CN103013140B - Carbon nano tube/collagen based composite material and preparation method thereof - Google Patents

Abstract

The invention discloses a carbon nanotube/collagen-based composite material and a preparation method thereof. The composite material is composed of collagen and carbon nanotubes, wherein the mass percentage of collagen is 96.0-99.8%, and the mass percentage of carbon nanotubes is The content is 0.2-4.0%. The carbon nanotube/collagen-based composite material obtained by the invention has good biocompatibility, tissue repair performance and extremely low immunogenicity, the required raw materials are easy to obtain, the preparation process conditions are relatively mild, and it has wide popularization and application value.

Description

A kind of carbon nanotube/collagen matrix composite material and preparation method thereof

technical field

The invention belongs to the technical field of nanocomposite materials and their preparation, and in particular relates to a carbon nanotube/collagen-based composite material and a preparation method thereof.

Background technique

In recent years, with the rapid increase of the social population and the acceleration of the pace of life, the high incidence of tissue deficiency (disease) damage caused by tumors, infections, decreased physiological functions, and other causes, the frequent occurrence of natural disasters, industrial accidents, and the development of the automobile industry and sports The popularity of sports has resulted in a sharp increase in human accidental injuries and tends to be younger. The repair of the above-mentioned tissue defects (diseases) that pose a great threat to human health requires tissue-guided regeneration materials.

Collagen is the main component of extracellular matrix (ECM) and belongs to structural protein. Its appearance brings the research of tissue-guided regenerative materials into a new stage. Collagen has: (1) a unique network structure that can guide cell growth; (2) non-antigenic; (3) can participate in the healing process and promote cell growth; (4) excellent physical and mechanical properties; (5) biological Good compatibility; (6) The advantages of degradability and absorption are unmatched by other natural materials. Therefore, collagen is a natural material with excellent performance and degradable medical tissue-guided regeneration.

Collagen exists in the form of collagen fibrils or collagen fibers in the body. The basic structural unit of collagen fibers is procollagen molecules, which are thin rods with a length of 300 nm, a diameter of 1.5 nm, and a relative molecular weight of 2.85×10 ⁵ . Each procollagen molecule is composed of 3 a-peptide chains with a left-handed helical structure, and the 3 peptide chains form a right-handed compound helix through mutual folding and coiling. The unique triple helical structure of collagen endows it with good biological properties, such as high tensile strength, biodegradability, low antigenic activity, low irritation and low cytotoxicity, and the ability to promote cell growth, and at the same time makes it compatible with other substances , Such as the effective combination of hydroxyapatite, carbon nanotubes and cytokines, these characteristics make it an ideal biomedical material and drug carrier.

In recent years, carbon nanotubes (CNTs) have attracted extensive attention due to their large specific surface area and good adhesion to polymer substrates, thereby effectively improving the mechanical properties of the substrate. It is a hollow tube formed by curling a planar hexagonal graphite monolayer formed by carbon atoms, and can be divided into multi-walled carbon nanotubes and single-walled carbon nanotubes. From the study of microstructure, it is found that the diameter of typical single-walled carbon nanotubes is 0.5-1.5 nm, and the length is about 100-300 nm, which is similar to the triple helical structure of collagen fibers in bones and cartilage (diameter 1.5 nm, length 300 nm). nm) are very similar. Studies at home and abroad have shown that: although CNTs as a single scaffold material has the advantages of good biomechanical properties, good cell attachment rate and proliferation rate, it also has the disadvantages of difficult plasticity and non-degradability. Therefore, the combination of CNTs and collagen scaffolds and other materials Composite promises to address its own shortcomings. In addition, CNTs have great application potential in nutrient and drug supply systems. They can form cylindrical channels between nutrient and drug supply systems and cells to transport substances such as peptides, proteins, plasmid DNA or oligonucleotides, Therefore, CNTs may be used as an effective carrier for delivering cytokines to repair tissue defects such as bone and cartilage.

The present invention combines the advantages of natural collagen and CNTs to prepare a carbon nanotube/collagen-based composite material with tissue-guided regeneration. The material has good biocompatibility, tissue repair performance and extremely low immunogenicity. It can be applied to the repair of tissue disease defects such as bone and cartilage.

Contents of the invention

The purpose of the present invention is to provide a carbon nanotube/collagen-based composite material and its preparation method. The composite material is a new type of tissue-guiding regeneration material with good biocompatibility and tissue repair performance.

To achieve the above object, the present invention adopts the following technical solutions:

A carbon nanotube/collagen-based composite material is composed of collagen and carbon nanotubes, wherein the mass percent content of collagen is 96.0-99.8%, and the mass percent content of carbon nanotubes is 0.2-4.0%.

The collagen is fish skin collagen or collagen of pigskin, cowhide, Achilles tendon.

The carbon nanotubes are modified single-walled carbon nanotubes, or covalently modified and non-covalently modified multi-walled carbon nanotubes.

A method for preparing the carbon nanotube/collagen-based composite material as described above comprises the following steps:

(1) The collagen-rich raw material is mechanically removed from subcutaneous muscle, fat and connective tissue impurities, cut into 1 mm × 1 mm pieces and cleaned;

(2) Wash and degrease with a mixed solution of 0.01 mol/L sodium hydroxide and 1% hydrogen peroxide and 10 wt% isopropanol solution;

(3) Dissolved in a dilute acid solution with pH=2.0-3.0, the acid being acetic acid, propionic acid or malonic acid;

(4) Add protease, the mass ratio of collagen to protease is 50-100:1, enzymolysis at 4-5°C for 16-24h;

(5) Add NaCl or KCl to the supernatant collected by centrifugation to make the final concentration reach 0.9 mol/L, let it stand for 10-12 h, centrifuge at 5000 r/min for 20 min, and collect the obtained precipitate;

(6) Dissolve the precipitate in 10 times the volume of 0.5 mol/L acetic acid solution, and collect the supernatant by centrifugation;

(7) Dialyze in 10 times the volume of 0.1 mol/L acetic acid solution for 12 hours, change the solution every 3 hours, and finally dialyze with purified water to neutrality;

(8) Mix the dialyzed collagen and carbon nanotubes, stir them magnetically for 10 h, freeze-dry and cross-link in the cross-linking agent solution;

(9) The cross-linked composite material was fully rinsed with purified water, freeze-dried again, and placed between two tetrafluoroethylene plates for 24 h to make it flat to obtain a carbon nanotube/collagen-based composite material.

The protease is pepsin, trypsin or papain.

The formula of the cross-linking agent is: 0.2 mol/L ribose + 10wt% acetone + 2wt% ammonia water, or one or more of formaldehyde, glutaraldehyde, carboimide, diepoxide, genipin, proanthocyanidins kind.

The mass percent content of the cross-linking agent in the cross-linking agent solution is 0.3-3%.

The beneficial effects of the present invention are: the carbon nanotube/collagen-based composite material obtained by the present invention has good biocompatibility, tissue repair performance and extremely low immunogenicity, the required raw materials are easily available, and the preparation process conditions are relatively mild. It has extensive promotion and application value.

Description of drawings

Fig. 1 is a field emission scanning electron microscope image of a carbon nanotube/collagen matrix composite material.

Detailed ways

Embodiment 1: prepare carbon nanotube/collagen matrix composite material, concrete steps are as follows:

(1) The collagen-rich fish skin is mechanically removed from impurities such as subcutaneous muscle, fat and connective tissue, cut into pieces of about 1mm x 1mm and cleaned;

(2) Wash and degrease with 0.01 mol/L sodium hydroxide, a mixed solution containing 1% hydrogen peroxide and 10% isopropanol solution;

(3) Dissolved in acetic acid solution with pH 2.0;

(4) Add protease to the above solution, the mass ratio of collagen to protease is 50:1, enzymolysis at 4°C for 24 h;

(5) Add NaCl to the supernatant collected by centrifugation to make the final concentration reach 0.9 mol/L, let it stand for 12 hours, centrifuge at 5000r/min for 20 minutes, and collect the obtained precipitate;

(5) Dissolve the precipitate in 10 times the volume of 0.5mol/L acetic acid solution, and collect the supernatant by centrifugation;

(7) Dialyze in 10 times the volume of 0.1mol/L acetic acid solution for 12 hours, change the solution every 3 hours, and finally dialyze with purified water to neutrality;

(8) Take the 96.0% dry weight collagen after dialysis and mix it with 4% carbon nanotubes, stir it magnetically for 10 h, freeze-dry it and cross-link it in the cross-linking agent solution, the formula of the cross-linking solution: 0.2 mol.L ^-1 ribose + 10% acetone + 2% ammonia;

(8) Rinse the cross-linked composite material fully with purified water, freeze-dry it again, take it out, place it between two tetrafluoroethylene plates and pressurize it for 24 hours to make it flat, and obtain a carbon nanotube/collagen-based composite material (see figure 1).

Embodiment 2: preparation of carbon nanotube/collagen-based composite material, the specific steps are as follows:

(1) Mechanically remove impurities such as subcutaneous muscle, fat and connective tissue from the fish skin rich in collagen, cut it into pieces of about 1 mm x 1 mm and clean it;

(2) Wash and degrease with 0.01 mol/L sodium hydroxide, a mixed solution containing 1% hydrogen peroxide and 10% isopropanol solution;

(3) Dissolved in malonic acid solution with pH 3.0;

(4) Add protease to the above solution, the mass ratio of collagen to protease is 100:1, enzymolysis at 5°C for 16 h;

(5) Add KCl to the supernatant collected by centrifugation to make the final concentration reach 0.9 mol/L, let it stand for 10 hours, centrifuge at 5000r/min for 20 minutes, and collect the obtained precipitate;

(6) Dissolve the precipitate in 10 times the volume of 0.5mol/L acetic acid solution, and centrifuge to collect the supernatant;

(7) Dialyze in 10 times the volume of 0.1 mol/L acetic acid solution for 12 hours, change the solution every 3 hours, and finally dialyze with purified water to neutrality;

(8) Take 99.8% dry weight collagen and 0.2% carbon nanotubes after dialysis, mix them with magnetic force for 10 h, freeze-dry and cross-link in 0.3% formaldehyde solution;

(9) The cross-linked composite material was fully rinsed with purified water, freeze-dried again, and placed between two tetrafluoroethylene plates for 24 h to make it flat to obtain a carbon nanotube/collagen-based composite material.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims (5)

1. carbon nanotube/collagen group composite material, is characterized in that: this matrix material is made up of collagen protein and carbon nanotube, and wherein the mass percentage content of collagen protein is 96.0-99.8%, and the mass percentage content of carbon nanotube is 0.2-4.0%;

Preparation method comprises the steps:

(1) the raw material machinery being rich in collagen protein is removed subdermal muscle, fat and reticular tissue impurity, clean up after cutting into 1 mm × 1 mm fragment;

(2) with 0.01 mol/L sodium hydroxide and volume fraction be the aqueous isopropanol washing degreasing of the mixing solutions of the hydrogen peroxide of 1%, 10wt%;

(3) be dissolved in the dilute acid soln of pH=2.0-3.0, described acid is acetic acid, propionic acid or propanedioic acid;

(4) add proteolytic enzyme, the mass ratio of collagen protein and proteolytic enzyme is enzymolysis 16-24h at 50-100:1,4-5 DEG C;

(5) in the supernatant liquor of collected by centrifugation, add NaCl or KCl, make final concentration reach 0.9 mo1/L, leave standstill centrifugal 20 min of 10-12 h, 5000 r/min, collect the throw out obtained;

(6) throw out is dissolved in 0.5 mol/L acetic acid solution of 10 times of volumes, collected by centrifugation supernatant liquor;

(7) dialyse 12 h in 0.1 mol/L acetic acid solution of 10 times of volumes, and every 3 h change liquid once, finally extremely neutral with purified water dialysis;

(8) after getting dialysis, collagen and carbon nanotube mix, magnetic agitation 10 h, crosslinked in cross-linking agent solution after lyophilize; Described linking agent formula is: 0.2 mol/L ribose+10wt% acetone+2wt% ammoniacal liquor, or formaldehyde, glutaraldehyde, carbonization imines, diepoxides, genipin, one or more in pycnogenols;

(9) by purified water, the matrix material after crosslinked is fully rinsed, lyophilize again, take out and be placed on 24 h that to pressurize between two pieces of tetrafluoroethylene flat boards and make it smooth, obtain carbon nanotube/collagen group composite material.

2. carbon nanotube/collagen group composite material according to claim 1, is characterized in that: described collagen protein is the collagen protein of collagen of fish skin or pigskin, ox-hide, heel string.

3. carbon nanotube/collagen group composite material according to claim 1, is characterized in that: described carbon nanotube is the Single Walled Carbon Nanotube of modification, or the multi-walled carbon nano-tubes of covalent modified and non-covalent modification.

4. carbon nanotube/collagen group composite material according to claim 1, is characterized in that: described proteolytic enzyme is stomach en-, trypsinase or papoid.

5. carbon nanotube/collagen group composite material according to claim 1, is characterized in that: in described cross-linking agent solution, the mass percentage of linking agent is 0.3-3%.