J Periodontol • March 2004

Effect of Systemic Matrix MetalloproteinaseInhibition on Periodontal Wound Repair:A Proof of Concept TrialR. Gapski,* J.L. Barr,* D.P. Sarment,* M.G. Layher,* S.S. Socransky,† and W.V. Giannobile*‡

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Background: The adjunctive use of matrix metalloproteinase(MMP) inhibitors with scaling and root planing (SRP) promotesnew attachment in patients with periodontal disease. This pilotstudy was designed to examine aspects of the biologicalresponse brought about by the MMP inhibitor low dose doxycy-cline (LDD) combined with access flap surgery (AFS) on themodulation of periodontal wound repair in patients with severechronic periodontitis.

Methods: Twenty-four subjects were enrolled into a 12-month,randomized, placebo-controlled, double-masked trial to evalu-ate clinical, biochemical, and microbial measures of disease inresponse to 6 months therapy of either placebo capsules + AFSor LDD (20 mg b.i.d.) + AFS. Clinical measures including prob-ing depth (PD), clinical attachment levels (CAL), and bleedingon probing (BOP) as well as gingival crevicular fluid bone markerassessment (ICTP) and microbial DNA analysis (levels and pro-portions of 40 bacterial species) were performed at baselineand 3, 6, 9, and 12 months.

Results: Patients treated with LDD + AFS showed more potentreductions in PD in surgically treated sites of >6 mm (P <0.05,12 months). Furthermore, LDD + AFS resulted in greater reduc-tions in ICTP levels compared to placebo + AFS. Rebounds inICTP levels were noted when the drug was withdrawn. No statis-tical differences between the groups in mean counts were foundfor any pathogen tested.

Conclusions: This pilot study suggests that LDD in combi-nation with AFS may improve the response of surgical therapyin reducing probing depth in severe chronic periodontal disease.LDD administration also tends to reduce local periodontal boneresorption during drug administration. The use of LDD did notappear to contribute to any significant shifts in the microbiotabeyond that of surgery alone. J Periodontol 2004;75:441-452.

KEY WORDSBone resorption/prevention and control; doxycycline/thera-peutic use; periodontal attachment; periodontal diseases/ther-apy; surgical flaps.

* Center for Craniofacial Regeneration and Department of Periodontics/Prevention/Geriatrics, School of Dentistry, University of Michigan, Ann Arbor, MI.

† The Forsyth Institute, Boston, MA.‡ Department of Bioengineering, College of Engineering, University of Michigan.

Editor’s note: A guest editorial commenting on this paper appears on page 493.

Although periodontitis is initiated bysubgingival microbiota, it is gener-ally accepted that mediators of

connective tissue breakdown are gener-ated to a large extent by the host’s res-ponse to the pathogenic infection.1 In asusceptible host, microbial virulence fac-tors trigger the release of host-derivedenzymes such as proteases (e.g., matrixmetalloproteinases [MMPs]) which can leadto periodontal tissue destruction.2-4 Colla-genases, a subclass of the MMP family, area group of enzymes capable of disruptingthe triple helix of type I collagen in phys-iological conditions, the primary structuralcomponent of the periodontium. Elevatedlevels of collagenases and other host-derived proteinases (e.g., cathepsins, elas-tase, tryptases/trypsin like proteinases)have been detected in inflamed gingiva,gingival crevicular fluid (GCF), and salivaof humans with periodontal disease.5-7

Studies have shown that tetracyclines(including the semi-synthetic analoguesminocycline and doxycycline) are capa-ble of inhibiting mammalian collagenaseactivity.8,9 Tetracycline’s inhibitory effecton collagenases appears to occur throughthe drug’s ability to bind to zinc cationsas well as calcium ions in the catalyticdomain of the MMP molecule.10,11 In fact,tetracycline also inhibits another class ofCa++ dependent metalloproteinases termedgelatinases (or type IV collagenases)12

and prevents the degradation of a serumprotein called α1-proteinase inhibitor (orα1-antitrypsin).13 Furthermore, adminis-tration of low doses of doxycycline (LDD;a tetracycline analog) given 20 mg b.i.d.

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rather than the 50 or 100 mg capsules, typically admin-istered as commercially available antibiotics, effectivelyinhibit collagenase activity in the gingival tissues aswell as in GCF14 without exerting direct effects on thesubgingival microorganisms.

Investigations have demonstrated the efficacy ofLDD therapy on promoting probing depth (PD) reduc-tion and clinical attachment level (CAL) gains whencombined with scaling and root planing (SRP) therapyin chronic periodontitis patients.15,16 Caton and co-workers, in a multi-center clinical trial, found that a9-month regimen of LDD + SRP provided statisticallysignificant improvements in CAL, PD, and bleeding onprobing (BOP) over a 9-month period compared toplacebo + SRP in periodontitis patients.17

Many of the crucial cellular responses of early post-surgical wound healing, such as inflammatory infiltra-tion, angiogenesis, and reepithelialization, are madepossible through the action of MMPs.18 In addition, theamount and organization of normal wound extracellu-lar matrix are determined by a dynamic balance amongoverall matrix synthesis, deposition, and degrada-tion.19,20 Expression of MMPs such as collagenasesand gelatinases are elevated in acute wounds and stillgreater levels are found in chronic wounds, indicatingthat uncontrolled proteolysis is a characteristic ofretarded healing.18,21,22 Porcine skin wound modelshave demonstrated that collagenase has early peakexpression at 1 to 7 days post-wounding, followed byslow reduction postoperatively with time, with thesharpest decline occurring after completion of epithe-lialization.23 The importance of collagen turnover kinet-ics mediated by MMP expression during wound healingcan be demonstrated in diabetes. It has been shownin both preclinical and human studies that uncontrolledhyperglycemia may impair wound healing, and this im-pairment seems to be directly associated with overex-pression of MMPs in the wound site.21,24,25 Furthermore,the utilization of CMT-1 (a chemically modified tetracy-cline that lacks antimicrobial properties but retainsdivalent cation binding and MMP inhibitory activity)inhibits diabetic connective tissue breakdown in strep-tozotocin-induced (STZ) diabetic rats by increasingboth steady-state levels of type I procollagen mRNAand collagen synthesis through mechanism(s) that areindependent of the antibacterial properties of tetracy-clines.26 In another preclinical study,27 STZ-induceddiabetic rats exhibited cessation of periosteal andcancellous bone formation. Interestingly, treatment ofdiabetic animals with 20 mg/day of minocycline(a semisynthetic tetracycline) showed increased boneformation rates comparable to control animals.27 More-over, growth plate thickness, reduced 43% in diabeticrats, were preserved in the diabetic animals treatedwith minocycline.27 Therefore, inhibition of elevatedlevels of specific MMPs during the early phases of tissue

repair may contribute to improved treatment outcomesby promoting a favorable wound healing in both con-nective tissue and bone.

The emphasis of this pilot, placebo-controlled,double-masked clinical investigation was to examineclinical and laboratory measures of periodontal statusresulting from a 6-month regimen of LDD on postsur-gical periodontal healing in patients with severe chronicperiodontitis. Clinical, microbiological, and biochemicalparameters were monitored over a 12-month period todetermine the biological response of LDD on surgicalperiodontal therapy.

MATERIALS AND METHODSStudy PopulationA written informed consent was provided for individualswilling to participate in this trial who met the inclusionand exclusion criteria outlined in a protocol approvedby the University of Michigan Institutional Review Board.Twenty-four subjects (aged 30 to 75 years) diagnosedwith severe chronic periodontitis were enrolled into apilot, double-masked, randomized, placebo-controlled,prospective, parallel arm trial. Pertinent inclusion crite-ria included patients with at least three teeth in the samesextant demonstrating both PD and CAL ≥5 mm to≤12 mm and BOP. The patients also had to have atleast 10 teeth in the functional dentition. Exclusion crite-ria included chronic treatment (i.e., 2 weeks or more)with any medication known to affect periodontal status(i.e., antibiotics or non-steroidal, anti-inflammatorydrugs); antibiotic treatment within 90 days of baseline;clinically significant or unstable organic diseases; com-promised healing potential such as connective tissuedisorders or bone metabolic diseases; conditions neces-sitating antibiotic prophylaxis; females who were preg-nant (as determined by positive urine pregnancy test atthe prescreening visit) or lactating, or who were of child-bearing potential and not utilizing birth control or absti-nence; documented allergies to tetracyclines; heavycigarette smokers (≥2 packs/day); active infectious dis-eases; immunocompromised; or taking steroid medica-tions. All dental care related to this trial was providedat no cost to eligible participants.

Study DesignPatients eligible to participate in the study received full-mouth SRP within 90 days of baseline (surgery). Four-teen days prior to surgery, subjects were again examined(screening visit) and inclusion/exclusion criteria reviewed.In this visit, two prescreened patients did not meet theinclusion criteria due to healing from SRP. Subjects whostill qualified for the study and required access flap surgery(AFS) in a minimum of one sextant were randomized toreceive either doxycycline hyclate 20 mg b.i.d.§ or

§ Periostat, CollaGenex Pharmaceuticals, Inc., Newtown, PA.

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� Vicryl, Ethicon, Inc., Somerville, NJ.

line appointment. Six sites weremeasured around each tooth inthe mouth (mesio-buccal, buc-cal, disto-buccal, disto-lingual,lingual, and mesio-lingual) forPD, CAL, and BOP. At the samevisit, microbiological parame-ters and gingival crevicular fluid(GCF) samples were collectedon teeth assigned to receivesurgery. Examiners (JLB andMGL) were trained and calibra-ted to minimize intra-rater (i.e.,intraexaminer) variability. Inaddition, in order to minimize theimpact of inter-rater variabilityon the sensitivity of this study,the same examiner assessed thesame patients for the durationof the trial.

Surgical procedures were thenperformed at baseline (BL) within14 days of the screening visitby one surgeon (RG). Patientsreturned for follow-up examina-

tions at months 1, 2, 3, 6, 9, and12 following baseline. Full-mouthmanual probing measurements,GCF, ICTP levels, and microbio-logical samples were assessed atBL and months 3, 6, 9, and 12.Patients received periodontal main-tenance therapy at 3, 6, 9, and12 months following randomizationand periodontal surgery. The studydesign is shown in Table 1.

Surgical ProcedureBriefly, following local anesthesia,treatment sextants received AFS,which consisted of submarginalprimary incisions (Fig. 1A) andfull-thickness mucoperiosteal flapreflection. The tooth root surfaceswere debrided, scaled and rootplaned using hand and sonic instru-mentation. Osseous recontouring(osteoplasty) when indicated wasperformed for flap adaptation(Fig. 1B). The tissues were reposi-tioned with polyglactin 910� orsilk suture materials (Fig. 1C). Thesurgical sites were then covered

with a periodontal dressing. After surgery, patients

Table 1.

Clinical Trial Longitudinal Measures

Month

Event Pre-Screening Screening Baseline 1 2 3 6 9 12

Medical history X

Informed consent X

SRP X

Full-mouth measurements X X X X X X X

Microbial sampling X X X X X

Urine pregnancy test X X

Surgery X

C-telopeptide level X X X X X X

Maintenance therapy X X X X

Dispense medication X X

Drug accountability X X

Figure 1.Access flap surgical procedure in a patient administered 20 mg b.i.d. LDD for a period of 6 months,followed for an additional 6 months. A) Scalloping incisions made to remove the collar of gingivaltissue. B) After flap reflection, the surgical site was debrided followed by SRP and osteoplasty wherenecessary. C) Flaps were repositioned utilizing interrupted sutures 4-0 polyglactin 910. Periodontaldressing was placed and 0.12% chlorhexidine rinses utilized for 14 days. D) LDD-treated sextant 1year post-LDD therapy + surgery. Note clinical appearance of normal gingival architecture andhealing.

placebo capsules b.i.d. for the 6-month treatmentperiod. Full-mouth manual clinical measurementswere recorded for all teeth within 2 weeks of the base-

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received pain control medication (an acetaminophen-containing analgesic) when needed and chemical plaquecontrol (0.12% chlorhexidine rinse) for 14 days.

Dispensing of Study Medication/Assessmentof ComplianceAt the screening visit, qualifying patients were assigneda code and were then randomized by a computer soft-ware program to the drug or placebo group. At thebaseline visit, each patient was issued a 3-month sup-ply of coded study medication including one containerfor morning and another for afternoon/evening intake.Three months following surgery, subjects returned resid-ual capsules and received an additional 3-month sup-ply. The residual capsules were counted and retained at3 and 6 months after baseline. Patient compliance (asa percentage) with the dosing schedule was recorded.

Microbiological MonitoringSubgingival plaque samples were collected from themesio-buccal sites of each tooth scheduled for surgeryusing sterile curets. Samples were then placed in ster-ile, labeled tubes containing 150 µL TE buffer (10 mMTris-HCl, 1 ml EDTA, pH 7.6) and vortexed. Microbialanalyses were performed for the presence and levelsof 40 subgingival pathogens (Table 2) utilizing wholegenomic DNA probes and checkerboard DNA-DNAhybridization as previously described.28 Signals wererecorded as 0: not detected; 1: <105 cells; 2: ∼105 cells;3: 105 to 106 cells; 4: ∼106 cells; and 5: >106 cells.

Bone Marker (ICTP) AnalysisGingival crevicular fluid was collected prior to clinicalmeasurements. Gingival tissue surrounding the site tobe sampled was dried with gauze, and supragingivalplaque was removed. A sterile methylcellulose strip¶

was gently placed into the mesio-buccal aspect of eachtooth sulcus or pocket until slight resistance was feltand the GCF collected for 10 seconds. Following col-lection, the sample was kept on ice for transport to thelaboratory and stored at −80°C. The frozen sampleswere then thawed at room temperature and proteinswere eluted through five centrifugations at 3,000 RPMfor 5 minutes with 20 µl phosphate buffered saline(PBS, pH 7.4) containing 15 nM aprotinin, 1 mM PMSF,and 0.1% human serum albumin as previouslydescribed.29 GCF ICTP levels were quantified usingradioimmunoassay.#

Gingival Biopsies (Protease Determination)Six patients (3 from test group, 3 from placebo group)scheduled to receive two sextants of surgery were eval-uated for changes in gingival proteinase activity, utiliz-ing gelatin zymography. Excised tissues from eachtreatment sextant were immediately placed on ice andsubsequently frozen (−80°C). Two months later, tissue

samples were harvested from another sextant, duringa subsequent surgery. To prepare tissue extracts, sam-ples were thawed on ice, homogenized in PBS, andcentrifuged (10,000 × g for 10 minutes at 4°C). Proteinconcentration in the extracts was determined using aprotein assay kit** using bovine serum albumin (BSA)as a standard. The supernatants were diluted with anequal volume of glycerol and stored at –80°C. Proteaseactivity was assessed by 12% gelatin zymography††

as previously described.30

Statistical AnalysesData available for each patient were subjected to anintent-to-treat analysis and included full-mouth clinicalmeasurements for three different parameters (PD, CAL,

¶ Periopaper, IDE-Interstate, Amityville, NY.# DiaSorin, Inc., Stillwater, MN.** BCA Protein Assay Reagent, Pierce, Rockford, IL.†† Bio-Rad Laboratories, Hercules, CA.

Table 2.

DNA Probes Used to Examine SubgingivalPlaque Samples

Actinobacillus actinomycetemcomitans Gemella morbillorum

Actinomyces. gerencseriae Leptotrichia buccalis

Actinomyces israelii Neisseria mucosa

Actinomyces naeslundii 1 Propionibacterium acne

Actinomyces naeslundii 2 Prevotella melaninogenica

Actinomyces odontolyticus Peptostreptococcus micros

Tannerella forsythus Porphyromonas gingivalis

Camphylobacter gracilis Prevotella intermedia

Camphylobacter rectus Prevotella nigrescens

Camphylobacter showae Selemonas noxia

Capnocytophaga gingivalis Streptococcus anginosus

Capnocytophaga ochracea Streptococcus constellatus

Capnocytophaga sputigena Streptococcus gordonii

Eikenella corrodens Streptococcus intermedius

Eubacterium nodatum Streptococcus mitis

Eubacterium saburreum Streptococcus oralis

Fusobacterium nucleatum ss nucleatum Streptococcus sanguis

Fusobacterium nucleatum ss Treponema denticolapolymorphum

Fusobacterium nucleatum ss vincentii Treponema socranskii

Fusobacterium periodonticum Veillonella parvula

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and BOP) examined at six sites per tooth.Sample size determination for this pilot studywas determined from Golub et al.8 using 80%power for differences expected in a primarybiological outcome measure, the bone resorp-tion marker, ICTP. The level and prevalenceof 40 species and ICTP levels (pg/site) wasmeasured from the surgically-treated quad-rant excluding third molars. Clinical, micro-bial, and ICTP data were averaged within apatient and then compared between groups.In other analyses, data were stratified accord-ing to baseline PD of 1 to 4, 5 to 6, and≥7 mm and averaged in a patient and thenacross the study population for clinical para-meters and ICTP levels. Differences betweendrug and placebo groups were performedusing the Mann-Whitney test. Differenceswith a P value less than 0.05 were consideredsignificant. Microbiologic data were analyzedseparately for sites receiving AFS + LDD andAFS + placebo. Mean levels (counts × 105)for each of 40 species were computed foreach patient in each treatment category ateach visit. Significant differences over time were deter-mined separately for each treatment category using theQuade test and the differences between groups weredetermined by the Mann-Whitney test. All analyses wereperformed using the patient as the unit of analysis.

RESULTSCalibrationThe results for calibration of the examiners revealed theintraclass correlation coefficients for PD were >0.90and for CAL measurements were >0.85. Intraexaminerreliability was determined utilizing the percent agree-ment within 1 mm between 2 passes and a weightedkappa statistic. The percent agreement within 1 mmwas ≥95.5% and ≥90% for PD and CAL measurements,respectively. The weighted kappa statistic was ≥0.92and ≥0.88 for PD and CAL measurements, respectively.

DemographicsA total of 24 patients entered into the study; 12 wererandomized to the LDD + AFS group and 12 to theplacebo + AFS group. Ten out of 12 patients in the druggroup and 11 out of 12 in the placebo group completedthe study for an overall drop-out rate of 12.5%. Onepatient in the LDD group moved away while the otherbegan administration of a calcium channel blocker thatconflicted with the inclusion criteria. The patient in theplacebo group reported lethargy after capsule intakeand requested exit from the study. At baseline, therewere no statistically significant differences (NS) betweengroups regarding age and gender (Table 3). There weretwo screening failures at baseline due to changes in PD

Table 3.

Baseline Mean (±± SD) Clinical Characteristicsand ICTP

Characteristic Placebo (n = 12) Drug (n = 12) P (Mann-Whitney)

AgeMean (SD) 53.3 (±9.8) 45.9 (±8.3) NS*Range 38-71 32-60

% males 67% 67% NS

Tobacco use (n [%])Current 3 (25%) 5 (41.7%)Past 6 (50%) 4 (33.3%)Never 3 (25%) 3 (25%)

BOP (%) 51.3 (±19.3) 56.6 (±15.9) NS

PD (mm) 3.0 (±0.5) 3.1 (±0.3) NS

CAL (mm) 3.8 (±0.8) 4.1(±0.8) NS

ICTP (pg/site) 34.0 (±44.5) 52.0 (±54.2) NS

* Not significant.

following SRP making the individuals ineligible for sur-gical therapy. Seventy-five percent of subjects reportedpast and current use of tobacco in both LDD andplacebo groups (Table 3). In addition, no significant dif-ferences were noted between the treatment groups withrespect to baseline clinical parameters (PD, BOP, andCAL) and ICTP levels (Table 3). Levels and distributionof subgingival species averaged per group at baselineare displayed in Figure 2. There were no significant dif-ferences between LDD and placebo groups for any ofthe 40 microorganisms analyzed at baseline.

ComplianceThe mean overall drug compliance for the LDD groupwas 80.6% (range 62% to 105%) while the placebogroup mean was 83.0% (range 18% to 100%); differencenon-significant (NS) between groups. Data also demon-strated that >90% of patients from both groups were>70% compliant during the study. Patients had a ten-dency to be slightly more compliant in the morningthan in the evening (84.1 ± 6.4% versus 79.4 ± 3.5%,respectively; P >0.05). The subjects also showed trendsof better compliance during the first 3 months of the trialcompared to the second 3 months (84.8 ± 5.0% versus79 ± 4.3%, respectively; P >0.05). One patient in thedrug group showed low compliance (18%).

Treatment Outcomes: Responses in ClinicalParameters and ICTP LevelsPooled surgical sites (all probing depth strata). Themean per-patient averages in BOP, PD, CAL, andICTP from baseline for all surgical sites are shown in

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Figure 3. As expected, both placebo and test groupsdemonstrated a statistically significant reduction in PDcompared to baseline (P <0.001); however, no differ-ences between groups were found. Surgical therapyresulted in mean CAL gains in both groups (P >0.05).The LDD group demonstrated a statistically significantreduction in percentage of BOP sites at 3 and 6 monthscompared to baseline (25% to 60%, P <0.05) while inthe placebo group, no statistical significance was found(∼15%, P >0.05). In addition, the LDD group demon-strated a decreased but not statistically significantexpression of GCF ICTP levels compared to placeboduring the drug administration period.

Moderate sites (baseline PD 5 to 6 mm). Meanper-patient averages in BOP, PD, CAL, and ICTP levelsfrom baseline at surgical sites with a baseline PD of 5to 6 mm are shown in Figure 4. Consistent with resultsfrom pooled PD strata, there were significant reductionsin PD and gains in CAL compared to baseline for bothdrug and control groups, while no statistical differencebetween treatments was found although trends for adrug effect for PD reduction were noted. ICTP levelswere reduced while patients were on LDD therapy, but

a rebound in ICTP levels in theLDD + AFS group was demon-strated after patients discontinuedthe drug. There was a statisticallysignificant reduction in the percentof BOP sites at 3 and 6 monthscompared to baseline in the LDD +AFS group (P <0.05), while no sucheffect was seen in patients receiv-ing placebo capsules (P >0.05).Both LDD + AFS and placebo +AFS groups showed comparablelevels in percentage of sites BOPafter the patients discontinued drugtherapy (P >0.05).

Deep sites (baseline PD ≥≥7 mm).Figure 5 shows the mean per-patient averages in BOP, PD, CAL,and ICTP levels for initially deepsites. Greater reductions in PD werenoted in this stratum for bothgroups. Drug therapy resulted inmore statistically significant reduc-tion in PD in deep sites comparedto controls (P <0.05) and this dif-ference was sustained for the dura-tion of the study. However, nodifferences were found betweengroups with respect to CAL. Inaddition, patients receiving LDD +AFS showed greater reductions inICTP levels compared to placebo +AFS during the first 6-month

observation period, but no statistical differences werefound (P = 0.09). This difference was also not signifi-cant when patients discontinued drug therapy. Again,LDD therapy resulted in a statistical reduction in thepercent of BOP sites (P <0.05) for 6 months while nosuch reductions were demonstrated in placebo controls(P >0.05). However, these differences between groupswere not significant by the end of the trial (P >0.05).

Microbial OutcomesComparisons between LDD + AFS and placebo + AFSwere performed utilizing mean microbial DNA countsof each microorganism averaged per patient and thenper group. When interanalysis was performed, no sta-tistically significant differences were found for any of the40 microorganisms tested at any given time point (datanot shown). There was an immediate reduction in redcomplex and orange complex species in both drug andplacebo groups after AFS was performed (Fig. 6).

Gelatin Zymography for Proteinase ExpressionFive patients (two LDD and three placebo) completedbaseline and 2 months harvest of gingival tissues for

Figure 2.Mean counts (±SEM) of species sampled at baseline. Microbial analyses were performed atbaseline for the presence and levels of 40 subgingival species utilizing whole genomic DNA probesand checkerboard DNA-DNA hybridization.The mean count for each species was computed foreach patient and then averaged across patients. Significant differences were determined using theMann-Whitney test. SEM = standard error of the means. Colors represent bacteria cluster groupingsdescribed by Socransky et al. 31

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gelatin zymography analysis. After tissue preparation,zymograms were run in a standard manner, utilizingcommercially available collagenases type IV as a posi-tive control. Results generated from all patients demon-strated that a downregulation in gelatinase activity (mean7.2%; range: 4.9% to 8.5%) in patients receiving adjunc-tive therapy, while an increase in activity was seen in theplacebo group (mean 5.6%; range: −10.4% to 17.5%).

DISCUSSIONThe purpose of the present investigation was to attemptto unravel biological changes that occur as a result ofa combination of periodontal therapies with antibac-terial effects (i.e., surgery) and host modulatory effects(i.e., LDD). The consideration of the bacterial-hostinteraction as a basic mechanism for periodontitispathogenesis has driven the concept that successfullong-term management of this disease may be depen-dent upon an effective control of bacterial load in com-bination with host modulatory therapies. To date, several

bone-sparing agents or host mod-ulatory drugs have been devel-oped to control pathwaysresponsible for periodontal tissuebreakdown such as regulation ofimmune32,33 and inflammatoryresponses,34-37 excessive pro-duction of matrix metallopro-teinases (MMPs),8,10,38 arachidonicacid metabolites,39-42 and regula-tion of bone metabolism.43,44

This pilot investigation soughtto evaluate the potential additiveeffect of LDD to modulate woundrepair in conjunction with peri-odontal surgery. The goal was toexplore the clinical, microbial, andbiochemical impact of this drugon the modulation of wound heal-ing post-surgically. Followingmechanical therapy in the form ofSRP, patients received access flapsurgery and were randomized toLDD or placebo groups for 6months of drug therapy, followedby a 6-month post-dosing evalu-ation. As expected, both therapiesled to significant clinical improve-ments in terms of PD reductionand CAL gain (Figs. 3, 4, and 5).Patients in the placebo + AFSshowed probing depth reductionsand clinical attachment level gainssimilar to previously reported lon-gitudinal studies, when the sametherapy was compared.45,46

Tetracycline analogues have been some of the mostextensively investigated host modulatory agents andspecifically, the low dose regimen of doxycyclinehyclate (20 mg b.i.d.).6 Both preclinical and humanclinical data have shown that tetracycline can sup-press MMPs,8,10,38 which are considered to be primaryenzymes responsible for the destruction of extracellularmatrix macromolecules. Investigators have demon-strated the effects of adjunctive LDD in conjunctionwith supragingival scaling and dental prophylaxis,47-49

and SRP.15,17,50 Caton et al.17 indicated that a 9-monthcourse of LDD with SRP resulted in statistically signif-icant improvements in PD reduction and CAL gainswhen compared with placebo controls. The effectswere maintained 12 months after initial therapy.50

In this study, clinical efficacy was examined at siteswith baseline PDs of 1 to 4 mm for shallow sulci/pockets(data not shown), 5 to 6 mm for moderate pockets(Fig. 4), and ≥7 mm for deep pockets (Fig. 5). Thisallowed analysis of the effects of LDD at sites fulfilling

Figure 3.Effect of LDD or placebo in combination with surgery on clinical parameters and ICTP for all sitesreceiving surgery. The shaded background indicates time of drug or placebo administration.The barsrepresent per-patient standard errors. P values indicate significant changes over time within atreatment as determined by the Quade test.

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the criteria for periodontal surgery (i.e., PD ≥5 mm). Inaddition, the patients exhibited moderate to severehorizontal bone loss that is generally not indicated fortraditional regenerative therapy. Therefore, patientsincluded in this trial exhibited bone loss patterns withlimited treatment options (non-surgical therapy or poc-ket reduction/elimination surgery). Although the presentinvestigation was not designed as a definitive clinicaltrial, our results suggested a number of intriguing leads.When LDD was administered in combination with AFS,a greater probing depth reduction was found in deepsites of AFS + LDD subjects compared to controls(Fig. 5, P <0.05). This result was maintained 6 monthsafter discontinuation of the drug (Fig. 5, P <0.05). How-ever, no statistically significant differences between ran-domized groups were noted for CAL in any of the probingdepth strata. There may be potential mechanisms thatexplain the PD reduction seen with LDD therapy: LDDtherapy may promote more rapid wound maturation by

inhibiting collagen disruption andindirectly encouraging collagen for-mation. Therefore, differences intiming, quality, and quantity ofwound contraction in the periodon-tal tissues might have promotedadditional PD reduction in the druggroup.

The results of this study re-vealed a significant reduction inpercentage of sites exhibiting BOPwhen patients were taking LDD ascompared to the placebo group(Figs. 3, 4, and 5). These resultsare in agreement with a previousinvestigation,17 where a reductionin bleeding sites was seen inpatients receiving adjunctive LDDcompared to placebo. By contrast,Golub et al.9 reported only mini-mal and non-significant reductionsin gingival inflammation and bleed-ing index51 with the use of LDD. Inthat study,9 the authors suggestthat the improvement in periodon-tal conditions by LDD therapywas mainly by inhibiting collagen-destructive enzymes rather thanaffecting microbially-induced in-flammation.9 However, it is import-ant to note that the effects of LDDon BOP in our investigation alsoappeared to be independent ofmicrobial shifts. Therefore, theeffects of LDD on gingival inflam-mation (i.e., BOP) need to be fur-ther evaluated. In addition, it should

be noted that both placebo and test groups experi-enced high BOP frequency at 12 months when deepprobing depths were considered. The reason for thatlies in the fact that the majority of patients were sur-gically treated only in the tested quadrant. In otherwords, remaining pockets after initial therapy on otherquadrants might have served as reservoirs of putativebacteria that potentially infected the surgically-treatedpockets. These findings have been confirmed by otherinvestigations which reported that a natural recolo-nization of pathogenic microorganisms occurs afterperiodontal therapy.52-54 Therefore, this aspect couldexplain the high frequency of BOP in deep sites aswell as recolonization of putative microorganisms foundin this investigation (e.g., Actinobacillus actino-mycetemcomitans, Porphyromonas gingivalis, and Pre-votella intermedia).

LDD + AFS resulted in a decrease in the putativebone resorption marker ICTP soon after the surgical

Figure 4.Effect of LDD or placebo in combination with surgery on clinical parameters and ICTP levelsfor initial pockets of 5 to 6 mm.The shaded background indicates period of drug or placeboadministration.The bars represent per-patient standard errors. P values indicate significant changesover time within a treatment as determined by the Quade test.

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therapy at 3 months while placebo controls demon-strated no change or increases in ICTP levels over the12-month observation period. ICTP reflects the amountof organic matrix being disrupted in alveolar bone(specifically, type I collagen) following osteoclasticbone resorption. Human histological data have shownthat osteoclastic activity peaks immediately after asurgical procedure is performed and is then followedby bone formation.55 Therefore, release of excessiveosteoclastic collagenases (or MMP-13) may haveincreased the disruption of collagen type I in bone andconsequently increased ICTP release in the placebo +AFS group. Golub et al.8 have demonstrated a directeffect of LDD therapy on the reduction of excessiveMMP-13 followed by a concomitant decline in levels ofcollagen degradation fragments. In addition, it has beendemonstrated that the increased circulating concen-tration of ICTP was most likely produced by action ofMMPs.56 In the same study, cathepsin K-mediated boneresorption (an osteoclastic cysteine protease) destroyedICTP antigenicity, further showing the specificity ofMMPs on the initial disruption of bone collagen.56

Hence, our findings have demon-strated that utilization of an MMPinhibitor may have a direct impacton postsurgical bone turnover.This result supports the notion thatdecreased collagenolytic activity(via LDD) can reduce boneresorption and act as a bone-spar-ing agent. Although these resultsare from only a small subset ofpatients from our investigation,these findings should encouragefurther exploration of the impactof host modulatory agents onbone maintenance.

With regard to the issue of low-dose antibiotics possibly resultingin direct microbial shifts, our studyfound no statistically significantdifferences between drug and con-trol groups at any time point forany of the 40 tested species.These results are in agreementwith previous human trials.57,58 Itshould also be noted that only sur-gical sites were evaluated formicrobial analysis, therefore,effects of this drug on other sitesare not available. When the datawere analyzed according to bac-terial complexes as proposed bySocransky et al.,31 a reduction inperiodontal pathogenic organismswas found in both groups, and this

outcome was attributed to the effects of periodontalsurgery. These findings are in concordance with recentreports59,60 that suggest that there is a beneficial effectof surgery on suppression of periodontal pathogensby eliminating reservoirs of virulent organisms. How-ever, effects of continuous administration or cyclicaldosing of LDD + surgery on microbial resistance orshifts were not evaluated in this study.

This investigation demonstrated no differencesbetween groups regarding drug compliance. The over-all compliance was approximately 80% with small ten-dencies for the patients to be more compliant in themorning and in the first 3 months of the drug admin-istration period (NS; P >0.05). In our study, a widerange of compliance was found (18% to 100%) but itwas also demonstrated that the majority of patientswere reasonably compliant with the 6-month b.i.d. reg-imen (over 90% were >70% compliant).

CONCLUSIONSSix-month administration of LDD suggests that thereis an enhanced postsurgical wound healing compared

Figure 5.Effect of LDD or placebo in combination with surgery on clinical parameters and ICTP for initialpocket of ≥7 mm.The shaded background indicates time of drug or placebo administration.The barsrepresent per-patient standard errors. P values indicate significant changes over time within atreatment as determined by the Quade test.

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to placebo controls with regard to PD reduction. Thispositive effect was most marked in deep sites (≥7 mm),where the differences in PD reduction were maintaineduntil the completion of the trial. Reductions in the boneresorption marker ICTP were also found in patients whileon the drug, suggesting the potential of LDD to act asa bone-sparing agent. In addition, the percentage ofBOP sites was affected by LDD therapy, but this effectwas only noticeable during the period of the drug admin-istration. Finally, no significant shifts on the periodon-tal microbiota beyond that attributable to the effects ofthe surgery could be seen with the utilization of LDD.

ACKNOWLEDGMENTSThe authors acknowledge the assistance of Drs. QimingJin, Amin ur Rahman, and Khalaf Al-Shammari duringearly stages of the investigation while at the Universityof Michigan, Ann Arbor, Michigan. The authors alsoappreciate the technical assistance of Denise Guerreroand Tina Yaskell, The Forsyth Institute, Boston, Massa-chusetts. We acknowledge Dr. Lorne Golub, State Uni-versity of New York at Stony Brook, for critically readingthe manuscript. This study was supported by NIH/NIDCR

grants 5T35 DE07101, DE10977,and DE12861; CollaGenex Phar-maceuticals, Inc., Newtown, Penn-sylvania; and the Delta Dental Fundof Michigan.

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Figure 6.Effect of surgery with and without adjunctive LDD therapy on the subgingival microflora. Microbialanalyses were performed for the presence and levels of 40 subgingival pathogens utilizing wholegenomic DNA probes and checkerboard DNA-DNA hybridization.The mean count for each specieswas computed per patient and then averaged across patients.Time points in each graph representbaseline; 3 months; 6 months; 9 months and 12 months, respectively.The shaded backgroundindicates time of drug or placebo administration. Significant differences over time were determinedusing the Quade test, and no differences between groups were detected by the Mann-Whitney test.SEM = standard error of the means.

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59. Levy RM, Giannobile WV, Feres M, Haffajee AD, Smith C,Socransky SS. The effect of apically repositioned flapsurgery on clinical parameters and the composition of thesubgingival microbiota: 12-month data. Int J PeriodonticsRestorative Dent 2002;22:209-219.

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Correspondence: Dr. William V. Giannobile, Department of Peri-odontics/Prevention/Geriatrics, University of Michigan Schoolof Dentistry, 1011 N. University Ave., Room 3397, Ann Arbor,MI 48109-1078. Fax: 734/763-5503; e-mail: wgiannob@umich.edu.

Accepted for publication July 22, 2003.

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