Inhibited Bacterial Biofilm Formation And Improved Osteogenic Activity On Gentamicin-Loaded Titania Nanotubes With Various Diameters

While Bing Yue.

We found that ‘NTG’ could significantly inhibit bacterial adhesion and biofilm formation compared to FlatTi or NT, and the ‘NT G’ with 160 nm and 200 nm diameters had stronger antibacterial activity extended because drug release time of ‘NTG’ with larger diameters. Our results showed that NT G and NT, especially those with 80 nm diameters, significantly promoted cell attachment, proliferation, spreading, and osteogenic differentiation when compared to FlatTi, and there was no significant difference between NTG and NT with identical diameter. That said, these nanotubes were loaded with 2 gentamicin mg using a lyophilization method and vacuum drying. Staphylococcus epidermidis, and two clinical isolates, aureus 376 and epidermidis 389, were selected to investigate the anti infective gentamicin ability loaded nanotubes. Human marrow derived mesenchymal stem cells were used to evaluate nanotubular effect pographies on the osteogenic differentiation of mesenchymal stem cells. Titania nanotubes loaded with antibiotics can deliver a high concentration of antibiotics locally at a specific site, thereby providing a promising strategy to prevent ‘implant associated’ infections. Make sure you scratch a few comments about it below. While nanotubes with 80 nm or 120 nm diameters had better effects, the NT also exhibited greater antibacterial ability than the FlatTi. In this study we have fabricated titania nanotubes with various diameters and 200 nm length via electrochemical anodization. Therefore. Nanotube modification and gentamicin loading can significantly improve the antibacterial ability and osteogenic activity of orthopedic implants. Considering the above said. Flat titanium and nanotubes without drug loading were also investigated and compared.

However, failure still occurs, Titanium and its alloys been widely applied for orthopedic implants because of their remarkable mechanical properties and biocompatibility.

Bacterial inhibition colonization and prevention of infection can also be beneficial to the osseointegration of implants. Infection and aseptic loosening remain two major complications for orthopedic implants. Needless to say, tissue cells will have difficulty adhering and proliferating on identical surface, once the bacteria have colonized and formed a biofilm on the implant. Remember, infection and aseptic loosening of implants usually occur because of poor integration with the surrounding bone tissue and bacterial contamination during the implant surgery. Lots of information can be found by going on the web. Whitehouse et al5 estimated that infections at orthopedic surgical sites prolong tal hospital stays by a median of 2 weeks per patient, approximately double rehospitalization rates, and increase health care costs by more than 300%. Make sure you drop some comments about it in the comment section. The competition for initial adhesion to the implant surface between bacteria and tissue cells begins immediately after the implant insertion. Also, in the United States, the most common causes of revision tal knee arthroplasty were infection and implant loosening in 20062 and the annual infection rate for orthopedic implants is 3percent.

Administration of perioperative antibiotic prophylaxis is a routine procedure in orthopedic surgery to prevent infection.

In this study, we further investigated and compared the antibacterial ability and osteogenic activity of gentamicin loaded and nonloaded nanotubes with four different diameters. The most effective method to resolve this problem is to deliver antibacterial agents locally from the implant surface. Popat et al1015 have loaded 80 nm diameter nanotubes with 200, 400, and 600 gentamicin μg and demonstrated that the gentamicin eluted from the nanotubes significantly reduced bacterial adhesion on their surfaces, and osteoblast differentiation was also enhanced on nanotubes filled with gentamicin. Eventually, gentamicin is an aminoglycoside antibiotic that is widely used to prevent implant related infections. They also demonstrated that 80 nm diameter nanotubes supported higher adhesion, proliferation, and osteogenic differentiation of marrow stromal cells compared to flat Ti surfaces. Titania nanotubes fabricated on Ti implants via electrochemical anodization have attracted increasing attention because of their good biocompatibility and chemical and mechanical properties. As a result, they demonstrated that the drug eluting Ti wires could reduce bone infection. Gulati et al16 have fabricated nanotube structures on a Ti surface wire and loaded the 170 nm diameter nanotubes with gentamicin. Systemic limitations administration such as systemic xicity may give rise to many complications. Local controlled drug delivery will maintain drugs optimal concentrations at the specific site over prolonged periods without high systemic levels. Nanotubes loaded with gentamicin can deliver high levels of antibiotics locally to inhibit bacterial adhesion on an implant without causing systemic xicity while maintaining excellent osseointegrative properties. Actually, nanotubes dimensions are controllable at a level that can mimic dimensions of the constituent the dimensions components of natural bone to enhance osteogenic activity. TNTs of properly controlled diameters and lengths can serve as carriers for antibacterial agents.

Staphylococcus aureus and epidermidis, gether account for two three out infection isolates. a standard strain, epidermidis were cultured on NT and NT G to evaluate nanotubular effect pographies on osteogenic cell functionality. Prior to the electrochemical anodization treatment, pure Ti discs of 995percent purity were sonicated with acetone to remove surface oil pollution. You see, flat Ti discs were used as a control in all the experiments. 40 V, 60 V, or 85 V, TNTs with length of 200 nm and diameters of 80, 120, 160, or 200 nm, respectively, were fabricated on the Ti discs, after anodization for 1 hour at a constant voltage of 25 V. The electrolyte consisted of 5percent weight NH4F and 10% volume distilled water in ethylene glycol. The anodization process was performed in a conventional two electrode cell. Oftentimes afterward, the specimens were cleaned with deionized water and dried. Both specimens sides were sterilized by ultraviolet irradiation before conducting the antibacterial and cell culture experiments. They were chemically polished in a mixed solution containing HNO3 and HF. Also, the TNTs surface morphologies with different diameters were studied using a scanning electron microscope. Pure Ti discs served as the anode electrode and stainless steel discs served as the cathode electrode. Anyway, all specimens were fabricated at room temperature.

Gentamicin was loaded into the nanotubes using a lyophilization method and vacuum drying. The surfaces were quickly rinsed by pipetting 1 PBS mL over the surface to remove any excess drug, right after the final drying step. Briefly, the TNT surfaces were cleaned with deionized water before loading. Consequently, the surfaces were then allowed to dry under vacuum at -45°C for 2 hours in freeze dry system. The rinse solutions were collected and stored for further analysis. This is the case. The loading step was repeated until the nanotubes were loaded with 2 gentamicin mg, after drying.

The clinical isolate aureus 376 and epidermidis 389 were kindly provided by Sad Jabbouri. The minimum inhibiting concentrations of gentamicin against ATCC 35984, aureus 376 and epidermidis 389 were determined by a microtiter broth dilution method as previously described. Our previous study demonstrated that the three tested strains were biofilm producing bacterial strains. The strains were propagated overnight on tryptone soy agar medium at 37° A sterile 10 μL loop was used to withdraw bacteria colonies from the TSA, which were then inoculated into 10 BBLTM mL TrypticaseTM soy broth and cultured for approximately 16 hours on a shaker at 250 rpm and 37° Cells were then harvested by centrifugation. These strains were stored at -80°C as glycerol stocks.

The specimens were incubated with bacterial suspensions of 1 × 106 CFUs/mL in TSB for 4, 24, and 48 hours. The discs were gently washed three times with PBS. The samples were subsequently freeze dried, sputter coated with gold, and observed using a SEM. Anyway, the hexamethyldisilazane was removed, and the surfaces were air dried for 30 minutes. The surfaces were fixed in 5% glutaraldehyde for 2 hours at 4°C, washed three times with cacodylate buffer, and dehydrated through a series of graded ethanol solutions for 10 minutes each. The surfaces were dried with hexamethyldisilazane for 10 minutes.

The bacterial adhesion and biofilm formation were also observed using CLSM. The specimens were removed at three different time points and were gently washed three times with PBS. In this study, we used ATCC 35984 for the CLSM and SEM observation assays. The viable and nonviable cells can be distinguished under the fluorescence microscope because viable bacteria with intact cell membranes appear fluorescent greenish, whereas nonviable bacteria with damaged membranes appear fluorescent redish. The samples were stained in a new 48well plate with 300 combination μL dye and analyzed with a CLSM. The images were acquired from random positions on the samples surfaces.

MSCs were cultured as described in our previous articles.

FlatTi, NT, and ‘NTG’ were placed in 48 well plates -25diphenyltetrazolium bromide solution was added to each well, and the plates were incubated at 37°C for 4 hours. The cells were observed using a fluorescence microscope, when the culture grew to approximately 80% confluence. Similarly, at each time point, the attached cells were fixed and stained with 4′,6′-diamidino2phenylindole. Usually, the supernatant was discarded and 1 dimethyl mL sulphoxide was added to the wells for 5 minutes to dissolve the formazan salts. The cells at passage 3 were used in the experiments. The mean absorbance obtained from the medium control well was determined from the test absorbance values. On p of this, in brief, cells were cultured in ‘α Modified’ Eagle’s Medium culture medium supplemented with 10percentage fetal bovine serum and 1% antibiotics. Basically, the plates were read at 570 nm using an automated plate reader. Actually, the cells were incubated at 37°C in a humidified atmosphere of 5percent CO2, and the growth medium was changed every 48 hours.

The hMSCs were seeded on the specimens placed in 24well plates at a density of 5 × 103 viable cells.

4, and 7 days, cell specimens proliferation was assessed using the MTT assay, after culturing for 1. The absorbance was measured at 570 nm. That’s right! In brief, at each time point, 100 MTT μL solution was added to each sample and incubated at 37°C for 4 hours. Another plate containing the α MEM culture medium and the specimens was used as a parallel control. The mean absorbance obtained from the medium control well was determined from the test absorbance values. The formazan was then dissolved using dimethyl sulphoxide.

NT and NTG on the osteogenic differentiation. 10, and 14 incubation days with the osteogenic induction medium in a 48well plate, the specimens were washed with PBS three times, and lysed in a 2% Triton X100 solution through four standard freeze thaw cycles, after 7. MEM culture medium was supplemented with 10% FBS, 1 μM dexamethasone, 50 μM ascorbate acid, and 10 mM βglycerophosphate sodium. Let me tell you something. The ALP activity in the lysis solution was determined through a colorimetric assay based on p nitrophenyl phosphate. Now let me tell you something. These media were renewed every 2 days throughout the study period. The medium was changed to the osteogenic induction medium, after culturing for 24 hours with the specimens. The ALP activity was determined following the procedures described in our previous article. The intracellular tal protein content was determined using a MicroBCA protein assay kit and the ALP activity was normalized to it. ALP staining was performed with a ALP staining kit on day 7 and day In brief, the cells were fixed with buffered formalin for 30 seconds, washed with distilled water two times, and then stained with a staining reagent for 45 minutes. Afterward, the specimens were washed three times with distilled water and then images were obtained using a scanner.

After 21 and 28 osteogenic days induction in 48well plates, the specimens were washed with PBS three times and fixed in 7% formaldehyde for 1 hour.


The results are expressed as mean ± standard deviation. The statistical analyses were performed using SPSS software version 131. All experiments were conducted in triplicate and repeated three times. The specimens were washed with distilled water until no more color appeared in the distilled water, and then images were obtained using a scanner. P005 was defined as significant and P001 was defined as highly significant, The one way analysis of variance and least significant difference post hoc tests were used to determine significance level. Although, they were then incubated with a 1% Alizarin Red solution for 45 minutes at room temperature.

Figure 1 shows SEM images of TNTs with different diameters. Figure 2A shows different loading efficiencies diameter NTThe results indicate that approximately 75%-80percentage of the gentamicin is retained in the nanotubes after the initial wash. In this study, we used anodization voltages of 25 V, 40 V, 60 V, or 85 V to fabricate nanotubes with length of 200 nm and diameters of 80 nm, 120 nm, 160 nm, or 200 nm. Remember, figure 1 shows SEM images of four different diameter nanotubular surfaces loaded with gentamicin. Prior to the drug release assay, the gentamicin loading efficiency in the nanotubes was evaluated. The loading efficiency of ‘NT G200’ was higher than that of NTG80 or ‘NTG120’.

On p of this, the nanotubes were filled with 2 gentamicin mg using the procedure described above. That’s where it starts getting very interesting, right? The drug surfaces loaded specimens retain the nanotubular structure with gentamicin incorporated into the nanotubes. However, nanotubes diameter can be controlled by altering the anodization parameters, as described above. Now pay attention please. The rinse concentrations solutions were measured by the previously described colorimetric assay. In this study, gentamicin was loaded into nanotubes using a lyophilization method and vacuum drying. Considering the above said. The loading efficiency was calculated from the formula η = /mo, where η is the loading efficiency, mo is gentamicin amount loaded in the nanotubes, and mr is gentamicin amount in the rinse solution.

Epidermidis 389 was susceptible to the gentamicin, while ATCC 35984 and aureus 376 exhibited a low level of susceptibility to the gentamicin, as shown in Table 2.

Living number bacteria on surface of the NT the surface was significantly lower than on the FlatTi, and the numbers on NT80 and NT120 were significantly lower than on NT160 and NT200. There was no difference between NT80 and NT120 or between NT160 and NT200. Viable number bacteria adhered to the nanotubes at the 4 hour time point was determined by the spread plate method. Living number three bacteria tested strains on the surface of the NTG was significantly lower than on the FlatTi or NT, and no difference was observed among NTG80, ‘NTG120’, NT G160″, and NTG200, as shown in Figure 3.

Bacterial adhesion and biofilm formation on the FlatTi surfaces, NT, and NT G were observed using SEM and CLSM at the 4, 24, and 48 hour time points. Similar to the SEM results observations, there were fewer bacterial colonies on the surfaces of NT80 and NT120 compared to those of NT160, NT200, and FlatTi. There were a few single bacterial colonies scattered on NT surfaces G80″ and ‘NTG120’ at the 4 and 24 hour time points, as shown in the SEM images in Figure 5. Additionally, at the 4 hour time point, there were fewer bacterial colonies on NT80 and NT120, and on NT80 and NT120 at the 48 hour time point, which indicated a low level of biofilm formation. Certainly, the bacterial colonies were sparsely distributed on the surface of ‘NT G80’ and ‘NT G120’ at 4 and 24 hours, which indicates no biofilm formation. In this study, we only evaluated the ATCC 35984 using SEM and CLSM. Ok, and now one of the most important parts. In contrast, it can be observed that many multiple bacterial colonies formed colony masses on the FlatTi surfaces, NT160, and NT200 at 24 and 48 hours. Nonetheless, the colonies on the ‘NT G80’ and NT G120″ surfaces were more obvious than those on ‘NTG160’ and NT G200 at the 48 hour time point.

Figure 8 shows that the hMSCs displayed significantly different shapes on the FlatTi surface and on the nanosurfaces. The cells on the nanotube surfaces displayed polygonal and clustering morphology. On p of that, the cell densities on the 80 surfaces and 120 nm nanotubes were higher than those on the 160 and 200 nm nanotubes and the FlatTi. I’m sure it sounds familiar.|Doesn’t it sound familiar?|Sounds familiar? Biomaterials. Acta Biomater.

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