CN100551449C - Tussah silk fibroin biomedical material and preparation method thereof - Google Patents

Abstract

The invention discloses a biomedical engineering material and a preparation method thereof, in particular to a three-dimensional tissue repair material and a preparation method thereof, which belong to the technical field of biomedical materials. It uses natural tussah silk as raw material. After the tussah silk fibroin is dissolved, the electrostatic spinning method is used to obtain a three-dimensional non-woven network structure composed of tussah silk fibroin fibers. The fiber diameter is 50nm-20μm, and the pores between the fibers are 1-500μm. , the thickness of the non-woven net is 50nm-20mm; the molecular weight of tussah silk fibroin is 10×10 ⁴ ～20×10 ⁵ D. This biomedical engineering material has the characteristics of non-toxic and harmless, good biocompatibility, and excellent cell adhesion and growth performance. It is rich in raw materials and low in cost. It is expected to replace high-cost biomedical materials such as collagen for artificial skin, Repair materials such as tendon, cartilage, and dura mater, especially tissue cell induction materials.

Description

Tussah silk fibroin biomedical material and preparation method thereof

technical field

The invention relates to a biomedical engineering material and a preparation method thereof, in particular to a three-dimensional tissue repair material and a preparation method thereof, which belong to the technical field of biomedical materials.

Background technique

Tussah silk contains a large amount of Arg-Gly-Asp (arginine-tyrosine-aspartic acid, RGD) tripeptide sequence, and this sequence has been proved to be beneficial for cell adhesion [Minoura N, Aiba S, Gotoh Y, Tsukada M, Imai Y. Attachment and growth of fibroblast cells on silkfibroin. Biochem Biophys Res Commun, 1995, 208 (2): 511-516; PierschbacherMD, Ruoslahti E, [J]. Nature, 1984, (309): 30- 33; Pierschbacher MD, Ruoslahti E. Pro Natl Acad Sci USA, 1984, (81): 5985-5988]. This point was proved in the in vitro cell culture experiment of Sofia [Sofia S, McCarthy MB, Gronowic G, et.al.Biomed Mater Res, 2001, (54): 139-148.]. The above disclosed research content shows that when the material is covalently modified with RGD, the activity of cells on the material is enhanced, the mineralization of osteoblast-like cells is promoted, and the induction of bone formation in vitro is enhanced. Therefore, tussah silk fibroin is expected to form greater application research results in cell culture substrates, wound covering membranes, artificial skin and tissue engineering scaffold materials, and biomedical material modification.

Tussah silkworm is an advantageous resource in my country. Tussah silk is composed of more than 80% silk fibroin and less than 20% sericin. It is a fairly pure natural protein fiber. The amino acid composition of silk fibroin is similar to human collagen , non-toxic, non-irritating, and has good biocompatibility. Therefore, tussah silk fibroin is an ideal raw material for preparing biomedical products, but because the diameter of natural tussah silk is more than 20 μm, and it is difficult to shape, the application range of natural tussah silk in the field of biomedicine is greatly limited.

The macromolecular chains of natural tussah silk fibroin have strong interactions and contain disulfide bonds, which are more difficult to dissolve than mulberry silk fibroin. Generally, the solvents that dissolve mulberry silk fibroin are difficult to dissolve tussah silk. Before the present invention was made, an article titled "Research on the Production Technology of Tussah Silk Peptide" [Liaoning Silk 2001, (3): 34-35] disclosed a method of dissolving tussah silk in phosphoric acid solution to prepare tussah silk Peptide method; the Chinese invention patent "Preparation Method of Tussah Silk Peptide" with publication number CN1415626A also discloses a method of adding tussah silk to phosphoric acid and dissolving it at a temperature of 100-130°C for 8-10 hours. A technical scheme for preparing tussah silk peptide. The principle of adopting the above technical solution is that silk fibroin is hydrolyzed and broken under the action of some strong acids, so that the molecular weight is greatly reduced, and a silk fibroin peptide with a molecular weight of about 200-5000D is produced, which is suitable for the fields of food and cosmetics. However, because the molecular weight is too small, it cannot meet the requirements for the shaping and mechanical properties of three-dimensional scaffolds prepared for biomedical materials.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art, and provide a tussah silk fibroin biomedical material that can meet the three-dimensional scaffold shaping and mechanical performance requirements, and is conducive to cell adhesion and growth, and a preparation method thereof.

The technical solution adopted in the present invention is: a tussah silk fibroin biomedical material, which is a three-dimensional non-woven network structure composed of tussah silk fibroin fibers, the fiber diameter is 50nm-20μm, and the pores between fibers are 1-500μm. The thickness of the spun net is 50nm-20mm; it contains 10-100% by weight of tussah silk fibroin with a molecular weight of 10×10 ⁴ ˜20×10 ⁵ D.

In the above-mentioned technical scheme, the balance is an additive; the additive is pure mulberry silk fibroin, polylactic acid or polyvinyl alcohol, polyethylene glycol, polyglycolic acid, polygalactone, cellulose, chitin, chitosan One or a combination of sugar, collagen, gelatin.

The preparation method of the above-mentioned tussah silk fibroin biomedical material, first degumming the tussah silk to obtain pure tussah silk fibroin, and then performing the following steps of processing:

①According to weight ratio, lithium thiocyanate: mercaptoethanol: urea=80～98:1～10:1～10 is made into ternary solution, and pure tussah silk fibroin is placed in above-mentioned ternary solution, at a temperature of 40～ Dissolve at 100°C for 5-70 hours, purify to obtain pure tussah silk fibroin protein aqueous solution, and then dry to obtain tussah silk fibroin protein with a molecular weight of 10×10 ⁴ to 20×10 ⁵ D;

② Dissolving the solute of the spinning stock solution in a solvent, the final concentration of the solution is 6-30%, and making the spinning stock solution; the solute of the spinning stock solution contains 10-100% by weight of the tussah silk fibroin obtained in step ① Protein: the solvent is one of formic acid and hexafluoroisopropanol;

③ Using the above-mentioned spinning dope by an electrospinning process to prepare a biomedical material with a three-dimensional non-woven network structure composed of tussah silk fibroin fibers.

In the above technical scheme, in the solute of the spinning stock solution, the balance is an additive; the additive is pure mulberry silk fibroin, polylactic acid or polyvinyl alcohol, polyethylene glycol, polyglycolic acid, polygalactone , cellulose, chitin, chitosan, collagen, gelatin or a combination thereof.

When the solvent described in the technical solution of the present invention is formic acid, the concentration of the formic acid solution is 60-99%, the dissolving temperature is 20-80°C, and the dissolving time is 0.5-5h. When the solvent is hexafluoroisopropanol, the dissolving temperature is 20-50° C., and the dissolving time is 24-240 h.

Compared with prior art, the advantage of the present invention is:

(1) The present invention uses tussah silk fibroin as the main raw material, which retains the advantages of good biocompatibility of natural tussah silk fibroin; it has a three-dimensional non-woven network structure, which can be directly used for the cultivation of tissue cells in vivo and in vitro And repair, it is an ideal biomedical material for tissue cell induction.

(2) The ternary solution provided by the present invention better solves the difficult problem of insoluble tussah silk fibroin fiber, and obtains tussah silk fibroin protein with a molecular weight of 10×10 ⁴ to 20×10 ⁵ D, which can meet the requirements of tissue preparation. The requirements for the shape and mechanical properties of three-dimensional scaffolds for repair materials provide abundant raw materials for biomedical materials.

(3) The tussah silk fibroin protein provided by the present invention can be blended with other biological materials, which is beneficial to give full play to the characteristics of each material and construct tissue repair materials suitable for various human bodies.

Description of drawings

Fig. 1 is the gel electrophoresis spectrum of the tussah silk fibroin protein solution prepared according to the technical scheme of the present invention, wherein, Marker1, Marker2 are standard sample, and swimming lane 1 sample is the tussah silk fibroin protein aqueous solution that concentration is 0.75%, and swimming lane 2 sample is concentration is a 1.5% tussah silk fibroin protein aqueous solution, the sample in swimming lane 3 is an aqueous tussah silk fibroin protein solution with a concentration of 2%, and the sample in swimming lane 4 is an aqueous tussah silk fibroin protein solution with a concentration of 3%.

Fig. 2 is a scanning electron micrograph of a three-dimensional non-woven network structure biomedical material composed of tussah silk fibroin fibers prepared according to the technical scheme of the present invention.

Detailed ways

The present invention will be further described below in conjunction with accompanying drawing and embodiment:

Embodiment one:

1. Put 0.6 kg of leftover tussah silk (a by-product of a silk reeling factory) into 10 liters of 0.05% sodium carbonate aqueous solution, boil for 0.5 hours, repeat the treatment three times, and remove the sericin around the fibroin to obtain pure tussah silk white;

2. Put the pure tussah silk fibroin after natural drying into a ternary solution with a weight ratio of lithium thiocyanate: mercaptoethanol: urea of 80:5:5 by weight, at a temperature of 60°C Dissolved under high temperature for 20 hours, purified by filtration and ultrafiltration, ultrasonic defoaming for 1 hour, and dried to make pure tussah silk fibroin;

3. Put the pure tussah silk fibroin protein in formic acid solution, dissolve it for 4 hours at a temperature of 30°C, the final concentration of the solution is 10%, filter, purify, and defoam to make spinning raw materials;

4. Using the electrospinning method, the distance from the receiving plate to the spinneret is 10cm, and the voltage is 10KV. The above-mentioned spinning stock solution is spray-spun to obtain a three-dimensional non-woven network structure biomedical material composed of pure tussah silk fibroin fibers .

Referring to accompanying drawing 1, it is the gel electrophoresis pattern of the tussah silk fibroin protein solution prepared by the technical scheme of the present embodiment, wherein, Marker1, Marker2 are standard sample, and swimming lane 1 sample is the tussah silk fibroin protein aqueous solution that concentration is 0.75%, The sample in swimming lane 2 is an aqueous solution of tussah silk fibroin protein with a concentration of 1.5%, the sample in swimming lane 3 is an aqueous solution of tussah silk fibroin protein with a concentration of 2%, and the sample in swimming lane 4 is an aqueous solution of tussah silk fibroin protein with a concentration of 3%. It can be seen from Figure 1 that compared with the standard sample, the molecular weight distribution of the tussah silk fibroin protein solution is between 100,000 and 2,000,000 D, and some of them are larger than 2,000,000 D. Requirements for shape and mechanical properties.

Embodiment two:

1. Put 1 kg of cocoon layer into 20 liters of neutral soap solution with a concentration of 0.5%, boil it for 2 hours, remove the sericin around the silk fibroin, wash it thoroughly with hot water to obtain pure tussah silk fibroin;

2. Place the dried pure tussah silk fibroin in a ternary solution of lithium thiocyanate: mercaptoethanol: urea = 85:5:10 by weight, and treat it for 50 hours at a temperature of 40°C. Dissolved into tussah silk fibroin protein solution, purified by filtration, dialysis, and dried into pure tussah silk fibroin protein;

3. Put the pure tussah silk fibroin and polylactic acid with a mass ratio of 90:10 in a formic acid solution with a concentration of 80%, dissolve them for 2 hours at a temperature of 50°C, purify by filtration, and defoam Make the spinning stock solution that the final concentration of solution is 10%;

4. Electrospinning is used to spray the above spinning stock solution according to conventional conditions to obtain a three-dimensional nonwoven web with tussah silk fibroin content of 90%.

Referring to accompanying drawing 2, the three-dimensional non-woven net structure biomedical material that the tussah silk fibroin protein fiber that prepares according to the technical scheme of this embodiment is made, the scanning electron micrograph of magnifying 5 thousand times at 16KV12cm, in the figure the diameter of the fiber is estimated to be about 50 times through 50 times. 0.62μm, the pore size is between 1.61 and 3.62μm. According to the technical scheme of the present invention, by controlling the final concentration of the spinning stock solution and the electrospinning process, it is possible to obtain tussah silk fibroin with a fiber diameter of 50 nm to 20 μm, interfiber pores of 1 to 500 μm, and a nonwoven web thickness of 50 nm to 20 mm. A three-dimensional non-woven network structure biomedical material composed of fibers.

Embodiment three:

1, prepare pure tussah silk fibroin by the method for step 1 in embodiment one;

2. Treat the dried pure tussah silk fibroin in a solution of lithium thiocyanate: mercaptoethanol: urea = 98:1:1 by weight at a temperature of 80°C for 5 hours, and obtain tussah silkworm after dissolving Silk fibroin solution, filtered, purified, and dried into pure tussah silk fibroin;

3. Put pure tussah silk fibroin and gelatin in a mass ratio of 80:20 in hexafluoroisopropanol solution, dissolve at 20°C for 150 hours, filter, purify, and defoam The final concentration of the solution is 5% spinning raw material;

4. Using the electrospinning method, the distance from the receiving plate to the spinneret is 15cm, and the voltage is 15KV to spray the above spinning stock solution to obtain a three-dimensional non-woven fabric composed of fibers with a tussah silk fibroin content of 80% and a gelatin content of 20%. Biomedical materials with spun mesh structure.

The three-dimensional non-woven mesh biomedical material of tussah silk fibroin protein fiber structure prepared according to the technical scheme provided by the present invention not only retains the good biocompatibility characteristics of the natural tussah silk fibroin protein, but also has abundant raw materials and low cost . In particular, its tissue cell induction function is expected to be used to replace high-cost collagen and other biomedical materials, as artificial skin, tendons, blood vessels, cartilage, ligaments, esophagus, dura mater and other repair scaffold materials or surgical repair materials.

Claims (6)

1. Antheraea pernyi silk fibrion biology medicine material is characterized in that: it is the three-D non-woven network structure that the tussah silk fibroin fiber constitutes, and fibre diameter is 50nm～20 μ m, and hole is 1～500 μ m between fiber, and nonwoven web thickness is 50nm～20mm; The molecular weight that it contains 10～100% weight is 10 * 10 ⁴～20 * 10 ⁵The tussah silk fibroin of D.

2. tussah silk protein biology medical material according to claim 1 is characterized in that: except that tussah silk fibroin, surplus is an additive; Described additive is a kind of in pure mulberry silk fibroin, polylactic acid or polyvinyl alcohol, Polyethylene Glycol, polyglycolic acid, polycaprolactone, cellulose, chitin, chitosan, collagen, the gelatin or their combination.

3. the preparation method of an Antheraea pernyi silk fibrion biology medicine material as claimed in claim 1 is come unstuck tussah silk earlier, obtains pure tussah silk peptide, it is characterized in that carrying out the processing of following steps again:

1. by weight, lithium rhodanate: mercaptoethanol: carbamide=80～98: 1～10: 1～10 makes ternary solution, pure tussah silk peptide is placed above-mentioned ternary solution, be to dissolve 5～70h under 40～100 ℃ the condition in temperature, make pure tussah silk fibroin aqueous solution after the purification, drying again, obtaining molecular weight is 10 * 10 ⁴～20 * 10 ⁵The tussah silk fibroin of D;

2. the solute with spinning solution dissolves in solvent, and the solution final concentration is 6～30%, makes spinning solution; In the solute of described spinning solution, contain the tussah silk fibroin that 1. step of 10～100% weight obtains; Described solvent is a kind of in formic acid, the hexafluoroisopropanol;

3. above-mentioned spinning solution is adopted electrostatic spinning process, make the cancellated bio-medical material of three-D non-woven that constitutes with the tussah silk fibroin fiber.

4. the preparation method of Antheraea pernyi silk fibrion biology medicine material according to claim 3, it is characterized in that: in the solute of described spinning solution, surplus is an additive; Described additive is a kind of in pure mulberry silk fibroin, polylactic acid or polyvinyl alcohol, Polyethylene Glycol, polyglycolic acid, polycaprolactone, cellulose, chitin, chitosan, collagen, the gelatin or their combination.

5. the preparation method of Antheraea pernyi silk fibrion biology medicine material according to claim 3, it is characterized in that: when described solvent was formic acid, formic acid solution concentration was 60～99%, and solution temperature is 20～80 ℃, and dissolution time is 0.5～5h.

6. the preparation method of Antheraea pernyi silk fibrion biology medicine material according to claim 3, it is characterized in that: when described solvent was hexafluoroisopropanol, solution temperature was 20～50 ℃, and dissolution time is 24～240h.

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