CN103861158B - Preparation method of silk fibroin and sulfated silk fibroin composite tubular scaffold - Google Patents
Preparation method of silk fibroin and sulfated silk fibroin composite tubular scaffold Download PDFInfo
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- CN103861158B CN103861158B CN201210533518.2A CN201210533518A CN103861158B CN 103861158 B CN103861158 B CN 103861158B CN 201210533518 A CN201210533518 A CN 201210533518A CN 103861158 B CN103861158 B CN 103861158B
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Abstract
The invention provides a preparation method of a silk fibroin and sulfated silk fibroin composite tubular scaffold. The composite tubular scaffold is characterized in that the composite tubular scaffold is composed of an elastic bonding layer, a fabric reinforcing layer and a compact coating layer, and the fabric reinforcing layer is embedded in the elastic bonding layer, and the outer surface of the compact coating layer is covered with the elastic bonding layer, wherein the compact coating layer and the fabric enhancing layer are made of silk fibroin, and the elastic bonding layer is made a silk fibroin and sulfated silk fibroin mixture. The elastic bonding layer can improve the compliance and the anticoagulation property of the tubular scaffold, the fabric reinforcing layer can improve the mechanical properties of the scaffold, and the compact coating layer can improve the impermeability of the scaffold. The tubular scaffold prepared in the invention has excellent mechanical properties, has good impermeability, cell compatibility and blood compatibility, has a controllable diameter, and can be used for restoring and reconstructing small caliber vessels and constructing a hemodialysis access. The preparation method has the advantages of simple operation, low cost, and commercial production realization.
Description
(1) technical field
The invention belongs to biomedical materials field, particularly relate to a kind of fibroin albumen and the sulphation fibroin albumen composite tube support with outstanding mechanical property and good permeability resistance, cell compatibility and blood compatibility.
(2) background technology
Society now, cardiovascular disease serious harm human body is healthy, reconstructing blood vessel occupies very consequence in Clinical Surgery, and the whole world needs to accept various vascular operation about more than 600,000 people every year, and wherein great majority all need suitable blood vessel graft.At present, the blood vessel graft applied clinically mainly comprises autologous vein, allogeneic blood vessels and synthetic blood vessel, due to the limited source of autologous vein and the rejection of allogeneic blood vessels, makes artificial vascular graft's attention.At present, the heavy caliber artificial blood vessel that diameter is greater than 6 millimeters achieves commercialization, and the clinical practice that diameter is less than the small-caliber artificial blood vessel of 6 millimeters is very disappointing.
The subject matter that small-caliber artificial blood vessel exists is exactly easily acute thrombus occurs after its implant into body, and the blood compatibility that vascular stent material has good biocompatibility, excellent mechanical property and a long-term stability is the key of its technological break-through.Anticoagulation modification to have become the outer scholar of Present Domestic research emphasis with the Long-term Anti thrombotic improving small-caliber tissue engineered blood vessels is carried out to timbering material.Material surface in conjunction with anticoagulant protein and plantation vascular endothelial cell be all the effective way improving timbering material anticoagulation function.Heparin is common anticoagulant protein, can improve hemorheology performance after the simple coating of heparin, but the insufficient strength combined, easily come off and cause too fast release.In recent years, some scholar's using plasmas irradiate and change material surface structure, material surface is generated and has the group of reactivity, heparin is fixed on material surface with covalent bond form, but its anticoagulant effect unsatisfactory.Be in timbering material in internal milieu due to the many factors such as body fluid, organic macromolecule, enzyme, free radical, cell will be touched, its biological environment is very complicated, adopt the method for surface modification comparatively limited to the improvement of material anticoagulation function, along with the degraded of timbering material and coming off of finishing coat, although its anticoagulation function can meet the needs of heavy caliber blood vessel of high blood flow, lower resistance, but cannot meet the needs to the higher small-caliber vascular of anticoagulation function requirement.On the other hand, thromboembolism preventing top layer can be formed at the inner surface plantation vascular endothelial cell of timbering material, the activity of the thrombinogen of engineering blood vessel inner surface can be reduced, thus improve the patency rate that the blood compatibility of material particularly improves small-caliber tissue engineered blood vessels, but endotheliocyte be subject to blood flow wash away after storage rate deficiency be perplex a great problem of Chinese scholars always.Research shows, when after the engineering blood vessel implant into body being implanted with endotheliocyte, being subject to blood flow impacts in initial 4 ~ 24 hours, have the endothelial denudation of 30% ~ 60%, and the reparation of Ink vessel transfusing cortex needs the regular hour, during this period of time the probability of vascularization thrombosis can greatly increase, therefore the storage rate improving engineering blood vessel endodermis is extremely important, and now the anticoagulation function of timbering material also seems particularly crucial, this just requires that the timbering material of small-caliber tissue engineered blood vessels should have good biocompatibility, there is anticoagulation function steady in a long-term again.
In recent years, fibroin albumen causes increasing concern with its good cell compatibility and mechanical property.Research worker has disclosed the technology (Chinese Patent Application No. 201010249479.4,201110135353.9) that a lot of method adopting fabric to strengthen prepares silk fibroin tubular bracket, the mechanical property of the silk fibroin tubular bracket prepared by the method significantly improves, but its blood compatibility is poor, thus limit it and apply further.In order to improve the blood compatibility of fibroin albumen, we carry out modification to fibroin albumen, have prepared sulphation fibroin albumen.Result of study shows, sulphation fibroin albumen not only has good blood compatibility, and can promote the sticking of vascular endothelial cell, breed and maintain its function.
In order to prepare desirable small-caliber vascular stent material, the two combines by the present invention, have employed three-layer composite structure: elastic bonding layer, fabric enhancement layer and coating compacted zone.Wherein, coating compacted zone and fabric enhancement layer are fibroin albumens, and elastic bonding layer is the mixture of fibroin albumen and sulphation fibroin albumen, is specially the blend solution of fibroin albumen and sulphation fibroin albumen.Elastic bonding layer can improve compliance and the anticoagulant property of tubular bracket, and fabric enhancement layer can improve the mechanical property of tubular bracket, and coating compacted zone can improve the anti-permeability performance of tubular bracket.
(3) summary of the invention
The object of the invention is to prepare a kind of intravascular tissue engineering support with outstanding mechanical property and good permeability resistance, cell compatibility and blood compatibility, and caliber is controlled, can be used for the reconstruction of small-caliber vascular and the foundation of hemodialysis path.
To achieve the above object of the invention, the technical solution used in the present invention is as follows:
A) preparation of fabric:
With braider, domestic silkworm silk is woven into tubulose, is placed in the Na of 0.1%
2cO
3in solution, in 98 ~ 100 DEG C of process 30 minutes, in triplicate, to slough the sericin on silk fiber surface, dry in air at room temperature.Then be inserted in the mould of corresponding size.
B) preparation of fibroin albumen:
Domestic silkworm silk is placed in the Na of 0.1%
2cO
3in solution, in 98 ~ 100 DEG C of process 30 minutes, in triplicate, to slough the sericin on silk fiber surface, dry in air at room temperature.Silk fiber is put into CaCl
2-CH
3cH
2oH-H
2dissolve in O (mol ratio is 1:2:8) solution, maintain the temperature at 78 ± 2 DEG C and constantly stir, obtaining silk fibroin protein solution, dialyse in distilled water with Dialysis tubing subsequently.Finally, silk fibroin protein solution being concentrated to mass body volume concentrations is 8% ~ 15%;
C) preparation of sulphation fibroin albumen:
The chlorosulfonic acid of 10mL is slowly joined in the pyridine of 60mL equably, then fibroin albumen sponge obtained for step (b) is added in the pyridine solution of chlorosulfonic acid, system temperature is progressively brought up to 80 DEG C and keeps certain hour.After reaction terminates, 200mL water is joined in system, and with appropriate sodium hydroxide solution neutralization reaction solution, insoluble matter in system is filtered, adding 500mL ethanol makes solvable sulphation fibroin albumen separate out, collected by centrifugation sulphation fibroin albumen, and makes it redissolve in a small amount of distilled water, dialyse in distilled water with Dialysis tubing subsequently, finally sulphation silk fibroin protein solution being concentrated to mass body volume concentrations is 8%;
D) preparation of elastic bonding layer:
By silk fibroin protein solution and sulphation silk fibroin protein solution blended, the mould with fabric is immersed in above-mentioned mixed solution, is placed in freezer dryer, obtain porous tubular scaffolds, soak support 30min with methanol;
E) preparation of compacted zone is applied:
The tubular bracket prepared above is impregnated in silk fibroin protein solution, forms coat and be placed on dry 0.5 ~ 3h in 40 DEG C ~ 70 DEG C baking ovens.Take out, with dehydrated alcohol process 10min.At least repeat this step 5 time, take out mould, obtain fibroin albumen and sulphation fibroin albumen composite tube support.
Advantage of the present invention is:
(1) fibroin albumen of the present invention and sulphation fibroin albumen composite tube support not only have good cell compatibility, and have outstanding blood compatibility.
(2) fibroin albumen of the present invention and sulphation fibroin albumen composite tube support have good tensile property.
(3) fibroin albumen of the present invention and sulphation fibroin albumen composite tube support have good compliance.
(4) fibroin albumen of the present invention and sulphation fibroin albumen composite tube support have good permeability resistance.
(5) the present invention has directly prepared fibroin albumen and sulphation fibroin albumen composite tube support, avoids the use of binding agent and cross-linking agent.
(6) present invention process is simple, and consumption is few, consuming time short, easily realizes commodity production.
(7) diameter of scalable silk fibroin small diameter blood vessel, length and thickness as required.
(8) fibroin albumen prepared by the present invention and sulphation fibroin albumen composite tube support can be used for the reconstruction of small-caliber vascular and the foundation of hemodialysis path.
(4) accompanying drawing explanation
Fig. 1 is the schematic cross-section of fibroin albumen of the present invention and sulphation fibroin albumen composite tube support.
1-elastic bonding layer 2-fabric enhancement layer 3-applies compacted zone
(5) detailed description of the invention
Specifically in conjunction with the embodiments, the present invention is further elaborated.These embodiments are only not used in for illustration of the present invention and limit the scope of the invention.In addition should be understood that those skilled in the art can make various change or amendment to invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Embodiment 1
As shown in Figure 1, be the schematic cross-section of composite tube support.Described composite tube support, comprises elastic bonding layer 1, fabric enhancement layer 2 and coating compacted zone 3 three layers composition.Described fabric enhancement layer 2 is embedded in elastic bonding layer 1, and coating compacted zone 3 covers on the outer surface of elastic bonding layer 2.Wherein, coating compacted zone 3 and fabric enhancement layer 2 are fibroin albumens, and elastic bonding layer 2 is mixture of fibroin albumen and sulphation fibroin albumen.The internal diameter of fibroin albumen and sulphation fibroin albumen composite tube support is 5mm.Wall thickness is 1mm.
The preparation method of above-mentioned fibroin albumen and sulphation fibroin albumen composite tube support is as follows:
A) preparation of fabric enhancement layer 2:
With braider, domestic silkworm silk being woven into internal diameter is 5mm, and wall thickness is 0.5mm tubular braid, is placed in the Na of 0.1%
2cO
3in solution, in 98 ~ 100 DEG C of process 30 minutes, in triplicate, dry in air at room temperature.Then be inserted in the mould of corresponding size.
B) preparation of fibroin albumen:
Domestic silkworm silk is placed in the Na of 0.1%
2cO
3in solution, in 98 DEG C of process 30 minutes, in triplicate, dry in air at room temperature.Silk fiber is put into CaCl
2-CH
3cH
2oH-H
2dissolve in O (mol ratio is 1:2:8) solution, maintain the temperature at 78 DEG C and constantly stir, obtaining silk fibroin protein solution, dialyse in distilled water with Dialysis tubing subsequently.Finally, silk fibroin protein solution polyglycol solution being concentrated to mass body volume concentrations is 8% ~ 15%;
C) preparation of sulphation fibroin albumen:
The chlorosulfonic acid of 10mL is slowly joined in the pyridine of 60mL equably, then fibroin albumen sponge obtained for step (b) is added in the pyridine solution of chlorosulfonic acid, system temperature is progressively brought up to 80 DEG C and keeps certain hour.After reaction terminates, 200mL water is joined in system, and with appropriate sodium hydroxide solution neutralization reaction solution, insoluble matter in system is filtered, adding 500mL ethanol makes solvable sulphation fibroin albumen separate out, collected by centrifugation sulphation fibroin albumen, and makes it redissolve in a small amount of distilled water, dialyse in distilled water with Dialysis tubing subsequently, finally sulphation silk fibroin protein solution being concentrated to mass body volume concentrations is 8%;
D) preparation of elastic bonding layer 1:
By silk fibroin protein solution and sulphation silk fibroin protein solution blended, then by the mould dipping with fabric wherein, be placed in freezer dryer, obtain porous tubular scaffolds, soak support 30min with methanol;
E) preparation of compacted zone 3 is applied:
The tubular bracket prepared above is impregnated in silk fibroin protein solution, forms coat and be placed on dry 0.5 ~ 3h in 40 DEG C of baking ovens.Take out, with dehydrated alcohol process 10min.At least repeat step e) 5 times, obtain fibroin albumen and sulphation fibroin albumen composite tube support.
The internal diameter of described fibroin albumen and sulphation fibroin albumen composite tube support is 5mm, and wall thickness is 1mm.
Embodiment 2
As shown in Figure 1, be the schematic cross-section of composite tube support.Described composite tube support, comprises elastic bonding layer 1, fabric enhancement layer 2 and coating compacted zone 3 three layers composition.Described fabric enhancement layer 2 is embedded in elastic bonding layer 1, and coating compacted zone 3 covers on the outer surface of elastic bonding layer 2.Wherein, coating compacted zone 3 and fabric enhancement layer 2 are fibroin albumens, and elastic bonding layer 2 is mixture of fibroin albumen and sulphation fibroin albumen.The internal diameter of fibroin albumen and sulphation fibroin albumen composite tube support is 4mm.Wall thickness is 0.9mm.
The preparation method of above-mentioned fibroin albumen and sulphation fibroin albumen composite tube support is as follows:
A) preparation of fabric enhancement layer 2:
With braider, domestic silkworm silk being woven into internal diameter is 4mm, and wall thickness is 0.5mm tubular braid, is placed in the Na of 0.1%
2cO
3in solution, in 98 ~ 100 DEG C of process 30 minutes, in triplicate, dry in air at room temperature.Then be inserted in the mould of corresponding size.
B) preparation of fibroin albumen:
Domestic silkworm silk is placed in the Na of 0.1%
2cO
3in solution, in 98 DEG C of process 30 minutes, in triplicate, dry in air at room temperature.Silk fiber is put into CaCl
2-CH
3cH
2oH-H
2dissolve in O (mol ratio is 1:2:8) solution, maintain the temperature at 78 DEG C and constantly stir, obtaining silk fibroin protein solution, dialyse in distilled water with Dialysis tubing subsequently.Finally, silk fibroin protein solution polyglycol solution being concentrated to mass body volume concentrations is 8% ~ 15%;
C) preparation of sulphation fibroin albumen:
The chlorosulfonic acid of 10mL is slowly joined in the pyridine of 60mL equably, then fibroin albumen sponge obtained for step (b) is added in the pyridine solution of chlorosulfonic acid, system temperature is progressively brought up to 80 DEG C and keeps certain hour.After reaction terminates, 200mL water is joined in system, and with appropriate sodium hydroxide solution neutralization reaction solution, insoluble matter in system is filtered, adding 500mL ethanol makes solvable sulphation fibroin albumen separate out, collected by centrifugation sulphation fibroin albumen, and makes it redissolve in a small amount of distilled water, dialyse in distilled water with Dialysis tubing subsequently, finally sulphation silk fibroin protein solution being concentrated to mass body volume concentrations is 8%;
D) preparation of elastic bonding layer 1:
By silk fibroin protein solution and sulphation silk fibroin protein solution blended, then by the mould dipping with fabric wherein, be placed in freezer dryer, obtain porous tubular scaffolds, soak support 30min with methanol;
E) preparation of compacted zone 3 is applied:
The tubular bracket prepared above is impregnated in silk fibroin protein solution, forms coat and be placed on dry 0.5 ~ 3h in 40 DEG C of baking ovens.Take out, with dehydrated alcohol process 10min.At least repeat step e) 5 times, obtain fibroin albumen composite tube support.The internal diameter of described fibroin albumen and sulphation fibroin albumen composite tube support is 4mm, and wall thickness is 0.9mm.
Embodiment 3
As shown in Figure 1, be the schematic cross-section of composite tube support.Described composite tube support, comprises elastic bonding layer 1, fabric enhancement layer 2 and coating compacted zone 3 three layers composition.Described fabric enhancement layer 2 is embedded in elastic bonding layer 1, and coating compacted zone 3 covers on the outer surface of elastic bonding layer 2.Wherein, coating compacted zone 3 and fabric enhancement layer 2 are fibroin albumens, and elastic bonding layer 2 is mixture of fibroin albumen and sulphation fibroin albumen.The internal diameter of fibroin albumen and sulphation fibroin albumen composite tube support is 3mm.Wall thickness is 0.8mm.
The preparation method of above-mentioned fibroin albumen and sulphation fibroin albumen composite tube support is as follows:
A) preparation of fabric enhancement layer 2:
With braider, domestic silkworm silk being woven into internal diameter is 3mm, and wall thickness is 0.5mm tubular braid, is placed in the Na of 0.1%
2cO
3in solution, in 98 ~ 100 DEG C of process 30 minutes, in triplicate, dry in air at room temperature.Then be inserted in the mould of corresponding size.
B) preparation of fibroin albumen:
Domestic silkworm silk is placed in the Na of 0.1%
2cO
3in solution, in 98 DEG C of process 30 minutes, in triplicate, dry in air at room temperature.Silk fiber is put into CaCl
2-CH
3cH
2oH-H
2dissolve in O (mol ratio is 1:2:8) solution, maintain the temperature at 78 DEG C and constantly stir, obtaining silk fibroin protein solution, dialyse in distilled water with Dialysis tubing subsequently.Finally, silk fibroin protein solution polyglycol solution being concentrated to mass body volume concentrations is 8% ~ 15%;
C) preparation of sulphation fibroin albumen:
The chlorosulfonic acid of 10mL is slowly joined in the pyridine of 60mL equably, then fibroin albumen sponge obtained for step (b) is added in the pyridine solution of chlorosulfonic acid, system temperature is progressively brought up to 80 DEG C and keeps certain hour.After reaction terminates, 200mL water is joined in system, and with appropriate sodium hydroxide solution neutralization reaction solution, insoluble matter in system is filtered, adding 500mL ethanol makes solvable sulphation fibroin albumen separate out, collected by centrifugation sulphation fibroin albumen, and makes it redissolve in a small amount of distilled water, dialyse in distilled water with Dialysis tubing subsequently, finally sulphation silk fibroin protein solution being concentrated to mass body volume concentrations is 8%;
D) preparation of elastic bonding layer 1:
By silk fibroin protein solution and sulphation silk fibroin protein solution blended, then by the mould dipping with fabric wherein, be placed in freezer dryer, obtain porous tubular scaffolds, soak support 30min with methanol;
E) preparation of compacted zone 3 is applied:
The tubular bracket prepared above is impregnated in silk fibroin protein solution, forms coat and be placed on dry 0.5 ~ 3h in 40 DEG C of baking ovens.Take out, with dehydrated alcohol process 10min.At least repeat step e) 5 times, obtain fibroin albumen composite tube support.The internal diameter of described fibroin albumen and sulphation fibroin albumen composite tube support is 3mm, and wall thickness is 0.8mm.
In addition, it should be noted that, elastic bonding layer gives silk fibroin tubular bracket anticoagulation function, and improves the compliance of tubular bracket, and the loose structure of elastic bonding layer is beneficial to adhesion and the increment of cell.Coating compacted zone improves the permeability resistance of silk fibroin tubular bracket, prevents from transplanting hourglass blood.The diameter of fibroin albumen and sulphation fibroin albumen composite tube support can select the mould of respective diameters as required.
Claims (6)
1. the preparation method of fibroin albumen and sulphation fibroin albumen composite tube support, is characterized in that the method is carried out as follows:
1) with silkworm boiled-off silk line for raw material, be woven into tubular braid with braider;
2) by fabric degumming process, be inserted in after drying in the mould of respective diameters;
3) be impregnated into by the mould with fabric in the blend solution of fibroin albumen and sulphation fibroin albumen, textile surface forms coat, the fabric scribbling coat is placed in freezer dryer and obtains elastic bonding layer, then with methanol process 30min;
4) tubular bracket prepared above is impregnated in silk fibroin protein solution, forms coat and be placed on dry 0.5 ~ 3h in 40 DEG C ~ 70 DEG C baking ovens, take out, with dehydrated alcohol process 10min;
5) step 4 is repeated) at least 5 times, take out mould, obtain fibroin albumen and sulphation fibroin albumen composite tube support.
2. the preparation method of fibroin albumen as claimed in claim 1 and sulphation fibroin albumen composite tube support, it is characterized in that: described fibroin albumen and sulphation fibroin albumen composite tube support comprise the cryodesiccated elastic bonding layer set gradually, fabric enhancement layer and coating compacted zone, and fabric enhanced layer packets is embedded in elastic bonding layer, coating compacted zone sticks on the outer surface of elastic bonding layer.
3. the preparation method of fibroin albumen as claimed in claim 1 and sulphation fibroin albumen composite tube support, it is characterized in that: in the blend solution of described fibroin albumen and sulphation fibroin albumen, fibroin albumen and sulphation fibroin albumen can adopt different mixed proportions, the mass fraction scope of sulphation fibroin albumen is 10% ~ 40%.
4. the preparation method of fibroin albumen as claimed in claim 2 and sulphation fibroin albumen composite tube support, is characterized in that: described fabric enhancement layer improves the mechanical property of tubular bracket, can adopt knitting, woven or braiding.
5. the preparation method of fibroin albumen as claimed in claim 1 and sulphation fibroin albumen composite tube support, is characterized in that: the pipe thickness scope of described fibroin albumen and sulphation fibroin albumen composite tube support is 0.8mm ~ 1mm.
6. the preparation method of fibroin albumen as claimed in claim 1 and sulphation fibroin albumen composite tube support, is characterized in that: described fibroin albumen and sulphation fibroin albumen composite tube support can be used for the reconstruction of small-caliber vascular and the foundation of hemodialysis path.
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| CN201210533518.2A CN103861158B (en) | 2012-12-11 | 2012-12-11 | Preparation method of silk fibroin and sulfated silk fibroin composite tubular scaffold |
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| CN103861158B true CN103861158B (en) | 2015-05-13 |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104524632B (en) * | 2015-01-21 | 2016-12-07 | 北京航空航天大学 | A kind of preparation method of the anticoagulation composite tube support with good conformability |
| CN106075596B (en) * | 2016-07-21 | 2021-02-09 | 南开大学 | Preparation technology of three-layer artificial blood vessel |
| CN106581752A (en) * | 2017-01-23 | 2017-04-26 | 苏州大学 | Degradable medicine slow release function composite enteric stent and making method thereof |
| CN109806771B (en) * | 2019-03-01 | 2021-09-14 | 东华大学 | Nanofiber-based composite hemodialysis membrane and preparation method thereof |
| CN112043877B (en) * | 2020-08-06 | 2022-10-14 | 苏州大学 | Silk anticoagulant tube stent tectorial membrane and preparation method thereof |
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