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Journal of Controlled Release 113 (2006) 102 110

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Review

Calcium phosphate cements as bone drug delivery systems: A review


M.P. Ginebra , T. Traykova, J.A. Planell
Biomedical Engineering Research Centre (CREB), Division of Biomaterials, Biomechanics and Tissue Engineering,
Department of Materials Science and Metallurgical Engineering, Technical University of Catalonia (UPC), Av. Diagonal 647, 08028 Barcelona, Spain
Received 22 November 2005; accepted 6 April 2006
Available online 5 June 2006

Abstract
Since calcium phosphate cements were proposed, several formulations have been developed, some of them commercialised, and they have
proven to be very efficient bone substitutes in different applications. Some of their properties, such as the injectability, or the low-temperature
setting, which allows the incorporation of different drugs, make them very attractive candidates as drug carriers. In this article, the performance of
calcium phosphate cements as carriers of different types of drugs, such as antibiotics, analgesics, anticancer, anti-inflammatory, as well as growth
factors is reviewed.
2006 Elsevier B.V. All rights reserved.

Contents
1.
2.
3.
4.
5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Why calcium phosphate cements? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Calcium phosphate cements as drug carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Drug-release kinetics from calcium phosphate cements . . . . . . . . . . . . . . . . . . . . . . . .
Calcium phosphate cements as carriers for antibiotics. . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Effect of antibiotic incorporation on the physico-chemical properties of CPC . . . . . . . . .
5.2. Antibiotic release kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. In vivo performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Calcium phosphate cements as carriers for other drugs: anti-inflammatory, analgesic and anticancer .
7. Calcium phosphate cements as carriers for growth factors and other proteins . . . . . . . . . . . .
8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: ACP, amorphous calcium phosphate; BMP, bone morphogenetic protein; CPC, calcium phosphate cement; DCP, dicalcium phosphate, CaHPO4;
DCPD, brushite, dicalcium phosphate dihydrate CaHPO42H2O; HA, hydroxyapatite, Ca10(PO4)6(OH)2; MCPM, monocalcium phosphate monohydrate Ca
(H2PO4)2H2O; MRSA, Staphylococcus aureus resistant to Meticilin-Cefem; PLGA, poly(lactic-co-glycolic acid); PMMA, poly(methyl methacrylate); rhTGF-1,
human recombinant-transforming growth factor-1; rhBMP-2, human recombinant-bone morphogenetic protein-2; SBF, simulated body fluid; -TCP, alphatricalcium phosphate, -Ca3(PO4)2; -TCP, beta-tricaclium phosphate, -Ca3(PO4)2; TTCP, tetracalcium phosphate, Ca4(PO4)2O; TGF, transforming growth factor;
TGF-SF, superfamily of transforming growth factor-beta.
Corresponding author. Tel.: +34 93 4011089; fax: +34 93 4016706.
E-mail address: maria.pau.ginebra@upc.edu (M.P. Ginebra).
0168-3659/$ - see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2006.04.007

M.P. Ginebra et al. / Journal of Controlled Release 113 (2006) 102110

1. Introduction
The increased life expectancy in the developed countries has
led to a serious rise in the number of musculoskeletal disorders,
such as osteoporosis and osteoarthritis. However, also the
number of medications to treat and even prevent these diseases
has expanded in recent years [1]. The development of new drugs
and active substances, allows treating some of these diseases
even in their initial stages.
A key issue in these treatments is to maximize the drug
access to specific bone sites, and to be able to control the
release of drugs, in order to maintain a desired drug
concentration level for long periods of time without reaching
a toxic level or dropping below the minimum effective level
[2]. For this reason, a major effort has been done focused on
the development of materials that are capable of releasing
drugs by a reproducible and predictable kinetics. Although
most of these drug carriers are polymers, in the specific field of
the pharmacological treatment of skeletal disorders, some
inorganic materials can also play a role. Indeed, calcium
phosphate based materials, which are bioactive, could have an
added value as drug carriers for the bone tissue. Moreover,
another relevant property of calcium phosphates is their unique
ability to adsorb different chemical species on their surfaces.
This property has been exploited for instance in the
hydroxyapatite (HA) chromatography, which has proved to
be very efficient for the purification and separation of proteins,
enzymes, nucleic acids and other macromolecules [3].
Especially relevant is the application of HA chromatography
to the purification of bone growth factors [4]. Indeed, the great
affinity of HA for these various active molecules can be
exploited within the field of the development of new matrixes
for drug delivery applications.
In this article we provide an insight in the application of one
family of calcium phosphate based materials, namely calcium
phosphate cements, as materials for controlled drug delivery.
2. Why calcium phosphate cements?
The possibility to obtain monolithic hydroxyapatite at
ambient or body temperature via a cementitious reaction was
put forward by LeGeros [5] and Brown and Chow [6] in the
early eighties. This was a significant step forward in the field of
bioceramics for bone regeneration, since it provided a material
which, in addition to being bioactive, was mouldable and had
the capacity of self-setting in vivo, within the bone cavity [7,8].
In addition, the development of injectable calcium phosphate
cement formulations established good prospects for minimally
invasive surgical techniques developed in recent years, less
aggressive than the classical surgical methods.
Since then, calcium phosphate cements have attracted much
attention and different formulations have been put forward
[6,913]. In general, all CPC are formed by a combination of
one or more calcium orthophosphates, which upon mixing with
a liquid phase, usually water or an aqueous solution, form a
paste which is able to set and harden after being implanted
within the body.

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Currently many commercial products exist on the market [8].


Most of them form hydroxyapatite upon setting, with low
crystallinity and high specific surface, which can incorporate
different ions in its lattice depending on the composition of the
starting materials. In general it can be stated that the formation
of hydroxyapatite through a cement reaction is a biomimetic
process, in the sense that it takes place at body temperature and
in a physiological environment. Indeed, the hydroxyapatite
formed in the setting of CPC is much more similar to biological
apatites than ceramic hydroxyapatite. Other cement formulations give other reaction products upon setting, such as brushite.
As mentioned, cement setting is a result of dissolution and
precipitation process. The interlocking between precipitated
crystals is responsible for cement hardening. Fig. 1 shows the
microstructure of an apatitic cement after setting. As it can be
seen, CPC develop a highly micro/nanoporous structure. Porosity
can vary between 30% and 50%, depending on the processing
conditions, e.g. liquid-to-powder ratio. The setting, rheological
and mechanical properties of the CPC can be adjusted by
modifying different factors, such as the chemical composition of
reactants, particle size or presence of nucleating agents [1417].
CPC are therefore very versatile materials, which can be adapted
to the different clinical requirements of various applications.
Different studies with CPCs have shown that they are highly
biocompatible and osteoconductive materials, which can
stimulate tissue regeneration [1824]. Most of the apatitic
cements are resorbed via cell-mediated processes. In these
processes, osteoclastic cells degrade the materials layer by layer,
starting at the bonecement interphase throughout its inner part.
Some of the advantages brought out by the development of
CPC in comparison with the use of calcium phosphates in the
form of ceramic granules or bulk materials can be summarized
as follows:
(a) In first place, the self-setting ability in vivo.
(b) Their injectability, which allows cement implantation by
means of minimally invasive surgical techniques, less
aggressive than the traditional surgical techniques.

Fig. 1. Microstructure of an apatitic calcium phosphate cement after setting,


showing the micro/nanoporous structure formed by the entanglement of the
precipitated crystals.

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M.P. Ginebra et al. / Journal of Controlled Release 113 (2006) 102110

(c) The perfect fit to the implant site, which assures good
bonematerial contact, even in geometrically complex
defects. This allows for an optimum tissuebiomaterial
contact, necessary for stimulating the bone ingrowth,
(d) The fact that the setting reaction which takes place under
in vivo conditions is a dissolutionprecipitation process,
resulting in most cases in a precipitated hydroxyapatite
with a high and interconnected microporosity, chemically
and structurally similar to biological apatites. These
factors contribute to an increased reactivity of CPC as
compared to calcium phosphate ceramics.
(e) Lastly, as mentioned above, the low-temperature setting
allows incorporation of different drugs: from antibiotics
and anti-inflammatory drugs to growth factors which are
able to stimulate certain biological responses. This aspect
gives a great potential to this type of materials for the
controlled drug delivery in target sites of the skeletal
system.
3. Calcium phosphate cements as drug carriers
In general, a potential substrate to be used as drug carrier
must have the ability to incorporate a drug, to retain it in a
specific target site, and to deliver it progressively with time in
the surrounding tissues. Additional advantages are provided if
the material is injectable and biodegradable.
The possibility to use CPCs not only as bone substitutes but
also as carriers for local and controlled supply of drugs is very
attractive and can be useful in treatments of different skeletal
diseases, such as bone tumours, osteoporosis or osteomyelitis,
which normally require long and painful therapies.
Unlike calcium phosphate ceramics employed as drug
delivery systems, where the drugs are usually absorbed on the
surface, in CPCs the drugs can be incorporated throughout the
whole material volume, by adding them into one of the two
cement phases. This fact can facilitate the release of drugs for
more prolonged times.
The studies about incorporation of drugs into CPC cover
different aspects. In the first place, it is necessary to verify that
the addition of the drug (either to the liquid or the solid phases
of the cement) does not interfere in the setting reaction,
modifying the physico-chemical properties, not only in terms of
the setting and hardening mechanisms but also with respect to
the rheological behaviour. Secondly, it is necessary to
characterize the kinetics of drug release in vitro. Subsequently,
the effectiveness of the cement to act as carrier for drug delivery
in vivo, must be assessed. And finally, the clinical performance
of the drug delivery system must be evaluated.
During the last decade, several studies related to the
application of both commercial and experimental CPCs as
drug carriers have been published. Major attention has been
paid to antibiotics, due to their wide areas of application: either
as prophylactics to prevent infections produced during surgical
interventions, or in general in the treatment of bone infections.
However, also anti-inflammatory of anticancer drugs or even
hormones has been studied. In addition, in the last years the
incorporation in CPCs of some other substances or factors able

to stimulate bone regeneration, such as bone morphogenetic


proteins (BMP) or transforming growth factors (TGF-), has
been considered.
4. Drug-release kinetics from calcium phosphate cements
The release of drugs from any drug delivery device depends
on different factors such as the microstructure, the drug
solubility, the type of bond between the drug and the matrix
which holds it, and the mechanism of degradation (if any) of the
matrix. [25,26]. Generally speaking, CPC could be ascribed to
the category of diffusion-controlled devices, where the drug is
incorporated into a non-biodegradable matrix, through which it
should diffuse. Indeed, although some CPC are resorbable, in
most of the CPC studied as drug-carriers, the rate of matrix
degradation (e.g. the cement itself) is much lower than the rate
of drug liberation. For that reason it is possible to assume that
the drug release is mainly controlled by the process of diffusion
through the cement matrix and not by the degradation of the
same.
In CPC the drugs can be introduced either in the liquid phase
or in the powder phase. During cement setting, dissolution of
calcium phosphates from the powder phase takes place, which is
followed by precipitation of a new phase. The new phase is in
most cases precipitated hydroxyapatite, and it can be assumed
that the drug is dispersed in the matrix formed by the set cement.
In this situation, normally the drug-release kinetics follows
Higuchi's law [27], at least at the initial stages (until around
60% drug is released):
Mt AM0 DCs 2C0 Cs t1=2

where Mt is the amount of drug released for time t; M0 is the


total amount of drug; A is the surface area of the device; D is the
diffusion coefficient of the drug in the matrix; Cs is the
solubility of the drug in the matrix; C0 is the initial
concentration of the drug in the matrix.
If we consider that, in the case of CPC, the matrix
containing the dispersed drug is very porous since it is formed
by a mesh of interlocking of crystals which create an open
micro or even nanoporosity, the diffusion coefficient D of the
matrix should be replaced by an effective diffusion coefficient
Deff:
Deff

D Ve
s

where Deff is the effective diffusion coefficient; D is the


diffusion coefficient of the crystals which form cement as such;
is the cement porosity; is the tortuosity of the cement.
By substituting (2) in (1) we obtain:

1=2
D Ve
Cs 2C0 eCs t
Mt AM0
3
s
Therefore, it is clear that porosity and other microstructural
parameters of the cement (e.g. tortuosity), play an important role
in the kinetics of drug release. This dependence has been shown
by Otsuka in several works [2832] and confirmed by other

M.P. Ginebra et al. / Journal of Controlled Release 113 (2006) 102110

authors [3335], in studies covering different CPC formulations


and different drugs.
It has to be pointed out, however, that in the cases when the
drug is poorly soluble, the release would not follow the law of
Higuchi, since it would be controlled by the dissolution of the
drug, the release being proportional to the time.
In most cases it can be considered that CPC do not degrade
while drug is released, or, in other words, that the porosity of the
matrix is maintained constant during drug delivery. However,
some authors have shown certain degree of degradation of CPC
during drug liberation. For example, Otsuka [36] showed an
increase of porosity in a carbonated hydroxyapatite cement
during the release of indomethacin, which was ascribed to the
cement degradation. In this case, the higher resorption rate of
the cement was explained by the fact that the addition of
different amounts of NaHCO3 to a TTCP/DCPD cement
resulted in a carbonated apatite more soluble than the
stoichiometric HA used in previous studies. Resorption of
carbonated HA with time produced an increase of porosity,
which increased the drug release rate. In summary, if porosity
does not remain constant, drug release kinetics does not follow
Higuchi's law, and the drug diffusion through the CPC matrix is
not any longer the only mechanism which controls drug
liberation. In any case, it should not be omitted that during
release of drugs, the speed of resorption of the CPC matrix
depends on various factors, such as chemical composition,
microstructure and crystallinity of the cement. In the case of
brushite cements, the degradation rate is much higher than that
of apatite cements, and therefore is more important to consider
these phenomena.
As it was mentioned previously, Higuchi's law is valid for
the initial stage of
pdrug release, being the mass of liberated drug
proportional to t . According to another model by Tung [37],
during the second stage, the release of drug is proportional to
the time, followed by a third stage at which the liberation is
stabilized as the concentration of the antibiotic in the
environment increases. In this model, the existence of the
second stage would depend on the type of antibiotic and its
solubility. Although not described by the author, the release
kinetics of the second stage could be explained by a low
solubility of the drug and hence a dissolution-controlled release,
proportional to the time.
5. Calcium phosphate cements as carriers for antibiotics
In the field of application of CPCs for drug delivery, the
antibiotics have been widely studied, due to their frequent use in
the treatment of infections of the skeletal system, or as a
prophylactic strategy. One of the key factors for the success of
surgical interventions aimed at the implantation of a prosthesis
or of an osteoconductive material is the prevention from
bacterial infections. Wound contamination, or postoperative
infections following fracture repair, implantation of joint
prosthesis or spine surgery, can cause serious problems. For
this reason antibiotics are often provided as prophylactics, either
orally or intravenously. However, the very little accessibility of
the site of infection very often prolongs the treatment of bone

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infections. A traditional method applied to control bone


infections is the implantation of poly-methylmethacrylate
spheres (PMMA) loaded with gentamicin sulphate in the
infection site [38]. However, PMMA spheres are nonresorbable
and must be removed after some months and replaced with new
spheres or other substitute material, able to facilitate bone
regeneration. Antibiotics can be also incorporated in calcium
phosphate ceramic blocks, although their resorption rates are
slow. Moreover, it is difficult to shape ceramics with complex
form in order to be fitted into any type and size of bone defect.
An alternative material that was proposed some decades ago
was calcium sulphate hemihydrate (Ca2SO41/2H2O) which can
be used in form of cement, giving as reaction product calcium
sulphate dihydrate (Ca2SO42H2O) [39]. The main drawbacks
of this material come from its low mechanical strength and very
high resorption rate. In this context, the combination of
antibiotics with CPC can have enhanced properties which
overcome some of the previously mentioned drawbacks.
5.1. Effect of antibiotic incorporation on the physico-chemical
properties of CPC
The setting reaction of the CPC can be affected or modified
by introducing a drug either to the powder phase or to its liquid
phase and, as a consequence, the physico-chemical and
mechanical properties can change [33,34,4042]. In general,
in apatitic cements antibiotics tend to increase their setting times
and reduce the mechanical strength [4042]. Takechi et al.
studied the effect of incorporating flomoxef sodium in different
concentrations (up to 10%) to the solid phase of a cement
formed by TTCP and DCPA [40]. Since this antibiotic is very
soluble, it was assumed that it dissolved fast in the cement liquid
phase. The authors noticed a strong reduction of mechanical
strength when the amount of antibiotic added increased. This
decrease of mechanical strength was attributed to increased
porosity and to some inhibition of the setting reaction, as
suggested by the presence of certain amount of reactants when
the antibiotic quantity increased. The drug release followed the
kinetics typical for these types of materials (as described in the
previous section), and after 72 h the total amount of liberated
antibiotic was between 55% and 60%.
In other cases, the modification of the CPC properties can be
due to some chemical interactions between the antibiotic and the
cement. Indeed, the addition of tetracycline to an apatitic
cement caused a strong reduction in mechanical properties
which was attributed to the ability of this antibiotic to chelate
Ca-atoms [41]. This was supported by the fact that when
antibiotic was introduced already complexed with calcium, the
interactions antibioticcement were limited. This allowed the
addition of bigger amounts of antibiotic without influencing
setting times and mechanical properties of the cement.
5.2. Antibiotic release kinetics
Hamanishi et al. [43] examined the incorporation of
vancomycin to a CPC formed by TTCP and DCPD for treating
osteomyelitis caused by MRSA. In in vitro studies they

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M.P. Ginebra et al. / Journal of Controlled Release 113 (2006) 102110

observed an effective release of vancomycin within 2 weeks, in


the case of CPCs containing 1% vancomycin, and within
9 weeks for CPCs containing a higher content (5% vancomycin). In both cases, the released concentrations were higher than
the effective concentration against different types of MRSA.
The rate of drug release depended on the crystallinity of the
cements, but for the two periods studied up to 95% of the
antibiotic was liberated in both cases.
The antibiotic release kinetics of brushite cements, more
resorbable than apatitic cements, was analysed by adding
gentamicine sulphate both in powder form and as a solution
[33]. The reactants used for the cement were MCPM and TCP. Once again the rheological and mechanical properties of
the cement changed by antibiotic addition, but in the opposite
direction of that reported by Takechi et al. They observed an
increase of the setting time, which was initially too short, as well
as an increase of mechanical strength, that was ascribed to the
presence of sulphate ions in the antibiotic. In the first few hours,
drug release waspvery rapid following Higuchi's law. It was
proportional to t until 50% of antibiotic was released, and
drug release tended to stabilise beyond this value. The total
amount of antibiotic was released within 7 days, and no
significant differences were observed depending on the way of
incorporation, as a powder or in solution.
An interesting aspect worth mentioning is the difference in
the rate of antibiotic release from CPC and from PMMA beads.
In a comparative study, the release kinetics of three antibiotics,
i.e. gentamicine, amikacine and ceftiofur incorporated either in
a commercial TTCP/DCP cement or in PMMA spheres, was
analysed [44]. The results after 30 days showed faster antibiotic
liberation from CPC than from the polymer. In both carriers, the
concentrations of liberated gentamicine and amikacine were
higher than the critical doses for preventing bacterial effects. On
the other hand, the liberation of ceftiofur from both materials
remained within the right levels only during 7 days, being
therefore inadequate when the bactericide effect has to be
prolonged in time.
In the cases where antibiotic release is considered to be
excessively fast, a possible strategy is to incorporate some
polymers in the CPC, in order to retard drug liberation. In this
sense some studies have been carried out aiming at the
formation of a gel into the cement pores, which served as
matrix for the antibiotic. Takechi et al. added sodium alginate or
chitosan [40,45]. Chitosan is found to stimulate bone formation,
osteoconductivity, endochondral ossification and membranous
osteoinduction. Thus, by combining sodium alginate and
chitosan with the cement, a slight decrease of antibiotic release
at the initial stage was observed, and liberation was maintained
to slightly higher levels to those obtained in commercial CPC.
Bohner et al. [34] applied a similar strategy by adding
polyacrylic acid (PAA) to brushite cement, and likewise
gentamicine delivery was prolonged in time, especially when
PAA had a high molecular weight. It was hypothesized that at
the initial stage the release kinetic would
p be controlled via
diffusion through pores (proportional to t ), and subsequently,
at the second stage, it would be controlled by the dissociation of
gentamicine sulphate from the complex formed with PAA

following a first order kinetics. Depending on the amount of


added PAA, the quantity of released gentamicine varied
between 58% and 100%. A constant delivery during 8 days
was achieved by using high molecular PAA, which practically
meant release of 100% antibiotic.
5.3. In vivo performance
In vivo release of vancomycin was studied by Hamanishi et
al. [43]. CPC were loaded with 1%, 2% and 5% of vancomycin
and implanted in tibial condyles of rabbits. After 3 weeks the
concentration of vancomycin in bone marrow was 20 times
higher than the minimum value clinically required, in the
cement with 5% vancomycin. Nevertheless, higher antibiotic
concentrations seemed to delay bone ingrowth on the implant
and thin fibrous capsules were formed.
In another in vivo study, Stallmann et al. [46] examined
and compared the effect of adding gentamicine and the
antibicrobian peptide hLF1-11 (a fragment of human lactoferrin) to a commercial CPC based in TTCP and DCP. These
materials were thereafter implanted in the femoral channel of
rabbits, vaccinated with Staphylococcus aureus. The development of osteomyelitis was reduced for both drugs, but this
reduction was more effective in the case of the gentamicineloaded CPC.
6. Calcium phosphate cements as carriers for other drugs:
anti-inflammatory, analgesic and anticancer
Besides their application as antibiotic carriers, CPC can be
appropriate matrixes for the incorporation of other drugs with
potential application in the musculoskeletal system.
As already mentioned, it is important to consider all
possible effects of drug addition on the setting reaction of the
CPC. Ginebra et al. studied the effect of the incorporation of
an amino salicylic acid derived methacrylamide in an apatitic
-TCP-based cement [47]. Salicylic acid is an analgesic and
anti-inflammatory, and moreover has a calcium complexation
ability. Strong effects were observed both in the rheological
and mechanical properties. The injectability was improved,
but simultaneously a decrease in the reaction rate of the
cement was observed. The mechanical strength increased, by
25% in compression and by 80% in flexion, which was
attributed to decreased porosity and smaller precipitated
crystals.
Otsuka et al. [32] carried out an in vitro study about the
release kinetics of aspirin from a TTCP/DCP/HA cement. The
rate of drug liberation was found to increase with higher
porosity of cement, which can be easily controlled by liquid-topowder ratio. Thus, the authors confirmed that kinetics of drug
release followed a modified Fick's law, which was controlled by
drug diffusion through pores.
Indomethacin is an anti-inflammatory, non-steroidal drug
with wide application in different pathologies of muscle
skeletal system, such as chronical joint rheumatism. Otsuka et
al. published different studies regarding the incorporation of
indomethacin in an apatite cement based in TTCP, DCPD and

M.P. Ginebra et al. / Journal of Controlled Release 113 (2006) 102110

HA [29,30,36,48]. Different concentrations of indomethacin


were combined with CPC and analyzed in vitro in simulated
body fluid (SBF) as well as in phosphate buffer solution (PBS).
In both cases the kinetics followed Higuchi's law, and the
amount of drug released increased with the amount of drug
incorporated in the cement. The release of indomethacin was
much slower in SBF than in PBS. This was attributed to the
formation of an HA layer on the surface upon immersing in
SBF, since the cement is a bioactive material [49]. This HA
layer reduced cement porosity and intervened the drug diffusion
to the surrounding fluid. There was a particular case where
indomethacin release from CPCs did not follow Higuchi's law,
i.e. when sodium bicarbonate was added to the cement, due to
the increase of the CPC resorption rate [36].
In vivo studies [30,48] were made where the cement was
implanted subcutaneously in rats and the concentration of
indomethacin in the plasma was measured. Initially indomethacin release was very fast. After 1 day it started to
decrease and in total it prolonged for 3 weeks. The higher
the initial amount of drug in the cement, the higher the
amount of liberated indomethacin. A relevant result was that
the half-life of indomethacin in plasma was much higher
when drug was introduced via cement implantation, rather
than when it was injected subcutaneously. The liberation of
the drug was prolonged over one month when it was added
in the cement. By comparing the concentrations of released
drug in vivo and in vitro, a linear relation at the initial stage
was observed, but liberation in vivo was much slower than
liberation in vitro during the last stage of the study. This
could be due to some surface changes in the cement caused
by the formation of an apatite layer due to the bioactive
character of the CPC, or by other changes due to protein
adsorption or cell activity.
Mercaptopurine is a drug used in the therapy of some
tumours, due to its ability to inhibit the proliferation of tumoral
cells. Otsuka et al. studied its incorporation to a CPC based in
TTCP and DCPD [28,31]. The setting reaction was not affected.
In vitro release of mercaptopurine showed a similar behaviour
to that observed for indomethacin. A clear dependence between
release rate and cement porosity was also observed, which can
be easily controlled by modifying the liquid-to-powder ratio of
the cement paste [31].
Estradiol is a feminine sexual hormone with estrogenic
activity, which can be used in the treatment of symptoms caused
by the deficit of estrogens during menopause, such as mineral
resorption and bone loss. Otsuka et al. studied the rate of release
of estradiol in vitro, incorporated in CPC and immersed in SBF,
and in vivo, by subcutaneous implantation in rats [50,51]. The
rate of estradiol liberation in vitro was inversely proportional to
calcium concentration in solution. Consistently, in vivo release
of estradiol was faster in rats which had lower concentrations of
vitamin D and Ca, compared to healthy rats, suggesting the
autoregulatory mechanism of estradiol liberation. The bone
mass of the recovery model rats was greater after the experiment
than before, suggesting that the severity of osteoporosis in these
animals could be reduced by the implantation of this estradiolloaded apatite cement.

107

7. Calcium phosphate cements as carriers for growth


factors and other proteins
Growth factors are a large group of polypeptides, able to
transmit signals which affect cellular activity [52]. Among
them, the superfamily of -transforming growth factors (TGF
-SF) is especially relevant for bone regeneration. It includes
the transforming growth factors 1, 2, 3 (TGF-1, 2 and 3),
and the bone morphogenetic proteins (BMP) among other
proteins. It is known that they play a role as activating agents for
the complex cascades of biological phenomena responsible for
bone formation, and therefore they can accelerate bone
ingrowth. A special feature of the subfamily of BMPs is that,
on the contrary to the other growth factors of the TGF-SF,
BMPs are osteoinductive. This means that BPMs can activate
the differentiation of pluripotential cells to bone forming cells,
thus leading to bone formation even outside bone tissue.
In the last years the recombination techniques have made
possible the industrial production of human growth factors in
big quantities and high purity. However, it is necessary to have
available materials which allow for the controlled administration of these factors at adequate therapeutic levels, and their
vectoring towards local tissue targets and cells. In fact, it is
known that injection of this type of substances alone cannot
induce tissue formation and regeneration, since protein diffuses
very fast from the implantation site. For that reason it is
necessary to select new materials which can act as substrates or
carriers for these biologically active factors. CPC possess a
great potential, because they set at room temperature, are
biocompatible and osteoconductive. Indeed, the idea of
incorporating growth factors into CPCs arose with the aim of
improving or increasing the osteoconductive capacity of
cements.
TGF- are multipotential regulators of bone cells metabolism and are capable of favouring bone formation in vivo,
depending on their concentration and way of application, as
shown in many studies. It has been shown also that TGF-1
stimulates proliferation of osteoblasts and collagen synthesis in
vitro [53], and can increase the thickness of cortical bone when
applied adjacent to periosteum in vivo [54]. Some studies in
vitro demonstrated that combinatory use of these factors with
calcium phosphate ceramics resulted in improved bone growth
due to adsorption of big doses of rhTGF-1 on the ceramic
surface [55,56]. Nevertheless, the use of resorbable CPC can
allow for a homogeneous distribution of the growth factor not
only on the surface but in the whole bulk of the cement, thus
making it possible the growth factor release for a more
prolonged time, while degradation of the cement takes place,
activating simultaneously the new bone formation.
Recently, Blom et al. studied the effect of the incorporation
of human recombinant TGF-1 (rhTGF-1) in a CPC, based in
-TCP, TTCP and DCPD, on the first stages of bone formation
in vitro, using primary rat bone cells [57]. The growth factor
was incorporated in the cement during setting, and results
showed that the addition of rhTGF-1 stimulated the differentiation of pre-osteoblastic cells in vitro. Other studies analysed
the effect of growth factor addition on the physico-chemical and

108

M.P. Ginebra et al. / Journal of Controlled Release 113 (2006) 102110

mechanical properties of two different cement formulations


[58,59], demonstrating that CPCs with TGF did not differ from
conventional cements, in terms of the setting parameters.
Preclinical studies in animals proved as well the stimulating
effect of TGF on bone growth [60]. In contrast to what
happened in most cases with the antibiotics, the drug release
kinetics of this osteogenic factor was quite slow. In both
cements an initial elution in the first days was followed by a
stabilization of the released amount after this time. These results
suggested that the growth factor was released only from the
superficial layer, in contact with the surrounding medium, and
not from the whole volume of the CPC. This was confirmed by
the fact that the enlargement of the area in contact with the
medium by fragmentation, resulted in another burst or rhTGF1 release. This behaviour can be related to the high binding
affinity of the protein for calcium phosphate ceramics, and puts
forward a different kinetics from that described for other
conventional drugs.
A similar trend was observed when human recombinant
BMP-2 loaded poly(DL-lactic-co-glycolic acid) (PLGA) microspheres were mixed with a CPC [61]. Release of rhBMP-2 was
very limited (mean of 3.1% after 28 days under neutral
conditions), much slower than the release of the protein in the
microspheres alone (18% after 28 days). This was attributed to
the physical entrapment of the microparticles within the
nanoapatitic porous cement. According to the authors, the
nanoporosity of the CPC not only did not facilitate the release of
the protein, but may have further limited it because of the high
binding affinity of the protein for CPC.
The in vivo performance of BMP-2 was assessed by
Seeherman et al. in a complete series of works where different
injectable osteogenic factor/carrier combinations was reviewed
in different large animal models [6265]. Among all the carriers
studied, a commercial resorbable CPC, based in ACP and
DCPD, was considered to be the best carrier in terms of bone
healing after 10 weeks, in a fibular osteotomy, in a non-human
primate model. One of the main advantages of this carrier is that
it can be implanted by a single percutaneous injection. Bone
healing was accelerated by approximately 40%, as compared to
untreated osteotomy sites. Ohura et al. obtained analogous
results by incorporating different amounts of recombinant
human BMP (rhBMP-2) to a cement formed by -TCP, MCPM,
calcium sulphate hemihydrate and granules of -TCP, after
setting [66], and implanting it in critical fractures in a rabbit
model. One of the main advantages of employing this CPCs as a
carrier with respect to other ceramics already used for the same
purpose [67] was supposed to be the faster rate of resorption of
this cement. In the fractures filled with cement with the
optimum rhBMP-2 concentration, bone formed very rapidly,
and after 3 weeks the fracture was already consolidated. Most of
the cement cylinders were resorbed, except from the -TCP
granules, and were completely replaced by new bone in
6 weeks. By the ninth week, the torsion resistance was 99%
recovered. None of the fractures consolidated when the
unloaded cement was used as a filling material. In conclusion,
the CPC turned out to be an adequate substrate for delivery of
rhBMP-2. Similar results are reported by Kamegai et al. [68].

Other proteins which have a relevant function in bone tissue


and which have been incorporated in CPC aiming at improving
their biological and mechanical properties are collagen I and
osteocalcin [69]. Osteocalcin is a non-collagenous protein
found in the extracellular bone matrix, which plays an important
role in the biomineralization process. The addition of
osteocalcin to an apatitic cement obtained from a mixture of
-TCP, DCP, calcium carbonate and HA seeds modified the
microstructure by reducing the size of precipitated hydroxyapatite crystals. On the other side osteocalcin modified the cellular
response in vitro of the osteosarcoma cell line SAOS-2,
improving their initial adhesion, but without significant effect
on cell proliferation.
Incorporation of insulin and bovine albumin in a CPC based
in TTCP and DCP was studied by Otsuka et al [70], in the case
of albumin as a model protein to test protein release. Their
results showed that these polypeptide drugs do not disturb the
setting reaction of cement. Moreover, their release in a
phosphate solution went on more than 3 weeks, following the
Higuchi's law.
8. Conclusions
It has been shown that the research carried out in the past
decade put forward the great potential of CPC as carriers for
controlled release and vectoring of drugs in the skeletal system.
Especially encouraging are the results obtained with osteogenic
factors for bone regeneration applications. However, one must
admit that the industrial use of CPC for drug delivery is not easy
for two main reasons. First, implant companies selling CPC do
not have the know-how to deal with drugs, whereas
pharmaceutical companies do not have any know-how with
CPC, and often do not have any interest in working with such
small markets. Second, infections are not always produced by
the same bacteria. Therefore, it would be of importance to
propose a system that could be combined with many different
drugs, in such a way that the surgeon could choose the drug just
before implantation. However, as it has been shown, various
drugs have various effects on CPC properties, and this
represents a serious drawback for the implementation of this
technology. A lot of work has still to be done to establish the
general laws that control the release profile of these types of
materials, in order to be able to adjust them to different
therapeutical needs and to obtain reproducible and predictable
drug delivery systems.
Acknowledgements
The authors thank the Science and Technology Spanish
Ministry for funding this work through project CICYT
MAT2002-04297. T. Traykova is grateful to the Spanish
Ministry of Education for her grant SB2003-0352.
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