Isolation of irradiated osteoblasts from megakaryocyte-conditioned marrow microenvironment. (A) Representative photomicrographs of in vitro–cultured osteoblasts selectively obtained from bones (Bo) in control (n = 6) and irradiated (TBI; n = 6) mice. The bones are harvested and completely depleted of hematopoietic marrow fraction. Left panels represent nonirradiated control (top) and irradiated (bottom) samples after 7 days of culture without PL (−PL). Osteoblasts in control samples begin to proliferate generating an adherent cell layer; conversely, after TBI the separation of irradiated osteoblasts () from marrow microenvironment is followed by a defective in vitro behavior. The addition of PL (+PL, right) increased the proliferation of osteoblasts from controls (top right) as well as from the irradiated animals (bottom right). Osteoblasts are indicated (
). Scale bar represents 200 μm. (B) Representative pictures of osteoblast colonies from irradiated or control mice, after 2 weeks in culture with or without PL. (C) In vitro osteoblast colony count (per 200 mg processed bone chips) from irradiated and control mice, with or without PL. A reduction of the average colony number is observed after irradiation (89.2 ± 23.7 vs 27 ± 12, P = .006), indicating a damage that is not restored because of the absence of megakaryocyte-derived growth factors in the cultures. PL was able to significantly increase the colony number both in control samples (89.2 ± 23.7 vs 118.7 ± 14.4, P = .04) and after TBI (27 ± 12 vs 55 ± 4.3, P = .03). Experiments were performed at least in triplicate.