Influence Cell Behavior Research Articles

Boosting bone regeneration using augmented melt-extruded additive-manufactured scaffolds

ABSTRACT Bone tissue engineering (BTE) is in active search of the ideal scaffold to give a clinical solution for bone regeneration in non-union fractures. During the last decades, the use of additive manufacturing (AM), and, in particular, melt extrusion AM (ME-AM), has been investigated towards this aim. ME-AM enables the fabrication of personalized 3D scaffolds, with a controlled and highly interconnected porosity, through the solvent-free processing of biodegradable and mechanically robust polymers. In addition to these properties matching the requirements for BTE scaffolds, the polymers used to fabricate these constructs are also more amenable for further functionalization than metals or ceramics, to influence cell behaviour, making thermoplastic materials a preferred choice for BTE. This review provides a comprehensive analysis of various ME-AM scaffolds developed for BTE, along with approaches used to augment their bioactivity, which includes architectural, surface physical and chemical modifications, the incorporation of secondary fibrous or hydrogel networks within the scaffold pores, and the use of composites for ME-AM scaffold fabrication.

Urea is a drop-in nitrogen source alternative to ammonium sulphate in Yarrowia lipolytica.

Media components, including the nitrogen source, are significant cost factors in cultivation processes. The nitrogen source also influences cell behavior and production performance. Ammonium sulfate is a widely used nitrogen source for microorganisms' cultivation. Urea is a sustainable and cheap alternative nitrogen source. We investigated the influence of urea as a nitrogen source compared to ammonium sulfate by cultivating phenotypically different Yarrowia lipolytica strains in chemostats under carbon or nitrogen limitation. We found no significant coherent changes in growth and lipid production. RNA sequencing revealed no significant concerted changes in the transcriptome. The genes involved in urea uptake and degradation are not upregulated on a transcriptional level. Our findings support urea usage, indicating that previous metabolic engineering efforts where ammonium sulfate was used are likely translatable to the usage of urea and can ease the way for urea as a cheap and sustainable nitrogen source in more applications.

Changes of Mutations and Copy-Number and Enhanced Cell Migration during Breast Tumorigenesis.

Although cancer stem cells (CSCs) play a major role in tumorigenesis and metastasis, the role of genetic alterations in invasiveness of CSCs is still unclear. Tumor microenvironment signals, such as extracellular matrix (ECM) composition, significantly influence cell behaviors. Unfortunately, these signals are often lost in in vitro cell culture. This study determines putative CSC populations, examines genetic changes during tumorigenesis of human breast epithelial stem cells, and investigates single-cell migration properties on ECM-mimetic platforms. Whole exome sequencing data indicate that tumorigenic cells have a higher somatic mutation burden than non-tumorigenic cells, and that mutations exclusive to tumorigenic cells exhibit higher predictive deleterious scores. Tumorigenic cells exhibit distinct somatic copy number variations (CNVs) including gain of duplications in chromosomes 5 and 8. ECM-mimetic topography selectively enhances migration speed of tumorigenic cells, but not of non-tumorigenic cells, and results in a wide distribution of tumorigenic single-cell migration speeds, suggesting heterogeneity in cellular sensing of contact guidance cues. This study identifies mutations and CNVs acquired during breast tumorigenesis, which can be associated with enhanced migration of breast tumorigenic cells, and demonstrates that a nanotopographically-defined platform can be applied to recapitulate an ECM structure for investigating cellular migration in the simulated tumor microenvironment.

Quantifying epithelial cell proliferation on curved surfaces

Out-of-plane curvature is an important, but poorly explored geometric parameter that influences cell behavior. We address the impact of curvature on epithelial proliferation through monitoring how MDCK cells proliferate on planar and curved toroidal hydrogel substrates with a broad range of Gaussian curvatures. We illustrate in detail the imaging processing methodology to characterize curved surfaces and quantify proliferation of cells. We find that MDCK cells grow readily on both curved and flat surfaces and can cover the entire surface of the toroidal structure as long as the initial seeding is uniform. Our analysis shows that proliferation does not depend on Gaussian curvature within the range probed in our experiment, but rather on cell density. Despite epithelial proliferation is insensitive to the curvature range presented in this study, the toroidal-construct fabrication technique and image processing methodology may find utility for probing cell processes like collective migration, as it involves long-range force transmission.

The solvent chosen for the manufacturing of electrospun polycaprolactone scaffolds influences cell behavior of lung cancer cells

The development of a trustworthy in vitro lung cancer model is essential to better understand the illness, find novel biomarkers, and establish new treatments. Polycaprolactone (PCL) electrospun nanofibers are a cost-effective and ECM-like approach for 3D cell culture. However, the solvent used to prepare the polymer solution has a significant impact on the fiber morphology and, consequently, on the cell behavior. Hence, the present study evaluated the effect of the solvent employed in the manufacturing on the physical properties of 15%-PCL electrospun scaffolds and consequently, on cell behavior of NCI-H1975 lung adenocarcinoma cells. Five solvents mixtures (acetic acid, acetic acid-formic acid (3:1, v/v), acetone, chloroform-ethanol (7:3, v/v), and chloroform-dichloromethane (7:3, v/v)) were tested. The highest cell viability (overline{x } = 33.4%) was found for cells cultured on chloroform-ethanol (7:3) PCL scaffolds. Chloroform-dichloromethane (7:3) PCL scaffolds exhibited a roughness that enhanced the quality of electrospun filament, in terms of cell viability. Our findings highlighted the influence of the solvent on fiber morphology and protein adsorption capacity of nanofilaments. Consequently, these features directly affected cell attachment, morphology, and viability.

Osteoglycin: An ECM Factor Regulating Fibrosis and Tumorigenesis

The extracellular matrix (ECM) is made up of noncellular components that have special properties for influencing cell behavior and tissue structure. Small leucine-rich proteoglycans (SLRPs) are nonfibrillar ECM components that serve as structural scaffolds and signaling molecules. osteoglycin (OGN), a class III SLRP, is a ubiquitous ECM component that not only helps to organize the extracellular matrix but also regulates a number of important biological processes. As a glycosylated protein in the ECM, OGN was originally considered to be involved in fiber assembly and was reported to have a connection with fibrosis. In addition to these functions, OGN is found in a variety of cancer tissues and is implicated in cellular processes linked to tumorigenesis, including cell proliferation, invasion, metastasis, and epithelial-mesenchymal transition (EMT). In this review, we summarize the structure and functions of OGN as well as its biological and clinical importance in the context of fibrotic illness and tumorigenesis. This review aims to improve our understanding of OGN and provide some new strategies for the treatment of fibrosis and cancer.

Investigating the effects of low intensity visible light on human keratinocytes using a customized LED exposure system

Photobiomodulation (PBM) refers to the use of light to modulate cellular processes, and has demonstrated utility in improving wound healing outcomes, and reducing pain and inflammation. Despite the potential benefits of PBM, the precise molecular mechanisms through which it influences cell behavior are not yet well understood. Inconsistent reporting of key light parameters has created uncertainty around optimal exposure profiles. In addition, very low intensities of light, < 0.1 J/cm2, have not been thoroughly examined for their use in PBM. Here, we present a custom-made compact, and modular LED-based exposure system for studying the effects of very low-intensity visible light (cell proliferation, migration, ROS production, and mitochondrial membrane potential) of three different wavelengths in a parallel manner. The device allows for six repeats of three different exposure conditions plus a non-irradiated control on a single 24-well plate. The immortalised human keratinocyte cell line, HaCaT, was selected as a major cellular component of the skin epidermal barrier. Furthermore, an in vitro wound model was developed by allowing the HaCaT to form a confluent monolayer, then scratching the cells with a pipette tip to form a wound. Cells were exposed to yellow (585 nm, 0.09 mW, ~ 3.7 mJ/cm2), orange (610 nm, 0.8 mW, ~ 31 mJ/cm2), and red (660 nm, 0.8 mW, ~ 31 mJ/cm2) light for 10 min. 48 h post-irradiation, immunohistochemistry was performed to evaluate cell viability, proliferation, ROS production, and mitochondrial membrane potential. The results demonstrate increased proliferation and decreased scratch area for all exposure conditions, however only red light increased the mitochondrial activity. Oxidative stress levels did not increase for any of the exposures. The present exposure system provides opportunities to better understand the complex cellular mechanisms driven by the irradiation of skin cells with visible light.

Establishment of fibroblast and myofibroblast phenotypes for use in in vitro co-culture models

Fibroblasts function to secrete and modify components of the extracellular matrix. During wound healing, fibroblasts migrate to the site of injury and differentiate into contractile myofibroblasts; this differentiation is characterised by an increased contractile capacity. Fully differentiated myofibroblasts can be distinguished from fibroblasts via the higher expression of α-smooth muscle actin as well as a denser cytoskeleton. Impaired wound healing has been characterised by a lack of myofibroblasts; as a result, tissue does not fully regain its strength and function. Under pathological conditions, this may be associated with the effect that a pro-inflammatory microenvironment has on fibroblast and skeletal muscle progenitor cell migration and differentiation.Given their distinct roles in tissue maintenance and repair, the communication between fibroblasts versus myofibroblasts with other cellular mediators of repair is likely to influence cell behaviour and the outcome of wound repair. An in vitro test model is required to investigate this intercellular influence, but the establishment of such a model is hampered by the difficulty in retaining the dedifferentiated fibroblastic phenotype under regular serum-containing cell culture conditions. We present a model that supports the establishment and retention in culture of fibroblast and myofibroblast phenotypes for use in a simple, inexpensive, yet relevant in vitro 2D assay. This model is then applied in a co-culture setting to determine whether the presence of myoblasts affects the ability of fibroblasts versus myofibroblasts to close an in vitro wound. Our results emphasize the importance of considering the impact of paracrine communication between all cells during wound healing.

Influence of loading rate and out of plane direction dependence on deformation and electro-mechanical failure behavior of a lithium-ion pouch cell

Understanding the behavior of lithium-ion battery cells under complex load situations that can occur in vehicle crash scenarios is important to integrate the cells in the best possible way in an electrified vehicle. Since individual cell components partly show highly anisotropic material behavior, this work investigates whether a direction-dependent behavior is present on cell level when loaded in the out-of-plane direction, which has not been investigated so far. Cylindrical indentation tests in out-of-plane direction were performed on a 41 Ah NMC pouch cell under laboratory conditions. Thereby, impactor orientation, loading speed and electrolyte quantity were varied in order to study the respective influences both, separately as well as in interaction with each other. Component tensile tests with different directions and speeds were carried out. The results were compared with those of the cell tests for a better understanding of the observed electro-mechanical behavior. It could be shown that the investigated cell shows a significant out-of-plane direction dependence at low-speed loading (1 mm/s). This results in an approximately 23 % lower failure strain when the impactor is aligned along the long side of the cell compared to its alignment along shot side. Strain rate influences cell behavior, resulting in higher stiffness and lower failure strain at higher loading speeds (3000 mm/s). Furthermore, the out-of-plane direction dependence is reduced at higher loading speed, which is in direct contrast to the observations of the tensile tests. This is attributed to viscosity effects during high-speed loading, since the liquid electrolyte affects the mechanical cell behavior, as shown by the experiments with reduced electrolyte quantity. This study shows that the out-of-plane direction dependence should be taken into account for the evaluation of the crash safety of a lithium-ion pouch cell.

Measuring dimensionality of cell-scaffold contacts of primary human bone marrow stromal cells cultured on electrospun fiber scaffolds.

The properties and structure of the cellular microenvironment can influence cell behavior. Sites of cell adhesion to the extracellular matrix (ECM) initiate intracellular signaling that directs cell functions such as proliferation, differentiation, and apoptosis. Electrospun fibers mimic the fibrous nature of native ECM proteins and cell culture in fibers affects cell shape and dimensionality, which can drive specific functions, such as the osteogenic differentiation of primary human bone marrow stromal cells (hBMSCs), by. In order to probe how scaffolds affect cell shape and behavior, cell-fiber contacts were imaged to assess their shape and dimensionality through a novel approach. Fluorescent polymeric fiber scaffolds were made so that they could be imaged by confocal fluorescence microscopy. Fluorescent polymer films were made as a planar control. hBSMCs were cultured on the fluorescent substrates and the cells and substrates were imaged. Two different image analysis approaches, one having geometrical assumptions and the other having statistical assumptions, were used to analyze the 3D structure of cell-scaffold contacts. The cells cultured in scaffolds contacted the fibers in multiple planes over the surface of the cell, while the cells cultured on films had contacts confined to the bottom surface of the cell. Shape metric analysis indicated that cell-fiber contacts had greater dimensionality and greater 3D character than the cell-film contacts. These results suggest that cell adhesion site-initiated signaling could emanate from multiple planes over the cell surface during culture in fibers, as opposed to emanating only from the cell's basal surface during culture on planar surfaces.

Dual-Crosslinked Alginate-Based Hydrogels with Tunable Mechanical Properties for Cultured Meat.

Cultured meat refers to the production of animal tissue by utilizing the same techniques as tissue engineering through cell culture. Various biomaterials have been designed to serve as in vitro supports for cell viability, growth, and migration. In this study, visible light and dual-crosslinked alginate hydrogels were designed to enable control of the physical and mechanical properties needed for the fabrication of cultured meat scaffolds. We hypothesized that a difference in hydrogel stiffness would influence cell behavior, indicating the efficacy of our processing methods to benefit the cultured meat field. Herein, we synthesized and created: (1) methacrylated alginate (AlgMA) to enable covalent crosslinking via visible light exposure, (2) Methacrylated alginate and arginyl-glycyl-aspartic acid RGD conjugates (AlgMA-RGD), using carbodiimide chemistries to provide cell-binding sites on the material, and (3) designer hydrogels incorporating different crosslinking techniques. The material and mechanical properties were evaluated to determine the structural integrity of the hydrogels, and in vitro cell assays were conducted to verify cytocompatibility and cell adhesion. Gelation, swell ratio, and weight loss calculations revealed longer gelation times for the AlgMA scaffolds and similar physical properties for all hydrogel groups. We showed that by adjusting the polymer concentration and the crosslinking methodology, the scaffold’s mechanical properties can be controlled and optimized within physiological ranges. Incorporating dual crosslinking significantly increased the compressive moduli of the AlgMA hydrogels, compared to visible-light crosslinking alone. Moreover, the muscle satellite cells responded favorably to the AlgMA scaffolds, with clear differences in cell density when cultured on materials with significantly different mechanical properties. Our results indicate the usefulness of the dual-crosslinking alginate hydrogel system to support in vitro meat growth.

Synergistic Effect of miR-200 and Young Extracellular Matrix-based Bio-scaffolds to Reduce Signs of Aging in Senescent Fibroblasts

Aging is defined as a complex, multifaceted degenerative process that causes a gradual decline of physiological functions and a rising mortality risk with time. Stopping senescence or even rejuvenating the body represent one of the long-standing human dreams. Somatic cell nuclear transfer as well as cell reprogramming have suggested the possibility to slow or even reverse signs of aging. We exploited miR-200 family ability to induce a transient high plasticity state in human skin fibroblasts isolated from old individuals and we investigated whether this ameliorates cellular and physiological hallmarks of senescence. In addition, based on the assumption that extracellular matrix (ECM) provides biomechanical stimuli directly influencing cell behavior, we examine whether ECM-based bio-scaffolds, obtained from decellularized ovaries of young swine, stably maintain the rejuvenated phenotype acquired by cells after miR-200 exposure. The results show the existence of multiple factors that cooperate to control a unique program, driving the cell clock. In particular, miR-200 family directly regulates the molecular mechanisms erasing cell senescence. However, this effect is transient, reversible, and quickly lost. On the other hand, the use of an adequate young microenvironment stabilizes the miR-200-mediated rejuvenating effects, suggesting that synergistic interactions occur among molecular effectors and ECM-derived biomechanical stimuli. The model here described is a useful tool to better characterize these complex regulations and to finely dissect the multiple and concurring biochemical and biomechanical cues driving the cell biological clock.Graphical

Viscoelastic properties of plasma-agarose hydrogels dictate favorable fibroblast responses for skin tissue engineering applications.

Dermal wound healing relies on the properties of the extracellular matrix (ECM). Thus, hydrogels that replicate skin ECM have reached clinical application. After a dermal injury, a transient, biodegradable fibrin clot is instrumental in wound healing. Human plasma, and its main constituent, fibrin would make a suitable biomaterial for improving wound healing and processed as hydrogels albeit with limited mechanical strength. To overcome this, plasma-agarose (PA) composite hydrogels have been developed and used to prepare diverse bioengineered tissues. To date, little is known about the influence of variable agarose concentrations on the viscoelastic properties of PA hydrogels and their correlation to cell biology. This study reports the characterization of the viscoelastic properties of different concentrations of agarose in PA hydrogels: 0%, 0.5%, 1%, 1.5%, and 2% (w/v), and their influence on the cell number and mitochondrial activity of human dermal fibroblasts. Results show that agarose addition increased the stiffness, relaxation time constants 1 (τ1) and 2 (τ2), and fiber diameter, whereas the porosity decreased. Changes in cell metabolism occurred at the early stages of culturing and correlated to the displacement of fast (τ1) and intermediate (τ2) Maxwell elements. Fibroblasts seeded in low PA concentrations spread faster during 14 d than cells cultured in higher agarose concentrations. Collectively, these results confirm that PA viscoelasticity and hydrogel architecture strongly influenced cell behavior. Therefore, viscoelasticity is a key parameter in the design of PA-based implants.

COLLOID–CELL INTERACTION ANALYSIS WITH ATOMIC FORCE MICROSCOPY — ζ CALCULATION AND ADHESION ANALYSIS

The interaction between cells and colloids is an important characteristic that influences cell behavior. Theoretically, much information could be revealed by analyzing the interactions in colloid–cell contact. In this study, in order to explore the interaction between cells and colloids, we developed a novel computational method able to obtain a zeta potential directly calculated from the force distance curve and apply to adhesion analysis, which used atomic force microscope (AFM), based on DLVO (Deryaguin–Landau–Verwey–Overbeek) theory and Mann Whitney U test, and combined with Zetasizer measurement. The calculation and analysis of [Formula: see text] of the cell surfaces of ncyc-1324 yeast, ncyc-1681 yeast and Pseudomonas fluorescens showed that pH affected the electrostatic distribution on the cell surface. Compared with the previous research methods, this method significantly reduces the computation and manual control, which is an effective method for multi-element surface analysis and comparison. For example, the reverse calculation and curve fitting method will significantly request more computation and manual control to set up the reference force curve that simulated with set zeta potential, while this method only need to calculate on one force curve. The deconvolution of different adhesion events from force curves showed that the heterogeneity of cell surface can be significantly displayed. This provides a method for determining the complexity of the cell surface. Furthermore, this method was used to study the effect of amoxicillin on cell surface interaction, which showed that the cells surface forces were influenced even the medicine concentration is not enough to make significant influence on microbials optical observation appearance. Thus, AFM force analysis is a more sensitive method to research the medicine influence compared to the traditional method.

Insight and Recent Advances into the Role of Topography on the Cell Differentiation and Proliferation on Biopolymeric Surfaces.

It is well known that surface topography plays an important role in cell behavior, including adhesion, migration, orientation, elongation, proliferation and differentiation. Studying these cell functions is essential in order to better understand and control specific characteristics of the cells and thus to enhance their potential in various biomedical applications. This review proposes to investigate the extent to which various surface relief patterns, imprinted in biopolymer films or in polymeric films coated with biopolymers, by utilizing specific lithographic techniques, influence cell behavior and development. We aim to understand how characteristics such as shape, dimension or chemical functionality of surface relief patterns alter the orientation and elongation of cells, and thus, finally make their mark on the cell proliferation and differentiation. We infer that such an insight is a prerequisite for pushing forward the comprehension of the methodologies and technologies used in tissue engineering applications and products, including skin or bone implants and wound or fracture healing.

Understanding thermal and mechanical effects on lithium plating in lithium-ion batteries

Lithium plating is one of the main concerns for cell durability, as cyclable lithium and cell capacity are decreased during the process. Thermal and mechanical loads can influence cell behavior, including lithium plating driving forces, as suggested by the Arrhenius equation and the Bruggeman relation, respectively. Hence, a 1D and a 3D model are employed to study the thermal and mechanical effects on lithium plating in a lithium-ion battery with a high areal capacity (4 mAh/cm2). The 1D model assumes uniform mechanical and temperature profile, while the 3D model is capable of capturing the nonuniformity of stress and temperature in the cell. The effects of mechanical deformation and different temperatures on the process of lithium plating are investigated in this study. With the help of the 1D charging simulations, it is observed that lower temperatures increase the risk of lithium plating and the presence of mechanical deformations can accelerate its initiation. Furthermore, 3D charging simulations indicated that folds and boundaries of jellyrolls are more susceptible to lithium plating due to the higher stress concentration and lower temperature, respectively.

Living Biointerfaces for the Maintenance of Mesenchymal Stem Cell Phenotypes

AbstractLiving interfaces are established as a novel class of active materials that aim to provide an alternative to traditional static cell culture methods by enabling users to accurately control cell behaviour in a precise, dynamic, and reliable system‐internal manner. To this day, the only reported biointerface has been a coculture between a biofilm of nonpathogenic genetically engineered bacteria and mammalian cells, where the recombinant proteins produced by the bacteria directly influence cell behaviour. In this work, a biointerface is presented between Lactococcus lactis (L. lactis) and human mesenchymal stem cells (hMSCs). L. lactis have been engineered to produce human C‐X‐C motif chemokine ligand 12, thrombopoietin, vascular cell adhesion protein 1, and the 7th–10th type III domains of human fibronectin, with the aim of recreating the native bone marrow conditions ex vivo. This active microenvironment has been shown to maintain key hMSC stemness markers, preventing their osteogenic and adipogenic differentiation, and maintaining high stem cell viability and physiological cell‐to‐substrate adhesion dynamics. This work presents proof of concept data that hMSC stemness can be regulated by living materials, using a system based on the symbiotic interaction between different engineered bacteria and mammalian cells.

In preprints: buckling under pressure during gastrulation.

In preprints: buckling under pressure during gastrulation.

Engineered Living Hydrogels.

Living biological systems, ranging from single cells to whole organisms, can sense, process information, and actuate in response to changing environmental conditions. Inspired by living biological systems, engineered living cells and nonliving matrices are brought together, which gives rise to the technology of engineered living materials. By designing the functionalities of living cells and the structures of nonliving matrices, engineered living materials can be created to detect variability in the surrounding environment and to adjust their functions accordingly, thereby enabling applications in health monitoring, disease treatment, and environmental remediation. Hydrogels, a class of soft, wet, and biocompatible materials, have been widely used as matrices for engineered living cells, leading to the nascent field of engineered living hydrogels. Here, the interactions between hydrogel matrices and engineered living cells are described, focusing on how hydrogels influence cell behaviors and how cells affect hydrogel properties. The interactions between engineered living hydrogels and their environments, and how these interactions enable versatile applications, are also discussed. Finally, current challenges facing the field of engineered living hydrogels for their applications in clinical and environmental settings are highlighted.

Cytokinin promotes growth cessation in the Arabidopsis root

The Arabidopsis root offers good opportunities to investigate how regulated cellular growth shapes different tissues and organs, a key question in developmental biology. Along the root's longitudinal axis, cells sequentially occupy different developmental states. Proliferative meristematic cells give rise to differentiating cells, which rapidly elongate in the elongation zone, then mature and stop growing in the differentiation zone. The phytohormone cytokinin contributes to this zonation by positioning the boundary between the meristem and the elongation zone, called the transition zone. However, the cellular growth profile underlying root zonation is not well understood, and the cellular mechanisms that mediate growth cessation remain unclear. By using time-lapse imaging, genetics, and computational analysis, we analyze the effect of cytokinin on root zonation and cellular growth. We found that cytokinin promotes growth cessation in the distal (shootward) elongation zone in conjunction with accelerating the transition from elongation to differentiation. We estimated cell-wall stiffness by using osmotic treatment experiments and found that cytokinin-mediated growth cessation is associated with cell-wall stiffening and requires the action of an auxin influx carrier, AUX1. Our measurement of growth and cell-wall mechanical properties at a cellular resolution reveal mechanisms via which cytokinin influences cell behavior to shape tissue patterns.