Improvements of Antibacterial and Cell Growth Activities for Hydroxyapatite Rods Modified Polyetheretherketone (PEEK) Implants with Direct Absorption of Antibiotics

Improvements of Antibacterial and Cell Growth Activities for Hydroxyapatite Rods Modified Polyetheretherketone (PEEK) Implants with Direct Absorption of Antibiotics | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Improvements of Antibacterial and Cell Growth Activities for Hydroxyapatite Rods Modified Polyetheretherketone (PEEK) Implants with Direct Absorption of Antibiotics Dave W. Chen, Ming-Kuang Chou, Ngi-Chiong Lau, Kong-Wei Cheng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4608945/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Demands of bone regeneration and fracture repairing technologies have become increasingly important due to aged society and limitation of autologous bone graft. Heterogeneous implants are always employed in clinical treatments but their antibacterial properties, biocompatibilities, and the mismatch elastic moduli with human bones have to be improved. Therefore, polyetheretherketone (PEEK)/ hydroxyapatite (HA) rod-like array samples were manufactured using hydrothermal method and then directly attached various types of Ampicillin, Vancomycin salts and their mixture onto HA arrays in order to improve their antibacterial properties and biocompatibility. Various ratios of ethylenediaminetetraacetic agent : calcium ions in the solution baths were set in order to obtain high values of specific area of HA rods for the loading of antibiotics. The specific surface area of the sample prepared with ethylenediaminetetraacetic agent : calcium ions ratio of 1:1 in solution bath showed the largest value. Samples through direct absorption of antibiotics can maintain their antibacterial activities up to 10 days. Sample (C) (direct absorption of Ampicillin + Vancomycin salt solution (w/w = 50/50)) showed good inhibition activities on E. coli and S. aureus . Cell growth activity of MC3T3-E1 onto the PEEK/HA/antibiotic samples also showed better performance than that of HA/PEEK sample, suggesting potential good application in polymer-made implants. Polyetheretherketone Hexagonal hydroxyapatite rods Antibacterial property Cell growth Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Demands of bone regeneration and fracture repairing technologies have become increasingly important due to aged society and limitation of autologous bone graft. Heterogeneous implants applied in the clinical treatments include metal-based implants such as stainless steel or titanium alloys [ 1 – 2 ], ceramic-based implants such as aluminum oxide or glass ceramic [ 3 – 5 ], bioactive glasses or polymer-based implants such as polymethylmethacrylate [ 3 – 6 ], respectively. While metal-based implants have many advantages such as inexpensive, easy to prepare and having good biocompatibility, their corrosion influences in the human body and the mismatch elastic moduli between human bone (7–30 GPa) and metal-made implant (~ 110 GPa for the Ti-based metal support) must be addressed in clinical treatments [ 1 , 3 , 5 , 7 ]. Additionally, poor medical image qualities of patients using the metal-based implants is also a major concern [ 8 ]. Ceramic-based implants offer high corrosion resistance, low thermal-expansion and good biological properties, but their high elastic moduli, easy to cause local stress, poor flexibility and difficult to machine limit their large scale applications in clinical treatments [ 5 ]. For clinical applications, heterogeneous implants with low-cost, easy production, fast manufacturing and similar physical properties to human bone are crucial. In comparison to metal-based or ceramic-based implants, polymer-based implants have the advantages of good biocompatibility, easy of machining, low-cost, good mechanical properties, non-toxicity and ability of producing porous structures. Therefore, the development of the polymer-based implants was then proposed and applied in the clinical treatments of skeletal wounds [ 1 , 6 – 10 ]. Also considering the requirement of fast production, a new technology called 3D-printing has been reported and applied for the preparation of heterogeneous polymer-based implants, facilitating fast manufacturing of implants or bone-tissue engineering [ 11 – 12 ]. Recently, an interesting polymer called polyetheretherketone (PEEK) has been reported for clinical bone regeneration or repairing treatments [ 1 , 2 , 3 , 6 , 13 – 14 ]. Heterogeneous PEEK implants have several advantages, including a similar elastic modulus (3–4 GPa) with human bone, low stress shielding, non-toxicity, good bio-stability in human body compared to traditional metal-based implants, and easy preparation with complex shapes using 3D printing technology [ 1 , 8 , 6 , 13 – 14 ]. Notably, the PEEK samples show no apparent influence on magnetic resonance imaging (MRI) compared to traditional metal-based implants [ 14 ] and have been approved by Food and Drug Administration (FDA) as implantable candidate since 1980 [ 14 ]. However, these PEEK-made implants are still uncommon in clinical treatments due to their low antibacterial properties, which may result in high possibility of periprosthetic joint infection (PJI) [ 3 , 6 , 13 – 14 ]. Tradition treatments for PJI involve adequate debridement, local infiltration of high concentration antibiotics and long term parenteral antibiotic injection. However, issues of drug wastage, long term antibiotic complications, cell toxicity, and drug resistance in the human body are always observed during the clinical treatments. Therefore, modifications are necessary to improve the antibacterial properties of these polymer-based implants. Various types of materials such as hydroxyapatite (HA) [ 15 – 18 ], metal tantalum [ 19 ] and niobium pentoxide [ 20 ], TiO 2 /ZnO [ 13 , 21 ], ZnO [ 3 ], Si 3 N 4 /Ta [ 22 ], Ag/peptide [ 23 ], and other composite antibacterial layers [ 24 – 26 ] have been employed to improve the bioactivities, antibacterial performances or attachment properties of bone cells for these polymer-based implants. Although coating of extra chemicals such as ZnO, Ag, metal Cu or tantalum onto polymer-based implants may inhibit the growth of microorganisms such as S. aureus on the heterogeneous implants [ 3 , 13 , 21 – 23 , 27 ], the decomposition of these thin film in the buffer solution has been observed, leading to cell toxicity for bone cells attached onto the heterogeneous implants [ 3 , 13 , 23 , 27 ]. Therefore, the development of high-safety, good biocompatible, non-toxic samples with suitable antibacterial properties and similarity to bone mineral is crucial. Recently, an interesting material called hydroxyapatite (HA) with chemical formula of Ca 10 (PO 4 ) 6 (OH) 2 has been developed due to its osteoconductivity, biocompatibility and similarity to the bone mineral phase [ 27 ]. However, the HA’s poor antibacterial and mechanical properties make it challenging for direct application in clinical treatment [ 27 ]. Incorporating HA with some minor ratios (~ 2–5%) of Ag, CeO 2 , Cu, ZnS, Nb 2 O 5 , Ag 2 O has been attempted to improve its antibacterial property with these heavy metal ions released from surface of implants [ 17 – 18 , 27 – 28 ]. Although these modifications can inhibit the microorganism growth, cell toxicity of these polymer-based implants with HA/metal ions coating has also been observed. Therefore, the incorporation of heavy metal ions into HA might not be an ideal approach. In our previous study [ 3 , 13 ], we deposited ZnO rod-like arrays [ 3 ] and TiO 2 /ZnO core-shell rod-like arrays [ 13 ] onto the PEEK disks using the solution growth method. These rod-like arrays with suitable surface area showed good capacities for loading of antibiotics and then inhibited the microorganism growth such as S. aureus on implants during clinical treatments. However, decomposition of the ZnO layer on PEEK sample in the buffer solution was observed, resulting in cell toxicity for the bone cells attached onto the surface of implants [ 3 ]. For the above discussion, HA-rod-like arrays might be a good candidate to replace the ZnO arrays on PEEK samples, which offer good specific surface area for loading of antibiotics. In the literature [ 29 – 30 ], HA showed good absorption ability for the ionic or organic molecular and can be used as a carrier for absorption agents or the substrates for loading catalysts [ 29 – 32 ]. If these HA/antibiotics grown onto PEEK implants are employed in the orthopedics patients, the antibiotics attached onto HA surface would release slowly into the human body, decreasing the possibility of microorganism infection during the recovery of clinical treatments. The HA on the PEEK surface, which is inorganic constituent that account around 60–70% in human bone, could also act as the gene and small molecule drug carrier to induce the osteogenic differentiation of stem cells and bone defect repair [ 33 – 34 ]. Huang et al. (2024) [ 34 ] employed hydrothermal method to prepare the selenium/strontium/zinc-doped hydroxyapatite (Se/ Sr/Zn-HA) powders. Their Se/Sr/Zn-HA powders exhibited banded crystal shape with a length of 2–10 µ m and a width of 1 µm. Incorporating Se, Sr and Zn ions into HA samples induced a transformation in their microstructures, transitioning from banded shape, the spherical type composed some sheets/ regular aggregate shapes to radial petal-like microstructures/micro-spherical structures with nano-flakes, respectively. Their HA samples were mixed with polycaprolactone (PCL) as ink to construct composite scaffolds using 3D-printing. The Se ions formed as the SeO 3 2− on samples surface and Zn ions released from samples enhanced antibacterial effects. Sr 2+ and Zn 2+ ions exhibited good osteogenic effects. The samples demonstrated effective antibacterial properties against S. aureus and E. coli , reducing microorganism growth of around 35% and 40% respectively, compared to pristine PCL sample after 24 hours of testing. All experimental groups affected the proliferation of MC3T3-E1 cells without cytotoxicity, with cell viability exceeding 100% over with a 7 days testing period. Swan et al. [ 35 ] incorporated cationic steroid antimicrobials (CSA13) into the strontium substituted HA micro- and nano-sized particles through low-temperature and high speed collision process. The resulting samples were implanted into the surface of N-carboxymethyl chitosan (N-CMCh) pre-embedded on PEEK substrate. CSA 13 antibacterial agents released from samples reduced the growth of Methicillin Resistant Staphylococcus Aureus by approximately 27%, compared to growth of Methicillin Resistant Staphylococcus Aureus on the PEEK substrate without the sample coating. The human osteoblast-like cells viability on these specimens exhibited significant cell growth even in the presence of CSA13. Morsy (2024) [ 36 ] fabricated and characterized 3D hybrid scaffolds composed of HA nanoparticles synthesized in situ in gelatin and PVA solutions, loaded with combustionally synthesized (10 wt %) zinc oxide nanoparticles (ZnO NPs). The samples showed high porosity (up to 78%) with a pore size ranging from 50–300 µm and good mechanical strength (approximately 55 kPa). Loading of ZnO into samples imparted good antibacterial performances against S. aureus and E. coil , with inhibition zone diameters of 7 mm and 6mm, respectively. Rachid et al. [ 37 ] prepared Zn-doped HA using traditional co-precipitation method, followed by dissolving these powders and chitosan in 1% acetic acid solution with cross-linked agent (glutaraldehyde) to obtain chitosan cross-linked Zn-doped HA composites. The resulting samples exhibited suitable antimicrobial activities against both Gram-positive and Gram-negative bacteria, with inhibition zone diameter of 20 mm and 16 mm, respectively. Yakufu et al. (2024) [ 38 ] prepared porous PEEK scaffolds with HA layer coated onto the pore wall surface using sacrificial porogen of HA-coated sodium chloride (NaCl) microspheres. With HA microspheres coated onto the pore wall of PEEK samples, their mechanical properties were comparable to pristine PEEK sample. The highly porous structure of PEEK@HA scaffolds and the HA coating layer promoted biomineralization in vitro and osseointegration in vivo. However, their antibacterial properties were not tested in this study. Therefore, the preparation of HA on PEEK substrates with diverse microstructures for loading suitable antibiotics might show good antibacterial properties and enhance bone cell growth on the PEEK implants in clinical orthopedic treatments. In this study, we prepared the HA films with different microstructures onto the 3D-printed PEEK disks using simple hydrothermal method. We evaluated the physical properties, short- and long-term antibacterial properties against Gram-positive and negative bacteria, and analyzed the influence of bone cell growth on the PEEK samples with various types of antibiotics directly absorbed onto HA thin films on the PEEK surface. 2. Experimental details 2 − 1 Preparation of HA layers on PEEK disks From the above discussion, the use of HA for the direct absorption of antibiotics is intriguing due to its good osteoconductivity, biocompatibility and similarity to the bone mineral phase. To achieve this, we deposited HA layers onto PEEK disks using traditional hydrothermal method. The PEEK disk (diameter of 14.6 mm and thickness of 1.8 mm) was obtained through our 3D-printer (Black Magic 3D, Prusa i3). The detailed procedures for preparing of 3D-printed PEEK disk were similar with those reported in our previous studies [ 1 , 3 ]. Subsequently, the PEEK was washed using ethanol, deionized water, acetone, and deionized water in the ultrasonic bath with time interval of 15 minute. Then the sample was blown dry with ultra-pure nitrogen gas. To ensure an uniform HA thin film on PEEK substrate and improve its attachment property, sulphonation process with SO 3 H functional group on PEEK sample was carried out to enable attachment of HA thin films on substrate [ 39 ]. This process involved immersing the PEEK into a 10 ml glass container containing concentrated sulfuric acid (98%, Honeywell, Fluka) for 10 minutes. After sulphonation, the samples were subjected to ultrasonic bath for 15 minutes to reduce any unreacted sulfuric acid on PEEK surface. This cleaning process was repeated at least three times, and the samples were then dried using ultra-pure nitrogen gas. For the growth of HA with various microstructures on PEEK substrates, similar procedures as reported by Suchanek et al. (2018) [ 30 ] were employed but the ratios of chelating agent : Ca ions in reaction solution were verified in order to obtained HA thin film with high surface area. The calcium nitrate (Ca(NO 3 ) 2 ⋅4H 2 O, UniRegion Bio-Tech, purity > 98%) with concentration of 0.14 M and ammonium phosphate monobasic (NH 4 H 2 PO 4 , J. T. Baker, purity > 98%) with concentration of 0.084 M and 30 mL of deionized water were mixed well in a glass container at 30 minutes interval. The ethylenediaminetetraacetic acid disodium salt dehydrate as the chelating agents (EDTA-Na 2 , C 10 H 14 N 2 O 8 Na 2 ⋅ 2H 2 O, Honeywell, Fluka, purity > 98%) with concentrations of 0.07, 0.14 and 0.28 M and monoethanolamine (MEA, C 2 H 7 NO, Aldrich Co., purity > 98%) with concentration of 0.28 M were then added into the reaction solution containing Ca 2+ ions in order to form Ca-complex ions and reduced the concentration of free Ca 2+ ions in the solution bath, which might make the HA become powders suspending in the solution bath. With most of free Ca 2+ ions formed as Ca-complex ions, a colorless reaction bath was obtained. Then ammonia hydroxide solution (NH 4 OH, J. T. Baker Co., purity = 37%) with volume of 5mL was added into the reaction bath to provide OH − ions in HA. The pH value of reaction solution was maintained at 10. Homogeneous reaction solution was then transferred into a 150 mL Teflon vessel and placed in a hydrothermal reactor. The autoclave was sealed and kept at the temperature of 180°C for 5 hours. Following the reaction, the samples were washed several times with deionized water to remove the unreacted chemicals attached to the PEEK substrates and then dried in an oven at 70°C for 24 hours. 2–2 Preparation of drug loaded on the samples Due to the poor inherent antibacterial property of pristine HA, it is imperative to enhance its antibacterial characteristic. In this study, antibiotics were directly absorbed onto the HA surface. To achieve this, concentration of 10000 µg/L for various types of antibiotics (pure Ampicillin salt, pure Vancomycin salt and the Ampicillin : Vancomycin of 50 : 50 in weight percentage) in the water bath were employed to prepare the HA/antibiotic samples through direct absorption process. From our previous studies [ 1 , 6 ], it was observed that the sample immersed in a water bath containing antibiotics for five days could result in 90% of antibiotics being absorbed onto the sample surface. Therefore, the HA/PEEK sample was placed in an aqueous solution containing different types of antibiotics with a total volume of 20 mL at room temperature for five days. Following the complete absorption of antibiotics onto the sample surface, the sample was put in a clean container to avoid any possible influence from other organisms or impurities. 2–3 Characterization of HA/PEEK samples The crystal phase of HA/PEEK sample was conducted using the X-ray diffractometer (XRD, D2-Phaser, Bruker, A26-X1-1) with CuK α (λ = 1.5405 Å) irradiation. The 2θ range in the XRD patterns of samples were in the range of 10–60° with scanning rate of 0.1°/s. A field-emission scanning electron microscope (FE-SEM, JOEL JSM-7500F) equipped with energy dispersive spectra (EDS, Horiba, 7021-H) with acceleration voltage of 10 kV and working distance of 15 mm was employed to analyze the surface and cross-section images of samples and their compositions. An UV-Visible spectrophotometer (UV-Vis, Varian Co., CARY50) was used to determine the concentration of antibiotics in the buffer solution bath. The optical densities of organisms in the solution bath were carried out using a microplate reader (SpectraMax M3, USA). 2–4 Antibacterial tests for samples In vitro analysis of concentrations for antibiotics in buffer solution was carried out to understand the drug release profiles from the samples into buffer solution. Similar approach can be observed in our previous studies [ 1 , 6 ]. 10 mL of the phosphate buffer solution (PBS, pH 7.4) was utilized for the analysis of drug release behavior from the sample into the PBS at 37°C with the shaking rate of 30 rpm. In vitro analysis test on the concentration of antibiotics in buffer solution was carried out within the suitable time interval using the UV-Vis spectrophotometer. Fresh buffer solution was replaced every testing procedure to avoid the saturated concentration of antibiotics in solution bath. The detected wavelengths for Ampicillin and Vancomycin salts were 220 nm and 280 nm using the UV-Vis spectrometer, respectively. The process for evaluation of the sample’s antibacterial property is similar to our previous studies [ 1 , 6 ], involving two types of antibacterial tests: Agar diffusion test and broth dilution test. For agar diffusion test, S. aureus (ATCC 29213) or E. coli (ATCC 25922) inoculum with a volume of 200 µL in the 100ml Nutrient broth solution (NB, Becton, Dickinson and Company, including beef extract 3g and peptone 5g) was seeded, and organisms were allowed to grow for 2.5 hours at 37 ° C with a constant wavering rate of 220 rpm. The bacterial concentration in the solution was adjusted to around 10 8 colony-forming unit (CFU)/mL. The antibiotic disk diffusion method was employed to observe antibacterial properties against S. aureus or E. coli in the agar containing NB at the Petri dish. An organism solution with a bacterial concentration of 10 8 CFU/ml and a total volume of 300 µl was seeded onto the agars in the Petri disks, and the inhibition zone test was analyzed at 37 ° C. Standard calibration curves of the inhibition zone on the organisms were created using the paper with loading of various concentrations of different types of antibiotics (1, 10, 100 and 1000 µg/mL), respectively. The concentration of antibiotic released from the sample was then obtained using interpreting these curves. For the broth dilution test, the optical density (OD) value of bacteria in NB solution was measured. NB solutions cultured with S. aureus or E. coil with a concentration of 10 8 CFU/ml were employed to evaluate their antimicrobial activities, respectively. Standard calibration curve with only organism in the solution was also tested to evaluate the growth rate of organism. The solution containing organism suspension was incubated at 37°C with the wavering rate of 150 rpm. The optical density (OD) values for these solutions containing organisms with antibiotics coated onto the samples were estimated using a microplate reader with the light wavelength set at 600 nm. The relative optical density value of the bacterial solution was calculated using the following equation: $$\text{R}\text{e}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e} \text{v}\text{a}\text{l}\text{u}\text{e} \text{o}\text{f} \text{O}\text{D}\left(\text{\%}\right)=\frac{OD value of solution }{OD value of standard solution }$$ 1 2–5 In vitro cytocompatibility of samples For the in vitro cell response tests, mouse osteoblast-like MC3T3-E1 cells (American Type Culture Collection, CRL-2593, USA) were cultured in a Minimum Essential Medium (α-MEM, Gibco BRL, Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, USA), 100 µg/mL penicillin and 100 µg/mL streptomycin sulfate (Gibco BRL, Thermo Fisher Scientific, USA) in a humidified atmosphere containing 5% CO 2 at 37°C. Before cell culture process, the specimens were sterilized under UV radiation, which was placed in 24-well plates. Then the cells were cultured on the specimens at a density of 2 × 10 6 cells per well to evaluate cell attachment, proliferation and cytotoxicity. The culture medium was replaced every 2 days. The morphology of the cell proliferation was observed using a confocal laser scanning microscopy (CLSM, Leica TCS SP8 X, USA) within the 3th, 7th, and 14th testing day, respectively. 3. Results and discussion 3.1 Physical characterization of HA/PEEK samples We deposited the HA layers onto the PEEK disks using traditional hydrothermal method to obtain the surface-modification PEEK samples with good antibacterial property, osteoconductivity, and biocompatibility. Traditional 3D-printer always uses polylactic acid (PLA) as the printing material, with temperature values for the bozzle and holder of below 190°C [ 1 , 40 – 41 ]. However, PEEK, being a high glass-transition temperature polymer, requires adjustments in the temperature settings for both the nozzle and holder in 3D-printer. Figure 1 (I) and (II) shows the analysis results of the thermogravimetric analyzer (TGA, TA TGA Q-50) and the differential scanning calorimeter (DSC, TA DSC 50) of PEEK fibers. Figure 1 (I) reveals that PEEK begins to loss its weight at the temperature exceeding 550°C. At the temperature higher than 600°C, the PEEK fibers lost approximately 45% of total weight, indicating decomposition starting at 550°C. Figure 1 (II) displays the DSC analysis results, showing T m (melting point) and T c (crystallization point) values for PEEK fibers at 338.74°C and 285.24°C, respectively, which are consistent with literature reports [ 6 , 13 , 42 – 43 ]. Based on the DSC and TGA analysis, the nozzle temperature in our 3D-printer must be maintained in the range of 286–550°C to ensure the PEEK sample in the nozzle at molten state. From the study proposed by Yang et al. (2017) [ 44 ], high temperature of the nozzle and holder in our fused deposition modelling printer can enhance the printing quality of PEEK disks. Therefore, we conducted various printing parameters such as temperature of nozzle, temperature of holder and printing speed for our 3D-printer. To prepare our PEEK disks, we set the temperature of holder at 280°C, printing speed at 10 mm/s, temperature of nozzle at 380±10°C and nozzle diameter at 1.0 mm for our 3D-printer. Figure 1 (III) presents the XRD pattern of our PEEK sample, which shows the XRD peaks for our 3D-printing PEEK are consistent with standard PEEK (JCPDS no. 52-2277). The XRD peaks for our samples located at 2θ of 19.03°, 21.01°, 23.06° and 29,15° corresponded to the crystal planes of (1 1 0), (1 1 1), (2 1 1) and (0 1 1) for PEEK, respectively. Three small and broad peaks at 2θ of around 33.26°, 39.19° and 47.65° are assigned to the PEEK phase. The result shown in Fig. 1 (III) confirmed that our sample was PEEK after the fused deposition printing process. For the growth of HA thin film on PEEK substrates, calcium nitrate, ammonium phosphate and ammonia hydroxide were utilized as sources of Ca, PO 4 2− and OH − ions, leading to the formation of HA through the following reaction [ 33 ]: 10Ca(NO) +6(NH)HPO + 2OH→Ca(PO)(OH) + 6NH +12 H +20 NO (1) To confirm the occurrence of the reaction (1) using the hydrothermal method, we collected the powders in the solution bath after hydrothermal process. Figure 1 (III) displays the XRD pattern of HA powders obtained after hydrothermal method, exhibiting the peaks of HA powders (red line) align well with standard HA peaks (JCPDS no. 74–566). The observed peaks at 2θ of around 29.5°, 32.23˚, 32.69˚, 33.34˚, 40.23˚, 47.11˚, 48.45˚, 49.82˚, 50.92˚, 51.65˚, 52.48˚ correspond to crystal planes of (2 1 0), (1 1 2), (3 0 0), (2 0 2), (2 2 1), (2 2 2), (2 3 0), (2 1 3), (3 2 1), (1 4 0), (3 0 3) for HA, respectively. These results indicate successful preparation of PEEK and HA samples using 3D printing and hydrothermal method. However, due to the high reaction rate of Eq. ( 1 ), most of HA formed as the powders suspended in the solution rather than the forming of thin film on the PEEK sample. To address this, the chelating agent and stabilizer were added to the solution bath [ 30 , 45 ]. We used EDTA-Na 2 as the chelating agent to form Ca-EDTA complexes, reducing the concentration of free Ca 2+ ions in the solution bath. Low concentration of free Ca 2+ ions in solution would reduce the ratio of HA powders formed in the solution bath and facilitated the HA thin film grown onto the PEEK substrate. With the reaction bath temperature of 180°C, the Ca-EDTA complexes would begin dissociation, releasing Ca 2+ ions that would be absorbed onto PEEK surface. These free Ca 2+ ions on the PEEK surface then reacted with PO 4 2− and OH − ions to form HA thin film on PEEK. Because the EDTA is the strong chelating agent on Ca 2+ ions, the parameters of reaction temperature, the ratio of EDTA : Ca 2+ and the pH values in the solution baths were important for the growth of HA thin film on PEEK substrate. In this study, MEA in the solution bath served as the stabilizer, morphologic control agents and the control unit for the release of Ca 2+ ions from EDTA agent in the solution bath [ 30 , 45 ]. Under high temperature and suitable pH value in solution bath, the HA thin film would form on the PEEK sample surface rather than powders suspending in the solution bath with suitable ratio of EDTA : Ca 2+ . High EDTA: Ca 2+ ratio in solution bath would reduce the growth of HA on PEEK substrate due to the low degree of super-saturation of HA. Conversely, low EDTA: Ca 2+ ratio in solution bath would cause most of HA to form powders rather than thin film on substrates. Therefore, we verified the ratios of EDTA : Ca 2+ ions in the solution bath to obtain a uniform and compact HA thin film on PEEK. Figure 2 (I) shows the XRD patterns of samples (i), (ii) and (iii), corresponding to the HA thin film on PEEK substrates prepared with the ratio of EDTA: Ca 2+ ions: of 0.5 : 1, 1 : 1 and 2 : 1, respectively. Sample (i) exhibited only two small peaks at 2θ of around 32.1° and 33.2°, which corresponded to the crystal planes of (1 1 2) and (2 0 2) for HA. Although we could observe a white HA thin film on PEEK, most of HA formed as the powders in the solution bath. This is because low ratio of EDTA : Ca in the solution bath and made free Ca 2+ , PO 4 2− and OH − ions approach to the supersaturation of HA. Sample (ii) showed peaks consistent with standard HA peaks, confirming the growth of HA thin film onto the PEEK substrate. The insert image shown in Fig. 2 (I) displayed the pictures of PEEK substrate before/after HA growth for sample (ii). A white thin film was covered on our PEEK disk after the hydrothermal treatment. In contrast, sample (iii) displays peaks corresponding to the PEEK sample, indicating absence or amorphous HA thin film growth. SEM images in Fig. 2 (II) revealed that sample (i) had a non-uniform thin film with crack and pinholes. Some layer/plate microstructures covered on the PEEK substrate were also observed. These layer or plate microstructures were similar to those reported in the literature [ 30 , 46 ]. When the ratio of EDTA: Ca 2+ in solution bath changed from 0.5 :1 to 1 : 1, the surface morphologies of samples transformed from plate/layer microstructures (sample (i)) to rod-arrays HA microstructures (sample (ii)). The diameter of hexagonal rod-like microstructure for sample (ii) was approximately 5.44 µm (Figure S1 ). The rod-array microstructures of HA were also reported in the literature [ 30 , 46 ]. When the ratio of EDTA : Ca 2+ was adjusted to 2 : 1 (sample (iii)), surface microstructures showed similar to pristine PEEK. Using the EDS (almost no Ca or P element was detected), SEM image and XRD analysis results, we could confirm that no HA thin film was grown onto the PEEK substrate using EDTA : Ca 2+ of 2 : 1 in solution bath (sample (iii)). The possible reason is the influence of EDTA chelating agent. The EDTA is a strong chelating agent on Ca 2+ ions. Most Ca 2+ ions in the solution bath were the stable EDTA-Ca complexes and reduced the concentration of free Ca 2+ ions in the solution bath. Although the temperature and pressure were high during our hydrothermal process, the dissolution of EDTA-Ca complexes was difficult and therefore made almost no HA thin film grow onto PEEK sample. Then we examined the microstructures of samples (i) and (ii). Their microstructures showed different with the change of the ratio for EDTA : Ca 2+ in the solution bath. According to the report proposed by Pu’ad et al. (2020) [ 47 ], the HA obtained using traditional chemical precipitation always low-crystallinity HA particles. With low ratio of EDTA : Ca 2+ in the solution bath, our approach was similar to chemical precipitation. Most of HA was formed as powders suspending rather than the thin film grown on the substrate. Therefore, a non-uniform, low-crystalline HA with crack or pinholes on PEEK substrate could be observed. When the EDTA : Ca 2+ ions of 1:1 was set, the concentration of free Ca 2+ ions in the solution decreased and the release rate of Ca 2+ ions from EDTA-Ca complexes could be controlled by adjusting the reaction temperature or pressure. Therefore the HA layers with rod-like microstructures could be obtained on substrates [ 30 , 46 – 47 ] and indicated that the HA layer with high surface area could be obtained on our sample (ii). Then we analyzed the composition of HA thin film on PEEK substrate (samples (i) and (ii)) using EDS analysis at 200 (X). For sample (i), the molar ratio of Ca : P of around 5 : 1 in HA indicated the element Ca was much higher than the theoretical ratio for HA (Ca/P = 1.67). The layer on PEEK substrate (sample (i)) was the low-crystalline HA layer with a large amount of free Ca 2+ ions absorbed onto sample surface. For sample (ii), its molar ratio of Ca : P was 62.54% :37.46%, which corresponded to the ratio of Ca/P of 1.67 for sample (ii). The EDS analysis results indicated that the rod-like HA thin film was grown onto PEEK substrate (sample (ii)). Figure 2 (III) shows the EDS mapping results for sample (ii), exhibiting the uniform distributions of Ca and P, which indicated that the uniform HA layer with rod-like microstructures was obtained using hydrothermal method with the EDTA : Ca 2+ of 1 : 1 in the solution bath. Because our topic was to prepare the HA thin film for the loading of antibiotics to improve its antibacterial property, it specific surface area was important. A specific surface area analyzer (Micomeritics, ASAP 2020) was employed to examine the change of specific surface area for samples (i) and (ii) with nitrogen gas pressure of 3 µm Hg. Using the absorption curves of samples, the effective values of surface area for samples (i) and (ii) of around 1.72 ± 0.02 m 2 /g and 2.21 ± 0.04 m 2 /g were obtained, respectively. The sample (ii) had higher specific surface area value than that of sample (i) due to it having rod-like microstructures and made it have good loading capacity for the antibiotics. The values of thickness for HA coated onto samples were around 5µm obtained from the cross-section FE-SEM images. 3.2 The antibacterial properties of samples Due to the poor antibacterial properties exhibited by the HA/PEEK samples, it is imperative to enhance their antibacterial efficacy. Generally, coating with biodegradable polymer such as PLGA (poly(D,L-lactide-co-gycolide)) containing antibiotics onto the sample surface was the major method to enhance their antibacterial properties [ 1 , 6 ]. The degradation of PLGA is employed as the control unit for the antibiotic released from sample surface into solution bath. However, the high cost of PLGA makes polymer-made implants become expensive. To address this issue, if our HA thin film could absorb antibiotics directly and maintain suitable antibacterial properties for at least ten days, these antibiotics/HA/PEEK samples could be employed in the clinical treatments. HA with the most stable Ca/P ratio of approximately 1.67 has been extensively studied and act as an excellent drug delivery carrier due to its high affinity and low translocation efficiency [ 48 – 50 ]. HA possess excellent drug carrier due to the active ions at the surface. The mesoporous structure at the surface of HA is attractive for chemical bonding with chemical, small molecules and generic drugs [ 51 ]. Ca 2+ at the surface of HA are the main bonding between HA and drug [ 52 ]. This electrostatic interaction between Ca 2+ and antibiotic contribute to the drug loading ability, drug release properties. Besides, the porosity and rod-like appearance of HA on PEEK surface increased the surface area of HA which affect its interaction with antibiotics. These characteristics largely contribute to slow and sustained release activity of antibiotics. [ 53 ] According to studies proposed by Zheng et al (2022) [ 54 ] and (2023) [ 55 ], Alendronate (ALN) is bound onto the nano-hydroxyapatite (nHA) through interaction with Ca 2+ on surface of nHA to establish slow and sustained release profle of ALN to facilitate bone formation. Liu et al. (2020) [ 49 ] also demonstrated the binding of the anticancer drug doxorubicin (DOX) to HA by electrostatic interaction of Ca 2+ at the surface of HAp which could prevent tumor recurrence. Then we selected sample (ii) for testing the direct absorption of various antibiotics, including pure Ampicillin sodium salt, pure Vancomycin hydrochloride salt and a mixture of Ampicillin : Vancomycin (w/w)=(50/50)). The design for the loading of mixture with weigh percentages of Ampicillin : Vancomycin = 50 : 50 aims to reduce the dosage of individual antibiotics absorbed onto the sample while maintaining adequate antibacterial activities against both Gram negative and positive bacteria. Sample (A), (B) and (C) correspond to sample (ii) loaded with pure Ampicillin sodium salt, pure Vancomycin hydrochloride salt and the mixture of Ampicillin + Vancomycin (w/w = 50/50), respectively. Following a five-day absorption period in a water bath, approximately 98.02%, 99.12% and 76.5% of antibiotics were fully absorbed onto the HA/PEEK sample surface for samples (A), (B) and (C), respectively. Nearly 100% of antibiotics for pure Ampicillin and Vancomycin salts were absorbed but only around 76.5% of the antibiotic mixture was absorbed onto HA sample surface. Detailed absorption results of the antibiotic mixture revealed that around 97.21% of Ampicillin salt was absorbed, but only around 56.2% of Vancomycin salt was absorbed onto the HA surface. This outcome aligns with the expectation due to the larger molecular weight of Vancomycin salt (M w = 1449.3 g/mol) compared to Ampicillin salt (M w = 349.41 g/mol). Small molecule are known to be more easily attached onto the sample surface, explaining the higher absorption ratio of Ampicillin salt onto the HA surface. Following the complete absorption of antibiotics onto sample surface, the samples were immersed in the PBS buffer solution to examine their drug-release profiles at 24-hour intervals. Figure 3 (I) and (II) display the drug-release profiles and cumulative values over a testing time interval of 24 hours using samples (A), (B) and (C), respectively. Additionally, Fig. 3 (I) included the values of MIC90 and MIC50 (minimum inhibition concentration for the inhibition of 90% and 50% organism growth) for E. coil (Gram-native bacteria) and S. aureus (Gram-positive bacteria) obtained from literature [ 1 , 6 ]. Concentrations of drug released from samples (A)-(C) in the buffer solution were higher than the values of MIC 90 and MIC 50 for S. aureus and MIC 50 for E. coil in testing time interval of 24 hours. Figure 3 (I) revealed that the concentrations of drug released from samples (A)-(C) could inhibit 90% growth for S. aureus (Gram-positive bacteria) and 50% for E. coil (Gram-negative bacteria), which indicated the samples had slightly poor antibacterial properties against Gram-negative bacteria. Cumulative values in PBS buffer solution for samples (A)-(C) shown in Fig. 3 (II) exhibit fast drug-release rates during the first 3 hour, attributed to the release of drug weakly physical-absorption on HA layer. Subsequently, stable drug-released profiles for samples (A)-(C) were observed between 3 and 24 hours in buffer solution. It was noted that around 60% of Ampicillin salt absorbed onto HA layer for sample (C) was released into buffer solution within the first 3 hours, contributing to higher cumulative values. Combined with the results for samples (A) and (C) shown in Fig. 3 (II), we could conclude that around 58.3 mg of Ampicillin salt was attached onto our samples with weakly physical absorption (total weight of 97.2 mg for Ampicillin salt was absorbed onto sample (C) and that of 196.04 mg for Ampicillin salt was absorbed onto sample (A)). Examining the drug-release profile of Vancomycin salt for samples (B) and (C), a similar drug-release profile was observed. Figure 3 (III) and (IV) shows the drug-release profiles and cumulative results for samples (A)-(C) within testing time interval of 10 days (0-240 hours). Sample (A) displayed drug-release profiles approaching the values of MIC 90 for E. coil , while other samples exhibited drug-released profiles higher than the values of MIC 50 and MIC 90 for S. aureus and MIC 50 for E. coil . These results indicate that the samples with direct absorption of antibiotics demonstrated good antibacterial properties against S. aureus and acceptable antibacterial properties against E. coil . Results presented in Fig. 3 (IV) suggest that approximately 55% of Ampicillin salt absorbed on sample (A), 40% of Vancomycin salt absorbed on sample (B) and 64% of Ampicillin salt + 53% of Vancomycin salt absorbed on sample (C) were released into buffer solution within testing time interval of 10 days. As only around 50–60% of antibiotics were released from samples within the 10-days testing interval, it implies that the samples might retain antibacterial properties for longer than 10 days. Figure 4 (I) and (II) illustrate drug-release profiles and cumulative results of antibiotics released from samples in buffer solution over a testing time interval of 28 days. For sample (A), concentration of drug in buffer solution remained higher than the values of MIC 50 and MIC 90 for S aureus and approach the value of MIC 90 for E. coil during the first 6 days. Subsequently, drug-released profiles for sample (A) were lower than MIC 90 value for E. coil between days 7th and 28th. Only on the 8th and 27th day for sample (A), drug concentrations in buffer solution slightly higher than the value of MIC 90 for E. coil were observed. For samples (B) and (C), drug-release profiles indicated concentrations higher than the values of MIC 50 for E. coil and MIC 50 and MIC 90 for S. aureus , suggesting good antibacterial properties against S. aureus and acceptable antibacterial properties against E. coil . Cumulative release profiles shown in Fig. 4 (II) reveal nearly liner drug-release profiles for samples (A)-(C) throughout the testing period of 1–28 days. Approximately 83% of Ampicillin salt absorbed onto sample (A) was released into buffer solution, along with around 48% of Vancomycin salt absorbed onto sample (B) was released into buffer solution in the 28-day testing period. Notably, around 78% of Ampicillin salt and almost 100% of Vancomycin salt absorbed onto sample (C) were released into buffer solution within the 28-day testing interval. Constant drug-release profiles of antibiotics from HA surface suggest that rod-like HA microstructures serve as suitable samples for loading antibiotic, consistent with previous literature [ 56 ]. Although we examined the release behaviors of various types of antibiotics via direct absorption onto the HA surface, the results suggest that the samples may exhibit good antibacterial properties on S. aureus and acceptable antibacterial properties on E. coil. within a time interval of at least 28 days. However, the concentration of antibiotic surpassing the MIC 90 value for the organism does not necessarily indicate genuine antibacterial properties, as the activity might diminish in a buffer solution. Therefore, we also tested their antimicrobial performances using traditional broth dilution and Agar diffusion tests. For the traditional broth dilution test, the OD values of S. aureus and E. coli in the solutions represents the amount of live bacteria [ 1 , 3 , 6 , 13 ]. Figure S2 illustrates the OD values changes over time for pristine PEEK and PEEK/HA samples on S. aureus and E. coil , respectively. At the testing time interval of 12 hours in solution bath containing organisms, the OD values of S aureus and E. coil in solutions containing pristine PEEK and PEEK/HA sample increased proportionally with testing time. There was no apparent difference in the growth of organisms in the solution bath for pristine PEEK or PEEK/HA samples. From the results shown in Figure S2, both pristine PEEK and PEEK/HA samples showed almost no antibacterial property against both E. coil and S. aureus . Then we examined their antibacterial properties on E. coil and S. aureus in the solution bath containing organisms with concentration of 10 8 CFU/ml and our samples. Figure 5 (I) and (II) shows the variation of relative optical densities of samples (A)-(C) over time for E. coil and S. aureus , respectively. The standard calibration curve was established using the OD values of organisms in the same solution with the PEEK/HA sample versus testing time. For E. coil with concentration of 10 8 CFU/mL in the solution (Fig. 5 (I)), the growth rates of E. coil were inhibited with samples (A)-(C) in the solution. Samples (A) (loading of Ampicillin salt on HA surface) and samples (C) (loading of mixture of antibiotic agents on HA, Ampicillin : Vancomycin = 50 :50) showed relatively good antimicrobial activities. However, the sample (B) (loading of Vancomycin salt on HA surface) exhibited very poor antimicrobial activity on E. coil . It is well known the Vancomycin salt has poor antimicrobial activity against E. coil. [ 1 , 3 , 6 ], which aligns with the expectations for sample (B). By examination the drug-released profiles of sample (B) in Fig. 4 (I) and (II), a relative low drug-release concentration in buffer solution during the testing time of 28 days was observed. Considering the results in Fig. 4 and the low antibacterial performance of Vancomycin salt on E. coil , the poor antibacterial activity on E . coil for sample (B) is reasonable. At the initial 6 hours of the test, samples (A) showed better antibacterial activities against E. coil . than sample (C). However, at the testing time interval of 12 hours, the difference in the inhibition of the growth on E. coil between sample (A) and (C) was not substantial. Since the loading of individual antibiotic on HA for sample (C) was only about 50% of samples (A) and (B), sample (C) serves as a good implant option, offering reduced cost, decreased cell toxicity and low drug resistance for these micro-organisms compared to samples (A) and (B). For S. aureu s with concentration of 10 8 CFU/mL in the solution (Fig. 5 (II)), the growth of S. aureus was inhibited with samples (A)-(C). Notably, samples (B) (loading of Vancomycin salt on HA surface) and (C) (loading of mixture of antibiotics on HA surface, Ampicillin : Vancomycin = 50 :50) demonstrated significant antimicrobial activities. The relative OD values of the solution bath containing S. aureus using samples (B) and (C) decreased to around 50% and 40%, respectively, compared to that for PEEK/HA sample in the testing time of 12 hours. Subsequently, we examined their drug-released profiles in the solution bath, as shown in Fig. 4 . All samples exhibited the drug concentration in buffer solution of higher than the MIC 90 values for S. aureus . However, sample (A) showed relatively poor antimicrobial property on S. aureus due to the poor antibacterial activity of Ampicillin salt against S. aureus [ 1 , 6 , 13 ]. Considering the testing results of samples (A)-(C) on organisms, the PEEK/HA sample with direct absorption of the antibiotic mixture (Ampicillin : Vancomycin = 50 :50) demonstrated good antibacterial activity against S. aureus and acceptable antibacterial activity against E. coil . However, the results of the broth dilution test only revealed short-term antibacterial activities for samples. To assess the long-term antibacterial properties, we conducted antimicrobial performances using the traditional disk diffusion method. Figure S3 (I)-(III) shows the antibacterial activities on E. coil with samples (A)-(C) at the time interval of 24 hours using traditional disk diffusion method, respectively. For sample (A) (HA/PEEK loading with Ampicillin salt), the diameter of its inhibition zone on E. coil was 2.95 cm at the testing time of 6th hour and increased to around 4.4 cm at the testing time of 24th hour. The antibacterial performance correlated with the paper reporting loading concentration of Ampicillin salt between 639-1000µg/ml, respectively. The result in Figure S3 (I) indicated that sample (A) exhibited good antibacterial performance on E. coil in the testing time of 24 hour, which was consistent with Fig. 5 (I). For sample (B) (HA/PEEK loading with Vancomycin salt only), the diameter of its inhibition zone was around 1.07 cm on E. coil at the 6th hour and increased to 2.4 cm at the 24th hour. The antibacterial performance for sample (B) on E. coil in the testing time interval of 24 hour corresponded to the paper loading with the concentration of Vancomycin salt between 10-1000µg/L, as reported in the literature. The result for sample (B) in Figure S3 (II) confirmed that HA/PEEK sample loaded with Vancomycin salt exhibited poor antibacterial performance on E. coil , which was also consistent with the results in Fig. 5 (I). For sample (C) shown in Figure S3 (III), the diameter of its inhibition zone on E. coil was around 3.43 cm at the testing time of 6th hour and increased to 4.2 cm at the testing time of 24th hour. The antibacterial performance of sample (C) on E. coil corresponded to paper loading with the concentration of antibiotic (Ampicillin : Vancomycin salt (w/w) = (50/50)) between 586–800 µg/L. The antibacterial performance of sample (C) showed similar activity to sample (A) and had better activity than that for sample (B) on E coil , consistent with the results shown in Fig. 5 (I). Subsequently, we evaluated their antibacterial performances on E. coil. over testing time interval of 10 days. Figure 6 (I)-(III) illustrates their antibacterial performances on E. coil . over testing time interval of 10 days, respectively. For sample (A) shown in Fig. 6 (I) (HA/PEEK sample with loading of Ampicillin salt only), it inhibition zone was 4.4 cm at the testing time of first day and remained almost the same in the testing time interval of 10 days. The antibacterial performance corresponded to the paper reporting Ampicillin salt with concentration of 800-1000µg/L, respectively. For sample (B) (HA/PEEK loading of Vancomycin salt only) shown in Fig. 6 (II), sample (B) exhibited relatively poor antibacterial activity on E. coil . at testing time interval of 10 days. Its inhibition zone was 2.4 cm at the first day and decreased to 2.0 cm at the 10th day. The results aligned with those shown in Fig. 5 (I) and those published in the literature [ 1 , 3 , 6 ]. For sample (C) (HA/PEEK with loading of Ampicillin : Vancomycin salt (w/w) = (50/50)) in Fig. 6 (III), its diameter for inhibition zone was 4.2 cm at the first testing day and increased to 4.4 cm at the 10th day. The antibacterial performance of sample (C) was much higher than sample (B) and showed a slightly higher than that for sample (A) on E. coil. It seems that the HA/PEEK sample with direct absorption of mixture of Ampicillin salt + Vancomycin salt has good antibacterial activity on E. coil. and can also reduce the cost of antibiotic and the influence of individual antibiotic on human body, such as cell toxicity or drug resistance for these microorganisms. Continuing to the evaluation of their antibacterial activities on S. aureus . Figure S4 (I)-(III) demonstrates the antibacterial activities on S. aureus with samples (A)-(C) at the time interval of 24 hours using traditional disk diffusion method, respectively. For sample (A), the diameter of its inhibition zone on S. aureus was 3.7 cm at the testing time of 6th hour and increased to around 4.4 cm at the testing time of 24th hour. Its antibacterial performance corresponded to the paper reporting loading concentration of Ampicillin salt between 1000–2218 µg/ml, respectively. The result shown in Figure S4 (I) also indicated that sample (A) exhibited good antibacterial performance on S. aureus in the testing time of 24 hour, consistent with that shown in Fig. 5 (II). For sample (B) (HA/PEEK loading with Vancomycin salt), the diameter of its inhibition zone was around 2.4 cm on S. aureus at the 6th hour and increased to 2.8 cm at the 24th hour. The antibacterial performance for sample (B) on S. aureus in the 24-hour testing interval corresponded to the paper loading with the concentration of Vancomycin salt between 100-1000µg/L, as reported in the literature. The result for sample (B) shown in Figure S4 (II) indicated that HA/PEEK sample loaded with Vancomycin salt exhibited better antibacterial performance on S. aureus than E. coil , consistent with the results shown in Fig. 5 (I) and (II). For sample (C) shown in Figure S4 (III), the diameter of its inhibition zone on S. aureus was around 4.0 cm at the testing time of 6th hour and increased to 4.5 cm at the testing time of 24th hour. The antibacterial performance of sample (C) on S. aureus corresponded to the paper with loading with concentrations of the antibiotic mixture (Ampicillin : Vancomycin salt (w/w) = (50/50)) between 636–839 µg/L. The antibacterial performance of sample (C) showed similar activity to sample (A) and had better activity than that for sample (B) on S. aureus . Subsequently, we evaluated their antibacterial performances on S. aureus at the testing time interval 10 days. Figure 6 (IV)-(VI) illustrates their antibacterial performances on S. aureus with testing time interval of 10 days. For sample (A) shown in Fig. 6 (IV) (HA/PEEK sample with loading of pristine Ampicillin salt), it inhibition zone was 4.4 cm at the testing time of first day and became a little larger in the testing time interval of 10 days (the diameter of its inhibition zone was 4.8 cm). The antibacterial performance correlated with the paper reporting Ampicillin salt concentration of 820-1540µg/L respectively. For sample (B) (HA/PEEK loading of pristine Vancomycin salt) shown in Fig. 6 (V), sample (B) exhibited almost the same antibacterial performance at testing time interval of 10 days. Its inhibition zone was 2.6–2.7 cm at the testing time of 10 days. For sample (C) (HA/PEEK with loading of Ampicillin : Vancomycin salt (w/w) = (50/50)) shown in Fig. 6 (VI), its diameter for inhibition zone on S. aureus was 4.5 cm at the first testing day and increased to 4.8 cm at the 10th day. The antibacterial performance of sample (C) was much higher than sample (B) and showed a slightly higher than that for sample (A) on S aureus. It seems that the HA/PEEK sample with direct absorption of mixture of Ampicillin salt + Vancomycin salt has good antibacterial activity on both E. coil. and S. aureus. These results also indicated the reduction of the cost for antibiotic, the influence of individual antibiotic on human body, such as cell toxicity or drug resistance for these microorganisms could be obtained using sample (C) as the implants.. 3.3 In vitro cytocompatibility of samples In the clinical orthopedic surgery, the major organisms caused PJI are S. aureus and coagulase-negative staphylococci. Sample (C) hold promise for clinical treatments, but its cytocompatibility remains a concern. Therefore, we conducted tests on the growth, adhesion and morphology of MC3T3-E1 cells on sample (C) at the 3th, 7th and 14th day interval. For comparison, PEEK disk, the PEEK/HA and samples (A) and (B) were also examined. Figure 7 presents their CLSM images at the 3th, 7th and 14th testing day. From the results shown in Fig. 7 , a few MC3T3-E1 cells were observed to grow and attached onto the pristine PEEK and HA/PEEK surface. This suggests that PEEK and HA/PEEK sample exhibited relatively poor cell growth activities. However, with the HA surface loaded with antibiotics (samples (A)-(C)), a relatively large number of MC3T3-E1 cells were observed to grow and attach onto the samples (PEEK/HA with loading of antibiotics) on the 3th day. In the images of samples (A)-(C) shown in Fig. 7 , the number of cells attached to the samples (B) was the highest and the sample (A) showed relatively poor cell attachment activity on the 3th testing day. However, in the CLSM images of samples (A)-(C) in Fig. 7 , the numbers of cells attached and grown onto the samples increased over the testing time. The cell growth and attachments on the samples (A)-(C) indicated low cell toxicity compared to the HA/PEEK sample. On the 7th and 14th days, the CLSM of all samples showed good cell growth, with samples (A) exhibiting better cell growth than samples (B) and (C). Even though samples (B) and (C) showed relatively poor cell growth activities compared to sample (A) but they still demonstrated much better cell growth activities than the HA/PEEK sample, suggesting the need to modify HA/PEEK with antibiotic loading. According to literature [ 57 – 58 ], surface morphology significantly influences cell growth, with rod or noddle-like microstructures inhibiting cell growth due to increased susceptibility to rupture by the particular surface nanorod microstructures. Poor cell growth activity for PEEK/HA samples was expected. Additionally, as reported by Galow et al. (2017) [ 59 ], MC3T3-E1 cells exhibit good cell growth activities in the solution with the pH value of 8.4. For sample (A) (HA/PEEK loading of Ampicillin salt only), the pH value of the solution containg Ampicillin salt is around 8.5, and that for Vancomycin is around 6.4. Therefore, the cell growth for sample (A) showed better cell growth activity than those for samples (B) and (C) due to the influence of antibiotic attached onto HA/PEEK sample surface. In summary, HA/PEEK samples with direct absorption of antibiotics, especially the mixture of Ampliciiln + Vancomycin salt demonstrate promising antibacterial properties against E. coil and S. aureus , with minimal cell toxicity on bone cells. These findings suggest potential applications for polymer-made implants. Conclusion In this study, we documented the growth of hydroxyapatite (HA) layers onto PEEK disk with various ratios of ehthylenediaminetetraacetic agent (EDTA) : calcium ions(Ca 2+ ) in the solution bath using hydrothermal method. XRD patterns and SEM images of the samples revealed that the HA layer with low-crystallinity could be obtained onto PEEK substrate with the EDTA : Ca 2+ of 0.5 : 1 in the solution bath. When the EDTA : Ca 2+ ratio was 1:1, the HA layer exhibited rod-like microstructure on the PEEK sample. However, with the EDTA : Ca 2+ of 2:1, no HA layer could be obtained on PEEK substrate due to the low concentration of free Ca 2+ ions in solution bath. Subsequently, various kinds of antibiotics were then directly absorbed onto the HA/PEEK samples from different types of antibiotics in water solution. Stable drug release behaviors in the PBS solution were observed, maintaining antibiotic concentrations in buffer solution greater than MIC 90 for S. aureus and MIC 50 for E. coli within a time interval of 1–28 days. Among the HA/PEEK samples, those loaded with mixture of Ampicillin + Vancomycin salt (w/w = 50/50) exhibited the most potent antibacterial properties on both E. coil and S. aureus . The suitable antimicrobial activity of the HA/PEEK sample against S. aureus and E. coli , which was the loading of mixture for Ampicillin and Vancomycin salts, could be maintained at least 10 days using the disk diffusion method. The use of relatively low dose of individual antibiotic also contributed to low cell cytotoxicity and minimized environmental pollution. Moreover, the loading of antibiotics onto the HA/PEEK samples resulted in good cell growth and proliferation. The samples with direct absorption of antibiotic agents exhibited enhanced long-term antibacterial activity and improved cell growth and proliferation on the sample surface compared to the HA/PEEK sample without antibiotics. This study introduced a simple technique for producing PEEK/HA/antibiotics materials, with potential applications in the biomedical technology within the human body. Declarations Supporting information: The SEM image for sample (ii) at 3 k(X); The change of OD values for pristine PEEK and PEEK/HA samples as a function of time on S. aureus and E. coil , respectively; The antibacterial properties on E. coil using samples with testing time of 1 day, and the antibacterial properties on S. aureus using samples with testing time of 1 day, Conflict of interest: The authors declare that they have no conflicts of interest. Funding: This study was funded by Chang Gung Memorial Hospital (CMRPG2N0331 and BMRP948). Author Contribution Dave W. Chen: Data curation woks on the Formal analysis, Investigation, Methodology, Writing – review & editing.; Ming-Kuang Chou focused on the Investigation, Methodology, Visualization; Ngi-Chiong Lau focused on the Methodology, Validation, Investigation. and Kong-Wei Cheng is Project administration, Supervision, Validation, Writing – review & editing. 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Sci Rep 7:2922 Mobarak MB, Uddin MN, Chowdhury F, Hossain MS, Mahmud M, Sarkar S, Tanvir SNI, Ahmed S (2024) Solid-state synthesis of poultry waste derived hydroxyapatite: Effect of calcination temperature on crystallographic parameters and biomedical competency. J Mol Struct 1301:1373211–1373214 Huang H, Qiang L, Fan M, Liu Y, Yang A, Chang D, Li J, Sun T, Wang Y, Guo Zhuang RH, Li X, Guo T, Wang J, Tan H, Zheng P, Wang J (2024) 3D-printed tri-element-doped hydroxyapatite/ polycaprolactone composite scaffolds with antibacterial potential for osteosarcoma therapy and bone regeneration. Bioactive Mater 31:18–37 Swain S, Misra RDK, Rautray TR (2023) Ceragenin-CSA13 loaded high strength coatings of nano- and micro-SrHA implanted N-carboxymethyl chitosan – polyetheretherketone by low temperature high speed collision approach: In vitro pro-osteogenicity and bactericidal activity against MRSA. Mater Chem Phys 309:1283611–1283618 Morsy R (2024) Development and characterization of antibacterial 3D porous hydroxyapatite-gelatin-PVA scaffolds containing zinc oxide nanoparticles. Mater Chem Phys 314:1288311–1288318 Rachid KB, Ahmed Z, Lahoucine B, Nurudeen AO, Zineb A, Azzeddine T, Hicham M, Jari SA, Abdessadik S, Eduardo AL, Małgorzata W, Noureddine E (2024) Multifunctional biocomposites based on cross-linked Shrimp waste-derived chitosan modified Zn 2+ @Calcium apatite for the removal of methyl orange and antibacterial activity. Mater Today Sustain 25:1006601–1006615 Yakufu M, Wang Z, Liu J, Zhang P (2022) Bionic manufacturing strategy of hydroxyapatite-coated polyether ether ketone scaffolds for promoting mineralization and osseointegration Mater. & Design 223:111193 1–11 Almasi D, Izman S, Assadian M, Ghanbari M, Abdul Kadir MR (2014) Crystalline ha coating on peek via chemical deposition. Appl Surf Sci 314:1034–1040 Elkasabgy NA, Mahmoud AA, Maged A (2020) 3D printing: an appealing route for customized drug delivery systems. Int J Pharm 588:119732 Chen CH, Yao YY, Tang CT, Lin TY, Chen DW, Cheng KW (2018) Long-term antibacterial performances of biodegradable polylactic acid materials with direct absorption of antibiotic agents. RSC Adv 8:16223–16231 Venkatraman P, Rader C, Bohmann N, Johan Foster E (2019) Structure-property-processing relationship of ethanol solvent exchanged PEEK. Polymer 169:154–159 Tsafack T, Bartolucci SF, Maurer JA (2020) An atomistic view of heat propagation from graphene to polyetheretherketone (PEEK). Compute Mater Sci 17:1095901–1095908 Yang C, Tian X, Li D, Cao Y, Zhao F, Shi C (2017) Influence of thermal processing conditions in 3D printing on the crystallinity and mechanical properties of PEEK material. J Mater Proce Tech 248:1–7 Cheng KW, Liang CJ (2010) Preparation of Zn–In–S film electrodes using chemical bath deposition for photoelectrochemical applications. Solar Energ Mater Solar Cells 94:1137–1145 Sadat-Shojai M, Khorasani MT, Dinpanah-Khoshdargi E, Jamshidi A (2013) Synthesis methods for nanosized hydroxyapatite with diverse structures. Acta Biomater 9:7591–7621 Pu’ad NASM, Haq RHA, Noh HM, Abdullah HZ, Idris MI, Lee TC (2020) Synthesis method of hydroxyapatite: A review Mater. Today: Proceedings 29: 233–239 Lara-Ochoa S, Ortega-Lara W, Guerrero-Beltrán CE (2021) Hydroxyapatite nanoparticles in drug delivery: physicochemistry and applications. Pharmaceutics 13:1642 Liu Y, Tang Y, Wu J, Sun J, Liao X, Teng Z, Lu G (2020) Facile synthesis of biodegradable flower-like hydroxyapatite for drug and gene delivery. J colloid interface Sci 570:402–410 Mushtaq A, Zhao R, Luo D, Dempsey E, Wang X, Iqbal MZ, Kong X (2021) Magnetic hydroxyapatite nanocomposites: The advances from synthesis to biomedical applications. Mater Design 197:109269 Kargozar S, Mollazadeh S, Kermani F, Webster TJ, Nazarnezhad S, Hamzehlou S, Baino F (2022) Hydroxyapatite nanoparticles for improved cancer theranostics. J Func Biomaterials 13:100 Sun H, Liu S, Zeng X, Meng X, Zhao L, Wan Y, Zuo G (2017) Morphology effect of nano-hydroxyapatite as a drug carrier of methotrexat. J Mater Science: Mater Med 28:1–8 Zhao Q, Zhang D, Sun R, Shang S, Wang H, Yang Y (2019) Adsorption behavior of drugs on hydroxyapatite with different morphologies: A combined experimental and molecular dynamics simulation study. Ceram Int 45:19522–19527 Zheng Y, Gao A, Bai J, Liao Q, Wu Y, Zhang W (2022) A programmed surface on polyetheretherketone for sequentially dictating osteoimmunomodulation and bone regeneration to achieve ameliorative osseointegration under osteoporotic conditions. Bioactive Mater 14:364–376 Zheng Z, Zhou H, Li M, Fu J, Dong J, Liu Y, Liu L (2023) Polyetheretherketone surface engineered with a degradable hybrid coating for accelerating osteogenesis Mater. Lett 331:133515 Wang G, Zhang H, He Q, Tong D, Ding C, Liu P P (2017) Micro-patterned titanium coatings with a grid-like structure doped with vancomycin against bacteria and affecting osteogenic differentiation. RSC Adv 7:19565–19575 Shi Z, Huang X, Cai Y, Tang R, Yang D (2009) Size effect of hydroxyapatite nanoparticles on proliferation and apoptosis of osteoblast-like cells. Acta Biomater 5:338–345 Hasan J, Crawford R, Ivanova EP (2013) Antibacterial surfaces: The quest for a new generation of biomaterials. Trends Biotechnol 31:295–304 Galow AM, Rebl A, Koczan D, Bonk SM, Baumann W, Gimsa J (2017) Increased osteoblast viability at alkaline pH in vitro provides a new perspective on bone regeneration. Biochem Biophys Rep 10:17–25 Additional Declarations No competing interests reported. Supplementary Files Figuresupporting1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4608945","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":320601170,"identity":"586c7373-9fb0-471f-97da-6e905dfe28a1","order_by":0,"name":"Dave W. Chen","email":"","orcid":"","institution":"Chang Gung Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Dave","middleName":"W.","lastName":"Chen","suffix":""},{"id":320601172,"identity":"16f2365d-9c43-4cf1-a79e-c2cd2f304b35","order_by":1,"name":"Ming-Kuang Chou","email":"","orcid":"","institution":"Chang Gung University","correspondingAuthor":false,"prefix":"","firstName":"Ming-Kuang","middleName":"","lastName":"Chou","suffix":""},{"id":320601176,"identity":"21816e0c-83e4-4c0c-920a-8f208dcc8214","order_by":2,"name":"Ngi-Chiong Lau","email":"","orcid":"","institution":"Chang Gung Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ngi-Chiong","middleName":"","lastName":"Lau","suffix":""},{"id":320601181,"identity":"bb87401f-b422-4f8d-bafb-2e8dc6e6efa5","order_by":3,"name":"Kong-Wei Cheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYBACCQYGNhAtx8DA2HgAKmhAlBZjoJYGJC0JhLUkNgAJ4rRIzkh+9uDjjtr0te2HgbbU1CY2sDdvk2D8cRinFmmJNHPDmWeO5247kwjUcux4YgPPsTIJhgTcWuSkc9ikeduO5W47ANTC2HAssUEixwyo5TZ+LX/bjqWbnX8I1SL/Br8WaZAWxraaBLMbYFtqgLbw4NciOf+ZmWRv2wHDbTeAtiQcO2DcxpNWbJGQ9h+nFokzh59J/Gyrkzc7n/7wwYeaOtl+9sMbb3ywScOpBQqg4ZMAZLBBGARBHQZjFIyCUTAKRgEcAADEIVu8d7NuAgAAAABJRU5ErkJggg==","orcid":"","institution":"Chang Gung University","correspondingAuthor":true,"prefix":"","firstName":"Kong-Wei","middleName":"","lastName":"Cheng","suffix":""}],"badges":[],"createdAt":"2024-06-20 03:53:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4608945/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4608945/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59996857,"identity":"9c37028b-c368-40ca-9ce6-56f5d6db158a","added_by":"auto","created_at":"2024-07-10 09:45:09","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":613650,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis results of PEEK using (I) TGA, (II) DSC measurements and (III) XRD patterns of PEEK and HA powders, respectively.\u003c/p\u003e","description":"","filename":"FigureJTICE2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4608945/v1/46615f52d4880b4e37b63fd0.jpg"},{"id":59996851,"identity":"c264395f-e1b6-4693-bd76-962a5c71dc1e","added_by":"auto","created_at":"2024-07-10 09:45:09","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1199942,"visible":true,"origin":"","legend":"\u003cp\u003e(I) The XRD patterns, (II) SEM images of PEEK/HA samples with various ratios of EDTA : Ca\u003csup\u003e2+\u003c/sup\u003e ions in precursor solutions and (III) the EDS mapping results for the sample with EDTA: Ca\u003csup\u003e2+\u003c/sup\u003e ion of 1: 1 in precursor solution. (Samples (i), (ii) and (iii) corresponded to the HA prepared with EDTA : Ca\u003csup\u003e2+\u003c/sup\u003e ion of 0.5:1, 1:1 and 2:1 in the precursor solutions, respectively.)\u003c/p\u003e","description":"","filename":"FigureJTICE4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4608945/v1/82cc37f4472749a23c20b30e.jpg"},{"id":59997477,"identity":"70d9c807-a5b5-403b-a9ce-638d9d9c860a","added_by":"auto","created_at":"2024-07-10 09:53:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":873573,"visible":true,"origin":"","legend":"\u003cp\u003e(I) Drug-release profiles for samples (A)-(C), (II) their cumulative values in the testing time of 24 hours, (III) drug-release profiles for samples (A)-(C) and their cumulative values in the testing time of ten days (240 hours) (Samples (A), (B) and (C) corresponded to the pristine Ampicillin salt, Vancomycin salt and their mixture with weight percentage of 50 :50 loading onto the sample (ii), respectively).\u003c/p\u003e","description":"","filename":"FigureJTICE6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4608945/v1/555634e8fa064c28c25c5ded.jpg"},{"id":59996855,"identity":"086aeb25-ed50-41bb-be56-cfebb9e6c2f3","added_by":"auto","created_at":"2024-07-10 09:45:09","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":360358,"visible":true,"origin":"","legend":"\u003cp\u003e(I) Drug-release profiles for samples (A)-(C), (II) their cumulative values in the testing time of 28 days (672 hours) (Samples (A), (B) and (C) corresponded to the pristine Ampicillin salt, Vancomycin salt and their mixture with weight percentage of 50 :50 loading onto sample (ii), respectively).\u003c/p\u003e","description":"","filename":"FigureJTICE8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4608945/v1/198f77b00db7cebe4c60404d.jpg"},{"id":59996853,"identity":"82e3da49-700e-4b53-afc0-8e17594739fb","added_by":"auto","created_at":"2024-07-10 09:45:09","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":280736,"visible":true,"origin":"","legend":"\u003cp\u003eVariation of relative optical densities in the solutions containing samples (A)-(C) as a function of time for (I)\u003cem\u003e E. coil\u003c/em\u003e and (II) \u003cem\u003eS. aureus\u003c/em\u003e, respectively.\u003c/p\u003e","description":"","filename":"FigureJTICE9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4608945/v1/5e0458371858d42103d8576d.jpg"},{"id":59996858,"identity":"5f033aef-c8f0-4110-89a1-34888e88f7bd","added_by":"auto","created_at":"2024-07-10 09:45:09","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1611281,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial properties for (I) sample (A), (II) sample (B), (III) sample (C) on \u003cem\u003eE. coil\u003c/em\u003e and (IV) sample (A), (V) sample (B), (VI) sample (C) on \u003cem\u003eS. aureus\u003c/em\u003e within testing time interval of 10 days, respectively.\u003c/p\u003e","description":"","filename":"FigureJTICE10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4608945/v1/086d3fde59ae606025454da7.jpg"},{"id":59997476,"identity":"fb040596-c10c-43cd-a8a3-87d00f75885f","added_by":"auto","created_at":"2024-07-10 09:53:09","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":552442,"visible":true,"origin":"","legend":"\u003cp\u003eThe CLSM images for samples within testing time of 3\u003csup\u003eth\u003c/sup\u003e, 7\u003csup\u003eth\u003c/sup\u003e and 14\u003csup\u003eth\u003c/sup\u003e day, respectively.\u003c/p\u003e","description":"","filename":"FigureJTICE13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4608945/v1/84cb32c189d3f75a48aaae24.jpg"},{"id":64611141,"identity":"294f628d-4ee6-406d-85bb-c8e8c8f6d388","added_by":"auto","created_at":"2024-09-16 14:04:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6179390,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4608945/v1/82e410e7-7341-4add-aeaf-70e27e015bf0.pdf"},{"id":59997478,"identity":"bb47c550-691d-4eef-8c99-1fc42a2e85e6","added_by":"auto","created_at":"2024-07-10 09:53:09","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":667032,"visible":true,"origin":"","legend":"","description":"","filename":"Figuresupporting1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4608945/v1/96595323734dc8beff9ba4e9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Improvements of Antibacterial and Cell Growth Activities for Hydroxyapatite Rods Modified Polyetheretherketone (PEEK) Implants with Direct Absorption of Antibiotics","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDemands of bone regeneration and fracture repairing technologies have become increasingly important due to aged society and limitation of autologous bone graft. Heterogeneous implants applied in the clinical treatments include metal-based implants such as stainless steel or titanium alloys [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], ceramic-based implants such as aluminum oxide or glass ceramic [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], bioactive glasses or polymer-based implants such as polymethylmethacrylate [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], respectively. While metal-based implants have many advantages such as inexpensive, easy to prepare and having good biocompatibility, their corrosion influences in the human body and the mismatch elastic moduli between human bone (7\u0026ndash;30 GPa) and metal-made implant (~\u0026thinsp;110 GPa for the Ti-based metal support) must be addressed in clinical treatments [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, poor medical image qualities of patients using the metal-based implants is also a major concern [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Ceramic-based implants offer high corrosion resistance, low thermal-expansion and good biological properties, but their high elastic moduli, easy to cause local stress, poor flexibility and difficult to machine limit their large scale applications in clinical treatments [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. For clinical applications, heterogeneous implants with low-cost, easy production, fast manufacturing and similar physical properties to human bone are crucial. In comparison to metal-based or ceramic-based implants, polymer-based implants have the advantages of good biocompatibility, easy of machining, low-cost, good mechanical properties, non-toxicity and ability of producing porous structures. Therefore, the development of the polymer-based implants was then proposed and applied in the clinical treatments of skeletal wounds [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Also considering the requirement of fast production, a new technology called 3D-printing has been reported and applied for the preparation of heterogeneous polymer-based implants, facilitating fast manufacturing of implants or bone-tissue engineering [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently, an interesting polymer called polyetheretherketone (PEEK) has been reported for clinical bone regeneration or repairing treatments [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Heterogeneous PEEK implants have several advantages, including a similar elastic modulus (3\u0026ndash;4 GPa) with human bone, low stress shielding, non-toxicity, good bio-stability in human body compared to traditional metal-based implants, and easy preparation with complex shapes using 3D printing technology [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Notably, the PEEK samples show no apparent influence on magnetic resonance imaging (MRI) compared to traditional metal-based implants [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and have been approved by Food and Drug Administration (FDA) as implantable candidate since 1980 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, these PEEK-made implants are still uncommon in clinical treatments due to their low antibacterial properties, which may result in high possibility of periprosthetic joint infection (PJI) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTradition treatments for PJI involve adequate debridement, local infiltration of high concentration antibiotics and long term parenteral antibiotic injection. However, issues of drug wastage, long term antibiotic complications, cell toxicity, and drug resistance in the human body are always observed during the clinical treatments. Therefore, modifications are necessary to improve the antibacterial properties of these polymer-based implants. Various types of materials such as hydroxyapatite (HA) [\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], metal tantalum [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and niobium pentoxide [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], TiO\u003csub\u003e2\u003c/sub\u003e/ZnO [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], ZnO [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], Si\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e/Ta [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], Ag/peptide [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and other composite antibacterial layers [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] have been employed to improve the bioactivities, antibacterial performances or attachment properties of bone cells for these polymer-based implants. Although coating of extra chemicals such as ZnO, Ag, metal Cu or tantalum onto polymer-based implants may inhibit the growth of microorganisms such as \u003cem\u003eS. aureus\u003c/em\u003e on the heterogeneous implants [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], the decomposition of these thin film in the buffer solution has been observed, leading to cell toxicity for bone cells attached onto the heterogeneous implants [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, the development of high-safety, good biocompatible, non-toxic samples with suitable antibacterial properties and similarity to bone mineral is crucial. Recently, an interesting material called hydroxyapatite (HA) with chemical formula of Ca\u003csub\u003e10\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e6\u003c/sub\u003e(OH)\u003csub\u003e2\u003c/sub\u003e has been developed due to its osteoconductivity, biocompatibility and similarity to the bone mineral phase [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, the HA\u0026rsquo;s poor antibacterial and mechanical properties make it challenging for direct application in clinical treatment [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Incorporating HA with some minor ratios (~\u0026thinsp;2\u0026ndash;5%) of Ag, CeO\u003csub\u003e2\u003c/sub\u003e, Cu, ZnS, Nb\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e, Ag\u003csub\u003e2\u003c/sub\u003eO has been attempted to improve its antibacterial property with these heavy metal ions released from surface of implants [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Although these modifications can inhibit the microorganism growth, cell toxicity of these polymer-based implants with HA/metal ions coating has also been observed. Therefore, the incorporation of heavy metal ions into HA might not be an ideal approach. In our previous study [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], we deposited ZnO rod-like arrays [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and TiO\u003csub\u003e2\u003c/sub\u003e/ZnO core-shell rod-like arrays [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] onto the PEEK disks using the solution growth method. These rod-like arrays with suitable surface area showed good capacities for loading of antibiotics and then inhibited the microorganism growth such as \u003cem\u003eS. aureus\u003c/em\u003e on implants during clinical treatments. However, decomposition of the ZnO layer on PEEK sample in the buffer solution was observed, resulting in cell toxicity for the bone cells attached onto the surface of implants [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. For the above discussion, HA-rod-like arrays might be a good candidate to replace the ZnO arrays on PEEK samples, which offer good specific surface area for loading of antibiotics. In the literature [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], HA showed good absorption ability for the ionic or organic molecular and can be used as a carrier for absorption agents or the substrates for loading catalysts [\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. If these HA/antibiotics grown onto PEEK implants are employed in the orthopedics patients, the antibiotics attached onto HA surface would release slowly into the human body, decreasing the possibility of microorganism infection during the recovery of clinical treatments. The HA on the PEEK surface, which is inorganic constituent that account around 60\u0026ndash;70% in human bone, could also act as the gene and small molecule drug carrier to induce the osteogenic differentiation of stem cells and bone defect repair [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Huang et al. (2024) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] employed hydrothermal method to prepare the selenium/strontium/zinc-doped hydroxyapatite (Se/ Sr/Zn-HA) powders. Their Se/Sr/Zn-HA powders exhibited banded crystal shape with a length of 2\u0026ndash;10 \u0026micro; m and a width of 1 \u0026micro;m. Incorporating Se, Sr and Zn ions into HA samples induced a transformation in their microstructures, transitioning from banded shape, the spherical type composed some sheets/ regular aggregate shapes to radial petal-like microstructures/micro-spherical structures with nano-flakes, respectively. Their HA samples were mixed with polycaprolactone (PCL) as ink to construct composite scaffolds using 3D-printing. The Se ions formed as the SeO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e on samples surface and Zn ions released from samples enhanced antibacterial effects. Sr\u003csup\u003e2+\u003c/sup\u003e and Zn\u003csup\u003e2+\u003c/sup\u003e ions exhibited good osteogenic effects. The samples demonstrated effective antibacterial properties against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e, reducing microorganism growth of around 35% and 40% respectively, compared to pristine PCL sample after 24 hours of testing. All experimental groups affected the proliferation of MC3T3-E1 cells without cytotoxicity, with cell viability exceeding 100% over with a 7 days testing period. Swan et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] incorporated cationic steroid antimicrobials (CSA13) into the strontium substituted HA micro- and nano-sized particles through low-temperature and high speed collision process. The resulting samples were implanted into the surface of N-carboxymethyl chitosan (N-CMCh) pre-embedded on PEEK substrate. CSA 13 antibacterial agents released from samples reduced the growth of \u003cem\u003eMethicillin Resistant Staphylococcus Aureus\u003c/em\u003e by approximately 27%, compared to growth of \u003cem\u003eMethicillin Resistant Staphylococcus Aureus\u003c/em\u003e on the PEEK substrate without the sample coating. The human osteoblast-like cells viability on these specimens exhibited significant cell growth even in the presence of CSA13. Morsy (2024) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] fabricated and characterized 3D hybrid scaffolds composed of HA nanoparticles synthesized in situ in gelatin and PVA solutions, loaded with combustionally synthesized (10 wt %) zinc oxide nanoparticles (ZnO NPs). The samples showed high porosity (up to 78%) with a pore size ranging from 50\u0026ndash;300 \u0026micro;m and good mechanical strength (approximately 55 kPa). Loading of ZnO into samples imparted good antibacterial performances against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coil\u003c/em\u003e, with inhibition zone diameters of 7 mm and 6mm, respectively. Rachid et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] prepared Zn-doped HA using traditional co-precipitation method, followed by dissolving these powders and chitosan in 1% acetic acid solution with cross-linked agent (glutaraldehyde) to obtain chitosan cross-linked Zn-doped HA composites. The resulting samples exhibited suitable antimicrobial activities against both Gram-positive and Gram-negative bacteria, with inhibition zone diameter of 20 mm and 16 mm, respectively. Yakufu et al. (2024) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] prepared porous PEEK scaffolds with HA layer coated onto the pore wall surface using sacrificial porogen of HA-coated sodium chloride (NaCl) microspheres. With HA microspheres coated onto the pore wall of PEEK samples, their mechanical properties were comparable to pristine PEEK sample. The highly porous structure of PEEK@HA scaffolds and the HA coating layer promoted biomineralization in vitro and osseointegration in vivo. However, their antibacterial properties were not tested in this study. Therefore, the preparation of HA on PEEK substrates with diverse microstructures for loading suitable antibiotics might show good antibacterial properties and enhance bone cell growth on the PEEK implants in clinical orthopedic treatments. In this study, we prepared the HA films with different microstructures onto the 3D-printed PEEK disks using simple hydrothermal method. We evaluated the physical properties, short- and long-term antibacterial properties against Gram-positive and negative bacteria, and analyzed the influence of bone cell growth on the PEEK samples with various types of antibiotics directly absorbed onto HA thin films on the PEEK surface.\u003c/p\u003e"},{"header":"2. Experimental details","content":"\n\u003ch3\u003e2 − 1 Preparation of HA layers on PEEK disks\u003c/h3\u003e\n\u003cp\u003eFrom the above discussion, the use of HA for the direct absorption of antibiotics is intriguing due to its good osteoconductivity, biocompatibility and similarity to the bone mineral phase. To achieve this, we deposited HA layers onto PEEK disks using traditional hydrothermal method. The PEEK disk (diameter of 14.6 mm and thickness of 1.8 mm) was obtained through our 3D-printer (Black Magic 3D, Prusa i3). The detailed procedures for preparing of 3D-printed PEEK disk were similar with those reported in our previous studies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Subsequently, the PEEK was washed using ethanol, deionized water, acetone, and deionized water in the ultrasonic bath with time interval of 15 minute. Then the sample was blown dry with ultra-pure nitrogen gas. To ensure an uniform HA thin film on PEEK substrate and improve its attachment property, sulphonation process with SO\u003csub\u003e3\u003c/sub\u003eH functional group on PEEK sample was carried out to enable attachment of HA thin films on substrate [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This process involved immersing the PEEK into a 10 ml glass container containing concentrated sulfuric acid (98%, Honeywell, Fluka) for 10 minutes. After sulphonation, the samples were subjected to ultrasonic bath for 15 minutes to reduce any unreacted sulfuric acid on PEEK surface. This cleaning process was repeated at least three times, and the samples were then dried using ultra-pure nitrogen gas.\u003c/p\u003e \u003cp\u003eFor the growth of HA with various microstructures on PEEK substrates, similar procedures as reported by Suchanek et al. (2018) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] were employed but the ratios of chelating agent : Ca ions in reaction solution were verified in order to obtained HA thin film with high surface area. The calcium nitrate (Ca(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026sdot;4H\u003csub\u003e2\u003c/sub\u003eO, UniRegion Bio-Tech, purity\u0026thinsp;\u0026gt;\u0026thinsp;98%) with concentration of 0.14 M and ammonium phosphate monobasic (NH\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, J. T. Baker, purity\u0026thinsp;\u0026gt;\u0026thinsp;98%) with concentration of 0.084 M and 30 mL of deionized water were mixed well in a glass container at 30 minutes interval. The ethylenediaminetetraacetic acid disodium salt dehydrate as the chelating agents (EDTA-Na\u003csub\u003e2\u003c/sub\u003e, C\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003eNa\u003csub\u003e2\u003c/sub\u003e \u0026sdot; 2H\u003csub\u003e2\u003c/sub\u003eO, Honeywell, Fluka, purity\u0026thinsp;\u0026gt;\u0026thinsp;98%) with concentrations of 0.07, 0.14 and 0.28 M and monoethanolamine (MEA, C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e7\u003c/sub\u003eNO, Aldrich Co., purity\u0026thinsp;\u0026gt;\u0026thinsp;98%) with concentration of 0.28 M were then added into the reaction solution containing Ca\u003csup\u003e2+\u003c/sup\u003e ions in order to form Ca-complex ions and reduced the concentration of free Ca\u003csup\u003e2+\u003c/sup\u003e ions in the solution bath, which might make the HA become powders suspending in the solution bath. With most of free Ca\u003csup\u003e2+\u003c/sup\u003e ions formed as Ca-complex ions, a colorless reaction bath was obtained. Then ammonia hydroxide solution (NH\u003csub\u003e4\u003c/sub\u003eOH, J. T. Baker Co., purity\u0026thinsp;=\u0026thinsp;37%) with volume of 5mL was added into the reaction bath to provide OH\u003csup\u003e\u0026minus;\u003c/sup\u003e ions in HA. The pH value of reaction solution was maintained at 10. Homogeneous reaction solution was then transferred into a 150 mL Teflon vessel and placed in a hydrothermal reactor. The autoclave was sealed and kept at the temperature of 180\u0026deg;C for 5 hours. Following the reaction, the samples were washed several times with deionized water to remove the unreacted chemicals attached to the PEEK substrates and then dried in an oven at 70\u0026deg;C for 24 hours.\u003c/p\u003e\n\u003ch3\u003e2–2 Preparation of drug loaded on the samples\u003c/h3\u003e\n\u003cp\u003eDue to the poor inherent antibacterial property of pristine HA, it is imperative to enhance its antibacterial characteristic. In this study, antibiotics were directly absorbed onto the HA surface. To achieve this, concentration of 10000 \u0026micro;g/L for various types of antibiotics (pure Ampicillin salt, pure Vancomycin salt and the Ampicillin : Vancomycin of 50 : 50 in weight percentage) in the water bath were employed to prepare the HA/antibiotic samples through direct absorption process. From our previous studies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], it was observed that the sample immersed in a water bath containing antibiotics for five days could result in 90% of antibiotics being absorbed onto the sample surface. Therefore, the HA/PEEK sample was placed in an aqueous solution containing different types of antibiotics with a total volume of 20 mL at room temperature for five days. Following the complete absorption of antibiotics onto the sample surface, the sample was put in a clean container to avoid any possible influence from other organisms or impurities.\u003c/p\u003e\n\u003ch3\u003e2–3 Characterization of HA/PEEK samples\u003c/h3\u003e\n\u003cp\u003eThe crystal phase of HA/PEEK sample was conducted using the X-ray diffractometer (XRD, D2-Phaser, Bruker, A26-X1-1) with CuK\u003csub\u003eα\u003c/sub\u003e (λ\u0026thinsp;=\u0026thinsp;1.5405 \u0026Aring;) irradiation. The 2θ range in the XRD patterns of samples were in the range of 10\u0026ndash;60\u0026deg; with scanning rate of 0.1\u0026deg;/s. A field-emission scanning electron microscope (FE-SEM, JOEL JSM-7500F) equipped with energy dispersive spectra (EDS, Horiba, 7021-H) with acceleration voltage of 10 kV and working distance of 15 mm was employed to analyze the surface and cross-section images of samples and their compositions. An UV-Visible spectrophotometer (UV-Vis, Varian Co., CARY50) was used to determine the concentration of antibiotics in the buffer solution bath. The optical densities of organisms in the solution bath were carried out using a microplate reader (SpectraMax M3, USA).\u003c/p\u003e\n\u003ch3\u003e2–4 Antibacterial tests for samples\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e analysis of concentrations for antibiotics in buffer solution was carried out to understand the drug release profiles from the samples into buffer solution. Similar approach can be observed in our previous studies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. 10 mL of the phosphate buffer solution (PBS, pH 7.4) was utilized for the analysis of drug release behavior from the sample into the PBS at 37\u0026deg;C with the shaking rate of 30 rpm. \u003cem\u003eIn vitro\u003c/em\u003e analysis test on the concentration of antibiotics in buffer solution was carried out within the suitable time interval using the UV-Vis spectrophotometer. Fresh buffer solution was replaced every testing procedure to avoid the saturated concentration of antibiotics in solution bath. The detected wavelengths for Ampicillin and Vancomycin salts were 220 nm and 280 nm using the UV-Vis spectrometer, respectively.\u003c/p\u003e \u003cp\u003eThe process for evaluation of the sample\u0026rsquo;s antibacterial property is similar to our previous studies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], involving two types of antibacterial tests: Agar diffusion test and broth dilution test. For agar diffusion test, \u003cem\u003eS. aureus (ATCC 29213)\u003c/em\u003e or \u003cem\u003eE. coli (ATCC 25922)\u003c/em\u003e inoculum with a volume of 200 \u0026micro;L in the 100ml Nutrient broth solution (NB, Becton, Dickinson and Company, including beef extract 3g and peptone 5g) was seeded, and organisms were allowed to grow for 2.5 hours at 37\u003csup\u003e\u0026deg;\u003c/sup\u003eC with a constant wavering rate of 220 rpm. The bacterial concentration in the solution was adjusted to around 10\u003csup\u003e8\u003c/sup\u003e colony-forming unit (CFU)/mL. The antibiotic disk diffusion method was employed to observe antibacterial properties against \u003cem\u003eS. aureus\u003c/em\u003e or \u003cem\u003eE. coli\u003c/em\u003e in the agar containing NB at the Petri dish. An organism solution with a bacterial concentration of 10\u003csup\u003e8\u003c/sup\u003e CFU/ml and a total volume of 300 \u0026micro;l was seeded onto the agars in the Petri disks, and the inhibition zone test was analyzed at 37\u003csup\u003e\u0026deg;\u003c/sup\u003eC. Standard calibration curves of the inhibition zone on the organisms were created using the paper with loading of various concentrations of different types of antibiotics (1, 10, 100 and 1000 \u0026micro;g/mL), respectively. The concentration of antibiotic released from the sample was then obtained using interpreting these curves.\u003c/p\u003e \u003cp\u003eFor the broth dilution test, the optical density (OD) value of bacteria in NB solution was measured. NB solutions cultured with \u003cem\u003eS. aureus\u003c/em\u003e or \u003cem\u003eE. coil\u003c/em\u003e with a concentration of 10\u003csup\u003e8\u003c/sup\u003e CFU/ml were employed to evaluate their antimicrobial activities, respectively. Standard calibration curve with only organism in the solution was also tested to evaluate the growth rate of organism. The solution containing organism suspension was incubated at 37\u0026deg;C with the wavering rate of 150 rpm. The optical density (OD) values for these solutions containing organisms with antibiotics coated onto the samples were estimated using a microplate reader with the light wavelength set at 600 nm. The relative optical density value of the bacterial solution was calculated using the following equation:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\text{R}\\text{e}\\text{l}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e} \\text{v}\\text{a}\\text{l}\\text{u}\\text{e} \\text{o}\\text{f} \\text{O}\\text{D}\\left(\\text{\\%}\\right)=\\frac{OD value of solution }{OD value of standard solution }$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003e2–5 In vitro cytocompatibility of samples\u003c/h3\u003e\n\u003cp\u003eFor the \u003cem\u003ein vitro\u003c/em\u003e cell response tests, mouse osteoblast-like MC3T3-E1 cells (American Type Culture Collection, CRL-2593, USA) were cultured in a Minimum Essential Medium (α-MEM, Gibco BRL, Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, USA), 100 \u0026micro;g/mL penicillin and 100 \u0026micro;g/mL streptomycin sulfate (Gibco BRL, Thermo Fisher Scientific, USA) in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. Before cell culture process, the specimens were sterilized under UV radiation, which was placed in 24-well plates. Then the cells were cultured on the specimens at a density of 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per well to evaluate cell attachment, proliferation and cytotoxicity. The culture medium was replaced every 2 days. The morphology of the cell proliferation was observed using a confocal laser scanning microscopy (CLSM, Leica TCS SP8 X, USA) within the 3th, 7th, and 14th testing day, respectively.\u003c/p\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Physical characterization of HA/PEEK samples\u003c/h2\u003e \u003cp\u003eWe deposited the HA layers onto the PEEK disks using traditional hydrothermal method to obtain the surface-modification PEEK samples with good antibacterial property, osteoconductivity, and biocompatibility. Traditional 3D-printer always uses polylactic acid (PLA) as the printing material, with temperature values for the bozzle and holder of below 190\u0026deg;C [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, PEEK, being a high glass-transition temperature polymer, requires adjustments in the temperature settings for both the nozzle and holder in 3D-printer. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (I) and (II) shows the analysis results of the thermogravimetric analyzer (TGA, TA TGA Q-50) and the differential scanning calorimeter (DSC, TA DSC 50) of PEEK fibers. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(I) reveals that PEEK begins to loss its weight at the temperature exceeding 550\u0026deg;C. At the temperature higher than 600\u0026deg;C, the PEEK fibers lost approximately 45% of total weight, indicating decomposition starting at 550\u0026deg;C. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (II) displays the DSC analysis results, showing T\u003csub\u003em\u003c/sub\u003e (melting point) and T\u003csub\u003ec\u003c/sub\u003e (crystallization point) values for PEEK fibers at 338.74\u0026deg;C and 285.24\u0026deg;C, respectively, which are consistent with literature reports [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Based on the DSC and TGA analysis, the nozzle temperature in our 3D-printer must be maintained in the range of 286\u0026ndash;550\u0026deg;C to ensure the PEEK sample in the nozzle at molten state. From the study proposed by Yang et al. (2017) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], high temperature of the nozzle and holder in our fused deposition modelling printer can enhance the printing quality of PEEK disks. Therefore, we conducted various printing parameters such as temperature of nozzle, temperature of holder and printing speed for our 3D-printer. To prepare our PEEK disks, we set the temperature of holder at 280\u0026deg;C, printing speed at 10 mm/s, temperature of nozzle at 380\u0026plusmn;10\u0026deg;C and nozzle diameter at 1.0 mm for our 3D-printer. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (III) presents the XRD pattern of our PEEK sample, which shows the XRD peaks for our 3D-printing PEEK are consistent with standard PEEK (JCPDS no. 52-2277). The XRD peaks for our samples located at 2θ of 19.03\u0026deg;, 21.01\u0026deg;, 23.06\u0026deg; and 29,15\u0026deg; corresponded to the crystal planes of (1 1 0), (1 1 1), (2 1 1) and (0 1 1) for PEEK, respectively. Three small and broad peaks at 2θ of around 33.26\u0026deg;, 39.19\u0026deg; and 47.65\u0026deg; are assigned to the PEEK phase. The result shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(III) confirmed that our sample was PEEK after the fused deposition printing process. For the growth of HA thin film on PEEK substrates, calcium nitrate, ammonium phosphate and ammonia hydroxide were utilized as sources of Ca, PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e and OH\u003csup\u003e\u0026minus;\u003c/sup\u003e ions, leading to the formation of HA through the following reaction [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]:\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e10Ca(NO) +6(NH)HPO + 2OH→Ca(PO)(OH) + 6NH +12 H +20 NO (1)\u003c/h3\u003e\n\u003cp\u003eTo confirm the occurrence of the reaction (1) using the hydrothermal method, we collected the powders in the solution bath after hydrothermal process. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (III) displays the XRD pattern of HA powders obtained after hydrothermal method, exhibiting the peaks of HA powders (red line) align well with standard HA peaks (JCPDS no. 74\u0026ndash;566). The observed peaks at 2θ of around 29.5\u0026deg;, 32.23˚, 32.69˚, 33.34˚, 40.23˚, 47.11˚, 48.45˚, 49.82˚, 50.92˚, 51.65˚, 52.48˚ correspond to crystal planes of (2 1 0), (1 1 2), (3 0 0), (2 0 2), (2 2 1), (2 2 2), (2 3 0), (2 1 3), (3 2 1), (1 4 0), (3 0 3) for HA, respectively. These results indicate successful preparation of PEEK and HA samples using 3D printing and hydrothermal method. However, due to the high reaction rate of Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), most of HA formed as the powders suspended in the solution rather than the forming of thin film on the PEEK sample. To address this, the chelating agent and stabilizer were added to the solution bath [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. We used EDTA-Na\u003csub\u003e2\u003c/sub\u003e as the chelating agent to form Ca-EDTA complexes, reducing the concentration of free Ca\u003csup\u003e2+\u003c/sup\u003e ions in the solution bath. Low concentration of free Ca\u003csup\u003e2+\u003c/sup\u003e ions in solution would reduce the ratio of HA powders formed in the solution bath and facilitated the HA thin film grown onto the PEEK substrate. With the reaction bath temperature of 180\u0026deg;C, the Ca-EDTA complexes would begin dissociation, releasing Ca\u003csup\u003e2+\u003c/sup\u003e ions that would be absorbed onto PEEK surface. These free Ca\u003csup\u003e2+\u003c/sup\u003e ions on the PEEK surface then reacted with PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e and OH\u003csup\u003e\u0026minus;\u003c/sup\u003e ions to form HA thin film on PEEK. Because the EDTA is the strong chelating agent on Ca\u003csup\u003e2+\u003c/sup\u003e ions, the parameters of reaction temperature, the ratio of EDTA : Ca\u003csup\u003e2+\u003c/sup\u003e and the pH values in the solution baths were important for the growth of HA thin film on PEEK substrate. In this study, MEA in the solution bath served as the stabilizer, morphologic control agents and the control unit for the release of Ca\u003csup\u003e2+\u003c/sup\u003e ions from EDTA agent in the solution bath [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Under high temperature and suitable pH value in solution bath, the HA thin film would form on the PEEK sample surface rather than powders suspending in the solution bath with suitable ratio of EDTA : Ca\u003csup\u003e2+\u003c/sup\u003e. High EDTA: Ca\u003csup\u003e2+\u003c/sup\u003e ratio in solution bath would reduce the growth of HA on PEEK substrate due to the low degree of super-saturation of HA. Conversely, low EDTA: Ca\u003csup\u003e2+\u003c/sup\u003e ratio in solution bath would cause most of HA to form powders rather than thin film on substrates. Therefore, we verified the ratios of EDTA : Ca\u003csup\u003e2+\u003c/sup\u003e ions in the solution bath to obtain a uniform and compact HA thin film on PEEK. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (I) shows the XRD patterns of samples (i), (ii) and (iii), corresponding to the HA thin film on PEEK substrates prepared with the ratio of EDTA: Ca\u003csup\u003e2+\u003c/sup\u003e ions: of 0.5 : 1, 1 : 1 and 2 : 1, respectively. Sample (i) exhibited only two small peaks at 2θ of around 32.1\u0026deg; and 33.2\u0026deg;, which corresponded to the crystal planes of (1 1 2) and (2 0 2) for HA. Although we could observe a white HA thin film on PEEK, most of HA formed as the powders in the solution bath. This is because low ratio of EDTA : Ca in the solution bath and made free Ca\u003csup\u003e2+\u003c/sup\u003e, PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e and OH\u003csup\u003e\u0026minus;\u003c/sup\u003e ions approach to the supersaturation of HA. Sample (ii) showed peaks consistent with standard HA peaks, confirming the growth of HA thin film onto the PEEK substrate. The insert image shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (I) displayed the pictures of PEEK substrate before/after HA growth for sample (ii). A white thin film was covered on our PEEK disk after the hydrothermal treatment. In contrast, sample (iii) displays peaks corresponding to the PEEK sample, indicating absence or amorphous HA thin film growth. SEM images in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (II) revealed that sample (i) had a non-uniform thin film with crack and pinholes. Some layer/plate microstructures covered on the PEEK substrate were also observed. These layer or plate microstructures were similar to those reported in the literature [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. When the ratio of EDTA: Ca\u003csup\u003e2+\u003c/sup\u003e in solution bath changed from 0.5 :1 to 1 : 1, the surface morphologies of samples transformed from plate/layer microstructures (sample (i)) to rod-arrays HA microstructures (sample (ii)). The diameter of hexagonal rod-like microstructure for sample (ii) was approximately 5.44 \u0026micro;m (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The rod-array microstructures of HA were also reported in the literature [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. When the ratio of EDTA : Ca\u003csup\u003e2+\u003c/sup\u003e was adjusted to 2 : 1 (sample (iii)), surface microstructures showed similar to pristine PEEK. Using the EDS (almost no Ca or P element was detected), SEM image and XRD analysis results, we could confirm that no HA thin film was grown onto the PEEK substrate using EDTA : Ca\u003csup\u003e2+\u003c/sup\u003e of 2 : 1 in solution bath (sample (iii)). The possible reason is the influence of EDTA chelating agent. The EDTA is a strong chelating agent on Ca\u003csup\u003e2+\u003c/sup\u003e ions. Most Ca\u003csup\u003e2+\u003c/sup\u003e ions in the solution bath were the stable EDTA-Ca complexes and reduced the concentration of free Ca\u003csup\u003e2+\u003c/sup\u003e ions in the solution bath. Although the temperature and pressure were high during our hydrothermal process, the dissolution of EDTA-Ca complexes was difficult and therefore made almost no HA thin film grow onto PEEK sample. Then we examined the microstructures of samples (i) and (ii). Their microstructures showed different with the change of the ratio for EDTA : Ca\u003csup\u003e2+\u003c/sup\u003e in the solution bath. According to the report proposed by Pu\u0026rsquo;ad et al. (2020) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], the HA obtained using traditional chemical precipitation always low-crystallinity HA particles. With low ratio of EDTA : Ca\u003csup\u003e2+\u003c/sup\u003e in the solution bath, our approach was similar to chemical precipitation. Most of HA was formed as powders suspending rather than the thin film grown on the substrate. Therefore, a non-uniform, low-crystalline HA with crack or pinholes on PEEK substrate could be observed. When the EDTA : Ca\u003csup\u003e2+\u003c/sup\u003e ions of 1:1 was set, the concentration of free Ca\u003csup\u003e2+\u003c/sup\u003e ions in the solution decreased and the release rate of Ca\u003csup\u003e2+\u003c/sup\u003e ions from EDTA-Ca complexes could be controlled by adjusting the reaction temperature or pressure. Therefore the HA layers with rod-like microstructures could be obtained on substrates [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and indicated that the HA layer with high surface area could be obtained on our sample (ii). Then we analyzed the composition of HA thin film on PEEK substrate (samples (i) and (ii)) using EDS analysis at 200 (X). For sample (i), the molar ratio of Ca : P of around 5 : 1 in HA indicated the element Ca was much higher than the theoretical ratio for HA (Ca/P\u0026thinsp;=\u0026thinsp;1.67). The layer on PEEK substrate (sample (i)) was the low-crystalline HA layer with a large amount of free Ca\u003csup\u003e2+\u003c/sup\u003e ions absorbed onto sample surface. For sample (ii), its molar ratio of Ca : P was 62.54% :37.46%, which corresponded to the ratio of Ca/P of 1.67 for sample (ii). The EDS analysis results indicated that the rod-like HA thin film was grown onto PEEK substrate (sample (ii)). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (III) shows the EDS mapping results for sample (ii), exhibiting the uniform distributions of Ca and P, which indicated that the uniform HA layer with rod-like microstructures was obtained using hydrothermal method with the EDTA : Ca\u003csup\u003e2+\u003c/sup\u003e of 1 : 1 in the solution bath. Because our topic was to prepare the HA thin film for the loading of antibiotics to improve its antibacterial property, it specific surface area was important. A specific surface area analyzer (Micomeritics, ASAP 2020) was employed to examine the change of specific surface area for samples (i) and (ii) with nitrogen gas pressure of 3 \u0026micro;m Hg. Using the absorption curves of samples, the effective values of surface area for samples (i) and (ii) of around 1.72 \u0026plusmn; 0.02 m\u003csup\u003e2\u003c/sup\u003e/g and 2.21 \u0026plusmn; 0.04 m\u003csup\u003e2\u003c/sup\u003e/g were obtained, respectively. The sample (ii) had higher specific surface area value than that of sample (i) due to it having rod-like microstructures and made it have good loading capacity for the antibiotics. The values of thickness for HA coated onto samples were around 5\u0026micro;m obtained from the cross-section FE-SEM images.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 \u003cem\u003eThe antibacterial properties of samples\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eDue to the poor antibacterial properties exhibited by the HA/PEEK samples, it is imperative to enhance their antibacterial efficacy. Generally, coating with biodegradable polymer such as PLGA (poly(D,L-lactide-co-gycolide)) containing antibiotics onto the sample surface was the major method to enhance their antibacterial properties [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The degradation of PLGA is employed as the control unit for the antibiotic released from sample surface into solution bath. However, the high cost of PLGA makes polymer-made implants become expensive. To address this issue, if our HA thin film could absorb antibiotics directly and maintain suitable antibacterial properties for at least ten days, these antibiotics/HA/PEEK samples could be employed in the clinical treatments. HA with the most stable Ca/P ratio of approximately 1.67 has been extensively studied and act as an excellent drug delivery carrier due to its high affinity and low translocation efficiency [\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. HA possess excellent drug carrier due to the active ions at the surface. The mesoporous structure at the surface of HA is attractive for chemical bonding with chemical, small molecules and generic drugs [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Ca\u003csup\u003e2+\u003c/sup\u003e at the surface of HA are the main bonding between HA and drug [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. This electrostatic interaction between Ca\u003csup\u003e2+\u003c/sup\u003e and antibiotic contribute to the drug loading ability, drug release properties. Besides, the porosity and rod-like appearance of HA on PEEK surface increased the surface area of HA which affect its interaction with antibiotics. These characteristics largely contribute to slow and sustained release activity of antibiotics. [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] According to studies proposed by Zheng et al (2022) [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] and (2023) [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], Alendronate (ALN) is bound onto the nano-hydroxyapatite (nHA) through interaction with Ca\u003csup\u003e2+\u003c/sup\u003e on surface of nHA to establish slow and sustained release profle of ALN to facilitate bone formation. Liu et al. (2020) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] also demonstrated the binding of the anticancer drug doxorubicin (DOX) to HA by electrostatic interaction of Ca\u003csup\u003e2+\u003c/sup\u003e at the surface of HAp which could prevent tumor recurrence. Then we selected sample (ii) for testing the direct absorption of various antibiotics, including pure Ampicillin sodium salt, pure Vancomycin hydrochloride salt and a mixture of Ampicillin : Vancomycin (w/w)=(50/50)). The design for the loading of mixture with weigh percentages of Ampicillin : Vancomycin\u0026thinsp;=\u0026thinsp;50 : 50 aims to reduce the dosage of individual antibiotics absorbed onto the sample while maintaining adequate antibacterial activities against both Gram negative and positive bacteria. Sample (A), (B) and (C) correspond to sample (ii) loaded with pure Ampicillin sodium salt, pure Vancomycin hydrochloride salt and the mixture of Ampicillin\u0026thinsp;+\u0026thinsp;Vancomycin (w/w\u0026thinsp;=\u0026thinsp;50/50), respectively. Following a five-day absorption period in a water bath, approximately 98.02%, 99.12% and 76.5% of antibiotics were fully absorbed onto the HA/PEEK sample surface for samples (A), (B) and (C), respectively. Nearly 100% of antibiotics for pure Ampicillin and Vancomycin salts were absorbed but only around 76.5% of the antibiotic mixture was absorbed onto HA sample surface. Detailed absorption results of the antibiotic mixture revealed that around 97.21% of Ampicillin salt was absorbed, but only around 56.2% of Vancomycin salt was absorbed onto the HA surface. This outcome aligns with the expectation due to the larger molecular weight of Vancomycin salt (M\u003csub\u003ew\u003c/sub\u003e = 1449.3 g/mol) compared to Ampicillin salt (M\u003csub\u003ew\u003c/sub\u003e = 349.41 g/mol). Small molecule are known to be more easily attached onto the sample surface, explaining the higher absorption ratio of Ampicillin salt onto the HA surface. Following the complete absorption of antibiotics onto sample surface, the samples were immersed in the PBS buffer solution to examine their drug-release profiles at 24-hour intervals. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (I) and (II) display the drug-release profiles and cumulative values over a testing time interval of 24 hours using samples (A), (B) and (C), respectively. Additionally, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(I) included the values of MIC90 and MIC50 (minimum inhibition concentration for the inhibition of 90% and 50% organism growth) for \u003cem\u003eE. coil\u003c/em\u003e (Gram-native bacteria) and \u003cem\u003eS. aureus\u003c/em\u003e (Gram-positive bacteria) obtained from literature [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Concentrations of drug released from samples (A)-(C) in the buffer solution were higher than the values of MIC 90 and MIC 50 for \u003cem\u003eS. aureus\u003c/em\u003e and MIC 50 for \u003cem\u003eE. coil\u003c/em\u003e in testing time interval of 24 hours. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(I) revealed that the concentrations of drug released from samples (A)-(C) could inhibit 90% growth for \u003cem\u003eS. aureus\u003c/em\u003e (Gram-positive bacteria) and 50% for \u003cem\u003eE. coil\u003c/em\u003e (Gram-negative bacteria), which indicated the samples had slightly poor antibacterial properties against Gram-negative bacteria. Cumulative values in PBS buffer solution for samples (A)-(C) shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (II) exhibit fast drug-release rates during the first 3 hour, attributed to the release of drug weakly physical-absorption on HA layer. Subsequently, stable drug-released profiles for samples (A)-(C) were observed between 3 and 24 hours in buffer solution. It was noted that around 60% of Ampicillin salt absorbed onto HA layer for sample (C) was released into buffer solution within the first 3 hours, contributing to higher cumulative values. Combined with the results for samples (A) and (C) shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (II), we could conclude that around 58.3 mg of Ampicillin salt was attached onto our samples with weakly physical absorption (total weight of 97.2 mg for Ampicillin salt was absorbed onto sample (C) and that of 196.04 mg for Ampicillin salt was absorbed onto sample (A)). Examining the drug-release profile of Vancomycin salt for samples (B) and (C), a similar drug-release profile was observed. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (III) and (IV) shows the drug-release profiles and cumulative results for samples (A)-(C) within testing time interval of 10 days (0-240 hours). Sample (A) displayed drug-release profiles approaching the values of MIC 90 for \u003cem\u003eE. coil\u003c/em\u003e, while other samples exhibited drug-released profiles higher than the values of MIC 50 and MIC 90 for \u003cem\u003eS. aureus\u003c/em\u003e and MIC 50 for \u003cem\u003eE. coil\u003c/em\u003e. These results indicate that the samples with direct absorption of antibiotics demonstrated good antibacterial properties against \u003cem\u003eS. aureus\u003c/em\u003e and acceptable antibacterial properties against \u003cem\u003eE. coil\u003c/em\u003e. Results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (IV) suggest that approximately 55% of Ampicillin salt absorbed on sample (A), 40% of Vancomycin salt absorbed on sample (B) and 64% of Ampicillin salt\u0026thinsp;+\u0026thinsp;53% of Vancomycin salt absorbed on sample (C) were released into buffer solution within testing time interval of 10 days. As only around 50\u0026ndash;60% of antibiotics were released from samples within the 10-days testing interval, it implies that the samples might retain antibacterial properties for longer than 10 days. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (I) and (II) illustrate drug-release profiles and cumulative results of antibiotics released from samples in buffer solution over a testing time interval of 28 days. For sample (A), concentration of drug in buffer solution remained higher than the values of MIC 50 and MIC 90 for \u003cem\u003eS aureus\u003c/em\u003e and approach the value of MIC 90 for \u003cem\u003eE. coil\u003c/em\u003e during the first 6 days. Subsequently, drug-released profiles for sample (A) were lower than MIC 90 value for \u003cem\u003eE. coil\u003c/em\u003e between days 7th and 28th. Only on the 8th and 27th day for sample (A), drug concentrations in buffer solution slightly higher than the value of MIC 90 for \u003cem\u003eE. coil\u003c/em\u003e were observed. For samples (B) and (C), drug-release profiles indicated concentrations higher than the values of MIC 50 for \u003cem\u003eE. coil\u003c/em\u003e and MIC 50 and MIC 90 for \u003cem\u003eS. aureus\u003c/em\u003e, suggesting good antibacterial properties against \u003cem\u003eS. aureus\u003c/em\u003e and acceptable antibacterial properties against \u003cem\u003eE. coil\u003c/em\u003e. Cumulative release profiles shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (II) reveal nearly liner drug-release profiles for samples (A)-(C) throughout the testing period of 1\u0026ndash;28 days. Approximately 83% of Ampicillin salt absorbed onto sample (A) was released into buffer solution, along with around 48% of Vancomycin salt absorbed onto sample (B) was released into buffer solution in the 28-day testing period. Notably, around 78% of Ampicillin salt and almost 100% of Vancomycin salt absorbed onto sample (C) were released into buffer solution within the 28-day testing interval. Constant drug-release profiles of antibiotics from HA surface suggest that rod-like HA microstructures serve as suitable samples for loading antibiotic, consistent with previous literature [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough we examined the release behaviors of various types of antibiotics via direct absorption onto the HA surface, the results suggest that the samples may exhibit good antibacterial properties on \u003cem\u003eS. aureus\u003c/em\u003e and acceptable antibacterial properties on \u003cem\u003eE. coil.\u003c/em\u003e within a time interval of at least 28 days. However, the concentration of antibiotic surpassing the MIC 90 value for the organism does not necessarily indicate genuine antibacterial properties, as the activity might diminish in a buffer solution. Therefore, we also tested their antimicrobial performances using traditional broth dilution and Agar diffusion tests. For the traditional broth dilution test, the OD values of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e in the solutions represents the amount of live bacteria [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Figure S2 illustrates the OD values changes over time for pristine PEEK and PEEK/HA samples on \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coil\u003c/em\u003e, respectively. At the testing time interval of 12 hours in solution bath containing organisms, the OD values of \u003cem\u003eS aureus\u003c/em\u003e and \u003cem\u003eE. coil\u003c/em\u003e in solutions containing pristine PEEK and PEEK/HA sample increased proportionally with testing time. There was no apparent difference in the growth of organisms in the solution bath for pristine PEEK or PEEK/HA samples. From the results shown in Figure S2, both pristine PEEK and PEEK/HA samples showed almost no antibacterial property against both \u003cem\u003eE. coil\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e. Then we examined their antibacterial properties on \u003cem\u003eE. coil\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e in the solution bath containing organisms with concentration of 10\u003csup\u003e8\u003c/sup\u003e CFU/ml and our samples. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (I) and (II) shows the variation of relative optical densities of samples (A)-(C) over time for \u003cem\u003eE. coil\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e, respectively. The standard calibration curve was established using the OD values of organisms in the same solution with the PEEK/HA sample versus testing time. For \u003cem\u003eE. coil\u003c/em\u003e with concentration of 10\u003csup\u003e8\u003c/sup\u003e CFU/mL in the solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (I)), the growth rates of \u003cem\u003eE. coil\u003c/em\u003e were inhibited with samples (A)-(C) in the solution. Samples (A) (loading of Ampicillin salt on HA surface) and samples (C) (loading of mixture of antibiotic agents on HA, Ampicillin : Vancomycin\u0026thinsp;=\u0026thinsp;50 :50) showed relatively good antimicrobial activities. However, the sample (B) (loading of Vancomycin salt on HA surface) exhibited very poor antimicrobial activity on \u003cem\u003eE. coil\u003c/em\u003e. It is well known the Vancomycin salt has poor antimicrobial activity against \u003cem\u003eE. coil.\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], which aligns with the expectations for sample (B). By examination the drug-released profiles of sample (B) in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (I) and (II), a relative low drug-release concentration in buffer solution during the testing time of 28 days was observed. Considering the results in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and the low antibacterial performance of Vancomycin salt on \u003cem\u003eE. coil\u003c/em\u003e, the poor antibacterial activity on \u003cem\u003eE\u003c/em\u003e. coil for sample (B) is reasonable. At the initial 6 hours of the test, samples (A) showed better antibacterial activities against \u003cem\u003eE. coil\u003c/em\u003e. than sample (C). However, at the testing time interval of 12 hours, the difference in the inhibition of the growth on \u003cem\u003eE. coil\u003c/em\u003e between sample (A) and (C) was not substantial. Since the loading of individual antibiotic on HA for sample (C) was only about 50% of samples (A) and (B), sample (C) serves as a good implant option, offering reduced cost, decreased cell toxicity and low drug resistance for these micro-organisms compared to samples (A) and (B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor \u003cem\u003eS. aureu\u003c/em\u003es with concentration of 10\u003csup\u003e8\u003c/sup\u003e CFU/mL in the solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (II)), the growth of \u003cem\u003eS. aureus\u003c/em\u003e was inhibited with samples (A)-(C). Notably, samples (B) (loading of Vancomycin salt on HA surface) and (C) (loading of mixture of antibiotics on HA surface, Ampicillin : Vancomycin\u0026thinsp;=\u0026thinsp;50 :50) demonstrated significant antimicrobial activities. The relative OD values of the solution bath containing \u003cem\u003eS. aureus\u003c/em\u003e using samples (B) and (C) decreased to around 50% and 40%, respectively, compared to that for PEEK/HA sample in the testing time of 12 hours. Subsequently, we examined their drug-released profiles in the solution bath, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. All samples exhibited the drug concentration in buffer solution of higher than the MIC 90 values for \u003cem\u003eS. aureus\u003c/em\u003e. However, sample (A) showed relatively poor antimicrobial property on \u003cem\u003eS. aureus\u003c/em\u003e due to the poor antibacterial activity of Ampicillin salt against \u003cem\u003eS. aureus\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Considering the testing results of samples (A)-(C) on organisms, the PEEK/HA sample with direct absorption of the antibiotic mixture (Ampicillin : Vancomycin\u0026thinsp;=\u0026thinsp;50 :50) demonstrated good antibacterial activity against \u003cem\u003eS. aureus\u003c/em\u003e and acceptable antibacterial activity against \u003cem\u003eE. coil\u003c/em\u003e. However, the results of the broth dilution test only revealed short-term antibacterial activities for samples. To assess the long-term antibacterial properties, we conducted antimicrobial performances using the traditional disk diffusion method. Figure S3 (I)-(III) shows the antibacterial activities on \u003cem\u003eE. coil\u003c/em\u003e with samples (A)-(C) at the time interval of 24 hours using traditional disk diffusion method, respectively. For sample (A) (HA/PEEK loading with Ampicillin salt), the diameter of its inhibition zone on \u003cem\u003eE. coil\u003c/em\u003e was 2.95 cm at the testing time of 6th hour and increased to around 4.4 cm at the testing time of 24th hour. The antibacterial performance correlated with the paper reporting loading concentration of Ampicillin salt between 639-1000\u0026micro;g/ml, respectively. The result in Figure S3 (I) indicated that sample (A) exhibited good antibacterial performance on \u003cem\u003eE. coil\u003c/em\u003e in the testing time of 24 hour, which was consistent with Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (I). For sample (B) (HA/PEEK loading with Vancomycin salt only), the diameter of its inhibition zone was around 1.07 cm on \u003cem\u003eE. coil\u003c/em\u003e at the 6th hour and increased to 2.4 cm at the 24th hour. The antibacterial performance for sample (B) on \u003cem\u003eE. coil\u003c/em\u003e in the testing time interval of 24 hour corresponded to the paper loading with the concentration of Vancomycin salt between 10-1000\u0026micro;g/L, as reported in the literature. The result for sample (B) in Figure S3 (II) confirmed that HA/PEEK sample loaded with Vancomycin salt exhibited poor antibacterial performance on \u003cem\u003eE. coil\u003c/em\u003e, which was also consistent with the results in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (I). For sample (C) shown in Figure S3 (III), the diameter of its inhibition zone on \u003cem\u003eE. coil\u003c/em\u003e was around 3.43 cm at the testing time of 6th hour and increased to 4.2 cm at the testing time of 24th hour. The antibacterial performance of sample (C) on \u003cem\u003eE. coil\u003c/em\u003e corresponded to paper loading with the concentration of antibiotic (Ampicillin : Vancomycin salt (w/w) = (50/50)) between 586\u0026ndash;800 \u0026micro;g/L. The antibacterial performance of sample (C) showed similar activity to sample (A) and had better activity than that for sample (B) on \u003cem\u003eE coil\u003c/em\u003e, consistent with the results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (I). Subsequently, we evaluated their antibacterial performances on \u003cem\u003eE. coil.\u003c/em\u003e over testing time interval of 10 days. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (I)-(III) illustrates their antibacterial performances on \u003cem\u003eE. coil\u003c/em\u003e. over testing time interval of 10 days, respectively. For sample (A) shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (I) (HA/PEEK sample with loading of Ampicillin salt only), it inhibition zone was 4.4 cm at the testing time of first day and remained almost the same in the testing time interval of 10 days. The antibacterial performance corresponded to the paper reporting Ampicillin salt with concentration of 800-1000\u0026micro;g/L, respectively. For sample (B) (HA/PEEK loading of Vancomycin salt only) shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (II), sample (B) exhibited relatively poor antibacterial activity on \u003cem\u003eE. coil\u003c/em\u003e. at testing time interval of 10 days. Its inhibition zone was 2.4 cm at the first day and decreased to 2.0 cm at the 10th day. The results aligned with those shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (I) and those published in the literature [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. For sample (C) (HA/PEEK with loading of Ampicillin : Vancomycin salt (w/w) = (50/50)) in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (III), its diameter for inhibition zone was 4.2 cm at the first testing day and increased to 4.4 cm at the 10th day. The antibacterial performance of sample (C) was much higher than sample (B) and showed a slightly higher than that for sample (A) on \u003cem\u003eE. coil.\u003c/em\u003e It seems that the HA/PEEK sample with direct absorption of mixture of Ampicillin salt\u0026thinsp;+\u0026thinsp;Vancomycin salt has good antibacterial activity on \u003cem\u003eE. coil.\u003c/em\u003e and can also reduce the cost of antibiotic and the influence of individual antibiotic on human body, such as cell toxicity or drug resistance for these microorganisms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eContinuing to the evaluation of their antibacterial activities on \u003cem\u003eS. aureus\u003c/em\u003e. Figure S4 (I)-(III) demonstrates the antibacterial activities on \u003cem\u003eS. aureus\u003c/em\u003e with samples (A)-(C) at the time interval of 24 hours using traditional disk diffusion method, respectively. For sample (A), the diameter of its inhibition zone on \u003cem\u003eS. aureus\u003c/em\u003e was 3.7 cm at the testing time of 6th hour and increased to around 4.4 cm at the testing time of 24th hour. Its antibacterial performance corresponded to the paper reporting loading concentration of Ampicillin salt between 1000\u0026ndash;2218 \u0026micro;g/ml, respectively. The result shown in Figure S4 (I) also indicated that sample (A) exhibited good antibacterial performance on \u003cem\u003eS. aureus\u003c/em\u003e in the testing time of 24 hour, consistent with that shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (II). For sample (B) (HA/PEEK loading with Vancomycin salt), the diameter of its inhibition zone was around 2.4 cm on \u003cem\u003eS. aureus\u003c/em\u003e at the 6th hour and increased to 2.8 cm at the 24th hour. The antibacterial performance for sample (B) on \u003cem\u003eS. aureus\u003c/em\u003e in the 24-hour testing interval corresponded to the paper loading with the concentration of Vancomycin salt between 100-1000\u0026micro;g/L, as reported in the literature. The result for sample (B) shown in Figure S4 (II) indicated that HA/PEEK sample loaded with Vancomycin salt exhibited better antibacterial performance on \u003cem\u003eS. aureus\u003c/em\u003e than \u003cem\u003eE. coil\u003c/em\u003e, consistent with the results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (I) and (II). For sample (C) shown in Figure S4 (III), the diameter of its inhibition zone on \u003cem\u003eS. aureus\u003c/em\u003e was around 4.0 cm at the testing time of 6th hour and increased to 4.5 cm at the testing time of 24th hour. The antibacterial performance of sample (C) on \u003cem\u003eS. aureus\u003c/em\u003e corresponded to the paper with loading with concentrations of the antibiotic mixture (Ampicillin : Vancomycin salt (w/w) = (50/50)) between 636\u0026ndash;839 \u0026micro;g/L. The antibacterial performance of sample (C) showed similar activity to sample (A) and had better activity than that for sample (B) on \u003cem\u003eS. aureus\u003c/em\u003e. Subsequently, we evaluated their antibacterial performances on \u003cem\u003eS. aureus\u003c/em\u003e at the testing time interval 10 days. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (IV)-(VI) illustrates their antibacterial performances on \u003cem\u003eS. aureus\u003c/em\u003e with testing time interval of 10 days. For sample (A) shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (IV) (HA/PEEK sample with loading of pristine Ampicillin salt), it inhibition zone was 4.4 cm at the testing time of first day and became a little larger in the testing time interval of 10 days (the diameter of its inhibition zone was 4.8 cm). The antibacterial performance correlated with the paper reporting Ampicillin salt concentration of 820-1540\u0026micro;g/L respectively. For sample (B) (HA/PEEK loading of pristine Vancomycin salt) shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (V), sample (B) exhibited almost the same antibacterial performance at testing time interval of 10 days. Its inhibition zone was 2.6\u0026ndash;2.7 cm at the testing time of 10 days. For sample (C) (HA/PEEK with loading of Ampicillin : Vancomycin salt (w/w) = (50/50)) shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (VI), its diameter for inhibition zone on \u003cem\u003eS. aureus\u003c/em\u003e was 4.5 cm at the first testing day and increased to 4.8 cm at the 10th day. The antibacterial performance of sample (C) was much higher than sample (B) and showed a slightly higher than that for sample (A) on \u003cem\u003eS aureus.\u003c/em\u003e It seems that the HA/PEEK sample with direct absorption of mixture of Ampicillin salt\u0026thinsp;+\u0026thinsp;Vancomycin salt has good antibacterial activity on both \u003cem\u003eE. coil.\u003c/em\u003e and \u003cem\u003eS. aureus.\u003c/em\u003e These results also indicated the reduction of the cost for antibiotic, the influence of individual antibiotic on human body, such as cell toxicity or drug resistance for these microorganisms could be obtained using sample (C) as the implants..\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.3 In vitro cytocompatibility of samples\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eIn the clinical orthopedic surgery, the major organisms caused PJI are \u003cem\u003eS. aureus\u003c/em\u003e and coagulase-negative staphylococci. Sample (C) hold promise for clinical treatments, but its cytocompatibility remains a concern. Therefore, we conducted tests on the growth, adhesion and morphology of MC3T3-E1 cells on sample (C) at the 3th, 7th and 14th day interval. For comparison, PEEK disk, the PEEK/HA and samples (A) and (B) were also examined. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e presents their CLSM images at the 3th, 7th and 14th testing day. From the results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, a few MC3T3-E1 cells were observed to grow and attached onto the pristine PEEK and HA/PEEK surface. This suggests that PEEK and HA/PEEK sample exhibited relatively poor cell growth activities. However, with the HA surface loaded with antibiotics (samples (A)-(C)), a relatively large number of MC3T3-E1 cells were observed to grow and attach onto the samples (PEEK/HA with loading of antibiotics) on the 3th day. In the images of samples (A)-(C) shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the number of cells attached to the samples (B) was the highest and the sample (A) showed relatively poor cell attachment activity on the 3th testing day. However, in the CLSM images of samples (A)-(C) in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the numbers of cells attached and grown onto the samples increased over the testing time. The cell growth and attachments on the samples (A)-(C) indicated low cell toxicity compared to the HA/PEEK sample. On the 7th and 14th days, the CLSM of all samples showed good cell growth, with samples (A) exhibiting better cell growth than samples (B) and (C). Even though samples (B) and (C) showed relatively poor cell growth activities compared to sample (A) but they still demonstrated much better cell growth activities than the HA/PEEK sample, suggesting the need to modify HA/PEEK with antibiotic loading. According to literature [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], surface morphology significantly influences cell growth, with rod or noddle-like microstructures inhibiting cell growth due to increased susceptibility to rupture by the particular surface nanorod microstructures. Poor cell growth activity for PEEK/HA samples was expected. Additionally, as reported by Galow et al. (2017) [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], MC3T3-E1 cells exhibit good cell growth activities in the solution with the pH value of 8.4. For sample (A) (HA/PEEK loading of Ampicillin salt only), the pH value of the solution containg Ampicillin salt is around 8.5, and that for Vancomycin is around 6.4. Therefore, the cell growth for sample (A) showed better cell growth activity than those for samples (B) and (C) due to the influence of antibiotic attached onto HA/PEEK sample surface. In summary, HA/PEEK samples with direct absorption of antibiotics, especially the mixture of Ampliciiln\u0026thinsp;+\u0026thinsp;Vancomycin salt demonstrate promising antibacterial properties against \u003cem\u003eE. coil\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e, with minimal cell toxicity on bone cells. These findings suggest potential applications for polymer-made implants.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eIn this study, we documented the growth of hydroxyapatite (HA) layers onto PEEK disk with various ratios of ehthylenediaminetetraacetic agent (EDTA) : calcium ions(Ca\u003csup\u003e2+\u003c/sup\u003e) in the solution bath using hydrothermal method. XRD patterns and SEM images of the samples revealed that the HA layer with low-crystallinity could be obtained onto PEEK substrate with the EDTA : Ca\u003csup\u003e2+\u003c/sup\u003e of 0.5 : 1 in the solution bath. When the EDTA : Ca\u003csup\u003e2+\u003c/sup\u003e ratio was 1:1, the HA layer exhibited rod-like microstructure on the PEEK sample. However, with the EDTA : Ca\u003csup\u003e2+\u003c/sup\u003e of 2:1, no HA layer could be obtained on PEEK substrate due to the low concentration of free Ca\u003csup\u003e2+\u003c/sup\u003e ions in solution bath. Subsequently, various kinds of antibiotics were then directly absorbed onto the HA/PEEK samples from different types of antibiotics in water solution. Stable drug release behaviors in the PBS solution were observed, maintaining antibiotic concentrations in buffer solution greater than MIC 90 for \u003cem\u003eS. aureus\u003c/em\u003e and MIC 50 for \u003cem\u003eE. coli\u003c/em\u003e within a time interval of 1\u0026ndash;28 days. Among the HA/PEEK samples, those loaded with mixture of Ampicillin\u0026thinsp;+\u0026thinsp;Vancomycin salt (w/w\u0026thinsp;=\u0026thinsp;50/50) exhibited the most potent antibacterial properties on both \u003cem\u003eE. coil\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e. The suitable antimicrobial activity of the HA/PEEK sample against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e, which was the loading of mixture for Ampicillin and Vancomycin salts, could be maintained at least 10 days using the disk diffusion method. The use of relatively low dose of individual antibiotic also contributed to low cell cytotoxicity and minimized environmental pollution. Moreover, the loading of antibiotics onto the HA/PEEK samples resulted in good cell growth and proliferation. The samples with direct absorption of antibiotic agents exhibited enhanced long-term antibacterial activity and improved cell growth and proliferation on the sample surface compared to the HA/PEEK sample without antibiotics. This study introduced a simple technique for producing PEEK/HA/antibiotics materials, with potential applications in the biomedical technology within the human body.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eSupporting information:\u003c/h2\u003e \u003cp\u003eThe SEM image for sample (ii) at 3 k(X); The change of OD values for pristine PEEK and PEEK/HA samples as a function of time on \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coil\u003c/em\u003e, respectively; The antibacterial properties on \u003cem\u003eE. coil\u003c/em\u003e using samples with testing time of 1 day, and the antibacterial properties on \u003cem\u003eS. aureus\u003c/em\u003e using samples with testing time of 1 day,\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflict of interest:\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis study was funded by Chang Gung Memorial Hospital (CMRPG2N0331 and BMRP948).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eDave W. Chen: Data curation woks on the Formal analysis, Investigation, Methodology, Writing \u0026ndash; review \u0026amp; editing.; Ming-Kuang Chou focused on the Investigation, Methodology, Visualization; Ngi-Chiong Lau focused on the Methodology, Validation, Investigation. and Kong-Wei Cheng is Project administration, Supervision, Validation, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAcknowledgments:\u003c/h2\u003e \u003cp\u003eThis work was sponsored by the Chang Gung Memorial Hospital with the grand numbers of CMRPG2N0331 and BMRP948. The authors thank the Chang Gung University Microscope Center for the analysis of SEM and EDS analysis.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLau NC, Tsai MH, Chen DW, Chen CH, Cheng KW (2020) Preparation and characterization for antibacterial activities of 3D printing polyetheretherketone disks coated with various ratios of ampicillin and vancomycin Salts. Appl Sci 10:971\u0026ndash;915\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalla VK, Bodhak S, Bose S, Bandyopadhyay A (2010) Porous tantalum structures for bone implants: fabrication, mechanical and in vitro biological properties. Acta Biomater 6:3349\u0026ndash;3359\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen DW, Lee KY, Tsai MH, Lin TY, Chen CH, Cheng KW (2019) Antibacterial applications on staphylococcus aureus using antibiotic agent/zinc oxide nonorod arrays/polyethylethylketone composite samples. Nanomaterials 9:7131\u0026ndash;7115\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCardoso DA, Jansen JA, Leeuwenburgh SCG (2012) Synthesis and application of nanostructured calcium phosphate ceramics for bone regeneration. Biomed Mater Res Part B 100B:2316\u0026ndash;2326\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Q, Yan J, Yang J, Li B (2016) Nanomaterials promise better bone repair. 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Biochem Biophys Rep 10:17\u0026ndash;25\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Polyetheretherketone, Hexagonal hydroxyapatite rods, Antibacterial property, Cell growth","lastPublishedDoi":"10.21203/rs.3.rs-4608945/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4608945/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDemands of bone regeneration and fracture repairing technologies have become increasingly important due to aged society and limitation of autologous bone graft. Heterogeneous implants are always employed in clinical treatments but their antibacterial properties, biocompatibilities, and the mismatch elastic moduli with human bones have to be improved. Therefore, polyetheretherketone (PEEK)/ hydroxyapatite (HA) rod-like array samples were manufactured using hydrothermal method and then directly attached various types of Ampicillin, Vancomycin salts and their mixture onto HA arrays in order to improve their antibacterial properties and biocompatibility. Various ratios of ethylenediaminetetraacetic agent : calcium ions in the solution baths were set in order to obtain high values of specific area of HA rods for the loading of antibiotics. The specific surface area of the sample prepared with ethylenediaminetetraacetic agent : calcium ions ratio of 1:1 in solution bath showed the largest value. Samples through direct absorption of antibiotics can maintain their antibacterial activities up to 10 days. Sample (C) (direct absorption of Ampicillin\u0026thinsp;+\u0026thinsp;Vancomycin salt solution (w/w\u0026thinsp;=\u0026thinsp;50/50)) showed good inhibition activities on \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e. Cell growth activity of MC3T3-E1 onto the PEEK/HA/antibiotic samples also showed better performance than that of HA/PEEK sample, suggesting potential good application in polymer-made implants.\u003c/p\u003e","manuscriptTitle":"Improvements of Antibacterial and Cell Growth Activities for Hydroxyapatite Rods Modified Polyetheretherketone (PEEK) Implants with Direct Absorption of Antibiotics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-10 09:45:04","doi":"10.21203/rs.3.rs-4608945/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"94f0cfd9-416d-4b8f-9e21-d3cefb4addb8","owner":[],"postedDate":"July 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-16T13:56:36+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-10 09:45:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4608945","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4608945","identity":"rs-4608945","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}