Tensile properties of engineered cartilage formed from chondrocyte- and MSC-laden hydrogels

Abstract

Objective: 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).

Design: 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: 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.

Conclusions: 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.

Figure 1. Tensile properties of acellular agarose hydrogels

Tensile ramp modulus (white squares) and ultimate strain (black diamonds) of acellular gels as a function of agarose content (% w/v). Data represents the mean and standard deviation of ten samples per group. * Indicates difference from 2% group, p<0.05; ** indicates greater than 3% group, p<0.05; *** indicates greater than 4% group, p<0.05.

Figure 2. Biochemical composition of tensile strips with variation in time in culture, media condition, cell type, and cell density

Top row: DNA content (µg/disk); middle row: sGAG content (%ww); bottom row: collagen content (%ww). * Indicates greater than all values lower in both CM− and CM+ conditions within cell and seeding density group (p<0.05); ** indicates greater than all values lower in both CM− and CM+ conditions within cell and seeding density group (p<0.05); # indicates greater than corresponding CH10M value at same time point and media condition (p<0.05); & indicates lower than corresponding CH10M value at same time point and media condition (p<0.05). Data represent the mean and standard deviation of seven to ten samples per group per time point.

Figure 3. Time-dependent tensile modulus (kPa), ultimate strain (%), and toughness (kPa) of constructs with culture in CM− or CM+ medium

* Indicates greater than all values lower in both CM− and CM+ conditions within cell and seeding density group (p<0.05); ** indicates greater than all values lower in both CM− and CM+ conditions within cell and seeding density group (p<0.05); *** indicates greater than all lower values within same cell type and seeding density group (p<0.05); # indicates greater than corresponding CH10M value at same time point and media condition (p<0.05). Data represent the mean and standard deviation of seven to ten samples per group per time point. Dotted line indicates corresponding property of 2% agarose from acelluluar studies.

Figure 4. Histologic appearance of constructs on day 56

H&E, Alcian Blue and Picrosirius Red staining of CH10M, CH30M and MSC10M constructs cultured in CM+ reveals no differences between groups at day 56. Constructs cultured in CM− conditions (not shown) showed lower staining intensities for chondrocyte groups, and absence of stain for MSCs. Scale bar: 200µm.

Figure 5. Immunohistochemical detection of amount and distribution of collagen type I and type II in day 56 constructs cultured in CM− or CM+ medium

Chondrocyte-laden constructs stained for both type I and type II collagen in CM− conditions, with an intense shift to predominantly type II collagen and loss of type I staining in CM+ conditions, regardless of seeding density. MSCs showed some pericellular staining of type I collagen and no type II collagen in CM− conditions, but a robust deposition of type II collagen throughout the construct with culture in CM+ conditions. Scale bar: 200µm.