PERIODONTAL INTRABONY DEFECT REGENERATION: ACCELL CONNEXUS ® VERSUS DEMINERALIZED FREEZE-DRIED BONE ALLOGRAFT

BY

RACHEL V DULEBOHN LCDR, DC, USN

A thesis submitted to the Faculty of the Periodontics Graduate Program

Naval Postgraduate Dental School Uniformed Services University of the Health Sciences

in partial fulfillment of the requirements for the degree of Master of Science in Oral Biology

June 2017

Naval Postgraduate Dental School Uniformed Services University of the Health Sciences

Bethesda, Maryland

CERTIFICATE OF APPROVAL

MASTER'S THESIS

This is to certify that the Master's thesis of

Rachel V Dulebohn

has been approved by the Examining Committee for the thesis requirement for the Master of Science degree in Oral Biology at the June 2017 graduation.

Research Committee: eyWessel, DDS, MS

Associate Professor, Periodontics ResearchCommittee Chair

~~~~ Dr. Matthew Miller, DDS, MS Program Director, Periodontics Resear. h Committee Member

D . an Roman, DDS, MS Chairman, Periodontics Research Committee Member

The author hereby certifies that the use of any copyrighted material in the thesis manuscript titled:

"PERIODONTAL INTRABONY DEFECT REGENERATION: ACCELL CONNEXUS ®VERSUS DEMINERALIZED FREEZE-DRIED BONE ALLOGRAFT"

is appropriately acknowledged and, beyond brief excerpts, is with the permission of the copyright owner.

RESIDENT SIGNATURE

~ Rachel V Dulebohn Periodontics Graduate Program Naval Postgraduate Dental School 09 JUNE 2017

NAVAL POSTGRADUATE DENTAL SCHOOL RACHEL V DULEBOHN

2017

This thesis may not be re-printed without the expressed written permission of the author.

Distribution  Statement    

Distribution  A:  Public  Release.      The  views  presented  here  are  those  of  the  author  and  are  not  to  be  construed  as  official  or  reflecting  the  views  of  the  Uniformed  Services  University  of  the  Health  Sciences,  the  Department  of  Defense  or  the  U.S.  Government.  

iv

ABSTRACT

PERIODONTAL INTRABONY DEFECT REGENERATION: ACCELL CONNEXUS ® VERSUS

DEMINERALIZED FREEZE-DRIED BONE ALLOGRAFT RACHEL V DULEBOHN

M.S, PERIODONTICS, 2017

Directed by: JEFFREY R WESSEL, ASSOCIATE PROFESSOR Naval Postgraduate Dental School Introduction: Guided tissue regeneration with demineralized freeze-dried bone allograft

(DFDBA) and a barrier membrane predictably improves clinical parameters in intrabony

defects. Accell Connexus®(AC) is a grafting material composed of DFDBA blended with

DFDBA processed for rapid growth factor release and a polymer putty.

Purpose: This randomized, double-blinded study compared AC to DFDBA for periodontal

regeneration in intrabony defects when used with a resorbable collagen membrane.

Methods: 25 subjects were enrolled and randomly assigned to receive AC or DFDBA for

treatment of an intrabony defect. Probing depth and clinical attachment level changes

were measured using custom stents at baseline, 6 and 12 months.

Results: 20 subjects completed the study. Both AC (n=10) and DFDBA (n=10) groups

improved significantly after treatment with no between-group differences for probing

depth reduction or clinical attachment gain.

Conclusion: Periodontal regeneration with Accell Connexus® is comparable to DFDBA for

probing depth reduction and clinical attachment level gain.

v

TABLE OF CONTENTS

LIST OF TABLES………………….……………………………………………………………………………….. vi LIST OF FIGURES………………...……………………………………………………………………………….. vii CHAPTER 1: LITERATURE REVIEW…………………………………………...…………………………. 1 CHAPTER 2: METHODOLOGY………………………………………………………………………………. 10 CHAPTER 3: RESULTS………………………………………………………………………………………….. 13 CHAPTER 4: DISCUSSION…….………………………………………………………………………………. 16 CHAPTER 5: CONCLUSION…..……………………………………………………………………………….. 20 REFERENCES……………………………………………………………………………………………………….. 21 APPENDICES……………………….……………………………………………………………………………….. 32

vi

LIST OF TABLES

Table Page

1. Baseline Demographic Data .................................................................................. 24

2. Surgical Data ............................................................................................................... 25

3. Probing Depth............................................................................................................. 26

4. Clinical Attachment Level ..................................................................................... 27

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LIST OF FIGURES

Figure Page

1. Comprehensive Inclusion and Exclusion Criteria Checklist ..................... 28

2. Probing Depth Graph ............................................................................................... 29

3. Clinical Attachment Level Graph ......................................................................... 30

4. Representative radiographs ................................................................................. 31

1

CHAPTER 1

LITERATURE REVIEW

Periodontal disease is characterized by the degeneration of the supporting

structures of the dentition, ultimately resulting in loss of teeth and occlusal function.

According to the latest NHANES data from 2010, which is considered the most accurate

estimate to date, 47% of the US population has periodontitis, with 30% having moderate

and 8.5% having severe periodontitis (Eke, 2012). The prevalence is even higher for

individuals over 65 with rates increasing to 53% of the public with moderate and 11% with

severe periodontitis. Periodontal disease represents a major health issue, not only due to

its intrinsic symptoms and complications but also because of potential substantial systemic

impacts. Periodontitis has been linked to diabetes, cardiovascular disease, rheumatoid

arthritis and other inflammatory diseases (Friedewald 2009, Iacopino 2001, Havemose-

Poulson 2006). Recognizing the importance of diagnosing and treating periodontitis is

paramount to promoting oral and systemic health.

Periodontitis is an inflammatory disease of the bone and periodontal ligament

supporting the teeth. Pathogenic bacteria invade the periodontal pocket and elicit a host

immune response. The resulting inflammatory response originates in the gingival pocket

as gingivitis. As the tissue destruction advances, it progressively damages the adjacent

periodontal ligament and alveolar bone. Chronic periodontitis is the most common form of

periodontitis. It typically follows a gradual, progressive course, sometimes with periods of

increased destruction.

One of the fundamental goals of periodontal therapy is to arrest the disease and

regenerate tissues that have been lost to disease. Bone destruction causes various defects

2

along with gingival recession and pocketing. In the history of periodontology, regeneration

is a fairly new treatment paradigm. Regeneration is defined as the “reproduction or

reconstitution of a lost or injured part”, with guided tissue regeneration (GTR) referring

specifically to “regeneration of the periodontal attachment apparatus” (AAP Glossary of

Periodontal Terms). This includes regeneration of not only lost bone, but also periodontal

ligament and cementum. Regeneration can be accomplished by osseous grafting or guided

tissue regeneration. Bowers, Freeman and other pioneers began experimenting with

grafting procedures in the 1960’s and 1970’s. Prior to that point, osseous resective surgery

was the principle treatment for management of periodontal disease. Resective surgery

consists of removing bone to eliminate defects and create bone contours that promote

healthy sulcus depth. Even though the average amount of bony support taken away is only

0.6 mm, resective surgery can result in significant clinical attachment loss and increased

tooth mobility (Selipsky, 1976). In the late 1980’s, Dr. Bowers published a series of studies

showing results of successful periodontal regeneration that were supported by histological

findings. He demonstrated that new cementum, periodontal ligament (PDL) and bone

formed on diseased root surfaces (Bowers I). Since then GTR procedures have progressed

and improved.

Guided tissue regeneration requires some type of barrier material and may

additionally utilize graft materials, root surface modification and biologic mediators. An

exclusive barrier is central to the concept of regeneration and performs multiple functions.

Most importantly, a barrier prevents invasion of the gingival epithelium and chorium onto

the root surface before PDL and cementum can form. Additionally, it maintains a space for

bone, cementum and PDL formation, stabilizes the clot, and in cases where a graft material

3

is used, contains and stabilizes that as well. Early GTR techniques only used a membrane

and were successful with average clinical attachment level gains of 4.7 mm in intrabony

defects that were initially 6.1 mm deep (Cortellini 1993). A small amount of crestal bone,

0.4 mm on average but up to 2 mm, is resorbed with remodeling, which contributes to the

alteration of the bony defect. PDL and bone cells are both sources for newly forming PDL,

cementum and bone (Wang 1998). These tissues form considerably slower than

epithelium, which begins migrating within two days, can be fully re-established after two

weeks and is capable of migrating under a replaced gingival flap. Bone does not begin to

proliferate until after four weeks following an initial period of resorption, and cementum

takes two months to be detected (Wilderman 1970). This major variance in tissue

formation rates necessitates membrane usage for regeneration. Without a membrane,

epithelial tissue rapidly populates all exposed surfaces and prevents bone, cementum and

PDL from forming.

Barrier membranes can be either non-resorbable or resorbable. Non-resorbable

membranes were the first membranes used for GTR. While they produce excellent

attachment gain, non-resorbable membranes require a second surgical procedure for

membrane removal and have a significant potential for exposure, which can potentially

compromise the outcome. If the membrane margins are covered with tissue, the

membrane can still provide all of its necessary functions. Examples of non-resorbable

membranes are porous expanded polytetrafluoroethylene (ePTFE), which is no longer

routinely used, and nonporous or dense polytetrafluoroethylene (PTFE). In cases where

space maintenance is more critical, for example where suprabony regeneration is being

4

attempted, titanium-reinforced PTFE membranes can be used to provide maximum stable

space maintenance (Rakhmatia 2013).

Resorbable membranes are desirable because they eliminate the need for a second

surgery for membrane removal. They also tend to be less stiff and are less likely to

undergo membrane exposure. However, if they do become exposed, they are rapidly

metabolized by salivary enzymes, P. gingivalis and other periodontal pathogens in the oral

environment and no longer provide protection to the graft and clot or exclude epithelium,

potentially resulting in significantly reduced GTR results (Sela 2009). While reduced

stiffness of resorbable membranes is beneficial for reducing membrane exposures, it also

renders them somewhat less effective for space maintenance if used without a graft.

Examples of resorbable membranes include collagen, polyglactin, polylactic acid and

calcium sulfate. Collagen membranes can also be crosslinked to increase resistance to

enzymatic degradation. However, crosslinking agents such as glutaraldehyde can cause a

significant, undesirable inflammatory reaction, as well as change the surface structure

making it more difficult for cells to attach (Grover 2012). Bio-Gide® is a porcine non-

crosslinked collagen resorbable bilayer membrane. It is composed of primarily Type I and

III collagen, similar to what is found in human gingival tissue. The outer layer is smooth

and prevents soft tissue invasion, whereas the inner layer is porous and fibrous to promote

cell migration and provide a framework. It is biocompatible and does not elicit any

detectable immune response (Schlegel 1997).

When used for regenerative procedures, Chen found that GTR results with Bio-

Gide® alone or combined with DFDBA were not significantly different (1995). However,

the mean bone fill was lower than other studies, 38-41%, and did not report intrabony

5

defect depth or morphology. More recently, a randomized controlled clinical trial reported

CAL gain with the use of membrane alone was 2.5 mm compared to 3.5 mm with use of

bone graft and membrane in intrabony defects that were initially ≥3 mm(Kher 2013).

Resorbable membranes have been shown to perform comparably to non-resorbable

membranes, particularly when used in conjunction with a graft material and do not

experience complications like early membrane exposures. Caffesse found that results for

GTR in furcations and intrabony defects that were randomly assigned e-PTFE or a

polylactic and polyglycolic membrane had equivalent results (Caffesse 1997). A meta-

analysis of 16 studies found that CAL gain was slightly higher but not statistically

significant different with resorbable membranes compared to non-resorbable membranes.

For intrabony defects of 5-7 mm, CAL gain averaged 3.7 mm with non-resorbable

membranes and 4.5 mm with resorbable membranes (Laurell 1998).

Graft materials are not necessary but may improve outcome and are used routinely

for GTR procedures in intrabony defects. A systematic review of different combinations of

graft materials and membranes found that there may be some added benefit of using a graft

in addition to a membrane (Sculean 2008). Graft materials include alloplasts, autografts,

allografts and xenografts. Alloplasts are synthetic materials such as hydroxyapatite and

methyl methacrylate, which are non-resorbable, and tricalcium phosphate and bioactive

glass, which are somewhat resorbable. Alloplasts have mixed outcomes when used for

GTR. However, histologic results frequently demonstrate alloplastic materials may incite a

reactive response and frequently become sequestered within a fibrous connective tissue

capsule.

6

Allografts are tissues originating from donors of the same species. These typically

come from cadavers and can be either calcified or decalcified. Decalcified freeze-dried

bone allograft (DFDBA) was histologically found to induce more new bone formation than

freeze-dried bone allograft (Bowers 1989, Reynolds 2003). DFDBA is the most commonly

used grafting material for GTR and is considered to be significantly more osteoinductive

than freeze-dried bone allograft (FDBA) (Zhang 1997). DFDBA showed increased vital

bone percentage of 38.42% compared to 24.63% with FDBA when used for socket

preservation (Wood, 2012).

Xenografts are tissue grafts derived from a different species. Bovine, porcine and

equine tissues have all been used for periodontal procedures. Xenografts are more heavily

processed than allografts for removal of all proteins and cells to prevent any immune

response, and therefore provide only a scaffold for native bone formation. Xenografts also

typically take longer to be metabolized by osteoclasts and replaced by native bone. A

histological analysis of bovine bone grafts in GTR treatments found that at 6 months, the

graft particles were still present and surrounded by native bone but the graft appeared to

be successful (Sculean 2004).

Chemical root surface modifiers do not seem to have much effect on GTR

procedures. In theory, these treatments improve outcomes by removal of the smear layer,

bacteria or matrix metalloproteinases (MMPs) or by exposure of collagen on the root

surface. However, application of ethylenediaminetetraacetic acid (EDTA), citric acid or

tetracycline to roots during GTR procedures demonstrate no significant increases in clinical

attachment gain (Mariotti 2003). Nevertheless, many providers still use them and claim

anecdotal efficacy.

7

On the other hand, biologic mediators, particularly growth factors, have been shown

in the literature to have more efficacy. Enamel matrix derivative (EMD), platelet-rich

plasma (PRP), and bone morphogenic proteins (BMPs) are some mediators that have been

used for GTR. EMD is a mixture of proteins, primarily amelogenin, that induce formation of

periodontal attachment structures during tooth development via induction of BMPs and

other intermediates which in turn stimulate PDL stem cells and osteoblasts. Emdogain®,

an extract of fetal porcine EMD, was found in a meta-analysis to significantly increase

attachment level gain by 1.08 mm and reduce pocket depths by 0.88 mm at one year post-

surgery compared to open flap debridement alone (Esposito 2009). BMPs are powerful

growth factors that stimulate bone growth by inducing mesenchymal stem cells to migrate

and differentiate into bone forming cells, but few studies have been conducted in humans

regarding periodontal regeneration. Because of their potency, BMPs also tend to cause

complications like excessive post-operative swelling, ectopic bone growth, tooth ankylosis

and root resorption. The latter effects are thought to be due the cytotoxic influences of

BMP on PDL cells that occur at higher than physiological doses and ultimately cause

apoptosis (Muthukuru 2013). BMPs have been used in soft tissue augmentation

procedures and significantly improved Miller class I and II recession defect coverage from

70% to 90% when used in conjunction with acellular dermal matrix allograft versus the

graft alone (Shepherd 2009). In contrast, BMPs have been shown to provide no increase in

regeneration when added to bone graft and membrane (Trombelli 2008). Regarding PRP,

usage appears to speed healing and reduce post-operative pain but these benefits might

still not justify the added cost and procedure of venipuncture for harvest. Platelet-derived

growth factor (PDGF) theoretically increases periodontal regeneration by stimulating

8

osteoblasts, fibroblasts and angiogenesis, increasing immune response and imparting

antimicrobial properties. However, meta-analysis of studies using PDGF found no

additional benefit when used for GTR or recession treatments (Massimo 2011).

Protocols for GTR have improved significantly over the years and continue to

evolve. There is always potential for further improvement in protocols, grafting materials,

membranes or surface modification techniques to make regeneration procedures more

successful, especially in defects that are less predictable. New grafting material blends

have been developed in an effort to increase the osteoinductive qualities of the product and

to improve handling characteristics. Much of the research and development was initially

done for orthopedic applications such as spinal fusions, treatment of non-union fractures

and to fill large intrabony voids (Pieske 2009, Schizas 2008). Some of these products have

since been marketed for other uses such as periodontal grafting.

Accell Connexus® is a bone matrix putty containing DFDBA made from both cortical

and cancellous bone. The final product is composed of an equal mixture of traditional

DFDBA and DFDBA that has been further processed to increase the availability of BMP-2.

BMP-2 release is increased fivefold compared to unprocessed DFDBA, as shown in vitro by

Khaliq 2007. The manufacturer claims that bone formation is greater with this product in

a sheep model tibia defect compared to DFDBA in the putty medium, but does not compare

it to DFDBA alone (Kay 2004). The only clinical study using Accell Connexus® for

periodontal applications was a histology study on extraction site preservation. Mandelaris

found that 12 core samples taken at 4 months post-socket preservation had an average

new vital bone formation of 53%, which was primarily woven bone, 9% residual graft

material and 38% connective tissue (2015).

9

Accell Connexus® also has excellent handling properties due to the proprietary

reverse phase carrier putty that solidifies at body temperature. The putty consists

primarily of poloxamer 407, an amphiphilic copolymer of polypropylene glycol and

polyethylene glycol. It is used for numerous applications including improving solubility of

hydrophobic drugs, creating time-release medications, implantable devices, and to provide

surfactant properties in products like toothpaste and contact lens cleaner (Dumortier

2006). The consistency of the material increases with increased temperature as the

polymer molecules begin to join one another. The process is also reversible, as lowering

the temperature will cause the molecules to dissociate. Fowler showed that addition of

similar copolymers to demineralized bone powder did not affect histologic bone fill in rat

calvarial defects (2002). Additionally, at low concentrations, poloxamer 407 has been

shown in vitro to stimulate human gingival fibroblast proliferation and attachment as well

as microvasculature formation, possibly due to a surfactant effect that promotes spreading

and adherence of the cells (Hokett 2000).

The coherent and malleable nature that poloxamer 407 imparts to Accell

Connexus® may improve graft stability and potentially increase the predictability of

regenerative treatment in poorly contained defects. Additionally, the increased BMP levels

may promote new bone formation. There are currently no human studies using Accell

Connexus® for the periodontal application of guided tissue regeneration.

10

CHAPTER 3

METHODOLOGY

The purpose of this study was to compare Accell Connexus® to DFDBA, both in

combination with a resorbable collagen membrane in a double-blinded, randomized

controlled clinical trial. Specifically, the hypothesis was that Accell Connexus® would

provide superior periodontal regeneration to DFDBA with greater clinical attachment level

gain and greater decrease in probing depth. Conversely, the null hypothesis was that Accell

Connexus® and DFDBA would produce equivalent results. This investigation is the only

clinical study to evaluate Accell Connexus® in this application.

Patient Selection

Subjects were selected from the Periodontics department at the Naval Postgraduate

Dental School who were 18 years or older with a diagnosis of generalized or localized

severe periodontitis and a site with probing depth of 6 mm or greater and a radiographic

vertical defect. All patients were in good general health without systemic conditions such

as diabetes. Pregnant females and smokers were excluded from the study. Table 1

provides a comprehensive list of study inclusion and exclusion criteria. Potential subjects

had an initial evaluation with a periodontist and underwent initial scaling and root planing

therapy. Four to six weeks following scaling and root planing, periodontal reevaluation

and full-mouth charting were completed. At this point, the study was explained to

qualifying patients, who signed consents if they agreed to participate. Patients who

declined to enroll in the study were still treated with the recommended therapy of guided

tissue regeneration. Once enrolled, subjects had impressions made to fabricate acrylic

11

measurement stents and also had a radiographic stent made. Using the measurement

stent, baseline values were recorded by study investigators, all of whom were all board

certified periodontists. Probing depth, clinical attachment level, bleeding-on-probing and

plaque were recorded and a periapical radiograph was taken with the stent.

Clinical Procedure

Surgeries were performed by second and third year periodontics residents. After

delivery of local anesthesia, sulcular incisions were made. Full thickness flaps were

elevated and defects were debrided of granulation tissue and calculus using hand scalers

and ultrasonic instrumentation. Defects were then confirmed to be graftable, with an

intrabony defect depth of at least 4mm. A study investigator then measured defect height

from both the cemento-enamel junction and the bony crest using the custom acrylic stent.

Defect depth and width measurements were also recorded. A numbered, sealed envelope

containing a randomized group assignment was opened to reveal the bone graft material

allocation. Bony modification was completed if needed and the defect was irrigated. 24%

EDTA gel was applied to the root surface for 2 minutes and then thoroughly rinsed. After

irrigation, intramarrow penetrations were made, the graft was placed and a trimmed Bio-

Gide ® membrane was placed over the defect and graft. The flaps were then re-

approximated for primary closure with non-resorbable monofilament PTFE suture.

Post-surgical care included pain medications, a 10 day course of antibiotics and

0.12% chlorhexidine mouth rinse. Post-surgical follow-up appointments for plaque

removal and reinforcement of oral hygiene instructions were made once a week for the

first two weeks, biweekly to two months, then monthly to four months. Periodontal

12

maintenance and oral hygiene instructions were performed at 6, 9 and 12 months post-

procedure. Study measurements by a blinded investigator and radiographs were

completed at 6 and 12 months. After the final 12 month appointment, patients were exited

from the study and followed for normal periodontal maintenance therapy by their

provider.

Statistical Analysis

The sample size for this study was calculated to be 14 subjects per group. Power

analysis was based on Hoidal et al, 2008, who compared DFDBA to DBDFA plus EMD for the

treatment of intrabony defects. For two independent study groups with continuous possible

outcomes receiving two different treatments, the sample size needed to find a clinically

significant mean difference of 1mm with at least one standard deviation at the 0.05 level with

power of 80% would be 14 subjects per group.

The first objective was to evaluate changes between the six and twelve month time

points as a result of the surgical treatments, using a T-test for equality of the means. The

second and chief objective was to evaluate any potential between-group differences for

probing depth and clinical attachment level. The Shapiro-Wilk test was chosen to evaluate

the normality of the baseline data. Finally, linear mixed models were attempted to

determine if there were any significant independent variables.

13

CHAPTER 4

RESULTS

For patient enrollment, a total of 51 subjects were assessed for eligibility. Twenty-

six were excluded for either not meeting inclusion criteria or declining to participate. The

selected 25 subjects were randomly assigned to a treatment group and 20 completed the

study. Two patients did not receive their assigned intervention. One of the patients had a

defect that was determined to be too shallow for grafting during surgical access and the

other patient was discovered to be a smoker on the day of the surgical procedure. Two

patients were disenrolled for poor hygiene compliance and one was lost to follow-up. After

attrition, 10 subjects per group remained, completed the study and were included in the

final analysis. Despite not reaching the desired sample size, post-hoc analysis revealed that

statistical significance would still not have been achieved with the additional 8 subjects.

All sites healed uneventfully and there were no adverse effects during treatment or

follow-up in either group. Bleeding on probing improved from 89% of sites to 50% of sites

after 12 months with no differences between groups. Plaque detection was similar before

and twelve months after treatment in both groups at 28%.

Baseline data

Baseline demographic data is listed in Table 1. There were no significant differences

between subjects assigned to the two groups. Mean age was 41.7 years for both groups and

ranged from 22 to 74 years. The baseline mean probing depth was 8.0 ± 1.5 mm for the

control group and 7.8 ± 2.6 mm for the test group with a p-value of 0.836 from the Shapiro-

14

Wilk analysis for normality. Baseline mean clinical attachment level was 10.5 ± 2.6 mm for

the control and 11.5 ± 2.8 mm for the test group with a p-value of 0.421.

Surgical Data

The surgical measurements are listed in Table 2. The primary defect type was 3-

walled, meaning that at least part of the defect was comprised of three walls. There was a

single 1-walled and two 2-walled defects in the Accell Connexus® group, and a single 2-

walled defect in the DFDBA group. All defects were graftable with no between-group

differences. The mean defect depth in the DFDBA group was 6.0 ± 2.2 mm and 5.7 ± 1.8

mm in the Accell Connexus® group. Mesial-distal width was 3.5 ± 1.6 mm for the DFDBA

group and 4.3 ± 3.2 mm for the test group with a p-value of 0.484. Defect buccal-lingual

widths were slightly wider in the control group at 8.7 ± 2.3 mm versus 6.9 ± 1.5 mm for the

test group and approached significance with a p-value of 0.048 from a t-test for equality of

means.

Probing depth

Both groups achieved significant improvement in probing depth with no significant

between group differences at either time point (Table 3, Figure 2). Mean probing depth

decreased from 7.8 mm to 4.1 mm in the test group and from 8 mm to 3.6 mm in the

DFDBA group by 12 months. There was a trend for continued improvement between the 6

and 12 month measurements for the DFDBA group only, with the mean improving from 4.4

± 1.3 mm to 3.6 ± 0.7 mm. This trend was not significant. The probing depth in the Accell

Connexus® group remained stable from 6 months (4.0 ± 0.9 mm) to 12 months (4.1 ± 1.7

mm).

15

Clinical attachment level

Mean clinical attachment level significantly improved in both groups with no

between group differences (Table 4, Figure 3). Clinical attachment level decreased from

11.5 mm to 8.7 mm in the test group after 12 months and decreased from 10.5 mm to 7.1

mm in the DFDBA group. DFDBA subjects saw slight continued improvement between 6

and 12 months, from 7.8 ± 1.8 mm to 7.1 ± 2.4 mm. Test subjects had a slight decline from

6 to 12 months from 7.1 ± 1.6 mm to 8.5 ± 1.0 mm. Neither of these trends were significant.

Radiographic bone levels

Radiographic data was not quantifiable and therefore not analyzed. Despite use of

the custom radiographic stents, several of the radiographs were misaligned and at slightly

different angulations preventing comparison measurements. Nevertheless, qualitative

radiographic bone fill was evident in all cases (Figure 4).

16

DISCUSSION

In this study, Accell Connexus® and DFDBA were compared for treatment of

intrabony periodontal defects in conjunction with a resorbable collagen membrane. The

results showed no difference in clinical response as measured by probing depth and clinical

attachment level improvement. A possible reason for lack of effect from the increased

BMP-2 levels from Accell Connexus® may be that it is released early and all at once. Khaliq

2007 found in vitro that with Accell Connexus® BMP-2 release spiked within 4 hours

versus a slower, ramping sustained release from DFDBA from days one to five. Perhaps

this rapid release is not sufficient for increased recruitment of mesenchymal stem cells and

induction of other BMPs. Inflammatory response could also be greater with Accell

Connexus®. Khorsand 2012 noted increased inflammatory reaction, foreign body

response and delayed bone formation in rabbit calvarial defects treated with Accell

Connexus® compared to Bio-Oss® and negative control subjects.

The target sample size for this study was not reached with four subjects fewer per

group than desired. The intended sample size was chosen to show significance at a

treatment response difference of 1 mm. Post-hoc analysis revealed that statistical

significance, if any, could have potentially been achieved with a sample size of 28 subjects

per group based on the effect difference.

Defect characterization also influences clinical response. The majority of the defects

in this study were three-walled defects, which are more predictable to treat than one- and

two-walled defects. Due to the sample size, there were not enough subjects to stratify by

defect type, depth or number of walls. Looking only at the defects with a three-walled

component, 9 of which were in the DFDBA group, and 7 in the test group, the baseline and

17

post-treatment means for probing depth and clinical attachment level were similar to the

overall analysis. For probing depth, there was a significant change over time in both

groups (P<0.001) but no overall difference in treatment groups (p=0.796) and no

difference in how the groups changed over time (p=0.189). Similarly for clinical

attachment level, both groups improved significantly over time (P=0.002) with no overall

difference in treatment groups (p=0.067) and no difference in how the groups changed

over time (p=0.457). This agreement between the overall and 3-walled analyses confirms

that the slight difference in defect types between groups did not skew the results. Perhaps

a larger sample size with a greater number of one- and two-walled defects, would have

been potentially shown difference between the groups.

Despite obvious radiographic defect fill, hard tissue analysis was not possible due to

several of the serial films not being correctly angulated. Perhaps a different stent could be

used that will more accurately replicate the original angulation and prevent the variation

that was observed in this study. Radiographic data is highly valuable when used to

compare and corroborate the clinical values of probing depth and clinical attachment level.

Quantitative analysis would have increased the robustness of this study.

Treatment gains in the current study agree with previous studies using DFDBA with

membranes for the treatment of intrabony defects. A twelve month study comparing

DFDBA alone to DFDBA with a non-resorbable ePTFE membrane reported a probing depth

change of 4 mm and a clinical attachment level improvement of 3.1 mm for the membrane

group (Trejo 2004). These values agree with the current reported DFDBA group probing

depth change of 3.6 mm and clinical attachment level change of 3.4 mm. Looking at

resorbable membranes, a recent study by Kher compared a collagen membrane alone to a

18

collagen membrane combined with DFDBA. The reported improvement at 6 months for

the DFDBA group was 4.06 ± 0.38 mm for probing depth and 3.54 ±0.36 mm for CAL.

Stratifying for 1- and 2- walled defects, use of a membrane with graft significantly

improved bone fill, whereas this was a still a trend, but not significant for the 3-walled

defects (Kher 2013).

Human studies on periodontal applications of putty grafts are sparse. This current

research is the only human study using Accell Connexus® for guided tissue regeneration.

The only other human study using Accell Connexus® for periodontal application is the

Mandelaris site preservation study (2015). There are also no studies for similar products

like Dynamatrix, a putty graft similar to Accell Connexus® with the same poloxamer carrier

but with mineralized bone chips instead of DFDBA. There is one intrabony regeneration

study on DBX®, a hyaluronate-based DFDBA grafting putty, used for intrabony defect

treatment without a membrane. Hyaluronate carriers may be less desirable than

poloxamers because after placement they can become diluted, lose their coherency and be

washed away. While there were no statistical differences between groups at reentry, DBX

subjects had bone that appeared more fibrous in nature and some of the encapsulated

portions had to be removed (Bender 2005).

Despite the lack of significant statistical or large clinical effect difference between

the groups, there is a significant price difference between the grafting materials. Accell

Connexus® is approximately three to four times more expensive than DFDBA. The current

results show that clinicians can confidently use DFDBA for intrabony grafting. This data is

also an important reminder that with any newer product, robust clinical evidence may be

19

lacking, and that marketing claims are often based on benchtop or animal studies that may

not accurately reflect how the product will perform clinically.

20

CONCLUSION

This controlled clinical study compared DFDBA to Accell Connexus® in conjunction

with a membrane for the treatment of intrabony defects. The findings revealed that there

is no additional clinical benefit or improvement in periodontal clinical parameters with use

of Accell Connexus® over traditional DFDBA grafting when combined with a resorbable

collagen membrane in the treatment of primarily 2- to 3- walled intrabony defects.

Therefore, angular intrabony defects can successfully be treated with either Accell

Connexus® or DFDBA in combination with a resorbable collagen membrane.

21

REFERENCES

AAP Glossary of Periodontal Terms. American Academy of Periodontology. 4th edition. 2001. Bender SA, et al. Evaluation of demineralized bone matrix paste and putty in periodontal intraosseous defects. J Periodontol. 2005; 76: 768-77. Bowers G, et al. Histologic evaluation of new attachment apparatus formation in humans: Part I. J Periodontol. 1989; 60: 664-674. Bowers G, et al. Histologic evaluation of new attachment apparatus formation in humans: Part II. J Periodontol. 1989; 60: 675-682. Bowers G, et al. Histologic evaluation of new attachment apparatus formation in humans: Part III. J Periodontol. 1989; 60: 683-693. Caffesse RG, et al. Clinical comparison of resorbable and non-resorbable barriers for guided periodontal tissue regeneration. J Clin Periodontol. 1997; 24: 727-752. Chen CC, et al. Evaluation of a collagen membrane with and without bone g4rafts in treating periodontal intrabony defects. J Periodontol. 1995; 66: 838-847. Clokie CM, Urist MR. Bone morphogenetic protein excipients, comparative observations on poloxamer. Plast. Reconstr. Surg. 2000; 105: 628-637. Cortellini P, Pini Prato G, Tonetti MS. Periodontal regeneration of human infrabony defects. II. Re-entry procedures and bone measures. J Periodontol. 1993; 64: 261-268. Dumortier G, et al. A review of poloxamer 407 pharmaceutical and pharmacological characteristics. Pharm Res. 2006; 23: 2709-28. Eke P, et al. Prevalence of Periodontitis in Adults in the United States: 2009-2010. J Dent Res. 2012; 91: 914-920. Esposito M, et al. Enamel matrix derivative (Emdogain ®) for periodontal tissue regeneration in intrabony defects. A Cochrane systematic review. Eur J Oral Implantol. 2009; 2: 247-266. Fowler EB, et al. Evaluation of pluronic polyols as carriers for grafting materials, study in rat calvaria defects. J Periodontol. 2002; 73: 191-197. Friedewald V, et al. The American Journal of Cardiology and Journal of Periodontology Editors’ Consensus: Periodontitis and Athersclerotic Cardiovascular Disease. J Periodontol. 2009; 80: 1012-1032.

22

Grover CN, et al. Crosslinking and composition influence the surface properties, mechanical stiffness and cell reactivity of collagen-based films. Acta Biomaterialia. 2012; 8: 2080-2090. Havemose-Poulson A, et al. Periodontal and hematological characteristics associated with aggressive periodontitis, juvenile idiopathic arthritis, and rheumatoid arthritis. J Periodontol. 2006; 77: 280-288. Hokett SD, et al. Pluronic polyol effects on human gingival fibroblast attachment and growth. J Periodontol. 2000; 71: 803-809. Iacopino A. Periodontitis and diabetes interrelationships: Role of inflammation. Ann Periodontol. 2001; 6: 125-137. Kay JF, et al. Effective design of bone graft materials using osteoinductive and osteoconductive components. Isotis Orthobiologicc. Poster presented at: Biomaterials in Regenerative Medicine, Philadelphia, PA. Oct 2004. Khaliq S, et al. Evaluation of a next generation DBM putty in a posterolateral spinal fusion model. 2009. Integra LifeSciences Corporation. Kher VK, et al. A comparative evaluation of the effectiveness of guided tissue regeneration by using a collagen membrane with or without decalcified freeze-dried bone allograft in the treatment of infrabony defects: a clinical and radiographic study. J Indian Soc Periodontol. 2013; 17: 484-489. Khorsand A, et al. Histological evaluation of Accell Connexus® and Bio-Oss® on quality and rate of bone healing: a single blind experimental study on rabbit’s calvarium. J Dent. (Tehran) 9: 116-127. Mandelaris GA, Lu M. Extraction socket preservation prior to implant placement. Dentistry Today 2015: Course # 184. Mariotti A. Efficacy of chemical root surface modifiers in the treatment of periodontal disease. A systematic review. Ann Periodontol. 2003; 8: 205-226. Massimo DF, et al. Is platelet concentrate advantageous for the surgical treatment of periodontal diseases? A systematic review and meta-analysis. J Periodontol. 2011; 82: 1100-1111. Muthukuru M. Bone morphogenic proein-2 induces apoptosis and cytotoxicity in periodontal ligament cells. J Periodontol. 2013; 84: 829-38. Pieske O, et al. Autologous bone graft versus demineralized bone matrix in internal fixation of ununited long bones. J Trauma Manag Outcomes. 2009; 3: 11.

23

Reynolds MA. et al. The efficacy of bone replacement grafts in the treatment of periodontal osseous defects. A systematic review. Ann Periodontol. 2003; 8- 227-265. Rakhmatia YD, et al. Current barrier membranes: titanium mesh and other membranes for guided bone regeneration in dental applications. J Prosthodont Res. 2013; 57: 3-14. Schizas C, et al. Posterolateral lumbar spine fusion using a novel demineralized bone matrix: a controlled case pilot study. Arch Ortho Trauma Surg. 2008; 128: 621-5. Schlegel AK, et al. Preclinical and clinical studies of a collagen membrane (Bio-Gide®). Biomaterials. 1997; 18: 535-538. Sculean A, Jepsen S. Biomaterials for the reconstructive treatment of periodontal intrabony defects. Part II. Guided tissue regeneration, biological agents and combination therapies. Perio. 2004; 2: 97-109. Sela MN, et al. Degradation of collagen0guided tissue regeneration membranes by proteolytic enzymes of Porphyromonas gingivalis and its inhibition by antibacterial agents. Clin. Oral Impl. Res. 2009; 20: 496-502. Selipsky H. Osseous surgery. How much need we compromise? Dent Clin North Am. 1976; 20: 79-106. Shepherd N, et al. Root coverage using acellular dermal matrix and comparing a coronally positioned tunnel with and without platelet-rich plasma: a pilot study in humans. J Periodontol. 2009; 80: 397-404. Trejo PM, Weltman RL. Favorable periodontal regenerative outcomes from teeth with presurgical mobility: a retrospective study. J Periodontol. 2004; 75:1532-1538. Trombelli L, Farina R. Clinical outcomes with bioactive agents alone or in combination with grafting or guided tissue regeneration. J Clin Periodontol. 2008. 35: 117-135. Wang J, et al. Expression of bone microsomal casein kinase II, bone sialoprotein, and osteopontin during repair of calvarial defects. Bone 1998; 22: 621-8. Wilderman M, Pennel B. Histogenesis of repair following osseous surgery. J Periodontol. 1970; 41: 551-565. Zhang M, et al. Effects of the demineralization process on the osteoinductivity of demineralized bone matrix. J Periodontol. 1997; 68: 1085-1092. Zitzmann NU, et al. Treatment of angular bone defects with a composite bone grafting material in combination with a collagen membrane. J Periodontol. 2003; 72: 687-694.

24

Table 1. Baseline Demographic Data (mean ±SD)

DFDBA Accell Connexus ® p-value

Males 6

7

1.000

Females

4

3

Mean age

41.7 ± 13.2

41.7 ± 14.9

1.000

Baseline PD

8.0 ± 1.5

7.8 ± 2.6

0.836

Baseline CAL 10.5 ± 2.6

11.5 ± 2.8

0.421

25

Table 2. Surgical Measurements (mean ±SD)

DFDBA Accell Connexus®

p-value

Defect type: 1-walled 0

1

.0582

2-walled

1

2

3-walled 9 7

Defect depth

6.0 ± 2.2

5.7 ± 1.8

0.741

Defect M-D width

3.5 ± 1.6

4.3 ± 3.2

0.484

Defect B-L width 8.7 ± 2.3

6.9 ± 1.5

0.048

26

Table 3. Probing Depth (mean ±SD)

Accell Connexus®

DFDBA

P-value

Baseline 7.8 ± 2.6

8.0 ± 1.5

0.836

6 mo 4.0 ± 0.9

4.4 ± 1.3

0.438

12 mo 4.1 ± 1.7

3.6 ± 0.7

0.408

27

Table 4. Clinical Attachment Level (mean ±SD)

Accell Connexus®

DFDBA

P-value

Baseline 11.5 ± 2.8

10.5 ± 1.5

0.421

6 mo 7.1 ± 1.6

7.8 ± 1.8

0.396

12 mo 8.5 ± 1.0

7.1 ± 2.4

0.107

28

Figure 1. Comprehensive Inclusion and Exclusion Criteria Checklist

Inclusion Criteria Yes (√) or No (X)

a. Patient 18 years or older b. Remaining in the Capital region for at least 12 months following the surgical

procedure for follow up appointments

c. Diagnosis of generalized or localized severe periodontitis d. Radiographic evidence of a vertical intrabony defect at one or more sites with a

probing depth ≥ 6 mm

Exclusion Criteria a. Under the age of 18 b. Patient moving from the Capital region area prior to 12 months following the surgical

treatment

c. Deep Grade II or Grade III furcation involvement in combination with the intrabony defect determined pre-surgically.

d. Restorations extending beyond the cementoenamel junction at the intrabony defect site

e. Indiscernible cementoenamel junction, either clinical or radiographic f. Periapical pathology, unrestored caries, defective restorations, root resorption,

or vertical root fracture

g. Patient requiring restorative dental care (fillings and crown and bridge work) that cannot be completed prior to fabrication of the customized stent

h. Female patients who are pregnant or nursing i. Current smoker or tobacco user. Former smokers will be excluded if they quit

smoking < 6 months prior to selection in the study.

j. Clinically significant systemic diseases, which may affect healing (e.g. uncontrolled diabetes).

k. Allergy to Chlorhexidine gluconate (Peridex) l. Allergy to tetracycline

m Poor oral hygiene unsuitable for periodontal surgery n. Cannot or will not sign consent form o. Receiving immunosuppressive therapy such as chemotherapy and systemic

corticosteroids. Does not include inhaled or topical steroids.

p. Severe endocrine-induced bone diseases (e.g. hyperthyroidism, altered parathyroid function)

q. Study tooth has mobility of Miller Class II or greater r. Bleeding disorders (e.g. hemophilia) s. Warfarin/ anticoagulant therapy t. History of osteoporosis or bisphosphonate use u. History of radiation therapy in head and neck region Any No (X) response to an inclusion criteria or a Yes (√) response to an exclusion criteria will disqualify an individual from study participation.

29

Figure 2. Probing Depth

0.0

2.0

4.0

6.0

8.0

10.0

Baseline 6 Months 12 Months

Prob

ing

Dep

th (m

m)

Accell

DFDBA

30

Figure 3. Clinical Attachment Level

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

Baseline 6 Months 12 Months

CAL

(mm

)

AccellDFDBA

31

Figure 4. Sample radiographs Baseline 6 months 12 months Baseline 6 months 12 months

32

Appendix 1 Between group data analysis for all subjects

Treatment Group

Accell Connexus® DFDBA

Mean Standard Deviation Valid N Mean

Standard Deviation Valid N

MaxBaseline_Probing_Depth 7.80 2.62 10 8.00 1.49 10 MaxSixMonth_Probing_Depth 4.00 .87 9 4.40 1.26 10 MaxTwelveMonth_Probing_Depth 4.10 1.73 10 3.60 .70 10 Change_SixMonth_Max_PD -3.67 3.00 9 -3.60 1.71 10 Change_TwelveMonth_Max_PD -3.70 3.50 10 -4.40 1.58 10 MaxBaseline_CAL 11.50 2.84 10 10.50 2.59 10 MaxSixMonth_CAL 7.11 1.62 9 7.80 1.81 10 MaxTwelveMonth_CAL 8.50 .97 10 7.10 2.42 10 Change_SixMonth_Max_CAL -4.44 3.32 9 -2.70 3.20 10 Change_TwelveMonth_Max_CAL -3.00 3.37 10 -3.40 2.63 10

33

Appendix 2

Independent Samples Test- All Subjects

Levene's Test

for Equality of

Variances t-test for Equality of Means

F Sig. t df

Sig.

(2-

tailed)

Mean

Differenc

e

SE

Differen

ce

95% Confidence

Interval of the

Difference

Lower Upper

Age Equal variances .024 .878 .000 18 1.000 .0000 6.2966 -13.2286 13.2286

Equal variances not .000 17.759 1.000 .0000 6.2966 -13.2415 13.2415

MaxBaseline_Probing_

Depth

Equal variances 1.806 .196 -.210 18 .836 -.20000 .95219 -2.20048 1.80048

Equal variances not -.210 14.287 .837 -.20000 .95219 -2.23841 1.83841

MaxSixMonth_Probing_

Depth

Equal variances .964 .340 -.795 17 .438 -.40000 .50332 -1.46192 .66192

Equal variances not -.811 15.949 .429 -.40000 .49329 -1.44600 .64600

MaxTwelveMonth_Probi

ng_Depth

Equal variances 1.785 .198 .848 18 .408 .50000 .58973 -.73897 1.73897

Equal variances not .848 11.868 .413 .50000 .58973 -.78650 1.78650

MaxBaseline_CAL Equal variances .059 .810 .823 18 .421 1.00000 1.21564 -1.55396 3.55396

Equal variances not .823 17.855 .422 1.00000 1.21564 -1.55545 3.55545

MaxSixMonth_CAL Equal variances .008 .929 -.870 17 .396 -.68889 .79182 -2.35949 .98171

Equal variances not -.876 17.000 .393 -.68889 .78677 -2.34884 .97106

MaxTwelveMonth_CAL Equal variances 2.997 .101 1.695 18 .107 1.40000 .82597 -.33529 3.13529

Equal variances not 1.695 11.819 .116 1.40000 .82597 -.40268 3.20268

MAX_Surgical_Depth Equal variances .931 .347 -.335 18 .741 -.30000 .89505 -2.18043 1.58043

Equal variances not -.335 17.523 .741 -.30000 .89505 -2.18410 1.58410

MAX_Surgical_MDWidt

h

Equal variances .662 .426 .715 18 .484 .80000 1.11853 -1.54995 3.14995

Equal variances not .715 13.231 .487 .80000 1.11853 -1.61215 3.21215

MAX_Surgical_BLWidth Equal variances 1.087 .311 -2.118 18 .048 -1.80000 .84984 -3.58544 -.01456

Equal variances not -2.118 15.318 .051 -1.80000 .84984 -3.60812 .00812

34

Appendix 3 Between group analysis for subjects with a 3-walled defect component

Treatment Group

Accell Connexus® DFDBA

Mean

Standard

Deviation Valid N Mean

Standard

Deviation Valid N

MaxBaseline_Probing_Depth 8.00 3.06 7 8.11 1.54 9

MaxSixMonth_Probing_Depth 3.83 .75 6 4.44 1.33 9

MaxTwelveMonth_Probing_Depth 4.29 1.89 7 3.67 .71 9

Change_SixMonth_Max_PD -4.00 3.58 6 -3.67 1.80 9

Change_TwelveMonth_Max_PD -3.71 4.07 7 -4.44 1.67 9

MaxBaseline_CAL 12.00 3.32 7 10.33 2.69 9

MaxSixMonth_CAL 7.50 1.64 6 7.56 1.74 9

MaxTwelveMonth_CAL 8.43 1.13 7 7.00 2.55 9

Change_SixMonth_Max_CAL -4.67 3.98 6 -2.78 3.38 9

Change_TwelveMonth_Max_CAL -3.57 3.95 7 -3.33 2.78 9

35

Appendix 4

Independent Samples Test- Three-walled Subjects

Levene's Test for Equality of Variances t-test for Equality of Means

F Sig. t df Sig. (2-tailed)

Mean Difference

Std. Error Difference

95% Confidence Interval of the Difference Lower Upper

Age Equal variances .543 .473 -1.139 14 .274 -7.0635 6.2000 -20.3611 6.2341 Equal variances not -1.180 14.000 .258 -7.0635 5.9878 -19.9060 5.7790

MaxBaseline_Probing_Depth Equal variances 2.415 .142 -.095 14 .925 -.11111 1.16556 -2.61099 2.38877 Equal variances not -.088 8.351 .932 -.11111 1.26320 -3.00289 2.78067

MaxSixMonth_Probing_Depth Equal variances 1.964 .185 -1.012 13 .330 -.61111 .60368 -1.91529 .69307 Equal variances not -1.131 12.798 .279 -.61111 .54035 -1.78034 .55812

MaxTwelveMonth_Probing_Depth Equal variances 2.255 .155 .911 14 .377 .61905 .67918 -.83765 2.07575 Equal variances not .823 7.313 .437 .61905 .75217 -1.14425 2.38234

MaxBaseline_CAL Equal variances .051 .825 1.111 14 .285 1.66667 1.49981 -1.55011 4.88344 Equal variances not 1.081 11.468 .302 1.66667 1.54175 -1.70988 5.04321

MaxSixMonth_CAL Equal variances .026 .874 -.062 13 .952 -.05556 .89779 -1.99512 1.88401 Equal variances not -.063 11.317 .951 -.05556 .88680 -2.00074 1.88963

MaxTwelveMonth_CAL Equal variances 1.546 .234 1.373 14 .191 1.42857 1.04079 -.80371 3.66085 Equal variances not 1.501 11.587 .160 1.42857 .95179 -.65341 3.51056

MAX_Surgical_Depth Equal variances .122 .732 -.099 14 .922 -.11111 1.11789 -2.50875 2.28653 Equal variances not -.100 13.324 .922 -.11111 1.11111 -2.50559 2.28337

MAX_Surgical_MDWidth Equal variances 1.320 .270 .952 14 .357 1.30159 1.36745 -1.63130 4.23448 Equal variances not .871 7.925 .409 1.30159 1.49459 -2.15062 4.75379

MAX_Surgical_BLWidth Equal variances 1.495 .242 -1.232 14 .238 -1.23810 1.00533 -3.39432 .91812 Equal variances not -1.327 12.633 .208 -1.23810 .93284 -3.25933 .78314