Engineered Coatings for Titanium Implants To Present Ultralow Doses of BMP-7

Abstract

The ongoing research to improve the clinical outcome of titanium implants has resulted in the implemetation of multiple approches to deliver osteogenic growth factors accelerating and sustaining osseointegration. Here we show the presentation of human bone morphogenetic protein 7 (BMP-7) adsorbed to titanium discs coated with poly(ethyl acrylate) (PEA). We have previously shown that PEA promotes fibronectin organization into nanonetworks exposing integrin- and growth-factor-binding domains, allowing a synergistic interaction at the integrin/growth factor receptor level. Here, titanium discs were coated with PEA and fibronectin and then decorated with ng/mL doses of BMP-7. Human mesenchymal stem cells were used to investigate cellular responses on these functionalized microenvironments. Cell adhesion, proliferation, and mineralization, as well as osteogenic markers expression (osteopontin and osteocalcin) revealed the ability of the system to be more potent in osteodifferentiation of the mesenchymal cells than combinations of titanium and BMP-7 in absence of PEA coatings. This work represents a novel strategy to improve the biological activity of titanium implants with BMP-7.

Figure 1

Characterization of engineered surfaces. (a) Scheme of a biomimetic system consisting of Ti layer coated with PEA functionalized with FN and specifically bound BMP-7 allowing synergistic signaling. (b) Thickness of polymer layer on Ti in comparison with glass tested by scratch assay; *** P < 0.001 (unpaired t test was used). (c) Static water contact angle and hysteresis of bare Ti surface and Ti/PEA, in comparison with glass surfaces; * P < 0.05; *** P < 0.001 (one-way ANOVA with Tukey’s post-test was used). (d) AFM images of FN network on Ti/PEA surfaces. (e) BMP-7 bound to Ti, Ti/PEA, and Ti/PEA/FN at two BMP-7 concentrations; the total amount of BMP-7 bound per cm² is shown; ns = nonsignificant; *** P < 0.001 (one-way ANOVA with Tukey’s post-test was used). (f) Cumulative BMP-7 release from materials over 2 weeks; results shown as percentage from the BMP-7 amount originally bound after 1 h of coating. Asterisk labels above the bar without any linking lines to other bars means this condition was significantly different from all other conditions in the graph with the same level of significance.

Figure 2

Focal adhesion (FA) analysis in hMSC on material surfaces: (a) Average cell size on Ti, Ti/PEA and with or without BMP-7 coating did not vary but significant differences were found for FA length—Ti with PEA coating had shorter FA than Ti alone or Ti/BMP-7; * P < 0.05; *** P < 0.001 (one-way ANOVA with Tukey’s post-test was used). (b) Distribution ratios for FA area and FA length. (c) Representative images of fluorescence labeled hMSC: Shorter FA are present more often on PEA surfaces in comparison with Ti or Ti/BMP-7 (inserts); vinculin representing FA is stained in red, cytoskeleton in green, and nuclei in blue; scale bar is 20 um.

Figure 3

Cell proliferation and mineralization on Ti surfaces: (a) Cell numbers on Ti/PEA/FN/BMP-7 are significantly smaller in comparison to three control surfaces (Ti alone, Ti/BMP-7, and Ti/PEA/FN) after 28 days (dark bars), corresponding to the differentiation process rather than cell proliferation; a positive differentiation control with hMSC growing on Ti surface in the presence of osteogenic medium (Ti + OM) also does not show any proliferation after 28 days; * P < 0.05; ** P < 0.01; *** P < 0.001 (two-way ANOVA with Bonferroni post-test was used). (b) Free calcium production assay at day 28 showed higher amount of Ca²⁺ in hMSC on Ti surface with PEA/FN/BMP-7 coating (dark green bar) in comparison with other samples in growth medium, suggesting this system to have the best potential for mineralization; differences between other samples in growth medium were not significant; *** P < 0.001 (one way ANOVA with Tukey’s post-test was done). An asterisk label above the bar without any linking lines to other bars means this condition was significantly different from all other conditions in the graph with the same level of significance.

Figure 4

Osteogenic differentiation of hMSC on engineered surfaces after 28 days of culture: (a) Fluorescence images of hMSC with osteogenic markers osteocalcin (OCN, top), and osteopontin (OPN, bottom) on Ti-functionalized substrate (Ti/PEA/FN/BMP-7) and three control surfaces; OCN and OPN are stained in green, cytoskeleton is in red and DAPI in blue. Scale bar represents 100 μm. (b) Quantification of OCN and OPN staining showing a significant increase in both markers on Ti/PEA samples functionalized with FN and BMP-7 (dark green bars); ** P < 0.01; *** P < 0.001 (one-way ANOVA with Tukey’s post-test was done).