Hashim, Nor Munira
(2018)
Differentiation and proliferation activities of human foetal osteoblast cell line (hFOB 1.19) treated with a polyphenol, tannic acid alone or in combination with pamidronate.
Masters thesis, Universiti Sains Malaysia.
Abstract
Bone continuously undergoes bone remodelling and imbalance in bone remodelling leads to bone pathogenesis, such as osteoporosis. Tannic acid (TA) is a polyphenol with antioxidant properties which may elevate osteoblast metabolism. Synergism is one of pharmacological effect that occur when drug combination is used. Hence, the aim of this study was to evaluate the effect of TA alone and combination of TA and pamidronate on differentiation, proliferation, mineralisation as well as morphology of human foetal osteoblast cells (hFOB 1.19). The hFOB 1.19 cells were treated with TA alone and combination of TA and pamidronate of different combination ratios at different concentrations (0.1 to 99.0 μg/ml). Half maximal effective concentration (EC50) for TA alone, pamidronate alone and different combination ratios of TA and pamidronate (25:75, 50:50, 75:25) were measured by using MTT assay. Analysis of synergistic effects was also performed on the different combination ratios based on Combination Index (CI) where CI < 1: synergism, CI = 1: additive effect and CI > 1: antagonism. The combination treatment with the lowest EC50 and CI<1 was then chosen for the next studies. Cell proliferation assay was conducted to compare the cell viability and morphology of hFOB 1.19 cells was observed by using inverted microscope after treatment from day 1 to day 7. In addition, detection of calcium and phosphate deposits were done by histochemical staining. Detection of bone specific genes [bone sialoprotein (BSP) and osterix (Osx)] was conducted by polymerase chain reaction (PCR) test. The EC50 of hFOB 1.19 cellstreated with TA alone (0.56 ± 0.22 μg/ml) and 75:25 ratio of TA and pamidronate (0.48 ± 0.03 μg/ml) were more effective compared to pamidronate alone (15.27 ± 0.22 μg/ml) and other combination ratios treatments [(25:75; 3.80 ± 0.13 μg/ml); (50:50; 2.25 ± 0.11 μg/ml)]. Analysis of synergistic result showed that only 75:25 combination ratio exhibited synergistic effect while the other two combination ratios manifested antagonistic effects. All groups showed significant different when compared between day 1 and day 7. However, all groups except combination group manifested significant different when comparing between day 3 and 7. When comparing on similar day which was day 7, both TA alone and pamidronate alone treated groups showed significantly increased in cell proliferation compared to control (untreated) and combination groups. The morphology of hFOB 1.19 cells treated with both TA and combination groups had uniform and elongated shape until the end of study. Treatment with TA alone demonstrated higher production of calcium and phosphate deposits compared to untreated control, pamidronate alone and combination groups. All groups manifested the expression of BSP and Osx until day 7 of treatment periods. In conclusion, TA alone rather than combination treatment of TA and pamidronate has greater ability in enhancing cell proliferation and morphology besides improved osteoblast mineralisation.
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