Occlusal Hypofunction Induces Atrophic Changes in Rat Gingiva
Objective: To clarify the influence of occlusal hypofunction on the integrity of gingival tissue and gingival extracellular matrix biosynthesis.
Materials and Methods: Thirteen-week-old male Wistar rats were divided into two groups. To eliminate occlusal forces, all the right maxillary molars were extracted in the hypofunctional group. The control group was anesthetized but not subjected to surgery. The rats were killed at 2 and 4 weeks after the procedure, and the lower right second molars were prepared for histological analysis. To investigate the effect of occlusal hypofunction on collagen biosynthesis, the expression of connective tissue growth factor (CTGF) and lysyl oxidase (LOX) was determined by immunohistochemistry as well as histological examination by hematoxylin and eosin staining.
Results: Disorientation of the collagen fibers, proliferation of the connective tissue fibroblasts, and enlargement of epithelial intercellular gaps were observed in gingival tissue of rat molars with experimental occlusal hypofunction. Immunohistochemically, the expression of CTGF and LOX was increased significantly (P < .05) in the hypofunctional group.
Conclusion: These results suggest that occlusal hypofunction can affect the structural integrity and the expression of CTGF and LOX in gingival tissue.Abstract
INTRODUCTION
Mechanical stimuli are well known to affect development, maintenance, and remodeling of the periodontal tissues. Clinically, occlusal hypofunctional condition occurs in teeth of malocclusion, such as open bite, missing teeth, or delayed restorations. Root resorption of these teeth during orthodontic treatment has been reported, and occlusal hypofunction was suggested to be one of the possible causes.12 Moreover, flare and swelling often occur in the gingiva of open-bite teeth, and gingival recession of high-displaced canine through orthodontic treatment has been experienced. Accordingly, it is interesting to understand the influence of occlusal stimuli on the periodontium.
There are many published reports on the relationship between occlusal stimuli and the homeostasis of periodontal tissues. We have reported previously that the loss of normal occlusal function leads to atrophic changes in the periodontal ligament (PDL), such as narrowing of the periodontal space, vascular constriction and alteration of the components, and deformation of the mechanoreceptor structure.3–6 Occlusal hypofunction is also known to decrease the alveolar bone mass and suppress the bone formation of the alveolar and jaw bones.7–9 Although there has been considerable focus on the changes in gingival homeostasis under occlusal hypofunction, the effect of the state on gingiva has received relatively little attention.
Gingiva overlies the surface of periodontal tissues to protect subepithelial tissues from a variety of external injury, such as bacterial infection and chemical/ physical stimuli. Therefore, it is important for clinicians to evaluate the involvement of gingiva in the maintenance of a sound periodontium and oral health. In this study, we hypothesize that occlusal hypofunction may cause atrophic changes of gingival tissue and alteration of collagen metabolism, such as PDL and alveolar bone.
Similar to other mucosal tissues, the gingiva undergoes remodeling to maintain homeostasis, and the attachment level was preserved to its original level through gingival remodeling during tooth movement in orthodontic treatment. Collagen metabolism is considered essential for remodeling of the gingival extracellular matrix (ECM). Also, collagen metabolism is a complex process that involves the coordinated actions of different cell types and is regulated by a multitude of growth factors. Among the many factors that are involved in collagen metabolism, connective tissue growth factor (CTGF) and lysyl oxidase (LOX) have recently become causative factors of interest in the pathogenesis of fibrotic disorders.
CTGF is a 38-kDa, cysteine-rich, matricellular protein, and it has diverse functions that include the regulation of cell adhesion, proliferation and differentiation, and the biosynthesis of ECM proteins.10–14 LOX is also a crucial enzyme in ECM formation15 and catalyzes the final enzymatic step of oxidative deamination in collagen. This deamination reaction is required for covalent cross-linking and the insolubilization of collagen precursors in the formation of a mature and functional ECM.
Therefore, the aim of this study was to investigate the influence of occlusal hypofunction on the integrity of gingival tissue by means of histological examination and gingival ECM biosynthesis using immunohistochemical analysis to measure the expression of CTGF and LOX.
MATERIALS AND METHODS
Hypofunctional Groups
All animals and the experimental procedures to which they were subjected in this study were approved by the Animal Ethics Committee of the Tokyo Medical and Dental University.
Twenty 13-week-old male Wistar rats were used in this investigation. All the animals were anesthetized by an intraperitoneal injection of chloral hydrate (400 mg/ kg). The animals were divided randomly into two groups of 10 rats: (1) a group of rats that were anesthetized only (control group) and (2) a test group of rats from which the right maxillary molars (M1, M2, M3) were extracted to create occlusal hypofunction of the lower right molars (hypofunctional group).46 Both groups of rats were fed a powdered diet (CE-2, Clea Japan, Shizuoka, Japan) and had access to tap water ad libitum. Their body weight was monitored during the experimental period.
Histomorphology
At 2 and 4 weeks after the procedure, five animals in each group were deeply anesthetized by chloral hydrate and were perfusion fixed transcardially with 4.0% paraformaldehyde in 0.1 M phosphate buffer pH 7.4 (Figure 1a). The mandibles were then removed, and these underwent further fixation by overnight immersion in the identical fixative solution at 4°C. The specimens were decalcified in 4.13% EDTA disodium solution pH 7.4 for 6 weeks at 4°C. The specimens were embedded in paraffin by conventional methods. Paraffin sections (4 μm thick) were frontally cut in such a manner as to show the crown and both apices of buccal and lingual roots of the lower right second molars (Figure 1b). The paraffin-embedded sections were then deparaffinized, rehydrated, and washed in phosphate-buffered saline (PBS). The sections were stained with hematoxylin and eosin (HE) for assessment of tissue histology using an EXLIPSE 80i microscope (Nikon, Tokyo, Japan).
Citation: The Angle Orthodontist 78, 6; 10.2319/092907-465.1
Immunohistochemistry
Immunohistochemical staining was performed on paraffin sections using the ABC method (Vector Laboratories, Burlingame, CA). The sections were deparaffinized with xylene and rehydrated in graded ethanol. The endogenous peroxidase activity was quenched by incubating the slides in 0.3% H2O2 in methanol for 30 minutes. The nonspecific binding sites were blocked using normal goat serum in Tris-buffered saline/0.1% Triton X-100 pH 7.6 (TBST) for 20 minutes. The sections were incubated overnight at 4°C with primary antibodies against CTGF (BioVision, Mountain View, CA), which was diluted 1:100 in PBS, and against LOX (IMGENEX, San Diego, CA), which was diluted 1:200 in PBS. The secondary antibody was diluted antibody against each IgG (VECTASTAIN ABC Staining Kit, Vector Laboratories) and was applied for 30 minutes at room temperature, respectively. The sections were then treated with an avidin-biotin macromolecular complex (VECTASTAIN ABC Staining Kit, Vector Laboratories) for 30 minutes at room temperature. For the purposes of visualization, the slides were stained in DAB and counterstained with methyl-green. Between each above-mentioned step, the sections were washed twice with TBST; each wash lasted 3 minutes. Specificity of immunohistochemical localization was verified using nonimmune IgG as the negative control.
The observation areas were defined as the free gingiva above the gingival grooves (Figure 1b) for quantitative analysis using computer-assisted image analysis (Image-Pro Plus 4.0, Media Cybernetics, Silver Spring, Md). To this end, the software automatically selects the positive cells based on similarities in the color of adjacent pixels. CTGF and LOX were evaluated by counting the number of stained cells. Data were collected from three serial sections per tissue specimen, and each set of data was counted on 3 different days to correct for systemic error in observation. No significance was noted in the measurements of these evaluations.
Statistical Analysis
All results are presented as the mean ± standard deviation. Data were statistically analyzed by a two-tailed Student's t-test using the Statistical Package for the Social Sciences software (SPSS, Chicago, Ill). Statistical significance was set at P = .05.
RESULTS
There were no significant differences between the body weights of two groups of rats during the experimental period (Figure 2). Neither obvious flare nor inflammation of the gingiva was evident in both groups (data not shown).
Citation: The Angle Orthodontist 78, 6; 10.2319/092907-465.1
Histological Analysis
HE-stained sections of all the specimens of gingival tissue revealed no gross evidence of inflammation, as determined by the low abundance of infiltrating inflammatory cells. In HE-stained sections of the gingival tissue that were prepared from the control group, small epithelial cells, a thin layer of epithelium, plus thick, well-orientated, and cross-weaved collagen fiber bundles and sparse fibroblasts with small nuclei were observed (Figure 3a, c). Otherwise, HE-stained sections of gingival tissue from the hypofunctional group were characterized by thickening of the epithelium, especially the junctional epithelium and crevicular epithelium, and the disorganization of collagen fiber bundles in connective tissue stroma, particularly in the dentogingival fibers (Figure 3b, d).
Citation: The Angle Orthodontist 78, 6; 10.2319/092907-465.1
Histological examination of the gingival epithelium also revealed that the number of epithelial cells was increased and the intercellular gaps were enlarged in the experimental rats. In the gingival lamina propria of the test rats, proliferation of the connective tissue fibroblasts has been induced, and the diameter of collagen fibers was thinned. Specifically, the collagen fibers had become mostly loose and dispersive. These findings in the gingival tissue of the hypofunctional group were more obvious after 4 weeks compared with the changes that were observed after 2 weeks.
CTGF Expression in Gingival Tissue
Figures 4a–d are representative images of CTGF immunoreactivity in the control and hypofunctional groups. Nonspecific staining for CTGF immunoreactivity for all groups was negligible. CTGF was localized predominantly in the epithelial layers, and the stratum spinosum epidermidis of the gingival epithelium appeared to express in both groups. In the hypofunctional group, immunoreactivity was also shown in the basal cells but not in the control group. CTGF immunoreactivity was higher in the hypofunctional group than in the control group. This observation was supported further by quantitative analysis of intracellular CTGF expression. The fibroblasts in the connective tissue were also positive in these slides, although the intensity of staining was weaker than that found in the epithelium. Thus, we confined the area for quantitative analysis of CTGF to the epithelial layers only. The intensity of intracellular CTGF staining was significantly higher in the hypofunctional group when compared with that in the control group (Figure 4e). The levels of CTGF expression at 4 weeks were significantly lower than those at 2 weeks in the hypofunctional group, and there were no significant differences in the control group (Figure 4e).
Citation: The Angle Orthodontist 78, 6; 10.2319/092907-465.1
LOX Expression in Gingival Tissue
Figures 5a–d are representative images of LOX immunoreactivity in the control and hypofunctional groups. In addition, nonspecific staining for LOX immunoreactivity for all groups was negligible. This analysis demonstrated elevated levels of expression in the gingival tissues from the rats with experimental occlusal hypofunction. Specifically, the elevated levels of LOX expression were confined to the cytoplasm and nucleus of connective tissue fibroblasts and endothelial layers of the blood vessels (Figure 5a–d). This result was confirmed by quantitative analysis of the intracellular expression of LOX. The intensity of intracellular LOX staining was significantly higher in the hypofunctional groups when compared with that in the control groups (Figure 5e). In contrast to the pattern of CTGF expression in the hypofunctional group, the levels of LOX expression were higher at 4 weeks when compared with those determined at 2 weeks in the hypofunctional group, and there were no significant differences in the control group (Figure 5e).
Citation: The Angle Orthodontist 78, 6; 10.2319/092907-465.1
DISCUSSION
This study indicates that the loss of occlusal function induces atrophic changes and alteration of collagen biosynthesis in the rat gingiva.
To avoid the influence of growth on our results, 13-week-old adult rats were used in the experiment. A powder diet was chosen to eliminate the mechanical stimuli caused by the bolus on mandibular molars and its attached gingiva. We focused on the effect of occlusal hypofunction on gingival metabolism and examined the expression of CTGF and LOX as indicators for collagen biosynthesis. CTGF and LOX were selected for the immunohistochemical analysis because CTGF is one of most important inducers of collagen biosynthesis and LOX is a critical factor in collagen maturation and accumulation.
The gingiva is composed of epithelium and connective tissue. In the connective tissue, one of the major components of ECM is interstitial collagen.16 These collagen fibers are functionally arranged to surround the teeth and thereby contribute to their support. In addition, the collagen provides mechanical properties and the scaffolding to the gingiva.17 Histological examination revealed that collagen bundles were thick and well orientated in the gingival tissue in the control group.
It is generally accepted that the configuration of an organism is closely related to its function. It is reported that the orientation of collagen fiber bundles alters continuously during the phase of tooth eruption, and after the tooth has come in contact with the opposite tooth and is functioning properly, the collagen fibers of periodontal tissue associate distinctly into groups of well-orientated supraalveolar fiber apparatus to support the tooth.
In our study, the hypofunctional condition may have caused the decreased demand for supporting tooth and so resulted in the disappearance of the functional arrangement of collagen bundles in the gingival tissue. During the orthodontic treatment, the tooth movement may start with the disruption of the gingival collagen fibers, and the reorganization of the collagen fiber network would be completed again to accommodate the new tooth position.18 Thus, the condition of collagen bundles before tooth movement may have some influence on the reorganization of the collagen fiber network. From this point of view, the disorganization of the functional arrangement of gingival collagen bundles induced by occlusal hypofunction could be one of the possible causes of unexpected gingival recession in the hypofunctional tooth such as a high-displaced canine during orthodontic tooth movement.
Many studies have mentioned that the expression of CTGF was detected only in mesenchymal and nonepithelial cell types, but recently, elevated CTGF levels in the epithelium have been reported in gingival overgrowth.19 Interestingly, our study showed that CTGF was expressed strongly in gingival epithelium under occlusal hypofunction with upregulation of LOX expression in the connective tissue. Moreover, we found that the expression of CTGF in gingival epithelium was limited to certain areas that coincided with the terminal position of dentogingival fibers that connect the gingiva to the tooth mechanically. These results indicated that the expression of CTGF may be triggered by the reduced mechanical stress on the teeth under hypofunctional condition.
On the other hand, LOX is reported to be a vital factor for the maturation of the collagen fiber bundles.15 As other researchers have reported,2021 the expressions of LOX were observed dominantly in the gingival lamina propria, particularly in the cytoplasm and nucleus of fibroblasts in this study. Proliferation of the fibroblasts and alteration in the collagen arrangement were evident in the gingiva under occlusal hypofunction. In addition, our data also suggested that a time lag may exist between the expressions of CTGF and LOX. As the production of CTGF in fibrotic gingival epithelium was highly significant, CTGF may play a role in the proliferation of basal gingival epithelial cells and promotes fibrosis in the connective tissue stroma.19
Accordingly, we propose that the hypofunctional condition induced the expression of CTGF in gingival epithelium and that those epithelial CTGF may affect LOX expression in connective tissue stroma, which is mediated by an interaction across the basement membrane. However, further studies are still needed to elucidate these mechanisms.
CONCLUSIONS
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Occlusal hypofunction caused atrophic changes of the rat gingiva with the expression of epithelial CTGF and LOX in the gingival tissue.
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These results demonstrate that occlusal function is an important factor in the maintenance and remodeling of gingiva.
The (a) experimental schedule and (b) schematic diagram of the frontal section of the mandible second molars. The investigation area for quantitative analysis was delimited as the free gingiva above the gingival grooves (inside the square area). AB indicates alveolar bone; P, dental pulp; PDL, periodontal ligament; B, buccal side; L, lingual side
The changes in body weight during the experimental period. Data are presented as the means ± SD (n = 5)
Representative frontal sections of free gingival tissue stained by hematoxylin and eosin in the control group at 2 weeks (a) and 4 weeks (c) and in the hypofunction group at 2 weeks (b) and 4 weeks (d) after extraction. In experimental occlusal hypofunction (b, d), thickening of the epithelium, disorientation of the collagen bundles, and more blood vessels were observed. Magnification: 100×, with a 400× inset. The bar represents 100 μm
Immunohistochemical analysis for connective tissue growth factor (CTGF). The distribution of CTGF in the gingival tissue of the control group at 2 weeks (a) and 4 weeks (c) and the hypofunction group at 2 weeks (b) and 4 weeks (d) after extraction. Arrowheads point to areas of strong CTGF expression in the epithelial cells. Magnification: 200×. The bar represents 200 μm. (e) Quantitative analysis of CTGF expression. The results are expressed as the mean ± SD (*P < .05, n = 5)
Immunohistochemical analysis for lysyl oxidase (LOX). The distribution of LOX in the gingival tissue of the control group at 2 weeks (a) and 4 weeks (c) and the hypofunction group at 2 weeks (b) and 4 weeks (d) after extraction. Arrowheads indicate the areas of strong LOX expression in connective tissue cells. Magnification: 100×, with a 400× inset. The bar represents 100 μm. (e) Quantitative analysis of LOX expression. The data are expressed as the mean ± SD (*P < .05, n = 5)
Contributor Notes
Corresponding author: Dr Yuji Ishida, Orthodontic Science, Department of Orofacial Development and Function, Division of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan (yjis.orts@tmd.ac.jp)