Collective effects in cellular structure formation mediated by compliant environments: a Monte Carlo study

Compliant environments can mediate interactions between mechanically active cells like fibroblasts. Starting with a phenomenological model for the behaviour of single cells, we use extensive Monte Carlo simulations to predict non-trivial structure formation for cell communities on soft elastic substrates as a function of elastic moduli, cell density, noise and cell position geometry. In general, we find a disordered structure as well as ordered string-like and ring-like structures. The transition between ordered and disordered structures is controlled both by cell density and noise level, while the transition between string- and ring-like ordered structures is controlled by the Poisson ratio. Similar effects are observed in three dimensions. Our results suggest that in regard to elastic effects, healthy connective tissue usually is in a macroscopically disordered state, but can be switched to a macroscopically ordered state by appropriate parameter variations, in a way that is reminiscent of wound contraction or diseased states like contracture.Comment: 45 pages, 7 postscript figures included, revised version accepted for publication in Acta Biomateriali

The convergence of haemodynamics, genomics, and endothelial structure in studies of the focal origin of atherosclerosis

The completion of the Human Genome Project and ongoing sequencing of mouse, rat and other genomes has led to an explosion of genetics-related technologies that are finding their way into all areas of biological research; the field of biorheology is no exception. Here we outline how two disparate modern molecular techniques, microarray analyses of gene expression and real-time spatial imaging of living cell structures, are being utilized in studies of endothelial mechanotransduction associated with controlled shear stress in vitro and haemodynamics in vivo. We emphasize the value of such techniques as components of an integrated understanding of vascular rheology. In mechanotransduction, a systems approach is recommended that encompasses fluid dynamics, cell biomechanics, live cell imaging, and the biochemical, cell biology and molecular biology methods that now encompass genomics. Microarrays are a useful and powerful tool for such integration by identifying simultaneous changes in the expression of many genes associated with interconnecting mechanoresponsive cellular pathways

Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells.

Although it is a central question in biology, how cell shape controls intracellular dynamics largely remains an open question. Here, we show that the shape of Arabidopsis pavement cells creates a stress pattern that controls microtubule orientation, which then guides cell wall reinforcement. Live-imaging, combined with modeling of cell mechanics, shows that microtubules align along the maximal tensile stress direction within the cells, and atomic force microscopy demonstrates that this leads to reinforcement of the cell wall parallel to the microtubules. This feedback loop is regulated: cell-shape derived stresses could be overridden by imposed tissue level stresses, showing how competition between subcellular and supracellular cues control microtubule behavior. Furthermore, at the microtubule level, we identified an amplification mechanism in which mechanical stress promotes the microtubule response to stress by increasing severing activity. These multiscale feedbacks likely contribute to the robustness of microtubule behavior in plant epidermis. DOI: http://dx.doi.org/10.7554/eLife.01967.001

Models of Mechanics and Growth in Developmental Biology: A Computational Morphodinamics approach

Recent evidence has revealed the role of mechanical cues in the development of shapes in organisms. This thesis is an effort to test some of the fundamental hypotheses about the relation between mechanics and patterning in plants. To do this, we develop mechanical models designed to include specific features of plant cell walls. These are heterogeneous stiffness and material anisotropy as well as rates and directions of growth, which we then relate to different domains of the plant tissue.In plant cell walls, anisotropic fiber deposition is the main controller of longitudinal growth. In our model, this is achieved spontaneously, by applying feedback from the maximal stress direction to the fiber orientation. We show that a stress feedback model is in fact an energy minimization process. This can be considered as an evolutionary motivation for the emergence of a stress feedback mechanism. Then we add continuous growth and cell division to the model and employ the strain signal directing large growth deformations. We show the advantages of strain-based growth model for emergence of plant-like organ shapes as well as for reproducing microtubular dynamics in hypocotyls and roots. We also investigate possibilities for describing microtubular patterns, at root hair outgrowth sites according to stress patterns. Altogether, the work described in this thesis, provides a new improved growth model for plant tissue, where mechanical properties are handled with appropriate care in the event of growth driven by either molecular or mechanical signals. The model unifies the patterning process for several different plant tissues, from shoot to single root hair cells, where it correctly predict microtubular dynamics and growth patterns. In a long-term perspective, this understanding can propagate to novel technologies for improvement of yield in agriculture and the forest industry

Combining Smart Material Platforms and New Computational Tools to Investigate Cell Motility Behavior and Control

Cell-extracellular matrix (ECM) interactions play a critical role in regulating important biological phenomena, including morphogenesis, tissue repair, and disease states. In vivo, cells are subjected to various mechanical, chemical, and electrical cues to collectively guide their functionality within a specific microenvironment. To better understand the mechanisms regulating cell adhesive, differentiation, and motility dynamics, researchers have developed in vitro platforms to synthetically mimic native tissue responses. While important information about cell-ECM interactions have been revealed using these systems, a knowledge gap currently exists regarding how cell responses in static environments relate to the dynamic cell-ECM interaction behaviors observed in vivo. Advances at the intersection of materials science, biophysics, and cell biology have recently enabled the production of dynamic ECM mimics where cells can be exposed to controlled mechanical, electrical or chemical cues to directly decouple cell-ECM related behaviors from cell-cell or cell-environmental factors. Utilization of these dynamic synthetic biomaterials will enable discovery of novel mechanisms fundamental in tissue development, homeostasis, repair, and disease. In this dissertation, the primary goal was to evaluate how mechanical changes in the ECM regulate cell motility and polarization responses. This was accomplished through two major aims: 1) by developing a modular image processing tool that could be applied in complex synthetic in vitro microenvironments to asses cell motility dynamics, and 2) to utilize that tool to advance understanding of mechanobiology and mechanotransduction processes associated with development, wound healing, and disease progression. Therefore, the first portion of this thesis (Chapters 2 and 3) dealt with proof of concept for our newly developed automated cell tracking system, termed ACTIVE (automated contour-based tracking for in vitro environments), while the second portion of this thesis (Chapter 4-7) addressed applying this system in multiple experimental designs to synthesize new knowledge regarding cell-ECM or cell-cell interactions. In Chapter 1, we introduced why cell-ECM interactions are essential for in vivo processes and highlighted the current state of the literature. In Chapter 2, we demonstrated that ACTIVE could achieve greater than 95% segmentation accuracy at multiple cell densities, while improving two-body cell-cell interaction error by up to 43%. In Chapter 3 we showed that ACTIVE could be applied to reveal subtle differences in fibroblast motility atop static wrinkled or static non-wrinkled surfaces at multiple cell densities. In Chapters 4 and 5, we characterized fibroblast motility and intracellular reorganization atop a dynamic shape memory polymer biomaterial, focusing on the role of the Rho-mediated pathway in the observed responses. We then utilized ACTIVE to identify differences in subpopulation dynamics of monoculture versus co-culture endothelial and smooth muscle cells (Chapter 6). In Chapter 7, we applied ACTIVE to investigate E. coli biofilm formation atop poly(dimethylsiloxane) surfaces with varying stiffness and line patterns. Finally, we presented a summary and future work in Chapter 8. Collectively, this work highlights the capabilities of the newly developed ACTIVE tracking system and demonstrates how to synthesize new information about mechanobiology and mechanotransduction processes using dynamic biomaterial platforms

Deciphering cell motility and spatial sensing of intestinal cell types using an ex vivo intestinal model

The intestine is a highly organized tissue with two distinct regions: the crypt and the villus. When stem cells divide at the crypt bottom, half of their progeny migrates upwards towards the villus, where they differentiate into various cell types, including the abundant absorptive enterocytes. However, the precise mechanisms governing this migration and tissue organization remain poorly understood. In this thesis, novel methodologies, such as long-term intravital imaging and decellularization of mouse intestine, are used to study cell type-specific motility within the tissue architecture. Moreover, work in this thesis probes the mechanisms mediating intestinal regeneration and aging, and the clonal competition during tumor development. In paper 1, we employ long-term intravital imaging to identify a greater number of longterm functioning intestinal stem cells (ISCs) in the small intestine compared to the colon. We further investigate this phenomenon by combining intravital imaging and the novel ex vivo live cell imaging assay to discover that stem cells in the small intestine display downward motility directed by Wnt-ligands. In Paper 2, the ex vivo live cell imaging assay was utilized to investigate active cell migration in several cell types. Our findings reveal that both ISCs and paneth cells possess an intrinsic ability to perceive positional cues embedded in the extracellular matrix (ECM), which guides them to their native location, the crypt. In contrast, enterocytes, lack this capability. Finally, we discovered that during aging ECM loses the signals guiding crypt homing of ISCs, and that the tumor-causing mutations render cells insensitive to ECM signals resulting in loss of crypt homing. In Paper 3, we introduce an optimized intestinal decellularization protocol and demonstrate its capacity to regenerate the intestinal epithelium from single-seeded stem cells, freshly isolated crypts, or organoids. During regeneration following damage, we discovered mesenchymally produced Asporin, which promotes Tgfβ-signaling and induces fetal-like reprogramming in intestinal tissue. Additionally, we observed that chronic upregulation of Asporin in the aged intestinal tissue hampers tissue repair. In Paper 4, we elucidate how Apc-mutant ISCs gain a clonal advantage over wild-type ISCs. We reveal that Apc-mutant ISCs secrete the Wnt-inhibitor Notum, which reduces the stemness and competitiveness of wild-type ISCs. Inhibition of Notum reverted the clonal advantage of Apc-mutant cells and reduced tumor burden. In conclusion, this thesis focused on highlighting the interplay between intestinal epithelial cells and the ECM, particularly the ability of ISCs and paneth cells to sense positional cues embedded in the ECM, guiding them to their native location. Additionally, key mechanisms disrupted during aging and in intestinal cancer are elucidated

Determinants of multi-scale patterning in growth plate cartilage

ABSTRACT Functional architectures of complex adaptive systems emerge by dynamic control over properties of individual components. During skeletal development, growth plate cartilage matches bone geometries to body plan requisites by spatiotemporally regulating chondrocyte actions. Bone growth potential is managed by the proximodistal patterning of chondrocyte populations into differentiation zones, while growth vectors are specified by the unique columnar arrangement of clonal groups. Chondrocyte organization at both tissue and cell levels is influenced by a cartilage-wide communication network that relies on zone-specific release and interpretation of paracrine signals. Despite genetic characterization of signaling interactions necessary for cartilage maturation, the regulatory mechanisms that couple positional information with polarized chondrocyte activities to coordinate skeletal morphogenesis remain poorly understood. Building on previous kinematic descriptions of column formation, the work contained in this dissertation suggests cytoskeletal regulation mediates crosstalk between long-range signaling and local cell behavior. Rearranging daughter chondrocytes specifically recruit actomyosin contractility to cortical surfaces, indicating a primary role for the actin cytoskeleton as the engine powering column formation kinetics. Disrupted chondrocyte contractility patterns are observed after genetic perturbation of planar cell polarity signaling, and after inhibiting integrin extracellular matrix binding, implicating actomyosin as a sensor able to integrate global with local signaling cues. To gain greater analytical control towards dissecting the mechanochemical patterning systems underlying cartilage architecture, an alginate hydrogel-based model of growth plate was developed. Daughter chondrocytes encapsulated in alginate beads deposit extracellular matrix in anisotropic and hierarchical configurations that resemble myosin localization in vivo, hinting cytoskeletal forces may sculpt the solid-state environment. Single-cell transcriptomic analysis of chondrocytes stimulated with recombinant ligands demonstrates the functionality of the IHH/PTHrP circuit in alginate beads, and points towards a novel role for PTHrP signaling gradients in transcriptional regulation of cytoskeletal and ECM proteins. Basal bead cultures tend towards resting/proliferative phenotypes driven by endogenous PTHrP expression, but activating IHH signaling induces position-dependent gene expression, consistent with a model of zone formation where concentration gradients generate spatial cues. Together, the work suggests that in addition to regulating chondrocyte differentiation, the tissue-wide signaling network in cartilage can influence cell-matrix interactions that may be important for cell behavior, and presents a novel culture model that can be used for future studies investigating how chondrocytes discern positional information to shape the growing tissue

3D Bioprinting Systems for the Study of Mammary Development and Tumorigenesis

Understanding the microenvironmental factors that control cell function, differentiation, and stem cell renewal represent the forefront of developmental and cancer biology. To accurately recreate and model these dynamic interactions in vitro requires both precision-controlled deposition of multiple cell types and well-defined three-dimensional (3D) extracellular matrix (ECM). To achieve this goal, we hypothesized that accessible bioprinting technology would eliminate the experimental inconsistency and random cell-organoid formation associated with manual cell-matrix embedding techniques commonly used for 3D, in vitro cell cultures. The first objective of this study was to adapt a commercially-available, 3D printer into a 3D bioprinter. Goal-based computer simulations were used to identify, evaluate, and optimize the performance of a 3D bioprinting system. Implementing these findings yielded a bioprinting system with reduced needle clogging and single cell print resolution. The minimal disruption of cell function was confirmed by the retention of pluripotency marker TRA-1-81 in bioprinted human induced pluripotent stem cells (hiPSCs) 7-days post-printing. This system was then used to investigate cell behavior during the initial stages of organoid-structure formation by generating 3D bioprinted arrays of individual, mammary epithelial cell (MEC) organoid-structures. This quantifiable, 3D bioprinting approach, was able to direct the ‘self-assembly’ of large MEC structures through organoid ‘fusion’ events among individual, bioprinted organoids along the printing template. Bioprinting maintained experimental consistency among multiple 3D scaffolds and experimental conditions, and presents the capability to generate high-fidelity, 3D arrays with multiple cell types. Compared to manual matrix embedding, bioprinted, co-culture experiments, containing normal MECs and breast cancer cell lines, significantly increased the ability to generate chimeric (tumor/normal) MEC structures. Thus, bioprinting stands highly qualified to investigate the role of microenvironmental processes related to cell fate determination and tissue formation