CN117797313A - Hydrogel scaffold material capable of continuously releasing hydrogen and magnesium ions as well as preparation method and application thereof - Google Patents
Hydrogel scaffold material capable of continuously releasing hydrogen and magnesium ions as well as preparation method and application thereof Download PDFInfo
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- CN117797313A CN117797313A CN202311848133.XA CN202311848133A CN117797313A CN 117797313 A CN117797313 A CN 117797313A CN 202311848133 A CN202311848133 A CN 202311848133A CN 117797313 A CN117797313 A CN 117797313A
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Abstract
The invention belongs to the technical field of bone defect repair materials, and particularly relates to a hydrogel scaffold material capable of continuously releasing hydrogen and magnesium ions, a preparation method and application thereof. The preparation method of the hydrogel stent material provided by the invention adopts the method based on the synthesis of magnesium hydride microspheres and methacryloyl gelatin coated by polylactic-co-glycolic acid (PLGA)The novel hydrogel stent material is obtained by combining the gas foaming technology and the photo-curing technology. Experiments prove that the stent material prepared by the invention, mgH coated by PLGA 2 The microsphere can reduce ROS production, promote macrophage transformation from M1 type polarization to M2 type polarization, enhance cell migration, improve local inflammation and oxidative stress, and promote angiogenesis and bone regeneration of diabetic bone defect. Therefore, the bracket material can provide a new treatment strategy and research direction for the diabetic bone defect, and has wide application prospect in the treatment of the diabetic bone defect patients.
Description
Technical Field
The invention belongs to the technical field of bone defect repair materials, and particularly relates to a hydrogel scaffold material capable of continuously releasing hydrogen and magnesium ions, a preparation method and application thereof.
Background
Diabetes is a chronic metabolic disease that severely threatens public health. In diabetics, the treatment of bone defects caused by trauma, tumors, infections or other causes is a significant clinical challenge. The diabetic environment is characterized by hyperglycemia, which not only disrupts the inflammatory response, but also promotes the production of Reactive Oxygen Species (ROS) and microvascular lesions, together with the prolongation of the healing process of bone defects, as compared to non-diabetic individuals. These conditions lead to a high incidence of bone nonunion in diabetics. Although there are several clinical interventions for treating bone defects, the unique complexity of the diabetic environment significantly limits its efficacy. Therefore, development of biological materials capable of regulating inflammatory environments, relieving oxidative stress and promoting angiogenesis, thereby enhancing regeneration and repair of bone defects in diabetic environments, is a technical problem that needs to be solved urgently in the medical field.
At present, clinical synthetic materials for bone defect reconstruction, such as bone cement, specific bioceramics and specific metals and their metals, have shown limitations in terms of vascularization, cell migration, degradation kinetics and biocompatibility. Hydrogels are emerging materials that mimic the natural extracellular matrix (ECM) and are very promising scaffold materials due to their good injectability, ductility, and compatibility with a variety of bioactive components.
Hydrogen (H) 2 ) As an antioxidant, active oxygen is selectively reduced without disrupting the physiological function of the active oxygen or other biomolecules, and is further converted to water. In vitro studies show that hydrogen can inhibit rankl-mediated NF- κB pathway activation and inhibit osteoclast differentiation, which is a promising approach to solve bone resorption. However, the low solubility and rapid dissipation of hydrogen present challenges to the sustained therapeutic concentrations for topical treatment, particularly of diabetic bone defects. Meanwhile, magnesium ions have been used to enhance bone stability and promote an anti-inflammatory environment. However, magnesium hydroxide produced during the application of magnesium materials can hinder the release of therapeutic magnesium ions under physiological conditions, further limiting the effectiveness of their application.
Therefore, the design of a biological material capable of regulating inflammatory response, scavenging ROS, promoting angiogenesis and providing mechanical support has important significance for promoting the treatment of bone defects of diabetics.
Disclosure of Invention
In order to solve the problems, one of the purposes of the invention is to provide a preparation method of a hydrogel scaffold material capable of continuously releasing hydrogen and magnesium ions, the scaffold material prepared by the method can reduce ROS, promote polarization of M2 macrophages, improve cell migration and reduce local inflammation, thereby promoting angiogenesis and bone regeneration, and a new feasible strategy can be provided for treatment of diabetic bone defects.
The second purpose of the invention is to provide the hydrogel stent material which is prepared by the preparation method and can release hydrogen and magnesium ions continuously.
It is a further object of the present invention to provide the use of the above-described hydrogel scaffold material which is sustained in release of hydrogen and magnesium ions.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method for preparing a hydrogel scaffold material capable of continuously releasing hydrogen and magnesium ions, which comprises the following steps:
(1) Dissolving polylactic acid-glycolic acid copolymer in solvent, adding magnesium hydride and mixing to obtain PLGA+MgH 2 Mixing the solutions; PLGA+MgH 2 Adding the mixed solution into polyethylene glycol solution, stirring, mixing, volatilizing solvent, centrifuging, collecting precipitate, and drying to obtain magnesium hydride microsphere coated with polylactic acid-glycolic acid copolymer, and recording as MgH 2 PLGA microspheres;
adding gelatin into water for swelling, adding methacrylic anhydride for mixing, dialyzing, centrifuging, and freeze drying to obtain methacryloyl gelatin; dissolving the methacryloyl gelatin to obtain a methacryloyl gelatin solution, and marking the methacryloyl gelatin solution as a GelMA solution;
(2) MgH obtained in the step (1) 2 Mixing the @ PLGA microspheres and the GelMA solution with air to foam to obtain a foam material, injecting the foam material into a mold, and performing crosslinking curing under ultraviolet conditions to obtain the hydrogel stent material capable of continuously releasing hydrogen and magnesium ions, which is marked as MgH 2 @PLGA/F-GM。
As a further preferable scheme, in the step (1), the solvent is one or more of 1, 4-dioxane, dimethyl sulfoxide, chloroform and dichloromethane. More preferably, the solvent is 1, 4-dioxane.
As a further preferable scheme, the dosage ratio of the polylactic acid-glycolic acid copolymer, the solvent and the magnesium hydride is (8-12) mg to (0.8-1.2) mL to (8-12) mg. More preferably, the dosage ratio of the polylactic acid-glycolic acid copolymer, the solvent and the magnesium hydride is 10mg to 1mL to 10mg.
As a further preferable scheme, in the step (1), the polyethylene glycol solution is dimethyl sulfoxide solution of polyethylene glycol; the concentration of polyethylene glycol in the polyethylene glycol solution is 2-10 mg/mL. Further preferably, the concentration of polyethylene glycol in the polyethylene glycol solution is 2mg/mL.
As a further preferable scheme, the mass ratio of polyethylene glycol, polylactic acid-glycolic acid copolymer and magnesium hydride is 1-3:1-5:1-5. It is further preferred that the mass ratio of polyethylene glycol, polylactic acid-glycolic acid copolymer, and magnesium hydride is 3:5:5.
As a further preferable scheme, in the step (1), the dosage ratio of the gelatin to the methacrylic anhydride is 8-12 g:5-7 mL. Further preferably, the ratio of gelatin to methacrylic anhydride is 10 g:6 mL.
As a further preferable mode, in the step (1), the dialysis is performed by using a dialysis bag of 8-14 kDa.
As a further preferred embodiment, in the step (1), the dissolution is to dissolve the methacryloyl gelatin in a solution of lithium phenyl (2, 4, 6-trimethylbenzoyl) phosphonate to obtain a solution of methacryloyl gelatin.
In a further preferable scheme, in the step (2), the foaming is performed by adopting a tee joint connected with at least two syringes, and the GelMA solution and MgH are realized by the reciprocating pushing of different syringes 2 And (3) mixing the PLGA microspheres to uniformly distribute air in the mixed hydrogel to form the foaming material.
In a further preferable embodiment, in the step (2), the wavelength of ultraviolet rays used for the crosslinking and curing is 400 to 410nm.
The hydrogel stent material capable of continuously releasing hydrogen and magnesium ions is prepared by the preparation method.
As a further preferred embodiment, the hydrogel scaffold material is an injectable scaffold.
Use of a hydrogel scaffold material that releases hydrogen and magnesium ions continuously as described above for the preparation of a scaffold material for repairing bone defects.
As a further preferred embodiment, the bone defect is a diabetic bone defect.
The technical scheme of the invention has the special advantages that:
currently, diabetes-related bone defects present a significant challenge to healing, and hyperglycemia-induced inflammation and oxidative stress exacerbate this challenge. Current treatments are inadequate to cope with this complex environment, and materials that regulate inflammation and promote angiogenesis are therefore highly desirable.
The preparation method of the hydrogel stent material capable of continuously releasing hydrogen and magnesium ions comprises the steps of firstly carrying out magnesium hydride microsphere (MgH) wrapped by polylactic acid-glycolic acid copolymer 2 PLGA), preparation of a methacryloyl gelatin (GelMA) solution, followed by MgH 2 Based on synthesis of PLGA microspheres and GelMA, a novel hydrogel scaffold material (MgH) is obtained by adopting a method combining gas foaming and photo-curing technologies 2 @ PLGA/F-GM). The preparation method is simple in process and easy to realize, and the prepared stent material consists of magnesium hydride microspheres wrapped by foamed methacryloyl gelatin mixed polylactic acid-glycolic acid copolymer, and has the characteristic of injectability.
In addition, the scaffold material prepared by the method can provide a certain mechanical support for the bone defect part, also can conform to the complex bone defect shape, forms a bone tissue engineering regeneration scaffold in situ, and adjusts the diabetes microenvironment. MgH when the scaffold degrades 2 Magnesium hydroxide (Mg (OH) 2 ) Simultaneously degrading PLGA to release lactic acid and glycolic acid. While the released acidic by-products promote Mg (OH) 2 Dissolution of the layer, thereby enhancing the therapeutic magnesium ion (Mg 2+ ) Is released. While Mg is 2+ Is a key for enhancing macrophage phenotype transformation, reducing inflammation and promoting vascularization, and is important for healing of diabetic bone defect. In one aspect, mg 2+ Can induce macrophage phenotype transition, relieve pro-inflammatory microenvironment, and regulate local inflammatory reaction. On the other hand, mg 2+ Helps to reduce microvascular complications associated with hyperglycemia, promotes angiogenesis and accelerates angiogenesis to support ossification. In addition, mgH in the stent material of the invention 2 Degradation of released hydrogen (H) 2 ) Can be achieved by reducing the activityThe generation of sexual oxygen (ROS) can thus counteract oxidative stress.
In combination, the scaffold material provided by the invention has the effects of reducing ROS, promoting polarization of M2 type macrophages, improving cell migration, relieving local inflammation, and promoting angiogenesis and bone regeneration. In particular, the invention adopts a plurality of groups of in-vitro and in-vivo experimental researches to prove that the biological stent material provided by the invention has MgH coated by PLGA in the bubble photo-curing hydrogel stent 2 The microspheres can reduce ROS production, promote macrophage transformation from M1 type polarization to M2 type polarization, enhance cell migration, and improve local inflammation and oxidative stress. These effects promote angiogenesis and bone regeneration in diabetic bone defects. Therefore, the stent material provided by the invention can provide a new treatment strategy and research direction for the diabetic bone defect, and has wide application prospect in the recovery treatment of the diabetic bone defect patients.
Drawings
FIG. 1 shows the magnesium hydride microsphere (MgH) coated by the polylactic acid-glycolic acid copolymer in the step (1) of the invention 2 Schematic of the preparation route of @ PLGA);
FIG. 2 shows the step (3) of the hydrogel scaffold material (MgH) with sustained release of hydrogen and magnesium ions according to the present invention 2 Schematic of the preparation route of @ PLGA/F-GM);
FIG. 3 shows MgH obtained in the step (1) of the present invention 2 PLGA microsphere (right panel) and MgH before encapsulation 2 SEM image of microspheres (left panel);
FIG. 4 shows MgH obtained in the step (1) of the present invention 2 @PLGA microspheres and MgH before encapsulation 2 XRD image of the microsphere;
FIG. 5 shows MgH according to example 1 of the present invention 2 PLGA/F-GM material, F-GM scaffold material of comparative example 1, mgH of comparative example 2 2 Appearance and SEM spectrogram of the/F-GM scaffold material;
FIG. 6 shows MgH according to example 1 of the present invention 2 PLGA/F-GM material, F-GM scaffold material of comparative example 1, mgH of comparative example 2 2 Mechanical property test result of the F-GM bracket material;
FIG. 7 shows MgH according to example 1 of the present invention 2 @PLGA/F-degradation rate of GM material in pure water, PBS, modified simulated body fluid (M-SBF);
FIG. 8 shows MgH prepared according to the present invention 2 PLGA/F-GM material and MgH of comparative example 2 2 In vitro release of H from/F-GM Material 2 And Mg (magnesium) 2+ Is a release profile of (2);
FIG. 9 is MgH of example 1 of the present invention 2 PLGA/F-GM material, F-GM scaffold material of comparative example 1, mgH of comparative example 2 2 Effect of/F-GM scaffold on cytotoxicity and viability;
FIG. 10 is MgH 2 MgH of powder, comparative example 2 2 F-GM Material, mgH according to example 1 of the present invention 2 Fluorescence imaging diagram of influence on ROS content after in vitro acting on Raw264.7 cells by the @ PLGA/F-GM material;
FIG. 11 is MgH 2 MgH of powder, comparative example 2 2 F-GM Material, mgH according to example 1 of the present invention 2 Quantitative results of ROS content after in vitro action of the @ PLGA/F-GM material on Raw264.7 cells;
FIG. 12 is MgH 2 MgH of powder, comparative example 2 2 F-GM Material, mgH according to example 1 of the present invention 2 SEM spectrogram of the PLGA/F-GM material after being acted on Raw264.7 cells in vitro;
FIG. 13 is MgH 2 MgH of powder, comparative example 2 2 F-GM Material, mgH according to example 1 of the present invention 2 The content measurement results of biomarkers IL-6, IL-1 beta and Arg-1 after the PLGA/F-GM material acts on Raw264.7 cells in vitro;
FIG. 14 is MgH 2 MgH of comparative example 2 2 F-GM Material, mgH according to example 1 of the present invention 2 Cell migration and void closure results after the scratch is treated by the PLGA/F-GM material;
FIG. 15 is MgH 2 MgH of comparative example 2 2 F-GM Material, mgH according to example 1 of the present invention 2 Vascularization promoting results after treatment of cells with PLGA/F-GM material;
FIG. 16 is MgH 2 MgH of comparative example 2 2 F-GM Material, mgH according to example 1 of the present invention 2 After cells are treated by PLGA/F-GM material, the healing area, node number and tube length are controlledQuantification of vascular markers;
FIG. 17 is a control group, mgH 2 Group, mgH 2 group/F-GM, mgH 2 Determination results of skull bone volume to tissue volume ratio of the group of diabetic mice @ PLGA/F-GM;
FIG. 18 is a control group, mgH 2 Group, mgH 2 group/F-GM, mgH 2 Determination results of defective area Osteogenesis (OCN) and angiogenesis (CD 31) after fluorescent staining of the skull of diabetic mice treated with PLGA/F-GM group.
Detailed Description
The invention will be further described with reference to the accompanying drawings and detailed description. It is to be understood that the following examples are merely illustrative of the present invention and are not intended to be limiting thereof. The reagents used in the examples below are all commercially available unless otherwise indicated.
Among them, polylactic acid-glycolic acid copolymer (PLGA) used in the following examples was obtained from Sigma Co., ltd, molecular weight of 38000-54000, and product number of 719900-5G; polyethylene glycol is from Ala-dine, has a molecular weight of 1000, and has a product number of P103719-500g; methacrylic anhydride was from Sigma-Alorich with a molar mass of 154.16g/mol; the molecular weight of the gelatin is 30 ten thousand to 100 ten thousand Da.
Example 1
The embodiment provides a hydrogel scaffold material capable of continuously releasing hydrogen and magnesium ions and a preparation method thereof, comprising the following steps:
(1) Polylactic acid-glycolic acid copolymer coated magnesium hydride microsphere (MgH 2 @ PLGA)
The operation of this step is schematically shown in fig. 1. The method comprises the following steps: 10mg of polylactic-co-glycolic acid (PLGA) was weighed into 1mL of 1, 4-dioxane and stirred for 30min, then 10mg of magnesium hydride (MgH) was added 2 ) Stirring is continued to obtain PLGA+MgH 2 The solution was mixed.
In addition, 6mg of polyethylene glycol (PEG) was weighed and dissolved in 3mL of dimethyl sulfoxide (DMSO), and stirred until dissolved, to obtain a PEG solution.
The PLGA+MgH obtained above is processed 2 Slowly dripping the mixed solution into PEG solution in high-speed stirringIn solution, the organic solvent in the resulting mixed solution was then evaporated overnight (> 6 h), and the mixed solution was centrifuged in a high speed centrifuge at room temperature the next day. Discarding the supernatant, drying the precipitate in a drying oven at 40 ℃ to obtain magnesium hydride microsphere coated with polylactic acid-glycolic acid copolymer, and marking as MgH 2 @PLGA。
(2) Preparation of methacryloyl gelatin (GelMA) solution
10g of Gelatin (Gelatin) was added to 100mL of double distilled water and left at room temperature for 1 hour to allow it to swell sufficiently, and stirred in a water bath at 60℃until the Gelatin was dissolved. Next, 6mL of Methacrylic Anhydride (MA) was added to the gelatin solution at 1mL/min, the resulting mixed solution was shaken in a shaker at 50℃for 3 hours, then 400mL of double distilled water was added, and the diluted solution was put into a dialysis bag of 8-14kDa for dialysis for 1 week. Changing the dialysate every 4 hours, centrifuging the solution at 3000rpm for 10 minutes, discarding the precipitated impurities, placing into a refrigerator at-80deg.C, and freeze-drying on a freeze dryer to obtain methacryloyl gelatin (GelMA). The lyophilized GelMA was dissolved in a solution of lithium phenyl (2, 4, 6-trimethylbenzoyl) phosphonate (LAP) to give a GelMA solution having a mass concentration of 10% (100 mg of GelMA was dissolved in 1mL of LAP solution at the time of preparation). Wherein the mass concentration of the LAP solution is 0.25%, and the LAP powder is dissolved in PBS.
(3)MgH 2 Preparation of @ PLGA/F-GM hydrogel scaffold
The operation of this step is schematically shown in fig. 2. Mixing the GelMA solution obtained in the step (2) with MgH obtained in the step (1) 2 The @ PLGA microspheres were foamed using a medical tee, and then crosslinked in a mold using a 405nm UV lamp to obtain the hydrogel scaffold material of example 1 (cylindrical, 6mm diameter, 2.5mm thickness, shape and size changeable as required).
Specifically, the foaming device used in the foaming process uses a medical tee (Ming An kang, china) to connect two syringes. A syringe containing the GelMA solution obtained in step (2) and air; the other syringe contains MgH obtained in the step (1) 2 PLGA microsphere powder and air. When the gas foaming is carried out, the two ends of the injector are pushed for about 100 timesGelMA solution and MgH 2 Mixing of the PLGA microsphere powder, the air was uniformly distributed in the hydrogel by pushing the mechanical shear force generated at both ends of the syringe multiple times, forming a foamed material, designated as F-GM.
F-GM was injected into the indicated mold and crosslinked using 405nm blue light. Since the blue light penetration of the crosslinked F-GM is weak, a large amount of F-GM cannot be injected simultaneously during the operation. Thus, only one thin layer of F-GM is injected at a time by the layering injection method, and then photo-curing is performed. Then injecting the next layer until the whole stent injection is completed, obtaining the hydrogel stent material with continuous release of hydrogen and magnesium ions, which is marked as MgH 2 @PLGA/F-GM。
Comparative example 1
This comparative example provides a methacryloyl gelatin hydrogel scaffold, designated as F-GM, prepared as follows:
in this comparative example, the foaming process was performed by referring to the method of example 1, and a medical three-way (Ming An kang, china) was used as the foaming device, and two syringes were connected. The preparation process of F-GM comprises the following steps: taking the GelMA solution with the mass concentration of 10% prepared in the step (2) of the example 1, sucking the GelMA solution by adopting one syringe, and leaving air in the other syringe. I.e. two syringes each containing GM-LAP solution and air. The two ends of the injector are alternately and continuously pushed for about 100 times, and the air is uniformly distributed in the hydrogel through the mechanical shearing force generated by pushing the two ends of the injector for multiple times, so that F-GM is formed. F-GM was injected into the indicated mold and crosslinked using 405nm blue light. Since the blue light penetrating power of the crosslinked F-GM is weak, a large amount of F-GM cannot be injected at the same time. Thus, only one thin layer of F-GM is injected at a time by the layering injection method, and then photo-curing is performed. The next layer is then injected until the entire stent injection is complete.
Comparative example 2
This comparative example provides a magnesium hydroxide-methacryloyl gelatin hydrogel scaffold, denoted MgH 2 The preparation method of the/F-GM comprises the following steps:
in this comparative example, the foaming process was carried out by the method of example 1, using a foaming deviceA medical tee (Ming An kang, china) was used to connect two syringes. MgH (MgH) 2 The preparation process of the/F-GM comprises the following steps: taking the GelMA solution with the mass concentration of 10% prepared in the step (2) of the example 1, sucking the GelMA solution by adopting one of the syringes, marking as GM, and reserving air and MgH in the other syringe 2 And (3) powder. Namely, the two syringes respectively contain GM-LAP solution, air and MgH 2 And (3) powder. The two ends of the injector are alternately and continuously pushed for about 100 times, and the air is uniformly distributed in the hydrogel through the mechanical shearing force generated by pushing the two ends of the injector for multiple times, so that MgH is formed 2 F-GM. MgH is processed 2 the/F-GM was injected into the indicated mold and crosslinked using 405nm blue light. Also adopts a layering injection method, and only injects a thin MgH layer at a time 2 F-GM, followed by photo-curing. The next layer is then injected until the entire stent injection is complete.
Test examples
In the following test examples, all data were collected in triplicate or five and expressed as mean and standard deviation. All data were evaluated using GraphPad except Image J for scratch area, angiogenesis data. * p <0.05, < p <0.01, < p <0.001, < p <0.0001 > is considered statistically significant, and ns indicates that the difference is not statistically significant.
Test example one, mgH 2 Characterization of @ PLGA microspheres
Step (1) of embodiment 1 of the present invention MgH 2 Wrapping in PLGA to obtain microsphere, protecting it from water to realize H 2 And Mg (magnesium) 2+ Is named MgH 2 @ PLGA. In this test example, mgH obtained by drying in the step (1) of example 1 2 @PLGA microspheres and MgH before encapsulation 2 Detection by electron scanning microscope (ZEISS GeminiSEM 300, germany) and XRD (Rigaku MiniFlex600, japan) were performed.
Wherein, the dried micro racket is photographed by a scanning electron microscope, and the particle size analysis is performed by Image J software. SEM image results are shown in fig. 3. FIG. 3 shows MgH 2 (left panel) and MgH 2 Spherical morphology of PLGA (right panel) was relatively uniform and particle size analysisThe average diameters of the latter two were 19.1 μm and 21.2. Mu.m.
Further, the invention utilizes XRD to further verify MgH 2 And MgH 2 Purity of PLGA, results are shown in figure 4. XRD results of FIG. 4 show MgH 2 And MgH 2 PLGA has a similar peak structure, indicating MgH 2 PLGA microspheres were successfully synthesized.
Test example II, morphological characterization of stent Material and Performance test
2.1 morphological observations
The MgH prepared in example 1 of the present invention after lyophilization was observed and analyzed by naked eyes and a scanning electron microscope 2 PLGA/F-GM scaffold Material, F-GM scaffold Material of comparative example 1, mgH of comparative example 2 2 Appearance and pore size of the/F-GM scaffold material. The porosity was calculated by analysis using ImageJ software. In addition, the surface morphology and microstructure of the lyophilized samples were observed by scanning electron microscopy (SEM, geminiSEM 300, zeiss). The appearance of the different scaffold materials (upper panel) and the corresponding SEM images (lower panel) are shown in fig. 5.
As can be seen from FIG. 5, the foaming and freeze-dried F-GM and MgH were observed by naked eyes 2 F-GM is white and MgH 2 PLGA/F-GM was white yellowish. This is related to the addition of PLGA. Under the scanning electron microscope, F-GM and MgH can be observed 2 F-GM and MgH 2 Air bubbles in PLGA/F-GM are relatively uniform, and F-GM and MgH are analyzed by Image J software 2 F-GM and MgH 2 Porosity of PLGA/F-GM. The porosity test result shows that MgH 2 F-GM and MgH 2 PLGA/F-GM has no significant difference compared with F-GM alone, and the larger pore structure is more favorable for oxygen, nutrient substance delivery, vascular ingrowth and cell migration.
2.2 evaluation of mechanical Properties
At the early stage MgH 2 Based on the synthesis of PLGA microballoons, a method combining gas foaming and photo-curing technology is adopted to generate foaming F-GM, which plays a role in providing a certain mechanical support for the bone defect part. To further evaluate the properties of the different F-GM, mechanical and degradation tests were carried out. By applying force The MgH prepared in example 1 of the present invention was tested by an optical tester (INSTRON-5542, china) 2 PLGA/F-GM scaffold Material, F-GM scaffold Material of comparative example 1, mgH of comparative example 2 2 the/F-GM scaffold material was subjected to compression tests, compression on a mechanic instrument at a rate of 5mm/s, all tests were performed at room temperature, three replicates per group. The results are shown in FIG. 6.
As can be seen from fig. 6, the foaming significantly increases the elasticity of the hydrogel, mgH when the hydrogel is compressed to 70% of the original height 2 F-GM and MgH 2 PLGA/F-GM has better elasticity than F-GM, especially MgH according to example 1 of the invention 2 The @ PLGA/F-GM material exhibits significantly better elasticity than other materials, and thus can impart good injectability to the scaffold material.
2.3 in vitro degradation test
To evaluate MgH obtained in example 1 of the present invention 2 Degree of in vitro degradation of PLGA/F-GM material scaffolds (n=3) were immersed in PBS, purified water and simulated body fluids for 30 days at room temperature. The initial weight (W 0 ). Then, after 5, 10, 15, 20, 25, 30 days of the action in PBS, pure water and simulated body fluid, the remaining material was taken out, and the material was vacuum-dried and weighed (W 1 ). All experiments were performed in triplicate. Degradation rate was calculated according to the following formula: d=w 0 -W 1 /W 0 X 100%. The results are shown in FIG. 7.
In FIG. 7, mgH was detected in pure water, PBS, modified simulated body fluid (M-SBF) 2 Degradation rate of PLGA/F-GM within 30 days. Wherein SBF is a metastable solution, is an apatite supersaturated solution containing calcium ions and phosphate ions, and is widely used for in vitro evaluation of bioactive materials. As can be seen from FIG. 7, mgH 2 At day 30, the material was almost completely degraded with the remaining hydrogel being 5.61% of the original weight, while MgH 2 The degradation rate of @ PLGA/F-GM in PBS and pure water was 18.74% and 16.17% by day 30, respectively. Thus, it is explained that MgH 2 The degradation rate of the @ PLGA/F-GM is matched with the inflammatory period after bone defect, and canBetter plays the role of reducing the generation of active oxygen by hydrogen and the role of promoting the conversion of macrophages from pro-inflammatory to anti-inflammatory by magnesium ions.
Compared with other materials for diabetic skin wound surface, the invention has the advantages of strong injectability, easy dispersion and promotion of magnesium ion and hydrogen release by adding PLGA microspheres and GelMA, and is more beneficial to playing a role in repairing diabetic bone defect.
Test example III, mgH 2 In vitro Release of PLGA/F-GM
In successful preparation of MgH 2 After PLGA, the invention was added with foamed gelatin methacrylic acid (F-GM), and MgH of comparative example 2 was studied 2 F-GM Material and MgH of example 1 2 H in the @ PLGA/F-GM Material 2 And Mg (magnesium) 2+ Release profile in vitro.
To test MgH 2 /F-GM、MgH 2 In vitro release of H by PLGA/F-GM 2 In the case of (2), mgH is contained in an amount of 3mg 2 MgH of (2) 2 /F-GM、MgH 2 PLGA/F-GM was immediately sealed in a penicillin bottle (15 mL volume) containing 10mL of purified water (pH=7.0). 1mL of gas was taken at various time points (0H, 5min, 10min, 30min, 1H, 3H, 6H, 12H, 1 day, 2 days, 3 days, 5 days, 7 days) and H was measured by a gas chromatograph (Agilent 8890, U.S.) 2 Is contained in the composition. And detecting the hydrogen content in the penicillin bottle, and detecting the hydrogen content in the liquid at different time points (0 h, 5min, 10min, 30min, 1h, 3h, 6h, 12h, 1 day, 2 days, 3 days, 5 days, 7 days, 9 days, 11 days, 13 days and 15 days). And carrying out statistical analysis on the obtained data.
To test MgH 2 /F-GM、MgH 2 In vitro release of Mg by PLGA/F-GM 2+ In the case of (2), mgH was measured in pure water (ph=7.0) 2 /F-GM、MgH 2 Mg in @ PLGA/F-GM 2+ Is a release profile of (2). The specific operation will contain 3mg MgH 2 MgH of (2) 2 /F-GM、MgH 2 @ PLGA/F-GM, immersed in 10mL of purified water. 2mL of liquid was collected at different time points (0 h, 5min, 10min, 30min, 1h, 3h, 6h, 12h, 1 day, 2 days, 3 days, 5 days, 7 days), and ICP-OES assay (U.S., Agilent 720ES (OES) quantitative determination of Mg 2+ For each sample, the liquid was replenished to 10mL and the cumulative release was detected. The results are shown in FIG. 8.
In FIG. 8A, the present invention uses MgH under the same test conditions 2 F-GM and MgH 2 Soaking PLGA/F-GM in pure water to examine Mg 2+ From FIG. 8A, it is observed that MgH was released on day 7 2 Mg produced by PLGA/F-GM 2+ Is MgH 2 1.46 times of/F-GM due to MgH 2 Mg (OH) formed on the surface of the particles 2 The layer hinders MgH 2 And MgH is hydrolyzed by 2 Acidic degradation products of PLGA in PLGA/F-GM are capable of dissolving Mg (OH) 2 Layer, thereby accelerating H 2 And soluble Mg 2+ Is generated. From this, it was demonstrated that the acidic environment caused by degradation of PLGA was H 2 And Mg (magnesium) 2+ The increase in generation of (c) creates opportunities. In addition, on the seventh day, mgH in the air was collected 2 H produced by PLGA/F-GM 2 Is MgH 2 1.45 times that of/F-GM (FIG. 8B). At the same time, the content of hydrogen dissolved in water was detected, and on day 14, mgH 2 H produced by PLGA/F-GM 2 Is MgH 2 1.86 times of/F-GM (FIG. 8C). Taken together, the results demonstrate that the presence of PLGA in the scaffold material of the present invention results in an acidic environment, H 2 And soluble Mg 2+ The generation of (c) increases.
Test example four, biocompatibility detection
The biocompatibility of the synthetic material is critical for the regeneration of tissue and bone. Next, the invention detects the biocompatibility of the synthetic material in vitro by a cell living and death test and a CCK-8 method, and examines MgH in vitro 2 Effect of PLGA/F-GM on cellular biocompatibility.
4.1, CCK8 detection
In the concentration-dependent test, bone marrow mesenchymal stem cells (BMSCs) were inoculated into 96-well plates, 5000 cells per well, and after the bone marrow mesenchymal stem cells were cultured in 100. Mu.L of low-sugar DMEM in 96-well plates for 12 hours, they were then incubated with MgH at different concentrations 2 Solutions (0, 0.5, 1, 1.5, 2, 2.5, 3. Mu.g/mL dissolved in complete medium) were co-processedCulturing for 3 days. On the third day, 10% cck8 solution was added to each well to interact with living cells. After 2 hours in an incubator at 37 ℃, the absorbance of the different groups in the 96-well plate was analyzed with a microplate reader (SpectraMAX iD 3) of a molecular apparatus.
Then MgH is added 2 MgH of powder, comparative example 2 2 Powder of/F-GM Material, mgH of example 1 2 The powder of PLGA/F-GM material was soaked in 10mL of PBS for 3 days to give a leaching solution for each group. Human Umbilical Vein Endothelial Cells (HUVECs) were inoculated into 96-well plates with 3000 cells per well and divided into 4 groups, i.e., control group, mgH 2 Powder, mgH 2 /F-GM、MgH 2 Leaching solution group of PLGA/F-GM, etc. After 1, 3 and 7 days of co-culture of the cells with the extract, the cell viability was measured by CCK-8 method using the same test procedure as described above. Each group was repeated 5 times. CCK-8 method for detecting MgH 2 、MgH 2 /F-GM、MgH 2 The effect of PLGA/F-GM on cytotoxicity and viability is shown in FIG. 9.
FIG. 9 shows the results of the test that MgH is present after 72h of treatment 2 The activity of bone marrow mesenchymal stem cells (BMSCs) of the @ PLGA/F-GM group is not obviously different from that of a control group, which indicates MgH 2 The @ PLGA/F-GM was not cytotoxic to bone marrow mesenchymal stem cells and had no effect on the proliferation activity of the cells (FIG. 9A). At the same time, mgH is determined 2 The optimal concentration of (C) is 1. Mu.g/mL. Thus, unless otherwise indicated, the following experiments used 1. Mu.g/mL MgH 2 A solution. Next, human Umbilical Vein Endothelial Cells (HUVECs) were treated with each set of extracts for 1, 3, 7 days, mgH over time 2 、MgH 2 /F-GM、MgH 2 There was no significant difference between the @ PLGA/F-GM group and the normal control group (control group), indicating MgH 2 PLGA/F-GM was also non-toxic to human umbilical vein endothelial cells and did not affect their normal growth (FIG. 9B). Therefore, the stent material of the invention has good biological safety.
4.2 staining of cell death
Human Umbilical Vein Endothelial Cells (HUVECs) are divided into 4 groups of 1-2×10 per dish 6 Individual cells were seeded in 12-well plates. The cells of each group are respectively combined with a normal cell culture medium (control group) and MgH 2 Powder, mgH 2 /F-GM、MgH 2 Co-culture of PLGA/F-GM extract for 1d, 3d, 7d. At the corresponding time the medium was aspirated, and 1mL of Calcein-AM/PI assay was added to each dish. At 37 ℃,5% CO 2 After incubation for 15min in the incubator, the different cell groups were photographed with a fluorescence microscope (Leica SP5, leica Camera AG, germany).
The photographing result of the cell living dying staining shows that the F-GM prepared by the gas foaming and photo-curing technology has no cytotoxicity and good biocompatibility. MgH at first and third days 2 、F-GM、MgH 2 /F-GM、MgH 2 There was no significant difference in cell viability for the @ PLGA/F-GM group, no dead cells were seen. Over time, by day five, only a few red spots (representing dead cells) were observed on the surface. Description of MgH 2 The @ PLGA/F-GM has good biocompatibility.
Test example five, in vitro MgH 2 @PLGA/F-GM reduces ROS production
At the time of verifying MgH 2 At prolonged H, PLGA/F-GM 2 After the effect of release, the present invention investigated whether it was able to reduce ROS production in vitro. First, raw264.7 cells were seeded in 6-well plates, lipopolysaccharide (LPS) was added to induce ROS overproduction, and then MgH was used 2 Powder (1. Mu.g/mL), mgH 2 /F-GM、MgH 2 Co-culture of the extract of PLGA/F-GM in normal cell culture medium (control). After 24h co-culture, raw264.7 cells were stained using ROS staining (DCFH-DA, green) and then imaged under a fluorescent microscope. Stained raw264.7 cells were collected for flow cytometry analysis and quantified. The results are shown in FIGS. 10 and 11.
The fluorescence imaging results of FIG. 10 show that MgH was used in comparison to untreated control 2 Solution, mgH 2 /F-GM、MgH 2 The leaching solution of PLGA/F-GM is MgH compared with the normal culture medium (Control) 2 The ROS reduction was most pronounced in the @ PLGA/F-GM extract.
To quantify the decrease in ROS production, stained raw264.7 cells were collected for flow cytometry analysis. Quantification of flow cytometry data, as shown11. FIG. 11 shows that MgH 2 、MgH 2 /F-GM、MgH 2 The PLGA/F-GM leaching solution group reduced the active oxygen to 84.46%, 74.92% and 40.10% of the original values, respectively, compared to the untreated group (Control group), confirming the MgH of the invention 2 PLGA/F-GM materials are indeed highly advantageous in reducing active oxygen levels.
Test example six, mgH in vitro 2 @P/F-GM promotes polarization of macrophages towards M2
After verifying the advantages of hydrogen for treating ROS, the invention then investigated MgH 2 @PLGA/F-GM causes the release of Mg 2+ While inducing a macrophage phenotype change function. To observe morphological changes in macrophages, 1×10 was used 5 Individual raw264.7 cells were seeded into 12-well plates containing sterilized silicon chips. With MgH 2 Solution (1. Mu.g/mL), mgH 2 /F-GM、MgH 2 After 24h co-culture of PLGA/F-GM extract with normal cell culture medium, the Raw264.7 cell silicon wafer was removed from the well plate, fixed with 2.5% glutaraldehyde for 30min, and dehydrated with an alcohol concentration gradient (30% 5min,50%5min,70%10min,80%10min,95%15min,100%15 min). Each group was then photographed by SEM (ZEISS GeminiSEM 300, germany) and the polarization of Raw264.7 cells was analyzed and the results are shown in FIG. 12.
The SEM image of FIG. 12 shows MgH compared to control (untreated) treated cells 2 Solution (1. Mu.g/mL), mgH 2 /F-GM、MgH 2 Explicit morphological changes were observed in Raw264.7 cells treated with PLGA/F-GM leach, indicating Mg 2+ Macrophage polarization is induced.
For more detailed studies, the present invention performed real-time fluorescent quantitative (RT-qPCR) analysis on raw264.7 cells treated for 3 days with each set of leach solutions. Untreated cells served as blank, LPS-induced cells served as control, and RT-qPCR was performed to detect the expression of mRNA levels of M1 macrophage biomarker (IL-6, IL-1β) and M2 macrophage biomarker (Arg-1). The results are shown in FIG. 13.
The results in FIG. 13 show that MgH 2 Expression of both IL-6 (FIG. 13B) and IL-1β (FIG. 13A) in the PLGA/F-GM leach set was statistically significant in the decreaseLow, indicating that polarization of M1 is suppressed after treatment. While MgH 2 Expression of Arg-1 from the PLGA/F-GM leaching solution group was significantly increased (FIG. 13C), indicating that M2 repolarization was simultaneously enhanced after treatment. From this, mgH 2 PLGA/F-GM can promote polarization of pro-inflammatory M1 macrophages to pro-healing M2 macrophages.
Test example seven, mgH in vitro 2 Vascularization promoting effect of @ P/F-GM
The test examples were evaluated for cell migration capacity in vitro by scratch and tube tests.
7.1 tube forming test
Sucking 50 mu L of matrigel into a 96-well plate, placing into a 37 ℃ incubator for 40-60 min, and spreading without bubbles. Then, the human umbilical vein endothelial cells are planted in 96-well plates, and the density of each well is 2-3 multiplied by 10 4 . Grouping cultured human umbilical vein endothelial cells, adding MgH 2 Powder, mgH 2 /F-GM、MgH 2 The extract of PLGA/F-GM was incubated at 37℃in an incubator for 4-6h, photographed with a microscope, and then analyzed for tube length and node number using Image J.
7.2 scratch test
Endothelial cells were seeded in 6-well plates at 1.2X10 per well 6 The cells were streaked out with the tip of a pipette in alignment with the sterilized ruler, then gently rinsed with PBS to remove floating cells, and replaced with serum-free medium and MgH 2 Powder (1. Mu.g/mL), mgH 2 /F-GM、MgH 2 The leaching solution of PLGA/F-GM is treated, and photographs are taken at the same position of 0h, 6h, 12h and 24h, and the migration condition of cells is observed.
The results of the above test are shown in FIGS. 14 to 16.
Using MgH 2 (1μg/mL)、MgH 2 /F-GM、MgH 2 The PLGA/F-GM leach was used to treat the interstices created by the scratches and the results are shown in FIG. 14. As can be seen from FIG. 14, each group healed to a different extent, but MgH of example 1 2 The healing effect after being treated by PLGA/F-GM material is best, and MgH is shown as time goes by 2 The healing effect of PLGA/F-GM is more obvious, even at 24 hours of the observation periodThe voids were closed, while the control group had very wide voids, indicating MgH 2 PLGA/F-GM has the advantage of good promotion of endothelial cell migration in human umbilical vein in vitro.
Subsequently, to verify MgH 2 The pro-vascularization effect of PLGA/F-GM in vitro was also tested. Human umbilical vein endothelial cells are respectively cultured with normal cell culture medium (control group) and MgH 2 (1μg/mL)、MgH 2 /F-GM、MgH 2 The PLGA/F-GM extract was incubated for 4-6 hours and photographed under a microscope, and the results are shown in FIG. 15. As is evident from FIG. 15, mgH is a higher concentration than the control group treated with the normal medium 2 (1μg/mL)、MgH 2 /F-GM、MgH 2 The tubing effect of the @ PLGA/F-GM leach solution set was much better, where MgH 2 The tube forming effect of the PLGA/F-GM leaching solution group is best and is obviously better than that of other groups.
In FIG. 16, the columns in the bar charts of FIGS. A-D represent, in order, a Control group, mgH 2 Group, mgH 2 group/F-GM, mgH 2 Group @ PLGA/F-GM. Quantitative analysis of healing area using Image J, mgH 2 (1μg/mL)、MgH 2 /F-GM、MgH 2 The void closure rate of the @ PLGA/F-GM leach groups was 1.03 times, 1.13 times and 1.21 times higher, respectively, than that of the control group (FIG. 16A). Subsequently, image J software was used to quantify the node and tube lengths of the different groups, as can be seen from the histogram, mgH 2 After treatment with PLGA/F-GM extract, the extract was compared with the Control group (Control group), mgH 2 Group, mgH 2 The number of nodes and tube length were increased by 2.43, 2.19, 1.78 times, respectively, compared to the @ PLGA group (FIGS. 16B, 16C). Finally, mgH was validated by real-time fluorescence quantification 2 The pro-vascularization effect of PLGA/F-GM in vitro is seen from the vascular markers such as platelet-endothelial cell adhesion molecule (Platelet endothelial cell adhesion molecule-1, PECAM-1/CD 31) and vascular endothelial growth factor (vascular endothelial growth factor, VEGF) as MgH 2 (1μg/mL)、MgH 2 /F-GM、MgH 2 Compared with the blank control group, the treated group of the PLGA/F-GM leaching solution has the expression of vascular markers with different degrees of expression rise, which indicates that magnesium ions have the effect of promoting vascularization, Most importantly, mgH 2 The vascular marker expression was highest in the PLGA/F-GM leach treated group (FIG. 16D).
To sum up, mgH 2 The advantage of PLGA/F-GM in promoting endothelial cell migration and vascularization in vitro was demonstrated and was expected to promote revascularization and further bone regeneration in vivo.
Test example eight, in vivo animal test investigation of MgH 2 PLGA/F-GM contributes to bone effects in vivo
In vitro, the biocompatibility, the inflammatory regulation and the vascularization promotion effect of the material are all verified. Next, mgH is verified 2 In vivo repair effects of PLGA/F-GM in diabetic mice skull defects.
8.1, micro CT scanning test
Test materials: male diabetic mice, strain Cas9-KO, purchased from Jiangsu province, jiugaokang biotechnology Co., ltd, were randomly divided into 4 groups of three. After the diabetic mice were anesthetized, the hair on the head of each mouse was shaved, and the external skin was cut to completely expose the skull. A model of the skull defect (3 mm diameter, one defect per diabetic mouse) was made with a bone drill. Blank groups are only defected and are not treated, mgH 2 Spraying powder, mgH 2 F-GM and MgH 2 The PLGA/F-GM group implanted a cylindrical material (3 mm diameter, about 1mm thickness) into the defect area. After the wound is sutured, antibiotics are sprinkled to prevent infection, and the skull is sampled at 4 weeks and 12 weeks. Three-dimensional computed tomography of the skull: after 4 and 12 weeks of implantation, diabetic mice were sacrificed by cervical dislocation, the entire skull was obtained, and the scan was performed using micro CT (Scanco, switzerland).
In the test, the invention mainly implants the foaming material into the skull defect of the diabetes mice for 4 weeks and 12 weeks, and takes the whole skull of the diabetes mice to observe the in-vivo osteogenesis effect. And taking the skull to carry out Micro-CT detection and determining the generation of new bone. As is clear from the results of the Micro CT scan, there was almost no bone regeneration at the defect site of the untreated control group at 4 weeks after molding, and the treatment group included MgH 2 MgH of group, comparative example 2 2 MgH of group/F-GM and example 1 2 The @ PLGA/F-GM group was superior to the control group, more importantly MgH at 4 weeks 2 The bone regeneration effect of the @ PLGA/F-GM group was optimal. As the implantation time was prolonged to 12 weeks, the area of regenerated bone was increased in the placebo group compared to 4 weeks, but the regeneration rate was slow, while MgH was used 2 、MgH 2 /F-GM、MgH 2 PLGA/F-GM bone regeneration effect was significantly better than that of the blank control group, and MgH of example 1 2 The repair effect of the PLGA/F-GM bone is obviously better than that of other groups. Next, according to the Micro CT report, the results of statistical analysis of the bone volume to tissue volume ratio of the skull of the 4-week and 8-week diabetic mice were calculated, and the results are shown in fig. 17.
FIG. 17 is a statistical result consistent with the Micro-CT result, mgH at 4 weeks 2 BV/TV ratio of regenerated bone of group @ PLGA/F-GM is higher than that of other three groups, mgH 2 PLGA/F-GM is control group, mgH 2 Group, mgH 2 2.04 times, 1.55 times and 1.28 times the group of/F-GM. And this trend remains unchanged over time, mgH by 12 weeks 2 PLGA/F-GM is control group, mgH 2 Group, mgH 2 2.49 times, 1.69 times and 1.29 times the group of/F-GM. These results all indicate MgH 2 PLGA/F-GM can reduce the generation of active oxygen, relieve oxidative stress, and promote vascularization in the environment of diabetes and hyperglycemia, thereby promoting bone repair and regeneration.
8.2, H & E staining, massa staining and immunofluorescent staining
Histological evaluation and immunohistochemical staining: the diabetic mouse skull obtained above was fixed in 4% paraformaldehyde solution for at least 24 hours. The fixed tissue was then decalcified by immersing in 10% edta solution for about one week. Then, paraffin-embedded samples were cut out at the center of the defect to give a defect cross section of 5 μm for hematoxylin and eosin (H & E) staining and masson staining, and Osteocalcin (OCN) and platelet endothelial cell adhesion molecule-1 (CD 31) were immunofluorescent stained.
H for defect areas implanted for 4 weeks and 12 weeks&E staining and masson staining to assess the area of regenerated tissue. The pale red areas of the defect are considered regenerated bone. In HE staining, implantation was performed for 4 weeks After that, all groups had a pale red dyeing area, in which MgH 2 The dyeing area of the @ PLGA/F-GM is higher than that of other groups, and the dyeing areas of all groups are increased along with the extension of implantation time, wherein MgH 2 The staining area of PLGA/F-GM was significantly increased. While only thin stained areas were observed for the blank and there were still large areas of unrepaired area.
In the case of the Maron staining, the results were similar to those of the HE staining, and the reaction of the Maron staining on collagen of bone tissue was related to the degree of collagen maturation, the bone tissue was red, the uncalcified cartilage was blue, and the Maron staining was consistent with the HE staining, whether it was 4 weeks or 12 weeks, mgH 2 The bone regeneration area of PLGA/F-GM was more.
The bone defect samples after 4 and 12 weeks of implantation were then immunofluorescent stained, and the effects of bone formation (OCN) and angiogenesis (CD 31) in the defect area were examined, and the results are shown in FIG. 18. Wherein, the Control group and MgH are sequentially arranged from left to right in the bar chart 2 Group, mgH 2 group/F-GM and MgH 2 Group @ PLGA/F-GM.
From FIG. 18, it can be seen that MgH was present from 4 weeks to 12 weeks 2 The positive expression areas of the vascular marker CD31 of PLGA/F-GM and the osteogenic marker OCN are more, the blank group is the least, and the quantitative result is consistent with the immunofluorescence staining result. Description of MgH 2 The PLGA/F-GM has better vascularization promoting effect, participates in blood and nutrition supply of bone repair, lays a good foundation for later ossification, and promotes regeneration and repair of bone defect in diabetes environment.
In summary, the invention researches the scaffold material required by the diabetic bone defect, prepares the foaming material combining the PLGA coated magnesium hydride microsphere and the photo-curing hydrogel by combining the gas foaming technology and the photo-curing technology, and establishes a model for promoting the regeneration and repair of the diabetic bone defect to verify the application effect of the scaffold material. In the stent material, hydrogen and magnesium ions generated when magnesium hydride encounters water can reduce the generation of active oxygen, reduce inflammatory reaction and promote vascularization so as to ossify, thereby achieving the aim of repairing the diabetic bone defect. And is also provided withPLGA degradation acid production promotes the release of magnesium ions in magnesium hydride, and can maintain vascularization effect. The foaming hydrogel is beneficial to the growth of tissue blood vessels, thereby providing oxygen and nutrition for bone repair. In summary, the experimental study results in vitro and in vivo show that: the scaffold material provided by the invention has the effects of reducing ROS, promoting polarization of M2 type macrophages, improving cell migration, relieving local inflammation, and promoting angiogenesis and bone regeneration. The injectable hydrogel MgH of the invention 2 The PLGA/F-GM material has great potential in the aspect of regeneration and repair of diabetic bone defect, and can provide a new reference method and a feasible strategy for clinical bone repair.
The above examples and test examples are only preferred embodiments of the present invention, and are merely for explaining the present invention, not limiting the present invention. Alterations, substitutions, modifications and the like will be apparent to those skilled in the art without departing from the spirit of the invention.
Claims (10)
1. A method for preparing a hydrogel scaffold material capable of continuously releasing hydrogen and magnesium ions, which is characterized by comprising the following steps:
(1) Dissolving polylactic acid-glycolic acid copolymer in solvent, adding magnesium hydride and mixing to obtain PLGA+MgH 2 Mixing the solutions; PLGA+MgH 2 Adding the mixed solution into polyethylene glycol solution, stirring, mixing, volatilizing solvent, centrifuging, collecting precipitate, and drying to obtain magnesium hydride microsphere coated with polylactic acid-glycolic acid copolymer, and recording as MgH 2 PLGA microspheres;
adding gelatin into water for swelling, adding methacrylic anhydride for mixing, dialyzing, centrifuging, and freeze drying to obtain methacryloyl gelatin; dissolving the methacryloyl gelatin to obtain a methacryloyl gelatin solution, and marking the methacryloyl gelatin solution as a GelMA solution;
(2) MgH obtained in the step (1) 2 Mixing the @ PLGA microspheres and the GelMA solution with air to foam to obtain a foam material, injecting the foam material into a mold, and performing crosslinking curing under ultraviolet conditions to obtain the hydrogel stent capable of continuously releasing hydrogen and magnesium ionsThe material, noted as MgH 2 @PLGA/F-GM。
2. The method for preparing a hydrogel scaffold material with sustained release of hydrogen and magnesium ions according to claim 1, wherein in the step (1), the solvent is one or more of 1, 4-dioxane, dimethyl sulfoxide, chloroform and dichloromethane; the dosage ratio of the polylactic acid-glycolic acid copolymer, the solvent and the magnesium hydride is (8-12) mg to (0.8-1.2) mL to (8-12) mg.
3. The method for preparing a hydrogel scaffold material capable of sustained release of hydrogen and magnesium ions according to claim 1, wherein in step (1), the polyethylene glycol solution is dimethyl sulfoxide solution of polyethylene glycol; the concentration of polyethylene glycol in the polyethylene glycol solution is 2-10 mg/mL; the mass ratio of polyethylene glycol, polylactic acid-glycolic acid copolymer and magnesium hydride is 1-3:1-5:1-5.
4. The method for producing a hydrogel scaffold material capable of sustained release of hydrogen and magnesium ions according to claim 1, wherein the ratio of gelatin to methacrylic anhydride in the step (1) is 8-12 g:5-7 mL.
5. The method for producing a hydrogel scaffold material which releases hydrogen and magnesium ions continuously according to claim 1, wherein in step (1), the dialysis is performed using a dialysis bag of 8-14 kDa; the dissolution is to dissolve the methacryloyl gelatin in a phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphonate solution to obtain a methacryloyl gelatin solution.
6. The method for preparing a hydrogel scaffold material capable of continuously releasing hydrogen and magnesium ions according to claim 1, wherein in the step (2), the foaming is performed by a tee joint connected with at least two syringes, and the GelMA solution and MgH are realized by reciprocating pushing of different syringes 2 Mixing the PLGA microspheres to uniformly distribute air in the mixed hydrogel to form the foaming materialThe method comprises the steps of carrying out a first treatment on the surface of the The wavelength of ultraviolet rays adopted by the crosslinking and curing is 400-410 nm.
7. A sustained release hydrogen and magnesium ion hydrogel scaffold material prepared by the method of any one of claims 1 to 6.
8. The sustained release hydrogen and magnesium ion hydrogel scaffold material of claim 7, wherein the hydrogel scaffold material is an injectable scaffold.
9. Use of a hydrogel scaffold material which releases hydrogen and magnesium ions continuously as claimed in claim 7 or 8 for the preparation of a scaffold material for repairing bone defects.
10. The use of a hydrogel scaffold material which is sustained release of hydrogen and magnesium ions according to claim 9, wherein the bone defect is a diabetic bone defect.
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