Cell microenvironment influences their behavior and functionality. It may be various soluble factors present in the close vicinity or the surrounding matrix that triggers multiple signaling pathways [208, 209]. All living tissues consist of a diverse population of cells embedded in specialized extracellular matrix (ECM) in a very intricate manner. This ECM provides the structural framework and determines cell polarity and arrangement. Biochemical composition and topographical organization of ECM are two key determining factors regulating cellular behavior and functionality. Hence, a lot of research has been focused on modulating cell-surface interactions by tuning surface topography [209]. One of the arguments about studying the co-relation between cell distribution pattern and their function is discrepancy among 2D vs 3D microenvironments.
However, the majority of studies have implemented 2D surfaces to study the fundamental principles of cell biology owing to its simplicity as compared to complex 3D microenvironments.
Most recently, structural control of surface topography using micro/nano technology has paved the way to mimic the 3D complex microenvironments while preserving the easiness of working with 2D surfaces [210, 211].
At cell substrate interface, several factors (including topography, wettability, roughness, chemistry and mechanical properties of the substrate) play a crucial role in determining cell behavior. The majority of human adherent cell types are spatially arranged innately in a unique manner that helps them maintain their function and overall tissue dynamics [212, 213]. Of note, vascular cells, typically endothelial cells (ECs) in tunica interna and smooth muscle cells (SMCs) in tunica media of blood vessels follow a unique topographical arrangement. ECs are aligned along the direction of blood flow and maintain vascular homeostasis [214], whereas SMCs are aligned radially contributing to the mechanical robustness and vasoactive responsiveness of blood vessels [215]. These design parameters become important for tissue engineering of vascular grafts where the main focus for success is to engineer the scaffold wall surface in such a way so that it recruits the progenitor vascular cells and provides a congenial microenvironment for their growth and functionality. Recently, several studies have shown the co-relation between vascular cell morphology/organization and their functionality in vitro [216] and in vivo [217, 218].
A functional and continuous monolayer of ECs maintains un-interrupted blood flow in the blood vessels. Any damage in ECs’ monolayer may lead to various pathological conditions like thrombosis, hyperplasia, stenosis and inflammation in the vessel wall, which alters the vascular
tone [219]. Besides that, cell morphology, shape, alignment and polarity are also other determining factors that regulate ECs’ functionality [220]. Hence, mimicking a native-like physiological environment becomes a point of interest. ECs present in blood vessels’ lumen lining are exposed to shear stress and cyclic circumferential strain due to continuous blood flow. Under these circumstances, these cells tend to adopt unidirectional alignment along the direction of blood flow.
The adjacent multiple layers of SMCs in the vascular wall (tunica media) align themselves radially (perpendicular to the direction of blood flow) [215]. Recently, several researchers have reported the positive effect of unidirectional alignment on vascular cell functionality. Majority of them focused on either engineering the cell culture surfaces (by printing grooved pattern) or by exposing the cultured cells to the flow shear stress [79, 213, 215, 221].
Surface wettability is another important parameter that influences cell morphology affecting their functionality [213]. It is well proven fact that cells prefer to spread better on moderate hydrophilic surfaces [222, 223]. Limited cell adhesion and spreading was reported on highly hydrophilic surfaces because such surfaces do not allow long-term retention of cell adhesion molecules [224]. The biochemical composition of the cell culture matrix or surface chemistry also regulates cell morphology and spreading [225, 226]. For instance, surface bio functionalization with cell binding RGD peptide enhances the cell attachment and proliferation via integrin (cell adhesion receptor) mediated process [227]. Apart from surface patterning (guiding cellular alignment), surface roughness also determines the functionality of vascular cells. Cellular response to surface roughness at micro scale is a bit controversial owing to the inconsistency noticed throughout several reports. For instance, endothelial cells showed superior growth on rougher PU- PEG (Polyurethane-polyethylene glycol) surface irrespective of presence of surface cell adhesion peptides (RGD) [228]. On the contrary, smoother solvent casted poly (L-lactic acid) (PLLA) surfaces supported better ECs’ functionality [229]. Nanostructured titanium surfaces favorably supported the adhesion of SMCs and ECs [230]. Such paradoxical behavior is generally attributed to undefined attributes of surface roughness and needs more specific definition that considers different nanostructured shapes, spacing between irregularities and sharpness of the peaks [224].
Among all cell-substrate interface properties, substrate rigidity is considered least important but recently it is identified as a very crucial factor in determining cells’ fate. The phenomenon of change in cell’s chemical microenvironment in response to mechanical signals is called
‘mechanotransduction’ [231]. Substrates with same chemical composition and different rigidity
have shown to modulate SMCs adhesion [224]. Studies have shown that substrates with low rigidity does not allow cellular spreading whereas cells spread better on stiffer substrates (depending on cell type) [232]. One of the plausible explanation of such cellular behavior might be related with imbalance between cell traction forces and corresponding extracellular matrix (ECM) response, a crucial parameter for assembly of cell-matrix adhesion complexes and cell spreading [224]. Several studies have reported the effect of individual factor on cellular functionality; however, in a native in vivo microenvironment, cells are exposed to multiple factors simultaneously. Hence, investigating the combinatorial effect of more than one factor becomes of great importance.
In the present study, we intend to explore the functionality of vascular cells (ECs and SMCs) when cultured on mulberry Bombyx mori-BM and non-mulberry Antheraea assama-AA silk films. Our selection of using different silk relied on following crucial parameters: (1) both proteins are highly biocompatible and have been explored for various tissue engineering applications [233, 234]; (2) their ability to be fabricated into myriad structural designs with nano level accuracy, allowing surface engineering to print any desirable pattern [14], and (3) structural variability in their native amino acid sequence and arrangement of secondary protein structures [235].The molecular composition of these silk proteins is quite different. Heavy chain of both silk proteins consists majorly of Ala (44.5% vs 28.8% for AA vs BM) and Gly (30.1% vs 43.7% for AA vs BM), nonpolar amino acids [126]. The alanine methyl groups are usually exposed to external microenvironment because of their arrangement outside of protein backbone [236].
Owing to presence of different percentage of hydrophobic amino acid residues in BM and AA silk proteins, the surface wettability of films made of these proteins is expected to be dissimilar. The crystalline core of AA is made-up of poly-alanine motifs, which makes 37.4% of total Ala out of 44.5%. In contrast, BM protein have multiple repeats of AGSGAG comprising 55% of total crystalline core [237]. Differential amino acid composition and arrangement leads to formation of unique secondary structures. For instance, the poly-alanine motifs present in AA silk prefer to form anti-parallel β-sheets via non-covalent inter-molecular interactions, which confers tensile strength [238]. Besides, it also provides more hydrophobicity, forming stronger β-sheets as compared with AGSGAG repeats of BM silk. In general, alanine motifs provide tensile strength to silk fibers whereas glycine rich sequences are responsible for fiber elasticity. It has also been reported that even the short polyalanine stretches (A)6 are able to confer remarkable tensile strength to AA
protein by forming strong β-nanocrystal, which is much higher than all other wild silk varieties [239]. Additionally, AA silk has intrinsic presence of RGD cell binding motif in its native amino acid structure making its surface chemically different from BM silk [126]. Owing to these aforementioned characteristics, surfaces of silk films obtained from different silks provide variable surface characteristics. Therefore, in the current endeavour, we cultured vascular cells on BM and AA silk films and looked into their functional behaviour in response to combinatorial effect of multiple physico-chemical factors acting at cell-substrate interface.
2.2 Materials and methods