Clinical Trial Details
— Status: Not yet recruiting
Administrative data
| NCT number |
NCT05711407 |
| Other study ID # |
Frag3 |
| Secondary ID |
|
| Status |
Not yet recruiting |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
March 3, 2023 |
| Est. completion date |
January 1, 2025 |
Study information
| Verified date |
February 2023 |
| Source |
Aristotle University Of Thessaloniki |
| Contact |
Ioannis Fragkioudakis |
| Phone |
+306973381496 |
| Email |
ifragkio[@]gmail.com |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
Peri-implantitis is defined as the pathological condition around dental implants
characterized by inflammation in the peri-implant mucosa and progressive bone loss,
eventually leading to implant loss. Peri-implantitis is thought to be a disease analogous to
periodontitis with a prevalence reaching 22%. Though peri-implantitis is readily recognized
as a part of modern dentistry, the exact etiology or an effective treatment regimen hasn't
been established yet. Thus, contemporary research is orientating toward acknowledging the
aetiologic and risk factors of the disease and of course establishing prognostic markers for
disease prevention. Microbiota residing in the subgingival plaque are considered the main
etiologic factor of the disease, however, current literature has not concluded on the exact
microbial composition of peri-implant lesions. In addition, genetic predisposition has been
recognized as a risk factor for disease initiation and progression and several observational
studies have addressed the potential association between various gene polymorphisms and the
occurrence of peri-implantitis. Lastly, to establish effective preventive measures, several
biomarkers have been evaluated as potential diagnostic and prognostic markers of disease
progression.
Objectives:
1. To identify the relationship of peri-implantitis with Cycloxygenase-2 (COX-2) and MMP-8
gene polymorphisms. Cyclooxygenase catalyzes the production of prostaglandins (PGs)
which are an important inflammatory mediator participating in the pathogenesis of
peri-implantitis. In addition, PGE2 expression in the peri-implant crevicular fluid will
be assessed.
2. To characterize the microbiota associated with peri-implantitis lesions, using novel
identification techniques enabling the identification of specific opportunistic bacteria
associated with the disease.
3. To test the diagnostic accuracy of a modern chairside test, using metalloproteinase-8
(MMP-8), an enzyme implicated in the pathogenesis of the disease, as a biomarker of
disease progression.
Description:
Dental implants have become an integrated part of modern dentistry during the last decades.
Since osseointegration (the process of an implant being incorporated into the jawbone) was
discovered, implant dentistry has gone substantial steps over time. Dental implants comprise
of an endosteal part made of titanium and an external prosthetic part, aiding in the
rehabilitation of edentulous patients. They offer the possibility to patients missing one or
several teeth to achieve the maximum functional and esthetic results, refraining from
traditional dentures that are quite unpleasant. Implant industry is gradually growing.
Overall, the global market of dental implants was valued at US$ 2,91 billion in 2016 with
more than 8.809 million implants placed annually in the US (1). However, the prevalence of
biological complications regarding dental implant supported restorations is growing in the
same rate (1).
The term "Peri-implantitis" was introduced in the 1st European Workshop of Periodontology in
1994 and since then numerous definitions have been proposed to describe the bone loss that
characterizes the aftermath of implant installation (2,3). Peri-implantitis and peri-implant
mucositis are covered under the term peri-implant diseases and are considered analogous to
periodontal diseases (4). Currently, peri-implantitis is defined as the pathological
condition around dental implants characterized by inflammation in the peri-implant mucosa and
progressive bone loss, eventually jeopardizing the fate of the implant (5). The number of
studies referring to peri-implantitis have risen in the last 30 years according to PubMed,
from 86 papers in the 90s' to a total of 1938 manuscripts until now (1). The prevalence of
peri-implantitis is also exhibiting growing rates. In a systematic review, Zitzman and
Berglundh in 2008 reported a prevalence ranging from 12 to 56% (3). More recent studies
reveal a prevalence of 22% (1-47%) (6), 20% (7), 26% (8), and 28% (9). It should be pointed
that in the previous studies the exact prevalence of the disease is difficult to estimate and
is dependent on several factors including the diagnostic criteria of the disease and the time
point chosen for the evaluation (10). However, it is obvious that peri-implantitis is a
growing problem affecting implant dentistry and will certainly be the topic of many future
studies.
Several protocols have been proposed for the treatment of peri-implantitis including,
non-surgical protocols, with implant decontaminating devices and lasers and surgical
protocols such as the use of regenerative materials (11,12) . However, if peri-implantitis is
already established, the proposed strategies and recommendations for its treatment can still
be considered as empirical. From the existing evidence it seems that nonsurgical therapy is
not effective, at least not in advanced cases. Surgical techniques may be necessary to
provide us with adequate access to degranulate the inflamed tissues effectively as well as to
decontaminate the implant surface (13).
The Consensus report of Workgroup 4 of the 2017 World Workshop on the Classification of
Periodontal and Peri-Implant Diseases and Conditions concluded that peri-implantitis is an
inflammatory process of a microbial origin that causes bone loss (4). In addition, several
risk factors have been proposed as potential co-drivers in this entity, including history of
periodontitis, smoking, diabetes and poor plaque control (5). Factors such as genetic
predisposition and release of titanium particles have also been indicated as potential risk
factors (14). The microbial involvement in peri-implantitis initiation and progression has
been established, however recent studies suggest that peri-implantitis may be a result of a
foreign body reaction, emphasizing the role of the host response in the disease initiation.
(15). In other words, the aetiology of the disease is yet to be elucidated. However, using
conventional DNA probe and cultural analyses, common periodontopathogenic bacteria have been
isolated at both healthy and diseased implant sites (16) and the distribution of the detected
species did not markedly differ by clinical implant status (17). However, when compared with
healthy implant sites alone, peri-implantitis was associated with higher counts of bacterial
species considered as consensus periodontal pathogens including Porphyromonas gingivalis and
Tannerella forsythia (18). Moreover, observational studies have indicated that
peri-implantitis was more frequently linked with opportunistic pathogens such as Pseudomonas
aeruginosa and Staphylococcus aureus ,(19,20) fungal organisms (e.g. Candida albicans,
Candida boidinii, Penicillum spp., Rhadotorula laryngis, Paelicomyces spp.),(21,22) and
viruses (i.e. human cytomegalovirus, Epstein-Barr virus), (23) thus pointing to a rather
complex and heterogenous infection. Therefore, the recognition of the exact bacterial
composition of peri-implant lesions is of paramount importance.
A significant observation about implant failures is that usually, a small number of patients
lose many implants. This clusterization phenomenon has been identified in many studies
(24,25). Weyant and his colleagues examined the survival rate of implants in 598 patients and
noticed that more than half of the cases that received multiple implants had more than one
failure. They estimated that patients who had one implant lost were 1.3 more likely to lose
more implants (26). These findings led to the hypothesis that host factors affect implant
survival and therefore genetic predisposition may play an important role in the development
of peri-implantitis. Many gene polymorphisms have been evaluated. Initially, most of the
studies referred to polymorphisms of cytokines which play a key role in the immune response
(27-29), such as the interleukins IL-1α, IL-1β, and their antagonist protein IL-1ra, IL-6,
IL-10, IL-17, TNF-α and Transforming Growth Factor-β1 (TGF-β1). Apart from these, various
other genes have been investigated, e.g. genes encoding CD14, receptor activator of nuclear
factor kappa B ligand (RANKL), microRNAs, bone morphogenetic proteins (BMPs), fibroblast
growth factor (FGF), TRAF family member-associated NF-kappa-β activator (TANK),
serine/threonine-protein kinase B-Raf (BRAF), calcitonin receptor (CTR), haptoglobin. There
are many discrepancies in the results of the above-mentioned studies. Some succeeded to
detect possible associations and others not. A more novel gene polymorphism mentioned in
genetic studies is that of the cyclooxygenase-2 (COX-2). A single nucleotide polymorphism of
COX-2 has been shown to alter the expression of the COX-2 gene. Several studies have found an
association among COX-2 gene polymorphisms and periodontitis (30-32). However, in the case of
peri-implantitis data is scarce.
The diagnosis of peri-implantitis is based on traditional clinical indices, similar to
assessment of periodontal disease. In specific, the diagnosis is established by the presence
of bleeding and/or suppuration on gentle probing, increased probing depth compared to
previous clinical examinations and the presence of bone loss beyond crestal bone level
changes resulting from initial bone remodeling (4). However, recent studies question the
diagnostic accuracy of clinical indices as such, supporting that the diagnosis of the disease
is far more complex (33). In these regards, novel diagnostic methods have been utilized in
order to achieve an early and proper diagnosis of the disease (34). A biomarker, or
biological marker is a measurable indicator of some biological state or condition. Biomarkers
are often measured and evaluated to examine normal biological processes, pathogenic
processes, or pharmacologic responses to a therapeutic intervention, and are useful in the
diagnosis and prognosis of a disease. In the case of peri-implantitis several biological
molecules have been evaluated as potential biomarkers aiding the diagnosis of the disease.
Molecules such as TNF-a, IL-1, RANKL have been associated with peri-implantitis in a variety
of studies (34). Lately, matrix metalloproteinase 8, (MMP-8),especially in its active form
(aMMP-8) has gained much attention of becoming promising biomarker candidate for diagnosing
and assessing the progression and course of these episodic oral inflammatory tissue
destructive and degenerative diseases (35,36).
Taken all together, peri-implantitis is a complicated disease with a high prevalence among
the population. In addition, current data on the etiology and diagnosis of the disease is
controversial.
PROPOSAL OBJECTIVES, STATE OF THE ART AND CHALLENGES
1. To address the relationship of peri-implantitis with COX-2 gene polymorphisms, as there
are no similar studies in the literature.
2. To identify the pathogenic microbiota that is associated with the disease using novel
bacterial identification methods.
3. To evaluate the diagnostic accuracy of an aMMP-8 Point-of-care chair-side test in
peri-implantitis.
The rationale in studying each of the studied objectives will be analyzed in a separate
section.
- COX-2 gene polymorphisms. The genetic predisposition is considered a potential risk factor
for peri-implantitis as already mentioned. A single nucleotide polymorphism (SNP) is single
nucleotide variation at a specific position of the genome. Each variation appears in some
appreciable degree within a population. SNPs may occur within coding and non-coding sequences
of genes, or in the intergenic regions. Considering degeneracy of the genetic code, SNP
within a coding sequence may change the amino acid sequence of the coding polypeptide
(nonsynonymous SNP), or not affect the protein sequence (synonymous SNP). SNPs outside
protein-coding regions may still regulate gene expression through affecting the transcription
factor binding, gene splicing, mRNA degradation, or the sequence of noncoding RNA. Therefore,
SNPs underlie differences in our susceptibility to disease. SNPs of inflammatory cytokine
genes may affect their expression levels or amino acid sequence and, consequently, the host
inflammatory response.
COX-2 converts arachidonic acid into prostaglandin H2, which is the precursor of
prostaglandin E2. Prostaglandin E2, which mediates proinflammatory and anti-inflammatory
reactions in many tissues (37), is also partly responsible for the activating processes
involved in resorption of the alveolar bone during the pathogenesis of periodontitis.
Associations of the cyclooxygenase 2(COX-2) gene with periodontitis were first identified in
Taiwanese and Chinese case-control populations (30,31) and subsequently validated in a
northwest European population. Studies as such involving the COX-2 gene have not been
conducted in the case of peri-implantitis yet. However, recent gene expression studied have
revealed an increased activity of the COX-2 pathway in the case of peri-implantitis (38,39),
implying that COX-2 may play an important role in the pathogenesis of peri-implantitis. The
association of peri-implantitis with certain gene polymorphisms will allow for a better
understanding of the disease, leading to more effective preventing and treatment strategies.
- Microbial factors associated with peri-implantitis Current data confirm that
peri-implant infections are dominated by Gram negative bacteria, similar to periodontal
infections, but some cases may harbor a distinct microbiota (20,40,41). Although early
reports showed similarities between the peri-implant and periodontal flora, later
studies demonstrated that peri-implantitis lesions may present not only consensus
periodontal pathogens but also opportunistic microorganisms, such as S.aureus, S.
anaerobius, Escherichia coli, Candida and Streptococci spp (18). Furthermore, sequencing
methods have also revealed other non-cultivable microorganisms associated with
peri-implant disease. Asaccharolytic anaerobic gram-positive rods (AAGPR) such as
Eubacterium nodatum, Eubacterium brachy, Slackia exigua, Gemella sanguinis and anaerobic
Gram-negative rods (OGNR) like Mitsuokella sp., Treponema lecithinolyticum have been
identified (42). The discrepancies among these studies may arise from the different
methods used for microbiological sampling and processing (43). The microbiological
profile of peri-implant diseases remains an issue of interest and many investigations
and reviews have been conducted in order to conclude whether the microbiota is different
from that of periodontitis; however, controversies still exist. The latest of the
studies conclude that there is insufficient evidence to support the distinct microbiota
between peri-implant and periodontal diseases (44,45) whereas others state that they may
be different entities in terms of microbiological profile (43). Therefore, further
studies need to be conducted, in order to define the microbiological profile of
peri-implant tissues. Newest technologies, such as shotgun sequencing of the whole
genome of bacteria involved in peri-implantitis would be very useful for this purpose,
but no such research has been published up to now. Thus, in this study Next Generation
Sequencing will be used will be utilized in order to identify the most purulent
microbiota associated with the disease. 16s rRNA sequencing will enable the
characterization of the whole taxonomic identity of the peri-implant lesions.
- Diagnostic accuracy of ImplantSafe MMP-8 Biomarker Test. Neutrophil collagenase, also
called matrix metalloproteinase (MMP)-8, polymorphonuclear (PMN) leukocyte collagenase,
or collagenase-2, has been identified and characterized as a major collagenolytic enzyme
that causes active periodontal and peri-implant degeneration (APD) in periodontitis and
peri-implantitis (46,47). MMP-8 can resolve and regulate inflammatory and immunological
cascades by processing nonmatrix bioactive substrates such as chemokines, cytokines,
serpins, and complement components. Physiological levels of MMP-8 can exert protective
and defensive anti-inflammatory characteristics (48). Increased levels of especially
active MMP-8 (aMMP-8), but not the latent, inactive proform, have been found in
periodontitis- and peri-implantitis-affected oral fluids (saliva, mouth rinse, gingival
crevicular fluid (GCF), and peri-implant sulcular fluid (PISF)) (49). A key
characteristic of active periodontal and peri-implant diseases is the sustained
pathological elevation and activation of aMMP-8 in periodontal and peri-implant tissues,
which are reflected in oral fluids (50). Consequently, aMMP-8 is a promising biomarker
candidate for diagnosing and assessing the progression and course of these episodic oral
inflammatory tissue destructive and degenerative diseases (46). More importantly, aMMP-8
in oral fluids can also serve as a predictive and preventive adjunctive biotechnological
tool to indicate (49,50) preventive interventions (secondary prevention or supportive
periodontal/peri-implant therapy (51,52) and to inhibit or reduce the conversion of
gingivitis and mucositis to periodontitis and peri-implantitis, respectively.
Recently, lateral-flow point-of-care (PoC)/chair-side tests (PerioSafe and ImplantSafe),
discovered in Finland and further developed in Germany (53), have been developed based on
earlier described technologies and monoclonal antibodies (53,54). The tests, PerioSafe and
ImplantSafe, and reader (ORALyser) have been developed and manufactured by Medix Biochemica
Ltd (Espoo, Finland) and Dentognostics GmbH (Jena, Germany) and are commercially available
from Dentognostics GmbH (Jena, Germany). In fact, the PoC/chair-side aMMP-8 lateral-flow
immunotests resemble the classical pregnancy and/or recently described HIV-PoC tests (55).
The aMMP-8 oral fluid tests can be used according to the manufacturer's instructions (47).
PerioSafe measures and analyses the levels of aMMP-8 in mouth rinse and ImplantSafe in PISF
and GCF; thus, PerioSafe is patient-specific and ImplantSafe is site-specific (47,53).
PerioSafe and ImplantSafe test-sticks can be quantitated by the ORALyser reader in 5 min
PoC/chair-side. PerioSafe and ImplantSafe with ORALyser quantitation are reliable,
quantitative, noninvasive, safe, and inexpensive adjunctive point-of-care diagnostic tools
for diagnosis, screening, monitoring, and prevention of periodontal and peri-implant diseases
(47). A pilot case-control peri-implantitis study shows both 100% sensitivity and specificity
for ImplantSafe test (56).
Methodology and Implementation:
To achieve the previous aims a case control study has been designed and will be carried on in
the department of Periodontology and Implant Biology, Dental School, Aristotle university of
Thessaloniki.
A study sample of minimum 100 patients attending the Postgraduate Clinic of Periodontology
and Implant Biology of Dental School Aristotle University of Thessaloniki for periodontal or
peri-implant treatment, will be recruited. Patients should be diagnosed with peri-implantitis
according the latest (2017)(4) criteria. The study protocol will be applied to the Ethical
Committee of the Faculty of Dentistry of the Aristotle University of Thessaloniki for
approval and all patients will be asked to sign their inform consent.
- Work Packages: i. COX-2 gene polymorphisms: Buccal swab will be obtained from all the
patients included in the study. After DNA extraction, a polymerase chain reaction (PCR)
process will be performed in order to identify the proposed polymorphisms, using commercial
primers specific for the gene. The gene polymorphism analyses, and relevant laboratory
process will be carried out with the collaboration of the school of Biology, Faculty of
Sciences, Aristotle University of Thessaloniki. More specifically, the samples will be
collected from Ioannis Fragkioudakis (Postgraduate student of the Department of
Periodontology and Implant Biology, Department of Dentistry, Aristotle University of
Thessaloniki) and will be stored in -80°C until further processing. The samples will be
analyzed in cooperation with the department of Biology, Aristotle university of Thessaloniki
under supervision of Professor Minas Arsenakis. The process will be carried out by Ioannis
Fragkioudakis and the PhD candidate Symela Koutounidou. The selection of this collaborating
organism is based on the previous experience of Professor Minas Arsenakis in gene
polymorphisms analysis and the fully equipped laboratory located in the department of Biology
AUTh. In addition, the PI, declares successful previous collaborations with the members of
the selected organization.
ii. Pathogenic Microbiota Samples will be obtained from the selected implants by the
insertion of sterile endodontic paper points into each peri-implant crevice. More
specifically, the samples will be collected from Ioannis Fragkioudakis (Postgraduate student
of the Department of Periodontology and Implant Biology, Department of Dentistry, Aristotle
University of Thessaloniki) and will be stored in -80°C until further processing The samples
will be analyzed using Next Generation Sequencing (NGS). More specifically ,16S rRNA
sequencing and HOMINGS will be used to identify the whole microbiota included in the samples,
enabling the characterization of all the taxonomic levels of bacteria. The NGS will be
conducted in collaboration with the department of microbiology, Medical School, A.U.Th. The
choice of the collaborating organism and members is based on the availability on the needed
laboratory equipment (Access provides by the associated professor Lemonia Skoura) in the
AHEPA university general Hospital of Thessaloniki. In addition, the needed laboratory work
will be conducted by Fani Chatzopoulou (PhD Student), familiar with the processes, under the
supervision of the Associate Professor Dimitrios Chatzidimitriou.
iii. MMP-8 Analysis ImplantSafe rapid aMMP8 analysis tests will be used for the evaluation of
the MMP-8 levels in the selected implants. The process will be as follows: Matrix
metalloproteinase (MMP)-8 (neutrophil collagenase-2) levels in its active form (aMMP-8) will
be collected from the Peri-implant Crevicular Fluid, with the aid of the paper strips
included in the test. Afterwards the samples will be analyzed quantitatively by the digital
reader ORALyzer® according to the manusfacturer's instructions giving an instant
quantification of the aMMP-8 levels. The ORALyzer has been in the possession of the
Department of Periodontology since 2019. All the samples will be collected and analyzed by
Ioannis Fragkioudakis.
