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The in vivo Levels of Matrix Metalloproteinase-1 and -8 in Gingival Crevicular Fluid during Initial Orthodontic Tooth Movement
1 Department of Pedodontics and Orthodontics, Institute of Dentistry, Biomedicum Helsinki (4th floor, C407b), POB 63, 00014 University of Helsinki, Helsinki, Finland; Correspondence: *corresponding author, Satu.Apajalahti{at}Helsinki.fi
Orthodontic force induces biochemical responses in the periodontal ligament (PDL), but the matrix metalloproteinase (MMP)-dependent molecular mechanisms in orthodontically induced periodontal remodeling have remained unclear. Previous studies indicate that mechanical stress induces MMP-1 production in human PDL cells in vitro. We tested the hypothesis whether the in vivo levels, molecular forms, and degree of activation of MMP-1 and MMP-8 in gingival crevicular fluid (GCF) reflect an early stage of orthodontic tooth movement. Molecular forms of MMP-1 and MMP-8 were analyzed by Western blot, and MMP-8 levels by quantitative immunofluoro-metric assay (IFMA). The results showed that GCF MMP-8 levels for orthodontically treated teeth were significantly higher at 4-8 hrs after force application than before activation, and when compared with the control teeth (p < 0.05). Analysis of our data indicates that the cells within the periodontium are up-regulated to produce MMP-8, and the increased expression and activation of GCF MMP-8 reflect enhanced periodontal remodeling induced by orthodontic force.
Key Words: matrix metalloproteinase (MMP) • gingival crevicular fluid • orthodontic force
During orthodontic tooth movement, the collagenous extracellular matrix of the periodontal ligament (PDL) and alveolar bone is remodeled (Nakagawa et al., 1994; Karimbux and Nishimura, 1995). Matrix metalloproteinases (MMPs) are enzymes that play a central role in PDL remodeling, both in physiological and in pathological conditions. Collagenase-1 (MMP-1) and collagenase-2 (MMP-8), because they share a unique ability to cleave native triple-helical interstitial collagens (Weiss, 1989; Knäuper et al., 1996; Konttinen et al., 1998), can initiate this tissue remodeling. MMP-8 degranulation by polymorphonuclear leukocytes (PMNs) is among the pivotal factors in pathological collagen destruction during periodontal diseases (Sorsa et al., 1994, 1999; Golub et al., 1995; Ingman et al., 1996). In inflammatory conditions such as periodontitis, 75-kDa MMP-8 is mainly PMN-derived, but certain non-PMN-lineage cells in the oral cavity—such as gingival and PDL fibroblasts as well as gingival sulcular epithelial cells—can express 55-kDa MMP-8 (Hanemaaijer et al., 1997; Tervahartiala et al., 2000; Kiili et al., 2002). MMP-1 is thought to be constitutively synthesized by many different cell types (Birkedal-Hansen, 1993; Ingman et al., 1996). In vitro studies on human gingival and periodontal fibroblasts under mechanical stress have shown elevated MMP-1 mRNA and protein production (Carano and Siciliani, 1996; Bolcato-Bellemin et al., 2000). More recently, it was found that orthodontic force significantly increases MMP-1 expression in the gingival tissues of the dog in vivo (Redlich et al., 2001). Moreover, it has been previously shown that total collagenase activity is elevated in the gingival crevicular fluid (GCF) of orthodontic patients (Sorsa et al., 1992). To our knowledge, the in vivo levels, molecular forms, and degree of activation of MMP-1 and MMP-8 have not yet been studied in humans. We thus analyzed, in this in vivo study, the levels, molecular forms, and degree of activation of fibroblast collagenase (MMP-1) and collagenase-2 (MMP-8) in the GCF of human teeth exposed to orthodontic force with fixed appliances. Furthermore, we addressed the time-dependent changes in MMP-1 and -8 in the initial phase of orthodontic tooth movement.
Orthodontic Patients GCF samples were obtained from 11 orthodontic patients (eight female, three male; aged 10-14 yrs, except for three adults aged 37-38) treated with fixed appliances in a private dental practice. GCF control samples came from six systemically healthy age-matched non-orthodontic patients. The patients had no systemic diseases, nor were they on any continuing medication. Gingival tissues around the teeth exposed to orthodontic force were found to be clinically healthy in the clinical periodontal examination, which included measurement of plaque index, gingival bleeding, and probing depth. No signs of periodontal diseases were evident in panoramic radiographs. None of the patients had used antibiotics within the preceding 6 mos. The study was carried out with the informed consent of the patients, and was approved by the Ethics Committee of the Institute of Dentistry, University of Helsinki.
GCF Collection
Western Blot and Immunofluorometric Assay (IFMA) Concentrations of MMP-8 in the GCF samples were determined from the elution buffer by a time-resolved immunofluorometric assay (IFMA) (Hanemaaijer et al., 1997; Mäntylä et al., 2003), and the amounts of monoclonal antibodies 8708 and 8706 for MMP-8 were 1.5 µg and 0.5 µg, respectively, per assay. Mean values were analyzed by the non-parametric Mann-Whitney U-test. A p value of less than 0.05 was considered significant.
Expression of Collagenase-2 (MMP-8) in GCF of Orthodontic and Control Patients GCF flow was not significantly affected by orthodontic treatment (not shown). The concentrations of MMP-8 obtained by IFMA-assay in the GCF of orthodontic and control patients are shown in Fig. 1  . Mean MMP-8 concentrations in the GCF of orthodontically treated teeth were significantly higher at 4-8 hrs after force application than before activation, and when compared with the control teeth (p < 0.05).
Immunoblots for MMP-8 in the GCF of 11 orthodontic patients taken at 0 to 8 hrs showed bands at > 100, 60-80, 40-55, and < 30 kDa (Table, Fig. 2A ). In 19% of the orthodontic samples, no staining for MMP-8 immunoreactivity was detectable; this usually included GCF samples taken at 0 or 1 hr. Immunoblots for MMP-8 in control GCF samples had hardly detectable or missing bands (Fig. 2B). The Table shows the mean percentages of different molecular species of collagenase-2 (MMP-8) isoforms detected in Western immunoblots of orthodontic GCF samples at 0 to 8 hrs. Bands in the range 60 to 80 kDa corresponding to PMN-type pro- and active enzymes made up the majority (42-62%) of the total MMP-8 immunoreactivity, except at 0 and 5 hrs. At 0 hr, in fact, only one patient had staining at 60 kDa and two patients at 75 kDa. From baseline to 1 hr, the amount of PMN-type active enzyme (60 kDa) was elevated in relation to total PMN-type (60-80 kDa) immunoreactivity from 31% to 79%, decreasing to 56% at 2 hrs, and thereafter being less than or nearly equal to the amount of PMN pro-enzyme (75-80 kDa). Of the total staining, the high-molecular-weight band (> 100 kDa), representing complexed enzyme, was the next most frequent. Low-molecular-weight staining at < 30 kDa, most likely representing degraded fragments of MMP-8, was seen in only one patient at 6-8 hrs. Bands around 40-55 kDa, representing fibroblast-type MMP-8, were visible in four patients from 2 to 8 hrs, with the maximum level at 2 hrs (9% of the total MMP-8 immunoreactivity).
Expression of Collagenase-1 (MMP-1) in GCF of Orthodontic and Control Patients The majority of collagenase-1 (MMP-1) Western immunoblots for experimental teeth failed to exhibit any detectable immunostaining (Fig. 2C  ). Of the 11 orthodontic samples, 3 showed immunoreactivity at > 100 kDa, whereas one patient showed staining at 52 kDa, corresponding to the MMP-1 proform, at 4 hrs. Overall, it was impossible for us to do any reliable analysis as to the distribution of MMP-1 expression at 0 to 8 hrs. For the control samples, no MMP-1 immuno-reactivity was detectable with the Western blot technique (Fig. 2D).
Our results in this in vivo study showed statistically significant elevated collagenase-2 (MMP-8), but not collagenase-1 (MMP-1), immunoreactivity in GCF of teeth under initial orthodontic stimuli. The orthodontic stimuli did not affect GCF flow. Mean total MMP-8 immunoreactivity increased significantly within 8 hrs after activation of fixed orthodontic appliances, so that at 4 hrs it was almost 2.5-fold and at 6 hrs 3.5-fold in relation to baseline (0 hr). In this respect, it is noteworthy that, clinically, the majority of orthodontic patients report pain at 4 to 24 hrs after the insertion of fixed appliances (Scheurer et al., 1996). Since no signs of gingival inflammation or pocket formation were evident among the orthodontic patients, we may assume that the cells within the periodontium were up-regulated to produce MMP-8, and that this increased expression of MMP-8 reflects enhanced PDL remodeling activity induced by orthodontic force. Recently, Redlich et al. (2001) have demonstrated an in vivo increase in MMP-1 mRNA and protein production in gingival tissue of dogs 3 days after force application. In vitro models of human fibroblasts under cyclic stretching have shown elevated MMP-1 synthesis after 4 days (Carano and Siciliani, 1996), and elevated mRNA expression for the enzyme after a 12-hour mechanical stimulation (Bolcato-Bellemin et al., 2000). In the present study, however, slight if any MMP-1 immunoreactivity was detectable by the Western blot technique in the GCF of experimental teeth. Differences in the methods as well as in the time scale for MMP-1 detection may, at least in part, explain the differences between our results and those of Redlich et al. (2001). Nonetheless, together with cathepsins, MMP-1 is involved in the degradation of collagenous bone matrix (Deláisse et al., 1993; Domon et al., 1999). In response to sustained force, monocytes in the PDL area are stimulated to form osteoclasts, which first appear within the compressed ligament in young humans after a period of 30 to 40 hrs after force application. Since there is a delay in the occurrence of bony response after force application, the elevated pattern of MMP-1 activity in GCF might be apparent only at the later stage of orthodontic tooth movement. Our laboratory has begun an in vivo study to analyze MMP-1 involvement in orthodontically induced bone remodeling in humans.
An elevated pattern of MMP expression and activation in inflammatory conditions of the PDL has been well-demonstrated (Sorsa et al., 1994, 1999; Golub et al., 1995; Ingman et al., 1996; Kiili et al., 2002; Mäntylä et al., 2003). MMP-8 species detected in periodontitis GCF may be derived from degranulating PMNs triggered by the periodontopathogenic bacteria and/or their virulence factors (Ding et al., 1995). In vitro studies on human gingival and periodontal fibroblasts have shown increased expression and synthesis of MMP-1 and MMP-8 by certain pro-inflammatory mediators, e.g., interleukin (IL)-1β and tumor necrosis factor- Molecular forms in the range 60 to 80 kDa, corresponding to PMN active and pro-enzyme, made up the majority of the total MMP-8 immunoreactivity, except at 0 and 5 hrs. We can only speculate on the cellular sources for the different MMP-8 isoforms detected in our orthodontic GCF. Yet, MMP-8 species from various cells clearly differ in molecular size and are influenced by the degree of glycosylation and activation (Hanemaaijer et al., 1997). The most likely cellular sources for the 60- to 80-kDa MMP-8 species are infiltrating leukocytes, i.e., neutrophils and monocytes/macrophages (Ding et al., 1995; Herman et al., 2001), but MMP-8 has recently also been shown to originate from gingival fibroblasts, sulcular epithelial cells, and even bone cells (Hanemaajier et al., 1997, Tervahartiala et al., 2000; Sasano et al., 2002). At 2 to 8 hrs, some MMP-8 immunoreactivity appeared as 40- to 55-kDa fibroblast-type MMP-8. These may well be less-glycosylated forms, of which de novo expression can be induced by pro-inflammatory cytokines in PDL fibroblasts. Thus, MMP-8 may have multiple potential cellular sources during orthodontic tooth movement. From baseline to 1 hr, the amount of PMN active enzyme (60 kDa) was elevated in relation to total immunoreactivity of the PMN-type (60-80 kDa) MMP-8 isoform, indicating that orthodontic force can also induce autoactivation of increased amounts of MMP-8. Importantly, our results demonstrate that the orthodontic force application quickly elevates MMP-8 autoactivation, i.e., in only 1 hr. In conclusion, our in vivo findings demonstrate a major role for collagenase-2 (MMP-8) in orthodontically induced PDL remodeling, with a significantly elevated and time-dependent pattern of enzyme expression occurring within 4 to 8 hrs after fixed appliance activation. Our findings further indicate that MMP-1 does not contribute to orthodontically induced PDL remodeling during initial tooth movement. Future studies may show that, similar to the situations with periodontitis-affected GCF, MMP-8 in GCF from teeth exposed to orthodontic force may have significant diagnostic implications (Sorsa et al., 1999, 2003; Mäntylä et al., 2003).
This study was supported by Finnish Women Dentists, the Finnish Dental Society Apollonia, the European Orthodontic Society, the Emil Aaltonen Foundation, Helsinki University Research Funds, the Else and Wilhelm Stockmann Foundation, and by a HUCH-EVO (TI020Y0002) grant. We thank Dr. Pirjo Savolainen for assistance in collecting the GCF samples. Received for publication March 7, 2003. Revision received July 25, 2003. Accepted for publication September 8, 2003.
Journal of Dental Research, Vol. 82, No. 12,
1018-1022 (2003) This article has been cited by other articles:
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