Bisphosphonate Enhances Osteogenic Differentiation of Pulp and Periodontal Cells

Bisphosphonate prevents bone loss by blocking osteoclast mediated bone degradation. Bisphosphonate treatment is associated with osteonecrosis of the jaws. However, its effect on cells that comprise jaw and teeth remains unclear. Objective: To evaluate the effect of Bisphosphonate on osteogenic capacity of Pulp and Periodontal (PDL) fibroblasts. Material and Method: Pulp and PDL tissue was isolated from non infected, impacted third molars of healthy individuals. Isolated cells were cultured in various concentration of bisphosphonate to check viability, growth and capacity to differentiate toward osteoblast lineage. Cytochemical, RT-PCR, western blot and in situ immunoflourescence analyses were used to determine expression of osteoblast genes and mineral deposition. Results: Pulp cells exhibited a 10-fold increased sensitivity to oral forms of bisphosphonate and an early susceptibility to the IV form of bisphosphonate when compared to PDL cells. Proliferation of both PDL and pulp cells was inhibited by bisphosphonate. Osteoblast master regulator Runx2 was expressed at higher levels (2-fold) in untreated PDL cells relative to pulp cells. Both PDL and pulp cells differentiated to osteoblasts when cultured in osteogenic media. Progressive increases in alkaline phosphatase activity (an early osteoblast marker), matrix formation (alcian blue staining) and mineral deposition (Von Kossa staining) were observed over 5 week time period. Bisphosphonate treatment of pulp and PDL cells resulted in enhanced osteoblast differentiation. To identify how bisphosphonate stimulated osteoblast differentiation RT-PCR was performed. At earlier time points, expression of Runx2 and Osterix, the two essential regulators of osteoblast differentiation was increased 2-fold. This coincided with increased expression of ALP, Osteopontin and Osteocalcin. Immunofluorscence studies demonstrated that bisphosphonate treatment did not alter the nuclear distribution of Runx2 or its association with splicing factor SC35 in PDL or pulp cells. Conclusion: Bisphosphonate induces expression of Runx2 and OSX to promote osteogenesis and cell differentiation of pulp and PDL cells. R01AG030228