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Bonding strength and Biocompatibility of HA/TiO2 Composite Coatings for Orthopaedic
Implant
C.M.Lin and S.K.Yen
Institute of Materials Engineering
National Chung Hsing University, Taichung, Taiwan, R. O. C.
Abstract
Insufficient bonding of juxtaposed bone to an orthopaedic/dental implant could be caused by
material surface properties that do not support new bone growth. For this reason, fabrication of
biomaterials surface properties which support osteointegration should be one of the key objectives
in the design of the next generation of orthopaedic/dental implants. Titanium and titanium alloy
have been widely used in several bioimplant application, but when implanted into the human body
it still contained some disadvantages, such as poor osteoinductive properties, wear debris and ions
release, which often lead to clinical failure. Electrolytic composite coatings of Titanium dioxide
(TiO2) and Hydroxyapatite (HA) were successfully deposited on titanium substrates in TiCl4
solution and subsequently in the mixed solution of Ca(NO3)2 and NH4H2PO4, respectively. The
coated specimens were evaluated by electrochemical polarization in 37℃ Hank’s solution, bonding
strength tests, surface morphology observations, and XRD analyses. The biocompatibility of
HA/TiO2 composite coatings were investigated by human G-292 osteoblast-like cells. Human G292 osteoblast-like cells were found to be a valid experimental model for primary human osteoblast.
The corrosion resistance, and bonding strength of the HA coated film were improved by the
intermediate coating of TiO2 from 11.3 MPa to 46.7 MPa. The crystallization of the HA/TiO2
composite coating specimens were further identified in details. The crystallization and the surface
morphology of the HA/TiO2 coated specimens annealed at 300℃ provide better support for the
differentiation and adhesion of osteoblast-like G-292 cells.
Introduction
Biomaterials must simultaneously possess many requirements and special properties such as nontoxicity, corrosion resistance, fatigue durability, biocompatibility, and good adhesion strength.
Titanium and titanium alloy biomaterials for implants have found widespread applications in the
orthopaedic and dental fields. It possessed excellent corrosion resistant, good mechanical properties
and biocompatibility. However, when titanium and titanium alloy is implanted into a complicated
and aggressive physiological in vivo environment, the oxide stability may be affected, resulting in
increasing metal ion release. Degasen et al. (1999) found that titanium ions (in concentration
ranging from 0.01 to 100 ng ml-1) did not determine damage (in terms of cell viability or cell injury)
but could inhibit phytohemagglutinin-induced T-cell, liposaccfaride-induce B-cell proliferation, and
influenced mineral formation. In addition, Nie et al. (2000) found that titanium exhibits poor
osteoinductive properties also caused its incomplete fixation to living bone, and formation of
fibrous tissue at the interface with the living bone. Therefore, we proposed a new concept used
electrolytic deposition technology to prepared HA/TiO2 composite coatings on Ti substrate.
Titanium dioxide (TiO2) at the substrate surface not only acted as a chemical barrier to against
release of metal ions from the implant, but also provided a chemical bonding between the titanium
substrate and the HA coating to increase the adhesion strength.
Method
Sample Preparation—An ASTM F67 Grade 1 Ti sheet, as received, was used as a substrate of the
deposition of the electrolytic HA/TiO2 composite coatings. It was cut into sheet thickness 0.8 mm.
The sheet was then cut into discs with a diameter of 15 mm for dynamic polarization tests, bonding
strength tests and cell culture.
Electrolytic Deposition and Annealing—The electrolytic deposition of TiO2 was carried out in a
TiCl4 solution. Subsequently, the TiO(OH)2 coated specimen was further deposited in a mixed
solution of Ca(NO3)2‧4H2O and NH4H2PO4 with Ca/P = 1.67. The titanium disc was the cathode,
platinum the anode, and saturated AgCl/Ag the reference electrode. The coated specimens were
then annealed for 1 hr in air at 300℃, 500℃, and 700℃, respectively.
Bonding strength—Bonding strength of HA/TiO2 layer to the substrate were measured by ASTM
C-633 methods (1993). Both side of the substrate were attached to cylindrical steel clamps 15 mm
in diameter and 20 mm in length by using Rapid-type Araldite glue. HA/TiO2 coated specimens
were prepared 5 samples, and the mean bonding strength were calculated from the fracture load and
surface area (π × 7.52 mm2). The fractured surfaces of the specimens were observed under a
scanning electron microscope (SEM/EDS).
Dynamic polarization—Uncoated Ti specimen, thermal oxide film, CaP coated and HA/TiO2 coated
specimens annealed at 300℃ were potentiodynamically polarized in aerated Hank’s solution. The
cyclic polarization test ranged from – 0.80 to + 0.80 V, then back to – 0.70 V at a scanning rate of
0.167 mV/sec at 36 ±1℃. If the oxidation is due to the corrosion of the electrode, this potential is
named “corrosion potential” Ecorr. The current density before pitting is nearly constant and defined
as the “passivation current density” ipass.
Cell culture－Human osteoblast-like cell line G-292 (ATCC CRL 1423) , originally isolated from a
human osteosarcoma. Cells were initially cultured in McCoy’5a medium containing 10﹪fetal
bovine serum (FBS), 100 units/ml of penicillin and 100 μg/ml of strepotmycin. The cells were then
thawed and cultured in 25 cm2 flasks and incubated at 37℃ in a humidified atmosphere with 5﹪
CO2. Media were changed every 2 days. Finally, cells were seeded onto samples (Ti substrate, CaP
coated and HA / TiO2 coated) in a 24-well plate at a density of 2×104 cells / well. The amount of
cells seeded on polystyrene (PS) control (2104 cells) was such as to obtain the same cell density
used for the coatings.
SEM and XRD— The surface morphology of specimens after electrolytic deposition, polarization
tests, bonding strength tests and cells culture were observed by scanning electron microscopy and
the composition energy dispersive spectroscopy (SEM/EDS). The crystal structure of HA/TiO2
composite on Ti substrate was analyzed by X-ray diffractomery (XRD).
Results
The XRD diagrams of HA/TiO2 as-coated specimens, only found HA (major) and octacalcium
phosphate (Ca8(HPO4)2(PO4)•5H2O, OCP, minor). Further annealing at 300℃, OCP disappeared
and only HA was clearly identified on the HA/TiO2 coated specimens. Annealing above 500℃,
composite coatings maintain HA crystal phase, didn’t occur phase transformation, as showed in
Fig.1. The potentiodynamic polarization curves of the electrolytic TiO2 coated, thermal growth
oxide film, CaP coated, HA/TiO2 composite coated and uncoated specimens in Hank’s solution at
37℃ are plotted in Fig.2. It was found that electrolytic TiO2 coated at the substrate surface have
higher corrosion resistance than the thermal growth oxide film. Furthermore, we also observed that
the corrosion potential and corrosion resistance of the HA/TiO2 composite coating were higher, and
the passivation current density and corrosion current density were lower than single CaP coated or
the uncoated. Apparently, the HA/TiO2 composite coatings exhibit the greatest corrosion resistance.
The higher Ecorr, lower icorr, lower ipass and higher polarization resistance Rp of the HA/TiO2 coated
specimens indicate that the coated film is more resistant to corrosion or more inert than the pure Ti
specimen. Fig.3 show the photographs and EDS mapping of the fractured surfaces of the CaP and
HA/TiO2 coated specimens. In the case of the CaP coated specimen, Ca and P images were weak on
whole surface of the substrate, as showed in Fig.3 (a). This indicated that the fracture occurred at
the interface between the substrate and the CaP coated. CaP coated specimen tensile bonding
strength only about 11.3 MPa. In the case of the HA/TiO2 coated specimens in Fig.3 (b), Ca and P
images were strong on the whole surface of the substrate. This indicates that the fracture occurred at
the HA-glue interface, leaving the HA layer on the substrate. HA/TiO2 coated specimen tensile
bonding strength increase to 46.7 MPa. In addition, osteoblast-like cells on HA/TiO2 coated surface
T(110)
T(102)
T(100)
O:OCP
H:HA
T:Ti
R:Rutile TiO2
pure Ti
electrolytic TiO 2 -300¢J
thermal oxide-300¢J
HA-TiO 2 -300¢J
CaP-300¢J
1000
H(213)
H(222)
R(101)
R(211)
800
H(211)
H(002)
R(110)
Intensity(arbitrary unit)
1000
T(002)
T(101)
exhibiting the polygonal and stellate morphology. The polygonal cells extended their broad
cytoplasmic form fine radiate-like pseudopodia it would enhance cells adhesion and future
migration activity, showed in Fig.4.
thermal oxide-300¢J
HA/TiO 2 -300¢J
electrolytic
TiO 2 -300¢J
pure Ti
CaP-300¢J
600
400
700¢J
O(322)
H (301)
H(201)
O(321)
500
E(mV)
H (210)
200
500¢J
0
-200
300¢J
-400
as-coated
-600
-800
0
10
Ti
20
30
40
50
60
70
-1000
-14
-13
-12
-11
-10
-9
-8
-7
-6
-5
-4
-3
2
log(I)(A/cm )
2£c
Fig.1 XRD diagrams of HA / TiO2 composite
coated specimens as coated and annealed at 300
℃, 500℃ and 700℃, respectively.
Fig.2 Polarization curves of the uncoated (pure
Ti), thermal growth oxide TiO2, electrolytic
deposition TiO2 coated, CaP coated and HA /
TiO2 coated specimens in Hank’s solution at 37
℃.
500μm
500μm
(a)
(b)
ALP (nmole p-nitrophenol/min)
Fig.3 SEM photographs and EDS images of fracture surface of (a) CaP coated, (b) HA / TiO 2
composite coated.
5μm
Fig.4 The surface morphology of the G-292
osteoblast-like cells after 3 days grown
on HA/TiO2 coated.
1
0.8
0.6
0.4
0.2
0
PS
Ti
CaP
HA/TiO2
Fig.5 Alkaline phosphatase activity of the G292 osteoblast-like cells after 14 days
grown on polystyrene (PS), pure Ti, CaP
coated and HA/TiO2 coated
Discussion
The DCPD that didn’t appear on HA/TiO2 coated specimens was attributed to the high
concentration of OH- supplied by the first deposition of TiO(OH)2. Yen et al. (2002) found that very
concentrated OH- environment could transform HPO42 into PO43 . The signification result indicate
that TiO(OH)2 gel not only promoted the HA synthesis, after annealing at 300℃ it would provide a
chemical bonding to increase bonding strength. The excellent adhesion was attributed to the
condensation of TiO(OH)2 with OH bonds adsorbed on Ti substrate and OH bonds of HA to form
the very strong Ti-TiO2-HA chemical bonding. After 3 days incubated, the cell morphology on PS
and Ti surface appeared cytoplasmic generate a more wide spread form. Furthermore, the microvilli
become longer and inter-cellular links. On the contrary, osteoblast-like cells on HA / TiO2 coated
surface exhibiting the polygonal and stellate morphology. The polygonal cells extended their broad
cytoplasmic form fine radiate-like pseudopodia it would enhance cell adhesion and future migration
activity. After 14 days cell culture, we could observe cell differentiation on the HA/TiO2 needle-like
coated specimens had evident increasing contrast with PS and mirror-polished Ti substrate. The
results indicated that roughness needle-like surfaces induced adhesion pseudopodia form a good cell
adhesion. The results presented here also support that materials characteristics may play an
important role, and explain why the reaction of the bio-active materials is better than that of bioinert material. Bio-active materials, such as HA/TiO2 may promote osteogenesis and establish an
interfacial bond with nearby tissues.
Conclusions
A novel electrolytic coatings method of HA/TiO2 composite coatings has been successfully
conducted on pure titanium to investigate its characteristics. Through the electrolytic deposition,
annealing, dynamic polarization tests, surface observations, XRD analysis, bonding strength tests
and cell culture assays, several conclusions are drawn:
1. The previous TiO2 coating and/or the further annealing had the effects on reducing the amount
of DCPD and OCP in HA, and the crystallization of HA on the CaP and HA/TiO2 coated
specimens annealed at 300℃ was the best. In addition to, it also stabilized the HA-coated film
to avoid high temperature phase transformation (Ca2P2O7) and β-Ca3(PO4)2).
2. The adhesion strength of electrolytic deposited HA on Ti substrate was dramatically improved
from the 11.3 MPa to 46.7 MPa by adding the intermediate electrolytic deposition of TiO2,
which showed the strong chemical bonding effects between Ti alloy substrate and HA coating.
3. The polarization tests in Hank’s solution revealed that the HA/TiO2 coated was the greatest
corrosion resistant.
4. HA/TiO2 composite coatings served as good substrates for the adhesion and spreading of the
osteoblast-like G-292 cell. From the cell activity assay, the mirror-polished surface evident
more cell proliferation. This proliferation rate can qualitatively be related smooth surfaces and
produce less adhesion pseudopodia, thus promoting their proliferation. In contrast to roughness
needle-like HA/TiO2 coated specimens, they induce adhesion pseudopodia form a good cell
adhesion and further cell differentiation.
References
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of roughness, fibronectin and vitronectin on attachment, spreading, and proliferation of human
osteoblast-like cells (Saos-2) on titanium surfaces, Calcified tissue international, 64:499-507, 1999
Nie X, Leyland A, Matthews A. Deposition of layered bioceramic hydroxyapatite/TiO2 coatings on
titanium alloys using a hybrid technique of micro-arc oxidation and electrophoresis, Surface
coatings technology, 125:407-414, 2000
Designation: C-633. Standard test method for adhesion of cohesive strength of flame-sprayed
coatings, Annual book of ASTM standards, Philadelphia, 3.01:665-669, 1993
Yen SK, Lin CM. Characterization of Electrolytic Al2O3/CaP Composite Coatings on Pure
Titanium, Journal of the Electrochemical Society, 149:79-87, 2002