Development and characterization of α-tricalcium phosphate/monocalcium aluminate composite bone cement ()
1. INTRODUCTION
Calcium phosphate cements (CPCs) are a clinical alternative to traditional bioceramics because they are easy to handle and shape, they mold themselves well to the contours of defective surfaces, and set in situ in the bone cavity to form a solid restoration [1]. Since they were developed in the mid-1980s, CPCs have also attracted great interest due to their chemical similarity to the mineral phase of bone tissue and their good osteoconductivity [2].
One of the most important formulations is based on α-tricalcium phosphate [α-Ca3(PO4)2; α-TCP], which sets in situ and forms a calcium-deficient hydroxyapatite [Ca9(HPO4)(PO4)5(OH); CDHA] when hydrated [3]. However, it is not very strong under compression [4] and its mechanical strength is low when compared to that of cortical bone [5] limiting its application to areas subjected to low mechanical loads [6].
In view of the excellent bioresorbability of CDHA, researchers have focused their efforts on overcoming the mechanical weakness of calcium phosphate cements by using different fillers, fibers and reinforcing additives that lead to the formation of various multiphase composites, based on the idea that the filler in the matrix may eliminate crack propagation [7]. Nevertheless, the presence of fillers prevents bone ingrowths into pores and produces a denser cement with a slower resorption rate and hence a slower bone substitution [8]. Therefore, it is difficult to increase the strength of these cements without negatively affecting other properties.
In the late 1990s, the Swedish company Doxa Certex AB proposed the use of calcium aluminate cements (CACs) as dental restorative materials in place of amalgam [9], and today the use of CACs has extended to several orthopedic applications [10,11]. The calcium aluminate system has two inherent features that make it suitable for load-bearing applications: fast setting and high consumption and turnover of water during the setting and reaction. The high water turnover gives the system a potentially high strength, several times that of normal CPCs. Moreover, CACs have good corrosion resistance in contact with body fluids and are biocompatible since the amount of Al ion leakage is very low [12,13]. In these materials, the main phase commonly used is monocalcium aluminate [CaAl2O4, CA] due to its optimal reaction rate compared with the other phases.
Although some calcium phosphates, as β-tricalcium phosphate, are used in combination with CACs in order to induce some biological activity in the resultant composites [14], the use of CA as a reinforced additive of traditional CPCs is not documented. Thus, the aim of this work was to design and study new α-TCP/CA formulations intended for biomedical applications. To this end, the chemical, mechanical and biological properties of α-TCP/CA cement after aging in simulated body fluid were investigated.
2. MATERIALS AND METHODS
2.1. Materials
α-TCP was prepared through solid state reaction, heating the appropriate mixture of Ca2HPO4·2H2O (Extra Pure, DyneÒ) and CaCO3 (Extra Pure, Nuclear) at 1300˚C for 5 h followed by quenching in air [15]. After calcination, the product was wet milled for 4 h in a polyethylene jar with alumina balls using an alcoholic medium (anhydrous ethanol, 99.5%, Cromoline) to an average particle size inferior to 10µm. The powder was composed of a mixture of 82% of α-TCP and 18% of b-TCP [16].
CA was synthesized through Pechini technique [17] using high purity Ca(NO3)2·4H2O (Synth, PA-ACS) and Al(NO3)3·9H2O (Synth, PA-ACS) in the presence of citric acid (C6H8O7·H2O) (Synth, PA-ACS) and ethylene glycol (C2H6O2) (Synth, PA). Suitable amounts of nitrate salts were dissolved, followed by the addition of citric acid and ethylene glycol. After gelification, the gel was heated at 150˚C for 24 h and calcined at 400˚C for 2 h to form the powder precursor, which was heat-treated at 1000˚C for 3 h. In order to obtain powders with similar average particle size, the same milling treatment as in the case of α-TCP was used.
2.2. Preparation of Composite Samples
Synthesized CA (7 µm; 11.88 m2/g) was mixed in powder ratios of 0, 5.0 and 10.0 mass % with α-TCP (10.71 µm; 5.52 m2/g). The liquid phase was a sodium phosphate buffer prepared from NaH2PO4 and Na2HPO4·12H2O and the liquid-to-powder ratios (L/P) employed were 0.4, 0.44 and 0.46 ml/g, respectively. Each powder sample was carefully weighed and mixed with the liquid phase in appropriate powder-to-liquid ratio, packed into silicon molds and aged at 36.5˚C with 100% humidity for 24 h.
2.3. Setting Time Measurement
The setting time of samples was measured according to ASTM C266-89 using a Gillmore Needles method [18]. Three specimens for each formulation were tested and standard deviation was used as a measure of the standard uncertainty. Initial setting time was determined as the end of moldability and final setting time was choosen as the time beyond which it is possible to touch the cement without serious damage [19].
2.4. In Vitro Tests
To assess in vitro bioactivity, the 24h-set pastes were soaked in simulated body fluid (SBF) at 36.5˚C [20] for 1, 7 and 14 days, after which they were rinsed gently with deionized water, dehydrated with ethanol, and dried.
For degradation tests, the disks were accurately weighed before and after immersion in SBF. The weight loss (WL) was calculated according to
(1)
being W0 the initial weight of the specimen and Wd the weight of the specimen dried after different degradation times (7, 14 and 21 days). All the measurements were taken in triplicate and the average values were calculated.
2.5. Cytotoxicity Test for Cements
The cell viability assay was performed by direct contact test according to ISO 10993-5 using peripheral blood mononuclear 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 was quantitatively assessed by MTT test. Experimental values were analyzed via one-way ANOVA test follow by Tukey’s Multiple Comparison Test.
2.6. Characterization Techniques
The phase composition of the samples was determined by X-ray diffraction (XRD) in a Philips® X’Pert MPD diffractometer equipped with a Cu-target. Diffractograms were recorded employing Ni-filtered radiation (λ = 1.5406 Å) with a step size of 0.05˚ and a time/step ratio of 1 second.
The powders’ specific surface area was determined by nitrogen gas sorption and obtained by five-point BET analysis using a Nova 1000 surface area analyzer, while the particle size distribution was determined in a CILAS 1180 particle size analyzer using isopropyl alcohol as dispersant.
The morphological variations of materials before and after soaking in SBF were characterized by Scanning Electron Microscopy (SEM) using a JEOL microscope (JSM-6060) on gold-coated samples.
Compressive strength (CS) was measured in a servohydraulic universal testing machine (MTS 810) equipped with a 10 kN load cell, at a loading rate of 1 mm/min. The number of replicas was n = 10 and Student’s Multiple Comparison Test was performed to compare mean values.
The pH value was measured during soaking in SBF and readings were taken in an mPA-210 pH meter at 36.5˚C.
3. RESULTS
Figure 1 shows the XRD pattern of CA where the presence of monoclinic CaAl2O4 (JCPDS 2310-36) as main phase, in addition to dicalcium aluminate [CaAl4O7, CA2] (JCPDS 2310-37) was found. The specific surface area of the powder was 9.12 m2/g and a slight increase to 11.88 m²/g was achieved after grinding.
Figure 2 shows the initial and final setting time of α-TCP and composites containing different CA mass%. For a-TCP-based cement the initial and final setting times were higher than those reported in the literature for similar compositions [22]. With the addition of CA, the setting times increased, this increase being directly proportional to the amount of CA added. There were no significant differences in the final setting times of composites containing CA.
Figures 3-5 show the powder XRD patterns of composites before and after soaking in SBF for 7 and 14 days. For all times and all formulations, the characteristic peaks of β-TCP (JCPDS 09-0169), which appears as a seconddary phase in a-TCP powder (JCPDS 29-0359), were detected. After 24 h setting (Figure 3), for a-TCP-based cement, mainly peaks of CDHA (JCPDS 46-0905) were observed. With the addition of CA, diffraction patterns were very different from those of a-TCP-based cement and apparently, only unreacted peaks of a-TCP in addition to β-TCP were present.
After 7 days of soaking (Figure 4) the intensity of a-TCP lines decreased in relation to set cements and CDHA lines appeared.
Fourteen days after, the hydration reaction seemed to be complete for a-TCP, whereas a great amount of unreacted a-TCP, in addition to CDHA, could be observed for
Figure 1. XRD pattern of CA obtained by the pechini route.
Figure 2. Initial setting and final setting time of the composites compared with α-TCP-based cement.
Figure 3. XRD pattern of cement pastes after 24 h of setting. na-Ca3(PO4)2; ob-Ca3(PO4)2; lCa9(HPO4)(PO4)5(OH).
Figure 4. XRD pattern of cement pastes after 7 days of soaking in SBF. na-Ca3(PO4)2; ob-Ca3(PO4)2; lCa9(HPO4)(PO4)5(OH).
composites containing CA (Figure 5). There were no peaks of CA and no proof of the presence of Ca3Al2O6·6H2O (C3AH6) (JCPDS 24-0217) or Al2O3·3H2O (AH3) (JCPDS 29-0041), the most likely phases during hydration of CA.
Figure 6 shows the SEM micrographs of the surface of composites after soaking in SBF. For conventional CPC (Figure 6(A)), a superficial layer of CDHA with a globular shape similar to some bioactive materials was deposited within 14 days [23].
Some bacterial contamination by Bacillis and Cocci colonies, represented by spherical and rod-shaped holes, were also observed [24].
For 5CA and 10CA (Figures 6(B) and (C)) small round shaped particles, spherulites-like cristals, of hydroxyapatite, were beginning to deposit on top of the leaf-like intermediary structure since the early stages (about 1day of soaking). Evidence of the formation of a new product containing phosphorus was formed on the surface of composites was confirmed by EDS analysis (Figure 7).
Figure 8 shows the compressive strength and porosity of a-TCP and a-TCP/CA composites before and after