Biofilm growth on the implant surface is the number one cause of the failure of the implants. Biofilms on implant surfaces are hard to eliminate by antibiotics due to the protection offered by the exopolymeric substances that embed the organisms in a matrix, impenetrable for most antibiotics and immune cells. Application of metals in nanoscale is considered to resolve biofilm formation. Here we studied the effect of iron-oxide nanoparticles over biofilm formation on different biomaterial surfaces and pluronic coated surfaces. Bacterial adhesion for 30 min showed significant reduction in bacterial adhesion on pluronic coated surfaces compared to other surfaces. Subsequently, bacteria were allowed to grow for 24 h in the presence of different concentrations of iron-oxide nanoparticles. A significant reduction in biofilm growth was observed in the presence of the highest concentration of iron-oxide nanoparticles on pluronic coated surfaces compared to other surfaces. Therefore, combination of polymer brush coating and iron-oxide nanoparticles could show a significant reduction in biofilm formation.
Biofilm growth on the surface of biomaterial implants is generally recognized as a cause of biomaterial-associated infection (BAI). These infections impose serious complications associated with the use of biomaterial implants. Regardless of the high sterile conditions and improved techniques in the operating theatre, both perioperative and postoperative contamination by microorganisms suspended in the air and from the skin flora continue to be the most common pathway for the contamination of biomaterial implants and medical devices [
BAI starts with the initial adhesion of microorganisms and then subsequently grows to form a biofilm. Bacterial adhesion on surfaces is influenced by physicochemical properties of the surface [
Nanoparticles are less than 100 nm in diameter and as a result properties such as surface area, chemical reactivity, and biological activity alter dramatically. The antibacterial efficacy of metal nanoparticles has been suggested to be due to their high surface-to-volume ratio rather than to the sole effect of metal-ion release [
Poly(methyl methacrylate) (PMMA) (Industrial Insulation, Chennai, India), polystyrene (PS) (Industrial Insulation, Chennai, India), tissue culture polystyrene well plates (TCPS) (NEST Biotech Co. Ltd., China), glass slide (GS, control), and surfaces (PMMA and TCPS) coated with a hydrophilic polyethylene oxide (PEO) layer were used. All samples except hydrophilic PEO coating and TCPS were rinsed thoroughly with ethanol (Jiangsu Huaxi International trade Co. Ltd., China) and washed with sterile water before use.
Hydrophilic PEO-coated surface (polymer brush coating) was prepared by first cleaning the surfaces in sterile water, ethanol, and water again and finally washing with sterile water. Surfaces were made hydrophobic by application of dimethyldichlorosilane coating. Exposure to a solution of 1 g/L pluronic F-68 solution (HIMEDIA Laboratories Pvt. Ltd., Mumbai, India) in phosphate-buffered saline (PBS: 10 mM potassium phosphate, 0.15 M NaCl, pH 7.0) for 20 min created a hydrophilic polymer brush coating over the surface.
The wettability of the surfaces was determined by water contact angle measurements at room temperature with an image analyzing system, using sessile drop technique. Each value was obtained by averaging five droplets on one sample.
4 mL of ferrous chloride and 1 mL of ferric chloride were added to a flask. Sodium hydroxide was added drop by drop and stirred continuously. Initially formed brown precipitate with time should be changed into a black precipitate, indicating the formation of iron-oxide nanoparticles. The size of the synthesized particles was determined using transmission electron microscopy (TEM). The optical measurement of the nanoparticles was studied by UV-visible spectrophotometer (UNICO) over the spectral range of 200–1000 nm.
Bacterial adhesion was performed on six different surfaces (GS, PS, PMMA, polymer brush coated PMMA, TCPS, and polymer brush coated TCPS). Samples were placed in the tissue culture polystyrene well plates. Each well was filled with 1 mL of bacterial suspension and allowed to adhere and grow aerobically at 37°C for 30 min. Bacterial adhesion on GS was considered as control. Subsequently, wells were washed with sterile phosphate buffer saline (10 mM potassium phosphate, 0.15 M NaCl, pH 7.0) to remove unbound bacteria and images were taken using phase contrast microscopy and the number of adherent bacteria per cm2 was determined using ImageJ software. Experiments were performed in triplicate with separately cultured bacteria.
Freshly prepared nutrient agar plates were used. Bacterial cultures were inoculated to the agar plates and incubated at 37°C for 30 min. Holes of 6 mm diameter were punched into the nutrient agar plates. Holes were filled with 100
In this study, TCPS and polymer brush coated TCPS were compared. 1 mL of bacterial suspension was added to each well and allowed to adhere and grow aerobically at 37°C for 30 min. Then, iron-oxide nanoparticles were introduced in different concentrations (0.01 mg/mL, 0.05 mg/mL, 0.10 mg/mL, and 0.15 mg/mL). Thereafter, biofilms were allowed to grow for 24 h. Subsequently, wells were washed with sterile water to remove unbound bacteria and biofilm development was assessed by measuring the optical density using spectrophotometer. To this end, 500
Experiments were performed in triplicate. Data are represented as a mean with standard deviation. For statistical analysis ANOVA was performed followed by a Tukey’s HSD post hoc test and a
The water contact angles of biomaterial and polymer brush coated surfaces are shown in Figure
Water contact angle of biomaterial surfaces (GS: glass slide, PS: polystyrene, TCPS: tissue culture polystyrene, and PMMA: poly(methyl methacrylate)) and pluronic coated surfaces.
The TEM images of synthesized iron-oxide nanoparticles are shown in Figure
(a) Transmission electron micrograph of iron-oxide nanoparticles. Bar denotes 5 nm. (b) UV-visible spectrum of iron-oxide nanoparticles [
Initial adhesion of bacteria after 30 min of incubation was significantly (
Number of adherent bacteria after 30 min on different biomaterial surfaces (GS: glass slide, PS: polystyrene, TCPS: tissue culture polystyrene, and PMMA: poly(methyl methacrylate)) and pluronic coated surfaces.
The antibacterial activity of iron-oxide nanoparticles is shown in Table
Antibacterial activity of iron-oxide nanoparticles.
Microorganisms | Zone of inhibition (mm) | |||
---|---|---|---|---|
Concentration of iron-oxide nanoparticles (mg/mL) | ||||
0.01 | 0.05 | 0.1 | 0.15 | |
|
10 | 12 | 17 | 26 |
|
11 | 13 | 16 | 28 |
|
13 | 16 | 19 | 29 |
Influence of iron-oxide nanoparticles at different concentrations against biofilm growth on polymer brush coated surface was shown in Figure
Optical density measurements of 24 h biofilm growth on pluronic coated TCPS surface in the presence of different concentrations (0.01 mg/mL, 0.05 mg/mL, 0.10 mg/mL, and 0.15 mg/mL) of iron-oxide nanoparticles.
This paper presents the experimental study on the bacterial adhesion and biofilm growth on various biomaterials including polymer brush coated surfaces and the strategy of using iron-oxide nanoparticles in eradication of biofilms. Biofilm growth on biomaterials is generally the cause of BAI.
Amongst other material properties, surface wettability plays a major role in bacterial adhesion to biomaterials. Wettability of biomaterial surfaces has been related to bacterial adhesion and biofilm growth [
Metals have been used as antibacterial agent for centuries [
In this study, influence of iron-oxide nanoparticles on biofilms formed on polymer brush coated biomaterial surface was evaluated. The study of combined effects of polymer brush coating and iron-oxide nanoparticles on biofilms is novel. A significant reduction (
This study demonstrates that wettability of a biomaterial surface influences bacterial adhesion and biofilm growth. Polymer brush coated surfaces showed reduced bacterial adhesion compared to bare surfaces. A significant reduction in biofilm growth was observed due to the influence of iron-oxide nanoparticles on biofilms formed on polymer brush coated biomaterial surfaces. Thus combinational strategies such as polymer brush coating to biomaterial surface and influence of iron-oxide nanoparticles could significantly reduce biomaterial-associated infections.
The authors declare no conflict of interests.
This research was funded by SSN College of Engineering, Kalavakkam, India.