(358f) Assessment and Control of ANTI-Microbial and ANTI-Inflammatory Responses of Macrophages to Different Titanium Nanomodifications

Bhardwaj, G., Northeastern University
Webster, T. J., Northeastern University
Yazici, H., Northeastern University


Bhardwaj1, Hilal Yazici
1,Thomas J. Webster1.

University, Boston, MA-02115

Introduction: To monitor the
immunological response of implants, the host response to materials must be
determined prior to implantation. This response is highly dominated by
macrophages which are the primary cells governing the immune response. Macrophages not only fuse to become multinucleated
foreign body giant cells, but they also activate T lymphocytes by expressing co-stimulatory
molecules (e.g., CD86 and CD80) and surface antigens (e.g., MHC II). T
lymphocyte activation and giant cell formation have been observed on
macrophages adherent to biomaterials and in retrieved adjacent tissues to failed
implants. They are major effectors in the defense against bacterial infection
and respond to interactions with gram-negative and gram-positive bacteria with
a marked enhancement of their functional activities. Thus, the assessment and
control of macrophage responses to various surface nano-modifications
on titanium implants is of vital importance. The current study aims at modifying the surface of titanium by coating it
with nanoscale hydroxyapatite, in a range of sizes, using electrophoretic
deposition with DC current. Macrophages are then seeded onto this surface and
their behavior observed concerning how to further improve the modification

Materials and Methods: Nanoscale hydroxyapatite was synthesized using a wet
chemical synthesis process in 4 different sizes ranging from 110-170 nm,
using Ca(NO3)24H2O, KH2PO4, distilled water
and ammonia in an acid digestion vessel for hydrothermal treatment followed by
drying in the oven. It was then coated onto a titanium mesh purchased from Alpha Aesar (Catalog no.7440-32-6) by electrophoretic deposition
(EPD) with a DC current at 151 V for a minute each. Macrophages purchased from
ATCC (RAW 264.7 (ATCC® TIB-71?)) were cultured using EMEM
(ATCC® 30-2003?), 10% FBS (ATCC® SCRR-30-2020?)
and a 1% penicillin-streptomycin solution (ATCC® 30-2300?).
Cell adhesion and proliferation was observed using MTS assay for 1, 3, 5 and 7
days. Levels of TNF-α, IL-1, IL-6
and nitrite released by the macrophages were studied using PCR.

Bacterial assays were conducted using Staphylococcus
(ATCC® 29740?), Ampicillin resistant E.coli and Pseudomonas aeruginosa (ATCC® 39324?) strains
of bacteria. 0.03% tryptic soy broth (TSB) and agar
plates (Sigma-Aldrich) were used as the media. A small amount of bacteria was
taken from the stock culture, streaked onto an agar plate, and then used as the
stock plate for further experiments. Colonies were scraped off from the stock
plate, added to 3 mL of 3% TSB and incubated at 37°C in humidified conditions
under a 5% carbon dioxide atmosphere for 18 hours. A small amount (0.1 mL) of
each sample was transferred to a few wells of a 96-well plate and absorption
was measured at 562 nm using a plate reader. A value of 0.52 to 0.54 was
obtained, indicating a density of 109 bacteria/mL. A dilution of 108 bacteria/mL was then prepared using 0.03%
TSB. The samples were sterilized with 70% ethanol for 20 minutes, transferred
into a 12-well plate, and rinsed once with PBS. They were then treated with 2
mL of the 108 bacteria/mL solution and incubated for 24 hours. The
bacteria solution was removed and the samples were rinsed twice with PBS. They
were transferred into 3 mL of PBS and sonicated for
10 minutes to create a first dilution (10-1) then three
subsequent dilutions (10-2, 10-3, and 10-4)
were created. Following this, 0.1 mL of each of the 10-3 and
10-4 dilutions were plated and incubated for 18 hours. The
number of bacterial colonies formed on each sample was counted and using these
values, the number of bacteria/mL was found. All experiments were conducted in
triplicate and differences between means were determined using analysis of
variance followed by Student's t-tests.

The particle size of the powders was determined using transmission electron
microscopy (TEM) and the sample composition was established using x-ray
diffraction (XRD). BET was used to measure the porosity of the powders. After
coating, samples were characterized using scanning electron microscopy (SEM),
atomic force microscopy (AFM) and contact angle analysis to confirm surface
roughness and wettability of the coatings, all according to standard

Results and Discussion: Particle size and distribution was determined using
TEM as shown in figure 1. After the samples were coated with the synthesized HA
powders, SEM was used for surface characterization as shown in figure 2. This
helped establish the presence of nano-crytalline
features on the surface. Contact angle measurements were made using distilled
water to test the hydrophobicity/ hydrophilicity of
the surface, as shown in figure 3. The sample coated with HA by EPD showed
complete wetting as compared to the hydrophobic behavior displayed by plain Ti.
Figure 4 shows the result of confocal and fluorescence microscopy performed on
the samples after seeding them with the 3 strains of bacteria and macrophages
and fixing them. There was a decreased proliferation of these cells on the
surface with reducing size of HA nanoparticles. Also, the change in the surface
topography of the material affected the adhesion and proliferation of the
macrophages leading to reduced activation of macrophages with increased
nanometer roughness. The level of TNF-α, IL-1, IL-6 and nitrite released
decreased with increasing hydrophilicity of surface
but increased overall with time1,2.
Bacterial activity decreased with smaller particle size of hydroxyapatite.

Figure 1: TEM image of
the 110 nm HA powder. Scale bar - 100 nm.

Figure 2: SEM image of the 110 nm HA powder coated onto
the titanium mesh using EPD. Scale bar- 1µm





Figure 3: A)
Contact angle image of the surface having 110 nm HA coated onto Ti using EPD.
Image shows complete wetting of the surface establishing its highly hydrophilic
nature. B) Contact angle image for the Plain Ti surface. Contact angle = 63.54º

Figure 4: Confocal and fluorescent  microscopy image showing bacterial and
macrophage density on Plain Ti, Plasma Sprayed HA on Ti and Ti coated with 100 nm HA
by EPD respectively. (a), (b), (c) represents S.aureus
; (d),(e), (f) represents P.aureginosa ; (g), (h), (i) represents Ampicillin resistant E.coli. The scale bar
for these images is 20 µm. (j),(k),(l) represents
macrophage density on day 1 of culture. The scale bar for these images is 60

Conclusions:  Controlling the size of the nanophase
hydroxyapatite, the coating procedure and its parameters can help influence the
fate of macrophage attachment, activation and functionality at the implant
surface by reducing levels of TNF-α,
IL-1, IL-6 and nitrite released with increased nanoscale topography. Bacterial activity is
influenced by the size of the particles, the roughness, the hydrophobicity/hydrophilicity and the coating procedure used.

The authors
would like to thank Northeastern University for funding.

References: 1.) Control of macrophage responses on hydrophobic and
hydrophilic carbon nanostructures, Y. Chun et al, Carbon 49 (2011) 2092?2103. 2.) Reduced responses of
macrophages on nanometer surface features of altered alumina crystalline phases,
D. Khang et al, Acta Biomaterialia 6 (2010) 3864?3872.