Effect of Local Alkaline Microenvironment on the Behaviors of Bacteria

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Biological and Medical Applications of Materials and Interfaces

Effect of Local Alkaline Microenvironment on the Behaviors of Bacteria and Osteogenic Cells Ji Tan, Donghui Wang, Huiliang Cao, Yuqin Qiao, Hongqin Zhu, and Xuanyong Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b15724 • Publication Date (Web): 09 Nov 2018 Downloaded from http://pubs.acs.org on November 11, 2018

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ACS Applied Materials & Interfaces

Effect of Local Alkaline Microenvironment on the Behaviors of Bacteria and Osteogenic Cells

Ji Tan,†,§ Donghui Wang,† Huiliang Cao,† Yuqin Qiao,† Hongqin Zhu† and Xuanyong Liu†,*

†State

Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai

Institute of Ceramics, Chinese Academy of Sciences, 200050, China. §University

of Chinese Academy of Science, Beijing 100049, China.

*Corresponding

Author

KEYWORDS:

antibacteria; osteogenesis; alkaline microenvironment; layered

double hydroxides, layered double oxides

ACS Paragon Plus Environment

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ABSTRACT The interactions between materials surfaces and bacteria/cells have been widely investigated, based on which biomaterials with antibacterial and osteogenic abilities can be designed to conquer implant failures. The pH of environments is known to affect bacterial growth and bone formation/resorption, it is possible to endow biomaterials with simultaneously antibacterial and osteogenic abilities by regulating their surface alkalinity. Herein we fabricated kinds of films with various alkalinity on titanium to explore the effect of local alkaline microenvironments around materials surfaces on the behaviors of bacteria and osteogenic cells. Both Gram-positive and negative bacteria were cultured on samples surfaces to investigate their antibacterial effects. The cell adhesion, proliferation and alkaline phosphatase activities were investigated by culturing both bone mesenchymal stem cell and osteoblast cell on samples surfaces. The results show that an appropriate local alkaline environment can effectively inhibit the growth of both Gram-positive and negative bacteria through inactivating ATP synthesis and inducing oxidative stress. Meanwhile, it can promote the bone mesenchymal stem cell osteogenic differentiation, and enhance the proliferation and alkaline phosphatase activities of osteoblast cell. In conclusion, materials surfaces endowed with an appropriate alkalinity can possess antibacterial and osteogenic properties, which provides a novel strategy to design multifunctional biomaterials for bone generation.


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INTRODUCTION The physical and chemical properties of biomaterials surfaces have direct effect on multiple cellular behaviors such as adhesion, migration, proliferation and differentiation. In the last decades, tremendous studies have been conducted to research the influences of micro-nanostructure,1-7 hardness,8-10 chemical functional groups,11-14 release elements,15-19 elastic modulus20-21 and electricity22-28 of materials surfaces on the behaviors of various cells including neural stem cell, mesenchymal stem cell, endothelial cell, osteoblast cell and bacteria. Exploring the effects of surface microenvironments on cells’ behaviors is significant to guide designing biomaterial with specific functions to solve the clinical problems. Nowadays, millions of patients worldwide benefit from orthopedic surgery, nevertheless, implant failures still occur at the bone/material interface due to the bacterial infections and deficient osseointegration.29 In view of this, numerous studies have focused on investigating the interaction between materials surfaces and bacteria or osteogenic cells, which aim to guide designing bifunctional biomaterials with antibacterial and osteogenic abilities.5-6, 30-31 Up to now, most previous studies have simply focused on doping antibacterial and osteogenic elements together into the surfaces of implants.32-33 However, excessive release of some antibacterial elements such as Ag, Cu and Zn from the surface would induce toxicity to normal cells. Therefore, scientists enthusiastically seek other surface cues such as physical isolation/puncture and electric stimulation to achieve preferable implant biomaterials. For instance, Lu et al. have disclosed that multilevel structures have selective inhibition to biofilm-positive



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S. epidermidis via dividing and trapping the bacteria in micro-pits with the size around 800

nm,

while

can

obviously

enhance

the

adhesion,

proliferation,

and

osteodifferentiation of rat bone mesenchymal stem cells.34 In addition, Li et al. have designed hybrid ZnO nanorods with an average diameter of 100 nm on titanium, which can not only effectively kill bacteria but also enhance the osteodifferentiation of osteoblast simultaneously by selectively physical puncturing bacteria.30 On the other hand, our group has revealed that materials surfaces with “schottky contact” characteristic could kill bacteria via inducing the charges of bacterial membrane transfer to materials, and have no adverse effects on osteoblast cells.25, 35-36 Wang et al. have recently discovered that the electrical interaction between the charging materials surfaces and bacteria could be applied to design new light-independent antibacterial materials without harm to the growth of osteoblast cells.22 Moreover, some studies have found that building an electric microenvironment on the materials surface could promote the mesenchymal stem cells osteogenic differentiation.26-28 Nevertheless, the above material systems are too complicated to fabricate. It is of great economic and scientific significance to find other simple cues, which could endow implant surface with selective antibacterial and osteogenic abilities. The pH, a common physical and chemical characteristic of the microenvironment around materials surfaces may work. Most non-extreme bacteria cannot grow well in a microenvironment with pH over 9.0.37 There exists a transmembrane proton electrochemical gradient across the membrane of bacteria, which is relevant to the production of the “energy currency” ATP (adenosine triphosphate):38 when H+ enters


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cell via the F-type H+-ATPase on membrane, ATP synthesis occurs within bacteria. In our previous work, we have discovered that consumption of H+ around the surfaces of titanium through continuous micro battery reaction could effectively restrain or even kill bacteria.23,

39-40

It can be inferred that a strong alkaline environment owning

abundant OH- may be another weapon to consume H+ and then kill bacteria. On the other hand, the balance between bone formation and resorption is significantly affected by the pH of microenvironment:41 alkalinity facilitates bone formation by stimulating the proliferation and differentiation of osteoblast cells, conversely, acidity facilitates bone resorption by enhancing the activity and differentiation of osteoclast cells.42-44 Besides, the activity of ALP (alkaline phosphatase), an ectoenzyme to hydrolyze pyrophosphate for deposition of hydroxyapatite, is also regulated by the pH of microenvironment. With an increase of pH from 7.4 to 8.5, the activity of ALP can be improved by 67%.45 Moreover, some studies have found that with the local pH increase to above 8.0, which was induced by degradation of bioactive degradable ceramics, both ALP activity and proliferation of osteoblast cells were dramatically enhanced.46-47 It can be seen that endowing implant biomaterial surface with alkalinity may be an effective strategy to simultaneously improve its antibacterial and osteogenic activities. However, some studies have implied that an overly alkaline surface not only kills bacteria but also gives rise to cell death.48 Consequently, controlling an applicable alkalinity level of surfaces is the key point to balance the antibacterial and osteogenic properties of biomaterials. LDHs (Layered double hydroxides) are a class of ionic lamellar materials with a



general formula [M2+1−xM3+x(OH)2][An−]x/n·mH2O, which are made up of positively charged brucite-like layers and interlayer containing various charge-balanced anions.49 Alkalinity is the most basic characteristic of LDHs, which is associated with the divalent metal hydroxide in the composition. In addition, its alkalinity level can be simply improved via calcination treatment.50 Above all, the LDHs are highly biocompatible, and have been widely applied in many medical fields such as antacid, drug-loading systems and corrosion resistant coatings. What mentioned above indicates that LDHs film is appropriate in regulating the alkalinity of biomaterials surfaces for investigating the effect of local alkaline microenvironment on cell/bacterial behaviors. In this work, considering that Mg-Al LDHs film can be easily grown on many kinds of materials surfaces (such as Mg, Al, Zn, Au, Ti and glass), it was selected as the model to regulate the surface alkalinity of biomedical titanium. The Mg-Al LDHs film was in situ grown on the titanium surface through a simple hydrothermal treatment, followed by moderate calcination treatment to transform it into LDOs (layered double oxides) film. With the increase of calcination temperature, the alkalinity of the film was enhanced. Then, the antibacterial and osteogenic abilities of various samples were investigated in vitro, and the underlying mechanism was further verified to primarily correlate to the surface alkalinity level.

EXPERIMENTAL SECTION Samples preparation First, commercially pure titanium (1 cm × 1 cm and 2 cm × 1 cm, thickness of 1


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mm) were ultrasonically cleaned with acetone, ethanol, and ultrapure water for 10 min, respectively. Then, the Mg-Al LDHs films were directly formed on titanium by hydrothermal treatment. Briefly, titanium was put in a Teflon-lined stainless with reaction solution consisting of 1.2 mM Al(NO3)3, 0.4 mM Mg(NO3)2 and 21.6 mM urea (5mL for each sample with 1 cm2 ), and then heated at 120 °C for 10 h. Finally, the obtained LDH samples were calcined for 2 h at 250 °C and 500 °C, and these acquired samples were marked as LDH-250 and LDH-500 in succession.

Samples characterization The surfaces morphologies of various samples were observed via the scanning electron microscopy (SEM, S3400, HITACHI, Japan). The elements type, content and distribution of samples surfaces were measured via X-ray photoelectron spectroscopy (XPS, PHI-5000C ESCA system PerkinElmer, USA) and electron energy disperse spectroscopy (EDS, IXRF-550i, IXRF SYSTEMS, USA). The phase composition of films was detected by X-ray diffraction (XRD; D/Max, RIGAKU, Tokyo, Japan). The types of surface functional groups were determined by Fourier transform infrared spectrometer (FTIR-7600, Lambda Scientific, Australia).

Surface wettability The surface wettability was detected by a contact angle measurement (SL200B, Solon, China). Briefly, 1 μL of ultrapure water was dropped on the samples surfaces, and then the equipped camera captured a photo. Finally, the contact angle was analyzed



via the corresponding software. Three replicate for each group were detected and the results were expressed as means ± SD (standard deviation).

Surface zeta potentials The surface zeta potential was measured using Surpass electro kinetic analyzer (Anton Parr, Austria), and the testing principle is described in previous publications.23 Briefly, two samples (2 cm × 1 cm, thickness of 1 mm) were fixed together on the holder with a gap of 100 ± 5 μm. A KCl solution (0.001 M) was chosen as the electrolyte solution and the pH of which was regulated by using HCl and NaOH (0.05 M). During the testing process, the KCl solution flow along the samples surfaces, thus the zeta potentials were acquired from the Helmholtz-Smoluchowski equation: ζ=

η dU × ×C dP ε × ε0

where, the ζ denotes the zeta potentials, dU/dP is the slope of the streaming potential and pressure, and η, ε, ε0, and C represent the electrolyte viscosity, dielectric constant, vacuum permittivity and conductivity, respectively.

Ions release The samples were immersed in 10 mL of normal saline at 37 °C without stirring for 1, 4, 8, and 16 days. The extract solution was collected at every time point. The amount of Mg and Al ions release were detected via inductively-coupled plasma atomic emission spectroscopy (ICP-AES, Varian Liberty 150, USA).


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Alkalinity evaluation To evaluate the effect of samples on the alkalinity of microenvironment around samples surfaces, the Ti, LDH, LDH-250 and LDH-500 samples were immersed with 5 mL normal saline for various times at 37 °C in the centrifuge tubes. At each time point of 0, 1, 4, 8 and 12 h, the pH of the normal saline immersing sample was measured via a pH meter (FE20 - FiveEasy™, METTLER TOLEDO). Before each measurement, the centrifuge tube was shaken up to make the solution homogeneous. To investigate the alkalinity holding capacities of different samples, the samples were successively immersed with normal saline for three times, each time for 1 day. At each time point, the pH of soak solution was measured and the soak solution was changed by a 5 mL fresh normal saline. Three replicate were detected and the results were expressed as means ± SD.

Bacterial responses Bacteria culture Escherichia coli (E. coli, ATCC 25922) and Staphylococcus (S. aureus, ATCC 25923) were cultured on samples surfaces to investigate their antibacterial effects. The E. coli and S. aureus were cultured by using Luria-Bertani (LB) and Nutrient Broth No 2 (NB) medium, respectively. Before culturing, the bacteria suspension was diluted to an appropriate concentration with normal saline. The samples were sterilized in 75 vol% alcohol for 2 hours and placed in a 24-well plate. Afterwards, a drop of bacterial suspension (60 μL with 107 cfu/ml) was dropped onto the samples surfaces, and then



were cultured at 37 °C in an incubator under the atmosphere. Cell viability of bacteria After cultured for 24 h, a 500 μL normal saline mixed with 10% (v/v) alamarBlue was added to each well. After incubation for 2 h, 100 μL reaction mixture was sucked up into a 96-well blank plate, and the fluorescence intensity (FI) of reduced alamarBlue in the mixture was detected with an enzyme-labeling instrument (BioTek, Cytation5) at 560 nm excitation wavelength and 590 nm emission wavelength. The cell viability was calculated as follows: (Ftest - Fblank)/(Fcontrol - Fblank) × 100%, where Ftest was the FI of LDH, LDH-250 and LDH-500 groups; Fblank was the FI of the blank hole, and Fcontrol was the FI of Ti control group. Four replicates for each group were detected and the results were expressed as means ± SD. Morphology of bacteria After incubation for 24 h, 0.5 mL of glutaraldehyde solution (2.5 vol%) was added to each well to immobilize the bacteria for 4 h. Then the bacteria were dehydrated by using the water and ethanol mixtures with volume fractions (ethanol vol%) 30%, 50%, 75%, 90%, 95% and 100%, respectively. After dehydration, the samples were dried by the ethanol and hexamethyldisilazane (HMDS) mixtures with volume ratio (ethanol: HMDS) of 2:1, 1:1, 1:2, respectively. Afterwards, the bacteria were observed with the scanning electron microscope. Bacteria live/dead staining The bacteria on the samples surfaces were stained using the LIVE/DEAD BacLight kit (L13152). Briefly, after cultured for 24 h, the bacteria were rinsed twice


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with 0.8 mL normal saline, and then 0.5 mL of dye was added to each sample, staining for 15 min at room temperature. Afterwards, the samples were rinsed with 0.8 mL normal saline, and then were observed with a fluorescence microscope. Bacteriostatic ring test The bacteriostatic ring test was conducted to identify whether it was the ions release caused the antibacterial effects. Briefly, 100 uL of bacteria suspensions (107 cfu/mL) containing E. coli or S. aureus was inoculated on the nutrient agar and the samples were putted on its surface (the modified surfaces contacting with the nutrient agar), and then cultured for 18 h. The antibacterial ability of the released ions was evaluated according to the area of inhibition zones around the samples (larger area indicates better antibacterial ability). Intracellular ROS assay The intracellular ROS levels of bacteria on various samples were assessed by using the Reactive Oxygen Species Assay Kit. Briefly, after cultured 24 h, 0.5 mL of DCFHDA (10 μM) was introduced to the 24 well-plate followed by incubation for 20 min at 37 °C. Then, 100 μL reaction solution was sucked up into a 96-well blank plate, and the fluorescence intensity of DCF (2′, 7′-dichlorofluorescein) in the medium was measured with an enzyme-labeling instrument at an extinction wavelength of 480 nm and an emission wavelength of 525 nm. The ROS level was correlated with the fluorescence intensity of DCF and the results were normalized to that of bacteria cultured on Ti surface and expressed as fold increase of ROS.



Osteogenic cells responses Cell culture The rat bone mesenchymal stem cells (rBMSCs) and the mouse osteoblasts cells (MC3T3-E1) were selected to investigate the samples’ osteogenic effect. The cells were kindly provided by Stem Cell Bank, Chinese Academy of Sciences and were cultured with 90% α-minimum essential medium (α-MEM), 10% fetal bovine serum (FBS) and 1% antimicrobial of penicillin and streptomycin at 37°C in a 5% CO2 incubator. The old culture medium was changed by the fresh culture medium after culturing cells every 3 days. Once the positive clone rate of cell lines reached 90%, a trypsin/EDTA solution was used to detach the cells, and then the cells were sub cultured or used for biological evaluation (only cells of passage 3-5). Cell initial adhesion The rBMSCs and MC3T3-E1 cells (5.0 × 104 cell/mL) were seeded on sterilized samples. After cultured for 1, 4 and 24 h, the cells were rinsed with PBS 3 times and then fixed with 4% paraformaldehyde (PFA) solution for 10 min. Afterwards, the cells were permeabilized with 0.1 (v/v) Triton X-100 and stained by DAPI and TRITCphalloidin. Finally, the cell nuclei and F-actin were observed with a fluorescence microscopy. Meanwhile, the cells were immobilized with 0.5 mL glutaraldehyde solution (2.5%) for 4 h, and then the cells were dehydrated by a series of H2O and ethanol mixture solution, and then dried by a series of ethanol and HMDS mixture solution following the steps described before. Afterwards, the morphologies of cells on various samples surfaces were observed with the scanning electron microscope.


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Cell proliferation and morphology The rBMSCs and MC3T3-E1 cells (2.0 × 104 cell/mL) were seeded on sterilized samples surfaces (four replicates). After cultured for 1, 4 and 7 days, the cells were rinsed with PBS 2 times followed by incubation for 2 h in 0.5 mL of medium mixed with 10% (vol) alamarBlue. Then, the fluorescence intensity of reduced alamarBlue was examined with an enzyme-labeling instrument. Besides, the cells were immobilized with 0.5 mL glutaraldehyde solution (2.5%) for 4 h. Afterwards, the cells were dehydrated by a series of mixture solution of water and ethanol, and dried by a series of mixture solution of ethanol and HMDS following the steps described before. Afterwards, the cells were observed with the scanning electron microscope. Cell live/dead staining The cells on the samples surfaces were stained using the live/dead cell staining kit (Biovision, USA). Briefly, the rBMSCs and MC3T3-E1 cells (2.0 × 104 cell/mL) were seeded on sterilized samples. After cultured for 4 days, the cells were rinsed with PBS 2 times. Afterwards, a 100 μL PBS solution mixed with calcein-AM (2 μM) and PI (propidium iodide, 5 μM) was introduced to each sample. After staining for 15 min at 37 °C, the live/dead stained cells were observed with a fluorescence microscope. Alkaline phosphatase activity assay The ALP activity cells on various samples was evaluated by the method as described in the literature.17 Briefly, the rBMSCs and MC3T3-E1 cells (1 × 104 cell/mL) were seeded on sterilized samples. After cultured for 7 days, the cells were rinsed with PBS 2 times, and then fixed with 4% PFA solution for 10 min. Afterwards, the ALP of



cells on samples were stained by a mix solution of naphthol AS-MX phosphate and fast blue RR salt. For the quantitative test, the cells were lysed from the samples (three replicates) followed by incubation with p-nitrophenyl phosphate for 30 min at 37°C. Afterwards, the ALP activity of cells was measured by detecting the absorbance at 405 nm wavelength. In addition, the total protein content was measured by using the BioRad protein assay kit (Bio-Rad, USA), and the results were revised by a range of BSA standards by detecting the absorbance at 570 nm. Finally, the relative ALP activities were normalized to the total protein and were presented as mM/mg total proteins.

Statistical analysis Statistical analysis was assessed by using a GraphPad Prism statistical software package. The statistical significance of the difference (p) was measured using two-way analysis of variance. A value of p < 0.05 was regard as statistically significant.

RESULTS AND DISCUSSION Surface structure and chemistry The Mg-Al LDHs film was directly grown on titanium surface by hydrothermal treatment, followed by moderate calcination to enhance surface alkalinity. As seen in Figure 1a, a sheet-like nanostructure appeared on the surface of pure titanium after hydrothermal treatment. A high magnification SEM image of LDH film reveals that the nanosheets are about 30-100 nm in diameter, and the adjacent distances are more than 200 nm on average. As shown in Figure 1b and c, the nanosheets became thinner and curlier after calcination treatment at 250 °C and 500 °C for 2 h, but the sheet-like


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structure was still maintained. The X-ray diffraction of LDH sample (Figure 2a) shows a series of characteristic peaks corresponding to [0 0 3], [0 0 6], [0 0 9], [0 1 2], [0 1 5], [0 1 8], [1 1 0] and [1 1 3] reflections of the Mg-Al LDHs.49 For LDH-250 and LDH-500 samples, the above mentioned diffraction peaks disappeared, and their XRD patterns are similar to that of pure Ti, which indicates that the layer structure of the LDH sample was destroyed after calcination. The FT-IR spectrums of the powders scraped from LDH, LDH-250 and LDH-500 samples are displayed in Figure 2b. For LDH sample, a sharp adsorption band appeared at 1355.6 cm-1 belongs to the characteristic stretching vibration peak of CO32anions in the interlayer.51 A strong infrared absorption band appeared in the range of 3200-3700 cm-1, which is relevant to the stretching vibration of hydroxyl groups in the lamellar and interlayer. The other infrared absorption bands in the range of 400-1000 cm-1 are mainly attributed to the lattice vibration of metal oxygen bonds Mg-O and AlO.52 For LDH-250 and LDH-500 samples, the peak at 1355.6 cm-1 gradually weakened and even completely disappeared, which indicates that most of CO32- anions in the interlayer were removed under calcination treatment. Meanwhile, the peak of -OH in the range of 3200-3700 cm-1 decreased, which results from the removal of H2O molecules and -OH group in lamellar. Moreover, the elements of all samples surfaces were detected by X-ray photoelectron spectrometer. As shown in Figure 2c and d, the Ti 2p3 peak disappeared, but the characteristic peaks of Mg 2p and Al 2p appeared on the modified Ti surfaces. The EDS elemental mapping of different samples (Figure 3) shows that all the modified Ti surfaces consist of Ti, O, Mg and Al elements. The



distribution area of Mg, Al and O is in accordance with the area of nanosheets, but the Ti is concentrated in the opposite areas. As a contrast, the Ti sample was also tested. The elements of Ti homogeneously distributed on the surface of pure Ti, and there exists a little O element owing to the native oxide layer. The compositions of samples surfaces analyzed by EDS are shown in Table 1. The contents of Ti, Mg and Al increased with the calcination temperature from 250 to 500 °C, while the content of O declined. The Mg/Al ratio of LDH, LDH-250 and LDH-500 samples surfaces is approximately equal to 2:1. During the hydrothermal process, the pH of reaction solution is in the range of 8.5 ~ 9.0 due to the decomposition of urea, in which the solubility of Al(OH)3 is much smaller, in comparison to that of Mg(OH)2. Therefore, the OH- tends to combine with Al3+ resulting in that the Mg/Al ratio of LDH is close to minimal (2:1). The formation of sheet-like morphology can be explained by the “evolution selection” growth mechanism, in line with the results reported in the literature.53 The morphology changing of samples after calcination is attributed to the removal of H2O and CO32from LDHs. With the temperature increasing to 250 °C, H2O and CO32- in the interlayer were removed and some hydroxyl condensation occurred in the lamellar, which made the nanosheets of LDH-250 sample became thinner. Prolonging to 500 °C, the H2O and CO32- were almost completely removed resulting in the slightly curling of the nanosheets. Higher calcination temperature making more H2O and CO2 be removed resulting in lower O content, which is in line with the EDS quantitative results. From the above discussion, the formation of the CO32- anion intercalated Mg-Al LDHs films


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on the pure titanium substrate can be confirmed. Meanwhile, the moderate calcination treatments with temperatures of 250 and 500 °C did not alter the main structural characteristic of LDH.

Surface physicochemical properties It is known that surface wettability can partly affect the biological properties of biomaterials. Hence, the water contact angles of samples were tested. As shown in Figure 4a, the water contact angles of Ti, LDH, LDH-250 and LDH-500 are 46.2 ± 4.1°, 31.9 ± 0.9°, 18.2 ± 1.1° and 23.1 ± 1.3° respectively, indicating that the surface of LDH sample is more hydrophilic than the surface of Ti sample. Moreover, the contact angles of LDH samples slightly decreased after calcination treatment. Zeta potentials test was conducted to measure the electrical charges on the samples surfaces. Seen from Figure 4b, the zeta potentials of all the samples surfaces decreased with the increasing pH value of KCl solution. The pure Ti surface presented lower zeta potentials than LDH, LDH-250 and LDH-500 samples surfaces. At pH 7.4, the trend of zeta potentials of various samples is as follows: Ti < LDH < LDH-250 < LDH-500, and the corresponding values are -68.5, -19.0 -14.6 and -11.1 mV, respectively. The zeta potential gap between Ti and the modified samples are much bigger than that among the three modified groups, which are probably attributed to their different electrochemical properties. For all the modified Ti samples, the positive lamellar and divalent Mg2+ and trivalent Al3+ cation resulted in their more positive zeta potentials. The amounts of releasing ions are shown in Figure 4c and d. The amount of Mg2+



released from LDH-250 and LDH-500 is higher than that from LDH at all periods. However, after immersion for 4 days, the amount of Al3+ released from LDH-250 and LDH-500 is lower than that from LDH. Furthermore, the cumulative amount of Mg2+ and Al3+ released from LDH-500 were higher than that of LDH-250 at all periods. The different zeta potentials of various samples are likely attributed to their differences in ions release. The pH values of solutions immersing various samples for 0, 1, 4, 8 and 12 h were measured and the results are shown in Figure 4e. At the beginning, the pH values of all groups are approximately 5.8. With time extended to 1 h, the pH value of Ti and LDH groups slightly changed to 5.58 and 6.05, respectively. However, the pH value of LDH250 and LDH-500 groups sharply increased to 7.29 and 9.51. Prolonging to 4 h, the pH value of Ti group decreased slightly to 5.38, the pH value of LDH group increased from 6.05 to 6.71, while the pH value of LDH-250 and LDH-500 groups reached up to 8.63 and 9.80, respectively. As time went by, the pH value of various samples groups tended to be constant and presented the following trend: Ti < LDH < LDH-250 < LDH-500. The difference is likely resulted from the different dissolution rate of various samples. For pure Ti, only a few titanic ions could release to the solution, which would slightly reduce the pH of the solution due to the hydrolysis of Ti cation. For LDH, LDH-250 and LDH-500 samples, along with reactions (1), (2), (3) and (4), the OH- accumulated on the samples surface, increasing the local alkalinity. Compared with LDH, some small pores existed on the LDH-250 and LDH-500 samples surfaces, which resulted in more active sites to react with H2O. Therefore, more


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hydroxyl ions would accumulate on the LDH-250 and LDH-500 surface leading to their stronger alkalinity. It should be reminded that with time going by, the Al3+ cation could react with excessive hydroxyl ions (Reaction (5)) which resulted in the slight decrease of pH for LDH-250 and LDH-500 samples. Mg(OH)2→Mg2 + + 2OH -

(1)

MgO + H2O→Mg2 + + 2OH Al(OH)3→Al3 + + 3OH -

(2) (3)

Al2O3 + 3H2O→2Al3 + + 6OH Al3 + + 4OH - →AlO2- + 2H2O

(4) (5)

To determine whether the alkalinity can maintain for a long time, we measured the pH value of NaCl solution after immersed various samples for 24 h and this procedure was recycled 3 times. Seen from Figure 4f, the pH values of the tested solutions were almost constant, indicating that the LDH, LDH-250 and LDH-500 samples can continuous produce OH- to keep their surface alkalinity even after 3 release circles. Through the above results and analysis, we can conclude that different alkaline microenvironments can be generated around various samples surfaces.

Antibacterial effects The morphology of bacteria cultured on each samples surface for 24 h were shown in Figure 5a. Plenty of S. aureus adhered, grown and gathered on the Ti surface, and exhibit a typical sphere cell morphology with a smooth surface. On the contrary, although collective S. aureus can be observed on LDH, LDH-250 and LDH-500



surfaces, the number of S. aureus gradually decline and the morphologies are smaller and irregular, compared with that on Ti surface. Besides, as is presented in Figure 5b, the cell viability of S. aureus on all modified Ti samples surfaces decreased. Particularly, few S. aureus can be observed on the LDH-500 surface, and the viability of S. aureus cultured on LDH-500 sample is the lowest, which indicates that the LDH-500 surface has excellent antibacterial activity against S. aureus. Similarly, the membranes of E. coli exhibit intact morphology on the Ti surface and the flagella of E. coli are clearly observed. However, the E. coli did not survive well on the LDH, LDH-250 and LDH500 surfaces, which can be confirmed by cell number reduction and cytosolic content leakage (Figure 5a). The above results were further confirmed by the Live/Dead bacterial staining results. As shown in Figure 5c and d, plenty of live S. aureus and E. coli (dyed green by SYTO 9) can be found on the Ti surface, and dead bacteria (dyed red by PI) can be hardly detected. On the LDH and LDH-250 samples surfaces, the amount of live bacteria decreased, but little dead bacteria can be found on these samples. On the LDH500 surface, both of live and dead bacteria reduced significantly. The results indicate that LDH-500 sample surface can effectively inhibit the growth of S. aureus and E. coli. In addition, as shown in Figure S1a and b, the colony amounts of both S. aureus and E. coli on LDH-500 sample are significantly less than that on other samples. The corresponding antibacterial rates of LDH-500 sample against S. aureus and E. coli are 86.27% and 88.44%, respectively. Nevertheless, the LDH and LDH-250 samples have little antibacterial ability, and even promote the proliferation of bacteria. As mentioned


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above, the cell viability and number of bacteria on LDH and LDH-250 surfaces declined, indicating that the growth of bacteria was suppressed. However, once the inhibited bacteria are placed in a nutrient-rich environment, they will recover and grow rapidly, which result in this indistinguishable colony counts results. The above results indicate that weak alkaline microenvironment only can restrain the growth of bacteria, but once the alkalinity of environment increase to an appropriate level, bacteria will be killed effectively.

Antibacterial mechanism Tremendous methods have been developed to endow biomaterials with antibacterial ability, based on two general strategies: (1). loading antibiotics or antimicrobial peptides; (2). incorporation of the inorganic antibacterial agents (Ag, Cu, Zn et al.). Due to problems of drug resistance and potential cytotoxicity, new strategies of modulating the physical and chemical properties of biomaterial surfaces have been proposed, and the underlying antibacterial mechanisms have been widely investigated. Firstly, it is reported that surface with different patterns at micro/nano meter scale can selectively inhibit bacteria but have little influence on mammalian cells because of the difference shape size between mammalian cells and bacteria. For instance, a micro porous structure with 5-11 μm in diameter can effectively inhibit bacterial activity1 and non-adhesive hydrogel arrays in nanometer scale can inhibit bacteria adhesion.6 For the materials system in our work, the surfaces of LDH, LDH-250 and LDH-500 have different antibacterial effects but possess a similar structure, indicating that some other



factors caused their antibacterial effects. Some researchers have discovered that the ‘‘sharp’’ edges of nanosheets of 2 D materials (such as graphene and graphene oxide) less than 1 nm in thickness are able to cut through the bacterial cell membrane leading to the bacteria death.54-55 However, the nanosheet array of LDH, LDH-250 and LDH500 samples surfaces are more than 100 nm in thickness, which is relatively large to induce negligible cytostatic or cytocidal effect to bacteria. Thus, we can conclude that the antibacterial effects of LDH, LDH-250 and LDH-500 films have not resulted from the nanosheet structure of samples. Secondly, the wettability of material surface has an influence on the adhesion of bacteria.56-57 However, there exists little difference in contact angle among LDH, LDH-250 and LDH-500 samples surfaces (Figure 4a), indicating that the different antibacterial effects are not attributed to their wettability. Thirdly, the surface charges can also affect the antibacterial activity of samples, because of that the cell membrane of bacteria is negatively charged.58 Positively charged surfaces would be attached more bacteria through electrostatic attraction. However, at the biological pH value, the trend of the zeta potentials of various samples (Figure 4b) are as follows: Ti < LDH < LDH-250 < LDH-500. The surface charges of LDH, LDH250 and LDH-500 films are more positive than that of Ti, indicating that the surface charge is not the dominated factor for their antibacterial activity. Moreover, the bacteriostatic ring test was conducted to investigate the influence of ion release on antibacterial behavior and the result is shown in Figure 6a. There is no inhibition zone appeared around all samples, so the release of Mg and Al ions does not contribute to the samples’ antibacterial capacity. Regardless of the above factors, the most obvious


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difference among these samples surface is alkalinity, and the LDH-500 surface owning the strongest alkalinity just has the highest antibacterial ability. Based on the above considerations, we believe that the surface alkalinity of the prepared samples is the key factor for their antibacterial activities. Furthermore, although there are few numbers of bacteria on the surface of LDH-500 sample, the intracellular ROS level of them shows about 6 times higher than that of bacteria on other samples surfaces, as shown in Figure 6b. Therefore, the antibacterial effect of LDH-500 may also relevant to the oxidative stress mediated by ROS production. As shown in Figure 7, the bacteria may be killed by the alkaline environment through the following two pathways: firstly, many hydroxide ions around the surface can consume the proton in the environment, thus hinder the energy metabolism of bacteria; secondly, excess hydroxide ions will induce the oxidative stress in bacteria and cause the damage of cell membrane. As displayed in Figure 7, there exist F-type H+-ATPase on the bacterial membrane to produce ATP (adenosine triphosphate) via utilizing the transmembrane proton motive force.38 Under normal microenvironment, the proton electrochemical gradient is established through the bacterial respiration process. As protons enter the cell along the electrochemical gradient via the proton pumps, ATP is successfully produced.59 Once the electrochemical potential of protons wears off, the driving force for the bacterial ATP synthesis will be weakened. ATP serves as an essential energy source for all living cells, so bacteria will be inhibited or even death resulting from lack of energy. It has previously been shown that S. aureus is able to survive an alkaline pH of 10 for



at least 6 h.60 However, the LDH-samples surfaces could continuously release OH-, which would constantly react with the bacterial extracellular H+. In this alkaline microenvironment, the proton between the cell membrane and the samples surface would be exceedingly consumed. As a result, a proton-depleted region would occur in the space between the bacteria and materials surfaces, leading to the disruption of bacterial proton electrochemical gradient. Then, the disruption would inactivate the ATP synthesis and the energy-dependent reactions,61 ultimately lead the bacteria maldevelopment or death. In addition, hydroxyl ions are highly oxidized free radicals and have extreme reactivity.62 They can react with several biomolecules of bacteria such as phospholipid and enzyme, which will generate many free radicals.63 Besides, ROS will be a by-product due to blockage of ATP synthesis and inactivation of enzyme of respiratory electron-transport chain.64 Thus, with the alkalinity of microenvironment increase to an appropriate level, bacterial intracellular ROS levels would sharply increase resulting in bacteria death. This hypothesis can be confirmed by the supplementary experiment about the effect of pH on the bacterial viability and intracellular ROS level (Figure S2).

Responses of osteogenic cells In this work, we selected two common osteogenic associated cells: rBMSCs with various differentiation potentials, and MC3T3-E1, the main functional cells for bone formation, to investigate the osteogenic effects of samples surfaces. Cell initial adhesion, proliferation, morphology, live/dead staining and alkaline phosphatase (ALP)


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activities were investigated to evaluate the cytocompatibility and osteogenic ability of alkaline microenvironment. The proliferation results of rBMSCs and MC3T3-E1 cells are shown in Figure 8a and b. After cultured for 1 and 4 days, the rBMSCs cells on Ti show significant high proliferation rate than that on modified Ti samples, and there exist no obvious difference among LDH, LDH-250 and LDH-500 samples. Prolonging to 7 days, the proliferation rate of rBMSCs cells on the LDH-500 is obviously lower than other groups. However, it should be reminded that the proliferation rate of cells on all samples distinctly increases with the elongation of culturing time. Compared with rBMSCs, MC3T3-E1 cells also present inhibited growth on the modified Ti samples surfaces after 1 day cultivation, but the cells restore their activeness gradually after 4 and 7 days. On the first day, MC3T3-E1 cells cannot grow well on the LDH, LDH-250 and LDH-500 samples compared with Ti. However, after 4 and 7 days incubation, the differences in proliferation rate between cells on Ti sample and that on modified Ti samples reduced visibly, and cells on LDH-500 sample shows slightly higher proliferation rate than that on LDH and LDH-250 samples. The corresponding SEM morphologies of cells cultured on the sample surfaces for 4 days are shown in Figure 9a and b (all the morphologies of cells cultured for 1, 4 and 7 days are shown in Figure S3 and S4). For rBMSCs cells, the surface of Ti was almost completely covered by cells, but only a few cells can be found on the LDH, LDH-250 and LDH-500 surfaces (Figure 9a). Despite all this, rBMSCs cells present a multipolar spindle morphology on surfaces of all samples, indicating they survived well on all samples. For MC3T3-E1 cells, one or more layers of cells can be found on all samples



surfaces, indicating that the cells grow well on all samples surfaces. To verify whether the alkaline surfaces of LDH, LDH-250 and LDH-500 are cytotoxic to rBMSCs and MC3T3-E1 cells, live/dead staining test was conducted. As shown in Figure 9c, more alive rBMSCs cells can be found on the surface of Ti, but the number of dead cells on all samples are nearly negligible. For MC3T3-E1 cells, all surfaces are completely covered by alive cells, and dead cells on all samples are hardly observed (Figure 9d). The above results indicate that comparing with pure Ti, cells on the alkaline surfaces of LDH, LDH-250 and LDH-500 samples present a slower cell proliferation rate but all samples have negligible cytotoxicity to cells. From the results of surface characterization, we can know that the obvious differences among the surfaces of various samples are their surface structure and alkalinity level. It is well known that the micro/nano structures on materials surfaces can affect cell proliferation through affecting cell adhesion. Thus, we investigate the initial cell adhesion and spreading activity by staining the cytoskeleton of rBMSCs and MC3T3-E1 cells cultured on samples surfaces for 1, 4 and 24 h, and the fluoroscopy images results are shown in Figure 10a and b. At the first hour, rBMSCs and MC3T3E1 cells on all samples present a round morphology. However, after 4 h, rBMSCs cells on Ti shows obvious spindle morphology with many filopodia, while most rBMSCs cells on LDH, LDH-250 and LDH-500 maintained round morphology. Prolonging to 24 h, rBMSCs cells on all samples show fibrous structure, but more F-actin, filopodia and lamellipodia can be observed from the cells on Ti. For MC3T3-E1 cells, the quantity of cells adhered to samples is larger than that of rBMSCs cells, but cells


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adhesion and spreading are similar to that of rBMSCs cells. The above results indicated that the nanosheet structures of LDH, LDH-250 and LDH-500 samples surfaces would inhibit cell adhesion and spreading, which can be further confirmed by the SEM morphology of cells cultured on various samples for 4 h, as shown in Figure S5. The ALP activities of rBMSCs and MC3T3-E1 cells cultured on the samples surfaces for 7 days were measured and the ALP staining images and quantitative results and are presented in Figure 11. For rBMSCs (Figure 11a and b), the ALP-positive areas on the alkaline LDH, LDH-250 and LDH-500 surface are larger than that on Ti. Some studies have shown that the large space between nanostructure will promote the MSCs cells differentiation2, indicating that the nanosheet structure of modified Ti samples may affect the ALP activity of rBMSCs cells. However, it should be noted that all of the modified Ti samples present similar surface morphology, but exhibit different ALP activity, indicating there are other factors affect cell differentiation except sample morphology. Previous studies have indicated that magnesium could stimulate bone formation via promoting osteogenic diﬀerentiation of the MSCs.18 However, the osteogenic effect of magnesium is mainly attributed to that they can promote the adhesion and up-regulate the integrin expression of osteogenic cells, and it has no significant effect on the ALP activity.40 Nevertheless, the pH could have influence on the osteogenic effects of elements, such as strontium and boron.47 An alkaline pH could play a positive role in Sr2+ affecting osteogenic cell activity. The alkaline microenvironment and releasing Mg2+ may produce a synergistic effect on ALP activities of rBMSCs, which need be verified by future studies. For MC3T3-E1 cells



(Figure 11c and d), the ALP activity of them on the LDH, LDH-250 and LDH-500 samples surfaces was also enhanced. Unlike rBMSCs cells, the MC3T3-E1 cells on LDH sample show slightly higher ALP activity compared to LDH-250 and LDH-500 surface, which is probably resulted from the different effect of alkaline microenvironment on different osteogenic cells. There exists an optimum space of surface nanoscale features for cells integrin clustering and focal adhesion, and the optimal length scale is less than 70 nm.2-3, 6, 65 Large separations between the adhesive protrusions will reduce the formation of focal adhesion and the clustering, inhibit the activation of integrin, and result in poor cell adhesion and spreading.66 In this study, the space of adjacent sheets of LDH, LDH-250 and LDH-500 structures is obviously larger than 70 nm, resulting in unfavorable effects on cell adhesion and spreading. Therefore, on the first day, the rBMSCs and MC3T3E1 cell proliferation on LDH, LDH-250 and LDH-500 are inferior to that on Ti. On the other hand, the different alkalinity level of various samples will affect the proliferation of rBMSCs and MC3T3-E1 cells as well. As shown in Figure S6, the results indicate that the medium with pH < 9.0 could promote the proliferation of MC3T3-E1 cells, and the medium with pH > 8.5 would inhibit the proliferation of rBMSCs cells. Thus, it is believed that the different proliferation behavior of rBMSCs and MC3T3-E1 cells on various samples is attributed to the combined effect of nanostructure and alkalinity of samples surface. Briefly, on the first day, the large space between adjacent sheets of LDH, LDH-250 and LDH-500 surfaces inhibited the cell adhesion and spreading, leading to poor cells proliferation. With culture time prolonging, the proliferation of


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rBMSCs cells was further inhibited, but the proliferation of MC3T3-E1 cells was promoted owing to the alkaline microenvironment around the LDH, LDH-250 and LDH-500 samples surfaces. Previous studies46-47, 67 have shown that a suitable alkaline environment around the implant surfaces could promote the osteogenic activities of osteoblasts and MSC cells. In our study, although the exact pH of the interface is difficult to measure, the alkalinity of microenvironments around various samples surfaces are obviously diverse. The trends are as follows (shown in Figure 4e and f): Ti < LDH < LDH-250 < LDH-500. In spite of the proliferation of rBMSCs cells was inhibited on the LDH-500 sample surface, but the ALP activity was significantly improved. ALP is an external enzyme of osteoblasts, and its activity is a distinct indicator of osteoblast differentiation. Therefore, it can be deduced that the alkaline microenvironments could promote rBMSCs differentiation towards osteoblast. Bone remodeling is a complicated process of simultaneous formation and resorption that involve kinds of cells including MSCs, osteoblast and osteoclast.41 From the above results, we hold the opinion that the bone remodeling process may be effectively promoted by creating a proper alkaline microenvironment: firstly, the alkaline environment will facilitate the differentiation of MSCs osteogenic toward osteoblast; secondly, it will promote the osteoblast proliferation to form new bone. Moreover, the alkaline microenvironment around LDH-500 sample surface shows excellent antibacterial ability against both gram-positive and negative bacteria, confirming the feasibility of endowing titanium with osteogenesis and antibacteria



effect simultaneously by regulating the alkalinity of materials surfaces. There should exist an optimum alkalinity to balance the antibacterial and osteogenic properties, and the exact pH value and the underlying mechanism still need be further studied.

CONCLUSION The alkaline Mg-Al LDHs film was directly grown on the surface of biomedical titanium by hydrothermal treatment. The alkalinity of Mg-Al LDHs film was enhanced by calcination treatment at 250 and 500 °C, but the main nanosheet morphology was maintained. In vitro antibacterial experiments show that LDH and LDH-250 films with weaker alkalinity are cytostatic to bacteria and LDH-500 films with stronger alkalinity can effectively kill bacteria. The antibacterial effect of alkaline microenvironment may result from the inactivation of ATP synthesis and the induction of oxidative stress in bacteria. On the other side, the nanosheet structures of LDH, LDH-250 and LDH-500 films would restrain the initial adhesion of osteogenic cells. However, the alkaline microenvironment generated by LDH, LDH-250 and LDH-500 samples can promote the mesenchymal stem cells osteogenic differentiation and enhance the proliferation and alkaline phosphatase activities of osteoblast cells. Our study provides a novel simple strategy of regulating the alkalinity of microenvironments around materials surface to endow biomaterials with selective antibacterial and osteogenic activities for bone generation.


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ASSOCIATED CONTENT Supporting information The results of re-cultivated S. aureus and E. coli colonies on agar; the effect of pH on bacteria; the SEM morphologies of cells cultured on samples for 1, 4 and 7 days; the SEM morphologies of cells cultured on samples for 4 h; the effect of pH on cells proliferation; supporting ﬁgures and text.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected]. Tel: +86 21 52412409. Fax: +86 21 52412409

ORCID Xuanyong Liu: 0000-0001-9440-8143

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS Financial support from the National Natural Science Foundation of China (51831011, 31670980), National Natural Science Foundation for Distinguished Young Scholars of China (51525207), Science and Technology Commission of Shanghai Municipality (18YF1426900, 18410760600) are acknowledged.



REFERENCES (1) Manabe, K.; Nishizawa, S.; Shiratori, S. Porous Surface Structure Fabricated by Breath Figures that Suppresses Pseudomonas Aeruginosa Biofilm Formation. ACS Appl. Mater. Interfaces 2013, 5, 11900-11905. (2) Oh, S.; Brammer, K. S.; Li, Y. S. J.; Teng, D.; Engler, A. J.; Chien, S.; Jin, S. Stem Cell Fate Dictated Solely by Altered Nanotube Dimension. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 2130-2135. (3) Park, J.; Bauer, S.; von der Mark, K.; Schmuki, P. Nanosize and Vitality: TiO2 Nanotube Diameter Directs Cell Fate. Nano Lett. 2007, 7, 1686-1691. (4) Puckett, S. D.; Taylor, E.; Raimondo, T.; Webster, T. J. The Relationship Between the Nanostructure of Titanium Surfaces and Bacterial Attachment. Biomaterials 2010, 31, 706-719. (5) Vargas-Alfredo, N.; Santos-Coquillat, A.; Martinez-Campos, E.; Dorronsoro, A.; Cortajarena, A. L.; Del Campo, A.; Rodriguez-Hernandez, J. Highly Efficient Antibacterial Surfaces Based on Bacterial/Cell Size Selective Microporous Supports. ACS Appl. Mater. Interfaces 2017, 9, 44270-44280. (6) Wang, Y.; Subbiahdoss, G.; Swartjes, J.; van der Mei, H. C.; Busscher, H. J.; Libera, M. Length-Scale Mediated Differential Adhesion of Mammalian Cells and Microbes. Adv. Funct. Mater. 2011, 21, 3916-3923. (7) Zhao, L.; Mei, S.; Chu, P. K.; Zhang, Y.; Wu, Z. The Influence of Hierarchical Hybrid Micro/nano-Textured Titanium Surface with Titania Nanotubes on Osteoblast Functions. Biomaterials 2010, 31, 5072-82.


Page 32 of 53

Page 33 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(8) Leipzig, N. D.; Shoichet, M. S. The Effect of Substrate Stiffness on Adult Neural Stem Cell Behavior. Biomaterials 2009, 30, 6867-6878. (9) Ye, K.; Wang, X.; Cao, L.; Li, S.; Li, Z.; Yu, L.; Ding, J. Matrix Stiffness and Nanoscale Spatial Organization of Cell-Adhesive Ligands Direct Stem Cell Fate. Nano Lett. 2015, 15, 4720-4729. (10) Hu, M.; Chang, H.; Zhang, H.; Wang, J.; Lei, W.-x.; Li, B.-c.; Ren, K.-f.; Ji, J. Mechanical Adaptability of the MMP-Responsive Film Improves the Functionality of Endothelial Cell Monolayer. Adv. Healthcare Mater. 2017, 6, 1601410. (11) Arima, Y.; Iwata, H. Effects of Surface Functional Groups on Protein Adsorption and Subsequent Cell Adhesion using Self-Assembled Monolayers. J. Mater. Chem. 2007, 17, 4079-4087. (12) Cao, B.; Peng, Y.; Liu, X.; Ding, J. Effects of Functional Groups of Materials on Nonspecific Adhesion and Chondrogenic Induction of Mesenchymal Stem Cells on Free and Micropatterned Surfaces. ACS Appl. Mater. Interfaces 2017, 9, 23574-23585. (13) Qiu, J.; Wang, D.; Geng, H.; Guo, J.; Qian, S.; Liu, X. How Oxygen-Containing Groups on Graphene Influence the Antibacterial Behaviors. Adv. Mater. Interfaces 2017, 4, 1700228. (14) Ren, Y. J.; Zhang, H.; Huang, H.; Wang, X. M.; Zhou, Z. Y.; Cui, F. Z.; An, Y. H. In Vitro Behavior of Neural Stem Cells in Response to Different Chemical Functional Groups. Biomaterials 2009, 30, 1036-1044. (15) Cao, H.; Qin, H.; Zhao, Y.; Jin, G.; Lu, T.; Meng, F.; Zhang, X.; Liu, X. NanoThick Calcium Oxide Armed Titanium: Boosts Bone Cells Against Methicillin-



Resistant Staphylococcus Aureus. Sci. Rep. 2016, 6, 21761. (16) Mei, S.; Wang, H.; Wang, W.; Tong, L.; Pan, H.; Ruan, C.; Ma, Q.; Liu, M.; Yang, H.; Zhang, L.; Cheng, Y.; Zhang, Y.; Zhao, L.; Chu, P. K. Antibacterial Effects and Biocompatibility of Titanium Surfaces with Graded Silver Incorporation in Titania Nanotubes. Biomaterials 2014, 35, 4255-4265. (17) Qiao, Y.; Zhang, W.; Tian, P.; Meng, F.; Zhu, H.; Jiang, X.; Liu, X.; Chu, P. K. Stimulation of Bone Growth Following Zinc Incorporation into Biomaterials. Biomaterials 2014, 35, 6882-6897. (18) Zhang, J.; Ma, X.; Lin, D.; Shi, H.; Yuan, Y.; Tang, W.; Zhou, H.; Guo, H.; Qian, J.; Liu, C. Magnesium Modification of a Calcium Phosphate Cement Alters Bone Marrow Stromal Cell Behavior via an Integrin-Mediated Mechanism. Biomaterials 2015, 53, 251-264. (19) Zhang, W.; Cao, H.; Zhang, X.; Li, G.; Chang, Q.; Zhao, J.; Qiao, Y.; Ding, X.; Yang, G.; Liu, X.; Jiang, X. A Strontium-Incorporated Nanoporous Titanium Implant Surface for Rapid Osseointegration. Nanoscale 2016, 8, 5291-5301. (20) Oda, H.; Konno, T.; Ishihara, K. The Use of the Mechanical Microenvironment of Phospholipid Polymer Hydrogels to Control Cell Behavior. Biomaterials 2013, 34, 5891-5896. (21) Saha, K.; Keung, A. J.; Irwin, E. F.; Li, Y.; Little, L.; Schaffer, D. V.; Healy, K. E. Substrate Modulus Directs Neural Stem Cell Behavior. Biophys. J. 2008, 95, 442638. (22) Wang, G.; Feng, H.; Hu, L.; Jin, W.; Hao, Q.; Gao, A.; Peng, X.; Li, W.; Wong,


Page 34 of 53

Page 35 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


K. Y.; Wang, H.; Li, Z.; Chu, P. K. An Antibacterial Platform based on Capacitive Carbon-Doped TiO2 Nanotubes after Direct or Alternating Current Charging. Nat. Commun. 2018, 9, 2055. (23) Cao, H.; Liu, X.; Meng, F.; Chu, P. K. Biological Actions of Silver Nanoparticles Embedded in Titanium Controlled by Micro-Galvanic Effects. Biomaterials 2011, 32, 693-705. (24) Jin, G.; Qin, H.; Cao, H.; Qiao, Y.; Zhao, Y.; Peng, X.; Zhang, X.; Liu, X.; Chu, P. K. Zn/Ag Micro-Galvanic Couples Formed on Titanium and Osseointegration Effects in the Presence of S. Aureus. Biomaterials 2015, 65, 22-31. (25) Li, J.; Wang, G.; Zhu, H.; Zhang, M.; Zheng, X.; Di, Z.; Liu, X.; Wang, X. Antibacterial Activity of Large-Area Monolayer Graphene Film Manipulated by Charge Transfer. Sci. Rep. 2014, 4, 4359. (26) Liu, Y.; Zhang, X.; Cao, C.; Zhang, Y.; Wei, J.; Li, Y. j.; Liang, W.; Hu, Z.; Zhang, J.; Wei, Y.; Deng, X. Built-In Electric Fields Dramatically Induce Enhancement of Osseointegration. Adv. Funct. Mater. 2017, 27, 1703771. (27) Ning, C.; Yu, P.; Zhu, Y.; Yao, M.; Zhu, X.; Wang, X.; Lin, Z.; Li, W.; Wang, S.; Tan, G.; Zhang, Y.; Wang, Y.; Mao, C. Built-In Microscale Electrostatic Fields Induced by Anatase-Rutile-Phase Transition in Selective Areas Promote Osteogenesis. NPG Asia Mater. 2016, 8, e234 (28) Zhang, X.; Zhang, C.; Lin, Y.; Hu, P.; Shen, Y.; Wang, K.; Meng, S.; Chai, Y.; Dai, X.; Liu, X.; Liu, Y.; Mo, X.; Cao, C.; Li, S.; Deng, X.; Chen, L. Nanocomposite Membranes Enhance Bone Regeneration through Restoring Physiological Electric



Microenvironment. ACS Nano 2016, 10, 7279-7286. (29) Neoh, K. G.; Hu, X.; Zheng, D.; Kang, E. T. Balancing Osteoblast Functions and Bacterial Adhesion on Functionalized Titanium Surfaces. Biomaterials 2012, 33, 28132822. (30) Li, J.; Tan, L.; Liu, X.; Cui, Z.; Yang, X.; Yeung, K. W. K.; Chu, P. K.; Wu, S. Balancing Bacteria–Osteoblast Competition through Selective Physical Puncture and Biofunctionalization of ZnO/Polydopamine/Arginine-Glycine-Aspartic Acid-Cysteine Nanorods. ACS Nano 2017, 11, 11250-11263. (31) Qian, S.; Qiao, Y.; Liu, X. Selective Biofunctional Modification of Titanium Implants for Osteogenic and Antibacterial Applications. J. Mater. Chem. B 2014, 2, 7475-7487. (32) Fielding, G. A.; Roy, M.; Bandyopadhyay, A.; Bose, S. Antibacterial and Biological Characteristics of Silver Containing and Strontium Doped Plasma Sprayed Hydroxyapatite Coatings. Acta Biomater. 2012, 8, 3144-3152. (33) Yu, Y.; Jin, G.; Xue, Y.; Wang, D.; Liu, X.; Sun, J. Multifunctions of Dual Zn/Mg Ion Co-Implanted Titanium on Osteogenesis, Angiogenesis and Bacteria Inhibition for Dental Implants. Acta Biomater. 2017, 49, 590-603. (34) Lu, T.; Li, J.; Qian, S.; Cao, H.; Ning, C.; Liu, X. Enhanced Osteogenic and Selective Antibacterial Activities on Micro-/Nano-Structured Carbon Fiber Reinforced Polyetheretherketone. J. Mater. Chem. B 2016, 4, 2944-2953. (35) Li, J.; Zhou, H.; Qian, S.; Liu, Z.; Feng, J.; Jin, P.; Liu, X. Plasmonic Gold Nanoparticles Modified Titania Nanotubes for Antibacterial Application. Appl. Phys.


Page 36 of 53

Page 37 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Lett. 2014, 104, 261110. (36) Tian, Y.; Cao, H.; Qiao, Y.; Meng, F.; Liu, X. Antibacterial Activity and Cytocompatibility of Titanium Oxide Coating Modified by Iron Ion Implantation. Acta Biomater. 2014, 10, 4505-4517. (37) Padan, E.; Bibi, E.; Ito, M.; Krulwich, T. A. Alkaline pH Homeostasis in Bacteria: New Insights. Biochim. Biophys. Acta (BBA), Biomembr. 2005, 1717 (2), 67-88. (38) Boyer, P. D. The ATP Synthase - A Splendid Molecular Machine. Annu. Rev. Biochem. 1997, 66, 717-749. (39) Cao, H.; Tang, K.; Liu, X. Bifunctional Galvanics Mediated Selective Toxicity on Titanium. Mater. Horiz. 2018, 5, 264-267. (40) Zhao, Y.; Cao, H.; Qin, H.; Cheng, T.; Qian, S.; Cheng, M.; Peng, X.; Wang, J.; Zhang, Y.; Jin, G.; Zhang, X.; Liu, X.; Chu, P. K. Balancing the Osteogenic and Antibacterial Properties of Titanium by Codoping of Mg and Ag: An in Vitro and in Vivo Study. ACS Appl. Mater. Interfaces 2015, 7, 17826-36. (41) Kaunitz, J. D.; Yamaguchi, D. T. TNAP, Trap, Ecto-Purinergic Signaling, and Bone Remodeling. J. Cell. Biochem. 2008, 105, 655-662. (42) Arnett, T. R. Extracellular pH Regulates Bone Cell Function. J. Nutr. 2008, 138, 415S-418S. (43) Meghji, S.; Morrison, M. S.; Henderson, B.; Arnett, T. R. pH Dependence of Bone Resorption: Mouse Calvarial Osteoclasts are Activated by Acidosis. Am. J. Physiol. 2001, 280, E112-E119. (44) Bushinsky, D. A. Metabolic Alkalosis Decreases Bone Calcium Efflux by



Suppressing Osteoclasts and Stimulating Osteoblasts. Am. J. Physiol. 1996, 271, F216F222. (45) Harada, M.; Udagawa, N.; Fukasawa, K.; Hiraoka, B. Y.; Mogi, M. Inorganic Pyrophosphatase Activity of Purified Bovine Pulp Alkaline Phosphatase at Physiological pH. J. Dent. Res. 2016, 65, 125-127. (46) Shen, Y.; Liu, W.; Lin, K.; Pan, H.; Darvell, B. W.; Peng, S.; Wen, C.; Deng, L.; Lu, W. W.; Chang, J. Interfacial pH: A Critical Factor for Osteoporotic Bone Regeneration. Langmuir 2011, 27, 2701-2708. (47) Shen, Y.; Liu, W.; Wen, C.; Pan, H.; Wang, T.; Darvell, B. W.; Lu, W. W.; Huang, W. Bone Regeneration: Importance of Local pH—Strontium-Doped Borosilicate Scaffold. J. Mater. Chem. 2012, 22, 8662-8670. (48) Li, J.; Wang, G.; Wang, D.; Wu, Q.; Jiang, X.; Liu, X. Alkali-Treated Titanium Selectively Regulating Biological Behaviors of Bacteria, Cancer Cells and Mesenchymal Stem Cells. J. Colloid Interface Sci. 2014, 436, 160-170. (49) Evans, D. G.; Slade, R. C. T. Structural Aspects of Layered Double Hydroxides. Springer, Berlin, Heidelberg, 2006, 119, 1-87. (50) Li, F.; Duan, X. Applications of Layered Double Hydroxides. Springer, Berlin, Heidelberg, 2006, 119, 193-223. (51) Hernandezmoreno, M. J.; Ulibarri, M. A.; Rendon, J. L.; Serna, C. J. IR Characteristics of Hydrotalcite-Like Compounds. Phys. Chem. Miner. 1985, 12, 34-38. (52) Hickey, L.; Kloprogge, J. T.; Frost, R. L. The Effects of Various Hydrothermal Treatments on Magnesium-Aluminium Hydrotalcites. J. Mater. Sci. 2000, 35, 4347-


Page 38 of 53

Page 39 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4355. (53) Wang, D.; Ge, N.; Li, J.; Qiao, Y.; Zhu, H.; Liu, X. Selective Tumor Cell Inhibition Effect of Ni–Ti Layered Double Hydroxides Thin Films Driven by the Reversed pH Gradients of Tumor Cells. ACS Appl. Mater. Interfaces 2015, 7, 7843-7854. (54) Mangadlao, J. D.; Santos, C. M.; Felipe, M. J. L.; de Leon, A. C. C.; Rodrigues, D. F.; Advincula, R. C. On the Antibacterial Mechanism of Graphene Oxide (GO) Langmuir–Blodgett Films. Chem. Commun. 2015, 51, 2886-2889. (55) Zou, X.; Zhang, L.; Wang, Z.; Luo, Y. Mechanisms of the Antimicrobial Activities of Graphene Materials. J. Am. Chem. Soc. 2016, 138, 2064-2077. (56) Dou, X.-Q.; Zhang, D.; Feng, C.; Jiang, L. Bioinspired Hierarchical Surface Structures with Tunable Wettability for Regulating Bacteria Adhesion. Acs Nano 2015, 9, 10664-10672. (57) Bruinsma, G. M.; van der Mei, H. C.; Busscher, H. J. Bacterial Adhesion to Surface Hydrophilic and Hydrophobic Contact Lenses. Biomaterials 2001, 22, 3217-3224. (58) Stoimenov, P. K.; Klinger, R. L.; Marchin, G. L.; Klabunde, K. J. Metal Oxide Nanoparticles as Bactericidal Agents. Langmuir 2002, 18, 6679-6686. (59) von Ballmoos, C. Alternative Proton Binding Mode in ATP Synthases. J. Bioenerg. Biomembr. 2007, 39, 441-445. (60) Anderson, K. L.; Roux, C. M.; Olson, M. W.; Luong, T. T.; Lee, C. Y.; Olson, R.; Dunman, P. M. Characterizing the Effects of Inorganic Acid and Alkaline Shock on the Staphylococcus Aureus Transcriptome and Messenger RNA Turnover. FEMS Immunol. Med. Microbiol. 2010, 60, 208-250.



(61) Trumpower, B. L.; Gennis, R. B. Energy Transduction by Cytochrome Complexes in Mitochondrial and Bacterial Respiration - the Enzymology of Coupling ElectronTransfer Reactions to Transmembrane Proton Translocation. Annu. Rev. Biochem. 1994, 63, 675-716. (62) Freeman, B. A.; Crapo, J. D. Free-Radicals and Tissue-Injury. Lab. Invest. 1982, 47, 412-426. (63) Siqueira, J. F.; Lopes, H. P. Mechanisms of Antimicrobial Activity of Calcium Hydroxide: a Critical Review. Int. Endod. J. 1999, 32, 361-369. (64) Bae, Y. S.; Oh, H.; Rhee, S. G.; Yoo, Y. D. Regulation of Reactive Oxygen Species Generation in Cell Signaling. Mol. Cells 2011, 32, 491-509. (65) Arnold, M.; Cavalcanti-Adam, E. A.; Glass, R.; Blümmel, J.; Eck, W.; Kantlehner, M.; Kessler, H.; Spatz, J. P. Activation of Integrin Function by Nanopatterned Adhesive Interfaces. ChemPhysChem 2004, 5, 383-388. (66) Lee, J.; Kang, B. S.; Hicks, B.; Chancellor Jr, T. F.; Chu, B. H.; Wang, H.-T.; Keselowsky, B. G.; Ren, F.; Lele, T. P. The Control of Cell Adhesion and Viability by Zinc Oxide Nanorods. Biomaterials 2008, 29, 3743-3749. (67) Li, Q.; Wang, D.; Qiu, J.; Peng, F.; Liu, X. Regulating the Local pH Level of Titanium via Mg–Fe Layered Double Hydroxides Films for Enhanced Osteogenesis. Biomater. Sci. 2018, 6, 1227-1237.


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Table 1. Elemental content of various samples surfaces. Ti (at. %)

O (at. %)

Mg (at. %) Al (at. %)

LDH

12.3

62.1

16.6

9.0

1.84

LDH-250

15.0

58.5

17.3

9.3

1.86

LDH-500

24.5

42.8

21.2

11.4

1.86


Mg/Al


Figure 1. SEM morphologies of Ti (a), LDH (b), LDH-250 (c) and LDH-500 (d) samples at low and high magnification. 80x56mm (300 x 300 DPI)


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Figure 2. XRD patterns of all samples (a); FT-IR spectrum acquired from the modified Ti samples (b); XPS full spectra of all samples (c) and the Mg 2p and Al 2p spectra (d) of modified Ti samples.



Figure 3. SEM images and corresponding EDS elemental mapping images of Ti, O, Mg and Al elements of all samples.


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Figure 4. Contact angles of various samples surfaces (a); zeta potential versus pH acquired from various samples surfaces (b); cumulative Mg (c) and Al (d) ion release amounts in saline solution after immersing LDH, LDH-250 and LDH-500 samples for various days; pH value of saline solution after immersing samples for various hours (e) and for 1 day with 3 times circulation (f). * p < 0.05, ** p < 0.01, *** p < 0.001. 80x91mm (300 x 300 DPI)



Figure 5. SEM morphologies (a) and cell viabilities (b) of S. aureus and E. coli cultured on various samples surfaces at low and high magnification; florescent microscopic images of the live/dead staining of S. aureus (c) and E. coli (d) after culturing on the samples surfaces for 24 h (the white scale bar is 100 μm). * p < 0.05, ** p < 0.01, *** p < 0.001.


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Figure 6. Inhibition rings around samples against E. coli and S. aureus (a); intracellular ROS levels of S. aureus and E. coli cultured on various samples surfaces for 24 h (b). ** p < 0.01*** p < 0.001.



Figure 7. Illustration for the possible antibacterial mechanism of the alkaline microenvironments generated by layered double hydroxides/oxides films on titanium surface.


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Figure 8. Fluorescent intensity of alamarBlue reduced by rBMSCs cells (a) and MC3T3-E1 cells (b) cultured on various samples surfaces for 1, 4 and 7 days. * p < 0.05, ** p < 0.01, *** p < 0.001, ****p < 0.0001.



Figure 9. SEM morphologies of rBMSCs cells (a) and MC3T3-E1 cells (b) cultured on samples surfaces for 4 days at low and high magnification; fluoroscopy images of live/dead (green/red) staining of rBMSCs cells (c) and MC3T3-E1 cells (d) cultured on various samples surfaces for 4 days (the white scale bar is 100 μm).


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Figure 10. Fluoroscopy images of F-actin stained with TRITC phalloidin (red) and the nucleus stained with DAPI (blue) of rBMSCs cells (a) and MC3T3-E1 cells (b) cultured on various samples surfaces for 1, 4 and 24 h.



Figure 11. ALP positive areas and the related qualitative ALP activity of rBMSCs cells (a) and (b), and MC3T3-E1 cells (c) and (d) cultured on various samples surfaces for 7 days. The green scale bar is 100 µm. * p < 0.05, ** p < 0.01, *** p < 0.001.


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TOC


Effect of Local Alkaline Microenvironment on the Behaviors of Bacteria

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