Received date: March 14, 2016; Accepted date: April 19, 2016
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Objective. The aim of the present study is to prepare a 3D porous silk fibroin scaffold with a hierachical structure that can meet the demands for bone tissue engineering. Materials and Methods. 3D fibroin scaffold was prepared by the methods of partial dissolution in acid solution and freeze drying fibroin solution. Results. The nets were composed of a mesh of randomly oriented fibers that ranged between 10 ?m and 30 ?m in diameter. Branchpoints and three dimensional open spaces were distributed throughout the structure with an average pore size of about 177.9 Â± 40.0 ?m. Conclusion. With the methods of non-woven silk fibroin net preparation and frozen-dried technics, it is possible to prepare a 3D porous silk fibroin scaffold with hierachical fine structure.
porous scaffold; fibroin; sponge; non-woven net; freeze drying
Silk fibroin is an important polymer that provides a set of material options for biomaterials because of the impressive biocompatibility, mechanical properties and biodegradability. Silk fibroin has been investigated intensively as biocompatibly and mechanically robust biomaterials for bone, cartilage, and ligament tissue engineering.
Porous silk fibroin structures, such as non-woven net, non-woven nanofibers and sponge, have been fabricated by various attempts, such as partial dissolution in acid solution, electrospinning, freeze drying and salt leaching [1,4,5,7]. Fibroin should be investigated more to prepare the structure with a desired shape and a controlled porous architecture for cell growth, tissue regeneration, and vascularization.
The aim of the present study is try to use partial dissolution and freeze drying methods to prepare a 3D porous bioscaffold with a structure consisting of silk fibroin net and silk fibroin sponge.
Preparation of non-woven silk nets
Raw B. mori silk fibers were boiled for half an hour in a 0.5% Na2CO3, and rinsed thoroughly with water to remove the glue-like sericin proteins surrounding the fibroin filaments and dried in air. Before using in next step, the silk will be dried in an electric drying chamber at 80 °C for 6 h. Degummed silk fibers were soaked into test tube containing the solution of 98% formic acid and 0.01 w/v% calcium chloride (material-to-fluid ratio, 1:200) at room temperature. The fiber suspension was shaken for 30min to achieve homogenous fiber distribution and kept still for 24 h. Finally, the acid solution was evaporated through water bath at 40 °C in an aerator, and the resulting non-woven material was repeatedly washed with distilled water to remove any residual salt and vacuum dried .
Preparation of fibroin solution
The degummed silks were completely dissolved after being soaken in a solution of calcium chloride/ethanol/ distilled water (1:2:8 mole ratio) at 80 °C for 4 h through stirring. The prepared solution was purified by being dialyzed against distilled water for 3 days. The concentration of silk fibroin aqueous solution was calculated by measuring the volume of solution and weighing the remaining solid after drying. The formula for the calculation of the concentration is as follows:
V2 = V1 × C1/C2,
where V2 is the volume of fibroin solution after evaporation, V1 is the volume of fibroin solution before evaporation, C1 is the concentration of the fibroin solution before evaporation and C2 is the concentration that will be needed in the experiment. At the present experiment, 20mL of fibroin solution (V1) with a concentration of 2.5 w/v% (C1) was held in a graduated flask. Finally, silk fibroin solution with various concentrations of 0.75, 1.5, 3, 6, 9 and 12 w/v% were prepared by dilution or evaporation.
Preparation of 3D fibroin scaffold
The dried non-woven fibroin net was immersed into the fibroin solution with various concentrations under the vacuum of 700mmHg for 10min to remove the air from the net. Then the fibroin net was kept soaking in the solution for 24 h before it was taken out of the fibroin solution and transferred into an aluminum vessel. Next, samples were frozen for 6 h at the temperature of −80 °C and vacuum dried for 48 h in a freeze dryer (LGJ-22, Beijing, China). All samples prepared in different concentrations were tested with SEM (JSM-5900 LV, Tokyo, Japan) in order to find a suitable concentration of fibroin solution to prepare a spongy porous silk fibroin scaffold. To measure the aperture of the porous structure, SEM photographs in bmp file format were tested with image analysis software (SMileView Ver. 2.1). The border of each pore in top layer was measured in the whole picture and the average pore size was calculated.
Usually the concentration of fibroin solution obtained was approximately 2.4–3 w/v% after being purified by dialysis against water for three days. Then it was stirred slowly at 37 °C to make it evaporate and concentrate up to a certain concentration according to experiment design. The fibroin solution with the concentration lower than 3 w/v% was light milky white. The liquid would be getting thicker, more white and more viscous along with the increase of concentration. When the concentration increased nearly to 6 w/v%, the solution became too viscous to be suitable for immersing the silk net into the solution. Furthermore, the solution turned into a semisolid gel as a result of protein crosslink when the concentration was up to 9 w/v% .
After soaking the net into fibroin solution and being frozen dried, the microstructure of the samples was tested with SEM for each group of different concentration.
Figure 1 shows the gross of silk fibroin net. The cylinder shape of fibroin net was prepared in a test tube. The nets were composed of a mesh of randomly oriented fibers that ranged between 10 μm and 30 μm in diameter. Branch points and three-dimensional open spaces were distributed throughout the structure with an average pore size of about 177.9 ± 40.0 μm . The individual fibers generally exhibited a smooth surface as revealed in Figure 2A. Threedimensional scaffolds are required in tissue engineering to support for the formation of tissue-relevant mimics as well as to promote cellular adherence, migration, formation of new extracellular matrix, tissue ingrowth and to foster the transport of nutrients and metabolic wastes .
The pore shape and size were strongly influenced by the solution concentration. When the concentration was lower than 0.75 w/v%, the whole surface of silk net was almost covered by a thin layer of fibroin film although pores could also be found somewhere under SEM test (see Figures 3A and 3B). The silk net soaking in 3 w/v% fibroin was able to form the pores with a complete sponge structure and the most homogenous distribution. The pore size ranged from 2 μm to 108.6 μm with an average size of 40 ± 20.4 μm, as shown in Figures 2B, 2C and 2D.
It is generally considered that pore sizes larger than 100 micron in diameter with an interconnected structure is a minimum requirement for such systems based on cell sizes and migration . The pore size of the silk fibroin net seems to be able to meet the needs for the potential use in tissue engineering. Kim et al. prepared a porous fibroin sponge with aqueous-derived silk fibroin by salt leaching and freeze-drying methods. The aqueous-derived scaffolds had highly homogenous and interconnected pores with pore sizes ranging from 470 μm to 940 μm, depending on the mode of preparation . The final structure prepared in the present experiment was more like a hierarchical structure. Although its pore was smaller than the ideal size for tissue engineering, this structure may be able to be used for drug deliver, biomembrane or other purposes. Further study will be needed to improve the structure. Its porosity, mechanical strength and other characteristics will be also investigated.
With the use of methods to prepare non-woven silk fibroin net and of freeze drying techniques, it is possible to fabricate a 3D porous silk fibroin scaffold with hierarchical fine structure, which may have a potential use as a bioscaffold or other biomaterials.