CN108114318B

CN108114318B - Egg membrane/hydroxyapatite composite material and preparation method and application thereof

Info

Publication number: CN108114318B
Application number: CN201810038767.1A
Authority: CN
Inventors: 罗丙红; 陈学兴; 文伟; 周长忍
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2018-01-16
Filing date: 2018-01-16
Publication date: 2021-03-19
Anticipated expiration: 2038-01-16
Also published as: CN108114318A

Abstract

The invention discloses an egg membrane/hydroxyapatite composite material and a preparation method and application thereof. The composite material consists of an egg membrane with a natural protein fiber network structure and hydroxyapatite subjected to in-situ mineralization deposition, wherein the mass percentage of the egg membrane is 5-95%, and the mass percentage of the hydroxyapatite is 95-5%. The invention fully utilizes the natural protein composition and fiber network structure of the egg membrane and good mechanical property, and adopts an in-vitro simulated mineralization method with mild reaction conditions to mineralize and deposit a layer of hydroxyapatite on the surface of the egg membrane in situ to construct a composite material imitating the human bone component and structure. The finally obtained egg membrane/hydroxyapatite composite material has good mechanical property, excellent biocompatibility and osteogenesis activity, and is expected to be applied to the field of bone tissue repair. The invention has the obvious advantages of simple preparation method, mild reaction condition, easily obtained raw materials, easily prepared materials in large batch and the like.

Description

Egg membrane/hydroxyapatite composite material and preparation method and application thereof

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to an egg membrane/hydroxyapatite composite material as well as a preparation method and application thereof.

Background

The high molecular bone repair material is mainly divided into an artificial synthetic material and a natural material, because the artificial synthetic material has the defects of general biocompatibility, lack of osteogenesis activity and the like, the research of the existing bone repair material still takes the natural material as the main part and takes the synthetic material as the auxiliary part, and the formation of an extracellular matrix structure induced bone is simulated by various processing means. Common natural materials are: collagen, cellulose, chitin, hyaluronic acid and the like. Since human bone is mainly composed of collagen and hydroxyapatite, collagen is the most ideal material for bone repair, but once extracted, the original structure and components are easily damaged, the extraction cost is expensive, and the collagen is insoluble, so that it is difficult to adopt an electrostatic spinning method to construct a fibrous structure simulating extracellular matrix.

Disclosure of Invention

The invention aims to provide an egg membrane/hydroxyapatite composite material.

The invention also aims to provide a preparation method of the egg membrane/hydroxyapatite composite material.

The invention further aims to provide the application of the egg membrane/hydroxyapatite composite material in the aspect of bone repair.

In order to achieve the purpose, the technical scheme of the invention is as follows:

an egg membrane/hydroxyapatite composite material, which consists of 5-95% of egg membrane and 95-5% of hydroxyapatite in percentage by mass; the egg membrane has a natural protein fiber network structure, and hydroxyapatite is formed on the surface of the egg membrane in situ by a biomineralization method.

The egg membrane is extracted by an acid method, and the hydroxyapatite is formed in situ on the surface of the egg membrane by a biomineralization method.

Preferably, the acid method for extracting the egg membrane comprises the following specific steps: opening a small opening at the blunt end of the egg, removing egg white and yolk, and washing the egg shell with water; then, the egg shells are placed in acid solution and soaked for 0.1-72h at room temperature, and the shell membranes are automatically separated; taking out the egg membrane, washing with deionized water to keep the egg membrane in an ellipsoidal shape, placing on a polytetrafluoroethylene plate, and vacuum drying.

Preferably, the biomineralization method comprises the specific steps of soaking the egg membrane in 1-10 times of simulated body fluid, statically soaking at a constant temperature of 37 ℃ for 3 h-14 d, taking out, washing with deionized water, and freeze-drying to obtain the egg membrane with hydroxyapatite minerals on the surface.

Preferably, the inorganic salt for configuring the simulated body fluid with 1-10 times is NaCl and NaHCO₃、KCl、K₂HPO₄·3H₂O、MgCl₂·6H₂O、Na₂SO₄And CaCl₂The 1-fold salt ion composition of the simulated body fluid is as follows: na (Na)⁺The concentration is 142.0 mmol/L; k⁺The concentration is 5.0 mmol/L; mg (magnesium)²⁺The concentration is 1.5 mmol/L; ca²⁺The concentration is 2.5 mmol/L; cl-, the concentration of which is 147.8 mmol/L; HCO-, the concentration of which is 4.2 mmol/L; HPO₄ ²-, the concentration thereof is 1.0 mmol/L; SO (SO)₄ ²-, the concentration thereof is 0.5 mmol/L.

Preferably, the change in the ratio of the concentration of the whole of the salt ions is configured to be a multiple of the simulated body fluid.

Preferably, the fold change of the simulated body fluid is configured to be the Ca²⁺、Cl^-、K⁺、HPO₄ ²-change in the fold of the concentration ratio.

Preferably, the acid solution used for extracting the egg membrane by the acid method is at least one of hydrochloric acid, sulfuric acid, acetic acid, nitric acid and ethylene diamine tetraacetic acid, and the concentration of the acid solution is 0.1-10 mol/L.

An application of an egg membrane/hydroxyapatite composite material in a bone tissue repair material.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention fully utilizes the natural protein composition of the egg membrane, the protein fiber network structure similar to the extracellular matrix structure and good mechanical property, and adopts an in-vitro simulated mineralization method with mild reaction conditions to mineralize and deposit a layer of hydroxyapatite on the surface of the egg membrane in situ to construct a novel composite material imitating the human bone components and structure.

(2) The egg membrane/hydroxyapatite composite material has good mechanical property, excellent biocompatibility and osteogenic activity. As the eggshell mineralized by the calcium carbonate is formed in the uterine part of the hen and can be formed within 24 hours, the eggshell membrane has good mineralization induction capability. In addition, hydroxyapatite has good osteogenic inductivity and is an ideal osteogenic active material.

(3) In vitro simulated mineralization is a process of simulating organisms to generate inorganic minerals under mild conditions through biomacromolecules, and is a process of selectively absorbing inorganic elements from the surrounding environment and depositing the inorganic elements on specific organic matters under the participation of organic matters in regulation and induction to construct highly ordered mineral substances with special functions in vivo. By preparing simulated body fluid with similar ion concentration to the inside of human tissue and carrying out simulated mineralization in vitro, hydroxyapatite mineral with an ordered structure is expected to be mineralized in situ on the surface of the material.

(4) The egg membrane as a natural biological scaffold material has the characteristics of wide source, low price and easy preparation, and related researches prove that the egg membrane dressing has good biocompatibility and low immunogenicity.

Drawings

Fig. 1 is a flow chart of the whole preparation process of the egg membrane/hydroxyapatite composite material.

FIG. 2 is a graph of the appearance and energy spectrum of the inner and outer sides of the egg membrane extracted by the acid method in example 1: wherein, the graph A is the topography of the inner side of the egg membrane, the graph B is the topography of the outer side of the egg membrane, the graph C is the energy spectrum graph of the inner side of the egg membrane, and the graph D is the energy spectrum graph of the outer side of the egg membrane.

FIG. 3 is a scanning electron micrograph of the egg membrane/hydroxyapatite composite material of example 2: wherein, the picture A is the scanning electron microscope picture of the inner side of the egg membrane/hydroxyapatite, and the picture B is the scanning electron microscope picture of the outer side of the egg membrane/hydroxyapatite.

Fig. 4 is a scanning electron microscope image and an energy spectrum image of hydroxyapatite on the surface of the egg membrane/hydroxyapatite composite material in example 3: wherein, the figure a is a scanning electron microscope image of the inner side of the egg membrane/hydroxyapatite, the figure aa is an energy spectrum image of the inner side of the egg membrane/hydroxyapatite, the figure b is a scanning electron microscope image of the outer side of the egg membrane/hydroxyapatite, and the figure bb is an energy spectrum image of the outer side of the egg membrane/hydroxyapatite.

Fig. 5 is a graph of the thermal weight loss of the eggshell membrane and the eggshell membrane/hydroxyapatite composite material prepared in example 5.

FIG. 6 is a graph showing the proliferation of mouse preosteoblasts (MC3T3-E1) on the inner and outer surfaces of the egg membrane/hydroxyapatite composite material prepared in example 6.

FIG. 7 is a confocal laser microscope photograph showing the adhesion and spreading of MC3T3-E1 cells on the inner and outer surfaces of the composite material of egg membrane and egg membrane/hydroxyapatite prepared in example 7 for 1 day: wherein, the picture A is the laser confocal microscope photo of the inner side of the egg membrane, the picture B is the laser confocal microscope photo of the outer side of the egg membrane, the picture C is the laser confocal microscope photo of the inner side of the egg membrane/hydroxyapatite, and the picture D is the laser confocal microscope photo of the outer side of the egg membrane/hydroxyapatite.

FIG. 8 is a graph comparing the secretion of alkaline phosphatase (ALP) in MC3T3-E1 cells cultured for 7 days and 14 days on the inner and outer surfaces of the eggshell membrane and eggshell membrane/hydroxyapatite composite prepared in example 8.

FIG. 9 is a graph comparing the expression levels of osteoblast genes associated with 7 days and 14 days in the culture of MC3T3-E1 cells on the inner and outer surfaces of the composite of egg membrane and egg membrane/hydroxyapatite prepared in example 8.

Detailed Description

The present invention will be described in further detail with reference to examples, in which all of the raw materials and reagents are commercially available and conventional, unless otherwise specified.

Example 1

(1) Opening a small opening at the blunt end of an egg, removing egg white and yolk, washing the egg shell with water, placing the obtained egg shell in 5.0mol/L acetic acid solution, soaking at room temperature for 0.5h, separating the shell from the membrane, taking out the egg membrane, washing with deionized water, slightly scrubbing, placing the egg membrane on a polytetrafluoroethylene plate, drying in a vacuum drying oven for 24h, and storing in a drying cabinet for later use. The internal and external appearances of the extracted egg membrane were observed by using a scanning electron microscope in combination with an energy spectrum, as shown in fig. 2. As can be seen from the figure, the inner side of the egg membrane is flat and dense, which is the limiting layer of the egg membrane, the limiting layer is spread on the fiber network structure of the egg membrane, the outer side of the egg membrane is provided with the fiber network structure with a plurality of protruding nodes, and the inner and outer element compositions of the egg membrane are both mainly C, H, O, N, S.

(2) Simulated Body Fluid (SBF) was formulated. At room temperature, putting 800mL of deionized water into a 1.0L beaker, continuously stirring by using a magnetic stirrer, and then sequentially and slowly adding inorganic salt according to the mass and sequence of SBF added inorganic salt prepared in the following table 1; and finally, when adding Tris, firstly dissolving Tris in 50mL of deionized water, then slowly dripping into a 1.0L beaker, and then fixing the volume in a 1.0L volumetric flask to control the final pH value to be 7.45, thus obtaining the clear and transparent SBF, wherein the salt ion composition of the SBF is as follows: na (Na)⁺，142.0mmol/L；K⁺，5.0mmol/L；Mg²⁺，1.5mmol/L；Ca²⁺，2.5mmol/L；Cl-，147.8mmol/L；HCO-，4.2mmol/L；HPO₄ ²-，1.0mmol/L；SO₄ ²-，0.5mmol/L。

TABLE 1 preparation of 1.0L SBF Mass and sequence of inorganic salt additions

(3) In vitro simulated mineralization was performed. Cutting the egg membrane prepared in the step (1) into pieces with the size of 1.5cm multiplied by 1.5cm, respectively putting the pieces into a 50mL centrifuge tube, then adding 50mL of the clear and transparent SBF solution prepared in the step (2) and heated to 37 ℃, screwing down a cover, putting the centrifuge tube into a 37 ℃ constant temperature water bath tank for mineralization and constant temperature for 14 days, and replacing the SBF solution at 37 ℃ every other day. Taking out after mineralization for 14 days, washing residual salt ions on the mineralized egg membrane with deionized water, and finally freeze-drying to obtain the egg membrane/hydroxyapatite composite material. The weight percentage of the hydroxyapatite in the prepared egg membrane/hydroxyapatite composite material is measured to be 56.5 percent through thermal gravimetric analysis.

Example 2

(1) Opening a small opening at the blunt end of an egg, removing egg white and yolk, washing the egg shell with water, placing the obtained egg shell in 0.1mol/L hydrochloric acid solution, soaking at room temperature for 48h, separating the shell from the membrane, taking out the egg membrane, washing with ions, slightly scrubbing, placing the egg membrane on a polytetrafluoroethylene plate, drying in a vacuum drying oven for 24h, and storing in a drying cabinet for later use.

(2) 1.5 times of simulated body fluid (1.5 × SBF) was prepared. At room temperature, 800mL of deionized water was placed in a 1.0L beaker, stirred continuously with a magnetic stirrer, and then the inorganic salts were slowly added in sequence according to the mass and sequence of 1.5 XSBF for adding inorganic salts as formulated in Table 2 below; and when adding Tris, dissolving Tris with 50mL of deionized water, slowly dripping into a 1.0L beaker, and fixing the volume in a 1.0L volumetric flask to control the final pH to 7.45 to obtain clear and transparent 1.5 xSBF with the inorganic salt K increased by 1.5 times₂HPO₄·3H₂O and CaCl₂。

TABLE 2 preparation of 1.0L of 1.5 XSBF inorganic salt additions in quality and order

(3) In vitro simulated mineralization was performed. Cutting the egg membrane prepared in the step (1) into pieces with the size of 1.5cm multiplied by 1.5cm, respectively putting the pieces into a 50mL centrifuge tube, then adding 50mL of the clear and transparent 1.5 multiplied by SBF solution which is prepared in the step (2) and is heated to 37 ℃, screwing down a cover, putting the centrifuge tube into a 37 ℃ constant temperature water bath box for mineralization for 5 days at constant temperature, replacing the clear and transparent 1.5 multiplied by SBF solution with the temperature of 37 ℃ every other day, then taking out, washing the residual salt ions on the mineralized egg membrane with deionized water, and finally freeze-drying to prepare the egg membrane/hydroxyapatite composite material. The microscopic morphology of the egg membrane/hydroxyapatite composite material was examined, as shown in fig. 3, a large amount of mineral salts were formed on both the inside and outside of the egg membrane. The weight percentage of the hydroxyapatite in the prepared egg membrane/hydroxyapatite composite material is measured to be 40.8 percent through thermal gravimetric analysis.

Example 3

(1) Opening a small opening at the blunt end of an egg, removing egg white and yolk, washing the egg shell with water, placing the obtained egg shell in 1.0mol/L hydrochloric acid solution, soaking at room temperature for 1.5h, automatically separating the shell membrane, taking out the egg membrane, washing with deionized water, slightly scrubbing, placing the egg membrane on a polytetrafluoroethylene plate, drying in a vacuum drying oven for 24h, and storing in a drying cabinet for later use.

(2) 1.5 times of simulated body fluid (1.5 × SBF) was prepared. At room temperature, 800mL of deionized water is put into a 1.0L beaker, and is continuously stirred by a magnetic stirrer, and then the inorganic salt is slowly added in sequence according to the mass and sequence of 1.5 XSBF added inorganic salt prepared in the table 2 in the example 2; and when adding Tris, dissolving Tris with 50mL of deionized water, slowly dripping into a 1.0L beaker, and fixing the volume in a 1.0L volumetric flask to control the final pH to 7.45 to obtain clear and transparent 1.5 xSBF, wherein the inorganic salt increased by 1.5 times is K₂HPO₄·3H₂O and CaCl₂。

(3) In vitro simulated mineralization was performed. Cutting two halves of the egg membrane prepared in the step (1), placing the cut egg membrane into 500mL beakers respectively, adding 500mL of the clear and transparent 1.5 xSBF solution prepared in the step (2) and heated to 37 ℃, covering the mouth of each beaker with a preservative film, placing the beakers into a 37 ℃ constant temperature water bath box for carrying out constant temperature mineralization for 2 days, taking out the beaker, washing the residual salt ions on the mineralized egg membrane with deionized water, and finally carrying out freeze drying to prepare the egg membrane/hydroxyapatite composite material. Fig. 4 is a scanning electron microscope appearance and EDS energy spectrum diagram of hydroxyapatite on the surface of the egg membrane/hydroxyapatite composite material. The result shows that after 2 days of mineralization, the inner surface and the outer surface of the egg membrane are covered by newly generated minerals, the minerals on the two sides of the egg membrane are point-scanned by combining an energy spectrometer attached to a scanning electron microscope, the Ca/P of the minerals is close to 1.67, and the result proves that the egg membrane/hydroxyapatite composite material is successfully prepared. The weight percentage content of the hydroxyapatite in the prepared egg membrane/hydroxyapatite composite material is measured to be 10.8 percent through thermal gravimetric analysis.

Example 4

(1) Opening a small opening at the blunt end of an egg, removing egg white and yolk, washing the egg shell with water, placing the obtained egg shell in 0.1mol/L nitric acid solution, soaking at room temperature for 1.0h, separating the shell from the membrane, taking out the egg membrane, washing with deionized water, slightly scrubbing, placing the egg membrane on a polytetrafluoroethylene plate, drying in a vacuum drying oven for 24h, and storing in a drying cabinet for later use.

(2) 1.5 times of simulated body fluid (1.5 × SBF) was prepared. At room temperature, 800mL of deionized water was placed in a 1.0L beaker, stirred continuously with a magnetic stirrer, and then the inorganic salts were slowly added in sequence according to the mass and sequence of 1.5 XSBF for adding inorganic salts as formulated in Table 3 below; and finally, when adding Tris, firstly dissolving Tris in 50mL of deionized water, then slowly dripping into a 1.0L beaker, then fixing the volume in a 1.0L volumetric flask, controlling the final pH value to be 7.30, and obtaining the clear and transparent 1.5 xSBF, wherein the inorganic salt which is increased by 1.5 times is NaCl and NaHCO₃、KCl、K₂HPO₄·3H₂O、MgCl₂·6H₂O、Na₂SO₄With CaCl₂。

TABLE 3 preparation of 1.0L of 1.5 XSBF inorganic salt additions in quality and order

(3) In vitro simulated mineralization was performed. Cutting the egg membrane prepared in the step (1) into pieces with the size of 1.5cm multiplied by 1.5cm, respectively putting the pieces into a 50mL centrifuge tube, then adding 50mL of the clear and transparent 1.5 multiplied by SBF solution which is prepared in the step (2) and is heated to 37 ℃, screwing down a cover, putting the centrifuge tube into a 37 ℃ constant temperature water bath box for mineralization at constant temperature for 4 days, replacing the clear and transparent 1.5 multiplied by SBF solution with the temperature of 37 ℃ every other day, then taking out, washing the residual salt ions on the mineralized egg membrane with deionized water, and finally freeze-drying to prepare the egg membrane/hydroxyapatite composite material. The weight percentage of the hydroxyapatite in the prepared egg membrane/hydroxyapatite composite material is measured to be 30.1 percent through thermal gravimetric analysis.

Example 5

(1) Opening a small opening at the blunt end of an egg, removing egg white and yolk, washing the egg shell with water, placing the obtained egg shell in 1.0mol/L sulfuric acid solution, soaking at room temperature for 12h, separating the shell from the membrane, taking out the egg membrane, washing with ions, slightly scrubbing, placing the egg membrane on a polytetrafluoroethylene plate, drying in a vacuum drying oven for 24h, and storing in a drying cabinet for later use.

(2) 5-fold mock body fluid (5 × SBF) was prepared. At room temperature, 800mL of deionized water was placed in a 1.0L beaker and stirred continuously with a magnetic stirrer, and then the inorganic salts were added slowly in sequence according to the mass and sequence of 5 XSBF for addition of inorganic salts as formulated in Table 4 below.

TABLE 4 preparation of 1.0L of 5 XSBF inorganic salt added in mass and sequence

Slowly dripping when adding 1.0mol/L hydrochloric acid and Tris at last to make the pH of the solution at 6.45, finally using a 1.0L volumetric flask to determine the volume, and finally obtaining the clear and transparent 5 xSBF with the pH of 6.40, wherein the inorganic salt increased by 5 times is NaCl and NaHCO₃、KCl、K₂HPO₄·3H₂O、MgCl₂·6H₂O、Na₂SO₄With CaCl₂。

(3) In vitro simulated mineralization was performed. Cutting two halves of the egg membrane prepared in the step (1), placing the two halves of the egg membrane into 500mL beakers respectively, then adding 500mL of the clear and transparent 5 xSBF solution prepared in the step (2) and heated to 37 ℃, covering the mouth of each beaker with a preservative film, placing the beaker into a 37 ℃ constant temperature water tank for mineralization and constant temperature for 12 hours, taking out the beaker, washing residual salt ions on the mineralized egg membrane with deionized water, and finally performing freeze drying to prepare the egg membrane/hydroxyapatite composite material. As shown in fig. 5, the weight percentage of hydroxyapatite in the prepared egg membrane/hydroxyapatite composite material was determined to be 32.5% by thermogravimetric analysis.

Example 6

(1) Opening a small opening at the blunt end of the egg, removing egg white and yolk, washing the egg shell with water, placing the obtained egg shell in 2.0mol/L EDTA solution, soaking at room temperature for 3.0h, separating the shell and the membrane, placing the egg membrane in a polytetrafluoroethylene plate, drying in a vacuum drying oven for 24h, and storing in a drying cabinet for later use.

(2) 10-fold mock body fluid (10 × SBF) was prepared. At room temperature, 800mL of deionized water was placed in a 1.0L beaker and stirred continuously with a magnetic stirrer, and then the inorganic salts were added slowly in sequence according to the mass and sequence of 10 XSBF addition inorganic salts as formulated in Table 5 below. Slowly adding 1.0mol/L hydrochloric acid and Tris, slowly adding dropwise until the pH of the solution is about 6.10, and diluting to constant volume with a 1.0L volumetric flask to obtain clear and transparent 10 xSBF with the final pH of 6.0, wherein the inorganic salt increased by 10 times is K₂HPO₄·3H₂O and CaCl₂。

TABLE 5 preparation of 1.0L of 10 XSBF inorganic salt addition quality and sequence

(3) In vitro simulated mineralization was performed. Cutting two halves of the egg membrane prepared in the step (1), placing the cut egg membrane into 500mL beakers respectively, adding 500mL of clear and transparent 10 xSBF solution prepared in the step (2) and heated to 37 ℃, covering the mouth of each beaker with a preservative film, placing the beakers into a 37 ℃ constant temperature water bath tank for mineralization and constant temperature for 24 hours, taking out the beakers, washing residual salt ions on the mineralized egg membrane with deionized water, and finally performing freeze drying to prepare the egg membrane/hydroxyapatite composite material. The weight percentage of the hydroxyapatite in the prepared egg membrane/hydroxyapatite composite material is 57.9 percent through thermal weight loss analysis.

The proliferation of MC3T3-E1 cells on the surface of the material was tested using the CCK-8 method. After cells are cultured for 1, 4 and 7 days, the culture medium is removed, the cells are washed twice by sterile PBS, 500 mu L of the prepared CCK-8 culture medium containing 10 percent is added for culturing for 3 hours, the cells are uniformly blown and beaten, 3 100 mu L of the cells are placed in each well and are measured at 450nm by an enzyme labeling instrument, and the higher the absorbance value is, the better the cell activity is. As shown in the results of fig. 6, the OD values of MC3T3-E1 cells on the surface of all the materials increased with time, and the OD values of the cells on the surface of the egg membrane/hydroxyapatite composite material were significantly better than that of the egg membrane group, and the OD values of the group of materials were the highest with the inner side of the egg membrane/hydroxyapatite composite material.

Example 7

(1) Opening a small opening at the blunt end of an egg, removing egg white and yolk, washing the egg shell with water, placing the obtained egg shell in 3.0mol/L hydrochloric acid solution, soaking at room temperature for 0.5h, automatically separating the shell membrane, taking out the egg membrane, washing with deionized water, slightly scrubbing, placing the egg membrane on a polytetrafluoroethylene plate, drying in a vacuum drying oven for 24h, and storing in a drying cabinet for later use.

(2) 5-fold mock body fluid (5 × SBF) was prepared. At room temperature, 800mL of deionized water was placed in a 1.0L beaker and stirred continuously with a magnetic stirrer, and then the inorganic salts were added slowly in sequence according to the mass and sequence of 5 XSBF inorganic salt addition as set forth in Table 6 below.

TABLE 6 preparation of 1.0L of 5 XSBF inorganic salt additions in quality and order

Slowly dripping until pH of the solution is 6.45 when 1.0mol/L hydrochloric acid and Tris are added, and metering volume with 1.0L volumetric flask to obtain clear and transparent 5 xSBF with inorganic salts of NaCl and NaHCO 5 times higher than that of the clear and transparent 5 xSBF₃、KCl、K₂HPO₄·3H₂O、MgCl₂·6H₂O、Na₂SO₄With CaCl₂。

(3) In vitro simulated mineralization was performed. Cutting two halves of the egg membrane prepared in the step (1), placing the cut egg membrane into 500mL beakers respectively, adding 500mL of the clear and transparent 5 xSBF solution which is prepared in the step (2) and is heated to 37 ℃, covering the openings of the beakers by preservative films, placing the beakers into a 37 ℃ constant temperature water bath box for mineralization and constant temperature for 4 days, replacing the clear and transparent 5 xSBF solution at 37 ℃ every other day, taking out the solution after 4 days, washing residual salt ions on the mineralized egg membrane by deionized water, and finally performing freeze drying to prepare the egg membrane/hydroxyapatite composite material. The weight percentage of the hydroxyapatite in the prepared egg membrane/hydroxyapatite composite material is measured to be 60.5 percent through thermal gravimetric analysis.

The adhesion and spreading of MC3T3-E1 cells after 1 day of culture were observed by laser focusing microscope. After 1 day of cell culture, the medium was removed and washed three times with PBS; adding 4% paraformaldehyde, fixing for 15 min, removing, and washing with PBS for three times; then, the mixture is shaken and cleaned for three times by 0.1 percent Tritonx-100, each time for 5min, and then the mixture is removed; 200 mu L of the freshly prepared rhodamine-labeled phalloidin (which is operated according to the purchased reagent specification) is added into each hole and is dyed in the dark for 1h, and then the wells are removed; cleaning twice with 0.1% Tritonx-100, and taking out, each time for 5 min; then 300 μ L of the in-situ prepared DAPI dye (operating according to the purchased instructions) was added to each well and stained in the dark for 20min, removed, washed twice with PBS, the cell-stained material was inverted in a confocal laser microscope petri dish and wetted with a small amount of PBS, and finally observed under a confocal laser microscope. The results in FIG. 7 show that MC3T3-E1 cells spread and adhere well on the inner surface of the egg membrane, and the spreading area of the cells is large; compared with the cell spreading on the egg membrane surface before mineralization, the cell is better adhered and spread on the egg membrane/hydroxyapatite composite material surface, and the cell spreading area is large.

Example 8

(1) Opening a small opening at the blunt end of an egg, removing egg white and yolk, washing the egg shell with water, placing the obtained egg shell in 1.5mol/L nitric acid solution, soaking at room temperature for 36h, separating the shell from the membrane, taking out the egg membrane, washing with ions, slightly scrubbing, placing the egg membrane on a polytetrafluoroethylene plate, drying in a vacuum drying oven for 24h, and storing in a drying cabinet for later use.

(2) 1.5 times of simulated body fluid (1.5 × SBF) was prepared. At room temperature, 800mL of deionized water was placed in a 1.0L beaker, stirred continuously with a magnetic stirrer, and then the inorganic salts were slowly added in sequence according to the mass and sequence of 1.5 XSBF for adding inorganic salts as formulated in Table 7 below; and when adding Tris, dissolving Tris with 50mL of deionized water, slowly dripping into a 1.0L beaker, and fixing the volume in a 1.0L volumetric flask to control the final pH to 7.45 to obtain clear and transparent 1.5 xSBF with the inorganic salt K increased by 1.5 times₂HPO₄·3H₂O and CaCl₂。

TABLE 7 preparation of 1.0L of 1.5 XSBF inorganic salt additions in quality and order

(3) In vitro simulated mineralization was performed. Cutting the egg membrane prepared in the step (1) into pieces with the size of 1.5cm multiplied by 1.5cm, respectively putting the pieces into a 50mL centrifuge tube, then adding 50mL of the clear and transparent 1.5 multiplied by SBF solution which is prepared in the step (2) and is heated to 37 ℃, screwing down a cover, putting the centrifuge tube into a 37 ℃ constant temperature water bath box to mineralize for 10 days at constant temperature, replacing the clear and transparent 1.5 multiplied by SBF solution with the temperature of 37 ℃ every other day, then taking out, washing the residual salt ions on the mineralized egg membrane with deionized water, and finally freeze-drying to prepare the egg membrane/hydroxyapatite composite material. The mass percentage of the hydroxyapatite in the prepared egg membrane/hydroxyapatite composite material is 49.2 percent through thermal weight loss analysis.

The ALP kit and BCA protein kit were used to evaluate the early differentiation of cells. After culturing the cells for 7 and 14 days, adding 300 mu L of 1.0% Tritonx-100 into each hole for cracking for 40min, and uniformly shaking every 10min to obtain uniform cell lysate; then, 30 μ L of cell lysate is taken from each well and put into a 96-well plate, the sample is loaded according to the instructions of an ALP kit, and then the absorbance is measured at 492 nm; simultaneously, 20 mu L of cell lysate is put into a 96-well plate, the operation and the sample adding are carried out according to the specification of the BCA protein kit, the absorbance is measured at 570nm, and a corresponding absorbance and BCA protein concentration standard curve is drawn according to the specification; finally, the amount of ALP per unit mass of protein was calculated using two absorbance values according to the specification formula. As shown in FIG. 8, the ALP amount secreted by MC3T3-E1 cells on the surface of different materials in 7 days and 14 days shows that the outer surface of the egg membrane is more favorable for osteogenic differentiation of the cells, and the mineralized surface of the composite material containing hydroxyapatite further promotes the osteogenic differentiation of the MC3T3-E1 cells.

Example 9

(1) Opening a small opening at the blunt end of the egg, removing egg white and yolk, washing the egg shell with water, placing the obtained egg shell in 3.0mol/L hydrochloric acid solution, soaking at room temperature for 0.5h, separating the shell and the membrane, keeping the egg membrane in the shape of an ellipsoid of the egg, placing the egg membrane on a polytetrafluoroethylene plate, placing the egg membrane in a vacuum drying oven, drying for 24h, and storing in a drying cabinet for later use.

(2) 2.5 times of simulated body fluid (2.5 × SBF) was prepared. At room temperature, 800mL of deionized water was placed in a 1.0L beaker and stirred continuously with a magnetic stirrer, and then the inorganic salts were added slowly in order according to the mass and sequence of 2.5 XSBF for addition of inorganic salts as formulated in Table 8 below. Slowly dripping until pH is about 7.35 when 1.0mol/L hydrochloric acid and Tris are added, and metering to volume with 1.0L volumetric flask to obtain clear and transparent 2.5 XSBF with inorganic salt K increased by 2.5 times and pH of 7.30₂HPO₄·3H₂O and CaCl₂。

TABLE 8 preparation of 1.0L of 2.5 XSBF inorganic salt additions in quality and order

(3) In vitro simulated mineralization was performed. Cutting two halves of the egg membrane prepared in the step (1), placing the cut egg membrane into 500mL beakers respectively, adding 500mL of clear and transparent 2.5 xSBF solution prepared in the step (2) and heated to 37 ℃, covering the mouth of each beaker with a preservative film, placing the beakers into a 37 ℃ constant temperature water bath box for mineralization at constant temperature for 3 days, taking out the beaker, washing residual salt ions on the mineralized egg membrane with deionized water, and finally performing freeze drying to prepare the egg membrane/hydroxyapatite composite material. The weight percentage of the hydroxyapatite in the prepared egg membrane/hydroxyapatite composite material is 37.9 percent through thermal weight loss analysis.

After culturing the cells of the same density on the material for 7 days and 14 days, respectively, the medium in the well plate was aspirated, washed three times with PBS, and the cells on the sample membrane were digested with trypsin and collected by centrifugation. The expression conditions of osteogenic differentiation related genes Runx-2, ALP, COL-I and OCN of MC3T3-E1 cells cultured on a sample membrane are detected by utilizing a reverse transcription-polymerase chain reaction (RT-PCR) technology and taking beta-actin as an internal reference. As shown in fig. 9, the results of the relative expression of different genes of MC3T3-E1 cells on the surfaces of different materials in 7 days and 14 days show that the expression effect of the egg membrane is slightly better than that of the blank sample, although the difference in gene expression between cells cultured on the inner and outer sides of the mineralized egg membrane is not obvious, the egg membrane/hydroxyapatite composite material prepared after the mineralization is more beneficial to the osteogenic gene expression of the cells, which further proves that the mineralized composite material containing hydroxyapatite can promote osteogenic differentiation of MC3T3-E1 cells.

It should be understood that the embodiments of the present invention are merely illustrative of the present invention and are not to be construed as limiting the scope of the present invention. The invention is not limited to the embodiments described above, but rather, the invention may be embodied in many different forms. The specific process parameters and the like in the following examples are also only one example in a suitable range, and a person skilled in the art can make appropriate modifications and selections through the description herein, and are not limited to the specific values in the following examples.

Claims

1. The egg membrane/hydroxyapatite composite material is characterized by comprising 5-95% of egg membrane and 95-5% of hydroxyapatite in percentage by mass; the egg membrane has a natural protein fiber network structure, and hydroxyapatite is formed on the surface of the egg membrane in situ by a biomineralization method;

the biomineralization method comprises the specific steps of soaking the egg membrane in 1-10 times of simulated body fluid, statically soaking for 3 h-14 d at a constant temperature of 15-40 ℃, then taking out and washing with deionized water, and then carrying out freeze drying to obtain the egg membrane with hydroxyapatite minerals on the surface.

2. The egg membrane/hydroxyapatite composite material according to claim 1, characterized in that the egg membrane is extracted by an acid method.

3. The egg membrane/hydroxyapatite composite material according to claim 2, characterized in that the specific steps of the acid method for extracting the egg membrane are as follows: opening a small opening at the blunt end of the egg, removing egg white and yolk, and washing the egg shell with water; then, the egg shells are placed in acid solution, the soaking time is 0.1-72h at room temperature, and the shell membranes are automatically separated; taking out the egg membrane, washing with deionized water to keep the egg membrane in an ellipsoidal shape, placing on a polytetrafluoroethylene plate, and vacuum drying.

4. The egg membrane/hydroxyapatite composite material according to claim 3, characterized in that the soaking time is 0.5-3 h.

5. The egg membrane/hydroxyapatite composite material according to claim 1, wherein the egg membrane is soaked in 1-3 times of simulated body fluid and statically soaked at a constant temperature of 37 ℃.

6. An egg according to claim 1The membrane/hydroxyapatite composite material is characterized in that the inorganic salt for preparing the simulated body fluid with the 1-10 times of the inorganic salt is NaCl and NaHCO₃、KCl、K₂HPO₄·3H₂O、MgCl₂·6H₂O、Na₂SO₄And CaCl₂The 1-fold salt ion composition of the simulated body fluid is as follows: na (Na)⁺The concentration is 142.0 mmol/L; k⁺The concentration is 5.0 mmol/L; mg (magnesium)²⁺The concentration is 1.5 mmol/L; ca²⁺The concentration is 2.5 mmol/L; cl^-The concentration is 147.8 mmol/L; HCO₃ ^-The concentration is 4.2 mmol/L; HPO₄ ^2-The concentration is 1.0 mmol/L; SO (SO)₄ ^2-The concentration was 0.5 mmol/L.

7. The egg membrane/hydroxyapatite composite material according to claim 6, wherein the change of the ratio of the simulated body fluid to the concentration ratio of the total salt ions is configured; or configuring the fold change of the simulated body fluid to the Ca²⁺、Cl^-、K⁺、HPO₄ ^2-Change of the concentration ratio multiple.

8. The egg membrane/hydroxyapatite composite material according to claim 2 or 3, wherein an acid solution adopted for acid extraction of the egg membrane is at least one of hydrochloric acid, sulfuric acid, acetic acid, nitric acid and ethylene diamine tetraacetic acid, and the concentration of the acid solution is 0.1-10 mol/L.

9. The egg membrane/hydroxyapatite composite material according to claim 8, wherein the concentration of the acid solution is 0.5-5 mol/L.

10. The use of the egg membrane/hydroxyapatite composite material according to claim 1 for preparing bone tissue repair materials.