CN103981561A - Method and device for preparing electro-polymerized silk fibroin hydrogel membrane and application of fibroin hydrogel membrane - Google Patents
Method and device for preparing electro-polymerized silk fibroin hydrogel membrane and application of fibroin hydrogel membrane Download PDFInfo
- Publication number
- CN103981561A CN103981561A CN201410221710.7A CN201410221710A CN103981561A CN 103981561 A CN103981561 A CN 103981561A CN 201410221710 A CN201410221710 A CN 201410221710A CN 103981561 A CN103981561 A CN 103981561A
- Authority
- CN
- China
- Prior art keywords
- membrane
- fibroin
- hydrogel membrane
- aquagel
- receiving
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The invention relates to a method and device for preparing an electro-polymerized silk fibroin hydrogel membrane and an application of the fibroin hydrogel membrane. The electro-polymerized silk fibroin hydrogel membrane is prepared by rapidly self-assembling silk fibroin on a nano-aperture barrier membrane under the action of electrophoresis; the device comprises a container for containing an electro-polymerized buffer solution, wherein the container is divided into an inner chamber and an outer chamber by the barrier membrane located in the middle of the container, the inner chamber is connected with a negative electrode, and the outer chamber is connected with a positive electrode; the silk fibroin injected into the inner chamber is negatively charged in the electro-polymerized buffer solution containing SDS (Sodium Dodecyl Sulphate), migrates towards the positive direction under the action of higher direct current electric field and is rapidly self-assembled at the barrier membrane to form the electro-polymerized silk fibroin hydrogel membrane. The hydrogel membrane is translucent, smooth, soft and elastic, and the water-holding capacity of the hydrogel membrane is about ten times of the weight of the hydrogel membrane, L929 cells can be adhered and grow on the surface of the hydrogel membrane, a drug encapsulated by the hydrogel membrane can be released sustainably, the hydrogel membrane can be slowly degraded after the hydrogel membrane is implanted into an animal body and no obvious immune or inflammatory response occurs. The gel membrane has application prospects in artificial skin, surgical implants or filler materials or cell culture substrates and other fields.
Description
Technical field
The invention provides a kind of method and processing unit (plant) that the Rapid self assembly under a DC electric field effect of the biopolymer with plus or minus electric charge is become to voltolisation fibroin aquagel membrane (ESFHM) in water or damping fluid.The present invention is also relevant with composition with the preparation method of medical tissue engineering materials.
Background technology
Utilizing the biocompatibility feature of natural silk fibroin to be processed into medical tissue engineering materials is one of current research and development focus.Silky fibre is common protein high molecular polymkeric substance, comprises spider silk, domestic silkworm silk, ricinus silk, tussah silk etc.
Conventional silky fibre typically refers to a kind of thiozell that some lepidopterous insects silkworm produces at present.This thiozell is mainly overlayed on its gluey sericin around by fibroin fiber and quilt and is formed.When being overlayed on after artificial removal of sericin of fiber outside, silk fibroin fiber is light because of having, comfortable, moisture-absorbing/releasing strong, softness, elasticity and the characteristic such as dyeing is gorgeous, is textile materials since ancient times, is often described as the queen of textile fibres.In recent years,, because of its biocompatibility, slow degradation and excellent mechanical characteristics, be usually used in the especially research and development of medical tissue engineering materials of bio-medical material.The research and development of these medical materials regeneration liquid fibroin that often fibroin fiber based on coming unstuck is made after high level salt solution dissolves is realized.This liquid fibroin is subject to fibroin biomaterial that the impact of envrionment conditions makes various forms as fibroin membrane, gel, regenerated fibre, powder or nano particle and 3D support etc.Through simple physical or chemical treatment, the structure of this regenerated silk protein is also easily transformed into alpha-helix (Silk I) or beta sheet (Silk II) structure from random coil, and these processing characteristics have been created advantageous favourable condition with regard to the bio-medical tissue engineering material that becomes excellent performance for silk fibroin exploitation.
Natural silk is a kind of silk fibroin of uniqueness, makes after liquid fibroin by dissolving, desalting and purifying, and it can again change and other different shape or form from its natural form.Utilize regeneration liquid fibroin to make the existing many reports of fibroin protein film and patent application by multiple physics or chemical process as moisture evaporation etc., after this liquid fibroin is freezed, carry out lyophilize and make the fibroin membrane of vesicular structure (CN1260363).Nearly ten years, to silk-protein particularly the exploitation of silk fibroin well beyond the classical purposes of its original traditional textile fibres and suture.For example, hydrogel (WO2005/012606; PCT/US08/65076), ultrathin membrane (WO2007/016524), thick film, conformal coating (WO2005/000483; WO2005/123114), microballoon (PCT/US2007/020789), 3D porous matrix (WO2004/062697), solid substance (WO2003/056297), microfluidic devices (PCT/US07/83646; PCT/US07/83634), electro-optical device (PCT/US07/83639), regenerated fibre from nano level (WO2004/0000915) to several centimetres (U.S. Pat. No. 6902932) are for bio-medical material and regeneration medicine (WO2006/042287; US20070212730; , or even immobilized enzyme/medicament nano particle (WO 2005/085327 of nano-scale particle or medicament slow release PCT/US08/55072); WO2007/112679).Especially under low voltage electric field effect, silk fibroin is transformed into fibroin coagulation glue near positive pole, and this coagulation glue mainly can be for fields (US20110171239A1 or WO2010036992A2) such as medical bio glue.
The fibroin biomaterial that silk fibroin solution is processed into certain form and quality requires specific exploration and individual character research and development technology.For example, electrospinning is a process that produces nano-scale fiber, and this fiber is often processed into the article of mat and tubular structure.In this electrospinning complete processing, the high voltage electric of conventional 10 KV～20 KV scopes is used for driving the quick injection of silk fibroin solution in syringe needle, thereby accelerated solidification becomes the solid fiber of sub-micron diameter.This technique can steadily be piled into the stratified material (Li et al., Biomaterials. 27:3115-24,2006) that is similar to nonwoven fabric construct.And in coagulation glue complete processing, be immersed in the positive and negative electrode in (typical concentrations 9%) regenerated silk solution of high density, low voltage electric field (direct current or alternating-current) as 24.8 V electric field actions under, fibroin molecule produces coagulation glue around positive pole, this fibroin coagulation glue can be used as medical bio tackiness agent (Yucel et al., J Struct. Biol. 170 (2010) 406 – 412), in the time that electric polarity is contrary, the anodal silk extract gel that around produces changes again and is called liquid silk fibroin solution.So in the coagulation glue course of processing, the transformation of silk fibroin solution and silk extract gel can be reversible.Also there is report to prepare aquagel membrane (Zhang et al., Journal of Materials Chemistry. 2011,21,7705) by electrophoretic deposition technique.Silk fibroin solution and chitosan solution are mixed into electrophoresis liquid, use titanium alloy sheet as electrode, at 4 DEG C, 5v/cm
-2condition under constant voltage 30 s, just can obtain Fibroin-Chitosan Blend Membranes gel coated material, these gel coat material can be for cell cultures, also can be used as a kind of effectively pharmaceutical carrier.
Summary of the invention
Problem to be solved by this invention is to overcome the deficiency that prior art exists, and a kind of preparation method, device and application thereof with the nanoporous of softness, elastic properties or the fibroin aquagel membrane of network structure is provided.
The technical scheme that realizes the object of the invention is to provide a kind of preparation method of fibroin aquagel membrane, and the interior chamber container that fills water-soluble fibroin protein solution is placed in to the mistress's container that fills voltolisation damping fluid, and the bottom of interior chamber container is barrier membranes; Interior chamber is connect to negative pole, and mistress connects positive pole, under DC electric field effect, obtains a kind of fibroin aquagel membrane on barrier membranes; Described barrier membranes is the pore membrane of receiving of holding back the above molecular weight of 1～100 kDa; The mass concentration of described water-soluble fibroin protein solution is 0.5～10%; Described voltolisation damping fluid is the salt-glycine system containing mass concentration 0.05～5% SDS, and the concentration of glycine is 1.0 mM～1000 mM, and the concentration of salt is 1.0 mM～1000 mM; Regulating the pH value of voltolisation damping fluid with HCl is 4.0～10.0; Described DC electric field is the constant current electric field of 10～200 mA, or the constant voltage electric field of 50～200 VDC; Be 10～100 min the action time of DC electric field.
The pore membrane of receiving of the present invention is the pore membrane of receiving of hydrophilic natural macromolecule material, and described hydrophilic natural macromolecule hydrophilic material is the one in agarose, dextran film.The described pore membrane of receiving can also be the pore membrane of receiving of wetting ability synthesized polymer material, and described wetting ability synthesized polymer material is polyacrylamide gel.The described pore membrane of receiving can be also the pore membrane of receiving of inorganic materials, and described inorganic materials is pottery.The described pore membrane of receiving is the dialysis membrane of hydrophilic high molecular material, and described dialysis membrane is regenerated cellulose dialysis membrane, cellulose ester dialysis membrane.
Salt of the present invention is inorganic salt phosphoric acid salt or carbonate.Also can be organic salt Citrate trianion or acetate.
Technical solution of the present invention can also comprise biologically active substance enzyme, polypeptide drugs in water-soluble fibroin protein solution, or small-molecule drug.
Prepare a device for above-mentioned fibroin aquagel membrane, its structure is: interior outer container is fixed in mistress's container, and interior chamber container bottom is barrier membranes, and described barrier membranes is the pore membrane of receiving of holding back the above molecular weight of 1～100 kDa; The negative pole of direct supply is connected in interior chamber, and mistress connects the positive pole of direct supply.
Technical solution of the present invention also comprises described fibroin aquagel membrane as medical tissue engineering materials artificial skin, surgical operation implanted stopping composition, cell culture substrate.
The present invention, in the course of processing of electropolymerization, introduces the electric field of a direct current constant current or constant voltage, and containing or do not contain in the Tris-glycine buffer of SDS, fibroin molecule is received Rapid self assembly on the barrier membranes of hole and become ESFHM impenetrable.In this container that is full of voltolisation damping fluid (pH6.50), received hole barrier membranes (only limiting the film that biopolymer passes through as fibroin) by one and be separated into inside and outside two Room, mistress inserts platinum filament or platinum plate anode, and interior chamber is inserted platinum filament or platinum plate negative electrode and additionally added silk fibroin protein solution.At constant voltage 13 VDC/cm
2or constant current 6 mA/cm
2in situation, fibroin molecule with a large amount of negative charges, moves to positive extreme direction equably under SDS parcel under electric field action, because of can not be through receiving hole barrier membranes, therefore in this nanometer film Rapid self assembly formation ESFHM.
The complete processing of silk fibroin voltolisation film of the present invention is simpler, silk fibroin molecular is combined a large amount of negative charge of rear band in voltolisation damping fluid with anionic detergent SDS, under higher DC electric field effect as other charged small molecules or ion, to positive extreme direction campaign.Charged small molecules or ion can penetrate because volume is little and be arranged on the middle nano aperture barrier membranes in inside and outside chamber, and huge silk fibroin molecular cannot penetrate and receives hole barrier membranes, thereby pile up and form the fibroin aquagel membrane with vesicular structure receiving on the barrier membranes of hole Rapid self assembly.
Barrier membranes be small molecules or ion can by and organic membrane or the mineral membrane of the nano aperture that biomacromolecule cannot penetrate, the hole organic membrane of receiving mainly refers to polyacrylamide gel film, dialysis membrane or other high molecular polymerization film etc.; The hole mineral membrane of receiving mainly refers to ceramic membrane etc.The pore size of polyacrylamide gel film is determined by gel content; The pore size of dialysis membrane can be selected and commercially availablely holds back the dialysis membrane of different molecular weight ranges and determine.The high molecular polymerization film that this restriction fibroin molecule passes through or mineral membrane have played the keying action of biopolymer electropolymerization.
Adding mass concentration 0.05～5%(preferred plan at Tris-glycine voltolisation damping fluid (preferred plan is pH6.5) is 0.2%) SDS liquid towards fibroin voltolisation become fibroin membrane to play equally crucial effect, but too much, very few or even there is no a SDS, although also can form aquagel membrane, affect the characteristic of ESFHM as elasticity, smooth degree or transparency etc.
Fibroin membrane or fibroin 3D material that the fibroin aquagel membrane that voltolisation forms forms than other method have more superiority, as the fibroin membrane that volatilization forms by moisture content.The fibroin membrane that the latter forms need just can become water-fast film after other materializations are processed, and this film elasticity in the aqueous solution, retractility, water-absorbent are limited; Hygrometric state voltolisation fibroin aquagel membrane (ESFHM) is very soft, high resilience, and water-retaining capacity is himself 10 times of left and right of weight, in vitro can be by proteasome degradation, can slowly decompose in vivo, without obviously immune response and inflammatory reaction; Simultaneously in vitro cell also easily in its surface adhesion, propagation and grow.
The fibroin aquagel membrane that voltolisation forms has more superiority, the particularly nanoporous of this aquagel membrane or network structure than the fibroin membrane that volatilization forms by moisture content.In voltolization process, moving because of charged micromolecular electrophoresis, the fibroin aquagel membrane that voltolisation is formed becomes nanoporous.
Time prepared by ESFHM, constant voltage or the constant current DC electric field particularly important of two ends application, its size directly affects the characteristic such as softness, elasticity of ESFHM.
If will add active substance as medicine etc. in ESFHM, these active substances can be joined in regeneration liquid fibroin.By the effect of electric field, these active substances just can be embedded in ESFHM.These ESFHM with active substance are having important using value during as artificial skin, 3D support as medical tissue engineering materials.
Brief description of the drawings
Fig. 1 is the structural representation of the preparation facilities of a kind of fibroin aquagel membrane of providing of the embodiment of the present invention;
Fig. 2 is the infrared absorption spectrum of (ESFHM-M) after (ESFHM) before the fibroin aquagel membrane that provides of embodiment of the present invention methyl alcohol dip treating;
The X-diffraction analysis collection of illustrative plates of (curve ESFHM-M) after (curve ESFHM) before methyl alcohol dip treating for the fibroin aquagel membrane that Fig. 3 embodiment of the present invention provides;
The thermogram spectrum of (ESFHM-M) after (ESFHM) before methyl alcohol dip treating for the fibroin aquagel membrane that Fig. 4 embodiment of the present invention provides;
Fig. 5 is the scanning electron microscope picture of the fibroin aquagel membrane that provides of the embodiment of the present invention; A figure is the fibroin aquagel membrane of preparing in the damping fluid without SDS, and b figure is at the fibroin aquagel membrane containing preparing in SDS damping fluid;
Fig. 6 is the fibroin aquagel membrane (ESFHM) that provides of embodiment of the present invention enzymolysis process graphic representation in vitro;
Fig. 7 is the releasing curve diagram of Rifampin in the fibroin aquagel membrane (ESFHM) that provides of the embodiment of the present invention;
Fig. 8 is the result figure of fibroin aquagel membrane TNF-alpha content in mice serum of providing of the embodiment of the present invention.
Embodiment
Below in conjunction with drawings and Examples, technical solution of the present invention is further elaborated.
Embodiment 1
1, regenerated silk solution preparation
The present invention mainly uses following three kinds of Degumming Procedures for Silkworm Silks:
(1) urea buffer solution Degumming method: silk cocoon is removed to clothing, cut open after pupa takes quality and add 30 times of amount volume 8M urea-Tris-H
2sO
4the urea come unglued liquid of-0.5M mercaptoethanol (pH7.00), in 80 DEG C of water-baths with 150 rpm vibration 2h.After taking-up, rinse well with deionized water, in 80 DEG C of dry 2 h, obtain the fibroin fiber coming unstuck completely.
(2) sodium carbonate Degumming method: silk cocoon is removed to clothing, cutting pupa open takes and adds the sodium carbonate solution that the massfraction of 25 times of amount volumes is 0.2% after quality, boil 30 min, after rinsing well with deionized water, 30 min that come unstuck again in sodium carbonate solution with same ratio, same concentration, to guarantee to remove the outer sericin adhering to of fibroin fiber.Finally rinse well with deionized water, in 80 DEG C of dry 2 h, obtain the fibroin fiber coming unstuck completely.
(3) strong basicity brine electrolysis method for degumming: silk cocoon is removed to clothing, cutting open after pupa takes quality adds the pH11.5 strong basicity brine electrolysis (being called for short: brine electrolysis) of 25 times of amount volumes to boil 30 min, change equally liquid and repeat to boil 1 time, to guarantee to remove the outer sericin adhering to of fibroin fiber.In 80 DEG C of dry 2 h, obtain the fibroin fiber coming unstuck completely.
The method that fibroin fiber dissolves has following two kinds:
(1) lithium-bromide solution dissolves: the fibroin fiber coming unstuck is dissolved in the lithium-bromide solution of 9.3M, bath raio 20:1 (V/W) dissolves to whole with 90 rpm vibrations in 25 DEG C of constant-temperature tables.
(2) calcium chloride ternary solvent is dissolved: the fibroin fiber coming unstuck is dissolved in to (CaCl
2-EOH-H
2o=1:2:8), in ternary solvent, bath raio 20:1 (V/W) dissolves to whole with 90 rpm vibrations in 70 DEG C of constant-temperature tables.The dialysis tubing for fiber solution (3.5K MWCO) obtaining is through water purification or deionized water dialysis 48h.Dialyzate separates 20min with filter paper filtering or centrifugal (8,000rpm), and getting supernatant liquor acquisition fibroin concentration is 4% aqueous solution (W/V), is placed in 4 DEG C and deposits for subsequent use.
From degumming silkworm cocoons to fibroin fiber, dissolve with purifying and be totally divided into following three classes:
(1) urea comes unstuck and lithium-bromide solution dissolving (being called for short: urea-lithiumbromide);
(2) strong basicity brine electrolysis comes unstuck and ternary solvent dissolving (being called for short: alkaline water-ternary solvent);
(3) sodium carbonate solution comes unstuck and dissolves with ternary solvent (being called for short: sodium carbonate-ternary solvent).
2, voltolisation fibroin aquagel membrane preparation
Referring to accompanying drawing 1, it is the structural representation of the preparation facilities of the present embodiment fibroin aquagel membrane, respectively with chamber and mistress in two container compositions, interior chamber container is fixed in mistress's container by interior chamber base, the bottom of interior chamber container is for receiving hole barrier membranes, supported and be fixed in interior chamber container bottom by barrier membranes, the hole barrier membranes of receiving is the pore membrane of receiving of holding back the above molecular weight of 1～100 kDa, the negative pole of direct supply is connected in interior chamber by electrode, mistress connects the positive pole of direct supply by electrode, by Tris-glycine buffer (0.2% SDS of certain volume, pH6.5) add the mistress of voltolisation device, and the regenerated silk solution of certain volume and the Tris-glycine buffer of certain volume (0.2% SDS, pH6.5) after mixing, join the interior chamber of voltolisation device, then (the present embodiment is selected Tanon EPS100 to open the controlled power of direct current of voltolisation device, Shanghai Tian Neng Science and Technology Ltd. manufactures), regulate volts DS 80v to start voltolisation, after 50 min, cut off the electricity supply, the voltolisation fibroin aquagel membrane forming on the barrier membranes of hole is received in taking-up, the translucent aquagel membrane of this softness after rinsing, be dipped in pure water, be placed in 4 DEG C for subsequent use.With methyl alcohol dip treating in contrast (be called for short: ESFHM-M), ESFHM is dipped in 80% methanol solution and processes 20min, after taking out with deionized water rinsing, be finally dipped in pure water, be placed in 4 DEG C for subsequent use.
In technical solution of the present invention, the hole barrier membranes of receiving can be the pore membrane of receiving for hydrophilic natural macromolecule material, as agarose, dextran film etc.; The hole barrier membranes of receiving also can be wetting ability synthesized polymer material, as polyacrylamide gel etc.; Can also be inorganic materials as pottery, and the dialysis membrane of hydrophilic high molecular material, as regenerated cellulose dialysis membrane, cellulose ester dialysis membrane etc.
In technical solution of the present invention, salt is inorganic salt phosphoric acid salt or carbonate, can be also organic salt Citrate trianion or acetate.Voltolisation damping fluid is the salt-glycine system containing mass concentration 0.05～5% SDS, and the concentration of glycine is 1.0 mM～1000 mM, and the concentration of salt is 1.0 mM～1000 mM; Regulating the pH value of voltolisation damping fluid with HCl is 4.0～10.0.In water-soluble fibroin protein solution, also can add biologically active substance enzyme, polypeptide drugs, or small-molecule drug.
In technical solution of the present invention, DC electric field is the constant current electric field of 10～200 mA, or the constant voltage electric field of 50～200 VDC; Be 10～100 min the action time of DC electric field.
3, the structure determination of voltolisation fibroin aquagel membrane
Adopt YG021 single thread force-machine (the present embodiment is selected Changzhou the second textile instrument Co., Ltd., Factory) to analyze the mechanical property of ESFHM, result is referring to table 1, test speed 10 mm/min; Adopt KES-FB3 type Compression Tester to analyze the compression performance of ESFHM, result is referring to table 2; (the present embodiment is selected Nicolet 6700 to adopt Varian Fourier infrared spectrometric analyzer; Thermo Fisher, USA) ESFHM is carried out to Infrared spectrum scanning, result is referring to accompanying drawing 2, scanning times 32 times, resolving power 1 cm
-1; The X-diffraction analysis result of ESFHM is referring to accompanying drawing 3; Adopt Pyris-1 D sodium carbonate, TGA, TMA-7 type thermal analyzer (PE company of the U.S.) to carry out ESFHM heat and analyze, N
2under protective condition, 20 DEG C/min of temperature rise rate, temperature is 50～350 DEG C, the D sodium carbonate curve of working sample is referring to accompanying drawing 4; ESFHM is rinsed repeatedly to the salt ion of removing remained on surface with deionized water, after cleaning, undertaken two fixing by glutaraldehyde-osmic acid stationary liquid, after phosphate buffered saline buffer rinses, shear fixing, after metal spraying, in scanning electron microscope, (the present embodiment is selected S-4700, HIT) upper, under 15 kV voltages, observe ESFHM ultrastructure referring to accompanying drawing 5; A figure is the fibroin aquagel membrane of preparing in the damping fluid without SDS, and b figure is at the fibroin aquagel membrane containing preparing in SDS damping fluid.The porous fibroin aquagel membrane providing can be used as medical tissue engineering materials artificial skin, surgical operation implanted stopping composition, cell culture substrate etc.
4, the vitro enzyme of voltolisation fibroin aquagel membrane degraded
In order to measure voltolisation fibroin aquagel membrane enzymolysis property in vitro, aquagel membrane sample immerses in neutral protease (concentration of enzymatic activity the is 4 U/ml) solution that molecular weight is 130,000, bath raio 1:50, slight vibration degraded (90 rpm) in 37 DEG C of constant-temperature tables.1,2,3,4 days time, to take out aquagel membrane and weigh, and the enzyme solution that contains degraded product is changed with the enzyme solution of new preparation, result is referring to accompanying drawing 6.
5, medicament slow release experiment
The present invention chooses this medicine of Rifampin as embedding object.After voltolisation, the voltolisation film surface of containing medicine is used to deionized water rinsing, be quantitatively placed in the EP pipe of 5mL, add the phosphate buffered saline buffer of pH7.4.Section is drawn supernatant liquor and is used Spectra Max M5 type microplate reader (Tuv Rheinland of North American. Inc.) to measure the absorbance of supernatant liquor at 475 nm places at a fixed time, draw drug release curve, result is referring to accompanying drawing 7.
6, cell culture experiments
Containing the DMEM-H30243.01B(Thermo HyClone of calf serum 10 %) add 1.8% Penstrep (invitrogen) in substratum and 1.1% L-Glutamine (invitrogen) is mixed with cell culture medium, for cultivating L-929 l cell.Voltolisation fibroin aquagel membrane uses 75% alcohol-pickled 24h sterilization, and PBS is added in 48 porocyte culture plates after rinsing, and adds substratum 500 μ L that configured in advance is good as the experimental group processing of spending the night before cell cultures.Control group only adds 500 μ L substratum, and the cell suspending liquid that cell concn is about to 1.5 × 104 cells/mL adds in culture plate, adds 100 μ L in every hole.All establish 3 multiple holes for every group, at 37 DEG C, 5%CO
2in cell culture incubator, cultivate.Adopt 3 48 orifice plates to cultivate respectively 3,4,5 days, result is referring to accompanying drawing 8.In the time cultivating corresponding time point, from incubator, take out culture plate, adopt the growthhabit of Te2000-U type inverted fluorescence microscope (Nikon, Japan) observation of cell.And adopt mtt assay to process sample, every hole adds 5 mg/mL MTT 500 μ L, continue to cultivate 4 h, abandon supernatant liquor, add dimethyl sulfoxide (DMSO) (DMSO) 500 μ L, concussion 10 min, use Spectra Max M5 type microplate reader (Tuv Rheinland of North American. Inc.) to measure absorbances at 570 nm places, draw cell growth curve, result is referring to table 3.
7, body is implanted into experiment
It is experimental subjects that the present invention adopts the male ICR mouse that clean level body weight is 35～40g, and experiment is divided into 3 groups, every group of 4 mouse.Blank group: mouse carries out sham-operation, sews up after incision again; Experimental group: the voltolisation fibroin aquagel membrane of the subcutaneous wadding warp 75% ethanol 24h sterilizing of mouse back; Positive controls: replant in mouse back skin through 75% ethanol 24h sterilizing after the freeze-drying of regenerated silk solution.After performing the operation 2,4,6,8 weeks, observe the outward appearance of mouse operative site, the degraded situation of aquagel membrane in body.Eyeball of mouse is got blood, and separation of serum detects the expression of inflammatory factor in serum, and result is referring to accompanying drawing 8.
The tensile properties of table 1. ESFHM
The compression performance of table 2. ESFHM
In table 2, preparation technology is: 0.1 mol/L Tris-HCl pH 6.52 (0.192 mol/L glycine; SDS); Constant voltage 80VDC; Voltolisation time 45min; 2mL 19 mg/mL fibroin samples.
Table 3 L929 l cell active testing result
In table 3, experimental group is on culture plate, to add the voltolisation fibroin aquagel membrane of preparation; Control group is on culture plate, not add voltolisation fibroin aquagel membrane.
Claims (10)
1. a preparation method for fibroin aquagel membrane, is characterized in that: the interior chamber container that fills water-soluble fibroin protein solution is placed in to the mistress's container that fills voltolisation damping fluid, and the bottom of interior chamber container is barrier membranes; Interior chamber is connect to negative pole, and mistress connects positive pole, under DC electric field effect, obtains a kind of fibroin aquagel membrane on barrier membranes;
Described barrier membranes is the pore membrane of receiving of holding back the above molecular weight of 1～100 kDa;
The mass concentration of described water-soluble fibroin protein solution is 0.5～10%;
Described voltolisation damping fluid is the salt-glycine system containing mass concentration 0.05 ~ 5% SDS, and the concentration of glycine is 1.0 mM～1000 mM, and the concentration of salt is 1.0 mM～1000 mM; Regulating the pH value of voltolisation damping fluid with HCl is 4.0～10.0;
Described DC electric field is the constant current electric field of 10～200 mA, or the constant voltage electric field of 50～200 VDC; Be 10～100 min the action time of DC electric field.
2. the preparation method of a kind of fibroin aquagel membrane according to claim 1, it is characterized in that: the described pore membrane of receiving is the pore membrane of receiving of hydrophilic natural macromolecule material, and described hydrophilic natural macromolecule hydrophilic material is the one in agarose, dextran film.
3. the preparation method of a kind of fibroin aquagel membrane according to claim 1, is characterized in that: the described pore membrane of receiving is the pore membrane of receiving of wetting ability synthesized polymer material, and described wetting ability synthesized polymer material is polyacrylamide gel.
4. the preparation method of a kind of fibroin aquagel membrane according to claim 1, is characterized in that: the described pore membrane of receiving is the pore membrane of receiving of inorganic materials, and described inorganic materials is pottery.
5. the preparation method of a kind of fibroin aquagel membrane according to claim 1, is characterized in that: the described pore membrane of receiving is the dialysis membrane of hydrophilic high molecular material, and described dialysis membrane is regenerated cellulose dialysis membrane, cellulose ester dialysis membrane.
6. the preparation method of a kind of fibroin aquagel membrane according to claim 1, is characterized in that: described salt is inorganic salt phosphoric acid salt or carbonate.
7. the preparation method of a kind of fibroin aquagel membrane according to claim 1, is characterized in that: described salt is organic salt Citrate trianion or acetate.
8. the preparation method of a kind of fibroin aquagel membrane according to claim 1, is characterized in that: water-soluble fibroin protein solution comprises biologically active substance enzyme, polypeptide drugs, or small-molecule drug.
9. a device of preparing fibroin aquagel membrane, is characterized in that: interior outer container is fixed in mistress's container, and interior chamber container bottom is barrier membranes, and described barrier membranes is the pore membrane of receiving of holding back the above molecular weight of 1～100 kDa; The negative pole of direct supply is connected in interior chamber, and mistress connects the positive pole of direct supply.
10. an application for fibroin aquagel membrane, is characterized in that: described fibroin aquagel membrane is used as to medical tissue engineering materials artificial skin, surgical operation implanted stopping composition, cell culture substrate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410221710.7A CN103981561B (en) | 2014-05-23 | 2014-05-23 | Preparation method, device and the application thereof of a kind of voltolisation fibroin aquagel membrane |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410221710.7A CN103981561B (en) | 2014-05-23 | 2014-05-23 | Preparation method, device and the application thereof of a kind of voltolisation fibroin aquagel membrane |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103981561A true CN103981561A (en) | 2014-08-13 |
| CN103981561B CN103981561B (en) | 2016-10-05 |
Family
ID=51273725
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410221710.7A Active CN103981561B (en) | 2014-05-23 | 2014-05-23 | Preparation method, device and the application thereof of a kind of voltolisation fibroin aquagel membrane |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103981561B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104436285A (en) * | 2014-12-12 | 2015-03-25 | 苏州大学 | Regenerated silk fibroin gel mask and preparation method thereof |
| CN104959042A (en) * | 2015-06-24 | 2015-10-07 | 苏州乔纳森新材料科技有限公司 | Dialysis membrane and preparation method thereof |
| CN105297112A (en) * | 2015-10-23 | 2016-02-03 | 闽南师范大学 | Method for preparing konjac glucomannan gel through direct current |
| CN106866996A (en) * | 2017-03-13 | 2017-06-20 | 武汉纺织大学 | A kind of fast preparation method of silk fibroin matter gel |
| CN107375994A (en) * | 2017-07-24 | 2017-11-24 | 青岛大学 | A kind of medical gel adhesive bandage product preparation method |
| CN107812233A (en) * | 2017-09-28 | 2018-03-20 | 王映 | A kind of cold compress dressing and preparation method thereof |
| CN112210260A (en) * | 2020-10-14 | 2021-01-12 | 东莞狐马商贸有限公司 | Preparation method of self-assembled fibroin nanofiber modified polymer coating |
| CN114702704A (en) * | 2022-01-29 | 2022-07-05 | 苏州大学 | Functional polymer film/hydrogel film based on one-way nanopore dehydration, and preparation method and device thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01308431A (en) * | 1988-06-06 | 1989-12-13 | Norin Suisansyo Sanshi Shikenjiyou | Silk fibroin hydrogel |
| CN2198286Y (en) * | 1994-05-10 | 1995-05-24 | 周考文 | Chlorine dioxide reinfoced disinfectant generator |
| CN101502670A (en) * | 2009-03-05 | 2009-08-12 | 苏州大学 | Method for preparing silk fibroin hydrogel |
| US20090234026A1 (en) * | 2003-04-10 | 2009-09-17 | Trustees Of Tufts College | Concentrated aqueous silk fibroin solution and use thereof |
| CN102220017A (en) * | 2011-05-05 | 2011-10-19 | 苏州大学 | Injectable moulded silk fibroin hydrogel and preparation method thereof |
-
2014
- 2014-05-23 CN CN201410221710.7A patent/CN103981561B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01308431A (en) * | 1988-06-06 | 1989-12-13 | Norin Suisansyo Sanshi Shikenjiyou | Silk fibroin hydrogel |
| CN2198286Y (en) * | 1994-05-10 | 1995-05-24 | 周考文 | Chlorine dioxide reinfoced disinfectant generator |
| US20090234026A1 (en) * | 2003-04-10 | 2009-09-17 | Trustees Of Tufts College | Concentrated aqueous silk fibroin solution and use thereof |
| CN101502670A (en) * | 2009-03-05 | 2009-08-12 | 苏州大学 | Method for preparing silk fibroin hydrogel |
| CN102220017A (en) * | 2011-05-05 | 2011-10-19 | 苏州大学 | Injectable moulded silk fibroin hydrogel and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| DEVID MANIGLIO ET AL: "electrodeposition of silk fibroin on metal substrates", 《JOURNAL OF BIOACTIVE AND COMPATIBLE POLYMERS》 * |
| ZHEN ZHANG ET AL: "low temperature electrophoretic deposition of porous chitosan/silk fibroin composite coating for titanium biofunctionalization", 《JOURNAL OF MATERIALS CHEMISTRY》 * |
| 李庆春 等: "丝胶蛋白水凝胶膜的合成及性能研究", 《轻纱工业与技术》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104436285A (en) * | 2014-12-12 | 2015-03-25 | 苏州大学 | Regenerated silk fibroin gel mask and preparation method thereof |
| CN106310349A (en) * | 2014-12-12 | 2017-01-11 | 苏州大学 | Regenerated fibroin protein gel mask |
| CN106310349B (en) * | 2014-12-12 | 2019-11-22 | 苏州大学 | A kind of regenerated silk fibroin gel mould |
| CN104959042A (en) * | 2015-06-24 | 2015-10-07 | 苏州乔纳森新材料科技有限公司 | Dialysis membrane and preparation method thereof |
| CN105297112A (en) * | 2015-10-23 | 2016-02-03 | 闽南师范大学 | Method for preparing konjac glucomannan gel through direct current |
| CN106866996A (en) * | 2017-03-13 | 2017-06-20 | 武汉纺织大学 | A kind of fast preparation method of silk fibroin matter gel |
| CN107375994A (en) * | 2017-07-24 | 2017-11-24 | 青岛大学 | A kind of medical gel adhesive bandage product preparation method |
| CN107812233A (en) * | 2017-09-28 | 2018-03-20 | 王映 | A kind of cold compress dressing and preparation method thereof |
| CN112210260A (en) * | 2020-10-14 | 2021-01-12 | 东莞狐马商贸有限公司 | Preparation method of self-assembled fibroin nanofiber modified polymer coating |
| CN114702704A (en) * | 2022-01-29 | 2022-07-05 | 苏州大学 | Functional polymer film/hydrogel film based on one-way nanopore dehydration, and preparation method and device thereof |
| CN114702704B (en) * | 2022-01-29 | 2023-11-24 | 苏州大学 | Functional polymer membrane/hydrogel membrane based on unidirectional nano-pore dehydration, preparation method and device |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103981561B (en) | 2016-10-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103981561B (en) | Preparation method, device and the application thereof of a kind of voltolisation fibroin aquagel membrane | |
| WO2015127711A1 (en) | Preparation method and use of sericin hydrogel | |
| ES2701478T3 (en) | Process for the production of a hybrid structure consisting of microfibres and coupled silk fibroin nanofibers, hybrid structure obtained in this way and its use as an implantable medical device | |
| CN105985529B (en) | Sericin-alginate composite hydrogel and preparation method thereof | |
| US10119202B2 (en) | Method for preparing electro-mechanically stretched hydrogel micro fibers | |
| CN102580160A (en) | Tissue engineering scaffold material of chemical bonding active material and preparation method thereof | |
| Landry et al. | Layers and multilayers of self-assembled polymers: tunable engineered extracellular matrix coatings for neural cell growth | |
| CN103211663A (en) | Method for preparing electrospun artificial blood vessel with micro-nano biomimic intima structure | |
| CN101837149A (en) | Anti-coagulation anti-bacterial biomedical material and preparation method thereof | |
| CN104667351A (en) | Nerve growth factor loaded silk fibroin nanofiber scaffold and preparation method | |
| CN102357264B (en) | Silk fibroin porous material and preparation method thereof and tissue engineering stent | |
| CN103418021A (en) | In-situ-crossly-linked electrospun fibrous membrane dressing made from collangen I and preparation method of in-situ crosslinking electrospun fibrous membrane dressing | |
| CN105664260A (en) | Method for preparing bone tissue engineering three-dimensional porous support based on graphene/silk fibroin | |
| Dutta et al. | Effect of collagen nanofibers and silanization on the interaction of HaCaT keratinocytes and 3T3 fibroblasts with alumina nanopores | |
| CN102652841A (en) | Method for preparing sulfated silk fibroin vascular tissue engineering scaffold by using electrostatic spinning | |
| Yang et al. | Minimally invasive bioprinting for in situ liver regeneration | |
| CN106492286B (en) | A kind of fibroin/bacteria cellulose composite hydrogel and its preparation method and application | |
| Grimmelsmann et al. | Needleless electrospinning of pure and blended chitosan | |
| KR100571672B1 (en) | Nanofiber containing -pga and process for preparing the same | |
| CN104971386A (en) | Silk protein scaffold material and preparation method thereof | |
| CN102154786A (en) | Mineralized corn protein fibrous membrane and preparation method thereof | |
| CN101397695B (en) | Method for preparing stable gelatine nano fiber for bionic stent material | |
| CN115804867A (en) | Fibrosis type III collagen nano-film, preparation method and application in skin regeneration | |
| CN105435306B (en) | The preparation method and purposes of biological nano sticking patch | |
| US20130280307A1 (en) | Encapsulation of matter in polymer structures |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |