CN110075352B

CN110075352B - Surface chemically modified heteronaphthalene polyarylene ether nitrile bone implant material and preparation method thereof

Info

Publication number: CN110075352B
Application number: CN201910370000.3A
Authority: CN
Inventors: 蹇锡高; 柳承德; 王锦艳; 刘程; 张守海; 翁志焕
Original assignee: Dalian University of Technology
Current assignee: Dalian University of Technology
Priority date: 2019-05-06
Filing date: 2019-05-06
Publication date: 2021-09-24
Anticipated expiration: 2039-05-06
Also published as: CN110075352A

Abstract

The invention provides a surface chemically modified heteronaphthalene biphenyl poly(arylene ether nitrile) bone implant material and a preparation method thereof. The poly(arylene ether nitrile) bone implant material includes a substrate and a coating, and the two are integrated into one through chemical bonds ; the substrate is a polyaryl ether nitrile containing a diazepine biphenyl structure, and the coating is a protein with osteogenic activity. The preparation method requires no equipment, has low cost, and can perform surface modification on complex-shaped bone implants. The modified coating prepared by the invention has a protein layer with osteogenic activity, has good biocompatibility and osteogenic activity, and can improve the performance of the polyarylene ether nitrile material without affecting the mechanical properties of the polyarylene ether nitrile. Biocompatibility and osteogenic activity have broad application prospects in bone implant materials. The excessive release of the protein layer can be controlled by chemically bonding the protein layer on the surface of the poly(arylene ether nitrile) base material.

Description

Surface chemically modified heteronaphthalene biphenyl poly (arylene ether nitrile) bone implant material and preparation method thereof

Technical Field

The invention relates to a medical polymer implant material, in particular to a polyaryl ether nitrile bone implant material with a phthalazinone biphenyl structure and a preparation method thereof.

Background

The medical implant material is one of biomedical materials with large clinical demand, and the bone implant material is taken as one of the medical implant materials and is always valued by people. Compared with metal bone implant materials, polymer bone implant materials have a higher percentage of bone implant materials than metal bone implant materials due to their unique properties and advantages. The basic requirements of the high polymer bone implant material are no toxicity, excellent biocompatibility, chemical stability, proper physical and mechanical properties, easy processing and forming, better performance/price ratio and the like. Polyarylether bone implant materials have been extensively studied for their mechanical properties matching bone. Polyetheretherketone (PEEK) is the most successful polyarylether material for industrial applications and is widely used as a bone implant material as a new semi-crystalline aromatic engineering thermoplastic. However, the problems of unsatisfactory osteogenic activity and the like are always important problems influencing the application of PEEK implants, and researchers carry out surface modification research on PEEK in order to improve the osteogenic activity of polyarylether bone implant materials. The preparation of the protein layer with osteogenic activity on the surface is a method with great application prospect, and can effectively improve the biocompatibility and osteogenic activity.

The phthalazine biphenyl monomer with full aromatic heterocyclic, twisted and non-coplanar structure is introduced into the molecular chain of the polyarylether to synthesize a series of naphthalene biphenyl polyarylethers, which are important members in high-performance engineering plastic families. Because the structure of the polyarylether is similar to that of PEEK, the polyarylether nitrile can be further modified by multiple functional groups of the phthalazinone polyarylether, and the polyarylether nitrile can be further used as a biomedical material. The phthalazinone polyarylether is novel high-performance thermoplastic resin with independent intellectual property rights, which is independently researched and developed by university of large-scale continuous processing industry, and in the early research work, the surface modification is carried out on the polyarylethersulfone ketone containing the phthalazinone structure, so that the biocompatibility and the osteogenic activity of the material are greatly improved. Park et al in TiO₂The surface of the nanotube is chemically bonded with BMP-2, so that the osteogenesis activity of the nanotube is improved, but the chemical bonding of the BMP-2 on the surface of the polyarylether material is not reported. The method adopted by the invention is to perform surface hydrophilic modification on the hydrophobic hetero-naphthalene biphenyl poly (arylene ether nitrile) material and endow the hydrophobic hetero-naphthalene biphenyl poly (arylene ether nitrile) material with a carboxyl active functional group with surface chemical modification, so that the material has the possibility of surface chemical modification, the surface pretreatment process of the material is improved, and the prepared coating with bioactivity is a protein layer with bioactivity, which is chemically bonded to the surface of the hetero-naphthalene biphenyl poly (arylene ether nitrile).

The hetero-naphthalene biphenyl poly (arylene ether nitrile) adopted by the invention is a thermoplastic poly (arylene ether) material with excellent performance, the mechanical property of the hetero-naphthalene biphenyl poly (arylene ether nitrile) is matched with the mechanical property of bones, and the surfaces of the hetero-naphthalene biphenyl poly (arylene ether nitrile) have cyano groups and can be hydrolyzed into carboxyl active functional groups. However, the application of the hetero-naphthalene biphenyl poly (arylene ether nitrile) as a bone implant material is limited by low biocompatibility and osteogenesis activity of the hetero-naphthalene biphenyl poly (arylene ether nitrile), and the invention aims to perform carboxylation modification on the surface of the hetero-naphthalene biphenyl poly (arylene ether nitrile) by a chemical method to improve the hydrophilicity of the material. The protein layer with biological activity is chemically bonded by a chemical bonding method so as to improve the biocompatibility and biological activity of the protein layer.

Disclosure of Invention

The invention aims to provide a novel naphthalene biphenyl polyarylether with osteogenesis activity, which is characterized in that a coating with osteogenesis activity is prepared on the surface of naphthalene biphenyl polyarylether nitrile, and the coating comprises a protein layer with bioactivity chemically bonded on a plane surface. The protein layer with biological activity is fixed on the surface of the heteronaphthalene biphenyl poly (arylene ether nitrile) by a chemical bonding method. The surface modified coating can improve the biocompatibility and the bioactivity of the heteronaphthalene biphenyl poly (arylene ether nitrile), and can play a role in a longer time, so that the long-term implant has longer lasting bioactivity.

The technical scheme of the invention is as follows:

the surface chemically modified heteronaphthalene biphenyl polyaryl ether nitrile bone implant material comprises a substrate and a coating, wherein the substrate and the coating are combined into a whole through a chemical bond; the substrate is polyaryl ether nitrile containing cyano and phthalazinone biphenyl structures, and the coating is protein with biological activity.

The protein comprises bone morphogenetic protein, collagen, osteopontin, plasma fibrin and the like.

The polyarylether nitrile containing the phthalazinone structure has the structural expression as follows:

the polyarylether nitrile containing the phthalazinone structure has the glass transition temperature of not less than 250 ℃, the thermal weight loss 5% decomposition temperature of not less than 480 ℃, and the intrinsic viscosity of the polyarylether is 0.1-0.9 dL/g;

wherein Ar is₁Is the main structure of the double-halogen monomer, and is the double-halogen monomer containing a cyano structure:

Ar₂is a main structure of bisphenol monomer, and is any one or combination of more than two of the following structures:

wherein R is₁、R₂、R₃、R₄Is hydrogen, halogen substituent, phenyl, phenoxy, straight-chain alkyl having at least 1 carbon atom, branched alkyl having at least 1 carbon atom or branched alkoxy having at least 1 carbon atom, R₁、R₂、R₃And R₄Are the same or different;

m is a positive integer;

n is 0 or a positive integer.

The preparation method of the surface chemically modified heteronaphthalene biphenyl poly (arylene ether nitrile) bone implant material comprises the following steps:

first step, carboxyl modification of polyarylene ether nitriles

Placing poly (arylene ether nitrile) (PPENK) with a plane structure or a three-dimensional surface structure and containing a phthalazinone biphenyl structure into a KOH solution of 1-6 mol/L, condensing, refluxing and magnetically stirring at 84-105 ℃, wherein the hydrolysis reaction time is 6 hours-3 days, after the reaction is finished, placing the material obtained by hydrolysis into a hydrochloric acid solution with the concentration of less than 2mol/L, and washing and drying the material after more than 5 minutes for later use;

second, preparing a bone implant material

Firstly, activating by using surface carboxylic acid groups activated by 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide EDC and an N-hydroxysuccinimide NHS coupling agent for at least 2 hours; wherein the molar ratio of EDC to NHS is greater than 2: 1; then washing for at least two times, placing the mixture in PBS solution of protein with osteogenesis activity, magnetically stirring for 12 hours to 2 days at room temperature, sequentially washing the mixture by PBS washing solution containing sodium tetraborate and sodium dodecyl sulfate and deionized water, drying the washed mixture at the temperature of not higher than 37 ℃, and placing the dried mixture in a refrigerator at the temperature of 4 ℃ for later use; wherein the pH value of the PBS solution is 7.4; in the PBS wash, the concentration of sodium tetraborate was 0.125M and the concentration of sodium dodecylsulfate was 0.367 mM.

Another method for preparing the heteronaphthalene biphenyl polyarylether nitrile with the carboxylic acid group on the surface is to utilize a plasma processing device to carry out surface treatment on the heteronaphthalene biphenyl polyarylether nitrile workpiece and introduce a carboxylic acid functional group on the surface.

The KOH solution is 4 mol/L.

The hydrolysis reaction time is 24 hours.

The coating has a layer thickness of less than 1 μm and has osteogenic activity.

The concentration of the protein solution with osteogenic activity is 100 ng/mL.

The beneficial results of the invention are as follows:

(1) the preparation method of the heteronaphthalene biphenyl polyarylether nitrile with the osteogenesis active coating on the surface does not need equipment, has low cost and can carry out surface modification on the bone implant with a complex shape.

(2) The modified coating prepared by the invention has a protein layer with osteogenic activity, has better biocompatibility and osteogenic activity, can improve the biocompatibility and osteogenic activity of the poly (arylene ether nitrile) material on the premise of not influencing the mechanical property of the poly (arylene ether nitrile), and has wide application prospect in the aspect of bone implant materials.

(3) The excessive release of the protein layer can be controlled by chemically bonding the protein layer on the surface of the poly (arylene ether nitrile) substrate material, so that the long-term implant can play a more long-acting role in vivo.

Drawings

FIG. 1 is an X-ray photoelectron spectrum of a sample of PPENK, HPPENK, PPENK/BMP-2.

FIG. 2 shows the contact angle of PPENK after modification.

FIG. 3 shows the PPENK/BMP-2 spectrum and the FITC characteristic spectrum under confocal microscope.

FIG. 4 shows the survival rate of cells cultured with BMP-2 modified slide extract measured by MTT method.

FIG. 5 shows the survival rate of cells cultured with BMP-2 modified slide extract measured by MTT method.

Detailed Description

The following further describes a specific embodiment of the present invention with reference to the drawings and technical solutions.

The invention is further illustrated with reference to specific examples.

The structural formula of PPENK is as follows:

example one

The method comprises the following steps of firstly, carrying out hot-press molding on PPENK powder, then sequentially using deionized water, ethanol, acetone and deionized water to clean a PPENK plate, and drying for later use. Putting the PPENK sheet into a KOH solution of 4mol/L, condensing and refluxing at 102 ℃, carrying out magnetic stirring, reacting for 24 hours, after the reaction is finished, putting the HPPENK sheet product into a hydrochloric acid solution of 1mol/L, and washing and drying the material for later use after more than 5 minutes;

secondly, putting HPPENK into 10mL of water, adding 0.7668g of EDC and 0.1151g of NHS, magnetically stirring for 2 hours at room temperature to activate carboxyl on the surface of the HPPENK sample, and after the reaction is finished, washing the sample with deionized water for three times. The surface-activated HPPENK tablets are put into 10mL PBS solution containing bone morphogenetic protein-2 with the concentration of 100ng/mL, reacted for 20 hours at room temperature, washed by 0.125M sodium tetraborate and 0.367mM sodium dodecyl sulfate PBS washing solution and deionized water in sequence, dried after washing and placed in a refrigerator at 4 ℃ for standby.

Example two

The bone morphogenetic protein-2 in example one was changed to BSA protein solution (2mg/mL), and the procedure was the same as in example one.

EXAMPLE III

The bone morphogenetic protein-2 in example I was changed to type I collagen (2mg/mL), and the procedure was the same as in example I.

Example four

The same procedure as in the first embodiment is followed except that the PPENK in the first embodiment is changed to PPENSK.

EXAMPLE five

The same procedure as in example one was repeated except that PPENK in example one was changed to PPENSK and bone morphogenetic protein-2 was changed to BSA protein solution (2 mg/mL).

EXAMPLE six

The same procedure as in example one was repeated except that PPENK in example one was changed to PPENSK and that bone morphogenetic protein-2 was changed to type I collagen (2 mg/mL).

And determining the hydrolysis condition of a cyano group on the surface by FTIR infrared spectroscopy analysis, representing the hydrophilicity and hydrophobicity of the surface of the sample by a water contact angle measuring instrument, and analyzing the change condition of the content of main elements before and after the PPENK surface modification by adopting an X-ray photoelectron spectrometer (XPS). The method comprises the steps of detecting the existence of BMP-2 on the surface of a sample by a mode of combining an antibody and an antigen and immunohistochemical staining, and determining the content of the BMP-2 on the surface by the loading efficiency and the in vitro release efficiency of the BMP-2. The biocompatibility of the material is characterized by taking MC3T3-E1 mouse embryo osteoblast precursor cells as test cells.

The PPENK tablet with the osteogenesis active protein on the surface, which is prepared by the method, has the following properties:

test example 1

X-ray photoelectron spectroscopy analysis is carried out on the PPENK/BMP-2 sample wafer to obtain the contents of carbon, nitrogen and oxygen elements, and XPS spectrograms of the three are shown in figure 1 when being compared with the PPENK and the HPPENK.

As shown in Table 1, the contents and ratios of the elements of the three groups of sample wafers can be used, after BMP-2 is connected, the contents of the elements on the surfaces of the sample wafers are obviously changed, and the increase of the O/C value is especially obvious, which shows that after reaction, the BMP-2 is effectively connected on the surfaces of the sample wafers.

TABLE 1 elemental contents and ratios of PPENK, HPPENK, PPENK/BMP-2 swatches

Test example two

And (3) respectively measuring the surface contact angles of the PPENK, HPPENK and PPENK/BMP-2 sample wafers by taking water as test liquid, and detecting the hydrophilicity of the PPENK, HPPENK and PPENK/BMP-2 sample wafers. The surface contact angles of the three samples are shown in fig. 2, after surface modification, the number of carboxylic acid groups on the surface is increased, the surface contact angle of HPPENK is reduced, and after BMP-2 modification, the surface contact angle is reduced, and the hydrophilicity is enhanced.

Test example three

FITC is marked on the surface of the BMP-2 modified PPENK plate in a mode of combining an antibody and an antigen, the fluorescence intensity of the processed sample is detected within the wavelength range of 500-624 nm, and the fluorescence intensity is compared with a characteristic spectrogram of the FITC (shown in figure 3). The experiment result shows that BMP-2 is successfully chemically bonded on the surface of the plate.

Test example four

The PPENK powder was hydrolyzed under the same experimental conditions to obtain HPPENK, which was then dissolved in chloroform at a ratio of 1% w/v and spin-coated onto the silicon wafer surface. The silicon wafer containing the HPPENK layer was incubated with EDC/NHS solution at room temperature for 45 minutes. Then BMP-2 protein was attached to the material surface and the silicon wafer surface was washed 2 times with PBS solution. Each swatch was incubated with rabbit anti-BMP-2 polyclonal antibody (1:100 dilution) for 1 hour and washed three times with PBS. The cells were incubated for 1 hour with biotin-labeled goat anti-rabbit polyclonal antibody (1:250 dilution) and washed three times with PBS. The cells were incubated for 45 min with streptavidin-alkaline phosphatase conjugate (1:100 dilution) and washed three times with PBS. And finally, dyeing the silicon wafer by using a Fast-red-staining kit. Use of

50003D measurement laser microscope observation of staining. The method of histochemical staining is adopted. Comparing the photos of the Si sheet, it can be observed that the dye is uniformly distributed on the surface of the Si sheet after fixing the BMP-2 by the chemical bond, which shows that the BMP-2 fixing effect is better.

Test example five

The BMP-2 loading efficiency and in vitro release efficiency of the material of example one were tested using the Langton BMP-2 kit.

BMP-2 immobilization efficiency in example one: BMP-2 is loaded in a chemical fixing mode, and the fixing efficiency when the concentration of a BMP-2 solution is 100ng/mL is 79.24 percent respectively.

TABLE 2 determination of BMP-2 Loading efficiency by chemical fixation method

Test example six

As shown in FIG. 4, the osteoblast precursor cells MC3T3-E1 were cultured with the leaching solutions of different samples for 3 days, and the relative proliferation rates of the cells were all greater than 75%, indicating that the samples had better cell compatibility.

Test example seven

Example two-six XPS test

The XPS tests were carried out on the materials obtained in examples two to six, and the results are shown in the table:

table 3 example two-six material sample wafer element content and ratio

When different kinds of proteins are chemically bonded on the surface of the material, the content of each element is changed, wherein the value of O/C is increased, which indicates that the proteins are successfully bonded on the surface of the material.

Test example nine:

examples two-six cytotoxicity assays

The material obtained in the second to sixth examples was subjected to cytotoxicity test by MTT method, and the leaching solution of the material was obtained by immersing the material in cell culture medium, and then co-cultured with mouse preosteoblasts using the leaching solution of different concentrations for one day, with the results shown in the following figure:

the cytotoxicity results show that the relative cell proliferation rates of the five materials obtained in the second to sixth examples reach more than 100 percent, the requirements of the international ISO 10993 on cytotoxicity are met, and the obtained materials are nontoxic.

Claims

1. A method for preparing a surface chemically modified heteronaphthalene biphenyl poly (arylene ether nitrile) bone implant material is characterized by comprising the following steps:

first step, carboxyl modification of polyarylene ether nitriles

Placing poly (arylene ether nitrile) containing a phthalazinone biphenyl structure with a planar structure or a three-dimensional surface structure in a 1-6 mol/L KOH solution, condensing, refluxing at 84-105 ℃, carrying out magnetic stirring, wherein the hydrolysis reaction time is 6 hours-3 days, after the reaction is finished, placing the material obtained by hydrolysis in a hydrochloric acid solution with the concentration of less than 2mol/L, and after more than 5 minutes, washing the material with water and drying the material at the temperature of below 37 ℃ for later use;

m is a positive integer;

n is 0 or a positive integer;

second, preparing a bone implant material

Firstly, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide EDC and surface carboxylic acid groups activated by an N-hydroxysuccinimide NHS coupling agent are adopted, and the reaction time is 2-24 hours; wherein the molar ratio of EDC to NHS is greater than 2: 1; then washing, placing the mixture in PBS solution of protein with osteogenesis activity, magnetically stirring the mixture for 12 hours to 2 days at room temperature, sequentially washing the mixture by PBS washing liquid containing sodium tetraborate and sodium dodecyl sulfate and deionized water, drying the washed mixture after washing, and placing the washed mixture in a refrigerator at 4 ℃ for later use; wherein the pH value of the PBS solution is 7.4; in the PBS washing solution, the concentration of the sodium tetraborate is 0.125M, and the concentration of the sodium dodecyl sulfate is 0.367 mM; the protein includes bone morphogenetic protein, collagen, osteopontin and plasma fibrin.

2. The method according to claim 1, wherein the concentration of the protein solution having an osteogenic activity in the second step is 100ng/mL to 1 mg/mL.

3. The method according to claim 1 or 2, wherein the KOH solution is not less than 4 mol/L.

4. The method of claim 3, wherein the KOH solution is 4M.

5. The method of claim 1, 2 or 4, wherein the hydrolysis reaction time is 24 hours.

6. A surface chemically modified hetero-naphthalene biphenyl poly (arylene ether nitrile) bone implant material obtained by the preparation method according to any one of claims 1 to 5, wherein the hetero-naphthalene biphenyl poly (arylene ether nitrile) bone implant material comprises a substrate and a coating which are bonded into a whole by chemical bonds; the substrate is polyaryl ether nitrile containing phthalazinone structure, and the coating is protein with osteogenic activity.

7. The heteronaphthalene biphenyl polyarylether nitrile bone implant material of claim 6, wherein the layer thickness of the coating is less than 1 μm.