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Discretizing Three-Dimensional Oxygen Gradients to Modulate and Investigate Cellular Processes.

ABSTRACT: With the increased realization of the effect of oxygen (O₂ ) deprivation (hypoxia) on cellular processes, recent efforts have focused on the development of engineered systems to control O₂ concentrations and establish biomimetic O₂ gradients to study and manipulate cellular behavior. Nonetheless, O₂ gradients present in 3D engineered platforms result in diverse cell behavior across the O₂ gradient, making it difficult to identify and study O₂ sensitive signaling pathways. Using a layer-by-layer assembled O₂ -controllable hydrogel, the authors precisely control O₂ concentrations and study uniform cell behavior in discretized O₂ gradients, then recapitulate the dynamics of cluster-based vasculogenesis, one mechanism for neovessel formation, and show distinctive gene expression patterns remarkably correlate to O₂ 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.

SUBMITTER: Blatchley MR

PROVIDER: S-EPMC8292886 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Bacterial bioﬁlms can form persistent infections on wounds and implanted medical devices and are associated with many chronic diseases, such as cystic fibrosis. These infections are medically difficult to treat, as biofilms are more resistant to antibiotic attack than their planktonic counterparts. An understanding of the spatial and temporal variation in the metabolism of biofilms is a critical component toward improved biofilm treatments. To this end, we developed oxygen-sensitive luminescent nanosensors to measure three-dimensional (3D) oxygen gradients, an application of which is demonstrated here with Pseudomonas aeruginosa biofilms. The method was applied here and improves on traditional one-dimensional (1D) methods of measuring oxygen profiles by investigating the spatial and temporal variation of oxygen concentration when bioﬁlms are challenged with antibiotic attack. We observed an increased oxygenation of biofilms that was consistent with cell death from comparisons with antibiotic kill curves for PAO1. Due to the spatial and temporal nature of our approach, we also identified spatial and temporal inhomogeneities in the biofilm metabolism that are consistent with previous observations. Clinical strains of P. aeruginosa subjected to similar interrogation showed variations in resistance to colistin and tobramycin, which are two antibiotics commonly used to treat P. aeruginosa infections in cystic fibrosis patients.IMPORTANCE Biofilm infections are more difficult to treat than planktonic infections for a variety of reasons, such as decreased antibiotic penetration. Their complex structure makes biofilms challenging to study without disruption. To address this limitation, we developed and demonstrated oxygen-sensitive luminescent nanosensors that can be incorporated into biofilms for studying oxygen penetration, distribution, and antibiotic efficacy-demonstrated here with our sensors monitoring antibiotic impacts on metabolism in biofilms formed from clinical isolates. The significance of our research is in demonstrating not only a nondisruptive method for imaging and measuring oxygen in biofilms but also that this nanoparticle-based sensing platform can be modified to measure many different ions and small molecule analytes.

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Discretizing Three-Dimensional Oxygen Gradients to Modulate and Investigate Cellular Processes.

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