CN113599578B - Composite electrostatic spinning fiber membrane containing dHAM and preparation method and application thereof - Google Patents

Abstract

The invention discloses a composite electrostatic spinning fibrous membrane containing dHAM and a preparation method and application thereof, belonging to the field of tissue engineering. The preparation method of the composite electrostatic spinning fiber membrane comprises the following steps: the method comprises the steps of amnion cleaning and acellular pretreatment to obtain dHAM, preparing soluble dHAM powder by adopting a solubilization step of freeze-drying grinding and digestive dialysis, preparing a blending/coaxial PLGA/PCL/dHAM composite electrostatic spinning solution by taking HFIP as an electrostatic spinning solvent, and performing blending electrostatic spinning or coaxial electrostatic spinning under the conditions that the environmental humidity is 30-50% and the temperature is 25-35 ℃.

Description

Composite electrospinning fiber membrane containing dHAM and its preparation method and application

technical field

The invention belongs to the field of tissue engineering, and in particular relates to a composite electrospinning fiber membrane containing dHAM and a preparation method and application thereof.

Background technique

Pelvic organ prolapse (POP) is common in middle-aged and elderly women. It is a kind of pelvic floor dysfunction due to the collapse and prolapse of organs caused by the weakening of pelvic floor levator ani muscle group and connective tissue and other supporting structures. A condition of multifactorial disease (Pelvic floor dysfunction, PFD). PFDs is a common disease. About 25% of women in the United States are affected by PFDs and feel pressured. In my country, up to 9.6% of adult women have clinical manifestations of POP, and the risk of surgery for women due to POP and other diseases is about 20%. It can be said that gynecological surgery common causes. In clinical practice, pelvic floor reconstruction surgery is a therapeutic strategy with good curative effect and high satisfaction.

Tissue engineering is a technology for repairing and reconstructing damaged tissues, including scaffolds, seed cells and cytokines. The specific steps are: seed cells with differentiation ability are planted on degradable biocompatible materials to form a composite biological network. The mesh is implanted into the site to be repaired, and the mesh disintegrates and fuses with the surrounding tissue to form a new tissue to achieve the effect of repair and reconstruction. Scaffold is a key element in tissue engineering, which provides sufficient space for tissue and organ regeneration. The choice of scaffold materials and preparation methods is directly related to the interaction of cells and growth factors with specific tissues, and is related to material implantation and subsequent damage to the tissue. The effect of functional fixes and improvements. Scaffolding materials prepared from biodegradable polymers are widely used in tissue engineering, but the high manufacturing cost and unsatisfactory biological activity of the raw materials still prompt researchers to find better alternatives to the raw materials. In recent years, tissue engineering methods have great potential in the field of POP treatment. In the field of reconstructive medicine, new tissue engineering biomesh has been favored by researchers. With the deepening of the understanding of the functional anatomy of the female pelvic floor and the improvement of pelvic floor reconstruction The increase in the demand for diseases has made the application of repair mesh more extensive. As one of the commonly used synthetic materials for pelvic floor reconstruction surgery, non-degradable polypropylene mesh was once considered an ideal model for repair mesh design, but it is prone to complications such as mesh exposure, perforation, infection and even bleeding after implantation. The FDA's 2011 safety notification update also attests to this. Therefore, tissue engineering applied to pelvic floor reconstruction also faces challenges in selecting ideal biomaterials, optimal seed cells and related inducing factors, and designing suitable scaffolds.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a method for preparing a dHAM-containing composite electrospinning fiber membrane, which adopts electrospinning technology to combine PCL, PLGA and dHAM under high-voltage static electricity to obtain a micron-scale fiber ordered arrangement. PCL/PLGA/dHAM electrospinning membrane, while fully retaining the physicochemical properties of PCL and PLGA, innovatively uses human amniotic membrane for electrospinning, with good physicochemical properties and biocompatibility, no obvious cytotoxicity to hUC-MSCs, It has potential clinical application value for POP.

The second object of the present invention is to provide a composite electrospinning fiber membrane containing dHAM obtained by the above preparation method.

The third object of the present invention is to provide the application of the above-mentioned dHAM-containing composite electrospinning fiber membrane in pelvic organ prolapse tissue engineering.

To achieve the above object, the present invention adopts the following technical solutions:

The preparation method of the composite electrospinning fiber membrane containing dHAM provided by the present invention comprises the following steps:

(1) Amniotic membrane washing and decellularization pretreatment to obtain dHAM;

(2) preparing soluble dHAM powder using a solubilization step comprising freeze-drying grinding, digestive dialysis and secondary freeze-drying grinding;

(3) Using hexafluoroisopropanol (HFIP) as the electrospinning solvent to prepare a blended PLGA/PCL/dHAM composite electrospinning solution or a coaxial PLGA/PCL/dHAM composite electrospinning solution;

(4) Connect the electrospinning device and turn on the power. The electrospinning solution obtained in step (3) is subjected to blending electrospinning or coaxial spinning under the conditions of an ambient humidity of 30%-50% and a temperature of 25-35°C Electrospinning to obtain a dHAM-containing composite electrospinning fiber membrane in which micron fibers are arranged in an orderly manner, ventilated for standing, and vacuum storage after the surface solvent is volatilized.

Preferably, in step (1), the amniotic membrane washing and decellularization pretreatment include the following steps:

(1-1) Fresh human amniotic membrane samples were laid flat on the ultra-clean workbench, washed with PBS solution prepared with deionized water to remove blood stains, and then rinsed with 75% ethanol to a semipermeable state;

(1-2) Soak in PBS solution containing 10% double antibody, while magnetic stirring, rinse and shake, and replace the PBS solution containing 10% double antibody every 12h for a total of 2 times;

(1-3) Soak in the surfactant Triton-100 solution that accounts for 1% by weight of PBS, and replace the surfactant Triton-100 solution that accounts for 1% by weight of PBS every 8h, for a total of 2 times;

(1-4) Soak in PBS solution containing 1000U pancreatic lipase/L, shake at 37°C for 24h, remove and wash;

(1-5) Soak in PBS solution containing 1000U DNase/L, shake at 37°C for 24h, remove and wash;

(1-6) Rinse in PBS solution containing 10% double antibody, and replace every 12h, for a total of 2 times, to obtain dHAM, which is stored in PBS solution containing double antibody at 4°C for use.

Preferably, in step (2), the preparation method of the soluble dHAM powder includes:

(2-1) Take the dHAM obtained in step (1), cut it into an appropriate size, spread it in a petri dish, cover it with a layer of plastic wrap with a hole, freeze it at -80°C in advance, and quickly transfer it to vacuum lyophilization. Freeze-dried in the machine for 48h to obtain freeze-dried dHAM; put the freeze-dried dHAM in a freezer grinder at -198°C, grind 3 times at 30Hz for 30s each time, and quickly transfer it to a 5mL centrifuge tube to obtain a 50-mesh sieve. Insoluble dHAM powder;

(2-2) Add 1 g of insoluble dHAM powder to 20 mL of an aqueous solution containing HCl (0.5M) and pepsin (≥1600 U/L) for digestion, and stir continuously for 3 days at 4°C and 200 r/min to make In order to obtain solubility, neutralize with NaOH (4N) to pH 7.4, prepare dHAM suspension, dialyze with deionized water for 24h, and change deionized water every 4h to obtain milky white dHAM viscous solution;

(2-3) The digested dHAM viscous solution was placed in a petri dish, covered with plastic wrap, and completely frozen at -80 °C, and then quickly transferred to a vacuum freeze dryer for lyophilization for 48 hours to obtain freeze-dried soluble dHAM; freeze-dried soluble dHAM was obtained; The dry soluble dHAM was placed in a freezer grinder at -198°C, and ground three times at 30 Hz for 30 s each time, and then quickly transferred to a 5 mL centrifuge tube to obtain a soluble dHAM powder that could pass through a 50-mesh sieve.

Preferably, in step (3), the preparation method of the blended PLGA/PCL/dHAM composite electrospinning solution is as follows: the electrospinning solvent HFIP is 10 mL, and 3 g of PCL is added, and magnetically stirred at a speed of 300-3000 rpm to Dissolving, after 12 hours, PCL electrospinning solution with a concentration of 30% was obtained, and the same method was used to obtain a PLGA electrospinning solution with a concentration of 15% and a dHAM electrospinning solution with a concentration of 5%. PCL, PLGA, dHAM were 1: 1:1 ratio of stirring and mixing to obtain;

And/or the preparation method of the coaxial PLGA/PCL/dHAM composite electrospinning solution is as follows: the electrospinning solvent HFIP is 10 mL, 3 g of PCL is added, and the solution is magnetically stirred at a speed of 300-3000 rpm until dissolved, and the concentration is obtained after 12 hours. 30% PCL electrospinning solution as the "core" part of the electrospinning solution, the same method was used to obtain 15% PLGA electrospinning solution and 5% dHAM electrospinning solution as "shell" Part of the electrospinning solution.

Preferably, in step (4), the electrospinning process parameters include: the voltage is 15-20 kV, the distance between the needle and the receiving rod is 18 cm, and the flow rate is 30 μL/min.

The present invention also provides the dHAM-containing composite electrospinning fiber membrane obtained by the above preparation method, which is a white and uniform laminated mesh fiber membrane.

The present invention also provides the application of the above-mentioned dHAM-containing composite electrospinning fiber membrane in pelvic organ prolapse tissue engineering.

Preferably, the dHAM-containing composite electrospinning fiber membrane is used as a tissue engineering composite biomesh or bioscaffold.

Compared with the prior art, the beneficial effects of the present invention are:

(1) The present invention adopts electrospinning technology to combine PCL, PLGA and dHAM under high-voltage static electricity. The high-voltage static electricity can rapidly stretch and thin the charged copolymer solution. Micro-fibers with a microporous structure can obtain PCL/PLGA/dHAM electrospinning membranes composed of micro-scale fibers arranged in an orderly manner. While fully retaining the physicochemical properties of PCL and PLGA, the human amniotic membrane is innovatively used for electrospinning. Good physicochemical properties and biocompatibility, no obvious cytotoxicity to hUC-MSCs, and microfiber structure similar to ECM, the layered electrospinning membrane is suitable for cell adhesion and proliferation, and is used for pelvic organ prolapse tissue engineering It has potential clinical application value.

(2) The present invention proposes for the first time to use the high-voltage electric field of electrospinning to prepare non-spinning pure dHAM into micron or nano-scale fibers, and adopt freeze-drying grinding, digestion dialysis, secondary freeze-drying grinding, etc., to maximize the retention of active ingredients. To increase the solubility of dHAM, and choose strong polar HFIP as solvent to dissolve dHAM uniformly. In addition, the irregular structure of dHAM is difficult to be wound into fibers under the traction of high-voltage electric field. In the present invention, two copolymers of PCL and PLGA, which are easy to be processed by electrospinning, are added into the electrospinning system, and HFIP is used as a solvent. A small amount of dHAM is added to the copolymer to obtain a laminated mesh composite electrospinning fiber membrane with micron-scale fibers arranged in an orderly manner.

The above and other features, aspects and advantages of the present invention may be better understood with reference to the following detailed description.

Description of drawings

Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

Figure 1 is the SEM images of HAM before (a) and after (b) decellularization;

Figure 2 is the DNA content of HAM and dHAM;

Fig. 3 is the SEM image of composite electrospinning fiber membrane;

Fig. 4 is the contact angle test of composite electrospinning fiber membrane;

Fig. 5 is the infrared spectrogram of G, T, GT and TH;

Fig. 6 is DSC curve (A is heating curve, B is cooling curve);

Figure 7 shows the mechanical properties of composite electrospun fiber membranes, where: (a) Young's modulus, (b) tensile strength (TS), (c) elongation at break (EAB) and (d) stress-strain Curve (n=3, bars are standard deviations);

Fig. 8 is the degradation retention rate of composite electrospinning fiber membrane;

Figure 9 is the cytotoxicity grade test of the composite electrospinning fiber membrane in different concentrations of material extracts;

Figure 10 is a biocompatibility-cell proliferation assay test of the composite electrospun fiber membrane.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are some, but not all, embodiments of the present invention. Based on the described embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

The amniotic membrane is a part of the human placenta and is mainly composed of a single layer of columnar epithelial cells, a dense basement membrane layer, acellular dense collagen layer, an underlying fibroblast and a spongy layer. It was originally an amniotic fluid-filled sac surrounding the developing fetus. The infants were discarded at birth, and fresh human amniotic membrane samples in the following examples were taken from the Obstetrics and Gynecology Department of the Sixth People's Hospital of Shanghai.

In tissue engineering, medical polymer materials have been certified by the US Food and Drug Administration as biodegradable and biocompatible poly(lactic-glycolic acid) PLGA, polycaprolactone (PCL) and other materials, PCL It is a high molecular polymer polymerized from ε-caprolactone monomer. Different polymerization conditions can obtain different molecular weights. The advantages are easy processing and good toughness, but poor hydrophilicity and long degradation time; PLGA is lactic acid and polyethanol. A non-toxic copolymer formed by the polymerization of two monomers of acid, the biodegradation rate can be controlled by the ratio of the two monomers, lactic acid and polyglycolic acid. Among the two monomers, lactic acid is a normal product of sugar metabolism, which can be degraded into H ₂ O and CO ₂ in the body, without interfering with the normal physiological function of human tissues; polyglycolic acid has good hydrophilicity, and blending with lactic acid can improve the degradation rate , Increase histocompatibility, because it has good biocompatibility and is widely used in medical materials, the disadvantage is that it is brittle, poor toughness, and the degradation cycle is relatively short, which cannot meet the requirements of long-term repair of the bottom of the pelvis. PCL has good toughness. Blending with PLGA can improve the mechanical parameters and prolong the degradation time. With the increase of PCL content, the fiber diameter decreases and the elongation at break of the fiber film increases. By adjusting the content of the polymer, the ratio of 30% PCL and 15% PLGA modulates the mechanical properties of electrospun fiber membranes.

Hexafluoroisopropanol (HFIP) is extremely volatile, and can fully extend the polymer into slender fibers when spinning in a high-voltage electric field. When it reaches the receiving device, the solvent has completely volatilized. Electrospinning with HFIP as a solvent The liquid can greatly increase the degree of dissolution of raw materials, especially the degree of dissolution of dHAM, and further reduce the fiber diameter. In the following examples, HFIP is used as the electrospinning solvent.

Example 1

PCL/PLGA/dHAM electrospinning film was prepared in the following examples, and the steps were as follows:

Step 1: Amniotic membrane decellularization

Preliminary cleaning: In the ultra-clean workbench, the amniotic membrane samples were laid flat, washed with PBS prepared with deionized water to remove blood stains, and then rinsed with 75% ethanol to a semipermeable state.

Antibacterial: prepare PBS containing 10% double antibody, soak the amniotic membrane in it, stir, rinse and shake with a magnetic stirrer, replace the soaking solution at intervals of 12 hours, and change it twice in total, and continue to shake and soak after replacement.

Surfactant rinsing: Prepare 1% surfactant Triton-100 solution by weight of PBS, and replace the soaking solution at 8h intervals, for a total of 2 times.

Pancreatic lipase rinse: prepare a PBS solution containing 1000 U of pancreatic lipase per liter, soak the amniotic membrane in it, shake it at 37°C for 24 hours, and then remove it for washing.

DNase rinsing: prepare a PBS solution containing 1000 U of DNase per liter, soak the amniotic membrane in it, shake it at 37°C for 3 hours, and then remove it for washing.

Secondary washing: The amniotic membrane will be rinsed with a PBS solution containing double antibodies, and replaced at an interval of 12 hours, for a total of 2 times, to obtain dHAM. Store in PBS solution containing double antibody at 4°C for later use.

Step 2: Preparation of Soluble dHAM Powder

Freeze-dried grinding: Cut dHAM into appropriate size, spread it in a petri dish, cover with a layer of plastic wrap with holes, put it in a -80°C refrigerator and freeze it completely, quickly transfer it to a vacuum freeze-drying machine, and freeze-dry it for 48 hours. Freeze-dried dHAM was made. The lyophilized dHAM was ground in a freezer at -198°C at 30 Hz for 3 times for 30 s each time, and quickly transferred to a 5 mL centrifuge tube with a spatula, and ground into an insoluble dHAM powder that could pass through a 50-mesh sieve.

Digestion dialysis: Take 1 g of insoluble dHAM powder and add it to 20 mL of an aqueous solution containing HCl (0.5M) and pepsin (≥1600 U/L) for digestion, and continue stirring at 200 revolutions per minute at 4°C for 3 days to make it Solubilization was obtained, neutralized to pH 7.4 with NaOH (4N), and formulated as a dHAM suspension. The dHAM suspension was dialyzed against deionized water for 24 h, and the deionized water was changed every 4 h to obtain a dHAM viscous solution.

Secondary freeze-drying and grinding: put the digested milky white dHAM viscous solution in a petri dish, cover it with a perforated plastic wrap, freeze it completely in a -80°C refrigerator, quickly transfer it to a vacuum freeze-drying machine, and freeze-dry it for 48 hours to make a freeze-dried product. Dissolve dHAM. The lyophilized soluble dHAM was ground in a freezer grinder at -198°C at 30 Hz for 3 times, each time for 30 s, quickly transferred to a 5 mL centrifuge tube with a spatula, and ground into a soluble dHAM powder that could pass through a 50-mesh sieve.

Detection and identification of the degree of decellularization: qualitative observation with scanning electron microscope, freeze-dried HAM before decellularization and dHAM after decellularization, and observe and take pictures with SEM at 500 times magnification under the condition of accelerating voltage of 5-6kV. The results are shown in Figure 1. .

Use the kit to determine the DNA concentration before and after decellularization to quantitatively identify the degree of amniotic membrane decellularization. Follow the instructions of the tissue DNA extraction kit to detect the DNA concentration of HAM and dHAM, and use the BCA kit to quantify the protein content in the dHAM digest, and analyze it at 562 nm. The absorbance value (Optical density, OD) was measured with a microplate reader, and the results are shown in Figure 2. Among them, the preparation of diluted bovine serum albumin standard solution (BSA) standard: take 50 μL of 2 mg/mL BSA standard in a 1 mL centrifuge tube, add 150 μL of deionized water, and mix to obtain 200 μL of 0.5 mg/mL diluted Add 0, 1, 2, 4, 8, 12, 16, 20 μL of the diluted standard to each well of the 96-well plate, add deionized water to make up to 20 μL, and add an appropriate amount of diluted dHAM to digest Add deionized water again to make up to 20 μL, take 6000 μL and 120 μL of each of Solution A and B in the kit and mix well to obtain BCA working solution, add 200 μL working solution to each well of 96-well plate, react at 60 ℃ for 30 min, cool to room temperature, The standard curve was obtained by measuring OD with a microplate reader at 562 nm, and the protein content in the dHAM digest after appropriate dilution was calculated by substituting the sample OD into the regression equation. Substituting the measured results into the regression equation of the standard curve, it is found that there is still a lot of protein in the amniotic membrane powder after decellularization. According to the results of the regression curve, it is calculated that each gram of decellularized amniotic membrane powder contains 81.693 mg of detectable protein components, which proves that the decellularized amniotic membrane powder contains 81.693 mg of detectable protein components. A series of solubilization steps such as cells, freeze-dried grinding, digestion dialysis, secondary freeze-dried grinding, etc., did not completely disappear the active protein components in dHAM, but became dispersed collagen fibers.

The combination of Triton X-100 and biological enzymes is simple, efficient, non-toxic, gentle and thorough. Before decellularization with biological enzymes, HAM was translucent and milky white, but after decellularization, dHAM became thinner and larger, clean and transparent, and retained a certain elasticity. The lyophilized dHAM is white paper-like, with a hard texture and a slightly rough surface. As shown in Figure 1, after SEM magnification 500 times, there are cell adhesion and filamentous tissue on the surface of HAM, while only wavy matrix can be seen on dHAM, the surface is clean and dense, and there is no cell residue. As shown in Figure 2, the average DNA concentration of HAM was 389.26 μg/mL, while the average DNA concentration of dHAM was 3.98 μg/mL, which was significantly lower than that before decellularization. The above SEM and DNA content detection results indicated that the amniotic membrane was decellularized more completely in the first step.

Step 3: Prepare Electrospinning Solution

When the electrospinning solvent is HFIP, the concentrations of PCL, PLGA, and dHAM are selected as 30%, 15%, and 5%, respectively, to prepare an electrospinning solution, as follows:

The solution of blending PLGA/PCL electrospinning film (referred to as G): the electrospinning solvent HFIP is 10mL, 3g of PCL is added, and the magnetic stirrer is stirred at a speed of 300-3000rpm until dissolved, and the concentration of 30% is obtained after 12h. PCL electrospinning solution, PLGA electrospinning solution with a concentration of 15% was obtained by the same method, and PCL and PLGA were stirred and mixed at a ratio of 1:1 to obtain a blended PLGA/PCL electrospinning solution.

Solution of coaxial PLGA/PCL electrospinning membrane (T for short): The electrospinning solvent HFIP is 10mL, 3g of PCL is added, and the magnetic stirrer is stirred at a speed of 300-3000rpm until dissolved, and the concentration of 30% is obtained after 12h. The PCL electrospinning solution was used as the "core" part of the electrospinning solution, and the PLGA electrospinning solution with a concentration of 15% was obtained by the same method as the "shell" part of the electrospinning solution.

The solution of blending PLGA/PCL/dHAM composite electrospinning membrane (GH for short): the electrospinning solvent HFIP is 10mL, 3g of PCL is added, and the magnetic stirrer is stirred at a speed of 300-3000rpm until dissolved, and the concentration after 12h is 30% PCL electrospinning solution, use the same method to obtain 15% PLGA electrospinning solution and 5% dHAM electrospinning solution, and mix PCL, PLGA, and dHAM in a ratio of 1:1:1. homogenized to obtain a blended PLGA/PCL/dHAM composite electrospinning solution.

Solution of coaxial PLGA/PCL/dHAM composite electrospinning membrane (TH for short): The electrospinning solvent HFIP is 10mL, 3g of PCL is added, and the magnetic stirrer is stirred at a speed of 300-3000rpm until dissolved. After 12h, the concentration is 30% PCL electrospinning solution was used as the "core" part of the electrospinning solution, and 15% PLGA electrospinning solution and 5% dHAM electrospinning solution were obtained in the same way as the "shell" part electrospinning solution.

Step 4: Electrospinning to prepare fiber scaffolds

Connect the electrospinning device, turn on the power, use different process parameters, observe the spinning shape under an inverted microscope and adjust it in time. After parameter optimization, the ambient humidity is 30%-50% and the temperature is 25-35 ℃. , the electrospinning process parameters include: voltage of 15–20 kV, distance between needles and receiver rods of 18 cm, and flow rate of 30 μL/min. The obtained fibers are in good condition. Four types of electrospinning films were collected and placed in a fume hood for 24 h. , after the surface solvent volatilizes, put it in a vacuum drying box for preservation.

Characterization and Testing

(1) Upright microscope observation

The fiber morphology was preliminarily characterized by an upright microscope. When stabilizing electrospinning, tweezers were used to clamp the glass slide for 3 minutes, and the obtained samples were used to observe the morphology under an upright microscope. The microscopic morphology of the electrospinning fiber membrane was observed by magnifying 40 times through the microscope. As shown in Figure 3, the four composite The electrospun fiber membranes are all white tissue paper, and microscopically exhibit a uniformly laminated microfiber membrane structure. G and T electrospinning scaffolds are pure white and flat, with a microscopic porous fiber structure, showing a disordered distribution; GH and TH electrospinning scaffolds have uneven surfaces, and their microstructures change significantly compared with G and T. The addition of dHAM shows as Rod-like or nearly bead-like structure. It can be seen that all four composite electrospun fibrous membranes are very similar in structure to the ECM.

(2) Scanning electron microscope observation

The microstructure of the electrospun membranes was further characterized by scanning electron microscopy, and fiber diameters were measured accordingly. The prepared electrospinning film appears to be a white and uniform laminated mesh film to the naked eye, and is smooth and tough to the touch. After the film was dried and the residual solvent was removed, it was cut into small pieces of 10mm×10mm with scissors, which were attached to the sample table and sprayed with gold for 90s, and observed with a scanning electron microscope at 5kV and magnified 1000 and 2000 times and took SEM images. SEM images were analyzed with Image J software, fiber diameter and distribution were measured, and mean diameter values were calculated (n=100).

The distribution trend and quantification of electrospun fibers are shown in Figure 3. The diameters of G and T fibers are concentrated in the region of 4–5 μm on average, and the diameters of GH and TH fibers added with dHAM are widely distributed, mainly concentrated in the region below 10 μm. A few fibers have larger diameters, which may be related to the incompletely dissolved dHAM components added to the electrospinning solution. For the fibers containing dHAM, a large number of fibers shrunk on the surface, and a small amount of fibers formed larger diameter fibers due to fusion with adjacent fibers. μm) and T (14.57 μm). The morphology of the coaxial electrospun fibers is the same as that of the blend fibers, but the fiber diameter is smaller, the minimum diameter is 1.62 μm, and there is a slight melting connection phenomenon at the intersection point. In addition, the average diameter of GH fibers was 9.3±0.21 μm, and the average diameter of TH fibers was 8.63±0.12 μm, which were both smaller than G (7.7±0.11 μm) and T (6.3±0.15 μm).

(3) Contact angle test

The contact angle (CA) test can judge the hydrophilicity of the electrospinning scaffold. The hydrophilicity can affect the protein adsorption and cell proliferation on the material. Clean the impurities on the surface of the electrospinning scaffold and keep it smooth and smooth. The measuring instrument measures the water contact angle of different fiber supports. A drop of deionized water is dropped on the fiber support at room temperature, and the angle between the water droplet and the surface of the support is instantaneously photographed with a high-resolution camera and observed.

The contact angle of the sample surface is analyzed by the image method. The larger the reading of the angle, the more hydrophobic and difficult to be wetted, that is, the liquid is easy to move on it. PCL surface has limited functional groups and is relatively hydrophobic. The pores on the surface of pure PCL microfiber scaffolds cannot pass water, lacking binding sites for cell adhesion, and the adsorption of intracellular proteins and natural biological activities on PCL materials are relatively weak. Water is beneficial to cell adhesion. By adding PLGA and dHAM to blend, the hydroxyl content and hydrophilicity on the surface of the material are increased, thereby improving the cell adhesion state of the material implanted in the body. Compared with PCL, PLGA and dHAM have lower processing performance, but their ability to direct biological reactions is higher. By adding PCL, the processing performance of PLGA and dHAM can be improved, and the bioactive materials that are easy to process can be prepared.

As shown in Figure 4, the contact angles (T and TH) of the coaxial electrospun fiber scaffolds were smaller than those of the blended fiber scaffolds (G and GH), and the water contact angles of different microfiber scaffolds decreased with increasing PLGA exposure It is small, indicating that PCL is hydrophobic, and PLGA can improve its hydrophilicity: the contact angle of T is 87.24°, and the contact angle of G is 102.52°, which is because PLGA is outside in coaxial electrospinning, and T has good affinity. water; the contact angles of TH and GH were 86.57° and 92.12°, respectively, indicating that the hydrophilicity of the electrospun fiber scaffold was better after adding dHAM. It can be seen that the performance of T and TH encapsulated by hydrophilic PLGA is better than that of G and GH, indicating that PLGA is more hydrophilic than PCL, and the coaxial electrospinning scaffold is more hydrophilic. After adding the dHAM component, most of the pores were filled with water. Therefore, the water contact angle of GH film and TH film is smaller than that of G film and T film.

(4) Fourier transform infrared spectroscopy

To determine the chemical structures of the four dHAM-containing composite electrospun fibrous membranes, samples were lyophilized and examined using standard operating procedures using Fourier transform infrared spectroscopy in the range of 400–4000 cm ^-1 to obtain the infrared spectra of the four materials. Spectrogram and analysis.

As shown in Fig. 5, the infrared spectra of the four electrospinning scaffolds G, T, GH and TH all show the main infrared characteristic peaks corresponding to PCL and PLGA, such as 1720 cm ^-1 and 1170 cm ^-1 corresponding to the ester carbonyl bond and Stretching of carbon-oxygen bonds. In addition, almost all PCL peaks coincide with all PLGA peaks, which is determined by the higher content of PCL than PLGA. As a world-recognized cell support carrier, PLGA has a large number of methyl groups, carbonyl groups and carboxyl groups, which support cell adhesion. and proliferation. In the GH spectrum, the characteristic bands of amide appeared at 1580cm ^-1 and 1545cm ^-1 , the existence of amide group confirmed the existence of dHAM in the electrospinning fiber, and the amide group in dHAM formed hydrogen bond to increase the hydrophilicity of the material; GH The characteristic peaks CCO and COC of polysaccharides were found in the fingerprint region of TH and TH, and the stretching range was between 1000 and 1200 cm ^-1 . In addition, the infrared spectra of GH and TH, the electrospun fiber scaffolds containing dHAM, behaved similarly to G and T, which may be due to the lower content of dHAM in GH and TH, while the higher content of PCL and PLGA, characterized by The intensity variation of the peaks is not exactly the same as that of the constituent content, which may be due to the non-uniform thickness of the electrospun film, while the intensity of the peaks is affected by the thickness.

(5) Characterization of thermal properties of electrospinning films

Differential scanning calorimeter (DSC) was used to observe the process of heating-up melting and cooling-down crystallization of different electrospinning films, and then characterize their thermodynamic properties. Mechanical refrigeration is equipped with nitrogen protection to prevent the sample from decomposing and oxidizing at high temperature. The amount of dry sample is 5 mg, the heating range is -80 °C to 250 °C, the heating rate is 10 °C/min, and the melting process of the sample is recorded; after holding for 5 minutes, the temperature is lowered The temperature range is from 250 °C to -80 °C, and the heating and cooling curves are drawn at a cooling rate of 10 °C/min to record the melting and crystallization process of the sample, as shown in Figure 6.

In the heating curve, the position of the melting endothermic peak can be seen. After heating to a certain temperature, the amorphous material is converted into a crystal, and the latent heat of crystallization is released. Thermal decomposition stage, after which the weight remains stable. The glass transition temperature, which is the decomposition peak, is about 55°C for both G and T, while GH is 57°C, indicating that the glass transition temperature is slightly increased after the addition of dHAM. The peak of TH is not obvious at about 33 °C, which may be due to the lower glass transition temperature due to the complete exposure of PLGA when wrapping dHAM. In the cooling curve, the peak generally represents the freezing point, T and G are both around 15°C, and GH is 25°C. For TH, there is no corresponding peak in the cooling curve, which may be due to the irreversible transformation from amorphous to crystalline, or slow cooling. has been fully crystallized in the process.

(6) Mechanical property test

In order to preliminarily characterize the mechanical properties of the composite electrospinning scaffold, four materials G, GH, T and TH were tested by uniaxial tensile test using a texture analyzer after equilibrating in a constant temperature and humidity environment for 24 hours. Before the tensile test, a fiber film with a thickness of about 3 mm was spun, and the sample was cut into a size of 1 cm in width and 2 cm in length, and the sample was tightly clamped between two sandpaper adhesive probes to generate maximum friction. The sample was initially gripped and pulled at a speed of 2 mm/min to ensure that the mechanical properties of the scaffold were recorded from the same starting point, and then pulled at a speed of 5 mm/min until the material broke, and a stress-strain curve was drawn from three parallel tests, as shown in shown in Figure 7.

The Young's modulus is determined by the slope of the stress-strain curve. The larger it is, the less deformable the material and the better the elasticity. G is not easy to deform. It may be because its PCL content is high and it is fully mixed with PLGA and added to the brittle PLGA. Flexible PCL can improve the mechanical strength of microfiber scaffolds, while PLGA-coated coaxial electrospinning has relatively poor elastic properties (Fig. 7A).

The tensile strength is the maximum average stress of the electrospinning scaffold before breaking in the tensile test. After changing the method of electrospinning and adding dHAM, TS was significantly reduced, and the maximum average stress of GH and TH were 2.088±0.856MPa and 3.498, respectively ±1.259MPa (Figure 7B).

The larger the elongation at break, the better the material tension, the blending or coaxial electrospinning method has little effect on the EAB, and higher EAB is observed in G and T (Fig. 7C), indicating that the addition of PCL keeps the fibers The diameter of dHAM makes the stent more stable, while the addition of dHAM weakens the fiber structure, destroys the mechanical properties, and the mechanical properties decrease significantly, but the mechanical properties of the pelvic floor tissue can still be met.

The nonlinear stress–strain curves show the changes of the scaffolds during the stretching process, and the differences among the four scaffolds are large due to the non-uniformity of the thickness of the electrospun scaffolds, indicating that the four scaffolds have unique mechanical properties and different fibers (Fig. 7D).

(7) In vitro degradation test

The degradation experiments of electrospinning membranes were carried out in PBS with a pH of 7.2–7.4. The electrospinning membranes were cut to 1 cm × 1 cm, and their initial mass W ₀ was recorded. in a wet incubator. The two samples were stored in an environment with a temperature of 23 ± 5°C and a relative humidity of 50 ± 10% for degradation as a blank control. Samples were taken every 2 weeks for a total of 12 weeks of testing.

In order to test the biodegradation performance of the scaffolds, the size of these scaffolds was cut to 1cm × 1cm, and then placed in an EP tube containing a degradation solution, which was a simulation of 1% porcine pancreatic lipase and 1% neutral protease. Body fluids (pH=7.2–7.4), called enzyme-containing simulated body fluids (eSBF), were then stored in a constant temperature and humidity incubator at 37°C.

Each group of 3 replicates was replaced with fresh degradation solution after each sampling to ensure enzymatic activity. The remaining mass (W _t ) of the composite electrospinning scaffold was weighed every 7 days, the sample was washed 3 times with DW, dried to equal weight at room temperature, and the retention rate (R%) was calculated after weighing. Finally, a degradation rate curve was drawn based on the results of three parallel experiments, as shown in Figure 8.

The two electrospinning films were stably degraded in the PBS environment, which conformed to the general hydrolytic degradation process of the copolymer, namely, moisture absorption, ester bond cleavage, soluble particle diffusion, and fragment dissolution in four stages. After being degraded in PBS for 12 weeks, the residual mass of the electrospinning film T was relatively small, with a retention of 86.77%, and the retention of the electrospinning film G was 90.21%, while the two blank control films were degraded under storage conditions. Coincidentally, the remaining mass after 12 weeks was significantly higher than the material degraded in PBS. It can be seen from the figure that beads are easily generated when adding dHAM containing electrospinning. Compared with the pure fibrous membrane, the membrane with beads degrades faster, and the degradation rate increases with the increase of the number of beads. The enzymatic degradation solution further confirmed that neutral proteases can degrade dHAM, since membranes containing dHAM degrade faster. Since PLGA is more hydrophilic and degrades faster than PCL, and pancreatic lipase degrades PLGA, the coaxial electrospinning membrane with PLGA as the shell degrades faster than the blended electrospinning membrane. After 6 weeks of degradation, TH with the fastest degradation rate still retained 65.21% of its mass, indicating that it can support the damaged tissue of the pelvic floor during a long biodegradation cycle, so as to achieve the purpose of tissue regeneration and restoration of anatomical structure to treat diseases. .

In summary, the contact angle test results show that the hydrophilic properties of the composite electrospinning fiber membranes T and TH wrapped with hydrophilic PLGA are better than those of G and GH, and the composite electrospinning fiber membranes GH and TH with the addition of dHAM components have better hydrophilic properties. The hydrophilic performance is better than G and T, and the better hydrophilic performance of the composite electrospinning fiber membrane containing dHAM is beneficial to the subsequent cell adhesion and growth. Through the characterization of infrared and thermodynamic properties, it was found that the electrospun membranes GH and TH were successfully spun into dHAM with characteristic groups and stable thermodynamic properties. The ideal biomaterial should exhibit mechanical properties that can maintain the formation of relevant tissues before complete degradation. Further evaluation of its mechanical properties and degradation rate shows that the stress-strain curves of the four composite electrospun fiber membranes are significantly different. The mechanical parameters such as Young's modulus, breaking strength and elongation at break of the silk films were better than those of the coaxial electrospinning films. Among them, the mechanical properties of electrospinning membrane G were the best, and the mechanical properties decreased after adding dHAM, but still could meet the requirements of pelvic floor tissue. After the 12-week hydrolysis test, the residual amount of the blended electrospinning film was 90.21%, and the coaxial electrospinning film degraded relatively quickly, and the residual amount was 86.77%, indicating that the coaxial electrospinning film with PLGA as the shell was better than the blend electrospinning film. Spinning membranes degrade faster. After 6 weeks of enzymatic hydrolysis test, TH with the fastest degradation rate still retained 65.21% of its mass, indicating that it could support damaged pelvic floor tissue during at least 6 weeks of biodegradation.

(8) Biological safety-cytotoxicity test

Preparation of electrospinning membrane extract: Cut each group of membranes to a size of 1 cm × 1 cm and weigh about 1 mg. For sterilization, put the front and back sides under ultraviolet light for 30 minutes, soak them in 75% ethanol for 30 minutes, wash with sterile PBS, and place them under ultraviolet light for 30 minutes each on the front and back sides. According to the ratio of electrospinning membrane:MSC culture solution=1mg:mL, the electrospinning membrane was completely immersed in the MSC culture solution, and placed in a constant temperature incubator for 3 days to filter and sterilize for use. After filtration and sterilization, four material extract.

Cell culture: Take pre-frozen MSCs and culture to P3. After digestion, make a suspension with a concentration of 500,000 cells per ml. Add 100 μL to each well of a 96-well plate, and culture for 24 hours to make the cells adhere to the wall. Aspirate and discard the old solution, and add 100 μL of 100%, 75%, 50%, 25%, and 12.5% extracts in different proportions to each well. Negative controls were broths without extract (n=6).

Detection of relative cell proliferation rate: After 3 days of culture, CCK8 method was used to detect the proliferation characteristics of cells on the four scaffolds. Add 100 μL of LCCCK-8 working solution to each well of a 96-well plate, take it out in time after incubation for 1 h, measure its OD at 450 nm, calculate the relative proliferation rate (P%) of cells, and convert the PGR of each group into material toxicity grades, grades 0 and 1. grade is non-toxic.

Cytotoxicity of materials: Before directly implanting hUC-MSCs into the scaffolds, the cytotoxicity of the scaffolds was tested with scaffold leaching solution. To investigate this, hUC-MSCs were cultured in MSCM medium containing different concentrations of extract and assayed with CCK8 to determine the effect of scaffolds on the viability of hUC-MSCs.

As shown in Figure 9, four different concentrations of electrospinning scaffold extracts all had a certain inhibitory effect on the proliferation of hUC-MSCs, and the degree of inhibition increased with the increase of the concentration of the material extract, but the calculated toxicity grades of the materials were all less than 1, indicating that the electrospinning scaffold has no obvious toxicity. Pure polymer electrospinning scaffolds had more obvious inhibitory effects on cell proliferation, such as G and T, while the material had better biocompatibility after adding dHAM. The inhibition of cell proliferation on the blended electrospinning membrane was more obvious, such as G and GH. The coaxial electrospinning membrane initially had a lower inhibition rate, and then because PLGA had better biocompatibility than PCL, the inhibition rate gradually increased after the gradual degradation of the surface PLGA. improve.

(9) Biocompatibility-Cell Proliferation Experiment

Quantitative test: A sterile electrospinning membrane (1cm×1cm) of the same size was placed in a 24-well plate to be fully infiltrated with PBS, and then sucked out. Soak in MSCM for 24h. The P3 generation MSCs were adjusted to a cell suspension of 2 × 10 ⁴ cells/mL, 100 μL of cell suspension per well, and hUC-MSCs were directly contacted with the four types of electrospinning scaffolds. After 7 days, the cell and scaffold complexes were taken out, soaked in CCK8 for 1 h and taken out, and the OD of the liquid at 450 nm was recorded (n=6).

Qualitative test: cells were cultured on sterile electrospinning scaffolds of G, T, GH, and TH at a density of 1×10 ⁴ cells/cm ² in a six-well plate, and MSCM without cells was used as a blank control Group. After 3d, cell viability was detected with a live dead cell kit. First, the cells were washed with PBS, and two staining solutions, calcein and propidium iodide were added successively, and the cells were incubated in the dark for 30 min at room temperature: calcein stimulated the esterase activity of living cells to appear green, and propidium iodide penetrated Permeabilized dead cells appeared red, and the staining solution was aspirated and observed with a laser scanning confocal microscope.

Biocompatibility of materials: The biocompatibility of fibers was determined by in vitro culture method. hUC-MSCs were implanted into scaffolds. In order to prevent contamination, both sides of the membrane were irradiated with ultraviolet light for 30 min, soaked in alcohol for 30 min, rinsed with PBS, and then placed in the membrane. 96-well plate, the early adhesion and spreading of cells on the material has a great influence on subsequent cell proliferation and differentiation. Three days after inoculating hUC-MSCs on the membrane, they were stained with a live dead cell kit and observed under a confocal microscope. Morphology of cells on the fiber surface, qualitative detection of cell viability and growth status.

As shown in Figure 10, the G and T groups had cell attachment, but the spreading was not obvious, while the dHAM-added scaffolds GH and TH groups showed normal polygonal morphology, few dead cells were noticed, while the vast majority of cells were alive , the membrane was intact, the green living cells on the GH and TH scaffolds grew well, and the cells still maintained normal metabolic activity and attachment morphology. The density of living cells in the GH and TH groups was significantly higher than that in the G and T groups. The results showed that the proliferation ability of hUC-MSCs on GH and TH was significantly higher than that on G and T, and the increase in the number of hUC-MSCs indicated that the biocompatibility of GH and TH was much stronger than that of G and T, that is, dHAM was not suitable for The cells responded well, indicating that GH and TH with high cytocompatibility are suitable for the repair and regeneration of pelvic floor tissue.

The cells adhered to the membrane surface to form a tissue-like structure. The hierarchical structure of the electrospinning scaffold was similar to that of the natural extracellular matrix. The cells showed good growth ability, and the dHAM-containing fiber scaffold had a good proliferation effect. The composite scaffolds prepared by electrospinning exhibited good biocompatibility and biodegradability, and could support the proliferation of hUC-MSCs on it, all of which cells successfully adhered to the membrane surface, displayed normal morphology, and exhibited normal morphology in a manner comparable to conventional two. Dimensional cultures proliferate at the same rate. Negligible numbers of dead cells were detected, indicating the compatibility of dHAM-containing three-dimensional membrane matrices for cell culture. Cells are easier to grow on scaffolds with high specific surface area and high porosity. Filamentous or widely spaced fibrous membranes provide high porosity suitable for cell growth. Low porosity fibrous membranes containing beads have a high specific surface area due to their reduced fiber diameter. Suitable for cell growth, electrospinning membranes containing bead-like structures have little effect on cell proliferation.

In summary, the present invention prepares a copolymer electrospinning film containing dHAM, innovatively uses dHAM as an extracellular matrix for electrospinning, and obtains degradation properties, mechanical properties and biocompatibility suitable for POP. Novel stent materials for treatment. Based on tissue engineering principles, PCL/PLGA-dHAM blends and coaxial electrospinning scaffolds were successfully prepared, which not only maintained the physical and chemical properties of chemically synthesized materials, but also improved biocompatibility by adding dHAM. Compared with the scaffolds synthesized by PCL/PLGA alone, the electrospun scaffolds containing dHAM could enhance the proliferation of MSCs. In addition, in the prepared electrospinning membrane material, the biocompatibility of GH and TH is much stronger than that of G and T, indicating that GH and TH with high cytocompatibility are suitable for pelvic tissue regeneration. PCL/PLGA/dHAM electrospinning Silk scaffolds can mimic the in vivo composition and structure of hUC-MSCs and promote the in vitro proliferation of hUC-MSCs.

Pelvic floor repair and remodeling is a dynamic equilibrium process, and cells are the manipulators. In pelvic floor tissue engineering, maintaining a stable microenvironment and maintaining the number of functional fibroblasts and smooth muscle cells are the main goals, and on the other hand. On the one hand, the induced differentiation of seed cells can produce a large number of biologically active factors, help connective and muscle tissue regeneration, and treat diseased pelvic floor. As the biomesh degrades at the repair site, newly formed tissue gradually replaces the mesh, supporting and correcting the normal anatomy of the pelvic floor to enable the treatment of POP. It was confirmed by biosafety-cytotoxicity test and biocompatibility-cell proliferation test that the scaffold had no cytotoxicity to hUC-MSCs, and had good biocompatibility, and the electrospinning membrane containing dHAM was more conducive to cell growth. Attachment and proliferation, showing its potential in the treatment of POP, can be used as a tissue-engineered composite biomesh for personalized medicine.

Claims (4)

1. the application of the composite electrospinning fibrous membrane containing dHAM in pelvic organ prolapse tissue engineering as biological mesh or biological scaffold material, it is characterized in that, the described composite electrospinning fibrous membrane containing dHAM is that the micron-scale fibers have The layered network structure arranged in order, its preparation method comprises the following steps: (1) Amniotic membrane washing and decellularization pretreatment, including: laying fresh human amniotic membrane samples on an ultra-clean workbench, washing with PBS solution prepared with deionized water to remove blood stains, and rinsing with 75% ethanol to a semipermeable state; Soak in the PBS solution containing 10% double antibody, while magnetic stirring, rinse and shake, and replace the PBS solution containing 10% double antibody every 12h for a total of 2 times; put in 1% of the weight of PBS The surfactant Triton Soak in -100 solution, and replace the surfactant Triton-100 solution, which accounts for 1% of the weight of PBS every 8h, for 2 times in total; soak in PBS solution containing 1000U pancreatic lipase/L, shake at 37°C for 24h , taken out and washed; soaked in PBS solution containing 1000U DNase/L, shaken at 37°C for 24h, taken out and washed; placed in PBS solution containing 10% double antibody, rinsed, replaced every 12h, a total of 2 times, to obtain dHAM, stored in PBS solution containing double antibody at 4°C; (2) Preparing soluble dHAM powder, including: taking the dHAM obtained in step (1), cutting it into an appropriate size and laying it on a petri dish, covering it with a layer of perforated plastic wrap, and placing it in -80°C for thorough freezing, Quickly transfer to a vacuum lyophilizer for lyophilization for 48h to obtain lyophilized dHAM; place lyophilized dHAM in a freezer grinder at -198°C, grind 3 times at 30Hz for 30s each time, and quickly transfer it to a 5mL centrifuge tube to obtain Insoluble dHAM powder that can pass through a 50-mesh sieve; take 1g of insoluble dHAM powder and add it to 20mL of an aqueous solution containing 0.5M HCl and not less than 1600U/L pepsin for digestion, and stir continuously for 3 days at 4°C and 200r/min. , to make it soluble, neutralized with 4N NaOH to pH 7.4, prepared into dHAM suspension, dialyzed with deionized water for 24h, and changed deionized water every 4h to obtain a milky white dHAM viscous solution; the digested dHAM viscous solution was placed in Put it in a petri dish, cover it with a perforated plastic wrap, freeze it at -80°C, and quickly transfer it to a vacuum freeze dryer for 48h to obtain freeze-dried soluble dHAM; place the freeze-dried soluble dHAM at -198°C for freeze-grinding. In the machine, grind 3 times at 30 Hz for 30 s each time, and quickly transfer to a 5 mL centrifuge tube to obtain soluble dHAM powder that can pass through a 50-mesh sieve; (3) Using hexafluoroisopropanol (HFIP) as the electrospinning solvent to prepare a blended PLGA/PCL/dHAM composite electrospinning solution or a coaxial PLGA/PCL/dHAM composite electrospinning solution; (4) Connect the electrospinning device and turn on the power. The electrospinning solution obtained in step (3) is subjected to blending electrospinning or coaxial spinning under the conditions of an ambient humidity of 30%-50% and a temperature of 25-35°C Electrospinning to obtain a dHAM-containing composite electrospinning fiber membrane with micron-sized fibers arranged in an orderly manner. 2. application according to claim 1 is characterized in that, in step (3), the compound method of described blending PLGA/PCL/dHAM composite electrospinning solution is: HFIP is 10mL, and the PCL of 3g is added, Stir magnetically at 300–3000 rpm until dissolved, and after 12 h, a PCL electrospinning solution with a concentration of 30% was obtained. The same method was used to obtain a PLGA electrospinning solution with a concentration of 15% and a 5% dHAM electrospinning solution. PCL, PLGA, and dHAM are obtained by stirring and mixing at a ratio of 1:1:1. 3. application according to claim 1, is characterized in that, the preparation method of described coaxial PLGA/PCL/dHAM composite electrospinning solution is: HFIP is 10mL, the PCL of 3g is added, magnetic force at 300-3000rpm speed Stir until dissolved. After 12 hours, PCL electrospinning solution with a concentration of 30% was obtained as the "core" part of the electrospinning solution. The same method was used to obtain PLGA electrospinning solution with a concentration of 15% and 5% dHAM electrospinning solution. Spinning dope, electrospinning dope as part of the "shell". 4 . The application according to claim 1 , wherein in step (4), the electrospinning process parameters include: a voltage of 15-20 kV, a distance between the needle and the receiving rod of 18 cm, and a flow rate of 30 μL/min. 5 .

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