Experimental study on the influence of hydrophilicity on bacterial adhesion in bioimplants

Titanium and its alloys are widely used as dental and orthopaedic implants because of their desirable mechanical properties and biocompatibility. However, implant failures due to bacterial infections occur in many cases unexpectedly. Formation of a biofilm on the implant surfaces facilitates growth of colony of bacteria which in turn reduces the effectiveness of antibacterial drugs. Hydrophilicity of implant surfaces diminishes the chances of biofilm formation. Micro surface features are created on the Ti6Al4V implant surface using a pulsed Nd-YAG laser and its influence on water wettability (and thus on hydrophilicity) has been studied and compared with a polished surface. Strains of Staphylococcus aureus have been inoculated on the test specimens and the bacterial adherence have been compared. The results show that micro-textured surfaces of Ti6Al4V have improved water wettability and thus are more hydrophilic than polished surface. This has reduced the formation of biofilm and bacterial growth.


Introduction
Ti6Al4V (Grade 5 Titanium alloy) is widely used for bioimplants due to its excellent properties such as low toxicity, corrosion resistance, high specific strength, MRI compatibility and high biocompatibility [1].In many cases implantation fails due to reasons such as poor osseointegration, lack of adoption of proper procedures in placement of implants and infections developed after placing the implants in human body. Patients suffer due to implant associated issues which increases chances of co morbid conditions. Huge financial burden is also placed on patients and society due to more frequent implant failures. Bacterial infections are identified as a major cause of implant failure and usually require replacement of infected implants or treatment with prolonged use of antibiotics [2]. There are further issues such as bacterial drug resistance which is emerging. Bacterial infections associated with implants are mainly attributed to the formation of a biofilm on the surface of implants. Biofilm consists of bacterial colonies, teichoic acids and extra cellular polymeric substances, which give immunity against antibiotics [3]. Staphylococcus aureus (S.aureus) is identified as the most common pathogen responsible to majority of the implant related infections. S.aureus is typically hydrophobic in nature due to the presence of highly negative charged teichoic and lipoteichoic acid as its cell wall constituents [

Selection of materials and surfa
Commercially procured Ti6Al4V (G samples are made in the form of tiny using wire cut EDM method (figure from 120 to 2000 grits using a po polished with lapping paste and w performance with textured surface. pulsed Nd-YAG laser system having samples were irradiated with the la 40W, scanning speed of 25mm/s and The laser-textured area on the spe dimples having a depth of 2.5μm. bath for 10 min, further rinsed in dis

Evaluation of Surface Topograp
The surface roughness and 3D topo using a laser confocal microscope ( maximum height of the profile (R z ) (a) fications techniques are available in order to impro texturing patterns with variable geometries, creati ring and coating etc. [8]. Surface coating tech disintegrate in the presence of biological fluids which ous surface topography modification techniques, most popular methods for creating a super hydrophi bricated using laser surface texturing which wil ree energy of the material. Laser texturing is f n of bacteria and reduced the formation of biof pulsed laser for surface texturing of grade 2 T ion of biofilm have also been investigated recently [1 ce texturing on altering the hydrophilicity of Ti s erial properties of Ti based implant surface have bee d depth measuring few microns) are created on the i ophilicity of surfaces is evaluated in comparison w en introduced on the prepared specimen surfaces an ace texturing Grade 5) specimens have been used in the experimen y blocks of flat surface area of 10mm x 10 mm and e 1). Surfaces were ground sequentially with a series olishing machine. Three surfaces each of Ti6Al4V were kept as polished surfaces for comparing th The laser surface texturing was performed using a g a power of 200W. The wave length of the laser wa aser beam using pre-selected processing parameters d a stand-off distance of 10mm.
um Grade5 specimens (a) Polished (b) Textured cimen samples were 10 x 10 mm which is fully c The laser textured samples were ultrasonically clea stilled water and air dried.
phy ography of the polished and laser-textured samples (Olympus LEXT OLS4000). Arithmetic mean roug ) were measured and compared with that of the polis ove the surface ing micro/nano hniques has a h will diminish laser surface ilic surface [9]. ll enhance the found to have film [10]

Wettability Analysis
The sessile drop method was used textured samples using a video-base and analysis were performed using testing liquid, and the volume of e Droplet images were captured in the intervals, counting since the start of least 60s. Average contact angle o sample at room temperature.

Bacteria strain
Specimens are inoculated with S.au (24 hrs) at 37 °C on a gyrotatory i was used to adjust the overnight cul fresh sterile MHB. This provided a (CFU)/ml. 1 ml of culture was ap CFU/ml, as verified by viable coun incubator with shaking at 100 rpm textured) were tested to ensure th compared by measuring the area cov

Influence of surface topography
The Surface topography images of influence of surface roughness on m adhesion to surface depends on sev energy, but the surface topography polished and laser-textured samples lying between 3.6 and 3.7μm. Bacteria commonly R a in the nanometre range, but also adhere to surfac e bacteria size [14]. It is found that surface roughne ion. Micro pattern supports the cell orientation, wou

Influence of water wettability
The smaller value of contact angle surface [16]. The values of contac shown in figure 3. Bacteria prefer to adhere on rou environmental disturbances such as roughness than polished. From the after laser texturing and the surfac cells is due to the presence of high acid [18].

Influence on Bacterial adherenc
The growth of Bactria on the spe florescence microscopy. The images polished (figure 4a) and laser te fluorescence indicates viable bacte dead bacteria with damaged memb factor for initial attachment of bacte The results show that bacterial adh than the polished specimens. Te colonization. This is assumed to be surface roughness (R a ) value. This section 3.2.Surface peaks act as a interaction energy of the bacteria. chemical composition [14].In Ti6A crystalline layer which reduces the Polymeric Substances considered a the biofilm maturation and coloniza ugher surfaces but the surface is protected by s shear force [17]. Laser textured specimen offers Table.1 it is clear that the contact angle is significa ce become more hydrophilic. The hydrophobic natu hly negatively charged and hydrophobic teichoic an herence is greatly reduced in laser-textured specime extured surfaces are found to be unfavourable due to the hydrophilic nature of the textured surface is evident from the wettability studies carried out a a spacer between the bacterial cell and surface, Ti6Al4V shows better antibacterial behaviour due Al4V, surface treatments convert the amorphous Tita chances of bacterial adhesion [9]. Bacteria produc as the major cell to cell connecting substance, which ation of dead cells on the textured surface [4].  Bacteria surface interaction throug interaction of bacteria to matching b these interactions, through reductio hydrophobic in nature so they pre hydrophilic in nature, which is liab maturation is found to be inhibited b

Conclusions
The present work is focused surfaces under the influence of mic textured samples are compared and performance of Ti6Al4V has been m of live/dead using the bacterial lum increased significantly after laser te between individual bacteria and tex greatly reduced by laser texturin significantly reduced the adhesion o is thus found to be a promising me antibacterial properties, reducing th