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Modelling cell connectivity in bone and its consequences for healthy ageing and regeneration.

Primary Supervisor: Dr Amy Naylor, Institute of Inflammation and Ageing

Secondary supervisor: Prof. Liam Grover

PhD project title: Modelling cell connectivity in bone and its consequences for healthy ageing and regeneration

University of Registration: University of Birmingham

Project outline:

Background: Maintenance of bone integrity is a key challenge for the ageing population and for regenerative biology. Bone is actively produced, remodelled and repaired throughout life and disruption of this process causes fragility, disability and failed regeneration.

Bone remodelling requires coordination between 3 cell-types: Osteoclasts, osteoblasts, osteocytes. The osteocyte is the master regulator of the process, coordinating the activities of all the others. Osteocytes are embedded within the bone matrix and are the longest-lived cell in the body (estimated life-span 25 years). They sense micro-damage and strain and send signals, via their extensive network of microscopic cellular extensions (canaliculi), to osteoclasts to trigger resorption of old/damaged bone tissue and to osteoblasts to repair it. The osteocyte canalicular network (OCN) allows communication between the entombed osteocytes and cells at the bone surface, allowing detection of stress, strain and damage to result in successful remodelling and repair of the tissue.

Methodology: Although osteocytes represent over 90% of cells in bone they are notoriously difficult to culture because, once isolated from the tissue, they retain phenotypic stability for just a few days. This has severely hampered interrogation of their roles and testing of drugs for bone regeneration. We have developed novel osteocyte culture methods1,2 to recapitulate osteocyte organisation and bone composition in vitro and have demonstrated that it supports osteocyte differentiation and survival for over a year (Fig.1).

Fig.1: Long-term culture of osteocytes under strain, supports osteocyte culture. A: Overview image of constructs. Over time the matrix (seeded with cells) contracts around the anchors. B-D: SEM at increasing magnification: B demonstrates mineralisation of the matrix. Scale bar 500µm; Cshows embedded osteocytes (pseudo-coloured yellow). Scale bar 30µm; D evidence of classic osteocyte morphology, with cells connected by dendrites. Scale bar 5µm.

Between the Institute of Inflammation and Ageing and the Healthcare Technologies Institute (both University of Birmingham) we have the capability to characterise construct structure from the molecules right through to the macroscopic tissue3 and are employing tissue clearing methodology and ultra-high resolution x-ray imaging (Diamond synchrotron facility) to map the OCN (Fig.2).

Fig.2: Nano-CT example (Eisenstein, UoB PhD thesis, 2017). These data can be used to construct models of the OCN, to explore how connectivity influences flow in the canaliculi and predict effects on bone healing.

Project objectives: Constructs, formed using osteocytes isolated from donor samples (healthy/unhealthy ageing and successful/failed regeneration) will be analysed using computational image analysis and network science to integrate whether and how the OCN is physically altered during the ageing process and in the context of successful compared to poor regeneration. Building on the osteocyte culture system, we will add osteoblasts and osteoclasts, allowing interactions between these key cell types to be identified and modelled and the effects of osteocyte age on connectivity and signalling to be explored. By combining osteocyte connectivity information with construct composition (micro-XRF, microCT and nano-indentation) and “omics” approaches (genomics, proteomics and metabolomics) we can model the effects of OCN changes on osteoblast mineralisation and osteoclast osteolysis, and explore their relationship to healthy regeneration and ageing. Besides enabling research into basic biological understanding of the role of osteocytes in coordinating bone remodelling, developing these co-culture platforms also supports replacement of in vivo experiments and development of high-throughput pharmaceutical screening platforms.

References:

  1. https://doi.org/10.1002/adbi.201700156
  2. https://doi.org/10.1038/s41526-021-00146-8
  3. https://doi.org/10.1002/adhm.201500617

BBSRC Strategic Research Priority: Understanding the Rules of Life: Structural Biology & Systems Biology & Integrated Understanding of Health: Ageing & Regenerative Biology

Techniques that will be undertaken during the project:

  • 3D cell and organoid culture
  • Scanning Electron Microscopy (SEM)
  • Tissue clearing (e.g. CLARITY) and imaging using light-sheet microscopy and confocal microscopy
  • Micro and nano-CT (including synchrotron imaging)
  • Network analysis (computational)
  • X-ray fluorescence imaging (XRF)
  • ‘Omics’ – e.g RNA Sequencing, metabolomics, proteomics

Contact: Dr Amy Naylor, University of Birmingham