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Polarized piezoelectric bioceramic composites exhibit antibacterial activity
Materials Chemistry and Physics 239 (2020) 122002
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Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Polarized piezoelectric bioceramic composites exhibit antibacterial activity Subhasmita Swain a, b, Rabindra Nath Padhy a, Tapash Ranjan Rautray b, * a
Central Research Laboratory, IMS and SUM Hospital, Siksha ‘O’ Anusandhan (Deemed to be University), Bhubaneswar, 751003, Odisha, India Biomaterials and Tissue Regeneration Laboratory, Centre of Excellence in Theoretical and Mathematical Sciences, Siksha ‘O’ Anusandhan (Deemed to be University), Khandagiri Square, Bhubaneswar, 751030, Odisha, India
b
H I G H L I G H T S
� Positively charged HA/BT composites showed antibacterial activity. � CLSM analysis confirmed the reduction of bacterial counts on polarized samples. � Positively polarized HA-BT composites rupture the bacteria membrane in vitro. A R T I C L E I N F O
A B S T R A C T
Keywords: piezoelectric Hydroxyapatite Polarization Anti-Bacterial Cell death
Electric charge is produced under the action of stress in a piezoelectric material. For enhanced bone growth and faster healing, biological piezoelectric materials can be effectively used. Since human bone has inherent electric charge for which it acts as a piezoelectric material, the substitutes of bone such as filler, cement or implant materials have attracted researchers in the field of biomedicine to investigate on effect of electrically active materials on bone growth. In this study, positively charged hydroxyapatite – barium titanate composites showed antibacterial activity against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa with remarkable inhibition zone and moreover Confocal Laser Scanning Microscopy confirmed the reduction of cell counts in polarized samples from qualitative and antibacterial rate percentage reduction studies. These bactericidal and bacteria repelling action may be attributed to the positively polarized surface of the hydroxyapatite – barium titanate composites that rupture the bacteria membrane during culture.
1. Introduction Bone is a natural composite material having a hierarchical structure formed spontaneously by its own components. The primary constituents of human bone are: collagen 30–40%, hydroxyapatite (HA) 60–70%, water 10–20%, some inherent proteins and essential minerals [1,2]. Human bone often shows piezoelectric actions against stress. In accor dance to Wolff’s law, the surface charge induced under stress is con nected with the production of bone functional shape [3,4]. Since HA is the primary inorganic constituent as compared to other components, its crystallographic structure controls the piezoelectricity [5]. Although vast research has been done in the field of biomaterials, less has been studied on the electrical properties of piezoelectric-bioceramic com posites [6]. The tetragonal, orthorhombic and the rhombohedral forms of BT are ferroelectric in nature. The tetragonal structure of BT shows ferroelec tricity, piezoelectricity having high dielectric constant for which it can
be used as a popular ferroelectric material applied in the fields of ca pacitors, energy storage devices and polarizable material [7]. Because of bacterial infections, most of the bioimplant procedures fail and need revision surgery [8,9]. Initially a biofilm is generated during growth and spreading of bacterial infection. Infections are caused by both Gram Positive (GP) and Gram negative (GN) bacteria [10]. GP bacteria are mostly Staphylococcus aureus, Streptococcus species, while GN bacteria are Enterococcus species, Enterobacter aerogens and Pseudomonas aerugi nosa [11]. Applications of antibiotics have been extensively used for bone infection management [12]. However, it requires the suitable antimicrobial stewardship program for complete control of infections. In this perspective, antibacterial agent would solve the persistent infection as long as in four weeks. Moreover, neither titanium nor calcium phosphate has antibacterial properties [13], implants with silver ion or TiO2 or ZnO or MgO as antibacterial agents are now-a-days used as coatings on implant materials [14,15]. To eradicate the infections completely, many methods have been adopted such as by changing the
* Corresponding author. E-mail address: [email protected] (T.R. Rautray). https://doi.org/10.1016/j.matchemphys.2019.122002 Received 16 April 2019; Received in revised form 5 August 2019; Accepted 8 August 2019 Available online 9 August 2019 0254-0584/© 2019 Elsevier B.V. All rights reserved.
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Materials Chemistry and Physics 239 (2020) 122002
compositional variation of implants containing Ag and ZnO ions [13, 15]; change in surface composition of implants such as by coating containing TiO2 nanowires [16], phospholipids [17] etc., changing the pH and concentration of NaCl, KCl etc. and by changing the roughness level of the coating and moreover by application of external factors such as electric and magnetic stimulation etc. are in effect continuously. Short or long term intake of antibiotics can be administered to a patient based on the drug used, the severity of an infection, and response to treatment. Due to deleterious effect of the use of antibacterial agents in bio materials simultaneously on human body, the use of polarized electric field has got great advantage that acts as a microbial inhibitor. Nega tively charged bacterial cells generally acquire charge on their surface due to ionization of functional groups such as phosphate-, amino-, hy droxyl- and carboxyl-groups. Recently the applications of external agents such as electric and magnetic fields have been employed to reduce bacterial populations [11]. Conversely, regulation of cell behavior is maintained by the electrical charge in the human body. The stimuli produced by intrinsic electrical charge in treating bacterial infection should be extensively researched that would control bacterial activities without any harm to the human body [18]. On the other hand, use of any organic or inorganic antimicrobial agents show adverse effect on the human body system. In the present study, we focus on the antibacterial study of polarized (100-x) HA-x BT (x ¼ 40, 60) i.e. the dense piezoelectric-bioceramic composites. The centre displacement of the positive and negative charges in the samples was caused by polarization as a result of which the negative and positive charges were developed on the opposite sur face respectively. The antibacterial effects of these HA-BT ceramics were evaluated.
2.1. Isolation, identification of bacterial strains and antibiotic sensitivity test GP bacteria (Staphylococcus aureus or S. aureus) and GN bacteria (Escherichia coli or E. coli, and Pseudomonas aeruginosa or P. aeruginosa) were extracted from patients suffering from bone fracture infections in the orthopedic department of IMS and SH, Bhubaneswar. Using a 4 mm thick Mueller–Hinton (MH) agar (HiMedia, Mumbai), antibiotic sensi tivity tests was performed [23]. On the polarized and non polarized 60BTþ40HA and 40BTþ60HA samples, the antibacterial activities were measured in terms of zones of inhibition produced by the samples. 2.2. Bacterial viability test One GP (S. aureus) and two GN (E. coli and P. aeruginosa) bacteria were used for the antibacterial study of 60HAþ40BT and 40HAþ60BT bioceramic-piezoelectric composites. All the bacterial strains were cultured in Mueller-Hinton Broth (MHB) medium [23]. MTT assay was used to measure in vitro bacterial viability. An average of three samples was taken from each composite sample. The samples were taken in a 24-well plate each well containing 1 mL bacterial suspension with a concentration of 1 � 106 CFU/mL and incubated at 37 � C. The in vitro bacteria culture was continued for 10 h, 20 h and 30 h and the adherent bacteria were evaluated for bacteria viability by adding 200 μL bacteria suspension with 200 μL MTT solution (5 mg/mL) and incubated at 37 � C for 6 h that formed formazan crystals. The optical density of the com posite solution was measured with a spectrophotometric microplate reader at a wavelength of 490 nm. 2.3. Antibacterial rate test
2. Materials and methods
The antibacterial rate test was performed to understand the comparative efficacy of the bacteria on the samples, hence no control was taken for that purpose. By counting bacteria number, the rate of antibacterial activity was performed for both the unpoled and poled samples in S. aureus, E. coli and P. aeruginosa bacteria suspension in 20 μL (1 � 106 CFU/mL) and incubated at 37 � C for 6 h. Immediately after incubation, the samples were rinsed with PBS solution for removal of non-adherent bacteria cells. Ultrasonic detachment of adherent cells was performed in 1 mL of sterile PBS solution and subsequently 100 μL of the bacteria suspension was spread onto the agar plates [24]. The bacteria number was counted on the samples after incubating the bacteria media at 37 � C. The antibacterial ratio was calculated using the following equation. Antibacterial ratio (%) ¼ AA B � 100. A is the average number of CFUs on control group and B is the average CFUs on experimental group.
Hydroxyapatite was prepared by precipitation method using ortho phosphoric acid and calcium hydroxide at a pH > 10. After ageing for 24 h, the slurry was washed thoroughly and filtered to obtain a sticky cake that was air dried at room temperature for 24 h and the dried cake was ground to fine powder and calcined at 1200 � C for 4 h to obtain hydroxyapatite [19]. Barium titanate was prepared by solid state route by using BaCO3 and TiO2 which were mixed in stoichiometric ratio and ground thoroughly in a ball mill in acetone milling medium for 12 h. The purpose of ball milling was to reduce the particle size as well as to obtain a homogeneous mixture. The dried powder mixture was then calcined at 1350 � C for 12 h in a platinum crucible to obtain barium titanate powder [20]. To obtain the pellets of composites in the ratio of 60 wt% BT þ 40 wt% HA (or 60BTþ40HA) and 40 wt% BT þ 60 wt% HA (or 40BTþ60HA), the said amount of the powder were thoroughly mixed with 1 wt% ethanol and 0.5 wt% urea as binder in deionized water and then the powder samples were pressed using a hydraulic press at 20 Ton pressure to obtain pellets of diameter 13 mm with thickness 2 mm and sintered at 1250 � C for 2 h. X-ray diffraction (XRD) analysis was per formed to confirm the presence of both HA and BT phases by using a Philips analytical X-ray diffractometer, X’Pert-MPD with PW 3020 ver tical goniometer and PW3710MPD control unit employing Bragg– Brentano parafocusing optics. The XRD data was collected at a scanning rate of 2� /min in 10–80� 2θ using CuKα-radiation (k ¼ 1.54056 Å) from an X-ray tube at 40 kV and 30 mA. For polarization, the samples were coated with silver at both the sides and polarized using positively charged electric field at a polari zation voltage of 2 kV/mm at 480 � C [21]. After polarization, the silver coating was removed and both the sides of the pellets were polished using 400 grit sized sand paper. After the removal of silver coating, the samples were checked using energy dispersive X-ray fluorescence (EDX) spectroscopic technique so as to ensure that no trace of silver is present on the sample surface [22]. Microbial activities were investigated in these samples in order to study their biological behavior towards the bacteria pathogens.
2.4. Bacteria adhesion observation The bacterial adhesion of all the three bacteria such as S. aureus, E. coli and P. aeruginosa were measured by taking a concentration of (1 � 106 CFU/mL) at 37 � C for 6 h. All the samples were affixed with paraformaldehyde (4% v/v) and were stained with Hoechst 33258 fluorescence dye at 4 � C for 5 min. Confocal laser scanning microscopy (CLSM) images were taken for the adhered bacteria on the samples. 2.5. Statistical analysis All the experimental results were expressed as mean � standard deviation. The differences in data were contrasted by one way analysis of variance (ANOVA). 3. Results XRD analysis confirmed the presence of phase pure HA and BT and is 2
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Materials Chemistry and Physics 239 (2020) 122002
Fig. 1. XRD pattern of HA/BT composite.
Fig. 2. Zone of inhibition of composites against S. aureusby disk diffu sion method.
depicted in Fig. 1. Antibiotic profiles of all the three pathogenic bacteria were identified by using antibiotic discs. While S. aureus showed resis tance to 13 out of 18 antibiotics, E. coli and P. aeruginosa were resistant to 11 and 12 respectively out of 18 antibiotics. Antibiotic resistance profile of most of the bacteria was found to be MDR (Table 1). Four composite samples (2 polarized þ 2 unpolarized) were studied for antibacterial test against all the three pathogenic bacterial species. The zone of inhibition using agar well diffusion method was carried out to quantify their antibacterial properties. Unpolarized samples showed very low level of zones of inhibition (Fig. 2). The highest zone of inhi bition obtained was for polarized 60BTþ 40HA sample that showed the zone of inhibition in the range of 25–29 mm (Table 2) for both GP and GN bacteria species. The polarized 40BTþ 60HA showed the zone of inhibition in the range of 20–26 mm for both GP and GN bacteria. On the other hand, the unpolarized composites showed very low level of zone of inhibition. The antibacterial study on the pure HA-BT samples and polarized samples were carried out for S. aureus, E. coli and P. aeruginosa patho genic bacteria. Initially the possible bacterial adhesion on the composite samples was quantitatively analyzed by using CLSM analysis from the live-dead cell images. Significantly poor antibacterial activity was observed for the unpolarized 60BTþ40HA and 40BTþ60HA samples as observed from the maximum number of green spots (live cells) in Fig. 3 for the S. aureus bacteria species cultured for 6 h. So, it can be inferred that S. aureus bacteria coming in contact with the unpolarized samples encountered negligible antibacterial activity. On the other hand, highest antibacterial activity was attributed to polarized 60BTþ40HA composite sample having more number of red spots (dead cells) followed by 40BTþ60HA sample for the same bacte rial species in same culture conditions (Fig. 4). After the CLSM
qualitative analysis, the antibacterial rate i.e. the percentage reduction of bacteria after culture for 6 h was quantified where it was found that the polarized 60BTþ40HA sample showed significant effect in reducing the bacteria number followed by 40BTþ60HA. On the other hand, the unpolarized samples showed insufficient killing efficiency for the bac terial species. From the quantitative analysis it was found that the polarized 60BTþ40HA sample against S. aureus, E. coli and P. aeruginosa showed significant effect in reducing the bacteria number by 47%, 46%, 41% respectively and polarized 40BTþ60HA sample against S. aureus, E. coli and P. aeruginosa showed significant effect in reducing the bac teria number by 43%, 35%, 34% respectively (Fig. 5). MTT assay was employed to measure the bacteria cell viability of S. aureus, E. coli and P. aeruginosa after 10 h, 20 h and 30 h of culture (Fig. 6, Fig. 7, Fig. 8). For all adherent bacteria cell types, polarized 60BTþ40HA and 40BTþ60HA showed restrained growth as compared to the unpolarized samples. These results are in consistent with the qualitative bacterial adhesion assay and quantitative zone of inhibition bacteriostasis rate results. Table 2 Zone of inhibition as observed on the bacterial species against the composites. Bacteria
Zone of inhibition
S. aureus E. coli P. aeruginosa
60BTþ40HA (Unpolarized)
60BTþ40HA (Polarized)
40BTþ60HA (Unpolarized)
40BTþ60HA (Polarized)
13 11 11
29 28 25
11 10 10
26 24 20
Table 1 Antibiotic susceptibility results of the used bacteria species. Bacterium
S. aureus E. coli P. aeruginosa
Susceptibility to prescribed antibiotics Amino-Glycosides
β-lactams
Cephalo-sporin
Fluoroquinolones
Glyco-peptides
Lincosamide
Sulfonamide
Stand alones
Ac
Ge
Ak
Am
Ox
Pt
Ce
Cf
Of
Le
Nx
Gt
Tei
Va
Cd
Cot
Ch
Lz
R R R
R R R
R R R
R R R
MS ND ND
R S R
R R R
R R R
R R R
R R R
R R R
R R MS
MS ND ND
MS ND ND
MS ND ND
R S R
R R R
S S S
Note: R: Resistant; S: Sensitive; MS: moderately sensitive; ND: not done. Antibiotics (μg/disc), Ac: amikacin 30; Ak: amoxyclav 30; Am: ampicillin 10; Cd: clindamycin 2; Cf: cefpodoxime 10; Ch: chloramphenicol 30; Cot: co-trimoxazole 25; Ce: ceftriaxone 30; Ge: gentamicin 10; Gt: gatifloxacin 5; Le: levofloxacin 5; Lz: linezolid 30; Of: ofloxacin 5; Ox: oxacillin 1; Pt: pipperacilin/tazobactam 100/10; Nx: norfloxacin 10; Tei: teichoplanin 5; Va: vancomycin 30. 3
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Materials Chemistry and Physics 239 (2020) 122002
Fig. 5. Anti-bacterial rates of composites. Fig. 3. CLSM image of unpolarized 60BTþ40HA sample.
Fig. 6. Viability of S. aureus adhered to composite. Fig. 4. CLSM image of polarized 60BTþ40HA sample.
4. Discussion The current investigation was carried out to understand the efficacy of polarized piezoelectric-bioceramics as antibacterial bone graft ma terials for biofilm prevention. Three pathogenic bacteria were cultured on both polarized and unpolarized HA þ BT composites taken in 60%:40% and 40%:60% w/w ratio respectively and the influence of these materials on the bacterial cells were assessed where it was found that the polarized composites show higher antibacterial activities. The primary fact behind the study is that the electric signal produced due to bone piezoelectricity plays a crucial role in bone tissue formation [25]. However, bacterial infection has posed a serious challenge on biocom patible implants. Many techniques have been adopted to eradicate the bacterial growth on implant surfaces that may lead to implant failure. In response to samples having antibacterial activity, both GP and GN bacteria have shown colony forming units (CFU) [26] and having zone of inhibition. For mimicking the natural bone, HA is used as a coating
Fig. 7. Viability of E. coli adhered to composite. 4
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incubated for 24 h showed 60% reduction in viability of S. aureus for pure HA while there was 70% reduction in same bacteria cells for HA-ZnO composites. In another study, glass ceramics of Cerec Blocs S3-M14 was polarized under a DC electric field of 0.1–1 kV/cm in air at 250 � C for 1 h; Streptococcus mutans incubated for 24 h reduced the bacterial adhesion on the substrates. From the present study, it can be summarized that the polarized materials have been found to inhibit pathogenic bacteria such as S. aureus, E. coli and P. aeruginosa. CLSM results confirmed that posi tively polarized HA þ BT composites reduced the adhesion ability of bacterial cells. MTT assay employed in this study was to quantify the viability of S. aureus, E. coli and P. aeruginosa adhered to the HA-BT composites after culture of 10, 20 and 30 h. The effective inhibition of bacteria cell viability suggested that the polarization of the composites had a synergistic bactericidal action on the cells. The positively charged HA þ BT may have blocked the pore channels of the adhered cells and the positive charge would rupture the bacteria membrane. The pores in the outer layer of bacterial cells are formed by proteins those are responsible for the charge transport. The positive electrical charge of the implant surface would neutralize the negative charges of bacteria membrane that could result in the structural change of the lipid layer and raise the permeability of bacterial cell membranes. The penetration and disruption of the cell membranes thus killed bacteria.
Fig. 8. Viability of P. aeruginosa adhered to composite.
material on implant surface but simultaneously HA attracts more bac teria in a bioenvironment [27]. Hence incorporation of some external agents such as antibacterial agent on implant surface can eliminate such issues. To kill bacteria using the antibacterial agents, various methods have been adopted which targets cell genome, respiration of cells, their division, metabolism and cell membrane as well. Due to lack of anti bacterial properties, HA cannot be used directly in prosthetic implants, rather HA has taken the assistance of external agents to be used in im plants. The external agents also reduce the use of antibiotics [28,29]. Recently external agents such as electric field, magnetic field and po larization have been used to control the formation of biofilm and bac terial adhesion to the implant surface [11]. Bacterial adhesion during the initial 6 h of implant surgery is a critical period that can continue bacterial infections [30] which may have deleterious effect on cell proliferation, colonization and formation of biofilms. Bacteria inhibition has been noticed by the use of external electric field, including its exposure time, pulse duration (in case of pulsed electric field [31]. The reason for the death of bacteria may be due to electrolysis of molecules on the surface of bacteria cells which gives rise to toxic substances such as H2O2, chlorine molecule and oxidizing rad icals [32]. A potential is induced on the cell membrane of the bacteria for short time that results in reducing membrane resistance. Generally electric field strength of 20kV–80kV is applied for short duration of time to kill bacteria cells [33]. For preventing biofilm formation and adhesion of bacteria cells, charge induction on biomaterial surface has now been adopted. The surface charge is induced at very high voltages and high temperature, for examples HA þ BT can be polarized by DC electric field of 2 kV/cm at 400 � C or above for 2 h duration. On the contrary it was decreased by 28% on negatively charged surface in similar conditions [34]. Water wetting characteristics of the poled surface is responsible for the antibacterial activity. When hydrophilicity is increased, the detachment of bacteria from the implant surface increases [34,35]. Hence it can be inferred that positively charged surface kills both GP and GN bacteria on biocomposite surface. It has been found from earlier studies that there is a decrease in cell numbers on HA surface by as much as 42.1% in S. aureus bacteria on a positively charged surface for 72 h incubation. On the other hand negatively charged surface enhanced the cell number by as much as 28% for similar conditions [36]. A low intensity electric field (1.5–30 V/cm) was applied on a glass substrate for the bacterial cells S. aureus incubated for 4 h and it was found that as high as 98% bacterial cells were decreased on the substrate [37]. In another study, composites of separately HA and HA with 10% ZnO was polarized with a DC electric field of 1 V/cm intensity and
5. Conclusion In this study positively charged HA þ BT composites showed anti bacterial activity against S. aureus, E. coli and P. aeruginosa with remarkable inhibition zone and CLSM confirmed the reduction of cell counts in polarized samples from qualitative and antibacterial rate percentage reduction studies. The HA þ BT polarized samples showed remarkable zones of inhibition against all the three types of bacteria. These bactericidal and bacteria repelling action may be attributed to the positively polarized surface of the HA þ BT composites that rupture the bacteria membrane during culture. The present findings show an alter native approach for effective topical antibacterial treatment in the implant sites. Acknowledgments This work was supported by Department of Science and Technology, Govt. of India, India with grant No. SB/SO/HS-113/2013 (A). References [1] K.H. Kim, T.R. Rautray, R. Narayanan, Surface Modification of Titanium for Biomaterial Applications, Nova Science Publisher, USA, 2010. [2] R. Rodriguez, D. Rangel, G. Fonseca, M. Gonzalez, S. Vargas, Piezoelectric properties of synthetic hydroxyapatite-based organic- inorganic hydrated materials, Results Phys. 6 (2016) 925–932. [3] S. Hu, F. Jia, C. Marinescu, F. Cimpoesu, Y. Qi, Y. Tao, A. Stroppa, W. Ren, Ferroelectric polarization of hydroxyapatite from density functional theory, RSC Adv. 7 (2017) 21375–21379. [4] H.M. Frost, Wolff’s Law and bone’s structural adaptations to mechanical usage: an overview for clinicians, Angle Orthod. 64 (1994) 175–188. [5] T. Leventouri, Synthetic and biological hydroxyapatites: crystal structure questions, Biomaterials 27 (2006) 3339–3342. [6] S.A.M. Tofail, A.A. Gandhi, M. Gregor, J. Bauer, Electrical properties of hydroxyapatite, Pure Appl. Chem. 87 (2015) 221–229. [7] M.H. Frey, D.A. Payne, Grain-size effect on structure and phase transformations for barium titanate, Phys. Rev. B. 54 (1996) 3158–3168. [8] S.D. Puckett, E. Taylor, T. Raimondo, T.J. Webster, The relationship between the nanostructure of titanium surfaces and bacterial attachment, Biomaterials 31 (2010) 706–713. [9] S.C. Ciobanu, L.S. Iconaru, P. Le Coustumer, L. Constantin, D. Predoi, Antibacterial activity of silver-doped hydroxyapatite nanoparticles against gram-positive and gram-negative bacteria, Nanoscale Res. Lett. 7 (2012) 324–334. [10] E.F. Berbari, A.D. Hanssen, M.C. Duffy, J.M. Steckelberg, D.M. Ilstrup, W. S. Harmsen, D.R. Osmon, Risk factors for prosthetic joint infection: case-control study, Clin. Infect. Dis. 27 (1998) 1247–1254. [11] A. Singh, A. Dubey, Various biomaterials and techniques for improving antibacterial response, ACS Appl. Bio Mater. 1 (2018) 3–20.
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Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Polarized piezoelectric bioceramic composites exhibit antibacterial activity Subhasmita Swain a, b, Rabindra Nath Padhy a, Tapash Ranjan Rautray b, * a
Central Research Laboratory, IMS and SUM Hospital, Siksha ‘O’ Anusandhan (Deemed to be University), Bhubaneswar, 751003, Odisha, India Biomaterials and Tissue Regeneration Laboratory, Centre of Excellence in Theoretical and Mathematical Sciences, Siksha ‘O’ Anusandhan (Deemed to be University), Khandagiri Square, Bhubaneswar, 751030, Odisha, India
b
H I G H L I G H T S
� Positively charged HA/BT composites showed antibacterial activity. � CLSM analysis confirmed the reduction of bacterial counts on polarized samples. � Positively polarized HA-BT composites rupture the bacteria membrane in vitro. A R T I C L E I N F O
A B S T R A C T
Keywords: piezoelectric Hydroxyapatite Polarization Anti-Bacterial Cell death
Electric charge is produced under the action of stress in a piezoelectric material. For enhanced bone growth and faster healing, biological piezoelectric materials can be effectively used. Since human bone has inherent electric charge for which it acts as a piezoelectric material, the substitutes of bone such as filler, cement or implant materials have attracted researchers in the field of biomedicine to investigate on effect of electrically active materials on bone growth. In this study, positively charged hydroxyapatite – barium titanate composites showed antibacterial activity against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa with remarkable inhibition zone and moreover Confocal Laser Scanning Microscopy confirmed the reduction of cell counts in polarized samples from qualitative and antibacterial rate percentage reduction studies. These bactericidal and bacteria repelling action may be attributed to the positively polarized surface of the hydroxyapatite – barium titanate composites that rupture the bacteria membrane during culture.
1. Introduction Bone is a natural composite material having a hierarchical structure formed spontaneously by its own components. The primary constituents of human bone are: collagen 30–40%, hydroxyapatite (HA) 60–70%, water 10–20%, some inherent proteins and essential minerals [1,2]. Human bone often shows piezoelectric actions against stress. In accor dance to Wolff’s law, the surface charge induced under stress is con nected with the production of bone functional shape [3,4]. Since HA is the primary inorganic constituent as compared to other components, its crystallographic structure controls the piezoelectricity [5]. Although vast research has been done in the field of biomaterials, less has been studied on the electrical properties of piezoelectric-bioceramic com posites [6]. The tetragonal, orthorhombic and the rhombohedral forms of BT are ferroelectric in nature. The tetragonal structure of BT shows ferroelec tricity, piezoelectricity having high dielectric constant for which it can
be used as a popular ferroelectric material applied in the fields of ca pacitors, energy storage devices and polarizable material [7]. Because of bacterial infections, most of the bioimplant procedures fail and need revision surgery [8,9]. Initially a biofilm is generated during growth and spreading of bacterial infection. Infections are caused by both Gram Positive (GP) and Gram negative (GN) bacteria [10]. GP bacteria are mostly Staphylococcus aureus, Streptococcus species, while GN bacteria are Enterococcus species, Enterobacter aerogens and Pseudomonas aerugi nosa [11]. Applications of antibiotics have been extensively used for bone infection management [12]. However, it requires the suitable antimicrobial stewardship program for complete control of infections. In this perspective, antibacterial agent would solve the persistent infection as long as in four weeks. Moreover, neither titanium nor calcium phosphate has antibacterial properties [13], implants with silver ion or TiO2 or ZnO or MgO as antibacterial agents are now-a-days used as coatings on implant materials [14,15]. To eradicate the infections completely, many methods have been adopted such as by changing the
* Corresponding author. E-mail address: [email protected] (T.R. Rautray). https://doi.org/10.1016/j.matchemphys.2019.122002 Received 16 April 2019; Received in revised form 5 August 2019; Accepted 8 August 2019 Available online 9 August 2019 0254-0584/© 2019 Elsevier B.V. All rights reserved.
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compositional variation of implants containing Ag and ZnO ions [13, 15]; change in surface composition of implants such as by coating containing TiO2 nanowires [16], phospholipids [17] etc., changing the pH and concentration of NaCl, KCl etc. and by changing the roughness level of the coating and moreover by application of external factors such as electric and magnetic stimulation etc. are in effect continuously. Short or long term intake of antibiotics can be administered to a patient based on the drug used, the severity of an infection, and response to treatment. Due to deleterious effect of the use of antibacterial agents in bio materials simultaneously on human body, the use of polarized electric field has got great advantage that acts as a microbial inhibitor. Nega tively charged bacterial cells generally acquire charge on their surface due to ionization of functional groups such as phosphate-, amino-, hy droxyl- and carboxyl-groups. Recently the applications of external agents such as electric and magnetic fields have been employed to reduce bacterial populations [11]. Conversely, regulation of cell behavior is maintained by the electrical charge in the human body. The stimuli produced by intrinsic electrical charge in treating bacterial infection should be extensively researched that would control bacterial activities without any harm to the human body [18]. On the other hand, use of any organic or inorganic antimicrobial agents show adverse effect on the human body system. In the present study, we focus on the antibacterial study of polarized (100-x) HA-x BT (x ¼ 40, 60) i.e. the dense piezoelectric-bioceramic composites. The centre displacement of the positive and negative charges in the samples was caused by polarization as a result of which the negative and positive charges were developed on the opposite sur face respectively. The antibacterial effects of these HA-BT ceramics were evaluated.
2.1. Isolation, identification of bacterial strains and antibiotic sensitivity test GP bacteria (Staphylococcus aureus or S. aureus) and GN bacteria (Escherichia coli or E. coli, and Pseudomonas aeruginosa or P. aeruginosa) were extracted from patients suffering from bone fracture infections in the orthopedic department of IMS and SH, Bhubaneswar. Using a 4 mm thick Mueller–Hinton (MH) agar (HiMedia, Mumbai), antibiotic sensi tivity tests was performed [23]. On the polarized and non polarized 60BTþ40HA and 40BTþ60HA samples, the antibacterial activities were measured in terms of zones of inhibition produced by the samples. 2.2. Bacterial viability test One GP (S. aureus) and two GN (E. coli and P. aeruginosa) bacteria were used for the antibacterial study of 60HAþ40BT and 40HAþ60BT bioceramic-piezoelectric composites. All the bacterial strains were cultured in Mueller-Hinton Broth (MHB) medium [23]. MTT assay was used to measure in vitro bacterial viability. An average of three samples was taken from each composite sample. The samples were taken in a 24-well plate each well containing 1 mL bacterial suspension with a concentration of 1 � 106 CFU/mL and incubated at 37 � C. The in vitro bacteria culture was continued for 10 h, 20 h and 30 h and the adherent bacteria were evaluated for bacteria viability by adding 200 μL bacteria suspension with 200 μL MTT solution (5 mg/mL) and incubated at 37 � C for 6 h that formed formazan crystals. The optical density of the com posite solution was measured with a spectrophotometric microplate reader at a wavelength of 490 nm. 2.3. Antibacterial rate test
2. Materials and methods
The antibacterial rate test was performed to understand the comparative efficacy of the bacteria on the samples, hence no control was taken for that purpose. By counting bacteria number, the rate of antibacterial activity was performed for both the unpoled and poled samples in S. aureus, E. coli and P. aeruginosa bacteria suspension in 20 μL (1 � 106 CFU/mL) and incubated at 37 � C for 6 h. Immediately after incubation, the samples were rinsed with PBS solution for removal of non-adherent bacteria cells. Ultrasonic detachment of adherent cells was performed in 1 mL of sterile PBS solution and subsequently 100 μL of the bacteria suspension was spread onto the agar plates [24]. The bacteria number was counted on the samples after incubating the bacteria media at 37 � C. The antibacterial ratio was calculated using the following equation. Antibacterial ratio (%) ¼ AA B � 100. A is the average number of CFUs on control group and B is the average CFUs on experimental group.
Hydroxyapatite was prepared by precipitation method using ortho phosphoric acid and calcium hydroxide at a pH > 10. After ageing for 24 h, the slurry was washed thoroughly and filtered to obtain a sticky cake that was air dried at room temperature for 24 h and the dried cake was ground to fine powder and calcined at 1200 � C for 4 h to obtain hydroxyapatite [19]. Barium titanate was prepared by solid state route by using BaCO3 and TiO2 which were mixed in stoichiometric ratio and ground thoroughly in a ball mill in acetone milling medium for 12 h. The purpose of ball milling was to reduce the particle size as well as to obtain a homogeneous mixture. The dried powder mixture was then calcined at 1350 � C for 12 h in a platinum crucible to obtain barium titanate powder [20]. To obtain the pellets of composites in the ratio of 60 wt% BT þ 40 wt% HA (or 60BTþ40HA) and 40 wt% BT þ 60 wt% HA (or 40BTþ60HA), the said amount of the powder were thoroughly mixed with 1 wt% ethanol and 0.5 wt% urea as binder in deionized water and then the powder samples were pressed using a hydraulic press at 20 Ton pressure to obtain pellets of diameter 13 mm with thickness 2 mm and sintered at 1250 � C for 2 h. X-ray diffraction (XRD) analysis was per formed to confirm the presence of both HA and BT phases by using a Philips analytical X-ray diffractometer, X’Pert-MPD with PW 3020 ver tical goniometer and PW3710MPD control unit employing Bragg– Brentano parafocusing optics. The XRD data was collected at a scanning rate of 2� /min in 10–80� 2θ using CuKα-radiation (k ¼ 1.54056 Å) from an X-ray tube at 40 kV and 30 mA. For polarization, the samples were coated with silver at both the sides and polarized using positively charged electric field at a polari zation voltage of 2 kV/mm at 480 � C [21]. After polarization, the silver coating was removed and both the sides of the pellets were polished using 400 grit sized sand paper. After the removal of silver coating, the samples were checked using energy dispersive X-ray fluorescence (EDX) spectroscopic technique so as to ensure that no trace of silver is present on the sample surface [22]. Microbial activities were investigated in these samples in order to study their biological behavior towards the bacteria pathogens.
2.4. Bacteria adhesion observation The bacterial adhesion of all the three bacteria such as S. aureus, E. coli and P. aeruginosa were measured by taking a concentration of (1 � 106 CFU/mL) at 37 � C for 6 h. All the samples were affixed with paraformaldehyde (4% v/v) and were stained with Hoechst 33258 fluorescence dye at 4 � C for 5 min. Confocal laser scanning microscopy (CLSM) images were taken for the adhered bacteria on the samples. 2.5. Statistical analysis All the experimental results were expressed as mean � standard deviation. The differences in data were contrasted by one way analysis of variance (ANOVA). 3. Results XRD analysis confirmed the presence of phase pure HA and BT and is 2
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Fig. 1. XRD pattern of HA/BT composite.
Fig. 2. Zone of inhibition of composites against S. aureusby disk diffu sion method.
depicted in Fig. 1. Antibiotic profiles of all the three pathogenic bacteria were identified by using antibiotic discs. While S. aureus showed resis tance to 13 out of 18 antibiotics, E. coli and P. aeruginosa were resistant to 11 and 12 respectively out of 18 antibiotics. Antibiotic resistance profile of most of the bacteria was found to be MDR (Table 1). Four composite samples (2 polarized þ 2 unpolarized) were studied for antibacterial test against all the three pathogenic bacterial species. The zone of inhibition using agar well diffusion method was carried out to quantify their antibacterial properties. Unpolarized samples showed very low level of zones of inhibition (Fig. 2). The highest zone of inhi bition obtained was for polarized 60BTþ 40HA sample that showed the zone of inhibition in the range of 25–29 mm (Table 2) for both GP and GN bacteria species. The polarized 40BTþ 60HA showed the zone of inhibition in the range of 20–26 mm for both GP and GN bacteria. On the other hand, the unpolarized composites showed very low level of zone of inhibition. The antibacterial study on the pure HA-BT samples and polarized samples were carried out for S. aureus, E. coli and P. aeruginosa patho genic bacteria. Initially the possible bacterial adhesion on the composite samples was quantitatively analyzed by using CLSM analysis from the live-dead cell images. Significantly poor antibacterial activity was observed for the unpolarized 60BTþ40HA and 40BTþ60HA samples as observed from the maximum number of green spots (live cells) in Fig. 3 for the S. aureus bacteria species cultured for 6 h. So, it can be inferred that S. aureus bacteria coming in contact with the unpolarized samples encountered negligible antibacterial activity. On the other hand, highest antibacterial activity was attributed to polarized 60BTþ40HA composite sample having more number of red spots (dead cells) followed by 40BTþ60HA sample for the same bacte rial species in same culture conditions (Fig. 4). After the CLSM
qualitative analysis, the antibacterial rate i.e. the percentage reduction of bacteria after culture for 6 h was quantified where it was found that the polarized 60BTþ40HA sample showed significant effect in reducing the bacteria number followed by 40BTþ60HA. On the other hand, the unpolarized samples showed insufficient killing efficiency for the bac terial species. From the quantitative analysis it was found that the polarized 60BTþ40HA sample against S. aureus, E. coli and P. aeruginosa showed significant effect in reducing the bacteria number by 47%, 46%, 41% respectively and polarized 40BTþ60HA sample against S. aureus, E. coli and P. aeruginosa showed significant effect in reducing the bac teria number by 43%, 35%, 34% respectively (Fig. 5). MTT assay was employed to measure the bacteria cell viability of S. aureus, E. coli and P. aeruginosa after 10 h, 20 h and 30 h of culture (Fig. 6, Fig. 7, Fig. 8). For all adherent bacteria cell types, polarized 60BTþ40HA and 40BTþ60HA showed restrained growth as compared to the unpolarized samples. These results are in consistent with the qualitative bacterial adhesion assay and quantitative zone of inhibition bacteriostasis rate results. Table 2 Zone of inhibition as observed on the bacterial species against the composites. Bacteria
Zone of inhibition
S. aureus E. coli P. aeruginosa
60BTþ40HA (Unpolarized)
60BTþ40HA (Polarized)
40BTþ60HA (Unpolarized)
40BTþ60HA (Polarized)
13 11 11
29 28 25
11 10 10
26 24 20
Table 1 Antibiotic susceptibility results of the used bacteria species. Bacterium
S. aureus E. coli P. aeruginosa
Susceptibility to prescribed antibiotics Amino-Glycosides
β-lactams
Cephalo-sporin
Fluoroquinolones
Glyco-peptides
Lincosamide
Sulfonamide
Stand alones
Ac
Ge
Ak
Am
Ox
Pt
Ce
Cf
Of
Le
Nx
Gt
Tei
Va
Cd
Cot
Ch
Lz
R R R
R R R
R R R
R R R
MS ND ND
R S R
R R R
R R R
R R R
R R R
R R R
R R MS
MS ND ND
MS ND ND
MS ND ND
R S R
R R R
S S S
Note: R: Resistant; S: Sensitive; MS: moderately sensitive; ND: not done. Antibiotics (μg/disc), Ac: amikacin 30; Ak: amoxyclav 30; Am: ampicillin 10; Cd: clindamycin 2; Cf: cefpodoxime 10; Ch: chloramphenicol 30; Cot: co-trimoxazole 25; Ce: ceftriaxone 30; Ge: gentamicin 10; Gt: gatifloxacin 5; Le: levofloxacin 5; Lz: linezolid 30; Of: ofloxacin 5; Ox: oxacillin 1; Pt: pipperacilin/tazobactam 100/10; Nx: norfloxacin 10; Tei: teichoplanin 5; Va: vancomycin 30. 3
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Fig. 5. Anti-bacterial rates of composites. Fig. 3. CLSM image of unpolarized 60BTþ40HA sample.
Fig. 6. Viability of S. aureus adhered to composite. Fig. 4. CLSM image of polarized 60BTþ40HA sample.
4. Discussion The current investigation was carried out to understand the efficacy of polarized piezoelectric-bioceramics as antibacterial bone graft ma terials for biofilm prevention. Three pathogenic bacteria were cultured on both polarized and unpolarized HA þ BT composites taken in 60%:40% and 40%:60% w/w ratio respectively and the influence of these materials on the bacterial cells were assessed where it was found that the polarized composites show higher antibacterial activities. The primary fact behind the study is that the electric signal produced due to bone piezoelectricity plays a crucial role in bone tissue formation [25]. However, bacterial infection has posed a serious challenge on biocom patible implants. Many techniques have been adopted to eradicate the bacterial growth on implant surfaces that may lead to implant failure. In response to samples having antibacterial activity, both GP and GN bacteria have shown colony forming units (CFU) [26] and having zone of inhibition. For mimicking the natural bone, HA is used as a coating
Fig. 7. Viability of E. coli adhered to composite. 4
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incubated for 24 h showed 60% reduction in viability of S. aureus for pure HA while there was 70% reduction in same bacteria cells for HA-ZnO composites. In another study, glass ceramics of Cerec Blocs S3-M14 was polarized under a DC electric field of 0.1–1 kV/cm in air at 250 � C for 1 h; Streptococcus mutans incubated for 24 h reduced the bacterial adhesion on the substrates. From the present study, it can be summarized that the polarized materials have been found to inhibit pathogenic bacteria such as S. aureus, E. coli and P. aeruginosa. CLSM results confirmed that posi tively polarized HA þ BT composites reduced the adhesion ability of bacterial cells. MTT assay employed in this study was to quantify the viability of S. aureus, E. coli and P. aeruginosa adhered to the HA-BT composites after culture of 10, 20 and 30 h. The effective inhibition of bacteria cell viability suggested that the polarization of the composites had a synergistic bactericidal action on the cells. The positively charged HA þ BT may have blocked the pore channels of the adhered cells and the positive charge would rupture the bacteria membrane. The pores in the outer layer of bacterial cells are formed by proteins those are responsible for the charge transport. The positive electrical charge of the implant surface would neutralize the negative charges of bacteria membrane that could result in the structural change of the lipid layer and raise the permeability of bacterial cell membranes. The penetration and disruption of the cell membranes thus killed bacteria.
Fig. 8. Viability of P. aeruginosa adhered to composite.
material on implant surface but simultaneously HA attracts more bac teria in a bioenvironment [27]. Hence incorporation of some external agents such as antibacterial agent on implant surface can eliminate such issues. To kill bacteria using the antibacterial agents, various methods have been adopted which targets cell genome, respiration of cells, their division, metabolism and cell membrane as well. Due to lack of anti bacterial properties, HA cannot be used directly in prosthetic implants, rather HA has taken the assistance of external agents to be used in im plants. The external agents also reduce the use of antibiotics [28,29]. Recently external agents such as electric field, magnetic field and po larization have been used to control the formation of biofilm and bac terial adhesion to the implant surface [11]. Bacterial adhesion during the initial 6 h of implant surgery is a critical period that can continue bacterial infections [30] which may have deleterious effect on cell proliferation, colonization and formation of biofilms. Bacteria inhibition has been noticed by the use of external electric field, including its exposure time, pulse duration (in case of pulsed electric field [31]. The reason for the death of bacteria may be due to electrolysis of molecules on the surface of bacteria cells which gives rise to toxic substances such as H2O2, chlorine molecule and oxidizing rad icals [32]. A potential is induced on the cell membrane of the bacteria for short time that results in reducing membrane resistance. Generally electric field strength of 20kV–80kV is applied for short duration of time to kill bacteria cells [33]. For preventing biofilm formation and adhesion of bacteria cells, charge induction on biomaterial surface has now been adopted. The surface charge is induced at very high voltages and high temperature, for examples HA þ BT can be polarized by DC electric field of 2 kV/cm at 400 � C or above for 2 h duration. On the contrary it was decreased by 28% on negatively charged surface in similar conditions [34]. Water wetting characteristics of the poled surface is responsible for the antibacterial activity. When hydrophilicity is increased, the detachment of bacteria from the implant surface increases [34,35]. Hence it can be inferred that positively charged surface kills both GP and GN bacteria on biocomposite surface. It has been found from earlier studies that there is a decrease in cell numbers on HA surface by as much as 42.1% in S. aureus bacteria on a positively charged surface for 72 h incubation. On the other hand negatively charged surface enhanced the cell number by as much as 28% for similar conditions [36]. A low intensity electric field (1.5–30 V/cm) was applied on a glass substrate for the bacterial cells S. aureus incubated for 4 h and it was found that as high as 98% bacterial cells were decreased on the substrate [37]. In another study, composites of separately HA and HA with 10% ZnO was polarized with a DC electric field of 1 V/cm intensity and
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