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Coating of Titanium with Electrically Polarized Hydroxyapatite Modulates Mesenchymal Stem Cell Adhesion Diana Olvera1, Keith Savino2, Jason A. Inzana1, Matthew Z. Yates2, Hani A. Awad1 1 Dept. of Biomedical Engineering; 2 Dept. of Chemical Engineering, University of Rochester, Rochester, NY, USA Introduction While orthopedic implants are generally successful, they are not without debilitating complications due to loosening. Success of any orthopedic implant relies on effective integration between the surface of the implant and bone, with no fibrous tissue interface. Efforts in this field show that improvements in implant surface topography enhances bone adherence. Because titanium (Ti) is endowed with characteristic corrosion resistance, biocompatibility, and mechanical properties, it is commonly used as a component of orthopedic implants. To improve osseointegration, the Ti surface can be coated with hydroxyapatite (Ca10(PO4)6(OH)2 or HAP)1. However, tissue growth on the synthetic HAP surface is still not as fast as that of natural bone, prolonging patient recovery after surgery and increasing the likelihood of failure2. We have developed a novel HAP coating technique3 with the ability to promote electrical polarization and strong charge storage. In this study, we hypothesize that this polarized HAP coating on Ti promotes attachment of osteogenic cells and mediates changes to their morphology and focal adhesions that may affect osseointegration. Materials and Methods Preparation of HAP substrates: HAP was deposited onto grade 2 Ti discs (0.25mm thick) using a published electrochemical-hydrothermal synthesis method3. Cell culture: Human bone marrow-derived MSCs were obtained from Lonza (Berkshire, UK) and cultured in vitro according to the manufacturer's protocol. Ti and HAP-coated Ti discs (n=3/group) were placed into 48well plates and glass bottom multi-well dishes were used as a control. The samples were seeded with MSCs at a low seeding density of 5000 cells/ cm2 to avoid artifacts from cell aggregation in cell (nuclear) and vinculin quantification. Immunofluorescence staining and SEM Imaging: After a 48 h incubation period, cells were washed in DPBS, fixed in 4% PFA, and then sequentially stained with mouse monoclonal primary antibodies against vinculin, F-actin, and nuclear To-Pro3. Cells were examined on a laser scanning confocal microscope (FV1000 Olympus) with a Zeiss 10× dry, and 40× oil objective lens using tiling and stacking modes for nucleus and vinculin quantification, respectively. Samples were then processed for scanning electron microscopy (SEM) to further characterize the morphology of the cells. Statistics: Quantitative cell attachment density and vinculin distribution per cell were contrasted among the experimental groups using ANOVA with Tukey’s post hoc test (GraphPad Prism, α=0.05). Results After 48 h, the immunostaining revealed cells containing many thick stress fibers in a parallel arrangement. Vinculin-labeled focal contacts did form short and dense patches, evenly distributed on the membrane surface, which were in contact with the different surfaces. These focal points appeared more frequent on the glass, Ti and negative polarized HAP surfaces (Fig 1). An intersurface comparison of cell (nuclear) density and focal adhesion showed that, although lower than the glass control, negative polarized HAP maintains the same quantity of cells compared to Ti, while positive polarized HAP has a significant reduction in attached cells (Fig 2A). The quality of cellular attachment, quantified by the number of focal adhesions per cell, tends to be improved with negative polarized HAP compared to Ti (Fig. 2B). Confocal observations showed that glass, Ti and negative polarized surfaces favored rapid cell attachment and denser focal adhesion points, while the positive polarized HAP surface had limited cellular response. These observations were also confirmed with SEM imaging. A B C D Figure 1. Immunofluorescent staining of MSCs for vinculin (green), F-actin (red) and nuclear To-Pro3 (blue) on (A) glassbottom, (B) Ti, (C) negative polarized HAP, (D) and positive polarized HAP. Figure 2. Intersurface comparison of cell (nuclear) density and focal adhesion. A) Cell (nuclear) density relatively equal for negative polarized HAP and Ti but significantly less for positive polarized HAP. B) Focal adhesion tends to be improved with negative polarized HAP. Discussion and Conclusions This study examined the effect of different surface topographies on the attachment of mesenchymal stem cells and the expression of focal adhesion. With this preliminary data, we hypothesize that the negative electrostatic charge trapped on the surface of polarized HAP has the potential promote bone cell attachment that can lead to enhanced osseointegration. The mechanism is as of yet unknown. Future studies will focus on gene expression analysis, and in vivo osseointegration. References 1. Suchanek, W. et al. 1998. J Mater Res. 13, 94-117. 2. Salgado, A. et al. 2004. Macromol Biosci. 4,743-765. 3. Liu, D. et al. 2009. Adv Funct Mater. 19, 3941-3947. Acknowledgements This work was supported by a University of Rochester Provost Multidisciplinary Award (Yates and Awad). Disclosures The authors have nothing to disclose.