Abstract
Objective
To assess the effect of fibronectin (Fn) and porcine type I collagen (PCOL) on odontoblast-like cells in vitro.
Material and Methods
Rat odontoblast-like cells (MDPC-23 cells) were inoculated and cultured on Fn-coated or type I collagen-coated substrates. Proliferation assay, alkaline phosphatase activity (ALP activity), mRNA expression of hard tissue-forming markers, and Alizarin red staining were investigated over a period of 10 days.
Results
Cells maintained a high proliferation activity on Fn and PCOL even at a low seeding concentration (0.5×104/mL) as demonstrated by CCK-8 assay. The proliferation activity of cells on Fn increases in a concentration-dependent manner while it reached a plateau after 10 µg/mL. Cells adopted long, thin and spindle shape on Fn(10-50) and PCOL. Parallel actin filaments were observed in MDPC-23 cells cultured on Fn and PCOL. ALP activity was markedly up-regulated on Fn and PCOL-coated surfaces. Importantly, gene expression of BSP (Fn10: 2.44±0.32; Fn20: 3.05±0.01; Fn30: 2.90±0.21; Fn40: 2.74±0.30; Fn50: 2.64±0.12; PCOL: 2.20±0.03) and OCN (Fn10: 2.52±0.23; Fn20: 2.28±0.24; Fn30: 2.34±0.21; Fn40: 2.34±0.25; Fn50: 2.20±0.22; PCOL: 1.56±0.16) was significantly enhanced on Fn and PCOL substrates as compared with control; moreover, expression of integrin beta 1 (ITGB1), an ubiquitous cell surface receptor was augmented in Fn(10-50) and PCOL groups simultaneously. In accordance with the ALP activity and gene expression data, calcific deposition in cells grown on Fn(10-50) and PCOL was observed as well.
Conclusion
Despite the limitation of this study, the findings indicate that a surface coating of Fn enhances the proliferation, differentiation and mineralization of odontoblast-like cells by activation of integrin beta 1 (ITG B1). The promoting effects of Fn on MDPC-23 cells were achieved at a comparatively lower coating concentration than type I collagen (300 µg/mL). Specifically, it is suggested that the optimum coating concentration of Fn to be 10 µg/mL.
Fibronectin; Odontoblasts; Cell differentiation; Cell proliferation
MDPC-23 cells were seeded to 12 well plate (non-treated tissue culture polystyrene) at the concentration of 1×104/mL in DMEM supplemented with 5% FBS. The photograph was taken 20 hours after inoculation. Cells adopted well spread and spindle shape in Fn(10-50) and PCOL-300, while those in control and Fn(0.1-1) were in round shapes. (Scale bar: 400 µm)
MDPC-23 cells in Fn(10) and PCOL-300 were well spread, F-actin formation was extensively activated in these two groups, while most of the cells grown in Fn(1) remain round in shape. (Scale bar: 200 µm)
MDPC-23 cells were seeded to 96 well plate (non-treated tissue culture polystyrene) at the concentration of 5×103/mL (Figure 5a) or 1×104/mL (Figure 5b) in DMEM supplemented with 5% FBS. Absorbance was read at wavelength of 450 nm. All data represents average±standard deviation (SD) from three separate experiments. Different symbols in each panel mean significant differences (p<0.05, Tukey’s test). OD: optical density. D1, D2, D4, D6 mean day 1, day 2, day 4, and day 6, respectively
Cells were seeded to 12 well plate (non-treated tissue culture polystyrene) at the concentration of 1×104/mL in DMEM supplemented with 5% FBS. ALP activity in all the experimental groups was enhanced, in particular, Fn(10) exhibited the highest ALP activity as compared with the other groups. All data represents average±standard deviation (SD) from three separate experiments. Different symbols mean significant differences (p<0.05, Tukey test). D5 means day 5
MDPC-23 cells were seeded to 12 well plate (non-treated tissue culture polystyrene) at the concentration of 1×104/mL in DMEM supplemented with 5% FBS. Odontogenesis related markers were significantly up-regulated in Fn and PCOL-300-treated substrates, especially, the expression of BSP and OCN was found to be markedly augmented in Fn(10-50). All data represent average±standard deviation (SD). Different symbols in each group mean significant differences (p<0.05, Tukey test). D7 means day 7
Cells were inoculated in 24 well plate (non-treated tissue culture polystyrene) at the concentration of 5×103/mL (upper) or 1×104/mL (lower). Odontogenic reagents were added in the same manner as Figure 6, and staining was conducted at 10 days of culture. Calcification nodules were clearly observed in Fn(10-50) and PCOL300 in both low and high cell seeding density