Single-phase, Antibacterial Tri-Magnesium Phosphate Hydrate

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Characterization, Synthesis, and Modifications

Single-phase, Antibacterial Tri-Magnesium Phosphate Hydrate Coatings on Polyetheretherketone (PEEK) Implants by Rapid Microwave Irradiation Technique Prabaha Sikder, Corey R. Grice, Boren Lin, Vijay K Goel, and Sarit B Bhaduri ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00594 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 7, 2018

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ACS Biomaterials Science & Engineering

Sikder et al. Antibacterial TMP coatings on PEEK

Single-phase, Antibacterial Tri-Magnesium Phosphate Hydrate Coatings on Polyetheretherketone (PEEK) Implants by Rapid Microwave Irradiation Technique Prabaha Sikdera*, Corey R. Griceb, Boren Linc, Vijay K. Goelc, Sarit B. Bhaduria a

Department of Mechanical, Industrial and Manufacturing Engineering, The University of Toledo, Toledo, OH 43606, USA b

Department of Physics & Astronomy, The University of Toledo, Toledo, OH 43606, USA c

Department of Bioengineering, The University of Toledo, Toledo, OH 43606, USA

Abstract This paper reports the fabrication and evaluation of single-phase, silver-doped tri-magnesium phosphate hydrate (Ag-TMPH) nanosheet coatings on polyetheretherketone (PEEK), a wellknown material used to fabricate orthopedic and spinal implants. While PEEK has better biomechanical compatibility with bone compared to metallic implants, it is also quite inert. Therefore, it is a common practice to coat PEEK implants with conventional calcium phosphates (CaPs) to enhance cell attachment, proliferation and differentiation. As opposed to well-studied CaP compounds, relatively less-explored magnesium phosphates (MgPs) are also becoming interesting orthopedic biomaterials and is the prime focus in this research. The novel aspects of this paper are as follows. First, we report developing TMPH coatings within minutes with the help of microwave irradiation technology. Microwave irradiation plays an important role in the coating formation with accelerated kinetics. Scanning electron microscopy (SEM) confirmed the fabrication of ⁓650 nm thick TMPH coatings. The coatings resulted in sub-micron level surface roughness and in vitro cell studies confirmed enhanced MC3T3 cell adhesion within 4 hours on such surfaces. Corresponding author: Tel.: +1 567-200-1995; E-mail address: [email protected] , [email protected] (Prabaha Sikder)

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The coatings also resulted in significant apatite formation after immersing in simulated body fluid for 7 days. Second, multi-functionality was achieved by doping TMPH coatings with Ag, thus rendering the coatings antibacterial. The antibacterial properties were evaluated against two most common infection causing bacterial strains – gram-negative Escherichia coli and grampositive Staphylococcus aureus. The results indicated good bacterial resistance and bactericidal properties of the Ag-TMPH coatings. Third, in spite of Ag doping, the single-phase nature of the coatings were retained (without forming composite systems) with the help of the low processing temperature of the microwave irradiation. The inductive coupled plasma technique confirmed that the doped single-phase TMPH coatings supported a uniform and controlled release of Ag+ ions over a period of 3 weeks. MTT assay evaluations and SEM micrographs confirmed no signs of cytotoxicity and healthy proliferation of cells in all cases. Quantitative real time PCR (qRTPCR) indicated a significant rise in collagen (Col1) and osteocalcin (OCN) gene expression levels in the case of TMPH coated PEEK. Thus, microwave irradiation was successfully employed in forming multi-functional i.e. bioactive, cytocompatible and antibacterial MgP coatings on PEEK. Keywords: Tri-magnesium phosphate hydrate, nanosheet coatings, bioactive, antibacterial, microwave irradiation. 1.0 Introduction Polyetheretherketone (PEEK) is well-recognized as an alternative material as opposed to its metallic counterparts in the implant industry. The chances of stress-shielding decreases significantly with the use of polymer PEEK, [1-2]. PEEK has a low Young’s modulus of 3.6 GPa and as opposed to titanium alloys with 110 GPa, it can be reinforced with carbon fibers and tailored to form the well-known carbon-fiber-reinforced PEEK (CFRPEEK) with an elastic 2 ACS Paragon Plus Environment

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modulus closely matching cortical bone (18 GPa) [1]. However, it is well known that unmodified PEEK is hydrophobic and bioinert in nature [1-2]. Indeed, in-vitro studies have proven insignificant cell proliferation on bare PEEK [2-4]. But most importantly, in the field of orthopedics, occurrences of surgical site infections (SSI) cause an array of problems like: morbidity of tissues at surgery sites, increased failure rates of the prostheses, second surgeries, and added medical costs. In a recent article published by the American College of Surgeons and Surgical Infection Society, it was reported that SSIs account for 20% of all hospital-acquired (HAI) or nosocomial infections. On a more destructive note, they result in a 2- to 11-fold increase in mortality risks. Further, SSI incidence has been calculated to be 2% to 5% in patients undergoing surgery and the estimated annual incidence lies in the range of 160,000 to 300,000 in the U.S. [5]. Finally, on grounds of financial burden, the annual cost for treating SSI is estimated at $3.5 to $10 billion in the U.S. alone [5]. Considering the primary two issues: bioinert nature of PEEK and SSI incidence, the broad theme of this research is to fabricate multi-functional i.e. bioactive, cytocompatible and antibacterial coatings on PEEK. Until now, calcium phosphate (CaP) based materials have been extensively used to enhance the bioactivity of CFRPEEK or PEEK. Reviews by Schwitalla et al. [6], Ma et al. [7], Najeeb et al. [2, 8], and Abdullah et al. [9], highlight the extensive research performed to enhance PEEK’s bioactivity. Bakar et al. incorporated hydroxyapatite (HA) into PEEK matrix and Wong et al. fabricated strontium (Sr)-containing HA/ PEEK composites to make PEEK bioactive [10, 11]. On the other hand, HA coated PEEK implants have exhibited enhanced cell proliferation and differentiation than uncoated ones both in-vitro and in-vivo. [12-15]. However, few drawbacks of the existing coating processes include: 1) severe damage of the PEEK substrate due to high processing temperature; 2) poor adhesion strength of coatings leading to delamination; 3) 3 ACS Paragon Plus Environment


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complex processing parameters, and 4) lack of cost-effectiveness [16-18]. Recently our group employed microwave irradiation technology to coat implant surfaces [13, 19-20]. However, as opposed to well-explored CaP coatings, biocompatibility/bioactivity of magnesium phosphate (MgP) coatings have not been explored much, as discussed in the following. The research in biomedical applications of MgPs is expanding fast and within the last decade, various MgP compounds (e.g., newberyite, struvite) have become important constituents in the development of newer biomaterials for orthopedic applications [21-24]. A recent review by Nabiyouni et al. highlights the great potential of MgPs in orthopedics [25]. Historically, the main focus was in fabricating self-setting MgP cements for orthopedics and our group made significant contributions in this field [26-28]. We are also the fist group to explore microwave assisted MgP coatings. For instance, Ren et al. efficiently developed biphasic MgPs to enhance corrosion resistance of Mg-AZ31 [29]. Bioactive amorphous MgP (AMP) was also successfully coated on sulfonated PEEK by microwaves [30]. However, nanostructured MgP coatings have not been explored on PEEK and recently Qi et al. reported commendable biocompatibility of bulk TMPH [31]. This is the reason for exploring nanostructured tri-magnesium phosphate hydrate (TMPH, Mg3(PO4)2.xH2O) as a coating material. . Besides, there are instances in the literature which have shown advantages of using MgPs as orthopedic materials over CaPs. Primarily, there are three main justifications which prove that MgPs are better than CaPs [32, 33]: 1) dissolution rates of MgPs are higher than those of CaPs which makes them more applicable as biodegradable materials with favorable resorption kinetics [23, 34]; 2) Mg2+ ions control several important intracellular activities and their presence stimulates better bone mineral metabolism (proliferation and differentiation) than CaPs [25, 35] and 3) Mg2+ ions inhibits HA crystal growth thus minimizing chances of unwanted crystallization into less soluble mineral 4 ACS Paragon Plus Environment

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phases in vivo [25, 36]. These are the prime motivations behind this present work which explores another such MgP application in orthopedics. Furthermore, with increasing chances of SSI, antibacterial MgPs need to be investigated as well. From a historical perspective, Ag-doped HA antibacterial coatings were well explored due to the outstanding bactericidal effect of Ag [37]. However, Ag-doped MgP coatings have neither been fabricated nor evaluated so far. Recently, our group successfully formed single-phase, antibacterial, silver-doped calcium deficient HA (Ag-CDHA) coatings on Ti6Al4V with the help of microwaves [38]. In the present effort, we investigate the formation of Ag-doped TMPH (AgTMPH) coatings and examine the antibacterial functions against two most popular infection causing strain - gram-negative Escherichia coli (E. coli) and gram-positive Staphylococcus aureus (S. aureus). It is noted that, in many cases, Ag-doping in HA have resulted in prominent cytotoxicity [39]. High processing temperatures and non-uniform material distribution often led to the formation of composite coatings with non-uniform and uncontrolled release of Ag [39]. In the present study, we employ an easy and scalable microwave irradiation technique, which is expected to promote uniform dopant distribution and form single-phase, Ag-TMPH coatings. Additionally, microwave irradiation is quite effective in coating polymers at low temperatures without any thermal degradation of substrates [13, 30, 40]. The major expectation was that the fabricated coatings will be multi-functional, i.e. bioactive, cytocompatible and antibacterial at the same time.

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2.0 Experimental 2.1 Material Preparation & Pre-treatment PEEK rods (medical grade), 304 mm long and 6.25 mm in diameter were cut into disk samples (Ø 6.25 x 3 mm3). Sample surfaces were polished with SiC papers to attain mirror finish, followed by subsequent ultrasonication in ethyl alcohol and ultra-pure (UP) water. After cleaning, they were dried at room temperature and immersed in 10 mol/L sodium hydroxide (NaOH) at 600C for 48 hours. Post alkali treatment, the samples were cleaned and kept ready for the coating process. 2.2 Coating Preparation Magnesium nitrate hexahydrate (Mg(NO3)2.6H2O, 98% purity) was used as the source of magnesium (Mg2+) ions, sodium hydrogen phosphate monobasic anhydrous (NaH2PO4, > 98% purity) was used for the phosphate (PO43-) ions source and silver nitrate (AgNO3, ≥ 99% purity) for the Ag+ ion source. The detailed coating solution compositions and respective specimen names are shown in Table 1. The reagents were added one by one into 200 ml UP water and were dissolute properly using a magnetic stirrer at 260 rpm. The pH value was adjusted to 8 by adding 1mol/L NaOH solution. After proper dissolution, three alkali treated PEEK discs were placed in 100 ml of the coating solution and were irradiated at the highest power in a 1200W microwave oven (Panasonic) for 5 minutes. The coating cycle was repeated with the remaining coating solution. Finally, the coated PEEK discs were retrieved, washed in UP water and dried at room temperature. Simultaneously, the precipitates from the coating bath were collected, centrifuged at 3000 rpm, washed and dried for further characterization.


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2.3 Coating Characterization The phase compositions of the precipitates and as-coated samples were identified by X-ray diffraction (XRD, Ultima III, Rigaku) with focused beam mono-chromated Cu Kα radiation (44 kV, 40 mA). Fourier transform infrared spectroscopy (FTIR, UMA-600 Microscope, Varian Excalibur Series) were used to characterize the functional groups of the coatings, using an ATR diamond crystal for 256 scans in the range between 4000 and 700 cm-1 with a resolution of 1 cm1

. Surface morphologies and elemental compositions of the coated samples were analyzed using

scanning electron microscopy (SEM, S4800, Hitachi) equipped with an energy dispersive X-ray spectroscopy (EDS, INCA, Oxford). Elemental analyses were performed at 20 kV with a 15 mm working distance. Cross-section samples and images were prepared using a dual-beam environmental SEM (FEI Quanta) equipped with gallium (Ga) ion source. Atomic force microscopy (AFM, Nanoscope V, Veeco Instruments) was operated in the tapping mode using a 1Ω Si probe tip over a 20µm x 20µm area. Optical profilometry (NV5000 5010, Zygo Instruments) was done using scanning white light inteteferometer over 356µm x 267µm area. The adhesion bond strengths of the coatings to the substrate were measured using tensile tests on a mechanical testing machine (858 Bionix, MTS). Aluminum studs were mounted and alligned carefully onto the specimens using epoxy glue and cured at 1500C for 1 hour. After cooling down to room temperature, the specimens were placed in specially designed fixtures in the machine and pulled with a speed of 0.5 mm/min until failure occurred. The recorded load at failure was used to calculate the adhesion strength [14, 16]. Water contact angle measurements were performed using a contact angle meter (CAM-MTCRO, Tantec) at room temperature.



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2.4 Ag+ Ion Release The Ag-TMPH coated PEEK samples were immersed in 5 ml phosphate buffered saline (PBS) at 370C over a period of 21 days to study the release kinetics of Ag+ ions [41]. At specific time points, the PEEK discs were taken out and the elemental concentrations were checked using inductive coupled plasma mass spectrometer (ICP-MS, XSeries2, Thermo Scientiﬁc). 2.5 In-vitro Antibacterial test The antibacterial functionality of the coatings were assessed against E. coli

(Strain: W3110,

ATCC 39936) and S. aureus (Strain: Seattle 1945, ATCC 25923). Super broth (SB) was used as the culture medium. 2.5.1 Zone of Inhibition (ZOI) Assay To assess the antibacterial activity distal to the coated implants, traditional ZOI tests were carried out on agar plates. Bacterial suspensions were spread uniformly on individual SB agar plates using circular beads. The coated specimens were then placed on the agar plates. TMP-0Ag served as the control in this assay. The agar plates were kept in an incubator for 2, 4 and 7 days at 370C and at specific time periods, ZOIs were measured using Vernier calipers. ZOIs were quantified using the following formula: ZOI coefficient = Total ZOI area / Specimen area. 2.5.2 OD600 Readings and Counting Colony forming Units (CFUs) Method Simultaneous OD600 recordings and counting CFUs method were used to determine the bactericidal property of the coatings [21]. The coated samples were placed in 24 well plate containing SB in a quantity which represented a medium volume/ samples surface area ratio of 0.33 ml/cm2. The well plate was kept covered in an incubator at 370C for 48 hours without any agitation. At the end of the time period, conditioned media or extracts were collected and used to culture E.coli and S. aureus. An overnight culture was diluted and added to the conditioned 8 ACS Paragon Plus Environment

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media to achieve a concentration of ∼108 CFUs/ml. Un-conditioned SB media (which contained no samples) with the specific concentration (∼108 CFUs/ml) was used as a control. The set-up was then moved into an incubator maintained at 370C. OD600 readings were recorded at specific time points (after 1, 5, 9, 12, 24 hours) using a spectrophotometer (BioTek, USA). Simultaneously, at specific time points, bacterial suspension was removed and serial dilutions were made using PBS and plated out on agar plates and incubated overnight at 370C to count the CFUs. 2.5.3 Morphology of Adhered Bacteria All the coated and PEEK samples were placed in a 24 well plate and seeded with 1 ml of bacterial concentration (∼108 CFUs/ml) of E. coli and S. aureus. After incubating them for 24 hours at 370C, the samples were retrieved, washed three times in PBS and immersed in 3 % glutaraldehyde fixing solution for 1 hour at room temperature. After fixation, they were dried sequentially in 30, 50, 70, 90, 95, 100 % ethyl alcohol followed by a hexamethyldisilazane (HMDS) treatment. Finally, they were dried overnight in a fume hood (Safeaire II, Thermo Scientific) with an air flow velocity 220 feet/min, coated with gold-palladium for 30 secs and imaged under SEM. 2.6 Simulated Body Fluid (SBF) Immersion In our studies related to SBF immersion, we have been using a modified version of the original Kokubo composition (c-SBF) [42] as per the suggestion of Tas [43]. This was done to mimic the HCO3- ion content presence in blood plasma and is termed as t-SBF. The ionic compositions of tSBF are tabulated in Table 2 [43]. Coated and bare alkali etched samples (served as control) were placed in individual 100 ml bottles filled with 30 ml t-SBF and kept in a thermostatic water 9 ACS Paragon Plus Environment


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bath at 37 ± 0.5 °C for 7 days. Moreover, t-SBF was replenished every 24 hours. Finally, the samples were retrieved, washed and dried at room temperature. XRD and SEM were performed to study the effect of t-SBF immersion on the samples. 2.7 In vitro Cytocompatibility Tests Pre-osteoblast MC3T3 mouse cells (CRL-2593™, ATCC, Manassas, VA, USA) were used for mammalian cell studies. The cells were cultured in complete minimum essential medium alpha (MEM-α, Thermo Scientific), supplemented with 10% fetal bovine serum (FBS, HyClone) and 1% penicillin/streptomycin (0.2g/ml) at 5% CO2 and 37⁓. All the samples were autoclaved before each assay and after cooled down to room-temperature, they were used for cell-culture. HA coated PEEK was chosen as a comparison material or control in the in-vitro cell proliferation assay. HA coatings were formed on PEEK as per the previous work published by our group [13]. 2.7.1 Cell Viability and Cell Proliferation - MTT Assay MTT assay was used to determine the cell viability of MC3T3 pre-osteoblasts cultured on coated and bare PEEK [44]. ⁓2.3 x 104 cells were seeded on each specimen and cultured in complete media for 3, 7, 14 or 21 days. At specific time periods, the specimens were retrieved, rinsed three times in PBS and prepared for the assay. After dissolving thiazolyl blue tetrazolium bromide (MTT, Sigma Aldrich, USA) powder in PBS to form the MTT stock solution, they were added to the specimens and control, followed by 4 hours incubation at 370C. Dimethyl sulfoxide (DMSO) was used to dissolve formazan and finally OD570 readings were recorded using a spectrophotometer.


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2.7.2 Osteogenic Gene Expression - Quantitative Real-Time (qRT) PCR In this assay, MC3T3 cells were seeded at 90% confluency in 10 cm dishes and further cultured with control or coated specimens for 3 or 7 days. At the end of specific time periods, RNA from these cells was extracted by TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and complementary DNA produced by reverse transcription using M-MLV reverse transcriptase (Promega, Madison, WI, USA). qRT PCR was performed using SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) by a 2-step amplification program (30s at 95°C and 60s at 62°C) on a thermal cycler (Eppendorf, Hamburg, Germany). The relative quantification of mRNA of the gene of interest was determined by the 2-∆∆CT method and presented as fold change compared to control samples. All reactions were run in triplicate. The forward and reverse primers for targeted

genes

are

listed

as

follows:

osteocalcin

GCAATAAGGTAGTGAACAGACTCC-3′andreverse

(OCN;

forward

5′-

5′-CTTTGTAGGCGGTCTTCAAGC-

3′), alkaline phosphatase (ALP; forward 5′-ATCTTTGGTCTGGCTCCCATG-3′ and reverse 5′TTTCCCGTTCACCGTCCAC-3′)

and

type

1

collagen

(COL1;

forward

5′-

GAGCGGAGTACTGGATCG-3′ and reverse 5′-GCTTCTTTTCCTTGGGGTT-3′). 2.7.3 Initial Cell Adhesion and Cell Morphology of Pre-osteoblasts Samples were kept in 48 well plate and seeded with ⁓6.5 x 104 cells. After 4 hours, samples were retrieved, washed three times in PBS and immersed in 3 % glutaraldehyde fixing solution for 1 hour at room temperature. After fixation, they were dried sequentially in 30, 50, 70, 90, 95, 100 % ethyl alcohol followed by a HMDS treatment. Finally, they were dried overnight in a fume hood and imaged under SEM. Simultaneously, MTT was performed in the same manner as described in 2.7.1. In another 48 well plate, bare PEEK and coated specimens were seeded with



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⁓ 2.4 x 104 cells/specimen. After 3, 7 or 14 days, the samples were retrieved from the culture medium and prepared for SEM imaging in the same manner as described above. 2.8 Statistical Analysis All test results represented means ± standard deviation in triplicates. One-way analysis of variance with Tukey test was conducted to determine the statistical difference between groups and ρ< 0.05 were considered significant. 3. Results 3.1 Coating characterization 3.1.1 XRD Analysis Figure 1a shows the XRD patterns of bare and coated PEEK specimens. The major peaks detected pertain to substrate PEEK in case of the coated samples. However, all the peaks from the XRD analysis of the powders i.e. the precipitates that were collected during the coating process, match with TMPH (Mg3(PO4)2.4H2O, PDF: 97-005-1490) as shown in Figure 1b [31]. No peaks related to other MgP or Ag compounds are present in the diffraction studies. 3.1.2 FTIR Analysis The FTIR results of bare and coated samples are shown in Figure 1c. The intensity of all the absorption bands pertaining to bare PEEK substrate appear much less in case of the coated specimens. Moreover, two significant absorption bands at 1014 cm-1 related to the phosphate group (PO43-) and 3150 cm-1 corresponding to structural water can be identified [31, 45]. Figure 1d which shows the FTIR results of the as-synthesized powders, give a much more comprehensible understanding about the coating composition. The broad absorption bands at 3438 cm-1 and 1653 cm-1 relates to the structural water present in MgP hydrates. The 12 ACS Paragon Plus Environment

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characteristic PO43- group is located in the range 1200-900 cm-1 and in the present case, five distinctive absorption bands at 1120 cm-1, 1068 cm-1, 1045 cm-1, 1014 cm-1 and 960 cm-1 can be resolved [45]. Additionally, the absorption peak at 775 cm-1 corresponds to symmetric stretching mode of P2O72- [31]. 3.1.3 SEM Analysis The set of data presented in Figure 2(a-d) shows the SEM micrographs, EDS results and crosssectional images of various kinds of TMPH coated PEEK specimens. The coatings comprise of randomly oriented polyhedral shaped thin sheet like structures. However, they mainly contain diamond shaped sheets. The dimension (longest diagonal) of the sheets range approximately from 100nm to 250nm with a thickness of about 20nm [31]. High instability of MgPs in aqueous medium (mainly alkaline) results in the wide size distribution of TMPH sheets. However, Agdoping at any level has no influence on coating morphology. The EDS results confirm the presence of Mg, P, O and Ag elements in un-doped and doped coatings respectively. The Mg/P atomic ratios as calculated lie in the range of 1.25 -1.50. The CS micrographs indicate partially compact coatings with presence of pores, successfully deposited by the microwave irradiation technique on PEEK. Stacking and random orientation of the as-deposited TMPH sheets are vivid in the CS micrographs. The substrate-coating interface show no presence of peeling and thermal degradation. Ag-doping at any level also does not influence the coating thickness. However, the coating thickness is not uniform over the whole surface area and lies in the range of 600-745 nm with a mean of 670 nm. 3.1.4 Surface Roughness& Adhesion Strength The roughness values and 3-dimensional images of bare and coated specimens as analyzed by AFM are presented in Table 3a and Figure 3a. Contrary to bare PEEK surfaces, the coatings exhibit roughness in the lower sub-micron range over 20µm x 20µm area. However, as shown in 13 ACS Paragon Plus Environment


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Table 3b and Figure 3b, optical profilometry analysis reveal the roughness of coatings to be much greater, in the higher end of the sub-micron scale. SEM images (Supplementary file, Figure S1) show the TMPH agglomerations over the actual coatings. Studies have reported about enhanced cell proliferation and gene expressions due to increase in roughness of the implant surface [46]. The set-up for adhesion testing of specimens is shown in Figure 3(c). The adhesion strengths of the TMPH coatings are recorded to be in the range of 11 MPa. However, the coating roughness and adhesion strength are not relevant to the level of Ag doping. 3.1.5 Water-Contact angle measurements The water-contact angle measurements (Supplementary file, Figure S2) denote a massive decrease (⁓75%) in case of the coated PEEK specimens with significant statistical difference with the un-treated ones. Irrespective of Ag doping, TMPH coatings exhibit a water contact angle in the range of ⁓200 suggesting high hydrophilicity of deposited TMPH. 3.1.6 Ion release The Ag+ ion release kinetics from the various coatings, as recorded by the ICP-MS technique is plotted in Figure 4. The nature of the release profile in case of three different kinds of AgTMPH coatings is quite similar. During the first 7 days, Ag+ release from all the coatings follows a steep rise. The effect is most prominent in TMP-5Ag due to the high concentration of doped Ag. However, after 7 days, in all cases, the release becomes gentle and tends to attain a steadystate. The moderate release profile of Ag+ after a week of incubation, indicates the long-term sustainable nature of Ag-TMPH coatings in terms of dopant release.


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3.2 In-vitro Antibacterial Properties 3.2.1 ZOI Test The formation of ZOI around a sample indicates its bacterial inhibition potential around itself. Figure 5a presents the representative photos of prominent ZOIs formed against E. coli by all the Ag-doped coated specimens in contrast to the control or un-doped ones (TMP-0Ag) after 2 days of incubation. The ZOIs formed by the Ag-doped coated specimens against S. aureus are shown in Figure 5b. It can be clearly seen that the extent of ZOIs formed against S. aureus is lesser than the ones formed by E. coli. The calculated ZOI coefficient helps in the better quantitative understanding of the ZOI areas. The ZOI coefficient results indicate two things: 1) the ZOI area is much smaller in case of S. aureus as compared to E. coli and; 2) the extent of ZOI formation or bacterial resistance is dependent on the level of Ag doping. Correspondingly, TMP-5Ag exhibits the largest ZOI with significant statistical difference with the ones formed by other Agdoped specimens. However, all of the doped samples showed distinct ZOIs even after 7 days. 3.2.2 Bactericidal Property Growth of bacteria corresponds to an increase in OD600 readings. Figure 6a shows the OD600 readings of E.coli and S. aureus as cultured in the coating extracts and control. During the first 1 hour, OD600 readings are almost similar for all. After 5 hours, a moderate increase is recorded in case of the control, TMP-0Ag and TMP-1Ag extracts. The increase is remarkable with prominent signs of turbidity after 9, 12 and 24 hours. OD600 readings also increase in case of TMP-1Ag, but not as much as the control. In contrast, OD600 readings in case of TMP-3Ag and TMP-5Ag extracts, do not elevate and remain nearly constant all throughout the assay time in case of E. coli. However, in case of S. aureus, a slight increase in readings is noticed but without any prominent signs of turbidity. 15 ACS Paragon Plus Environment


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Bactericidal property of the coatings are assessed by counting the CFUs formed by live/viable bacteria. Figure 6b shows the average number of CFUs formed by viable E.coli and S. aureus cultured in the coating extracts and control after 1, 5, 9, 12 and 24 hours. As expected, bacteria grow exponentially when cultured in un-conditioned media (control) and TMP-0Ag extracts. The average CFUs are also high when cultured in TMP-1Ag extracts, thus it does not exhibit satisfactory bactericidal effect. Whereas, the number of CFUs decrease significantly over increase in incubation time when both the bacterial strains is cultured in TMP-3Ag and TMP5Ag extracts. The antibacterial effect is the most prominent in the extracts containing higher Agdoping extracts, with much better effects to be seen in case of E. coli. Approximately 3Log kill to 5Log kill is recorded in case of E. coli and 2Log kill to 4 Log kill is recorded in case of S. aureus. 3.2.3 Morphology of Adhered Bacteria Figures 7(a-b) show the typical morphology of adherent E.coli and S. aureus onto bare PEEK and various kinds of TMPH coatings. The capsule-like morphology of E.coli and spherical shape of S. aureus is evident in the SEM micrographs. When considering several different scan areas in SEM, visually reduced number of both the bacterial strains are seen to adhere the Ag-TMPH coatings as compared to un-doped and bare PEEK. The insets which contain the high magnification images of the bacteria reveal the damaged cell walls of lysed E.coli and S. aureus (red arrows) on the Ag-doped samples as opposed to well-rounded ones on bare PEEK and TMP-0Ag [38] In some cases, the effect is not so evident with only irregular cell walls (TMP1Ag), while in some cases, the effect is very prominent with Ag+ ions completely destroying the cell membranes thus killing the bacteria (TMP-3Ag & TMP-5Ag).


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3.3 SBF Immersion The bioactivity of the coated samples are assessed in-vitro by SBF immersion for 7 days and the results are presented in Figure 8(a-c) [42]. In contrast to the scarce and inconsistent amount of spherical particles formed on the surface of alkali-etched specimens, overwhelming amount of spherical particles can be seen on the TMPH coated specimens. The as-deposited coatings cannot be figured out in the SEM micrographs as the spherical particles, about 3µm in size, completely covered the surface. Higher magnification micrographs reveal the characteristic flake-like morphology of the particles. [42, 43]. The EDS spectra (Figure 8b & Supplementary Fig S3) recorded from the particles formed on the t-SBF immersed specimens reveal the presence of high amounts of Ca, P and C. The average Ca/P ratio calculated is about ⁓1.52, which is approximately the same as that of the bone mineral. XRD results of all the samples immersed in t-SBF for 7 days are plotted in Figure 8c. No additional peaks are seen in the case of bare and alkali etched PEEK samples immersed in SBF. However, broadened peaks pertaining to apatite are vividly observed at ⁓ 250 and ⁓ 330 in case of all the TMPH coated samples. Combined results of SEM, EDS and XRD analysis substantiate the copious formation of bone-like, carbonated, calcium-deficient apatite on the coated specimens indicating high bioactivity of TMPH nanosheets [42]. 3.4 In-vitro cell-studies 3.4.1 Cell Proliferation Figure 9a shows the results of cell viability on the specimens as measured by the MTT assay. After 3 or 7 days, all un-doped and Ag-doped specimens show higher MC3T3 cell viability than bare PEEK denoting enhanced cytocompatibility. Ag-doping at all levels in the present study does not induce any cytotoxic effect. Even though TMP-0Ag exhibit higher cell viability at every 17 ACS Paragon Plus Environment


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time periods, TMP-3Ag and TMP-5Ag show statistically significant increase in MC3T3 proliferation after 3 and 7 days respectively. However, after 14 and 21 days, cell viability recorded on all the specimens are same as by this time they reach a saturation on the specimen surface area. Further, in all cases, TMP-0Ag yielded results almost equal to HA coated PEEK specimens (supplementary file, Fig S4). 3.4.2 Gene expressions The osteogenic differentiation properties of the coated PEEK samples as assessed by qRT-PCR of ALP, collagen (Col 1), Osteoclacin (OCN) mRNA expressions are presented in Figure 9(bd). In case of all the three gene markers, mRNA expression levels show no significant difference as compared to the control after 3 days. After 7 days, TMPH coated specimens show an increase in ALP expressions, though not statistically significant with the control (Figure 9b). However, in case of Col1, after 7 days (Figure 9c), statistically significant increase in mRNA expressions are observed with TMP-0Ag and TMP-1Ag samples in contrast to coatings doped with higher wt.% of Ag. Interestingly, a significant enhancement in OCN expression levels can be observed after 7 days in case of all the coated samples except TMP-5Ag (Figure 9d). Further, as per previous reports [22], MgP results in healthy pre-osteoblast cell differentiation almost equal to CaPs. 3.4.3 Initial cell adhesion and Cell-morphology SEM images and MTT assay in Figure 10a show the numerous amount of MC3T3 preosteoblast cells attached during the first 4 hours on the TMPH coatings. Bare PEEK’s bioinert nature is evident from the scanty number of attached cells. Interestingly, the cells attach preferably all over the randomly oriented TMPH sheets highlighting their favorable biocompatibility [31]. The growth morphology of MC3T3 over 3, 7 and 14 days are shown in 18 ACS Paragon Plus Environment

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Figure 10b & Supplementary Fig S5. Cells on all coated samples exhibit comparable morphology (typical fibroblastic pre-osteoblast) to those attached on bare PEEK. The preosteoblasts possess a flattened morphology, forming a healthy network on all the coated specimens by cytoplasmic extension and filopodia attachment. Additionally, copious deposits of extra cellular matrix (ECM) completely covered the surfaces for which the coatings cannot be seen. However, at the end of 14 days, individual MC3T3 cell structures cannot be distinguished on bare and coated PEEK specimens, as they reach a saturation and connect to each other forming sheet-like layers. 4. Discussion This paper has significant interdisciplinary attributes as noted before. An important feature of this study is the use of MgP, specifically TMPH as a multi-functional coating. So far, TMPH have received much less attention with respect to newberyite or struvite. Precipitation of TMPH from an aqueous solution was first studied by Tamimi et al. [22]. No other studies explored this new material until Qi et al. formed TMPH nanosheets with the help of microwaves [31]. We chose this as our coating material as it is comparatively more reliable to attain a uniform coating over PEEK with nanostructured MgP, in this case TMPH. The mechanism of formation and deposition of TMPH coatings over PEEK in the present study follows a dissolution-precipitation mechanism as shown in Figure 11a. Interestingly, Qi et al formed TMPH nanosheets with a starting solution which precipitated AMP at room temperature [31]. Similarly, Ostrowski et al. precipitated AMP from a highly basic, saturated solution at room temperature and heat treated it up to 7500C to form highly crystalline, anhydrous tri-magnesium phosphates (TMP, Mg3(PO4)2) [47]. In contrast, Tamimi et al. formed TMPH by elevating the temperature of the solution which precipitated cattite (Mg3(PO4)2.22H2O) at room temperature 19 ACS Paragon Plus Environment


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[22]. However, the synthesis took 24 hours. In the present case, cattite precipitates instantaneously at room temperature due to similar ionic concentrations of starting solutions to that of Tamimi et al [22]. Aqueous precipitation of MgPs under various conditions have been studied previously by researchers [48, 49]. Divalent Mg can coordinate with high levels of H2O molecules because of its high charge and small ionic radii. Moreover, the Gibbs free energy required to remove the H2O molecules from the outer coordination shell in cattite is relatively low for which the water molecules stay coordinated to Mg3(PO4)2 at low temperatures (≤ 200C). With the increase in processing temperature, cattite gains energy and easily dissociates the outer H2O molecules thus forming TMPH [22]. Due to the high instability of cattite at slightly higher temperature than 200C, it is believed that TMPH starts to form right (⁓30 secs) after the exposure of microwaves. At this point, it is also important to understand the coating deposition mechanism of the TMPH sheets on the surface of alkali etched PEEK. Previous reports have demonstrated about how NaOH treatment aids in the formation of potential apatite nuclei on polymeric surfaces when immersed in SBF [50]. Briefly, immersion in NaOH at 600C first results in the addition of Na to the existing PEEK structure. When the alkaline PEEK surfaces are immersed in the aqueous coating bath, the Na+ ions release with a simultaneous and rapid deposition of OH- on the surface. This results in an overall negative charge on PEEK which then attracts the abundant Mg2+ ions from the coating solution. Finally, the overall positive charge due to Mg2+ on the surface, attracts the negatively charged PO42- ions and combine with them resulting in the formation of TMPH [30]. Over processing time, more and more TMPH sheets form and stack up to increase the coating thickness. Here, microwave irradiation plays two significant roles: 1) It accelerates the dissolution of irregular, large cattite sheets and helps in


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forming and freezing well-defined diamond shaped TMPH nanosheets within minutes, and, 2) It also accelerates the deposition of TMPH on PEEK. In the diffraction studies, XRD did identify some TMPH peaks but with the high intensity signals from PEEK substrate, those get concealed. The nano-crystalline nature and nanometer level thickness of the TMPH coatings also reduces sharpness. This is why the precipitates are analyzed distinctively to obtain a better understanding of coatings’ composition. Whole pattern fitting (WPF) of XRD peaks reveal the crystallite size of TMPH precipitates to be in the nanometer range (Table S1). The presence of single-phasic TMPH is ascertained with good matches of all compositions with PDF database and previous reports (Fig 1b) [31]. Studies have emphasized about inhomogeneous material distribution in Ag-HA composite coatings leading to undesirable in-vitro scenarios [39]. On the contrary, in the present case, homogeneous Ag distribution is achieved and single phase nature of Ag-TMPH coatings might be retained due to low processing temperature (⁓1200C) and effect of microwaves. Yet another helpful feature of microwave exposure is that the low processing temperature also helps in preventing any thermal degradation of the substrate [13, 30]. Even though PEEK is a high temperature polymer, degradation can result from high processing temperature. When plasma spraying was used to coat HA on CFRPEEK, it resulted in a low density, chemically inhomogeneous coating with poor adhesion strength. [18]. As opposed to that, CS micrographs in the present study signify three features: 1) the substrate is devoid of any thermal or chemical degradation because of the benign processing conditions in microwave irradiation technique; 2) the TMPH sheets are bonded to PEEK chemically without the absence of any peeling and; 3) a



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variation in coating thickness is noted which is mainly due to the uncontrolled rate of TMPH deposition and their random orientation of stacking on the PEEK substrate. Random stacking and orientation of the TMPH sheets deposited by microwaves also lead to nano-level surface roughness when AFM is employed to scan a small area. But, when OP is performed over a much greater area, the roughness values increase. Fig S1 shows the TMPH agglomerates at some places over the actual coatings. It is believed that these congregations are the main reasons for influencing the drastic increase in roughness. Moreover, non-uniform substrate (PEEK) topography also play a major role in influencing the coatings roughness then. Also, adhesion strength between the coating and the substrate is an important criteria for determining the quality of the coatings [51]. Adhesion strengths of TMPH coatings synthesized by microwave irradiation lie in the range of 11 MPa which is in well accordance with previously reported HA coatings on PEEK by other technologies [14, 51]. Be it enhancing corrosion resistance or bioactivity, MgP coatings have shown promising results [29, 30]. However, MgPs are not bactericidal by itself [24]. With increasing chances of SSIs and wide usage of PEEK in orthopedics and dental applications, neither antibacterial MgPs nor other bactericidal coatings on PEEK have been explored. Even though HA coatings have been used much on PEEK, few efforts have been made to incorporate Ag into it [52-53]. Reactive oxygen species (ROS) resulting from oxidation of titanium nanoparticles showed no antibacterial effect against E.coli [54]. In case of sulfonated PEEK, E.coli survived the localized acidic environment [55]. Thus, two possible antibacterial mechanisms rendered ineffective against E.coli, let alone S. aureus which is much more difficult to kill. On the contrary, antibacterial effectiveness here is based on Ag+ ions release from the doped TMPH coatings. ICP-MS results recorded for every 22 ACS Paragon Plus Environment

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coatings during the 1st day is more than 0.1 ppm which is required for a prominent antibacterial effect [56]. Further, the trend of Ag+ release with a steep rise during the 1st week followed by a sustained nature has been reported by previous researchers [36, 56-57].

Interestingly, after 3

weeks, the highest Ag+ concentration recorded in the present case is ⁓ 1.2 ppm, which is less than the cytotoxic concentration (1.6 ppm) [56]. Countless contemporary research have been using Ag in coatings to form antibacterial coatings [41, 56-58]. However, strict attention demands to regulate the controlled release of Ag. For instance, Xu et al. doped Ag nanoparticles into one-dimensional titanate nanowires followed by a chitosan nanofilm deposition with the help of spin-assisted layer by layer assembly method [58]. Chitosan acted as a controlling material to check Ag release. On the contrary, in the present case, no additional material was used and the sustainable release of Ag+ ions resulted in antibacterial effect with no signs of cytotoxicity. Ag+ ions is bactericidal because of its capability to interfere and disrupt the bacteria’s breathing mechanisms [37]. Successful diffusion of Ag+ ions into the agar surfaces result in prominent ZOIs around the samples. Constant OD600 readings indicate no growth in E.coli and S. aureus over 24 hours, however, it is not sufficient to determine their bactericidal effect as spectrophotometer picks up signals from both dead and live bacteria. The CFU method confirms that even though the low Ag+ release from TMP-1Ag can somewhat resist only the E.coli growth, but it is not sufficient enough to reveal a prominent bactericidal effect. Whereas, high release of Ag+ ions from TMP-3Ag and TMP-5Ag lead to remarkable reduction of bacteria. In case of both strains of bacteria, ⁓2 - 3 log kill and ⁓4 - 5 log kill are recorded at the end of 9h and 12h respectively; results on basis of which the coatings can be claimed antibacterial in the U.S. Subsequent to any surgery, the first 24 hours are very critical due to the compromised 23 ACS Paragon Plus Environment


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nature of the immune system. If bacterial colonization can be inhibited at the implants and surgical site within that time point, the chances of SSIs diminishes drastically. In the present experiment, by the end of 24 hours, E.coli no longer could shield against the Ag+ ions and completely abated. However, the scenario was different when the gram-positive strain was used. The antibacterial functionality attained in case of S. aureus was less compared to E. coli for the very reason that gram-positive S. aureus possess a much thicker cell wall as compared to E. coli, thus making it more difficult for the Ag+ ions to penetrate through and kill them [38]. Finally, bacterial anti-adhesion surfaces is much needed to prevent biofilm formation [5]. Ag+ from doped TMPH sheets inhibited bacterial attachment and biofilm formation. The broken cell walls of lysed cells validate the fact Ag+ penetrated through E.coli and S. aureus membranes, somehow confirming the antibacterial mechanism [38]. On the same note, considering the concept of “Race for the surfaces”, as soon as an implant is embedded inside the body, its fate is determined by a race between bacteria and osteoblasts. Defeating bacterial colonization, good initial adhesion of bone cells onto the implant surface is highly significant for eventual tissue integration and their successful working [44, 59]. In the present case, MC3T3 pre-osteoblasts adhere well within the first 4 hours and continue spreading with a flattened morphology for 14 days on the TMPH sheets, signifying high affinity of the preosteoblast to them (Fig S5 & Fig 10(a-b)). Further, the SBF immersion studies confirming the presence of copious apatite suggest high surface mineralization capability of the TMPH coatings as opposed to etched PEEK [42, 43]. Higher rate of apatite forming ability in SBF predicts a faster in vivo bone binding capability of the TMPH coated PEEK implants [42]. Previous studies have shown the in vitro dissolution and re-precipitation of primary MgP phases like newberyite, struvite or AMP into hydrated TMP, 24 ACS Paragon Plus Environment

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typically bobierrite (Mg3(PO4)2.8H2O) [26-27, 29]. Interestingly, in the present case, TMPH do not dissolve in SBF, but gets concealed by a thick layer of apatite as detected by XRD. Indeed, studies have confirmed about the greater stability of TMPH as compared to well-known MgP compounds (newberyite, AMP) due to its lower solubility in SBF [22, 29]. However, it demands an explanation for the formation of CaP globules on TMPH. The apatite formation mechanism is pictorially shown in Figure 11b. It is believed that TMPH with as low as four bonded molecules of water is much more stable in the physiological medium [22, 29]. When the coatings are immersed in SBF, along with the continuous sustained release of Mg2+ ions from TMPH, negatively charged OH- groups accumulate on the coated surfaces. This results in an overall negative charge which readily attracts the Ca2+ ions from the SBF solution. The settling down of Ca2+ ions gives the surface a positive charge which is responsible for pulling the PO43- ions, thus finally forming apatite globules on the TMPH sheets. The continuous consumption of Ca2+ and PO43- ions from SBF helps the globules to grow in size (Z direction) over the immersion time [20, 42]. A final comment is about the effect of Mg2+ ions on apatite structure. It is well known that Mg2+ ions inhibit apatite crystallization. Zhao et al. substituted 50 mol% of Ca2+ with Mg2+ and observed highly amorphous apatite structure as opposed to crystallized apatite yielded by the un-substituted one [20]. In the present study, we attain similar results with broadened XRD peaks at ⁓ 250 and ⁓ 330 after SBF immersion (Figure 8c). This signifies that Mg2+ release from the coatings might have inhibited complete crystallization which resulted in the formation of nanocrystalline apatite on the TMPH coatings. However, it is believed that in the present case, the slow dissolution of Mg2+ ions from TMPH do not create a pronounced inhibition effect on 1) dissolution of the coatings and, 2) crystallization of apatite globules.



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Enhancement in cell proliferation in vitro indicates more bone formation in vivo around the implant [44, 46]. In the present study, with enhanced pre-osteoblast proliferation at every time frame, the TMPH coated specimens are expected to witness a strong bone-implant integration. Considering the biodegradability of MgPs, presence and release of Mg2+ is one of the prime reasons to stimulate cell proliferation [21-23], however, excess might cause cytotoxic effects [47]. Favorable biocompatibility and hydrophilicity of TMPH coatings are additional factors [31]. Surface morphology of implants plays an important factor in cytocompatibility. Although the relationship is unclear, in the present case, random orientation of TMPH sheets resulting in sub-micron surface roughness provide larger surface / interfacial contact area for filopodia attachment and cytoplasmic extension. Thus, this factor also leads to enhanced MC3T3 adhesion and proliferation [31, 46]. Finally, single-phase Ag-TMPH support sustainable Ag+ release which result in no cytotoxicity as opposed to composite Ag-CaPs [39]. To investigate the effects of various TMPH coating on the differentiation of MC3T3 preosteoblast cells, expression of common makers of osteoblast differentiation such as ALP, Col1 and OCN were assessed. Previous studies reported by Tamimi et al. suggested that Col1 and OCN, both indicating a later stage marker of osteoblast differentiation, were highly expressed in case of newberyite and cattite, in a pattern similar to hydroxyapatite or brushite. Whereas, ALP expression, a marker for the earlier stage of osteoblast differentiation was significantly lower in case of MgPs as compared to CaPs [22]. ALP expressions were not further induced as reported by Ren et al. when MC3T3 were cultured on AMP coated PEEK samples, which may be due to the rapid release of magnesium and consumption of calcium during biomineralization [30]. Consistently, TMPH coatings do not promote ALP expression in pre-sosteoclast MC3T3 cells compared to bare PEEK after a 7-day culturing but markedly augment Col1 and OCN 26 ACS Paragon Plus Environment

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expressions, which to some effect was interfered by Ag content incorporated in the coatings. The robust ECM formation during MC3T3 cell proliferation (Fig 10b) also support the observation of high Col1 expression enhanced by TMPH coatings [44]. The highest level of Ag-doping i.e. TMP-5Ag specimens significantly reduce both Col1 and OCN expressions indicating that this specific Ag content inhibits osteoblastic differentiation. However, TMP-3Ag show higher OCN expressions as opposed to Col1. An important point of consideration is that the Ag-doping level needed to achieve satisfactory antibacterial effectiveness might sometimes interfere with specific expressions levels of osteoblastic differentiation. A critical doping trade-off always remains in achieving good bactericidal effect and also enhanced osteoblastic activities. In this case, TMP3Ag kills favorable number of S. aureus and E. coli while showing accelerated biominearlization in vitro [22, 44]. Thus, Ag-doped MgP coatings prove to be reliable systems which can significantly reduce SSI in orthopedics while safe-guarding the actual work of the implants. 5.0 Conclusion In this study, we successfully fabricated multi-functional coatings on PEEK via microwave irradiation. The prime advantages of the present fabrication technique are as follows: 1) it involves low processing temperature which prevents thermal degradation of PEEK, 2) the whole process takes place in minutes highlighting the enhanced kinetic acceleration of coating deposition and 3) the low temperature and effect of microwaves help in uniform dopant distribution which helps in retaining single phase nature of Ag-TMPH. The doped coatings resulted in sustained release of Ag+ prompting in satisfactory antibacterial effect against E.coli. Factors like hydrophilicity, sub-micron surface roughness and good cytocompatibility resulted in enhanced pre-osteoblast (MC3T3) adhesion, proliferation. qRT-PCR results revealed an upregulation in Col1 and OCN gene expressions. However, in some cases the increase was not as 27 ACS Paragon Plus Environment


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high as un-doped coatings. Considering Ag’s cytotoxic effects, a trade-off should be maintained in the Ag-doping level to get the best antibacterial and cytocompatibility results. In the present study, TMPH coatings doped with 3 wt. % Ag resulted in enhanced pre-osteoblast cell proliferation, differentiation and also terminated E.coli and S. aureus growth. Supporting Information Additional information about physical and in vitro characterization of various specimens are present in the supporting information document of this paper. Acknowledgements The work is funded by NSF grant no 1312211. Reference [1] S.M. Kurtz, J.N. Devine, PEEK biomaterials in trauma, orthopedic, and spinal implants, Biomaterials 28 (2007) 4845-4869. http://dx.doi.org/10.1016/j.biomaterials.2007.07.013 [2] S. Najeeb, M.S. Zafar, Z. Khurshid, F. Siddiqui, Applications of polyetheretherketone (PEEK) in oral implantology and prosthodontics, J Prosthodont Res. 60 (2016) 12-19. http://dx.doi.org/10.1016/j.jpor.2015.10.001 [3] L.M. Wenz, K. Merritt, S.A. Brown, A. Moet, A.D. Steffee, In vitro biocompatibility of polyetheretherketone and polysulfone composites, J Biomed Mater Res A. 24 (1990) 207215. http://dx.doi.org/10.1002/jbm.820240207 [4] M. Zhao, M. An, Q. Wang, X. Liu, W. Lai, X. Zhao, S. Wei, J. Ji, Quantitative proteomic analysis of human osteoblast-like MG-63 cells in response to bioinert implant material titanium and polyetheretherketone, J. Proteom 75 (2012) 3560-3573. http://dx.doi.org/10.1016/j.jprot.2012.03.033 [5] K.A. Ban, J.P. Minei, C. Laronga, B.G. Harbrecht, E.H. Jensen, D.E. Fry, K.M. Itani, E.P. Dellinger, C.Y. Ko, T.M. Duane. American College of Surgeons and Surgical Infection Society: surgical site infection guidelines, 2016 update. Journal of the American College of Surgeons 224 (2017) 59-74. http://dx.doi.org/10.1016/j.jamcollsurg.2016.10.029 [6] A. Schwitalla, W.D. Müller, PEEK dental implants: a review of the literature, J Oral Implantol. 38 (2013) 743-749. http://dx.doi.org/10.1563/AAID-JOI-D-11-00002 [7] R. Ma, T. Tang, Current strategies to improve the bioactivity of PEEK, Int. J. Mol. Sci. 15 (2014) 5426-5445. http://dx.doi.org/10.3390/ijms15045426 [8] S. Najeeb, Z. Khurshid, J.P. Matinlinna, F. Siddiqui, M.Z. Nassani, K. Baroudi, Nanomodified peek dental implants: Bioactive composites and surface modification—A review, Int J Dent (2015) 1-7. http://dx.doi.org/10.1155/2015/381759 28 ACS Paragon Plus Environment

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List of Tables Table 1: Compositions of coating solutions. Table 2: Ion concentrations of t-SBF and human blood plasma. Table 3: Roughness values as calculated by (a) Atomic force microscopy (AFM) and (b) Optical Profilometry (OP)

List of Figures Fig 1: XRD analysis of (a) bare and coated PEEK specimen, (b) precipitates collected after the microwave-irradiation process. FTIR analysis of (c) bare and coated PEEK specimen (d) precipitates collected after microwave irradiation process. Nomenclature: (i) TMP-0Ag, (ii) TMP-1Ag, (iii) TMP-3Ag and (iv)TMP-5Ag. Fig 2: SEM morphology, EDS spectra and cross-sectional micrographs of (a) TMP-0Ag, (b) TMP-1Ag, (c) TMP-3Ag and (d) TMP-5Ag specimens. Fig 3: Three dimensional images showing roughness as analyzed by (a) Atomic force microscopy (AFM) and (b) Optical profilometry (OP). (c) Set-up showing adhesion testing of coatings. Fig 4: ICP results showing the Ag+ ion release kinetics from the doped TMPH coatings. Fig 5: Representative zone of inhibition (ZOI) images and ZOI coefficients of coated PEEK specimens after 2 days incubation against (a) E. coli (b) S. aureus *indicates statistically significant (Tukey test, ρ< 0.05) Fig 6: (a) OD600 readings of E.coli and S. aureus as cultured in control and coating extracts; (b) Corresponding number of CFUs representing live E.coli and S. aureus as cultured in the same extracts. *indicates statistically significant (Tukey test, ρ< 0.05). Fig 7: SEM images showing adherent (a) E. coli (b) S. aureus on various surfaces. The insets show the high magnification images of individual bacteria. Red arrows indicate the effect of Ag+ ions on the bacterial morphology. Fig 8: (a) Sporadic spherical apatite particles formed on alkali etched PEEK as opposed to overwhelming apatite formed on coated specimens after immersing in t-SBF for 7 days. (b) EDS results of apatite globules formed on TMP-5Ag. (c) XRD analysis of SBF immersed specimens. Fig 9: (a) MTT assay results of MC3T3 pre-osteoblasts cultured on specimens over 21 days. qRT-PCR results showing mRNA expressions of (b) ALP, (c) COL1 and (d) OCN gene markers of MC3T3 cells after culturing on the specimens over 7 days. *indicates statistically significant with respect to Bare PEEK, indicates statistically significant with respect to TMP-0Ag, indicates statistically significant with respect to TMP-0Ag and TMP-1Ag (Tukey test, ρ< 0.05)



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Fig 10: (a) MC3T3 cell adhesion on various surfaces within 4 hours of incubation.*indicates statistically significant with respect to Bare PEEK (Tukey test, ρ< 0.05). (b) Growth morphology of MC3T3 cells over 14 days. Fig 11: Mechanism showing the formation of (a) Ag-TMPH coatings on PEEK via microwaveirradiation and (b) CaP apatite globules formed on the TMPH coatings after immersing them in t-SBF for 7 days at 370C.


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Specimen Name

Mg(NO3)2.6H2O (gms) 2.3076 2.284 2.238 2.192

TMP-0Ag TMP-1Ag TMP-3Ag TMP-5Ag

NaH2PO4 (gms) 1.607 1.607 1.607 1.607

AgNO3 wt.% 0 1 3 5

H2 O (ml) 200 200 200 200

Table 1 Ion

Concentration (mM) Human blood plasma t-SBF (pH 7.4)

Na+ K+ Mg2+ Ca2+ ClHCO3HPO42SO42-

142.0 5.0 1.5 2.5 103.0 27.0 1.0 0.5

142.0 5.0 1.5 2.5 125.0 27.0 1.0 0.5

Table 2 Serial No

Specimen Name

Roughness Values (Ra)

RMS Values

1. 2. 3. 4. 5.

Bare PEEK TMP-0Ag TMP-1Ag TMP-3Ag TMP-5Ag

44.9 nm 128 nm 265 nm 318 nm 210 nm

60.6 nm 163 nm 338 nm 415 nm 300 nm

Table 3a Serial No

Specimen Name

Roughness Values (Ra)

RMS Values

1. 2. 3. 4. 5.

Bare PEEK TMP-0Ag TMP-1Ag TMP-3Ag TMP-5Ag

124.5 ± 0.01 nm 494 ± 0.072 nm * 608 ± 0.055 nm * 738.33 ± 0.017 nm *a 801.33 ± 0.144 nm *a

170.5 ± 0.01 nm 699.33 ± 0.1 nm * 1104.67 ± 0.136 nm * 1043.67 ± 0.024 nm * 1486.33 ± 0.35 nm *a

Table 3b 35 ACS Paragon Plus Environment


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Table of Contents graphic Title: Single-phase, Antibacterial Tri-Magnesium Phosphate Hydrate Coatings on Polyetheretherketone (PEEK) Implants by Rapid Microwave Irradiation Technique Authors: Prabaha Sikder, Corey R. Grice, Boren Lin, Vijay K. Goel, Sarit B. Bhaduri


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Revised Graphical Abstract 304x166mm (120 x 120 DPI)



Figure 1(a) : XRD analysis of bare and coated PEEK specimen 264x217mm (120 x 120 DPI)


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Figure 1(b) : XRD analysis of precipitates collected after the microwave-irradiation process. 199x170mm (120 x 120 DPI)



Figure 1(c) : FTIR analysis of (c) bare and coated PEEK specimen 289x229mm (120 x 120 DPI)


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Figure 1(d): FTIR analysis of precipitates collected after microwave irradiation process 285x228mm (120 x 120 DPI)



Fig 2: SEM morphology (a) TMP-0Ag 221x153mm (120 x 120 DPI)


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Fig 2: SEM morphology (b) TMP-1Ag 216x151mm (120 x 120 DPI)



Fig 2: SEM morphology (c) TMP-3Ag 220x155mm (120 x 120 DPI)


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Fig 2: SEM morphology (d) TMP-3Ag 219x154mm (120 x 120 DPI)



Figure 2 extension: EDS spectra of (a) TMP-0Ag 163x99mm (120 x 120 DPI)


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Figure 2 extension : cross-sectional micrographs of (a) TMP-0Ag 186x157mm (120 x 120 DPI)


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Figure 2 extension : cross-sectional micrographs of (b) TMP-1Ag 247x218mm (120 x 120 DPI)



Figure 2 extension : cross-sectional micrographs of (c) TMP-3Ag 245x215mm (120 x 120 DPI)


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Figure 2 extension : cross-sectional micrographs of (d) TMP-5Ag 263x212mm (120 x 120 DPI)



Three dimensional images showing roughness as analyzed by (a) Atomic force microscopy (AFM) 197x93mm (120 x 120 DPI)


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Three dimensional images showing roughness as analyzed by (b) Optical profilometry (OP). 216x135mm (120 x 120 DPI)





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Figure 3(c): Set-up showing adhesion testing of coatings. 330x91mm (150 x 150 DPI)


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Fig 4: ICP results showing the Ag+ ion release kinetics from the doped TMPH coatings. 212x169mm (120 x 120 DPI)



Fig 5: Representative zone of inhibition (ZOI) images of coated PEEK specimens after 2 days incubation against (a) E. coli 212x193mm (150 x 150 DPI)


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Fig 5: Representative zone of inhibition (ZOI) images of coated PEEK specimens after 2 days incubation against (b) S. aureus 188x187mm (150 x 150 DPI)



ZOI coefficients of coated PEEK specimens after 2 days incubation against (a) E. coli 227x177mm (101 x 101 DPI)


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ZOI coefficients of coated PEEK specimens after 2 days incubation against (b) S. aureus 204x163mm (120 x 120 DPI)



(a) OD600 readings of E.coli and S. aureus as cultured in control and coating extracts 221x177mm (120 x 120 DPI)


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(a) OD600 readings of E.coli and S. aureus as cultured in control and coating extracts 225x176mm (120 x 120 DPI)



(b) Corresponding number of CFUs representing live E.coli and S. aureus as cultured in the same extracts. 218x168mm (120 x 120 DPI)


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(b) Corresponding number of CFUs representing live E.coli and S. aureus as cultured in the same extracts. 233x176mm (120 x 120 DPI)



SEM images showing adherent (a) E. coli on various surfaces. The insets show the high magnification images of individual bacteria. Red arrows indicate the effect of Ag+ ions on the bacterial morphology. 332x169mm (150 x 150 DPI)


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SEM images showing adherent (b) S. aureus on various surfaces. The insets show the high magnification images of individual bacteria. Red arrows indicate the effect of Ag+ ions on the bacterial morphology. 334x168mm (150 x 150 DPI)



Fig 8: (a) Sporadic spherical apatite particles formed on alkali etched PEEK as opposed to overwhelming apatite formed on coated specimens after immersing in t-SBF for 7 days. 207x145mm (120 x 120 DPI)


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Figure 8: (b) EDS results of apatite globules formed on TMP-5Ag. 160x94mm (120 x 120 DPI)


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Figure 8: (c) XRD analysis of SBF immersed specimens 230x190mm (150 x 150 DPI)



Fig 9: (a) MTT assay results of MC3T3 pre-osteoblasts cultured on specimens over 21 days. 206x165mm (120 x 120 DPI)


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Fig 9: qRT-PCR results showing mRNA expressions of (b) ALP 275x217mm (120 x 120 DPI)



Fig 9: qRT-PCR results showing mRNA expressions of (c) Col1 276x221mm (120 x 120 DPI)


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Fig 9: qRT-PCR results showing mRNA expressions of (d) OCN 268x223mm (120 x 120 DPI)



Fig 10: (a) MC3T3 cell adhesion on various surfaces within 4 hours of incubation 282x197mm (120 x 120 DPI)


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Fig 10 (b) Growth morphology of MC3T3 cells over 14 days. 213x155mm (120 x 120 DPI)


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Fig 11: Mechanism showing the formation of (a) Ag-TMPH coatings on PEEK via microwave-irradiation 314x176mm (120 x 120 DPI)



Fig 11(b): CaP apatite globules formed on the TMPH coatings after immersing them in t-SBF for 7 days at 370 C. 230x167mm (120 x 120 DPI)


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Single-phase, Antibacterial Tri-Magnesium Phosphate Hydrate

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