Electrospinning And Dry-spinning Of Biomimetic Regenerated Silk Fibroin Aqueous Solutions

Natural silk fibers,such as spider silk or silkworm silk,exhibit exceptional mechanical properties that rival those of synthetic high performance fibers such as Kevlar.Silks also possess impressive biological properties that extend their usefulness to biomaterials as sutures and scaffolds in tissue engineering.Recently,much attention has been paid to biomimetic spinning of natural silk fibers.Different methods,such as wet spinning and electrospinning,have been used to spin silk fibers artificially from recombinant spidroin solution or regenerated silk fibroin solution,but the mechanical properties of artificial silk fibers are still less than that of natural silk fibers.The result shows that the mechanism of spider or silkworm spinning are very similar and both of them spinning by using high concentrated liquid crystal solution as spinning dope with water as solvent under ambient temperature and pressure, despite their independent evolution.The spinning process for spider silk or silkworm silk essentially includes the conformational transition from random coil /α-helix conformation in silk dope intoβ-sheet conformation in solid fiber.With the further understanding of the mechanism of spider or silkworm spinning,it is found that the processing parameters,such as protein concentration,pH,the kinds and contents of metallic ions and elongational flow,play very important role in the natural spinning process.Therefore,it is key step for biomimetic spinning to investigate in vitro the influence of the processing parameters on properties and fiber forming ability of silk fibroin solution.In this paper,silk fibroin of silkworm is selected as a model system. Concentrated regenerated silk fibroin（RSF） aqueous solution was prepared by a mild method.The molecular weight of RSF was analyzed by SDS-PAGE.It was found that the molecular weights of RSF were between 40 kDa and 86 kDa,which were smaller than that of the native silk fibroin（350 kDa）.The RSF aqueous solution prepared by our experiment procedure was weak alkalescence.In order to quickly monitoring the change of concentrated RSF aqueous solution;we established a convenience method to obtain the concentration of RSF aqueous solution by measuring the refraction index of RSF aqueous solution.Electrospinning is an attractive method of producing fibers with diameters ranging from micrometers down to tens of nanometers.In our case,the electrospinning process was somewhat analogous to the spinning process of silkworm.After undergoing a shear flow in the tube and capillary,the jet of the solution was elongated many times and water was evaporated in air.Firstly,the high concentration RSF aqueous solutions at neutral（pH6.9） were prepared,and then the effects of electrospinning parameters on the morphology,diameter and diameter distribution of regenerated silk fibers were investigated by Orthogonal design.The results showed that voltage,concentration and the distance between tip and collection plate were the most dominant factor to the morphology,diameter and diameter distribution of regenerated silk fibers,respectively.The structure of electrospun mats was characterized by Raman spectroscopy（RS）,wide-angle X-ray diffraction（WAXD） and Modulated differential scanning calorimetry（MDSC）.It was found that electrospun mats prepared by RSF aqueous solutions at neutral（pH6.9） involved a little silk I structure and were predominantly amorphous structure.In vivo of the silkworm B.mori,there is a decrease in pH from 6.9 to 4.8 as the silk dope flows through the silk gland from the posterior to anterior divisions.In order to mimic the environmental condition in the gland of silkworm,RSF aqueous solutions at pH 6.9,6.0,5.6,5.2 and 4.8,which are similar to the pH in the different divisions of the gland of B.mori,were prepared,respectively.The effects of pH on theological properties and the electrospinnablity of RSF aqueous solutions have been investigated.The results indicted that with the increase in concentration of the solutions,the apparent viscosities of the solutions increased.With the decrease in pH and the increase in concentration,the rheological behavior of the solutions exhibited a transition from Newtonion fluid to non-Newtonion fluid and a stress induced phase separation and crystallization could be observed at higher shear rates.Lowering the pH promoted the evaporation of water from the solutions and induced gel formation. RSF aqueous solutions had the optimal pH value for gel formation,fairly close to the isoelectric point of H-chain fibroin（4-5）.With the decreased in concentration and in pH,the electrospinnable concentration of RSF solutions and the average diameter of the electrospun silk fibers decreased.However,the pH of the solution had limited influence on crystallization of the electrospun silk fibers,the electrospun silk fibers were still predominantly random coil/silkâ… conformation.In this paper,by adding K⁺,Ca²⁺,Mg²⁺ ions,which were important ions in the silk gland,into RSF aqueous solutions with a high concentrations of 30%at a pH of 6.0,the effects of single kind of metallic ions or mixed metallic ions on rheological properties and the electrospinnablity of RSF aqueous solutions have been investigated. The results showed that metallic ions had influence on rheological properties of RSF aqueous solutions.At the same content of K⁺,Ca²⁺,Mg²⁺ ions,the apparent viscosities of the solutions with K⁺ were higher than that of the solutions with Ca²⁺,Mg²⁺.With the increase in the adding amounts of metallic ions,the apparent viscosities of the solutions were decreased at the same concentrations.In addition,the kind of metallic ions had influence on the morphology of regenerated silk fibers.There were a lot of micro particles in the surface of regenerated silk fibers with K⁺,while the surface of regenerated silk fibers with Ca²⁺and Mg²⁺ was smooth.The adding amounts of mixed metallic ions had influence on the electrospinnablity of RSF aqueous solutions.When the adding amounts of mixed metallic ions was 0.15 mol/L and 0.225 mol/L,the RSF aqueous solutions could be electrospinning,but when the adding amounts of mixed metallic ions was 0.3 mol/L,the RSF aqueous solutions could not be electrospun.For RSF aqueous solution with the addition of 0.225 mol/L mixed metallic ions,the electrospinnable concentration of RSF aqueous solutions were between 27%and 35%. With the increasing of concentration of RSF aqueous solutions,the average diameters and standard deviations of the electrospun silk fibers were increased.The results also showed that the humidity anneal had a remarkable influence on solubility of electrospun mats in water.Before the humidity annealing,the electrospun mats was soluble in water;but after the humidity annealing,the electrospun mats was insoluble in water.The structure analysis of electrospun mats showed that the electrospun mats was predominantly amorphous structure before the humidity annealing,while after the humidity annealing the electrospun mats was had conformational transition from amorphous structure to the Silkâ… and Silkâ…¡structure.On the other hand,the humidity annealing could improve the mechanical properties of electrospun mats.After investigation the effects of protein concentration,pH,the kinds and contents of metallic ions on rheological properties and the electrospinnablity of RSF aqueous solutions,we would like to know how these parameters interact with RSF aqueous solutions,and the parameter of storage time was also considered.It was found that rheological properties of RSF aqueous solutions changed greatly under the cooperation effects of metallic ions,fibroin concentration,pH,water content and storage time.And the weak gel could be formed from RSF aqueous solutions with Mg²⁺ and Ca²⁺ ions.By using the weak gel as the spinning dope,regenerated silk fibers were successfully obtained by dry spinning technique.The morphology and structure of regenerated silk fibers were investigated.In addition,the as-spun regenerated fibers were treated with methanol solution,the morphology,structure and mechanical properties of the fibers before and after treated with methanol solution were also assessed.The results showed that Mg²⁺ and Ca²⁺ ions were more efficient ions than K⁺ ion for producing gel at a pH of 6.0.With the decrease in water content and the increase in storage time,RSF aqueous solutions with Mg²⁺ and Ca²⁺ ions transferred gradually from silk fibroin solution to a gel state,and this gel state could be only formed in weak acidic RSF aqueous solutions.Storage had a remarkable influence on rheological properties of RSF aqueous solutions.After storing about 3 days,the apparent viscosities of the 40%RSF aqueous solution with the addition of 0.3 M Mg²⁺ and Ca²⁺ at a pH of 6.0 or 5.2 nearly increased 3 orders of magnitude.In this case,RSF aqueous solutions could be used as the dry spinning dope,which have an apparent viscosity that approached to viscosity of the native dope in the silkworm gland,the filament could be obtained continuously in air.These results reveal that the rapid aggregation of RSF molecules could be achieve by adding an amount of divalent cations at particular pH and the solution could transfer to the gel after storing, despite RSF solution is obtained by treating silk fibers with heat and powerful chaotropic agents,under which conditions molecular weight of silk fibroin is reduced and the glycoprotein P-25 is likely to be denatured.The results also showed that the processing parameters had a cooperation effects in assemble of silk molecules.It has been found that the as-spun fiber with Ca²⁺ ions was thin and had a smooth surface; the diameters of the fiber were 30±5μm.The structure analysis of the as-spun fibers showed that Silkâ…¡-related state（distortedβ-sheet） conformation existed but the as-spun regenerated fiber were predominantly in random-coil or silkâ… conformation. Temperature and humidity had influence on the mechanical properties of the as-spun fibers.Compared with degummed silk fiber,the as-spun fibers were weak and brittle. The as-spun fibers were treated with methanol solution,the morphology,structure and mechanical properties of the fibers had changed.After treated with methanol solution, the fibers showed banded features along the fiber axis and exhibited an opaque white color.The structure analysis of the fibers showed that Silkâ…¡structure has been formed in the fibers.Compared with degummed silk fiber,the as-spun fibers treated with methanol solution had higher module,but the breaking elongation was low,the fibers were still brittle,which due to low tropism degree and no sufficiently extending of silk molecular.The results of these studies assist us further understanding the processing environment interacts with the silk fibroin molecules in the natural spinning processing and provide useful information for the silk biomimetic spinning.We believe that it is possible to obtain regenerated silk protein fibers with high performance by biomimetic spinning.