Expression of proteins of elastic fibers and collagen type I in orthodontically rotated teeth in rats

Introduction

The aims of this study were to investigate the expression of proteins of elastic fibers and collagen type I in the supra-alveolar structure of orthodontically rotated teeth in rats and to elucidate whether circumferential supracrestal fiberotomy diminishes relapse.

Methods

The rats’ maxillary left first molars were rotated by couple of force. Specimens were divided into groups according to different orthodontic procedures. A1-3 and B1-3 were blank control groups and operation control groups. Group C underwent rotation only, and group D was treated with rotation and retention. Groups E and F were treated with rotation, retention, and release of retention; additionally, circumferential supracrestal fiberotomy was performed in group F before the release of retention. The animals were killed, and the jaws were processed for histologic evaluation using the immunohistochemical method to evaluate the protein expressions of elastin, fibrillin-1, fibrillin-2, and collagen type I in supra-alveolar structures (around and below the gingival sulcus) between the maxillary left first and second molars. The degree and percentage of relapse were measured by a series of impressions.

Results

The degree and percentage of relapse in group F were much lower than those in group E ( P <0.05). Collagen type I was increased in group C ( P <0.05) and at normal levels in groups D, E, and F. Elastin below the gingival sulcus and fibrillin-1 showed the same patterns of expression and were consistently elevated in groups C, D, E, and F ( P <0.05). No positive staining for elastin was found around the gingival sulcus in any specimen. The difference in the expression of fibrillin-2 between the experimental groups (C, D, E, and F) and their matching control groups was not statistically significant ( P >0.05).

Conclusions

Circumferential supracrestal fiberotomy can alleviate the relapse of rotated teeth. Collagen fibers of supra-alveolar structures might contribute to relapse in a short time, whereas elastic fibers may be the reason that rotated teeth relapse to their original positions after retention.

Highlights

  • Circumferential supracrestal fiberotomy can alleviate relapse of rotated teeth.

  • Supra-alveolar collagen fibers might contribute to relapse in the short term.

  • Elastic fibers may cause rotated teeth to relapse over the long term.

After active orthodontic treatment, teeth move to their ideal places, but remodeling and rearrangement of the supporting tissues around the teeth have not been completely balanced, leading to instability and relapse. The phenomenon that rotated teeth return to pretreatment positions is much more frequent. The relapse of corrected tooth rotations was usually attributed to the gingival and transseptal fibers of the periodontium. Reitan ascribed the relapse of rotated teeth after long-term retention to contraction of the displaced gingival fibers and other supra-alveolar structures; Boese advocated that a significant proportion of the relapse was caused by the stretched principal fibers during the initial 8 weeks of retention. The increased oxytalan fibers and collagen in the supra-alveolar tissue, combined with the stable attachments of the transseptal fibers, seemed responsible for the relapse after the first 8 weeks. Edwards had similar findings. However, Redlich et al showed disorganized, split, and ripped collagen fibers, along with an increase in elastic fibers after orthodontic rotation and retention. They assumed that the changing elastic properties of all gingival tissues caused the relapse rather than “stretched” collagen fibers.

Collagen and elastic fibers coexist in the extracellular matrix of the gingiva and periodontal membrane. Collagen, mainly collagen type I (Col-I), has a high turnover rate, and centrifugal force can change the transcription of Col-I and collagenase in vitro. The half life of elastic fibers is supposed to be 70 years because of 1 component of its composition—elastin. Elastin, arising through lysine-mediated cross-linking of tropoelastin, can be broken down and metabolized by elastases; its degradation is extremely slow because of the extensive cross-linking. Under pathologic conditions (eg, abdominal aortic aneurysm ) or change in the physiologic environment, the synthesis and degradation of elastin may alter partly, since the extracellular matrix undergoes some transformation. Redlich et al reported that the mRNA levels of tropoelastin increased significantly in a time-dependent manner after simulation of orthodontic force; this implies that the gene of tropoelastin can respond to applied force. Microfibrillar, consisting of fibrillins (FBN), is another major component of elastic fibers. FBN has 2 isoforms in rodents (FBN-1 and FBN-2); FBN-1 is the most abundant isoform.

Many attempts have been made to prevent a rotated tooth from relapse: injection of relaxin, low-level laser therapy, overcorrection, prolonging the retention, early correction, reproximation, and gingivectomy. Among these, circumferential supracrestal fiberotomy (CSF) is one of the most studied methods for alleviating relapse. Jahanbin et al reported that relapse in the control group (27.8%) was significantly greater than in the CSF group (9.7%) over 1 month. CSF, transecting the free gingival and transseptal fibers to increase their adaptation to the new tooth position, is indicated for moderately to severely rotated teeth, and also when the supragingival fibers have been markedly displaced.

Our objective was to elucidate the possible impact of elastic and collagen fibers in supra-alveolar structures on rotational relapse by analyzing the expressions of elastin, FBN-1, FBN-2, and Col-I under various orthodontic procedures. We also evaluated the efficacy and potential mechanism of CSF in preventing rotational relapse.

Material and methods

One hundred fifteen male Sprague-Dawley rats (age, 6-7 weeks; weight, 150-160 g) were housed in separate cages, supplied with powdered food and tap water, air conditioning, and lighting in a 12-to-24 hour light and dark cycle. All animal care and experimental procedures were performed according to the Guidelines for Animal Experimentation of Sichuan University. All animal experiments were approved by the Animal Welfare Committee of Sichuan University. After adaptation to the new situation for 1 week, the rats were randomly divided into 10 groups, as shown in Table I .

Table I
Group assignment
Group Procedure Number Execution time
A1 Blank control group 5 T1
B1 Operation control group 5 T1
C Rotation 20 T1
A2 Blank control group 5 T2
B2 Operation control group 5 T2
D Rotation and retention 20 T2
A3 Blank control group 5 T3
B3 Operation control group 5 T3
E Rotation and retention and subsequent relapse 20 T3
F Rotation and retention, subsequent CSF, and relapse 25 T3

A1-3, B1-3, and C-F are groups having different orthodontic procedures.
Number means how many rats in the group.
T1 , After rotation for 5 weeks; T2 , after rotation for 5 weeks and retention for 8 weeks; T3 , after rotation for 5 weeks, retention for 8 weeks, and relapse for 2 weeks.

The rats, except those in groups A1 through 3, were anesthetized with an intraperitoneal injection of 10% chloral hydrate (0.35 ml/100 g). Then they were placed in a supine position with a mouth gag. In groups C through F, nickel-titanium coil springs (Ming Xing Spring, Chengdu, China), whose force levels were set to approximately 20 g with an electric force gauge (Yueqing Handpi Instruments, Zhejiang, China) and bonded to the mesial-palatal and distal-buccal sides of the maxillary left first molar, respectively, to rotate the experimental teeth. Indirect anchorage was obtained from 1 appliance, made of 0.7 mm of steel wire and fixed firmly around the maxillary incisors with dental adhesive resin (3M, St Paul, Minn) ( Fig 1 ). To prevent detachment of the appliance from the maxillary incisors, occlusal interference was eliminated by grinding the incisal edge of the mandibular incisors. Groups B1 through 3 underwent the same procedures, with the exception that the nickel-titanium coil springs exerted no force. The appliances were checked every day and reinstalled if there was any detachment or damage.

Fig 1
A, Intraoral photograph of orthodontic appliance used to create rotational couple forces; B, diagram of rotating maxillary left first molar; C, diagram of retention; D, dental cast used to evaluate rotation and relapse; black line , crossing the dots that were marked in the shallow pits of the maxillary right and left third molars on the dental cast; green line , perpendicular to the black line; yellow line , going through the buccal-distal groove of the maxillary left first molar. Degrees of the angles between the green and yellow lines were recorded to calculate rotation and relapse. M, Maxillary molars; Sp, springs to exert couple forces; SS1, 0.7-mm stainless steel wires for anchorage; SS2, double strand of 0.1-mm stainless steel wires to retain the rotated teeth.

After 5 weeks of rotation, fixed retaining appliances were used to replace the rotational device in the rats from groups D through F and were kept in the mouth for 8 weeks. Double-strand 0.1-mm stainless steel wires were fixed between the indirect anchorage and the mesial-palatal side of the maxillary left first molar to keep the experimental teeth in place ( Fig 1 ).

After retention, retainers in groups E and F were removed, and the experimental teeth were monitored for 2 weeks to investigate the stability of movements. Before the relapse, CSF, well documented by Brain, was used on the rats in group F. A surgical blade was inserted into the gingival sulcus, and a circumferential incision tracing the alveolar crest was made along the long axis of the tooth. The procedure involved severing all fibrous attachments surrounding the tooth to a depth approximately to the alveolar crest.

To record the status of the teeth, impressions were taken with alginate materials and poured with stone. These dental casts were used for designated measurements. The tooth positions were recorded before rotational force application (T0), before the retainer (T1), before the relapse (T2), and after the relapse (T3).

The dental casts were trimmed so that the occlusal surface of the posterior teeth was parallel to the horizon. The photographs with constant magnification were prepared from the occlusal view of the dental models from the same height and angle of each cast. The digital images were then imported into the Digimizer image analysis software (version 4.3.4.0; MedCalc Software, Ostend, Belgium), and 3 lines were drawn to calculate the degrees of rotation and relapse: line 1, a line crossing the dots marked in the shallow pits of the maxillary right and left third molars on the dental cast; line 2, a line perpendicular to line 1; and line 3, a line through the buccal-distal groove of the maxillary left first molar.

The lower right angles between lines 2 and 3 were recorded to calculate rotation and relapse ( Fig 1 ): the degree of rotation, T1−T0, assessed the effect of rotation; the degree of relapse during retention, T1−T2, assessed the effect of retention; and the degree of relapse during relapse, T2−T3, recorded the amount of relapse in groups E and F to evaluate whether CSF could reduce relapse after rotation. The percentage of relapse = (T2−T3)/(T2−T0)*100%.

The rats were killed with an overdose of anesthetic. Maxillary segments were dissected, fixed in 4% paraformaldehyde solution for 24 hours at 4°C, and placed in 10% ethylenediaminetetraacetic acid at room temperature for 4 weeks of decalcification; This solution (pH: 7.4) was changed twice a week. Fully decalcified samples were dehydrated in a graded ethanol series and then fixed in paraffin. Horizontal serial sections, 4μm thick, were cut perpendicular to the longitudinal axis of the experimental teeth. Two separate sections were chosen from these qualified ones: one from the area around the gingival sulcus, and the other from the area below the gingival sulcus. These sections were mounted on polylysine-coated glass slides.

The specimens were examined under light microscopy after hematoxylin and eosin staining and immunohistochemistry. Immunohistochemistry was performed according to the manual of the immunohistochemical assay kit (Zhongshan Jinqiao Biological Technology, Beijing, China) (streptavidin/peroxidase method). The paraffin-embedded sections were dewaxed with xylene and dehydrated with graded ethanol. Endogenous peroxidase activity was blocked and inactivated with 3% hydrogen peroxide at 37°C for 30 minutes and 0.1% trypsin (HyClone Laboratories, Logan, Utah) was used to retrieve antigen for 20 minutes at 37°C. Sections were incubated with goat serum at 37°C for 20 minutes to seal the nonspecific site and then incubated with primary antibodies—rabbit antirat elastin, FBN-1, FBN-2, and Col-I polyclonal antibodies (Abcam, Cambridge, United Kingdom), overnight at 4°C. Negative controls were incubated with phosphate-buffered saline solution (supplementation). Secondary goat antirabbit antibody was added for 30 minutes at room temperature, followed by horseradish peroxidase for 15 minutes. Diaminobenzidine tetrahydrochloride was used to visualize the staining. Sections were counterstained with hematoxylin. Finally, the sections were mounted with neutral balsam and observed under light microscopy. No primary antibody was applied in the negative controls ( Supplemental Figure ).

An inverted microscope (IX71; Olympus, Tokyo, Japan) was used to observe hematoxylin and eosin and immunohistochemistry staining. The supra-alveolar structure was divided into 2 parts: around the gingival sulcus, and below the gingival sulcus by the bottom of the gingival sulcus. Two visual fields (400 times magnification) between the maxillary left first and second molars were randomly selected at each part, and images were collected with the ACT-1 cell image analysis system (Nikon, Tokyo, Japan). Positive immunohistochemistry staining in each group was semiquantitatively determined using the Image-pro Plus software (version 6.0; Media Cybernetics, Shanghai, China), and the mean optical density was calculated.

Statistical analysis

Data were expressed as means and standard deviations. Intergroup comparisons of the mean optical density values of immunohistochemistry staining around and below the gingival sulcus were achieved with 1-way analysis of variance (ANOVA), as well as the degrees of rotation and relapse and the percentage of relapse. The Bonferroni test was used to compare all pairs of means after the 1-way ANOVA. Values of P <0.05/n were considered to have statistical significance (n means the number of groups brought into Bonferroni test). That is, if 3 groups are included in the statistical analysis, P <0.017 means significant between-group differences in the comparison. All statistical testing was performed with SPSS software (version 20.0; IBM, Armonk, NY).

Results

Table II gives descriptive statistics for degree of rotation (T1−T0) for groups C through F, degree of relapse during retention (T1−T2) for groups D through F, and degree and percentage of relapse (T2−T3), (T2−T3)/(T2−T0)*100%, during relapse for groups E and F. Statistical analysis indicated no significant intergroup difference in the angle of rotation of the experimental molars (T1−T0) for groups C through F and relapse during retention (T1−T2) for groups D through F. But there were significant differences in the degrees (T2−T3) and percentages of relapse (T2−T3)/(T2−T0)*100% during relapse between groups E and F. The mean amounts of relapse after removing the retainer were 6.85° ± 1.56° and 1.70° ± 0.78° for groups E and F, respectively. A higher percentage of relapse (51.25%) was observed in group E compared with group F (13.69%).

Table II
Descriptive statistics and results of statistical analysis for comparison of amount and percentage of relapse in the study groups
Group n T1−T0 T1−T2 T2−T3 (T2−T3)/(T2−T0)*100%
Mean ± SD (°) Pairwise comparisons Mean ± SD (°) Pairwise comparisons Mean ± SD (°) Pairwise comparisons Mean ± SD (%) Pairwise comparisons
C 18 13.71 ± 2.20 a
D 18 14.88 ± 2.51 a 0.72 ± 0.74 b
E 17 15.48 ± 3.60 a 1.32 ± 1.57 b 6.85 ± 1.56 c 51.25 ± 16.12 e
F 22 14.39 ± 2.74 a 1.06 ± 1.48 b 1.70 ± 0.78 d 13.69 ± 6.18 f
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Dec 10, 2018 | Posted by in Orthodontics | Comments Off on Expression of proteins of elastic fibers and collagen type I in orthodontically rotated teeth in rats
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