INTRODUCTION

Pressure in the periodontal ligament (PDL) induced by orthodontic forces results in vascular changes that lead to cellular activation and release of proinflammatory molecules such as prostaglandins, cytokines, and proteinases.1 These are known to initiate or regulate the biologic processes related to alveolar bone remodeling. Although orthodontic force itself does not involve any particular pathogens, orthodontic loading and bacteria-induced tissue destruction share common inflammatory mediators that may cause tissue destruction.23

Interleukin (IL)-1 is a key mediator in a variety of activities associated with immune and inflammatory responses.4 IL-1 is triggered by various stimuli, including neurotransmitters, bacterial products, other cytokines, and mechanical forces.5 The amount of IL-1β in human gingival fibroblasts increases during orthodontic movement.6 The periodontal ligament and the gingival fibroblasts exposed to IL-1 in vitro release prostaglandin (PG)E₂ in a dose-dependent manner and secrete matrix metalloproteinases (MMPs).78

MMPs have been implicated as major proteolytic enzymes in the degradation of a variety of collagens and other extracellular matrix (ECM) molecules. Among the various subtypes of MMPs, MMP-2 and MMP-9 (gelatinases) are expressed during inflammatory responses of tissues.9–12 Tissue inhibitors of matrix metalloproteinases (TIMPs) inhibit most MMPs by binding to their active parts.13 Especially TIMP-1 and TIMP-2 have a strong inhibitory effect on fibroblast-derived MMPs and polymorphonuclear leukocyte-derived MMPs, respectively.14 The suppression of MMP-9 by TIMP-1 has been shown. According to Cao, TIMP-1 has an inhibitory effect on proMMP-2. TIMP-3 has an inhibitory effect on proMMP-2 and proMMP-9.15 The above factors have been expected to provide important clues to understanding inflammatory tissue changes.

In addition to the periodontal ligament and alveolar bone, the soft tissue surrounding the tooth participates in remodeling of the periodontal structure during tooth movement. However, the direct effects of orthodontic loading on soft tissue have gained little attention. A recent study revealed increased tumor necrosis factor-α (TNF-α) and IL-1α in rat gingival tissues under orthodontic loading, suggesting a possible force-induced direct increase in cytokines in the soft tissue.16 Hence, a comprehensive understanding of localized remodeling would include knowledge of the role of surrounding soft tissue, as well as hard tissue.

The purpose of this study was to evaluate the effects of orthodontic loading on levels of IL-1β, MMP-9, and TIMP-1 mRNAs in gingival soft tissue through the real-time reverse transcription-polymerase chain reaction (RT-PCR).

MATERIALS AND METHODS

Animals and Appliance Installation

Fourteen 9-week-old Wistar male rats (mean weight, 280 ± 10 g) were used in this study. They were kept in stainless steel cages under a standard 12-hour light/ dark cycle and were fed a pellet diet (8811M0001, Extrusion, Superfeed Co Ltd, Gangwon, Korea) and tap water ad libitum. Three rats served as the untreated control group and another three as the sham appliance control group. In the remaining eight rats, a constant calibrated force of 20 g was applied to move the left maxillary first molar mesially with the use of a closed nickel-titanium (Ni-Ti) coil spring (Sentalloy, 0.009 × 0.036, Tomy, Tokyo, Japan). Proximal line angles on the maxillary left first molar and incisors were notched with the use of a high-speed diamond point. The springs were engaged with a stainless steel ligature wire and were light cured on the tooth surface with a self-etching primer and light-cured resin (3M Unitek, Monrovia, Calif) (Figure 1). Reactivation was not performed during the experimental period. The right maxillary first molars were not given any force and served as the contralateral control side. In the sham appliance group, the springs were attached in the same way but were not activated.

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The weight of the animals was recorded on the day of appliance insertion and at sacrifice. All procedures were performed with animals under general anesthesia with a subcutaneous injection per animal of 0.04 mL of Zoletil (tiletamine 125 mL, zolazepam 125 mL, Virbac, Carros Cedex, France) and 0.01 mL of Rompun (xylazine hydroxychloride 23.32 mg/1 mL, 2036D, Bayer AG, Leverkeusen, Germany). On day 7 and day 14 following orthodontic loading, four rats were sacrificed with the use of ether. The gingiva on the mesial (pressure) side of the maxillary left and right first molar was excised and frozen in liquid nitrogen for later use.

Real-Time RT-PCR

TRIzol reagent (Molecular Research Center Inc, Cincinnati, Ohio) was used to extract the total RNA from each sample, as described by Ribaudo et al.17 One microgram of the total RNA was reverse transcribed into cDNA with the use of Promega's Reverse Transcription System (Promega Corp, Madison, Wis). Reverse transcriptase was added to the mixture that contained RT POX buffer, 2.5 mM MgCl₂, Oligo (dT) primer, dNTP Mix, and Rnasin. The reagents were incubated at 42°C for 15 minutes and then were heated to 95°C for 5 minutes.

A real-time RT-PCR was performed with the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, Calif). Primers and probes of IL-1β, MMP-9, and TIMP-1 are shown in Table 1. PCRs were performed in a total volume of 25 μL, which contained 50 ng cDNA sample, 1X TaqMan Universal PCR Master Mix, 300 nM of each primer, and 250 nM TaqMan probe. Cycle conditions for IL-1β, MMP-9, TIMP-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 30 seconds at 95°C, 30 seconds at 60°C, then 30 seconds at 72°C. cDNAs for IL-1β, MMP-9, TIMP-1, and GAPDH then were amplified for 40 cycles. Oligonucleotide hybridization probes labeled with 6-carboxyfluorescein as reporter fluorescence and 6-carboxy-tetramethyl-rhodamine as quencher fluorescence were used in PCR. The levels of IL-1β, MMP-9, and TIMP-1 were normalized according to GAPDH mRNA levels.

Table 1.  Genes Analyzed by Quantitative TaqMan Polymerase Chain Reaction

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RESULTS

Notable inflammatory signs such as bleeding and/or swelling were not found during the experiment. Experimental rats weighed 310 ± 10 g (7th day) and 330 ± 15 g (14th day), and control rats weighed 315 ± 5 g (7th day) and 340 ± 10 g (14th day), respectively, with no significant difference observed at any time point.

In the sham activation control group, mRNA levels showed no significant difference between right (no appliance) and left (sham activation) sides (Figure 2). Hence in the experiment group, the no appliance side was directly compared with the appliance side at each time point, because bilateral placement of springs often seriously affects the viability of the animal. IL-1β mRNA on the pressure side was significantly increased compared with that on the control side on day 7 (2.57 ± 0.48 vs 0.35 ± 0.02) (P < .05), and it remained significantly high on day 14 with a moderate decrease (1.10 ± 0.19 vs 0.33 ± 0.02) (P < .05) (Figure 3). In contrast, MMP-9 mRNA on the pressure side was constantly upregulated on both day 7 and day 14 (0.31 ± 0.08 vs 0.06 ± 0.01 and 0.31 ± 0.19 vs 0.04 ± 0.01, respectively) (P < .05) (Figure 4). TIMP-1 mRNAs displayed a similar increase on both day 7 and day 14 (292.48 ± 99.12 vs 7.16 ± 0.75 and 285.38 ± 29.08 vs 9.11 ± 0.68, respectively) (P < .05) (Figure 5). The mRNA levels at the no appliance control side remained stable during the experimental period, showing no significant changes.

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DISCUSSION

A primary concern of orthodontists has been the cellular or subcellular changes that occur in the periodontal ligament and alveolar bone. Various inflammatory cytokines and proteases therefore have been detected, mainly in the periodontal ligament or in gingival crevicular fluid.18–20 However, it should be noted that remarkable changes in gingival soft tissue contour should always follow tooth movement. Furthermore, undesired pathologic changes in the soft tissue may interfere with orthodontic treatment. This implies that gingival soft tissues are biologically active during orthodontic treatment.

Gingival inflammation is associated with increased inflammatory cytokines and/or MMPs in the PDL,21 and mutual activation of inflammatory tissue destruction by gingival and periodontal ligament fibroblasts has been suggested.22 In spite of these regional and functional connections, the role of the gingival tissue in orthodontic treatment has been neglected. To focus on biologic events in the gingival tissue, isolation of gingival tissue under orthodontic loading and observation of primary subcellular changes (ie, specific gene transcription) were prerequisites for the present study. A real-time RT-PCR was recruited in this study because it enables a relatively reliable RNA quantification regardless of the number of PCR cycles by quickly displaying real-time results, unlike conventional RT-PCR.2324

In this study, the mRNA levels of IL-1β, MMP-9, and TIMP-1 were significantly elevated at all pressure sides for a relatively long term (2 weeks), and no remarkable change was noted in the sham activation group, indicating that changes were largely due to pressure stimulation of the gingival tissue. Although many studies have shown an increase in proinflammatory cytokines in the PDL,16192526 the direct effect of mechanical loading on the IL-1β from gingival fibroblasts is not yet clear. Skutek et al27 demonstrated a stretch-induced increase in secretion of IL-6 in the tendon fibroblasts. Yang et al28 reported that the inflammatory responses of tendon fibroblasts were dependent on the magnitude of stretching, with anti-inflammatory response at a low level and proinflammatory responses at a high level of force. Changes in IL-1β mRNA levels in this study are somewhat consistent with those described in a previous report on IL-1β secretion from gingival crevicular fluid.19 However, a relatively constant elevation is notable.

The decrease in IL-1β on day 14 may be attributed to a decrease in force level caused by lack of reactivation, but Lee et al25 have shown that cytokines may be refractory to appliance reactivation. Another possible cause is the inherent periodicity of IL-1β. Iwasaki et al29 reported that IL-1β concentration in the gingival crevicular fluid was elevated and returned to baseline in 14 to 28 days. However, a sustained elevation of IL-1β mRNA in pressure side gingival tissue compared with the control side was a novel finding in our study, implying a possible role of gingival soft tissue in tissue remodeling.

In our study, MMP-9 also was increased by orthodontic loading. MMP-2 and MMP-9 were previously detected on both pressure and tension side PDL during orthodontic movement to aid the degradation of collagen.20 Takahashi et al20 reported that MMP-9 mRNA in PDL increased within 7 days after orthodontic loading, then decreased at day 14 through in situ hybridization. In contrast, primary MMP-9 production in gingival tissue is an interesting finding, whereas MMP-3 has been localized in the gingiva by Beklen et al.22 The sustained level of MMP-9 at day 14 may be associated with the stimulatory or synergistic effects of mechanical loading and of ILs on the secretion of MMP-9, which has been shown previously in cardiac fibroblasts,30 in rabbit tendon cells,31 and in murine macrophages.32 Therefore, it is not surprising to see the constant elevation in MMP-9, although the possible role of increased MMP-9 in the gingival tissue has yet to be clarified.

A more striking finding was the remarkable elevation of TIMP-1. The behavior of TIMPs in response to mechanical loading is somewhat contradictory. Redlich et al33 reported an elevation of TIMP-1 in the gingiva under orthodontic loading only after appliance removal. In contrast, Tsuji et al34 showed that PDL cells under tensile stress expressed increased levels of TIMP-1 and -2, with no change noted in MMPs. Furthermore, effects of IL-1β on TIMP-1 production can be stimulatory in the cardiac fibroblast or refractory in the gingival fibroblast, varying according to cell type.35 A relevant finding with our results was shown by Gaultier et al.36 In their clinical study, a significant increase in both MMP-9 and its antagonist TIMP-1 was characteristic in cases of severe gingival inflammation. The reason why the pressure side gingiva in this study displayed similar responses to those seen with severe gingival inflammation is not clear. Considering the inhibitory role of TIMP-1 against MMP-9, gingival inflammation may be associated with homeostasis of local tissue in the maintenance of healthy status during tooth movement.

Taken together, primary elevation of those key mRNAs in the gingiva suggests the following possibilities: (1) Those mRNAs may be directly involved in the gingival inflammation and/or soft tissue destruction that surround the moving tooth; or (2) inflammatory mediators produced in the gingiva may play a regulatory role in the induction of bone remodeling in the PDL, as proposed by Beklen et al.22 In any case, an increase in inflammatory mediators under orthodontic loading in the gingiva suggests that active biologic effects of pressure on the gingival soft tissue are evident.

Unfortunately, because of technical and anatomic limitations of study animals, only the pressure side gingiva was excised in this study. Dudic et al37 and Garlet et al38 demonstrated differences in cytokines and other metabolites between pressure and tension sides with the use of gingival crevicular fluid and PDL. A comparative observation on pressure and tension side gingiva, as well as on PDL cells, therefore will be very useful for comprehensive substantiation of the findings described here. An in vitro cell culture study also will help to elucidate the mutual action between mediators.

CONCLUSIONS

• Application of an orthodontic force leads to a significant increase in IL-1β mRNA in pressure side gingiva on both day 7 and day 14 after loading, with a slight decrease noted at day 14.

• After orthodontic loading, MMP-9 and TIMP-1 mRNAs in pressure side gingiva were significantly elevated on both day 7 and day 14, with no significant difference reported between days 7 and 14.

• These results imply that the primary elevation of inflammatory mediators in soft tissue is induced by orthodontic pressure.