Date of Award
MCh by research (Master of Surgery by research)
Dr Garry Duffy
Bone grafts are required in the treatment of many different orthopaedic and rheumatological pathologies and worldwide bone is the second most commonly transplanted material after blood products. This thesis looks at tissue-engineered alternatives to the traditional bone autografts and allografts, particularly methods of upregulating human cells to produce new bone.
Recently ephrinB2, a human cell-surface ligand, and its receptor, EphB4, have been shown to play a role in bone growth and remodeling. This thesis looks at the potential of ephrinB2 ligand overexpression to upregulate osteogenesis by human Mesenchymal Stem Cells (hMSCs). Polyethyleneimine (PEI) is well-established nonviral, transfection vector and thus a possible method of overexpressing ephrinB2 ligand in hMSCs. Gene Activated Matrices (GAMs) consisting of a suitable matrix or scaffold, carrying genes and a transfection vector designed to upregulate osteogenesis are a combination of tissue engineering and gene therapy.
Transfection efficiency of ephrinB2 plasmid using PEI on mono layer of hMSCs proved adequate to study the effects of ephrinB2 ligand overexpression. This was then repeated on a 3D GAM using a collagen-nano-hydroxyapatate(nHa) scaffold, this also resulted in significant overexpression of ephrinB2 by hMSCs seeded on the scaffold. Cells overexpressing ephrinB2 in both the monolayer and on the GAMs resulted in a relative increase in calcium deposition.
To further investigate the molecular mechanism involved downstream of the ephrinB2-EphB4 cell surface interaction qRT-PCR was carried out. . Two genes known to be related to osteogenic differentiation, Osterix and Dlx5, were elevated in cells overexpressing ephrinB2. However the extensively studied, genes BMP2 or Runx2 demonstrated no change in activation in the transfected cells indicating the increased ossification observed with ephrinB2 overexpression is independent of this traditional pathway.
To assess the transfection efficiency and safety of GAMs in vivo using an established animal model Green Fluorescent Protein was used in place of ephrinB2. This involved implanting GAMs consisting of a collagen-nHa scaffold carrying PEI-GFP polypexes into a critical defect in rat calvarium. This GAM relied on migration of native rat cells onto it in order to be transfected with GFP. After only 7 days in vivo there was evidence of significant cell migration onto the GAM and transfection of these cells to express GFP. This demonstrated the ability of native cells to migrate onto the GAM along with transfection efficiency of PEI of those cells. This in vivo is ideal for further assessment of ephrinB2 over expression along with other osteogenic cell signals.
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McSorley K. Osteogenic Potential and Safety of ephrinB2 transfected with Polyethyleneimine Gene Activated Matrices for Use in Bone Tissue Engineering [MCh Thesis]. Dublin: Royal College of Surgeons in Ireland; 2011.