Enamel matrix derivative in combination with bone grafts

475

Q U I N T E S S E N C E I N T E R N AT I O N A L

VOLUME 45 • NUMBER 6 • JUNE 2014

Enamel matrix derivative in combination

with bone grafts: A review of the literature

Richard J. Miron, MSc, PhD1/Vincent Guillemette2/Yufeng Zhang, Prof, DMD, Dr Med Dent, MSc, PhD3/ Fatiha Chandad, Prof, MSc, PhD4/Anton Sculean, Prof, DMD, Dr Med Dent, MS, PhD, Dr hc5

Objective: Over 15 years have passed since an enamel matrix derivative (EMD) was introduced as a biologic agent capable of periodontal regeneration. Histologic and controlled clinical studies have provided evidence for periodontal regeneration and substantial clinical improvements following its use. The purpose of this review article was to perform a systematic review comparing the effect of EMD when used alone or in combination with various types of bone grafting material. Data Sources: A literature search was conducted on several medical databases including Medline, EMBASE, LILACS, and CENTRAL. For study inclusion, all studies that used EMD in combination with a bone graft were included. In the initial search, a total of 820 articles were found, 71 of which were

selected for this review article. Studies were divided into in vitro, in vivo, and clinical studies. The clinical studies were sub-divided into four subgroups to determine the effect of EMD in combination with autogenous bone, allografts, xenografts, and alloplasts. Results: The analysis from the present study demonstrates that while EMD in combination with certain bone grafts is able to improve the regeneration of periodontal intrabony and furcation defects, direct evidence supporting the combination approach is still missing. Conclusion: Further controlled clinical trials are required to explain the large variability that exists amongst the conducted studies. (Quintessence Int 2014;45:475–487; doi: 10.3290/j.qi.a31541)

Key words: animals, bone grafts, Emdogain, enamel matrix derivative, intrabony periodontal defects, osteoinduction, preclinical studies, systematic review

PERIODONTOLOGY

Richard J. Miron

Periodontitis, an infectious disease characterized by

progressive attachment and bone degeneration, may

ultimately result in tooth loss if left untreated. Results

from a national survey conducted in the United States

in 2009 and 2010 demonstrated that over 47% of the

adult population aged 30 years and above had peri-

odontitis, distributed as 8.7%, 30.0%, and 8.5% with

mild, moderate, and severe periodontitis, respectively.1

Such surveys clearly demonstrate the prevalence and

importance of developing clear guidelines aimed at

restoring a functional periodontal apparatus. Regenera-

tive periodontal surgery aims to predictably restore the

tooth’s supporting apparatus (ie, root cementum, peri-

1 Research Fellow, Faculté de Medecine Dentaire, Pavillon de Médecine Dentaire,

Université Laval, Québec, Canada; and The State Key Laboratory Breeding Base

of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Bio-

medicine Ministry of Education, School & Hospital of Stomatology, Wuhan Uni-

versity, Wuhan 430079, People’s Republic of China.

2 Research Student, Faculté de Medecine Dentaire, Pavillon de Médecine Den-

taire, Université Laval, Québec, Canada.

3 Associate Professor, The State Key Laboratory Breeding Base of Basic Science of

Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of

Education, School & Hospital of Stomatology, Wuhan University, Wuhan 430079,

People’s Republic of China.

4 Head of Research, Groupe de Recherche en Écologie Buccale, Faculté de Mede-

cine Dentaire, Pavillon de Médecine Dentaire, Université Laval, Québec, Canada.

5 Head, Department of Periodontology, School of Dental Medicine, University of

Bern, Switzerland.

Correspondence: Dr Richard J. Miron, Faculté de Medecine Dentaire, Pavillon de Médecine Dentaire, 2420 Rue de la Terrasse, Université Laval, Québec, G1V 0A6, Canada. Email: [email protected]

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odontal ligament, and bone) that has been lost follow-

ing periodontal disease or trauma.2-5 Results from lon-

gitudinal clinical studies have provided some evidence

that chronic periodontal disease can be successfully

treated.6 Patient education and training in personal

hygiene combined with meticulous plaque control fol-

lowed by nonsurgical and surgical periodontal treat-

ment aiming to disrupt the subgingival plaque biofilm

has been reported. All of these methods are aimed at

reducing periodontal pocket depth (PD).6,7

One well-established method to enhance periodon-

tal regeneration is the use of an enamel matrix deriva-

tive (EMD).8 The use of EMD has been shown to

enhance regeneration of root cementum, periodontal

ligament, and alveolar bone, and to result in substantial

clinical improvements evidenced by probing depth

reduction, gain in clinical attachment, and bone fill.9

This review article plans to give insight into the

advances made with respect to the combination of

EMD with a bone grafting material. A number of in

vitro, in vivo, and clinical studies are discussed eliciting

the use of this combination for bone and periodontal

regeneration.

BIOLOGIC RATIONALE OF THE USE OF EMD

Over 15 years ago, EMD was first introduced as an

adjunctive to periodontal surgery. It was originally

developed at BIORA, in Malmö, Sweden, but has since

been commercially available from Straumann, Basel,

Switzerland. The development of Emdogain for peri-

odontal regeneration mimics the normal development

of periodontal tissues.10 The histologic observation that

amelogenin, which until then was considered an

enamel-specific protein, is deposited onto the surface

of developing tooth roots prior to cementum forma-

tion, led to the hypothesis that amelogenin might be

responsible for the differentiation of periodontal tis-

sues.10 This hypothesis was the basis of a number of

biologic and clinical studies thereafter.10-16 Based on

these observations, the purified amelogenin fraction

was given the working name EMD, and this formulation

has been the basis for numerous publications support-

ing its use in periodontal regeneration.

The major components of EMD are amelogenins, a

family of hydrophobic proteins derived from different

splice variants and controlled post-secretory from the

expression of a single gene that accounts for more than

95% of the total protein content.17 These proteins self-

assemble into supramolecular aggregates that form an

insoluble extracellular matrix that functions to control

the ultrastructural organization of the developing

enamel crystallites.17 Other proteins found in the

enamel matrix include enamelin, ameloblastin (also

called amelin or sheathlin), amelotin, apin, and various

proteinases.18,19

Previous in vitro research has documented and

characterized the role of EMD in many cell types. Boss-

hardt20 has published a review article of the biologic

mediators associated with EMD at the cellular and

molecular levels. It was demonstrated that EMD has a

significant influence on the cell behavior of many cell

types by mediating cell attachment, spreading, prolif-

eration, and survival, as well as expression of transcrip-

tion factors, growth factors, cytokines, extracellular

matrix constituents, and other molecules involved in

the regulation of bone remodeling.20

Fig 1 Periodontal regeneration follow-ing treatment of a human intrabony defect with a combi-nation of Emdogain + natural bone mineral (eg, Bio-Oss spongio-sa). D, dentin; G, graft; NB, new bone; NC, new cementum.

D

NB

NB

GNC

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COMBINATION OF BONE GRAFT + EMD

Although histologic and controlled clinical studies have

provided evidence for periodontal regeneration and

substantial clinical improvements following the use of

EMD in intrabony defects (Fig 1), concerns have been

expressed regarding the viscous nature of EMD, which

may not be suffi cient to prevent fl ap collapse in peri-

odontal defects with a complicated anatomy. A fl ap

collapse may subsequently lead to a limitation of the

space available for regeneration, thus limiting the clin-

ical outcomes.21,22 In order to overcome this potential

limitation and improve the clinical results obtained

with EMD, various combinations of EMD with diff erent

types of grafting materials have been utilized.

CLASSIFICATION OF BONE GRAFTS

The primary goal of bone grafts is to facilitate recruit-

ment of bone-forming osteoblasts capable of prolifer-

ating and diff erentiating on an osteoconductive sur-

face promoting the formation of new bone.23 A variety

of biologic and synthetic materials are available for the

surgical treatment of alveolar bone loss (Fig 2).

Although autogenous bone (AB) graft is considered

the gold standard, many other materials, including

bone allografts, xenografts, and alloplasts, are exten-

sively being studied in order to avoid the drawbacks

and limitations of AB, which include donor site morbid-

ity and additional surgical costs.24-28 The ideal bone

grafting material should generate and sustain suffi -

cient alveolar bone for long-term function, have low

Figs 2a to 2h Scanning electron micros-copy (SEM) images of various bone grafts that are commonly used in dentistry at low and high magnifi cation: (a and b) Autog-enous bone (AB) harvested via a bone scraper; (c and d) demineralized freeze-dried bone allograft (DFDBA); (e and f) a natural bone graft from bovine origine (Bio-Oss xenograft); (g and h) a synthetic calcium phosphate.

a

d

g

b

e

h

c

f

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morbidity and complication rate, as well as a short

healing time.

The regenerative potential of bone grafts is gov-

erned by three fundamental mechanisms.23 The ideal

grafting material should provide:

• an osteoconductive matrix, which allows vascular

invasion and cellular infiltration

• osteoinductive factors, which recruit and induce

mesenchymal cells to differentiate into mature

bone-forming cells

• osteogenic cells contained inside the bone graft

capable of laying new bone matrix.

Autogenous bone

AB (often termed autologous bone) grafting involves

the harvesting of bone obtained from the same indi-

vidual receiving the graft.29 Common in oral and maxil-

lofacial surgery is the harvesting from the mandibular

symphysis (chin area) or anterior mandibular ramus

(the coronoid process). Commonly employed harvest-

ing techniques include grinding of bone blocks with a

bone mill, harvesting with piezosurgery, collection of

drilling particles (bone slurry) with a bone trap, or using

a bone scraper.29,30 The advantages of AB grafts are

their osteoinductive potential, their ability to be used

as blocks or particulate, and the incorporation of osteo-

genic cells located inside their bone matrix.30 Their

limitations are their morbidity from the donor site and

unpredictable resorption.31

Allografts

Allografts involve the harvesting of bone obtained from

another human individual receiving the graft. These are

commonly obtained from bone banks built from

donated cadavers.32 There are three types of bone

allografts available on the market:

• fresh or fresh-frozen bone

• freeze-dried bone allograft (FDBA)

• Demineralized freeze-dried bone allograft (DFDBA).

Currently, the use of allografts is permitted in the United

States; however, their use in most countries in Europe

and Asia is not permitted. The advantages of allografts

are their availability when compared to AB, their lack of

donor site morbidity, and their ability to contain osteo-

inductive growth factors and cytokines embedded in

the bone matrix.23 Disadvantages are their possible

transmission of disease and lack of osteogenic cells.

Xenografts

Xenografts are bone substitutes derived from a species

other than human, such as animals and plants. One

well-documented xenograft is natural bone mineral

(NBM), which is a highly purified anorganic bone matrix

mineral from bovine origin ranging in size from

0.25 mm to 1 mm under the trademark name Bio-Oss®

(Geistlich Biomaterials).33,34 A clinical case is presented

in Figs 3 to 7, demonstrating its combination with EMD.

The advantages of xenografts include their availability,

safety, surface characteristics that are comparable to

AB, and little to no long-term resorption. Their limita-

tions include limited mechanical stability, inability to

alter resorbability, lack of osteoinductive potential, and

patients’ fear of “animal origin”.33,34

Alloplasts

Alloplasts are synthetically fabricated of hydroxyapatite

(HA), beta-tricalcium phosphate (β-TCP), polymers, and

bioactive glasses.35-38 They are osteoconductive and

recent research suggests that some may also be osteo-

inductive.23,39 Recently, alloplasts have been fabricated

with the incorporation of growth factors and/or cyto-

kines.23 Their advantages are their availability, safety,

and modifiable resorbability. Their limitations include

surface characteristics, limited clinical cases, and lack of

osteoinduction.

RESOURCES SELECTION

In order to gather all available biologic data relevant to

the topic, a systematic approach was applied by search-

ing databases including MEDLINE, PubMed, Embase,

and Cochrane Databases up until 28 May 2013. The

initial search criteria for “enamel matrix proteins”,

“enamel matrix derivative”, and “emdogain” generated

820 initial articles (Fig 3). Of these, an individual hand

search was established and screened for all in vitro, in

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vivo, and clinical studies utilizing EMD with any type of

bone grafting material. A total of 6 in vitro, 15 in vivo,

and 19 clinical studies were selected after careful

review and are presented in the remainder of this arti-

cle. In addition, a secondary search of possible articles

relevant to the topic was undertaken in other data-

bases to identify possible additional studies that may

have been missed.

In vitro studies

To date, only six studies have tested the combination of

EMD with various bone grafting materials. In the first in

vitro study conducted by Reichert et al,40 the combina-

tion of EMD with seven various bone grafts was tested

for the proliferative ability of a commercially available

hip-bone derived osteoblastic cell line (HHOBc, Promo-

Cell). In this study, the proliferation of osteoblasts was

only significantly upregulated on Bio-Oss particles fol-

lowing 7 days of incubation. The authors report that no

clear correlation between bone grafts coated with EMD

could be observed and that further investigations were

necessary to understand the exact modus of interac-

tion between EMD and bone grafts.

Further research from our laboratory was con-

ducted to develop the regenerative potential of Bio-Oss

in combination with EMD.41,42 Recently, we demon-

strated that EMD significantly increased cell attach-

ment, proliferation, and differentiation of human pri-

mary osteoblasts and periodontal ligament (PDL) cells

on EMD-coated Bio-Oss particles when compared to

control particles in vitro.41 EMD also stimulated the

release of growth factors, cytokines, and differentiation

markers including bone morphogenetic protein 2

(BMP-2), transforming growth factor beta 1 (TGF-β1),

Fig 7 Postoperative radiograph at 1 year.

Fig 3 Preoperative radiograph of intrabony defect to be treated with EMD + a natural bone mineral (Bio-Oss).

Fig 4 Intraoral image of intrabony defect.

Fig 5 Application of Emdogain (EMD) to the intrabony defect.

Fig 6 Defect filled with EMD + a natural bone mineral (Bio-Oss).

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collagen1α1, alkaline phosphatase, and osteocalcin.41

Further research analyzing this combination utilizing a

predesigned gene array revealed that EMD also pro-

motes gene expression of various osteoblast differen-

tiation markers including a number of collagen types

and isoforms, SMAD intracellular proteins, osteopontin,

cadherin, and a variety of growth factors and cytokines

including BMPs, vascular endothelial growth factors,

insulin-like growth factor, TGF, and their associated

receptor proteins.42

The combination of EMD with other bone grafts

including DFDBA and a new biphasic calcium phos-

phate further support its combination, although obvi-

ous variations between each combination have been

reported and reasons remain unknown.43,44 In the only

other in vitro combination of EMD with a grafting ma-

terial, Mrozik et al45 combined Straumann Bone

Ceramic with EMD and tested the proliferation and dif-

ferentiation of postnatal mesenchymal stromal cells

(MSCs), bone marrow stromal cells (BMSCs), and peri-

odontal ligament fibroblasts (PDLFs). The results from

this study demonstrated that collagen-I mRNA was

upregulated in both MSC populations over the 72-hour

time course with EMD. Expression of BMP-2 and the

osteogenic transcription factor Cbfa-1/Runx2 showed

early stimulation in both MSC types after 24 hours. Both

osteopontin and periostin mRNA were upregulated in

BMSCs, and higher levels of bone sialoprotein were

observed in only PDLFs following treatment with EMD.

In contrast, they found that expression of BMP-4 was

consistently downregulated by both MSC types follow-

ing treatment with EMD. In conclusion, it was sug-

gested that early upregulation of several important

bone-related genes suggests that EMD may have a

significant stimulatory effect in the commitment of

mesenchymal cells to osteogenic differentiation. How-

ever, future research was suggested to determine the

effects of EMD on late differentiation and mineraliza-

tion of osteoblasts.

In vivo animal studies

A number of in vivo studies with various defect models

are now available demonstrating the effect of EMD in

combination with a bone graft (Table 1). Boyan et al46

were the first to test the effects of EMD in vivo by test-

ing the ability for EMD with/without DFDBA to form

ectopic bone formation in nude mice. While EMD was

not able to induce ectopic bone formation alone, it was

shown that it had an osteopromotive effect on bone

regeneration when it was combined with DFDBA.46

Since then, three additional studies have studied the

effects of EMD on ectopic bone formation. All studies

were unable to find any evidence that EMD promoted

ectopic bone formation in pectoralis muscles, thigh

muscles, and the dorsal surface in either a rat or mouse

model.45,47,48

The effect of EMD on bone formation has also been

tested in a number of animal pure bone defect models.

Two studies in rat calvaria demonstrated nonsignificant

improvements of bone formation when combining

EMD with either deproteinized bovine bone mineral

(DBBM) or bioglass (BG).49,50 In rabbit calvarial defects,

Murai et al51 showed little advantage for the use of EMD

in titanium caps containing either β-TCP or β-TCP +

EMD. The other rabbit calvaria study showed statisti-

cally significant improvements in bone formation when

EMD was combined with Bio-Oss.52 In the remaining

two studies, EMD had a significantly positive improve-

ment in a rat femur defect when combined with Bio-

Oss when compared to Bio-Oss alone,53 as well as

improved healing of bone defects following extraction

when combined with AB when compared to AB alone.54

To date, five studies have demonstrated the ability

for EMD to enhance periodontal regeneration in both

rat and dog intrabony defects. Fernandes et al55 created

Class III furcation defects in five mongrel dogs and

showed that the combination of EMD with BG was non-

significantly superior to either the use of EMD alone or

BG alone. Donos et al56 studied the effects of bone for-

mation under periosteal flaps of the mandibular ramus

and found no advantage of a combination of GBR +

DBBM + EMD when compared to either GBR + DBBM or

GBR + EMD alone. In a one-wall intrabony defect, Shi-

rakata et al57 showed that EMD in combination with a

CaP cement generated significantly new bone forma-

tion when compared to EMD alone. Yamamoto et al58

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demonstrated a significant advantage (P < .05) when

EMD was combined with bovine-derived xenograft

(BDX) when compared to BDX alone for the treatment

of intrabony defects in dogs. Most recently, Oortgiesen

et al59 demonstrated that EMD in combination with CaP

was able to significantly increase new bone formation

Table 1 Chronological list demonstrating the various animal models testing the efficacy of EMD in combina-tion with a bone graft when compared to either a bone graft alone or EMD alone

Study Models Study design Duration Treatment groups Outcome P value

Boyan et al46 32 miceEctopic bone formation in calf muscle

56 days

DFDBA Ectopic BF

< .05EMD No ectopic BF

DFBDA + EMD Greater ectopic BF

Donos et al49 40 ratsBilateral calvarial defects

120 days

EMD W = 2.96; L = 1.70

NSDBBM W = 1.78; L = 0.33

EMD + DBBM W = 1.76; L = 1.36

Donos et al56 20 ratsBone formation under periosteal flap of man-dibular rasmus

60, 120 days

GBR + DBBM Bone fill = 19%

NSGBR +EMD Bone fill = 15%

GBR + DBBM + EMD Bone fill = 9.5%

Fernandes et al55 5 dogsTreatment of Class III furcation defects

90 days

BG PR = 1.5%

NSEMD PR = 1.0%

EMD + BG PR = 6.3%

Murai et al51 14 rabbits Calvaria defects 30, 90 daysβ-TCP New BF 36.6%

NSΒ-TCP + EMD New BF 42.2%

Donos et al47 16 ratsEctopic bone formation in pectoralis muscles

60, 120 daysDBBM No ectopic BF

NSEMD + DBBM No ectopic BF

Prata et al54 10 ratsIntrabony defects fol-lowing extraction

7, 21, 42 daysAB BF = 62%

< .05AB + EMD BF = 72%

Shirakata et al57 4 dogsOne-wall intrabony defects

70 days

EMD BF = 2.33 mm

< .0001CaP cement BF = 3.50 mm

EMD + CaP cement BF = 3.07 mm

Yamamoto et al58 24 dogs Intrabony defects 56 daysBDX New BF 31.6%

< .05BDX + EMD New BF 52.2%

Potijanyakul et al50 20 rats Calvaria defects 14, 28, 56 daysBG BF = 6%

NSBG + EMD BF = 18%

Chan et al48 20 miceEctopic bone formation in thigh muscle

28, 56 daysHA-TCP No ectopic BF

NSHA-TCP + EMD No ectopic BF

Mrozik et al45 4 miceEctopic bone formation in dorsal surface

42–56 daysBone ceramic No ectopic BF

NSBone ceramic + EMD No ectopic BF

Shahriari et al52 20 rabbitsCalvaria cranial bone defects

14, 28, 56, 84 days

Bio-Oss BF = 70%

< .001EMD BF = 83%

Bio-Oss + EMD BF = 96%

Miron et al53 27 rats Femur defects 14, 28, 56 daysNBM BF = 14%

< .05NBM + EMD BF = 16%

Oortgiesen et al59 15 rats Three-wall intrabony defects

84 daysEMD Bone reg. score = 1.9

< .01EMD + CaP Bone reg. score = 2.9

BF, bone formation; Bone reg. score, bone regeneration score; L, length (mm); NS, not significant; PR, periodontal regeneration; W, width (mm).

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in three-wall intrabony defects in a rat model. In con-

clusion, the results from the current in vivo studies sug-

gest that EMD was unable to induce ectopic bone for-

mation. Although the combination of EMD with a bone

grafting material may lead to improved regeneration of

new bone formation and/or new periodontal regenera-

tion, a lack of consistent and predictably significant

findings was commonly observed.

Clinical studies

To date, a number of clinical trials have evaluated the

use of EMD in combination with a bone grafting ma-

terial either compared to EMD alone or bone graft

alone. Tables 2 to 5 present relevant studies utilizing

EMD when combined with AB, allografts, xenografts,

and alloplasts respectively.

Two studies report the combination of EMD with AB

(Table 2).60,61 Guida et al60 in 2007 showed in a parallel

study of 28 intraosseous lesions that the combination

of EMD with AB did not offer a statistically significant

advantage when compared to EMD alone. Yilmaz et al61

also studied in two- and three-wall intrabony defects

the effects of EMD with AB and found that the combi-

nation approach led to statistically inferior results for

the combination approach clinical attachment levels

(CALs).61 The mean PD and CAL changes in the EMD-

alone group were 4.9 and 4.6 mm respectively, and in

the EMD + AB group the mean changes were 4.2 and

3.5 mm respectively.61

The combination of bone allografts with EMD has

been investigated in four clinical studies (Table 3).

Gurinsky et al62 found in a split-mouth study of 40

patients that EMD + DFDBA offered no statistical differ-

ence in mean PD or CAL levels after a 6-month healing

period. Furthermore, Hoidal et al63 also demonstrated

in a parallel study with 32 patients that the combina-

Table 2 Clinical studies comparing the effects of EMD in combination with autogenous bone to that of either autogenous bone alone or EMD alone

Study

Study design

(no. patients) Clinical defects

Healing

period

Treatment

groups

Mean PD

change (mm) P value

Mean CAL

change (mm) P value

Guida et al60 Parallel (27)28 intraosseous lesions

12 monthsEMD 5.6

NS4.6

NSEMD + AB 5.1 4.9

Yilmaz et al61 Parallel (40)40 two- or three-wall intrabony defects

12 monthsEMD 4.9

< .0014.2

< .001EMD + AB 4.6 3.5

NS, not significant.

Table 3 Clinical studies comparing the effects of EMD in combination with allogenic bone to that of either allogenic bone alone or EMD alone

Study

Study design


Healing

period Treatment groups

Mean PD

change (mm) P value

Mean CAL

change

(mm) P value

Gurinsky et al62

Split mouth (40)67 intrabony defects ≥ 3 mm

6 monthsEMD 4.0

NS3.2

NSEMD + DFDBA 3.6 3.0

Hoidal et al63

Parallel (32)41 intrabony defects ≥ 3 mm

6 monthsDFDBA 2.45

NS1.63

NSDFDBA + EMD 2.56 1.47

Aspriello et al64

Parallel (56)56 intraosseous defects

12 monthsDFDBA 3.75

< .053.5

< .05DFDBA + EMD 5.0 4.0

Jaiswal and Deo65

Parallel (30)30 Class II furcation defects

12 monthsDFDBA + GTR 0.81

< .051.5

< .05DFDBA + GTR + EMD 1.74 2.12


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tion did not lead to any significant changes after a

6-month healing period when compared to the control

group receiving DFDBA alone. In a parallel study with

56 intraosseous defects, Aspriello et al64 demonstrated

after a 6-month healing period that the combination of

DFDBA with EMD led to significant improvements in

mean CAL and PD reduction. Furthermore, recently

Jaiswal and Deo65 also demonstrated in 30 Class II fur-

cation defects that the combination of DFDBA + EMD +

guided tissue regeneration (GTR) led to statistically

improved mean PD and CAL changes when compared

to DFDBA + GTR alone.

The combination of EMD with an NBM (also known

as BDX or Bio-Oss) has been investigated in five clinical

studies (Table 4). Lekovic et al66 were the first to show in

a split-mouth study with 21 patients having intrabony

defects > 6 mm that the combination of EMD with NBM

led to statistically improved mean PD and CAL change

after a 6-month healing period when compared to EMD

alone. Since then, four other studies have analyzed the

effects of EMD in combination with NBM for the treat-

ment of intrabony defects. In each of these studies, no

statistical advantage could be observed for the combi-

nation approach when compared to either NBM alone

or EMD alone.67-70

The use of EMD in combination with a variety of

synthetic alloplasts including BG, β-TCP, biphasic cal-

cium phosphate (BCP), and HA has been investigated in

a number of clinical studies. In two parallel studies

evaluating intrabony defects > 6 mm, the combination

of EMD with a synthetic BG did not significantly change

the mean PD or CAL levels when compared to either BG

alone or EMD alone following a 12-month healing

period.71,72 Kuru et al73 also tested the effects of EMD in

combination with BG. After an 8-month healing period,

a statistically significant increase in PD reduction and

CAL levels was observed for the combination group

when compared to EMD alone.73 Bokan et al74 found

that the combination of EMD with β-TCP did not lead to

statistically superior results in mean PD change and

CAL levels after a 12-month healing period when com-

pared to EMD alone for the treatment of intrabony

defects greater or equal to 3 mm. A similar result was

also found for the combination of EMD with BCP. It was

shown that after a 6-month healing period, no signifi-

cant difference could be observed for the treatment of

73 intrabony defects.75 Meyle et al76 found that the

combination of EMD with bone ceramic did not

improve the clinical parameters when compared to

EMD alone. Two recent studies evaluated the use of

EMD in combination with a HA/β-TCP bone graft.77,78 It

was demonstrated that EMD statistically improved the

mean PD and CAL change for the treatment of

intrabony defects greater or equal to 3 mm,77 but there

Table 4 Clinical studies comparing the effects of EMD in combination with a xenograft to that of either a xeno-graft alone or EMD alone

Study

Study design


Healing

period

Treatment

groups

Mean PD

change (mm) P value

Mean CAL

change (mm) P value

Lekovic et al66 Split mouth (21)42 intrabony defects > 6 mm

6 monthsEMD 1.91

< .0011.71

< .001EMD + NBM 3.43 3.31

Velasquez-Plata et al69

Split mouth (16)32 intrabony defects ≥ 5 mm

6–8 monthsEMD 3.8

NS2.9

NSEMD + NBM 4.0 3.4

Sculean et al68 Parallel (24)24 intrabony defects

12 monthsBDX 6.5

NS4.9

NSBDX + EMD 5.7 4.7

Scheyer et al67 Split mouth (17)34 intrabony defects ≥ 5 mm

6 monthsBDX 3.9

NS3.7

NSBDX + EMD 4.2 3.8

Zucchelli et al70 Parallel (16)16 intrabony defects > 5 mm

12 monthsEMD 5.8

NS5.8

< .01EMD + NBM 6.2 4.9


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was no clear statistically significant advantage for the

treatment of 30 furcation defects greater or equal to

5 mm.78

DISCUSSION

The results from the present systematic review do not

provide conclusive support for the clinical combination

of EMD with a bone grafting material. Although the

majority of in vitro cell culture studies demonstrate that

the addition of EMD to a bone graft led to significantly

improved cell attachment, proliferation, and differen-

tiation, in vivo and clinical investigation has only dem-

onstrated significant benefit in a small number of stud-

ies.

One phenomenon that merits much more investi-

gation is the role of bacterial pathogens on degrada-

tion of enamel matrix proteins. As many pathogens are

present in gingivitis and periodontitis, including gin-

gipains and lipopolysaccharide (LPS) capable of peri-

odontal breakdown, if left untreated their ability to

degrade enamel matrix proteins found in EMD should

also be considered. Currently a number of studies have

investigated the role of bacterial pathogens on EMD.79-

84 In these investigations, the main conclusion is that

EMD is capable of reducing bacterial growth, namely

showing that EMD suppresses the growth of Porphy-

romonas gingivalis. This finding was largely attributed

to the carrier molecule found in EMD, that of propylene

glycol alginate (PGA). In one of these studies, PDL cells

were grown with EMD, and thereafter P gingivalis was

co-cultured to determine if bacterial pathogens were

able to influence the behavior of PDL cells treated with

EMD.79 It was shown that P gingivalis diminishes the

effect of EMD on PDL cells in vitro through a coopera-

tive action of gingipains.79 Although it is clear that the

effects of pathogens on EMD negatively affects PDL cell

behavior, it remains unknown to what extent the action

of gingipains and other degradation proteases have on

the degradation of the protein molecules found in

EMD. Furthermore, it remains unknown to what extent

the concentration of proteases should be minimally

Table 5 Clinical studies comparing the effects of EMD in combination with synthetic alloplasts to that of either an alloplast alone or EMD alone

Study

Study design


Healing

period

Treatment

groups

Mean PD

change (mm) P value

Mean CAL

change (mm) P value

Sculean et al71 Parallel (28)28 intrabony defects ≥ 6 mm

12 monthsBG 4.22

NS3.07

NSEMD + BG 4.15 3.22

Sculean et al72 Parallel (30)30 intrabony defects ≥ 6 mm

12 monthsEMD 4.5

NS3.9

NSEMD + BG 4.2 3.2

Kuru et al73 Parallel (23)23 intrabony defects ≥ 6 mm

8 monthsEMD 5.03

< .054.06

< .05EMD + BG 5.73 4.17

Bokan et al74 Parallel (56)56 intrabony defects ≥ 3 mm

12 monthsEMD 3.9

NS3.7

NSEMD + β-TCP 4.1 4.0

Jepsen et al75 Parallel (73)73 intrabony defects ≥ 4 mm

6 monthsEMD 2.55

NS1.83

NSEMD + BCP 1.93 1.31

Meyle et al76 Parallel (73)73 intrabony defect ≥ 4 mm

12 monthsEMD 2.9

NS1.9

NSEMD + BC 2.8 1.7

De Leonardis and Paolantonio77

Parallel (34)34 intrabony defects ≥ 3 mm

12 monthsEMD 3.51

< .0012.73

< .001EMD + HA/β-TCP 4.00 3.47

Peres et al78 Parallel (30)30 furcation defects ≥ 5 mm

6 monthsHA/β-TCP 2.37

NS1.47

NSHA/β-TCP + EMD 2.63 1.57


485


Miron et al


reduced in a clinical setting prior to the application of

EMD in order to optimize the effects of EMD and

improve periodontal regeneration. It may be that some

of the observed variability between clinical studies may

be directly linked to the clinician’s ability to remove

periodontal pathogens and treat periodontal disease

prior to any regenerative approach taking place. This

fundamental characterization merits further investiga-

tion.

Another highly neglected area of research that is

currently being investigated in our laboratories is the

ability for EMD to adsorb to the surface of bone graft-

ing materials. As such, results from our laboratory have

demonstrated that the ability for EMD to bind to vari-

ous grafting materials is vastly influenced by surface

characteristics of the grafting materials (unpublished

data). We are presently determining how these grafting

materials are thereafter able to influence the release

kinetics of the proteins found in EMD and how these

released amelogenin proteins may also affect cell

behavior of periodontal cells thereafter. It is plausible

that the variations observed in clinical trials may have

to do with the ability for EMD to adsorb to the grafting

material and/or be released from the grafting material.

It is also noteworthy to mention that the delivery vehi-

cle of EMD has changed in recent years. Originally, EMD

was delivered as a liquid up until the mid-2000s, but

more recently the proteins found in EMD have been

packaged in a PGA in order to optimize the delivery

and adsorption of their containing proteins. It remains

to be determined what influence either of these carriers

has on the adsorption of proteins to various grafting

materials and not solely on root surfaces as illustrated

in the original studies.

With respect to further research involving clinical

trials, it is currently difficult to compare results between

previous studies. Not only do the defined set of patient

parameters vary with respect to their starting defect

type (intrabony vs furcation), defect morphology (two-

wall vs three-wall), and defect size (intrabony defects

> 3 mm, > 5 mm or > 6 mm), but also a large number of

the studies compare the combination of EMD + bone

graft to either EMD alone or bone grafting material

alone. Since the results also vary between EMD alone

and bone grafting material alone, it becomes difficult

to fully critique the optimal regenerative approach to

utilize. Further clinical trials are necessary with better

defined patient inclusion criteria, and it would be of

scientific value to include two control groups (EMD

alone and bone grafting material alone) in order to

further elucidate the ideal regenerative approach for

future treatment of periodontal defects.

CONCLUSION

The findings from the present systematic literature

review do not provide strong support for combining

EMD with a bone graft. Although the combination of

EMD with various bone grafts has led to significant

improvements in a number of studies, a larger number

of studies show that the combination led to limited

nonsignificant improvements in mean PD reduction

and CAL change. Future studies with larger patient

numbers and better defined parameters are necessary

to fully characterize whether the clinical combination

of EMD with a bone grafting material is suitable for

predictable regeneration of intrabony and furcation

defects.

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Enamel matrix derivative in combination with bone grafts