Publications

Human mesenchymal stromal cells (hMSCs) seeded on calcium phosphate (CaP) bioceramics are extensively explored in bone tissue engineering and have recently shown effective clinical outcomes. In previous pre-clinical studies, hMSCs-CaP-mediated bone formation was preceded by osteoclastogenesis at the implantation site. The current study evaluates to what extent phase composition of CaPs affects the osteoclast response and ultimately influence bone formation. To this end, four different CaP bioceramics were used, hydroxyapatite (HA), beta-tricalcium phosphate (beta-TCP) and two biphasic composites of HA/beta- TCP ratios of 60/40 and 20/80 respectively, for in vitro osteoclast differentiation and correlation with in vivo osteoclastogenesis and bone formation. All ceramics allowed osteoclast formation in vitro from mouse and human precursors, except for pure HA, which significantly impaired their maturation. Ectopic implantation alongside hMSCs in subcutis sites of nude mice revealed new bone formation at 8 weeks in all conditions with relative amounts for beta-TCP > biphasic CaPs > HA. Surprisingly, while hMSCs were essential for osteoinduction, their survival did not correlate with bone formation. By contrast, the degree of early osteoclastogenesis (2 weeks) seemed to define the extent of subsequent bone formation. Together, our findings suggest that the osteoclastic response could be used as a predictive marker in hMSC-CaPbased bone regeneration and strengthens the need to understand the underlying mechanisms for future biomaterial development. Statement of significance The combination of mesenchymal stromal cells (MSCs) and calcium phosphate (CaP) materials has demonstrated its safety and efficacy for bone regeneration in clinical trials, despite our insufficient understanding of the underlying biological mechanisms. Osteoclasts were previously suggested as key mediators between the early inflammatory phase following biomaterial implantation and the subsequent bone formation. Here we compared the affinity of osteoclasts for various CaP materials with different ratios of hydroxyapatite to beta-tricalcium phosphate. We found that osteoclast formation, both in vitro and at early stages in vivo, correlates with bone formation when the materials were implanted alongside MSCs in mice. Surprisingly, MSC survival did not correlate with bone formation, suggesting that the number or phenotype of osteoclasts formed was more important. (c) 2024 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )

JTD Keywords: Acid phosphatase tartrate resistant isoenzyme, Animal, Animal cell, Animal experiment, Animal tissue, Animals, Article, Beta-tricalcium phosphate, Bioceramics, Biocompatible materials, Biomaterial, Bone, Bone development, Bone formation, Bone regeneration, Calcium phosphate, Calcium phosphate materials, Calcium phosphates, Cd14 antigen, Cell differentiation, Cell engineering, Cell maturation, Cell survival, Ceramics, Chemical composition, Controlled study, Correlation analysis, Correlation coefficient, Data correlation, Durapatite, Engraftment, Flowcharting, Human, Human cell, Human mesenchymal stromal cell, Human mesenchymal stromal cells, Humans, Hydroxyapatite, Hydroxyapatites, In vitro study, In vivo study, In-vitro, In-vivo, Mammals, Marrow stromal cells, Material composition, Material compositions, Mesenchymal stroma cell, Mesenchymal stromal cells, Mice, Mice, nude, Monocyte, Mouse, Nonhuman, Nude mouse, Ossification, Osteoclast, Osteoclastogenesis, Osteoclasts, Osteogenesis, Osteoinduction, Phase composition, Regeneration strategies, Resorption, Scaffolds, Stem-cells, Subcutaneous tissue, Tissue engineering, Transmission control protocol, Tri-calcium phosphates, Vimentin

Simple Summary Neuroblastoma (NB), a prevalent childhood cancer, presents challenges in treatment due to its cellular diversity and the presence of tumor-derived endothelial cells (TECs) associated with chemoresistance. We lack specific biomarkers for TECs, hindering effective therapies. We developed a stiffness-based in vitro platform simulating arterial and venous conditions to address this gap. Notably, adrenergic NB cells transdifferentiated into TECs where there was an arterial-like stiffness, while mesenchymal cells did not. This platform facilitated the identification of Globotriaosylceramide (GB3) as a novel TEC biomarker. Moreover, we harnessed Shiga toxin-functionalized nanoparticles for the specific targeting of GB3-positive cells, showing promise for future therapeutic strategies. Our study provides insights into NB heterogeneity, offers a predictive tool for assessing aggressiveness, and introduces potential targets for precision therapies.Abstract Neuroblastoma (NB) is a childhood cancer in sympathetic nervous system cells. NB exhibits cellular heterogeneity, with adrenergic and mesenchymal states displaying distinct tumorigenic potentials. NB is highly vascularized, and blood vessels can form through various mechanisms, including endothelial transdifferentiation, leading to the development of tumor-derived endothelial cells (TECs) associated with chemoresistance. We lack specific biomarkers for TECs. Therefore, identifying new TEC biomarkers is vital for effective NB therapies. A stiffness-based platform simulating human arterial and venous stiffness was developed to study NB TECs in vitro. Adrenergic cells cultured on arterial-like stiffness transdifferentiated into TECs, while mesenchymal state cells did not. The TECs derived from adrenergic cells served as a model to explore new biomarkers, with a particular focus on GB3, a glycosphingolipid receptor implicated in angiogenesis, metastasis, and drug resistance. Notably, the TECs unequivocally expressed GB3, validating its novelty as a marker. To explore targeted therapeutic interventions, nanoparticles functionalized with the non-toxic subunit B of the Shiga toxin were generated, because they demonstrated a robust affinity for GB3-positive cells. Our results demonstrate the value of the stiffness-based platform as a predictive tool for assessing NB aggressiveness, the discovery of new biomarkers, and the evaluation of the effectiveness of targeted therapeutic strategies.

JTD Keywords: Alternative vasculature, Angiogenesis, Cells, Differentiation, Gb3, Neuroblastoma, Origin, Tumor-derived endothelial cells

Recently, organoids have emerged as revolutionizing tools with the unprecedented potential to recreate organ-specific microanatomy in vitro. Upon their derivation from human pluripotent stem cells (hPSCs), organoids reveal the blueprints of human organogenesis, further allowing the faithful recapitulation of their physiology. Nevertheless, along with the evolution of this field, advanced research exposed the organoids' shortcomings, particularly regarding poor reproducibility rates and overall immatureness. To resolve these challenges, many studies have started to underscore the relevance of mechanical cues as a relevant source to induce and externally control hPSCs differentiation. Indeed, established organoid generation protocols from hPSCs have mainly relyed on the biochemical induction of fundamental signalling pathways present during kidney formation in mammals, whereas mechanical cues have largely been unexplored. This review aims to discuss the pertinence of (bio) physical cues within hPSCs-derived organoid cultures, while deciphering their effect on morphogenesis. Moreover, we will explore state-of-the-art mechanobiology techniques as revolutionizing means for understanding the underlying role of mechanical forces in biological processes in organoid model systems.

JTD Keywords: development, hpscs, mechanobiology, nephrogenesis, Activated ion-channel, Development, Extracellular-matrix viscoelasticity, Forces, Hpscs, Ips cells, Mechanical regulation, Mechanobiology, Nephrogenesis, Nephron progenitors, Organoids, Pluripotent stem-cells, Self-renewal, Substrate mechanics, Tissue

The function of organs such as lungs, kidneys and mammary glands relies on the three-dimensional geometry of their epithelium. To adopt shapes such as spheres, tubes and ellipsoids, epithelia generate mechanical stresses that are generally unknown. Here we engineer curved epithelial monolayers of controlled size and shape and map their state of stress. We design pressurized epithelia with circular, rectangular and ellipsoidal footprints. We develop a computational method, called curved monolayer stress microscopy, to map the stress tensor in these epithelia. This method establishes a correspondence between epithelial shape and mechanical stress without assumptions of material properties. In epithelia with spherical geometry we show that stress weakly increases with areal strain in a size-independent manner. In epithelia with rectangular and ellipsoidal cross-section we find pronounced stress anisotropies that impact cell alignment. Our approach enables a systematic study of how geometry and stress influence epithelial fate and function in three-dimensions.© 2023. The Author(s).

JTD Keywords: cell, forces, morphogenesis, tension, E-cadherin

Immunoassays show great potential for the detection of low levels of cytokines, due to their high sensitivity and excellent specificity. There is a particular demand for biosensors that enable both high-throughput screening and continuous monitoring of clinically relevant cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα). To this end, we here introduce a novel bioluminescent immunoassay based on the ratiometric plug-and-play immunodiagnostics (RAPPID) platform, with an improved intrinsic signal-to-background and an >80-fold increase in the luminescent signal. The new dRAPPID assay, comprising a dimeric protein G adapter connected via a semiflexible linker, was applied to detect the secretion of IL-6 by breast carcinoma cells upon TNFα stimulation and the production of low concentrations of IL-6 (∼18 pM) in an endotoxin-stimulated human 3D muscle tissue model. Moreover, we integrated the dRAPPID assay in a newly developed microfluidic device for the simultaneous and continuous monitoring of changes in IL-6 and TNFα in the low-nanomolar range. The luminescence-based read-out and the homogeneous nature of the dRAPPID platform allowed for detection with a simple measurement setup, consisting of a digital camera and a light-sealed box. This permits the usage of the continuous dRAPPID monitoring chip at the point of need, without the requirement for complex or expensive detection techniques.

JTD Keywords: cells, code, elisa, il-6, inflammation, kits, pathogenesis, procalcitonin, release, Interleukin-6

Sprouting angiogenesis is a core biological process critical to vascular development. Its accurate simulation, relevant to multiple facets of human health, is of broad, interdisciplinary appeal. This study presents an in-silico model replicating a microfluidic assay where endothelial cells sprout into a biomimetic extracellular matrix, specifically, a large-pore, low-concentration fibrin-based porous hydrogel, influenced by chemotactic factors. We introduce a novel approach by incorporating the extracellular matrix and chemotactic factor effects into a unified term using a single parameter, primarily focusing on modelling sprouting dynamics and morphology. This continuous model naturally describes chemotactic-induced sprouting with no need for additional rules. In addition, we extended our base model to account for matrix sensing and degradation, crucial aspects of angiogenesis. We validate our model via a hybrid in-silico experimental method, comparing the model predictions with experimental results derived from the microfluidic setup. Our results underscore the intricate relationship between the extracellular matrix structure and angiogenic sprouting, proposing a promising method for predicting the influence of the extracellular matrix on angiogenesis.Copyright © 2023 Ferre-Torres, Noguera-Monteagudo, Lopez-Canosa, Romero-Arias, Barrio, Castaño and Hernandez-Machado.

JTD Keywords: angiogenesis, biomimmetic, chemotaxis, endothelial cells, filopodia, growth, in silico model, mathematical models, mechanisms, metalloproteinase, migration, morphogenesis, phase field, pore-size, simulation, Angiogenesis, Biomimmetic, Chemotaxis, Endothelial cells, Extracellular matrix, In silico model, Mathematical models, Phase field, Tip cells

The symptoms of Myotonic Dystrophy Type 1 (DM1) are multi-systemic and life-threatening. The neuromuscular disorder is rooted in a non-coding CTG microsatellite expansion in the DM1 protein kinase (DMPK) gene that, upon transcription, physically sequesters the Muscleblind-like (MBNL) family of splicing regulator proteins. The high-affinity binding occurring between the proteins and the repetitions disallow MBNL proteins from performing their post-transcriptional splicing regulation leading to downstream molecular effects directly related to disease symptoms such as myotonia and muscle weakness. In this study, we build on previously demonstrated evidence showing that the silencing of miRNA-23b and miRNA-218 can increase MBNL1 protein in DM1 cells and mice. Here, we use blockmiR antisense technology in DM1 muscle cells, 3D mouse-derived muscle tissue, and in vivo mice to block the binding sites of these microRNAs in order to increase MBNL translation into protein without binding to microRNAs. The blockmiRs show therapeutic effects with the rescue of mis-splicing, MBNL subcellular localization, and highly specific transcriptomic expression. The blockmiRs are well tolerated in 3D mouse skeletal tissue inducing no immune response. In vivo, a candidate blockmiR also increases Mbnl1/2 protein and rescues grip strength, splicing, and histological phenotypes.

JTD Keywords: antisense oligonucleotides, aon, blockmir, brain, expression, genes, mbnl, mir-218, mir-23b, mirna, muscleblind, myotonic dystrophy 1, phenotypes, proteins, type-1, Messenger-rna, Muscleblind, Myotonic dystrophy 1

Surface curvature both emerges from, and influences the behavior of, living objects at length scales ranging from cell membranes to single cells to tissues and organs. The relevance of surface curvature in biology is supported by numerous experimental and theoretical investigations in recent years. In this review, first, a brief introduction to the key ideas of surface curvature in the context of biological systems is given and the challenges that arise when measuring surface curvature are discussed. Giving an overview of the emergence of curvature in biological systems, its significance at different length scales becomes apparent. On the other hand, summarizing current findings also shows that both single cells and entire cell sheets, tissues or organisms respond to curvature by modulating their shape and their migration behavior. Finally, the interplay between the distribution of morphogens or micro-organisms and the emergence of curvature across length scales is addressed with examples demonstrating these key mechanistic principles of morphogenesis. Overall, this review highlights that curved interfaces are not merely a passive by-product of the chemical, biological, and mechanical processes but that curvature acts also as a signal that co-determines these processes.© 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH.

JTD Keywords: biological systems, butterfly wing scales, cubic membranes, extracellular-matrix, geometry, mechanotransduction, membrane curvature, morphogenesis, neotissue growth, pattern-formation, soft materials, surface curvature, tissue-growth, Biological systems, Collective cell-migration, Surface curvature

Direct lineage reprogramming of one somatic cell into another without transitioning through a progenitor stage has emerged as a strategy to generate clinically relevant cell types. One cell type of interest is the pancreatic insulin-producing β cell whose loss and/or dysfunction leads to diabetes. To date it has been possible to create β-like cells from related endodermal cell types by forcing the expression of developmental transcription factors, but not from more distant cell lineages like fibroblasts. In light of the therapeutic benefits of choosing an accessible cell type as the cell of origin, in this study we set out to analyze the feasibility of transforming human skin fibroblasts into β-like cells. We describe how the timed-introduction of five developmental transcription factors (Neurog3, Pdx1, MafA, Pax4, and Nkx2-2) promotes conversion of fibroblasts toward a β-cell fate. Reprogrammed cells exhibit β-cell features including β-cell gene expression and glucose-responsive intracellular calcium mobilization. Moreover, reprogrammed cells display glucose-induced insulin secretion in vitro and in vivo. This work provides proof-of-concept of the capacity to make insulin-producing cells from human fibroblasts via transcription factor-mediated direct reprogramming.© 2023. The Author(s).

JTD Keywords: adult, beta-cells, differentiation, direct conversion, genes, in-vivo, islets, maturation, pancreatic progenitors, Pluripotent stem-cells

Tissue morphogenesis occurs in a complex physicochemical microenvironment with limited experimental accessibility. This often prevents a clear identification of the processes that govern the formation of a given functional shape. By applying state-of-the-art methods to minimal tissue systems, synthetic morphogenesis aims to engineer the discrete events that are necessary and sufficient to build specific tissue shapes. Here, we review recent advances in synthetic morphogenesis, highlighting how a combination of microfabrication and mechanobiology is fostering our understanding of how tissues are built.Copyright © 2022 Elsevier Ltd. All rights reserved.

JTD Keywords: cell dynamics, elongation, endothelial-cells, epithelium, growth, lumen, mechanical tension, patterns, self-organization, synthetic morphogenesis, tissue folding, tissue mechanics, topological defects, Stem-cells, Tissue shape

Biomolecular and physical cues of the extracellular matrix environment regulate collective cell dynamics and tissue patterning. Nonetheless, how the viscoelastic properties of the matrix regulate collective cell spatial and temporal organization is not fully understood. Here we show that the passive viscoelastic properties of the matrix encapsulating a spheroidal tissue of breast epithelial cells guide tissue proliferation in space and in time. Matrix viscoelasticity prompts symmetry breaking of the spheroid, leading to the formation of invading finger-like protrusions, YAP nuclear translocation and epithelial-to-mesenchymal transition both in vitro and in vivo in a Arp2/3-complex-dependent manner. Computational modelling of these observations allows us to establish a phase diagram relating morphological stability with matrix viscoelasticity, tissue viscosity, cell motility and cell division rate, which is experimentally validated by biochemical assays and in vitro experiments with an intestinal organoid. Altogether, this work highlights the role of stress relaxation mechanisms in tissue growth dynamics, a fundamental process in morphogenesis and oncogenesis.© 2022. The Author(s), under exclusive licence to Springer Nature Limited.

JTD Keywords: in-vitro, migration, morphogenesis, stiffness, Intestinal stem-cell

The extracellular matrix (ECM) of the lung is a filamentous network composed mainly of collagens, elastin, and proteoglycans that provides structural and physical support to its populating cells. Proliferation, migration and overall behaviour of those cells is greatly determined by micromechanical queues provided by the ECM. Lung fibrosis displays an aberrant increased deposition of ECM which likely changes filament organization and stiffens the ECM, thus upregulating the profibrotic profile of pulmonary cells. We have previously used AFM to assess changes in the Young’s Modulus (E) of the ECM in the lung. Here, we perform further ECM topographical, mechanical and viscoelastic analysis at the micro- and nano-scale throughout fibrosis development. Furthermore, we provide nanoscale correlations between topographical and elastic properties of the ECM fibres. Firstly, we identify a softening of the ECM after rats are instilled with media associated with recovery of mechanical homeostasis, which is hindered in bleomycin-instilled lungs. Moreover, we find opposite correlations between fibre stiffness and roughness in PBS- vs bleomycin-treated lung. Our findings suggest that changes in ECM nanoscale organization take place at different stages of fibrosis, with the potential to help identify pharmacological targets to hinder its progression.

JTD Keywords: atomic force microscopy, cells, deposition, extracellular matrix, idiopathic pulmonary fibrosis, mechanisms, mechanosensing, membranes, micromechanical properties, pathogenesis, stiffness, tissues, viscoelasticity, Extracellular matrix, Induced lung fibrosis, Mechanosensing

Abstract Oxidative stress, which occurs when an organism is exposed to an adverse stimulus that results in a misbalance of antioxidant and pro-oxidants species, is the common denominator of diseases considered as a risk factor for SARS-CoV-2 lethality. Indeed, reactive oxygen species caused by oxidative stress have been related to many virus pathogenicity. In this work, simulations have been performed on the receptor binding domain of SARS-CoV-2 spike glycoprotein to study what residues are more susceptible to be attacked by ·OH, which is one of the most reactive radicals associated to oxidative stress. The results indicate that isoleucine (ILE) probably plays a crucial role in modification processes driven by radicals. Accordingly, QM/MM-MD simulations have been conducted to study both the ·OH-mediated hydrogen abstraction of ILE residues and the induced modification of the resulting ILE radical through hydroxylation or nitrosylation reactions. All in all, in silico studies show the importance of the chemical environment triggered by oxidative stress on the modifications of the virus, which is expected to help for foreseeing the identification or development of antioxidants as therapeutic drugs. Graphic abstract

JTD Keywords: atom abstraction, damage, density functionals, hydrogen abstraction, isoleucine, molecular dynamics, pathogenesis, protein, reactive oxygen species, receptor binding domain, residues, spike protein, Amino-acids, Hydrogen abstraction, Isoleucine, Molecular dynamics, Reactive oxygen species, Receptor binding domain, Spike protein

Mesenchymal condensation is a prevalent morphogenetic transition that is essential in chondrogenesis. However, the current understanding of condensation mechanisms is limited. In vivo, progenitor cells directionally migrate from the surrounding loose mesenchyme towards regions of increasing matrix adherence (the condensation centers), which is accompanied by the upregulation of fibronectin. Here, we focused on the mechanisms of cell migration during mesenchymal cell condensation and the effects of matrix adherence. Dendrimer-based nanopatterns of the cell-adhesive peptide arginine-glycine-aspartic acid (RGD), which is present in fibronectin, were used to regulate substrate adhesion. We recorded collective and single-cell migration of mesenchymal stem cells, under chondrogenic induction, using live-cell imaging. Our results show that the cell migration mode of single cells depends on substrate adhesiveness, and that cell directionality controls cell condensation and the fusion of condensates. Inhibition experiments revealed that cell-cell interactions mediated by N-cadherin (also known as CDH2) are also pivotal for directional migration of cell condensates by maintaining cell-cell cohesion, thus suggesting a fine interplay between cell-matrix and cell-cell adhesions. Our results shed light on the role of cell interactions with a fibronectin-depositing matrix during chondrogenesis in vitro, with possible applications in regenerative medicine. This article has an associated First Person interview with the first author of the paper.© 2022. Published by The Company of Biologists Ltd.

JTD Keywords: alpha-v-beta-3, arginine-glycine-aspartic acid, chondrogenesis, dynamics, expression, fibronectin, gastrulation, involvement, mechanisms, mesenchymal condensation, model, nanopatterned substrates, rgd, Arginine-glycine-aspartic acid, Cell migration, Chondrogenesis, Mesenchymal condensation, N-cadherin, Nanopatterned substrates, Rgd

Upon the initiation of collective cell migration, the cells at the free edge are specified as leader cells; however, the mechanism underlying the leader cell specification remains elusive. Here, we show that lamellipodial extension after the release from mechanical confinement causes sustained extracellular signal-regulated kinase (ERK) activation and underlies the leader cell specification. Live-imaging of Madin-Darby canine kidney (MDCK) cells and mouse epidermis through the use of Förster resonance energy transfer (FRET)-based biosensors showed that leader cells exhibit sustained ERK activation in a hepatocyte growth factor (HGF)-dependent manner. Meanwhile, follower cells exhibit oscillatory ERK activation waves in an epidermal growth factor (EGF) signaling-dependent manner. Lamellipodial extension at the free edge increases the cellular sensitivity to HGF. The HGF-dependent ERK activation, in turn, promotes lamellipodial extension, thereby forming a positive feedback loop between cell extension and ERK activation and specifying the cells at the free edge as the leader cells. Our findings show that the integration of physical and biochemical cues underlies the leader cell specification during collective cell migration.Copyright © 2022 Elsevier Inc. All rights reserved.

JTD Keywords: activation, c-met, contact inhibition, focal adhesions, heparan-sulfate, mechanical forces, morphogenesis, rho, stress fibers, Collective cell migration, Erk, Feedback regulation, Fret, Growth-factor receptor, Hgf, Lamellipodia, Leader cell specification, Signal transduction, Traction force, Wound healing

Blood-vessel formation generates unique vascular patterns in each individual. The principles governing the apparent stochasticity of this process remain to be elucidated. Using mathematical methods, we find that the transition between two fundamental vascular morphogenetic programs-sprouting angiogenesis and vascular remodeling-is established by a shift of collective front-to-rear polarity of endothelial cells in the mouse retina. We demonstrate that the competition between biochemical (VEGFA) and mechanical (blood-flow-induced shear stress) cues controls this collective polarity shift. Shear stress increases tension at focal adhesions overriding VEGFA-driven collective polarization, which relies on tension at adherens junctions. We propose that vascular morphogenetic cues compete to regulate individual cell polarity and migration through tension shifts that translates into tissue-level emergent behaviors, ultimately leading to uniquely organized vascular patterns.Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.

JTD Keywords: activation, angiogenesis, dynamics, flow, forces, image, mechanisms, vinculin, Angiogenesis, Cell polarity, Fluid shear, Mechanobiology, Morphogenesis, Shear stress

Most of the conventional in vitro models to test biomaterial-driven vascularization are too simplistic to recapitulate the complex interactions taking place in the actual cell microenvironment, which results in a poor prediction of the in vivo performance of the material. However, during the last decade, cell culture models based on microfluidic technology have allowed attaining unprecedented levels of tissue biomimicry. In this work, we propose a microfluidic-based 3D model to evaluate the effect of bioactive biomaterials capable of releasing signalling cues (such as ions or proteins) in the recruitment of endogenous endothelial progenitor cells, a key step in the vascularization process. The usability of the platform is demonstrated using experimentally-validated finite element models and migration and proliferation studies with rat endothelial progenitor cells (rEPCs) and bone marrow-derived rat mesenchymal stromal cells (BM-rMSCs). As a proof of concept of biomaterial evaluation, the response of rEPCs to an electrospun composite made of polylactic acid with calcium phosphates nanoparticles (PLA+CaP) was compared in a co-culture microenvironment with BM-rMSC to a regular PLA control. Our results show a significantly higher rEPCs migration and the upregulation of several pro-inflammatory and proangiogenic proteins in the case of the PLA+CaP. The effects of osteopontin (OPN) on the rEPCs migratory response were also studied using this platform, suggesting its important role in mediating their recruitment to a calcium-rich microenvironment. This new tool could be applied to screen the capacity of a variety of bioactive scaffolds to induce vascularization and accelerate the preclinical testing of biomaterials. STATEMENT OF SIGNIFICANCE: : For many years researchers have used neovascularization models to evaluate bioactive biomaterials both in vitro, with low predictive results due to their poor biomimicry and minimal control over cell cues such as spatiotemporal biomolecule signaling, and in vivo models, presenting drawbacks such as being highly costly, time-consuming, poor human extrapolation, and ethically controversial. We describe a compact microphysiological platform designed for the evaluation of proangiogenesis in biomaterials through the quantification of the level of sprouting in a mimicked endothelium able to react to gradients of biomaterial-released signals in a fibrin-based extracellular matrix. This model is a useful tool to perform preclinical trustworthy studies in tissue regeneration and to better understand the different elements involved in the complex process of vascularization.Copyright © 2022. Published by Elsevier Ltd.

JTD Keywords: angiogenesis, bioactive materials, bone regeneration, bone-formation, calcium-phosphate, extracellular calcium, in-vitro, interstitial flow, ion release, microfluidic model, signalling gradient, substitutes, tissue engineering, vascularization, vegf, Ion release, Mesenchymal stem-cells, Tissue engineering, Vascularization

Growing evidence suggests that the physical properties of the cellular microenvironment influence cell migration. However, it is not currently understood how active physical remodelling by cells affects migration dynamics. Here we report that cell clusters seeded on deformable collagen-I networks display persistent collective migration despite not showing any apparent intrinsic polarity. Clusters generate transient gradients in collagen density and alignment due to viscoelastic relaxation of the collagen networks. Combining theory and experiments, we show that crosslinking collagen networks or reducing cell cluster size results in reduced network deformation, shorter viscoelastic relaxation time and smaller gradients, leading to lower migration persistence. Traction force and Brillouin microscopy reveal asymmetries in force distributions and collagen stiffness during migration, providing evidence of mechanical cross-talk between cells and their substrate during migration. This physical model provides a mechanism for self-generated directional migration on viscoelastic substrates in the absence of internal biochemical polarity cues.; Cell clusters mechanically reorganize viscoelastic collagen networks, resulting in transient gradients in collagen density, alignment and stiffness that promote spontaneous persistent migration.

JTD Keywords: Cell-migration, Design, Invasion, Limits, Mechanics, Microscopy, Morphogenesis, Motility, Rear, Rigidity

The differentiation of human pluripotent stem cells (hPSCs) towards organoids is one of the biggest scientific advances in regenerative medicine. Kidney organoids have not only laid the groundwork for various organ-like tissue systems but also provided insights into kidney embryonic development. Thus, several protocols for the differentiation of renal progenitors or mature cell types have been established. Insights into the interplay of developmental pathways in nephrogenesis and determination of different cell fates have enabled the in vitro recapitulation of nephrogenesis. Here we first provide an overview of kidney morphogenesis and patterning in the mouse model in order to dissect signalling pathways that are key to define culture conditions sustaining renal differentiation from hPSCs. Secondly, we also highlight how genome editing approaches have provided insights on the specific role of different genes and molecular pathways during renal differentiation from hPSCs. Based on this knowledge we further review how CRISPR/Cas9 technology has enabled the recapitulation and correction of cellular phenotypes associated with human renal disease. Last, we also revise how the field has positively benefited from emerging technologies as single cell RNA sequencing and discuss current limitations on kidney organoid technology that will take advantage from bioengineering solutions to help standardizing the use of this model systems to study kidney development and disease.Copyright © 2022 Safi, Marco, Moya, Prado, Garreta and Montserrat.

JTD Keywords: crispr, directed differentiation, epithelial-cells, expression, kidney engineering, kidney organoids, model, mouse, nephrogenesis, nephron number, podocytes, progenitor, Crispr, Kidney engineering, Kidney organoids, Nephrogenesis, Pluripotent stem cells, Pluripotent stem-cells

Liver fibrosis staging is a key element driving the prognosis of patients with chronic liver disease. Currently, biopsy is the only technique capable of diagnosing liver fibrosis in patients with alcohol-related liver disease (ArLD) and non-alcoholic fatty liver disease (NAFLD) unequivocally. Non-invasive (e.g. plasma-based) biomarker assays are attractive tools to diagnose and stage disease, yet must prove that they are reliable and sensitive to be used clinically. Here we demonstrate 1 H nuclear magnetic resonance as a method to rapidly quantify the endogenous concentration of ammonium ions from human plasma extracts and show their ability to report upon early and advanced stages of ArLD and NAFLD. We show that, irrespective of the disease aetiology, ammonium concentration is a more robust and informative marker of fibrosis stage than current clinically assessed blood hepatic biomarkers. Subject to validation in larger cohorts, the study indicates that the method can provide accurate and rapid staging of ArLD and NAFLD without need for an invasive biopsy.This article is protected by copyright. All rights reserved.

JTD Keywords: ammonium quantification, blood biomarkers, chronic liver disease, disease biomarkers, hepatic dysfunction, nmr, pathogenesis, Ammonium quantification, Hepatic dysfunction, Hepatic-encephalopathy

Gene duplications occur in prokaryotic genomes at a detectable frequency. In many instances, the biological function of the duplicates is unknown, and hence, the significance of the presence of multiple copies of these genes remains unclear.; Gene duplications significantly impact the gene repertoires of both eukaryotic and prokaryotic microorganisms. The genomes of pathogenic Escherichia coli strains share a group of duplicated genes whose function is mostly unknown. The irmA gene is one of the duplicates encoded in several pathogenic E. coli strains. The function of its gene product was investigated in the uropathogenic E. coli strain CFT073, which contains a single functional copy. The IrmA protein structure mimics that of human interleukin receptors and likely plays a role during infection. The enteroaggregative E. coli strain 042 contains two functional copies of the irmA gene. In the present work, we investigated their biological roles. The irmA_4509 allele is expressed under several growth conditions. Its expression is modulated by the global regulators OxyR and Hha, with optimal expression at 37 degrees C and under nutritional stress conditions. Expression of the irmA_2244 allele can only be detected when the irmA_4509 allele is knocked out. Differences in the promoter regions of both alleles account for their differential expression. Our results show that under several environmental conditions, the expression of the IrmA protein in strain 042 is dictated by the irmA_4509 allele. The irmA_2244 allele appears to play a backup role to ensure IrmA expression when the irmA_4509 allele loses its function. IMPORTANCE Gene duplications occur in prokaryotic genomes at a detectable frequency. In many instances, the biological function of the duplicates is unknown, and hence, the significance of the presence of multiple copies of these genes remains unclear. In pathogenic E. coli isolates, the irmA gene can be present either as a single copy or in two or more copies. We focused our work on studying why a different pathogenic E. coli strain encodes two functional copies of the irmA gene. We show that under several environmental conditions, one of the alleles dictates IrmA expression, and the second remains silent. The latter allele is only expressed when the former is silenced. The presence of more than one functional copy of the irmA gene in some pathogenic E. coli strains can result in sufficient expression of this virulence factor during the infection process.

JTD Keywords: 042, aec69, enteroaggregative e. coli, gene duplications, 042, Adaptation, Aec69, Aggregative adherence, Chromosomal genes, Coli, Duplication, Enteroaggregative e, Escherichia-coli, Evolution, Gene duplications, Hha/ymoa, Irma, Mechanism, Outer-membrane, Protein

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an important regulator of extracellular matrix turnover that has been traditionally regarded as a potential tumor suppressor owing to its inhibitory effects of matrix metal-loproteinases. Intriguingly, this interpretation has been challenged by the consistent observation that increased expression of TIMP-1 is associated with poor prognosis in virtually all cancer types including lung cancer, supporting a tumor-promoting function. However, how TIMP-1 is dysregulated within the tumor micro-environment and how it drives tumor progression in lung cancer is poorly understood. We analyzed the expression of TIMP-1 and its cell surface receptor CD63 in two major lung cancer subtypes: lung adenocarci-noma (ADC) and squamous cell carcinoma (SCC), and defined the tumor-promoting effects of their interac-tion. We found that TIMP-1 is aberrantly overexpressed in tumor-associated fibroblasts (TAFs) in ADC compared to SCC. Mechanistically, TIMP-1 overexpression was mediated by the selective hyperactivity of the pro-fibrotic TGF-61/SMAD3 pathway in ADC-TAFs. Likewise, CD63 was upregulated in ADC compared to SCC cells. Genetic analyses revealed that TIMP-1 secreted by TGF-61-activated ADC-TAFs is both nec-essary and sufficient to enhance growth and invasion of ADC cancer cells in culture, and that tumor cell expression of CD63 was required for these effects. Consistently, in vivo analyses revealed that ADC cells co-injected with fibroblasts with reduced SMAD3 or TIMP-1 expression into immunocompromised mice attenu-ated tumor aggressiveness compared to tumors bearing parental fibroblasts. We also found that high TIMP1 and CD63 mRNA levels combined define a stronger prognostic biomarker than TIMP1 alone. Our results identify an excessive stromal TIMP-1 within the tumor microenvironment selectively in lung ADC, and implicate it in a novel tumor-promoting TAF-carcinoma crosstalk, thereby pointing to TIMP-1/CD63 interaction as a novel therapeutic target in lung cancer. (c) 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

JTD Keywords: cancer-associated fibroblast, cd63, fibrosis, smad3, tgf-β1, timp-1, Angiogenesis, Cancer cells, Cancer-associated fibroblast, Cd63, Expression, Fibrosis, Hepatocellular-carcinoma, Metalloproteinases, Nintedanib, Prognostic-significance, Protein, Smad3, Squamous-cell carcinoma, Tgf-? 1, Tgf-β1, Timp-1, Tissue inhibitor, Tumor microenvironment

Collagen VI-related dystrophies (COL6-RDs) are a group of rare congenital neuromuscular dystrophies that represent a continuum of overlapping clinical phenotypes that go from the milder Bethlem myopathy (BM) to the severe Ullrich congenital muscular dystrophy, for which there is no effective treatment. Mutations in one of the three Collagen VI genes alter the incorporation of this protein into the extracellular matrix (ECM), affecting the assembly and the structural integrity of the whole fibrillar network. Clinical hallmarks of COL6-RDs are secondary to the ECM disruption and include muscle weakness, proximal joint contractures, and distal hyperlaxity. Although some traits have been identified in patients’ ECMs, a correlation between the ECM features and the clinical phenotype has not been established, mainly due to the lack of predictive and reliable models of the pathology. Herein, we engineered a new personalized pre-clinical model of COL6-RDs using cell-derived matrices (CDMs) technology to better recapitulate the complexity of the native scenario. We found that CDMs from COL6-RD patients presented alterations in ECM structure and composition, showing a significantly decreased Collagen VI secretion, especially in the more severe phenotypes, and a decrease in Fibrillin-1 inclusion. Next, we examined the Collagen VI-mediated deposition of Fibronectin in the ECM, finding a higher alignment, length, width, and straightness than in patients with COL6-RDs. Overall, these results indicate that CDMs models are promising tools to explore the alterations that arise in the composition and fibrillar architecture due to mutations in Collagen VI genes, especially in early stages of matrix organization. Ultimately, CDMs derived from COL6-RD patients may become relevant pre-clinical models, which may help identifying novel biomarkers to be employed in the clinics and to investigate novel therapeutic targets and treatments. Copyright © 2022 Almici, Chiappini, López-Márquez, Badosa, Blázquez, Caballero, Montero, Natera-de Benito, Nascimento, Roldán, Lagunas, Jiménez-Mallebrera and Samitier.

JTD Keywords: alpha-3 chain, binding, collagen vi related muscular dystrophy, decellularisation, decellularized matrices, deficiency, expression, extracellular matrix, fibroblasts, fibronectin, in vitro model, patient-derived ecms, skeletal-muscle, ullrich, Cell-derived matrices, Collagen, Collagen vi related muscular dystrophy, Decellularisation, Decellularization, Extracellular matrices, Extracellular matrix, Genes, In vitro model, In-vitro, In-vitro models, Matrix, Matrix model, Muscular dystrophy, Pathology, Patient-derived ecm, Patient-derived ecms, Pre-clinical

During development, organs reach precise shapes and sizes. Organ morphology is not always obtained through growth; a classic counterexample is the condensation of the nervous system during Drosophila embryogenesis. The mechanics underlying such condensation remain poorly understood. Here, we characterize the condensation of the embryonic ventral nerve cord (VNC) at both subcellular and tissue scales. This analysis reveals that condensation is not a unidirectional continuous process but instead occurs through oscillatory contractions. The VNC mechanical properties spatially and temporally vary, and forces along its longitudinal axis are spatially heterogeneous. We demonstrate that the process of VNC condensation is dependent on the coordinated mechanical activities of neurons and glia. These outcomes are consistent with a viscoelastic model of condensation, which incorporates time delays and effective frictional interactions. In summary, we have defined the progressive mechanics driving VNC condensation, providing insights into how a highly viscous tissue can autonomously change shape and size.

JTD Keywords: actomyosin, central nervous system, drosophila, glia, mechanics, morphogenesis, neuron, ventral nerve cord, Collagen-iv, Contraction, Forces, Gene, Glial-cells, Migration, Morphogenesis, Quantification, System, Tissue, Viscolelastic model

Several studies are aimed at developing systems based on naturaland biocompatible polymers for bone tissue engineering. Here, weemphasized how the bio-activation of chitosan (CS)-based scaffoldsby organic and inorganic signals is able to promote osteogenesis,angiogenesis and to modulate the inflammation response by usingin vitro models to mimic bone fracture microenvironment. CSscaffolds by using two different approaches based on inorganic andorganic compounds, were bio-activated respectively1. The expres-sion of inflammatory mediators was investigated (TGF-band IL-6).Additionally, to assess the effect of CS scaffold on angiogenesis,CD31 expression, cell adhesion, proliferation, migration and tubeformation by HUVECs were detected. The results highlighted thatinorganic and organic signals promote cell proliferation and differ-entiation without significant differences between the material groups.In particular, scaffolds bio-activated by using inorganic signals(hydroxyapatite nanoparticles) inhibit pro-inflammatory mediator’sproduction (IL-1band IL-6), induce anti-inflammatory cytokinegeneration (IL-10) and reduce nitric monoxide metabolites (nitrites).Conversely, scaffolds bio-activated by using organic signals (BMP-2mimicking peptide) were able to decrease pro-inflammatory markerswithout any effect on anti-inflammatory cytokines levels and on ni-trites. Furthermore, scaffolds promote angiogenesis by increasingcell proliferation, migration and tube formation with best resultsobtained for BMP based-scaffolds. Our results support the conceptthat CS biomaterials may be novel multi-target devices to treat bonerelated inflammation stimulating neo-vascularization of tissue-en-gineered constructs.ACKNOWLEDGEMENTS: The study was supported by OPENLAB. The authors thank Mariarosaria Bonetti for lab technicalsupport & data elaboration and Dr. Roberta Marzella for support toproject management.

JTD Keywords: Angiogenesis, Chitosan/pegda based scaffolds, Osteogenesis

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JTD Keywords: cancer, cells, cellular basis, delivery, encapsulation, in-vitro, inflammation, macrophage, methotrexate, pathogenesis, polymersome, polymersomes, synoviocyte, Arthritis, Rheumatoid-arthritis

Transmission of a plasmid from one bacterial cell to another, in several instances, underlies the dissemination of antimicrobial resistance (AMR) genes. The process requires well-characterized enzymatic machinery that facilitates cell-to-cell contact and the transfer of the plasmid.

JTD Keywords: antimicrobial resistance, bacterial ig-like proteins, bacterial lg-like proteins, chromosomal genes, identification, inca/c, mutational analysis, plasmid conjugation, products, r-factors, resistance plasmids, salmonella-enterica, sequence, Antimicrobial resistance, Bacterial ig-like proteins, Escherichia-coli, Plasmid conjugation

JTD Keywords: augmentation, calcium-phosphate, expansion, fabrication, hydroxyapatite, made artificial bones, osteogenesis, reconstruction, regeneration, 3-d printing, 3d printers, 3d printing, 3d-printing, Abstracting, Additives, Biomaterial, Bone, Bone regeneration, Bone substitution, Bone tissue engineering, Ceramic, Ceramics, Clinic, Controlled drug delivery, Personalized medicines, Surgical planning, Targeted drug delivery, Three-dimensional-printing, Tricalcium phosphate scaffolds

During kidney development the emergence of complex multicellular shapes such as the nephron (the functional unit of the kidney) rely on spatiotemporally coordinated developmental programs. These involve gene regulatory networks, signaling pathways and mechanical forces, that work in concert to shape and form the nephron(s). The generation of kidney organoids from human pluripotent stem cells now represent an unprecedented experimental set up to study these processes. Here we discuss the potential applications of kidney organoids to advance our knowledge of how mechanical forces and cell fate specification spatiotemporally interact to orchestrate nephron patterning and morphogenesis in humans. Progress in innovative techniques for quantifying and perturbing these processes in a controlled manner will be crucial. A mechanistic understanding of the multicellular dynamic processes occurring during nephrogenesis will pave the way to unveil new mechanisms of human kidney disease. © 2021

JTD Keywords: differentiation, dynamics, induction, lumen formation, models, mouse, organogenesis, reveals, tubules, Divergent features

Neuroblastoma (NB) is the most common extracranial pediatric solid cancer originating from undifferentiated neural crest cells. NB cells express EZH2 and GLI1 genes that are known to maintain the undifferentiated phenotype of cancer stem cells (CSC) in NB. Recent studies suggest that tumor-derived extracellular vesicles (EVs) can regulate the transformation of surrounding cells into CSC by transferring tumor-specific molecules they contain. However, the horizontal transfer of EVs molecules in NB remains largely unknown. We report the analysis of NB-derived EVs in bioengineered models of NB that are based on a collagen 1/hyaluronic acid scaffold designed to mimic the native tumor niche. Using these models, we observed an enrichment of GLI1 and EZH2 mRNAs in NB-derived EVs. As a consequence of the uptake of NB-derived EVs, the host cells increased the expression levels of GLI1 and EZH2. These results suggest the alteration of the expression profile of stromal cells through an EV-based mechanism, and point the GLI1 and EZH2 mRNAs in the EV cargo as diagnostic biomarkers in NB.

JTD Keywords: exosomes, genes, lines, maintenance, pathway, proliferation, rna, stemness, tumor, Cancer

In contrast to sintered calcium phosphates (CaPs) commonly employed as scaffolds to deliver mesenchymal stromal cells (MSCs) targeting bone repair, low temperature setting conditions of calcium deficient hydroxyapatite (CDHA) yield biomimetic topology with high specific surface area. In this study, the healing capacity of CDHA administering MSCs to bone defects is evaluated for the first time and compared with sintered beta-tricalcium phosphate (β-TCP) constructs sharing the same interconnected macroporosity. Xeno-free expanded human bone marrow MSCs attached to the surface of the hydrophobic β-TCP constructs, while infiltrating the pores of the hydrophilic CDHA. Implantation of MSCs on CaPs for 8 weeks in calvaria defects of nude mice exhibited complete healing, with bone formation aligned along the periphery of β-TCP, and conversely distributed within the pores of CDHA. Human monocyte-osteoclast differentiation was inhibited in vitro by direct culture on CDHA compared to β-TCP biomaterials and indirectly by administration of MSC-conditioned media generated on CDHA, while MSCs increased osteoclastogenesis in both CaPs in vivo. MSC engraftment was significantly higher in CDHA constructs, and also correlated positively with bone in-growth in scaffolds. These findings demonstrate that biomimetic CDHA are favorable carriers for MSC therapies and should be explored further towards clinical bone regeneration strategies. Statement of significance: Delivery of mesenchymal stromal cells (MSCs) on calcium phosphate (CaP) biomaterials enhances reconstruction of bone defects. Traditional CaPs are produced at high temperature, but calcium deficient hydroxyapatite (CDHA) prepared at room temperature yields a surface structure more similar to native bone mineral. The objective of this study was to compare the capacity of biomimetic CDHA scaffolds with sintered β-TCP scaffolds for bone repair mediated by MSCs for the first time. In vitro, greater cell infiltration occurred in CDHA scaffolds and following 8 weeks in vivo, MSC engraftment was higher in CDHA compared to β-TCP, as was bone in-growth. These findings demonstrate the impact of material features such as surface structure, and highlight that CDHA should be explored towards clinical bone regeneration strategies.

JTD Keywords: beta-tricalcium phosphate, bone regeneration, calcium deficient hydroxyapatite, differentiation, engraftment, human bone marrow mesenchymal stromal cells, hydroxyapatite scaffolds, in-vitro, inhibition, osteogenesis, osteoinduction, stem-cells, surface-topography, tissue, Beta-tricalcium phosphate, Bone regeneration, Calcium deficient hydroxyapatite, Engraftment, Human bone marrow mesenchymal stromal cells

Pulmonary endarterectomy (PEA) resected material offers a unique opportunity to develop an in vitro endothelial cell model of chronic thromboembolic pulmonary hypertension (CTEPH). We aimed to comprehensively analyze the endothelial function, molecular signature, and mitochondrial profile of CTEPH-derived endothelial cells to better understand the pathophysiological mechanisms of endothelial dysfunction behind CTEPH, and to identify potential novel targets for the prevention and treatment of the disease. Isolated cells from specimens obtained at PEA (CTEPH-EC), were characterized based on morphology, phenotype, and functional analyses (in vitro and in vivo tubule formation, proliferation, apoptosis, and migration). Mitochondrial content, morphology, and dynamics, as well as high-resolution respirometry and oxidative stress, were also studied. CTEPH-EC displayed a hyperproliferative phenotype with an increase expression of adhesion molecules and a decreased apoptosis, eNOS activity, migration capacity and reduced angiogenic capacity in vitro and in vivo compared to healthy endothelial cells. CTEPH-EC presented altered mitochondrial dynamics, increased mitochondrial respiration and an unbalanced production of reactive oxygen species and antioxidants. Our study is the foremost comprehensive investigation of CTEPH-EC. Modulation of redox, mitochondrial homeostasis and adhesion molecule overexpression arise as novel targets and biomarkers in CTEPH.

JTD Keywords: angiogenesis, cd31, dysfunction, expression, pathogenesis, thrombus, C-reactive protein

Intestinal organoids capture essential features of the intestinal epithelium such as crypt folding, cellular compartmentalization and collective movements. Each of these processes and their coordination require patterned forces that are at present unknown. Here we map three-dimensional cellular forces in mouse intestinal organoids grown on soft hydrogels. We show that these organoids exhibit a non-monotonic stress distribution that defines mechanical and functional compartments. The stem cell compartment pushes the extracellular matrix and folds through apical constriction, whereas the transit amplifying zone pulls the extracellular matrix and elongates through basal constriction. The size of the stem cell compartment depends on the extracellular-matrix stiffness and endogenous cellular forces. Computational modelling reveals that crypt shape and force distribution rely on cell surface tensions following cortical actomyosin density. Finally, cells are pulled out of the crypt along a gradient of increasing tension. Our study unveils how patterned forces enable compartmentalization, folding and collective migration in the intestinal epithelium. Perez-Gonzalez et al. explore the mechanical properties of intestinal organoids, and report the existence of distinct mechanical domains and that cells are pulled out of the central crypt along a gradient of increasing tension.

JTD Keywords: Forces, Growth, Gut, Monolayers, Morphogenesis, Reveal, Stem-cells, Tension

Objective: Wound healing is a complex process that involves the interaction between different cell types and bioactive factors. Impaired wound healing is characterized by a loss in synchronization of these interactions, resulting in nonhealing chronic wounds. Chronic wounds are a socioeconomic burden, one of the most prominent clinical manifestations of diabetes, however, they lack satisfactory treatment options. The objective of this study was to develop polymeric composites that deliver ions having wound healing properties and evaluate its performance using a pressure ulcer model in diabetic mice. Approach: To develop a polymeric composite wound dressing containing ion-releasing nanoparticles for chronic wound healing. This composite was chemically and physically characterized and evaluated using a pressure ulcer wound model in diabetic (db/db) mice to explore their potential as novel wound dressing. Results: This dressing exhibits a controlled ion release and a goodin vitrobioactivity. The polymeric composite dressing treatment stimulates angiogenesis, collagen synthesis, granulation tissue formation, and accelerates wound closure of ischemic wounds created in diabetic mice. In addition, the performance of the newly designed composite is remarkably better than a commercially available dressing frequently used for the treatment of low-exuding chronic wounds. Innovation: The developed nanoplatforms are cell- and growth factor free and control the host microenvironment resulting in enhanced wound healing. These nanoplatforms are available by cost-effective synthesis with a defined composition, offering an additional advantage in potential clinical application. Conclusion: Based on the obtained results, these polymeric composites offer an optimum approach for chronic wound healing without adding cells or external biological factors.

JTD Keywords: angiogenesis, bioactive dressings, chronic wounds, Angiogenesis, Bioactive dressings, Bioactive glass, Bioglass, Cells, Chronic wounds, Diabetes, Endothelial growth-factor, Expression, Hydrogel, Induction

© 2020 Elsevier Ltd Protein phase transitions are particularly amenable for cell signalling as these highly cooperative processes allow cells to make binary decisions in response to relatively small intracellular changes. The different processes of condensate formation and the distinct material properties of the resulting condensates provide a dictionary to modulate a range of decisions on cell fate. We argue that, on the one hand, the reversibility of liquid demixing offers a chance to arrest cell growth under specific circumstances. On the other hand, the transition to amyloids is better suited for terminal decisions such as those leading to apoptosis and necrosis. Here, we review recent examples of both scenarios, highlighting how mutations in signalling proteins affect the formation of biomolecular condensates with drastic effects on cell survival.

JTD Keywords: amyloid, cell death, deep mutagenesis, llps, rna-binding proteins, Amyloid, Cell death, Deep mutagenesis, Llps, Rna-binding proteins

Tissue engineering and regenerative medicine approaches use biomaterials in combination with cells to regenerate lost functions of tissues and organs to prevent organ transplantation. However, most of the current strategies fail in mimicking the tissue's extracellular matrix properties. In order to mimic native tissue conditions, we developed cell-derived matrix (CDM) microtissues (MT). Our methodology uses poly-lactic acid (PLA) and Cultispher(R) S microcarriers' (MCs') as scaffold templates, which are seeded with rat bone marrow mesenchymal stem cells (rBM-MSCs). The scaffold template allows cells to generate an extracellular matrix, which is then extracted for downstream use. The newly formed CDM provides cells with a complex physical (MT architecture) and biochemical (deposited ECM proteins) environment, also showing spontaneous angiogenic potential. Our results suggest that MTs generated from the combination of these two MCs (mixed MTs) are excellent candidates for tissue vascularization. Overall, this study provides a methodology for in-house fabrication of microtissues with angiogenic potential for downstream use in various tissue regenerative strategies.

JTD Keywords: angiogenesis, cell-derived matrix, cultispher® s, microtissue, poly-lactic acid microcarriers, Angiogenesis, Cell-derived matrix, Cultispher (r) s, Microtissue, Poly-lactic acid microcarriers, Rat bone marrow mesenchymal stem cells

Plaques of the amyloid beta (A beta) peptide are a pathological hallmark of Alzheimer's disease (AD), the most common form of dementia. Mutations in A beta also cause familial forms of AD (fAD). Here, we use deep mutational scanning to quantify the effects of >14,000 mutations on the aggregation of A beta. The resulting genetic landscape reveals mechanistic insights into fibril nucleation, including the importance of charge and gatekeeper residues in the disordered region outside of the amyloid core in preventing nucleation. Strikingly, unlike computational predictors and previous measurements, the empirical nucleation scores accurately identify all known dominant fAD mutations in A beta, genetically validating that the mechanism of nucleation in a cell-based assay is likely to be very similar to the mechanism that causes the human disease. These results provide the first comprehensive atlas of how mutations alter the formation of any amyloid fibril and a resource for the interpretation of genetic variation in A beta.

JTD Keywords: aggregation, kinetics, oligomers, onset, rates, state, Aggregation, Alzheimer's, Amyloid, Computational biology, Deep mutagenesis, Genetics, Genomics, Kinetics, Nucleation, Oligomers, Onset, Precursor protein, Rates, S. cerevisiae, State, Systems biology

© 2020 Elsevier B.V. In the current work, our purpose was based on the assessment of bioactive chitosan (CS)/Poly(ethylene glycol) diacrylate (PEGDA) based scaffolds ability to stimulate in vitro angiogenesis process. The bioactivation of the scaffolds was accomplished by using organic (BMP-2 peptide) and inorganic (hydroxyapatite nanoparticles) cues. In particular, the properties of the materials in terms of biological response promotion on human umbilical vein endothelial cells (HUVECs) were studied by using in vitro angiogenesis tests based on cell growth and proliferation. Furthermore, our interest was to examine the scaffolds capability to modulate two important steps involved in angiogenesis process: migration and tube formation of cells. Our data underlined that bioactive signals on CS/PEGDA scaffolds surface induce a desirable effect on angiogenic response concerning angiogenic marker expression (CD-31) and endothelial tissue formation (tube formation). Taken together, the results emphasized the concept that bioactive CS/PEGDA scaffolds may be novel implants for stimulating neovascularization of tissue-engineered constructs in regenerative medicine field.

JTD Keywords: angiogenesis, bmp-2 peptide, chitosan/pegda based scaffolds, human umbilical vein endothelial cells huvecs, Angiogenesis, Bmp-2 peptide, Chitosan/pegda based scaffolds, Human umbilical vein endothelial cells huvecs, Osteogenesis

Goel and colleagues show that CDK4/6 inhibition induces global chromatin changes mediated by AP-1 factors, which mediate key biological and clinical effects in breast cancer. Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) were designed to induce cancer cell cycle arrest. Recent studies have suggested that these agents also exert other effects, influencing cancer cell immunogenicity, apoptotic responses and differentiation. Using cell-based and mouse models of breast cancer together with clinical specimens, we show that CDK4/6 inhibitors induce remodeling of cancer cell chromatin characterized by widespread enhancer activation, and that this explains many of these effects. The newly activated enhancers include classical super-enhancers that drive luminal differentiation and apoptotic evasion, as well as a set of enhancers overlying endogenous retroviral elements that are enriched for proximity to interferon-driven genes. Mechanistically, CDK4/6 inhibition increases the level of several activator protein-1 transcription factor proteins, which are in turn implicated in the activity of many of the new enhancers. Our findings offer insights into CDK4/6 pathway biology and should inform the future development of CDK4/6 inhibitors.

JTD Keywords: Abemaciclib, Androgen receptor, Animal experiment, Animal model, Animal tissue, Apoptosis, Article, Breast cancer, C-jun, Cancer cell, Carcinoembryonic antigen related cell adhesion molecule 1, Caspase 3, Cell cycle arrest, Cells, Chromatin, Chromatin immunoprecipitation, Controlled study, Cyclin dependent kinase 4, Cyclin dependent kinase 6, Dna damage, Epidermal growth factor receptor 2, Estrogen receptor, Female, Flow cytometry, Fulvestrant, Hla drb1 antigen, Human, Human cell, Immunoblotting, Immunogenicity, Immunoprecipitation, Interferon, Luciferase assay, Mcf-7 cell line, Mda-mb-231 cell line, Microarray analysis, Morphogenesis, Mouse, Nonhuman, Palbociclib, Protein, Protein expression, Rb, Resistance, Rna polymerase ii, Rna sequence, Selective-inhibition, Senescence, Short tandem repeat, Signal transduction, Tamoxifen, Transcription elongation, Transcription factor, Transcription factor ap 1, Transcriptome, Tumor biopsy, Tumor differentiation, Tumor spheroid, Tumor xenograft, Vinculin, Whole exome sequencing

Scaffolds based on bioconjugated hydrogels are attractive for tissue engineering because they can partly mimic human tissue characteristics. For example, they can further increase their bioactivity with cells. However, most of the hydrogels present problems related to their processability, consequently limiting their use in 3D printing to produce tailor-made scaffolds. The goal of this work is to develop bioconjugated hydrogel nanocomposite inks for 3D printed scaffold fabrication through a micro-extrusion process having improved both biocompatibility and processability. The hydrogel is based on a photocrosslinkable alginate bioconjugated with both gelatin and chondroitin sulfate in order to mimic the cartilage extracellular matrix, while the nanofiller is based on graphene oxide to enhance the printability and cell proliferation. Our results show that the incorporation of graphene oxide into the hydrogel inks considerably improved the shape fidelity and resolution of 3D printed scaffolds because of a faster viscosity recovery post extrusion of the ink. Moreover, the nanocomposite inks produce anisotropic threads after the 3D printing process because of the templating of the graphene oxide liquid crystal. The in vitro proliferation assay of human adipose tissue-derived mesenchymal stem cells (hADMSCs) shows that bioconjugated scaffolds present higher cell proliferation than pure alginate, with the nanocomposites presenting the highest values at long times. Live/Dead assay otherwise displays full viability of hADMSCs adhered on the different scaffolds at day 7. Notably, the scaffolds produced with nanocomposite hydrogel inks were able to guide the cell proliferation following the direction of the 3D printed threads. In addition, the bioconjugated alginate hydrogel matrix induced chondrogenic differentiation without exogenous pro-chondrogenesis factors as concluded from immunostaining after 28 days of culture. This high cytocompatibility and chondroinductive effect toward hADMSCs, together with the improved printability and anisotropic structures, makes these nanocomposite hydrogel inks a promising candidate for cartilage tissue engineering based on 3D printing.

JTD Keywords: 3D printing, Chondrogenesis, Graphene oxide, Hydrogels, Liquid crystals

Tackling the first stages of the chondrogenic commitment is essential to drive chondrogenic differentiation to healthy hyaline cartilage and minimize hypertrophy. During chondrogenesis, the extracellular matrix continuously evolves, adapting to the tissue adhesive requirements at each stage. Here, we take advantage of previously developed nanopatterns, in which local surface adhesiveness can be precisely tuned, to investigate its effects on prechondrogenic condensation. Fluorescence live cell imaging, immunostaining, confocal microscopy and PCR analysis are used to follow the condensation process on the nanopatterns. Cell tracking parameters, condensate morphology, cell–cell interactions, mechanotransduction and chondrogenic commitment are evaluated in response to local surface adhesiveness. Results show that only condensates on the nanopatterns of high local surface adhesiveness are stable in culture and able to enter the chondrogenic pathway, thus highlighting the importance of controlling cell–substrate adhesion in the tissue engineering strategies for cartilage repair.

JTD Keywords: Dendrimer, Nanopatterning, RGD, Mesenchymal cell condensation, Cell–cell interactions, YAP, Chondrogenesis

Molecular and cellular research modalities for the study of liver pathologies have been tremendously improved over the recent decades. Advanced technologies offer novel opportunities to establish cell isolation techniques with excellent purity, paving the path for 2D and 3D microscopy and high-throughput assays (e.g., bulk or single-cell RNA sequencing). The use of stem cell and organoid research will help to decipher the pathophysiology of liver diseases and the interaction between various parenchymal and non-parenchymal liver cells. Furthermore, sophisticated animal models of liver disease allow for the in vivo assessment of fibrogenesis, portal hypertension and hepatocellular carcinoma (HCC) and for the preclinical testing of therapeutic strategies. The purpose of this review is to portray in detail novel in vitro and in vivo methods for the study of liver cell biology that had been presented at the workshop of the 8th meeting of the European Club for Liver Cell Biology (ECLCB-8) in October of 2018 in Bonn, Germany.

JTD Keywords: Fibrogenesis, Hepatic stellate cells, Hepatocellular cancer, In vitro models, Steatosis

Bone apatite consists of carbonated calcium-deficient hydroxyapatite (CDHA) nanocrystals. Biomimetic routes allow fabricating synthetic bone grafts that mimic biological apatite. In this work, we explored the role of two distinctive features of biomimetic apatites, namely, nanocrystal morphology (plate vs needle-like crystals) and carbonate content, on the bone regeneration potential of CDHA scaffolds in an in vivo canine model. Both ectopic bone formation and scaffold degradation were drastically affected by the nanocrystal morphology after intramuscular implantation. Fine-CDHA foams with needle-like nanocrystals, comparable in size to bone mineral, showed a markedly higher osteoinductive potential and a superior degradation than chemically identical coarse-CDHA foams with larger plate-shaped crystals. These findings correlated well with the superior bone-healing capacity showed by the fine-CDHA scaffolds when implanted intraosseously. Moreover, carbonate doping of CDHA, which resulted in small plate-shaped nanocrystals, accelerated both the intrinsic osteoinduction and the bone healing capacity, and significantly increased the cell-mediated resorption. These results suggest that tuning the chemical composition and the nanostructural features may allow the material to enter the physiological bone remodeling cycle, promoting a tight synchronization between scaffold degradation and bone formation.

JTD Keywords: Biomimetic, Calcium phosphate, Carbonated apatite, Foaming, Nanostructure, Osteogenesis, Osteoinduction

Calcium phosphate (CaP) substrates are successfully used as bone grafts due to their osteogenic properties. However, the influence of the physicochemical features of CaPs in angiogenesis is frequently neglected despite it being a crucial process for bone regeneration. The present work focuses on analyzing the effects of textural parameters of biomimetic calcium deficient hydroxyapatite (CDHA) and sintered beta-tricalcium phosphate (β-TCP), such as specific surface area, surface roughness, and microstructure, on the behavior of rat endothelial progenitor cells (rEPCs) and their crosstalk with rat mesenchymal stem cells (rMSCs). The higher reactivity of CDHA results in low proliferation rates in monocultured and cocultured systems. This effect is especially pronounced for rMSCs alone, and for CDHA with a fine microstructure. In terms of angiogenic and osteogenic gene expressions, the upregulation of particular genes is especially enhanced for needle-like CDHA compared to plate-like CDHA and β-TCP, suggesting the importance not only of the chemistry of the substrate, but also of its textural features. Moreover, the coculture of rEPCs and rMSCs on needle-like CDHA results in early upregulation of osteogenic modulator, i.e., protein deglycase 1 might be a possible cause of overexpression of osteogenic-related genes on the same substrate.

JTD Keywords: Angiogenesis, Calcium phosphates, Cocultures, Osteogenesis

Biomaterial implantation triggers inflammatory reactions. Understanding the effect of physicochemical features of biomaterials on the release of inflammatory cytokines from immune cells would be of great interest in view of designing bone graft materials to enhance the healing of bone defects. The present work investigated the interactions of two chemically and texturally different calcium phosphate (CaPs) substrates with macrophages, one of the main innate immune cells, and its further impact on osteogenic differentiation of bone forming cells. The behaviour of macrophages seeded on biomimetic calcium deficient hydroxyapatite (CDHA) and sintered β-tricalcium phosphate (β-TCP) was assessed in terms of the release of inflammatory cytokines and osteoclastogenic factors. The osteogenic differentiation of bone progenitor cells (bone marrow stromal cells (BMSCs) and osteoblastic cell line (SaOS-2)) were subsequently studied by incubating with the conditioned medium induced by macrophage-CaPs interaction in order to reveal the effect of immune cell reaction to CaPs on osteogenic differentiation. It was found that the incubation of macrophages with CaPs substrates caused a decrease of pro-inflammatory cytokines, more pronounced for β-TCP compared with CDHA showing significantly decreased IL-6, TNF-a, and iNOS. However, the macrophage-CDHA interaction resulted in a more favourable environment for osteogenic differentiation of osteoblasts with more collagen type I production and osteogenic genes (Runx2, BSP) expression, suggesting that osteogenic differentiation of bone cells is not only determined by the nature of biomaterials, but also significantly influenced by the inflammatory environment generated by the interaction of immune cells and biomaterials. Statement of Significance: The field of osteoimmunology highlights the importance of the cross-talk between immune and bone cells for effective bone regeneration. This tight interaction opens the door to new strategies that encompass the development of smart cell-instructive biomaterials which performance covers the events from early inflammation to osteogenesis. The present work links the anti-inflammatory and osteoimmunomodulatory features of synthetic bone grafts to their chemistry and texture, focussing on the cross-talk between macrophages and two major orchestrators of bone healing, namely primary mesenchymal stem cells and osteoblasts. The results emphasize the importance of the microenvironment created through the interaction between the substrate and the immune cells as it can stimulate osteogenic events and subsequently foster bone healing.

JTD Keywords: Calcium phosphates, Immunomodulation, Inflammation, Osteogenesis, Osteoimmunomodulation

The immobilization of natural molecules on synthetic bone grafts stands as a strategy to enhance their biological interactions. During the early stages of healing, immune cells and osteoclasts (OC) modulate the inflammatory response and resorb the biomaterial, respectively. In this study, heparin, a naturally occurring molecule in the bone extracellular matrix, was covalently immobilized on biomimetic calcium‐deficient hydroxyapatite (CDHA). The effect of heparin‐functionalized CDHA on inflammation and osteoclastogenesis was investigated using primary human cells and compared with pristine CDHA and beta-tricalcium phosphate (β-TCP). Biomimetic substrates led to lower oxidative stresses by neutrophils and monocytes than sintered β-TCP, even though no further reduction was induced by the presence of heparin. In contrast, heparinized CDHA fostered osteoclastogenesis. Optical images of stained TRAP positive cells showed an earlier and higher presence of multinucleated cells, compatible with OC at 14 days, while pristine CDHA and β-TCP present OC at 21–28 days. Although no statistically significant differences were found in the OC activity, microscopy images evidenced early stages of degradation on heparinized CDHA, compatible with osteoclastic resorption. Overall, the results suggest that the functionalization with heparin fostered the formation and activity of OC, thus offering a promising strategy to integrate biomaterials in the bone remodelling cycle by increasing their OC-mediated resorption.

JTD Keywords: Biomaterial, Heparin, Hydroxyapatite, Inflammation, Osteoclastogenesis

Extracellular matrix remodeling plays a pivotal role during mesenchyme patterning into different lineages. Tension exerted from cell membrane receptors bound to extracellular matrix ligands is transmitted by the cytoskeleton to the cell nucleus inducing gene expression. Here, we used dendrimer-based arginine–glycine–aspartic acid (RGD) uneven nanopatterns, which allow the control of local surface adhesiveness at the nanoscale, to unveil the adhesive requirements of mesenchymal tenogenic and osteogenic commitments. Cell response was found to depend on the tension resulting from cell–substrate interactions, which affects nuclear morphology and is regulated by focal adhesion size and distribution.

JTD Keywords: Arginine–glycine–aspartic acid (RGD), Nanopattern, Mesenchymal stem cells, Tenogenesis, Osteogenesis, Cell nuclei, Focal adhesions

Reactive oxygen species (ROS) contribute to tissue damage and remodelling mediated by the inflammatory response after injury. Here we show that ROS, which promote axonal dieback and degeneration after injury, are also required for axonal regeneration and functional recovery after spinal injury. We find that ROS production in the injured sciatic nerve and dorsal root ganglia requires CX3CR1-dependent recruitment of inflammatory cells. Next, exosomes containing functional NADPH oxidase 2 complexes are released from macrophages and incorporated into injured axons via endocytosis. Once in axonal endosomes, active NOX2 is retrogradely transported to the cell body through an importin-β1–dynein-dependent mechanism. Endosomal NOX2 oxidizes PTEN, which leads to its inactivation, thus stimulating PI3K–phosporylated (p-)Akt signalling and regenerative outgrowth. Challenging the view that ROS are exclusively involved in nerve degeneration, we propose a previously unrecognized role of ROS in mammalian axonal regeneration through a NOX2–PI3K–p-Akt signalling pathway.

JTD Keywords: Adult neurogenesis, Endocytosis, Exocytosis, Monocytes and macrophages, Stress signalling

Immune cells are sensitive to the microstructural and textural properties of materials. Tuning the structural features of synthetic bone grafts could be a valuable strategy to regulate the specific response of the immune system, which in turn modulates the activity of bone cells. The aim of this study was to analyse the effect of the structural characteristics of biomimetic calcium deficient hydroxyapatite (CDHA) on the innate immune response of macrophages and the subsequent impact on osteogenesis and osteoclastogenesis. Murine RAW 264.7 cells were cultured, under standard and inflammatory conditions, on chemically identical CDHA substrates that varied in microstructure and porosity. The impact on osteogenesis was evaluated by incubating osteoblastic cells (SaOS-2) with RAW-CDHA conditioned extracts. The results showed that macrophages were sensitive to different textural and structural properties of CDHA. Under standard conditions, the impact of inflammatory cytokine production by RAW cells cultured on CDHA played a significant role in the degradation of substrates, suggesting the impact of resorptive behaviour of RAW cells on biomimetic surfaces. Osteoblast differentiation was stimulated by the conditioned media collected from RAW cells cultured on needle-like nanostructured CDHA. The results demonstrated that needle-like nanostructured CDHA was able to generate a favourable osteoimmune environment to regulate osteoblast differentiation and osteogenesis. Under inflammatory conditions, the incubation of RAW cells with less porous CDHA resulted in a decreased gene expression and release of pro-inflammatory cytokines.

JTD Keywords: Calcium phosphates, Biomimetic hydroxyapatite, Osteoimmunomodulation, Inflammation, Osteogenesis, Osteoclastogesis

There is an urgent need of synthetic bone grafts with enhanced osteogenic capacity. This can be achieved by combining biomaterials with exogenous growth factors, which however can have numerous undesired side effects, but also by tuning the intrinsic biomaterial properties. In a previous study, we showed the synergistic effect of nanostructure and pore architecture of biomimetic calcium deficient hydroxyapatite (CDHA) scaffolds in enhancing osteoinduction, i.e. fostering the differentiation of mesenchymal stem cells to bone forming cells. This was demonstrated by assessing bone formation after implanting the scaffolds intramuscularly. The present study goes one step forward, since it analyzes the effect of the geometrical features of the same CDHA scaffolds, obtained either by 3D-printing or by foaming, on the osteogenic potential and resorption behaviour in a bony environment. After 6 and 12 weeks of intraosseous implantation, both bone formation and material degradation had been drastically affected by the macropore architecture of the scaffolds. Whereas nanostructured CDHA was shown to be highly osteoconductive both in the robocast and foamed scaffolds, a superior osteogenic capacity was observed in the foamed scaffolds, which was associated with their higher intrinsic osteoinductive potential. Moreover, they showed a significantly higher cell-mediated degradation than the robocast constructs, with a simultaneous and progressive replacement of the scaffold by new bone. In conclusion, these results demonstrate that the control of macropore architecture is a crucial parameter in the design of synthetic bone grafts, which allows fostering both material degradation and new bone formation. Statement of Significance: 3D-printing technologies open new perspectives for the design of patient-specific bone grafts, since they allow customizing the external shape together with the internal architecture of implants. In this respect, it is important to design the appropriate pore geometry to maximize the bone healing capacity of these implants. The present study analyses the effect of pore architecture of nanostructured hydroxyapatite scaffolds, obtained either by 3D-printing or foaming, on the osteogenic potential and scaffold resorption in an in vivo model. While nanostructured hydroxyapatite showed excellent osteoconductive properties irrespective of pore geometry, we demonstrated that the spherical, concave macropores of foamed scaffolds significantly promoted both material resorption and bone regeneration compared to the 3D-printed scaffolds with orthogonal-patterned struts and therefore prismatic, convex macropores.

JTD Keywords: Osteogenesis, Pore architecture, 3D-printing, Foaming, Calcium phosphate

Immune cells play a vital role in regulating bone dynamics. This has boosted the interest in developing biomaterials that can modulate both the immune and skeletal systems. In this study, calcium phosphates discs (i.e., beta-tricalcium phosphate, β-TCP) are functionalized with heparin to investigate the effects on immune and stem cell responses. The results show that the functionalized surfaces downregulate the release of hydrogen peroxide and proinflammatory cytokines (tumor necrosis factor alpha and interleukin 1 beta) from human monocytes and neutrophils, compared to nonfunctionalized discs. The macrophages show both elongated and round shapes on the two ceramic substrates, but the morphology of cells on heparinized β-TCP tends toward a higher elongation after 72 h. The heparinized substrates support rat mesenchymal stem cell (MSC) adhesion and proliferation, and anticipate the differentiation toward the osteoblastic lineage as compared to β-TCP and control. The coupling between the inflammatory response and osteogenesis is assessed by culturing MSCs with the macrophage supernatants. The downregulation of inflammation in contact with the heparinized substrates induces higher expression of bone-related markers by MSCs.

JTD Keywords: Calcium phosphates, Heparinization, Inflammation, Osteogenesis

There is growing recognition that the mechanical interactions between cells and their local extracellular matrix (ECM) are central regulators of tissue development, homeostasis, repair and disease progression. The unique ability of atomic force microscopy (AFM) to probe quantitatively mechanical properties and forces at the nanometer or micrometer scales in all kinds of biological samples has been instrumental in the recent advances in cell and tissue mechanics. In this review we illustrate how AFM has provided important insights on our current understanding of the mechanobiology of cells, ECM and cell-ECM bidirectional interactions, particularly in the context of soft acinar tissues like the mammary gland or pulmonary tissue. AFM measurements have revealed that intrinsic cell micromechanics is cell-type specific, and have underscored the prominent role of β1 integrin/FAK(Y397) signaling and the actomyosin cytoskeleton in the mechanoresponses of both parenchymal and stromal cells. Moreover AFM has unveiled that the micromechanics of the ECM obtained by tissue decellularization is unique for each anatomical compartment, which may support both its specific function and cell differentiation. AFM has also enabled identifying critical mechanoregulatory proteins involved in branching morphogenesis (MMP14) and acinar differentiation (α3β1 integrin), and has clarified the role of altered tissue mechanics and architecture in a variety of pathologic conditions. Critical technical issues of AFM mechanical measurements like tip geometry effects are also discussed.

JTD Keywords: Atomic force microscopy, Beta1 integrin, Elastic modulus, Extracellular matrix, Morphogenesis, Tissue decellularization

The development and maturation of cortical circuits relies on the coordinated actions of long and short range axonal guidance cues. In this regard, the class 3 semaphorins and their receptors have been seen to be involved in the development and maturation of the hippocampal connections. However, although the role of most of their family members have been described, very few data about the participation of Semaphorin 3E (Sema3E) and its receptor PlexinD1 during the development and maturation of the entorhino-hippocampal (EH) connection are available. In the present study, we focused on determining their roles both during development and in adulthood. We determined a relevant role for Sema3E/PlexinD1 in the layer-specific development of the EH connection. Indeed, mice lacking Sema3E/PlexinD1 signalling showed aberrant layering of entorhinal axons in the hippocampus during embryonic and perinatal stages. In addition, absence of Sema3E/PlexinD1 signalling results in further changes in postnatal and adult hippocampal formation, such as numerous misrouted ectopic mossy fibers. More relevantly, we describe how subgranular cells express PlexinD1 and how the absence of Sema3E induces a dysregulation of the proliferation of dentate gyrus progenitors leading to the presence of ectopic cells in the molecular layer. Lastly, Sema3E mutant mice displayed increased network excitability both in the dentate gyrus and the hippocampus proper.

JTD Keywords: Adult neurogenesis, Axon and dendritic guidance

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

JTD Keywords: Bioengineering, Dendrimer, Nanopattern, Arginine-Glycine-Aspartic Acid (RGD), Atomic Force Microscopy (AFM), Cell Adhesion, Mesenchymal Stem Cells (Mscs), Chondrogenesis

Embryo-scale morphogenesis arises from patterned mechanical forces. During Drosophila gastrulation, actomyosin contractility drives apical constriction in ventral cells, leading to furrow formation and mesoderm invagination. It remains unclear whether and how mechanical properties of the ectoderm influence this process. Here, we show that Neuralized (Neur), an E3 ubiquitin ligase active in the mesoderm, regulates collective apical constriction and furrow formation. Conversely, the Bearded (Brd) proteins antagonize maternal Neur and lower medial–apical contractility in the ectoderm: in Brd-mutant embryos, the ventral furrow invaginates properly but rapidly unfolds as medial MyoII levels increase in the ectoderm. Increasing contractility in the ectoderm via activated Rho similarly triggers furrow unfolding whereas decreasing contractility restores furrow invagination in Brd-mutant embryos. Thus, the inhibition of Neur by Brd in the ectoderm differentiates the mechanics of the ectoderm from that of the mesoderm and patterns the activity of MyoII along the dorsal–ventral axis.

JTD Keywords: Drosophila, Gastrulation, Morphogenesis

The principles underlying the biomechanics of morphogenesis are largely unknown. Epiboly is an essential embryonic event in which three tissues coordinate to direct the expansion of the blastoderm. How and where forces are generated during epiboly, and how these are globally coupled remains elusive. Here we developed a method, hydrodynamic regression (HR), to infer 3D pressure fields, mechanical power, and cortical surface tension profiles. HR is based on velocity measurements retrieved from 2D+T microscopy and their hydrodynamic modeling. We applied HR to identify biomechanically active structures and changes in cortex local tension during epiboly in zebrafish. Based on our results, we propose a novel physical description for epiboly, where tissue movements are directed by a polarized gradient of cortical tension. We found that this gradient relies on local contractile forces at the cortex, differences in elastic properties between cortex components and the passive transmission of forces within the yolk cell. All in all, our work identifies a novel way to physically regulate concerted cellular movements that might be instrumental for the mechanical control of many morphogenetic processes.

JTD Keywords: Epiboly, Hydrodynamics, Mechanics, Morphogenesis, Zebrafish

Insufficient angiogenesis remains a major hurdle in current bone tissue engineering strategies. An extensive body of work has focused on the use of angiogenic factors or endothelial progenitor cells. However, these approaches are inherently complex, in terms of regulatory and methodologic implementation, and present a high cost. We have recently demonstrate the potential of electrospun poly(lactic acid) (PLA) fiber-based membranes, containing calcium phosphate (CaP) ormoglass particles, to elicit angiogenesis in vivo, in a subcutaneous model in mice. Here we have devised an injectable composite, containing CaP glass-ceramic particles, dispersed within a (Hydroxypropyl)methyl cellulose (HPMC) matrix, with the capacity to release calcium in a more sustained fashion. We show that by tuning the release of calcium in vivo, in a rat bone defect model, we could improve both bone formation and increase angiogenesis. The bone regeneration kinetics was dependent on the Ca2+ release rate, with the faster Ca2+ release composite gel showing improved bone repair at 3 weeks, in relation to control. In the same line, improved angiogenesis could be observed for the same gel formulation at 6 weeks post implantation. This methodology allows to integrate two fundamental processes for bone tissue regeneration while using a simple, cost effective, and safe approach. Statement of Significance In current bone tissue engineering approaches the achievement of sufficient angiogenesis, during tissue regeneration, is a major limitation in order to attain full tissue functionality. Recently, we have shown that calcium ions, released by the degradation of calcium phosphate ormoglasses (CaP), are effective angiogenic promoters, in both in vitro and in a subcutaneous implantation model. Here, we devised an injectable composite, containing CaP glass-ceramic particles, dispersed within a HPMC matrix, enabling the release of calcium in a more sustained fashion. We show that by tuning the release of calcium in vivo, in a rat bone defect model, we could improve both bone formation and increase angiogenesis. This simple and cost effective approach holds great promise to translate to the clinics.

JTD Keywords: Angiogenesis, Bone regeneration, Calcium phosphate ormoglasses

The success of scaffold implantation in acellular tissue engineering approaches relies on the ability of the material to interact properly with the biological environment. This behavior mainly depends on the design of the graft surface and, more precisely, on its capacity to biodegrade in a well-defined manner (nature of ions released, surface-to-volume ratio, dissolution profile of this release, rate of material resorption, and preservation of mechanical properties). The assessment of the biological behavior of temporary templates is therefore very important in tissue engineering, especially for composites, which usually exhibit complicated degradation behavior. Here, blended polylactic acid (PLA) calcium phosphate ORMOGLASS (organically modified glass) nanofibrous mats have been incubated up to 4 weeks in physiological simulated conditions, and their morphological, topographical, and chemical changes have been investigated. The results showed that a significant loss of inorganic phase occurred at the beginning of the immersion and the ORMOGLASS maintained a stable composition afterward throughout the degradation period. As a whole, the nanostructured scaffolds underwent fast and heterogeneous degradation. This study reveals that an angiogenic calcium-rich environment can be achieved through fast-degrading ORMOGLASS/PLA blended fibers, which seems to be an excellent alternative for guided bone regeneration.

JTD Keywords: Angiogenesis, Calcium release, Electrospinning, Fast degradation, Nanofibers, ORMOGLASSES

Ribonucleotide reductases (RNR) catalyze the last step of deoxyribonucleotide synthesis, and are therefore essential to DNA-based life. Three forms of RNR exist: classes I, II, and III. While eukaryotic cells use only class Ia RNR, bacteria can harbor any combination of classes, granting them adaptability. The opportunistic pathogen Pseudomonas aeruginosa surprisingly encodes all three classes, allowing it to thrive in different environments. Here we study an aspect of the complex RNR regulation whose molecular mechanism has never been elucidated, the well-described induction through oxidative stress, and link it to the AlgZR two-component system, the primary regulator of the mucoid phenotype. Through bioinformatics, we identify AlgR binding locations in RNR promoters, which we characterize functionally through EMSA and physically through AFM imaging. Gene reporter assays in different growth models are used to study the AlgZR-mediated control on the RNR network under various environmental conditions and physiological states. Thereby, we show that the two-component system AlgZR, which is crucial for bacterial conversion to the mucoid phenotype associated with chronic disease, controls the RNR network and directs how the DNA synthesis pathway is modulated in mucoid and non-mucoid biofilms, allowing it to respond to oxidative stress.

JTD Keywords: Bacterial genes, Bacteriology, Pathogens

Pseudomonas aeruginosa strain PAO1 has become the reference strain in many laboratories. One enzyme that is essential for its cell division is the ribonucleotide reductase (RNR) enzyme that supplies the deoxynucleotides required for DNA synthesis and repair. P. aeruginosa is one of the few microorganisms that encodes three different RNR classes (Ia, II and III) in its genome, enabling it to grow and adapt to diverse environmental conditions, including during infection. In this work, we demonstrate that a lack of RNR activity induces cell elongation in P. aeruginosa PAO1. Moreover, RNR gene expression during anaerobiosis differs among P. aeruginosa strains, with class III highly expressed in P. aeruginosa clinical isolates relative to the laboratory P. aeruginosa PAO1 strain. A single point mutation was identified in the P. aeruginosa PAO1 strain class III RNR promoter region that disrupts its anaerobic transcription by the Dnr regulator. An engineered strain that induces the class III RNR expression allows P. aeruginosa PAO1 anaerobic growth and increases its virulence to resemble that of clinical strains. Our results demonstrate that P. aeruginosa PAO1 is adapted to laboratory conditions and is not the best reference strain for anaerobic or infection studies.

JTD Keywords: Bacterial genes, Cellular microbiology, Pathogens

Abstract In current bone tissue engineering strategies the achievement of sufficient angiogenesis during tissue regeneration is still a major limitation in order to attain full functionality. Several strategies have been described to tackle this problem, mainly by the use of angiogenic factors or endothelial progenitor cells. However, when facing a clinical scenario these approaches are inherently complex and present a high cost. As such, more cost effective alternatives are awaited. Here, we demonstrate the potential of electrospun poly(lactic acid) (PLA) fiber-based membranes, containing calcium phosphate ormoglass (CaP) particles, to elicit angiogenesis in vivo, in a subcutaneous model in mice. We show that the current approach elicited the local expression of angiogenic factors, associated to a chemotactic effect on macrophages, and sustained angiogenesis into the biomaterial. As both PLA and CaP are currently accepted for clinical application these off-the-shelf novel membranes have great potential for guided bone regeneration applications. Statement of significance In current bone tissue engineering approaches the achievement of sufficient angiogenesis, during tissue regeneration, is a major limitation in order to attain full tissue functionality. Recently, our group has found that calcium ions released by the degradation of calcium phosphate ormoglasses (CaP) are effective angiogenic promoters. Based on this, in this work we successfully produced hybrid fibrous mats with different contents of CaP nanoparticles and thus with different calcium ion release rates, using an ormoglass – poly(lactic acid) blend approach. We show that these matrices, upon implantation in a subcutaneous site, could elicit the local expression of angiogenic factors, associated to a chemotactic effect on macrophages, and sustained angiogenesis into the biomaterial, in a CaP dose dependent manner. This off-the-shelf cost effective approach presents great potential to translate to the clinics.

JTD Keywords: Angiogenesis, Bone regeneration, Calcium phosphate ormoglass

Chronic lung infections by the ubiquitous and extremely adaptable opportunistic pathogen Pseudomonas aeruginosa correlate with the formation of a biofilm, where bacteria grow in association with an extracellular matrix and display a wide range of changes in gene expression and metabolism. This leads to increased resistance to physical stress and antibiotic therapies, while enhancing cell-to-cell communication. Oxygen diffusion through the complex biofilm structure generates an oxygen concentration gradient, leading to the appearance of anaerobic microenvironments. Ribonucleotide reductases (RNRs) are a family of highly sophisticated enzymes responsible for the synthesis of the deoxyribonucleotides, and they constitute the only de novo pathway for the formation of the building blocks needed for DNA synthesis and repair. P. aeruginosa is one of the few bacteria encoding all three known RNR classes (Ia, II, and III). Class Ia RNRs are oxygen dependent, class II are oxygen independent, and class III are oxygen sensitive. A tight control of RNR activity is essential for anaerobic growth and therefore for biofilm development. In this work we explored the role of the different RNR classes in biofilm formation under aerobic and anaerobic initial conditions and using static and continuous-flow biofilm models. We demonstrated the importance of class II and III RNR for proper cell division in biofilm development and maturation. We also determined that these classes are transcriptionally induced during biofilm formation and under anaerobic conditions. The molecular mechanism of their anaerobic regulation was also studied, finding that the Anr/Dnr system is responsible for class II RNR induction. These data can be integrated with previous knowledge about biofilms in a model where these structures are understood as a set of layers determined by oxygen concentration and contain cells with different RNR expression profiles, bringing us a step closer to the understanding of this complex growth pattern, essential for P. aeruginosa chronic infections.

JTD Keywords: Pseudomonas aeruginosa, Ribonucleotide Reductases, Vitamin B 12, Anaerobic metabolism, Biofilm formation, DNA Synthesis, Oxygen diffusion, nrd genes.

Electrospinning is a method that can be used to efficiently produce scaffolds that mimic the fibrous structure of natural tissue, such as muscle structures or the extracellular matrix of bone. The technique is often used as a way of depositing composites (organic/inorganic materials) to obtain bioactive nanofibers which have the requisite mechanical properties for use in tissue engineering. However, many factors can influence the formation and collection of fibers, including experimental variables such as the parameters of the solution of the electrospun slurry. In this study, we assessed the influence of the polymer concentration, glass content and glass hydrolysis level on the morphology and thickness of fibers produced by electrospinning for a PCL-(Si-Ca-P2) bioactive ormoglass—organically modified glass—blend. Based on previous assays, this combination of materials shows good angiogenic and osteogenic properties, which gives it great potential for use in tissue engineering. The results of our study showed that blend preparation directly affected the features of the resulting fibers, and when the parameters of the blend are precisely controlled, fibers with a regular diameter could be produced fairly easily when 2,2,2-trifluoroethanol was used as a solvent instead of tetrahydrofuran. The diameter of the homogeneous fibers ranged from 360 to 620 nm depending on the experimental conditions used. This demonstrates that experimental optimization of the electrospinning process is crucial in order to obtain a deposit of hybrid nanofibers with a regular shape.

JTD Keywords: Si-based glasses, Ormoglass, Electrospinning, Hybrid materials, Bioactivity, Angiogenesis

Regenerative medicine strategies to promote recovery following traumatic brain injuries are currently focused on the use of biomaterials as delivery systems for cells or bioactive molecules. This study shows that cell-free biomimetic scaffolds consisting of radially aligned electrospun poly-l/dl lactic acid (PLA70/30) nanofibers release l-lactate and reproduce the 3D organization and supportive function of radial glia embryonic neural stem cells. The topology of PLA nanofibers supports neuronal migration while l-lactate released during PLA degradation acts as an alternative fuel for neurons and is required for progenitor maintenance. Radial scaffolds implanted into cavities made in the postnatal mouse brain fostered complete implant vascularization, sustained neurogenesis, and allowed the long-term survival and integration of the newly generated neurons. Our results suggest that the endogenous central nervous system is capable of regeneration through the invivo dedifferentiation induced by biophysical and metabolic cues, with no need for exogenous cells, growth factors, or genetic manipulation.

JTD Keywords: Lactate, Nanofibers, Neural stem cells, Neurogenesis, Regeneration, Vascularization

In bone regeneration, silicon-based calcium phosphate glasses (Bioglasses) have been widely used since the 1970s. However, they dissolve very slowly because of their high amount of Si (SiO2 > 45%). Recently, our group has found that calcium ions released by the degradation of glasses in which the job of silicon is done by just 5% of TiO2 are effective angiogenic promoters, because of their stimulation of a cell-membrane calcium sensing receptor (CaSR). Based on this, other focused tests on angiogenesis have found that Bioglasses also have the potential to be angiogenic promoters even with high contents of silicon (80%); however, their slow degradation is still a problem, as the levels of silicon cannot be decreased any lower than 45%. In this work, we propose a new generation of hybrid organically modified glasses, ormoglasses, that enable the levels of silicon to be reduced, therefore speeding up the degradation process. Using electrospinning as a faithful way to mimic the extracellular matrix (ECM), we successfully produced hybrid fibrous mats with three different contents of Si (40, 52, and 70%), and thus three different calcium ion release rates, using an ormoglass–polycaprolactone blend approach. These mats offered a good platform to evaluate different calcium release rates as osteogenic promoters in an in vivo subcutaneous environment. Complementary data were collected to complement Ca2+ release analysis, such as stiffness evaluation by AFM, ζ-potential, morphology evaluation by FESEM, proliferation and differentiation analysis, as well as in vivo subcutaneous implantations. Material and biological characterization suggested that compositions of organic/inorganic hybrid materials with a Si content equivalent to 40%, which were also those that released more calcium, were osteogenic. They also showed a greater ability to form blood vessels. These results suggest that Si-based ormoglasses can be considered an efficient tool for calcium release modulation, which could play a key role in the angiogenic promoting process.

JTD Keywords: Biological materials, Blood vessels, Calcium, Electrospinning, Glass, Hybrid materials, Silicon oxides, Sol-gel process, Sol-gels, Angiogenesis, Biological characterization, Calcium phosphate glass, Calcium-sensing receptors, Degradation process, Extracellular matrices, Organic/inorganic hybrid materials, ormoglasses, Silicon

In this work we have evaluated the capacity of bone morphogenetic protein-2 (BMP-2) and fibrin-binding platelet-derived growth factor-BB (PDGF-BB) to support cell growth and induce bone regeneration using two different imaging technologies to improve the understanding of structural and organizational processes participating in tissue repair. Human mesenchymal stem cells from adipose tissue (hAMSCs) expressing two luciferase genes, one under the control of the cytomegalovirus (CMV) promoter and the other under the control of a tissue-specific promoter (osteocalcin or platelet endothelial cell adhesion molecule), were seeded in fibrin matrices containing BMP-2 and fibrin-binding PDGF-BB, and further implanted intramuscularly or in a mouse calvarial defect. Then, cell growth and bone regeneration were monitored by bioluminescence imaging (BLI) to analyze the evolution of target gene expression, indicative of cell differentiation towards the osteoblastic and endothelial lineages. Non-invasive imaging was supplemented with micro-computed tomography (μCT) to evaluate bone regeneration and high-resolution μCT of vascular casts. Results from BLI showed hAMSC growth during the first week in all cases, followed by a rapid decrease in cell number; as well as an increment of osteocalcin but not PECAM-1 expression 3 weeks after implantation. Results from μCT show that the delivery of BMP-2 and PDGF-BB by fibrin induced the formation of more bone and improves vascularization, resulting in more abundant and thicker vessels, in comparison with controls. Although the inclusion of hAMSCs in the fibrin matrices made no significant difference in any of these parameters, there was a significant increment in the connectivity of the vascular network in defects treated with hAMSCs.

JTD Keywords: Angiogenesis, Bioluminescence imaging, Bone regeneration, Fibrin, Mesenchymal stem cell

The angiogenic capacity of a new biomaterial composite of poly(lactic acid) and calcium phosphate glass (PLA/CaP) was analyzed by noninvasive bioluminescence imaging (BLI) and histological procedures. Human adipose tissue-derived mesenchymal stromal cells expressing cytomegalovirus (CMV) promoter regulated Photinus pyralis luciferase (hAMSC-PLuc) grew up to 30 times the initial cell load, in vitro, when seeded in PLA/CaP scaffolds, but suffered an initial growth crisis followed by recovery when the scaffolds were subcutaneously implanted in SCID mice. To analyze changes in gene expression, hAMSC-PLuc cells were double labeled with a CMV promoter regulated Renilla reniformis luciferase and a Photinus pyralis luciferase reporter regulated by either the PECAM promoter or a hypoxia response element (HRE) artificial promoter and seeded in PLA/CaP and PLA scaffolds implanted in SCID mice. Analysis by BLI showed that hAMSCs in scaffolds were induced to differentiate to the endothelial lineage and did this faster in PLA/CaP than in PLA scaffolds. Endothelial differentiation correlated with a decrease in the activity of HRE regulated luciferase expression, indicative of a reduction of hypoxia. Histological analysis showed that PLA/CaP scaffolds were colonized by a functional host vascular system. Moreover, colonization by isolectin B4 positive host cells was more effective in PLA/CaP than in PLA scaffolds, corroborating BLI results.

JTD Keywords: Scaffold, Bioluminescence imaging, Cell differentiation, Angiogenesis, Mesenchymal stromal cells

Smart biomaterials play a key role when aiming at successful tissue repair by means of regenerative medicine approaches, and are expected to contain chemical as well as mechanical cues that will guide the regenerative process. Recent advances in the understanding of stem cell biology and mechanosensing have shed new light onto the importance of the local microenvironment in determining cell fate. Herein we report the biological properties of a bioactive, biodegradable calcium phosphate glass/polylactic acid composite biomaterial that promotes bone marrow-derived endothelial progenitor cell (EPC) mobilisation, differentiation and angiogenesis through the creation of a controlled bone healing-like microenvironment. The angiogenic response is triggered by biochemical and mechanical cues provided by the composite, which activate two synergistic cell signalling pathways: a biochemical one mediated by the calcium-sensing receptor and a mechanosensitive one regulated by non-muscle myosin II contraction. Together, these signals promote a synergistic response by activating EPCs-mediated VEGF and VEGFR-2 synthesis, which in turn promote progenitor cell homing, differentiation and tubulogenesis. These findings highlight the importance of controlling microenvironmental cues for stem/progenitor cell tissue engineering and offer exciting new therapeutical opportunities for biomaterialbased vascularisation approaches and clinical applications.

JTD Keywords: Calcium phosphate glass composite, Smart biomaterial, Endothelial progenitor cell, Angiogenesis, Mechanosensing, Calcium-sensing receptor

The self-assembly of peptides and proteins into amyloid fibrils of nanometric thickness and up to several micrometres in length, a phenomenon widely observed in biological systems, has recently aroused a growing interest in nanotechnology and nanomedicine. Here we have applied atomic force microscopy and single molecule force spectroscopy to study the amyloidogenesis of a peptide derived from human amylin and of its reverse sequence. The spontaneous formation of protofibrils and their orientation along well-defined directions on graphite and DMSO-coated graphite substrates make the studied peptides interesting candidates for nanotechnological applications. The measured binding forces between peptides correlate with the number of hydrogen bonds between individual peptides inside the fibril structure according to molecular dynamics simulations.

JTD Keywords: Amyloid fibril, Amyloidogenesis, Binding forces, Fibril structure, Graphite substrate, Molecular dynamics simulations, Nanometrics, Protofibrils, Single molecule force spectroscopy, Spontaneous formation, Atomic force microscopy, Atomic spectroscopy, Graphite, Hydrogen bonds, Medical nanotechnology, Molecular dynamics, Molecular physics, Self assembly, Thickness measurement, Peptides

For a monolayer sheet to migrate cohesively, it has long been suspected that each constituent cell must exert physical forces not only upon its extracellular matrix but also upon neighboring cells. The first comprehensive maps of these distinct force components reveal an unexpected physical picture. Rather than showing smooth and systematic variation within the monolayer, the distribution of physical forces is dominated by heterogeneity, both in space and in time, which emerges spontaneously, propagates over great distances, and cooperates over the span of many cell bodies. To explain the severe ruggedness of this force landscape and its role in collective cell guidance, the well known mechanisms of chemotaxis, durotaxis, haptotaxis are clearly insufficient. In a broad range of epithelial and endothelial cell sheets, collective cell migration is governed instead by a newly discovered emergent mechanism of innately collective cell guidance - plithotaxis.

JTD Keywords: Positional information, Drosophila embryo, Sheet migration, Dpp gradient, Cells, Force, Morphogenesis, Transition, Identification, Proliferation

Bacillus anthracis is a severe mammalian pathogen encoding a class Ib ribonucleotide reductase (RNR). RNR is a universal enzyme that provides the four essential deoxyribonucleotides needed for DNA replication and repair. Almost all Bacillus spp. encode both class Ib and class III RNR operons, but the B. anthracis class III operon was reported to encode a pseudogene, and conceivably class Ib RNR is necessary for spore germination and proliferation of B. anthracis upon infection. The class Ib RNR operon in B. anthracis encodes genes for the catalytic NrdE protein, the tyrosyl radical metalloprotein NrdF, and the flavodoxin protein NrdI. The tyrosyl radical in NrdF is stabilized by an adjacent Mn(2)(III) site (Mn-NrdF) formed by the action of the NrdI protein or by a Fe(2)(III) site (Fe-NrdF) formed spontaneously from Fe(2+) and O(2). In this study, we show that the properties of B. anthracis Mn-NrdF and Fe-NrdF are in general similar for interaction with NrdE and NrdI. Intriguingly, the enzyme activity of Mn-NrdF was approximately an order of magnitude higher than that of Fe-NrdF in the presence of the class Ib-specific physiological reductant NrdH, strongly suggesting that the Mn-NrdF form is important in the life cycle of B. anthracis. Whether the Fe-NrdF form only exists in vitro or whether the NrdF protein in B. anthracis is a true cambialistic enzyme that can work with either manganese or iron remains to be established.

JTD Keywords: Escherichia-coli, Corynebacterium-ammoniagenes, Crystal-structure, Cofactor, Cubunit, Growth, Genes

Most eukaryotic cells sense and respond to the mechanical properties of their surroundings. This can strongly influence their collective behavior in embryonic development, tissue function, and wound healing. We use a deformable substrate to measure collective behavior in cell motion due to substrate mediated cell-cell interactions. We quantify spatial and temporal correlations in migration velocity and substrate deformation, and show that cooperative cell-driven patterns of substrate deformation mediate long-distance mechanical coupling between cells and control collective cell migration.

JTD Keywords: Movement, Morphogenesis, Stiffness, Forces, Flocks

The role of amyloid beta (A beta) peptide in the onset and progression of Alzheimer's disease is linked to the presence of soluble A beta species. Sulfated glycosaminoglycans (GAGs) promote A beta fibrillogenesis and reduce the toxicity of the peptide in neuronal cell cultures, but a satisfactory rationale to explain these effects at the molecular level has not been provided yet. We have used circular dichroism, Fourier transform infrared spectroscopy, fluorescence microscopy and spectroscopy, protease digestion, atomic force microscopy (AFM), and molecular dynamics simulations to characterize the association of the 42-residue fragment A beta(42) with sulfated GAGs, hyaluronan, chitosan, and poly(vinyl sulfate) (PVS). Our results indicate that the formation of stable A beta(42) fibrils is promoted by polymeric GAGs with negative charges placed in-frame with the 4.8-angstrom separating A beta(42) monomers within protofibrillar beta-sheets. Incubation of A beta(42) with excess sulfated GAGs and hyaluronan increased amyloid fibril content and resistance to proteolysis 2- to 5-fold, whereas in the presence of the cationic polysaccharide chitosan, A beta(42) fibrillar species were reduced by 25% and sensitivity to protease degradation increased similar to 3-fold. Fibrils of intermediate stability were obtained in the presence of PVS, an anionic polymer with more tightly packed charges than GAGs. Important structural differences between A beta(42) fibrils induced by PVS and A beta(42) fibrils obtained in the presence of GAGs and hyaluronan were observed by AFM, whereas mainly precursor protofibrillar forms were detected after incubation with chitosan. Computed binding energies per peptide from -11.2 to -13.5 kcal/mol were calculated for GAGs and PVS, whereas a significantly lower value of -7.4 kcal/mol was obtained for chitosan. Taken together, our data suggest a simple and straightforward mechanism to explain the role of GAGs as enhancers of the formation of insoluble A beta(42) fibrils trapping soluble toxic forms.

JTD Keywords: Alzheimer's disease, Amyloid fibril structure, Fibrillogenesis enhancers and inhibitors, Polysaccharides

We have studied the motor abilities and associative learning capabilities of adult mice placed in different enriched environments. Three-month-old animals were maintained for a month alone (AL), alone in a physically enriched environment (PHY), and, finally, in groups in the absence (SO) or presence (SOPHY) of an enriched environment. The animals' capabilities were subsequently checked in the rotarod test, and for classical and instrumental learning. The PHY and SOPHY groups presented better performances in the rotarod test and in the acquisition of the instrumental learning task. In contrast, no significant differences between groups were observed for classical eyeblink conditioning. The four groups presented similar increases in the strength of field EPSPs (fEPSPs) evoked at the hippocampal CA3-CA1 synapse across classical conditioning sessions, with no significant differences between groups. These trained animals were pulse-injected with bromodeoxyuridine (BrdU) to determine hippocampal neurogenesis. No significant differences were found in the number of NeuN/BrdU double-labeled neurons. We repeated the same BrdU study in one-month-old mice raised for an additional month in the above-mentioned four different environments. These animals were not submitted to rotarod or conditioned tests. Non-trained PHY and SOPHY groups presented more neurogenesis than the other two groups. Thus, neurogenesis seems to be related to physical enrichment at early ages, but not to learning acquisition in adult mice.

JTD Keywords: Long-term potentiation, Adult neurogenesis, Synaptic transmission, Cell proliferation, CA3-CA1 synapse, Granule cells

Tissue engineering aims to regenerate tissues and organs by using cell and biomaterial-based approaches. One of the current challenges in the field is to promote proper vascularization in the implant to prevent cell death and promote host integration. Bone marrow endothelial progenitor cells (BM-EPCs) and mesenchymal stem cells (MSCs) are bone marrow resident stem cells widely employed for proangiogenic applications. In vivo, they are likely to interact frequently both in the bone marrow and at sites of injury. In this study, the physical and biochemical interactions between BM-EPCs and MSCs in an in vitro co-culture system were investigated to further clarify their roles in vascularization. BM-EPC/MSC co-cultures established close cell-cell contacts soon after seeding and self-assembled to form elongated structures at 3 days. Besides direct contact, cells also exhibited vesicle transport phenomena. When co-cultured in Matrigel, tube formation was greatly enhanced even in serum-starved, growth factor free medium. Both MSCs and BM-EPCs contributed to these tubes. However, cell proliferation was greatly reduced in co-culture and morphological differences were observed. Gene expression and cluster analysis for wide panel of angiogenesis-related transcripts demonstrated up-regulation of angiogenic markers but down-regulation of many other cytokines. These data suggest that cross-talk occurs in between BM-EPCs and MSCs through paracrine and direct cell contact mechanisms leading to modulation of the angiogenic response.

JTD Keywords: Bone marrow, Endothelial progenitor cell, Co-culture, Mesenchymal stem cell, Angiogenesis

Angiogenesis is a complex process regulated by many cell types and a large variety of biochemical signals such as growth factors, transcription factors, oxygen and nutrient diffusion among others. In the present study, we found out that Flk-1(+) CD34(+) progenitor cells (bone marrow resident cells with an important role in angiogenesis) were responsive to changes in extracellular calcium concentration through a membrane bound, G-protein-coupled receptor sensitive to calcium ions related to the calcium-sensing receptor (CaSR). Calcium was able to induce progenitor cell migration in Boyden chamber experiments and tubulogenesis in Matrigel assays. Addition of anti-CaSR antibodies completely blocked the effect, while CaSR agonist Mg2+ produced a similar response to that of calcium. Real time RT-PCR for a wide array of angiogenesis-related genes showed increased expression of endothelial markers and signaling pathways involved in angiogenesis. These results suggest calcium could be a physiological modulator of the bone marrow progenitor cell-mediated angiogenic response.

JTD Keywords: Endothelial progenitor cell, Calcium-sensing receptor, Angiogenesis, Chemotaxis, Calcium, Bone marrow

Fundamental biological processes including morphogenesis, tissue repair and tumour metastasis require collective cell motions(1-3), and to drive these motions cells exert traction forces on their surroundings(4). Current understanding emphasizes that these traction forces arise mainly in 'leader cells' at the front edge of the advancing cell sheet(5-9). Our data are contrary to that assumption and show for the first time by direct measurement that traction forces driving collective cell migration arise predominately many cell rows behind the leading front edge and extend across enormous distances. Traction fluctuations are anomalous, moreover, exhibiting broad non-Gaussian distributions characterized by exponential tails(10-12). Taken together, these unexpected findings demonstrate that although the leader cell may have a pivotal role in local cell guidance, physical forces that it generates are but a small part of a global tug-of-war involving cells well back from the leading edge.

JTD Keywords: Focal adhesions, Granular matter, Bead packs, Morphogenesis, Sheets, Actin, Fluctuations, Fibroblasts, Microscopy, Diversity

The influence of surface chemistry -substrates with controlled surface density of -OH groups- on fibronectin conformation and distribution is directly observed by Atomic Force Microscopy (AFM). FN fibrillogenesis, which is known to be a process triggered by interaction with integrins, is shown in our case to be induced by the substrate (in absence of cells), which is able to enhance FN-FN interactions leading to the formation of a protein network on the material surface. This phenomenon depends both on surface chemistry and protein concentration. The level of the FN fibrillogenesis was quantified by calculating the fractal dimension of the adsorbed protein from image analysis of the AFM results. The total amount of adsorbed FN is obtained by making use of a methodology which employs western-blotting combined with image analysis of the corresponding protein bands, with the lowest sensitivity threshold equal to 15 ng of adsorbed protein. Furthermore, FN adsorption is correlated to human osteoblast adhesion through morphology and actin cytoskeleton formation. Actin polymerization is in need of the formation of the protein network on the substrate's surface. Cell morphology is more rounded (as quantified by calculating the circularity of the cells by image analysis) the lower the degree of FN fibrillogenesis on the substrate.

JTD Keywords: Cell-adhesion, Conformational-changes, Electron-microscopy, Protein adsorption, Fractal dimension, Integrin binding, Biocompatibility, Monolayers, Matrix, Fibrillogenesis

The Streptococcus pyogenes genome harbors two clusters of class Ib ribonucleotide reductase genes, nrdHEF and nrdF*I*E*, and a second stand-alone nrdI gene, designated nrdI2. We show that both clusters are expressed simultaneously as two independent operons. The NrdEF enzyme is functionally active in vitro, while the NrdE*F* enzyme is not. The NrdF* protein lacks three of the six highly conserved iron-liganding side chains and cannot form a dinuclear iron site or a tyrosyl radical. In vivo, on the other hand, both operons are functional in heterologous complementation in Escherichia coli. The nrdF*I*E* operon requires the presence of the nrdI* gene, and the nrdHEF operon gained activity upon cotranscription of the heterologous nrdI gene from Streptococcus pneumoniae, while neither nrdI* nor nrdI2 from S. pyogenes rendered it active. Our results highlight the essential role of the flavodoxin NrdI protein in vivo, and we suggest that it is needed to reduce met-NrdF, thereby enabling the spontaneous reformation of the tyrosyl radical. The NrdI* flavodoxin may play a more direct role in ribonucleotide reduction by the NrdF*I*E* system. We discuss the possibility that the nrdF*I*E* operon has been horizontally transferred to S. pyogenes from Mycoplasma spp.

JTD Keywords: Group-a streptococcus, Bacillus-subtilis genes, Escherichia-coli, Corynebacterium-ammoniagenes, Mycobacterium-tuberculosis, Expression analysis, Genome sequence, Small-subunit, Salmonella-typhimurium, Iron center

The H-NS protein plays a significant role in the modulation of gene expression in Gram-negative bacteria. Whereas isolation and characterization of hns mutants in Escherichia coli, Salmonella and Shigella represented critical steps to gain insight into the modulatory role of H-NS, it has hitherto not been possible to isolate hns mutants in Yersinia. The hns mutation is considered to be deleterious in this genus. To study the modulatory role of H-NS in Yersinia we circumvented hns lethality by expressing in Y. enterocolitica a truncated H-NS protein known to exhibit anti-H-NS activity in E. coli (H-NST(EPEC)). Y. enterocolitica cells expressing H-NST(EPEC) showed an altered growth rate and several differences in the protein expression pattern, including the ProV protein, which is modulated by H-NS in other enteric bacteria. To further confirm that H-NST(EPEC) expression in Yersinia can be used to demonstrate H-NS-dependent regulation in this genus, we used this approach to show that H-NS modulates expression of the YmoA protein.

JTD Keywords: Bacterial Proteins/biosynthesis/genetics/ physiology, DNA-Binding Proteins/biosynthesis/genetics/ physiology, Electrophoresis, Gel, Two-Dimensional, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Genes, Essential, Proteome/analysis, RNA, Bacterial/biosynthesis, RNA, Messenger/biosynthesis, Reverse Transcriptase Polymerase Chain Reaction, Sequence Deletion, Yersinia enterocolitica/chemistry/genetics/growth & development/ physiology

In this study we report on an experimental method based on dielectrophoretic analysis to identify changes in four Escherichia coli isogenic strains that differed exclusively in one mutant allele. The dielectrophoretic properties of wild-type cells were compared to those of hns, hha, and hha hns mutant derivatives. The hns and hha genes code respectively for the global regulators Hha and H-NS. The Hha and H-NS proteins modulate gene expression in Escherichia coli and other Gram negative bacteria. Mutations in either hha or hns genes result in a pleiotropic phenotype. A two-shell prolate ellipsoidal model has been used to fit the experimental data, obtained from dielectrophoresis measurements, and to study the differences in the dielectric properties of the bacterial strains. The experimental results show that the mutant genotype can be predicted from the dielectrophoretic analysis of the corresponding cultures, opening the way to the development of microdevices for specific identification. Therefore, this study shows that dielectrophoresis can be a valuable tool to study bacterial populations which, although apparently homogeneous, may present phenotypic variability.

JTD Keywords: H-NS, Dielectric behaviour, Hemolysin genes, Cells, Separation, Expression, Proteins, HHA, Electrorotation, Polarization