Effects of EMD liquid(Osteogain) on periodontalhealing in class III furcationdefects in monkeysShirakata Y, Miron RJ, Nakamura T, Sena K, Shinohara Y, Horai N, Bosshardt DD,Noguchi K, Sculean A. Effects of EMD liquid (Osteogain) on periodontal healing inclass III furcation defects in monkeys. J Clin Periodontol 2017; 44: 298–307. doi: 10.1111/jcpe.12663

AbstractAim: To evaluate the effect of a novel liquid carrier system of enamel matrixderivative (Osteogain) soaked on an absorbable collagen sponge (ACS) upon peri-odontal wound healing/regeneration in furcation defects in monkeys.Materials and Methods: The stability of the conventional enamel matrix deriva-tive (Emdogain) and Osteogain adsorbed onto ACS was evaluated by ELISA.Chronic class III furcation defects were created at teeth 36, 37, 46, 47 in threemonkeys (Macaca fascicularis). The 12 defects were assigned to one of the follow-ing treatments: (1) open flap debridement (OFD) + ACS, (2) OFD+Emdogain/ACS, (3) OFD+Osteogain/ACS, and (4) OFD alone. At 16 weeks followingreconstructive surgery, the animals were killed for histological evaluation.Results: A 20–60% significantly higher amount of total adsorbed amelogenin wasfound for ACS-loaded Osteogain when compared to Emdogain. The histomor-phometric analysis revealed that both approaches (OFD + Emdogain/ACS andOFD + Osteogain/ACS) resulted in higher amounts of connective tissue attach-ment and bone formation compared to treatment with OFD + ACS and OFDalone. Furthermore, OFD + Osteogain/ACS group showed higher new attach-ment formation, cementum, and new bone area.Conclusions: Within their limits, the present data indicate that Osteogain pos-sesses favourable physicochemical properties facilitating adsorption of amelogeninon ACS and may additionally enhance periodontal wound healing/regenerationwhen compared to Emdogain.

Yoshinori Shirakata1,Richard J. Miron2,3,

Toshiaki Nakamura1, Kotaro Sena1,Yukiya Shinohara1, Naoto Horai4,

Dieter D. Bosshardt5,Kazuyuki Noguchi1 and

Anton Sculean6

1Department of Periodontology, Kagoshima

University Graduate School of Medical and

Dental Sciences, Kagoshima, Japan;2Department of Periodontology, Nova

Southeastern University, Fort Lauderdale, FL,

USA; 3Department of Periodontics and Oral

Medicine, University of Michigan School of

Dentistry, Ann Arber, MI, USA; 4Shin Nippon

Biomedical Laboratories, Ltd, Kagoshima,

Japan; 5Robert K. Schenk Laboratory of Oral

Histology, University of Bern, Bern,

Switzerland; 6Department of Periodontology,

School of Dental Medicine, University of

Bern, Bern, Switzerland

Key words: absorbable collagen sponge;

carrier; class III furcation defect; enamel

matrix proteins; periodontal regeneration

Accepted for publication 5 December 2016

Pre-clinical and clinical studies haveprovided substantial evidence on thebiologic potential of an enamelmatrix derivative (EMD) to promoteperiodontal wound healing/regenera-tion and improve the clinical out-comes in intra-bony, furcation, andrecession-type defects (Miron et al.2016a). However, due to its fluid

Conflict of interest and source of funding statementThe authors report no conflicts of interest related to this study.This study was partly supported by a Grant-in-Aid for Scientific Research C (No.25463052 to Dr. Y. Shirakata) and a Grant-in-Aid for Scientific Research B (No.15H05036 to Dr. K. Noguchi) from Japan Society for the Promotion of Science.The authors declare no conflict of interests regarding the materials tested in thisstudy. The materials were kindly provided by Straumann, Basel Switzerland, andBotiss, Berlin Germany.

© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd298

J Clin Periodontol 2017; 44: 298–307 doi: 10.1111/jcpe.12663

consistency, the use of EMD gel(Emdogain) appears to possess lim-ited space-making potential which,in defects with a more complicatedanatomy (e.g. so called non-con-tained-type defects), may not be ableto prevent the collapse of a mucope-riosteal flap thus limiting the avail-able space for regeneration(Mellonig 1999, Lekovic et al. 2000,Cochran et al. 2003, Shirakata et al.2007). In order to overcome thesepotential shortcomings, cliniciansoften combine various types of bonegrafting materials with Emdogain,especially when treating non-con-tained-type defects (Sculean et al.2011). The combination of Emdo-gain and different types of graftingmaterials has been extensively evalu-ated in pre-clinical and clinical stud-ies (Lekovic et al. 2000, Cochranet al. 2003, Shirakata et al. 2007,Gurinsky et al. 2004, Bokan et al.2006, Kuru et al. 2006, Yilmaz et al.2010). Although a recent systematicreview reported that the combinationof Emdogain with bone graftingmaterials may lead to statisticallysignificantly higher clinical improve-ments, the analysis also revealed ahigh heterogeneity between the clini-cal outcomes obtained with the vari-ous combination approaches(Matarasso et al. 2015). One possiblereason for the high clinical variabil-ity in the outcomes when using thecombination of Emdogain and graft-ing materials may be related to sub-stantial differences in enamel matrixproteins (EMPs) adsorption and sub-sequent cell proliferation betweenthe utilized bone grafting materials(Miron et al. 2015). In this respect,very recent data from a series ofin vitro studies revealed that the useof EMD without its propylene-gly-col-alginate (PGA) carrier markedlyimproved protein adsorption whencompared to conventional EMD(e.g. Emdogain) (Miron et al. 2015).Further advantages of using EMDwithout its PGA carrier is related toits more fluid consistency and subse-quent improvement of surface coat-ing and penetration of EMPs withinthe bone biomaterials coupled withthe capacity for a gradual release ofEMPs over time (Miron et al. 2015).These prominent findings led to thedevelopment of a new liquid carriersystem for EMD (Osteogain) specifi-cally designed for mixing it with

different biomaterials including bonegrafts and collagen matrices/scaf-folds. Very recently, Miron et al.(2016b) reported that pre-coatingOsteogain onto a natural bovinemineral (NBM) significantlyincreased cell adhesion, proliferation,and differentiation of osteoblastsin vitro (Miron et al. 2016b) andimproved new bone formation in arat femur bone defect model (Zhanget al. 2016). However, despite theseencouraging data, it is still unclearto what extent Osteogain is effectivein promoting periodontal woundhealing/regeneration in periodontaldefects.

An important aspect that is stillcontroversially discussed in the liter-ature is the need of using bone graft-ing materials for enhancingperiodontal regeneration. Since mostfindings from histological studies inanimals and humans indicate thatthe healing following the use of bonegrafting materials is frequently char-acterized by persistence of graftingresidues embedded either in bone orencapsulated in connective tissue, thebiologic rationale of using suchmaterials needs to be questioned(Ivanovic et al. 2014, Sculean et al.2015). Thus, although clinical conse-quences of persisting graft residuesare still unclear, from a biologicalpoint of view, it would be desirablethat a biomaterial used as a carrierfor biologics (e.g. growth factors orEMD) or to stabilize the wound bysupporting the flap, is completelyresorbed and replaced by regener-ated periodontal tissues (e.g. cemen-tum, periodontal ligament, andbone). During the last decades vari-ous types of collagens matrices havebeen successfully used as carriers forgrowth factors and Emdogain, point-ing to the potential biological advan-tages of this combination to supportwound healing/regeneration (Susinet al. 2015, St€ahli et al. 2016).

However, at present, it isunknown whether the combinationof EMD and an absorbable collagensponge (ACS) may represent apotential option for reconstructivesurgery in complex non-containeddefects such as class III furcationdefects. Therefore, the aim of thepresent pre-clinical study was toevaluate the effects of Osteogainwith ACS in chronic class III furca-tion defects in non-human primates.

Materials and Methods

Emdogain, Osteogain, and ACSs

Emdogain and Osteogain (0.3 mlvials, concentration 30 mg/ml; Strau-mann AG, Basel, Switzerland) andnative type I and III porcine absorb-able collagen sponges (ACS: Colla-cone�; Botiss, Berlin, Germany)were utilized in this study.

Quantification of amelogenin adsorption

to ACSs

To determine the quantity of Emdo-gain/Osteogain adsorption to the sur-face of ACSs, ELISA quantificationassay was utilized for amelogenin, themain protein found in EMD encom-passing 90–95% of the total proteincontent. Briefly, 0.3 ml of Emdogain/Osteogain was poured onto cylindri-cal ACS scaffolds in six-well dishesfor 10 min at 37�C. After the coatingperiod incubation, the samples weresimply washed with phosphate-buf-fered saline (PBS), and the remainingPBS solution containing unattachedenamel matrix proteins was collectedand quantified by a QuantikineColorimetric Sandwich ELISA (Por-cine Amelogenin, X isoform(AMELX) ELISA Kit; MyBioSourceInc, San Diego, CA, USA) accordingto the manufacturer’s protocol. Sub-traction of total coated protein fromthe amount of unadsorbed proteinwas used to determine the amount ofadsorbed material to the surface ofACS as previously described (Mironet al. 2015). Furthermore, in order todetermine the quantity of amelogeninprotein being released from ACS overtime, coated ACSs were soaked in5 ml of PBS and samples were col-lected at various time points including15 min, 1 h, 8 h, 1, 3, and 10 days.All samples were quantified in tripli-cate and three independent experi-ments were performed.

Experimental animals

Three 7–8-year old male monkeys(Macaca fascicularis), weighing 6.91–7.02 kg, were used. The animalsexhibited intact dentition with healthyperiodontium. They were kept in indi-vidual cages at 23–29�C, relativehumidity of 30–70%, and a 12-hourlight/dark cycle. Approximately,108 g of solid food (HF Primate J

© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Periodontal healing with EMD liquid 299

12G 5K9J, Purina Mills, LLC, GraySummit, MO, USA) was provided toeach animal daily and water wasavailable ad libitum. All proceduresduring the in-life phase for about9 months (from 6 November 2014 to3 August 2015) were approved by theethical committee of the AnimalResearch Center of KagoshimaUniversity, Japan (approval no.D14026) and were performed inaccordance with standards publishedby the National Research Council(Guide for the Care and Use of Labo-ratory Animals, NIH OACU) of theNational Institutes of Health Policyon Human Care and Use of Labora-tory Animals.

Preparation of experimental defects

One surgeon (Y. S) performed all sur-gical procedures under general andlocal anaesthesia using aseptic rou-tines. Before the operation, buprenor-phine hydrochloride (Lepetan injection0.2 mg; Otsuka Pharmaceutical Co.,Ltd, Tokyo, Japan, 0.1 ml/Kg), toameliorate pain, and an antibiotic(Mycilinzol Meiji, 0.05 ml/kg; MeijiSeika Pharma Co., Ltd, Tokyo,Japan), to prevent infection, wereadministered intramuscularly. Generalanaesthesia was achieved with a keta-mine hydrochloride (0.2 ml/kg IM;Supriya Lifescience Ltd, Mumbai,India)/medetomidine hydrochloride(Domitor, 0.08 ml/kg IM; Orion Cor-poration, Espoo, Finland) combina-tion maintaining spontaneousbreathing. Local anaesthesia was per-formed using lidocaine HCl/epinephr-ine (2%, 1:80,000; Xylocaine, FujisawaInc., Osaka, Japan). After the opera-tion, atipamezole hydrochloride (Anti-sedan, 0.08 ml/kg, Orion Corporation)was administered intramuscularly.Ketoprofen (Capisten IM 50 mg,2 mg/kg, 0.1 ml/kg; Kissei Pharma-ceutical Co., Ltd, Matsumoto, Japan),to ameliorate pain, and an antibiotic(Mycilinzol Meiji, 0.05 ml/kg; MeijiSeika Pharma Co., Ltd), to preventinfection, were intramuscularly admin-istered to all animals for 2 days afteroperation.

As a pre-treatment, the secondpremolars and third molars in themandible were extracted for flap man-agement to prevent material exposureand obtain primary closure as previ-ously described (Donos et al.2003,Gkranias et al. 2012). After a healing

period of 2 months, the mucope-riosteal flaps were raised and class IIIfurcation defects were surgically cre-ated at the first and the secondmandibular molars with the use ofbone chisels and slowly rotating dia-mond burs (12 defects in total). Thedimensions of the exposed furcationdefects were 5 mm wide and 5 mmhigh. In order to prevent spontaneoushealing and induce plaque accumula-tion, the defects were filled withimpression materials (EXAFINEPUTTY TYPE, GC Corporation,Tokyo, Japan) (Fig. 1a). Subse-quently, the flaps were repositionedand stabilized with 4-0 silk sutures(MersilkTM; Ethicon Ltd, Edinburgh,UK). Sutures were removed at10 days following surgery. During8 weeks following the first surgery,no oral hygiene measures were per-formed and the animals were fed asoft diet. After 8 weeks, the impres-sion material was removed from thedefects and a plaque control regimen,consisting of oral cavity flushing witha chlorhexidine gluconate solution(5% HIBITANE�, 25 ml of a 2%solution; Sumitomo DainipponPharma Co., Ltd., Osaka, Japan) wasperformed for a period of 4 weeks.

Reconstructive surgery

At 16 weeks following creation of thedefects (Fig. 1b), intra-sulcular inci-sions were performed and full-thick-ness buccal and lingual flaps wereelevated in order to expose the furca-tion defects. All granulation tissue(Fig. 1c) was removed and theexposed root surface was carefullyscaled and planed. Cementum wasremoved using Gracey curettes and achisel (Fig. 1d). Reference notcheswere made using a #1 round bur onthe root surface at the base of thedefects for histometric analysis. ClassIII furcation defects received one ofthe following treatments: ACS alone,Emdogain with ACS (Emdogain/ACS), Osteogain with ACS (Osteo-gain/ACS), and open flap debride-ment (OFD) as a surgical control.The experimental conditions wererotated between defect sites in subse-quent animals. Emdogain/ACS andOsteogain/ACS were not placed inthe same unilateral side in the sameanimal. In the ACS group, ACS wasmixed with sterile saline before beingapplied to the defect. Root surfaces

that received Emdogain or Osteogainwere conditioned with a 24% EDTAgel (PrefGel�; Straumann AG) for2 min and then, along with the adja-cent mucoperiosteal flaps, thoroughlyrinsed with sterile saline. Prior to theplacement of Emdogain/ACS orOsteogain/ACS, the ACS was fullysaturated with Emdogain or Osteo-gain and the constructs were allowedto rest for 10 min (Fig. 1e). The con-structs were then filled in the defectwith moderate pressure (Fig. 1f).Maximum care was taken during sur-gery to prevent mixing of Emdogainor Osteogain to the other site in thesame side of the mandible.

A periosteal releasing incision wasmade to allow coronal displacementof the flap, followed by suturing(Gore-Tex CV-6 Suture; W.L. Gore& Associates Inc., Flagstaff, AZ,USA) slightly coronal to the CEJ(Fig. 1g). After reconstructive sur-gery, the animals received similartreatments of the antibiotics and anal-gesics which were used during prepa-ration of experimental defects.Sutures were removed after 14 daysof healing and postoperative plaquecontrol was maintained as previouslydescribed. Then, 4 months after thereconstructive surgery (Fig. 1h) theanimals were anesthetized by anintravenous injection of sodium pen-tobarbital (64.8 mg/ml, 0.4 ml/kg;Tokyo Chemical Industry Co., Ltd,Tokyo, Japan) and killed by exsan-guination.

Histological processing

All the defects, including the experi-mental and control sites, were thendissected with the surrounding softand hard tissues. The tissue blockswere fixed in 10% buffered formalin,trimmed, and rinsed in PBS. Thesamples were decalcified in Kalki-toxTM solution (Wako Pure ChemicalIndustries Ltd., Osaka, Japan) for3 weeks, dehydrated, and embeddedin paraffin. Step serial sections of6 lm thickness were then preparedalong the mesio-distal plane, stainedwith haematoxylin/eosin or withAzan–Mallory at intervals of 90 lm.

Histometric analysis

All the specimens were analysed his-tometrically under a light microscope(Eclipse E800; Nikon Inc., Tokyo,

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300 Shirakata et al.

Japan) equipped with a computerizedimaging system (Image-pro PlusMedia Cybernetics, Silver Spring,MD, USA). For the histometric anal-ysis, three sections approximately

90 lm apart were selected from themost central area of each class IIIfurcation defect, identified by thelength of the root canal and the refer-ence notches. A line connecting both

notches defined the apical limit of thedefect and the following parameterswere measured by the same experi-enced and masked examiner (T. N.).The mean value of each histometricparameter was then calculated foreach site.

Area measurements (in mm2 and %)

1 Bone defect area (BDA): area lim-ited by the apical line and the rootsurface in the furcation region;

2 Non-filled area (NFA): portion ofthe BDA not filled with any tissue,partially filled with plaque depos-ited on the root surface;

3 Epithelium tissue area (ETA): por-tion of the BDA filled with epithe-lium tissue;

4 Connective tissue area (CTA):portion of the BDA filled withconnective tissue;

5 New bone area (NBA): portion ofthe BDA filled with new bone

Area measurements, except forBDA, were calculated as the percent-age of the BDA within each defect.

Linear measurements (in mm and %)

1 Length of the root surface (LRS):length of the root surface from themesial notch to the distal notch;

2 Tissue-free defect length (TFL):portion of the LRS with the absenceof any new tissue formation;

3 Junctional epithelial migration(JE): total linear extensions of theroot surface covered by epithelialtissue;

4 Connective tissue adhesion (CT):total linear extensions of the rootsurface covered by connective tis-sue without cementum;

5 New cementum formation (NC):total linear extensions of the rootsurface coved by new cementum:

6 New attachment formation (NA):total linear extensions of the rootsurface covered by NC adjacent tonewly formed bone, with function-ally oriented collagen fibres

Linear measurements, except forLRS, were also expressed as the per-centage of the LRS within each defect.

Examiner calibration

Thirty-six sections from all sites wereread by the examiner without cali-bration before the measurements.

(a) (b)

(c) (d)

(e) (f)

(g) (h)

Fig. 1. Clinical appearance of the mandibular buccal aspect of Macaca fascicularis. (a)Induction of chronic inflammation. After fabrication of Class III furcation defects,impression materials were placed to encourage growth of oral microflora along theexposed root surfaces. (b) Prior to reconstructive surgery. (c) Immediately after flapreflection. Note the excessive granulation tissue in the chronic defects. (d) Defects wereexposed and debrided again at the time of reconstructive surgery. (e) Osteogain/ab-sorbable collagen sponge (ACS) construct before surgical implantation. (f) left (secondmolar): ACS alone, right (first molar): placement of Osteogain/ACS. (g) Flaps werecoronally repositioned and sutured. (h) 16 weeks after reconstructive surgery.

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Periodontal healing with EMD liquid 301

Forty-eight hours later, the sameexaminer read all 36 sections againto evaluate intra-examiner repro-ducibility. Inter-calibration of theexaminer was accepted at the 90%level. Because of low sample size dueto the selected model chosen, the sta-tistical analysis was restricted todescriptive statistics.

Statistical analysis

For the amelogenin ELISA quantifi-cation experiments, means and stan-dard errors (SE) were calculated.Statistically significant differenceswere examined by multiple t-testsbetween both groups. Statistical sig-nificance was defined as a p-value of0.05 corrected using the Bonferroni–Dunn method utilizing GraphPadPrism software (La Jolla, CA, USA).

Results

Ability to adsorb and release Emdogain

and Osteogain over time

ELISA was utilized to investigatethe amount of adsorbed amelogeninwhen Emdogain or Osteogain wereloaded onto ACS (Fig. 2). While itwas first found that both carriersefficiently loaded amelogenin ontothe collagen sponges at time point 0,a simple saline rinse with PBS signif-icantly removed over 20% more(from >90% to 70%) of the totalamelogenin content from Emdogainwhen compared to Osteogain wherethe total protein content remained<90% of the initial concentrations(Fig. 2). At each of the remainingtime points thereafter, a 20–60% sig-nificantly higher amount of totaladsorbed amelogenin was found forcollagen sponges loaded with Osteo-gain when compared to Emdogain

(Fig. 2). After a 10-day period,nearly 60% of the initial amelogeninprotein content found in Osteogainremained present within the collagensponges, whereas in the Emdogainsamples, no remaining amelogenincould be quantified as valuesapproached 0% (Fig. 2).

Clinical observations

All surgical treatments were well tol-erated by the animals, and clinicalhealing was uneventful at all 12 sites.No visible adverse reactions includ-ing material exposure, infection, andsuppuration were observed through-out the experimental period.

Histological observations

OFD group

In the OFD group, apical migrationof the junctional epithelium consid-erably occurred (Fig. 3a,e,i) withvarying degrees of new attachment,and new bone formation wasobserved. In one defect, considerablenew cementum formation and mod-erate new attachment formationoccurred (Fig. 3a). Artefacts (separa-tions between the new cementumand the root surface) were consis-tently detected in all three defects.Thick new cellular cementum withor without collagen fibres obliquelyoriented to the root surface was seenin the lower portion of the defect(Fig. 4a,b). The collagen fibresappeared to be sparser than thoseobserved in the Emdogain/ACS andOsteogain/ACS groups (Figs 3e,iand 4b).

ACS group

The healing pattern in the ACSgroup was characterized by limitedperiodontal regeneration (Fig. 3b, f

and j). Junctional epitheliummigrated to the most coronal exten-sion of new cementum with less con-nective tissue adhesion (Fig. 4c,d). Asmall amount of new bone formedin the lower portion of the defect(Fig. 3b,f,j). New cementum forma-tion and new attachment formationwere minimal in one defect, andrestricted to the mid portion of therest of two defects. Moderate thickand thin cellular cementum with orwithout collagen fibres obliquely ori-ented to the root surface wasobserved (Fig. 4c,d).

Emdogain/ACS group

In the Emdogain/ACS group, apicalextension of the junctional epithe-lium was more restrained than in theOFD and ACS groups. A greateramount of new cementum wasobserved in the EMD group than inthe control and ACS groups. Newbone formation was noted extendingfrom the apical notch towards thecoronal region of the defect (Fig. 3g,k). Thin acellular cementum andthick cellular cementum, with colla-gen fibres obliquely oriented to theroot surfaces, were observed(Fig. 4e,f). Furthermore, the collagenfibres appeared to be denser thanthose observed in the OFD and ACSgroups. Many blood vessels wereobserved within the newly formedperiodontal ligament (Fig. 4e,f).

Osteogain/ACS group

In general, the healing pattern in theOsteogain/ACS group was similar tothe Emdogain/ACS group. However,migration of junctional epitheliumwas more restrained in the Osteo-gain/ACS group than in the OFD,ACS, and Emdogain/ACS groups.Furthermore, new attachment andnew bone formation were consis-tently noted extending from theapical notches towards the coronalregion of the defect in all defects(Fig. 3d,h, l). Moderately thick newcellular and thin acellular cemen-tum, with dense collagen fibresobliquely or perpendicular orientedto the denuded root surface wasmore consistently observed (Fig. 4g,h). Highly vascularized new peri-odontal ligament-like tissue, tightlyconfined to between the new cemen-tum and new bone, maintained itswidth up to the coronal portion(Fig. 4g,h).

Fig. 2. In vitro release profiles of Emdogain and Osteogain from absorbable collagensponge. (*, p values < 0.05 was considered significant).

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302 Shirakata et al.

ACS appeared to be completelyresorbed after 16 weeks of healing inthe ACS, Emdogain/ACS, andOsteogain/ACS groups. Completedefect resolution of furcation defectswas not achieved in any of defectsin all four treatment groups. Therewas neither extensive root resorptionnor ankylosis, irrespective of theexperimental group.

Histometric analysis

The results of histometric analysis aresummarized in Tables 1 and 2. TheETA/BDA in the Emdogain/ACS(14.6 � 2.7%) and Osteogain/ACS(14.3 � 1.5%) groups were lowerthan those in the OFD (19.2 � 3.9%)and ACS (21.7 � 3.2%) groups.Osteogain/ACS group showed the

greatest amount of newly formedbone (NBA/BDA) among the groupsexamined. The length of junctionalepithelium migration observed in theOsteogain/ACS group was shorterthan those in the OFD, ACS, andEmdogain/ACS groups. The Emdo-gain/ACS and Osteogain/ACS groupsshowed greater cementum formationthan the ACS group. The amount of

(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

Fig. 3. Overview photomicrographs of all Class III furcation defects in different groups (Azan-Mallory staining). (a), (e), and (i):Open flap debridement (OFD) group Overview. (scale bar: 1 mm). (b), (f), and (j): Absorbable collagen sponge (ACS) group Over-view. (scale bar: 1 mm). (c), (g), and (k): Emdogain/ACS group Overview. (scale bar: 1 mm). (d), (h), and (l): Osteogain/ACS groupOverview. (scale bar: 1 mm). Arrowhead: notch (apical extent of root planing).

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Periodontal healing with EMD liquid 303

new cementum in the Osteogain/ACSgroup (40.5 � 7.2%) was twice asgreat as in the ACS group (19.4 �7.5%). Moreover, new attachmentformation was most extensive in theOsteogain/ACS (37.4 � 4.6%) groupwhen compared to the OFD(19.4 � 6.9%), ACS (14.1 � 9.6%),and Emdogain/ACS (25.0 � 1.8%)groups.

Discussion

To the best of our knowledge, thisis the first report evaluating thepotential effects on periodontalwound healing/regeneration of anew liquid carrier system for EMD(Osteogain) in chronic class III fur-cation defects in non-human pri-mates. Many investigators have

become interested in the plausiblereasons for the high clinical variabil-ity for studies reporting the combi-nation of EMD with bone graftingmaterials (Tu et al. 2010, Mironet al. 2014). Previous data haveshown that the use of Emdogain,although ideal for root surfaceadsorption, displayed drasticallyincreased thickness of coating to thebone grating surface which waseasily dissolved following a simplePBS rinse. Contrarily, the use ofOsteogain (EMD dissolved inacetic acid solution) exhibited morefavourable surface coating bydemonstrating an increase and morecomplete surface loading of porousgraft materials and tighter and morestable surface coatings with enamelmatrix proteins (Miron et al. 2015).

In this study, we used ACS as aputative carrier for EMD since ACShas been extensively used as anappropriate carrier including highclinical applicability, biocompatibil-ity, and uneventful biodegradation inbone and periodontal surgeries(McPherson 1992, Cochran et al.2000, Yamashita et al. 2010, Kimet al. 2013). In addition, it has beenreported that the fast resorption of aresidual material is desirable to avoidthe risk for infection and to increasethe amount of regenerated tissues inbone/periodontal defects (MacNeillet al. 1999, Shirakata et al. 2002,2007, Potijanyakul et al. 2010, Yosh-inuma et al. 2012). Histological find-ings demonstrated that ACS wascompletely resorbed after 16 weeks ofhealing in the ACS, Emdogain/ACS,

(a) (b) (c) (d)

(e) (f) (g) (h)

Fig. 4. Representative photomicrographs of Class III furcation defects in open flap debridement (OFD) group (Azan-Mallory stain-ing). (a) Higher magnification of the framed area (left) in Fig. 3i (scale bar: 200 lm), (b) Higher magnification of the framed area(right) in Fig. 3i (scale bar: 200 lm), in ACS group (c) Higher magnification of the framed area (left) in Fig. 3f (scale bar: 200 lm),(d) Higher magnification of the framed area (right) in Fig. 3f (scale bar: 200 lm), in Emdogain/ACS group (e) Higher magnificationof the apical framed area (left) in Fig. 3g, (f) Higher magnification of the framed area (right) in Fig. 3g (scale bar: 200 lm), and inOsteogain/ACS group (g) Higher magnification of the framed area (left) in Fig. 3d (scale bar: 200 lm), (h) Higher magnification ofthe framed area (right) in Fig. 3d (scale bar: 200 lm). JE: junctional epithelium, NB: new bone, D: root dentin, NC: new cemen-tum, PDL: periodontal ligament.

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304 Shirakata et al.

and Osteogain/ACS groups. Com-plete resolution of all furcationdefects was not achieved in any of thespecimens. On the one hand, theincomplete regeneration may beexplained by the large chronic-type,

furcation defects exhibiting a lowerhealing potential and by the difficul-ties to ensure a plaque-free healingenvironment in this animal model(Caton et al. 1994, Donos et al.2003). It has been extensively

demonstrated that periodontalwound healing and the outcomes fol-lowing conventional and regenerativeperiodontal surgery are negativelyinfluenced by plaque accumulation ofthe wound area (Rosling et al. 1976,

Table 1. Histomorphometric area measurement in each group (mean � SD in mm2 and %; n = 3 animals, n = 12 sites)

Histometric parameter Animal No Experimental condition

Control ACS Emdogain/ACS Osteogain/ACS

BDA (mm2) 1 12.3 11 16.2 12.72 13.1 10.2 11.6 14.53 12.1 18 11.6 17.5

Mean � SD 12.5 � 0.5 13.1 � 4.3 13.1 � 2.7 15.0 � 2.4NFA in mm2 and (%) 1 0 (0.4) 2.7 (25.0) 4.9 (30.3) 3.2 (25.6)

2 5.9 (45.4) 3.1 (30.5) 3.4 (28.8) 2.6 (17.7)3 1.5 (12.4) 5.2 (28.6) 1.1 (9.5) 4.2 (24.3)

Mean � SD 2.5 � 3.1 (19.4 � 23.4) 3.7 � 1.3 (28.0 � 2.8) 3.1 � 1.9 (22.9 � 11.6) 3.4 � 0.8 (22.5 � 4.3)ETA in mm2 and (%) 1 2.5 (20.5) 2.6 (23.8) 2.4 (14.7) 1.6 (13.0)

2 1.9 (14.8) 1.8 (17.9) 1.4 (11.9) 2.1 (14.1)3 2.7 (22.4) 4.2 (23.3) 2.0 (17.2) 2.8 (15.9)

Mean � SD 2.4 � 0.4 (19.2 � 3.9) 2.9 � 1.2 (21.7 � 3.2) 1.9 � 0.5 (14.6 � 2.7) 2.2 � 0.6 (14.3 � 1.5)CTA in mm2 and (%) 1 2.0 (16.7) 1.4 (13.1) 2.4 (14.7) 1.2 (9.5)

2 1.3 (9.6) 1.9 (18.4) 1.4 (11.9) 1.8 (12.1)3 2.4 (20.1) 3.8 (21.0) 2.9 (25.3) 3.2 (18.2)

Mean � SD 1.9 � 0.6 (15.4 � 5.4) 2.4 � 1.2 (17.5 � 4.0) 2.2 � 0.8 (17.3 � 7.1) 2.1 � 1.0 (13.3 � 4.5)NBA in mm2 and (%) 1 3.2 (26.3) 3.8 (34.5) 5.4 (33.4) 5.4 (42.4)

2 3.2 (24.1) 2.8 (27.1) 4.3 (37.0) 6.9 (47.2)3 4.1 (34.3) 3.1 (17.2) 3.6 (30.9) 5.3 (30.2)

Mean � SD 3.5 � 0.6 (28.3 � 5.3) 3.2 � 0.5 (26.3 � 8.7) 4.4 � 0.9 (33.8 � 3.0) 5.8 � 0.9 (39.9 � 8.8)

ACS, absorbable collagen sponge; BDA, bone defect area; NFA, non-filled area; ETA; epithelium tissue area; CTA, connective tissue area;NBA, new bone area.

Table 2. Histomorphometric linear measurement in each group (mean � SD in mm and %; n = 3 animals, n = 12 sites)

Histometric parameter Animal No Experimental condition

Control ACS Emdogain/ACS Osteogain/ACS

LRS (mm) 1 12.3 10.1 13.1 122 11.3 10.7 11.7 12.33 12.2 14.5 10.9 16.5

Mean � SD 11.9 � 0.5 11.8 � 2.4 11.9 � 1.1 13.6 � 2.5TFL in mm and (%) 1 1.4 (11.6) 5.8 (57.2) 7.0 (53.8) 5.4 (44.6)

2 7.9 (69.8) 5.5 (51.7) 5.7 (48.6) 5.5 (45.1)3 3.8 (31.0) 7.3 (50.5) 4.6 (42.2) 7.3 (44.3)

Mean � SD 4.4 � 3.3 (37.5 � 29.6) 6.2 � 1.0 (53.1 � 3.6) 5.8 � 1.2 (48.2 � 5.8) 6.1 � 1.1 (44.7 � 0.4)JE in mm and (%) 1 6.2 (50.7) 2.0 (19.9) 1.6 (11.9) 1.5 (12.5)

2 0.9 (7.6) 2.7 (25.4) 1.6 (14.1) 1.5 (12.5)3 5.0 (40.6) 4.9 (33.9) 3.7 (33.9) 3.6 (22.0)

Mean � SD 4.0 � 2.8 (33.0 � 22.5) 3.2 � 1.5 (26.4 � 7.0) 2.3 � 1.2 (19.9 � 12.1) 2.2 � 1.2 (15.7 � 5.5)CT in mm and (%) 1 0.4 (2.9) 0 (0) 0.2 (1.1) 0.1 (1.1)

2 0.1 (0.4) 0.3 (2.5) 0.6 (5.5) 0.2 (1.6)3 0.1 (1.2) 0.1 (0.7) 0.3 (2.8) 0.4 (2.4)

Mean � SD 0.2 � 0.2 (1.5 � 1.3) 0.1 � 0.1 (1.1 � 1.3) 0.4 � 0.3 (3.1 � 2.2) 0.2 � 0.1 (1.7 � 0.7)NC in mm and (%) 1 5.1 (41.6) 2.8 (27.3) 3.9 (29.9) 5.0 (41.3)

2 2.6 (23.1) 2.0 (18.5) 3.8 (32.6) 5.8 (47.2)3 3.3 (26.9) 1.8 (12.4) 4.3 (39.7) 5.4 (32.9)

Mean � SD 3.7 � 1.3 (30.5 � 9.8) 2.2 � 0.5 (19.4 � 7.5) 4.0 � 0.3 (34.1 � 5.1) 5.4 � 0.4 (40.5 � 7.2)NA in mm and (%) 1 3.3 (26.9) 2.4 (23.4) 3.0 (23.1) 4.9 (40.8)

2 2.1 (18.1) 1.6 (14.7) 3.1 (26.6) 4.8 (39.2)3 1.6 (13.2) 0.6 (4.2) 2.8 (25.4) 5.3 (32.3)

Mean � SD 2.3 � 0.9 (19.4 � 6.9) 1.5 � 0.9 (14.1 � 9.6) 3.0 � 0.2 (25.0 � 1.8) 5.0 � 0.3 (37.4 � 4.6)

ACS, absorbable collagen sponge; LRS, length of the root surface; TFL, tissue-free defect length, JE, junctional epithelial migration; CT,connective tissue adhesion; NC, new cementum; NA, new attachment formation.

© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Periodontal healing with EMD liquid 305

Nyman et al. 1977, Lindhe et al.1995, Tonetti et al. 1996, Rossa et al.2000, Gkranias et al. 2012). On theother hand, it cannot be excludedthat the used ACS did not possess theoptimal mechanical characteristics toensure sufficient stability of thewound, subsequently resulting in acollapse of the mucoperiosteal flapand more limited space for regenera-tion (Susin et al. 2015). Although theexpenses and demanding mainte-nance may restrict the broad use ofnon-human primates, the microbio-logical, immunological, and morpho-logical features are quite similar tothose of humans (Pellegrini et al.2009). Furthermore, chronic peri-odontal defects with minimal sponta-neous repair are valuable forevaluating new medical formulationsor drugs as a putative periodontalregenerative therapy prior to clinicalapplication in humans (Caton et al.1994, Giannobile et al. 1994). Theamount of new tissue formationobtained in the Emdogain/ACS andOsteogain/ACS groups was greaterthan those in the OFD and ACSgroups in the present animal model.Furthermore, the migration of thejunctional epithelium was morerestricted in the Emdogain/ACS andOsteogain/ACS groups compared tothe OFD and ACS groups. For thenewly formed cementum, no distinctqualitative differences were observedbetween the Emdogain/ACS and theOsteogain/ACS group, they werecomposed of mixed cellular/acellularcementum (Araujo & Lindhe 1998,Donos et al. 2003, Shirakata et al.2007, Gkranias et al. 2012). Func-tionally oriented collagen fibres withmany blood vessels in some partsalong the denuded root surface wereobserved, and they appeared to bedenser than those observed in thecontrol and ACS groups. These find-ings are comparable to those of previ-ous studies reporting that EMDhistologically presented positiveregeneration results in animals andhuman biopsies (Hammarstr€om et al.1997, Heijl et al. 1997, Mellonig1999, Donos et al. 2003, Hovey et al.2006, Shirakata et al. 2007, Gkraniaset al. 2012, Ivanovic et al. 2014, Scu-lean et al. 2015).

Interestingly, the amount of theregenerated tissue (i.e. NBA, NC, andNA) was the greatest in the Osteo-gain/ACS group, superior to that

obtained in the Emdogain/ACSgroup. This may be due to the factthat the ELISA assay demonstratedthat a 20–60% significantly higheramount of total adsorbed amelogeninwas found for the Osteogain/ACSgroup when compared to Emdogain/ACS. Furthermore, the ACS loadedwith Emdogain started to degrade inPBS by 3 days, whereas those pre-coated with Osteogain showed morestable properties. These findings sug-gest that Osteogain-adsorbed ACSnot only maintains the sustainedrelease of amelogenin but also pro-vides an environment conducive toaccelerating periodontal regeneration.Furthermore, the positive effects ofOsteogain on periodontal regenera-tion may be explained by previousfindings indicating that Osteogain sig-nificantly increased cell adhesion,proliferation, and differentiation ofosteoblasts in vitro (Miron et al.2016b), as well as promoted the upre-gulation of genes encoding BMP-2,TGF-b1, collagen 1 and osteocalcin(Miron et al. 2016c).

Within their limits, the presentdata indicate that Osteogain pos-sesses favourable physicochemicalproperties facilitating adsorption ofamelogenin on ACS and may addi-tionally enhance periodontal woundhealing/regeneration when comparedto Emdogain. None of the treat-ments achieved complete regenera-tion, that is, class III furcation stillpersisted after treatment. Furtherpre-clinical and clinical studies arethus warranted to evaluate the bio-logic and clinical value of this novelEMD formulation on periodontalwound healing/regeneration.

Acknowledgements

The authors wish to thank Mr.Makoto Tanoue for animal technicalsupport; Mr. Shinya Maeda, Mr. Kei-suke Ishimaru, and Ms. Maya Iwasefor the histological preparation of thespecimens; Shin Nippon Biomedi-cal Laboratories, Ltd, Kagoshima,Japan. Institut Straumann AG, (Basel,Switzerland) provided the Osteogainand Emdogain used in this study. Theauthors thank Dr. Stefano Tuguluand Dr. Benjamin E. Pippenger (Insti-tut Straumann AG, Basel, Switzer-land) for their help and scientificcomments in conducting this study.Absorbable collagen sponge

(Collacone�) was provided free ofcharge by Botiss Dental, Berlin, Ger-many.

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Address:Yoshinori ShirakataDepartment of PeriodontologyKagoshima University Graduate School ofMedical and Dental Sciences8-35-1 SakuragaokaKagoshima 890-8544JapanE-mail: syoshi@dent.kagoshima-u.ac.jp

Clinical Relevance

Scientific rationale for the study: Anew liquid carrier system forenamel matrix derivative (EMD)(Osteogain) has recently beendeveloped to facilitate bone bioma-terial mixing. This study evaluatedthe effect of Osteogain withabsorbable collagen sponges (ACS)

in chronic class III furcation defectsin monkeys.Principal findings: Statistically signifi-cantly higher amount of totaladsorbed amelogenin was detectedfor Osteogain/ACS when comparedto Emdogain, which yielded higheramounts of new connective tissue

attachment and bone formation infurcation defects.Practical implications: Osteogain/ACS promoted periodontal woundhealing/regeneration in class IIIfurcation defects, thus warrantingfurther evaluation in preclinicaland clinical settings.

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