Methods of characterising the process of early biomineralisation

McGuinness, Adam John Anthony ORCID: 0000-0003-4659-9648 (2024). Methods of characterising the process of early biomineralisation. University of Birmingham. Ph.D.

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Abstract

Mature bone is a highly ordered structure composed of hydroxyapatite crystals uniformly arranged within regularly spaced fibrils of collagen, populated with several cell types. This arrangement produces a material with a unique combination of strength and resistance to fracture, as well as the ability to remodel, repair and regenerate in response to injury or changing load. Intrafibrillar mineralisation present in mature bone is a thermodynamically unfavourable process, suggesting highly controlled processes during early mineralisation, current knowledge of which is poorly understood. Evidence suggests extracellular vesicles (EVs) are involved in this early mineralisation process, potentially acting as a nucleating and stabilising agent in mineralisation, but the heterogeneity and poor characterisation of these nanoparticles limits the understanding of their role.

Whilst the heterogeneity of EVs as a population is unquestioned, identification of the presence or absence of subgroups within these populations has not been adequately investigated. Interrogation of individual EVs would allow the construction of a highly resolved map of EV characteristics. Collecting this data is the first step in identifying distinct populations within the EV secretome. From there, identification of the impact of these distinct populations and their attendant molecular characteristics, upon mineralisation would be a significant boost to therapeutic treatments for mineralisation abnormalities, such as osteoporosis, non-union fractures, and heterotopic ossification.

This thesis investigates new techniques for investigating the biochemical and physical characterisation of EVs, as well as the early mineralisation stages of osteoblast like cells, and the role of EVs therein. Here we apply the methods of x ray fluorescence (XRF), Raman spectroscopy and transmission electron microscopy (TEM) to mineralising cell cultures and EVs to elucidate the processes and characteristics of the early mineralisation process.

We utilise XRF to detect the presence of elemental ions in control compounds, with signal strength reliably corresponding to the proportion of elements present. When applied to mineralising cell cultures, a changing elemental composition across time points is observed. We have documented the presence of a calcium deficient, phosphorous dense material in early mineralisation, maturing towards an increased calcium presence in later time points. This suggests intermediate mineral phases are present in mineralising bone prior to the hydroxyapatite present in mature tissue. XRF was also used to quantify characteristics of mineralising cell culture, using automated image analysis to identify mineral nodule number and size, and observe the rapid mineralisation induced by coculture of osteoblasts with EVs.

In addition to these results, we have shown the correlation of Raman signal with the mineral content of bone, making it another potentially useful tool in the investigation of mineralisation. Attempts to incorporate molecular tweezers with Raman spectroscopy for the characterisation of individual osteoblast EVs were ultimately unsuccessful, however, lessons were identified for future application of both techniques.

Finally, improvements to current TEM techniques of EV imaging were demonstrated in this thesis, using gold nanoparticles to label exosome associated surface proteins, and a methylcellulose supporting film to maintain a spherical morphology.

The findings presented here offer insight into the early mineralisation process, as well as developing techniques to explore these findings further. Clarification of potential intermediate mineral stages can be further explored using the XRF and Raman spectroscopy techniques developed in this manuscript. The TEM protocols developed here may provide a more physiological insight into EVs, whilst the labelling process can be used to identify the presence of specific proteins. Automated imaging analysis techniques may be useful for high throughput imaging systems, identifying the impact of drugs or changes in conditions (e.g. hypoxia) upon mineralisation progression. Together, these techniques and results contribute to the current and future understanding of early mineralisation and may one day be exploited to improve medical outcomes for implants, cements, and diseases such as osteogenesis imperfecta.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):