Effects of Hypertonic (NaCl) Two-Dimensional and Three-Dimensional Culture Conditions on the Properties of Cartilage Tissue Engineered from an Expanded Mature Bovine Chondrocyte Source

Abstract

Clinically relevant mature cartilage cells (chondrocytes) present challenges for use in cartilage tissue engineering applications, given their low capacity for cell division and tissue production. Since the in situ environment of chondrocytes is hypertonic relative to standard culture medium conditions, in this study we tested the hypothesis that using culture medium of a hypertonic, more physiologic osmolarity during both two-dimensional (2D) expansion of mature bovine chondrocytes (MBCs) and their subsequent encapsulation culture in three-dimensional (3D) agarose hydrogel constructs produces improved engineered tissue construct mechanical and biochemical properties. Results demonstrate that 2D expansion of MBCs in hypertonic (NaCl) medium before encapsulation yielded improved construct mechanical properties. However, 3D encapsulation culture of cells in hypertonic (NaCl) medium yielded poorer construct mechanical properties. Osmolarity-related differences in construct biochemical content and organization may have contributed to differences in mechanical properties, as construct glycosaminoglycan content correlated moderately with construct mechanical properties, and construct collagen distribution varied between 3D osmotic culture groups. Results of this study suggest that application of hypertonic (NaCl) medium during 2D mature chondrocyte expansion, but not 3D encapsulated chondrocyte culture, may serve as a convenient and inexpensive method for improving mechanical properties of expanded cell-seeded constructs.

Promoting increased mechanical properties of tissue engineered neocartilage via the application of hyperosmolarity and 4α-phorbol 12,13-didecanoate (4αPDD)

“…The present study revealed that hyperosmolarity (450mOsm) applied to self-assembled chondrocytes reduced only collagen content. This result is in contrast to the increased collagen content seen in agarose-embedded chondrocytes (Sampat et al, 2013), but in agreement with the decreases seen with expanded chondrocytes (Oswald et al, 2011). The discrepancy in hyperosmolarity’s effects on ECM production suggests that further optimization of hyperosmotic loading levels in neocartilage engineering is needed.…”

“…Applied to agarose-encapsulated articular chondrocytes, hyperosmolarity (400mOsm) elicited significant increases in tensile stiffness and GAG and DNA content (Sampat et al, 2013). In contrast, when applied to 2D-expanded chondrocytes in hydrogels, hyperosmolarity (400mOsm) decreased Young’s modulus, and GAG, collagen, and DNA content (Oswald et al, 2011). The present study revealed that hyperosmolarity (450mOsm) applied to self-assembled chondrocytes reduced only collagen content.…”

Insulin, Ascorbate, and Glucose Have a Much Greater Influence Than Transferrin and Selenous Acid on theIn VitroGrowth of Engineered Cartilage in Chondrogenic Media

“…While modulating glucose concentration affects the medium osmolarity, and osmotic environment changes have been shown to have an impact on chondrocyte metabolism, 57,58 the osmolarity change elicited in this study by reducing glucose was low compared to osmolarities previously studied, and therefore the resulting changes in tissue properties are likely attributed to the concentration of glucose. Due to their lower medium concentrations, altering the concentrations of insulin, transferrin, selenous acid, or ascorbate does not elicit substantial changes in the medium osmolarity.…”

Redifferentiation of dedifferentiated chondrocytes in a novel three‐dimensional microcavitary hydrogel

“…It is well known that chondrocytes would undergo a dedifferentiation process which is considered as progressive change of their rounded morphology 22 and gradually loss of their chondrogenic capacity with further monolayer culture. 23 During the 27,28 adopting various cell densities 29,30 or ameliorating cell culture conditions like hypoxic 31 or hypertonic 32 environments.…”