Intracellular Calcium and Sodium Modulation of Self-Assembled Neocartilage Using Costal Chondrocytes

Ion signaling through Ca²⁺ and Na⁺ plays a key role in mechanotransduction and encourages a chondrogenic phenotype and tissue maturation. In this study, we propose that the pleiotropic effects of Ca²⁺ and Na⁺ modulation can be used to induce maturation and improvement of neocartilage derived from redifferentiated expanded chondrocytes from minipig rib cartilage. Three ion modulators were employed: (1) 4α-phorbol-12,13-didecanoate (4-αPDD), an agonist of the Ca²⁺-permeable transient receptor potential vanilloid 4 (TRPV4), (2) ouabain, an inhibitor of the Na⁺/K⁺ pump, and (3) ionomycin, a Ca²⁺ ionophore. These ion modulators were used individually or in combination. While no beneficial effects were observed when using combinations of the ion modulators, single treatment of constructs with the three ion modulators resulted in multiple effects in structure-function relationships. The most significant findings were related to ionomycin. Treatment of neocartilage with ionomycin produced 61% and 115% increases in glycosaminoglycan and pyridinoline crosslink content, respectively, compared with the control. Moreover, treatment with this Ca²⁺ ionophore resulted in a 45% increase of the aggregate modulus, and a 63% increase in the tensile Young's modulus, resulting in aggregate and Young's moduli of 567 kPa and 8.43 MPa, respectively. These results support the use of ion modulation to develop biomimetic neocartilage using expanded redifferentiated costal chondrocytes. Impact Statement New cost-effective, replicable, and highly controllable strategies are required to develop neocartilage with biomimetic properties akin to native tissue. Ion signaling plays a key role in mechanotransduction, promoting chondrogenic phenotype. Using rib cartilage, we proposed that Ca²⁺ and Na⁺ modulation could be used to induce maturation of neotissue derived from redifferentiated, expanded costal chondrocytes, improving its mechanical properties. Our results indicate that Ca²⁺ modulation with ionomycin, which stimulated extracellular matrix deposition and collagen crosslinking, improved morphological and mechanical features of neocartilage constructs, and holds potential as a powerful tool to engineer hyaline-like tissues.