Mechanical strain triggers differentiation of dental mesenchymal stem cells by activating osteogenesis-specific biomarkers expression

Abstract

Dental stem cell biotechnology has been used as a potential method to treat the dental diseases. This study aimed to investigate effects of mechanical stimulation on osteogenic properties of rat dental mesenchymal stem cells (DMSCs). DMSCs were isolated from rat teeth root tissues and identified by detecting vimentin and keratin expression. Flexcell FX4K tension system that mediating cyclic strain was used to treat DMSCs. MTT assay was used to observe DMSCs viability. Alkaline phosphatase (ALP) staining and alizarin red staining were conducted. Osteogenesis-specific biomarkers, such as receptor activator for nuclear factor-kB ligand (RANKL), osteoprotegerin (OPG), dentin sialoprotein (DSP) and bone sialoprotein (BSP), were evaluated using RT-PCR, western blot and immunohistochemistry assay, respectively. Positive ALP staining and alizarin red staining confirmed DMSCs phenotype. There were no significant morphology differences between mechanical stimuli treated cells and normal control cells. MTT results showed no significant differences between normal control cells and mechanically stimulated DMSCs. RT-PCR, western blot and immunohistochemistry assay indicated that 10% cyclic strain could trigger an obvious change of mRNA and protein expression of RANKL, OPG, DSP and BSP, respectively. Mechanical stimulation could trigger relative higher levels of calcium deposition in DMSCs. Mechanical strain triggered bone formation mainly through activating RANKL gene expression. In conclusion, 10% cycle mechanical strain could stimulate higher amounts of ALP and calcium deposition by activating RNAKL, and could trigger dramatically changes of mRNA and protein expression of osteogenesis-specific biomarkers, such as OPG, BSP and DSP.

Keywords: Dental mesenchymal stem cells; biomarker; mechanical strain; osteogenesis.

Figure 1

Primary culture and identification for the isolated dental mesenchymal cells (DMSCs). A. Isolated and primary cultured DMSCs cells observed under light microscopy. B. Identification for the isolated DMSCs by detecting Vimentin expression and immunohistochemistry assay. C. Identification for the isolated DMSCs by detecting Keratin expression and immunohistochemistry assay. The cells incubated with rabbit anti-Vimentin polyclonal antibody and rabbit anti-Keratin polyclonal antibody. The amplification has been illustrated in the graphs.

Figure 2

DMSCs morphology observation and the examination of DMSCs cell viability. A. DMSCs morphology observation. B. DMSCs cell viability examination by using MTT assay. C. Image of the Flexcell FX-4000 Tension System.

Figure 3

Immunohistochemistry staining for the alkaline phosphatase (ALP) and alizarin red. A. ALP staining for examining the ALP activity. B. Alizarin red staining for evaluating calcium deposition. The amplification has been illustrated in the graphs.

Figure 4

Observation for the osteogenesis specific biomarkers undergoing the effects of mechanical stimulation. The mRNA of OPG (A), RANKL (B), DSP (C) and BSP (D) were normalized to the internal control of actin mRNA expression. The statistically significant differences between two groups were illustrated in the graphs.

Figure 5

Western bolt assay results for the osteogenesis specific biomarkers. A. OPG expression and statistical analysis. B. RANKL expression and statistical analysis. C. DSP expression and statistical analysis. D. BSP expression and statistical analysis. The amplification has been illustrated in the graphs.

Figure 6

Examination for the osteogenesis-specific biomarkers by using immunohistochemistry. A. OPG, RANKL, DSP and BSP expression determination. B. Statistical analysis for biomarkers.

Figure 7

Effects of RANKL small interfering RNA (RANKL siRNA) on the alkaline phosphatase (ALP) and alizarin red. A. ALP staining for examining the ALP activity. B. Statistical analysis for ALP activity. C. Alizarin red staining for evaluating calcium deposition. D. Statistical analysis for calcium deposition. The amplification has been illustrated in the graphs.