PVDF bionic periosteum doped with curcumin-loaded magnesium-based MOF and preparation method of PVDF bionic periosteum

Abstract

The invention belongs to the technical field of bone repair materials, and particularly discloses a PVDF bionic periosteum doped with curcumin-loaded magnesium-based MOF and a preparation method of the PVDF bionic periosteum. The PVDF bionic periosteum is prepared by taking an electrospinning 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). According to the preparation method, the piezoelectric PVDF film is constructed by utilizing an electrostatic spinning technology, and the curcumin-loaded magnesium-based MOF is doped, so that osteogenic differentiation can be promoted in three aspects of promoting revascularization, promoting neural differentiation and regulating inflammation, and a new solution is provided for repairing large-size bone defects.

Description

Technical Field

[0001] The invention belongs to the technical field of bone repair materials, and particularly relates to a PVDF bionic periosteum doped with magnesium-based MOF loaded with curcumin and a preparation method thereof. Background Art

[0002] Bone defects, especially large ones, remain a difficult problem in orthopedic clinical practice. Although traditional bone defect treatments such as autologous bone transplantation, allogeneic bone transplantation, and synthetic bone materials have been used, they each have their limitations. Autologous bone transplantation is prone to cause damage to the donor site, and donors are limited; allogeneic bone faces the risk of immune rejection and infection; synthetic bone materials still have much room for improvement in terms of osteoconductivity and osteoinductivity. These methods are often not ideal when faced with large or complex bone defects. In particular, the repair of nerve and vascular damage, as well as the control of local inflammatory responses, further increase the difficulty of bone regeneration.

[0003] The periosteum is an important component of the surface of bone tissue and has multiple functions, including promoting bone formation, regulating inflammation, and promoting blood vessel and nerve regeneration. Studies have shown that the role of the periosteum in bone defect repair is not only to provide physical support, but also that its rich osteoblasts, angiogenic factors, and nerve growth factors play a key role in promoting bone tissue reconstruction. The lack of functional periosteum often leads to delayed bone regeneration, insufficient blood supply, and poor recovery of nerve function. Therefore, reconstructing the function of the periosteum is of great significance for the effective repair of large-sized bone defects.

[0004] In recent years, periosteum tissue engineering has gradually become a research hotspot in the field of bone defect repair, aiming to reconstruct the function of the periosteum through bionic means. At present, a variety of materials have been tried to construct bionic periosteum, common materials such as collagen, chitosan, polylactic acid, etc. These materials simulate the physical properties of natural periosteum to a certain extent and promote the attachment and proliferation of osteoblasts. However, existing materials are still insufficient in promoting nerve and blood vessel regeneration and regulating local inflammation, and cannot effectively achieve the comprehensive repair effect of multifunctional periosteum.

[0005] 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 materials, electrospun bionic periosteum can generate electric charges under mechanical stimulation, simulate bioelectric signals in the human body, and promote nerve regeneration. In addition, electrospinning technology can design bionic periosteum with different functions by regulating fiber diameter, porosity and surface chemical properties. Compared with traditional materials, electrospun bionic periosteum performs well in multifunctional integration.

[0006] Although some existing bionic periosteums have shown certain effects in bone defect repair, there is still much room for improvement in promoting nerve and blood vessel regeneration. The functions of existing materials are still relatively single, and it is difficult to promote bone defect repair from multiple aspects. The electrospun piezoelectric bionic periosteum of the present invention overcomes these limitations by combining nanostructures with piezoelectric effects. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, the present invention provides a multifunctional bionic periosteum that can effectively generate bioelectric signals under mechanical stimulation, promote blood vessel and nerve regeneration, and has the ability to regulate inflammatory response, providing a new solution for the repair of large-size bone defects.

[0008] In order to achieve the above object, the specific technical solution adopted by the present invention is as follows:

[0009] In the first aspect, the present invention provides a PVDF biomimetic periosteum doped with a magnesium-based MOF loaded with curcumin, which is prepared by electrospinning using an electrospinning solution as a raw material; the electrospinning solution comprises the following components: 15-20% mass volume ratio of PVDF, 0.3-3% solid mass ratio of a magnesium-based metal organic framework (magnesium-based MOF) loaded with curcumin, and a 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 the magnesium-based MOF loaded with curcumin / the sum of the mass of the magnesium-based MOF loaded with curcumin and the mass of PVDF.

[0010] Furthermore, in the electrospinning solution, 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 and the best piezoelectric performance.

[0011] In a second aspect, the present invention provides a method for preparing the above-mentioned PVDF bionic periosteum doped with magnesium-based MOF loaded with curcumin, comprising the following steps:

[0012] (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;

[0013] (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;

[0014] (3) Weighing PVDF and adding it to the solution obtained in step (2), heating and stirring until the PVDF is dissolved;

[0015] (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 electrospinning is performed to obtain an electrospun bionic periosteum.

[0016] 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 a magnesium-based MOF.

[0017] Furthermore, the molar ratio of magnesium acetate to 2,5-dihydroxyterephthalic acid is 8:6, and the volume ratio of DMF, ethanol and water in the mixture is 15:1:1.

[0018] Furthermore, during the preparation of the magnesium-based MOF, the heating reaction conditions are: 60-80°C for 48 hours, and then 120-130°C for 2 hours; the centrifugal speed is 9000-12000 rpm.

[0019] Furthermore, in step (1), the stirring speed is 100-300 rpm, and the centrifugal speed is 9000-12000 rpm.

[0020] Furthermore, in step (3), the heating temperature is 40 to 50° C. and the heating time is 8 to 12 hours.

[0021] 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.

[0022] The present invention has the following beneficial effects:

[0023] 1. The bionic periosteum of the present invention is a PVDF film doped with 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.

[0024] 2. The bionic periosteum of the present invention utilizes electrospinning technology to construct a film with directional fiber arrangement, which helps the directional migration and extension of nerve cells and promotes nerve growth.

[0025] 3. The bionic periosteum of the present invention increases the relative fraction of the β phase in PVDF and its piezoelectric properties by optimizing the dosage of the magnesium-based MOF loaded with curcumin, thereby further promoting neural differentiation.

[0026] 4. The bionic periosteum of the present invention has good sustainability in the release of magnesium ions and curcumin, can exert its effects continuously and stably during bone repair, and has a long-lasting efficacy.

[0027] 5. The bionic periosteum of the present invention promotes vascular regeneration, neural differentiation, and inflammation as a whole. As a periosteum substitute, it regulates the microenvironment of bone defects, thereby promoting osteogenic differentiation and promoting the repair of large bone defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : SEM image of the magnesium-based MOF prepared in Example 1.

[0029] Figure 2 : Physical picture of the PVDF-MOF / Cur (PMC) membrane prepared in Example 1.

[0030] Figure 3 : SEM images and fiber diameter measurement results of the PMC membrane and pure PVDF membrane prepared in Example 1.

[0031] Figure 4 : Results of XRD test (left), infrared test (middle) and β-phase content calculation (right) of the films prepared with different amounts of magnesium-based MOF loaded with curcumin in Example 2.

[0032] Figure 5 : Voltage and current output results of the membranes prepared by 3% curcumin-loaded magnesium-based MOF (top) and magnesium-based MOF without curcumin loading (bottom) under 30N force in Example 2.

[0033] Figure 6 : The release rate test results of magnesium ions and curcumin from the PMC membrane in Example 2.

[0034] Figure 7 : PC12 cell experiment flow chart of Example 3.

[0035] Figure 8 : Immunofluorescence images and quantitative results of PC12 cells seeded on the periosteum surface in Example 3.

[0036] Fig. 9 : Cytoskeleton staining images of PC12 cells seeded on the periosteum surface and neurite length measurement in Example 3.

[0037] Fig.10 : Experimental results of PC12 cells co-cultured with periosteum supernatant to induce osteogenic differentiation in Example 3. In the figure, A is an immunofluorescence image; B is an ALP and ARS staining image; C is the rt-qPCR result; D is the immunofluorescence quantitative result; E is the ALP and ARS staining intensity quantitative result.

[0038] Fig.11 : Flow chart of the HUVEC cell experiment of Example 4.

[0039] Fig.12 : Pictures of the experimental results of HUVEC cells after co-culture with periosteum in Example 4. In the figure, A is a scratch test; B is a tube formation test; C is an immunofluorescence test.

[0040] Fig.13 : Quantitative results of HUVEC cell-related experiments after co-culture with periosteum in Example 4. A is a scratch test; B is angiogenesis test; C is a rt-qPCR test; D is an immunofluorescence test.

[0041] Fig.14 : Experimental results of HUVEC cells co-cultured with periosteum to induce osteogenic differentiation in Example 4. Fig.10 .

[0042] Fig.15 : Flow chart of the RAW264.7 cell experiment of Example 5.

[0043] Fig.16 : Results of the RAW264.7 cell polarization experiment after co-culture with periosteum in Example 5. In the figure, A is the expression of protein markers of M1 and M2 macrophages (the left side of the figure 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.

[0044] Fig.17 : Experimental results of the co-culture of RAW264.7 cells with periosteum to induce osteogenic differentiation in Example 5. Fig.10 .

[0045] Fig.18 : Animal experiment results of PMC periosteum in Example 6. DETAILED DESCRIPTION

[0046] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0047] Example 1: PMC periosteum preparation

[0048] In this embodiment, the PVDF biomimetic periosteum doped with magnesium-based MOF loaded with curcumin was prepared according to the following steps:

[0049] 1. Synthesis of magnesium-based MOF

[0050] 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 15:1:1); transferred to a reactor, reacted at 65°C for 48 hours, and then reacted at 125°C for 2 hours; after naturally cooling to room temperature, centrifuged at 9000 rpm for washing, and then dried to obtain magnesium-MOF-74, the scanning electron microscope image of which is shown in FIG. Figure 1 shown.

[0051] 2. Construction of magnesium-based MOF loaded with curcumin

[0052] 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.

[0053] 3. Preparation of electrospinning solution

[0054] 60 mg of magnesium-based MOF loaded with curcumin was weighed and put into 10 ml of a solution of DMF:acetone (volume ratio) = 3:2, and ultrasonicated for 30 minutes until uniformly dispersed; then 2 g of PVDF was weighed and added to the solution, and stirred overnight at 700 rpm and 50 °C to obtain a mixed solution in which the drug-loaded magnesium-based MOF accounted for 3% of the solid mass (drug-loaded magnesium-based MOF + PVDF), i.e., the electrospinning solution.

[0055] 4. Electrospinning

[0056] 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 electrospun bionic periosteum (PVDF-MOF / Cur, PMC). The physical picture and electron microscope picture of the electrospun membrane are shown in Figure 2-3 As shown, the electrospun fibers are uniform in thickness, with no significant difference in diameter from the pure PVDF membrane, and the fibers run in the same direction, which is beneficial to the growth and extension of nerve cells.

[0057] Example 2: PMC periosteum performance test

[0058] 1. Piezoelectric performance

[0059] In Example 1, the curcumin-loaded magnesium-based MOF adopts a ratio of 3%, which can increase the relative content of the β phase in the PVDF and improve the overall piezoelectric properties of the membrane. The following is the optimization process of the dosage:

[0060] PVDF-MOF / Cur periosteum was prepared according to the method of Example 1 with the dosage of 0%, 0.3%, 1%, 3%, 5% and 7% of magnesium-based MOF loaded with curcumin, and infrared and XRD tests were performed on the prepared groups of membranes. The results are as follows Figure 4 As shown, the corresponding peak of PVDF's β phase in the infrared spectrum increases, and the same is true for the XRD spectrum, the corresponding β phase increases, and the corresponding α phase decreases. The β phase content is calculated by the infrared image. The addition of 0.3% to 3% of curcumin-loaded magnesium-based MOF can increase the β phase content compared to pure PVDF, and the 3% group is the highest, indicating that the membrane prepared by 3% curcumin-loaded magnesium-based MOF has the best piezoelectric property (β phase and piezoelectric property are positively correlated), and has the best effect on improving neural differentiation. Figure 5 The voltage and current output of the membrane prepared by the magnesium-based MOF with 3% curcumin loading under a force of 30N are shown. Compared with the pure PVDF group, both the voltage and current are increased (indicating enhanced piezoelectric properties).

[0061] 2. Drug release rate

[0062] The PVDF-MOF / Cur membrane prepared in Example 1 was immersed in ddH 2 At different time points, the liquid was aspirated and supplemented with new ddHO. 2 O. Mg was determined by ICP and absorbance at 480 nm, respectively. 2+ The release rate and Cur release rate. Figure 6 As shown, the release of magnesium ions and curcumin is sustained, which can continuously promote angiogenesis and maintain an anti-inflammatory microenvironment during bone repair.

[0063] Example 3: PC12 cell experiment to evaluate PMC periosteum function

[0064] This example uses PC12 cell experiments (experimental process as shown in Figure 7 The results show that the PMC membrane prepared in Example 1 can promote nerve growth and differentiation, and that nerve differentiation can promote osteogenic differentiation / bone repair, as follows:

[0065] 1. PVDF, PM (PVDF-MOF), and PMC biomimetic periosteum were spread on 6-well plates, and PC12 was seeded on the biomimetic periosteum 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 membrane-free control group (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.

[0066] Immunofluorescence results Figure 8As 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. Fig. 9 As shown, 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.

[0067] 2. PC12 was planted on the periosteum of PMC and divided into two groups: ultrasound / no ultrasound. After three days, ultrasound was removed and fresh culture medium was replaced for another day to eliminate the interference of drug release in PMC. The supernatant of the culture medium was extracted and prepared as conditioned medium with α-MEM complete culture medium at a ratio of 1:1, and then osteogenic inducers were added, which were recorded as PMCP-CM and PC12-CM, respectively. BMSC (rat bone marrow mesenchymal stem cells) were co-cultured with HA@GelMA scaffolds, and the cells were cultured with PMCP-CM and PC12-CM, respectively. On the 7th day of osteogenic induction, rt-qPCR and OPN and Runx2 immunofluorescence were used to detect the expression of osteogenic-related genes, ALP staining was performed on the 7th and 14th days to detect the activity of osteoblasts, and ARS staining was performed on the 21st day to detect the mineralized nodules of osteoblasts.

[0068] The results are as follows Fig.10 As shown in Figure 2, the expression levels of bone-related genes Runx2, Col-1, OCN, ALP, and OPN in the PMCP-CM group were significantly higher than those in the PC12-CM group ( Fig.10 C); The protein expression levels of osteogenic genes Runx2 and OPN in the PMCP-CM group were significantly higher than those in the PC12-CM group ( Fig.10 A and D); Compared with PC12-CM, the ALP staining and ARS staining areas in PMCP-CM group ( Fig.10 B) and strength ( Fig.10 E) is larger, showing stronger osteoblast activity and matrix mineralization level. These results all indicate that the PMC membrane of the present invention can generate electrical stimulation under ultrasound to promote neural differentiation and then osteogenic differentiation, thereby helping bone repair.

[0069] Example 4: HUVEC cell experiment to evaluate PMC periosteum function

[0070] This example uses HUVEC cell experiments (experimental process as shown in Fig.11 The results show that the PMC membrane prepared in Example 1 can promote angiogenesis and promote osteogenic differentiation, as follows:

[0071] 1. HUVECs were seeded in 6-well plates at a density of 8w cells / well, cultured in DMEM medium, and co-cultured with PVDF, PM, and PMC biomimetic periosteum using transwell, and a membrane-free control group (Control) was set up. In terms of cell scratch experiments, 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. In terms of tube formation experiments, 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.

[0072] The results are as follows Figure 12-13 As shown, it can be concluded that PMC membrane can promote the migration of vascular endothelial cells ( Fig.12 A, 13A), pipe ( Fig.12 B, 13B), and the expression of related proteins CD31 and Hif-1α was upregulated ( Fig.12 C, 13D), CD31, Hif-1α, and Ang-1 were significantly upregulated at the molecular expression level ( Fig.13 C), indicating that PMC membrane can promote angiogenesis, and this effect is mainly achieved by releasing magnesium ions.

[0073] 2. HUVEC was co-cultured with / without PMC for 3 days, then PMC was removed and replaced with fresh medium for another day to eliminate the interference of drug release in PMC. The supernatant of the culture medium was extracted and prepared as conditioned medium with α-MEM complete medium at a ratio of 1:1, and then osteogenic inducers were added, which were recorded as PMCH-CM and HUVEC-CM, respectively. BMSCs were co-cultured with HA@GelMA scaffolds, and cells were cultured with PMCH-CM and HUVEC-CM, respectively. rt-qPCR and OPN and Runx2 immunofluorescence were performed on the 7th day of osteogenic induction, ALP staining was performed on the 7th and 14th days, and ARS staining was performed on the 21st day.

[0074] The results are as follows Fig.14 As shown in Figure 2, the expression levels of bone marrow genes Runx2, Col-1, OCN, ALP, and OPN in the PMCH-CM group were significantly higher than those in the HUVEC-CM group ( Fig.14 C); The protein expression levels of osteogenic genes Runx2 and OPN in PMCH-CM group were significantly higher than those in HUVEC-CM ( Fig.14A and D); Compared with the HUVEC-CM group, the ALP staining and ARS staining areas in the PMCH-CM group ( Fig.14 B) and strength ( Fig.14 E) is larger, showing stronger osteoblast activity and matrix mineralization level. These results all indicate that the PMC membrane of the present invention can promote osteogenic differentiation by promoting angiogenesis and thus help bone repair.

[0075] Example 5: RAW264.7 cell experiment to evaluate PMC periosteum function

[0076] This example uses RAW264.7 cell experiments (experimental process as shown in Fig.15 The results show that the PMC membrane prepared in Example 1 can regulate inflammation, and that regulating inflammation can promote osteogenic differentiation, as follows:

[0077] 1. 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 in DMEM complete medium. After the cells adhered, DMEM complete medium containing 2μg / ml LPS was replaced for 24 hours, and then transwell was used to co-culture with PVDF, PM, and PMC biomimetic periosteum 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.

[0078] The results are as follows Fig.16 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.16 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.16 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.

[0079] 2. After Raw264.7 adhered to the wall, it was cultured in DMEM complete medium containing 2μg / ml LPS for 24 hours, then replaced with fresh medium and co-cultured with PMC membrane for three days, then PMC was removed and replaced with fresh medium for another day to eliminate the interference of drug release in PMC. The supernatant of the culture medium was extracted and prepared as conditioned medium with α-MEM complete medium at a ratio of 1:1, and then osteogenic inducers were added, which were recorded as PMCR-CM and Raw264.7-CM, respectively. BMSCs were co-cultured with HA@GelMA scaffolds, and cells were cultured with PMCR-CM and Raw264.7-CM, respectively. rt-qPCR and OPN and Runx2 immunofluorescence were performed on the 7th day of osteogenic induction, ALP staining was performed on the 7th and 14th days, and ARS staining was performed on the 21st day.

[0080] The results are as follows Fig.17 As shown in Figure 2, the expression levels of bone marrow genes Runx2, Col-1, OCN, ALP, and OPN in the PMCR-CM group were significantly higher than those in the RAW264.7-CM group ( Fig.17 C); The protein expression levels of osteogenic genes Runx2 and OPN in the PMCR-CM group were significantly higher than those in the RAW264.7-CM group ( Fig.17 A and D); Compared with the RAW264.7-CM group, the ALP staining and ARS staining areas in the PMCR-CM group ( Fig.17 B) and strength ( Fig.17 E) is larger, showing stronger osteoblast activity and matrix mineralization level. These results all indicate that the PMC membrane of the present invention can promote osteogenic differentiation and help bone repair by regulating inflammation.

[0081] Example 6: Animal experiment to evaluate the periosteum function of PMC

[0082] 4-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, the 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:

[0083] Control (blank control): no membrane implantation;

[0084] HA@GelMA (positive control): hydrogel material known to have tissue repair ability;

[0085] PMCG: PMC+HA@GelMA composite membrane (HA@GelMA is compounded with PMC membrane by extrusion printing, and HA@GelMA is included during implantation);

[0086] US+PMCG: Ultrasound+(PMC+HA@GelMA composite film);

[0087] 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, and the bone repair effect was observed by skull CT.

[0088] The results are as follows Fig.18 As shown, compared with the HA@GelMA group, the US+PMCG and PMCG groups had the implantation of PMC membrane, and the wound healing effect was significantly better, which demonstrated that the PMC membrane of the present invention has a good bone defect repair effect, and the repair effect is better under ultrasonic stimulation and piezoelectricity.

[0089] In summary, the electrospun bionic periosteum prepared by the present invention promotes neural differentiation, angiogenesis, and regulates inflammation through electrical stimulation, magnesium ion release, and curcumin release, thereby reshaping the osteogenic microenvironment and promoting osteogenic differentiation, and can be used as a bone repair material.

[0090] 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. A PVDF biomimetic periosteum doped with magnesium-based MOF loaded with curcumin, characterized in that: The electrospinning liquid is used as a raw material and is prepared by electrostatic spinning; the electrospinning liquid comprises the following components: 15-20% by mass volume of PVDF, 0.3-3% by solid mass of 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 PVDF biomimetic periosteum doped with magnesium-based MOF loaded with curcumin according to claim 1, characterized in that: The solid mass ratio of the curcumin-loaded magnesium-based MOF is 3%.

3. The method for preparing the PVDF biomimetic periosteum doped with magnesium-based MOF loaded with curcumin according to claim 1, characterized in that: The following steps are involved: (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, and the electrospinning machine and the roller collector are turned on for electrospinning to obtain electrospun bionic periosteum.

4. The method for preparing the PVDF biomimetic periosteum doped with magnesium-based MOF loaded with curcumin according to claim 3, 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.

5. The method for preparing the PVDF biomimetic periosteum doped with magnesium-based MOF loaded with curcumin according to claim 4, characterized in that, The molar ratio of the magnesium acetate to 2,5-dihydroxyterephthalic acid is 8:6, and the volume ratio of DMF, ethanol and water in the mixture is 15:1:

1.

6. The method for preparing the PVDF biomimetic periosteum doped with magnesium-based MOF loaded with curcumin according to claim 4, characterized in that, During the preparation of magnesium-based MOF, the heating reaction conditions are: 60-80°C for 48 hours, then 120-130°C for 2 hours; the centrifugal speed is 9000-12000rpm.

7. The method for preparing the PVDF biomimetic periosteum doped with magnesium-based MOF loaded with curcumin according to claim 3, characterized in that, In step (1), the stirring speed is 100-300 rpm, and the centrifugal speed is 9000-12000 rpm.

8. The method for preparing the PVDF biomimetic periosteum doped with magnesium-based MOF loaded with curcumin according to claim 3, characterized in that, In step (3), the heating temperature is 40 to 50° C. and the heating time is 8 to 12 hours.

9. The method for preparing the PVDF biomimetic periosteum doped with magnesium-based MOF loaded with curcumin according to claim 3, 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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