascularized bone regeneration of bone defect. BACKGROUND

[0002] Electrophysiological microenvironment is of great significance to maintain normal physiological activities of human body. Endogenous electric field has been found in heart, nerve and bone tissue, and is involved in organ development, wound healing and tissue regeneration. Electret material is a kind of dielectric material characterized by permanent polarization. After being polarized by external electric field, the polarization charge of electret material remains inside or on the surface, forming a stable built-in potential, which provides endogenous electric stimulation. At the same time, electret material has the advantages of wide selection range and low cost. Due to the natural electret characteristics of bone tissue and the positive effect of electret on promoting bone growth, bone repair materials based on electret electric stimulation have potential in the application of critical size bone defect repair.

[0003] Silicon dioxide (SiO2) as a representative of inorganic silicon-based electret material is widely studied and applied due to its high stability and long service life. At the same time, SiO2 has good biological safety and can be used as a food additive. In addition, SiO2 can provide stable and persistent surface potential at the implant site after polarization, thereby simulating the endogenous electrophysiological microenvironment, which is conducive to the activation of osteoblasts and bone tissue regeneration.

[0004] Due to the fact that the bone tissue in most parts of the craniofacial region has no strong attachment of masticatory muscles, the use of piezoelectric stimulation to promote bone in these areas often requires external stimulation such as ultrasound or magnetic field, which increases the complexity and uncertainty of treatment. At this time, the stable potential of electret after polarization can provide continuous electric stimulation and reconstruct the electrophysiological microenvironment of the bone defect site, which can help to promote bone regeneration without the need for external stimulation.

[0005] During bone healing, early blood vessel formation can transport nutrients necessary for cell survival and clean metabolic products; however, in the treatment of large area bone defect, the central region of the graft often has insufficient angiogenesis and hypoxia, which affects cell survival and differentiation, thereby leading to treatment failure. In order to promote the rapid and effective vascularization of the graft material, a series of strategies have been developed, mainly including the application of pro-angiogenic factors, cell co-culture system, biological fabrication of blood vessel network and "bioreactor" combining tissue engineering with surgery.

[0006] Angiogenic growth factors are most commonly used due to their relatively simple use, availability of commercial products, and the ability to combine them with various osteogenic strategies. In recent decades, a number of angiogenic growth factors have been discovered, such as the VEGF family, FGF, Angiopoietin (ANG), Platelet-derived growth factor (PDGF), etc. In order to induce in vivo angiogenesis in tissue engineering applications, angiogenic growth factors are often mixed on various scaffold materials (such as hydrogels or solid scaffolds), which can act as drug release systems after implantation. Angiogenic growth factors are often used in combination with osteogenic factors to enhance bone tissue formation. Although many studies have shown that the use of angiogenic growth factors has significant benefits, the therapeutic effect is often affected due to the short half-life of recombinant angiogenic growth factors and the problems in achieving the correct spatial and temporal release kinetics. At the same time, VEGF also has the risk of stimulating hemangioma or tumor recurrence, especially for patients after radiotherapy or tumor resection. In addition, such growth factors are often expensive.

[0007] In order to overcome the shortcomings of the above-mentioned growth factors, a large number of studies are actively seeking alternative drugs. Statins are structural analogs of 3-hydroxy-3-methylglutaryl coenzyme A, which can competitively inhibit HMG-CoA reductase, which is responsible for the first step of sterol biosynthesis. Atorvastatin (AVT) is the fifth statin approved by the US FDA for the treatment of patients with high cholesterol, which is found to reduce major cardiovascular events in patients with coronary heart disease, ischemic stroke, diabetes, and cardiovascular disease in patients at high risk of hypertension, promote angiogenesis, and has good safety. This efficacy and safety have been confirmed in more than 400 clinical trials and 2.3 billion patients in clinical experience. In addition, atorvastatin has no oral-related side effects under low-dose local application.

[0008] Therefore, based on the potential of SiO2 as an electret in promoting osteogenesis and the advantages of AVT in promoting angiogenesis, the combination of the two is expected to be applied in the repair of critical-sized bone defects. SUMMARY

[0009] In order to overcome the above-mentioned defects of the prior art, the application prepares a novel coaxial electrospinning scaffold (SiO2 / AVT) coupled with electret electric stimulation and angiogenic drugs by coaxial electrospinning technology, loads the clinical drug atorvastatin AVT which promotes angiogenesis in the fiber shell layer, loads the electret material SiO2 which promotes osteogenic differentiation in the fiber core layer, and after electric field polarization, uses the electric stimulation provided by the core layer SiO2 electret to promote osteogenesis, uses the AVT in the fiber shell layer to promote early blood vessel formation, and finally realizes vascularized bone regeneration, which is a good strategy for repairing critical bone defects.

[0010] The difficulties to be overcome by the application mainly include poor hydrophilicity of the scaffold material, early loss of functional components caused by degradation, unstable electric stimulation caused by the interference of the body fluid environment on the electret, difficulties in short half-life of angiogenic active factors and insufficient early vascularization, and screening of SiO2 and AVT concentrations which meet good biological safety and have the best osteogenic / angiogenic effect. Conventional electric stimulation equipment is cumbersome, inconvenient to use, cannot be well worn (such as the craniofacial region), and electric stimulation is not easy to control, and for most craniofacial bones (such as the skull, maxilla, zygoma, etc.) without strong muscle attachment, scaffolds using piezoelectric materials or conductive materials need additional power, ultrasonic or magnetic field stimulation, which increases the complexity and uncertainty of treatment. The electret material SiO2 has high stability and long service life, and good biological safety, so it has application potential as an electrically active material in bone defect repair. By polarizing SiO2 with suitable biological safety concentration, a stable and long-lasting surface potential can be provided at the implant site, thereby simulating the endogenous electrophysiological microenvironment, which is beneficial to the activation of osteoblasts and bone tissue regeneration. The clinical drug AVT has been proven to be safe for the human body in extensive clinical experiments and applications, and low-dose application not only can promote angiogenesis, but also has no side effects of systemic application, can avoid the shortcomings of expensive, short half-life and potential safety of various pro-angiogenic factors (such as VEGF), and has good application prospect. When the application proposes this technical scheme, the slow-degrading PCL material is used as the matrix, which can maintain the necessary growth space for bone regeneration during the critical period of bone defect repair, but has the disadvantage of hydrophobicity. By using the sequential degradation characteristics of the core-shell structure of coaxial electrospinning (slow degradation of the core layer and relatively fast degradation of the shell layer), AVT with good pro-angiogenic effect is loaded in the shell layer, which can increase the hydrophilicity of the scaffold, and its early release behavior can promote early angiogenesis; SiO2 is loaded in the core layer of the coaxial electrospinning, which avoids the influence of the surrounding body fluid environment and continuously provides electret electric stimulation to promote osteogenesis, and finally promotes vascularized bone regeneration of skull critical bone defects.

[0011] The application provides a pharmaceutical composition, which at least comprises AVT and SiO2.

[0012] The ratio of the AVT and SiO2 is 0.25%-4%:1%-2%, preferably 4%:2%.

[0013] In the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0014] Further, the pharmaceutical composition can be used alone and / or in combination with other drugs.

[0015] Preferably, the pharmaceutically acceptable carrier refers to substances that do not produce adverse, allergic or other untoward reactions when administered to an animal or human in the appropriate amount. The pharmaceutically acceptable carrier can include, but is not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methyl cellulose, ethyl cellulose and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; coloring agents; flavoring agents; tabletting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffer solutions, etc. These substances are used as needed to help the stability of the formulation or to help improve the activity or its bioavailability or to produce an acceptable taste or smell in the case of oral administration.

[0016] Specifically, the pharmaceutical composition can further comprise a physiologically compatible excipient, which includes a buffer, a diluent, an excipient, a filler, a binder, a humectant, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, etc.

[0017] Specifically, the pharmaceutical composition can be prepared into an injection, a sterile powder for injection, a tablet, a pill, a capsule, a lozenge, a liquor, a powder, a granule, a syrup, a solution, a tincture, an aerosol, a powder aerosol, or a suppository, etc. The pharmaceutical composition in the above-mentioned various dosage forms can be prepared according to the conventional methods in the pharmaceutical field.

[0018] Specifically, the pharmaceutical composition can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spraying, nose drops, eye drops, penetration, absorption, physical or chemical mediated methods; or is mixed or wrapped with other substances and introduced into the body. Preferably, the administration is by injection. The pharmaceutical composition can also be used in combination with other treatment methods, which include surgery, radiotherapy, chemotherapy, targeted therapy.

[0019] The application also provides a coaxial electrospun scaffold coupled with electret electric stimulation and angiogenic drugs, comprising a core layer and a shell layer, wherein the core layer comprises SiO2 and a first matrix material; and the shell layer comprises AVT and a second matrix material.

[0020] Preferably, the first matrix material is polycaprolactone (PCL).

[0021] Preferably, the first matrix material such as PCL serves as the matrix material of the scaffold and simultaneously as the carrier of SiO2 and AVT.

[0022] Preferably, the number average molecular weight Mn of the PCL is 60000-100000; more preferably, the number average molecular weight Mn of the PCL is 60000-80000, or 75000-95000, or 80000-100000; and most preferably, the number average molecular weight Mn of the PCL is 800000.

[0023] Preferably, the second matrix material is polycaprolactone (PCL).

[0024] Preferably, the second matrix material such as PCL serves as the matrix material of the scaffold and simultaneously as the carrier of SiO2 and AVT.

[0025] Preferably, the number average molecular weight Mn of the PCL is 60000-100000; more preferably, the number average molecular weight Mn of the PCL is 60000-80000, or 75000-95000, or 80000-100000; and most preferably, the number average molecular weight Mn of the PCL is 800000.

[0026] Preferably, the mass ratio of the SiO2 and the first matrix material is 1%-2% (w / w): 16%; and more preferably, the mass ratio of the SiO2 and the first matrix material is 2%: 16% PCL.

[0027] Preferably, the mass ratio of the AVT and the second matrix material is 0.25%-4% (w / w): 16%; and more preferably, the mass ratio of the AVT and the second matrix material is 4%: 16%.

[0028] Preferably, the coaxial electrospun scaffold has a thickness of 0.1 mm.

[0029] Preferably, the coaxial electrospun scaffold has an elastic modulus (Young's modulus) of 44.24 MPa ± 2.22 MPa.

[0030] Preferably, the coaxial electrospun scaffold has a tensile strength of 2.38 MPa ± 0.12 MPa.

[0031] The degradation rate of the coaxial electrospinning scaffold is: 0.49%±0.16% when immersed in PBS at 37 DEG C for 7 days; the degradation rate is: 2.04%±0.84% when immersed in PBS for 28 days.

[0032] The application further provides a preparation method of the coaxial electrospinning scaffold coupled with electret electric stimulation and angiogenic drugs, and specifically comprises the following steps:

[0033] The raw material of the core layer is dispersed in a first dispersing agent to obtain a core layer solution;

[0034] The raw material of the shell layer is dispersed in a second dispersing agent to obtain a shell layer solution;

[0035] The core layer solution and the shell layer solution are coaxially electrospun to form the core layer and the shell layer, thereby obtaining the coaxial electrospinning scaffold.

[0036] In the application, the coaxial electrospinning technology is as follows: the shell layer solution and the core layer solution are respectively loaded into two different syringes, a spinning system is composed of two coaxial but different inner diameter capillary tubes, under the action of a high voltage electric field, the shell layer solution and the core layer solution converge, are stretched and received by a receiving device roller, and then a fiber filament bundle with a core-shell structure is formed and is wound on the surface of the roller to form an electrospinning scaffold.

[0037] The core layer comprises SiO2 and a first matrix material; and the shell layer comprises AVT and a second matrix material.

[0038] The first matrix material is one or more of polycaprolactone (PCL), poly-L-lactic acid (PLLA), polylactic acid (PLA), etc.; and preferably, the first matrix material is polycaprolactone (PCL).

[0039] The first matrix material such as PCL serves as a matrix material of the scaffold and simultaneously serves as a carrier of SiO2 and AVT.

[0040] The number average molecular weight Mn of the PCL is 60000-100000; preferably, the number average molecular weight Mn of the PCL is 60000-80000, or 75000-95000, or 80000-100000; and further preferably, the number average molecular weight Mn of the PCL is 800000.

[0041] The second matrix material is one or more of polycaprolactone (PCL), poly-L-lactic acid (PLLA), polylactic acid (PLA), etc.; and preferably, the second matrix material is polycaprolactone (PCL).

[0042] The second matrix material such as PCL serves as a matrix material of the scaffold and simultaneously serves as a carrier of SiO2 and AVT.

[0043] The PCL has a number average molecular weight of Mn of 60000-100000; preferably, the PCL has a number average molecular weight of Mn of 60000-80000, or 75000-95000, or 80000-100000; further preferably, 800000.

[0044] The mass ratio of the SiO2 and the first matrix material is 1%-2% (w / w): 16%; preferably, 2%: 16% PCL.

[0045] The mass ratio of the AVT and the second matrix material is 0.25%-4% (w / w): 16%; preferably, 4%: 16%.

[0046] The ratio of the use amount of the first matrix material and the first dispersant = 16% (w / v).

[0047] The ratio of the use amount of the second matrix material and the second dispersant = 16% (w / v).

[0048] The first dispersant is hexafluoroisopropanol.

[0049] The second dispersant is hexafluoroisopropanol.

[0050] The added amount of the SiO2 in the first dispersant is 3.2 g / L.

[0051] The added amount of the AVT in the second dispersant is 6.4 g / L.

[0052] The needle voltage is +7 kV.

[0053] The receiving roller voltage is -8 kV.

[0054] The receiving distance is 15 cm.

[0055] The roller rotation speed is 10 rpm / min.

[0056] The propelling speed of the shell layer injector is set to 1 mL / h.

[0057] The propelling speed of the core layer injector is set to 0.5 mL / h.

[0058] The spinning time is 3 h.

[0059] In one specific embodiment, the preparation of the coaxial electret electrostimulation and angiogenic drug coupled electrospun scaffold includes the following steps:

[0060] Prepare a light-proof glass bottle, weigh 0.064 g of atorvastatin (AVT) into the bottle, then add 10 mL of hexafluoroispropanol (HFIP) into the bottle respectively, and use a magnetic stirrer to stir at room temperature for 20 min until completely dissolved, then add 1.6 g of polycaprolactone (PCL), and prepare a 16% (w / v) PCL / HFIP solution containing 4% (w / w) AVT / PCL concentration, and stir overnight at room temperature on a magnetic stirrer until the PCL particles are completely dissolved as the electrospinning working solution.

[0061] Prepare a light-proof glass bottle, weigh 0.064 g of atorvastatin (AVT) into the bottle, then add 10 mL of hexafluoroispropanol (HFIP) into the bottle respectively, and use a magnetic stirrer to stir at room temperature for 20 min until completely dissolved, then add 1.6 g of polycaprolactone (PCL), and prepare a 16% (w / v) PCL / HFIP solution containing 4% (w / w) AVT / PCL concentration, and stir overnight at room temperature on a magnetic stirrer until the PCL particles are completely dissolved as the electrospinning working solution.

[0062] Use a 5 mL syringe to draw the above working solution and install it into the electrospinning machine, use a coaxial electrospinning needle (shell layer 22G, core layer 17G) as the positive electrode, and an aluminum foil wrapped around the roller-shaped collector as the receiving substrate and connected as the negative electrode. The needle voltage is +7 kV, the receiving roller voltage is -8 kV, the receiving distance is 15 cm, and the roller speed is 10 rpm / min. Set the push speed of the shell layer syringe to 1 mL / h, and the push speed of the core layer syringe to 0.5 mL / h, and the spinning time is 3 h, to prepare a SiO2 / AVT coaxial electrospinning scaffold with a 4% AVT shell layer and a 2% SiO2 core layer.

[0063] The application also provides the use of the composition or the coaxial electrospinning scaffold in the preparation of a product for promoting angiogenesis and / or osteogenic differentiation.

[0064] The application also provides the use of the coaxial electrospinning scaffold in the preparation of a product for promoting bone defect repair.

[0065] The application also provides the use of the coaxial electrospinning scaffold in the preparation of a product for promoting vascularized bone regeneration of skull defects.

[0066] The beneficial effects of the application include: the application utilizes the characteristics of the time sequence degradation of the core-shell structure by coaxial electrospinning technology (the core layer degrades slowly, and the shell layer degrades relatively fast), the slowly degradable PCL material is used as the matrix of the scaffold, which is beneficial to maintain the space necessary for bone regeneration, the AVT with good effect of promoting angiogenesis is loaded in the shell layer, the hydrophilicity of the PCL scaffold is increased, and the early ordered release of the AVT in the shell layer promotes early angiogenesis, the SiO2 is loaded in the core layer of the coaxial electrospinning, the influence of the surrounding body fluid environment is avoided, and the electrostimulation for promoting osteogenesis is continuously provided. The hydrophilic scaffold with the integrated core-shell fiber structure prepared by the application has good overall hydrophilicity, slow degradation, continuous piezoelectric stimulation for promoting osteogenesis, and early release of AVT for promoting early vascularization, and finally promotes the vascularized bone regeneration of the skull critical bone defect. BRIEF DESCRIPTION OF DRAWINGS

[0067] Figure 1 Schematic diagram of the coaxial electrospun scaffold with electret electrostimulation coupled with AVT promoting the vascularized bone regeneration of the skull defect; wherein, A. preparation and polarization of the coaxial electrospun scaffold with SiO2 loaded in the core layer and AVT loaded in the shell layer; B. the coaxial electrospun scaffold promotes the osteogenic differentiation of BMSCs and the vascular differentiation of HUVECs through electrostimulation and AVT, and finally realizes the vascularized bone regeneration of the skull critical defect.

[0068] Figure 2 CCK-8 experiment of HUVECs under different AVT concentrations.

[0069] Figure 3 Tube formation experiment of HUVECs under different AVT concentrations, A. tube staining; B. semi-quantitative analysis.

[0070] Figure 4 Surface potential of the coaxial electrospun scaffold loaded with different concentrations of SiO2.

[0071] Figure 5 CCK-8 experiment of BMSCs cultured on the surface of the coaxial electrospun scaffold loaded with different concentrations of SiO2.

[0072] Figure 6 ALP detection of BMSCs cultured on the surface of the coaxial electrospun scaffold loaded with different concentrations of SiO2, A. ALP staining on the 4th day of culture; B. semi-quantitative detection of ALP activity on the 4th day of culture; C. ALP staining on the 7th day of culture; D. semi-quantitative detection of ALP activity on the 7th day of culture.

[0073] Figure 7 Surface morphology, internal structure and fiber diameter distribution of the coaxial electrospun scaffold,

[0074] A. Scanning electron microscope image; B. Transmission electron microscope image; C. Fiber diameter distribution.

[0075] Figure 8 Hydrophilicity characterization of coaxial electrospun scaffolds,

[0076] A. Water contact angle at 2s; B. Water contact angle at 25s.

[0077] Figure 9 Mechanical property characterization of coaxial electrospun scaffolds,

[0078] A. Stress-strain curve; B. Elastic modulus; C. Tensile strength.

[0079] Figure 10 Degradation curve of coaxial electrospun scaffolds at 28 days.

[0080] Figure 11 AVT release curve of coaxial electrospun scaffolds at 21 days.

[0081] Figure 12 Surface potential of coaxial electrospun scaffolds.

[0082] Figure 13 Biocompatibility of coaxial electrospun scaffolds,

[0083] A. BMSCs adhesion on coaxial electrospun scaffolds after 24h culture; B. Effect of coaxial electrospun scaffolds on BMSCs proliferation; C. Effect of coaxial electrospun scaffolds on HUVECs proliferation.

[0084] Figure 14 Effect of coaxial electrospun scaffolds on HUVECs tube formation ability,

[0085] A. Tube formation experiment; B. Quantitative analysis of tube number per field; C. Quantitative analysis of tube cross number.

[0086] Figure 15 Effect of coaxial electrospun scaffolds on BMSCs ALP activity,

[0087] A. ALP staining at 4th day culture; B. Semi-quantitative detection of ALP activity at 4th day culture; C. ALP staining at 7th day culture; D. Semi-quantitative detection of ALP activity at 7th day culture.

[0088] Figure 16 HE staining of organs after 4 weeks of subcutaneous implantation of coaxial electrospun scaffolds,

[0089] (Liver: liver; Heart: heart; Lung: lung; Spleen: spleen; Kidney: kidney).

[0090] Figure 17 Surgical procedure of rat calvarial critical size bone defect model.

[0091] Figure 18 Micro-CT images of rat calvaria, quantitative analysis of new bone and VG staining at 8 weeks after operation,

[0092] A. Micro-CT three-dimensional reconstruction image; B. Micro-CT coronal image; C. VG staining image; D. BV / TV; E. Quantitative analysis of osteogenesis area.

[0093] Figure 19 Masson and HE staining of calvaria at 8 weeks after operation;

[0094] A. Masson staining; B. HE staining; the arrow shows the bone marrow cavity and red blood cells.

[0095] Figure 20 Sequential fluorescence labeling and quantitative analysis of bone regeneration in calvarial defects at 8 weeks after operation;

[0096] A. Sequential fluorescence labeling image; B. Quantitative analysis of fluorescence area. DETAILED DESCRIPTION

[0097] The present application will be further described in conjunction with the following specific examples and drawings, and the protection scope of the present application is not limited to the following examples. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and are protected by the appended claims. The process, conditions, reagents, experimental methods, etc. for implementing the present application, except for the following specifically mentioned contents, are the general knowledge and common sense in the art, and the present application has no special limitation.

[0098] The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way intended to limit the application or its application or uses. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0099] In all examples shown and discussed here, any specific value should be interpreted as merely exemplary, not as a limitation. Therefore, other examples of exemplary embodiments can have different values.

[0100] Example 1: Coaxial electrospun scaffolds with different concentrations of AVT in the shell layer and different concentrations of SiO2 in the core layer were prepared, and through in vitro biological experiments, the concentrations of AVT and SiO2 that exhibit the best pro-angiogenic and pro-osteogenic properties were selected, respectively.

[0101] 1.1 Preparation of AVT electrospinning working solution with different concentrations

[0102] Five light-proof glass bottles were prepared and labeled for grouping. 0 g, 0.004 g, 0.016 g, 0.064 g, and 0.256 g of atorvastatin (AVT) were weighed into the bottles, respectively. Then 10 mL of hexafluoroispropanol (HFIP) was added to each bottle, and the mixture was stirred at room temperature for 20 min using a magnetic stirrer until the AVT was completely dissolved. Then 1.6 g of polycaprolactone (PCL) was added to each bottle to prepare 16% (w / v) PCL / HFIP solutions containing 0%, 0.25%, 1%, 4%, and 16% (w / w) AVT / PCL, respectively. The solutions were stirred overnight at room temperature on a magnetic stirrer until the PCL particles were completely dissolved, and then used as the electrospinning working solution.

[0103] 1.2 Preparation of SiO2 electrospinning working solution with different concentrations

[0104] Four light-proof glass bottles were prepared and labeled for grouping. 0 g, 0.1 g, 0.2 g, and 0.4 g of SiO2 nanoparticles were weighed into the bottles, respectively. Then 10 mL of HFIP was added to each bottle, and the mixture was subjected to ultrasonic treatment for 20 min to ensure uniform dispersion of the SiO2. Then 1.6 g of PCL was added to each bottle to prepare 16% (w / v) PCL / HFIP solutions containing 0%, 1%, 2%, and 4% (w / v) SiO2 / HFIP, respectively. The solutions were stirred overnight at room temperature on a magnetic stirrer until the PCL particles were completely dissolved, and then used as the electrospinning working solution.

[0105] 1.3 Preparation of coaxial electrospinning scaffolds with different AVT or SiO2 concentrations

[0106] A 5 mL syringe was used to draw the working solution described above and installed on the electrospinning machine. A coaxial electrospinning needle (shell layer 22G, core layer 17G) was used as the positive electrode, and an aluminum foil wrapped around a roller-shaped collector was used as the receiving substrate and connected as the negative electrode. The needle voltage was +7 kV, the receiving roller voltage was -8 kV, the receiving distance was 15 cm, and the roller rotation speed was 10 rpm / min. The push speed of the shell layer syringe was set to 1 mL / h, and the push speed of the core layer syringe was set to 0.5 mL / h. The spinning time was 3 h. The grouping of coaxial electrospinning scaffolds with different drug or nanoparticle concentrations is shown in Table 1.

[0107] Table 1 Grouping of coaxial electrospinning scaffolds with different AVT or SiO2 concentrations

[0108]

[0109] 1.4 Polarization of coaxial electrospinning scaffolds

[0110] Coaxial electrospun scaffold was cut into a size of 14mm in diameter or 34mm in diameter, and the cut material was placed on the metal negative plate of the direct current power supply with the front face upward. The positive electrode of the direct current power supply was adjusted to be fixed at a distance of 5cm from the coaxial electrospun film. The power switch was turned on, the voltage was set to 18kV, and the polarization time was set to 20min. After the polarization was completed, the material was collected for subsequent experiments, and the direct current power supply was turned off.

[0111] 1.5 In vitro biocompatibility detection of AVT-loaded coaxial electrospun scaffold

[0112] CCK-8 experiment was used to detect the proliferation of human umbilical vein endothelial cells (HUVECs) on coaxial electrospun scaffolds loaded with different concentrations of AVT. During the 5-day culture period, HUVECs on PCL (0% AVT), 0.25% AVT, 1% AVT, and 4% AVT groups all showed similar cell proliferation curves, and there was no obvious cytotoxicity. However, the OD value of the 16% AVT group was lower than that of the other four groups on the 3rd and 5th days of culture, and the difference was significant, indicating that the 16% AVT group had cytotoxicity, and subsequent experiments did not use this group, see Figure 2 .

[0113] 1.6 In vitro pro-angiogenic ability detection of AVT-loaded coaxial electrospun scaffold

[0114] The present application further detects the influence of coaxial electrospun scaffolds loaded with different concentrations of AVT on the tube formation ability of HUVECs. After 4h of culture on Matrigel, Calcein AM was used to stain HUVECs, and the results showed that only a small amount of tubes were formed in the PCL group, while in the 0.25% AVT, 1% AVT and 4% AVT groups, the number of tubes gradually increased, and among them, the 4% AVT group had the most tube formation. Quantitative analysis also showed consistent results, see Figure 3 .

[0115] Therefore, the present application considers that coaxial electrospun scaffolds loaded with 0.25%-4% AVT have good biocompatibility and pro-angiogenic effect, and 4% AVT has the best pro-angiogenic performance.

[0116] 1.7 Surface potential detection of SiO2-loaded coaxial electrospun scaffold

[0117] The surface potential detection results of coaxial electrospun scaffolds loaded with different concentrations of SiO2 are as follows Figure 4The surface potential of the coaxial electrospun scaffolds with different concentrations of SiO2increased with the increase of SiO2concentration, while no surface charge distribution was detected in the coaxial electrospun scaffold without SiO2. The coaxial electrospun scaffolds with different concentrations of SiO2had higher surface potential immediately after polarization, which were 1% SiO2(-0.41±0.02 kV), 2% SiO2(-0.58±0.03 kV), and 4% SiO2(-0.96±0.06 kV), respectively. The surface potential gradually decreased to 1% SiO2(-0.19±0.02 kV), 2% SiO2(-0.33±0.04 kV), and 4% SiO2(-0.66±0.05 kV) within 12 h, and then remained stable for up to 21 days, which were 1% SiO2(-0.18±0.04 kV), 2% SiO2(-0.28±0.03 kV), and 4% SiO2(-0.59±0.06 kV), respectively.

[0118] 1.8 In vitro biocompatibility detection of SiO2-loaded coaxial electrospun scaffolds

[0119] The proliferation of BMSCs on the coaxial electrospun scaffolds with different concentrations of SiO2in the core layer was detected by CCK-8, and the results are shown in Figure 5 During the 5-day culture period, BMSCs on the coaxial electrospun scaffolds with PCL (0% SiO2), 1% SiO2, 2% SiO2, and 4% SiO2all exhibited similar cell proliferation curves, i.e., all groups had good biocompatibility.

[0120] 1.9 In vitro osteogenic ability detection of SiO2-loaded coaxial electrospun scaffolds

[0121] The effect of coaxial electrospun scaffolds with different concentrations of SiO2on the osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) was evaluated by detecting ALP activity. On the 4th and 7th days of culture, BMSCs in the 1% and 2% SiO2groups exhibited enhanced ALP staining, and the 2% SiO2group had the best ALP staining result, which was consistent with the ALP semi-quantitative determination result Figure 6 B, D), which was statistically different from other groups.

[0122] Therefore, the present application believes that 1%-2% concentration of SiO2has good biocompatibility and promotes osteogenesis, and 2% concentration of SiO2has the best osteogenic performance. Therefore, 2% SiO2is selected as the best optimized concentration of SiO2for subsequent experiments.

[0123] Example 2 SiO2 / AVT coaxial electrospun scaffolds with 4% AVT shell layer and 4% SiO2core layer were prepared using PCL as the matrix.

[0124] Prepare a light-proof glass bottle, weigh 0.064 g of atorvastatin (AVT) into it, then add 10 mL of hexafluoroispropanol (HFIP) respectively and use a magnetic stirrer to stir at room temperature for 20 min until completely dissolved, then add 1.6 g of polycaprolactone (PCL), respectively, to prepare a 16% (w / v) PCL / HFIP solution containing 4% (w / w) AVT / PCL concentration, and stir overnight at room temperature on a magnetic stirrer until the PCL particles are completely dissolved as the electrospinning working solution.

[0125] Prepare a light-proof glass bottle, weigh 0.064 g of atorvastatin (AVT) into it, then add 10 mL of hexafluoroispropanol (HFIP) respectively and use a magnetic stirrer to stir at room temperature for 20 min until completely dissolved, then add 1.6 g of polycaprolactone (PCL), respectively, to prepare a 16% (w / v) PCL / HFIP solution containing 4% (w / w) AVT / PCL concentration, and stir overnight at room temperature on a magnetic stirrer until the PCL particles are completely dissolved as the electrospinning working solution.

[0126] Use a 5 mL syringe to draw the above working solution and install it on the electrospinning machine, use a coaxial electrospinning needle (shell layer 22G, core layer 17G) as the positive electrode, and aluminum foil wrapped around the roller-shaped collector as the receiving substrate and connected as the negative electrode. The needle voltage is +7 kV, the receiving roller voltage is -8 kV, the receiving distance is 15 cm, and the roller speed is 10 rpm / min. Set the push speed of the shell layer syringe to 1 mL / h and the push speed of the core layer syringe to 0.5 mL / h, and the spinning time is 3 h to prepare a SiO2 / AVT coaxial electrospinning scaffold with a 4% AVT shell layer and a 2% SiO2 core layer.

[0127] Table 2 Grouping of coaxial electrospinning scaffolds coupled with electret electric stimulation and atorvastatin

[0128]

[0129] Example 3 Material characterization of SiO2 / AVT coaxial electrospinning scaffolds.

[0130] 1.1 Use scanning electron microscope (SEM) and transmission electron microscope (TEM) to characterize the surface morphology and internal features of the coaxial electrospinning scaffold, respectively. The SEM image shows that the fibers of each group are randomly arranged, the surface is loose and porous, the fibers are uniform in thickness and the surface is smooth and flat. Use Image J to measure the diameter of the fibers in each group, and the results are as follows: Figure 7As shown in Figure C, the diameters of all fibers are in the micro-nano size range. TEM images show that each group of fibers has a core-shell structure, and the core layers of the SiO2 group and the SiO2 / AVT group show clearly visible and uniformly dispersed SiO2 nanoparticles.

[0131] 1.2 The hydrophilicity of the material was characterized using water contact angle testing, such as... Figure 8 As shown, when water droplets contact the material surface for 2 seconds, the water droplets on all four groups of materials exhibit a typical elliptical shape. At this time, the water contact angles are: PCL group (125.40°±2.49°), SiO2 group (125.20°±1.82°), AVT group (126.00°±4.62°), and SiO2 / AVT group (124.10°±3.27°). Quantitative results show no significant differences. When the water droplets were in contact with the material surface for 25 seconds, the morphology of the water droplets in the PCL group and the SiO2 group was similar to that at 2 seconds, with water contact angles of 123.00°±3.79° and 123.70°±1.62°, respectively. However, the water droplets in the AVT group and the SiO2 / AVT group showed obvious spreading, and the water contact angles were significantly smaller, at 78.37°±7.13° and 84.90°±8.81°, respectively. There was no significant difference between the two groups, but the differences were statistically significant compared with the PCL group and the SiO2 group.

[0132] 1.3 The mechanical property test results of the coaxial electrospun film are as follows: Figure 9 As shown in the figure. The results of the elastic modulus (Young's modulus) test were as follows: PCL group (64.54 MPa ± 3.99 MPa), AVT group (76.64 MPa ± 11.23 MPa), SiO2 group (42.62 MPa ± 1.33 MPa), and SiO2 / AVT group (44.24 MPa ± 2.22 MPa). The results indicate that the AVT group had the highest elastic modulus, but there was no statistically significant difference compared to the PCL group. The PCL and AVT groups showed statistically significant differences compared to the SiO2 and SiO2 / AVT groups. The results of the tensile strength test were as follows: PCL group (2.74 MPa ± 0.44 MPa), AVT group (2.80 MPa ± 0.21 MPa), SiO2 group (2.39 MPa ± 0.17 MPa), and SiO2 / AVT group (2.38 MPa ± 0.12 MPa), with no significant differences among the groups.

[0133] 1.4 The coaxial electrospun scaffolds were immersed in PBS at 37°C for 28 days to investigate their degradation performance. The degradation rates of each group were PCL group (0.33% ± 0.29%), Si02group (0.53% ± 0.11%), AVT group (0.67% ± 0.23%), and Si02 / AVT group (0.49% ± 0.16%) after 7 days of immersion in PBS at 37°C. The degradation rates of each group were PCL group (1.51% ± 0.12%), Si02group (1.70% ± 0.72%), AVT group (2.10% ± 0.63%), and Si02 / AVT group (2.04% ± 0.84%) after 28 days of immersion in PBS at 37°C, as shown in Figure 10 .

[0134] 1.5 The AVT release results of the coaxial electrospun scaffolds are shown in Figure 11 . The AVT in the shell layer of the AVT group and the Si02 / AVT group showed an initial rapid release and a slow sustained release later, with the cumulative release of AVT on the first day being 43.32% (AVT group) and 42.04% (Si02 / AVT group), respectively. Subsequently, in the AVT in vitro release for 21 days, the AVT group reached a cumulative release of 62.76%, and the cumulative release of the Si02 / AVT group was 68.54%.

[0135] 1.6 The surface potential detection results of the prepared coaxial electrospun scaffolds after polarization for 20 min under an 18 kV electric field are shown in Figure 12 . No surface potential was detected at all time points for the coaxial electrospun scaffolds without Si02, while similar surface potential sizes and change curves were detected for the Si02group (2% Si02) and the Si02 / AVT group (2% Si02 / AVT) coaxial electrospun scaffolds. The Si02group and the Si02 / AVT group had a high surface potential immediately after polarization, being -0.62 ± 0.04 kV for the Si02group and -0.62 ± 0.06 kV for the Si02 / AVT group. The surface potential gradually decreased to -0.33 ± 0.01 kV for the Si02group and -0.31 ± 0.01 kV for the Si02 / AVT group within 12 h. The surface potential was then basically stable for 21 days, being -0.29 ± 0.04 kV for the Si02group and -0.26 ± 0.05 kV for the Si02 / AVT group.

[0136] Example 4 The Si02 / AVT coaxial electrospun scaffold has good biocompatibility in vitro and can effectively promote angiogenesis and osteogenesis differentiation.

[0137] 1.1 The in vitro biocompatibility of the coaxial electrospun scaffold was detected by 24 h cell adhesion and cell proliferation experiments. The 24 h BMSCs cell adhesion is shown inFigure 13 As shown, BMSCs were in close contact with randomly arranged fibers on the surface of coaxial electrospun scaffolds, exhibiting a spread cell morphology, in which BMSCs in the SiO2 group, the AVT group and the SiO2 / AVT group all exhibited more cell tentacles and greater spread morphology than the PCL group.

[0138] The proliferation of rat BMSCs and HUVECs on the coaxial electrospun scaffolds was detected using CCK-8. During the 5-day culture period, BMSCs exhibited good cell proliferation trends on the electrospun scaffolds in each group, and there was no statistical difference between each group. HUVECs cultured on the SiO2 / AVT coaxial electrospun scaffold exhibited a higher cell proliferation rate at 5 days of culture, and the difference was statistically significant compared with the PCL group. The results showed that the SiO2 / AVT coaxial electrospun scaffold had good biocompatibility in vitro.

[0139] 1.2 The effect of coaxial electrospun scaffolds on the tube formation ability of HUVECs was detected by tube formation experiment. HUVECs cultured in the PCL group formed a small number of small tubes, and the number of small tubes in the SiO2 group was more than that in the PCL group. More small tubes were formed in the AVT group and the SiO2 / AVT group. Quantitative analysis showed that the number of small tubes in the SiO2 group was significantly increased compared with the PCL group, and the AVT group and the SiO2 / AVT group had more small tube numbers and tube intersections than the PCL group and the SiO2 group, and the difference was statistically significant, as shown in Figure 14 .

[0140] 1.3 The effect of coaxial electrospun scaffolds on the osteogenic differentiation of BMSCs was evaluated by detecting ALP activity. The results of semi-quantitative detection of ALP activity and ALP staining at 4 and 7 days of culture showed that the ALP expression level in the PCL group and the AVT group was low, and there was no obvious difference. The ALP activity in the SiO2 group and the SiO2 / AVT group was significantly increased, and the difference was statistically significant compared with the other two groups, as shown in Figure 15 .

[0141] The above results showed that the SiO2 / AVT coaxial electrospun scaffold could effectively promote angiogenesis and osteogenesis in vitro.

[0142] Example 5 The SiO2 / AVT coaxial electrospun scaffold had good biocompatibility in vivo, and could effectively promote the repair of rat skull defects.

[0143] 1.1 Four weeks after the subcutaneous implantation of the scaffold, HE staining of the visceral paraffin sections of the Blank group, the PCL group, the SiO2 group, the AVT group and the SiO2 / AVT group showed that the tissue and cell morphology was normal, the arrangement was orderly, the structure was clear, and there was no necrotic tissue and inflammatory cell infiltration, indicating that the scaffold had good in vivo biocompatibility, as shown inFigure 16 .

[0144] 1.2 The effect of the coaxial electrospun scaffold coupled with AVT and EES on the in vivo vascularization of the bone regeneration was evaluated using the rat calvarial critical-sized bone defect model (see Figure 17 ). A 5-mm diameter critical-sized bone defect was created on the rat calvaria and implanted with the coaxial electrospun scaffold. The results of the micro-CT scanning and the quantitative analysis of the new bone formation of the rat calvaria at 8 weeks postoperatively (see Figure 18 ) showed that the Blank group had a small amount of new bone tissue around the defect edge, while the other four groups had better bone formation area and BV / TV values. Compared with the PCL group, the AVT group, the SiO2 group and the SiO2 / AVT group had an increasing amount of new bone formation in the bone defect area, especially in the SiO2 / AVT group, where more than 80% of the bone defect area was filled with new bone tissue. The results of the quantitative analysis were consistent with the results observed from the micro-CT images and the VG-stained images of the hard tissue sections, indicating that the SiO2 / AVT coaxial electrospun scaffold could effectively promote the repair of the rat calvarial defect.

[0145] 1.3 The new blood vessels in the bone regeneration area of the rat calvarial defect at 8 weeks postoperatively were observed using Masson staining, and the results are shown in the figure. The Blank group had only a small amount of new bone tissue around the edge of the bone defect, and no obvious bone marrow cavity and new blood vessels were observed. The PCL group had a larger bone formation area than the Blank group, but had fewer new blood vessels. The AVT group, the SiO2 group and the SiO2 / AVT group had an increasing amount of bone formation area, and more obvious bone marrow cavities and blood vessels were observed, especially in the SiO2 / AVT group, where a large number of bone marrow cavities, blood vessels and red blood cells (red arrows) were observed in the bone formation area, indicating active angiogenesis. Figure 19

[0146] 1.4 The rats were injected intraperitoneally with tetracycline, alizarin red and calcein at 2, 4 and 6 weeks postoperatively as fluorescent markers for new bone growth. The hard tissue sections of the calvaria at 8 weeks postoperatively were observed using a laser confocal microscope, and the yellow (tetracycline, TE), red (alizarin red, AL) and green (calcein, CA) fluorescent markers were observed to have a hierarchical and spaced distribution, indicating the process of new bone growth layer by layer. As shown in Figure 20 A, the Blank group had only a small amount of three-color fluorescent markers around the edge of the defect, while the fluorescent areas of the PCL group, the AVT group, the SiO2 group and the SiO2 / AVT group increased in turn, and the SiO2 / AVT group exhibited the largest TE, AL and CA fluorescent areas. The results of the quantitative analysis of the fluorescent areas were consistent with the observation results of the fluorescent marker images (see Figure 20 ).

[0147] ​The results of micro-CT, VG staining, HE staining, Masson staining and fluorescence labeling and analysis of the rat skull at 8 weeks after the operation show that the SiO2 / AVT scaffold significantly promotes the vascularized bone regeneration of the mandibular bone defect of the rat.

[0148] Discussion

[0149] Electrophysiological microenvironment has far-reaching significance for maintaining normal physiological activities of the human body. Endogenous electric field has been found in heart, nerve and bone tissue, and is involved in organ development, wound healing and tissue regeneration. Changes in endogenous electric field affect cell metabolism, leading to different proliferation and differentiation behaviors. In the environment of bone defect, the piezoelectric or electret properties of collagen and hydroxyapatite in natural bone will generate internal electric field, and negative charges will gradually accumulate in the bone defect area, thereby guiding the regeneration of bone. Therefore, the reconstruction of the electrophysiological microenvironment of bone tissue by electret can be used as a useful tool for critical-sized bone defect regeneration.

[0150] SiO2 electret, as a representative of inorganic silicon-based electret, has the advantages of high stability and long service life. SiO2 can generate stable and persistent surface potential after polarization, simulating the electrophysiological microenvironment of bone and being conducive to bone regeneration. Based on the potential of SiO2 as electret in promoting osteogenesis and the advantage of AVT in promoting angiogenesis, the present application proposes to use coaxial electrospinning technology to prepare a new coaxial electrospinning scaffold (SiO2 / AVT) coupling electret electric stimulation and AVT. The clinical drug AVT promoting angiogenesis is loaded in the fiber shell layer, and SiO2 nanoparticles are loaded in the fiber core layer. After electric field polarization, the electric stimulation provided by the core layer SiO2 electret promotes osteogenesis, the shell layer AVT promotes early vascularization through early and rapid release, SiO2 simulates the electrophysiological microenvironment of bone to continuously promote osteogenesis, and finally realizes the vascularized bone regeneration of critical-sized bone defect.

[0151] The coaxial electrospinning scaffold with different concentrations of AVT in the fiber shell layer was used for HUVECs surface culture, and the effects of AVT on cell proliferation, cell migration, tube formation and angiogenesis gene expression were detected. At 3 days and 5 days of culture, the cell proliferation level of the 16% AVT group was significantly down-regulated, proving that it has cytotoxicity, while the 0.25%-4% AVT group has consistent cell growth level with the control group, proving that it has good biocompatibility. The tube formation experiment results show that the 4% AVT group has the best effect on promoting HUVECs tube formation in vitro. Therefore, 4% AVT has good biocompatibility and the best effect on promoting HUVECs angiogenesis differentiation, and thus it is used as the optimized concentration of AVT for subsequent experiments.

[0152] The BMSCs were cultured on the surface of the coaxial electrospun scaffolds with different concentrations of SiO2 in the fiber core layer, and the cell proliferation and osteogenic differentiation ability were detected to screen the optimal concentration of SiO2. The results showed that the coaxial electrospun scaffolds with 1%-4% SiO2 loaded in the core layer had good biocompatibility in the 5-day culture period. ALP staining and semi-quantitative results showed that the 2% SiO2 group exhibited the highest ALP expression, indicating that the electrical stimulation of the coaxial electrospun scaffold with 2% SiO2 had the best osteogenic differentiation promoting ability. Therefore, the coaxial electrospun scaffold with 2% SiO2 loaded in the core layer had good biocompatibility and the best effect of electrical stimulation in promoting osteogenic differentiation, and was selected as the optimal concentration of SiO2.

[0153] According to the foregoing results, the coaxial electrospun scaffold (SiO2 / AVT) with electret electrical stimulation coupled AVT was prepared with 4% AVT as the fiber shell layer and 2% SiO2 as the fiber core layer, and material characterization was performed. SEM results showed that the fibers in each group were randomly arranged, the surface was porous, the morphology was similar to the natural extracellular matrix, and the surface of the single fiber was smooth and uniform in thickness, with an average diameter in the micro-nanometer size range. TEM detection showed the successful construction of the coaxial electrospun core-shell structure and the successful loading of SiO2 nanoparticles in the core layer. Four-week degradation experiment showed that the coaxial electrospun scaffold degraded slowly, indicating that it could act as a scaffold material in the body and stably and continuously play a role in the bone repair period. The drug release experiment results showed that the AVT in the shell layer was rapidly released in the initial stage, with a cumulative release of more than 40% on the first day, and then slowly and continuously released, with a cumulative release of more than 62% at 3 weeks, and the release of AVT met the demand for early vascularization in large-area bone defect repair

[10] . Subsequently, the surface potential of the electrospun scaffold was detected after polarization, and the results showed that the SiO2 group and the SiO2 / AVT group had almost the same surface potential size and maintenance level, which indicated that the loading of AVT in the fiber shell layer did not interfere with the distribution and maintenance ability of the surface charge after polarization of SiO2. Therefore, the coaxial electrospun scaffold (SiO2 / AVT) coupled with electret electrical stimulation and AVT met our original intention: the early rapid release of AVT in the shell layer induced rapid vascularization, and the slow degradation of SiO2 in the core layer stably maintained the surface potential for at least 3 weeks after polarization, which met the requirements of inducing vascularization in the early stage and providing continuous electrical stimulation to simulate the bone electrophysiological microenvironment and promote osteogenesis in large-area bone defect repair.

[0154] The good biocompatibility of the scaffold is the guarantee for the success of bone repair. We first detected the in vitro biocompatibility of the coaxial electrospun scaffold through cell proliferation and cell adhesion experiments. The CCK-8 was used to detect the effect of the coaxial electrospun scaffold on the proliferation of BMSCs and HUVECs. The results showed that there was no statistical difference in the proliferation rate of BMSCs on the coaxial electrospun scaffold in each group, while HUVECs in the SiO2 / AVT group had a higher cell proliferation level than that in the PCL group at 5 days of culture, indicating that the coaxial electrospun scaffold loaded with SiO2 and AVT had good biocompatibility, and the combination of SiO2 electric stimulation and AVT helped to promote the proliferation of HUVECs. Cell adhesion experiments showed that cells were in close contact with fibers, showing a spread cell morphology. BMSCs in the AVT group, the SiO2 group and the SiO2 / AVT group exhibited more cell tentacles and spread morphology. The above results showed that the SiO2 / AVT electrospun scaffold had good biocompatibility, and the loading of AVT and the electric stimulation of SiO2 were beneficial to cell adhesion.

[0155] Since early rapid vascularization is the key to the successful regeneration of critical size bone defects, we evaluated the in vitro blood vessel formation ability of the coaxial electrospun scaffold through tube formation experiment. The results showed that the tube formation in the SiO2 group increased compared with PCL, while more tube formation was observed in the AVT group and the SiO2 / AVT group.

[0156] Promoting the osteogenic differentiation of BMSCs is the most important property of bone repair materials. The in vitro osteogenic ability of the coaxial electrospun scaffold was evaluated by ALP activity detection. The SiO2 group and the SiO2 / AVT group showed the strongest ALP activity, which proved that the electric stimulation of SiO2 electret could significantly promote the osteogenic differentiation of BMSCs, and the loading of AVT did not affect the osteogenic ability of SiO2 electric stimulation.

[0157] To further verify the in vivo biological safety and vascularized bone regeneration ability of the coaxial electrospun scaffold coupled with electret electric stimulation and atorvastatin (SiO2 / AVT), rat subcutaneous implantation model and rat critical size skull defect model were constructed respectively. The histological staining results of the organs after 4 weeks of rat subcutaneous implantation proved that the coaxial electrospun scaffold had excellent in vivo biocompatibility.

[0158] In the rat calvarial critical-size defect model, the results of Micro-CT analysis at 8 weeks postoperatively showed that there was very little new bone formation at the edge of the Blank group, indicating the successful establishment of the calvarial critical-size defect. Compared with the Blank group, the rest of the groups had different degrees of new bone formation, indicating that the coaxial electrospun scaffold could promote bone defect repair. Compared with the PCL group, the defect area of the AVT group, the SiO2 group and the SiO2 / AVT group could see the new bone formation increasing in turn, especially the SiO2 / AVT group, which had more than 80% of the bone tissue in the bone defect area, proving that the coaxial electrospun scaffold (SiO2 / AVT) coupling electret electric stimulation and AVT also achieved the best in situ vascularized bone regeneration in the repair of rat calvarial critical-size defect.

Claims

1. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises at least AVT and SiO2.

2. The pharmaceutical composition of claim 1, wherein, The ratio of the amount of AVT to SiO2 is 0.25%-4%:1%-2%.

3. An electrospun coaxial scaffold coupling electret electrostimulation and angiogenic drugs, characterized in that, The coaxial electrospun scaffold comprises a core layer and a shell layer, wherein the core layer comprises SiO2 and a first matrix material; and the shell layer comprises AVT and a second matrix material.

4. The coaxial electrospun scaffold of claim 3, wherein, The first matrix material is one or more of polycaprolactone (PCL), poly-L-lactic acid (PLLA) and polylactic acid (PLA); and / or, the second matrix material is one or more of polycaprolactone (PCL), poly-L-lactic acid (PLLA) and polylactic acid (PLA); and / or, the mass ratio of SiO2 to the first matrix material is 1%-2%(w / w):16%; and / or, the mass ratio of AVT to the second matrix material is 0.25%-4%(w / w):16%(w / v).

5. The coaxial electrospun scaffold of claim 4, wherein, The number average molecular weight (Mn) of the PCL is 60000-100000.

6. The coaxial electrospun scaffold of claim 3, wherein, The coaxial electrospun scaffold has a thickness of 0.1mm; and / or, the coaxial electrospun scaffold has an elastic modulus of 44.24MPa±2.22MPa; and / or, the coaxial electrospun scaffold has a tensile strength of 2.38MPa±0.12MPa.

7. A method of making a coaxial electrospun scaffold coupling electret electrostimulation and angiogenic drugs as claimed in any one of claims 3-6, characterized in that, The method comprises the following steps: The raw materials of the core layer are dispersed in a first dispersant to obtain a core layer solution; The raw materials of the shell layer are dispersed in a second dispersant to obtain a shell layer solution; The core layer solution and the shell layer solution are prepared into the core layer and the shell layer by using a coaxial electrospinning technology to obtain the coaxial electrospun scaffold.

8. The method of claim 7, wherein, The first dispersant is hexafluoroisopropanol; and / or, the second dispersant is hexafluoroisopropanol; and / or, the addition amount of SiO2 in the first dispersant is 3.2g / L; and / or, the addition amount of AVT in the second dispersant is 6.4g / L.

9. The method of claim 7, wherein, The coaxial electrospinning technology comprises one or more of the following technical features: The needle voltage is +7kV; The receiving roller voltage is -8kV; The receiving distance is 15cm; The roller rotation speed is 10rpm / min; The pushing speed of the shell layer injector is set to 1mL / h; The pushing speed of the core layer injector is set to 0.5mL / h; The spinning time is 3h.

10. Use of the composition of claim 1 or 2 or the coaxial electrospun scaffold of claim 3-6 in the preparation of a product for promoting angiogenesis and / or osteogenesis differentiation.

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