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Title: Characteristics and differentiation of cells involved in bone formation
Author: Maybee, Sarah Helen
ISNI:       0000 0001 3621 9918
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1984
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Postnatal marrow stronal cells culture in vitro contain cells which are osteogenic when implanted in vivo in diffusion chambers, but these have never formed bone in vitro. The purpose of this work was to develop in vitro culture methods to enhance the number and osteoblastic characteristics of rabbit marrow stromal cells and to investigate the expression of alkaline phosphatase as a possible sign of early osteogenic differentiation. Alkaline phosphatase (AP) is considered a phenotypic marker for the osteoblast line, and culture conditions were adjusted to maximize this enzyme's activity in stromal cells in vitro. Hydrocortisone (1018 to 10-6 M) caused dose-dependent changes in seme characteristics of the adherent cells from marrow: growth rate was higher; AP activity was increased up to 100 times the control level and an extracellular matrix was produced by the cells at confluence. However, there was no difference in the amount of bone formed in diffusion chambers frcm stromal cells grown with or without hydrocortisone. By culturing marrow explants rather than dissociated cells, growth rate and AP activity of the confluent cells were further increased. Some calcification occurred in these explant cultures but appeared to be non-specific. When explants were cultured on collagen lattices, much larger areas of calcification occurred in association with AP-positive stromal cells. Migrating out of the lattice onto the culture flask were cells with high AP activity and low proliferative potential. The degree of inhibition by hcmoarganine and phenylalanine, heat lability and electrqphoretic mobility of the AP activity in crude extracts of cultured marrow cells, bone, kidney, liver, intestine, spleen, thymus, lung and brain were compared. AP in bone, stromal cells, spleen and lung were indistinguishable using these criteria. AP from other tissues each had unique characteristics. Monoclonal antibodies to AP extracted frcm bone were produced. These reacted with AP on the surface of a subpopulation of marrow stromal cells, which can be isolated using the fluorescence-activated cell sorter. The antibodies will also be useful for AP staining at the light and electron microscope levels. In conclusion, AP present on the surface of some strcmal cells has properties of the bone isoenzyme, although this AP form may not be tissue-specific. AP activity in cultured stromal cells is increased in the presence of hydrocortisone. Monoclonal antibodies can be used to isolate AP+ stromal cells for further investigation of their osteogenicity.
Supervisor: Triffitt, Jim Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Bones ; Growth ; Skeleton ; Bone cells ; Bone marrow cells