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RESEARCH REPORT |
1 Department of Pedodontics and Orthodontics, Institute of Dentistry, Biomedicum Helsinki (4th floor, C407b), POB 63, 00014 University of Helsinki, Helsinki, Finland;
2 Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital (HUCH), Institute of Dentistry, University of Helsinki, the Orton Research Institute and the Orthopedic Hospital of the Invalid Foundation, Helsinki, Finland; and
3 Department of Health, City of Helsinki, Finland;
*corresponding author, Satu.Apajalahti{at}Helsinki.fi
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: matrix metalloproteinase (MMP) • gingival crevicular fluid • orthodontic force
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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
For each patient, the permanent upper incisor, upper canine, or lower central incisor undergoing orthodontic tooth movement served as the experimental tooth. Orthodontic force was applied through an arch wire. GCF samples were collected from each experimental tooth immediately before fixed appliance activation and every hour for 8 hrs following application of the orthodontic force. The surfaces of the teeth were dried gently and kept dry with cotton rolls. Two filter-paper strips were kept at the gingival margin in the sulcus for 3 min. We measured the GCF flow volume by weighing the strips in the polypropylene tubes according to the Mettler AJ 100/GWB scale. Thereafter, the absorbed fluid was eluted from each strip into 25 µL of 0.2 mol/L NaCl-1.0 mmol/L CaCl2-50 mmol/L Tris-HCl, pH 7.5, and stored at -20°C prior to analysis. Control GCF samples, also weighed, were collected by an identical method from the upper central incisors every hour for 8 hrs.
Western Blot and Immunofluorometric Assay (IFMA)
The molecular forms of MMP-1 and MMP-8 in the GCF from experimental and control teeth were analyzed by the Western blot method, with specific antibodies for MMP-1 and -8 (both used at 2 µg/mL final concentrations), and by quantitated computer image scanning as previously described (Hanemaaijer et al., 1997; Prikk et al., 2001; Kiili et al., 2002; Apajalahti et al., 2003). Human PMN and rheumatoid synovial culture media (Hanemaaijer et al., 1997; Kiili et al., 2002) were used as positive controls for MMP-1 and -8.
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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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. 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).
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). 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).
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). 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). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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)-1ß and tumor necrosis factor-
(TNF-) (Hanemaaijer et al., 1997; Wu et al., 1999; Abe et al., 2001). Since no visible plaque and gingival inflammation could be detected indicating healthy infection- and inflammation-free periodontium, we may assume that, during orthodontically induced PDL remodeling, the evident participation of pro-inflammatory mediators plays a key role in MMP regulation.
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).
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ACKNOWLEDGMENTS |
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Received March 7, 2003; Last revision July 25, 2003; Accepted September 8, 2003
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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