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E
FFECTS OF
D
ISSOLVED
C
ALCIUM AND
P
HOSPHOROUS ON
O
STEOBLAST
R
ESPONSES
S. Ma, BS
Y. Yang, PhD
D. L. Carnes, PhD
K. Kim, PhD
S. Park, PhD, DDS
S. H. Oh, PhD
J. L. Ong, PhD
K
EY WORDS
Hydroxyapatite
Materials characterization
Dissolution
Alkaline phosphatase
Specific activity
Osteocalcin
S. Ma, BS, and D. L. Carnes, PhD, ar e
withtheSchoolofDentistryatthe
University of Texas Health Science Center
at San Antonio.
Y.Yang,PhD,S.H.Oh,PhD,and J. L.
Ong, PhD, are with the Departments of
Biomedical Engineering and Orth opedic
Surgery , University of Tennessee Health
Science Center, Memphis, Tenn. Address
correspondence to Dr Yang at 920 Madison
A venue, Suite 1005, Memphis, TN 38163
(e-mail: yyang19@utmem.ed u).
K. Kim, PhD, is with the Department of
Dental Biomaterials, College of Dentistry
and Institute of Biomaterials Research and
Development, Kyungpook National
University , Jung-Gu, Daegu, Korea.
S. Park, PhD, DDS, is with the
Department of Prosthodontic s,
College of Dentistry , Chonnam
National University, Dong-Gu,
Gwangju, Korea.
The dissolution behavior of hydroxyapatite (HA) and its effect on the
initial cellular response is of both fundamental and clinical importance.
In this study, plasma-sprayed HA coatings were characterized by X-ray
diffraction and Fourier transform infrared spectroscopy (FTIR).
Calcium (Ca) and inorganic phosphorous (Pi) ions released from
plasma-sprayed HA coatings within 3 weeks were measured by flame
atomic absorption and colorimetrically molybdenum blue complex,
respectively. To investigate the effect of dissolution of HA coatings on
osteoblast response, additional Ca and Pi were added into the cell
culture media to simulate the dissolution concentrations. Human
embryonic palatal mesenchyme cells, an osteoblast precursor cell line,
were used to evaluate the biological responses to enhanced Ca and Pi
media over 2 weeks. Osteoblast differentiation and mineralization were
measured by alkaline phosphatase–specific assay and 1,25 (OH)
2
vitamin D
3
stimulated osteocalcin production. The coatings exhibited
an HA-type s tructure. FTIR indicated t he possible pres ence of
carbonates on the coatings. A dissolution study indicated a continual
increase in Ca and Pi over time. In the cell culture study, enhanced
osteoblast differentiation occurred in the presence of additional Ca
concentration in the cell culture media. However, additional Pi
concentration in the cell culture media was suggested to slow down
osteoblast differentiation and mineralization.
I
NTRODUCTION
D
ental and ortho-
pedic implant sur-
faces have been
altered with hy-
droxyapatite (HA)
and calcium phos-
phate (CaP) coatings, with the
assumption that osteointegration
of the implants can be improved.
As such, properties of HA and
CaP ceramics and coatings have
been ext ensively studied.
1–5
In
particular, t he crystallinity of
HA and other CaP ceramics are
of great interest because of their
dissolution properties.
6
It is
known that amorphous or
smaller imperfect crystals have a
higher dissolution rate compared
with crystalline compounds.
6
It
has also been reported that the
incorporation of sodium and car-
bonate ions in the HA and CaP
structure greatly increases the
dissolution rate.
7
Journal of Oral Implantology 61
RESEARCH
Despite the slow dissolution
rate of highly crystalline HA
ceramics, it was also reported that
the in vivo cellular response could
be compromised by its high crys-
tallinity, indicating that some
amorphous or more soluble
phases in the coatings would be
more desirable and result in
a more stable interface with the
biological environment.
8
How-
ever, the science of the bone-
implant interface is still not fully
understood. As reported in our
previous studies, the ultimate in-
terfacial strength and bone-im-
plant contact length were higher
for more amorphous CaP-coated
implants when compared with
more crystalline CaP-coated im-
plants.
9
As such, in this study, it
was hypothesized that osteoblast
differentiation and onset of min-
eralization would be affected by
the dissolved calcium (Ca) and
inorganic phosphorous (Pi) con-
centration in the tissue culture
media. Thus, the objective of this
study was to evaluate the proper-
ties of plasma-sprayed HA coat-
ings and to measure the effect of
additional Ca and Pi concentra-
tion on osteoblast response.
M
ATERIALS AND METHODS
Hydroxyapatite
Sterile plasma-sprayed HA-
coated disks (10.1 mm diameter 3
2.15 mm thick) were obtained
from Friatec AG (Mannheim,
Germany).
X-ray diffraction
X-ray diffraction (XRD) was per-
formed to evaluate the structure
of the HA coatings before the
experiment. A Siemens D500 dif-
fractometer (New York, NY) us-
ing Cu K
a
radiation with energies
of 40 keV and 30 mA was used.
The incident X rays passed
through 38- and 18-slits before
impinging upon the CaP coatings.
Diffracted X rays passed through
18-, 0.68-, and 0.058-slits at the X-
ray counter. Three HA samples
were analyzed and the data were
collected from 208 to 558 2h at 0.18
per minute scan rate. Crystalline
coatings were identified by
matching the peaks with standard
synthetic HA. The lattice param-
eters and crystallite size (1 SD)
were calculated based on the 410
reflection for the a-lattice spacing
and 004 reflection for the c-lattice
spacing. The crystallite size was
calculated by the Scherrer equa-
tion.
Fourier transform infrared
spectroscopy
The molecular composition and
structure of HA coatings was
evaluated with a Magna-IR Spec-
trometer 550 (Nicolet Instrument
Corp, Madison, Wis) interfaced
with a SpectraTech microscope
(Stamford, Conn). Triplicate sam-
ples were collected at a resolution
of 4 cm
1
and a scan number
of 100. The Ti surface was used
as a reference for background
subtraction.
Dissolution study
Hydroxyapatite-coated disks were
immersed in a 2.5-mL 1.0-M Tris
buffer containing 80 lM NaCl,
with pH of the solution balanced
at 7.4 before the study. The study
was performed in triplicates in
a sterile and humidified atmo-
sphere of 95% O
2
air and 5% CO
2
at 378C for 21 days. The buffer
medium was changed daily. As
the buffer medium was collected
each day, the volume withdrawn
and pH were recorded. Each
withdrawn buffer medium was
saved for subsequent analysis of
Ca and Pi ions released.
Measurement of Pi
Released Pi ions were measured
colorimetrically by using the re-
action of ammonium molybdate
and ascorbic acid with the Pi to
obtain a molybdenum blue com-
plex. The reaction was done in
a 96-well microtiter plate. Each
sample was diluted 10-fold to
make a 100-lL solution. A work-
ing solution was made by com-
bining 2 parts double-deionized
water, 1 part 5.0 N H
2
SO
4
(Baker
analyzed), 1 part 0.01 M ammo-
nium molybdate tetrahydrate
(Sigma Chemical Co, St Louis,
Mo) in water, and 1 part 10%
ascorbic acid (Sigma). The work-
ing solution was made fresh for
each assay. A 100-lL working
solution was added to 100-lL
sample. After 1 hour at room
temperature, the complex was
read at 750 nm on a Dynatech
MR5000 microplate reader (Dy-
nex, Middlesex, UK).
10–12
The 1.0-
M Tris buffer containing 80 lM
NaCl (pH 7.4) was used as a base-
line reference. At an a level of
0.05, statistical analyses for Pi
release was carried out by analy-
sis of variance (ANOVA).
Measurement of inorganic
Ca ions
Released Ca ions were measured
with a flame atomic absorption
spectrophotometer. The blank
was prepared by adding 90.0 mL
double-distilled deionized water
to 10.0 mL 103 1% LaCl
3
20%
HNO
3
. Samples were prepared by
combining 2.0 mL of sample from
dissolution media to 0.2 mL 103
1% LaCl
3
20% HNO
3
.Samples
were diluted with 0.1% LaCl
3
2.0% HNO
3
. The samples were
measured at 422.7 nm and energy
of 49 keV by using a Perkin Elmer
3030 atomic absorption spectro-
photometer (Wellesley, MA) with
a Perkin Elmer intenistron calcium
lampwitha slit of0.78 and acurrent
of 10 A. The 1.0-M Tris buffer
containing 80 lMNaCl(pH7.4)
was used as a baseline reference.
OSTEOBLAST RESPONSES TO DISSOLVED CALCIUM AND PHOSPHOROUS
62 Vol. XXXI/No. Two/2005
At an a level of 0.05, statistical
analyses for Ca release was carried
out by ANOVA.
Preparation of cell culture
medium
Four groups of Dulbelco modi-
fied eagles media (DMEM) con-
taining different Ca and Pi
concentrations were prepared.
The low and high Ca and Pi
correspond to the dissolution
measured at day 3 and day 21,
respectively (Table). In this study,
the low and high Ca media were
prepared by adding 0.1 and 0.2
lg/mL of CaCl
2
into DMEM
media, respectively. The low and
high Pi media were prepared by
adding 2.3 and 6.1 lg/mL of
NaH
2
PO
4
into the DMEM, re-
spectively. In addition, a DMEM
without additional Ca or Pi was
used as control.
Cell culture study
Titanium (Ti) disks of 13 mm in
diameter and 2 mm thick were
ground to 600 grits, ultrasonically
cleaned with acetone and ethanol,
and passivated with 40% (vol-
ume) HNO
3
at room tempera-
ture. The disks were then steril-
ized with ultraviolet light for 48
hours before placing them in 24-
well tissue culture plates. Amer-
ican type culture collection 1486
human embryonic palatal mesen-
chyme cells, an osteoblast pre-
cursor cell line, were then seeded
on the Ti surfaces at a concentra-
tion of 20 000 cells/mL. Five
groups of media were used for
cell culture, and the media was
changed twice a week. Triplicate
samples were analyzed for cellu-
lar differentiation by measuring
the alkaline phosphatase (ALP)-
specific activity over 9 days post-
confluency. Differences in cellular
responses to dissolved Ca and Pi
were statistically compared by
the ANOVA test.
ALP-specific assay
On the day of the assay, medium
was removed from the cell cul-
tures and the cell layers were
lysed with 1 mL Triton X-100
(0.2%). An aliquot of the triton
lysate (50 lL) was added to 50 lL
of working reagent containing
equal parts (1:1:1) of 1.5-M 2-amino-
2-methyl-1-propanol (Sigma), 20
mM p-nitrophenyl phosphate
(Sigma), and 1 mM magnesium
chloride. The samples were then
incubated for 1 hour at 378C. After
incubation, the reaction was stop-
ped with 100 lL of 1 N NaOH, and
the absorbance was read at 410 nm
with a microplate reader. Alkaline
phosphatase activity was deter-
mined from the absorbance by
a standard curve prepared from
p-nitrophenol stock standard
(Sigma). The ALP-specific activity
was statistically compared by
ANOVA.
1,25 (OH
2
) vitamin D
3
stimulated osteocalcin
production
1,25 (OH
2
) vitamin D
3
stimulated
osteocalcin production was mea-
sured with a commercially avail-
able midtact human osteocalcin
EIA kit (Biomedical Technologies
Inc, Staughton, Mass). On the day
of the assay, the medium was
removed from the cultures and
stored at 48C until assayed, when
the samples were thawed to room
temperature. The samples (25 lL)
or human osteocalcin standard
(25 lL) were added to the micro-
titer plate that came with the kit.
This was followed by the addition
of the osteocalcin antiserum (100
lL). The microtiter plate was then
swirled gently for 1 minute and
covered, followed by incubation
at 378C for 2½ hours. The solu-
tion was then aspirated and the
plate was washed 3 times with 0.3
mL of phosphate buffer solution.
After washing, 100 lL of strepta-
vidin-horseradish peroxidase re-
agent was added to all wells,
swirled, and incubated at room
temperature for 30 minutes. The
media was again aspirated and
the plate was washed 3 times
with 0.3 mL of phosphate buffer
solution. A mixture of 100 lLof
3,39,5,59 tetramethylbenzidine and
hydrogen peroxide solution (1:1)
was then added to all wells and
incubated in the dark at room
temperature for 15 minutes. This
was followed by the addition of
H
2
SO
4
(100 lL) to stop the re-
action. Absorbance was then im-
mediately read at 450 nm.
Osteocalcin concentrations were
determined by a standard curve
prepared from the osteocalcin kit.
Differences in 1,25 (OH
2
) vitamin
D
3
stimulated osteocalcin pro-
duction were statistically com-
pared by the ANOVA test at an
a value of 0.05.
R
ESULTS
X-ray diffraction
As shown in Figure 1, HA coat-
ings were observed to have HA-
type structure, with the peaks
matching Joint Committee on
Powder Diffraction Standards
(JCPDS) 9-0432. The sharp and
distinct peaks indicated a more
crystalline coating. However,
a slight shift in the XRD peaks
was observed as compared with
the peak positions reported in the
TABLE
Release of calcium (Ca) and
inorganic phosphorous (Pi) ions
from hydroxyapatite surfaces after
21 days in solution
Day
Released Ca
(lg/mL)
Released Pi
(lg/mL)
3 0.14 6 0.01 2.55 6 0.09
7 0.17 6 0.01 3.70 6 0.13
14 0.22 6 0.01 5.48 6 0.11
21 0.27 6 0.02 6.82 6 0.03
S. Ma et al
Journal of Oral Implantology 63
JCPDS index. The a- and c-lattice
spacings for the HA coatings
were 9.39 6 0.002 A
˚
and 6.88 6
0.0004 A
˚
, respectively. Crystallite
size was 2779 6 3A
˚
in the c
direction and 461 6 11 A
˚
in the
a direction.
Fourier transform infrared
spectroscopy
A representative Fourier trans-
form infrared spectroscopy
(FTIR) spectrum of HA coatings
is shown in Figure 2. Broad
absorption bands in the range
865 to 1039 cm
1
and 1108 to
1414 cm
1
were observed for the
coatings, indicating presence of
PO
4
. The broad band in the region
of 1400 cm
1
also indicates possi-
ble traces of CO
3
. A strong OH
band at 3568 cm
1
was also
observed for the coatings.
Ca release
During the 21-day period, no
significant change from the initial
pH of 7.4 was observed. As
shown in the Table, a continual
increase in Ca release was ob-
served for HA coatings immersed
in solution. Released Ca was
observed to increase from 0.14 6
0.01 lg/mL at day 3 to 0.27 6 0.02
lg/mL at day 21.
Pi release
Similarly, a continual increase in
Pi was also observed for HA
coatings immersed in Tris buffer
(Table). Released Pi was observed
to increase from 2.55 6 0.09 lg/
mL at day 3 to 6.82 6 0.03 lg/mL
at day 21.
ALP-specific activity
From the dissolution study, 0.2 lg
CaCl
2
/mL (high Ca), 0.1 lgCaCl
2
/
mL (low Ca), 6.1 lg NaH
2
PO
4
/mL
(high Pi), or 2.3 lg NaH
2
PO
4
/mL
(low Pi) were added to DMEM. As
shown in Figure 3, osteoblast pre-
cursor cells were observed to in-
duce statistically higher ALP-
specific activity when cultured
with media containing additional
Ca compared with the cells cul-
tured with media containing ad-
ditional Pi and control media. The
ALP-specific activity for cells cul-
tured on high and low Pi media
was observed to remain signifi-
cantly low compared with cells
cultured with the control media.
1,25 (OH
2
) vitamin D
3
stimulated osteocalcin
production
Similar to the ALP-specific activ-
ity, the 1,25 (OH)
2
vitamin D
3
stimulated osteocalcin produc-
tion for cells cultured in high
and low Pi media was observed
to remain significantly low com-
pared with cells cultured with the
control media (Figure 4). No
significant difference of 1,25
(OH)
2
vitamin D
3
stimulated os-
teocalcin produced by the cells in
control media and media contain-
ing additional Ca was observed.
D
ISCUSSION
In many dissolution studies re-
ported in the literature, acidic,
basic, and buffered physiological
salt solutions have been
used.
7,13,14
In addition, the suc-
cess of an implant was recently
suggested to depend on its ability
to resorb or degrade, thereby
allowing cellular penetration.
15,16
All CaP coatings will degrade or
dissolve to some degree, regard-
less of the degree of crystallinity.
17
Because amorphous phases are
expected to dissolve more rapidly
than crystalline phases,
18
it is
possible that the amorphous CaP
phases could control the initial
biological response.
In this study, the dissolution
properties of HA were investi-
gated with a commercially avail-
FIGURE 1–2. FIGURE 1. X-ray diffraction of a representative plasma-sprayed hydroxyapatite (HA) surface before the study. FIGURE 2.
Fourier transform infrared spectroscopy spectrum of a representative plasma-sprayed HA surface before the study.
OSTEOBLAST RESPONSES TO DISSOLVED CALCIUM AND PHOSPHOROUS
64 Vol. XXXI/No. Two/2005
able plasma-sprayed HA coating.
Because it is known that sur-
face properties of implants play
critical roles in bone-implant
interactions, the HA coatings
were characterized before the cell
culture study.
By using XRD analyses, the
HA coatings were observed to
exhibit a crystalline HA-type
structure. No significant differ-
ence in the c-lattice spacings was
observed between the coatings
and the spacings reported in the
JCPDS index; however, the a-
lattice spacings observed in the
coatings was 9.36 6 0.002 A
˚
compared with the 9.41 A
˚
re-
ported in the JCPDS index. The
slight contraction in the a-lattice
spacing has been attributed to
many factors, such as the pres-
ence of carbonates.
19
The crystal-
lite size of the coatings in the c and
a directions was 2779 6 3A
˚
and
461 6 11 A
˚
, respectively, suggest-
ing a hexagonal structure. A
crystallite size of about 5000 A
˚
has been reported for HA pow-
ders.
20,21
These differences in
crystallite size and lattice spac-
ings have been associated with
the alteration of structural prop-
erties during the plasma-spraying
process. In addition, a slight shift
in the XRD peak positions of the
coatings, as compared with the
JCPDS index, suggested a strain
associated with interfacial inter-
actions between the Ti substrate
and the coatings and also the
presence of other contaminants.
19
FTIR analyses indicated broad
absorption bands in the range 865
to1039 cm
1
and 1108 to 1414 cm
1
for the coatings, suggesting the
presence of PO
4
. The presence of
a band in the region of 1400 cm
1
also indicated the presence of CO
3
in the coatings. A strong absorp-
tion band at about 3568 cm
1
was
observed in the HA coatings, in-
dicating bounded crystalline
OH.
14
This strong OH absorption
band indicated that the hydroxyl
group was not lost during the
plasma-spraying process.
During the 21-day immersion
study, a daily change in buffer was
used to minimize or eliminate any
changes in pH. As expected, the
pH of the media remained stable
at 7.4 6 0.1. In a dissolution study,
HA was reported to continue to
dissolve as long as it is subjected
to an undersaturated environ-
ment, regardless of the crystalline
phase.
22
This continuous release
of Ca and Pi was observed in this
study. As with this study and in
previous studies, Pi released as
a result of dissolution of HA into
an undersaturated solution con-
sequently yielded a continual in-
crease in Pi in the solutions.
23
However, as observed in this
study, the rate of Pi dissolution
from HA coatings was different
from the dissolution rate of Ca.
Difference in rate of Ca and Pi
released may be due to the bind-
ing of Ca and Pi in the form of
a phosphate and the equilibrium
possibility of different ion species
in solution. It is worth nothing
that the Pi release is enhanced in
the presence of protein.
24
In in vitro cell culture studies, 2
other biochemical markers, the
ALP-specific activity and osteo-
FIGURES 3–4. FIGURE 3. Alkaline phosphatase–specific activity of osteoblast precursor cells in control media without additional
calcium (Ca) or inorganic phosphorous (Pi) addition, in media with additional high and low Ca addition, and in media with
additional high and low Pi addition. F
IGURE 4. Osteocalcin production of osteoblast precursor cells in control media (without
additional Ca or Pi addition), in media with additional high and low Ca addition, and in media with additional high and low
Pi addition.
S. Ma et al
Journal of Oral Implantology 65
calcin level, are used as markers
for determining osteoblast pheno-
type and are considered to be
important factors in determining
bone mineralization.
25–27
Cells
grown on media containing high
and low Ca
2þ
were observed to
exhibit a significantly higher ALP-
specific activity over the course of
the study, indicating significantly
greater cellular differentiation. It
has been suggested that the low
ALP-specific activity and 1,25
(OH)
2
vitamin D
3
simulated os-
teocalcin production on media
containing high and low Pi could
be attributed to many factors,
including apoptosis of osteoblast
cells in culture. In other studies,
Pi-treated cells have been re-
ported to display profound loss
of mitochondrial membrane po-
tential, suggesting that Pi acti-
vated the death program through
the induction of a mitochondrial
membrane permeability transi-
tion.
28,29
However, it has also been
suggested that cell apoptosis oc-
curs only when cells are close to
the elevated Pi levels, whereas
increased osteoblast proliferation,
biosynthetic, and mineralization
activities will occur when ele-
vated levels of Pi are at a distance
from the site of active bone re-
sorption. In addition, a recent in
vivo study reported that the
poorly crystalline HA and b-TCP
ceramics inhibited bone regener-
ation when compared with crys-
talline HA in a healing tibial
wound.
30
The inhibition of bone
regeneration is probably attrib-
uted to the local elevated Pi con-
centration released from poorly
crystalline HA and resorbable b-
TCP in vivo.
These observations indicate
that the HEPM cells displayed
a more differentiated osteoblast-
like phenotype on biomaterials
surfaces that can release Ca,
suggesting that surfaces capable
of releasing more Ca may be more
advantageous for bone-biomate-
rial interface reactions. In addi-
tion, this study shows the
importance of characterizing HA
surfaces and the governing effect
of Ca and Pi released on the
expression of osteoblast charac-
teristics in vitro.
C
ONCLUSIONS
Osteoblast cells were observed to
respond differently to the differ-
ent concentration of Ca and Pi in
the media. In this study, en-
hanced osteoblast differentiation
occurred in the presence of addi-
tional Ca concentration in the cell
culture media. However, addi-
tional Pi concentration in the cell
culture media was suggested to
slow down osteoblast differentia-
tion and mineralization.
A
CKNOWLEDGMENT
This study was funded by NIH/
NIAMS 1RO1AR46581. We also
thank the postdoctoral fellowship
program of the Korea Science
and Engineering Foundation
(KOSE) for its support.
R
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