Materials Science and Engineering C 43 (2014) 65–75
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Antibiotic-free composite bone cements with antibacterial and bioactive properties. A preliminary study Marta Miola a,⁎,1, Matteo Bruno a, Giovanni Maina c, Giacomo Fucale b, Giovanni Lucchetta d, Enrica Vernè a a
Applied Science and Technology Department, Politecnico di Torino, Italy Chemical, Clinical and Microbiological Analyses Dept., CTO, Turin, Italy Department of Clinical and Biological Sciences, University of Turin, Turin, Italy d Department of Innovation in Mechanics and Management, University of Padova, Padova, Italy b c
a r t i c l e
i n f o
Article history: Received 30 September 2013 Received in revised form 21 May 2014 Accepted 30 June 2014 Available online 8 July 2014 Keywords: Antibacterial Bioactive glass Bone cement Composite Antibiotic-free
a b s t r a c t Two bone cements (Palacos R® and Palacos LV®) based on polymethylmethacrylate (PMMA), clinically used in several cemented prosthetic devices, have been enriched with silver containing bioactive glass powders and compared with the plain commercial ones. The obtained composite cements have been subjected to a preliminary characterization by means of morphological and compositional analyses, compression mechanical tests, bioactivity test (by soaking into simulated body fluids), leaching tests and in vitro antibacterial test (count of colonies forming units, McFarland index evaluation, inhibition zone evaluation). The glass powders appeared uniformly dispersed inside the PMMA matrix and good mechanical properties (in compression) have been reached. The composite cements showed a bioactive behavior (since they developed hydroxyapatite on their surface after soaking in simulated body fluid) and a good antibacterial performance. The release of silver ions, which is the principal reason of antibacterial properties, is mainly reached after the first hours of contact with the leaching solution, as it is expected for a reasonable prevention of bacterial colonization during in vivo applications. © 2014 Published by Elsevier B.V.
1. Introduction Post-surgical infections are still one of the most frequent adverse events in the prosthetic surgery. They are often caused by the adhesion of bacteria on the implant surfaces, which is responsible of an invasive inflammatory process along the bone/implant interface. This situation, followed by bacteria proliferation, is enhanced by the formation of a biofilm, a three-dimensional self-produced structure that protects bacteria against patient own defense system and from systemic antibiotic therapy [1]. It is estimated that infection rates in orthopedic surgery is about 1% (for primary hip and knee arthroplasties) while in trauma surgery they can reach 12–53% for open fractures (open fractures Gustilo grade III) [2]. Most of the infections often cause implant failure [3,4] and, in turn, the need for a revision surgery, which causes prolonged hospitalization and other severe complications [5]. Aiming to avoid bacterial adhesion and to extend the longevity of bone
⁎ Corresponding author at: Politecnico di Torino, Applied Science and Technology Department, DISAT, Corso Duca degli Abruzzi 24, 10129 Torino, Italy. Tel.: + 39 0110904717; fax: +39 0110904624. E-mail address: [email protected] (M. Miola). 1 Present affiliation: Department of Health Sciences, Università del Piemonte Orientale “A. Avogadro”, Novara, Italy.
http://dx.doi.org/10.1016/j.msec.2014.06.026 0928-4931/© 2014 Published by Elsevier B.V.
implants, different prevention solutions, like strict antiseptic operative procedures (i.e. laminar flow and systemic antibiotic prophylaxis) have been proposed. However, even if the infection development rate has been significantly reduced, the problem of septic loosening in joint arthroplasty is still open. For this reason, beside antiseptic procedures, several surgical treatments have been proposed to reduce bacterial contamination of implant surfaces and different strategies have been investigated in biomaterial formulations. A simply way to reduce the bacterial adhesion on the implant surface is to favor the direct bonding between the prosthesis and the bone, by a selective adhesion and proliferation of osteogenic cells, rather than bacteria, promoting a fast osteointegration, without the formation of soft tissue layers. Although PMMA bone cements are known to assure a fast primary fixation, mainly by a mechanical anchoring to the bone trabeculae, it is established that the bone/cement interface is a weaklink zone in the prosthesis/bone interface, because it does not adhere actively to the bone [6]. The improvement of bone-bonding ability of PMMA bone cement by using different kinds of bioactive fillers (e.g., Ceravital® [7], hydroxyapatite [8] and Bioglass® [9]) is discussed in the literature; however, is still not demonstrated that a fast bone healing means the absolute absence of bacterial adhesion to the implant surface. For this reason, more specific formulations have been investigated, aiming to impart bacteriostatic or antibacterial properties to different bone cements. Antibiotic loaded PMMA-based bone cements
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have been widely used since '70. During the last decades several formulations have been compared, like antibiotic loaded cements with different antibiotics (tobramycin, vancomycin, cephazolin) [10,11] industrially or manually added to the polymeric matrix and bone cements loaded with a combination of two antibiotics [11–13]. However, the principal drawbacks of these formulations can be individuated as follows: i) the large size of antibiotic molecules leads to a release, which is only a surface phenomenon; ii) commonly the highest release is observed during the few first hours, that means, in turn, a long term low concentration around the implant (under the antibiotic MIC) with a subsequent high risk of antibiotic-resistant strain development [1,14,15]. For the second reason, antibiotic-loaded bone cements are mainly indicated for revision surgery, rather than for prophylaxis, since their local action is not indicated for infection prevention, but for its treatment, if prevention failed. The increasing percentage of total joint infections with multiresistant bacteria stimulated the scientific community on the research of alternative strategies to give antibacterial properties to these materials, without the use of antibiotics, to be used as a prevention approach, more efficient of both plain bone cements and antibiotic loaded ones. At this purposes, bone cements loaded with Ag nanoparticles have been recently described in the literature [16,17]; the Ag nanoparticle utilization is proposed to prevent infections caused by multiresistant bacteria, taking into account the well-known broad antimicrobial effect of silver [18,19], however, the antibacterial properties of nanosilver in these formulations are not yet completely proven and the different efficacy of silver ions vs metallic silver is still debated. Moojen et al. [20] state that metallic Ag nanoparticles contained in the cement are not released and need to be ionized before they exhibit their antimicrobial activity. Therefore, the cement predominantly exhibits an antimicrobial effect at the direct cement surface and not in the surrounding bone tissue; this provides the possibility of bacteria to replicate several times before the silver starts to interfere with its proliferation. Aiming to improve the implant longevity by means of both fast osteointegration and low bacterial adhesion, a new approach has been reported by some authors [21,22] by using both antibacterial and bioactive fillers in the same bone cement. This aim seems to be reached, but with quite complex solutions, which imply the addition of two different solid phases into the polymeric matrix (one to impart bioactivity and the second one to impart antibacterial properties) with some concerns on the mechanical properties of the final cement as well as on its industrial and clinical feasibility. Moreover, in several cases this approach still used antibiotics as antibacterial phase, without avoiding the risk of resistant bacterial strain development and still maintaining the incomplete efficacy of drug release. In this work, the synthesis and characterization of composite bone cement, based on a PMMA matrix containing a single additional solid phase with both bioactive and antibacterial properties are proposed for the first time. The additional phase is a bioactive glass, containing silver ions, so the same filler shows at the same time the ability of promoting bone ingrowth and a bacteriostatic effect. The presence of silver ions instead of metallic Ag nanoparticles implies an immediate availability of Ag ions, which can be released and limit not only the bacterial adhesion, but also their proliferation in the surrounding tissue. Bioactive glasses and glass–ceramics are widely studied and developed in several commercial formulations [23] for bone restoration. The term “bioactive” refers to their ability of producing a chemical bond to the living bone by a well-known mechanism, which involves ion leaching from their amorphous network in the surrounding biological fluids and the precipitation of hydroxyapatite on their surfaces that is considered a sufficient requirement to bond with the living bone [24, 25]. They have been also described as osteoinductive materials, since they are able to elicit both intracellular and extracellular responses, promoting osteogenic stem cell colonization, by the release of specific ions (Si4+, Mg2+, Ca2+). The “genetic design” of bioactive glasses has become one of the most challenging fields of bone tissue engineering
[26] doping the glass with a variety of active ions (Sr, Cu, Fe, B, Zn, etc.), useful both for stimulating bone regeneration and for therapeutic purposes. In this contest, silver containing bioactive glasses and glass–ceramics have been produced in different ways [27–29] and their biocompatibility with osteoblast-like cells as well as their antibacterial properties have been reported to demonstrate both their efficacy and safety. The formulation described in this work has been specifically developed to be used as filler for composite bone cement, and represents a preliminary solution for a new family of antibiotic-free, osteoinductive and antibacterial bone cements, which could be proposed as a valid alternative to the existing ones, with potential indications mainly for prophylaxis of implant-related infections. 2. Materials and methods 2.1. Glass synthesis and characterization The glass synthesized in this research work belongs to the SiO2– Na2O–CaO–P2O5–B2O3–Al2O3–Ag2O system. The glass, named SBAG from now on, was produced by melting the reactants in a Pt crucible at 1450 °C and by quenching the melt in water to obtain a frit that was subsequently milled and sieved to achieve a glass powders with a grain size below 20 μm. This grain size was selected after preliminary experimental test, which evidenced good handling properties. The glass powders were subjected to the differential thermal analysis (DTA 404 PC NETZSCH) with a heating rate of 10 °C/min from 25 °C to 1300 °C to individuate the glass characteristic temperatures and to check if any further phase was present within the amorphous one. Moreover, SBAG powders were analyzed by scanning electron microscopy (SEM — FEI, QUANTA INSPECT 200), and energy dispersion spectrometry (EDS — EDAX PV 9900) to evaluate the powders morphology and composition. Finally, X-ray diffraction (XRD — X'Pert Philips diffractometer) was performed in order to assess the amorphous nature of the obtained powders, using the Bragg Brentano camera geometry and CuKα incident radiation. Diffraction patterns were detected by using a step of 0.02° (2θ) with a fixed counting time of 1 s per step from 10° to 70° (2θ); the pattern analysis was carried out using X'Pert High Score software and PCPDF data bank. 2.2. Composite cements preparation and characterization Composite bone cements (named CBCs from now on) were prepared using two commercial PMMA-based cements with different viscosity (purchased by Heraeus Medical): Palacos® R (high viscosity) and Palacos® LV (low viscosity). Palacos® has been selected over different commercial formulations because it is one of the most commonly used PMMA-based bone cements. Since the addition of the bioactive and antibacterial glass-phase (SBAG powders) does not imply any modification into the polymerization mechanism of the organic matrix, it can be ideally extended to any other commercial or experimental formulation, taking into account their different component ratios and rheology and properly modifying the amount and grain size distribution of the glass powders. So this work does not suggest any improvement of the commercial polymeric phase, but it is only intended to investigate the role of the new inorganic additional phase (SBAG) on the final properties of common PMMA-based bone cement. As most of the commercial formulations, Palacos® (LV and R) is sold in a kit with one sachet of a solid phase and one ampoule of liquid. The solid phase has a complex formulation and, for Palacos®, contains powders of the co-polymer poly(methyl acrylate, methyl methacrylate), zirconium dioxide as radio-opaque agent and benzoyl peroxide as initiator (as reported on the data sheets). The liquid phase contains the monomer methyl methacrylate (MMA), N,N-dimethyl-toluidine as activator and hydroquinone as inhibitor. Chlorophyll is present both in the liquid and in the solid phase to impart green color to the cement.
M. Miola et al. / Materials Science and Engineering C 43 (2014) 65–75 Table 1 Component's proportion of CBCs.
Palacos® LV30 Palacos® R30 Palacos® LV50 Palacos® R50
Glass (wt.%)
Solid phase (wt.%)
Liquid phase/solid phase + glass ratio
30
70
0.5
50
50
0.5
The SBAG glass was added to the solid phase in two different proportions: 30% and 50% by weight. The amounts of glass powders incorporated in the PMMA have been selected based on the literature data and preliminary experimental tests. In our experience, an amount higher than 50% by weight does not allow a good handling of the cements during curing. The powders were mechanically mixed for about half hour, in order to assure a good dispersion of glass particles in the PMMA pre-polymerized particles. Subsequently the mixed solid phases were manually mixed with the liquid phase (containing the monomer MMA) under a laminar flow cabinet, maintaining the same liquid/solid phase ratio of commercial bone cements. The mixture was stirred for about 30 s and, as soon as the mass did not stick to the rubber gloves of the operators, it was transferred into a polished aluminium mold (100 × 100 × 5 mm with 25 holes of 10 mm in diameter or 90 × 90 × 24 mm with 25 holes of 12 mm in diameter). The obtained cements were analyzed by SEM–EDS analyses in order to verify the morphology and the glass distribution in the polymerized matrix. Table 1 resumes the component's proportion of CBCs. 2.3. In vitro formation of apatite layer The SBAG powders and the CBCs were soaked in 25 mL of simulated body fluid (SBF — Kokubo [30]) for period up to 1 month in order to verify the sample bioactivity. The SBF volume was selected on the basis of Kokubo protocol using a volume (V)/surface (S) ratio of about: 2 VðmLÞ ¼ S mm =10: The test was performed in triplicate. Every 3 days a refresh of SBF solution was carried out to mimic the renewal of physiological fluid and pH measurements were carried out during the refresh process. Afterwards the samples were analyzed by means of SEM–EDS and XRD analyses. 2.4. Leaching test To evaluate the silver release, the CBCs were soaked in 30 mL of SBF at 37 °C for 28 days. At fixed time periods (3 h, 1, 3, 7, 14, 28 days) 1 mL
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of solution was picked out and analyzed with a graphite furnace atomic absorption spectroscopy (GF-AAS) [PERKIN ELMER (mod. 4100 ZL)] employing a Zeeman-effect background corrector with an autosampler (mod. AS/71), programmed to dispense 20 μL of the sample. Two matrix modifiers have been employed in order to obtain a higher absorption signal: Pd(NO3)2 and Mg(NO3)2 have been directly added on the graphite tube by means of the auto-sampler. Standard solutions were daily prepared from AG MERCK standard solution traceable to SRM from NIST to calibrate the instrument. The calibration standard solutions were 25.0 and 50.0 μg/L. The limit of detection was 0.2 μg/L (CV 5.3%). The picked solution was not replaced by fresh SBF and the obtained result was calculated considering the volume decrease of solution and the sample surface. The test was performed in triplicate. 2.5. Antibacterial property evaluation The antibacterial properties of CBCs were evaluated through three different antimicrobial tests: the McFarland index estimation, the count of colonies forming unit (CFU) [31] and the inhibition halo evaluation [32], using a Staphylococcus aureus standard stock (ATCC 29213). The McFarland index and the CFU count were performed by dipping both composites and commercial cements as control in a standard bacterial broth containing approximately 5 · 105 CFU/mL and incubated 24 h at 35 °C. Subsequently the samples were removed from the broth, gently rinsed in a physiological solution (0.9% P/V of NaCl in distilled water) and vortexed 1 min at 50 Hz, always in the physiological solution, to detach the bacteria colonies adhered on sample surface. In order to quantify the bacteria proliferated in the culture broth and their adhesion on the cement surface, both the culture broth and the vortexing solutions were serially diluted and spread on agar plate, which were incubated overnight at 35 °C to allow the growth and the following count of CFU. Moreover the McFarland index was evaluated by measuring the turbidity of cultured bacterial broth (spectrophotometer Phoenix 237 Spec BD); the broth turbidity and, as a consequence, the McFarland index increase in the case of bacteria proliferation. For the inhibition halo test, samples were placed in contact with an agar plate (Mueller Hinton agar) uniformly covered with a bacterial broth, previously prepared with a standard procedure, and incubated overnight at 35 °C, as described in [33]. Afterwards the inhibition zone was observed and measured. For CBCs containing 50% of SBAG the inhibition halo was repeated up to 3 days: at the end of the first incubation the samples were removed and re-placed in a new agar plate containing fresh bacterial inoculum. This test was repeated for three days. All antibacterial tests were performed in triplicate.
Fig. 1. SEM image and EDS analyses of SBAG powders.
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Fig. 2. SEM micrographs of Palacos® R50 surface (a) and section (b) and Palacos® R30 surface (c) and section (d).
2.6. Compression test CBCs have been subjected to a simple and preliminary compression test, with the purpose of verifying if the addition of a glass in the
polymeric matrix could negatively affect their cohesion properties. CBCs containing both 30 wt.% and 50% of inorganic phase have been compared with the commercial composition (as control) by means of compressive test in accordance with ASTM D 695-96 standard [34].
Fig. 3. SEM image of SBAG powders (a) and EDS analyses (b) after 28 days of SBF treatment, atomic percentage variation (c) and XRD analyses (d) of glass powders soaked in SBF.
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Fig. 4. XRD analysis of CBCs containing 50 wt.% of glass (Palacos® R50 and Palacos® LV50) after SBF treatment.
Samples for compression test have been cured into an aluminium mould of 90 × 90 × 24 mm with 25 holes of 12 mm in diameter. Then, cured samples were polished with SiC abrasive papers to remove all superficial roughness. Compressive test was performed on five specimens for each CBC composition, using an Instron machine at 20 mm/min crosshead speed; Palacos R® and Palacos LV® were used as sample control. 2.7. Statistical analysis The results of antibacterial and compression tests were expressed as mean ± standard deviation. All results were analyzed by one-way analysis of variance (ANOVA) and Tukey's test: p b 0.05 was considered significant. The Tukey's test was used as post-hoc test in order to individuate significant differences considering that this test presents some limitation for sample number n b 7. 3. Results and discussions 3.1. Glass synthesis and characterization The aim of the work is to impart bioactivity and antibacterial properties to a polymeric bone cement, by the addition of a single inorganic phase with both of these properties: a bioactive glass doped with silver ions. As reported in the Introduction, the presence of silver in the ionic form assures an efficient release of this ion and, in turn, good antibacterial properties. Moreover, the amorphous nature of bioactive glasses is a requirement for a good bioactive behavior. The thermal analyses of SBAG glass (DTA picture not reported) revealed only the signals that were expected from a completely amorphous phase: a transition temperature (at 560 °C), a crystallisation temperature (at 686 °C) and liquidus temperature (at 1217 °C). Any other signal, associated with the eventual presence of different phases, was detected.
The preliminary characterizations of SBAG glass revealed a completely amorphous microstructure (detected by XRD, picture not reported), confirming that silver was introduced in the ionic form: any crystalline phase containing silver and any metallic silver were detected. SEM–EDS analyses (Fig. 1) show silver-containing glass powders with irregular shape, typical of particles obtained by milling of a brittle materials. The powders are sieved below 20 μm, but, as it can be observed, the majority of the glass particles show a grain-size below 5 μm, while only few particles present a grater size (about 10 ÷ 20 μm).
3.2. Composite cements preparation and characterization As previously described, CBCs were synthesized by mechanically mixing the commercial pre-polymerized poly(methyl acrylate, methyl methacrylate) powders with SBAG particles. Afterwards, the composite solid phase was manually mixed with the commercial liquid phase to allow the cement polymerization. From now on, the low viscosity CBCs containing respectively 30% and 50% of SBAG were named Palacos® LV30 and Palacos® LV50, and the high viscosity ones Palacos® R30 and Palacos® R50. SEM–EDS characterizations of CBC generally show a good dispersion of the glass particles into the PMMA matrix (Fig. 2), considering the high amount of glass added to PMMA matrix the CBC surface and section appear homogeneous. As expected, the amount of exposed glass is higher for CBCs containing 50% of glass; this aspect affects the antibacterial and leaching properties of composites, as it will be reported in the following paragraphs. In Fig. 2 a comparison between Palacos® R30 and Palacos® R50 is reported as example. A little tendency to form glass agglomerates can be still observed, mostly in the cements containing 50% of SBAG, due to the high amount of glass phase. This feature must be taken into account in view of a future optimization of the CBC mechanical properties.
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Fig. 5. HAp and precipitates of Ca and P on CBCs surface after in vitro test in SBF solution (28 days).
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Fig. 6. Leaching test: amount of Ag+ released from Palacos® LV30 and Palacos® LV50 samples in SBF solution up to one month.
3.3. In vitro formation of apatite layer The bioactivity test was performed by soaking in SBF both the SBAG powders and the CBCs for period up to one month. The sample bioactivity, in terms of growth of hydroxyapatite on their surface, was estimated by SEM–EDS and XRD analyses. Fig. 3 shows the SBAG glass powder morphology and compositional analyses after 28 days of SBF treatment, as
well as the XRD spectra and atomic percentage variation of glass element during the SBF treatment. The EDS semi-quantitative analysis on the glass surface reveals an increase of Si and a depletion of Na already after one day; moreover an increase of P content was observed after 7 days, while the Ca amount seems to remain unchanged. The XRD analyses confirm the presence of signals that can be related to silica gel (after 7 days of SBF treatment, 2θ 20°–25°) and the precipitation of HAp or its precursors
Fig. 7. Evaluation of the CFU proliferation (a) and adhesion (b) for 30 wt.% SBAG-containing cements (Palacos® R30 and Palacos® LV30).
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Fig. 8. Evaluation of the CFU proliferation (a) and adhesion (b) for 50 wt.% SBAG-containing cements (Palacos® R50 and Palacos® LV50).
(after 14 days); in fact the peaks at 32.04 and 39.67 are characteristic of hydroxyapatite (ref. code 00-001-1008 PCPDF data bank). The nucleation and precipitation of HAp on CBCs are difficult to investigate with XRD analyses, since the commercial cements contain
ZrO2 as radio-opaque agent, which is characterized by a XRD pattern with peaks of high intensities. For this reason the XRD pattern registered on Palacos® LV30 and Palacos® R30 did not show the presence of peaks different from those of ZrO2, either after 28 days of soaking in SBF. The
Fig. 9. McFarland index evaluation for 30 wt.% SBAG-containing cements (a) and 50 wt.% SBAG-containing cements (b).
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fixed time periods with GF-AAS. Fig. 6 reports, as an example, the release trend of low viscosity CBCs; the release trend is similar for both CBCs; as expected Palacos® LV50 releases a greater amount of silver than Palacos® LV30. The high values of the obtained CV are due to the very complex real matrix of the release test: the release of silver ions could be affected in some way by the complex ion exchange connected with glass bioactivity mechanism; moreover the release was performed in a very complex solution (SBF) and the aliquots of the solution were highly diluted according to the analytical sensitivity for the specific element. Finally the obtained Ag values (μg/mm2) were very low, so minimal changes produced SD high values. For both the composites the greatest amount of Ag+ ions were released during the first 3–7 days, which are known as the most critical for infection development after an implant surgery. Subsequently, a slower and continuous silver release was observed. Taking into account that the maintenance of an antibacterial effect for prolonged time is important to prevent latent infections, this trend appears of interest for the foreseen application. Palacos® R composites showed similar release curves and the total amount of released silver ions is of the same order of magnitude (data not reported). These results let one to assess that the silver leaching profile is not affected by the properties (i.e. the viscosity) of the polymeric matrix, and that the amount of leached silver mainly depends on the amount of Ag-doped glass introduced in the composite. 3.5. Antibacterial property evaluation
Fig. 10. S. aureus inhibition halo for Palacos® R30 and Palacos® LV30 (a), for Palacos® R50 (a) and Palacos® LV50 (b) up to 3 days.
XRD patterns registered on Palacos® LV50 and Palacos® R50 showed the appearance of some peaks after 14 days of SBF referable to hydroxyapatite or its precursors (Fig. 4). SEM–EDS analyses showed that after one day of soaking in SBF, few precipitates rich in Ca and P are visible on Palacos® LV50 and Palacos® R50 surfaces, while no precipitates were detected on Palacos® LV30 and Palacos® R30. After 7 days Ca and P precipitates were individuated on both 30% and 50% SBAG containing bone cements. After 14 and 28 days of SBF treatment the amount of precipitates increases with the typical globular morphology of in vitro-grown HAp. At this purpose, Fig. 5 reports some examples of precipitates individuated after 28 days of soaking in SBF for all CBCs. These results confirm that the glass particles were exposed to the cement surface conferring bioactive properties to the CBCs, with kinetics similar to those of the pure glass particles. 3.4. Leaching test The amount and profile of silver release were investigated by dipping samples in SBF and analyzing an aliquot of spilled solution at
In order to investigate the antimicrobial effect of the CBCs, the count of CFU was carried out: this is a quantitative test that allows the quantification of bacterial colonies proliferated around the sample and adhered on its surface. Figs. 7 and 8 show the obtained results for Palacos® LV30, Palacos® R30, Palacos® LV50 and Palacos® R50. For the estimation of CFU proliferation, a bacterial broth and a broth containing commercial cements were used as references, while for the evaluation of adhered CFU on sample surface only a broth containing commercial cements was utilized as control. As far as Palacos® LV30 and Palacos® R30 are concerned (Fig. 7) only a slight decrease of CFU proliferated in the broth containing CBCs (especially for Palacos® LV30) was detected. From the bacterial adhesion evaluation test it seems that CBCs containing 30% of SBAG seem not able to limit the bacteria adhesion (see Fig. 7b). The Palacos® LV50 and Palacos® R50 were able to reduce the CFU proliferation of about 1–1.5 magnitude order respect control broth and commercial cement containing broth (Fig. 8a); moreover a slight reduction of bacteria adhesion was also observed for both low and high viscosity cements (Fig. 8b). Statistical analysis (one-way analysis of variance and Tukey's test, p b 0.05) evidenced a significant difference on the CFU proliferation for both CBC compositions. The McFarland index evaluation (Fig. 9) confirmed this behavior, in fact a slight decrease of McFarland index, and of the bacteria proliferation, was observed for CBCs, in particular for Palacos® LV50 and Palacos® R50. In order to better investigate the antibacterial effect of the CBCs, the inhibition halo test was also performed: a discontinuous halo (b1 mm) is observed for CBCs containing 30% of SBAG, particularly for Palacos® LV30 (Fig. 10a). Fig. 10b reports the inhibition halo test performed for Palacos® LV50 and Palacos® R50 up to 3 days. As can be observed, both high and low viscosity cements produce a significant inhibition zone, of about 2 mm, after one day in contact with agar substrate. For this reason the test was prolonged up to 3 days. The halo decreases gradually during the subsequent days: at the end of the second day the inhibition zone measures about 1–1.5 mm and, at the third day, a discontinuous halo less than 1 mm can be detected. These results are considered of clinical interest by the Standard “SNV 195920-1992”
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Fig. 11. Compressive strength of high (a — Palacos® R30 and Palacos® R50) and low viscosity (b — Palacos® LV30 and Palacos® LV50) bone cements.
[35,36], that relates the dimension of the inhibition halo with the entity of the antibacterial capability. According to this standard an inhibition zone N 1 mm is considered good for antibacterial activity, while an inhibition zone b 1 mm is considered fairly good. Then, the results of the inhibition halo test suggest that also Palacos® LV30 and Palacos® R30 possess the ability to limit the bacterial contamination. The different antibacterial behavior between 30% SBAG and 50% SBAG containing cements is due to the different amount of glass incorporated in the polymer matrix and, as a consequence, to the silver incorporation and release. In fact, Palacos® LV50 releases about a double amount of silver respect Palacos® LV30 (Fig. 6). On the contrary, any significant difference can be noticed between the halo dimension of low and high viscosity CBCs (results are in agreement with the leaching test). The water drops condensed on the samples, visible in Fig. 10, normally are present when it is impossible the overturn the agar plate (for the possible samples fall) during the incubation, but do not interferes with the results of the test.
3.6. Compression test The compressive tests were performed, in accordance to ASTM D 695-96 on five cylindrical specimens for each cement. Even if the ISO 5833 is the rigorous standard to evaluate the mechanical properties of acrylic cements, this test was performed to preliminarily compare the mechanical performances of the CBC, containing different amounts of brittle phase (i.e. the SBAG glass), with the plain bone cements. As far as the high viscosity cements are concerned (Fig. 11a) a slight decrease of compression strength was detected for Palacos® R30 with respect to the commercial cement, while a significant reduction was observed for Palacos® R50 (one-way analysis of variance and Tukey's test, p b 0.05). A similar result was obtained for low viscosity cements (Fig. 11b): in this case any difference was detected between commercial cements and
Palacos® LV30, whereas a significant compression resistance decrease was always observed for Palacos® LV50 (one way ANOVA, p b 0 · 05). Then, from the mechanical point of view, it can be asserted that the introduction of 50 wt.% of glass in the PMMA matrix compromises the compression strength of the cements, while the synthesis of composites containing 30% of glass does not. This preliminary experimental evidence let us asses that a further optimization of the CBC is needed, in terms of glass particle amount and distribution, before planning a complete mechanical characterization (including flexural strength evaluation, fatigue test and bending modulus) using the appropriate standards for acrylic bone cements.
4. Conclusions This work explored the innovative idea to impart bioactive and antibacterial properties to PMMA-based bone cements by adding a single inorganic phase composed by silver containing bioactive glass powders (SBAG). CBCs were prepared by introducing 30% and 50% by weight of SBAG in the polymer matrix of two commercial cements with different viscosity: Palacos® LV (low) and Palacos® R (high). All CBC formulations showed a good dispersion of the glass inside the PMMA matrix, with a few tendency to glass particle agglomeration in the formulation with 50% of glass phase. All the formulations revealed a bioactive behavior. The silver ions were released with similar profile both for 30% and 50% SBAG containing cements, but, as expected, in different amounts. Palacos® LV50 and Palacos® R50 demonstrate a good ability of bacterial contamination inhibition, both in terms of adhesion and proliferation, whereas a slightly lower effect was observed for Palacos® LV30 and Palacos® R30. From the mechanical point of view, Palacos® LV30 and Palacos® R30 showed good compression strength, comparable with the commercial cement reference, while a decrease of mechanical strength was observed for Palacos® LV50 and Palacos® R50.
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Taking into account these results, in this work a good compromise between bioactivity, antibacterial effect and preserved compression strength was achieved only with the formulations containing 30% of bioactive and antibacterial glass (Palacos® LV30 and Palacos® R30). A further characterization in terms of biocompatibility and mechanical properties in accordance with ISO 5833 (bending strength, fatigue test, bending modulus) is needed and will be focused on the CBC formulations containing 30% glass powders. An optimization of the CBC properties will be also explored by modifying the glass powder size distribution, amount and silver content, in order to achieve the best antibacterial effect, while maintaining bioactivity and the required mechanical properties, also in other polymeric bone cements of different formulations. Acknowledgment The authors would like to acknowledge Prof. Enrico Prenesti (Università degli Studi di Torino, Department of Chemistry) for the helpful discussion. References [1] M. Diefenbeck, T. Mückley, G.O. Hofmann, Prophylaxis and treatment of implantrelated infections by local application of antibiotics. Injury, Int. J. Care Injured 37 (2006) S95–S104. [2] I. Uçkay, P. Hoffmeyer, D. Lew, D. Pittet, Prevention of surgical site infections in orthopaedic surgery and bone trauma: state-of-the-art update review article, J. Hosp. Infect. 84 (1) (2013) 5–12. [3] S.M. Kurtz, K.L. Ong, J. Schmier, F. Mowat, K. Saleh, E. Dybvik, J. Kärrholm, G. Garellick, L.I. Havelin, O. Furnes, H. Malchau, E. Lau, Future clinical and economic impact of revision total hip and knee arthroplasty, J. Bone Joint Surg. Am. 89 (Suppl. 3) (2007) 144–151. [4] J.E. Phillips, T.P. Crane, M. Noy, T.S. Elliott, R.J. Grimer, The incidence of deep prosthetic infections in a specialist orthopaedic hospital: a 15-year prospective survey, J. Bone Joint Surg. (Br.) 88 (7) (2006) 943–948. [5] F. Anagnostou, A. Debet, G. Pavon-Djavid, Z. Goudaby, G. Hélary, V. Migonne, Osteoblast functions on functionalized PMMA-based polymers exhibiting Staphylococcus aureus adhesion inhibition, Biomaterials 27 (2006) 3912–3919. [6] W.F. Mousa, M. Kobayashi, S. Shinzato, M. Kamimura, M. Neo, S. Yoshihara, T. Nakamura, Biological and mechanical properties of PMMA-based bioactive bone cements, Biomaterials 21 (2000) 2137–2146. [7] W. Hennig, B.A. Blencke, H. BroKmer, K.K. Deutscher, A. Gross, W. Ege, Investigations with bioactivated polymethylmethacrylate, J. Biomed. Mater. Res. 13 (1979) 89–99. [8] K. Serbetci, F. Korkusuz, N. Hasirci, Thermal and mechanical properties of hydroxyapatite impregnated acrylic bone cements, Polym. Test. 23 (2004) 145–155. [9] J.T. Heikkila, A.J. Aho, I. Kangasniemi, A. Yli-Urpo, Polymethylmethacrylate composites: disturbed bone formation at the surface of bioactive glass and hydroxyapatite, Biomaterials 17 (1996) 1755–1760. [10] W. Matthew, M.D. Squire, J. Brian, M.D. Ludwig, R. Jonathan, B.S. Thompson, B.S.J. Jagodzinski, B.S.D. Hall, M.D.D. Andes, Premixed antibiotic bone cement an in vitro comparison of antimicrobial efficacy, J. Arthroplast. 23 (6) (2008) 110–114. [11] A. Bistolfi, G. Massazza, E. Verné, A. Massè, D. Deledda, S. Ferraris, M. Miola, F. Galetto, M. Crova1, Antibiotic-loaded cement in orthopedic surgery: a review, ISRN Orthod. 2011 (2011) 1–8. [12] E. Bertazzoni Minelli, T. Della Bora, A. Benini, Different microbial biofilm formation on polymethylmethacrylate (PMMA) bone cement loaded with gentamicin and vancomycin, Anaerobe 17 (6) (2011) 380–383.
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[13] http://www.tecres.it/products_dett.php?lang=en&cat=ortopedia&id_cat=29&id_ prod=48 . [14] B. Thornes, P. Murray, D. Bouchier-Hayes, Development of resistant strains of Staphylococcus epidermidis on gentamicin-loaded bone cement in vivo, J. Bone Joint Surg. (Br.) 84-B (2002) 758–760. [15] D. Campoccia, L. Montanaro, P. Speziale, C.R. Arciola, Antibiotic-loaded biomaterials and the risks for the spread of antibiotic resistance following their prophylactic and therapeutic clinical use, Biomaterials 31 (2010) 6363–6377. [16] V. Alt, T. Bechert, P. Steinrucke, M. Wagener, P. Seidel, E. Dingeldein, E. Domann, R. Schnettler, An in vitro assessment of the antibacterial properties and cytotoxicity of nanoparticulate silver bone cement, Biomaterials 25 (2004) 4383–4391. [17] P. Prokopovich, R. Leech, C.J. Carmalt, I.P. Parkin, S. Perni, A novel bone cement impregnated with silver–tiopronin nanoparticles: its antimicrobial, cytotoxic, and mechanical properties, Int. J. Nanomedicine 8 (2013) 2227–2237. [18] Q.L. Feng, J. Wu, G.Q. Chen, F.Z. Cui, T.N. Kim, J.O. Kim, A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus, J. Biomed. Mater. Res. 52 (4) (2000) 662–668. [19] J.S. Kim, E. Kuk, K.N. Yu, J.H. Kim, S.J. Park, H.J. Lee, S.H. Kim, Y.K. Park, Y.H. Park, C.Y. Hwang, Y.K. Kim, Y.S. Lee, D.H. Jeong, M.H. Cho, Antimicrobial effects of silver nanoparticles, Nanomedicine 3 (1) (2007) 95–101. [20] D.J. Moojen, H.C. Vogely, A. Fleer, A.J. Verbout, R.M. Castelein, W.J. Dhert, No efficacy of silver bone cement in the prevention of methicillin-sensitive Staphylococcal infections in a rabbit contaminated implant bed model, J. Orthop. Res. 27 (8) (2009) 1002–1007. [21] D. Arcos, C.V. Ragel, M. Vallet-Regi, Bioactivity in glass/PMMA composites used as drug delivery system, Biomaterials 22 (2001) 701–708. [22] S. Cavalu, V. Simon, G. Goller, I. Akin, Bioactivity and antimicrobial properties of PMMA/Ag2O acrylic bone cement collagen coated, Dig. J. Nanomater. Biostruct. 6 (2) (2011) 779–790. [23] L.C. Gerhardt, A.R. Boccaccini, Bioact. Glas. Glas-Ceram. Scaffolds Bone Tissue Eng. Mater. 3 (2010) 3867–3910. [24] L.L. Hench, Bioceram. J. Am. Ceram. Soc. 81 (1993) 705–1728. [25] W. Cao, L.L. Hench, Bioactive materials, Ceram. Int. 22 (1996) 493–507. [26] A. Hoppe, N.S. Güldal, A.R. Boccaccini, A review of the biological response to ionic dissolution products from bioactive glasses and glass–ceramics, Biomaterials 32 (2011) 2757–2774. [27] M. Catauro, M.G. Raucci, F. de Gaetano, A. Marotta, Antibacterial and bioactive silvercontaining Na2O·CaO·2SiO2 glass prepared by sol–gel method, J. Mater. Sci. Mater. Med. 15 (2004) 831–837. [28] E. Verne', M. Miola, C. Vitale-Brovarone, M. Cannas, S. Gatti, G. Fucale, G. Maina, A. Masse', S. Di Nunzio, Surface silver-doping of biocompatible glass to induce antibacterial properties. Part I: massive glass, J. Mater. Sci. Mater. Med. 20 (2009) 733–740. [29] M. Miola, S. Ferraris, S. Di Nunzio, P.F. Robotti, G. Bianchi, G. Fucale, G. Maina, M. Cannas, S. Gatti, A. Masse', C. Vitale-Brovarone, E. Verne', Surface silver-doping of biocompatible glasses to induce antibacterial properties. Part II: plasma sprayed glass-coatings, J. Mater. Sci. Mater. Med. 20 (3) (2009) 741–749. [30] T. Kokubo, H. Takadama, How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 27 (2006) 2907–2915. [31] Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, Approved Standard M7-A6, 6th edn NCCLS, Villanova, PA, USA, 2003. [32] Performance standards for antimicrobial disk susceptibility tests, approved standard M2-A9, 9th ed. NCCLS, Villanova, PA, USA, 2003. [33] C. Vitale-Brovarone, M. Miola, C. Balagna, E. Vernè, 3D-glass–ceramic scaffolds with antibacterial properties for bone grafting, Chem. Eng. J. 137 (2008) 129–136. [34] ASTM D 695–96 “Standard Test Method for Compressive Properties of Rigid Plastics”, 1996. [35] M. Pollini, F. Paladini, M. Catalano, A. Taurino, A. Licciulli, A. Maffezzoli, A. Sannino, Antibacterial coatings on haemodialysis catheters by photochemical deposition of silver nanoparticles, J. Mater. Sci. Mater. Med. 22 (2011) 2005–2012. [36] S-14-63 SN 195920:1992 Textile Flächengebilde: Prüfung der antibakteriellen Wirkung; Agar diffusions test, 1992.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Antibiotic-free composite bone cements with antibacterial and bioactive properties. A preliminary study Marta Miola a,⁎,1, Matteo Bruno a, Giovanni Maina c, Giacomo Fucale b, Giovanni Lucchetta d, Enrica Vernè a a
Applied Science and Technology Department, Politecnico di Torino, Italy Chemical, Clinical and Microbiological Analyses Dept., CTO, Turin, Italy Department of Clinical and Biological Sciences, University of Turin, Turin, Italy d Department of Innovation in Mechanics and Management, University of Padova, Padova, Italy b c
a r t i c l e
i n f o
Article history: Received 30 September 2013 Received in revised form 21 May 2014 Accepted 30 June 2014 Available online 8 July 2014 Keywords: Antibacterial Bioactive glass Bone cement Composite Antibiotic-free
a b s t r a c t Two bone cements (Palacos R® and Palacos LV®) based on polymethylmethacrylate (PMMA), clinically used in several cemented prosthetic devices, have been enriched with silver containing bioactive glass powders and compared with the plain commercial ones. The obtained composite cements have been subjected to a preliminary characterization by means of morphological and compositional analyses, compression mechanical tests, bioactivity test (by soaking into simulated body fluids), leaching tests and in vitro antibacterial test (count of colonies forming units, McFarland index evaluation, inhibition zone evaluation). The glass powders appeared uniformly dispersed inside the PMMA matrix and good mechanical properties (in compression) have been reached. The composite cements showed a bioactive behavior (since they developed hydroxyapatite on their surface after soaking in simulated body fluid) and a good antibacterial performance. The release of silver ions, which is the principal reason of antibacterial properties, is mainly reached after the first hours of contact with the leaching solution, as it is expected for a reasonable prevention of bacterial colonization during in vivo applications. © 2014 Published by Elsevier B.V.
1. Introduction Post-surgical infections are still one of the most frequent adverse events in the prosthetic surgery. They are often caused by the adhesion of bacteria on the implant surfaces, which is responsible of an invasive inflammatory process along the bone/implant interface. This situation, followed by bacteria proliferation, is enhanced by the formation of a biofilm, a three-dimensional self-produced structure that protects bacteria against patient own defense system and from systemic antibiotic therapy [1]. It is estimated that infection rates in orthopedic surgery is about 1% (for primary hip and knee arthroplasties) while in trauma surgery they can reach 12–53% for open fractures (open fractures Gustilo grade III) [2]. Most of the infections often cause implant failure [3,4] and, in turn, the need for a revision surgery, which causes prolonged hospitalization and other severe complications [5]. Aiming to avoid bacterial adhesion and to extend the longevity of bone
⁎ Corresponding author at: Politecnico di Torino, Applied Science and Technology Department, DISAT, Corso Duca degli Abruzzi 24, 10129 Torino, Italy. Tel.: + 39 0110904717; fax: +39 0110904624. E-mail address: [email protected] (M. Miola). 1 Present affiliation: Department of Health Sciences, Università del Piemonte Orientale “A. Avogadro”, Novara, Italy.
http://dx.doi.org/10.1016/j.msec.2014.06.026 0928-4931/© 2014 Published by Elsevier B.V.
implants, different prevention solutions, like strict antiseptic operative procedures (i.e. laminar flow and systemic antibiotic prophylaxis) have been proposed. However, even if the infection development rate has been significantly reduced, the problem of septic loosening in joint arthroplasty is still open. For this reason, beside antiseptic procedures, several surgical treatments have been proposed to reduce bacterial contamination of implant surfaces and different strategies have been investigated in biomaterial formulations. A simply way to reduce the bacterial adhesion on the implant surface is to favor the direct bonding between the prosthesis and the bone, by a selective adhesion and proliferation of osteogenic cells, rather than bacteria, promoting a fast osteointegration, without the formation of soft tissue layers. Although PMMA bone cements are known to assure a fast primary fixation, mainly by a mechanical anchoring to the bone trabeculae, it is established that the bone/cement interface is a weaklink zone in the prosthesis/bone interface, because it does not adhere actively to the bone [6]. The improvement of bone-bonding ability of PMMA bone cement by using different kinds of bioactive fillers (e.g., Ceravital® [7], hydroxyapatite [8] and Bioglass® [9]) is discussed in the literature; however, is still not demonstrated that a fast bone healing means the absolute absence of bacterial adhesion to the implant surface. For this reason, more specific formulations have been investigated, aiming to impart bacteriostatic or antibacterial properties to different bone cements. Antibiotic loaded PMMA-based bone cements
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have been widely used since '70. During the last decades several formulations have been compared, like antibiotic loaded cements with different antibiotics (tobramycin, vancomycin, cephazolin) [10,11] industrially or manually added to the polymeric matrix and bone cements loaded with a combination of two antibiotics [11–13]. However, the principal drawbacks of these formulations can be individuated as follows: i) the large size of antibiotic molecules leads to a release, which is only a surface phenomenon; ii) commonly the highest release is observed during the few first hours, that means, in turn, a long term low concentration around the implant (under the antibiotic MIC) with a subsequent high risk of antibiotic-resistant strain development [1,14,15]. For the second reason, antibiotic-loaded bone cements are mainly indicated for revision surgery, rather than for prophylaxis, since their local action is not indicated for infection prevention, but for its treatment, if prevention failed. The increasing percentage of total joint infections with multiresistant bacteria stimulated the scientific community on the research of alternative strategies to give antibacterial properties to these materials, without the use of antibiotics, to be used as a prevention approach, more efficient of both plain bone cements and antibiotic loaded ones. At this purposes, bone cements loaded with Ag nanoparticles have been recently described in the literature [16,17]; the Ag nanoparticle utilization is proposed to prevent infections caused by multiresistant bacteria, taking into account the well-known broad antimicrobial effect of silver [18,19], however, the antibacterial properties of nanosilver in these formulations are not yet completely proven and the different efficacy of silver ions vs metallic silver is still debated. Moojen et al. [20] state that metallic Ag nanoparticles contained in the cement are not released and need to be ionized before they exhibit their antimicrobial activity. Therefore, the cement predominantly exhibits an antimicrobial effect at the direct cement surface and not in the surrounding bone tissue; this provides the possibility of bacteria to replicate several times before the silver starts to interfere with its proliferation. Aiming to improve the implant longevity by means of both fast osteointegration and low bacterial adhesion, a new approach has been reported by some authors [21,22] by using both antibacterial and bioactive fillers in the same bone cement. This aim seems to be reached, but with quite complex solutions, which imply the addition of two different solid phases into the polymeric matrix (one to impart bioactivity and the second one to impart antibacterial properties) with some concerns on the mechanical properties of the final cement as well as on its industrial and clinical feasibility. Moreover, in several cases this approach still used antibiotics as antibacterial phase, without avoiding the risk of resistant bacterial strain development and still maintaining the incomplete efficacy of drug release. In this work, the synthesis and characterization of composite bone cement, based on a PMMA matrix containing a single additional solid phase with both bioactive and antibacterial properties are proposed for the first time. The additional phase is a bioactive glass, containing silver ions, so the same filler shows at the same time the ability of promoting bone ingrowth and a bacteriostatic effect. The presence of silver ions instead of metallic Ag nanoparticles implies an immediate availability of Ag ions, which can be released and limit not only the bacterial adhesion, but also their proliferation in the surrounding tissue. Bioactive glasses and glass–ceramics are widely studied and developed in several commercial formulations [23] for bone restoration. The term “bioactive” refers to their ability of producing a chemical bond to the living bone by a well-known mechanism, which involves ion leaching from their amorphous network in the surrounding biological fluids and the precipitation of hydroxyapatite on their surfaces that is considered a sufficient requirement to bond with the living bone [24, 25]. They have been also described as osteoinductive materials, since they are able to elicit both intracellular and extracellular responses, promoting osteogenic stem cell colonization, by the release of specific ions (Si4+, Mg2+, Ca2+). The “genetic design” of bioactive glasses has become one of the most challenging fields of bone tissue engineering
[26] doping the glass with a variety of active ions (Sr, Cu, Fe, B, Zn, etc.), useful both for stimulating bone regeneration and for therapeutic purposes. In this contest, silver containing bioactive glasses and glass–ceramics have been produced in different ways [27–29] and their biocompatibility with osteoblast-like cells as well as their antibacterial properties have been reported to demonstrate both their efficacy and safety. The formulation described in this work has been specifically developed to be used as filler for composite bone cement, and represents a preliminary solution for a new family of antibiotic-free, osteoinductive and antibacterial bone cements, which could be proposed as a valid alternative to the existing ones, with potential indications mainly for prophylaxis of implant-related infections. 2. Materials and methods 2.1. Glass synthesis and characterization The glass synthesized in this research work belongs to the SiO2– Na2O–CaO–P2O5–B2O3–Al2O3–Ag2O system. The glass, named SBAG from now on, was produced by melting the reactants in a Pt crucible at 1450 °C and by quenching the melt in water to obtain a frit that was subsequently milled and sieved to achieve a glass powders with a grain size below 20 μm. This grain size was selected after preliminary experimental test, which evidenced good handling properties. The glass powders were subjected to the differential thermal analysis (DTA 404 PC NETZSCH) with a heating rate of 10 °C/min from 25 °C to 1300 °C to individuate the glass characteristic temperatures and to check if any further phase was present within the amorphous one. Moreover, SBAG powders were analyzed by scanning electron microscopy (SEM — FEI, QUANTA INSPECT 200), and energy dispersion spectrometry (EDS — EDAX PV 9900) to evaluate the powders morphology and composition. Finally, X-ray diffraction (XRD — X'Pert Philips diffractometer) was performed in order to assess the amorphous nature of the obtained powders, using the Bragg Brentano camera geometry and CuKα incident radiation. Diffraction patterns were detected by using a step of 0.02° (2θ) with a fixed counting time of 1 s per step from 10° to 70° (2θ); the pattern analysis was carried out using X'Pert High Score software and PCPDF data bank. 2.2. Composite cements preparation and characterization Composite bone cements (named CBCs from now on) were prepared using two commercial PMMA-based cements with different viscosity (purchased by Heraeus Medical): Palacos® R (high viscosity) and Palacos® LV (low viscosity). Palacos® has been selected over different commercial formulations because it is one of the most commonly used PMMA-based bone cements. Since the addition of the bioactive and antibacterial glass-phase (SBAG powders) does not imply any modification into the polymerization mechanism of the organic matrix, it can be ideally extended to any other commercial or experimental formulation, taking into account their different component ratios and rheology and properly modifying the amount and grain size distribution of the glass powders. So this work does not suggest any improvement of the commercial polymeric phase, but it is only intended to investigate the role of the new inorganic additional phase (SBAG) on the final properties of common PMMA-based bone cement. As most of the commercial formulations, Palacos® (LV and R) is sold in a kit with one sachet of a solid phase and one ampoule of liquid. The solid phase has a complex formulation and, for Palacos®, contains powders of the co-polymer poly(methyl acrylate, methyl methacrylate), zirconium dioxide as radio-opaque agent and benzoyl peroxide as initiator (as reported on the data sheets). The liquid phase contains the monomer methyl methacrylate (MMA), N,N-dimethyl-toluidine as activator and hydroquinone as inhibitor. Chlorophyll is present both in the liquid and in the solid phase to impart green color to the cement.
M. Miola et al. / Materials Science and Engineering C 43 (2014) 65–75 Table 1 Component's proportion of CBCs.
Palacos® LV30 Palacos® R30 Palacos® LV50 Palacos® R50
Glass (wt.%)
Solid phase (wt.%)
Liquid phase/solid phase + glass ratio
30
70
0.5
50
50
0.5
The SBAG glass was added to the solid phase in two different proportions: 30% and 50% by weight. The amounts of glass powders incorporated in the PMMA have been selected based on the literature data and preliminary experimental tests. In our experience, an amount higher than 50% by weight does not allow a good handling of the cements during curing. The powders were mechanically mixed for about half hour, in order to assure a good dispersion of glass particles in the PMMA pre-polymerized particles. Subsequently the mixed solid phases were manually mixed with the liquid phase (containing the monomer MMA) under a laminar flow cabinet, maintaining the same liquid/solid phase ratio of commercial bone cements. The mixture was stirred for about 30 s and, as soon as the mass did not stick to the rubber gloves of the operators, it was transferred into a polished aluminium mold (100 × 100 × 5 mm with 25 holes of 10 mm in diameter or 90 × 90 × 24 mm with 25 holes of 12 mm in diameter). The obtained cements were analyzed by SEM–EDS analyses in order to verify the morphology and the glass distribution in the polymerized matrix. Table 1 resumes the component's proportion of CBCs. 2.3. In vitro formation of apatite layer The SBAG powders and the CBCs were soaked in 25 mL of simulated body fluid (SBF — Kokubo [30]) for period up to 1 month in order to verify the sample bioactivity. The SBF volume was selected on the basis of Kokubo protocol using a volume (V)/surface (S) ratio of about: 2 VðmLÞ ¼ S mm =10: The test was performed in triplicate. Every 3 days a refresh of SBF solution was carried out to mimic the renewal of physiological fluid and pH measurements were carried out during the refresh process. Afterwards the samples were analyzed by means of SEM–EDS and XRD analyses. 2.4. Leaching test To evaluate the silver release, the CBCs were soaked in 30 mL of SBF at 37 °C for 28 days. At fixed time periods (3 h, 1, 3, 7, 14, 28 days) 1 mL
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of solution was picked out and analyzed with a graphite furnace atomic absorption spectroscopy (GF-AAS) [PERKIN ELMER (mod. 4100 ZL)] employing a Zeeman-effect background corrector with an autosampler (mod. AS/71), programmed to dispense 20 μL of the sample. Two matrix modifiers have been employed in order to obtain a higher absorption signal: Pd(NO3)2 and Mg(NO3)2 have been directly added on the graphite tube by means of the auto-sampler. Standard solutions were daily prepared from AG MERCK standard solution traceable to SRM from NIST to calibrate the instrument. The calibration standard solutions were 25.0 and 50.0 μg/L. The limit of detection was 0.2 μg/L (CV 5.3%). The picked solution was not replaced by fresh SBF and the obtained result was calculated considering the volume decrease of solution and the sample surface. The test was performed in triplicate. 2.5. Antibacterial property evaluation The antibacterial properties of CBCs were evaluated through three different antimicrobial tests: the McFarland index estimation, the count of colonies forming unit (CFU) [31] and the inhibition halo evaluation [32], using a Staphylococcus aureus standard stock (ATCC 29213). The McFarland index and the CFU count were performed by dipping both composites and commercial cements as control in a standard bacterial broth containing approximately 5 · 105 CFU/mL and incubated 24 h at 35 °C. Subsequently the samples were removed from the broth, gently rinsed in a physiological solution (0.9% P/V of NaCl in distilled water) and vortexed 1 min at 50 Hz, always in the physiological solution, to detach the bacteria colonies adhered on sample surface. In order to quantify the bacteria proliferated in the culture broth and their adhesion on the cement surface, both the culture broth and the vortexing solutions were serially diluted and spread on agar plate, which were incubated overnight at 35 °C to allow the growth and the following count of CFU. Moreover the McFarland index was evaluated by measuring the turbidity of cultured bacterial broth (spectrophotometer Phoenix 237 Spec BD); the broth turbidity and, as a consequence, the McFarland index increase in the case of bacteria proliferation. For the inhibition halo test, samples were placed in contact with an agar plate (Mueller Hinton agar) uniformly covered with a bacterial broth, previously prepared with a standard procedure, and incubated overnight at 35 °C, as described in [33]. Afterwards the inhibition zone was observed and measured. For CBCs containing 50% of SBAG the inhibition halo was repeated up to 3 days: at the end of the first incubation the samples were removed and re-placed in a new agar plate containing fresh bacterial inoculum. This test was repeated for three days. All antibacterial tests were performed in triplicate.
Fig. 1. SEM image and EDS analyses of SBAG powders.
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Fig. 2. SEM micrographs of Palacos® R50 surface (a) and section (b) and Palacos® R30 surface (c) and section (d).
2.6. Compression test CBCs have been subjected to a simple and preliminary compression test, with the purpose of verifying if the addition of a glass in the
polymeric matrix could negatively affect their cohesion properties. CBCs containing both 30 wt.% and 50% of inorganic phase have been compared with the commercial composition (as control) by means of compressive test in accordance with ASTM D 695-96 standard [34].
Fig. 3. SEM image of SBAG powders (a) and EDS analyses (b) after 28 days of SBF treatment, atomic percentage variation (c) and XRD analyses (d) of glass powders soaked in SBF.
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Fig. 4. XRD analysis of CBCs containing 50 wt.% of glass (Palacos® R50 and Palacos® LV50) after SBF treatment.
Samples for compression test have been cured into an aluminium mould of 90 × 90 × 24 mm with 25 holes of 12 mm in diameter. Then, cured samples were polished with SiC abrasive papers to remove all superficial roughness. Compressive test was performed on five specimens for each CBC composition, using an Instron machine at 20 mm/min crosshead speed; Palacos R® and Palacos LV® were used as sample control. 2.7. Statistical analysis The results of antibacterial and compression tests were expressed as mean ± standard deviation. All results were analyzed by one-way analysis of variance (ANOVA) and Tukey's test: p b 0.05 was considered significant. The Tukey's test was used as post-hoc test in order to individuate significant differences considering that this test presents some limitation for sample number n b 7. 3. Results and discussions 3.1. Glass synthesis and characterization The aim of the work is to impart bioactivity and antibacterial properties to a polymeric bone cement, by the addition of a single inorganic phase with both of these properties: a bioactive glass doped with silver ions. As reported in the Introduction, the presence of silver in the ionic form assures an efficient release of this ion and, in turn, good antibacterial properties. Moreover, the amorphous nature of bioactive glasses is a requirement for a good bioactive behavior. The thermal analyses of SBAG glass (DTA picture not reported) revealed only the signals that were expected from a completely amorphous phase: a transition temperature (at 560 °C), a crystallisation temperature (at 686 °C) and liquidus temperature (at 1217 °C). Any other signal, associated with the eventual presence of different phases, was detected.
The preliminary characterizations of SBAG glass revealed a completely amorphous microstructure (detected by XRD, picture not reported), confirming that silver was introduced in the ionic form: any crystalline phase containing silver and any metallic silver were detected. SEM–EDS analyses (Fig. 1) show silver-containing glass powders with irregular shape, typical of particles obtained by milling of a brittle materials. The powders are sieved below 20 μm, but, as it can be observed, the majority of the glass particles show a grain-size below 5 μm, while only few particles present a grater size (about 10 ÷ 20 μm).
3.2. Composite cements preparation and characterization As previously described, CBCs were synthesized by mechanically mixing the commercial pre-polymerized poly(methyl acrylate, methyl methacrylate) powders with SBAG particles. Afterwards, the composite solid phase was manually mixed with the commercial liquid phase to allow the cement polymerization. From now on, the low viscosity CBCs containing respectively 30% and 50% of SBAG were named Palacos® LV30 and Palacos® LV50, and the high viscosity ones Palacos® R30 and Palacos® R50. SEM–EDS characterizations of CBC generally show a good dispersion of the glass particles into the PMMA matrix (Fig. 2), considering the high amount of glass added to PMMA matrix the CBC surface and section appear homogeneous. As expected, the amount of exposed glass is higher for CBCs containing 50% of glass; this aspect affects the antibacterial and leaching properties of composites, as it will be reported in the following paragraphs. In Fig. 2 a comparison between Palacos® R30 and Palacos® R50 is reported as example. A little tendency to form glass agglomerates can be still observed, mostly in the cements containing 50% of SBAG, due to the high amount of glass phase. This feature must be taken into account in view of a future optimization of the CBC mechanical properties.
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Fig. 5. HAp and precipitates of Ca and P on CBCs surface after in vitro test in SBF solution (28 days).
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Fig. 6. Leaching test: amount of Ag+ released from Palacos® LV30 and Palacos® LV50 samples in SBF solution up to one month.
3.3. In vitro formation of apatite layer The bioactivity test was performed by soaking in SBF both the SBAG powders and the CBCs for period up to one month. The sample bioactivity, in terms of growth of hydroxyapatite on their surface, was estimated by SEM–EDS and XRD analyses. Fig. 3 shows the SBAG glass powder morphology and compositional analyses after 28 days of SBF treatment, as
well as the XRD spectra and atomic percentage variation of glass element during the SBF treatment. The EDS semi-quantitative analysis on the glass surface reveals an increase of Si and a depletion of Na already after one day; moreover an increase of P content was observed after 7 days, while the Ca amount seems to remain unchanged. The XRD analyses confirm the presence of signals that can be related to silica gel (after 7 days of SBF treatment, 2θ 20°–25°) and the precipitation of HAp or its precursors
Fig. 7. Evaluation of the CFU proliferation (a) and adhesion (b) for 30 wt.% SBAG-containing cements (Palacos® R30 and Palacos® LV30).
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Fig. 8. Evaluation of the CFU proliferation (a) and adhesion (b) for 50 wt.% SBAG-containing cements (Palacos® R50 and Palacos® LV50).
(after 14 days); in fact the peaks at 32.04 and 39.67 are characteristic of hydroxyapatite (ref. code 00-001-1008 PCPDF data bank). The nucleation and precipitation of HAp on CBCs are difficult to investigate with XRD analyses, since the commercial cements contain
ZrO2 as radio-opaque agent, which is characterized by a XRD pattern with peaks of high intensities. For this reason the XRD pattern registered on Palacos® LV30 and Palacos® R30 did not show the presence of peaks different from those of ZrO2, either after 28 days of soaking in SBF. The
Fig. 9. McFarland index evaluation for 30 wt.% SBAG-containing cements (a) and 50 wt.% SBAG-containing cements (b).
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fixed time periods with GF-AAS. Fig. 6 reports, as an example, the release trend of low viscosity CBCs; the release trend is similar for both CBCs; as expected Palacos® LV50 releases a greater amount of silver than Palacos® LV30. The high values of the obtained CV are due to the very complex real matrix of the release test: the release of silver ions could be affected in some way by the complex ion exchange connected with glass bioactivity mechanism; moreover the release was performed in a very complex solution (SBF) and the aliquots of the solution were highly diluted according to the analytical sensitivity for the specific element. Finally the obtained Ag values (μg/mm2) were very low, so minimal changes produced SD high values. For both the composites the greatest amount of Ag+ ions were released during the first 3–7 days, which are known as the most critical for infection development after an implant surgery. Subsequently, a slower and continuous silver release was observed. Taking into account that the maintenance of an antibacterial effect for prolonged time is important to prevent latent infections, this trend appears of interest for the foreseen application. Palacos® R composites showed similar release curves and the total amount of released silver ions is of the same order of magnitude (data not reported). These results let one to assess that the silver leaching profile is not affected by the properties (i.e. the viscosity) of the polymeric matrix, and that the amount of leached silver mainly depends on the amount of Ag-doped glass introduced in the composite. 3.5. Antibacterial property evaluation
Fig. 10. S. aureus inhibition halo for Palacos® R30 and Palacos® LV30 (a), for Palacos® R50 (a) and Palacos® LV50 (b) up to 3 days.
XRD patterns registered on Palacos® LV50 and Palacos® R50 showed the appearance of some peaks after 14 days of SBF referable to hydroxyapatite or its precursors (Fig. 4). SEM–EDS analyses showed that after one day of soaking in SBF, few precipitates rich in Ca and P are visible on Palacos® LV50 and Palacos® R50 surfaces, while no precipitates were detected on Palacos® LV30 and Palacos® R30. After 7 days Ca and P precipitates were individuated on both 30% and 50% SBAG containing bone cements. After 14 and 28 days of SBF treatment the amount of precipitates increases with the typical globular morphology of in vitro-grown HAp. At this purpose, Fig. 5 reports some examples of precipitates individuated after 28 days of soaking in SBF for all CBCs. These results confirm that the glass particles were exposed to the cement surface conferring bioactive properties to the CBCs, with kinetics similar to those of the pure glass particles. 3.4. Leaching test The amount and profile of silver release were investigated by dipping samples in SBF and analyzing an aliquot of spilled solution at
In order to investigate the antimicrobial effect of the CBCs, the count of CFU was carried out: this is a quantitative test that allows the quantification of bacterial colonies proliferated around the sample and adhered on its surface. Figs. 7 and 8 show the obtained results for Palacos® LV30, Palacos® R30, Palacos® LV50 and Palacos® R50. For the estimation of CFU proliferation, a bacterial broth and a broth containing commercial cements were used as references, while for the evaluation of adhered CFU on sample surface only a broth containing commercial cements was utilized as control. As far as Palacos® LV30 and Palacos® R30 are concerned (Fig. 7) only a slight decrease of CFU proliferated in the broth containing CBCs (especially for Palacos® LV30) was detected. From the bacterial adhesion evaluation test it seems that CBCs containing 30% of SBAG seem not able to limit the bacteria adhesion (see Fig. 7b). The Palacos® LV50 and Palacos® R50 were able to reduce the CFU proliferation of about 1–1.5 magnitude order respect control broth and commercial cement containing broth (Fig. 8a); moreover a slight reduction of bacteria adhesion was also observed for both low and high viscosity cements (Fig. 8b). Statistical analysis (one-way analysis of variance and Tukey's test, p b 0.05) evidenced a significant difference on the CFU proliferation for both CBC compositions. The McFarland index evaluation (Fig. 9) confirmed this behavior, in fact a slight decrease of McFarland index, and of the bacteria proliferation, was observed for CBCs, in particular for Palacos® LV50 and Palacos® R50. In order to better investigate the antibacterial effect of the CBCs, the inhibition halo test was also performed: a discontinuous halo (b1 mm) is observed for CBCs containing 30% of SBAG, particularly for Palacos® LV30 (Fig. 10a). Fig. 10b reports the inhibition halo test performed for Palacos® LV50 and Palacos® R50 up to 3 days. As can be observed, both high and low viscosity cements produce a significant inhibition zone, of about 2 mm, after one day in contact with agar substrate. For this reason the test was prolonged up to 3 days. The halo decreases gradually during the subsequent days: at the end of the second day the inhibition zone measures about 1–1.5 mm and, at the third day, a discontinuous halo less than 1 mm can be detected. These results are considered of clinical interest by the Standard “SNV 195920-1992”
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Fig. 11. Compressive strength of high (a — Palacos® R30 and Palacos® R50) and low viscosity (b — Palacos® LV30 and Palacos® LV50) bone cements.
[35,36], that relates the dimension of the inhibition halo with the entity of the antibacterial capability. According to this standard an inhibition zone N 1 mm is considered good for antibacterial activity, while an inhibition zone b 1 mm is considered fairly good. Then, the results of the inhibition halo test suggest that also Palacos® LV30 and Palacos® R30 possess the ability to limit the bacterial contamination. The different antibacterial behavior between 30% SBAG and 50% SBAG containing cements is due to the different amount of glass incorporated in the polymer matrix and, as a consequence, to the silver incorporation and release. In fact, Palacos® LV50 releases about a double amount of silver respect Palacos® LV30 (Fig. 6). On the contrary, any significant difference can be noticed between the halo dimension of low and high viscosity CBCs (results are in agreement with the leaching test). The water drops condensed on the samples, visible in Fig. 10, normally are present when it is impossible the overturn the agar plate (for the possible samples fall) during the incubation, but do not interferes with the results of the test.
3.6. Compression test The compressive tests were performed, in accordance to ASTM D 695-96 on five cylindrical specimens for each cement. Even if the ISO 5833 is the rigorous standard to evaluate the mechanical properties of acrylic cements, this test was performed to preliminarily compare the mechanical performances of the CBC, containing different amounts of brittle phase (i.e. the SBAG glass), with the plain bone cements. As far as the high viscosity cements are concerned (Fig. 11a) a slight decrease of compression strength was detected for Palacos® R30 with respect to the commercial cement, while a significant reduction was observed for Palacos® R50 (one-way analysis of variance and Tukey's test, p b 0.05). A similar result was obtained for low viscosity cements (Fig. 11b): in this case any difference was detected between commercial cements and
Palacos® LV30, whereas a significant compression resistance decrease was always observed for Palacos® LV50 (one way ANOVA, p b 0 · 05). Then, from the mechanical point of view, it can be asserted that the introduction of 50 wt.% of glass in the PMMA matrix compromises the compression strength of the cements, while the synthesis of composites containing 30% of glass does not. This preliminary experimental evidence let us asses that a further optimization of the CBC is needed, in terms of glass particle amount and distribution, before planning a complete mechanical characterization (including flexural strength evaluation, fatigue test and bending modulus) using the appropriate standards for acrylic bone cements.
4. Conclusions This work explored the innovative idea to impart bioactive and antibacterial properties to PMMA-based bone cements by adding a single inorganic phase composed by silver containing bioactive glass powders (SBAG). CBCs were prepared by introducing 30% and 50% by weight of SBAG in the polymer matrix of two commercial cements with different viscosity: Palacos® LV (low) and Palacos® R (high). All CBC formulations showed a good dispersion of the glass inside the PMMA matrix, with a few tendency to glass particle agglomeration in the formulation with 50% of glass phase. All the formulations revealed a bioactive behavior. The silver ions were released with similar profile both for 30% and 50% SBAG containing cements, but, as expected, in different amounts. Palacos® LV50 and Palacos® R50 demonstrate a good ability of bacterial contamination inhibition, both in terms of adhesion and proliferation, whereas a slightly lower effect was observed for Palacos® LV30 and Palacos® R30. From the mechanical point of view, Palacos® LV30 and Palacos® R30 showed good compression strength, comparable with the commercial cement reference, while a decrease of mechanical strength was observed for Palacos® LV50 and Palacos® R50.
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Taking into account these results, in this work a good compromise between bioactivity, antibacterial effect and preserved compression strength was achieved only with the formulations containing 30% of bioactive and antibacterial glass (Palacos® LV30 and Palacos® R30). A further characterization in terms of biocompatibility and mechanical properties in accordance with ISO 5833 (bending strength, fatigue test, bending modulus) is needed and will be focused on the CBC formulations containing 30% glass powders. An optimization of the CBC properties will be also explored by modifying the glass powder size distribution, amount and silver content, in order to achieve the best antibacterial effect, while maintaining bioactivity and the required mechanical properties, also in other polymeric bone cements of different formulations. Acknowledgment The authors would like to acknowledge Prof. Enrico Prenesti (Università degli Studi di Torino, Department of Chemistry) for the helpful discussion. References [1] M. Diefenbeck, T. Mückley, G.O. Hofmann, Prophylaxis and treatment of implantrelated infections by local application of antibiotics. Injury, Int. J. Care Injured 37 (2006) S95–S104. [2] I. Uçkay, P. Hoffmeyer, D. Lew, D. Pittet, Prevention of surgical site infections in orthopaedic surgery and bone trauma: state-of-the-art update review article, J. Hosp. Infect. 84 (1) (2013) 5–12. [3] S.M. Kurtz, K.L. Ong, J. Schmier, F. Mowat, K. Saleh, E. Dybvik, J. Kärrholm, G. Garellick, L.I. Havelin, O. Furnes, H. Malchau, E. Lau, Future clinical and economic impact of revision total hip and knee arthroplasty, J. Bone Joint Surg. Am. 89 (Suppl. 3) (2007) 144–151. [4] J.E. Phillips, T.P. Crane, M. Noy, T.S. Elliott, R.J. Grimer, The incidence of deep prosthetic infections in a specialist orthopaedic hospital: a 15-year prospective survey, J. Bone Joint Surg. (Br.) 88 (7) (2006) 943–948. [5] F. Anagnostou, A. Debet, G. Pavon-Djavid, Z. Goudaby, G. Hélary, V. Migonne, Osteoblast functions on functionalized PMMA-based polymers exhibiting Staphylococcus aureus adhesion inhibition, Biomaterials 27 (2006) 3912–3919. [6] W.F. Mousa, M. Kobayashi, S. Shinzato, M. Kamimura, M. Neo, S. Yoshihara, T. Nakamura, Biological and mechanical properties of PMMA-based bioactive bone cements, Biomaterials 21 (2000) 2137–2146. [7] W. Hennig, B.A. Blencke, H. BroKmer, K.K. Deutscher, A. Gross, W. Ege, Investigations with bioactivated polymethylmethacrylate, J. Biomed. Mater. Res. 13 (1979) 89–99. [8] K. Serbetci, F. Korkusuz, N. Hasirci, Thermal and mechanical properties of hydroxyapatite impregnated acrylic bone cements, Polym. Test. 23 (2004) 145–155. [9] J.T. Heikkila, A.J. Aho, I. Kangasniemi, A. Yli-Urpo, Polymethylmethacrylate composites: disturbed bone formation at the surface of bioactive glass and hydroxyapatite, Biomaterials 17 (1996) 1755–1760. [10] W. Matthew, M.D. Squire, J. Brian, M.D. Ludwig, R. Jonathan, B.S. Thompson, B.S.J. Jagodzinski, B.S.D. Hall, M.D.D. Andes, Premixed antibiotic bone cement an in vitro comparison of antimicrobial efficacy, J. Arthroplast. 23 (6) (2008) 110–114. [11] A. Bistolfi, G. Massazza, E. Verné, A. Massè, D. Deledda, S. Ferraris, M. Miola, F. Galetto, M. Crova1, Antibiotic-loaded cement in orthopedic surgery: a review, ISRN Orthod. 2011 (2011) 1–8. [12] E. Bertazzoni Minelli, T. Della Bora, A. Benini, Different microbial biofilm formation on polymethylmethacrylate (PMMA) bone cement loaded with gentamicin and vancomycin, Anaerobe 17 (6) (2011) 380–383.
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