Binding activity of BMP-2_GFP on bone graft by protein concentration.

<p>A. Representative fluorescence images. B. Fluorescence intensity. HA bone grafts were placed in 24-well plates and adsorbed with a various concentrations of BMP-2_GFP (0–35 μg) in SBF at 37°C for 2 h. The fluorescent image of BMP-2_GFP adsorption to the granules was captured under fluorescence microscopy and quantified. Results represent the mean ± SD (n = 3).</p

ALP activity assay of pre-released BMP-2 incorporated bone graft.

<p>ALP activity was quantified in C2C12 cells exposed to BMP-2 incorporated HA bone grafts after pre-release in SBF for indicated times or HA bone grafts only (control). Results represent the mean ± SD (n = 3).</p

Binding activity of BMP-2_GFP on bone graft by temperature.

<p>A. Representative fluorescence images. B. Fluorescence intensity. HA bone grafts were placed in 24-well plates and adsorbed with 30 μg of BMP-2_GFP in SBF at 4°C, 20°C, and 37°C. The fluorescent image of BMP-2_GFP adsorption to the granules was captured under fluorescence microscopy and also quantified. Results represent the mean ± SD (n = 3).</p

Schematic representation of the BMP-2_GFP fusion proteins and Western blotting analysis of BMP-2_GFP shown at 45 kDa.

<p>Schematic representation of the BMP-2_GFP fusion proteins and Western blotting analysis of BMP-2_GFP shown at 45 kDa.</p

The release profile of BMP-2_GFP on bone graft by fluorescence intensity assay.

<p>The release profile of BMP-2_GFP from bone graft was conversely expressed as the retention profile of BMP-2_GFP. A. Representative fluorescence images by fluorescence-based retention assay. HA bone grafts were placed in 24-well plates and incubated with 30 μg of BMP-2_GFP in SBF at 20°C for 1 day. Then, the sustained release profile of BMP-2_GFP from bone grafts was measured by fluorescence microscopy for 14 weeks. The fluorescence image was captured and the fluorescence intensity was normalized with respect to the initial fluorescence. B. Release profile using fluorescence-based retention assay and ELISA. HA bone grafts were placed in 24-well plates and incubated with 30 μg of BMP-2_GFP in SBF at 20°C for 1 day. The amount of released BMP-2_GFP was quantified via ELISA according to the manufacturer’s instructions. Results represent the mean ± SD (n = 3).</p

Protein attachment activity of FN9-10_ELP 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.</p

Cell proliferation activity of hMSCs on the FN9-10_ELP-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.</p

ALP activity of hMSCs on the FN9-10_ELP-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.</p

Cell adhesion activity of hMSCs according to incubation time on the FN9-10_ELP-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.</p

Expression of chimeric FN9-10_ELP 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.</p