Clinical trial NCT03452891
Conditions: Periodontal Diseases
Sponsor: University of Nebraska
Start date: 2019-03-01; Completion date: 2020-12-31
Enrollment: 50 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this study is to determine of local application of commericially-available, FDA-approved preparation of simvastatin is effective in increasing clinical attachment levels (primary outcome), as well as alveolar bone (secondary outcome) compared to standard mechanical therapy in patients on periodontal maintenance therapy (PMT). Subjects undergoing PMT at the UNMC College of Dentistry clinics will be recruited to participate in the randomized one-year clinical trial based on the following eligibility criteria: 1) diagnosis of chronic advanced periodontitis (generalized or localized), 2) participating in regular PMT visits (3-6) month intervals), 3) no systemic diseases or medication which significantly impact periodontal inflammation or bone turnover (e.g. steroids, bisphosphonates, \> 325 mg aspirin/day and in good general health, 4) one experimental quadrant of the mouth with an inflamed 6-9 mm interproximal posterior periodontal pocket with history of bleeding on probing (BOP), 5) willingness to sign consent form. Subjects will be divided into two groups for additional therapy in a 6-9 mm interproximal periodontal pocket at baseline: 1) local anesthesia and mini-flap reflection with subgingival mechanical debridement plus application of the simvastatin-methylcellulose gel or 2) local anesthesia and mini-flap reflection with subgingival mechanical debridement plus application of methylcellulose gel alone. Samples/measurement will be obtained at the designated experimental site at baseline, 2 weeks, 6 and 12 months during PMT: 1) digital radiographs (baseline and 12 months only; bone height measurements), 2) presence of explorer-detectable supragingival plaque, 3) 30-second gingival crevicular fluid (GCF) sample (markers of inflammation, bone turnover), 4) recession from the cemento-enamel junction, 5) probing pocket depth and bleeding on probing (BOP). Following the 12-month visit, the research-specific intervention and measurements in the experiment quadrant will be removed from routine PMT.

Detailed description:
Dental records of patients attending routine periodontal maintenance therapy visits at the UNMC College of Dentistry clinics are reviewed as part of the direct supervision by faculty, including investigators involved int his study. Assessment includes review of medical and dental histories and previous oral hygiene and periodontal charting. Subjects who appear to qualify (40-85 years old, diagnosis of advanced chronic adult periodontitis, one quadrant with at least 3 posterior teeth and one 6-9 mm periodontal pocket, no systemic diseases or medications which significantly impact periodontal inflammation and bone turnover) will be informed of the study, given an opportunity to ask questions, and given an informed consent form to take home. Once signed consent is obtained, subjects will be randomized to one of the two groups: 1) SRP + SIM or 2) SRP + methylcellulose.

At the baseline visit, all women subjects will be given a urine pregnancy test. Once the pregnancy test is determined to be negative, the following sequence of procedures will be performed by the faculty investigator (AK, RR, RH): 1) assignment of the experimental site (6-9 mm pocket with previous bleeding on probing); 2) remove supraginigval plaque from the experimental tooth with explorer \[record positive sites\]; 3) insert paper strip into facial and lingual of experimental site sulcus for 30 seconds for gingival crevicular fluid (GCF) sample, remove and place into sterile vial, and freeze at -80º C for testing inflammation markers; 4) measure recession on experimental teeth and adjacent interproximal tooth at mesial-facial, mid-facial, distal-facial, mesial-lingual, mid-lingual and distal lingual with UNC 15 probe; 5) measure probing pocket depths at same surfaces as recession; 6) record sites that bleed within 30 seconds. After the faculty investigator (primary or secondary investigator) leaves, the participating personnel (LK) will; 7) take a baseline radiograph, administer local anesthesia to the experimental site via local injection, 9) surgical mini-flap reflection localized to the experimental site (papilla reflection), 10) remove most of the remaining interproximal soft tissue to allow access to the root and measure markers of inflammation, 11) scaling/root planing of site to remove supra- and subgingival bacterial plaque and calculus; 12) placement of SIM-methylcellulose gel, 13) repositioning of papilla to initial location and placement of intra-oral cyanoacrylate; 14) finish full-mouth measurement of probing depths and bleeding on probing; 15) perform full-mouth scaling to remove supra- and subgingival bacterial plaque and calculus; 16) review oral hygiene instructions.

It is possible that steps 14, 15 and 16 could be completed prior to steps 1-13 on all teeth except experimental and adjacent teeth due to availability of faculty investigators at the time of the PMT appointment.

It is also possible that steps 1-13 could be completed at a separate appointment.

Subjects will return for postoperative visit in two weeks. Subjects will return for subsequent PMT visits after 3, 6, 9 and 12 months. Steps 14-16 will be completed at each visit as part of routine PMT. At 6 and 12 months, steps 2-6 will also be completed. At 12 months, a radiograph will also be taken.

Dental record data that will be recorded at each visit includes probing depths (PD), recession, bleeding on probing (BOP) and presence of plaque (+/-) at test site and adjacent interproximal sites.

The expected amount of time for the baseline visit in 90-120 minutes. The expected amount of time for subsequent visits in 60-90 minutes.

Arms: arm 1: simvastatin in methylcellulose gel arm 2: Methylcellulose gel
Interventions: intervention 1: Methylcellulose intervention 2: Simvastatin-Methylcellulose
Intervention descriptions: intervention 1: Methylcellulose gel will be placed in a deep (6-9 mm) periodontal pocket. intervention 2: Simvastatin-Methylcellulose gel will be placed in a deep (6-9 mm) periodontal pocket.

Protocol documents:
Prot_SAP_002.pdf: 
Study Protocol 
 
 
 
Effect of Locally-Applied Simvastatin on Clinical Attachment Level and 
Alveolar Bone in Periodontal Maintenance Patients 
 
NCT: 03452891 
 
March 3, 2022 
 
 


Patient Population  
This one year, randomized, and double-masked clinical trial was conducted from March 2019-
December 2020.   Fifty patients, who were receiving periodontal maintenance therapy at the University 
of Nebraska Medical Center (UNMC) College of Dentistry were screened and identified by faculty and 
investigators involved in this study (RR, AK, MC and HR).  Assessment included a review of medical and 
dental histories and previous oral hygiene and periodontal charting.  Inclusion criteria for the study 
included subjects between the ages of 40-85 years, a periodontal diagnosis of advanced chronic 
periodontitis (stage III-IV, Grade B (Tonetti et al. 2018)), one quadrant with at least three posterior teeth 
and one 6-9 mm periodontal pocket with a history of BOP and no radiographic vertical bony defect ≥1.5 
mm, overall good systemic health, and a history of regular PMT.  Exclusion criteria consisted of subjects 
with systemic diseases that significantly affect periodontal inflammation and bone turnover (e.g., 
chronic use of steroids or non-steroidal anti-inflammatory drugs, estrogens, bisphosphonates, 
calcitonin, methotrexate, antibiotics, >325mg aspirin/day), surgical periodontal therapy within the past 
year, and pregnant or breast-feeding females.  Patients who met the inclusion criteria had the protocol 
explained and had all questions answered prior to obtaining consent.   
 
 
 
 
 
 
 
 
Figure 1: Study Design Flowchart 


 
 
  
  
 
 
 
 
 
 
Randomization 
 
 
                                
 
 
 
 
 
 
Follow-up 
 
 
         
 
Analysis 
 
 
 
 
 
 
 
 
 
 
 
Analyzed (n=26) 
Excluded from analysis (n=0) 
Clinical screening 
(n=100) 
Excluded (n=50) 

Declined participation (n=27)  

Inadequate experimental site (n=15) 

Medical history disqualification (n=3) 

Failure to schedule (n=5) 
Randomized (n=50) 
Randomized to PR/PR + SIM/MCL (n=27) 
Initiated intervention (n=27) 
Randomized to PR/PR + MCL (n=23) 
Initiated intervention (n=23) 
Analyzed (n=22) 
Excluded from analysis (n=0) 
Patient dropout (n=1) 

Reason: Lung cancer 
o
Discontinued intervention 
(after 6-months) 
Patient dropout (n=1) 

Reason: Failure to keep 
appointments 
o
Discontinued intervention 
(after 6-months) 


 
Data Collection 
Clinical measures and gingival crevicular fluid (GCF) collection were collected by one of three 
calibrated dentists (RR, AK, and RH).  During GCF collection, experimental site (6-9 mm interproximal 
pocket with no vertical bony ≥ 1.5 mm, and history of bleeding on probing) was isolated with cotton rolls 
and gently dried with gauze.  Supragingival plaque was removed from the test with a dental explorer.  
For GCF collection, an absorbent paper strip (PerioPaper Strips, Oraflow, Hewlett, NY) was inserted into 
the facial and lingual/palatal sulcus of the experimental site (Figure 2).  After 30 seconds, the paper 
strips were immediately placed into a sterile vial and frozen at -80°C until further analysis.  Strips 
contaminated by blood were discarded and were retaken after two minutes.  Next, supragingival plaque, 
recession, probing depths, and bleeding on probing (BOP) within 30 seconds were recorded on 6 sites 
(MF, F, DF, ML, L, DL) on the experimental tooth and adjacent tooth.  
Treatment Protocol  
After the clinical data and GCF collection was completed, the investigator involved with data 
collection left, and the surgical / drug application phase of treatment was completed by a single clinician 
(LK, MB) and assistants (MC, LA) not involved with clinical measurements as described previously (Jasa 
et al. 2020).   Following administration of local anesthesia to the experimental site, a #12B blade was 
used to reflect both the facial and lingual/palatal papilla including in the experimental 6-9 mm 
interproximal pocket.  Interproximal soft tissue was removed to allow access to the root.  To measure 
activation of gene markers of inflammation and bone turnover, an approximately 2x2x2 mm piece of the 
interproximal tissue was placed in a sterile vial of 1.5 mL RNAlater solution (ThermoFisher Scientific, 
Waltham, MA) (Figure 3) and frozen at -20°C until further analysis.  Scaling and root planning was 
performed interproximally on the test site and on the adjacent interproximal tooth surface.  Verification 


of a clean and smooth root surface was done using an 11/12 explorer and the Perioscope (Perioscopy 
Unit, Zest Dental Solutions, Carlsbad, CA) by the clinician (Figure 4).   
After mechanical therapy was completed, the unmasked clinician (LK or MB) randomly assigned 
patients to test simvastatin in methylcellulose (SIM/MCL) or control (MCL) groups.  The root surface was 
etched for 2 minutes with ethylenediaminetetraacetic acid (EDTA, Pref-Gel, Straumann, Andover, MA) 
followed by irrigation with sterile saline.  SIM and MCL were prepared by local compounding pharmacy 
(Pharmacy Solutions Lincoln, NE) and mixed immediately prior placement to achieve 2.2 mg simvastatin 
(SIM) suspended in 0.15 ml methylcellulose gel (MCL) (test group) or 0.15 ml of methylcellulose gel 
alone (control group)(Figure 5).  
Gels were placed at the base of the pocket and deposited up the interproximal root surface of 
the experimental tooth (Figure 6).  The papilla were re-approximated under pressure and sealed using 
cyanacrylate (PeriAcryl, Glustitch, Delta, BC, Canada).  Routine periodontal maintenance therapy (PMT) 
then was completed by MC or LA, including full mouth periodontal charting, debridement, and root 
planing of inflamed pockets avoiding the experimental area.  Patients were instructed to avoid brushing 
and flossing of the experimental site for 6 weeks.  They were dispensed Listerine (Johnson & Johnson 
Consumer, Inc., New Brunswick, New Jersey) and instructed to be used twice a day for 30 seconds for 6 
weeks.  Patients were asked to return for postoperative visits after 2 and 6 weeks along with PMT recalls 
at 3, 6, 9, and 12 months.  GCF collection was repeated at 2- weeks and 12-month PMT visits and clinical 
measurements were repeated at 12 months (Figure 7) by one of the three calibrated examiners (AK, RR, 
and HR).  Collection of approximately 2x2x2 mm interproximal tissue was repeated at 2 weeks by either 
LK or MB.  During the 6-week follow up, patients were given Gel-Kam® preventative treatment gel 
(Colgate Oral Pharmaceuticals, NY, NY) and a GUM® Proxabrush® (Sunstar Americas, Inc., Schaumburg, 
IL) and patients were instructed to brush the experimental site twice a day using the provided 


interproximal brush.   Participants were questioned about adverse events at 2-weeks and 6-months, and 
12-months PMT visits.    
   Figure 2: Baseline GCF Sample  
                     Figure 3: Baseline Interproximal tissue 
 
 
 
 
       
Figure 4: 
Perioscope use                                                 Figure 5: Mixing SIM & MCL 
    
 
 
 
 
 
 
 
 
 
Figure 6: Placement of SIM 
                                  Figure 7: 12-months post-therapy 
 
 
 
 
 
 
 
 
 
 


 
Analysis of GCF samples  
Each GCF sample containing two paper strips was eluted using 85 μl of 1× PBS by gently 
agitating the samples on a rocker plate for 1 h at 4°C.  Analyte concentrations were measured using 
magnetic bead panels (Millipore, Billerica, MA) and read on a MAGPIX with Luminex xPONENT software 
(Luminex Corporation, Austin, TX) per the manufacturers’ recommendations.   
Nine analytes were measured:  Fibroblast growth factor (FGF-2), interleukin (IL)-1β, IL-6, IL-10, 
IL-17A, insulin-like growth factor 1 (IGF-1), tumor necrosis factor (TNF)-α, receptor activator of nuclear 
factor kappa-Β ligand (RANKL), and vascular endothelial growth factor A (VEGF-A).  The amounts of 
cytokines are reported in picograms per ml, then mathematically adjusted by multiplying 0.085 to 
achieve pg per sample.  
 Analysis of interproximal tissue samples 
 Interproximal tissue samples were collected between the interproximal sulcus of the 
experimental site and adjacent tooth and were stored in 1.5 mL RNAlater solution (ThermoFisher 
Scientific, Waltham, MA).  RNA extraction was conducted using the NucleoSpin RNA XS—complete kit 
for isolation and purification of total RNA from extremely small samples (Marcherey – Nagel, Düren, 
Germany).  DNA digestion and cDNA synthesis were done using the QuantiTect Reverse Transcription Kit 
from QIAGEN© (Germantown, MD) per the manufacturers’ recommendations.  
Samples were diluted with water and placed in 96-well custom array plates in technical 
triplicate; qPCR was executed with SsoAdvanced Universal SYBR Green Supermix from Bio-Rad 
(Hercules, CA) reagents.  PCR conditions were 39 cycles at 95°C for 3 minutes (1 cycle) 95°C for 15 
seconds, and at 58°C for 30 seconds (39 cycles).  At the end point of rt-PCR analysis, the threshold cycle 


or ct vaules were recorded.  To analyze the relative changes in gene expression, the 2^ΔΔct method was 
done, as described by Livak et al (2001). 
Statistical analyses  
A sample size of 22 per group was needed to achieve at least 80% power to detect a difference 
of 1.0 mm in clinical attachment level between groups with a common estimated group standard 
deviation of 1.1 mm with a significance level of 0.05 using a two-sided two-sample t-test.  This is based 
on mean data from most relevant previous studies (Killeen et al., 2012 and Jasa et al., 2019).   
For the experimental interproximal treatment sulcus, BOP was considered present at baseline if 
at least one buccal or lingual interproximal site had the condition present.  The follow-up variables for 
BOP were determined as follows: if the patient started without BOP and ended without BOP or showed 
a reduction (I.e. presence of BOP at baseline to absence of BOP at 12 months), that patient was 
considered to have a good outcome.  If the patient began with BOP and showed no improvement or 
they developed BOP, that patient was considered to have a poor outcome.  Associations between 
categorical variables were assessed using Chi-Square tests, or Fisher’s exact tests when 
expected cell counts were low.  Medians and inter-quartile ranges (IQRs; the range of the 
middle 50% of the data (25th percentile, 75th percentile)) were calculated for each treatment 
condition, and Wilcoxon Rank Sum tests were used to examine differences in distributions of 
BOP between the two treatment conditions (i.e. SIM/MCL or MCL) for baseline BOP values.  For 
the change in BOP outcome, logistic regression models were used, which included group and 
adjusted for worst side. Adjusted odds ratios are presented with 95% confidence intervals.   
Descriptive statistics for raw GCF and RT-PCR continuous data are given as medians and 
interquartile ranges (IQRs, representing the range of the middle 50% of the data).  For analysis, 
data was log transformed due to skew.   


General estimating equations were run with each measure of interest as the outcome, 
and each model included the variables (analyte) group and time, as well as the interaction 
between group and time to assess if change over time differed by group. If the interaction was 
not significant, main effects models were run.  P-values for post hoc comparisons were adjusted 
using simulation methods.  Pearson correlations were also run to assess for association 
between GCF and interproximal tissue samples at a given time point.  Clinical attachment level 
(CAL) change between baseline and 12-months was the primary clinical outcome measure for 
this clinical trial.  For change in CAL baseline CAL values were subtracted from 12 months CAL 
value, and difference scores were plotted against GCF and rt-PCR measures at two weeks (i.e. > 
negative values indicate reduction in clinical attachment loss).   Associations between change in 
CAL and measures at 2-weeks were assessed using Pearson correlations.  All analyses were 
performed using SAS software version 9.4 (SAS Institute Inc., Cary, NC).