In vitro biodegradation of three brushite calcium phosphate cements by a macrophage cell-line.

Abstract

Depending upon local conditions, brushite (CaHPO4 x 2 H2O) cements may be largely resorbed or (following hydrolysis to hydroxyapatite) remain stable in vivo. To determine which factors influence cement resorption, previous studies have investigated the solution-driven degradation of brushite cements in vitro in the absence of any cells. However, the mechanism of cell-mediated biodegradation of the brushite cement is still unknown. The aim of the current study was to observe the cell-mediated biodegradation of brushite cement formulations in vitro. The cements were aged in the presence of a murine cell line (RAW264.7), which had the potential to form osteoclasts in the presence of the receptor for nuclear factor kappa B ligand (RANKL) in vitro, independently of macrophage colony stimulating factor (M-CSF). The cytotoxicity of the cements on RAW264.7 cells and the calcium and phosphate released from materials to the culture media were analysed. Scanning electron microscopy (SEM) and focused ion beam (FIB) microscopy were used to characterise the ultrastructure of the cells. The results showed that the RAW264.7 cell line formed multinucleated TRAP positive osteoclast-like cells, capable of ruffled border formation and lacunar resorption on the brushite calcium phosphate cement in vitro. In the osteoclast-like cell cultures, ultrastructural analysis by SEM revealed phenotypic characteristics of osteoclasts including formation of a sealing zone and ruffled border. Penetration of the surface of the cement, was demonstrated using FIB, and this showed the potential demineralising effect of the cells on the cements. This study has set up a useful model to investigate the cell-mediated cement degradation in vitro.

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Biomaterials 27 (2006) 4557–4565
In vitro biodegradation of three brushite calcium phosphate
cements by a macrophage cell-line
Zhidao Xiaa, Liam Michael Groverb,1, Yizhong Huangc, Iannis E. Adamopoulosa,
Uwe Gbureckd, James T. Triffitta, Richard M. Sheltone, Jake E. Barraletb,?
aNuffield Department of Orthopaedic Surgery, The Botnar Research Centre, Institute of Muskuloskeletal Sciences,
University of Oxford, Oxford OX3 7LD, UK
bFaculty of Dentistry, McGill University, Montre ´al, Que ´bec, Canada H3A 2B2
cThe Department of Materials, University of Oxford, Oxford OX1 3PH, UK
dDepartment of Functional Materials in Medicine and Dentistry, University of Wu ¨rzburg, Wu ¨rzburg D-97070, Germany
eBiomaterials Unit, School of Dentistry, University of Birmingham, Birmingham B6 4NN, UK
Received 8 February 2006; accepted 6 April 2006
Available online 23 May 2006
Abstract
Depending upon local conditions, brushite (CaHPO4?2H2O) cements may be largely resorbed or (following hydrolysis to
hydroxyapatite) remain stable in vivo. To determine which factors influence cement resorption, previous studies have investigated the
solution-driven degradation of brushite cements in vitro in the absence of any cells. However, the mechanism of cell-mediated
biodegradation of the brushite cement is still unknown. The aim of the current study was to observe the cell-mediated biodegradation of
brushite cement formulations in vitro. The cements were aged in the presence of a murine cell line (RAW264.7), which had the potential
to form osteoclasts in the presence of the receptor for nuclear factor kappa B ligand (RANKL) in vitro, independently of macrophage
colony stimulating factor (M-CSF). The cytotoxicity of the cements on RAW264.7 cells and the calcium and phosphate released from
materials to the culture media were analysed. Scanning electron microscopy (SEM) and focused ion beam (FIB) microscopy were used to
characterise the ultrastructure of the cells. The results showed that the RAW264.7 cell line formed multinucleated TRAP positive
osteoclast-like cells, capable of ruffled border formation and lacunar resorption on the brushite calcium phosphate cement in vitro. In the
osteoclast-like cell cultures, ultrastuctural analysis by SEM revealed phenotypic characteristics of osteoclasts including formation
of a sealing zone and ruffled border. Penetration of the surface of the cement, was demonstrated using FIB, and this showed the
potential demineralising effect of the cells on the cements. This study has set up a useful model to investigate the cell-mediated cement
degradation in vitro.
r 2006 Elsevier Ltd. All rights reserved.
Keywords: Brushite; Calcium phosphate cements; Biodegradation; Macrophage; Osteoclast; Focused ion beam microscopy (FIB)
1. Introduction
Although bone has the ability to repair itself following
damage, extensive tissue loss due to trauma, surgical
removal or disease may require the placement of a bone
graft to prevent fibrous tissue in-growth and to preserve
mechanical integrity. The most frequently used bone graft
is autogenous tissue which is usually harvested from the
iliac crest or the ribs [1]. While autografts remain the most
frequently used bone grafting material, this is associated
with a number of serious drawbacks including lack of
availability, donor site morbidity and possible permanent
gait disturbance as a result of the harvesting procedure [2].
The disadvantages associated with autografts have resulted
in a large amount of research activity to find synthetic
alternatives. In the past 20 years, perhaps the most
frequently investigated synthetic bone grafts have been
the calcium orthophosphate ceramics. One class of calcium
ARTICLE IN PRESS
www.elsevier.com/locate/biomaterials
0142-9612/$-see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2006.04.030
?Corresponding author. Tel.: +15143987203.
E-mail address: jake.barralet@mcgill.ca (J.E. Barralet).
1Present address: Chemical Engineering, University of Birmingham,
Birmingham B15 2TT, UK.

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orthophosphate used in bone grafting applications are the
calcium phosphate cements (CPCs) [3]. They typically set
following the combination of a solid component containing
one or more calcium orthophosphate powders with an
aqueous solution. Depending upon the pH value of the
cement paste the end-product of the cement setting
reaction may be either brushite (pHp4.2) [4] or hydro-
xyapatite (pH44.2) [5]. Perhaps because they are usually
set at a pH value closer to the physiological environment
than brushite cements, hydroxyapatite cements have been
more frequently studied. However, brushite is more soluble
than hydroxyapatite in physiological conditions and as a
consequence has been shown to be more completely
resorbed following implantation in animal models [6].
The degradation of brushite cement in vitro has been
tested using phosphate-buffered saline, serum and simu-
lated body fluid [7]. These studies have demonstrated that
degradation involves erosion, fragmentation and dissolu-
tion, all of which release calcium and phosphate ions into
solution. The rate at which dissolution may occur from the
brushite cement is dependent upon the chemical equili-
brium between dissolution and recrystallisation. Whole
blood is supersaturated with respect to calcium and
phosphate ions to such an extent that spontaneous
precipitation of hydroxyapatite in vivo is prevented only
by the presence of a number of ionic and protein
crystallisation inhibitors [8]. Supersaturation of the in vivo
milieu with regard to calcium and phosphate ions means
that the dissolution of brushite from the cement ought to
occur slowly and so cannot entirely explain the complete
degradation of brushite cements in vivo. A recent study has
reported that in vivo brushite cement degradation is aided
by macrophages which phagocytose cement particles [9].
Previous studies have investigated the solution-driven
degradation of the brushite cements in vitro in the absence
of any of the cells encountered in vivo [7]. The aim of the
current study was to observe the cell-mediated biodegrada-
tion of three different brushite cement formulations in
vitro. The selected cements set to form matrices consisting
of different proportions of brushite and either b-tricalcium
phosphate (b-TCP) or nanocrystalline hydroxyapatite. To
investigate the potential roles of macrophages and osteo-
clasts in cement degradation, RAW264.7, a murine
monocyte/macrophage cell line, which has the potential
to form osteoclasts in vitro independently of macrophage
colony stimulating factor (M-CSF) was selected for the
study. The cytotoxicity of the cements on RAW264.7 cells,
the calcium and phosphate released from materials and
ultrastructure of the cells on materials were also analysed.
2. Materials and methods
2.1. Specimen preparation
Three different brushite cements were examined in this study. Two of
the cements set following the combination of either b-TCP (Plasma-Biotal,
Derbyshire, UK) or nanocrystalline hydroxyapatite, precipitated in
accordance with the method reported previously [10], with orthopho-
sphoric acid solution (Table 1). The third cement set following the
combination of b-TCP (Plasma-Biotal, Derbyshire, UK) with an
orthophosphoric acid/pyrophosphoric acid blend (Rhodia, West-Mid-
lands, UK) and double-distilled water. All cements were mixed to a
powder to liquid ratio of 1.75g/mL. The cement formulations and the
abbreviations used throughout the paper are listed in Table 1. To form
cylindrical specimens of diameter 6mm and height 12mm, the cement
pastes were cast into a PTFE split mould and once set were removed from
the mould before storage for 24h at 37711C and 100% relative humidity.
The set specimens were sectioned by using a low speed diamond saw
(Isomet, Buehler, IL, USA) at a speed of 100 revolutions per minute using
DP blue (Struers, Glasgow, UK) to lubricate the blade. The resulting
nominally identical discs of height 2mm were then sterilised using ethylene
oxide. The cement discs were washed in 25mL 100mM phosphate buffered
saline (PBS; Sigma-Aldrich, Gillingham, UK) for 1h and were stored in
PBS for 12h. The discs were washed in a-MEM (Gibco BRL, Paisley, UK)
twice for 1h and were stored in a-MEM for an additional 24h. A single
cement disc was placed in each well of a 96-well tissue culture plate
(Fahrenheit, Milton-Keynes, UK).
2.2. Cell culture
The RAW264.7 murine macrophage cells were recovered from stock as
previously reported [11] and cultured in 10% foetal calf serum (FCS) (MB
Meldrum Ltd., Buckinghamshire, UK) a-MEM. Cells were removed from
culture substrates for further experiments using a disposable cell scraper.
The seeding density for RAW264.7 cells was 4?105cells/cm2. The cells
were fed using 10% FCS a-MEM supplement with 25ng/mL M-CSF
(R&D Systems Europe Ltd., Oxfordshire, UK) to maintain their
macrophage phenotype, or with 10ng/mL receptor for nuclear factor
kappa B ligand (RANKL) (Peprotech Europe Ltd., Oxfordshire, UK) to
induce osteoclast differentiation. Culture medium was replaced on days 1,
4, 7, 11 and 14. Culture medium was collected and frozen in sealed vials at
?201C before replenishment on days 1, 7 and 14. The frozen medium was
thawed for 6h at ambient temperature (24711C) for measurement of pH
and calcium and phosphorus concentrations. Cells cultured on cement
were analysed on days 7 and 14 using the LIVE/DEAD assay (Molecular
Probe, Leiden, The Netherland).
ARTICLE IN PRESS
Table 1
The formulations, abbreviations and the proportion of the hardened cement contributed by brushite for the three brushite cement formulations used in
this study
CementNature of solid componentAcid componentAdditivesProportion of brushite in set
cement (wt%)
BR
NA
b-TCP
Nanocrystalline
hydroxyapatite
b-TCP
2 M H3PO4
3.5 M H3PO4
50mM trisodium citrate
None
34
70
NO 1.5 M H4P2O7
2.7 M H3PO4
None84
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2.3. Media characterisation
The pH value of the culture medium at each time-point was determined
using a pH meter (420, Mettler-Toledo, Leicestershire, UK) from the
average of three measurements. An inductively coupled mass spectrometer
(Varian, Darmstadt, Germany) was used to determine the Ca2+and PO4
contents of the culture media. Following dilution to 1:1000 using de-
ionised water about 15mL of this solution was transferred to a
polyethylene tube for measurement. To determine the precise Ca2+and
PO4
standard solutions containing Ca2+and PO4
100 and 1000 parts per billion.
3?
3?ion concentrations the diluted culture media were compared with
3?at concentrations of 0, 10,
2.4. Tartrate-resistant acid phosphatase (TRAP) staining
TRAP staining was used to identify whether the RAW264.7 macro-
phages differentiated to form osteoclasts following the addition of
RANKL to the culture. Briefly, cells were fixed using 10% formaldehyde
for 10min and then using a 1:1 volume mixture of ethanol and acetone for
1min. The cells were washed once using 100mM PBS. Fast-red salt
(Sigma-Aldrich, Gillingham, UK) was then added to TRAP solution
(containing 59.3 M sodium tartrate, 165.7mM sodium acetate, and 0.56mg/
mL naphthol AS-MX phosphate) and the cells were incubated in the
resultant solution for 5min at 251C.
2.5. Cell viability assay
On day 7, cells were washed twice with PBS to remove foetal calf serum
in culture medium which would interfere with the staining procedures to
assess the cell viability of RAW264.7 cells. Samples were stained using a
LIVE/DEAD stain kit (Molecular Probes, Leiden, The Netherland). The
kit contained two fluorescent dyes: calcein to stain living cells green and
ethidium homodimer-1 (Ethd-1) to stain damaged or dead cells red.
Samples were stained using 4mM calcein and 2mM Ethd-1 (final
concentration) in PBS for 30min at 37711C. Samples were rinsed twice
using PBS to remove any dye and fixed either in 4% formaldehyde (Sigma-
Aldrich, Dorset, UK) or 4% glutaraldehyde (Sigma-Aldrich, Dorset, UK)
in 100mM PBS at 4711C to prevent cell detachment from the surface of
the cement. Samples were washed again in PBS to remove the fixative
before being observed using fluorescence microscopy. Images were
captured using a colour video camera (JVC 3-CCD, KY-F55B,
Yokohama, Japan) at 100? magnification using the Optimas 5.1 software
(Optimas Corp., Seattle, USA). For each sample, three images were
captured. Images were opened in Adobe photoshop 6.0 (San Jose, CA,
USA) and each image (1.18mm2) was divided into 50 squares using a grid.
Live and dead cells were counted in five squares to estimate the cell
numbers in an image. Three indices were selected for analysis as follows:

Number of cells per disc ¼
X
3
i?1
Ni
!
=3=CA,(1)
Total cell number per disc ¼ ðLive cells ðgreenÞ per disc
þ Dead cells ðredÞ per discÞ,
ð2Þ
Dead cell ratio ð%Þ
Average number of dead cells ðredÞ
Total number of cells ðred and greenÞ
where N is the number of cells per image, C is the calibrated area of an
image at given magnification, in this case C ¼ 1:18mm2, and A is the
surface area of the cement disc (28.26mm2).
¼
??
? 100,
ð3Þ
2.6. Scanning electron microscopy (SEM) and focused ion beam
(FIB) microscopy
The cements with cells were fixed in 4% glutaraldehyde, 100mM PBS
and dehydrated using a graded series of ethanol up to absolute, which was
finally substituted with hexamethyldisilazane (Sigma-Aldrich Company
Ltd., Dorset, UK) and left to dry in ambient conditions overnight. The
samples were mounted on stubs and sputter-coated with gold prior to
examination using a JEOL JSM 5300 scanning electron microscope
(JEOL, Tokyo, Japan). A focused ion beam microscope [12] (FIB
200TEM, FEI UK Ltd., Cambridge, UK) was used to examine the
ultrastructure of the cells. When a target cell was selected, a rectangle
covering the desired surface was chosen. Ion milling was initiated with
a beam current of 1000–3000picoamperes (pA) on the chosen area to etch
a given depth (d) on the surface. The image was revealed and recorded
at a sample tilt of 451, in which a beam current of 10pA was used in order
to minimise the possible damage during ion beam scanning.
2.7. Statistical analysis
Multiple comparisons of the data were performed using a one-way
analysis of variance (ANOVA) within a statistical analysis package
(GraphPad InStats, v.3.06 for Windows, San-Diego, USA). A Tukey
multiple range post hoc test was used to compare paired groups of data.
Statistical tests were performed at a 95% significance level (po0:05).
3. Results
3.1. Cement cytotoxicity
The morphology of RAW264.7 cells are shown in Fig. 1.
RAW264.7 cells were 6–10mm in diameter (Fig. 1a). By
RANKL treatment, osteoclast-like multinuclear giant cells
were observed (Fig. 1b). RANKL treated cultures of
RAW264.7 cells contained TRAP+cells (Fig. 1c), whereas
no TRAP+cells were noted in control cultures. The
RAW264.7 cells on the cements after LIVE/DEAD
ARTICLE IN PRESS
Fig. 1. RAW264.7 cells on glass coverslips, images captured using light microscopy: (a) control RAW264.7 cells; (b) osteoclast-like cells formed in vitro in
RANKL treated cultures; and (c) TRAP positive staining of the osteoclast-like cells in RANKL treated cultures.
Z. Xia et al. / Biomaterials 27 (2006) 4557–4565
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fluorescent staining are shown in supplement Fig. 1. The
total cell numbers on the three cements are shown in Fig. 2.
In control cultures (RANKL-) cell numbers on the NA
cement declined significantly from day 7 to day 14
(po0:01), and the cell number on this cement was
significantly lower than those on either the BR or NO
cements (po0:01) at any time-point. There was no
significant difference between the cell numbers on the BR
and NO cements at any time-point (Fig. 2A). In RANKL
treated cultures, the total cell numbers on the NA, BR and
NO cements all declined significantly between 7 and 14
days of culture (po0:001, o0.001 and o0.01, respec-
tively). Of the three cements studied, in the presence of
RANKL the total cell number on the NA cement after
both day 7 and 14 was significantly (po0:001) lower than
that on either the BR or NO cement. There was no
significant difference in cell numbers on the BR and NO
cements cultured in the presence of RANKL after either 7
or 14 days of culture (Fig. 2B).
The dead cell ratio, reflecting the cytotoxicity of the
cements, is shown in Fig. 3. In all control cultures
(RANKL-), except for the BR cement at day 7 which
showed a lower dead cell ratio than the NA cement, there
were no significant differences between day 7 and day 14
and between cements in all the three groups (Fig. 3A). In
RANKL treated cultures the dead cell ratio on the NA
cement remained at the same level as seen at day 7, whereas
there was an increase on the BR cement (po0:5) and a
decrease on the NO cement (po0:01).
3.2. Analysis of culture media
The PO4
in Table 2. PO4
increased by 4 times at day 7 (po0:001), and then halved
by day 14 (p40:05). There was an increase in PO4
BR group at day 14 (po0:05) compared with day 1 and day
7 but there was no significant change in the NO group at
three time points. In RANKL treated cultures, there was
also a dramatic increase of PO4
There was no significant change of PO4
at all time points, but there were decreases of PO4
3?concentrations in the culture media are shown
3?in the NA group control cultures had
3?in the
3?at day 7 in the NA group.
3?in the BR group
3?in NA
ARTICLE IN PRESS
0
10000
20000
30000
40000
50000
60000
70000
Cell numbers/disc
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
Cell numbers/disc
RANKL (-)
##
##
##
##
###
###
###
###
***
***
RANKL (+)
Day 7
Day 14
Day 7Day14
(A)
(B)
Fig. 2. The total cell number on the NA (&), BR ( ) and NO (’) cement
discs after 7 and 14 days of culture in (A) the absence and (B) presence of
RANKL (*, ***, Comparison with the same materials at day 7 and day
14, po0:05 and o0.001, respectively. ]], ]]], comparison with NA at the
same time point, po0:01, o0.001, respectively).
0
2
4
6
8
10
12
14
16
18
Dead cells ratio (%)
0
5
10
15
20
25
Dead cell ratio (%)
RANKL (-)
##
$$$
*
##
**
##
#
RANKL (+)
Day 7
Day 14
Day 7
Day 14
(A)
(B)
Fig. 3. The dead/live cell ratios of RAW264.7 cells cultured on the NA
(&), BR( ) and NO (’) cements for 7 and 14 days in (A) the absence of
RANKL and (B) the presence of RANKL (*, **, comparison with the
same materials at day 7 and day 14, po0:05 and o0.01, respectively. ], ]],
comparison with NA at the same time point, po0:05 and o0.01,
respectively. $$$, comparison with BR at the same time point, po0:001).
Z. Xia et al. / Biomaterials 27 (2006) 4557–4565
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and NO groups (po0:01) at day 14 compared with day 1
(po0:01). The Ca2+concentrations in culture media are
shown in Table 2. In all control cultures (RANKL-), the
average concentration of Ca2+in media increased more
than threefold at day 7 and day 14 compared with day 1;
however, only in the NO group at day 14 the difference was
statistically significant compared with day 1 (po0:01) and
the NA group at day 14 (po0:01). The dramatic differences
in RANKL treated cultures in the three groups were the
decrease in average Ca2+concentration at day 14 however
the decreases were not significant compared with day 1.
After one day of culture the NO cement caused the pH
of the culture medium to drop to 6.2770.05 in control
cultures and 6.2370.04 (Table 2) in RANKL treated
cultures. Of the cements studied the BR cement reduced the
pH of the culture medium the least (6.7570.02) in the
absence of RANKL and 6.8070.03 in the presence of
RANKL. With time the pH of the culture media in which
the BR and NO cements were immersed increased towards
pH 7–7.25, however, in the case of the NA cement the
culturemedium reacheda pH
6.3070.05 and 6.1170.04 after 14 days of culture.
value minimumof
3.3. SEM and FIB microscopy
Scanning electron micrographs of the RAW264.7 in
control cultures on the NO cement are shown in Fig. 4.
RAW264.7 cells were 5–10mm diameter with fine cell
processes and surface folds (Fig. 4a–c). Morphologically
there were two different types of giant cells: (a) well-
developed giant cells of 20–30mm in diameter, with a few
long (10–15mm) cell processes and abundant surface folds
(Fig. 4a); and (b) those formed by aggregation of
RAW264.7 cells with very short and fine cell processes
(Fig. 4b). A number of dead giant cells were evident as the
giant cells became fragmentary with crystal-like structures
formed inside cells (Fig. 4d).
By evenly milling a thin layer from the chosen area of
dead giant cells on the cements (Fig. 5a and b) using FIB
microscopy, no phagocytosed particles were observed.
However, in situ cross-sections of selected giant cells
showed that the cell processes had penetrated deeply into
the surface of the cement and had engulfed cement particles
(Fig. 5c and d). A gap between cell and cement particles
was evident (Fig. 5e).
In RANKLtreatedcultures,
RAW264.7 cells differentiated into osteoclast-like cells
and these cells were TRAP positive. These giant cells were
of diameter 30–50mm with abundant microfilaments on the
cell surface (Fig. 6a and b). The well-developed osteoclast-
like cells formed an apparent sealing-zone where the outer
aspect of the cell membrane contacted the cement
substratum (Fig. 6b). The cell morphologies in RANKL
treated cultures were of non-differentiated cells (Fig. 6c),
differentiated osteoclast-like cells (Fig. 6d), apoptotic
osteoclast-like cells (Fig. 6e) and dead osteoclast-like cells
with crystal formation (Fig. 6f).
The FIB milling analysis (Fig. 7a) confirmed that the cell
processes of the osteoclast-like cells penetrated deeply into
the cements (Fig. 7b–d). At the cell–cement interface, the
cell membranes of osteoclast-like cells formed ruffled-
borders and extended into the gaps between the cement
particles (Fig. 7e and f), and there were abundant vesicles
in the cytoplasm (Fig. 7c, e and f).
lessthan 30%of
4. Discussion
Although brushite cements were originally reported
almost 20 years ago [13] the literature contains few
references to the cellular reactions to these cements in
vitro. One possible reason for this is that the cements set by
an acid/base reaction, which reduces the pH value of the
cement mix to as low as 3 [4]. As cell culture medium has a
relatively low inherent buffering capacity such pH altera-
tions are likely to have an acute toxic effect on the cells.
Indeed in this study although the cement samples were
extensively washed in phosphate-buffered saline and
a-MEM prior to culture, the medium had a pH of
ARTICLE IN PRESS
Table 2
The measured pH values and calcium and phosphorous concentrations of the minimal essential medium after 1, 7 and 14 days of culture on NA, BR and
NO cement in the presence and absence of RANKL (standard deviations given in brackets)
CementCulture
RANKL
+/?
1 day 7 days 14 days
pH Ca conc.
(ppm)
P
conc.
(ppm)
pH Ca
conc.
(ppm)
P
conc.
(ppm)
pH Ca
conc.
(ppm)
P
conc.
(ppm)
NA
?
+
6.35 (0.03)
6.40 (0.04)
26 (10)
40 (3)
414 (71)
554 (24)
6.45 (0.02)
6.35 (0.03)
259 (68)
206 (64)
2072 (46)
2332 (205)
6.30 (0.05)
6.11 (0.04)
191 (149)
0 (0)
912 (242)
109 (3)
BR
?
+
6.75 (0.02)
6.8 (0.03)
96 (13)
163 (37)
138 (1)
158 (23)
6.95 (0.04)
6.75 (0.04)
176 (88)
606 (184)
116 (27)
221 (42)
7.10 (0.04)
7.00 (0.05)
283 (95)
9 (1)
711 (33)
41 (1)
NO
?
+
6.27 (0.05)
6.23 (0.04)
98 (31)
188 (139)
371 (47)
506 (57)
6.93 (0.03)
6.65 (0.02)
456 (93)
224 (121)
527 (162)
431 (84)
7.25 (0.03)
7.10 (0.03)
607 (72)
48 (40)
324 (26)
0 (0)
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6.2370.04 (Table 2) after the first day in culture. This pH
rose subsequently after 7 and 14 days as a consequence of
further medium changes and replenishment. Despite the
low pH of brushite cements immediately after setting there
have been a number of in vivo studies which have reported
favourable host responses after implantation [14,15]
ARTICLE IN PRESS
Fig. 5. Focussed ion beam electron images showing: (a) a giant cell that showed cell fragmentation with crystal-like structures within the cell was chosen
for milling to demonstrate internal structure; (b) the milled giant cell, except for cell membranes (arrows with tail), the crystal-like structure (arrow without
tail) was actually empty; (c) an area of interest on a live giant cell was chosen for milling; (d) after milling the giant cell was tilted by 451 exposing the
interface between the macrophage and the cement; and (e) a cell process of the giant macrophage had penetrated the cement ( ). The gaps between cells
and cement were visible (arrows without tail).
Fig. 4. Scanning electron micrographs of macrophages on NO cement: (a) well differentiated giant macrophage at 14 days. Notice the cell size of giant
cells compared with the undifferentiated cells around; (b) the fusion of RAW264.7 cells to form giant cells at 14 days; (c) undifferentiated RAW264.7 cells
at day 7; and (d) dead giant cells at 14 days showing cell fragmentation with crystal-like structures within cells. Bar ¼ 10mm.
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demonstrating the functional buffering capacity of the
in vivo milieu.
The same mechanism of decomposition is presumed for
CPCs [16] as for calcium phosphate ceramics [17], although
the cell type involved in their breakdown varies according
to the type of cement. CPCs that are resorbed relatively
quickly are removed by macrophages and giant cells, whilst
the more slowly resorbed CPCs are degraded by osteoclast-
like cells [18]. Our study demonstrates that RAW264.7 cells
in control and RANKL treated cultures formed macro-
phages and multinucleated giant cells on the cements. The
increase in calcium concentrations in the RANKL(?)
groups evidently showed the biodegradation of the
cements. There was no evidence in the current study to
show whether or not the cement particles were phagocy-
tosed by small-sized single nuclear macrophages. By evenly
milling a thin layer from an area of dead giant cells present
on the cements, it was demonstrated that there were no
cement particles within the dead multinuclear giant cells
(Fig. 5a and b). There was not sufficient evidence to show
phagocytosis of the giant cells in the current study,
however, cell processes were observed to have penetrated
deeply into the surface of the cements (Fig. 5c and d).
Mouse macrophages and giant cells are capable of
phagocytosing biomaterial particles less than the size of
the cells [19,20]. For biodegradation to be mediated by
phagocytosis of particles from the cement surface, particles
should have been found within the cells. However, this
was not the case and any biodegradation of the cement in
the current study was likely to be due to extracellular
dissolution rather than phagocytosis.
Osteoclasts have been shown to form resorption lacunae
on the surface of CPCs [21] and the present study has
demonstrated the presence of multinucleated TRAP
positive osteoclast-like cells, capable of ruffled border
formation and lacunar resorption on the surface of the
brushite cement in RANKL treated cultures. The degrada-
tion of brushite by osteoclasts may be expected to cause a
local increase in calcium concentration. However, in this
study this was not the case as in the presence of RANKL
the activity of osteoclast-like cells did not result in increases
of Ca and P concentration in the culture medium over and
above that seen in the absence of RANKL. On the
contrary, a trend of decreases in Ca and P (although not
significant for Ca concentrations) was observed in the
RANKL treated group although the cause of this trend
is unknown. Possible reasons may be: (1) 30ng/mL
RANKL only stimulated less than 30% of RAW264.7
cells to differentiate to osteoclast-like cells; (2) the life
span of osteoclast-like cells differentiated from RAW264.7
cells were 11–14 days so after 14 days most osteoclastic
cells had undergone apoptosis. The decrease in total cell
number in RANKL(+) samples at day 14 reflected this cell
death and was also shown by the SEM observations;
(3) there was recrystallisation of mineral in the cement
matrix and in apoptotic osteoclast-like cells. The minerals
accumulated in apoptotic osteoclasts before transport
out may become a source of Ca2+to trigger remineralisa-
tion, as seen in Fig. 6. The variable calcium and phosphate
contents of the media at various time points may reflect
this crystal deposition and the lowest values seen at
14 days in the RANKL treated cultures signify greatest
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Fig. 6. Scanning electron micrographs of osteoclast-like cells on NO cement: (a) 7 days after seeding; (b) well-developed osteoclast-like cell 14 days after
seeding. Notice the sealing zone formed by cell membrane along the border of the cell and the cell size compared with nearby undifferentiated cells;
(c) undifferentiated RAW264.7 cells 7 days after seeding; (d) differentiated cells 7 days after seeding; (e) an apoptotic osteoclast-like cell at 7 days after
seeding. Notice the damaged cell membrane; and (f) a dead osteoclast-like cell 14 days after seeding, notice the crystals formed on the dead cell.
Bar ¼ 10mm.
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precipitation at this time point resulting in the lowest
Ca?P products.
It has been reported that osteoclasts may not resorb
brushite cements as rapidly as hydroxyapatite cements
because at a pH of 3.9–4.2, brushite is actually less soluble
than hydroxyapatite [22]. Osteoclast mediated resorption,
therefore, may be expected to occur more slowly on
brushite cement than on hydroxyapatite cements. With
time, the brushite in the cement may hydrolyse to form
hydroxyapatite, as this change occurs it would be expected
that osteoclasts could play a more dominant role in
material degradation.
The hydrolysis reaction (Eq. (4)) is generally considered
to be undesirable since the formation of hydroxyapatite in
the cement matrix can result in a reduction in the rate at
which the cement may be resorbed [9].
10CaHPO4? 2H2O
! Ca10ðPO4Þ6OH2þ 4HPO2?
4þ 6Hþþ H2O:
ð4Þ
As Eq. (4) illustrates, the hydrolysis of brushite (molar
calcium to phosphorous ratio ¼ 1) to stoichiometric
hydroxyapatite (theoretical molar calcium to phosphorous
ratio ¼ 1.67) results in the liberation of acid phosphate and
hydrogen ions. It may be that the marked increase in
phosphorous concentration (Table 2) and accompanying
reduction in pH of the medium in which the NA brushite
cement was aged (Table 2) may be attributed to the
hydrolysis of the brushite component of the cement to
hydroxyapatite.
It is generally accepted that cement resorption occurs
through extracellular liquid dissolution with cement disin-
tegration and particle formation, and phagocytosis of the
cement particles by macrophages [9]. The phagocytosis of
cement particles was not supported in the current study
using RAW264.7 cells. However, by using focused ion
beam milling, the multinuclear giant cells were found to
partly engulf the cements via cell pseudopodia penetrating
deeply into the cements to form a sealed compartment. In
addition, the osteoclast-like cells formed a sealing-zone and
ARTICLE IN PRESS
Fig. 7. Focussed ion beam electron images showing the interface between an osteoclast-like cell and NO cement: (a) shows the area on an osteoclast-like
cell chosen for milling; (b) the chosen area after milling, the part of cell labelled with a white star penetrating into the cement; (c) the sample tilted by 451 to
highlight the cell penetration and the cell part and cell–material interface; (d) an additional area of interest selected for milling; (e) following milling of (c)
the remaining cell organelles were removed and the interface between cell and material was exposed; and (f) a higher magnification of (e) to highlight the
ruffled-border of the cells. Oc, osteoclast-like cells; V, vesicles within the osteoclast-like cells; P, cement particles; and R, the ruffled border of the
osteoclast-like cells.
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ruffled border and resorption lacunae-like structures. This
suggests that extracellular degradation of cement by
multinuclear giant cells may also play a role in the cement
resorption.
5. Conclusion
In conclusion, the RAW264.7 cell line formed macro-
phage/multinucleated giant cells and osteoclast-like cells on
the brushite CPCs in vitro. The significant release of calcium
in the NO cement showed biodegradation of the cement in
the presence of macrophage/multinucleated giant cells.
Although calcium levels were not elevated in the culture
media from the osteoclast-like cell cultures, ultra-structural
analysis by SEM revealed phenotypic characteristics of
osteoclasts including formation of a sealing zone and ruffled
border. Penetration deep inside the surface of the cements
showed the potential for their demineralisation by cellular
activities. This study has developed a useful model to
investigate the cell-mediated cement degradation in vitro
and demonstrates the value of focused ion beam microscopy
in investigation of cell–biomaterial interactions.
Acknowledgement
The authors acknowledge the financial support of
EPSRC (CASE studentship, L. Grover), Smith and
Nephew (York, UK and Memphis, TN) and BBSRC.
Appendix A. Supplementary material
Supplementary data associated with this article can be
found in the online version at: doi:10.1016/j.biomaterials.
2006.04.030.
References
[1] Goulet JA, Senunas LE, DeSilva GL, Greenfield ML. Autogenous
iliac crest bone graft. Complications and functional assessment. Clin
Orthop Relat Res 1997;339:76–81.
[2] Fowler BL, Dall BE, Rowe DE. Complications associated with
harvesting autogenous iliac bone graft. Am J Orthop 1995;24:
895–903.
[3] Jansen J, Ooms E, Verdonschot N, Wolke J. Injectable calcium
phosphate cement for bone repair and implant fixation. Orthop Clin
North Am 2005;361:89–95.
[4] Bohner M, Van Landuyt P, Merkle HP, Lemaitre J. Composition
effects on the pH of a hydraulic calcium phosphate cement. J Mater
Sci-Mater Med 1997;8:675–81.
[5] Brown PW, Chow LC. A calcium phosphate water setting cement.
Cements research progress. In: Proceedings of the American
Ceramics Society, 1986. p. 352–79.
[6] Klein CP, de Groot K, Driessen AA, van der Lubbe HB. Interaction
of biodegradable beta-whitlockite ceramics with bone tissue: an in
vivo study. Biomaterials 1985;6:189–92.
[7] Grover LM, Knowles JC, Fleming GJP, Barralet JE. In vitro
ageing of brushite calcium phosphate cement. Biomaterials 2003;24:
4133–41.
[8] Terkeltaub RA, Santoro DA, Mandel G, Mandel N. Serum and
plasma inhibit neutrophil stimulation by hydroxyapatite crystals.
Evidence that serum alpha 2-HS glycoprotein is a potent and specific
crystal-bound inhibitor. Arthritis Rheum 1988;31:1081–9.
[9] Theiss F, Apelt D, Brand B, Kutter A, Zlinszky K, Bohner M, et al.
Biocompatibility and resorption of a brushite calcium phosphate
cement. Biomaterials 2005;26:4383–94.
[10] Barralet JE, Lilley KJ, Grover LM, Farrar DF, Ansell C, Gbureck U.
Cements from nanocrystalline hydroxyapatite. J Mater Sci-Mater
Med 2004;15:407–11.
[11] Itonaga I, Sabokbar A, Sun SG, Kudo O, Danks L, Ferguson D,
et al. Transforming growth factor-beta induces osteoclast formation
in the absence of RANKL. Bone 2004;34:57–64.
[12] Huang YZ, Lozano-Perez S, Langford RM, Titchmarsh JM, Jenkins
ML. Preparation of transmission electron microscopy cross-section
specimens of crack tips using focused ion beam milling. J Microsc
2002;207:129–36.
[13] Lemaitre J, Mirtchi A, Mortier A. Calcium phosphate cements for
medical use: state of the art and perspectives of development. Silic Ind
1987;10:141–6.
[14] Frayssinet P, Gineste L, Conte P, Fages J, Rouquet N. Short-term
implantation effects of a DCPD-based calcium phosphate cement.
Biomaterials 1998;19:971–7.
[15] Constantz BR, Barr BM, Ison IC, Fulmer MT, Baker J, McKinney
L, et al. Histological, chemical, and crystallographic analysis of four
calcium phosphate cements in different rabbit osseous sites. J Biomed
Mater Res 1998;43:451–61.
[16] Jarcho M. Calcium phosphate ceramics as hard tissue prosthetics.
Clin Orthop Relat Res 1981;157:259–78.
[17] Damien CJ, Parsons JR. Bone graft and bone graft substitutes: a
review of current technology and applications. J Appl Biomater
1991;2:187–208.
[18] Ooms EM, Wolke JG, van der Waerden JP, Jansen JA. Trebecular
bone response to injectable calcium phosphate (Ca-P) cement.
J Biomed Mater Res 2002;61:9–18.
[19] Xia ZD, Zhu TB, Du JY, Zheng QX, Wang L, Li SP, et al.
Macrophages in degradation of collagen/hydroxylapatite(CHA),
beta-tricalcium phosphate ceramics (TCP) artificial bone graft. An
in vivo study. Chin Med J (England) 1994;107:845–9.
[20] Lam KH, Schakenraad JM, Esselbrugge H, Feijen J, Nieuwenhuis P.
The effect of phagocytosis of poly(L-lactic acid) fragments on
cellular morphology and viability. J Biomed Mater Res 1993;27:
1569–77.
[21] Schilling AF, Linhart W, Filke S, Gebauer M, Schinke T, Rueger JM,
et al. Resorbability of bone substitute biomaterials by human
osteoclasts. Biomaterials 2004;25:3963–72.
[22] Tas AC, Bhaduri SB. Chemical processing of CaHPO4?2H2O: its
conversion to hydroxyapatite. J Am Ceram Soc 2004;87:2195–200.
ARTICLE IN PRESS
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