The Intei-nat.oniil Joj,-nal oí PeriudorT. cs S. Rcstoiative Dentstry

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The Influence of Sulfate-ReducingBacteria Colonization of 2 DifferentBioresorbable Barrier Membranes forGTR. An 18-Month Case-ControlledMicrobiologie and Clinical Study

W.-D. Grimm, Dr med dent hab'il'/P. Cichon, Dr med dent hab(7"/H. van derHoeven, PhD'*VR S. Langendijk, PhD""/F. Smith, MS***'VM. G. Worley""-"'/I. Schmitz, PhD**""*/S. Offenbacher; DOS, PhD, MMSc****""

The purpose of the present microbiologie and case-controlled clinical study was toexamine the co'odization of 2 different resorbable barrier mernbranes by stitfate-reduc-ing bacteria [SRBJ. The barrier membranes tested were Guidor matrrx barrier andResolut regenerative material. Ten patients exhibiting 3 Class II furcation defects and 7intrabony defects were included in the study Tfie probing depth and the clinical attach-ment level at 4 surfaces per tooth were taken at tfie beginning oi^thesludy. Microbolo-gic samples were taken irom tfie expérimentai sites and from the approxrmai sites of theadjacent teeth. Both types of resorbable membranes were positive for 5RB colonization.The detection of SRB in 2 of 7 intrabony defects and in all defects witfi furcatjon involve-ment before the membrane placemer^t indicated that these organisms are a commoninhabitant ol sites showing periodontal destruction and are associated with guided tis-sue regeneration (GTR). According to the clinical criteria for healing tendencies used inthissludy, tfie GTR procedures were less successful iri the presence oi SRB. There wereno significant clinical effects of different resorbable membrane materials or membranelayout on attachrnent level changes for either the intrabony defect or furcation groupsafter 18 months. There were no statistical differences for sites that became exposed toSRB when compared to sites that remained unexposed after ) 8 months. The numericsignificance of SRB in relation to the total microbial count needs to be determined togain insight into the écologie role of membrane résorption rates. (Int J PeriodonticsRestorative Dent 2000;20:93-99.)

'Chair and Professor of Perbdontology, University of Witten, Germany; and AdjunctProfessor of Pen odontology. University of North Carolina at Chapel Hill.

"Professor of Pen odontology. University of WItten, Germany."•Professor of Oral Microbiology, Univeisity of Witten, Germany; and Explore Research

Laboratones, Nijmegen, The Netherlands.""Explore Research Laboratories, Nijmegen, The Netherlands.

"•'•Laboratory Manager, Center of Oral and Systemic Diseases, University of North Csroliraat Chapel Hill.

•"•"Information Manager, Center of Oral and Systemic Diseases, University of NorthCarolina at Chapel Hill.

" " " ' Inst i tu t of Pathology, Bergmanns h eil Hospital, University Clinic, Bochum, Geimany.•"'""•Department of Peri odontology. Center of Oral and Systemic Diseases, University of

North Carolina at Chapel Hill.

Reprint requests. Prof Dr Grimm, Department of Periodontology, University ofWitten/Herdecke, Alfred-Herrhausen-Strasse 50, 58448 Witten, Germany

Guided tissue regeneration (GTR)

has been used to regenerate perj-

odontal tissues lost as a result of

periodontal diseases. The objective

of a GTR procedure is to promote

formation of new connective tissue

attachment on a root surface that

has been detached because of

plaque-induced inflammation. Ny-

man et aP pioneered GTR using mil-

lipore (paper) filters. This impractical

material was soon replaced by ex-

panded polytetrafluoroethylene (e-

PTFE) membranes^ and by re-

sorbable materials (for review see

Greenstein and Caton- ), including

collagen,''-^ polyglycolic acid,* poly-

lactic add, and copolymers of the

latter 2.''-'2 The ability of both non-

resorbable and resorbable mem-

branes to elicit neoformation of con-

nective tissue attachment has been

fully documented.^•^•"' Infection is a

major complicating factor in GTR.'^

Bactériologie analyses of retrieved e-

PTFE barriers have demonstrated

bacterial contamination.^^"'^ Al-

though the histologie and clinical

events of GTR are well documented,

the role of bacterial enzymes in the

case of guided periodontal wound

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94

healing and regeneration is stilllargely poorly understood. The ex-tent of bacterial contamination ofthe barrier membranes correlatedinversely with clinical assessment ofattachment gain.^° One third ofall cultivable organisms were gram-negative anaerobic rods. This in-cluded black-pigmented rods andFusobacterium sp. ^ With respect toproteoglycan destruction, bacterialenzymes may play a significant role.Porphyromonas gingivalis strainsappear to be capable of degradingType I collagen.^^ In addition todirect tissue damage, indirect effectsof enzymes on proteoglycans have

The term sulfate-reducing bac-teria (SRB) describes a heteroge-neous groupé" of microorganismsthat has in common the dissimila-tory reduction of sulfate and oblig-ate anaerobiosis, A broad spectrumof organic substrates can serve aselectron donors; nearly all knownfermentation products from thedegradation of proteins, carbohy-drates, lipids, and components ofpoiylactic acid membranes could beused in a similarway. Sulfate-reduc-ing bacteria thereby produce hydro-gen sulfide. The detection of SRB inperiodontal pockets^^ indicated thatthese bacteria are common inhabi-tants of sites showing periodontaldestruction. The search for SRB wasundertaken because several condi-tions required for the growth ofthese bacteria are met in the peri-odontal pocket: (1) an anaerobicenvironment with a low redox poten-tial, (2) the presence of low-molecu-lar-weight fermentation products.

and (3) a neutral to slightly alkalinepH. Little is known ofthe influence ofSRB on barrier membrane degrada-tion in periodontal wound healingfollowing GTR.

The purpose ofthe present mi-crobiologie and clinical study was toexamine the colonization by SRS of2 different resorbable barrier mem-branes for periodontal regenerationand to follow up on the clinical out-comes over a period of 18 months.

Method and materials

In the study 2 types of commerciallyavailable barrier membranes wereused: Resolut regenerative material(3i/WL Gore) and Guidor matrix bar-rier (John O. Butler). The occlusivefilm in Resolut is composed of acopolymer of 85% polylactide and15% polyglycolide. The randomfiber matrix on both the inner andouter surfaces of the material isdesigned to provide rapid tissueintegration. A composition of 100%polyglycolic acid (PGA) was chosenbecause it satisfied the résorptiontime requirements. The polymermolecular weight and copolymerratio ofthe occlusive film were cho-sen after testing many combinations.The 85:15 composition resorbs moreslowly than the pure PGA fibers, pro-viding the 4- to 0-week occlusive-ness required for a GTR device. TheGuidor membrane is composed ofpoiylactic acid with a multilayerdesign, softened with citric acid toachieve malleability. The résorptionprocess is programmed to ensurebarrier function for a minimum of 6

weeks, after which it slowly resorbswithout interfering with the regen-erative healing process.

Patients

Patients enrolled in this study^* hadclinical evidence of either Glass IIfurcation invasions or intrabonydefects. They were in good physicalhealth and had no previous peri-odontal treatment on the teeth con-sidered for treatment. Ten patientsexhibiting 3 Glass II furcation defeirts(2 Guidor matrix barriers and oneResolut regenerative material) and 7intrabony defects (3 Guidor and 4Resolut) were enrolled in the study.The patients' ages ranged from 28 to49 years. Initial therapy, deep scal-ing, and root planing were per-formed on teeth scheduled forregenerative therapy consideringthe treatment protocol of the au-thors' department (University ofWitten).

Surgical protocol

The basic surgical protocol was com-parable to that already described inrecent studies.''^^ The buccal andlingual intrasulcular incisions werecarefully performed for preservingthe interdental papilla (modifiedpapilla presentation technique'*). Afull-thicknessflap was raised past themucogingival junction to providebetter access to the defect. Verticalreleasing incisions were performedas needed to increase defect ac-cessibility. The epithelium and the

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granulation tissue ofthe inner sur-face of the flap were removed andthe root surface and/or the furcationarea were cleaned using curettes andan ultrasonic device with a flame-shaped diamond working tip Re-sorbable membranes were adaptedto cover the furcation area or theintrabony defect, extending up to 3mm apical to the bone crest. Thefull-thickness flap was coronally dis-placed for complete coverage ofthemarginal border of the membrane.To accomplish this, a fenestraticnincision ofthe periosteum was per-formed at the base of the flap. Themembrane was tightly attached tothe tooth by a resorbable slingsuture. The flap was sutured.

Soft tissue clinical measure-ments were taken the day of surgery(baseline) and 6 weeks after surgery.To evaluate wound healing, mea-surements were made using a scoreof visible gingival inflammation andexposure ofthe barrier membrane (3grades): normal wound healing with-out visible gingival inflammation(grade 1}; delayed wound healingwith visible signs of gingival inflam-mation (grade 2); and delayedwound healing with barrier mem-brane exposure (grade 3). The fol-lowing clinical measurements weretaken at the time of surgery and 6and 18 months after surgery^^: gin-gival recession (GR), probing depth(PD), and clinical attachment level(CAL), Furcations were probed usinga PCP-11 probe (Hu-Friedy); PD andCAL were measured on 4 surfaces ofthe experimental sites.

Patients were instructed to rinsetwice daily with 0.2% chlorhexidine

and to use modified oral hygieneprocedures for 2 weeks in the treat-ment groups. Professional toothcleaning was performed weekly forthe first 6 weeks.

Microbiologie monitoring

The samples were taken from theexperimental sites and from theapproximal sites of the adjacentteeth before membrane placementand after 6 weeks. Sterile paperpoints were inserted one after theother into the area of the furcationentrance or into the pocket. After20 s, the points were withdrawntogether (pooled samples) anddropped into a 2-mL screw-cap vialwith a semisynthetic medium. Thissemisynthetic basal medium with apH of 7.2 and a redox potentialbelow -100 mV^^ was used forenrichment of SRB in the peri-odontal plaque samples. The sam-ples were incubated in an anaerobicchamber (Braun) at 35°C in an at-mosphere of 91% Nj , 4% Hj, and5% COj, and an oxygen concen-tration below 5 ppm, Agar plateswere prepared with the samemedium, supplemented with 20 g/Lof agar

Sulfate reduction in the primaryenrichment cultures was indicatedby strong blackening caused by theprecipitation of FeS, Tenfold serialdilutions ofthe enrichment culturewere plated onto agar medium andincubated for 21 days. Blackeningofthe plates indicated the presenceof colonies of SRB, All incubationwas done in the anaerobic chamber.

Results

In a preliminary study,^^ the authorsinvestigated the in vitro colonizationof resorbable barrier membranes.Figure 1 shows Resolut regenerativematerial colonized in vitro by SRB,The résorption process programmedto ensure barrier function for a mini-mum of 6 weeks was not disturbed orinfluenced by the presence of SRB,and the barrier membranes slowlyresorbed. Figure 2 shows in vitro SRBcolonization of the Guidor matrix bar-rier. In a further in vitro study,^' theauthors investigated the colonizationof 3 types of of resorbable nonim-pregnated and tetracycline-impreg-nated membranes by strains of SRBand Pgingivalis. The study evaluatedwhether the tetracycline-impreg-nated membranes could inhibit bac-terial adhesion and penetration intomembranes. Strains of SRB wereoriginally isolated from periodontalpockets and were characterized byphylogenetic analysis of 16 S rDNAsequences. Scanning electron micro-scopy (SEM) analysis revealed differ-ences in the accumulation of SRBstrains and P gingivalis on the mem-branes. The tetracycline-impreg-nated membranes showed fewerbacteria attached and less evidenceof colony formation than the non-treated membranes. In general, theaffinity of the bacteria to the mem-branes was low. Cell aggregateswere found primarily on rough areasof the membranes. These in vitrostudies underscore the importance ofcontrolling putative periodonto-pathogens in barrier membranes andthe associated surgical sites in GTR.

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96

Fig 1 Résolut regeneratii/e matenal covered by in vitro SRßdeposits. (Original magnification x 200.}

Fig 2 In vitro SRB colonization on ffie Cuidor matrix barrier.(Orrgma) magnilication x 200.)

2.5-

2.0-

S 1.0-

0.5-

Furcalion intrabony Furcation Inlrabony2 wh 6 wk

Fig 3 Effect of SRB exposure on healing. CAL = cfínrcaí attach-ment ievel.

Occurrence of SRB

Sulfate-reducing bacteria weredetected in 2 of 7 intrabony defectsand in all defects with furcationinvolvement before the membraneswere placed. There was no evidenceof SRB after 6 weeks. No differenceswere seen with the different types ofresorbable membranes composedof polymers (D, L-lactic acid) orcopolymers of polylactic acid.

Case-controlied clinical study

The case-controlled clinical studyevaluated healing tendencies andCAL changes in deep intrabony peri-odontal defects and in Class II fur-cations. Figure 3 shows that theoccurrence of SRB has an influenceon healing tendencies. The presenceof SRB on the resorbable mem-branes has been shown to preventhealing with GTR in the first 6 weeks.

According to the clinical critera forhealing tendencies used in thisstudy, the GTR procedures were lesssuccessful in the presence of SRB.hlowever, there were no significantclinical effects of different resorbablemembrane materials or membranelayout on attachment level changesforeitherthe intrabony defect or fur-cation groups after 18 months. Thedata (Table 1) relating to changes inPD, CAL, and GR met or exceeded

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97

Table 1

Treatment

ResolutGuidor

Results of soft and hard tissue measurements with both types of resorbable barriermembrane (mean ± standard deviation, in mm)*

AttachmentInitial 18 mo Change level change

7.9 ± 1.8 5.6 ± 0,8 2.3 ± 0,8 3,5 ±1.5ö,6±1.8 3,8+1.1 2,8±0.7 3.2 + 1.7

Bonelevel change

0.6 ± 1,60,5 ± 1,4

Gingivalrecession change

1,2±1,21.3 + 1.8

the success criteria of2 mm or greater attachment levelgain^^ independent of the occur-rence of SRB before membraneplacement or of the type of mem-brane used. There were no statisticaldifferences for sites that becameexposed to SRB when comparedwith sites not exposed after 18months.

Discussion

This report is the first clinical study todescribe the presence of SRB onbioresorbable membranes used forGTR. The detection of SRB in 2 of 7intrabony defects and in all defectswith furcation involvement beforemembrane placement was taken toindicate that these organisms are acommon inhabitant of sites show-ing periodontal destruction and areassociated with GTR,

The numbers of SRB seem tobe limited by the energy sourcesulfate.^° Sources of sulfate in the

periodontium after surgical proce-dures include the fluid, mainly a tran-sudate of periodontal tissues, theconstituents of which are derivedfrom serum, inflammatory cells, andhost tissue. The mean concentrationof free sulfate in serum is 0.3 mol/L,and sulfate from serum is availableduring periodontal wound healingand the soft connective tissue regen-eration, Proteoglycan synthesis richin chondroitin sulfate provides a suit-able matrix for cellular infiltration. Atthe same time the matrix matura-tion takes place, dermatan sulfateincreases. It is tempting to assumethat the sulfate-containing proteo-glycans represent a source of sulfatefor the SRB. Proteoglycans consti-tute a major component in the extra-cellular matrix of connective tissues.They consist of a central protein coreto which highly anionic heteropoly-saccharide chains called glycos-aminoglycans are linked. Sulfate isbound to glucosamine and galacto-samine residues in glycosamino-glycans. Proteoglycan species are

characteristically distributed amongthe periodontal tissues, Chondroitinsulfate with a he pa ran su If ate werereported to be predominant increvicular fluid associated with sitesof active bone remodeling duringorthodontic treatment.

Grosso et aP' reported alteredprotease profiles of periodontal lig-ament cells as a result of exposureto bacterial products during thehealing phase after GTR proce-dures. They have also shown thatthese cells produce more mRNAforseveral cytokines, i-iigh-level pro-duction of cytokines such as inter-leukin-Tß was associated with cellsisolated from regenerative proce-dures,^^ Production of both pro-teases and cytokines by all cells inthe healing wound likely modulateshealing and affects the degradationprocess of the resorbable mem-branes. With respect to bone matrixdeposition in guided periodontalwound healing and regeneration,the usual sequence is an initialdeposition of a hyaluronan-rich

Volume 20, Number 1, 2000

matrix with plasma proteins. Sulfate-

reducing bacteria require free sul-

fate for their growth, but can also

liberate the sugar-bound sulfate (P,

S. Langendijk, unpublished data).

Sulfatase activity—most likely from

lysozomal origin—in oral bacteria

has been detected so far in Strepto-

cocci and Campy/obacter,^^ which

are found in periodontal pockets.

Sulfate-reducing bacteria produce

equimolar amounts of sulfide from

the reduction of sulfate. Hydrogen

sulfide is considered to be toxic to

mammalian cells by inactivation of

cytochrome oxidase,^'' its ability to

split disulfide bonds in proteins and

binding of various metal ions.

Further, H S in periodontal sites

during wound healing may well ori-

ginate from the degradation of cys-

teine by SRB. The numeric sig-

nificance of SRB in relation to the

total microbial count needs to be

determined to gain insight into the

écologie role of membrane résorp-

tion rates.

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