Elsevier

Biomaterials

Volume 26, Issue 15, May 2005, Pages 2775-2785
Biomaterials

Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin

https://doi.org/10.1016/j.biomaterials.2004.07.044Get rights and content

Abstract

A new all-aqueous process is described to form three-dimensional porous silk fibroin matrices with control of structural and morphological features. The result of this process are scaffolds with controllable porosity and pore sizes that fully degrade in the presence of proteases, unlike prior methods to generate silk-based biomaterials that required the use of organic solvent treatments to impart control of structure and stability in aqueous environments, with low rates of proteolytic hydrolysis. A mechanism is proposed for this novel process that imparts physical stability via hydrophobic interactions. Adjusting the concentration of silk fibroin in water, and the particle size of granular NaCl used in the process, leads to the control of morphological and functional properties of the scaffolds. The aqueous-derived scaffolds had highly homogeneous and interconnected pores with pore sizes ranging from 470 to 940 μm, depending on the mode of preparation. The scaffolds had porosities >90% and compressive strength and modulus up to 320±10 and 3330±500 KPa, respectively, when formed from 10% aqueous solutions of fibroin. The scaffolds fully degraded upon exposure to protease during 21 days, unlike the scaffolds prepared from organic solvent processing. These new silk-based three-dimensional matrices provide useful properties as biomaterial matrices due to the all-aqueous mode of preparation, control of pore size, connectivity of pores, degradability and useful mechanical features. Importantly, this process offers an entirely new window of materials properties when compared with traditional silk fibroin-based materials.

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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