Stability And Reprogramming Of Hoxa Expression In Regionally Distinct Osteoblasts

Methods: Primary rat osteoblast cultures from 4 matched calvariae and femurs were established by cell explantation and collagenase digestion. Cells were expanded in medium supplemented with FGF-2 and characterised by Alkaline phosphatase (ALP) staining and Osteocalcin (OCN) expression. CellTracker™ Green and Red fluorescent dyes were used to label calvarial and femoral cells respectively for subsequent sorting by flow cytometry. Hoxa gene expression was assessed by qRT-PCR (Taqman). Results: In monolayer culture, osteoblasts derived from femurs consistently showed persisting high levels of expression of Hoxa7 and lower expression levels of Hoxa4, Hoxa5, Hoxa10, and Hoxa13. Calvarial osteoblasts showed noHoxa gene expression when tested at passage 5 and passage 10.

However when Hoxa –ve calvarial and Hoxa +ve femoral osteoblasts were grown in co-culture for periods of up to 7 days and subsequently sorted by flow cytometry, calvarial osteoblasts progressively switched to express high levels of Hoxa7 and also expressed Hoxa4, Hoxa5, Hoxa10, and Hoxa13. This Hoxa expression persisted in previously Hoxa –ve calvarial cells for at least 2 passages after co-cultures.

Conclusion: The pattern of Hoxa gene expression in adult primary osteoblasts was consistent with their regional origins and persisted in culture up to passage 10, but co-culture induced Hoxa expression in calvarial cells. These observations require further functional investigation to understand the implications for bone grafting and tissue engineering procedures.

This work is supported in part by Guy's & St. Thomas' NHS Foundation Trust and Thai Government Science and Technology Scholarship to S. Prajaneh.