the international journal of periodontics & restorative ......basis.12,15 in a multicenter...

The International Journal of Periodontics & Restorative Dentistry

Human histologic evidence ofregeneration of the periodontal sup-porting tissues—including cemen-tum, connective tissue attachment,and alveolar bone—has beendemonstrated with various treat-ment materials including autoge-nous bone1–3; demineralized freeze-dried bone allograft (DFDBA)4,5;Bio-Oss (Geistlich) alone,6 in combi-nation with Bio-Gide (Geistlich),7 orin combination with autogenousbone and Bio-Gide8; and enamelmatrix derivative (EMD; Emdogain,Biora).9–14

Several reports have demon-strated that EMD can yield peri-odontal regeneration on previouslydiseased root surfaces inhumans,10–14 but on an inconsistentbasis.12,15 In a multicenter 10-caseseries, histologic evaluation of theregion coronal to the base of thecalculus notch showed evidence ofregeneration in 3 of 10 intrabonydefects treated with citric acid con-ditioning and EMD.12 In these 3cases, connective fiber insertion intoboth new bone and new cemetumwas demonstrated. A parallelarrangement of the connective tis-

Two Patterns of Histologic Healing in an Intrabony Defect FollowingTreatment with Enamel MatrixDerivative: A Human Case Report

Zeina Majzoub, DCD, DMD, MScD*Marina Bobbo, DDS**Fady Atiyeh, DCD***Giampiero Cordioli, MD, DDS****

Human histologic evidence of periodontal regeneration following treatment ofintrabony defects with enamel matrix derivative (EMD) has yielded inconsistentresults in recent case reports. A 46-year-old woman presenting one deep intra-bony defect at the distal root of a mandibular first molar scheduled for extractionwas selected for EMD therapy. During surgery, a notch was placed at the mostapical level of calculus on the experimental root. Nine months postsurgery, ablock section including the distal root and surrounding periodontal tissues wasobtained and processed in a mesiodistal plane for histologic evaluation.Histologic analysis demonstrated two different patterns of healing along theproximal and furcal surfaces. Regeneration with new cellular cementum, bone,and periodontal ligament with functional fiber orientation was observed on thedistal aspect of the root, whereas the furcal surface healed through ankylosis. Thisreport underlines the biologic variability in wound healing following EMD therapyin periodontal intrabony defects and within the same defect. Host-specific intrin-sic and/or extrinsic factors accounting for this variability remain to be investigat-ed. (Int J Periodontics Restorative Dent 2005;25:xxx–xxx.)

*Assistant Professor, Department of Periodontology, Institute of ClinicalDentistry, University of Padova, Italy.

**Clinical Fellow, Department of Oral Surgery, Institute of Clinical Dentistry,University of Padova, Italy.

***Associate Professor, Department of Periodontology, School of Dentistry,Lebanese University, Beirut.

****Professor and Chair, Department of Periodontology, Institute of ClinicalDentistry, University of Padova, Italy.

Correspondence to: Dr Giampiero Cordioli, Università Degli Studi diPadova, Istituto di Clinica Odontoiatrica, Via Giustiniani 2, 35100 Padova,Italy. Fax: + 39 (049) 8218229. e-mail: [email protected]

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Volume 25, Number 2, 2005

sue fibers of the periodontal liga-ment (PDL) was also observed. Bothcellular and acellular new cementumwere identified, deposited on bothold cementum and dentin. Threeother defects yielded connective tis-sue attachment/adhesion, whereashealing in the remaining 4 sitesresulted in a long junctional epithe-lium.12

Sculean et al11 report the for-mation of 1.88 mm and 4.82 mm ofnew cementum of a mixed cellularand acellular type with inserting col-lagen fibers covering the previouslydiseased root surface in two deep,combined one- and two-walleddefects at two maxillary centralincisors extracted 6 months follow-ing treatment with EMD. These twodefects showed 1.77 mm of newbone and no bone regrowth, respec-tively.

In two human biopsies evalu-ated 6 and 9 months following treat-ment of one- and two-walled intra-bony defects with EMD, only slightregeneration was evidenced at themost apical level of the notch pre-pared at the bottom of the intrabonydefect.15 A slight amount of newbone was observed, but there wasno new cementum formation. Noroot resorption or ankylosis wasobserved in any of the above-men-tioned human histologic reports.

The aim of the present casereport is to present histologic woundhealing observed 9 months follow-ing treatment of a human periodon-tal intrabony defect with EMD.

Method and materials

The patient selected for treatmentwith EMD was a 46-year-old femalenonsmoker in good physical healthand with no contraindication for sur-gical periodontal therapy. Thepatient was diagnosed with gener-alized moderate chronic periodonti-tis and presented one deep intra-bony defect at the mandibular firstmolar that was considered to have ahopeless prognosis. The patient hadreceived comprehensive periodontaltherapy, including scaling and rootplaning in all quadrants and apicallypositioned flaps in the maxillary andmandibular left quadrants.

The initial therapy was per-formed on all teeth in the mandibu-lar right quadrant, but not on theexperimental tooth. No occlusaladjustment or odontoplasty was per-formed on the experimental toothprior to therapy, as fremitus was notdetected, nor were contactsobserved in centric occlusion orexcursive or lateral mandibularmovements. The mandibular firstmolar was scheduled for extractionbecause of advanced bone loss,through-and-through furcationinvolvement, and a history of multi-ple periodontal abscesses. Thepatient was informed about thenature of the project, and she gaveinformed consent to have the distalroot of the molar surgically removed,together with the surrounding peri-odontal tissues, following EMD ther-apy. This procedure was approvedby the Ethics Committee of theUniversity of Padova Institute ofClinical Dentistry.

Pre- and postsurgical documen-tation consisted of clinical pho-tographs, standardized periapicalradiographs, and clinical measure-ments made just prior to surgery and9 months postoperative. Clinicalparameters included probing depth,recession, and clinical attachmentlevel (CAL), with the cementoenameljunction (CEJ) as the reference point.All clinical measurements were car-ried out using a periodontal probe(XP23/UNC15, Hu-Friedy) androunded to the nearest 0.5 mm(Table 1). Radiographic measure-ments to detect bone level gainbetween the two examinations weremade by superimposing a transpar-ent millimeter grid on the radi-ographs, using the amalgam apicalmargin and the roof of the furcationas reference points at the distal andfurcal aspects, respectively. The radi-ographic distance between theselandmarks and the bottom of thedefect was measured and roundedto the nearest 0.5 mm (Table 1).

The surgical procedure and clin-ical measurements were performedby the same clinician. Reverse bevelsulcular incisions were made at thebuccal and lingual surfaces of thefirst and second molars, with releas-ing incisions at the mesial line anglesof the first premolar, and full-thick-ness flaps were reflected. Thoroughremoval of granulation tissue withinthe defect was performed, avoidingdisturbance of the calculus presenton the root surface. A notch servingas a landmark for the histologic mea-surements was made into the distalroot through and along the apicalextent of calculus with a No. 1/4

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round bur, using fiberoptic illumina-tion and magnifying loops toenhance visibility. Following notchpreparation, scaling and root planingwere carried out using hand andultrasonic instruments in an attemptto create a hard root surface coronalto the reference notch. Special carewas taken to preserve the integrity ofthe notch during root surface instru-mentation.

Defect morphology (Fig 1) andmeasurements from the CEJ to thealveolar crest and to the depth of theosseous defect were documented.The flaps were tested for closure,and the tissues were presuturedusing a horizontal internal mattresssuture in the defect-associated dis-tal space.16 The root surface wastreated for 15 seconds with topicalapplication of 37% orthophosphoric

surgery. The patient was seen 7 daysposturgical for wound inspectionand removal of nonstabilizingsutures. Remaining sutures wereremoved at 3 weeks. The patientwas asked to avoid mechanicalplaque control until healing had pro-gressed sufficiently to allow normaloral hygiene measures. She wasrecalled for oral hygiene reinforce-ment and professional supragingi-val plaque control on a monthly basisfor the first 6 months and every 3months thereafter. Subgingivalinstrumentation was not performedat any of the recall appointments.

Nine months postoperative (Fig2), a second surgical procedure wascarried out to remove the distal rooten bloc. Full-thickness flaps werereflected on the buccal and lingualaspects of the tooth, leaving undis-

acid, then thoroughly rinsed withphysiologic solution according tothe manufacturer’s recommenda-tions. Subsequently, the EMD wasapplied to coat the entire extent ofthe exposed root surface and slightlyoverfill the defect. The suture (No. 5-0 monofilament; Ethilon, Ethicon/Johnson & Johnson) was then tight-ened and supplemented by inter-rupted sutures16 to achieve com-plete coverage over the EMD sites,flap positioning at the CEJ, and mar-ginal tissue adaptation to the rootsurface.

The patient was given a nons-teroidal antiinflammatory drug for 3days and antibiotics (doxycycline100 mg two times daily) for 10 dayspostoperative and was instructed torinse with chlorhexidine 0.2% threetimes daily for 6 weeks following

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Table 1 Clinical and radiographic measurements (mm) at baseline and 9 months following EMD therapy

Baseline clinical Intraoperative osseous Postoperative clinical RadiographicSurface PPD REC CAL CEJ–crest CEJ–bottom of defect PPD REC CAL Baseline 9 mo

Distal 10.0 1.0 11.0 3.5 12.0 3.0 3.0 6.0 13.0 8.5Furcal 12.0 0.0 12.0 5.5 13.0 3.0 3.5 6.5 9.5 4.0

EMD = enamel matrix derivative; PPD = probing pocket depth; REC = recession; CAL = clinical attachment level; CEJ = cementoenamel junction.

Fig 1 (left) Intraoperative buccal view ofcircumferential intrabony defect at distalroot of mandibular first molar associatedwith through-and-through furcationinvolvement.

Fig 2 (right) Buccal view of mandibularfirst molar 9 months postoperative, justprior to biopsy procedure. Note slightmarginal gingival inflammation in inter-radicular area and soft tissues partially fill-ing buccal entrance of furcation.

turbed the marginal buccal, distal,and furcal tissues. The two rootswere then separated, and the mesialroot was extracted (Fig 3a). Verticalincisions converging apically weremade down to the bone surfacethrough the marginal distal and fur-cal gingival tissues at approximately1 mm and 2 mm, respectively, fromthe distal and mesial line angles ofthe root. The vertical incisions delim-ited a triangular area extendingapproximately 10 mm apical to thegingival margin. These incisionswere subsequently extendedthrough most of the buccolingualwidth of the alveolar bone up to thelingual surface of the distal root witha No. 700L cross-cut fissure burmounted on a high-speed hand-piece under sterile water irrigation.The root with the surrounding tis-sues, including the buccal alveolarplate along with wedges of proxi-mal and furcal hard and soft tissues,

were removed (Fig 3b). Bio-Oss wasplaced into the biopsy and extrac-tion sites and covered with a Gore-Tex membrane (WL Gore) to aug-ment the residual ridge and preparethe edentulous area for futureimplant placement. The buccal andlingual flaps were released throughapical periosteal incisions and coro-nally respositioned to allow for com-plete coverage of the membrane.

The block specimen was imme-diately rinsed with sterile saline andfixed in 10% neutral buffered for-malin solution for histologic pro-cessing. After fixation, the specimenwas decalcified in 5% formic acid forabout 4 weeks, embedded in paraf-fin, and sectioned step serially at lev-els 50 µm apart, along a plane par-allel to the long axis of the root in amesiodistal direction at a thicknessof 5 to 6 µm. For the histologic analy-sis, four sections 50 µm apart, rep-resenting the most mid-distal area of

the root, were stained with hema-toxylin-eosin and trichrome stainsand examined under a light micro-scope. Linear measurements weremade to assess the newly formedcementum, connective tissue attach-ment or adhesion, and boneregrowth coronal to the referencenotch. Qualitative histologic para-meters, including root resorption,ankylosis, direction of fibers of newlyformed connective tissue, type ofcementum formation, and presenceof inflammation, were also evalu-ated.

Results

Clinical and radiographic findings

The patient consistently demon-strated good compliance with peri-odontal maintenance throughout

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Fig 3a (left) Buccal view of rightmandibular area during biopsy procedure,following extraction of mesial root.Attempt was made to maintain the integri-ty of the marginal soft tissues at buccal,mesial, and distal aspects of distal root.

Fig 3b (right) Distal root immediately fol-lowing its removal en bloc with surround-ing periodontal hard and soft tissues.

the 9-month postoperative healingphase. She maintained adequatelevels of plaque control, except inthe furcation area. This was partiallyfilled with interradicular soft tissues,allowing only limited access for oralhygiene tools (Fig 2).

The intraoperative morphologiccharacterization of the defect at thedistal root demonstrated a supra-bony component associated with adeep intrabony circumferentialdefect involving all four surfaces ofthe root (Fig 1). Preoperative, CALwas 11.0 mm and 12.0 mm on thedistal and furcal aspects, respec-tively. The corresponding gains inCAL at 9 months postoperative were5.0 mm and 5.5 mm. Probing depthreduction of 7.0 mm and 9.0 mmwas observed at the distal and furcalsites, respectively. Significant newhard tissue formation was observedradiographically, with a gain in boneheight from the base of the defect of4.5 mm and 5.5 mm at the distaland furcal surfaces, respectively (Fig4). A well-defined crestal lamina durawas radiographically evident in thedistal interproximal area. Tooth vital-ity was positive prior to surgery and9 months postoperative. Increasedmobility of degree 2 was observedat baseline and reduced to less than

degree 1 prior to the biopsy proce-dure.17

Histologic and histomorpho-metric findings

Two distinct histologic healing pat-terns were evidenced along theproximal and furcal aspects of thedistal root (Fig 5): regeneration atthe distal surface and ankylosis atthe furcal surface. [AU: Edit OK?]

Regeneration with new cemen-tum, bone, and connective tissueattachment was observed coronalto the notch at the proximal aspectof the distal root (Figs 6 to 8). Thenewly formed cementum was uni-formly thick, predominantly cellularin nature with embedded cemento-cyte-like cells, and deposited overthe old cementum. In the coronalaspect of the root, an artifactual sep-aration of the newly formed cemen-tum from the root surface was pre-sent, most likely related to thehistologic processing. No resorptionlacunae were observed at the old-new cementum interface, indicatingthat the new cementum formationwas not preceded by a superficialresorption of the old cementum sur-face. The intervening dense con-

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Fig 4a (left) Baseline periapical radi-ograph.

Fig 4b (right) Nine-month postoperativeradiograph shows bone fill in both distaland furcal components of intrabony defectand well-defined crestal lamina dura in dis-tal interproximal area.

Fig 5 Histologic overview of a centralsection shows both proximal (left) and fur-cal (right) aspects of distal root. Proximalsurface demonstrates regeneration withnew cementum, bone, and connective tis-sue attachment in area demarcated by redarrows. Healing through ankylosis can beseen at furcal aspect in area delineated byblue arrows (hematoxylin-eosin stain; origi-nal magnification � 3).

nective tissue layer between the newcementum and newly formed alve-olar bone was inserted into both tis-sues and displayed functional fiberorientation. Although the most coro-nal extension of the newly formedcementum could not be clearly iden-tified on any of the examined sec-tions, new bone formation seemedto parallel cementum depositionthroughout most of the defect.Histomorphometrically, new cemen-tum covered nearly 3.22 mm,whereas bone extended 3.63 mmcoronal to the base of the referencenotch.

The marginal gingival connec-tive tissue in correspondence withthe junctional epithelium at the fur-cal root surface exhibited an inflam-matory cellular infiltrate extendingin isolated clusters within thesupracrestal transseptal fibers (Figs9 to 11). New dense cortical bonepresenting a multilayered arrange-ment with numerous embeddedosteocytes was observed in directcontact with the old cementum sur-face up to 4.17 mm coronal to thereference notch. In one specimenstained with hematoxylin-eosin, asmall area of mineralized cellular

tissue within the notch area was ini-tially interpreted as cementum-likeformation. The same root-associ-ated cementum-like tissue ap-peared partially confluent with andindistinguishible from the alveolarbone in a section 50 µm away.Numerous small, shallow resorp-tion lacunae on the old cementumsurface and matching interdigita-tions at the bone–old cementuminterface were observed, indicat-ing that a resorptive activity pre-ceded the subsequent mineralizedtissue formation against the rootsurface.

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Fig 6 Higher magnification of distal sur-face demonstrates new attachment appa-ratus coronal to base of calculus notch (N).[AU: Please indicate where “N” shouldbe shown on the figure. (Also in Fig 9.)]Note artifactual separation between newlyformed cementum and root surface incoronal portion of defect. Although themost coronal extension of new cementumcannot be clearly identified, bone regener-ation appears to parallel new cementumdeposition (between arrows) (hematoxylin-eosin stain; original magnification � 8).

Fig 7 Higher magnification of area withinbottom frame in Fig 6 shows new cellularcementum, with inserting connective tis-sue fibers deposited over old cementum(hematoxylin-eosin stain; original magnifi-cation � 62).

Fig 8 Higher power of artifactual splitarea (top frame in Fig 6) demonstratesnewly formed cellular cementum separat-ed from root surface. Inserting connectivetissue fibers can be observed at newcementum surface (hematoxylin-eosinstain; original magnification � 62).

Discussion

This human histologic case reportdemonstrated significant improve-ment of CAL and radiographic bonefill following treatment of a peri-odontal intrabony defect with EMD,but a dual histologic healing patternwith regeneration at the proximalaspect and ankylosis through mostof the furcal surface of the same root.

The regeneration at the proxi-mal aspect of the distal root can becompared to the histologic out-come described in other humancase reports.10–14 The functionalfiber orientation of the PDL, pre-dominantly cellular nature of thenewly formed cementum, and itsdeposition on old cementum in thepresent case are in line with earlierfindings.11–13 The split between thenewly formed and old cementum inthe coronal aspect of the defectcan be attributed to an artifactualseparation. This is confirmed by thefact that the new cementum wasfirmly attached to the root surfacein the apical portion of the defect.Nevertheless, it cannot beexcluded that the artifactual micro-scopic split could have resultedfrom a weak attachment betweenthe regenerative cementum androot hard tissues, similar to whathas been observed with cementumformed under guided tissue regen-eration conditions in humanteeth.18 In contrast with previousresults that have demonstrated adiscrepancy between boneregrowth and connective tissueattachment formation,11–14 boneregeneration in the present report

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Fig 10a Higher magnification of areawithin bottom frame in Fig 9 shows slenderlayer of what appears to be cellularcementum attached to root surface andthinning out coronally (between arrows).Connective tissue with parallel fiber orien-tation separates this tissue from newlyformed bone (hematoxylin-eosin stain;original magnification � 25).

Fig 10b Pinpoint fusion of bone andcementum-like tissue in isolated areas innotch (arrows) within section 50 µm away[AU: Away/apart from what point?](trichrome stain; original magnification �25).

Fig 9 Furcal surface of distal root showsregenerated bone ankylosed to root sur-face (arrows) coronal to calculus notch (N)(hematoxylin-eosin stain; original magnifi-cation � 6).

appeared to parallel new cemen-tum deposition.

The ankylotic pattern observedin the present report does notappear to be a transient phenome-non like the bony union demon-strated during early healing inrodents19,20 following treatment ofsurgically induced fenestrationdefects and in monkeys21 followingreplantation of extracted incisors.Unlike the early ankylosis that dis-appears at the later stages of woundhealing with restoration of the PDLspace, the ankylosis in this reportwas still detected 9 months postop-erative along the entire root surface

coronal to the reference notch, prob-ably indicating its irreversible nature.The permanent character of thisankylosis and the unlikely possibilityof developing reparative cementumand reestablishing a normal PDLspace were further confirmed by theestablishment of a regenerationhealing pattern at the correspondingproximal surface of the same root atthe same postoperative interval.Conversely, the absence of multinu-cleated cells at the root-bone inter-face in the present case suggeststhat the irregular root surface profilewas established through a resorp-tive process at the earlier healingperiod and may corroborate thenonprogressive nature of the resorp-tion-ankylosis phenomenon at thebiopsy time point.

Bone tissue formation in directcontact with denuded root surfacesis not only limited to injury to thedentoalveolar structures,22–25 butrather a common finding followingcertain periodontal proce-dures.1,26–40 Various degrees of rootresorption and ankylosis have beenreported with a variety of therapeu-tic modalities and materials used inthe treatment of periodontal defects,including fresh iliac bone mar-row,1,26–29 platelet-derived growthfactor (PDGF) with citric acid appli-cation,30 bone morphogenetic pro-tein (BMP)-2,31,32 BMP-3 (osteo-genin) combined with DFDBA,33

osteogenic protein (OP)-1,34 androot surface demineralization.35–40

Beyond the well-known re-versible or irreversible damage tothe PDL cells implicated in ankylosisassociated with dental trauma, a

number of hypotheses were pro-posed to explain the ankylosis-inducing mechanisms in periodontaltherapy-related studies. Replace-ment ankylosis observed in fresh iliacbone marrow–treated sites has beenattributed to the presence of viablebone cells transferred with the graftinto the periodontal defect and act-ing directly or indirectly on theregenerating periodontal tissues.28

In biologically modulated periodon-tal therapy,30–34 the occurrence ofankylosis is complicated to interpretbecause of the lack of cellular speci-ficity, wide array of biologic activitiesof most molecular mediators, andcomplex interaction between thevarious molecular signals at a woundsite. In citric acid therapy in animalmodels,35–37,40 various factors havebeen advocated as potential anky-losis-inducing mechanisms. The inci-dence of ankylosis is greater at citricacid–treated than non–acid treatedteeth,40 at increased distance fromthe base of the treated lesion,35,36

and in larger defects.35 Furthermore,root resorption and ankylosis are sig-nificantly correlated with the magni-tude of connective tissue attach-ment, suggesting that rootresorption and ankylosis frequentlyoccur following the accomplishmentof connective tissue repair overextended portions of the treatedroot surfaces.36,38

In addition to the factors inher-ent to the surgical procedure and/orregenerative material, external stim-uli such as occlusal forces and mas-ticatory function have been impli-cated as potential regulators of cellturnover in the PDL and modifiers of

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Fig 11 Higher magnification (top framein Fig 9) shows ankylotic union betweennewly formed bone arranged in multilayersand root surface. Old cementum showsnumerous small, shallow resorption lacu-nae in perfect interdigitation with osseousprofile (hematoxylin-eosin stain; originalmagnification � 50).

the healing response.19,41–43 Finally,research-related considerations suchas the animal and defect models37

and the submerged toothmodel31,33,44 have been reported tobe more frequently associated withankylosis.

The ankylosis at the furcal aspectis difficult to interpret in light of theexisting in vitro, animal, and humanhistologic data related to EMD ther-apy. The in vitro findings relative tothe mechanisms of action of EMDassessed in a variety of cells45–58

have been, at least in part, contra-dictory. The discrepancies in thesestudies have been attributed to dif-ferences in EMD concentrations, celllines and densities, growing condi-tions and experimental designs, andmethods applied.59 EMD has beenshown to significantly stimulate pro-liferation of human PDL cells,45,46,57

modulate matrix synthesis by humanPDL cells,46,47 enhance attachmentand spreading of human PDL fibrob-lasts to culture plates coated withEMD,48 and promote proliferationof transgenic mice cementoblasts.49

Although caution should be appliedwhen extrapolating from in vitro find-ings to actual wound healingprocesses, these effects of EMD onthe PDL cellular lines, coupled withEMD’s inhibitory effect on epithelialproliferation,51,57 are in line with therequirements of periodontal regen-eration and the results of clinical andanimal studies showing that EMDmay prevent ankylosis in trans-planted and replanted teeth.60–63

Inconsistent results have alsobeen reported relative to the specificeffect of EMD on the differentiation,

nants. Root surface preparation hasbeen conventionally performed withorthophosphoric acid 37%,15,74–79

citric acid80,81 at pH 1, or ethylene-diaminetetraacetic acid (EDTA) 24%at neutral pH.15,79,82–92 The favor-able therapeutic results obtainedwith all three agents and positiveoutcomes demonstrated withoutany chemical conditioning suggestthat specific chemical root prepara-tion may not be required to achievesignificant adjunctive clinicalimprovements in EMD therapy.Further studies are needed to inves-tigate the potential effects of rootsurface conditioning combined withEMD therapy.

Second, marginal inflammationin the furcation area could havealtered the phenotypic expressionof mesenchymal cells or altered themultifunctional effect of EMD onthese cells. While the patient’s over-all interproximal plaque and bleed-ing scores were optimal throughoutthe follow-up period, the interradic-ular furcation site of the first molarshowed persistent signs of inflam-mation because of access limita-tions.

Third, the root surface in the fur-cation area could have, as a result ofdifferent inherent characteristicsrelated to the nature of cementum93

and presence of concavities,94 pro-vided a different substratum for therepair tissues.

The ankylosis pattern at the ref-erence notch in this report appearssimilar to the “pinpoint” fusion ofbone and cementum observed inisolated areas following grafting ofhuman intrabony defects with

mitogenesis, and biosynthesis ofbone cell lineages.59 However, themajority of in vitro49,50,52–56 and ani-mal studies64–68 suggest osteopro-motive properties of EMD.Considering that the inherent multi-functional characteristics of EMD donot justify its selective behavior at asingle wound site with a combinedregeneration-ankylosis healing pat-tern, analysis of the extrinsic and/orintrinsic factors that could have gen-erated different histologic outcomesat two adjacent aspects of the sameroot suggests that the ankylosis inthe furcal site may be related to thefollowing considerations.

First to be examined is thepotential effect of acid remnants atthe base of the defect at its furcationaspect to inflict severe chemicalinjury on the coronal portion of thePDL and interradicular crest, whichprovide the cellular elements forregeneration of cementum andPDL.69,70 [AU: Edit OK?] This couldhave been caused by the difficultaccess to the bottom of the furca-tion-associated defect, resulting inpartial washout of the orthophos-phoric acid used to condition theroot surface. The necrotizing effectof low-pH orthophosphoric acid mayhave impaired the vitality and sub-sequently the healing potential ofprecursor cells involved in cemen-tum and connective tissue forma-tion.71–73 Root surface conditioningwas used in the present study, as ithas been advocated as a prerequi-site of the EMD protocol to allowremoval of the smear layer and pre-cipitation of the EMD proteins ontoa root surface free of organic rem-

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DFDBA and osteogenin.33 Similar tothe present report, those authorsindicate that bone and cementumremain fused for three to four serialsections and then become sepa-rated by a PDL space. They point outthat this type of ankylosis is differentfrom replacement-type ankylosisand speculate that it might occurwhen the PDL formation fails to keeppace with the rapidly formingcementum and bone.

With the limited information thatcan be drawn from this case report,it may be concluded that the use ofEMD in intrabony defects yields sat-isfactory clinical and radiogaphicoutcomes. However, under the exist-ing clinical conditions and surgicalmodality applied in the present case,healing at 9 months postoperativewas mediated through a dual histo-logic healing pattern, with regener-ation at the proximal aspect andankylosis through most of the furcalsurface of the same root. Host-spe-cific intrinsic and/or extrinsic factorsaccounting for this variability need tobe further investigated.

Acknowledgments

The authors would like to thank Mr GiorgioMichelotto, Histology Technician, Instituteof Dermatology, University of Padova, forhis help in the histologic preparation of thespecimens. The authors also acknowledgethe assistance of Drs Luca Landi andGabriella Grusovin in data analysis and inter-pretation.

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