Application of electrostatic spinning membrane in preparation of tissue repair product

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses application of an electrostatic spinning membrane in preparation of a tissue repair product, and the tissue repair product is a product with at least one of the following effects: i) promoting neural differentiation, ii) promoting revascularization, and iii) regulating and controlling inflammatory response. The electrostatic spinning membrane is prepared by taking an electrostatic spinning liquid as a raw material through electrostatic spinning; the electrospinning liquid comprises the following components: PVDF with a mass-volume ratio of 15-20%, magnesium-based MOF loaded with curcumin with a solid mass ratio of 0.3-3%, and a solvent DMF / acetone solution; the volume ratio of DMF to acetone in the solvent is 3: (1.5-2.5). The PVDF electrostatic spinning membrane doped with the curcumin-loaded magnesium-based MOF is prepared by utilizing an electrostatic spinning technology, and the electrostatic spinning membrane is proved to have three effects of promoting neural differentiation, promoting vascular regeneration and regulating and controlling inflammatory response, and can be used as a multifunctional high-quality material to be applied to repair of tissues such as skin and bones.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application of an electrospinning membrane in the preparation of a tissue repair product. Background Art

[0002] Tissue repair involves complex biological processes, including neural differentiation, angiogenesis, and inflammation regulation. Tissue repair, neural differentiation, and angiogenesis are intertwined and promote each other. During neural regeneration, peripheral nerves release neurotrophic factors (such as NGF and BDNF) to promote cell proliferation and differentiation in the damaged area, thereby accelerating tissue repair. Angiogenesis is essential for tissue repair, especially in the process of wound healing and neural differentiation; new blood vessels not only provide oxygen and nutrients, but also help remove metabolic waste, reduce local oxidative stress and inflammatory response, and promote tissue repair and regeneration. M2 macrophages promote the migration and proliferation of surrounding cells, especially fibroblasts and endothelial cells, by secreting a variety of growth factors (such as VEGF, FGF, PDGF, etc.), helping to rebuild damaged tissue structures; they can also promote angiogenesis by secreting factors such as vascular endothelial growth factor (VEGF).

[0003] Using medical repair materials to promote the repair of skin, bone and other tissues by external application or implantation in the damaged area is a relatively common method. However, the current medical materials have relatively single functions and it is difficult to promote tissue repair from multiple aspects such as neural differentiation, vascular regeneration, and inflammation regulation. Therefore, the effect and speed of tissue repair need to be improved.

[0004] In recent years, electrospinning technology has been widely used in the field of tissue engineering because it can prepare materials with nanofiber structures, simulate natural extracellular matrix, and provide a good environment for cells to attach and proliferate. By introducing piezoelectric substances, electrospinning materials can generate electric charges under mechanical stimulation, simulate bioelectric signals in the human body, and promote nerve regeneration. In addition, electrospinning technology can design spinning membranes with different functions by regulating fiber diameter, porosity, surface chemical properties, and introducing drug release systems. Compared with traditional materials, electrospinning biomimetic membranes perform well in multifunctional integration. It can be seen that the use of electrospinning technology to prepare high-quality tissue repair materials has great potential, but related research is currently rare. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides an application of an electrospinning membrane in the preparation of a tissue repair product. The application of the electrospinning membrane to tissue repair can promote wound healing in many aspects and has a better repair effect than existing repair materials.

[0006] In order to achieve the above object, the specific technical solution adopted by the present invention is as follows:

[0007] The present invention provides an application of an electrospinning membrane in the preparation of a tissue repair product, wherein the tissue repair product is a product having at least one of the following functions: i) promoting neural differentiation, ii) promoting angiogenesis, and iii) regulating inflammatory response; the electrospinning membrane is prepared by electrospinning using an electrospinning liquid as a raw material; the electrospinning liquid comprises the following components: 15-20% mass volume ratio of PVDF, 0.3-3% solid mass ratio of magnesium-based MOF loaded with curcumin, and solvent DMF / acetone solution; the volume ratio of DMF and acetone in the solvent is 3: (1.5-2.5). Among them, the mass volume ratio refers to the mass of PVDF / solvent volume, and the solid mass ratio refers to the mass of magnesium-based MOF loaded with curcumin / the sum of the mass of magnesium-based MOF loaded with curcumin and PVDF.

[0008] Furthermore, the promotion of neural differentiation includes upregulating the expression of β-tubulin-III and GAP43 proteins, and promoting the directional extension of nerve cells; the promotion of angiogenesis includes promoting the migration and tube formation of vascular endothelial cells, and upregulating the expression of proteins CD31 and Hif-1α; the regulation of inflammatory response includes upregulating the expression of protein CD206 and anti-inflammatory factors IL-10 and IL-1ra, thereby regulating the transformation of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type.

[0009] Furthermore, the tissue repair includes but is not limited to skin, bone, and muscle tissue repair.

[0010] Furthermore, the product is in the form of a single-layer electrospun membrane structure or a composite membrane structure of an electrospun membrane and a hydrogel (such as GelMA, PVA, etc.).

[0011] Furthermore, in the electrospinning solution of the electrospinning membrane, the solid mass ratio of the magnesium-based MOF loaded with curcumin is 3%. The PVDF membrane prepared with the magnesium-based MOF loaded with curcumin in this amount has the highest β-phase content, the best piezoelectric performance, and is more conducive to neural differentiation.

[0012] Furthermore, the preparation method of the electrospinning membrane is as follows:

[0013] (1) Dissolving curcumin in methanol, adding magnesium-based MOF, stirring, and centrifuging to obtain a precipitate to obtain a magnesium-based MOF loaded with curcumin;

[0014] (2) weighing the curcumin-loaded magnesium-based MOF obtained in step (1), adding it to a DMF / acetone solution, and ultrasonically treating it until it is uniformly dispersed;

[0015] (3) Weighing PVDF and adding it to the solution obtained in step (2), heating and stirring until the PVDF is dissolved;

[0016] (4) The electrospinning liquid obtained in step (3) is loaded into a syringe, the tin foil is fixed on the roller collector, the positive and negative electrodes of the power supply are connected to the syringe needle and the roller respectively, the electrospinning machine and the roller collector are turned on, and an electrospinning membrane is obtained.

[0017] Furthermore, in step (1), the magnesium-based MOF is prepared by the following method: magnesium acetate and 2,5-dihydroxyterephthalic acid are weighed and dissolved in a mixture of dimethylformamide (DMF), ethanol and water, transferred to a reactor, heated for reaction, naturally cooled to room temperature, centrifuged, washed and dried to obtain magnesium MOF.

[0018] Furthermore, in step (3), the heating temperature is 40 to 50° C. and the heating time is 8 to 12 hours.

[0019] Furthermore, in step (4), the specification of the syringe needle is 19-21G, the distance between the needle and the roller collector is 15-18 cm, the voltage is 15-18 kV, the syringe injection speed is 1.5-2.0 ml / h, and the rotation speed of the roller collector is 3000 rpm.

[0020] The present invention has the following beneficial effects:

[0021] 1. The electrospun membrane of the present invention uses PVDF as a matrix and is doped with a magnesium-based MOF loaded with curcumin, which promotes angiogenesis through the release of magnesium ions; regulates inflammation through the release of magnesium ions and curcumin; and promotes neural differentiation through the release of magnesium ions and the electrical stimulation generated by PVDF under low-intensity pulsed ultrasound.

[0022] 2. The present invention uses electrospinning technology to construct a film with directional fiber arrangement, which helps the directional migration and extension of nerve cells and promotes nerve growth; by optimizing the amount of magnesium-based MOF loaded with curcumin, the relative fraction of β phase in PVDF is increased, the piezoelectric properties of the film are improved, and nerve differentiation is promoted.

[0023] 3. The present invention proves that the electrospun membrane has three functions: promoting nerve differentiation, promoting angiogenesis, and regulating inflammatory response. It can promote tissue repair from many aspects and can be used as a multifunctional high-quality material for skin, bone, muscle and other tissue repair. In addition, the electrospun membrane of the present invention can also be expanded to the treatment of diseases with any one or more symptoms of nerve damage, vascular damage, and inflammation, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : The β-phase content of the membranes prepared by using different amounts of magnesium-based MOF loaded with curcumin in Example 1.

[0025] Figure 2 : Actual picture of the electrospinning membrane prepared in Example 1.

[0026] Figure 3 : Electron microscopy images and fiber diameter measurement results of the electrospun membrane (containing 3% magnesium-based MOF loaded with curcumin) and pure PVDF membrane prepared in Example 1.

[0027] Figure 4 : The release rate test results of magnesium ions and curcumin from the electrospinning membrane prepared in Example 1.

[0028] Figure 5 : Immunofluorescence images and quantitative results of PC12 cells in Example 2.

[0029] Figure 6 : Cytoskeleton staining images of PC12 cells and neurite length measurement in Example 2.

[0030] Figure 7 : Pictures of HUVEC cell-related experimental results in Example 3. In the figure, A is a scratch test; B is angiogenesis test; C is an immunofluorescence test.

[0031] Figure 8 : Quantitative results of HUVEC cell-related experiments in Example 3. A is a scratch test; B is angiogenesis test; C is a rt-qPCR test; D is an immunofluorescence test.

[0032] Fig. 9 : Experimental results of RAW264.7 cells in Example 4. In the figure, A is the expression of protein markers of M1 and M2 macrophages (the left side is the immunofluorescence image, and the right side is the immunofluorescence quantitative analysis result); B is the expression of released factors of M1 and M2 macrophages.

[0033] Fig.10 : Results of HE staining (upper) and Masson staining (lower) in Example 5.

[0034] Fig.11 : Result diagram of immunofluorescence detection of bone tissue repair-related protein expression in Example 5.

[0035] Fig.12 : Quantitative results of the immunofluorescence detection of bone tissue repair-related protein expression in Example 5. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0037] Example 1: Preparation of PMC electrospinning membrane

[0038] 1. Synthesis of magnesium-based MOF

[0039] Magnesium acetate and 2,5-dihydroxyterephthalic acid were weighed in a molar ratio of 8:6 and dissolved in a mixture of DMF, ethanol and water (volume ratio of 15:1:1); the mixture was transferred to a reactor and reacted at 65°C for 48 hours and then at 125°C for 2 hours; after naturally cooling to room temperature, the mixture was centrifuged and washed at 9000 rpm and then dried to obtain magnesium-MOF-74.

[0040] 2. Construction of magnesium-based MOF loaded with curcumin

[0041] 100 mg of curcumin was weighed and put into 20 ml of methanol solution, and ultrasonicated until clear; 200 mg of synthesized magnesium-MOF-74 was added and stirred at 200 rpm for 24 hours; then centrifuged at 9000 rpm, the supernatant was discarded, and dried to obtain magnesium-based MOF loaded with curcumin.

[0042] 3. Preparation of electrospinning solution

[0043] Weigh 60 mg of magnesium-based MOF loaded with curcumin, put it into 10 ml of DMF: acetone (volume ratio) = 3:2, and ultrasonicate for 30 minutes until it is evenly dispersed; then weigh 2 g of PVDF and add it to the solution, stir overnight at 700 rpm and 50 ° C to obtain a mixed solution in which the drug-loaded magnesium-based MOF accounts for 3% of the solid mass (drug-loaded magnesium-based MOF + VDF), that is, the electrospinning solution. In this step, 3% of the magnesium-based MOF loaded with curcumin is the best amount. Figure 1 The β-phase content of PVDF membranes prepared with different dosages of magnesium-based MOF loaded with curcumin (calculated by infrared spectra) was the highest in the 3% group at concentrations of 0%, 0.3%, 1%, 3%, 5%, and 7%, indicating the best piezoelectric performance (β-phase and piezoelectric performance are positively correlated) and the best effect on enhancing neural differentiation.

[0044] 4. Electrospinning

[0045] The electrospinning solution was sucked into a 10 ml syringe, a 20G needle was used, the distance from the syringe to the receiver was 18 cm, the voltage was 18 kV, the injection speed was 1.8 ml / h, the rotating drum receiver rotation speed was 3000 rpm, aluminum foil was laid, and then electrospinning was performed, and the obtained film was collected, which was the PVDF-MOF / Cur electrospinning membrane (PMC membrane). The actual picture is as follows Figure 2 As shown, the electron microscope image and fiber diameter measurement data are shown in Figure 3 As shown in the figure, the electrospun fibers are uniform in thickness, with no significant difference in diameter from the pure PVDF membrane, and the fibers are oriented in the same direction, which is beneficial to the growth and extension of nerve cells. The drug release rate of the membrane was tested, and the results are as follows: Figure 4 As shown, the release of magnesium ions and curcumin is sustained, and they can continue to play a role during bone repair, with long-lasting efficacy.

[0046] Example 2: Experiment on promoting neural differentiation by PMC electrospinning membrane

[0047] PVDF, PM (PVDF-MOF), and PMC membranes were spread on 6-well plates, and PC12 was seeded on the membranes at a density of 8w cells / well. One PMC group was ultrasonicated for 15 minutes every day to generate electrical stimulation, recorded as US+PMC, and a control group without membrane (Control) was set up. RNA was extracted by Trizol method on the 3rd day, and reverse transcription and rt-qPCR were used to evaluate the expression of genes related to neural differentiation. Skeleton staining and immunofluorescence staining of β-tubulin III and GAP43 were performed on the 3rd day.

[0048] Immunofluorescence results Figure 5 As shown in Figure 2, it can be concluded that the US+PMC group promotes the expression of functional proteins in nerve cells such as β-tubulin-III and GAP43, and promotes neural differentiation. Figure 6 As shown, under ultrasound stimulation, the directionally arranged fibers of the PMC membrane can make nerve cells extend in a directional manner and promote nerve growth, and the electrical stimulation generated by ultrasound increases the length of neurites, providing the necessary structural basis for nerve repair.

[0049] Example 3: Experiment on promoting angiogenesis by PMC electrospinning membrane

[0050] 1. HUVECs were seeded in 6-well plates at a density of 8w cells / well, cultured with DMEM medium, and co-cultured with PVDF, PM, and PMC membranes using transwell, and a membrane-free control group (Control) was set up. For the cell scratch test, when the cells were full in the 6-well plate, a straight line was drawn with a 200μl gun tip, and then the scratched cells were washed off with PBS, and then cultured with serum-free medium, and photographed under an inverted microscope at 0, 6, 12, and 24 hours, and cell migration was analyzed. For the tube formation experiment, cells were seeded in 96 wells covered with matrix gel at a density of 5000 cells / well, and photographed at a certain time between 4 and 8 hours to analyze their tube formation. On the third day of cell culture, RNA was extracted by Trizol method, reverse transcribed, and the expression of angiogenesis-related genes was evaluated using rt-qPCR. Immunofluorescence staining of CD31 and HIF-1a was performed on the third day of cell culture.

[0051] The results are as follows Figure 7-8 As shown, it can be concluded that PMC membrane can promote the migration of vascular endothelial cells ( Figure 7 A, 8A), pipe ( Figure 7 B, 8B), and the expression of related proteins CD31, Hif-1α, and Ang-1 was upregulated ( Figure 7C, 8D), CD31, Hif-1α, and Ang-1 were significantly upregulated at the molecular expression level ( Figure 8 C), indicating that PMC membrane can promote angiogenesis, and this effect is mainly achieved by releasing magnesium ions.

[0052] Example 4: PMC electrospinning membrane regulation of inflammation experiment

[0053] Raw264.7 cells were used to simulate macrophages in vitro. The cells were seeded in 6-well plates at a density of 8w / well and cultured with DMEM complete medium. After the cells adhered, DMEM complete medium containing 2μg / ml LPS was replaced and cultured for 24 hours. Then, transwell was used to co-culture with PVDF, PM, and PMC membranes for three days, and a membrane-free control group (Control) was set up. On the third day, RNA was extracted using Trizol method, and reverse transcription and rt-qPCR were used to evaluate the expression of pro-inflammatory and anti-inflammatory related factor genes. Immunofluorescence staining of CCR7 and CD206 was performed on the third day.

[0054] The results are as follows Fig. 9 As shown, the expression of CCR7, a protein marker of M1 macrophages, was downregulated, while the expression of CD206, a protein marker of M2 macrophages, was upregulated ( Fig. 9 A); The expression of pro-inflammatory factors IL-1β, iNOS, and IL-6 released by M1 macrophages was downregulated, while the expression of anti-inflammatory factors IL-10 and IL-1ra released by M2 macrophages was upregulated ( Fig. 9 B). These results indicate that PMC membrane can inhibit macrophage M1 polarization and promote M2 polarization, regulating the transition of macrophages from pro-inflammatory M1 to anti-inflammatory M2 through the release of magnesium ions and curcumin.

[0055] Example 5: Tissue repair experiment of PMC electrospinning membrane

[0056] Examples 2-4 confirm that the electrospun membrane of the present invention has three functions: promoting neural differentiation, promoting angiogenesis, and regulating inflammatory response, reflecting the multi-faceted promotion of tissue repair and effectively improving the tissue repair effect. This example further lists the electrospun membrane repair experiment on bone damage, intuitively showing the bone tissue repair effect of the electrospun membrane, as follows:

[0057] Four-week-old SD male rats weighing approximately 200-220 g were used. The experimental rats were anesthetized by inhalation of isoflurane gas. After complete shaving and disinfection, a longitudinal incision was made in the center of the surgical site, and the soft tissue was carefully separated to expose the skull. After removing the periosteum, two bilateral defects with a diameter of 5 mm were made on the skull, and the electrospun membrane was implanted into the defect and the skin was sutured. Penicillin was injected intraperitoneally every day after surgery for three days. There were 4 groups in total:

[0058] Control (blank control): no membrane implantation;

[0059] HA@GelMA (positive control): hydrogel material known to have tissue repair ability;

[0060] PMCG: PMC+HA@GelMA composite membrane (HA@GelMA is compounded with PMC membrane by extrusion printing, and HA@GelMA is included during implantation);

[0061] US+PMCG: Ultrasound+(PMC+HA@GelMA composite film);

[0062] Among them, the US+PMCG group used ultrasound to stimulate the defect for 15 minutes every day starting from the third day after surgery until samples were taken at 4 and 8 weeks. The bone tissue repair was analyzed by histological staining (HE staining, Masson staining) and immunofluorescence protein detection.

[0063] HE staining and Masson staining results are as follows Fig.10 As shown in the figure, the new bone formation in the US+PMCG group was the most significant, followed by PMCG, HA@GelMA and Control. At 8 weeks, the US+PMCG was almost completely filled with new bone tissue, while the control group was still in the early stage of osteogenesis and was covered by thinner fibrous tissue.

[0064] Immunofluorescence protein detection results Figure 11-12 As shown in the figure, the immunofluorescence staining of the smooth muscle cell marker α-SMA showed the highest number of blood vessels and fluorescence intensity in the PMCG and US+PMCG groups, which was significantly higher than that in the HA@GelMA group, indicating that the PMC membrane effectively promoted the formation of new blood vessels. CGPR is a key protein secreted by nerve cells to promote osteogenic differentiation. The US+PMCG group had the highest CGRP fluorescence intensity, followed by PMCG, while the Control and HA@GelMA groups had less, indicating that the PMC membrane effectively promoted neural differentiation. CD68 is a membrane protein co-expressed by macrophages, while CD163 is only expressed on M2 macrophages. In CD68 / CD163 staining, both the US+PMCG and PMCG groups had more CD163 expression, while the Control and HA@GelMA groups had fewer corresponding macrophages and only less fluorescence; the analysis of the proportion of CD163 in the total fluorescence also concluded that the US+PMCG and PMCG groups had the highest proportion of M2 macrophages, indicating that the PMC membrane effectively promoted inflammatory regulation. In the fluorescence staining of the osteogenesis-related protein OPN, the fluorescence intensity of the PMCG and US+PMCG groups were higher than that of the HA@GelMA group.

[0065] The above results show that the US+PMCG and PMCG groups with additional PMC membrane implantation have obvious advantages over the HA@GelMA group, reflecting that the electrospun membrane of the present invention has good effects in promoting angiogenesis, promoting neural differentiation, regulating inflammation, and has a good effect on bone tissue repair, and the repair effect is better under ultrasonic stimulation and piezoelectricity.

[0066] This specific implementation is only an explanation of the present invention, not a limitation of the present invention. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. An application of an electrospinning membrane in the preparation of a tissue repair product, characterized in that: The tissue repair product is a product having at least one of the following effects: i) promoting nerve differentiation, ii) promoting angiogenesis, iii) regulating inflammatory response; The electrospinning membrane is prepared by electrospinning using an electrospinning solution as a raw material; the electrospinning solution comprises the following components: 15-20% by mass volume of PVDF, 0.3-3% by solid mass of a magnesium-based MOF loaded with curcumin, and a solvent DMF / acetone solution; the volume ratio of DMF to acetone in the solvent is 3:(1.5-2.5).

2. The use of the electrospinning membrane according to claim 1 in preparing tissue repair products, characterized in that: The promotion of neural differentiation includes upregulating the expression of β-tubulin-III and GAP43 proteins, and promoting the directional extension of nerve cells; the promotion of angiogenesis includes promoting the migration and tube formation of vascular endothelial cells, and upregulating the expression of proteins CD31 and Hif-1α; the regulation of inflammatory response includes upregulating the expression of protein CD206 and anti-inflammatory factors IL-10 and IL-1ra, thereby regulating the transformation of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type.

3. The use of the electrospinning membrane according to claim 1 in preparing tissue repair products, characterized in that: The tissue repair includes skin, bone and muscle tissue repair.

4. The use of the electrospinning membrane according to claim 1 in preparing tissue repair products, characterized in that: The product is in the form of a single-layer structure of an electrospun membrane or a multi-layer structure of an electrospun membrane and a hydrogel.

5. The use of the electrospinning membrane according to claim 1 in preparing tissue repair products, characterized in that: In the electrospinning solution of the electrospinning membrane, the solid mass ratio of the magnesium-based MOF carrying curcumin is 3%.

6. Use of the electrospinning membrane according to claim 1 or 5 in preparing tissue repair products, characterized in that: The preparation method of the electrospinning membrane is as follows: (1) Dissolving curcumin in methanol, adding magnesium-based MOF, stirring, and centrifuging to obtain a precipitate to obtain a magnesium-based MOF loaded with curcumin; (2) weighing the curcumin-loaded magnesium-based MOF obtained in step (1), adding it to a DMF / acetone solution, and ultrasonically treating it until it is uniformly dispersed; (3) Weighing PVDF and adding it to the solution obtained in step (2), heating and stirring until the PVDF is dissolved; (4) The electrospinning liquid obtained in step (3) is loaded into a syringe, the tin foil is fixed on the roller collector, the positive and negative electrodes of the power supply are connected to the syringe needle and the roller respectively, the electrospinning machine and the roller collector are turned on, and an electrospinning membrane is obtained.

7. Use of the electrospinning membrane according to claim 6 in preparing tissue repair products, characterized in that: In step (1), the magnesium-based MOF is prepared by the following method: magnesium acetate and 2,5-dihydroxyterephthalic acid are weighed and dissolved in a mixture of DMF, ethanol and water, transferred to a reactor, heated for reaction, naturally cooled to room temperature, centrifuged, washed and dried to obtain the magnesium-based MOF.

8. The use of the electrospinning membrane according to claim 6 in preparing tissue repair products, characterized in that: In step (3), the heating temperature is 40 to 50° C. and the heating time is 8 to 12 hours.

9. Use of the electrospinning membrane according to claim 6 in preparing tissue repair products, characterized in that: In step (4), the specification of the syringe needle is 19-21G, the distance between the needle and the roller collector is 15-18 cm, the voltage is 15-18 kV, the syringe injection speed is 1.5-2.0 ml / h, and the rotation speed of the roller collector is 3000 rpm.

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