Effect of Cellular Positional Identity on Bone Regenerative Capacity for Tissue Engineering
The aim of this study was to investigate the stability of positional identity markers and phenotypic differences in isolated osteoblasts from distinct anatomic regions. In addition, the ability of heterotypic co-cultures to reprogramme site-specific Hoxa gene expression also tested. Rat osteoblastic cells from femurs and calvariae were harvested as matched pairs of cultures from 4 male rats. Cells were expanded extensively in medium supplemented with FGF-2, and were shown to maintain their osteoblastic phenotype as characterised by alkaline phosphatase (ALP) staining, osteopontin (OPN), osteocalcin (OCN) expression and osteoblast-associated gene expression in long term culture. Gene expression of cells was determined by quantitative RT-PCR. Differences in Hoxa gene expression as markers of positional identity were maintained for up to at least 10 passages, with calvarial cells remaining Hoxa-ve throughout. The transcription factors Msx2 and Irx5 were consistently more highly expressed in calvarial cells, whereas Tbx3 expression was elevated in femoral cells. Expression of the osteoblast-associated genes Bglap and Sppl were elevated in femoral cells, and also associated with increased osteopontin secretion and bone nodule formation. Runx2 was elevated in calvarial cells. Cells were also pre-labelled with fluorescent vital staining and co-cultured for 7 days prior to separating by fluorescence activated cell sorter to investigate the possibility of re-programming of Hoxa negative cells by direct contact with Hoxa +ve cells. However no evidence was seen of modulation of positional identity genes and phenotypes in these heterotypic cultures. In conclusion, the results demonstrate persistence of expression of positional identity gene markers and phenotypic differences between femoral and calvarial osteoblasts for prolonged periods in culture. These data suggest that these differences in regionally defined osteoblasts are inherently programmed in the cells as a result of their embryological position. The results may have considerable implications when considering the transplantation of autologous cells in tissue engineering.
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