Agrin-Matrix Metalloproteinase-12 axis confers a mechanically competent microenvironment in skin wound healing.

An orchestrated wound healing program drives skin repair via collective epidermal cell proliferation and migration. However, the molecular determinants of the tissue microenvironment supporting wound healing remain poorly understood. Herein we discover that proteoglycan Agrin is enriched within the early wound-microenvironment and is indispensable for efficient healing. Agrin enhances the mechanoperception of keratinocytes by augmenting their stiffness, traction stress and fluidic velocity fields in retaliation to bulk substrate rigidity. Importantly, Agrin overhauls cytoskeletal architecture via enhancing actomyosin cables upon sensing geometric stress and force following an injury. Moreover, we identify Matrix Metalloproteinase-12 (MMP12) as a downstream effector of Agrin's mechanoperception. We also reveal a promising potential of a recombinant Agrin fragment as a bio-additive material that assimilates optimal mechanobiological and pro-angiogenic parameters by engaging MMP12 in accelerated wound healing. Together, we propose that Agrin-MMP12 pathway integrates a broad range of mechanical stimuli to coordinate a competent skin wound healing niche.

An integrated platform to facilitate the calculation, validation and visualization of optical flow velocities in biological images.

Optical flow algorithms have seen poor adoption in the biological community compared with particle image velocimetry for quantifying cellular dynamics because of the lack of proper validation and an intuitive user interface. To address these challenges, we present OpFlowLab, an integrated platform that integrates our motion estimation workflow. Using routines in our workflow, we demonstrate that optical flow algorithms are more accurate than PIV in simulated images of the movement of nuclei. Qualitative assessment with actual nucleus images further supported this finding. Additionally, we show that refinement of the optical flow velocities is possible with a simple object-matching procedure, opening up the possibility of obtaining reasonable velocity estimates under less ideal imaging conditions. To visualize velocity fields, we employ artificial tracers to allow for the drawing of pathlines. Through the adoption of OpFlowLab, we are confident that optical flow algorithms will allow for the exploration of dynamic biological systems in greater accuracy and detail.

Spherical microwell arrays for studying single cells and microtissues in 3D confinement.

Microwell arrays have emerged as three-dimensional substrates for cell culture due to their simplicity of fabrication and promise for high-throughput applications such as 3D cell-based assays for drug screening. To date, most microwells have had cylindrical geometries. Motivated by our previous findings that cells display 3D physiological characteristics when grown in the spherical micropores of monodisperse foam scaffolds (Lee et al 2013 Integr. Biol. 5 1447-55 and Lin et al 2011 Soft Matter 7 10010-6), here we engineered novel microwells shaped as spherical caps with obtuse polar angles, yielding narrow apertures. When used as bare substrates, these microwells were suitable for culturing cell spheroids; the narrow apertures sterically hindered unattached cultured cells from rolling out of microwells under agitation. When only the walls of the microwell were conjugated with extracellular matrix proteins, cells remained confined in the microwells. Epithelial cells proliferated and burst out of the aperture, and cell polarity was oriented based on the distribution of extracellular matrix proteins in the microwells. Surprisingly, single fibroblast cells in spherical wells of various diameters (40-100 µm) underwent cell-cycle arrest, while cells in circular cylindrical microwells continued to proliferate. Spatial confinement was not sufficient to cause cell-cycle arrest; however, confinement in a constant negative-curvature microenvironment led to cell-cycle arrest. Overall, these investigations demonstrate that this spherical microwell substrate constitutes a novel basic research tool for elucidating how cells respond to dimensionality and microenvironment with radii of curvature at the cellular length scale.

Revealing the dependence of cell spreading kinetics on its spreading morphology using microcontact printed fibronectin patterns.

Since the dawn of in vitro cell cultures, how cells interact and proliferate within a given external environment has always been an important issue in the study of cell biology. It is now well known that mammalian cells typically exhibit a three-phase sigmoid spreading on encountering a substrate. To further this understanding, we examined the influence of cell shape towards the second rapid expansion phase of spreading. Specifically, 3T3 fibroblasts were seeded onto silicon elastomer films made from polydimethylsiloxane (PDMS), and micro-contact printed with fibronectin stripes of various dimensions. PDMS is adopted in our study for its biocompatibility, its ease in producing very smooth surfaces, and in the fabrication of micro-contact printing stamps. The substrate patterns are compared with respect to their influence on cell spreading over time. Our studies reveal, during the early rapid expansion phase, 3T3 fibroblasts are found to spread radially following a t≃¹·8 law; meanwhile, they proliferated in a lengthwise fashion on the striped patterns, following a t≃¹ law. We account for the observed differences in kinetics through a simple geometric analysis which predicted similar trends. In particular, a t² law for radial spreading cells, and a t¹ law for lengthwise spreading cells.

In situ real-time investigation of cancer cell photothermolysis mediated by excited gold nanorod surface plasmons.

The photothermolysis of living EMT-6 breast tumor cells triggered by gold nanorods (AuNRs) with two-photon irradiation was conducted in situ and under real-time observation. The morphology and plasma membrane permeability of the cells were key indicators to the phenomena. AuNRs with an aspect ratio of 3.92, and a longitudinal absorption peak at 800 nm were synthesized with a seed-mediated method. The nanorods surfaces were further modified with polystyrenesulfonate (PSS) for biocompatibility. The prepared nanorods displayed excellent two-photon photoluminescence imaging. In situ real-time results revealed cavities internal to the cells were created from thermal explosions triggered by AuNRs localized photothermal effect. The cavitation dynamic is energy dependent and responsible for the perforation or sudden rupture of the plasma membrane. The energy threshold for cell therapy depended significantly on the number of nanorods taken up per cell. For an ingested AuNR cluster quantity N approximately 10-30 per cell, it is found that energy fluences E larger-than 93 mJ/cm(2) led to effective cell destruction in the crumbled form within a very short period. As for a lower energy level E = 18 mJ/cm(2) with N approximately 60-100, a non-instant, but progressive cell deterioration, is observed.