A Micro-scale Humanized Ventilator-on-a-Chip to Examine the Injurious Effects of Mechanical Ventilation.

Patients with compromised respiratory function frequently require mechanical ventilation to survive. Unfortunately, non-uniform ventilation of injured lungs generates complex mechanical forces that lead to ventilator induced lung injury (VILI). Although investigators have developed lung-on-a-chip systems to simulate normal respiration, modeling the complex mechanics of VILI as well as the subsequent recovery phase is a challenge. Here we present a novel humanized in vitro ventilator-on-a-chip (VOC) model of the lung microenvironment that simulates the different types of injurious forces generated in the lung during mechanical ventilation. We used transepithelial/endothelial electrical resistance (TEER) measurements to investigate how individual and simultaneous application of the different mechanical forces alters real-time changes in barrier integrity during and after injury. We find that compressive stress (i.e. barotrauma) does not significantly alter barrier integrity while over-distention (20% cyclic radial strain, volutrauma) results in decreased barrier integrity that quickly recovers upon removal of mechanical stress. Conversely, surface tension forces generated during airway reopening (atelectrauma), result in a rapid loss of barrier integrity with a delayed recovery relative to volutrauma. Simultaneous application of cyclic stretching (volutrauma) and airway reopening (atelectrauma), indicate that the surface tension forces associated with reopening fluid-occluded lung regions is the primary driver of barrier disruption. Thus, our novel VOC system can monitor the effects of different types of injurious forces on barrier disruption and recovery in real-time and can be used to identify the biomechanical mechanisms of VILI.

Substrate stiffness directs diverging vascular fates.

Embryonic stem cells (ESC) are excellent cell culture systems for elucidating developmental signals that may be part of the stem cell niche. Although stem cells are traditionally induced using predominately soluble signals, the mechanical environment of the niche can also play a role in directing cells towards differential cell lineages. Interested in diverging vascular fates, we set out to examine to what extent mechanical signaling played a role in endothelial cell and/or smooth muscle fate. Using chemically-defined staged vascular differentiation methods, vascular progenitor cells (VPC) fate was examined on single stiffness polyacrylamide hydrogels of 10 kPa, 40 kPa and >0.1 GPa. Emergence of vascular cell populations aligned with corresponding hydrogel stiffness: EC-lineages favoring the softer material and SMC lineages favoring the stiffest material. Statistical significance was observed on both cell lines on almost all days. Transcriptome analysis indicated that the populations on the varying stiffness emerge in distinct categories. Lastly, blocking studies show that αvß1, and not αvß6, activation mediates stiffness-directed vascular differentiation. Overall, these studies indicate that softer materials direct VPCs into a more EC-like fate compared to stiffer materials. STATEMENT OF SIGNIFICANCE: Although stem cells are traditionally induced using predominately soluble signals, the mechanical environment of the niche also plays a role in directing cell fate. Several studies have examined the stiffness-induced cell fate from mesenchymal stem cells (MSCs) and undifferentiated embryonic stem cells (ESCs). This is the first study that rigorously examines the role of matrix stiffness in diverging vascular fates from a purified population of vascular progenitor cells (VPCs). We show that the emergence of endothelial cell (EC) versus smooth muscle cell (SMC) populations corresponds with hydrogel stiffness: EC-lineages favoring the softness material and SMC lineages favoring the stiffest material, and that αvß1 activation mediates this stiffness-directed vascular differentiation.

Leaf-inspired microcontact printing vascular patterns.

The vascularization of tissue grafts is critical for maintaining viability of the cells within a transplanted graft. A number of strategies are currently being investigated including very promising microfluidics systems. Here, we explored the potential for generating a vasculature-patterned endothelial cells that could be integrated into distinct layers between sheets of primary cells. Bioinspired from the leaf veins, we generated a reverse mold with a fractal vascular-branching pattern that models the unique spatial arrangement over multiple length scales that precisely mimic branching vasculature. By coating the reverse mold with 50 µg ml-1 of fibronectin and stamping enabled selective adhesion of the human umbilical vein endothelial cells (HUVECs) to the patterned adhesive matrix, we show that a vascular-branching pattern can be transferred by microcontact printing. Moreover, this pattern can be maintained and transferred to a 3D hydrogel matrix and remains stable for up to 4 d. After 4 d, HUVECs can be observed migrating and sprouting into Matrigel. These printed vascular branching patterns, especially after transfer to 3D hydrogels, provide a viable alternative strategy to the prevascularization of complex tissues.