CN106693050B - A preparation method of composite scaffold material based on collagen and collagen fibers - Google Patents

Abstract

The invention discloses a preparation method of a composite scaffold material based on collagen and collagen fibers, which is characterized in that collagen is organically mixed with its aggregated structure collagen fibers, and functional oxidized polysaccharides and graphene oxide are used to compound collagen-collagen fibers The solution is compounded and synergistically modified, and then the collagen-collagen fiber composite solution is blended with a certain amount of chitosan and polyvinyl alcohol, and then undergoes processes such as 3D printing, microwave drying, electrospraying, and sterilization to obtain A method for preparing a composite scaffold material based on collagen and collagen fibers. The material uses collagen fibers with a stronger biomimetic natural collagen structure, and uses 3D printing technology to biodesign the microstructure and morphology of the scaffold, fully imitating natural active tissues from the chemical composition and natural structure, and the obtained scaffold material has a stable structure, It has superior physical and chemical properties, good cell compatibility and biodegradability, and can induce tissue regeneration and repair, and can be used as various types of tissue engineering scaffolds.

Description

A preparation method of composite scaffold material based on collagen and collagen fibers

technical field

The invention relates to a composite support material based on collagen and collagen fibers, belonging to the field of biomedical material preparation.

Background technique

Tissue engineering scaffold is the most basic structure of tissue engineering tissue, which provides a suitable environment for the growth of cells and tissues, and gradually degrades and disappears with the construction of tissues, thus providing new space for tissues and cells. This structure is the place where cells obtain nutrients, gas exchange, waste excretion and growth metabolism, and is the basis for forming new tissues and organs with certain shapes and functions. For decades, living tissue has been produced through a "two-step process". The first step is to design a scaffold for the tissue using some kind of biodegradable material. To fabricate a designed scaffold using traditional methods, researchers model it, sculpt it, or use chemicals to etch a porous shape. The second step is to grow a scaffold using living cells. A large number of studies have proved that these technologies have many disadvantages, such as a large amount of highly toxic organic solvents, long manufacturing cycles, labor-intensive processes, inability to completely remove residues in the polymer matrix, poor reproducibility, and irregular pores. The space cannot be fully connected, the structure is too thin, and so on. Furthermore, most of these methods cannot control the shape.

Compared with this method, 3D printing can precisely print the shape and structure of the desired tissue. 3D printed forms of inanimate restorations such as bone implants, dental crowns, contact lenses and hearing aids are already in the bodies of thousands of people around the world. Currently, 3D printed body parts use a single material such as metal, ceramic or plastic. They make commercial sense because their market value comes from their ability to fit snugly to a specially shaped body. However, these materials can only become inanimate replacement parts, and the real 3D bioprinting should be to create living tissues. Bioprinting, the use of "living ink," a printable gel with living cells suspended inside. Living cells are printed with "gap fillers" (such as collagen, to temporarily fill the space until they fuse with other cells) to create many special and precise shapes of human tissue. The living cells secrete substances into the hydrogel, which ultimately forms a supporting matrix. . As the cells grow, the matrix develops into living tissue.

Collagen and collagen fibers have good biocompatibility and biodegradability, as well as the ability to promote cell growth and adhesion, and are pH and temperature sensitive, which is good for molding. They have been used in the field of 3D printing living tissues. However, the mechanical properties of 3D printed active tissue of pure collagen or collagen fibers are not enough to support a certain shape of tissue, so adding other biomaterials or a small amount of cross-linking agent becomes a necessary element in the 3D printing process.

Therefore, the method of a composite scaffold material based on collagen and collagen fibers obtained in this patent has broad application prospects in the field of 3D printing of living tissues.

Contents of the invention

The object of the present invention is to provide a composite scaffold material based on collagen and collagen fibers to address the deficiencies of the prior art. The scaffold material has excellent cytocompatibility, which is very conducive to the growth, proliferation and migration of cells. Cells can grow into the scaffold well, and has certain anti-inflammatory, anti-oxidation, antibacterial and other effects. It has good biological activity and can be used as a scaffold for tissue engineering.

The object of the present invention can be realized by following preparation technology, and its preparation method step is as follows:

(1) Preparation of collagen-collagen fiber composite swelling solution based on oxidized polysaccharide and graphene oxide: mix 1-2 parts by weight of collagen and collagen fiber with a mass ratio of 1-9:9-1, and heat at 4-10°C Swell it in an acetic acid-sodium acetate buffer system with a pH of 4.0 under stirring to obtain a collagen-collagen fiber composite swelling solution with a concentration of 1 to 2 wt %; then add 0.05 to 0.4 parts by weight of oxidized polysaccharide to the above composite swelling solution , react in the dark at 4-10°C for 10-24h, after the reaction is complete, add 0.01-0.2 parts by weight of graphene oxide to the composite swelling solution, and continue the reaction at 4-10°C for 24-48h to obtain a polysaccharide based on oxidized polysaccharide and Graphene oxide collagen-collagen fiber composite swelling solution;

(2) Preparation of stock solution of composite scaffold material based on collagen and collagen fibers: add 0.5-1.5 parts by weight of chitosan to the above-mentioned collagen-collagen fiber composite swelling solution, stir at 4-10°C until completely dissolved, and obtain a mixed Solution A: Dissolve 6-12 parts by weight of polyvinyl alcohol in deionized water at 40-80°C to obtain an aqueous solution of polyvinyl alcohol with a concentration of 6-12wt%; the volume ratio will be 1-10:0.01-2 The mixed liquid A is mixed with polyvinyl alcohol aqueous solution, and stirred at 4-10°C until fully co-dissolved to obtain a stock solution of a composite scaffold material based on collagen and collagen fibers;

(3) Preparation of composite scaffold materials based on collagen and collagen fibers: According to the required scaffold shape and structure, computer-aided design software was used to compile a program to control the movement of the platform, and the stock solution of the composite scaffold materials based on collagen and collagen fibers was injected into the 3D Bioprinter, 3D printing to obtain a collagen-collagen fiber composite scaffold, and transfer the composite scaffold to a microwave dryer to form a film; then 1 to 2 parts by weight of collagen solution in 0.05 to 0.5M acetic acid solution to prepare a concentration of 1-2wt% collagen solution collagen, spray the above-mentioned collagen solution on the front and back sides of the collagen-collagen fiber composite scaffold with an electrostatic sprayer, and finally freeze-dry and sterilize it with gamma rays produced by 60KGy/h ⁶⁰ Co Bacteria, molding and packaging to obtain the final product of the composite scaffold material based on collagen and collagen fibers.

In the above preparation method, the oxidized polysaccharide used in step (1) is obtained by oxidizing carboxymethyl cellulose, hyaluronic acid, chitosan, dextran, etc. with sodium periodate. For the specific preparation method, please refer to our research group Early published patents [Dan Weihua, Liu Xinhua, Dan Nianhua, etc. Collagen Aggregate Composite Medical Fiber with Antibacterial/Bacteriostatic Effects. Chinese Invention Patent. 201510127304.9; Dan Nianhua, Dan Weihua, Liu Xinhua, etc. Sugar and its preparation method. Patent authorization number: CN104004112 A; Dan Weihua, Liu Xinhua, Dan Nianhua, etc. Oxidized chitosan grafted modified porcine dermal collagen micro-nano fiber membrane and its preparation method. Patent authorization number: CN 104013995 A]; the graphene oxide, polyvinyl alcohol, and chitosan described in step (1) are all medical grade; the 3D printing procedure of the composite scaffold material of collagen and collagen fibers in step (1) needs to be based on the specific scaffold use, Depends on shape and fine structure.

The present invention has the following advantages:

(1) Different from most of the collagen-based scaffold materials reported, the present invention uses collagen and collagen fibers, in which collagen fibers are aggregates of collagen, and its structure is more complex, which can better imitate the existing structure of collagen in living tissues , studies have shown that its physical and chemical properties, biological activity, tissue induction regenerative repair ability are significantly stronger;

(2) The present invention uses oxidized polysaccharides and graphene oxide as collagen modifiers. On the one hand, oxidized polysaccharides can be combined with collagen to achieve Schiff base bonds to achieve effective cross-linking effects, and the polysaccharides can still maintain their bulk after oxidation. Functionality is very beneficial to the preparation of functionalized collagen; on the other hand, graphene oxide can significantly enhance the physical and chemical properties of collagen without affecting the biological activity of collagen, and can endow collagen with certain antibacterial/bacteriostatic properties;

(3) The use of 3D printing technology can carry out bionic biological design on the shape and microstructure of the scaffold, and control the ingrowth and growth of cells by regulating the microstructure and shape of the scaffold.

Detailed ways

The present invention will be specifically described below by implementing it. It is necessary to point out that this embodiment is only used to further illustrate the present invention, and can not be interpreted as limiting the protection scope of the present invention. Make non-essential improvements and adjustments to the content.

Example 1

(1) Preparation of collagen-collagen fiber composite swelling liquid based on oxidized polysaccharide and graphene oxide: Mix 1 part by weight of collagen and collagen fiber with a mass ratio of 1:9, stir at 4°C and swell it at a pH of In the acetic acid-sodium acetate buffer system of 4.0, a collagen-collagen fiber composite swelling solution with a concentration of 1wt% was obtained; then, 0.05 parts by weight of oxidized polysaccharide was added to the above composite swelling solution, and reacted in the dark at 4°C for 24 hours until the reaction was completed Finally, add 0.2 parts by weight of graphene oxide to the composite swelling solution, and continue to react for 48 hours at 4°C to obtain a collagen-collagen fiber composite swelling solution based on oxidized polysaccharides and graphene oxide;

(2) Preparation of the stock solution of the composite scaffold material based on collagen and collagen fibers: add 0.5 parts by weight of chitosan to the above-mentioned collagen-collagen fiber composite swelling solution, stir at 4°C until completely dissolved, and obtain the mixed solution A; Dissolve 6 parts by weight of polyvinyl alcohol in deionized water at 40°C to obtain a polyvinyl alcohol aqueous solution with a concentration of 6wt%; mix the mixed liquid A with a volume ratio of 1:2 with the polyvinyl alcohol aqueous solution, and Stir until fully co-dissolved to obtain a stock solution of a composite scaffold material based on collagen and collagen fibers;

(3) Preparation of composite scaffold materials based on collagen and collagen fibers: According to the required scaffold shape and structure, computer-aided design software was used to compile a program to control the movement of the platform, and the stock solution of the composite scaffold materials based on collagen and collagen fibers was injected into the 3D Bioprinter, 3D printing to obtain a collagen-collagen fiber composite scaffold, and transfer the composite scaffold to a microwave dryer to form a film; then 1 weight part of collagen solution in 0.05M acetic acid solution to prepare a concentration of 1wt% Collagen solution collagen, using an electrostatic sprayer to spray the above collagen solution on the front and back sides of the collagen-collagen fiber composite support, and finally freeze-dried, sterilized and sterilized by gamma rays produced by 6KGy/h ⁶⁰ Co, molded and packaged, and obtained based on The final product of the composite scaffold material of collagen and collagen fibers.

Example 2

(1) Preparation of collagen-collagen fiber composite swelling liquid based on oxidized polysaccharide and graphene oxide: mix 1.5 parts by weight of collagen and collagen fiber with a mass ratio of 3:7, stir at 5°C and swell it at a pH of In the acetic acid-sodium acetate buffer system of 4.0, a collagen-collagen fiber composite swelling solution with a concentration of 1.5wt% was obtained; then, 0.2 parts by weight of oxidized polysaccharide was added to the above-mentioned composite swelling solution, and reacted in the dark at 5°C for 15 hours. After completion, add 0.1 parts by weight of graphene oxide to the composite swelling solution, and continue the reaction at 5°C for 24 to 48 hours to obtain a collagen-collagen fiber composite swelling solution based on oxidized polysaccharide and graphene oxide;

(2) Preparation of stock solution of composite scaffold material based on collagen and collagen fibers: add 1 part by weight of chitosan to the above-mentioned collagen-collagen fiber composite swelling solution, stir at 5°C until completely dissolved, and obtain mixed solution A; Dissolve 8 parts by weight of polyvinyl alcohol in deionized water at 60°C to obtain a polyvinyl alcohol aqueous solution with a concentration of 8wt%; mix the mixed liquid A with a volume ratio of 5:1 with the polyvinyl alcohol aqueous solution, and Stir until fully co-dissolved to obtain a stock solution of a composite scaffold material based on collagen and collagen fibers;

(3) Preparation of composite scaffold materials based on collagen and collagen fibers: According to the required scaffold shape and structure, computer-aided design software was used to compile a program to control the movement of the platform, and the stock solution of the composite scaffold materials based on collagen and collagen fibers was injected into the 3D Bioprinter, 3D printing to obtain a collagen-collagen fiber composite scaffold, and transfer the composite scaffold to a microwave dryer to form a film; then 1.5 parts by weight of collagen solution in 0.1M acetic acid solution to prepare a concentration of 1.5wt% Collagen solution collagen, using an electrostatic sprayer to spray the above collagen solution on the front and back sides of the collagen-collagen fiber composite support, finally freeze-dried, sterilized and sterilized by γ-rays produced by 15KGy/h ⁶⁰ Co, molded and packaged, and obtained The final product of the composite scaffold material based on collagen and collagen fibers.

Example 3

(1) Preparation of collagen-collagen fiber composite swelling solution based on oxidized polysaccharide and graphene oxide: Mix 2 parts by weight of collagen and collagen fiber with a mass ratio of 9:1, stir at 10°C and swell it at a pH of In the acetic acid-sodium acetate buffer system of 4.0, a collagen-collagen fiber composite swelling solution with a concentration of 2wt% was obtained; then, 0.4 parts by weight of oxidized polysaccharide was added to the above composite swelling solution, and reacted in the dark at 10°C for 10 hours until the reaction was completed Finally, add 0.01 parts by weight of graphene oxide to the composite swelling solution, and continue to react for 24 hours at 4-10° C. to obtain a collagen-collagen fiber composite swelling solution based on oxidized polysaccharide and graphene oxide;

(2) Preparation of stock solution of composite scaffold material based on collagen and collagen fibers: Add 1.5 parts by weight of chitosan to the above-mentioned collagen-collagen fiber composite swelling solution, stir at 10°C until completely dissolved, and obtain mixed solution A; Dissolve 6-12 parts by weight of polyvinyl alcohol in deionized water at 80°C to obtain a polyvinyl alcohol aqueous solution with a concentration of 12wt%; mix the mixed liquid A with a volume ratio of 10:0.01 with the polyvinyl alcohol aqueous solution, Stir at ℃ until fully co-dissolved to obtain a stock solution of a composite scaffold material based on collagen and collagen fibers;

(3) Preparation of composite scaffold materials based on collagen and collagen fibers: According to the required scaffold shape and structure, computer-aided design software was used to compile a program to control the movement of the platform, and the stock solution of the composite scaffold materials based on collagen and collagen fibers was injected into the 3D Bioprinter, 3D printing to obtain a collagen-collagen fiber composite scaffold, and transfer the composite scaffold to a microwave dryer to form a film; then 2 parts by weight of collagen solution in 0.5M acetic acid solution to prepare a concentration of 2wt% Collagen solution collagen, using an electrostatic sprayer to spray the above collagen solution on the front and back sides of the collagen-collagen fiber composite support, and finally freeze-dried, sterilized and sterilized by γ-rays produced by 30KGy/h ⁶⁰ Co, molded and packaged, and obtained based on The final product of the composite scaffold material of collagen and collagen fibers.

Claims (3)

1. a preparation method based on collagen and collagen fiber composite support material, it is characterized in that the method may further comprise the steps: (1) Preparation of collagen-collagen fiber composite swelling liquid based on oxidized polysaccharide and graphene oxide: mix 1-2 parts by weight of collagen and collagen fibers with a mass ratio of 1-9:9-1, and mix them at 4-10 Stir at ℃, swell it in an acetic acid-sodium acetate buffer system with a pH of 4.0 to obtain a collagen-collagen fiber composite swelling solution with a concentration of 1 to 2 wt %; then add 0.05 to 0.4 parts by weight to the above composite swelling solution The oxidized polysaccharides were reacted in the dark at 4-10°C for 10-24 hours. After the reaction was completed, 0.01-0.2 parts by weight of graphene oxide was added to the composite swelling solution, and the reaction was continued for 24-48 hours at 4-10°C. Collagen-collagen fiber composite swelling solution of oxidized polysaccharide and graphene oxide; (2) Preparation of stock solution of composite scaffold material based on collagen and collagen fibers: add 0.5-1.5 parts by weight of chitosan to the above-mentioned collagen-collagen fiber composite swelling solution, stir at 4-10°C until completely dissolved, and obtain a mixed Solution A: Dissolve 6-12 parts by weight of polyvinyl alcohol in deionized water at 40-80°C to obtain an aqueous solution of polyvinyl alcohol with a concentration of 6-12wt%; the volume ratio will be 1-10:0.01-2 The mixed solution A is mixed with polyvinyl alcohol aqueous solution, and stirred at 4-10°C until fully co-dissolved to obtain a stock solution of a composite scaffold material based on collagen and collagen fibers; (3) Preparation of composite scaffold materials based on collagen and collagen fibers: According to the required scaffold shape and structure, computer-aided design software was used to compile a program to control the movement of the platform, and the stock solution of the composite scaffold materials based on collagen and collagen fibers was injected into the 3D Bioprinter, 3D printing to obtain a collagen-collagen fiber composite scaffold, and transfer the composite scaffold to a microwave dryer to form a film; then dissolve 1-2 parts by weight of collagen in 0.05-0.5M acetic acid solution, and prepare a concentration of 1-2wt% collagen solution, use an electrostatic sprayer to spray the above-mentioned collagen solution on the front and back sides of the collagen-collagen fiber composite scaffold, and finally freeze-dry and sterilize with gamma rays produced by 60KGy/h ⁶⁰ Co , molding and packaging to obtain the final product of the composite scaffold material based on collagen and collagen fibers; the composite scaffold material based on collagen and collagen fibers mainly includes four base materials of collagen, collagen fibers, chitosan and polyvinyl alcohol, the key The performance indicators are as follows: Average pore size: ≥20μm; Heavy metal content: ≤10μg/g(m/m); Cytotoxicity: the cytotoxic reaction is not greater than grade 1; Sterility test: sterile; Sensitization test: no delayed hypersensitivity reaction; Intradermal test: primary irritation index PII<0.4. 2. the preparation method of the composite support material based on collagen and collagen fibers according to claim 1, is characterized in that, described collagen and collagen fibers are all obtained from pigskin, pig tendon, oxhide, ox tendon, donkey skin or mouse Extracted from any one of the tail tendons. 3. the preparation method of the composite support material based on collagen and collagen fiber described in claim 1, is characterized in that, described oxidized polysaccharide is polysaccharide gained through sodium periodate oxidation, selects oxidized chitosan, oxidized hyaluronic acid for use , oxidized dextran, and oxidized carboxymethylcellulose.