University of Southern California Dissertations and Theses

Characterization of cytokine/chemokine and microbiology profiles of peri-implant sulci and implant-supported ridge lap pontics

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Characterization of cytokine/chemokine and microbiology profiles of peri-implant sulci and implant-supported ridge lap pontics

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CHARACTERIZATION OF CYTOKINE/CHEMOKINE AND MICROBIOLOGY
PROFILES OF PERI-IMPLANT SULCI AND IMPLANT-SUPPORTED
RIDGE LAP PONTICS

by

Yvonne Tam

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(CRANIOFACIAL BIOLOGY)

December 2012

Copyright 2012 Yvonne Tam

ii

Acknowledgements

It would not have been possible to write this master’s thesis without the help and
support of family, friends, and mentors around me. I would like to extend my gratitude to
Dr. Hessam Nowzari for his advice and invaluable knowledge of periodontology and
implantology, to Dr. Maria Villacres for her expertise in microbiology and immunology,
to Dr. Homah Zadeh for his guidance and patience, to Dr. Mahvash Navazesh for her
support, as well as Dr. Tae Hyung Kim and Domenico Cascione for sharing their
photographs and advice. Lastly, but most importantly, I would like to thank Dr. Sandra
Rich for her insight and encouragement in writing this thesis for which I am extremely
grateful.

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Table of Contents

Acknowledgements

ii
List of Tables

iv
List of Figures

v
Abstract

vi
Chapter 1: Introduction

1
Chapter 2: Materials and Methods

14
Chapter 3: Results

18
Chapter 4: Discussion and Conclusion

24
References 27

iv

List of Tables

Table 1. Frequency and percentage of microbiota detected in positive
sites.

18

Table 2. Summary of cytokine and chemokine concentrations detected in
implants, teeth, and pontic sites.

20

v

List of Figures

Figure 1. Microbiologica profile of implant, teeth, and pontic sites.

20

Figure 2. Comparison of IL-8 concentrations from BD CBA Human
Inflammatory Cytokine (a) and Human Chemokine kits (b).

21
Figure 3. IL-1ß Concentrations.

22
Figure 4. IL-6 Concentrations.

23
Figure 5. Concentrations of RANTES (a), MIG (b), MCP-1 (c), and IP-
10 (d).
24

vi

Abstract

Background: Some investigators have detected edema, swelling and histological changes
in the soft tissue under pontics, while others have demonstrated that exceptional plaque
control can lead to healthy soft tissue at pontic sites. A new pontic design by Kim et al.
(2009) attempts to address the issue of esthetics and oral hygiene by creating a ridge lap
pontic with circumferential pressure, and was hypothesized to be associated with
favorable microbiological and inflammatory profiles.
Objective: The aim of this study was to explore the microbiological and
cytokine/chemokine profiles of implants as well as the “pressure pontic” site in implant-
supported fixed partial dentures and contrast them to that of healthy teeth.
Materials and Methods: Gingival crevicular fluid was sampled from 6 patients, each with
an implant-supported fixed prosthesis and a healthy tooth. One implant, the adjacent
pontic site, and the contralateral tooth from the implant sampled were sampled whenever
possible with Periopaper strips and subgingival plaque was collected with paper points.
Both were placed separately into the peri-implant sulcus, periodontal sulcus as well as the
pontic-mucosa interface on the mesio- and disto-lingual surfaces. Microbial samples were
analyzed for periodontopathic bacteria via selective anaerobic culture techniques. Pro-
inflammatory cytokines were quantified by flow cytometry analysis of GCF with two
separate kits. The first kit tested for IL-8, IL-6, IL-1β, TNF-α, and IL-10 while the
second one detected the concentration of IL-8, IP-10, RANTES, MCP-1, and MIG.
Results: The number of sites along with the cultivable levels of periodontopathic bacteria
detected in pontic sites, implant sulci, and periodontal sulci were similar. There were no

vii

significant differences detected in cytokines and chemokines due to a wide range of
values and limited number of samples. The results of the chemokine values for patients 5
and 6, who were sampled after one month, appeared higher than those of patients 1-4,
who were sampled one year after implant restoration.
Conclusion: The ridge lap pontic with circumferential pressure merits further evaluation.
The microbiological profile of the pontic site was comparable to that of other pontic
research designs observed by other researchers (Wang et al. 1998). The pontic area also
provided esthetic results, a lack of clinical inflammation, as well as a decrease in immune
response over time.

1

Chapter 1: Introduction
The ovate pontic design is a common selection for the restoration of fixed partial
dentures in the anterior region due to its high esthetic profile in comparison to other
designs such as ridge lap, modified ridge lap, and hygienic pontics. A histologic
observation of the healing of soft tissue in contact with the ovate pontic has revealed mild
inflammation after 12 months in select patients. Mucosa under pontic sites was also
found to have a thinner keratinized layer and a greater amount of inflammatory cells
versus that of mucosa taken from outside of the restoration (Zitzmann et al. 2002). This
finding may implicate a possible mild immune response, even though the authors
concluded that long-term mucosal health can be maintained under pontic sites. Some
investigators have detected edema, swelling and histological changes in the soft tissue
under pontics, while others have demonstrated that exceptional plaque control can lead to
healthy soft tissue at pontic sites (Cavazos et al. 1968, Tolboe et al. 1988, Podshadley
1968, Silness et al. 1982).
When selecting the design of a pontic, esthetics and function, as well as the ability
to maintain oral hygiene and healthy gingiva must be carefully considered. Ovate, ridge
lap, modified ridge lap, and hygienic pontic designs have been created to satisfy these
requirements. However, there are advantages and disadvantages of each. As a result, Kim
et al. (2009) developed the circumferential pressure-ridge lap pontic design to improve
esthetics and function in an implant-supported fixed partial denture. In a case report, they
hypothesized that the pressure exerted in this design could prevent plaque accumulation
under the pontic. The assumption being that the design could minimize inflammation

2

that has been noted with other designs. In their case study, they reported that in removing
a provisional with a “pressure pontic,” after ten months, they were unable to detect any
clinical signs of inflammation under the pontic.
Thus, the circumferential pressure ridge lap pontic as well as the implants
adjacent to them are hypothesized to be associated with favorable microbiological and
inflammatory profiles (Kim et al. 2009). The aim of this study was to explore the
microbiological and cytokine/chemokine profiles of healthy implants and “pressure
pontics” in implant-supported fixed parital dentures and contrast them to that of healthy
teeth.
Immunological responses triggered by infection are controlled by cytokines and
chemokines, mediating the progression as well as suppression of inflammation. As some
initiate the inflammatory cascade, others are responsible for chemotaxis, angiogenesis,
and induction of enzyme secretion in order to assist the body in diminishing the traumatic
agent and stimulating the correct immune response. The following reviews the literature
on the immunological response of gingiva surrounding natural teeth versus that
associated with the peri-implant muscosa surrounding a dental implant. A pontic may
exist between natural teeth, implants or a combination of the two, and there is limited
data that shows the immunological and microbiological relationship between the three
variables in a partially edentulous patient. Thus the gingival beneath and around a pontic
may be affected by either or both immunologic reactions and vice versa resulting in a
possible impact on periodontal disease and/or systemic diseases.

3

Chemokines
Chemokines are a family of chemotactic cytokines that bind to specific receptors.
They selectively attract different cell subsets to the site of inflammation and interact with
classical cytokines to modulate a local immune response. They are responsible for the
migration and maintenance of inflammatory cells such as polymorphonuclar leukocytes
(PMNs), macrophages, dendritic cells, natural killer cells, and other lymphocytes in the
gingival tissues. Leukocytes are activated through specific receptors of the seven-
transmembrane spanning G protein-coupled proteins. (Rossi and Zlotnik 2000) Altered
chemotactic behavior has been observed and documented in patients with periodontitis.
(Sigusch et al. 2001)

Interleukin-8 (IL-8)
Interleukin-8 (IL-8) is a potentially important regulator of PMN activity in
gingival tissues as it induces chemotaxis (Ribeiro et al. 1991) and modifies the activity of
other leukocytes including basophils (Kieger et al. 1992), lymphocytes (Larsen et al.
1989), eosinophils (Schroder et al. 1990), and monocytes (Neuner et al. 1994). Because
of its potential to regulate the cellular inflammatory response in the periodontium, many
researchers have explored the levels on IL-8 found in healthy periodontium and
compared it to that of periodontitis patients, hoping to map the cascade of periodontal
disease. Chung et al. evaluated the concentrations of IL-8 in 14 control subjects and 30
periodontitis patients and found a highly significant increase of IL-8 concentration
(p<0.005) in the healthy control group. They observed a lower concentration of IL-8 in

4

patients with deeper mean probing depths and greater percentage of bleeding on probing
site, suggesting an inverse relationship between IL-8 activity and PMN recruitment. The
group also compared the concentration of IL-8 before and after nonsurgical periodontal
treatment in the periodontitis group, and did not discover a significant difference between
levels detected at baseline and 2 weeks after treatment. (Chung et al. 1991). Other studies
have also resulted in similar observations where IL-8 levels were found to be
significantly higher in healthy periodontal tissues and related to low clinical
inflammation. (Jin et al. 2000, Mathur et al. 1996, Payne et al. 1993). However, Garlet et
al. found that IL-8 expression was similar in biopsied tissues of control and periodontitis
patients (Garlet et al. 2003) while others have detected an increase in IL-8 expression in
diseased tissue. (Tsai et al. 1995). Possible explanations for differences in these
observations may be the stage of the disease or the method of detection. Most research
groups cited sampled crevicular fluid while Garlet et al. (2003) analyzed the mRNA
expression of the chemokines within gingival tissue.
Although inflamed peri-implant mucosa was observed immunohistochemically to
consist of lymphocytes, macrophages and plasma cells, resembling that of inflamed
gingival tissue (Seymour et al. 1989), studies have found a significant increase in
cytyokine and chemokine expression in peri-implant inflammation (Bordin et al. 2009,
Venza et al. 2010), possibly differentiating peri-implantitis from periodontitis. Petkovic
et al. (2010) also found significantly lower IL-8 concentration in peri-implant crevicular
fluid of healthy control subjects compared to early and advanced peri-implantitis groups.
Mean PICF values were found to be significantly higher in the group with advanced

5

mucositis in comparison to the group with early mucositis as well (Petkovic et al. 2010).
Nowzari et al. (2008) compared cytokine and bacteria levels between teeth and implants
in healthy subjects, and discovered a 2-fold concentration of IL-8 in healthy implants. In
contrast, however, Severino et al. (2011) did not find a significant difference between the
peri-implantitis and healthy control group. Another study that biopsied for cytokine
expression (TNF-α, IL-1β, IL-8) with PCR of periodontal and peri-implant soft tissue,
also did not find any significant difference in pro- and anti-inflammatory cytokines
(Schierano et al. 2010). Their observations are consistent with the tissue biopsy
conducted by Garlet et al. (2003) pertaining to healthy controls and periodontitis patients,
which may suggest that IL-8 levels secreted may contrast with the amount of gene
expression within the tissue.

Interleukin-6 (IL-6)
Interleukin-6 (IL-6) is a cytokine with the potential for activation of osteoclasts
with an increased expression in macrophages, fibroblasts as well as epithelial cells of
patients with peri-implantitis (Konttinen et al. 2006, Johnson and Serio 2007)
Significantly higher expression was also observed in periodontal and peri-implant
inflammation (Bordin et al. 2009, Giannelli et al. 2009). Liskmann et al. (2006) found
increased concentrations of IL-6 in the crevicular fluid of patients with peri-implant
disease defined by bleeding, pocket depth, and gingival index in 30 patients. Others have
also found statistically higher IL-6 levels in peri-implantitis when compared to healthy
subjects (Konttinen et al. 2006). However, Mengel et al. (1996) did not observe a

6

significant difference in IL-6 concentration in implants placed in patients with
periodontal disease and those without. The results from Severino and his group analyzed
IL-6 levels in peri-implantitis patients and healthy subjects showed a lack of difference as
well (Severino 2011). Salcetti et al. (1997) measure IL-6 levels from patients with failed
implants and compared that to those with stable implants, and their results also did not
reveal any significant difference between the two groups. Another method of analysis is
to determine the polymorphic gene encoding for IL-6 was a genetic risk factor for early
implant loss, and Campos et al. (2005) showed it was negligible. Other studies have also
shown a lack of relationship between IL-6 levels and peri-implant disease (Lopez et al.
2006, Duarte et al. 2009). Without consistent results, it is difficult to conclude whether
IL-6 expression is significant in the disease process of periodontitis or peri-implantitis.

Interleukin-10 (IL-10)
Interleukin-10 (IL-10) is known to function as an inhibitor of inflammation. IL-10
expression was found to be significantly higher in both mucositis and peri-implantitis
(Duarte 2009). Liksmann et al. (2006) also reported higher concentrations IL-10 in
patients with peri-implantitis. It is suggested that increased IL-10 expression in
periodontal disease may indicate an attempt to control the potentially destructive
response in inflamed soft tissue around dental implants (Sasaki et al. 2000, Garlet et al.
2003). However, Duarte et al. (2009) did not find a difference in IL-10 levels between
healthy subjects and peri-implantitis patients. In a separate study, they also discovered
that IL-10 expression did not change 3 months after mechanical treatment for mucositis

7

and peri-implantitis (Duarte 2009). Other studies also support the lack of relationship
between IL-10 and peri-implant disease. (Lopez et al. 2006, Salcetti et al. 1997, Mengel
et al. 1996). It continues to be unclear whether IL-10 plays a significant role in
periodontal or peri-implant inflammation.

Interleukin-12 (IL-12)
Within the inflammatory response, interleukin-12 (IL-12) is known as the T cell-
stimulating factor and induces secretion of INF-γ from activated T cells and natural killer
cells (Hou et al. 2003). Concentrations of IL-12 have been found to be elevated in
advanced peri-implantitis, with increased plaque accumulation, bleeding and pocket
depth (Duarte et al. 2009). Fokkema et al. (2003), however, evaluated the change of
chemokines before and after periodontal therapy and discovered that levels of IL-12 were
significantly increased after periodontal therapy. Its exact role in peri-implantitis remains
unidentified.

RANTES
RANTES (regulated upon activation, normal T cell expressed, and secreted) is
also known has CCL5, a chemokine that stimulates chemotaxis and activation for
monocytes, eosinophils, basophils, and T lymphocytes (Graves 1999). A study by
Gamonal et al. (2000) analyzed RANTES concentrations in the gingival crevicular fluid
(GCF) sampled from periodontitis patients and healthy control subjects. GCF was
collected from probing depths of < 3 mm, 4-6 mm, and > 6 mm from both groups.

8

RANTES expression was detected only in the periodontitis group, and was found to be
significantly higher in concentration in probing depths < 3 mm compared to probing
depths > 6 mm. RANTES was not found to have a significant increase in active
periodontal sites compared to inactive sites, but a positive correlation was observed in
periodontitis patients. Another group found a 2-fold increase of RANTES in patients with
untreated periodontal disease that supports the findings mentioned above. They also
evaluated the chemokine levels after periodontal therapy was provided and no changes
were observed. (Fokkema 2003). However, Gemmell et al. (2001) found weak expression
of chemokines in keratinocytes from gingival biopsies, and found that levels of RANTES
were reduced with increasing inflammation. The methodology of detection and analysis
differs and research remains inconsistent in revealing the significance of RANTES in oral
inflammation.

Monocyte chemotactic protein-1/Chemokine ligand 2 (CCL2)
Chemokine ligand 2 (CCL2) is also known as monocyte chemotactic protein-1
(MCP-1) and it functions to recruit monocytes, memory T cells, and dendritic cells to
sites of inflammation. (Carr et al. 1994, Xu et al. 1996). MCP-1 is found to be more
frequently detected and in higher expression in chronic periodontitis (Garlet et al. 2003).
Fokkema et al. evaluated the change of chemokines before and after periodontal therapy
and observed that MCP-1 trended towards higher levels in untreated periodontitis patients
(p < 0.07), but did significantly decrease after periodontal therapy (Fokkema et al. 2003).
Others found MCP-1 to be associated with bacterial infection. When compared to

9

minimally inflamed tissue, the MCP-1 expression in bacteria-associated gingivitis and
osseous inflammation was upregulated. (Yu et al. 1993, Yu and Graves 1995)

Interleukin-1b (IL-1β) and Tumor necrosis factor-alpha (TNF-α)
Interleukin-1b (IL-1β) and TNF-α act synergistically to initiate a cascade of
inflammatory mediators (Ejeil et al. 2004) and to stimulate bone resorption, prostaglandin
synthesis and protease production by cells such as fibroblasts and osteoblasts.
(Billingham 1987). Both have little direct chemotactic activity for leukocytes but these
cytokines stimulate the recruitment of inflammatory cells by inducing chemokine
expression and cell-adhesion molecules. It has been shown by Rogers et al. (2002) that
ions from implants can stimulate peripheral blood mononuclear cells to produce IL-1β
and TNF-α in vitro. IL-1β was found in elevated levels in gingival crevicular fluid of
periodontitis and peri-implantitis compared to healthy controls (Curtis et al. 1997,
Gamonal et al. 2000). Ataoglu et al. (2002) observed that implants with inflamed mucosa
had higher levesl of IL-1β compared to implants with slightly or non-inflamed mucosa,
supporting the findings of the previous studies. TNF-α and IL-1β concentrations were
also found to be significantly lower than that of early and advanced peri-implant
mucositis (Petkovic et al. 2010). When comparing diseased tissue and healthy, there
appears to be a consistent finding of elevated IL-1β and TNF-α expression. However, a
study by Nowzari et al. (2010) comparing the levels of IL-1 b and TNF-α in healthy
periodontal sulci to that of healthy peri-implant sulci, a significant difference was not
detected for the levels of IL-1β while TNF-α concentration was greater in implant sites

10

than in periodontal sites. This finding may show that although the two cytokines are
synergistic, they provide different functions in activating an immune response and may
be present at different times.

Monokine induced by gamma interferon (MIG)
Monokine induced by gamma interferon (MIG) is a chemokine induced by INF-γ
and functions as a chemotactic factor for human T cells, B cells, and dendritic cells (Park
et al. 2002). Garlet et al. (2003) found MIG levels were more prevalent and higher in
aggressive periodontitis and associated with higher INF-γ expression and lower IL-10
expression. Limited research has been conducted to show how MIG relates to periodontal
disease and peri-implant mucositis.

Microbiology
Subgingival microbiota around implants affected by pocketing and bone loss
present with high levels of periodontal pathogens, and periodontally involved teeth may
serve as reservoirs (Hultin et al. 2002). Cosyn et al. (2011) observed a significant link
between periodontal probing depth and sulcular levels of bacteria. They found that ¾ of
the samples from peri-implant sulcus consisted of A. actinomycetemcomitans, F.
nucleatum, L. buccalis, P. micra, P. melaninogenica, and Treponema denticola, all
species that have been found to be associated with periodontal disease. However,
Nowzari et al. (2008) observed that the frequency and levels of periodontopathic bacteria
were higher around teeth than implants including P. gingivalis, T. forsythia,

11

fusobacterium spp., and enteric gram negative rods. They concluded that pro-
inflammatory cytokine production was unrelated to heavy bacterial challenge, but that
cytokine levels increased at sites where periodontopathic bacteria were detected. Another
study that compared the microflora of healthy periodontium with that of the peri-implant
sulci found they were similar and consisted of Gram-positive cocci and a few Gram-
negative rods (Schierano et al. 2003). It was also determined by other groups that the
surface properties of implants do not influence bacterial flora or plaque maturation
(D’Ercole et al. 2008, Quirynen and Listgarten 1990, Gerber et al. 2006), but these
studies do not prove whether the bacteria are transmissible within the same oral cavity
and affect disease progression in adjacent areas.
Tabanella et al. (2009) described the indications of peri-implantitis as a loss of
lamina dura, presence of T. forsythia, Campylobacter species, P. micros, Fusobacterium
species, and pain. Other studies have observations of similar microbial species including
Campylobacter rectus, Fusobacterium species, Prevotella intermedia, Porphyromonas
gingivalis, and spirochetes, and A. actinomycetemcomitans evident in peri-implantitis
patients (Rams et al. 1983, Rams and Roberts 1984, Alcoforado et al. 1991, Mombelli et
al. 1995, Leonardt et al. 93). Tabanella et al. (2009) compared the number of ailing
implants in the mandible to the maxilla, and in the anterior with the posterior regions. His
findings included an absence of lamina dura in every case associated with peri-implant
bone loss. Sites with exposed threads but no increased pocket depth showed increased
levels of P. micros and Campylobacter species. Pain was more frequently found in
partially edentulous patients, and the total viable microbial counts in diseased sites were

12

2-fold (4.89 million vs. 2.46 million) that of healthy sites while the percentage of
periodontopathic bacteria in diseased sites were nearly 4-fold that of the healthy (41.8%
vs. 11.3%). The percentage of major periopathogens in diseased sites was 6.8% vs 5.37%
in healthy sites. Their results may indicate that periodontal pathogens are responsible for
implant failures. In a different perspective, Nowzari et al. (2008) also observed a greater
number of periodontal disease-inducing bacteria in healthy implants when compared to
healthy teeth. This may implicate that implants either harbor more bacteria or are more
susceptible to periodontopathic pathogens. However, Schierano et al. (2010) did not find
a significant difference in microbiological composition of plaque sampled from implant
sites and that of teeth.
Studies have explored the interaction between cytokines and chemokines with
bacteria-specific immune cells. Oido-Mori et al. (2001) found that P. gingivalis
gingipain-B enhances IL-8 production while indirectly inhibiting IP-10 production. This
mechanism my impact the inflammatory cell infiltrate since IL-8 preferentially recruits
neutrophils and IP-10 attracts activated T cells to the site of injury. It has also been found
that P. gingivalis-specific T cells, monocytes, and B cells produce chemokines in
response to P. gingivalis outer membrane antigens, shedding light to the mechanism of
periodontal and/or peri-implant disease.
Wang et al. (1998) evaluated the frequency and species of bacteria that may be
harbored under a pontic site. They found 97% of pontic sites positive for P. intermedia
while 64% of inflamed pontic sites were positive for P. gingivalis and 85% were positive
for B. forsythus. It is also interesting to note more P. intermedia was detected under

13

inflamed metallic pontics compared to inflamed porcelain pontics. The authors concluded
that both pontic material and health status of mucosa affect the composition of associated
microbiota. (Wang et al. 1998) Another study by Lee et al. (1999) discovered that the
microbiota of remaining teeth had a major influence on the peri-implant microbiota, and
that patients with a history of periodontitis had a greater impact on peri-implant
microbiota. It may be possible that pontics are another influential site for microbial
retention. The extent of the periodontal or peri-implant infection and the microorganisms
that are found within oral fluid secreted by adjacent tissue may vary significantly, and
this paper seeks to shed some light on the microbial and immunological relationship of
soft tissue surrounding a healthy implant, natural dentition, and a pontic site. This paper
explores the effect of the pontic site in relation to periodontopathic bacteria as well as the
immune response based on the hypothesis that the “pressure pontic” and implants
adjacent to them are associated with favorable microbiological and inflammatory profiles
(Kim et al. 2010).

14

Chapter 2: Materials and Methods
This was a proof of concept study utilizing fluid sampled from the oral cavity and
the variables tested are the microbial, chemokine and cytokine profile of implant-
supported fixed partial dentures in comparison to periodontally healthy teeth.
The study was approved by the Institutional Review Board at University of
Southern California. Study patients were from USC Advanced Periodontics, Advanced
Prosthodontics, and the Oral Health Center including those who had received implants
for implant-supported fixed partial dentures designed with the circumferential pressure-
ridge lap pontic (Kim 2009). Informed consent was obtained and Health Insurance
Portability and Accountability Act (HIPAA) were reviewed and signed. Six partially
edentulous patients, with one or more clinically healthy dental implants restored with a
fixed partial denture utilizing the circumferential pressure-ridge lap pontic, participated in
this study. Each one had clinically healthy periodontium without a history of
periodontitis. All patients were non-smokers. Patients had probing depths ranging from 2-
4 mm, no radiographic bone loss beyond the first thread of the implant or around teeth. A
CP12 (Hu-Friedy, Chicago, IL, USA) periodontal probe and conventional peri-apical
radiographs were used to determine the clinical parameters. Exclusion criteria included
periodontal disease and peri-implant mucositis defined by radiographic bone loss,
bleeding on probing, clinical attachment loss > 3 mm, and a probing depth > 4 mm.
Patients who were pregnant or lactating, taking antibiotics within the last 3 months,
taking non-steroidal anti-inflammatory drugs 2 weeks prior to sample collection were

15

also excluded from the study. A single clinician conducted sampling and clinical
examinations.

Pontic Design
The circumferential pressure-ridge lap pontic was fabricated according to the
protocol stated in a previous article (Kim 2009). A wax up was completed and a cast
duplicated. The gingival margin was traced onto the cast and the pontic contour was
carved out on the cast. The result was a square-shaped concave pontic instead of a convex
pontic shaped like that of an ovate pontic. Provisional restorations were seated onto the
implants with a minimum of 2 mm of soft tissue between the osseous crest and gingival
margin, and confirmed with sounding of the tissue with an endodontic file. Of the 6
patients in this study, 4 patients had the provisional restorations in place for 1 year or
longer and 2 patients had them for about 4 weeks.

Microbiologic Sampling and Analysis
Microbiologic sampling was completed after isolating sampling sites with cotton
rolls and removing supragingival plaque with gauze. Two sterile paper points were
placed into the mesial and distal sites of the peri-implant sulcus of one implant,
periodontal pocket a contralateral tooth, and the pontic-mucosal space for 30 seconds for
each patient. Bleeding was avoided during sampling, and the paper points were
transferred immediately after to a vial of VMGA III. The samples were processed for
bacterial culture and analyzed for periodontopathic bacteria at the Oral Microbiology

16

Testing Laboratory at USC Ostrow School of Dentistry within 24-48 hours at 25 C
according to the protocol proposed by (Slots). Samples were incubated in CO2 anaerobic
culture and brucella blood agar medium at 35 C in an anaerobic container for 7 days.
TSBV (Tryptic Soy Serum Bacitracin Vancomycin Agar) medium was incubated in 10%
CO2 at 37 C for 4 days to detect the following periodontopathogens: Aggregatibacter
actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia/ Eikenella
corrodens, Capnocytophaga sp., Dialister pneumosintes, Gram negative enteric rods,
Staphylococci spp., and yeasts. Frequency detection of each pathogen and levels as the
mean percentage (%) + standard deviation in positive sites were recorded.

Oral Fluid Sampling and Analysis
Gingival crevicular fluid around implants (PICF), teeth (GCF) as well as pontic-
mucosa fluid (PMF) were collected with Periopaper strips (Oraflow Inc., NY, USA).
Sterile Periopaper strips were placed into the mesio-buccal and disto-buccal sites until
they were saturated measuring 2 uL in the same isolated sites used for subgingival plaque
sampling. The strips were then placed into sterile 1.5 mL low protein-binding centrifuge
tubes pre-labeled and filled with 100ml sterile phosphate buffered saline, and
immediately placed on dry ice for transportation. The samples were stored at -70 C until
bead array flow cytometry processing. Two cytometric bead array kits were used for
detection and quantification of cytokines and chemokines within the samples. The first
kit used was BD Cytometric Bead Array (CBA) Human Inflammatory Cytokines Kit
which detected for levels of IL-8, IL-1β, IL-6, IL-10, TNF-α, and IL-12. The second kit,

17

BD CBA Human Chemokines Kit, detected for IL-8, MCP-1, IP-10, RANTES, and MIG.
Periopaper strips were incubated at 4 C for 12 hours and vortexed for 20 min prior to
processing according to manufacturer’s instructions. Data was acquired in a FACS
Caliber flow cytometer (Kecton Dickinson, Franklin Lakes, NJ, USA).

Statistical Analysis
The concentration of cytokines and chemokines is depicted as the mean +
standard error of the mean. Differences for the concentration of cytokines between
implants and teeth were assessed with the independent t-test. The detection of each
pathogen is presented as the frequency of detection (# of sites) and the cultivable
microbiota as the mean % in positive sites.

18

Chapter 3: Results

A total of 6 subjects with a mean age of 45 years, each with an implant-supported
fixed partial denture participated in the study. Six healthy teeth contralateral to the 6
implants were selected for sampling. All 6 implants selected were Dentium Implantium
Implants, and 6 adjacent pontic sites were also tested.
The frequency of detection (# of sites) and percent of periodontopathic bacteria
found in positive sites are presented in Table 1. Fusobacterium species and Enteric gram
negative rods appear to be the most frequently detected periodontopathic bacteria. The
number of sites along with the cultivable levels of periodontopathic bacteria detected in
pontic sites, implant sulci, and periodontal sulci were similar. However, the red complex
bacteria, P. gingivalis and P. intermedia, were only detected at pontic sites.

Table 1. Frequency and percentage of microbiota detected in positive sites.
Pontics Implants Teeth
Microorganism
Freq
(# of sites)
% Micro
(+) sites
Freq
(# of sites)
% Micro
(+) sites
Freq
(# of sites)
% Micro
(+) sites
Aggregetibacter
actinomycetemcomitans 0 0 0 0 0 0
Porphyromonas
gingivalis 1 2.3 0 0 0 0
Prevotella intermedia 1 3.1 0 0 0 0
Tanerella forsythia 0 0 0 0 0 0
Campylobacter sp. 1 3.8 1 3.1 2 3.45
Eubacterium sp. 0 0 0 0 0 0
Fusobacterium sp. 3 5.63 3 5.67 4 5.55
Micromonas micros 0 0 1 2.3 1 1.5
Enteric rods 4 4.83 3 2.7 3 3.6
Yeast 0 0 1 0.03 2 0.17
Eikenella corrodens 0 0 0 0 0 0
Dialister pneumosintes 1 2.3 0 0 0 0
Staphylococcus sp. 0 0 0 0 1 4

19

Figure 1. Microbiologica profile of implant, teeth, and pontic sites.

Due to the timing of sampling and the execution of the laboratory testing for
cytokines and chemokines, 5 samples were measured with the BD CBA Human
Inflammatory Cytokines kit, and 6 samples were used for the BD CBA Human
Chemokine kit. Both kits included beads for the detection of IL-8. The results and values
for IL-8 concentration determined from both kits were comparable (Figure 2a, 2b). No
significant difference detected between the three groups due to a wide range of values
and limited number of samples (Table 2).

20

Table 2. Summary of cytokine and chemokine concentrations detected in implants, teeth,
and pontic sites.

Cytokine/ Implants Pontics Teeth
Chemokine pg/mL + SEM
p
pg/mL + SEM
p
pg/mL + SEM p
IL-8 (a) 466 + 505.51
0.71
638.88 + 885+87
0.73
370.74 + 312.67 0.54
IL-8 (b) 719.5 + 1131.39
0.71
1018.25 + 1546.72
0.69
503.8 + 600.39 0.47
IL-6 118.44 + 118.99
0.84
136.94 + 164.32
0.76
97.82 + 87.36 0.65
IL-1β 10.24 + 20.14
0.42
1.62 + 3.62
0.42
45.08 + 89.22 0.31
RANTES 1.82 + 2.84
0.32
10.52 + 20.01
0.31
4.15 + 4.56 0.46
IP-10 2.45 + 4.35
0.34
422.27 + 1027.59
0.52
4.6 + 6.71 0.34
MCP-1 1.28 + 3.14
0.32
9.5 + 18.88
0.73
0.75 + 1.84 0.50
MIG 5.3 + 5.28
0.25
190.22 + 368.71
0.50
12.97 + 26.16 0.27
* comparison of concentration values for each of the immune mediators amongst implants, pontics, and
teeth were not significant statistically.

Figure 2. Comparison of IL-8 concentrations from BD CBA Human Inflammatory
Cytokine (a) and Human Chemokine kits (b).

(a) IL-8 (BD CBA Human Inflammatory Cytokines Kit)

21

Figure 2. Continued
(b) IL-8 (BD CBA Human Chemokines Kit)

Table 2 as well as figures 3 and 4 show the average trends and the p-values of IL-
6 and IL-1β. There are no significant differences determined due to the high range in
values and small sample size. IL-6 trends are higher for implant and pontic sites while IL-
1β showed a higher trend in the periodontal sulcus and the pontic site with the lowest
detectable concentration. Values for TNF-α and IL-10 were not included because few
values were detected for these variables.

Figure 3. IL-1β Concentrations

22

Figure 4. IL-6 Concentrations

The mean values for RANTES, IP-10, MCP-1, and MIG are listed in Table 2.
There is a trend for higher levels of RANTES, IP-10, and MIG in pontic sites although no
significant difference were detected. The results of the chemokine values for patients 5
and 6, who were sampled after one month, appeared higher than those of patients 1-4,
who were sampled one year after implant restoration (Figure 5). However, due to the
small number of subjects, statistical significance cannot be determined. These
observations were consistent with a reduced immune response with the increase of time.

23

Figure 5. Concentrations of RANTES (a), MIG (b), MCP-1 (c), and IP-10 (d).

(a) (b)

(c) (d)

24

Chapter 4: Discussion and Conclusion
A goal of this study was to satisfy the proof of concept phase of research in order
to determine whether the pontic design merited further evaluation. Levels of cytokines
and chemokines were highly variable amongst implants, teeth, and pontic sites.
Therefore, any investigation aiming to demonstrate significance in levels of these
mediators will likely require large number of samples to reach statistical significance.
Limitations and difficulties encountered during this study included difficulty in
identifying and recruiting subjects, in particular since one of the requirements was to be
able to sample the site after a minimum of one year. Due to the inadequate number of
subjects who satisfied this latter requirement, two subjects were sampled one month after
implant restoration. Each patient was required to have the same implant system utilized
as well as the same pontic design to restore a partial edentulous area in the maxillary
anterior region.
Two different BD CBA kits were utilized for the detection of cytokines and
chemokines in the current study. However, both kits contained beads for IL-8 detection,
and comparison of the same sample ran by both kits showed that the kits were consistent
in their results. Also, due to the difficulty of recruiting patients and the timing of running
the flow cytometry, 5 of the 6 samples were analyzed with the BD CBA Cytokines Kit
while all 6 samples were analyzed with the BD CBA Chemokines Kit.
In our study, P. gingivalis and P. intermedia were only detected in pontic sites.
Although there are not enough samples to reach a statistical significance, these findings

25

agree with the study by Wang et al. (1998) where they found 97% of pontic sites positive
for P. intermedia and 64% of inflamed pontic sites were positive for P. gingivalis.
Tripodakis and Constandtinides (1990) found that inflammation occurred under
pontics when flossing was neglected. However, the subjects in this study did not have any
clinical signs of inflammation at the time of sampling. As described by Kim et al. (2009),
the ridge lap pontic with circumferential pressure in contact with tissue prevented
clinically visible plaque accumulation and tissue inflammation. As a result, the goal in
this study was to observe inflammation at a molecular level.
A trend of reduced immune response was observed with the increase of time in
the present study. Inconsistency of the research on chemokines and cytokines in relation
to periodontal disease and peri-implant mucositis shows a continued lack of
understanding of these proteins. One of the challenges of conducting cross-sectional
studies is the heterogeneity of subjects, who may be at different points of their immune
response. This heterogeneity may result in varying immune mediators. Similar results
have been observed within the crevicular fluid showing a time-dependent decline of
chemokine values in implant sites from the time of implant placement to one year after
fitting the prostheses (Spyrou et al. 2002). Another study evaluated the cytokine levels 24
hours after implant placement and found a significant increase in the levels of IL-6 and
IL-8, which may reflect the inflammation due to trauma from surgery (Pietruski et al.
2001). They evaluated the same sites after 4 months and noticed a decrease in the
cytokine levels. Schierano et al. (2000, 2003) also observed a decrease in levels of IL-6,
IL-8, and IL-10 eight months after the restorations were placed. It appears that the

26

cytokine and chemokine levels detected may be determined by time of sampling which is
indicative of the stage of inflammatory response at which the sample was taken. Further
research that focuses on the detection of cytokine and chemokine concentrations at
different time intervals may reveal the extent of tolerance the immune system has on the
different materials placed next to soft tissue.
In conclusion, the circumferential pressure ridge-lap pontic merits further
evaluation. The microbiological profile of the pontic site was comparable to that of other
pontic designs observed by other researchers (Wang et al. 1998). The pontic area also
provided esthetic results, a lack of clinical inflammation, as well as a decrease in immune
response over time.

27

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Abstract (if available)

Abstract Background: Some investigators have detected edema, swelling and histological changes in the soft tissue under pontics, while others have demonstrated that exceptional plaque control can lead to healthy soft tissue at pontic sites. A new pontic design by Kim et al. (2009) attempts to address the issue of esthetics and oral hygiene by creating a ridge lap pontic with circumferential pressure, and was hypothesized to be associated with favorable microbiological and inflammatory profiles. ❧ Objective: The aim of this study was to explore the microbiological and cytokine/chemokine profiles of implants as well as the “pressure pontic” site in implantsupported fixed partial dentures and contrast them to that of healthy teeth. ❧ Materials and Methods: Gingival crevicular fluid was sampled from 6 patients, each with an implant-supported fixed prosthesis and a healthy tooth. One implant, the adjacent pontic site, and the contralateral tooth from the implant sampled were sampled whenever possible with Periopaper strips and subgingival plaque was collected with paper points. Both were placed separately into the peri-implant sulcus, periodontal sulcus as well as the pontic-mucosa interface on the mesio- and disto-lingual surfaces. Microbial samples were analyzed for periodontopathic bacteria via selective anaerobic culture techniques. Proinflammatory cytokines were quantified by flow cytometry analysis of GCF with two separate kits. The first kit tested for IL-8, IL-6, IL-1β, TNF-α, and IL-10 while the second one detected the concentration of IL-8, IP-10, RANTES, MCP-1, and MIG. ❧ Results: The number of sites along with the cultivable levels of periodontopathic bacteria detected in pontic sites, implant sulci, and periodontal sulci were similar. There were no significant differences detected in cytokines and chemokines due to a wide range of values and limited number of samples. The results of the chemokine values for patients 5 and 6, who were sampled after one month, appeared higher than those of patients 1-4, who were sampled one year after implant restoration. ❧ Conclusion: The ridge lap pontic with circumferential pressure merits further evaluation. The microbiological profile of the pontic site was comparable to that of other pontic research designs observed by other researchers (Wang et al. 1998). The pontic area also provided esthetic results, a lack of clinical inflammation, as well as a decrease in immune response over time.

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Asset Metadata

Creator Tam, Yvonne (author)

Core Title Characterization of cytokine/chemokine and microbiology profiles of peri-implant sulci and implant-supported ridge lap pontics

School School of Dentistry

Degree Master of Science

Degree Program Craniofacial Biology

Publication Date 09/04/2012

Defense Date 09/24/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemokines,cytokines,implants,OAI-PMH Harvest,pontics

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Zadeh, Homah (committee chair), Navazesh, Mahvash (committee member), Rich, Sandra (committee member)

Creator Email yvonne.tam@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-93541

Unique identifier UC11288268

Identifier usctheses-c3-93541 (legacy record id)

Legacy Identifier etd-TamYvonne-1182.pdf

Dmrecord 93541

Document Type Thesis

Rights Tam, Yvonne

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA