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IOP PUBLISHING BIOMEDICAL MATERIALS
Biomed. Mater. 8 (2013) 025010 (8pp) doi:10.1088/1748-6041/8/2/025010

Effects of DCPD cement chemistry on

degradation properties and
cytocompatibility: comparison of
MCPM/β-TCP and MCPM/HA
formulations
Daniel L Alge 1,5 , W Scott Goebel 2,3 and Tien-Min Gabriel Chu 4,6
1
Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47908, USA
2
Herman B Wells Center for Pediatric Research, Indiana University School of Medicine,
Indianapolis, IN 46202, USA
3
Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202, USA
4
Department of Restorative Dentistry, Division of Dental Biomaterials, Indiana University School of
Dentistry, Indianapolis, IN 46202, USA
E-mail: [email protected]

Received 10 December 2012

Accepted for publication 29 January 2013
Published 22 February 2013
Online at stacks.iop.org/BMM/8/025010

Abstract
Dicalcium phosphate dihydrate (DCPD) cements are attractive biomaterials for bone repair,
and a number of different DCPD cement formulations have been proposed in the literature. In
this study, we have specifically compared monocalcium phosphate monohydrate
(MCPM)/hydroxyapatite (HA) and MCPM/β-tricalcium phosphate (β-TCP) formulations to
test the hypothesis that DCPD cement chemistry affects the degradation properties and
cytocompatibility of the cement. Using simple in vitro models we found that MCPM/β-TCP
formulations degraded primarily by DCPD dissolution, which was associated with a slight pH
drop and relatively low mass loss. Cytocompatibility testing of cement conditioned culture
media revealed no significant change in cell viability relative to the negative control for all of
the MCPM/β-TCP formulations. In contrast, the MCPM/HA formulations were prone to
undergo rapid conversion of DCPD to HA, resulting in a sharp pH drop and extensive mass
loss. A stoichiometric excess of HA in the cement was found to accelerate the conversion
process, and significant cytotoxicity was observed for the MCPM/HA formulations containing
excess HA. Collectively, these results show that, although the product of the setting reaction is
the same, DCPD cements produced with MCPM/HA and MCPM/β-TCP formulations differ
significantly in their degradation properties and cytocompatibility. These differences may have
important implications for the selection of a DCPD cement formulation for clinical
application.

1. Introduction repair. Calcium phosphate cements in general are

osteoconductive and bioactive due to their compositional
Dicalcium phosphate dihydrate (DCPD, also known as similarity to bone mineral [1, 2]. Furthermore, because
brushite) cements are attractive biomaterials for bone
of their cementitious nature, they can be molded
5
intraoperatively to conform to irregularly shaped bone
Present address: Department of Chemical and Biological Engineering,
University of Colorado, Boulder, CO 80309, USA. defects, which is particularly advantageous for cranio- and
6 Author to whom any correspondence should be addressed.
maxillofacial reconstruction [3, 4], and even fabricated

1748-6041/13/025010+08$33.00 1 © 2013 IOP Publishing Ltd Printed in the UK & the USA
Biomed. Mater. 8 (2013) 025010 D L Alge et al

into complex 3D scaffold architectures for tissue Table 1. DCPD cement formulationsa.
engineering [5–7]. The principal advantage of DCPD MCPM:HA MCPM:β-TCP Equivalents of excess baseb
cements, however, is the excellent solubility of DCPD at
physiologic pH. This property translates to superior 4:1 1:1 0
4:1.5 1:1.5 0.5
biodegradability and resorbability compared to calcium
4:2 1:2 1
phosphate cements that set to form hydroxyapatite [8]. 4:3 1:3 2
DCPD cements have conventionally been prepared by a
mixing β-tricalcium phosphate (β-TCP) with water and an Presented as molar ratios.
b
Calculated according to equations (1) and (2).
acidic phosphate source, such as monocalcium phosphate
monohydrate (MCPM) or phosphoric acid [9–11]. The
reaction governing the formation of DCPD from MCPM and β-TCP-based formulations. We previously studied the in
β-TCP is vitro degradation properties of DCPD cements prepared from
MCPM and HA and found conversion of DCPD to HA to be a
Ca (H2 PO4 )2 · H2 O + β − Ca3 (PO4 )2
key mechanism [27]. This result was in stark contrast to what
+7H2 O → 4CaHPO4 · 2H2 O. (1) has been reported for DCPD cements prepared using β-TCP
Alternatively, our group and others have shown that β-TCP can [17–19], suggesting that even though the reaction product is
be replaced with hydroxyapatite (HA) [12–14]. The reaction the same, the chemistry plays an important role in determining
governing the formation of DCPD from MCPM and HA is the final properties of the cement. To test this hypothesis,
4Ca (H2 PO4 )2 · H2 O + Ca10 (PO4 )6 (OH)2 the objective of this study was to directly assess the effects
of the cement chemistry on degradation properties through
+22H2 O → 14CaHPO4 · 2H2 O. (2) a head-to-head comparison of DCPD cements prepared with
MCPM/HA and MCPM/β-TCP formulations. To this end, we
Using HA instead of β-TCP has some potential subjected cement specimens from both formulations to static
advantages. For example, the solubility differences between degradation in PBS for 14 days and monitored changes in pH,
HA and β-TCP could make a wider range of setting and mass loss and composition. In addition, because conversion
resorption properties accessible [15]. Tuning the DCPD of DCPD to HA produces an acidic environment that could
cement resorption rate by incorporating more basic calcium potentially be cytotoxic, we used an in vitro assay to directly
phosphates is of particular interest, as rapid resorption of compare the cytocompatibility of MCPM/HA and MCPM/β-
DCPD can limit bone apposition [8] and potentially result in TCP formulations.
sub-optimal bone healing. Finally, HA has superior mechanical
properties compared to β-TCP [16], which could be leveraged
2. Materials and methods
to improve the mechanical properties of biphasic DCPD-based
cements. 2.1. DCPD cement preparation
Because the principal advantage of DCPD is its
degradability under physiologic conditions, there has been DCPD cements were prepared with the MCPM/β-TCP
considerable interest in studying the degradation properties and MCPM/HA systems according to table 1. Briefly, for
of DCPD cements. For example, Bohner et al and Grover the MCPM/HA system, MCPM (crystallinity = 91.5%;
et al both studied the degradation properties of β-TCP-based Strem Chemicals, Newburyport, MA) was dry mixed with
DCPD cement formulations in vitro using methods based poorly crystalline HA (percent crystallinity = 74.8%; Strem
on soaking in solutions simulating the conditions of the in Chemicals) in MCPM:HA molar ratios of 4:1, 4:1.5, 4:2
vivo environment [17–19]. The in vivo degradation properties and 4:3, and then combined with 100 mM sodium citrate
of β-TCP-based DCPD cement formulations have also been (Fisher Scientific, Pittsburgh, PA) in a powder to liquid
studied, with an emphasis on understanding compositional mass ratio (P/L) of 1.0 g/g. Sodium citrate was added
changes [20–24]. Of particular interest in these studies as a setting regulator to retard cement hardening during
is whether the DCPD dissolves, is resorbed through cell- sample preparation [13, 14]. Similarly, MCPM and β-TCP
mediated degradation or is converted to HA. Conversion of (percent crystallinity = 91.8%; Fluka Chemical Corporation,
DCPD to HA occurs because DCPD dissolution produces a Ronkonkoma, NY) were dry mixed in MCPM:β-TCP molar
solution that is supersaturated with respect to HA, which is ratios of 1:1, 1:1.5, 1:2 and 1:3 and mixed with 100 mM
the most stable calcium orthophosphate phase at pH greater sodium citrate in a P/L of 1.0 g/g. The molar ratio groups
than about 4, thereby leading to HA precipitation [25]. While for the MCPM/β-TCP system were chosen in order to have
this process typically occurs slowly due to the slow crystal an equivalent molar excess of base reactant compared to the
growth kinetics of HA, it has important implications. First and MCPM/HA groups, as can be seen from equations (1) and
foremost, because HA has low solubility at physiologic pH and (2). It should be noted, however, that this experimental design
is slowly resorbed [8], conversion of DCPD to HA negates the resulted in similar but not identical Ca:P ratios due to the
advantage of biodegradability [9, 26]. In addition, conversion different basicities of HA and β-TCP. The percent crystallinity
of DCPD to HA produces phosphoric acid [25], which may of all reactant powders was determined by x-ray diffraction
have ramifications for biocompatibility. analysis. The data were acquired using a Bruker D8 Focus
The degradation properties of DCPD cements prepared instrument equipped with a Cu Kα source and 1D high speed
with HA have not been well characterized compared to Lynxeye detector (Bruker AXS, Madison, WI). Analysis was

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Biomed. Mater. 8 (2013) 025010 D L Alge et al

performed using Bruker DiffracPlus EVA software and the stem cells, which were isolated and cultured as we previously
ICDD PDF2 database for indexing patterns. described [28]. The cells were added to a 96-well plate at
Cylindrical specimens for each experimental group were 5000 cells per well. After the cells had adhered to plate,
prepared by manually pressing the unhardened cement paste 100 μL of the conditioned media was added to each well. Non-
into TeflonR
molds which had nominal dimensions of 3 mm conditioned medium which had not been exposed to DCPD
diameter and 7 mm height (mold tolerance: diameter = 3.05 cement was used as a negative control. After 24 h, the media
± 0.06 mm). After allowing the cements to set at room was removed, the plate was rinsed with PBS and the Cell
temperature in air for approximately 30 min, the specimens Titer-Glo Luminescent Cell Viability Assay (Promega) was
were removed from the mold and dried under vacuum in a used to assess viability after treatment. Importantly, this assay
desicator chamber for 2 days prior to use in order to obtain measures intracellular ATP, which is an indirect measure of
initial masses prior to degradation. cell number. Thus, the luminescence data are presented as a
percentage of the negative control (n = 5).
2.2. Evaluation of degradation properties
2.4. Statistical analysis
Cement degradation was characterized using an in vitro model
based on static soaking in PBS (pH = 7.4; from Fisher Quantitative data for pH and mass loss are presented as the
Scientific). To characterize changes in pH and mass, individual mean plus or minus the standard deviation. An analysis of
samples from each experimental group were placed separately variance (ANOVA) two factor mixed effects model was used
into glass vials containing 3 ml of PBS, resulting in a to determine the effects of base reactant and equivalents
cement surface area to liquid volume ratio of approximately of excess base reactant on the pH and mass loss at day
50 mm2 ml−1. The submerged cement samples were then 14, and cytocompatibility. Significance between experimental
incubated at 37 ◦ C for up to 14 days with no soaking media groups was determined by post hoc comparisons using Tukey’s
changes. Three specimens from each group were removed method (α = 0.05).
from the incubator at 2, 4, 6, 8, 10, 12 and 14 days. The pH of
the PBS was measured with a pH meter (Denver Instruments, 3. Results
Arvada, CO), which was calibrated with the appropriate buffer
solutions prior to each use. Cement specimens were then dried 3.1. Evolution of pH and mass loss during degradation
under vacuum in a desicator chamber for 2 days and weighed
to determine the percent change in mass. The MCPM/HA and MCPM/β-TCP cements showed
In addition, to characterize changes in composition, markedly different pH profiles over the 14-day degradation
samples of each molar ratio were removed at 6 and 14 days, period (figure 1), and the effect of base reactant (i.e. HA or
dried for 2 days in a vacuum desicator chamber, and then β-TCP) on final pH was statistically significant (p < 0.05 from
analyzed by powder x-ray diffraction. Day 0 cement specimens ANOVA). On day 2, the 4:1, 4:1.5, 4:2 and 4:3 MCPM:HA
were also analyzed for comparison. Briefly, the dried cement groups had pHs of 6.28 ± 0.12, 5.83 ± 0.09, 5.61 ± 0.09
was crushed to a fine powder using a mortar and pestle, and 5.61 ± 0.02, respectively. After this sharp initial drop,
dispersed on a glass slide in acetone, and then analyzed on the pH leveled off to the day 14 values of 5.86 ± 0.16, 5.41
a Siemens D5000 automated powder x-ray diffractometer ± 0.02, 5.25 ± 0.04 and 5.28 ± 0.07 for the 4:1, 4:1.5, 4:2
equipped with a Cu tube and graphite monochromator (Bruker and 4:3 groups, respectively. The 4:1 and 4:1.5 groups were
AXS). The powder was scanned at 40 kV and 30 mA from significantly different from all other groups (p < 0.05). In
5◦ to 40◦ (2θ ) in 0.02◦ increments at a scan speed of contrast, the pH values for the MCPM/β-TCP cements were
1◦ min−1. The resultant x-ray diffraction patterns were much higher and showed no trend with the amount of excess
compared to Joint Committee on Powder Diffraction base reactant. The final pH values for the 1:1, 1:1.5, 1:2 and 1:3
Standards–Powder Diffraction Files (JCPDSPDF) in order to groups were 6.56 ± 0.07, 6.96 ± 0.09, 7.06 ± 0.05 and 6.64
determine the phases present. ± 0.23, respectively. These values were significantly higher
compared to MCPM/HA groups with equivalent amounts of
2.3. Cytocompatibility assay excess base (p < 0.05).
Mass loss followed a nearly identical trend to pH
To test the effects of cement chemistry on cytocompatibility, (figure 2), with the effect of base reactant on final mass loss
cylindrical specimens from each experimental group were being statistically significant (p < 0.05 from ANOVA). After
sterilized by soaking in 70% ethanol for 30 min, dried and 2 days the 4:1, 4:1.5, 4:2 and 4:3 MCPM:HA groups had lost
then soaked individually in cell culture medium (Dulbecco’s 4.27 ± 2.34%, 9.10 ± 0.45%, 11.58 ± 2.18% and 13.77 ±
modified eagles medium (Invitrogen, Carlsbad, CA) with 10% 0.05% of their masses, respectively. As with pH, the values
fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, leveled off after a sharp initial change, with the final mass loss
GA)). As in the degradation study, each cement specimen values being 8.94 ± 0.91%, 15.87 ± 1.22%, 17.01 ± 3.05%
was soaked in 3 ml of media to maintain a consistent cement and 17.32 ± 1.64% for the 4:1, 4:1.5, 4:2 and 4:3 groups,
surface area to liquid volume ratio between experiments respectively. The 4:1 group was significantly different from
(∼ 50 mm2 ml−1). After 24 h, the conditioned media was all other groups (p < 0.05). Mass loss in the MCPM/β-TCP
removed with a pipette. The effects of this conditioned media cements was much lower and showed no trend with the amount
on cell viability were then evaluated on murine mesenchymal of excess base reactant. The final mass loss values for the 1:1,

3
Biomed. Mater. 8 (2013) 025010 D L Alge et al

(A) (A)

(B) (B)

Figure 2. Comparison of MCPM/HA and MCPM/β-TCP cement

Figure 1. Comparison of MCPM/HA and MCPM/β-TCP cement mass loss during in vitro degradation. (A) MCPM/HA cements.
pH change during in vitro degradation. (A) MCPM/HA cements. (B) MCPM/β-TCP cements.
(B) MCPM/β-TCP cements.

MCPM:HA group consisted of pure HA at day 6. By day 14,

1:1.5, 1:2 and 1:3 groups were 5.75 ± 1.11%, 1.93 ± 0.70%, the relative fraction of HA had increased further. DCPD was
0.93 ± 0.84% and 4.79 ± 1.27%, respectively. These values still the predominant phase in the 4:1 group, although small
were significantly lower compared to MCPM/HA groups with amounts of HA and anhydrous dicalcium phosphate (DCP)
equivalent amounts of excess base (p < 0.05). were present. Similarly, DCPD and a small amount of DCP
were also present in the 4:1.5 group at 14 days, but the relative
3.2. Compositional changes during degradation amount of HA was markedly increased compared to day 6.
The 4:2 and 4:3 groups consisted of pure HA at day 14.
Phase composition of the MCPM/HA and MCPM/β-TCP While the appearance of substantial amounts of poorly
cements was evaluated at day 0 to determine the phases initially crystalline HA was observed in the MCPM/HA cements, the
present and their relative amounts. Initially, the 4:1 MCPM:HA MCPM/β-TCP cements consisted of predominantly DCPD
group consisted of pure DCPD. The other MCPM/HA groups and unreacted β-TCP at both day 6 and day 14. The major
were biphasic, with the relative amount of HA increasing change compared to the day 0 results was a decrease in the
with increasing stoichiometric excess, as expected (figure 3). percent of DCPD and a concomitant increase in the percent
Similarly, the 1:1 MCPM/β-TCP group consisted of only of β-TCP. Small amounts of HA were noted in the 1:2 and
DCPD, whereas the other MCPM/β-TCP groups contained 1:3 groups at day 14, but the relative fraction of HA was
increasing amounts of β-TCP with increasing stoichiometric substantially lower than in the MCPM/HA cements.
excess (figure 4).
All experimental groups were analyzed again at days
3.3. Cell viability after exposure to conditioned media
6 and 14 in order to qualitatively evaluate compositional
changes during degradation. First considering the MCPM/HA The pH of the cell culture media was clearly affected by the
cements, it is clear that by day 6 the relative fraction of HA in all MCPM/HA cements. The medium used to soak the 4:1.5,
four experimental groups was increased compared to the day 0 4:2 and 4:3 MCPM:HA cements was orange-yellow in color.
results. As expected, a greater amount of HA was present as the Cell culture medium contains phenol red as a pH indicator.
stoichiometric excess of HA increased. Interestingly, the 4:3 Thus, this color change was indicative of a drop in pH. In

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Biomed. Mater. 8 (2013) 025010 D L Alge et al

(A) (B )

(C) (D)

Figure 3. Powder x-ray diffraction patterns for MCPM/HA cements at days 0, 6, and day 14 of the degradation study. (A) 4:1, (B) 4:1.5,
(C) 4:2, and (D) 4:3 MCPM:HA molar ratios. Note: DCPD (00-009-0077), +HA (00-009-0432), #DCP (00-009-0080).

(A) (B )

(C) (D)

Figure 4. Powder x-ray diffraction patterns for MCPM/β-TCP cements at days 0, 6 and day 14 of the degradation study. (A) 1:1, (B) 1:1.5,
(C) 1:2, and (D) 1:3 MCPM:β-TCP molar ratios. Note: DCPD (00-009-0077), oβ-TCP (00-009-0169), +HA (00-009-0432).

contrast, no color change in the media used to soak the 4:1 Addition of the DCPD cement conditioned media to
MCPM:HA or any of the MCPM:β-TCP formulations was murine mesenchymal stem cells for 24 h had a significant
apparent, suggesting minimal change in pH. effect on cell viability (figure 5). The luminescent signals

5
Biomed. Mater. 8 (2013) 025010 D L Alge et al

to mechanical properties. Once the solubility limit is reached

and the solution is saturated with respect to DCPD, further
dissolution can only proceed if calcium and phosphate are
removed from the solution. For a closed system, such as our
in vitro static degradation model, this removal process can
only occur via a precipitation reaction. Precipitation can occur,
since DCPD is only metastable at pH greater than about 4 and
its dissolution supersaturates the solution with respect to HA
[31]. As a result, the third potential degradation mechanism
for DCPD cements is conversion of DCPD to a more basic
species such as HA. Importantly, after conversion to HA begins
to occur in the cement, DCPD dissolution can resume, which
again supersaturates the solution with respect to HA, leading
to further conversion. The overall reaction for the conversion
Figure 5. Comparison of MCPM/HA and MCPM/β-TCP cement of DCPD to HA is
cytocompatibility. Note: ∗ indicates a statistically significant 10CaHPO4 · 2H2 O → Ca10 (PO4 )6 (OH)2
decrease compared to the other MCPM/HA formulations, as well as
the equivalent MCPM/β-TCP formulation (p < 0.05). +4H3 PO4 + 18H2 O. (4)

measured for cells treated with 1:1, 1:1.5, 1:2 and 1:3 Importantly, this process results in both a progressive pH
MCPM:β-TCP were 90.16 ± 7.50, 90.03 ± 6.24, 91.72 drop and a net decrease in mass (HA/DCPD mass ratio for
± 6.21 and 97.37 ± 2.88 percent of the negative control, conversion is ≈ 0.58), and will proceed until the DCPD-HA
indicating minimal cytotoxicity. The differences between these singularity point is reached. This process can be understood
experimental groups were not statistically significant. The by considering the solubility isotherms of DCPD and HA (for
results for the MCPM:HA cements were markedly different. more in depth discussion we recommend [15] and [25]).
The 4:1, 4:1.5, 4:2 and 4:3 MCPM:HA groups had relative Analyzing the pH and mass loss data in figures 1 and 2,
luminescent signals of 121.12 ± 9.29, 70.57 ± 5.53, 40.61 differences between the MCPM/HA cements and MCPM/β-
± 7.00 and 3.33 ± 0.96 percent compared to the negative TCP cements are clear. Notably, pH drop and mass loss were
control. The differences between the four MCPMA:HA relatively low for the MCPM/β-TCP cements, and there was
experimental groups were statistically significant (p < 0.05). no trend with molar excess of β-TCP. These data indicate
The values for the 4:1.5, 4:2 and 4:3 MCPM:HA cements were dissolution as the key degradation mechanism, as all four
also significantly lower compared to the stoichiometrically MCPM/β-TCP groups appear to have reached the solubility
equivalent MCPM:β-TCP formulations (i.e. 1:1.5, 1:2 and 1:3 limit, with no further dissolution occurring. This conclusion
MCPM:β-TCP, respectively; p < 0.05). is supported by the powder x-ray diffraction data presented
in figure 4. All groups showed an increase in the fraction of
4. Discussion β-TCP at days 6 and 14 due to DCPD dissolution. Only very
small amounts of HA were found to be present in the 1:2 and
Characterizing and understanding the degradation properties 1:3 groups at day 14. Overall, these results are consistent with
of calcium phosphate cements is critical to assess their utility the published literature on in vitro degradation of β-TCP-based
as biomaterials for bone repair. In this study we tested the DCPD cement formulations. For example, Grover et al studied
hypothesis that DCPD cement chemistry affects the in vitro the degradation of DCPD cements prepared from β-TCP and
degradation properties and cytocompatibility of the cement orthophosphoric acid and reported roughly 8% mass loss and
product. We specifically investigated the effects of the base minimal conversion to HA after 14 days of static soaking in
reactant in the formulation by directly comparing MCPM/β- PBS [18]. In a subsequent study of DCPD cements prepared
TCP and MCPM/HA formulations. from β-TCP and pyrophosphoric acid published by the same
In order to evaluate the results of this study, it is group, conversion of DCPD to HA was not noted over a 90-day
important to recognize that three mechanisms can contribute period in either PBS or serum [19].
to the degradation of DCPD cements in our in vitro In contrast to what was observed for the MCPM/β-
system: dissolution, disintegration and conversion [18, 29, TCP, the MCPM/HA cements showed significant pH drop
30]. Dissolution occurs when DCPD is placed in a solution and underwent extensive mass loss, suggesting accelerated
undersaturated in calcium and phosphate ions. It directly leads conversion of DCPD to HA as the key degradation mechanism
to mass loss in the cement and proceeds according to the in this formulation. As alluded to earlier, the only way for pH
following equation: drop and mass loss to be increased in the MCPM/HA cements,
assuming that saturation was reached for the MCPM/β-TCP
CaHPO4 · 2H2 O → Ca2+ + HPO2−
4 + 2H2 O. (3) cements, is if conversion to HA were occurring. Indeed,
High degrees of dissolution can also lead to disintegration powder x-ray diffraction revealed the presence of substantial
of the cement because of microstructural changes [18, 19], amounts of HA in all of the MCPM/HA cements at days
which further increases mass loss and can also be detrimental 6 and 14. The 4:1 MCPM:HA group initially consisted of

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Biomed. Mater. 8 (2013) 025010 D L Alge et al

pure DCPD. The presence of a comparatively larger fraction the MCPM/HA cements could have significant implications
of HA in this group alone compared to the MCPM/β-TCP for biocompatibility because of the phosphoric acid produced.
cements is indicative of accelerated conversion to HA in the We hypothesized that the increased acidity resulting from
MCPM/HA system. However, further consideration of the accelerated conversion of DCPD to HA in the MCPM/HA
other MCPM/HA cements provides additional confirmation system would have a cytotoxic effect compared to MCPM/β-
of accelerated conversion of DCPD to HA compared to TCP formulations. To test this hypothesis, we used an in
MCPM/β-TCP cements and also provides insight to the vitro biocompatibility assay to directly compare the effects
mechanism. of MCPM/HA and MCPM/β-TCP cements on the viability
The 4:1.5, 4:2, and 4:3 MCPM:HA cements had a of murine mesenchymal stem cells. After 24 h in cell
significantly lower pH and significantly higher mass loss culture media conditioned with DCPD cement prepared with
compared to the 4:1 group. Furthermore, they contained an the MCPM/β-TCP system, the percent of viable cells was
even greater fraction of HA at days 6 and 14, with the 4:3 virtually unaffected (figure 5). These results agree with
group consisting of pure HA after just 6 days (figure 3). published data on MCPM/β-TCP formulations for DCPD
Importantly, the approximately 15% mass loss observed over cements, which have shown good cytocompatibility with
the 2-week degradation period cannot account for the extent osteoblast and macrophage cell lines [33–35]. In contrast,
of disappearance of DCPD observed by XRD. Although no while the 4:1 MCPM:HA group did not negatively impact
significant differences in pH or mass loss were observed cell viability, the 4:1.5, 4:2 and 4:3 MCPM:HA groups
between the 4:1.5, 4:2 and 4:3 MCPM:HA groups, these results showed significantly lower luminescence values compared to
suggest that conversion to HA is accelerated by the presence the negative control (p < 0.05 for comparisons between all
of unreacted HA in the cement. Recognizing that crystal MCPM/HA groups and for comparisons between the groups
nucleation is the rate-limiting step in the conversion of DCPD with equivalent excess of base). Cell viability was reduced to
to HA [21], it is likely that unreacted HA facilitates conversion nearly zero for the 4:3 group. This sharp decline in cell viability
by providing nucleation sites for HA precipitation. It is is most likely the result of rapid phosphoric acid production
possible that a small amount of unreacted HA, undetectable and acidification of the cell culture media. Considering that
by x-ray diffraction, was present in the 4:1 MCPM:HA group this acidification occurred over just 24 h in vitro, this result
and led to the accelerated conversion to HA compared to the suggests that the propensity toward rapid conversion of DCPD
MCPM/β-TCP cements, which lacked inherent nucleation to HA in MCPM/HA formulations may have an effect on in
sites for HA precipitation, but at a slower rate compared to the vivo biocompatibility.
4:1.5, 4:2 and 4:3 MCPM:HA groups. Interestingly, a small
amount of HA formation was observed in the MCPM/β-TCP
cements, but did not lead to rapid conversion to HA over the 5. Conclusions
course of our experiments. While epitaxial crystal growth of
In this study we have used simple in vitro models to evaluate the
HA from DCPD does not occur, HA precipitation is thought
effects of DCPD cement chemistry on the cement degradation
to slow conversion by creating a barrier to further dissolution
properties and cytocompatibility. We have specifically tested
[32]. Thus, it appears that that in MCPM/HA cements the
MCPM/HA and MCPM/β-TCP formulations for DCPD
presence of unreacted HA crystals throughout the cement
cements in a head-to-head comparison. Our results clearly
provides readily accessible nucleation sites for precipitation
without hindering further DCPD dissolution. show that major differences exist between the MCPM/HA and
Accelerated conversion of DCPD to HA in the MCPM/β-TCP formulations. Specifically, while conversion
MCPM/HA formulations compared to the MCPM/β-TCP to HA occurs in DCPD cements prepared with β-TCP, it is
formulations is an important finding, as rapid conversion slow due to the slow crystal growth kinetics of HA. In contrast,
to HA could potentially nullify the principal advantage of our results definitively show that rapid conversion of DCPD to
DCPD cements, which is their excellent resorbability [19]. HA is a key factor in the MCPM/HA cement system, and that
Additionally, the observation that excess HA in MCPM/HA excess HA further accelerates the conversion process. Because
formulations further accelerates conversion of DCPD to HA is conversion to HA could limit the resorption of the cement and
also important. Currently available commercial DCPD cement potentially have a negative effect on biocompatibility, future
formulations are biphasic and contain excess base reactant. in vivo characterization of MCPM/HA cement formulations
For example, ChronOS InjectTM (Synthes, Inc., West Chester, will be essential in assessing their potential for clinical use as
PA), which is an MCPM/β-TCP formulation, consists of bone repair materials.
approximately 15 wt% of unreacted β-TCP powder plus an
additional 30 wt% β-TCP granules after the setting reaction is Acknowledgments
complete [26]. Excess base reactant is desirable for modulating
cement properties such as acidity of the paste during setting, We thank Professor Jeffery Swope of the IUPUI Department
mechanical properties and resorption rate. However, tuning of of Geological Science for the use of his x-ray diffractometer,
the cement properties through a biphasic approach may not be and Ms. Patricia Metcalf of the Purdue University School
feasible in the MCPM/HA system due to the role of unreacted of Materials Science and Engineering for her assistance
HA in accelerating the conversion of DCPD to HA. in determining the percent crystallinity of the calcium
In addition to affecting the cement properties during phosphate powders. This work was supported in part by
degradation, a propensity toward rapid conversion to HA in the Indiana University School of Dentistry Professional

7
Biomed. Mater. 8 (2013) 025010 D L Alge et al

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General BioTechnology, LLC, The Genesis Bank, LLC, and
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Renovocyte, LLC. and Barralet J E 2006 Biologically mediated resorption of
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