Table 1.
Sequence of primer used in the real-time PCR.
Fig 1.
Expression of chimeric FN9-10ELP and schematic illustration of bio-functionalization for enhanced cellular responses on titanium discs.
(A) The purity and molecular weight of chimeric FN9-10ELP were measured by 12% SDS-PAGE and Western blotting. The molecular weight shown is approximately 38 kDa. (B) Bio-functionalization of titanium discs using chimeric FN9-10ELP to induce osteogenic differentiation of hMSCs.
Fig 2.
Protein attachment activity of FN9-10ELP on the titanium discs.
The titanium discs were immersed overnight in various concentrations of FN9-10ELP (0−25 μg) in 6-well plates at 4°C. The absorbance of FN9-10ELP was quantified by ELISA using a His-tag probe and reported the mean ± SD (n = 3). p< 0.001.
Fig 3.
In vitro cumulative release profiles of FN9-10ELP from titanium discs.
The titanium discs were coated with FN9-10ELP (10 μg·mL−1) and a control substance (5% BSA) in a volume of 500 μL/disc overnight at 4°C. The amount of FN9-10ELP absorbed from titanium discs was measured using an ELISA. The cumulative release was evaluated as the initial attachment amount (100%). The Release profiles indicate the mean ± SD (n = 3).
Fig 4.
Cell adhesion activity of hMSCs according to incubation time on the FN9-10ELP-titanium discs.
The titanium discs were immersed overnight in 10 μg·mL−1 of FN9-10ELP at 4°C, and while the non-coated titanium discs served as the control. The hMSCs were seeded at a density of 1 × 105 cells/disc on the titanium discs and incubated at 37°C for up to 150 min. The hMSCs adhesion activities were evaluated by crystal violet assay and are presented as the mean ± SD (n = 3). p < 0.001.
Fig 5.
Cell proliferation activity of hMSCs on the FN9-10ELP-titanium discs for 0, 4 and 8 days.
The titanium discs were immersed in 0 or 10 μg·mL−1 FN9-10ELP overnight at 4°C then, hMSCs were seeded at a density of 1 × 104 cells/disc on titanium discs and incubated for 0, 4 and 8 days at 37°C. The absorbance of formazan contained in the cells was used as a measure of cell proliferation. The cell proliferation activities are expressed as the mean ± SD (n = 3). p < 0.001.
Fig 6.
ALP activity of hMSCs on the FN9-10ELP-titanium discs for 0, 5 and 10 days.
The titanium discs were immersed in 10 μg·mL−1 FN9-10ELP overnight at 4°C, while the non-coated discs served as the control. The hMSCs were seeded at a density of 5 × 103 cells/disc and incubated for 0, 5 and 10 days at 37°C. The ALP activities were normalized to the control and are reported as the mean ± SD (n = 3). p < 0.01.
Fig 7.
Mineralization activity of hMSCs on the FN9-10ELP-titanium discs for 0, 5 and 10 days.
The titanium discs were immersed in 10 μg·mL−1 FN9-10ELP overnight at 4°C, while the non-coated discs served as the control. The hMSCs were seeded at a density of 5 × 103 cells/disc and incubated for 0, 5 and 10 days at 37°C. The mineralization activities are reported as the mean ± SD (n = 3). p < 0.05.
Fig 8.
Osteogenic differentiation activities of hMSCs on the FN9-10ELP-titanium discs for 20 days.
The titanium discs were immersed overnight in 10 μg·mL−1 of FN9-10ELP at 4°C, while the non-coated discs served as the control. The hMSCs were seed at a density of 5 × 103 cells/disc and incubated for 20 days at 37°C. (A) The mRNA levels of osteogenesis-related genes (Col I, RUNX2, OPN, OCN, BSP, TAZ) were analyzed via real-time PCR, and the comparative mRNA levels of each gene were evaluated relative to that of the β-actin as an internal control. The osteogenic differentiation activities are presented as the mean ± SD (n = 3). p < 0.05 and p < 0.01. (B) The protein levels of Col I, OPN, and OCN were analyzed via western blotting. Band density values of Col I, OPN, and OCN were normalized with β-actin and these proteins were represented as an arbitrary ratio compared to control titanium.