J Periodontal Implant Sci.  2012 Dec;42(6):224-230.

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

• Vaziri S
• Vahabi S
• Torshabi M
• Hematzadeh S

Abstract

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.

Keyword

Alizarin red; Bone graft; Cell differentiation; Cell proliferation; Regeneration

MeSH Terms

Anthraquinones
Bone Substitutes
Cell Differentiation
Cell Line
Cell Proliferation
Durapatite
Gene Expression
Osteopontin
Polymerase Chain Reaction
Regeneration
Reverse Transcription
Sarcoma
Transplantation, Homologous
Transplants
Anthraquinones
Bone Substitutes
Durapatite
Osteopontin

Figure

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  Figure 1 The effect of A, B and C test groups on saos-2 cell viability and proliferation. Cells were treated with 8 mg/mL (A) and 16 mg/mL (B) concentration of test groups. After 24 and 48 hours, the cell viability and proliferation were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results are presented as mean±standard deviation (SD) of three independent experiments (n=3; a)p<0.001, b)P<0.01).

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  Figure 2 The effect of test groups A, B and C on bone mineralization in sarcoma osteogenic (SaOS-2) cell. Optical image of alizarin red S in SaOS-2 osteoblast-like cells for day 5 after treatment with 8 mg/mL and 16 mg/mL concentration of test groups.

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  Figure 3 The effect of A, B and C test groups on expression of osteogenic markers in sarcoma osteogenic (SaOS-2) cells. SaOS-2 cells were treated with 8 mg/mL (A) and 16 mg/mL (B) concentration of test groups. The mRNA expression of 3 osteogenic markers was evaluated 3 and 5 day after treatment and compared with positive (differentiated cells) and negative control (undifferentiated cells). β-actin was used as internal control. OP: osteopontin, OC: osteocalcin, ON: osteonectin.

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