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

In vivo and in vitro biofilm formationon two different titanium implantsurfaces

Ralf BurgersTill GerlachSebastian HahnelFrank SchwarzGerhard HandelMartin Gosau

Authors’ affiliations:Ralf Burgers, Sebastian Hahnel, Gerhard Handel,Department of Prosthetic Dentistry, UniversityMedical Center Regensburg, Regensburg, GermanyTill Gerlach, Martin Gosau, Department ofCranio-Maxillo-Facial Surgery, University MedicalCenter Regensburg, Regensburg, GermanyFrank Schwarz, Department of Oral Surgery,Heinrich Heine University, Dusseldorf, Germany

Correspondence to:Ralf BurgersDepartment of Prosthetic DentistryUniversity Medical Center Regensburg93042 RegensburgGermanyTel.: þ 49 941 944 6059Fax: þ 49 941 944 6171e-mail: [email protected]

Key words: biofilm, dental implant, live/dead staining, surface free energy, surface rough-

ness, Streptococcus sanguinis

Abstract

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 (371C, 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 (Ra) was determined with a perthometer,

and surface free energy (SFE) was measured with a goniometer.

Results: Prom showed a significantly higher median Ra (0.95mm) and a significantly lower

median SFE (18.3 mJ/m2) than Pt (Ra¼0.15mm; SFE¼39.6 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 Ra, 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.

The success of dental implants depends on

the osseointegration between the implant

and the surrounding bone as well as on an

inflammation-free contact healing with

the mucosal connective tissue. While the

problems in osseous healing of implants

appear to be largely solved, the sealing of

the implant surface through soft tissue may

be crucial for long-term therapeutic success

(Abrahamsson et al. 1998). Microbial ad-

hesion and the accumulation of pathogenic

biofilms are considered to play major roles

Date:Accepted 22 June 2009

To cite this article:Burgers R, Gerlach T, Hahnel S, Schwarz F, Handel G,Gosau M. In vivo and in vitro biofilm formation on twodifferent titanium implant surfaces.Clin. Oral Impl. Res. 21, 2010; 156–164doi: 10.1111/j.1600-0501.2009.01815.x

156 c� 2009 John Wiley & Sons A/S

mailto:[email protected]

in the pathogenesis of peri-implantitis and

implant loss (Scarano et al. 2004; Elter et al.

2008). Once the clean implant surface is

exposed to the oral cavity, it is immediately

covered by salivary pellicle and colonized

by microorganisms (Elter et al. 2008). Be-

sides sufficient oral hygiene through me-

chanical and chemical cleaning, it is

therefore most important to develop im-

plant surfaces around the transmucosal por-

tion that reduce the number of initially

adhering bacteria, which in turn minimizes

plaque formation and subsequently the in-

flammation of soft tissues (Grossner-

Schreiber et al. 2001, 2004).

The physico-chemical characteristics of

specific material surfaces are known to

significantly influence the bacterial adhe-

sion process (Wu-Yuan et al. 1995; An &

Friedman 1998; Rasperini et al. 1998;

Teughels et al. 2006). Both surface free

energy (SFE) and surface roughness are

known to play a major role in this process,

whereas surface roughness has been sug-

gested to be the predominant of both para-

meters (Nakazato et al. 1989; Esposito et al.

1998; Quirynen et al. 1999, 2002). The

bony and connective tissue interfaces of

most dental implant materials are porous

or micro-textured to support osseointegra-

tion. Therefore, titanium implant surfaces

that are too smooth prevent cell attach-

ment. On the other hand, roughly textured

implant substrata enhance plaque accumu-

lation, whereas high-polished materials

with reduced surface roughness limit initial

biofilm formation in vivo (An & Friedman

1998; Quirynen et al. 1999; Teughels et al.

2006). In this context, a value of 0.2mm

has been generally accepted as the average

roughness threshold below which the

amount of bacterial adhesion cannot be

reduced any further (Bollen et al. 1997;

Grossner-Schreiber et al. 2001; Elter et al.

2008). Therefore, to aid the clinical treat-

ment of bacteria-induced peri-implant de-

structions, exposed implant threads with

rough micro-structure are burnished and

polished (implantoplasty) to prevent exces-

sive plaque accumulation (Lang et al.

2004). The SFE of a solid substratum has

been shown to have crucial impact on the

initial adhesion of oral microorganisms

(Busscher et al. 1984; Weerkamp et al.

1985; Pratt-Terpstra et al. 1988, 1989). In

the thermodynamic model of microbial

adhesion, bacterial strains with high SFE

have a negative interfacial free energy of

adhesion (DFadho0) at substratum surfaces

with high SFE. Therefore, these strains are

expected to preferentially adhere to such

substrata (Weerkamp et al. 1985; Pratt-

Terpstra et al. 1988, 1989). Surface rough-

ness and SFE may be easily modified on

titanium dental implants by sand-blasting,

plasma nitriding, acid etching, titanium

vacuum plasma spraying, or by applying

various chemical coatings (Browne & Greg-

son 1994; Del et al. 2005). As a conse-

quence, a more detailed understanding of

the role of different implant surface proper-

ties in bacterial adhesion and an optimiza-

tion of these physico-chemical

characteristics seems necessary.

For this purpose, a large number of ex-

perimental systems for studying microbial

adhesion in vivo and in vitro are available,

most of which are based on direct colony

counting (Bollen et al. 1996; Drake et al.

1999; Barbour et al. 2007) and scanning

electron microscopy (SEM) observations

(Gatewood et al. 1993; Drake et al. 1999;

Rimondini et al. 2002; Kuula et al. 2004;

Del et al. 2005; Barbour et al. 2008). In

contrast to these traditional microbial

quantification methods, systems based on

biofluorescence have gained increasing im-

portance, because they have become ac-

cepted as simple, precise, reproducible,

and highly sensitive tools for the quantifi-

cation of adhering microorganisms (Grivet

et al. 1999; Grossner-Schreiber et al. 2001;

Gaines et al. 2003; Pier-Francesco et al.

2006; Muller et al. 2007; Buergers et al.

2008; Hahnel et al. 2008). Newly devel-

oped two-color live/dead staining techni-

ques complement the investigative

possibilities by providing a visual differen-

tiation between living and dead bacteria

(Boulos et al. 1999; Hannig et al. 2007;

Al-Ahmad et al. 2008). In this context,

SYTO 9 and propidium iodide staining

has proven superior in comparison with

other assays, because it provides a clear

differentiation between dead and active

microorganisms without interference of

background fluorescence (Decker 2001).

In general, fluorescence microscopy ap-

proaches offer the opportunity to visualize

bacteria directly on the substrata and in the

state of adherence (Hannig et al. 2007).

The present study was conducted to

evaluate the in vitro and in vivo microbial

adhesion to sand-blasted and acid-etched

Promotes

titanium surface in comparison

with machined pure titanium by means of

two highly sensitive fluorescence techni-

ques (Resazurin and live/dead staining)

and to correlate these findings to differ-

ences in surface roughness, SFE, and sur-

face morphology.

Materials and methods

Characterization of titanium surfaces

Two different types of titanium specimens

measuring 9 mm in diameter and 2 mm in

thickness were used. The textured Promotes

titanium surface (Camlog Biotechnologies

AG, Basel, Switzerland) is sand-blasted and

acid-etched and is used for the Camlogs

dental implant system (Camlog Biotechnol-

ogies AG). Promotes

(Prom) surfaces were

compared with machined pure titanium

specimens (Pt; Camlog). The surface rough-

ness (Ra) of five specimens of each of the

two materials on three different sites was

determined with a stylus instrument (Perth-

ometer S6P; Perthen, Gottingen, Germany).

The total SFE, its dispersion, and polar

components were calculated from auto-

mated contact angle measurements (OCA

15 plus; Dataphysics Instruments, Filder-

stadt, Germany) as described before (Hahnel

et al. 2008). SEM was conducted for three

specimens of the two titanium materials.

The specimens were mounted directly on

aluminum stubs and imaged with SEM

(magnification � 600 and � 8000)

(Quanta FEG 400; FEI Company, Eindho-

ven, the Netherlands).

In vivo biofilm formation

Three healthy women (A, B, and C) and

three healthy men (D, E, and F) volun-

teered to participate in the present study

(age 22–38 years, mean: 28 years; all non-

smokers). None of the volunteers had used

antibacterial mouth rinses or systemic

antibiotics for 12 months before the start

of the study. Oral examination was carried

out by an experienced dentist. All volun-

teers had physiological salivary flow rates

(1–1.5 ml/min) and excellent oral hygiene

(plaque index and sulcus bleeding index

close to zero). Informed written consent

had been given by the subjects and the

study had been approved by the Ethics

Committee of the University of Regens-

burg (application 08/131). All specimens

Burgers et al �Microbial adhesion to implant surfaces

c� 2009 John Wiley & Sons A/S 157 | Clin. Oral Impl. Res. 21, 2010 / 156–164

were disinfected by ultrasonication in 3%

sodium hypochlorite (Pharmacy, Univer-

sity Hospital Regensburg, Regensburg,

Germany) for 20 min and then washed in

distilled water. Samples were free of micro-

organisms after this treatment as proven by

SEM and live/dead staining. For in vivo

biofilm formation, the specimens were

fixed to individual removable acrylic upper

jaw splints (Fig. 1), with which the tita-

nium specimens were positioned in the

buccal region of the premolars and first

molars, so that biofilm growth was

not disturbed by tongue movements. Each

volunteer had two Prom and two Pt speci-

mens inserted. Food or beverage consump-

tion and oral hygiene were forbidden during

carriage. The splints were worn for 12 h.

Then, the plaque-covered specimens were

removed from the splints and immediately

processed for fluorescence staining.

All specimens were transferred to well

plates and washed threefold in phosphate-

buffered saline (PBS) to remove non-adher-

ing cells. The Live/Dead BacLight bacterial

viability kit (Molecular Probes, Eugene,

OR, USA) was used to determine the

proportion of live or active cells (fluores-

cent green) and dead or inactive cells (fluor-

escent red). The live/dead stain was

prepared by diluting 18 ml of staining com-

ponent A (SYTO 9) and 18 ml of staining

component B (propidium iodide) in 15 ml

of distilled water. Five hundred microliters

of the reagent mixture were added to each

well, and specimens were incubated at

room temperature and in darkness for

15 min. Each specimen was carefully posi-

tioned on a glass slide covered with com-

ponent C (mounting oil) and stored in the

dark at 41C until further processing. Fluor-

escence emission was determined with a

fluorescence microscope (BX61; Olympus

GmbH, Hamburg, Germany) in combina-

tion with the image processing software

cell ^ P (Olympus GmbH). In each speci-

men, the fluorescent microscopic images

of five randomly selected sites were cap-

tured with a digital camera (U-CMAD3;

Olympus GmbH) that was connected to

the microscope. Living and dead cells in

the same microscopic field were viewed

separately with different fluorescence filter

sets (FITC/F41-054 and Alexa594/

F41-027; AHF Analysetechnik, Tubingen,

Germany) and digitally combined to one

picture. The areas covered by dead cells

(fluorescent red), viable cells (fluorescent

green), and total cells were calculated as

percentage of specific standard microscopic

fields (420 � 320mm¼0.13 mm2) with

the image analysis software Optimas 6.2

(Meyer Instruments, Houston, TX, USA).

In vitro biofilm formation

Streptococcus sanguinis (strain 20068;

DSMZ, Braunschweig, Germany) was

used in this study for in vitro biofilm

formation. After a frozen (�601C) pre-

culture had been established, the bacteria

were exposed on an agar plate and incu-

bated at 371C for 48 h. Cultures from a

single colony were cultivated in sterile

trypticase soy broth (Trytic-Soy Broth; BD

Diagnostics, Sparks, MD, USA) supple-

mented with yeast extract (Sigma-Aldrich,

St Louis, MO, USA) at 371C for 16 h. The

day before the experiment, 1 ml of each

bacterial suspension had been inoculated

with 250 ml of sterile trypticase soy broth

(BD Diagnostics) and incubated at 371C for

12 h. The bacterial suspensions were cen-

trifuged at 896 g at 181C for 5 min and

washed twice in PBS (Sigma-Aldrich). Op-

tical density of the suspensions was ad-

justed to 0.3 at 540 nm.

As described before, the oxidation–

reduction fluorescence dye Alamar Blue/

Resazurin (0.75 g/ml aqua dest) (Sigma-

Aldrich) was used to determine the total

quantity of adhering bacteria (Buergers

et al. 2007). Fluorescence intensities were

recorded by an automated multi-detection

reader (Fluostar optima; BMG Labtech,

Offenburg, Germany) at wavelengths of

530 nm excitation and 590 nm emission.

Unstimulated human saliva was collected

from one healthy donor aged 31 years who

did not show any active carious lesions

or periodontal diseases. The saliva was

sterilized by means of single-use filtration

devices with pore sizes 0.45 and 0.2 mm

(Vacuflo PV050/2 and PV050/3; Schleicher

& Schull Microscience GmBH, Dassel,

Germany). Incubated at 371C for 120 min,

the filtered saliva formed a salivary pellicle

on all specimens (Hahnel et al. 2008). After

removal of saliva, PBS (1 ml) was added to

each well (15 specimens for each material)

and determined the autofluorescence of

each specimen. After the removal of the

buffer, 1 ml of bacterial suspension was

added to each well, and the well plates

were incubated with Resazurin (15ml) at

371C for 120 min. The mixture of bacterial

suspension and Resazurin was extracted by

suction, the wells were subsequently

washed twice, and 1 ml of PBS was added.

Fluorescence intensities after bacterial adhe-

sion were determined as described above.

The fluorescence values of pure PBS (0-

control), buffer and Resazurin (dye-control),

and pure bacterial solution (bacteria-control)

served as control references. High relative

fluorescence intensities (RFIs) indicated

high streptococcal adhesion.

The in vitro pellicle and biofilm forma-

tion was repeated with further five speci-

mens of each material, but without the

addition of Resazurin. After 120-min in-

cubation on an orbital shaker (semi-static

conditions) with S. sanguinis, the propor-

tion of live or active streptococci (fluores-

cent green) and dead or inactive

streptococci (fluorescent red) was deter-

mined with the Live/Dead BacLight bac-

terial viability kit (Molecular Probes) as

described above (please see ‘In vivo biofilm

formation’).

Statistical analysis

Continuous data were summarized by

using medians and interquartile ranges

(25th–75th percentile). Global between-

group comparisons were conducted by the

Brunner–Langer test for the in vivo testing

and by the Kruskal–Wallis rank analysis of

variance for the in vitro testing. The detec-

tion of differences between Ra and SFE, and

the amount of adhering bacteria on the

tested titanium surfaces (n¼ 2) were de-

tected by the pair-wise Mann–Whitney U-

test in combination with the Bonferroni

adjustment (two-sided a¼0.025). Calcula-

tions were done with statistical soft-

ware SPSS 15.0 for Windows (SPSS Corp.,

Fig. 1. Intra-oral micrograph of the individual upper

jaw splint in situ with two titanium specimens in

place.


158 | Clin. Oral Impl. Res. 21, 2010 / 156–164 c� 2009 John Wiley & Sons A/S

Chicago, IL, USA) and SAS (SAS Institute,

Cary, NC, USA).

Results

Surface characterization of titaniumsubstrata

The Kruskal–Wallis rank analysis of var-

iance revealed significant differences be-

tween Pt and Prom in surface roughness

(Ra) and SFE values (Po0.001 for both

comparisons). Significantly higher median

roughness values were found for Prom

(0.95mm) than for Pt (0.15mm). The med-

ian SFE of Pt was significantly higher (total

SFE: 39.6 mJ/m2; dispersion component:

35.9 mJ/m2; polar component: 3.7 mJ/m2)

than the median SFE of Prom (total

SFE: 18.3 mJ/m2; dispersion component:

18.1 mJ/m2; polar component: 0.2 mJ/m2).

SEM images of Prom discs showed a

homogenous roughened micro-structure

(Fig. 2), whereas at a higher magnification,

Pt surfaces showed a circular configuration

of alternating plane and flattened and rough

surface areas (Fig. 3). The adhesion of

microorganisms – both in vitro and in

vivo – corresponded to these circular sur-

face modifications on Pt surfaces, and

bacteria were arranged in orbital patterns

(Fig. 4). On Prom surfaces, microorganisms

in vivo and S. sanguinis in vitro adhesion

tests were uniformly distributed in small

aggregates and did not follow any pattern

(Fig. 5).

In vivo determination of microbial adhe-sion (live/dead staining)

The Brunner–Langer statistical test revea-

led significant differences between the tes-

ted materials for total cells (P¼0.00478),

vital cells (P¼ 0.00215), and dead cells

(P¼ 0.03322). Calculations from live and

dead stainings are given in Figs 6 and 7 and

in Table 1. Examples for the in vivo fluor-

escent microscopic images are shown in

Figs 8, 4, and 5. In general, the six volun-

teers showed significant differences in mi-

crobial adhesion (total bacteria, dead, and

vital bacteria). The assessment of the fluor-

escence micrographs of all volunteers

showed that 1.1% (median) of the surface

of reference material Pt was covered by

microorganisms after 12 h in situ. Signifi-

cantly larger areas were covered by micro-

organisms on Prom (8.5%) (P¼0.00478).

Figure 7 and Table 1 show the percentage of

dead cells among all adhering cells (living

and dead cells). 29.1% of detected micro-

organisms on Pt and 23.7% of those on

Prom were dead (P¼0.03322).

The microorganisms colonized the sur-

face as monolayer or isolated aggregates of

cells, or both, which were randomly dis-

tributed but well defined (cf. Fig. 4). Ecto-

pic solitary epithelial cells from the oral

mucosa – strongly adherent to the substra-

tum – were found on each Prom specimen,

whereas not a single cell was found on Pt

surfaces (cf. Fig. 8).

In vitro determination of microbial adhesion(live/dead and Resazurin staining)

Calculations from the in vitro live/dead

stainings are given in Table 2. Examples

for the in vitro fluorescent microscopic

images are shown in Figs 9 and 10. Accord-

ing to the in vivo results, a significantly

higher amount of adhering S. sanguinis

was found on Prom than on Pt. 11.2%

(median) of the surface of Prom discs was

covered by streptococci, out of which 0.5%

were dead (fluorescent red) and 10.7% vital

(fluorescent green). Significantly smaller

Fig. 2. Scanning electron microscopy (magnification � 8000) of Prom titanium micro-structure.

Fig. 3. Scanning electron microscopy ( � 8000) of machined Pt. Plane (P) areas alternate with rough (R) areas.



areas were covered by S. sanguinis on Pt

(3% in total: 0.4% dead bacteria, 2.7%

vital bacteria) (Po0.001).

Table 2 shows the results from the in

vitro Resazurin quantification method as

RFIs on the two test titanium materials. In

general, statistically significant differences

were found after the Kruskal–Wallis rank

analysis of variance (Po0.001). Higher

RFI, representing higher amounts of adher-

ing streptococci, were found for Prom

[median fluorescence intensity: 13489 re-

lative fluorescence units (RFU)] and signif-

icantly lower RFI, meaning lower amounts

of adhering bacteria, for Pt (9945 RFU)

(Po0.001).

Discussion

The adhesion of oral microorganisms and

the subsequent formation of pathogenic

biofilms on the surface of dental implants

result in infections of the peri-implant

tissues and finally in implant failure.

Peri-implantitis has been reported in 16%

of implant patients after 9–14 years (Roos-

Jansaker et al. 2006) When peri-implant

inflammatory reactions lead to an exposi-

tion of micro-structured implant parts to

the oral cavity, the typical and instinctive

recommendation is to debride these parts

and obtain a smoother surface (Del et al.

2005; Barbour et al. 2007). Scientific

knowledge on the fundamental processes

of bacterial adhesion to implant surfaces is

poor and therapy recommendations on

peri-implantitis are mainly based on em-

pirical values rather than on well-founded

research results. Therefore, the main objec-

tive of the present study was to investigate

in vitro and in vivo microbial adhesion to

two different titanium surfaces by means of

two highly sensitive fluorescence techni-

ques (Resazurin and live/dead staining) and

to correlate these findings to specific sur-

face characteristics [surface roughness (Ra)

and SFE].

S. sanguinis, formerly known as S. san-

guis, was used as a test microorganism for

the in vitro part of the study because of its

common presence in the human oral cav-

ity. Moreover, S. sanguinis is known as a

pioneer bacterium in the oral biofilm

(Grossner-Schreiber et al. 2001; Rimondini

et al. 2002; Mabboux et al. 2004; Del et al.

2005). Additionally, Drake et al. (1999)

Fig. 4. Fluorescence micrograph (live/dead staining) of Pt surface after 12 h in situ. Circular arrangement of

microorganisms following the substratum surface configuration as seen in Fig. 3 (scale bar¼ 100mm).

Fig. 5. Fluorescence micrograph (live/dead staining) of Prom titanium surface after 12 h in situ biofilm

formation. Vital (green) and dead (red) streptococci (scale bar¼ 100mm).

Fig. 6. Areas (%) covered by bacteria after 12 h of in vivo biofilm formation in the oral cavity of six volunteers

(A–F) on two different titanium implant surfaces (medians and 25/75 percentiles).



showed that hydrophobic titanium surfaces

were preferentially colonized by S. sangui-

nis (rather hydrophobic). The tested tita-

nium is the most frequently used material

for the construction of implant systems in

modern dentistry. Tailor-made modifica-

tions of titanium implant surfaces have

been invented to optimize the bone binding

potential of dental implants. In general,

these surface modifications lead to increased

Ra and variable changes in SFE, which in

turn changes the adhesion potential of such

titanium surfaces (Pier-Francesco et al.

2006).

In in vitro testing, a significantly higher

microbial quantity of adhering S. sanguinis

was found on Prom than on Pt (Resazurin

technique: 1.4-fold; live/dead staining: 3.7-

fold). This phenomenon was even more

pronounced in in vivo testing (7.7-fold

higher plaque accumulation on Prom than

on Pt), probably because of the sheltering

effect of rough surfaces against removal

forces, which do not occur under semi-

static in vitro conditions (Rimondini et al.

2002). In general, the higher number of

adhering microorganisms on Prom can be

explained by different surface characteris-

tics such as Ra and SFE. Many studies have

been conducted on the influence of surface

characteristics on restorative and prosthetic

materials. In contrast, little information

is available on fundamental interactions

between microorganisms and implant sur-

faces. Additionally, detailed observations of

the corresponding implant substrata and

their specific surface properties have often

been disregarded.

One reported risk for peri-implant micro-

bial adhesion and resulting peri-implantitis

is high surface roughness of the transmu-

cosal component of an implant (Quirynen

et al. 2002). Current opinion indicates that

low SFE materials with reduced Ra limits

initial microbial adhesion (An & Friedman

1998; Quirynen et al. 1999). Our median

SFE values of the two tested titanium

materials are consistent with SFE values

found by Grossner-Schreiber et al. (2001)

(who reported SFE values of different

titanium surfaces between 34.3 and

38.71mJ/m2 including low polar compo-

nents) and Mabboux et al. (2004) (SFE

values of 43.8 and 44.6 mJ/m2 also with

low polar components). In the present

study, Prom showed significantly higher

Ra values, lower SFE values, and a higher

in vitro and in vivo microbial adhesion

than Pt. Therefore, the influence of Ra on

plaque accumulation appears to be more

important than the influence of SFE. Many

studies postulated a predominant influence

of Ra in contrast to SFE, which could be

affirmed by our investigation (Bollen et al.

1996, 1997; Quirynen et al. 1999; Pier-

Francesco et al. 2006; Teughels et al. 2006).

Some in vivo studies indicated a threshold

Table 1. In vivo microbial colonization on two different titanium implant surfaces after12 h indicated by live/dead fluorescence staining (medians and 25/75 percentiles)

Resazurin(in vitro)

Live/dead staining (in vitro)

Relativefluorescenceintensity (RFI)]

Totalbacterian

Deadbacterian

Vitalbacterian

Ratio deadto totalbacteria (%)

Prom (micro-

structure)

13,489

(11,979/14,878)

11.2 (8.7/13.7) 0.5 (0.4/0.6) 10.7 (8.3/13.2) 3.7 (3.5/4.3)

Pt (machined) 9945

(8264/10,907)

3 (1.7/3.6) 0.4 (0.2/0.4) 2.7 (1.5/3.2) 11.4 (10.8/12.9)

nSurface area covered by bacteria (%).

Fig. 8. Fluorescence micrograph (live/dead staining) of Prom titanium surface after 12 h in situ. Adherent oral

mucosal epithelial cells and low bacterial colonization with vital (green) and dead (red) bacteria (scale

bar¼ 100 mm).

Fig. 7. Percentage of dead to total bacteria after 12 h of in vivo biofilm formation on two different titanium

implant surfaces (medians and 25/75 percentiles) in the oral cavity of six volunteers (A–F).



Ra of 0.2mm, below which no further

reduction of bacterial accumulation could

be expected (Bollen et al. 1996, 1997). The

enhanced bacterial adhesion on Prom sup-

ported this thesis. According to Verran &

Boyd (2001), Prom (median Ra¼ 0.95mm)

can be described as micro-rough and Pt

(median Ra¼ 0.15mm) as nano-rough. Mi-

croorganisms on rough surfaces are assumed

to be more protected against shear forces,

and initial colonization has been shown to

start from surface irregularities (Nyvad &

Fejerskov 1987; Bollen et al. 1997; Hannig

1999; Quirynen et al. 1999). Surprisingly, a

higher retention of S. sanguinis on rough

surface areas was also shown in the semi-

static in vitro part of our study, in which no

significant shear forces can be expected.

Additionally, rough and micro-textured sur-

faces not only seem to accumulate more

plaque, but this plaque also contains more

pathogenic flora (Bollen et al. 1996).

In summary, it was clearly shown that

rough surfaces enhance bacterial adhesion

both in vivo and in vitro and that the

influence of Ra outweighs the influence

of SFE. As a consequence, all micro-struc-

tured parts that are exposed to the oral

cavity and its microorganisms should be

highly polished to generate an anti-adher-

ent or plaque-reducing effect. The rele-

vance of this procedure was affirmed by

the in vitro and in vivo results of this study.

The amount of adhering bacteria in vivo

showed high intra-individual and inter-

individual variability, which was also

shown by Hannig et al. (2007) even after

shorter incubation times. However, all six

volunteers showed the same differences in

microbial load on the two tested titanium

surfaces (Prom vs. Pt) (Prom4Pt). Addi-

tionally, these differences correlated well to

the in vitro results, which in turn proved the

significance of our in vitro adhesion model.

For the first time, in vivo and in vitro

bacterial adhesion on dental implants was

investigated by means of live/dead staining

to study the influence of different surface

textures on the viability of adhering bac-

teria. Fluorometric techniques in adhesion

studies offer the opportunity to quantita-

Table 2. In vitro colonization of Streptococcus sanguinis on two different titanium implant surfaces indicated by Resazurin and live/deadfluorescence staining (medians and 25/75 percentiles)

Patient Implant material Total cells Dead cells Vital cells Ratio dead to total (%)

A–F Pt (pure titanium) 1.1 (0.7/2.5) 0.4 (0.1/0.6) 0.7 (0.4/1.9) 29.1 (21.8/40.1)Prom (Promote

s

) 8.5 (5.1/12.2) 2.1 (0.9/4.3) 6.6 (3.4/8.2) 23.7 (18/37.4)A Pt 2.8 (1.1/3.8) 0.7 (0.4/0.8) 2.2 (0.5/3.2) 25.8 (24.6/32.2)

Prom 11.5 (9.4/14) 2.9 (1.9/4.4) 10 (7.1/13.9) 23.2 (20.6/28)B Pt 0.6 (0.4/0.7) 0.1 (0.1/0.3) 0.4 (0.3/0.5) 26.9 (18.1/44.5)

Prom 5.8 (4.9/7.5) 1.2 (1/2.8) 4.5 (3.3/5.3) 23.4 (17.7/37.3)C Pt 1.3 (0.4/1.8) 0.3 (0.1/0.4) 0.9 (0.4/1.3) 28.2 (14/37.3)

Prom 12.7 (8.1/18.6) 5.5 (3.3/7.7) 7.2 (4.6/11.1) 40.4 (29.4/40.8)D Pt 3.6 (1.9/5.9) 1.1 (0.6/2.1) 1.9 (1.4/4.2) 31.4 (28.9/39.6)

Prom 13.4 (10.4/17.9) 4.6 (2.4/7.8) 9.5 (7.8/10.1) 35.6 (25.9/41.9)E Pt 1.1 (0.9/1.8) 0.5 (0.4/0.5) 0.7 (0.5/1.3) 31.8 (26.4/49.6)

Prom 5.6 (3.4/9) 1.1 (0.7/2.4) 3.4 (2.8/7.3) 21 (18.7/23.4)F Pt 1 (0.7/1.1) 0.2 (0.1/0.4) 0.7 (0.6/0.8) 28.2 (15.4/30.6)

Prom 3.4 (2.8/5.1) 0.5 (0.3/0.8) 2.7 (2.2/4.8) 14.1 (8.4/20.8)

Fig. 9. Fluorescence micrograph (live/dead staining) of Pt surface after in vitro biofilm formation. Vital (green)

and dead (red) streptococci (scale bar¼25 mm).

Fig. 10. Fluorescence micrograph (live/dead staining) of Pt surface after in vitro biofilm formation. Dense

monolayer of vital (green) and dead (red) streptococci (scale bar¼100 mm).



tively investigate a high number of speci-

mens in a short period of time and, at the

same time, to study detailed morphological

aspects by microscopic techniques. No

statistical differences were found between

Prom and Pt with regard to the percentage

of dead and vital bacteria. Therefore, differ-

ences in surface characteristics such as Ra

or SFE – which differed significantly be-

tween Prom and Pt – may not influence

the viability of oral biofilms. Interestingly,

a high percentage of dead bacteria among

the adhering cells (Pt: 29.1% and Prom:

23.7%) could be already found after 12 h of

in vivo incubation. Our findings corre-

spond to the results of Hannig et al.

(2007), who found about 40% of dead

bacteria after different intra-oral incubation

times. In conclusion, the role of dead

biological material in the formation process

of (oral) biofilms may have been under-

estimated in the past and should be further

investigated. In general, the information

obtained from fluorescence techniques is

mainly quantitative in nature. Therefore,

the species composition of the microbial

communities on the tested specimens was

not determined. In contrast to previously

conducted molecular methods, no conclu-

sions on biofilm composition could be

drawn (Amann et al. 1995).

In vitro models are frequently used in

studies on microbial adhesion on specific

substrata, because they are reproducible and

cost-effective. Additionally, the interpreta-

tion of results is simplified by the user-

defined inclusion and exclusion of specific

influencing factors. In comparison, in vivo

models offer the opportunity to evaluate

materials in realistic clinical conditions, pre-

senting composite plaque, co-adhering mi-

croorganisms, salivary pellicle, and removal

forces by salivary flow and chewing activ-

ities (Rimondini et al. 2002). With the

benefit of hindsight, our in vitro results are

in strong accordance with our in vivo results.

Therefore, our semi-static in vitro model in

combination with the Resazurin and the

live/dead techniques represent realistic and

reproducible models for testing the bacterial

adhesion on dental implants.

Adherent solitary epithelial cells from

the oral mucosa were found on each

Prom specimen, whereas not a single cell

was found on Pt surfaces. This secondary

finding showed that material surface char-

acteristics, which have a pivotal influence

on plaque accumulation, also influence the

formation of epithelial attachment. The

effect of Ra and SFE on attachment, orien-

tation, and proliferation of human gingival

fibroblasts has been demonstrated in various

other studies (Kononen et al. 1992; Mustafa

et al. 1998; Lauer et al. 2001; Rimondini et

al. 2002). Although this hypothesis was not

tested in our study, the high number of

ectopic epithelial cells on Prom – strongly

adherent to the surface – seems to suggest

that Prom may be a promising material

capable of enhancing cell attachment for-

mation and osseointegration.

Biological contamination is difficult to

remove from structured implant surfaces,

e.g. by the use of plastic curettes or sonic/

ultrasonic scalers (Schwarz et al. 2009).

Thus, in addition to making dental implants

with high bone binding potentials, it is

important to develop implant surfaces that

reduce the number of initially adhering

bacteria. At this point in time, these two

requests for the construction of implant

materials unfortunately contradict each

other. In general, manufacturers’ modifica-

tions of implant surfaces and the treatment

recommendations on exposed implant struc-

tures should be based on research results.

Therefore, further investigations are needed

into the essential proceedings of bacterial

adhesion on implant surfaces. As titanium

itself does not show any antibacterial activ-

ity, anti-adhesive coatings or alloys to pro-

vide bactericidal surfaces may be an

alternative to conventional invasive peri-

implantitis therapies.

Conclusions

Within the limitations of the present study,

our findings indicate that in vitro and

in vivo adhesion of bacteria to differently

textured titanium surfaces is primarily in-

fluenced by surface roughness. In contrast,

the influence of SFE is of lesser impor-

tance. Therefore, any micro-structured

part that is exposed to the oral cavity

should be highly polished to generate an

anti-adherent and plaque-reducing effect.

A high percentage of dead cells among the

adhering bacteria could be observed on all

titanium specimens. This observation af-

firms the pivotal role of dead biological

material in the formation process of (oral)

biofilms. At last, the two fluorometric

techniques (Resazurin and live/dead stain-

ing) used in the present study proved to be

reproducible, sensitive, and precise tools to

study in vivo and in vitro initial biofilm

formation on titanium implant surfaces.

Acknowledgements: We are grateful

to the Camlog Biotechnologies AG

(Basel, Switzerland) for the supply of

titanium specimens. The authors

declare that they have no conflict of

interest.

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