Ion modulatory treatments toward functional self-assembled neocartilage

Signals that recapitulate in vitro the conditions found in vivo, such as hypoxia or mechanical forces, contribute to the generation of tissue-engineered hyaline-like tissues. The cell regulatory processes behind hypoxic and mechanical stimuli rely on ion concentration; iron is required to degrade the hypoxia inducible factor 1a (HIF1α) under normoxia, whereas the initiation of mechanotransduction requires the cytoplasmic increase of calcium concentration. In this work, we propose that ion modulation can be used to improve the biomechanical properties of self-assembled neocartilage constructs derived from rejuvenated expanded minipig rib chondrocytes. The objectives of this work were 1) to determine the effects of iron sequestration on self-assembled neocartilage constructs using two doses of the iron chelator deferoxamine (DFO), and 2) to evaluate the performance of the combined treatment of DFO and ionomycin, a calcium ionophore that triggers cytoplasmic calcium accumulation. This study employed a two-phase approach. In Phase I, constructs treated with a high dose of DFO (100 µM) exhibited an 87% increase in pyridinoline crosslinks, a 57% increase in the Young's modulus, and a 112% increase in the ultimate tensile strength (UTS) of the neotissue. In Phase II, the combined use of both ion modulators resulted in 150% and 176% significant increases in the Young's modulus and UTS of neocartilage constructs, respectively; for the first time, neocartilage constructs achieved a Young's modulus of 11.76±3.29 MPa and UTS of 4.20±1.24 MPa. The results of this work provide evidence that ion modulation can be employed to improve the biomechanical properties in engineered neotissues. STATEMENT OF SIGNIFICANCE: The translation of tissue-engineered products requires the development of strategies capable of producing biomimetic neotissues in a replicable, controllable, and cost-effective manner. Among other functions, Fe²⁺ and Ca²⁺ are involved in the control of the hypoxic response and mechanotransduction, respectively. Both stimuli, hypoxia and mechanical forces, are known to favor chondrogenesis. This study utilized ion modulators to improve the mechanical properties self-assembled neocartilage constructs derived from expanded and rejuvenated costal chondrocytes via Fe²⁺ sequestration and Ca²⁺ influx, alone or in combination. The results indicate that ion modulation induced tissue maturation and a significant improvement of the mechanical properties, and holds potential as a tool to mitigate the need for bioreactors and engineer hyaline-like tissues.