Piezoelectric stimulation and AVT coupled coaxial electrostatic spinning stent, preparation and application in mandible defect vascularized bone regeneration

Abstract

The invention discloses a novel coaxial electrostatic spinning stent coupling piezoelectric stimulation and AVT, the morphology of the stent simulates an extracellular matrix, adhesion, growth and differentiation of cells are facilitated, and a space is provided for growth of new blood vessels and new bones; the coaxial electrostatic spinning stent takes an artificial high polymer material as a matrix, is good in biological safety and can be degraded in vivo, and a degradation product is non-toxic; a clinical drug AVT for promoting angiogenesis is loaded in a fiber shell layer, and the defects that various angiogenesis promoting factors are high in price, short in half-life period, insufficient in potential safety and the like are overcome; the piezoelectric material ZnO for promoting osteogenic differentiation is loaded on the fiber core layer, various defects of external electrical stimulation equipment are avoided, the osteogenic differentiation is continuously promoted by collecting masticatory muscle force and converting the masticatory muscle force into piezoelectric stimulation, meanwhile, angiogenesis is promoted in combination with early release of AVT, and finally, vascularized bone regeneration of lower jawbone defects is promoted. According to the invention, the source of required materials is sufficient, the preparation technology is simple, the cost is low, the biological safety is good, and the potential clinical application prospect is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and relates to a coaxial electrospun scaffold coupled with piezoelectric stimulation and AVT atorvastatin, preparation and application in promoting vascularized bone regeneration of mandibular defects. BACKGROUND

[0002] In the repair of critical bone defects, the rapid and effective vascularization of implanted materials is a prerequisite for successful bone regeneration. Although various types of pro-angiogenic growth factors have shown outstanding advantages in many studies, due to their short half-life, difficulty in controlling the correct spatiotemporal release, and potential risks of use (such as immunogenicity and risk of stimulating the growth or recurrence of hemangioma), it is difficult to be widely used in clinical practice. Therefore, we choose the clinical drug atorvastatin (AVT) as the angiogenesis inducer. AVT is the fifth statin approved by the US FDA for the treatment of patients with high cholesterol. It is found to reduce major cardiovascular events in patients with coronary heart disease, ischemic stroke, diabetes, and high-risk patients with cardiovascular diseases such as hypertension, and has good safety. This efficacy and safety have been confirmed in more than 400 clinical trials and 230 million patients in clinical practice. In recent years, studies have found that AVT is associated with the regulation of serine / threonine protein kinase Akt in endothelial cells, which regulates various angiogenic processes in endothelial cells, and has effects such as changing endothelial function, controlling inflammatory response, and inhibiting thrombosis. Therefore, AVT is a good pro-angiogenic regulator. At the same time, AVT was found to have a potential effect of promoting osteogenesis. Therefore, the strategy of using AVT to induce angiogenesis has potential application prospects in bone defect repair.

[0003] Zinc oxide (ZnO) is a unique material with semiconductor and piezoelectric double properties. Due to its piezoelectric properties, ZnO nanosheets will generate a local electric field under the action of cell intrinsic mechanical force, thereby promoting Saos-2 cell osteogenic differentiation. In addition, ZnO has been found to have effects such as promoting tissue repair, promoting osteogenesis, antibacterial, and regulating immune response. At the same time, compared with piezoelectric materials with high piezoelectric coefficients such as PVDF and its copolymer, potassium sodium niobate, lead zirconate titanate, etc., the piezoelectric coefficient of ZnO is 0.4-9.5 pC / N, which is closer to the natural bone tissue (0.7-2.3 pC / N), and thus it may be a more suitable piezoelectric material for bone defect repair.

[0004] The mandible has a different biomechanical environment from other parts of the craniofacial skeleton due to the presence of strong attachments of the masseter, medial pterygoid, and temporalis muscles. Some scholars used micro-CT and histological staining to track the healing process of a 3mm full-thickness defect in the rat mandible and found that more new bone growth occurred in the direction of masticatory muscle movement. In another study, the authors injected botulinum toxin into the masticatory muscles of adult rats to atrophy the muscles on one side and found that the bone mineral content, cortical bone thickness, and trabecular bone thickness of the mandible on that side were significantly reduced. This shows that the presence of masticatory muscles creates a different biomechanical environment, which in turn has a different impact on the regeneration of mandibular bone tissue. This natural biomechanical environment also provides a suitable scenario for the application of piezoelectric materials, which can convert the biomechanical force generated by the physiological movement of the masticatory muscles into piezoelectric stimulation to promote bone regeneration. This piezoelectric stimulation induced by the physiological movement of the masticatory muscles is in line with the physiological rhythm and does not require external ultrasonic, light, and magnetic field excitation.

[0005] Therefore, the strategy of combining ZnO piezoelectricity to promote osteogenesis and AVT to promote angiogenesis is expected to be applied to the repair of mandibular defects. SUMMARY

[0006] To overcome the above-mentioned defects mentioned in the prior art, the present application prepares a novel coaxial electrospun scaffold with a core-shell structure that couples piezoelectric stimulation and angiogenic drugs by coaxial electrospinning technology. The morphology simulates the extracellular matrix, which is conducive to cell adhesion, growth, differentiation, and provides space for the growth of new blood vessels and new bone. The coaxial electrospun scaffold uses artificial polymer materials as the matrix, which has good biological safety, can be degraded in vivo, and the degradation products are non-toxic. The clinical drug AVT, which promotes angiogenesis, is loaded in the fiber shell layer to avoid the shortcomings of expensive, short half-life, and potential safety of various pro-angiogenic factors (such as VEGF). The piezoelectric material ZnO, which promotes osteogenic differentiation, is loaded in the fiber core layer, avoiding the various shortcomings of external electric stimulation devices. By collecting the masticatory muscle force to convert it into piezoelectric stimulation to continuously promote osteogenic differentiation, combined with the early release of AVT to promote angiogenesis, the vascularized bone regeneration of mandibular defects is ultimately promoted. The required materials are abundant in source, the preparation technology is simple, the cost is low, and the biological safety is good, which has potential clinical application prospects.

[0007] The difficulties to be overcome by the present application mainly include rational utilization / avoidance of the biological force during masticatory muscle movement, poor hydrophilicity of the scaffold material, early loss of functional components caused by degradation, instability of piezoelectric stimulation caused by interference of the body fluid environment on the piezoelectric material, short half-life of angiogenic factors, and insufficient early vascularization, while meeting the selection of the optimal concentration of ZnO and AVT that satisfies good biological safety and promotes the best bone / angiogenic effect. The natural biomechanical environment of the mandible provides a suitable scenario for the application of piezoelectric materials, however, high piezoelectric coefficient materials (such as PVDF, KNN, barium titanate, etc.) that do not match the piezoelectric coefficient of bone tissue may not be suitable for bone defect repair. The piezoelectric coefficient of ZnO is close to that of bone tissue, which can convert the masticatory muscle force into piezoelectric stimulation to promote bone regeneration. Other biological effects of ZnO are another advantage of piezoelectric materials for repairing jaw defects (such as biological safety, antibacterial properties, etc.), emphasizing the harmless or even potential benefits of the body during the metabolic process of ZnO after the degradation of the electrospun scaffold. 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 promotes angiogenesis but also has no systemic side effects, avoiding the shortcomings of various pro-angiogenic factors (such as VEGF) such as high price, short half-life, and potential safety issues, and has good application prospects. In the design of this technical solution, slow-degrading PCL material is used as the matrix to maintain the necessary growth space for bone regeneration during the critical period of bone defect repair. At the same time, by taking advantage of the sequential degradation characteristics of coaxial electrospun core-shell structure (slow degradation of the core layer and relatively fast degradation of the shell layer), ZnO is loaded in the core layer of the coaxial electrospun fiber to avoid the influence of the surrounding body fluid environment and provide sustained piezoelectric stimulation during the critical period of bone defect repair (the first three weeks of repair) to continuously promote bone regeneration; AVT with good pro-angiogenic effect is loaded in the shell layer to increase the hydrophilicity of the scaffold while its early release behavior can promote early angiogenesis, ultimately promoting vascularized bone regeneration of the mandibular critical bone defect.

[0008] The present application provides a pharmaceutical composition comprising AVT and ZnO.

[0009] Preferably, the ratio of AVT to ZnO is 4:4.

[0010] In the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0011] Further, the pharmaceutical composition can be used alone and / or in combination with other drugs.

[0012] Preferably, the pharmaceutically acceptable carrier means that when the drug is properly administered to animals or humans, they do not produce adverse, allergic or other adverse reactions. The pharmaceutically acceptable carriers can include, but are 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; tragacanth powder; 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 oil; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavorings; tabletting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help the stability of the formulation or to help improve the activity or its bioavailability or in the case of oral to produce an acceptable taste or smell.

[0013] Specifically, the pharmaceutical composition can further comprise a physiologically compatible auxiliary material, including 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.

[0014] 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 mist, or a suppository, etc. The above various dosage forms of the pharmaceutical composition can be prepared according to the conventional methods in the pharmaceutical field.

[0015] 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 before being introduced into the body. Preferably, it is administered by injection. The pharmaceutical composition can also be used in combination with other treatment methods, including surgery, radiotherapy, chemotherapy, targeted therapy.

[0016] The present application also provides a coaxial electrospun scaffold of core-shell structure coupled with piezoelectric stimulation and angiogenic drugs, comprising a core layer and a shell layer, wherein the core layer comprises ZnO, a first matrix; the shell layer comprises AVT, a second matrix;

[0017] The first matrix is polycaprolactone (PCL), poly-L-lactic acid (PLLA), polylactic acid (PLA), etc.; preferably, polycaprolactone (PCL).

[0018] The number average molecular weight of the PCL is Mn 60000-100000; preferably, the number average molecular weight of the PCL is Mn 60000-80000, or 75000-95000, or 80000-100000; further preferably, 800000.

[0019] The second matrix is polycaprolactone (PCL), poly-L-lactic acid (PLLA), polylactic acid (PLA), etc.; preferably, polycaprolactone (PCL).

[0020] The number average molecular weight of the PCL is Mn 60000-100000; preferably, the number average molecular weight of the PCL is Mn 60000-80000, or 75000-95000, or 80000-100000; further preferably, 800000.

[0021] The use amount ratio of the ZnO and the first matrix is 1%-4% (w / w): 16%; preferably, 4%: 16%.

[0022] The use amount ratio of the AVT and the second matrix is 0.25%-4% (w / w): 16%; preferably, 4%: 16%.

[0023] The thickness of the coaxial electrospun scaffold is 0.1 mm.

[0024] The elastic modulus (Young's modulus) of the coaxial electrospun scaffold is: 57.82 MPa ± 4.85 MPa;

[0025] The tensile strength of the coaxial electrospun scaffold is: 2.33 MPa ± 0.49 MPa.

[0026] The application also provides a preparation method of the coaxial electrospun scaffold with a core-shell structure coupling piezoelectric stimulation and angiogenic drugs, specifically comprising the following steps:

[0027] The raw materials of the core layer are dispersed in a first dispersant to obtain a core layer solution;

[0028] The raw materials of the shell layer are dispersed in a second dispersant to obtain a shell layer solution;

[0029] The core layer solution and the shell layer solution are coaxially electrospun to form a core layer and a shell layer, thereby obtaining the coaxial electrospun scaffold.

[0030] In the present application, the coaxial electrospinning technology is as follows: the shell layer solution and the core layer solution are respectively filled in two different syringes, the spinning system is composed of two coaxial but different inner diameter capillary tubes, under the action of high voltage electric field, the shell layer solution and the core layer solution converge, after stretching and receiving of the receiving device roller, the fiber tows with core-shell structure are formed and wound on the surface of the roller to form the electrospinning scaffold.

[0031] The core layer comprises ZnO, a first matrix;

[0032] The shell layer comprises AVT, a second matrix;

[0033] The first matrix is polycaprolactone (PCL), poly-L-lactic acid (PLLA), polylactic acid (PLA) or the like; preferably, polycaprolactone (PCL).

[0034] The number average molecular weight of the PCL is Mn 60000-100000; preferably, the number average molecular weight of the PCL is Mn 60000-80000, or 75000-95000, or 80000-100000; further preferably, 800000.

[0035] The second matrix is polycaprolactone (PCL), poly-L-lactic acid (PLLA), polylactic acid (PLA) or the like; preferably, polycaprolactone (PCL).

[0036] The number average molecular weight of the PCL is Mn 60000-100000; preferably, the number average molecular weight of the PCL is Mn 60000-80000, or 75000-95000, or 80000-100000; further preferably, 800000.

[0037] The amount ratio of the ZnO and the first matrix is 1%-4% (w / w): 16%; preferably, 4%: 16%.

[0038] The first matrix: the first dispersant = 16% (w / v);

[0039] The amount ratio of the AVT and the second matrix is 0.25%-4% (w / w): 16%; preferably, 4%: 16%.

[0040] The second matrix: the second dispersant = 16% (w / v);

[0041] The first dispersant is hexafluoroisopropanol.

[0042] The second dispersant is hexafluoroisopropanol.

[0043] The addition amount of the ZnO in the first dispersant is 6.4 g / L.

[0044] The AVT is added in the second dispersant in an amount of 6.4 g / L.

[0045] The needle voltage is +7 kV.

[0046] The receiving roller voltage is -8 kV.

[0047] The receiving distance is 15 cm.

[0048] The roller rotation speed is 10 rpm / min.

[0049] The pushing speed of the shell layer injector is set to 1 mL / h.

[0050] The pushing speed of the core layer injector is set to 0.5 mL / h.

[0051] The spinning time is 3 h.

[0052] In one embodiment, the preparation of the coaxial electrospinning scaffold of the core-shell structure coupled with piezoelectric stimulation and angiogenic drugs comprises the following steps: two light-proof glass bottles are prepared, 0.064 g of Atorvastatin (AVT) and 0.064 g of Zinc oxide (ZnO) nanoparticles are weighed and added into the bottles respectively, then 10 mL of Hexafluoroispropanol (HFIP) is added into each bottle and stirred at room temperature for 20 min using a magnetic stirrer until completely dissolved, 1.6 g of Polycaprolactone (PCL) is added to prepare a 16% (w / v) PCL / HFIP solution containing 4% (w / w) AVT / PCL and a 16% (w / v) PCL / HFIP solution containing 4% (w / w) ZnO / PCL respectively, and the PCL particles are completely dissolved after overnight stirring at room temperature on a magnetic stirrer, which is used as the electrospinning working solution.

[0053] The working solution is taken up using a 5 mL syringe and installed on an electrospinning machine, a coaxial electrospinning needle (shell layer 22G, core layer 17G) is used as the positive electrode, an aluminum foil is 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 rotation speed is 10 rpm / min. The pushing speed of the shell layer injector is set to 1 mL / h, the pushing speed of the core layer injector is set to 0.5 mL / h, and the spinning time is 3 h, to prepare a ZnO / AVT coaxial electrospinning scaffold with a 4% AVT shell layer and a 4% ZnO core layer.

[0054] The application also provides the use of the composition or the coaxial electrospinning scaffold in the preparation of a product for promoting bone defect repair.

[0055] The application also provides use of the composition or the coaxial electrospun scaffold in preparation of a product for promoting vascularized bone regeneration of mandibular bone defects.

[0056] The application also provides use of the composition or the coaxial electrospun scaffold in preparation of a product for promoting osteogenic differentiation.

[0057] The application also provides use of the composition or the coaxial electrospun scaffold in preparation of a product for promoting angiogenic differentiation.

[0058] The application can be used for repair of bone defects with strong muscle attachment (such as mandibular bone defects, femoral bone defects), and can convert autologous muscle physiological mechanical stimulation into piezoelectric stimulation for promoting bone defect repair. If combined with exogenous stimulation (such as ultrasonic stimulation) to induce piezoelectric stimulation, it can be used for bone defect repair at almost all sites.

[0059] The beneficial effects of the present application include: the present application focuses on the unique biomechanical environment of the mandible in the craniofacial skeleton and the key step of early vascularization in the process of critical bone defect repair. The natural biomechanical environment of the mandible provides a suitable scenario for the application of piezoelectric materials. The piezoelectric material ZnO with a matching bone piezoelectric coefficient can convert the masticatory muscle force into a piezoelectric stimulus that promotes bone regeneration. This piezoelectric stimulus induced by the physiological movement of the autologous masticatory muscle is in accordance with the physiological rhythm and does not require an additional excitation source, avoiding the complexity and uncertainty brought by the application of external excitation sources such as ultrasound, light and magnetic field. In the repair of critical bone defects, promoting early vascularization is the key to successful treatment. The present application uses the clinical drug AVT to promote angiogenesis, which can avoid the shortcomings of expensive, short half-life and potential safety of various pro-angiogenic factors (such as VEGF), and has good application prospects. The present application uses the characteristics of the coaxial electrospinning technology of the core-shell structure time sequence degradation (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. AVT with good pro-angiogenic effect is loaded in the shell layer to increase the hydrophilicity of the PCL scaffold and promote early angiogenesis by the early and orderly release of AVT in the shell layer. ZnO piezoelectric material with a matching bone piezoelectric coefficient and biological safety is loaded in the core layer to avoid the influence of the surrounding body fluid environment and convert the muscle force of the masticatory muscle physiological movement into a piezoelectric stimulus with physiological and rhythmic characteristics to promote osteogenesis. The hydrophilic scaffold of the "piezoelectric + drug" integrated core-shell fiber structure prepared by the present application significantly increases the hydrophilicity of the PCL base scaffold with AVT loaded in the shell layer, which is beneficial to cell adhesion and growth, and the early release of AVT in the shell layer promotes early angiogenesis. The ZnO loaded in the core layer is wrapped by the hydrophobic and slowly degradable PCL, avoiding the contact and interference of the surrounding body fluid environment, and can provide a piezoelectric stimulus to promote osteogenesis during the bone defect repair period by the physiological movement of the masticatory muscle. The scaffold prepared by the present application has good overall hydrophilicity, slow degradation, sustained piezoelectric stimulation to promote osteogenesis, and early release of AVT to promote early vascularization, ultimately promoting vascularized bone regeneration of mandibular critical bone defects. BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1 Schematic diagram of coaxial electrospun scaffold with piezoelectric stimulation coupled with AVT promoting vascularized bone regeneration of mandibular defects, wherein A. preparation of coaxial electrospun scaffold, core layer loaded with ZnO, shell layer loaded with AVT; B. coaxial electrospun scaffold stimulates BMSCs osteogenic differentiation and HUVECs angiogenic differentiation through piezoelectric stimulation and AVT stimulation; C. coaxial electrospun scaffold with piezoelectric stimulation and AVT promotes vascularized bone regeneration of mandibular critical defects.

[0061] Figure 2 Piezoelectric properties of coaxial electrospun scaffolds loaded with different concentrations of ZnO;

[0062] A-C. Voltage and current output of coaxial electrospun scaffolds with different ZnO concentration under different resistance (10 MΩ, 50 MΩ and 100 MΩ) loading; D. Voltage and current output of 1%, 2%, 4% and 8% ZnO scaffolds under 0.1 MΩ-1000 MΩ resistance loading.

[0063] Figure 3 CCK-8 experiment.

[0064] Figure 4 Tube formation experiment; A. Tube formation staining; B. Semi-quantitative analysis.

[0065] Figure 5 Angiogenic gene expression qRT-PCR experiment.

[0066] Figure 6 CCK-8 experiment.

[0067] Figure 7 ALP detection; A. ALP semi-quantitative detection; B. ALP staining.

[0068] Figure 8 Osteogenic gene expression qRT-PCR experiment.

[0069] Figure 9 Surface morphology and internal characteristic characterization of coaxial electrospun scaffolds; A. Scanning electron microscope image; B. Transmission electron microscope image; C. Fiber diameter distribution.

[0070] Figure 10 Hydrophilicity characterization of coaxial electrospun scaffolds; A. Water contact angle when water droplets contact for 2s; B. Water contact angle when water droplets contact for 25s.

[0071] Figure 11 Mechanical property characterization of coaxial electrospun scaffolds; A. Stress-strain curve; B. Elastic modulus; C. Tensile strength.

[0072] Figure 12 21-day AVT release curve of coaxial electrospun scaffolds.

[0073] Figure 13 28-day degradation curve of coaxial electrospun scaffolds.

[0074] Figure 14 Piezoelectric property of coaxial electrospun scaffolds; A. Voltage output under different frequency compression deformation with 100 MΩ loading; B. Voltage output under different frequency bending deformation with 100 MΩ loading; C. Voltage output when immersed in PBS at 37℃ for 3 weeks; D. Voltage output when immersed in PBS at 37℃ for 6 weeks; E. Voltage output curve when immersed in PBS at 37℃ for 6 weeks.

[0075] Figure 15 Effect of coaxial electrospun scaffolds on BMSCs and HUVECs proliferation; A. BMSCs; B. HUVECs ( / F means cultured in Flexcell system, i.e. dynamic culture environment).

[0076] Figure 16 Tube formation experiment; Tube formation staining; B-C. Semi-quantitative analysis.

[0077] Figure 17 Effect of coaxial electrospun scaffolds on BMSCs osteogenic gene expression; ( / F means cultured in Flexcell system, i.e. dynamic culture environment.

[0078] Figure 18 Skin histological staining after 4 weeks of subcutaneous implantation of coaxial electrospun scaffolds; A. HE staining; B. Masson staining; the arrow shows the newly formed blood vessels.

[0079] Figure 19 HE staining of organs after 4 weeks of subcutaneous implantation of coaxial electrospun scaffolds; (Liver: liver; Heart: heart; Lung: lung; Spleen: spleen; Kidney: kidney).

[0080] Figure 20 In vivo test of piezoelectric properties of coaxial electrospun scaffolds; A. Simple piezoelectric nanogenerator (PENG); B. Exposure of masseter muscle (yellow arrow); C. PENG sutured to the deep surface of the masseter muscle; D. Piezoelectric test (load resistance 100 MΩ); E, F. Exposure of femoral muscle group (yellow arrow); G. PENG sutured to the deep surface of the femoral muscle group; H. Voltage output of PCL film fixed on the masseter muscle; I. Voltage output of ZnO / AVT film fixed on the masseter muscle; J. Voltage output of ZnO / AVT film fixed on the femoral muscle group.

[0081] Figure 21 Surgical procedure of rat mandibular critical-sized bone defect model (5 mm);

[0082] Micro-CT image of rat mandible, quantitative analysis of new bone and VG staining 8 weeks after surgery.

[0083] Figure 22 A. Micro-CT three-dimensional reconstruction image; B. VG staining image; C. Quantitative analysis of osteogenic area; D. Bone tissue volume ratio to total tissue volume (BV / TV).

[0084] Figure 23 HE and Masson staining of rat mandible 8 weeks after surgery; A. HE staining; B. Masson staining; the arrow shows the bone marrow cavity and red blood cells.

[0085] Figure 24 Sequence fluorescent labeling and quantitative analysis of bone regeneration in the mandibular defects of rats 8 weeks after operation; A. Sequence fluorescent image; B. Quantitative analysis of fluorescent area. DETAILED DESCRIPTION

[0086] The present application is further described in conjunction with the following specific examples and accompanying drawings, which are intended to illustrate and not to limit the scope of the present application. Any changes and modifications that can occur to those skilled in the art are intended to be included within the scope of the present application as defined by the appended claims, and the scope of protection is not limited to the following examples. The process, conditions, reagents, experimental methods, etc. for implementing the present application are generally known in the art and are not specifically limited, except for the following specifically mentioned contents.

[0087] The following description of at least one example embodiment is merely illustrative in nature and is in no way intended to limit the scope of the application or its applications or uses. Any other embodiments, which can be derived from the embodiments in the present application by those skilled in the art without creative work, are within the scope of protection of the present application.

[0088] In all examples shown and discussed here, any specific value should be interpreted as merely exemplary, and not as a limitation. Thus, other examples of the example embodiments can have different values.

[0089] Example 1: Coaxial electrospinning scaffolds with different concentrations of AVT in the shell layer and different concentrations of ZnO in the core layer were prepared, and through in vitro biological experiments, the concentrations of AVT and ZnO that exhibit the best pro-angiogenic and pro-osteogenic properties were selected respectively.

[0090] 1.1 Preparation of electrospinning working solution with different concentrations of AVT

[0091] 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 each bottle, and then 10 mL of hexafluoroispropanol (HFIP) was added to each bottle. The bottles were then placed on a magnetic stirrer and stirred at room temperature for 20 min 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 concentrations, respectively. The bottles were then placed on a magnetic stirrer and stirred at room temperature overnight until the PCL particles were completely dissolved. The resulting solutions were used as the electrospinning working solution.

[0092] 1.2 Preparation of electrospinning working solution with different concentrations of ZnO

[0093] Five light-proof glass bottles were prepared and labeled for grouping. 0 g, 0.016 g, 0.032 g, 0.064 g, and 0.128 g of zinc oxide (ZnO) nanoparticles were weighed and added to the bottles, respectively. Then 10 mL of HFIP was added to each bottle, and the ZnO was uniformly dispersed under the action of an ultrasonic working instrument for 20 min. Then 1.6 g of PCL was added to each bottle to prepare 16% (w / v) PCL / HFIP solutions containing 0%, 1%, 2%, 4%, and 8% (w / w) ZnO / PCL concentrations, respectively. After the PCL particles were completely dissolved at room temperature on a magnetic stirrer overnight, the solutions were used as the electrospinning working solution.

[0094] 1.3 Preparation of coaxial electrospun scaffolds with different AVT or ZnO concentrations

[0095] A 5 mL syringe was used to draw the working solution described above and install it into 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 electrospun scaffolds with different drug or nanoparticle concentrations is shown in Table 1.

[0096] Table 1 Grouping of coaxial electrospun scaffolds with different AVT or ZnO concentrations

[0097]

[0098]

[0099] 1.4 Piezoelectric performance characterization of coaxial electrospun scaffolds with different concentrations of ZnO

[0100] Under different loading resistances, approximately 0.1 v and -10 nA voltage and current outputs were detected on the PCL group (0% ZnO), which was due to the fine triboelectric signal. However, increased output voltage and short-circuit current were detected on the remaining coaxial electrospun scaffolds as the ZnO content increased. The test results using 0.1 MΩ-1000 MΩ resistors also supported the above conclusion Figure 2 D). Under different loading resistances (10 MΩ, 50 MΩ, and 100 MΩ), the coaxial electrospun scaffolds loaded with different concentrations of ZnO produced output voltages ranging from 0.2 v to 0.9 v, and output currents ranging from -10 nA to -85 nA Figure 2 A-C). The piezoelectric performance test results showed that the piezoelectric properties of the coaxial electrospun scaffolds were positively correlated with the content of ZnO.

[0101] 1.5 In vitro biological experiment screening the optimal working concentration of ZnO or AVT

[0102] The proliferation of human umbilical vein endothelial cells (HUVECs) on the coaxial electrospun scaffold loaded with different concentrations of AVT was detected using CCK-8. During the 5-day culture period, HUVECs on the electrospun scaffolds in the PCL (0% AVT), 0.25% AVT, 1% AVT, and 4% AVT groups all exhibited similar cell proliferation curves, and there was no obvious cytotoxicity. However, on the 3rd and 5th days of culture, the OD value of the 16% AVT group was lower than that of the other four groups, and the difference was statistically significant, indicating that the 16% AVT group had cytotoxicity, and this group was not used in subsequent experiments (see Figure 3 ).

[0103] The present application further detects the effect of coaxial electrospun scaffolds loaded with different concentrations of AVT on the tube formation ability of HUVECs. As shown in Figure 4 , after 4 hours of culture on Matrigel, HUVECs were stained with Calcein AM, and the results showed that only a small amount of tube formation occurred in the PCL group, while in the 0.25% AVT, 1% AVT, and 4% AVT groups, tube formation gradually increased, with the 4% AVT group having the most tube formation, and quantitative analysis also yielded consistent results.

[0104] The present application uses qRT-PCR to detect the effect of coaxial electrospun scaffolds loaded with different concentrations of AVT on the expression of angiogenic genes in HUVECs. As shown in Figure 5 , after 4 days of culture of HUVECs on coaxial electrospun membranes loaded with PCL (0% AVT), 0.25% AVT, 1% AVT, and 4% AVT, the expression of VEGF and HIF-1 in the 0.25% AVT, 1% AVT, and 4% AVT groups was significantly upregulated compared to the PCL group, and the expression of ANGIO-1 in the 0.25% AVT and 4% AVT groups was significantly upregulated compared to the PCL group, with the 4% AVT group having the most significantly increased HIF-1 expression.

[0105] Therefore, the present application believes that coaxial electrospun scaffolds loaded with 0.25%-4% AVT have good biocompatibility and pro-angiogenic effects, and AVT at a concentration of 4% has the best pro-angiogenic performance.

[0106] The present application uses CCK-8 to detect the proliferation of rat bone marrow mesenchymal stem cells (BMSCs) on coaxial electrospun scaffolds loaded with different concentrations of ZnO. As shown in Figure 6As shown, during the 5-day culture period, BMSCs exhibited similar cell proliferation curves on electrospun membranes in the PCL (0% ZnO), 1% ZnO, 2% ZnO, and 4% ZnO groups, indicating no significant cytotoxicity. However, on days 3 and 5 of culture, the OD value of the 8% ZnO group was significantly lower than that of the other four groups, indicating that the 8% ZnO group was cytotoxic, and this group was not used in subsequent experiments.

[0107] The effects of coaxial electrospun scaffolds loaded with different concentrations of ZnO on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) were evaluated by detecting alkaline phosphatase (ALP) activity and osteogenic gene expression. To further clarify the role of the piezoelectric effect derived from ZnO in promoting BMSC osteogenic differentiation, all groups were further divided into static and dynamic culture groups. The static culture group underwent conventional material surface culture, while the dynamic culture group received 8 hours of tension stimulation daily using the Flexcell system. After 7 days of culture, there was no significant difference in ALP expression among the PCL (0% ZnO), 1% ZnO, 2% ZnO, and 4% ZnO groups in the static culture group. However, ALP expression in all groups of the dynamic culture group was significantly upregulated compared to the static culture group. Furthermore, within the dynamic culture group, the 4% ZnO group exhibited the highest ALP activity, with a statistically significant difference compared to other groups (see [link to dynamic culture group]). Figure 7 ).

[0108] This invention further uses qRT-PCR to evaluate the effect of coaxial electrospun membranes loaded with different concentrations of ZnO on osteogenic gene expression in BMSCs. Figure 8 As shown, after culturing BMSCs on coaxial electrospun membranes of PCL (0% ZnO), 1% ZnO, 2% ZnO, and 4% ZnO for 7 days, there were no significant differences in the expression of osteogenic marker genes Runx2, BMP2, OPN, and BSP among the groups in the static culture group. However, the expression of Runx2, BMP2, and OPN in all groups of the dynamic culture group was significantly upregulated compared to the static culture group. In the dynamic culture group, the 1%, 2%, and 4% ZnO groups all showed significantly higher expression of Runx2, BMP2, OPN, and BSP than the PCL group, with the 4% ZnO group exhibiting the highest osteogenic gene expression.

[0109] Therefore, this invention suggests that ZnO with a concentration of 1%-4% has good biocompatibility and promotes bone growth in sports environments, with 4% ZnO exhibiting the best bone-promoting properties.

[0110] Example 2: Using PCL as the matrix, a ZnO / AVT coaxial electrospinning scaffold with a 4% AVT shell and a 4% ZnO core was prepared.

[0111] Two light-proof glass bottles were prepared, 0.064 g of atorvastatin (AVT) and 0.064 g of zinc oxide (ZnO) nanoparticles were weighed into the bottles respectively, then 10 mL of hexafluoroispropanol (HFIP) was added into each bottle and stirred at room temperature for 20 min until completely dissolved, then 1.6 g of polycaprolactone (PCL) was added to prepare 16% (w / v) PCL / HFIP solution containing 4% (w / w) AVT / PCL and 4% (w / w) ZnO / PCL respectively, and stirred overnight at room temperature on a magnetic stirrer until the PCL particles were completely dissolved as the electrospinning working solution.

[0112] The above working solutions were taken with 5 mL syringes and installed on the electrospinning machine respectively, using coaxial electrospinning needle (shell layer 22G, core layer 17G) as the positive electrode, aluminum foil wrapped around the roller-shaped collector 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, the push speed of the core layer syringe was set to 0.5 mL / h, and the spinning time was 3 h to prepare ZnO / AVT coaxial electrospinning scaffolds with 4% AVT shell layer and 4% ZnO core layer.

[0113] Table 2 Grouping of coaxial electrospinning scaffolds coupled with piezoelectric stimulation and AVT

[0114]

[0115] Example 3 Material characterization of ZnO / AVT coaxial electrospinning scaffolds.

[0116] The coaxial electrospinning scaffolds mentioned in the following experiments refer to the coaxial electrospinning scaffolds prepared in Example 2 of the present application.

[0117] 1.1 The surface morphology and internal characteristics of the coaxial electrospinning scaffolds were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) respectively. The SEM images showed that the fibers of each group were randomly arranged, the fibers were uniform in thickness and had smooth surfaces. The diameter distribution of the fibers of each group was analyzed using Image J, and all the fiber diameters were in the micro-nanometer size range. The TEM images showed that the fibers of each group had a core-shell structure, and the ZnO nanoparticles of the core layer of the ZnO group and the ZnO / AVT group were clearly visible (see Figure 9 ).

[0118] 1.2 The hydrophilicity of the material was characterized by detecting the water contact angle. As Figure 10As shown, when water droplets contact the material surface for 2 seconds, the water droplets on the four material surfaces exhibit a typical elliptical shape. At this time, the water contact angles are: PCL group (129.40°±1.88°), ZnO group (127.80°±4.87°), AVT group (120.20°±3.66°), and ZnO / AVT group (121.90°±3.78°). 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 ZnO group was similar to that at 2 seconds, with water contact angles of 129.70°±2.11° and 130.10°±1.62°, respectively. However, the water droplets in the AVT group and the ZnO / AVT group showed obvious spreading and significantly smaller water contact angles, of 70.99°±7.44° and 77.85°±0.92°, respectively. There was no significant difference between the two groups, but the differences were statistically significant compared with the PCL group and the ZnO group.

[0119] 1.3 The mechanical property test results of the coaxial electrospinning support are as follows: Figure 11 As shown in the figure. The results of the elastic modulus (Young's modulus) test are as follows: PCL group (64.53MPa±4.96MPa), AVT group (72.69MPa±4.75MPa), ZnO group (42.31MPa±2.92MPa), and ZnO / AVT group (57.82MPa±4.85MPa). The results show that the AVT group has the highest elastic modulus, which is statistically significantly different from the ZnO group and the ZnO / AVT group, but not statistically different from the PCL group; the ZnO group has the lowest elastic modulus, which is statistically significantly different from the other three groups. The tensile strength test results were as follows: PCL group (2.87MPa±0.37MPa), AVT group (2.49MPa±0.31MPa), ZnO group (2.49MPa±0.14MPa), and ZnO / AVT group (2.33MPa±0.49MPa), with no significant differences among the groups.

[0120] 1.4 AVT release results of coaxial electrospinning scaffolds are as follows Figure 12 As shown, the AVT in the shells of the AVT group and the ZnO / AVT group exhibited rapid initial release followed by slow and sustained release. The cumulative AVT release in the first 12 hours was 41.27% (AVT group) and 39.34% (ZnO / AVT group), respectively. Subsequently, during a sustained 21-day in vitro release, the AVT group reached 81.05% of the cumulative release, while the ZnO / AVT group achieved 75.6%.

[0121] 1.5 The coaxial electrospinning scaffold was immersed in PBS at 37°C for 28 days to study its degradation performance. Figure 13The degradation rates of each group were as follows: PCL group (0.52% ± 0.25%), ZnO group (0.47% ± 0.23%), AVT group (0.60% ± 0.32%), and ZnO / AVT group (0.44% ± 0.2%) when immersed in PBS at 37°C for 7 days. The degradation rates of each group were as follows: PCL group (1.44% ± 0.32%), ZnO group (1.96% ± 0.38%), AVT group (1.69% ± 0.24%), and ZnO / AVT group (1.48% ± 0.6%) when immersed in PBS for 28 days.

[0122] 1.6 The piezoelectric performance of the coaxial electrospun scaffold was characterized, as shown in Figure 14 At a load resistance of 100 MΩ, the PCL group and the AVT group could detect voltage and current outputs of about 0.1 v and -10 nA under compression and bending deformation at different frequencies, because the encapsulated simple nanogenerator could generate slight triboelectricity. The ZnO group stably output voltage and current signals of about 0.8 v and -42 nA under compression at different frequencies, and a voltage output of about 0.9 v under bending deformation. The ZnO / AVT group output voltage and current signals were slightly weaker than those of the ZnO group, about 0.78 v and -37 nA under compression, but produced a voltage output (about 0.9 v) consistent with that of the ZnO group under bending deformation. To detect the ability of the coaxial electrospun scaffold to maintain piezoelectricity in vivo, the film was immersed in PBS at 37°C for 3 weeks and 6 weeks, and its piezoelectric performance was detected Figure 14 E), the results showed that the ZnO group and the ZnO / AVT group could maintain the initial voltage output of about 0.9 v when immersed for 3 weeks, but the output voltage was about 0.45 v when immersed for 6 weeks, i.e., a decrease of about 50%.

[0123] Example 4 The ZnO / AVT coaxial electrospun scaffold has good biocompatibility in vitro and can effectively promote angiogenesis and osteogenesis differentiation.

[0124] The coaxial electrospun scaffold mentioned in the following experiments refers to the coaxial electrospun scaffold prepared in Example 2 of the present application.

[0125] 1.1 CCK-8 was used to detect the proliferation of HUVECs and rat BMSCs on the coaxial electrospun scaffold, and all groups were further subdivided into static culture groups and dynamic culture groups to avoid interference of the cell force instrument on the subsequent experimental results. As shown in Figure 15 HUVECs and BMSCs showed good cell proliferation trends on the electrospun films in each group during a 5-day culture period, and there was no statistically significant difference between groups, indicating that the scaffold had good biocompatibility.

[0126] 1.2 The effect of each group of coaxial electrospun scaffolds on the tube formation ability of HUVECs was further detected by tube formation experiment. As shown in Figure 16 Figure 2B, only a small amount of tube formation was observed in the PCL group and the ZnO group in the static culture group, while the AVT group and the ZnO / AVT group showed significantly increased tube formation. In the dynamic culture group, more tube formation was observed in the ZnO group, the AVT group and the ZnO / AVT group, among which the ZnO / AVT group showed the most obvious ability to promote tube formation of HUVECs. Quantitative analysis also showed consistent results, indicating that the ZnO / AVT scaffold could effectively promote blood vessel formation in vitro.

[0127] 1.3 The effect of coaxial electrospun scaffolds on the expression of osteogenic genes of BMSCs was evaluated by qRT-PCR. As shown in Figure 17 Figure 3B, after 7 days of culture of BMSCs on each group of coaxial electrospun scaffolds, the expression of osteogenic marker genes Runx2, Col1 and OPN did not show significant up-regulation or down-regulation among each group in the static culture group, while the expression of BSP in the AVT group and the ZnO / AVT group was significantly up-regulated compared with the PCL group. In the dynamic culture group, the expression of Runx2, Col1, OPN and BSP in the ZnO, AVT and ZnO / AVT groups was significantly up-regulated compared with the other groups, among which the ZnO / AVT group showed the highest expression of Runx2, Col1, OPN and BSP, indicating that the ZnO / AVT scaffold could effectively promote bone formation in vitro.

[0128] Example 5 The ZnO / AVT coaxial electrospun scaffold has good biocompatibility in vivo, can convert the mechanical force of the masseter and femoral muscle group into piezoelectric stimulation, and effectively promotes the repair of rat mandibular bone defects.

[0129] 1.1 The effect of coaxial electrospun scaffolds on promoting angiogenesis in vivo was verified by subcutaneous implantation experiment on the back of rats. As shown in Figure 18 Figure 1B, at 4 weeks after the operation, the full-thickness skin on the back was removed, and HE staining and Masson staining were performed to observe the subcutaneous neovascularization. As shown in the figure, only a small amount of neovascularization was observed in the Blank group and the PCL group, while the ZnO group showed slightly more neovascularization than the former two groups. The AVT group and the ZnO / AVT group showed significantly increased subcutaneous neovascularization, and a large number of red blood cells were observed in the dense blood vessels.

[0130] At 4 weeks after the subcutaneous implantation of the scaffold, as shown in Figure 19 Figure 1C, HE staining of the visceral paraffin sections of the Blank group, the PCL group, the ZnO group, the AVT group and the ZnO / AVT group showed normal tissue and cell morphology, orderly arrangement, clear structure, and no necrotic tissue or inflammatory cell infiltration, indicating that the scaffold had good in vivo biocompatibility.

[0131] 1.2To evaluate the ability of coaxial electrospun scaffold to collect muscle force into piezoelectric stimulation, electrospun films were sutured between the masseter muscle and mandible and between the femoral muscle group and femur of rats, respectively, as shown in FIG. 2A and FIG. 2B, and the output voltage of the films was measured under the conditions of opening and closing the mouth (masseter muscle movement) and stretching the leg (femoral muscle group movement), respectively. As shown in FIG. 2C and FIG. 2D, the results showed that the PCL group generated only about 0.1 v of voltage output under the action of the masseter muscle at a load resistance of 100 MΩ, which was a slight triboelectric signal. The ZnO / AVT group generated 0.32 v and 0.5 v of voltage output under the action of the masseter muscle and the femoral muscle group, respectively. Figure 21 Figure 20

[0132] The results of micro-CT scanning and quantitative analysis of new bone of the mandible of rats 8 weeks after surgery are shown in FIG. 3A and FIG. 3B. The Blank group only had a small amount of new bone tissue around the edge of the defect, while the other four groups had better osteogenesis area and BV / TV value. Compared with the PCL group, the bone defect area of the AVT group, the ZnO group and the ZnO / AVT group all showed more obvious new bone formation, especially the ZnO / AVT group, which repaired more than 70% of the bone defect area, indicating that the ZnO / AVT scaffold can effectively promote the repair of the mandibular bone defect of rats. Figure 22

[0133] 1.3 Further observation of the new blood vessels in the new bone regeneration area of the mandibular defect of rats 8 weeks after surgery was performed using HE staining and Masson staining, and the results are shown in FIG. 4A and FIG. 4B. The Blank group only had a small amount of new bone tissue around the edge of the defect, and no obvious bone marrow cavity and new blood vessels were observed. The PCL group had a larger osteogenesis area than the Blank group, but the new blood vessels around the osteogenesis edge were few, and a dense cortical bone was observed around the edge. The osteogenesis area of the AVT group, the ZnO group and the ZnO / AVT group all showed more obvious bone marrow cavity and new blood vessels, especially the ZnO / AVT group, and the observation of the osteogenesis area in close contact with the film showed that there were enlarged bone marrow cavities, a large number of blood vessels and red blood cells (red arrows), indicating active blood vessel formation. Figure 23

[0134] The rats were injected intraperitoneally with tetracycline (TE), alizarin red (AL) and calcein (CA) as fluorescent markers for new bone growth at 2, 4 and 6 weeks after surgery, respectively. Observation of the hard tissue sections of the mandible 8 weeks after surgery using a laser confocal microscope showed that the yellow (TE), red (AL) and green (CA) three-color fluorescence was distributed in layers, indicating the process of new bone growth layer by layer. As shown in FIG. 5A, the Blank group only had a small amount of three-color fluorescent markers, and the fluorescent area of the PCL group, the AVT group, the ZnO group and the ZnO / AVT group increased in turn, and the ZnO / AVT group exhibited the largest TE, AL and CA fluorescent area. The quantitative results of the fluorescent area were consistent with the observation results of the fluorescent staining images (FIG. 5B). Figure 24 ​​​​​

[0135] The results of micro-CT, VG staining, HE staining, Masson staining and fluorescence labeling and analysis of the rat mandible at 8 weeks after operation show that the ZnO / AVT scaffold significantly promotes the vascularized bone regeneration of the rat mandible bone defect.

[0136] Discussion

[0137] The repair of critical bone defects is still a challenge. In the early stage of bone healing, the ingrowth of blood vessels provides essential oxygen and nutrients for cell colonization, proliferation and differentiation and helps to clean up the corresponding metabolic products, which is a prerequisite for bone regeneration. Early vascularization is of great significance for the regeneration of critical bone defects.

[0138] The electrospinning technology is simple and controllable, and the prepared fibers are in the micro-nano diameter range and have a morphology similar to the natural extracellular matrix, but it also has the shortcomings of weak bioactivity. Therefore, the bioactivity of the electrospinning scaffold can be improved by loading active factors or drugs and other ingredients. In the present application, we selected:

[0139] (1) The FDA-certified artificial synthetic polymer material PCL is used as the base material for electrospinning. PCL has the advantages of sufficient source, good biocompatibility, in vivo degradation and non-toxic degradation products, and has been widely used in the biomedical field.

[0140] (2) Atorvastatin (AVT) is a statin drug that has been clinically used to treat patients with high cholesterol. It can also reduce major cardiovascular events in patients at high risk of cardiovascular disease such as coronary heart disease, ischemic stroke, diabetes, and hypertension, and has good safety. At low doses, it has excellent ability to promote angiogenesis.

[0141] (3) ZnO is a classic semiconductor piezoelectric material with good biocompatibility. The ZnO-based nanogenerator has been shown to promote osteogenic differentiation. The piezoelectric coefficient of ZnO and natural bone tissue is closer, which may be a more suitable piezoelectric material for bone defect repair. In addition, zinc is a trace mineral that also has the effects of promoting growth, repairing damage, regulating immune response, and scavenging free radicals. Therefore, ZnO as a piezoelectric material has broad prospects for application in the biomedical field.

[0142] The mandibular surface has strong attachment of masticatory muscles, and the biomechanical force of masticatory muscles will affect the healing of mandibular defects under normal physiological activities, but at the same time, it also provides a suitable biomechanical scenario for piezoelectric stimulation to promote bone repair, and the piezoelectric stimulation generated in this scenario has more physiological rhythm. Therefore, based on the importance and timing of early angiogenesis in bone regeneration, the faster controlled release ability of the coaxial electrospun shell layer, and the effect of piezoelectricity on promoting osteogenesis: a new coaxial electrospun scaffold coupled with piezoelectric stimulation and AVT is prepared by using coaxial electrospun technology, AVT, a clinical drug that promotes angiogenesis, is loaded into the fiber shell layer, and ZnO nanoparticles with piezoelectric properties are loaded into the fiber core layer. Through the early and rapid release of AVT to promote early angiogenesis, ZnO collects the masticatory muscle force under physiological movement to convert piezoelectric stimulation to continuously promote osteogenesis, and ultimately achieve vascularized bone regeneration of mandibular defects.

[0143] The coaxial electrospun scaffold with different concentrations of AVT in the fiber shell layer was used to culture HUVECs on the surface, and the effects of AVT on cell proliferation, cell migration, tube formation, and angiogenic 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, indicating that it had cytotoxicity, while the 0.25%-4% AVT group had consistent cell growth levels with the control group, indicating that it had good biocompatibility. The tube formation experiment and angiogenic gene expression experiment results showed that the 4% AVT group had the best effect on promoting HUVECs tube formation and angiogenic gene expression in vitro. Therefore, 4% AVT has good biocompatibility, the best ability to promote HUVECs migration, tube formation, and angiogenic differentiation, and thus it is used as the optimal concentration of AVT for subsequent experiments.

[0144] Then, the coaxial electrospun scaffold with different concentrations of ZnO in the fiber core layer was used to culture BMSCs on the surface, and the cell proliferation and osteogenic differentiation ability were detected. The BMSCs in the 8% ZnO group showed cell proliferation inhibition at 3 days and 5 days of culture, while the 1%-4% ZnO group coaxial electrospun scaffold had good biocompatibility. A cell force instrument was used to provide rhythmic stretching deformation to the electrospun membrane to promote the generation of piezoelectric stimulation, and the activity of ALP was detected to screen the optimal concentration of ZnO piezoelectric stimulation to promote osteogenic differentiation. The ALP detection results showed that all groups in the dynamic culture group had higher ALP expression than the static culture group, indicating that the movement stimulation had the effect of promoting osteogenic differentiation. At the same time, among the dynamic culture group, the electrospun membrane loaded with ZnO had higher ALP activity and osteogenic gene expression, confirming the effect of piezoelectricity on promoting osteogenic differentiation, and the 4% ZnO group showed the highest ALP activity and osteogenic gene expression. Therefore, according to the good biocompatibility and the best effect of piezoelectricity on promoting osteogenic differentiation of the 4% ZnO group, it is selected as the optimal concentration of ZnO for subsequent experiments.

[0145] According to the above results, a coaxial electrospun scaffold (ZnO / AVT) with piezoelectric stimulation coupling AVT was prepared with 4% AVT as the shell layer and 4% ZnO as the core layer, and material characterization was performed. SEM showed that the fibers of each group were randomly arranged, the surface was loose and 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-nano size range. TEM showed the successful construction of the coaxial electrospun core-shell structure and the successful loading of the core layer ZnO nanoparticles. The drug release experiment results showed that the shell layer AVT was rapidly released at the initial stage, with a cumulative release amount of about 40% in the first 12 h, and then slowly and continuously released, with a cumulative release amount of about 80% at 3 weeks. The release of AVT meets the needs of early vascularization in large-area bone defect repair. The 4-week degradation experiment showed that the coaxial electrospun scaffold degrades slowly, which means that it can act as a scaffold material in the body and play a stable and continuous role in bone repair period. Subsequently, the piezoelectric properties of the coaxial electrospun scaffold were verified under different frequencies of compression and bending test, and the results showed that the piezoelectric properties of the ZnO / AVT group were slightly lower than those of the ZnO group, which may be due to the loading of the shell layer AVT. The piezoelectric properties of the coaxial electrospun scaffold were consistent with the initial ones after PBS immersion for 3 weeks, and decreased by nearly 50% after PBS immersion for 6 weeks, which may be related to the partial degradation of the fiber shell layer and the exposure of the core layer fibers, resulting in the loss of ZnO. Studies have shown that the 2-3 weeks after initial transplantation is a critical period for bone regeneration, so the piezoelectric property test meets our original intention of designing the coaxial electrospun scaffold with piezoelectric stimulation coupling AVT. The above experiments prove that the shell layer AVT can induce early angiogenesis by early and rapid release, and the core layer ZnO degrades slowly and maintains its piezoelectric properties during the critical period of bone repair, which meets the characteristics of inducing early vascularization and continuously providing piezoelectric stimulation to promote osteogenesis in bone defect repair.

[0146] BMSCs and HUVECs were cultured on the coaxial electrospun scaffold, and static and dynamic cultures were performed. Cell proliferation experiments showed that there was no statistical difference in cell proliferation of BMSCs and HUVECs in each group, indicating that the coaxial electrospun scaffold loaded with ZnO and AVT at the same time had good biocompatibility.

[0147] Since early rapid angiogenesis is the key to the successful repair of large-area bone defects, we evaluated the in vitro angiogenesis capacity of the ZnO / AVT coaxial electrospun scaffold through tube formation experiment. The results showed that the coaxial electrospun scaffold with piezoelectric stimulation coupling AVT could exert the synergistic effect of piezoelectric stimulation and AVT, and promote angiogenesis to the greatest extent.

[0148] The most important thing for bone repair materials is the ability to promote BMSCs osteogenic differentiation. The in vitro osteogenic ability of coaxial electrospun scaffolds was evaluated by detecting the expression level of osteogenic genes. qRT-PCR was used to detect the expression of osteogenic marker genes Runx2, OPN, BSP and Col1 at the mRNA level, and the results showed that the expression levels of Runx2, OPN and Col1 in each group of the static culture group were at a low level, while the ZnO group in the dynamic culture group had significantly up-regulated osteogenic gene expression. The above results show that the piezoelectric stimulation of coaxial electrospun scaffolds with ZnO-loaded fiber core layer significantly promotes the osteogenic differentiation of BMSCs. Interestingly, in the detection of osteogenic gene expression, the AVT group in the dynamic culture group showed similar ability to stimulate osteogenic gene expression as the ZnO group, and the AVT group could also significantly up-regulate the expression of BSP in static culture, indicating that AVT has potential to promote osteogenic differentiation. At the same time, the ZnO / AVT group in the dynamic culture group showed the highest osteogenic gene expression, proving that piezoelectric stimulation and AVT have obvious synergistic effect in promoting the osteogenic differentiation of BMSCs.

[0149] To further verify the in vivo biological safety and vascularized bone regeneration ability of coaxial electrospun scaffolds coupled with piezoelectric stimulation and atorvastatin, rat subcutaneous implantation models and rat mandibular critical defect models were constructed. The histological staining results of organs after 4 weeks of rat subcutaneous implantation proved that coaxial electrospun scaffolds loaded with ZnO / AVT had excellent in vivo biocompatibility.

[0150] In the rat mandibular critical defect model, micro-CT results showed that there was almost no new bone formation in the Blank group, indicating the successful establishment of critical bone defects. Compared with the Blank group, the remaining groups had different degrees of new bone formation, which showed that coaxial electrospun scaffolds as a kind of bone repair scaffold material could improve the repair of bone defect area. Compared with the PCL group, the defect area of AVT, ZnO and ZnO / AVT groups showed an increasing new bone formation, especially the ZnO / AVT group, which repaired more than 70% of the bone defect area. HE and Masson staining and fluorescence labeling results supported the micro-CT results. The experimental results showed that the piezoelectric stimulation of ZnO group and the pro-angiogenic effect of atorvastatin could promote bone defect repair to some extent, and the two could synergistically produce excellent effect of promoting bone regeneration.

[0151] CONCLUSION

[0152] (1) Coaxial electrospun scaffolds with a shell layer loaded with pro-angiogenic drug AVT and a core layer loaded with piezoelectric material ZnO were successfully prepared by coaxial electrospun technology.

[0153] (2) The optimal concentration of shell layer 4% AVT is to promote HUVECs angiogenesis, and the optimal concentration of core layer 4% ZnO is to promote BMSCs osteogenesis under piezoelectric stimulation, and both have good in vitro biocompatibility.

[0154] (3) The surface morphology of coaxial electrospun scaffold is similar to extracellular matrix, and the AVT loaded in fiber shell layer has the behavior of early rapid release and late slow release. The coaxial electrospun scaffold degrades slowly, and the piezoelectric performance of coaxial electrospun scaffold loaded with ZnO in the core layer can be stably maintained in PBS at 37℃ for at least 3 weeks, and the AVT loaded in the fiber shell layer has only a weak effect on its electrical properties.

[0155] (4) The fiber shell layer loaded with 4% AVT can significantly promote HUVECs angiogenesis, and the piezoelectric stimulation of the electrospun membrane with 4% ZnO loaded in the fiber core layer can significantly promote BMSCs osteogenesis, and the piezoelectric stimulation and AVT synergistically most significantly promote BMSCs osteogenesis and HUVECs angiogenesis.

[0156] (5) The coaxial electrospun scaffold (ZnO / AVT) coupled with piezoelectric stimulation and AVT has good in vivo biocompatibility, can convert the mechanical force of masseter muscle and femoral muscle group into piezoelectric stimulation in vivo, and exhibits the best result of promoting vascularized bone regeneration in the rat mandibular defect model.

Claims

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

2. The pharmaceutical composition of claim 1, wherein, The use ratio of the AVT and ZnO is 0.25%-4%:1%-4%.

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 ZnO and a first matrix; and the shell layer comprises AVT and a second matrix.

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

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 57.82MPa±4.85MPa; and / or, the coaxial electrospun scaffold has a tensile strength of 2.33MPa±0.49MPa.

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, Specifically comprising 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 by using a coaxial electrospinning technology to form the core layer and the shell layer, thereby obtaining 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 the ZnO in the first dispersant is 6.4g / L; and / or, the addition amount of the AVT in the second dispersant is 6.4g / L; and / or, the first matrix:the first dispersant=16% (w / v); and / or, the second matrix:the second dispersant=16% (w / v).

9. The method of claim 7, wherein, In the coaxial electrospinning technology, one or more of the following technical features is included: The needle voltage is +7kV; The receiving roller voltage is -8kV; The receiving distance is 15cm; The roller rotation speed is 10rpm / min; The advancing speed of the shell layer injector is set to 1mL / h; The advancing 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 the repair of osteogenesis defects, promoting the vascularized bone regeneration of mandibular bone defects, promoting osteogenic differentiation, and promoting hemangiogenic differentiation.

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