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Acta Biomaterialia 5 (2009) 2161–2168

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Preparation and properties of macroporous brushite bone cements

G. Cama a,*, F. Barberis a, R. Botter a, P. Cirillo a, M. Capurro a, R. Quarto b, S. Scaglione b,E. Finocchio c, V. Mussi d, U. Valbusa d

a Department of Civil , Environmental and Architectural Engineering (DICAT), University of Genova, Area of Materials Engineering,

P.zale Kennedy 1, Fiera del Mare, Pad D, 16129 Genova, Italyb Advanced Biotechnology Center (CBA), Largo Rosanna Benzi 10, 16133 Genova, Italy

c Department of Chemical and Process Engineering, University of Genova , P.zale Kennedy 1, Fiera del Mare, Pad D, 16129 Genova, Italyd Department of Physic, University of Genova via Dodecaneso, 3 16146, Genova and Nanomed Labs, Advanced Biotechnology Center (CBA), Largo Rosanna

Benzi 10 16133 Genova, Italy

Received 31 July 2008; received in revised form 26 January 2009; accepted 3 February 2009Available online 13 February 2009

Abstract

In the present work a macroporous brushite bone cement for use either as an injected or mouldable paste, or in the shape of pre-formed grafts, has been investigated. Macropores have been introduced by adding to the powder single crystals of mannitol whichworked as a porogen. The size of the crystals was in the range of 250–500 lm in diameter, suitable for cell infiltration, with a shape ratiobetween 3 and 6. From compression tests on cylindrical samples an elastic modulus in the range 2.5–4.2 GPa and a compressive strengthin the range 17.5–32.6 MPa were obtained for a volume fraction of macropores varying between 15 and 0%. Thus the compressivestrength exceeded in all tests the maximum value currently attributed to cancellous bone.� 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Bone cement; Brushite; Cell adhesion; Porosity; Mechanical properties

1. Introduction

Calcium phosphate bone cements are used as mouldableor injectable pastes to fill bone cavities, defects or disconti-nuities [1,2]. These cements, which set and harden shortlyafter implantation, form highly biocompatible and osteo-conductive scaffolds [3].

The chemical reaction which produces the biomaterial isactivated by mechanically or manually mixing several cal-cium phosphate phases with a liquid phase. Dependingon the pH of the reaction, two types of cements areobtained: for pH higher than ca. 4.2 the reaction productis hydroxyapatite (HA) [4], in the other case brushite(DCPD) [5] is obtained.

Brushite cements have raised considerable interest in thelatest decade, because they are metastable under physiolog-

1742-7061/$ - see front matter � 2009 Acta Materialia Inc. Published by Else

doi:10.1016/j.actbio.2009.02.012

* Corresponding author. Tel.: +39 010 353 6037.E-mail address: giuseppe.cama@unige.it (G. Cama).

ical conditions and can be resorbed more quickly than HAcements that are stable [6]. Cements of the first type mayimprove, therefore, the ingrowth of bone tissue from thesurface to the inside of the implant, which means that theyare more osteoconductive.

Unfortunately the setting time of brushite cements isgenerally too short, typically ranging between 30 and 60 s[7], to be efficiently used by surgeons.

To make such cements suitable for orthopaedic applica-tions specific setting retardants, such as pyrophosphateions or citrates [8,9] are usually added to slow down the set-ting process. The action of retardants essentially consists ininhibiting both nucleation and growth of calcium phos-phate crystals [10,11]. In a recent work [8], a reduction ofgrain growth rate has been attributed to the action of pyro-phosphate ions in concentration of about 4.3 wt.%. As aconsequence smaller crystals with a better packing factorwere formed, improving the tensile diametral strength ofthe cement as set.

vier Ltd. All rights reserved.

2162 G. Cama et al. / Acta Biomaterialia 5 (2009) 2161–2168

There are different philosophies concerning the choice ofporogens. In some instances fast dissolving porogens (ase.g. mannitol) are used, while in other cases fibers ofresorbable polymers are preferred to preserve mechanicalstrength in the initial stage of implantation. Inclusions ofboth types have been used by other authors to obtain anoptimum compromise between the two requirements[12–14]. As to the pore size, which has to be large enoughto admit bone cells as macrophages and/or osteoblasts,some authors [15] compared the amount of bone ingrowthin hydroxyapatite implants containing pores of differentsizes from 100 to 600 lm and found best results when poreswere interconnected and with a diameter in the range of300–400 lm. For implants of resorbable materials theresorption time is the target parameter depending on thethickness of the pore walls. In this case the problem is quitecomplex, involving as inter-correlated variables the poresize and shape, as well as the resorption rate which, in turn,is related to the mechanisms of cell activation. Accordingto the theoretical model by Bohner and Baumgart [16],considering macropores of spherical shape distributed ina CFC lattice, the optimum pore radius depends on theimplant size and ranges between 200 and 350 lm.

Finally the in vivo ageing strongly depends on a numberof factors having to do not only with the formulation of thereactants but also with characteristics of the implant, suchas the animal model, the type of bone to treat, the size ofthe defect to heal [17] and the flow of the surrounding bio-logical fluids [18].

This work is aimed at obtaining a macroporous resorb-able cement that can be in situ injected or moulded. Thefirst step was to prepare a microporous brushite cementwith a nearly ideal composition, a setting time suitablefor in vivo implantation and mechanical properties suffi-ciently high to allow for the introduction of macroporesin a certain volume fraction, while preserving a compres-sive strength comparable to that of cancellous bone. Suc-cessively a macroporous cement was studied, obtained byadding to the calcium phosphate powder mannitol crystalsas a biocompatible and water-soluble porogen, in differentvolume fractions. The experimental techniques used tocharacterize the brushite cement were IR spectroscopyand XRD, to check the degree of conversion of the reac-tants into the final product, and SEM analysis to investi-gate microstructure. Compression tests were carried outon samples with different volume fractions of macropores.Preliminary checks of biocompatibility and cellular adhe-sion on cements without and with macropores were per-formed with positive results.

2. Materials and methods

Macroporous cements were prepared with a solid phasemade of equimolar quantities of b-tricalcium phosphate(b-TCP, assay P96%) (FLUKA, Germany) and monocal-cium phosphate monohydrate (MCPM, assay min. 98%)(Sharlau, Spain) wherein mannitol single crystals in volume

fractions between 0 and 15 vol.% were dispersed for use asa porogen. The liquid phase consisted of a sodium citrate(C6H7O7Na) solution of molar concentration C equal to0.5 M, saturated with mannitol to avoid an anticipated dis-solution of the porogen. The whole was stirred manuallyfor ca. 3 min. The ratio between the amounts of the solidand the liquid phase was fixed at a value of R = 3 g ml�1.The mannitol crystals were of acicular shape with shapefactors between 3 and 6 and were sieved to keep the diam-eter in the range of 250–500 lm.

The pH of the sodium citrate solution was measuredwith a pH meter (WTW, pH 539 microprocessor pHmeter).

The setting time of cements containing different frac-tions of mannitol was evaluated by a Vicat needle followingthe directions of ASTM C-191, on samples held at a tem-perature of 37 �C with 100% relative humidity.

Successively to mixing, the paste was poured into a Tef-lon mould of cylindrical shape to obtain samples 16 mm inlength and 10 mm in diameter.

The samples, once hardened at a temperature of 37 �Cwith 100% relative humidity and kept in this conditionfor 24 h, were immersed in deionized water (which wasrefreshed every day) for 5 days, in order to let the porogendissolve.

After this time the samples, in number of six for eachmacroporosity class, were tested in compression, as stillwet, by a servo-hydraulic machine INSTRON 8501, witha crosshead speed of 1 mm min�1 starting from a preloadof 20 N.

Identification of brushite was achieved by means ofX-ray diffraction on powder, using a Philips PW3710 dif-fractometer. The sample was run in the interval of 2hbetween 3� and 80�, with a generator potential of 30 kV,a generator current of 22 mA (using a CuKa radiation), aNi filter, and a scan speed of 1 min�1. The software usedfor XRD data reduction was Philips PC-APD DiffractionSoftware.

FT-IR spectra of samples in form of powder diluted inKBr (0.1%w/w) were recorded by a FT instrument Nicolet6700, DTGS-KBr detector, 100 scans, and displayed inabsorbance units.

Both XRD patterns and FT-IR spectra were obtainedon samples without and with macropores after incubationin deionized water and revealed that no transformation ofbrushite could be observed. Moreover, micrometric mea-surements of sample dimensions were taken before andafter incubation, to exclude any phenomenon of surfacedegradation.

The microstructure of a brushite cement without poro-gen and the disposition of macropores inside the cementmatrix were observed by SEM.

To estimate the percolation threshold of the macroporescontained in the investigated samples, some of them werecharged with graphite rods similar in size and shape to themannitol crystals and the electric conductivity between elec-trodes fixed to the bases was measured by an ohmmeter. The

G. Cama et al. / Acta Biomaterialia 5 (2009) 2161–2168 2163

electric percolation threshold, i.e. the value of porosity cor-responding to a sudden rise of electric conductivity by atleast one or more orders of magnitude, turned out to beabout 30 vol.%. This parameter is surely a useful indicationalso for osteoconductivity, but the two cases are rather dif-ferent. Unlike electric conductivity which demands at leasta conductive path across the pores, osteoconductivity maybe effective even with closed pores, because cells can maketheir way through by resorbing pore walls, but the poresmust be so close to each other that bone tissue ingrowthspreads to the whole bulk. This argument suggests that agood osteoconductive cement might have a porosity some-what lower than the electric threshold (say 20% instead of30%), with an improvement of mechanical strength.

2.1. Cell adhesion

The osteoblast cell line MG63 was used to check cellularaffinity to the surfaces of two different scaffolds withoutand with macropores (in a volume fraction of 15%), after2 and 48 h. A total number of four samples were examined.For each scaffold type, one sample was sterilized andexposed to cell adhesion 24 h after formation at 37 �C100% humidity, the other was previously immerged inwater for 5 days. The test at 24 h was aimed at evaluatingthe influence of mannitol (in samples containing it as aporogen) and of sodium citrate on cell activity. All sampleswere sterilized by gamma irradiation (5000 rad) for 60 h.

For each sample, cells were suspended in Dulbecco med-ium supplemented with 10% FCS, 100 IU ml�1 penicillinand 100 mg ml�1 streptomycin and plated at a density of106 cells cm�2. Medium was changed 2 days after the origi-nal plating and then twice a week. When culture disheswere nearly confluent, cells were detached with 0.05% tryp-sin–0.01% EDTA. An amount of 105 cells cm�2 were stat-ically loaded onto scaffolds (dishes 2 mm in height and10 mm in diameter) previously pre-wetted in culture med-ium. Cytotoxicity and cells adhesion efficiency of differentsamples were qualitatively assessed through optical micros-copy by using toluidine blue (TB) staining. Respectivelyafter 2 and 48 h of cells culture onto scaffolds, samples werefixed in 4% formalin for 10 min, rinsed in PBS and stainedwith toluidine blue for 5 min at room temperature. Sampleswere then rinsed in water and analysed by optical micros-copy at different magnifications.

Fig. 1. Comparison of the XRD pattern of brushite (0% mannitol) asmeasured in the present work with the relevant spikes (in blue) of thereference pattern for brushite (Ref. Code: 72-0713) and with thecharacteristic spikes (in red) of monetite (Ref. Code: 71-1760). (Forinterpretation of the references to colour in this figure legend, the reader isreferred to the web version of this article.)

3. Results

3.1. Degree of conversion of reactants into brushite

The stoichiometric chemical reaction for the formationof DCPD made of a mixture of MCPM, b-TCP and wateris as follows [19]:

Ca3ðPO4Þ2 þ CaðH2PO4Þ2 �H2Oþ 7H2O

! 4CaHPO4 � 2H2O ð1Þ

XRD analyses and FT-IR spectrography have been car-ried out to ascertain the degree of conversion of the reac-tants into the final product.

The X-ray diffraction measurements evidenced a degreeof conversion into brushite higher than 95%. In Fig. 1 theXRD pattern for the reaction product (0% mannitol)obtained in the present work is compared both with the rel-evant spikes (in blue) of the reference pattern for brushite(JCPDS 72-0713) and with the characteristic spikes (inred) of monetite (JCPDS 71-1760).

FT-IR spectra of brushite are characterized by the O–Hstretching modes of the crystallization water, with twopeak doublets at, respectively, 3540, 3485 cm�1 and 3289,3165 cm�1. The H–O–H bending gives rise to an absorp-tion at 1651 cm�1. The main IR bands characterizing thePO4 group can be detected at 1140, 1066 and 990 cm�1,due to PO stretching modes of the PO3 fragment. Weakersharp bands at 1210 and 875 cm�1 are due, respectively,to the P–O(H) stretching mode and the in-plane P–O–Hbending mode [20]. Bands at frequencies below 650 cm�1

are assigned mainly to PO deformation modes of the tetra-hedral PO4 group. All the corresponding peaks are well evi-dent in the spectrum of Fig. 2.

The pH value of the retardant solution was 3.5 ± 0.1.

3.2. Microstructure analysis

The typical microstructure of a microporous brushitecement formed with water as liquid phase is shown inFig. 3a, where the brushite grains have the typical shapeof thick platelets and are rather homogeneous in size.Fig. 3b, which refers to a cement with setting retardant(sodium citrate), shows a different microstructure consist-ing of thinner platelets relatively inhomogeneous in size,with large voids. Fig. 4 shows a number of mannitol single

Fig. 2. Infrared spectra of brushite sample measured in KBr powder.

Fig. 3. SEM micrographs of the fracture surface of Brushite cement: (a) without retardant, (b) with retardant in the liquid phase.

Fig. 4. Stereograph of mannitol single crystals used as porogen.

2164 G. Cama et al. / Acta Biomaterialia 5 (2009) 2161–2168

crystals used as porogen and Fig. 5 is the SEM image of thefracture surface of a macroporous cement.

3.3. Compressive strength and static elastic modulus of the

calcium phosphate cements

The setting time for cements which did not include man-nitol in their formulation was about 10 min, but it wasobserved to significantly increase with the content of poro-gen. Cements with the highest volume fraction of mannitol(15 vol.%) exhibited a setting time of ca. 30 min. Otherauthors observed the same effect in a calcium phosphatecement with the same type of porogen [21].

The samples containing mannitol were kept in water for5 days to ensure complete dissolution of the porogen: inthis way the volume fraction of mannitol is the same asthe volume fraction of macropores in the material aftertreatment.

The results of the compressive tests, made on six samplesfor each composition, are given in Table 1 as mean value

Fig. 6. Compressive strength (rc) and elastic modulus (E) plottedlogarithmically vs. (1 � fp) (fp = volume fraction of macropores).

Fig. 5. SEM micrograph of the surface fracture of macroporous brushitecement.

G. Cama et al. / Acta Biomaterialia 5 (2009) 2161–2168 2165

and standard deviation of both compressive strength (rc)and static elastic modulus (E). It is seen that the compres-sive strength of the cement with the highest volume fractionof porogen (15 vol.%) is on average 17 MPa, superior tothat of cancellous bone (2–12 MPa) [22]. To discuss thedependence of such mechanical properties on the volumefraction of macropores (fp), the data have been fitted to alaw of the type [13,23].

Y ¼ Yoð1� fpÞK ð2Þwhere Y represents the considered property (rc or E) andYo the corresponding value for a non-porous cement; K

is a constant exponent expected to depend on the shapeand distribution of the macropores. The results of the fit-ting are shown in the logarithmic plot of Fig. 6, wherethe slopes of the straight lines represent the estimated val-ues of exponent K, respectively Kr ffi 3.7 for the compres-sive strength and KE ffi 3 for the elastic modulus.

3.4. Cellular adhesion

Fig. 7 reports the results of tests of cellular affinity ondifferent scaffolds of brushite cements without and withmacropores. Cells, stained with toluidine blue, were ableto adhere onto all samples, with no significant differencesboth at short time (after 2 h, first row) and after prolongedtime (48 h, second row). Photos marked with a ‘‘minus”

sign refer to samples kept in water for 5 days after setting,those marked with a ‘‘plus” sign to samples which did notundergo such a treatment. Although the toluidine blue is a

Table 1Mechanical properties of macroporous cements.

% v/vmannitol

<rc>(MPa)

Standard deviationrc (MPa)

<E>(GPa)

Standarddeviation E (GPa)

0 32.6 0.8 4.2 0.35 24 1.1 2.8 0.6

10 20.4 1.7 2.6 0.415 17.5 1.6 2.5 0.2

qualitative test, it displays very well the capacity of differ-ent structures to allow/favor cell adhesion. Both scaffoldtypes displayed a high efficiency of cell seeding alreadyafter 2 h. Cells adhered and spread onto surfaces, main-taining their activity with time. It is seen, however (blockdiagram in Fig. 7), that best results were obtained for sam-ples where sodium citrate and mannitol are still present inthe scaffold and are released during the test of cellularadhesion.

4. Discussion

Macropores are introduced in a resorbable bone cementto increase the surface area available for cell adhesion,which is the first step in the process of osteoconductivity.The total surface area of a sample containing macroporeswith a volume fraction fp can be calculated as

A ¼ Ao þ fpVV p

Ap ð3Þ

where Ao is the area of the external surface, V is the samplevolume, and Ap and Vp are respectively the surface areaand the volume of a single macropore. For a cubic sampleof side length L, containing macropores of cylindricalshape of average diameter d and length l = wd, being wthe shape factor, Eq. (3) yields:

AAo¼ 1þ fp

32þ 1

w

� �Ld

ð4Þ

Applying this figure to a scaffold with L = 10 mm con-taining macropores with a mean diameter of 0.3 mm anda shape factor w = 4 (similar to the ones studied in thepresent work), having a volume fraction fp = 0.15, the sur-face area would increase by about 375% with respect to anon-porous sample. Lower values of the shape factor orspherical shapes should be preferred, but the influence ofw is relatively small (with a shape factor equal to 1, theincrement in surface area would be 500%, the same as forspherical pores). The advantage of having macropores witha higher shape factor is that they are fewer in number, thepore diameter, sample volume and porosity being the same.In fact the pore number is np = fp

VV p¼ 4f pV

pd3w, inversely pro-

Fig. 7. Cell adhesion onto different scaffolds, observed at the optical microscope through the toluidine blue staining (10� magnification). First row ofimages: results after 2 h; second row: results after 48 h. Symbols (+) and (�) refer, respectively, to cements containing mannitol (crystals or in saturatedsolution) and to cements where the mannitol was dissolved prior to test (samples kept in water for 5 days after setting). Block diagram: percentage ofcellular adhesion in the different tests.

2166 G. Cama et al. / Acta Biomaterialia 5 (2009) 2161–2168

portional to the shape factor. To obtain an effective osteo-conductivity in the case of not interconnected macropores,cells will have to make their own way inside the scaffold byresorbing the walls separating adjacent pores. Clearly, thetime required for this step is the shorter the lower is thepore number.

The problem of controlling the time of resorption of ascaffold containing macropores is, in general, a tough oneand involves several variables, such as porosity, diameterand shape factor of macropores and their spatial distribu-tion. A model of spherical pores ordered in a cubic latticewas outlined by Bohner and Baumgart [16]. They were ableto derive optimum sets of the variables for the formation ina given time of break-through channels capable of admit-ting cell conduction between adjacent pores. An analogousmodel for elongated cylindrical pores is more difficult to bestudied, because the relative orientation of the pore axishas to be taken into account. This is a statistical variablewhich can be also influenced by the way of injecting or stir-ring the cement paste.

Among the variables determining the behaviour of a mac-roporous scaffold, the volume fraction of macroporesdeserves a special comment. Of course, if one could reachthe percolation threshold, the scaffold would be osteocon-ductive from the beginning. Experiments made with graphiterods included in a brushite paste to simulate the porogencrystals used in the present research (see Section 2), indicatedthat the electrical percolation threshold is above 30%. Thisvalue would reduce the mechanical strength by too much:

following Eq. (2) with Kr = 3.7, the compressive strengthof the scaffold would become about one half the oneobtained with 15% of macropores. On the other hand, a per-colate macropore network may be unnecessary and even det-rimental, because brushite begins to dissolve before theinitiation of cell activity and in certain conditions a gap(up to 400 lm of thickness) was found to exist between thenewly formed bone and the resorbing bone cement [24]. Thismight eventually bring a macroporous scaffold to collapseshortly after implantation.

The ideal calcium–phosphate bone cement ought to beresorbed gradually along with the ingrowth of bone tissue.Simultaneously it is important to preserve a load-bearingcapacity of the implant, which can be useful in the first per-iod of convalescence. The former goal is solved by optimiz-ing the macropore pattern, as discussed above.Unfortunately, the introduction of macropores in thecement tends to reduce its mechanical strength accordingto a law of the type rc = rco(1 � fp)K

r, i.e. Eq. (2).According to this equation, for a given porosity rc canbe enhanced either by reducing exponent Kr or by increas-ing rco. The way that exponent Kr depends on the macro-pore characteristics is not known with precision. Xu et al.[13] report Kr ffi 3.3 for bending strength of chitosan-rein-forced HA samples containing macropores similar to ourown. In the present work we found Kr ffi 3.7 for compres-sive strength. This exponent represents the sensitivity of thecompression strength to porosity. In the case of a closedporosity, its value is expected to increase with the pore

Table 2Influence of R ratio and molar fraction of retardant on mechanical properties of microporous cement.

R (g ml�1) Molar fractionof retardantliquid (M)

Compressive strength<rc> (MPa)

Standard deviationrc (MPa)

Young’s modulus<E> (GPa)

Standard deviationE (GPa)

2 0.5 27.2 2.3 3.1 0.463 0.5 32.6 0.8 4.2 0.33 0.8 33 3.4 3.7 0.683 1.1 36.3 3.5 3.5 0.363 1.5 12.5 0.7 1.77 0.544 0.8 46 3.4 5.4 0.48

G. Cama et al. / Acta Biomaterialia 5 (2009) 2161–2168 2167

aspect ratio, because the failure mechanism is crack prop-agation starting at the pore surface. For cellular solidsand foams with open pores [25], which have different fail-ure mechanisms, this parameter is nearly independent ofthe pore shape and considerably lower (1.5 or 2 accordingto the type of failure).

The matrix strength rco can be raised by acting either onthe P/L ratio (R) or on the molar concentration (C) of theretardant solution. To this purpose further compressivetests were made with the results reported in Table 2.Firstly, by fixing C = 0.5 M and letting R vary through val-ues 2, 3 and 4 g ml�1, the value of rco increased from27.2 MPa (R = 2 g ml�1) to 32.6 MPa (R = 3 g ml�1).R = 4 g ml�1 gave a cement paste too dry to be moulded.

Successively, having fixed R = 3 g ml�1, compressivetest were run with C = 0.8 M, C = 1.1 M and C = 1.5 M.The obtained values of strength raised from 33 MPa(C = 0.8 M) to 36.3 MPa (C = 1.1 M), but dropped to12.5 MPa with C = 1.5 M, showing that beyondC = 1.1 M a reduction of strength is to be expected. Thisis due to the presence of monetite which, as reported bysome authors [26,9], reduces the mechanical strength byincreasing the volume fraction of micropores.

An attempt with R = 4 g ml�1 and C = 0.8 M was suc-cessfully made and provided a compressive strength of46 MPa. Raising the molar concentration of retardantfrom 0.5 M to 0.8 M should not change appreciably thepH of the liquid phase, so there is no risk of reducing cellactivity in the case of in vivo implantation.

These encouraging results need be confirmed by furtherexperiment because we noticed that the standard deviation(see Table 2) tends to increase with C higher than 0.5 M.

As rule-of-thumb, adopted in the present work, one maychoose a molar concentration of retardant equal to 0.5 M(which seems to yield a lower scattering of compressivestrength) with a P/L ratio such as to produce a macropo-rous cement with a compressive strength not lower thanthat of cancellous bone [22]. The results obtained in thepresent paper show that this is feasible, with a margin leftto increasing the macroporosity even beyond 15 vol.%.

The results of in vitro cell adhesion tests show (Fig. 7block diagram) that the choice of mannitol as a porogenseems to be successful for two reasons. On the one hand,it enhances the degree of cell adhesion in the whole; 2 hfrom implantation.

5. Conclusions

To improve osteoconductivity, brushite cements withmacropores of elongated shape were prepared by addingmannitol crystals as a porogen to the powder. Samples withdifferent values of porosity in the range 0–15 vol.% weretested in compression and the compressive strength wasfound to depend on the porosity through an equation ofthe type rc = rco (1 � fp)K

r where Kr ffi 3.7. The experi-mental matrix strength rco of 33 MPa has been increasedup to 46 MPa by acting on the powder to liquid ratioand on the molar concentration of retardant. Brushite bonecements prepared in this way may provide a strength com-parable to that of cancellous bone with a porosity evenhigher than the percolation threshold of 30%. In vitro celladhesion proved to be enhanced by the presence of manni-tol used as a porogen.

Acknowledgements

The authors are indebted to Prof. Roberto Cabella andLab Technician Antonio Cucchiara, University of Genova,DIPTERIS, for identification of brushite by means ofXRD.

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