Effect of Sodium Bicarbonate on Extracellular pH, Matrix Accumulation, and Morphology of Cultured Articular Chondrocytes

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Abstract
Tissue engineering is a promising approach for articular cartilage repair; however, attempts to develop tissue that mimic native cartilage have been problematic. The synthesis and accumulation of extracellular matrix macromolecules may be adversely affected by a change in pH of the media during cell culture. Typically, zwitterion buffers (e.g., HEPES) are used in culture media because they can perform under both slightly acidic and alkaline conditions. However, because chondrocytes tend to rapidly acidify the media, better pH maintenance may be achieved by utilizing additional buffering agents that operate under acidic conditions (e.g., bicarbonate). The purpose of this study was to investigate the combined effect of HEPES and sodium bicarbonate (NaHCO3) on extracellular pH and matrix accumulation by cultured articular chondrocytes. Isolated bovine articular chondrocytes were seeded on Millicell filters and cultured in HEPES-buffered media containing 0, 7, or 14 mM NaHCO3. Throughout the 4-week culture period, the extracellular pH was more neutral with increasing NaHCO3 concentration. Addition of NaHCO3 increased synthesis (140 ± 29%) and accumulation (20 ± 2%) of proteoglycans whereas collagen was unaffected. There was also an increase in tissue cellularity (41 ± 13%). Morphologically, increasing numbers of flattened cells, resembling superficial zone chondrocytes, localized to the surface of the in vitro-formed tissue with increasing NaHCO3 supplementation were observed by light and transmission electron microscopy. Experiments are underway to determine whether these effects are due to pH maintenance or to the increased concentration of bicarbonate ions, which regulate intracellular pH.