Accepted Manuscript Bisphosphonates enhance bacterial adhesion and biofilm formation on bone hydroxyapatite Dr. Marcin Kos, MD, DDS, PhD, Adam Junka, PhD, Danuta Smutnicka, MSc, Patrycja Szymczyk, M Eng, Karolina Gluza, M Eng, Marzenna Bartoszewicz, MD, PhD PII:
S1010-5182(15)00122-5
DOI:
10.1016/j.jcms.2015.04.018
Reference:
YJCMS 2042
To appear in:
Journal of Cranio-Maxillo-Facial Surgery
Received Date: 5 October 2014 Revised Date:
21 March 2015
Accepted Date: 22 April 2015
Please cite this article as: Kos M, Junka A, Smutnicka D, Szymczyk P, Gluza K, Bartoszewicz M, Bisphosphonates enhance bacterial adhesion and biofilm formation on bone hydroxyapatite, Journal of Cranio-Maxillofacial Surgery (2015), doi: 10.1016/j.jcms.2015.04.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Bisphosphonates enhance bacterial adhesion and biofilm formation on bone hydroxyapatite. a
b
b
c
Marcin Kos MD, DDS, PhD, Adam Junka PhD, Danuta Smutnicka MSc, Patrycja Szymczyk M Eng, d
b
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Karolina Gluza M Eng, Marzenna Bartoszewicz MD, PhD.
a
Department of Maxillofacial and Plastic Surgery. Klinikum Oldenburg. (Head: PD Dr. med, Dr. med.
dent. Lei Li). Rachel-Straus-Strasse 10, 26 133 Oldenburg, Germany. E-Mail : [email protected] b
Department of Pharmaceutical Microbiology and Parasitology. Medical University of Wroclaw. (Head:
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Dr hab. n. med. Marzenna Bartoszewicz). Borowska 211a 4, 50 556 Wroclaw, Poland. E-mail: [email protected] b
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Department of Pharmaceutical Microbiology and Parasitology. Medical University of Wroclaw. (Head:
Dr hab. n. med. Marzenna Bartoszewicz). Borowska 211a 4, 50 556 Wroclaw, Poland. E-mail: [email protected] c
Mechanics Department of Wroclaw University of Technology, The Centre for Advanced Material
Technologies. (Head: Prof. dr hab. Edward Chlebus ). Krasinskiego 13a, 50 449 Wroclaw, Poland. E-mail: [email protected] d
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Department of Bioorganic Chemistry, Faculty of Chemistry. Wroclaw University of Technology. (Head:
Dr hab. Piotr Mlynarz). C.K. Norwida 4/6, 50 373 Wroclaw, Poland. E-mail: [email protected] b
Department of Pharmaceutical Microbiology and Parasitology. Medical University of Wroclaw. ((Head:
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Dr hab. n. med. Marzenna Bartoszewicz). Borowska 211a 4, 50 556 Wroclaw, Poland. E-mail:
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[email protected]
Corresponding author: Dr. Marcin Kos
Department of Maxillofacial and Plastic Surgery. Klinikum Oldenburg. Rachel-Straus-Strasse 10, 26 133 Oldenburg, Germany. Phone: +49 441 403 77668 Fax: +49 441 403 2625, E-Mail: [email protected]
ACCEPTED MANUSCRIPT Summary Background: Because of the suspicion that bisphosphonates enhance bacterial colonization, this study evaluated adhesion and biofilm formation by Streptococcus mutans 25175, Staphylococcus aureus 6538, and Pseudomonas aeruginosa 14454 reference strains on
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hydroxyapatite coated with clodronate, pamidronate, or zoledronate.
Material and Methods: Bacterial strains were cultured on bisphosphonate-coated and
noncoated hydroxyapatite discs. After incubation, nonadhered bacteria were removed by
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centrifugation. Biofilm formation was confirmed by scanning electron microscopy. Bacterial colonization was estimated using quantitative cultures compared by means with Kruskal-
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Wallis and post-hoc Student-Newman-Keuls tests. Modeling of the interactions between bisphosphonates and hydroxyapatite was performed using the Density Functional Theory method.
Results: Bacterial colonization of the hydroxyapatite discs was significantly higher for all
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tested strains in the presence of bisphosphonates vs. controls. Adherence in the presence of pamidronate was higher than with other bisphosphonates. Density Functional Theory analysis showed that the protonated amine group of pamidronate, which are not present in clodronate
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or zoledronate, forms two additional hydrogen bonds with hydroxyapatite. Moreover, the reactive cationic amino group of pamidronate may attract bacteria by direct electrostatic
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interaction.
Conclusion: Increased bacterial adhesion and biofilm formation can promote osteomyelitis, cause failure of dental implants or bisphosphonate-coated joint prostheses, and complicate bone surgery in patients on bisphosphonates.
INTRODUCTION
ACCEPTED MANUSCRIPT The introduction of bisphosphonates (BPs) into clinical practice has considerably reduced the number of pathological fractures and metastases to bones, and has diminished the risk of hypercalcemia in oncologic patients. BPs have been quickly applied to prevent bone loss in osteoporosis, to treat bone dysplasia and metabolic bone diseases, and to improve the
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stability of endoprostheses (Charpulat et al., 2004; Drake et al., 2008; Graham and Russel, 2011; Wilkinson and Little, 2011). Some of the adverse effects of using BPs were known from the beginning, such as increased temperature, atrial fibrillation, irritation of the digestive tract,
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and toxic effects on the kidneys (Drake et al., 2008).
Many years later, additional complications associated with the use of the BPs were
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recognized, such as atypical fractures of the femur shaft and necrosis and inflammation of the jaw bone (Marx, 2003, Ruggiero et al., 2004; Isaacs et al., 2010; Thompson et al., 2012). Necrosis of large and therapy-resistant jaw bone exposures in patients receiving BPs is termed bisphosphonate-related osteonecrosis of the jaw (BRONJ). The pathogenesis of BRONJ still
explained.
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draws the attention of scientists, because many aspects of this phenomenon have not yet been
Marx (2003) hypothesized that the inhibition of bone metabolism plays an important
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role; however, some pathological issues still cannot be explained on this basis. It is still not clear why necrosis of bone affiliated with the use of BPs develops only in the area of the jaws,
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or why the vast majority of cases result from exposure of the jaw bones following tooth extractions, oral surgery, pressure from poorly fitting dentures, or periodontitis, or why the disease is characterized by a high recurrence rate (Hasegawa et al., 2013, Barba-Recero et al., 2014). In trying to explain these problems, BPs may adversely affect wound healing through toxic actions on epithelial cells in addition to inhibiting bone metabolism (Acil et al., 2012), lowering the pH of the environment (Otto et al., 2010) and inhibiting angiogenesis (Stressing et al., 2011). At the same time, infection of the denuded bone with creation of biofilm
ACCEPTED MANUSCRIPT composed of Gram-positive and Gram-negative strains as well as anaerobes has been documented in pathological and microbiological reports concerning BRONJ (Sedghizadeh et al., 2008; Wei et al., 2012). Moreover, microbial colonization in BRONJ cases was also significantly higher than in bone necrosis occurring in the absence of BPs, raising the
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possibility that BPs could enhance bacterial colonization and biofilm formation in patients treated with these drugs (Kos et al., 2010). In a recent report, we demonstrated that one of the BPs, namely, pamidronate, increases the adhesion to hydroxyapatite (HA) and facilitates the
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colonization of clinical and reference strains of Pseudomonas aeruginosa and Staphylococcus aureus, species frequently associated with bone infections (Kos et al., 2013).
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To investigate this characteristic in a broader group of BPs, we compared the adhesion and growth of Gram-positive and Gram-negative bacterial strains on hydroxyapatite discs coated with commonly used BPs including clodronate, pamidronate, and zoledronate, and we compared the results with those obtained with noncoated discs. Moreover, we applied
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molecular modeling based on the Density Functional Theory method to visualize the structure of the coated HA to explain the interactions of the hydroxyapatite surface with BPs as well as with bacterial receptors (Rimola et al., 2008).
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The results of this study may help to answer questions about the potential risk of BPlinked bacterial adhesion causing infection after tooth extraction or in open fractures,
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osteotomies, or dental implants in patients on BPs, as well as the safety of using BPs to improve stability in joint arthroplasty.
MATERIAL AND METHODS
Bacterial strains
ACCEPTED MANUSCRIPT For experimental purposes, the following ATCC reference strains were used: Streptococcus mutans 25175, Staphylococcus aureus 6538, and Pseudomonas aeruginosa 14454. Biofilms of S. aureus and P. aeruginosa were incubated in liquid Tryptic Soya Broth medium, whereas S. mutans was incubated in liquid Brain–Heart Infusion medium. These
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strains are part of the strain collection of the Department of Microbiology of the Medical University of Wroclaw.
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Hydroxyapatite discs
The HA discs used for the experiments had the following characteristics:
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0.38-inch diameter, 0.06- to 0.02-inch thickness, and purity greater than 95%; x-ray diffraction patterns that conformed to Joint Committee on Powder Diffraction Standard 9-432 (International Centre for Diffraction Data, Newtown Square, PA); total heavy metal as lead less than 40 ppm; and a trace element concentration that conformed to ASTM standard
Bisphosphates
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F1185-88 (ASTM International, West Conshohocken, PA).
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The following BPs were used for experimental purposes: disodium clodronate (Bonefos; Bayer Pharma), disodium pamidronate (pamidronate; Medac) and zoledronic acid
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(Vipharm SA). The controls consisted of culture medium with 0.9% saline solution. Coating of hydroxyapatite discs with bisphosphonates The HA discs were coated with 200-µL aliquots of BPs, reaching a final concentration
of 0.8 mg/L per disc. This concentration provides full saturation of the hydroxyapatite discs, which was tested in the series of preliminary experiments and our previous works (Kos et al., 2013, Junka et al., 2014). The HA discs were dried under a controlled, constant, heated airflow between applications. The presence of the coating was confirmed using a Zeiss EVO
ACCEPTED MANUSCRIPT MA25 scanning electron microscope (Carl Zeiss Poland, Poland) with an energy dispersive xray microanalysis attachment and an accelerating voltage of 20 kV. Confirmation by scanning electron microscopy of the investigated strains ability to form
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biofilms on hydroxyapatite discs The S. aureus and P. aeruginosa strains, which were cultured on appropriate agar plates, were transferred to liquid media and incubated at 37°C for 24 hours under aerobic
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conditions. S. mutans was incubated at the same temperature and time, but in facultatively anaerobic conditions. After incubation, the densities of the bacterial suspensions were
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measured using a densitometer (Biomerieux Poland, Warsaw Poland) and diluted to 105 cells/mL. The bacterial dilutions were incubated in the presence of the HA discs at 37°C for 24 hours. After incubation, the discs were thoroughly rinsed using physiologic saline to remove nonadherent bacteria and to leave only biofilm-forming microorganisms.
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Subsequently, HA discs with biofilm on it were fixed using 3% glutarate (Poch, Gliwice, Poland) for 15 minutes at room temperature. Then, the samples were rinsed twice with phosphate buffer (Sigma Aldrich Poland, Poznan, Poland) to remove the fixative. The next
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step was dehydration in increasing concentrations of ethanol (25%, 60%, 95%, and 100%) for 5 minutes in each solution. After rinsing off the ethanol, the samples were dried. Then, the
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samples were covered with gold and palladium (60:40; sputter current, 40 mA; sputter time, 50 seconds) using a Quorum machine (Quorum International, Fort Worth, TX) and examined under a Zeiss EVO MA25 scanning electron microscope. X-ray computed microtomography To estimate the exact surface of the HA discs and to exclude the possibility that the differences in disc surface influence the results, computed microtomography methods (µCT) were used. Scaffolds were scanned using the µCT system (Metrotom 1500; Carl Zeiss,
ACCEPTED MANUSCRIPT Oberkochen, Germany). The system consisted of a flat panel detector with a resolution of 1024 × 1024 px (400-µm pixel size) and a 16-bit grayscale, rotary table, and microfocus x-ray tube with a maximum accelerating voltage of 225 kV and maximum current of 1000 µA. To achieve maximum resolution, the tube voltage was fixed at 220 kV and the current at 120 µA.
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The number of projections carried out during the rotation of the sample of 360º was 800 with a 1-second integration time for each. The result matched the parameters permitted to achieve a voxel size of 31 µm. The obtained data were analyzed using VG Studio MAX (Volume
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Graphics GmbH, Heidelberg, Germany) software. It was demonstrated that average size of
Biofilm culture under static conditions
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randomly chosen HA discs was constant and amounted 186,898 mm2 ± 0,358.
Strains were incubated in the presence of HA discs, either coated or not coated with BPs, under the conditions described previously.
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Biofilm culture under flow conditions
Flow conditions were used to force bacterial adhesion to occur and to compare it with static
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culture conditions. Strains were incubated in liquid media as described earlier. Subsequently, the bacterial suspensions were diluted to 105 colony-forming units per microliter (cfu/µL).
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Then, 30 mL of the suspension was introduced into a cubic box containing the HA disc using a peristaltic pump (Ismatec, Reglo Digital) at 1 mL/min for 30 minutes then left for another 3.5 hours under static conditions. Initially, all bacterial species were subjected to this experiment. However, the adherence of S. mutans to the HA discs was not reproducible; therefore, this strain was excluded from further flow condition experiments (Fig. 1). Calculation of molecular interactions between bisphosphonates and hydroxyapatite surface Quantum mechanical simulations according to the Density Functional Theory method were used to determine the pattern of interactions approximate the ground-state wave function
ACCEPTED MANUSCRIPT and ground-state energy of the quantum electronic structure of the interaction between HA and BP ions. All calculations were performed using the Gaussian 09 software package. The structures of complexes between commercially available BPs and the HA surface were optimized using the wb97xd functional, which includes empirical dispersion with 6-
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31++g(d,p) basis stet. Calculations were carried using a polarizable continuum solvation
model. For these calculations, the HA structure was chosen as described by Rimola et al., (2008).
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Statistical analysis
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The results were expressed as mean cfu/mm2 ± standard error of the mean (SEM). A power analysis was performed for sample size estimation prior to experimentation. KruskalWallis one-way analysis of variance on ranks with post-hoc all pairwise multiple comparison procedure (Student-Newman-Keuls method) tests were used to determine the differences
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between treatment groups. Statistical significance was defined as p ≤ 0.05. All calculations were performed using SigmaPlot statistical software, version 12.5.
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RESULTS
Static conditions
All experimental strains were able to form strong biofilm structures on the surface of
manufactured HA discs (Fig. 2). The number of cfu/mm2 formed by P. aeruginosa and S. mutans incubated with HA discs coated with clodronate, pamidronate, or zoledronate was significantly higher in the presence of each of these BP when compared with noncoated controls. In the case of S. aureus, the difference was significant for pamidronate, but not clodronate or zoledronate.
ACCEPTED MANUSCRIPT Additionally, adherence in the presence of pamidronate was significantly higher in most cases than in the presence of clodronate or zoledronate. The distributions of the values for the tested strains are presented in Table 1 and Fig. 3.
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Flow conditions
Flow conditions were projected to emphasize bacterial adhesion compared with
ambiguous conditions during static cultures of S. aureus. The number of cfu/mm2 formed by
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S. aureus and P. aeruginosa under these conditions were significantly higher for HA discs coated with clodronate, pamidronate, and zoledronate compared with noncoated controls.
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Moreover, the bacterial strains adhered significantly stronger in the presence of pamidronate than with the other two BPs. Table 2 and the Fig. 4 show the distribution of results for S. aureus and P. aeruginosa. It was not possible to obtain repeatable measurements for S.
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mutans under the flow conditions.
Interactions of bisphosphonates with the hydroxyapatite surface
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Analyses performed using the Density Functional Theory method showed that pamidronate, clodronate, and zoledronate can all form strong electrostatic interactions with
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the surface of HA. The distance between the oxygen atom in the phosphonic group and the calcium ions in HA was comparable for all BPs and amounted to 2.38 Å for pamidronate and 2.4 Å for clodronate and zoledronate. Moreover, the protonated amine group of pamidronate could form two additional hydrogen bonds with oxygen atoms from the phosphate ion within the HA (Fig. 5, above), whereas the chlorine atoms in clodronate and the imidazol ring in zoledronate were too far from the HA surface to form any reasonable interactions, moreover the imidazole ring of the zoledronate possess a nitrogen atom with free electron pair which could even push back the negative charged oxygen ions of the hydroxyapatite (Fig. 5, below).
ACCEPTED MANUSCRIPT Diverse atomic structures on BPs, such as phosphate residues, carbon atoms, protons, chloride ions, or an amine ring, could serve as “hooks” for bacteria, facilitating bacterial adhesion. Moreover, the reactive cationic amino group of pamidronate could attract bacteria by direct
DISCUSSION
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electrostatic interaction.
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The results of this study show that BPs commonly used clinically, represented by non-
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aminobisphosphonates such as clodronate and aminobisphosphonates such as pamidronate and zoledronate, can enhance adhesion and promote the formation of biofilms by a broad spectrum of bacterial strains on HA discs coated with these substances, compared with nontreated control HA discs. Moreover, adhesion in the presence of pamidronate was
microorganism tested.
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significantly stronger in most cases than that for clodronate or zoledronate, regardless of the
Under stationary conditions, the adhesion of S. mutans and P. aeruginosa in the
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presence of BPs was significantly stronger; however, it was not possible to confirm the significantly higher adhesion of S. aureus strains, because of strong deviation of
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measurements, in this set. Therefore, we performed the experiment under flow conditions, which favors adhesion. Under flow conditions, both S. aureus and P. aeruginosa adhered much more strongly to discs saturated with BPs than to noncoated discs; however, under flow conditions, it was not possible to obtain repeatable results for S. mutans. The observation of this phenomenon strongly emphasizes the necessity of using different research models in order to obtain a fuller and more realistic picture of the relationship between HA and different microorganisms after treatment with BPs. The diversity of bacterial species adhering more strongly to BP-treated HA suggests a nonspecific rather than specific (i.e., restricted to one species or family) mechanism of the
ACCEPTED MANUSCRIPT interaction of these drugs with the HA surface. The importance of bacterial biofilm formation in the pathogenesis of BRONJ was reported previously, but the mechanisms responsible for bacterial colonization of denuded bone in patients on BPs were unclear (Sedghizadeh et al., 2008; Kos et al., 2010; Wei et al., 2012). Based on our experiments, one may suppose that
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BP-mediated bacterial adhesion plays an important role in this process.
Using the Density Functional Theory method to model the interactions showed that pamidronate may attach to HA through 4 bonds, whereas clodronate and zoledronate could be
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anchored with only 2 bonds, namely, phosphonic groups. When combined with HA,
pamidronate could allow the bacteria to anchor through 2 phosphate residues and 2 carbon
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atoms, as well as through hydrogen bonding. Clodronate exhibits similar interactions with bacteria, but does so through chloride ions, whereas the structure of zoledronate provides even greater possibilities for bacterial adhesion through the presence of an amine ring (Ragunath et al., 2008; Chen et al., 2011; Hinterdorfer et al., 2012). Despite these interactions, pamidronate
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has the greatest potential to create bonds with HA, more strongly promoting the formation of a biofilm. This suggests that the adhesion of bacteria to HA through a BP is accomplished indirectly. Consequently, BPs with the greatest potential for bonding to HA would promote
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the greatest extent of bacterial adhesion. The steps described in bacterial adhesion mechanisms probably favor the cells approaching closer to the bone surface then facilitating
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binding through typical interactions via adhesins. An expanded model of binding such as this could explain the higher adhesion rates in cases in which BPs were used. The results of this study are paramount in explaining issues in the pathophysiology of
BRONJ that are still unclear. The currently accepted theory of the origin of BRONJ assumes that impaired osteoclast function is followed by the impaired osteoblast activity due to the feedback between these two cell groups. Thus, the arrest of osteoclast function reflects not only diminished bone resorption but also reduced bone formation, both of which lead to decreased bone turnover and consequently to bone necrosis. Additional superinfection with
ACCEPTED MANUSCRIPT microorganisms leads to treatment-resistant osteomyelitis. However, although the BPs that are generally administered are deposited throughout the entire skeleton, the disease exclusively affects the jaws and has a high recurrence rate. Furthermore, although it can occur spontaneously, in the majority of cases it is provoked by dental surgery such as tooth
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extractions, root-tip resections, or cystectomies (Marx, 2003; Ruggiero et al., 2004).
Sometimes clinical symptoms are preceded by local trauma, pressure from ill-fitting dentures, or periodontal infection. These issues could be explained by increased adherence of
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microorganisms to the bone in the presence of BPs.
Exposure of bone during dental surgery acts as a trigger, opening the door to bacterial
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invasion. The presence of BPs promotes bacterial adhesion and biofilm formation on the surface of the affected bone, which is an essential step in the development of persistent osteomyelitis. In light of the above findings, jaw bones are especially subject to infection compared with other parts of the skeleton because they are more often subject to direct
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contact with the external environment due to the thin epithelial lining covering their surface, are susceptibility to trauma, and contain the teeth. Enhanced adhesion and biofilm formation should be considered when planning dental
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implant treatments in patients on BPs. There are reports confirming an increased infection rate in these patients with bacterial species known to be etiological factors of bone infections
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(Sedghizadeh et al., 2008; Wei et al., 2012). Based on the results of this study, one can assume that the other areas of BP
application (joint arthroplasty, treatment of open fractures, metabolic bone diseases) may result in an increased infection rate. Some clinical issues seem to have already confirmed this suspicion. Howe et al. (2013) reported a case of serious osteomyelitis after operative treatment of a femur shaft fracture in a patient on BPs. Nelson et al. (2005) questioned whether aseptic loosening of endoprosthesis was truly aseptic. The theory of increased bacterial adherence and biofilm formation could allow a better understanding of these
ACCEPTED MANUSCRIPT pathological conditions that was not previously clear. Thus, an identification and correct understanding of the pathology of osteomyelitis in patients on BPs may show that bone infection in patients on BPs is not restricted to the oral cavity. Furthermore, a better understanding of the pathophysiology connected with the use of BPs is paramount for
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continued safe clinical use of these drugs.
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CONCLUSION
The evidence of enhanced, bisphosphonate-linked bacterial adhesion to bone hydroxyapatite
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and biofilm formation could explain some unclear aspects of the pathophysiology of bisphosphonate-related osteonecrosis of the jaw, including the prevalent appearance after bone exposure and the recurrent nature of osteomyelitis accompanying BRONJ, and would justify antimicrobial treatment in BRONJ. Close attention should be paid to the potential for
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infection of open fractures, osteotomies, or implantations in patients on BPs and the safety of
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using BPs for improvement of stability in joint arthroplasty.
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Acknowledgements
Modeling calculations were carried out using resources provided by the Wroclaw Centre for Networking and Supercomputing (Wroclaw, Poland).
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ACCEPTED MANUSCRIPT Nelson CL, McLaren AC, McLaren SG, Johnson JW, Smeltzer MS: Is aseptic loosening truly
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ACCEPTED MANUSCRIPT Fig. 1. System for testing bacterial adherence under flow conditions. The system consisted of a box with two insets (A) for placement of the hydroxyapatite discs (B), and openings for installing
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the peristaltic pump hoses (C). The suspension of the bacterial strain being tested (1) was pumped into the box. Excess fluid was removed to the collection vessel (2). The whole system was maintained at a temperature of 37°C with a thermostat (for better viewing, this is not
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shown in the picture). Fig. 2.
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Ability of the tested strains to form a biofilm on the surface of the hydroxyapatite. (A) Sterile and clean hydroxyapatite disc. (B) S. aureus forming multilayer structure with visible coccal cells (x947), (C) P. aeruginosa biofilm embedded within extracellular slime (x3390), (D) Dense structure of Str. mutans biofilm (dental plaque) with visible streptococcal cells (x1530). The
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representative images under different magnification were chosen because the size of cells and biofilm structure differs among different bacterial species.
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Fig. 3.
Comparison of the number of colony forming units / mm2 formed by the different bacterial
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strains in the presence of the tested bisphosphonate-coated or noncoated (control) hydroxyapatite discs under static conditions. Fig. 4.
Comparison of the number of colony forming units / mm2 formed by the different bacterial strains in the presence of the tested bisphosphonate-coated or noncoated (control) hydroxyapatite discs under flow conditions.
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Fig. 5. Simulation of the molecular structure of the surface of hydroxyapatite coated with different
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bisphosphonates using Density Functional Theory analysis. (Above). A particle of pamidronate in the NH+; PO2H* protonation state is presented. HP, Hydroxyapatite structure. Oxygen atoms (red) from phosphoric groups of pamidronate are
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palced over 2 calcium atoms (green) of hydroxyapatite and can form strong electrostatic interactions with the hydroxyapatite surface. The distance between the oxygen atom of the
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pamidronate phosphonic group and the calcium ions of hydroxyapatite is ~ 2.38 Å. The side chain of pamidronate containing the reactive NH3+ group protrudes over the hydroxyapatite structure and forms 2 additional bonds (green dashed lines), which may be reason for the increased level of bacterial adhesion. The distance NH1-O1 amounts ~ 1,852 Å, the distance
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NH2-O2 amounts ~1,849 Å. N-H angles to O from PO43- are γ N-H1-O1~142.4° and γ N-H1O1~137.7° respectively. Nitrogen atoms (blue), carbon atoms (gray), phosphorous molecules (orange), hydrogen atoms (white). (Below). Interactions of clodronate and zoledronate with
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the hydroxyapatite surface are presented. Both bisphosphonates, like pamidronate, can form
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strong electrostatic interactions with the hydroxyapatite surface. The distance between oxygen atoms in the phosphonic groups and calcium ions in hydroxyapatite is ~2.40 Å, but the chlorine atoms in the side chain of the clodronate and the imidazole ring of the zoledronate are too far from the hydroxyapatite to form any reasonable interactions. Moreover, the imidazole ring of the zoledronate possesses a nitrogen atom with free electron pair which could even push back the negative charged oxygen ions of the hydroxyapatite.
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Table 1. Distribution of statistically significant differences in adherence and biofilm formation of different bacterial strains on the hydroxyapatite discs coated with clodronate (B), pamidronate (P), or zolendronate (Z) and uncoated discs (K) during culturing under static conditions. P.aeruginosa Yes Yes Yes No No No
Str. mutans Yes Yes Yes No No No
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S. aureus Yes No No Yes Yes No
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P vs. K B vs. K Z vs. K P vs. B P vs. Z B vs. Z
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Table 2. Distribution of statistically significant differences in adherence and biofilm formation of different bacterial strains on the hydroxyapatite discs coated with clodronate (B), pamidronate (P), or zolendronate (Z) and uncoated discs (K) during culturing under flow conditions. P. aeruginosa Yes Yes Yes No Yes Yes
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S. aureus Yes Yes Yes Yes Yes Yes
P vs. K B vs. K Z vs. K P vs. B P vs. Z B vs. Z
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ACCEPTED MANUSCRIPT Bisphosphonates enhance biofilm formation on the surface of the bone hydroxyapatite.
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Bisphosphonates can promote osteomyelitis in treated patients.
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Bone surgery in the presence of bisphosphonates threatens with osteomyelitis.
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S1010-5182(15)00122-5
DOI:
10.1016/j.jcms.2015.04.018
Reference:
YJCMS 2042
To appear in:
Journal of Cranio-Maxillo-Facial Surgery
Received Date: 5 October 2014 Revised Date:
21 March 2015
Accepted Date: 22 April 2015
Please cite this article as: Kos M, Junka A, Smutnicka D, Szymczyk P, Gluza K, Bartoszewicz M, Bisphosphonates enhance bacterial adhesion and biofilm formation on bone hydroxyapatite, Journal of Cranio-Maxillofacial Surgery (2015), doi: 10.1016/j.jcms.2015.04.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Bisphosphonates enhance bacterial adhesion and biofilm formation on bone hydroxyapatite. a
b
b
c
Marcin Kos MD, DDS, PhD, Adam Junka PhD, Danuta Smutnicka MSc, Patrycja Szymczyk M Eng, d
b
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Karolina Gluza M Eng, Marzenna Bartoszewicz MD, PhD.
a
Department of Maxillofacial and Plastic Surgery. Klinikum Oldenburg. (Head: PD Dr. med, Dr. med.
dent. Lei Li). Rachel-Straus-Strasse 10, 26 133 Oldenburg, Germany. E-Mail : [email protected] b
Department of Pharmaceutical Microbiology and Parasitology. Medical University of Wroclaw. (Head:
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Dr hab. n. med. Marzenna Bartoszewicz). Borowska 211a 4, 50 556 Wroclaw, Poland. E-mail: [email protected] b
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Department of Pharmaceutical Microbiology and Parasitology. Medical University of Wroclaw. (Head:
Dr hab. n. med. Marzenna Bartoszewicz). Borowska 211a 4, 50 556 Wroclaw, Poland. E-mail: [email protected] c
Mechanics Department of Wroclaw University of Technology, The Centre for Advanced Material
Technologies. (Head: Prof. dr hab. Edward Chlebus ). Krasinskiego 13a, 50 449 Wroclaw, Poland. E-mail: [email protected] d
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Department of Bioorganic Chemistry, Faculty of Chemistry. Wroclaw University of Technology. (Head:
Dr hab. Piotr Mlynarz). C.K. Norwida 4/6, 50 373 Wroclaw, Poland. E-mail: [email protected] b
Department of Pharmaceutical Microbiology and Parasitology. Medical University of Wroclaw. ((Head:
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Dr hab. n. med. Marzenna Bartoszewicz). Borowska 211a 4, 50 556 Wroclaw, Poland. E-mail:
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[email protected]
Corresponding author: Dr. Marcin Kos
Department of Maxillofacial and Plastic Surgery. Klinikum Oldenburg. Rachel-Straus-Strasse 10, 26 133 Oldenburg, Germany. Phone: +49 441 403 77668 Fax: +49 441 403 2625, E-Mail: [email protected]
ACCEPTED MANUSCRIPT Summary Background: Because of the suspicion that bisphosphonates enhance bacterial colonization, this study evaluated adhesion and biofilm formation by Streptococcus mutans 25175, Staphylococcus aureus 6538, and Pseudomonas aeruginosa 14454 reference strains on
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hydroxyapatite coated with clodronate, pamidronate, or zoledronate.
Material and Methods: Bacterial strains were cultured on bisphosphonate-coated and
noncoated hydroxyapatite discs. After incubation, nonadhered bacteria were removed by
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centrifugation. Biofilm formation was confirmed by scanning electron microscopy. Bacterial colonization was estimated using quantitative cultures compared by means with Kruskal-
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Wallis and post-hoc Student-Newman-Keuls tests. Modeling of the interactions between bisphosphonates and hydroxyapatite was performed using the Density Functional Theory method.
Results: Bacterial colonization of the hydroxyapatite discs was significantly higher for all
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tested strains in the presence of bisphosphonates vs. controls. Adherence in the presence of pamidronate was higher than with other bisphosphonates. Density Functional Theory analysis showed that the protonated amine group of pamidronate, which are not present in clodronate
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or zoledronate, forms two additional hydrogen bonds with hydroxyapatite. Moreover, the reactive cationic amino group of pamidronate may attract bacteria by direct electrostatic
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interaction.
Conclusion: Increased bacterial adhesion and biofilm formation can promote osteomyelitis, cause failure of dental implants or bisphosphonate-coated joint prostheses, and complicate bone surgery in patients on bisphosphonates.
INTRODUCTION
ACCEPTED MANUSCRIPT The introduction of bisphosphonates (BPs) into clinical practice has considerably reduced the number of pathological fractures and metastases to bones, and has diminished the risk of hypercalcemia in oncologic patients. BPs have been quickly applied to prevent bone loss in osteoporosis, to treat bone dysplasia and metabolic bone diseases, and to improve the
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stability of endoprostheses (Charpulat et al., 2004; Drake et al., 2008; Graham and Russel, 2011; Wilkinson and Little, 2011). Some of the adverse effects of using BPs were known from the beginning, such as increased temperature, atrial fibrillation, irritation of the digestive tract,
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and toxic effects on the kidneys (Drake et al., 2008).
Many years later, additional complications associated with the use of the BPs were
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recognized, such as atypical fractures of the femur shaft and necrosis and inflammation of the jaw bone (Marx, 2003, Ruggiero et al., 2004; Isaacs et al., 2010; Thompson et al., 2012). Necrosis of large and therapy-resistant jaw bone exposures in patients receiving BPs is termed bisphosphonate-related osteonecrosis of the jaw (BRONJ). The pathogenesis of BRONJ still
explained.
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draws the attention of scientists, because many aspects of this phenomenon have not yet been
Marx (2003) hypothesized that the inhibition of bone metabolism plays an important
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role; however, some pathological issues still cannot be explained on this basis. It is still not clear why necrosis of bone affiliated with the use of BPs develops only in the area of the jaws,
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or why the vast majority of cases result from exposure of the jaw bones following tooth extractions, oral surgery, pressure from poorly fitting dentures, or periodontitis, or why the disease is characterized by a high recurrence rate (Hasegawa et al., 2013, Barba-Recero et al., 2014). In trying to explain these problems, BPs may adversely affect wound healing through toxic actions on epithelial cells in addition to inhibiting bone metabolism (Acil et al., 2012), lowering the pH of the environment (Otto et al., 2010) and inhibiting angiogenesis (Stressing et al., 2011). At the same time, infection of the denuded bone with creation of biofilm
ACCEPTED MANUSCRIPT composed of Gram-positive and Gram-negative strains as well as anaerobes has been documented in pathological and microbiological reports concerning BRONJ (Sedghizadeh et al., 2008; Wei et al., 2012). Moreover, microbial colonization in BRONJ cases was also significantly higher than in bone necrosis occurring in the absence of BPs, raising the
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possibility that BPs could enhance bacterial colonization and biofilm formation in patients treated with these drugs (Kos et al., 2010). In a recent report, we demonstrated that one of the BPs, namely, pamidronate, increases the adhesion to hydroxyapatite (HA) and facilitates the
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colonization of clinical and reference strains of Pseudomonas aeruginosa and Staphylococcus aureus, species frequently associated with bone infections (Kos et al., 2013).
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To investigate this characteristic in a broader group of BPs, we compared the adhesion and growth of Gram-positive and Gram-negative bacterial strains on hydroxyapatite discs coated with commonly used BPs including clodronate, pamidronate, and zoledronate, and we compared the results with those obtained with noncoated discs. Moreover, we applied
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molecular modeling based on the Density Functional Theory method to visualize the structure of the coated HA to explain the interactions of the hydroxyapatite surface with BPs as well as with bacterial receptors (Rimola et al., 2008).
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The results of this study may help to answer questions about the potential risk of BPlinked bacterial adhesion causing infection after tooth extraction or in open fractures,
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osteotomies, or dental implants in patients on BPs, as well as the safety of using BPs to improve stability in joint arthroplasty.
MATERIAL AND METHODS
Bacterial strains
ACCEPTED MANUSCRIPT For experimental purposes, the following ATCC reference strains were used: Streptococcus mutans 25175, Staphylococcus aureus 6538, and Pseudomonas aeruginosa 14454. Biofilms of S. aureus and P. aeruginosa were incubated in liquid Tryptic Soya Broth medium, whereas S. mutans was incubated in liquid Brain–Heart Infusion medium. These
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strains are part of the strain collection of the Department of Microbiology of the Medical University of Wroclaw.
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Hydroxyapatite discs
The HA discs used for the experiments had the following characteristics:
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0.38-inch diameter, 0.06- to 0.02-inch thickness, and purity greater than 95%; x-ray diffraction patterns that conformed to Joint Committee on Powder Diffraction Standard 9-432 (International Centre for Diffraction Data, Newtown Square, PA); total heavy metal as lead less than 40 ppm; and a trace element concentration that conformed to ASTM standard
Bisphosphates
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F1185-88 (ASTM International, West Conshohocken, PA).
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The following BPs were used for experimental purposes: disodium clodronate (Bonefos; Bayer Pharma), disodium pamidronate (pamidronate; Medac) and zoledronic acid
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(Vipharm SA). The controls consisted of culture medium with 0.9% saline solution. Coating of hydroxyapatite discs with bisphosphonates The HA discs were coated with 200-µL aliquots of BPs, reaching a final concentration
of 0.8 mg/L per disc. This concentration provides full saturation of the hydroxyapatite discs, which was tested in the series of preliminary experiments and our previous works (Kos et al., 2013, Junka et al., 2014). The HA discs were dried under a controlled, constant, heated airflow between applications. The presence of the coating was confirmed using a Zeiss EVO
ACCEPTED MANUSCRIPT MA25 scanning electron microscope (Carl Zeiss Poland, Poland) with an energy dispersive xray microanalysis attachment and an accelerating voltage of 20 kV. Confirmation by scanning electron microscopy of the investigated strains ability to form
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biofilms on hydroxyapatite discs The S. aureus and P. aeruginosa strains, which were cultured on appropriate agar plates, were transferred to liquid media and incubated at 37°C for 24 hours under aerobic
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conditions. S. mutans was incubated at the same temperature and time, but in facultatively anaerobic conditions. After incubation, the densities of the bacterial suspensions were
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measured using a densitometer (Biomerieux Poland, Warsaw Poland) and diluted to 105 cells/mL. The bacterial dilutions were incubated in the presence of the HA discs at 37°C for 24 hours. After incubation, the discs were thoroughly rinsed using physiologic saline to remove nonadherent bacteria and to leave only biofilm-forming microorganisms.
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Subsequently, HA discs with biofilm on it were fixed using 3% glutarate (Poch, Gliwice, Poland) for 15 minutes at room temperature. Then, the samples were rinsed twice with phosphate buffer (Sigma Aldrich Poland, Poznan, Poland) to remove the fixative. The next
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step was dehydration in increasing concentrations of ethanol (25%, 60%, 95%, and 100%) for 5 minutes in each solution. After rinsing off the ethanol, the samples were dried. Then, the
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samples were covered with gold and palladium (60:40; sputter current, 40 mA; sputter time, 50 seconds) using a Quorum machine (Quorum International, Fort Worth, TX) and examined under a Zeiss EVO MA25 scanning electron microscope. X-ray computed microtomography To estimate the exact surface of the HA discs and to exclude the possibility that the differences in disc surface influence the results, computed microtomography methods (µCT) were used. Scaffolds were scanned using the µCT system (Metrotom 1500; Carl Zeiss,
ACCEPTED MANUSCRIPT Oberkochen, Germany). The system consisted of a flat panel detector with a resolution of 1024 × 1024 px (400-µm pixel size) and a 16-bit grayscale, rotary table, and microfocus x-ray tube with a maximum accelerating voltage of 225 kV and maximum current of 1000 µA. To achieve maximum resolution, the tube voltage was fixed at 220 kV and the current at 120 µA.
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The number of projections carried out during the rotation of the sample of 360º was 800 with a 1-second integration time for each. The result matched the parameters permitted to achieve a voxel size of 31 µm. The obtained data were analyzed using VG Studio MAX (Volume
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Graphics GmbH, Heidelberg, Germany) software. It was demonstrated that average size of
Biofilm culture under static conditions
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randomly chosen HA discs was constant and amounted 186,898 mm2 ± 0,358.
Strains were incubated in the presence of HA discs, either coated or not coated with BPs, under the conditions described previously.
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Biofilm culture under flow conditions
Flow conditions were used to force bacterial adhesion to occur and to compare it with static
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culture conditions. Strains were incubated in liquid media as described earlier. Subsequently, the bacterial suspensions were diluted to 105 colony-forming units per microliter (cfu/µL).
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Then, 30 mL of the suspension was introduced into a cubic box containing the HA disc using a peristaltic pump (Ismatec, Reglo Digital) at 1 mL/min for 30 minutes then left for another 3.5 hours under static conditions. Initially, all bacterial species were subjected to this experiment. However, the adherence of S. mutans to the HA discs was not reproducible; therefore, this strain was excluded from further flow condition experiments (Fig. 1). Calculation of molecular interactions between bisphosphonates and hydroxyapatite surface Quantum mechanical simulations according to the Density Functional Theory method were used to determine the pattern of interactions approximate the ground-state wave function
ACCEPTED MANUSCRIPT and ground-state energy of the quantum electronic structure of the interaction between HA and BP ions. All calculations were performed using the Gaussian 09 software package. The structures of complexes between commercially available BPs and the HA surface were optimized using the wb97xd functional, which includes empirical dispersion with 6-
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31++g(d,p) basis stet. Calculations were carried using a polarizable continuum solvation
model. For these calculations, the HA structure was chosen as described by Rimola et al., (2008).
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Statistical analysis
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The results were expressed as mean cfu/mm2 ± standard error of the mean (SEM). A power analysis was performed for sample size estimation prior to experimentation. KruskalWallis one-way analysis of variance on ranks with post-hoc all pairwise multiple comparison procedure (Student-Newman-Keuls method) tests were used to determine the differences
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between treatment groups. Statistical significance was defined as p ≤ 0.05. All calculations were performed using SigmaPlot statistical software, version 12.5.
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RESULTS
Static conditions
All experimental strains were able to form strong biofilm structures on the surface of
manufactured HA discs (Fig. 2). The number of cfu/mm2 formed by P. aeruginosa and S. mutans incubated with HA discs coated with clodronate, pamidronate, or zoledronate was significantly higher in the presence of each of these BP when compared with noncoated controls. In the case of S. aureus, the difference was significant for pamidronate, but not clodronate or zoledronate.
ACCEPTED MANUSCRIPT Additionally, adherence in the presence of pamidronate was significantly higher in most cases than in the presence of clodronate or zoledronate. The distributions of the values for the tested strains are presented in Table 1 and Fig. 3.
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Flow conditions
Flow conditions were projected to emphasize bacterial adhesion compared with
ambiguous conditions during static cultures of S. aureus. The number of cfu/mm2 formed by
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S. aureus and P. aeruginosa under these conditions were significantly higher for HA discs coated with clodronate, pamidronate, and zoledronate compared with noncoated controls.
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Moreover, the bacterial strains adhered significantly stronger in the presence of pamidronate than with the other two BPs. Table 2 and the Fig. 4 show the distribution of results for S. aureus and P. aeruginosa. It was not possible to obtain repeatable measurements for S.
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mutans under the flow conditions.
Interactions of bisphosphonates with the hydroxyapatite surface
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Analyses performed using the Density Functional Theory method showed that pamidronate, clodronate, and zoledronate can all form strong electrostatic interactions with
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the surface of HA. The distance between the oxygen atom in the phosphonic group and the calcium ions in HA was comparable for all BPs and amounted to 2.38 Å for pamidronate and 2.4 Å for clodronate and zoledronate. Moreover, the protonated amine group of pamidronate could form two additional hydrogen bonds with oxygen atoms from the phosphate ion within the HA (Fig. 5, above), whereas the chlorine atoms in clodronate and the imidazol ring in zoledronate were too far from the HA surface to form any reasonable interactions, moreover the imidazole ring of the zoledronate possess a nitrogen atom with free electron pair which could even push back the negative charged oxygen ions of the hydroxyapatite (Fig. 5, below).
ACCEPTED MANUSCRIPT Diverse atomic structures on BPs, such as phosphate residues, carbon atoms, protons, chloride ions, or an amine ring, could serve as “hooks” for bacteria, facilitating bacterial adhesion. Moreover, the reactive cationic amino group of pamidronate could attract bacteria by direct
DISCUSSION
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electrostatic interaction.
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The results of this study show that BPs commonly used clinically, represented by non-
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aminobisphosphonates such as clodronate and aminobisphosphonates such as pamidronate and zoledronate, can enhance adhesion and promote the formation of biofilms by a broad spectrum of bacterial strains on HA discs coated with these substances, compared with nontreated control HA discs. Moreover, adhesion in the presence of pamidronate was
microorganism tested.
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significantly stronger in most cases than that for clodronate or zoledronate, regardless of the
Under stationary conditions, the adhesion of S. mutans and P. aeruginosa in the
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presence of BPs was significantly stronger; however, it was not possible to confirm the significantly higher adhesion of S. aureus strains, because of strong deviation of
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measurements, in this set. Therefore, we performed the experiment under flow conditions, which favors adhesion. Under flow conditions, both S. aureus and P. aeruginosa adhered much more strongly to discs saturated with BPs than to noncoated discs; however, under flow conditions, it was not possible to obtain repeatable results for S. mutans. The observation of this phenomenon strongly emphasizes the necessity of using different research models in order to obtain a fuller and more realistic picture of the relationship between HA and different microorganisms after treatment with BPs. The diversity of bacterial species adhering more strongly to BP-treated HA suggests a nonspecific rather than specific (i.e., restricted to one species or family) mechanism of the
ACCEPTED MANUSCRIPT interaction of these drugs with the HA surface. The importance of bacterial biofilm formation in the pathogenesis of BRONJ was reported previously, but the mechanisms responsible for bacterial colonization of denuded bone in patients on BPs were unclear (Sedghizadeh et al., 2008; Kos et al., 2010; Wei et al., 2012). Based on our experiments, one may suppose that
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BP-mediated bacterial adhesion plays an important role in this process.
Using the Density Functional Theory method to model the interactions showed that pamidronate may attach to HA through 4 bonds, whereas clodronate and zoledronate could be
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anchored with only 2 bonds, namely, phosphonic groups. When combined with HA,
pamidronate could allow the bacteria to anchor through 2 phosphate residues and 2 carbon
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atoms, as well as through hydrogen bonding. Clodronate exhibits similar interactions with bacteria, but does so through chloride ions, whereas the structure of zoledronate provides even greater possibilities for bacterial adhesion through the presence of an amine ring (Ragunath et al., 2008; Chen et al., 2011; Hinterdorfer et al., 2012). Despite these interactions, pamidronate
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has the greatest potential to create bonds with HA, more strongly promoting the formation of a biofilm. This suggests that the adhesion of bacteria to HA through a BP is accomplished indirectly. Consequently, BPs with the greatest potential for bonding to HA would promote
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the greatest extent of bacterial adhesion. The steps described in bacterial adhesion mechanisms probably favor the cells approaching closer to the bone surface then facilitating
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binding through typical interactions via adhesins. An expanded model of binding such as this could explain the higher adhesion rates in cases in which BPs were used. The results of this study are paramount in explaining issues in the pathophysiology of
BRONJ that are still unclear. The currently accepted theory of the origin of BRONJ assumes that impaired osteoclast function is followed by the impaired osteoblast activity due to the feedback between these two cell groups. Thus, the arrest of osteoclast function reflects not only diminished bone resorption but also reduced bone formation, both of which lead to decreased bone turnover and consequently to bone necrosis. Additional superinfection with
ACCEPTED MANUSCRIPT microorganisms leads to treatment-resistant osteomyelitis. However, although the BPs that are generally administered are deposited throughout the entire skeleton, the disease exclusively affects the jaws and has a high recurrence rate. Furthermore, although it can occur spontaneously, in the majority of cases it is provoked by dental surgery such as tooth
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extractions, root-tip resections, or cystectomies (Marx, 2003; Ruggiero et al., 2004).
Sometimes clinical symptoms are preceded by local trauma, pressure from ill-fitting dentures, or periodontal infection. These issues could be explained by increased adherence of
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microorganisms to the bone in the presence of BPs.
Exposure of bone during dental surgery acts as a trigger, opening the door to bacterial
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invasion. The presence of BPs promotes bacterial adhesion and biofilm formation on the surface of the affected bone, which is an essential step in the development of persistent osteomyelitis. In light of the above findings, jaw bones are especially subject to infection compared with other parts of the skeleton because they are more often subject to direct
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contact with the external environment due to the thin epithelial lining covering their surface, are susceptibility to trauma, and contain the teeth. Enhanced adhesion and biofilm formation should be considered when planning dental
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implant treatments in patients on BPs. There are reports confirming an increased infection rate in these patients with bacterial species known to be etiological factors of bone infections
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(Sedghizadeh et al., 2008; Wei et al., 2012). Based on the results of this study, one can assume that the other areas of BP
application (joint arthroplasty, treatment of open fractures, metabolic bone diseases) may result in an increased infection rate. Some clinical issues seem to have already confirmed this suspicion. Howe et al. (2013) reported a case of serious osteomyelitis after operative treatment of a femur shaft fracture in a patient on BPs. Nelson et al. (2005) questioned whether aseptic loosening of endoprosthesis was truly aseptic. The theory of increased bacterial adherence and biofilm formation could allow a better understanding of these
ACCEPTED MANUSCRIPT pathological conditions that was not previously clear. Thus, an identification and correct understanding of the pathology of osteomyelitis in patients on BPs may show that bone infection in patients on BPs is not restricted to the oral cavity. Furthermore, a better understanding of the pathophysiology connected with the use of BPs is paramount for
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continued safe clinical use of these drugs.
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CONCLUSION
The evidence of enhanced, bisphosphonate-linked bacterial adhesion to bone hydroxyapatite
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and biofilm formation could explain some unclear aspects of the pathophysiology of bisphosphonate-related osteonecrosis of the jaw, including the prevalent appearance after bone exposure and the recurrent nature of osteomyelitis accompanying BRONJ, and would justify antimicrobial treatment in BRONJ. Close attention should be paid to the potential for
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infection of open fractures, osteotomies, or implantations in patients on BPs and the safety of
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using BPs for improvement of stability in joint arthroplasty.
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Acknowledgements
Modeling calculations were carried out using resources provided by the Wroclaw Centre for Networking and Supercomputing (Wroclaw, Poland).
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ACCEPTED MANUSCRIPT Kos M, Junka A, Smutnicka D, Bartoszewicz M, Kurzynowski T, Gluza K: Pamidronate enhances bacterial adhesion to bone hydroxyapatite. Another puzzle in the pathology of bisphosphonate-related osteonecrosis of the jaw? J Oral Maxillofac Surg 71: 1010-
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Turniak M, Sedghizadeh PP: Microbial biofilms are able to destroy hydroxyapatite in the absence of host immunity in vitro. J Oral Maxillofac Surg doi: 10.1016/j.joms.2014.09.019.
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1232-48, 2008
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Chen Y, Busscher HJ, van der Mei HC, Norde W: Statistical analysis of long- and shortrange forces involved in bacterial adhesion to substratum surfaces as measured using atomic force microscopy. Appl Environ Microbiol 77: 5065-70, 2011
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ACCEPTED MANUSCRIPT Nelson CL, McLaren AC, McLaren SG, Johnson JW, Smeltzer MS: Is aseptic loosening truly
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aseptic? Clin Orthop Relat Res 437: 25-30, 2005
ACCEPTED MANUSCRIPT Fig. 1. System for testing bacterial adherence under flow conditions. The system consisted of a box with two insets (A) for placement of the hydroxyapatite discs (B), and openings for installing
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the peristaltic pump hoses (C). The suspension of the bacterial strain being tested (1) was pumped into the box. Excess fluid was removed to the collection vessel (2). The whole system was maintained at a temperature of 37°C with a thermostat (for better viewing, this is not
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shown in the picture). Fig. 2.
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Ability of the tested strains to form a biofilm on the surface of the hydroxyapatite. (A) Sterile and clean hydroxyapatite disc. (B) S. aureus forming multilayer structure with visible coccal cells (x947), (C) P. aeruginosa biofilm embedded within extracellular slime (x3390), (D) Dense structure of Str. mutans biofilm (dental plaque) with visible streptococcal cells (x1530). The
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representative images under different magnification were chosen because the size of cells and biofilm structure differs among different bacterial species.
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Fig. 3.
Comparison of the number of colony forming units / mm2 formed by the different bacterial
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strains in the presence of the tested bisphosphonate-coated or noncoated (control) hydroxyapatite discs under static conditions. Fig. 4.
Comparison of the number of colony forming units / mm2 formed by the different bacterial strains in the presence of the tested bisphosphonate-coated or noncoated (control) hydroxyapatite discs under flow conditions.
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Fig. 5. Simulation of the molecular structure of the surface of hydroxyapatite coated with different
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bisphosphonates using Density Functional Theory analysis. (Above). A particle of pamidronate in the NH+; PO2H* protonation state is presented. HP, Hydroxyapatite structure. Oxygen atoms (red) from phosphoric groups of pamidronate are
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palced over 2 calcium atoms (green) of hydroxyapatite and can form strong electrostatic interactions with the hydroxyapatite surface. The distance between the oxygen atom of the
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pamidronate phosphonic group and the calcium ions of hydroxyapatite is ~ 2.38 Å. The side chain of pamidronate containing the reactive NH3+ group protrudes over the hydroxyapatite structure and forms 2 additional bonds (green dashed lines), which may be reason for the increased level of bacterial adhesion. The distance NH1-O1 amounts ~ 1,852 Å, the distance
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NH2-O2 amounts ~1,849 Å. N-H angles to O from PO43- are γ N-H1-O1~142.4° and γ N-H1O1~137.7° respectively. Nitrogen atoms (blue), carbon atoms (gray), phosphorous molecules (orange), hydrogen atoms (white). (Below). Interactions of clodronate and zoledronate with
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the hydroxyapatite surface are presented. Both bisphosphonates, like pamidronate, can form
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strong electrostatic interactions with the hydroxyapatite surface. The distance between oxygen atoms in the phosphonic groups and calcium ions in hydroxyapatite is ~2.40 Å, but the chlorine atoms in the side chain of the clodronate and the imidazole ring of the zoledronate are too far from the hydroxyapatite to form any reasonable interactions. Moreover, the imidazole ring of the zoledronate possesses a nitrogen atom with free electron pair which could even push back the negative charged oxygen ions of the hydroxyapatite.
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Table 1. Distribution of statistically significant differences in adherence and biofilm formation of different bacterial strains on the hydroxyapatite discs coated with clodronate (B), pamidronate (P), or zolendronate (Z) and uncoated discs (K) during culturing under static conditions. P.aeruginosa Yes Yes Yes No No No
Str. mutans Yes Yes Yes No No No
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S. aureus Yes No No Yes Yes No
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P vs. K B vs. K Z vs. K P vs. B P vs. Z B vs. Z
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Table 2. Distribution of statistically significant differences in adherence and biofilm formation of different bacterial strains on the hydroxyapatite discs coated with clodronate (B), pamidronate (P), or zolendronate (Z) and uncoated discs (K) during culturing under flow conditions. P. aeruginosa Yes Yes Yes No Yes Yes
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S. aureus Yes Yes Yes Yes Yes Yes
P vs. K B vs. K Z vs. K P vs. B P vs. Z B vs. Z
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ACCEPTED MANUSCRIPT Bisphosphonates enhance biofilm formation on the surface of the bone hydroxyapatite.
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Bisphosphonates can promote osteomyelitis in treated patients.
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Bone surgery in the presence of bisphosphonates threatens with osteomyelitis.
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