An analysis of craniosynostotic ooprogenitor cells and their potential for bone tissue engineering.
Doctoral thesis, UCL (University College London).
The limited availability of autologous bone for grafting post-surgical defects in patients with craniosynostosis, necessitates the use of alloplastic materials which are prone to rejection or infection. The use of bio-composite materials consisting of alloplastic bioabsorbable scaffolds and autologous osteoprogenitor cells to avoid rejection, could provide a valuable approach to the repair of critical bone defects in the skull. However, in patients with syndromic craniosynostosis, autologous osteoprogenitors carry the mutations which cause premature ossification and consequent malformations requiring cranioplasty. The aim of this study was to develop strategies for growing effectively mutated osteoprogenitors with the view to developing autologous therapies for patients with craniosynostosis. To this purpose, the behaviour of mouse osteoprogenitor cell lines carrying either a common human mutation (FGFR2-C278F) for syndromic craniosynostosis or the human wild type FGFR2 (WT-FGFR2) was investigated. In particular the focus was on i) how the mutation affected proliferation and osteogenisis; ii) the effect of exogenous fibroblast growth factor (FGF) 18 on cell growth and morphology; iii) analysis of osteoprogenitor attachment and growth on bioabsorbable scaffolds uncoated or coated with extracellular matrix molecules (fibronectin, laminin). RT-PCR analysis of cells carrying the FGFR2-C278F mutation and wild type FGFR2 cells, revealed differential expression of FGF18 and molecules important in the terminal differentiation of mineralising osteoblasts: Osteocalcin was expressed at greater levels in cells carrying the mutation compared to wild type cells at 1.8 fold (p < 0.01) and 1.4 fold (p = 0.02) in pre-confluent and day 5 confluent cells respectively. Wild type cells expressed alkaline phosphatase from day 5 compared to mutated cells which did not express until day 30. Most striking was the greater expression of FGF18 in the mutated cells; 1.8 fold (p < 0.01) at pre confluence, 1.3 fold (p = 0.01) at day 5 and 1.5 fold (p < 0.01) at day 30. In the early phase of cell cultures, cells carrying the mutation demonstrated higher mitotic activity than wild type cells as determined by p-H3 staining (18.6% +ve and 11.2% +ve respectively; p < 0.01, day 2 post plating). However, mutated cells demonstrated altered attachment behaviour, clustering forming early nodules and did not reach full confluence. Subsequent treatment of mutated cells with FGF18 [10-9M] appeared to allow cells to reach confluence and “rescue” the phenotype. A concentration dependent increase in cellular mitosis for all cell lines was observed. This was significantly greater at day 1 in mutated cells compared to wild type cells (51% and 29% respectively; p <0.01 at FGF18 [10-9M], and 60% and 43% respectively; p < 0.01 at FGF18[10-8M]). Attachment and growth of osteoblasts to a commercially available, clinically licensed scaffold could be enhanced by modification of the surface with the extracellular matrix (ECM) molecules, fibronectin and laminin. Both substrates significantly enhanced attachment at all time points up to 48 hours post plating (e.g. C278F cells at 1 hour post plating: 10,020 cells [-ve]; 37,600 [fibronectin]; 41,670 [laminin]; p < 0.01). These findings indicated that the use of osteoprogenitor cells carrying mutations could present a feasible strategy in bone tissue engineering, which warrants further development towards the overall multidisciplinary approach in the management of these complex clinical challenges.
|Title:||An analysis of craniosynostotic ooprogenitor cells and their potential for bone tissue engineering|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Population Health Sciences > Institute of Child Health > Department of Neurosciences and Mental Health > ICH - Developmental Biology Unit|
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