The objective of this study was to determine the capacity of chondrocyte- and mesenchymal stem cell (MSC)-laden hydrogel constructs to achieve native tissue tensile properties when cultured in a chemically defined medium supplemented with transforming growth factor-beta3 (TGF-beta3).
Cell-laden agarose hydrogel constructs (seeded with bovine chondrocytes or MSCs) were formed as prismatic strips and cultured in a chemically defined serum-free medium in the presence or absence of TGF-beta3. The effects of seeding density (10 vs 30 million cells/mL) and cell type (chondrocyte vs MSC) were evaluated over a 56-day period. Biochemical content, collagenous matrix deposition and localization, and tensile properties (ramp modulus, ultimate strain, and toughness) were assessed biweekly.
Results show that the tensile properties of cell-seeded agarose constructs increase with time in culture. However, tensile properties (modulus, ultimate strain, and toughness) achieved on day 56 were not dependent on either the initial seeding density or the cell type employed. When cultured in medium supplemented with TGF-beta3, tensile modulus increased and plateaued at a level of 300-400 kPa for each cell type and starting cell concentration. Ultimate strain and toughness also increased relative to starting values. Collagen deposition increased in constructs seeded with both cell types and at both seeding densities, with exposure to TGF-beta3 resulting in a clear shift toward type II collagen deposition as determined by immunohistochemical staining.
These findings demonstrate that the tensile properties, an important and often overlooked metric of cartilage development, increase with time in culture in engineered hydrogel-based cartilage constructs. Under the free-swelling conditions employed in the present study, tensile moduli and toughness did not match that of the native tissue, though significant time-dependent increases were observed with the inclusion of TGF-beta3. Of note, MSC-seeded constructs achieved tensile properties that were comparable to chondrocyte-seeded constructs, confirming the utility of this alternative cell source in cartilage tissue engineering. Further work, including both modulation of the chemical and mechanical culture environment, is required to optimize the deposition of collagen and its remodeling to achieve tensile properties in engineered constructs matching the native tissue.