CN100551449C - Antheraea pernyi silk fibrion biology medicine material and preparation method thereof - Google Patents
Antheraea pernyi silk fibrion biology medicine material and preparation method thereof Download PDFInfo
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- CN100551449C CN100551449C CNB2006101559704A CN200610155970A CN100551449C CN 100551449 C CN100551449 C CN 100551449C CN B2006101559704 A CNB2006101559704 A CN B2006101559704A CN 200610155970 A CN200610155970 A CN 200610155970A CN 100551449 C CN100551449 C CN 100551449C
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Abstract
The present invention discloses a kind of bio-medical engineering material and preparation method thereof, and three-dimensional tissue's repair materials of the easy tactophily of particularly a kind of cell and preparation method thereof belongs to the bio-medical material technical field.It is raw material with the natural tussah silk, after the tussah silk fibroin dissolving, adopts electrospinning process, obtain the three-D non-woven network structure that the tussah silk fibroin fiber constitutes, 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 of tussah silk peptide is 10 * 10
4～20 * 10
5D.This bio-medical engineering material has nontoxic, biocompatibility is good, the characteristics of cell tactophily function admirable, and abundant raw material, with low cost, be expected to replace expensive bio-medical materials such as collagen, as repair materials, particularly histiocyte induced material such as artificial skin, tendon, cartilage, cerebral dura mateies.
Description
Technical field
The present invention relates to a kind of bio-medical engineering material and preparation method thereof, three-dimensional tissue's repair materials of the easy tactophily of particularly a kind of cell and preparation method thereof belongs to the bio-medical material technical field.
Background technology
Tussah silk contains a large amount of Arg-Gly-Asp (arginine-aminoacetic acid-aspartic acid, RGD) tripeptide sequence, and favourable [the Minoura N of sticking of verified this sequence pair cell, 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, RuoslahtiE.Pro Natl Acad Sci USA, 1984, (81): 5985-5988].This point is at Sofia[Sofia S, McCarthy MB, Gronowic G, et.al.Biomed Mater Res, 2001, (54): 139-148.] etc. cell culture experiments in vitro in proved.Above-mentioned disclosed research contents shows, with material during with the RGD covalent modification, the increased activity of cell on material, and promoted the mineral nitrogenization of analogy osteoblast strengthened inducing that external skeleton forms.Therefore tussah silk peptide is expected to forming bigger applied research achievement as aspects such as cell culture substrate, wound covering film, artificial skin and tissue engineering bracket material and bio-medical material modifications.
Antherea pernyi Guerin-Meneville is the superior resources of China, tussah silk by about more than 80% fibroin and formed less than 20% sericin, be quite pure natural protein fiber, the aminoacid of fibroin is formed with human collagen albumen certain similarity, avirulence, has no stimulation, has excellent biological compatibility.Therefore, the tussah silk fibroin is the more satisfactory raw material of preparation bio-medical goods, but because the natural tussah silk diameter is more than the 20 μ m, and more difficult plastotype, so the natural tussah silk element is greatly limited in the range of application of biomedical sector.
Between natural tussah silk cellulose fiber macromolecular chain very strong interaction is arranged, contains disulfide bond, than Bombyxmori Linnaeus fibroin more indissoluble separate, the solvent that generally dissolves the Bombyxmori Linnaeus fibroin is difficult to the dissolving tussah silk peptide.Before the present invention makes, be entitled as in the literary composition of " peptide of tussur silk production technology research " [Liaoning silk 2001, (3): 34-35], a kind of method that makes peptide of tussur silk in the phosphoric acid solution that tussah silk is dissolved in is disclosed; Publication number is in the Chinese invention patent " peptide of tussur silk preparation method " of CN1415626A, also discloses a kind of tussah silk to be joined in the phosphoric acid, dissolves 8～10h under 100～130 ℃ temperature conditions, the technical scheme of preparation peptide of tussur silk.To be fibroin albumen issue unboiled water in the effect of part strong acid to the principle that adopts technique scheme separates chain rupture, and molecular weight is reduced greatly, generates the silk polypeplide of molecular weight about 200～5000D, is applicable to the foods and cosmetics field.But, can't satisfy the requirement of the moulding and mechanical property of preparation bio-medical material three-dimensional rack because molecular weight is too little.
Summary of the invention
The objective of the invention is to overcome the deficiency that prior art exists, provide a kind of and can satisfy the moulding and mechanical property requirements of three-dimensional rack, help Antheraea pernyi silk fibrion biology medicine material of cell tactophily and preparation method thereof.
The technical solution used in the present invention is: a kind of Antheraea pernyi silk fibrion biology medicine material, it is the three-D non-woven network structure that the tussah silk fibroin fiber constitutes, 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.
In the technique scheme, surplus is an additive; Described additive is a kind of in pure mulberry silk fibroin, polylactic acid or polyvinyl alcohol, Polyethylene Glycol, polyglycolic acid, poly-several lactones, cellulose, chitin, chitosan, collagen, the gelatin or their combination.
The preparation method of above-mentioned Antheraea pernyi silk fibrion biology medicine material is come unstuck tussah silk earlier, obtains pure tussah silk peptide, carries 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.
In the technique scheme, 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, poly-several lactones, cellulose, chitin, chitosan, collagen, the gelatin or their combination.
When the described solvent of technical solution of the present invention was formic acid, formic acid solution concentration was 60～99%, and solution temperature is 20～80 ℃, and dissolution time is 0.5～5h.When described solvent was hexafluoroisopropanol, solution temperature was 20～50 ℃, and dissolution time is 24～240h.
Compared with prior art, advantage of the present invention is:
(1) the present invention is a primary raw material with the tussah silk fibroin, has kept the advantage of natural tussah silk fibroin matter excellent biological compatibility; The three-D non-woven network structure form that has can be directly used in histiocytic cultivation in inside and outside and reparation, is that ideal histiocyte is induced bio-medical material.
(2) ternary solution provided by the present invention has solved the insoluble difficult problem of tussah silk peptide fiber preferably, has obtained molecular weight 10 * 10
4～20 * 10
5The tussah silk fibroin of D can satisfy the requirement of the moulding and mechanical property of preparation tissue renovation material three-dimensional rack, for bio-medical material provides abundant raw material.
(3) tussah silk fibroin provided by the invention can with other biomaterial blend, help giving full play to each properties of materials, make up to be fit to the tissue renovation material of various human bodies.
Description of drawings
Fig. 1 is the gel electrophoresis spectrum by the tussah silk fibroin solution of technical solution of the present invention preparation, wherein, Marker1, Marker2 are standard specimen, swimming lane 1 sample is that concentration is 0.75% tussah silk fibroin aqueous solution, swimming lane 2 samples are that concentration is 1.5% tussah silk fibroin aqueous solution, swimming lane 3 samples are that concentration is 2% tussah silk fibroin aqueous solution, and swimming lane 4 samples are that concentration is 3% tussah silk fibroin aqueous solution.
Fig. 2 is the sem photograph by the three-D non-woven network structure bio-medical material of the tussah silk fibroin fiber formation of technical solution of the present invention preparation.
The specific embodiment
Below in conjunction with drawings and Examples the present invention is further described:
Embodiment one:
1,0.6 kilogram of leftover bits and pieces tussah silk (reeling mill's side-product) being put into 10 liters of concentration is 0.05% aqueous sodium carbonate, boils 0.5 hour, and the sericin of most fibroin periphery is taken off in reprocessing three times, obtains pure tussah silk fibroin;
2, with the pure tussah silk fibroin after drying naturally, put into by weight lithium rhodanate: mercaptoethanol: carbamide is dissolving 20 hours in 80: 5: 5 the ternary solution, under 60 ℃ temperature conditions by weight, after filtration, ultrafiltration purifies, and makes pure tussah silk fibroin after ultrasound wave froth breaking 1 hour, the drying;
3, pure tussah silk fibroin being placed formic acid solution, is dissolving 4 hours under 30 ℃ the condition in temperature, and the solution final concentration is 10%, after filtration, purify, makes spinning material behind the froth breaking;
4, adopt electrospinning process, dash receiver is 10cm to the spinning nozzle distance, and voltage is 10KV, above-mentioned spinning solution is sprayed spin, and obtains the cancellated bio-medical material of three-D non-woven that pure tussah silk fibroin fiber constitutes.
Referring to accompanying drawing 1, it is the gel electrophoresis spectrum by the tussah silk fibroin solution of present embodiment technical scheme preparation, wherein, Marker1, Marker2 are standard specimen, swimming lane 1 sample is that concentration is 0.75% tussah silk fibroin aqueous solution, swimming lane 2 samples are that concentration is 1.5% tussah silk fibroin aqueous solution, and swimming lane 3 samples are that concentration is 2% tussah silk fibroin aqueous solution, and swimming lane 4 samples are that concentration is 3% tussah silk fibroin aqueous solution.Can see by Fig. 1, test control, the molecular weight distribution of tussah silk fibroin solution also has part greater than 2000000D between 100000～2000000D, the tussah silk fibroin raw material that molecular weight is bigger can satisfy the requirement of the moulding and mechanical property of preparation bio-medical material three-dimensional rack.
Embodiment two:
1,1 kilogram of cocoon layer being put into 20 liters of concentration is 0.5% neutral soap solution, boils 2 hours, takes off the sericin of most fibroin periphery, obtains pure tussah silk fibroin behind the hot water thorough washing;
2, dried pure tussah silk fibroin is placed by weight lithium rhodanate: mercaptoethanol: the ternary solution of carbamide=85: 5: 10, be to handle through 50 hours under 40 ℃ the condition in temperature, be dissolved into tussah silk fibroin solution, after filtration, dialysis purifies, is dried to pure tussah silk fibroin;
3, will be 90: 10 pure tussah silk fibroin and polylactic acid by mass ratio, placing concentration is 80% formic acid solution, is dissolving 2 hours under 50 ℃ the condition in temperature, purifies after filtration, makes the solution final concentration behind the froth breaking and be 10% spinning solution;
4, adopt method of electrostatic spinning, according to normal condition above-mentioned spinning solution is sprayed and spin, acquisition tussah silk peptide content is 90% three-D non-woven net.
Referring to accompanying drawing 2, press the three-D non-woven web frame bio-medical material of the tussah silk fibroin fiber formation of present embodiment technical scheme preparation, amplify 5,000 times sem photograph at 16KV12cm, fiber is about 0.62 μ m through 50 budgetary estimate diameters among the figure, and the aperture is between 1.61～3.62 μ m.By technical solution of the present invention, final concentration and electrostatic spinning process by the control spinning solution, can obtain fibre diameter is 50nm～20 μ m, hole is 1～500 μ m between fiber, and nonwoven web thickness is the three-D non-woven web frame bio-medical material that the tussah silk fibroin fiber of 50nm～20mm constitutes.
Embodiment three:
1, the method by step 1 among the embodiment one prepares pure tussah silk fibroin;
2, with dried pure tussah silk fibroin by weight lithium rhodanate: mercaptoethanol: in the solution of carbamide=98: 1: 1, be to handle 5 hours under 80 ℃ the condition in temperature, obtain tussah silk fibroin solution after the dissolving, after filtration, purify, be dried to pure tussah silk fibroin;
3, will be that 80: 20 pure tussah silk fibroin and gelatin places hexafluoroisopropanol solution by mass ratio, be dissolving 150 hours under 20 ℃ the condition in temperature, after filtration, purify, makes the solution final concentration behind the froth breaking and be 5% spinning material;
4, using electrospinning process, be 15cm receiving plate to the spinning nozzle distance, and voltage 15KV sprays above-mentioned spinning solution and spins, the bio-medical material of the three-D non-woven web frame that acquisition tussah silk peptide content is 80%, gelatine content is 20% fiber formation.
Press the three-D non-woven gauze bio medical material of the tussah silk fibroin fibre structure of technical scheme preparation provided by the invention, the protein that has not only kept the natural tussah silk element has the characteristic of excellent biological compatibility, and abundant raw material, and is with low cost.Particularly its histiocyte inducing function is expected to be used to replace expensive bio-medical materials such as collagen, and as artificial skin, tendon, blood vessel, cartilage, ligament, esophagus, cerebral dura mater etc. are repaired with timbering material or surgical repair material.
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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