Case Report

Clinical and Histologic Evaluation of Non-SurgicalPeriodontal Therapy With Enamel MatrixDerivative: A Report of Four Cases

James T. Mellonig,* Pilar Valderrama,* Holly J. Gregory,* and David L. Cochran*

Background: Enamel matrix derivative (EMD) is acomposite of proteins that was demonstrated histolog-ically to work as an adjunct to periodontal regenera-tive surgical therapy. The purpose of this study wasto evaluate the clinical and histologic effects of EMDas an adjunct to scaling and root planing.

Methods: Four patients with severe chronic peri-odontitis and scheduled to receive complete dentureswere accrued. Probing depth and clinical attachmentlevels were obtained. Unlimited time was allowed forhand and ultrasonic instrumentation. A notch wasplaced in the root ‡1 to 2 mm from the apical extentof root planing. EMD was inserted into the pocket,and a periodontal dressing was placed. Patientswere seen every 2 weeks for plaque control. At 6months post-treatment, soft tissue measurementswere repeated, and the teeth were removed en blocand prepared for histomorphologic analysis.

Results: Probing depth reduction and clinical at-tachment level gain were obtained in three-fourthsof the specimens. Three of the four specimens ana-lyzed histologically demonstrated new cementum,bone, periodontal ligament, and connective tissue at-tachment coronal to the notch. In one specimen, thegingival margin had receded below the notch.

Conclusions: The results were unexpected andmay represent an aberration. However, the substan-tial reduction in deep probing depths and clinical at-tachment level gain in three of four specimens, inaddition to the histologic findings of new cementum,new bone, a new periodontal ligament, and a newconnective tissue attachment, suggest that EMDmay be useful as an adjunct to scaling and root plan-ing in single-rooted teeth. J Periodontol 2009;80:1534-1540.

KEY WORDS

Case report; dental scaling; histology; root planing;therapy.

Enamel matrix derivative (EMD) was intro-duced in 19971 and has become one of themost heavily researched dental products.2,3

EMD is a composite of enamel matrix proteins,4 90%of which is composed of porcine-derived ameloge-nin.5 EMD was demonstrated to be safe for humanuse.6 Cohort studies,7 clinical controlled trials,8,9 andhistologic evaluations all attest to its value as anadjunct to periodontal regenerative therapy.10 How-ever, EMD’s greatest benefit may be its potentbiologic effects. EMD stimulates fibroblast prolifera-tion, the growth of periodontal ligament (PDL) cells,osteogenesis, and the proliferation and differentia-tion of osteoblasts;11,12 it also prolongs osteoblastgrowth and enhances trabecular bone regenera-tion,13-15 promotes osteoprotegerin production,12

and enhances osteopontin expression and transform-ing growth factor-beta1 production.16 EMD stimulatesbone sialoprotein, signal transduction of bone mor-phogenetic protein, release of vascular endothelialgrowth factor, and angiogenesis.17 EMD also has anti-inflammatory properties. It limits the release ofproinflammatory cytokines, modulates tumor necrosisfactor-alpha and prostaglandin, and inhibits caspaseactivation.18,19 EMD has a negative effect on thegrowth of periodontal pathogens and might be usefulas an antiadhesive agent for breast cancer cells.20,21

The benefits and limitations of scaling and rootplaning have been well documented.22-24 Several ar-ticles25-27 analyzed the combination of scaling androot planing with EMD. After scaling and root planingand treating the root surfaces with EDTA, EMD wasinserted into one-half of the pockets in 28 subjects.25

After 3 weeks of healing, there was no difference inprobing depth (PD) reduction between test and con-trol sites; however, there was less postoperative painin sites treated with EMD.25 A similar study26 com-pared scaling and root planing alone to scaling androot planing with EMD. After 3 months, there wasno difference in PD reduction and clinical attachmentlevel (CAL) gain. Mombelli et al.27 also compared

* Department of Periodontics, Dental School, University of Texas HealthScience Center at San Antonio, San Antonio, TX. doi: 10.1902/jop.2009.090160

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scaling and root planing with and without EMD;one-half of the subjects also received antibiotics. Atthe 12-month evaluation period, sites treated withscaling and root planing plus EMD plus antibioticsgained a larger amount of clinical attachment. Theabove studies do not support the use of EMD as anadjunct to scaling and root planing. There may bean increased CAL gain if antibiotics are used.

Several studies28-30 reported on the histology ofwound healing after scaling and root planing alone.In 1937, Skillen and Lundquist28 evaluated the histo-logic healing response in one subject. Two root sur-faces were analyzed at 4 weeks. In both specimens,epithelium proliferated and covered the root surfaceso rapidly that reattachment of connective tissue(CT) was prevented. Dragoo29 performed a histologicexamination of wound healing after the scaling androot planing of 20 teeth in 10 subjects. Healing wasobserved at 1, 6, 8, and 12 weeks. In all specimens,the junctional epithelium migrated past the root notchplaced at the apical extent of root planing. No new ce-mentum (NC) was noted in the root notch of any of thespecimens. In another study, five subjects with a totalof 12 teeth and 24 root surfaces were analyzed aftertreatment with scaling and root planing, followed bychlorhexidine rinses in one-half of the subjects. Anotch was placed in the root at the apical extent of in-strumentation. En bloc biopsies were removed after 8weeks. In all instances, healing was by a long junc-tional epithelium, sometimes extending beyond theroot notch.30 Only a single study31 histologicallyevaluated scaling and root planing with EMD in hu-mans; 16 teeth in 16 subjects were removed en bloc6 months after scaling and root planing, 10 with andsix without EMD. No notch was placed in the root sur-face to indicate the apical extent of root planing. All sixspecimens treated by scaling and root planing alonehealed by a junctional epithelium. Two of the 10 spec-imens healed with a histologically insignificant amountof NC (0.5 and 0.2 mm) and new bone (NB; 0.3 and 0.2mm). The purpose of this study was to further investi-gate the histologic response of EMD combined withscaling and root planing.

MATERIALS AND METHODS

Four subjects were recruited who had been diagnosedwith severe chronic periodontitis (PD and CAL >7.0mm) and scheduled for complete dentures (one fe-male and three males; mean age: 58 years; range:37 to 77 years). The subjects were recruited fromMay 2000 until June 2002. Each patient signed a Uni-versity of Texas Health Science Center at San Antonioinstitutional review board–approved consent formthat each individual read and then received a verbalexplanation concerning the details of their participa-tion, including risks and benefits and that they were

free to remove themselves from the study at any time.All of the patients were in good health, and nonesmoked. One clinician (Dr. Mellonig) performed allof the procedures mentioned below. All teeth under-went soft tissue measurements using calibrated Uni-versity of North Carolina 15-mm probes, including

Table 1.

Clinical and Histologic Evaluation of theNon-Surgical Use of EMD at Baseline and6 Months Post-Treatment

Baseline (mm) Post-Treatment (mm)

Patient Site PD REC CAL PD REC CAL

1 #8 M 10 0 10 2 2 4

2 #11 D 15 1 16 5 3 8

3 #8 D 13 2 15 9 3 11

4 #23 D 7 0 7 4 5 9

M = mesial; D = distal.

Table 2.

Clinical and Histologic Evaluation of theNon-Surgical Use of EMD: Histologic andPost-Treatment Clinical Results

Patient Site

PD

Reduction

(mm)

REC

(mm)

CAL

(mm)

Histologic Healing/

Regeneration

1 #8 M 8 2 6 +

2 #11 D 10 2 8 +

3 #8 D 4 1 3 +

4 #23 D 3 5 -2 -M = mesial; + = regeneration; D = distal; - = no regeneration.

Table 3.

Clinical and Histologic Evaluation of theNon-Surgical Use of EMD: HistologicPost-Treatment Measurements

Patient NC (mm) NB (mm)

New PDL

(mm) CT (mm) JE (mm)

1 2.18 1.07 1.07 0.43 0.98

2 2.88 3.21 2.88 0.00 2.86

3 0.42 1.33 0.42 1.02 2.67

4 0.00 0.00 0.00 0.00 -2.59*

CT = new CT attachment; JE = length of junctional epithelium* JE below notch.

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assessment of mobility.Complete supragingivalscaling and root planingwas performed with handand ultrasonic instrumen-tation. No oral hygiene in-structions were given atthis time. Within 1 weekof supragingival scalingand root planing, onetooth in the anterior partof the mouth with exten-sive PD and clinicalattachment loss thatprovided easy access foralveolar ridge augmenta-tion was selected as thetest tooth (Table 1). Thistooth received a secondround of meticulous sub-gingival scaling and rootplaning with ultrasonicinstrumentation using adiamond-tip insert andwithno time limit.Theend-point was reached whenthe operator decided thatthe subgingival root sur-face was clean, and noaccretions could be de-tected with the sharp endof an explorer. No anes-thesia was administered.No splinting or othermethod of tooth stabiliza-tion was used.

At the conclusion ofinstrumentation, a round1/2 bur was inserted intothe pocket as far as itwould reach or until slightresistance was met at thebase of the pocket. Thebur was backed off fromthe base of instrumen-tation ‡2 mm. A notchwas placed on the rootsurface to serve as thehistologic marker for his-tomorphometric measure-ment. EMD,† 30 mg/ml,was inserted into thepocket to the base of the lesion and was allowed to ex-ude from the pocket. No EDTA or other root surfacemodification was used to avoid additional variables.Aperiodontaldressingwasplaced toassist in retention

of the EMD. No antibiotics or analgesics were pre-scribed. The patients returned at 1 week for dressing

Figure 1.Patient 1. A) A notch is evident on the right, outlined by the rectangle. B) High-power view of rectangle in A.C) Magnification of rectangle in A. D) High-power view of square in C showing new CT inserting into newcementum (C). B = new bone; AN = apical border of the notch; CN = coronal border of the notch. (H&E;original magnification: A, ·2; B, ·20; C, ·4; D, ·40.)

† Emdogain, Institute Straumann, Basel, Switzerland.

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removal and supragingivalscaling. Each subject wasseen every 2 weeks forsupragingival scaling andocclusal adjustment, asneeded, for 6 months. At6 months, PD, CAL, andgingival recession (REC)measurements were re-peated (Tables 1 and 2).Each test tooth was re-moveden bloc asdescribedby Bowers et al.,32 with oneexception. The crown of thetest tooth was cut off 2 to 3mmabovethegingivalmar-gin, and a groove was cutinto the occlusal surface ina buccal-lingual direc-tion. After the en bloc bi-opsy was removed, theresidual alveolar ridge de-fects were reconstructedwith the guided bone re-generation technique usingfreeze-dried bone allograft‡

and a bioabsorbable physi-cal barrier.§

The patient was referredfor prosthetic rehabilita-tion after 3 to 4 monthsof wound healing. The pa-tients received completedentures or implant-sup-ported overdentures. Thespecimens were fixed in10% neutral buffer for-malin with 1% CaCl2, de-calcified in EDTA, andembedded in paraffin. Thebuccal-lingual grooveserved to orient the speci-men in the paraffin blockto ensure a cut perpendic-ular to the notch. Thespecimens were sectionedat 6 to 8 mm. Once thenotch was identified, everytenth sectioned histologic section was captured andstained with hematoxylin and eosin (H&E). Onlystained sections in which the entire notch area wasclearly identifiable were subjected to histomorpho-logic analysis using a light microscope with a camera.The digitized image was saved and analyzed usingspecialized image software.i The program was cali-brated to obtain a 1:1 ratio with the linear measure-

ments on the slides. The amount of NB, NC, newPDL, and new CT and the length of junctional epithe-lial attachment were recorded (Table 3).

Figure 2.Patient 2. A) New bone (B), separated from the main body of new bone (B), can be seen in close proximity tothe notch. B) Notch area in large square in A. A thin layer of new cementum (C) is separated from the rootsurface by an artifact (A). C) A higher-power view of the large square in B. New CT inserting into newcementum (C). D = dentin. D) Area seen in small square in A, showing the termination of the junctionalepithelium (JE), artifact (A), and CT fibers inserting into a thin layer of new cementum (C). (H&E; originalmagnification: A, ·10; B, ·20; C and D, ·40.)

‡ Musculoskeletal Transplant Foundation, Edison, NJ.§ Bio-Gide, Osteohealth, Shirley, NY.i Image Pro-Plus Version 6.0, Media Cybernetics, Bethesda, MD.

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RESULTS

The clinical and histol-ogicresultsarepresentedin Tables 1 through 3.

Patient 1The tooth included inthe study was the upperright central incisor,which had an initial PDof 10 mm on the mesialaspect and Miller ClassII mobility. Bone loss,as determined radio-graphically, was almosthorizontal except for aslight <1.0-mm angulardefect. At 6 months,PD was 2.0 mm. Therewas a CAL gain of 6mm. This site healed byregeneration and newattachment (Fig. 1).

Patient 2The tooth included in the analyses was the left maxil-lary canine that initially had an angular bony defect of4.0 mm, with a PD of 15 mm and Class II mobility.There was a notable 10-mm reduction in PD and aCAL gain of 8.0 mm (Table 2). Histologically, healingwas by regeneration. The junctional epithelium ex-tended apical to the alveolar crest by 0.33 mm (Table3; Fig. 2).

Patient 3The tooth included in the study was the right maxillarycentral incisor. This tooth presented a radiographicangular bone defect ;3.0 mm and a PD of 13 mm.At the end of treatment, a significant PD of 9 mm re-mained (Table 1). Wound healing in this patientoccurred by a slight amount of regeneration. NC ex-tended coronal to the root notch by 0.50 mm. New at-tachment of 1.02 mm was by CT adhesion to thedentinal root surface without any NC (Table 3; Fig. 3).

Patient 4The analyzed tooth was the lower left lateral incisor.This tooth presented a CAL of 7.0 mm, a PD of7.0 mm, and Class I mobility (Table 1). There was nonew attachment or regeneration because the gingivalmargin migrated below the root notch (Table 3). Thistooth was not removed for histologic examination.

DISCUSSION

The results of this study suggest that EMD can beused as an adjunct to scaling and root planing in deep

periodontal pockets in which other surgery is notfeasible or is refused by the patient. The findings ofNB, NC, and new PDL were not expected. There areseveral possible explanations for the results in thisstudy.

1) The histologic reference notch was placed in anon-contaminated zone on the root. This is certainlypossible, but every effort was made to put the notch‡2.0 mm above the base of instrumentation. Further-more, the pattern of NB formation suggests that if thiswere true, there should be some evidence of a burwound in bone and subsequent repair evidenced bya reversal line. This was not the case in any of thethree specimens.

2) There was a lack of controls, but this study wasmeant to be a case report of unusual observations.However, there are numerous historic histologic con-trols.28-31 There is a total of 61 human block sectionsof scaling and root planing in humans, all healed byjunctional epithelium. In addition, there are ;39 blocksections in humans of wound healing after treatmentby open flap debridement.32-35 All of these sectionshealed by junctional epithelium to the apex of thenotch and sometimes beyond, with no evidence of ce-mentum. The evidence seems to be overwhelmingthat treatment by scaling and root planing results inan epithelial attachment.28-31 Therefore, includingcontrols in this study seems superfluous. Further-more, the power of a randomized controlled clinicalstudy would be much higher than four subjects. Thiswould make such a study prohibitive from a financialand subject point of view.

Figure 3.Patient 3. A) PDL is in close proximity to the notch. B) Magnification of area of notch in lower left square in A. Alayer of new cementum (C) is separated from the dentin (D) of the root surface by an artifact space (A). CT canbe seen inserting into new cementum (C). C) Magnification of upper right square in A, demonstrating new boneformation (B) and CT fibers inserting into new bone. (H&E; original magnification: A, ·2; B and C, ·40.)

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3) There was a bias toward favorable results; how-ever, just the opposite was true because biologicevidence indicates that to regenerate the periodon-tium, the fiber attachment at the base of the defectmust be removed to allow the egress of cells fromthe PDL.36,37

4) There was no limit on the time for scaling androot planing. Every study22,23 that evaluated the re-moval of subgingival calculus on single-rooted teethindicated that a significant percentage of calculus re-mained. This is also more than likely for the cases inthis report.

5) EMD promotes apoptosis of epithelial cells.5

Anything that delays the downgrowth of epithelium al-lows time for the regeneration of the attachment appa-ratus.

6) EMD has a negative effect on the growth of pu-tative periodontal pathogens.20 This would be similarto the effect of antibiotics and would promote subgin-gival wound healing.

7) EMD has a myriad of biologic effects. EMD pro-motes cementogenesis and osteogenesis13,38 andstimulates the release of bone inductive, growth,and anti-inflammatory factors from cells.18

8) A 2-week recall would help to control bacterialplaque and, therefore, promoted a better outcome.39

9) It is also possible that the results reported heremay be an aberration because the results in other ar-ticles25,26,31 after scaling and root planing with andwithout EMD showed little or no difference with regardto clinical or histologic parameters.

10) Creating a root notch for histologic observa-tions may have fostered NC in the notch. This seemsunlikely because after scaling and root planing, aswell as open flap debridement, histologic findings inhumans showed healing with epithelium to or beyondthe notch, with no NC.33-35

11) The use of a diamond-tipped instrument dur-ing root planing may have removed epithelium, ex-posed CT, and induced attachment. This also seemsunlikely because histologic findings in humans inwhich epithelium removal was attempted with aninternal bevel or crestal incision showed that it was in-effective in removing pocket epithelium.40 Further-more, there is no human histologic evidence thatthe removal of all pocket epithelium will result inNB, NC, and new PDL.

The results obtained in this study are not just a sim-ple matter of scaling and root planing and placingEMD into the pocket and in no way should be consid-ered a substitute for surgery where it is appropriate.Each test tooth received subgingival scaling and rootplaning with a diamond-tipped ultrasonic instrumentin an aggressive manner as possible without anesthe-sia. A strong attempt was made to instrument to the

base of the pocket. The average instrumentation timein this study ranged from 9 to 11 minutes for a single-rooted tooth. Therefore, this procedure is just as de-manding as surgery; the duration of treatment maybe longer, and the results may be less predictable.

CONCLUSIONS

This study demonstrated that periodontal regenera-tion may be possible after the use of EMD combinedwith scaling and root planing. This technique mayhave use in patients in whom surgery is not an optionor in those who refuse surgical intervention. The re-sults of this study may also be viewed as an aberrationbecause other studies25,26,28-31 did not confirm theclinical or histologic results reported here.

ACKNOWLEDGMENTS

This study was funded by a grant from Biora, Malmo,Sweden, now Institute Straumann, Basel, Switzerland.Dr. Cochran has conducted research using productsmade by the Straumann Company and has lecturedon those products. Some lectures may have beensponsored by the company.Drs. Mellonig,Valderrama,and Gregory report no conflicts of interest related tothis case report.

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31. Sculean A, Windisch P, Keglevich T, Gera I. Histologicevaluation of human intrabony defects followingnon-surgical periodontal therapy with and withoutapplication of an enamel matrix protein derivative.J Periodontol 2003;74:153-160.

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Correspondence: Dr. James T. Mellonig, Department ofPeriodontics, The University of Texas Health ScienceCenter at San Antonio, 7703 Floyd Curl Dr., San Antonio,TX 78229. E-mail: mellonig@uthscsa.edu.

Submitted March 20, 2009; accepted for publication May13, 2009.

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