Finlay, Melissa (2023). Developing, characterising, and testing a humanised 3D in vitro bone model, capable of long-term osteocyte culture. University of Birmingham. Ph.D.
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Finlay2023PhD.pdf
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Abstract
Introduction: Bone research, particularly into bone drug therapeutics, requires large-scale animal studies because complete in vitro bone models do not exist; primarily due to the complex and dynamic nature of bone tissue and the inability to culture osteocytes, (the main cell responsible for bone health maintenance). With an aging population, the number of people at risk and living with debilitating and enduring bone conditions, chiefly osteoporosis, is expected to rise. An ensuing increased pressure for effective therapies, along with the movement to replace animals in research, warrants greater effort to develop in vitro models that successfully recapitulate the cellular and extracellular complexities of bone tissue. Recent developments in 3D cell culture methods have enabled the creation of a self-structuring bone model (SSBM) that possesses physiochemical characteristics comparable to native bone, furthermore, it is the first model of its kind to house osteocytes long-term. However, this method used freshly isolated rat periosteal cells for production of each batch and the high variability in the resulting cell populations decreases reproducibility and reliability.
Aim: The aim of this project was to adapt and develop this SSBM culture method to utilise a secondary human osteoblast cell line to improve the reproducibility and relevance to human disease. Resulting constructs were structurally and biologically characterised, then their feasibility as a novel bone drug screening platform was investigated.
Methods: The human osteoblast cell line, hFOB 1.19, was seeded on a fibrin hydrogel, cast between two calcium phosphate anchors. Resulting SSBM constructs were cultured and analysed after 4-, 8- and 12-weeks post-cell seeding. Methods traditionally used for 2D cell culture and whole tissue analysis were adapted and developed specifically for SSBM construct analysis. These methods were then used to structurally and biologically characterise SSBM constructs, as well as measure their responsivity after exposure to bone formation modulators (BFMs): vitamin D, parathyroid hormone and Betulin.
Results: The hFOB 1.19 cell line successfully produced bone model constructs composed of a collagen-containing matrix that mineralised over time. Structural analysis using alizarin red and picrosirius red staining approaches along with x-ray fluorescence revealed heterogenous distribution and maturation of this mineralising matrix; demonstrative of osteoid production, and its subsequent mineralisation to bone-like tissue. Polymerase chain reaction (qPCR) analysis showed distinct changes in gene expression over a 12-week culture period, indicative of maturing osteoblasts and osteocytogenesis. Immunofluorescent (IF) staining and ELISAs further supported these findings, confirming the presence of podoplanin (PDPN) (an early osteocytic marker), as well as prolonged cell viability and activity. qPCR, ELISAs and IF staining analysis also showed that SSBM cultures were responsive to the presence to each individual BFM.
Summary: A versatile, simple, and humanised osteocyte 3D culture method, with an optimised analytical pipeline, has been established. Confirmation of their responsivity to BFMs endorses the feasibility of using the method as a high-throughput assayable platform for bone research and drug development. Preliminary steps have been taken to assess the potential of 3D bioprinting to produce such a platform and its development has the potential to make a significant impact on how bone research is approached, as well as significantly reducing and replacing the reliance and use of animals in this research field.
Type of Work: | Thesis (Doctorates > Ph.D.) | |||||||||
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Award Type: | Doctorates > Ph.D. | |||||||||
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Licence: | All rights reserved | |||||||||
College/Faculty: | Colleges (2008 onwards) > College of Medical & Dental Sciences | |||||||||
School or Department: | Institute of Inflammation and Ageing | |||||||||
Funders: | Other | |||||||||
Other Funders: | NC3Rs | |||||||||
Subjects: | Q Science > QP Physiology | |||||||||
URI: | http://etheses.bham.ac.uk/id/eprint/14343 |
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