In vivo and in vitro biofilm formation on two different titanium implant surfaces

Objectives: The aim of the present in vitro and human in vivo study was twofold: first, to evaluate the initial biofilm formation on different titanium implant surfaces by means of two highly sensitive fluorescent techniques and, second, to correlate these findings to different surface properties. Materials and methods: In vivo biofilm formation was induced on purely machined (Pt) and on sand‐blasted and acid‐etched titanium (Prom) specimens, which were mounted buccally on individual splints and worn by six study participants for 12 h. In vitro bacterial adhesion was also investigated after incubation with Streptococcus sanguinis suspension (37°C, 2 h). Adherent bacteria were quantified by the following fluorescence techniques: Resazurin staining in combination with an automated fluorescence reader or live/dead cell labeling and fluorescence microscopy. Surface roughness (R_a) was determined with a perthometer, and surface free energy (SFE) was measured with a goniometer. Results: Prom showed a significantly higher median R_a (0.95 μm) and a significantly lower median SFE (18.3 mJ/m²) than Pt (R_a=0.15 μm; SFE=39.6 mJ/m²). The in vitro and in vivo tests showed a significantly higher bacterial adhesion to Prom than to Pt, and the initial biofilm formation on Pt corresponded to the circular surface modifications on the machined substratum. Both observations may be attributed to the predominant influence of surface roughness on bacterial adhesion. No significant differences in the percentage of dead cells among all adhering bacteria were found between Prom (23.7%) and Pt (29.1%). Ectopic solitary epithelial cells from the oral mucosa – strongly adhering to the substratum – were found on each Prom specimen, but not on any of the Pt surfaces. Conclusions: Initial bacterial adhesion to differently textured titanium surfaces is primarily influenced by R_a, whereas the influence of SFE seems to be of only minor importance. Therefore, the micro‐structured parts of an implant that are exposed to the oral cavity should be highly polished to prevent plaque accumulation. Both tested fluorometric techniques proved to be highly sensitive and reproducible in the quantification of biofilm formation on titanium implant surfaces. To cite this article:
Bürgers R, Gerlach T, Hahnel S, Schwarz F, Handel G, Gosau M. In vivo and in vitro biofilm formation on two different titanium implant surfaces.
Clin. Oral Impl. Res. 21 , 2010; 156–164
doi: 10.1111/j.1600‐0501.2009.01815.x     

Chemical debridement of contaminated titanium surfaces: An in vitro study

Abstract

Objective. To compare the efficacy of different chemical solutions when used for chemical debridement of biofilm contaminated titanium surfaces in an in-vitro experimental study. Materials and methods. Commercially pure titanium discs with a diameter of 6.2 mm and height of 2 mm, mirror-polished with a measured surface amplitude value S_A = 0.037 μm ± 0.009 were used as test-surfaces. A biofilm was simulated with multi-layers of Staphylococcus epidermidis ATCC359844 covering the entire titanium surface. The chemical agents tested were: 3% H₂O₂, 0.2% Chlorhexidine, 24% EDTA-gel, 3% H₂O₂ mixed with 1.6 g/L TiO₂ and sterile saline solution. The decontamination effect was evaluated by optical density analysis using spectrophotometry and with scanning electron microscopy (SEM) images of the remaining biofilm. Results. The suspensions of 3% H₂O₂ and 1.6 g/L TiO₂ or 3% H₂O₂ alone were the most effective in removing S. epidermidis biofilms (p < 0.05), whereas 0.2% chlorhexidine or 24% EDTA gel had no significant effects. SEM images of the remaining biofilms supported the quantitative results indicating the higher efficacy of 3% H₂O₂ and 1.6 g/L TiO₂ or 3% H₂O₂ alone. It also revealed that EDTA, despite a non-significant effect on reducing the amount of established biofilms, was able to alter the biofilm architecture, as demonstrated by increased interspaced regions. Conclusions. In this in vitro study the decontamination potential of a suspension of 3% H₂O₂ and 1.6 g/L TiO₂ or 3% H₂O₂ alone were encouraging. Whether such procedures would have a similar effect in vivo remains to be determined.     

In vitro osteogenesis on a microstructured titanium surface with additional submicron-scale topography

The present study aimed to evaluate key parameters of in vitro osteogenesis on (1) commercially pure titanium (cpTi) discs with 20-200-microm-scale microtopography patterned with additional micron- and submicron-scale topography (0.5-20 microm; Plus surface, Dentsply Friadent), (2) control cpTi discs with 20-200-microm-scale microtopography (DPS, Deep Profile Surface, Dentsply Friadent), and (3) a machined surface. Using calvaria-derived osteogenic cultures, the following parameters were assessed: cell adhesion and spreading, growth curve and cell viability, alkaline phosphatase (ALP) activity and total protein content, immunolocalization of fibronectin, bone sialoprotein (BSP) and osteopontin (OPN), and bone-like tissue formation. The results showed no major differences between surfaces in terms of cell adhesion, growth curve, cell viability (days 4 and 11), ALP activity, or total protein content (days 11 and 17). At day 11, cultures grown on Plus exhibited small, well-defined nodular areas of calcified matrix, which were only rarely observed on DPS and absent on the machined surface. Such areas were larger at day 17 and were not associated with the typical mineralized bone-like nodules (with BSP- and OPN-positive osteoblastic cells on top). At day 17, the total mineralized area was significantly larger on DPS than on a Plus or machined surface (DPS>Plus>machined; Kruskal-Wallis test, P<0.05). Direct fluorescence allowed the straightforward observation of higher amounts of apoptotic bodies associated with mineralized nodules for Plus. The results suggested the occurrence of an additional, early pattern of matrix mineralization mostly for the Plus microstructured surface, which did not necessarily translate into larger bone-like tissue formation in vitro.     

Photo-induced hydrophilicity enhances initial cell behavior and early bone apposition

Objective: The anatase form of titanium dioxide (TiO₂) exhibits photo‐induced hydrophilicity when it is irradiated with ultraviolet (UV) light. In the present study, the effect of photo‐induced hydrophilicity on initial cell behavior and bone formation was evaluated. Materials and methods: Plasma source ion implantation method and post‐annealing were employed for coating the anatase form of TiO₂ to the surface of the titanium disk and implant. Half of the disks and implants were illuminated with UV for 24 h beforehand, whereas the other halves were blinded and used as controls. Photo‐induced hydrophilicity was confirmed by a static wettability assay. The effects of this hydrophilicity on cell behavior were evaluated by means of cell attachment, proliferation and morphology using pluripotent mesenchymal precursor C2C12 cells. Thereafter, bone formation around the hydrophilic implant inserted in the rabbit tibia was confirmed histomorphometrically. Results: The water contact angle of the photo‐induced hydrophilic disk decreased markedly from 43.5° to 0.5°. Cell attachment and proliferation on this hydrophilic disk showed significant improvement. The cell morphology on this hydrophilic disk was extremely flattened, with an elongation of the lamellipodia, whereas a round/spherical morphology was observed on the control disk. The photo‐induced hydrophilic implant enhanced the bone formation with the bone‐to‐metal contact of 28.2% after 2 weeks of healing (control: 17.97%). Conclusion: The photo‐induced hydrophilic surface used in the current study improves the initial cell reactions and enhances early bone apposition to the implant.     

The effect of zirconia and titanium surfaces on biofilm formation and on host-derived immunological parameters

The aim of this study was to analyse the effect of zirconia and titanium surfaces on biofilm formation and host-derived parameters. Studies comparing zirconia and titanium surfaces were selected up to September 1, 2019. The outcome measures were surface roughness, contact angle, bacterial count, bacterial adherence, biofilm thickness, bacterial distribution, and specifically investigated biofilm and specific host-derived immunological parameters. Random-effects meta-analyses of in vitro and in vivo studies were conducted. A total of 39 studies were included for data extraction. In the systematic review data, 10 studies stated that zirconia accumulated less initial oral biofilm parameters, 16 investigations showed negligible inter-material differences, and only one study showed that zirconia attracted the most biofilm. However, in the meta-analysis, the bacterial coverage was found to be significantly superior for zirconia surfaces (P <  0.00001); the other outcome measures did not show any statistically significant differences between zirconia and titanium for the remaining parameters and the studies presented a substantial degree of heterogeneity. Overall, on the basis of the meta-analysis, the current data situation does not allow a clear preference for the use of zirconia or titanium.     

不同pH对变异链球菌体外生物膜形成的影响

目的：了解变异链球菌临床株体外生物膜形成规律以及不同pH对生物膜形成的影响。方法：采用微孔板培养,染色、分光光度测定法（A630）绘制体外不同pH条件下（pH=7.0～5.0）593号、18号菌株以及变异链球菌标准株（ATCC25175）的生物膜生长曲线。结果：体外变异链球菌各株在pH=5.0时均不能形成生物膜;pH=7.0时细菌生物膜形成表现为缓慢的非线性生长,12～24h生物膜开始成熟,24～36h出现一相对的生长停滞期;pH=5.0时对已形成12h的变链菌生物膜生长有明显的抑制作用,但经历12h的酸休克后各菌株的生物膜均能恢复生长。结论：变异链球菌在体外pH=7.0时于12～24h形成稳定的生物膜,该生物膜能抵抗一定程度的酸（pH=5.0）攻击,而浮游状态的细菌则不能。     

Analysis of early biofilm formation on oral implants in man

Biofilm formation on oral implants can cause inflammation of peri-implant tissues, which endangers the long-term success of osseointegrated implants. It has been reported previously that implants revealing signs of peri-implantitis contain subgingival microbiota similar to those of natural teeth with periodontitis. The purpose of the first part of this study was an atraumatic, quantitative investigation of biofilm formation on oral implant abutments; the objective of the second part was to investigate whether Haemophilus actinomycetemcomitans and Porphyromonas gingivalis were present in the crevicular fluid around oral implants. Biofilm formation on 14 healing abutments, inserted for 14 days in 10 patients, was analysed quantitatively by use of secondary-electron and Rutherford-backscattering-detection methods. A 16S rRNA-based polymerase chain reaction detection method was used to detect the presence of H. actinomycetemcomitans and P. gingivalis in the crevicular fluid. For this investigation, samples of sulcus fluid were collected with sterile paper points at four measurement points per abutment. The difference between biofilm coverage of supragingival surfaces (17.5 +/- 18.3%) and subgingival surfaces (0.8 +/- 1.0%) was statistically significant (P < 0.05). By use of universal primers, bacteria were found in all the samples taken, although the two periodontal pathogens were not found in any of the samples. The absence of periodontal pathogens from the sulcus fluid during initial bacterial colonization, despite massive supragingival biofilm formation, substantiates the assumption that cellular adherence of peri-implant tissue by means of hemidesmosoma, actin filaments and microvilli reduces the risk of formation of anaerobic subgingival pockets.     

Inhibitory effects of cranberry juice on attachment of oral streptococci and biofilm formation

Cranberry juice is known to inhibit bacterial adhesion. We examined the inhibitory effect of cranberry juice on the adhesion of oral streptococci strains labeled with [3H]-thymidine to saliva-coated hydroxyapatite beads (s-HA). When the bacterial cells were momentarily exposed to cranberry juice, their adherence to s-HA decreased significantly compared with the control (P < 0.01). Their hydrophobicity also decreased dependently with the concentration of cranberry juice. We also evaluated the inhibitory effect of cranberry juice on biofilm formation. By using a microplate system, we found that the high molecular mass constituents of cranberry juice inhibited the biofilm formation of the tested streptococci. The inhibitory activity was related to the reduction of the hydrophobicity. The present findings suggest that cranberry juice component(s) can inhibit colonization by oral streptococci to the tooth surface and can thus slow development of dental plaque.     

不同培养条件对口腔细菌生物膜生长的影响

目的：研究5种口腔细菌在体外形成生物膜的能力、形成过程及不同培养条件对生物膜形成的影响,并通过激光共聚焦扫描显微镜（CISM）观察细菌生物膜的形成及结构特征。方法：选择与龋病发生关系密切的5种口腔细菌,分别接种在含牛心脑浸液培养基（BHI）、人工唾液（BM5）以及人唾液培养基的标准96孔板中,在培养6h、8h、12h、24h、36h、48h和60h后取出相应的微孔板,11％Crystal Violet染色生物膜,95％乙醇复吸Crystal Violet,HTS7000PLUS多孔板高效分析仪测定各时段细菌生物膜的生长情况,并绘制生长曲线。CLSM观察细菌生物膜形成的结构变化。结果：在BHI及BM5中5种口腔细菌均能形成生物膜,在人唾液中没有形成生物膜。细菌生物膜形成表现为缓慢的非线性生长,出现了一个相对的生长停滞期,这个时期出现在36～48h之间,不同细菌略有差别。激光共聚焦扫描显微镜（asM）观察发现在培养6h后仅有少量细菌粘附形成散在微菌落,24h后出现生物膜的基本结构,48h形成成熟的生物膜。结论：研究发现在体外细菌形成生物膜的能力与培养基条件密切相关,细菌生物膜形成缓慢,其间有一个相对生长停滞期。细菌定植,粘附,共集聚是生物膜形成的重要步骤。     

In situ analysis of multispecies biofilm formation on customized titanium surfaces

Many studies to identify surfaces that enhance the incorporation of dental implants into bone and soft-tissue have been undertaken previously. However, to succeed in the clinical situation, an implant surface must not support development of microbial biofilms with a pathogenic potential. As a first step in investigating this, we used two-species and three-species biofilm models with 16S ribosomal RNA fluorescence in situ hybridization and confocal laser scanning microscopy to examine the effect of surface characteristics on biofilm formation by species that can colonize titanium implants in vivo: Streptococcus sanguinis, Actinomyces naeslundii and Lactobacillus salivarius. Surfaces blasted with Al(2) O(3) (S(a) = 1.0-2.0 μm) showed a seven-fold higher bacterial adhesion after 2 h than turned surfaces (S(a) = 0.18 μm) whereas porous surfaces, generated by anodic oxidation (S(a) = 0.4 μm), showed four-fold greater adhesion than turned surfaces. Hence, increased roughness promoted adhesion, most likely through protection of bacteria from shear forces. Chemical modification of the blasted and oxidized surfaces by incorporation of Ca(2+) ions reduced adhesion compared with the corresponding non-modified surfaces. After 14 h, biofilm growth occurred in the three-species model but not in the two-species consortium (containing S. sanguinis and A. naeslundii only). The biofilm biovolume on all surfaces was similar, suggesting that the influence of surface characteristics on adhesion was compensated for by biofilm development.