Identification of Bacteria in Periapical Lesions Using DNA Probes

1
41486
Bacterial Adhesion on Integrated Abutment Crowns
TM. In Vitro Study (I)
ABSTRACT. Objectives The goal of the present
investigation was to determine if the
Diamond-crown material, used to make Bicon's
Integrated Abutment Crown (IAC), is less
susceptible to harbor/attract bacterial plaque
than All Ceramic (AC) or Metal Ceramic (MC)
crowns. A secondary goal was to compare the
composition of the plaque attracted on tested
surfaces. Methods 6 IAC, 6 AC and 6 MC crowns
were equally divided in 2 test groups. The AC and
MC crowns were cemented on titanium abutments.
Group I (3 IAC,3 AC, 3 MC)and group II were
incubated for 10 min in a bacterial solution
containing 10 different oral bacteria at O.D.1
Tanerella forsythensis, Prevotella intermedia,
Campylobacter rectus, Fusobacterium nucleatum,
Actinomyces odontololyticus, A. naeslundii,
Streptococcus intermedius, S. oralis,
Actinobacillus actinomycetemcomitans serotype b,
Porphyromonas gingivalis. After a brief wash in
PBS to eliminate the unbound plaque, the crown
samples in group I were incubated in 500µl of
Tris-EDTA buffer with 500µl of NaOH. The samples
were then hybridized with 10 whole chromosomal
probes to the above mentioned microorganisms. The
microbiological analysis was completed using the
checkerboard DNA-DNA hybridization method. The
samples in group II were briefly washed in PBS
and fixed in 4 formalin for scanning electron
microscopy (SEM) Results All experimental
crowns showed bacterial adhesion. There was no
statistical difference in the microbial
compositions when comparing crowns. The SEM
showed that the AC crowns were harboring the
heaviest bacterial deposits. MC and IAC showed
the least bacterial deposits especially at the
abutment/crown interface. Conclusions The IAC
and MC crowns appear to be less prone to
bacterial colonization, in an in vitro setting
then an all ceramic crown. IAC and MC crowns
harbored very few bacteria at the abutment/crown
interface This study was supported by a research
grant from Bicon, Inc.
S. DIBART1, M. MARINCOLA2, M.L. WARBINGTON1, and
Z. SKOBE3
1 Boston University, MA, USA, 2 University of
Cartagena, AISI, Italian Association of
Restorative Implant Dentistry, Rome, Italy, 3
Forsyth Institute, Boston, MA
INTRODUCTION
The trauma surrounding the partial or total loss
of the natural dentition to periodontal diseases
or caries has been alleviated in the last decades
by the introduction of the concept of predictable
osseointegration to the dental profession 1,2.
Successfully osseointegrated dental implants have
revolutionized the practice of late 20th century
dentistry. They provided patients with the
ability to have fixed restorations instead of
removable devices, and help avoid the mutilation
of adjacent natural teeth when a 3 unit fixed
partial restoration was envisioned. As with all
prosthetic restorations in the oral cavity, they
are subject to factors impacting esthetics,
function, and periodontal health. Periodontal
health or peri-implant health is the most
critical aspect of this trilogy, since
compromising it could
mean potentially disastrous effects on esthetics
and function. The tissues supporting dental
implants are susceptible to disease
(peri-implantitis), which in turn could lead to
bone loss and implant failure. The disease
process is initiated by microorganisms that are
present in the periodontal plaque. These
bacteria and their by-products (enzymes, toxins,
metabolic products), in the susceptible host,
will start a whole cascade of events that will
lead to periodontal tissue damage. bacteria need
to adhere to a solid surface (i.e. prosthetic or
natural crown, soft tissue etc.) in their primary
phase of colonization before causing the disease.
A restoration material that would repel or cause
bacteria to adhere minimally would be a plus in
preventing disease.
In June 2001, Bicon Inc. (Boston, MA) introduced
the Integrated Abutment Crown (IAC). This is a
new concept where the implant abutment and the
crown material are one integral unit (Fig.1). A
poly-ceramic material such as Diamond Crown
(DRM Research Labs Inc., Branford, CT) is fused
onto the coronal post of a titanium alloy
abutment. The IAC is then placed directly into
the well of the implant, there is no need for
cementation or screw retention. The goal of the
present investigation was to determine if the
Diamond Crown material used to make Bicons IAC
is less susceptible to harbor/attract bacterial
plaque than All Ceramic (AC) or Porcelain Fused
to Metal (PFM) crowns.
Figure 1. Clinical photograph of a Bicon
Integrated Abutment Crown. The porcelain is
fused to the abutment, there is no cementation.


A
RESULTS
All experimental crowns (IAC, AC, PFM) showed
bacterial adhesion. All 10 bacterial species
were found to adhere to each individual crowns.
There was no statistical difference in the
microbial composition found on the crowns
investigated. This is probably due to the small
size of the sample that did not show any
statistically significant changes. The SEM
analysis showed a clear difference in terms of
amount and location of the microorganisms on the
various crowns. All ceramic (AC) crowns harbored
the heaviest bacterial deposits (Fig 2) and this
was true for all areas studied, from occlusal
surface to crown margins. Also more than one type
of microorganism could be differentiated
morphologically. This was confirmed by the
microbiological analysis of the samples, all of
the 10 microbial species could be detected using
DNA probes (data not shown). At the
abutment/crown interface a lack of cement was
noticed as well as porosity in the ceramic
structures (Fig 4). Many bacterial colonies were
noticed in that area including in the micro-gap.
Bacteria were also present, albeit at a much
lower number, on porcelain fused to metal (PFM)
and integrated abutment crowns (IAC). Bacterial
colonization was also distributed differently. A
few clusters of bacteria were present on the
occlusal third of the IAC and extremely few on
the PFM. Of much interest was the observations
carried out at the crown margins (PFM) and
abutment/crown interface (IAC). There were very
few microorganisms observed at the abutment/crown
interface for PFM and IAC. These 2 entities
appeared to show the least amount of bacterial
deposits (Figs. 3 and 4).
METHODS
Figure 3. Scanning electron micrograph of a
Porcelain fused to metal crown. Top of the
micrograph shows crown abutment junction, with
cement closing the gap. Lower half of the
micrograph shows the porcelain margin at high
magnification, with very few or no bacteria
present.
In the in vitro phase we tested bacterial
presence and composition comparing the Integrated
Abutment Crown to All Ceramic (porcelain Noritake
Cerabien, Noritake Co., Nagoya, Japan) and
Porcelain Fused to Metal (Noritake Super
Porcelain EX-3, Noritake Co., Nagoya, Japan)
crowns in a laboratory setting. The All Ceramic
and the Porcelain Fused to Metal crowns were
cemented on the abutments using Fuji Plus cement
(GC Corporation, Tokyo, Japan). A total of 18
crowns were analyzed for bacterial
presence/adhesion. All the crowns were provided
by the Bicon corporation for our testing. The 18
crowns were divided in 2 groups. Group 1 which
underwent microbiological analysis and group 2
which underwent SEM analysis. These groups were
further sub-divided in 1A (3 IAC), 1B (3 All
Ceramic Crowns), 1C (3 Porcelain Fused to Metal
Crowns) and 2A, 2B, 2C. The 9 crowns of group 1
were incubated for 10 minutes in a 100 ml PBS
solution containing 10 oral bacteria at optical
density of 1 Tannerella forsythensis, Prevotella
intermedia, Campylobacter rectus, Fusobacterium
nucleatum, Actynomyces odontolyticus, A.
naeslundii, Streptococcus intermedius, S. oralis,
Actinobacillus actinomycetemcomitans serotype b,
Porphyromonas gingivalis. The crowns were
immersed in a beaker containing the bacterial
solution, with a stir bar. The crowns were
removed and rinsed in sterile PBS for 30 seconds
and put in a 15 ml tube containing 0.5 ml of TE
(10 mM Tris-HCl, 1 mM EDTA, pH 7, 6). The
bacterial DNA was denatured by adding 0.5 ml of a
0.5M solution of NaOH. The samples were then
hybridized with 10 whole chromosomal DNA probes
to these microorganisms according to the protocol
described by Socransky et al. The 9 crowns of
group 2 were also incubated for 10 minutes in the
above mentioned bacterial mixture and rinsed in
sterile PBS for 30 seconds. They were then fixed
in 4 formalin at room temperature overnight,
metal coated and prepared for scanning electron
microscopy.





Figure 2. Scanning electron micrograph of an all
ceramic crown-abutment junction. Notice the gap
and lack of cement between prosthesis (top) and
implant abutment (bottom).
Figure 4. Scanning electron micrograph of an
Integrated Abutment Crown. Top half of the
micrograph shows crown-abutment interface, with
no gap. Lower half of the micrograph shows the
crown margin at high magnification, with very few
or no bacteria present.

CONCLUSION
The results of this in vitro study seem to show
that bacterial presence is inevitable on any type
of prosthetic restoration. However the IAC
design seems to go toward reducing this
eventuality by eliminating the gap between
implant abutment and crown, and providing a
smooth cervical interface.