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1. Continuum interpretation of mechano-adaptation in micropatterned epithelia informed by in vitro experiments

   https://doi.org/10.1093/intbio/zyad009  

   Cook, Bernard L. ; Alford, Patrick W. ( August 2023 , Integrative Biology)

   Epithelial tissues adapt their form and function following mechanical perturbations, or mechano-adapt, and these changes often result in reactive forces that oppose the direction of the applied change. Tissues subjected to ectopic tensions, for example, employ behaviors that lower tension, such as increasing proliferation or actomyosin turnover. This oppositional behavior suggests that the tissue has a mechanical homeostasis. Whether attributed to maintenance of cellular area, cell density, or cell and tissue tensions, epithelial mechanical homeostasis has been implicated in coordinating embryonic morphogenesis, wound healing, and maintenance of adult tissues. Despite advances toward understanding the feedback between mechanical state and tissue response in epithelia, more work remains to be done to examine how tissues regulate mechanical homeostasis using epithelial sheets with defined micropatterned shapes. Here, we used cellular microbiaxial stretching (CμBS) to investigate mechano-adaptation in micropatterned tissues of different shape consisting of Madin–Darby canine kidney cells. Using the CμBS platform, tissues were subjected to a 30% stretch that was held for 24 h. We found that, following stretch, tissue stresses immediately increased then slowly evolved over time, approaching their pre-stretch values by 24 h. Organization of the actin cytoskeletal was found to play a role in this process: anisotropic ally structured tissues exhibited anisotropic stress patterns, and the cytoskeletal became more aligned following stretch and reorganized over time. Interestingly, in unstretched tissues, stresses also decreased, which was found to be driven by proliferation-induced cellular confinement and change in tissue thickness. We modeled these behaviors with a continuum-based model of epithelial growth that accounted for stress-induced actin remodeling and proliferation, and found this model to strongly capture experimental behavior. Ultimately, this combined experimental-modeling approach suggests that epithelial mechano-adaptation depends on cellular architecture and proliferation, which can be modeled with a field-averaged approach applicable to more specific contexts in which change is driven by epithelial mechanical homeostasis.

   Insight box Epithelial tissues adapt their form and function following mechanical perturbation, and it is thought that this ‘mechano-adaptation’ plays an important role in driving processes like embryonic morphogenesis, wound healing, and adult tissue maintenance. Here, we use cellular microbiaxial stretching to probe this process in vitro in small epithelial tissues whose geometries were both controlled and varied. By using a highly precise stretching device and a continuum mechanics modeling framework, we revealed that tissue mechanical state changes following stretch and over time, and that this behavior can be explained by stress-dependent changes in actin fibers and proliferation. Integration of these approaches enabled a systematic approach to empirically and precisely measure these phenomena.

    

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2. Directing Multicellular Organization by Varying the Aspect Ratio of Soft Hydrogel Microwells

   https://doi.org/10.1002/advs.202104649  

   Pahapale, Gayatri J. ; Tao, Jiaxiang ; Nikolic, Milos ; Gao, Sammy ; Scarcelli, Giuliano ; Sun, Sean X. ; Romer, Lewis H. ; Gracias, David H. ( April 2022 , Advanced Science)

   Multicellular organization with precise spatial definition is essential to various biological processes, including morphogenesis, development, and healing in vascular and other tissues. Gradients and patterns of chemoattractants are well‐described guides of multicellular organization, but the influences of 3D geometry of soft hydrogels are less well defined. Here, the discovery of a new mode of endothelial cell self‐organization guided by combinatorial effects of stiffness and geometry, independent of protein or chemical patterning, is described. Endothelial cells in 2 kPa microwells are found to be ≈30 times more likely to migrate to the edge to organize in ring‐like patterns than in stiff 35 kPa microwells. This organization is independent of curvature and significantly more pronounced in 2 kPa microwells with aspect ratio (perimeter/depth) < 25. Physical factors of cells and substrates that drive this behavior are systematically investigated and a mathematical model that explains the organization by balancing the dynamic interaction between tangential cytoskeletal tension, cell–cell, and cell–substrate adhesion is presented. These findings demonstrate the importance of combinatorial effects of geometry and stiffness in complex cellular organization that can be leveraged to facilitate the engineering of bionics and integrated model organoid systems with customized nutrient vascular networks.

    

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3. A self-exciting point process to study multicellular spatial signaling patterns

   https://doi.org/10.1073/pnas.2026123118  

   Verma, Archit ; Jena, Siddhartha G. ; Isakov, Danielle R. ; Aoki, Kazuhiro ; Toettcher, Jared E. ; Engelhardt, Barbara E. ( August 2021 , Proceedings of the National Academy of Sciences)

   Multicellular organisms rely on spatial signaling among cells to drive their organization, development, and response to stimuli. Several models have been proposed to capture the behavior of spatial signaling in multicellular systems, but existing approaches fail to capture both the autonomous behavior of single cells and the interactions of a cell with its neighbors simultaneously. We propose a spatiotemporal model of dynamic cell signaling based on Hawkes processes—self-exciting point processes—that model the signaling processes within a cell and spatial couplings between cells. With this cellular point process (CPP), we capture both the single-cell pathway activation rate and the magnitude and duration of signaling between cells relative to their spatial location. Furthermore, our model captures tissues composed of heterogeneous cell types with different bursting rates and signaling behaviors across multiple signaling proteins. We apply our model to epithelial cell systems that exhibit a range of autonomous and spatial signaling behaviors basally and under pharmacological exposure. Our model identifies known drug-induced signaling deficits, characterizes signaling changes across a wound front, and generalizes to multichannel observations. 

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4. The form-finding and fabrication of an ultra-thin funicular bridge prototype made up of hollow glass units

   LU, Yao ; CREGAN, Matthew ; CHHADEH, Philipp A ; SEYEDAHMADIAN, Alireza ; BOLHASSANI, Mohammad ; Schneider, Jens ; YOST, Joseph R ; AKBARZADEH, Masoud ( October 2021 , International Association of Shell and Spatial Structures)

   The recent development of three-dimensional graphic statics (3DGS) has greatly increased the ease of designing complex and efficient spatial funicular structural forms [1]. The reciprocal diagram based 3DGS approaches not only generate highly efficient funicular structures [2], but also result in planarity constraints due to the polyhedron nature of the reciprocal diagrams [3]. Our previous research has shown the feasibility of leveraging this planarity by using planar glass sheets to materialize the 10m-span, double-layer glass bridge [3]. This paper is framed as a proof of concept for the 10m bridge and explores the form-finding, detail configuration, fabrication constraints, and assembly logic by designing and constructing a small-scale bridge prototype with a span of 2.5m. The prototype is designed in a modular approach, where each polyhedral cell of the form is materialized using a hollow glass unit (HGU) (Figure 1a), which can be prefabricated and preassembled, and therefore, greatly simplifies the assembly of the whole bridge. The compression-only form of the prototype is generated using the PolyFrame beta [4] plug-in for Rhinoceros [5]. The form-finding is carried out with a comprehensive consideration of a variety of parameters, including fabrication constraints, assembly ease, construction cost, and practicality. To start the form-finding process, a group of closed convex force polyhedrons is aggregated, controlling the topology of the form diagram and the orientations of the form elements. By manipulating the face tilting angles of the force diagram, the supported edges at the end of the bridge are all made horizontal, reducing the difficulty of the support design. Then, vertex locations and edge lengths of the form diagram are constrained, determining the final dimensions of both the bridge and the cells. After getting the geometry of the bridge, the detail developments are streamlined. Each of the 13 HGUs consists of two flat deck plates and a series of side plates (Figure 1b). To interlock the adjacent cells and prevent possible sliding, a male-female connection mechanism is introduced to the conjoint side plates of the HGUs (Figure 1b). Additionally, to eliminate the direct contact of the glass parts and prevent the stress concentration, two softer transparent materials are involved for connecting purposes. Within each HGU, silicon-based binding agent is used to hold the glass parts together; between the neighboring HGUs, plastic sheets are placed as interface materials (Figure 1b). Figure 1. a) The 2.5m-span small-scale prototype dome, b) Exploded view showing deck plates, side plates, male-female connection, and interface material For the fabrication of the glass parts, 5-axis Waterjet cutting techniques are applied. While the glass sheets for the deck plates can be purchased from the market, the irregular side plates with male-female connections need to be made from kiln-cast glass. In terms of the Waterjet cutting constraints, there is a max cutting angle of 60 degrees from vertical. With respect to this, all the glass parts are examined during the design process to ensure they all satisfy the cutting angle requirements. Aiming to achieve a fast and precise assembly, several assistant techniques are developed. On the local HGU level, assembly connectors are designed and 3D-printed to help locate the glass parts. On the global prototype level, the assembly sequence of the HGUs are simulated to avoid interference. Besides, a labeling system is also established to organize the fabricated parts and guide the entire assembly process. The design and construction of this small-scale prototype provide important information for the future development of the full-scale bridge regarding the interlocking detail design, the fabrication constraints, and assembly logic. The actual structural performance of the prototype awaits further investigation through-loading experiments. 

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5. A kidney proximal tubule model to evaluate effects of basement membrane stiffening on renal tubular epithelial cells

   https://doi.org/10.1093/intbio/zyac016  

   Wang, Dan ; Sant, Snehal ; Lawless, Craig ; Ferrell, Nicholas ( December 2022 , Integrative Biology)

   The kidney tubule consists of a single layer of epithelial cells supported by the tubular basement membrane (TBM), a thin layer of specialized extracellular matrix (ECM). The mechanical properties of the ECM are important for regulating a wide range of cell functions including proliferation, differentiation and cell survival. Increased ECM stiffness plays a role in promoting multiple pathological conditions including cancer, fibrosis and heart disease. How changes in TBM mechanics regulate tubular epithelial cell behavior is not fully understood. Here we introduce a cell culture system that utilizes in vivo-derived TBM to investigate cell–matrix interactions in kidney proximal tubule cells. Basement membrane mechanics was controlled using genipin, a biocompatibility crosslinker. Genipin modification resulted in a dose-dependent increase in matrix stiffness. Crosslinking had a marginal but statistically significant impact on the diffusive molecular transport properties of the TBM, likely due to a reduction in pore size. Both native and genipin-modified TBM substrates supported tubular epithelial cell growth. Cells were able to attach and proliferate to form confluent monolayers. Tubular epithelial cells polarized and assembled organized cell–cell junctions. Genipin modification had minimal impact on cell viability and proliferation. Genipin stiffened TBM increased gene expression of pro-fibrotic cytokines and altered gene expression for N-cadherin, a proximal tubular epithelial specific cell–cell junction marker. This work introduces a new cell culture model for cell-basement membrane mechanobiology studies that utilizes in vivo-derived basement membrane. We also demonstrate that TBM stiffening affects tubular epithelial cell function through altered gene expression of cell-specific differentiation markers and induced increased expression of pro-fibrotic growth factors.

    

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