Human bone marrow stromal cell responses on electrospun silk fibroin mats

Abstract

Fibers with nanoscale diameters provide benefits due to high surface area for biomaterial scaffolds. In this study electrospun silk fibroin-based fibers with average diameter 700±50 nm were prepared from aqueous regenerated silkworm silk solutions. Adhesion, spreading and proliferation of human bone marrow stromal cells (BMSCs) on these silk matrices was studied. Scanning electron microscopy (SEM) and MTT analyses demonstrated that the electrospun silk matrices supported BMSC attachment and proliferation over 14 days in culture similar to native silk fibroin (∼15 μm fiber diameter) matrices. The ability of electrospun silk matrices to support BMSC attachment, spreading and growth in vitro, combined with a biocompatibility and biodegradable properties of the silk protein matrix, suggest potential use of these biomaterial matrices as scaffolds for tissue engineering.

Introduction

Electrospinning for the formation of fine fibers has been explored for applications such as high performance filters [1], [2], [3] and biomaterials for vascular grafts, wound dressings or tissue engineering scaffolds [4], [5], [6]. Electrospinning offers an alternative approach to protein fiber formation that can potentially generate nanometer scale diameter fibers. This would be a useful feature in some biomaterial and tissue engineering applications to enhance surface area while maintaining high porosity [7]. Electrospinning has been utilized to generate nanometer diameter fibers from recombinant elastin protein [7], silk-like protein [8], [9], [10], silkworm silk [11], [12], type I collagen [4], [13], [14], [15] and spider dragline silk [11].

Interest in the use of reprocessed silks such as fibroin in biotechnological materials and in biomedical applications derives from the unique mechanical properties of these fibers and their biocompatibility and biodegradability [16], [17], [18]. For example, we have reported silk protein-based matrices developed for ligament and bone tissue engineering [19], [20]. We have shown that silk fibroin, after proper extraction of the contaminating sericin proteins, is non-immunogenic, biocompatible and capable of supporting bone marrow stromal cell (BMSC) attachment, spreading, growth and differentiation as well as eliciting a negligible response during standard in vitro macrophage assays [21]. Systematic in vivo studies indicate that silk induces a foreign body response comparable to most commonly used degradable synthetic and natural polymers such as poly(glycolic acid)-poly(lactic acid) (PGA-PLA) copolymers and collagen [20], [22].

One goal in the field of biomaterials is to fabricate matrices that mimic the structure and biological function of the extracelluar matrix (ECM) [4], [5], [6]. ECM is composed of two main groups of macromolecules, proteoglycans and collagen, that together form a composite-like structure. Fibrous collagens embedded in proteoglycans maintain structural and mechanical properties. The collagen is organized in a three-dimensional network composed of collagen fibers that form hierarchical structure from nanometer-scale multi-fibrils to macroscopic tissue architecture [23], [24]. Ideally, the dimensions of the building blocks of a tissue-engineered scaffold should be on a similar scale as those of natural ECM. The scaffold should also have mechanically supportive properties for tissue regeneration while at the same time guiding cell differentiation and function. Furthermore, biocompatibility and biodegradability, with non-toxic and non-inflammatory degradation products during replacement in vivo by cellular ECM components are key criteria. To achieve this goal, natural and synthetic polymers have been electrospun to produce 3-D nano size fibers for cell culture [4], [6].

We previously reported the formation of electrospun scaffolds from aqueous silkworm silk (Bombyx mori) solutions with poly(ethylene oxide) (PEO). Fiber sizes ranged from 500 nm to 1 μm which are about 40 times smaller than degummed native silk fibroin [12]. The purpose of the present study was to investigate stromal cell responses on these fibers to determine the potential utility of this novel silk fibroin protein matrix for scaffolding.