Human liver organoid derived intra-hepatic bile duct cells support SARS-CoV-2 infection and replication.

Although the main route of infection for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the respiratory tract, liver injury is also commonly seen in many patients, as evidenced by deranged parenchymal liver enzymes. Furthermore, the severity of liver damage has been shown to correlate with higher mortality. Overall, the mechanism behind the liver injury remains unclear. We showed in this study that intra-hepatic bile duct cells could be grown using a human liver organoid platform. The cholangiocytes were not only susceptible to SARS-CoV-2 infection, they also supported efficient viral replication. We also showed that SARS-CoV-2 replication was much higher than SARS-CoV. Our findings suggested direct cytopathic viral damage being a mechanism for SARS-CoV-2 liver injury.

Identification of a wide spectrum of ciliary gene mutations in nonsyndromic biliary atresia patients implicates ciliary dysfunction as a novel disease mechanism.

BACKGROUND: Biliary atresia (BA) is the most common obstructive cholangiopathy in neonates, often progressing to end-stage cirrhosis. BA pathogenesis is believed to be multifactorial, but the genetic contribution, especially for nonsyndromic BA (common form: > 85%) remains poorly defined. METHODS: We conducted whole exome sequencing on 89 nonsyndromic BA trios to identify rare variants contributing to BA etiology. Functional evaluation using patients' liver biopsies, human cell and zebrafish models were performed. Clinical impact on respiratory system was assessed with clinical evaluation, nasal nitric oxide (nNO), high speed video analysis and transmission electron microscopy. FINDINGS: We detected rare, deleterious de novo or biallelic variants in liver-expressed ciliary genes in 31.5% (28/89) of the BA patients. Burden test revealed 2.6-fold (odds ratio (OR) [95% confidence intervals (CI)]= 2.58 [1.15-6.07], adjusted p = 0.034) over-representation of rare, deleterious mutations in liver-expressed ciliary gene set in patients compared to controls. Functional analyses further demonstrated absence of cilia in the BA livers with KIF3B and TTC17 mutations, and knockdown of PCNT, KIF3B and TTC17 in human control fibroblasts and cholangiocytes resulted in reduced number of cilia. Additionally, CRISPR/Cas9-engineered zebrafish knockouts of KIF3B, PCNT and TTC17 displayed reduced biliary flow. Abnormally low level of nNO was detected in 80% (8/10) of BA patients carrying deleterious ciliary mutations, implicating the intrinsic ciliary defects. INTERPRETATION: Our findings support strong genetic susceptibility for nonsyndromic BA. Ciliary gene mutations leading to cholangiocyte cilia malformation and dysfunction could be a key biological mechanism in BA pathogenesis. FUNDING: The study is supported by General Research Fund, HMRF Commissioned Paediatric Research at HKCH and Li Ka Shing Faculty of Medicine Enhanced New Staff Start-up Fund.

Advanced reconfigurable scaffolds fabricated by 4D printing for treating critical-size bone defects of irregular shapes.

While scaffold-based tissue engineering has been widely used to treat bone critical-size defects, challenges such as implantation of scaffolds in defects with irregular shapes and implantation of scaffolds through minimally invasive surgery remain in the tissue engineering field. Customized bioactive bone tissue engineering scaffolds with reconfigurable capability for both easy scaffold implantation and perfect shape fitting in irregularly shaped bone defects are therefore needed. Herein, applying 4D printing, photothermal-responsive shape memory bone tissue engineering scaffolds are constructed by incorporating black phosphorus nanosheets and osteogenic peptide into ß-tricalcium phosphate/poly(lactic acid-co-trimethylene carbonate) (TCP/P(DLLA-TMC)) nanocomposite scaffolds. When near-infrared irradiation is applied to customized scaffolds on-demand, scaffold temperature rapidly increases to 45 °C, enabling scaffold shape reconfiguration for easy scaffold implantation and precise fitting in irregular bone defects. Once the implantation is finished, scaffold temperature rapidly decreases to 37 °C and scaffolds display mechanical properties comparable to those of human cancellous bone. The improved osteogenesis in bone defect sites is then initiated through pulsed peptide release from scaffolds. Compact integration of reconfigurable scaffolds in rat cranial bone defects and improved new bone formation are demonstrated through micro-computed tomography and histochemical analyses. This study shows a facile method to clinically treat bone defects of irregular shapes.

Biohybrid Triboelectric Nanogenerator for Label-Free Pharmacological Fingerprinting in Cardiomyocytes.

The development of new drugs requires high-throughput and cost-effective pharmacological assessment in relevant biological models. Here, we introduce a novel pharmacological screening platform that combines a biohybrid triboelectric nanogenerator (TENG) and informatic analysis for self-powered, noninvasive, and label-free biosensing in cardiac cells. The cyclic mechanical activity of functional cardiomyocytes is dynamically captured by a specially designed biohybrid TENG device and is analyzed by a custom-made machine learning algorithm to reveal distinctive fingerprints in response to different pharmacological treatment. The core of the TENG device is a multilayer mesh substrate with microscale-gapped triboelectric layers, which are induced to generate electrical outputs by the characteristic motion of cardiomyocytes upon pharmaceutical treatment. Later bioinformatic extraction from the recorded TENG signal is sufficient to predict a drug's identity and efficacy, demonstrating the great potential of this platform as a biocompatible, low-cost, and highly sensitive drug screening system.

Vascularized neural constructs for ex-vivo reconstitution of blood-brain barrier function.

Ex-vivo blood-brain barrier (BBB) model is of great value for studying brain function and drug development, but it is still challenging to engineer macroscale three-dimensional (3D) tissue constructs to recapitulate physiological and functional aspects of BBB. Here, we describe a delicate 3D vascularized neural constructs for ex-vivo reconstitution of BBB function. The tissue-engineered tissue construct is based on a multicomponent 3D co-culture of four types of cells, which typically exist in the BBB and were spatially defined and organized to mimic the in vivo BBB structure and function. A porous polycaprolactone/poly (d,l-lactide-co-glycolide) (PCL/PLGA) microfluidic perfusion system works as the vasculature network, which was made by freeze-coating a 3D-printed sacrificial template. Endothelial cells were seeded inside the channels of the network to form 3D interconnected blood vessels; while other types of cells, including pericytes, astrocytes, and neurons, were co-cultured in a collagen matrix wrapping the vasculature network to derive a vascularized neural construct that recapitulates in vivo BBB function with great complexity and delicacy. Using this model, we successfully reconstituted BBB function with parameters that are similar to the in vivo condition, and demonstrated the identification of BBB-penetrating therapeutics by examining the molecular delivery to neuronal cells when relevant biologic molecules were applied to the vasculature circulation system of the neural construct.

Cryogenic 3D printing of heterogeneous scaffolds with gradient mechanical strengths and spatial delivery of osteogenic peptide/TGF-ß1 for osteochondral tissue regeneration.

Due to the increasing aging population and the high probability of sport injury among young people nowadays, it is of great demand to repair/regenerate diseased/defected osteochondral tissue. Given that osteochondral tissue mainly consists of a subchondral layer and a cartilage layer which are structurally heterogeneous and mechanically distinct, developing a biomimetic bi-phasic scaffold with excellent bonding strength to regenerate osteochondral tissue is highly desirable. Three-dimensional (3D) printing is advantageous in producing scaffolds with customized shape, designed structure/composition gradients and hence can be used to produce heterogeneous scaffolds for osteochondral tissue regeneration. In this study, bi-layered osteochondral scaffolds were developed through cryogenic 3D printing, in which osteogenic peptide/ß-tricalcium phosphate/poly(lactic-co-glycolic acid) water-in-oil composite emulsions were printed into hierarchically porous subchondral layer while poly(D,L-lactic acid-co-trimethylene carbonate) water-in-oil emulsions were printed into thermal-responsive cartilage frame on top of the subchondral layer. The cartilage frame was further filled/dispensed with transforming growth factor-ß1 loaded collagen I hydrogel to form the cartilage module. Although the continuously constructed osteochondral scaffolds had distinct microscopic morphologies and varied mechanical properties at the subchondral zone and cartilage zone at 37 °C, respectively, the two layers were closely bonded together, showing excellent shear strength and peeling strength. Rat bone marrow derived mesenchymal stem cells (rBMSCs) exhibited high viability and proliferation at both subchondral- and cartilage layer. Moreover, gradient rBMSC osteogenic/chondrogenic differentiation was obtained in the osteochondral scaffolds. This proof-of-concept study provides a facile way to produce integrated osteochondral scaffolds for concurrently directing rBMSC osteogenic/chondrogenic differentiation at different regions.

Organic electrochemical transistor arrays for real-time mapping of evoked neurotransmitter release in vivo.

Though neurotransmitters are essential elements in neuronal signal transduction, techniques for in vivo analysis are still limited. Here, we describe an organic electrochemical transistor array (OECT-array) technique for monitoring catecholamine neurotransmitters (CA-NTs) in rat brains. The OECT-array is an active sensor with intrinsic amplification capability, allowing real-time and direct readout of transient CA-NT release with a sensitivity of nanomolar range and a temporal resolution of several milliseconds. The device has a working voltage lower than half of that typically used in a prevalent cyclic voltammetry measurement, and operates continuously in vivo for hours without significant signal drift, which is inaccessible for existing methods. With the OECT-array, we demonstrate simultaneous mapping of evoked dopamine release at multiple striatal brain regions in different physiological scenarios, and reveal a complex cross-talk between the mesolimbic and the nigrostriatal pathways, which is heterogeneously affected by the reciprocal innervation between ventral tegmental area and substantia nigra pars compacta.

Cells in the nervous system pass messages using a combination of electrical and chemical signals. When an electrical impulse reaches the end of one cell, it triggers the release of chemicals called neurotransmitters, which pass the message along. Neurotransmitters can be either activating or inhibitory, determining whether the next cell fires its own electrical signal or remains silent. Currently, researchers lack effective methods for measuring neurotransmitters directly. Instead, methods mainly focus on electrical recordings, which can only tell when cells are active. One new approach is to use miniature devices called organic electrochemical transistors. Transistors are common circuit board components that can switch or amplify electrical signals. Organic electrochemical transistors combine these standard components with a semi-conductive material and a flexible membrane. When they interact with certain biological molecules, they release electrons, inducing a voltage. This allows organic electrochemical transistors to detect and measure neurotransmitter release. So far, the technology has been shown to work in tissue isolated from a brain, but no-one has used it to detect neurotransmitters inside a living brain. Xie, Wang et al. now present a new device that can detect the release of the neurotransmitter, dopamine, in real-time in living rats. The device is a miniature microarray of transistors fixed to a blade-shaped film. Xie, Wang et al. implanted this device into the brain of an anaesthetised rat and then stimulated nearby brain cells using an electrode. The device was able to detect the release of the neurotransmitter dopamine, despite there being a range of chemicals released inside the brain. It was sensitive to tiny amounts of the neurotransmitter and could distinguish bursts that were only milliseconds apart. Finally, Xie, Wang et al. also implanted the array across two connected brain areas to show that it was possible to watch different brain regions at the same time. This is the first time that transistor arrays have measured neurotransmitter release in a living brain. The new device works at low voltage, so can track brain cell activity for hours, opening the way for brand new neuroscience experiments. In the future, adaptations could extend the technology even further. More sensors could give higher resolution results, different materials could detect different neurotransmitters, and larger arrays could map larger brain areas.

Flexible and fully implantable upconversion device for wireless optogenetic stimulation of the spinal cord in behaving animals.

Wireless optogenetics based on the upconversion technique has recently provided an effective and interference-free alternative for remote brain stimulation and inhibition in behaving animals, which is of great promise for neuroscience research. However, more versatile upconversion devices are yet to be implemented for neural tissues other than the brain. In this study, a flexible and fully implantable upconversion device was developed for epidural spinal cord stimulation. The upconversion device was fabricated via a straightforward, two-step, heat-pulling process using biocompatible thermoplastic polypropylene as a backbone, which is mixed with upconversion nanoparticles (UCNPs) to form a flexible optrode device that converts near-infrared (NIR) irradiation to visible light for the optogenetic manipulation of spinal cord tissues. In this system, the flexible upconversion device is fully implantable within the rigid spine structure, and shows excellent long-term biocompatibility even after a four-month experiment. In anesthetized mice, the UCNP device implanted at the L4 vertebra can be used to reliably evoke hindlimb muscular activity upon NIR triggering. In behaving mice, neural modulation by the same UCNP devices effectively inhibits the animals' movement as a result of remote spinal cord stimulation. We believe that the flexible upconversion device provides new possibilities for wireless neural modulation in spinal cord tissues, and will become a valuable supplement to the current tool sets of upconversion based wireless optogenetics.

Injectable Nanoreinforced Shape-Memory Hydrogel System for Regenerating Spinal Cord Tissue from Traumatic Injury.

Traumatic injury in the central nervous system can lead to loss of functional neurons. Transplantation of neural progenitors is a promising therapeutic strategy. However, infusion of dissociated cells often suffers from low viability, uneven cell distribution, and poor in vivo engraftment that could be reinforced by a better cell delivery system. Here, we develop an injectable composite hydrogel system for use as a minimally invasive treatment of spinal cord injury (SCI) using motor neurons (MNs) derived from embryonic stem cells (ESCs). The composite hydrogel is based on a modified gelatin matrix integrated with shape-memory polymer fibers. The gelatin matrix creates a local microenvironment for cell assembly and also acts as a lubricant during injection through a fine catheter. Notably, shape-memory fiber scaffolds are able to recover to maintain the microstructures even after dramatic deformation from injection operation, providing the necessary support and guidance for motor neuron differentiation. We find that the composite hydrogel with an aligned fiber scaffold greatly improves the viability of ESCs and their differentiation toward MNs both in vitro and in vivo. When transplanted to SCI animals by injection, the ESC-loaded composite hydrogels are identified to significantly enhance tissue regeneration and motor function recovery in mice. With this proof-of-concept study, we believe that the injectable composite hydrogel system provides a promising solution for in vivo cell delivery with minimum invasiveness and can be readily extended to other stem-cell-based regenerative treatments.