Abdul Qader Al-Kukasih, Sarah Talib
(2015)
The effects of biphasic calcium phosphate and simvastatin on human dental pulp cells in vitro on dentin regeneration.
Masters thesis, Universiti Sains Malaysia.
Abstract
This study was conducted to prepare an appropriate biphasic calcium phosphate (BCP)
scaffold in combination with optimal concentration of simvastatin to induce human
dental pulp cells differentiation and dentin tissue regeneration. BCP scaffold of 20/80
hydroxyapatite (HA) to β-tricalcium phosphate (β-TCP) ratio with micropores < 10
μm, macropores of 300 μm, and porosity of 65 % was successfully synthesized using
wet precipitation method and polyethylene microspheres as pore-creating agents. The
scaffold was characterized using x-ray diffraction (XRD), fourier transform infra-red
spectroscopy (FTIR), x-ray fluorescence (XRF), field emission scanning electron
microscope (FESEM), linear shrinkage, and total porosity measurements. BCP sample
extract was prepared and combined with four different concentrations of simvastatin
(2.0, 1.5, 1.0 and 0.5 μM) for the assessment of cell viability using MTT (3-(4,5-
dimethyl-thiazoyl)-2,5-diphenyl-tetrazolium bromide) assay. The alkaline phosphatase
activity was assessed using alkaline phosphatase assay. The groups which showed the
best cell viability and alkaline phosphatase activity were selected. They were assessed
for both odontogenic differentiation potential of collagen type I alpha 1 (COL1A1),
bone sialoprotein (BSP), dentin matrix protein-1 (DMP-1), dentin sialophosphoprotein
(DSPP), and Runt-related transcription factor-2 (RUNX-2) genes expression analysis
using reverse transcription-polymerase chain reaction (RT-PCR) and extracellular
matrix mineralization detection using Alizarin Red S staining. The results showed that
combination groups of BCP +1.5 μM, BCP + 1.0 μM, and BCP + 0.5 μM had higher
mean cell viability index, except BCP + 2.0 μM that showed cytotoxicity. For alkaline
phosphatase activity, the combination groups of BCP + 1.5 μM, BCP +1.0 μM, and
BCP + 0.5 μM showed higher mean alkaline phosphatase activity index with BCP +
1.5 μM the highest. For the odontogenic differentiation potential, BCP + 1.5 μM
combination group showed up-regulation of COL1A1, DMP-1, BSP, and DSPP genes
and down-regulation of RUNX-2 gene. For the extracellular matrix mineralization,
BCP + 1.5 μM combination group showed the highest ability to induce the mineral
deposition. In conclusion, the combination of BCP and 1.5 μM simvastatin achieved
together a preferable induction of human dental pulp cells differentiation toward
dentin tissue regeneration.
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