A smart electronically controlled spraying device and a method for preparing a nanofiber bone repair membrane.

Abstract

This application discloses an intelligent electronically controlled spraying device and a method for preparing a nanofiber bone repair membrane. The device includes a controllable nozzle, a nozzle solution tank, a moving guide rail, a speed-changing gearbox, a negative pressure suction device, and a material placement box. This application achieves the loading of growth factors with a gradient concentration onto the repair material by controlling the spraying speed, travel speed, and direction. Negative pressure suction allows the loaded growth factors to penetrate the repair material in situ. The absorbable nanofiber bone repair membrane, functionalized with VEGF in a bidirectional gradient, has a high VEGF concentration in the central region, gradually decreasing towards both ends. This chemotactic effect guides newly formed vascular endothelial cells from the bone defect ends to migrate directionally to the high concentration in the central region of the repair membrane, accelerating the directional vascularization of bone tissue from both ends towards the central region. Simultaneously, the newly generated blood vessels act as a scaffold to guide cells to rapidly migrate into the fibrous membrane, thereby efficiently promoting bone tissue regeneration and repair.

Description

Technical Field

[0001] This application belongs to the fields of regenerative tissue engineering and vascular tissue engineering technology, and more specifically, relates to an intelligent electronically controlled spraying device for loading gradient concentration growth factors into bone and ligament tissue repair materials and a method for preparing nanofiber bone repair membranes. Background Technology

[0002] Following tissue injury, vascular endothelial cells are activated. In cases of contusion, endothelial cells can migrate along the intact intima structure. However, recent research indicates that transverse injuries disrupt the intima-intima connections, at which point endothelial cell migration relies on regenerated blood vessels as a scaffold. Directional vascular growth is crucial for guiding the directional migration of endothelial cells and the directional regeneration of bone.

[0003] For long segmental defects, fibrous membranes are needed for bridging to achieve structural connection. Therefore, the key to accelerating defect repair lies in how to promote the rapid directional vascularization of the fibrous membrane and guide endothelial cells to migrate directionally and rapidly into the interior of the fibrous membrane from both proximal and distal ends of the injury, thereby accelerating the vascular connection and bridging area and promoting functional recovery.

[0004] Vascular endothelial growth factor (VEGF) can chemotactically attract endothelial cell migration and promote angiogenesis. Numerous studies have confirmed that high expression of VEGF through genetic engineering has protective and regenerative effects on tissues. However, these studies have failed to address how to accelerate vascularization of fibrous membranes to promote rapid vascular bridging at joint sites. Research by Ma Fukai et al. found that VEGF-functionalized fibrous membranes significantly promoted tissue regeneration compared to ordinary collagen fiber membranes. Other studies have indicated that simple concentration gradient guidance cannot achieve rapid directional vascularization in the central region of the fibrous membrane, limiting the polarized migration of endothelial cells to high-VEGF concentration areas and the rate of angiogenesis. Therefore, fibrous membranes lacking directional concentration gradients cannot effectively guide the rapid and directional migration of endothelial cells into the membrane, thus limiting the rate at which VEGF promotes vascular bridging of the fibrous membrane.

[0005] In existing technologies, the method for creating gradient cell membranes involves reacting ethylenediamine with PCL to generate NH2 groups, which are then coated onto the cell membrane with a concentration gradient or uniform distribution of NGF. In short, the cell membrane is folded vertically along its fiber orientation, and the fold is placed in an ethylenediamine solution. The solution is continuously and gently stirred while distilled water is gradually added. Through the continuous decrease in ethylenediamine concentration and the linear change in reaction time, the density of NH2 groups exhibits a gradient distribution with a steeper axial direction. To obtain a uniform distribution of NH2 groups, the cell membrane is saturated only in the ethylenediamine solution. Then, the catheter is immersed in a mixture of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and sodium heparin, followed by immersion in 2-(N-morpholine)ethanesulfonic acid (MES, Sigma) buffer and brief rotation at room temperature to ensure covalent bonding of the heparin carboxyl groups to the gradient NH2 groups on the PCL surface, forming a gradient heparin distribution. Finally, the gradient heparinized cell membrane was reacted with VEGF at 4°C to obtain a gradient VEGF-ordered nanofiber osteoblast membrane. In existing membrane fabrication techniques, all steps require manual operation, and it is difficult to precisely and easily create the concentration gradient of the loaded factors on the membrane. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this application aims to provide an intelligent electronically controlled spraying device and a method for preparing a growth factor nanofiber bone repair membrane with a gradient concentration. This aims to solve the problems of existing membrane-making technologies being cumbersome, difficult to manufacture, and unable to be automated due to the lack of automated and convenient methods for producing VEGF (or other factor) loaded cell membranes and biomembranes, as well as the inability to accurately and easily create a gradient of factor concentrations on the membrane.

[0007] This application provides an intelligent electronically controlled spraying device, comprising: a controllable nozzle, a nozzle solution tank, a moving guide rail, a speed-changing gearbox, a negative pressure suction device, and a material placement box; the controllable nozzle is connected to the nozzle solution tank and fixed on the moving guide rail, and is used to spray a strip-shaped mist; the spraying range is greater than the width of the fiber membrane; the nozzle solution tank is used to store the spraying solution and enable the spraying solution to be replaceable; the speed-changing gearbox is used to infinitely adjust the spray volume of the controllable nozzle and control the movement of the controllable nozzle on the moving guide rail; the material placement box includes upper and lower layers, the upper layer is used to support the PCL fiber membrane that needs to be loaded with gradient concentration growth factors, and the lower layer is connected to the negative pressure suction device, which is used to extract liquid and gas, and can also provide positive pressure to prevent the solution in the upper layer from flowing into the lower layer. The bottom of the upper layer can be connected to the lower layer. Through electronically controlled strip-shaped mist spraying and negative pressure suction wetting, the sprayed growth factors can be accurately immersed locally into the material to be loaded.

[0008] Furthermore, the blade-shaped controllable nozzle has a small sprayable thickness, which ensures that the spray range on the fiber membrane always remains a strip with the width of the fiber membrane as the length of the spray range, and the width is less than 0.5 mm.

[0009] Furthermore, the gearbox has dual gears, which are used to control the spray speed and travel speed and direction of the controllable nozzle.

[0010] Furthermore, the transmission gearbox can be computer-controlled to adjust speed and acceleration, adjusting the gearbox's acceleration to move it from the center to the left and right ends with an acceleration matched to the membrane. This repeated operation forms a fiber membrane with a high concentration of the substance in the middle and low concentrations at both ends. Specifically, this acceleration can be set to match different membranes, and can be 1... ~3 .

[0011] Furthermore, a storage plate with densely distributed micropores is provided at the bottom of the upper layer of the material placement box. The storage plate is used to place the fiber membrane. When the negative pressure aspirator is turned on, the liquid can be quickly drawn into the pore plate. When the solution needs to remain in the upper layer to soak the fiber membrane, the lower negative pressure aspirator can provide positive pressure to prevent the solution from flowing into the lower layer.

[0012] More preferably, the aperture of the bottom shelf of the upper layer can be 1 mm.

[0013] Furthermore, the controllable nozzle and the nozzle solution tank can be connected via a hose.

[0014] This application also provides a method for preparing nanofiber bone repair membranes based on the above-mentioned intelligent electronically controlled spraying device, comprising the following steps:

[0015] S1 Preparation of electrospun PCL fiber membranes:

[0016] PCL was dissolved in a 10% methanol / chloroform mixed solution to obtain an electrospinning solution. The flow rate of the electrospinning solution was controlled at 0.01 ml / h-0.30 ml / h, and the PCL fiber membrane was obtained by drying to remove the residual solvent.

[0017] S2 performs bidirectional gradient ammonolysis on the PCL fiber membrane:

[0018] The fiber membrane is firmly attached to the tank wall using a negative pressure suction device. The initial position of the controllable nozzle is set in the exact center of the fiber membrane, and the controllable nozzle is positioned so that it extends from the left and right sides at a 1-degree angle from the initial position. The spraying is accelerated towards both ends to obtain a gradient fiber membrane with a high -NH2 density in the central part and a gradually decreasing -NH2 density towards both ends.

[0019] S3 prepares a bidirectional gradient heparinized fibrous membrane via a heparin coupling reaction:

[0020] A buffer solution containing morpholine ethanesulfonic acid is uniformly sprayed onto the gradient fiber membrane and allowed to fully impregnate it for a period of time. Then, a solution containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide / heparin is sprayed onto the membrane to covalently bind heparin to the amino-carrying fiber membrane. After washing away the unbound heparin, a bidirectional gradient heparinized fiber membrane is obtained.

[0021] S4 preparation of bidirectional gradient VEGF fiber membrane:

[0022] VEGF solution was uniformly sprayed onto a heparinized fibrous membrane with a bidirectional gradient to obtain a VEGF-functionalized nanofiber bone repair membrane with a bidirectional gradient concentration.

[0023] This application also provides a method for promoting bone regeneration using the above-mentioned nanofiber bone repair membrane loaded with gradient concentration of growth factors, comprising: releasing a high concentration of VEGF in the central part of the VEGF-functionalized fiber membrane with a bidirectional gradient, thereby accelerating the migration of endothelial cells at both ends of the damaged area to the high concentration in the central part of the VEGF-functionalized fiber membrane with a bidirectional gradient through chemotaxis.

[0024] Accelerating the directional vascularization of the fibrous membrane from both ends towards the center, the generated blood vessels act as a scaffold, guiding vascular endothelial cells to migrate rapidly into the fibrous membrane, thereby accelerating regeneration and extending distally along the vascular endothelial cells.

[0025] Among them, the VEGF-functionalized fiber membrane with bidirectional gradient has a high amount of VEGF loaded in the center and relatively low amount of VEGF loaded at both ends.

[0026] In summary, compared with the prior art, the technical solutions conceived in this application have the following technical effects:

[0027] (1) Because the intelligent electronically controlled spraying device has a blade-shaped controllable nozzle that automatically adjusts the spray size and a moving guide rail that controls the nozzle to move at a certain acceleration on the guide rail, the nozzle can automatically control the spray size, or the computer can control the acceleration of the moving guide rail, thereby forming the required concentration gradient; because it has a negative pressure suction device, when the device provides negative pressure, the lower layer can be in a negative pressure environment, so that the solution at the bottom of the upper layer can be quickly drawn away through the small aperture on the plate. At the same time, if the negative pressure suction device is adjusted to provide a certain positive pressure, so that the lower layer is in a positive pressure environment, the solution at the bottom of the upper layer can remain in the upper layer. Thus, when the solution needs to stay and react fully with the membrane, the solution can stay in the upper layer, and when the solution needs to leave the membrane, the solution can be quickly drawn away; because this device can be automatically controlled by computer to adjust the solution gradient, the spraying range of the loaded solution, and the residence time of the sprayed solution on the fiber membrane during the fiber membrane manufacturing process, it can achieve automated computer control of the manufacturing of biomembranes loaded with various factors.

[0028] (2) Compared with existing technologies, the biofilm produced by this device can precisely control the concentration of the loaded factors on the membrane and infinitely adjust the gradient of the loaded factors on the membrane. At the same time, it eliminates the tedious process of manual membrane production, making the production of various biofilms more refined and automated.

[0029] (3) The VEGF-functionalized fibrous membrane with bidirectional gradient provided in this application has a high VEGF loading in the center and a relatively low VEGF loading at both ends. This allows endothelial cells at the damaged ends to migrate towards the high concentration in the center of the fibrous membrane through chemotaxis, accelerating the directional vascularization of the fibrous membrane from both ends to the center. At the same time, the generated blood vessels can act as a scaffold to guide the rapid migration of vascular endothelial cells into the fibrous membrane, thereby promoting regeneration and extending distally along the vascular endothelial cells.

[0030] (4) This application achieves the loading of growth factors with gradient concentration on the repair material by controlling the spraying speed, travel speed and direction. The loading growth factors are in situ immersed in the repair material by negative pressure attraction. The absorbable nanofiber bone repair membrane with VEGF functionalized by bidirectional gradient has a high concentration of VEGF in the central region and gradually decreases the concentration of VEGF towards both ends. It can guide the newly formed vascular endothelial cells at the bone defect ends to migrate in a directional manner to the high concentration in the central region of the repair membrane through chemotaxis, accelerate the directional vascularization of bone tissue in the defect area from both ends to the central region, and at the same time, the newly generated blood vessels can act as a scaffold to guide the cells to migrate rapidly into the fiber membrane, thereby efficiently promoting bone tissue regeneration and repair. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the structure of the intelligent electronically controlled spraying device provided in the embodiments of this application;

[0032] 1 is a controllable nozzle; 2 is a variable speed gearbox; 3 is a PCL fiber membrane; 4 is a computer; 5 is a moving guide rail; 6 is a negative pressure suction device; and 7 is a material placement box.

[0033] Figure 2 This is a flowchart of the method for preparing a fiber membrane of bidirectional gradient vascular endothelial growth factor provided in the embodiments of this application;

[0034] Figure 3 This is a schematic diagram of the gradient of the PCL fiber membrane produced using the intelligent electronically controlled spraying device provided in this application;

[0035] Figure 4 These are images showing the detection of cell migration on bone repair membranes using three types of immunofluorescence staining on fiber membranes and the detection of cell proliferation on bone repair membranes using EDU kits provided in the embodiments of this application.

[0036] Where AC represents the cell migration assay, DF represents the EDU assay, G is a statistical diagram showing the differences in cell number after immunofluorescence staining on three membranes: PCL, PCL + homogeneous VEGF, and PCL + gradient VEGF; H is a statistical diagram showing the differences in cell proliferation on three membranes: PCL, PCL + homogeneous VEGF, and PCL + gradient VEGF. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0038] This application provides an intelligent electronically controlled spraying device for loading gradient concentrations of growth factors into bone and ligament tissue repair materials, and a method for preparing nanofiber bone repair membranes. By preparing nanofiber bone repair membranes with bidirectional gradient concentrations of VEGF, the directional vascularization of newly formed tissue guided by the fiber membrane can be accelerated, and vascular endothelial cells can be guided to migrate rapidly from both ends of the bone defect to the central region, thereby promoting bone regeneration and repair, and solving the problem that the vascularization of bone regenerated tissue guided by fiber repair membranes without concentration gradients of VEGF lacks directionality.

[0039] The application principle of this application will be described in detail below with reference to the accompanying drawings.

[0040] Figure 1This application discloses an intelligent electronically controlled device for loading gradient concentration growth factors onto bone and muscle tissue repair materials. The device includes a blade-shaped controllable nozzle 1, a replaceable nozzle solution tank, a moving guide rail 5, a speed-changing gearbox 2, a negative pressure suction device 6, and a material placement box 7. The blade-shaped controllable nozzle 1 is connected to the solution tank via a flexible hose and fixed to the controllable nozzle moving guide rail. The speed-changing gearbox 2 can control the speed and acceleration via computer to move the nozzle on the nozzle moving guide rail. The assemblable membrane material placement box has two layers: the upper layer holds the PCL fiber membrane 3 to be loaded with gradient concentration growth factors, and the lower layer is connected to the negative pressure suction device 6 to extract liquids and gases. The blade-shaped controllable nozzle 1 can spray a thin strip-shaped mist with a spray range greater than the width of the fiber membrane and a small spray thickness, ensuring that the spray range on the fiber membrane always remains a rectangle with the width of the fiber membrane as the length of the spray range, and the width is less than 0.5 mm. The controllable nozzle 1 can infinitely adjust the spray volume via the computer 4. The solution tank is replaceable for easy solution replacement.

[0041] A storage plate with densely distributed micropores is provided at the bottom of the upper layer of the material placement box. Specifically, the plane on which the fiber membrane in the middle of the assemblable membrane material placement box is placed can be a perforated mesh plate with extremely fine pores. This mesh plate can prevent the liquid from flowing quickly to the lower layer when the negative pressure suction device in the lower layer of the placement box provides positive pressure, so that the liquid slowly stays on the perforated plate on which the fiber membrane is placed due to the positive pressure in the lower layer. When the negative pressure suction device provides negative pressure, the liquid can be quickly drawn into the perforated plate.

[0042] Preferably, the controllable nozzle can be a blade-shaped controllable nozzle, and the sprayed mist is a strip-shaped mist.

[0043] This intelligent electronically controlled device for loading gradient concentrations of growth factors into bone and muscle tissue repair materials allows for solution replacement via a solution tank and spraying speed control via a variable speed gearbox. The solution tank contains 200 μL of 0.1 M ethylenediamine solution, and the spraying speed is controlled from the center outwards at a rate of 1... The membrane was repeatedly sprayed with accelerated coating, followed by PBS spraying to wash away unreacted ethylenediamine; resulting in a gradient fiber membrane with a high -NH2 density in the central part and a gradually decreasing -NH2 density towards both ends.

[0044] like Figure 2 As shown, the method for preparing a fibrous membrane of bidirectional gradient vascular endothelial growth factor according to an embodiment of this application includes the following steps:

[0045] Preparation of S1 electrospun PCL fiber membrane: At room temperature, PCL was dissolved in a 10% methanol / chloroform mixed solution to prepare an electrospinning solution. The flow rate of the electrospinning solution was controlled by a micro-injection pump at 0.01 ml / h-0.30 ml / h. The voltage between the two electrodes of the electrospinning device was set to 10 kV. The distance between the syringe and the receiver was 15 cm. A 21-G needle nozzle was used. The receiver was a PCL fiber membrane with a diameter of 8 cm and a length of 25 cm. The membrane was dried in a vacuum drying oven to remove residual solvent and obtain the PCL fiber membrane.

[0046] S2 PCL fiber membrane bidirectional gradient ammonolysis: At room temperature, a 25cm cell membrane is laid flat in the placement tank of the electronically controlled device. The negative pressure aspirator is turned on to ensure the fiber membrane adheres firmly to the tank wall. 200µl of 0.1M ethylenediamine solution is added to the solution tube of the spray gun. The spray gun is positioned in the center of the membrane, and the spray gun is turned on, allowing the spray gun to spray from both sides at a 1:1 ratio. The spraying is accelerated towards both ends to obtain a gradient fiber membrane with a high -NH2 density in the central part and a gradually decreasing -NH2 density towards both ends.

[0047] Preparation of bidirectional gradient heparinized fibrous membranes via S3 heparin coupling reaction: At room temperature, disassemble the spray gun solution tank containing ethylenediamine solution and replace it with a solution tank containing morpholine ethanesulfonic acid buffer. Turn on the spray gun and spray evenly to completely impregnate the PCL fibrous membrane with bidirectional gradient NH2 obtained in step two with morpholine ethanesulfonic acid for 15 min. Remove the morpholine ethanesulfonic acid and continue spraying as above, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide / heparin solution. Decolorize in the dark for 12 h to covalently bind heparin to the amino-carrying fibrous membrane. Turn off the aspirator, provide positive pressure to keep the solution in the upper layer, and then spray in a large amount of PBS to wash away unbound heparin. Ultraclean.

[0048] Preparation of S4 bidirectional gradient VEGF fiber membrane: At room temperature, the bidirectional gradient heparinized fiber membrane was sprayed with 5 ml of VEGF solution with a concentration of 1000 ng / ml, and then immersed in a 4℃ refrigerator for 12 h. The VEGF solution was then aspirated, and the membrane was rinsed with PBS solution. The membrane was then removed and dried in a clean bench to obtain a VEGF-functionalized fiber membrane with a bidirectional gradient.

[0049] Figure 3 A schematic diagram of the gradient of the PCL fiber membrane produced using the intelligent electronically controlled spraying device provided in this application is shown. As can be seen from the VEGF concentration diagram, the darker the color, the higher the concentration. The VEGF concentration decreases as the coating moves from the center towards both ends, with the center being the darkest color and the ends becoming lighter.

[0050] In S1, PCL is dissolved in a 10% methanol / chloroform mixed solution. The methanol / chloroform accounts for 10% of the mixed solution by volume, and the volume ratio of methanol to chloroform is 1:5.

[0051] In S2, PCL fiber membrane undergoes bidirectional gradient ammonolysis: A 25cm cell membrane is laid flat in the placement tank of the electronically controlled device. The negative pressure suction device is turned on to ensure the fiber membrane adheres firmly to the tank wall. 200µl of 0.1M ethylenediamine solution is added to the solution tube of the spray gun, positioning the spray gun in the center of the membrane. The spray gun is then turned on, allowing the spray gun to spray from both the left and right sides at a 1... The coating is accelerated towards both ends and sprayed repeatedly to obtain a gradient fiber membrane with a high -NH2 density in the central part and a gradually decreasing -NH2 density towards both ends. The application principle of this application will be further described below with reference to the accompanying drawings.

[0052] Besides polycaprolactone (PCL), many natural or synthetic polymers can be used to prepare fibrous membrane structures by electrospinning, such as collagen, fibronectin, laminin, chitosan, silk fibroin, poly(L-lactic acid) (PLA), poly(glycolic acid) (PGA), polylactic-co-glycolic acid copolymer (PLGA), and polyethylene glycol (PEG).

[0053] This bidirectional gradient controlled-release VEGF fiber membrane can be used not only in regenerative tissue engineering but also in vascular tissue engineering. The endothelialization process of the fiber membrane can be accelerated by controlling the amount of VEGF loaded on the fiber membrane by changing the ammonolysis time.

[0054] The bidirectional gradient VEGF method described in this application can not only prepare 25cm fiber membranes, but also be used to prepare bone and ligament tissue repair materials rich in other factors.

[0055] This application also provides a method for promoting the regeneration of a VEGF-functionalized fibrous membrane with a bidirectional gradient. The VEGF-functionalized fibrous membrane with a bidirectional gradient provided by this application has a high VEGF loading in the center and a relatively low VEGF loading at both ends. Therefore, the VEGF concentration released in the central part of the fibrous membrane is high. Through chemotaxis, it accelerates the migration of endothelial cells at the damaged ends to the high concentration in the center of the fibrous membrane, and accelerates the directional vascularization of the fibrous membrane from both ends to the center. The generated blood vessels act as a scaffold to guide the rapid directional migration of vascular endothelial cells into the fibrous membrane, thereby accelerating regeneration and extending distally along the vascular endothelial cells.

[0056] To further verify that the nanofiber bone repair membrane prepared using the intelligent electronic spraying device of this application can efficiently promote bone tissue regeneration and repair, cell migration experiments and EDU kits were used to detect the cell proliferation of the bone repair membrane.

[0057] The experimental steps of the cell migration experiment include: (1) For gradient materials, a small material should be laid at the bottom according to the gradient direction, and the yellow membrane should be sealed around the edges. Then, a layer of ordinary material should be added on the material, and the cell planting wells should be cut off at both ends. (2) Washing: Discard the culture medium, slowly add room temperature TBS to the cells, wash twice, 5 seconds each time. (3) Fixation: Cover the cells with 4% neutral formaldehyde fixative (prepared with TBS buffer), place at 4℃, and fix for 15 minutes; the fixative should be sufficient. (4) Washing: Remove the fixative, use 4℃ pre-cooled TBS buffer, rinse 3 times, 5 minutes each time. (5) Blocking: Completely cover the sample with 5% blank goat serum. The slides should be placed in a humidified box. For cell well plates, the well plates can be directly sealed and incubated in a 37℃ constant temperature and humidity incubator for 30 minutes. (6) Primary antibody dilution: Dilute the antibody in antibody dilution solution according to the instructions. (7) Primary antibody incubation: Aspirate the blocking solution, add the diluted primary antibody, and incubate at 4℃ overnight. (8) Warming: Place the sample at room temperature and warm for 15 minutes. (9) Washing: Remove the antibody working solution, wash once with TBST buffer for 5 minutes; wash three times with TBS buffer for 5 minutes each time. (10) Secondary antibody dilution: Dilute the antibody in antibody diluent according to the instructions. (11) Secondary antibody incubation: Incubate at room temperature in the dark for 1 hour. (12) Washing: Remove the secondary antibody working solution, wash once with TBST buffer for 5 minutes; wash three times with TBS buffer for 5 minutes each time. (13) Nucleus staining / mounting: Add DAPI working solution to the sample, incubate at room temperature in the dark for 10 minutes; remove the DAPI working solution, wash once with TBST buffer for 5 minutes; wash three times with TBS buffer for 5 minutes each time; add anti-fluorescence attenuation mounting medium, observe and acquire images under a fluorescence microscope. (14) Endothelial cells were seeded onto the low-concentration side (approximately 3 mm wide) of ordered nanofiber membranes (2 cm long, 1 cm wide) loaded with different VEGF gradients and concentrations, with fiber diameters of 300 nm, 600 nm, and 1000 nm, respectively. Immunofluorescence staining was performed: Two days after seeding, the endothelial cells were fixed with 4% paraformaldehyde at 4°C for 15 min, treated with 0.2% Triton X-100 at 37°C for 10 min, and incubated with blocking solution (10% normal goat serum) at room temperature for 30 min. The specimens were incubated overnight at 4°C with primary antibodies of rabbit anti-S100 polyclonal antibody (1:200) and mouse anti-NF200 monoclonal antibody (1:200). The next day, the specimens were incubated at 37°C for 1 hour with secondary antibodies of goat anti-rabbit IgG TRITC (1:500) and goat anti-mouse IgG FITC (1:500). The specimens were rinsed and mounted. The specimens were observed and photographed under a fluorescence confocal microscope. ImageJ software was used to perform statistical analysis on the number of migrating cells, average distance, and maximum migration distance on the bone regeneration membrane.

[0058] The specific steps for detecting cell proliferation with the EDU kit include:

[0059] 1. Preparation of heparinized ordered nanofiber membranes by heparin coupling reaction:

[0060] After encapsulating the ammonified ordered membrane with a coverslip, it was placed in a morpholine ethanesulfonic acid buffer. The morpholine ethanesulfonic acid was removed, and a 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide / heparin solution was added. The membrane was then decolorized and incubated overnight in the dark on a shaker to covalently bind heparin to the amino-carrying ordered membrane. Unbound heparin was washed away with PBS, and the membrane was dried in a clean bench to obtain the heparinized ordered membrane.

[0061] 2. Preparation of VEGF ordered nanofiber membranes:

[0062] A heparinized ordered membrane was wrapped around a coverslip, placed in a beaker, and 100 ng / ml of VEGF solution was added. The mixture was soaked overnight at 4°C. The VEGF solution was then removed, and the membrane was rinsed with PBS solution and dried in a clean bench to obtain a VEGF-functionalized ordered bone regeneration membrane cell proliferation model.

[0063] 3. Cell resuscitation:

[0064] (1) Preheat the water bath to 37°C.

[0065] (2) Put on gloves, mask and cap, take out the cryovial containing HUVEC (containing 1 mL of cell mixture) from the liquid nitrogen tank, and immediately put it into a 37°C water bath. Gently shake the cryovial to thaw it quickly within 1 minute.

[0066] (3) After complete dissolution, wipe the outer wall of the cryovial with 75% alcohol for disinfection before bringing it into the laminar flow hood.

[0067] (4) In a clean bench, add 10 mL of freshly prepared culture medium to a 15 mL sterile centrifuge tube, open the cryopreservation tube, add all the frozen and thawed cell suspensions to the centrifuge tube, centrifuge at 1000 rpm for 3 minutes at room temperature, and remove the upper layer of culture medium.

[0068] (5) Resuspend the cell pellet in 1 mL of culture medium, gently pipette to mix, add to T25 cell culture flask, add culture medium to 5 mL, and incubate at 37℃ in a 5% CO2 incubator.

[0069] (6) Change the medium after 48 hours. When the cells are nearly 80% fused, they can be passaged.

[0070] 4. Cell plating:

[0071] HUVEC cells in the logarithmic growth phase and in good growth condition were digested with trypsin, and after cell counting, they were seeded into a bone repair membrane wrapped with a round coverslip in a 12-well plate at a density of 4×104 cells / well. The plates were then placed in a 37 ℃ constant temperature cell culture incubator for 24 hours to allow the cells to adhere.

[0072] 5. EDU tag:

[0073] (1) Dilute EDU to 20µM with complete medium. Add 200µl of diluted EDU working solution to each well of a 12-well plate to make a final concentration of 10µM (a suitable concentration for most cells). Continue incubation at 37°C for 2 hours. The length of cell incubation time depends on the cell growth rate. For common cell types, 2 hours of incubation is generally sufficient.

[0074] (2) Remove the cell culture medium and wash with PBS 1-2 times, 3 minutes each time.

[0075] 6. Cell fixation:

[0076] (1) Cell fixation: Remove the washing solution, add 1 ml of 4% paraformaldehyde to each well and fix the cells at room temperature for 15 min.

[0077] (2) Remove the fixative and wash with PBS 3 times, 3-5 minutes each time.

[0078] (3) Remove PBS, add 1 ml of permeation buffer to each well and incubate at room temperature for 10-15 minutes.

[0079] (4) Remove the permeation solution and wash with PBS 1-2 times, 3-5 minutes each time.

[0080] 7. Staining (avoiding light):

[0081] (1) Prepare the reaction solution according to the kit instructions. For a 12-well plate, add 200µl of reaction solution to each well, gently shake the plate to ensure that the reaction solution evenly covers the sample, and incubate at room temperature in the dark for 30 minutes.

[0082] (2) Remove the reaction solution and wash with PBS 3 times, each time for 3-5 minutes.

[0083] 8. Nuclear staining (protected from light):

[0084] (1) Dilute Hoechst33342 reaction solution to 1X with deionized water, remove PBS washing solution, add 200µl of Hoechst33342 to each well, and stain at room temperature in the dark for 10 minutes.

[0085] (2) Remove Hoechst by washing with PBS three times for 3-5 minutes each time.

[0086] 9. Counting and taking photos:

[0087] Observe EDU-labeled and unlabeled cells under a fluorescence microscope or confocal microscope, take pictures and count them.

[0088] The experiment needs to be repeated three times, and each group must have at least three replicates. Statistical software is then used to analyze the data.

[0089] Figure 4 The image shows three types of immunofluorescence staining for detecting cell migration on the bone repair membrane and the effect of the EDU kit on detecting cell proliferation on the bone repair membrane. At each detection time point (day 1, day 3, day 5), the number of proliferating cells labeled with fluorescent gold in the PCL + bidirectional gradient VEGF group was significantly higher than that in the PCL + no gradient VEGF group and the PCL group. *P<0.05, **P< 0.01 The study suggests that VEGF-functionalized fibrous membranes with bidirectional gradients have a greater advantage in repair than VEGF-functionalized fibrous membranes without gradients or simple fibrous membranes, and can better promote bone regeneration.

[0090] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. An intelligent electronically controlled spraying device, characterized in that, include: Controllable nozzle, nozzle solution tank, moving guide rail, speed gearbox, negative pressure suction device and material placement box; The controllable nozzle is connected to the nozzle solution tank and fixed on the moving guide rail, and is used to spray a strip-shaped mist; and the spraying range is greater than the width of the fiber membrane; The nozzle solution tank is used to store the spraying solution and to make the spraying solution replaceable; The variable speed gearbox is used to infinitely adjust the spray volume of the controllable nozzle and control the movement of the controllable nozzle on the moving guide rail; The material placement box includes upper and lower layers. The upper layer is used to hold the PCL nanofiber membrane that needs to be loaded with gradient concentrations of growth factors. The lower layer is connected to the negative pressure aspirator, which is used to extract liquids and gases and can also provide positive pressure to prevent the solution in the upper layer from flowing into the lower layer. The bottom of the upper layer can be connected to the lower layer. Through electronically controlled strip-shaped mist spraying and negative pressure aspiration wetting, the sprayed growth factors can be accurately immersed in the material to be loaded locally. A storage plate with densely distributed micropores is provided at the bottom of the upper layer of the material placement box. The storage plate is used to support the PCL fiber membrane that needs to be loaded with gradient concentrations of growth factors. When the negative pressure aspirator is turned on, the liquid can be quickly drawn into the pore plate. When the solution needs to remain in the upper layer to soak the fiber membrane, the lower negative pressure aspirator can provide positive pressure to prevent the solution from flowing into the lower layer.

2. The intelligent electronically controlled spraying device as described in claim 1, characterized in that, The controllable nozzle is a blade-shaped controllable nozzle. The blade-shaped controllable nozzle has a small spray thickness, so that the spray range on the fiber membrane always remains a strip with the width of the fiber membrane as the length of the spray range, and the width is less than 0.5 mm.

3. The intelligent electronically controlled spraying device as described in claim 1, characterized in that, The gearbox has two gears, which are used to control the spraying speed and travel speed and direction of the controllable nozzle.

4. The intelligent electronically controlled spraying device as described in claim 3, characterized in that, The variable speed gearbox can control the speed and acceleration via computer, and control the gearbox acceleration so that the gearbox moves from the middle to the left and right ends with an acceleration that matches the membrane. This operation is repeated to form a fiber membrane with a high concentration of the substance in the middle and a low concentration of the substance at both ends.

5. The intelligent electronically controlled spraying device as described in claim 1, characterized in that, The aperture of the upper bottom shelf is 1mm.

6. The intelligent electronically controlled spraying device as described in claim 1, characterized in that, The controllable nozzle is connected to the nozzle solution tank via a hose.

7. A method for preparing a gradient concentration growth factor nanofiber bone repair membrane based on the intelligent electronically controlled spraying device according to any one of claims 1-6, characterized in that, Includes the following steps: S1 Preparation of electrospun PCL fiber membranes: PCL was dissolved in a 10% methanol / chloroform mixed solution to obtain an electrospinning solution. The flow rate of the electrospinning solution was controlled at 0.01 ml / h-0.30 ml / h. After removing the residual solvent by drying, a PCL nanofiber membrane was obtained. S2 performs bidirectional gradient ammonolysis on the PCL fiber membrane: The fiber membrane is firmly attached to the tank wall by a negative pressure suction device. The initial position of the controllable nozzle is set in the middle of the fiber membrane. The controllable nozzle accelerates the spraying from the left and right sides at an acceleration of 1 cm / s towards both ends from the initial position to obtain a gradient fiber membrane with high -NH2 density in the central part and gradually decreasing -MH2 density towards both ends. S3 prepares a bidirectional gradient heparinized fibrous membrane via a heparin coupling reaction: A solution containing morpholine ethanesulfonic acid buffer is uniformly sprayed onto the gradient fiber membrane and allowed to fully impregnate it for a period of time. Then, a solution containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxydiimide / heparin is sprayed onto the membrane to covalently bind heparin to the amino-carrying fiber membrane. After washing away the unbound heparin, a bidirectional gradient heparinized fiber membrane is obtained. S4 preparation of bidirectional gradient VEGF fiber membrane: VEGF solution was uniformly sprayed onto a heparinized fibrous membrane with a bidirectional gradient to obtain a VEGF-functionalized nanofiber bone repair membrane with a bidirectional gradient concentration.

8. The application of the nanofiber bone repair membrane according to claim 7 in the preparation of a product that promotes bone regeneration, characterized in that, include: The central part of the VEGF-functionalized fiber membrane with a bidirectional gradient releases a high concentration of VEGF, which accelerates the migration of endothelial cells at both ends of the damaged area to the high concentration in the central part of the VEGF-functionalized fiber membrane through chemotaxis. Accelerating the directional vascularization of the fibrous membrane from both ends towards the center, the generated blood vessels act as a scaffold, guiding vascular endothelial cells to migrate rapidly into the fibrous membrane, thereby accelerating regeneration and extending distally along the vascular endothelial cells.

9. The application as described in claim 8, characterized in that, The VEGF-functionalized fiber membrane with bidirectional gradient has a high VEGF loading in the center and a relatively low VEGF loading at both ends.

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