Pseudotyped zoonotic thogotoviruses exhibit broad entry range in mammalian cells.

Viruses in the thogotovirus genus of the family Orthomyxoviridae are much less well-understood than influenza viruses despite documented zoonotic transmission and association with human disease. This study therefore developed a cell-cell fusion assay and three pseudotyping tools and used them to assess envelope function and cell tropism. Envelope glycoproteins of Dhori (DHOV), Thogoto (THOV), Bourbon, and Sinu viruses were all revealed to exhibit pH-dependent triggering of membrane fusion. Lentivirus vectors were robustly pseudotyped with these glycoproteins while influenza virus vectors showed pseudotyping compatibility, albeit at lower efficiencies. Replication-competent vesicular stomatitis virus expressing DHOV or THOV glycoproteins were also successfully generated. These pseudotyped viruses mediated entry into a wide range of mammalian cell lines, including human primary cells. The promiscuousness of these viruses suggests the use of a relatively ubiquitous receptor and their entry into numerous mammalian cells emphasize their high potential as veterinary and zoonotic diseases.

Hydrogel-based 3D human iPSC-derived neuronal culture for the study of rabies virus infection.

Background: Rabies is a highly fatal infectious disease that poses a significant threat to human health in developing countries. In vitro study-based understanding of pathogenesis and tropism of different strains of rabies virus (RABV) in the central nervous system (CNS) is limited due to the lack of suitable culture models that recapitulate the complex communication pathways among host cells, extracellular matrices, and viruses. Therefore, a three-dimensional (3D) cell culture that mimics cell-matrix interactions, resembling in vivo microenvironment, is necessary to discover relevant underlying mechanisms of RABV infection and host responses. Methods: The 3D collagen-Matrigel hydrogel encapsulating hiPSC-derived neurons for RABV infection was developed and characterized based on cell viability, morphology, and gene expression analysis of neuronal markers. The replication kinetics of two different strains of RABV [wild-type Thai (TH) and Challenge Virus Standard (CVS)-11 strains] in both 2D and 3D neuronal cultures were examined. Differential gene expression analysis (DEG) of the neuropathological pathway of RABV-infected 2D and 3D models was also investigated via NanoString analysis. Results: The 3D hiPSC-derived neurons revealed a more physiologically interconnected neuronal network as well as more robust and prolonged maturation and differentiation than the conventional 2D monolayer model. TH and CVS-11 exhibited distinct growth kinetics in 3D neuronal model. Additionally, gene expression analysis of the neuropathological pathway observed during RABV infection demonstrated a vast number of differentially expressed genes (DEGs) in 3D model. Unlike 2D neuronal model, 3D model displayed more pronounced cellular responses upon infection with CVS-11 when compared to the TH-infected group, highlighting the influence of the cell environment on RABV-host interactions. Gene ontology (GO) enrichment of DEGs in the infected 3D neuronal culture showed alterations of genes associated with the inflammatory response, apoptotic signaling pathway, glutamatergic synapse, and trans-synaptic signaling which did not significantly change in 2D culture. Conclusion: We demonstrated the use of a hydrogel-based 3D hiPSC-derived neuronal model, a highly promising technology, to study RABV infection in a more physiological environment, which will broaden our understanding of RABV-host interactions in the CNS.

Dual-Functional Drug Delivery System for Bisphosphonate-Related Osteonecrosis Prevention and Its Bioinspired Releasing Model and In Vitro Assessment.

Clindamycin (CDM)/geranylgeraniol (GGOH)-loaded plasma-treated mesoporous silica nanoparticles/carboxymethyl chitosan composite hydrogels (CHG60 and CHG120) were developed for the prevention of medication-related osteonecrosis of the jaw associated with bisphosphonates (MRONJ-B). The pore structure and performances of CHGs, e.g., drug release profiles and kinetics, antibacterial activity, zoledronic acid (ZA)-induced cytotoxicity reversal activity, and acute cytotoxicity, were evaluated. The bioinspired platform mimicking in vivo fibrin matrices was also proposed for the in vitro/in vivo correlation. CHG120 was further encapsulated in the human-derived fibrin, generating FCHG120. The SEM and µCT images revealed the interconnected porous structures of CHG120 in both pure and fibrin-surrounding hydrogels with %porosity of 75 and 36%, respectively, indicating the presence of fibrin inside the hydrogel pores, besides its peripheral region, which was evidenced by confocal microscopy. The co-presence of GGOH moderately decelerated the overall releases of CDM from CHGs in the studied releasing fluids, i.e., phosphate buffer saline-based fluid (PBB) and simulated interstitial fluid (SIF). The whole-lifetime release patterns of CDM, fitted by the Ritger-Peppas equation, appeared nondifferentiable, divided into two releasing stages, i.e., rapid and steady releasing stages, whereas the biphasic drug release patterns of GGOH were observed with Phase I and II releases fitted by the Higuchi and Ritger-Peppas equations, respectively. Notably, the burst releases of both drugs were subsided with lengthier durations (up to 10-12 days) in SIF, compared with those in PBB, enabling CHGs to elicit satisfactory antibacterial and ZA cytotoxicity reversal activities for MRONJ-B prevention. The fibrin network in FCHG120 further reduced and sustained the drug releases for at least 14 days, lengthening bactericidal and ZA cytotoxicity reversal activities of FCHG and decreasing in vitro and in ovo acute drug toxicity. This highlighted the significance of fibrin matrices as appropriate in vivo-like platforms to evaluate the performance of an implant.

Engineered aligned endothelial cell structures in tethered collagen hydrogels promote peripheral nerve regeneration.

Vascularisation is important in nerve tissue engineering to provide blood supply and nutrients for long-term survival of implanted cells. Furthermore, blood vessels in regenerating nerves have been shown to serve as tracks for Schwann cells to migrate along and thus form Bands of Büngner which promote axonal regeneration. In this study, we have developed tissue-engineered constructs containing aligned endothelial cells, or co-cultures of both endothelial cells and Schwann cells to test whether these structures could promote regeneration across peripheral nerve gaps. Type I rat tail collagen gels containing HUVECs (Human Umbilical Vein Endothelial Cells, 4 × 106 cells/ml) were cast in perforated tethering silicone conduits to facilitate cellular self-alignment and tube formation for 4 days of culture. For co-culture constructs, optimal tube formation and cellular alignment was achieved with a ratio of 4:0.5 × 106 cells/ml (HUVECs:Schwann cells). An in vivo test of the engineered constructs to bridge a 10 mm gap in rat sciatic nerves for 4 weeks revealed that constructs containing only HUVECs significantly promoted axonal regeneration and vascularisation across the gap, as compared to conventional aligned Schwann cell constructs and those containing co-cultured HUVECs and Schwann cells. Our results suggest that tissue-engineered constructs containing aligned endothelial cells within collagen matrix could be good candidates to treat peripheral nerve injury. STATEMENT OF SIGNIFICANCE: Nerve tissue engineering provides a potential way to overcome the limitations associated with current clinical grafting techniques for the repair of severe peripheral nerve injuries. However, the therapeutic cells within engineered nerve tissue require effective vascularisation in order to survive. This work therefore aimed to develop engineered nerve constructs containing aligned tube-like structures made from endothelial cells. Not only did this provide a method to improve vascularisation, it demonstrated for the first time that aligned endothelial cells can outperform Schwann cells in promoting nerve regeneration in the rat sciatic nerve model. This has introduced the concept of developing pre-vascularised engineered nerve tissues, and indicated the potential usefulness of endothelial cell structures in tissue engineering for peripheral nerve repair.

Rapidly formed stable and aligned dense collagen gels seeded with Schwann cells support peripheral nerve regeneration.

OBJECTIVE: Gel aspiration-ejection (GAE) has recently been developed for the rapid production of dense, anisotropic collagen gel scaffolds with adjustable collagen fibrillar densities. In this study, a GAE system was applied to produce aligned Schwann cells within a type-1 collagen matrix to generate GAE-engineered neural tissues (GAE-EngNT) for potential nerve tissue engineering applications. APPROACH: The stability and mechanical properties of the constructs were investigated along with the viability, morphology and distribution of Schwann cells. Having established the methodology to construct stable robust Schwann cell-loaded engineered neural tissues using GAE (GAE-EngNTs), the potential of these constructs in supporting and guiding neuronal regeneration, was assessed both in vitro and in vivo. MAIN RESULTS: Dynamic mechanical analysis strain and frequency sweeps revealed that the GAE-EngNT produced via cannula gauge number 16 G (∼1.2 mm diameter) exhibited similar linear viscoelastic behaviors to rat sciatic nerves. The viability and alignment of seeded Schwann cells in GAE-EngNT were maintained over time post GAE, supporting and guiding neuronal growth in vitro with an optimal cell density of 2.0 × 106 cells ml-1. An in vivo test of the GAE-EngNTs implanted within silicone conduits to bridge a 10 mm gap in rat sciatic nerves for 4 weeks revealed that the constructs significantly promoted axonal regeneration and vascularization across the gap, as compared with the empty conduits although less effective regeneration compared with the autograft groups. SIGNIFICANCE: Therefore, this is a promising approach for generating anisotropic and robust engineered tissue which can be used with Schwann cells for peripheral nerve repair.

Vascularization Strategies for Peripheral Nerve Tissue Engineering.

Vascularization plays a significant role in treating nerve injury, especially to avoid the central necrosis observed in nerve grafts for large and long nerve defects. It is known that sufficient vascularization can sustain cell survival and maintain cell integration within tissue-engineered constructs. Several studies have also shown that vascularization affects nerve regeneration. Motivated by these studies, vascularized nerve grafts have been developed using various different techniques, although donor site morbidity and limited nerve supply remain significant drawbacks. Tissue engineering provides an exciting alternative approach to prefabricate vascularized nerve constructs which could overcome the limitations of grafts. In this review article, we focus on the role of vascularization in nerve regeneration, discussing various approaches to generate vascularized nerve constructs and the contribution of tissue engineering and mathematical modeling to aid in developing vascularized engineered nerve constructs, illustrating these aspects with examples from our research experience. Anat Rec, 301:1657-1667, 2018. © 2018 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.