(67h) Reduced Adhesion of Macrophages On Nanotubular PDMS Molds for Catheter Applications

Introduction: Polydimethylsiloxane (PDMS) has
been used for short- and long-term catheters. However, PDMS shunt tubing has
been scrutinized recently because of their extremely high failure rates. While
there are many reasons why PDMS shunt systems fail, one is tissue occlusion of
the ventricular catheter, most often caused by macrophages. It is hypothesized
that nanotextured and nanotubular surfaces can be carefully manipulated to
inhibit immune cell
(specifically, macrophages) and improve the fibroblast adhesion due to their unique
surface energy properties which have the ability to control initial protein
absorption and subsequent cell behavior. The objective of this in vitro study
was to create polydimethylsiloxane (PDMS) molds of titanium (Ti) anodized to
possess nanotubes and test inflammatory cell and fibroblast responses on such substrates.

Materials and Methods: 99.2% pure titanium foils (Alfa
Aesar, Ward Hills, MA) were cut into 1cm×1cm squares and cleaned with acetone,
70% ethanol, deionized water. The samples were etched for 1min with a solution
of 1.5% nitric acid and 1.5% hydrofluoric acid (HF) to remove the thin oxidized
layer. The cleaned Ti sample was used as an anode, while a platinum (Pt) mesh
served as a cathode. Both were immersed in an electrolyte solution consisting
of 1.5% HF. The Ti and Pt samples were connected to a DC power supply. A
constant voltage of 20V was applied for 10 minutes and the distance between Ti
anode and Pt cathode was kept constant at 1cm. After anodization Ti samples
were rinsed with large amounts of deionized water immediately and air dried.

Next, the PDMS monomer
and cross-linking agents (Sylgard 184, Dow Chemical Co.) were mixed at 10:1
(weight ratios) for 15min and were then placed in a vacuum chamber for 30min to
remove air bubbles. The mixture was cast onto the nanotubular Ti master mold
and then was placed into a vacuum chamber for another 1h. The PDMS was cured at
60 °C for 2h followed by cooling and was gently peeled away from the Ti
template. To improve the peeling off ability, a gold film was sputtered onto
the top surface of the PDMS template and the PDMS slurry was again poured onto
the first PDMS. The desired PDMS replica was obtained after curing.

The IC-21 macrophage cell
line was used (TIB-186; ATCC, Manassas, VA) for determining an immune response.
Macrophages were cultured according
to ATCC instructions in RPMI-1640 medium (Invitrogen, Carlsbad, CA) with 10%
fetal bovine serum and 1% penicillin/streptomycin (HyClone Laboratories Inc,
Logan, UT) under standard incubator conditions (37°C, humidified, 5% CO₂/95%
air environment). Macrophages were seeded at 3500 cells/cm² per substrate and
were cultured in a humidified 5% CO₂/95% air environment at 37°C for
24 hours. After 24 hours, nonadherent macrophages were removed by rinsing in
phosphate-buffered saline (PBS). Macrophages adherent on the substrates were
fixed with 4% formalin in PBS and stained with both rhodamine phalloidin (R415)
(Molecular Probes, Eugene, OR) and DAPI (4', 6-diamidino-2-phenylindole)
(33258) (Sigma-Aldrich). F-actin filaments and cell nuclei were visualized
using tetramethyl rhodamine iso-thiocyanate and DAPI, respectively.

Also, to determine the
adhesion and proliferation of skin fibroblasts (ATCC, CCL-110), a cell
proliferation assay (CellTiter 96, Promega) was used. Briefly, for cell
adhesion, cells were seeded at 3500
cells/cm² in standard cell culture
media and were incubated for 4 hours. For proliferation studies, cells were
seeded at 3500 cells/cm² for 1, 3 and 5 days. The
dye solution were added to the cells after the end of the prescribed period for
4h, then the stop solution was added and incubated overnight. A plate reader
was used to test cell density. Numerical data were analyzed with a Student's
t-test to make pair-wise comparisons. Statistical significance was considered
at p<0.05.

 Results and Discussion: As expected, compared with
unmodified Ti (Figure 1. a), nano-sized tubes were distributed uniformly on the
Ti surface after anodization (Figure 1, c). The uniform pores, as observed by
scanning electron microscopy, were estimated to have a diameter of 60-70nm and
a depth of 200nm. After pouring the PDMS slurry onto the surface of the
nanotubular Ti and peeling off the first PDMS template, a nanopatterned structure
was observed (Figure 1, d). Most importantly, the PDMS replica showed similar
nanostructures as anodized Ti.

Figure 1.  SEM images of

a) Unanodized Ti     b) PDMS
based on unanodized Ti

c) Anodized Ti         d) PDMS
based on anodized Ti

Scale bars are 200nm.

The results also demonstrated
decreased numbers of macrophages adhered to nanotubular Ti and PDMS replica
surfaces. Most importantly, the increased fibroblast adhesion, proliferation,
and long-term functions on nanotubular substrates indicated the nanoscale
topographies of polymers should be strongly considered for catheter
applications.

Conclusions: A PDMS replica was obtained
from anodized nanotubular Ti and demonstrated a promising ability to control macrophage and fibroblast functions.

Acknowledgements:
The authors would
like to thank Northeastern University for funding.