There are about 250,000 to 400,000 patients in the US suffering from spinal cord injury [SCI] [1
], usually due to trauma or traffic accidents, which could lead to death or life-long paralysis. According to the National Multiple Sclerosis Society, there are about 400,000 multiple sclerosis [MS] patients in the US, and this number is steadily growing: about 200 people are diagnosed each week. Currently, there is no effective cure for SCI or MS since adult humans do not fully regenerate their damaged neurons and axons. The inability for the body to regenerate and re-innervate target neuronal axons greatly limits therapy feasibility [1
]. The unique abilities of human embryonic stem cells [hESCs] - namely, their self-renewal and potency - hold great promise for regenerative medicine. For SCI and MS patients, the capacity of hESCs to differentiate into specific neuronal lineages through effective induction is highly encouraging. The unique appeal of hESC-based transplantation for SCI and MS is the possibility of those transplanted cells to repair damaged neuronal tissues. However, the harsh microenvironment and the lack of supportive substrates during transplantation result in a low survival rate of transplanted cells and diminish the feasibility of stem cell-related cell therapy [3
]. In regenerative medicine and tissue engineering, both naturally derived and synthetic materials have been extensively explored and provided their respective advantages [4
]. For instance, nanofibers incorporated with the pentapeptide epitope, isolucine-lysin-valine-alanine-valine, were constructed to induce rapid differentiation of cells into neurons. Biomaterials synthesized with synthetic or natural polymers were fabricated to facilitate the complex tissue formations [4
]. Generally, due to their inherent properties of biological recognition, extracted natural proteins present better cell-triggered proteolysis degradation and biocompatibility; synthetic materials provide more flexible material properties with specific designs. In this study, we aimed to integrate natural silk fibroin protein with synthetic carbon nanotubes [CNTs] to construct scaffolds for neuronal developments.
CNTs are a conductive biomaterial with sizes comparable to extracellular matrix molecules such as collagens and laminins, which have been reported to favor neuronal growth [10
]. In addition, due to their excellent mechanical strength and flexibility, CNTs can contribute to the structural integrity of scaffolds. Substrates prepared with CNTs have been demonstrated to be biocompatible and can support neuronal growth and differentiation [10
]. It has also been proposed that neurons grown on a CNT meshwork displayed better signal transmission, possibly due to tight contacts between the CNTs and neural membranes, favoring electrical shortcuts [12
]. All of the above characteristics make CNTs a promising biomaterial to repair damaged neuronal tissues.
Silks are natural polymers (protein) that have been widely used as biomaterials for many years. Fibroin protein is extracted from silk (Bombyx mori
), consisting of 90% of amino acids such as glycine, alanine, and serine. Various ratios of amino acids are distributed on the supramolecular structure of fibroin, consisting of a hydrophobic heavy chain (350 kD to 370 kD) and hydrophilic light chain (25 kD) [13
]. Due to its mechanically robust and flexible nature in thin film form, biocompatibility, and in vivo
reabsorbing and water-dissolvable properties, fibroin protein has been used as a building material for scaffolds for various tissue engineering applications and stem cell researches [8
]. For instance, fibroin scaffolds have been successfully applied to human mesenchymal stem cell differentiation, especially for ligament, bone, or cartilage tissue engineering [17
]. In addition, successful bio-integrated electronics has been developed based on dissolvable silk fibroin films [20
Unmodified CNTs tend to aggregate rather than disperse in aqueous solutions due to their hydrophobic nature. These heterogeneous aggregations of CNTs not only bring about difficulties in scaffold preparation, but also limit their applications. In an effort to resolve this problem, various surfactants were adapted to disaggregate and uniformly disperse CNTs in different solvents. However, the bio-toxicity of residuals still remains as one of the major concerns for cell scaffolding fabrication [21
]. Since fibroin consists of 75% of nonpolar hydrophobic amino acids [14
], it has been shown that the amphiphilic fibroin protein can effectively serve as a dispersant for CNTs [24
]. Here, we used fibroin extraction to disperse CNTs homogeneously to build silk-CNT composite scaffolds. This study aims to combine the unique advantages of these two biocompatible materials to build silk-CNT scaffolds in order to acquire sufficient neuronal differentiation efficiency from hESCs for effective neuronal cell transplantation.