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Journal of Biological Sciences

Year: 2008 | Volume: 8 | Issue: 3 | Page No.: 506-516
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Authors

Z.A. Intan Zarina

Z.A. Shahrul Hisham

M.A.W. Rohaya

S. Sahidan

Z.A. Zaidah

Keywords

The aim of this study is to differentiate peripheral blood mononucleated cells that have been isolated from ICR mice`s (Mus musculus)peripheral blood into mature osteoblast and osteoclast cells with addition of growth factors. The mononucleated cells were isolated by density centrifugation using Ficoll-PaqueTM Plus. The culture mediums were then supplemented with growth factors; 50 ng mL-1 RANKL and 25 ng mL-1 M-CSF to differentiate into osteoclast cells. On the other hand, osteoblast assay, 50μg mL-1 ascorbic acid and 10 mM β-glycerophosphate were added to support differentiation. For control, the same cells were used without supplementation of respective growth factors. Biochemical analysis for osteoblast, i.e., Alkaline Phosphatase (ALP) activity was determined on day 3-14. The results showed that the ALP activity in the differentiation medium is significantly increased (p<0.05) on day 10 and day 14 as compared to control, i.e., cells without growth factors. Tartrate Resistant Acid Phosphatase (TRAP) activity that represented as osteoclast biomarker also showed significant increased (p<0.05) from day 3 until day 10 in the present of RANKL and M-CSF. The viability of differentiated cells also showed that the cells were able to survive until 10 to 14 days in the presence of respective growth factors without significant increased in respective differentiation medium. RT-PCR analysis on isolated RNA from mononucleated cells after 14 and 10 days in their differentiation medium showed that the osteoblast and osteoclast were expressing ALP (~373 bp) and TRAP (~281 bp) gene, respectively. Mononucleated cells originated from peripheral blood have the potential to differentiate into osteoblast and osteoclast cells in the presence of specific growth factors. The respective cells are primitive enough to differentiate into two distinct types of mature cells hence can be categorized as multipotent stem cells.
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