Erratum: Publisher's Note: "A suspended layer additive manufacturing approach to the bioprinting of tri-layered skin equivalents" [APL Bioeng. 5, 046103 (2021)].

[This corrects the article DOI: 10.1063/5.0061361.].

NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency.

Autophagy is a homeostatic process critical for cellular survival, and its malfunction is implicated in human diseases including neurodegeneration. Loss of autophagy contributes to cytotoxicity and tissue degeneration, but the mechanistic understanding of this phenomenon remains elusive. Here, we generated autophagy-deficient (ATG5-/-) human embryonic stem cells (hESCs), from which we established a human neuronal platform to investigate how loss of autophagy affects neuronal survival. ATG5-/- neurons exhibit basal cytotoxicity accompanied by metabolic defects. Depletion of nicotinamide adenine dinucleotide (NAD) due to hyperactivation of NAD-consuming enzymes is found to trigger cell death via mitochondrial depolarization in ATG5-/- neurons. Boosting intracellular NAD levels improves cell viability by restoring mitochondrial bioenergetics and proteostasis in ATG5-/- neurons. Our findings elucidate a mechanistic link between autophagy deficiency and neuronal cell death that can be targeted for therapeutic interventions in neurodegenerative and lysosomal storage diseases associated with autophagic defect.

A detailed methodology for the long-term in vitro culture and analysis of three-dimensional, self-structuring bone models generated from cell lines or primary osteoblastic cell populations.

Background: There are insufficient in vitro bone models that accommodate long-term culture of osteoblasts and support their differentiation to osteocytes. The increased demand for effective therapies for bone diseases, and the ethical requirement to replace animals in research, warrants the development of such models.Here we present an in-depth protocol to prepare, create and maintain three-dimensional, in vitro, self-structuring bone models that support osteocytogenesis and long-term osteoblast survival (>1 year). Methods: Osteoblastic cells are seeded on a fibrin hydrogel, cast between two beta-tricalcium phosphate anchors. Analytical methods optimised for these self-structuring bone model (SSBM) constructs, including RT-qPCR, immunofluorescence staining and XRF, are described in detail. Results: Over time, the cells restructure and replace the initial matrix with a collagen-rich, mineralising one; and demonstrate differentiation towards osteocytes within 12 weeks of culture. Conclusions: Whilst optimised using a secondary human cell line (hFOB 1.19), this protocol readily accommodates osteoblasts from other species (rat and mouse) and origins (primary and secondary). This simple, straightforward method creates reproducible in vitro bone models that are responsive to exogenous stimuli, offering a versatile platform for conducting preclinical translatable research studies.

A suspended layer additive manufacturing approach to the bioprinting of tri-layered skin equivalents.

Skin exhibits a complex structure consisting of three predominant layers (epidermis, dermis, and hypodermis). Extensive trauma may result in the loss of these structures and poor repair, in the longer term, forming scarred tissue and associated reduction in function. Although a number of skin replacements exist, there have been no solutions that recapitulate the chemical, mechanical, and biological roles that exist within native skin. This study reports the use of suspended layer additive manufacturing to produce a continuous tri-layered implant, which closely resembles human skin. Through careful control of the bioink composition, gradients (chemical and cellular) were formed throughout the printed construct. Culture of the model demonstrated that over 21 days, the cellular components played a key role in remodeling the supporting matrix into architectures comparable with those of healthy skin. Indeed, it has been demonstrated that even at seven days post-implantation, the integration of the implant had occurred, with mobilization of the adipose tissue from the surrounding tissue into the construct itself. As such, it is believed that these implants can facilitate healing, commencing from the fascia, up toward the skin surface-a mechanism recently shown to be key within deep wounds.

Oxygen Consumption Evaluation: An Important Indicator of Metabolic State, Cellular Function, and Cell Fate Along Neural Deregulation.

Depletion of oxygen (O2) levels and reduction in the ATP synthesis (or even its complete blockage) are important characteristics of mitochondrial dysfunction; features that are often correlated with neurodegeneration. The measurement of oxygen consumption rate (OCR) is thus essential to evaluate cellular metabolism, survival, and neuroprotective strategies. In the present chapter, we describe the oxygen consumption assay using a Clark-type oxygen electrode in different types of samples named cells suspension (from primary and established cell culture), brain slices (ex vivo), and fresh brain tissues. In addition, we demonstrate herein how the program Oxygraph can be used in order to analyze the data and different approaches to normalize it.

Controlled self-assembly of chemical gardens enables fabrication of heterogeneous chemobrionic materials.

Chemical gardens are an example of a chemobrionic system that typically result in abiotic macro-, micro- and nano- material architectures, with formation driven by complex out-of-equilibrium reaction mechanisms. From a technological perspective, controlling chemobrionic processes may hold great promise for the creation of novel, compositionally diverse and ultimately, useful materials and devices. In this work, we engineer an innovative custom-built liquid exchange unit that enables us to control the formation of tubular chemical garden structures grown from the interface between calcium loaded hydrogel and phosphate solution. We show that systematic displacement of phosphate solution with water (H2O) can halt self-assembly, precisely control tube height and purify structures in situ. Furthermore, we demonstrate the fabrication of a heterogeneous chemobrionic composite material composed of aligned, high-aspect ratio calcium phosphate channels running through an otherwise dense matrix of poly(2-hydroxyethyl methacrylate) (pHEMA). Given that the principles we derive can be broadly applied to potentially control various chemobrionic systems, this work paves the way for fabricating multifunctional materials that may hold great potential in a variety of application areas, such as regenerative medicine, catalysis and microfluidics.

Chemobrionic structures in tissue engineering: self-assembling calcium phosphate tubes as cellular scaffolds.

A diverse range of complex patterns and mineralised hierarchical microstructures can be derived from chemobrionic systems, with formation driven by complex reaction-diffusion mechanisms far from thermodynamic equilibrium. In these experiments, self-assembling calcium phosphate tubes are generated using hydrogels made with 1 M calcium solutions layered with solutions of dibasic sodium phosphate over a range of concentrations between 0.2-1 M. Self-assembling structures prepared using 0.8 M dibasic sodium phosphate solutions were selected to assess cell-material interactions. Candidate chemobrionic scaffolds were characterised by micro-X-Ray fluorescence (µ-XRF) spectroscopy, Raman spectroscopy, powder X-ray diffraction (XRD), helium pycnometry and scanning electron microscopy (SEM). As prepared tubes were formed from non-stoichiometric hydroxyapatite (HA, Ca10-x(PO4)6-x(HPO4)x(OH)2-x (0 ≤x≤ 1)), which was confirmed as calcium deficient hydroxyapatite (CDHA, Ca9(PO4)5HPO4OH). Thermal treatment of tubes in air at 650 °C for 4 h converted the structures to beta tricalcium phosphate (ß-TCP, ß-Ca3(PO4)2). The potential of these scaffolds to support the attachment of bone marrow derived mesenchymal stem cells (BMSCs) was investigated for the first time, and we demonstrate cell attachment and elongation on the fabricated tubular structures.

Chemical Screening Approaches Enabling Drug Discovery of Autophagy Modulators for Biomedical Applications in Human Diseases.

Autophagy is an intracellular degradation pathway for malfunctioning aggregation-prone proteins, damaged organelles, unwanted macromolecules and invading pathogens. This process is essential for maintaining cellular and tissue homeostasis that contribute to organismal survival. Autophagy dysfunction has been implicated in the pathogenesis of diverse human diseases, and therefore, therapeutic exploitation of autophagy is of potential biomedical relevance. A number of chemical screening approaches have been established for the drug discovery of autophagy modulators based on the perturbations of autophagy reporters or the clearance of autophagy substrates. These readouts can be detected by fluorescence and high-content microscopy, flow cytometry, microplate reader and immunoblotting, and the assays have evolved to enable high-throughput screening and measurement of autophagic flux. Several pharmacological modulators of autophagy have been identified that act either via the classical mechanistic target of rapamycin (mTOR) pathway or independently of mTOR. Many of these autophagy modulators have been demonstrated to exert beneficial effects in transgenic models of neurodegenerative disorders, cancer, infectious diseases, liver diseases, myopathies as well as in lifespan extension. This review describes the commonly used chemical screening approaches in mammalian cells and the key autophagy modulators identified through these methods, and highlights the therapeutic benefits of these compounds in specific disease contexts.

In Vitro Screening Platforms for Identifying Autophagy Modulators in Mammalian Cells.

Autophagy is a vital homeostatic pathway essential for cellular survival and human health. It primarily functions as an intracellular degradation process for the turnover of aggregation-prone proteins and unwanted organelles. Dysregulation of autophagy underlying diverse human diseases reduces cell viability, whereas stimulation of autophagy is cytoprotective in a number of transgenic disease models including neurodegenerative disorders. Thus, therapeutic exploitation of autophagy is considered a potential treatment strategy in certain human diseases, and therefore, chemical inducers of autophagy have tremendous biomedical relevance. In this review, we describe the in vitro screening platforms to identify autophagy modulators in mammalian cells using various methodologies including fluorescence and high-content imaging, flow cytometry, fluorescence and luminescence detection by microplate reader, immunoblotting, and immunofluorescence. The commonly used autophagy reporters in these screening platforms are either based on autophagy marker like LC3 or autophagy substrate such as aggregation-prone proteins or p62/SQSTM1. The reporters and assays for monitoring autophagy are evolving over time to become more sensitive in measuring autophagic flux with the capability of high-throughput applications for drug discovery. Here we highlight these developments and also describe the stringent secondary autophagy assays for characterizing the autophagy modulators arising from the primary screen. Since autophagy is implicated in myriad human physiological and pathological conditions, these technologies will enable identifying novel chemical modulators or genetic regulators of autophagy that will be of biomedical and fundamental importance to human health.