Intracellular Na(+) and Ca(2+) modulation increases the tensile properties of developing engineered articular cartilage

Abstract

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.

Fig 1

Experimental designs for each phase in this study. In Phase I, a low and high concentration of several intracellular Na⁺ and Ca²⁺ modulators (ouabain, bumetanide, ionomycin, and histamine) were examined at 14 days. In Phase II, effects of the Na⁺ modulators were examined at 4 wks, and the effects of continuous treatment with 0.3 µM ionomycin from days 10–14 were compared to treating only 1 hr per day on days 10–14. Phase III experiments were also 4 wks in duration. Based on Phase II results, combined treatment with 0.3 µM ionomycin and 20 µM ouabain for 1 hr per day on days 10–14 of culture was compared to individual treatment with these agents.

Fig 2

Representative data from construct tensile testing. A) Stress versus axial strain, B) digital images taken during the test for determination of Poisson’s ratio, and C) linear regression of axial strain versus the negative of lateral strain. In B, note the subtle decrease in the lateral dimension of the construct. “Time-stamps” are provided in each subfigure to demonstrate the way in which lateral strain was matched in time to axial strain.

Fig 3

Gross morphological (A–D) and immunohistochemistry (IHC) images for type II collagen (E–H) and type I collagen (I–L) from the Phase III experiment. Note the smaller size of ouabain treated constructs. All groups stained for type II collagen, while groups treated with ionomycin also showed trace collagen type I staining. The scale Bar in L = 100 µm and applies to all IHC images.

Fig 4

Phase III A) tensile stiffness and B) total collagen normalized to construct wet weight. All three treatment groups resulted in an ~95% increase in tensile stiffness compared to control, while groups treated with ouabain contained significantly more total collagen per wet weight than controls.