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Title: The role of hypoxia in regulating the formation and activity of bone cells
Author: Utting, Jennifer Caroline
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2006
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Hypoxia occurs when the blood supply to tissues is reduced or disrupted. In normal tissues pC > 2 is in the range 5 - 12% however in diseased tissue this value can fall to below 1% O2. Much pathology associated with bone loss is also associated with decreased local or systemic p02, such as infection, inflammation, and cancer. The aim of this thesis was to determine the effects of decreased pC > 2 on the formation and activity of osteoclasts (OC), the bone resorbing cells, and osteoblasts (OB), the bone forming cells. Exposure of murine bone marrow monocytes, cultured in the presence of M-CSF and RANKL, to pC > 2 in the range 20% to 0.2% led to dramatic increases in osteoclast formation at 1 - 2% O2, with significant increases even at pC > 2 as low as 0.2% (severe hypoxia), over those cultured at 20% O2 (normoxia). Decreased O2 did not activate mature OC isolated from neonatal rat bones to resorb bone, indicating that hypoxia has no effect on osteoclast activity. When the effects of hypoxia were tested on human OC formation from peripheral blood monocytes, a similar trend was found to that observed in rodents, indicating that hypoxia is acting directly on OC rather than stromal cells present in the marrow cultures, in support of this result, the effects of hypoxia were dependent on permissive levels of M-CSF and RANKL. In contrast to its stimulatory effects on OC, hypoxia inhibited the production of mineralised nodules in primary rat OB cultures in a dose dependent manner, with almost complete abolition of nodule formation at 0.2% O2. Proliferation of rat OB, measured by tritiated thymidine incorporation at days 6, 12 and 18 of culture, was inhibited by 50 - 70% in 2% O2 as opposed to culture at 20% O2 however, apoptosis was not altered by culturing OB at 2% O2. Transmission electron microscopy of nodules revealed dense, regular fibrils of type I collagen in 20% O2 cultures, whereas this ultrastructure was disorganised in collagen formed in hypoxia. Collagen production was decreased and reduced lysine hydroxylation within the deposited collagen was observed when OB were cultured at 2% O2. Furthermore OB expressed lower levels of the lysyl hydroxylase enzymes in cultures maintained at 2% O2. The percentage of proline hydroxylation in deposited collagen was unaffected by decreased O2 availability. Expression of collagen type lal was unaffected by hypoxia, as was expression of all transforming growth factor P isoforms however expression of the differentiation markers osteocalcin and alkaline phosphatase were delayed in hypoxic cultures, and OB alkaline phosphatase activity failed to increase with time in hypoxic cultures, suggesting that differentiation of OB is delayed by hypoxia. Together these results indicate that hypoxia induces in OB a state of 'suspended animation' where cellular energy is directed towards survival rather than bone formation. The reciprocal nature of the responses of OC and OB to hypoxia would lead to marked bone loss in situations where oxygen tension is reduced and may explain the bone loss associated with a variety of pathological situations. These results emphasise the importance of the vasculature in maintaining bone health.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available