Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin
Introduction
Three-dimensional scaffolds are required in tissue engineering to support for the formation of tissue-relevant mimics as well as to promote cellular migration, adherence, formation of new extracellular matrix, tissue ingrowth and to foster the transport of nutrients and metabolic wastes. Pore sizes >100 μm in diameter that are also interconnected are generally considered a minimum requirement for such systems based on cell sizes and migration [1], [2], [3]. Sufficient mechanical properties of the scaffolds are also necessary to support tissue function and integration, as is degradation at a rate comparable with new tissue growth.
A number of methods, such as salt leaching, gas forming or freeze-drying, have been reported to generate porous three-dimensional matrices from natural and synthetic polymers [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12]. Scaffolds from natural polymers have potential advantages of biocompatibility, versatility of chemistry, and cell-controlled degradability and intrinsic cellular interactions [13]. To enhance mechanical properties a number of physical and chemical modifications have been reported, including crosslinking by chemical and physical means. Synthetic polymers often provide useful mechanical properties for biomaterials for tissue engineering applications, however, many of these polymers do not degrade under physiological conditions [13].
Silkworm silk fibroin has been used commercially as biomedical sutures for decades, and in textile production for centuries. Silkworm silk from Bombyx mori consists primarily of two protein components. Fibroin is the structural protein of silk fibers and sericins are the water-soluble glue-like proteins that bind the fibroin fibers together [14]. Silk fibroin consists of heavy and light chain polypeptides of ∼350 and ∼25 kDa, respectively, connected by a disulfide link [15], [16]. Fibroin is a protein dominated in composition by the amino acids glycine, alanine and serine which form antiparallel β-sheets in the spun fibers, leading to the stability and mechanical features of the fibers [17], [18], [19].
Silk fibroin provides an important set of material options for biomaterials and scaffolds for tissue engineering because of the impressive mechanical properties, biocompatibility and biodegradability [20], [21], [22], [23], [24], [25], [26], [27]. In recent studies we have reported the formation of three-dimensional porous silk fibroin scaffolds, however, the process was based on the use of hexafluoroisopropanol (HFIP) as well as methanol [28]. The use of HFIP was required to optimize solubility and the methanol to induce an amorphous to β-sheet conformation transition in the fibroin, in order to generate water-stable silk structures.
In the present study, we sought to develop a new strategy for silk fibroin processing for biomaterials that avoided the use of organic solvents or harsh chemicals. To accomplish this goal, a new mechanism was employed to promote water solubility and then stability of the assembled silk fibroin without the use of HFIP or methanol. A new process was developed and then used successfully to generate three-dimensional silk scaffolds with control of pore size and functional features. Importantly, the very slow degrading matrices prepared previously with the organic solvent processing become rapidly degradable under these new all-aqueous processing conditions, reflecting differences in surface structure of the scaffolds.
Section snippets
Preparation of silk fibroin aqueous solution
Cocoons of B. mori were boiled for 20 min in an aqueous solution of 0.02 m Na2CO3, and then rinsed thoroughly with distilled water to extract the glue-like sericin proteins and wax. The extracted silk fibroin was then dissolved in 9.3 m LiBr solution at 60 °C for 4 h, yielding a 20 w/v% solution. This solution was dialyzed in distilled water using a Slide-a-Lyzer dialysis cassette (MWCO 3500, Pierce) for 2 days. The final concentration of silk fibroin aqueous solution was ca. 8 w/v%, which was
Preparation of aqueous-derived scaffolds
Porous silk fibroin scaffolds were prepared using a salt leaching method that has been previously used in the preparation of porous scaffolds from other polymers such as collagen and polylactic acid [1], [10], [31]. The pore size and the porosity of the scaffolds were regulated by the addition of granular NaCl with particle sizes of diameter 300–1180 μm to the silk fibroin aqueous solution. In this process the surface of the NaCl particles dissolved in the silk fibroin aqueous solution, while
Conclusions
Porous silk fibroin scaffolds were prepared directly from silk fibroin aqueous solutions by a salt leaching method, in the complete absence of any organic solvents or chemical crosslinking. The formation of these water-stable but aqueous-derived scaffolds included a structural transition from random coil to β-sheet. This transition provides a mechanistic basis for the transition, as the salt promotes water loss from the hydrophobic domains that dominate the fibroin structure, leading to
Acknowledgements
The authors thank the NIH (EB003210) for supporting this program. The authors also thank Hyoung-Joon Jin (Tufts U.) for helpful discussions and Vassilis Karageorgiou (Tufts U.) for technical input.
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