university of zurich zurich open ......the cell or tissue to regulate bone resorption. an increased...
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University of ZurichZurich Open Repository and Archive
Winterthurerstr. 190
CH-8057 Zurich
http://www.zora.uzh.ch
Year: 2011
Oral biofilm challenge regulates the RANKL-OPG system inperiodontal ligament and dental pulp cells
Belibasakis, G N; Meier, A; Guggenheim, B; Bostanci, N
http://www.ncbi.nlm.nih.gov/pubmed/21075196.Postprint available at:http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch
Originally published at:Belibasakis, G N; Meier, A; Guggenheim, B; Bostanci, N (2011). Oral biofilm challenge regulates theRANKL-OPG system in periodontal ligament and dental pulp cells. Microbial Pathogenesis, 50(1):6-11.
http://www.ncbi.nlm.nih.gov/pubmed/21075196.Postprint available at:http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch
Originally published at:Belibasakis, G N; Meier, A; Guggenheim, B; Bostanci, N (2011). Oral biofilm challenge regulates theRANKL-OPG system in periodontal ligament and dental pulp cells. Microbial Pathogenesis, 50(1):6-11.
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Oral biofilm challenge regulates the RANKL-OPG system inperiodontal ligament and dental pulp cells
Abstract
Inflammatory bone destruction triggered by oral bacteria is a hallmark of chronic and apicalperiodontitis. Receptor activator of NF-κB ligand (RANKL) activates bone resorption, whereasosteoprotegerin (OPG) blocks its action. These are members of the tumor necrosis factor ligand andreceptor families, respectively. Although individual oral pathogens are known to regulate RANKL andOPG expression in cells of relevance to the respective diseases, such as periodontal ligament (PDL) anddental pulp (DP) cells, the effect of polymicrobial oral biofilms is not known. This study aimed toinvestigate the effect of the Zürich in vitro supragingival biofilm model on RANKL and OPG geneexpression, in human PDL and DP cell cultures, by quantitative real-time polymerase chain reaction.RANKL expression was more pronouncedly up-regulated in DP than PDL cells (4-fold greater),whereas OPG was up-regulated to a similar extent. The RANKL/OPG ratio was increased only in DPcells, indicating an enhanced capacity for inducing bone resorption. The expression of pro-inflammatorycytokine interleukin-1β was also increased in DP, but not PDL cells. Collectively, the highresponsiveness of DP, but not PDL cells to the supragingival biofilm challenge could constitute aputative pathogenic mechanism for apical periodontitis, which may not crucial for chronic periodontitis.
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University of ZurichZurich Open Repository and Archive
Winterthurerstr. 190
CH-8057 Zurich
http://www.zora.uzh.ch
Year: 2011
Oral biofilm challenge regulates the RANKL-OPG system inperiodontal ligament and dental pulp cells
Belibasakis, G N; Meier, A; Guggenheim, B; Bostanci, N
Belibasakis, G N; Meier, A; Guggenheim, B; Bostanci, N (2011). Oral biofilm challenge regulates theRANKL-OPG system in periodontal ligament and dental pulp cells. Microbial Pathogenesis, 50(1):6-11.Postprint available at:http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch
Originally published at:Microbial Pathogenesis 2011, 50(1):6-11.
Belibasakis, G N; Meier, A; Guggenheim, B; Bostanci, N (2011). Oral biofilm challenge regulates theRANKL-OPG system in periodontal ligament and dental pulp cells. Microbial Pathogenesis, 50(1):6-11.Postprint available at:http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch
Originally published at:Microbial Pathogenesis 2011, 50(1):6-11.
-
Oral biofilm challenge regulates the RANKL-OPG system inperiodontal ligament and dental pulp cells
Abstract
Inflammatory bone destruction triggered by oral bacteria is a hallmark of chronic and apicalperiodontitis. Receptor activator of NF-κB ligand (RANKL) activates bone resorption, whereasosteoprotegerin (OPG) blocks its action. These are members of the tumor necrosis factor ligand andreceptor families, respectively. Although individual oral pathogens are known to regulate RANKL andOPG expression in cells of relevance to the respective diseases, such as periodontal ligament (PDL) anddental pulp (DP) cells, the effect of polymicrobial oral biofilms is not known. This study aimed toinvestigate the effect of the Zürich in vitro supragingival biofilm model on RANKL and OPG geneexpression, in human PDL and DP cell cultures, by quantitative real-time polymerase chain reaction.RANKL expression was more pronouncedly up-regulated in DP than PDL cells (4-fold greater),whereas OPG was up-regulated to a similar extent. The RANKL/OPG ratio was increased only in DPcells, indicating an enhanced capacity for inducing bone resorption. The expression of pro-inflammatorycytokine interleukin-1β was also increased in DP, but not PDL cells. Collectively, the highresponsiveness of DP, but not PDL cells to the supragingival biofilm challenge could constitute aputative pathogenic mechanism for apical periodontitis, which may not crucial for chronic periodontitis.
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Oral biofilm challenge regulates the RANKL-OPG system in
periodontal ligament and dental pulp cells
Georgios N. Belibasakis *, Andre Meier, Bernhard Guggenheim,
Nagihan Bostanci
Section of Oral Microbiology and General Immunology, Institute of Oral
Biology, Center for Dental Medicine, Faculty of Medicine, University of
Zürich, Switzerland
* Corresponding Author:
Dr. Georgios N. Belibasakis
Institute of Oral Biology
Center for Dental Medicine
University of Zürich
Plattenstrasse 11
8032 Zürich
Switzerland
e-mail: [email protected]
Tel: +41-44-6343329
mailto:[email protected]
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Abstract
Inflammatory bone destruction triggered by oral bacteria is a hallmark of chronic and
apical periodontitis. Receptor activator of NF-?�B ligand (RANKL) activates bone
resporption, whereas osteoprotegerin (OPG) blocks its action. These are members of
the tumor necrosis factor ligand and receptor families, respectively. Although
individual oral pathogens are known to regulate RANKL and OPG expression in cells
of relevance to the respective diseases, such as periodontal ligament (PDL) and dental
pulp (DP) cells, the effect of polymicrobial oral biofilms is not known. This study
aimed to investigate the effect of the Zürich in vitro supragingival biofilm model on
RANKL and OPG gene expression, in human PDL and DP cell cultures, by
quantitative real-time polymerase chain reaction. RANKL expression was more
pronouncedly up-regulated in DP than PDL cells (4-fold greater), whereas OPG was
up-regulated to a similar extent. The RANKL/OPG ratio was increased only in DP
cells, indicating an enhanced capacity for inducing bone resorption. The expression of
pro-inflammatory cytokine interleukin-1� was also increased in DP, but not PDL
cells. Collectively, the high responsiveness of DP, but not PDL cells to the
supragingival biofilm challenge could constitute a putative pathogenic mechanism for
apical periodontitis, which may not crucial for chronic periodontitis.
Keywords: Oral biofilm, periodontal ligament, dental pulp, receptor activator of NF-
k� ligand; osteoprotegerin, interleukin-1�
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1. Introduction
Periodontal diseases are perhaps the most common chronic inflammatory diseases in
humans. Their main trait is the inflammatory destruction of the tooth-supporting
(periodontal) tissues, as a result of oral bacteria colonizing the tooth surfaces in the
form of polymicrobial biofilm communities [1]. Depending on the localization of the
biofilm in relation to the gingival margin, this can be either “supragingival” or
“subgingival”. Biofilms or their released products can cause an inflammatory
response by the periodontal tissues, aiming to eliminate this bacterial challenge [2].
However, an excessive inflammatory response would induce tissue damage, rather
than being protective [3]. Gingivitis is a clinical condition where the host
inflammatory response to the biofilm is restricted to the superficial gingival tissue.
However, when the inflammation expands into the deeper periodontal tissues, it will
develop into periodontitis, which is characterised by the destruction of the tooth-
surrounding alveolar bone and interconnecting periodontal ligament (PDL). If left
untreated, periodontitis will eventually lead to tooth loss.
Apical periodontitis results from the spread of the inflammatory material from
an infected dental pulp (DP) into the periradicular tissues. The etiological agent is
mixed bacterial species that reach the dental pulp as a result of carious lesions,
endodontic infection or trauma [4]. In the initial phase of apical periodontitis, the
inflammation is restricted to the apical PDL space without concomitant bone
resorption. However, the prolonged presence of bacterial challenge in the region
could eventually develop into chronic apical periodontitis. This condition is
associated with periapical bone resorption and potentially the formation of a cyst,
depending on the virulence of the invading bacterial species [5]. For the resolution of
this condition endodontic treatment is required.
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Bone resorption in physiological and pathological conditions, such as
periodontitis, is regulated by the interplay of a system of two molecules belonging to
the tumor necrosis factor (TNF) ligand and receptor superfamilies. These are
respectively Receptor Activator of NF-?�B Ligand (RANKL) and osteoprotegerin
(OPG). RANKL is expressed as a membrane bound or secreted ligand by osteobasts,
fibroblasts and activated T- and B-cells. By activating its cognate RANK receptor on
osteoclast precursors (cells of the monocyte/macrophage lineage), it triggers their
fusion and differentiation into multi-nucleated osteoclasts, which will resorb bone [6].
RANKL can be cleaved and released from the cell surface by a metalloprotease
known as tumour necrosis factor-a converting enzyme (TACE)/ADAM17 [7]. The
soluble decoy receptor OPG acts as an inhibitor of RANKL by binding to it and thus
preventing the RANK-RANKL interaction and downstream events [8]. Changes in
the relative RANKL/OPG expression ratio are considered indicative of the capacity of
the cell or tissue to regulate bone resorption. An increased ratio can be indicative of
enhanced bone resorption in pathological inflammatory conditions, such as
periodontitis [9, 10]. The RANKL-OPG system is regulated by a number of local and
systemic factors, including the pro-inflammatory cytokine interleukin(IL)-1� [11].
Excessive production of IL-1� is considered detrimental for tissue destruction in
periodontal disease [12, 13] and apical periodontitis [5].
Both the PDL and DP contain mesenchymal cells that express RANKL and
OPG and can support osteoclastogenesis [14]. The balanced expression of these
factors is decisive for the regulation of bone resorption by DP cells [15] or PDL cells
[16]. Evidence demonstrates that RANKL is expressed in both PDL and DP tissue in
periapical lesions [17, 18] and during root resorption [19].
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The current knowledge on the pathogenic mechanisms of periodontal diseases
comes mostly from studies using bacteria in planktonic state, due to the complication
and high technical demands in employing multi-species in vitro biofilm models.
Nevertheless, the use of oral biofilms rather than individual oral bacterial species
provides a more accurate view of the pathogenic events that take place in periodontal
or periapical diseases, such as bone resorption. The present study employs an in vitro
supragingival biofilm model, aiming to investigate its effects on RANKL, OPG,
TACE and IL-1� gene expression in human primary PDL and DP cell cultures.
2. Materials and Methods
2.1 In vitro biofilm model
The 6-species suprgagingival Zürich biofilm model [20] used in this study consisted
of Veillonella dispar ATCC 17748 (OMZ 493), Fusobacterium nucleatum KP-F8
(OMZ 598), Streptococcus oralis SK248 (OMZ 607), Actinomyces naeslundii (OMZ
745), Streptococcus mutans UAB159 (OMZ 918) and Candida albicans (OMZ 110).
Biofilms were grown in 24-well cell culture plates on sintered hydroxyapatite (HA)
discs, resembling natural tooth surfaces, and were pre-conditioned for pellicle
formation with foetal calf serum (FCS) at 1:1 dilution, for 4 h. To initiate biofilm
formation, HA discs were covered for 16.5 h with 1.6 ml of growth medium
consisting of 70% FCS (dilution 1:10), 30% FUM culture medium [21] containing
0.3% glucose, and 200 µl of a bacterial cell suspension containing equal volumes and
density from each strain. After 16.5 h of anaerobic incubation at 37°C, the inoculum
suspension was removed from the discs by 'dip-washing' using forceps, transferred
into wells with fresh medium (70% FCS dilution 1:10, and 30% FUM containing
0.15% glucose and 0.15% sucrose), and incubated for further 48 h in anaerobic
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atmosphere. During this time-period, the discs were dip-washed 3x and given fresh
medium once daily. After a total 64.5 h of incubation, one biofilm-carrying HA disc
was placed per cell culture well, with the biofilm-coated surface facing towards the
cell monolayer. A plastic ring support ensured a distance of 1 mm between the
biofilm and the cell monolayer, allowing fluid flow. This form of biofilm challenge
was maintained in the cell culture for up to 24 h [22].
2.2 Cell cultures
Human PDL and DP tissue were obtained from a periodontally healthy young
individual who had their first premolar extracted in the course of orthodontic
treatment. The procedure was performed with the consent of the donor and the
approval of the Ethical Committee of the Center of Dental Medicine, University of
Zürich, abiding by the guidelines for studies with human cells of irreversibly
anonymized origin and not subject to any form of storage in a bio-bank. After
removal from the tooth, the tissues biopsies were dissected into smaller specimens
and maintained in culture. The cells were cultured in Minimum Essential Medium
(MEM) Alpha medium (Gibco), supplemented with 10 % heat-inactivated FCS
(Sigma), 50 U/ml penicillin, and 50 µ�g/ml streptomycin (Sigma). The explanted PDL
and DP cells were then passaged. Both cell types exhibited spindle-shaped fibroblast-
like morphology. Further characterisation was performed by analysing gene
expression levels of collagen type Ia�1, alkaline phosphatase and nestin in these cells
by quantitative real-time PCR. Both PDL and DP cells constitutively expressed these
markers. Collagen type Ia�1 and nestin were expressed 2.0-fold and 4.1-fold higher,
respectively, in PDL compared to DP cells. Alkaline phosphatase expression in DP
cells was only 0.2-fold higher than PDL cells (data not shown). For the experiments,
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DP or PDL cells at passage 3 were seeded at concentration 20 x 103 cells/cm2 in
antibiotics-free and 5% FBS culture medium, and were allowed to attach for 24 h,
maintaining a sub-confluent status. Thereafter, the cells were co-cultured for up-to 24
h in the presence or absence of the supragingival biofilm, or the non-biofilm coated
hydroxyapatite, again in antibiotics-free and 5% FBS culture medium.
2.3 Cytotoxicity assay
The putative cytotoxic effects of the biofilm on PDL and dental pulp cell cultures
were evaluated by measurement of the extracellularly released cytosolic lactate
dehydrogenase (LDH), using the CytoTox96® Non-Radioactive Cytotoxicity Assay
(Promega). In brief, PDL or DP cell cultures were exposed to the established biofilm
for 6 h and 24 h. Cell culture supernatants were thereafter collected, and the cell
monolayer was lysed in equal volume of lysis buffer, provided in the kit. The
collected suspensions, either cell-culture supernatant or cell lysate, were centrifuged
at 1000 rpm for 5 min to pellet down any cell debris, and thereafter 50 µ�l/well of each
were transferred into an optically clear 96-well plate. Fifty µl of the reaction solution
was added to each well and incubated for 30 min in the dark. The reaction was then
stopped and the absorbance was measured at 490 nm, subtracting background values
from all samples.
2.4 RNA extraction and cDNA synthesis
Upon termination of the experiments, the culture media were discarded, and the cell
monolayers were washed twice in PBS before being lysed. The collected cell lysate
was homogenized with QIAshredder (QIAGEN), and total RNA was extracted by
using the RNeasy Mini Kit (QIAGEN), according to the manufacturer’s instructions.
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The extracted RNA was finally eluted in 40 µ�l RNase free water and its concentration
was determined spectrophotometrically. One µ�g of total RNA was reverse transcribed
into single-stranded cDNA by using M-MLV Reverse Transcriptase (RNase H Minus,
Point Mutant), Oligo(dT)15 Primers, and PCR Nucleotide Mix according to the
manufacturer’s protocol (all from Promega), at 40°C for 60 min, and 70°C for 15 min.
The resulting cDNA was stored at -20°C until further use.
2.5 Quantitative real-time PCR
For RANKL, OPG, TACE and IL-1� gene expression analyses, quantitative real-time
PCR was performed in an ABI Prism 7000 Sequence Detection System and software
(Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and
RNA Polymerase II (RP2) were used as housekeeping genes of the samples. The
amplification reactions were performed with qPCR SYBR Master Mix (Applied
Biosystems). The standard PCR conditions were 10 min at 95ºC, followed 40 cycles
at 95ºC for 15 seconds, 60ºC for 1 min, and 72°C for 30 seconds. The expression
levels of RANKL, OPG, TACE and IL-1� transcripts in each sample were calculated
by the comparative Ct method (2-?�Ct formula) after normalization to the respective
average Ct value of the two housekeeping genes. The primer sequences were for
RANKL (NM003701) forward: GTCTGCAGCGTCGCCCTGTT and reverse:
ACCATGAGCCATCCACCATCGC, for OPG (NM002546) forward:
CGCCTCCAAGCCCCTGAGGT and reverse: CAAGGGGCGCACACGGTCTT, for
TACE (NM003183) forward: GGCGTGTCCTACTGCACAGGT and reverse:
GGGCACACAGCGGCCAGAAA, for IL-1� (NM000576) forward:
ACGCTCCGGGACTCACAGCA and reverse: TGAGGCCCAAGGCCACAGGT,
for collagen type Ia�1 (NM000088) forward: AAGATGGACTCAACGGTCTC and
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reverse: CAGGAAGCTGAAGTCGAAAC, for alkaline phosphatase (BC136325)
forward: ATGAAGGAAAAGCCAAGCAG and reverse:
ATGGAGACATTCTCTCGTTC, for nestin: (NM006617) forward:
CCTGCAAAAGGAGAATCAAG and reverse: GTTCTCAATGTCTCTTGGTC, for
GAPDH (NM002046) forward: CAGCCTCCCGCTTCGCTCTC and reverse:
CCAGGCGCCCAATACGACCA, and for RP2 (NM000937) forward:
CCCGCTGCGCACCATCAAGA and reverse: CCCCTGCCTCGGGTCCATCA.
2.6 Statistical analysis
The Student’s t-test for unpaired values was used to compare the differences between
control and test groups, and data were considered significant at P
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On the next step, the effect of biofilm challenge on RANKL gene expression by the
cells was investigated. After 3 h, RANKL expression was not affected in PDL cells,
but after 6 h this was significantly increased by 2.3-fold, compared to the control (Fig.
1A). However, in DP cells RANKL expression was already increased by 8.0-fold and
12.5-fold at 3 h and 6 h, respectively (Fig. 1B).
3.3 Effects of the biofilm on OPG gene expression
The expression of OPG in response to the biofilm was also investigated in this
experimental system. There were no changes after 3 h of challenge, but after 6 h this
was increased by 1.7-fold in PDL cells (Fig. 2A) and 1.9-fold in DP cells (Fig. 2B).
3.4 Regulation of the RANKL/OPG ratio by the biofilm
These regulations in RANKL and OPG expression resulted in changes in the relative
RANKL/OPG expression ratio, which is a measure indicative of the capacity of the
cells to induce osteoclastogenesis and bone resorption. Compared to the control, this
ratio was increased after 6 h of biofilm challenge by approximately 1.3-fold in PDL
cells (Fig. 3A) and 7.0-fold in DP cells (Fig. 3B). This indicates that the supragingival
biofilm has the capacity to regulate the RANKL-OPG system favourably for bone
resorption in DP cells, but not in PDL cells.
3.5 Effects of the biofilm on TACE gene expression
The gene expression of TACE was also investigated in response to the supragingival
biofilm challenge. The results indicate that there were no changes in TACE
expression after 3 h of challenge, but after 6 h this was increased by approximately
1.4-fold in PDL cells (Fig. 4A), and 1.6-fold in DP cells (Fig. 4B).
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3.6 Effects of the biofilm on IL-1β gene expression
Finally, the gene expression of IL-1� was also investigated in response to the
supragingival biofilm challenge. The results indicate that there were no significant
changes in IL-1� expression after 3 h of challenge. After 6 h, IL-1� expression was
increased by approximately 1.5-fold in PDL cells (Fig. 5A), and 1.9-fold in DP pulp
cells (Fig. 5B), but only the latter proved to be statistically significant.
4. Discussion
This is the first to study to employ an in vitro biofilm to investigate the regulation of
the RANKL-OPG system. Most host-bacteria interactions models have so far
employed single bacterial species to address such questions. The obvious advantage
of the biofilm approach is that several bacterial species are represented in a
conformation that resembles their natural state, when attached on the tooth surface.
An in vitro subgingival oral biofilm model has so far been used to study apoptotic and
pro-inflammatory cytokine stimulating effects in gingival epithelial cells [22].
The present findings demonstrate that the supragingival biofilm challenge
increases both RANKL and OPG gene expressions in both PDL cells and DP cells.
None of the six bacterial species present in this in vitro biofilm have been previously
studied as for their capacity to regulate the RANKL-OPG system in host cells.
Putative periodontal pathogens, such as Aggregatibacter actinomycetemcomitans and
Porphyromonas gingivalis, have been studied individually and have been shown to
up-regulate RANKL expression in PDL cells [23, 24], but DP cells have not been
studied in this respect. In the present experimental system, although the magnitude of
OPG induction was similar between the two cell types, there were quantitative
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differences in RANKL expression. In particular, the supragingival biofilm caused 4-
fold greater RANKL up-regulation in DP cells compared to PDL cells, indicating that
the former cell type is more responsive to this challenge. Such differences have been
previously considered, for instance in the case of PDL cells and gingival fibroblasts
[23, 24], and could indicative of distinct roles of the cells in the pathogenesis of
periodontitis. This may reflect the nature of the respective diseases, in which these
cells are involved. To this extent, RANKL in periapical lesions has been particularly
associated with fibroblastic cells, rather than T-cells [25], as opposed to periodontitis,
where cells of the immune system are considered a major source of RANKL [9, 26,
27]. When the relative RANKL/OPG ratio was calculated, this was significantly
increased in DP cells, compared to PDL cells, indicating a higher capacity for
inducing bone resorption by the former cells. PDL cells could be better adapted to
tackle bacterial challenges, as they are frequently exposed to their products within the
continuously colonized periodontal environment. On the contrary, DP cells are highly
protected within the specialized niche of the pulp cavity and thus less adapted to
bacterial challenges, as they are not directly exposed to them. A potential bacterial
invasion of this tissue (i.e. through a dental caries lesion) may therefore cause a more
detrimental response by DP cells.
This study has also investigated the effects of the biofilm challenge on TACE
expression. TACE is a global “sheddase”, with the capacity to enzymatically cleave
and release the ectodomain of several cell-membrane bound cytokines [28], exhibiting
high specificity for RANKL [7]. Hence, TACE can mobilize cytokines into the local
microenvironment, thus amplifying their potential inflammatory effects. In the present
experimental model, TACE expression was moderately (approximately 1.5-fold) up-
regulated in both DP and PDL cells in response to the biofilm challenge. In a previous
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study, the putative periodontal pathogen P. gingivalis was shown to more strongly
induce TACE expression in a T-cell line, after 6 h of challenge [29]. However, it is
difficult at this stage to make safe interpretations on the potential of supragingival and
subgingival species for TACE induction, as there may also be differential responses
depending on cell types. In fact, T-cells are shown to be the major TACE-producing
cell population in periodontitis [30].
Interleukin-1� is a major pro-inflammatory cytokine with a primary role in the
pathogenesis of periodontal disease, and particularly in the induction of bone
resorption [31]. Putative periodontal pathogens have individually the capacity to
induce the production of this cytokine [32-35]. In the present study, the biofilm
challenge caused a significant up-regulation of IL-1� expression in DP cells, but not
in PDL cells. This may indicate that supragingival biofilms induce stronger pro-
inflammatory responses in DP than in PDL, and may therefore be more detrimental
for the development of pulpitis or periapical inflammation, rather than chronic
periodontitis. In contrast, it has recently been shown that subgingival biofilm
challenge induced IL-1� production in gingival epithelial cells, which may be of
direct relevance to the pathogenesis of periodontitis [22].
Collectively, the major finding of this study is the demonstration that
supragingival biofilm challenge enhances the RANKL/OPG ratio and IL-1�
expression in DP, but not in PDL cells. The high responsiveness of DP cells denotes
an enhanced capacity of these cells for inducing bone resorption, which could indicate
a putative pathogenic role for apical periodontitis. On the contrary, the low
responsiveness of PDL cells may reflect their potential adaptation to the bacterial
species represented in this supragingival biofilm, but also corroborates the low
virulence nature of these species for the pathogenesis of initial chronic periodontitis.
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5. Acknowledgements
The authors would like to thank Mrs Verena Osterwalder and Mrs Elpida Plattner for
their excellent technical assistance. This study was supported by the authors’ Institute.
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Tables
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Table 1. Cytotoxic effects of supragingival biofilm on PDL and dental pulp cells.
PDL cells Control Biofilm Ring only
6 hours 13.5 ± 0.5 14.6 ± 1.3 16.1 ± 2.7
24 hours 17.6 ± 1.3 42.6 ± 7.0 * 19.8 ± 3.0
Dental pulp cells Control Biofilm Ring only
6 hours 18.3 ± 6.5 21.5 ± 5.3 20.4 ± 4.6
24 hours 22.2 ± 2.3 41.1 ± 9.5 * 20.2 ± 1.4
PDL or DP cells were challenged with the biofilm for 6 and 24 h. Extracellularly
released LDH, representing the relative number of dead cells, is expressed as
percentage of total LDH (extracellularly released + intracellular content) in each
group. Numbers represent mean percentage values ± SD of three independent cell
cultures in each group. The “Ring only” group represents cell cultures in presence of
ring and hydroxyapatatite disc alone (without biofilm). The asterisk represents
statistically significant difference compared to the respective control group.
Figure Legends
Figure 1
Regulation of RANKL expression in PDL and DP cells. PDL cell (A) or DP cell (B)
cultures were challenged with the supragingival biofilm for 3 h and 6 h. The gene
expression levels of RANKL were measured by qPCR analysis, and normalized
against the expression levels of the GAPDH and RP2 (housekeeping genes). The
results are expressed as the 2-?�CT formula. Bars represent mean values ± SEM from
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20
20
three independent cell cultures in each group. The asterisk represents statistically
significant difference compared to the respective control group.
Figure 2
Regulation of OPG expression in PDL and DP cells. PDL cell (A) or DP cell (B)
cultures were challenged with the supragingival biofilm for 3 h and 6 h. The gene
expression levels of OPG were measured by qPCR analysis, and normalized against
the expression levels of the GAPDH and RP2 (housekeeping genes). The results are
expressed as the 2-?�CT formula. Bars represent mean values ± SEM from three
independent cell cultures in each group. The asterisk represents statistically
significant difference compared to the respective control group.
Figure 3
Regulation of the RANKL/OPG expression ratio in PDL and DP cells. PDL cell (A)
or DP cell (B) cultures were challenged with the supragingival biofilm for 3 h and 6 h.
The relative RANKL/OPG gene expression ratio was calculated based on the RANKL
and OPG gene expression values measured by qPCR. Bars represent mean values ±
SEM from three independent cell cultures in each group. The asterisk represents
statistically significant difference compared to the respective control group.
Figure 4
Regulation of TACE expression in PDL and DP cells. PDL cell (A) or DP cell (B)
cultures were challenged with the supragingival biofilm for 3 h and 6 h. The gene
expression levels of TACE were measured by qPCR analysis, and normalized against
the expression levels of the GAPDH and RP2 (housekeeping genes). The results are
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21
21
expressed as the 2-?�CT formula. Bars represent mean values ± SEM from three
independent cell cultures in each group. The asterisk represents statistically
significant difference compared to the respective control group.
Figure 5
Regulation of IL-1β expression in PDL and DP cells. PDL cell (A) or DP cell (B)
cultures were challenged with the supragingival biofilm for 3 h and 6 h. The gene
expression levels of IL-1� were measured by qPCR analysis, and normalized against
the expression levels of the GAPDH and RP2 (housekeeping genes). The results are
expressed as the 2-?�CT formula. Bars represent mean values ± SEM from three
independent cell cultures in each group. The asterisk represents statistically
significant difference compared to the respective control group.