Flemmig1998- Long-Term Maintenance of Alveolar Bone Gain After Implantation of Autolyzed, Antigen-Extracted, Allogenic Bone in Periodontal Intraosseous Defects

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Long-Term Maintenance of AlveolarBone Gain After Implantation ofAutolyzed, Antigen-Extracted,Allogenic Bone in PeriodontalIntraosseous DefectsThomas F. Flemmig* Benjamin Ehmke* Katja Bolz* Norbert R. Kühler,1Helge Kareh* Jürgen F. Reuther,'' and Bernd Klaiber4

This randomized controlled trial assessed the long-term maintenance of alveolarbone gain after implantation of autolyzed, antigen-extracted, allogenic (AAA) bone.AAA bone is a demineralized freeze-dried bone allograft processed after previously

described methods. In each of 14 patients, AAA bone was implanted into the intraos-seous defect of 1 tooth (test); a second tooth with an intraosseous defect was treatedby modified Widman flap surgery alone (control). All patients were offered supportiveperiodontal therapy at 3- to 6-month intervals following treatment. Clinical measure-ments were taken prior to surgery, 6 months, and 3 years following surgery. Of the14 patients enrolled, 11 patients completed the 6-month and 8 patients the 3-yearexamination. In test teeth, bone gain was significantly greater compared to controlteeth at 6 months (2.2±0.5 mm and 1.2±0.5 mm, respectively) and 3 years (2.3±0.7mm and 1.1 ±0.8 mm, respectively) (P < 0.05). Also, more probing attachment wasgained in test compared to control teeth at 3 years (2.0±0.7 mm and 0.8±0.5 mm,respectively; < 0.05). At 3 years, Porphyromonas gingivalis was detected in 3 testand 2 control teeth by Polymerase chain reaction, whereas no Actinobacillus actino-

mycetemcomitans was found. Due to the low detection frequency, there was no clearcorrelation between the maintenance of alveolar bone during supportive periodontaltherapy and subgingival infection with P. gingivalis. The data indicated that alveolarbone gain after implantation of AAA bone may be maintained over a minimum of 3years in patients receiving periodontal supportive therapy. J Periodontal 1998;69:47-53.

Key Words: Alveolar ridge augmentation; grafts, bone; periodontitis/therapy; bone,demineralized; bone, freeze-dried; bone regeneration; periodontal regeneration.

In

periodontal intraosseous

defects, regeneration of

peri-odontal tissues may be achieved by implantation of au-togenous or allogenic bone and/or guided tissue regen-eration.1-3 Demineralized freeze-dried bone allografts(DFDBA) appear to have high osteogenetic potential4 andhistologie analysis in humans has indicated that partialperiodontal regeneration; i.e., alveolar bone and cemen-timi apposition, and formation of functionally oriented

*Department of Periodontology, Julius Maximilian University, Würz-burg, Germany.'Clinic for Oral and Maxillofacial Surgery.'Institute for Hygiene and Microbiology.

5Clinic for Operative Dentistry and Periodontology.

periodontal ligament fibers, may occur

following implan-tation of DFDBA in intraosseous defects.3 Controlledclinical studies have repeatedly demonstrated that im-

plantation of DFDBA in periodontal intraosseous defectsresults in significantly greater alveolar bone and/or prob-ing attachment gain compared to open debridementalone.56 This has also been shown for autolyzed, antigen-extracted, allogenic (AAA) bone.7-8

AAA bone is a type of DFDBA processed accordingto the methods of Urist et al.9 with minor modifications.In comparison to DFDBA, the procedure of AAA bonepreparation includes the extraction of cell-surface glyco-

proteins which represent major antigens responsible for


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48 ALLOGENIC BONE GRAFTS IN PERIODONTAL DEFECTSJ Periodontol

January 1998

bone allograft reactivity. In addition, the collagen matrixof AAA bone is shrunk during preparation, allowing bet-ter diffusion of bone morphogenetic proteins.10 The effi-cacy of AAA bone has been investigated in animals andhumans. It has been demonstrated that AAA bone mayinduce

heterotopic new bone formation in rodents10 and

non-human primates." In addition, in humans, AAA bonehas been clinically applied for the treatment of bone de-fects resulting from excison of benign bone tumors,12 seg-mentai defects of long bones,13 and to perform intertrans-verse process spinal fusion14 and cranioplasties.15

To evaluate the clinical value of regenerative tech-niques, information on the long-term maintenance of re-generated periodontal tissues in periodontal intraosseousdefects appears to be crucial. Following guided tissue re-generation using expanded polytetrafluoroethylene (e-PTFE) membranes in periodontal intraosseous defects,

probing attachment gain 1 year

following periodontal sur-

gery has been shown to be maintained in some patients,while lost in others during the subsequent years. Factorsinfluencing the maintenance of periodontal attachmentgain include oral hygiene, frequency of supportive peri-odontal therapy, subgingival infection with Porphyro-monas gingivalis, and smoking.16-18

Although there are case reports indicating that alveolarbone gain after implantation of DFDBA in combinationwith ePTFE membranes may be maintained for up to 5

years,19 the observation time of all previous randomizedcontrolled trials assessing DFDBA or AAA bone werelimited to a maximum of 1 year. Therefore, the presentrandomized controlled trial assessed the maintenance ofalveolar bone gain over 3 years following implantation ofAAA bone in periodontal intraosseous defects.

MATERIALS AND METHODSThe preparation of AAA bone slightly difffered from theoriginal AAA bone procedure described by Urist et al.9Briefly, human diaphyseal cortical bone ground in liquidnitrogen to a particle size less than 2 mm was deminer-alized and acid-soluble matrix proteins were extracted in0.6 M hydrochloric acid at 4°C. After demineralization,the bone was washed in distilled water at 4°C for 30 min-

utes. Autolytic digestion of bone cells was performed byincubation in 0.1 M phosphate buffer, pH 7.4, containing3 mM N-ethylmaleimide and 10 mM NaN3 at 37°C underagitation for 3 days. The bone was then washed in stirred,de-ionized water for 6 hours at 4°C. Collagen fibrils wereshrunk and high-molecular-weight protein Polysaccha-rides were extracted in 6 M lithium chloride and low-

molecular-weight protein Polysaccharides were extractedby incubation in 0.3 M calcium chloride for 24 hours at4 °C in the presence of 3 mM NaN3. Thereafter, the bonewas washed extensively in deionized water at 4 °C for 12hours in the presence of 10 mM NaN3 and 3 mM NEM.

Lipids, intracellular components as well as cell membrane

lipoproteins were extracted by incubation in chloroform-methanol (1:1) for 24 hours at room temperature. Afterthe chloroform-methanol was decanted, the bone was airdried. The bone was then washed in sterile, deionizedwater at 4°C for 4 hours and deep frozen. Using a cen-

trifugal mill, the frozen bone was

ground in the

presenceof liquid nitrogen to a defined particle size of 250 to 500µ . Thereafter, the particles were washed with 95% eth-anol for 1 hour at room temperature followed by rinsingwith sterile deionized water at 4°C for 1 hour, deep frozen,lyophilized for 10 days, and finally sterilized in ethyl-eneoxide after packing.10

Study PopulationA total of 14 adult patients with radiographie signs ofinterdental periodontal intraosseous defects and probingattachment loss of more than 6 mm on at least 2 teethwere enrolled into the

study. Patients representing a con-

secutive sample were recruited from the Department ofPeriodontology, School of Dental Medicine, Bavarian Ju-lius Maximilian University, Würzburg, Germany. Patientswith any of the following conditions were excluded fromthe study: bleeding disorders; cardiovascular diseases;agranulocytosis; leukemia; diabetes mellitus; use of ni-fedipine, phenytoin, cyclosporine A, or non-steroidal anti-inflammatory drugs; allergies against tetracycline, neo-mycin, or local anesthetics; and/or pregnancy. Smokingwas not an exclusion criterion. All patients signed theinformed consent approved by the Ethics Committee ofthe Medical Faculty of Julius Maximilian University,Würzburg.

Periodontal TherapyPeriodontal surgery. After completion of full-mouth scal-ing and root planing as well as oral hygiene instructions,1 tooth with a periodontal intraosseous defect was ran-domly assigned to receive AAA bone (test), and a secondtooth with a periodontal intraosseous defect was treatedby open debridement alone (control) in each patient. Ran-domization was performed using a randomization list es-tablished before the beginning of the study.

In test teeth, sulcular incisions were made to the crest

of the alveolar bone and mucoperiosteal flaps were re-flected. After removal of granulation tissues, the surgi-cally-exposed root surfaces were scaled and planed withGracey curets and/or fine diamond burs with 40 µ grain.AAA bone was reconstituted in a solution of 3250 LU.

neomycinsulfate and 250 LU. bacitracin11 (1 ml) andplaced into the intraosseous defect up to the alveolar crest.For stabilization, the implanted AAA bone was coveredwith fibrinogen*1 and a periosteal pedicle flap prepared aspreviously described to ensure maximum graft coverage.20

'Nebacitin, Bocardes, Heidelberg, Germany.'Tissucol Duo, Immuno GmbH, Heidelberg, Germany.


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The buccal and lingual mucosal flaps were then reposi-tioned using horizontal mattress sutures. Control teeth re-ceived open debridement; i.e., modified Widman flap sur-gery, alone.21 A periodontal dressing* was placed on bothtest and control teeth. Periodontal dressing and sutureswere removed after 7

days.Postoperative medication. Patients were prescribed250 mg tetracycline q.i.d. for 14 days and instructed torinse with 15 ml 0.2% Chlorhexidine digluconate solutionb.i.d. for 4 weeks. In addition, 500 mg paracetamole q.i.d.was prescribed for patient's comfort.

Supportive periodontal therapy. All patients receivedsupportive periodontal therapy; i.e., full mouth supra- andsubgingival scaling as well as supragingival polishing, 3and 6 months following periodontal surgery. However,scaling at 3 months was limited to the supragingival areain test and control teeth to prevent damaging the newlyformed periodontal tissues. Between the 6-month and 3-year examinations, patients were offered periodontal sup-portive therapy at 6-month intervals.

MeasurementsClinical parameters. During the entire study all clinicalmeasurements were performed by the same examinerblinded to the type of surgical therapy rendered. The fol-lowing clinical measurements were performed at 6 sitesper tooth immediately prior to and at 3 months, 6 months,and 3 years following periodontal surgery: plaque index,gingival index,22 probing depth, and relative probing at-tachment level. Relative alveolar bone level was also

measured preoperatively at 6 months and 3 years underlocal anesthesia at 6 sites per tooth; the periodontal probewas advanced apically until a hard resistance was felt. Inaddition, intraoperatively; i.e., after reflection of the peri-odontal flap and soft tissue degranulation, alveolar bonelevel, intraosseous defect depth from the base of the de-fect to the crest of the alveolar bone, and the number of

bony defect walls were assessed. Probing attachment leveland alveolar bone level were measured using a calibratedprobe** and a customized stent. For reproducible mea-surements at the same sites during the course of the study,grooves were cut into the stent at the mesio-buccal, mid-

buccal, disto-buccal, mesio-lingual, mid-lingual, and dis-to-lingual of test and control teeth.

Microbiological analysis. At 36 months, subgingivalplaque samples were taken from the deepest sites of testand control teeth. Following removal of supragingivalplaque, subgingival plaque was harvested using a sterilecuret. Plaque samples were analyzed by polymerase chainreaction (PCR) for Actinobacillus actinomycetemcomitansand P. gingivalis with minor modifications as previouslydescribed.23-24 The detection limit of the PCR for A. ac-

*Coe Pak, GC America Inc., Chicago, IL.

**North Carolina Periodontal Probe, Hu-Friedy, Chicago, IL.

Table 1. Mean ± SEM Periodontal Parameters of Test and Control Teeth at Baseline of 8 Patients Completing the 36-Month Examination

Test Control

Plaque index 0.7 ± 0.2 0.7 ± 0.2Gingival index 0.5 ± 0.1 0.7 ± 0.1

Probing depth (mm) 5.3 ± 0.6 6.0 ± 0.5Defect depth (mm) 4.7 ± 0.7 5.3 ± 0.6

tinomycetemcomitans and P. gingivalis was 101 CFU and102 CFU, respectively.24-25 However, since only 15 µ othe 1 ml plaque suspension were analyzed, the detectiolimits per ml suspension were approximaly 103 CFU/mand 104 CFU/ml for A. actinomycetemcomitans and Pgingivalis, respectively. Precautions as described byKwok and Higuchi25 were used to prevent contamination

Statistical

AnalysisIn each tooth, the 2 deepest sites of the periodontal intraosseous defect were used for analysis. Correlatedpaired f-test, which adjusts for site-to-site dependencieswas used to assess treatment effects between test and control teeth and long-term changes (from 6 to 36 monthswithin each group.26 As a primary outcome variable, bone

probing level was used. Correlations between pre- andintraoperative alveolar bone level measurement as well abetween plaque and gingival index scores and maintenance of alveolar bone level from 6 to 36 months were

assessed using Spearman's correlation coefficient. Aldata are presented as means ± standard error of the mean

RESULTS

Study Population and Periodontal IntraosseousDefectsEleven of the 14 patients enrolled into the study completed the 6-month examination and 8 patients (4 femaleand 4 males with a mean age of 47.3 ± 4.1 years) werere-examined at 36 months. There were no significant differences in the clinical parameters between test and control teeth at baseline (Table 1). In test defects, 3 had bony wall, 1 had 2 bony walls, and 4 had 3 bony wallsin control defects, 1 had 1 bony wall, 2 had 2 bony wallsand 5 had 3 bony walls.

Short-Term Clinical Treatment Outcome FollowingImplantation of AAA BoneIn 11 patients, implantation of AAA bone in periodontaintraosseous defects resulted in significantly greater gaiof alveolar bone level (2.2±0.5 mm) compared to opedebridement alone (1.2±0.5 mm) at 6 months (P < 0.05(Fig. 1); compared to the initial intraosseous defect depthosseous defect fill in test and control teeth was 46.1% an

19.2%, respectively. The probing attachment gain wa

also significantly higher in test (2.3 ±0.5 mm) than in con


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January 1998

n=ll n=8 Test Control

^ (

BL

36 months

Figure 1. Mean alveolar bone level (BL) changes in test (T) and control(C) teeth. Error bars indicate SEM and positive values gain; * signifi-cant difference between and C, < 0.05.

n=ll n=8

AL

36 months

PD

Figure 2. Mean attachment level (CAL) and probing depth (PD) changesin test (T) and control (C) teeth. Error bars indicate SEM, positive val-ues gain and negative values reduction; * significant difference between and C, < 0.05.

trol teeth (0.8 ±0.3 mm) at 3 months (P < 0.05). How-ever, both test and control teeth demonstrated great vari-

ability in bone level and probing attachment. Althoughthere was a trend towards greater reduction in probingdepth in test compared to control teeth, the differencebetween groups did not reach statistical significance (P =0.09, at 6 months) (Fig. 2).

Long-Term Maintenance of Treatment OutcomeFollowing Implantation of AAA BoneIn the 8 patients completing the study, significantly great-er alveolar bone gain and probing attachment gain wasfound in test (2.3±0.7 mm and 2.0±0.7 mm, respective-ly) compared to control teeth (1.1±0.8 mm and 0.8 ±0.5 mm, respectively) at 36 months (P < 0.05). From 6

to 36 months, no significant changes in alveolar bone lev-

36 months7-P.g.-SmokerSmoker

Figure 3. Mean alveolar bone level changes in each individual test andcontrol tooth, indicates patient number; "P.g." subgingival detectionof P. gingivalis at 36 months; and "smoker" an active smoking habit;-indicates 2 coinciding values;^^^~indicates 4 coinciding values.

Test Controln=ll n=8

36 months7-P.g.-Smoker

3-P.g.3'6 monlhs

7-P.g.-Smoker

Figure 4. Mean probing attachment level changes in each individual testtooth, t indicates patient number; "P.g." subgingival detection of P.gingivalis at 36 months; and "smoker" an active smoking habit;-indicates 2 coinciding values;^^^~indicates 4 coinciding values.

el, probing attachment level, and probing depth occurredin test or control teeth (Figs. 1 through 5).

Parameters Influencing Long-Term TreatmentOutcomeAt 36 months, subgingival P. gingivalis was found in 3test and 2 control teeth, whereas A. actinomycetemcomi-tans was not detected in any of the investigational teeth.Subgingival infection with P. gingivalis did not have aconsistent influence on the maintenance of alveolar bonelevel in test or control teeth (Figs. 3 and 4).

Only 1 patient (number 7) was an active smoker duringthe course of the study and the only patient who lostalveolar bone level in the test tooth after 6 months. Al-though the alveolar bone level was stable in this patient'scontrol tooth after 6 months, there had been alveolar boneloss immediately after periodontal surgery. In this patient,both test and control teeth were also infected subgingi-vally with P. gingivalis (Figs. 3 and 4).

In test and control teeth, mean plaque index scores didnot change significantly over the course of the study; i.e.,after completion of initial therapy. In addition, there wasno significant correlation of plaque index and gingivalindex scores at 6 or 36 months with the maintenance of

alveolar bone level.


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Test

baseline 6 months 36 months

Control

baseline 6 months 36 months

Figure 5. Representative radiographs of intraosseous defects treated by implantation of AAA bone (test) ormodified Widman flap surgery alone (control) at baseline and 6 and 36 months following therapy.


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January 1998

Correlation Between Pre- and IntraoperativeAlveolar Bone Level Measurements

Preoperative alveolar bone level measurements showedhigh correlation with the alveolar bone level assessed in-traoperatively (r = 0.97, < 0.0001).

Compliance With Supportive Periodontal TherapyThe 8 patients completing the study attended supportiveperiodontal therapies with an average interval of 6.5 ±3months and a range of 4 to 9 months.

DISCUSSIONThe short-term results of this study confirm previous re-ports indicating that implantation of AAA bone in peri-odontal intraosseous defects may result in significantlymore alveolar bone gain compared to open debridementalone. The amount of osseous defect fill after implantationof AAA bone fill; i.e., 46%, was similar to that found in

other studies following implantation of AAA bone orDFDBA where the defect fill ranged from 48% to 65%.5-7Although there was significantly more alveolar bone gainfollowing implantation of AAA bone compared to opendebridement alone, the mean difference in alveolar bone

gain between test and control teeth was only 1.0 ±0.5 mm.A risk-benefit assessment for the use of AAA bone in

periodontal regeneration is necessary in light of potentialdisease transmission by allografts which is estimated tobe minimal but not risk free.27 In addition, mean alveolarbone gain results were highly variable, ranging from 0.5mm to 4.5 mm, indicating a low predictability of treat-ment outcome. Highly variable results have been also re-ported for other regenerative techniques; e.g., guided tis-sue regeneration.2829

The long-term results of this randomized controlled tri-al demonstrated for the first time that alveolar bone and

probing attachment gain after implantation of AAA bonein periodontal intraosseous defects may be maintainedover a minimum of 3 years in patients receiving sup-portive periodontal therapy. Maintenance of alveolar boneand probing attachment levels was similar in both test andcontrol defects. This indicates that the newly formed boneand periodontal attachment after implantation of AAA

bone may react similarly to supportive periodontal ther-apy as non-grafted defects following open debridementand supports previous reports demonstrating a compara-ble behavior of probing attachment levels in sites treatedby GTR and sites treated conventionally during support-ive periodontal therapy.18 Since the 3 patients not com-plying with supportive periodontal therapy did not com-plete the 36-month examination, it remains unknownwhether the short-term treatment outcome after implan-tation of AAA bone can be maintained without supportiveperiodontal therapy.

Subgingival detection of P. gingivalis 4 years follow-

ing periodontal regeneration using ePTFE membranes has

been previously associated with a significantly increasedrisk for probing attachment loss during supportive peri-odontal therapy.17 In the present study, the only patientdemonstrating loss of alveolar bone and probing attach-ment in the test tooth during supportive periodontal ther-apy had subgingival P. gingivalis and was an activesmoker. Since no other patient experienced alveolar boneloss at the test site, the statistical power was too low to

identify any single factor that adversely influenced main-tenance of the short-term treatment outcome. Another ma-

jor risk factor associated with probing attachment lossafter guided tissue regeneration (GTR); i.e., high plaquescores, was absent in this study;16 18 all patients performedgood oral hygiene during the course of the study (meanplaque index of 0.9±0.3 or less). Thus, gained alveolarbone and probing attachment following the implantationof AAA bone and following GTR using ePTFE mem-branes may be maintained over a long period in patientspracticing good oral hygiene and receiving supportiveperiodontal therapy.18-30

Bone level measurements by sounding under local an-esthesia were found to correlate highly with intraoperativebone level measurements (r = 0.97, < 0.0001). Thisfinding indicates that re-entry procedures to determine al-veolar bone gain after regenerative procedures may beunnecessary, thereby facilitating patient recruitment andcompliance for clinical trials assessing long-term treat-ment outcomes of regenerative procedures. In addition,the risk of losing bone resulting from the reflection of amucoperiosteal flap31 may be avoided. In Class II furca-

tions, it has been reported that gained alveolar bone fol-lowing reconstructive periodontal surgery was lost afterre-entry procedure.32-34

The results of this study indicated that implantation ofAAA bone in intraosseous periodontal defects may resultin higher alveolar bone and probing attachment gain com-pared to open debridement alone. In patients receivingsupportive periodontal therapy, the short-term treatmentoutcome may be maintained over a minimum of 3 years.However, due to the low, but statistically significant in-cremental alveolar bone and probing attachment gain fol-lowing implantation of AAA bone compared to open de-

bridement alone, the high variability of results, and theminimal but potential risk for disease transmission, theclinical relevance of this regenerative technique needs tobe questioned.

AcknowledgmentsThe authors are indebted to C. Kopp for excellent tech-nical assistance, Dr. I. Haubitz for conducting the statis-tical analysis, and D.M.S. Flemmig for reviewing themanuscript.

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Send reprint requests to: Dr. Thomas F. Flemmig, Department of Per-iodontology, Pleicherwall 2, 97070 Würzburg, Germany; fax: +49-931-201-7268; e-mail: [email protected]

Accepted for publication May 19, 1997.

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Flemmig1998- Long-Term Maintenance of Alveolar Bone Gain After Implantation of Autolyzed, Antigen-Extracted, Allogenic Bone in Periodontal Intraosseous Defects