Control of crystallinity of hydrated products

in a calcium phosphate bone cement

Xiupeng Wang,1,2 Jiandong Ye,1,2 Yingjun Wang,1,2 Xianpei Wu,3 Bo Bai3

1
Key Laboratory of Specially Functional Materials and Advanced Manufacturing Technology,
South China University of Technology, Ministry of Education, Guangzhou 510641, China
2
School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, China
3
The First Affiliated Hospital, Guangzhou Medical College, Guangzhou 510120, China

Received 20 January 2006; revised 1 August 2006; accepted 15 August 2006

Published online 16 January 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31059

Abstract: In this study, a calcium phosphate cement (CPC),
consisting of partially crystallized calcium phosphate (PCCP),
was synthesized. X-ray diffraction (XRD), Fourier transform
infrared spectrometry (FTIR) and scanning electronic micro-
scope (SEM) were used to characterize the cement. The results
showed that by changing the ratio of amorphous calcium
phosphate (ACP) to PCCP in the cement, hydrated products
of controllable crystallinity were obtained. With increase in
the relative amount of PCCP, the hydrated products changed
gradually from very poor crystallinity with little needle-like
hydroxyapatite (Hap) crystallites to relatively high crystallin-
ity with more needle-like Hap crystallites; the compressive
strength of the cement increased, and the degradation of the
cement decreased. The cement was implanted into the tibia
tubercle of healthy mature Zelanian white rabbits and the his-
tological specimens were obtained after 4 and 16 weeks of im-
plantation. The result revealed that this bone cement was bio-
compatible and showed very early osteoconductive proper-
ties. Thus, the CPC has potential for use in orthopedic surgery
for filling non- load-bearing bone defects. Ó 2007 Wiley Peri-
odicals, Inc. J Biomed Mater Res 81A: 781–790, 2007
Key words: calcium phosphate; crystallinity; biodegrada-
tion; bone remodeling

INTRODUCTION CPCs were first formulated by Brown and Chow6,7

Because of its similarity to the mineral phase of
bone and the nature of set in situ, a calcium phos-
phate cement (CPC) is regarded as a promising ma-
terial for use in the repair of bone defects or voids.1
CPCs are suitable for the repair and reconstruction
of bone2,3 because they show excellent biocompati-
bility, bioactivity, and osteotransductive, that is, after
implantation in bone defects they are rapidly inte-
grated into the bone structure, after which they are
transformed into new bone.4 CPCs are composed of
one or several calcium phosphates that, once mixed
with a liquid phase, give a moldable paste. Within a
few minutes, the paste sets because of the in situ for-
mation of a solid calcium phosphate. The setting
reaction provokes hardening of the paste through
entanglements of the crystals of the precipitate.5
Correspondence to: J. Ye; e-mail: jdye@scut.edu.cn
Contract grant sponsor: National Natural Science Foun-
dation of China; contract grant number: 50172015
Contract grant sponsor: Science and Technology Pro-
gram of Guangzhou; contract grant number: 200523-D2041
' 2007 Wiley Periodicals, Inc.
as being composed of tetracalcium phosphate (TTCP)
and dicalcium phosphate anhydrous (DCPA). From
then on, CPCs have been prepared using different
methods or from different precursor calcium phos-
phate compounds,8–14 such as H3PO4 þ a-tricalcium
phosphate (a-TCP), a-TCP þ TTCP þ dicalcium phos-
phate dihydrate (DCPD), a-TCP þ b-TCP þ hydroxy-
apatite (HAp), monocalcium phosphate monohydrate
(MCPM) þ CaO, a-TCP þ MCPM þ CaCO3, a-TCP þ
DCPD, and b-TCP þ MCPM. However, most of these
cements degraded either too fast or too slow and did
not match with the new bone ingrowth in vivo. In par-
ticular, some of those transformed into relatively high
crystalline hydroxyapatite after hydration, which was
still quite different from the bone mineral of human
body and essentially non-resorbable in vivo. CPC was
degraded through a dissolution process associated
with a cellular process in vivo.15 Studies show that
osteoclast mediated degradation of hydroxyapatite ce-
ramic by simultaneous resorption and phagocytosis in
vivo.16 Although the degradation process can be chemi-
cal or biological, it is rare for cells to be mediated by bi-
ological degradation.17 The process of biodegradation
is considered to be directly influenced by the type of
material crystallization.15 Therefore, the resorbability
782
of CPC should be closely related to its dissolubility,
which is mainly controlled by the crystallinity of the
hydrated products. A high crystallinity leads to a low
dissolution rate as well as a low resorption. For this
reason, several attempts have been made to improve
the resorption behavior of CPCs, for example, by
increasing the porosity of the material. Some authors
have proposed to mix water-soluble phases with the
cements, such as mannitol,18 Na2HPO4,19 calcium sul-
phate hemihydrate,20 absorbable fibers,21,22 and surfac-
tant molecules,23 with the cement. When the cement is
implanted, these phases dissolve, creating pores in the
matrix and thus assuring new bone ingrowth.
In the present report, we have formulated CPCs
that have controllable crystallinity of HA in the
hydrated products. They are to be used as bone defect
filler. Bone defect fillers are usually non-load-bearing;
therefore, their mechanical properties are not critical.
Nonetheless, there may be some load-bearing applica-
tions. Thus, in the present study, the diametral tensile
strength (DTS) and compressive strength of the cements
were determined. Furthermore, the phase evolution,
porosity, setting time, mass loss, and microstructure of
the cement are described together with a histological
evaluation of the cement when implanted in healthy
and mature Zelanian white rabbits.
MATERIALS AND METHODS
Materials
The CPC was prepared by mixing amorphous calcium
phosphate (ACP) (media diameter 22.3 mm), partially crys-
tallized calcium phosphate (PCCP), (media diameter 23.5
mm) and DCPA (media diameter 5.8 mm). ACP with a Ca/
P ratio of 1.5 and containing carbonate was synthesized by
a chemical precipitation method from an aqueous solution
of Ca(NO3)2
4H2O, (NH4)2HPO4
12H2O and NH4HCO3.
PCCP was produced by calcining ACP at the temperature
of 4508C for 2 h. DCPA was commercially obtained
(Shanghai No.4 Reagent and H.V. Chemical Co., China), as
were NH4HCO3, Ca(NO3)2
4H2O, and (NH4)2HPO4

12H2O (Guangzhou Chemical Reagent Factory, China). All
the commercially obtained reagents were AR grade pure.
The liquid phase (de-ionized water) and the cement pow-
ders were mixed in a mortar to obtain a paste with work-
able consistency, using a liquid/powder ratio of 0.45 mL/
g. Five variants of the CPC were formulated and the
details of their compositions are given in Table I. Steel
molds were used to prepare cement columns with a diam-
eter of 6 mm and a height of 12 mm for the compressive
strength tests and with the same diameter and a height of
3 mm for the DTS tests. After pouring the cement into the
steel molds, the cement was pressed by a steel column
with a diameter of 5.6 mm under a stress of about 700 kPa
for 5 s to eliminate big air bubbles. All experiments were
done at about 24–268C and 50–60% humidity. Sample
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
WANG ET AL.
TABLE I
Experimental Arrangements
Composition
(wt %) CPC1 CPC2 CPC3 CPC4 CPC5
ACP 50 37.5 25 12.5 0
PCCP 0 12.5 25 37.5 50
DCPA 50 50 50 50 50
dimensions were 12  6 mm2 for compressive strength test
and 3  6 mm2 for DTS test. The cement specimens were
stored in an incubator at 378C and 97% humidity for 24 h.
Then the samples were ready for tests.
Characterization
ACP, PCCP, DCPA, and the hydrated specimens (milled
into powders) were analyzed using XRD (X‘Pert Pro, PAN-
alytical, The Netherlands) and FTIR (AVATAR 360, NICO-
LET, USA). The cyrstallinity in the HA powders can be
evaluated by the following equation:24
Crystallinity ¼ ½1  ðV112=300 =I300 Þ  100% ð1Þ
where I300 is the intensity of (300) diffraction peak and
V112/300 is the intensity of the hollow between (112) and
(300) diffraction peaks of HA. Microstructure of the hy-
drated specimens was observed with SEM (HITACHI H-
800, Hitachi, Japan) on the gold-coated samples at a magni-
fication of 5000. AR grade pure DCPA used in this study
was commercially obtained; and the XRD and FTIR spectra
of the DCPA were similar to the standard spectra. There-
fore, there is no mention of the XRD and FTIR spectra of
the DCPA in this article. The strengths of the columns were
measured using a universal material testing machine
(Instron 5567, Instron Corp., USA) at a crosshead speed of
0.5 mm/min for compressive strength tests and 10 mm/
min for the DTS tests. The porosity of the hardened speci-
mens was determined using the Archimedes method, as
described by Yang et al.25 Setting time of the cements was
measured according to ASTM C191-03. After pouring the
cement into the conical steel ring with an inside diameter
of (70 6 3) mm at bottom and (60 6 3) mm at top and a
height of (40 6 1) mm, the indenter ((300 6 0.5) g in mass,
(1 6 0.05) mm in diameter of the needle) was carefully low-
ered vertically onto the surface of the newly shaped cement
samples and allowed to remain there for 5 s. The indenta-
tion was repeated at intervals of 30 s until the cement was
hardened. Initial setting occurs when a 1-mm needle pene-
trates 25 mm into cement paste. Final set occurs when there
is no visible penetration. For the determination of each
property, four specimens were used.
In vitro degradation measurement
The specimens with a diameter of 6 mm and a height of
about 6 mm were weighed and then placed in polystyrene
vials containing 200 mL de-ionized water at 37.08C, in a
shaking bath for predetermined intervals of 6 h, 12 h, and
1, 3, 7, 14, and 21 days at a liquid-to-solid ratio of 1.50 mg/
CONTROL OF CRYSTALLINITY OF HYDRATED PRODUCTS IN A CP BONE CEMENT 783

mL. The water was refreshed every 24 h. After each soaking

period, the soaking water was filtrated and the specimen
was dried at 37.08C and weighed again. Each measurement
was repeated 4 times and the average value was calculated.
One hundred and forty samples were used for the mass loss
test.

Histological evaluation

Eight healthy and mature Zelanian white rabbits (2.3 6

0.3 kg) were used in this study. The animals were housed
in a stable. National guidelines for the care and use of labo-
ratory animals were observed. In each rabbit, two implant
areas (diameter ¼ depth ¼ 7 mm) were prepared on both
sides of the tibia tubercle. In this article, we intend to focus
on investigating the composition, phase evolution, micro- Figure 2. FTIR spectra of PCCP and ACP.
structure, mechanical strength, setting time of the novel
CPC system. Therefore only one cement (CPC4) was used
to examine the biocompatibility and bioactivity of the ACP RESULTS
þ PCCP þ DCPA system cement; and CPC4 was chosen by
random. CPC4 with 0.2 wt % rhBMP2 was also tested for
The XRD patterns of the ACP and PCCP that were
comparison. Four weeks and 16 weeks after the last surgi-
cal session, the rabbits were killed, and the implants with
used to prepare the cement powders are shown in
their surrounding tissues were immediately excised. The Figure 1. In the pattern for the ACP, the weak peaks
specimens were fixed, decalcified, and embedded in paraf- of ACP reveal that it is composed of very poorly
fin. Then 4-mm-thin sections were prepared from each spec- crystalline calcium phosphate. In the pattern for the
imen, using a microtome. These sections were stained with PCCP, the diffraction peaks are narrow and intense
hematoxylin-eosin (HE) and examined by light microscopy. which suggests that the material’s crystallinity is
In addition, we used a Nikon E2000-E inverted biological higher than that for the ACP.
microscope, Nikon digital camera, and Image Pro Plus 5.1 The FTIR spectra of ACP and PCCP are shown in
(Media Cybernetics Ins., USA) image analysis software to Figure 2. The band at 1647 cm1 corresponds to the
quantify the total area of cement as maintained and the adsorbent water. The broken-up bands at 1430 cm1
new bone ingrowth in the rabbits 16 weeks implantation.
and 870 cm1 correspond to the CO32 bands. The
PO43 bands appear in the follow range: 1090–1030
Statistical analysis cm1, 957 cm1, and 600–500 cm1.
The XRD patterns for the as-prepared CPCs are
shown in Figure 3. The diffraction peaks correspond-
One-way ANOVA was performed by SPSS (SPSS, USA)
to detect significant (a ¼ 0.05) effects of the content of PCCP ing to DCPA are narrow and intense, while those cor-
on the compressive strength, porosity and setting time of responding to ACP or PCCP are broad. Because of the
the cement. Newman–Keuls multiple comparison tests were
conducted if a significant difference (p < 0.05) existed.

Figure 1. XRD patterns of PCCP and ACP. Figure 3. XRD patterns of the as-prepared cements.

Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a

784 WANG ET AL.

intense DCPA peaks, there is almost no difference in

the XRD patterns of the as-prepared cements (as-
CPC1, as-CPC2, as-CPC3, as-CPC4, and as-CPC5).
The XRD patterns of the hydrated cement are
shown in Figure 4, together with the pattern of bone
mineral. The peaks corresponding to DCPA, ACP,
and PCCP disappeared, as these materials had con-
verted into HA.
The calculated crystallinities of the cements are
shown in Figure 5. With the increase of PCCP from
0 to 50 wt %, the crystallinity of the hydrated cement
increased from 11 to 41% (Fig. 5).
The FTIR spectra of the hydrated cement samples
are shown in Figure 6. The band at 1653 cm1 corre-
sponds to the adsorbent water. The CO32 bands
appeared at 1472 cm1 and 1413 cm1. The PO43 Figure 5. The calculated crystallinities of the cements.
bands appeared in the follow range: 1090–1030 cm1,
957 cm1, and 600–500 cm1.
The SEM micrographs of ACP, PCCP, DCPA and tial and finial setting time were (23 6 1.3) and (33 6
the cross-section of the hydrated cement specimens 1.5) min, respectively. With increase in the relative
are given in Figure 7. The ACP, PCCP, and DCPA amount of PCCP, the initial and finial setting time
granules observed in SEM were 5–20 mm in diameter decreased significantly (p ¼ 0.05). For the cement with
(Fig. 7). After hydration, the morphology of the mate- 50 wt % PCCP (CPC5) the initial and finial setting
rials changed, with a large number of precipitated time were (11 6 1.2) and (20 6 1.5) min, respectively.
Hap grains with length and width of about 1000 nm The development of the compressive strength of
and 100 nm, respectively, being evident (Fig. 7). In CPC4 is listed in Figure 9. Because the compressive
CPC1, only a limited number of needle-like Hap strength results for CPC1–CPC5 were almost the
grains fragmentarily located in the hydrated cement same, only compressive strength for CPC4 was listed
can be seen. With increase in the amount of PCCP in in this article. The compressive strength of the speci-
the cement, more needle-like Hap grains were precipi- mens increased fast within the first 24 h. The com-
tated (Fig. 7) into a network structure. In Figure 7(h) pressive strength of CPC4 was (23.2 6 2.5) MPa at
(corresponding to CPC5), a large number of precipi- 24 h. Then, with increase in aging time, there was a
tated needle-like Hap grains tightly interlaced into a small increase in compressive strength.
network structure were seen. The DTS and porosity with different content of
Both the initial and finial setting times of the PCCP are shown in Figure 10. For the cement with-
cements decreased with an increase in PCCP content out PCCP (CPC1) the DTS was only (3.28 6 0.2)
(Fig. 8). For the cement without PCCP (CPC1) the ini- MPa, and the porosity was (45.8 6 1.9)%. With

Figure 4. XRD patterns of the hydrated cements. Figure 6. FTIR spectra of the hydrated cements.

Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a

CONTROL OF CRYSTALLINITY OF HYDRATED PRODUCTS IN A CP BONE CEMENT 785

Figure 7. SEM micrographs of (a) ACP, (b) PCCP, (c) DCPA and the hydrated samples. (d–h) correspond to CPC1,
CPC2, CPC3, CPC4, and CPC5, respectively.

Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a

786
Figure 8. Setting time of the cements.
increase in the relative amount of PCCP, the DTS
increased significantly (p ¼ 0.05) while the decrease in
porosity was not significant (p ¼ 0.05) (Fig. 10). For the
cement with 50 wt % PCCP (CPC5) the DTS reached
6.5 6 0.6 MPa, and the porosity was (39.4 6 2.1)%.
An ideal calcium phosphate must have a biodegra-
dation rate designed to match the cell/tissue growth
rates. Therefore, the biodegradation rate of the cement
was characterized by means of mass loss test. The
mass loss (wt %) versus soaking time curves for the
hydrated cements with different amount of PCCP
were plotted in Figure 11. With increase in the relative
amount of PCCP, the mass loss of the cement speci-
mens, at a given soaking time, decreased (Fig. 11).
Histological analysis of the results after 4 weeks of
implantation showed that a small amount of cartilage
and fibroblast was formed with very little inflamma-
tory cells around the CPC4 cement (Fig. 12). For the
Figure 9. Compressive strength of CPC for different con-
serving time.
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
WANG ET AL.
Figure 10. Variation of DTS and porosity with the content
of PCCP.
cement with rhBMP-2, after 4 weeks of implantation,
a large amount of cartilage had formed at interface
between the cement and the tissue, and part of the
material was ossified. No inflammatory cells and fiber
vesicles were found around the material (Fig. 13).
After 16 weeks, no inflammatory cells and fiber
vesicles was observed (Fig. 14). Fibrocartilage was
formed and parts of them had ossified, and a small
amount of trabecula bone was detected at this time.
While, in the cement with rhBMP-2, after 16 weeks of
implantation, histological findings showed that the
quantity of new bone increased and most of the carti-
lage were transformed into mature bone tissue. Inte-
grated trabecula bone and Haversian system were
formed (Fig. 15). Furthermore, the percentages of
bone ingrowth in the rabbits 16 weeks after implanta-
tion for CPC4 cement without and with BMP-2 were
(41.7 6 16.6)% and (71.7 6 21.0)%, respectively.
Figure 11. In vitro mass loss tests of the hydrated
cements.
CONTROL OF CRYSTALLINITY OF HYDRATED PRODUCTS IN A CP BONE CEMENT
Figure 12. Light microscopy photo of the CPC4 cement
after implantation for 4 weeks. (HE stain, original magnifi-
cation 100). [Color figure can be viewed in the online
issue, which is available at www.interscience.wiley.com.]
DISCUSSION
PCCP has a higher crystallinity than ACP; thus, the
diffraction peaks of PCCP were much narrow and
intense than those in ACP (Fig. 1). The broad absorp-
tion bands at 1030 and 567 cm1 revealed the poor
crystallinity of ACP and PCCP. The CO absorption
bands detected at about 1430 cm1 and 870 cm1
showed the presence of CaCO3 (Fig. 2). Thus for both
the ACP and PCCP, their XRD and FTIR results are
consistent.
For CPCs, liquid-to-powder ratio (L/P) is a very im-
portant variable. Cements prepared with higher L/P
ratio, exhibit much longer initial and final setting
time,26 much lower compressive strength,27 and much
Figure 13. Light microscopy photo of the cement with
rhBMP-2 after implantation for 4 weeks. (HE stain, original
magnification 200). [Color figure can be viewed in the
online issue, which is available at www.interscience.wiley.
com.]
787
Figure 14. Light microscopy photo of the CPC4 cement
after implantation for 16 weeks. (HE stain, original magni-
fication 200). [Color figure can be viewed in the online
issue, which is available at www.interscience.wiley.com.]
bigger porosity. For CPCs, the liquid with which the
powder is mixed is also a very important variable. For
instance, Na2HPO4 was reported as accelerator on the
initial and final setting time.28 Hydroxyethyl starch,
sodium dextran sulfate, cyclodextrine, and alginic
acid have been reported to prolong the initial and fin-
ial setting time and to decrease the compressive
strength of the cement.29
In this study, in order to evaluate the influence of
the amount of PCCP on the properties of the cement,
the cement powders were homogeneously mixed with
de-ionized water at a liquid to powder ratio of 0.45 mL/
g. After storing the cement specimens in an incubator
at 378C and 97% humidity for 24 h, the phase compo-
sition of the hydrated cement was ascertained from
X-ray diffraction. As the cement hydrated, DCPA,
Figure 15. Light microscopy photo of the cement with
rhBMP-2 after implantation for 16 weeks. (HE stain, origi-
nal magnification 100). [Color figure can be viewed in
the online issue, which is available at www.interscience.
wiley.com.]
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
788
TABLE II
Percentage of Bone Ingrowth in the Rabbits 16 Weeks
After Implantation
Without BMP-2 With BMP-2
Percentages of bone ingrowth 41.7616.6 71.7621.0
ACP and PCCP disappeared clearly and turned into
HA after hydration (Figs. 3 and 4) because diffraction
peak intensity and width are greatly affected by crys-
tallite size and lattice order. Large and more highly
ordered crystallites have many more uniformly
spaced lattice planes available to contribute to dif-
fraction count rate and peak sharpness than smaller
and low ordered crystallites.30 With the increase in
the relative amount of PCCP, the diffraction peaks of
the hydrated cements became more narrow and
intense (Fig. 4). Diffraction peak intensity and width
are greatly affected by crystallite size and lattice
order. Large and more highly ordered crystallites
have many more uniformly spaced lattice planes
available to contribute to diffraction count rate and
peak sharpness than smaller and low ordered crystal-
lites.30 Thus with increase in the relative amount of
PCCP, the crystallinity of the hydrated cement
increased gradually from a low amount of Hap (11%)
to a much higher one (41%) (Figs. 4 and 5). It can be
seen that the XRD patterns of the samples in this
study were very similar to that of natural bone min-
eral. It is known that the formation of bone-like hy-
droxyapatite benefits the improvement of biocompat-
ibility and bioactivity and accelerates the degradation
rate of the cement. The broken-up bands at 1472
cm1 and 1413 cm1 (Fig. 6), which were different
from the single band of carbonate at the range
between 1320 cm1 and 1530 cm1, revealed the dope
of CO32 in the HAp crystal lattice.31,32 All FTIR bands
were broad, indicating poor crystallinity of HA. These
results are consistent with the XRD patterns (Fig. 4).
The setting reaction of CPC involves dissolution of
calcium phosphate followed by nucleation and growth
of hydroxyapatite. More crystals were obtained in
PCCP by calcining ACP (Fig. 1). For CPC with PCCP,
the crystallized components in PCCP provide nuclea-
tion sites for Hap crystal growth, thus accelerating the
rate of Hap nucleation and precipitation in the cement
(Figs. 4, 5, and 7). Yang et al.33 showed that addition of
apatite seeds into the starting CPC powder mixture
promotes the formation of deposited apatite as these
seeds provide nucleation sites for Hap crystal growth.
This may be responsible for the higher reactivity of the
PCCP compared to the ACP. Thus with increase in the
relative amount of PCCP, setting time of the cement
decreased (Fig. 8).
The mechanical strength and porosity are of great in-
terest to surgeons, as these data directly affect the selec-
tion of indications and the postoperative care. With
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
WANG ET AL.
increase in the relative amount of PCCP, the DTS
increased significantly (p ¼ 0.35) while the decrease in
porosity was not significant (p ¼ 0.72) (Fig. 10). The crys-
tallinity of the hydrated cement increased with increase
in the relative amount of PCCP, which may account for
the increased compressive strength of the cement.
With increase in the relative amount of PCCP, the
mass loss of the cement specimens, at a given soak-
ing time, decreased (Fig. 11). This is attributed to the
decrease in the solubility of HA that accompanies
the increase in the crystallinity of the hydrated
cements (Fig. 4). Thus, the biodegradation rate of the
cement may be easily controlled by changing the ra-
tio of ACP and PCCP in the as-prepared cement.
The in vitro degradation tests were performed in de-
ionized water in this article based on the following
considerations: If the in vitro degradation tests were
performed in simulated body fluid or other aggressive
medium, the calcium, phosphate and other ions in the
liquid phase may precipitate on the surface of the
cement (so-called biomimetic mineralization). At the
same time calcium phosphates in the cement may dis-
solve into the liquid phase. Therefore, under the influ-
ence of the mineralization it will be difficult to evalu-
ate the degradation of the materials. Choosing de-ion-
ized water as degradation medium was intended to
evaluate the degradation of the cement caused by dis-
solution in this study. The bone cement is degraded
through a dissolution process associated with a cellu-
lar process.15 Study showed that osteoclast mediated
degradation of hydroxyapatite ceramic by simultane-
ous resorption and phagocytosis in vivo.16 The degra-
dation process can be chemical or biological.34–36 But
it has been noted, however, that cell mediated biologi-
cal degradation is rare.36 The process of biodegrada-
tion was considered to be directly influenced by the
type of material crystallization.15 Therefore, the main
difference between the in vitro and in vivo degradation
of the material was the degradation without or with
the effect of cell. The mechanism of the in vitro degra-
dation was only the dissolution of the materials into
the liquid phase. This process is controlled by the
crystallinity of the material, as a material with high
crystallinity leads to a low dissolution rate.
Histological studies in light microscopy confirmed
that CPC4 was both biocompatible and bioactive.
Oreffo et al. have shown that apatite provided an excel-
lent surface for osteoblast proliferation and differentia-
tion.37 This phenomenon was caused by precipitation
of biological apatite by secondary nucleation and het-
eroepitaxy on residual crystals.38 CPC4 showed very
early osteoconductive properties, because viable new
cartilage were clearly detected only 4 weeks after im-
plantation (Figs. 12 and 13) and integrated trabecula
bone and Haversian system were formed only 16
weeks after implantation (Figs. 14 and 15). This was
particularly noteworthy because osteoconduction usu-
CONTROL OF CRYSTALLINITY OF HYDRATED PRODUCTS IN A CP BONE CEMENT
ally requires several months in vivo.39 Furthermore,
CPC4 with rhBMP-2 exhibited better early osteocon-
ductive properties and osteoinductive properties than
that without rhBMP-2 (Figs. 12–15), indicating that this
cement is a good carrier for rhBMP-2.
In this article, we intended to focus our research on
the relationship between the composition and phase
evolution and the microstructure, mechanical strength,
and setting time of the CPC system. Only short-term
animal studies have been carried out, with the results
showing that the cement has excellent biocompatibility.
Nonetheless, long-term biocompatibility evaluations
and the cytotoxicity of the cements should be carried
out. Also, the values of Affinity Index in the histological
analysis should be computed and, especially, the
resorbability tests of the CPCs in vivo should be carried
out in the further work. The flexural strength of the
cements should also be determined if load-bearing
applications are anticipated. Furthermore, the consis-
tency of the cement pastes should be tested according to
ASTM C 187-98.
CONCLUSIONS
By changing the ratio between partially crystallized
calcium phosphate (PCCP) and amorphous calcium
phosphate (ACP), a calcium phosphate cement with
controlled crystallinity was prepared. With the
increase in the relative amount of PCCP, the crystal-
linity of the hydrated cement increased gradually
from a very poor crystallinity to a higher crystallinity
of Hap. With the increase of PCCP, more needle-like
HA were precipitated in the cement and at the same
time the compressive strength of the cements raised
markedly. The results of the mass loss tests showed
that the biodegradation rate of the materials may be
controlled by changing the ratio of ACP and PCCP in
the as-prepared cement. Histological studies in light
microscopy confirmed that this cement was both bio-
compatible and bioactive. Furthermore, the cement
with rhBMP-2 exhibited excellent early osteoconduc-
tive properties and osteoinductive properties. A con-
venient way of adjusting the crystallinity and degra-
dation performance of a CPC was shown in this
study, whose results indicate that the CPC formu-
lated has the potential for use as a bone defect filler.
References
1. Yuan HP, Li YB, de Bruijn JD, de Groot K, Zhang XD. Tissue
responses of calcium phosphate cement: A study in dogs.
Biomaterials 2000;21:1283–1290.
2. Friedman CD, Constantino PD, Jones K, Chow LC, Pelzer HJ.
Sisson GA. Hydroxyapatite cement II obliteration and recon-
struction of the cat frontal sinus. Arch Otolaryngol Head
Neck Surg 1991;117:385–389.
789
3. Hong YC, Wang JT, Hong CY, Brown WE, Chow LC. The
periapical tissue reactions to a calcium phosphate cement in
the teeth of monkeys. J Biomed Mater Res 1991;25:485–498.
4. Jansen JA, de Ruijter JE, Schaeken HG, Van der Waerden JPCM,
Planell JA, Driessens FCM. Evaluation of tricalcium phosphate/
hydroxyapatite cement for tooth replacement: An experi-
mental anomaly study. J Mater Sci Mater Med 1995;6:653–657.
5. Fernandez E, Gil FJ, Ginebra MP, Driessens FCM, Planell JA,
Best SM. J Mater Sci Mater Med 1999;10:177–183.
6. Brown WE, Chow LC. A new calcium phosphate setting cement.
J Dent Res 1983;62:672.
7. Brown WE, Chow LC. Combinations of sparingly soluble cal-
cium phosphates in slurries and pastes as mineralizers and
cements. US Patent 4,612,053, September 16, 1986.
8. Khairoun I, Driessens FCM, Boltong MG, Planell JA, Wenz R.
Addition of cohesion promoters to calcium phosphate cements.
Biomaterials 1999;20:393–398.
9. Niedhart C, Maus U, Redmann E, Siebert CH. In vivo testing
of a new in situ setting b-tricalcium phosphate cement for
osseous reconstruction. J Biomed Mater Res 2001;55:530–537.
10. Wang XP, Ye JD, Wang H. Effects of additives on the rheo-
logical pProperties and injectability of a calcium phosphate
bone substitute material. J Biomed Mater Res B: Appl Bio-
mater 2006;8:259–264.
11. Frayssinet P, Gineste L, Conte P, Fages J, Rouquet N. Short-
term implantation effects of a DCPD-based calcium phos-
phate cement. Biomaterials 1998;19:971–977.
12. Bai B, Jazrawi LM, Kummer FJ. The use of an injectable, bio-
degradable calcium phosphate bone substitute for the pro-
phylactic augmentation of osteoporotic vertebrae and the
management of vertebral compression fractures. Spine 1999;
24:1521–1526.
13. Kurashina K, Kurita H, Kotani A, Takeuchi H, Hirano M.
In vivo study of a calcium phosphate cement consisting of a-
tricalcium phosphate/dicalcium phosphate dibasic/tetracal-
cium phosphate monoxide. Biomaterials 1997;18:147–151.
14. TenHuisen KS, Brown PW. Formation of calcium-deficient hy-
droxyapatite from a-tricalcium phosphate. Biomaterials 1998;
19:2209–2217.
15. Lu JX, Descamps M, Dejou J, Koubi G, Hardouin P, Lemaitre
J, Proust JP. The biodegradation mechanism of calcium phos-
phate biomaterials in bone. J Biomed Mater Res B: Appl Bio-
mater 2002;63:408–412.
16. Wenisch S, Stahl JP, Horas U, Heiss C, Kilian O, Trinkaus K,
Hild A, Schnettler R. In vivo mechanisms of hydroxyapatite
ceramic degradation by osteoclasts: Fine structural micros-
copy. J Biomed Mater Res A 2003;67:713–718.
17. Bauer TW, Geesink RGT, Zimmerman R, McMahon JT.
Hydroxyapatite-coated femoral stems. Histological analysis of
components retrieved at autopsy. J Bone Joint Surg Am 1991;
73:1439–1452.
18. Markovic M, Takagi S, Chow LC. Formation of macropores
in calcium phosphate cements through the use of mannitol
crystals. Key Eng Mater 2001;192–195:773–776.
19. Takagi S, Chow LC. Formation of macropores in calcium phos-
phate cement implants. J Mater Sci Mater Med 2001;12:135–139.
20. Nilsson M, Fernández E, Sarda S, Lidgren L, Planell JA.
Characterization of a novel calcium phosphate/sulphate bone
cement. J Biomed Mater Res 2002;61:600–607.
21. Xu HHK, Simon CG Fast-setting and anti-washout calcium
phosphate scaffolds with high strength and controlled macro-
pore formation rates. J Biomed Mater Res A 2004;68:725–734.
22. Zhang Y, Xu HHK. Effects of synergistic reinforcement and
absorbable fiber strength on hydroxyapatite bone cement.
J Biomed Mater Res A 2005;75:832–840.
23. Sarda S, Nilsson M, Balcells M, Fernández E. Influence of
surfactant molecules as air-entraining agent for bone cement
macroporosity. J Biomed Mater Res A 2003;65:215–221.
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
790
24. Landi E, Tampieri A, Celotti G, Sprio S. Densification behav-
ior and mechanisms of synthetic hydroxyapatite. J Ceram Soc
2000;20:2377–2387.
25. Yang J, Shi GX, Bei JZ, Wang SG, Cao YL, Shang QX, Yang
GH, Wang WJ. Fabrication and surface modification of mac-
roporous poly(L-lactic acid) and poly(L-lactic-co-glycolic acid)
(70/30) cell scaffolds for human skin fibroblast cell culture.
J Biomed Mater Res 2002;62:438–446.
26. Bigi A, Bracci B, Panzavolta S. Effect of added gelatin on the
properties of calcium phosphate cement. Biomaterials 2004;
25:2893–2899.
27. Takagi S, Chow LC, Hirayama S, Eichmiller FC. Properties of
elastomeric calcium phosphate cement-chitosan composites.
Dent Mater 2003;19:797–804.
28. Del Real RP, Wolke JGC, ValletRegi M, Jansen JA. A new
method to produce macropores in calcium phosphate
ceramics. Biomaterials 2002;23:3673–3680.
29. Khairoun I, Driessens FCM, Boltong MG, Planell JA, Wenz R.
Addition of cohesion promoters to calcium phosphate cements.
Biomaterials 1999;20:393–398.
30. Constantz BR, Ison IC, Fulmer MT, Poster RD, Smith ST,
Wagoner MV, Ross J, Goldstein SA, Jupiter JB, Rosenthal DI.
Skeletal repair by in situ formation of the mineral phase of
bone. Science 1995;267:1796–1799.
31. Komath M, Varma HK. Development of a fully injectable cal-
cium phosphate cement for orthopedic and dental applica-
tions. Bull Mater Sci 2003;26:415–422.
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
WANG ET AL.
32. Filho OP, Torre GP, Hench LL. Effect of crystallization on an
apatite layer formation of bioactive glass s45S5. J Biomed
Mater Res 1996;30:509–514.
33. Yang QZ, Troczynski T, Liu DM. Influence of apatite seeds
on the synthesis of calcium phosphate cement. Biomaterials
2002;23:2751–2760.
34. Manley MT.Calcium phosphate biomaterials. In: Geesink RGT,
Manley MT, editors. Hydroxyapatite Coatings in Orthopaedic
Surgery. New York:Raven; 1993. pp 1–23.
35. Jarcho M. Calcium phosphate ceramics as hard tissue pros-
thetics. Clin Orthop Relat Res 1981;157:259–278.
36. Bauer TW, Geesink RGT, Zimmerman R, McMahon JT.
Hydroxyapatite-coated femoral stems. Histological analysis of
components retrieved at autopsy. J Bone Joint Surg Am 1991;
73:1439–1452.
37. Oreffo ROC, Driessens FCM, Planell JA, Triffitt JT. Growth
and differentiation of human bone marrow osteoprogenitors
on novel calcium phosphate cements. Biomaterials 1998;19:
1845–1854.
38. Daculsi G, LeGeros RZ, Heugheaert M, Barbieux I. Formation
of carbonate apatite crystals after implantation of calcium
phosphate ceramics. Calcif Tissue Int 1990;46:20–27.
39. Jansen JA, de Ruijter DE, Schaeken HG, van der Waerden
JPCM, Planell JA, Driessens FCM. Evaluation of tricalcium
phosphate hydroxyapatite cement for tooth replacement: An
experimental animal model study. J Mater Sci Mater Med
1995;6:653–657.