1 Introduction implants, one of the most devastating complications of orthopedic surgery, is prominent. Implants are now a main component of medical practice, Unfortunately, when metal Ti exposed to air, an oxide promoting patient well-being and saving countless lives ﬁlm on its surface would spontaneous format for the strong each year; however, along with these beneﬁts comes some afﬁnity between titanium (Ti) and oxygen (O). In addition, negative outcomes. Postoperative infection associated with under in vivo conditions, the oxide ﬁlm will quickly * Yungang Luo Yungangl@hotmail.com Department of Orthopedics, Second Hospital, Jilin University, 130041 Changchun, China Department of Stomatology, Second Hospital, Jilin University, Department of Oral Medicine, West China Hospital of 130041 Changchun, China Stomatology, Sichuan University, Chengdu, China College of Chemistry, Jilin University, Qianjin Street, 130012 Changchun China 1234567890();,: 1234567890();,: 50 Page 2 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:50 transform into a bioﬁlm by combining with amylase and Nanosized TiO , owing to its remarkable NP size effect, protein, which exist throughout bodily ﬂuid. This bioﬁlm has always been the focus of biomaterial research; however, then acts as a protective shield against both host defenses the potential biological toxicity of NPs hinder its practical and antibacterial agents [1–3]. Antibiotic concentrations application in clinical settings. NP-constructed micro- might be as much as 1000-fold higher than needed to pre- spheres, which have both bulk size and the unique proper- vent the growth of inhibited bacteria in bioﬁlms as to the ties of NPs, do provide an alternate route to making TiO planktonic bacteria [3, 4]. Given this, coating the surface of safer to use in biomaterials. implant device with antibacterial agents would be an In this study, NP-constructed microspheres Ag/TiO was effective solution . prepared by a facile one-step process of homogeneous Various functional antimicrobial coatings have been precipitation. Ag at different concentrations doped into available, such as the FDA-approved INFUSE® bone graft TiO was fabricated and used to estimate its antibacterial (Medtronic) , CarriGen porous bone substitute material properties against gram-positive S. aureus and gram- (ETEX),  Cerasorb (Curasam) , and Spineplex P Bone negative E. coli. To investigate the cytocompatibility of Cement (Stryker) . In general, antimicrobial coating Ag/TiO , MC3T3-E1 cells were used for the cytocompat- materials can be classiﬁed into two categories depending on ibility test. The results demonstrated that adding Ag parti- their method of intervention during infection and bioﬁlm cles to TiO reduces bacterial growth while being nontoxic formation: coatings that physically prevent bacterial adhe- to mammalian cell growth. sion and those that release antimicrobial agents and kill adherent bacteria. The former always involve materials that could dissolve after a short period of time. Such as polymer 2 Material and methods brushes  and a layer-by-layer technique used in degradable multilayer coatings . Though they have 2.1 Sample preparation shown great promise toward time-release coatings, the use of organics also brings some health risks in clinical The Ag-loaded TiO was prepared using the homogeneous applications. precipitation method. Silver nitrate (AgNO , 99.99%) was In contrast, materials that kill adherent bacteria are rela- used as the silver precursor with glycol as the solvent. To tively conventional. Antimicrobial peptides (AMPs) and 32.0 mL glycol were sequentially added 16.0 mL 1.25-mol/ quaternary ammonium salts (QASs) have exhibited positive L titanium sulfate (Ti[SO ] ), 0.05 mol/L AgNO , 24.0 g 4 2 3 effect in infection preventing [12, 13]. However, the potential urea (CO[NH ] ), and 2.0 mL 1.0-g/L polyvinylpyrrolidone 2 2 cytotoxicity limits their clinical usefulness [14, 15]. (PVP), followed by vigorous stirring to obtain a uniform Thus, antibacterial biomaterials for implant should solution. The volume of the mixture solution was ﬁxed at simultaneously provide excellent antibacterial activity and 200 mL and the amount of AgNO was varied so that the cell compatibility. Among those are inorganic antibacterial molar ratio of Ag to TiO in the catalysts was within the agents, including silver (Ag), zinc oxide (ZnO), copper range of 0–2.5%. Subsequently, the reaction mixtures were oxide (Cu O, CuO), and Ti dioxide (TiO ), and so on. kept in an oscillating water bath at 90 °C for 4.0 h, with an 2 2 The antibacterial effect of TiO origin from its excellent oscillation frequency of 90–110 rpm. Finally, white pre- photo-catalytic activity ; however, the effect after light cipitates were collected, washed three times with deionized excitation will diminish in the dark [17, 18]. This is a cri- water, dried at 80 °C in an oven for ~4.0 h, and ground with tical ﬂaw limiting its biomedical applications, since no light a mortar. The entire experiment process was done under exist around as soon as implants are placed in bone. For- darkened conditions. tunately, this situation can be improved by noble metal doping in TiO . But researches on the antibacterial 2.2 Sample characterization ability in dark still not the masses. Ag has been known as disinfectant for its broad spectrum The elementary components and Ag concentrations in the of antimicrobial activities . The extremely difﬁculty of samples were determined by energy-dispersive X-ray Ag resistance developed by bacteria is one of the greatest spectroscopy (EDS; EDAX-Falcon, Mahwah, NJ, USA) challenges in traditional antibiotics research, and does well with a resolution of 129 eV. in vivo applications [21, 22]. Giglio et al.  demonstrated The morphology was characterized by JSM-5500 LV the substantial antibacterial activity of hydrogel coatings of ﬁeld-emission scanning electron microscope (SEM; JEOL, electro-synthesized Ag nanoparticle (AgNP-)modiﬁed poly Ltd., Tokyo, Japan) at an accelerating voltage of 30 kV. The (ethylene glycol diacrylate-)co-acrylic acid (PEGDA-AA) planar view was investigated using the TECNAI F20 high- on a Ti substrate against gram-positive (S. aureus) and resolution transmission electron microscopy (HR-TEM; FEI, gram-negative (Pseudomonas aeruginosa) bacteria. Hillsboro, OR, USA) at 200 kV. Distribution of Ag atoms in Journal of Materials Science: Materials in Medicine (2018) 29:50 Page 3 of 10 50 the TiO was monitored by EDS elemental mapping with an of the Ag/TiO powder. Cells were cultured with α-MEM 2 2 EDX-ray spectroscope attached to the HRTEM. (Gibco) supplemented with 10% fetal calf serum (Gibco) Crystal form of the powder was studied using the D/ and 100 mg/mL streptomycin coupled with 100 units/mL Max-2550 X-ray diffractometer (XRD; Rigaku Corporation, penicillin at 37 °C in a humidiﬁed atmosphere of .0% CO . Tokyo, Japan) ﬁtted with a Cu Kα (λ = 1.5418 Å) source at The cells, which were ~70% conﬂuent, were harvested by 50 kV and 200 mA, within the range of 2θ = 20 ~ 80° at a mild trypsinization, centrifuged, and resuspended in the scan speed of 10°/min. Nitrogen (N ) adsorption on the complete medium of α-MEM, and reseeded. The culture surface of samples was measured to calculate the speciﬁc media were refreshed every 2.0 d and all experiments were surface area using the Brunauer–Emmett–Teller (BET) repeated three times. equation . A micrometrics ASAP 2420 surface area and porosity analyzer was used as described elsewhere . The ESCALAB™ 250Xi X-ray Photoelectron Spectro- 2.4.2 Cell viability assay meter (XPS, ESCLAB 250, Thermo Scientiﬁc, Waltham, MA, USA) with an Al Kα radiation source (hν 1000 eV) was CCK-8 (Dojindo Laboratories, Kumamoto, Japan) was used used to identify the chemical constituents of the different to assess the cytotoxicity of the MC3T3-E1 cells in vitro. prepared samples and elemental states of the Ag particles. After counting, MC3T3-E1 cells were seeded at a density of 5000 cells/cm into 12-well plates. After incubation for 2.3 Antibacterial performance test 24.0 h, MC3T3-E1 cells were rinsed thoroughly with phosphate-buffered saline. The resuspended cells were then 2.3.1 Bacterial strains and growth conditions exposed to the Ag/TiO powders at 0.0885 g/cm having different amounts of Ag. Control groups involved the use of Strains of bacteria used for this evaluation were S. aureus α-MEM as the blank and the MC3T3-E1 cells not exposed (ATCC6538P) and E. coli (ATCC25922) purchased from to the samples as controls. The MC3T3-E1 cells used as a the www.bnbio.com. The bacteria were inoculated with positive control were cultured with all other conditions Luria-Bertani (LB) solid plate or liquid medium. being identical. After incubating for 1.0, 3.0, 5.0, 7.0 day, respectively, CCK-8 was added to every well and incubated 2.3.2 The disc diffusion test for 2.0 h; supernatant was transferred to new 12-well cell culture plates. The supernatant’s absorbance value of The antibacterial activity of Ag/TiO powder was evaluated optical density (OD) thereafter was measured using the using the disc diffusion test. The test is performed using the Varioskan Flash Multimode Reader (Thermo Scientiﬁc, guidelines of the Clinical and Laboratory Standards Institute Waltham, MA, USA) at a wavelength of 450 nm accom- . Solutions of Ag/TiO samples with a concentration of panied by a reference wavelength of 630 nm. Finally, the 7.0 mg/mL were prepared using Ag/TiO powder in double- viability of MC3T3-E1 cells was expressed as a percentage distilled water. These solutions were then doped onto of relative growth rate (RGR) according to the following Whatman ﬁlters and sterilized with moist heat. Melted LB formula: medium (90 mL) was poured into petri dishes (Iwaki, RGRðÞ 100%¼ ðOD OD Þ=ðOD OD Þ test blank control blank Japan) and solidiﬁed. The agar surfaces were then inocu- 100% lated using a glass swab dipped in the bacterial cell sus- 5 6 pension. The suspension was adjusted to 10 –10 colony ð1Þ forming units (CFU) of the previous test organisms. After drying for approximately 5.0 min, the inoculated agar plates where, OD is the OD of the Ag/TiO sample and OD test 2 control were planted with the Ag/TiO dampened ﬁlter papers, and OD are the ODs of the MC3T3-E1 cells without 2 blank incubated for 16–18 h at 37.0 °C in dark, and examined for samples and the blank control sample (α-MEM medium). the diameter of the inhibitory zones where no visible bac- terial growth could be observed. Each sample was made in 2.5 Statistical analyses triplicate. The assays were performed in triplicate and data were 2.4 Cell viability expressed as the mean ± standard deviation. Each experi- ment was repeated three times with data of a typical 2.4.1 Cell culture experiment shown. A one-way analysis of variance com- bined with a Student-Newman-Keuls post hoc test was used Newborn mouse calvaria-derived preosteoblastic cells to determine the level of signiﬁcance. p < 0.05 was regarded (MC3T3-E1 subclone 14) were used in the biological assays as signiﬁcant. 50 Page 4 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:50 3 Results The elemental constituents of the different prepared samples and chemical states of the samples were determined 3.1 Sample characterization by XPS. The XPS full survey spectra of TiO and Ag/TiO 2 2 samples and HR spectra of O 1 s, Ag 3d, and Ti 2p are EDS analysis showed that the samples were composed shown in Fig. 1m–p. The C signal is ascribed to adventi- mainly of the Ti, Ag, and O (Fig. 1a–g). Atomic con- tious contamination. Ag can be obviously conﬁrmed in the centrations listed under the images in Fig. 1a–g revealed full spectrum of the 1.89 Ag/TiO sample shown in Fig. 1m that the atomic ratio percentage (at%) of Ag to Ti in the in addition to Ti and O. From the HR spectra in Fig. 1n, Ti Ag/TiO powder varied from 0 to 2.25%. The Ag/TiO 2p exhibits two peaks centered at 464.2 and 458.5 eV, 2 2 powder with different content of Ag is referred to in the assigned to the binding energies of Ti (2p ) and Ti (2p ), 1/2 3/2 following text as TiO , 0.12 Ag/TiO , 0.56 Ag/TiO , 0.82 respectively . The split between Ti (2p ) and Ti (2p ) 2 2 2 1/2 3/2 4+ Ag/TiO , 1.1 Ag/TiO , 1.89 Ag/TiO , and 2.25 Ag/TiO is 5.7 eV, indicative of Ti in the anatase phase of TiO 2 2 2 2 2 where the numerals represent the atomic percentage of Ag [28, 29]. The binding energies of Ti2p were slightly lower to Ti. in the presence of Ag. The lower Fermi level, compared to Moreover, the anatase phase of TiO and tetragonal that of TiO , makes Ag responsible for the shift in binding 2 2 structure of Ag can be observed in the interior HR-TEM energy because of the transition of electrons from TiO to lattice pattern (Fig. 1h), in which the interplanar spacing of Ag and the subsequent outer cloud density changes in TiO ~0.352 and ~0.189 nm account for 101 and 200 planes of [30, 31]. The O 1 s peak (Fig. 1o) consists of three sub- anatase, respectively. In addition, the ~0.204 nm corre- peaks. The O 1s peak at binding energy of 529.7 and sponds to the interplanar spacing of 200 planes of Ag. 531.45 eV are assigned to the Ti-O bonds, which have the Surface morphology of the TiO and 1.89 Ag/TiO largest peak area . The O 1s peak at 532.6 eV conﬁrms 2 2 powder is shown in Fig. 1i. TiO powder was entirely the presence of the surface chemisorbed –OH of TiO 2 2 composed of spherical crystal units, whose diameter varied matrix . The Ag 3d doublets at 367.71 eV (Ag 3d ) 5/2 from 2.0 to 5.0 μm. When loaded with Ag, the morphology and 373.80 eV (Ag 3d ) (Fig. 1p) with a spin energy 3/2 as well as diameter of the crystal units were similar to that separation of 6.09 eV correspond to the binding energy of of TiO alone. Ag , conﬁrming the existence of the oxidized state of Ag Figure 1j exhibits the XRD patterns of the 1.89 Ag/TiO . in the Ag/TiO sample. 2 2 A typical XRD pattern of a TiO sample is shown in the image, and all characteristic diffraction peaks at 2θ = 25.3° 3.2 The disc diffusion test (101), 37.8° (004), 48.0° (200), and 55.1° (211) are readily indexed to the anatase phase of TiO according to JCPDS The results of the disc diffusion test were observed in Ag/ card No. 84-1285; however, the characteristic peaks of Ag TiO powder with different contents of Ag (Fig. 2). The were missing in the patterns. The absence of Ag peaks is samples show the inhibitory effect of Ag/TiO against both very likely a result of the small amount of added Ag, which E. coli (Fig. 2a1–a7) and S. aureus (Fig. 2b1–b7), which can be conﬁrmed by the results of the HR-TEM lattice exhibit observable inhibition zones with a diameter pattern. >7.0 mm (Table 2), with the exception of TiO and 0.1 Ag/ The BET surface area is another important aspect that TiO against E. coli because of their smaller or no Ag can provide the basis for the antibacterial properties. The N content. Of the samples enhanced with increasing Ag con- adsorption-desorption isotherm of 1.89 Ag/TiO,as tent in the powder, 2.25 Ag/TiO showed the most promi- 2 2 observed in Fig. 1k, was used to study the pore structure of nent inhibiting effect (exhibits the largest inhibition zone) Ag/TiO samples, displaying a higher surface area of owing to the largest Ag content, which highlights the sig- 362.8 m /g. The Barrett-Joyner-Halenda pore size distribu- niﬁcance of the amount of Ag incorporated into the powder tion curve corresponding to the isotherm is shown in the on preventing the colonization of bacteria. The antibacterial insert in Fig. 1k and indicates the presence of holes in the activity of the samples against S. aureus was signiﬁcantly spherical crystal unit’s surface. The BET surface area of the stronger than that against E. coli. The diameter of the samples is as 355–365 m /g (Table 1). As the amount of Ag inhibition area of the S. aureus group was signiﬁcantly particles on Ag/TiO increases, the speciﬁc surface area of different (P < 0.05) from that of the E. coli group, which the Ag/TiO powders decreases slightly. could be attributed to the disparity between the two bacteria The distributions of Ti, O, and Ag in the spherical crystal on the structure. For the E. coli group, there always exists units obtained by EDS elemental mapping are shown in an area in which there is a density reduction of bacterial Fig. 1l The elemental maps reveal that Ag is homo- colonies instead of a clear zone of inhibition as with the S. geneously dispersed throughout the spherical TiO crystal aureus group. This might be related to different antibacterial units. effects of Ag ions on the two bacteria. All of the above Journal of Materials Science: Materials in Medicine (2018) 29:50 Page 5 of 10 50 Fig. 1 Continued 50 Page 6 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:50 Fig. 1 a–g EDS data obtained from Ag/TiO powder with different amount of Ag showing changes in Ag content with a range of 0–2.25% (at%). HR- TEM images of the 1.89 Ag/ TiO powder (h). Image I showing the structure of 1.89 Ag/TiO powder. j XRD of 1.89 Ag/TiO powder was shown. Nitrogen adsorption-desorption isotherm and the corresponding BJH pore size distribution curve (the insert in the bottom right- hand corners) acquired from 1.89 Ag/TiO (k). l The distributions of Ti, O, and Ag in the spherical crystal units of the 1.89 Ag/TiO . The XPS spectra of TiO and 1.89Ag/TiO 2 2 samples. The XPS full survey spectra (m) and the high- resolution spectra of Ti 2p (n), O 1s (o) of TiO and 1.89 Ag/TiO 2 2 samples and the high-resolution spectra of Ag 3d (p) of 1.89 Ag/ TiO Table 1 The BET surface area of the samples of 92–99%. According to the standard , when RGR is 90–100%, the cytotoxicity scale of a biomedical material is Sample name Speciﬁc surface area (m /g) noted as Grade 0, and when 75–90%, as Grade 1. A material TiO 359.98 that has a cytotoxicity of either Grade 0 or 1 means “no 0.56 Ag/TiO , 363.28 toxicity to the cell”. All data indicate that nearly all of the 1.10 Ag/TiO 359.17 TiO and Ag/TiO samples had no adverse impact on cell 2 2 1.89 Ag/TiO 354.06 viability. No signiﬁcant difference in cell viability was 2.25 Ag/TiO 362.80 observed among the 0.12 Ag/ TiO , 0.56 Ag/ TiO , or 0.82 2 2 Ag/ TiO throughout the culturing period. highlights the distinct advantage that Ag has over other antibacterial agents, ones that might be effective on only 4 Discussion one of the two kinds of germ (gram-positive bacteria or gram-negative bacteria). The battle between surgeons and bacteria is a protracted war. Considering the susceptibility of artiﬁcial implant 3.3 Cell viability and proliferation surfaces to adhesion and colonization by microorganisms, implant-coating material having proper antibacterial prop- Figure 2c shows the viability of MC3T3-E1 cells cultured erties as well as cytocompatibility is being actively pur- in vitro with the TiO and Ag/TiO samples in α-MEM for sued. In this study, Ag/TiO antibacterial microspheres 2 2 2 1.0, 3.0, 5.0, and 7.0 d assessed by CCK-8 assay. When were produced using the conventional impregnation MC3T3-E1 cells were co-cultured with the sample, cell method. In this process, Ag particles attach to the TiO viability increased with time, especially on the seventh day, microsphere. The amount of Ag was adjusted by varying which exhibited a sharp increase. The initial relatively lower the amount of AgNO in the reaction solution. The TiO 3 2 viability at 1.0 and 3.0 d might be a result of the short microsphere powder with the proper amount of Ag exhibits adaptation period. The RGR of cells on the TiO and Ag/ relatively high antibacterial properties and no cytotoxic TiO samples at 5.0 and 7.0 days were >100% with the effects on MC3T3 cells. This study describes a simple exception of the 1.89 Ag/TiO and 2.25 Ag/TiO groups, approach by which to fabricate such Ag/TiO antibacterial 2 2 2 which showed relatively lower RGR values within the range coatings. Journal of Materials Science: Materials in Medicine (2018) 29:50 Page 7 of 10 50 The facile one-step homogeneous precipitation method was used in this work for preparing Ag/TiO antibacterial material. The crystal nucleation and growth of TiO involves the following reactions: COðÞ NH þ3H O ! CO "þ2NH H O ð2Þ 2 2 2 3 2 TiðÞ SO þ4NH H O ! TiO #þ2NðÞ H SO 4 3 2 2 4 4 2 2 ð3Þ þ2H O When heated up to 60 C, urea began to hydrolyze into CO and NH � H O. With the accumulation of NH � H O, 2 3 2 3 2 pH value of reaction solution increased which provides a favorable environment for the hydrolysis of Ti (SO ) . 4 2 TiO formed in the reaction precipitate and growth as a center of the nuclei. The formation of Ag can be given in sequence as fol- lows: 2AgNO ! 2Ag #þ2NO "þO"ð4Þ 2 2 The NH � H O formed in (1) also works as morphology 3 2 of catalyst in reaction system. Namely, the existence of NH � H O always lead to spherical morphology . With the help of NH � H O, TiO and Ag nanoparticles self- 3 2 2 assemble into Ag/TiO microspheres, shown in Fig. 3a. And, at the same time, the gap between nanoparticles makes a hole net-like structure in the Ag/TiO microspheres, which lead to an excellent microstructure with a surface area of >355 m /g (Table 1). The high surface area makes it an ideal carrier for many drug-delivery applications. PVP in this reaction system acts as a stabilizer, inhibiting TiO and Ag nanoparticles from agglomerating and limiting nuclei size Fig. 2 The results of the disc diffusion test of Ag/TiO powder with . With the help of dispersing agent ethylene glycol in different content of Ag against E. coli (a1–a7) and S. aureus (b1–b7) in dark. 1–7 correspond to samples of TiO , 0.12 Ag/TiO , 0.56 Ag/ 2 2 this study, a regular spherical TiO with a desirable range of TiO , 0.82 Ag/TiO , 1.1 Ag/TiO , 1.89 Ag/TiO , and 2.25 Ag/TiO , 2 2 2 2 2 Ag content can be produced in the medium. respectly. c RGR values of Ag/ TiO samples (*p < 0.05) and the Samples were highly effective against both gram-positive proliferation growth curve of the MC3T3 cells with incubation dura- bacteria (S. aureus) and gram-negative bacteria (E. coli), tion for 1, 3, 5 and 7 days which are the dominant bacteria of infections related to medical devices. The inhibition zone displayed in the disc diffusion test provides us clear evidence that the sur- Table 2 Averaged inhibition zones for TiO , 0.12 Ag/TiO , 0.56 Ag/ 2 2 rounding area of the material can also be protected. Cao TiO , 0.82 Ag/TiO , 1.1 Ag/TiO , 1.89 Ag/TiO , and 2.25 Ag/TiO 2 2 2 2 2 et al.  has conﬁrmed that the physical Schotty contact Sample name Inhibition zone (mm) structure related to those boundaries at Ag/TiO is the main S. aureus E. coli reason for the antibacterial activity of Ag/TiO in dark. That is, in this structure, TiO is a semiconductor, and Ag is TiO 7.03 6.00 metal particle possessing electron storage behavior. The 0.12 Ag/TiO 7.48 6.00 electrons exist on bacterial membrane are electron donors. 0.56 Ag/TiO 9.48 8.34 Therefore, as suggested in Fig. 3b, in the dark, electrons 0.82 Ag/TiO 10.16 9.54 generated in bacteria metabolism are readily transferred 1.10 Ag/TiO 11.99 11.10 along the route of “bacterial membrane -TiO surface—Ag 1.89 Ag/TiO 12.63 11.41 /TiO interface—Ag metal particles” owing to the Schottky 2.25 Ag/TiO 13.48 12.00 barrier effect. This electron transfer mode blocks electron- 50 Page 8 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:50 statement. As evidenced by the test, the Ag/TiO powder with the designated amount of Ag, 0.12 Ag/TiO , 0.56 Ag/ TiO , 0.82 Ag/TiO , or 1.1 Ag/TiO , demonstrated nearly 2 2 2 no opposing forces and even had a positive impact on the enhanced cell activities of MC3T3 cells. In addition, other samples with a higher content, such as 1.89 Ag/TiO and 2.25 Ag/TiO , although not similar to those of lower Ag content in promoting cell growth, exhibit no evident dele- terious effects on the basic ability of multiplication and survival of MC3T3-E1 cells. Furthermore, experimental evidence shows that biomaterials that contain a proper amount of Ag are compatible with mammalian cells, including osteoblasts [1, 41]. Generally, the Ag/TiO powder does have both good cytocompatibility and excel- lent antibacterial ability. 5 Conclusion By using Ti(SO ) as the Ti source, anatase microspheres 4 2 modiﬁed with Ag NPs were successfully produced using a Fig. 3 a The preparation reaction mechanism of Ag/TiO microsphere. urea-based homogeneous precipitation method. In the dark, b Illustration for electron transfer stimulated antibacterial action of Ag/ TiO microsphere in the dark. Namely, electrons are transferred along 2 the transferring route of “bacterial membrane -TiO surface the route of “bacterial membrane -TiO surface—Ag /TiO interface— 2 2 —Ag /TiO interface—Ag metal particles” for electrons Ag metal particles”, and lead to accumulation of valence-band hole (h + along owing to the Schottky barrier effect is the main reason ) at the TiO side that explains cytosolic and content leakage for the antibacterial effect. With a high speciﬁc surface area (oxidation) the NP-constructed Ag/TiO microspheres improves anti- hole recombination in TiO . As a result, redundant valence- bacterial performance against both S. aureus and E. coli. band holes (h ) at the TiO side adjacent the boundaries After incubation for 1.0 week, the Ag/TiO microspheres VB 2 2 can lead to biocide action through directly electrostatic showed an excellent cytocompatibility to MC3T3-E1 cells, effects-based reaction with the membrane lipids or stimu- as observed through the CCK8 test. NP-constructed Ag/ lating catalytic oxidation process. Consequently, pores TiO microspheres with high speciﬁc surface area prepared present on the outer membrane and eventually cytoplasm in this work provide us with a new choice for the applica- leak out and cell crack. tion of Ag/TiO biomaterial in medicine. Furthermore, the larger surface area provides Ag/TiO Funding This work was jointly supported by Science and Technology structure with more opportunities for contact with micro- Agency of Jilin Province (NO.20130206056GX) and Finance depart- organisms, which makes the sample a suitable vehicle in ment of Jilin Province (Research on dental implant coating materials with bacteriostatic activity). favor of the antibacterial properties of Ag. In addition, it is worth noting that, within our assay, samples were designed to be subjected to intense attack from bacteria with a con- Compliance with ethical standards 5 6 centration of 10 –10 CFU/mL. Such critical conditions are Conﬂict of interest The authors declare that they have no conﬂict of much harsher than those under normal circumstances interest. in vivo. It is also necessary to determine whether the Ag/TiO material possesses good cytocompatibility. 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Journal of Materials Science: Materials in Medicine – Springer Journals
Published: Apr 23, 2018