Electroactive Mg2+-Hydroxyapatite Nanostructured Networks against

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Biological and Medical Applications of Materials and Interfaces

Electroactive Mg2+- Hydroxyapatite Nanostructured Networks against Drug - Resistance Bone Infection Strains Nancy C. Andrés, Juan Manuel Sieben, Monica Baldini, Carlos Hernan Rodriguez, Angela Famiglietti, and Paula V. Messina ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b06055 • Publication Date (Web): 25 May 2018 Downloaded from http://pubs.acs.org on May 26, 2018

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ACS Applied Materials & Interfaces

1

Electroactive Mg2+- Hydroxyapatite Nanostructured Networks against Drug - Resistance Bone Infection Strains Nancy C. Andrés *a, Juan M. Siebenb, Mónica Baldini c, Carlos H. Rodríguez d, Ángela Famiglietti d, Paula V. Messinaa (a) INQUISUR – CONICET, Department of Chemistry, Universidad Nacional del Sur, B8000CPB, Bahía Blanca, Argentina. (b) INIEC– CONICET, Department of Chemistry Engineering, Universidad Nacional del Sur, B8000CPB Bahía Blanca, Argentina. (c) Department of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur, B8000ICN, Bahía Blanca, Argentina. (d) Laboratory of Bacteriology, Department of Clinical Biochemistry, Hospital de Clínicas “José de San Martín”, Faculty of Pharmacy and Biochemistry, Universidad de Buenos Aires, C1113AAD CABA, Buenos Aires, Argentina. * Author to whom correspondence should be addressed. Tel.: +54 291 4595159. Fax: +54 291 4595160. Electronic mail: [email protected]

Abstract. Surface colonization competition between bacteria and host cells, is ones of the critical factors involved during tissue / implant integration. Current biomaterials are evaluated both for their ability to withstand favourable responses of host tissue cells and to resist bacterial contamination. In this work, the antibacterial ability of biocompatible Mg2+ - substituted nanostructured hydroxyapatite (HA) was investigated. The densities of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli strains was significantly decreased after culture in the presence of Mg-substituted HA materials in a direct correlation with Mg2+- Ca2+ switch in the HA lattice. It was notice that this diminution was accompanied with a minimal alteration of bacterial environments, therefore the Mg2+ - HA antibacterial effect was associated with the material surface topography and it electroactive behaviour. 2.23 Wt% Mg2+- HA samples exhibited the best antibacterial

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2 performance, it decreased two-folds the initial population of E. coli, P. aeruginosa and S. aureus at the intermediate concentration (50 mg / mL of broth). Our results reinforce the aptitude of Mg-HA nanostructured materials to be used in antibacterial coatings for implantable devices and/or medicinal materials to prevent bone infection and to promote wound healing.

Keywords: Magnesium substitution; hydroxyapatite; implant, infections, antibacterial effects

1. Introduction The high incidence of bacterial infection in orthopaedic implantable devices during reconstructive surgery is one of the biggest challenges that researchers must to confront. biochemical methods rely on oral biocide substances therapy, Bacterial resistance

6

5

1-4

Conventional

that are sometimes unsuccessful.

in addition to antibiotics unfavourable ratio of dosage efficiency and

chemical toxicity create extra complications that usually demand surgical revision

7

and even the

removal of the implant. 8 These facts have led to the progress of further attempts to treat bacterial diseases by stimulation of body’s natural defence system; to mention some examples, delivering biological molecules like cytokines , 9 antimicrobial peptides, 10-11 antibacterial nanoparticles, 12 and a combination of these lines.

13-14

Alternatively, biophysical approaches provide a promising tactic

to win the war against bacteria thanks to the advance of surface manipulation techniques. 15-17 It has been proved that surface features regulate cell / substrate interactions, modulates cell adhesion, proliferation and fate.

18-19

Recently, super-hydrophobic nano-pillar structures of cicada’s wings

were found to have antibacterial properties against Pseudomonas aeruginosa based solely on their surface physical assembly. A series of recent studies have demonstrated that a lot of useful information for drug development can be obtained by conducting various studies, either experimentally or theoretically. However, different targets would need different approaches .20-21 To study the drug targets or inhibitors, the molecular docking approach was used.

22-23

For predicting drug-target interactions in cellular


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3 networking systems,

24

the bioinformatics/system biology approach was adopted.

25-26

To identify

various posttranslational modification sites in proteins, DNA, and RNA so as to find novel strategy to treat cancers and other major diseases ,27 the general PseAAC approach approach and 28 PseKNC approach was adopted .29-30 To study multi-target drugs or classifying drugs according to the Anatomical Therapeutic Chemical system recommended by the

31-32

World Health Organization,

the multi-label theory and approach were used. In this study, we are

34

33

to the design novel

nanostructured surfaces with potential antibacterial capacity based on inorganic elements that have already been applied to regenerative medicine: metal nanoparticles,

35

salts, and metal oxides. 36 In

a previous work, we have successfully tailored the chemical composition, degree of crystallinity, hydrophilicity, and morphology of Mg2+- substituted hydroxyapatite (Mg-HA) nanostructured materials achieving an optimal response of osteoblast in vitro.

37

Mg2+ - Ca2+ substitution on

biogenic HA lattice plays a pivotal role in its natural performances associated with the formation, regulation and maintenance of calcified tissues; antibacterial activity.

39

38

additionally Mg0 oxidation products revealed

These characteristics suggest that Mg2+ - based bioceramics can provide

clinical utility as an orthopaedic implant both stimulating the osteogenic cells response and preventing their attendant infections. The goal of this study is to confirm the antimicrobial abilities of four Mg-HA materials which, in combination with their osteogenic characteristics,

37

makes

them suitable for their potential use in regenerative medicine. The present work was organized as follows, firstly the effect of Mg2+ - Ca2+ exchange on the HA topography, hydration, surface charge and electroactivity was evaluated. Following, these physicochemical characteristics were correlated with the antibacterial capacity of the tested materials. HA sample with 2.36 Wt% Mg2+ displayed specific electroactive properties that in addition to an accurate surface nanotopography acted against the survival of two drug-resistance strains responsible of habitual bone infections: Staphylococcus aureus and Pseudomonas aeruginosa.

40

The use of an ion-substituted bioceramic as antibiotics

offers multiple advantages compared to local delivery systems of pharms or biological molecules, i.e. reduced cost effective preparation, long-term storage permanence, stability against sterilization



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4 processes, rapid and sustained antibacterial effect, broad-spectrum activity, and in some cases, increased potency compared with traditional drugs.

41

The results obtained from this work lead

forward the design of nanostructured biomaterials exhibiting biophysical cues that simultaneously exert a positive osteogenic cellular stimulation and avoid bacterial contamination.

2. Materials and methods 2.1 Test materials Nanostructured Mg2+ - substituted apatites denoted as MgI-HA, MgII-HA, MgIII-HA and MgIVHA, containing 0.72; 1.23; 2.46 and 12.3 Wt% Mg2+ respectively, were prepared and characterized as previously described.

37

Smooth magnesium (Mg0) turnings (Carlo Erba, CAS N° 7439-95-4)

were used as control (C+). 2.2 Zeta potential (ζ) measurements A Malvern Zeta Sizer Nano (ZS90) with a He-Ne laser (λ = 633 nm) was used for Zeta potential (ζ) measurements. Malvern's software provides the zeta potential from electrophoretic mobilities (µE), using the 42 Henry equation. 2.3 Surface characterization The topography of each sample was analysed from SEM microphotographs.

37

The micrographs

were taken at two randomly chosen areas on each specimen (one in a central position and one at 1.5 mm from the outer edge). To evaluate the symmetry of the roughness profile about the mean line, the profile skewness (Rsk) and kurtosis (Rku) were computed:

R sk =

R sk =

1 Rq

3

∫

−∞

1 NR q

∞

3

43

y 3 p ( y )dy

(1)

 N 3  ∑ Yi   i =1 

(2)

where Yi is the height of the profile at i point of a total N number of points, and Rq is the root mean square roughness: 43


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5

Rq =

1 l 2 { y( x )} dx ∫ l 0

Rq =

1 n 2 ∑ yi n i =1

Rku =

1

Rku =

Rq

4

∫

−∞

1 NRq

∞

4

(3)

(4)

y 4 p ( y )dy

(5)

 N 4  ∑ Yi   i =1 

(6)

3D surface plots, (Rsk) and (Rku) parameters were obtained using the image visualization software Image J 1.34 s, NIH Image, USA 44 with an uncertainty of 5 %. 2.4 Near infrared spectroscopy (FTIR – NIR). A Nicolet iS50 FTIR - NIR spectrophotometer (Thermo Scientific, Waltham, MA, USA) associated to a diffuse reflectance accessory (DRA, also called an integrating sphere) was used. The spectra were obtained in air atmosphere and at room temperature (RT). In the present study the integrating sphere was operated in the reflectance mode in the region of 1000 – 2500 nm. A Gold NIR Diffuse Reflection Standard (99.9 % reflective) was used as a reference to calibrate the baseline. The powder samples were supported inside flat bottom glass vials to form pellets of 10 mm diameter and 5 mm thick for measurements. Integrated software was used to convert the spectra from reflectance to absorbance and to perform additional analysis such as peak resolution. 2.4 Cyclic voltammetry study. Electroactive behavior of Mg-HA materials was tested against L-Ascorbic acid redox probe (AA, A92902 Sigma - Aldrich). The concentration of AA for the determination of the electrochemical behavior of the Mg-HA materials was 10.0 mM in buffered solution. Phosphate buffer solution (PBS, pH = 7.2) was prepared by mixing 600 mL of a 0.25 M Na2HPO4 (106559 Merck) solution with 400 mL 0.25 M NaH2PO4 (106346 Merck). Conventional three-compartment glass cells were used to run the electrochemical experiments at RT with a Princeton Applied Research VersaSTAT 3



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6 potentiostat / galvanostat. The counter electrode was a Pt wire, while a saturated calomel electrode (SCE, +0.241 vs. NHE) served as reference electrode. All potentials mentioned in this work are referred to this electrode. The solution was thoroughly de-aerated by bubbling N2 gas for 30 min. After bubbling, an inert nitrogen atmosphere was maintained over the electrolyte during the experiments. Cyclic voltammograms (CV) were recorded at different scan rates, ranging from 10 to 500 mV s−1. All experiments were performed in triplicate to ensure reproducibility. Electrodes were prepared as follows: 20.0 mg of each Mg-HA sample was dispersed ultrasonically in 1 mL ethanol for 45 min, then 20 µL of the slurry was pipetted and spread on a mirror polished glassy carbon rod (GC, 3 mm diameter), followed by air-drying at room temperature. Afterwards, 10 µL of a Nafion/ethanol solution (0.05 Wt. %) was pipetted on the Mg-HA modified-GC electrode. The electrode was left to dry in air for 1 h prior to use. The semi-integral convolution of the voltammetric data was done using the eL-ChemViewer based freeware package. Deakin et al.

46

45

According to

the heterogeneous rate constant (k(E)) for quasi-reversible systems on the positive

sweep can be expressed as follows: () = /

(7)

() /( )

where i is the current at the time t, IL is the semi-integral diffusional limiting current, I(E) is the semiintegral current at an applied potential, n is the number of electrons in the rate-determining step, F is the Faraday constant, D is the diffusion coefficient and Eo’ is the formal potential of the twoelectron process. For electrochemically irreversible systems, as in the case of AA oxidation, the relationship for the forward wave in the voltammograms is:

%

()

ln() = ln + "# $

(8)

All calculations were made with the diffusion coefficient obtained from an electrochemistry handbook. 47 Furthermore, the scan rate dependence with the peak heights for the anodic wave was evaluated by the Randles-Ševčik equation:

48

&' = 2.687 - 100 1#2/ (3)/ 4

(9)


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7 In this expression, Ip is the peak current, A is the electroactive area, C is the concentration of the electroactive specie, n is the number of exchanged electrons, and v is the scan rate, respectively. 2.5 Bacterial viability quantification Antimicrobial effects of nanostructured Mg2+ - substituted apatites were performed against three bacterial strains which were chosen for representing a clinically relevant spectrum of organisms.

40

Gram-negative, Escherichia coli (E. coli, ATCC 25922) and Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853) and; Gram-positive, Staphylococcus aureus (S. aureus, ATCC 29213) bacteria which were stored at Clinics José de San Martín Hospital, Buenos Aires, Argentina were used in this study. Mg0 turnings were used as positive control (C+) due to it recognized antibacterial properties.

39

Nanostructured Mg2+ - substituted apatites and Mg0 turnings (C+) were size reduced

by mortar. The obtained powders were placed into 9.5 mL of Mueller Hinton broth (Sigma-Aldrich, CAS N° 70192) at a concentration set of 25, 50 and 100 mg / mL and they were sterilized in an autoclave at 121°C during 20 min. Then, the mixture was vigorously sonicated during 10 min to avoid aggregation, stirring for another 3 h at 220 rpm in a rotatory shaker. pH of the broth were measured prior to the bacteria addition using ION 510 Benchtop Meter (OAKTON Instruments, USA). 0.5 mL of a single bacteria dilution of 1 × 105 Colony Forming Units per mL (CFU mL-1, equivalent to 0.5 McFarland turbidity standards) was added to each material-broth mixture at 37°C; they were exposed to the materials for 60 min with continuous stirring at 220 rpm in a rotary shaker. After material contact time, 0.5 mL broth supernatants aliquots were taken and diluted as required accordingly to the method described by Miles et al.

49

From each of these serial dilutions,

0.5 mL was poured into sterile culture petri dishes containing Mueller Hinton agar (Sigma-Aldrich, CAS N° 70191). The plates were incubated for 24 h at 37 °C and then those containing between 30 and 300 colonies were counted and multiplied by the proper dilution value to attain the final bacteria number. Results are expressed as percentage of bacteria colony counts reduction, 5% = 5 − 58 ⁄5 × 100 where Ri and Rf are the initial and final bacterial amounts expressed in CFU mL-1.



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8

2.7 Statistical analysis All quantitative tests were carried out in triplicate, and then mean values with standard deviations (SD) were calculated. At each time point the distributions of CFU mL-1 were compared for each pair of groups using the least-significant difference method to protect against Type I error increase, 50

followed by pairwise Wilcoxon rank sums test for nonparametric data.

51

Statistical significance

was set at p < 0.05.

3. Results and discussion 3.1 Mg2+ - enriched HA surfaces. At low and moderate fluid rates, such as in an implant / physiological fluid interface and as in our experimental conditions, non-motile bacteria (S. aureus) adhere to surfaces; motile bacteria (E. coli and P. aeruginosa) attach regardless of fluid velocity. 55 In addition, the surfaces of our investigated Mg-HA materials released soluble compounds (Ca2+ and Mg2+ ions

23

) that stimulate bacteria, in

those conditions attraction may begin before bacteria come into direct contact with the surface.

52

Under those concepts, surface features like topography, hydration, surface charge and the presence of electroactive sites must modulate the bacterial adherence and their subsequent survival. These aspects are discussed below. 3.1.1 Roughness, charge and hydration The topography of each Mg-HA material surface is described by a series of peaks and valleys that can be represented using 3D surface plots and quantified by Rku and Rsk coefficients, figure 1. It was recognized that Rku and Rsk are particularly important to describe surface roughness of calcified tissues implants

53

because osteogenic cells’ adherence is sensitive to the stress concentrations

which come from the presence of sharp peaks at the implant / tissue interface; 54 likewise they can be considered against bacterial proliferation. 36 All materials presented positive values of Rku and Rsk coefficients indicating the existence of a spiky surface profile and a predominance of peaks over


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9 troughs, figure 1. However, such asymmetry was different in each material, and highly dependent of Mg2+ - Ca2+ substitution, figure 2a.

Figure 1, 3D surface profiles of Mg- substituted HA frameworks: (a) MgI-HA, (b) MgII-HA, (c) MgIII-HA, (d) MgIV-HA.

It was determined that differences of surface topography were a direct consequence of the morphological alteration of their ion-substituted nanoparticles. In a preceding investigation,

37

it

was demonstrated that Mg2+- Ca2+ replacement into the crystal unit-cell of HA associated to the



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10 accumulation of Mg2+ in mineral surface stabilized amorphous calcium phosphate (ACP) phase and delayed HA crystallization. Increased amount of superficial Mg2+ generated smaller, irregular and agglomerated HA crystals that molded HA nanoparticles of larger dimensions. In addition, higher Mg2+ concentrations activated an ion diffusion mechanism similar to those occurred in biomineral formation which caused a bicontinuous porous structure inside Mg2+ - HA nanorods. 37

Figure 2, (a) variation of roughness parameters, Rku and Rsk, and (b) zeta potential, ξ, as a function of Mg2+ amount on HA-materials. Statistical analysis was performed among different materials; pure HA was used as reference, *p < 0.05. Significant differences between the samples are indicated with brackets.

Figure 2a shows the variation of Rku and Rsk as a function of Mg2+ concentration. As we had predicted Mg2+ - Ca2+ exchange altered the material roughness, all Mg2+ - substituted HA surfaces exhibited Rku values superior than pure HA (Rku = 2.12

55

) while no statistically significant

differences related to Rsk values were detected. Mg2+ concentrations up to Wt% Mg2+ ≈ 2 exhibited a platykurtoic distribution, Rku < 3, where a fairly high peaks and low valleys surface existed. Greater Mg2+ concentrations, Wt% Mg2+ > 2, showed a predominance of high peaks surfaces, Rku > 3.


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11

Figure 3, FT-NIR adsorption bands of (a) LHBW, (b) IHBW and (c) HHBW chemisorbed on Mg2+-HA surfaces. (d) IHBW/LHBW and HHBW/IHBW maximum absorbance intensities ratio as a function of Mg2+ concentration.

Changes on surface roughness enhanced the wettability caused by the chemistry of the surface,

56

therefore rich Mg2+- HA surfaces favored superficial water adsorption by a dual effect: electrostatic interaction and surface roughness. Water is involved as a prime interfacial host in living environments, affecting protein adsorption capacity

57

and cellular adhesion

58

, so it must be

analyzed to interpret the bacterial behavior. As a consequence of the superior charge to size ratio, Mg2+ - Ca2+ substitution provoked an extra positive charge density at surface, figure 2b, which strongly hold water molecules chemisorbed on the acidic cationic sites.

37

Figure 3 shows the

characteristic FT-NIR adsorptions bands of bonded water molecules on Mg2+ - HA samples. Quantification of the amounts of each type of adsorbed water was performed by computing the ratio between maximum absorbance intensities of bands ascribed to high hydrogen bonded water



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12 (HHBW, ν = 4320 cm-1), intermediate hydrogen bonded water (IHBW, ν = 6950 - 7100 cm-1) and less hydrogen bonded water (LHBW, ν = 5150 - 5250 cm-1); results are summarized in figure 3d. Mg2+ exchange is energetically favored on Ca (1) position of apatite lattice, 23 where it was reported that an equally four-fold coordination among water molecules and phosphate oxygen atoms existed. 59

Therefore, water mobility dramatically decreased as the Mg2+- Ca2+ substitution increased, figure

3d. At maximum Mg2+ concentration, a compact and solid-like character of the interfacial water molecules was observed due to strongly electrostatic / hydrogen bonding forces among Mg2+ and PO4-3 ions. 3.1.2 Electroactive behaviour Electroactive performance of Mg-HA materials was tested against L-Ascorbic acid (AA) (also known as Vitamin C) redox probe. L-Ascorbic acid is an essential antioxidant and a cofactor associated with the regulation, development, and maintenance of several cell types in the body, including bone.

60

The activity of AA at living organisms depends on its redox skills, given by the

relations between ascorbic acid, semidehydroascorbic acid, and dehydroascorbic acid, so it is currently used to examine the biological redox status.

61

Figure 4a shows the cyclic voltammetry

curves obtained for the modified-GC electrodes at 50 mV s-1. The irreversible electrochemical oxidation of AA is characterized by the presence of a single oxidation peak at the potential region between 0.0 V and 0.8 V.


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13

Figure 4, (a) steady CV of the electrodes in 10 mM AA + pH 7.2 PBS solution at a scan rate of 50 mV s-1. The arrows indicate the scan direction. (b) Randles-Ševčik plot obtained from the CV data for MgII-HA electrode. Scan rates ranging from 10 and 500 mV s-1. (c) Semi-integral cyclic voltammogram (anodic branch) for the electrooxidation of AA at the Mg-HA modified-GC electrodes. (d) Variation of apparent heterogeneous rate constant, k(E), as a function of Mg2+ amount, E = 300 mV. 46

This anodic peak is associated with the irreversible transformation of Vitamin C in

dehydroascorbic acid via a multistep process that involves the transfer of two electrons and one proton at neutral pH. It was observed that the peak current increased with the Mg2+ content in the hydroxyapatite. Moreover, the anodic peak potential (Ep) occurred at 0.45 V for unsubstituted HA electrode and it is shifted gradually to less positive potentials with the increase of Mg2+ content. The peak potential located at 0.41, 0.36 and 0.34 V for MgI-HA, MgII-HA and MIII-HA, respectively. However, the peak potential for MgIV-HA sample was very similar to that of un-substituted HA electrode. Therefore, except for MIV-HA electrode, the presence of Mg2+ in the hydroxyapatite enhanced the electrochemical response of the modified GC electrodes towards the electrooxidation



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14 of L-ascorbic acid. Figure 4b shows the Randles-Ševčik plot for MII-HA; the plot of the peak current linearly varied with the square root of the scan rate over the entire range investigated, demonstrating that the irreversible oxidation of AA at the electrodes was controlled by a diffusion process. Similar plots were obtained for the other samples. The direct kinetic analysis of AA oxidation from voltammetric experiments is not possible because the reduction peak is absent in the reverse scan. However, the apparent heterogeneous rate constant, k(E), can be estimated from the semi-integral analysis of the anodic branch of the voltammograms. Figure 4c displays the semiintegral CV curves for the electrooxidation of AA at the different Mg-HA modified-GC electrodes. The rate determining step (rds) of the reaction could be determined by analyzing the slope of the semi-integral CV plot. The transference of a first electron in the rds corresponded to a 0.5F/RT slope, while the second electron transference in the rds is associated to a 1.5F/RT slope. The slopes determined from the Randles-Ševčik plots were between 0.34F/RT and 0.49F/RT indicating that the transference of the first electron is the rds, i.e. the formation of the ascorbyl radical anion.

62

The

calculated apparent heterogeneous rate constants for the electrooxidation of AA at the Mg-HA modified-GC electrodes were summarized in figure 4d. It could be noted that k(E) is strongly influenced by the presence of Mg2+ in the hydroxyapatite structure. The apparent rate constant for AA oxidation linearly increased with the hydroxyapatite Mg2+ content to reach a maximum value for the 2.5 Wt. % Mg2+ sample (MgIII-HA). Notwithstanding, the heterogeneous rate constants of the reaction were between 2 and 4 times higher in MgIV-HA electrode than in unsubstituted HA electrode.

3.2 Antibacterial and toxicity effect of Mg2+ - substituted HA powders In the preceding sections, we have demonstrated that Mg2+ – Ca2+ switch altered topography, hydration, charge and electroactivity of Mg-HA surfaces in a dose dependent manner. Following the antibacterial activity of Mg-HA nanostructured materials, i.e. their abilities to exert an action on at last one of the following points was analysed: (i) modification of bacterial micro-environment,


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15 (ii) disruption of their physicochemical adsorption capacity and (iii) interference of bacterial metabolic state.

Figure 5, antibacterial activity of different Mg2+-HA powders against (a) Staphylococcus aureus, (b) Escherichia coli and (c) Pseudomonas aeruginosa as a function of material concentration. Results are expressed as reduction percentage (R%); smooth magnesium (Mg0) turnings were used as positive antibacterial control (C+). Bacterial exposed time to materials: 1h.

Figure 5 shows proliferation reduction percentage of different bacteria after 24h of incubation in the presence of Mg-HA samples. All Mg-HA materials displayed a degree of antibacterial activity against all studied microorganisms. S. aureus and E. coli exhibited a similar reduction profile regardless of Gram-positive or Gram-negative status, bacillus or coccus shape and motility (presence of flagella). From 50 mg of material / mL broth, the antibacterial behaviour augmented linearly with ionic substitution up to 2.46 Wt% Mg2+ and then slightly decreased, figure 6. In contrast, the antibacterial capacity of the Mg-HA powders against P. aeruginosa evidenced an



Page 16 of 37

16 almost null power until 1.23 Wt% Mg2+ and then an abrupt bacterial reduction occurred since 2.46 Wt% Mg2+. High concentrations of unsubstituted and poor Mg2+ substituted HA materials (100 mg / mL of HA, MgI-HA and MgII-HA) were necessary to attain an antibacterial activity similar to C+ against P. aeruginosa, figure 6. On the other hand, higher Mg2+ substituted HA samples (Mg IIIHA and MgIV-HA) were efficient to attain an antibacterial effect similar to C+ for P. aeruginosa strain at all tested concentrations.

Figure 6, bacterial density against Mg2+ substitution on HA samples. The amount of microorganisms was estimate after 1 h of contact with 50 mg mL-1 of material. Initial inoculum: 1 × 105 CFU mL-1. The first effect to be analyzed was the influence of Mg2+ – HA samples on the microbial culture media. Designed to be biodegradable, materials could release ions affecting the physical and chemical properties of broth; these would potentially alter the optimum conditions of bacteria growth and proliferation. Both HA and Mg-substituted derivatives behave as insoluble salts under physiological conditions;

37

according to their solubility products, the released ionic concentration

are shown in figure 7. The release of OH- and PO4-3 ions slightly alkalized broth giving rise to pH ≈ 8, table 1.


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17

Figure 7, ionic concentration released after 4.3 h degradation of the different Mg-HA materials at pH = 7.4 and 37°C.

pH Material concentration (mg / mL broth) 0 25 50 100 7.4 ± 0.1 10.2 ± 0.1 11.4 ± 0.1 11.4 ± 0.1 8.1 ± 0.1 8.1 ± 0.1 8.1 ± 0.1 8.2 ± 0.1 8.2 ± 0.1 8.2 ± 0.1 8.1 ± 0.1 8.1 ± 0.1 8.1 ± 0.1 8.1 ± 0.1 8.1 ± 0.1 8.1 ± 0.1 8.1 ± 0.1 8.1 ± 0.1 8.1 ± 0.1

Broth + Mg0 shavings + HA + MgI-HA + MgII-HA + MgIII-HA + MgIV-HA

Table 1. Broth physicochemical properties against material concentration pH is an important parameter to consider in terms of bacterial proliferation; antibiotic therapy effectiveness,

63

49

also relates to

and skeletal defects healing. 64 The measured pH values of broth

in the presence of materials were similar to those experimented by bacteria in a typical wound: open wounds have slightly acidic to alkaline pH (6.5 to 8.5) while in the chronic wounds a pH range of about 7.2 to 8.9 exists.68 Therefore, there was not a significant variation of the environmental pH to affect the tested microorganism progresses. In fact, literature results demonstrated that a slightly alkaline wound environment promoted bacterial growth rising proteolytic activity, obstructing fibroblasts action and decreasing oxygen supply.

63-64

Degradation of materials also released 2.5 -

3.5 mM of Ca2+ and 1.8 - 2.4 mM of Mg2+ ions during bacteria culture; again these concentrations



Page 18 of 37

18 were within the range of physiological concentrations.

65

Organisms must maintain physiological

levels of Mg2+ and Ca2+ because these divalent ions are critical for the stabilization of membranes, ribosomes, nucleic acids, and a variety of enzymatic reactions.

66

Still lacking of toxicity, the ions

can exert an action on water availability by restraining it accessibility to organisms.

67

The amount

of ions released from the material was not enough to alter the bacteria culture media to prevent their progress. Therefore it cannot be associated to the experimental results summarized in figures 5 and 6, where a clear antibacterial effect was appreciated. The following point to consider is related to the surface properties of the material and its link with the adhesion of microorganisms. The surface energy of bacteria is typically smaller than the surface energy of the broth in which they are suspended; this mismatch causes that cells preferentially attach to lower surface energies materials (i.e. slightly hydrophobic surfaces). Water adsorption and the subsequent formation of hydration layers would be a cause of bacterial adsorption disruption. Accordingly to the water contact angles, θw, S. aureus cells are moderately hydrophobic (θw = 72° ± 8)

58

while E. coli cells and P.

aeruginosa are moderately hydrophilic (θw = 33.4 ± 4 and θw = 43.3° ± 8, respectively)

72

so the

adsorption of both type of strains on Mg-HA surfaces that are slightly hydrophilic, figure 3, would be favored than to be suspended in broth. Furthermore, E. coli, S. aureus and P. aeruginosa with the zeta potential value, ξ, of approximately -38.42, - 35.6 and -14.4 mV

68

are electronegative, so it

may be expected that some degree of electrostatic repulsion would occur among bacterial cells and the negatively charged materials surfaces, figure 2b.


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19

Figure 8, bacterial density variation as a function of (a) apparent electron transfer constant, k(E, 300 mV), and (b) roughness parameter, Rku. The amount of microorganisms was estimate after 1 h of contact with 50 mg mL-1 of each Mg-HA sample.

No direct effect was evidenced between the surface charge and surface water adsorption layers formation on the reduction of bacterial proliferation. Therefore, in terms of thermodynamics and electrostatic cues, no disruption of bacteria adhesion onto the Mg-HA surfaces was detected. Finally, we consider the capacity of Mg-HA materials to influence the redox mechanism by which electron-transport chain (ETC) enables prokaryote cells to generate the energy that they need to survive. Analysing the variation of electron apparent heterogeneous rate constant, k(E, 300 mV), and the reduction profile of S. aureus and E. coli strains as a function of Mg2+ concentration on Mg-HA materials, it could be observed that they display an entirely correlated tendency, figure 8a. There was a bacterial reduction with the electron transfer rate increases from HA to MgIII-HA materials



Page 20 of 37

20 following by a new augment in bacterial proliferation for the MIV-HA material where the effect on electron transfer declines. Literature findings revealed that it could be possible an extracellular electron transfer (EET) from S. aureus to an inorganic surface.

69

If the material was capable to

receive electrons, as in our case, it was observed that such process lead to bacteria death.

69

Agreeing to such mechanism, 69 the bacterial transferences took place without external requirements and microorganism did not die from reactive oxygen species production but from electron loss on the surface. Therefore, the bacterial annihilation was limited to its adsorption on the substrate surface. 73 This mechanism was reasonably adapted to our results at exception of P. aeruginosa; this could be due to its very versatile energetic metabolism.

70

The reduction of P. aeruginosa was

investigated as a function of surface topographic parameters; from analysis of figure 8b it can be inferred that predomination of high peaks on Mg-HA surfaces (Rku > 3) could be well correlated with a high reduction of P. aeruginosa viability. Nano-patterns are common on nature surfaces, a number of research groups have attempted to establish a direct link between the self-cleaning and the ability to prevent attachment and proliferation of microorganism.

34

Recently it was

demonstrated that self-cleaning surfaces are not necessary inherently antibiofouling in nature. 34 For example, P. aeruginosa are unrestricted adhered onto the surface of the wings of the Clanger cicada (Psaltoda claripennis) and as soon as they adhere, they are efficiently killed by the wing surface. 34 A physical mechanism of antibacterial effect was proposed, by which the pattern of the cicada wing surface may lead to a drastic increase of the bacterial total area adsorption, accompanied by a nonuniform stretching of the bacterial membrane, which may lead to the irreversible membrane rupture and death of the bacteria.

34

We hypothesize that a similar mechanism was operating in our

experimental conditions.


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21 Conclusion Antibacterial implantable materials must locally kill bacteria or slow down their growth, without being toxic to host tissue cells. Based on preliminary screening results, Mg2+ - HA materials improved the adhesion and proliferation of primary rat osteoblast (rOBs) and endothelial cell (rECs) in an ionic-exchange dependent manner.

37

Here we have demonstrated their antibiotic effect on

pathogen microorganism direct involved on implant infections: Staphylococcus aureus and Pseudomonas aeruginosa. These bactericide Mg-HA samples should be capable of inhibiting both Gram negative and Gram positive organisms, having a general local effect in the lowest dose. Incorporation of 2.23 Wt% of Mg2+ in the HA structure (MgIII-HA) decreased two-folds the initial number of E. coli, P. aeruginosa and S. aureus at the intermediate concentration (50 mg of material / mL of broth), granting antibacterial properties in addition to the promotion of new bone growth formation skills.

37

The optimal bactericide characteristics exhibited by MgIII-HA sample are a

consequence of a synergic effect between its surface roughness features (predomination of high peaks) and its electroactive response (ability to accept electrons). 70

User-friendly and publicly accessible web-servers represent the future direction for developing

practically more useful prediction methods and computational tools series of recent publications. impacts on medical science,

74

73

71-72

as is demonstrated in a

Actually, many practically useful web-servers have increasing

driving medicinal chemistry into an unprecedented revolution , we

shall make efforts in our future work to provide a web-server to display the findings that can be manipulated by users according to their need as those established to use the predictor called

75

“pLoc-mGneg" for predicting the subcellular localization of Gram-negative bacterial proteins with both single and multiple locations. This will give us a closer approach about how the nanomaterials are affecting the biological functions this organisms.

Conflict of interest The authors disclose no potential conflicts of interest.



Page 22 of 37

22 Acknowledgements. The authors acknowledge Universidad Nacional del Sur (PGI 24/Q064), Concejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina (CONICET, PIP – 11220130100100CO) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, PICT − 201-0126). We also thank Drs. Marcos Fernández-Leyes and Hernán Ritacco from IFISURCONICET for their assistance with ζ-potential measurements. NCA has postdoctoral fellowship of CONICET. JMS and PVM are researchers of CONICET.


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Broth + Mg0 shavings + HA + MgI-HA + MgII-HA + MgIII-HA + MgIV-HA

pH Material concentration (mg / mL broth) 0 25 50 100 7.4  0.1 10.2  0.1 11.4  0.1 11.4  0.1 8.1  0.1 8.1  0.1 8.1  0.1 8.2  0.1 8.2  0.1 8.2  0.1 8.1  0.1 8.1  0.1 8.1  0.1 8.1  0.1 8.1  0.1 8.1  0.1 8.1  0.1 8.1  0.1 8.1  0.1


Electroactive Mg2+-Hydroxyapatite Nanostructured Networks against

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