Gallium-doped titania nanotubes elicit anti-bacterial efficacy in vivo against Escherichia coli and Staphylococcus aureus biofilm

Materialia 5 (2019) 100209

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Gallium-doped titania nanotubes elicit anti-bacterial efficacy in vivo against Escherichia coli and Staphylococcus aureus biofilm Junjie Dong a, Dong Fang b, Lei Zhang c,∗, Quan Shan b,∗∗, Yunchao Huang a,d,∗∗∗ a

Department of Orthopedics, The First Affiliated Hospital of Kunming Medical University, Kunming 650032, China School of Materials Science and Engineering, Kunming University of Science and Technology, Kunming 650093, China c Department of Mechanical & Industrial Engineering, Northeastern University, Boston, Massachusetts 02115, United States d Department of Thoracic Surgery, The Third Affiliated Hospital of Kunming Medical University, Tumor Hospital of Yunnan Province, Kunming 650118, China b

a r t i c l e Keywords: Titania nanotubes Gallium Biofilm Escherichia coli Staphylococcus aureus

i n f o

a b s t r a c t In this work, the modification of titanium surface with TiO2 nano-tubes (TNTs), and a solvent casting of biocompatible polymer (poly-DL-lactic acid) film combines gallium (III), as a factor of anti-inflammatory and bone resorption inhibitors, was assessed in the spinal infection rat model. Under in vitro condition, it is found that S. aureus and E. coli can attach and competitively survive on the TiO2 nano-tube surface and form the mixed bacteria biofilm. By contrasting the in vivo implantation of Cp Ti, TNTs and Ga-Cp Ti scaffolds, Ga-doped TNTs showed distinct and excellent anti-bacterial property and reduced inflammation and favorable compatibility with osteoblasts, which reveal a large potential of exploiting and modifying the multi-biofunction of implantable biomaterials. The described in vivo work expands the fundamental understanding of the advantages of gallium (III) and titania nano-array on antiresorptive and antimicrobial properties and the designing strategy of the composite coating is broadly applicable to a wide range of multifunctional biomaterials.

1. Introduction Titanium and its alloys have been playing a significant role in dental and orthopedic implants due to their excellent corrosion resistance, good mechanical properties and biocompatibility [1,2]. However, the biomedical materials do not have an intrinsic antibacterial ability which hindering the development and clinical application [3,4]. In fact, bacterial biofilms often form on the inert surfaces of implanted devices such as catheters, prosthetic cardiac valves and intrauterine devices. Infections associated with bacterial biofilm, especially with dual-species bacterial [5] growth usually are one of the most serious challenges that need to be eradicated after surgery that lead to not only implant failure, but also a complication, morbidity, and mortality [6,7]. In recent years, several strategies [8,9] have been proposed to endow titanium implants with antibacterial characteristics. In particular, titanium embedded with microelements and surface modification has drawn much attention [10]. Introduction of agents such as Ag, Cu and Zn to the Ti materials can produce antibacterial properties, and the role of electron transfer between the modified surface and bacteria have been suggested [9,11–15]. As an anti-tumor and anti-hypercalcemia drug approved by FDA (Food and Drug Administration), many studies [16–19] have shown that

gallium nitrate (Ga(NO3 )3 ) has a broad-spectrum antimicrobial activity for Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Baumanii and Escherichia coli. It is mostly due to the research from Hedley et al. [20] and Kaneko et al. [21] that Gallium (III) displays as an antagonist to the actions of Fe2+ ions in processes of cellular metabolism, which inhibits replicative DNA synthesis and redox ability of bacteria. In addition to that, the action of gallium in Ga(NO3 )3 on bone metabolism decreases the hypercalcemia associated with cancer [22]. Ga ions can prevent the breakdown of bone through the inhibition of osteoclastic activity and therefore decreases hydroxyapatite (Ca5 (PO4 )3 (OH), HA) crystal formation and lowers the amount of free Ca ions in the blood, with adsorption of Ga onto the surfaces of HA crystals [23,24]. The increased concentration of Ga in the bone leads to increasing the synthesis of collagen as well as the formation of the bone tissue inside the cell [25–27]. However, the bioactivity of Ga(NO3 )3 is low and thus requires a continuous and stable osteointegration property [28,29]. For this reason, we investigated the possibility to encapsulated Ga(NO3 )3 with Titanium dioxide nanotubes (TNTs) to form a composite coating on biomedical Ti bone plate for the development of a local delivery system of Ga (III) ions in osteoporotic sites.

∗

Corresponding author at: Department of Mechanical & Industrial Engineering, Northeastern University, Boston, Massachusetts 02115, United State. Corresponding author at: School of Materials Science and Engineering, Kunming University of Science and Technology, Kunming 650093, China. ∗∗∗ Corresponding author at: Department of Orthopedics, The First Affiliated Hospital of Kunming Medical University, Kunming 650032, China.; Department of Thoracic Surgery, The Third Affiliated Hospital of Kunming Medical University, Tumor Hospital of Yunnan Province, Kunming, 650118, China. E-mail addresses: [email protected] (L. Zhang), [email protected] (Q. Shan), [email protected] (Y. Huang). ∗∗

https://doi.org/10.1016/j.mtla.2019.100209 Received 30 September 2018; Accepted 13 January 2019 Available online 15 January 2019 2589-1529/© 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

J. Dong, D. Fang and L. Zhang et al.

TNTs have drawn wide attention and been extensively applied in the field of orthopedic and dental implant materials, due to the good corrosion resistance, superior biocompatibility and enhanced bioactivity [30,31]. Moreover, taking advantages of the nano-hierarchy of bionic structures, the rough nano-topography, large specific surface area and their ability to integrate bio-function can not only provide a favorable environment for cell adhesion and sustainable adsorption of extracellular proteins, but also stimulate and regulate the physiological responses through their characteristic compatibility with the biological system [32–34]. In addition, a large amount of research [35–38] has focused on the wide applications of surface modified TNTs in the biomedical and photoelectrocatalytic field. For instance, Kodama et al. [39] loaded HA on the TNTs to promote the integration of implants to bone tissue. Khan et al. [40] mixed Ga and N with TNTs samples provided the good photocatalytic activity, enhanced optical absorption and effective separation between photoexcited carries. Domínguez-Espíndola et al. [41] introduced Ag to decorated TNTs to improve the bacterial resistance against Pseudomonas aeruginosa through photoelectrocatalytic inactivation mechanism. Several surface treatment techniques have been proposed to endow the bone implants with antibacterial agents, such as ion implantation, sputtering and plasma spray [42–44]. Although these techniques have been employed for the introduction of Ag, Cu and Zn, the rate of release antibacterial metal ion in the physiological environment were showed lower than that prepared by biodegradable and self-adhesive gel, which might limit the antibacterial ability of implants, particularly for resisting the formation of the mixed bacteria biofilm. Poly-DL-lactic acid (PDLLA) is a biodegradable and bioactive supermolecular acid, which has been accepted by FDA on the application of surgical suture, bone fixation implants, and drug release system [45]. So far, there are few publications about Ga-PDLLA-TNTs coating for the resistance of mixed bacteria biofilm, and a lack of studies on the bacterial resistance and osseointegration of Ga ions delivery system in osteoporotic sites by building the mixed bacteria biofilm spinal infection rat model. Herein, we describe a simple protocol with a combination of anodic oxidation and solvent casting to integrate Ga(NO3 )3 with TiO2 nanotubes coatings for the development of the bacterial resistance, osteogenesis and osseointegration of biomedical Ti bone plate materials. The formation mechanism of E. coli and S. aureus biofilm on the TiO2 nanotubes coatings were investigated. Besides, by building the mixed bacteria biofilm spinal infection rat model, the ability and properties of Ga ions delivery system in osteoporotic sites was investigated under in vivo implantation. 2. Materials and methods 2.1. Preparation of Ga-TNTs samples and the characterization The ordered TiO2 nanotubes (TNTs) were prepared by electrochemical anodization of the Ti metal sheets (TA1 GB/T3260-1994, Baoji Sanli Nonferrous Metals Co., China) in an NH4 F-ethylene glycol solution and the Ga-TNTs was fabricated by solvent casting technique. The Ti sheet was used as substrate material with a dimension of 20 × 20 × 0.9 mm3 grounded with abrasive paper (from 400 to 2000-grit), cleaned ultrasonically in acetone, alcohol, and deionized water, and dried in nitrogen. The cleaned Ti sheets were anodized in the electrolyte contains 0.55 g of NH4 F, 5 mL of deionized water, and 95 mL of ethylene glycol for 1 h at room temperature (25 °C). The anodization potential was 60 V using a direct current (D.C.) power source with the positive terminal connected to Ti sheet and the negative terminal connected to graphite. The distance between the two electrodes was 2 cm. After anodization, the Ti sheets were thoroughly washed in distilled water, dried in air and annealed at 450 °C for 2 h in a tube furnace in air at a heating rate of 10 °C min−1 . The annealed TNTs samples were cut into 0.5 × 0.5 cm2 for the subsequent solvent casting process. 2 g of poly-DL-lactic acid (PDLLA) and 0.4 g of Ga(NO3 )3 (gallium nitrate) were weighed and dissolved in 20 mL of ethyl acetate, stirred

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and ultrasonically shake until the ingredients were thoroughly mixed. Afterward, the prepared annealed TNTs samples were fully immersed in Ga(NO3 )3 -PDLLA coating solution for 5 min at 37 °C and dried in air. The immersion was repeated 3 times for obtaining the uniform Ga-PDLLA coating. In this work, pure Ti sheets with a size of 0.5 × 0.5 cm2 were also coated by Ga-PDLLA composites for the reference after grounded and polished, cleaned ultrasonically in acetone, alcohol, and deionized water, and dried in nitrogen. The microstructure of the post-anodization surface and as-obtained coating were observed using scanning electron microscopy (SEM, QUANTA200, FEI, Holland) combined with Energy Dispersive Spectrometer (EDS, GENESIS, EDAX, USA). All the samples coating with 15 nm gold metal via plasma sputter (S150L-1, Cowaq Company, Germany) prior to imaging. In order to study the releasing behaviors of Ga ions with PDLLA on Ti-TNTs bone plate, rectangular samples of 5 mm × 5 mm × 0.9 mm3 were prepared. The surface of the sample was mechanically polished by emery papers with 320 up to 2000 grit, rinsing with ethanol and deionized water by ultrasonic cleaner and drying in air. Three samples per group were immersed in 50 mL PBS (phosphate buffered saline, GE Healthcare Life Sciences, USA) in a hermetic beaker and kept under thermostatic conditions in a water bath at 37±1 °C for 14 days. 10 mL solution was sucked from the beaker after the sample immersed for 1 day, 3 days, 7 days, 10 days and 14 days, respectively. At the same time, fresh PBS solution was added into the beaker to keep the total volume (50 mL) constant. The Ga ions in sucked liquid samples were analyzed with Inductively Coupled Plasma Optical Emission Spectrometer (ICPOES) (Optima 700 DV, PerkinElmer, USA). 2.2. Preparation of mixed bacterial suspension and in vitro investigation The antibacterial activity of the sterilized samples was assessed with Staphylococcus aureus (S. aureus, ATCC25923) and Escherichia coli (E. coli, ATCC25922) because the S. aureus and E. coli are the most prevalent species that seriously induce a variety of clinical-acquired infections. For the bacteria incubation, 15 g of tryptone soy broth (TSB) was weighed and 500 mL of distilled water was added, stirred until the TSB was completely dissolved. Put the TSB solution into a microwave oven and heated it to boil and sterilized by high-pressure steam at 121 °C for 30 min. The prepared incubation TSB medium was put into a fridge and kept at 4 °C before use. S. aureus and E. coli in 12 mL TSB medium were cultivated overnight in a rotating shaker at 37 °C and kept for 150 r/min. The mixed bacteria solution with a concentration of 1 × 107 CFU/mL was prepared for the anti-bacterial biofilm test. All the experimental samples were immersed in 75% alcohol for 12 h for sterilization and dried in nitrogen. The sterilized sample sheets of 0.5 × 0.5 cm2 were cleaned with phosphate-buffered saline (PBS) and put on the wells of a 24-well plate. 2 mL of the mixed bacteria solution were spread on the surface and placed the 24-well plate containing bacteria into an incubator overnight at 37 °C. After co-cultured for 24 h, the materials were gently washed with 2.5 mL of PBS, added 3.5% pentanediol and 1% osmic acid solution, dehydrated with ethyl alcohol and placed into a fridge at −20 °C for the fixation. The microstructure of mixed bacteria biofilm was observed by using SEM and laser scanning confocal microscope (LSCM, FV1200, Olympus, Japan) after CO2 critical point drying and spray-gold process. 2.3. In vivo spinal infection model and biocompatibility Male SD rats were used in the present work. The work consisted of four groups (n = 15 per group) corresponding to four kinds of scaffolds, for example, Cp Ti (Group A), TNTs (Group B), Ga-TNTs (Group C) and Ga-Cp Ti (Group D). Surgical procedures are shown in Supplementary Fig. S1. For anesthetization, the experimental rats were injected intraperitoneally with 3% sodium pentobarbital 1–1.5 mL/kg. After surface disinfection, a 2 cm incision was made between L4 and L6 according to the spinous process positioning. The two pieces of sterilized scaffolds

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Fig. 1. Representative sample characterization on structure and antibacterial biofilm in vitro: (A) SEM image of TiO2 nanotube layer showing the corresponding enlarged and cross-sectional images; (B) optical images of Ga-PDLLA, Cp Ti, Ga-Cp Ti, TNTs and Ga-TNTs; (C) SEM image of Ga-TNTs coating fabricated by using solvent casting; (D) in vitro investigation of antibacterial biofilm (E. coli and S. aureus) on TNTs surface.

from the same group are inserted into both sides of the spinous process, and 50 μL of S. aureus-E. coli mixed bacteria (1 × 107 CFU/mL) was also instilled on both sides of each sample, respectively. The incision was closed by using nylon sutures followed by the application of a topical antiseptic. Each rat was then caged individually for 2 weeks and monitored for local reactions such as erythema, inflammation, mobility, water, and food intake and body weight at 1st, 3rd, 7th, and 14th day after surgery. For histological analysis, antibacterial and osseointegration assessment, fixed scaffolds with the surrounding tissue were cleaned with normal saline (NS) after sacrificing the animals. LSCM and SEM were used to observe the adhesion of bacteria on the scaffolds and the formation of bacterial biofilm. HE staining of the tissue sections was performed as per standard protocol and histological examination was using an optical microscope (OM, DM4000B, Leica, German). The surgical protocol followed NIH guide-lines for the care and use of laboratory animals and was approved by animal care and use committee in the first affiliated hospital of Kunming medical university (Kunming, Yunnan, China) 3. Results 3.1. Sample and bacteria biofilm characterization Fig. 1(A) shows in the SEM image a typical of a TiO2 nanotube layer formed on the Cp Ti (Pure Titanium) substrates. The SEM morphology and the corresponded EDS mapping image of Cp Ti sheet were shown in (Supplementary Fig. S2). Laterally spaced and vertically aligned morphology shows a well-ordered tube array with an outer diameter of 146.72±13.14 nm, and nanotube length of 7.12±0.13 μm. The optical images of the Ga-PDLLA coating and Ga-TNTs sample are shown in Fig. 1(B). Cp Ti, Ga-Cp Ti and TNTs are also presented as a comparison. The color of the TNTs sample shows dark yellow. After the clear Ga-PDLLA layer was attached on the surface of the Ti-base plates, it would not change the color of the samples obviously. The rough

and relatively uniform layer with a certain thickness was observed on the TNTs sample, and the porous nanoarray was also totally covered (as is shown in the SEM images, Fig. 1(C)). Besides, EDS mapping scanning image of Ga-PDLLA coating surface was shown in Supplementary Fig. S3. It is found that Ga ions were distributed uniformly with PDLLA gel. In fact, rough structure, increased surface area and the overall effect of the nano-characteristics could provide favorable conditions for protein adsorption, mineral deposition and cell adhesion [46]. According to some researches [36,47], they demonstrated that the nano TiO2 surface may be more useful for reducing the adhesion of bacteria. For reference, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were co-cultured in vitro on the TNT samples (146.72±13.14 nm of outer diameter and 7.12±0.13 μm of length) in this work (Fig. 1(D)). It was found that bacterial biofilm was formed on the surface of the TNTs sample during co-cultured. When the coculture time was 24 h, rod-shaped morphology with the length of about 1.64 μm could be observed, as obtained by SEM (Supplementary Fig. S4). The dead number of two bacteria species increased with the coculture time increasing from 48 to 72 h. The research from Sauer et al. [48] demonstrated that the mixed bacterial biofilm with different species exists competitive viability. In this work, we observed that the number of E. coli on the surface of the TNT sample increased at the initial 16–18 h of co-cultured if compared to S. aureus. It is inferred that E. coli has a shorter reproduction time and dominates in forming the bacteria biofilm. 3.2. In vivo implantation and pathology assessments We examined the effect of gallium nitrate doped TNTs composites (Ga-TNTs, Group C) on E. coli and S. aureus biofilm by implanting in the rat spine. For reference, Cp Ti (Group A), TNTs (Group B) and gallium nitrate doped Cp Ti (Ga-Cp Ti, Group D) were also studied in vivo by using the same animal model. After 1 day of surgery, all the experimental

J. Dong, D. Fang and L. Zhang et al.

animals survived and no secreta were observed out either from surgical incision or mouth, nose and urethra. The body weight of all the animals decreased slightly every week during the experimental period because of the inflammation from the mixed bacteria biofilm, and there was no significant difference between the body weights of the four groups (data is collected in Supplementary Fig. S5). On the first day of surgery, decreased ability to move and feed autonomously was observed in all the experimental animals, and there was little difference in the moving ability between the four group experimental animals. On the three days after surgery, the moving and autonomous feeding ability of all the experimental animals recovered, but the animals in group C shows better moving recovery ability. Only the experimental animals in group C can climb over the cardboard box with 18 cm height, while there was no difference between the animals in the rest of the three groups. From 7

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days after surgery, all the experimental animals in four groups have recovered back and exhibit a similar moving and feeding autonomously ability. Inflammatory response of E. coli and S. aureus biofilm together with implants was estimated by visual observations after sacrificing the animals at a different time point (see Fig. 2(A)). During the first three days after surgery, inflammation signs such as redness and swelling formed at or around the incision site. The experimental animals in group A have the largest infection area if compared to that of the rest three groups (Fig. 2(B)). At days 7, it was found that dot-shaped scar formed on the skin surface around the surgical incision sites, and the experimental animals in group C have the smallest infection area (Fig. 2(C)). After the two weeks of surgery, the incision scar together with pus was formed on the experimental animals in group A. The experimental animals in

Fig. 2. Inflammatory response of mixed bacteria together with implants after sacrificing the animals: (A) surgery incision infection by visual observations; (B) incisional infection area at 3 days after the surgery; (C) incisional infection area at 7 days after the surgery; (D) incisional infection area at 14 days after the surgery; ∗ Compared to the control group: ∗ indicates P < 0.05; ∗ ∗ indicates P < 0.005.

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Fig. 3. Histological interaction stained using H&E on the effect of E. coli and S. aureus biofilm together with scaffolds at different implantation time.

group C and group D have slighter infection response than the animals in group A and B (Fig. 2(D)). To further elucidate the effect of E. coli and S. aureus biofilm together with implants on the histological interaction, the implantation tissue were stained using H&E (Hematoxylin–Eosin) (Fig. 3). On the 3 days after surgery, a large number of inflammatory cells infiltration together with varying degrees of necrosis of muscle cells were observed in the striated muscle histology of all the experimental animals in the four groups. Visual observation of implantation sites was supported with histological analysis (Supplementary Fig. S6). On days 3, inflammatory signs such as redness, swelling and pus formation around the local muscle and soft tissue were observed in the four groups of experimental animals. The experimental rats in group A and B showed heavier necrosis of soft tissues with dark red than those in the group C and D. On the 7 days after surgery, necrosis tissues with pus formation turned from dark red into dark yellow, which was observed in the experimental animals of group A and B. In the histology stained using H&E, the experimental rats in group A and B still present a mass of in-

flammatory cells infiltration, while granulation tissue, a small number of capillaries and normal striated muscle tissues have been observed on those in the group C and D. On days 14, the experimental rats in group A showed the most severe tissue necrosis. Granulation tissue was dominated in the rats of group C and D, and collagen micro-formation was seen in group C. In group B, the inflammatory decreased slightly while dominated as chronic, and the granulation tissues began to proliferate. 3.3. Anti-bacteria biofilm and osseointegration assessments To investigated the antibacterial effect of gallium nitrate doped into TNTs, at each cultivation time point, all the experimental animals from each group after sacrificing were examined for the viability of S. aureus and E. coli bacteria biofilm using live (green)-dead (red) staining and scanning laser confocal microscopy (Fig. 4). On the three days after surgery, cultivated Cp Ti scaffolds (Group A) showed a large

Fig. 4. Viability of S. aureus and E. coli bacteria biofilm on the implants after different days of surgery.

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Fig. 5. The SEM morphology of the mixed bacteria and soft tissues growing on the surface of scaffolds after implanted for 14 days from sacrificing experimental animals: (A) Cp Ti; (B) TNTs; (C) Ga-TNTs and (D) Ga-Cp Ti samples.

concentration of bacteria which proliferate nicely and cover the sample surface. However, the situation is different for the mixed bacteria in group B (TNTs), group C (Ga-TNTs) and D (Ga-Cp Ti). The density of fluorescent signal (live bacteria) is less weak than the samples in group A. After 7 days, the viability of S. aureus and E. coli bacteria reduced and the number of dead bacteria increased on the scaffolds in the four groups. When the cultivation time is prolonged to 14 days, scattered live bacteria were seen on the samples in group C and D. The group C samples maintained a low concentration of live bacteria during the cultivation, which provides a clue that the implanted gallium nitrate doped into TNTs elicit strong anti-bacterial efficacy against E. coli and S. aureus. Fig. 5 shows the morphology of the mixed bacteria and soft tissues growing on the surface of Cp Ti (Fig. 5(A)), TNTs (Fig. 5(B)), Ga-TNTs (Fig. 5(C)) and Ga-Cp Ti samples (Fig. 5(D)) respectively after implanted for 14 days from sacrificing experimental animals. Although some of the soft tissues peeled off when the scaffolds take out from the surgery, SEM images present stretched cells with pseudopodia-like features together with mixed bacteria attached on the surface of all the samples in the four groups. The stretched cells with pseudopodia-like features demonstrated good activity as well as proliferation states. From Fig. 5(B) and (C), cells were prone to aggregate, and much higher cell density generated. Plenty of cells covered on the nano-tubes surfaces were gathered together, resulting in cellular outline to be ambiguous. The positive results demonstrated that TNTs and Ga-TNTs samples possess improved bioactivity mainly due to their higher surface wettability from the nanoscale-featured appearance. 4. Discussion TNTs implants are favorable for the anti-bacterial properties, even some researches still remained different and conflicting results when using different bacterial species under different experiment [36,49,50]. S. aureus and E. coli are among the most prevalent species of gram-positive and gram-negative bacteria, respectively, that serious induce a variety of clinical-acquired infections. Herein, we cultured E. coli and S. aureus on the TNTs samples. It is found that mixed bacterial biofilm could form on the surface of the TNTs sample within 24 h co-culture time, and the number of bacteria decreased with the culturing time increasing. It is demonstrated that the bacteria biofilm can form on the surface of TNTs due to their nano-topography and nanoscale roughness, as the

similar mechanism that TNTs can provide the rough surface and topographic signals for cell adhesion. Meanwhile, TNTs provide a certain of inhibiting effect for the bacterial activity when prolongs the incubation time of E. coli together with S. aureus. Given this, using electrochemical anodization and solvent casting to integrate Ga ions with TNTs to make biomedical Ti or TNTs elicits anti-bacterial biofilm efficacy in vivo, while also exhibiting excellent bioactivity and osseointegration, which is the dominant objective in this work. It is worth noting, however, that when both E. coli and S. aureus exist in the physiological environment and attach the surface of implants, the mixed bacteria show a competitive viability relationship, that resulting in the reduced bio-activity of bacteria. To support this inference, we also co-cultured E. coli and S. aureus with the same scaffolds respectively as a comparison experiment (Supplementary Fig. S7). It is found that the viability of mixed bacteria higher than that of the single bacteria for 12 h incubation time. However, as the incubation time prolonged from 24 h, the viability activity of the mixed bacteria tended to decrease. At this point, in addition, Release rate values and Cumulative values released by Ga ion from the Ga-PDLLA coating on Ti-TNTs base as a function of immersion time in PBS were shown in (Supplementary Fig. S7). Overall, it is shown an initial burst of Ga ion release during the first 3 days (48 h) of immersion test followed by a gradual increase at a much lower rate. Due to the controlled biodegradability of PDLLA gel, a large amount of Ga ion releasing becomes the key factor of inhibiting the forming of the mixed bacteria biofilm. Some studies [51,52] have shown that bacteria have cooperative, antagonistic or neutral interaction. In this respect, the mixed bacteria biofilm (E. coli and S. aureus) on the TNTs samples seem to have a competitive survival interaction instead of a pure antagonistic and extinct. It is found that E. coli species gradually gained the viability advantages during the antagonistic incubation of the mixed bacteria. In the preparation process of introducing Ga ions into TNTs, polyDL-lactic acid (PDLLA) plays an important “bridge” role in integrating Ga ions with a well-ordered TNTs array by using solvent casting. PDLLA is a biodegradable and bioactive supermolecular acid, which has been accepted by FDA on the application of surgical suture, bone fixation implants, and drug release system. Verrier et al. [45] combined PDLLA with bio-glass to fabricate a composite film to provide the extracellular matrix for intervertebral disc annular cells. It is found that the PDLLA/Bioglass composite film stimuli and promote the production of the cellular

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matrix with a large number of sulfate glycosaminoglycans (sGAG) and collagen. In this work, Ga ions were doped into PDLLA bio-carrier and then become a composite coating integrate with TNTs by solvent casting technique. The work provides a strategy of chemically combine the bio-function with bionic structure in a modified bone fixation implant materials. Establishing an animal infection model for investigating the effect of Ga-doped TNTs against E. coli and S. aureus biofilm is one of the most dominate work in this study. In recent decades, bone infection is still one of the most serious complications of spinal surgery. The postoperative infection rates significantly increase by 2–8 times when using metal implants during surgery. Postoperative spinal infections can lead to severe inflammatory reactions, tissue necrosis, and bone absorption, resulting in increased mortality and medical costs. Ofluoglu et al. [53] reported an approach to building the infection model in the rat spine with a Titanium implant. In their work, 3 mm titanium microscrew was implanted in the thoracolumbar area (T10-L1) after laminar decortications along the spinous processes. 50 μL S. aureus solution with a different concentration of 1 × 102 CFU/mL, 1 × 103 CFU/mL and 1 × 106 CFU/mL were squirted on the decorticated lamina site. All experimental animals were sacrificed after 2 weeks. Poelstra et al. [54] studied a multiple-sites (vertebrae T13, L3, and L6) spinal implant infection model in rabbits. The middle vertebrae sites (L3) were used as sterile control sites and the outer sites (T13, L6) were challenged with different amounts of methicillin-resistant S. aureus. The animals were sacrificed after 7 days. Thereupon, according to the literature, we modified the approach to building the spinal infection model in SD rats. The implantation sites were selected in the thoracolumbar area (L4–L6). The skin and subcutaneous tissue were been laminar decortications along the spinous processes. CpTi, TNTs, Ga-TNTs, and GaCpTisamples were implanted between the erector spinae and the spinous processes of the experimental rat respectively. 50 μl mixed bacteria of E. coli and S. aureus solution with a concentration of 1 × 107 CFU/mL was squirted on both sides of the implantation plates. The experimental rats of different groups were sacrificed at the different postoperative time of 3 days, 7 days and 14 days respectively. Herein, the route of implantation between the erector spinae and the spinous processes simulated the spinal posterior approach surgery. It is worth mentioning that the implants loose were found at the preliminary in vivo trials. However, by carefully stripping the muscles to make the materials have sufficient implant space and the incision was tightly closing layer by layer, no implants loose were observed during the in vivo experiment. According to the in vitro and postoperative assessment, E. coli and S. aureus can obviously attach and form bacteria biofilm on TNTs. Compare to the Cp Ti and TNTs group, the experimental rats in Ga-doped TNTs implants have the lowest inflammation and smallest trauma area in the spinal infection model. As the reference, the experimental animals in GaCp Ti group have superior anti-inflammatory property than the group of Cp Ti and TNTs, while not better than the group of Ga-TNTs. When the Ga3+ ion provides from an external source to the live bacteria, it will combine with the transferring and lactoferrin, which inhibits the synthesis of DNA and protein, thereby disrupting the metabolism of bacteria. It is demonstrated that Ga ions play a more dominated role on reducing the activity of E. coli and S. aureus mixed bacteria biofilm, while TNTs and the rough nanoscale feature play a more important role on osteoblasts attachment and osseointegration. In conclusion, this work developed an effective and rapid protocol to build a dense Ga-TNTs coating for the development of the bacterial resistance and osseointegration of biomedical Ti bone plate materials. On the basis of the control experiment building, in vitro biological analyses, in vivo infection model implantation and animal postoperative evaluation produces bacteria death, prevents biofilm formation and reduces inflammation. The distinct antibacterial results and good compatibility with osteoblasts reveal the large potential of exploiting and modifying the multi-biofunction of implantable biomaterials.

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5. Conclusion A titania layer with an array of nanotube structures (146 nm in diameter, and 7 μm in length) was synthesized on titanium surfaces by electrochemical anodization. Under in vitro conditions, S. aureus and E. coli were found can attach and competitively survive on the titania nanotubes (TNTs) surface and form the mixed bacteria biofilm. To improve properties of titanium for orthopedic applications, this work explored that gallium (III) ions, which are known for the antiresorptive and antimicrobial properties, can be integrated with TNTs to significantly improve the anti-bacterial ability (S. aureus and E. coli) by using a PDLLA biodegradable polymer solvent casting method. By setting up the spinal infection animal model and designing the control experiment, in vivo implantation and the postoperative reaction of Ga-TNTs scaffold showed distinct anti-bacterial property and reduced inflammation. Declaration of interest The authors declare no competing financial interest. Acknowledgments This work was jointly supported by the National Natural Science Foundation of China: the effect of Escherichia coli density regulating factor QseC in biomaterials implantation (30872555) and Study on the effect and mechanism of staphylococcal epidermidis-agrC specific binding polypeptide in the infection of cardiothoracic biomaterials (81460278). We also would like acknowledge the support by China Scholarship Council (CSC) under the CSC Number 201708530250. All authors thank Yongmin Liu ([email protected]), Guohao Dai ([email protected]) and Sandra Shefelbine ([email protected]) from Northeastern University (Boston, MA, United States) for stimulating discussions. Supplementary material Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.mtla.2019.100209. References [1] A. Biesiekierski, J. Wang, M.A.-H. Gepreel, C. Wen, A new look at biomedical Ti-based shape memory alloys, Acta Biomater. 8 (5) (2012) 1661–1669. [2] J. Qiao, J. Fan, F. Yang, X. Shi, H. Yang, A. Lan, The corrosion behavior of Ti-based metallic glass matrix composites in the H2 SO4 solution, Metals 8 (1) (2018) 52. [3] L. Zhang, J. Tan, Z. Meng, Z. He, Y. Zhang, Y. Jiang, R. Zhou, Low elastic modulus Ti-Ag/Ti radial gradient porous composite with high strength and large plasticity prepared by spark plasma sintering, Mater. Sci. Eng.: A 688 (2017) 330–337. [4] Y. Zheng, B. Zhang, B. Wang, Y. Wang, L. Li, Q. Yang, L. Cui, Introduction of antibacterial function into biomedical TiNi shape memory alloy by the addition of element Ag, Acta Biomater. 7 (6) (2011) 2758–2767. [5] A. Besinis, S.D. Hadi, H. Le, C. Tredwin, R. Handy, Antibacterial activity and biofilm inhibition by surface modified titanium alloy medical implants following application of silver, titanium dioxide and hydroxyapatite nanocoatings, Nanotoxicology 11 (3) (2017) 327–338. [6] M. Freire, A. Devaraj, A. Young, J. Navarro, J. Downey, C. Chen, L. Bakaletz, H. Zadeh, S. Goodman, A bacterial‐biofilm‐induced oral osteolytic infection can be successfully treated by immuno‐targeting an extracellular nucleoid‐associated protein, Mol. Oral Microbiol. 32 (1) (2017) 74–88. [7] L. Vishwanat, R. Duong, K. Takimoto, L. Phillips, C.O. Espitia, A. Diogenes, S.B. Ruparel, D. Kolodrubetz, N.B. Ruparel, Effect of bacterial biofilm on the osteogenic differentiation of stem cells of apical papilla, J. Endod. 43 (6) (2017) 916–922. [8] Y. Yan, X. Zhang, Y. Huang, Q. Ding, X. Pang, Antibacterial and bioactivity of silver substituted hydroxyapatite/TiO2 nanotube composite coatings on titanium, Appl. Surf. Sci. 314 (2014) 348–357. [9] G. Wang, W. Jin, A.M. Qasim, A. Gao, X. Peng, W. Li, H. Feng, P.K. Chu, Antibacterial effects of titanium embedded with silver nanoparticles based on electron-transfer-induced reactive oxygen species, Biomaterials 124 (2017) 25–34. [10] A. Jastrzębska, K. Jastrzębski, W. Jakubowski, Can titanium anodization lead to the formation of antimicrobial surfaces? Acta Innov. 26 (2018) 21–27. [11] L. Zhang, Z.Y. He, J. Tan, M. Calin, K.G. Prashanth, B. Sarac, B. Völker, Y.H. Jiang, R. Zhou, J. Eckert, Designing a multifunctional Ti-2Cu-4Ca porous biomaterial with favorable mechanical properties and high bioactivity, J. Alloys Compd. 727 (2017) 338–345.

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