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Idiopathic pulmonary fibrosis (IPF) is a chronic disease of the lung caused by a rampant inflammatory response that results in the deposition of excessive extracellular matrix (ECM). IPF patient lungs also develop fibroblastic foci that consist of activated fibroblasts and myofibroblasts. In concert with ECM deposition, the increased cell density within fibroblastic foci imposes confining forces on lung fibroblasts. In this work, we observed that increased cell density increases the incidence of the fibroblast-to-myofibroblast transition (FMT), but mechanical confinement imposed by micropillars has no effect on FMT incidence. We found that human lung fibroblasts (HLFs) express more α-SMA and deposit more collagen matrix, which are both characteristics of myofibroblasts, in response to TGF-ß1 when cells are seeded at a high density compared with a medium or a low density. These results support the hypothesis that HLFs undergo FMT more readily in response to TGF-ß1 when cells are densely packed, and this effect could be dependent on increased OB-cadherin expression. This work demonstrates that cell density is an important factor to consider when modelling IPF in vitro, and it may suggest decreasing cell density within fibroblastic foci as a strategy to reduce IPF burden.

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Mesenchymal stem cells (MSCs) and tumor cells have the unique capability to migrate out of their native environment and either home or metastasize, respectively, through extremely heterogeneous environments to a distant location. Once there, they can either aid in tissue regrowth or impart an immunomodulatory effect in the case of MSCs, or form secondary tumors in the case of tumor cells. During these journeys, cells experience physically confining forces that impinge on the cell body and the nucleus, ultimately causing a multitude of cellular changes. Most drastically, confining individual MSCs within hydrogels or confining monolayers of MSCs within agarose wells can sway MSC lineage commitment, while applying a confining compressive stress to metastatic tumor cells can increase their invasiveness. In this review, we seek to understand the signaling cascades that occur as cells sense confining forces and how that translates to behavioral changes, including elongated and multinucleated cell morphologies, novel migrational mechanisms, and altered gene expression, leading to a unique MSC secretome that could hold great promise for anti-inflammatory treatments. Through comparison of these altered behaviors, we aim to discern how MSCs alter their lineage selection, while tumor cells may become more aggressive and invasive. Synthesizing this information can be useful for employing MSCs for therapeutic approaches through systemic injections or tissue engineered grafts, and developing improved strategies for metastatic cancer therapies.

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Histatin 5 (Hst-5) is an antimicrobial peptide with strong antifungal activity against Candida albicans, an opportunistic pathogen that is a common cause of oral thrush. The peptide is natively secreted by human salivary glands and shows promise as an alternative therapeutic against infections caused by C. albicans. However, Hst-5 can be cleaved and inactivated by a family of secreted aspartic proteases (Saps) produced by C. albicans. Single-residue substitutions can significantly affect the proteolytic resistance of Hst-5 to Saps and its antifungal activity; the K17R substitution increases resistance to proteolysis, while the K11R substitution enhances antifungal activity. In this work, we showed that the positive effects of these two single-residue modifications can be combined in a single peptide, K11R-K17R, with improved proteolytic resistance and antifungal activity. We also investigated the effect of additional single-residue substitutions, with a focus on the effect of addition or removal of negatively charged residues, and found Sap-dependent effects on degradation. Both single- and double-substitutions affected the kinetics of proteolytic degradation of the intact peptide and of the fragments formed during degradation. Our results demonstrate the importance of considering proteolytic stability and not just antimicrobial activity when designing peptides for potential therapeutic applications.

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Mechanical cues such as stiffness have been shown to influence cell gene expression, protein expression, and cell behaviors critical for tissue engineering. The mechanical cue of confinement is also a pervasive parameter affecting cells in vivo and in tissue-engineered constructs. Despite its prevalence, the mechanical cue of confinement lacks assays that provide precise control over the degree of confinement induced on cells, yield a large sample size, enable long-term culture, and enable easy visualization of cells over time. In this study, we developed a process to systematically confine cells using micropillar arrays. Using photolithography and polydimethylsiloxane (PDMS) molding, we created PDMS arrays of micropillars that were 5, 10, 20, or 50 µm in spacing and between 13 and 17 µm in height. The tops of micropillars were coated with Pluronic F127 to inhibit cell adhesion, and we observed that mesenchymal stem cells (MSCs) robustly infiltrated into the micropillar arrays. MSC and nucleus morphology were altered by narrowing the micropillar spacing, and cytoskeletal elements within MSCs appeared to become more diffuse with increasing confinement. Specifically, MSCs exhibited a ring of actin around their periphery and punctate focal adhesions. MSC migration speed was reduced by narrowing micropillar spacing, and distinct migration behaviors of MSCs emerged in the presence of micropillars. MSCs continued to proliferate within micropillar arrays after 3 weeks in culture, displaying our assay's capability for long-term studies. Our assay also has the capacity to provide adequate cell numbers for quantitative assays to investigate the effect of confinement on gene and protein expression. Through deeper understanding of cell mechanotransduction in the context of confinement, we can modify tissue-engineered constructs to be optimal for a given purpose. Impact Statement In this study, we developed a novel process to systematically confine cells using micropillar arrays. Our assay provides insight into cell behavior in response to mechanical confinement. Through deeper understanding of how cells sense and respond to confinement, we can fine tune tissue-engineered constructs to be optimal for a given purpose. By combining confinement with other physical cues, we can harness mechanical properties to encourage or inhibit cell migration, direct cells down a particular lineage, induce cell secretion of specific cytokines or extracellular vesicles, and ultimately direct cells to behave in a way conducive to tissue engineering.

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Mechanosensing of the mechanical microenvironment by cells regulates cell phenotype and function. The nucleus is critical in mechanosensing, as it transmits external forces from the cellular microenvironment to the nuclear envelope housing chromatin. This study aims to elucidate how mechanical confinement affects nuclear deformation within several cell types, and to determine the role of cytoskeletal elements in controlling nuclear deformation. Human cancer cells (MDA-MB-231), human mesenchymal stem cells (MSCs), and mouse fibroblasts (L929) were seeded within polydimethylsiloxane (PDMS) microfluidic devices containing microchannels of varying cross-sectional areas, and nuclear morphology and volume were quantified via image processing of fluorescent cell nuclei. We found that the nuclear major axis length remained fairly constant with increasing confinement in MSCs and MDA-MB-231 cells, but increased with increasing confinement in L929 cells. Nuclear volume of L929 cells and MSCs decreased in the most confining channels. However, L929 nuclei were much more isotropic in unconfined channels than MSC nuclei. When microtubule polymerization or myosin II contractility was inhibited, nuclear deformation was altered only in MSCs in wide channels. This work informs our understanding of nuclear mechanics in physiologically relevant spaces, and suggests diverging roles of the cytoskeleton in regulating nuclear deformation in different cell types.

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The in vivo microenvironment is critical for providing physico-chemical signaling cues which ultimately regulate human mesenchymal stem cell (hMSC) behavior in clinically-relevant applications. hMSCs experience mechanical confinement of the cell body and nucleus in three dimensional (3D) tissues during homing and in porous tissue engineered scaffolds, yet the effects of this mechanical cue on hMSC migration are not known. Here, we use a microchannel device to systematically examine the effect of confinement on hMSC migration and cytoskeletal organization. Notably, we show that hMSC actin and microtubules change from filamentous in unconfined spaces to a more diffuse network in confinement, and that confinement abrogates the presence of paxillin-rich focal adhesions seen in 2D. Furthermore, several morphological parameters of the hMSC body are altered in confinement. Interestingly, hMSC speed displays a biphasic trend as a function of confinement, and increasing hMSC passage number decreases speed in all but the narrowest microchannels. Confinement also alters the relative contributions of cytoskeletal (i.e., actin and microtubule) and contractile (i.e., myosin II and Rho kinase) machinery in hMSC migration in unconfined and confined spaces. These results provide an improved understanding of how hMSCs navigate mechanical confinement, which is a central component of complicated 3D microenvironments. Hence, this work may provide insight towards more effective control of hMSC localization in porous tissue engineered scaffolds and mobilization to distinct tissue sites during homing after clinical therapy.

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