Tooth and periodontal regeneration is one of the most exciting research fields in dentistry (Nakahara, 2006, 2008, 2011; Nakahara and Ide, 2007). In recent years, miniature pigs have often been used as laboratory animal models for medical research (Van Dorp et al., 1998; Polejaeva et al., 2000; Schwartz and Kagan, 2002; Xu et al., 2003; Watanabe et al., 2004; Svendsen, 2006) because they are smaller than pigs (livestock) and because the morphology and size of their organs are similar to those of humans (Bustad, 1966). Miniature pigs are also frequently used in the dental field for basic research. Miniature pigs are frequently used for tooth and periodontal regeneration research (Sonoyama et al., 2006; Ibi et al. 2007; Liu et al., 2008; Abukawa et al., 2009; Ando et al., 2009; Honda et al. 2009; Zhang et al., 2009; Zheng et al., 2009; Ding et al., 2010), because they simulate application to humans. However, despite the fact that miniature pigs are valuable laboratory animals, there is little information on miniature pig teeth. Although there have been some reports on the number of teeth and tooth eruption stages (Weaver et al., 1966, 1969; Schumacher et al., 1989; Li, 1993; Oltramari et al., 2007; Wang et al., 2007), the conclusions drawn are quite different among them; e.g., the definitions of deciduous and permanent teeth and the number of teeth (dentition) differ. The controversial results were based on observations of the directly visible tooth crown. The aspects of tooth roots and tooth germs before eruption have been ignored because it is difficult to observe the tissues in which they are planted in the jawbone. Although some studies reported simple two-dimensional (2D) X-ray analysis of the jawbones, neither the number of tooth roots nor the morphology of the dentition has been examined. Therefore, precise information such as the number and morphology of teeth and the tooth eruption formula should be redefined. The purpose of this study was to examine the time-course changes of the dentition and 3D tooth structure (especially the root) of the miniature pig mandibular cheek teeth during postnatal tooth development, i.e., from the neonatal stage of the deciduous dentition to the completion stage of the permanent dentition (2 weeks to 29 months of age), through X-ray analyses using soft X-ray and micro-CT. This is the first study to use micro-CT for this purpose. This study offers valuable data for dental research using miniature pigs.
The miniature pig is a useful large laboratory animal model. Various tissues and organs of miniature pigs are similar to those of humans in terms of developmental, anatomical, immunological, and physiological characteristics. The oral and maxillofacial region of miniature pigs is often used in preclinical studies of regenerative dentistry. However, there is limited information on the dentition and tooth structure of miniature pigs. The purpose of this study was to examine the time-course changes of dentition and tooth structure (especially the root) of the miniature pig mandibular cheek teeth through X-ray analyses using soft X-ray for two-dimensional observations and micro-CT for three-dimensional observations. The mandibles of male Clawn strain miniature pigs (2 weeks and 3, 5, 7, 9, 11, 14, 17, and 29 months of age) were used. X-ray analysis of the dentition of miniature pig cheek teeth showed that the eruption pattern of the miniature pig is diphyodont and that the replacement pattern is vertical. Previous definitions of deciduous and permanent teeth often varied and there has been no consensus on the number of teeth (dentition); however, we found that three molars are present in the deciduous dentition and that four premolars and three molars are present in the permanent dentition. Furthermore, we confirmed the number of tooth roots and root canals. We believe that these findings will be highly useful in future studies using miniature pig teeth. Anat Rec, 2013. © 2013 Wiley Periodicals, Inc.
MATERIALS AND METHODS
Clawn strain miniature pigs (Fig. 1) were originally established in the 1980s and bred at the Japan Farm CLAWN Institute (Kagoshima, Japan) (Nakanishi et al., 1991; Watanabe et al., 2004). Briefly, this pig was developed by crossbreeding two types of F1: the F1 crossbreed of the Göttingen strain miniature pig and the Ohmini strain miniature pig, and the F1 crossbreed of the Landrace strain pig and the Great Yorkshire strain pig (Fig. 2). In general, male Clawn strain miniature pigs weigh 0.6 kg at birth, 11 kg at 3 months, 21 kg at 6 months, and 40 kg at 12 months, and become capable of breeding at ∼6 months.
Male Clawn strain miniature pig mandibles in 11 stages (2 days, 2 and 3 weeks, and 3, 5, 7, 9, 11, 14, 17, and 29 months of age) were supplied by the Japan Farm CLAWN Institute (Kagoshima, Japan). The mandibles in nine stages (2 weeks, and 3, 5, 7, 9, 11, 14, 17, and 29 months of age) were used for X-ray analysis (Soft X-ray and Micro-CT) representing each typical stage. On the other hand, the mandibles of four stages (2 days, 3 weeks, and 3 and 5 months of age) were used for histological analysis. One sample was prepared for each stage (data not shown).
Soft X-Ray Analysis
To observe the 2D time-course changes of dentition of the mandibular cheek teeth of the miniature pig, all samples fixed in 10% formalin neutral buffered solution (Wako Pure Chemical Industries, Osaka, Japan) were radiographed using a soft X-ray device (M-100W; SOFTEX, Tokyo, Japan). The images were obtained from all samples using a tube voltage of 100 kV, tube current of 5 mA, and exposure time of 1 min. High Precision Photo Plate (PXSO, Konica Minolta, Tokyo, Japan) was used as the film.
To observe the three-dimensional (3D) sequential changes of the dentition and morphology of the mandibular cheek teeth of the miniature pig, a micro-CT device (HMX-225 Actis 4; TESCO, Yokohama, Japan) was used. Slice images were obtained from each sample using a tube voltage of 100 kV, tube current of 70 µA, and slice thickness of 50–140 µm. The sample was placed at the center of the material table so that the jawbone including the teeth was contained within the imaging range. Computerized 3D reconstruction was performed using 3D analytic software (TRI/3D-BON; Ratoc System Engineering, Tokyo, Japan). The teeth were extracted and spuriously stained upon the slice image. Thereafter, 3D images of the teeth were reconstructed.
The mandibles (2 days, 3 weeks, and 3 and 5 months) were fixed in 10% formalin neutral buffered solution. They were then decalcified with formic acid-sodium citrate reagent (Morse, 1945) by moderate stirring at room temperature for at least 1 month depending on the developmental stage of each sample, until the radiopacity of the samples could not be detected using a digital X-ray system (Compuray, Yoshida, Tokyo, Japan). Finally, they were embedded in paraffin. The serial frontal sections of mandibles were cut at 5 µm thickness and stained with hematoxylin and eosin (HE).
Results of X-ray analysis of the dentition of miniature pig cheek teeth are shown in Fig. 3. For detailed analysis of tooth development, we observed samples using micro-CT (Fig. 4). The dentition of miniature pigs was diphyodont. The deciduous teeth Dp2, Dp3, and Dp4 were shed and replaced by the successional teeth P2, P3, and P4, while P1 was an additional tooth that was not replaced. The molars (M1, M2, and M3) were additional teeth located in the posterior region of the deciduous teeth. To clarify whether Dp1 exists in the anterior edentulous region of Dp2 (i.e., the diastema), histological examination was performed with HE staining. No tooth structure was detected from 2 days to 5 months of age (data not shown). Based on these results, we found that P1 is an additional (nonshedding) tooth and that the putative Dp1 appears to be missing. In summary, three molars (Dp2, Dp3, and Dp4) exist in the deciduous dentition. Four premolars (P1, P2, P3, and P4) and three molars (M1, M2, and M3) are present in the permanent dentition.
Calcification was observed in Dp2, Dp3, Dp4, and M1 by 2 weeks (Figs. 3 and 4). At 5 months, the deciduous dentition was complete, and calcified P1 and M2 were observed. At 7 months, with eruption of M1 it became a mixed dentition. Moreover, the roots of Dp2, Dp3, and Dp4 had completely developed and P3 and P4 could be observed under Dp3 and Dp4, respectively. At 9 months, eruption of P1 and calcification of P2 and M3 were observed. Dp3 and Dp4 were shed between the age of 11 and 14 months. The approximate times of the beginning of calcification of miniature pig teeth, completion of teeth, and replacement in the dentition are described in Fig. 5.
The tooth roots of the miniature pig cheek teeth were examined in 3D using micro-CT images (Fig. 6). The number of roots of each tooth was as follows: Dp2 = 2, Dp3 = 2, Dp4 = 5 (deciduous molar); P1 = 1, P2 = 2, P3 = 2, P4 = 2 (premolar); M1 = 4, M2 = 4, M3 = 5 (molar) (Fig. 6a). In Dp2, Dp3, P2, P3, and P4, the two roots were situated on the mesial and distal. The four roots in M1 and M2 were mesiobuccal, mesiolingual, distobuccal, and distolingual and the five roots were situated on the mesiobuccal, mesiolingual, middlebuccal, distobuccal, and distolingual sides in Dp4 or on the mesiobuccal, mesiolingual, distobuccal, distolingual, and distal sides in M3. It is difficult to observe the 3D morphology of the pulp cavity because it is hollow. Therefore, a 3D reconstruction of the pulp cavity was made by staining the pulp cavity through computer processing (Fig. 6b). The form of the pulp cavity resembled the shape of the tooth. The number of root canals was as follows: Dp2 = 2, Dp3 = 2, Dp4 = 5; P1 = 1, P2 = 2, P3 = 2, P4 = 3; M1 = 4, M2 = 4, M3 = 5. These results are summarized in Table 1.
|Number of roots||2||2||5||1||2||2||2||4||4||5|
|Number of root canals||2||2||5||1||2||2||3||4||4||5|
Pigs are regarded as a useful model for biomedical research, especially in regenerative medicine, because of their anatomic, physiologic, and immunologic similarities to human. In particular, miniature pigs are used extensively for xenotransplantation because of their smaller body size and well-defined genetic background as compared to pigs (livestock) (Ando et al., 2005; Tudor et al., 2010). Miniature pigs are used in the field of regenerative dentistry. Some studies have demonstrated that miniature pigs serve as an excellent translational model for autologous stem cells in transplantation (Sonoyama et al., 2006; Oltramari et al., 2007). In addition, miniature pigs are frequently used for tooth and periodontal regeneration research (Ibi et al., 2007; Liu et al., 2008; Abukawa et al., 2009; Ando et al., 2009; Honda et al., 2009; Zhang et al., 2009; Zheng et al., 2009; Ding et al., 2010). However, despite the fact that miniature pigs are useful laboratory animals, there is little information on miniature pig teeth. It is important to collect basic dental data. In this study, the time-course changes in the dentition and the morphology of the roots of the mandibular cheek teeth of miniature pigs were investigated through 3D observation by X-ray analysis. We examined one sample for every stage; it is difficult to examine many samples in each stage, because miniature pigs are expensive and large in size. As pre-examination, we had already investigated many other stages (from birth to the time permanent dentition is complete) using a soft X-ray prior to conducting this research. Therefore, we believe that the data shown here have credibility without inconsistency.
We found that the dentition of the miniature pig is diphyodontic and that there were three molars in the deciduous dentition as well as four premolars and three molars in the permanent dentition. In previous reports, the number and denomination of the deciduous molars, premolars, and molars have been controversial, which has caused confusion in conducting research using miniature pig teeth (Wang et al., 2007). One possible reason for these discrepancies is the existence of the first premolar (P1), located posterior to the canine (Figs. 3 and 4). Weaver et al. (1966, 1969) and Li (1993) defined the tooth located posterior to the canine as Dp1. On the other hand, McKean et al. (1971), Schumacher et al. (1989) and Oltramari (2007) defined the tooth as P1. These observations led to confusion in the tooth number. In this study, we found that P1 is not replaced by a permanent tooth during postnatal tooth development, while other premolars change from Dp2, Dp3, and Dp4 to P2, P3, and P4, respectively. Calcification begins and P1 erupts much earlier than P2, P3, and P4, but closer to the respective time points in Dp2, Dp3, and Dp4, and this fact seems to create confusion among many researchers; i.e., P1 has been defined as Dp1. Unlike deciduous teeth, a permanent tooth is not normally shed because of the presence of an additional tooth that may grow out later. We examined whether any additional tooth existed in the jawbone in the region of P1 by X-ray analysis, but none was found. Therefore, we defined this tooth as a permanent tooth that is not replaced and redefined it as P1. The observations we have defined match those in the reports on pigs by Tonge and McCance (1973) and Ikeda (1979), and the reports on wild boars by Imoto (1977) and Hayashi et al. (1977). Our definition is in agreement with the reports on miniature pigs by McKean et al. (1971), Schumacher et al. (1989) and Oltramari (2007).
Miniature pigs are developed by breeding pigs to downsize for laboratory use. Like wild boars, they belong to the wild boar family of the order Artiodactyla, and their cheek teeth are bunodont. Teeth of miniature pigs are thought to have characteristics similar to those of pigs and wild boars. According to Ikeda's (1979) report on pigs and Imoto's (1977) report on wild boars, the mandibular P1 located in the diastema, which is an edentulous region between the canine tooth and P2, has an irregular morphology and is sometimes congenitally missing. In other words, they suggest that P1 in the diastema shows a tendency toward degeneration. To clarify whether the Dp1 exists before the eruption of P1, we carried out a histological examination through HE staining to find the putative Dp1 tooth germ in the diastema region during the period from just after birth (2 days) to the initiation of P1 eruption (5 months). However, no sign of Dp1 was detected (data not shown). These results are consistent with the above-mentioned findings based on the X-ray analyses. Putative Dp1, which appeared to be missing, was not observed in the diastema, while a permanent tooth (i.e., P1), which is not replaced, existed.
In this study, a micro-CT device was used to examine the morphology of the mandibular cheek teeth in miniature pigs. Such an analysis has never been reported previously. Previous reports on the morphology of miniature pig teeth merely concern the tooth crown, and there have been no reports regarding the root. This is because the root area is surrounded by bone and cannot be observed. Although 2D observation using X-rays was previously reported (Weaver et al., 1966; Schumacher et al., 1989; Oltramari et al., 2007), the number of roots could not be accurately determined. In the present study, using a micro-CT device, the tooth was extracted from the jawbone nondestructively on the computer and the root area was observed in 3D. To observe the roots of the deciduous teeth, an image of the teeth of a 7-month-old miniature pig was used, and to observe the roots of the permanent teeth, an image of the teeth of a 29-month-old miniature pig was used. Furthermore, the morphology of the pulp cavity was examined through computerized 3D reconstruction by staining the extracted pulp cavity spuriously (the M3 image of the 29-month-old miniature pig was not reconstructed because its root formation was not complete). The form of the pulp cavity resembled the shape of the tooth but was reduced in size, as in human teeth. The number of roots and the number of root canals were the same for each tooth with the exception of P4. Miniature pigs are useful for research as they are large animals with characteristics very similar to those of humans. In this study, we presented the changes in dentition and the morphology of roots using nine samples from miniature pigs aged 2 weeks to 29 months. The results of our study will be highly useful in further research using miniature pig teeth.
The authors thank Dr. Hiroshi Ishikawa of the Department of NDU Life Sciences, School of Life Dentistry at Tokyo, The Nippon Dental University for providing scientific advice and support for this study. They also thank Naho Fushimi for excellent technical assistance. Finally, they thank Drs. Takashi Ohashi, Ken Nakahara, Hideaki Kinoshita and Kosaku Sawada of the Department of Anatomy, Tokyo Dental College for their support and for the use of the micro-CT device.