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.