The original version of this Article contained an error in the author affiliations. The affiliation of Marjan Enayati with 'Ludwig Boltzmann Cluster for Cardiovascular Research at the Center for ...Biomedical Research, Medical University of Vienna, Austria' was inadvertently omitted. This has now been corrected in both the PDF and HTML versions of the Article.
In the original version of this Article the dataset identifier in the Data Availability statement was incorrect. The correct dataset identifier is PXD009500. This has been corrected in the HTML and ...PDF versions of this Article.
Damage to osteochondral (OC) tissues can lead to pain, loss of motility, and progress to osteoarthritis. Tissue engineering approaches offer the possibility of replacing damaged tissues and restoring ...joint function; however, replicating the spatial and functional heterogeneity of native OC tissue remains a pressing challenge. Chondrocytes in healthy cartilage exist in relatively low-oxygen conditions, while osteoblasts in the underlying bone experience higher oxygen pressures. Such oxygen gradients also exist in the limb bud, where they influence OC tissue development. The cellular response to these spatial variations in oxygen pressure, which is mediated by the hypoxia inducible factor (HIF) pathway, plays a central role in regulating osteo- and chondrogenesis by directing progenitor cell differentiation and promoting and maintaining appropriate extracellular matrix production. Understanding the role of the HIF pathway in OC tissue development may enable new approaches to engineer OC tissue. In this review, we discuss strategies to spatially and temporarily regulate the HIF pathway in progenitor cells to create functional OC tissue for regenerative therapies.
Growing interest in exploring mechanically mediated biological phenomena has resulted in cell culture substrates and 3D matrices with variable stiffnesses becoming standard tools in biology labs. ...However, correlating stiffness with biological outcomes and comparing results between research groups is hampered by variability in the methods used to determine Young's (elastic) modulus, E, and by the inaccessibility of relevant mechanical engineering protocols to most biology labs. Here, we describe a protocol for measuring E of soft 2D surfaces and 3D hydrogels using atomic force microscopy (AFM) force spectroscopy. We provide instructions for preparing hydrogels with and without encapsulated live cells, and provide a method for mounting samples within the AFM. We also provide details on how to calibrate the instrument, and give step-by-step instructions for collecting force-displacement curves in both manual and automatic modes (stiffness mapping). We then provide details on how to apply either the Hertz or the Oliver-Pharr model to calculate E, and give additional instructions to aid the user in plotting data distributions and carrying out statistical analyses. We also provide instructions for inferring differential matrix remodeling activity in hydrogels containing encapsulated single cells or organoids. Our protocol is suitable for probing a range of synthetic and naturally derived polymeric hydrogels such as polyethylene glycol, polyacrylamide, hyaluronic acid, collagen, or Matrigel. Although sample preparation timings will vary, a user with introductory training to AFM will be able to use this protocol to characterize the mechanical properties of two to six soft surfaces or 3D hydrogels in a single day.
Display omitted
•Human induced pluripotent stem cells in self-renewing or differentiating conditions.•Simple live phase images analysed using CellProfiler and frame to frame ...subtraction.•Convolutional Neuronal Network predicts culture condition from spheroid morphology.
High-throughput imaging methods can be applied to relevant cell culture models, fostering their use in research and translational applications. Improvements in microscopy, computational capabilities and data analysis have enabled high-throughput, high-content approaches from endpoint 2D microscopy images. Nonetheless, trade-offs in acquisition, computation and storage between content and throughput remain, in particular when cells and cell structures are imaged in 3D. Moreover, live 3D phase contrast microscopy images are not often amenable to analysis because of the high level of background noise.
Cultures of Human induced pluripotent stem cells (hiPSC) offer unprecedented scope to profile and screen conditions affecting cell fate decisions, self-organisation and early embryonic development. However, quantifying changes in the morphology or function of cell structures derived from hiPSCs over time presents significant challenges. Here, we report a novel method based on the analysis of live phase contrast microscopy images of hiPSC spheroids. We compare self-renewing versus differentiating media conditions, which give rise to spheroids with distinct morphologies; round versus branched, respectively. These cell structures are segmented from 2D projections and analysed based on frame-to-frame variations. Importantly, a tailored convolutional neural network is trained and applied to predict culture conditions from time-frame images.
We compare our results with more classic and involved endpoint 3D confocal microscopy and propose that such approaches can complement spheroid-based assays developed for the purpose of screening and profiling. This workflow can be realistically implemented in laboratories using imaging-based high-throughput methods for regenerative medicine and drug discovery.
Glass ionomer cements (GIC) can be used to protect dentine following caries removal. However, GIC have little biological activity on biological repair processes, which means that neo-dentine ...formation remains reliant on limited endogenous regenerative processes. Wnt/β-catenin signalling is known to play a central role in stimulating tertiary dentine formation following tooth damage and can be stimulated by a range of glycogen synthase kinase (GSK3) antagonists, including lithium ions.
Here, we created lithium-containing bioactive glass (BG) by substituting lithium for sodium ions in 45S5 BG. We then replaced between 10 and 40% of the powder phase of a commercial GIC with the lithium-substituted BG to create a range of formulations of ‘LithGlassGIC’. In vitro physical properties of the resulting glasses were characterised and their ability to stimulate reactionary dentine formation in mouse molars in vivo was tested.
Lithium release from LithGlassGIC increased with increasing lithium content in the cement. In common with unmodified commercial GIC, all formations of LithGlassGIC showed in vitro toxicity when measured using an indirect cell culture assay based on ISO10993:5, precluding direct pulp contact. However, in a murine non-exposed pulp model of tooth damage, LithGlassGIC quickly released lithium ions, which could be transiently detected in the saliva and blood. LithGlassGIC also enhanced the formation of tertiary dentine, resulting in a thickening of the dentine at the damage site that restored lost dentine volume. Dentine regeneration was likely mediated by upregulation of Wnt/β-catenin activity, as LithGlassGIC placed in TCF/Lef:H2B-GFP reporter mice showed enhanced GFP activity.
We conclude that LithGlassGIC acts as a biological restorative material that promotes tertiary dentine formation and restores tooth structure.
To coordinate cell fate with changes in spatial organization, stem cells (SCs) require specific and adaptable systems of signal exchange and cell-to-cell communication. Pluripotent embryonic stem ...cells (ESCs) use cytonemes to pair with trophoblast stem cells (TSCs) and form synthetic embryonic structures in a Wnt-dependent manner. How these interactions vary with pluripotency states remains elusive. Here we show that ESC transition to an early primed ESC (pESC) state reduces their pairing with TSCs and impairs synthetic embryogenesis. pESCs can activate the Wnt/β-catenin pathway in response to soluble Wnt ligands, but their cytonemes form unspecific and unstable interactions with localized Wnt sources. This is due to an impaired crosstalk between Wnt and glutamate receptor activity and reduced generation of Ca2+ transients on the cytonemes upon Wnt source contact. Induced iGluR activation can partially restore cytoneme function in pESCs, while transient overexpression of E-cadherin improves pESC-TSC pairing. Our results illustrate how changes in pluripotency state alter the mechanisms SCs use to self-organize.
The accumulation of calcified material in cardiovascular tissue is thought to involve cytochemical, extracellular matrix and systemic signals; however, its precise composition and nanoscale ...architecture remain largely unexplored. Using nano-analytical electron microscopy techniques, we examined valves, aortae and coronary arteries from patients with and without calcific cardiovascular disease and detected spherical calcium phosphate particles, regardless of the presence of calcific lesions. We also examined lesions after sectioning with a focused ion beam and found that the spherical particles are composed of highly crystalline hydroxyapatite that crystallographically and structurally differs from bone mineral. Taken together, these data suggest that mineralized spherical particles may play a fundamental role in calcific lesion formation. Their ubiquitous presence in varied cardiovascular tissues and from patients with a spectrum of diseases further suggests that lesion formation may follow a common process. Indeed, applying materials science techniques to ectopic and orthotopic calcification has great potential to lend critical insights into pathophysiological processes underlying calcific cardiovascular disease.
PDF
