Toughening mechanisms for the attachment of architectured materials: The mechanics of the tendon enthesis

Architectured materials offer tailored mechanical properties but are limited in engineering applications due to challenges in maintaining toughness across their attachments. The enthesis connects tendon and bone, two vastly different architectured materials, and exhibits toughness across a wide range of loadings. Understanding the mechanisms by which this is achieved could inform the development of engineered attachments. Integrating experiments, simulations, and novel imaging that enabled simultaneous observation of mineralized and unmineralized tissues, we identified putative mechanisms of enthesis toughening in a mouse model and then manipulated these mechanisms via in vivo control of mineralization and architecture. Imaging uncovered a fibrous architecture within the enthesis that controls trade-offs between strength and toughness. In vivo models of pathology revealed architectural adaptations that optimize these trade-offs through cross-scale mechanisms including nanoscale protein denaturation, milliscale load-sharing, and macroscale energy absorption. Results suggest strategies for optimizing architecture for tough bimaterial attachments in medicine and engineering.

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'Off-the-shelf’ allogeneic antigen-specific adoptive T-cell therapy for the treatment of multiple EBV-associated malignancies

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001608 ◽

2021 ◽

Vol 9 (2) ◽

pp. e001608

Author(s):

Debottam Sinha ◽

Sriganesh Srihari ◽

Kirrliee Beckett ◽

Laetitia Le Texier ◽

Matthew Solomon ◽

...

Keyword(s):

T Cell ◽

Cell Therapy ◽

Adoptive T Cell Therapy ◽

Nuclear Antigen ◽

T Cell Therapy ◽

In Vivo Models ◽

Hla Alleles ◽

Cell Therapies ◽

Wide Range

BackgroundEpstein-Barr virus (EBV), an oncogenic human gammaherpesvirus, is associated with a wide range of human malignancies of epithelial and B-cell origin. Recent studies have demonstrated promising safety and clinical efficacy of allogeneic ‘off-the-shelf’ virus-specific T-cell therapies for post-transplant viral complications.MethodsTaking a clue from these studies, we developed a highly efficient EBV-specific T-cell expansion process using a replication-deficient AdE1-LMPpoly vector that specifically targets EBV-encoded nuclear antigen 1 (EBNA1) and latent membrane proteins 1 and 2 (LMP1 and LMP2), expressed in latency II malignancies.ResultsThese allogeneic EBV-specific T cells efficiently recognized human leukocyte antigen (HLA)-matched EBNA1-expressing and/or LMP1 and LMP2-expressing malignant cells and demonstrated therapeutic potential in a number of in vivo models, including EBV lymphomas that emerged spontaneously in humanized mice following EBV infection. Interestingly, we were able to override resistance to T-cell therapy in vivo using a ‘restriction-switching’ approach, through sequential infusion of two different allogeneic T-cell therapies restricted through different HLA alleles. Furthermore, we have shown that inhibition of the programmed cell death protein-1/programmed death-ligand 1 axis in combination with EBV-specific T-cell therapy significantly improved overall survival of tumor-bearing mice when compared with monotherapy.ConclusionThese findings suggest that restriction switching by sequential infusion of allogeneic T-cell therapies that target EBV through distinct HLA alleles may improve clinical response.

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An optimized live bacterial delivery platform for the production and delivery of therapeutic nucleic acids and proteins

10.1101/2021.10.17.464697 ◽

2021 ◽

Author(s):

Darcy S.O. Mora ◽

Madeline Cox ◽

Forgivemore Magunda ◽

Ashley B. Williams ◽

Lyndsey Linke

Keyword(s):

Epithelial Cells ◽

Mouse Model ◽

Nucleic Acids ◽

Endosomal Escape ◽

Therapeutic Window ◽

Target Cells ◽

Full Potential ◽

Wide Range ◽

Delivery Platform

There is an unmet need for delivery platforms that realize the full potential of next-generation therapeutic and vaccine technologies, especially those that require intracellular delivery of nucleic acids. The in vivo usefulness of the current state-of-the-art delivery systems is limited by numerous intrinsic weaknesses, including lack of targeting specificity, inefficient entry and endosomal escape into target cells, undesirable immune activation, off-target effects, a small therapeutic window, limited genetic encoding and cargo capacity, and manufacturing challenges. Here we present our characterization of a delivery platform based on the use of engineered live, tissue-targeting, non-pathogenic bacteria (Escherichia coli strain SVC1) for intracellular cargo delivery. The SVC1 bacteria are engineered to specifically bind to epithelial cells via a surface-expressed targeting ligand, to escape the endosome upon intracellularization, and to have minimal immunogenicity. Here we report findings on key features of this system. First, we demonstrated that bacterial delivery of a short hairpin RNA (shRNA) can target and silence a gene in an in vitro mammalian respiratory cell model. Next, we used an in vivo mouse model to demonstrate that SVC1 bacteria are invasive to epithelial cells of various tissues and organs (eye, nose, mouth, stomach, vagina, skeletal muscle, and lungs) via local administration. We also showed that repeat dosing of SVC1 bacteria to the lungs is minimally immunogenic and that it does not have adverse effects on tissue homeostasis. Finally, to validate the potential of SVC1 bacteria in therapeutic applications, we demonstrated that bacterial delivery of influenza- targeting shRNAs to the respiratory tissues can mitigate viral replication in a mouse model of influenza infection. Our ongoing work is focused on further refining this platform for efficient delivery of nucleic acids, gene editing machinery, and therapeutic proteins, and we expect that this platform technology will enable a wide range of advanced therapeutic approaches.

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Immortalized neurons for the study of hypothalamic function

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00649.2010 ◽

2011 ◽

Vol 300 (5) ◽

pp. R1030-R1052 ◽

Cited By ~ 22

Author(s):

Prasad S. Dalvi ◽

Anaies Nazarians-Armavil ◽

Stephanie Tung ◽

Denise D. Belsham

Keyword(s):

Neuronal Cell ◽

Glucose Sensing ◽

T Antigen ◽

Molecular Techniques ◽

Cell Models ◽

Hypothalamic Neurons ◽

In Vivo Models ◽

Wide Range ◽

Hypothalamic Function

The hypothalamus is a vital part of the central nervous system: it harbors control systems implicated in regulation of a wide range of homeostatic processes, including energy balance and reproduction. Structurally, the hypothalamus is a complex neuroendocrine tissue composed of a multitude of unique neuronal cell types that express a number of neuromodulators, including hormones, classical neurotransmitters, and specific neuropeptides that play a critical role in mediating hypothalamic function. However, neuropeptide and receptor gene expression, second messenger activation, and electrophysiological and secretory properties of these hypothalamic neurons are not yet fully defined, primarily because the heterogeneity and complex neuronal architecture of the neuroendocrine hypothalamus make such studies challenging to perform in vivo. To circumvent this problem, our research group recently generated embryonic- and adult-derived hypothalamic neuronal cell models by utilizing the novel molecular techniques of ciliary neurotrophic factor-induced neurogenesis and SV40 T antigen transfer to primary hypothalamic neuronal cell cultures. Significant research with these cell lines has demonstrated their value as a potential tool for use in molecular genetic analysis of hypothalamic neuronal function. Insights gained from hypothalamic immortalized cells used in conjunction with in vivo models will enhance our understanding of hypothalamic functions such as neurogenesis, neuronal plasticity, glucose sensing, energy homeostasis, circadian rhythms, and reproduction. This review discusses the generation and use of hypothalamic cell models to study mechanisms underlying the function of individual hypothalamic neurons and to gain a more complete understanding of the overall physiology of the hypothalamus.

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Fabrication and Modeling of Dynamic Multipolymer Nanofibrous Scaffolds

Journal of Biomechanical Engineering ◽

10.1115/1.3192140 ◽

2009 ◽

Vol 131 (10) ◽

Cited By ~ 63

Author(s):

Brendon M. Baker ◽

Nandan L. Nerurkar ◽

Jason A. Burdick ◽

Dawn M. Elliott ◽

Robert L. Mauck

Keyword(s):

Mechanical Properties ◽

Rational Design ◽

Cell Infiltration ◽

Tissue Properties ◽

Matrix Deposition ◽

Native Tissue ◽

Nanofibrous Scaffolds ◽

Wide Range ◽

Anisotropic Mechanical Properties

Aligned nanofibrous scaffolds hold tremendous potential for the engineering of dense connective tissues. These biomimetic micropatterns direct organized cell-mediated matrix deposition and can be tuned to possess nonlinear and anisotropic mechanical properties. For these scaffolds to function in vivo, however, they must either recapitulate the full dynamic mechanical range of the native tissue upon implantation or must foster cell infiltration and matrix deposition so as to enable construct maturation to meet these criteria. In our recent studies, we noted that cell infiltration into dense aligned structures is limited but could be expedited via the inclusion of a distinct rapidly eroding sacrificial component. In the present study, we sought to further the fabrication of dynamic nanofibrous constructs by combining multiple-fiber populations, each with distinct mechanical characteristics, into a single composite nanofibrous scaffold. Toward this goal, we developed a novel method for the generation of aligned electrospun composites containing rapidly eroding (PEO), moderately degradable (PLGA and PCL/PLGA), and slowly degrading (PCL) fiber populations. We evaluated the mechanical properties of these composites upon formation and with degradation in a physiologic environment. Furthermore, we employed a hyperelastic constrained-mixture model to capture the nonlinear and time-dependent properties of these scaffolds when formed as single-fiber populations or in multipolymer composites. After validating this model, we demonstrated that by carefully selecting fiber populations with differing mechanical properties and altering the relative fraction of each, a wide range of mechanical properties (and degradation characteristics) can be achieved. This advance allows for the rational design of nanofibrous scaffolds to match native tissue properties and will significantly enhance our ability to fabricate replacements for load-bearing tissues of the musculoskeletal system.

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Altered canonical hedgehog-gli signalling axis in pesticide-induced bone marrow aplasia mouse model

Archives of Industrial Hygiene and Toxicology ◽

10.2478/10004-1254-63-2012-2255 ◽

2012 ◽

Vol 63 (3) ◽

pp. 271-282 ◽

Cited By ~ 9

Author(s):

Malay Chaklader ◽

Prosun Das ◽

Jacintha Archana Pereira ◽

Samaresh Chaudhuri ◽

Sujata Law

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Pesticide Exposure ◽

Receptor Expression ◽

Human Beings ◽

Bone Marrow Aplasia ◽

Hedgehog Signalling ◽

Pesticide Treatment ◽

Wide Range

The mechanistic interplay between pesticide exposure and development of marrow aplasia is not yet well established but there are indices that chronic pesticide exposure in some instances causes marrow aplasia like haematopoietic degenerative condition in human beings. Canonical Hedgehog (Hh) signalling has multiple roles in a wide range of developmental processes, including haematopoiesis. The present study was designed to explore the status of four important components of the canonical Hedgehog signalling cascade, the Sonic Hedgehog (Shh), Ptch1, Smo, and Gli1, in a mouse model of chronic pesticide-induced bone marrow aplasia. We used 5 % aqueous mixture of pesticides (chlorpyriphos, prophenophos, cypermethrin, alpha-methrin, and hexaconazole) for inhalation and dermal exposure of 6 hours per day and 5 days a week up to 90 days. Murine bone marrow aplasia related to chronic pesticide treatment was confi rmed primarily by haemogram, bone marrow cellularity, short term bone marrow explant culture for cellular kinetics, bone marrow smear, and fl ow cytometric Lin-Sca-1+C-kit+ extracellular receptor expression pattern. Later, components of hedgehog signalling were analysed in the bone marrow of both control and pesticide-treated aplastic groups of animals. The results depicted pancytopenic feature of peripheral blood, developmental anomaly of neutrophils, depression of primitive stem and progenitor population along with Shh, Ptch1, Smo and Gli1 expression in aplasia group. This investigation suggests that pesticide-induced downregulation of two critically important proteins - Ptch1 and Gli1 - inside the haematopoietic stem and progenitor cell population impairs haematopoietic homeostasis and regeneration mechanism in vivo concurrent with bone marrow aplasia.

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Analysis of an open source, closed-loop, realtime system for hippocampal sharp-wave ripple disruption

10.1101/298661 ◽

2018 ◽

Author(s):

Shayok Dutta ◽

Etienne Ackermann ◽

Caleb Kemere

Keyword(s):

Open Source ◽

Neural Activity ◽

Closed Loop ◽

Detection System ◽

Detection Accuracy ◽

Sharp Wave ◽

Trade Offs ◽

Wide Range ◽

Realtime System

AbstractTransient neural activity pervades hippocampal electrophysiological activity. During more quiescent states, brief ≈100 ms periods comprising large ≈150–250 Hz oscillations known as sharp-wave ripples (SWR) which co-occur with ensemble bursts of spiking activity, are regularly found in local field potentials recorded from area CA1. SWRs and their concomitant neural activity are thought to be important for memory consolidation, recall, and memory-guided decision making. Temporally-selective manipulations of hippocampal neural activity upon online hippocampal SWR detection have been used as causal evidence of the importance of SWR for mnemonic process as evinced by behavioral and/or physiological changes. However, though this approach is becoming more wide spread, the performance trade-offs involved in building a SWR detection and disruption system have not been explored, limiting the design and interpretation of SWR detection experiments. We present an open source, plug-and-play, online ripple detection system with a detailed performance characterization. Our system has been constructed to interface with an open source software platform, Trodes, and two hardware acquisition platforms, Open Ephys and SpikeGadgets. We show that our in vivo results — approximately 80% detection latencies falling in between ≈20–66 ms with ≈2 ms closed-loop latencies while maintaining <10 false detections per minute — are dependent upon both algorithmic trade-offs and acquisition hardware. We discuss strategies to improve detection accuracy and potential limitations of online ripple disruptions. By characterizing this system in detail, we present a template for analyzing other closed-loop neural detection and perturbation systems. Thus, we anticipate our modular, open source, realtime system will facilitate a wide range of carefully-designed causal closed-loop neuroscience experiments.

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Galectin-3 levels are elevated following nintedanib treatment

Therapeutic Advances in Chronic Disease ◽

10.1177/2040622320968412 ◽

2020 ◽

Vol 11 ◽

pp. 204062232096841

Author(s):

Gali Epstein Shochet ◽

Alon Pomerantz ◽

David Shitrit ◽

Becky Bardenstein-Wald ◽

Kjetil Ask ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Mouse Model ◽

Quantitative Polymerase Chain Reaction ◽

Lung Fibroblasts ◽

Mrna Levels ◽

Serum Samples ◽

In Vivo Models ◽

Galectin 3 ◽

The Impact

Background and Aims: Idiopathic pulmonary fibrosis (IPF) is a common and severe form of pulmonary fibrosis. Nintedanib, a triple angiokinase inhibitor, is approved for treating IPF. Galectin 3 (Gal-3) activates a variety of profibrotic processes. Currently, the Gal-3 inhibitor TD139 is being tested in phase II clinical trials. Since this treatment is given ‘on top’ of nintedanib, it is important to estimate its effect on Gal-3 levels. Therefore, we evaluated the impact of nintedanib on Gal-3 expression using both in vitro and in vivo models, in addition to serum samples from patients with IPF. Methods: Gal-3 levels were evaluated in IPF and control tissue samples, primary human lung fibroblasts (HLFs) following nintedanib treatment (10–100 nM, quantitative polymerase chain reaction), and in a silica-induced fibrosis mouse model with/without nintedanib (0.021–0.21 mg/kg) by immunohistochemistry. In addition, Gal-3 levels were analyzed in serum samples from 41 patients with interstitial lung disease patients with/without nintedanib treatment by ELISA. Results: Nintedanib addition to HLFs resulted in significant elevations in Gal-3, phospho-signal transducer and activator of transcription 3 (pSTAT3), as well as IL-8 mRNA levels ( p < 0.05). Gal-3 expression was higher in samples from IPF patients compared with non-IPF controls at the protein and mRNA levels ( p < 0.05). In the in vivo mouse model, Gal-3 levels were increased following fibrosis induction and even further increased with the addition of nintedanib, mostly in macrophages ( p < 0.05). Patients receiving nintedanib presented with higher Gal-3 serum levels compared with those who did not receive nintedanib ( p < 0.05). Conclusion: Nintedanib elevates Gal-3 levels in both experimental models, along with patient samples. These findings highlight the possibility of using combined inhibition therapy for patients with IPF.

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Microfluidic neural axon diode

TECHNOLOGY ◽

10.1142/s2339547816500102 ◽

2016 ◽

Vol 04 (04) ◽

pp. 240-248 ◽

Cited By ~ 8

Author(s):

Sangcheol Na ◽

Myeongwoo Kang ◽

Seokyoung Bang ◽

Daehun Park ◽

Jinhyun Kim ◽

...

Keyword(s):

Neural Circuit ◽

Axon Growth ◽

In Vivo Models ◽

Starting Point ◽

Wide Range ◽

Reproducible Method ◽

Physical And Chemical ◽

Biology Research

Neural circuits, groups of neurons connected in directional manner, play a central role in information processing. Advances in neuronal biology research is limited by a lack of appropriate in vitro methods to construct and probe neuronal networks. Here, we describe a microfluidic culture platform that directs the growth of axons using “neural diode” structures to control neural connectivity. This platform is compatible with live cell imaging and can be used to (i) form pre-synaptic and postsynaptic neurons by directional axon growth and (ii) localize physical and chemical treatment to pre- or postsynaptic neuron groups (i.e. virus infection and etc.). The “neural diode” design consist of a microchannel that split into two branches: one is directed straight toward while the other returns back toward the starting point in a closed loop to send the axons back to the origin. We optimized the “neural diode” pattern dimension and design to achieve close to 70% directionality with a single unit of the “diode”. When repeated 3 times, near perfect (98–100% at wide range of cell concentrations) directionality can be achieved. The living neural circuit was characterized using Ca imaging and confirmed their function. The platform also serves as a straightforward, reproducible method to recapitulate a variety of neural circuit in vitro that were previously observable only in brain slice or in vivo models. The microfluidic neural diode may lead to better models for understanding the neural circuit and neurodegenerative diseases.

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A Brief Review of the Modelling of the Time Dependent Mechanical Properties of Tissue Engineering Scaffolds

Journal of Biomimetics Biomaterials and Tissue Engineering ◽

10.4028/www.scientific.net/jbbte.6.19 ◽

2010 ◽

Vol 6 ◽

pp. 19-33 ◽

Cited By ~ 11

Author(s):

N.K. Bawolin ◽

W.J. Zhang ◽

Xiong Biao Chen

Keyword(s):

Mechanical Properties ◽

Tissue Engineering ◽

Treatment Process ◽

Effective Properties ◽

Critical Role ◽

Time Dependent ◽

Tissue Engineering Scaffolds ◽

Wide Range ◽

Scaffold Degradation

The functionality of tissue scaffolds in vivo plays a critical role in the treatment process. Due to the time dependent nature of the mechanical properties of the constituent phases of the scaffold, a wide range of mechanical property histories may be observed during the treatment process, possibly influencing outcomes. The critical nature of the mechanical properties in load bearing applications indicates a need for the simultaneous modelling of both scaffold degradation and tissue regeneration with time, and the resulting effective properties of the tissue engineering construct. To this end, a review of the literature is conducted to identify the various existing approaches to modelling scaffold degradation, tissue behavior, and the dependency of the two processes on one another.

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RB depletion is required for the continuous growth of tumors initiated by loss of RB

PLoS Genetics ◽

10.1371/journal.pgen.1009941 ◽

2021 ◽

Vol 17 (12) ◽

pp. e1009941

Author(s):

Alex Doan ◽

Julia Arand ◽

Diana Gong ◽

Alexandros P. Drainas ◽

Yan Ting Shue ◽

...

Keyword(s):

Mouse Model ◽

Cancer Cells ◽

Mechanisms Of Action ◽

Thyroid Tumors ◽

Continuous Growth ◽

Knock Out ◽

Cycle Arrest ◽

Wide Range ◽

Knock Out Mice

The retinoblastoma (RB) tumor suppressor is functionally inactivated in a wide range of human tumors where this inactivation promotes tumorigenesis in part by allowing uncontrolled proliferation. RB has been extensively studied, but its mechanisms of action in normal and cancer cells remain only partly understood. Here, we describe a new mouse model to investigate the consequences of RB depletion and its re-activation in vivo. In these mice, induction of shRNA molecules targeting RB for knock-down results in the development of phenotypes similar to Rb knock-out mice, including the development of pituitary and thyroid tumors. Re-expression of RB leads to cell cycle arrest in cancer cells and repression of transcriptional programs driven by E2F activity. Thus, continuous RB loss is required for the maintenance of tumor phenotypes initiated by loss of RB, and this new mouse model will provide a new platform to investigate RB function in vivo.

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