Engineering microscale topographies to control the cell-substrate interface

a b s t r a c t Cells in their in vivo microenvironment constantly encounter and respond to a multitude of signals. While the role of biochemical signals has long been appreciated, the importance of biophysical signals has only recently been investigated. Biophysical cues are presented in different forms including topography and mechanical stiffness imparted by the extracellular matrix and adjoining cells. Microfabrication technologies have allowed for the generation of biomaterials with microscale topographies to study the effect of biophysical cues on cellular function at the cellesubstrate interface. Topographies of different geometries and with varying microscale dimensions have been used to better understand cell adhesion, migration, and differentiation at the cellular and sub-cellular scales. Furthermore, quantification of cellgenerated forces has been illustrated with micropillar topographies to shed light on the process of mechanotransduction. In this review, we highlight recent advances made in these areas and how they have been utilized for neural, cardiac, and musculoskeletal tissue engineering application

Molecular and cellular analysis of topography-induced mechanotransduction

Edited Abstract: Mechanotransduction is the process by which cells convert mechanical stimuli into an adaptive gene- and protein-level response, via signalling cascades or direct physical effects of the cytoskeleton on the nucleus, and appropriate mechanosignalling is crucial for tissue development and function. Most techniques currently used to study cellular mechanoresponses are relatively damaging to the cells. In contrast, topographically structured substrates, such as microgrooves, have great potential for use as non-invasive mechanostimuli. In this study, quartz microgrooved substrata (2 μm depth x 25 μm pitch) were used as platforms for the confinement and alignment of cells. A multi-layered approach was adopted to begin to integrate the changes induced by the topographical mechanostimulus at the chromosome, small RNA, transcript, protein and structural levels. Together, the results provide insight into multiple facets of topography-induced mechanotransduction, which should contribute to understanding of mechanotransduction and cell-material interactions

Heading in the right direction : guiding cellular alignment by substrate anisotropy

Energie en entropie sturen cellen in de zelfde richtin

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

3D printing approaches for guiding endothelial cell vascularization and migration

3D printing technology is rapidly advancing and is being increasingly used for biological applications. The spatial control of 3D printing makes it especially attractive for fabricating 3D tissues and for studying the role of geometry in biology. We utilized two different types of 3D printing to engineer vascularized tissues with complex vascular architectures, to use engineered vasculature to treat ischemia, and to study directional endothelial cell migration on curved wave topography. To engineer 3D tissues, perfusable vascular networks must be embedded within the tissue to supply nutrients and oxygen to cells. 3D-printed sugar filaments have previously been used as a cytocompatible sacrificial template to rapidly cast vascular networks. We improved upon the 3D-printed sugar method and used it to fabricate complex vascular geometries that were not previously possible, such as a branched channel geometry, with controlled fluid flow through the channels. We also integrated an approach utilizing vascular self-assembly to generate thick tissues with dense, capillary-scale vessel networks. The vascularized tissues fabricated using 3D-printed sugar successfully integrated with a host vasculature upon implantation and restored perfusion in two different animal models of ischemia. Cell migration critical to numerous biological processes can be guided by surface topography. However, fabrication limitations constrain topography studies to geometries that may not adequately mimic physiological environments. Direct Laser Writing (DLW) provides the necessary 3D flexibility and control to create well-defined curved waveforms similar to those found in physiological settings, such as the lumen of blood vessels. We found that endothelial cells migrated fastest along square waves, intermediate along triangular waves, and slowest along sine waves and that directional cell migration on sine waves decreased at longer sinusoid wavelengths. Interestingly, inhibition of Rac1 decreased directional migration on 3D sine waves but not on 2D micropatterned lines, suggesting that cells may utilize different molecular pathways to sense curved topographies. Our study demonstrates that DLW can be employed to investigate directional migration on a wide array of surfaces with curvatures that are unattainable using conventional manufacturing techniques.2020-10-22T00:00:00

Active matter: from collective self-propelled rods to cell-like particles

Active matter comprises systems with sustained energy uptake and dissipation of its constituents. This applies to systems across many scales. We study ensembles of self-propelled rods (SPRs) in periodic boundaries and in confinement to mimic the collective behavior and dynamics of bacteria, and active filaments. Our models describe active systems that allow the propulsion to adapt to its environment. While SPRs with density-dependent slowing down partially capture the behavior observed for bacteria, SPRs in ring-like confinements can be considered as a minimal, soft matter model for cell motility. Phoretic microswimmers and genetically modified E. coli show density-dependent reduced propulsion.This motivates the investigation of the collective behavior and dynamics of SPRs with density-dependent propulsion force. The density-dependent slowing down enhances polar ordering and cluster formation, and induces rod perpendicularity at cluster borders. As a model of cellular motility due to cytoskeletal activity, SPRs inside mobile, rigid circular confinement are considered, which build complex self-propelled rings. The rod self-organization gives rise to complex motility patterns, such as run-and-tumble and run-and-circle motion. Motility patterns observed for self-propelled rigid rings are also observed for motile cells. Taking a further step towards a more realistic modeling of cell motility, we study SPRs inside mobile, deformable rings. In addition to ring motility, also ring deformability plays a crucial role in SPR alignment and cluster formation. Here, pulling forces at the back of the rings are crucial to recovering cell-like shapes and motion. While our models do not take into account biochemical aspects of biological systems, they allow the identification of crucial mechanical aspects, and help to test different underlying mechanisms to interpret microscopic observations

Os efeitos dos polímeros piezoeléctricos na diferenciação neuronal

Mestrado em Biomedicina MolecularO crescimento de neurites é crucial para o desenvolvimento neuronal, bem como para a plasticidade e reparação na fase adulta. Após uma lesão neuronal, o sucesso da reparação é determinando pelas propriedades plásticas constitutivas dos neurónios afetados e pelo seu potencial de regeneração, que é influenciado por sinais externos físicos (ex.: cicatriz glial) e químicos (ex.: moléculas inibitórias). Recentemente, o desenvolvimento de materiais à nano-escala, que interagem com os sistemas biológicos a nível molecular, prometem revolucionar o tratamento das lesões do Sistema Nervoso Central e Periférico. Os scaffolds de nanomateriais podem suportar e promover o crescimento de neurites e consequentemente, intervir nas complexas interações moleculares que ocorrem a após o dano neuronal, entre as células e o seu ambiente extracelular. Vários estudos têm demonstrado que os materiais piezoeléctricos, que geram carga elétrica em resposta ao stress mecânico, podem ser usados para a preparação de scaffolds eletricamente carregados que devem influenciar o comportamento celular. Este estudo centrou-se nos efeitos dos materiais baseados em PLLA (ácido poli (L – láctico)) sob a forma de filmes, nanofibras orientadas aleatória e alinhadamente, e da sua polarização, na diferenciação neuronal. A linha celular de neuroblastoma (SH-SY5Y) foi utilizada para avaliar o efeito dos materiais-baseados em PLLA na adesão, viabilidade, morfologia celular, bem como na diferenciação tipo-neuronal. A análise proteómica baseada em espectrometria de massa das células cultivadas em nanofibras de PLLA foi também efetuada. Os neurónios corticais embriónicos foram seguidamente utilizados para avaliar os efeitos das nanofibras de PLLA alinhadas e da sua polarização no crescimento de neurites. Nesta análise, descobrimos que os materiais de PLLA parecem inibir parcialmente a proliferação celular, enquanto promovem a diferenciação, alterando os níveis das proteínas que intervêm nestes processos. Ocorrem alterações significativas do citoesqueleto, particularmente ao nível do citoesqueleto de actina, que não induzem mas parecem potenciar o crescimento de neurites sob exposição a um sinal extracelular como o ácido retinóico. Este efeito parece ser particularmente evidente para as nanofibras de PLLA alinhadas, que induzem efeitos intermédios na restruturação do citoesqueleto. Em geral, a polarização das amostras de PLLA tem efeitos benéficos na proliferação celular e potencia o crescimento de neurites, particularmente nos neurónios. Acreditamos que as nanofibras de PLLA alinhadas serão um bom scaffold para regeneração neuronal, uma vez que mimetiza o ambiente mecânico natural das células. Contudo, futuras experiências in vitro e in vivo são necessárias para comprovar a eficácia deste potencial scaffold.Neuritic growth is crucial for neural development, as well as for adaptation and repair in adulthood. Upon neuronal injury, the successful neuritic regrowth is determined by the constitutive plastic properties of neurons and by their regenerative potential, which is influenced by physical (e.g. glial scar) and chemical (e.g. inhibitory molecules) extrinsic cues. Recently, the development of nanometer-scale materials, which can interact with biological systems at a molecular level, provide hope to revolutionize the treatment of central and peripheral nervous system injuries. Nanomaterial scaffolds can support and promote neuritic outgrowth and consequently, take part in the complex molecular interactions between cells and their extracellular environment after neuronal injury. Several studies have shown that piezoelectric materials, which generate electrical charge in response to mechanical strain, may be used to prepare bioactive electrically charged scaffolds that may influence cell behavior. This study focused on the effects of PLLA (poly-L-lactic acid) – based materials in the form of films, random and aligned nanofibers, and of their polarization, on neuronal-like and neuronal differentiation. The neuroblastoma SH-SY5Y cell line was used to evaluate the effect of PLLA – based materials on cellular adhesion, viability, morphology and neuron-like differentiation. Mass spectrometry-based proteomic analysis of cells grown on PLLA nanofibers was also conducted. Primary embryonic cortical neurons were further used to evaluate the effect of PLLA aligned nanofibers and their polarization on neuritic outgrowth. In this analysis, we found that PLLA materials seem to partially inhibit cell proliferation, while promoting neuronal differentiation, altering the levels of proteins that intervene in these processes. Dramatic cytoskeleton remodeling occurs, particularly at the actin cytoskeleton level, which does not induce but may potentiate neuritic outgrowth upon exposure to an extracellular cue, such as Retinoic Acid. This effect seems to be particularly evident for PLLA aligned nanofibers, which induce intermediate effects in the cytoskeleton remodeling. In general, polarization of the PLLA polymers has beneficial effects on cell proliferation and potentiates the neuritic outgrowth, particularly in neurons. We believe that polarized PLLA aligned nanofibers would be a good scaffold for neuronal regeneration, since it mimics the natural mechanical cell environment and enhances neuritic outgrowth. However, further in vitro and in vivo investigations are required to prove the efficacy of this potential scaffold