Collagen Tubular Airway-on-Chip for Extended Epithelial Culture and Investigation of Ventilation Dynamics.

The lower respiratory tract is a hierarchical network of compliant tubular structures that are made from extracellular matrix proteins with a wall lined by an epithelium. While microfluidic airway-on-a-chip models incorporate the effects of shear and stretch on the epithelium, week-long air-liquid-interface culture at physiological shear stresses, the circular cross-section, and compliance of native airway walls have yet to be recapitulated. To overcome these limitations, a collagen tube-based airway model is presented. The lumen is lined with a confluent epithelium during two-week continuous perfusion with warm, humid air while presenting culture medium from the outside and compensating for evaporation. The model recapitulates human small airways in extracellular matrix composition and mechanical microenvironment, allowing for the first time dynamic studies of elastocapillary phenomena associated with regular breathing and mechanical ventilation, as well as their impacts on the epithelium. A case study reveales increasing damage to the epithelium during repetitive collapse and reopening cycles as opposed to overdistension, suggesting expiratory flow resistance to reduce atelectasis. The model is expected to promote systematic comparisons between different clinically used ventilation strategies and, more broadly, to enhance human organ-on-a-chip platforms for a variety of tubular tissues.

A human model of arteriovenous malformation (AVM)-on-a-chip reproduces key disease hallmarks and enables drug testing in perfused human vessel networks.

Brain arteriovenous malformations (AVMs) are a disorder wherein abnormal, enlarged blood vessels connect arteries directly to veins, without an intervening capillary bed. AVMs are one of the leading causes of hemorrhagic stroke in children and young adults. Most human sporadic brain AVMs are associated with genetic activating mutations in the KRAS gene. Our goal was to develop an in vitro model that would allow for simultaneous morphological and functional phenotypic data capture in real time during AVM disease progression. By generating human endothelial cells harboring a clinically relevant mutation found in most human patients (activating mutations within the small GTPase KRAS) and seeding them in a dynamic microfluidic cell culture system that enables vessel formation and perfusion, we demonstrate that vessels formed by KRAS4AG12V mutant endothelial cells (ECs) were significantly wider and more leaky than vascular beds formed by wild-type ECs, recapitulating key structural and functional hallmarks of human AVM pathogenesis. Immunofluorescence staining revealed a breakdown of adherens junctions in mutant KRAS vessels, leading to increased vascular permeability, a hallmark of hemorrhagic stroke. Finally, pharmacological blockade of MEK kinase activity, but not PI3K inhibition, improved endothelial barrier function (decreased permeability) without affecting vessel diameter. Collectively, our studies describe the creation of human KRAS-dependent AVM-like vessels in vitro in a self-assembling microvessel platform that is amenable to phenotypic observation and drug delivery.

Engineered human cardiac microtissues: The state-of-the-(he)art.

Due to the integration of recent advances in stem cell biology, materials science, and engineering, the field of cardiac tissue engineering has been rapidly progressing toward developing more accurate functional 3D cardiac microtissues from human cell sources. These engineered tissues enable screening of cardiotoxic drugs, disease modeling (eg, by using cells from specific genetic backgrounds or modifying environmental conditions) and can serve as novel drug development platforms. This concise review presents the most recent advances and improvements in cardiac tissue formation, including cardiomyocyte maturation and disease modeling.

Endothelium-mediated contributions to fibrosis.

Fibrosis, characterized by abnormal and excessive deposition of extracellular matrix, results in compromised tissue and organ structure. This can lead to reduced organ function and eventual failure. Although activated fibroblasts, called myofibroblasts, are considered the central players in fibrosis, the contribution of endothelial cells to the inception and progression of fibrosis has become increasingly recognized. Endothelial cells can contribute to fibrosis by acting as a source of myofibroblasts via endothelial-mesenchymal transition (EndoMT), or by becoming senescent, by secretion of profibrotic mediators and pro-inflammatory cytokines, chemokines and exosomes, promoting the recruitment of immune cells, and by participating in vascular rarefaction and decreased angiogenesis. In this review, we provide an overview of the different aspects of fibrosis in which endothelial cells have been implicated.

De-epithelialization of porcine tracheal allografts as an approach for tracheal tissue engineering.

Replacement of large tracheal defects remains an unmet clinical need. While recellularization of acellular tracheal grafts appeared to be a viable pathway, evidence from the clinic suggests otherwise. In hindsight, complete removal of chondrocytes and repopulation of the tracheal chondroid matrix to achieve functional tracheal cartilage may have been unrealistic. In contrast, the concept of a hybrid graft whereby the epithelium is removed and the immune-privileged cartilage is preserved is a radically different path with initial reports indicating potential clinical success. Here, we present a novel approach using a double-chamber bioreactor to de-epithelialize tracheal grafts and subsequently repopulate the grafts with exogenous cells. A 3 h treatment with sodium dodecyl sulfate perfused through the inner chamber efficiently removes the majority of the tracheal epithelium while the outer chamber, perfused with growth media, keeps most (68.6 ± 7.3%) of the chondrocyte population viable. De-epithelialized grafts support human bronchial epithelial cell (BEAS-2B) attachment, viability and growth over 7 days. While not without limitations, our approach suggests value in the ultimate use of a chimeric allograft with intact donor cartilage re-epithelialized with recipient-derived epithelium. By adopting a brief and partial decellularization approach, specifically removing the epithelium, we avoid the need for cartilage regeneration.

Optimal biomaterials for tracheal epithelial grafts: An in vitro systematic comparative analysis.

Tracheal injury, stenosis, and malignancy demand tracheal reconstruction, which often fails due to the lack of a functioning epithelium. We performed an extensive comparative analysis to determine optimal biomaterials for developing tracheal epithelial grafts with mucociliary function. We screened Hyaluronan-Poly(Ethylene Glycol), Chitosan-Collagen, Collagen Vitrigel Membrane, Fibrin Glue, Silk Fibroin, and Gelatin based on various parameters including mechanical strength, bulk degradation, cell attachment, spreading, metabolic activity, focal adhesion formation, and differentiation into ciliated and goblet cells. Silk Fibroin had significantly higher tensile strength (21.23 ±â€¯4.42 MPa), retained 50% of its mass across 5 weeks, allowed 80-100% cell spreading and increasing metabolic activity across 10 days, focal adhesion formation within 2 h, and differentiation into 5.9 ±â€¯2.6% goblet cells. Silk Fibroin, however, led to poor ciliation, producing 5.5 ±â€¯3.9% ciliated cells, whereas Collagen Vitrigel Membrane promoted excellent ciliation. To capitalize on the mechanical and differentiation benefits of its respective components, we developed a composite biomaterial of Silk Fibroin and Collagen Vitrigel Membrane (SF-CVM), which demonstrated enhanced maturation into 20.6 ±â€¯1.7% ciliated and 5.6 ±â€¯1.0% goblet cells. Development of biomaterials-based airway epithelial grafts that provide desirable mechanics and differentiation is a major step towards treatment of airway disease. STATEMENT OF SIGNIFICANCE: Tracheal blockage, injury, and malignancy greater than 50% of the adult tracheal length cannot be safely resected. Tracheal replacement is one approach, but a major cause of transplant failure is the lack of a functioning epithelium. While tissue engineering for tracheal regeneration using biomaterials is promising, there is currently no gold standard. Therefore, we performed a systematic comparative study to characterize relevant materials for generating a biomaterials-based airway epithelial graft. We developed a composite biomaterial intended for surgical implantation providing tensile strength, slow biodegradation, and optimal support for differentiation of mature epithelia. This is a significant step augmenting current state-of-the-art methods for airway surgeries, laryngeal reconstruction, and tracheal tissue engineering.