Clinical, Microbiological and Histological Effects of Cemented Implant Restorations. A Cross-over Controlled Clinical Study
Phase: NA
Conditions: Gingivitis and Periodontal Diseases, Tooth Loss
Sponsor: University of Zurich

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Clinical, microbiological and histological effects of 
cemented implant restorations.  
A cross-over controlled clinical study 
(Kliniska, mikrobiologiska och histologiska effekter av cementerad 
implantat restaurering.  
En cross-over kontrollerad klinisk studie) 
 
Nadja Naenni, Sigrun Eick, Niklaus P.Lang, Christoph H.F. 
Hämmerle, Björn Gjelvold, Jenö Kisch, Ann Wennerberg 
 
 

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Abstract  
Aim:  
The primary aim of this study is to test whether or not cement residues in the 
submucosal environment of implants lead to a change in the microbiota and induce 
inflammation of the periimplant tissues. 
Material and Methods:  
24 patients in need of a single tooth replacement will be enrolled in this cross-over 
controlled clinical study. All patients will receive a two-piece dental implant, which will 
be restored with both a cemented and a screw-retained single crown. At the time of 
impression taking, patients will be randomized into two groups.  
Patients in group A will receive a screw-retained crown. Every 4 weeks 
microbiological samples using sterile paper points will be collected and analyzed for 
bacterial content by real-time PCR. Additionally, two host markers (MMP8, IL-1ß) will 
be determined by ELISA. Following this first period of 16 weeks, the screw-retained 
crown will be replaced by a new intraorally cemented crown. Cement removal will be 
preformed according to best clinical procedure. These crowns will again be left for 
another period of 16 weeks and followed up for the harvesting of microbiological 
samples every 4 weeks. After the second 16-week the crowns will be removed to 
evaluate any excess cement. All patients will be fitted with the orginial screw-retained 
crown. Clinical parameters for inflammation and probing depths will be obtained after 
each 16 week-period. 
In group B the crowns will be incorporated in a reverse pattern. During the first 16 
weeks any possible cement residues will be removed according to best clinical 
procedure, while for the second period of 16 weeks patients will be fitted with a 
screw-retained single crown. Again, microbiological and clinical parameters will be 

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obtained at the same intervals as in Group A.  
After the second 16 week period the screw-retained crowns will be (re-) inserted in all 
patients, single tooth x-rays taken and clinical baseline values obtained. Additionally, 
a soft tissue biopsie will be harvested at the time of insertion of the final screw-
retained crown. Patients will be followed up for another 16-week period. 
Results: 
 
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Introduction 
 
The relationship between dental restorations and periodontal conditions has been 
studied, and it has consistently been demonstrated that dental restorations may 
constitute a risk factor for the initiation and progression of marginal periodontitis 
(Bjorn et al., 1969, Than et al., 1982, Claman et al., 1986, Jansson et al., 1994). 
Periodontitis is an opportunistic infection involving pathogenic bacteria in a 
susceptible host. The pathogenecity of the microbiota is determined by the 
composition of the biofilms (Haffajee and Socransky, 2005). Gram-negative strictly 
anaerobic micro-organisms, such as Prophyromonas gingivalis, Prevotella 
intermedia, Fusobacterium nucleatum sp., Tannerella forsythensis and Treponema 
denticola belong to the putative periodontal pathogens. Moreover, Aggregatibacter 
actinomycetemcomitans is occasionally found in significant numbers. Consequently 
the conversion of a health-associated microbiota predominated by gram-positive 
facultative cocci and rods to a gram-negative strictly anaerobic microbiota may initiate 
the disease process. Such conversions are commonly observed in the presence of 
biofilm retaining factors (Brunsvold and Lane, 1990, Nassar et al., 1995, Lang et al., 
1983) such as calculus or overhanging restoration margins. Already half a century 
ago, the role of potentially harmful effects of imprecise dental restorations impinging 
on periodontal health have been described (Waerhaug, 1956).  
In an experimental study with deliberately placed overhanging margins of overlays in 
human volunteers it was demonstrated that gingival inflammation resulted 2-4 months 
after the placement of such overlays (Lang et al., 1983) concomittantly with a 
significant conversion from a health-associated to a disease-associated microbiota 

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predominated by gram-negative anaerobic bacteria. 
 This gingival inflammation was reverted to health soon after removal of the 
overhanging margins and the replacement of the overlays by clinically perfect 
margins. Again, this happened in accordance with a shift of the gram-negative 
anaerobic microbiota towards a health-associated microbiota.  
In a cross-over design the study was repeated and similar outcomes were reported. 
At the sites of the overhanging margins the predominant cultivable flora was 
determined using continuous anaerobic culturing techniques. When restorations with 
overhanging margins were in situ an increase in gingival index scores and a 
subgingival microbiota closely resembling that of chronic periodontitis was detected. 
The presumptive gram-negative anaerobic pathogens proportionally increased from a 
few percent (2-4%) to substantial proportions (> 25%). Following replacement of the 
restorations with clinically perfect margins, a microbiota characteristic for gingival 
health was re-established.  
Clinically, an increase in gingival index scores was detected when overhanging 
margins were placed. Bleeding on probing always preceded the peak level of black 
pigmenting gram-negative anaerobic bacteria. During the limited duration of the 
experiment, no loss of attachment was detected at any site using subtraction 
radiography. The microbiota observed at the filling sites with perfect margins first 
represented a gram-positive, predominantly coccoid and health-associated 
microbiota, while in the second half of the experiment, when restorations with 
overhanging margins were placed, the microbiota resembled that of a 
periodontopathic gram-negative anaerobic biofilm. Thus, a potential mechanism for 
the initiation of periodontal disease associated with iatrogenic factors had 

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convincingly been demonstrated (Lang et al., 1983). 
 
Periodontal and peri-implant diseases represent opportunistic infections triggered by 
a gram-negative anaerobic microbiota (Mombelli and Decaillet, 2011, Haffajee and 
Socransky, 2005). Although the microflora around teeth is very similar to that around 
implants, differences on a histological level exist between the two entities. 
(Shahabouee et al., 2012, Berglundh et al., 2011) Biofilm retaining factors such as 
calculus, overhanging margins and cement excess may contribute to the conversion 
of a health-associated to a disease-associated microbiota. While established for 
teeth, a similar association has not yet been demonstrated at implant-crown sites. 
Peri-implant diseases consist of two recognized entities, peri-implant mucositis and 
peri-implantitis (Jepsen et al., 2015). The former may appear to be the precursor of 
the latter which is characterized by loss of alveolar bone around an osseointegrated 
implant (Berglundh et al., 2011, Heitz-Mayfield, 2008). However, the factors 
influencing the conversion of peri-implant mucositis to peri-implantitis are still 
unknown. As it has been observed that dental restorations that were inaccessible for 
oral hygiene practices resulted in an initiation and progression of peri-implantitis 
(Serino and Strom, 2009) implant reconstructions, respectively excess cement may 
act as a biofilm retaining factor similar to dental restorations, hereby triggering 
disease progression (Wilson, 2009, Staubli et al., 2016, Korsch et al., 2014).  
 
Hence, the primary aim of the present clinical experiment is to test whether or not 
cement residues in the submucosal environment of implants lead to a conversion of a 
health-associated to a disease-associated microbiota and consequently induces 

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inflammation of the peri-implant tissues.  
Additionally, histopathological evaluation of the soft tissues around both cemented 
and screw-retained reconstructions will be performed to determine whether there are 
differences regarding host responses at implant sites as compared to teeth.  
 
Material and Methods 
 
Ethical aspects  
The protocol of this study will be submitted to the regional ethical committee for 
human research in Malmö, Sweden. Patients will be informed of the risks and benefits 
in participating in this study and consent forms will be signed.  
All patients will receive a screw-retained single crown. The implant material as well as 
the reconstruction will be provided according to an existing fee while the implant 
installation will be charged regulary. Furthermore, regular annual recall visits with a 
dental hygienist will be scheduled for two years following the conclusion of the study.  
 
 
Experimental subjects   
From the patient pool of the specialist clinic (Folktandvården AB Skåne, 
Specialisttandvård, Lund, Sweden), 24 adult patients with single tooth gaps in the 
posterior region of either jaw (no antagonist needed) will be recruited. Patients have 
to be systemically healthy and eligible for implant placement. 
 
 
 

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Inclusion criteria: 
- patient older than 18 years 
 
- systemically healthy subject 
- periodontally healthy individuals  
- absence of peri-implantitis 
- no bone loss 
- good oral hygiene  (PCR ≤ 20%) 
- healthy periodontal tissues (BoP≤ 20%) 
- patients with a single tooth gap in the posterior area of either jaw  
 (premolars and molars) 
- at least 8mm in mandible; at least 6mm in maxilla (summers technique) 
Exlusion criteria: 
- ongoing periodontal disease 
- bruxism 
- unwilling to comply with study procedures 
- heavy smokers (≥10 cig/d) 
- ongoing periodontitis/implantitis 
 
 
Clinical procedures  
All subjects will receive a thorough baseline examination including a patient history, 
an examination of all oral tissues encompassing tooth vitality, hard structures (caries), 
as well as gingival and periodontal tissues and a radiopraphic status. Depending on 
this examination the patient will receive a hygienic phase following which an oral 

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implant will be placed.  
 
 
Surgical procedure  
A two-piece dental implant (Astra EV, DentsplySirona Implants, Mölndal, Sweden) will 
be placed according to the manufacturers recommendation. The implant shoulder will 
be placed flush with the bone. In cases of minimal soft tissue thickness it may be 
placed subrestally in order to allow for the biological width to be established. Before 
suturing the soft tissue thickness will be measured with a calibrated periodontal probe 
(UNC 15). After primary wound closure a periapical radiograph of the implant site will 
be obtained. Following a period of submerged healing of 10-12 weeks, abutment 
connection (AC) will be performed, soft tissue measured with a periodontal probe 
(UNC 15 with a point diameter of 0.4mm) and a standardized healing abutment 
(DentsplySirona Implants, Mölndal, Sweden) inserted. 
 
Prosthetic procedure  
In order to establish maturated soft tissue condition (Berglundh et al., 2007, Salvi et 
al., 2015) 4 weeks after AC a digital impression with an implant scan-body will be 
taken. Healing abutments will be re-inserted and left until the try-in of the 
abutment/crown.  

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A titanium abutment (Atlantis® Abutment, DentsplySirona Implants, Mölndal, 
Sweden) will be designed according to the respective soft tissue resulting in a 
physiological emergence profile and a submucosal location of 1-1.5mm of the future 
abutment/crown transition. At the time of impression taking patients will be 
randomized into two groups (A and B). 
Every patient will receive two identical CAD/CAM-manufactured titanium abutments 
(Atlantis® Abutment, Dentsply Implants, Mölndal, Sweden). The respective full-
contour zirconia crowns will be designed by the dental technician and will further be 
extraorally or intraorally cemented.  
 
 
screening
prosthetic phase
implant placement
surgical phase
impression
X-ray
X-ray
10-12 weeks

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Group A: 
12 subjects will receive a screw-retained single crown. A titanium abutment will be 
digitally designed by the dental technician and milled (Atlantis® Abutment, 
DentsplySirona, Mölndal, Sweden). After try-in of the abutment, a milled full-contour 
zirconia crown will be tried in (temporarily glued to the abutment). If occlusal and 
interdental contacts as well as form and color are satisfactory, the crown (A-) will be 
cemented extraorally by the dental technician using a resin cement (Multilink, Ivoclar 
Vivadent, Solna, Sweden) (Fig.2). Any cement excess will be removed, checked with 
binoculars and the subgingival part of the reconstruction polished before insertion. 
The reconstruction will be left in situ for 16 weeks. After the first period of 16 weeks, a 
new titanium abutment (duplicate) will be milled (Atlantis® Abutment, DentsplySirona, 
Mölndal, Sweden), allowing for cementation of a single crown. A new full-contour 
zirconia crown (A+) will be designed (by the dental technician), milled and cemented 
intraorally again using the same resin cement (Multilink, Ivoclar Vivadent, Solna, 
Sweden) (Fig.2). After setting of the cement, the seat of the crown as well as the 
occlusion will be checked. Great care will be given in order to remove any excess 

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cement. Absence of cement will be verified by taking a single-tooth x-ray. Again, this 
reconstruction will be followed up every 4 weeks and the reconstruction will be left in 
situ for a second period of 16 weeks. Subsequently, all patients will be restored with 
the original screw-retained clinically perfect crown (A-). Hence, group A yields no 
cement residues in the first period, but may do so in the second time-period (A-/+).  
 
Group B: 
12 subjects will undergo the same, but reverse order of therapy: 
In the first period the subjects will receive a cement-retained full-contour zirconia 
crown (B+) on an individualized titanium abutment (Atlantis® Abutment, 
DentsplySirona, Mölndal, Sweden). Again, great care will be given in order to remove 
any excess cement. After cementation, a single-tooth x-ray will be taken to control the 
fit of the crown and the absence of any possible cement residues. In the second 
period of 16 weeks a new full-contour zirconia crown on an identically designed 
titanium abutment (Atlantis® Abutment, DentsplySirona, Mölndal, Sweden) will be 
extraorally cemented and fixed by screw-retention leaving no cement residues (B-). 
Subsequently, all patients will be fitted with the screw-retained crown (B-).  
 

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Follow up 
Clinical parameters  
The following clinical indices will be obtained at determined timepoints: 
t-1 = at the time of crown insertion  
• before crown-insertion: 
 Mucosa thickness (MT) using a calibrated periodontal probe (UNC 15) 
(measuring from the margo mucosae to the crown/abutment conversion) 
t0 = 1week after crown insertion (Baseline)  
• Probing depth (D) using a calibrated periodontal probe (UNC 15 with a point 
diameter of 0.4mm) and a probing pressure of approximately 0.25 N 
• modified PLI (mPLI) (Mombelli et al., 1988)  
• modified Bleeding index (mBI) (Mombelli et al., 1988) 
• Microbiological sampling using sterile paper points at four sites per implant 
t1 = 8 weeks (±1w) after crown insertion  
• Microbiological sampling 
t2 = before insertion of new crown (at the end of the 1st 16week-period) 
• PD 
• modified PLI (mPLI) (Mombelli et al., 1988)  
• modified Bleeding index (mBI) (Mombelli et al., 1988) 
• Recession of the soft tissue using a calibrated periodontal probe (UNC 15) 
• Microbiological sampling 
t3 = 8 weeks (±1w) after insertion crown of second crown 
• Microbiological sampling 
t4 = before insertion of the final screw-retained crown in all patients  

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(at the end of the 2nd 16week-period) 
• PD 
• modified PLI (mPLI) (Mombelli et al., 1988)  
• modified Bleeding index (mBI) (Mombelli et al., 1988) 
• Recession of the soft tissue using a calibrated periodontal probe (UNC 15)  
• Microbiological sampling 
t5 = termination of experiment  
 
 
(after 16 weeks with final screw-retained crown) 
• PD 
• modified PLI (mPLI) (Mombelli et al., 1988)  
• modified Bleeding index (mBI) (Mombelli et al., 1988) 
• Recession of the soft tissue using a calibrated periodontal probe (UNC 15) 
• Microbiological sampling 
 
Attachement levels will be calculated by deducting recessions (negative) from the 
probing depths. All patients will be followed up one year after insertion of the screw-
retained crowns (t2) for a regular check-up. 
 
Radiographs 
A single-tooth x-ray will be obtained at the time of AC to ensure sufficient 
osseointegration of the implant before entering the prosthetic phase. At the time of 
insertion of the respective reconstruction in both groups, another periapical 
radiograph will be obtained to ensure the fit of the reconstruction and to check for 
eventual cement residue.  

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Microbiological sampling and analysis  
8 weeks after the placement of the first reconstruction (t=1) and every  
(8±1w) weeks thereafter as well as 16 weeks after insertion of the final screw-retained 
crown, microbiological samples will be obtained.  
Gingival crevicular fluid (GCF) will be collected using standardized filter paper strips 
(Periopaper®, Harco, Winnipeg, Canada) from the implant site. The site will be first 
isolated with cotton rolls and air dried. The sterile paper strip is then placed at the 
entrance of the sulcus (superficial intracrevicular method (Griffiths, 2003) and left in 
place for 30s. After that, the strip(s) is (are) placed in a new Eppendorf tube. 
Thereafter, sterile paper points will be inserted until resistance is felt at four sites in 
the peri-implant sulcus. Without removing of supragingival plaque, sterile paper point 
(ISO 50) will be inserted into the periodontal pocket for 30 s. 
Subsequently, the samples will be frozen and sent to the laboratory of oral 
microbiology at the University of Berne (Prof. Sigrun Eick). A battery of 10 putative 
periodontal pathogens will be analyzed using real-time PCR. Moreover, the samples 
will be assayed for host markers such as MMP 8 and IL-1ß using ELISA.  
Bleeding on sampling will be obtained at the same time intervals. 
 
Harvesting of biopsies  
At the time of insertion of the screw-retained final crown (t=4) and in case of a 
sufficient amount of keratinized mucosa, a crestal full-thickness soft tissue biopsy will 
be harvested. Two parallel crestal incisions will be connected to sulcular incisions at 
both the implant and adjacent tooth. The dimensions of the biopsies will be 2 mm in 

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width and a length ranging from the distal/mesial aspect of the implant site to the 
mesial/distal aspect of the adjacent tooth. The vertical dimension will extended from 
the mucosal margin to the bone crest.  
1-2 single sutures will be used to adapt the wound margins and to prevent the 
exposure of the bone. All patients will be re-instructed to clean the implant sites using 
0.2% chlorhexidine rinsing solution. No manual cleaning will be allowed for another 7 
days i.e. until suture removal.   
 
The biopsies will be used for histomorphometric measurements as well as the 
determination of inflammation and/or infection markers applying light-microscopy. 
Tissue samples will further be processed with a (ribonucleid acid) RNA solution to 
allow for RNA extraction. This will then be analyzed via polymerase chain reaction 
(PCR) to extract the inflammation genome and identify inflammation markers such as 
Interleukin (IL-4, IL-3, IL-1alfa, IL-1beta) and tumor necrose factor (TNF-alfa). A part 
ot these biopsies will further be used for the determination of inflammatory markers 
using PCR. 
 
Evaluation of cement residues 
Retrieved crowns will be examined using light-microscopy/electron microscopy to 
determine the area of submucosal excess cement. 
 
Statistical analysis  

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The clinical and microbiological data will be analysed longitudinally with paired t-test 
testing the hypothesis with no difference between the two timepoints and between 
groups within the experimental periods of 16 weeks.   
_____________________________________________________________________________________________ 
 
 
 
 

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