Matrix Metalloproteinase Levels inChildren With Aggressive PeriodontitisBarnett Alfant,* Luciana M. Shaddox,*† Jeffrey Tobler,* Ingvar Magnusson,* Ikramuddin Aukhil,†

and Clay Walker*

Background: Matrix metalloproteinases (MMPs) are a fam-ily of host-derived proteinases reported to mediate multiplefunctions associated with periodontal destruction and inflam-mation. Most of the existing data have been gathered fromadults with chronic periodontitis. The purpose of this studywas to determine the MMP levels in a cohort of African Amer-ican children with and without aggressive periodontitis.

Methods: Gingival crevicular fluid (GCF) was collected in acohort of 44 African American children, 7 to 19 years of age,with and without aggressive periodontitis (AgP) and com-pared to healthy unrelated children and to adults with chronicperiodontitis (CP). GCF volume was determined with a cali-brated gingival fluid meter. The samples were assayed forMMP-1, -2, -3, -8, -9, -12, and -13 using fluorimetric substrates.

Results: The MMP levels from diseased sites in the subjectswith AgP were statistically higher (P <0.05) in almost all in-stances than those associated with the unrelated controls orwith the subjects with CP. MMP-8 was significantly elevatedin the diseased sites of the children with AgP relative to non-diseased sites in the same children (P = 0.002), as well asthe siblings, non-diseased controls, and subjects with CP (P £0.0001). There was no positive correlation between probingdepth and any MMP level.

Conclusions: MMP levels were elevated in AgP sites relativeto non-diseased sites in the same subjects, in siblings, and inunrelated controls. MMPs associated with the AgP sites in chil-dren were generally elevated compared to an adult cohort witha history of CP. J Periodontol 2008;79:819-826.

KEY WORDS

Children; metalloproteinases; periodontitis.

Matrix metalloproteinases (MMPs),collectively known as matrixins,form a family of highly homolo-

gous zinc- and calcium-dependent en-dopeptidases that regulate cell matrixcomposition. This family includes 23human MMPs that are commonly dividedinto six subgroups: collagenases, gela-tinases (or type IV collagenases), stro-melysins, matrilysins, membrane-typemetalloproteinases, and others. EachMMP has distinct but often overlappingsubstrate specificities, which togethercan cleave virtually all components ofthe extracellular matrix (ECM) and base-ment membrane.1 In addition to ECMdegradation, MMP proteolysis can: createspace for cells to migrate; produce spe-cific cleavage fragments with indepen-dent biologic activity; regulate tissuearchitecture through effects on the ECMand intercellular junctions; and activate,deactivate, or modify the activity of sig-naling molecules directly and indirectly.2

Additional substrates include peptidegrowth factors, cell surface receptors,cell-adhesion molecules, cytokines, andchemokines, as well as other MMPs andunrelated proteases.3 Thus, MMPs par-ticipate in a wide range of physiologicprocesses, including morphogenesis,wound healing, tissue remodeling, angio-genesis, and normal immune responsesto infection.4 To prevent tissue destruc-tion that is due to excessive proteolyticactivity, MMP activity is tightly regu-lated.5 The activities of most MMPs arenormally low in steady-state tissues;

* Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, FL.† Department of Periodontology, College of Dentistry, University of Florida.

doi: 10.1902/jop.2008.070513

J Periodontol • May 2008

819

however, MMP expression can be detected in repair orremodeling processes, in diseased or inflamed tis-sues, and in cell types grown in culture.6 The samefunctions of MMPs that are beneficial under normalphysiologic conditions turn into key mechanisms ofdisease pathogenesis where aberrant ECM turnoverpredominates, such as rheumatoid arthritis, athero-sclerosis, tumor metastasis, and periodontal disease.

Periodontitis is an inflammatory disease of the sup-porting tissues of the teeth, resulting in progressivedestruction of the periodontal ligament (PDL) and al-veolar bone with pocket formation, recession, or both.The pathogenic processes are largely a response tomicrobial-induced destructive mechanisms. Theseprocesses are initiated by the microbial biofilm butare undertaken by the host cells in chronic diseaseprogression; thus, it is the host tissue itself that resultsin the destruction observed.7 The host initiates andcontrols the release of enzymes, including MMPs, toallow the tissues to retreat from the microbial destruc-tive lesions. Recently, there has been increasing evi-dence implicating MMPs as key mediators in thetissue destruction associated with the various formsof periodontal disease, including the progression fromgingivitis to periodontitis.8-10 Under non-pathologicconditions, controlled phagocytosis and intracellu-lar digestion of collagen fibrils are observed at highlevels in dynamic soft tissues, such as gingiva andPDL, during normal tissue remodeling. However, un-der pathologic conditions, the balance between colla-gen synthesis and degradation is disrupted, resultingin progressive tissue destruction that is due to exces-sive collagen breakdown. This condition begins inearly gingivitis, and if it becomes chronic, periodonti-tis may occur, leading to breakdown of the PDL andeventually the alveolar bone. In the progression froma healthy periodontium to a diseased site, collagenbreakdown switches from the well-controlled intracel-lular pathway to a metalloproteinase-mediated extra-cellular pathway.11 In periodontal tissues, MMPs areexpressed by inflammatory cells (monocytes, macro-phages, lymphocytes, and polymorphonuclear cells)and by resident cells (fibroblasts, epithelial cells, andendothelial cells).12 Previous studies, mostly involv-ing subjects with chronic periodontitis (CP), demon-strated elevated levels of various MMPs associatedwith inflammation, as well as correlations betweenthese levels and the extent of tissue destruction.13

In particular, active forms of MMPs in the gingivalcrevicular fluid (GCF) were shown to be associatedwith progressive periodontal destruction.14-17

Although there have been a number of reports15-17,19

concerning various MMP levels in chronic periodonti-tis, little information is available about the levels ofvarious MMPs associated with aggressive periodontitis(AgP) in children. In this study, we report on the MMP

levels of the collagenases (MMP-1, -8, and -13), thegel-atinases (MMP-2 and -9), stromelysin (MMP-3), andmacrophage elastase (MMP-12) in a cohort of children,7 to 19 years of age, with and without localized AgP(LAgP). Because certain MMP levels may be higher inchildren as a result of the oral tissue and bone remod-eling associated with growth, wealso included, for com-parison purposes, MMP levels obtained for a cohort ofadults with CP.

MATERIALS AND METHODS

Study PopulationForty-four African American participants between theages of 7 and 19 years were recruited from the LeonCounty Health Department, Tallahassee, Florida,over a period of 1 year between August 2006 and July2007. After clinical evaluations, 23 subjects with AgP,diagnosed based on probing depths (PDs), clinicalattachment levels, and radiographs, nine siblings withno evidence of periodontitis, and 12 healthy controls,who were unrelated to the diseased subjects (healthycontrols were obtained from the Leon County clinicand the University of Florida College of Dentistrypediatric clinics) were entered into this study. The17 adults with CP were between 35 and 65 years ofage and were in good health with no known underlyingsystemic diseases; most were white and were non-smokers. The subjects with CP were drawn from pa-tients in the general dental clinics and the periodonticsclinic at the University of Florida College of Dentistry.

Pertinent information concerning the study pro-tocol was explained to each patient, and informedconsent was obtained from all participants, or theirparents in the case of underage patients, as requiredin the study protocol approved by the University ofFlorida Institutional Review Board. Complete medicaland dental histories were taken from all participants.All subjects were in good general health, were non-obese, and were considered to be free of any underly-ing systemic diseases. Exclusion criteria included:any history of systemic disease that could interferewith the clinical characteristics, incidence, or progres-sion of periodontal disease; periodontal treatmentwithin the previous 6 months; and chronic treatmentwith any medication known to affect periodontal statuswithin the previous 3 months (i.e., antibiotics, non-steroidal anti-inflammatory drugs, or contraceptives).Clinical diagnosis and the selection of subjects wasbased on the clinical and radiographic criteria pro-posed by the American Academy of Periodontology.18

Sample CollectionFor each of the subjects with AgP, GCF samples werecollected from two deep sites (PD ‡4 mm), almost al-ways from the permanent first molars, and from a non-diseased site (PD £2 mm), which was usually from the

MMP Levels in Children With Aggressive Periodontitis Volume 79 • Number 5

820

permanent anterior teeth. However, a few samplesfrom non-diseased sites were collected from primaryteeth. Samples from the siblings and the healthy con-trols were usually collected from two sites (PD £2 mm)from the permanent first molars. In addition, for com-parison purposes, GCF samples were collected fromtwo sites (PD ‡4 mm) in each adult diagnosed withCP. GCF samples were collected first to avoid any ten-dency the site may have for bleeding on/after plaquesampling. Samples with any gingival bleeding wereexcluded. The sampling area was isolated with cottonrolls to avoid contact with saliva, and supragingivalplaque was removed to eliminate the risk for plaquecontamination. After gentle air-drying, GCF was col-lected using a filter paper strip‡ by inserting the strip1 mm into the sulcus and allowing the GCF to wickup the strip. The sample collection was not timed be-cause it required more time to collect measurable fluidfrom healthy sites than from the diseased sites. A vol-ume was collected that fell on the calibration curve ofthe gingival fluid meter, and the time to collect a mea-surable amount of fluid from healthy sites in the chil-dren varied significantly. Timing the collection wouldhave resulted in too little fluid or an oversaturation ofthe strips depending on the site sampled. The volumeof GCF collected was immediately measured chair-side with a calibrated gingival fluid meter.§ The stripwas placed in a dry microcentrifuge tube and kepton ice. Upon return to the laboratory, the sampleswere stored at -80�C until used for measurement ofthe MMPs. The GCF readings were converted to vol-ume based on standards curves generated for the in-strument used.

GCF Processing and MMP Enzymatic AssaysThe GCF absorbed to the strip was eluted with 50 mlMMP buffer (50 mM Tris-HCl; 0.2 M NaCl; 5 mMCaCl2, pH 7.5) followed by centrifugation to minimizeretention of proteins on the filter paper. The activitiesof MMP-1, -2, -3, -8, -9, -12, and -13 were assessedusing commercially available fluorimetric MMP kitsspecific for each MMPi in accordance with the manu-facturer’s instructions. The detection limits of thesekits are given by the manufacturer as 0.1 to 1.0 ng de-pending on the particular assay kit. Briefly, each spe-cific MMP substrate (50 ml) was diluted in 5 ml assaybuffer (provided with the kit), and 50 ml diluted sub-strate was added to the desired well of a black fluori-metric 96-well microtiter plate.¶ Two microliters of thesample were added to each well and mixed by gentlyshaking for 15 minutes in the dark, followed by incu-bation for an additional 45 minutes in the dark. Eachof the seven MMP substrates was arranged on eachplate in rows, and the samples were applied in col-umns, with the first two wells of each row serving ascontrols for background fluorescence. Fluorescence

intensity was measured at excitation/emission (Ex/Em) = 490 nm/520 nm using a fluorimetric readerand associated software.# The data, initially expressedas relative fluorescence units, were converted to nano-grams based on standard curves (R2 >0.99) generatedwith the purified recombinant MMP** and its specificsubstrate. This value was adjusted to concentration(ng/ml) based on the volume of the GCF collected.

The MMPs were assayed without activating the pro-enzyme MMP form for all of the samples collectedfrom the children groups. The MMP activity previouslyobtained for the chronic periodontitis group was mea-sured following incubation for 1 hour with 1 mM4-aminophenylmercuric acetate (APMA).

Statistical AnalysisBecause of the distribution of MMP values obtained,non-parametric statistics were applied to avoid theeffect that outliers might exert. Statistical differencesamong the five groups were sought using the Kruskal-Wallis test. Statistical differences between the diseasedgroup and each of the other groups were detected us-ing the Mann-Whitney test (a non-parametric versionof the unpaired t test). We chose to consider the sam-ples from the deep sites and the shallow sites withinthe children with AgP to be unpaired because GCFwas collected from sites that were clearly clinically dif-ferent, even though they were from the same subjects.Based on the data obtained, we treated each site asbeing independent, rather than considering the sub-ject to be independent. A P value £0.05 was consid-ered statistically significant. All statistical analyseswere performed using a software program.††

RESULTS

A summary of the subjects included in the five groupsis given in Table 1. The deep sites in the AgP grouphad PDs from 4 to 11 mm. PD did not correlatestrongly with age (R2 = 0.318). Children as youngas 7 years and as old as 18 years presented with4- and 5-mm pockets, whereas some of the deepestpockets (PD >8 mm) were found in children aged12 to 14 years.

A graphic summary of the MMP levels (mean – SD)is presented in Figure 1. Comparisons were only madewithin each MMP. As is apparent in Figure 1, the MMPlevels associated with the deep sites from the AgPgroup are consistently elevated relative to the otherfour groups. For statistical purposes, the deep (PD‡4 mm) and shallow (PD £2 mm) sites within the

‡ Periopaper, ProFlow, Amityville, NY.§ Periotron, ProFlow.i Enzolyte 520, AnaSpec, San Jose, CA.¶ Fisher Scientific, Ocala, FL.# Synergy HT, BioTek, Winooski, VT.** AnaSpec.†† StatView, SAS Institute, Cary, NC.

J Periodontol • May 2008 Alfant, Shaddox, Tobler, Magnusson, Aukhil, Walker

821

children with AgP were considered two separategroups relative to MMP activity (Table 2). Statisticaldifferences (P <0.025) were found among the fivegroups for all MMPs tested, with the exception ofMMP-13. Highly significant differences (P <0.0001)in MMP-8 were detected between the deep sites fromthe children with AgP relative to all of the other groups,with the exception of the shallow sites in these samechildren (P = 0.02) Statistically significant differencesbetween the AgP deep sites and the healthy, unrelatedchildren were detected for each of the MMPs. With theexception of MMP-1, significant differences were alsodetected between the AgP deep sites and the sitesfrom the adults with CP.

Although no significant differences were detectedbetween the shallow sites in the children with AgP rel-ative to their siblings (P >0.35), MMP levels associ-ated with the shallow sites and the siblings werehigher than those obtained from the healthy, unre-lated children, and statistically significant differences(P £0.05) were found for MMP-3, -9, and -12. A trendtoward significance was detected for MMP-1 (P =0.054) and possibly for MMP-2 (P = 0.076), but notfor MMP-8 (P >0.10) or MMP-13 (P >0.61).

Although the numbers of siblings and the childrenwith AgP (nine and 23, respectively) are not balanced,additional statistical testing (using multivariate anal-ysis of variance to test differences among the threegroups and multivariate paired t test for differencesbetween two groups of log-transformed data) yieldedsimilar P values for statistical differences between theMMPs for the deep sites associated with the AgP grouprelative to the shallow sites in this AgP group as well asthe siblings.

The sites sampled in the AgP group, including thedeep and shallow sites, were examined to determinewhether differences were apparent in the MMP levelsbased on PD. The pockets were separated into deep(PD 7 to 11 mm), moderate (PD 4 to 6 mm), and shal-low (PD £2 mm) (Fig. 2). The deepest pockets (PD7 to 11 mm) had slightly higher mean levels of theMMPs, with the exception of MMP-8. This was slightlyelevated in the moderate pockets (PD 4 to 6 mm) rel-

ative to the deeper pockets. The meanMMP levels associated with the shallowpockets (PD <2 mm) were consistentlythe lowest. Statistically significant dif-ferences in the MMP levels associatedwith these three PD groups were de-tected for MMP-3 (P = 0.0458), MMP-8(P = 0.0143), and MMP-12 (P =0.0015). No differences were detectedbetween the deep and moderate sites.

A coefficient correlation matrix wasrun to determine whether any positiverelationships existed between PD andMMP levels or between individual MMPlevels for the AgP sites with PD 4 to 11mm (Table 3). No significant correla-tions were detected between PD andany particular MMP level. However,strong positive correlations were foundbetween the levels associated with cer-tain MMPs. The collagenases (MMP-1,-8, and -13) correlated strongly (R2 >0.90) with each other, as did the gelatin-ases (MMP-2 and -9; R2 = 0.928).MMP-3 (stromelysin) yielded a positivecorrelation (R2 >0.80) with each of thetwo gelatinases.

Table 1.

Characteristics of the Five Subject Groups

Group

PD (mm;

mean – SD)

Age (years;

mean – SD)

Subjects

(n)

Sites

(n)

AgP deep sites 6.2 – 1.7 14.1 – 4.0 23 46

AgP shallow sites 2 14.1 – 4.0 23 23

Siblings of AgP 2 12.6 – 5.2 9 18

Healthy controls 2 13.6 – 4.43 12 23

Chronic periodontitis 5.2 – 0.6 Not available 17 34

Figure 1.MMP levels (mean – SD) for each of the five subject groups: AgP deep sites (purple),AgP shallow sites (white), siblings (green), healthy controls (light blue), and adults withchronic periodontitis (blue).

MMP Levels in Children With Aggressive Periodontitis Volume 79 • Number 5

822

DISCUSSION

This article presents two interesting findings. One isthe presence of AgP in a cohort of children as youngas 7 years of age. The second is the relatively highlevels of MMPs present in the GCF from the diseasedsites of these children with AgP relative to non-dis-eased sites within the same children, their siblings,healthy unrelated children, and subjects with CP.AgP in children, particularly young children, is ex-tremely rare, and there is very limited information re-garding the disease process. AgP in older children,e.g., LAgP, has been investigated more thoroughly.However, even with the latter, there is little information

concerning MMP levels. Almost all of thedata on MMP levels in periodontal dis-eases have been derived from adultswith CP. Tissue extracts and culturedtissue explants of inflamed gingiva con-tained more collagenase activity thanextracts from healthy human gin-giva.19-21 Collagenase activity in theGCF was reported to be increased andto show correlation with the severityof periodontal disease.22 In particular,MMP-8 has been implicated with the de-struction associated with periodontitis.This proteolytic enzyme was reported tobe elevated, relative to healthy tissues,in chronic periodontitis,23-26 diseasedperi-implant sulcular fluid,27 and LAgP.28

The latter study, by Tervahartiala et al.,28

reported higher numbers of MMP-8– and-13–positive cells in the gingiva of dis-eased sites in adults and juveniles. How-ever, only seven subjects with an agerange of 18 to 22 years were examined.

In this study, the active levels of sevenMMPs (1, 2, 3, 8, 9, 12, and 13) in GCF

were determined in a cohort of 44 African Americanchildren with and without AgP. To our knowledge, thisis the first study to examine a wide range of MMPs inthe GCF of such children. The principal finding of thisstudy was that active MMPs in the GCF of diseasedsites of this group were significantly higher than inhealthy subjects within the same population. In addi-tion, these sites contained significantly higher MMPlevels, with the exception of MMP-1, relative to adultCP diseased sites, despite the fact that the adult MMPswere activated with APMA before the assay. We read-ily acknowledge that our data would be cleaner if theMMP samples collected from the CP group had not

Figure 2.MMP levels (mean – SD) in deep (PD 7 to 11 mm; purple), moderate (PD 4 to 6 mm; lightblue), and shallow (PD £2 mm; blue) pockets in the AgP group.

Table 2.

Statistically Significant Differences in MMP Levels Among the Five Groups(Kruskal-Wallis rank test) and Differences Between the AgP Sites Relative to Eachof the Other Groups (Mann-Whitney test)

MMP-1 MMP-2 MMP-3 MMP-8 MMP-9 MMP-12 MMP-13

Among groups 0.0027 0.0210 0.0210 <0.0001 0.0016 <0.0001 0.0621

Deep versus shallow 0.0870 0.2585 0.0298 0.0020 0.1395 0.0014 0.1180

Deep versus sibs 0.0115 0.2367 0.0534 <0.0001 0.1157 0.0056 0.2031

Deep versus controls 0.0005 0.0043 <0.0001 <0.0001 0.0003 <0.0001 0.0280

Deep versus adults 0.0889 0.0143 <0.0001 <0.0001 0.0056 <0.0001 0.0132

Deep = sites with PD ‡4 mm in children with AgP; shallow = sites with PD £2 mm in children with AgP; sibs = siblings of children with AgP, with no evidence ofdisease; controls = healthy unrelated children; adults = sites with PD ‡4 mm from adults with CP.

J Periodontol • May 2008 Alfant, Shaddox, Tobler, Magnusson, Aukhil, Walker

823

been activated. However, a previous study29 using afluorigenic MMP assay noted a three-fold increase influorescent substrate activity following APMA activa-tion. Thus, MMP levels in our CP group were likely el-evated as a result of activation with APMA, but theywere still significantly lower than those associatedwith the diseased sites in the AgP group. Without ac-tivation, the MMP samples from the CP group would beexpected to be even lower and would show an evengreater difference. Although this study, as well as sev-eral other studies, suggested that MMP activity may beassociated with the severity of periodontitis, it is notclear whether the elevation of these enzymes exacer-bates the disease process or is elevated as a result ofthe disease.

Although the principal findings of this study aremostly congruent with previous studies,8,15-17,20-22

the most significant results may be the similarities ofMMP activity between the shallow sites of subjects withAgP and their siblings; no statistically significant dif-ferences were found for any of the MMPs betweenthese sites. In addition, the levels of MMP-3, -9, and-12 in the shallow sites in the AgP and sibling groupsweresignificantly highercompared to thehealthy con-trols, including a strong trend toward significance forMMP-1. MMP-3, -9, and -12 have been suspected ofhaving roles in bone resorption. MMP-3 can be pro-duced by osteoblasts, and its synthesis was shownto be upregulated in estrogen-deficient mouse osteo-blasts, leading to the implication that itmayhavea rolein the early stages during the pathogenesis of osteopo-rosis.30 Additionally, MMP-3 production by osteo-blasts and osteocytes of human neonatal rib hasbeen proposed to facilitate cellular migration and ex-pansion of bone.31 MMP-3 activation of proMMP-1,also produced by osteoblasts, and their coordinatedupregulation upon osteoblast stimulation during bone

formation were shown to be key in the processing ofcollagen on bone surfaces, allowing osteoclast re-cruitment leading to bone resorptionduringbone mor-phogenesis.32 MMP-9 and -12 were shown to beproduced by osteoclasts and are believed to playkey roles in osteoclastic bone resorption by facilitatingmigration of osteoclastic cells toward bone surfacesthrough matrices.33 Osteoclasts express MMP-9 atvery high levels, and its inhibition was shown to havean inhibitory effect on osteoclastic bone resorption.34

Osteoclast-derived MMP-12 is believed to play a rolein osteoclast attachment, spreading, and resorption;however, gene knockout experiments33 demon-strated that its activity is not critical in osteoclasticbone resorption. However, MMP-12 is primarily pro-duced by macrophages, and a study35 examining itsin vivo substrates suggested strong roles in osteoclastrecruitment. In vivo, MMP-12 was shown to be impor-tant in activating other MMPs, such as MMP-2 and -3,by which MMP-12 exaggerates the cascade of proteo-lytic processes.35

Our study represents a contribution aimed at bettercharacterization of the hyperinflammatory responseassociated with localized sites in children with AgP.GCF is an inflammatory exudate that drains fromthe infected pocket and is believed to provide insightinto the disease process currently operating in adja-cent tissue.36 It should be emphasized that our assaymeasured the active forms of MMPs. MMPs can exist inlatent or active forms, but the active form is moreclosely related to progressive periodontal disease,whereas the latent form is more strongly associatedwith gingival inflammation alone.22 In addition, wechose to evaluate the MMP levels in GCF because thisfluid provides a non-invasive means to investigate dis-ease activity. However, because of the episodic natureof disease activity associated with CP, it is very likely

Table 3.

Correlation Between PD and MMP Levels

PD MMP-1 MMP-2 MMP-3 MMP-8 MMP-9 MMP-12 MMP-13

PD 1.0000

MMP-1 -0.0391 1.0000

MMP-2 -0.0382 0.7410 1.0000

MMP-3 -0.0084 0.6325 0.8085 1.0000

MMP-8 -0.1046 0.9295 0.6646 0.5590 1.0000

MMP-9 0.0433 0.7039 0.9277 0.8517 0.6106 1.0000

MMP-12 -0.0121 0.5264 0.6334 0.7509 0.4750 0.7590 1.0000

MMP-13 -0.0628 0.9504 0.7959 0.5905 0.9188 0.6893 0.4636 1.0000

MMP Levels in Children With Aggressive Periodontitis Volume 79 • Number 5

824

that many of the CP sites sampled were in remission.This would be in clear contrast to our AgP group,which reflects a more consistently active diseasestate, and may skew differences between the groups.In addition, we used a fluorimetric assay to determinea wide range of MMPs. Thus, substrate differencesmake it difficult, if not impossible, to compare our ab-solute values with previous studies or even betweenMMP types within this study. However, our results con-sistently demonstrated significant differences be-tween groups for each MMP.

Although all MMPs assayed were elevated in theAgP diseased sites relative to shallow sites withinthe same subjects and to the siblings, the levels asso-ciated with these two groups tended to be elevated rel-ative to the unrelated, healthy children. The elevatedlevels of MMP activity found within shallow sites in thechildren with AgP and their siblings may provide aunique opportunity to further evaluate the potentialprognostic capability of MMPs. Although previous re-search points to high heterogeneity of host immuno-logic risk factors in patients with periodontitis,37 inaddition to the hormonal variations that likely exist,such differences would be expected to be lower be-tween siblings if disease development follows a similarpattern. In addition, environmental and behavioralcharacteristics are likely to be more similar than innon-related subjects. Previous studies7,37,38 indicatedthat parents, offspring, and siblings of children diag-nosed with AgP have a 50% chance of having hador of developing this disease. Although the clinicalusefulness of GCF-based diagnostics in adult CPhas not been fully implemented, their potential sensi-tivity in assessing the treatment responses of AgP inchildren, as well as possibly indicating sites at risk,may provide a more appropriate stage at which toassess their clinical usefulness.

AgP in children is a rare form of periodontal disease,with prevalence rates <1% reported in North Americanpopulations.38 Although the cohort reported here isdrawn from one rural area in North Florida, it is verylikely that other areas may have similar AgP cohorts,particularly African Americans in rural, dentally under-served areas. We believe that AgP in children in theserural areas is vastly underdiagnosed and deserves fur-ther study.

ACKNOWLEDGMENTS

Theauthors thankDrs. EdwardZapert and JohnBidwelland assistant Tiffany Davis of the Leon County HealthDepartment for their cooperation and assistance. With-out theirhelp, thisprojectwouldnothavebeenpossible.We also acknowledge Ms. Karla Kontax, College ofDentistry, University of Florida, who collected theMMP data from the adults with CP as her summer re-search project. This research was funded in part by a

seed grant from the University of Florida College ofDentistry. The authors report no conflicts of interestrelated to this study.

REFERENCES1. Nagase H, Visse R, Murphy G. Structure and function

of matrix metalloproteinases and TIMPs. CardiovascRes 2006;69:562-573.

2. Sternlicht MD, Werb Z. How matrix metalloproteinasesregulate cell behavior. Annu Rev Cell Dev Biol 2001;17:463-516.

3. Page-McCaw A, Ewald AJ, Werb Z. Matrix metal-loproteinases and the regulation of tissue remodeling.Nat Rev Mol Cell Biol 2007;8:221-233.

4. Uitto VJ, Overall CM, McCulloch C. Proteolytic hostcell enzymes in gingival crevice fluid. Periodontol 20002003;31:77-104.

5. Leppert D, Lindberg RL, Kappos L, Leib SL. Matrixmetalloproteinases: Multifunctional effectors of inflam-mation in multiple sclerosis and bacterial meningitis.Brain Res Rev 2001;36:249-257.

6. Parks WC, Wilson CL, Lopez-Boado YS. Matrix metal-loproteinases as modulators of inflammation and in-nate immunity. Nat Rev Immunol 2004;4:617-629.

7. Kinane D, Podmore M, Murray M, Hodge P, Ebersole J.Etiopathogenesis of periodontitis in children and ado-lescents. Periodontol 2000 2001;26:54-91.

8. Reynolds J, Hembry R, Meikle M. Connective tissuedegradation in health and periodontal disease and theroles of matrix metalloproteinases and their naturalinhibitors. Adv Dent Res 1994;8:312-319.

9. Golub LM, McNamara TF, Ryan ME, et al. Adjunctivetreatment with subantimicrobial doses of doxycycline:Effects on gingival fluid collagenase activity and at-tachment loss in adult periodontitis. J Clin Periodontol2001;28:146-156.

10. Garlet G, Martins W, Fonseca B, Ferreira B, Silva J.Matrix metalloproteinases, their physiological inhibi-tors and osteoclast factors are differentially regulatedby the cytokine profile in human periodontal disease.J Clin Periodontol 2004;31:671-679.

11. Kinane D. Regulators of tissue destruction and ho-meostasis as diagnostic aids in periodontology. Peri-odontol 2000 2000;24:215-225.

12. Hannas A, Pereira J, Granjeiro J, Tjaderhane L. Therole of matrix metalloproteinases in the oral environ-ment. Acta Odontol Scand 2007;65:1-13.

13. Golub LM, Siegel K, Ramamurthy NS, Mandel ID.Some characteristics of collagenase activity in gingi-val crevicular fluid and its relationship to gingivaldiseases in humans. J Dent Res 1976;55:1049-1057.

14. Golub L, Stakiw J, Singer D. Collagenolytic activity ofhuman gingival crevice fluid. J Dent Res 1974;53:1501.

15. Kiili M, Cox S, Chen H, et al. Collagenase-2 (MMP-8)and collagenase-3 (MMP-13) in adult periodontitis:Molecular forms and levels in gingival crevicular fluidand immunolocalisation in gingival tissue. J Clin Peri-odontol 2002;29:224-232.

16. Ilgenli T, Vardar-Sengul S, Gurkan A, et al. Gingivalcrevicular fluid matrix metalloproteinase-13 levels andmolecular forms in various types of periodontal dis-eases. Oral Dis 2006;12:573-579.

17. Hernandez M, Valenzuela M, Lopez-Otin C, et al.Matrix metalloproteinase-13 is highly expressed in

J Periodontol • May 2008 Alfant, Shaddox, Tobler, Magnusson, Aukhil, Walker

825

destructive periodontal disease activity. J Periodontol2006;77:1863-1870.

18. Armitage GC. Development of a classification systemfor periodontal diseases and conditions. Ann Peri-odontol 1999;4:1-6.

19. Korostoff J, Wang J, Sarment D, Stewart J, FeldmanR, Billings P. Analysis of in situ protease activity inchronic adult periodontitis patients: Expression of ac-tivated MMP-2 and a 40 kDa serine protease. J Peri-odontol 2000;71:353-360.

20. Sarment DP, Korostoff J, D’Angelo M, Polson AM,Feldman RS, Billings PC. In situ localization and char-acterization of active proteases in chronically inflamedand healthy human gingival tissues. J Periodontol 1999;70:1303-1312.

21. Ejeil A, Igondjo-Tchen S, Ghomrasseni S, Pellat B,Godeau G, Gogly B. Expression of matrix metallo-proteinases (MMPs) and tissue inhibitors of metallo-proteinases (TIMPs) in healthy and diseased humangingiva. J Periodontol 2003;74:188-195.

22. Sorsa T, Tjaderhane L, Salo T. Matrix metalloprotein-ases (MMPs) in oral diseases. Oral Dis 2004;10:311-318.

23. Chen H, Cox S, Eley B, Mantyla P, Ronka H, Sorsa T.Matrix metalloproteinase-8 levels and elastase activitiesin gingival crevicular fluid from chronic adult periodon-titis patients. J Clin Periodontol 2000;27:366-369.

24. Haerian A, Adonogianaki E, Mooney J, Docherty J,Kinane D. Gingival crevicular stromelysin, collagen-ase and tissue inhibitor of metalloproteinases levels inhealthy and diseased sites. J Clin Periodontol 1995;22:505-509.

25. Kinane D, Darby I, Said S, et al. Changes in gingivalcrevicular fluid matrix metalloproteinase-8 levels dur-ing periodontal treatment and maintenance. J Peri-odontal Res 2003;38:400-404.

26. Kumar M, Vamsi G, Sripriya R, Sehgal P. Expression ofmatrix metalloproteinases (MMP-8 and -9) in chronicperiodontitis patients with and without diabetes melli-tus. J Periodontol 2006;77:1803-1808.

27. Kivela-Rajamaki M, Maisi P, Srinivas R, et al. Levels andmolecular forms of MMP-7 (matrilysin-1) and MMP-8(collagenase-2) in diseased human peri-implant sulcularfluid. J Periodontal Res 2003;38:583-590.

28. Tervahartiala T, Pirila E, Ceponis A, et al. The in vivoexpression of the collagenolytic matrix metalloprotein-ases (MMP-2, -8, -13, and -14) and matrilysin (MMP-

7) in adult and localized juvenile periodontitis. J DentRes 2000;79:1969-1977.

29. Bhide V, Smith L, Overall C, Birek P, McCulloch C. Useof a fluorogenic septapeptide matrix metalloproteinaseassay to assess responses to periodontal treatment.J Periodontol 2000;71:690-700.

30. Breckon JJ, Papaioannou S, Kon LW, et al. Stromelysin(MMP-3) synthesis is up-regulated in estrogen-deficientmouse osteoblasts in vivo and in vitro. J Bone MinerRes 1999;14:1880-1890.

31. Zeitler P, Pahnke J, Marx A. Expression of stromelysin-1(MMP-3), gelatinase B (MMP-9), and plasminogen ac-tivator system during fetal calvarial development.Histopathology 2004;44:360-366.

32. Sasaki K, Takagi M, Konttinen YT, et al. Upregulationof matrix metalloproteinase (MMP)-1 and its activatorMMP-3 of human osteoblast by uniaxial cyclic stimu-lation. J Biomed Mater Res B Appl Biomater 2007;80:491-498.

33. Hou P, Troen T, Ovejero MC, et al. Matrix metallo-proteinase-12 (MMP-12) in osteoclasts: New lesson onthe involvement of MMPs in bone resorption. Bone 2004;34:37-47.

34. Ishibashi O, Niwa S, Kadoyama K, Inui T. MMP-9antisense oligodeoxynucleotide exerts an inhibitoryeffect on osteoclastic bone resorption by suppressingcell migration. Life Sci 2006;79:1657-1660.

35. Chen YE. MMP-12, an old enzyme plays a new role inthe pathogenesis of rheumatoid arthritis? Am J Pathol2004;165:1069-1070.

36. Lamster IB, Ahlo JK. Analysis of gingival crevicularfluid as applied to the diagnosis of oral and systemicdiseases. Ann N Y Acad Sci 2007;1098:216-229.

37. Takahashi K, Ohyama H, Kitanaka M, et al. Hetero-geneity of host immunological risk factors in patientswith aggressive periodontitis. J Periodontol 2001;72:425-437.

38. Albandar JM, Tinoco EM. Global epidemiology ofperiodontal diseases in children and young persons.Periodontol 2000 2002;29:153-176.

Correspondence: Dr. Clay Walker, Box 100424, Universityof Florida, Gainesville, FL 32610-0424. Fax: 352/392-2361; e-mail: walkerc1@ufl.edu.

Submitted September 20, 2007; accepted for publicationNovember 26, 2007.

MMP Levels in Children With Aggressive Periodontitis Volume 79 • Number 5

826