University of Nebraska Medical Center DigitalCommons@UNMC
Theses & Dissertations Graduate Studies
Spring 5-4-2019
The Effect of Emdogain Periodontal Regenerative Material on Inflammation in eriodontalP Maintenance Patients
Jessica M. Gradoville University of Nebraska Medical Center
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THE EFFECT OF EMDOGAIN PERIODONTAL REGENERATIVE MATERIAL ON INFLAMMATION IN PERIODONTAL MAINTENANCE PATIENTS
By
Jessica Marie Gradoville, D.D.S.
A THESIS
Presented to the Faculty of
the University of Nebraska Graduate College
in Partial Fulfillment of Requirements
for the Degree of Master of Science
Medical Sciences Interdepartmental Area (Oral Biology)
Under the Supervision of Professor Amy C. Killeen
University of Nebraska Medical Center Omaha, Nebraska
May 2019
Advisory Committee:
Richard A. Reinhardt, D.D.S., Ph.D.
Jeffrey B. Payne, D.D.S., M. Dent. Sc.
James K. Wahl III, Ph.D.
Sung K. Kim, D.D.S. i
ACKNOWLEDGMENTS
Research has played an integral role in my education and career for many years, so when beginning my periodontal residency the decision to continue being involved in research was a simple one. My journey in research began while pursuing my undergraduate degree and progressed through dental school; however, I was never conducting the research I was investigating. That all changed thanks to the support I received from my periodontal faculty.
From the very start, I was excited about the end goal because I knew this was a strong project and one that pertains well to the field of periodontics. I cannot thank enough all of those who have provided unlimited guidance along my journey.
Dr. Killeen sets a perfect example of what it takes to be a researcher, mentor, instructor, and role model, and my heartfelt thanks goes out to her. Dr. Killeen supported me in every aspect of my life, from clinical work, to research, to personal life. She was not only there for the big moments, but the small moments as well. Whenever I had a question or was unsure of the next step, she was available and willing to help. She never wavered in her support. Without her guidance, this thesis would not have been possible. I thank her for her valuable advice and inspiration during my residency.
My research committee was an integral part of my success and I would like to extend my gratitude to each member. Dr. Richard Reinhardt, who helped keep me on track and provided extensive knowledge and advice throughout my residency. Dr. Jeffrey Payne for his insightful suggestions and unparalleled knowledge, your contributions were invaluable. Dr. James Wahl and
Dr. Sung Kim, thank you for offering constructive criticism and assistance throughout the study.
I cannot begin to express my thanks to my co-resident and friend, Dr. Erica Jasa. Dr. Jasa supported and challenged me each day of residency. Her clinical skills and assistance played a pivotal role in this research project. Without her encouragement, advice, and companionship, I would not be the person I am today. ii
Especially helpful to me during this time were Mrs. Megan Christiansen, Mrs. Marian
Schmid, and Kaeli Samson. Without Mrs. Megan Christiansen, this research project could not have been completed. Thank you for playing the largest role in treating patients and providing the greatest care. Mrs. Marian Schmid was instrumental in the lab, working tirelessly and providing loyal support when evaluating the lab samples. Kaeli Samson helped us with the statistical analysis and ultimately interpreted our data.
To the patients who took part in this yearlong study, they also deserve recognition. Without your participation and interest, we would not have been able to accomplish this project.
Funding for this study was provided by Windsweep Farm (Lincoln, NE), the late Dr.
Mick Dragoo and his wife, Mary, as well as the Nebraska Society of Periodontology and the
University of Nebraska Medical Center College of Dentistry Surgical Specialties Department.
Your contributions and aid in this project are appreciated.
Special thanks goes to my husband, CJ, my son, Theodore, and my parents, Kirk and
Rachele. CJ has stood by my side, through the good and the bad, the last twelve years. He understands my passion and love for knowledge and I would not be the person I am without his support. Theodore was born in the thick of my thesis development and writing, I hope one day I make him proud. My parents have given my constant love and encouragement throughout my education. I thank them for believing in me, from the very beginning. I love you all.
THE EFFECT OF EMDOGAIN PERIODONTAL REGENERATIVE MATERIAL ON
INFLAMMATION IN PERIODONTAL MAINTENANCE PATIENTS
Jessica M. Gradoville, D.D.S., M.S.
University of Nebraska, 2019
Advisor: Amy C. Killeen, D.D.S., M.S.
The purpose of this double-blinded, randomized, controlled clinical trial was to determine if local application of enamel matrix protein derivative (Emdogain:EMD), combined with minimally-invasive papilla reflection is effective in reducing inflammation in periodontal pockets in patients on periodontal maintenance therapy (PMT). Fifty patients diagnosed with advanced chronic periodontitis presenting with a 6-9mm interproximal probing depth were included in the trial. Experimental (EMD; n=24) and control (saline, n=26) therapies were randomly allocated.
Roots were treated with mini-flaps and root planing assisted with endoscope evaluation before
EMD or saline application. Inflammation was assessed by bleeding on probing (BOP) at baseline,
6-, and 12-months and gingival crevicular fluid (GCF) samples at baseline, 2-weeks, 6-, and 12- months. GCF was evaluated for change in IL-1β and PGE2 levels using ELISA. A significant reduction in BOP at the treatment site was seen for both groups after 12-months. After adjustments, patients with BOP present at baseline had a higher risk of a poor BOP outcome at
12-months (AOR = 5.68, p = 0.048). At 2-weeks, there was a significant reduction in IL-1β with
EMD (mean = -40.15 pg, p = 0.05) and at 12-months a trend for reduction in IL-1β with EMD
(mean = -32.85 pg, p = 0.07). Differences in IL-1β between groups were not significant. The addition of EMD does not significantly improve BOP; however, EMD does significantly decrease
IL-1β in the short-term compared to a control in periodontal maintenance patients. iv
TABLE OF CONTENTS
ACKNOWLEDGEMENTS...... i
ABSTRACT...... iii
TABLE OF CONTENTS...... iv
LIST OF FIGURES...... vi
LIST OF TABLES...... vii
LIST OF ABBREVIATIONS…………………………………………………………………...viii
CHAPTER 1: INTRODUCTION...... 1
CHAPTER 2: LITERATURE REVIEW: PERIODONTITIS...... 3
CHAPTER 3: LITERATURE REVIEW: ENAMEL MATRIX DERIVATIVE...... 6
CHAPTER 4: LITERATURE REVIEW: MICROSURGERY AND PERIOSCOPE...... 10
CHAPTER 5: LITERATURE REVIEW: INTERLEUKIN-1β AND PROSTAGLANDIN E2.....13
CHAPTER 6: RESEARCH HYPOTHESIS AND SPECIFIC AIMS………………...... 17
CHAPTER 7: MATERIALS AND METHODS……...... 18
Patient population and study design…………………………………………………………...18
Data collection and treatment protocol………………………………………………………..21
Analysis of GCF samples………………………………………………………………………...24
Statistical analyses………………………………………………………………………………..25
CHAPTER 8: RESULTS…...... 28
Patient characteristics……………………………………………………………………………28
Clinical outcomes…………………………………………………………………………………29
Inflammatory biomarker outcomes………………………………………………………….….34
CHAPTER 9: DISCUSSION……………………………………………………………………..40 v
CHAPTER 10: CONCLUSION……………………………………………………………….…48
BIBLIOGRAPHY...... 49
APPENDIX A: RAW CLINICAL DATA- PATIENT CHARACTERISTICS...... 59
APPENDIX B: RAW CLINICAL DATA- BOP…………………………………………………61
APPENDIX C: RAW CLINICAL DATA- Il-1β..………………………………………………..63
APPENDIX D: RAW CLINICAL DATA- PGE2…………………………………………….…..65
APPENDIX E: CONSENT FORM………………………………………………………………67
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LIST OF FIGURES Figure 1: Study Design Flowchart ...... 20
Figure 2: Baseline probing depth………………………………………………………………...22
Figure 3: Baseline GCF sampling………...... 22
Figure 4: Perioscope use………………………………………………………………………....23
Figure 5: Placement of EMD ...... 23
Figure 6: PR closure……...... 23
Figure 7: 2-weeks post-therapy ...... 23
Figure 8: 6- months post-therapy……...... 23
Figure 9: 12-months post-therapy… ...... 23
Figure 10- Change in BOP at 6 months ...... 31
Figure 11- Change in BOP at 12 months ...... 32
Figure 12: Change in BOP over time compared to initial IL-1β levels ...... 37
Figure 13: Change in BOP over time compared to change in IL-1β levels from baseline to 2- weeks ...... 37
Figure 14: Change in BOP over time compared to change in IL-1β levels from baseline to 6- months ...... 38
Figure 15: Change in BOP over time compared to change in IL-1β levels from baseline to 12- months ...... 38
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LIST OF TABLES
Table 1: Differences in demographics between groups ...... 28
Table 2: Experimental-teeth BOP measurements between groups ...... 29
Table 3: Experimental-teeth BOP Change ...... 30
Table 4: Presence or Absence of BOP at treatment-site at specific time points ...... 30
Table 5: 6-month treatment site BOP change (modeling odds of poor outcome) ...... 33
Table 6: 12-month treatment site BOP change (modeling odds of poor outcome) ...... 33
Table 7: IL-1β levels ...... 34
Table 8: IL-1β level changes compared to baseline ...... 35
Table 9: Presence or absence of PGE2 at treatment sites ...... 36
Table 10: AOR of poor BOP outcome compared to IL-1β at baseline and IL-1β changes ...... 39
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LIST OF ABBREVIATIONS
BOP bleeding on probing
CAL clinical attachment level
PD probing depth
PI plaque index
EMD Emdogain
ELISA enzyme-linked immunosorbent assay
GCF gingival crevicular fluid
PR papilla reflection
RP root preparation
IBL interproximal bone loss
IL-1β interleukin-1 beta
PGE2 prostaglandin E2
PMT periodontal maintenance therapy
MIST minimally invasive surgical technique
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CHAPTER 1: INTRODUCTION
Periodontitis is a chronic inflammatory disease that is characterized by an irreversible loss of the hard and soft tissues of the periodontium. The periodontium is made up of the following tissues: gingiva, cementum, alveolar bone, and the periodontal ligament. This chronic disease is of importance because an estimated 42% of adults over thirty years of age in the United
States are affected (Eke et al. 2018) and it is one of the principal causes of tooth loss.
Periodontal destruction is driven by the inflammatory response to bacterial biofilm. Signs of inflammation include gingival bleeding on probing (BOP), suppuration, increased gingival crevicular fluid (GCF), and biomarkers of inflammation including interleukin-1β (IL-1β) (Masada et al. 1990) and prostaglandin (PG) E2 (Nakashima et al. 1994).
The treatment of periodontitis varies and may include non-surgical and/or surgical therapies. Non-surgical therapy includes the removal of bacterial biofilm, calculus, and contaminated cementum through scaling and root planing (SRP) with hand instruments such as curettes and ultrasonic scalers. Following this instrumentation, a decrease in spirochetes and rods is noted (Lavanchy et al.1987) along with improvement in PD, BOP, and CAL (Kaldahl et al.
1996a, Becker et al. 2001). Other factors (e.g., occlusal trauma, iatrogenic restorations, tooth crowding, smoking) that may be contributing to the disease are also addressed at this point in therapy. With severe periodontitis, surgery is recommended to treat sites with deep pockets
(≥6mm). Once a patient is deemed periodontally stable, they are placed into a periodontal maintenance therapy (PMT) program at patient-specific intervals. The purpose of PMT is to help the patient maintain their oral health and to monitor their condition. If non-surgical or surgical therapy is completed without the appropriate maintenance, therapy results often do not have long- lasting effects (Becker et al. 1984). The recurrence of deep pockets at any point during PMT often leads to the recommendation of additional surgery, but patient acceptance of this option is
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limited. The reason surgery is recommended is because the presence of deep pockets (6 mm or greater) is indicative of further disease progression (Renvert et al. 2002).
Adjunctive therapies to augment periodontal treatment are used for patients who have not responded favorably to conventional treatment. Adjunctive therapies include systemic antibiotics
(Zandbergen et al. 2013), lasers (Cobb 2016), subgingival irrigants like PVP-Iodine (Rosling et al. 2001), chlorhexidine (Wennstrom et al. 1987), use of an endoscope (Kuang et al. 2017), local chemotherapeutics including Atridox (Garrett et al. 2000), and regenerative materials such as
Emdogain (EMD). Each therapy has varying degrees of success but overall the same goal of improving the patient’s periodontal outcome. The use of minimally-invasive surgical techniques with the endoscope and the addition of a regenerative material in conjunction with SRP may result in lower morbidity and better patient acceptance. The hypothesis of this research is that minimally-invasive papilla reflection/root preparation and EMD would better reduce inflammation as compared to a minimally-invasive papilla reflection/root preparation and saline in residual deep pockets during PMT.
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CHAPTER 2: LITERATURE REVIEW: PERIODONTITIS
Periodontitis is a complex disease caused by the host’s inflammatory response to a plaque biofilm at the interface between the gingiva and the tooth surface. At this interface, up to 700 different bacterial species can be present (Socransky and Haffajee 2008) however, just a few of these indigenous bacteria are causing the destruction and thus progressing from a diagnosis of gingivitis to periodontitis (Theilade 1986). The bacteria that were found to be associated with the clinical findings of periodontal disease, PD, and BOP, were Bacteroides forsythus,
Porphyromonas gingivalis, and Treponema denticola (Socransky et al. 1998). These bacteria form the red complex and possess virulence factors which alter the host’s immune response, causing periodontal destruction. As has been previously stated, the host’s immune system is key to the breakdown of connective tissue and alveolar bone, or periodontal destruction. Some individuals possess these specific microorganisms but do not show evidence of disease progression (Haffajee et al. 2004). Detecting these susceptible individuals is difficult, but genetic factors like neutrophil deficiency (Kalkwarf et al. 1984) and antibody avidity (Whitney et al.
1992) have been thought to contribute to periodontal breakdown. The host immune response to these bacteria is of fundamental importance and not only varies amongst people, but can vary amongst sites in the same individual.
As demonstrated by Page and Schroeder (1976), gingival disease begins within two to four days of plaque accumulation adjacent to the gingival tissues. The initial lesion of periodontitis shows loss of collagen along with vasculitis due to the release of chemotactic and antigenic substances by the plaque (Page and Schroeder 1976). At this stage, the main inflammatory response is neutrophil-mediated. With a lack of oral hygiene, this stage progresses to the early lesion within four to ten days, leading to a host response composed primarily of lymphocytes. A continued loss of connective tissue substance is seen and as the lesion becomes more established, the loss of collagen and fibroblast alteration increases and plasma cells are the
4
predominant inflammatory response. This development occurs in two to three weeks and can either remain stable for years or become progressive. If the established lesion converts into the advanced lesion, it manifests clinically as periodontitis since now bone loss has occurred. This destruction is dependent on the concentration of inflammatory mediators present in the gingival tissues and the penetration of these mediators within the gingival tissue to reach a critical distance from the alveolar bone (Graves and Cochran 2003). The critical distance was found to be between
2 mm (Waerhaug 1979) and 2.5 mm (Page and Schroeder 1981). At a distance greater than 2.5 mm, bone resorption should be absent due to the increased distance created between the bacteria and the alveolar bone.
Bone resorption due to periodontal destruction often results in bone loss patterns including horizontal bone loss, vertical bone loss, or a combination of the two. The pattern of bone destruction is determined by probing depths and bitewing radiographs. It is important to determine the pattern of bone loss because this aids in the diagnosis and treatment of the disease.
Horizontal bone loss is more common and is characterized by the osseous crest remaining perpendicular to the tooth surface. Vertical bone loss (also known as angular defects or intrabony defects) occurs alongside the root where the base of the defect is apical to the surrounding bone.
These intrabony defects are classified based on the number of osseous walls present (one, two or three). Vertical defects appear most often on the mesial and distal surfaces and roughly 60% of people with these defects have only a single defect (Nielsen et al. 1980).
The ultimate goal of treating periodontal disease is resolving inflammation, preventing further progression of destruction, and restoring the patient’s comfort and function. Therapeutic approaches can either halt progression of periodontal attachment loss or regenerate the lost periodontal tissue. Depending on the approach chosen, multiple modalities can be utilized; including surgical therapy, non-surgical therapy, or a combination of the two. The purpose of both surgical and non-surgical therapy is to cause a permanent microbial shift which can lead to a
5
decrease in PD and increase in the CAL (Haffajee et al. 1997). In addition to professional treatment, patient compliance is paramount to long-term periodontal stability. Long-term tooth retention is related to oral hygiene/compliance and well-maintained patients. Those who follow the maintenance therapy recommended have lower rates of tooth mortality (Hirschfeld et al.
1978, Wood et al. 1989). In a study by Wilson et al. (1987), it was found that patients who comply with their suggested maintenance interval retained more teeth than those who were erratic. Patients who were compliers lost no teeth whereas those who were erratic lost an average of 0.06 teeth per year. Patients with periodontal disease that go untreated lose even more teeth, as was shown by Becker et al. (1979), who noted the average tooth loss per non-compliant patient was 0.6 teeth per year, 10x more than erratic compliers. To stress the point, patients considered to be at a high-risk for periodontitis lost significantly more teeth (2.59 ± 3.9) than patients with moderate- (1.02 ± 1.8) or low-risk (1.18 ± 1.9) during supportive periodontal therapy (Matuliene et al. 2010). The study additionally found patients with erratic compliance lost significantly more teeth (3.11 ± 4.5) than compliant patients on supportive periodontal therapy (1.07 ± 1.6). These studies stress the importance of maintenance in regards to tooth loss.
During non-surgical therapy, teeth are debrided through SRP to eliminate the plaque and calculus present as well as reduce the bacterial load. Scaling and root planing has long been regarded as the “gold standard” of periodontal disease treatment (Cobb 2002). Non-surgical periodontal therapy has been shown to reduce tooth mortality rate by 58% compared to no therapy (Hujoel et al. 2000). Shortly following SRP, improvements in periodontal health are expected, such as a reduction in PD, BOP, PI, and a gain in CAL (Kaldahl et al. 1996, Becker et al. 2001). Additionally, an increase in the bone density has also been noted (Hwang et al. 2008).
In some cases of non-responding sites, often noted during PMT, surgical therapy or use of additional regenerative materials/biologics may be indicated.
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CHAPTER 3: LITERATURE REVIEW: ENAMEL MATRIX DERIVATIVE
Regenerative periodontal therapy aims to bring back the periodontal tissues that have been lost following an inflammatory insult. Regeneration can only be confirmed histologically and includes the regeneration of the tooth’s alveolar bone, periodontal ligament, and cementum over a previously diseased root surface. Since human histologic specimens are difficult to obtain, clinical criteria such as bone fill of osseous defects and gain of CAL are the best ways to determine what is believed to be true periodontal regeneration (Consensus Report, 1998).
Regeneration of periodontal defects has been shown with enamel matrix derivative (EMD) and the classic option of guided tissue regeneration (GTR). GTR utilizes barrier membranes along with autografts, allografts, and xenografts, to aid in the formation of the periodontal attachment apparatus while excluding epithelial migration. Guided tissue regeneration has proven to be successful in regards to gain in clinical attachment level (Cortellini et al. 1995, Cortellini et al.
1996) with gains up to 4.6 mm. Although the most important element to the long-term success of regeneration is supportive periodontal maintenance, other standards that need to be met for a more favorable outcome include patient and defect factors. A patient should have good oral hygiene to minimize the amount of inflammation present. Less inflammation allows for better flap reflection and suturing. Ideally, a deeper and narrower defect is preferable, as Tonetti et al.
(1993) found greater bone gains in deeper pockets (>3 mm) and pockets with an angle of 25 degrees or less (1.6 mm more attachment than defects with an angle of 36 degrees or more). Additionally, the more adjacent bony walls that are present the better the outcome.
Regeneration was found to average 1.5, 1.7, and 2.3 mm for one-, two- and three-wall defects, respectively (Kim et al. 2004). These intrabony periodontal defects that have a more favorable regenerative potential are diagnosed based off of periodontal probing with the aid of radiographs.
For example, different attachment levels between two interproximal surfaces represent the intrabony component of the defect. What is important to understand is not all bony defects are
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able to utilize guided tissue regeneration. The addition of growth factors, like those in EMD, could aid or enhance in the promotion of regeneration. However, clinically it appears that EMD and GTR are comparable to one another. A study by Sanz et al. (2004) found that clinically there were no significant differences between EMD and GTR at 1 year, except that EMD did demonstrate less recession. Sculean et al. (2001) found that a combination therapy of EMD and
GTR as compared to either regenerative procedure alone, showed no significant clinical improvement. However, all three regenerative treatment modalities led to a higher CAL gain than the flap surgery (control). In a study that disagreed with Sculean, Siciliano et al. (2011), showed the use of EMD in the treatment of non-contained intrabony defects led to less CAL gain as compared to GTR. A frequent complication found with GTR is the inability to achieve primary closure. This lack of primary closure of the flap in the interdental area allows for contamination of the barrier membrane and/or grafting material and reduced clinical outcomes (DeSanctis et al.
1996).
Emdogain, a commercially available product made by Straumann (Andover, MA) , is an extract of porcine enamel matrix in a polymeric matrix found to be present on root surfaces for greater than 4 weeks after its application (Sculean et al. 2002). Amelogenins constitute the majority (90%) of Emdogain (Lnygstadaas et al. 2009) with the remaining proteins including tuft protein, enamelin, proteases, and albumin (Bartlett et al. 2006, Margolis et al. 2006). Porcine enamel matrix proteins are considered “self” when encountered in the human body because they have a very similar gene pattern. Enamel matrix contains amelogenins which are involved in the formation of enamel and in turn aid in the development of acellular cementum (Slavkin, et al.
1997). Cementum deposition is needed for the formation of both the periodontal ligament and the alveolar bone (Armitage 1991). Other studies have demonstrated that EMD stimulates growth of multiple types of mesenchymal cells including periodontal ligament fibroblasts (Hoang et al.
2000, Gestrelius et al. 1997), cementoblasts, and osteoblasts (He et al. 2004). Downgrowth of the junctional epithelium onto the dental root surface interferes with regeneration but it an in vitro
8
study verified that EMD is a cytostatic agent, suppressing the proliferation of oral epithelial cells
(Kawase et al. 2003). A study on cultured PDL cells showed that the attachment rate, growth, and metabolism of these PDL cells was significantly increased when EMD was present (Lyngstadaas et al. 2001). Furthermore, Parkar et al. (2004) showed that EMD down-regulates inflammatory genes (IL-6, interferon γ, IL-13) while concurrently up-regulating genes coding for growth factors
(platelet-derived growth factor and bone morphogenetic proteins-1 and -4). EMD may also have certain antibacterial effects on key periodontal bacteria including Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia (Spahr et al.
2002). However, periodontal bacteria present in the pocket could alter the effect of EMD, making this product better utilized in maintenance patients who are considered more “well-controlled”.
For example, Inaba et al. (2004) found that P. gingivalis diminishes the effect of EMD on periodontal ligament cells through the action of their gingipains. Gingipains are proteinases and are virulent factors found in P. gingivalis.
Histologic and clinical studies on humans utilizing EMD have shown its great regenerative potential. Heijl (1997a) conducted a case report that showed the formation of new acellular cementum histologically after 4 months. This cementum was firmly attached to the underlying dentin and covered 73% of the defect. Yukna and Mellonig (2000) found that EMD showed some regenerative potential. Limitations to these studies include lack of a control. EMD along with non-surgical therapy (SRP) was found to result in similar treatment outcomes as non- surgical therapy alone (Mombelli et al. 2005). When compared to open flap surgery alone, Froum et al. (2001) showed a defect fill three times greater when EMD was used. Another study utilizing a modified widman flap (MWF) in conjunction with either EMD or a placebo found increased bone fill of the osseous defects in 93% of sites treated with EMD but no bone fill in the control group. Other statistically significant improvements with EMD over placebo were found in pocket reduction (3.1 mm versus 2.3 mm, respectively) and attachment level (2.2 mm versus 1.7 mm, respectively) (Heijl et al. 1997b). The long-term stability of EMD is an important clinical
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parameter and multiple studies (Rasperini et al. 2005, Sculean et al. 2004) have confirmed this stability as long as a strict maintenance protocol is followed. Contrary to these studies, Rosing et al. (2005) found no significant differences clinically or radiographically when EMD was used with an open flap debridement after 1 year. Overall, studies on the use of EMD are contradictory, limited, and few discuss its role on inflammatory biomarkers. Because EMD is relatively expensive, it is important to have a more accurate representation of the regenerative effects that can be achieved.
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CHAPTER 4: LITERATURE REVIEW: MICROSURGERY AND PERIOSCOPE
Traditional periodontal surgical techniques include more aggressive flap designs to gain access to the root surface and alveolar bone. Access is necessary because complete removal of the subgingival biofilm and calculus is not easily accomplished. When comparing closed root planing to open flap root planing, the mean percent stained surface area covered by residual plaque and calculus was 54.3% and 33%, respectively (Wylam et al. 1993). Other studies have found that
SRP effectiveness decreases as the PD increases (Brayer et al. 1989). In addition, when an open flap procedure is implemented, the effectiveness goes up, but a 100% calculus-free surface is never obtained. In addition to clinicians not being completely effective at calculus removal, anatomic factors such as root grooves, concavities, cemento-enamel projections, and the CEJ, can pose problems to successful instrumentation. Aggressive flap designs, which involve a larger number of teeth, can cause an increased risk of infection, soft tissue damage, bone loss, and recession. Since intrabony periodontal defects are typically localized, often a microsurgical approach is favorable. Microsurgery can result in improved soft tissue healing, minimal soft tissue damage, less recession, and a higher probability of achieving primary closure (Cortellini and Tonetti 2001) allowing for less contamination. Minimally invasive periodontal surgery (MIS) was first reported and described by Harrel and Rees in 1995 and has been refined many times since that initial introduction. Modifications include the minimally invasive surgical technique
(MIST), modification-MIST (Cortellini et al. 2009) and videoscope associated minimally invasive surgery (VMIS). MIS incorporates the elevation of the interdental papilla associated with the intrabony defect, so a small buccal and lingual flap limited to the teeth neighboring the defect, are elevated. Elevation of the mucoperiosteal flap is full thickness without involvement of the mucogingival junction but apical enough to expose the osseous crest. A traditional full thickness mucoperiosteal flap results in bone loss (0.8 mm total bone loss) (Wilderman et al.
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1970), along with increased surgical and healing time (Levin et al. 1977). Although a true MIST approach was not implemented in this study because we were not exposing the alveolar crest, the papilla reflection (PR) reflects a similar microsurgical approach. Due to the difficulty in visualization of these small surgical sites, the use of a visual aid has been proposed.
Various visualization aids have been introduced into the dental market, with the most common being surgical loupes worn by many dentists and dental specialists. An additional noninvasive tool, the PerioscopeTM (Perioscopy Inc., Oakland, CA), which is a minimally invasive dental endoscope, has also been introduced. The Perioscopy System is a miniature camera that is attached to an endoscope (fiber) which can be placed subgingivally to magnify the specific root of interest. The Perioscope allows for a 20-40x magnification of the site. Without the aid of the Perioscope, practitioners must confirm an adequately debrided root surface via hand instruments and tactile sensation. In a study by Sherman et al. (1990), it was demonstrated that clinical detection of subgingival calculus was difficult to accomplish. The addition of this magnification allows a supplementary verification of the root surface debridement through direct visualization. A study by Osborn et al. (2014) found more calculus was detected using the
Perioscope as compared to tactile detection. As has been stated previously, inflammation from the biofilm or calculus present can negatively affect the regenerative potential and thus it is imperative to have a thoroughly cleaned root. It is also known that the main etiologic factor for the initiation and progression of periodontal disease is bacterial biofilm. Wilson et al. (2008a) used a periodontal endoscope and found a significant relationship between calculus covered with biofilm and inflammation in the pocket wall. Wilson et al (2008b) went on to show that when calculus was removed, with the aid of an endoscope in conjunction with SRP, no histologic signs of inflammation were noted at 6 months. Geisinger et al. (2007) found that the use of the periodontal endoscope resulted in a statistically significant improvement in calculus removal during SRP. It was additionally noted that the removal was more apparent in deeper pockets. In a systematic review by Kuang et al. (2017) it was concluded that the endoscope may provide
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additional benefits for calculus removal compared to traditional SRP. However, it's important to note that using the endoscope often takes more time (6.01 minutes) to perform than non-aided
SRP.
When adding EMD to MIST, contradictory findings are apparent and many studies are simply case series. One such case series found the combination of MIS and EMD resulted in significant reductions in probing depths, an improvement in attachment levels, and little or no increase in recession (Harrel et al. 2005). Cortellini and Tonetti (2007) found similar results with
MIST and EMD in isolated, deep intrabony defects with limited intra- and post-operative morbidity. A randomized clinical trial by Ribeiro et al. (2011) found that EMD with MIST was similar to MIST alone in all measured clinical parameters at 3 and 6 month post operative appointments. An endoscope was not used with the MIST procedure, and could be an effective addition to the procedure. Additional limitations to these studies include short follow-up periods and no control site or groups.
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CHAPTER 5: LITERATURE REVIEW: IL-1β AND PGE2
The host inflammatory response of periodontal disease involves elements of both innate and adaptive immunity (Hajishengallis and Krostoff 2017). Interactions between the innate and adaptive immune cells are due, in large part, to cytokine interactions. Cytokines are cell signaling molecules produced by immune cells that activate effector mechanisms. Cytokines also aid in cell-to-cell communication in immune responses and stimulate the movement of cells towards the sites of inflammation and infection. A balance of cytokine regulation is needed. Heightened cytokine production could result in more destruction or progression of disease. Further study of cytokines is needed because it may explain why some individuals are more susceptible to periodontal disease than others.
It is possible to measure cytokines in the GCF of diseased periodontal pockets to study inflammation with the use of enzyme-linked immunosorbent assay (ELISA) techniques. GCF is an inflammatory exudate originating from the subgingival microvasculature that can be collected within the gingival sulcus. Although GCF originates from the vasculature, systemic cytokine levels of the blood serum do not accurately depict the inflammatory state in the periodontium, or vice versa, as most of the cytokines are released locally and not systemically in periodontal disease (Orozco et al. 2006). Several inflammatory markers have been identified in the GCF of periodontally involved teeth, including IL-1β (Masada et al. 1990) and PGE2 (Nakashima et al.
1994).
IL-1β is a pro-inflammatory cytokine that is released upon activation of the host inflammatory response to bacteria and their by-products (Cochran 2008). It is produced predominantly by monocytes and macrophages, along with fibroblasts, dendritic cells,
Langerhans cells, B cells, endothelial cells, neutrophils, epithelial cells, and bone cells (Horowitz
1993, Oppenheim et al. 1986). Some of IL-1β’s pro-inflammatory effects include: release and/or activation of nociceptive molecules like prostaglandin and interleukin-6 (Samad et al. 2001);
14
activation of matrix metalloproteinases that degrade extracellular matrix proteins (Kawasaki et al.
2008); stimulation of T-lymphocytes and lymphokine production (Mizel 1987); proliferation of
B-lymphocytes and antibody production (Chiplunkar et al. 1986); and enhancement of neutrophil chemotaxis and activation (Sauder et al. 1984). Specifically important for periodontal disease, IL-
1β promotes osteoclast formation and is a potent inducer of bone demineralization (Dewhirst et al. 1985).
IL-1β can be predictably measured in GCF and has been shown to be present at higher levels in inflamed/periodontally-involved sites compared to healthy sites. The amount of IL-1β from diseased sites was 3-fold greater than that from non-inflamed sites (Hou et al. 1995). A study by Preiss and Meyle (1994) sampling diseased sites from 19 untreated patients with moderate to severe periodontitis and from 14 healthy control patients found a difference in the concentration of IL-1β. In the healthy patients the concentration range between 22.8 ng/ml and
150 ng/ml while in the periodontal patients the concentration was between 85.8 ng/ml and 882.2 ng/ml. Another study by Yaghobee et al. (2013) showed as probing depth increased, the concentration of IL-1β also increased. A probing depth of 3 mm correlated with a concentration of 67.33 pg/µl whereas a probing depth of 5 mm correlated with a concentration of 537.98 pg/μl.
Clinically, there has been a significant correlation between the level of IL-β in GCF and clinical parameters such plaque index, gingival index, probing depth, and bone levels (Priess et al.
1994, Stashenko et al. 1991). Ishihara et al. (1997) evaluated the correlation of IL-1β levels in
GCF and the clinical status of patients with slight, moderate, or severe levels of periodontitis and healthy controls. No IL-1β was detected in the GCF obtained from non-inflamed sites of healthy subjects. The total amount of IL-1β was correlated with alveolar bone loss. Finally, Engebretson et al. (2002) found that probing depth and attachment level were each strongly correlated with IL-
1β levels. Therefore, it is generally concluded that measuring the IL-1β biomarker in GCF may be valuable in detecting the activity of inflammation and breakdown in periodontal tissues.
15
Following scaling and root planing, it has been shown that inflammation is reduced as well as the amount of IL-1β in periodontally diseased sites (Engebretson et al. 2002, Hou et al.
1995). Engebretson also found that IL-1β returned to near baseline levels in patients with severe disease after 24 weeks. Smoking status was found to influence IL-1β levels (Goutoudi et al. 2004) following therapy and smokers were found to have higher levels of IL-1β. These facts stress the importance of non-surgical therapy and modification of patient lifestyle factors.
Prostaglandins are lipids derived from arachidonic acid metabolism and play a key role in the generation of an inflammatory response. Arachidonic acid is a 20-carbon unsaturated fatty acid that is released from the plasma membrane by phospholipases (Ricciotti and FitzGerald
2012). PGE2 production from arachidonic acid depends on the COX enzymes. COX-1 is expressed constitutively in most cells whereas COX-2 is induced by inflammatory stimuli, hormones, and growth factors (Dubois et al. 1998). PGE2 is one of four bioactive prostaglandins generated in vivo by macrophages and fibroblasts and its presence leads to the classic signs of inflammation (redness, edema, and pain) through arterial dilation, increased microvascular permeability, and activation of peripheral sensory neurons and central sites within the spinal cord and brain (Funk 2001). In addition to redness, edema, and pain, PGE2 can induce bone resorption and increase the number of osteoclasts present (Chambers et al. 1983) as well as decrease collagen synthesis by fibroblasts (Tipton et al. 2003). Increased bone resorption and decreased collagen synthesis are classic findings in periodontal disease.
PGE2 levels in GCF have been reported to have a positive correlation with periodontal inflammation (Nakashima et al. 1994, Tsai et al. 1998) and are typically associated with the onset of inflammation (Rajakariar et al. 2006). A study by Kumar et al. (2013) aimed at estimating the levels of PGE2 in GCF in periodontal health, chronic periodontitis, and after periodontal therapy found that the mean PGE2 concentrations in GCF increased progressively from health (56.28 pg/ml) to periodontitis (326.62 pg/ml). Following non-surgical periodontal treatment (SRP), a significant reduction of PGE2 (107.643 pg/ml) was noted. Leibur (1999) had similar results. The
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mean GCF PGE2 concentrations in the periodontitis group were higher compared to the concentrations following non-surgical treatment. This decrease in the concentration of PGE2 could be due to the subsequent decrease in gram-negative bacteria present following SRP. Hessle et al. (2003) found that gram-negative bacteria were able to induce a strong PGE2 production in monocytes.
Due to the inflammatory alterations that PGE2 elicits, clinical manifestations, like BOP, in its presence are expected. GCF levels of PGE2 were found to have a strong positive correlation with periodontal parameters including gingival index, plaque index, probing depth, and clinical attachment loss (Kumar et al. 2013). When comparing PGE2 levels between smokers to non- smokers with chronic periodontitis, no differences were seen (Heasman et al. 1998).
Overall, GCF levels of IL-1β and PGE2 have been found to be significantly associated with clinical signs of periodontal disease (Sanchez et al. 2013) making these inflammatory markers adequate choices for assay.
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CHAPTER 6: RESEARCH HYPOTHESIS AND SPECIFIC AIMS
The central research hypothesis is that the addition of EMD to PR/RP will decrease the number of
BOP positive treatment sites and decrease the amount of inflammatory biomarkers present in the
GCF at the conclusion of treatment relative to a saline control.
Specific Aims
1. To determine whether EMD reduces the amount of IL-1β and PGE2 in the GCF of
periodontal patients relative to a saline control.
2. To determine whether EMD reduces the frequency of BOP at EMD treated sites versus a
saline control.
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CHAPTER 7: MATERIALS AND METHODS
Patient population and study design
The clinical phase of this double-blinded, randomized controlled study was conducted from February 2017 to July 2018. Patients regularly attending the University of Nebraska
Medical Center College of Dentistry for PMT were screened and identified by faculty and investigators involved in this study (RR, AK, MC, and JP). Recruitment for this clinical trial occurred between September 2016 and April 2017. The inclusion criteria for the study included subjects between the ages of 40-85 years, a periodontal diagnosis of advanced chronic periodontitis (Stage III, Grade B (Tonetti et al. 2018)), one quadrant with at least three posterior teeth and one 6-9 mm periodontal pocket, overall good systemic health, and a history of regular
PMT. Exclusion criteria consisted of subjects with systemic diseases that significantly affect periodontal inflammation and bone turnover (e.g., chronic use of steroids or non-steroidal anti- inflammatory drugs, estrogens, bisphosphonates, calcitonin, methotrexate, antibiotics, >325 mg aspirin/day), surgical periodontal therapy within the past year, and pregnant or breast-feeding females. Individuals meeting the inclusion criteria had the protocol explained and questions answered prior to obtaining consent. Following consent, patients were randomly assigned by a randomization table to test (EMD) or control (saline) groups with randomization stratified by gender and smoking status by a clinician not involved with clinical measurements (EJ, JG).
Clinical measurements and GCF samples were obtained at baseline, 2-week, 6-month, and 12- month visits by one of three calibrated clinicians (RR, AK, JP). Next, the test site (EMD) received papilla reflection and root planing, visualization for confirmation of removal of calculus/plaque with use of the Perioscope followed by administration of etching gel (Pref-Gel,
Straumann, Andover, MA ) and EMD in the experimental site by EJ or JG. The control site
(saline) received papilla reflection and root planing, visualization for confirmation of removal of calculus/plaque with use of Perioscope followed by administration of saline in the experimental
19
site by EJ or JG. Papilla were reapproximated and sealed with cyanoacrylate cement (PeriAcryl,
Glustitch Inc, Delta, BC, Canada). After completion of surgical phase of treatment, PMT was completed by MC. Periodontal maintenance therapy was performed at baseline, 3-month, 6- month, 9-month, and 12-month appointments. The study was registered with ClinicalTrials.gov
(NCT02972788) and approved by the University of Nebraska Medical Center Institutional
Review Board (#783-16-FB).
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Figure 1: Study Design Flowchart
21
Data Collection and Treatment Protocol
Consent was discussed with patient and signed after answering any questions. Clinical data and GCF collection were collected by one of three trained and calibrated dentists (RR, AK,
JP). During data collection, the experimental site was isolated with the aid of cotton rolls, supragingival plaque was removed (and recorded) from the test teeth with a dental explorer, then an absorbent paper strip (PerioPaper strips, Oraflow, Hewlett, NY) was inserted into the facial and lingual sulcus of the experimental sites for 30 seconds for collection of GCF. The strip was placed in the pocket until resistance was felt. The paper strip was then immediately placed into a sterile vial and frozen at -80ᵒC until further analysis. GCF strips contaminated with blood were discarded and collection was repeated. PDs were then measured at the same sites. BOP was recorded as positive for sites that bled within 30 seconds. After completion of necessary measurements and departure of the faculty investigator, the surgical phase of treatment was completed by a single clinician (JG or EJ) not involved with clinical measurements. Following administration of local anesthesia to the experimental site via local injection, a surgical mini-flap reflection (papilla-reflection on buccal and lingual/palatal) was completed using a #12B blade.
Removal of the interproximal soft tissue allowed for access to the root and improved visualization with the Perioscope (Perioscopy Unit, Zest Dental Solutions, Carlsbad, CA). Scaling and root planing was performed circumferentially on the test and adjacent interproximal tooth as to prevent disturbances to the reapproximated papilla during the subsequent periodontal maintenance appointment. Verification of a clean and smooth root surface was done using an
11/12 explorer and Perioscope (SC1) by both the clinician and MC. Irrigation of the site using sterile saline removed debris from the pocket. Following scaling and root planing, the treatment assignment was completed by EJ or JG. The root surface was then etched for 2 minutes with ethylenediaminetetraacetic acid (EDTA, Pref-Gel, Straumann, Andover, MA) followed by irrigation with sterile saline. Randomization determined whether the site received EMD (0.3 mL)
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or sterile saline (0.3 mL), which was placed at the base of the pocket and deposited up the root surface of the experimental and adjacent interproximal tooth. Excess was removed using a damp
2x2 gauze and compression of the buccal and lingual/palatal papilla. The papillae were reapproximated under pressure and sealed using cyanoacrylate (PeriAcryl, GluStitch, Delta, BC,
Canada). Damp gauze pressure for 3-5 minutes was done to set the Periacryl and to minimize clot formation. Routine periodontal maintenance therapy, including full-mouth periodontal charting, full-mouth debridement and root planing of inflamed pockets (excluding experimental site) was performed by MC. Patients were instructed to avoid flossing and brushing of the experimental site for 6 weeks. Listerine (Johnson & Johnson Consumer, Inc., New Brunswick, New Jersey) mouth rinse was dispensed to aid in maintaining a low level of plaque accumulation at the test sites. It was asked to be used twice daily for 30 seconds (morning and night) for 6 weeks.
Patients were asked to return for postoperative visits at 2- and 6-weeks along with periodontal maintenance recalls at 3-, 6-, 9-, and 12-months. GCF collection was repeated at 2- week, 6-, and 12-month visits. The same measurements completed at baseline were recorded at 6- and 12-months by one of three calibrated examiners. One dental hygienist (MC) completed the periodontal maintenance therapies. Figures 2-9 below are photos depicting select steps of data collection and treatment rendered.
Figure 2: Baseline probing depth Figure 3: Baseline GCF sampling
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Figure 4: Perioscope use Figure 5: Placement of EMD
Figure 6: PR closure Figure 7: 2-weeks post-therapy
Figure 8: 6- months post-therapy Figure 9: 12-months post-therapy
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Analysis of GCF samples
GCF samples were collected at both the buccal and lingual interproximal treatment sites and pooled at baseline, 2-week, 6-, and 12- month visits. GCF samples were analyzed for IL-1β and PGE2 using standard ELISA techniques. The IL-1β ELISA kit utilized the quantitative sandwich enzyme immunoassay technique (R&D Systems, Human IL-1β/IL-1F2 Quantikine®
ELISA, Minneapolis, MN) whereas the PGE2 ELISA kit was a competitive assay (Cayman
Chemical, Prostaglandin E2 ELISA Kit-Monoclonal, Ann Arbor, MI). All assay procedures were performed at the same time by individuals (JG, MS) without knowledge of the therapy allocation.
The ELISA tests were performed according to the manufacturer’s instructions and protocol. The stored GCF samples were allowed to thaw at room temperature. Sample strips were then eluted in
1 mL of phosphate buffer solution and gently agitated for 1 hour.
IL-1β
Two-hundred microliters of each standard and reconstituted GCF sample were aliquoted in duplicate to wells that were pre-coated with a monoclonal antibody specific for human IL-1β.
This was allowed to incubate for 2 hours at room temperature. The wells were then aspirated and washed 3 times before the addition of 200 microliters of human IL-1β conjugate was added to each well and allowed to incubate again for 2 hours at room temperature. Aspiration and washes were once more performed. Two-hundred microliters of substrate solution was added to each well, this incubated for 20 minutes at room temperature, protected from light. Fifty microliters of stop solution were added to each well and gently agitated until a yellow color was obtained. The microplate was read at a wavelength of 450 nm.
The IL-1β standard calibration curves were generated. The minimum detectable concentration was 3.9 pg/mL and the maximum detectable concentration was 250 pg/mL.
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Each GCF sample was analyzed separately. The optical density and relative IL-1β concentration of each sample were estimated from the standard curve. The IL-1β concentration of a GCF sample from each site was the average of each sample’s duplicate.
PGE2
Fifty microliters of sample were added to each well followed by the addition of 50 microliters of the prostaglandin E2 ACHe Tracer and 50 microliters of the prostaglandin E2 monoclonal antibody. The plate was then incubated for 18 hours at 4℃. The wells were rinsed 5 times with wash buffer and 200 microliters of Ellman’s Reagant was added to each well. The plate was placed on an orbital shaker and covered to allow for development in the dark for 60 minutes. The plate was read at a wavelength of 405 nm.
A log of the PGE2 standard calibration curves was generated. The minimum detectable concentration was 7.8 pg/mL and the maximum detectable concentration was 500 pg/mL.
Each GCF sample was analyzed separately. The optical density and relative PGE2 concentration of each sample were estimated from the standard curve. The PGE2 concentration of a GCF sample from each site was the average of each sample’s duplicate.
Statistical analyses
The ideal sample size to ensure adequate power was calculated to find a difference in
CAL (primary outcome), to ensure a difference of 1.0 mm between groups with a common estimated group standard deviation of 1.1 with a significance level of 0.05 using a two-sided two- sample t-test. It was determined that a sample size of 22 treatment sites per group was necessary to achieve at least 80% power. The sample size selected was not specific for a difference in either inflammatory biomarker between groups and was based on the primary outcome for this clinical trial.
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For treatment site BOP measurements, both lingual and buccal interproximal sites were assessed, regardless of site of deepest pocket. BOP was considered present at baseline if at least one interproximal treatment site had the condition present. The follow-up variables for BOP were determined as follows: if the patient started without a condition (i.e., no BOP) and ended without a condition, or showed a reduction (either two sites with the condition to only one site, or one site to none), that patient was considered to have a good outcome; if the patient started with a condition (i.e., BOP) and showed no improvement (e.g. one site, and one site), or if they had more sites with the condition than at baseline, that patient was considered to have a poor outcome.
BOP and PGE2 measurements were dichotomized as being present or absent. PGE2 was dichotomized given the large percentage of patients who did not have any detectable PGE2.
Mean and standard error (SE) were calculated for each treatment condition. Wilcoxon
Rank Sum tests and T-tests were used to examine differences in distributions of numeric variables between the two treatment conditions (i.e. Emdogain or placebo). Associations between categorical variables were assessing using Chi-Square tests or Fisher’s exact tests when expected cell counts were low. Vertical bar charts were generated to show associations between changes in measurements and biochemical markers. Inferential statistics on these associations were made using linear models where the outcome was the change in measurement, and the model adjusted for the initial measurement, group, and site of worst pocket, as well as any potential interactions involving group. Similar models were also used to assess significant differences in changes of measurements over time using model estimated means, as well as differences in changes observed between the two treatment groups, while controlling for site of worst pocket and initial measurement. When initial measurement was significantly associated with the change in measurement, model adjusted change in measurement means were calculated for three different values of initial measurement: the 10th percentile, mean, and 90th percentile. Significant main
27
effects of variables with more than two levels were further assessed using post-hoc pairwise comparisons which used stimulation to adjusted p-values for multiple comparisons.
Differences in the proportion of patients with BOP at baseline vs. six months or twelve months were assessed using McNemar’s tests, separately for Emdogain and placebo patients. All analyses were performed using SAS software version 9.4 (SAS Institute Inc., Cary, NC). We considered p-values less than or equal to 0.05 to be statistically significant.
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CHAPTER 8: RESULTS
Patient Characteristics
Following screening of the patients, 50 eligible patients were invited to participate and consented to participate in the study. Intervention was initiated on all 50 subjects and 48 completed the 12-month PMT (4% dropout rate, both being from the saline group). One patient did not return following the 6-month post-treatment exam due to the test tooth being extracted because of root caries. The other patient was deceased following the 3-month post-treatment exam. Both reasons for patient dropout were not believed to be related to the therapy administered.
Baseline characteristics of patients included in this clinical trial are represented in Table
1. There were no significant differences between groups at baseline (age p = 0.41; sex p = 0.40; smoking status p = 0.33).
Table 1: Differences in demographics between groups
Emdogain Control
n % n % P-value
Gender 0.40
Female 13 54.2 11 42.3
Male 11 45.8 15 57.7
Smoking 0.33 Status
Non-smoker 21 87.5 21 76.9
Smoker 3 12.5 6 23.1
Mean Age 66.92 64.96 0.41 (years)
*Indicates significant difference (p ≤0.05)
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Clinical Outcomes:
The mean baseline and 12-month post-therapy BOP measurements of experimental-teeth
(test tooth and adjacent tooth) BOP are presented in Table 2.
Table 2: Experimental-teeth BOP measurements between groups
PR/RP + EMD PR/RP + Saline P-value
N 24 24
Baseline BOP 36% (0.05) 41% (0.05) 0.47 Mean (SE)
12-MO POST- 29% (0.05) 25% (0.04) 0.53 THERAPY BOP Mean (SE)
*Indicates significant difference (p ≤0.05)
The mean baseline BOP of the experimental-teeth for PR/RP + EMD and PR/RP + saline was not statistically different (p = 0.47). The difference in experimental-teeth BOP at the 12- month post-therapy visit between both groups was also not statistically different (p = 0.53).
However, compared to baseline, BOP of the experimental-teeth was reduced for both groups following 12 months of therapy.
In Table 3, the change in BOP was observed after adjusting for initial BOP and worst site. In the EMD group, a mean reduction of 8% was seen at 12-months post-therapy from baseline, this was not significant (p = 0.0965). Contrary to this, the saline group showed a mean
BOP reduction of 14% and this was statistically significant (p = 0.006). This reduction was not statistically significant between groups.
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Table 3: Experimental-teeth BOP Change
Model Adjusted P-value for Mean P-value for Variable Mean Change (Final- Change (Change over (Differences between Initial) (±SE) time) variable levels)
0.381
EMD 8% (5%) 0.0965
Saline 14% (5%) 0.006*
*Indicates significant difference (p ≤0.05)
The distribution of BOP at baseline, 6-, and 12-months is represented in Table 4. BOP was recorded for both the buccal and lingual/palatal sites of all interproximal treatment sites following probing depth measurement. At baseline, 6-, and 12-month time points, there were no significant differences between the groups in regards to the number of treatment site/s with BOP
(p = 0.75, 0.44, and 0.38, respectively).
Table 4: Presence or Absence of BOP at treatment-site at specific time points
Emdogain Saline P-value
Baseline BOP 0.75
Absent 6 (25%) 7 (29.2%)
Present 18 (75%) 17 (70.8%)
BOP 6-months 0.44
Absent 11 (45.8%) 8 (34.8%)
Present 13 (54.2%) 15 (65.2%)
BOP 12- 0.38 months Absent 12 (50%) 15 (62.5%)
Present 12 (50%) 9 (37.5%)
*Indicates significant difference (p ≤0.05)
31
Assessment of the proportion of patients with BOP at baseline to patients with BOP at 6- and 12-months post-operatively was completed for each group (EMD and saline) and is present in
Figures 10 and 11. With the EMD group, the proportion of patients with BOP at 6-months did not significantly differ from the proportion of patients with BOP at baseline (p = 0.13, Figure 10).
The same outcome was seen at 6-months with the saline group (p = 0.74, Figure 10). However, at
12-months, the proportion of EMD and saline patients with BOP was lower than the proportion with BOP at baseline (p = 0.03; p = 0.01, Figure 11, respectively). Overall, a significant reduction in BOP at the treatment site after 12-months of therapy was found, irrespective of the treatment rendered.
Figure 10- Change in BOP at 6 months
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Figure 11- Change in BOP at 12 months
After adjusting for variables including initial treatment site BOP, treatment group, and worst site, an adjusted odds ratio (AOR) was determined for 6- and 12-month treatment site BOP
(see Tables 5 and 6, respectively). At 6-months, after controlling for the treatment site initial BOP and the worst site, there was not a significant difference in the odds of having a poor BOP outcome between the EMD and saline groups (p = 0.75). The same was found at 12-months (p =
0.16). After adjusting for group and worst site, patients who had BOP present at baseline had a higher adjusted odds of a poor BOP outcome at 12-months, this was significant (AOR = 5.68, p =
0.048). This was not seen at 6-months (p =0.76). Finally, after controlling for treatment site initial
BOP and group, whether the worst site was on the buccal, lingual, or both, there was not an increased risk of a poor BOP outcome at either 6-months (p = 0.78) or 12-months (p = 0.67).
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Table 5: 6-month treatment site BOP change (modeling odds of poor outcome)
AOR 95% CI for AOR P-value
Initial Treatment Site BOP 0.76
Absent 1 Reference
Present 1.23 0.32, 4.76
Group 0.75
Placebo 1 Reference
Emdogain 0.83 0.26, 2.65
Worst Site 0.78
Both 1 Reference
Buccal 1.14 0.16, 8.08
Lingual 1.64 0.32, 8.39
*Indicates significant difference (p ≤0.05)
Table 6: 12-month treatment site BOP change (modeling odds of poor outcome)
AOR 95% CI for AOR P-value
Initial Treatment Site BOP 0.048*
Absent 1 Reference
Present 5.68 1.02, 31.76
Group 0.16
Placebo 1 Reference
Emdogain 2.53 0.70, 9.08
Worst Site 0.67
Both 1 Reference
Buccal 0.96 0.11, 8.09
Lingual 1.79 0.33, 8,38
*Indicates significant difference (p ≤0.05)
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Inflammatory Biomarker Outcomes:
GCF samples from 50 patients were analyzed. Comparisons of IL-1β levels for PR/RP +
EMD and PR/RP + saline taken at baseline, 2-week, 6-, and 12-months post-therapy are shown in
Table 7. Table 8 then shows the IL-1β level changes compared to baseline at each of the subsequent time points that GCF was collected.
Table 7: IL-1β levels
Therapy PR/RP + EMD PR/RP + Saline P-value Mean pg/sample Mean pg/sample (SE) (SE) N 24 26
IL-1β initial 141.10 (25.44) 109.38 (20.85) 0.34
IL-1β 2-week 99.21 (17.16) 104.62 (16.93) 0.82
IL-1β 6-month 125.11 (21.74) 153.91 (20.66) 0.34
IL-1β 12-month 114.02 (18.31) 111.35 (18.93) 0.92
*Indicates significant difference (p ≤0.05)
35
Table 8: IL-1β level changes compared to baseline
Therapy PR/RP + EMD PR/RP + Saline
N 24 26
BASELINE Mean 141.10 pg (25.44) 109.38 pg (20.85) (SE)/sample
2 WK POST-THERAPY -40.15 pg (19.57) -23.64 pg (17.87) Mean Change (SE)/sample P-Value for Mean Change 0.05* 0.19 (Change over Time) 6-MO POST-THERAPY -32.66 pg (21.66) 15.51 pg (20.08) Mean (SE)/sample P-Value for Mean Change 0.14 0.44 (Change over Time) 12-MO POST-THERAPY -32.85 pg (17.99) -14.13 (16.78) Mean (SE)/sample P-Value for Mean Change 0.07 0.40 (Change over Time)
*Indicates significant difference (p ≤0.05)
At each time point that GCF was collected, IL-1β levels were not significantly different between the groups (Table 7).
When assessing the change in IL-1β levels compared to baseline (Table 8), a statistically significant reduction in IL-1β with PR/RP + EMD was seen at 2- weeks post-therapy (mean =
-40.15 pg, p = 0.05). At 12-months post-therapy, a trend for reduction of IL-1β levels was seen with the EMD group (mean = -32.85, p = 0.07) compared to PR/RP + saline.
Roughly 70% of PGE2 samples were below detectable levels. The presence or absence of
PGE2 in both groups is shown in Table 9. Differences between the EMD and saline group were not significant at any time point. When detectable and after controlling for initial treatment site
BOP, group, and worst site, patients who had PGE2 present at baseline had an increased risk of a
36
poor BOP outcome compared to those patients who did not have PGE2 present [AOR = 4.6, 95%
CI (0.906, 23.299)]. However, this was not statistically significant (p = 0.066).
Table 9: Presence or absence of PGE2 at treatment sites
EMD (N=24) Placebo (N=26) P-value
Initial PGE2 0.14
Absent 16 (66.67%) 22 (84.62%)
Present 8 (33.33%) 4 (15.38%)
2- week PGE2 0.55
Absent 10 (41.67%) 13 (50%)
Present 14 (58.33%) 13 (50%)
6-month PGE2 1.00
Absent 20 (83.33%) 19 (79.17%)
Present 4 (16.67%) 5 (20.83%)
12-month PGE2 0.50
Absent 18 (75%) 21 (84%)
Present 6 (25%) 4 (16%)
*Indicates significant difference (p ≤0.05)
Figures 12-15 are box and whisker plots that illustrate the raw IL-1β data that was plotted but not adjusted for; these are for descriptive purposes only. Instead, Table 10 shows the association between IL-1β and the AOR of a poor BOP outcome (adjusting was done for initial treatment site BOP, group, and worst site). It was found that there was no significant association between an increase in IL-1β at baseline and the change observed in BOP (p = 0.127) (Figure 12,
Table 10). In addition, no statistically significant difference was found for any IL-1β change
(Figures 13-15, Table 10) and the change observed in BOP at the treatment site.
37
Figure 12: Change in BOP over time compared to initial IL-1β levels
Figure 13: Change in BOP over time compared to change in IL-1β levels from baseline to 2- weeks
38
Figure 14: Change in BOP over time compared to change in IL-1β levels from baseline to 6- months
Figure 15: Change in BOP over time compared to change in IL-1β levels from baseline to 12- months
39
Table 10: AOR of poor BOP outcome compared to IL-1β at baseline and IL-1β changes
Predictor Adjusted Odds 95% CI for AOR P-value Ratio (AOR) of Poor BOP Outcome at Treatment Site
IL-1β Baseline 1.005 0.998, 1.012 0.127
IL-1β Change (2 week - Baseline) 0.997 0.990, 1.003 0.338
IL-1β Change (6 months - Baseline) 0.998 0.991, 1.005 0.511
IL-1β Change (12 months - Baseline) 1.000 0.992, 1.008 0.941
*Indicates significant difference (p ≤0.05)
40
CHAPTER 9: DISCUSSION
This double-blinded, placebo-randomized controlled clinical trial aimed to compare clinical and inflammatory biomarkers measurements of two therapies, PR/RP + EMD (test) and
PR/RP + saline (control), for non-resolving, 6-9 mm pockets in periodontal maintenance patients over a 12-month period. Patients included in the study were those who regularly attended a 3- month maintenance program and had previously received periodontal therapy at the University of
Nebraska Medical Center College of Dentistry. Every precaution was taken to eliminate any bias by compartmentalizing the various aspects of this study protocol as follows: masked examiners
(RR, AK, JP) collected all data and patients did not know which group they were in. The clinicians (JG, EJ) performed all papilla-reflections, scaling and root planing, and then randomized/performed placement of either EMD or saline.
The primary outcome measured in this study was change in clinical attachment level with secondary outcomes including change in alveolar bone height and GCF inflammatory biomarkers.
The current study demonstrated no significant differences between groups at baseline in regards to age (p = 0.41), sex (p = 0.40), and smoking status (p = 0.33). The current study also demonstrated inflamed pockets treated with PR/RP + EMD or PR/RP + saline showed BOP reduction of the treatment-teeth 12-months post therapy in a periodontal maintenance population
(Table 2). Baseline BOP means of the experimental-teeth between the groups were not significantly different (p = 0.57) as was 12-month treatment-teeth BOP means (p = 0.53).
Experimental-teeth encompassed the test tooth along with the adjacent tooth and included the average BOP of 12 sites (6 per tooth). To assess if the change in BOP was significant between groups, initial BOP, and worst site were adjusted for in Table 3. The mean change in BOP from baseline to 12-months post-therapy for EMD showed an 8% reduction whereas the BOP reduction for the saline group was 14%. The reduction in BOP was not significant for the EMD group (p = 0.0965) but it was significant for the saline group (p = 0.006). Although the saline
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group showed a statistically significant reduction, the difference between the groups was not significant (p = 0.381). Treatment site was also evaluated. This included only the site where either the EMD or saline was placed interproximally, therefore, only 1 BOP measurement was taken into consideration. Table 4 shows the presence or absence of BOP in each group at the treatment-site at specific time points (baseline, 6-months, and 12-months). No significant difference between the groups, in regards to the number of treatment site/s with BOP, was found
(Table 4). The proportion of patients with BOP present at the treatment site at baseline versus
BOP present at 6-months and 12-months was evaluated (Figures 10 and 11). Overall, the proportion of patients with BOP present at 6-months was not significantly different from the proportion of patients with BOP present at baseline for the EMD and saline group (p = 0.13 and
0.74, respectively). On the other hand, the proportion of patients with BOP present at 12-months was significantly different from the proportion of patients with BOP present at baseline. This was significant for both the EMD group (p = 0.03) and the saline group (p = 0.01). As a result, BOP was reduced after 12-months regardless of the treatment. When looking at the risk of a poor BOP outcome at the treatment site, the EMD group still did not show any improvement over saline in regards to BOP at 6-months (p = 0.75) and 12-months (p = 0.16). The only significance found was patients who had BOP present at baseline had a higher AOR of a poor BOP outcome at 12- months (Table 6). This information can go hand in hand with a study by Joss et al. (1994) who stated that patients with a higher mean BOP have a higher risk for further attachment loss at single sites. Wilson et al. (2008) confirmed the presence of BOP indicates histologic inflammation or presence of bacteria. Therefore, the presence of BOP could be important clinically because it could aid as a prognostic tool for future BOP and periodontal attachment loss. Disagreeing with this statement, Chavez et al. (1990) stated that the presence of BOP is not necessarily a reliable predictor of disease. One reason for this includes the variability in pressure amongst clinicians when probing.
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Periodontal studies that are clinically-based tend to evaluate PD, CAL, and recession in addition to BOP. One such study compared EMD + MIST (similar to our papilla reflection) to saline + MIST (Ribeiro et al. 2011). Unlike the current study, this study only investigated their patients for 6 months and did not use a Perioscope to aid in calculus/biofilm removal.
Nevertheless, the results showed that EMD + MIST was similar to MIST alone in all measured clinical parameters at 3 and 6 month post-operative appointments, similar to the clinical findings in our study. They assessed clinical inflammation by looking at full mouth bleeding scores, not
BOP specific to the treatment site. No difference in full mouth bleeding scores was noted between the two groups in the Ribeiro et al. study. Additionally, a randomized clinical split mouth trial by
Jentsch et al. (2018) investigated the effects of flapless EMD administration in combination with
SRP in periodontal maintenance patients. Similarly to our study, patients were followed for 1 year and received PMT every three months. It was found that the adjunctive use of EMD in sites with residual probing depths ≥ 5 and ≤ 9 mm after initial non-surgical therapy resulted in enhanced treatment outcomes (PD, BOP) compared to SRP alone. Another 12 month study assessing a microsurgical flap with application of EMD (Fickl et al. 2009) found this combination to be superior to open flap debridement in regards to PD reduction, CAL gain, and radiographic bone fill. Finally, Cortellini (2007) treated forty isolated intra-bony defects utilizing a microscope in conjunction with the MIST and EMD application. Comparison to a control site was not implemented. It was found, after 1 year, that the MIST, in combination with EMD, resulted in improved clinical attachment gain (4.9 ± 1.7 mm, p < 0.0001) and residual pocket depths that were only 3 ± 0.6 mm. The clinical effects of EMD have been evaluated a number of times with varying results in regards to BOP outcome. The effect this has on inflammatory biomarkers, specifically IL-1β and PGE2, is more limited.
IL-1β is a pro-inflammatory cytokine released by various cells, including monocytes, neutrophils, and fibroblasts, in the periodontium. In the current study, when evaluating baseline
IL-1β, no significant differences were found between the groups (PR/RP + EMD= 141.10
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pg/sample, PR/RP + saline= 109.38 pg/sample; p= 0.34). In addition, no significant differences in
IL-1β were found between the groups at any of the subsequent GCF collection time points (2- weeks, 6-, and 12-months) either. Furthermore, when evaluating the change in IL-1β from baseline to 2-week, 6-, and 12-month post-therapy visits, a significant reduction in IL-1β in the
PR/RP + EMD group (mean change = -40.15 pg; p = 0.05) compared to the PR/RP + saline group
(mean change = -23.64 pg, p = 0.19) at 2-weeks post-therapy and a trend for reduction in the
EMD group at 12-months post-therapy (mean change = -32.85 pg; p = 0.07) was seen. This is contrary to a previous study by Giannopoulou et al. (2006), who found that EMD did not affect the expression of inflammatory mediators, including IL-1β, after 12 months. An in-vitro study, in addition, found EMD did not promote IL-1β secretion as the monocyte activity increased
(Khedmat et al. 2010).
We would assume that the pro-inflammatory effects of IL-1β would result in a higher chance of poor BOP outcome; this however, was not the case (Table 10). Initial IL-1β and the change in IL-1β over time did not have an effect on the BOP outcome after adjusting for baseline treatment site BOP, group, and worst site. Like it has been previously stated, the only time an increased risk of a poor BOP outcome was significant was if the patient had BOP present at baseline (Table 6).
PGE2 is often associated with inflammation, correlating with BOP in periodontal disease.
Periodontitis is a chronic inflammatory disease resulting in alveolar bone destruction but inflammation shows beneficial effects in the initial stages of wound healing (angiogenesis). EMD has also been shown to play a significant role in wound healing allowing for soft tissue regeneration and angiogenesis (Miron et al. 2014). In our sample, the PGE2 production was often below the limits of ELISA detection, which is comparable to a study by Sato et al. (2008).
Nonetheless, when monocytes were exposed to EMD, Sato et al. showed a significant increase in the production of PGE2 compared to monocytes not treated with EMD. Similarly, Takayanagi et al. (2006) was able to find that COX2 mRNA levels were increased following EMD treatment.
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This is important because the COX2 pathway leads to prostaglandin production. Both studies found that EMD increased PGE2, whether directly or indirectly. In this study, when PGE2 was detectable, patients in either group who had PGE2 present at baseline had a 4.6x adjusted odds of a poor BOP outcome compared to those patients who did not have PGE2 present (not statistically significant, p = 0.066). This worsened BOP outcome could be beneficial in regards to providing a matrix for regenerative cells to populate, or could be problematic, leading to periodontal destruction. The role of PGE2 in regeneration warrants further study.
Although reports evaluating EMD’s role on IL-1β and PGE2 expression are limited, other biomarkers have been studied. Myhre et al. (2006) found overall that EMD limits the release of
TNF-훼 and IL-8, while it had no effect on IL-10. This study was completed by collecting blood from healthy donors and suggests that EMD may be anti-inflammatory. Ribeiro et al. (2011) did collect GCF samples but these were assayed for transforming growth factor - β1, osteoprotegerin, and osteocalcin, of which none showed any difference between the test group (EMD) and control group. Another study evaluated EMD’s potential on matrix metalloproteinase-8 expression and found it down-regulated its release from polymorphonuclear leukocytes when stimulated by bacteria (Karima and Van Dyke 2012). By controlling the inflammation that collagenases induce,
EMD could promote early healing. It also needs to be noted that EMD can be degraded by the proteases in the GCF of periodontitis patients (Laaksonen et al. 2010), ultimately reducing the regenerative potential.
Plaque control was poor in the recruited maintenance patients. Plaque was present at most treated sites at both baseline and post-therapy exams, resulting in minimal improvement in PI.
The presence of plaque was expected at the 2- and 6-week time points because EMD’s protocol specifies no brushing, flossing, etc. for this time frame. Also, a majority of our study sites were in posterior sextants which have been shown to be difficult for patients to clean effectively
(Cumming and Loe 1972, Prasad et al. 2011). This can help explain the presence of BOP seen in the treatment-teeth and at the specific treatment sites after 12-months of PMT. However, even
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with plaque present, IL-1β was significantly reduced after 2-weeks in the EMD group (Table 8).
Improved PI may have resulted in better clinical results, like BOP reduction, and a reduction in the inflammatory biomarkers present for both groups because it’s known that EMD is less effective in sites with periodontal bacteria present (Inaba et al. 2004).
The use of EMD is comparable to other adjunctive therapies (minocycline, doxycycline, povidone-iodine, and lasers) and guided tissue regeneration in its benefits and limitations. The purpose of the adjunctive therapies is to reduce subgingival bacterial flora and clinical signs of periodontitis where the purpose of guided tissue regeneration is a gain in CAL. A systematic review from the AAP Regeneration Workshop concludes that the use of biologics, like EMD,
“generally increase bone fill and improve CAL and reduce PD compared with open flap debridement procedures in the treatment of intrabony defects.” This review states that these improvements are comparable to GTR (Kao et al. 2015). Unfortunately, little is published in regards to its effect on inflammatory biomarkers like IL-1β and PGE2. Ultimately, the use of
PR/RP + EMD in practice to treat non-resolving periodontal pockets in maintenance patients is safe but showed no additional benefits to PR/RP + saline. The adjunctive use of EMD utilizing a strict protocol ensures safe outcomes and does not cause any harm or immunogenic potential to the patient, as shown in the current study and others (Zetterstrom et al. 2005). The PR/RP completed for each patient is also considered safe, as very few post-operative complications were mentioned. However, because similar clinical and cytokine measurement outcomes were noted for both the test group and control group, a papilla-reflection to aid in SRP may be all that is needed to see positive results, yet EMD-induced inflammation reduction (IL-1β) may be a further positive outcome.
There are several limitations in this study that should be addressed. First, only two inflammatory molecules (IL-1β and PGE2) were measured, both pro-inflammatory, representing only a small fraction of all biomarkers in an inflamed pocket. Furthermore, only a small amount of PGE2 was found in the samples taken, with a majority of sites not detectable. Although we are
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unsure as to why the levels were so low, it could have been due to error while preparing for the assay. Two, to allow for healing following papilla-reflection, we cannot evaluate BOP at 2-weeks post-therapy. This is important to note because a significant change in IL-1β took place in the
EMD group at the 2-week GCF collection. Third, the poor oral hygiene/lack of brushing of participants likely impacted the BOP and pro-inflammatory biomarker results seen. Fourth, a
Perioscope was used. Were additional benefits noted in both groups because of the visualization added by the Perioscope? Like the additional use of the Perioscope, did the EDTA application supplement or inhibit the results seen? EDTA is a chelating agent that binds with calcium and is not commonly applied following standard non-surgical or regenerative therapy. It is a root conditioner which allows for the removal of the smear layer created after scaling and root planing or exposure of the root to the external environment. EDTA works by demineralizing the acellular cementum and exposing the collagen present in the dentinal tubules. Some studies do not support the use of a root conditioner prior to EMD use (Guzman-Martinez et al. 2009). However, a study by Saito et al. (2014) found that EMD in combination with 24% EDTA showed an additional benefit in terms of population of cells expressing alkaline phosphatase activity on the root surface compared to EMD alone. Expression of alkaline phosphatase is characteristic of periodontal ligament cells and is thought to be involved in bone calcification but further studies are needed to better confirm this. Finally, the history of each study site was not investigated. Although all sites were inflamed, some may have been experiencing active attachment loss while others may have been periodontally stable. The activity, or lack thereof, could influence the response and inflammatory condition of each pocket because we know active periodontitis can influence the effect of EMD.
Additional studies could look more closely at the way EMD affects cytokines and other inflammatory cells rather than clinical outcomes. Although clinical outcomes are important, the role of the immune response is essential. If key inflammatory cells are present, therapies can be introduced to help control the response seen, ultimately improving the clinical outcomes. Also, if
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EMD aids in early healing (decreased IL-1β at 2-weeks), with what other therapies could this product be used? Does this early decrease in IL-1β have long term effects?
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CHAPTER 11: CONCLUSIONS
Both papilla reflection with root planing + EMD and papilla reflection with root planing
+ saline resulted in an improvement in clinical outcomes (BOP) in non-resolving periodontal pockets in maintenance patients. The addition of EMD to papilla reflection with root planing does significantly decrease IL-1β in the short-term compared to papilla reflection with root planing alone in periodontal maintenance patients.
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APPENDIX A: RAW DATA- PATIENT CHARACTERISTICS
Patient Group Age Gender Smoking N1 2 59 1 1 N2 1 67 2 0 N3 1 74 1 0 N4 2 70 2 0 N5 1 67 2 0 N6 1 76 1 1 N7 2 55 1 0 N8 2 63 2 0 N9 2 80 2 0 N10 1 69 1 0 N11 1 62 1 0 N12 2 67 1 0 N13 2 76 2 0 N14 1 58 2 1 N15 2 83 2 0 N16 1 68 2 0 N17 2 47 1 0 N18 2 44 1 1 N19 2 65 1 1 N20 1 67 1 0 N21 1 59 2 0 N22 1 65 1 0 N23 1 80 2 0 N24 2 62 1 1 N25 1 65 2 0 N26 1 62 1 0 N27 1 67 2 1 N28 2 75 2 0 N29 2 76 1 0 N30 1 62 2 0 N31 2 68 1 0 N32 2 69 1 1 N33 2 66 1 1 N34 2 52 2 0 N35 2 60 1 0 N36 1 69 2 0 N37 2 79 1 0 N38 1 69 1 0 N39 2 65 2 0
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N40 2 73 1 0 N41 2 67 2 0 N42 2 73 2 0 N43 1 68 2 0 N44 2 78 1 0 N45 2 48 2 0 N46 1 58 1 0 N47 1 61 2 0 N48 1 75 1 0 N49 1 68 1 0 N50 1 70 2 0 1= EMD 1= male 0= non- smoker 2= Saline 2= female 1 = smoker
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APPENDIX B: RAW CLINICAL DATA- BOP
Pt Grp Baseline Baseline 6 month 6 month 12 month 12 month treatmen treatmen treatmen treatmen treatmen treatmen t site t site t site t site t site t site Buccal Lingual Buccal Lingual Buccal Lingual BOP BOP BOP BOP BOP BOP
N1 2 0 1 1 0 1 1 N2 1 0 1 1 0 1 1 N3 1 0 0 1 1 1 1 N4 2 1 1 0 0 0 0 N5 1 1 1 0 0 1 1 N6 1 0 0 0 0 0 0 N7 2 0 0 0 1 1 0 N8 2 0 0 0 0 0 0 N9 2 1 1 1 1 1 1 N10 1 1 1 1 0 1 1 N11 1 1 1 1 1 0 1 N12 2 1 1 0 1 0 0 N13 2 0 1 0 1 0 0 N14 1 1 1 1 0 0 0 N15 2 0 1 0 1 N16 1 0 1 0 1 0 1 N17 2 1 1 1 1 0 1 N18 2 1 1 0 1 0 0 N19 2 0 1 0 1 0 0 N20 1 1 0 1 1 0 0 N21 1 1 1 1 0 0 1 N22 1 0 1 1 1 1 1 N23 1 0 0 0 0 0 0 N24 2 0 1 0 0 1 1 N25 1 0 1 1 1 1 1 N26 1 1 1 0 0 0 0 N27 1 1 0 0 0 0 0 N28 2 0 1 1 0 1 1 N29 2 1 1 1 0 0 0 N30 1 0 1 0 0 0 0 N31 2 1 1 0 0 1 0 N32 2 1 1 0 0 0 0 N33 2 0 0 0 0 1 0 N34 2 0 0 1 1 0 0
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N35 2 1 1 1 1 N36 1 0 1 1 1 1 1 N37 2 1 1 0 1 0 0 N38 1 1 1 1 1 1 1 N39 2 0 1 0 1 0 1 N40 2 1 1 1 1 0 1 N41 2 1 1 0 1 0 1 N42 2 1 1 0 1 1 1 N43 1 1 1 0 1 0 1 N44 2 1 1 N45 2 0 0 0 1 1 0 N46 1 1 0 0 0 0 0 N47 1 1 1 1 1 0 0 N48 1 0 1 0 1 0 1 N49 1 1 1 0 0 0 1 N50 1 0 0 1 1 0 0
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APPENDIX C: RAW CLINICAL DATA- IL-1β
Patient Group IL-I β IL-I β 2 IL-I β 6 IL-I β initial week mo 12 mo
N1 2 161.277 303.295 200.983 367.08 N2 1 320.199 134.938 87.664 142.702 N3 1 376.22 97.001 179.263 157.15 N4 2 32.626 8.842 22.405 19.162 N5 1 171.4 18.08 16.606 169.435 N6 1 34.887 185.651 17.884 35.083 N7 2 120.883 126.486 192.531 163.341 N8 2 8.744 146.437 31.152 37.147 N9 2 26.434 12.577 132.579 129.433 N10 1 214.055 48.056 237.053 170.024 N11 1 108.563 5.057 32.405 32.513 N12 2 439.235 67.161 87.535 303.479 N13 2 78.927 80.78 42.973 6.038 N14 1 49.074 71.519 45.588 49.728 N15 2 43.627 146.696 238.652 N16 1 87.971 80.998 178.293 20.747 N17 2 117.606 21.182 41.339 18.35 N18 2 15.408 94.508 101.481 86.336 N19 2 11.703 110.088 152.362 211.85 N20 1 100.5 87.535 49.074 137.653 N21 1 0 13.288 53.793 17.119 N22 1 99.909 197.615 75.141 76.372 N23 1 4.119 72.13 51.057 0.562 N24 2 154.51 16.572 5.488 15.204 N25 1 48.32 96.899 70.078 100.594 N26 1 23.825 69.667 21.225 0 N27 1 251.531 81.983 170.794 307.637 N28 2 57.993 28.018 61.59 86.442 N29 2 73.035 93.745 75.324 92.764 N30 1 149.411 23.966 60.141 100.769 N31 2 226.77 87.857 197.162 86.076 N32 2 0 135.608 246.249 143.4 N33 2 228.106 127.372 254.486 177.349 N34 2 24.634 70.27 253.707 60.141
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N35 2 120.137 76.392 158.649 N36 1 36.878 34.54 154.754 136.276 N37 2 70.493 95.203 289.437 65.929 N38 1 245.687 46.707 270.468 252.066 N39 2 267.436 331.532 376.389 179.605 N40 2 143.636 59.778 198.426 158.483 N41 2 279.042 233.663 282.388 26.422 N42 2 49.426 31.546 79.33 23.077 N43 1 1.223 93.551 74.102 34.055 N44 2 86.441 214.424 N45 2 5.719 0 129.834 56.641 N46 1 78.599 122.41 24.854 6.138 N47 1 323.899 85.918 166.221 118.018 N48 1 14.83 110.422 235.008 198.166 N49 1 285.653 200.835 361.12 254.152 N50 1 359.562 402.193 370.025 219.424
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APPENDIX D: RAW CLINICAL DATA- PGE2
Patient Group PGE2 PGE2 2 PGE2 6 PGE2 initial week mo 60 12 mo 60 60 60
N1 2 4.753 397.338 0 0 N2 1 427.01 0 0 95.291 N3 1 135.615 0 0 0 N4 2 0 0 0 0 N5 1 0 0 0 0 N6 1 0 277.889 0 0 N7 2 0 0 35.947 0 N8 2 0 370.709 0 0 N9 2 52.685 0 0 0 N10 1 32.369 0 0 81.82 N11 1 143.987 0 0 0 N12 2 78.288 0 0 110.784 N13 2 0 140.454 0 0 N14 1 225.933 273.265 8.35 0 N15 2 0 148.932 21.733 N16 1 0 0 50.03 119.261 N17 2 6.938 0 0 0 N18 2 0 151.757 213.924 15.415 N19 2 0 31.445 0 0 N20 1 0 462.784 0 0 N21 1 0 62.761 0 0 N22 1 108.989 40.393 0 0 N23 1 0 343.485 0 11.686 N24 2 0 0 0 0 N25 1 0 32.936 0 0 N26 1 0 81.774 0 0 N27 1 98.178 74.691 0 50.086 N28 2 0 0 93.356 0 N29 2 0 29.689 0 0 N30 1 0 0 0 0 N31 2 0 0 0 0 N32 2 0 0 0 0 N33 2 0 0 0 0 N34 2 0 3.158 0 0
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N35 2 0 43.32 0 N36 1 0 426.502 0 0 N37 2 0 0 0 37.294 N38 1 70.731 0 0 302.34 N39 2 0 110.691 0 0 N40 2 0 0 0 324.359 N41 2 0 299.893 47.896 0 N42 2 0 23.43 0 0 N43 1 0 193.875 0 0 N44 2 0 495.62 0 N45 2 0 0 0 0 N46 1 0 0 0 0 N47 1 0 0 0 0 N48 1 0 206.117 53.468 0 N49 1 0 203.967 0 0 N50 1 0 487.074 18.301 0
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APPENDIX E: CONSENT FORM
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