Novel Nanostructured Hydroxyapatite Coating for Dental and Orthopedic Implants:

Understanding Coating Structure, Integrity and Cell Attachment for Next Generation Implants

Huinan Liu,1 Wenping Jiang,1 and Ajay Malshe1,2*

1
NanoMech LLC, Springdale, AR; 2Materials and Manufacturing Research Laboratories
(MMRL), University of Arkansas, Fayetteville, AR
* Contacting Author: Tel 479-575-6561; E-mail apm2@uark.edu

ABSTRACT

A novel hybrid coating process, combining NanoSpray® (built on electrostatic spray

coating, ESC) technology with microwave sintering process, was developed for synthesizing
hydroxyapatite (HA) based nanostructured coating with favorable properties for dental and
orthopedic implants. Specifically, HA nanoparticles were deposited on commercially pure (CP
grade) titanium substrates using NanoSpray® technique to produce the HA coating, which was
then sintered in a microwave furnace under controlled conditions. The produced HA coating
were characterized for grain size and pore size using an environmental scanning electron
microscope (ESEM), the composition and calcium (Ca) / phosphorous (P) ratio using energy
dispersive X-ray (EDX) analysis, and crystalline phases using X-ray diffraction (XRD). The
results demonstrated that a nanocrystalline HA coating with a grain size from 50 to 300 nm and a
gradient of nano-to-micron pore sizes was fabricated successfully using this novel hybrid coating
process. The controlled nano-scale grain size and a gradient of pore sizes are expected to
promote bone cell functions and to facilitate bone healing. The initial in vitro study demonstrated
that the HA nanocoatings promoted human palatal mesenchymal cell functions. Besides
biological properties, such HA coating was also characterized for its mechanical properties, such
as adhesion strength, hardness and toughness. Microscratch test results showed that the critical
load of coating reached as high as 10 N. In conclusion, this study demonstrated that the novel
hybrid coating process including electrostatic NanoSpray® followed by microwave sintering
results in nanoscale HA coating for dental/orthopedic application.

INTRODUCTION

Hydroxyapatite (HA) has been widely used as a coating material for orthopedic/dental
applications due to its similar chemical composition to natural bone mineral and its capability to
promote bone regeneration [1]. It has been reported that HA with nano-scale crystalline features
and controlled porosity and pore size could promote osseointegration (that is, direct bonding to
natural bone) [2, 3]. So far, a number of methods have been developed or used commercially to
deposit HA on metal implants, such as electrophoretic deposition [4], sputter [5], dip coating [6],
spin coating and plasma spray [7]. It has been shown, however, it is very challenging to produce
a crystalline HA coating with desirable coating functional features, such as surface roughness as
well as controlled pore size and porosity, retained at nanoscale. In addition, it is also necessary
for nano-HA coating to have good adhesion strength to metallic substrates and sufficient
mechanical properties for load-bearing conditions. Therefore, in this study, a novel hybrid
coating process, combining electrostatic spray coating, NanoSpray® [8] with a microwave
sintering process [9], was developed to meet mechanical and biological requirements for next-
generation dental/orthopedic implants.

MATERIALS AND METHODS

HA nanoparticles with average particle size of 100 nm were deposited on titanium (Ti)
substrates using Nanospray® coating system which was developed based on the electrostatic
spray coating technology (Figure 1). The HA nanoparticles were generally electrically insulating
in nature and can carry the static surface charge over a distance of a few tens of centimeters. The
HA particles were charged when they exit the powder spray gun and exposed to an electrostatic
field generated by a pointed electrode, which is typically of a few tens of kilovolts (- 60~-80 kV).
The charged particles followed the electric field lines toward the grounded objects (titanium
substrates in this study) and formed a uniform and conformal coating perform [10]. The coating
preform can be consolidated with desired chemistry and surface morphology, and reasonable
adhesion by use of variety of processes such as laser [10]. Considering about the requirement for
minimized grain growth and readiness for process scale-up, in this work, the produced HA
coating preform was sintered in a microwave furnace. The sintering of HA-coated Ti implants
was performed in an air environment in order to achieve desirable nano-HA chemistry (Ca/P
ratio of 1.60 ± 0.06 to mimic natural bone mineral). The parameters for the sintering were set at a
temperature of 1000-1300 ºC for 5-20 minutes, based upon initial optimization.

Figure 1: A schematic sketch showing the mechanism of NanoSpray® for HA nanoparticles

deposition.

The deposited HA coating was characterized for grain size and pore size using an
environmental scanning electron microscope (ESEM), the chemical composition and Ca/P ratio
using energy dispersive x-ray (EDX) analysis, and crystalline phases using X-ray diffraction
(XRD). Such HA coating was further characterized for its mechanical properties, such as
adhesion strength (scratch resistance), hardness and toughness. The microscratch test method is
commonly used to measure the critical load of a coating, which is directly correlated to the
coating adhesion. Thus, microscratch testing according to ASTM C1624 was carried out for the
HA-coated samples. The diamond stylus was drawn on top of each sample by using an
increasing load, between 0.03N to 30 N, at constant velocity of 0.75 mm/min, until some well-
defined failure occurred. The normal load under which the de-lamination of the coating from the
Ti implants occurred is defined as critical load, which is typically determined by optical
observation in combination with acoustic emission technique.

Human palatal mesenchymal cells (HEPM 1486; ATCC) were cultured in EMEM with
10% FBS. The specimens of four different surfaces (textured titanium- TiP, HA coating on
textured titanium - HATiP, HA coating on machined titanium- HATiM and a control TCP) were
UV sterilized for 10 min on each side. SEM was utilized for the study of cell morphology and its
interaction with the coating surfaces after 72 hours of culture. Early matrix expression was
measured using a key transcription factor for bone differentiation, cbfa-1, an early marker for the
capacity for organic mineral formation, alkaline phosphatase and a late differentiation matrix-
related protein, osteocalcin.

RESULTS

Figure 2 shows the results of HA nanocoatings deposited by NanoSpray® system before

the microwave sintering. The HA coating surface morphology was characterized using SEM
(Figure 2a-c). The chemical composition of HA coating before microwave sintering was
characterized using EDX. As shown in Figure 2e in comparison with original HA particles
(Figure 2d), the chemical composition of the deposited HA coating preform is in consistence
with that of the as-received HA particles. The coating thickness variation was thoroughly
characterized using cross sections and statistical results showed the thickness of 60 ± 2.1 µm. A
representative cross sections of the deposited HA nanocoatings is shown in Figure 2f.

(a) (b) (c)

(d) (e) (f)

Figure 2: (a-c) SEM images of HA coating preform deposited by NanoSpray® system (before
microwave sintering) on titanium substrates from low to high magnifications. (d) EDX results of
HA particles before the coating process. (e) EDX results of HA nanocoatings on titanium
substrates. (f) SEM micrograph of the cross-section of the HA nanocoating on titanium
substrates to show the coating thickness (59.4 µm) and uniformity. The scale bars are 100 µm for
(a) and (f), 20 µm for (b), 10 µm for (c).

After the microwave sintering, the results demonstrated that a nanocrystalline HA coating
with a grain size from 50 to 300 nm and a gradient of nano-to-micron pore sizes were fabricated
successfully using this novel coating process (Figure 3). The controlled nano-scale grain size and
a gradient of pore size are expected to promote bone cell functions and to facilitate bone healing.
EDX results showed that the nano-HA coating had a Ca/P ratio of 1.6 (Figure 3d), very close to
natural bone, favorable for bone cell growth. XRD results confirmed that the nano-HA coating
was highly crystalline after sintering (Figure 3e).

(a) (b) (c)

(d) 400 (e)

350
300
Ca/P ratio=1.60 ± 0.06 250
200
150
100
50
0 2θ
20 30 40 50 60

Figure 3: (a-c) SEM micrographs of HA nanocoating after microwave sintering. (a) Low
magnification to show uniformity. (b) High magnification to show nano-scale features and nano-
porosity. (c) Cross-section of HA coating to show the thickness of coating (~ 60 μm) and its
uniformity. (d) EDX results of HA nanocoatings after microwave sintering showed the Ca/P ratio
of 1.60 ± 0.06 (mimic natural bone mineral). (e) XRD results of HA nanocoatings after
microwave sintering confirmed presence of highly crystalline HA.

Optical examination at the end of the microscratch test coupled with both acoustic
emission response and frictional properties variation during the test provided complete insight
into the coating adhesion. Microscratch test results showed that the critical load of coating de-
lamination reached as high as 10 N.

Human palatal mesenchymal cell attachment on HA nanocoatings were very high, with
an average of 88.20 ± 2.03 % for the HATiP and 86.5 ± 1.35 % for HATiM (Figure 4a), which
suggests nano crystalline and quasi-stoichiometric HA surfaces were capable of high degrees of
cell attachment and did not result in early cytotoxic cellular necrosis. Cell differentiation assays
indicated that HA nanocoatings were capable of high levels of cell adhesion, which, in turn, led
to high level of early osteoblast (bone forming cells) gene expression. Initial in vitro results
suggested positive effects of HA nanocoatings on cell functions, but further in-depth in vivo
biological studies are necessary in order to understand tissue interactions with HA nanocoatings.

(a)
(b)

HATiP TiP HATiM TCP

Figure 4: Human palatal mesenchymal cell attachment on HATiP, TiP, HATiM and a control
TCP. (a) The percentage of cell adhesion after 2 hr of culture. (b) SEM image of cell
morphology and cell interaction with HA-coated surface.

CONCLUSIONS

This study demonstrated that this novel hybrid coating process (NanoSpray® coating
followed by a microwave sintering) is very promising for synthesizing nanostructured HA
coating for commercial dental/orthopedic implants. The coating process offered high deposition
rate, suitability for various composite coatings, compatibility with simple and complex
geometries, flexibility, low energy consumption and low cost. The results based on this study
demonstrated that the application of this coating process could reduce or even eliminate the
formation of amorphous phase HA, which is soluble in body fluids and results in subsequent
dissolution of the material before natural bone tissue integrates. Technically, the HA
nanocoatings fabricated by this coating process have the following benefits: improved adhesion
strength prevents coating delamination; biomimetic chemistry to natural bone tissues (Ca/P ratio
very close to natural bone); large effective surface areas enhance cell attachment and growth;
nano-scale roughness enabled by nanoparticles of HA promotes implant-tissue integration; nano-
to-micron pores provide more anchor sites for inducing enhanced cell activities; and highly
crystalline HA coating reduces HA dissolution in body fluids.

ACKNOWLEDGMENTS

The authors would like to acknowledge the financial support from the SBIR program of
National Institute of Health (NIH, R43EB003335-01).
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