Periosteum-bone double-layer stent as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bone repair, and particularly discloses a periosteum-bone double-layer stent as well as a preparation method and application thereof. The preparation method comprises the following steps: preparing a PVDF (Polyvinylidene Fluoride) electrostatic spinning membrane of curcumin-loaded magnesium-based MOF by adopting an electrostatic spinning technology, taking the PVDF electrostatic spinning membrane as a periosteum layer, and preparing hydroxyapatite-loaded hydrogel on the periosteum layer by adopting a bio-printer to serve as a bone layer, so as to form the periosteum-bone double-layer stent. In the constructed double-layer stent, the bone layer promotes osteogenic differentiation and local mineralization of stem cells by virtue of degradation and release of hydroxyapatite in the bone layer, and the bone defect repair speed is increased; the periosteum layer plays roles in promoting neural differentiation, promoting revascularization and regulating and controlling inflammation by virtue of electrical stimulation, magnesium ion release and curcumin release, an osteogenesis microenvironment is remodeled, and bone defect repair is further accelerated on the basis that the bone scaffold promotes the repair effect.

Description

Technical Field

[0001] The invention belongs to the technical field of bone repair, and specifically relates to a periosteum-bone double-layer scaffold and a preparation method and application thereof. Background Art

[0002] Bone defects, especially large ones, remain a difficult problem in orthopedic clinics. 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; and 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.

[0003] Periosteum is an important component of the surface of bone tissue, and has multiple functions such as promoting osteogenesis, regulating inflammation, and promoting blood vessel and nerve regeneration. Studies have shown that the role of 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. Lack of functional periosteum often leads to delayed bone regeneration, insufficient blood supply, and poor recovery of nerve function. Therefore, reconstructing the function of periosteum is of great significance for the effective repair of large-sized bone defects. 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 periosteum through bionic means. At present, a variety of materials have been tried to construct bionic periosteum, 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. Although some bionic periosteum have shown certain effects in bone defect repair, there is still a lot of room for improvement in promoting nerve and blood vessel regeneration, and the comprehensive repair effect of multifunctional periosteum cannot be effectively achieved.

[0004] In recent years, the implantation of tissue engineering scaffolds has been considered an effective strategy for treating osteochondral defects and plays an important role in the bone reconstruction process. Hydrogel is currently the most commonly used bone repair scaffold material. It has a three-dimensional network structure, can simulate the extracellular matrix (ECM), and provide a suitable microenvironment for cell proliferation and adhesion. It also has the advantages of good biocompatibility, can absorb a large amount of exudate from damaged tissues, provides a platform for sustained drug release, and has good hydrophilicity. However, it is far from enough to rely solely on the properties of hydrogels to promote bone repair. Adding active ingredients to hydrogels to promote the repair of bone defects is one of the current research strategies. However, the existing hydrogels loaded with active ingredients are not ideal for repairing bone defects due to various reasons such as low bioavailability of active ingredients, and cannot reconstruct the multiple functions of the periosteum.

[0005] In view of this, the present invention proposes a double-layer scaffold of multifunctional periosteum and hydrogel composite, which overcomes the shortcomings of existing hydrogel scaffolds and bionic periosteum and greatly improves the level of bone repair. Summary of the invention

[0006] The purpose of the present invention is to provide a periosteum-bone double-layer scaffold and its preparation method and application. The double-layer scaffold is constructed with hydrogel material as the inner layer and PVDF bionic periosteum as the outer layer. It has multiple functions of regulating inflammation and promoting blood vessel and nerve regeneration, and provides a new solution for the repair of large-sized bone defects.

[0007] In order to achieve the above object, the specific technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for preparing a periosteum-bone double-layer scaffold, which specifically comprises the following steps:

[0009] (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;

[0010] (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;

[0011] (3) weighing PVDF and adding it to the solution obtained in step (2), heating and stirring until the PVDF is dissolved to obtain an electrospinning solution;

[0012] (4) The electrospinning liquid 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 to obtain the electrospinning bionic periosteum;

[0013] (5) Weighing GelMA and placing it into the photoinitiator solution, heating and dissolving it to form a GelMA solution;

[0014] (6) Weigh hydroxyapatite and add it to the GelMA solution, mix well and filter;

[0015] (7) Printing the solution obtained in step (6) on the electrospinning membrane using a bio-extrusion printer, and irradiating the membrane with ultraviolet light while printing to cross-link the membrane, thereby obtaining a periosteum-bone double-layer scaffold.

[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 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] Further, in step (3), the electrospinning solution 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. More preferably, the solid mass ratio of the magnesium-based MOF loaded with curcumin is 3%.

[0021] Furthermore, in step (3), the heating temperature is 40 to 50° C. and the heating time is 8 to 12 hours.

[0022] 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.

[0023] Furthermore, in step (5), the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, and the mass volume ratio of GelMA in the photoinitiator solution is 8-12%; the heating temperature of the GelMA solution is 40-50° C., and the heating time is 20-40 minutes.

[0024] Furthermore, in step (6), the hydroxyapatite is added at 25-35% of the mass of GelMA.

[0025] Furthermore, in step (7), a 200-300 μm sterile print head is used for printing, the printer barrel temperature is controlled at 15-20° C., the receiving platform temperature is controlled at 8-12° C., and the extrusion pressure is controlled at 0.05-0.2 MPa.

[0026] In a second aspect, the present invention provides a periosteum-bone double-layer scaffold prepared according to the above preparation method. The double-layer scaffold includes a periosteum layer and a bone layer: the periosteum layer is a PVDF electrospun membrane doped with a magnesium-based MOF loaded with curcumin, denoted as PVDF-MOF / Cur, abbreviated as PMC; the bone layer is a GelMA hydrogel doped with hydroxyapatite, denoted as HA@GelMA.

[0027] In a third aspect, the present invention provides the use of the periosteum-bone double-layer scaffold prepared by the above preparation method as a bone defect repair material.

[0028] The present invention has the following beneficial effects:

[0029] 1. In the double-layer scaffold of the present invention, the periosteum layer is a PVDF film (PMC) 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. In this way, the multiple functions of the periosteum are reconstructed and the bone repair effect is significantly improved.

[0030] 2. The present invention adopts electrospinning technology to construct PVDF periosteum with directional fiber arrangement, which helps the directional migration and extension of nerve cells and promotes nerve growth; by optimizing the dosage 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.

[0031] 3. The release of magnesium ions and curcumin from the periosteum layer of the PVDF of the present invention is sustained, and can play a role continuously and stably during bone repair, with long-lasting efficacy.

[0032] 4. In the double-layer scaffold of the present invention, the bone layer is a GelMA hydrogel material doped with hydroxyapatite (HA@GelMA), which promotes the osteogenic differentiation and local mineralization of stem cells and accelerates the speed of bone defect repair through the degradation and release of hydroxyapatite.

[0033] 5. The double-layer stent of the present invention has no toxicity to internal organs and can ensure safety in use.

[0034] In summary, the double-layer scaffold constructed by the present invention, in which the HA@GelMA bone layer promotes the osteogenic differentiation and local mineralization of stem cells by virtue of the degradation and release of hydroxyapatite therein, and accelerates the speed of bone defect repair; the PMC periosteum layer promotes neural differentiation, promotes angiogenesis, and regulates inflammation by virtue of electrical stimulation, magnesium ion release, and curcumin release, reshapes the osteogenic microenvironment, and further accelerates the bone defect repair on the basis of the bone scaffold promoting the repair effect. Therefore, the double-layer scaffold of the present invention is a multifunctional high-quality bone defect repair material, which has obvious advantages compared to the single-layer HA@GelMA scaffold. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 : Comparison results of electron microscopy images and fiber diameter measurements of the periosteum (PVDF-MOF / Cur) and pure PVDF membrane prepared in Example 1.

[0036] Figure 2 : Actual pictures of the bone layer (HA@GelMA) and periosteum-bone double-layer scaffold (PMCG) prepared in Example 1.

[0037] Figure 3 : Results of XRD test (left), infrared test (middle) and β-phase content calculation (right) of periosteum prepared with different amounts of magnesium-based MOF loaded with curcumin in Example 2.

[0038] Figure 4 : The voltage and current output results of the periosteum (upper) and pure PVDF membrane (lower) prepared with 3% curcumin-loaded magnesium-based MOF in Example 2 under a force of 30N.

[0039] Figure 5 : The magnesium ion and curcumin release rate test results of the periosteum in Example 2.

[0040] Figure 6 : Immunofluorescence images and quantitative results of PC12 cells in Example 3.

[0041] Figure 7 : Cytoskeleton staining images of PC12 cells and neurite length measurement in Example 3.

[0042] Figure 8 : 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.

[0043] Fig. 9 : 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.

[0044] Fig.10 : Experimental results of RAW264.7 cells in Example 3. 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.

[0045] Fig.11 : Experimental flow chart for testing the bone defect repair effect of the double-layer scaffold in Example 4.

[0046] Fig.12: The skull CT images in Example 4 and the quantitative results of bone volume (BV / TV) and bone density (BMD) obtained from the CT images.

[0047] Fig.13 : Results of HE staining (upper) and Masson staining (lower) in Example 4.

[0048] Fig.14 : Result diagram of immunofluorescence detection of bone repair-related protein expression in Example 4.

[0049] Fig.15 : Quantitative results of immunofluorescence detection of bone repair-related protein expression in Example 4.

[0050] Fig.16 : HE staining results of the double-layer stent safety test in Example 5. DETAILED DESCRIPTION

[0051] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0052] Example 1: Preparation of periosteum-bone double-layer scaffold

[0053] This embodiment prepares the periosteum-bone double-layer scaffold according to the following steps:

[0054] 1. Synthesis of magnesium-based MOF

[0055] 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.

[0056] 2. Construction of magnesium-based MOF loaded with curcumin

[0057] 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.

[0058] 3. Preparation of electrospinning solution

[0059] Weigh 60 mg of magnesium-based MOF loaded with curcumin, put it into 10 ml of a solution 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 it at 700 rpm and 50 ° C overnight 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 + PVDF), that is, the electrospinning solution.

[0060] 4. Electrospinning

[0061] 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 to obtain electrospun bionic periosteum (PVDF-MOF / Cur, PMC). The electron microscope image and fiber diameter measurement data are shown in Figure 1 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.

[0062] 5. Prepare bone layer solution

[0063] Dissolve phenyl (2,4,6-trimethylbenzoyl) lithium phosphate with deionized water, and heat in a water bath in a light-proof environment to obtain a photoinitiator solution; weigh GelMA and put it into the photoinitiator solution to form a 10% mass volume ratio GelMA solution; heat in a water bath at 45°C for 30 minutes until dissolved, weigh 30% of the mass of GelMA hydroxyapatite and add it to the solution, blow evenly with a rubber-tipped dropper, and then use a 0.22μm sterile filter head to filter and sterilize.

[0064] 6. Printing bone layer

[0065] A bio-extrusion printer was used to print the above solution on the electrospinning membrane. A 300μm sterile print head was used for printing, and the temperature of the printer barrel was controlled at 18°C ​​and the receiving platform temperature was 10°C. The extrusion pressure was 0.12MPa, and printing was performed according to the designed path. UV light was irradiated while printing to make it cross-linked; the UV light irradiation conditions were: light source wavelength of 405nm, power of 3W, and time of 15s. After cross-linking, a periosteum-bone double-layer scaffold (PMCG) was obtained. The actual picture of the PMCG scaffold and the HA@GelMA bone layer is shown in the figure. Figure 2 shown.

[0066] Example 2: Double-layer scaffold periosteal layer performance test

[0067] 1. Piezoelectric performance

[0068] In Example 1, the curcumin-loaded magnesium-based MOF uses a 3% ratio, which can increase the relative content of the β phase in PVDF and improve the piezoelectric properties of the periosteum. The following is the optimization process of the dosage:

[0069] PVDF 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 3As shown, the corresponding peak of the β phase of PVDF in the infrared spectrum increases, and the same is true for the XRD spectrum. The corresponding peak of β increases and the corresponding peak of α decreases. The β phase content is calculated by the infrared image. The addition of 0.3% to 3% of the magnesium-based MOF loaded with curcumin can increase the β phase content compared with pure PVDF, and the 3% group is the highest, indicating that the periosteum prepared by 3% magnesium-based MOF loaded with curcumin has the best piezoelectric performance (the β phase is positively correlated with the piezoelectric performance).

[0070] Figure 4 The voltage and current output of the periosteum prepared by 3% curcumin-loaded magnesium-based MOF under 30N force are shown. Compared with the pure PVDF group, both the voltage and current are increased, indicating that the piezoelectric properties are enhanced, which can further promote neural differentiation and improve the bone defect repair effect.

[0071] 2. Drug release rate

[0072] The PVDF-MOF / Cur membrane prepared in step 4 of Example 1 was immersed in ddH2O, the liquid was aspirated at different time points, and new ddH2O was added. The Mg content was measured by ICP and absorbance at 480 nm. 2+ The release rate and Cur release rate. Figure 5 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.

[0073] Example 3: Double-layer scaffold periosteum cell experiment

[0074] 1. PC12 cell experiments prove that the periosteum promotes neural differentiation

[0075] 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.

[0076] Immunofluorescence results Figure 6 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 7As 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.

[0077] 2. HUVEC cell experiments prove that the periosteum promotes angiogenesis

[0078] 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 the cell scratch experiment, 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 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.

[0079] The results are as follows Figure 8-9 As shown, it can be concluded that the membrane prepared in Example 1 can promote the migration of vascular endothelial cells ( Figure 8 A, 9A), pipe ( Figure 8 B, 9B), and the expression of related proteins CD31 and Hif-1α was upregulated ( Figure 8 C, 9D), CD31, Hif-1α, and Ang-1 were significantly upregulated at the molecular expression level ( Fig. 9 C), indicating that PMC membrane can promote angiogenesis, and this effect is mainly achieved by releasing magnesium ions.

[0080] 3. RAW264.7 cell experiments prove that the periosteum can regulate inflammation

[0081] 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 and cultured for 24 hours. 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.

[0082] The results are as follows Fig.10 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.10 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.10 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.

[0083] In summary, this example confirms that the double-layer scaffold periosteum layer has multiple functions of promoting neural differentiation, promoting angiogenesis and regulating inflammation, and can reshape the osteogenic microenvironment, thereby promoting osteogenic differentiation and improving bone repair effects.

[0084] Example 4: Experiment on repairing bone defects with periosteum-bone double-layer scaffold

[0085] The periosteum-bone double-layer scaffold (PMCG) prepared in Example 1 was subjected to an animal experiment for bone defect repair. The experimental method and flow are as follows: Fig.11 As shown, the details are as follows: 4-week-old SD male rats weighing approximately 200-220g 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 5mm were made on the skull, and the periosteum-bone implants were implanted into the defects and the skin was sutured. Penicillin was injected intraperitoneally every day after surgery for three days. It was divided into 4 groups:

[0086] Control (blank control): no membrane implantation;

[0087] HA@GelMA (positive control);

[0088] PMCG: PMC+HA@GelMA double-layer scaffold;

[0089] US+PMCG: ultrasound+(PMC+HA@GelMA double-layer scaffold);

[0090] 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 tissue repair status was analyzed through skull CT, HE staining, Masson staining, immunofluorescence protein detection, and bone mass and bone density obtained by CT.

[0091] Skull CT and the bone mass and bone density results obtained from it are as follows Fig.12As shown in the figure, BV / TV proves that US+PMCG has the fastest bone growth rate, while the Control group is the slowest, and HA@GelMA is only faster than Control. There is a consistent trend in bone density. The new bone in the US+PMCG group has the highest bone density, and HA@GelMA is only higher than the Control group.

[0092] HE staining and Masson staining results are as follows Fig.13 As shown in the figure, HE and Masson staining results further confirmed the previous conclusions. 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.

[0093] Immunofluorescence protein detection results Figure 14-15 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, indicating that the PMCG and US+PMCG groups 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. 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 group had less fluorescence due to less new tissue and correspondingly less macrophages; analysis of the proportion of CD163 in total fluorescence also concluded that the US+PMCG and PMCG groups had the highest proportion of M2 macrophages. 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, indicating that the bionic periosteum in the periosteum-bone double-layer scaffold further promoted the bone repair of the overall defect site by regulating vascular regeneration, neural differentiation, and inflammation.

[0094] The above results show that after the PMCG double-layer scaffold of the present invention is combined with electrical stimulation treatment, the healing effect of the skull injury is the best and the bone defect repair effect is good.

[0095] Example 5: Safety test of periosteum-bone double-layer scaffold

[0096] At 8 weeks, the four experimental rats in the control, HA@GelMA, PMCG and US+PMCG groups in Example 4 were sacrificed, and then the key internal organs (heart, liver, spleen, lung and kidney) were taken for HE section staining. The results of HE staining of internal organs are shown in Figure 2. Fig.16As shown, it can be seen that the stent did not cause any damage to the kidney, lung, spleen, liver, and heart tissues, indicating that the stent has no toxicity to the internal organs and has good safety.

[0097] 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 method for preparing a periosteum-bone double-layer scaffold, 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 to obtain an electrospinning solution; (4) The electrospinning liquid 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 to obtain the electrospinning bionic periosteum; (5) Weighing GelMA and placing it into the photoinitiator solution, heating and dissolving it to form a GelMA solution; (6) Weigh hydroxyapatite and add it to the GelMA solution, mix well and filter; (7) Printing the solution obtained in step (6) on the electrospinning membrane using a bio-extrusion printer, and irradiating the membrane with ultraviolet light while printing to cross-link the membrane, thereby obtaining a periosteum-bone double-layer scaffold.

2. The method for preparing the periosteum-bone double-layer scaffold according to claim 1, 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.

3. The method for preparing the periosteum-bone double-layer scaffold according to claim 1, characterized in that: In step (3), the electrospinning solution 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).

4. The method for preparing the periosteum-bone double-layer scaffold according to claim 1, characterized in that: In step (3), the heating temperature is 40 to 50° C. and the heating time is 8 to 12 hours.

5. The method for preparing the periosteum-bone double-layer scaffold according to claim 1, 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.

6. The method for preparing the periosteum-bone double-layer scaffold according to claim 1, characterized in that: In step (5), the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, and the mass volume ratio of GelMA in the photoinitiator solution is 8-12%; the heating temperature of the GelMA solution is 40-50° C., and the heating time is 20-40 minutes.

7. The method for preparing the periosteum-bone double-layer scaffold according to claim 1, characterized in that: In step (6), the hydroxyapatite is added at 25-35% of the mass of GelMA.

8. The method for preparing the periosteum-bone double-layer scaffold according to claim 1, characterized in that: In step (7), a 200-300 μm sterile print head is used for printing, the printer barrel temperature is controlled at 15-20° C., the receiving platform temperature is controlled at 8-12° C., and the extrusion pressure is controlled at 0.05-0.2 MPa.

9. The periosteum-bone double-layer scaffold prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the periosteum-bone double-layer scaffold according to claim 9 as a bone defect repair material.

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