Differential maturation and structure-function relationships in mesenchymal stem cell- and chondrocyte-seeded hydrogels

Abstract

Degenerative disease and damage to articular cartilage represents a growing concern in the aging population. New strategies for engineering cartilage have employed mesenchymal stem cells (MSCs) as a cell source. However, recent work has suggested that chondrocytes (CHs) produce extracellular matrix (ECM) with superior mechanical properties than MSCs do. Because MSC-biomaterial interactions are important for both initial cell viability and subsequent chondrogenesis, we compared the growth of MSC- and CH-based constructs in three distinct hydrogels-agarose (AG), photocrosslinkable hyaluronic acid (HA), and self-assembling peptide (Puramatrix, Pu). Bovine CHs and MSCs were isolated from the same group of donors and seeded in AG, Pu, and HA at 20 million cells/mL. Constructs were cultured for 8 weeks with biweekly analysis of construct physical properties, viability, ECM content, and mechanical properties. Correlation analysis was performed to determine quantitative relationships between formed matrix and mechanical properties for each cell type in each hydrogel. Results demonstrate that functional chondrogenesis, as evidenced by increasing mechanical properties, occurred in each MSC-seeded hydrogel. Interestingly, while CH-seeded constructs were strongly dependent on the 3D environment in which they were encapsulated, similar growth profiles were observed in each MSC-laden hydrogel. In every case, MSC-laden constructs possessed mechanical properties significantly lower than those of CH-seeded AG constructs. This finding suggests that methods for inducing MSC chondrogenesis have yet to be optimized to produce cells whose functional matrix-forming potential matches that of native CHs.

FIG. 1.

Calcein AM staining of live cells in construct cross sections on day 42 for CHs (A–C) and MSCs (D–F) in AG (left), MeHA (middle), and Pu (right) hydrogels. Magnification, 40×; scale bar, 50 μm. Color images available online at www.liebertonline.com/ten.

FIG. 2.

Biochemical content of CH- and MSC-seeded constructs as a function of time over an 8-week culture period. (A) DNA content, (B) s-GAG as a percentage of the wet weight (ww), and (C) collagen as a percentage of the wet weight. Data represent the mean ± SD of three to four samples from one of two replicate studies. *p < 0.05 for day 56 comparisons between hydrogels within cell type. **Greater value (p < 0.05) for comparisons on day 56 within hydrogel between cell types. ^†No significant increase from day 0 (p > 0.05).

FIG. 3.

Histological analysis of CH- and MSC-seeded constructs on day 56. Alcian blue staining of deposited PGs for CH-seeded (A–C) and MSC-seeded (D–F) AG (top), MeHA (middle), and Pu (bottom) hydrogels. Picrosirius red staining of deposited collagen for CH-seeded (G–I) and MSC-seeded (J–L) AG (top), HA (middle), and Pu (bottom) hydrogels. Magnification, 100×; scale bar, 200 μm. Color images available online at www.liebertonline.com/ten.

FIG. 4.

(A) Equilibrium modulus, E_Y, and (B) dynamic modulus, G*, of AG, MeHA, and Pu hydrogels seeded with CHs or MSCs measured biweekly over a 56-day culture period. Data represent the mean ± SD of three to four samples from one of two replicate studies. *p < 0.05 for day 56 comparisons between hydrogels within cell type. **Greater value (p < 0.05) for comparisons on day 56 within hydrogel between cell types. ^†No significant increase from day 0 (p > 0.05).

FIG. 5.

Correlation plots relating measured mechanical properties to biochemical constituents. (A) Plots for CH-seeded hydrogels. (B) Plots for MSC-seeded hydrogels. Dashed line shows linear curve fit for each gel type.