Download aderência bacteriana e formação de biofilme em superfície de

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. This means
that rough surfaces of dental biomaterials can facilitate bacterial colonization and biofilm
formation in the oral environment and become a niche of persistent infection.
5. References
1. Aykent F; Yondem I; Ozyesil AG; Gunal SK; Avunduk MC; Ozkan S. Effect of different
finishing techniques for restorative materials on surface roughness and bacterial adhesion.
Journal of Prosthetic Dentistry 2010;103: 221-227.
2. Braga FJC; Marques RFC; Filho EA; Guastaldi AC. Surface modification of Ti dental
implants by Nd:YVO4 laser irradiation. Applied Surface Science 2007;253:9203-9208.
3. Bollen L; Lambrecht P; Quirynen M. Comparison of surface roughness or oral hard
materials to the threshold surface roughness for bacterial plaque retention: a review of the
literature. Dental Materials 1997;13:258-269.
4. Burgers R; Gerlach T; Hahnel S; Schwartz F; Handel G; Gosau M. In vivo and in vitro
biofilm formation on two different titanium implant surfaces. Clinial Oral Implants
Research 2010;21:156-164.
5. Busscher HJ; Rinastiti M; Siswomihardjo W, van der Mei HC Biofilm formation on dental
restorative and implant materials. Journal of Dental Research 2010;89:657-665.
6. Busscher HJ; van der Mei HC. Physico-chemical interactions in initial microbial adhesion
and relevance for biofilm formation. Advances in Dental Research 1997;11(1):24-32.
9
7. Busscher HJ; Weerkamp AH, van der Mei HC, van Pelt AWJ, de Jong HP, Arends J.
Measurements of the surface free energy of bacterial cell surfaces and its relevance for
adhesion. Applied and Environmental Microbiology 1984;51:910-914.
8. Cisar J; Takahashi Y; Ruhl S; Donkerloot J; Sandberg A. Specific inhibition of bacterial
adhesion: observation from study of gram-positive bacteria that initiate biofilm formation
on the tooth surface. Advances in Dental Research 1997;11:168-175.
9. Costerton JW; Stewart PS; Greenberg EP Bacterial biofilms: A common cause of persistent
infections. Science 1999;284:1318-1322.
10. Dewhirst FE;Chen T; Izard J; Paster BJ; Tanner ACR; Yu WH; Lakshmanan A; Wade
WG. The human Oral Microbiome. Journal of Bacteriology 2010;192(19):5002-5017.
11. Donlan RM; Costerton JW. Biofilms: survival mechanisms of clinically relevant
microorganisms. Clinical Microbiology Reviews 2002;15(2): 167-193.
12. Dhir S; Biofilm and dental implant: The microbial link. Journal of Indian Society
Periodontology 2013;17(1): 5-11.
13. Doyle RJ; Rosenberg M; Drake D. Hydrophobicity of oral bacteria. In: Doyle R;
Rosenberg M, editors. Microbial cell surface hydrophobicity. Washington: American
Society for Microbiology 1990. p. 387-419.
14. Faeda RS; Tavares HS; Sartori R; Guastaldi AC; Marcantonio Jr E. Biological
performance of chemical hydroxyapatite coating associated with implant surface
modification by laser beam: biomechanical study in rabbit tíbias. Journal Oral
Maxillofacial Surgery 2009;67:1706-1715.
15. Faltermeier A, Bürgers R, Rosentritt M. Bacterial adhesion of Streptococcus mutans to
esthetic bracket materials. American Journal of Orthodontics and Dentofacial
Orthopedics 2008; 133:S99-S103.
10
16. Foster J; Kolenbrander P. Developmentof multispecies oral bacteria community in a
saliva-conditioned flow cell. Applied and Environmental Microbiology 2004;70:43404348.
17. Fujioka-Hirai Y; Akagawa Y; Minagi S; Tsuru H; Miyake Y; Suginaka H. Adherence of
Streptococcus mutans to implant materials. Journal of Biomedical and Materials
Research. 1987; 21(7):913-920.
18. Guéhennec LL; Soueidan A; Layrolle P; Amouriq Y. Surface treatments of titanium
dental implants for rapid osseointegration. Dental Materials 2007;23:844-854.
19. Gupta A; Dhanraj M; Sivagami G. Status of surface treatment in endosseous implant: a
literary overview. Indian Journal of Dental Research 2010; 21(3):433-438.
20. Gurenlian, J.R. The role of dental plaque biofilm in oral health. Journal Dental Hygiene
2007;81(5):1-11.
21. Gristina AG; Biomaterial-centered infection: Microbial adhesion versus tissue integration.
Science 1987;237(25): 1588-1595.
22. Größner-Schreiber B; Teichmann J; Hannig M; Dorferv C; Wenderoth D; Ott S. Modified
implant surfaces show different biofilm composition under in vivo conditions. Clinical
Oral Implants Research 2009;20:817-826.
23. Gyorgy E;Perez Del Pino A; Serra P; Morenza JL. Surface nitridation of titanium by
pulsed Nd:YAG laser irradiation. Applied Surface Science 2002;186:130-134.
24. Hamada S; Slade HD. Biology, Immunology, and Cariogenicity of Streptococcus mutans.
Microbiological Reviews 1980;44(2):331-384.
25. Kolenbrander PE; London J Adhere today here tomorrow: oral bacterial adherence.
Journal of Bacteriology 1993;175:3247-3252.
11
26. Kreth J; Merrit J; Qi F. Bacterial and host interactions or oral streptococci. DNA and Cell
Biology 2009;28(8):397-403.
27. Larsson K; Glantz PO. Microbial adhesion to surfaces with different surface charges. Acta
Odontologica Scandinavica 1981; 39:79-82.
28. Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiology Reviews
1986; 50: 353–380.
29. Lüdecke C; Jandt KD; Siegismund D; Kujau J; Zang E; Rettenmayr M; Bossert J;
Roth M. Reproducible biofilm cultivation of chemostat-grown Escherichia coli and
investigation of bacterial adhesion on biomaterials using a non-constant depth film
fermenter. PlosOne 2014;9(1):e84837.
30. Montanaro, L.; Campoccia, D.; Rizzi, S.; Donati, M.E.; Breschi, L.;Prati, C.; Arciola C.R.
Evaluation of bacterial adhesion of Streptococcus mutans on dental restorative materials.
Biomaterials 2004; 25: 4457-4463.
31. Nakazato G;Tsuchiya H; Sato M; Yamauchi M. In vivo plaque formation on dental
implants materials. International Journal Oral Maxillofacial Implants 1989;4(4):321326.
32. Novaes Jr, AB; Souza, SLS; Barros, RRM.; Pereira, KKY; Iezi,G; Piattelli, A Influence of
implant surfaces on osseointegration. Brazilian Dental Journal 2010;21(6):471-481.
33. Pier-Francesco A; Adams R; Waters M; Williams D. Titanium surface modification and
its effects on the adherence of Porphyromonas gingivalis: an in vitro study. Clinical Oral
Implants Research 2006;17:633-637.
34. Pratt-Terpstra IH;Weerkamp AH, Busscher HJ. The effects os pellicle formation on
streptococcal adhesion to human enamel and artificial substrata with various surface free
energies. Journal Dental Research 1989;68(3):463-467.
12
35. Quirynen, M.; Bollen, CML. The influence of surface roughness and surface-free energy
on supra-and subgingival plaque formation in man. Journal Clinical Periodontology
1995;22:1-14.
36. Quirynen M.; Bollen C; Papaioannou W; Van Eldere J; Van Steenberghe D. The influence
of titanium abutments surface roughness on plaque accumulation and gingivitis. Shortterm observations. International Journal Oral Maxillofacial Implants 1996;11:169-178.
37. Quirynen M; van der Mei HC; Bollen CM; Schotte A; Marechal M; Doornbusch GI;
Naert I; Busscher HJ, Van Steenberghe D. An in vivo study of the influence of the
surface roughness of implants on the microbiology of supra and subgingival plaque.
Journal of Dentistry Research 1993;72(9):1304-1309.
38. Rocha SS; Bernardi ACA; Pizzolitto AC; Adabo GL; Pizzolitto EL. Streptococcus mutans
attachment on a cast titanium surfaces. Materials Research 2009;12(1):41-44.
39. Rimondini L, Cerroni L, Carrassi A, Torricelli P. Bacterial colonization of Zirconia
Ceramic Surface: an in vitro and in vivo study. International Journal of Oral
Maxillofacial Implants 2002; 17:793-798.
40. Rimondini L; Fare S; Brambilla E; Felloni A; Consoni C; Brossa F; Carassi A. The effect
of surface roughness on early in vivo plaque colonization on titanium. Journal of
Periondontology 1997;68:556-562.
41. Satou, J; Fukunaga, A; Satou N; Shintani H; Okuda K. Streptococcal adherence on
various restorative materials. Journal of Dental Research 1988;67(3):588-591.
42. Steinberg D; Eyal S. Early formation of Streptococcus sobrinus biofilm on various dental
restorative materials. Journal of Dentistry 2002; 30: 47-51.
13
43. Tanner, J.; Vallitu, P.K.; Soderling, E. Adherence of Streptococcus mutans to an E-glass
fiber-reinforced composite and conventional restorative materials used in
prosthetic
dentistry. Journal Biomedical Materials Research 2000;49(2):250-256.
44. Teughels W; Assche NV; Sliepen I; Quirynen M. Effect of material characteristics and/or
surface topography on biofilm development. Clinical Oral Implants Research
2006;17(2):68-81.
45. Ozcan M; Hammerle C. Titanium as a reconstruction and implant material in dentistry:
advantages and pitfalls. Materials 2012;5:1528-1545.
46. Vogler EA Structure and reactivity of water at biomaterials surfaces. Advances Colloid
Interface Science 1998;74:69-117.
47. Wennerberg, A; Albrektsson, T. 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.