Postmenopausal osteoporosis is a skeletal disorder characterised by bone loss. Declining oestrogen levels postmenopause disrupt bone remodelling by overstimulating resorption. Although the disorder is currently studied in animals, we should aim to minimise their use. Therefore, this thesis explored the feasibility of developing an in vitro model of postmenopausal osteoporosis using tissue engineering principles.
The response of three osteoblast cell lines, MC3T3-E1, MLOA5, and IDG-SW3, to oestrogen was explored, finding only MC3T3-E1 was stimulated by the hormone. The ability of RAW264.7 to undergo osteoclastogenesis was strongly influenced by seeding density and proliferation. Additionally, tartrate-resistant acid phosphatase (TRAP) activity could be suppressed by oestrogen exposure. Due to its ability to support osteoclastogenesis in co-culture, IDG-SW3 was the most suitable osteoblast cell line for the model.
Bone-matrix deposition over 28 days on three scaffolds (PolyHIPE, polyurethane, Biotek) was compared to select the most appropriate for the model. PolyHIPE and polyurethane scaffolds supported significantly more matrix deposition than the Biotek. Mineralisation on the scaffold could be detected by micro-computed tomography; however, the presence of PBS interfered with this. Due to its cellular performance and ease of manufacture, the polyurethane scaffold was identified as the most suitable for the model.
Changes in mineral content, TRAP and alkaline phosphatase activity were confirmed as markers for osteoclast and osteoblast activity in co-culture. RAW264.7 pretreatment with oestrogen to mimic pre-menopause had lasting effects on their ability to undergo osteoclastogenesis. 2D co-cultures using oestrogen withdrawal to mimic menopause resulted in increased resorption, analogous to the effect seen in vivo. From the conditions assessed in 3D co-cultures, no equivalent response was observed. This thesis demonstrates it is possible to imitate the onset of postmenopausal osteoporosis in vitro. However, a 3D system that uses human cells and longer time periods is necessary to provide a valid alternative to animal models.
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