Correspondence to: Masahiro Tsuchiya, DDS, PhD, Division of Aging and Geriatric Dentistry, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980–8575, Japan. Fax: +81-22-717–8396. E-mail: email@example.com
Physiological migration of human teeth with aging frequently results in esthetic and functional problems (Richardson, 1995; Brunsvold, 2005). Although some investigators have hypothesized an important role of high occlusal forces in accelerating teeth migration (Wei et al., 2008; Holla et al., 2012), there is little distinct evidence that occlusal force within the physiological range alters teeth migration with age. Rat molars also physiologically migrate in the distal direction with aging, which results in several histological characteristics in periodontal tissues similar to those observed in humans (Sicher and Weinmann, 1944; Cleall et al., 1968; Roberts and Morey, 1985). Therefore, investigation of the physiological and pathological migration of human teeth could be achieved by examining rats due to their common histological features, especially in cementum (Kagayama et al., 1994, 1996; Kagayama and Sasano, 2000; Tsuchiya et al., 2008).
Cementum is a mineralized tissue covering the root dentin surface and serves as the attachment sites of the periodontal ligament (Kagayama and Sasano, 2000; Saygin et al., 2000; Grzesik and Narayanan, 2002). Cementum formed by cementoblasts is distinguished into acellular or cellular cementum based on whether cementocytes are absent or present in their tissues. Acellular cementum is formed on a coronal half of roots whereas cellular cementum is persistently formed on the apical half with aging. Recent studies indicate a constructive adaptation in cementum against the functional load as observed in the periodontal ligament (Niver et al., 2011; Leong et al., 2012). Interestingly, acellular cementum on the distal side of rat molar roots is hypomineralized (Kagayama et al., 1994, 1996). Considering the localization of more osteoclasts in the distal periodontium and a lower bone formation in the distal side of alveolar bone facing molar roots, the distal periodontium could suffer more compressive stress due to the physiological distal drift of rat molars (Sicher and Weinmann, 1944; Roberts and Morey, 1985; Kagayama et al., 1994). These histological features indicate that cementogenesis is possibly suppressed by compressive stress in vivo. Although many in vitro studies have recently shown that cementoblasts are responsive to mechanical forces (Bosshardt, 2005; Huang et al., 2009; Yu et al., 2009; Rego et al., 2011; Tian et al., 2011; Diercke et al., 2012), this has yet to be well established in vivo. Indeed, it has not been determined whether small mechanical stresses caused by molar drift could modify cementum formation. Investigation of the physiological distal drift in rat molars could help elucidate the relationship between cementogenesis and physiological mechanical stress.
Occlusal force may be the most influential mechanical factor that leads to the physiological distal drift in rat molars because of the simultaneous appearance of physiological distal drift in rat molars with occlusal contact (Sicher and Weinmann, 1944; Roberts and Morey, 1985). The same may also occur in the human. The aims of this study were to morphologically evaluate whether (1) extraction of the upper first molar (M1) slows the physiological distal drift in rat molars and (2) whether the slowing of this physiological drift results in the modification of cementogenesis on the roots of the molars.
MATERIALS AND METHODS
Animals and Tissue Preparation
This study was approved by the Tohoku University Animal Use and Care Committee. All experimental protocols followed the guidelines for animal use and care issued by the Tohoku University Animal Use and Care Committee. Male Wistar rats at 2 weeks of age were used in this study and were given an intraperitoneal injection (i.p.) of tetracycline at 1 day before the operation. The upper right first molar (M1) in the experimental group was extracted under sodium pentobarbital (i.p., 0.4 mg/kg) anesthesia and supplemented with ether inhalation. All rats had free access to food and water throughout the study period. Body weights showed no significant difference between the control and the experimental groups during the study. At the end of the study, the rats will killed sacrificed with the overdose of sodium pentobarbital (60 mg/kg; i.p. injection). The heads of rats were dissected out and immersed in 4% paraformaldehyde in a 0.1 M phosphate buffer, pH 7.4, overnight at 4°C for tissue fixation. After taking X-ray radiographs, the samples were divided into three groups: extracted side group (right maxillae from rats with extracted molar, shown as Ext-side), intact side group (left maxillae from rats with extracted molar, shown as Intact-side) and control group (shown as Control).
Radiographic and Craniometric Analysis Using soft X-Ray
Craniums at 2, 4, 8, 12, 16, and 32 weeks of age were radiographed by means of a microradiography unit (Softex CMR Unit; Softex, Tokyo, Japan) with X-ray film (FR; Fuji Photo Film, Tokyo, Japan) under standardized conditions (25 kV, 3.6 mA, 1 min). In order to examine the migration of rat molars and jaw deformities, the distance between reference points on rat maxillae or mandibles were measured. The measurement points are shown in Fig. 1C,D, and definitions are as previously described (Fukazawa and Sakamoto, 1982; Furuta et al., 1999; Morita, 2001; Maki et al., 2002). Measurements were performed three times for each sample to obtain mean values.
The measurement points:
Na: the most anterior point of the nasal bone,
Oc: the most posterior point of occipital bone,
rZy, lZy: the most exterior point of the zygomatic arch (right and left),
Ss: the closest point on sagittal suture from r(l)Zy,
Id: Infradentale (lingual side) of upper incisor
M2d: the highest point of the mesial alveolar bone at the second molar (M2)
Cd: the most posterior point of condylar process,
Cd': the perpendicular point on the mandibular plane from Cd,
Me: the lowest anterior point on the mandibular plane,
Go: the most posterior point of angular process,
OP: the line on occlusal surfaces of lower dentition,
MP: the line of the mandibular plane
Na–Oc: the length of the cranium,
r(l)Zy–Ss: the width of the unicranium (right or left),
Id-M2d: the antero-posterior position of M2,
Cd-Cd': the height of mandibular ramus,
Go-Me: the length of mandibular body,
Molar length: the distance from the mesial cusp on OP to the apex of the mesial root (red in Fig. 1D),
Root length: the distance from the mesial cervix to the apex of the mesial root (blue in Fig. 1D),
Crown length: the difference between the molar and root lengths,
Note that the asymmetry of Id-M2d is given as the absolute difference of Id-M2d between right and left sides (Fig. 2B), and that the width of molar roots is given as the most mesial-distal length of total root region of M2 or of the third molar (M3) on sagittal radiographs of rat maxillae (Fig. 4B).
Demineralized Specimens and Histological Measurements
Five specimens at 8 weeks of age were decalcified in Morse's Solution (10% sodium citrate and 22.5% formic acid) for 2 weeks at 4°C. After dehydration through a graded series of ethanol solutions, specimens were embedded in paraffin. Serial horizontal sections, 5-µm thick, were cut, deparaffinized and stained with hematoxylin-eosin. To determine whether physiological distal drift of molars influences cementum formation, the thickness of acellular cementum on half of the distal roots (at about 1 mm from the highest point of alveolar bone) of upper M2 were measured on at least 20 sections from each sample (Groeneveld et al., 1995; Huang et al., 2005). Cementum thickness was given by the mean distance from three measurements between the most outer cementum surface and the most inner cementum surface on M2 distal root. Measurements were performed by two investigators and mean values were obtained.
Vital staining and undemineralized specimens
For vital staining with fluorescent dyes, rats were injected intraperitoneally with alizarin red S (30 mg/kg, Wako Chemicals, Osaka, Japan) at 6 weeks after molar extraction (8 weeks of age) (Akiba et al., 2006). After tissue preparation, specimens were dehydrated through a graded series of ethanol solutions, cleared in xylene, and then embedded in resin (Osteoresin embedment kit: Wako Pure Chemical Industries, Japan). The specimens were cut horizontally at a thickness of 200 μm using a saw microtome (BS-300CL: Exakt, Norderstedt, Germany). The surface of each section was further ground and polished using a micromilling system (MG-4000: Exakt) to a thickness of 30 μm and observed by fluorescence microscopy.
Data are shown as means ± standard error (S.E.). Statistical analysis was performed by Student's unpaired t-test to assess differences between two experimental groups or by one-way analysis of variance (ANOVA) followed by the Bonferroni procedure for multiple comparisons using SPSS v.14 software (Chicago, IL). With respect to results involving two factors, molar extraction and time, two-way ANOVA was performed for time-by-operation analysis to confirm their interactions before post hoc test. Appearance of the loss of proximal contact between M2 and M3 was compared using the chi-squared test with the significance level at P < 0.01 (Table 1).
Oral features and cranium morphology after M1 extraction
At 16 weeks of age, the position of M2 in Ext-side was more anterior than that in Intact-side (as shown in Fig. 1A). Interestingly, the loss of proximal contact between M2 and M3 was observed only in Ext-side, but not in Intact-side (in enlarged view of Fig. 1B). To evaluate possible cranium deformation that could affect M2 position, we performed cephalometric analysis with soft X-ray radiographs of rat craniums and mandibles (Fukazawa and Sakamoto, 1982; Morita, 2001; Maki et al., 2002). M1 extraction had no significant effects on the growth of rat cranium including the mandible (Table 2). The loss of proximal contact between M2 and M3 was significant only in Ext-side (four per six rats at 16 weeks of age), but not in Intact-side or in Control (Table 1). The distance between M2 and M3 in Ext-side was also significantly wider than those in other groups. As this phenomenon was observed in one of six rats at 8 weeks of age and in two of six rats at 12 weeks of age, the distance tends to increase with age after M1 extraction.
Table 1. Loss of proximal contact between M2 and M3 in maxilla
Number of rats (16 weeks of age) was 6.
The value of distance, the length between the closest points on proximal surface of each molar, represented the mean ± S.E.
Significant difference among three groups with chi-squared test (P < 0.01).
Significant difference (P <0.01) to others with Bonferroni post hoc test.
Each value represented the mean ± S.E. from six rats (16 weeks of age). NS: not significant between groups.
Change in the anterior-posterior position of M2 in maxilla after M1 extraction
The anterior-posterior positions of M2 in maxillae were expressed as the lengths of Id-M2d on microradiographs. After 4 weeks of age, the increment of Id-M2d in Ext-side was significantly smaller than those in other groups, and the difference between Id-M2d in Ext-side and in others increased with age (Fig. 2A). There was no significant difference at all points of age between Control and Intact-side. Indeed, asymmetry of Id-M2d in the experimental group deteriorated with age compared with the Control group (Fig. 2B). Taken together, the extraction of upper M1 resulted in the slowing of physiological distal drift in upper M2 without cranium deformation.
Conversely, the anterior-posterior position of lower M1 did not indicate any significant differences among the three groups except for 4 weeks of age (Fig. 3A), while the physiological distal drift was consistent. Interestingly, the molar length of lower M1 in Ext-side was significantly longer than that in Control. Both the crown length and the root length of lower M1 in Ext-side were also significantly longer than those in the other groups (Fig. 3B).
Effect on cementum formation in relation to the change in molar distal drift
In general, cementum thickness on the distal side of upper M2 root was less than that on the mesial side as shown in Intact-side (right panel in Fig. 4A) (Kagayama et al., 1994, 1996). In Ext-side, however, thick cementum was distinctly observed on the distal side (left panel in Fig. 4A). Therefore, to examine whether the cementum formation was affected in relation to the change in physiological drift, microradiographic and histological measurements of the molar root was carried out. At 16 weeks of age, the root width, the most mesial-distal distances of root surface on sagittal microradiographs, in Ext-side was significantly greater than those in the other groups (Fig. 4B). Meanwhile, the upper M3 root showed no significant difference in the root width among three groups, though there was a similar trend.
Histomorphometry of dentin and cementum of the disto-buccal root of upper M2 are summarized in Table 3. First, the thickness of root dentin at the middle of the disto-buccal root in M2 shows no difference among the three groups. Conversely, distal cementum on the disto-buccal root of M2 was significantly increased in Ext-side (Fig. 4C). Furthermore, the ratio of cementum thickness on the mesial side to that on the distal side in Ext-side was the smallest among the three groups (P < 0.001) (Table 3). By contrast, the change in molar distal drift had no effect on the formation of mesial cementum of the same root. Note that these differences among the three groups were consistent in other roots of upper M2, including two mesial roots (arrowheads in Fig. 4A).
Table 3. Cementum thickness on the distal buccal root of M2
Number of rats (16 weeks of age) was 6.
The value represented the mean ± S.E.
Dentin thickness was indicated with the length of the major axis of root dentin. -Cementum thickness was indicated with the closest length from the surface of cementum to root dentin on each direction.
Histological distribution of the vital staining in molar root
Representative images taken from undemineralized horizontal sections at the middle of M2 root at 8 weeks of age are shown in Fig. 5. In Control (Fig. 5A, right panel) and Intact-side (Fig. 5C, right panel), there was an accumulation of alizarin red on the mesial side of alveolar bone around the molar root, indicating that actively forming calcified tissues were typically dominant, but almost negative on the distal side because of the physiological distal drift. However, the fluorescence in distal cementum of the molar root of M2 (arrowheads in Fig. 5) was more distinct in Ext-side (Fig. 5B) than in the other groups (Fig. 5A,C).
This study shows that upper M1 extraction distinctly induces the slowing of physiological distal drift in rat maxillae molars. Considering the growth and development of M1 root that can push M2 and M3 back, the removal of occlusion by grinding molar crowns might be a more suitable method for the purpose than M1 extraction. However, periodic grinding is necessary for the prevention of potential occlusion due to active molar extrusion in rodent and the risk of periodontal infectious disease via exposed pulp is frequent. Therefore, we chose upper M1 extraction that resulted in a persistent loss of occlusal force, followed by the severe asymmetry of rat upper dentitions at 16 weeks of age. We believe that M1 extraction is more effective for suppressing the physiological distal drift and more reliable at verifying the key contribution of M1 to the occlusal development (Leong et al., 2012).
The proximal contact loss following tooth extraction frequently leads to the migration of adjacent teeth as observed here. The previous study suggested that occlusal loads within dentition are balanced by the existence of proximal contact (Oh et al., 2004). Hence, the loss of proximal contacts followed by the change of occlusal load distribution may directly contribute to the slow-down of physiological distal drift (Dorfer et al., 2000). Although lower M1, the opposing molar to the extracted tooth, was extruded with the elongation of root length, the mandibular dentition in the experimental group did not show serious asymmetry as shown in the maxilla. More clarification about the participation of occlusal force in the physiological drift is required. A further experiment with the extraction of lower molar(s) could bring us crucial information in the future.
The slow-down of rat molar drift resulted in the activation of cementum formation only on the distal side of rat molars, but did not significantly change the mesial cementum of the same dental root. These findings support a previous in vitro study that cementoblasts are more sensitive to the compressive force than the tensile stress (Huang et al., 2009). The histological feature of newly formed cementum observed in the current study indicates acellular cementum (Fig. 4A), which is known to be formed during early root formation (Kagayama and Sasano, 2000; Saygin et al., 2000; Grzesik and Narayanan, 2002). Activation of acellular cementum formation is also seen in the regenerative process after dental root resorption or periodontium injury (Kagayama et al., 1996; King and Hughes, 1999). However, the root surface of upper M2 in Ext-side showed less root resorption, a major histological indicator of excessive mechanical stress (Bergamo, 1969; Holla et al., 2012). It is conceivable that the changes in mechanical stress distribution in the periodontium after upper M1 extraction modified cementogenesis during the growth period. Thus, compressive forces that lead to physiological distal drift may suppress cementogenesis in the distal side of rat molars.
In conclusion, our results show that the physiological distal drift in rat molars is driven by occlusal force and leads to an imbalance of the acellular cementum thickness as observed between the mesial and distal sides of the root surface of rat molars. The present results provide further insights into the mechanisms and modifiers of human teeth migration.
The authors thank Dr. Yoshiaki Shimizu (Division of Oral Pathology, Tohoku University Graduate School of Dentistry) and Ms. Miho Oikawa (Graduate School of Biomedical Engineering, Tohoku University) for supporting our research. However, the founding bodies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This study received no additional external funding.