Shear stress magnitude and duration modulates matrix composition and tensile mechanical properties in engineered cartilaginous tissue

Abstract

Cartilage tissue-engineering strategies aim to produce a functional extracellular matrix similar to that of the native tissue. However, none of the myriad approaches taken have successfully generated a construct possessing the structure, composition, and mechanical properties of healthy articular cartilage. One possible approach to modulating the matrix composition and mechanical properties of engineered tissues is through the use of bioreactor-driven mechanical stimulation. In this study, we hypothesized that exposing scaffold-free cartilaginous tissue constructs to 7 days of continuous shear stress at 0.001 or 0.1 Pa would increase collagen deposition and tensile mechanical properties compared to that of static controls. Histologically, type II collagen staining was evident in all construct groups, while a surface layer of type I collagen increased in thickness with increasing shear stress magnitude. The areal fraction of type I collagen was higher in the 0.1-Pa group (25.2 +/- 2.2%) than either the 0.001-Pa (13.6 +/- 3.8%) or the static (7.9 +/- 1.5%) group. Type II collagen content, as assessed by ELISA, was also higher in the 0.1-Pa group (7.5 +/- 2.1%) compared to the 0.001-Pa (3.0 +/- 2.25%) or static groups (3.7 +/- 3.2%). Temporal gene expression analysis showed a flow-induced increase in type I and type II collagen expression within 24 h of exposure. Interestingly, while the 0.1-Pa group showed higher collagen content, this group retained less sulfated glycosaminoglycans in the matrix over time in bioreactor culture. Increases in both tensile Young's modulus and ultimate strength were observed with increasing shear stress, yielding constructs possessing a modulus of nearly 5 MPa and strength of 1.3 MPa. This study demonstrates that shear stress is a potent modulator of both the amount and type of synthesized extracellular matrix constituents in engineered cartilaginous tissue with corresponding effects on mechanical function.

Figure 1

Schematic of Bioreactor (Flow Configuration). The bioreactor consists of a rectangular Upper and Lower Chamber which contain nutrient media. Nutrient media recirculates through the upper chamber (length L of 4.57 cm, width b of 1.27 cm, height h of 350 μm) at a flow rate Q, applying a fluid-induced shear stress (τ) to the tissue, which resides on a semi-permeable membrane. Additional bioreactor details, including to-scale drawings, can be found in previous reports (Gemmiti and Guldberg 2006; Pierre et al. 2007).

Figure 2

Histology. Representative cross-sections for static, flow at 0.001-Pa and flow at 0.1-Pa when stained for type I, II, and IX collagen, total collagen (picrosirius red, birefringent light) and sulfated glycosaminoglycans (Safranin-O). Note the increase in type I collagen content co-localized with flattened, fibroblastic cells.

Figure 3

Matrix Composition. Tissues flowed at 0.1-Pa possessed a greater number of cells (A), but less sGAG on a per-cell (B) and per-dry and per-wet weight (C) basis than the other groups. More sGAG was present in the media across all groups (D, significance not shown). However, no difference was found between groups when media and tissue contributions were summed (not shown). [N= 8-11 for A, B and C; N=4 per day for D] Star represents p≤0.05. Overall ANOVA values for the treatment of flow was p<0.0001 for all groups (A, total DNA; B, sGAG per-cell; C, sGAG per-weight).

Figure 4

Collagen Content. 0.1-Pa flow tissues contained more total collagen on a dry and wet weight basis (A) than the other groups, but the static group contained the most total collagen on per cell basis (B). The increase in total collagen was from contributions of both type I (C) and type II (wet and dry) (D) collagen; both types were significantly increased in 0.1-Pa flow compared to other groups. Star represents p≤0.05. Overall ANOVA values for the treatment of flow was p=0.012 (A, total collagen per-weight) and p<0.0001 (B, total collagen per-cell; C, type I collagen; D, type II collagen).

Figure 5

Mechanical Properties. Both flow conditions resulted in higher Young's modulus (A) and ultimate strength (B) than the static condition. The higher flow group (0.1) was also significantly increased compared to the lower flow group (0.001). Shaded stars denote significance p<0.0001 whereas open stars denote significance p=0.046 for Young's modulus (A) and p=0.005 for ultimate strength (B). Overall ANOVA values for the treatment of flow was p<0.0001 for both Young's modulus and ultimate strength. [N=9-14] Star represents p≤0.05.

Figure 6

Gene Expression. Type I (A) and type II (B) collagen are modulated by shear stress magnitude and duration of application, most notably at the 24- and 72-hour time points. Open stars denote significance (p<0.05) for 0.1-Pa compare to other groups at that time point, whereas shaded stars represent significance (p<0.05) between all groups at that time point. [N=3-4 for 2, 24, 72h; N=7-9 for 168h]