Apart from providing mechanical robustness to the blood vessels by secreting the structural components, SMCs also contribute to tissue remodeling by secreting ECM degrading enzymes- MMP-2 (matrix metalloproteinase-2) and MMP-9. Considering this, we explored the effect of surface topography and hydrophilicity on matrix remodeling capability of SMCs. Our gelatin zymography results suggested that cellular alignment on patterned silk surfaces boost the secretion of MMP-2 and MMP-9 irrespective of cell surface hydrophilicity. Very bright and sharp gel bands were spotted for MMP-2 (at 62 kDa) as compared with MMP-9 (92 kDa) (Figure 2.13A-i). On quantifying the relative band density for MMP-9, TCP cultured cells showed minimal secretion followed by flat films (3 times higher) and patterned films (8-9 times higher) (Figure 2.13A-ii).
Similarly, SMCs cultured on TCP showed minimum MMP-2 production and AAP with maximum production of MMP-2 (3 times higher) (Figure 2.13A-iii).
Blood vessel walls are always exposed to continuous contraction and relaxation; hence the most important aspect for SMC functionality is their contractile behavior. In order to speculate about the impact of different surface topography and hydrophilicity on SMCs contractile behavior in 3D culture conditions, we encapsulated SMCs in collagen hydrogels post 4 days of 2D culture on silk film surfaces (Figure 2.13B-i). Gel contraction was quantified in terms of its size after 24 h culture. Maximum contraction was observed for BMP and AAP groups (~60%); which was significantly higher than flat film surfaces (~30%) and TCP (~10%) (p<0.01) (Figure 2.13B-ii).
SMC contraction was solely found to be affected by cellular patterning and no effect was observed for variable surface hydrophilicity.
traits. By using proteins from different variety of silk worms (either mulberry or non-mulberry), we fabricated surfaces having different properties without any chemical modification. These inherent physico-chemical properties of BM and AA silk films owing to the compositional difference of the silk fibroins, provided surfaces with variable roughness, wettability, chemistry (presence of RGD motif on AA film) and stiffness (BMF and AAF). Moreover, we also engineered the silk surfaces by printing a microgroove pattern to induce unidirectional alignment of porcine primary vascular cells.
Several studies have recently reported the unidirectional alignment of SMCs [79, 213, 221, 251-253] and ECs [214, 220, 254-257]. The rationale behind using such specifically designed topographical surfaces is to improve the cell functionality. SMCs are capable of adopting two contrasting phenotypes: contractile and synthetic. Cell behavior in both of these phenotypes differs drastically. Under healthy physiological conditions, SMCs remain in the contractile phenotype.
However, a phenotype switch (towards synthetic) leads to development of multiple pathological conditions including atherosclerosis, intimal hyperplasia and restenosis after angioplasty.
Unidirectional alignment of SMCs is primarily aimed to induce contractile phenotype in order to prevent the diseased condition [258]. In blood vessels, ECs’ monolayer in the lumen works as barrier between blood and underlying tissue. It also acts as natural anticoagulant surface that allows uninterrupted channeling of blood. Because of direct contact with bloodstream, ECs are regularly exposed to fluid shear stress. It induces their alignment along the direction of blood flow and ultimately help them maintaining their function [220, 256].
In order to assess the cumulative effect of surface physico-chemical properties and unidirectional cell alignment, we used flat and micro grooved silk films from both silk varieties.
Surface roughness of AA films was superior to BM counterparts as depicted by AFM analysis.
The higher surface roughness might be attributed to distinct presence of inherent amino acid stretches and their arrangement [126, 236]. In contrast, for patterned films, no significant difference was observed between two silk types; possibly due to dominance of microstructural surface features over nanostructures. By convention, any surface with a water contact angle less than 90 degrees is considered hydrophilic. BMF and AAF films showed different degrees of hydrophilicity (water contact angle 69.92 ± 1.75 and 46.76 ± 1.39 degrees respectively). AAF surfaces were more hydrophilic than BMF surfaces. One plausible justification for AA surfaces being more hydrophilic might be related with the presence of higher percentage of Ala amino acid
residues, which is more hydrophobic than Gly. When in aqueous solution, the non-polar amino acids tend to form hydrophobic core, leaving polar amino acid residues in contact with outer environment, hence more hydrophilic surface. This hypothesis was further substantiated from our FTIR deconvolution data, wherein we observed higher β-sheet content in AA groups in comparison to BM groups. The β-sheet rich crystalline region in silk fibroin forms the intact hydrophobic core. The predominantly higher β-sheet content noticed in AAF contributes for more polar groups exposed outwards, which favored the relatively hydrophilic surface of AAF. We further analyzed the contact angle for patterned silk films. For more precise measurement, we recorded the contact angle from both directions; i.e. parallel and perpendicular to the microgrooves. A similar hydrophilicity trend was recorded for patterned silk films with no significant difference between the contact angles from two different directions. Silk films’
hydrophilicity range is also relevant for this study owing to better EC adhesion on surfaces with a contact angle 40-45 degrees as compared to surfaces with a contact angle of 60-65 degrees’
surfaces [259, 260]. Although these prior studies report the cell adhesion percentage on surfaces with different wettability, the functional behavior of ECs was not investigated. On further analysis of mechanical properties of silk films, both film types showed significant difference. AA films were mechanically stiffer and showed higher tensile modulus. Load-displacement curves also suggested significantly higher elongation of AA films at the breaking point. The higher tensile strength and elongation of AA silk is because of presence of distinct polyalanine stretches in its native structure; conferring formation of higher amounts of anti-parallel β-sheet structures [238].
We further evaluated the biological reaction of vascular cells in response to variable cell- substrate interface features of silk films. We first looked into the proliferation of vascular cells.
Surface pattern induced ECs’ alignment is reported to enhance the cellular proliferation [261, 262]
but in the present study, irrespective of surface patterning, ECs cultured on AA films showed significantly higher proliferation than BM films in a 6 days experiment (p<0.01). As mentioned earlier, interactions at cell-substrate interface determine the cell behavior; therefore, superior proliferation of ECs on AA films might be attributed to either surface wettability (allowing better cell adhesion) [259, 260], presence of inherent RGD cell binding motifs on the surface of AA films (improving cell adhesion and proliferation) [126, 263] or substrate stiffness (stiffer substrates are reported to support better ECs proliferation) [264]. As suggested by AFM data, AAF and AAP films’ surface roughness showed no significant difference, eliminating influence of roughness
factor on ECs’ proliferation. These findings suggest that favorable substrate chemistry, wettability and stiffness of AA films cumulatively dominate the influence of surface patterning and subsequent cell alignment on ECs’ proliferation. When compared between BMF and BMP films, surface patterning altered the ECs’ proliferation profile. ECs cultured on BMP films showed comparatively lesser proliferation. This contrasts with a few prior reports, where ECs’ alignment was found to improve cellular proliferation [261, 262]. The plausible reason for such cellular behavior might relate with the size of grooves (depth and peak distance) that might alter cell adhesion and proliferation. On the other hand, the proliferation profile of SMCs showed a contrasting behavior than ECs and surface pattern induced alignment was observed to be the dominating factor over other surface properties. In line with the previous reports, SMCs’ alignment suppressed the proliferation rate, one of the features observed during phenotype transition from synthetic (high proliferation index) to contractile (low proliferation index) [258]. The underlying mechanism of alignment-induced switch of SMCs’ contractile phenotype has not been explored in details but several studies suspect the possible role of integrin for signaling across the plasma membrane. The two downstream signaling molecules FAK (focal adhesion kinase) and ERK (extracellular signal regulated kinase) might be involved with proliferation alteration of SMCs cultured on flat and patterned surfaces [251].
The progression of cell cycle is directly co-related with cell proliferation [258, 265]. On that account we further investigated the cell cycle profile of vascular cells under the influence of various surface properties. In native blood vessels, SMCs remain in a non-proliferative quiescent state (G0 phase) and maintain a contractile phenotype [266]. Hence, we evaluated the percentage cell population in G0 phase only. SMCs cultured on patterned silk surfaces (BMP and AAP) showed increment in quiescent phase (G0 phase) cell population when compared with flat surface counterparts (p<0.01). Based on these results, it may be inferred that unidirectional alignment of SMCs induce the non-proliferative (contractile) phenotype via induction of quiescent phase cells.
On the other hand, no significant difference was observed for G0 phase cell population for ECs in response to variable cell-substrate interfacial properties. However, in line with proliferation data, a higher G1 phase cell population was recorded for ECs cultured on AA films, irrespective of their surface patterning. It suggests that on AA films, more number of cells are entering in cell cycle leading to higher proliferation. Important to note here is that under in vivo conditions, ECs are exposed to a very dynamic microenvironment where they are attached as a monolayer on basement
membrane (topographical factor) and remain in direct contact with blood flow (dynamic mechanical loading) [267]. Both of these factors synergistically modulate the ECs’ cell cycle progression. It is evidenced by a previous report where three-fold reduction (compared to randomly aligned cells) in Ki67 expression (a proliferation associated protein expressed during cell cycle phases but absent from G0 phase) was observed for endothelial cells cultured on aligned nanofibers and exposed to orthogonal shear stress [268]. Such observations clearly indicate that modulation of cell-substrate interface might not be enough to recapitulate the in-vivo like microenvironment rather fluid shear stress is equally important.
Functional aspect of vascular cells is further assessed by investigating the expression of functional genes and proteins. A healthy monolayer of ECs is needed to maintain the vascular homeostasis by releasing nitric oxide (NO), which regulates vasodilation and suppress platelet activation and aggregation [269]. The NO molecule is synthesized by nitric oxide synthase (eNOS) enzyme. On analyzing the eNOS expression by ECs cultured on different silk films, we observed that BMF group showed the maximum expression while the patterned surfaces were found to suppress the eNOS expression. In order to further verify at the protein level, we quantified the production of NO by ECs. TCP cultured ECs produced maximum NO, but in order to normalize these values we calculated fold increase in NO production from Day 1 to Day 4. In agreement with the gene expression data, BMF group observed maximum fold increase (p<0.01). This is in contrast with previous reports where shear stress was observed to induce eNOS expression and NO production [254]. In a previous report it was observed that substrate compliance induced modulation of ECs’ NO production is mediated by fluid shear stress [270]. Surface microgroove induced alignment of ECs has also shown to influence the production of NO. As suspected in a previous report, microgroove size also matters when it comes to alignment induced functional manipulation of ECs. Microgrooves measuring 5 μm suppressed the eNOS expression significantly as compared to higher dimensions [271]. In addition, eNOS expression remained unchanged on flat and microgrooved surfaces [271, 272]. In yet another report, researchers have shown the effect of ratio of groove to ridge width influencing the ECs’ cytokine and chemokine profile related with remodeling and inflammation [254]. It is obvious from the prior studies that microgroove dimension also affects ECs’ functionality crucially. The suppressed expression of eNOS and downregulation of NO production from ECs cultured on patterned silk surfaces might be related with the microgroove width of patterned silk films used in this study. However, an in
depth investigation on effect of microgroove dimensions on silk films is needed to ascertain any such possibility. Angiopoietins are another class of proteins that regulate the vascular homeostasis.
Angiopoietin-1 (ANGPT1) stabilizes the vascular endothelium by activating Tie2 receptor whereas Angiopoietin-2 (ANGPT2) acts as an antagonist for the former protein [273]. Considering the importance of ANGPT1 for the maintenance of healthy vascular endothelium, we further enquired if its expression is affected by surface physico-chemical cues of silk films. A drastic downregulation of ANGPT1 was observed on patterned silk films. As per our best understanding, there is no such report investigating the effect of biophysical factors on ANGPT1 expression. A detailed molecular analysis is needed to justify these observations. Such paradoxical behavior might be related to the fact that ECs’ alignment and other substrate properties (roughness, wettability and chemistry) are not the only determining factors of cell functionality; dynamic microenvironment created by blood flow shear stress is also of crucial importance.
SMCs present in tunica media primarily hold the mechanistic responsibility by maintaining their contractile phenotype and producing extracellular matrix (ECM) proteins – collagen and elastin. Therefore, we further aimed to investigate the expression of contractile phenotype related genes and collagen synthesis in response to variable cell-substrate interfacial properties.
Expression of two contractile genes is studied – αSMA and SM-MHC. Cellular alignment on patterned surfaces (BMP and AAP) upregulated the expression of contractile genes, which is in agreement with previous reports [215]. However, BMF group (having a comparatively less hydrophilic surface, lesser surface roughness and without any presence of RGD cell binding motifs) also exhibited the enhanced expression of both of these genes. Such behavior of SMCs might be related to the cell clustering and self-assembly of cells on BMF films. Owing to the inferior hydrophilicity of BMF films, after reaching an optimal cell density, SMCs favored more cell-cell interaction compared to cell-substrate interaction and started forming cell clusters mimicking 3D micro tissues that might be one of the plausible justifications for improved contractile gene expression on BMF films [274-276]. A similar trend is observed with the collagen quantification analysis. Including patterned silk films (BMP and AAP); SMCs cultured on BMF films showed enhanced collagen production. Increased collagen production from SMCs expressing upregulation of contractile genes is in agreement with a previous report [277]. The underlying mechanism for such cellular behavior relies on increased gap junction communication mediated by Wnt3a and subsequent modulation of Wnt signaling [277]. Our findings suggest that
although surface patterning induced cellular alignment is sufficient to induce SMCs’ contractile phenotype and collagen production but other surface properties cumulatively might also lead to a similar cellular response.
The functional aspect of SMCs is further elaborated and investigated in terms of their matrix remodeling capability (production of matrix metalloproteinase – MMP-2 and MMP-9) and physical contraction ability using collagen gels. For any tissue engineered vascular graft (TEVG), matrix remodeling is very crucial. In this regard, SMCs are known to produce MMP-2 and MMP- 9 in blood vessels undergoing remodeling post injury [278]. Increased expression of MMP-2 and MMP-9 is observed for BMP and AAP groups independent of other surface properties. Cell alignment induced upregulation of MMPs might relate to the SMCs’ contractile phenotype [277].
We further looked into the contraction ability of SMCs cultured on different silk films by encapsulating them in collagen hydrogel [246]. SMCs cultured on patterned surfaces showed better contraction capability. However, the superior expression of contractile genes for BMF group is not reflected here. It indicates that SMCs’ alignment and cell-cell gap junction mediated communication is important for their functional contractile behavior. These outcomes indicate that predominant unidirectional alignment of SMCs lead to induction of their contractile phenotype, allows more collagen and MMP production and shows better contraction capability when compared with cells cultured on flat silk films growing in random orientation. Another key observation is that SMCs’ contractile phenotype and ECM production might also be altered depending on synergistic effect of surface wettability, surface chemistry and substrate stiffness, as observed in case of BMF group.
Our findings in this work suggest that depending on cell type, surface properties of silk films synergistically determine cellular functionality. Most of the prior research is focused on extrapolating the effect of a single factor at the molecular level in a highly controlled microenvironment, however unveiling the effect of complex dynamic environment is of much clinical relevance. Although this study is limited to few standard substrate properties of silk films without any consideration of dynamic mechanical microenvironment (due to blood flow shear stress) but a detailed molecular and functional analysis by mimicking the complex native-like microenvironment is much needed to understand the vascular cell dynamics under various physio pathological conditions.