The effect of concentration, thermal history and cell seeding density on the initial mechanical properties of agarose hydrogels

Agarose hydrogels are commonly used for cartilage tissue engineering studies and to provide a three dimensional environment to investigate cellular mechanobiology. Interpreting the results of such studies requires accurate quantification of the mechanical properties of the hydrogel. There is significant variation in the reported mechanical properties of agarose hydrogels, and little is reported on the influence of factors associated with mixing these hydrogels with cell suspensions on their initial mechanical properties. The objective of this study was to determine the influence of agarose concentration, the cooling rate during gelation, the thermal history following gelation and the cell seeding density on the initial mechanical properties of agarose hydrogels. The average ramp modulus of 2% agarose gel in tension was 24.9 kPa (+/-1.7, n=10), compared with 55.6 kPa (+/-0.5, n=10) in compression. The average tensile equilibrium modulus was 39.7 kPa (+/-5.7, n=6), significantly higher than the compressive equilibrium modulus of 14.2 kPa (+/-1.6, n=10). The equilibrium and dynamic compressive modulus of agarose hydrogels were observed to reduce if maintained at 37 ( composite function)C following gelation compared with samples maintained at room temperature. Depending on the methodology used to encapsulate chondrocytes within agarose hydrogels, the equilibrium compressive modulus was found to be significantly higher for acellular 2% agarose gels compared with 2% agarose gels seeded at approximately 40 x 10(6) cells/mL. These results have important implications for interpreting the results of chondrocyte mechanobiology studies in agarose hydrogels.