Surface and bulk stresses drive morphological changes in fibrous microtissues

Engineered fibrous tissues consisting of cells encapsulated within collagen gels are widely used three-dimensional in vitro models of morphogenesis and wound healing. Although cell-mediated matrix remodeling that occurs within these scaffolds has been extensively studied, less is known about the mesoscale physical principles governing the dynamics of tissue shape. Here, we show both experimentally and by using computer simulations how surface contraction through the development of surface stresses (analogous to surface tension in fluids) coordinates with bulk contraction to drive shape evolution in constrained three-dimensional microtissues. We used microelectromechanical systems technology to generate arrays of fibrous microtissues and robot-assisted microsurgery to perform local incisions and implantation. We introduce a technique based on phototoxic activation of a small molecule to selectively kill cells in a spatially controlled manner. The model simulations, which reproduced the experimentally observed shape changes after surgical and photochemical operations, indicate that fitting of only bulk and surface contractile moduli is sufficient for the prediction of the equilibrium shape of the microtissues. The computational and experimental methods we have developed provide a general framework to study and predict the morphogenic states of contractile fibrous tissues under external loading at multiple length scales.Published versio

Mechanical principles of engineering fibrous tissues with programmable shape

The form and structure of biological tissues define their function. The emergence of tissue morphology during development is one of the wonders of nature. Cells mechanically probe and manipulate their surroundings while constructing structures from the extracellular matrix that they produce. Self-assembly of tissues is essentially a mechanical process, touching various engineering disciplines including solid and fluid mechanics as well as thermodynamics. Recent advancements in materials science, molecular biology, and microtechnology enabled the engineering of living tissues in vitro as a means to discover the physical principles of morphogenesis. The quest is far from being completed, particularly for the cases where cells are interacting through a fibrous matrix. There is no algorithm or protocol that can prescribe long-lasting shapes to these constructs with arbitrary complexity. Considering the potential impact of this endeavor in regenerative medicine and drug screening, novel perspectives are urgently needed. The objective of this thesis is to provide an experimental and computational framework that would together enable the researcher to perform system identification and sculpturing on small scale biological samples. System identification involves dissecting the contributions of forces and motion on the evolution of the tissue shape. Sculpturing living matter means guiding the morphogenesis process through pre-programmed mechanical perturbations. Computational modeling of tissue mechanics aid both the interrogation and engineering phases. As the biological sample, a very simple composition that is based on a cell-laden collagen gel was used. Two novel technologies are introduced: (1) a fully-motorized robotic micromanipulation system and (2) a spatiotemporally resolved optochemical stimulation protocol. The platforms are integrated with modern imaging systems for real-time recording and automation. The thesis starts with a detailed investigation of constrained fibrous microtissues where the emphasis was placed on the controlled perturbation of the mechanical state. Experiments revealed an opportunity and an important challenge. The study showed that surface stresses were as important as bulk stresses in the equilibrium configuration of the tissues. A physics-based computational model was developed that accurately captured tissue morphology by only considering bulk and surface contraction. The challenge is that tissues under the influence of these contractile stresses are inherently unstable and they are always inclined to deviate from the prescribed shape. Epithelial tissues undergo a fluid to solid transition. In the second part, this transition was harnessed to stabilize the tissue shape and enable reshaping upon mechanically induced fluidization. Unconstrained fibrous microtissues with an epithelial shell were successfully morphed into prescribed shapes and re-shaped under the guidance of precise mechanical manipulations. The final part of the thesis suggests various future directions that would capitalize on the presented results and advance the concept towards a robust and versatile tissue engineering solution

Synthetic Biology Platform for Sensing and Integrating Endogenous Transcriptional Inputs in Mammalian Cells

One of the goals of synthetic biology is to develop programmable artificial gene networks that can transduce multiple endogenous molecular cues to precisely control cell behavior. Realizing this vision requires interfacing natural molecular inputs with synthetic components that generate functional molecular outputs. Interfacing synthetic circuits with endogenous mammalian transcription factors has been particularly difficult. Here, we describe a systematic approach that enables integration and transduction of multiple mammalian transcription factor inputs by a synthetic network. The approach is facilitated by a proportional amplifier sensor based on synergistic positive autoregulation. The circuits efficiently transduce endogenous transcription factor levels into RNAi, transcriptional transactivation, and site-specific recombination. They also enable AND logic between pairs of arbitrary transcription factors. The results establish a framework for developing synthetic gene networks that interface with cellular processes through transcriptional regulators

A model for 3D deformation and reconstruction of contractile microtissues

Tissue morphogenesis and regeneration are essentially mechanical processes that involve coordination of cellular forces, production and structural remodeling of extracellular matrix (ECM), and cell migration. Discovering the principles of cell–ECM interactions and tissue-scale deformation in mechanically-loaded tissues is instrumental to the development of novel regenerative therapies. The combination of high-throughput three-dimensional (3D) culture systems and experimentally-validated computational models accelerate the study of these principles. In our previous work [E. Mailand, et al., Biophys. J., 2019, 117, 975–986], we showed that prominent surface stresses emerge in constrained fibroblast-populated collagen gels, driving the morphogenesis of fibrous microtissues. Here, we introduce an active material model that allows the embodiment of surface and bulk contractile stresses while maintaining the passive elasticity of the ECM in a 3D setting. Unlike existing models, the stresses are driven by mechanosensing and not by an externally applied signal. The mechanosensing component is incorporated in the model through a direct coupling of the local deformation state with the associated contractile force generation. Further, we propose a finite element implementation to account for large deformations, nonlinear active material response, and surface effects. Simulation results quantitatively capture complex shape changes during tissue formation and as a response to surgical disruption of tissue boundaries, allowing precise calibration of the parameters of the 3D model. The results of this study imply that the organization of the extracellular matrix in the bulk of the tissue may not be a major factor behind the morphogenesis of fibrous tissues at sub-millimeter length scales

Deconstructing body axis morphogenesis in zebrafish embryos using robot-assisted tissue micromanipulation

Classic microsurgical techniques, such as those used in the early 1900s by Mangold and Spemann, have been instrumental in advancing our understanding of embryonic development. However, these techniques are highly specialized, leading to issues of inter-operator variability. Here we introduce a user-friendly robotic microsurgery platform that allows precise mechanical manipulation of soft tissues in zebrafish embryos. Using our platform, we reproducibly targeted precise regions of tail explants, and quantified the response in real-time by following notochord and presomitic mesoderm (PSM) morphogenesis and segmentation clock dynamics during vertebrate anteroposterior axis elongation. We find an extension force generated through the posterior notochord that is strong enough to buckle the structure. Our data suggest that this force generates a unidirectional notochord extension towards the tailbud because PSM tissue around the posterior notochord does not let it slide anteriorly. These results complement existing biomechanical models of axis elongation, revealing a critical coupling between the posterior notochord, the tailbud, and the PSM, and show that somite patterning is robust against structural perturbations

Engineered Extracellular Matrices with Integrated Wireless Microactuators to Study Mechanobiology

Mechanobiology explores how forces regulate cell behaviors and what molecular machinery are responsible for the sensing, transduction, and modulation of mechanical cues. To this end, probing of cells cultured on planar substrates has served as a primary experimental setting for many decades. However, native extracellular matrices (ECMs) consist of fibrous protein assemblies where the physical properties spanning from the individual fiber to the network architecture can influence the transmission of forces to and from the cells. Here, a robotic manipulation platform that allows wireless, localized, and programmable deformation of an engineered fibrous ECM is introduced. A finite‐element‐based digital twin of the fiber network calibrated against measured local and global parameters enables the calculation of deformations and stresses generated by different magnetic actuation schemes across a range of network properties. Physiologically relevant mechanical forces are applied to cells cultured on the fiber network, statically or dynamically, revealing insights into the effects of matrix‐borne forces and deformations as well as force‐mediated matrix remodeling on cell migration and intracellular signaling. These capabilities are not matched by any existing approach, and this versatile platform has the potential to uncover fundamental mechanisms of mechanobiology in settings with greater relevance to living tissues.An in vitro biomimetic platform that provides independent control over geometry, mechanics, and structure over extracellular matrices is introduced. Remote application of forces using robotically controlled magnetic microactuators triggers native mechanobiology responses including intracellular signaling and directed migration. A digital twin complements the platform by reporting stresses generated upon actuation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/170830/1/adma202102641-sup-0001-SuppMat.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/170830/2/adma202102641_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/170830/3/adma202102641.pd

Complete inclusion of bioactive molecules and particles in polydimethylsiloxane: a straightforward process under mild conditions

By applying a slow curing process, we show that biomolecules can be incorporated via a simple process as liquid stable phases inside a polydimethylsiloxane (PDMS) matrix. The process is carried out under mild conditions with regards to temperature, pH and relative humidity, and is thus suitable for application to biological entities. Fluorescence and enzymatic activity measurements show that the biochemical properties of the proteins and enzyme tested are preserved, without loss due to adsorption at the liquid-polymer interface. Protected from external stimuli by the PDMS matrix, these soft liquid composite materials are new tools of interest for robotics, microfluidics, diagnostics and chemical microreactors.ISSN:2045-232