Background/objective: Annatto-derived tocotrienol (AnTT) contains 90% δ-tocotrienol and 10% γ-tocotrienol, and have been shown to improve bone formation, structure and strength in animal models of osteoporosis. In this study, we aimed to elucidate the effects of AnTT on proliferation and differentiation of osteoblasts using murine preosteoblast cells, MC3T3-E1.
Methods: MC3T3-E1 cells were plated (1x104 cells/mL) and incubated overnight in growth media (α-MEM + 10% bovine serum). At day 0, the cells were cultured in osteoblast differentiation media (ODM; growth media + 3 mM sodium phosphate + 50 µg/mL ascorbic acid) and treated with various concentrations of AnTT (0.001 – 20 µg/mL). Cell viability and cell proliferation were measured after 1 day, 3 days and 6 days of AnTT treatment by MTS and BrdU assays respectively. Expression of bone formation-related genes (collagen1α1, alkaline phosphatase, osteocalcin) were measured. RNA extraction was carried out after every time-point (3 - 24 days) of AnTT treatment and Real-time PCR was performed using iQ SYBR Green kit after RNA was converted into cDNA.
Results: For cell viability and cell proliferation, AnTT treatment for all time points didn’t show any significant difference compared to control. 4 doses (0.001, 0.01, 0.1 and 1 µg/mL) was selected to be used in the gene expression studies. Col1α1 and ALP gene expression (early markers of osteoblastogenesis), were significantly increased by AnTT at day 6 compared to control and reached maximum level at day 9 for col1α1 gene and at day 12 for ALP gene. OPN gene expression (late marker of osteoblastogenesis) was significantly increased from day 9 until day 21 in AnTT-treated groups compared to control.
Conclusion: In conclusion, annatto tocotrienol showed no effect on osteoblast proliferation but enhanced osteoblast differentiation in preosteoblast cells by increasing the expression of osteoblast differentiation genes; col1α1, ALP and OPN.