Intelligent electric control spraying device and preparation method of nanofiber bone repair membrane

Abstract

The invention discloses an intelligent electric control spraying device and a nanofiber bone repair membrane preparation method. The device comprises a controllable nozzle, a nozzle solution box, a movable guide rail, a speed change gear box, a negative pressure aspirator and a material containing box. Growth factors in gradient concentration distribution are loaded on the repair material by controlling the spraying speed, the advancing speed and the advancing direction, in-situ immersion of the loaded growth factors in the repair material is achieved through negative pressure suction, and through the bidirectional gradient VEGF functionalized absorbable nanofiber bone repair membrane, the concentration of VEGF loaded in the central area is high, and the absorbable nanofiber bone repair membrane has good biocompatibility. The concentration of VEGF loaded to the two ends is gradually reduced, so that neovascular endothelial cells of bone defect broken ends can be guided to directionally migrate to the high-concentration part of the central area of the repair membrane through chemotactic action, and the directional vascularization of bone tissues in a defect area from the two ends to the central area is accelerated; meanwhile, the newly generated blood vessel can be used as a stent to guide cells to quickly migrate and enter a fibrous membrane, so that bone tissue regeneration and repair are efficiently promoted.

Description

Technical Field

[0001] The present application belongs to the technical field of regenerative tissue engineering and vascular tissue engineering, and more specifically, relates to an intelligent electrically controlled spraying device for loading gradient concentration growth factors into bone and ligament tissue repair materials and a method for preparing a nanofiber bone repair membrane. Background Art

[0002] Following tissue injury, vascular endothelial cells are activated. In the case of contusions, endothelial cells are able to migrate along the intact intimal structure. However, recent studies have shown that transection injuries disrupt intimal junctions, requiring endothelial cell migration to rely on regenerated blood vessels as a scaffold. Directional vascular growth is crucial for guiding endothelial cell migration and bone regeneration.

[0003] For long-segment defects, bridging with a fiber membrane is necessary to achieve structural connectivity. Therefore, the key to accelerating defect repair lies in promoting rapid directional vascularization of the fiber membrane and guiding endothelial cells to migrate rapidly and directionally into the fiber membrane from both the distal and proximal ends of the injury, thereby accelerating vascularization through the bridging area and promoting functional recovery.

[0004] Vascular endothelial growth factor (VEGF) can chemotactically attract endothelial cells and promote angiogenesis. Numerous studies have demonstrated that overexpressing VEGF through genetic engineering can protect tissues and promote regeneration. However, these studies have failed to address the question of how to accelerate the vascularization of fiber membranes to promote rapid vascularization across the bridged site. Ma Fukai et al. found that fiber membranes functionalized with VEGF significantly promoted tissue regeneration compared to conventional collagen fiber membranes. Other studies have shown that simple concentration gradient guidance cannot achieve rapid directional vascularization in the central region of the fiber membrane, limiting the polarized migration of endothelial cells toward the region with high VEGF concentrations and the rate of blood vessel formation. Therefore, fiber membranes lacking a directional concentration gradient are unable to effectively guide the rapid and directional migration of endothelial cells into the membrane, thereby limiting the rate at which VEGF promotes vascularization across the fiber membrane.

[0005] In existing methods, gradient cell membranes are fabricated by reacting ethylenediamine with PCL to generate NH2 groups, which are then coated with a gradient or uniform distribution of NGF. Briefly, the cell membrane is folded in half, oriented in the direction of fiber alignment, and the fold is placed in an ethylenediamine solution. The solution is gently stirred and distilled water is gradually added. As the concentration of the ethylenediamine solution decreases and the reaction time is linearly varied, the density of NH2 groups exhibits a steeper axial gradient. To achieve a uniform distribution of NH2 groups, the cell membrane is saturated exclusively with the ethylenediamine solution. The catheter is then immersed in a mixture of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and sodium heparin, then immersed in 2-(N-morpholino)ethanesulfonic acid (MES, Sigma) buffer and briefly rotated at room temperature to ensure covalent binding of the carboxyl groups of the heparin 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 nanofibrous bone cell membrane. In existing membrane-making techniques, all steps require manual manipulation, and it is difficult to accurately and easily create a concentration gradient of the loading factor on the membrane. Summary of the Invention

[0006] In response to the defects of the existing technology, the purpose of this application is to provide an intelligent electric-controlled spraying device and a method for preparing a nanofiber bone repair membrane loaded with gradient concentration growth factors, aiming to solve the problems in the existing technology that the existing membrane-making technology is cumbersome, difficult to produce, and cannot be automated due to the lack of automated and convenient production of cell membranes and biological membranes loaded with VEGF (or other factors), and the inability to accurately and simply produce a device with a gradient concentration of the factors loaded on the membrane.

[0007] The present application provides an intelligent electrically-controlled spraying device, comprising: a controllable nozzle, a nozzle solution tank, a movable guide rail, a speed-changing gear box, a negative pressure aspirator and a material placement box; the controllable nozzle is connected to the nozzle solution tank and fixed on the movable guide rail for spraying 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 realize the replaceable spraying solution; the speed-changing gear box is used to steplessly adjust the spraying amount of the controllable nozzle and control the movement of the controllable nozzle on the movable guide rail; the material placement box comprises an upper and a lower layer, the upper layer is used to carry the PCL fiber membrane that needs to be loaded with gradient concentration growth factors, and the lower layer is connected to the negative pressure aspirator, which is used to extract liquid and gas, and can also provide positive pressure to prevent the upper solution from flowing into the lower layer. The bottom of the upper layer can be connected to the lower layer, and the electrically-controlled strip-shaped mist spraying and negative pressure suction infiltration are used to achieve local and precise immersion of the sprayed growth factors in the required loaded materials.

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

[0009] Furthermore, the speed change gear box has dual speed gears, which are used to adjust the spraying speed and the travel speed and direction of the controllable nozzle respectively.

[0010] Furthermore, the speed change gearbox can be controlled by computer to control the speed and acceleration, so that the gearbox moves from the middle to the left and right ends at an acceleration that matches the membrane. This operation is repeated to form a fiber membrane with a high concentration of substances in the middle and low concentration of substances at both ends. Specifically, the acceleration can be set according to different membranes and can be 1 ~3 .

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

[0012] More preferably, the hole diameter of the upper bottom storage plate may be 1 mm.

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

[0014] The present application also provides a method for preparing a nanofiber bone repair membrane based on the above-mentioned intelligent electronically controlled spraying device, comprising the following steps: S1 Preparation of electrospun PCL fiber membrane: PCL was dissolved in a 10% methanol / chloroform mixed solution to obtain an electrospinning solution, the flow rate of the electrospinning solution was controlled to be 0.01 ml / h-0.30 ml / h, and the residual solvent was removed by drying to obtain a PCL fiber membrane; S2 performs bidirectional gradient aminolysis on the PCL fiber membrane: The fiber membrane is firmly attached to the groove wall by a negative pressure suction device, and the initial position of the controllable nozzle is set in the middle of the fiber membrane, so that the controllable nozzle is moved from the left and right sides by 1 The acceleration is accelerated to spray toward both ends to obtain a gradient fiber membrane with high -NH2 density in the center and gradually decreasing -NH2 density toward both ends; S3 Preparation of bidirectional gradient heparinized fiber membrane by heparin coupling reaction: A morpholineethanesulfonic acid buffer solution is evenly sprayed on the gradient fiber membrane and allowed to fully soak for a period of time; 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide / heparin solution is then added to the gradient fiber membrane to covalently bind heparin to the fiber membrane carrying amino groups, and unbound heparin is washed away to obtain a bidirectional gradient heparinized fiber membrane; S4 Preparation of bidirectional gradient VEGF fiber membrane: VEGF solution was evenly sprayed on the bidirectional gradient heparinized fiber membrane to obtain a VEGF functionalized nanofiber bone repair membrane with a bidirectional gradient concentration.

[0015] The present application also provides a method for promoting bone regeneration using the above-mentioned nanofiber bone repair membrane loaded with gradient concentration growth factors, comprising: releasing a high concentration of VEGF from the central part of the bidirectional gradient VEGF-functionalized fiber membrane, and accelerating the migration of endothelial cells at both ends of the damaged area to the high concentration area in the central part of the bidirectional gradient VEGF-functionalized fiber membrane through chemotaxis; The vascularization of the fiber membrane is accelerated from both ends to the center. The generated blood vessels act as scaffolds to guide the rapid directional migration of vascular endothelial cells into the fiber membrane, thereby accelerating regeneration and extending toward the distal end along the vascular endothelial cells.

[0016] Among them, the amount of VEGF loaded in the center of the bidirectional gradient VEGF-functionalized fiber membrane is high, while the amount of VEGF loaded at both ends is relatively small.

[0017] In summary, the above technical solutions conceived by this application have the following technical effects compared with the prior art: (1) Since the intelligent electronically controlled spraying device has a blade-shaped controllable nozzle that automatically adjusts the spray size and a movable guide rail that can control 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 movable guide rail, thereby forming the required concentration gradient; since 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 bottom solution of the upper layer can be quickly drawn away through the tiny aperture on the placement plate. At the same time, if the negative pressure suction device is adjusted to provide a certain positive pressure, the lower layer can be in a positive pressure environment and the bottom solution of the upper layer can be retained in the upper layer, so that when the solution needs to stay and fully react with the membrane, the solution can be retained in the upper layer, and when the solution needs to leave the membrane, the solution can be quickly drawn away; since this device can be controlled by a computer, it can automatically adjust the solution gradient, the spray range of the loaded solution, and the residence time of the sprayed solution on the fiber membrane in the fiber membrane production process, thereby achieving automatic computer control to produce biofilms loaded with various factors.

[0018] (2) Compared with the existing technology, the biofilm produced by this device can accurately control the concentration of the factors loaded on the membrane and infinitely adjust the gradient of the factors loaded on the membrane. At the same time, it eliminates the tedious process of manual membrane making, making the production of various biofilms more refined and automated.

[0019] (3) The bidirectional gradient VEGF functionalized fiber membrane provided by the present application has a high amount of VEGF loaded in the center and a relatively low amount of VEGF loaded at the two ends. This can induce endothelial cells at the damaged ends to migrate to the high concentration area in the center of the fiber membrane through chemotaxis, thereby accelerating the vascularization of the fiber membrane from the two 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 fiber membrane, thereby promoting regeneration and extending along the vascular endothelial cells to the distal end.

[0020] (4) The present application realizes loading of growth factors with gradient concentration distribution on the repair material by controlling the spraying speed, travel speed and direction, and realizes in situ immersion of the loaded growth factors in the repair material by negative pressure attraction. The absorbable nanofiber bone repair membrane functionalized with bidirectional gradient VEGF has a high concentration of VEGF loaded in the central area, while the concentration of VEGF loaded toward the two ends gradually decreases. It can guide the newly formed vascular endothelial cells at the ends of the bone defect to migrate toward the high concentration in the central area of the repair membrane through chemotaxis, accelerate the directional vascularization of the bone tissue in the defect area from the two ends to the central area, and at the same time, the newly generated blood vessels can act as a scaffold to guide the cells to quickly migrate into the fiber membrane, thereby effectively promoting the regeneration and repair of bone tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the structure of the intelligent electronically controlled spraying device provided in an embodiment of the present application; 1 is the controllable nozzle, 2 is the speed change gear box, 3 is the PCL fiber membrane, 4 is the computer, 5 is the moving guide rail, 6 is the negative pressure suction device, and 7 is the material placement box. Figure 2 This is a flow chart of the method for preparing a bidirectional gradient vascular endothelial growth factor fiber membrane provided in an embodiment of the present application; Figure 3 Schematic diagram of a PCL fiber membrane gradient produced using the intelligent electronically controlled spraying device provided in this application; Figure 4 This is a diagram showing the detection of cell migration on bone repair membranes using immunofluorescence staining on three fiber membranes provided in the examples of the present application, and the detection of cell proliferation on bone repair membranes using an EDU kit; AC is a cell migration experiment, DF is an EDU experiment, G is a statistical diagram of the difference in the number of cells stained by immunofluorescence on the three membranes of PCL, PCL+uniform VEGF, and PCL+gradient VEGF; H is a statistical diagram of the difference in the number of cell proliferation in the EDU experiment on the three membranes of PCL, PCL+uniform VEGF, and PCL+gradient VEGF. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of this application more clearly understood, the present application is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0023] The present application provides an intelligent electrically controlled spraying device for loading gradient concentration growth factors on bone and ligament tissue repair materials and a method for preparing a nanofiber bone repair membrane. By preparing a nanofiber bone repair membrane with a bidirectional gradient concentration of VEGF, the directional vascularization of new tissue guided by the fiber membrane can be accelerated, and the vascular endothelial cells can be guided to migrate rapidly from the two ends of the bone defect to the central area, thereby promoting bone regeneration and repair, and solving the problem of non-directional vascularization of bone regeneration tissue guided by the VEGF fiber repair membrane without a concentration gradient.

[0024] The application principle of this application is described in detail below with reference to the accompanying drawings.

[0025] Figure 1 The present application discloses an intelligent electronically controlled device for loading bone and muscle tissue repair materials with gradient concentration growth factors. The device comprises a blade-shaped controllable nozzle 1, a replaceable nozzle solution tank, a movable guide rail 5, a speed change gear box 2, a negative pressure aspirator 6, and a material storage box 7. The blade-shaped controllable nozzle 1 is connected to the solution tank via a hose and fixed to the controllable nozzle movable guide rail. The speed change gear box 2 can control the nozzle's movement on the nozzle movable guide rail by computer-controlled speed and acceleration. The assembleable membrane material storage box has two layers, the upper layer for holding the PCL fiber membrane 3 to be loaded with gradient concentration growth factors, and the lower layer connected to the negative pressure aspirator 6 for extracting liquid and gas. The blade-shaped controllable nozzle 1 can spray a thin mist strip. The spray range is larger than the fiber membrane width and the spray thickness is small, so that the spray range and the fiber membrane always maintain a rectangular shape with the fiber membrane width as the spray range length and a width of less than 0.5 mm. The controllable nozzle 1 can infinitely adjust the spray volume via computer 4. The solution tank is replaceable, making it convenient to change solutions.

[0026] 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 assembled membrane material placement box is placed can be a layer of perforated mesh with extremely fine pores. This mesh 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, and the liquid will slowly stay on the aperture plate where the fiber membrane is placed due to the positive pressure of the lower layer. When the negative pressure suction device provides negative pressure, the liquid can be quickly drawn out of the aperture plate.

[0027] Preferably, the controllable nozzle may be a blade-shaped controllable nozzle, and the sprayed mist is a strip-shaped mist.

[0028] The intelligent electronic control device for loading gradient concentration growth factors into bone and muscle tissue repair materials can replace the solution through the solution box and control the spraying speed through the speed change gear box. 200ul of 0.1M ethylenediamine solution is added to the solution box and sprayed from the middle to both ends at a speed of 1 The acceleration was repeatedly sprayed, and then PBS was sprayed to wash away the unreacted ethylenediamine; a gradient fiber membrane was obtained with a high -NH2 density in the center and a gradually decreasing -NH2 density toward the two ends.

[0029] like Figure 2 As shown, the method for preparing the fiber membrane of the bidirectional gradient vascular endothelial growth factor in the embodiment of the present application includes the following steps: S1 Preparation of electrospun PCL fiber membrane: PCL was dissolved in a 10% methanol / chloroform mixed solution at room temperature to prepare an electrospinning solution. The flow rate of the electrospinning solution was controlled by a microinjection 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 an 8 cm diameter roller. The PCL fiber membrane with a length of 25 cm was dried in a vacuum drying oven to remove the residual solvent to obtain a PCL fiber membrane. S2 PCL fiber membrane bidirectional gradient ammonia hydrolysis: at room temperature, spread 25cm cell membrane in the placement tank of the electronic control device, turn on the negative pressure suction device to make the fiber membrane firmly attached to the tank wall, add 200ul, 0.1M ethylenediamine solution to the solution tube of the spray gun, place the spray gun in the middle of the membrane, turn on the spray gun, and spray the membrane from the left and right sides at 1 The acceleration is accelerated to spray towards both ends to obtain a gradient fiber membrane with high -NH2 density in the center and gradually decreasing -NH2 density towards both ends; S3 Heparin coupling reaction to prepare bidirectional gradient heparinized fiber membrane: at room temperature, remove the spray gun solution tank containing ethylenediamine solution and replace it with a tube tank containing morpholineethanesulfonic acid buffer solution. Turn on the spray gun and spray evenly to allow the PCL fiber membrane with bidirectional gradient NH2 obtained in step 2 to be completely infiltrated with morpholineethanesulfonic acid for 15 minutes. Remove morpholineethanesulfonic acid and continue spraying as above to add 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide / heparin solution. Decolorize in the dark for 12 hours to covalently bind heparin to the fiber membrane carrying amino groups. Turn off the aspirator and provide positive pressure to maintain the solution in the upper layer. Then spray a large amount of PBS to wash away unbound heparin and clean it thoroughly. Preparation of S4 bidirectional gradient VEGF fiber membrane: At room temperature, the bidirectional gradient heparinized fiber membrane was sprayed with 5 ml of 1000 ng / ml VEGF solution as above, and then placed in a 4°C refrigerator to soak for 12 hours. The VEGF solution was aspirated and rinsed with PBS solution. The membrane was taken out and dried in a clean bench to obtain a fiber membrane with bidirectional gradient VEGF functionalization.

[0030] Figure 3 A schematic diagram of a PCL fiber membrane gradient produced using the intelligent electronically controlled spraying device provided by this application is shown. As can be seen from the VEGF concentration graph, the darker the color, the higher the concentration. The VEGF concentration decreases as you move from the center toward the ends, with the color being darkest in the center and becoming lighter at the ends.

[0031] In S1, PCL was dissolved in a 10% methanol / chloroform mixed solution, wherein the volume mass ratio of methanol / chloroform accounted for 10% of the mixed solution, and the volume ratio of methanol:chloroform was 1:5.

[0032] In S2, PCL fiber membrane bidirectional gradient aminolysis: 25cm cell membrane was spread flat in the placement tank of the electronic control device, and the negative pressure suction device was turned on to make the fiber membrane firmly attached to the tank wall. 200u1 of 0.1M ethylenediamine solution was added to the solution tube of the spray gun. The spray gun was placed in the middle of the membrane and turned on. The spray gun was sprayed from the left and right sides at 1 The acceleration is accelerated to spray towards both ends, and repeated several times to obtain a gradient fiber membrane with high -NH2 density in the center and gradually decreasing -NH2 density towards both ends; the application principle of this application is further described in conjunction with the accompanying drawings.

[0033] In addition to polycaprolactone (PCL), many natural or synthetic polymers can be used to prepare fiber membrane structures by electrospinning, such as collagen, fibronectin, laminin, chitosan, silk protein, poly (L-lactic acid) (PLA), poly (glycolic acid) (PGA), poly lactose-glycolic acid copolymer (PLGA), polyethylene glycol (PEG) This bidirectional gradient controlled release of 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 changing the aminolysis time to control the amount of VEGF loaded on the fiber membrane.

[0034] The method for preparing bidirectional gradient VEGF in this application can not only be used to prepare 25 cm fiber membranes, but can also be used to prepare bone and ligament tissue repair materials rich in other factors.

[0035] The present application also provides a method for promoting regeneration of a bidirectional gradient VEGF-functionalized fiber membrane. The bidirectional gradient VEGF-functionalized fiber membrane provided by the present application has a high amount of VEGF loaded in the center, while the amount of VEGF loaded at the two ends is relatively small. Therefore, the VEGF concentration released in the central part of the fiber membrane is high. Through chemotaxis, the endothelial cells at the damaged ends are accelerated to migrate to the high concentration area in the center of the fiber membrane, and the vascularization of the fiber membrane is accelerated from the two ends to the center. The generated blood vessels act as a scaffold to guide the rapid directional migration of vascular endothelial cells into the fiber membrane, thereby accelerating regeneration and extending along the vascular endothelial cells to the distal end.

[0036] In order to further verify that the nanofiber bone repair membrane prepared by the intelligent electric-controlled spraying device of the present application can effectively promote the regeneration and repair of bone tissue, the cell migration experiment and EDU kit are used to detect the proliferation of bone repair membrane cells.

[0037] Among them, the experimental steps of the cell migration experiment include: (1) For gradient materials, a smaller material must be laid on the bottom layer in the gradient direction, and then a layer of ordinary material is added on the material after the yellow film is sealed around it, and the cell planting holes are cut at both ends. (2) Washing: Discard the culture medium, slowly add room temperature TBS to the cells, and wash twice, each time for 5 seconds. (3) Fixation: Cover the cells with 4% neutral formaldehyde fixative (prepared with TBS buffer), place at 4℃, and fix for 15 minutes; the fixative must be sufficient. (4) Washing: Remove the fixative, use 4℃ pre-cooled TBS buffer, rinse three times, each time for 5 minutes. (5) Blocking: Cover the sample completely with 5% blank goat serum, and the slices must be placed in a humidified box. The cell well plate can be directly sealed and placed in a 37℃ constant temperature and humidity incubator for incubation for 30 minutes. (6) Primary antibody dilution: Dilute the antibody in the antibody diluent according to the instructions. (7) Primary antibody incubation: Aspirate the blocking solution, add the diluted primary antibody, and incubate at 4℃ overnight. (8) Rewarming: Place the sample at room temperature and rewarm 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. (10) Secondary antibody dilution: Dilute the antibody in the antibody diluent according to the instructions. (11) Secondary antibody incubation: Incubate at room temperature for 1 hour in the dark. (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. (13) Nuclear 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. After adding anti-fluorescence decay mounting medium, observe and collect images under a fluorescence microscope. (14) Endothelial cells were seeded on the low-concentration side of an ordered nanofiber membrane (2 cm long and 1 cm wide) loaded with different VEGF gradients and different VEGF concentrations, with fiber diameters of 300 nm, 600 nm, and 1000 nm, respectively. Immunofluorescence staining: Two days after seeding the endothelial cells, they were fixed with 4% paraformaldehyde at 4°C for 15 minutes, treated with 0.2% Triton X-100 at 37°C for 10 minutes, and incubated with blocking solution (10% normal goat serum) at room temperature for 30 minutes. The specimens were incubated overnight at 4°C with rabbit anti-S100 polyclonal antibody (1:200) and mouse anti-NF200 monoclonal antibody (1:200). The next day, they were incubated with goat anti-rabbit IgG TRITC (1:500) and goat anti-mouse IgG FITC (1:500) secondary antibodies at 37°C for 1 hour. The sections were rinsed and mounted. The sections were observed and photographed under a fluorescence confocal microscope. ImageJ software was used to perform statistical analysis on the number, average distance and maximum migration distance of the migrating cells on the bone regeneration membrane.

[0038] The specific steps of the EDU kit for detecting cell proliferation include: 1. Preparation of heparinized ordered nanofiber membrane by heparin coupling reaction: The aminated ordered membrane was wrapped with a coverslip and placed in a morpholineethanesulfonic acid buffer. The morpholineethanesulfonic acid was removed and a 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide / heparin solution was added. The membrane was shaken overnight in the dark to decolorize and covalently bind heparin to the ordered membrane carrying amino groups. Unbound heparin was washed with PBS and the membrane was dried in a clean bench to obtain a heparinized ordered membrane.

[0039] 2. Preparation of VEGF ordered nanofiber membrane: The heparinized ordered membrane was wrapped around the coverslip, placed in a beaker, and 100 ng / ml VEGF solution was added. The membrane was immersed at 4°C overnight, the VEGF solution was aspirated, the membrane was rinsed with PBS solution, and the membrane was dried in a clean bench to prepare a VEGF-functionalized ordered bone regeneration membrane cell proliferation model.

[0040] 3. Cell recovery: (1) Preheat the water bath to 37°C.

[0041] (2) Wearing gloves and a mask, take out the cryotube containing HUVEC (containing 1 mL of cell mixture) from the liquid nitrogen tank, immediately place it in a 37°C water bath, and gently shake the cryotube to dissolve it quickly within 1 minute.

[0042] (3) After complete dissolution, wipe the outer wall of the cryopreservation tube with 75% alcohol for disinfection and then bring it into the clean bench.

[0043] (4) In a clean bench, add 10 mL of freshly prepared culture medium to a 15 mL sterile centrifuge tube. Open the cryovial and add all the frozen-thawed cell suspension to the centrifuge tube. Centrifuge at 1000 rpm for 3 minutes at room temperature and remove the upper culture medium.

[0044] (5) Resuspend the cell pellet in 1 mL of culture medium, gently pipette to mix, and add to a T25 cell culture flask. Make up the volume to 5 mL of culture medium and place in a 37°C, 5% CO2 incubator for culture.

[0045] (6) Change the medium after 48 hours, and the cells can be passaged when the fusion reaches nearly 80%.

[0046] 4. Cell plating: HUVEC cells in the logarithmic growth phase and in good growth condition were digested with trypsin, and after counting the cells, they were seeded onto the bone repair membrane wrapped with a round cover glass in a 12-well plate at a density of 4×104 cells / well. The plates were then placed in a 37°C constant temperature cell culture incubator and cultured for 24 hours until the cells adhered.

[0047] 5.EDU tag: (1) Dilute EDU to 20µM with complete culture medium. Add 200µl of diluted EDU working solution to each well of a 12-well plate to make the final concentration 10µM (a suitable concentration for most cells). Continue incubating at 37°C for 2 hours. The length of cell incubation time depends on the cell growth rate. For common cell types, incubation for 2 hours is generally sufficient.

[0048] (2) Remove the cell culture medium and wash with PBS 1-2 times, 3 minutes each time.

[0049] 6. Fixing cells: (1) Cell fixation: Remove the washing solution and add 1 ml of 4% paraformaldehyde to each well to fix the cells at room temperature for 15 minutes.

[0050] (2) Remove the fixative and wash with PBS three times, 3 to 5 minutes each time.

[0051] (3) Remove PBS and add 1 ml of permeabilization solution to each well and incubate at room temperature for 10-15 minutes.

[0052] (4) Remove the permeabilization solution and wash with PBS 1 to 2 times, each time for 3 to 5 minutes.

[0053] 7. Dyeing (avoid light): (1) Prepare the reaction solution according to the kit instructions. For a 12-well plate, add 200 µl of reaction solution to each well. Gently shake the culture plate to ensure that the reaction solution evenly covers the sample. Incubate at room temperature in the dark for 30 minutes.

[0054] (2) Remove the reaction solution and wash with PBS three times, each time for 3 to 5 minutes.

[0055] 8. Nuclear staining (protect from light): (1) Dilute the Hoechst 33342 reaction solution to 1X with deionized water. After removing the PBS washing solution, add 200µl of Hoechst 33342 to each well and stain for 10 minutes at room temperature in the dark.

[0056] (2) Remove Hoechst and wash with PBS three times, 3 to 5 minutes each time.

[0057] 9. Counting and taking photos: The EdU-labeled and unlabeled cells were observed under a fluorescence microscope or a confocal microscope, photographed, and counted.

[0058] The experiment was repeated three times, and each group in the experiment had to have at least three replicate wells. Finally, statistical software was used to analyze the data.

[0059] Figure 4The graph shows the detection of cell migration on bone repair membrane by immunofluorescence staining of three fiber membranes and the detection of cell proliferation on bone repair membrane by EDU kit. 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 PCL group ( *P<0.05, **P< 0.01 ), suggesting that the bidirectional gradient VEGF functionalized fiber membrane has more advantages in repair than the non-gradient VEGF fiber membrane and simple fiber membrane, and can better promote bone regeneration.

[0060] It is easy for those skilled in the art to understand that the above is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. An intelligent electronically controlled spraying device, characterized in that: include: Controllable nozzle, nozzle solution box, moving guide rail, speed change gear box, negative pressure aspirator and material placement box; The controllable nozzle is connected to the nozzle solution tank and fixed on the movable guide rail, and is used to spray strip-shaped mist; and the spraying range is larger than the width of the fiber membrane; The nozzle solution tank is used to store the spray solution and realize the replacement of the spray solution; The speed change gear box is used to steplessly adjust the spray volume of the controllable nozzle and control the movement of the controllable nozzle on the movable guide rail; The material placement box includes two layers, the upper layer is used to carry the PCL nanofiber membrane that needs to be loaded with gradient concentration growth factors, and the lower layer is connected to the negative pressure aspirator. The negative pressure aspirator is used to extract liquid and gas, and can also provide positive pressure to prevent the upper layer solution from flowing into the lower layer. The bottom of the upper layer can be connected to the lower layer, and the sprayed growth factors can be precisely immersed in the local area of the material to be loaded through electric-controlled strip mist spraying and negative pressure suction infiltration.

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

3. The intelligent electronically controlled spraying device according to claim 1, characterized in that: The speed change gear box has dual speed change gears, which are used to adjust the spraying speed and the traveling speed and direction of the controllable nozzle respectively.

4. The intelligent electronically controlled spraying device according to claim 3, characterized in that: The speed change gearbox can control the speed and acceleration through a computer, control the gearbox acceleration, and move the gearbox from the middle to the left and right ends at an acceleration matching 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 according to any one of claims 1 to 4, characterized in that: A placement plate with densely distributed micropores is provided at the bottom of the upper layer of the material placement box. The placement plate is used to carry the PCL fiber membrane that needs to be loaded with gradient concentration growth factors. When the negative pressure aspirator is turned on, the liquid can be quickly drawn out of the aperture plate; when the solution needs to stay 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.

6. The intelligent electronically controlled spraying device according to claim 5, characterized in that: The aperture of the upper bottom storage plate is 1 mm.

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

8. 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 to 7, characterized in that: The steps include: S1 Preparation of electrospun PCL fiber membrane: PCL was dissolved in a 10% methanol / chloroform mixed solution to obtain an electrospinning solution, the flow rate of the electrospinning solution was controlled to be 0.01 ml / h-0.30 ml / h, and the residual solvent was removed by drying to obtain a PCL nanofiber membrane; S2 performs bidirectional gradient aminolysis on the PCL fiber membrane: The fiber membrane is firmly attached to the groove wall by a negative pressure suction device, and the initial position of the controllable nozzle is set in the middle of the fiber membrane, so that the controllable nozzle is moved from the left and right sides by 1 The acceleration is accelerated to spray toward both ends to obtain a gradient fiber membrane with high -NH2 density in the center and gradually decreasing -NH2 density toward both ends; S3 Preparation of bidirectional gradient heparinized fiber membrane by heparin coupling reaction: A morpholineethanesulfonic acid buffer solution is evenly sprayed on the gradient fiber membrane and allowed to fully soak for a period of time; 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide / heparin solution is then added to the gradient fiber membrane to covalently bind heparin to the fiber membrane carrying amino groups, and unbound heparin is washed away to obtain a bidirectional gradient heparinized fiber membrane; S4 Preparation of bidirectional gradient VEGF fiber membrane: VEGF solution was evenly sprayed on the bidirectional gradient heparinized fiber membrane to obtain a VEGF functionalized nanofiber bone repair membrane with a bidirectional gradient concentration.

9. A method for promoting bone regeneration using the nanofiber bone repair membrane according to claim 8, characterized in that: include: The central part of the bidirectional gradient VEGF-functionalized fiber membrane releases high concentrations of VEGF, which accelerates the migration of endothelial cells at both ends of the damaged membrane to the high concentration area in the central part of the bidirectional gradient VEGF-functionalized fiber membrane through chemotaxis; The vascularization of the fiber membrane is accelerated from both ends to the center. The generated blood vessels act as scaffolds to guide the rapid directional migration of vascular endothelial cells into the fiber membrane, thereby accelerating regeneration and extending toward the distal end along the vascular endothelial cells.

10. The method according to claim 9, wherein The amount of VEGF loaded in the center of the bidirectional gradient VEGF-functionalized fiber membrane is high, while the amount of VEGF loaded at both ends is relatively small.

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