Download Coating of Titanium with Electrically Polarized

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.