A nanocomposite hydrogel based on Mg-MOFs and its preparation method and application

Abstract

The present invention provides a nanocomposite hydrogel based on Mg-MOFs, wherein the nanocomposite hydrogel has hollow mesoporous silica nanoparticles (HMSNs) as the core and magnesium-gallic acid metal organic framework (Mg-MOFs) as the shell to form a core-shell nanocomposite HMMs; after the core-shell nanocomposite HMMs is subjected to an amination treatment, naringin (Nar) is loaded to obtain a drug-loaded core-shell nanocomposite (N-HMMs@Nar); the drug-loaded core-shell nanocomposite (N-HMMs@Nar) is subjected to a cross-linking reaction with methacrylate gelatin and PEGDA to form a nanocomposite hydrogel. The N-HMMs@Nar@GP hydrogel prepared by the present invention has great application potential in bone defect repair.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a Mg-MOFs-based nanocomposite hydrogel and a preparation method thereof, and application of the nanocomposite hydrogel in preparing a bone defect repair material. Background Art

[0002] Metal-organic frameworks (MOFs) are porous crystalline materials composed of metal nodes and organic bridges. They are ideal materials for metal ion storage and release due to their rich metal active sites, high specific surface area, and high porosity. MOFs have been shown to have unique biological activity due to surface group structure modification and the release of metal ions when the framework disintegrates. However, the biocompatibility of MOFs limits their therapeutic effects, so it is extremely important to improve their biostability and cell affinity.

[0003] Gelatin methacrylate (GelMA) hydrogel is widely used in biomaterials due to its drug sustained release performance and excellent biocompatibility. As a drug carrier, GelMA hydrogel can improve the therapeutic effect of drugs in the body through drug sustained release. Therefore, GelMA hydrogel plays an important role in tissue engineering.

[0004] Although studies have been conducted on the use of hydrogels as carriers for loading protein drugs, current research has not been able to provide effective treatment for bone defects. Therefore, existing hydrogel drug delivery platforms are difficult to effectively restore bone defects, have poor efficacy, and a long recovery period. This problem urgently needs to be solved. Summary of the invention

[0005] The present invention provides a nanocomposite hydrogel based on Mg-MOFs. The hydrogel has good biocompatibility, can promote osteogenic differentiation, is beneficial to angiogenesis, and effectively improves the repair ability of tibia in vivo.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A Mg-MOFs-based nanocomposite hydrogel, wherein the nanocomposite hydrogel has hollow mesoporous silica nanoparticles (HMSNs) as the core and magnesium-gallic acid metal organic frameworks (Mg-MOFs) as the shell to form a core-shell nanocomposite material (HMMs);

[0008] The core-shell nanocomposite material (HMMs) is subjected to an amination treatment and then loaded with naringin (Nar) to obtain a drug-loaded core-shell nanocomposite material (N-HMMs@Nar);

[0009] The drug-loaded core-shell nanocomposite material (N-HMMs@Nar) is cross-linked with methacrylate gelatin and PEGDA to form a nanocomposite hydrogel.

[0010] In some embodiments, the preparation process of the core-shell nanocomposite material (HMMs) includes: dissolving hollow mesoporous silica nanoparticles (HMSNs), gallic acid, and anhydrous magnesium chloride in water and mixing them, reacting at 100° C. to 150° C., and cooling to room temperature to prepare the core-shell nanocomposite material (HMMs).

[0011] In some embodiments, the molar ratio of gallic acid to anhydrous magnesium chloride is 1-3:1, preferably, the molar ratio of gallic acid to anhydrous magnesium chloride is 2-3:1, and more preferably, the molar ratio of gallic acid to anhydrous magnesium chloride is 2.13:1. In some embodiments, the mass ratio of hollow mesoporous silica nanoparticles (HMSNs) to magnesium-gallic acid is 1-10:1. Preferably, the mass ratio is 2-5:1. More preferably, the mass ratio is 2.5:1.

[0012] Wherein, the magnesium-gallic acid is a mixture of gallic acid and anhydrous magnesium chloride.

[0013] In some embodiments, the core-shell nanocomposites (HMMs) are subjected to an amination treatment to prepare aminated core-shell nanocomposites (N-HMMs).

[0014] In some embodiments, the step of preparing the core-shell nanocomposite material (HMMs) by amination treatment comprises: dissolving HMMs in an organic solvent, heating to 60°C to 100°C, adding APTES, and reacting under argon protection to prepare an amination core-shell nanocomposite material (N-HMMs). The organic solvent is selected from methanol and ethanol. Preferably, ethanol.

[0015] In some embodiments, the preparation process of the hollow mesoporous silica nanoparticles (HMSNs) comprises:

[0016] (1) Add tetraethyl orthosilicate to anhydrous ethanol, ammonia and deionized water, react for 1-2 hours and then centrifuge to collect sSiO 2 , and resuspended in deionized water for later use;

[0017] (2) Add hexadecyltrimethylammonium bromide, triethanolamine and sSiO prepared in step (1) into deionized water. 2 , stir until completely mixed, slowly add tetraethyl orthosilicate, react at 60℃~70℃ for 5-10 hours, collect the lower aqueous phase solution to obtain CTAB / mSiO 2 @sSiO 2;

[0018] (3) Add water to the lower aqueous phase to make up the volume, then add sodium carbonate and vigorously stir for 0.5-2h, and extract with deionized water and methanol solution of sodium chloride in turn to obtain hollow mesoporous silica nanoparticles (HMSNs).

[0019] In some embodiments, the preparation process of the drug-loaded core-shell nanocomposite (N-HMMs@Nar) includes: mixing the aminated core-shell nanocomposite (N-HMMs) with naringin (Nar), slowly stirring for 5-8 hours, centrifuging, and drying to prepare the drug-loaded core-shell nanocomposite (N-HMMs@Nar).

[0020] In some embodiments, the mass ratio of the core-shell nanocomposite material to naringin is 1:0.5 to 4. Preferably, the mass ratio is 1:2.

[0021] In some embodiments, the preparation step of the nanocomposite hydrogel includes: dissolving methacrylate gelatin, PEGDA and the drug-loaded core-shell nanocomposite in a buffer solution, protecting from light, adding a photoinitiator, and irradiating with ultraviolet light for cross-linking reaction to obtain a nanocomposite hydrogel. Preferably, methacrylate gelatin, PEGDA and the drug-loaded core-shell nanocomposite are dissolved in PBS buffer solution for dissolution.

[0022] Furthermore, the prepared nanocomposite hydrogel is further immersed in PBS buffer for 1-2 hours and then freeze-dried to obtain the nanocomposite hydrogel.

[0023] In some embodiments, the UV light irradiation time is 8-10 s.

[0024] In some embodiments, the concentration of the methacrylate gelatin is 5%-20% (w / v). Preferably, the concentration of the methacrylate gelatin is 5%-15% (w / v). More preferably, the concentration of the methacrylate gelatin is 10% (w / v).

[0025] In some embodiments, the concentration of PEGDA is 1%-10% (w / v), preferably, the concentration of methacrylate is 2%-5% (w / v), and more preferably, the concentration of methacrylate is 5% (w / v).

[0026] In some embodiments, the mass ratio of methacrylate gelatin to PEGDA is 2-5:1. Preferably, the mass ratio is 2:1.

[0027] In some embodiments, the mass ratio of the methacrylate gelatin, PEGDA and the drug-loaded core-shell nanocomposite material is 10-30:5-20:1. Preferably, the mass ratio is 20:10:1.

[0028] In some embodiments, the photoinitiator is Irgacure 2959.

[0029] In some embodiments, the preparation step of the methacrylate gelatin includes: dissolving gelatin in PBS until it is completely dissolved, adding methacrylic anhydride, reacting for 2-5 hours, adding a stop solution to terminate the reaction, and obtaining methacrylate gelatin. Preferably, the stop solution is selected from PBS buffer.

[0030] Furthermore, the mass volume ratio of gelatin to methacrylic anhydride is 1:5-10, preferably, the mass volume ratio is 1:8.

[0031] On the other hand, the present invention also provides a method for preparing a nanocomposite hydrogel based on Mg-MOFs, the preparation method comprising the following steps:

[0032] A) HMSNs, gallic acid and anhydrous magnesium chloride were dissolved in water and mixed, reacted at 100°C to 150°C for 26-30 hours, and cooled to room temperature to prepare core-shell nanocomposites (HMMs);

[0033] B) HMMs were dissolved in an organic solvent, heated to 60°C~100°C, APTES was added, and the reaction was carried out under argon protection for 4-8 hours to prepare an amino core-shell nanocomposite material (N-HMMs);

[0034] C) dissolving the aminated core-shell nanocomposite (N-HMMs) and naringin (Nar) in deionized water and mixing them, then slowly stirring for 5-8 hours, centrifuging, and drying to prepare the drug-loaded aminated core-shell nanocomposite;

[0035] D) Methacrylate gelatin, PEGDA and drug-loaded amino core-shell nanocomposite are dissolved in a buffer solution, protected from light, a photoinitiator is added, and ultraviolet light is irradiated for cross-linking reaction to obtain a nanocomposite hydrogel.

[0036] In some embodiments, the organic solvent in step B) is selected from methanol and ethanol, preferably ethanol.

[0037] On the other hand, the present invention also provides the use of the Mg-MOFs-based nanocomposite hydrogel as described above in the preparation of a bone tissue repair material.

[0038] Beneficial effects:

[0039] The present invention uses hollow mesoporous silica nanoparticles (HMSNs) as the core and magnesium-gallic acid metal organic frameworks (Mg-MOFs) as the shell to prepare a core-shell nanocomposite material. After further amino treatment, the small molecule bone-inducing drug naringin (Nar) is loaded to prepare a core-shell nanocomposite material (N-HMMs@Nar). N-HMMs@Nar is loaded into gelatin methacrylate / polyethylene glycol diacrylate hydrogel (GelMA / PEGDA, GP) to construct a nanocomposite hydrogel (N-HMMs@Nar@GP) for synergistic osteogenesis and angiogenesis in bone regeneration. Specifically, nanoscale N-HMMs not only serve as a carrier of Nar, but also can slowly release bioactive magnesium ions (Mg) through the natural degradation of Mg-MOFs. 2+ ) and antioxidant gallic acid. In order to adapt to the traumatic characteristics of bone defects and create conditions for in situ treatment, N-HMMs@Nar was completely encapsulated in an injectable photoreactive GP gel. In vitro studies have shown that N-HMMs@Nar@GP has good biocompatibility and can promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and angiogenesis of human umbilical vein endothelial cells (HUVECs). Micro-CT and histopathological examinations confirmed the ability of N-HMMs@Nar@GP to promote osteogenesis and angiogenesis in an in vivo tibial defect model. Therefore, the N-HMMs@Nar@GP hydrogel prepared by the present invention has great application potential in bone defect repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the characterization spectrum of HMSNs / HMMs / N-HMMs / N-HMMs@Nar in Example 1 ( Figure 1 A in Figure 1 B in Figure 1 The C in them are sSiO 2 , TEM images of HMSNs and N-HMMs; Figure 1 D in the equation is sSiO 2 / Particle size distribution curve of HMSNs / HMMs; Figure 1 E in it is the XRD pattern of HMSNs / Mg-MOFs / HMMs; Figure 1 F in it is the FT-IR spectrum of HMSNs / Mg-MOFs / HMMs / N-HMMs / N-HMMs@Nar; Figure 1 G in it is the nitrogen adsorption-desorption curve of HMSNs and HMMs; Figure 1 H in Figure 1 I in the figure are the surface pore size distribution diagrams of HMSNs and HMMs respectively);

[0041] Figure 2Zeta potential diagram of HMSNs / Mg-MOFs / N-HMMs / Nar / N-HMMs@Nar of Example 1;

[0042] Figure 3 is the characterization spectrum of GelMA / GP hydrogel in Example 1 ( Figure 3 A in the figure is the H of GelMA 1 - NMR spectra; Figure 3 B is the apparent morphology of GP hydrogels with different mass ratios after UV cross-linking; Figure 3 C in the figure is the FT-IR spectra of GelMA, PEGDA and GP hydrogels; Figure 3 D in the figure is the in vitro swelling curve of GP hydrogels with different mass ratios; Figure 3 E in the figure is the in vitro degradation rate curve of GP hydrogels with different mass ratios; Figure 3 F in the figure is the compression resistance curve of GP hydrogels with different mass ratios);

[0043] Figure 4 is the characterization spectrum of N-HMMs@Nar@GP in Example 1 ( Figure 4 A in Figure 4 B in the figure are SEM images of GP hydrogel and N-HMMs@Nar@GP hydrogel, respectively; Figure 4 C in N-HMMs and N-HMMs@GP for Mg 2+ Release rate curve graph; Figure 4 D in the figure is the curve of Nar release rate of N-HMMs@Nar and N-HMMs@Nar@GP);

[0044] Figure 5 Figure 2 shows the cytotoxicity study of N-HMMs@GP hydrogel. Figure 5 A in Figure 5 B in the figure shows the effects of N-HMMs, GP hydrogel and N-HMMs@GP hydrogel on the survival rates of BMSCs and HUVECs; Figure 5 C in Figure 5 D in the figure are live / dead cell staining images of N-HMMs, GP hydrogel and N-HMMs@GP hydrogel respectively);

[0045] Figure 6 Figure 2 shows the in vitro osteogenic and angiogenic functions of N-HMMs@Nar@GP hydrogel. Figure 6 A in Figure 6 C in the figure are the staining images of Alizarin Red (ARS) and Alkaline Phosphatase (ALP), respectively; Figure 6 B in the figure is the change of ARS activity of osteogenic mineralized nodules by N-HMMs@Nar@GP hydrogel; Figure 6D in the figure is the change of ALP activity in promoting osteogenic differentiation of BMSCs by N-HMMs@Nar@GP hydrogel);

[0046] Figure 7 Figure 2 shows the in vitro angiogenic activity of N-HMMs@Nar@GP hydrogel. Figure 7 A in the figure is a microscopic image of the migration efficiency of endothelial cells; Figure 7 Microscope image of the B tubule formation effect; Figure 7 C in the figure is the relative migration efficiency area diagram of endothelial cells; Figure 7 D in Figure 7 E in the figure are statistical diagrams of tubule formation effect);

[0047] Figure 8 Figure 2 shows the in vivo tibial repair effect of N-HMMs@Nar@GP hydrogel ( Figure 8 A in the figure is the micro-CT image of tibial repair in vivo; Figure 8 B in Figure 8 C in the figure are quantitative analysis of new bone volume (BV) and new bone volume / tissue volume (BV / TV) analysis graphs; Figure 8 D in Figure 8 Figures E and B are histological analyses of the defect sites using H&E staining and Masson's trichrome staining, respectively). DETAILED DESCRIPTION

[0048] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0049] Unless otherwise specified, the reagents or consumables used in the present invention are commercially available.

[0050] Abbreviations:

[0051] CTAB: hexadecyltrimethylammonium bromide; APTES: 3-aminopropyltriethoxysilane; PEGDA: polyethylene glycol diacrylate.

[0052] Test Materials:

[0053] Tetraethyl orthosilicate, hexadecyltrimethylammonium bromide, triethanolamine, sodium carbonate, gallic acid, anhydrous magnesium chloride, 3-aminopropyltriethoxysilane, gelatin, methacrylic anhydride, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, polyethylene glycol diacrylate, naringin, methanol, anhydrous ethanol and ammonia water: Shanghai Aladdin Biochemical Technology Co., Ltd. Rat bone marrow mesenchymal stem cells, penicillin-streptomycin solution (double antibody), phosphate buffer, recombinant trypsin digestion solution (without phenol red): Wuhan Punosai Life Science Technology Co., Ltd. Human umbilical vein endothelial cells, human umbilical vein endothelial cell special culture medium: Shanghai Saibaikang Biotechnology Co., Ltd. Mesenchymal stem cell special serum, α-MEM (containing glutamine and phenol red): Shanghai Xiaopeng Biotechnology Co., Ltd. 4% paraformaldehyde fixative, CCK-8 kit, Calcein / PI cell viability and cytotoxicity detection kit, BCIP / NBT alkaline phosphatase colorimetric kit, Alizarin Red S staining solution (0.2%, pH 8.3), immunostaining blocking buffer, hematoxylin and eosin (HE) staining kit, Masson trichrome staining kit: Shanghai Bio-Tech Biotechnology Co., Ltd. Matrigel: Corning Incorporated, USA.

[0054] Example 1 Preparation of N-HMMs@Nar@GP hydrogel

[0055] 1.1 Synthesis of N-HMMs:

[0056] 5 mL of tetraethyl orthosilicate was added to 180 mL of anhydrous ethanol, 4 mL of ammonia water and 25 mL of deionized water, and then centrifuged to collect sSiO 2 And resuspend in deionized water for later use. Add 30g hexadecyltrimethylammonium bromide, 0.9mL triethanolamine and sSiO 2 Stir until completely mixed, slowly add 10 mL of tetraethyl orthosilicate, react at 60 °C for 6 hours, collect the lower aqueous phase solution to obtain CTAB / mSiO 2 @sSiO 2 The obtained aqueous solution was fixed to 360 mL with water, and then 7.64 g of sodium carbonate was added and stirred vigorously for 1 h. The solution was extracted with deionized water and methanol solution of sodium chloride for several times to completely remove sSiO 2and hexadecyltrimethylammonium bromide to obtain hollow mesoporous silica nanoparticles (HMSNs). Add 0.5g HMSNs to 30mL deionized water, add 0.2g of gallic acid and anhydrous magnesium chloride with a molar ratio of 2.13:1, place in a polytetrafluoroethylene-lined autoclave, react at 110℃ for 24h, cool to room temperature to obtain the product HMSNs@Mg-MOFs (abbreviated as HMMs). Wash with deionized water and anhydrous ethanol. Add 0.1g HMMs to 150mL anhydrous ethanol, add 1mL APTES after stabilization at 80℃, react for 6h under argon protection, and obtain amino HMMs (N-HMMs) by high-speed centrifugation.

[0057] 1.2 Synthesis of N-HMMs@Nar:

[0058] 0.5 g N-HMMs and 1 g naringin (Nar) were slowly stirred in deionized water for 6 h, and the solid was collected by centrifugation and dried at 45 °C to obtain N-HMMs@Nar.

[0059] 1.3 Synthesis of Gelatin Methacrylate (GelMA):

[0060] Take 5g of gelatin and dissolve it in 50mL of phosphate buffer (PBS, pH7.4, 50℃), stir until completely dissolved, then slowly add 4mL of methacrylic anhydride, continue the reaction for 3h, then add 150mL of PBS to terminate the reaction to obtain methacrylated gelatin (GelMA).

[0061] 1.4 Synthesis of Gelatin Methacrylate / Polyethylene Glycol Diacrylate Hydrogel (GelMA / PEGDA, GP):

[0062] GelMA was dialyzed (14 kDa) for one week and then freeze-dried for storage. 0.1 g of GelMA and 0.05 g of polyethylene glycol diacrylate (PEGDA) were dissolved in 1 mL of PBS, 0.0025 g of photoinitiator (Irgacure 2959) was added in a dark place, and then irradiated with ultraviolet light (UV, 365 nm) for 10 s to obtain GP hydrogel (10% (w / v) GelMA / 5% (w / v) PEGDA, G10P5). The GP hydrogel was soaked in PBS for 2 h, and after removing toxic substances, it was freeze-dried for use.

[0063] The above method was used to prepare GP hydrogels with different ratios: G10P0 (10% (w / v) GelMA), G10P2 (10% (w / v) GelMA / 2% (w / v) PEGDA), G15P0 (15% (w / v) GelMA), G15P2 (15% (w / v) GelMA / 2% (w / v) PEGDA), and G15P5 (15% (w / v) GelMA / 5% (w / v) PEGDA).

[0064] 1.5 Synthesis of N-HMMs@Nar@GP hydrogel:

[0065] 0.1 g GelMA, 0.05 g PEGDA and 0.005 g N-HMMs@Nar were dissolved in 1 mL PBS and protected from light. After adding 0.0025 g photoinitiator (Irgacure 2959), the mixture was irradiated with ultraviolet light (UV, 365 nm) for 10 s to obtain N-HMMs@Nar@GP hydrogel. The N-HMMs@Nar@GP hydrogel was soaked in PBS for 2 h, and after removing toxic substances, it was freeze-dried for later use.

[0066] Example 2 Characterization and performance testing of N-HMMs / N-HMMs@Nar / GP hydrogel / N-HMMs@Nar@GP hydrogel

[0067] 2.1 Characterization of N-HMMs

[0068] The structure of N-HMMs was observed by transmission electron microscopy (TEM). The results showed that HMSNs are monodispersed uniform nanoparticles with mesoporous and hollow structures, and Mg-MOFs can be uniformly coated on the surface of HMSNs with the help of the mesoporous structure ( Figure 1 A. sSiO 2 , Figure 1 B. HMSNs, Figure 1 C. N-HMMs in the sample). Particle size analysis shows that the coating of Mg-MOFs has a slight effect on the particle size of HMSNs, which may be due to the change in the mesoporous part, which is consistent with the TEM results ( Figure 1 D in the figure). XRD analysis showed that HMMs exhibited characteristic peaks of both HMSNs and Mg-MOFs, indicating that HMMs were successfully synthesized ( Figure 1 E in the figure). The FT-IR spectrum also verifies that the synthesized HMMs and N-HMMs ( Figure 1 F in ). Furthermore, the nitrogen adsorption-desorption curves and surface pore size distributions of HMSNs and HMMs were determined by BET, as shown in Figure 1 G in Figure 1 H in Figure 1The results show that the specific surface area and mesopore size of HMMs are reduced compared with those of HMSNs. This is because the Mg-MOF coating makes part of the mesopores on the surface of HMSNs replaced by smaller pores of MOF.

[0069] 2.2 Characterization of N-HMMs@Nar

[0070] The Zeta potential of the material and drug was measured by DLS. The results showed that the surfaces of HMMs and Nar had negative potentials of −22.53±1.26 mV and −24.97±0.86 mV, respectively, while the surface of N-HMMs modified by amino group had a positive potential of 8.17±0.78 mV, indicating that Nar could be adsorbed by N-HMMs to obtain N-HMMs@Nar. Figure 2 shown.

[0071] 2.3 Characterization of GelMA / GP hydrogels

[0072] Using H 1 -NMR verification of the synthesis of GelMA hydrogels, such as Figure 3 As shown in A, the results show that a represents the acrylic acid proton peak of GelMA, and b represents the methyl group in GelMA, proving that GelMA was synthesized and prepared. PEGDA was introduced to prepare GelMA / PEGDA (GP) hydrogel. The apparent morphology of the hydrogels with different mass ratios was observed after UV cross-linking. The results showed that the content of GelMA and PEDGA had no effect on the apparent morphology of the hydrogel ( Figure 3 B). GelMA, PEGDA and GP hydrogels were characterized by FT-IR, and the prepared GP hydrogels were verified. Figure 3 As shown in C.

[0073] Furthermore, the in vitro swelling, degradation and compressive properties of GP hydrogel were studied.

[0074] (1) In vitro swelling test of GP hydrogel:

[0075] Weigh the freeze-dried GP and record its mass as W0. Place it in a small beaker and add 10.00 mL of phosphate buffer solution (pH 7.4, 37°C) to immerse the hydrogel. Take out the hydrogel at different times and weigh it as Wt until the weight of the hydrogel no longer changes. The calculation formula is as follows:

[0076] Swelling rate %=Wt / W0×100%

[0077] Wherein, W0 is the initial freeze-dried hydrogel weight, and Wt is the hydrogel weight at different times.

[0078] (2) In vitro degradation assay of GP hydrogel:

[0079] Weigh the freeze-dried GP and record its mass as W0. Place it in a small beaker and add 10.00 mL of phosphate buffer solution (pH 7.4, 37°C) to immerse the hydrogel. Replace the buffer solution every 2 days. Take out the hydrogel at different times, freeze-dry it, and weigh it, record it as Wt. The calculation formula is as follows:

[0080] In vitro degradation rate %=(W0-Wt) / W0×100%

[0081] Wherein, W0 is the initial freeze-dried hydrogel weight, and Wt is the hydrogel weight at different times.

[0082] (3) The compression resistance of the gel block was measured using a universal mechanical testing machine.

[0083] In vitro swelling experiments showed that the increase of GelMA and PEGDA could significantly reduce the swelling of hydrogels ( Figure 3 D in the figure). The results of in vitro degradation test showed that the hydrogel had long-term stability under PBS (37°C, pH 7.4) conditions. The higher the content of GelMA and PEGDA, the lower the in vitro degradation efficiency ( Figure 3 E in the figure). The compression test results show that the 10% GelMA / 5% PEGDA composite hydrogel (w / v) has strong mechanical properties ( Figure 3 F in.

[0084] 2.4 Characterization of N-HMMs@Nar@GP

[0085] Before UV crosslinking, N-HMMs@Nar was added to the GP prepolymer solution and then crosslinked to obtain N-HMMs@Nar@GP hydrogel. SEM images show that the addition of N-HMMs@Nar increases the roughness and particle adhesion ( Figure 4 A and Figure 4 The Mg content of N-HMMs and N-HMMs@GP was investigated in PBS at pH 7.4. 2+ The release efficiency of GP was shown to be able to reduce the Mg 2+ The release rate was significantly reduced, but the total release amount was not significantly affected. This delayed release behavior can 2+ The concentration is maintained at a moderate level, thus providing a good and stable antioxidant effect ( Figure 4 C in the figure). Similarly, the release efficiency of Nar from N-HMMs@Nar and N-HMMs@Nar@GP hydrogels was studied, and the results also showed that N-HMMs@Nar@GP hydrogel had more stable drug release properties than N-HMMs@Nar ( Figure 4 D in.

[0086] Example 3 Cytotoxicity study of N-HMMs@GP hydrogel

[0087] Cell culture: Rat bone marrow mesenchymal stem cells (BMSCs) were cultured in α-MEM supplemented with 10% fetal bovine serum for mesenchymal stem cells and 1% penicillin-streptomycin solution; human umbilical vein endothelial cells (HUVECs) were cultured in a special culture medium for human umbilical vein endothelial cells. The culture environment was 37°C and 5% CO 2 .

[0088] Cytotoxicity: CCK-8 kit was used to detect the cytotoxicity of N-HMMs@GP hydrogel to BMSCs and HUVECs. BMSCs and HUVECs were incubated at 1×10 4 Cells were seeded in 96-well plates at a density of 100 cells / well. After cell attachment, BMSCs and HUVECs were treated with N-HMMs (100, 200 and 300 μg / mL), GP hydrogels and N-HMMs@GP hydrogels (containing 100, 200 and 300 μg / mL of N-HMMs) for 7 days, and the cell activity on days 1, 3, 5 and 7 was compared using the CCK-S kit. Cell viability was detected according to the method described in the kit.

[0089] The results showed that for BMSCs, the cell survival rate of each experimental group was greater than 80% on the first and third days, with no significant difference from the control group. For HUVECs, all groups showed good biosafety ( Figure 5 A and Figure 5 B in. # P<0.05, ## P<0.01, ### P<0.001). Based on the above results, N-HMMs (200 μg / mL) and N-HMMs@GP hydrogel (containing N-HMMs 200 μg / mL) were selected for subsequent experiments to ensure biosafety and effectiveness.

[0090] (2) Live / dead cell staining: Calcein / PI cell viability and cytotoxicity detection kit was used to detect the toxicity of N-HMMs@GP hydrogel to BMSCs and HUVECs. BMSCs and HUVECs were seeded in 24-well plates at a density of 1×104 cells / well. After cell attachment, they were co-cultured with different concentrations of N-HMMs (200 μg / mL) and N-HMMs@GP hydrogel (200 μg / mL) extracts for 1, 3, 5, and 7 days, respectively. The cells were stained according to the method described in the kit, and then the fluorescence images were captured using an inverted fluorescence microscope.

[0091] The results showed that the cytotoxicity of N-HMMs, GP hydrogels, and N-HMMs@GP hydrogels was evaluated using the Calcein-AM / PI cell viability / cytotoxicity assay kit. During the co-culture with the materials, the cell density continued to increase and the number of dead cells was low, proving that each experimental group had good in vitro biocompatibility ( Figure 5 C and Figure 5 D in.

[0092] Example 4 Study on the osteogenic and angiogenic functions of N-HMMs@Nar@GP hydrogel in vitro

[0093] (1) In vitro osteogenic activity study: ALP activity detection and the formation of osteogenic mineralized nodules were used to evaluate the osteogenic differentiation ability of BMSCs. 4 The cells were seeded into 24-well plates at a density of 100 cells / well and co-cultured with the hydrogel extract after the cell density reached 80%. The osteogenic differentiation of BMSCs was detected by alkaline phosphatase staining (day 7 and day 14) and alizarin red staining (day 10 and day 21) according to the method described in the kit.

[0094] The results showed that the formation of mineralized nodules was evaluated by Alizarin Red S staining on the 10th and 21st days of co-culture of materials and cells. After treatment with N-HMMs@Nar@GP hydrogel, the number of mineralized nodules increased significantly ( Figure 6 A and Figure 6 B). ALP staining was performed on the 7th and 14th days of co-culture of materials and cells. Compared with the control group and GP composite hydrogel, ALP staining in the N-HMMs@Nar@GP hydrogel group was significantly enhanced, indicating increased ALP activity. The above results show that N-HMMs@Nar@GP hydrogel can promote the osteogenic differentiation of BMSCs ( Figure 6 C and Figure 6 D in.

[0095] (2) In vitro angiogenesis activity study: Bone tissue contains a rich vascular network, so early angiogenesis is a key link in the bone formation process. By co-culturing HUVECs with a culture medium containing a hydrogel extract, the effect of the composite material on angiogenesis was explored. The migration efficiency of endothelial cells (scratch test) and tube-forming activity were observed to evaluate the angiogenesis effect. The migration ability of HUVECs was analyzed by a scratch test. HUVECs were cultured at 5×10 5The cells were planted at a density of 100 cells / well in a 12-well plate. After the cell density reached 80%, they were co-cultured with the hydrogel extract for 12 hours and scratched with a 200uL pipette tip. Then, the hydrogel extract was used to continue the culture, and the closure of the scratch was observed at 6h, 12h and 24h using an optical microscope. The tubule formation effect of HUVECs, the cells were incubated with the hydrogel extract for 24h, and after coating Matrigel on the 24-well plate, 8×10 4 Cells / well were inoculated into the wells, and tubule formation was observed under a light microscope after 6 h.

[0096] The results showed that compared with the control group, there was no significant difference in the migration of HUVECs in the GP hydrogel group, while the cell migration efficiency in the N-HMMs@Nar@GP hydrogel group was significantly enhanced, indicating that N-HMMs@Nar plays a major role in cell migration ( Figure 7 A and Figure 7 C in the figure). Furthermore, in the tubule formation test, similar results were found. Compared with the control group, the tubule formation effect of the N-HMMs@Nar@GP hydrogel group was significantly improved ( Figure 7 B in Figure 7 D and Figure 7 E in.

[0097] Example 5 Study on the in vivo tibial repair ability of N-HMMs@Nar@GP hydrogel

[0098] Thirty-six 6-week-old male SD rats were anesthetized and a circular bone defect (2.4 mm in diameter) was created above the tibia of the right hind limb. Sterile saline was used to completely remove the hematoma in the defect area. The rats were randomly divided into three groups (n=12): (A) control group, (B) GP hydrogel injection group, and (C) N-HMMs@Nar@GP hydrogel injection group. After injection, the hydrogel was exposed to UV light for gelation. Two weeks after implantation, six rats in each group were randomly killed; four weeks after implantation, the remaining rats were killed. The tibia was harvested for micro-CT and histological analysis.

[0099] Micro-CT was used to evaluate the bone repair level of each group at 2 and 4 weeks. The results showed that 2 weeks after surgery, there was little difference between the GP hydrogel group and the control group, while the N-HMMs@Nar@GP hydrogel group had a slight increase in new bone tissue at the edge of the injury. 4 weeks after surgery, the difference was more obvious. Compared with 2 weeks, the new bone tissue in the same group increased. In addition, after GP hydrogel treatment, the amount of new bone tissue formed was significantly higher than that in the control group, while the N-HMMs@Nar@GP hydrogel group had the largest amount of new bone tissue formation and the bone defect was almost completely repaired ( Figure 8 The above results were also confirmed by quantitative analysis of the new bone volume (BV) and the new bone volume / tissue volume (BV / TV) ratio. Figure 8 B and Figure 8 As shown in C.

[0100] H&E staining and Masson's trichrome staining were used for histological analysis of the defect site. Similar to the micro-CT results, at 2 weeks, the newly formed bone tissue was less and mostly located at the edge of the bone defect, growing along the hydrogel or into the hydrogel. More new bone tissue and collagen were observed in the N-HMMs@Nar@GP hydrogel group. Four weeks after surgery, a large amount of new bone tissue was produced at the defect site in the N-HMMs@Nar@GP hydrogel group, and its growth range basically covered the defect area. The above results prove that GP hydrogel has a basic repair effect on bone defects, and the effect is better than that of the control group. After the incorporation of N-HMMs@Nar, due to the osteogenic and angiogenic multifunctionality of each component of the nanoparticles, the N-HMMs@Nar@GP hydrogel can significantly enhance the bone repair function ( Figure 8 D and Figure 8 E in.

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and substitutions can be made without departing from the technical principles of the present invention. These improvements and substitutions should also be regarded as the scope of protection of the present invention.

Claims

1. A nanocomposite hydrogel based on Mg-MOFs, characterized in that: The nanocomposite hydrogel has hollow mesoporous silica nanoparticles as the core and a magnesium-gallate metal organic framework as the shell to form a core-shell nanocomposite material; The molar ratio of gallic acid to anhydrous magnesium chloride is 1-3:1; the preparation process of the core-shell nanocomposite material comprises: dissolving hollow mesoporous silica nanoparticles, gallic acid and anhydrous magnesium chloride in water, mixing, reacting at 100° C.-150° C., cooling to room temperature, and preparing the core-shell nanocomposite material; The core-shell nanocomposite material is subjected to an amination treatment and then loaded with naringin to obtain a drug-loaded core-shell nanocomposite material; the mass ratio of the core-shell nanocomposite material to naringin is 1:0.5-4; Methacrylate gelatin, PEGDA and a drug-loaded core-shell nanocomposite material are dissolved in a buffer solution, protected from light, a photoinitiator is added, and ultraviolet light is irradiated to perform a cross-linking reaction to obtain a nanocomposite hydrogel; the mass ratio of the methacrylate gelatin, PEGDA and the drug-loaded core-shell nanocomposite material is 10-30:5-20:

1.

2. The Mg-MOFs-based nanocomposite hydrogel according to claim 1, characterized in that: The preparation steps of the core-shell nanocomposite material subjected to amination treatment include: dissolving the core-shell nanocomposite material in an organic solvent, heating it to 60° C. to 100° C., adding APTES, and reacting under argon protection to prepare the amination core-shell nanocomposite material.

3. The Mg-MOFs-based nanocomposite hydrogel according to claim 1, characterized in that: The preparation process of the drug-loaded core-shell nanocomposite material comprises: mixing the amino-modified core-shell nanocomposite material with naringin, slowly stirring for 5-8 hours, centrifuging, and drying to prepare the drug-loaded core-shell nanocomposite material.

4. The Mg-MOFs-based nanocomposite hydrogel according to claim 1, characterized in that: The photoinitiator is Irgacure 2959.

5. Use of a Mg-MOFs-based nanocomposite hydrogel as described in any one of claims 1 to 4 in the preparation of a bone tissue repair material.

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