CN102716513B - Method for modifying RGD on Ca-P ceramic material surface - Google Patents

Abstract

The invention discloses a method for modifying RGD on the surface of a calcium-phosphorus ceramic material, comprising the following steps: a) providing calcium-phosphorus ceramics; b) placing the calcium-phosphorus ceramics in aqueous oxalic acid to make the surface of the calcium-phosphorus ceramics and The oxalic acid aqueous solution is reacted, and the calcium-phosphorus ceramics are taken out after the reaction; c) the calcium-phosphorus ceramics reacted with the oxalic acid aqueous solution are placed in the chloroform solution of EDC/NHS for reaction, and the calcium-phosphorus ceramics are taken out after the reaction; d) the calcium-phosphorus ceramics are combined with the The calcium-phosphorus ceramics after the reaction of the chloroform solution of EDC/NHS is placed in the ethanol aqueous solution of RGD for reaction, and the calcium-phosphorus ceramics with enhanced RGD modification effect are obtained. According to the method of the embodiment of the present invention, the RGD is combined with the surface of the calcium-phosphorus ceramic material through a chemical bond, and the binding force is effectively improved; the method does not introduce toxic groups on the surface of the calcium-phosphorus ceramic material, and does not affect its use in the biomedical field; and The method for modifying RGD in the present invention is simple and easy to operate, and can reduce costs.

Description

A method for modifying RGD on the surface of calcium phosphorus ceramic material

technical field

The invention relates to the technical field of ceramic materials, and more specifically, the invention relates to a method for modifying RGD on the surface of a calcium-phosphorus ceramic material.

Background technique

Calcium-phosphorus ceramics are widely used in the field of bone repair due to their inorganic composition similar to bone and good biocompatibility. Since the preparation of ceramic materials usually undergoes a high-temperature sintering process, and the sintered materials have poor surface activity, which is not conducive to cell adhesion and growth, so surface modification is required. RGD polypeptide is widely used in the surface modification of biological materials because it can be specifically recognized by protein receptors on the cell surface, thereby promoting cell adhesion, growth and other processes. The surface modification of calcium-phosphorus ceramic materials by RGD has also been widely studied. The current research mainly focuses on simple physical adsorption modification and chemical modification. Through physical adsorption modification, the cell adhesion on the surface of the material can be promoted to a certain extent, but due to the weak intermolecular force, the binding force between RGD and the surface of the material is also weak, so that RGD cannot continue to play a role effectively; while RGD is modified by chemical bonds. The method has received attention, but at present it is mainly realized through the coupling of silane coupling agents and other organic molecules. This modification process introduces certain toxic organic molecules, which has defects in medical applications.

Contents of the invention

The present invention aims to solve at least one of the above-mentioned technical problems.

Therefore, an object of the present invention is to propose a method for improving the surface binding force and adhesion of calcium-phosphorus ceramics, thereby enhancing the effect of RGD modification on the surface of calcium-phosphorus ceramics.

The method for modifying RGD on the surface of a calcium-phosphorus ceramic material according to an embodiment of the present invention includes the following steps:

a) Provide calcium phosphorus ceramics;

b) placing the calcium-phosphorus ceramics in an aqueous oxalic acid solution to react the surface of the calcium-phosphorus ceramics with the aqueous oxalic acid solution, and taking out the calcium-phosphorus ceramics after the reaction;

c) placing the calcium-phosphorus ceramics reacted with the oxalic acid aqueous solution in a chloroform solution of EDC/NHS for reaction, and taking out the calcium-phosphorus ceramics after the reaction;

d) The calcium-phosphorus ceramics reacted with the chloroform solution of EDC/NHS are placed in the ethanol aqueous solution of RGD to react, and the calcium-phosphorus ceramics with enhanced RGD modification effect are obtained.

According to the method for modifying RGD on the surface of the calcium-phosphorus ceramic material in the embodiment of the present invention, the RGD is combined with the surface of the calcium-phosphorus ceramic material through a chemical bond, and the binding force is effectively improved; the modification method does not introduce toxic groups on the surface of the calcium-phosphorus ceramic material, and does not It affects its use in the biomedical field; and the method for modifying RGD in the present invention is simple and easy to operate and can reduce costs.

In addition, the method for modifying RGD on the surface of a calcium-phosphorus ceramic material according to the above-mentioned embodiments of the present invention may also have the following additional technical features:

According to an embodiment of the present invention, the calcium-phosphorus ceramic is in the form of flakes, blocks, porous or powder.

According to an embodiment of the present invention, the calcium phosphorus ceramic is hydroxyapatite, β-tricalcium phosphate or a mixture thereof.

According to an embodiment of the present invention, the step b) includes:

b-1) preparing aqueous oxalic acid solution;

b-2) putting calcium phosphorus ceramics into the oxalic acid aqueous solution to react;

b-3) Take out the reacted calcium phosphorus ceramics, rinse with deionized water and dry.

According to an embodiment of the present invention, the concentration of the aqueous oxalic acid solution is 10 ^-5 -10 ^-3 mol/L.

According to an embodiment of the present invention, in step b-2), calcium phosphorus ceramics are reacted with the oxalic acid aqueous solution for 0.5-2 hours.

According to an embodiment of the present invention, the step c) includes:

c-1) Weighing 1-9 parts by weight of EDC and 1-9 parts by weight of NHS dissolved in chloroform, stirring under ventilated conditions and then standing still for more than 1 hour to dissolve the EDC and NHS;

c-2) placing the calcium-phosphorus ceramics reacted with the oxalic acid aqueous solution in the chloroform solution of EDC/NHS, and stirring continuously to fully react the calcium-phosphorus ceramics with the chloroform solution of EDC/NHS;

c-3) Take out the reacted calcium phosphorus ceramics, wash them with chloroform and acetone successively, and dry them in the air.

According to an embodiment of the present invention, stirring is carried out for 6-36 hours in step c-2).

According to one embodiment of the present invention, said step d) includes:

d-1) Dissolve absolute ethanol in deionized water to prepare an aqueous ethanol solution with a volume fraction of 10-100%;

d-2) dissolving RGD in the aqueous ethanol solution to form an RGD solution with a concentration of 1-5 mg/mL;

d-3) placing the calcium phosphorus ceramics reacted with the EDC/NHS chloroform solution in the RGD solution;

d-4) Take out the reacted calcium-phosphorus ceramics, rinse the surface of the calcium-phosphorus ceramics with ethanol solution, and dry to obtain the calcium-phosphorus ceramics with enhanced RGD modification effect.

According to an embodiment of the present invention, in step d-3), the calcium-phosphorus ceramics are reacted in the RGD solution for 6-36 hours.

Additional aspects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Description of drawings

The above and/or additional aspects and advantages of the present invention will become apparent and comprehensible from the description of the embodiments in conjunction with the following drawings, wherein:

1 is a schematic flow diagram of a method for modifying RGD on the surface of a calcium-phosphorus ceramic material according to an embodiment of the present invention;

Fig. 2 is a schematic diagram of the Fourier transform infrared spectrum of a sheet-shaped calcium-phosphorus ceramic, wherein Fig. 2 (a) shows the Fourier transform of the surface of the calcium-phosphorus ceramic obtained by modifying the surface of the calcium-phosphorus ceramic material according to an embodiment of the present invention. Schematic diagram of transform infrared spectrum, Figure 2(b) shows the schematic diagram of Fourier transform infrared spectrum of the surface of calcium-phosphorus ceramics modified by simple physical adsorption;

Fig. 3 is a schematic diagram of the Fourier transform infrared spectrum of bulk calcium-phosphorus ceramics, wherein Fig. 3 (a) and Fig. 3 (b) respectively represent the schematic diagrams of the Fourier transform infrared spectra of the two faces of the bulk calcium-phosphorus ceramics;

Figure 4 is a schematic diagram of the Fourier transform infrared spectrum of powdered calcium-phosphorus ceramics, wherein Figure 4 (a) represents the Fu of the surface of the porous calcium-phosphorus ceramic material modified according to the method of modifying RGD on the surface of the calcium-phosphorus ceramic material according to an embodiment of the present invention Schematic diagram of the Fourier transform infrared spectrum. Figure 4(b) shows the schematic diagram of the Fourier transform infrared spectrum of the porous calcium-phosphorus ceramic material obtained by simple physical adsorption modification after grinding and passing through a 60-mesh sieve.

Detailed ways

Embodiments of the present invention are described in detail below, examples of which are shown in the drawings, wherein the same or similar reference numerals designate the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the figures are exemplary only for explaining the present invention and should not be construed as limiting the present invention.

Firstly, the flow of the method for modifying RGD on the surface of calcium-phosphorus ceramic material involved in the present invention will be described with reference to FIG. 1 .

Concretely, the method for modifying RGD on the surface of the calcium-phosphorus ceramic material involved in the present invention comprises the following steps:

a) Provide calcium phosphorus ceramics;

b) placing the calcium-phosphorus ceramics in an aqueous oxalic acid solution to react the surface of the calcium-phosphorus ceramics with the aqueous oxalic acid solution, and taking out the calcium-phosphorus ceramics after the reaction;

c) placing the calcium-phosphorus ceramics reacted with the oxalic acid aqueous solution in a chloroform solution of EDC/NHS for reaction, and taking out the calcium-phosphorus ceramics after the reaction;

d) The calcium-phosphorus ceramics reacted with the chloroform solution of EDC/NHS are placed in the ethanol aqueous solution of RGD to react, and the calcium-phosphorus ceramics with enhanced RGD modification effect are obtained.

Therefore, according to the method for modifying RGD on the surface of the calcium-phosphorus ceramic material according to the embodiment of the present invention, the RGD is combined with the surface of the calcium-phosphorus ceramic material through a chemical bond, and the binding force is effectively improved; the modification method does not introduce toxic radicals on the surface of the calcium-phosphorus ceramic material. The group does not affect its use in the field of biomedicine; and the method for modifying RGD of the present invention is simple and easy to operate and can reduce costs.

Regarding the calcium-phosphorus ceramics, it should be understood that the shape of the calcium-phosphorus ceramics is not particularly limited, for example, it may be in the form of flakes, blocks, porous or powder. The composition of the calcium phosphorus ceramic mainly includes hydroxyapatite and β-tricalcium phosphate, wherein the mass percentage of the hydroxyapatite is 0-100%.

The preparation method of the calcium-phosphorus ceramics is not particularly limited. For example, the preparation method of the sheet-shaped calcium-phosphorus ceramics can be: respectively weigh 35g of hydroxyapatite and 65g of β-tricalcium phosphate, add 50mL of ethanol and 50mL of water, and use planetary The ball mill is ground at 110r/min for 24 hours, dried at 70°C for 24 hours, passed through an 80-mesh sieve, and pressed into tablets with a vulcanizer under a pressure of 10MPa. The size of the tablets is Sintering at 1250°C and holding for 2 hours to obtain calcium phosphorus ceramic material.

There are no special restrictions on the specific operation method of step b), for example, it may include:

b-1) preparing aqueous oxalic acid solution;

b-2) putting calcium phosphorus ceramics into the oxalic acid aqueous solution to react;

b-3) Take out the reacted calcium phosphorus ceramics, rinse with deionized water and dry.

As for the preparation method of the oxalic acid aqueous solution, conventional reagent preparation methods can be adopted, and the concentration of the oxalic acid aqueous solution is preferably but not limited to 10 ^-5 ~ 10 ^-3 mol/L. In step b-2), in order to fully react the calcium phosphorus ceramics with the aqueous oxalic acid solution, the reaction time can be limited to 0.5-2 hours.

The specific operation steps of step c) may include:

c-1) Weigh 1-9 parts by weight of EDC and 1-9 parts by weight of NHS and dissolve them in chloroform, stir under ventilated conditions and let stand for more than 1 hour to dissolve the EDC and NHS;

c-2) placing the calcium-phosphorus ceramics reacted with the oxalic acid aqueous solution in the chloroform solution of the EDC/NHS, and continuously stirring to fully react the calcium-phosphorus ceramics with the EDC/NHS chloroform solution;

c-3) Take out the reacted calcium-phosphorus ceramics, rinse them with chloroform and acetone successively, and dry them in the air.

There is no special limitation on the control of ventilation conditions in step c-1), as long as the air circulation can be ensured, for example, the EDC and NHs can be stirred and dissolved in a fume hood.

In order to ensure that the calcium phosphorus ceramics can fully react with the chloroform solution of EDC/NHS, the reaction solution can be continuously stirred for 6-36 hours in step c-2).

The specific operation steps of step d) may include:

d-1) Dissolving absolute ethanol in deionized water to prepare an aqueous ethanol solution with a volume fraction of 10-100%;

d-2) Dissolving RGD in the aqueous ethanol solution to form an RGD solution with a concentration of 1-5 mg/mL;

d-3) placing the calcium phosphorus ceramics reacted with the EDC/NHS chloroform solution in the RGD solution for reaction;

d-4) Taking out the reacted calcium-phosphorus ceramics, washing the surface of the calcium-phosphorus ceramics with an ethanol solution, and drying to obtain calcium-phosphorus ceramics with enhanced RGD modification effect.

In order for RGD to fully play a modifying effect, in step d-3), the reaction time of the calcium-phosphorous ceramics in the RGD solution is preferably 6-36 hours.

Thus, calcium phosphorus ceramics with enhanced RGD modification effect can be obtained.

The method for modifying RGD on the surface of a calcium-phosphorus ceramic material according to an embodiment of the present invention will be described below with reference to specific examples.

Example 1

Weigh 35g of hydroxyapatite and 65g of β-tricalcium phosphate respectively, add 50mL of ethanol and 50mL of water, grind with a planetary ball mill at 110r/min for 24h, dry at 70°C for 24h, pass through an 80-mesh sieve, and The machine presses the tablet under the pressure of 10MPa, and the size of the tablet is Sintering at 1250°C and holding for 2 hours to obtain calcium phosphorus ceramic material.

The calcium-phosphorous ceramic material obtained by sintering was treated in an aqueous solution of oxalic acid with a concentration of 5×10 ^-4 mol/L for 2 hours, and the surface was rinsed with deionized water and dried.

Place the oxalic acid-treated material in 20 mL of chloroform solution with a concentration of 1 mgEDC+1 mg NHS/mL, stir continuously for 24 h, rinse the surface and dry it.

The above materials were placed in 10 mL of ethanol solution with a volume fraction of 50% and RGD solution with a concentration of 1 mg/mL for 24 hours, and the surface was rinsed with a 50% ethanol solution and dried.

Fig. 2 is a comparison chart of Fourier transform infrared spectra of the calcium-phosphorus ceramic material surface (a) obtained by RGD modification by the above method and the calcium-phosphorus ceramic surface (b) obtained by simple physical adsorption modification. Comparing Figure 2(a) and Figure 2(b), it can be seen that after modification by the above chemical bonding method, the C=O peak of the carboxyl group at 1715cm ^-1 on the surface of the material, the NH peak at 1580cm ^-1 , and the amide bond at 1640cm ^-1 The CN peaks in all have obvious enhancements, indicating that the RGD on the surface of the material has increased significantly. It shows that the modification effect of RGD on the surface of the material is greatly enhanced by the method of chemical bonding.

Example 2

Weigh 100g of hydroxyapatite, add 50mL of ethanol and 50mL of water, grind it with a planetary ball mill at 110r/min for 24h, dry at 70°C for 24h, pass through a 80-mesh sieve, and briquette with a vulcanizer under a pressure of 10MPa. The size of the block is 10×10×10mm, and it is sintered at 1250° C. and kept for 2 hours to obtain a calcium-phosphorus ceramic material.

The calcium-phosphorous ceramic material obtained by sintering was treated in an aqueous solution of 2×10 ^-5 mol/L oxalic acid for 2 hours, and the surface was rinsed with deionized water and dried.

Place the oxalic acid-treated material in 20 mL of chloroform solution with a concentration of 1 mgEDC+0.75 mg NHS/mL, stir continuously for 24 h, rinse the surface and dry it.

The above material was treated in 10 mL of RGD absolute ethanol solution with a concentration of 2 mg/mL for 24 hours, the surface was rinsed and dried, and both sides of the block material were randomly analyzed by Fourier transform infrared spectroscopy to obtain Figure 2.

It can be seen from Figure 3 that the C=O peak of the carboxyl group appears at 1715cm ^-1 on both sides (Figure 3(a) and Figure 3(b)), and the CN peak in the amide bond appears at 1640cm ^-1 , indicating that the More RGD was present on both surfaces tested. That is, this method is also applicable to the modification of bulk materials.

Example 3

Weigh 70g of hydroxyapatite and 30g of β-tricalcium phosphate, add 50mL of ethanol, 50mL of water, PVA, dextrin and polyethylene glycol, grind for 24h with a planetary ball mill at 110r/min to make a slurry, soak the The slurry was sintered at 1250° C. after natural drying, and kept for 2 hours to obtain a porous calcium-phosphorus ceramic material.

The calcium-phosphorus ceramic material obtained by sintering was treated in an aqueous solution of oxalic acid with a concentration of 2×10 ^-4 mol/L for 2 hours, and the surface was rinsed with deionized water and dried.

Place the oxalic acid-treated material in 20 mL of chloroform solution with a concentration of 0.5 mgEDC+1 mgNHS/mL, stir continuously for 36 h, rinse the surface and dry it.

The above materials were placed in 10 mL of ethanol solution with a volume fraction of 20% and RGD solution with a concentration of 3 mg/mL for 36 h, and the surface was rinsed with a 20% ethanol solution and dried.

Fig. 4 is a comparison chart of Fourier transform infrared spectra of the porous calcium-phosphorus ceramic material obtained by RGD modification by the above method and the porous calcium-phosphorus ceramic material obtained by simple physical adsorption modification after grinding and passing through a 60-mesh sieve. It can be seen from Figure 4 that after RGD modification of the porous material by the above method, the C=O peak of the carboxyl group appeared at 1715 cm ^-1 , and the CN peak of the amide bond appeared at 1640 cm ^-1 , indicating that there are more on the inner surface of the porous material. RGD exists. That is to say, this method can also modify the inner surface of porous materials.

In the description of this specification, descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or characteristic is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications, substitutions and modifications can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the invention is defined by the claims and their equivalents.

Claims (6)

1. A method for modifying RGD on the surface of a calcium-phosphorus ceramic material, characterized in that it may further comprise the steps: a) providing calcium-phosphorus ceramics, the calcium-phosphorus ceramics being hydroxyapatite, β-tricalcium phosphate or a mixture thereof; b) placing the calcium-phosphorus ceramics in an aqueous oxalic acid solution so that the surface of the calcium-phosphorus ceramics reacts with the aqueous oxalic acid solution, and taking out the calcium-phosphorus ceramics after the reaction, the concentration of the aqueous oxalic acid solution is 10 ^-5 to 10 ^-3 mol /L, calcium phosphorus ceramics react with the oxalic acid aqueous solution for 0.5～2h; c) placing the calcium-phosphorus ceramics reacted with the oxalic acid aqueous solution in a chloroform solution of EDC/NHS for reaction, and taking out the calcium-phosphorus ceramics after the reaction; d) putting the calcium-phosphorus ceramics reacted with the chloroform solution of EDC/NHS in the ethanol aqueous solution of RGD for reaction, and reacting the calcium-phosphorus ceramics in the RGD solution for 6-36 hours to obtain calcium-phosphorus ceramics with enhanced RGD modification effect . 2 . The method for modifying RGD on the surface of a calcium-phosphorus ceramic material according to claim 1 , wherein the calcium-phosphorus ceramic is in the form of sheet, block, porous or powder. 3. the method for modifying RGD on the calcium phosphorus ceramic material surface according to claim 1, is characterized in that, described step b) comprises: b-1) preparing aqueous oxalic acid solution; b-2) putting the calcium phosphorus ceramic into the oxalic acid aqueous solution to react; b-3) Take out the reacted calcium phosphorus ceramics, rinse with deionized water and dry. 4. the method for modifying RGD on the calcium phosphorus ceramic material surface according to claim 1, is characterized in that, described step c) comprises: c-1) Weigh 1-9 parts by weight of EDC and 1-9 parts by weight of NHS and dissolve them in chloroform, stir under ventilated conditions and let stand for more than 1 hour to dissolve the EDC and NHS; c-2) placing the calcium-phosphorus ceramics reacted with the oxalic acid aqueous solution in the chloroform solution of the EDC/NHS, and continuously stirring to fully react the calcium-phosphorus ceramics with the EDC/NHS chloroform solution; c-3) Take out the reacted calcium-phosphorus ceramics, rinse them with chloroform and acetone successively, and dry them in the air. 5 . The method for modifying RGD on the surface of a calcium-phosphorus ceramic material according to claim 4 , characterized in that, in step c-2), stirring for 6-36 hours. 6. the method for modifying RGD on the calcium phosphorus ceramic material surface according to claim 1, is characterized in that, described step d) comprises: d-1) Dissolving absolute ethanol in deionized water to prepare an aqueous ethanol solution with a volume fraction of 10-100%; d-2) Dissolving RGD in the aqueous ethanol solution to form an RGD solution with a concentration of 1-5 mg/mL; d-3) placing the calcium phosphorus ceramics reacted with the EDC/NHS chloroform solution in the RGD solution for reaction; d-4) Taking out the reacted calcium-phosphorus ceramics, washing the surface of the calcium-phosphorus ceramics with an ethanol solution, and drying to obtain calcium-phosphorus ceramics with enhanced RGD modification effect.