Ionic osmolytes and intracellular calcium regulate tissue production in chondrocytes cultured in a 3D charged hydrogel

• A charged synthetic hydrogel is employed to study chondrocyte mechanobiology.
• A link among ionic osmolytes, dynamic loading, and intracellular calcium is shown.
• Dynamic changes in osmolarity enhanced chondrocyte synthesis of proteoglycans.
• Intracellular calcium mediates tissue production under loading and no loading.

The goal of this study was to investigate the role of fixed negative charges in regulating cartilage-like tissue production by chondrocytes under static and dynamic three-dimensional culture, and to determine whether intracellular calcium ([Ca2 +]i) is involved in mediating this response. Initial experiments using the 3D neutral hydrogel were conducted in static isotonic culture with ionic and non-ionic osmolytes added to the culture medium. Tissue production by bovine chondrocytes with non-ionic osmolytes was 1.9-fold greater than with ionic osmolytes, suggesting that the ionic nature of the osmolyte is an important regulator of tissue production. To investigate fixed negative charges, a 3D culture system containing encapsulated chondrocytes was employed based on a synthetic and neutral hydrogel platform within which negatively charged chondroitin sulfate was incorporated in a controlled manner. Incorporation of negative charges did not affect the mechanical properties of the hydrogel; however, intracellular ion concentration was elevated from the culture medium (330 mOsm) and estimated to be similar to that in ~ 400 mOsm culture medium. With dynamic loading, GAG synthesis decreased by 26% in neutral hydrogels cultured in 400 mOsm medium, and increased by 26% in charged gels cultured in 330 mOsm. Treatment of chondrocyte-seeded hydrogels with the Ca2 + chelator BAPTA-AM decreased GAG synthesis by 32–46% and was similar among all conditions, suggesting multiple roles for Ca2 + mediated tissue production including with ionic osmolytes. In conclusion, findings from this study suggest that a dynamic ionic environment regulates tissue synthesis and points to [Ca2 +]i signaling as a potential mediator.