CN111135344A

CN111135344A - Scaffold for repairing carbon nano tube/collagen-based cartilage of composite albumin and preparation method thereof

Info

Publication number: CN111135344A
Application number: CN202010125955.5A
Authority: CN
Inventors: 王建华; 黄晨光; 陈杨燕; 丁自凤; 陈立
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2020-02-27
Filing date: 2020-02-27
Publication date: 2020-05-12

Abstract

The invention discloses a composite albumin carbon nanotube/collagen-based cartilage repair scaffold for articular cartilage defect repair and a preparation method thereof. The repair scaffold is prepared by using carboxyl functionalized single-walled carbon nanotubes, albumin powder and freeze-dried collagen as main raw materials. After the repairing scaffold is implanted into an animal model with articular cartilage defect for 12 weeks, the repairing scaffold can better repair the articular cartilage defect, wherein the repairing effect of the repairing scaffold containing 0.5 wt% of carbon nano tubes is the best. The cartilage repairing scaffold prepared by the invention has the characteristics of good mechanical property, biocompatibility, biodegradability and the like, has a good repairing effect on articular cartilage defects, and is a good tissue regeneration material which can be popularized and used in cartilage tissue engineering.

Description

Scaffold for repairing carbon nano tube/collagen-based cartilage of composite albumin and preparation method thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a carbon nanotube/collagen-based cartilage repair scaffold compounded with albumin and a preparation method thereof.

Background

Articular cartilage is a very complex hyaline-like cartilage connective tissue that plays a major role in weight bearing and movement in joint activity. Since the cartilage tissue has no blood vessels, lymphatic vessels and nervous tissues, the nutrition is mainly absorbed by synovial fluid secreted by synovium, synovial blood vessel osmosis and diffusion mode. When cartilage is damaged, the condition of the damaged part is gradually worsened due to the lack of blood supply and limited nutrient absorption, so that the cartilage is extremely difficult to repair by itself after being damaged. At present, the clinical methods for repairing the articular cartilage mainly comprise: subchondral bone microarthrosis, autologous chondrocyte transplantation, allogeneic cartilage transplantation, and the like, but the above methods are not satisfactory in practice, and therefore, tissue engineering has rapidly grown in recent years. In cartilage tissue engineering, the scaffold material serves as a carrier for growth of chondrocytes and plays a decisive role in growth of the chondrocytes, so that research on repair materials is very important. For the repair materials currently under development, poor mechanical properties are one of the main disadvantages of such materials.

Carbon nanotubes are cylindrical graphite tubes having a large aspect ratio, and are often classified into single-walled carbon nanotubes and multi-walled carbon nanotubes according to their tube wall structures, which have received wide attention in various fields such as medicine, tissue engineering, drug administration, etc. due to their unique chemical, mechanical, electrical conductivity and low toxicity. However, a large number of studies have confirmed that carbon nanotubes may have potential cytotoxicity, and have not been found to be used in vivo studies for cartilage defect repair in the field of cartilage repair. Therefore, there is a need for animal or organism in vivo studies on carbon nanotube-containing repair materials.

Serum albumin is the earliest and clearest component in plasma research, and has the advantages of safety, no toxicity, biodegradability, good biocompatibility and the like. The combination of serum albumin and multi-wall carbon nano-tubes can reduce the potential toxic effect of the multi-wall carbon nano-tubes, but the effect on the single-wall carbon nano-tubes is not reported. Collagen, which accounts for 50-80% of the articular cartilage, is a main component of the articular cartilage, has the advantages of good biocompatibility, degradability, low immunogenicity and the like, and has been widely used for the preparation of tissue regeneration materials.

The invention synthesizes the carbon nano tube to effectively improve the mechanical property of the material, the potential cytotoxicity of the single-walled carbon nano tube can be counteracted by the combined serum albumin, and the composite albumin-carbon nano tube/collagen-based cartilage repair bracket which can effectively repair cartilage defect is prepared by cooperating with the excellent performance of natural collagen.

Disclosure of Invention

The invention aims to provide a scaffold for repairing a carbon nano tube/collagen-based cartilage, which is compounded with albumin, has good mechanical strength and biological performance, and shows a good repairing effect in the in-vivo repair of cartilage defects.

In order to achieve the purpose, the invention adopts the following technical scheme:

the scaffold for repairing the carbon nanotube/collagen-based cartilage of the composite albumin is prepared by taking a carboxyl functionalized single-walled carbon nanotube, albumin powder and freeze-dried collagen as main raw materials, wherein the mass ratio of the carboxyl functionalized single-walled carbon nanotube to the albumin powder to the freeze-dried collagen is (0-4): 4: 200. the freeze-dried collagen is derived from fish skin, pig skin, cow skin or beef tendon.

The preparation method of the albumin-compounded carbon nanotube/collagen-based cartilage repair scaffold comprises the following steps:

(1) dissolving the freeze-dried collagen in water to prepare a collagen solution;

(2) dissolving albumin powder in ultrapure water to prepare a protein water solution;

(3) adding the carboxyl functionalized single-walled carbon nanotubes into the protein aqueous solution obtained in the step (2), and performing ultrasonic treatment to uniformly disperse the carboxyl functionalized single-walled carbon nanotubes;

(4) mixing the uniformly dispersed solution obtained in the step (3) with the collagen solution obtained in the step (1), and stirring overnight by magnetic force;

(5) after freeze-drying, adding a cross-linking agent according to 350 mL/g for cross-linking; the cross-linking agent is prepared by dissolving 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in ethanol solution with volume concentration of 95% according to the amount of 50 mmol/L and 8 mmol/L respectively and mixing;

(6) repeatedly soaking in distilled water after crosslinking until cleaning, and lyophilizing again; soaking with distilled water for 30min, and repeating soaking for 6 times.

The invention has the following beneficial effects:

1. the cartilage repair stent for treating articular cartilage defects, which is prepared by the invention, has the advantages of simple preparation process, low price, easy obtainment and mature process. The repair scaffold is of a three-dimensional net structure, the mechanical property of the carbon nanotube reinforced material is utilized, the potential toxicity of the carbon nanotube is counteracted by albumin, and an excellent cell microenvironment is provided for the repair material by utilizing collagen, so that the repair scaffold is an excellent repair material suitable for articular cartilage defect treatment.

2. Animal experiments prove that the repair scaffold prepared by the invention has good effect of repairing cartilage defect.

Drawings

Fig. 1 is a scanning electron microscope image of the albumin-complexed carbon nanotube/collagen-based cartilage repair scaffold obtained in example 3 at different magnifications (wherein black arrows indicate carbon nanotubes in the repair scaffold and gray arrows indicate collagen fibers in the repair scaffold).

FIG. 2 is a graph showing the compressive strength of the prosthetic stents obtained in examples 1-4.

FIG. 3 is a graph showing cytotoxicity tests of the scaffolds prepared in examples 1-4.

FIG. 4 is a comparison graph of toluidine blue staining of pathological specimen detection at 12 weeks for the repaired stent with carbon nanotube content of 0 (A), 0.5 wt% (B) and 2.0 wt% (C), respectively.

Detailed Description

In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.

The preparation method of the carboxyl functionalized single-walled carbon nanotube comprises the following steps: firstly, preparing a single-walled carbon nanotube by adopting a chemical vapor deposition method, wherein the purity of the single-walled carbon nanotube is more than 95 wt%, the inner diameter of the single-walled carbon nanotube is 0.8-1.6 nm, and the outer diameter of the single-walled carbon nanotube is 1-2 nm; and then adding 120 mL of 2.6 mol/L dilute nitric acid into a single-neck round-bottom flask, adding 50 mg of the single-walled carbon nanotube, carrying out ultrasonic treatment for 30min, heating to a reflux state, stirring for 24 h, cooling after the reaction is finished, washing with ultrapure water to be neutral, collecting, and drying to obtain the purified carboxyl functionalized single-walled carbon nanotube.

The cross-linking agent is prepared by dissolving 5 mmol of 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide and 0.8 mmol of N-hydroxysuccinimide in 100 mL of 95 volume percent ethanol solution and mixing.

Example 1

(1) Accurately weighing 0.2 g of bovine type I collagen freeze-dried powder, adding 15 mL of deionized water (containing 0.1% glacial acetic acid) into a magnetic stirrer, and uniformly stirring to obtain a collagen solution (O);

(2) accurately weighing 4mg of bovine serum albumin powder, dissolving the bovine serum albumin powder in 10 mL of ultrapure water, and placing the bovine serum albumin powder on a magnetic stirrer to be stirred and dissolved to prepare a bovine serum albumin solution with the concentration of 0.4 mg/mL;

(3) accurately weighing 2 mg of carboxyl functionalized single-walled carbon nanotubes, adding the carboxyl functionalized single-walled carbon nanotubes into the bovine serum albumin solution prepared in the step (2), and performing ultrasonic treatment for 30min (during which the solution is vibrated every 5 min) to disperse the carboxyl functionalized single-walled carbon nanotubes, thereby finally obtaining a suspension in which the carboxyl functionalized single-walled carbon nanotubes are uniformly dispersed;

(4) adding a collagen solution (O) into the suspension in which the carboxyl functionalized single-walled carbon nanotubes are uniformly dispersed, violently stirring for 30min, slowly stirring overnight, freeze-drying, taking out, and adding 70 mL of a cross-linking agent for cross-linking;

(5) and soaking the cross-linked scaffold in distilled water for 30min, repeating the soaking for 6 times, and freeze-drying again to obtain the repair scaffold with the carbon nanotube content of 1.0 wt%, which is marked as A.

Example 2

Replacing the using amount of the carboxyl functionalized single-walled carbon nanotube in the step (3) with 1 mg, and performing the other steps in the same way as in the example 1 to prepare a repair scaffold with the carbon nanotube content of 0.5 wt%, which is marked as B.

Example 3

Replacing the using amount of the carboxyl functionalized single-walled carbon nanotube in the step (3) with 4mg, and preparing the repairing scaffold with the carbon nanotube content of 2.0 wt% by the same steps as the example 1, wherein the repairing scaffold is marked as C.

Example 4

Replacing the using amount of the carboxyl functionalized single-walled carbon nanotube in the step (3) with 0 mg, and performing the other steps in the same manner as in the example 1 to obtain a repair scaffold without the carbon nanotube, which is marked as D.

1. And (3) testing mechanical properties:

the prosthetic scaffolds prepared in examples 1-4 were trimmed to cylinders 5 cm in diameter and 5 cm in height and tested for mechanical properties on a universal material testing machine, respectively, with an instrument chuck speed of 1 mm/min during the test. And taking the corresponding compressive stress when the deformation reaches 60% as the compressive modulus of the stent material, and taking the stress corresponding to the 60% deformation rate to calculate the compressive strength.

As can be seen from fig. 2, the compressive strength of the repair scaffold is increased with the addition of the carbon nanotubes, which proves that the addition of the carbon nanotubes has the effect of improving the mechanical properties of the repair scaffold.

2. Cytotoxicity test:

the prosthetic scaffolds prepared in examples 1-4 were added to DMEM/F-12 medium at 5 mg/mL (scaffold/medium) and extracted in an incubator at 37 ℃ for 48 h. And filtering the obtained leaching liquor by a 0.22 mu m filter membrane, and adding 10% FBS into the leaching liquor to obtain a sample solution to be detected corresponding to each repair bracket.

BMSCs at 1X 10⁴Cell/well Density in 96-well plates at 37 ℃ with 5% CO₂After culturing for 24 h, removing non-adherent cells from the pore plate, and adding the sample solution to be detected into the pores respectivelyIn the plate, the culture was continued, and the culture medium was changed every two days. Samples were taken on

days

1, 3, 5, and 7 of the culture to measure the absorbance at 450 nm, and the results are shown in FIG. 3 (the plate to which no sample solution was added was used as a blank).

As can be seen from fig. 3, compared with the blank control, the cell viability of the sample solution with the repair scaffold added thereto is not significantly inhibited, and on day 7, each group shows a cell viability trend of D > B > a > C > control (the cell viability gradually decreases to a normal level with the increase of the content of the carbon nanotube), which proves that the addition amount of the carbon nanotube is within 2.0 wt% without cytotoxicity. Meanwhile, the comparison between D and control shows that the addition of the bovine serum albumin composite collagen-based material into cells is helpful for improving the cell viability.

3. Animal in vivo test:

operation: 24 clean-grade New Zealand rabbits were randomly divided by weight into 6 groups of 4, 8 joints per group. The grouping is as follows: model control group (group a): the defect part is not treated; defect group (group B): implanting the sterile repair scaffold prepared in example 1 into the defect; defect group (group C): implanting the sterile repair scaffold prepared in example 2 into the defect; defect group (group D): implanting the sterile repair scaffold prepared in example 3 into the defect; defect group (group E): implanting the sterile repair scaffold prepared in example 4 into the defect; blank control group (group F): normal group without defect. After grouping, anaesthetizing with fast-sleep new II (0.3 mL/kg) and constructing cartilage and subchondral bone defect models, wherein the diameter of a defect area is 4 mm, and the depth is about 4 mm.

According to the grouping, the repair stents sterilized by gamma rays are respectively implanted into the articular cartilage defect model, and the wounds are sutured layer by layer. Each rabbit was injected intramuscularly with 10 ten thousand units of penicillin sodium for 1 week after surgery to prevent infection.

Post-operative observation and specimen handling: at 12 weeks after surgery, groups of experimental animals were sacrificed by otolimbic intravenous injection of excess hypnotic neo-II, gross observation, HE staining, toluidine blue staining.

As a result: the albumin-compounded carbon nanotube/collagen-based cartilage repair scaffold has a good repair effect on articular cartilage defects, and when the content of the carbon nanotubes is 0.5 wt%, the repair effect is optimal (as shown in figure 4), and the boundary between the new cartilage tissue and the surrounding tissue is not obvious. With the increase of the content of the carbon nano tube, the mechanical property of the repair material is better improved, the transparent cartilage tissue can be still generated at the defect part, but the repair period is delayed because the degradation period of the repair scaffold is prolonged. Therefore, the albumin-compounded carbon nanotube/collagen-based cartilage repair scaffold for repairing articular cartilage defects of animals does not have the best effect when the content of the carbon nanotubes is higher and is 0.5 wt%.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims

1. A scaffold for repairing carbon nano tube/collagen-based cartilage of composite albumin is characterized in that: the cartilage repair scaffold is prepared from carboxyl functionalized single-walled carbon nanotubes, albumin powder and freeze-dried collagen serving as main raw materials, wherein the mass ratio of the carboxyl functionalized single-walled carbon nanotubes to the albumin powder to the freeze-dried collagen is (0-4): 4: 200.

2. the scaffold for carbon nanotube/collagen-based cartilage repair of composite albumin according to claim 1, wherein: the freeze-dried collagen is derived from fish skin, pig skin, cow skin or beef tendon.

3. A method for preparing the albumin-complexed carbon nanotube/collagen-based cartilage repair scaffold according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:

(5) adding a cross-linking agent for cross-linking after freeze-drying;

(6) repeatedly soaking in distilled water after crosslinking, cleaning, and lyophilizing again.

4. The method for preparing the scaffold for repairing the carbon nanotube/collagen-based cartilage, which is compounded with albumin according to claim 3, wherein the method comprises the following steps: the cross-linking agent in the step (5) is prepared by dissolving and mixing 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in ethanol solution with volume concentration of 95% according to the amount of 50 mmol/L and 8 mmol/L respectively.

5. The method for preparing the scaffold for repairing the carbon nanotube/collagen-based cartilage, which is compounded with albumin according to claim 3, wherein the method comprises the following steps: in the step (6), the time for soaking once by using distilled water is 30min, and the repeated soaking times are 6 times.