a-Tricalcium phosphate cements modified with b-dicalcium silicate and tricalcium aluminate: Physicochemical characterization, in vitro bioactivity and cytotoxicity  l Garcıa Carrodeguas,2 Luis Alberto dos Santos,3 Daniel Correa,1 Amisel Almirall,1 Rau 2 4  n,5 Jose  Angel Delgado1 Antonio H. De Aza, Juan Parra, Lizette Morejo 1

 micas y Composites, Centro de Biomateriales, Universidad de La Habana, 10400 La Habana, Cuba Departamento de Cera  mica, Instituto de Cera mica y Vidrio, CSIC, Madrid, Spain Departamento de Cera 3 Labiomat-Departamento de Materiales, Escuela de Ingenierıas, Universidad Federal de Rıo Grande del Sur, 91509-900, Porto Alegre, Rio Grande do Sul, Brazil 4   n Clınica y Biopatologıa Experimental, Hospital Provincial de Avila, n Unidad de Investigacio Centro de Investigacio  en Red-Bioingenierıa, Biomateriales y Nanomedicina (CIBER-BBN), Avila, Spain 5 Departamento de Quımica Macromolecular, Centro de Biomateriales, Universidad de La Habana, 10400, La Habana, Cuba 2

Received 26 November 2013; revised 18 March 2014; accepted 30 March 2014 Published online 25 April 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33176 Abstract: Biocompatibility, injectability and in situ self-setting are characteristics of calcium phosphate cements which make them promising materials for a wide range of clinical applications in traumatology and maxillo-facial surgery. One of the main disadvantages is their relatively low strength which restricts their use to nonload-bearing applications. aTricalcium phosphate (a-C3P) cement sets into calciumdeficient hydroxyapatite (CDHA), which is biocompatible and plays an essential role in the formation, growth and maintenance of tissue-biomaterial interface. b-Dicalcium silicate (bC2S) and tricalcium aluminate (C3A) are Portland cement components, these compounds react with water to form hydrated phases that enhance mechanical strength of the end products. In this study, setting time, compressive strength (CS) and in vitro bioactivity and biocompatibility were evaluated to determine the influence of addition of bC2S and C3A to a-C3P-based cement. X-ray diffraction and

scanning electron microscopy were used to investigate phase composition and morphological changes in cement samples. Addition of C3A resulted in cements having suitable setting times, but low CS, only partial conversion into CDHA and cytotoxicity. However, addition of b-C2S delayed the setting times but promoted total conversion into CDHA by soaking in simulated body fluid and strengthened the set cement over the limit strength of cancellous bone. The best properties were obtained for cement added with 10 wt % of b-C2S, which showed in vitro bioactivity and cytocompatibility, makC 2014 Wiley ing it a suitable candidate as bone substitute. V Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 72– 83, 2015.

Key Words: calcium phosphate cement, a-tricalcium phosphate, b-dicalcium silicate, tricalcium aluminate, cytocompatibility

 n L, Delgado JA. How to cite this article: Correa D, Almirall A, Carrodeguas RG, dos Santos LA, De Aza AH, Parra J, Morejo 2015. a-Tricalcium phosphate cements modified with b-dicalcium silicate and tricalcium aluminate: Physicochemical characterization, in vitro bioactivity and cytotoxicity. J Biomed Mater Res Part B 2015:103B:72–83.

INTRODUCTION

Different materials such as bioactive glasses, glass ceramics, bioceramics, synthetic and natural polymers, and composites have been studied and tested as biomaterials for bone substitution procedures. A biomaterial intended to repair hard tissue should be biocompatible, bioactive, osteoconductive, preferably bioreabsorbable and its strength and elasticity should match those of the tissue to be substituted.1,2 Calcium phosphate ceramics are widely used in clinical

practice (e.g., dentistry, traumatology, and maxillofacial surgeries) due to its similarity to the mineral component of bone tissue.3–5 However, ceramics are hard to fit the shape of the tissue defect to be repaired. Calcium phosphate cements (CPCs) are presented as an alternative to ceramics because they have the advantage to perfectly adapt to the shape of implant site. CPCs comprise one or more calcium phosphate salts, which upon mixing with water or aqueous solution form a moldable paste that sets and hardens as

Correspondence to: J. A. Delgado, Ave. Universidad e/Ronda y G, Vedado, La Habana, Cuba, CP 10400 (e-mail: [email protected]) Contract grant sponsor: Ministry of Foreign Affairs and Cooperation of Spain for the MAEC-AECID Scholarships Program granted to members of the Cuban team Contract grant sponsor: Ministry of Economy and Competitiveness of Spain; contract grant number: Project CICYT MAT2010-17753 Contract grant sponsor: Program JAE-CSIC from Spain for supporting R. G. Carrodeguas; contract grant number: JAEDOC087-2009 Contract grant sponsor: INNPACTO from Spain for supporting R. G. Carrodeguas; contract grant number: IPT-2012-0560-010000 Contract grant sponsor: CAPES Program of Brazil; contract grant number: Project No. 032/07

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result of the precipitation of interlocked crystals of a different calcium phosphate.6,7 There are two main kinds of CPCs, depending on the end product of the setting reaction, apatite cements and dicalcium phosphate dihydrate (DCPD or brushite) cements. DCPD is formed below pH 4.2 while apatite is formed to pH>4.2.2 a-Tricalcium phosphate (a-C3P, Ca3(PO4)2) mixed with an aqueous solution of dibasic sodium phosphate (Na2HPO4) yields a cement paste that sets into calcium-deficient hydroxyapatite (CDHA, Ca10 2 x(HPO4)x(PO4)6 2 xOH2 2 x, with 0  x  1),8,9 both in air at room temperature and under physiological conditions. CDHA is biocompatible, bioactive, osteoconductive (bone ingrowth potential) and comparable to the mineral constituent of bone in terms of chemical constitution and crystallinity.3,10–12 However, the clinical use of CPCs is restricted to filling nonload-bearing bone defects due to its low mechanical properties.7,13,14 Hence, a lot of attempts to improve their mechanical properties have been conducted involving cement composition, liquid/powder ratio, reinforcing additives (e.g., fibers, polymers), and so forth.10,14 Calcium silicates (C3S, Ca3SiO5 and b-C2S, b-Ca2SiO4) and tricalcium aluminates (C3A, Ca3Al2O6) are the main constituents of Portland cement clinker.15 They react with water and set by forming hydration products with high mechanical strength.16,17 Mineral trioxide aggregate (MTA) is a modified Portland cement approved in 1998 by FDA for pulp capping and sealing root canals in Endodontics.18–20 MTA is biocompatible and has good sealing ability and the capacity to promote regeneration of original tissue when is placed in direct contact with dental pulp and periradicular tissues.21,22 It has been well established that several materials containing CaO-SiO2 have high bioactive potential and they are able to bond tightly to living bone and soft tissues.23 Particularly, b-C2S, the main hydraulic component of Portland cement, is bioactive in vitro and biocompatible. When mixed with an aqueous solution in the proper liquid/powder ratio it yields an injectable paste that sets and hardens by forming hydrated phases known as CASAH.22,24–26 For these reasons b-C2S is a promising candidate as bioactive self-setting material in the biomedical field (bone/dental repair).23,24 On the other hand, cements based in monocalcium aluminate (CaAl2O4, CA) and other calcium aluminates have been studied as biomedical materials. It has been reported that these cements are bioactive in vitro and biocompatible and have good strength. They have been proposed for several applications in dentistry and traumatology.27–30 On the other hand, C3A is the fastest setting component of Portland cement, yielding an hydrated network mainly composed of hydrogarnet (Ca3Al2(OH)12).17 C3A cement evolves a considerable amount of heat during setting and becomes hard and strong rapidly.16,17 So, C3A might accelerate the setting and improve the short-term compressive strength (CS) if added to CDHA cement.17,25,31 In this study, b-C2S and C3A were studied as reinforcing additives for a-C3P cement. Setting time, biodegradability, in vitro bioactivity and biocompatibility were evaluated to

determine whether b-C2S and C3A could also affect these properties when compared with conventional a-C3P-based cement. MATERIALS AND METHODS

Preparation of the components of the cements a-C3P (a-Ca3(PO4)2) was synthesized by neutralizing an aqueous suspension of Ca(OH)2 with H3PO4 solution and further transformation of the precipitated amorphous calcium phosphate (ACP) into a-C3P by heating at high temperature. Thus, 500 mL of 0.500 M orthophosphoric acid (prepared from H3PO4 Panreac, PA, 85% min) were slowly poured onto 21.0 g (0.375 mol) of calcium oxide (prepared from CaCO3, Panreac, PA, 99.0% min, CaCO3 was calcined at 900 C for 4 h) previously suspended in 1000 mL of deionized water under stirring. The resulting suspension of ACP was further stirred for 30 min and vacuum filtered. The filtered cake was washed with water and ethanol and dried at 100 6 10 C overnight. Dried cake was crushed and sieved (150 mesh). The powder of ACP was transformed into aC3P by heating in zirconia crucible at 1300 C at a heating rate of 10 C/min, maintained for 6 h at this temperature, and then cooling at room temperature at the same rate. b-C2S (b-Ca2SiO4) was prepared by sol–gel process using Ca(NO3)24H2O (Panreac, PA, 99% min) and Si(OC2H5)4 (TEOS; Merck Millipore, for synthesis, 99% min) at Ca/Si molar ratio of 2. First, TEOS was added and dissolved in a mixture of ethanol (Panreac, PA, 99.8%) and distilled and deionized water (molar ratio TEOS:ethanol:H2O 5 1:5:25) and 10 mL of 0.1 M HNO3 (Panreac, PA, 53%) were added as catalyst. The TEOS solution was hydrolyzed at room temperature under stirring overnight. Further, 0.35 mol of Ca(NO3)24H2O were added under continuous stirring for 6 h at 60 C, then the solution was aged at room temperature for 24 h while gelation occurred. The obtained hydrogel was dried at 80 C in oven for 48 h, further the gel was calcined at 800 C (10 C/min) for 3 h. C3A (Ca3Al2O6) was synthesized by citric acid precursor method (also known as citrate route).32,33 In this case, 0.3 mol of Ca(NO3)24H2O and 0.2 mol of Al(NO3)39H2O (Panreac, PA, 98% min) were dissolved in distilled and deionized water and then mixed with aqueous solution of citric acid (C6H8O7H2O, Aldrich, PA, 99.5% min) under continuous stirring for 2 h at 60 C. After, an adequate volume of ethylene glycol (Panreac, PA, 99.5%; ethylene glycol/citric acid molar ratio of 2) was added and the temperature was risen to 80 C, the stirring was maintained for 4 h. The resultant mixture was kept at room temperature overnight and then was dried at 150 C in oven. Finally, the obtained product was calcined at 950 C (10 C/min) for 3 h. Synthetic powders (a-C3P, b-C2S and C3A) were wet milled in attrition mill for 2 h using isopropyl alcohol. The particle size distribution of synthetic powders was measured in a Master Sizer S laser light-dispersion analyzer size provided with a wet sample dispersion unit Hydro SM (Malvern Instruments). Powders were previously dispersed in ethanol and deflocculated with DolapixV with the aid of an ultrasound bath.

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Specific surface area of synthetic powders previously degasified at 100 C for 12 h was determined with a Monosorb Surface Area Analyzer MS-13 (Quantachrome Corp.) using the single point N2-B.E.T. approach. X-ray fluorescence spectrometry (XRF; MagiX Super Q version 3.0 Phillips X-ray fluorescence spectrometer, Philips) was used to determine the chemical composition of synthesized powders. Qualitative X-ray diffraction phase analysis Crystalline phases present in the raw materials and set cements were identified by XRD analysis. XRD powder patterns were recorded on an automated diffractometer (D5000, Siemens) using CuKa radiation (1.5418 Å). Data were collected in Bragg–Brentano (h/2h) vertical geometry (flat reflection mode) between 5 and 70 in 0.05 steps (time/step ratio of 1.5 s) and samples were rotated at p/2 rad/s. Cements preparation a-C3P powder was homogeneously mixed with 10 wt % of b-C2S or C3A powders. Pure a-C3P powder was used as control. The cement powder was thoroughly mixed with 2.5 wt % Na2HPO4 aqueous solution at a liquid to powder (L/P) ratio of 0.40 mL/g for pure a-C3P and 0.50 mL/g for cements with additives (b-C2S or C3A powders). All cement pastes were spatulated for 3 min before casting in the corresponding molds. Setting time Initial and final setting times were measured according to the standard UNE-EN 196-3:20051A1 “Methods of Testing Cements. Part 3: Determination of setting times and soundness” with the aid of a Vicat needle and using a cylindrical mold (Ø 5 15 mm; h 5 10 mm) instead the standardized mold. The relative humidity and temperature were kept at 97.5 6 2.5% and 36.5 6 1.5 C during measurements. Setting temperature The setting temperatures of cements were recorded with the aid of a specifically designed adiabatic chamber34 with a T-type thermocouple inserted in the bulk of the cement pastes and connected to a temperature data logger TESTO 177-T4 (Testo AG). Experiments were conducted at 36.5 6 1.5 C and 97.5 6 2.5% of relative humidity. The maximum temperature reached during setting was measured for at least two replicas for each composition and the mean value was calculated. Compressive strength CS of set cements were measured on cylindrical probes (Ø 5 6 mm; h 5 12 mm). Probes were prepared by casting cement pastes into cylindrical Teflon molds, aging for 24 h at 97.5 6 2.5% relative humidity and 36.5 C, finishing parallel faces with sand paper and carefully unmolding. The CS measurements were carried out in an universal testing machine model 1114 (Instron Corp.) provided with a load cell of 5 kN at a loading rate of 0.5 mm/min. Load-

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displacement data were collected at 100 ms intervals and processed with the software SCM 3000 v. 14.7.9 (MicroTest). Seven replicas were measured for each group and the mean value and standard deviation were calculated. In vitro bioactivity Cement samples were prepared by casting cement pastes into cylindrical Teflon molds (Ø 5 6 mm; h 5 2 mm), and aging for 24 h at 97.5 6 2.5% relative humidity and 36.5 6 1.5 C. In vitro bioactivity of cements samples was assessed according to the standard ISO 23317:2007 “Implants for surgery. In vitro evaluation for Apatite-forming ability of implant materials” procedure proposed by Kokubo.35 Cement discs were placed in polystyrene vials containing simulated body fluid (SBF; volume/area ratio 5 0.1 mL/ mm2) and maintained at 36.5 C for 7 and 21 days without refreshing the soaking medium. Further, the samples were gently rinsed with deionized water to remove SBF solutions, and dried at room temperature. Morphological variations in the disc surfaces were analyzed by field-emission scanning electron microscopy (FE-SEM S-4700, Hitachi, Japan). The concentrations of Ca, P, and Si soluble species released to the SBF by cements samples were determined by inductively coupled plasma atomic emission spectroscopy (Thermo Jarrel Ash-IRIS Advantage) at 0, 1, 7, and 14 days and changes in pH of soaking solutions were also measured by an electrolyte-type pH meter (MPA-210). Cytotoxicity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay] The cell proliferation assay was performed by the extraction method with fetal human osteoblast cells (HOb; Health Protection Agency Culture Collections-HPAC-406-05f). A culture medium of fetal osteoblast human cells was used to evaluate the in vitro biological response of the cement samples. The culture medium was supplemented with 10% fetal bovine serum (FBS; Gibco), 200 mM L-glutamine (Sigma), 100 U/mL penicillin (Sigma), and 100 lg/mL streptomicyn (Sigma) in humidified atmosphere of 5% carbon dioxide and 95% air at 37 C. ThermanoxV discs (13 mm in diameter, TMX; Nunc) were used as negative control and a 0.5% Triton-X100 (Merck) solution was used as positive control. Circular cement samples (14 mm in diameter and 3 mm in thickness) were used to quantify the cytotoxicity of delivery products for 7 days, as well as for the surface microscopic studies of these materials. All samples were sterilized with ethylene oxide. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method was used to assess the cell proliferation levels. This assay relies upon the ability of living cells to reduce a tetrazolium salt into a soluble colored formazan product. To determine the toxicity of leached, the samples were introduced in 5 mL-free FBS-culture medium and maintained in rotation at 37 C for 1, 3, and 7 days. The culture medium was then removed and stored. The cell suspension was adjust to a density of 1 3 105 cell/mL (1 3 104 cell/cm2) and 100 mL cell suspension was R

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TABLE I. Mean Particle Size and Specific Surface Area of Synthesized Powders (Standard Deviation Between Parentheses)

Mean particle size (lm) Specific surface area (m2/g)

a-C3P

b-C2S

C3A

4.4 (0.3) 1.8 (0.1)

2.5 (0.2) 11.0 (0.2)

2.9 (0.2) 8.0 (0.1)

added to 96-well plate (Sarsted) and incubated at 37 C for 24 h. Then the medium was removed from the plate wells, adding in place of these 100 mL of 1, 3, and 7 days eluted extracts to each well, which were kept in contact with the cultures at 37 C and 5% CO2 for another 24 h. Later, 100 mL of 0.5 mg/ mL MTT solution was added and incubated for 4 h at 37 C. Then, 100 mL dimethyl sulfoxide (Scharlau) was added to each well, the plate was shaken for 10 min and the optical density (OD) at 570 nm was measured with a detector Biotek ELX808IU. The relative cell viability was calculated by the following equation: Relative Cell Viability ð%TMX Þ 5 ðODs 2OD R Þ=ðOD C 2OD R Þ3100

(1) Being ODS, ODR, and ODC optical density of formazan production from samples, reference (culture medium without cells) and negative control TMX, respectively. The data were analyzed via one-way analysis of variance test and p value < 0.05 was used to establish statistical significance. Cell morphology Cement and TMX discs (n 5 2) were placed in the wells of 24-well culture plates and 1 mL of PBS were added and incubated at 37 C for 24 h under 5% CO2 atmosphere. Overnatant solution was removed and 1 mL of cells suspension (105 cell/mL) was added on samples and negative control and incubated at 37 C for 24 h under 5% CO2 atmosphere. The culture medium was removed and samples were fixed with 1 mL of 2.5% glutaraldehyde (Fluka) in 0.1 M sodium cacodylate buffer (Sigma) for 4 h at 4 C in darkness. Samples were washed twice with sterile water and dried at 37 C. After gold coating the surface of samples with cells was examined under the scanning electron microscope (Hitachi TM-1000). RESULTS

Characterization of powders The values of mean particle size and specific surface area for the synthesized powders are showed in Table I. The

three powders presented a particle size smaller than that reported in the literature (10 mm) for the calcium phosphate cements preparation.36 The results of the XRF analysis for the synthesized powders are presented in Table II. Figure 1 shows the XRD patterns of synthesized powders. The patterns exhibited a sharp peaks and low background signal typical of highly crystalline materials. Only the characteristic diffraction peaks of a-C3P [Figure 1(a)], bC2S [Figure 1(b)], and C3A [Figure 1(c)], respectively, were observed. No secondary crystalline phases were detected for any of three synthesized powders. Setting time and maximum temperature during setting of the cements The effect of the addition of b-C2S and C3A on the initial and final setting time of a-C3P cement is displayed in Figure 2. The initial and final setting time of TSi-10 paste was the highest of the three pastes studied. Addition of b-C2S delayed initial and final setting. Initial setting time increased from 19 min for T, to 41 min for TSi-10. On the other hand, final setting time of TSi-10 rose to 62 min when compared with the 23 min obtained for T paste. However, neither initial no final setting time were significantly affected by the addition of 10% of C3A (TAl-10) to the C3P cement (T) as can be seen in Figure 2. Figure 3 displays the evolution of temperature of the cement paste with time for the three cement pastes. Setting process of the three cement pastes was exothermic. An increase in the temperature of the cement paste was always observed, however the peak temperature in any cases overcame the normal body temperature. The maximum temperatures reached during setting and the time elapsed from mixing to maximum temperature were 35.2, 35.9, and 35.4 C; and 17, 10, and 58 min for T, TAl-10, and TSi-10, respectively. The position of the peak temperatures in the curves T versus t agreed with the results of setting time. The larger the setting time the more delayed the maximum temperature was reached. Characterization of set cements Specimens of the set cements aged for 24 h at 36.5 6 1.5 C in an atmosphere saturated in water vapor were crushed and powdered and their X-ray diffraction patterns were obtained. They are displayed in Figures 4–6. CDHA (Ca9HPO4(PO4)5OH, JCPDS 46–0905) formed during the hydrolysis of a-C3P was the main crystalline phase identified in the patterns of the three experimental cements.7,9,37–39 In the pattern of cement T, (Figure 4), CDHA was the main crystalline phase detected, however a very slow intensity peak at

TABLE II. X-Ray Fluorescence Analysis of the Powders (Standard Deviation Between Parentheses) wt % Powder A-C3P B-C2S C3A

CaO

SiO2

Al2O3

P2O5

Na2O

Ignition Loss

54.2 (0.05) 65.4 (0.06) 37.4 (0.05)

– 33.5 (0.03) –

– – 62.4 (0.06)

44.6 (0.03) 0.01 (0.003) 0.05 (0.007)

0.03 (0.008) – 0.01 (0.004)

0.8 (0.02) 1.0 (0.03) 0.07 (0.003)

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FIGURE 1. XRD patterns of a-C3P, b-C2S, and C3A powders.

FIGURE 3. Maximum temperature during setting of cement samples.

30.8 (2h), corresponding to the 100% intensity reflection (1 7 0) of a-C3P (JCPDF 09–0348), was also detected, indicating that the setting reaction has not still been completed. On the other hand, many peaks corresponding to a-C3P were also detected in cements TSi-10 (Figure 5) and TAl-10 (Figure 6), indicating that a certain amount of unreacted aC3P remained after 24 h of setting in these materials. Neither peaks corresponding to crystalline calcium–silicate–hydrate (C–S–H), nor peaks corresponding to hydrogarnet (C3AH6), were detected in the diffraction patterns of TSi-10 nor TAl-10 set cements, respectively.

and 6). No unreacted a-C3P was detected in TSi-10 after soaking for 21 days in SBF (Figure 5), however, a minimum amount of unreacted a-C3P still remained in TAl-10 cement after 21 days in SBF (Figure 6). Characteristic peaks of C–S–H or C3AH6 were not detected in the diffraction patterns of TSi-10 (Figure 5) or TAl-10 (Figure 6), respectively, by soaking in SBF 7 for 21 days. SEM images of the surface of set cements before and after immersion in SBF are shown in Figure 7. In all cases soaking in SBF led to distinctive surface remodeling. The surfaces of T and TSi-10 set cements before soaking exhibited some particles of unreacted a-C3P surrounded by apatite microcrystals. However, at the surface of TAl-10 set cement an amorphous substance may be noticed in addition to residual a-C3P particles and apatite crystals. This amorphous substance is probably Al(OH)3 gel resulting of the hydration reaction of Ca3Al2O6 (C3A).16,17 After soaking in SBF, a-C3P-based cement surface was covered by entangled

In vitro reactivity in SBF Specimens of set cements were soaked in SBF for 7 and 21 days and their X-ray diffraction powder patterns were obtained (Figures 4–6). For cement T only CDHA peaks were detected after soaking in SBF for 7 and 21 days (Figure 4). In the added cements (TSi-10 and TAl-10) the peaks of unreacted a-C3P were observed in set cements considerably weakened after soaking in SBF for 7 days (Figures 5

FIGURE 2. Initial and final setting time of cement samples [* denotes statistically significant (p < 0.05) differences with regard to sample T].

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FIGURE 4. XRD patterns of T cement before and after soaking in SBF for 7 and 21 days.

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21 days to less than 50% of its original value, the CS of TSi10 increased over 30% by soaking for 21 day in SBF. After 21 days in SBF, TSi-10 was significantly stronger than T. On the other hand, TAl-10 cement exhibited very poor CS, which was no significantly influenced by soaking in SBF. The internal microstructure of the experimental cements was studied by SEM on their fracture surfaces (Figure 11). After setting, the microstructure of cement T is mainly constituted of plate-like crystals, whereas in TSi-10 and TAl-10 microstructures were observed needlelike structures formed onto nonreacted a-C3P particles. By soaking 21 days in SBF TSi-10 cement developed a microstructure constituted of needle-like crystals randomly oriented in all directions, which is denser when compared with T and TAl-10.

FIGURE 5. XRD patterns of TSi-10 cement before and after soaking in SBF for 7 and 21 days.

plate-like apatite crystals,39,40 whereas globular structures with typical bone-like apatite morphology similar to those formed in bioactive materials under physiological conditions1,22,23,40–43 were deposited and observed onto TSi-10 and TAl-10 surfaces. The evolution of pH of the SBF solution in contact with samples of experimental cements was displayed in Figure 8. An initial increase in pH was observed for all experimental cements. The magnitude of pH increase obeyed the order T > TSi-10 > TAl-10. The maximum pH value of 8.2 was reached for cement TAl-10 after 7 days in SBF. This value is still mild enough to cause not harm to surrounding tissues during a hypothetical in vivo implantation. Furthermore, after reaching its maximum value the pH of SBF in contact with TAl-10 soaking diminished to 7.8 at day 21st of soaking. On the other hand, pH values for SBF soaking T and TSi-10 cements reached a maximum (7.5 and 7.7, respectively) in the first hour and then were stabilized at 7.1 and 7.6, respectively, after 21 days. Figure 9(a,b) shows the concentration profiles of Ca and P in SBF during soaking the cement specimens for various periods of time. Both, Ca and P concentrations gradually decreased during soaking for the three experimental cements. CDHA formation at the surface and inside the cement samples may be responsible for the observed decrease in Ca and P concentrations during soaking in SBF.

Compressive strength The CS of experimental cements, as prepared and after immersion in SBF was shown in Figure 10. As prepared cement T was significantly stronger than added cements TSi-10 and TAl-10. However, addition of 10 wt % of b-C2S to a-C3P-cement had a remarkable effect on strength improve during soaking in SBF. Whereas the strength of nonadded cement T decayed during immersion in SBF for

In vitro biocompatibility The results of the MTT assay are displayed in Figure 12. The soluble products extracted from T, TSi-10, and TAl-10 cements by incubation 1 day in culture medium were moderately cytotoxic with regard to the negative control (TMX), being the extracts fromTSi-10 the least cytotoxic. The values of relative cell viability obtained for T and TSi-10 extracts were over 70% for all analyzed periods, suggesting that these cement samples were no cytotoxic toward human fetal osteoblast cells, whereas TAl-10 extracts had a cytotoxic response due to its cell viability was less 60% all time (Figure 12).44 TSi-10 was the material whose extracts showed the highest relative cell viability for all studied periods. Cell morphology SEM images (Figure 13) show the morphological features of osteoblast cells cultured on cement discs for 1 day. The cells were observed to adhere on cement surfaces.

FIGURE 6. XRD patterns of TAl-10 cement before and after soaking in SBF for 7 and 21 days.

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FIGURE 7. Scanning electron micrographs of the surface of cement samples before and after immersion in SBF. T: (a) before SBF, (b) 7 days in SBF, (c) 21 days in SBF; TSi-10: (d) before SBF, (e) 7 days in SBF, (f) 21 days in SBF; TAl-10: (g) before SBF, (h) 7 days in SBF, and (i) 21 days in SBF.

DISCUSSION

In this study quenching was not necessary to obtain the pure a-C3P phase during cooling at room temperature, because the b ! a transition (1125 C) is of reconstruc-

FIGURE 8. Changes in pH of the SBF solutions after soaking the T, TSi-10, and TAl-10 cement samples.

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tive type and high activation energy would require surpassing to the reverse a ! b transformation occurs. Hence, the a phase can be retained at room temperature in a metastable state.45 On the other hand, b-C2S is an unstable phase and can undergo transformation to a more stable g phase; therefore chemical stabilizers were often used to prevent the morphological transformation b ! g. Here, it was used a sol–gel process that preserved the b-C2S phase without use of chemical stabilizers, and there were no impurities in the prepared powders such as SiO2, CaO, and other silicates.15,24 To obtain C3A, citric acid precursor method was used. This method is a useful powder preparation technique used successfully in the preparation of highly dispersed mixed oxides or oxide solid solutions because the citric acid has the ability to form polybasic acid chelates with various cations. These chelates can undergo polyesterification when heated in a polyhydroxyl alcohol (ethylene glycol) to form a polymeric glass with the cations uniformly distributed throughout its network.33 The initial setting time (Figure 3) of cement T (19 min) was higher than that reported in the literature (3–8 min)8,46–48 for calcium phosphate cements for clinical

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3a-Ca 3 ðPO 4 Þ2ðsÞ 1 H 2 O ! Ca 9 ðHPO 4 ÞðPO 4 Þ5 OH ðsÞ

FIGURE 9. (a) Profiles of [Ca] in SBF during soaking of the experimental cements. (b) Profiles of [P] in SBF during soaking of the experimental cements.

applications when a neutral phosphate solution such as disodium hydrogen phosphate solution (Na2HPO4) was used as liquid phase and liquid to powder (L/P) ratio of 0.32 mL/g. In our case a workable cement paste wasonly reached with L/P ratio of 0.40 mL/g, this higher L/P of cement T probably delayed the setting reaction and its setting time was higher than traditional calcium phosphatebased cements. With the addition of C3A setting time of TAl-10 was decreased to 13 , because C3A reacts rapidly and exothermically with aqueous solutions which is also responsible for higher strength at early stage of setting.16,17 However, the difference in setting time observed TAl-10 when compared with T was not statistically significant. In contrast, b-C2S alone hydrates very slowly,16 hence the longer setting time was obtained for TSi-10 cement. The reaction of Equation 2 takes place during the setting of a-C3P-based cement. a-C3P dissolves and CDHA precipitates when saturation is reached.7,35,38 The growing CDHA crystals progressively become entangled, causing the hardening of the cement paste and providing the set cement with stiffness and strength.5,9,37

(2)

The characteristic peaks of CDHA were detected for cement T (Figure 4) in XRD patterns, both at 24 h, a little peak of a-C3P was also detected at this time after setting and after soaking in SBF. By adding 10 wt % of b-C2S and C3A no significant changes appeared in the XRD patterns of TSi-10 and TAl-10 (Figures 5 and 6, respectively) before immersion in SBF. The intensities of CDHA peaks increased by prolonging soaking from 7 to 21 days indicating the formation of bone-like apatite due to hydrolysis of remaining a-C3P and deposition from SBF on the cement surfaces. However, for TAl-10 a small peak at 30.8 (2h) is still visible for samples treated in SBF 7 day and 21 day. This peak, corresponding to the 100% intensity reflection (1 7 0) of aC3P (JCPDF 09-0348), indicates that a small amount of aC3P particles remained unreacted. This fact can be related to the frankly alkaline pH (over 8.0) developed during setting of TAl-10 (Figure 8), which causes a decrease in the solubility of a-C3P driving to retard of hydration reaction and further CDHA formation.4 It is considered that the bone-like apatite layer formed on the surface of bioactive materials plays an essential role in chemical bonding at the living tissue–biomaterial interface. This apatite layer can be reproduced in vitro by immersion in SBF.24,49–51 Hench51,52 proposed a sequence of reactions involved in the formation of bone-like apatite layer on the surface of bioactive glasses containing CaO– SiO2. The development of a hydrated silica layer on these bioactive glasses, which provide specific sites favorable for CDHA nucleation, is one decisive step in this reaction sequence.23,24 On the other hand, Kokubo38,52 has shown that in vitro formation of the apatite layer is very sensitive to the pH at the interface biomaterial-solution, and an increase in pH of the solution would favor the deposition of HA on the surface of apatite/wollastonite glass–ceramics. The surfaces of all cement samples became coated by a typical apatite layer after soaking in SBF suggesting all three

FIGURE 10. Compressive strength of cement samples before and after soaking in SBF. [* denotes statistically significant (p < 0.05) differences from sample T].

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FIGURE 11. Fracture scanning electron micrographs of cement specimens before (0 day) and after (21 days) immersion in SBF. T (a—0 day, b and c—21 days), TSi-10 (d—0 day, e and f—21 days), TAl-10 (g—0 day, h and i—21 days).

materials are biaoctive (Figure 7). Surface of a-C3P-based cement was covered by entangled plate-like structures, whereas globular structures with typical bone-like apatite morphology were deposited onto TSi-10 and TAl-10

FIGURE 12. Relative cell viability of the different cement extracts. * denotes statistically significant differences from negative control TMX (p < 0.05).

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surfaces. Ions releasing to SBF by cement samples taking place at the early stage of soaking makes the solution supersaturated in Ca21 and PO432 [Figure 9(a,b)]. Ca–P clusters are nucleated at the surface of the materials and they growth to confluence into a biological-like apatite layer onto cement surfaces consuming part of the ionic Ca and P species existing in solution. At the early stage of soaking in SBF, jointly with Ca and P ions release, the pH (Figure 8) reached a maximum 1 day (except for solution containing TAl-10 where the maximum pH was attained after 7 days), and then gradually decreased due to the consume OH2 ions in the formation of the apatite layer. Low mechanical strength is one of main handicaps of a-C3P-based cements that prevents they be used in loadbearing clinical applications. The entanglement of CDHA crystals is the major responsible for the mechanical strength of a-C3P-based cement. CS of set cement T (Figure 10) was higher than that of TSi-10 and TAl-10 probably due to practically all a-C3P was converted into CDHA, while in TSi-10 and TAl-10 a certain amount of a-C3P particles remained unreacted after setting. In addition, fracture surface SEM images [Figure 11(a)] revealed the entanglement of the plate-like structures of CDHA conforming the microstructure of a-C3P-based cement while

a-C3P CEMENTS WITH b-DICALCIUM SILICATE AND TRICALCIUM ALUMINATE

ORIGINAL RESEARCH REPORT

FIGURE 13. Scanning electron micrographs of osteoblast cells adhered to negative control TMX and cement surfaces after 1 day of incubation. TMX (a and b); T (c); TAl-10 (d), and TSi-10 (e and f).

an incipient formation of CDHA crystals on fracture surfaces of TSi-10 and TAl-10 [Figure 11(d,g)] was observed. The smaller crystal sizes conforming the microstructure of these cements drastically reduce the CS of them at early stage. On the other hand, the entanglement of the needlelike crystals enhanced the mechanical strength of TSi-10 cement after soaking in SBF [Figure 11(e,f)], resulting in a CS of 36 MPa (Figure 10) which was higher than that reported for human trabecular bone (10–30 MPa).9 However, smaller and less interlocked needle-like crystals were observed for TAl-10 [Figure 11(h,i)] when compared with TSi-10, hence its CS is the lowest of the three samples.

Plate-like structures were developed in T microstructure [Figure 11(b,c)] and provide this cement with a CS of 27 MPa, after soaking in SBF for 21 days, comparable to that of trabecular bone. In vitro cytotoxicity studies are powerful and simple tools to assess biocompatibilty of new biomaterials. Some studies have demonstrated that a-C3P ceramics favor osteoblasts proliferation and expression, possibly because of the release of calcium and phosphate ions in such concentrations that stimulate the osteogenic activity of cells.53,54 In addition, other studies revealed that the dissolution extracts of bioactive silicate-containing materials such as bioactive

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glasses and calcium silicate bioceramics and cements stimulated cell proliferation and gene expression.22,55,56 The cytotoxicity assay used in this work measured the relative viability of cells cultured in the presence of aqueous extracts of the tested materials with regard to cultures made with extracts of the negative control (TMX). The higher the relative cell viability the lower the cytotoxicity of the soluble products extracted from the tested material. The results obtained indicated that the leachates obtained from TAl-10 by treating them with culture medium for 1 day were cytotoxic toward human fetal osteoblasts (relative cell viability less than 60%) (Figure 12), whereas those from T and TSi-10 were no cytotoxic (over 70% of relative cell viability).44 The cytotoxicity of leachates obtained by subsequent extractions (1–3 days and 3–7 days in Figure 12) diminished progressively for the three experimental cements. TSi-10 was the material whose extracts showed the highest relative cell viability for all extraction periods. The results suggested that certain products are dissolved from the cements samples in the first stage of the treatment with culture medium in enough amounts to evoke a frankly cytotoxic response toward human fetal osteoblasts for TAl-10. However, T and TSi-10 extracts were no cytotoxic. The cytotoxicity of the extracts obtained in subsequent treatments diminished, which indicates that most soluble products responsible for toxicity are almost completely removed during the first periods of soaking in culture medium. The highest cell viability observed for extracts from sample TSi-10 could be related to the ability to stimulate osteoblast cells activity attributed to silicate ions.55–60 The highest cytotoxicity (lowest cell viability) found for the extracts from TAl-10 could be related to the frankly alkaline pH produced by this material (Figure 8). The cell morphology results showed osteoblast cells adhere on cement surfaces after incubation for 1 day (Figure 13). The cells exhibited typical osteoblast morphologies with spread polygonal shape. In concordance to cytotoxicity results, an intimate contact between osteoblast cells and TSi-10 surface was observed [Figure 13(f)], suggesting that cement surface represents a good substrate for cell attachment, growth, and proliferation.

CONCLUSIONS

Addition of 10 wt % of b-C2S (TSi-10) and C3A (TAl-10) to a-C3P cement (T) did not affect bioactivity of the end materials, proved by deposition of biological-like apatite layer on cement surfaces by soaking in SBF. Both additives drastically diminished the CS of set cements; however the addition of 10 wt % of b-C2S prevented the degradation of the mechanical strength during immersion in SBF for 21 day. The CS for the cement added with 10 wt % of b-C2S (TSi-10) after soaking in SBF was higher than that measured for human trabecular bone. TSi-10 cement extracts rendered the highest cell viability of the three studied cements. By adding 10 wt % b-C2S to a-C3P cement (TSi-10) was possible to improve CS, bioactivity, and biocompatibility

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