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Unforeseen decreases in dissolved oxygen levels affect tube formation kinetics in collagen gels.

ABSTRACT: The availability of oxygen (O(2)) is a critical parameter affecting vascular tube formation. In this study, we hypothesize that dissolved oxygen (DO) levels in collagen gels change during the three-dimensional (3D) culture of human umbilical vein endothelial cells (HUVECs) in atmospheric conditions and that such changes affect the kinetics of tube formation through the production of reactive oxygen species (ROS). We demonstrate a decrease in O(2) tension during 3D cultures of HUVECs. Noninvasive measurements of DO levels during culture under atmospheric conditions revealed a profound decrease that reached as low as 2% O(2) at the end of 24 h. After media replacement, DO levels rose rapidly and equilibrated at ?15% O(2), creating a reoxygenated environment. To accurately estimate DO gradients in 3D collagen gels, we developed a 3D mathematical model and determined the Michaelis-Menten parameters, V(max) and K(m), of HUVECs in collagen gels. We detected an increase in ROS levels throughout the culture period. Using diphenyliodonium to inhibit ROS production resulted in the complete inhibition of tube formation. Interference RNA studies further showed that hypoxia-inducible factors (HIFs)-1? and -2? are not involved in the formation of 3D tubes in collagen gels. We conclude that ROS affect the tubulogenesis process through HIF?-independent pathways, where the levels of ROS are influenced by the uncontrolled variations in DO levels. This study is the first demonstration of the critical and unexpected role of O(2) during 3D in vitro culture models of tubulogenesis in atmospheric conditions.

SUBMITTER: Abaci HE

PROVIDER: S-EPMC3154554 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Unforeseen decreases in dissolved oxygen levels affect tube formation kinetics in collagen gels.

Abaci Hasan Erbil HE Truitt Rachel R Tan Scott S Gerecht Sharon S

American journal of physiology. Cell physiology 20110504 2

The availability of oxygen (O(2)) is a critical parameter affecting vascular tube formation. In this study, we hypothesize that dissolved oxygen (DO) levels in collagen gels change during the three-dimensional (3D) culture of human umbilical vein endothelial cells (HUVECs) in atmospheric conditions and that such changes affect the kinetics of tube formation through the production of reactive oxygen species (ROS). We demonstrate a decrease in O(2) tension during 3D cultures of HUVECs. Noninvasive ...[more]

PMID: 21543738

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Project description:Biofilms on wound surfaces are treated topically with hyperosmotic agents, such as medical-grade honey and cadexomer iodine; in some cases, these treatments are combined with antibiotics. Tissue repair requires oxygen, and a low pH is conducive to oxygen release from red blood cells and epithelialization. We investigated the variation of dissolved oxygen concentration and pH with biofilm depth and the variation in oxygen consumption rates when biofilms are challenged with medical-grade honey or cadexomer iodine combined with vancomycin or ciprofloxacin. Dissolved oxygen and pH depth profiles in Staphylococcus aureus biofilms were measured using microelectrodes. The presence of cadexomer iodine with vancomycin or ciprofloxacin on the surface of the biofilm permitted a measurable concentration of oxygen at greater biofilm depths (101.6 ± 27.3 μm, P = 0.02; and 155.5 ± 27.9 μm, P = 0.016, respectively) than in untreated controls (30.1 μm). Decreases in pH of ∼0.6 and ∼0.4 units were observed in biofilms challenged with medical-grade honey alone and combined with ciprofloxacin, respectively (P < 0.001 and 0.01, respectively); the number of bacteria recovered from biofilms was significantly reduced (1.26 log) by treatment with cadexomer iodine and ciprofloxacin (P = 0.002) compared to the untreated control. Combining cadexomer iodine and ciprofloxacin improved dissolved oxygen concentration and penetration depth into the biofilm, while medical-grade honey was associated with a lower pH; not all treatments established a bactericidal effect in the time frame used in the experiments.IMPORTANCE Reports about using hyperosmotic agents and antibiotics against wound biofilms focus mostly on killing bacteria, but the results of these treatments should additionally be considered in the context of how they affect physiologically important parameters, such as oxygen concentration and pH. We confirmed that the combination of a hyperosmotic agent and an antibiotic results in greater dissolved oxygen and reduced pH within an S. aureus biofilm.

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Unforeseen decreases in dissolved oxygen levels affect tube formation kinetics in collagen gels.

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Unforeseen decreases in dissolved oxygen levels affect tube formation kinetics in collagen gels.

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