Discretizing Three‐Dimensional Oxygen Gradients to Modulate and Investigate Cellular Processes

Abstract

With the increased realization of the effect of oxygen (O 2 ) deprivation (hypoxia) on cellular processes, recent efforts have focused on the development of engineered systems to control O 2 concentrations and establish biomimetic O 2 gradients to study and manipulate cellular behavior. Nonetheless, O 2 gradients present in 3D engineered platforms result in diverse cell behavior across the O 2 gradient, making it difficult to identify and study O 2 sensitive signaling pathways. Using a layer-by-layer assembled O 2 -controllable hydrogel, the authors precisely control O 2 concentrations and study uniform cell behavior in discretized O 2 gradients, then recapitulate the dynamics of cluster-based vasculogenesis, one mechanism for neovessel formation, and show distinctive gene expression patterns remarkably correlate to O 2 concentrations. Using RNA sequencing, it is found that time-dependent regulation of cyclic adenosine monophosphate signaling enables cell survival and clustering in the high stress microenvironments. Various extracellular matrix modulators orchestrate hypoxia-driven endothelial cell clustering. Finally, clustering is facilitated by regulators of cell-cell interactions, mainly vascular cell adhesion molecule 1. Taken together, novel regulators of hypoxic cluster-based vasculogenesis are identified, and evidence for the utility of a unique platform is provided to study dynamic cellular responses to 3D hypoxic environments, with broad applicability in development, regeneration, and disease.

Tissue Engineering Approaches to Uncover Therapeutic Targets for Endothelial Dysfunction in Pathological Microenvironments

“…Further, adding an external crosslinking agent enhances branching and sprouting from the ECFC clusters, thus highlighting the importance of matrix stiffness in vascular network formation [74]. A subsequent study [75] found that generating distinct Gel-HI layers allows for mechanistic studies. RNA sequencing analysis revealed the time-dependent regulation of numerous biological functions that govern the hypoxic-cluster formation, including cell survival, apoptosis, cell cycle progression, and carbohydrate metabolism.…”

“…The downregulation of cyclic adenosine monophosphate signaling allowed for cell survival in a high-stress microenvironment generated by the rapid upregulation of oxidative stress and ROS (Figure 1A). Further cluster stabilization was facilitated through the upregulation of vascular cell adhesion molecule 1 (VCAM-1) and the carbohydrate metabolism in hypoxic conditions [75]. Therefore, the targeted delivery of Gel-HI with discretized oxygen gradients holds promise as the therapeutic system for eliciting angiogenic responses in ischemic diseases.…”

Hydrogels to Recapture Extracellular Matrix Cues That Regulate Vascularization

“…Pyrogallol-mediated oxygen scavenger reaction in a microfluidic chamber led to oxygen gradient-driven directional 3D endothelial network formation in fibrinogen/hyaluronic acid hydrogels. 84 Additionally, enzymatic crosslinked hydrogel platforms presenting hypoxic gradient 85 or a distinct layered assembly of predictable O 2 tension (Figure 3B) 86 have further unveiled the molecular mechanisms mediated by HIFs-independent reactive oxygen species production for cluster-based vasculogenesis.…”

“…Adapted from Freeman et al 67 B , Modulation of cluster formation of endothelial colony forming cells using hypoxic hydrogel constructs. Adapted from Blatchley et al 86 C , A microfluidic hydrogel platform that maintains interstitial flow to promote in vitro human umbilical vein endothelial cell (HUVEC) microvascular networks. Millimeter length of perfusable vessels in a fibrin gel.…”

Improved Osteogenesis by Mineralization Combined With Double-Crosslinked Hydrogel Coating for Proliferation and Differentiation of Mesenchymal Stem Cells

“…In consideration of a layer-by-layer (LBL) assembly technique as a successful measurement, it has been explored as an appealing strategy in establishing the multi-functional coating for osteogenesis ( Blatchley et al, 2021 ; Piluso et al, 2021 ). It could not only regulate local and extended drug release on the surface of PLLA acting as seal barrier but it could also orient the osteogenic differentiation of bone mesenchymal stem cells (BMSCs) and bone formation ( Lin, Hsieh, Tseng, & Hsu, 2016 ; Tsai et al, 2015 ).…”