In vitro assay for osteoinductive activity of
different demineralized freeze-dried bone

Shahram Vaziri1, Surena Vahabi1, Maryam Torshabi2, Somayeh Hematzadeh3,*
1Department of Periodontology, Dental school, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2Department of Dental Material, Dental school, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3Department of Periodontology, Dental school, Qazvin University of Medical Sciences, Qazvin, Iran
Purpose: Various bone graft materials have been used for periodontal tissue regeneration. Demineralized freeze-dried bone
allograft (DFDBA) is a widely used bone substitute. The current widespread use of DFDBA is based on its potential osteoinductive
ability. Due to the lack of verifiable data, the purpose of this study was to assess the osteoinductive activity of different
DFDBAs in vitro.
Methods: Sarcoma osteogenic (SaOS-2) cells (human osteoblast-like cells) were exposed to 8 mg/mL and 16 mg/mL concentrations
of three commercial types of DFDBA: Osseo+, AlloOss, and Cenobone. The effect of these materials on cell proliferation
was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The osteoinductive ability
was evaluated using alizarin red staining, and the results were confirmed by evaluating osteogenic gene expression using reverse
transcription polymerase chain reaction (RT-PCR).
Results: In the SaOS-2 cells, an 8 mg/mL concentration of Osseo+ and Cenobone significantly increased cell proliferation in
48 hours after exposure (P<0.001); however, in these two bone materials, the proliferation of cells was significantly decreased
after 48 hours of exposure with a 16 mg/mL concentration (P<0.001). The alizarin red staining results demonstrated that the
16 mg/mL concentration of all three tested DFDBA induced complete morphologic differentiation and mineralized nodule
production of the SaOS-2 cells. The RT-PCR results revealed osteopontin gene expression at a 16 mg/mL concentration of all
three test groups, but not at an 8 mg/mL concentration.
Conclusions: These commercial types of DFDBA are capable of decreasing proliferation and increasing osteogenic differentiation
of the SaOS-2 cell line and have osteoinductive activity in vitro.