EFFECT OF RHBMP-2 ON THE OSTEOGENIC POTENTIAL OF BONE-MARROW STROMAL CELLS FROM AN OSTEOGENESIS IMPERFECTA MOUSE (OIM)

To understand whether osteogenesis imperfecta (OI) could result from d efective differentiation of osteoprogenitor cells, we investigated the osteogenic potential of bone marrow stromal cells from a mouse model of human OI (oim). Bone marrow was flushed from the femurs and tibias of oim and normal littermates using a syringe with Dulbecco's modified Eagle's medium, and cells were allowed to adhere to flasks, Adherent cells were trypsinized and passaged weekly at a 1:4 split, The establi shed stromal cells were assessed for collagen synthesis, alkaline phos phatase, and osteocalcin production in the presence or absence of rhBM P-2. The stromal cells were also assessed for mineralization by Von-Ko ssa staining and for exogenous gene transfer using adeno-lacZ and a re troviral vector, The bone marrow stromal cells from oim mice synthesiz ed alpha 1(I) homotrimers as expected, whereas the stromal cells from the normal littermates synthesized alpha 1(I)(2) alpha 2(I) heterotrim ers. The bone marrow stromal cells exhibited low levels of alkaline ph osphatase activity under basal conditions; upon treatment with rhBMP-2 , the level of the alkaline phosphatase activity increased approximate ly 40-fold, Cytochemical staining of the cells confirmed the expressio n of alkaline phosphatase by the oim stromal cells and its augmentatio n by rhBMP-2, Osteocalcin production in the stromal cells was also enh anced approximately threefold by rhBMP-2. oim stromal cells grown in t he presence of beta-glycerophosphate and ascorbic acid demonstrated Vo n-Kossa-positive solid deposits after 3 weeks in culture, Ten days aft er infection with adeno-lacZ, approximately 70% of oim stromal cells e xpressed the transgene product, and after infection with a retrovirus, approximately 20% of the cells expressed the transgene, These data in dicate that bone marrow stromal cells have osteogenic potential, and a lso the potential to be transduced with exogenous genes, Under basal c onditions, however, the stromal cells from oim mice exhibited signific antly lower levels of alkaline phosphatase activity than their normal littermates. (C) 1997 by Elsevier Science Inc. All rights reserved.