The most appropriate (Ca+Zn)/P molar ratio to minimize

the zinc content of ZnTCP/HAP ceramic used in the

promotion of bone formation

Yu Sogo, Tokoha Sakurai, Kazuo Onuma, Atsuo Ito

Tissue Engineering Research Center, National Institute of Advanced Industrial Science and Technology, Central 4,
1-1-1 Higashi, Tsukuba-shi, Ibaraki 305-8562, Japan

Received 6 July 2001; revised 6 December 2001; accepted 19 December 2001

Abstract: The (Ca+Zn)/P molar ratio of zinc-containing when the (Ca+Zn)/P molar ratio is increased to 1.64. The
tricalcium phosphate and hydroxyapatite composite ceram- relative growth rate of MC3T3-E1 cells cultured on ZnTCP/
ics (ZnTCP/HAP) was investigated in the range from 1.50 to HAP1.64 had a maximum value of 1.3 at a zinc content of
1.66 to reveal the minimum zinc content possible in ceramics 0.51 wt %; however, a 1.20 Zn wt % is required to attain the
used in the promotion of osteoblastic cell proliferation and same value in relative growth rate in the case of ZnTCP/
bone formation. The release rate of zinc increased with an HAP1.60. A reduction in zinc content at a certain zinc release
increase in the (Ca+Zn)/P molar ratio up to 1.64 (ZnTCP/ rate results from an increase in zinc content of the ZnTCP
HAP1.64) and then decreased over 1.64 even when the zinc phase in ZnTCP/HAP. The ZnTCP/HAP1.64 would be bet-
content of ZnTCP/HAP was the same. The zinc release rate ter than any other ZnTCP/HAP relative to material safety
of ZnTCP/HAP1.64 with a 0.21 Zn wt % nearly coincided because the risk of an accidental burst of zinc from the ma-
terial is minimized due to the low zinc content. © 2002 Wiley
with that of ZnTCP/HAP1.60 with a 0.316 Zn wt %, which
Periodicals, Inc. J Biomed Mater Res 62: 457–463, 2002
was found be the most effective for promoting bone forma-
tion in rabbit femora. This means that the zinc content Key words: zinc; tricalcium phosphate; hydroxyapatite; cell
needed to promote bone formation can be reduced by 34% proliferation

INTRODUCTION content, with maximum bone formation shown at a

zinc content of 0.316 wt %. However, there was no
Zinc-containing ␤-tricalcium phosphate (ZnTCP) statistical significance between the ZnTCP and TCP
functions as a carrier of zinc, an essential element that relative to the amount of bone formed.4
has stimulatory effects on bone formation in vitro and The zinc content of zinc-releasing calcium phos-
in vivo. 1,2 Using the ZnTCP, two types of zinc- phate ceramics can be reduced without losing the
releasing calcium phosphate ceramics have been de- stimulatory effects on bone formation by changing the
veloped for promoting bone formation: one is a com- (Ca+Zn)/P molar ratio. The (Ca+Zn)/P molar ratio
posite of ZnTCP and hydroxyapatite (HAP) with a ranges from 1.50 to 1.67; the former corresponds to
(Ca+Zn)/P molar ratio of 1.60 (ZnTCP/HAP1.60), and TCP-monophasic material and the latter to HAP-
the other is a monophasic ZnTCP ceramic with a monophasic material. A change in the (Ca+Zn)/P mo-
(Ca+Zn)/P molar ratio of 1.50. ZnTCP/HAP1.60 sig- lar ratio results in a change in the distribution of zinc
nificantly promoted mouse osteoblastic MC3T3-E1 cell between the TCP and the HAP phases even if the over-
proliferation in vitro at a zinc content of 1.20 wt %.3 all zinc content of ZnTCP/HAP is unchanged.
The bone formation around a ZnTCP/HAP1.60 im- The zinc content of ZnTCP controls the solubility of
plant in rabbit femora increased by 51% (p = 0.0509) at the ZnTCP phase.5 Moreover, it is well established
a zinc content of 0.316 wt % in comparison with that that the solubility of TCP/HAP biphasic ceramics de-
around a TCP/HAP implant with a Ca/P molar ratio pends on their Ca/P ratios.6–9 From the viewpoint of
of 1.60. The bone formation around a ZnTCP implant material safety, the less the zinc content is, the less the
in rabbit femora also appeared to be a function of zinc risk of an accidental burst of zinc from the material
there will be. However, only limited information is
Correspondence to: Y. Sogo; e-mail: [email protected]
available with regard to the effects of (Ca+Zn)/P mo-
© 2002 Wiley Periodicals, Inc. lar ratio on the kinetics of zinc release and thus on the
458 SOGO ET AL.

optimum zinc content for promoting osteoblastic cell and CaZn2(PO4)2 phases because the solid solution limit of
proliferation and bone formation. zinc in TCP is 12 mol %.11
The purpose of the present study was to determine ZnTCP powder was mixed with stoichiometric and pure
the effect of (Ca+Zn)/P molar ratio on the release ki- HAP and TCP powders (Advance Co.) with the chemical
netics of zinc and to discover the optimum (Ca+Zn)/P compositions listed in Table I. The mixture was ground,
molar ratio that would minimize zinc content and still supplemented with 3 wt % poly(vinyl alcohol) and 1 wt %
promote osteoblastic cell proliferation and bone for- poly(ethylene glycol), sieved to under 75 ␮m, pressed at 98
MPa, and sintered at 1100°C for 1 h to obtain ceramic disks
mation.
with (Ca+Zn)/P ratios of 1.50 (ZnTCP), 1.60 (ZnTCP/
HAP1.60), 1.64 (ZnTCP/HAP1.64), and 1.66 (ZnTCP/
HAP1.66) [Table I]. The size of the ceramics was 13.5 ± 0.1
mm in diameter and 1.0 ± 0.1 mm in thickness.
MATERIALS AND METHODS
Powder X-ray diffraction patterns were measured using a
powder X-ray diffractometer (Model Rint 2400; Rigaku, Ja-
ZnTCP powders with zinc contents of 10 and 30 mol % pan). The contents of HAP and ZnTCP or TCP phases in the
were prepared by the solution method described else- ceramics were determined from the ratio of TCP (0210) peak
where.10 Briefly, starting materials were high-purity calcium intensity to HAP (211) peak intensity. For calibration, the
carbonate (99.99 wt %, Ube Materials, Ube, Japan), reagent- intensity ratios of the mixtures consisting of known amounts
grade phosphoric acid (85 wt %, Nacalai Tesque, Kyoto) and of the stoichiometric TCP and HAP powders also were mea-
reagent-grade zinc nitrate hexahydrate (98 wt %, Kanto sured. Prior to the mixing, the stoichiometric TCP and HAP
Chemical Co., Tokyo). A suspension of calcium hydroxide powders were heated at 1100°C for 1 h to adjust their peak
(0.6 Ca mol/L) was prepared using calcium oxide obtained sharpness to those of the ZnTCP/HAP ceramics. All peak
by heating the calcium carbonate at 1000°C for 3 h. The intensities were determined after deconvolution of peaks.
suspension was mixed with a zinc nitrate hexahydrate so- The zinc contents of the ZnTCP phase in the ceramics
lution (0.5 Zn mol/L) and a phosphoric acid solution (0.5 P were determined from the positional shift of the ZnTCP
mol/L), followed by filtration and heat treatment at 850°C (0210) peak (n = 6). Silicon (NBS X-ray powder diffraction
for 1 h. The 10 mol % ZnTCP powder is a monophasic pow- reference, 630b) was used as the external standard of the
der, but the 30 mol % ZnTCP powder is a mixture of ZnTCP peak position. For calibration, ZnTCP powders containing 0,

TABLE I
The Samples Used in the Present Study with Their Chemical Composition and Starting Materials
Sample Chemical Composition (wt %) Starting Materials (wt %)
Total
Zinc content
Series (wt %) CaO ZnO P2O5 Measured Theoretical TCP ZnTCP HAP
ZnTCP/HAP1.64
0.00 55.13 <0.01 43.77 98.89 98.48 15.00 0.00a 85.00
0.05 55.27 0.06 43.77 99.10 98.48 13.66 1.36a 84.98
0.13 55.27 0.16 43.77 99.20 98.48 13.09 1.94a 84.96
0.21 55.27 0.26 43.99 99.52 98.48 12.14 2.92a 84.94
0.25 55.13 0.31 43.77 99.20 98.48 11.19 3.89a 84.92
0.31 55.13 0.39 43.77 99.28 98.48 10.24 4.86a 84.90
0.32 55.13 0.40 43.77 99.29 98.48 9.28 5.83a 84.88
0.51 54.85 0.63 43.77 99.25 98.48 7.38 7.78a 84.85
0.65 54.85 0.81 43.77 99.42 98.48 5.48 9.72a 84.81
0.89 54.71 1.11 43.99 99.81 98.48 1.67 13.61a 84.73
1.09 54.43 1.36 43.77 99.55 98.48 8.60 6.79b 84.61
2.01 53.73 2.50 43.77 100.00 98.49 2.21 13.58b 84.21
ZnTCP
0.32 53.59 0.40 46.74 100.73 100.00 95.14 4.85a 0.00
ZnTCP/HAP1.60
0.33 54.43 0.41 44.68 99.52 98.89 33.39 4.86a 61.75
ZnTCP/HAP1.66
0.27 55.27 0.34 43.54 99.14 98.28 2.13 1.70b 96.17
HAP
0.00 55.41 <0.01 42.39 97.80 96.6 — — —
TCP
0.00 54.15 <0.01 46.06 100.21 100.00 — — —
10 mol % ZnTCP
6.52 46.73 8.12 45.60 100.45 100.00 — — —
a b
ZnTCP powder containing zinc at 10 mol% was used; ZnTCP powder containing zinc at 30 mol% was used.
MINIMIZING ZINC IN ZNTCP/HAP CERAMIC 459

2, 4, 6, 8, and 10 mol % of zinc were used as the standard of

the positional shift of the peak. The standard ZnTCP pow-
ders were prepared by heating the mixture of the stoichio-
metric TCP and the 10 mol % ZnTCP at 1100°C for 1 h. The
zinc content of the ZnTCP phase in the ceramics was ana-
lyzed statistically by one-way analysis of variance (ANOVA)
followed by the Fisher’s protected least significant difference
test.
The ceramic disks were immersed in 20 mL of a sodium
acetate–acetic acid buffer solution (0.1 mol/L) at 37°C for 8
days. The initial pH of the buffer solution was adjusted to
4.9. Every 2 days the pH value of the solution was measured
and 2 mL of the solution was pipetted out to analyze for
released zinc, calcium, and phosphorus using inductively
coupled plasma–atomic emission spectroscopy (ICP; SPS-
7800, Seiko Instruments, Tokyo).
The control was ZnTCP/HAP1.60 with a zinc content of
0.316 wt % that had been proven to promote bone formation
in vivo.3 The release tests were carried out three times inde-
pendently for each kind of ZnTCP/HAP. Values of ion ac-
tivity products for HAP, octacalcium phosphate, dicalcium
phosphate dihydrate, dicalcium phosphate anhydrous, ho-
peite [Zn3(PO4)2⭈H2O], ZnTCP, and amorphous calcium
phosphate were calculated from the measured concentration
and pH in order to check whether the solutions were satu-
rated with respect to these phases during the release test.
ZnTCP/HAP1.64 ceramic disks (n = 8) were sterilized by
heating at 160°C for 2 h. One mL of alpha-modified mini-
mum essential medium supplemented with 10% fetal bovine
serum (␣MEM + 10% FBS) containing mouse osteoblastic
MC3T3-E1 cells at a concentration of 15,000 cells per mL was
poured onto the disks. The control was TCP/HAP disks
with a Ca/P molar ratio of 1.64 prepared under the same
conditions.
The MC3T3-E1 cells were cultured for 4 days followed by
fixation with 0.5 mL of calcium-free magnesium-free phos-
phate-buffered saline [PBS(−)] solution containing 2.5% glu-
taraldehyde and staining with crystal violet. A blind count-
ing of the number of cells on the disks was carried out using
an optical reflection microscope in which the types of the
disks were unidentified to the counter. The number of cells
was analyzed statistically by one-way ANOVA followed by
the Fisher’s protected least significant difference test. The
␣MEM + 10% FBS after the cell culture was analyzed for zinc
using an ICP spectrometer.

RESULTS
Figure 1. (A) Release of zinc from the ZnTCP/HAP with
zinc content nearly equal to 0.3 wt % immersed in the so-
Under a constant zinc content nearly equal to 0.30 dium acetate–acetic acid buffer solution. (B) Release of cal-
wt %, the release rate of zinc increased with an in- cium and zinc from the ZnTCP/HAP, with zinc content
crease in (Ca+Zn)/P molar ratio in the range of 1.50 to nearly equal to 0.3 wt % immersed in the sodium acetate–
1.64 [Fig. 1(A)]. As a result, the initial release rate of acetic acid buffer solution. Solid lines show the release of
calcium and dotted lines show the release of phosphorus.
ZnTCP/HAP1.64 was 1.7–3.8 times higher than those
of ZnTCP and ZnTCP/HAP1.60. However, when the
(Ca+Zn)/P molar ratio exceeded this range, the re- HAP1.66. This can be attributed to the precipitation of
lease rate of zinc decreased, as shown in the case of zinc that contained poorly crystallized apatite because
ZnTCP/HAP1.66. After 4 days, the release rate of zinc the solution had become saturated with respect to apa-
decreased to zero or less, except in the case of ZnTCP/ tite except in the case of ZnTCP/HAP1.66.
460 SOGO ET AL.

The saturation was revealed by the values of ionic

activity product, (Ca2+)10(PO43−)6(OH−)2, which are in
the range of the solubility product value of HAP,
10−118.7–10−115.0 (Table II).12–14 Throughout the release
tests, all solutions were undersaturated with respect to
octacalcium phosphate, dicalcium phosphate dihy-
drate, dicalcium phosphate anhydrous, hopeite,
ZnTCP, and amorphous calcium phosphate. Calcium
and phosphorus concentrations continued to increase
even after the saturation [Fig 1(B)]. The (Ca+Zn)/P
molar ratios of the buffer solutions were maintained in
the range from 1.49 to 1.57 during the release tests
regardless of the (Ca+Zn)/P molar ratios of the ceram-
ics (Fig. 2). Thus zinc was released insofar as the so-
lution was undersaturated with respect to apatite,
with the release rate of zinc in a decreasing order:
ZnTCP/HAP1.64 > ZnTCP/HAP1.66 > ZnTCP/
HAP1.60 > ZnTCP.
The release rate of zinc in the ZnTCP/HAP1.64 in-
creased in the sodium acetate–acetic acid buffer solu-
tion with an increase in zinc content in the ceramics Figure 2. Changes in (Ca+Zn)/P molar ratio of the sodium
(Fig. 3). At a zinc content of 0.21 wt %, the release rate acetate–acetic acid buffer solution during release tests for the
of zinc nearly coincided with that of ZnTCP/HAP1.60 ZnTCP and the ZnTCP/HAP, with zinc content nearly equal
to 0.3 wt %.
with a zinc content of 0.316 wt %, which had been
found in a previous study to be the most effective zinc cell proliferation.3 The ZnTCP/HAP1.64, with a zinc
content for promoting bone formation in rabbit content higher than 1.09 wt %, contained a zinc oxide
femora.4 After 4 days, the release rate of zinc de- phase and showed cytotoxicity.
creased to zero or less because the solution had be- The zinc content of the ZnTCP phase was higher for
come saturated with respect to apatite. (Ca+Zn)/P the ZnTCP/HAP1.64 than it was for the ZnTCP/
molar ratios of the buffer solution ranged from 1.49 to HAP1.60 (Fig. 6). As a result, the zinc content of the
1.55 regardless of immersion times and the zinc con- ZnTCP phase was nearly equivalent for the ZnTCP/
tents of the ceramics (Fig. 4). HAP1.64 (with 0.21 Zn wt %) and the ZnTCP/
The relative growth rate of MC3T3-E1 cells cultured HAP1.60 (with 0.316 Zn wt %) and for the ZnTCP/
on ZnTCP/HAP1.64 had a maximum value of 1.3 at a HAP1.64 (with 0.51 Zn wt %) and the ZnTCP/
zinc content of 0.51 wt % (Fig. 5). This value nearly HAP1.60 (with 1.20 Zn wt %). With a zinc content
coincided with that of ZnTCP/HAP1.60 with a zinc nearly equal to 0.30 wt %, the zinc content of the
content of 1.20 wt %, which in a previous study was ZnTCP phase was the highest at a (Ca+Zn)/P molar
found to be the most effective in promoting MC3T3-E1 ratio of 1.64 (Fig. 7).

TABLE II
Negative Logarithms of Ionic Activity Products for Hydroxyapatite for the Sodium Acetate–Acetic Acid Buffer
Solution after Immersion of the Zinc-Containing Calcium Phosphate Ceramic Disks
Immersion Time/Day
2 4 6 8
Zinc content nearly equal to 0.3 wt % Series
ZnTCP 118.28 ± 1.08 115.10 ± 0.34 114.70 ± 0.13 114.02 ± 0.33
ZnTCP/HAP1.60 119.50 ± 0.29 116.11 ± 0.64 115.48 ± 0.35 114.81 ± 0.69
ZnTCP/HAP1.64 119.85 ± 0.85 117.36 ± 0.50 116.78 ± 0.59 116.60 ± 0.21
ZnTCP/HAP1.66 124.39 ± 0.37 122.85 ± 1.68 120.77 ± 0.19 119.54 ± 0.14
ZnTCP/HAP1.64
Zinc content/wt %
0.05 118.69 ± 0.32 115.31 ± 0.51 114.07 ± 0.34 113.08 ± 0.37
0.13 119.30 ± 0.76 116.42 ± 0.44 115.29 ± 0.15 114.75 ± 0.58
0.21 119.10 ± 0.70 116.74 ± 0.19 115.87 ± 0.44 115.40 ± 0.50
0.25 119.81 ± 1.06 117.37 ± 0.68 116.29 ± 0.17 116.14 ± 0.14
0.30 119.85 ± 0.81 117.36 ± 0.28 116.78 ± 0.32 116.60 ± 0.23
MINIMIZING ZINC IN ZNTCP/HAP CERAMIC 461

Figure 5. Relative growth rate of the osteoblastic MC3T3-

E1 cells cultured on the ZnTCP/HAP1.64 with various zinc
contents. Significant differences are at *p < 0.01, **p < 0.001,
and ***p < 0.0001, compared with the control.
Figure 3. Release of zinc from the ZnTCP/HAP1.64 im-
mersed in the sodium acetate–acetic acid buffer solution in
ZnTCP/HAP1.60 with 0.316 Zn wt %, which already
comparison with that from ZnTCP/HAP1.60, with 0.33 Zn
wt %. had been proved to promote bone formation in vivo.4
The release rate of zinc in vivo is controlled not by
physicochemical dissolution but by osteoclastic and/
DISCUSSION
or phagocytic activities in which ZnTCP is dissolved
in an acidic fluid. The pH of the acidic fluid ranges
The zinc content required for promoting bone for- from 3.9 to 6.0, which is indicated by the optimum pH
mation could be reduced by 34% when the (Ca+Zn)/P values of lysosomal enzymes produced by these
molar ratio of ZnTCP/HAP was increased to 1.64. The cells.15–18
rate of zinc release at pH 4.9 from ZnTCP/HAP1.64 Whenever these cells resorb bone apatite, the acidic
with 0.21 Zn wt % best coincided with that from fluid is absolutely undersaturated with respect to bone

Figure 4. Changes in (Ca+Zn)/P molar ratio of the sodium Figure 6. Zinc content of the ZnTCP phase in the ZnTCP/
acetate–acetic acid buffer solution during the release tests for HAP1.60 and 1.64 as a function of zinc content in the ceram-
ZnTCP/HAP1.64. ics.
462 SOGO ET AL.

of the ZnTCP/HAP. The HAP phase can contain a

much smaller amount of zinc than the TCP phase al-
though the solid solution limit of zinc in HAP remains
to be clarified. Therefore, the higher the (Ca+Zn)/P
molar ratio of ZnTCP/HAP, the higher the zinc con-
tent in the ZnTCP phase except in the case of ZnTCP/
HAP1.66. As a result, the zinc content of ZnTCP is
nearly equivalent between ZnTCP/HAP1.60 (with
0.316 Zn wt %) and ZnTCP/HAP1.64 (with 0.21 Zn wt
%), and they both release the required amount of zinc
needed to promote bone formation.
The zinc content of ZnTCP also is nearly equivalent
between ZnTCP/HAP1.60 with 1.20 Zn wt % and
ZnTCP/HAP1.64 with 0.51 Zn wt %, both of which
showed maximum cell proliferation. The extraordi-
nary decrease in the zinc content of the ZnTCP phase
for ZnTCP/HAP1.66 may indicate the formation of a
zinc-containing third phase, additional to the ZnTCP
and the HAP phases, although no such phase is de-
Figure 7. Zinc content of the ZnTCP phase in the ZnTCP/ tected by X-ray diffractometry. Whatever the reason
HAP, with zinc content nearly equal to 0.3 wt % as a func-
tion of (Ca+Zn)/P molar ratio. Significant differences are at for the extraordinary decrease in the zinc content of
*p < 0.001 and **p < 0.0001. the ZnTCP phase, this decrease causes the release of
less zinc from ZnTCP/HAP1.66 than it does from
ZnTCP/HAP1.64.
apatite. Thus the coincidence in the release rate of zinc
between ZnTCP/HAP1.64 (with a Zn wt % of 0.21)
and ZnTCP/HAP1.60 (with a Zn wt % of 0.316) in the
solution undersaturated with respect to apatite indi- CONCLUSIONS
cates that the optimum zinc content of ZnTCP/
HAP1.64 for promoting bone formation is at or near It is suggested that a (Ca+Zn)/P molar ratio of 1.64
0.21 wt %. The zinc contents of 0.316 and 0.21 wt % is the most appropriate for minimizing the zinc con-
correspond to 16–25 and 11–17 times that of human tent of ZnTCP/HAP used in the promotion of osteo-
bone, respectively. blastic cell proliferation and bone formation. ZnTCP/
The zinc content required for promoting osteoblas- HAP1.64 is better than any other ZnTCP/HAP rela-
tic cell proliferation in vitro was reduced by 60% when tive to material safety because the risk of an accidental
the (Ca+Zn)/P molar ratio of ZnTCP/HAP was in- burst of zinc from the material is minimized due to the
creased to 1.64. The relative growth rate of MC3T3-E1 material’s low zinc content.
cells was maximized at a zinc content of 0.51 wt % on
ZnTCP/HAP1.64 ceramics although it has been re-
ported that ZnTCP/HAP1.60 has a maximum MC3T3-
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