In the recent past, research on non-mulberry silk fibroin based biomaterials has gained momentum due to superior mechanical and biological properties and various other advantages that they offer. Previously, Li et al (2008) reported the fabrication of porous 3D scaffold from regenerated A. pernyi fibroin and Fang et al (2009) reported the preparation of braided fibrous scaffold from A. pernyi cocoon derived fibroin. While, Mandal and Kundu (2008a, 2008b) reported the fabrication of 2D films and porous 3D scaffolds from A. mylitta gland derived fibroin. However, there are no any reports on the development of non-mulberry silk fibroin based porous 3D scaffolds with fibrous and nonwoven biomimetic architecture. Such scaffolds were previously reported from B. mori fibroin, for example, Unger et al (2004a, 2004b) reported the preparation of nonwoven scaffold and its positive interactions with as many as 11 human cells and subsequent endothelialization of the scaffold. Later, Dal Pra et al (2005, 2006) reported the de novo engineering of reticular connective tissue and preparation of dermo-epidermal equivalents using B. mori fibroin based nonwoven scaffold.
Keeping in view of the advantages offered by non-mulbery fibroin over its counterpart and the biomimetic features offered by fibrous nonwoven scaffolds, in the current study, the first of its kind, we describe the fabrication of A. assama silk fibroin based porous 3D scaffolds with fibrous and nonwoven biomimetic architecture. The fabrication process is simple, efficient and reproducible, and does not involve any toxic cross linking agents. Initially, cocoons of A. assama were subjected to degumming process to yield pure fibroin fibers. They were then suspended in lithium bromide / formic acid solution, homogenized and subsequently cast in a mold. Figure 2.1 shows the physical appearance of muga silkworm cocoon, silk fibroin fibers obtained after degumming process and the nonwoven scaffold obtained after fabrication process. The SEM pictures of raw and degummed fibers clearly demonstrated the successful removal of sericin, calcium oxalate salts and other external contaminants (Figure 2.2). Also, SEM examination suggested the successful fabrication of a highly interconnected, homogenously porous 3D nonwoven scaffold (Figure 2.2). The scaffold was found to have good physical properties viz. a pore size of around 150 µm, porosity of about 90%
and water uptake capacity of nearly 90% which conveniently favor cell infiltration and exchange of gases/ nutrients/ waste products.
Striking changes in the FT-IR spectra of raw and degummed fibroin fibers clearly depicted the chemical / structural changes due to initial processing and degumming of raw fibers which removed the external sericin layer. However, there were no significant changes in the spectra of degummed silk fiber and scaffold (Figure 2.3). Both displayed peaks at around 1600-1690 cm-1, 1480-1575 cm-1, 1229-1301 cm-1, 625-767 cm-1 and 640-800 cm-1 which are distinctive of amide I-V respectively (Dal Pra et al., 2005;
Wilson et al., 2000). Besides, the degummed fiber and the scaffold exhibited an intense signal at 965 cm-1
Thermal property analysis of A. assama fibroin based scaffold revealed the onset of thermal degradation and endothermic decomposition peak at around 330
which is characteristic of β(3a) type conformation of non-mulberry fibroin (Keten et al, 2010). The FT-IR spectra of scaffold represented a characteristic and stable conformation of silk fibroin and were in good agreement with the distinctive spectrum displayed by A. assama silk fibroin (Wilson et al., 2000; Hazarika et al., 1998).
oC and 375 oC respectively (Figure 2.4). While from the literature, the onset of thermal degradation and endothermic decomposition peak of B. mori fibroin based scaffold were found to be at around 289 oC and 291 o
Since it is the blood that first comes into contact with the scaffold surface, the blood compatibility of a scaffold is very crucial in determining the fate of an implant (Sharma, 2001). We performed in vitro thrombogenecity, hemolysis, platelet and leukocyte count, protein adsorption and platelet adhesion experiments to evaluate the blood compatibility of the scaffold. The results of the thrombogenecity and hemolysis assay were very encouraging with a negligible variation in the thrombus formation and red blood cell lysis among heparinized blood incubated with scaffold and without scaffold. We did not find any significant difference in the number of platelets and leukocytes (both total and differential) of the heparinized blood sample incubated with scaffold in comparison to control (heparinized blood alone, without scaffold). Protein adsorption and platelet adhesion properties of a biomaterial, which trigger coagulation, are two critical parameters to evaluate its blood compatibility. Our studies show that there C respectively (Dal Pra et al., 2006; Taddei et al. 2007).
Similarly, tensile strength studies of A. assama fibroin based scaffold revealed an yield point at approximately 4% elongation with a yield plateau of up to 7 % in comparison to 5 % elongation of B. mori fibroin based scaffold (Dal Pra et al., 2006). Hence, the results suggest that the fibroin from A. assama has superior thermal stability and mechanical strength over B. mori fibroin.
was approximately 10.57 μg/cm2
TERM strategies generally involve, building of a cell-scaffold construct in vitro, followed by implantation in vivo. Therefore, it is desired that scaffold should facilitate attachment, migration and growth of cells resulting in the formation of a functional tissue.
In this study, we found that the scaffold produced using A. assama fibroin has good cyto- compatibility with all the four human cell lines tested (A 549, KB, HepG2 and HeLa).
The SEM examination of cell-scaffold construct revealed the successful attachment and spreading of cells on and within the scaffold (Figure 2.6 and 2.7). The cell viability and growth at different time intervals was assessed by MTT assay - a simple, sensitive, rapid, reliable and efficient assay recognized to estimate the cell viability within the scaffold (Pabbruwe et al., 2005; Khattak et al., 2006). The results reveal that the cell proliferation was normal in 2D cultures and reached confluence after a week of incubation, but thereafter, there was no increase in the cell densities due to contact inhibition; and prolonged arrest of cell proliferation might have probably lead to cell death. However, the cell growth and proliferation was exponential on the scaffold since the cells have the freedom to grow in three dimensions (Figure 2.8). Additionally, a Picro-Sirius red assay was performed for in situ quantification of collagen deposited by growing cells in cell- scaffold constructs. In 3D cultures, there was an exponential increase in the collagen content with time due to the increased growth and spreading of cells on scaffold. But in case of 2D cultures, there was an initial increase, which decreased with time due to reduction in cell viability (Figure 2.9). Hence, the results of cell attachment and spreading, cell migration, viability and growth, and collagen content, collectively suggest a very good cellular compatibility of the scaffold. These results also hold promise and encouragement for progressing towards generation of functional tissues with this novel material.
of protein adsorption (Figure 2.5), and about 2.9 % of platelet adhesion over the scaffold. Although the results of thrombogenecity, hemolysis, TLC, DLC and platelet count suggest that A. assama fibroin based nonwoven scaffold is safe for blood, the results of protein adsorption and platelet adhesion studies indicate that the scaffold needs further modification to make it more suitable for use in TERM applications.
A critical factor for successful TERM is neo-vascularization that provides continuous support of oxygen, nutrients and biochemical cues to the cells in the 3D matrix. Therefore, the intrinsic pot ential of a biomaterial to promote angiogenesis is
crucial for good performance as a scaffold. Generally, the angiogenesis potential of a scaffold is assessed by its compatibility with endothelial cells in vitro, which is time consuming, expensive and cannot be extrapolated to in vivo results. We followed a simple, rapid, reproducible and efficient ex ovo CAM assay to evaluate the angiogenesis properties of the scaffold. The CAM assay is considered to be a reliable method, as results are comparable with in vivo animal model studies (Valdes et al., 2002; Azzarello et al., 2007; Klueh et al., 2003; Ribatti et al., 1996). In the present study, the results revealed the ability of the A. assama fibroin based nonwoven scaffold to promote angiogenesis (Figure 2.10). The presence of blood vessels in and around the scaffold suggests that the biological properties of the scaffold were congenial to support neo- vascularization. To the best of our knowledge, this is the first report suggesting the angiogenesis potential of fibroin based scaffolds based on CAM assay.
Nitric oxide (NO) is generated by many cell types in a variety of tissues, utilized throughout the animal kingdom as a signaling or toxic molecule and involved in many physiological and pathological processes (Wink and Mitchell, 1998). In mammals, it acts as a vascular relaxing agent, a neurotransmitter, an inhibitor of platelet aggregation and also has a role in inflammation and immunity (Wink and Mitchell, 1998). However, there has been substantial evidence about the possible role of NO as a critical mediator of angiogenesis by enhancing endothelial cell survival, proliferation and migration and thus is said to be pro-angiogenic (Cooke, 2003). In the current study, a plateau in NO content of 3D cultures after 1-2 weeks suggests a saturation point, whereas, a drastic drop in NO content of 2D cultures was due to cell senescence after 1 week (Figure 2.11). The present study is first of its kind to the report presence of NO in fibroin based 3D cell cultures.
From an angiogenesis perspective, it is suggested that the NO found in cellular compatibility experiments would supplement the ability of the scaffold to promote angiogenesis and tissue homeostasis.
Biodegradable materials are preferred candidates for use as scaffolds in TERM applications since upon degradation they create void space which will be occupied by the growing cells and natural ECM material deposited by colonizing cells. Though the US Pharmacopeia defined the fibroin as non-degradable, many in vitro and in vivo studies suggest the biodegradability of fibroin (Wang et al., 2008; Taddei et al. 2006; Horan et al., 2005; Arai et al., 2004). In the present study, we evaluated the in vitro biodegradability of A. assama fibroin based nonwoven scaffold, whereby the scaffold was
found to lose weight with time upon treatment with trypsin (Figure 2.12a). The scaffold degradation was comparatively more than that of pure fibers, which may be due to changes caused to fibroin upo n treatment with formic acid / lithium bromide during the fabrication process (Dal Pra et al., 2006). Additionally, the results of SEM examination of trypsin digested scaffold revealed decrease in fiber diameter, increase in fiber roughness and loss of micro-architecture and thus suggest the degradation of scaffold (Figure 2.12b). Hence, being proteinaceous in nature, the fibroin from A. assama would be amenable for proteolytic degradation by cellular proteases when implanted in vivo and the degradation products would be metabolized in the body; this is an additional advantage of preferring it as a biomaterial for use as scaffold in TERM applications.