OBJECTIVE: Significant collagen content and tensile properties are difficult to achieve in tissue-engineered articular cartilage. The aim of this study was to investigate whether treating developing tissue-engineered cartilage constructs with modulators of intracellular Na(+) or Ca(2+) could increase collagen concentration and construct tensile properties. METHODS: Inhibitors of Na(+) ion transporters and stimulators of intracellular Ca(2+) were investigated for their ability to affect articular cartilage development in a scaffoldless, 3-dimensional chondrocyte culture. Using a systematic approach, we applied ouabain (Na(+)/K(+)-ATPase inhibitor), bumetanide (Na(+)/K(+)/2Cl(-) tritransporter inhibitor), histamine (cAMP activator), and ionomycin (a Ca(2+) ionophore) to tissue-engineered constructs for 1 hour daily on days 10-14 of culture and examined the constructs at 2 weeks or 4 weeks. The gross morphology, biochemical content, and compressive and tensile mechanical properties of the constructs were assayed. RESULTS: The results of these experiments showed that 20 microM ouabain, 0.3 microM ionomycin, or their combination increased the tensile modulus by 40-95% compared with untreated controls and resulted in an increased amount of collagen normalized to construct wet weight. In constructs exposed to ouabain, the increased percentage of collagen per construct wet weight was secondary to decreased glycosaminoglycan production on a per-cell basis. Treatment with 20 microM ouabain also increased the ultimate tensile strength of neo-tissue by 56-86% at 4 weeks. Other construct properties, such as construct growth and type I collagen production, were affected differently by Na(+) modulation with ouabain versus Ca(2+) modulation with ionomycin. CONCLUSION: These data are the first to show that treatments known to alter intracellular ion concentrations are a viable method for increasing the mechanical properties of engineered articular cartilage and identifying potentially important relationships to hydrostatic pressure mechanotransduction. Ouabain and ionomycin may be useful pharmacologic agents for increasing tensile integrity and directing construct maturation.