Disuse induced by botulinum toxin affects the bone marrow expression profile of bone genes leading to a rapid bone loss.

OBJECTIVES: Molecular events occurring in the bone marrow microenvironment of an immobilized mouse limb after Botulinum toxin (BTX) injection haven't been characterized. BTX injection induces a localized disuse in which the tissue events have well been characterized. METHODS: BTX injection was performed in the right quadriceps; saline injection in the left side was used as control. Mice were sacrificed at 0, 7, 14, 21 and 28 days; tibias were used for microCT analysis; bone marrow from femurs for RT-PCR analysis. RESULTS: MicroCT revealed bone loss and microarchitectural damages on the immobilized side as from 7d; cortical area tended to be lower on the immobilized limb at 28d. Gene expression of formation factors was altered as from 7 days post-BTX: alkaline phosphatase, Tgfß1, Lrp5, Sfrp2. Only Sfrp2 and Lrp5 were maintained altered until 28d. Expression of Dkk1 increased from 21d and represented a late inhibitor of formation. Gene expression of resorption markers increased as from 7d (Rankl, Tracp, Il1α, Il1ß and Il6) and was maintained until 28d for Tracp and Il6. CONCLUSION: A localized disuse induces rapid modifications in the bone marrow gene expression leading to bone loss due to an early decrease of formation associated with an increase in resorption.

Isolation of osteoprogenitors from murine bone marrow by selection of CD11b negative cells.

Selection of cells having the most osteogenic potential is a strategy used in bone tissue engineering. Preclinical studies using murine bone marrow cells must consider the large amount of hematopoietic cells in the adherent fraction. The aim of this study was to enrich a murine bone marrow cell population with osteoprogenitors by using a simple and reliable method. Bone marrow from C57Bl/6 mice was extracted and cells which adhered onto plastic were expanded in primary culture for 14 days. Immunolabeling of the CD11b surface antigen was performed and the CD11b(-) cell fraction was isolated by FACS. Sorted and unsorted populations were analyzed for gene expression of osteoblast differentiation, alkaline phosphatase (AlkP) activity and matrix mineralization capacities. Selection of CD11b(-) cells increased the number of AlkP(+) cells from the plastic adherent fraction from 6.3% +/- 0.8 to 56% +/- 3.3 with a sevenfold increase in AlkP activity. mRNA analysis revealed a significant increase in the CD11b(-) fraction for Osterix (41-fold), RANKL (17-fold), M-CSF (8-fold) and Runx-2 (8-fold). An osteogenic population was obtained with improved capacities to produce a mineralized extracellular matrix in vitro, independently of the presence of glucocorticoids in the culture medium.