Antibacterial Behavior of Additively Manufactured Porous Titanium

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Antibacterial behavior of additively manufactured porous titanium with nanotubular surfaces releasing silver ions Saber Amin Yavari, Loek Loozen, Fernanda L. Paganelli, Sadra Bakhshandeh, Karel Lietaert, James de Groot, Ad C. Fluit, C.H. Edwin Boel, Jacqueline Alblas, Charles Vogely, Harrie Weinans, and Amir Abbas Zadpoor ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03152 • Publication Date (Web): 14 Jun 2016 Downloaded from http://pubs.acs.org on June 15, 2016

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ACS Applied Materials & Interfaces

Antibacterial behavior of additively manufactured porous titanium with nanotubular surfaces releasing silver ions S. Amin Yavari1,2*, L. Loozen1, F.L. Paganelli3, S. Bakhshandeh2, K. Lietaert4,5, J.A. Groot3, A.C. Fluit3, C.H.E. Boel3, J. Alblas1, H.C. Vogely1, H. Weinans1,2,6, A.A. Zadpoor2 1

Department of Orthopedics, University Medical Centre Utrecht, Utrecht, The Netherlands Department of Biomechanical Engineering, Delft University of Technology, Delft, The Netherlands 3 Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands 4 3D Systems - LayerWise NV, Leuven, Belgium 5 Department of Metallurgy and Materials Engineering, KU Leuven, Leuven, Belgium 6 Department of Rheumatology, University Medical Centre Utrecht, Utrecht, The Netherlands 2

*

Corresponding author, email: [email protected], tel: +31-88-7559025

ACS Paragon Plus Environment


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ABSTRACT Additive manufacturing (3D printing) has enabled fabrication of geometrically complex and fully interconnected porous biomaterials with huge surface areas that could be used for biofunctionalization to achieve multi-functional biomaterials. Covering the huge surface area of such porous titanium with nano-tubes has been already shown to result in improved bone regeneration performance and implant fixation. In this study, we loaded TiO2 nano-tubes with silver antimicrobial agents to equip them with an additional bio-functionality, i.e. antimicrobial behavior. An optimized anodizing protocol was used to create nano-tubes on the entire surface area of direct metal printed porous titanium scaffolds. The nano-tubes were then loaded by soaking them in three different concentrations (i.e. 0.02, 0.1, and 0.5 M) of AgNO3 solution. The antimicrobial behavior and cell viability of the developed biomaterials were assessed. As far as the early time points (i.e. up to 1 day) are concerned, the biomaterials were found to be extremely effective in preventing biofilm formation and decreasing the number of planktonic bacteria particularly for the middle and high concentrations of silver ions. Interestingly, nano-tubes not loaded with antimicrobial agents also showed significantly smaller numbers of adherent bacteria at day 1, which may be attributed to the bactericidal effect of high aspect ratio nano-topographies. The specimens with the highest concentrations of antimicrobial agents adversely affected cell viability at day 1, but this effect is expected to decrease or disappear in the following days as the rate of release of silver ions was observed to markedly decrease within the next few days. The antimicrobial effects of the biomaterials particularly the ones with the middle and high concentrations of anti-microbial agents continued until two weeks. The potency of the developed biomaterials in decreasing the number of planktonic bacteria and hindering the formation of biofilms make them promising candidates for combating peri-operative implant-associated infections. Keywords: Anti-bacterial surfaces/coatings, porous implants, additive manufacturing, multifunctional biomaterials, nano-topography. 2


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1. INTRODUCTION Additive manufacturing techniques have enabled fabrication of geometrically complex yet fully interconnected porous biomaterials based on rationally designed repeating unit cells that can vary spatially both in terms of dimensions and topology1-5. The freedom offered by additive manufacturing techniques to design and fabricate such topologically complex biomaterials has multiple functional consequences among which freedom in adjusting the static

4, 6

and fatigue

7-9

mechanical behavior of porous biomaterials is just one. Perhaps

equally important is the large inter-connected pore space that allows for bone ingrowth and drug delivery

12-13

10-11

and the huge surface area of such porous biomaterials that could be

used for bio-functionalization 14-16. The surface area of additively manufactured porous biomaterials is up to several orders of magnitude larger than their equivalent solid biomaterials. This is a major advantage when these biomaterials are used as bone substituting biomaterials and as a part of orthopaedic implants. That is because the huge surface area of porous biomaterials could be used for inducing two major bio-functionalities, namely enhanced bone regeneration performance and resistance against implant-associated infection. In a recent study

17

, we demonstrated that

covering the entire surface of porous titanium biomaterials with TiO2 nano-tubes could lead to up-regulation of osteogenic markers in vitro and significantly improved biomechanical performance in vivo. Other researchers have found similar effects not only for nano-tubular surfaces 18-19 but also for surfaces covered with other types of nano-topographical features 2021

.

In the current study, we take additively manufactured porous titanium biomaterials with nanotubular titanium oxide (TiO2) surfaces another step further and add a new functionality, i.e. resistance against implant-associated infection. Implant-associated infection is a major clinical problem that is notoriously difficult and expensive to treat, requires repeated surgeries

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and long-term treatment, and causes high morbidity

22-25

. Once the bacteria adhere to the

surface, they start to divide and protect itself against the immune system through formation of the so-called bio-films. Systemic antibiotic therapy is very inefficient in killing bacteria protected by bio-films. Due to such limitations, implants equipped with active release strategies have emerged as an alternative concept in treatment of implant-associated infections

26

. In this study, we used an active release strategy to locally release silver-based

antimicrobial agents from the nano-tubular surface created on additively manufactured porous titanium biomaterials, thereby combating biofilm formation and implant-associated infection. Silver-based anti-microbial agents are used in this study, because as non-specific bactericides, they target a wide range of bacteria and could as well work around the problem of multidrugresistant strains 27. It has been shown that the antibacterial effect of silver-based antimicrobial agents is exclusively due to the production of silver ions (and not the shape or size of the particles)

28

. In killing bacteria, silver ions primarily work through three major mechanisms

including membrane damage, production of reactive oxygen species (ROS), and causing cellular uptake of silver ions 29. The application of surfaces covered by nano-tubes is motivated by several reasons. First, as previously mentioned, nano-tubes have been shown to enhance the bone regeneration performance of porous titanium biomaterials 30-31. Second, nano-tube surfaces created on solid surfaces and containing anti-microbial agents have exhibited strong anti-microbial effects in a number of recent studies

32-34

. Third, a few recent studies have demonstrated the anti-

microbial effects of high aspect ratio nano-features

35-36

. As high aspect ratio nano-

topographical constructs, nano-tubes may contribute towards the anti-microbial effect of porous titanium biomaterials even without delivery of anti-microbial agents or even after the depletion of the source of anti-microbial agents. Finally, covering the surface of porous titanium biomaterials with hollow nano-tubes adds an additional layer of hierarchy to the

4


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already huge surface area. It is expected that the geometrical features of nano-tubes such as diameter and length determine the release profile of the antimicrobial agents. We first performed a preliminary parametric study to understand the effects of the geometrical parameters of the nano-tubular structure on the release profile and antimicrobial performance of additively manufactured porous titanium biomaterials. Based on the parametric study, a specific combination of nano-tube diameter and nano-tube length were chosen for in-depth analysis. The anti-microbial performance and cell culture response of additively manufactured porous titanium biomaterials with different concentrations of antimicrobial agents were then studied and compared with those of as-manufactured specimens. 2. MATERIALS AND METHODS 2.1. Materials and manufacturing Porous structures were manufactured by Direct Metal Printing (DMP) on a ProX™ DMP 320 machine (3D Systems, Belgium). The STL file was created in Magics (Materialise, Belgium) by filling a disc (Figure 1a) with dodecahedron unit cells (Figure 1b). The state of specimens after the manufacturing process is called the as-manufactured (AsM) condition. The nominal dimensions of the discs were as follows: diameter = 8 mm and height = 3 mm. Slicing and hatching was performed with DMP Explorer (3D systems, US). Pure Ti powder with spherical shape according to ASTM F67 was used. The production was performed under an inert gas atmosphere with oxygen concentration below 50 ppm. After production, the samples were removed from the Ti baseplate by wire electrical discharge machining. Loose powders in the porous structures were removed by ultrasonic cleaning in demineralized water. The relative density of 10 samples was calculated by measuring the actual dimensions of the samples. The actual height and diameter of the samples were respectively 3.15±0.03 mm and 8.13±0.02 mm, resulting in a relative density of 37.19±0.83%. The density of the solidified material from which the struts of the porous structure were made is desired to be as close as

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possible to 100% to ensure no pores exists in the bulk of the struts. The density of the bulk strut material was therefore measured by applying the Archimedes technique to 10 specimens. An OHAUS Pioneer PA214C balance with the density kit was used for that purpose. The strut density was found to be 98.78±0.63%. 2.2. Electrochemical surface treatment An electrochemical surface treatment technique, namely anodizing, was used to cover the surface of additively manufactured porous titanium biomaterials with nano-tube arrays. In that process, the porous disks and an inert platinum mesh were respectively the anode and cathode. A 60V/20A power supply (CPX400SP; Aim TTI) was used for applying the electric potential through copper wires. The separation distance of anode and cathode was ≈3 cm. A Teflon beaker (VWR) was used as the container of the entire setup in which cathode and anode were submerged in an electrolytic solution made from mixing 0.27 M NH4F (Sigma– Aldrich) water-based solution with glycerol (1,2,3-propanetriol) (Sigma–Aldrich) in a volume ratio of 16.7:83.3%37. It is very challenging to uniformly cover the entire surface of additively manufactured porous biomaterials (including the internal surfaces) with nano-tubular surfaces. We started from the values provided in the literature37 for solid titanium and performed an extensive parametric study. The surfaces resulting from the different set of parameters considered in the parametric study were carefully evaluated under SEM and three sets of parameters that resulted in uniform coverage of the internal and external surfaces of the porous structures were selected for a preliminary study. To study the effects of nano-tube diameter and length on the release profile and anti-microbial behavior, a preliminary study was performed in which three combinations of electrical potential and anodizing time were used: 20V, 3h (NT 203), 30V, 2h (NT 302) and 40V, 3h (NT 403). The specimens were washed after the anodizing process with ultra-pure water while being sonicated, and dried overnight at 40°C. Based on a

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preliminary study of the release profile and anti-bacterial behavior of different abovementioned groups, one combination of anodizing voltage and time was chosen for the final anti-bacterial evaluations conducted in the remainder of the study. The additively manufactured porous titanium biomaterials covered with nano-tubular surfaces were then soaked in AgNO3 (Sigma–Aldrich) solutions prepared with three different concentration, i.e. 0.02, 0.1, and 0.5 M) for 30 min. The specimens labeling (NT-0.02Ag, NT0.1Ag, and NT-0.5Ag) reflects the concentration of this solution. The specimens were then washed with ultra-pure water and dried. The process concluded with 30 min of UV irradiation. The above-mentioned process for loading the specimens with anti-microbial agents was performed two times. 2.3. Surface characterization A JEOL (JSM-6500F, Japan) scanning electron microscope (SEM) with energy-dispersive xray spectroscopy (EDS) was used for observing the surface of additively manufactured porous titanium biomaterials both after anodizing and after loading the nano-tubular surfaces with anti-microbial agents. 2.4. Ag+ release The release of silver ions from the specimens of different groups was measured using inductively coupled plasma mass spectroscopy (ICP-OES, Spectro Arcos). Towards that end, three specimens from each group were immersed in 25 ml of daily-refreshed ultra-pure water that was kept at 37±0.5°C. The water was sampled at specific time points (i.e. 1, 3, 5, 7, and 15 days) to measure the concentration of silver ions. The measurements were repeated three times for each time point. 2.5. Inhibition zone Trypticase soy agar (TSA) plates evenly cultured with 10 µL of Staphylococcus aureus (ATCC 6538) suspension (cultured overnight) and kept at 37°C for 24 h were used for

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measuring the inhibition zone of one specimen from every group. The details of the protocol for preparing the bacterial suspension are presented below. 2.6. Antibacterial assay One strain of Staphylococcus aureus (ATCC 6538) (Sigma–Aldrich) was used to assess the anti-microbial potential of the additively manufactured porous titanium biomaterials covered with nano-tubular surfaces and loaded with silver-based anti-microbial agents. A trypticase soy broth (TSB) medium supplemented with 1% glucose was used to culture the bacteria at 37°C for 18 h. After dilution to OD600 0.01, 8 mL of the bacteria suspension was used for incubating four specimens from each group (2 mL per specimen) at 37°C for 1 day. The number of planktonic (free living, non-adherent) was determined by serially diluted using the plate counting method

33

. To determine the number of adherent (biofilm) bacteria, first the

specimens were rinsed with phosphate buffered saline (PBS) three times to remove the nonadherent bacteria. Subsequently, every specimen was subjected to 30 seconds of vortexing followed by 15 minutes shaking while being immersed in 2 mL PBS. The number of bacteria in the resulting suspension was then determined using the same technique as in the case of planktonic bacteria. The same specimens were used for re-incubation before they were thoroughly cleaned and dried and subjected to a sterilization protocol. Using the abovedescribed procedure, the numbers of planktonic and adherent bacteria were determined also at days 7 and 14. Between two subsequent time points, specimens were kept in 3 mL of PBS that was refreshed on a daily basis. The number of planktonic and adherent bacteria could be compared with their reference values to determine how effective the specimens of different groups were in reducing the number of planktonic and adherent bacteria. The normalized number of planktonic bacteria, Rp, was calculated by subtracting the number of planktonic bacteria found in the incubation

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medium of different specimens from the average number of bacteria in the culture medium in which no specimen was present, divided by the latter number: ܴ‫ ݌‬ሺ%ሻ =

ሺ‫݈݋ݎݐ݊݋ܿ ݂݋ ܷܨܥ‬ሺ ‫ݐ݅ݓ‬ℎ‫݊݁݉݅ܿ݁݌ݏ ݐݑ݋‬ሻ − ‫ݏ݌ݑ݋ݎ݃ ݈ܽݐ݊݁݉݅ݎ݁݌ݔ݁ ݂݋ ܷܨܥ‬ሻ × 100 ‫݈݋ݎݐ݊݋ܿ ݂݋ ܷܨܥ‬ሺ ‫ݐ݅ݓ‬ℎ‫݊݁݉݅ܿ݁݌ݏ ݐݑ݋‬ሻ

The reference number in the case of adherent bacteria is the number of bacteria found on the surface of as-manufactured specimens. The normalized number of adherent bacteria, Ra, was calculated with respect to this reference number by subtracting the number of adherent bacteria found in the suspensions of the specimens from other group from the reference number and dividing the result by the reference number: ܴܽ =

ሺ‫݈݋ݎݐ݊݋ܿ ݂݋ ܷܨܥ‬ሺ‫ܯݏܣ‬ሻ − ‫ݏ݌ݑ݋ݎ݃ ݈ܽݐ݊݁݉݅ݎ݁݌ݔ݁ ݂݋ ܷܨܥ‬ ‫݈݋ݎݐ݊݋ܿ ݂݋ ܷܨܥ‬ሺ‫ܯݏܣ‬ሻ

2.7. Scanning electron Microscopy The specimens retrieved from the bacterial cultures were observed under SEM (JEOL JSM6500F, Japan). To prepare specimens for observation under SEM, they first went through fixation and dehydration processes. After washing the specimen with PBS three times, they were fixed by 2% glutaraldehyde at 4°C for 2h and underwent dehydration process. During the dehydration process, the specimens were immersed consecutively in 3 mL of the following solutions: 25% and 50% ethanol-PBS solutions, 75% and 90% ethanol-water solutions, 100% ethanol (repeated two times), 50% ethanol-hexamethyldisilazane, and 100% hexamethyldisilazane. The specimens were then allowed to dry overnight and sputtering of gold with a thickness 1.7 nm was applied on the specimen surfaces. 2.8. Cell culture and live/dead assay Human MSCs (hMSCs) were isolated from the bone marrow of patients, who had given written consent with approval of the local medical ethical committee (UMC Utrecht). The procedure involved aspiration from the iliac crest of patients and collecting the samples in heparin-coated tubes. MSCs were isolated by adherence to tissue culture plastic and cultured in an expansion medium consisting of αMEM and supplemented with 10% fetal calf serum 9



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(Cambrex, Charles City, IA, USA), 100 U/ml penicillin, 0.2 mM L-ascorbic acid-2-phosphate (AsAP) (Sigma) and 1 ng/ml bFGF (R&D Systems). A humidified incubator was used to keep the culture at 37˚C and 5% CO2. hMSCs were trypsinized and seeded on four specimens from each group (0.2×106 cells/specimen). To ensure homogenous dispersion, cells were repeatedly pipetted on the scaffolds after which scaffolds were incubated in the expansion medium. After 24 hours, the presence of hMSCs on the specimens was assessed using a live/dead assay (Invitrogen, L3224, USA). The living cells attached to the scaffolds were visualized using a fluorescence microscope (E600, Nikon). For observation under SEM, all specimens were rinsed with PBS after one day of culture and were fixed with 2.5% glutaraldehyde, dehydrated in graded ethanol series, freeze-dried, sputter-coated with thin gold layers, and observed by SEM (JEOL JSM-6500F, Japan). 2.9. Statistical analysis Statistical analysis was performed using MATLAB (Mathworks, US) in which case one-way ANOVA together with Tukey–Kramer post-hoc analysis was used to compare different groups at different time points. A significance threshold of 0.05 was used. 3. RESULTS The mean diameter of the nano-tubes varied between 68 and 113 nm depending on the anodizing parameters, while the mean length of the nano-tubes varied between 370 and 646 nm (Table 1, Figure 2). The internal surface of additively manufactured porous titanium biomaterials was covered with uniform nano-tube arrays (Figure 2). The standard deviations of the diameter and length of the nano-tubes were small as compared to the mean diameter and length values and were respectively in the ranges 8-16 nm and 11-26 nm (Table 1). For all measurement points (i.e. 1, 3, and 5 days), the concentration of silver ions increased as the diameter and length of the nano-tubes increased (Figure 3a). All specimens exhibited a sizable inhibition zone and the specimen with the middle range of nano-tube diameter and length was

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chosen for further study with three different concentrations of silver-based anti-microbial agents (Figure 3). The internal walls of the nano-tubes present on the surface of additively manufactured porous titanium biomaterials were lined by a silver-containing phase (Figure 4). Nano-tubular surfaces immersed in solutions with different concentrations of silver-based anti-microbial agents had a clearly different presence of the Ag phase on their surface with the weight percentage increasing from 3.8 to 14.9%, as the Ag concentration in the solution increased from 0.02 to 0.5 M (Figure 4). The amount of Ag+ released in the unit time was larger for samples loaded with higher concentrations of silver (Figure 5). The rate of release of silver ions was the highest within the first few days and slowed down in the later days (Figure 5). At day 1, the specimens covered with nano-tubular surfaces containing anti-microbial agents had significantly lower numbers of planktonic and adherent bacteria regardless of the concentration of silver (Figure 6a-b, 7). Even when the nano-tubular surfaces were not loaded with anti-microbial agents, there was significantly lower number of adherent bacteria at day 1 as compared to as-manufactured specimens (Figure 6b, 7). At day 7, the specimens with two highest silver concentrations had significantly lower number of planktonic and adherent bacteria (Figure 6a-b, 7). At day 14, the specimens with the highest silver concentration exhibited significantly smaller numbers of planktonic bacteria (Figure 6a, 7). The normalized numbers of planktonic and adherent bacteria showed that the efficacy of the specimens with two highest concentrations is very close to 100% at day 1 (Figure 6c-d). As far as the adherent bacteria are concerned, the specimens with the lowest silver concentration and the ones without any silver-based anti-microbial agents exhibit efficacies close to 80% (Figure 6d). A relatively large number of bacteria were found on the surface of as-manufactured specimens as well as on the surface of specimens with nano-tubes but no silver (NT group) (Figure 8). Very few bacteria were found on the surface of the specimens with the low and

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middle silver concentrations (NT-0.02Ag and NT-0.1Ag) (Figure 8). Virtually no bacteria were found on the surface of the specimens with the highest silver concentration (NT-0.5Ag) (Figure 8). The specimens with lowest silver concentration (NT-0.02Ag) were not qualitatively different from nano-tube covered specimens (NT) in terms of the live cells revealed by the live-dead assay (Figure 9). The density of live cells was clearly lower for specimens with higher silver concentrations (NT-0.1Ag and NT-0.5Ag). Similar observations could be made for the cells observed under SEM: the density of cells attached to the specimens with the lowest silver concentration (NT-0.02Ag) did appear to be different from those attached to the nano-tube covered specimens (NT) (Figure 10). Cell density on the specimens with the higher silver concentrations (NT-0.1Ag and NT-0.5Ag) was clearly lower than those on the specimens covered with nano-tubes only (NT) (Figure 10). 4. DISCUSSION Additively manufactured porous titanium biomaterials covered with nano-tubular surface containing silver-based anti-microbial agents were developed in this study. The anti-microbial performance and cell viability on the surface of the developed biomaterials were assessed in vitro. The results clearly showed the strong anti-microbial behavior of the developed biomaterials particularly for the early time points and higher concentrations of silver-based anti-microbial agents indicating a clear concentration-dependent antimicrobial behavior. The strong early time-point anti-microbial effect held both for planktonic bacteria and adherent bacteria: even the lowest concentration of silver-based anti-microbial agents resulted in significantly lower number of both planktonic and adherent bacteria at day 1, while the two higher concentrations resulted in almost complete prevention of bio-films formation and resulted in negligible number of planktonic bacteria as compared to the control group. The highest concentration of silver-based anti-microbial agents, however, adversely affected the viability of hMSCs.

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Implant-associated infection as it relates to orthopaedic implants can be categorized into three categories: peri-operatively, hematogenously, and contiguously

24

. Early implant-associated

infection occurs peri-operatively, manifests itself up to 3 months after surgery, and is usually caused by relatively virulent microorganisms such as Staphylococcus aureus formation of biofilm occurs soon after surgery

24

. Indeed,

22

. Once the biofilm is formed, combating

infection will be extremely difficult 38-40. Some studies have shown that the antibiotic dosage required for killing bacteria protected by a biofilm is up to 1000 times higher than the dosage required for killing the same strain in a suspension

38-40

. It is therefore not realistic to expect

systematically administer antibiotics to reach the local concentration level required for killing the bacteria protected by a biofilm. Even locally delivered antibiotics will not reach the required concentrations. The most effective approach to minimize the risk of per-operative infection may therefore be to try preventing the formation of biofilms within the first few hours to first days after surgery. The additively manufactured porous titanium biomaterials developed in the current study are good candidates for that approach, because they could be tuned to release any dosage of nonspecific silver-based anti-microbial agents from a huge surface area within the first few hours to first few days after surgery. The results of the anti-microbial assay clearly show the potency of the developed biomaterials in preventing the formation of biofilms as well as killing planktonic bacteria within the first day after surgery (Figure 6d). The results of the present study show that high concentrations of silver-based anti-microbial agents could cause certain levels of cytotoxicity within the first day after cell culture. Further research is required to determine short-term and long-term cytotoxicity of the developed biomaterials particularly in vivo. Moreover, future research using bacteria and cell co-cultures similar to the ones reported in some recent studies41-42 are recommended for a more thorough (micro-) biological evaluation of the additively manufactured porous biomaterials developed in the current study.

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It can be envisaged that the nano-tubular surfaces could be loaded with antibiotics or antimicrobial peptides instead of silver-based anti-microbial agents to circumvent the cytotoxic effects even within the first few days after surgery. However, a non-specific anti-microbial agent such as those based on silver nano-particles may be more effective in combating a wide spectrum of virulent microorganisms, including the strains showing multidrug-resistance. Even though the concentration of silver ions is locally high during the first few hours to first few days post-implantation, the systemic concentration remains quite low. To put the numbers in perspective, the amount of silver released from the specimens with the middle silver concentration (NT-0.1Ag) in the first day is within the range of oral silver intake of an average adult person per day (i.e. 20-80 µg/day

43

). The amount released rapidly decreases

during the next few days and reaches levels that are well below the oral intake level even for the specimens with the highest silver concentration. Interestingly, additively manufactured porous titanium biomaterials covered with nanotubular surfaces had significantly smaller number of adherent bacteria at day 1 even when they were not loaded with silver-based anti-microbial agents. This suggests that nano-tubes alone could be helpful in preventing the formation of biofilms within the first few hours to the first day post-implantation. This is in line with the findings of a few recent studies that have demonstrated the bactericidal properties of high aspect ratio nano-topographies

35-36, 44

. The

effect of nano-tubes in decreasing the number of adherent bacteria vanishes at days 7 and 14, presumably because of coverage and leveling of high aspect ratio nano-topographies by (cell and bacteria) residues. In addition to working as a reservoir for storing and ultimately releasing anti-microbial agents, nanotubes facilitate the release of silver ions. Covering nano-tubular surfaces with nano-tubes adds another dimension (i.e. the depth of the nanotube) to the already huge surface area of additively manufactured porous titanium biomaterials. Since the internal surfaces of the nano-

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tubes were covered by the silver-containing phase (Figure 4), this additional dimension drastically increases the chance of silver oxidation and, subsequently, production of silver ions. Given the fact that the anti-microbial effects of silver are shown to be exclusively related to concentration of silver ions (and not the size and shape of silver particles) 28, facilitating the production of silver ions could result in strong anti-bacterial effect. The biomaterials developed in the current study can be considered as a part of a larger plan for preventing implant-associated infections. Even if the proposed biomaterials are fully effective in preventing peri-operative infections, additional strategies are needed for combating hematogenous and contiguous infections. Additional local release mechanisms may therefore be required for long-term release of anti-microbial agents that either have to occur at smaller concentrations to prevent cytotoxicity and ensure sustainability or have to be linked to a triggering mechanism that enables on-demand delivery of the anti-microbial agents. Equipping additively manufactured porous titanium biomaterials with the proposed antimicrobial surfaces may offer an additional advantage. Despite the fact that these materials have been developed recently, they have already made their way to actual clinical applications, see e.g. 45-46. As previously mentioned, huge surface area is one of the features of this type of biomaterials. A large part of this surface is the internal surfaces within pores that cannot be easily reached from outside, for example, by UV light. The large surface area together with unreachability of a significant portion of this surface area makes the sterilization process much more challenging than the case of solid implants where the surface area is limited and can be easily reached from outside. The nano-tubular surfaces containing anti-microbial agents may therefore be particularly useful for prevention of pre- and peri-operative contaminations in this kind of biomaterials and implants. It should be, however, noted that addition of silver changes the nanotopographical features of the surface of the additively manufactured porous biomaterials. The effects of such changes in the nanotopographical

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features on the bone regeneration performance of the developed biomaterials needs to be studied in the future possibly using a bacteria-cell co-culture protocol. CONCLUSIONS Additively manufactured porous titanium biomaterials covered with nano-tubular surfaces and loaded with silver-based anti-microbial agents were developed and assessed in vitro using both anti-microbial and cell viability assays. The developed biomaterials were found to be extremely effective in killing planktonic bacteria and preventing biofilm formation at the early time points (up to 1 day). The developed biomaterials are therefore promising candidates for combating peri-operative implant-associated infections. However, for the groups with the highest silver concentrations cell viability was adversely affected. The rate of release of silver ions decreased during the next few days. The specimens covered with nanotubes showed significantly decreased number of adherent bacteria at day 1 even when they were not loaded with any anti-microbial agents. This shows the anti-bacterial effect of nanotubes as high aspect ratio nano-topographies. ACKNOWLEDGMENT This work was supported by the Topsector Life Science Health (LSH) program of the Dutch ministry of Education, and the Zimmer Biomet Group. The supports of Eline Kolken for drawing graphical abstract is also acknowledged.

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Table captions Table 1. The parameters used in the anodizing process and the dimensions of the resulting TiO2 nano-tubes. Figure captions Figure 1. Macrographs of the additively manufactured porous specimens (scale bar: 1 mm) (a) and an SEM image of its micro-architecture (b). Figure 2. SEM images of anodized porous titanium with the following parameters: 20 V, 3h (a and d), 30 V, 2 h (b and e), 40 V, 3 h (c and f) Figure 3. Silver ion release from different NT-0.5Ag specimens (non-cumulative) (a), diffusion inhibition zone measurement of different NT-0.5Ag specimens (b), and inhibition zone images of NT203-0.5Ag (c) NT302-0.5Ag (d) and NT403-0.5Ag (e) after 1 day incubation, (scale bar: 1 cm). Figure 4. SEM images and EDS map of anodized porous titanium after immersion in AgNO3, NT-0.02Ag (a and d) NT-0.1Ag (b and e) NT-0.5Ag (c and f). Figure 5. Cumulative Ag+ release profiles from NT-0.02Ag, NT-0.1Ag and NT-0.5Ag specimens. Figure 6. (Normalized) number of planktonic bacteria and its antibacterial rate (a and c) (normalized) number of adhered bacteria (biofilm) and its antibacterial rate (b and d) on different surfaces. Figure 7. Antibacterial performance of AsM (a and f), NT (b and g), NT-0.02Ag (c and h), NT-0.1Ag (d and i), and NT-0.5Ag (e and j) against Staphylococcus aureus after 7 days. The first row (a to e) corresponds to planktonic bacteria and the second row (f to j) to adherent bacteria. Figure 8. SEM images of bacteria on AsM (a and f), NT (b and g), NT-0.02Ag (c and h), NT0.1Ag (d and i) and NT-0.5Ag (e and j) after incubation for 1 day.

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Figure 9. Fluorescence images after live/dead staining of hMSCs cells cultured for 1 day on AsM (a), NT (b), NT-0.02Ag (c), NT-0.1Ag (d) and NT-0.5Ag (e) (scale bar: 200 µm). Figure 10. Morphology of cells and cell attachment on AsM (a, f, k), NT (b, g, l), NT-0.02Ag (c, h, m), NT-0.1Ag (d, i, n) and NT-0.5Ag (e, j, o) specimens after incubation for 1 day.

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Table 1 Samples

Applied voltage (V)

Time (h)

NT 203 NT 302 NT 403

20 30 40

3 2 3

23

Diameter of NT (nm; Mean±SD) 68.1±16.0 100.8±9.3 112.6±8.4


Length of NT (nm; Mean±SD) 369.6±16.7 501.6±26.2 645.6±11.4


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

Figure 1 a

b


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Figure 2 NT 203

NT 302

NT 403

a

b

c

d

e

f

25 Environment ACS Paragon Plus


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Figure 3

a

c

b

d


e

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Figure 4 NT-0.02Ag

NT-0.1Ag

a

b

NT-0.5Ag

c Ti

Ti

O

140

Element

Wt.%

Element

Wt.%

Ti Ag N O

58.45 3.86 1.07 36.62

Ti Ag N O

57.59 9.74 0.85 31.82

O N

N Ag

160

Ag

Ti

Element

Wt.%

Ti Ag N O

61.92 14.88 0.92 22.88

120 100

CPS/eV

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


80

O

60

Ti

Ag

N

40

Ti

Ti

20 0 0

d

e


1

2 3 Binding energy (keV)

f

4

5


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Figure 5


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Figure 6 a

b

c

d

29 Environment ACS Paragon Plus


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Figure 7 AsM

NT

NT-0.02 Ag

NT-0.1 Ag

NT-0.5 Ag

a

b

c

d

e

f

g

h

i

j


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Figure 8 ASM

NT

NT-0.02Ag

NT-0.1Ag

NT-0.5Ag

a

b

c

d

e

f

g

h

i

j



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Figure 9 AsM

NT

NT-0.02Ag

NT-0.1Ag

NT-0.5Ag

a

b

c

d

e

32 ACS Paragon Plus Environment

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Figure 10 AsM

NT

NT-0.02Ag

NT-0.1Ag

NT-0.5Ag

a

b

c

d

e

f

g

h

i

j

k

l

m

n

o

33 ACS Paragon Plus Environment


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

297x209mm (300 x 300 DPI)


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Antibacterial Behavior of Additively Manufactured Porous Titanium

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