Prostaglandin E (PGE) and interleukin-l[3 (IL-lfi) levels in gingival crevicular fluid during human orthodontic tooth movement

William G. Grieve III, DDS, MS," Georgia K. Johnson, DDS, MS, ~ Robert N. Moore, DDS, PhD, EdD, c Richard A. Reinhardt, DDS, PhD, ~ and Linda M. DuBois, DDS, PhD ~ Eugene, Ore., Iowa City, Iowa, and Lincoh~, Neb. The purpose of this study was to examine gingival crevicular fluid (GCF) levels of two potent bone resorbing mediators, prostaglandin E (PGE) and interleukin-113 (IL-113), during human orthodontic tooth movement. The study included 10 patients, each having one treatment tooth undergoing orthodontic movement and a contralateral control tooth. The GCF was sampled at control sites and treatment (compression) sites before activation and at 1, 24, 48, and 168 hours. Prevention of plaque-induced inflammation allowed this study to focus on the dynamics of mechanically stimulated PGE and IL-113 GCF levels. The PGE and IL-113 levels were determined with radioimmunoassay. At 1 and 24 hours, mean GCF IL-113 levels were significantly elevated at treatment teeth (8.9 _+ 2.0 and 19.2 ___6.0 pg, respectively) compared with control teeth (2.0 _+ 1.1 pg, p = 0.0049, and 2.9 +_ 1.0 pg, p = 0.0209, respectively). The GCF levels of PGE for the treatment teeth were significantly higher at 24 and 48 hours (108.9 4- 11.9 and 97.9 4- 7.3 pg) than the control teeth (61.8 -- 7.2 pg, p = 0.0071, and 70.8 _ 7.4 pg, p = 0.0021, respectively). The GCF levels of PGE and IL-113 remained at baseline levels throughout the study for the control teeth, whereas significant elevations from baseline in GCF IL-113 (24 hours) and PGE levels (24 and 48 hours) were observed over time in the treatment teeth (p -< 0.05). These results demonstrate that bone-resorbing PGE and IL-113 produced within the periodontium are detectable in GCF during the early phases of tooth movement and return to baseline within 7 days. (AMJ ORTHOD DENTOFACORTHOP 1994;105:369-74.)

S u c c e s s f u l orthodontic tooth movement requires the remodeling of the periodontium, particularly the alveolar bone. When a small orthodontic force is applied for a prolonged period, an inflammatory event occurs within the periodontium, resulting in bone resorption that accommodates movement of the tooth. The mechanism of bone resorption may be related to release of inflammatory mediators, such as prostaglandin E2 (PGFa) and interleukin-I (IL-1), which interact with bone cells. Prostaglandin E (PGE) has long been recognized as a potent stimulator of bone resorption, 2 and its production is modulated in part by IL-1) Interleukin-I exists in two forms, alpha and beta, 3 of which interleukin-1 (IL-113) is most involved in bone
"In private practice, Eugene, Ore. bAssociate Professor, Department of Periodontics and Dows Institute for Dental Research, University of Iowa, Iowa City, Iowa. "Professor and Chairperson, Department of Orthodontics, UNMC College of Dentistry, Lincoln, Neb. eProfessor and Vice-Chairperson, Department of Surgical Specialties, UNMC College of Dentistry, Lincoln, Neb. CAssociate Professor, Department of Adult Restorative Dentistry, UNMC College of Dentistry, Lincoln, Neb. Copyright 9 1994 by the American Association of Orthodontists. 0889-5406/94/$3.00 + 0.I0 811139963

metabolism. Interleukin-113 stimulates bone resorption and concomitantly inhibits bone formation: The bulk of the research related to the rote of PGE and IL-113 in orthodontic teeth movement has been done in vitro and in animal models. Both human fibroblasts and bone cells have been shown to respond to mechanical stress with increased production of P G E ) '6 Furthermore, Saito and coworkers7 have demonstrated that the addition of IL-113 to mechanically stressed peri. odontal ligament fibroblasts increased PGE production in a synergistic fashion. With a cat model, researchers have localized PGE ~'8 and IL-1137 in the periodontium of teeth undergoing movement. Taken together, these studies report that production of PGE and IL-113 is increased during mechanical stress in in vitro and animal models during tooth movement. Little information is available concerning the production of these mediators during orthodontic tooth movement in human subjects. As a result of the application of mechanical forces, the cells in the periodontal ligament may produce sufficient amounts of PGE and IL-113 to diffuse into the gingival crevicular fluid (GCF). Therefore the concentration of these substances in the GCF may increase during tooth movement and
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Fig. 1. Force of labial activation in unilateral custom formed orthodontic wire was measured with calibrated force gauge.

provide a noninvasive model for the study of the dynamics of mediator production. The purpose of this study was to test the hypothesis that levels of PGE and IL-113 in the G C F increase during orthodontic tooth movement. Careful control of bacterially induced soft tissue inflammation allowed focus on mechanically stimulated PGE and IL-I production and ultimate expression in the GCF.

MATERIAL AND METHODS Experimental subjects

Ten adult orthodontic patients (5 females, mean age

27.8 --- 3.9 years and 5 males, mean age 24.6 --- 1.5 years) were selected to participate in this study. These patients met the following criteria: (1) good general health; (2) lack of antibiotic therapy within the past 6 months; (3) no use of antiinflammatory drugs in the month preceding the study; (4) periodontally healthy, with generalized probing depths --<3 mm and no radiographic evidence of periodontal bone loss; and (5) requirement of buccal/labial tooth movement as part of their orthodontic treatment plan

The patients were not allowed to take any drugs during the study that potentially could affect the production or the activity of PGE and IL-II3, such as nonsteroidal antiinflammatory medications. Since some discomfort is a normal part of orthodontic tooth movement, codeine was prescribed as needed in 15 mg tablets. To insure optimal control of bacterial plaque, patients received oral hygiene instructions at baseline, and each patient rinsed with 0.5 ounces 0.12% chlorhexidine (Peridex, Procter & Gamble, Cincinnati, Ohio) twice daily. At the mesiofacial aspect of both treatment and control teeth, GCF was collected and clinical parameters recorded immediately before activation (baseline), and at the following time periods following activation: 1 hour; 24 hours; 48 hours; and 168 hours.

GCF collection
The GCF sampling was performed in an air conditioned clinic maintained at approximately 21 ~ C with 30% relative humidity. The sites under study were isolated with cotton rolls and gently dried with an air syringe.9 Paper strips (Periopaper, Harco, Tustin, Calif.) were carefully inserted 1 mm into the gingival crevice and allowed to remain for 30 seconds ~~(Fig. 2). Fluid volumes were measured with a Periotron 6000 (Siemens Medical Systems, Inc., !selin, N.J.), which had been calibrated with human serum. Immediately after measurement in the Periotron, the periopaper strips from the individual sites were wrapped in foil, placed in sealed plastic tubes, and snap frozen in liquid nitrogen.9The samples then were stored at - 80~ C until analysis.

Experimental design
A treatment tooth undergoing a buccal/labial tipping movement and a contralateral control tooth were identified in each subject. Orthodontic brackets were placed on both the treatment and control teeth, and the treatment tooth was activated with a unilateral orthodontic wire (0.014 Nitinol, Activ-arch, Unitek/3M, Monrovia, Calif.). The orthodontic wire was custom formed for each patient with varying amounts of buccal/labial offset to produce approximately 100 gm or 0.980 N of force at baseline as measured with a calibrated Dontrix orthodontic force gauge (ETM, Monrovia, Calif.) (Fig. 1).

Clinical parameters
After GCF collection, the following parameters were assessed at the control and treatment teeth at each visit: probing depth, presence or absence of plaque, and presence or absence of bleeding on iarobing.

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Fig. 2. Within each patient, GCF was sampled at mesiofacial line angle of tooth undergoing labial
movement, as well as at contralateral control tooth.

PGE and IL-113 determination

The crcvicular fluid was eluted from the paper strips by centrifugal filtration with 50 ILl aliquots of radioimmunoassay (RIA) buffer as described by Offenbacher and coworders. 9 To quantify the levels of PGE and IL-113 in the GCF, duplicate samples from each site were evaluated with radioimmunoassay (RIA) (Advanced Magnetics, Cambridge, Mass.). The principle of RIA is based on competition of the PGE (or IL- 113) in the sample with radioactively labeled PGE or (IL-113) for a limited number of sites on antibodies to PGE (or IL-I13). These assays used iodinated human PGE (or IL113) and rabbit antisera raised against PGE2 (or IL-113). Antibody bound PGE or IL-113 was separated from unbound PGE or IL-113 with magnetic goat antirabbit immunoglobulin G (lgG) through centrifugation. The antibody-bound labeled PGE or IL-113 was quantified in a gamma counter, and PGE or IL-113 concentrations were determined by interpolation from a standard curve. The total PGE and IL-I13 in each 30 second GCF sample was determined by calculating for the dilution of the sample used in the assays. According to the manufacturer, the PGE2 antisera had 50% cross reactivity with PGE~, and the IL-113 antisera had no cross reactivity with other human lymphokines. The sensitivity of these assays ranged from 4.1 to 1000 pg/0.1 ml for PGE, and 5 to 1000 pg/0.1 ml for IL-113.

perimposed on one another. The amount of tooth movement for each tooth was measured with a Boley gauge.

Statistical analysis
The mean and standard error of measurement for PGE and IL-113 values were calculated for control and treatment sites at each sampling time. Mediator values were analyzed with the repeated measures two-way analysis of variance with sampling time and treatment as the independent variables. When significant interactions were found, paired t tests were performed as post hoc procedures to compare treatment and control teeth mean values at different times. The PGE and IL- 113mean values were analyzed over time with separate repeated measures one-way analyses of variance. Significant differences were identified with Tukey's procedure. '2 The number of control and treatment sites positive for plaque and bleeding on probing for the 10 subjects were compared at baseline and each time point with the Cochran test." Probing depth values were compared at baseline for the control and treatment teeth, and probing depth was analyzed across all the sampling times with separate repeated measures one-way analyses of variance and the Tukey's post hoc procedure. :2

RESULTS
All 10 subjects completed the study, and the treatment teeth consisted of seven maxillary lateral incisors and three maxillary first premolars. Contralateral teeth provided control sites.

Verification of tooth movement

Orthodontic study models were made at baseline and 7 days. To evaluate changes in tooth position for both treatment and control teeth, cephalometric radiographs were taken of these models as described by Van Horn and coworkers." Briefly each model, with its occlusal surface against an 8 x 10-inch radiographic cassette, was secured with tape. The radiographs were exposed in a cephalometer, and tracings of the pretreatment and posttreatment radiographs were su-

Clinical parameters
Plaque accumulation was minimal, with -<6% of the sites harboring plaque throughout the entire study. Gingival health was excellent with a lack of gingival

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American Journal of Orthodontics and DentofaciaI Orthopedics April 1994

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bleeding or probing depths > 3 mm at any time point in the study. Treatment and control values did not vary significantly at baseline or during the study.
G C F P G E and IL-11~ levels

movement of the treatment teeth was 1.0 +__ 0.3 mm, whereas no movement was detected for the control teeth.
DISCUSSION

After orthodontic activation, significant differences were demonstrated between control and treatment teeth for both PGE and IL-I[3 values. The mean PGE values of the treatment teeth were significantly higher than the control teeth at 24 hours (108.9 11.9 pg versus 61.8 7.2 pg, p = 0.0071) and 48 hours (97.9 7.3 pg versus 70.8 -4- 7.4 pg, p = 0.0021) (Fig. 3). For IL-113, significant differences between treatment and control teeth were noted at 1 (8.9 - 2.0 pg versus 2.0 _+_ 1.I pg, p = 0.0049) and 24 hours (19.2 +_ 6.0 pg versus 2.9 1.0 pg, p = 0.0209) (Fig. 4). Over time the mean value for the PGE at treatment sites was significantly higher at 24 hours (108.9 +__ 11.9 pg) than at baseline (64.8 9.2 pg), 1 (71.3 - 13.2 pg) and 168 hours (63.9 8.8 pg) (p --< 0.05). The mean value for the total PGE at the treatment sites was significantly elevated at 48 hours (97.9 +__ 7.3 pg) compared with baseline (.64.8 9.2 pg) and 168 hours (63.9 8.8 pg) (p--< 0.05), (Fig. 3). Within the treatment sites, the mean total IL-113 value was significantly higher at 24 hours (19.2 6.0 pg) than at baseline (2.5 --+ 1.4 pg), 48 (2.2 1.6pg) and 168 hours (2.1 1.2 pg) (p --< 0.05), (Fig. 4). At control sites, PGE and IL-113 levels did not change significantly over time.
Model analysis

Evaluation of the radiographic tracings made of orthodontic study models demonstrated that the average

The finding of increased levels of GCF PGE and IL-II3 adjacent to teeth undergoing orthodontic tooth movement indicates that the cells within the periodontium are producing increased PGE and IL-113 in response to an orthodontic force and that these mediators can be detected noninvasively in the GCF. Careful control of bacterially induced inflammation allowed the current research to focus on mediators associated with mechanically induced inflammation within the bone and periodontal ligament. Previous reports have demonstrated reduced or absent quantities of PGE and IL-113 in GCF at noninflamed s i t e s . 9"t4J5 Although IL-113 and PGE levels were consistently low in healthy control sites in the current study, the sensitive RIA did detect measurable levels of IL-113 and PGE in 44% and 98% of sites, respectively. However, control of bacterially induced inflammation clearly separated mechanically activated from nonactivated sites on the basis of GCF IL-113 and PGE levels. On activation, GCF IL-113 levels increased rapidly in 90% of the treatment sites, becoming significantly elevated at 1 and 24 hours. The PGE production p e a k e d later (24 and 48 hours) than IL-113, suggesting a stimulatory effect of IL-113 on PGE. This supports previous in vitro studies that have demonstrated IL-113 enhanced PGE production in mechanically stressed periodontal ligament fibroblasts at 24 hours. 7 The decrease in IL113 at 48 hours noted in the present study could be indicative of feedback inhibition by increased levels of PGE as described by Kunkel and coworkers. '6

American Journal of Orthodontics and Dentofacial Orthopedics

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Potential sources of PGE and IL-113 during tooth movement include cells associated with both the periodontal ligament andalveolar bone, such as fibroblasts, macrophages, cementoblasts, cementoclasts, osteoblasts, and osteoclasts. With a cat model, Lynch and associates" reported that in the early stages of tooth movement (at 12 and 24 hours), many periodontal ligament cell types stained positively for IL-113. Photometric quantification of the intensity of staining for both PGE and IL-113 has shown a significant increase in intensity at 24 hours after activation. 7 These findings are consistent with the increased amounts of IL-113 and PGE at 1 to 24 hours reported in the present study. Lynch and coworkers ~7also reported that at 14 days when both bone apposition and resorption are ongoing, only osteoclasts and adjacent mononucleated cells in resorption sites stained distinctly for IL-II3. The decreased number of IL-113 positive cells and their association with bone would account for the return of GCF IL-113 levels to baseline levels in the present study. Perphaps at this stage of tooth movement it is not possible to detect more subtle alterations in mediator levels that are occurring primarily in the bone. Because the appliance design did not provide for continuous forces after initial tooth movement, the lack of active forces at 168 hours may also explain why mediators were not elevated at this point in time. Another limitation of GCF based studies is the inherent variability of mediator quantities and GCF volume. According to Lamster and others, I~ measurement of the total quantity of mediator collected for a standardized time will allow for more sensitive detection of site-to-site and patient-to-patient GCF differences

without significant contamination by serum. Therefore the present study has reported total values, although results were similar whether total (pg) or concentration (pg/I.tl) GCF values (data not shown) of the mediators were compared. The clinical significance of research regarding the mechanism of bone metabolism in orthodontic tooth movement is related to its potential pharmacologic modulation. This includes the effects of adjunctive PGE or IL-113, as well as prostaglandin-inhibiting drugs, on the rate of tooth movement. In both monkeys and human beings, local administration of PGE almost doubled the rate of tooth movement as compared with sham treated controls, with an absence of macroscopic, radiographic or local side effects. '8";9 Although the potential for local or systemic deletorious effects would seem to exist with local administration of PGE or IL-I, current research has not confirmed this. 2~ On the other hand, there is concern regarding the effect of antiinflammatory drugs on the rate of tooth movement. These drugs, commonly used to control the discomfort associated with tooth movement, inhibit cyclooxygenase activity and therefore the synthesis of prostaglandins. 22 This in turn may affect the underlying mechanism of tooth movement (inflammation) resulting in an inhibition of osseous remodeling. Animals treated with prostaglandin inhibitors have been shown to experience decreased osteoclastic activity 23 and a significant decrease in the rate of tooth movement. 2~ However, these studies used higher than therapeutic doses of antiinflammatory drugs for periods of several days. In clinical practice, these drugs are used at lower doses for only 1 to 2 days after activation.

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T h e present study has demonstrated that P G E and IL-l[3 p r o d u c e d during orthodontic tooth m o v e m e n t in h u m a n beings can be m e a s u r e d with a n o n i n v a s i v e technique, analysis o f G C F . Increases in IL-13 levels at I and 24 hours p r e c e d e d p e a k P G E levels w h i c h occurred at 24 and 48 hours after-activation. Control o f bacterially induced i n f l a m m a t i o n a l l o w e d focus on the dynamics o f m e d i a t o r production resulting f r o m m e c h a n ically induced i n f l a m m a t i o n in the periodontal ligament and bone. This m o d e l could also be used to study the d y n a m i c s o f other cytokines implicated in b o n e resorption, sucfl as t u m o r necrosis factor and interleukin-6. Future research should e x a m i n e the effect o f therapeutic r e g i m e n s o f nonsteroidal antiinflammatory drugs on prostaglandin p r o d u c t i o n and tooth m o v e m e n t in h u m a n subjects. As w e learn m o r e about alterations in the levels o f these m e d i a t o r s , a m o r e e f f e c t i v e and prudent p h a r m a c o l o g i c r e g i m e n during orthodontic treatment m a y be i m p l e m e n t e d . REFERENCES 1. Storey E. The nature of tooth movement. AM J ORTttOD 1973;63:292-314. 2. Klein DC, Raisz LG. Prostaglandins: stimulation of bone resorption in tissue culture. Endocrinology 1970;86:1436-40. 3. Dinarello CA. Biology of interleukin 1. FASEB J 1988;2:10815. 4. Stashenko P, Obernesser MS, Dewhirst FE. Effect of immune cytokines on bone. Immunological Investigations 1989;18:23949. 5. Ngan PW, Crock B, Varghese J, Lanese R, Shanfeld J, Davidovitch Z. lmmunohistochemical assessment of the effect of chemical and mechanical stimuli on cAMP and prostaglandin E revels in human gingival flbroblasts in vitro. Arch Oral Biol 1988;33:163-74. 6. Yeh C, Rodan GA. Tensile forces enhances prostaglandin E synthesis in osteoblastic cells grown on collagen ribbon. Calcif Tiss Int 1984;36:$67-$71. 7. Saito M, Saito S, Ngan PW, Shanfeld J, Davidovitch Z. Interleukin-I beta and prostaglandin E are involved in the response of periodontal cells to mechanical stress in vivo and in vitro. At4 J ORTIIODDENTOFAC ORTti'OP 1991;99:226-40. 8. Davidovitch Z, Shanfeld J. PGE levels in alveolar bone of orthodontically treated cats. J Dent Res 1980;59:Abstr#362. 9. Offenbacher S, Farr DH, Goodson JM. Measurement of prostaglandin E in crevicular fluid. J Clin Periodont 1981;8:359-67. 10. Lamster IB, Hartley LJ, Vogel RI. Development o.f a biochemical profile for gingival crcvicular fluid. Methodological considera-

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