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Bioactive composite bone cement based on a-tricalcium phosphate/

tricalcium silicate

Loreley Morejon-Alonso,1 Oscar Jacinto Bareiro Ferreira,1 Raul Garcia Carrodeguas,2

Luis Alberto dos Santos1

1Escola de Engenharia, Departamento de Materiais, Universidade Federal do Rio Grande do Sul, Porto Alegre (RS), Brasil2Departamento de Ceramica, Instituto de Ceramica y Vidrio - CSIC, Madrid, Spain

Received 7 January 2011; revised 13 May 2011; accepted 25 May 2011

Published online 17 October 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.31926

Abstract: Silicon compounds are known as bioactive materials

that are able to bond to the living bone tissue by inducing an

osteogenic response through the stimulation and activation of

osteoblasts. To improve the bioactive and mechanical proper-

ties of an a-Ca3PO4-based cement, the effects of the addition of

Ca3SiO5 (C3S) on physical, chemical, mechanical, and biologi-

cal properties after soaking in simulated body fluid (SBF) were

studied. The morphological and structural changes of the ma-

terial during immersion were analyzed by X-ray diffraction and

scanning electron microscopy. The results showed that it is

possible to increase the compressive strength of the cement

by adding 5% of C3S. Higher C3S contents enhance bioactivity

and biocompatibility by the formation of a dense and homoge-

neous hydroxyapatite layer within 7 days; however, compres-

sive strength decreases drastically as a consequence of

delayed hydrolysis of a-Ca3(PO4)2. An increment in setting

times and degradation rate of composites containing C3S was

also observed. VC 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part

B: Appl Biomater 100B: 94102, 2012.

Key Words: calcium phosphates cements, hydroxyapatite, tri-

calcium silicate, in vitro

How to cite this article: Morejon-Alonso L, Bareiro Ferreira OJ, Garca Carrodeguas R, Santos LA. 2012. Bioactive composite

bone cement based on a-tricalcium phosphate/tricalcium silicate. J Biomed Mater Res Part B 2012:100B:94102.

INTRODUCTION

Calcium phosphate cements (CPCs) represent a clinicalalternative to the use of traditional bioceramics as they can

be easily manipulated and shaped, they provide intimateadaptation to the contours of defect surfaces, and set in situin the bone cavity to form a solid restoration.1 They alsohave attracted great interest since they were developedin the mid 80s owing to their chemical similarity to themineral phase of bone tissue and good osteoconductivity.2

One of the most important formulations is that based ona-tricalcium phosphate [a-Ca3(PO4)2; a-TCP] which sets insitu and forms a calcium deficient hydroxyapatite [Ca9(H-PO4)(PO4)5(OH); CDHA] when hydrated.

3 However, it is onlystrong enough under compression4 and has low mechanicalstrength when compared with cortical bone5; consequently,its application is restricted to places that require low me-

chanical loads.6

Being aware of the excellent bioresorbability of CDHA,researchers are concentrating their efforts in trying to over-come the mechanical weakness of calcium phosphatescements by using different fillers, fibers, and reinforcingadditives that lead to the formation of various multiphasecomposites based on the idea that a filler in the matrixmight stop crack propagation.7 Nevertheless, adding fillers

affects the capacity to allow bone ingrowths into pores andproduces a denser cement with a slower resorption rateand hence a slower bone substitution.8 Therefore, it is diffi-

cult to increase the strength of cements without having anegative impact on the other properties.

Taking into account that silicon plays an important role inbone formation9-11 and its presence improves the bioactivity ofmaterials by enhancing mesenchymal cell differentiation andincreasing osteoblasts activity12,13; the addition of calcium sili-cates could be an effective way to improve the biocompatibilityand bioactivity of calcium phosphates cements. On the otherhand, since silicates have spontaneous development of strengthtowards water14 (spontaneous consolidation), the introductionof these additives in traditional formulations, could alsoenhance the mechanical properties of these materials.

Due to the rapid development of strength during the

first stages of hydration and the higher content of siliconwith respect to Ca2SiO4, dicalcium silicate (C2S), Ca3SiO5(C3S) was chosen as additive. Different formulations ofa-TCP/Ca3SiO5 were prepared and the effects of C3S addi-tion on setting times, chemical composition, bioactivity, andmechanical properties before, during, and after ageing insimulated body fluid (SBF) were studied. Cytotocixity anddegradability were also determined.

Correspondence to: L. Morejon-Alonso; e-mail: [email protected]

Contract grant sponsors: Coordenacao de Aperfeicoamento de Pessoal de Nvel Superior (CAPES), Brasil

94VC

2011 WILEY PERIODICALS, INC.


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MATERIALS AND METHODS

Materials

To prepare the a-TCP/C3S cement, all chemicals of analyticalgrade were used. a-TCP was prepared as described byMonma et al.15 using c-Ca2P2O7 and CaCO3 as raw materialsin stoichiometric amounts. After calcination, the productwas wet milled for 4 h in a polyethylene jar with alumina

balls using an alcoholic medium (anhydrous ethanol). Trical-cium silicate powders were synthesized by solgel route,using Ca(NO3)2.4H2O and Si(OC2H5)4 (TEOS).

16 Repeatedgrindings and calcinations at 1400C were necessary toreach the product as monophasic as possible. To obtainpowders with similar particle size distributions, the samemilled treatment was made that in the case ofa-TCP.

Preparation of composite samples

Synthesized C3S (7.11 lm, average diameter) was mixedwith a-TCP (10.71 lm, average diameter) in powder ratiosof 0, 5.0, and 10.0 mass%. The liquid phase was a sodiumphosphate buffer prepared from NaH2PO4 and Na2H-

PO4.12H2O, and the liquid/powder ratios (L/P) was depend-ent of the content of C3S added ranging from 0.4 to0.44 mL/g. Each powder sample was carefully weighed andmixed with the liquid phase in appropriate liquid-to-powderratio, packed into silicon molds and aged at 36.5C withcontrolled humidity for 24 h.

Setting time measurement

Setting time of samples was measured according to ASTMC266-89 using a Gillmore Needles method.17 Three speci-mens for each formulation were performed and standarddeviation was used as a measure of the standard uncertainty.Initial setting time was determined as the end of moldability

without serious damage to the cement structure, and thefinal setting as the time beyond which it is possible to touchthe cement without causing serious damage.18

In vitro tests

To assess in vitro bioactivity, the 24 h set pastes were soak-ing in SBF at 36.5C19,20 for 14 days and afterward, gentlyrinsed with deionized water followed by ethanol dehydra-tion and drying in atmospheric temperature.

For degradation tests, the disks were accurately weighedbefore and after immersion in SBF. The weight loss (WL)was calculated according to Eq. (1), being W0 the initialweight of the specimen and Wd the weight of the specimendried after different degradation times (7, 14, and 21 days).Each measurement was performed three times and the av-erage value was calculated.

WL% W0 Wd=W0 100 (1)

Cytotoxicity test for cements

The cell viability assay was performed by direct contact testaccording to ISO 10993-5 using peripheral blood mononu-clear cells (PBMCs) and a procedure described elsewhere.21

Latex (1 cm2) and culture medium were used as positive

and negative controls and the number of viable cells wasquantitatively assessed by MTT test. Experimental valueswere analyzed via one-way ANOVA test follow by Tukeysmultiple comparison test.

Characterization techniques

Phase composition of the samples was determined by X-raydiffraction (XRD) in a PHILLIPSV

R

diffractometer (XPertMPD) and Cu-target. Diffractograms were recorded usingNi-filtered radiation (k 1.5406 ) with a step size of0.05 and a time/step ratio of 1 s.

Morphological differences before and after soaking inSBF were characterized by Scanning Electron Microscopy(SEM) using a JEOL microscope (JSM-6060) on gold-coated

samples.Compressive strength (CS) was measured in servohy-

draulic Universal Testing Machine (MTS 810) with a loadmeasuring cell of 10 kN and a loading rate of 1 mm/min.The number of replicas was n 10 and student multiplecomparison test was used to compare mean values.

pH measures were carried out during soaking in SBFand lectures were made in an mPA-210 pH meter at 36.5C.

RESULTS

Effect of C3S on the setting time ofa-TCP cement

Figure 1 shows the initial and final setting times of compo-sites containing 0% (a-TCP), 5% (C3S) and 10% (10C3S).

The initial and final setting times ofa-TCP were higher thanthose reported in the literature for similar materials22 andthe addition of C3S produced a remarkable increase in set-ting times. No significant differences were found when theamount of C3S added was raised.

Characterization of CPC/C3S composites cement

Figures 24 show the powder XRD patterns of compositesafter setting (24 h setting) and after soaking in SBF for 7and 14 days. After 24 h setting (Figure 1), for a-TCP-basedcement, mainly peaks of CDHA (JCPDS 46-0905) were

FIGURE 1. Initial setting and final setting time of the paste samples

with various contents of C3S [L/P) 0.4 mL/g].

ORIGINAL RESEARCH REPORT

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS

|JAN 2012 VOL 100B, ISSUE 1 95


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observed. With the addition of C3S, the diffraction peaks ofCHDA appear less intense and unreacted peaks of a-TCPwere also detected. The greater the amount of C3S added,the greater the intensity of the peaks of unreacted a-TCPand the lower the CDHA formed. After 7 days of soaking(Figure 2) the intensity of a-TCP lines decreased in relationto set cements and CDHA lines appeared stronger. Within

14 days of SBF soaking, the hydration reaction seemed tobe complete for a-TCP and 5C3S, whereas great amount ofunreacted a-TCP in addition to CDHA can be observed for10C3S (Figure 3). For all times and all formulations, thecharacteristic peaks of b-TCP (JCPDS 09-0169), whichappeared as a secondary phase in a-TCP powder and is notinvolved in the hydration process, were present.

FIGURE 2. XRD patterns of cements 24h set. n a-Ca3(PO4)2; ~ b-Ca3(PO4)2; Ca9(HPO4)(PO4)5(OH).

FIGURE 3. XRD patterns of cements after 7 days in SBF. n a-Ca3(PO4)2; ~ b-Ca3(PO4)2; Ca9(HPO4)(PO4)5(OH).

96 MOREJON-ALONSO ET AL. a-TRICALCIUM PHOSPHATE/TRICALCIUM SILICATE COMPOSITE CEMENT


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Furthermore, no calcium-silicate-hydrate (C-S-H) or calciumhydroxide [Ca(OH)2] resulting from hydration of C3S weredetected by this method.

The bone-like apatite formation: Microstructural

changes during soaking

Since the main hydration product of a-TCP-based cement

systems is CDHA, it is difficult to account for the ability ofthe materials to induce the deposition of apatite in SBF;however, the observation by SEM is an effective way to esti-mate the bioactivity of materials as the apatite grains andlayers that are formed have characteristic features. Figure 5shows the SEM surface images of a-TCP, 5C3S, and 10C3S atdifferent times of immersion. For a-TCP-based cement [Fig-ure 5(a)] a fine layer of CDHA crystals deposited onto thea-TCP grains was observed before immersion, while a net-work of entangled plate-like apatite crystals was detectedfor subsequent immersion times. Fourteen days after soak-ing, a layer of bone-like apatite, precipitated under physio-logical conditions, was clearly observed. With the additionof 5C3S, the formation of a gelatinous coating covering the

surface of unreacted grains at early times of hydration wasdetected [Figure 5(b)]. After soaking in SBF, the morphologyof 5C3S surface varied slightly and a needle microstructureappeared the same as type I C-S-H23; whereas a homogene-ous layer of CDHA with globular shape morphology, typicalof bioactive materials after immersion in SBF was detectedwithin 14 days.24 With larger additions of C3S [Figure 5(c)],the surface before immersion appeared covered or speckledwith small distinct features that could be identified as smallcrystals of dry C-S-H. After immersion for 7 days, a welldefined bone-like apatite layer is formed and transformed

into a more dense and homogeneous layer with increasingimmersion time. Although measures were taken to maintainaseptic conditions, bacterial contamination by Bacillus andCocci colonies was observed at the surface of samples.25

Different microstructural features were found in thefracture surface of different composites (Figure 6). Sinceearly stages, a-TCP showed the typical petal-like plates cov-ering the biggest grains and the growth of these plates with

time, at the expense of smaller grains, within the intersticesof fracture surface [Figure 6(a)].

With the addition of C3S neither petal or needle-likecrystals distinctive of the beginning of setting and hardeningof the CPC, nor the microstructure of rod-like characteristicsof more aged systems,22 were observed. With low contentsof C3S [Figure 6(b)], the deposition of small CDHA crystalson the top of the larger a-TCP particles could be seen. Thesecrystals evolve and grow into acicular crystals encasingsome individual grains ofb-TCP. The presence of some char-acteristic hollow-shells (Hadley grains),26 showing an emptyshell of hydration products inside which an a-TCP or C3Shad fully reacted, could be clearly identified in both the a-

TCP and the 5C3S cement. For greater additions of C3S [Fig-ure 6(c)] no significant differences with soaking time werefound and the most common morphology observed was amixed result of fibrillar and amorphous type I C-S-H whichare characteristic of the early stages of C3S hydration.

Mechanical strength

Figure 7 shows the compressive strength of a-TCP and a-TCP/C3S composites before and after soaking in SBF for 7and 14 days. The results indicate that the compressivestrength of a-TCP decreased slightly with soaking, whereas

FIGURE 4. XRD patterns of cements after 14 days in SBF. n a-Ca3(PO4)2; ~ b-Ca3(PO4)2; Ca9(HPO4)(PO4)5(OH).





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the compressive strength of 5C3S increased with ageingtime, reaching values similar to those of a-TCP after harden-ing (16.86 MPa). For 10C3S, values of compressive strengthwere very low and no significant changes were observedduring immersion.

pH analysis on in vitro immersion

Figure 8 shows the changes in pH values of SBF caused by thecement pastes. The results showed that for a-TCP, the pH value

decreased slightly in the first 24 h after immersion and thenranges from 7.3 to 7.5. With the addition of C3S the pHincreased rapidly, reaching a maximum of 10.4 for a 10% addi-tion, and then decreased gradually and stabilized after 72 h.

In vitro degradation

Figure 9 shows the degradation of the a-TCP and a-TCP/C3Scomposite pastes with different contents of C3S after soak-ing in SBF solution for various time periods. It was foundthat the weight of a-TCP increased with time, whereas theweight of composites containing C3S experienced a mass

loss during the first 7 days and then began to gain mass. Itwas also found that the degradation rate of the compositescontaining C3S was higher than that ofa-TCP, while the deg-radation rate raised proportionate to the increase in theamount of C3S added.

Cytotocixity test

Figure 10 shows the result of a direct cytotoxicity test forcomposites against the PBMCs after 24 and 48 h of incuba-

tion. It was found that the cytotoxicity of the compositescontaining C3S was significantly lower than the positive con-trol (p < 0.05) at 24 h of incubation, and biocompatibilitywas higher than in traditional a-TCP. It was also found thatthe viability of PBMCs decreased in all compositions afterincubating for 48 h.

DISCUSSION

Setting times for CPCs usually ranged from 5 to 8 min whena neutral phosphate such as disodium hydrogen phosphate(Na2HPO4) or sodium dihydrogen phosphate (NaH2PO4) was

FIGURE 5. SEM micrographs of surface after soaking in SBF (A) a-TCP, (B) 5C3S, and (C) 10C3S.



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FIGURE 6. SEM micrographs of fracture surface after soaking in SBF (A) a-TCP, (B) 5C3S, and (C) 10C3S.

FIGURE 7. Compressive strength of the a-TCP a-TCP/C3S composites

after soaking in SBF for various times.

FIGURE 8. Changes in pH value of SBF soaked with a-TCP and a-TCP/

C3S composite.





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added to the liquid phase.22 When b-TCP is present, settingtimes are often delayed due to the non-participation ofsame in the hydration reaction [Eq. (2)]; and in this case, alarge quantity of b-TCP (18%) was present in the originalpowder as a result of the doping of Mg2 raw materials(0.28% as MgO).21 With the addition of C3S, the solubility ofa-TCP decreases due to the formation of Ca(OH)2 duringCa3SiO5 hydrolysis [Eq. (3)], which results in alkaline cir-cumstances and delayed dissolution of a-TCP. Furthermore,the formation of a dense calcium silicate hydrate gel (C-S-H)on the surface of a-TCP particles [Figure 5(b)] also retardsdissolution ofa-TCP grains and the precipitation of CDHA.

3a Ca3 PO4 2 s H2O Ca9 HPO4 PO4 5OH s (2)Ca3SiO5 s 3H2O CaO:SiO2:H2O gel 2Ca OH 2 s (3)

As in the case with C3S hydrolysis,27 the hydration

mechanism of a-TCP-based cements is diffusion dependentof the reactive species through the layer of formed product.Therefore, the degree of extension of the a-TCP hydration,usually 80% at 24 h,28 is lower as evidenced by the pres-ence of intense unreacted peaks in the diffraction patternsof composites containing C3S (Figure 2). Even when thepresence of silicate ions formed from C3S promotes apatitenucleation,29 the main effect of adding C3S at early stagesseems to be the delay in the dissolution of a-TCP and the

precipitation of CDHA. While initially retarded by the pres-ence of C3S, hydration reaction occurs when soaking time isincreased and the unreacted peaks of a-TCP disappearresulting in more intense peaks of CDHA. For formulationswith higher content of C3S, a-TCP peaks can be found within14 days and the CDHA peaks appear more intense, probablydue to its precipitation from SBF solution (Figures 3 and 4).

When compared with bioactive bone substitution mate-rials such as A-W glass ceramic and Bioglass

VR

, the CPCsappear to be less capable of inducing an homogeneousbone-like apatite layer on the surface to form chemical

bond to bone tissue at early stages of implantation.30 Theresults of SEM analyses (Figure 5) showed that a-TCP/C3Scomposite pastes induce homogeneous apatite depositionon the paste surface due to the presence of C3S, and the for-mation time of same was dependent on the content of C3S.As is generally accepted, the deposition of apatite on thesurface of calcium phosphate cement is largely dependent

on supersaturation of Ca2 and PO43 ions and indeed suchprocess proceeds with low rate resulting in the absence ofhomogeneous apatite layer formation at early stages of im-plantation.24 In the presence of C3S, the HSiO

3 ions arereleased during hydration of the composite paste, acting assites for nucleation of apatite crystals and hence accelerat-ing the deposition of apatite on the surface. The apatitelayer in a-TCP-based cement was formed within 14 days.This is the time which is reported as required to the deposi-tion of new bone on the surface of the implant during invivo experiments,31 which shows the superior bioactivity ofcomposites.

A critical problem that limits wider clinical application

of CPCs is their mechanical properties. Since C3S had a neg-ative impact on setting times and the hydration rate of a-TCP, the mechanical strength of composites at early stageswas not improved. Another factor that could explain thisbehavior is related to the crystal growth of hydroxyapatite.The SEM results show (Figure 5) thata-TCP set by entangle-ment of plate-like crystals, while composites containing C3Shad a gelatinous coating of C-S-H on the surface coveringthe a-TCP particles, also showed an incipient precipitationof CDHA crystals on the fracture surface in the first 24 h,which caused the fall of the mechanical properties since theprecipitation of CHDA is responsible for the adherence andinterlocking of the crystalline grains, which results inhardening.

With the consumption of C3S and the growth of CHDAneedles-like crystals on composites, the mechanical proper-ties begin to increase reaching values similar to those of a-TCP-based cement after setting. Adding more C3S producesmore C-S-H, which delays the solubilization of the a-TCPgrains, and consequently, the precipitation of CDHA. In thiscase, the C-S-H does not act as filler does not bond with the

FIGURE 9. Weight loss of the a-TCP and a-TCP/C3S composites after

soaking in SBF for various times.

FIGURE 10. Cell viability of PBMCs on the different pastes after cultur-

ing for 24 and 48 h. *p< 0.05 compared with () control group.



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apatite formed during the setting, thus the compressivestrength is not enhanced.

The dissolution of calcium ions into body fluids plays animportant role in the nucleation and growth of hydroxyapa-tite layers on a materials surface. When immersed in SBF,the pH of the a-TCP-based cement decreases to values closeto 7.0 probably due to the formation of some H3PO4 during

hydrolyzation of Ca3PO4 into Ca10(PO4)6(OH)232 and afterthis time values remain almost unchanged. With the addi-tion of C3S, OH

ions are released to SBF solution raisingthe pH of SBF solution in the first 72 h, after which the pHstabilizes implying that OH ions are no longer released tothe medium.

Conventional CPCs usually have a slow degradability andoften experience a mass gain after long soaking periods as aresult of hydrolysis ofa-TCP or due to the formation of apa-tite on the surface of the samples.33 Considering the factthat the degradability is primarily governed by the chemicalcomposition and the physical characteristics of the material,the reason for the higher degradation rate of the a-TCP/C3S

composites could be the higher solubility of the C-S-H ascompared with CDHA. A mixture of two crystallized calciumsilicate hydrated identified by DRX as 1.5CaO.SiO2.H2O(JCPDS 33-0306) and 2CaO.SiO2.0.5H2O (JCPDS 12-0199)

34

were found in the supernatant solution during the first 72h, indicating the solubilization of the silicate hydrate at thattime. After 7 days of soaking, a reversal behavior can beseen and the amount of apatite deposited on the cementsurface and formed due to the hydrolysis of a-TCP waslarger than dissolution of C-S-H and/or other phases, so theweight of samples increased.

It is generally accepted that the in vitro cellmaterialinteraction is a useful criterion in the evaluation of new bio-materials. The results of cell culture showed that compo-sites containing C3S are less cytotoxic and more compatiblethan pure a-TCP-based cement and the positive effect in cy-totoxicity could be attributed to the dissolution of silicateions present in a-TCP/C3S pastes that stimulate cell prolifer-ation.35,36 The slight increase in cytotoxicity 48 h after cul-turing may be explained due to the occurrence of somechemical transformations of the material in culture mediumsince the pastes continue hydrating after 7 days in aqueousmedia. Nevertheless, the study indicated that the compositecement was not only biocompatible but also non-cytotoxic.

CONCLUSIONS

In this article, novel bioactive composite cements were pre-

pared by adding Ca3SiO5 into the traditional a-Ca3(PO4)2-based cement. The composites thus obtained showed anincrease in setting times, and a considerable delay in therate ofa-TCP hydration, which caused the loss of initial me-chanical properties together with the microstructural differ-ences found due to the formation of a C-S-H gel. Further-more, the presence of high contents of C3S increases thedegradability of cements and raises the pH of medium atearly stages, compromising the precipitation of CDHA. How-ever, the a-TCP/C3S composites possessed excellent bioactiv-ity, as indicated by the formation of bone-like apatite in SBF

and were not only more biocompatible but also more non-cytotoxic as showed by the in vitro cytotoxic assay.

The results showed that it is only possible to incorpo-rate up to 5% with an improvement of the bioactivity andbiocompatibility without compromising significantly the me-chanical strength of materials.

The Brazilian Government entity dedicated to the train-

ing of human resources.

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