(397v) Increased Osteoblast Adhesion and Proliferation On Electrophoretic Deposition Coated Nano Hydroxyapatite On Titanium-6,4

INCREASED
OSTEOBLAST ADHESION AND PROLIFERATION ON ELECTROPHORETIC DEPOSITION COATED NANO
HYDROXYAPATITE ON TITANIUM-6,4

Garima Bhardwaj¹, Dennis
Mathew¹, Linlin Sun², Thomas J.Webster² and Geetha
Manivasagam³.

¹Department of Biomedical Engineering, VIT University,Vellore-632014,Tamil
Nadu,India.

²Department of Chemical Engineering, Northeastern
University, Boston, MA-02115, USA.

³Department of Mechanical Engineering, VIT
University,Vellore-632014,Tamil Nadu,India.

Introduction:Improving the performance of
titanium implants for orthopedic applications has been a concern for many
years.This can be completed by designing surface properties to promote
osseointegration.   One manner to improve orthopedic implant
performance is to coat metals with bioactive ceramics, such as nanocrystalline
hydroxyapatite, which have been shown to improve bone formation compared to
micron crystalline hydroxyapatite and other ceramics. However, current
hydroxyapatite coating methods are time consuming, may be expensive, and involve
high heat which transforms nanocrystalline hydroxyapatite into micron
crystalline hydroxyapatite decreasing bioactivity and decreasing coating
adhesion strength.The goal of this present research was to develop a novel,
quick, and inexpensive implant coating process to improve bone cell responses.

Materials
and Methods:All of the tests in this
study were performed on a titanium alloy, Ti-6Al-4V, which was cut into small
pieces with dimensions of 1cm X 1 cm. 
Nanophase hydroxyapatite (HAP) was prepared by wet chemical synthesis
using Ca
(NO₃)₂4H₂O,
KH₂PO₄,distilled water, 
ammonia and acetone with a stirring time of 1 hour  and an aging time of 24 hours followed by
filtering then sintering at 900 ͦC. 
The HAP was then coated onto the alloy using electrophoretic deposition
(EPD). The electrolyte was prepared by dissolving HAP in isopropyl alcohol and
stirring for 10 minutes followed by overnight settling. The mixture was again
stirred for 2 hours before starting the coating and Ti-6,4 was used as a
working electrode(-ve) and 316 stainless steel was used as a counter
electrode(+ve).Voltage varying from 40 to 80 V was used for 2 to 5 minutes and
an optimal coating was achieved at 60 V for 4 minutes. Samples were
characterized using X-ray Diffraction (XRD), SEM, and contact angle analysis to
confirm crystallinity, surface roughness, and chemistry of the coatings.  Osteoblasts (ATCC-C12720) were cultured in
growth medium supplemented with 1% penicillin.A 4 hour MTT adhesion assay was
performed to determine cell viability.A MTT proliferation assay was performed
after 1,3 and 5 days of culture.All experiments were conducted in triplicate
and repeated three times each.

Results
and Discussion XRD results of the
nanophase HAP (Figure 1) and SEM images of the samples taken after the coating
process (Figure 2) confirmed the uniformity and nanoscale character of the
coatings. Contact angle results of the coated samples showed an increased
hydrophilic nature of the coated samples. Results from the MTT assay showed
increased osteoblast adhesion (Figure 3) and proliferation (Figure 4) on the
coated samples compared to controls (tissue culture polystyrene and uncoated
Ti-6Al-4V).

Figure
1: XRD of nanocrystalline HA used in the present study

Figure
2. SEM of the EPD nanocrystalline HA on titanium-6,4

Figure
3. Increased osteoblast adhesion on HA (EPD) coated Ti compared to Ti control.
Data = mean +/- SEM; n = 3 * p < 0.01 compared to Ti control,F-test
value=0.000356.

Figure 4: Osteoblasts proliferation results, day 1, 3
and 5 respectively.Data = mean +/- SEM; n = 3; * p < 0.01 compared to Ti
control,F value for Day 1=0.001872,F value for Day 3=0.0017608,F value for Day
5=0.007834

Figure 5:Contact angle analysis
results(actual picture and values of drop and surface contact) for uncoated
Ti-6,4 obtained using distilled water as a fluid.

Figure 6:Contact angle analysis
results(actual picture and values of drop and surface
contact) for Ti-6,4 coated with nano-HA by EPD obtained using distilled water
as a fluid.

Conclusions:  Electrophoretic deposition is a novel, inexpensive
and quick coating process that leads to a strong and optimal coating to
increase bone cell compatibility. Hence, this coating process should be further
explored for enhancing orthopedic implant efficacy.

Acknowledgements: The authors thank
Northeastern University  and VIT
University for funding.