A Clinical Use of Er,Cr:YSGG Laser: an Anti-biofilm Treatment
Phase: NA
Conditions: Endodontic Disease, Root Canal Infection
Sponsor: University of Pennsylvania

Protocol ID: 853701
Title: Erbium, Chromium: Yttrium, Scandium, Gallium, Garnet (Er,Cr:YSGG) Laser in 
Root Canal Disinfection 
NCT05959629
 5/9/2023
Protocol Template V3.0 (09Nov2020)

Protocol [Insert Study Number] - Er,Cr:YSGG laser in Endodontics. Waterlase Express™, 
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CLINICAL RESEARCH PROTOCOL 
INVESTIGATIONAL 
PRODUCT(S): 
Waterlase Express™, BIOLASE®. 
Root canal disinfection protocol 
STUDY 
NUMBER(S): 
IRB 
Number 
Other 
Protocol 
Identifiers 
N/A 
PROTOCOL(S) TITLE: 
A clinical use of Er,Cr:YSGG laser: an anti-biofilm treatment 
REGULATORY 
SPONSOR: 
Penn Dental Medicine – Endodontic Department 
Evans Building 
4001 Spruce St 
Philadelphia, PA 
19104-6003 
FUNDING SPONSOR(S): 
Penn Dental Medicine – Endodontic Department 
MEDICAL DIRECTOR 
Dr. Bekir Karabucak (Chair of Endodontic Department) 
ORIGINAL PROTOCOL 
DATE: 
VERSION NUMBER: 
V1.0 
VERSION DATE:  
05.09.2023 

 
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Confidential and Proprietary Information of Penn Dental Medicine – Endodontic Department 
 
1 
Study Summary .......................................................................................................... 9 
1.1 
Synopsis .................................................................................................................. 9 
1.2 
Key Roles and Study Governance ..................................................................... 11 
1.3 Schema ....................................................................................................................... 12 
2 
Introduction and RationalE .................................................................................... 14 
2.1 
Study Rationale ................................................................................................... 14 
2.2 
Background .......................................................................................................... 14 
2.2.1 
Pharmacokinetics, Pharmacodynamics and Toxicology ........................... 16 
2.2.2 
Assessment for Potential Study Products Drug-Drug, Drug-Device, 
Device-Device Interactions ........................................................................... 16 
2.2.3 
Clinical Adverse Event Profile ..................................................................... 16 
2.2.4 
Dosing Rationale ........................................................................................... 16 
2.3 
Risk/Benefit Assessment ..................................................................................... 16 
2.3.1 
Known Potential Risks .................................................................................. 16 
2.3.2 
Known Potential Benefits ............................................................................. 17 
2.3.3 
Assessment of Potential Risks and Benefits ................................................ 17 
3 
Study Objectives and Endpoints............................................................................. 18 
4 
Study Plan ................................................................................................................. 20 
4.1 
Study Design ........................................................................................................ 20 
4.2 
Scientific Rationale for Study Design ................................................................ 21 
4.3 
Justification for Dose .......................................................................................... 21 
4.4 
End of Study Definition ...................................................................................... 21 
5 
Study Population ...................................................................................................... 22 
5.1 
Inclusion Criteria ................................................................................................ 22 
5.2 
Exclusion Criteria ............................................................................................... 22 
5.3 
Lifestyle Considerations ..................................................................................... 22 
5.4 
Screen Failures .................................................................................................... 22 
5.5 
Strategies for Recruitment and Retention ........................................................ 23 
6 
Study Intervention ................................................................................................... 24 
6.1 
Study Intervention(s) Administration ............................................................... 24 
6.1.1 
Study Intervention Description (attachment 3) .......................................... 24 
6.1.2 
Dosing and Administration .......................................................................... 25 
6.2 
Preparation/Handling/Storage/Accountability ................................................ 25 
6.2.1 
Acquisition and accountability .................................................................... 25 
6.2.2 
Formulation, Appearance, Packaging, and Labeling ................................ 25 
6.2.3 
Product Storage and Stability ...................................................................... 25 
6.2.4 
Preparation .................................................................................................... 25 
6.3 
Measures to Minimize Bias: Randomization and Blinding ............................. 26 
6.4 
Study Intervention Compliance ......................................................................... 26 
6.5 
Concomitant Therapy ......................................................................................... 27 
6.5.1 
Rescue Medicine ............................................................................................ 27 
 

 
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7 
Study Intervention Discontinuation and Participant 
Discontinuation/Withdrawal ................................................................................... 27 
7.1 
Discontinuation of Study Intervention .............................................................. 27 
7.2 
Participant Discontinuation/Withdrawal from the Study ............................... 27 
7.3 
Lost To Follow-Up .............................................................................................. 28 
8 
Study Assessment and Procedures ......................................................................... 29 
8.1 
Efficacy Assessments ........................................................................................... 29 
8.2 
Safety and Other Assessments ........................................................................... 30 
8.3 
Adverse Events and Serious Adverse Events ................................................... 31 
8.3.1 
Definition of Adverse Events (AE) .............................................................. 31 
8.3.2 
Definition of Serious Adverse Events (SAE) ............................................... 31 
8.3.3 
Unanticipated Adverse Device Effect (UADE) ........................................... 31 
8.3.4 
Classification of an Adverse Event .............................................................. 32 
8.3.5 
Time Period and Frequency for Event Assessment and Follow-Up ......... 34 
8.3.6 
Adverse Event Reporting ............................................................................. 34 
8.3.7 
Serious Adverse Event Reporting ................................................................ 35 
8.3.8 
Reporting Events to Participants ................................................................. 35 
8.3.9 
Events of Special Interest ............................................................................. 35 
8.3.10 
Reporting of Pregnancy ................................................................................ 35 
8.4 
Unanticipated Problems ..................................................................................... 35 
8.4.1 
Definition of Unanticipated Problems (UP) ................................................ 35 
8.4.2 
Unanticipated Problem Reporting .............................................................. 36 
8.4.3 
Reporting Unanticipated Problems To Participants ................................. 37 
8.5 
Device Reporting ................................................................................................. 37 
9 
Statistical Considerations ........................................................................................ 37 
9.1 
Statistical Hypotheses ......................................................................................... 37 
9.2 
Sample Size Determination ................................................................................ 38 
9.3 
Populations for Analyses .................................................................................... 38 
9.4 
Statistical Analyses .............................................................................................. 38 
9.4.1 
General Approach ......................................................................................... 38 
9.4.2 
Analysis of the Primary Efficacy Endpoint(s) ............................................ 38 
9.4.3 
Analysis of the Secondary Endpoint(s) ....................................................... 39 
9.4.4 
Safety Analyses .............................................................................................. 39 
9.4.5 
Baseline Descriptive Statistics ...................................................................... 40 
9.4.6 
Planned Interim Analyses ............................................................................ 40 
9.4.7 
Sub-Group Analyses ..................................................................................... 40 
9.4.8 
Tabulation of Individual Participant Data ................................................. 40 
9.4.9 
Exploratory Analyses .................................................................................... 40 
10 Supporting Documentation and Operational Considerations ............................. 40 
10.1 
Regulatory, Ethical, and Study Oversight Considerations ............................. 40 
10.1.1 
Informed Consent Process ............................................................................ 40 

 
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10.1.2 
Study Discontinuation and Closure ............................................................. 41 
10.1.3 
Confidentiality and Privacy ......................................................................... 42 
10.1.4 
Future Use of Stored Specimens and Data ................................................. 42 
10.1.5 
Safety Oversight ............................................................................................ 43 
10.1.6 
Clinical Monitoring ....................................................................................... 43 
10.1.7 
Quality Assurance and Quality Control ..................................................... 43 
10.1.8 
Data Handling and Record Keeping ........................................................... 43 
10.1.9 
Protocol Deviations ....................................................................................... 44 
10.1.10 
Publication and Data Sharing Policy .......................................................... 45 
10.1.11 
Conflict of Interest Policy ............................................................................. 45 
10.2 
Additional Considerations .................................................................................. 45 
10.3 
Protocol Amendment History ............................................................................ 45 
11 REFERENCES ......................................................................................................... 46 
12 APPENDIX ............................................................................................................... 48 
12.1 
Schedule of Activities (SoA) ............................................................................... 48 
 
 
 
 
 

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PRINCIPAL INVESTIGATOR SIGNATURE
STUDY 
SPONSOR:
Penn Dental Medicine – Endodontic Department
STUDY TITLE:
A clinical use of Er,Cr:YSGG laser: an anti-biofilm treatment
STUDY ID
PROTOCOL 
VERSION
[Insert IRB #]
V1.0
I have read the referenced protocol. I agree to conduct the study in accordance to this protocol, in 
compliance with the Declaration of Helsinki, Good Clinical Practices (GCP), and all applicable 
regulatory requirements and guidelines.
Principal 
Investigator Name 
Dr. Bekir Karabucak/Dr. 
Flavia Teles
Signature       
Affiliation:
Penn Dental Medicine
Date
05/09/2023
pp

 
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Abbreviations 
AE 
Adverse Event 
ANCOVA 
Analysis of Covariance 
CFR 
Code of Federal Regulations 
CLIA 
Clinical Laboratory Improvement Amendments 
MP 
Monitoring Plan 
COC 
Certificate of Confidentiality 
CONSORT 
Consolidated Standards of Reporting Trials 
CRF 
Case Report Form 
DCC 
Data Coordinating Center 
DHHS 
Department of Health and Human Services 
DSMB 
Data Safety Monitoring Board 
DRE 
Disease-Related Event 
EC 
Ethics Committee 
eCRF 
Electronic Case Report Forms 
FDA 
Food and Drug Administration 
FDAAA 
Food and Drug Administration Amendments Act of 2007 
FFR 
Federal Financial Report 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practices 
GMP 
Good Manufacturing Practices 
GWAS 
Genome-Wide Association Studies 
HIPAA 
Health Insurance Portability and Accountability Act  
IB 
Investigator’s Brochure 
ICH 
International Conference on Harmonization  
ICMJE 
International Committee of Medical Journal Editors 
IDE 
Investigational Device Exemption 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
ISO 
International Organization for Standardization 

 
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ITT 
Intention-To-Treat 
LSMEANS 
Least-Squares Means 
MedDRA 
Medical Dictionary for Regulatory Activities 
MOP 
Manual of Procedures 
MSDS 
Material Safety Data Sheet 
NCT 
National Clinical Trial 
NIH  
National Institutes of Health 
NIH IC 
NIH Institute or Center 
OHRP 
Office for Human Research Protections 
PI 
Principal Investigator 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
DSMC 
Data Safety Monitoring Committee 
SoA 
Schedule of Activities 
SOC 
System Organ Class 
SOP 
Standard Operating Procedure 
UADE 
Unanticipated Adverse Device Effect 
UP 
Unanticipated Problem 
US 
United States 
 
 
 
 

 
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1 STUDY SUMMARY 
1.1 
Synopsis 
Title:  
A clinical use of Er,Cr:YSGG laser: an anti-biofilm treatment 
Short Title: 
Er,Cr:YSGG laser in Endodontics 
Study 
Description:  
The purpose of this study is to develop a protocol for biofilms disinfection 
with a clinically approved and commercially available Er,Cr:YSGG laser 
treatments. This protocol will be testing local single topical application of 
Lasers within the canal system in patients going through routine endodontic 
treatment, evaluate its potential as anti-biofilm treatment and compare it to 
other currently used antibacterial protocols. 
Objectives:  
The purpose of this study is to develop a better, more effective disinfection 
protocol against biofilms within the root canal system, subsequently, 
improve the success of root canal treatment in teeth with apical 
periodontitis. This protocol will be testing local single topical application 
of a very low dose of 2780 nm Er,Cr:YSGG laser within the canal system 
in patients going through routine endodontic treatment, will evaluate its 
potential as an advanced antibacterial, anti-biofilm treatment by intra-canal 
bacterial sampling and compare it to other currently used antibacterial 
protocols. 
Primary 
Endpoint:  
The difference in bacterial reduction between the experimental group 
(laser) and the comparison group (routinely used irrigation protocol) by 
measuring reduction in colony forming units (CFU). Friedman's and 
Wilcoxon signed-rank tests will be used to compare the amount of bacteria 
before and after treatment in both groups, with significance levels set at 5% 
(P < 0.05). 
Secondary 
Endpoints: 
Secondary endpoints: 
- 
Measurement of pain: Following a previously published study 
conducted at the Department of Endodontics, University of 
Pennsylvania1. Patients will be asked to rate the intensity of 
preoperative pain on a numeric rating scale (NRS) from 0 (no pain) 
to 10 (worst pain) before receiving root canal treatment. Along with 
NRS, the Wong-Baker facial grimace scale will also presented to 
the patients to help them in scoring the pain. At the end of each 
visit, the patients will be given a survey and asked to rate the 

 
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intensity of postoperative pain at 4, 24, and 48 h after the procedure. 
Patients will be instructed to take 1000-mg acetaminophen as 
needed. If acetaminophen was consumed, the patients will be asked 
to record the dose and time on the survey. They will be provided 
stamped return envelopes to mail surveys back to the Department 
of Endodontics (attachment 1). 
- 
Treatment success determined clinically and radiographically: 
Radiographically, Following periapical (PAI) index by Órstavik2, 
description of radiographic fndings: 1. Normal periapical 
structures. 2. Small changes in the bone structure. 3. Change in the 
bone structure with mineral loss. 4. Periodontitis with a well-
defined radiolucent area. 5. Severe periodontitis with exacerbating 
features. Success is defined as either complete (radiographic 
resolution of a periapical lesion - the radiographic sign of 
inflammatory processes surrounding a root tip) or incomplete 
healing (scar tissue formation) and failure includes uncertain 
healing (radiographic reduction of a periapical lesion or same lesion 
size) or unsatisfactory healing (increase in lesion size) as 
determined on the radiograph. Clinically, success is defined by the 
absence of pain, swelling, percussion sensitivity or sinus tracts. 
Clinical failure is defined as the persistent presence of any of the 
symptoms mentioned above. Treatment success/failure measured at 
the standard of care 6 month, 1 year and 2 year follow up (± 7 days) 
post root canal filling. 
Study 
Population: 
76 Patients presenting to the Department of Endodontics, School of Dental 
Medicine, University of Pennsylvania for evaluation and routine 
endodontic treatment of infected, necrotic teeth with chronic apical 
periodontitis.  
Phase: 
Postmarket 
Description of 
Sites/Facilities  
Department of Endodontics, School of Dental Medicine, University of 
Pennsylvania. 
Enrolling Sites: 
Patients presenting to the Department of Endodontics, School of Dental 
Medicine, University of Pennsylvania for evaluation and routine 
endodontic treatment of infected, necrotic teeth with chronic apical 
periodontitis. 

 
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Description of 
Study 
Intervention: 
An ER:Cr;YSSG laser 2780 nm (Waterlase Express™, BIOLASE®) with 
300μm tip (EdgePro #3, RFT 3) will be placed into the mid-root of the 
canal. The tip will be activated and slowly withdrawn to the orifice (1-
2mm/sec) following the manufacturer settings (energy 15mJ, repetition 
rate 50Hz, 0% air, 0% water). 
Study Duration: 
The projected time frame for this research project is from June 2023 to June 
2027. Enrollment is from June 2023 to June 2025. Followed by 2 years for 
the last follow up, which is the date of the second visit for the last subject 
enrolled +2 years (± 7 days). The recruitment, treatment and analysis of 76 
subjects with teeth matching the inclusion criteria can be reasonably 
achieved within these 4 years. 
Participant 
Duration: 
The subjects will participate in the study for 2 years. This is the standard 
of care timeline for routine root canal therapy with 6 months, 1 year, and 2 
year follow up.   
 
 
 
1.2 
Key Roles and Study Governance 
Sponsor 
Medical Director 
Endodontic Department 
Bekir Karabucak, DMD, MS. 
Chair and Professor of Endodontics, 
Postdoctoral Endodontics Program, Director. 
Penn Dental Medicine 
Penn Dental Medicine 
Evans Building 
4001 Spruce St 
Philadelphia, PA 
19104-6003 
Evans Building, 3rd floor 
4001 Spruce St 
Philadelphia, PA 
19104-6003 
(215) 898-4617 
215-898-4927 
mhajis@upenn.edu 
bekirk@upenn.edu 
 
 

 
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1.3 Schema 
Prior to 
Randomization 
(Screening) 
Day -1 
 
Visit 1 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Visit 2 
After 1 week 
(± 3 days)  
 
Visit 3 
 
 
Visit 4  
 
 
Visit 5 
Total 76 participants: Obtain informed consent. Screen potential 
participants by inclusion and exclusion criteria; clinical assessment, obtain 
radiographs, medical history/medications, document. 
Obturation of the root canal, final root canal filling following our standard 
root canal treatment procedures 
Patient will be prepared and then 
S0: sample will be taken from the tooth surface. 
S1: After initial access to the root canal orifices. 
S2: After the final 30.04 taper apical size is reached and irrigation using 
(group 1 or group 2). 
S3: Upon completion of final routine irrigation protocol. 
The root canals will be dried with paper points, a medication (calcium 
hydroxide) will be placed and the teeth sealed with a temporary restoration. 
Pain assessments: preoperative, and at 4, 24, and 48 h after the procedure. 
Randomize 
Group 2: Er,Cr:YSGG laser + 
standard of care (NaOCl) 
38 participants 
Group 1: Standard of care (NaOCl) 
38 participants 
Standard of care/routine 6m follow up (± 7 days) clinical and radiographic 
evaluation (Refer to section 3) 
Standard of care/routine 1 year follow up (± 7 days) clinical and 
radiographic evaluation (Refer to section 3)  
Standard of care/routine 2 year follow up (± 7 days) clinical and 
radiographic evaluation (Refer to section 3) 
Optional pre-screening through medical records review/phone script  

 
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Visit 1 Sampling Protocol 
* Before all samplings, solutions will be removed with 5cc of sterile saline and canal content will 
be neutralized with 5% sodium thiosulfate solution.
Step 
Followed by* 
Rubber dam application. 
Chlorhexidine 2% will be used to disinfect the tooth and the rubber dam.  
The removal of caries and the endodontic access will be carried out by sterile 
high-speed carbide burs. 
Chlorhexidine 2% will be used to disinfect the tooth and the rubber dam again. 
Sample 0 
After initial access to the root canal orifices, a bacteriological sample will be 
taken from the targeted canals. 
Sample 1 
Canals will be instrumented up to size 30/0.04 taper using either (NaOCl) or 
(laser). (Refer to study design section 4.1)  
Sample 2 
All canals in all groups will be irrigated with 5cc of 3% NaOCl with the 
ultrasonic irrigant activation following the standard endodontic procedure 
protocol.  
Sample 3 

 
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2 INTRODUCTION AND RATIONALE 
2.1 
Study Rationale 
The purpose of this study is to develop a better, more effective disinfection protocol against 
biofilms within the root canal system, subsequently, improve the success of root canal treatment 
in teeth with apical periodontitis. This protocol will be testing local single topical application of a 
very low dose of an ER:Cr;YSSG laser 2780 nm (Waterlase Express™, BIOLASE®) within the 
canal system in patients going through routine endodontic treatment, will evaluate its potential as 
an advanced antibacterial, anti-biofilm treatment by intra-canal bacterial sampling and compare it 
to other currently used antibacterial protocols. 
2.2 
Background 
Endodontic research has established the importance of microorganisms in the pathogenesis 
of apical periodontitis3-5. The reduction or elimination of microorganisms is a logical aim for 
successful endodontic treatment. Sjörgen et al6 evaluated the long-term outcome after one step 
endodontic treatment of teeth with negative or positive cultures after instrumentation. 94% of the 
teeth filled after a negative culture succeeded, whereas only 68% of the canals filled after a positive 
culture had been detected were successful. Based on this study the optimal clinical success will be 
reached if a tooth is filled when the bacterial load of the canal is undetectable with culturing 
methods. Because culturing is as yet only effective as a research tool, the clinician should expect 
optimal success when treating an infected tooth with a technique or protocol that has been shown 
experimentally to result in consistent negative cultures. Byström et al,7-9 published a series of 
studies to evaluate the antibacteriological effects of the individual steps in the endodontic 
procedure. Only after instrumentation using endodontic instruments with NaOCl and EDTA 
irrigation and a dressing of calcium hydroxide it was possible to obtain a predictable negative 
culture7-9. However, Histo-bacteriological studies have shown that even after meticulous chemo-
mechanical preparation using endodontic instruments with NaOCl and EDTA and obturation of 
the root canal system, a significant amount of bacterial biofilm is often present in the anatomical 
complexities of the root canal system (isthmuses, apical ramification, accessory canals, and 
dentinal tubules) and on the walls of the main root canal that was not engaged by the root canal 
instruments10-12. 
Endodontic biofilms may play a key role in infections persisting after primary or secondary 
endodontic treatment, as microorganisms growing in biofilms are better protected from adverse 
environmental changes and other antimicrobial agents. Ricucci et al,13 evaluated the prevalence of 
bacterial biofilms in untreated root canals of teeth showing apical periodontitis in 80% of the cases. 
Nevertheless, the persistence of endodontic infections is also attributed to the presence of the 
aforementioned anatomical complexities, which can harbor biofilm and protect it from the effects 
of the current disinfection solutions14. Based on these facts, advanced disinfection strategies with 
the ability to disrupt biofilm are needed to achieve the goal of endodontic treatment and to 

 
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overcome the persistence of endodontic infections, if possible, using less harsh chemicals as 
currently used (e.g. NaOCl). 
The great evolutionary leap in solving this problem is the association of biomechanical 
preparation with mechanized instrumentation, including abundant ultrasonic irrigation of 
antiseptic solutions associated with surfactants, laser ablation, and three-dimensional filling of the 
root canal system15. For this evolutionary leap to be fully achieved, all clinical steps, that is, 
instrumentation, irrigation, ablation, and filling, must be used, since the absence of one of them 
makes the complete disinfection of the root canal system unfeasible. The literature demonstrates 
the limitations of irrigating solutions, instrumentation and the partial effect of the action of 
ultrasound, resulting in a filling in a still infected medium. In addition to this, during the 
instrumentation, a contaminated residual layer called the “smear layer” is formed16. 
The laser has been used with importance in most medical and dental specialties, especially 
in Endodontics in the last decade. Its properties are known in the incision of tissues, in cases of 
pulpotomy, and reduction of postoperative pain. Regarding the use inside the root canal, there are 
still some doubts about its effectiveness. Lasers, regardless of types, are individually unable to 
eliminate contaminations in the root canal since the root canal temperature will reach the limit to 
where damage to adjacent tissues and nerves occurs before bacteria are ablated17. In order for lasers 
to ablate all contaminants in the root canal, the absorption efficiency of laser energy at the root 
canal surface has to increase greatly at lower laser power without substantially raising the 
temperature in the root canal. Due to the high affinity of water with Erbium laser, different Erbium 
family lasers (Er:YAG 2940nm and Er,Cr:YSGG 2780nm) represent the cutting-edge technology 
for laser-activated irrigant (LAI) in Endodontics. These two wavelengths are the most appropriate 
for different irrigant solutions, considered safe and respecting accepted clinical parameters18. 
An FDA cleared ER:Cr;YSSG laser machine (Waterlase Express™, BIOLASE®) will be 
tested (clearance number: K161669, attachment 2). This machine has different settings which are 
clinically suitable to be used in root canal disinfection protocols. Many studies have been published 
to test the efficacy of ER:Cr;YSSG laser in biofilm removal inside the root canal system19. 
Qualitative and quantitative in vitro studies showed that ER:Cr;YSSG laser activation of 4% 
NaOCl resulted in greater E. faecalis biofilm reduction within the dentinal tubules20, and improved 
antimicrobial effect of NaOCl in comparison to syringe irrigation21. One blind randomized clinical 
trial concluded that ER:Cr;YSSG laser can achieve promising endodontic outcome, however larger 
sample size with longer follow-up periods are needed22. This study is to validate the method of 
using (Waterlase Express™, BIOLASE®) to ablate or disinfect microbe contaminants, and 
achieve cleaning of the root canal in association with standard endodontic procedures. 
Proof-of-concept experiments in endodontic treatment could open the door for laser to be 
used in numerous clinical anti-biofilm applications. The assessment of bacterial reduction during 
endodontic therapy by bacterial sampling and culturing is an established technique and has been 
used to evaluate treatment protocols or variations thereof23-26. There is a current need for a better 
disinfecting agent that is capable of overcoming the limitations of the currently used irrigation 
solutions. 

 
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Based on our preliminary findings, testing local single topical application of a very low 
dose of Er,Cr:YSGG 2780nm (Waterlase Express™, BIOLASE®) has a potential as an advanced anti-
biofilm clinical therapy with its potent antibacterial and anti-biofilm activities. 
The purpose of this study is to develop an irrigation disinfection protocol for eradicating 
biofilms with a clinically approved and commercially available Er,Cr:YSGG 2780nm laser. This 
protocol will be testing local application of (Waterlase Express™, BIOLASE®), within the root 
canal system in patients going through routine endodontic treatment, will evaluate its potential as 
an advanced anti-biofilm disinfection technique and compare it to other currently used 
antibacterial protocols. 
2.2.1 
Pharmacokinetics, Pharmacodynamics and Toxicology 
N/A 
2.2.2 
Assessment for Potential Study Products Drug-Drug, Drug-Device, 
Device-Device Interactions 
N/A 
2.2.3 
Clinical Adverse Event Profile 
There are no known adverse effects associated with the (Waterlase Express™, BIOLASE®) 
device being used in this study. The Waterlase Express™ device complies with Industry Canada 
license-exempt RSS standard(s) and installed, operated, and maintained according to the current 
revision of the guidelines specified in the Canadian standard CAN/CSA-Z386:2014 safe use of 
lasers in health care. It also complies with FDA laser standards. All these light sources have been 
independently tested by regulatory authorities and used both clinically and experimentally for 
over a decade. Since the device is Class IV laser, participants, providers and observers will wear 
appropriate laser protection eyewear (OD 3 or greater) for the 2780 nm wavelength whenever the 
laser is in use. 
2.2.4 
Dosing Rationale 
Tip: RFT3, 15mJ 50Hz 0.75W 0% air 0% water, according to manufacturer instructions 
(attachment 3). 
2.3 
Risk/Benefit Assessment 
2.3.1 
Known Potential Risks 
Only the risks or discomforts that can arise during or after any routine root canal treatment 
procedure apply. Bacterial sampling will not have any adverse effect on the treatment or on 
treatment outcome. There are no potential risks expected from the noninvasive bacteriological 

 
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Confidential and Proprietary Information of Penn Dental Medicine – Endodontic Department 
 
sampling, including any potential physical, psychological, social, economic, monetary, legal or 
other risks. Identifying the antibacterial benefits of Er,Cr:YSGG laser is the goal of our experiment.  
Er,Cr:YSGG laser has been used in dentistry as an alternative technology to overcome the 
limitations of current chemical-mechanical instrumentation techniques and improve root canal 
disinfection. Er,Cr:YSGG laser used in this experiment is limited within the root canal system and 
applied on dentine surfaces only. It will not be used systemically. 300μm laser tip will be kept 
within the root canal system, placed into the mid-root of the canal. The tip will be activated and 
slowly withdrawn to the orifice (1-2mm/sec) following the manufacturer settings as proposed in 
this protocol. Experiment was designed that the final root canal disinfection is not relying on be 
irrigated with 5cc of 3% NaOCl with the ultrasonic irrigant activation following the standard 
endodontic procedure protocol and then antibacterial medication (CaOH2) will placed as done 
routinely to treat cases with pulp necrosis and apical periodontitis. 
Provider, patient and assistant will wear appropriate laser protection eyewear for the 2780 nm 
wavelength (OD 3 or greater) whenever the laser is in use. 
Data that will be collected include: demographic data, medical history/medications, tooth number, 
diagnosis and the PDM chart number (for potential verification of study correctness). All treatment 
procedures at the PDM, including the routine root canal treatment of the participants, during which 
the samples will be taken, are subject to strict HIPPA (privacy) and OSHA (hygiene) regulations. 
2.3.2 
Known Potential Benefits 
The participant is not expected to get any personal benefit from taking part in this proposed 
research study. Microbial elimination is a key to the successful treatment of infected, necrotic 
teeth. Finding a better protocol to reduce intracanal infection will increase the successful outcome 
of endodontically treated necrotic, infected teeth and will result in an increased retention of natural 
teeth for the general population. The proposed study will verify by the means of microbial 
sampling and culturing, whether Er,Cr:YSGG laser can provide better, effective disinfection on 
root canal surfaces and a significant reduction of intra-canal infection in-vivo. If this can be 
demonstrated by the proposed investigation it will have clinical significance as an improvement 
of root canal therapy. 
2.3.3 
Assessment of Potential Risks and Benefits 
The risks or discomforts that can arise during or after any routine root canal treatment procedure 
apply. All subjects will receive the same standard of care disinfection protocol, with one arm 
receiving the additional Waterlase Express™ laser. However, this device do involve Class IV 
lasers that can harm eyes. Participants, providers and observers will wear appropriate laser 
protection eyewear (OD 3 or greater) whenever the laser is in use. Our consent forms (for routine 
root canal procedures and for the study) contain clinic and investigator contact information 
(attachment 4 and 5). Subjects' concerns or complaints will be reviewed routinely. 

 
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3 STUDY OBJECTIVES AND ENDPOINTS 
The purpose of this study is to develop a better, more effective disinfection protocol against 
biofilms within the root canal system, subsequently, improve the success of root canal treatment 
in teeth with apical periodontitis. This protocol will be testing local single topical application of a 
very low dose of 2780 nm Er,Cr:YSGG laser within the canal system in patients going through 
routine endodontic treatment, will evaluate its potential as an advanced antibacterial, anti-biofilm 
treatment by intra-canal bacterial sampling and compare it to other currently used antibacterial 
protocols. 
OBJECTIVES 
ENDPOINTS 
Primary 
objective: 
Look 
at 
microbial colonization before 
and after treatment. 
Secondary objective: 
Measurment of pain, survival of 
the tooth, periapical bone 
healing and resolution of signs 
and symptoms. 
 
Primary endpoint: The difference in bacterial reduction 
between the experimental group (laser) and the comparison 
group (routinely used irrigation protocol) by measuring 
reduction in colony forming units (CFU). Friedman's and 
Wilcoxon signed-rank tests will be used to compare the 
amount of bacteria before and after treatment in both groups, 
with significance levels set at 5% (P < 0.05). 
Secondary endpoints: 
Measurement of pain: Following a previous published 
study done at the Department of Endodontics, University of 
Pennsylvania1.  Patients will be asked to rate the intensity of 
preoperative pain on a numeric rating scale (NRS) from 0 
(no pain) to 10 (worst pain) before receiving root canal 
treatment. Along with NRS, the Wong-Baker facial grimace 
scale will also presented to the patients to help them in 
scoring the pain. At the end of each visit, the patients will 
be given a survey and asked to rate the intensity of 
postoperative pain at 4, 24, and 48 h after the procedure. 
Patients will be instructed to take 1000-mg acetaminophen 
as needed. If acetaminophen was consumed, the patients 
will be asked to record the dose and time on the survey. They 
will be provided stamped return envelopes to mail surveys 
back to the Department of Endodontics (attachment 1). 
Treatment 
success 
determined 
clinically 
and 
radiographically: Radiographically, Following periapical 
(PAI) index by Órstavik2, description of radiographic 
fndings: 1. Normal periapical structures. 2. Small changes 
in the bone structure. 3. Change in the bone structure with 
mineral loss. 4. Periodontitis with a well-defined radiolucent 
area. 5. Severe periodontitis with exacerbating features. 

 
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OBJECTIVES 
ENDPOINTS 
Success is defined as either complete (radiographic 
resolution of a periapical lesion - the radiographic sign of 
inflammatory processes surrounding a root tip) or 
incomplete healing (scar tissue formation) and failure 
includes uncertain healing (radiographic reduction of a 
periapical lesion or same lesion size) or unsatisfactory 
healing (increase in lesion size) as determined on the 
radiograph. Clinically success is defined by the absence of 
pain, swelling, percussion sensitivity or sinus tracts. Clinical 
failure is defined as the persistent presence of any of the 
symptoms mentioned above. Treatment success/failure 
measured at 6 month, 1 year and 2 year follow up (± 7 days) 
post root canal filling. 

 
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4 STUDY PLAN 
4.1 
Study Design 
  
All the steps and procedures described are part of the standard of care root canal procedure 
except for sampling and the addition of Waterlase Express™ laser which are specific study 
procedures. All study specific procedures are in bold in this section. 
The patient will be anesthetized, and the tooth isolated with rubber dam. Chlorhexidine 2% 
will be used to disinfect the tooth and the rubber dam. The removal of caries and the endodontic 
access will be carried out by sterile high-speed carbide burs. Chlorhexidine 2% will be used to 
disinfect the tooth and the rubber dam one more time and bacteriological sample (S0) will 
be taken from the tooth surface. After initial access to the root canal orifices, a bacteriological 
sample will be taken from the targeted canals (S1). Sterile paper points will be placed into 
the canal orifices, allowed to saturate and then transferred to a vial containing reduced 
transport fluid (RTF).  
For NaOCl group, canals will be instrumented up to size 30/0.04 taper using 1.5cc of 3% 
NaOCl in between files. For Er,Cr:YSGG laser group, canals will be instrumented up to size 
30/0.04 taper using 1.5cc of normal saline in between files, then rinse each canal with 1cc 
normal saline and flood the access. Then ER:Cr;YSSG laser 2780nm (Waterlase Express™, 
BIOLASE®) with 300μm tip (EdgePro #3, RFT 3) will be placed into the mid-root of the 
canal. The tip will be activated and slowly withdrawn to the orifice (1-2mm/sec) following 
the manufacturer settings (energy 15mJ, repetition rate 50Hz, 0% air, 0% water). When the 
final 30.04 taper apical size is reached, a second bacterial sample will be taken (S2).  
Standard root canal procedures: 
All canals in all groups will be irrigated with 5cc of 3% NaOCl with the ultrasonic irrigant 
activation following the standard endodontic procedure protocol. Upon completion of irrigation, a 
third bacterial sample will be taken (S3). Before all samplings, solutions will be removed with 
5cc of sterile saline and canal content will be neutralized with 5% sodium thiosulfate solution. 
The remaining treatment sequences of the routine root canal therapy will be carried out after these 
procedures including further root-end enlargement and final routine irrigation protocol. The root 
canals will be dried with paper points, a medication (calcium hydroxide) will be placed and the 
teeth sealed with a temporary restoration. Patients will return after one week (± 3 days) for 
completion of the root filling. For both groups, the treatment procedures carried out during this 
investigation do not differ from the standard root canal treatment protocol with the exception of 
additional irrigation step with Er,Cr:YSGG laser and the bacteriologic sampling procedures. 
The paper points used to take the bacteriological sampling will be transferred to the 
microbiology laboratory using a vial containing 1ml of reduced transport fluid (RTF). The 
laboratory procedures will be performed at the University of Pennsylvania Leon Levy Oral Health 
Sciences Building of the School of Dental Medicine in the Microbiology Laboratory (Dr. Teles’ 
laboratory). Vial labels will contain information on tooth number, sample number (S0-S1-S2-S3) 
and the experimental group. Patient information will not be used in any case. RTF vials with 
samples will be vortexed before preparing aliquots. Samples with dilutions of 10, 100, and 1000-

 
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fold will be prepared under anaerobic conditions using sterile glassware. Cell culture dishes with 
anaerobic sheep blood agar will be inoculated with 0.25 ml of undiluted sample, as well as each 
of the three dilutions. The culture plates will be incubated at 37°C in an anaerobic glove box 
containing 5% hydrogen, 10% nitrogen, and 85% CO2 for 7 days. After incubation, the number of 
colony forming units will be determined by using a stereoscope. Independent t-test and Wilcoxon-
Mann-Whitney test will be used for statistical analysis. 
4.2 
Scientific Rationale for Study Design 
For standard irrigation protocol group (NaOCl/control group): canals will be instrumented 
up to size 30/0.04 taper using 1.5cc of 3% NaOCl in between files following the standard 
endodontic procedure protocol. 
For Er,Cr:YSGG laser group: canals will be instrumented up to size 30/0.04 taper using 1.5cc 
of normal saline in between files, then rinse each canal with 1cc normal saline and flood the access. 
Then ER:Cr;YSSG laser 2780nm (Waterlase Express™, BIOLASE®) with 300μm tip (EdgePro 
#3, RFT 3) will be placed into the mid-root of the canal. The tip will be activated and slowly 
withdrawn to the orifice (1-2mm/sec) following the manufacturer settings (energy 15mJ, repetition 
rate 50Hz, 0% air, 0% water). 
When the final 30.04 taper apical size is reached. All canals in all groups will be irrigated with 5cc 
of 3% NaOCl with the ultrasonic irrigant activation following the standard endodontic procedure 
protocol.  
4.3 
Justification for Dose 
Tip: RFT3, 15mJ 50Hz 0.75W 0% air 0% water, following the manufacturer instructions 
(attachment 3). 
4.4 
End of Study Definition 
A participant is considered to have completed the study if he or she has completed all phases of 
the study including the last visit or the last scheduled procedure shown in the Schedule of 
Activities (SoA), Appendix Section 12.1. 

 
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5 STUDY POPULATION 
5.1 
Inclusion Criteria 
In order to be eligible to participate in this study, an individual must meet all of the following 
criteria: 
1. Provision of signed and dated informed consent form. 
2. Stated willingness to comply with all study procedures and availability for the duration of the 
study. 
3. Male or female (Gender is not an inclusion or exclusion criteria), aged 18 years old or above. 
4. In good general health as evidenced by medical history or non-contributory medical history 
(Patient can be seen for regular dental appointment in PDM; ASA classes I and II). 
5. Radiographic presence of periapical radioluceny. 
6. Negative response to thermal sensitivity testing (difluordichlormethane at -50 °C, Endo-Ice, 
Coltène/Whaledent Inc., Cuyahoga Falls, Ohio) or electric pulp testing. 
7. Enough tooth structure for adequate isolation with rubber dam. 
8. No history of previous endodontic treatment on the tooth. 
9. Teeth with single canal and lower molars with two mesial canals. 
5.2 
Exclusion Criteria 
Economic status, gender, race or ethnicity are not of concern for the proposed investigation and 
therefore for study exclusion. The cut-off age of 18 years was only chosen so as to limit the need 
for parental consent. Patients will not be eligible to participate in the study if any of the following 
exclusion criteria applies:. 
1. Patients who report they are pregnant. 
2. Teeth affected by dental trauma. 
3. Periodontal changes (pockets 3 mm, mobility I or gingival edema). 
4. Radiographic presence of resorptive processes. 
5. Per the investigator's discretion, unable or unlikely to comply with study procedure. 
6. Presence of any condition which, in the opinion of the investigator, makes participation in the 
study not in the individual's best interest. 
5.3 
Lifestyle Considerations 
N/A 
5.4 
Screen Failures 
Screen failures are defined as participants who consent to participate in the clinical trial but are 
not subsequently randomly assigned to the study intervention or entered in the study. A minimal 
set of screen failure information is required to ensure transparent reporting of screen failure 
participants, to meet the Consolidated Standards of Reporting Trials (CONSORT) publishing 

 
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requirements and to respond to queries from regulatory authorities. Minimal information includes 
demography, screen failure details, eligibility criteria, and any serious adverse event (SAE). 
Individuals who do not meet the criteria for participation in this trial (screen failure) may be 
rescreened. Rescreened participants should be assigned the same participant number as for the 
initial screening. 
5.5 
Strategies for Recruitment and Retention 
Patients may be prescreened (through medical records/phone script) and will be contacted and 
informed about the study ahead of time, at least 1 day before their appointment. We will try our 
best to contact patients and inform them about the study ahead of time, but sometimes it may not 
be possible. The Endodontics clinic is a referral based clinic, sometimes we see new external 
referrals or emergency patients. We don’t have any direct contact with those patient ahead of time, 
and sometimes emergency patients would show up without prior appointments. Thus, patient may 
be included even if they haven’t been contacted prior. 
Patients  presenting to the Department of Endodontics, School of Dental Medicine (PDM), 
University of Pennsylvania for evaluation and routine endodontic treatment of infected, necrotic 
teeth with chronic apical periodontitis will be asked to take part in the study if they appear to meet 
the inclusion criteria specified above. Patients will be asked if they were interested to participate 
in this investigation after a clinical diagnosis matching is confirmed and the patient/tooth meets 
the inclusion criteria specified above. Initial clinical diagnosis will be made by a postgraduate 
resident and verified by the supervising faculty. The PI, a full-time faculty, and the co-investigator, 
endodontic resident, will be informed about a potential study participant, verify the diagnosis and 
ask for participation (including consent and alternatives). Any recruitment materials that are 
developed will be submitted to the IRB for approval prior to use.  No non-registered PDM patients 
will be asked to participate in the study. 

 
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6 STUDY INTERVENTION 
6.1 
Study Intervention(s) Administration 
6.1.1 
Study Intervention Description (attachment 3) 
- 
 Dimensions: 
• Laser console (W x L x H) 9 x 18 x 12 in (23 x 46 x 30.5 cm) 
• Tablet (W x H x D) 6.7 in x 9.3 in x 0.2 in (16.9 cm x 23.8 cm x 0.6 cm) 
• Screen size 9.7 in (24.6 cm) 
• Weight laser console (with water) 27.2 lbs (12.3 kg) 
• Weight Tablet 0.9 lbs (390 g) 
- 
Electrical: 
Class I Medical Electrical (ME) Equipment 
• Operating voltage 100 - 240 VAC 
• Frequency 50 / 60 Hz 
• Current rating 6A / 3A 
• Main control Main Power Switch 
• On / Off control Keyswitch 
• Remote interruption Remote interlock connector 
- 
Air and water spray: 
• Water type Distilled or De-Ionized only 
• External air source 60-120 psi. (4.1 – 8.2 bar) 
• Water 0 - 100% 
• Air 0 - 100% 
- 
Optical: 
• Laser classification IV (4) 
• Medium Er,Cr:YSGG 
(Erbium, Chromium: Yttrium, Scandium, Gallium, Garnet) 
• Wavelength 2.78 μm (2780nm) 
• Mode Free-running Pulsed 
• Frequency (Pulse Rate) 5 – 50 Hz 
• Average power 0.1- 4.0 W 
• Power accuracy ± 20% 
• Pulse energy 10– 250 mJ 
• Pulse duration for “H” mode (Short pulse) 60 μs 
• Pulse duration “S” mode (Long pulse) 700 μs 
• Handpiece head angles 70° contra-angle 
• Fiber Tip diameter range (Spot size) 200 – 1200 μm 
• Output divergence ≥ 8° per side 
• Aiming beam 625-670 nm (red) laser, 1mW max (Laser Class 1) 
• Nominal ocular hazard distance (NOHD) 5cm 
• Maximum permissible exposure (MPE) 3.46 X 10⁵W/m2 

 
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6.1.2 
Dosing and Administration 
For standard irrigation protocol group (NaOCl group): canals will be instrumented up to size 
30/0.04 taper using 1.5cc of 3% NaOCl in between files following the standard endodontic 
procedure protocol. 
For Er,Cr:YSGG laser group: canals will be instrumented up to size 30/0.04 taper using 1.5cc 
of normal saline in between files, then rinse each canal with 1cc normal saline and flood the access. 
Then ER:Cr;YSSG laser 2780nm (Waterlase Express™, BIOLASE®) with 300μm tip (EdgePro 
#3, RFT 3) will be placed into the mid-root of the canal. The tip will be activated and slowly 
withdrawn to the orifice (1-2mm/sec) following the manufacturer settings (energy 15mJ, repetition 
rate 50Hz, 0% air, 0% water). 
When the final 30.04 taper apical size is reached. All canals in all groups will be irrigated with 5cc 
of 3% NaOCl with the ultrasonic irrigant activation following the standard endodontic procedure 
protocol.  
6.2 
Preparation/Handling/Storage/Accountability 
6.2.1 
Acquisition and accountability 
The study intervention and control products (both drugs and devices) are regularly available and 
routinely used in the Endodontic clinic, and will be provided to the investigator as needed. 
6.2.2 
Formulation, Appearance, Packaging, and Labeling 
N/A 
6.2.3 
Product Storage and Stability 
Control (NaOCl): Will be stored and used as regulated by the Endodontic clinic. 
Waterlase Express™ and accessories will be stored in a cool dry place when not in use. Storage 
temperature should be 5° to 45°C (41°F to 113°F), relative humidity 10% to 90%, non-condensing. 
Cover the system when not in use for extended periods of time and store in a place where it will 
not be accidentally bumped or banged. 
6.2.4 
Preparation 
Control (NaOCl): 12cc syringe will be filled with 3% NaOCl to be dispensed using 30 gauge 
needle as used routinely in the Endodontic clinic. 
Waterlase Express™: 
- 
Daily start-up: 
• Power up the Tablet. Verify that all connections have been properly secured; turn the main power 
switch at the back of the console ON (I) (when the power switch is OFF, LEDs will not light up); 
insert the key into the Keyswitch and rotate it clockwise to the ON (I) position. The LED light on 

 
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the Tablet Holder lights up when the Tablet is properly powered on and docked in the Tablet 
Holder, connected and active. 
• Make sure the air supply is connected and verify that the patient water bottle is no less than ⅓ 
full with distilled or de-ionized water. It is suggested to completely fill the patient water bottle at 
the start of the day. 
 
- 
Initiating the Laser From off Status: 
• Turn the main power switch at the back of the console ON (I) (when the power switch is OFF, 
LEDs will not light up); insert the key into the Keyswitch and rotate it clockwise to the ON (I) 
position 
• Press and hold the power switch on the right side of the Tablet to turn it on 
• The startup screen will appear, and the system software will begin to load (approximately 30 
seconds) 
• Verify that the Footswitch and laser are paired 
• Attach the Handpiece to the Fiber Optic Cable 
• Ensure the tablet is properly docked in the Tablet Holder and secured with the Tablet Latch. 
• All users configured to the system listed on the User selection screen; select the proper account. 
A screen will appear giving the user the option to prim the Handpiece or skip this step (if the 
Handpiece is already primed). If "prime" is chosen, a message will appear confirming that priming 
is taking place (approximately 6 seconds). 
After priming, set-up is complete; the system will now transition to the home (Procedure) screen 
in Standby mode. 
 
- 
Activating the Waterlase Express™: 
Place the system into Ready mode after selecting a procedure (Endodontic) by pressing the 
Function Control Button on the Tablet Holder; wait approximately 2 seconds, then press down on 
the Footswitch when ready to fire the laser. 
6.3 
Measures to Minimize Bias: Randomization and Blinding 
Simple randomization by generating simple allocation sequence, allocation concealment, and 
implementation of the random allocation sequence upon enrollment. 
As this is part of a procedure, the investigator and study participant cannot be blinded to the 
treatment.  To minimize bias, the investigator will provide the same information regarding 
reporting pain to all study participants. 
6.4 
Study Intervention Compliance 
The study intervention will occur during a routine endodonic procedure. Administration of the 
study intervention will be done by qualified personnel and recorded in the case report forms. 

 
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6.5 
Concomitant Therapy 
For this protocol, a prescription medication is defined as a medication that can be prescribed only 
by a properly authorized/licensed clinician. Medications to be reported in the Case Report Form 
(CRF) are concomitant prescription medications, over-the-counter medications and supplements. 
6.5.1 
Rescue Medicine 
All participants will be provided with the Post Endodontic Treatment Instructions form 
(attachment 6). This form will include those information regarding rescue medicine: 
Take 1000-mg acetaminophen as needed. (Tylenol; 2 tablets of 500mg as needed). 
If acetaminophen was consumed, the patients will be asked to record the dose and time on the 
survey. 
7 STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL 
7.1 
Discontinuation of Study Intervention 
If the subject is discontinued from the study intervention, they will be withdrawn from the study.  
The reason for withdrawal will be recorded in the case report forms. 
7.2 
Participant Discontinuation/Withdrawal from the Study 
Participants are free to withdraw from participation in the study at any time upon request. 
An investigator may discontinue or withdraw a participant from the study for the following reasons: 
• Significant non-compliance to attend scheduled visits 
• If any clinical adverse event (AE), laboratory abnormality, or other medical condition or 
situation occurs such that continued participation in the study would not be in the best 
interest of the participant 
• Disease progression which requires discontinuation of the study 
• If the participant meets an exclusion criterion (either newly developed or not previously 
recognized) that precludes further study participation 
The reason for participant discontinuation or withdrawal from the study will be recorded on the 
Case Report Form (CRF). Subjects who sign the informed consent form and are randomized but 
do not receive the treatment may be replaced. Subjects who sign the informed consent form and 
are randomized and receive the treatment, and subsequently withdraw, or are withdrawn or 
discontinued from the study, will not be replaced. 

 
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7.3 
Lost To Follow-Up 
A participant will be considered lost to follow-up if he or she fails to return for 1 scheduled visit 
and is unable to be contacted by the study site staff. 
The following actions must be taken if a participant fails to return to the clinic for a required study 
visit: 
• The site will attempt to contact the participant and reschedule the missed visit (1 month) 
and counsel the participant on the importance of maintaining the assigned visit schedule 
and ascertain if the participant wishes to and/or should continue in the study. 
• Before a participant is deemed lost to follow-up, the investigator or designee will make 
every effort to regain contact with the participant (where possible, 3 telephone calls and, if 
necessary, a certified letter to the participant’s last known mailing address or local 
equivalent methods). These contact attempts should be documented in the participant’s 
medical record or study file.  
• Should the participant continue to be unreachable, he or she will be considered to have 
withdrawn from the study with a primary reason of lost to follow-up. 

 
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8 STUDY ASSESSMENT AND PROCEDURES 
8.1 
Efficacy Assessments 
Primary objective: Look at microbial colonization before and after treatment. 
The treatment and sampling procedures will largely follow the protocol described by Shuping et 
al.24. Briefly, the patient will be anesthetized, and the tooth isolated with rubber dam. 
Chlorhexidine 2% will be used to disinfect the tooth and the rubber dam. The removal of caries 
and the endodontic access will be carried out by sterile high-speed carbide burs. Chlorhexidine 2% 
will be used to disinfect the tooth and the rubber dam one more time and bacteriological sample 
(S0) will be taken from the tooth surface. After initial access to the root canal orifices, a 
bacteriological sample will be taken from the targeted canals (S1). Sterile paper points will be 
placed into the canal orifices, allowed to saturate and then transferred to a vial containing reduced 
transport fluid (RTF). For NaOCl group, canals will be instrumented up to size 30/0.04 taper using 
1.5cc of 3% NaOCl in between files. For Er,Cr:YSGG laser group, canals will be instrumented up 
to size 30/0.04 taper using 1.5cc of normal saline in between files, then rinse each canal with 1cc 
normal saline and flood the access. Then ER:Cr;YSSG laser 2780nm (Waterlase Express™, 
BIOLASE®) with 300μm tip (EdgePro #3, RFT 3) will be placed into the mid-root of the canal. 
The tip will be activated and slowly withdrawn to the orifice (1-2mm/sec) following the 
manufacturer settings (energy 15mJ, repetition rate 50Hz, 0% air, 0% water). When the final 30.04 
taper apical size is reached, a second bacterial sample will be taken (S2). All canals in all groups 
will be irrigated with 5cc of 3% NaOCl with the ultrasonic irrigant activation following the 
standard endodontic procedure protocol. Upon completion of irrigation, a third bacterial sample 
will be taken (S3). Before all samplings, solutions will be removed with 5cc of sterile saline and 
canal content will be neutralized with 5% sodium thiosulfate solution. The remaining treatment 
sequences of the routine root canal therapy will be carried out after these procedures including 
further root-end enlargement and final routine irrigation protocol. The root canals will be dried 
with paper points, a medication (calcium hydroxide) will be placed and the teeth sealed with a 
temporary restoration. Patients will return after one week (± 3 days) for completion of the root 
filling. For both groups, the treatment procedures carried out during this investigation do not differ 
from the standard root canal treatment protocol with the exception of additional irrigation step 
with the experimental solution and the bacteriologic sampling procedures. 
The paper points used to take the bacteriological sampling will be transferred to the 
microbiology laboratory using a vial containing 1ml of reduced transport fluid (RTF). The 
laboratory procedures will be performed at the University of Pennsylvania Leon Levy Oral Health 
Sciences Building of the School of Dental Medicine in the Microbiology Laboratory (Dr. Teles’ 
laboratory). Vial labels will contain information on tooth number, sample number (S0-S1-S2-S3) 
and the experimental group. Patient information will not be used in any case. RTF vials with 
samples will be vortexed before preparing aliquots. Samples with dilutions of 10, 100, and 1000-
fold will be prepared under anaerobic conditions using sterile glassware. Cell culture dishes with 
anaerobic sheep blood agar will be inoculated with 0.25 ml of undiluted sample, as well as each 

 
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of the three dilutions. The culture plates will be incubated at 37°C in an anaerobic glove box 
containing 5% hydrogen, 10% nitrogen, and 85% CO2 for 7 days. After incubation the number of 
colony forming units will be determined by using a stereoscope. Independent t-test and Wilcoxon-
Mann-Whitney test will be used for statistical analysis. 
Secondary objectives: Measurement of pain, survival of the tooth, periapical bone healing 
and resolution of signs and symptoms. 
Measurement of pain: Following a previously published study conducted at the Department of 
Endodontics, University of Pennsylvania1. Patients will be asked to rate the intensity of 
preoperative pain on a numeric rating scale (NRS) from 0 (no pain) to 10 (worst pain) before 
receiving root canal treatment. Along with NRS, the Wong-Baker facial grimace scale will also 
presented to the patients to help them in scoring the pain. At the end of each visit, the patients will 
be given a survey and asked to rate the intensity of postoperative pain at 4, 24, and 48 h after the 
procedure. Patients will be instructed to take 1000-mg acetaminophen as needed. If acetaminophen 
was consumed, the patients will be asked to record the dose and time on the survey. They will be 
provided stamped return envelopes to mail surveys back to the Department of Endodontics 
(attachment 1). 
Treatment success determined clinically and radiographically: Radiographically, Following 
periapical (PAI) index by Órstavik2, description of radiographic fndings: 1. Normal periapical 
structures. 2. Small changes in the bone structure. 3. Change in the bone structure with mineral 
loss. 4. Periodontitis with a well-defined radiolucent area. 5. Severe periodontitis with 
exacerbating features. Success is defined as either complete (radiographic resolution of a periapical 
lesion - the radiographic sign of inflammatory processes surrounding a root tip) or incomplete 
healing (scar tissue formation) and failure includes uncertain healing (radiographic reduction of a 
periapical lesion or same lesion size) or unsatisfactory healing (increase in lesion size) as 
determined on the radiograph. Clinically, success is defined by the absence of pain, swelling, 
percussion sensitivity or sinus tracts. Clinical failure is defined as the persistent presence of any 
of the symptoms mentioned above. Treatment success/failure measured at the standard of care 6 
month, 1 year and 2 year follow up (± 7 days) post root canal filling. 
8.2 
Safety and Other Assessments 
The resident performing the endodontic treatment will evaluate and treatment plan the case as 
routinely performed through a clinical and radiographic examination of the involved teeth. At least 
one periapical radiograph of the involved tooth will be obtained during the consultation as routine 
protocol requires for every root canal treatment. In order to be eligible to participate in this study, 
an individual must meet all of the previously mentioned criteria (check section 5.1 and 5.2). On 
the day of procedure, the resident will conduct and complete the root canal treatment as standard 
protocol requires, including medical history review/update, clinical assessment, vital signs (blood 
pressure). Participants, providers and observers will wear appropriate laser protection eyewear 
(OD 3 or greater) whenever the laser is in use. 

 
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8.3 
Adverse Events and Serious Adverse Events 
8.3.1 
Definition of Adverse Events (AE) 
An adverse event (AE) is any untoward medical occurrence associated with the use of an 
intervention in humans, whether or not considered intervention related. Intercurrent illnesses or 
injuries should be regarded as adverse events. 
A pre-existing condition should be recorded as an adverse event if the frequency, intensity or the 
character of the condition changes. 
8.3.2 
Definition of Serious Adverse Events (SAE)  
Serious Adverse Events (SAE) 
Adverse events are classified as serious or non-serious. A serious adverse event is any AE that, in 
the view of the investigator is: 
• fatal 
• life-threatening 
• requires or prolongs hospital stay 
• results in persistent or significant disability or incapacity 
• a congenital anomaly or birth defect 
• an important medical event when the event does not fit the other outcomes, but the event 
may jeopardize the patient and may require medical or surgical intervention (treatment) to 
prevent one of the other outcomes. 
• required intervention to prevent permanent impairment or damage  
Important medical events are those that may not be immediately life threatening but are clearly of 
major clinical significance. They may jeopardize the subject and may require intervention to 
prevent one of the other serious outcomes noted above. For example, drug overdose or abuse, a 
seizure that did not result in in-patient hospitalization, or intensive treatment of bronchospasm in 
an emergency department would typically be considered serious. 
8.3.3 
Unanticipated Adverse Device Effect (UADE) 
A UADE is any serious adverse effect on health or safety, or any life-threatening problem or death 
caused by, or associated with, a device, if that effect, problem, or death was not previously 
identified in nature, severity, or degree of incidence in the investigational plan or application, or 
any other unanticipated serious problem associated with a device that relates to the rights, safety, 
or welfare of subjects. 

 
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8.3.4 
Classification of an Adverse Event 
8.3.4.1 
Severity of Event 
For adverse events (AEs) not included in the protocol defined grading system, the following 
guidelines will be used to describe severity. 
• Mild – Events require minimal or no treatment and do not interfere with the participant’s 
daily activities.  
• Moderate – Events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning. 
• Severe – Events interrupt a participant’s usual daily activity and may require systemic drug 
therapy or other treatment. Severe events are usually potentially life-threatening or 
incapacitating. Of note, the term “severe” does not necessarily equate to “serious”. 
8.3.4.2 
Relationship to Study Intervention 
All adverse events (AEs) must have their relationship to laser assessed by the clinician who 
examines and evaluates the participant based on temporal relationship and his/her clinical 
judgment. The degree of certainty about causality will be graded using the categories below. In a 
clinical trial, the study product must always be considered. 
• Related – The AE is known to occur with the laser, there is a reasonable possibility that the 
laser caused the AE, or there is a temporal relationship between the laser and event. 
Reasonable possibility means that there is evidence to suggest a causal relationship 
between the laser and the AE. 
• Not Related – There is not a reasonable possibility that the administration of the laser 
caused the event, there is no temporal relationship between the laser and event onset, or an 
alternate etiology has been established. 
OR 
• Definitely Related – There is clear evidence to suggest a causal relationship, and other 
possible contributing factors can be ruled out. The clinical event, including an abnormal 
laboratory test result, occurs in a plausible time relationship to laser administration and 
cannot be explained by concurrent disease or other drugs or chemicals. The response to 
withdrawal of the laser group (dechallenge) should be clinically plausible. The event must 
be pharmacologically or phenomenologically definitive, with use of a satisfactory 
rechallenge procedure if necessary. 
• Probably Related – There is evidence to suggest a causal relationship, and the influence of 
other factors is unlikely. The clinical event, including an abnormal laboratory test result, 
occurs within a reasonable time after administration of the laser, is unlikely to be attributed 
to concurrent disease or other drugs or chemicals, and follows a clinically reasonable 

 
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response on withdrawal (dechallenge). Rechallenge information is not required to fulfill 
this definition. 
• Possibly Related – There is some evidence to suggest a causal relationship (e.g., the event 
occurred within a reasonable time after administration of the laser). However, other factors 
may have contributed to the event (e.g., the participant’s clinical condition, other 
concomitant events). Although an AE may rate only as “possibly related” soon after 
discovery, it can be flagged as requiring more information and later be upgraded to 
“probably related” or “definitely related”, as appropriate. 
• Unlikely to be related – A clinical event, including an abnormal laboratory test result, 
whose temporal relationship to laser administration makes a causal relationship improbable 
(e.g., the event did not occur within a reasonable time after administration of the laser) and 
in which other drugs or chemicals or underlying disease provides plausible explanations 
(e.g., the participant’s clinical condition, other concomitant treatments). 
• Unrelated – The AE is completely independent of laser administration, and/or evidence 
exists that the event is definitely related to another etiology. There must be an alternative, 
definitive etiology documented by the clinician. 
8.3.4.3 
Expectedness  
Expected adverse events include those that are expected as part of routine root canal treatment 
such as: the possibility of instruments breaking within the canals; perforations (extra opening) of 
the crown or root of the tooth; damage to existing fillings, crown or bridges; fractures of the tooth; 
discomfort; jaw muscle cramps and spasms; temporomandibular (jaw) joint difficulty; swelling 
and pain. During and after the treatment, complications may be discovered which make treatment 
impossible or which may require endodontic surgery or extraction of the tooth. Complications that 
anesthesia, injection, prescribed analgesics (pain relievers) may include, but are not limited to: 
swelling, infection, bleeding, discoloration of the face, discomfort, pain, nausea, drowsiness, 
allergic reactions, numbness or tingling of the lip, gum, or tongue (this condition is usually 
temporary). 
A separate consent form explaining all expected adverse events for patients participating in the 
study will be discussed and signed by each participant (attachment 5). Waterlase Express™:  
Water from spray may splash during laser use. This device do involve Class IV lasers that can 
harm eyes. Participants, providers and observers will wear appropriate laser protection eyewear 
(OD 3 or greater) whenever the laser is in use. No other known risks are expected when using 
Waterlase Express™ for root canal disinfection (attachment 3). 
Investigator will be responsible for determining whether an adverse event (AE) is expected or 
unexpected. An AE will be considered unexpected if the nature, severity, or frequency of the event 
is not consistent with the risk information previously described. 

 
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8.3.5 
Time Period and Frequency for Event Assessment and Follow-Up 
Safety will be assessed by monitoring and recording potential adverse effects using the CTCAE at 
each study visit. Participants will be monitored by medical histories, physical examinations. If 
CTCAE grading does not exist for an adverse event, the severity of mild, moderate, severe, life-
threatening, and death, corresponding to Grades 1-5, will be used whenever possible. 
At each contact with the subject, the investigator will seek information on adverse events by non-
directive questioning and, as appropriate, by examination. Adverse events may also be detected 
when they are volunteered by the subject during the screening process or between visits, or through 
physical examination, laboratory test, or other assessments. Information on all adverse events will 
be recorded in the source documentation. To the extent possible, adverse events will be recorded 
as a diagnosis and symptoms used to make the diagnosis recorded within the diagnosis event. 
As much as possible, each adverse event or follow-up information will be evaluated to determine: 
1. Severity grade (CTCAE Grade 1-5) 
2. Duration (start and end dates) 
3. Relationship to the study treatment or process – [Reasonable possibility that AE is related: 
No (unrelated/ not suspected) or Yes (a suspected adverse reaction)]. If yes (suspected) - 
is the event possibly, probably or definitely related to the investigational treatment? 
4. Expectedness to study treatment or process – [Unexpected – if the event severity and/or 
frequency is not described in the investigator brochure or protocol]. 
5. Action taken with respect to study or investigational treatment or process (none, dose 
adjusted, temporarily interrupted, permanently discontinued, unknown, not applicable) 
6. Whether medication or therapy taken (no concomitant medication/non-drug therapy, 
concomitant medication/non-drug therapy) 
7. Whether the event is serious 
Once an adverse event is detected, it should be followed until its resolution or until it is judged to 
be permanent, and assessment should be made at each visit (or more frequently, if necessary) of 
any changes in severity, the suspected relationship to the study treatment, the interventions 
required to treat it, and the outcome. 
 
8.3.6 
Adverse Event Reporting 
Reporting Period 
Adverse events will be reported from the time of informed consent until study completion. 
Investigator Reporting: Notifying the Study Sponsor 
N/A 

 
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Investigator Reporting: Local Reporting Requirements 
The investigator will report AEs and SAEs to the IRB/EC of record and other local regulatory 
groups per the local requirements. 
New information regarding the SAE will be reported as it becomes available and in the same 
manner that the initial SAE (i.e. SAE form). The investigator will follow the event to resolution or 
until the event is deemed and documented irreversible, whichever is longer. 
8.3.7 
Serious Adverse Event Reporting  
The study investigator shall complete an Unanticipated Adverse Device Effect Form and submit 
to the reviewing Institutional Review Board (IRB) . The study investigator is responsible for 
conducting an evaluation of an unanticipated adverse device effect and shall report the results of 
such evaluation to the Food and Drug Administration (FDA) and to all reviewing IRBs and 
participating investigators per the applicable regulation. 
8.3.8 
Reporting Events to Participants  
N/A 
8.3.9 
Events of Special Interest  
N/A 
8.3.10 
Reporting of Pregnancy 
Pregnancy, in and of itself, is not regarded as an AE unless there is suspicion that study device or 
process may have interfered with the effectiveness of a contraceptive medication or method. When 
a pregnancy has been confirmed in a subject, and the fetus is exposed to study device and/or 
process (maternally or paternally), the following procedures should be followed to ensure subject 
safety: 
Data on fetal outcome are collected for regulatory reporting and device safety evaluation. Follow-
up should be conducted for each pregnancy to determine outcome, including spontaneous or 
voluntary termination, details of the birth, and the presence or absence of any birth defects, 
congenital abnormalities, or maternal and/or newborn complications. 
8.4 
Unanticipated Problems 
8.4.1 
Definition of Unanticipated Problems (UP) 
The Office for Human Research Protections (OHRP) considers unanticipated problems involving 
risks to participants or others to include, in general, any incident, experience, or outcome that 
meets all the following criteria: 
• Unexpected in terms of nature, severity, or frequency given (a) the research procedures 
that are described in the protocol-related documents, such as the Institutional Review Board 

 
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(IRB)-approved research protocol and informed consent document; and (b) the 
characteristics of the participant population being studied; 
• Related or possibly related to participation in the research (“possibly related” means there 
is a reasonable possibility that the incident, experience, or outcome may have been caused 
by the procedures involved in the research); and 
• Suggests that the research places participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or 
recognized. 
8.4.2 
Unanticipated Problem Reporting 
Unanticipated problems (UPs) such as: 
• 
Post-marketing withdrawal of a drug, device, or biologic used in a research protocol due 
to safety concerns. 
• 
FDA ban of a drug, device, or biologic used in a research protocol due to safety concerns. 
• 
Complaint of a participant when the complaint indicates unexpected risks, or the 
complaint cannot be resolved by the research team 
• 
Breach of confidentiality 
• 
Incarceration of a participant when the research was not previously approved under 
Subpart C and the investigator believes it is in the best interest of the subject to remain on 
the study 
• 
Premature closure of a study (e.g., due safety, lack of efficacy, feasibility, financial 
reasons, etc.) 
should be reported by the investigator to the reviewing Institutional Review Board (IRB) and to 
the Data Coordinating Center (DCC)/lead principal investigator (PI). The UP report will include 
the following information: 
• Protocol identifying information: protocol title and number, PI’s name, and the IRB 
project number; 
• A detailed description of the event, incident, experience, or outcome;  
• An explanation of the basis for determining that the event, incident, experience, or 
outcome represents an UP; 
• A description of any changes to the protocol or other corrective actions that have been 
taken or are proposed in response to the UP. 
To satisfy the requirement for prompt reporting, UPs will be reported using the following 
timeline: 
• UPs that are serious adverse events (SAEs) will be reported as any other SAE. 

 
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• Any other UP will be reported to the IRB and to the DCC/study sponsor the investigator 
becoming aware of the problem. 
• All UPs should be reported to appropriate institutional officials (as required by an 
institution’s written reporting procedures), the supporting agency head (or designee), and 
the Office for Human Research Protections (OHRP) after the IRB’s receipt of the report 
of the problem from the investigator. 
 
8.4.3 
Reporting Unanticipated Problems To Participants  
N/A 
8.5 
Device Reporting 
Safety reporting for the device will be according to 21 CFR 812.150. 
9 STATISTICAL CONSIDERATIONS 
9.1 
Statistical Hypotheses 
H0: There is no difference between the laser group and the control group in microbial load 
reduction based on CFU. 
Ha: There is a difference between the laser group and the control group in microbial load reduction 
based on CFU. 
Primary endpoint: The difference in bacterial reduction between the experimental group (laser) 
and the comparison group (routinely used irrigation protocol) by measuring reduction in colony 
forming units (CFU). Friedman's and Wilcoxon signed-rank tests will be used to compare the 
amount of bacteria before and after treatment in both groups, with significance levels set at 5% (P 
< 0.05). 
Secondary endpoints: 
Measurement of pain: Following a previous published study done at the Department of 
Endodontics, University of Pennsylvania1.  Patients will be asked to rate the intensity of 
preoperative pain on a numeric rating scale (NRS) from 0 (no pain) to 10 (worst pain) before 
receiving root canal treatment. Along with NRS, the Wong-Baker facial grimace scale will also 
presented to the patients to help them in scoring the pain. At the end of each visit, the patients will 
be given a survey and asked to rate the intensity of postoperative pain at 4, 24, and 48 h after the 
procedure. Patients will be instructed to take 1000-mg acetaminophen as needed. If acetaminophen 
was consumed, the patients will be asked to record the dose and time on the survey. They will be 
provided stamped return envelopes to mail surveys back to the Department of Endodontics 
(attachment 1). 

 
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Treatment success determined clinically and radiographically: Radiographically, Following 
periapical (PAI) index by Órstavik2, description of radiographic fndings: 1. Normal periapical 
structures. 2. Small changes in the bone structure. 3. Change in the bone structure with mineral 
loss. 4. Periodontitis with a well-defined radiolucent area. 5. Severe periodontitis with 
exacerbating features. Success is defined as either complete (radiographic resolution of a periapical 
lesion - the radiographic sign of inflammatory processes surrounding a root tip) or incomplete 
healing (scar tissue formation) and failure includes uncertain healing (radiographic reduction of a 
periapical lesion or same lesion size) or unsatisfactory healing (increase in lesion size) as 
determined on the radiograph. Clinically success is defined by the absence of pain, swelling, 
percussion sensitivity or sinus tracts. Clinical failure is defined as the persistent presence of any 
of the symptoms mentioned above. Treatment success/failure measured at 6 month, 1 year and 2 
year follow up (± 7 days) post root canal filling. 
9.2 
Sample Size Determination 
Decrease in microbial load based in CFU. 
Sample size calculation, with 80% power (β = 0.20), indicated a minimum sample size of 52 teeth 
(26 per group) to show a 5% (α = 0.05) difference between groups. The calculations were made in 
the G*Power 3.1 program. 
Test: Independent t test. 
Two tails. 
Statistical test: Means: Wilcoxon-Mann-Whitney test (two groups). 
Effect size: 0.8 (large) 
α = 0.05, β = 0.20 
Attrition rate of 30% was considered, indicated a minimum sample size of 76 teeth (38 per group). 
9.3 
Populations for Analyses 
Patients presenting to the Department of Endodontics, School of Dental Medicine, University of 
Pennsylvania for evaluation and routine endodontic treatment of infected, necrotic teeth with 
chronic apical periodontitis who meet the inclusion criteria specified above. 
9.4 
Statistical Analyses 
9.4.1 
General Approach 
For descriptive statistics, categorical data will be presented as present/absent or divided into groups. 
Continuous data will be presented as percentages, means with standard deviations, median, range. 
9.4.2 
Analysis of the Primary Efficacy Endpoint(s) 
Primary endpoint: The difference in bacterial reduction between the experimental group (laser) 
and the comparison group (routinely used irrigation protocol) by measuring reduction in colony 
forming units (CFU). Friedman's and Wilcoxon signed-rank tests will be used to compare the 

 
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amount of bacteria before and after treatment in both groups, with significance levels set at 5% (P 
< 0.05). 
9.4.3 
Analysis of the Secondary Endpoint(s) 
Secondary endpoint: 
Measurement of pain: Following a previous published study done at the Department of 
Endodontics, University of Pennsylvania1.  Patients will be asked to rate the intensity of 
preoperative pain on a numeric rating scale (NRS) from 0 (no pain) to 10 (worst pain) before 
receiving root canal treatment. Along with NRS, the Wong-Baker facial grimace scale will also 
presented to the patients to help them in scoring the pain. At the end of each visit, the patients will 
be given a survey and asked to rate the intensity of postoperative pain at 4, 24, and 48 h after the 
procedure. Patients will be instructed to take 1000-mg acetaminophen as needed. If acetaminophen 
was consumed, the patients will be asked to record the dose and time on the survey. They will be 
provided stamped return envelopes to mail surveys back to the Department of Endodontics 
(attachment 1). 
Treatment success determined clinically and radiographically: Radiographically, 
Following periapical (PAI) index by Órstavik2, description of radiographic fndings: 1. Normal 
periapical structures. 2. Small changes in the bone structure. 3. Change in the bone structure with 
mineral loss. 4. Periodontitis with a well-defined radiolucent area. 5. Severe periodontitis with 
exacerbating features. Success is defined as either complete (radiographic resolution of a periapical 
lesion - the radiographic sign of inflammatory processes surrounding a root tip) or incomplete 
healing (scar tissue formation) and failure includes uncertain healing (radiographic reduction of a 
periapical lesion or same lesion size) or unsatisfactory healing (increase in lesion size) as 
determined on the radiograph. Clinically success is defined by the absence of pain, swelling, 
percussion sensitivity or sinus tracts. Clinical failure is defined as the persistent presence of any 
of the symptoms mentioned above. Treatment success/failure measured at 6 month, 1 year and 2 
year follow up (± 7 days) post root canal filling. 
9.4.4 
Safety Analyses 
Primary endpoint: The difference in bacterial reduction between the experimental group (laser) 
and the comparison group (routinely used irrigation protocol) by measuring reduction in colony 
forming units (CFU). Friedman's and Wilcoxon signed-rank tests will be used to compare the 
amount of bacteria before and after treatment in both groups, with significance levels set at 5% (P 
< 0.05). 
The resident performing the endodontic treatment will evaluate and treatment plan the case as 
routinely performed through a clinical and radiographic examination of the involved teeth. At least 
one periapical radiograph of the involved tooth will be obtained during the consultation as routine 
protocol requires for every root canal treatment. In order to be eligible to participate in this study, 
an individual must meet all of the previously mentioned criteria (check section 5.1 and 5.2). On 
the day of procedure, the resident will conduct and complete the root canal treatment as standard 

 
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protocol requires, including medical history review/update, clinical assessment, vital signs (blood 
pressure). Participants, providers and observers will wear appropriate laser protection eyewear 
(OD 3 or greater) whenever the laser is in use. 
9.4.5 
Baseline Descriptive Statistics 
The baseline demographic and clinical characteristics of the study population, including age, 
gender, pre-op pain and endodontic diagnosis will be analyzid using descriptive statistics. 
Descriptive statistics will be presented as counts, proportions (%), mean and standard deviation 
whenever appropriate. The demographic and clinical characteristics of the study population will 
be analyzed using the Fisher’s Exact Test. P-value of  ≤0.05 will be considered statistically 
significant. 
9.4.6 
Planned Interim Analyses  
N/A 
9.4.7 
Sub-Group Analyses 
N/A 
9.4.8 
Tabulation of Individual Participant Data 
N/A 
9.4.9 
Exploratory Analyses 
N/A 
10 SUPPORTING DOCUMENTATION AND OPERATIONAL 
CONSIDERATIONS 
10.1 Regulatory, Ethical, and Study Oversight Considerations 
10.1.1 
Informed Consent Process 
10.1.1.1 
Consent/Assent and Other Informational Documents Provided To 
Participants 
Consent forms describing in detail the laser device, study procedures, and risks are given to the 
participant and written documentation of informed consent is required prior to enrolling the subject 
in the study. The following consent materials are submitted with this protocol (attachment 5). 
10.1.1.2 
Consent Procedures and Documentation 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in the 
study and continues throughout the individual’s study participation. Consent forms will be 

 
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Institutional Review Board (IRB)-approved and the participant will be asked to read and review 
the document. The investigator will explain the research study to the participant and answer any 
questions that may arise. A verbal explanation will be provided in terms suited to the participant’s 
comprehension of the purposes, procedures, and potential risks of the study and of their rights as 
research participants. Participants will have the opportunity to carefully review the written consent 
form and ask questions prior to signing. The participants should have the opportunity to discuss 
the study with their family or surrogates or think about it prior to agreeing to participate. The 
participant will sign the informed consent document prior to any procedures being done 
specifically for the study. Participants will have the option of consenting in REDCap. Participants 
must be informed that participation is voluntary and that they may withdraw from the study at any 
time, without prejudice. A copy of the informed consent document will be given to the participants 
for their records. The informed consent process will be conducted and documented in the source 
document (including the date), and the form signed, before the participant undergoes any study-
specific procedures. The rights and welfare of the participants will be protected by emphasizing to 
them that the quality of their medical care will not be adversely affected if they decline to 
participate in this study. 
10.1.2 
Study Discontinuation and Closure 
This study may be temporarily suspended or prematurely terminated by the Sponsor or the PI at 
any site if there is sufficient reasonable cause. Written notification, documenting the reason for 
study suspension or termination, will be provided by the suspending or terminating party to study 
participants, investigator, funding agency, the Investigational New Drug (IND) or Investigational 
Device Exemption (IDE) sponsor and regulatory authorities. If the study is prematurely terminated 
or suspended, the Principal Investigator (PI) will promptly inform study participants, the 
Institutional Review Board (IRB), and sponsor and will provide the reason(s) for the termination 
or suspension. Study participants will be contacted, as applicable, and be informed of changes to 
study visit schedule. 
Circumstances that may warrant termination or suspension include, but are not limited to: 
• Determination of unexpected, significant, or unacceptable risk to participants 
• Demonstration of efficacy that would warrant stopping 
• Insufficient compliance to protocol requirements 
• Data that are not sufficiently complete and/or evaluable 
• Determination that the primary endpoint has been met 
• Determination of futility 
Study may resume once concerns about safety, protocol compliance, and data quality are addressed, 
and satisfy the sponsor, IRB and/or Food and Drug Administration (FDA). 

 
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In terminating the study, the Sponsor and the Principal Investigator will assure that adequate 
consideration is given to the protection of the subjects’ interests. 
10.1.3 
Confidentiality and Privacy 
Participant confidentiality and privacy is strictly held in trust by the participating investigators, 
their staff, and the sponsor(s) and their interventions. This confidentiality is extended to cover 
testing of biological samples and genetic tests in addition to the clinical information relating to 
participants. Therefore, the study protocol, documentation, data, and all other information 
generated will be held in strict confidence. No information concerning the study or the data will 
be released to any unauthorized third party without prior written approval of the sponsor.  
All research activities will be conducted in as private a setting as possible. 
The study monitor, other authorized representatives of the sponsor, representatives of the 
Institutional Review Board (IRB), regulatory agencies or pharmaceutical company supplying 
study product may inspect all documents and records required to be maintained by the 
investigator, including but not limited to, medical records (office, clinic, or hospital) and 
pharmacy records for the participants in this study. The clinical study site will permit access to 
such records. 
The study participant’s contact information will be securely stored at each clinical site for 
internal use during the study. At the end of the study, all records will continue to be kept in a 
secure location for as long a period as dictated by the reviewing IRB, Institutional policies, or 
sponsor requirements. 
Study participant research data, which is for purposes of statistical analysis and scientific 
reporting, will be transmitted to and stored at password-protected database. This will not include 
the participant’s contact or identifying information. Rather, individual participants and their 
research data will be identified by a unique study identification number. The study data entry and 
study management systems used by clinical sites and by University of Pennsylvania School of 
Dental Medicine research staff will be secured and password protected. At the end of the study, 
all study databases will be de-identified and archived at password-protected database. 
10.1.4 
Future Use of Stored Specimens and Data  
Data collected for this study will be analyzed and stored at password-protected database. After 
the study is completed, the de-identified, archived data will be transmitted to and stored at 
password-protected database, for use by other researchers including those outside of the study. 
Permission to transmit data to the Teles lab will be included in the informed consent. 
With the participant’s approval and as approved by local Institutional Review Boards (IRBs), de-
identified biological samples will be stored at the Teles lab with the same goal as the sharing of 
data with the Teles lab. These samples could be used to research the causes of pulp disease, its 
complications and other conditions for which individuals with pulp disease are at increased risk, 
and to improve treatment. The Teles lab will also be provided with a code-link that will allow 

 
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linking the biological specimens with the phenotypic data from each participant, maintaining the 
blinding of the identity of the participant. 
During the conduct of the study, an individual participant can choose to withdraw consent to 
have biological specimens stored for future research. However, withdrawal of consent regarding 
biosample storage may not be possible after the study is completed. 
When the study is completed, access to study data and/or samples will be provided through the 
Teles lab. 
10.1.5 
Safety Oversight 
The PI’s; Dr. Bekir Karabucak (Chair of Endodontic Department), and Dr. Flavia Teles will be 
responsible for safety oversight. 
10.1.6 
Clinical Monitoring 
Dr. Bekir Karabucak (Chair of Endodontic Department) 
10.1.7 
Quality Assurance and Quality Control 
All monitoring and audits are to be performed according to ICH GCP E6(R2). 
Each clinical site will perform internal quality management of study conduct, data and biological 
specimen collection, documentation and completion. An individualized quality management plan 
will be developed to describe a site’s quality management.  
Quality control (QC) procedures will be implemented beginning with the data entry system and 
data QC checks that will be run on the database will be generated. Any missing data or data 
anomalies will be communicated to the site(s) for clarification/resolution. 
Following written Standard Operating Procedures (SOPs), the monitors will verify that the 
clinical trial is conducted and data are generated, and specimens are collected, documented 
(recorded), and reported in compliance with the protocol, International Conference on 
Harmonisation Good Clinical Practice (ICH GCP), and applicable regulatory requirements (e.g., 
Good Laboratory Practices (GLP), Good Manufacturing Practices (GMP)). 
The investigational site will provide direct access to all trial related sites, source data/documents, 
and reports for the purpose of monitoring and auditing by the sponsor, and inspection by local 
and regulatory authorities. 
10.1.8 
Data Handling and Record Keeping  
10.1.8.1 
Data Collection and Management Responsibilities  
Data collection is the responsibility of the clinical trial staff at the site under the supervision of 
the site investigator. The investigator is responsible for ensuring the accuracy, completeness, 
legibility, and timeliness of the data reported. 

 
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All source documents should be completed in a neat, legible manner to ensure accurate 
interpretation of data and follow ALCOAC standards (attributable, legible, contemporaneous, 
original, accurate, and complete). 
Hardcopies of the study visit worksheets will be provided for use as source document worksheets 
for recording data for each participant enrolled in the study. Data recorded in the electronic case 
report form (eCRF) derived from source documents should be consistent with the data recorded 
on the source documents. 
Clinical data (including adverse events (AEs), concomitant medications, and expected adverse 
reactions data) and clinical laboratory data will be entered in Axium and red cap, a 21 CFR Part 
11-compliant data capture system provided by the Axium and red cap. The data system includes 
password protection and internal quality checks, such as automatic range checks, to identify data 
that appear inconsistent, incomplete, or inaccurate. Clinical data will be entered directly from the 
source documents. 
10.1.8.2 
Study Records Retention  
Study documents should be retained for a minimum of 2 years after the last approval of a 
marketing application in an International Conference on Harmonization (ICH) region and until 
there are no pending or contemplated marketing applications in an ICH region or until at least 2 
years have elapsed since the formal discontinuation of clinical development of the Waterlase 
Express device. These documents should be retained for a longer period, however, if required by 
local regulations. No records will be destroyed without the written consent of the sponsor, if 
applicable. It is the responsibility of the sponsor to inform the investigator when these documents 
no longer need to be retained. 
10.1.9 
Protocol Deviations 
The PI and the study team should document all scenarios where the protocol is not followed and 
provide, in particular: 
• Who deviated from the protocol 
• What was the deviation 
• When did the deviation occur 
• How did the deviation happen 
• What is the impact of the deviation 
• A root cause analysis of why the deviation occurred 
If the assessment results in a determination that any of the following are potentially affected, the 
deviation would be considered of significant impact: 
• having the potential to adversely affect subject safety; OR 

 
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• increases risks to participants; OR 
• adversely affects the integrity of the data; OR 
• violates the rights and welfare of participants, OR 
• affects the subject’s willingness to participate in research.  
• there is a potential for an overall impact on the research that should be shared with the 
IRB for consideration and development of next best steps to address it 
10.1.10 
Publication and Data Sharing Policy 
This study will comply with the data sharing agreement. 
The Sponsor must approve all sharing of information/data prior to its occurrence. 
10.1.11 
Conflict of Interest Policy 
The independence of this study from any actual or perceived influence, such as by the 
pharmaceutical industry, is critical. Therefore, any actual conflict of interest of persons who have 
a role in the design, conduct, analysis, publication, or any aspect of this trial will be disclosed 
and managed. Furthermore, persons who have a perceived conflict of interest will be required to 
have such conflicts managed in a way that is appropriate to their participation in the design and 
conduct of this trial. 
10.2 Additional Considerations 
N/A 
10.3 Protocol Amendment History 
Version 
Date 
Description of Change  
Brief Rationale 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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Version 
Date 
Description of Change  
Brief Rationale 
 
 
 
 
 
 
 
 
 
11 REFERENCES 
1. 
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pain after root canal filling with warm vertical obturation with resin-based sealer and sealer-
based obturation with calcium silicate-based sealer: a prospective clinical trial. Clin Oral 
Investig. Aug 2021;25(8):5033-5042. doi:10.1007/s00784-021-03814-x 
2. 
Orstavik D, Kerekes K, Eriksen HM. The periapical index: a scoring system for 
radiographic assessment of apical periodontitis. Endod Dent Traumatol. Feb 1986;2(1):20-34. 
doi:10.1111/j.1600-9657.1986.tb00119.x 
3. 
Kakehashi S. The effect of surgical exposures of dental pulps in germ-free and 
conventional laboratory rats. 1965; 
4. 
G. B. Micro-organisms from necrotic pulp of traumatized teeth. Odontol Revy 1974; 
25:347-58 1974; 
5. 
Kerekes K, Tronstad L. Long-term results of endodontic treatment performed with a 
standardized technique. J Endod. Mar 1979;5(3):83-90. doi:10.1016/S0099-2399(79)80154-5 
6. 
al. Se. Influence of infection at the time of root filling on the outcome of endodontic 
treatment of teeth with apical periodontitis. 1997; 
7. 
Bystrom A, Sundqvist G. The antibacterial action of sodium hypochlorite and EDTA in 
60 cases of endodontic therapy. Int Endod J. Jan 1985;18(1):35-40. doi:10.1111/j.1365-
2591.1985.tb00416.x 
8. 
Bystrom A, Sundqvist G. Bacteriologic evaluation of the effect of 0.5 percent sodium 
hypochlorite in endodontic therapy. Oral Surg Oral Med Oral Pathol. Mar 1983;55(3):307-12. 
doi:10.1016/0030-4220(83)90333-x 
9. 
Bystrom A, Sundqvist G. Bacteriologic evaluation of the efficacy of mechanical root 
canal instrumentation in endodontic therapy. Scand J Dent Res. Aug 1981;89(4):321-8. 
doi:10.1111/j.1600-0722.1981.tb01689.x 
10. 
Nair PN, Henry S, Cano V, Vera J. Microbial status of apical root canal system of human 
mandibular first molars with primary apical periodontitis after "one-visit" endodontic treatment. 
Oral Surg Oral Med Oral Pathol Oral Radiol Endod. Feb 2005;99(2):231-52. 
doi:10.1016/j.tripleo.2004.10.005 
11. 
Ricucci D, Siqueira JF, Jr., Bate AL, Pitt Ford TR. Histologic investigation of root canal-
treated teeth with apical periodontitis: a retrospective study from twenty-four patients. J Endod. 
Apr 2009;35(4):493-502. doi:10.1016/j.joen.2008.12.014 

 
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12. 
Vera J, Siqueira JF, Jr., Ricucci D, et al. One- versus two-visit endodontic treatment of 
teeth with apical periodontitis: a histobacteriologic study. J Endod. Aug 2012;38(8):1040-52. 
doi:10.1016/j.joen.2012.04.010 
13. 
Ricucci D, Siqueira JF, Jr. Biofilms and apical periodontitis: study of prevalence and 
association with clinical and histopathologic findings. J Endod. Aug 2010;36(8):1277-88. 
doi:10.1016/j.joen.2010.04.007 
14. 
Kishen A. Advanced therapeutic options for endodontic biofilms. Endodontic Topics. 
2010;22(1):99-123. doi:10.1111/j.1601-1546.2012.00284.x 
15. 
Zorita-Garcia M, Alonso-Ezpeleta LO, Cobo M, et al. Photodynamic therapy in 
endodontic root canal treatment significantly increases bacterial clearance, preventing apical 
periodontitis. Quintessence Int. 2019;50(10):782-789. doi:10.3290/j.qi.a43249 
16. 
Wang X, Cheng X, Liu B, Liu X, Yu Q, He W. Effect of Laser-Activated Irrigations on 
Smear Layer Removal from the Root Canal Wall. Photomed Laser Surg. Dec 2017;35(12):688-
694. doi:10.1089/pho.2017.4266 
17. 
Bordea IR, Hanna R, Chiniforush N, et al. Evaluation of the outcome of various laser 
therapy applications in root canal disinfection: A systematic review. Photodiagnosis Photodyn 
Ther. Mar 2020;29:101611. doi:10.1016/j.pdpdt.2019.101611 
18. 
Meire MA, Coenye T, Nelis HJ, De Moor RJ. Evaluation of Nd:YAG and Er:YAG 
irradiation, antibacterial photodynamic therapy and sodium hypochlorite treatment on 
Enterococcus faecalis biofilms. Int Endod J. May 2012;45(5):482-91. doi:10.1111/j.1365-
2591.2011.02000.x 
19. 
Josic U, Mazzitelli C, Maravic T, Fidler A, Breschi L, Mazzoni A. Biofilm in 
Endodontics: In Vitro Cultivation Possibilities, Sonic-, Ultrasonic- and Laser-Assisted Removal 
Techniques and Evaluation of the Cleaning Efficacy. Polymers (Basel). Mar 25 
2022;14(7)doi:10.3390/polym14071334 
20. 
Seet AN, Zilm PS, Gully NJ, Cathro PR. Qualitative comparison of sonic or laser 
energisation of 4% sodium hypochlorite on an Enterococcus faecalis biofilm grown in vitro. Aust 
Endod J. Dec 2012;38(3):100-6. doi:10.1111/j.1747-4477.2012.00366.x 
21. 
Suer K, Ozkan L, Guvenir M. Antimicrobial effects of sodium hypochlorite and 
Er,Cr:YSGG laser against Enterococcus faecalis biofilm. Niger J Clin Pract. Sep 
2020;23(9):1188-1193. doi:10.4103/njcp.njcp_632_18 
22. 
Martins MR, Carvalho MF, Pina-Vaz I, Capelas JA, Martins MA, Gutknecht N. Outcome 
of Er,Cr:YSGG laser-assisted treatment of teeth with apical periodontitis: a blind randomized 
clinical trial. Photomed Laser Surg. Jan 2014;32(1):3-9. doi:10.1089/pho.2013.3573 
23. 
Dalton BC, Orstavik D, Phillips C, Pettiette M, Trope M. Bacterial reduction with nickel-
titanium rotary instrumentation. J Endod. Nov 1998;24(11):763-7. doi:10.1016/S0099-
2399(98)80170-2 
24. 
Shuping GB, Orstavik D, Sigurdsson A, Trope M. Reduction of intracanal bacteria using 
nickel-titanium rotary instrumentation and various medications. J Endod. Dec 2000;26(12):751-
5. doi:10.1097/00004770-200012000-00022 

 
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25. 
McGurkin-Smith R, Trope M, Caplan D, Sigurdsson A. Reduction of intracanal bacteria 
using GT rotary instrumentation, 5.25% NaOCl, EDTA, and Ca(OH)2. J Endod. May 
2005;31(5):359-63. doi:10.1097/01.don.0000145035.85272.7c 
26. 
Wang CS, Arnold RR, Trope M, Teixeira FB. Clinical efficiency of 2% chlorhexidine gel 
in reducing intracanal bacteria. J Endod. Nov 2007;33(11):1283-9. 
doi:10.1016/j.joen.2007.07.010 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
12 APPENDIX  
12.1  Schedule of Activities (SoA) 

 
 
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Procedures 
Pre-Screening 
Up to Day 0 
Enrollment/Baseline 
Visit 1, Day 0 
Study Visit 2 
Day 7 +/-3 days 
Study Visit 3 
6 months +/-7 days 
Study Visit 4 
1 year +/-7 days 
Study Visit 5 
2 yeras +/-7 days 
Prescreening (medical records, phone script) 
X 
 
 
 
 
 
Study informed consent 
 
X 
 
 
 
 
Demographics 
 
X 
 
 
 
 
Medical history review/update 
 
X 
X 
X 
X 
X 
Clinical assessment 
 
X 
X 
X 
X 
X 
Vital signs (Blood pressure) 
 
X 
X 
X 
X 
X 
Randomization 
 
X 
 
 
 
 
Administer study intervention 
 
X 
 
 
 
 
Sample collection   
 
X 
 
 
 
 
Pain Assessments (pre-op, and 4, 24, and 48 hours 
post-op) 
 
X 
 
 
 
 
Adverse event review and evaluation 
 
X 
X 
X 
X 
X 
Radiologic/Imaging assessment 
 
X 
X 
X 
X 
X 
Completion of the study 
 
 
 
 
 
X 
 
END OF DOCUMENT

 
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