Figure 5

Tenocytes from Ink4a/Arf^−/− mice can produce 3D tendon-like constructs similar to wild type (wt) cells. (a) The macroscopic appearance of the silicone plate with the tendon-like constructs before harvesting (left) with a close-up of the tendons in their wells (right). (b)–(e) Histology of H&E stained tendon-like constructs obtained from wild-type (wt) cells in a low passage (b) and Ink4a/Arf^−/− cells in low (c), intermediate (d) and high (e) passages (top row: scale bar 400 µm, bottom row: scale bar 100 µm). (f) 3D tendon-like constructs of Ink4a/Arf^−/− tenocytes in an intermediate passage following freeze and thaw cycle showed analogous cell and fiber arrangement to 3D tendon-like constructs of wt or Ink4a/Arf^−/− cells, which had not undergone a freeze–thaw cycle (left micrograph: scale bar = 400 µm, right micrograph: scale bar = 100 µm). (g) qPCR analysis of tendon markers- Col1, Col3, Col5, Scx, Tnmd and Tnc presented as fold change (FC) in gene expression between 3 and 2D, for wt and Ink4a/Arf^−/− cells individually. The dotted line in each graph represents a FC of 1 (no difference between 2 and 3D gene expression). The expression in Col1, Col3, Col5 and Tnc, but not in Tnmd and Scx, was significantly higher in 3D than 2D culture for both wt and Ink4a/Arf^−/− cells (for mean normalized mRNA expression and p-values, see Table 2).