The mesio-angulated impaction of mandibular third molars makes them unsuitable as donor teeth for tooth autotransplantation. However, uprighted molars may be applicable for autotransplantation. This study aimed to determine the amount of periodontal ligament (PDL) on the root surfaces of extracted third molars after an application of uprighting force and to examine the amount of PDL at the tension and compression sites.
In this prospective cohort study, 15 mesio-angulated mandibular third molars (iM8s) from 15 patients planned for orthodontic extraction, were uprighted using springs connected to miniscrews, whereas 15 nonopposing and fully erupted mandibular third molars from 15 other patients served as controls. The altered angulation was monitored and assessed from panoramic radiographs. All 30 molars, removed by simple extraction, were stained with 0.04% (w/v) toluidine blue to analyze the percentages of stained PDL on the root surfaces.
An average period of 3.4 months was necessary to upright the iM8s at a mean rate of 8.3° per month. The mean percentage of stained PDL on the loaded iM8s was significantly greater than that on the unloaded molars ( P <0.05). The mean percentages of stained PDL were significantly increased at the cervical and middle thirds and the buccal, mesial, and distal surfaces of the loaded iM8s compared with those of the unloaded molars ( P <0.05), whereas the apical third and the lingual surface, corresponding to the compression sites, showed no significant increases.
Orthodontic uprighting leads to significantly increased proliferative PDL on certain radicular portions and surfaces of iM8s, which might be useful for tooth autotransplantation.
The average period of uprighting mesio-angulated mandibular third molars was 3.4 months.
The mean rate of reduced angulation was 8.3° per month.
The reduced angulation was correlated with an increased period of uprighting.
The mean percentage of stained periodontal ligament on uprighted molars was significantly increased.
Significant periodontal ligament enhancement was observed at the tension sites.
Maintaining cell viability within human periodontal ligaments (PDLs) and good tissue adaptation of PDLs are among the most important considerations for successful tooth autotransplantation. However, PDLs can be damaged mechanically during extraction. Complete PDL healing after tooth replantation can be expected if damage to the PDL of the donor tooth during extraction is reduced or limited, whereas the development of root resorption is regarded as the beginning of failure, resulting from trauma to the PDL at the time of replantation. PDL cells possess an ability to produce and secrete a wide range of regulatory molecules, which are crucial components of PDL tissue remodeling and homeostasis. The multilineage differentiation potential of PDL stem cells essentially partakes in PDL homeostasis by giving rise to various types of progenitor cells. Despite cellular, genetic, biochemical, and mechanical factors working together to maintain this homeostasis, much of the research investigating PDL homeostasis has dealt extensively with mechanical stress from applied physical force and its consequent biochemical signaling molecules, leading to changes in the metabolism and organization of the soft connective tissue within PDLs.
Both animal and clinical studies have shown that applying orthodontic force to the donor tooth is beneficial for autotransplantation in terms of facilitating extraction and of stimulating PDL proliferation, consequently resulting in reduced rates of root resorption. An animal study by Suzaki et al reported that preloading of light orthodontic force for 7 days before extraction significantly increased the PDL space and the width of the alveolar socket, as observed histologically, resulting in rich PDL tissues attached to the root surface of the extracted teeth. Moreover, the length of active root resorption lacunae was shorter than that in the contralateral control unloaded teeth, thus suggesting that the increased width of the PDL space and the alveolar socket may prevent crushing of the PDL during extraction. Cho et al applied preloading orthodontic forces to third molars in 2 patients to increase tooth mobility before autotransplantation, using uprighting springs and removable appliances. The teeth were easily extracted and then successfully transplanted. The authors concluded that the preapplication of orthodontic force reduces the risks of PDL damage and, therefore, lessens the risk of ankylosis. In a retrospective study, Choi et al observed the effects of intentional PDL stimulation through the application of orthodontic extrusive forces applied to the donor teeth before autotransplantation. A higher success rate was observed in the teeth with preoperative orthodontic extrusion than in the teeth without orthodontic extrusion. Collectively, these studies support the use of orthodontic force application before transplantation; such force application contributes significantly to successful autotransplantation. However, direct assessment of enhanced PDL on the root surfaces and evaluation of PDL amounts at the tension and compression sites have not yet been performed.
Although mandibular third molars have been reported to be the most frequently used donor teeth for autotransplantation, , , they are often present as impacted teeth, especially at the mesio-angulated position, thus making them unsuitable candidates for autotransplantation. In this study, the orthodontic extraction approach for atraumatic extraction of mesio-angulated impacted mandibular third molars (iM8s) was used to evaluate the effects of controlled orthodontic preloading force on human PDLs. It was hypothesized that the use of springs to upright iM8s, providing controlled orthodontic preloading forces, would make the clinical application of iM8s as suitable donor teeth for tooth autotransplantation possible. Therefore, the purposes of this study were (1) to determine the amount of PDL on the root surfaces of extracted third molars after an application of uprighting force and (2) to examine the amount of PDL at the tension and the compression sites after tooth displacement.
Material and methods
In this prospective cohort study, 30 patients, at the graduate clinic, Department of Orthodontics, Faculty of Dentistry, Bangkokthonburi University, consisting of 22 women and 8 men, who were referred for the removal of their mandibular third molars as part of their orthodontic treatment plan, were recruited from January to September, 2018, during the period of orthodontic treatment plan. The 30 patients were categorized into 2 groups; the experimental group including 15 patients (11 women and 4 men; aged 20-36 years) who had at least 1 iM8 in their mouth and the control group consisting of 15 patients (11 women and 4 men; aged 20-34 years) who had at least 1 nonopposing and fully erupted mandibular third molar. The sample size calculation (n ≥ 10 for each group) was determined by G∗Power software (version 18.104.22.168; Franz Faul, University of Kiel, Kiel, Schleswig-Holstein, Germany) with the effect size (Glass’s Δ) = 1.8 derived from preliminary data, α = 0.05 and 1−β = 0.95. The inclusion criteria were (1) patients with good general health; (2) no radiographic sign of periodontal bone loss as shown by full-mouth periapical radiographs; (3) noncarious mandibular third molars with complete root formation; (4) partial eruption of iM8s with depth A or B according to the Pell and Gregory classification and the Winter classification, respectively, for the experimental group; (5) similar root form and shape between the experimental and the control groups, as evidenced by the full-mouth periapical radiographs; and (6) all 30 selected roots were free from dilacerations or other malformations that might have hindered the extraction. Exclusion criteria were (1) patients with systemic diseases, (2) patients requiring any medications, (3) patients with a smoking habit, (4) presence of periodontal disease or periapical lesions, (5) teeth with extensive caries or restorations, (6) teeth with incomplete root formation, and (7) teeth with abnormal root form and shape. Approval for the use of dental tissues for research activities was received from the human ethics committee of the Bangkokthonburi University (approval number: 11/2561). Written informed consent was obtained from all patients before initiation of the study.
A total of 200 g of force was applied to 15 mandibular third molars in the experimental group that met the inclusion criteria, using a custom-made uprighting device, Smart Spring, that was developed by E.Y.S. and adjusted for each individual patient ( Fig 1 ). The Smart Spring contained a closed stainless-steel coil spring ( Fig 1 , A, asterisk ; Dynaflex, St. Ann, Mo) and 150-g open nickel-titanium coil spring ( Fig 1 , A, arrowhead ; Sentalloy; Tomy Orthodontics, Tokyo, Japan), wrapping around a 0.017 × 0.025-inch stainless-steel rectangular wire ( Fig 1 , A, arrow ; Highland Metals Inc, Franklin, Ind), which was bended to form a hook and a helical loop at the mesial and the distal ends, respectively. The wire connected to a single 1.6 × 8.0-mm miniscrew anchorage (Jeil Medical, Seoul, Korea) using an elastomeric ligature to prevent the wire from being rotated around the buccal miniscrew. The purposes of the hook at the mesial end of the Smart Spring were (1) to allow the application of additional force, if necessary, using an elastomeric chain or ligature tie connecting the hook to the head of miniscrew; (2) to limit the maximal amount of distal movement for the iM8s to prevent their excessive distal movement as a “fail-safe” mechanism, albeit the relatively heavy distalizing force (150 g of force) used in this study; (3) to protect soft tissues in the oral cavity from being injured to maximize comfort for each patient; and (4) to hold the spring. In addition to the adjustable hook, the Smart Spring could be bended and contoured to adapt to the intraoral anatomy of each patient. On the distal end of the wire, the helical loop provided increased flexibility and served as a stopper of the main wire. The helical loop was adjusted to deliver controlled tip back force (50 g of force) to upright the iM8s. The distal portion of the Smart Spring was inserted into a 0.022-inch Roth prescription buccal tube (Tomy Orthodontics; Fig 1 , A ) that was bonded at the buccal surface of each iM8 with Super-Bond C&B cement (Sun Medical, Shiga, Japan). When the Smart Spring is inserted into the buccal tube, a stable connection is obtained, thus avoiding any undesirable rotation of the wire. The orthodontic miniscrew was precisely placed inter-radicularly in the premolar area ( Fig 1 , A ), using a 3-dimensional surgical guide, following the protocol by Suzuki and Suzuki for safe placement. The changes in position and angulation ( light gray in Fig 1 , B vs dark gray in Fig 1 , C ) of the iM8s were assessed using pre- and postoperative panoramic radiographs ( Fig 1 , D-F ). Careful positioning of the patients was verified to reduce positioning errors in panoramic radiography, as previously described. Loading duration was recorded from the beginning of orthodontic force application until each tooth was uprighted to an angulation, at which the iM8s could be removed by simple and atraumatic exodontia, on the basis of clinical judgment of an experienced oral surgeon. Smart Springs were reactivated monthly by adjusting the closed coil spring ( Fig 1 , A, asterisk ) to ensure adequate tooth movement. Passive and activated stages of the Smart Spring device are illustrated in Figure 2 .
The 15 control mandibular third molars received no force application. Before tooth extraction, a total of 1.7 mL for the inferior alveolar nerve, the lingual nerve, and the long buccal nerve blocks using 4% articaine with epinephrine 1:100,000 (Septanest SP; Septodont, Co, Paris, France) was administered by S.L. using a conventional aspirated dental syringe and a disposable 27-gauge and 30-mm-long needle (Terumo, Co, Tokyo, Japan). Both the experimental and control teeth were removed by gentle separation of gingiva using a straight elevator number EL3S (Hu-Friedy Mfg. Co, LLC, Chicago, Ill), followed by the tooth forceps number 151 (Hu-Friedy Presidential; Hu-Friedy Mfg. Co, LLC) to avoid injury to the PDL tissue. Subsequently, the extracted teeth were first checked for the complete integrity of their root without any root tip fracture before being immersed in normal saline to remove blood, fixed, and assessed for remaining PDL. After the extraction of third molars in both the loaded and unloaded groups, all 30 patients showed up for a 1-week follow-up to check their extraction wounds.
The extracted molars, consisting of 15 loaded and 15 unloaded teeth, were used for assessment of remaining PDL on the root surfaces, following the protocol of Nakdilok et al. Briefly, the extracted molars were soaked in 10% neutral buffered formalin solution for 24 hours to fix the periodontal tissue. Then, the teeth were rinsed gently with phosphate buffered saline (PBS; pH 7.2) for 30 seconds to remove residual formalin solution. The remaining PDL on the root surfaces of the extracted teeth was stained using 0.04% (w/v) toluidine blue (Sigma-Aldrich, St. Louis, Mo), freshly prepared for each tooth, for 10 minutes. Then, the teeth were destained in 20 mL of PBS (pH 7.2) with a daily replacement of PBS for 14 days. The buccal, lingual, mesial, and distal radicular surfaces were observed by an Olympus SZX7 stereomicroscope (Olympus America Inc, Center Valley, Pa), with each surface digitally photographed perpendicular to the tooth axis. The stained PDL area against the total area of each radicular surface on each root surface was analyzed using ImageJ2 software (National Institute of Mental Health, Bethesda, Md) with fixed values of hue, saturation, and brightness for every digitized image. The percentage of stained PDL area in each tooth was derived from an average of the percentage of stained PDL area from the 4 radicular surfaces. For further image analysis of the radicular portion, the roots were divided equally, according to its length, into 3 portions, including apical third, middle third, and cervical third ( Fig 3 ). The percentage of stained PDL area in each portion of the root was calculated from an average of the percentage of stained PDL area from the 4 radicular surfaces. The intraexaminer reliability of the measurements was 0.968 as determined by the intraclass correlation coefficient.
Comparisons of mean age and the proportion of the sexes between the experimental and control groups were analyzed using the Independent t test and the chi-square test, respectively. The data of all variables studied, including loading condition, different tooth surfaces, and different tooth portions, were first tested for normality by the Shapiro-Wilk test, and their distributions were found to be normal. The percentages of overall stained PDL tissue in each tooth, of stained PDL tissue on each surface, and of stained PDL tissue on each portion of the root between the experimental and control teeth were analyzed and compared using the Independent t test. The percentages of stained PDL tissue on the 4 surfaces, on the 3 portions, and on the 4 surfaces and the 3 portions were compared using 1-way analysis of variance (ANOVA) followed by multiple comparisons using the Dunnett T3 test because of unequal variances of all tested groups. The Pearson method was used for analyzing a correlation between reduced angulation and increased loading duration. Data were analyzed using SPSS statistical software (version 19.0; SPSS Inc, Chicago, Ill). The results were considered statistically significant if P values were <0.05.
The mean age in the experimental loaded group (23.6 ± 1.1 years; 95% confidence interval, 21.33-25.87; Table ) was not different from that in the control unloaded group (24.5 ± 1.0 years; 95% confidence interval, 22.41-26.66; P = 0.525). The proportion of sex in both groups was also not different (11 women vs 4 men; P = 1.0).