Characterization and manufacture of an agarose hydrogel system for loading embedded chondrocytes

Abstract

Studying chondrocyte biology in osteoarthritis in vitro is challenging due to the requirement to simulate the three-dimensional matrix of cartilage. A commonly used in vitro scaffold for chondrocyte culture is agarose, and although the physical properties of agarose are known to align with the pericellular matrix of cartilage, little is known about its thermal properties. This is especially important because joint temperature affects chondrocyte metabolic processes. In this research, the thermal characteristics of agarose hydrogels were characterized for the first time. The thermal properties of agarose can be an accurate in vitro representation of the pericellular matrix of chondrocytes for modeling the specific heat capacity and thermal conductivity. The viscoelastic heat generated by cyclically compressing these hydrogels was also shown to have a negligible effect on the temperature of the gels. To provide physiological loading to these cell- impregnated hydrogels, a custom displacement-controlled bioreactor was designed, manufactured, and programmed. This bioreactor met required design specifications in multi-modal loading capabilities both in the compression and shear directions. This bioreactor was used to apply cyclical compressive and shear loading to chondrocytes in agarose. This applied cyclical loading promoted changes in central carbon metabolism based on the duration and type of loading, as determined using targeted mass spectrometry-based metabolomics. Specifically, chondrocytes loaded in high durations of compression and shear showed an increase in glycolytic and TCA reactions. Cells which were loaded only in shear showed an increase in succinate buildup, which suggests energy dysregulation. Finally, a relationship between heat generated by chondrocytes and available carbon sources was established through the measurement of temperature changes using a microcalorimeter. This data suggests that chondrocytes encapsulated in agarose generate a measurable quantity of heat during the operation of their central carbon metabolic pathways, and this heat is dependent on the presence of carbon sources. With the validation of agarose hydrogels as a viable thermal mimic of cartilage, the creation of a bioreactor which capable of applying well- controlled deformations to chondrocytes in agarose, and the demonstrated connection between heat generation and metabolic activity, this project has advanced knowledge of metabolic processes and their relationship to chondrocyte mechanotransduction.