Application of a metal phenolic gelatin-coated modified polyarylether bone graft in bone repair under oxidative stress

Abstract

The present invention provides a polyarylether bone implant material modified with a metal phenolic gelatin coating for use in bone repair under oxidative stress. The polyarylether bone implant material modified with a metal phenolic gelatin coating comprises a polyarylether base material containing a diazinone biphenyl structure and a metal phenolic gelatin coating, the two being integrated into one by imitating the physical adhesion of mussels; the metal phenolic gelatin coating precursor solution comprises a multifunctional phenolic monomer containing diazinone biphenyl, FeCl3, gelatin (Gel), and a solvent. The preparation process of the present invention is relatively simple, and the resulting hydrogel-coated polyarylether material modified with a diazinone biphenyl does not require the addition of an additional anti-inflammatory component, and has excellent ROS scavenging efficiency, antioxidant and anti-inflammatory activity, while also having an osteogenesis-promoting effect, providing a good environment for the osteogenic differentiation of stem cells, and helping the implant material to better promote bone regeneration and bone fusion in vivo. The invention has broad application prospects in the field of polymer bone implant materials.

Description

Technical Field

[0001] The invention belongs to the technical field of functional polymer bone implant materials and relates to the application of a polyarylether bone implant material modified with a metal phenolic gelatin coating for bone repair under oxidative stress. Background Art

[0002] Bone tissue defects caused by trauma, tumor resection, degenerative diseases, etc. usually require bone transplantation. This process is accompanied by the accumulation of excessive reactive oxygen species (ROS) and the occurrence of oxidative stress response, which promotes osteoclast activity by increasing the expression of RANKL and reducing the content of OPG, thereby affecting the bone healing process. In addition, excessive ROS hinders the synthesis and mineralization of extracellular matrix by osteoblasts, changes the balance between bone resorption and bone deposition, and thus seriously affects the patient's postoperative repair. Although autologous bone transplantation is considered a "gold standard" technology in clinical practice, the secondary damage, postoperative bleeding in the donor site, pain and other problems caused by this method may be the main reason hindering its clinical development. Therefore, the design of bone implant materials should not only have excellent biocompatibility, but also use intelligent technology to regulate its antioxidant properties, effectively utilize and control the inflammatory response, and thus improve the bone integration effect.

[0003] Compared with traditional polyetheretherketone (PEEK), the polyarylether resin containing a diazinone biphenyl structure independently developed by Dalian University of Technology has both high temperature resistance and solubility. At the same time, the nitrogen heterocyclic structure therein has good biocompatibility, and is expected to become a domestic alternative to PEEK bone implant materials. In recent years, hydrogel coating modification technology has been frequently used for the surface modification of diazinone biphenyl polyarylether resins. Combined with the excellent biocompatibility and functional properties of the hydrogel itself, it solves the problem of biological inertness of diazinone biphenyl polyarylether resins. On this basis, how to better achieve the osteogenic effect of bone grafts in the oxidative stress environment of high ROS accumulation at the defect site remains a challenge and requires further exploration. Summary of the Invention

[0004] To address the above-mentioned technical problems, the present invention proposes to prepare a phthalazinone-biphenyl phenolic hydrogel coating with ROS scavenging ability on the surface of a poly(aryl ether) phthalazinone, thereby improving the antioxidant and anti-inflammatory activities of the bone graft and better achieving the bone-forming effect of the bone graft under the oxidative stress environment of high ROS accumulation at the defect site. The present invention aims to provide a poly(aryl ether) bone graft modified with a metal phenolic gelatin coating for bone repair under oxidative stress. A multifunctional hydrogel coating is prepared on the surface of the poly(aryl ether) phthalazinone. Gelatin, a multifunctional phenolic monomer containing phthalazinone biphenyl, and ferric chloride are uniformly mixed and then coated on the surface of the poly(aryl ether) phthalazinone. The cyclic lactam-like metal phenolic hydrogel coating material is prepared through hydrogen bonding, metal coordination, and Schiff base cross-linking. The phthalazinone-biphenyl phenolic structure in the coating imparts ROS scavenging ability to the material, thereby achieving the antioxidant, anti-inflammatory, and osteogenic effects of the poly(aryl ether) phthalazinone bone graft.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0006] A polyarylether bone implant material modified with a metal phenolic gelatin coating is used for bone repair under oxidative stress. The polyarylether bone implant material modified with a metal phenolic gelatin coating comprises a polyarylether base material containing a diazinone biphenyl structure and a metal phenolic gelatin coating, which are integrated into one by physical adhesion mimicking that of mussels. A precursor solution of the metal phenolic gelatin coating comprises a multifunctional phenolic monomer containing diazinone biphenyl, ferric chloride (FeCl3), gelatin (Gel), and a solvent. The precursor solution comprises 1g of Gel: multifunctional phenolic monomer containing diazinone biphenyl: FeCl3: solvent in a ratio of 1ml: 0.01-0.5M: 0.00324g-0.0162g: 5mL-20mL.

[0007] The structural formula of the multifunctional phenolic monomer containing diazinone biphenyl is as follows:

[0008]

[0009] Wherein R1, R2, R3, and R4 are hydrogen or hydroxyl groups, and there is at least one hydroxyl group in the structures of R1, R2, R3, and R4.

[0010] The solvent is a mixed solution of dimethylformamide and deionized water, and the volume ratio of dimethylformamide to deionized water is 4-6:1.

[0011] The preparation method of the polyarylether bone implant material modified with a metal phenolic gelatin coating comprises the following steps:

[0012] (1) hot-pressing a polyarylether material containing a diazinone biphenyl structure, ultrasonically washing it with acetone, ethanol, and deionized water, and drying it for later use;

[0013] (2) Soaking the sheet substrate obtained in step (1) in a metal phenolic gelatin coating precursor solution, taking it out after 5 minutes, and placing it in an oven at 37°C to 60°C for 1 to 4 hours to form a gel, thereby obtaining a metal phenolic gelatin coating modified polyarylene ether bone implant material (hydrogel coating modified polyarylene ether material containing diazinone biphenyl structure).

[0014] The polyarylether material containing a phthalazinone biphenyl structure includes phthalazinone polyarylether sulfone (PPES), phthalazinone polyarylether ketone (PPEK), phthalazinone polyarylether nitrile (PPEN), phthalazinone polyarylether sulfone ketone (PPESK), phthalazinone polyarylether nitrile ketone (PPENK) or phthalazinone polyarylether nitrile sulfone ketone (PPENSK); the molecular chain thereof contains a phthalazinone biphenyl structure, and the specific structure is as follows:

[0015]

[0016] Wherein, Ar1 and Ar3 are the main structures of the dihalogen monomer, and Ar1 and Ar3 are the same or different and are any one or more of the following structures:

[0017]

[0018] Ar2 is the main structure of the bisphenol monomer, which is any one or more of the following structures:

[0019]

[0020] Wherein, R1, R2, R3, and R4 are hydrogen, a halogen substituent, a phenyl group, a phenoxy group, a straight-chain alkyl group containing at least 1 carbon atom, a branched alkyl group containing at least 1 carbon atom, or a branched alkoxy group containing at least 1 carbon atom, and the structures of R1, R2, R3, and R4 are the same or different.

[0021] The bone defect repair under oxidative stress includes various bone defect-related diseases accompanied by inflammation. The oxidative stress environment includes bone defect repair caused by diabetes.

[0022] Beneficial effects of the present invention:

[0023] The present invention uses a one-step method to modify the substrate surface with a hydrogel coating and physically fix the coating using the mussel-like adhesion properties of polyphenols. The preparation process is relatively simple, and the resulting hydrogel-coated poly(phenylene ether) material, modified with a phthalate coating, exhibits excellent ROS scavenging efficiency, antioxidant and anti-inflammatory activity without the need for additional anti-inflammatory components. It also exhibits an osteogenesis-promoting effect, providing a favorable environment for the osteogenic differentiation of stem cells. This helps the implant material better promote bone regeneration and bone fusion in vivo, and has broad application prospects in the field of polymer bone implant materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Time-dependent relationship curve between THPZB monomer concentration change and ROS clearance rate at different times.

[0025] Figure 2 Confocal image of the material obtained in Example 1 after staining with DPBF fluorescent probe.

[0026] Figure 3 HUVEC cell migration images of the material obtained in Example 1 under oxidative stress.

[0027] Figure 4 Western blotting images of the material obtained in Example 1 after co-culture with HUVEC cells under oxidative stress.

[0028] Figure 5 Example 1 PCR detection of inflammation-related factors after the obtained materials were co-cultured with RAW264.7 cells under an inflammatory environment.

[0029] Figure 6 The obtained materials in Example 1 affect the proliferation of RAW264.7 cells under inflammatory conditions.

[0030] Figure 7 Example 1 PCR detection of the osteogenic differentiation of rBMSC cells using the obtained materials. DETAILED DESCRIPTION

[0031] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and technical solutions.

[0032] The structural formula of poly(phenylene ether nitrile ketone) (PPENK) is as follows:

[0033]

[0034] Example 1

[0035] In the first step, PPENK powder was hot-pressed and then cut into cubes with a length × width × thickness of 1 cm × 1 cm × 0.1 cm. The PPENK plates were ultrasonically cleaned with acetone, ethanol, and deionized water in sequence and dried at 100°C for later use.

[0036] In the second step, 0.2 g of gelatin was weighed and added to 1 mL of DMF / deionized water (0.8 mL / 0.2 mL) mixed solvent, stirred at 60°C until completely dissolved, and then 0.0744 g of diazinone-containing triphenylphenol benzaldehyde (THPZB) powder and 0.0026 ferric chloride powder were added. After complete dissolution, the PPENK sample obtained in the first step was immersed in the mixed solution, taken out after 5 minutes, and placed in a 37°C oven for 1 hour for self-crosslinking to obtain a hydrogel coating-modified diazinone-containing biphenyl structure polyarylether material.

[0037] The THPZB synthesis route is as follows: specifically, phthalazinone trimethoxybenzene (TMPZ): p-fluorobenzaldehyde: cesium fluoride (CsF) in a ratio of 0.1 mol: 1.2 mol: 1.2 mol is dissolved in 200 mL of dimethylacetamide (DMAc), refluxed for 12 hours under nitrogen, and immersed in deionized water to obtain an intermediate product. 0.1 mol of the dried intermediate product is dissolved in 200 mL of 4°C anhydrous dichloromethane, 3 mol of anhydrous aluminum chloride is slowly added in batches, and the reaction is carried out at 55°C under nitrogen atmosphere for 7 days. Finally, it is immersed in ice water and filtered to obtain the final THPZB product.

[0038]

[0039] In order to explore the effects of the diazinone biphenyl polyphenol benzaldehyde monomer in the hydrogel coating of the present invention on the antioxidant, anti-inflammatory and osteogenesis of the material, other conditions remained unchanged and the addition amount of THPZB was changed. The specific amount is shown in Table 1.

[0040] Table 1 Material name and amount

[0041]

[0042] The 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) assay was used to determine the ROS scavenging efficiency of THPZB monomers in vitro. Human umbilical vein endothelial cells (HUVECs) were used as test cells, and the antioxidant and migration abilities of the material under oxidative stress were characterized using the DPBF fluorescence probe assay, cell wound healing assay, and Western blotting. Real-time fluorescence quantitative PCR was used to characterize the anti-inflammatory activity of the material in macrophages (RAW264.7) as test cells. PCR was used to characterize the effect of the material on the osteogenic differentiation of rat mesenchymal stem cells (rBMSCs).

[0043] The properties of the hydrogel coating modified polyarylether material containing a phthalazinone biphenyl structure prepared by the above method are as follows:

[0044] Test Example 1

[0045] Solutions with different THPZB concentrations were added dropwise to the DPPH solution, and the OD values ​​of the solutions at 517 nm were measured at 0, 30, 60, 120, and 150 minutes, respectively. According to the formula: Calculate the ROS removal efficiency of different samples at different times. Figure 1 As shown in Figure 3, the ROS clearance rate of 0.2 M THPZB reached 80% at 150 minutes, and the concentration of THPZB was positively correlated with the ROS clearance rate.

[0046] Test Example 2

[0047] Tert-butyl hydroperoxide (tBHP) was added to the cell culture dish to create an oxidative stress environment, and 1,3-diphenylisobenzofuran (DPBF) fluorescent probe was used to detect the oxidative stress in HUVEC cells co-cultured with the material obtained in Example 1. 1 O2 distribution was specifically stained and observed using single-photon laser confocal microscopy, such as Figure 2 As shown in the figure, tBHP as the positive control group has the highest fluorescence intensity. With the increase of THPZB content in the hydrogel coating, the fluorescence intensity of the modified PPENK material group decreased significantly, indicating that it can effectively remove the 1 O2, has excellent antioxidant properties.

[0048] Test Example 3

[0049] The migration ability of HUVEC cells co-cultured with the material obtained in Example 1 under oxidative stress was determined by cell scratch method. Figure 3 As shown. Within 24 hours, the modified PPENK groups showed obvious cell migration, especially G 20 / THPZB 0.2 / Fe 16 The / PPENK group basically reached the same level as the blank control group, indicating that it can effectively remove excess ROS in the system and promote cell migration.

[0050] Test Example 4

[0051] Western Blotting was used to detect the protein expression of angiogenesis-related factors such as FGF and VEGF in HUVEC cells co-cultured with the materials obtained in Example 1 under oxidative stress. The results were as follows: Figure 4As shown in the figure. Under oxidative stress, protein expression in the positive control tBHP and PPENK groups was significantly reduced compared to the blank control group. In contrast, protein expression in the PPENK group modified with the hydrogel coating was significantly increased and positively correlated with the THPZB content in the hydrogel coating. This is mainly due to the antioxidant effect of the polyphenol structure in the THPZB monomer, which neutralizes the excess ROS in the system, thereby eliminating the inhibitory effect of excessive ROS on angiogenesis.

[0052] Test Example 5

[0053] Lipopolysaccharide was used to stimulate RAW264.7 cells to create an in vitro inflammatory model. PCR technology was used to detect the expression of pro-inflammatory (such as IL-1, IL-6, TNF, COX-2) and anti-inflammatory (such as TGF-β, IL-10) related genes in RAW264.7 cells co-cultured with the materials obtained in Example 1 under an inflammatory environment. The results are as follows. Figure 5 Compared with the positive control group without samples, all the materials in Example 1 can significantly inhibit the expression of pro-inflammatory genes and promote the expression of anti-inflammatory genes, indicating that they have good anti-inflammatory activity.

[0054] Test Example 6

[0055] In the inflammatory environment of Test Example 5, the CCK-8 method was used to determine the effect of the material obtained in Example 1 on the proliferation of RAW264.7 cells. The results are as follows: Figure 6 The hydrogel-coated modified PPENK material can significantly inhibit the proliferation of RAW264.7 cells after 7 days of co-culture.

[0056] Test Example 7

[0057] The rBMSC rat mesenchymal stem cells were cultured using the RAW264.7 macrophage conditioned medium in Test Example 5. The expression of osteogenic related genes (such as OCN, Runx2, Col-1, ALP) after 10 days of co-culture of the material obtained in Example 1 with rBMSC cells was verified using PCR technology. The results are as follows: Figure 7 As shown, all samples in Example 1 can promote the osteogenic differentiation of rBMSC cells under inflammatory conditions.

Claims

1. A polyarylether bone implant material modified with a metal phenolic gelatin coating for bone repair under oxidative stress, characterized in that: The metal phenolic gelatin coating modified polyarylether bone implant material comprises a polyarylether base material containing a diazinone biphenyl structure and a metal phenolic gelatin coating, which are integrated into one by imitating the physical adhesion of mussels; the metal phenolic gelatin coating precursor solution comprises a diazinone biphenyl multifunctional phenolic monomer, FeCl3, Gel and a solvent, wherein the precursor solution comprises Gel: diazinone biphenyl multifunctional phenolic monomer: FeCl3: solvent in the following ratios: 1g: 0.01-0.5M: 0.00324g-0.0162g: 5mL-20mL; The structural formula of the multifunctional phenolic monomer containing diazinone biphenyl is as follows: Wherein R1, R2, R3, and R4 are hydrogen or hydroxyl, and at least one of the structures of R1, R2, R3, and R4 is hydroxyl.

2. The polyarylether bone implant material according to claim 1, wherein: The solvent is a mixed solution of dimethylformamide and deionized water, and the volume ratio of dimethylformamide to deionized water is 4-6:

1.

3. The polyarylether bone implant material according to claim 1, wherein: The preparation method of the polyarylether bone implant material modified with a metal phenolic gelatin coating comprises the following steps: (1) hot-pressing a polyarylether material containing a diazinone biphenyl structure, ultrasonically washing it with acetone, ethanol, and deionized water, and drying it for later use; (2) Immerse the sheet substrate obtained in step (1) in a metal phenolic gelatin coating precursor solution, take it out after 5 minutes, and place it in an oven at 37°C to 60°C for 1 to 4 hours to form a gel, thereby obtaining a metal phenolic gelatin coating modified polyarylether bone implant material.

4. The polyarylether bone implant material according to claim 1, wherein: The polyarylether material containing a diazolinone biphenyl structure includes phthalazinone polyarylether sulfone, phthalazinone polyarylether ketone, phthalazinone polyarylether nitrile, phthalazinone polyarylether sulfone ketone, phthalazinone polyarylether nitrile ketone or phthalazinone polyarylether nitrile sulfone ketone; the molecular chain thereof contains a diazolinone biphenyl structure, and the specific structure is as follows: Wherein, Ar1 and Ar3 are the main structures of the dihalogen monomer, and Ar1 and Ar3 are the same or different and are any one or more of the following structures: Ar2 is the main structure of the bisphenol monomer, which is any one or more of the following structures: Wherein, R1, R2, R3, and R4 are hydrogen, a halogen substituent, a phenyl group, a phenoxy group, a straight-chain alkyl group containing at least 1 carbon atom, a branched alkyl group containing at least 1 carbon atom, or a branched alkoxy group containing at least 1 carbon atom, and the structures of R1, R2, R3, and R4 are the same or different.

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