Protein Micropatterning in 2.5D: An Approach to Investigate Cellular Responses in Multi-Cue Environments.

The extracellular microenvironment is an important regulator of cell functions. Numerous structural cues present in the cellular microenvironment, such as ligand distribution and substrate topography, have been shown to influence cell behavior. However, the roles of these cues are often studied individually using simplified, single-cue platforms that lack the complexity of the three-dimensional, multi-cue environment cells encounter in vivo. Developing ways to bridge this gap, while still allowing mechanistic investigation into the cellular response, represents a critical step to advance the field. Here, we present a new approach to address this need by combining optics-based protein patterning and lithography-based substrate microfabrication, which enables high-throughput investigation of complex cellular environments. Using a contactless and maskless UV-projection system, we created patterns of extracellular proteins (resembling contact-guidance cues) on a two-and-a-half-dimensional (2.5D) cell culture chip containing a library of well-defined microstructures (resembling topographical cues). As a first step, we optimized experimental parameters of the patterning protocol for the patterning of protein matrixes on planar and non-planar (2.5D cell culture chip) substrates and tested the technique with adherent cells (human bone marrow stromal cells). Next, we fine-tuned protein incubation conditions for two different vascular-derived human cell types (myofibroblasts and umbilical vein endothelial cells) and quantified the orientation response of these cells on the 2.5D, physiologically relevant multi-cue environments. On concave, patterned structures (curvatures between κ = 1/2500 and κ = 1/125 µm-1), both cell types predominantly oriented in the direction of the contact-guidance pattern. In contrast, for human myofibroblasts on micropatterned convex substrates with higher curvatures (κ ≥ 1/1000 µm-1), the majority of cells aligned along the longitudinal direction of the 2.5D features, indicating that these cells followed the structural cues from the substrate curvature instead. These findings exemplify the potential of this approach for systematic investigation of cellular responses to multiple microenvironmental cues.

Cellular Contact Guidance Emerges from Gap Avoidance.

In the presence of anisotropic biochemical or topographical patterns, cells tend to align in the direction of these cues-a widely reported phenomenon known as "contact guidance." To investigate the origins of contact guidance, here, we created substrates micropatterned with parallel lines of fibronectin with dimensions spanning multiple orders of magnitude. Quantitative morphometric analysis of our experimental data reveals two regimes of contact guidance governed by the length scale of the cues that cannot be explained by enforced alignment of focal adhesions. Adopting computational simulations of cell remodeling on inhomogeneous substrates based on a statistical mechanics framework for living cells, we show that contact guidance emerges from anisotropic cell shape fluctuation and "gap avoidance," i.e., the energetic penalty of cell adhesions on non-adhesive gaps. Our findings therefore point to general biophysical mechanisms underlying cellular contact guidance, without the necessity of invoking specific molecular pathways.

Functional peptide presentation on different hydrogen bonding biomaterials using supramolecular additives.

Supramolecular biomaterials based on hydrogen bonding units can be conveniently functionalized in a mix-and-match approach using supramolecular additives. The presentation of bioactive additives has been sparsely investigated in supramolecular-based elastomeric biomaterials. Here it was investigated how cell adhesive peptides are presented and affect the surface in supramolecular biomaterials based either on ureido-pyrimidinone (UPy) or bisurea (BU) moieties. Polycaprolactone modified with UPy or BU moieties served as the base material. RGD or cyclic (c)RGD were conjugated to complementary supramolecular motifs, and were mixed with the corresponding base materials as supramolecular additives. Biomaterial surface morphology changed upon bioactivation, resulting in the formation of random aggregates on UPy-based materials, and fibrous aggregates on BU-materials. Moreover, peptide type affected aggregation morphology, in which RGD led to larger cluster formation than cRGD. Increased cRGD concentrations led to reduced focal adhesion size and cell migration velocity, and increased focal adhesion numbers in both systems, yet most prominent on functionalized BU-biomaterials. In conclusion, both systems exhibited distinct peptide presenting properties, of which the BU-system most strongly affected cellular adhesive behavior on the biomaterial. This research provided deeper insights in the differences between supramolecular elastomeric platforms, and the level of peptide introduction for biomaterial applications.

Entropic Forces Drive Cellular Contact Guidance.

Contact guidance-the widely known phenomenon of cell alignment induced by anisotropic environmental features-is an essential step in the organization of adherent cells, but the mechanisms by which cells achieve this orientational ordering remain unclear. Here, we seeded myofibroblasts on substrates micropatterned with stripes of fibronectin and observed that contact guidance emerges at stripe widths much greater than the cell size. To understand the origins of this surprising observation, we combined morphometric analysis of cells and their subcellular components with a, to our knowledge, novel statistical framework for modeling nonthermal fluctuations of living cells. This modeling framework is shown to predict not only the trends but also the statistical variability of a wide range of biological observables, including cell (and nucleus) shapes, sizes, and orientations, as well as stress-fiber arrangements within the cells with remarkable fidelity with a single set of cell parameters. By comparing observations and theory, we identified two regimes of contact guidance: 1) guidance on stripe widths smaller than the cell size (w ≤ 160 µm), which is accompanied by biochemical changes within the cells, including increasing stress-fiber polarization and cell elongation; and 2) entropic guidance on larger stripe widths, which is governed by fluctuations in the cell morphology. Overall, our findings suggest an entropy-mediated mechanism for contact guidance associated with the tendency of cells to maximize their morphological entropy through shape fluctuations.

An automated quantitative analysis of cell, nucleus and focal adhesion morphology.

Adherent cells sense the physical properties of their environment via focal adhesions. Improved understanding of how cells sense and response to their physical surroundings is aided by quantitative evaluation of focal adhesion size, number, orientation, and distribution in conjunction with the morphology of single cells and the corresponding nuclei. We developed a fast, user-friendly and automated image analysis algorithm capable of capturing and characterizing these individual components with a high level of accuracy. We demonstrate the robustness and applicability of the algorithm by quantifying morphological changes in response to a variety of environmental changes as well as manipulations of cellular components of mechanotransductions. Finally, as a proof-of-concept we use our algorithm to quantify the effect of Rho-associated kinase inhibitor Y-27632 on focal adhesion maturation. We show that a decrease in cell contractility leads to a decrease in focal adhesion size and aspect ratio.

Test-retest reliability of skeletal muscle oxygenation measurements during submaximal cycling exercise in patients with chronic heart failure.

The potential purpose of near-infrared spectroscopy (NIRS) as a clinical application in patients with chronic heart failure (CHF) is the identification of limitations in O2 delivery or utilization during exercise. The objective of this study was to evaluate absolute and relative test-retest reliability of skeletal muscle oxygenation measurements in patients with CHF. Thirty patients with systolic heart failure (left ventricular ejection fraction 31 ± 8%) performed 6-min constant-load cycling tests at 80% of the anaerobic threshold (AT) with tissue saturation index (TSI) measurement at the vastus lateralis. Tests were repeated after 10 ± 5 days to evaluate reliability. Absolute reliability was assessed with limits of agreement (LoA, expressed as bias ± random error) and coefficients of variation (CV) for absolute values (LoA range: 0·4 ± 6·2% to 0·6 ± 7·9%; CV range: 4·7-7·1%), amplitudes (LoA range -0·5 ± 5·8% to -0·7 ± 6·8%; CV range: 26·2-42·1%), onset and recovery kinetics (mean response times; LoA 0·4 ± 9·5 s, CV 23·5% and LoA -5·8 ± 50·8 s, CV 67·4% respectively) and overshoot characteristics (CV range 45·7-208·6%). Relative reliability was assessed with intraclass correlation coefficients for absolute values (range 0·74-0·90), amplitudes (range 0·85-0·92), onset and recovery kinetics (0·53 and 0·51, respectively) and overshoot characteristics (range 0·17-0·74). In conclusion, absolute reliability of absolute values and onset kinetics seems acceptable for serial within-subject comparison, and as such, for evaluation of treatment effects. Absolute reliability of amplitudes and recovery kinetics is considered unsatisfactory. Relative reliability of absolute values and amplitudes is sufficient for purposes of physiological distinction between patients with CHF. Despite lower relative reliability, kinetics may still be useful for clinical application.

Heading in the Right Direction: Understanding Cellular Orientation Responses to Complex Biophysical Environments.

The aim of cardiovascular regeneration is to mimic the biological and mechanical functioning of tissues. For this it is crucial to recapitulate the in vivo cellular organization, which is the result of controlled cellular orientation. Cellular orientation response stems from the interaction between the cell and its complex biophysical environment. Environmental biophysical cues are continuously detected and transduced to the nucleus through entwined mechanotransduction pathways. Next to the biochemical cascades invoked by the mechanical stimuli, the structural mechanotransduction pathway made of focal adhesions and the actin cytoskeleton can quickly transduce the biophysical signals directly to the nucleus. Observations linking cellular orientation response to biophysical cues have pointed out that the anisotropy and cyclic straining of the substrate influence cellular orientation. Yet, little is known about the mechanisms governing cellular orientation responses in case of cues applied separately and in combination. This review provides the state-of-the-art knowledge on the structural mechanotransduction pathway of adhesive cells, followed by an overview of the current understanding of cellular orientation responses to substrate anisotropy and uniaxial cyclic strain. Finally, we argue that comprehensive understanding of cellular orientation in complex biophysical environments requires systematic approaches based on the dissection of (sub)cellular responses to the individual cues composing the biophysical niche.