Encapsulation of Chondrocytes in High-Stiffness Agarose Microenvironments for In Vitro Modeling of Osteoarthritis Mechanotransduction

Abstract

In articular cartilage, chondrocytes reside within a gel-like pericellular matrix (PCM). This matrix provides a mechanical link through which joint loads are transmitted to chondrocytes. The stiffness of the PCM decreases in the most common degenerative joint disease, osteoarthritis. To develop a system for modeling the stiffness of both the healthy and osteoarthritic PCM, we determined the concentration-stiffness relationships for agarose. We extended these results to encapsulate chondrocytes in agarose of physiological stiffness. Finally, we assessed the relevance of stiffness for chondrocyte mechanotransduction by examining the biological response to mechanical loading for cells encapsulated in low- and high-stiffness gels. We achieved agarose equilibrium stiffness values as large as 51.3 kPa. At 4.0% agarose, we found equilibrium moduli of 34.3 ± 1.65 kPa, and at 4.5% agarose, we found equilibrium moduli of 35.7 ± 0.95 kPa. Cyclical tests found complex moduli of ~100-300 kPa. Viability was >96% for all studies. We observed distinct metabolomic responses in >500 functional small molecules describing changes in cell physiology, between primary human chondrocytes encapsulated in 2.0 and 4.5% agarose indicating that the gel stiffness affects cellular mechanotransduction. These data demonstrate both the feasibility of modeling the chondrocyte pericellular matrix stiffness and the importance of the physiological pericellular stiffness for understanding chondrocyte mechanotransduction.

Combining Targeted Metabolomic Data with a Model of Glucose Metabolism: Toward Progress in Chondrocyte Mechanotransduction

“…For example, the PCM can release FGF2, a fibroblast growth factor, to initiate diverse signaling in response to external stimuli [25]. This study used previously published data involving SW1353 chondrocytes subjected to compression in agarose of physiological stiffness [10, 14]. SW1353 cells were selected because they are a commonly used in chondrocyte and osteoarthritis research [26–28].…”

“…daily activity [9], have the potential to alter the energy metabolism of chondrocytes via mechanotransduction. We have previously observed changes in concentrations of central energy metabolites as a result of dynamic compression [10]. Other studies demonstrate that chondrocytes sense compression and can distinguish between static compression, dynamic compression, and even shear.…”

Mechanotransduction in primary human osteoarthritic chondrocytes is mediated by metabolism of energy, lipids, and amino acids

“…cellular metabolites smaller than ∼1000 Da). Building upon previous methodology to encapsulate chondrocytes in agarose similar in stiffness to the human PCM, we profiled the metabolomic responses of primary human OA chondrocytes in response to applied compression at physiological levels [20, 21]. …”

Metabolic responses induced by compression of chondrocytes in variable-stiffness microenvironments

“…While healthy cartilage has PCM stiffness values of ~40kPa, the stiffness of osteoarthritic PCM is ~24kPa (Alexopoulos et al, 2005). Current studies have analyzed 3D cultures in 1–3% (v/v) agarose hydrogels, which has a stiffness ranging from 15–19kPa (Jutila et al, 2015), falling to the lower boundary for OA PCM environments and providing limited data on the progression of chondrocyte mechanotransduction pathways from healthy to OA. However, recent advances have characterized mechanical properties of higher concentration agarose hydrogels and found that the stiffness of 4.5% (v/v) agarose hydrogels approaches that of healthy PCM.…”

“…However, recent advances have characterized mechanical properties of higher concentration agarose hydrogels and found that the stiffness of 4.5% (v/v) agarose hydrogels approaches that of healthy PCM. Therefore, comparing 2 and 4.5% agarose hydrogels provide an opportunity for studying altered mechanotransduction in OA disease progression (Jutila et al, 2015). …”