processing and characterisation of a novel electropolymerized silk fibroin hydrogel membrane - pearl necklace and earring set

by:JINGLIXIN     2019-08-15
processing and characterisation of a novel electropolymerized silk fibroin hydrogel membrane  -  pearl necklace and earring set
Silk protein has excellent properties and can be made into a variety of forms of biocompatible medical materials, including gels.
Here, we report a new method for the preparation of electro-polymerization silk gel film (ESFHM)
, It is formed on the nano-hole film as a barrier to the use of homemade equipment at a higher DC voltage.
Solution of regenerated silk in Tris buffer (pH 6. 55–7. 55)
Added to the reservoir with negative electricity, silk molecules migrate to positive electricity at 80VDC, resulting in the formation of ESFHM on the Barrier membrane.
The barrier membrane of MWCO for 10 kda kd is conducive to the formation of ESFHM. Semi-
Transparent ESFHM with an expansion ratio of 1056.
4% mainly by beta-sheets and α-
Spiral crystal structure.
SEM studies have shown that ESFHM consists of a 3D mesh woven from a silk protein nanoparticles chain of approximately 30 nm in size, similar to a pearl necklace.
In vitro studies have shown that ESFHM is biodegradable enough to promote cell adhesion and growth.
Therefore, ESFHM, as an artificial skin or for transplant, is a promising candidate for loading biologically active proteins and appropriate cells.
According to the procedure described in our previous study, silk protein solution was prepared from silkworm cocoons.
Three kinds of silk refining methods and two kinds of silk fiber dissolution methods were tested.
Urea buffer (UB)
Heat up in 8 μm water urea containing 0. 04u2005M Tris-SO (pH 7. 0)and 0.
5 m ethanol 2 u2005 h at 80 °c
In boiling 0, the shell was degassed twice. 2% (w/v)NaCO solution (SC)for 0. 5u2005h.
Refining method of strong alkaline electrolytic water (SAEW)
25 times (v/w)in SAEW (pH 11. 50)
For 30 minutes at 100 °c.
After these degumming treatments, the resulting silk material is repeatedly washed in deionised water and then air-dried.
Add 20 times to the dense fiber (v/w)9. 3u2005M LiBr (LiBr)
Reagents for Ajisawa (
CaCl/ethanol/water, 1: 2: 8 mole ratio, short TS).
After 10 min of de-ionic water filtration or centrifuge at 8000 rpm, the filtered solution or supernatant is continuously dialysis in the running water for 48 h to remove the CaCl, using cellulose half molecules and some impurities
Permeable membrane (cut-off 5u2005kDa).
ESFHM was prepared with six aqueous solutions of silk protein.
The experimental device consists of two electrodes made of platinum wire.
Negative electrode (cathode)
Inner chamber and positive electrode (anode)
Being Nano in an external chamber
Membrane and its support ().
Before electric polymerization, 20 ml of electric polymerization buffer and 2 ml of silk protein aqueous solution (20u2005mg/mL)
Was mixed and added to the inner chamber while adding 30 ml of the same buffer to the outer chamber.
The electrode is electrically connected to the programmable direct on the top of the platinum wire using a crocodile clip-current (DC)power supply.
The closed circuit consists of the following parts: Power supply → (+)
Electrode → Silk liquid → (−)
Electrode → power supply.
When these positive charges on the surface of the silk molecule are combined with SDS as negative charges, the silk molecule migrate to the Yang pole in the electric polymerization buffer.
30-40 minutes after running, half
A transparent gel film is formed on a barrier film with nano holes (nano film). Three post-
In order to improve the performance of ESFHM, the processing method is adopted.
ESFHM soak 80% (v/v)
Methanol for 20 minutes and then discharged on paper towels (ESFHM-M).
ESFHM dry in a smoky cupboard (AD-ESFHM).
ESFHM is frozen and frozen at-20 °c (L-ESFHM).
As a control, the silk solution is dripped onto the PVC plate using a liquid transfer tube and then air-dried at 25°C (SFM).
SFM bubble 80% (v/v)
Methanol of 30 m/s and then discharged with a paper towel (SFM-M).
To evaluate the expansion of ESFHM, the sample was immersed in deionic water for 24 hours at 37 °c.
After removing the excess surface de-ionic water with the filter paper, the expansion weight of each membrane is recorded.
The sample is then dried in the fume hood to obtain dry weight.
Calculate the expansion ratio of each sample using the following equation: % universal testing machine, INSTRON, US).
The length between grip anchors is 0. 5u2005cm.
The test was performed at a strain rate of 10 mm/min.
The sample deformation and strain as well as the stress-strain curves were obtained by software analysis.
Compression Performance Analyzed using KES-FB3-
Compression tester (
Kato Technology Co. , Ltd).
The infrared spectrum of ESFHM was obtained using an infrared spectrometer (Nicolet 6700;
Thermo Fisher, United States of America)
The molecular conformations were determined in the spectral region of 4000-400.
Repeated scans 16 times per measurement with a resolution of 4 cm. Ten-
Point smoothing for infrared spectrum.
Micro-focal Raman spectrometer (
LabRAM HR800 of Horiba JobinYvon)
LMPLFL optical microscope (
50 x mesh with a Aperture of 400 u2005 μm)
Used to analyze the surface texture of ESFHM in the wavelength range of 400-4000 at 4 cm resolution.
The wavelength of the laser is 633 nm, and the sample is irradiated by 20 m/s during the spectral recording process.
Adjacent ten-
For point smoothing of Raman spectrum.
Thermal analysis of ESFHM was performed using differential scanning Heat Meter (DSC)instrument (
DSC 2010 for TA Instruments)
In the temperature range of 50 to 350 °c, the heating rate at 20 °c/minX-
Analysis of ESFHM using X-ray diffraction
Ray diffraction (X'Pert-
P. mpd, panalyical)
Recording the spectrum in an angle range of 5 to 50 degrees ().
To characterize the internal microstructure of ESFHM, propolis-
Copper oxide fixing agent.
After rinsing with PBS, cut the sample to expose the Cross
The stage and the sputtering meter coated with Pt/Pd (SP-
2 AJA sputtering system).
Observe samples using a scanning electron microscope (Hitachi S-4700, Japan)
At an acceleration voltage of 15 kV.
At least five images are used for quantification in each group of samples.
In order to characterize the enzyme degradation performance of ESFHM, gel film samples were dried in the fume hood and wet-
Calculate drying.
Next, put the wet sample into the Erlenmeyer flask containing 0.
05 u2005 M phosphate buffer (PBS), pH 8.
1 u/mL neutral protease at 0,37 °c (
1: 50 Bath ratio, wet W/V).
A sample of each condition was deleted for a predefined period of time.
The loss of quality is determined by the ratio of the final weight to the original dry weight.
The sample size of each group is three membranes. L-
From the Institute of Cell Biology of the Chinese Academy of Sciences, 929 mouse epithelial cells were obtained (Shanghai, P. R. China).
Cells are maintained in Dulbecco-modified Eagle medium (DMEM)with DMEM-H30243. 01B (Thermo HyClone)
With 10% bovine serum protein and 1% penicillin/L-
Cultured in a standard cell incubator (HERAcell 240i)
In the atmosphere of 5% CO at 37 °c
Change cell media every 2 days.
When fusion is reached, these cells are transmitted twice a week.
Cell metabolic activity on ESFHM surface was determined by MTT staining.
Cut the film into a diameter of 1 cm and immerse it in 75% (v/v)
Ethanol for 48 h, thoroughly rinsed with sterilized DPBS.
Then add the sterilized film to 48-
Overnight before Cell plating, pretreatment with cell culture medium.
Next, 100 µL-
929 mouse fiberglass cells were suspended on the surface of the membrane with a density of 1.
Good 5x10 cells.
There are 3 wells in each group.
Change the medium every day.
Remove and visualize the tablet using a fluorescence microscope at a predetermined time (
Nikon Eclipse TE 2000-U, Japan).
5 mg/mL 3-500 u2005 μ l after observation(4, 5-Dinazol-2-yl)-2,5-
Dibenzol (MTT)
Solutions are added to each well. After a 4-
After H incubation, the supernatant was carefully removed and the formazan Crystal was dissolved with a solution of 500 u2005 μ l px.
After 10 minutes of stirring, the absorbance is read at 570 nm using a microplate reader (
SpectrMax M5, Molecular Devices, Sunrise, United States of America).
Each membrane sample is repeated three times on average.
Samples of all quantifiable analyses were n = 4-5 and there was a technical duplication for each biological replication.
ESFHM and cell morphology analysis were performed at n = 3, showing only representative images.
Unless otherwise stated, the data is expressed as mean ± standard deviation (SD)
And use one-
Way ANOVA with post-event t-
Test, using a 5% (P < 0. 05)
Degree of importance.
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