Trimethylamine N-oxide as a media supplement for cartilage tissue engineering

Abstract

Supplements added to the culture media (e.g., growth factors and dexamethasone) have been successful in improving mechanical and biochemical properties of engineered cartilage towards native values. Trimethylamine N-oxide (TMAO), a natural osmolyte found in shark cartilage, is thought to induce protein folding, and counteracts the destabilizing effect of the high concentrations of urea stored by sharks. The objective of this study was to investigate the use of TMAO as a media supplement for promoting growth of functional engineered cartilage in culture. In the first study, TMAO was added to the culture media for the first 14 days in culture and concentrations of 0-200 mM were evaluated. In the second study, TMAO was supplemented to the culture media following chondroitinase ABC digestion, which has been previously shown to mediate an increased collagen content in engineered cartilage. A dose-dependent response was observed with improved mechanical and biochemical properties for engineered constructs cultured with TMAO at concentrations of 5-100 mM. The Young's modulus of digested constructs cultured in TMAO was 2× greater than digested constructs cultured in the control medium and recovered to undigested control levels by day 42. In conclusion, these initial studies with TMAO as a media supplement show promise for improving the compressive mechanical properties, increasing extracellular matrix production, and increasing the recovery time following chABC digestion.

Figure 1

Schematic of the two experiments. Note that culture media used in both studies was supplemented with 10 ng/ml of TGF-β3 for the first 14 days in culture. In Study 1, the culture media was supplemented with different concentrations of TMAO for the first 2 weeks of culture. In Study 2, engineered cartilage cultured in CM was digested at day 14 then cultured either in CM or CM supplemented with 5 mM of TMAO.

Figure 2

Strong linear correlation between the TMAO concentration and the media osmolarity.

Figure 3

(A) The equilibrium Young's modulus and (B) dynamic modulus for engineered constructs from Study 1. *p < 0.05 compared to the control group (0 mM TMAO).

Figure 4

(A) Collagen and (B) GAG content normalized to wet weight (WW) for engineered cartilage in Study 1. *p < 0.05 compared to the control group (0 mM TMAO).

Figure 5

Immunohistochemistry of collagen type II (A,C) and type VI (B,D) for the control (top row) and the 50 mM TMAO group (bottom row). Collagen is shown in green and the cell nuclei are red. Bar = 100 μm. Type VI collagen staining was more prominent throughout engineered cartilage for constructs cultured in TMAO supplemented media (D). [Color figure can be seen in the online version of this article, available at http://wileyonlinelibrary.com/journal/jor]

Figure 6

Young's modulus of samples digested with chABC at day 14. Following the digestion, samples were cultured in CM (square) or CM supplemented with 5 mM of TMAO (triangle). Undigested samples served as the control (circle). *Significant difference between the undigested control and the chABC-digested constructs (p ≤ 0.05).

Figure 7

(A) Collagen and (B) GAG normalized to wet weight for the control group (white bar) and digested samples cultured in CM (black) or TMAO media (diagonal) at day 42. *Significant difference to the control group (p ≤ 0.05).

Figure 8

Picrosirius Red staining for collagen in the (A) edge and (B) center of chABC-digested constructs cultured in CM supplemented with 5 mM TMAO at day 42. Alcian Blue staining for proteoglycans in the (C) edge and (D) center of chABC-digested constructs cultured in CM supplemented with 5 mM TMAO. Inset represents staining of constructs at day 0. The asterisk represents regions of lighter Alcian Blue staining at the edge and darker staining at the center. Bar = 500 μm. [Color figure can be seen in the online version of this article, available at http://wileyonlinelibrary.com/journal/jor]