Download hej Research Protocol for

Regenerative Treatment of Peri-implantitis
A randomized clinical trail.
Catrine Isehed
Supervisor: Professor Stefan Renvert
Background
Biological complications in implant dentistry are referred to as peri-implant mucositis
and peri-implantitis. At the first European Workshop on Periodontology, periimplantitis was defined as an inflammatory process affecting the tissues around an
osseointegrated implant in function, resulting in loss of supporting bone and periimplant mucositis was defined as reversible inflammatory changes of the peri-implant
soft tissues without any bone loss (Albrektsson & Isidor 1994).
Therapies proposed for the management of peri-implant diseases appear to be
based on the evidence available for treatment of periodontitis. Most publications on
treatment of peri-implant lesions in humans report individual cases treated by
combined procedures, aimed at reducing the bacterial load within the peri-implant
pocket (for review see. Jansåker et al. 2003. The concept that bacteria play a major
role in the aetiology of peri-implant mucositis and peri-implantitis is well documented
(Berglundh et al. 1992, Pontoriero et al. 1994, Mombelli et al. 1998, Augthun and
Conrads 1997, Salcetti et al. 1997, Leonhardt et al. 1999, Quirynen et al. 2002,
2006). Mombelli (2002) reviewed the role of bacteria in causation of peri-implantitis
and found support for the concept that the microflora present in the oral cavity before
implant placement influences the microflora developing on implants. On the surface
of the implant the sub-gingival microbial flora causes the development of a tightly
fixed layer of plaque, the so called bio-film that protects the bacteria against antimicrobial agents (Lamont and Jenkinson 2000). Eradication of pathogens by
mechanical means on the implant surface with threads and often a rough surface
structure may not be possible. Thus if the goal is to eliminate or significantly reduce
the levels of pathogens with mechanical interception and adjunct anti-microbial
medication it might be required to improve our ability to obtain a significant clinical
impact on the pathogens, to reduce and control the inflammatory response. Several
reports have indicated a healing potential of peri-implant tissues following
suppression of the peri-implant microbiota by a combination of mechanical and
chemical means (see reviews by Mombelli 2002 and Klinge et al. 2002).
Due to technical difficulties in decontaminating implants by mechanical means alone,
the use of adjunctive antimicrobial components have been proposed for the
treatment of peri-implant infection (Flemmig 1994, Kao et al. 1997, Lang et al. 1997).
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The presence of exposed threads and in many cases a rough surface structure
makes it difficult to clean using conventional non surgical treatment options. It may
accordingly be impossible to access the implant surface in order to remove hard and
soft deposits without surgical intervention. Animal research has demonstrated that it
may be possible to regenerate bone and even to obtain re-osseointegration on a
previously infected implant surface (Kolonidis et al. 2003, Schou et al. 2003).
Different regenerative therapies have been proposed for humans and many case
reports are available in the literature (for review see Roos- Jansåker et al 2003).
However limited data exist from comparative clinical trails.
In a clinical trail evaluating osseous grating with or without the use of a resorbable or
a non-resorbable membrane, Khury and Buchmann (2001) reported an average bone
fill in the range of 2-3 mm using the different treatment models. No difference was
found between groups. Their technique included bone to be taken elsewhere in the
oral cavity causing the patient additional surgical trauma and also submerging the
implants resulting in additional prosthetic treatments if the suprastructure needs to be
replaced during the healing period.
Emdogain is a protein extract purified from porcine enamel and has been introduced
in clinical practice to promote periodontal tissue regeneration. EMD is composed
mainly of amelogenins (90%), while the remaining 10% is composed of nonamelogenin enamel matrix proteins such as enamelins, tuftelin, amelin and
ameloblastin. EMD has been reported to promote proliferation, migration, adhesion
and differentiation of cells associated with healing periodontal tissues in vivo
(Schwartz et al. 2000). In a recent review it was concluded that “overall the data
support the positive effect of EMDs on osteoblastdifferentiation” (Jiang et al 2006,
Hughes at al 2006).
Few studies are available evaluating the results of EMDs in conjunction with implant
treatment. In a study that histometrically evaluated the effect of enamel matrix
derivative (EMD) on bone healing after guided bone regeneration (GBR) in
dehiscence-type osseous defects around dental implants; i.e., in the absence of
periodontal ligament cells it was concluded that EMD may positively influence bone
healing after GBR around titanium implants (Casati et al 2002). The effect of new
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trabecular bone to titanium implants was evaluated in a rat model. The authors
reported that EMD was found to be an effective biological matrix for enhancing new
trabecular bone induction and resulting attachment of orthopedic prostheses to the
recipient bone (Shimizu-Ishiura et al. 2002) and Schwarz et al. (2004) in an in vitro
study reported that EMD enhanced cell proliferation and viability of human SaOs(2)
osteoblasts on SLA titanium implants in a concentration-dependent manner.
If the loss of osseointegration is more pronounced it is not possible to either treat the
infection or to accomplish new bone regeneration in the defects using a non-surgical
approach. Surgery may be needed to obtain access to the infected surface and to
use a regenerative approach. As the literature regarding treatment of peri-implantitis
is scarce, randomized clinical trails of peri-implantitis treatment are needed. The aim
with the proposed study is to; determine whether healing of peri-implantitis lesions
using surgical debridement, and detoxification of the implant surface is affected by
the use of enamel matrix derivate.
Screening
A preliminary screening will identify subjects with peri-implant disease and an entry
form will be completed if the subject meets the inclusion criteria.
Objective
Aims of study. The study is a single-center case control study over 12 months
We intend to assess:
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the clinical effects of enamel matrix proteins on peri-implant defects.

the microbial outcome of a regenerative surgical procedure

the anti-inflammatory efficacy of such treatment assessed clinically and
biochemistry analysis of key cytokine markers of inflammation in gingival fluid.
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Outcome variables:
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The primary outcome measures are; 1) clinical probing pocket depth values, 2)
Changes in “bone levels” (presence of mineralization in conjunction with the
implant
measured
at
periapical
radiograph
3)the
extent
of
gingival
inflammation (bleeding on probing- BOP) 4) recession of the peri-implant
mucosal margin at test and control sites between baseline and at study
endpoint (12 months)

The secondary outcome measures are the effects on the presence and
proportions of Porphyromonas gingivalis, Tannerella forsythia, Treponema
denticola, Streptococci spp. and Staphylococcus aureus in peri-implant
pockets and from the dorsum of the tongue.

The tertiary outcomes measures are the impact on cytokine expression in
gingival fluid.

The forth outcome measure is the prevalence of implant loss
Patient screening
Patients will be recruited at a specialist clinic for Periodontology therapy. A
preliminary evaluation will identify patients with implants diagnosed as having periimplant infections. Patient enrolment into the study will be completed within 18
months from the beginning of the study. If a PD reduction of 1mm is to be detected at
 = 0.05 and a power of  = 0.2, the appropriate number of subjects per group would
be around n = 25. Hence, it is foreseen to incorporate approximately 50 subjects in
the study.
Exclusion criteria

Subjects with uncontrolled diabetes mellitus (HbA1c > 7,0)

Subjects requiring antibiotic prophylaxis

Subjects taking prednisolone or other anti-inflammatory prescription drug

Subjects taking medications known to have effects on gingival growth

Subjects with a history of taking systemic antibiotics in the preceding 3 months
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Inclusion criteria - Presence of peri-implantitis
Following a review of the medical history (see exclusion criteria) a full mouth routine
periodontal examination including analysis of available radiographs will be performed.
1) Subjects who consent to participate and have a minimum of one osseointegrated
implant with angular peri-implant bone loss
2) Peri-implant marginal bone loss≥ 2mm as determined from a comparison of the
bone level one year following implant reconstruction with the bone level at screening
(screening radiograph), or
3)  3 mm in depth as determined from a periapical radiograph). (This includes the
total loss of bone).
In combination with probing pocket depth ≥5 mm together with bleeding and/or pus
on probing using 0.2 N probing force.
Patient entry
The study will be submitted to the Ethics committee of Lund and written consent will
be obtained from all subjects to be entered in the study. Once the entry criteria have
been confirmed the subject will be entered to the study and assigned a patient
number.
After having been entered into the study, subjects will be randomly assigned to one
of the two treatment regimens. Random assignment will be performed according to
pre-defined randomisation.
Subject protection – monitoring of adverse events
At each visit the clinician will evaluate patients for any adverse events. Should a
patient require any treatment during the course of the study, the necessary treatment
will be provided at the discretion of the clinician and according to the current standard
of care. All adverse events related to the treatment provided will be recorded on an
adverse event form. The investigation will be performed according to the principles of
the Declaration of Helsinki on experimentation involving human subjects.
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Investigator training and calibration
The two participating investigators will attend training and calibration session aimed
at 1) instruction and calibration in the measurement techniques to be used 2)
instruction in the compilation of the data collection sheets.
Pre-treatment
Any periodontal infection in the remaining dentition should be treated prior to
baseline measures.
Measurements
One and the same examiner, unaware of treatment group for the patient, will
perform all the measurements. Before treatment the following baseline
measurements will be recorded:
1. Intraoral photographs of the implant site
2. Full set of Intraoral radiographs (if radiographs not older than 3 months
are not available)
3. Presence/absence of hyperplasia
4. Full mouth plaque score (FMPS) Presence of dental plaque along the
gingival/mucosal margin recorded after use of disclosing dye and
expressed as a percentage of examined sites within each patient (four
sites per tooth and implant).
5. Local Plaque Score. Presence of dental plaque along the mucosal
margin at four sites of each treated implant recorded after use of
disclosing dye and expressed as a percentage of implant sites within
each patient.
6. Microbial and cytokine sampling, the deepest site of each qualifying
implant will be isolated with cotton rolls. Supragingival plaque will be
removed with sterile cotton pellets. Four paper points will be inserted
submucosally until resistance is met and left for 30 seconds. Two paper
points will be placed in a sterile dry Eppendorf tube to be used for DNA
technique. The samples should be frozen to -70 and sent to
microbiology laboratory at the University of Berne, Berne, Switzerland
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where they will be analyzed using the checkerboard DNA-DNA
hybridization technique for the presence and levels of 40 subgingival
species (Socransky et al. 2004). The other two will be put in Eppendorf
tubes and frozen for analysis of cytokines. Another sample will be
obtained from the site adjacent to the deepest implant site on a
neighbouring tooth/implant).
7. Probing pocket depths (PD) at the implant (4 sites/implant). Recorded
at four sites of each treated implant to the nearest mm using a plastic
probe with a standardized force of 0.2 N (Hawe Click-Probe, Hawe
Neos Dental, Switzerland). At baseline and end-point 12 months
measuring after removal of screw-retained superstructure.
8. Recession of the mucosal margin relative to the restoration margin
(REC) at the implant (4 sites/implant). Position of the mucosal margin
apical to the restoration margin is positive recession (+), position of the
mucosal margin coronal to the restoration margin is negative recession
(-).
9. Presence /absence bleeding on probing (BOP) at the implant (4
sites/implant). Bleeding appearing after measurement of probing depth
and expressed as a percentage of examined sites (4 sites/implant).
10. Presence /absence suppuration (SUP) at the implant (4 sites/implant).
11. Probing depth (PD) at all teeth/implants (4 sites).
12. Presence/absence of bleeding on probing (BOP) at all teeth/implants (4
sites)
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All probing measurements will be made using a manual probe (Hawe Click
probe) using a force of 0.25 N.

Clinical indices will be recorded at 4 sites per tooth/implant; mesial and distal
interproximal sites, midbuccal and mid-lingual.

Full mouth plaque scores (FMPS) will be recorded as the percentage of total
surfaces (4 aspects per tooth/implant) that revealed the presence of plaque
(O'Leary et al. 1972).
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
Bleeding on probing (BOP) will be assessed dichotomously at a force of 0.25
N with a manual pressure sensitive probe (Hawe Click probe) at 4 sites per
tooth/implant. Full mouth bleeding scores (FMBS) will be calculated as the
percentage of total surfaces that bleed on probing.
Microbial analysis - Enumeration of organisms using DNA probes
Samples will be individually placed in labelled Eppendorf tubes and frozen
containing 0.15ml TE (10mM Tris-HCL, 1mM EDTA, pH 7,6). The vials will be
stored at -20° C during three to four weeks and then processed. All samples will
be analyzed by the checkerboard DNA-DNA hybridization technique. A total of 40
bacterial strains will be included in the checkerboard panel1. Whole genomic
DNA-probes and sample DNA precipitation will be obtained as described
elsewhere2. The checkerboard DNA-DNA hybridization will be performed, in detail
as described elsewhere (Socransky et al 2004, Katsoulis et al 2005). Briefly,
bacterial DNA was extracted, concentrated on nylon membranes (Roche
Diagnostics GmbH, Mannheim, Germany) and fixed by cross-linking using
ultraviolet light (Stratalinker 1800, Stratagene, La Jolla, CA). The membranes with
fixed DNA will be placed in a Miniblotter 45 (Immunitics, Cambridge MA USA).
Signals will be detected by chemiluminescence using the Storm Fluor-Imager
(Storm 840, Amersham Biosciences, Piscataway, NJ, USA) with a setup of 200
Microns and 600 Volt. The digitized information will be analyzed by a software
program
(ImageQuant,
Amersham
Pharmacia,
Piscataway
NJ)
allowing
comparison of the density 19 sample-lanes against the 2 standard-Ianes (105 or
106 cells). Signals will be converted to absolute counts by comparisons with these
standards (Socransky et al 2004).The checkerboard DNA-DNA hybridization
assays will be used to identify the presence of 78 species (see Table 2).
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Microbial samples for microbiological analysis will be taken immediately before
surgery, at two week before suture removal, at 6 weeks and at 3, 6, 12, 36 and 60
months (total of 8 time points) after surgery.
Analysis of Cytokines
The gingival fluid samples will be analyzed for levels of Il-1, Il-6, Il-8, TNF , and
PGE2 . ELISA assays will be performed using ELISA (R&D Systems) and quantified
by the Storm Fluorimager (Storm 840, Amershamn Bioscience, Piscataway, NJ) and
by a software program (Image Quant (Amersham Pharmacia, Piscataway, NJ).
ELISA (enzyme-linked immunosorbent assay) serves to analyse the kind of protein
contained in a specific sample and to measure its quantity. In performing ELISA
standardized methods are being used. The absorption is being put into a spectrophotometer with 570nm. Concentrations of proteins are calculated on the basis of
standard curves and given in μg/μl. IN addition RT-PCR (Applied Biosystems model
7500) will be used to verify the results.
Gingival fluid samples for analysis of cytokines will be taken at the same time interval
as microbial samples - immediately before surgery, at two week before suture
removal, at 6 weeks and after 3, 6, 12, 36 and 60 months (total of 8 time points)
Statistical analysis
Descriptive statistics will be used to describe the study population. ROC (Receiver
Operation Characteristic) curves will be studied to identify factor responses that a
distinctively different between test and control groups. Such factors will be used in
logistic forward regression analysis. Non-parametric Kruskal-Wallis test will be used
to assess changes over time. It is anticipated that none of the variables studied will
present with normal distribution characteristics.
Intraoral radiographs
Intraoral standardised radiographs of the site of interest will be taken at baseline, 12,
36 and 60 months. A parallel technique will be used.
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Treatment procedure

Intrasulcular incision and vertical releasing incisions when needed (at a
distance of about 10mm from the implant) to required adequate access.

Removal of chronic inflammatory tissue using titanium curettes.

Surface decontamination – by rubbing the implant surface with saline soaked
foam pellets, followed by extensive rinsing with saline 2 x 20 ml.
Now randomization is performed and depending on the result of the
randomization application of Emdogain will be used or not.

The flaps are replaced and sutured with 5/0 polyamid non-resorbable sutures
without tension. If needed a periosteal releasing incision is made.
Intrasurgical measurements will be made following debridement of the defect at 4
aspects.

The distance from the restoration margin to the base of the defect (mm)
In the event that the restoration has been removed to improve surgical access,
the restoration margin reference point is substituted with the implant shoulder, or
(if an abutment is interposed between restoration margin and implant shoulder)
the abutment-restoration junction.

The distance from the alveolar bone crest to the base of the defect (mm)

Horizontal width of the defect i.e. the distance from the implant surface to the
bony wall (mm).

The defect morphology will be described (circumferential, crater, wide,
angular, narrow).
Post-operative pain will be controlled with ibuprofen as required. All patients will be
instructed to rinse twice daily with chlorhexidine mouthrinse (0.12% or 0,2%) and to
use modified oral hygiene procedures for the first 6 postoperative weeks following
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access flap surgery. Patients will be advised not to chew on the treated area during
the first two weeks post-operative.
Patients will be given special oral hygiene instructions for the treated area: starting on
two week after surgery. Patients will be asked to gently brush the treated area with a
ultra-soft toothbrush (TePe Gentle care, TePe, Malmö, Sweden). The toothbrush will
be immersed in chlorhexidine solution and used to wipe the mucosal area with light
vertical strokes.
Sutures will be removed at the 2 week post-operative visit. (Microbial samples)
No interproximal cleaning will be allowed in the first 3 weeks.
At the 3 week post-operative visit the healing is evaluated and the patient is
instructed in using a super soft interproximal brush (TePe super soft interdental
brush, TePe, Malmö, Sweden
At the 6 week post-operative visit a microbiological sample is taken and the patients
will be instructed to resume normal oral hygiene procedures including interproximal
cleaning and to discontinue chlorhexidine mouthrinsing.
Maintenance care
During year 1 a maintenance care program will be provided every 3 rd month. After
the first year maintenance will be performed at 3-6 months as required. At each
maintenance care appointment FMPS will be recorded. Patients will receive
professional prophylaxis as required.
Post-surgical re-evaluation:
Clinical measurements 6, 12, 36 and 60 months
1.
2.
3.
4.
5.
6.
7.
PD (4 sites/implant)
Recession of the mucosal margin (4 sites/implant)
BOP (4 sites/implant)
Presence/absence of plaque (4 sites/implant)
Presence/absence of suppuration (4 sites/implant)
FMPS (4 sites per tooth/implant)
FMBS (4 sites per tooth/implant)
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Standardised intra-oral radiographs (12, 36 and 60 months)
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enamel matrix derivative to titanium implantation in rat femurs. J Biomed Mater
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and Goodson, J.M. (2004) Use of checkerboard DNA-DNA hybridization to study
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. List of Pathogens in the checkerboard DNA DNA hybridization panel.
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Actinobacillus actinomycetemcomitans serotype a
Actinobacillus actinomycetemcomitans serotype Y4
Actinomyces israelii
Actinomyce neuii
Actinomyces naeslundii type I, and type II
Actinomyces odontolyticum
Actinomyces viscosus
Bifidobacatreiumureolyticus
Bifodobacterium biavatii
Bifodobacterium breve
Campylobacter gracilis
Campylobacter rectus
Campylobacter showae
Capnocytophaga gingivalis
Capnocytophaga ochracea
Capnocytophaga sputigena
Echerichia coli
Eikenella corrodens
Enterococcus faecalis
Eubacterium nodatum
Eubacterium saburreum
Fusobacterium nucleatum sp.nucleatum
Fusobacterium nucleatum sp. polymorphum
Fusobacterium nucleatum sp. vincentii
Fusobacterium periodonticum
Gemella morbillorum
Lactobacillus acidophilus
Lactobacillus gasseri
Lactobacillus iners
Lacotbacilus jenseri
Leptotrichia buccalis
Peptostreptococcus micros
Peptostreptococcus anaerobicus
Proteus mirabilis
Pseudomonas aeruginosa
Neisseria mucosa
Prevotella bivia
Prevotell disiens
Prevotella intermedia
Prevotella melaninogenica (isolate A)
Prevotella melaninogenica (isolate B)
Prevotella nigrescens
Porphyromonas gingivalis
Propionybacterium acnes (type I+II)
Selenomonas noxia
Staphylococcus aureus (FB055)
Staphylococcus aureus (FB095)
Staphylococcus haemolyticus
Staphylococcus epidermis
Streptococcus anginosus
Streptococcus constellatus
Streptococcus gordonii
Streptococcus intermedius
Streptococcus mitis
Streptococcus oralis
Streptococcus sanguis
Streptococcus mutans
Streprtococcis agalactiae
Tannerella forsythia
Treponema denticola
Treponema socranskii
Veillonella parvula
18