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1 Bacterial Interactions With Biomaterials Metallic Surfaces Elisabeth Loshchagin Pizzolitto*, Milena Mioralli; Antonio Carlos Guastaldi Universidade Estadual Paulista – UNESP, Faculdade de Ciências Farmacêuticas/Instituto de Química, Araraquara (SP), Brazil. The concern with the extended use of implanted biomaterials are the bacterial adhesion, biofilm formation and the infection. The objective of this study was to verify bacterial adhesion on pure titanium used in dentistry. For this study was selected Streptococcus mutans ATCC 25175. Titanium coupons (diameter, 12mm; thickness 2mm) with smooth surface were prepared by laser beam Nd:YAG to obtain rough surfaces. The surfaces were assessed by physicochemical methods and bacteria counting by conventional microbiology culture. Each coupon was placed in Mueller Hinton broth medium (1x108) colony forming units (CFU)/mL of Streptococcus mutans bacteria and incubated for 1, 6, 24, 48 and 72 hours. After, they were washed with saline, and transferred to a sterile glass tubes with 5 mL of saline and subjected to ultrasonic bath (40kHz) for 8 minutes. For viable cells counts an aliquot of the suspension was serially diluted and spread on Mueller Hinton agar plates, incubated for 18 hours. The CFUs counted and results were expressed as CFU/mL. Data showed that smooth surface was hydrophobic and rough surface hydrophilic and SEM observations shown bacterial adhesion on both surfaces. These findings shown that the titanium metallic surfaces are susceptible to Streptococcus mutans biofilm formation. Keywords: Streptococcus mutans, bacterial adhesion, commercially pure titanium, laser beam Nd:YAG, biofilm 2 1. Introduction In modern medicine metallic biomaterials can be used to restore various functions in the human body, including dental restorations, appliances and implants20,29. The main concern with dental implants is the integration with the surrounding tissues, required for the sucess of extended use of biomaterials12,18,19,21,29,32. The bacterial adhesion and biofilm development on the surface of the dental biomaterial can be a clinical problem9,20, due to sistemic infections and bacteria in this lifestyle is resistant to antibiotics and immune system9,11,20. Nowadays, dental plaque is considered a biofilm, which is a complex, organized microbial community. An uncontrolled accumulation of bacteria on the teeth, dentures or endosseous implants allow the formation of thick biofilms, and can lead to dental caries, gingivitis and periodontites20,44. This solid surfaces can allow the oral streptococci colonization, as Streptococcus mutans and form biofilm, which can be associated with caries9,10,24,25,28. The commercial pure titanium (cpTi) can be a metallic biomaterial for use as oral implants due to its biocompatibility and osseointegration45,47, and its surface can be modified in order to alter structural properties. So, to improve the biological performance of cpTi with the host a laser beam (Nd: YAG-Neoymium – Yttrium Aluminum Garnet) can be one of approach to modify surfaces of titanium23. Sometimes, an implant failure is due to biofilm formation and not due to mechanical adjustments. The aim of this study was to evaluate bacterial adhesion on commercially pure titanium used in dentistry. 2-Material and Methods 2.1 Preparation of MetallicCoupons 3 Discs (12 mm in diameter, 0.2 mm in thickness) of commercially pure titanium (cp Ti) were used in this study. One surface was smoothed in a polishing machine (MAXIPLAN) and water abrasive sandpaper (3M) particle size 180, rinsed with distilled water and dried at room temperature. The machined titanium was used as control. And, another one, considered rough surface, received a laser treatment that was carried out by means a pulsed Nd:YAG laser (DigiLaser, DML-100)2,14, from the Department of Physics-Chemistry from Institute of Chemistry-UNESP. Titanium discs were cleaned in ultrasonic bath with mild detergent solutions distilledwater, acetone, ethanol and ultra pure water and dried at room temperature. After laser ablation, the Ti coupons were autoclaved at 121ºC and then immersed in the bacterial suspension. The surface of the coupons was observed by scanning electron microscopy (LEO 440), and the wettability of the surfaces was determined by means of goniometer OCA-20 Contact Angle System (DataPhysics Instruments) at room temperature. A profilometer (Mitutoyo SJ-301) was used to measure the surface roughness14 and results were expressed in µm as average roughness Ra (arithmetic mean of sampling area roughness). 2.2. Bacterial Strain And Adherence Assay. Streptococcus mutans (S. mutans) reference 25175 from Instituto Adolfo Lutz (São Paulo) was selected for the in vitro assays. The bacteria obtained from stock was transferred into tubes containing 7 mL of Thioglycollate medium U.S.P. (Oxoid, England) and incubated at 35-37ºC for 24 hours. One aliquot was used to inoculate 5.0 mL of BHI (Brain Heart Infusion, Oxoid, England) untill to obtain a suspension of approximately 108cells/mL of Streptococcus mutans, 4 an optical density of 1.0 at 540nm. Thereafter, 200 L of this cell suspension was transferred to conical tubes of polystyrene, sterile, containing 15 mL of Mueller-Hinton broth and the coupons of the Ticp. And, incubated at 37ºC, under constant agitation of 100 rpm, during 1, 6, 24, 48 and 72 hours. Then, the coupons were washed with 5 mL of sterile 0.9% NaCl solution, and introduced, separately, in a sterile glass tube with 5 mL of sterile 0.9% NaCl solution and subjected to ultrasonic bath cleaner (Unique, Indaiatuba, Brazil) at frequency of 40kHz for 8 minutes in order to leaves bacteria in suspension. The coupons were removed from the 0.9% NaCl solution and examined by means of scanning electron microscopy (SEM), and the bacterial suspension was serially diluted in 0.9% NaCl solution and seeded according to bacteriological techniques. 2.3. Electron Microscopic Procedures. The samples discs of the Ticp were fixed with 2.5% glutaradehyde in 0.1M phosphate buffer (pH7.1) for 15 min, dehydrated with series of aqueous ethanol solutions (15, 30, 50, 95 and 100%) for 15 min each, and dried at 37ºC, coated with gold (1KV, 15mAp, S150 B Edwards) during 2 minutes and examined by using a JEOL-JSM (T330A) SEM. 2.4. Viability assay. An aliquot (0.1 mL) of each dilution of the bacterial suspension was seeded on TSA (Tryptic Soy Agar). Plates were incubated at 37ºC in bacteriological incubator during 24 h. The bacterial growth on each plate was counted, calculated and the total colony forming units expressed in CFU / mL. 2.5. Statistical Analysis. 5 Data were analyzed by one-way analysis of variance (ANOVA). Student t-test was conducted to compare the samples. Statistical analysis used OriginPro software (version 5.0) at a confidence interval of 95%. 3. Results and Discussion Table 1. Shows the value of surface roughness and wettability, while Table 2 shows the quantification of viable cells recovered from surfaces cpTi determined in this study. The Streptococcus mutans biofilm observed by SEM on the smooth and rough surfaces of the cpTi following 48 hours exposure to Mueller Hinton broth, as showed in Figure 1 A, B. Table 1Table 2Figure 1A, B Data showed that the smooth surface is hydrophobic while the roughened surface is hydrophilic. There is a statistical difference (P<0.01)14. Vogler et al. (1998) reported that on surfaces that water contact angle is greater than 65º is called hidrophobic and less than 65º is considered hydrophilic. The parameters that may explain the bacterial adhesion to solid surface include the physical and chemical interactions, such as hydrophobicity13,27 and thermodynamics34. The surface characteristics of the solid substratrum and the adhering bacteria can affect the adherence process5,6. In the present study data showed that S. mutans adhere on both surfaces, notwithstanding, the cpTi smooth surface was hydrophobic and the rough surface was hydrophilic. Although, Busccher et al (1984)7 reported that due to thermodynamics S. mutans can adhere better to hydrophilic substrate. However, Satou et al. (1988)41 found no relationship between the hydrophobicity of the substrate surface and 6 adhesion of S. mutans. By the other hand, Fujioka-Hirai et al. (1987)17 suggested that S. mutans adhere to titanium alloy due to hydrophobic interactions. It is well known that a bacteria retained on grooves in rough surface can be protected of shear forces33. Although Aykent et al. (2010)1 observed a positive correlation between surface roughness and S. mutans adhesion. Several studies shown the oral bacterial adherence on the rough surfaces of dental implant biomaterials3,4,22,31,36,37,38. But, the main concern is with the implant surface that comes in contact with the host tissue, due to osseointegration 14,35-37,44 , and nevertheless there are no studies on bacterial adhesion and his effects on titanium surface modified by laser ablation process as Nd: YAG. Bacterial adhesion to the smooth and rough surfaces had a similar pattern. Viable cell counts of S. mutans detached from cpTi smooth surface showed that the average standard deviation was equal to 1.66±1.67x106 and from cpTi rough surface was equal to 1.06±1.07x106. One Way - ANOVA showed that the 0.05 level averages are not significantly different. But, SEM observations showed that the rough surface retained more bacteria and the biofilm formed was thicker (Fig.1B). In this current study the results of the bacteriological tests revealed that the amount of viable S. mutans cells retrieved from the rough and polished surface were not different (Table 2). The viable cells in biofilms of Streptococcus mutans are a significant factor in the pathogenesis of dental plaque42. Data from the present study showed that S. mutans adhesion and viable counts in the biofilm are related, according with Steinberg & Eyal (2002)42. Several studies have shown that S. mutans has been used in tests on bacterial adhesion because it is the main organism in the process of dental caries and, in dental biofilm formation, in which several bacterial species in the human oral cavity are involved 8,15,16, 25. 7 In the present study data obtained from SEM showed a positive relationship between the S. mutans growth and the cpTi surface modified by a laser ablation process and polished surface (Figure 1AB). There was no statistical difference between the bacterial attachment on these surfaces. S. mutans adhered on titanium surfaces, which is in agreement with Rimondini et al.39 found that pathogenic bacteria adhere to titanium. The present investigation evaluate the adherence of S. mutans on smootth and rough surfaces, after immersion in a liquid culture inoculated with S. mutans and a pH 7.0, under this conditions both surfaces examined exhibited a bacterial adherence. The explanation of the bacterial adhesion in culture broth may be due to physicochemical interactions of the surface and extracellular polysaccharide production from bacteria30. The results of the present investigation are consistent with Montanaro et al.30 reported that biofilm of S. mutans can be formed in an aqueous environment and in the absence of physiological proteins. By the other hand, Tanner et al.43 conducted a Streptococcus mutans adherence test to biomaterial surfaces that included titanium, cobalt chromium and gold alloys using scanning electron microscopy and report that the initial adhesion was observed after immersion of each of these surfaces in suspension of S. mutans. In the present study, SEM observations demonstrated S. mutans adhesion on both surfaces of cpTi, and data obtained are consistent with the observations of Rimondini et al.40. And, Tanner et al.43 reported that S. mutans is used as a model in experimental studies of bacterial adhesion. The findings in the present study shown the Streptococcus mutans adhesion on solid surface in aqueous environment and biofilm formation. 4. Conclusions The bacterial adhesion occurred during 1, 6, 24, 48 and 72 hours after immersion in the Mueller Hinton broth. There was no difference on S. mutans adhesion on both surfaces. The 8 adhesion and biofilm formation was demonstrated by scanning electron microscope on smooth and rough surface of cp Ti in the conditions of the study. The laser irradiation of high intensity Nd: YAG applied to modify the surface of the commercially pure titanium for dental implants used in particular for improving the osseointegration does not reduced bacterial adhesion. The presence of bacteria on the implant surface may lead to inflammatory reactions and this event can be a risk factor for the failure of implantation due to infection. 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Effects of titanium surface topography on bone integration: a systematic review. Clinical Oral Implants Research 2009;20 (4):172-184. 14 Table 1. Characteristics of the investigated surfaces of cpTi Titanium (cpTi) Contact angle (º) Roughness (Ra)14 Smooth 75.26°±0.70 0.33±0.06 µm Rough <7º 1.38± 0.23 µm 15 Table 2. Viable cells of S. mutans (Cfu/mL) retrieved from cpTi smooth and rough surfaces Period of time Viable cells: cfu/mL from Viable cells: cfu/mL from rough smooth surfaces surfaces 1 7.00 x 103 1.40 x 104 6 4.90 x 105 5.30 x 105 24 1.02 x 106 2.80 x 106 48 2.80 x 106 1.28 x 106 72 4.00 x 106 6.80 x 105 16 Figure 1. Scanning electron micrograph of cpTi surfaces with adhered S. mutans cells after 48 h exposure to Mueller Hinton Broth, as shown by arrow. A) cpTi smooth surface showing the attachment and formation of S. mutans biofilm. B) cp Ti rough surface showing S. mutans biofilm. Original magnification:X5000. Bar corresponds to 5µm.