Characterisation of the intimate relationship between collagen and mineral during skeletal biomineralisation.
Lead Research Organisation:
University of Edinburgh
Department Name: The Roslin Institute
Abstract
The development and growth of the skeleton depends on the integration of events directing cells derived from the primitive mesenchyme to produce cartilage and bone matrix, and to mineralise and remodel that matrix. Bone is a composite material made up of mostly collagen and mineral whose combined material properties make bone strong and tough, providing rigidity and resistance to fracture. Changes in the composition and organisation of these building blocks affect bone's mechanical integrity and can lead to some diseases of great public health concern, e.g. osteoarthritis, rickets and osteoporosis. Understanding the mineralisation process better will also allow a better understanding of soft tissue ossification abnormalities such as arterial calcification. The initiation of the first mineral crystals with the collagenous extracellular matrix is pivotal for ensuring that mineralisation progresses physiologically but unfortunately there are significant gaps in our knowledge on how this process occurs and is regulated. Therefore, this inter-disciplinary studentship will use various biology and chemistry approaches to determine how a protein (PHOSPHO1) essential for bone formation, regulates bone mineralisation and the establishment of the intimate relationship between the collagen and mineral phases. Ultimately this studentship will tell us how PHOSPHO1 regulates fibrillar collagen mineralisation.
Organisations
People |
ORCID iD |
| Colin Farquharson (Primary Supervisor) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M010996/1 | 01/10/2015 | 31/03/2024 | |||
| 1803936 | Studentship | BB/M010996/1 | 01/09/2016 | 31/08/2020 |
| Description | To date this studentship has generated new knowledge surrounding the biology of the PHOSPHO1 phosphatase (encoded by the Phospho1 gene) and its function in bone mineralisation during skeletal development. I have established that gene expression of Phospho1 and other phosphatases including Alpl (tissue non-specific alkaline phosphatase; TNAP) are correlated during skeletal development, and this coincides with changes in the expression of other mineralisation-related proteins. Both PHOSPHO1 and TNAP are expressed at sites which are actively mineralising during embryonic development and they are found in close proximity at the membranes of cells at these regions. When PHOSPHO1 is genetically knocked-out, mineralisation of the developing skeleton is significantly reduced. I have examined these changes at the smallest scales using powerful electron microscopy and shown that this is due to an accumulation of small extracellular vesicles which, in the wild-type, are the initial locus of mineral formation. PHOSPHO1 is thought to reside inside these matrix vesicles and generate phosphate ions which are used to make mineral crystals. In the absence of PHOSPHO1 therefore mineral formation is much slower. It is currently unknown how PHOSPHO1 substrates are generated inside matrix vesicles. I have introduced and validated a primary cell culture model of matrix vesicle production and isolation to our lab in order to investigate this question. I have so far established that the number of vesicles released by stimulated cells increases over time and contain both PHOSPHO1 and TNAP. Current studies are aiming to investigate the biochemical pathway upstream of PHOSPHO1 using proteomic, lippidomic and metabolimic approaches. These data will be combined with cryo-transmission electron microscopy of vesicle isolates to examine changes in the events leading to vesicular mineralisation with and without PHOSPHO1 present. In collaboration with Prof Nicholas Morton (Queen's Medical Research Institute, University of Edinburgh), we have also attempted to validate potential upstream candidates, namely ectonucleotide pyrophosphatase phosphodiesterase 6 (ENPP6). We found that mice which were genetically ablated for ENPP6 exhibited mildly hypomineralised bones in young animals, which subsequently recovered over time. Futhermore, ENPP6 was expressed in mature osteoblasts at mineralising sites in vivo, and was upregulated over a mineralising time course in isolated osteoblasts in vitro. This provides evidence that ENPP6 may act upstream of PHOSPHO1 to provide its substrates, but this is likely not the only pathway through which this occurs. To further interrogate the biochemistry of MVs a primary osteoblast cell culture model of MV generation and isolation was characterised relative to the more commonly used MC3T3 cell line. Primary osteoblasts generated vesicles were largely consistent with those from MC3T3s and contained both PHOSPHO1 and TNAP. These data confirm that primary osteoblasts represent a suitable model for the investigation of MVs. This model was further used to characterise the protein and lipid cargo of MVs to investigate both their biogenesis and to examine whether the biochemical mechanism hypothesised to deliver PHOSPHO1 substrates within vesicles, and therefore the generation of intravesicular phosphate, is disrupted in its absence. Proteomics data implicated the role of depolymerisation in the osteoblast cytoskeleton during the release of MVs into the extracellular matrix, and also indicated that vesicle biogenesis may be mediated by GTPase signalling. Lipidomic analysis of wild-type and Phospho1-null vesicles furthermore revealed perturbations in the pattern of lipids present in the absence of PHOSPHO1. |
| Exploitation Route | These studies surrounding the function of the PHOSPHO1 phosphatase have elucidated aspects of its basic biology and in doing so have opened potential therapeutic avenues for correcting a lack of mineralisation across a range of musculoskeletal conditions. Before treatments are realised however, further research is needed to fully explicate the PHOSPHO1 pathway of phosphate generation, and how it interacts synergistically with other mechanisms at play within and around the matrix vesicle. Interventions in these processes may be of interest in the healthcare and pharmaceutical sectors. |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | A tale of two phosphatases: How do matrix vesicles generate phosphate for healthy bone mineralisation? |
| Amount | £17,684 (GBP) |
| Funding ID | M847 |
| Organisation | Rosetrees Trust |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 06/2019 |
| End | 05/2020 |
| Description | Biochemical Society Sponsored Event Grant |
| Amount | £300 (GBP) |
| Organisation | Biochemical Society |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 12/2018 |
| End | 12/2018 |
| Description | Bone Research Society Sponsored Event |
| Amount | £500 (GBP) |
| Organisation | Bone Research Society |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 12/2018 |
| End | 12/2018 |
| Description | Roslin Institute Early Career Grant |
| Amount | £1,414 (GBP) |
| Organisation | University of Edinburgh |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 01/2019 |
| End | 07/2019 |
| Description | Anatomy Nights: Matters of the Heart - public anatomy outreach workshop |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Public/other audiences |
| Results and Impact | Anatomy Nights: Matters of the Heart is an international anatomy outreach event which aims to bring anatomists to the general public. I ran the Edinburgh event in 2020 which involved a short talk and pig heart dissection, followed by discussion of veterinary specimens and histology of the heart. ~35 members of the public attended including interested adults, school teachers, and an entire biology class from Inverness. Many of the participants reported an increased appreciation of and interest in the heart and anatomy in general. A large proportion of the school students who attended reported an increased interest in studying anatomy or biomedical sciences/medicine at University. |
| Year(s) Of Engagement Activity | 2020 |
| Description | Easter Bush Campus Open Day |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Public/other audiences |
| Results and Impact | We participated in the Easter Bush Campus Open Day 2017 which attracted hundreds of visitors across all age groups. Our activities surrounding bone biology provoked lots of questions and discussion, especially around musculoskeletal diseases of ageing such as osteoporosis and osteoarthritis. |
| Year(s) Of Engagement Activity | 2017 |
| URL | https://www.ed.ac.uk/roslin/news-events/archive/2017/easter-bush-campus-open-day |
| Description | Pint of Science Festival |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Public/other audiences |
| Results and Impact | Pint of Science is an international science festival which aims to bring research scientists to the public in an informal setting. I worked as an Event Manager with the Edinburgh team over two years which co-ordinated 36 events over 12 venues across the city in each year. These ranged from those focussed on life sciences to societal issues. |
| Year(s) Of Engagement Activity | 2018,2019 |
| Description | Science Insights Sessions |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Schools |
| Results and Impact | Students from local high schools were invited into our lab to do some hands-on bone histology, including staining and light microscopy, over two afternoons. The small group sizes enabled in-depth and wide-ranging discussions about the science, along with an exploration of career paths into science. |
| Year(s) Of Engagement Activity | 2017,2018 |
| URL | https://www.ed.ac.uk/medicine-vet-medicine/outreach/science-insights |