Transient hypoxia improves matrix properties in tissue engineered cartilage

Abstract

Adult articular cartilage is a hypoxic tissue, with oxygen tension ranging from <10% at the cartilage surface to <1% in the deepest layers. In addition to spatial gradients, cartilage development is also associated with temporal changes in oxygen tension. However, a vast majority of cartilage tissue engineering protocols involves cultivation of chondrocytes or their progenitors under ambient oxygen concentration (21% O2), that is, significantly above physiological levels in either developing or adult cartilage. Our study was designed to test the hypothesis that transient hypoxia followed by normoxic conditions results in improved quality of engineered cartilaginous ECM. To this end, we systematically compared the effects of normoxia (21% O2 for 28 days), hypoxia (5% O2 for 28 days) and transient hypoxia—reoxygenation (5% O2 for 7 days and 21% O2 for 21 days) on the matrix composition and expression of the chondrogenic genes in cartilage constructs engineered in vitro. We demonstrated that reoxygenation had the most effect on the expression of cartilaginous genes including COL2A1, ACAN, and SOX9 and increased tissue concentrations of amounts of glycosaminoglycans and type II collagen. The equilibrium Young’s moduli of tissues grown under transient hypoxia (510.01 ± 28.15 kPa) and under normoxic conditions (417.60 ± 68.46 kPa) were significantly higher than those measured under hypoxic conditions (279.61 ± 20.52 kPa). These data suggest that the cultivation protocols utilizing transient hypoxia with reoxygenation have high potential for efficient cartilage tissue engineering, but need further optimization in order to achieve higher mechanical functionality of engineered constructs.

Functional consequences of glucose and oxygen deprivation on engineered mesenchymal stem cell-based cartilage constructs

“…On its own, hypoxic culture (in the presence of high glucose) resulted in constructs with slightly lower GAG content and mechanical properties compared to normoxia, consistent with similar studies conducted with chondrocytes [38]. Glucose consumption was greatest in TGF-β3 conditions, likely due to their higher anabolic state under this chondrogenically differentiated condition.…”

Synergistic Effects of Hypoxia and Morphogenetic Factors on Early Chondrogenic Commitment of Human Embryonic Stem Cells in Embryoid Body Culture

“…2). A possible explanation is that at early stages of development, it is beneficial to have a longer period of hypoxia to promote the expression of cartilaginous genes, while at later stages the transfer to normoxic, energy-rich conditions enhances the production and functional assembly of cartilaginous matrix [23]. …”

“…For hypoxic culture (Group 1), EBs were placed inside a Billups-Rothenber modular chamber supplied with low-oxygen gas (5 % O 2 + 5 % CO 2 + 95 % N 2 ). Humidity in the chamber was maintained by a Petri dish with 20 ml distilled water [23]. …”

Nutrient channels and stirring enhanced the composition and stiffness of large cartilage constructs

“…A number of culture techniques have been shown to possess temporal effects on the properties of engineered cartilage, such as transient TGF-β supplementation (Byers et al, 2008), delayed dynamic compressive loading (Lima et al, 2007), transient hypoxia (Yodmuang et al, 2013), and transient perfusion (Davisson et al, 2002). In light of these findings, the effects of transient or delayed media stirring in this culture system could prove to be a worthwhile investigation.…”