Fig. 7. The response of periosteal cells to exogenous Ihh. (A) Comparison of proliferation rates in control (RCAS-GFP infected) and experimental (RCAS-Ihh infected) micromass cultures after 4 days. Values shown are means±s.d. of BrdU labelled cells per unit area after a 30-minute pulse. (B) Phase contrast image of control micromass after 11 days in culture, showing polygonal osteoblast-like cells, but no nodules. (C) Ihh-infected micromass from the same series as B, again showing polygonal cells at day 11. (D) Same micromass as in C, shown as an example of the precocious bone nodules found in Ihh infected micromasses. (E) Day 11 control micromass displaying alkaline phosphatase (AP) activity (red). (F) Ihh-infected micromass additionally demonstrates mineralised bone matrix (black stain). (G,H,I,J) Cultures at day 14. (G) AP activity is evident at the periphery of the control micormass. (H) AP activity is increased in Ihh-infected cultures. (I,J) Sections stained additionally for von Kossa, demonstrating mineralised bone matrix at the centre of both micromasses; the matrix is increased in Ihh-treated cultures (J). Scale bars: B-D, 50 µm; E-J, 1 mm.

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