CN109985277B

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Publication number: CN109985277B
Application number: CN201910239541.2A
Authority: CN
Inventors: 廖俊威; 邵润哲
Original assignee: Guangdong University of Technology
Current assignee: Guangdong University of Technology
Priority date: 2019-03-27
Filing date: 2019-03-27
Publication date: 2021-09-14
Anticipated expiration: 2039-03-27
Also published as: CN109985277A

Abstract

Translated fromChinese

本发明提供了一种骨组织工程用的多孔羟基磷灰石/壳聚糖复合材料支架及其制备方法。所述的制备方法通过对用于制备的固相和液相进行改进，制备得到抗压强度好的的大孔径和高孔隙率的多孔羟基磷灰石/壳聚糖复合材料支架；进一步地，所述的制备方法利用壳聚糖和海藻酸钠带相反电荷的性质在所述多孔羟基磷灰石/壳聚糖复合材料支架表面涂层，增强其力学性能，同时增加对骨细胞的黏附。

The invention provides a porous hydroxyapatite/chitosan composite material scaffold for bone tissue engineering and a preparation method thereof. The preparation method is to prepare a porous hydroxyapatite/chitosan composite scaffold with a large pore size and a high porosity with good compressive strength by improving the solid phase and the liquid phase used for the preparation; further, The preparation method utilizes the oppositely charged properties of chitosan and sodium alginate to coat the surface of the porous hydroxyapatite/chitosan composite scaffold to enhance its mechanical properties and increase the adhesion to bone cells at the same time.

Description

Porous hydroxyapatite/chitosan composite material scaffold for bone tissue engineering and preparation method thereof

Technical Field

The invention relates to the technical field of artificial bone scaffolds, in particular to a porous hydroxyapatite/chitosan composite scaffold for bone tissue engineering and a preparation method thereof.

Background

Hydroxyapatite is the main inorganic phase component of natural bone, and has good biocompatibility, osteoconductivity and chemical stability, without damaging biological tissues. However, the hydroxyapatite artificial bone scaffold with a single component has high brittleness and poor mechanical strength. Inspired by the unique combination mode of inorganic phase and organic phase of natural bone, in recent years, the composite material combining hydroxyapatite and organic phase is widely researched to prepare the artificial bone scaffold with excellent performance.

Based on the above, researchers found that the porous artificial bone scaffold is more bioactive. The porous artificial bone scaffold is more beneficial to promoting the large-scale adhesion, proliferation and differentiation of osteocytes, provides nutrition and signal regulation for bone remodeling and construction, and is more beneficial to the reconstruction and differentiated growth of bone tissues when the porosity of the porous artificial bone scaffold is higher. However, the higher the porosity, the lower the mechanical strength of the porous artificial bone scaffold. In addition, high-temperature treatment is mostly needed to prepare the porous artificial bone scaffold with high porosity, and the high-temperature treatment has great damage to organic matters in the porous artificial bone scaffold, so that the porous artificial bone scaffold formed by compounding hydroxyapatite and organic phase cannot be obtained.

Disclosure of Invention

The invention aims to provide a preparation method of a porous hydroxyapatite/chitosan composite material scaffold for bone tissue engineering, which can effectively compound hydroxyapatite and chitosan to obtain an artificial bone scaffold with higher porosity and compressive strength.

The invention also aims to provide the porous hydroxyapatite/chitosan composite material scaffold prepared by the preparation method. The porous hydroxyapatite/chitosan composite material scaffold has high porosity and compressive strength.

In order to solve the technical problems, the invention adopts the technical scheme that:

a preparation method of a porous hydroxyapatite/chitosan composite material scaffold for bone tissue engineering comprises the following steps:

s1, preparing a solid phase: mixing and grinding the following components in parts by weight:

47-67 parts of a first hydroxyapatite/chitosan composite material, 19-20 parts of sodium bicarbonate, 6-9 parts of magnesium oxide and 6-24 parts of a second hydroxyapatite/chitosan composite material; the mass fraction of chitosan in the first hydroxyapatite/chitosan composite material is 30%, and the mass fraction of chitosan in the second hydroxyapatite/chitosan composite material is 10%;

preparing a liquid phase: mixing a citric acid solution with the mass fraction of 30% with chitosan to obtain a chitosan citric acid solution with the mass fraction of 0.5% of chitosan;

s2, mixing the solid phase and the liquid phase according to the mass ratio of the solid to the liquid of 7: 10-3: 4, pouring the mixture into a support mold, reacting at room temperature for 2-6 hours, and drying to obtain the porous hydroxyapatite/chitosan composite support for bone tissue engineering.

The chitosan has better biocompatibility and is beneficial to the adhesion and activation of bone cells. Meanwhile, charges exist on the surface of the chitosan, so that the chitosan has an adsorption effect on growth factors and the like, and is beneficial to the structural remodeling and construction of new tissues.

The first hydroxyapatite/chitosan composite material is closer to the proportion of organic and inorganic components of human bones, and has more plasticity during the forming of the bracket compared with the hydroxyapatite/chitosan composite material with low chitosan content, so as to be beneficial to obtaining larger porosity. The inventor finds that the second hydroxyapatite/chitosan composite material brings elasticity and keeps more rigidity due to the fact that the second hydroxyapatite/chitosan composite material contains a small amount of chitosan, and the second hydroxyapatite/chitosan composite material is added on the basis of the first hydroxyapatite/chitosan composite material according to a certain proportion, so that the prepared composite material support has higher compressive strength while better plasticity is ensured.

In the solid phase, sodium bicarbonate is added, and contains bicarbonate radical, and the bicarbonate radical can generate carbon dioxide when meeting acid, and can generate tension to the surrounding closed environment, so that pores can be generated in the compact and plastic composite material bracket. And the reaction of the sodium bicarbonate and the acid is mild and controllable.

The chitosan is beta- (1, 4) -2-amino-2 deoxy-D-glucose, and the molecular weight is 30 ten thousand.

Preferably, in S1, a solid phase is prepared: mixing and grinding the following components in parts by weight:

57 parts of a first hydroxyapatite/chitosan composite material, 20 parts of sodium bicarbonate, 8 parts of magnesium oxide and 15 parts of a second hydroxyapatite/chitosan composite material.

Preferably, in S2, the solid phase and the liquid phase are mixed at a solid-liquid mass ratio of 3: 4.

Further, the preparation method of the porous hydroxyapatite/chitosan composite scaffold further comprises the following steps:

and S3, sequentially coating sodium alginate and chitosan on the porous hydroxyapatite/chitosan composite material support prepared in the step S2. The chitosan and sodium alginate with opposite charges are coated on the surface of the composite material bracket, so that the mechanical property of the composite material bracket can be enhanced, the adhesion to osteocytes can be increased, and the cell proliferation and differentiation growth are facilitated.

The method for sequentially coating the sodium alginate and the chitosan comprises the following steps: and (2) immersing the porous hydroxyapatite/chitosan composite material support in a sodium alginate solution, vacuumizing in a sealed environment, taking out the porous hydroxyapatite/chitosan composite material support, drying in vacuum until no bubbles exist in pores of the porous hydroxyapatite/chitosan composite material support, immersing in a chitosan acetic acid solution with the pH value of 4, vacuumizing in the sealed environment, drying in vacuum, fumigating with ammonia gas for 6-12 hours in the sealed environment, drying at 40-80 ℃, washing with deionized water to be neutral, and drying.

In the above steps, the concentrations of the sodium alginate solution and the chitosan acetic acid solution can be selected according to actual conditions. Preferably, the mass fraction of sodium alginate in the sodium alginate solution is 0.5%; the mass fraction of chitosan in the chitosan acetic acid solution is 0.5%.

Wherein the vacuumizing time is 5 min-10 min, and the ammonia fumigation time is 6 h. The temperature for vacuum drying was 50 ℃.

According to the invention, an alkaline gas environment is provided to remove acid radical ions combined with chitosan in the coating and carboxylate ions in residual citric acid by using an ammonia gas fumigation mode, so that the biocompatibility of the composite material scaffold is ensured. Meanwhile, the mode can avoid the damage to the composite material bracket caused by adopting alkaline solution, and reduce the influence on the compressive strength.

The preparation method of the first hydroxyapatite/chitosan composite material or the second hydroxyapatite/chitosan composite material comprises the following steps: mixing a calcium nitrate solution and a chitosan solution, dropwise adding a disodium hydrogen phosphate solution while stirring, controlling the calcium-phosphorus ratio to be 1.67, adjusting the pH value by using ammonia water, keeping the pH value to be 10, stirring for 0.5-2 h, aging for 12h at room temperature, washing with deionized water to be neutral, drying, grinding and sieving.

Wherein the concentration of the calcium nitrate solution is 0.5mol/L, and the concentration of the disodium hydrogen phosphate solution is 0.3 mol/L.

The porous hydroxyapatite/chitosan composite material bracket prepared by the preparation method is provided.

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

(1) according to the preparation method, hydroxyapatite/chitosan composite materials with different chitosan contents, sodium bicarbonate and magnesium oxide are selected as solid phases, so that the porous hydroxyapatite/chitosan composite material support with excellent mechanical properties, large aperture and high porosity is prepared, and meanwhile, the porous hydroxyapatite/chitosan composite material support has good mechanical strength and biological properties.

(2) Furthermore, the method of the invention also utilizes the property that chitosan and sodium alginate have opposite charges to coat on the surface of the material, which can enhance the mechanical property, increase the adhesion to osteocytes and be more beneficial to the proliferation and differentiation growth of the cells. And acid radical ions combined with chitosan in the coating and carboxylate ions in residual citric acid are removed by ammonia gas fumigation, so that the biocompatibility of the composite material scaffold is ensured, and the influence on the compressive strength of the composite material scaffold can be reduced.

Drawings

Fig. 1 is an SEM micrograph of the porous hydroxyapatite/chitosan composite scaffold of example 1.

Fig. 2 is a SEM high-magnification image of the porous hydroxyapatite/chitosan composite scaffold of example 1.

Fig. 3 is an SEM micrograph of the porous hydroxyapatite/chitosan composite scaffold of example 2.

Fig. 4 is a SEM high magnification view of the porous hydroxyapatite/chitosan composite scaffold of example 2.

Detailed Description

The present invention is further illustrated by the following specific examples in the specification, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

The preparation method of the hydroxyapatite/chitosan composite material adopted in the following examples and comparative examples is as follows: mixing a calcium nitrate solution and a chitosan solution, slowly dropwise adding a disodium hydrogen phosphate solution while stirring, controlling the calcium-phosphorus ratio to be 1.67, adjusting the pH value by using ammonia water, keeping the pH value to be 10, stirring for 1h, aging at room temperature for 12h, washing with deionized water to be neutral, performing suction filtration, drying at 50 ℃ to constant weight, and grinding and sieving with a 200-mesh sieve.

Example 1

57 parts of a first hydroxyapatite/chitosan composite material, 20 parts of sodium bicarbonate, 8 parts of magnesium oxide and 15 parts of a second hydroxyapatite/chitosan composite material; wherein the chitosan mass fraction of the first hydroxyapatite/chitosan composite material is 30%, and the chitosan mass fraction of the second hydroxyapatite/chitosan composite material is 10%;

s2, mixing the solid phase and the liquid phase according to the mass ratio of the solid to the liquid being 3:4, pouring the mixture into a support mold, reacting for 2 hours at room temperature, and drying at 40 ℃ to obtain the porous hydroxyapatite/chitosan composite support for bone tissue engineering.

Example 2

In this embodiment, the porous hydroxyapatite/chitosan composite scaffold is sequentially coated with sodium alginate and chitosan.

S3, immersing the porous hydroxyapatite/chitosan composite material support in a sodium alginate solution, vacuumizing for 10min in a sealed environment, taking out the porous hydroxyapatite/chitosan composite material support, drying in vacuum at 50 ℃ until no bubbles exist in pores of the porous hydroxyapatite/chitosan composite material support, immersing the porous hydroxyapatite/chitosan composite material support in a chitosan acetic acid solution with the pH value of 4, vacuumizing for 5min in the sealed environment, drying in vacuum at 50 ℃, then fumigating for 6-12 h in ammonia gas in the sealed environment, washing with deionized water to be neutral, and drying at 50 ℃.

Wherein the mass fraction of sodium alginate in the sodium alginate solution is 0.5%; the mass fraction of chitosan in the chitosan acetic acid solution is 0.5%.

Examples 3 to 5 and comparative examples 1 to 2

In examples 3 to 5 and comparative examples 1 to 2, the preparation method of the porous hydroxyapatite/chitosan composite scaffold is substantially the same as that of example 2, and the main differences are shown in table 1:

TABLE 1

Comparative example 3

In this comparative example, a porous hydroxyapatite/chitosan composite scaffold was prepared substantially in the same manner as in example 2, with the main difference being that components of the solid phase were prepared. The method comprises the following specific steps:

preparing a solid phase: mixing and grinding the following components in parts by weight: 72 parts of a first hydroxyapatite/chitosan composite material, 20 parts of sodium bicarbonate and 8 parts of magnesium oxide; wherein the chitosan mass fraction of the first hydroxyapatite/chitosan composite material is 30%.

The comparative example only adopts the hydroxyapatite/chitosan composite material with the chitosan mass fraction of 30 percent.

Comparative example 4

preparing a solid phase: mixing and grinding the following components in parts by weight: 72 parts of second hydroxyl apatite/chitosan composite material, 20 parts of sodium bicarbonate and 8 parts of magnesium oxide; wherein the second hydroxyl apatite/chitosan composite material has a chitosan mass fraction of 10%.

The comparative example only adopts hydroxyapatite/chitosan composite material with the chitosan mass fraction of 10 percent.

Comparative example 5

preparing a solid phase: mixing and grinding the following components in parts by weight: 57 parts of a first hydroxyapatite/chitosan composite material, 8 parts of magnesium oxide and 15 parts of a second hydroxyapatite/chitosan composite material; wherein the chitosan mass fraction of the first hydroxyapatite/chitosan composite material is 30%, and the chitosan mass fraction of the second hydroxyapatite/chitosan composite material is 10%;

sodium bicarbonate was not used in the solid phase of this comparative example.

Comparative example 6

In this comparative example, a porous hydroxyapatite/chitosan composite scaffold was prepared substantially in the same manner as in example 1, with the main difference being that components of the solid phase were prepared. The method comprises the following specific steps:

preparing a solid phase: mixing and grinding the following components in parts by weight: 57 parts of a first hydroxyapatite/chitosan composite material, 20 parts of sodium bicarbonate and 15 parts of a second hydroxyapatite/chitosan composite material; wherein the chitosan mass fraction of the first hydroxyapatite/chitosan composite material is 30%, and the chitosan mass fraction of the second hydroxyapatite/chitosan composite material is 10%;

no magnesium oxide was used in the solid phase of this comparative example.

Comparative example 7

preparing a solid phase: mixing and grinding the following components in parts by weight: 57 parts of a first hydroxyapatite/chitosan composite material and 15 parts of a second hydroxyapatite/chitosan composite material; wherein the chitosan mass fraction of the first hydroxyapatite/chitosan composite material is 30%, and the chitosan mass fraction of the second hydroxyapatite/chitosan composite material is 10%;

sodium bicarbonate and magnesium oxide were not used in the solid phase of this comparative example.

Comparative example 8

In this comparative example, the preparation method of the porous hydroxyapatite/chitosan composite scaffold is substantially the same as that of example 2, except that S3 is defined as follows:

s3, drying the chitosan coating, then soaking in 0.1mol/L sodium hydroxide solution for 6h, drying at 40 ℃ for 6h, washing with deionized water to be neutral, and drying at 40 ℃.

The ammonia fumigation of this comparative example was replaced by soaking in sodium hydroxide solution.

Testing

The porosity and compressive strength of the porous hydroxyapatite/chitosan composite material scaffold prepared in the above examples and comparative examples are detected, and the test method is as follows:

porosity in the sample was measured according to the drainage (ethanol) method. First, a pycnometer filled with ethanol (anhydrous) is weighed W₁Weighing the sample to obtain a dry sample, weighing the dry sample to obtain a specific gravity bottle filled with absolute ethanol and placed in a support fully saturated with ethanol, and weighing the specific gravity bottle₂Finally, the specific gravity bottle after the bracket is taken out is weighed₃. The open porosity of the scaffold ε ═ W₂-W₃-Ws)/(W₁-W₃)。

And (3) carrying out a compression strength test on the prepared bracket by using an Inspekt Table Blue 5KN electronic universal experiment machine, wherein 4-5 non-damaged samples are selected for each test point. And (3) polishing the test samples up and down by using sand paper, observing the parallelism of the upper bottom surface and the lower bottom surface by using flat tongs, measuring the diameter and the height of each test sample, detecting at a compression rate of 1mm/min, removing extreme values of results, and averaging.

The results are as follows:

TABLE 2

FIGS. 1 and 2 are low-power and high-power views of a scanning electron microscope of example 1, and FIG. 1 shows that the prepared sample has a pore size of about 100 μm to 500 μm, which is an advantageous pore size of a porous scaffold for bone tissue engineering and also contains a small number of through holes. FIG. 2 is a partial enlargement of the material of FIG. 1, showing that the particles are irregular in shape, and the particles are directly bonded to enhance mechanical properties, and have a surface roughness which is favorable for adhesion of cells and nutrients.

FIGS. 3 and 4 are the scanning electron microscope macroscopic and macroscopic images of example 2, and the surface of FIG. 3 is smoother than that of FIG. 1, and is a sodium alginate chitosan coating film. Fig. 4 can also be seen in comparison to fig. 2 that the surface particles are covered by a film, indicating that the coating works well.

It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A preparation method of a porous hydroxyapatite/chitosan composite material scaffold for bone tissue engineering is characterized by comprising the following steps:

s2, mixing the solid phase and the liquid phase according to the mass ratio of the solid to the liquid of 7: 10-3: 4, pouring the mixture into a support mold, reacting at room temperature for 2-6 hours, and drying to obtain the porous hydroxyapatite/chitosan composite support for bone tissue engineering;

and S3, sequentially coating sodium alginate and chitosan on the porous hydroxyapatite/chitosan composite material support prepared in the step S2.

2. The method according to claim 1, wherein in S1, a solid phase: mixing and grinding the following components in parts by weight:

3. The production method according to claim 1, wherein in S2, the solid phase and the liquid phase are mixed at a solid-liquid mass ratio of 3: 4.

4. The preparation method of claim 1, wherein the sequential coating of sodium alginate and chitosan is as follows: and (2) immersing the porous hydroxyapatite/chitosan composite material support in a sodium alginate solution, vacuumizing in a sealed environment, taking out the porous hydroxyapatite/chitosan composite material support, drying in vacuum until no bubbles exist in pores of the porous hydroxyapatite/chitosan composite material support, immersing in a chitosan acetic acid solution with the pH value of 4, vacuumizing in the sealed environment, drying in vacuum, fumigating with ammonia gas for 6-12 hours in the sealed environment, drying at 40-80 ℃, washing with deionized water to be neutral, and drying.

5. The method according to claim 1, wherein in S1, the first hydroxyapatite/chitosan composite material or the second hydroxyapatite/chitosan composite material is prepared by: mixing a calcium nitrate solution and a chitosan solution, dropwise adding a disodium hydrogen phosphate solution while stirring, controlling the calcium-phosphorus ratio to be 1.67, adjusting the pH value by using ammonia water, keeping the pH value to be 10, stirring for 0.5-2 h, aging for 12h at room temperature, washing with deionized water to be neutral, drying, grinding and sieving.

6. The porous hydroxyapatite/chitosan composite material scaffold prepared by the preparation method of any one of claims 1 to 5.