Preparation method of GelMA / ZIF-8 / MEL hydrogel coating on porous titanium alloy surface

Abstract

This invention discloses a method for preparing a GelMA / ZIF-8 / MEL hydrogel coating on a porous titanium alloy surface, belonging to the field of surface modification of porous titanium alloy implants for biomedical applications. A ZIF-8-loaded MEL system is constructed and loaded onto the surface of a titanium alloy prosthesis via GelMA, enabling the porous titanium alloy implant to regulate the oxidative stress environment at the prosthesis-bone interface. This invention is the first to apply a GelMA / ZIF-8 / MEL hydrogel coating to the surface modification of porous titanium alloy implants, constructing a bioactive implant interface. MEL loaded onto ZIF-8 and applied to the surface of the porous titanium alloy implant via GelMA helps prolong the drug's half-life, reduce local drug concentration, alleviate side effects, and produce a synergistic osteogenic differentiation effect on osteoblasts, thereby enhancing the bioactivity of the porous titanium alloy implant. Furthermore, the preparation of the GelMA / ZIF-8 / MEL hydrogel coating in this invention is not limited by the substrate shape and can be used to prepare bioactive coatings on substrates with complex shapes.

Description

Technical Field

[0001] The invention belongs to the field of surface modification of porous titanium alloy implants of biomedical materials, and particularly relates to a method for preparing a GelMA / ZIF-8 / MEL hydrogel coating on the surface of a porous titanium alloy. Background Art

[0002] In the field of orthopedic implants, porous titanium alloy is a commonly used material with a series of advantages, such as open pore structure, reduced "stress shielding", and favorable bone ingrowth. However, the insufficient biological activity of titanium alloy leads to poor integration with bone under certain pathological conditions, such as osteoporosis.

[0003] Insufficient bone regeneration and excessive bone loss are the main causes of osteoporosis. Studies have shown that oxidative stress plays an important role in this process. According to tests, serum antioxidant enzyme activity is reduced and oxides are increased in postmenopausal osteoporosis patients. When osteoporosis occurs, excessive ROS (reactive oxygen species) increases the expression of pro-apoptotic genes, thereby promoting cell apoptosis and inhibiting bone formation. This oxidative stress environment destroys the integration of implants and bones, becoming an important cause of implant loosening and falling off.

[0004] Melatonin (MEL) is one of the hormones synthesized, stored and secreted by the pineal gland, and is also the most effective endogenous free radical scavenger discovered so far. MEL may repair mitochondrial function by targeting mitochondria, clearing ROS in mitochondria, and promote the transcriptional expression of antioxidant enzymes, thereby enhancing the osteogenic capacity of bone marrow mesenchymal stem cells. However, changes in MEL concentration will produce different results, that is, high concentrations inhibit osteogenic differentiation, and medium and low concentrations promote osteogenic differentiation, making it difficult for local application of MEL to effectively exert a regulatory effect.

[0005] ZIF-8 is a polyhedral metal organic framework obtained by ionic coordination bonds between zinc ions and 2-methylimidazole. It has good thermal and chemical stability and has been used as a drug carrier. It is worth noting that ZIF-8 has a good osteogenic effect. GelMA hydrogel is a modified gelatin obtained by the reaction of gelatin and methacrylic anhydride (MA). It has the characteristics of photocrosslinking, excellent biocompatibility, low immune response and degradability. More importantly, GelMA hydrogel retains the Arg-Gly-Asp (RGD) sequence that can promote cell adhesion. The combined use of GelMA hydrogel and ZIF-8 effectively reduces the concentration of MEL and prolongs the half-life.

[0006] At present, there has been no research on the application of ZIF-8 / MEL in porous titanium alloy surface coating. Based on the above advantages, it is proposed to use ZIF-8 as a carrier to load MEL, and load it onto the surface of porous titanium alloy through GelMA, so as to realize the biological function of titanium alloy implants to regulate the homeostasis of their surrounding microenvironment and improve the long-term stability of the implants. Summary of the invention

[0007] The present invention provides a method for preparing a GelMA / ZIF-8 / MEL hydrogel coating on a porous titanium alloy surface, constructs a ZIF-8 loaded MEL system, and loads it onto the surface of a titanium alloy prosthesis through GelMA, thereby realizing the biological function of the porous titanium alloy implant in regulating the oxidative stress environment at the interface between the prosthesis and the bone.

[0008] A method for preparing a GelMA / ZIF-8 / MEL hydrogel coating on a porous titanium alloy surface comprises the following steps:

[0009] Step (1), preparation of ZIF-8 / MEL system:

[0010] Dissolve 2-methylimidazole in methanol solution, and stir and dissolve at room temperature for 30 minutes to obtain solution A, so that the concentration of 2-methylimidazole in solution A is 32.8 mg / ml;

[0011] Dissolve Zn(NO3)2⋅6H2O and MEL in methanol solution, stir and dissolve at room temperature for 30 minutes to obtain solution B; make the concentration of Zn(NO3)2⋅6H2O 29.4mg / ml, and the concentration of MEL 3.5mg / ml;

[0012] Take 2 ml of solution A and add it to 1 ml of solution B. Stir the mixture at room temperature for 60 minutes and centrifuge for 15-20 minutes to obtain a white precipitate, which is ZIF-8 / MEL nanoparticles.

[0013] The obtained ZIF-8 / MEL nanoparticles were washed several times with methanol solution and dried under ventilation at room temperature for later use;

[0014] Step (2), preparation of GelMA loaded ZIF-8 / MEL:

[0015] 1 g of GelMA powder was dissolved in 10 ml of ultrapure water containing 2.5 mg of LAP (phenyl-2,4,6-trimethylbenzoylphosphinate), and then 1 mg of ZIF-8 / MEL nanoparticles was added. The mixture was stirred for 20 min in a dark environment and ultrasonically vibrated for 10 min to obtain the GelMA / ZIF-8 / MEL hybrid hydrogel.

[0016] Step (3), attachment of GelMA / ZIF-8 / MEL hydrogel coating on the surface of porous titanium alloy implant:

[0017] The porous titanium alloy implant was immersed in the GelMA / ZIF-8 / MEL mixed hydrogel obtained in the above step (2), stirred for 30 min, ultrasonically vibrated for 10 min, and then irradiated under ultraviolet light for 30 s to complete the attachment of the GelMA / ZIF-8 / MEL hydrogel coating on the surface of the porous titanium alloy implant.

[0018] The diameter of the ZIF-8 / MEL nanoparticles described in step (1) is 100 nm.

[0019] The centrifugal speed described in step (1) is 15000 rpm / min.

[0020] In step (3), the porous titanium alloy has a pore size of 500 μm and a porosity of 70%.

[0021] Beneficial effects of the present invention:

[0022] 1. This invention is the first to apply GelMA / ZIF-8 / MEL hydrogel coating to the surface modification of porous titanium alloy implants and construct a bioactive implant interface.

[0023] 2. MEL is loaded on ZIF-8 and applied to the surface of porous titanium alloy implants through GelMA, which is beneficial to prolong the drug half-life, reduce local drug concentration, and alleviate side effects.

[0024] 3. MEL loaded on ZIF-8 produces a synergistic osteogenic differentiation effect on osteoblasts and enhances the bioactivity of porous titanium alloy implants.

[0025] 4. The preparation of the GelMA / ZIF-8 / MEL hydrogel coating in the present invention is not limited by the shape of the substrate, and a bioactive coating can be prepared on a substrate with a complex shape. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 These are SEM images of ZIF-8 and ZIF-8 / MEL in Example 1 of the present invention.

[0027] Figure 2 TEM images of ZIF-8 and ZIF-8 / MEL in Example 1 of the present invention.

[0028] Figure 3 FTIR images of ZIF-8 and ZIF-8 / MEL in Example 1 of the present invention.

[0029] Figure 4These are the XRD images of ZIF-8 and ZIF-8 / MEL in Example 1 of the present invention.

[0030] Figure 5 This is a SEM image of the untreated surface of the porous titanium alloy implant of Example 1 of the present invention.

[0031] Figure 6 This is a SEM image of the functionalized surface coating of the porous titanium alloy implant in Example 1 of the present invention.

[0032] Figure 7 This is the ALP staining of each group in Example 2 of the present invention.

[0033] Figure 8 This is the ARS staining of each group in Example 2 of the present invention.

[0034] Fig. 9 This is the ROS staining of each group in Example 2 of the present invention. DETAILED DESCRIPTION

[0035] See also Figures 1 to 9 Shown is an embodiment of the present invention.

[0036] Example 1

[0037] A method for preparing a GelMA / ZIF-8 / MEL hydrogel coating on a porous titanium alloy surface comprises the following steps:

[0038] Step (1), preparation of ZIF-8 / MEL system:

[0039] Dissolve 2-methylimidazole in methanol solution, and stir and dissolve at room temperature for 30 minutes to obtain solution A, so that the concentration of 2-methylimidazole in solution A is 32.8 mg / ml;

[0040] Dissolve Zn(NO3)2⋅6H2O and MEL in methanol solution, stir and dissolve at room temperature for 30 minutes to obtain solution B; make the concentration of Zn(NO3)2⋅6H2O 29.4mg / ml, and the concentration of MEL 3.5mg / ml;

[0041] 2 ml of solution A was added to 1 ml of solution B, and the mixture was stirred at room temperature for 60 minutes. The mixture was centrifuged for 20 minutes at a speed of 15000 rpm to obtain a white precipitate, which was ZIF-8 / MEL nanoparticles with a diameter of 100 nm.

[0042] The obtained ZIF-8 / MEL nanoparticles were washed three times with methanol solution and dried under ventilation at room temperature for later use;

[0043] Step (2), preparation of GelMA loaded ZIF-8 / MEL:

[0044] 1 g of GelMA powder was dissolved in 10 ml of ultrapure water containing 2.5 mg of LAP (phenyl-2,4,6-trimethylbenzoylphosphinate), and then 1 mg of ZIF-8 / MEL nanoparticles was added. The mixture was stirred for 20 min in a dark environment and ultrasonically vibrated for 10 min to obtain the GelMA / ZIF-8 / MEL hybrid hydrogel.

[0045] Step (3), attachment of GelMA / ZIF-8 / MEL hydrogel coating on the surface of porous titanium alloy implant:

[0046] The porous titanium alloy implant with a pore size of 500 μm and a porosity of 70% was immersed in the GelMA / ZIF-8 / MEL mixed hydrogel obtained in the above step (2), stirred for 30 min, ultrasonically vibrated for 10 min, and then irradiated under ultraviolet light for 30 s to complete the attachment of the GelMA / ZIF-8 / MEL hydrogel coating on the surface of the porous titanium alloy implant.

[0047] After the ZIF-8 / MEL nanoparticles were dried, the ion shape and particle size were observed using a scanning electron microscope. The ZIF-8 / MEL nanoparticles were diluted and placed on a copper mesh and air-dried, and then observed and analyzed for element distribution using a transmission electron microscope. The characteristic functional groups of ZIF-8 and ZIF-8 / MEL were detected using FT-IR spectroscopy. The crystal structure of the nanoparticles was observed using XRD.

[0048] All the results suggested that ZIF-8 / MEL was successfully synthesized and filled into the micropores of porous titanium alloy by GelMA.

[0049] Example 2

[0050] The ability of the GelMA / ZIF-8 / MEL hydrogel coating constructed on the surface of porous titanium alloy implants to regulate osteogenic differentiation was verified by Example 2. Figure 7 , Figure 8 and Fig. 9 .

[0051] Experiments related to regulating the osteogenesis process are as follows:

[0052] (1) ALP detection: BMSCs were seeded into 24-well plates and cultured for 2 days, then the osteogenic induction conditioned medium was replaced. After 7 days of culture, the cells were washed 3 times and treated with tissue fixative for 30 minutes. BCIP / NBT staining working solution was prepared and reacted with the cells for 10 minutes, and the reaction was terminated by washing 3 times with double distilled water, and then observed under an inverted microscope.

[0053] (2) Alizarin red (ARS) detection: BMSCs were seeded into 24-well plates and cultured for 2 days, and then the osteogenic induction conditioned medium was replaced. After 14 days of culture in the osteogenic induction conditioned medium, the cells were fixed for 30 minutes and then stained with Alizarin red staining solution for 10 minutes. The cells were washed several times until the washing solution became colorless. The cells were observed and photographed under a microscope.

[0054] The experiments related to regulating antioxidant (ROS) are as follows:

[0055] (1) BMSCs were seeded into 24-well plates containing each group of scaffolds and cultured for 2 days, and then the cells were washed three times. Dichlorodihydrofluorescein diacetate (DCFH-DA) staining solution was prepared according to the manufacturer's instructions and added to the well plate for 30 minutes. The cells were washed three times and Hoechst 33342 was added for 30 minutes to mark the cell nucleus. After washing the cells, they were observed under a fluorescence microscope.

[0056] (2) All the results showed that ZIF-8 and MEL had a synergistic effect on the osteogenic differentiation of BMSCs and jointly enhanced the degree of differentiation. The GelMA / ZIF-8 / MEL hydrogel coating provided an antioxidant effect for titanium alloy implants, which was beneficial for long-term regulation of the antioxidant microenvironment around the implants.

[0057] The method of obtaining BMSCs (bone marrow mesenchymal stem cells) was disclosed in the October 2024 issue of Volume 37 of Medical Theory and Practice.

Claims

1. A method for preparing a GelMA / ZIF-8 / melatonin hydrogel coating on a porous titanium alloy surface, wherein The method is characterized in that it comprises the following steps: Step (1), preparation of ZIF-8 / melatonin system: Dissolve 2-methylimidazole in methanol solution, and stir and dissolve at room temperature for 30 minutes to obtain solution A, so that the concentration of 2-methylimidazole in solution A is 32.8 mg / ml; Dissolve Zn(NO3)2⋅6H2O and melatonin in methanol solution, stir and dissolve at room temperature for 30 minutes to obtain solution B; make the concentration of Zn(NO3)2⋅6H2O 29.4mg / ml, and the concentration of melatonin 3.5mg / ml; 2 ml of solution A was added to 1 ml of solution B, and the mixture was stirred at room temperature for 60 minutes. The mixture was centrifuged for 15-20 minutes to obtain a white precipitate, which was the ZIF-8 / melatonin nanoparticles. The obtained ZIF-8 / melatonin nanoparticles were washed several times with methanol solution and dried under ventilation at room temperature for later use; Step (2), preparation of GelMA loaded with ZIF-8 / melatonin: 1 g of GelMA powder was dissolved in 10 ml of ultrapure water containing 2.5 mg of lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and then 1 mg of ZIF-8 / melatonin nanoparticles was added. The mixture was stirred for 20 min in a dark environment and ultrasonically vibrated for 10 min to obtain a GelMA / ZIF-8 / melatonin mixed hydrogel. Step (3), attachment of GelMA / ZIF-8 / melatonin hydrogel coating on the surface of porous titanium alloy implant: The porous titanium alloy implant was immersed in the GelMA / ZIF-8 / melatonin mixed hydrogel obtained in the above step (2), stirred for 30 min, ultrasonically vibrated for 10 min, and then irradiated under ultraviolet light for 30 s to complete the attachment of the GelMA / ZIF-8 / melatonin hydrogel coating on the surface of the porous titanium alloy implant.

2. The method for preparing a GelMA / ZIF-8 / melatonin hydrogel coating on a porous titanium alloy surface according to claim 1, characterized in that: The diameter of the ZIF-8 / melatonin nanoparticles described in step (1) is 100 nm.

3. The method for preparing a GelMA / ZIF-8 / melatonin hydrogel coating on a porous titanium alloy surface according to claim 1, characterized in that: The centrifugal speed described in step (1) is 15000 rpm / min.

4. The method for preparing a GelMA / ZIF-8 / melatonin hydrogel coating on a porous titanium alloy surface according to claim 1, characterized in that: In step (3), the porous titanium alloy has a pore size of 500 μm and a porosity of 70%.

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