Biomimetic Highly Ordered Apatite
Coatings for Dental Implants
Sherif Elsharkawy1,2,3,4,5*, Sara
Gamea1,6, Natalia Karpukhina5,
Maisoon Al-Jawad5,7*
- Centre for Oral, Clinical, and Translational Sciences,
Faculty of Dentistry, Oral & Craniofacial Sciences, King’s College
London, London, SE1 9RT, United Kingdom
- Restorative Dentistry, Dental Directorate, Guy’s and St
Thomas’ NHS Trust, London, SE1 9RT, United Kingdom
- London Centre of Nanotechnology, London, WC1H 0AH,
United Kingdom
- Institute of Bioengineering, School of Engineering and
Materials Science, Queen Mary University of London, London, E1 2DP,
United Kingdom
- Barts and The London School of Medicine and Dentistry,
Queen Mary University of London, London, E1 2DP, United Kingdom
- Tanta University, Faculty of Dentistry, Department of
Restorative Dentistry, Tanta, 31111, Egypt
- School of Dentistry, University of Leeds, Leeds, LS2
9JT, UK
Correspondence:
*Email :
sherif.elsharkawy@kcl.ac.uk
*Email :
m.al-jawad@leeds.ac.uk
KEYWORDS: dental implants, coatings, hydroxyapatite,
fluorapatite, hierarchical mineralization
ABSTRACT:
Bioactive coatings on metallic ‘titanium’ or ceramic ‘sapphire’ implants
are known for their potential to promote bone biocompatibility,
osseointegration, and long-term survival. In this study, we have
investigated the effect of the surface topography of titanium and
sapphire at different temperatures on the chemistry, morphology,
organization, and coverage of the synthesized apatite coatings. We use a
wet chemical method with a fluoride rich calcium-phosphate solution, to
induce bioactive coatings onto etched titanium, non-etched titanium,
polished and unpolished sapphire at 37, 70, and 90˚C. Fluoridated
hydroxyapatite formation is detected across all temperatures using FTIR,
synchrotron XRD, and MAS-NMR. Surface topography and temperature changes
play a crucial role in the organization and coverage of the apatite
crystals. Well-defined hexagonal nanocrystals are observed across each
of the conditions, in the range between 35-81 nm. At lower temperatures,
self-assembled organized nanocrystals appear to grow out from spherical
structures, creating highly-ordered apatite architectures. However, at
90˚C, the nanocrystals seem to lack the hierarchical organization and
appear to be arranged randomly. This work demonstrates a promising
avenue for modifying implant surfaces with highly-ordered apatite-based
coatings at physiological conditions.
INTRODUCTION
The use of implants to repair and replace damaged hard tissues is a
growing field of research. Even though implants have a very high success
rate, implant failures do still happen. Their success is governed by
early osseointegration stage, which in turn is responsible for long-term
clinical success.1 Different materials have been used
as dental implant material including, pure titanium, Ti6Al4V alloy,
stainless steel, vitreous carbon, and single crystal aluminium oxide
(alumina).2
In order to improve the biological response at the bone-implant
interface and subsequently promote healing, the implant surface is
typically changed on a variety of lengthscales.3Numerous research studies 4–7 supported the
hypothesis that titanium implant roughness enhances osteoblastic
development, bone apposition, mechanical interlocking, and stress
distribution around implant, leading to faster osseointegration.
Nevertheless, several issues with corrosion, wear, and unfavourable
tissue interactions have been documented.8 It is
crucial to prevent corrosion of human body metals since it can
compromise their mechanical integrity and
biocompatibility.9 Unwanted ions from implants
could enter the body through corrosion and surface film breakdown
causing adverse biological reactions and lead to mechanical failure of
the device.10 On the other hand, the bioactivity of
titanium, in biological environment is poor.11
Single-crystal zirconia and alumina endosteal dental implants have been
investigated over the last 30 years in animal12,13 and
human studies14,15 Akagawa et al.16found that single-crystal alumina exhibits less tissue response compared
to titanium and therefore showed more favourable biocompatibility, due
to it being highly inert, non-toxic, more corrosion-resistant, and in a
high chemical oxidation state. Moreover, they found uniformly organized
collagen fibers at bone- alumina implant interface that resemble those
found in the periodontal ligaments, hence alumina implants exhibited a
good peri-mucosal seal. On the other hand, concerns have been raised by
clinicians regarding the high risk of alumina implant fracture during
surgery.17
There are multiple physical techniques that can be employed in order to
modify the implant surface including, plasma-spraying, grit blasting,
and acid etching. Furthermore, implant surfaces can be biochemically
modified by a hydroxyapatite (HAp) coating. Coating of metallic implants
with thin layers of bioactive materials merges mechanical advantages of
the metal with the excellent biocompatibility of the surface layer.
Moreover, the coatings can shield the implants against corrosion,
limiting the release of metallic ions into the body.18Coating dental implants with a material such as HAp has greatly enhanced
the bone healing process around the implant due to its bioactivity,
osteoconductivity, and osteoinduction properties and it has a similar
chemical composition to bone tissue forming a chemical
bond.19 Aebli et al.20 concluded
that HAp coating of dental implants significantly enhances their early
fixation and osseointegration, as they found that bone-implant contact
with HAp coated implants was significantly higher after 2 and 4 weeks
(139 % and 48 % higher respectively) compared to uncoated ones.
Hierarchical mineralized structures are found in biomineralized tissues
such as teeth and bones,21 Wu et
al.22 have grown ordered apatite crystals that show
improved mechanical properties, excellent bioactivity, and
biocompatibility, however the mechanisms that enhance the
biocompatibility of ordered over disordered surfaces are not yet well
understood. Additionally,23,24 synthesized
successfully ordered (two-level hierarchy) fluorapatite (FAp) crystals
on metallic substrates, which were aligned parallel to each other, and
their c-axes were perpendicular to the substrate. Moreover, they
investigated its effects on cell adhesion, growth and mineralization.
They found that ordered FAp crystals have higher cellular attachment and
bond strength to the substrate compared to disordered crystals.
Moreover, FAp ordered crystals were found to stimulate higher expression
of bone mineralization markers compared to the uncoated metallic
surfaces. Hence, it can significantly accelerate the
osseointegration.25
A simple wet chemical method is considered a novel biomimetic method to
coat dental implants,26 this method has many benefits
such as coating the implant with fluoridated HAp, which is thought to
reduce osteoclastic maturation, inhibit phagocytosis23and it is less soluble than HAp.27 Furthermore, it has
the ability to synthesize ordered crystals, which are self-assembled
into higher hierarchical structures mimicking the biomineralized tissues
such as bone.28 Ordered crystals showed better
mechanical properties, excellent bioactivity and biocompatibility
compared to disordered.24 Additionally, the wet
chemical method can be processed at near physiological conditions, so it
is cost-effective, and it has a great potential to be applied clinically
compared to hydrothermal methods.29 However, the
crystal growth behaviour is dependent on various parameters that control
the wet chemical method including temperature, substrate, and surface
topography used to grow the apatite crystals on.30Therefore, there is a need to develop inexpensive, dense, highly
organized apatite-based implant coatings with improved mechanical,
biological and physicochemical properties, as well as controlled
morphology.
In this study, we aim to optimize the synthesis of ordered biomimetic
fluoridated HAp crystals on titanium and sapphire substrates, study the
effect of substrate topography and temperature on the morphology,
chemistry, organization, and coverage of the synthesized apatite
crystals.
MATERIALS AND METHODS
Materials:
This study includes commercially pure titanium discs; 99.6% pure, 0.7
mm thick and 15 mm in diameter supplied from Goodfellow Corporation
(Coraopolis, PA, USA). Sapphire discs (length: 5 mm/ width: 5 mm /
thickness: 0.5 mm) supplied from MTI Corporation with
<0001> orientation (c-plane) and polished to 1 Å.
The isoelectric point of titanium is approximately
6.2,31 while sapphire is a single crystal, where its
isoelectric point is about 5.5.32
Discs Preparation:
For etching titanium discs, a 1:1 ratio of hydrogen peroxide 50% GPR
RECTAPUR (H₂O₂) (VWR International Ltd, Leicestershire, UK) and H₂SO₄
49-51% (Fluka Analytical®, Sigma-Aldrich®, St. Louis, MO) was used; by
adding 10 ml H₂SO₄ very slowly to a beaker of H₂O₂ then left for 24
hours. Then the discs were removed, rinsed thoroughly with plenty of
water and dried using air and filter papers.33
Cleaning of Sapphire
Sapphire single crystal substrates were cleaned as follows, discs were
placed in a Teflon holder inside a (40 ml) beaker filled with deionized
water (DW), then left in a sonicator bath (Kerry Ultrasonics, Guyson
Int. LTD, North Yorkshire, UK) for 15 minutes. Then, discs were placed
in sodium dodecyl sulphate (SDS) 4% w/v solution and left in the
sonicator for 15 minutes. Subsequently the discs were washed and left in
DW for a further 15 minutes in the sonicator in order to remove any last
traces of contamination. Finally, discs were air dried and sterilized by
using ultraviolet ozone cleaner (UVOCS® Inc., Lansdale, PA, USA) for 30
minutes.
Wet chemical method
This method was undertaking following;34 (0.1046 g) of
HAp powder was added to a (100 ml) of deionized water, then (0.0084 g)
of sodium fluoride was added with continuous stirring. Nitric acid
(69%) was added drop wisely into the solution very slowly until the
powder was completely dissolved. Ammonium hydroxide solution (30%) was
added drop wisely until the pH was readjusted to pH 6.0, then each
substrate was placed in the bottom of beaker and incubated for eight
days using a temperature-controlled incubator (LTE Scientific, Oldham,
UK) at 37, 70 and 90˚C using a water bath (Fisherbrand®, Leicestershire,
UK). After incubation of solutions, precipitation on the base of the
beakers were collected for further analysis.
Fourier transform infrared spectroscopy (FTIR)
FTIR analysis was conducted using the FTIR Spectrum GX (PerkinElmer®,
Waltham, MA, USA). The powder sample was placed over the IR window to
cover it with a metal cover and the sample was scanned. The program was
set to take the average of ten scans, to analyse the samples at a
wavenumber of 4000 cm-1 to 450 cm-1 in respect to % of transmittance.
Synchrotron X-Ray Diffraction (S-XRD)
The specimens were examined using the S-XRD beamline XMaS (BM28) at the
European Synchrotron Radiation Facility (ESRF, Grenoble, France) using
an X-ray energy of 15 keV (equivalent to a wavelength of λ = 0.82 Å) and
a beam spot size of 50 μm. Two-dimensional diffraction images were
collected every 30 seconds with a 2048 × 2048 pixel MAR CCD detector
mounted behind the specimen, perpendicular to the X-ray beam and centred
on it. A sample to detector distance of 132 mm allowed a 2 theta (2θ)
range of 5-30° which is equivalent to 2θ range of 9-58° on a lab
diffraction machine with CuKα radiation of λ=1.54 Å. A diffraction
pattern from a calibration standard sample (LaB6) was also collected to
allow data reduction and analysis.
After diffraction images were collected for each specimen,
two-dimensional diffraction patterns from the powder were pre-processed
using the FIT2D software.35 Each 2D diffraction image
was normalized to the incident flux and integrated over the entire 360°
azimuthal range to create 1D Intensity vs. 2θ diffraction patterns for
Rietveld refinement with the GSAS Rietveld refinement software.
Instrument parameters of X-ray wavelength, sample-to-detector distance,
detector tilts, and intrinsic peak shape were determined by refinement
of a LaB6 powder standard. Then for each specimen the phases present,
their relative proportions and lattice parameters were refined, and
values extracted. The quality of the refinement was determined by least
squares methods where the goodness of fit increases as chi-square (χ2)
approached unity, where χ2 is the ratio of the weighted R-factor and the
expected R-factor squared.
Magic angle spinning-nuclear magnetic resonance (MAS-NMR)
In order to investigate the fluoride (F-)
interactions, present in the precipitates, solid-state 19F MAS-NMR
analysis was conducted using a 14.1 Tesla spectrometer (600 MHz Bruker,
Coventry, UK) at a Larmor frequency of 564.5 Mega Hertz (MHz) under
spinning conditions of 22 kilo Hertz (kHz) in 2.5 mm rotor. The spectra
were acquired with a single-pulse experiment of 60 seconds recycle
duration, by using a fluorine free background probe. The 19F chemical
shift scale was calibrated using the -120 ppm peak of 1M of NaF solution
along with trichloro-fluoro-methane, as a second reference. Spectra were
acquired for 4 hours with accumulation of 240 scans. Data was
deconvoluted using DMFIT program.36
Substrate Preparation
Commercially pure titanium discs (Goodfellow Corporation, Coraopolis,
PA, USA) were either unetched or etched using a 1:1 ratio of hydrogen
peroxide 50% and sulphuric acid 50% for 24 hours, then the discs were
removed, rinsed thoroughly with plenty of water and dried using air and
filter papers.21 On the other hand, polished Sa (P-Sa)
and (U-Sa) single crystal substrates were cleaned using a sonicator bath
(Kerry Ultrasonics, North Yorkshire, UK) and were sterilized using
ultraviolet ozone cleaner (UVOCS® Inc., Lansdale, PA,
USA).
Scanning Electron Microscope (SEM)
Substrates were mounted on aluminium stubs via self-adhesive tape and
were coated by Agar auto sputter coating machine (Agar Scientific,
Essex, UK) using gold/palladium target disk. The morphology of synthetic
apatite surface along with the organization of crystals and their
dimensions were analyzed using SEM (FEI Inspect F, Hillsboro, OR, USA)
at the following magnifications; 300X, 1000X, 2000X, 5000X, 10 000X, 40
000X, 80 000X and 150 000X, with working distance (WD) about 10 mm and
3.5 spot size. Images were then transferred to ImageJ (NIH, US) for
quantitative analysis.
Transmission electron microscope (TEM)
Samples were analyzed using JEOL JEM 2010 (JEOL Ltd., Tokyo, Japan) to
assess the morphology of the crystals and their assembly. D-spacings
(lattice fringes) belonging to HAp crystal structures were determined
and calculated, a plot of a surface retrieved from ImageJ software shows
the peaks in graph that represents the lattice periodicities in the form
of alternation between white and grey value which has been counted.
RESULTS AND DISCUSSION
FTIR Characterization
After 8 days of mineralization (Figure 1), the precipitates were
collected from the different temperatures 37, 70, and 90°C and measured
together with commercially available HAp powder (HAp
Captal® R). FTIR analysis was used to characterize the
chemical composition of the synthesized powders as shown in Table 1. We
observed a wide range of phosphates
(PO43-) bands that correspond to the
typical apatite structures.