Hypoxia in Static and Dynamic 3D Culture Systems for Tissue Engineering of Bone

Abstract

Tissue engineering of sizeable cell-scaffold constructs is limited by gradients in tissue quality from the periphery toward the center. Because homogenous delivery of oxygen to three-dimensional (3D) cell cultures remains an unsolved challenge, we hypothesized that uneven oxygen supply may impede uniform cellular growth on scaffolds. In this study we challenged static and dynamic 3D culture systems designed for bone tissue engineering applications with a well-growing subclone of MC3T3-E1 preosteoblasts and continuously measured the oxygen concentrations in the center of cell-seeded scaffolds and in the surrounding medium. After as little as 5 days in static culture, central oxygen concentrations dropped to 0%. Subsequently, cells died in central regions of the scaffold but not in its periphery, where oxygen levels were approximately 4%. The use of perfusion bioreactors successfully prevented cell death, yet central oxygen concentrations did not rise above 4%. We conclude that 3D culture in vitro is associated with relevant oxygen gradients, which can be the cause of inhomogeneous tissue quality. Perfusion bioreactors prevent cell death but they do not entirely eliminate 3D culture-associated oxygen gradients. Therefore, we advise continuous oxygen monitoring of 3D culture systems to ensure tissue quality throughout engineered constructs.

Involvement of Angiopoietin-like 4 in Matrix Remodeling during Chondrogenic Differentiation of Mesenchymal Stem Cells

“…Induction of ANGPTL4 Expression in MSCs Requires Hypoxia or Micromass Culture-Hypoxia has been shown to occur inside three-dimensional culture systems (21) and is a well known inducer of ANGPTL4 expression in various cell types (22,23). Therefore, we examined the contribution of hypoxia, threedimensional culture, and TGF-␤-3 in the induction of ANGPTL4 expression in MSCs.…”

Numerical simulation of fluid field and in vitro three-dimensional fabrication of tissue-engineered bones in a rotating bioreactor and in vivo implantation for repairing segmental bone defects

“…Currently, static culture systems that are most commonly used for in vitro amplification of seeded cells in tissueengineered bones and in the construction of engineered tissues have many shortcoming and disadvantages (Collins et al 1998;Volkmer et al 2008;da Costa Goncalves et al 2012;Zhu et al 2010;Wang et al 2005). Hydrodynamic advantages that are observed in bioreactor cultures of tissueengineered bones have led to a significant assistance to this field (Rauh et al 2011;Yeatts & Fisher 2011;Salter et al 2012;Yu et al 2004;Liu et al 2012).…”

Tracking Hypoxic Signaling in Encapsulated Stem Cells

“…Studies in 3D culture revealed that the oxygen concentration decreases from the periphery toward the center of the scaffolds, which correlates with cell density and cell viability. 43,44 The oxygen concentrations in the media and inside the scaffolds have been measured using oxygensensing dyes, oxygen microelectrodes, fluorescent probes, and microparticles. [45][46][47][48][49][50] Mathematical modeling approaches have been developed accounting for scaffold geometry and local oxygen tension to predict oxygen gradients inside the scaffolds.…”

“…The material(s) selected for the scaffold and how it is synthesized influence the diffusion of oxygen and nutrients to the cells and this is a major limitation in tissue engineering. 43 Gradients of oxygen concentration can form within materials and this will affect cellular differentiation or cell fate decisions. [57][58][59] This marker system was developed to identify hypoxic signaling within these environments and to track this response at a cellular level without the need to disrupt the integrity of the scaffold or cells.…”