An in-silico Investigation Into the Role of Strain and Structure on Vascular Smooth Muscle Cell Growth.

The orientation of vascular cells can greatly influence the in vivo mechanical properties and functionality of soft vascular tissues. How cell orientation mediates the growth response of cells is of critical importance in understanding the response of soft tissues to mechanical stimuli or injury. To date, considerable evidence has shown that cells align with structural cues such as collagen fibers. However, in the presence of uniaxial cyclic strain on unstructured substrates, cells generally align themselves perpendicularly to the mechanical stimulus, such as strain, a phenomenon known as "strain avoidance." The cellular response to this interplay between structural cues and a mechanical stimulus is poorly understood. A recent in vitro experimental study in our lab has investigated both the individual and collective response of rat aortic smooth muscle cells (RASMC) to structural (collagenous aligned constructs) and mechanical (cyclic strain) cues. In this study, a 2D agent-based model (ABM) is developed to simulate the collective response of RASMC to varying amplitudes of cyclic strain (0-10%, 2-8%, 4-6%) when seeded on unstructured (PDMS) and structured (decellularized collagenous tissue) constructs. An ABM is presented that is fit to the experimental outcomes in terms of cellular alignment and cell growth on PDMS substrates, under cyclic strain amplitudes of (4-6%, 2-8%, 0-10%) at 24 and 72 h timepoints. Furthermore, the ABM can predict RASMC alignment and change in cell number on a structured construct at a cyclic strain amplitude of 0-10% after 10 days. The ABM suggests that strain avoidance behavior observed in cells is dominated by selective cell proliferation and apoptosis at these early time points, as opposed to cell re-orientation, i.e., cells perpendicular to the strain increase their rate of proliferation, whilst the rate of apoptosis simultaneously increases in cells parallel to the strain direction. The development of in-silico modeling platforms, such as that presented here, allow for further understanding of the response of cells to changes in their mechanical environment. Such models offer an efficient and robust means to design and optimize the compliance and topological structure of implantable devices and could be used to aid the design of next-generation vascular grafts and stents.

A simple method to align cells on 3D hydrogels using 3D printed molds.

Vascular smooth muscle cells align circumferentially around the vessel lumen, which allows these cells to control vascular tone by contracting and relaxing. It is essential that this circumferential alignment is recapitulated in tissue engineered blood vessels. While many methods have been reported to align cells on 2D polymeric substrates, few techniques enable cell alignment on a 3D physiologically relevant hydrogel substrate. We hypothesized that the ridges inherent to the sides of fused deposition modeling 3D printed molds could be used to topographically pattern both stiff and soft substrates and thereby align cells on flat and curved surfaces. Flat and curved molds with 150, 250, and 350 µm ridges were 3D printed and used to topographically pattern polydimethylsiloxane and gelatin-methacryloyl. The ridges transferred to both substrates with less than 10% change in ridge size. Vascular smooth muscle cells were then seeded on each substrate, and nuclear and actin alignment were quantified. Cells were highly aligned with the molded ridges to a similar extent on both the stiffer polydimethylsiloxane and the softer gelatin-methacryloyl substrates. These data confirm that fused deposition modeling 3D printed molds are a rapid, cost-effective way to topographically pattern stiff and soft substrates in varied 3D shapes. This method will enable investigators to align cells on 3D polymeric and hydrogel structures for tissue engineering and other applications.

Resident multipotent vascular stem cells exhibit amplitude dependent strain avoidance similar to that of vascular smooth muscle cells.

Atherosclerosis is one of the leading causes of mortality worldwide, and presents as a narrowing or occlusion of the arterial lumen. Interventions to re-open the arterial lumen can result in re-occlusion through intimal hyperplasia. Historically only de-differentiated vascular smooth muscle cells were thought to contribute to intimal hyperplasia. However recent significant evidence suggests that resident medial multipotent vascular stem cells (MVSC) may also play a role. We therefore investigated the strain response of MVSC since these resident cells are also subjected to strain within their native environment. Accordingly, we applied uniaxial 1 Hz cyclic uniaxial tensile strain at three amplitudes around a mean strain of 5%, (4-6%, 2-8% and 0-10%) to either rat MVSC or rat VSMC before their strain response was evaluated. While both cell types strain avoid, the strain avoidant response was greater for MVSC after 24 h, while VSMC strain avoid to a greater degree after 72 h. Additionally, both cell types increase strain avoidance as strain amplitude is increased. Moreover, MVSC and VSMC both demonstrate a strain-induced decrease in cell number, an effect more pronounced for MVSC. These experiments demonstrate for the first time the mechano-sensitivity of MVSC that may influence intimal thickening, and emphasizes the importance of strain amplitude in controlling the response of vascular cells in tissue engineering applications.

Primary cilia are sensors of electrical field stimulation to induce osteogenesis of human adipose-derived stem cells.

In this study, we report for the first time that the primary cilium acts as a crucial sensor for electrical field stimulation (EFS)-enhanced osteogenic response in osteoprogenitor cells. In addition, primary cilia seem to functionally modulate effects of EFS-induced cellular calcium oscillations. Primary cilia are organelles that have recently been implicated to play a crucial sensor role for many mechanical and chemical stimuli on stem cells. Here, we investigate the role of primary cilia in EFS-enhanced osteogenic response of human adipose-derived stem cells (hASCs) by knocking down 2 primary cilia structural proteins, polycystin-1 and intraflagellar protein-88. Our results indicate that structurally integrated primary cilia are required for detection of electrical field signals in hASCs. Furthermore, by measuring changes of cytoplasmic calcium concentration in hASCs during EFS, our findings also suggest that primary cilia may potentially function as a crucial calcium-signaling nexus in hASCs during EFS.-Cai, S., Bodle, J. C., Mathieu, P. S., Amos, A., Hamouda, M., Bernacki, S., McCarty, G., Loboa, E. G. Primary cilia are sensors of electrical field stimulation to induce osteogenesis of human adipose-derived stem cells.

Primary cilium mechanotransduction of tensile strain in 3D culture: Finite element analyses of strain amplification caused by tensile strain applied to a primary cilium embedded in a collagen matrix.

Human adipose-derived stem cells (hASC) exhibit multilineage differentiation potential with lineage specification that is dictated by both the chemical and mechanical stimuli to which they are exposed. We have previously shown that 10% cyclic tensile strain increases hASC osteogenesis and cell-mediated calcium accretion. We have also recently shown that primary cilia are present on hASC and that chemically-induced lineage specification of hASC concurrently results in length and conformation changes of the primary cilia. Further, we have observed cilia length changes in hASC cultured within a collagen I gel in response to 10% cyclic tensile strain. We therefore hypothesize that primary cilia may play a key mechanotransduction role for hASC exposed to tensile strain. The goal of this study was to use finite element analysis (FEA) to determine strains occurring within the ciliary membrane in response to 10% tensile strain applied parallel, or perpendicular, to cilia orientation. To elucidate the mechanical environment experienced by the cilium, several lengths were modeled and evaluated based on cilia lengths measured on hASC grown under varied culture conditions. Principal tensile strains in both hASC and ciliary membranes were calculated using FEA, and the magnitude and location of maximum principal tensile strain determined. We found that maximum principal tensile strain was concentrated at the base of the cilium. In the linear elastic model, applying strain perpendicular to the cilium resulted in maximum strains within the ciliary membrane from 150% to 200%, while applying strain parallel to the cilium resulted in much higher strains, approximately 400%. In the hyperelastic model, applying strain perpendicular to the cilium resulted in maximum strains within the ciliary membrane around 30%, while applying strain parallel to the cilium resulted in much higher strains ranging from 50% to 70%. Interestingly, FEA results indicated that primary cilium length was not directly related to ciliary membrane strain. Rather, it appears that cilium orientation may be more important than cilium length in determining sensitivity of hASC to tensile strain. This is the first study to model the effects of tensile strain on the primary cilium and provides newfound insight into the potential role of the primary cilium as a mechanosensor, particularly in tensile strain and potentially a multitude of other mechanical stimuli beyond fluid shear.

Cytoskeletal and focal adhesion influences on mesenchymal stem cell shape, mechanical properties, and differentiation down osteogenic, adipogenic, and chondrogenic pathways.

Mesenchymal stem cells (MSCs) hold great potential for regenerative medicine and tissue-engineering applications. They have multipotent differentiation capabilities and have been shown to differentiate down various lineages, including osteoblasts, adipocytes, chondrocytes, myocytes, and possibly neurons. The majority of approaches to control the MSC fate have been via the use of chemical factors in the form of growth factors within the culture medium. More recently, it has been understood that mechanical forces play a significant role in regulating MSC fate. We and others have shown that mechanical stimuli can control MSC lineage specification. The cytoskeleton is known to play a large role in mechanotransduction, and a growing number of studies are showing that it can also contribute to MSC differentiation. This review analyzes the significant contribution of actin and integrin distribution, and the smaller role of microtubules, in regulating MSC fate. Osteogenic differentiation is more prevalent in MSCs with a stiff, spread actin cytoskeleton and greater numbers of focal adhesions. Both adipogenic differentiation and chondrogenic differentiation are encouraged when MSCs have a spherical morphology associated with a dispersed actin cytoskeleton with few focal adhesions. Different mechanical stimuli can be implemented to alter these cytoskeletal patterns and encourage MSC differentiation to the desired lineage.

A stretching device for high-resolution live-cell imaging.

Several custom-built and commercially available devices are available to investigate cellular responses to substrate strain. However, analysis of structural dynamics by microscopy in living cells during stretch is not readily feasible. We describe a novel stretch device optimized for high-resolution live-cell imaging. The unit assembles onto standard inverted microscopes and applies constant magnitude or cyclic stretch at physiological magnitudes to cultured cells on elastic membranes. Interchangeable modular indenters enable delivery of equibiaxial and uniaxial stretch profiles. Strain analysis performed by tracking fluorescent microspheres adhered onto the substrate demonstrated reproducible application of stretch profiles. In endothelial cells transiently expressing enhanced green fluorescent protein (EGFP)-vimentin and paxillin-DsRed2 and subjected to constant magnitude equibiaxial stretch, the two-dimensional strain tensor demonstrated efficient transmission through the extracellular matrix and focal adhesions. Decreased transmission to the intermediate filament network was measured, and a heterogeneous spatial distribution of maximum stretch magnitude revealed discrete sites of strain focusing. Spatial correlation of vimentin and paxillin displacement vectors provided an estimate of the extent of mechanical coupling between the structures. Interestingly, switching the spatial profile of substrate strain reveals that actin-mediated edge ruffling is not desensitized to repeated mechanostimulation. These initial observations show that the stretch device is compatible with live-cell microscopy and is a novel tool for measuring dynamic structural remodeling under mechanical strain.