A controllable hydrogen production-promoting bone defect repair hydrogel and preparation method thereof

Abstract

This patent application discloses a controllable hydrogen production to promote bone defect repair hydrogel and its preparation method, which is made by mixing two components: solid powder A and liquid reagent B: the solid powder A is magnesium particles with a polymer coating, and the polymer coating adopts water-free polyethylene glycol; the liquid reagent B is polylactic acid-glycolic acid hydrogel. The hydrogel of the present invention can significantly delay the degradation time of magnesium particles in vivo while avoiding the degradation of magnesium particles during the coating preparation process. The hydrogel has antioxidant, anti-inflammatory and bone regeneration promoting properties, is suitable for irregular osteoporotic bone defects, and has good mechanical properties and is easy to minimally invasive implantation. At the same time, the preparation method adopted is simpler to operate and convenient for widespread clinical use.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and in particular to a hydrogel capable of controllably producing hydrogen to promote bone defect repair and a preparation method thereof. Background Art

[0002] Osteoporosis is a systemic disease that affects bone metabolism and is characterized by decreased bone mass and destruction of bone microstructure. Currently, bone repair in osteoporotic patients remains a huge challenge, mainly because the local injury is often accompanied by abnormal levels of reactive oxygen species (ROS) and inflammation, which impairs bone regeneration and slows the healing of bone defects.

[0003] Injectable hydrogels are currently widely studied bone repair materials. On the one hand, this type of hydrogel can fill and repair bone defects through minimally invasive injection, effectively avoiding the damage caused by open surgery; on the other hand, by integrating substances with different biological activities into the hydrogel, the hydrogel can be given antibacterial, anti-inflammatory, and promotion of stem cell osteogenic differentiation and other functions, thereby meeting the various needs of clinical treatment. For the repair of osteoporotic bone defects, researchers have tried to introduce various antioxidants into the hydrogel to improve inflammation and promote bone regeneration by removing ROS in the damaged area. Among them, commonly used antioxidants include MnO 2 , resveratrol, etc. However, these antioxidants have poor tissue permeability and are difficult to fully play the role of eliminating ROS after implantation. At the same time, these antioxidants can not only remove cytotoxic ROS such as hydroxyl free radicals, but also indiscriminately remove ROS with normal physiological functions such as oxidative metabolism (such as nitric oxide, etc.). In addition, for antioxidants containing transition metals such as Mn, their potential biological toxicity and side effects have always been a concern.

[0004] Hydrogen (H 2 ) is an emerging antioxidant with excellent antioxidant and anti-inflammatory properties, showing significant advantages in the treatment of ROS-related diseases. 2 It has good tissue and cell membrane permeability and can effectively diffuse in mitochondria, nuclei and other organelles that are the main sources of ROS. 2 It can selectively react with highly cytotoxic hydroxyl radicals without damaging oxidative metabolism or cell signaling systems, and alleviate the oxidative stress and inflammatory response caused by them. In addition, hydrogen, as an endogenous inert gas, has high safety and excellent biocompatibility. However, due to the 2 Due to its short residence time and extremely low solubility in body fluids, it is difficult to achieve H 2 The long-term delivery has limited therapeutic effect.

[0005] Magnesium and magnesium alloys have excellent biocompatibility and biodegradability, and can be degraded to produce magnesium ions and hydrogen after implantation in the body. Studies have shown that magnesium ions at appropriate concentrations can promote the proliferation, migration and osteogenic differentiation of stem cells and accelerate bone defect repair. Recently, Xu et al. used magnesium particles to react with H 2 O reacts to produce hydrogen, and a device that can generate H in situ after implantation in vivo is designed. 2 Hyaluronic acid-magnesium microspheres (Mg-HA) were used in this study. The experimental results showed that after intra-articular injection, the microspheres could continuously produce H 2 , significantly improving the inflammatory environment and cartilage destruction caused by osteoarthritis. However, due to the powdery physical properties of the microspheres, the mechanical properties are poor and irregular bone defects cannot be fully filled. At the same time, the reactivity of magnesium particles with water is very high, and it is necessary to prepare a coating on the surface to improve its corrosion resistance, reduce the degradation rate in the body, and prolong the hydrogen release time. However, in the process of preparing the surface coating of magnesium particles, rapid degradation of magnesium particles is prone to occur, and it is difficult to prepare the surface coating. Patent CN117258034A discloses a magnesium-based injectable composite hydrogel. Although a dense coating (including polydopamine, silica, organosilane and metal organic framework coating) is prepared on the surface of magnesium-based powder particles, the corrosion resistance and hydrogen production time of magnesium particles are improved, but the coating preparation reaction system still contains water (no more than 10vol%), which cannot avoid the degradation of magnesium particles, which is not conducive to the efficient preparation of the coating and subsequent conversion. At the same time, the above-mentioned hydrogel needs to be prepared by photocrosslinking or ionic crosslinking, which is cumbersome to operate and is not conducive to widespread clinical application. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a controllable hydrogen-producing hydrogel for promoting bone defect repair and a preparation method thereof. The hydrogel of the present invention can significantly delay the degradation time of magnesium particles in vivo while avoiding the degradation of magnesium particles during the coating preparation process. The hydrogel has antioxidant, anti-inflammatory and bone regeneration promoting properties, is suitable for irregular osteoporotic bone defects, has good mechanical properties, and is convenient for minimally invasive implantation. At the same time, the preparation method adopted is simpler to operate and is convenient for widespread clinical application.

[0007] The technical solution adopted by the present invention is as follows:

[0008] A controllable hydrogen production-promoting bone defect repair hydrogel is prepared by mixing two components: solid powder A and liquid reagent B:

[0009] The solid powder A is magnesium particles with a high molecular polymer coating, and the high molecular polymer coating is made of polyethylene glycol without water;

[0010] The liquid reagent B is polylactic acid-glycolic acid hydrogel.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] 1. The magnesium particle high molecular polymer coating of the present invention utilizes a non-aqueous polyethylene glycol solution to prepare an asymmetric protective coating on the surface of the magnesium particles, that is, a PEG coating with openings (about 15 μm in diameter) is formed on the surface of the magnesium particles, which slows down the degradation rate in vivo, significantly delays the degradation time of the magnesium particles in vivo, and avoids the degradation of the magnesium particles during the coating preparation process.

[0013] 2. The hydrogel of the present invention has excellent injectability, conformability and liquid-solid phase transition properties. After local injection in vivo, the in-situ solidification of the gel can be achieved through the solvent exchange reaction mediated by N-methylpyrrolidone in the gel and water molecules in the surrounding environment, thereby achieving full filling of bone defects under minimally invasive injection, and is suitable for repairing various deep bone defects and irregular bone defects. In addition, the applicant has found that after the hydrogel of the present invention is implanted in vivo, during the liquid-solid phase transition process and the hydrogen generated by the degradation of magnesium particles, a loose and porous scaffold-like structure will be formed in situ, which is conducive to the growth of tissues and cells, and provides effective mechanical support for the implantation site, accelerating the repair of bone defects.

[0014] 3. The applicant also found that the hydrogel of the present invention can not only form effective mechanical support for the bone defect site after implantation in vivo, but also accelerate the repair of bone defects by inhibiting the generation of osteoclasts and promoting the regeneration of new bone tissue. In addition, the hydrogel can achieve biodegradation, gradually degrade after implantation in vivo and can guide the growth of new bone tissue, achieving nearly half the area of ​​osteoporotic bone defects repair at 4 weeks. 8 weeks after implantation, the hydrogel is completely degraded and filled with a large amount of remodeled bone tissue, promoting the repair effect of osteoporotic bone defects.

[0015] 3. After the hydrogel of the present invention is implanted in vivo, it can achieve the effects of ROS removal, inflammation relief, osteoclast inhibition, and osteogenic differentiation promotion through local sustained release of hydrogen and magnesium ions, thereby accelerating the repair and reconstruction of osteoporotic bone defects.

[0016] As a preferred embodiment of the present invention, in the solid powder A, the magnesium particles are spherical particles with a diameter of 5-500 μm.

[0017] As a preferred embodiment of the present invention, the diameter of the magnesium particles is 30-70 μm, and the molecular weight of the polyethylene glycol is 1500-2500.

[0018] As a preferred embodiment of the present invention, in the liquid reagent B, the components of the polylactic acid-glycolic acid hydrogel are polylactic acid-glycolic acid polymer and an organic solvent.

[0019] As a preferred embodiment of the present invention, the molecular weight of the polylactic acid-glycolic acid polymer is 35000-45000, and the organic solvent is N-methylpyrrolidone. N-methylpyrrolidone can fully dissolve the polylactic acid-glycolic acid polymer, and after contacting the aqueous solution, the N-methylpyrrolidone precipitates to achieve in-situ precipitation of the polylactic acid-glycolic acid polymer, that is, to give the hydrogel the performance of liquid-solid phase transition.

[0020] As a preferred embodiment of the present invention, the mass volume ratio of the polylactic acid-glycolic acid to N-methylpyrrolidone is 0.5-3 g:1 mL.

[0021] The present invention also provides a method for preparing the hydrogel capable of controlling hydrogen production to promote bone defect repair as described in any one of the above embodiments, comprising the following steps:

[0022] (1) preparing solid powder A: preparing a polymer coating on the surface of magnesium particles;

[0023] (2) preparing liquid reagent B: adding polylactic acid-glycolic acid to N-methylpyrrolidone, and shaking and mixing on a shaker to obtain polylactic acid-glycolic acid hydrogel;

[0024] (3) The solid powder A and the liquid reagent B are evenly mixed in a mass volume ratio of 0.5-5 mg:1 mL to prepare a hydrogel with controllable hydrogen production to promote bone defect repair.

[0025] Compared with the prior art, the preparation method of the present invention has the following advantages:

[0026] During the preparation process, the hydrogel of the present invention, by introducing N-methyl pyrrolidone as a solvent, can realize the solvent exchange after the hydrogel is implanted in vivo (N-methyl pyrrolidone is very soluble in water), so that the polylactic acid-glycolic acid polymer is precipitated in situ, that is, without the need to introduce a cross-linking agent or an external stimulus, the in situ solidification of the hydrogel is realized, which is convenient for clinical wide application. When the hydrogel of the present invention is used, the operation is simple, fast and efficient, and it is suitable for a variety of clinical application scenarios. At the same time, the hydrogel of the present invention can form a loose and porous scaffold-like structure at the implant site through the hydrogen generated by the gel curing reaction and the degradation of magnesium particles, which provides favorable conditions for nutrient exchange and cell growth, and can provide a certain strength of mechanical support.

[0027] As a preferred embodiment of the present invention, in the solid powder A, the magnesium particles with a polymer coating are prepared by the following steps:

[0028] (1) Spin-coat 50 μL of polyvinyl pyrrolidone solution on a glass slide. The solvent of the polyvinyl pyrrolidone solution is anhydrous ethanol. After drying, spin-coat once more, and then spray 9-12 mg of Mg particles;

[0029] (2) Spray 90-120 μL of polyethylene glycol solution on the surface of the above slide glass. The solvent of the polyethylene glycol solution is ethyl acetate. After drying overnight, scrape the particles on the surface of the slide glass to obtain magnesium particles with polyethylene glycol coating.

[0030] This method can be used to prepare a polyethylene glycol coating with an asymmetric structure on the surface of magnesium microspheres, that is, the coating has openings on one side, which can retain the ability of magnesium particles to react with water to produce hydrogen.

[0031] As a preferred embodiment of the present invention, in the polyvinyl pyrrolidone solution, the mass volume ratio of polyvinyl pyrrolidone to anhydrous ethanol is 18-22 mg:1 mL; in the polyethylene glycol solution, the mass volume ratio of polyethylene glycol to ethyl acetate is 9-12 mg:1 mL.

[0032] As a preferred embodiment of the present invention, the monomer ratio of polylactic acid-glycolic acid is 50:50; the temperature for shaking and mixing is 35-38° C.; and the time for shaking and mixing is 48 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The characterization results of PEG@Mg microspheres are as follows: A is the scanning electron microscope image of PEG@Mg microspheres; B is the element distribution diagram of PEG@Mg microspheres; C is the particle size distribution diagram of Mg particles and PEG@Mg microspheres.

[0034] Figure 2 The following are the results of characterization of PEG@Mg-PLGA hydrogel: A is the evaluation of the injectability of PEG@Mg-PLGA hydrogel using a 1mL syringe; B is the evaluation of the liquid-solid phase transition ability of PEG@Mg-PLGA hydrogel using deionized water; C is the result of testing the conformability of PEG@Mg-PLGA hydrogel using molds of different shapes; D is the SEM image of the surface of solid PLGA and PEG@Mg-PLGA hydrogel; E is the SEM image of the interior of solid PEG@Mg-PLGA hydrogel; F is the element distribution map of the interior of solid PEG@Mg-PLGA hydrogel; G is the elastic modulus analysis result of solid PLGA and PEG@Mg-PLGA hydrogel. **, P < 0.01.

[0035] Figure 3 These are the results of the hydrogen release experiment of PEG@Mg-PLGA hydrogel: A is a general picture of 1 mL PLGA and PEG@Mg-PLGA hydrogel after being immersed in deionized water for 6 hours; B is a result chart of the hydrogen content released within 7 days after 1 mL PLGA and PEG@Mg-PLGA hydrogel were immersed in deionized water.

[0036] Figure 4The results of the PEG@Mg-PLGA hydrogel cell compatibility experiment are shown in Figure 1. A shows the results of the cell activity analysis after MEFs cells were co-cultured with PLGA or PEG@Mg-PLGA hydrogel for 24h and 48h; B shows the results of the cell activity analysis after RAW264.7 cells were co-cultured with PLGA or PEG@Mg-PLGA hydrogel for 24h and 48h. *, P < 0.05.

[0037] Figure 5 The results of the experiment of PEG@Mg-PLGA hydrogel scavenging intracellular reactive oxygen species: A is the fluorescence image of ROS in MEFs and RAW264.7 cells; B is the flow cytometry quantitative analysis result of ROS in MEFs and RAW264.7 cells. **, P < 0.01; ***, P < 0.001.

[0038] Figure 6 The results of the in vitro anti-inflammatory experiment of PEG@Mg-PLGA hydrogel are shown in Figure 1: A is a fluorescence image of cells labeled with iNOS, Arg-1 and DAPI in RAW264.7 cells; B is a result of the determination of cytokine (TNG-α, IL-1β, TGF-β and IL-1O) concentrations in the culture medium of RAW264.7 cells. **, P < 0.01; ***, P < 0.001.

[0039] Figure 7 The results of the osteoclast inhibition and osteogenic induction experiments of PEG@Mg-PLGA hydrogels are shown in Figure 1: A is the TRAP staining of RAW264.7 cells after osteoclast induction; B is the result of osteoclast counting in the TRAP staining; C is the ALP staining of MEFs cells after osteogenic induction; D is the result of ALP activity determination after osteogenic induction of MEFs cells; E is the ARS staining of MEFs cells after osteogenic induction; F is the quantitative analysis result of ARS staining. **, P < 0.01; ***, P < 0.001.

[0040] Figure 8 The results of the PEG@Mg-PLGA hydrogel repairing osteoporotic bone defect experiment: A is a flowchart of the construction of osteoporotic rat bone defect model and the implantation of PEG@Mg-PLGA hydrogel into bone defects; B is a micro CT 3D reconstruction of the bone defect area at 4 and 8 weeks; C is a HE staining image of the bone defect area at 4 and 8 weeks; D is a ROS fluorescence staining image of the bone defect area at 4 weeks; E is a ROS fluorescence quantitative analysis result of the bone defect area at 4 weeks. ***, P < 0.001.

[0041] Fig. 9The results of the in vivo biosafety experiment of PEG@Mg-PLGA hydrogel are shown in Figure 1. A is the HE staining of the visceral tissue of the experimental rats at 8 weeks; B is the serological analysis result of the experimental rats at 8 weeks. DETAILED DESCRIPTION

[0042] Typical embodiments that embody the features and advantages of the present invention will be described in detail in the following description. It should be understood that the present invention can have various changes in different embodiments without departing from the scope of the present invention, and the descriptions and illustrations therein are essentially used for illustration purposes rather than for limiting the present invention.

[0043] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0044] In the following specific examples, PLGA is polylactic acid-glycolic acid, the molecular weight of PLGA is preferably 40000, and the monomer ratio of PLGA is preferably 50:50; PEG is polyethylene glycol, the molecular weight of PEG is preferably 2000; NMP is N-methylpyrrolidone. The molecular weight and dosage ratio of each component in the preparation process are within the selected range, and the preparation effect is equivalent, so a detailed example is specifically disclosed below.

[0045] In the embodiment of the present invention, the room temperature is 20-30°C.

[0046] Example

[0047] A method for preparing a hydrogel capable of controlling hydrogen production to promote bone defect repair comprises the following specific steps:

[0048] (1) Preparation of solid powder A (PEG@Mg microspheres)

[0049] First, 20 mg of polyvinyl pyrrolidone (PVP) was weighed and dissolved in 1 mL of anhydrous ethanol to obtain a PVP-ethanol solution. 50 μL of the solution was evenly spin-coated on the surface of a glass slide, and then 10 mg of Mg particles were evenly sprayed.

[0050] Weigh 10 mg of PEG and dissolve it in 1 mL of ethyl acetate. Take 100 μL of the polyethylene glycol solution and evenly apply it on the top surface of the above slide and dry it at room temperature overnight. Use a scraper to collect the microspheres on the surface of the slide to obtain PEG@Mg microspheres.

[0051] (2) Preparation of liquid reagent B (PLGA hydrogel)

[0052] 1.5 g of polylactic-co-glycolic acid (PLGA particles) was added to 1 mL of NMP solution and shaken in a constant temperature shaker at 37° C. for 48 hours until PLGA was fully dissolved to obtain PLGA hydrogel.

[0053] (3) Preparation of hydrogels with controllable hydrogen production to promote bone defect repair

[0054] 2 mg of PEG@Mg microspheres were added to the PLGA hydrogel and stirred evenly to obtain a hydrogel with controllable hydrogen production to promote bone defect repair (PEG@Mg-PLGA hydrogel).

[0055] Hydrogel experimental verification

[0056] The following are characterization experiments, controllable hydrogen release experiments, cell compatibility experiments, experiments on eliminating intracellular reactive oxygen species, anti-inflammatory experiments, experiments on promoting osteoblastic differentiation and inhibiting osteoclast differentiation, osteoporotic bone defect repair experiments, and biosafety experiments on the hydrogel prepared in the embodiment of the present invention, as follows:

[0057] 1. Characterization Experiments and Results

[0058] 1. Characterization of solid powder A (PEG@Mg microspheres)

[0059] The solid powder A (PEG@Mg microspheres) prepared in step (1) was subjected to characterization experiments. The characterization experimental methods and experimental results are shown below:

[0060] Characterization experimental methods:

[0061] The morphology of PEG@Mg microspheres was observed using a scanning electron microscope (SEM, ZEISS Sigma 300), and the distribution of elements on the surface of the microspheres was detected by X-ray energy dispersive spectroscopy (EDS). At the same time, the sizes of pure Mg particles and PEG@Mg microspheres were measured and analyzed and compared.

[0062] Experimental results:

[0063] According to the SEM image results ( Figure 1 A), PEG@Mg microspheres have an asymmetric coating, that is, one side of the coating has a small opening (about 15 μm in diameter). Combined with EDS results analysis ( Figure 1 B), the interior of PEG@Mg microspheres is Mg from Mg particles, while the surface coating is C, N and O from PEG. In addition, the particle size distribution results show that ( Figure 1 C), compared with pure Mg particles (49.49±5.14μm), PEG@Mg microspheres have a larger particle size (55.78±4.65μm), which may be related to the formation of its surface coating. The above results prove that PEG@Mg microspheres with asymmetric PEG coating are successfully prepared.

[0064] 2. Characterization of hydrogel with controllable hydrogen production to promote bone defect repair (PEG@Mg-PLGA hydrogel)

[0065] The controllable hydrogen production-promoting bone defect repair hydrogel (PEG@Mg-PLGA hydrogel) prepared in step (3) was subjected to a characterization experiment. The characterization experimental method and experimental results adopted are as follows:

[0066] Characterization experimental methods:

[0067] 0.5 mL of PEG@Mg-PLGA hydrogel was filled into the barrel of a 1 mL syringe (needle inner diameter 1.2 mm), and the hydrogel was injected by squeezing to test its injectability.

[0068] 0.5 mL of PEG@Mg-PLGA hydrogel was added dropwise into deionized water through the syringe, taken out with tweezers after 5 minutes, and photographed to record the change process of its physical properties and analyze its liquid-solid phase transition ability.

[0069] PEG@Mg-PLGA hydrogel was injected into molds with different shapes. After 5 minutes, the mold filled with PEG@Mg-PLGA hydrogel was immersed in deionized water for 5 minutes, then taken out and the conformability of PEG@Mg-PLGA hydrogel was observed.

[0070] The cured PLGA and PEG@Mg-PLGA hydrogels were subjected to SEM and EDS examinations to observe the surface and internal morphology and element distribution of the materials.

[0071] The elastic modulus of the cured PLGA and PEG@Mg-PLGA hydrogels was measured using an electronic universal testing machine (INSTRON 5982).

[0072] Experimental results:

[0073] like Figure 2 As shown in A, PEG@Mg-PLGA hydrogel can be smoothly extruded through a 1 mL syringe, which means it is injectable.

[0074] like Figure 2 As shown in B, after the PEG@Mg-PLGA hydrogel was added to deionized water for 5 minutes, it was observed that it changed from a liquid gel to a solid state, indicating that the hydrogel can achieve a rapid liquid-solid phase transition in an aqueous solution environment.

[0075] like Figure 2 As shown in Figure C, after the PEG@Mg-PLGA hydrogel was dropped into molds of different shapes, it was found that the shape of the cured PEG@Mg-PLGA hydrogel was completely consistent with the internal contour of the mold, indicating that the hydrogel has excellent conformability and can fill defects of different shapes after injection and in situ curing.

[0076] like Figure 2As shown in D, after solidification, the PEG@Mg-PLGA hydrogel containing PEG@Mg microspheres (2 mg / mL) has more and larger pores formed on its surface than the solid PLGA hydrogel.

[0077] The SEM and EDS results of solid-state PEG@Mg-PLGA hydrogel showed that ( Figure 2 E, 2F), the interior of which is a loose and porous scaffold-like structure, and the Mg element can be evenly distributed in the hydrogel. The mechanical detection analysis found that ( Figure 2 G), the elastic modulus of the solid PEG@Mg-PLGA hydrogel was slightly lower than that of PLGA, which was about 26.72±2.92Mpa.

[0078] The above experimental results show that the hydrogel of the present invention can be used to fill irregular bone defects through minimally invasive injection, and form a loose and porous scaffold-like structure in situ through liquid-solid phase transition, which is conducive to the growth of tissues and cells and provides effective mechanical support for the implanted site. At the same time, the hydrogel of the present invention can be implanted without light or ionic crosslinking agents, which is simple, fast and efficient, and is suitable for a variety of clinical application scenarios.

[0079] II. Experiment on the Controllable Release of Hydrogen by the Hydrogel of the Present Invention in Vitro

[0080] Experimental methods:

[0081] 1 mL of PEG@Mg-PLGA hydrogel was added to the methylene blue probe solution (MB-Pt). Based on the absorbance change of the MB-Pt probe solution at 664 nm and combined with the MB standard concentration curve, the hydrogen released by the hydrogel within 7 days can be quantitatively detected. In addition, 1 mL of PEG@Mg-PLGA hydrogel was added to deionized water, and photos were taken to record the bubble generation on the hydrogel surface.

[0082] Experimental results:

[0083] like Figure 3 As shown in A, after the hydrogel was added to deionized water for 6 hours, no obvious bubbles were observed on the surface of the PLGA hydrogel, while a large number of bubbles were observed on the surface of the PEG@Mg-PLGA hydrogel. Figure 3 B shows that PEG@Mg-PLGA hydrogel can continuously release hydrogen within 7 days, and its release rate is faster within the first 24 hours, while the release rate gradually decreases within 24 hours to 7 days.

[0084] 3. Experiment on Cytocompatibility of Hydrogels of the Present Invention

[0085] Experimental methods:

[0086] To evaluate the cytocompatibility of the hydrogel, mouse mononuclear macrophage RAW264.7 cells (5×10 3 ) or mouse embryonic fibroblasts MEFs (5×10 3 ) were inoculated in a 24-well plate. After culturing for 12 hours, 50 μL of different hydrogels were added and cultured for another 48 hours, while the wells without hydrogels were set as the control group. After culturing for 24 and 48 hours, Cell Counting Kit-8 (CCK-8) reagent (10% volume fraction) was added to each well and incubated at 37°C for 1 hour. Subsequently, the absorbance of the cell supernatant in each well was measured at 450 nm using an ELISA reader. The absorbance values ​​are shown as percentages of the control values.

[0087] Experimental Results

[0088] like Figure 4 As shown in the figure, both PLGA and PEG@Mg-PLGA showed good cell compatibility. At the same time, PEG@Mg-PLGA hydrogel had a certain promoting effect on the proliferation of MEFs cells.

[0089] IV. Experiment on Eliminating Intracellular Reactive Oxygen Species by the Hydrogel of the Present Invention

[0090] Experimental methods:

[0091] Mouse mononuclear macrophage RAW264.7 (5×10 3 ) or mouse embryonic fibroblasts MEFs (5×10 3 ) were inoculated in 24-well plates and cultured for 12 h. To simulate the environmental stimulation of oxidative stress, hydrogen peroxide solution (H 2 O 2 ) to a final concentration of 200 μM, and the culture was continued for 12 h. The wells to which 50 μL of PLGA and PEG@Mg-PLGA hydrogels were added were designated as the PLGA group and the PEG@Mg-PLGA group, respectively, while the wells without hydrogels were designated as the H 2 O 2 The group was placed in a cell culture incubator and cultured for 12 hours. 2 O 2 The wells containing normal culture medium were set as negative control groups. DCFH-DA and DAPI reagents were used to label ROS and cell nuclei in cells, respectively, and then fluorescence microscopy and flow cytometry were used to analyze the ROS levels in cells in each well.

[0092] Experimental Results

[0093] like Figure 5 As shown in A, compared with the negative control group, H 2 O 2In the group, obvious green fluorescence (DCF) was observed in the cells, indicating that the added H 2 O 2 This results in the generation of a large amount of reactive oxygen species (ROS) in cells. 2 O 2 The green fluorescence intensity in the PEG@Mg-PLGA group was significantly reduced, indicating that the PEG@Mg-PLGA hydrogel reduced the intracellular ROS level under oxidative stress relief. Figure 5 As shown in (B), flow cytometric analysis results further confirmed the ability of PEG@Mg-PLGA hydrogel to eliminate intracellular ROS.

[0094] V. In vitro anti-inflammatory experiment of the hydrogel of the present invention

[0095] Experimental methods:

[0096] Mouse mononuclear macrophages (RAW264.7) were cultured at a rate of 5×10 3 Each well was inoculated in a 24-well plate, and H 2 O 2 The final concentration was 200 μM and the culture was continued for 12 hours. The wells to which 50 μL of PLGA and PEG@Mg-PLGA hydrogel were added were designated as the PLGA group and the PEG@Mg-PLGA group, and the wells without hydrogel were designated as the H 2 O 2 group. And will use the H 2 O 2 The wells containing ordinary culture medium were set as negative control groups. After culturing in a cell culture incubator for 3 days, the cell supernatant in each well was collected, and the cells were fixed with 4% paraformaldehyde. On the one hand, the M1 and M2 macrophages were stained and marked with iNOS and Arg-1 antibodies, respectively, by cell immunofluorescence technology, and then the polarization of macrophages in each group was analyzed using fluorescence microscopy. On the other hand, the cytokines secreted by M1 and M2 macrophages (M1: TNF-α, IL-1β; M2: TGF-β, IL-10) were detected by enzyme-linked immunosorbent assay (ELISA) kits to analyze the inhibition of cell inflammation by each group of hydrogels.

[0097] Experimental Results

[0098] like Figure 6 As shown in A, compared with H 2 O 2 In the PEG@Mg-PLGA group, the red fluorescence intensity of iNOS in the cells was significantly reduced, while the green fluorescence intensity of Arg-1 was significantly increased. Figure 6 B) Tips, compared to H 2 O2 In the PEG@Mg-PLGA group, the M1 cell-related factors were significantly reduced, while the M2 cell-related factors were significantly increased. Combined with the above results, PEG@Mg-PLGA hydrogel can promote the polarization of RAW264.7 cells into M2 macrophages under oxidative stress and inhibit their polarization into M1 macrophages, thereby achieving the effect of inhibiting inflammation.

[0099] VI. Experiment on the hydrogel of the present invention promoting osteogenic differentiation and inhibiting osteoclast differentiation in vitro

[0100] Experimental methods:

[0101] Mouse mononuclear macrophages (RAW264.7) were cultured at a rate of 5×10 3 Each well was inoculated in a 24-well plate. After overnight culture in a cell culture incubator, the normal culture medium was replaced with osteoclast induction medium, and 50 μL of PLGA and PEG@Mg-PLGA hydrogels were added to the PLGA group and PEG@Mg-PLGA group, respectively. The wells without hydrogel were set as osteoclast induction groups. In addition, the wells that continued to use normal culture medium were set as negative control groups. After 7 days of osteoclast induction, the cells in the wells were fixed with 4% paraformaldehyde and stained with tartrate-resistant acid phosphatase (TRAP), and the osteoclast differentiation of each group was analyzed by counting TRAP-positive cells under a microscope.

[0102] Mouse embryonic fibroblasts (MEFs) with multidirectional differentiation potential were cultured at a rate of 5×10 3 Each well was inoculated in a 24-well plate. After overnight culture in a cell culture incubator, the normal culture medium was replaced with osteogenic induction medium, and 50 μL of PLGA and PEG@Mg-PLGA hydrogels were added to the PLGA group and PEG@Mg-PLGA group, respectively. The wells without hydrogel were set as the osteogenic induction group. In addition, the wells that continued to use the normal culture medium were set as the negative control group. After 7 days of osteogenic induction culture, alkaline phosphatase (ALP) staining and ALP activity detection were performed on the cells in the well plate to analyze the osteogenic differentiation of MEFs; after 14 days of osteogenic induction culture, the cells in the well plate were fixed with 4% paraformaldehyde, stained with alizarin red (ARS) and photographed. In addition, the complex of calcium salt and alizarin red in each well was dissolved with 20% acetic acid, and the absorbance of the complex at 405nm was measured by a spectrophotometer to quantitatively analyze the biomineralization in the well.

[0103] Experimental results:

[0104] like Figure 7As shown in A and 7B, compared with the osteoclast induction group, the number of osteoclasts in the PEG@Mg-PLGA group was significantly reduced, indicating that the PEG@Mg-PLGA hydrogel has the effect of inhibiting osteoclastogenesis. In addition, ALP staining ( Figure 7 C) Quantification of ALP activity ( Figure 7 D) ARS staining and quantitative analysis results ( Figure 7 E, 7F), it was found that PEG@Mg-PLGA can promote osteogenic differentiation and in vitro biomineralization of MEFs cells, indicating that it has the effect of promoting osteogenesis. In summary, the hydrogel of the present invention has the effect of inhibiting osteoclastogenesis and promoting osteogenic differentiation, and is expected to correct the osteoblast-osteoclast imbalance caused by osteoporosis after implantation in vivo.

[0105] VII. Experimental study on repairing osteoporotic bone defects using hydrogel of the present invention

[0106] Experimental methods

[0107] Establishment of SD rat osteoporosis model: Female SD rats (10 weeks old, 250-300 g) were anesthetized by intraperitoneal injection of sodium pentobarbital solution (2 wt%, 40 mg / kg), and bilateral ovaries were removed under aseptic surgical conditions. After 3 months, the rat osteoporosis model was established.

[0108] Establishment of osteoporotic bone defect model in SD rats: The osteoporotic rats obtained above were anesthetized by intraperitoneal injection of 2wt% sodium pentobarbital (40 mg / kg), and the lateral femoral condyle was exposed using surgical instruments. A cylindrical bone defect with a diameter of 3 mm and a depth of 3 mm was drilled on the lateral femoral condyle of the rat using an orthopedic drill with a diameter of 3 mm. During the drilling process, 0.9% saline was continuously perfused to prevent thermal damage. After the defect was completed, about 20 μL of different materials were injected through a 1 mL syringe to completely fill the bone defect, and 0.9% saline was added to immerse the hydrogel for 5 minutes. Then, the wound was sutured in layers with 3-0 surgical sutures and disinfected. The experimental rats were divided into three groups according to the different injected materials: (1) control group (20 μL PBS); (2) PLGA group (20 μL PLGA hydrogel); (3) PEG@Mg-PLGA group (20 μL PEG@Mg-PLGA hydrogel). At 4 and 8 weeks after implantation, three rats in each group were sacrificed for micro CT scanning and histopathological analysis (HE and ROS staining).

[0109] Experimental Results

[0110] like Figure 8 As shown in A, PEG@Mg-PLGA hydrogel can fully fill the bone defect and transform into a solid state after being soaked in saline. Figure 8 B), at 4 and 8 weeks, only a small amount of new bone tissue was generated at the defect edge in the control group and PLGA group. In the PEG@Mg-PLGA group, a large amount of new bone tissue was observed in the middle area of ​​the defect at 4 weeks; at 8 weeks, the interior of the bone defect was almost completely filled with new bone tissue. The results of quantitative analysis of new bone showed that the amount of new bone in the PEG@Mg-PLGA group increased significantly compared with the control group and PLGA group. Figure 8 C), the bone defects in the control group were mainly filled with fibrous tissue at the 4th and 8th weeks, indicating that the osteoporotic bone defects could not be repaired naturally. In the PLGA group, only a small amount of new bone was formed at the edge of the defect 8 weeks after implantation. In the PEG@Mg-PLGA group, nearly half of the bone defect area was repaired at 4 weeks, and a small amount of incompletely degraded hydrogel and newly grown bone tissue were visible, indicating that the hydrogel of the present invention can be gradually degraded after implantation in the body and guide the growth of new bone tissue. 8 weeks after implantation, the PEG@Mg-PLGA hydrogel was completely degraded, and the bone defect area was filled with a large amount of remodeled bone tissue, indicating that the hydrogel of the present invention can promote the repair of osteoporotic bone defects. In addition, Figure 8 It can be seen from D that PEG@Mg-PLGA hydrogel can partially remove ROS in the implanted area and alleviate the local oxidative stress in osteoporotic bone defects.

[0111] In summary, the hydrogel of the present invention can be implanted and filled in osteoporotic bone defects in a minimally invasive manner. After implantation in the body, it can gradually degrade, alleviate local ROS and significantly promote the generation and ingrowth of new bone tissue, ultimately achieving the repair of osteoporotic bone defects.

[0112] 8. In vivo biosafety experiment of the hydrogel of the present invention

[0113] Experimental methods

[0114] For the experimental rats in Example 8, material biosafety testing was performed at 8 weeks, including serological analysis (ALT, AST, BUN, CREA and magnesium ion detection), as well as sectioning and HE staining of important organs such as the rat heart, liver, spleen, lung, kidney and brain.

[0115] Experimental Results

[0116] Serological results of experimental rats ( Fig. 9 A) showed that the ALT, AST, BUN and CREA indicators of the PEG@Mg-PLGA group were all within the normal range, and the concentration of magnesium ions in the serum was not abnormal, indicating that the hydrogel of the present invention did not affect the liver and kidney functions of rats. In addition, the tissue sections of important organs were stained ( Fig. 9B) shows that the hydrogel of the present invention does not cause pathological changes in various organs after implantation in vivo. In summary, the hydrogel of the present invention has excellent biosafety and good clinical transformation potential.

[0117] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A hydrogel capable of controlling hydrogen production to promote bone defect repair, characterized in that: It is made by mixing two components: solid powder A and liquid reagent B: The solid powder A is magnesium particles with a high molecular polymer coating, and the high molecular polymer coating is made of polyethylene glycol without water; The liquid reagent B is polylactic acid-glycolic acid hydrogel, and the organic solvent used in the polylactic acid-glycolic acid hydrogel is N-methylpyrrolidone; The solid powder A is prepared by the following steps: (1) Spin coat 50 μL of polyvinyl pyrrolidone solution on a glass slide. The solvent of the polyvinyl pyrrolidone solution is anhydrous ethanol. After drying, repeat the spin coating once, and then spray 9-12 mg of Mg particles; (2) Spray 90-120 μL of polyethylene glycol solution onto the surface of the glass slide. The solvent of the polyethylene glycol solution is ethyl acetate. After drying overnight, scrape the particles on the surface of the glass slide to obtain magnesium particles with a polyethylene glycol coating.

2. The controllable hydrogen production-promoting bone defect repair hydrogel according to claim 1, characterized in that: In the solid powder A, the magnesium particles are spherical particles with a diameter of 5-500 μm.

3. The controllable hydrogen production-promoting bone defect repair hydrogel according to claim 2, characterized in that: The diameter of the magnesium particles is 30-70 μm, and the molecular weight of the polyethylene glycol is 1500-2500.

4. The controllable hydrogen production-promoting bone defect repair hydrogel according to claim 2, characterized in that: In the liquid reagent B, the components of the polylactic acid-glycolic acid hydrogel are polylactic acid-glycolic acid polymer and an organic solvent.

5. The controllable hydrogen production-promoting bone defect repair hydrogel according to claim 4, characterized in that: The molecular weight of the polylactic acid-glycolic acid polymer is 35000-45000, and the organic solvent is N-methylpyrrolidone.

6. The controllable hydrogen production-promoting bone defect repair hydrogel according to claim 5, characterized in that: The mass volume ratio of the polylactic acid-glycolic acid to N-methylpyrrolidone is 0.5-3 g:1 mL.

7. A method for preparing a hydrogel capable of controlling hydrogen production to promote bone defect repair according to any one of claims 1 to 6, characterized in that: The steps include: (1) Preparing solid powder A: Preparing a polymer coating on the surface of magnesium particles; (2) preparing liquid reagent B: adding polylactic acid-glycolic acid to N-methylpyrrolidone and mixing by shaking on a shaker to obtain polylactic acid-glycolic acid hydrogel; (3) The solid powder A and the liquid reagent B are evenly mixed in a mass volume ratio of 0.5-5 mg:1 mL to prepare a hydrogel with controllable hydrogen production to promote bone defect repair.

8. The controllable hydrogen production-promoting bone defect repair hydrogel according to claim 1, characterized in that: In the polyvinyl pyrrolidone solution, the mass volume ratio of polyvinyl pyrrolidone to anhydrous ethanol is 18-22 mg:1 mL; in the polyethylene glycol solution, the mass volume ratio of polyethylene glycol to ethyl acetate is 9-12 mg:1 mL.

9. The method for preparing the hydrogel capable of controlling hydrogen production to promote bone defect repair according to claim 7, characterized in that: The monomer ratio of polylactic acid-glycolic acid is 50:50; the temperature of shaking and mixing is 35-38° C.; and the time of shaking and mixing is 12-96 hours.

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