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Microscopic analysis of molar–incisor malformation
Vol. 119 No. 5 May 2015
Microscopic analysis of molareincisor malformation Hyo-Seol Lee, DDS, MSD,a Soo-Hyun Kim, DDS,a Seong-Oh Kim, DDS, PhD,a,b Byung-Jai Choi, DDS, PhD,a,b Sung-Won Cho, DDS, PhD,c Wonse Park, DDS, PhD,d and Je Seon Song, DDS, PhDa,b Objective. Molareincisor malformation (MIM) is a newly discovered type of dental anomaly that involves a characteristic root malformation of the permanent first molars. The aim of this study was to reveal the microstructure of MIM teeth in order to determine their origin. Study Design. Four MIM teeth were extracted from a 9-year-old girl due to severe mobility. The detailed microstructure of the teeth was determined by examinations with micro-computed tomography (micro-CT), hematoxylin and eosin (H&E) staining, immunohistochemical staining, and scanning electron microscopy to reveal the detailed microstructure. Results. Micro-CT and H&E staining revealed the pulpal floor comprising three layers: upper, middle, and lower. Amorphous hard tissues and hyperactive cells were observed in the middle layer of the pulpal floor, and the cells stained positively for dentin sialoprotein and osteocalcin, but not for collagen XII. Conclusion. The results of the present study imply that MIM-affected molars probably result from inappropriate differentiation of the apical pulp and dental follicle. (Oral Surg Oral Med Oral Pathol Oral Radiol 2015;119:544-552)
After crown formation, root development begins through the interaction between the Hertwig root sheath and the dental papilla, which originates from the ectomesenchyme.1 The dental papilla differentiates into odontoblasts and forms dentin and pulp, and the Hertwig root sheath is associated with the number of roots and their morphology.1,2 However, as with the mechanism underlying crown formation, the way in which root formation occurs has yet to be established. Root malformations occur as a result of various genetic and developmental factors (Table I). Hereditary dentin malformations in teeth, including dentin dysplasia (DD) and dentinogenesis imperfecta, are characterized by pulp obliteration and short roots. This type of root malformation is most obvious in DD type 1 (DD I), in which the root is missing or short and the pulp chamber is obliterated before eruption.3 This obliterated pulp chamber exhibits a cascade, or waterfall, form at high magnification due to the hard tissue preventing the formation of normal dentin.4 Apical abscess without dental caries often occurs and is treated with tooth extraction or endodontic therapy.5 In This research was supported by the Basic Science Research Program of the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2011-0022160 and 2012 R1 A1 A2041910). a Department of Pediatric Dentistry, College of Dentistry, Yonsei University, Seoul, Republic of Korea. b Oral Science Research Center, College of Dentistry, Yonsei University, Seoul, Republic of Korea. c Division in Anatomy & Developmental Biology, Department of Oral Biology, College of Dentistry, Yonsei University, Seoul, Republic of Korea. d Department of General Dentistry, College of Dentistry, Yonsei University, Seoul, Republic of Korea. Received for publication Aug 19, 2014; returned for revision Oct 10, 2014; accepted for publication Oct 19, 2014. Ó 2015 Elsevier Inc. All rights reserved. 2212-4403/$ - see front matter http://dx.doi.org/10.1016/j.oooo.2014.10.013
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addition, unlike DD type 2 and dentinogenesis imperfecta, the gene mutation responsible for DD I has not yet been identified.6 Other types of root malformation occur in regional odontodysplasia,7,8 segmental odontomaxillary dysplasia,9 vitamin-D-resistant rickets,10,11 hypoparathyroidism,12,13 and pseudohypoparathyroidism.14 It is also found in Schimke immuno-osseous dysplasia, which is an autosomal recessive disorder that has prominent features of spondyloepiphyseal dysplasia, renal dysfunction, T-cell immunodeficiency, and facial dysmorphism.15 Oral manifestations include peg lateralis, oligodontia, and hypodeveloped molar roots.16 The characteristics and etiology of most root malformations are generally revealed by microstructure studies in case reports.17-22 Two case reports of a new type of root malformation have been published recently. Witt el al.23 reported a distinct form of root malformation associated with a cervical mineralized diaphragm (CMD), and Lee et al.24 named this condition molareincisor malformation (MIM), based on their discovery in 12 patients of a dental anomaly that was characterized by root malformation of the permanent first molars and deciduous second molars and a cervical notch in the permanent maxillary central incisors. The etiology of these symptoms is thought to be systemic diseases contracted at or medications taken at 1 to 2 years of age.23 In a
Statement of Clinical Relevance A molareincisor malformation (MIM) is considered a developmental disorder of teeth, originating from a systemic metabolic disorder contracted at or medication taken at 1 to 2 years after birth. These abnormalities cause clinical problems, such as impaction, space loss, early exfoliation, and spontaneous pain.
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Table I. Comparison of the microscopic characteristics of various root malformations MIM23,24 Etiology Genetic Developmental* Location Generalized Localized Radiographic features Crown Pulp
Root Other characteristics
Histologic features
SEM
DD I3-6
ROD7,8
SOD9
Yes Yes
Yes
Normal Broad and thick furcation, narrow and twisted root canal Aplastic, short, or thin Occasionally periapical abscess
Normal Complete obliteration before eruption, “crescent appearance” Short, blunted Often periapical abscess
Dysplastic root dentin, amorphous hard tissue in furcation area with odontoblastic cell with hyperactivity Crystallized structure, loose dentinal tubule
Globular dysplastic dentin or amorphous atubular dentin mass
“Cascade” or “waterfall” appearance, dysplastic dentin
SIOD15 Yes
Yes
Yes
Yes (several adjacent teeth)
Yes (unilateral of the maxillary teeth)
Thin enamel and dentin Large and diffusely calcified pulp chamber
Thin enamel and dentin Large pulp chamber and root canal
Normal Large pulp chamber
Normal Obstructed
Poorly defined “Ghost teeth” appearance
Short Missing one or both premolars, delayed tooth eruption, irregular bone and fibrous hyperplasia Tubular dentin defects in coronal dentine, pulp fibrosis, lack of an odontoblastic layer
Short, blunted Frequent periapical abscess, rachitic changes at the epiphyses of the radius, ulna, tibia, and fibula
Molar root hypoplasia Microdontia, oligodontia
Yes Yes (mainly permanent first molar)
Vit-D rickets10,11
Yes
Thin surface layer of regular dentin and pulpal obliteration by irregular dentinal mass, wide predentin, denticle Poorly organized enamel and coronal dentin, more normal radicular dentin
Yes (several teeth)
Hypomineralized dentin, increased width to predentin, disorganized odontoblast
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MIM, molar-incisor malformation; DD I, dentin dysplasia type I; ROD, regional odontodysplasia; SOD, segmental odontomaxillary dysplasia; Vit-D rickets, vitamin-D-resistant rickets; SIOD, Schimke immuno-osseous dysplasia; SEM, scanning electron microscopy. *Disorders occurred during the development of the teeth with no known reason.
ORAL AND MAXILLOFACIAL PATHOLOGY 546 Lee et al.
previous study, we observed the root malformation but did not determine its origin. The aim of the present study was to elucidate the origin of the root malformation and the components of the thickened pulpal floor by using several microscopy-based experiments.
MATERIALS AND METHODS Patient information A 6-year-old girl was first brought to the Yonsei University Dental Hospital with the chief complaint of bilateral space loss of the upper deciduous second molars. The patient had a medical history of premature birth at 28 weeks with a low birthweight of 1.1 kg; she was kept in an intensive care unit for 8 weeks following a diagnosis of perinatal asphyxia. She had also suffered further brain injury (right frontal intracerebral hemorrhage) and a zygomaticomaxillary fracture during a car accident at the age of 17 months. She was subsequently diagnosed with left hemiplegia and had been receiving an anticonvulsant agent, sodium valproate (Orfil Syrup, Bukwang Pharmaceutical, Seoul, Korea) for 22 months. Clinical and radiographic examinations revealed that the upper and lower permanent first molars had a normal crown contour and texture, but they exhibited the so-called MIM (Figure 1, A). The upper and lower permanent first molars on both sides were extracted when the patient was 9 years old during orthodontic treatment. Among the extracted teeth, upper and lower permanent first molars on the right side were subjected to microscopic analysis after informed consent had been obtained from the patient and her parents. The study protocol was approved by the institutional review board of Yonsei University Dental Hospital (approval no. 2-2013-0020). Micro-computed tomography (micro-CT) The extracted upper and lower right permanent first molars were fixed in 10% neutral buffered formalin (Sigma-Aldrich, St. Louis, MI) for 1 day, washed with saline, and then scanned using micro-CT (NFR PolarisG90 MVC-Cardiac Gating Model, NanoFocus Ray, Jeonju, Korea). Scanning was performed at a resolution of 35 mm. Digital microradiographic images were acquired (n ¼ 512 images for each molar) at 65 kVp and 115 mA. The area of interest was reconstructed using Invivo Dental software (version 5.3, Anatomage Korea, Seoul, Korea). Scanning electron microscopy (SEM) image analysis After micro-CT analysis, organic material was removed from the extracted upper and lower permanent first molars by soaking them in 5% sodium
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hypochlorite (Duksan Pure Chemicals, Asan, Korea) for 1 hour. After saline irrigation, the lower right permanent first molar was sectioned longitudinally in the distobuccal-to-mesiolingual direction using a diamond bur (Diamond Point FG Hi Tech, Shofu, Kyoto, Japan). Similarly, the upper right permanent first molar was sectioned longitudinally in the midbuccal-to-distopalatal direction. The sectioned half of each tooth was then analyzed. Each obtained sample was placed into a dry oven (TD700, Dosaka EM, Kyoto, Japan) for 24 hours and then sputter-coated with gold (IB-3, Eiko, Tokyo, Japan) at 6 mA for 6 minutes before being examined with SEM (FE SEM S-800, Hitachi, Tokyo, Japan) at an acceleration voltage of 20 kV.
Hematoxylin eosin (H&E) staining After SEM analysis, the remaining sectioned halves of the upper and lower right permanent first molars were decalcified by using 10% ethylene diamine tetra-acetic acid (pH 7.4; Wako Pure Chemical Industries, Osaka, Japan) for 8 weeks and then embedded in paraffin. Serial sections were cut at a thickness of 3 mm, mounted onto glass slides, deparaffinized, and then stained with H&E. The sections were examined by optical microscopy (Bx-40, Olympus, Tokyo, Japan) and stereoscopic microscopy (M165 FC, Leica Microsystems, Wetzlar, Germany).
Immunohistochemical staining The sections were deparaffinized in xylene, rehydrated, rinsed with distilled water, and then subjected to immunohistochemical staining, using antibodies raised against dentin sialoprotein (DSP), collagen XII, and osteocalcin (OC). Antigen retrieval for DSP and collagen XII staining required no additional treatment, but sections were treated with protease K (ready to use; Dako, Carpinteria, CA) for 10 minutes at room temperature to enable OC staining. The sections were immersed in 3% hydrogen peroxide for 10 minutes to inactivate endogenous peroxidase activity and then incubated with one or other of the following primary antibodies overnight: antihuman DSP (1:1500 dilution; rabbit polyclonal antibody, sc-33586; Santa Cruz Biotechnology, Santa Cruz, CA), antihuman collagen XII (1:2000 dilution; rabbit polyclonal antibody, sc68862; Santa Cruz Biotechnology), and antihuman OC (1:5000 dilution; rabbit polyclonal antibody, #AB10911, Millipore, Temecula, CA). The sections were subsequently incubated for 20 minutes with horseradish peroxidaseelabeled polymer conjugated with a secondary rabbit antibody in an EnVisionþsystem kit (Dako). The color was developed using
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ORIGINAL ARTICLE Lee et al. 547
Fig. 1. Radiographic and clinical appearance. A, Panoramic radiograph obtained at the age of 6 years and 8 months. The upper and lower permanent first molars have vestigial and thin roots and exhibit constricted pulp cavities in the crowns (arrows). B, Extracted upper right permanent first molar. C, Extracted lower right permanent first molar. The furcation floor has a convex appearance.
3,3’-diaminobenzidine substrate (Dako), counterstained with Gill’s hematoxylin solution (Merck, Darmstadt, Germany), and then examined using a stereoscopic microscope.
RESULTS Micro-CT Compared with normal teeth (Supplemental Figure 1), the crowns of the upper and lower right permanent MIM teeth had a normal contour and texture. Micro-CT revealed that the morphology and radiopacity of the enamel and dentin of the crown were normal. The pulpal floor comprised three layers: upper layer of the pulpal floor (UPF), middle layer of the pulpal floor (MPF), and lower layer of the pulpal floor (LPF). Although the radiopacities of the UPF and LPF were similar to those of dentin, the MPF exhibited a higher radiopacity and porosity than both the UPF and LPF, but a lower radiopacity compared with the enamel. In the upper right permanent molar, the mesiobuccal and distobuccal roots were partially obliterated (Figure 2, A), and the lingual root of the lower right permanent molar was barely developed. The pulpal floor structures of the three layers were readily observed. Furthermore, accessory canals were visible from the LPF to the MPF (see Figure 2, B). SEM images In the longitudinal section of the extracted teeth, the MPF appeared as a yellowish layer (Figure 3, A and D); columnar crystal-like structures were observed at higher magnification (see Figure 3, B, C, E, and F), and the number of dentinal tubules was markedly reduced compared with normal dentin (Supplemental Figure 2).
Fig. 2. Three-dimensional reconstruction and micro-CT appearance. A, Upper right permanent first molar. The arrow indicates the middle layer of the pulpal floor (MPF); its root canal is partially obliterated by calcified matrix. B, Lower right permanent first molar. The arrow indicates the MPF; its radiopacity is between those of enamel and dentin. L, lingual; B, buccal; P, palatal.
H&E staining H&E staining revealed that the UPF was composed of two parts: The UPF near the pulp had a normal dentin structure, whereas that toward the MPF appeared amorphous (Figure 4, C and G). Hematoxylin is used extensively as a nuclear counterstain and for the staining of specific intracellular and extracellular substances, and many strongly stained osteodentin-like hard tissues and hyperactive cells were observed in the MPF, whereas the LPF contained cementocyte-like cells within the lacunae (see Figure 4, D and H).
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Fig. 3. Scanning electron microscopy images. A, Longitudinal section of the upper right permanent first molar. B, Highermagnification view of A, focusing on the middle layer of the pulpal floor (MPF) and the upper layer of the pulpal floor (UPF). C, Higher-magnification view of B, showing columnar crystal-like structures (closed arrow) and very few dentinal tubules (open arrow). D, Longitudinal section of the lower right permanent first molar. E, Higher-magnification view of D, focusing on the MPF and UPF. Many small nodular structures are evident on both the MPF and UPF. F, Higher-magnification view of E, showing columnar crystal-like tissues (closed arrow) and very few dentinal tubules (open arrow). Scale bars: 600 mm in B and E, and 15 mm in C and F.
Immunohistochemical staining DSP is used to identify the mean active mineralization of dentin and was strongly expressed in the MPF, moderately expressed in the upper part of the UPF and normal dentin, but not expressed at all in the LPF (Figure 5, AeD). Similarly, OC is used to identify osteoblast-like cells and the cellular cementum and its associated cells and was strongly expressed in the LPF, moderately expressed in the MPF, but barely expressed in the UPF and normal dentin (see Figure 5, EeH). Collagen XII is expressed mainly in dense connective tissues of tendons, ligaments, and blood vessel walls, and is the most abundant collagen in periodontal tissues, and it was expressed strongly in the periodontal ligament (PDL; see Figure 5, IeL) but weakly in some areas around the blood vessels in the MPF and LPF.
DISCUSSION Microscopic analysis of two of the four permanent first molars with MIM extracted from a 9-year-old girl revealed that the MIM teeth were characterized by normal crowns, malformed roots, and a thickened pulpal floor. Hypermineralized hard tissues and hyperactive cells, believed to be odontoblast-like cells derived from the apical pulp, were found at the center of the thickened pulpal floor.
The teeth of this patient were diagnosed as having MIM based on the definition of this malformation provided by Lee et al.24 Those authors studied 12 patients with molar root and incisor malformation and defined MIM as a newly discovered type of dental anomaly with root malformations of the permanent first molars and deciduous second molars and crown defects in the incisors, which were associated with neurologic systemic disease at the age of 1 to 2 years after birth.24 In the present study, all of the patient’s permanent first molars exhibited root malformation. It was suspected that the premature exfoliation and space loss of the upper deciduous second molars was caused by MIM of those teeth. The notch at the cervical third on the first incisors seemed to be absent. In addition, the patient had a medical history of premature birth (28 weeks) and low birth weight. Furthermore, at the age of 18 months, she had suffered a car accident that had resulted in brain damage, and she had been on antiepileptic medication since then. The diagnosis of MIM in her teeth was based on all of these factors. The conclusions drawn herein both agree and disagree with the findings of Witt et al.23 First, the findings of both studies indicated that MIM is caused not by genetic factors but by environmental factors and that it is related to past medical history, such as medication and central nervous systemerelated
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ORIGINAL ARTICLE Lee et al. 549
Fig. 4. Histologic findings (H&E staining). A, Histologic findings of the extracted upper right permanent molar, showing partial discontinuation of the root canal at the pulpal floor; amorphous hard tissues can be seen. B, Higher-magnification view of the labeled inset in A, focusing on the normal dentin and pulp, and the upper layer of the pulpal floor (UPF). C, Higher-magnification view of the labeled inset in A, focusing on the UPF and middle layer of the pulpal floor (MPF). D, Higher-magnification view of the labeled inset in A, focusing on the MPF and the lower layer of the pulpal floor (LPF). Instead of root dentin, amorphous hard tissueelike cellular cementum (open arrow) is evident in the LPF. Osteodentin-like hard tissue (closed arrow) and pulp-like hyperactive cells can be seen in the MPF. E, Histologic findings of the extracted lower right permanent molar, showing pulp-like cells and an amorphous tissue layer at the pulpal floor. F, Higher-magnification view of the labeled inset in E, focusing on the normal dentin, and the odontoblast layer and pulp tissue. G, Higher-magnification view of the labeled inset in E, focusing on the UPF. Dentinal tubules were seldom observed. H, Higher-magnification view of the labeled inset in E, focusing on the UPF, MPF, and LPF. Amorphous hard tissueelike cellular cementum (open arrow) can be observed in the LPF. Osteodentin-like hard tissue (closed arrow) and pulp-like hyperactive cells can be seen in the MPF. Scale bars: 0.752 mm in A, 100 mm in BeD and FeH, and 1.03 mm in E. P, pulp; ND, normal dentin.
disease. There is also agreement regarding the symmetric location involving the permanent first molars. In addition, Lee et al.24 analyzed multiple cases and concluded that the lower permanent first molars are always involved and that in 50% of cases, there are associated symptoms in the deciduous second molars and upper central incisors. Both Witt et al.23 and the present study found hypermineralized tissues in a thickened pulpal floor and observed the tissues using SEM and transmission electron microscopy. SEM in the present study revealed unique crystals that are thought to be formed by precipitation in a supersaturated solution, rather than by cell-controlled deposition. Using transmission electron microscopy, Witt et al. concluded that the mineralized globules in CMD consisted of hydroxyapatite. Witt et al.23 named the hypermineralized tissues cervical mineralized diaphragm and noted that it has a density between that of enamel and dentin and that it has the same constituents, including hydroxyapatite.
Those authors asserted that the CMD has a soft-tissue canal that originates from the dental follicle and forms collagen bundle like the PDL. However, in contrast to the study by Witt et al.,23 the present study revealed that the thickened pulpal floor comprises three layersdUPF, MPF, and LPFdand that the MPF comprises the CMD. Moreover, the MPF originates mainly from the apical pulp and partially from the dental follicle, and the MPF and LPF seem to communicate via blood vessels, as evidenced in the present immunohistochemical study. The internal cells of the MPF expressed DSP and OC, and the blood vessels of the MPF and LPF were stained by collagen XII; together, these results suggest that dentinal DSP facilitates initiation of hydroxyapatite formation along or inside the collagen fibril, leading to conversion of predentin to dentin at the mineralization front. The expression of DSP strongly indicates the mean active mineralization of dentin.25 Osteoblasts and osteocytes were immunostained for OC but not for DSP;
ORAL AND MAXILLOFACIAL PATHOLOGY 550 Lee et al.
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Fig. 5. Immunohistochemical staining. A and B, Dentin sialoprotein (DSP) immunostaining in the upper and lower right permanent molars. DSP is strongly expressed in the middle layer of the pulpal floor (MPF) but very weakly expressed in the upper layer of the pulpal floor (UPF) and lower layer of the pulpal floor (LPF). C and D, Higher-magnification views of the labeled insets in B. DSP expression is weaker in the UPF than in normal dentin and is very weak in the LPF. E and F, Osteocalcin (OC) immunostaining in the upper and lower right permanent molars. OC is strongly expressed in the MPF, LPF, and cellular cementum but very weakly expressed in the UPF and normal dentin. G and H, Higher-magnification views of the labeled insets in F. OC is expressed in the MPF, but barely in the UPF. OC is strongly expressed in the LPF and MPF. I and J, Collagen XII immunostaining in the upper and lower right permanent molars. Collagen XII is strongly expressed in the periodontal ligament but is very weakly expressed in dentin, cementum, and pulp tissue. K and L, Higher-magnification views of the labeled insets in J. Collagen XII is not expressed in the UPF and is weakly expressed in the blood vessels of the MPF and LPF. Scale bars: 0.85 mm in A and E, 0.711 mm in B and F, 0.897 mm in I, 0.742 mm in J, and 100 mm in C, D, G, H, K, and L.
accordingly, the synthesis of OC can be regarded as the expression of a cell with an osteoblast-like phenotype. Cellular cementum and its associated cells express OC but not DSP.26 Collagen XII is expressed mainly in the dense connective tissues of tendons, ligaments, dermis, cornea, blood vessel walls, meninges, and developing membranous bones.27 The expression of collagen XII has been compared with that of type I collagen, which is the most abundant collagen in periodontal tissues.28 The presence of two origins was suspected on the basis of the proximity of the apical pulp and dental follicle. It is known that DSP and OC are expressed in
locations of active mineralization in teeth,29 whereas collagen XII is distributed around the PDL.30,31 In our study, DSP was seen to be strongly expressed in odontoblastic layer and OC in the cellular cementum (Supplemental Figure 3). Thus, the internal cell tissues of the MPF appear to comprise mostly odontoblast-like cells that originate from the apical pulp, and the adjacent hard tissues are thought to be dysplastic or amorphous dentin, the so-called osteodentin. The UPF comprises dysplastic and amorphous dentin, and the LPF is considered to be cellular cementum, as reported by Witt et al.23
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It is suspected that the etiology of the MPF is abnormal differentiation in the apical pulp and is caused by external factors, such as neurologic systemic disease or medication, at an age of 1 to 2 years. The apical pulp is composed of loose cellular tissues located under a developing tooth, including mesenchymal stem cells from the apical papillae (SCAP).28 SCAP is known to be associated with root development because they differentiate into odontoblasts.29 The apical pulp is segregated from the dental papilla by an apical cellerich zone and has a location and shape similar to those of the MPF. In addition, the SCAP are activated by external stimulation and rapidly differentiate into replacement or tertiary dentin, forming odontoblast-like cells; this is the hypothetical cause of excessive MPF mineralization.32,33 Further study is needed into whether systemic disease or medication can affect the root development in permanent first molars or deciduous second molars and whether the internal cell tissues in the MPF are identical to the odontoblast-like cells derived from the SCAP. Root malformation might arise from the intervention of epigenetic factorsdsuch as systemic disease or medicationdin the signaling pathways involved in normal root development. Recent animal testing revealed that a specific gene variation in root odontoblasts is able to affect root formation.34 The genes Nfic,35 Msx2,36 Shh,37 Sp6,38 Noggin,39 b-catenin,40 smadd4,41 Ntl,42 and Rankl43,44 are related to the shape, length, and number of roots. Among these, the most relevant to the present study is the Wnt/b-catenin signaling associated with root and cementum formation.45 Depending on the expression rate of the signaling, MIM-like symptoms, such as rootless molars, thin dentin, hyperplastic cementum, and incisor disruption, may occur.42,43 Further studies on signaling pathways and genes are warranted. Clinically, MIM teeth have various characteristics and are only extracted when absolutely necessary. Teeth are exfoliated in cases of apical abscess not related with dental caries in the permanent first molars and of spontaneous mobility during orthodontic treatment, as in the patient in the present study. Witt et al.23 had extracted a tooth with severe caries in dentin. DD I is associated with apical abscess without dental caries. Although the etiology has not yet been identified unequivocally, such abscesses are thought to be caused by pulp necrosis induced by pulp chamber obliteration or by pulp exposure induced by dentinal defects.46 Conventional treatments include exfoliation, follow-up, and apicoectomy, although a recent case report demonstrated successful endodontic treatment with a conventional endodontic procedure.47 Successful orthodontic treatment for DD I has also been reported.48 The malformed roots of MIM teeth render it difficult to
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apply endodontic treatments, but conservative pulp treatment can be applied with detailed and accurate information regarding the shape of the root and crown shape obtained using radiographic tools, such as computed tomography.
CONCLUSION MIM-affected molars are characterized by a normal crown, a malformed root, and a thickened pulpal floor in the permanent first molars. The thickened pulpal floor can be divided into three layers. The most distinctive layer, the MPF, comprises hard tissueelike amorphous dentin and odontoblast-like cells that originate mainly from the apical pulp and partially from the dental follicle, whereas the UPF comprises dysplastic and amorphous dentin derived from the dental papilla, and the LPF is considered to be cellular cementum from the dental follicle. Although the differentiation anomaly of the apical pulp and dental follicle as a result of external stimulation is thought to be the main cause of MIM, this needs to be confirmed in further studies. REFERENCES 1. Bei M. Molecular genetics of tooth development. Curr Opin Genet Dev. 2009;19:504-510. 2. Huang XF, Chai Y. Molecular regulatory mechanism of tooth root development. Int J Oral Sci. 2012;4:177-181. 3. O Carroll MK, Duncan WK, Perkins TM. Dentin dysplasia: review of the literature and a proposed subclassification based on radiographic findings. Oral Surg Oral Med Oral Pathol. 1991;72: 119-125. 4. Sauk JJ Jr, Lyon HW, Trowbridge HO, Witkop CJ Jr. An electron optic analysis and explanation for the etiology of dentinal dysplasia. Oral Surg Oral Med Oral Pathol. 1972;33: 763-771. 5. Tidwell E, Cummingham CJ. Dentinal dysplasia: endodontic treatment, with case report. J Endod. 1979;5:372-376. 6. Kim JW, Simmer JP. Hereditary dentin defects. J Dent Res. 2007;86:392-399. 7. Fanibunda KB, Soames JV. Odontodysplasia, gingival manifestations, and accompanying abnormalities. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1996;81:84-88. 8. Crawford PJ, Aldred MJ. Regional odontodysplasia: a bibliography. J Oral Pathol Med. 1989;18:251-263. 9. Azevedo RS, da Silveira LJ, Moliterno LF, Miranda AM, de Almeida OP, Pires FR. Segmental odontomaxillary dysplasia: report of a case emphasizing histopathological, immunohistochemical and scanning electron microscopic features. J Oral Sci. 2013;55:259-262. 10. Goodman JR, Gelbier MJ, Bennett JH, Winter GB. Dental problems associated with hypophosphataemic vitamin D resistant rickets. Int J Paediatr Dent. 1998;8:19-28. 11. Chaussain-Miller C, Sinding C, Wolikow M, Lasfargues JJ, Godeau G, Garabedian M. Dental abnormalities in patients with familial hypophosphatemic vitamin D-resistant rickets: prevention by early treatment with 1-hydroxyvitamin D. J Pediatr. 2003;142:324-331. 12. Kamarthi N, Venkatraman S, Patil PB. Dental findings in the diagnosis of idiopathic hypoparathyroidism. Ann Saudi Med. 2013;33:411-413.
ORAL AND MAXILLOFACIAL PATHOLOGY 552 Lee et al. 13. Kelly A, Pomarico L, de Souza IP. Cessation of dental development in a child with idiopathic hypoparathyroidism: a 5-year follow-up. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009;107:673-677. 14. Assif D. Dental changes in hypoparathyroidism. Refuat Hapeh Vehashinayim. 1977;26:13-19. 15. Schimke RN, Horton WA, King CR. Chondroitin-6-sulphaturia, defective cellular immunity, and nephrotic syndrome. Lancet. 1971;2:1088-1089. 16. Morimoto M, Kerouredan O, Gendronneau M, et al. Dental abnormalities in Schimke immuno-osseous dysplasia. J Dent Res. 2012;91:29 S-37 S. 17. Morimoto M, Yu Z, Stenzel P, et al. Reduced elastogenesis: A clue to the arteriosclerosis and emphysematous changes in Schimke immuno-osseous dysplasia? Orphanet J Rare Dis. 2012;7:70. 18. Toomarian L, Mashhadiabbas F, Mirkarimi M, Mehrdad L. Dentin dysplasia type I: a case report and review of the literature. J Med Case Rep. 2010;4:1. 19. Gondim JO, Pretel H, Ramalho LT, Santos-Pinto LA, Giro EM. Regional odontodysplasia in early childhood: a clinical and histologic study. J Indian Soc Pedod Prev Dent. 2009;27:175-178. 20. Armstrong C, Napier SS, Boyd RC, Gregg TA. Histopathology of the teeth in segmental odontomaxillary dysplasia: new findings. J Oral Pathol Med. 2004;33:246-248. 21. Murayama T, Iwatsubo R, Akiyama S, Amano A, Morisaki I. Familial hypophosphatemic vitamin D-resistant rickets: dental findings and histologic study of teeth. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2000;90:310-316. 22. Melnick M, Levin LS, Brady J. Dentin dysplasia type I: a scanning electron microscopic analysis of the primary dentition. Oral Surg Oral Med Oral Pathol. 1980;50:335-340. 23. Witt CV, Hirt T, Rutz G, Luder HU. Root malformation associated with a cervical mineralized diaphragmda distinct form of tooth abnormality? Oral Surg Oral Med Oral Pathol Oral Radiol. 2014;117:e311-e319. 24. Lee HS, Kim SH, Kim SO, et al. A new type of dental anomaly: Molar-incisor malformation (MIM). Oral Surg Oral Med Oral Pathol Oral Radiol. 2014;118:101-109.e3. 25. Suzuki S, Sreenath T, Haruyama N, et al. Dentin sialoprotein and dentin phosphoprotein have distinct roles in dentin mineralization. Matrix Biol. 2009;28:221-229. 26. Bronckers AL, Farach-Carson MC, Van Waveren E, Butler WT. Immunolocalization of osteopontin, osteocalcin, and dentin sialoprotein during dental root formation and early cementogenesis in the rat. J Bone Miner Res. 1994;9:833-841. 27. Oh SP, Griffith CM, Hay ED, Olsen BR. Tissue-specific expression of type XII collagen during mouse embryonic development. Dev Dyn. 1993;196:37-46. 28. MacNeil RL, Berry JE, Strayhorn CL, Shigeyama Y, Somerman MJ. Expression of type I and XII collagen during development of the periodontal ligament in the mouse. Arch Oral Biol. 1998;43:779-787. 29. Maciejewska I, Chomik E. Hereditary dentine diseases resulting from mutations in DSPP gene. J Dent. 2012;40:542-548. 30. Bartold PM, Walsh LJ, Narayanan AS. Molecular and cell biology of the gingiva. Periodontol 2000. 2000;24:28-55. 31. Walchli C, Koch M, Chiquet M, Odermatt BF, Trueb B. Tissuespecific expression of the fibril-associated collagens XII and XIV. J Cell Sci. 1994;107:669-681. 32. Tziafas D, Kodonas K. Differentiation potential of dental papilla, dental pulp, and apical papilla progenitor cells. J Endod. 2010;36: 781-789. 33. La Noce M, Paino F, Spina A, et al. Dental pulp stem cells: state of the art and suggestions for a true translation of research into therapy. J Dent. 2014;42:761-768.
OOOO May 2015 34. Bae CH, Kim TH, Chu JY, Cho ES. New population of odontoblasts responsible for tooth root formation. Gene Expr Patterns. 2013;13:197-202. 35. Steele-Perkins G, Butz KG, Lyons GE, et al. Essential role for NFI-C/CTF transcription-replication factor in tooth root development. Mol Cell Biol. 2003;23:1075-1084. 36. Satokata I, Ma L, Ohshima H, et al. Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nat Genet. 2000;24:391-395. 37. Nakatomi M, Morita I, Eto K, Ota MS. Sonic hedgehog signaling is important in tooth root development. J Dent Res. 2006;85:427431. 38. Nakamura T, de Vega S, Fukumoto S, Jimenez L, Unda F, Yamada Y. Transcription factor epiprofin is essential for tooth morphogenesis by regulating epithelial cell fate and tooth number. J Biol Chem. 2008;283:4825-4833. 39. Plikus MV, Zeichner-David M, Mayer JA, et al. Morphoregulation of teeth: modulating the number, size, shape and differentiation by tuning Bmp activity. Evol Dev. 2005;7: 440-457. 40. Bae CH, Lee JY, Kim TH, et al. Excessive Wnt/beta-catenin signaling disturbs tooth-root formation. J Periodontal Res. 2013;48:405-410. 41. Huang H, Constante M, Layoun A, Santos MM. Contribution of STAT3 and SMAD4 pathways to the regulation of hepcidin by opposing stimuli. Blood. 2009;113:3593-3599. 42. Lu X, Rios HF, Jiang B, et al. A new osteopetrosis mutant mouse strain (ntl) with odontoma-like proliferations and lack of tooth roots. Eur J Oral Sci. 2009;117:625-635. 43. Kong YY, Yoshida H, Sarosi I, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature. 1999;397:315-323. 44. Li J, Sarosi I, Yan XQ, et al. RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc Natl Acad Sci U S A. 2000;97:1566-1571. 45. Chen J, Lan Y, Baek JA, Gao Y, Jiang R. Wnt/beta-catenin signaling plays an essential role in activation of odontogenic mesenchyme during early tooth development. Dev Biol. 2009;334:174-185. 46. Van Waes H, Luder HU. [Hypophosphatemic rickets. Hereditary disorder of metabolism and dentin dysplasia]. Schweiz Monatsschr Zahnmed. 2013;123:410-411. 47. Ravanshad S, Khayat A. Endodontic therapy on a dentition exhibiting multiple periapical radiolucencies associated with dentinal dysplasia Type 1. Aust Endod J. 2006;32:40-42. 48. Bespalez-Filho R, Couto Sde A, Souza PH, et al. Orthodontic treatment of a patient with dentin dysplasia type I. Am J Orthod Dentofacial Orthop. 2013;143:421-425. Reprint requests: Je Seon Song, DDS, PhD Department of Pediatric Dentistry Yonsei University College of Dentistry Seoul, Republic of Korea 250 Seongsanno, Seodaemun-gu Seoul 120-752, Korea [email protected]
SUPPLEMENTARY DATA Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.oooo.2014. 10.013
Microscopic analysis of molareincisor malformation Hyo-Seol Lee, DDS, MSD,a Soo-Hyun Kim, DDS,a Seong-Oh Kim, DDS, PhD,a,b Byung-Jai Choi, DDS, PhD,a,b Sung-Won Cho, DDS, PhD,c Wonse Park, DDS, PhD,d and Je Seon Song, DDS, PhDa,b Objective. Molareincisor malformation (MIM) is a newly discovered type of dental anomaly that involves a characteristic root malformation of the permanent first molars. The aim of this study was to reveal the microstructure of MIM teeth in order to determine their origin. Study Design. Four MIM teeth were extracted from a 9-year-old girl due to severe mobility. The detailed microstructure of the teeth was determined by examinations with micro-computed tomography (micro-CT), hematoxylin and eosin (H&E) staining, immunohistochemical staining, and scanning electron microscopy to reveal the detailed microstructure. Results. Micro-CT and H&E staining revealed the pulpal floor comprising three layers: upper, middle, and lower. Amorphous hard tissues and hyperactive cells were observed in the middle layer of the pulpal floor, and the cells stained positively for dentin sialoprotein and osteocalcin, but not for collagen XII. Conclusion. The results of the present study imply that MIM-affected molars probably result from inappropriate differentiation of the apical pulp and dental follicle. (Oral Surg Oral Med Oral Pathol Oral Radiol 2015;119:544-552)
After crown formation, root development begins through the interaction between the Hertwig root sheath and the dental papilla, which originates from the ectomesenchyme.1 The dental papilla differentiates into odontoblasts and forms dentin and pulp, and the Hertwig root sheath is associated with the number of roots and their morphology.1,2 However, as with the mechanism underlying crown formation, the way in which root formation occurs has yet to be established. Root malformations occur as a result of various genetic and developmental factors (Table I). Hereditary dentin malformations in teeth, including dentin dysplasia (DD) and dentinogenesis imperfecta, are characterized by pulp obliteration and short roots. This type of root malformation is most obvious in DD type 1 (DD I), in which the root is missing or short and the pulp chamber is obliterated before eruption.3 This obliterated pulp chamber exhibits a cascade, or waterfall, form at high magnification due to the hard tissue preventing the formation of normal dentin.4 Apical abscess without dental caries often occurs and is treated with tooth extraction or endodontic therapy.5 In This research was supported by the Basic Science Research Program of the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2011-0022160 and 2012 R1 A1 A2041910). a Department of Pediatric Dentistry, College of Dentistry, Yonsei University, Seoul, Republic of Korea. b Oral Science Research Center, College of Dentistry, Yonsei University, Seoul, Republic of Korea. c Division in Anatomy & Developmental Biology, Department of Oral Biology, College of Dentistry, Yonsei University, Seoul, Republic of Korea. d Department of General Dentistry, College of Dentistry, Yonsei University, Seoul, Republic of Korea. Received for publication Aug 19, 2014; returned for revision Oct 10, 2014; accepted for publication Oct 19, 2014. Ó 2015 Elsevier Inc. All rights reserved. 2212-4403/$ - see front matter http://dx.doi.org/10.1016/j.oooo.2014.10.013
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addition, unlike DD type 2 and dentinogenesis imperfecta, the gene mutation responsible for DD I has not yet been identified.6 Other types of root malformation occur in regional odontodysplasia,7,8 segmental odontomaxillary dysplasia,9 vitamin-D-resistant rickets,10,11 hypoparathyroidism,12,13 and pseudohypoparathyroidism.14 It is also found in Schimke immuno-osseous dysplasia, which is an autosomal recessive disorder that has prominent features of spondyloepiphyseal dysplasia, renal dysfunction, T-cell immunodeficiency, and facial dysmorphism.15 Oral manifestations include peg lateralis, oligodontia, and hypodeveloped molar roots.16 The characteristics and etiology of most root malformations are generally revealed by microstructure studies in case reports.17-22 Two case reports of a new type of root malformation have been published recently. Witt el al.23 reported a distinct form of root malformation associated with a cervical mineralized diaphragm (CMD), and Lee et al.24 named this condition molareincisor malformation (MIM), based on their discovery in 12 patients of a dental anomaly that was characterized by root malformation of the permanent first molars and deciduous second molars and a cervical notch in the permanent maxillary central incisors. The etiology of these symptoms is thought to be systemic diseases contracted at or medications taken at 1 to 2 years of age.23 In a
Statement of Clinical Relevance A molareincisor malformation (MIM) is considered a developmental disorder of teeth, originating from a systemic metabolic disorder contracted at or medication taken at 1 to 2 years after birth. These abnormalities cause clinical problems, such as impaction, space loss, early exfoliation, and spontaneous pain.
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Table I. Comparison of the microscopic characteristics of various root malformations MIM23,24 Etiology Genetic Developmental* Location Generalized Localized Radiographic features Crown Pulp
Root Other characteristics
Histologic features
SEM
DD I3-6
ROD7,8
SOD9
Yes Yes
Yes
Normal Broad and thick furcation, narrow and twisted root canal Aplastic, short, or thin Occasionally periapical abscess
Normal Complete obliteration before eruption, “crescent appearance” Short, blunted Often periapical abscess
Dysplastic root dentin, amorphous hard tissue in furcation area with odontoblastic cell with hyperactivity Crystallized structure, loose dentinal tubule
Globular dysplastic dentin or amorphous atubular dentin mass
“Cascade” or “waterfall” appearance, dysplastic dentin
SIOD15 Yes
Yes
Yes
Yes (several adjacent teeth)
Yes (unilateral of the maxillary teeth)
Thin enamel and dentin Large and diffusely calcified pulp chamber
Thin enamel and dentin Large pulp chamber and root canal
Normal Large pulp chamber
Normal Obstructed
Poorly defined “Ghost teeth” appearance
Short Missing one or both premolars, delayed tooth eruption, irregular bone and fibrous hyperplasia Tubular dentin defects in coronal dentine, pulp fibrosis, lack of an odontoblastic layer
Short, blunted Frequent periapical abscess, rachitic changes at the epiphyses of the radius, ulna, tibia, and fibula
Molar root hypoplasia Microdontia, oligodontia
Yes Yes (mainly permanent first molar)
Vit-D rickets10,11
Yes
Thin surface layer of regular dentin and pulpal obliteration by irregular dentinal mass, wide predentin, denticle Poorly organized enamel and coronal dentin, more normal radicular dentin
Yes (several teeth)
Hypomineralized dentin, increased width to predentin, disorganized odontoblast
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MIM, molar-incisor malformation; DD I, dentin dysplasia type I; ROD, regional odontodysplasia; SOD, segmental odontomaxillary dysplasia; Vit-D rickets, vitamin-D-resistant rickets; SIOD, Schimke immuno-osseous dysplasia; SEM, scanning electron microscopy. *Disorders occurred during the development of the teeth with no known reason.
ORAL AND MAXILLOFACIAL PATHOLOGY 546 Lee et al.
previous study, we observed the root malformation but did not determine its origin. The aim of the present study was to elucidate the origin of the root malformation and the components of the thickened pulpal floor by using several microscopy-based experiments.
MATERIALS AND METHODS Patient information A 6-year-old girl was first brought to the Yonsei University Dental Hospital with the chief complaint of bilateral space loss of the upper deciduous second molars. The patient had a medical history of premature birth at 28 weeks with a low birthweight of 1.1 kg; she was kept in an intensive care unit for 8 weeks following a diagnosis of perinatal asphyxia. She had also suffered further brain injury (right frontal intracerebral hemorrhage) and a zygomaticomaxillary fracture during a car accident at the age of 17 months. She was subsequently diagnosed with left hemiplegia and had been receiving an anticonvulsant agent, sodium valproate (Orfil Syrup, Bukwang Pharmaceutical, Seoul, Korea) for 22 months. Clinical and radiographic examinations revealed that the upper and lower permanent first molars had a normal crown contour and texture, but they exhibited the so-called MIM (Figure 1, A). The upper and lower permanent first molars on both sides were extracted when the patient was 9 years old during orthodontic treatment. Among the extracted teeth, upper and lower permanent first molars on the right side were subjected to microscopic analysis after informed consent had been obtained from the patient and her parents. The study protocol was approved by the institutional review board of Yonsei University Dental Hospital (approval no. 2-2013-0020). Micro-computed tomography (micro-CT) The extracted upper and lower right permanent first molars were fixed in 10% neutral buffered formalin (Sigma-Aldrich, St. Louis, MI) for 1 day, washed with saline, and then scanned using micro-CT (NFR PolarisG90 MVC-Cardiac Gating Model, NanoFocus Ray, Jeonju, Korea). Scanning was performed at a resolution of 35 mm. Digital microradiographic images were acquired (n ¼ 512 images for each molar) at 65 kVp and 115 mA. The area of interest was reconstructed using Invivo Dental software (version 5.3, Anatomage Korea, Seoul, Korea). Scanning electron microscopy (SEM) image analysis After micro-CT analysis, organic material was removed from the extracted upper and lower permanent first molars by soaking them in 5% sodium
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hypochlorite (Duksan Pure Chemicals, Asan, Korea) for 1 hour. After saline irrigation, the lower right permanent first molar was sectioned longitudinally in the distobuccal-to-mesiolingual direction using a diamond bur (Diamond Point FG Hi Tech, Shofu, Kyoto, Japan). Similarly, the upper right permanent first molar was sectioned longitudinally in the midbuccal-to-distopalatal direction. The sectioned half of each tooth was then analyzed. Each obtained sample was placed into a dry oven (TD700, Dosaka EM, Kyoto, Japan) for 24 hours and then sputter-coated with gold (IB-3, Eiko, Tokyo, Japan) at 6 mA for 6 minutes before being examined with SEM (FE SEM S-800, Hitachi, Tokyo, Japan) at an acceleration voltage of 20 kV.
Hematoxylin eosin (H&E) staining After SEM analysis, the remaining sectioned halves of the upper and lower right permanent first molars were decalcified by using 10% ethylene diamine tetra-acetic acid (pH 7.4; Wako Pure Chemical Industries, Osaka, Japan) for 8 weeks and then embedded in paraffin. Serial sections were cut at a thickness of 3 mm, mounted onto glass slides, deparaffinized, and then stained with H&E. The sections were examined by optical microscopy (Bx-40, Olympus, Tokyo, Japan) and stereoscopic microscopy (M165 FC, Leica Microsystems, Wetzlar, Germany).
Immunohistochemical staining The sections were deparaffinized in xylene, rehydrated, rinsed with distilled water, and then subjected to immunohistochemical staining, using antibodies raised against dentin sialoprotein (DSP), collagen XII, and osteocalcin (OC). Antigen retrieval for DSP and collagen XII staining required no additional treatment, but sections were treated with protease K (ready to use; Dako, Carpinteria, CA) for 10 minutes at room temperature to enable OC staining. The sections were immersed in 3% hydrogen peroxide for 10 minutes to inactivate endogenous peroxidase activity and then incubated with one or other of the following primary antibodies overnight: antihuman DSP (1:1500 dilution; rabbit polyclonal antibody, sc-33586; Santa Cruz Biotechnology, Santa Cruz, CA), antihuman collagen XII (1:2000 dilution; rabbit polyclonal antibody, sc68862; Santa Cruz Biotechnology), and antihuman OC (1:5000 dilution; rabbit polyclonal antibody, #AB10911, Millipore, Temecula, CA). The sections were subsequently incubated for 20 minutes with horseradish peroxidaseelabeled polymer conjugated with a secondary rabbit antibody in an EnVisionþsystem kit (Dako). The color was developed using
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ORIGINAL ARTICLE Lee et al. 547
Fig. 1. Radiographic and clinical appearance. A, Panoramic radiograph obtained at the age of 6 years and 8 months. The upper and lower permanent first molars have vestigial and thin roots and exhibit constricted pulp cavities in the crowns (arrows). B, Extracted upper right permanent first molar. C, Extracted lower right permanent first molar. The furcation floor has a convex appearance.
3,3’-diaminobenzidine substrate (Dako), counterstained with Gill’s hematoxylin solution (Merck, Darmstadt, Germany), and then examined using a stereoscopic microscope.
RESULTS Micro-CT Compared with normal teeth (Supplemental Figure 1), the crowns of the upper and lower right permanent MIM teeth had a normal contour and texture. Micro-CT revealed that the morphology and radiopacity of the enamel and dentin of the crown were normal. The pulpal floor comprised three layers: upper layer of the pulpal floor (UPF), middle layer of the pulpal floor (MPF), and lower layer of the pulpal floor (LPF). Although the radiopacities of the UPF and LPF were similar to those of dentin, the MPF exhibited a higher radiopacity and porosity than both the UPF and LPF, but a lower radiopacity compared with the enamel. In the upper right permanent molar, the mesiobuccal and distobuccal roots were partially obliterated (Figure 2, A), and the lingual root of the lower right permanent molar was barely developed. The pulpal floor structures of the three layers were readily observed. Furthermore, accessory canals were visible from the LPF to the MPF (see Figure 2, B). SEM images In the longitudinal section of the extracted teeth, the MPF appeared as a yellowish layer (Figure 3, A and D); columnar crystal-like structures were observed at higher magnification (see Figure 3, B, C, E, and F), and the number of dentinal tubules was markedly reduced compared with normal dentin (Supplemental Figure 2).
Fig. 2. Three-dimensional reconstruction and micro-CT appearance. A, Upper right permanent first molar. The arrow indicates the middle layer of the pulpal floor (MPF); its root canal is partially obliterated by calcified matrix. B, Lower right permanent first molar. The arrow indicates the MPF; its radiopacity is between those of enamel and dentin. L, lingual; B, buccal; P, palatal.
H&E staining H&E staining revealed that the UPF was composed of two parts: The UPF near the pulp had a normal dentin structure, whereas that toward the MPF appeared amorphous (Figure 4, C and G). Hematoxylin is used extensively as a nuclear counterstain and for the staining of specific intracellular and extracellular substances, and many strongly stained osteodentin-like hard tissues and hyperactive cells were observed in the MPF, whereas the LPF contained cementocyte-like cells within the lacunae (see Figure 4, D and H).
ORAL AND MAXILLOFACIAL PATHOLOGY 548 Lee et al.
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Fig. 3. Scanning electron microscopy images. A, Longitudinal section of the upper right permanent first molar. B, Highermagnification view of A, focusing on the middle layer of the pulpal floor (MPF) and the upper layer of the pulpal floor (UPF). C, Higher-magnification view of B, showing columnar crystal-like structures (closed arrow) and very few dentinal tubules (open arrow). D, Longitudinal section of the lower right permanent first molar. E, Higher-magnification view of D, focusing on the MPF and UPF. Many small nodular structures are evident on both the MPF and UPF. F, Higher-magnification view of E, showing columnar crystal-like tissues (closed arrow) and very few dentinal tubules (open arrow). Scale bars: 600 mm in B and E, and 15 mm in C and F.
Immunohistochemical staining DSP is used to identify the mean active mineralization of dentin and was strongly expressed in the MPF, moderately expressed in the upper part of the UPF and normal dentin, but not expressed at all in the LPF (Figure 5, AeD). Similarly, OC is used to identify osteoblast-like cells and the cellular cementum and its associated cells and was strongly expressed in the LPF, moderately expressed in the MPF, but barely expressed in the UPF and normal dentin (see Figure 5, EeH). Collagen XII is expressed mainly in dense connective tissues of tendons, ligaments, and blood vessel walls, and is the most abundant collagen in periodontal tissues, and it was expressed strongly in the periodontal ligament (PDL; see Figure 5, IeL) but weakly in some areas around the blood vessels in the MPF and LPF.
DISCUSSION Microscopic analysis of two of the four permanent first molars with MIM extracted from a 9-year-old girl revealed that the MIM teeth were characterized by normal crowns, malformed roots, and a thickened pulpal floor. Hypermineralized hard tissues and hyperactive cells, believed to be odontoblast-like cells derived from the apical pulp, were found at the center of the thickened pulpal floor.
The teeth of this patient were diagnosed as having MIM based on the definition of this malformation provided by Lee et al.24 Those authors studied 12 patients with molar root and incisor malformation and defined MIM as a newly discovered type of dental anomaly with root malformations of the permanent first molars and deciduous second molars and crown defects in the incisors, which were associated with neurologic systemic disease at the age of 1 to 2 years after birth.24 In the present study, all of the patient’s permanent first molars exhibited root malformation. It was suspected that the premature exfoliation and space loss of the upper deciduous second molars was caused by MIM of those teeth. The notch at the cervical third on the first incisors seemed to be absent. In addition, the patient had a medical history of premature birth (28 weeks) and low birth weight. Furthermore, at the age of 18 months, she had suffered a car accident that had resulted in brain damage, and she had been on antiepileptic medication since then. The diagnosis of MIM in her teeth was based on all of these factors. The conclusions drawn herein both agree and disagree with the findings of Witt et al.23 First, the findings of both studies indicated that MIM is caused not by genetic factors but by environmental factors and that it is related to past medical history, such as medication and central nervous systemerelated
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Fig. 4. Histologic findings (H&E staining). A, Histologic findings of the extracted upper right permanent molar, showing partial discontinuation of the root canal at the pulpal floor; amorphous hard tissues can be seen. B, Higher-magnification view of the labeled inset in A, focusing on the normal dentin and pulp, and the upper layer of the pulpal floor (UPF). C, Higher-magnification view of the labeled inset in A, focusing on the UPF and middle layer of the pulpal floor (MPF). D, Higher-magnification view of the labeled inset in A, focusing on the MPF and the lower layer of the pulpal floor (LPF). Instead of root dentin, amorphous hard tissueelike cellular cementum (open arrow) is evident in the LPF. Osteodentin-like hard tissue (closed arrow) and pulp-like hyperactive cells can be seen in the MPF. E, Histologic findings of the extracted lower right permanent molar, showing pulp-like cells and an amorphous tissue layer at the pulpal floor. F, Higher-magnification view of the labeled inset in E, focusing on the normal dentin, and the odontoblast layer and pulp tissue. G, Higher-magnification view of the labeled inset in E, focusing on the UPF. Dentinal tubules were seldom observed. H, Higher-magnification view of the labeled inset in E, focusing on the UPF, MPF, and LPF. Amorphous hard tissueelike cellular cementum (open arrow) can be observed in the LPF. Osteodentin-like hard tissue (closed arrow) and pulp-like hyperactive cells can be seen in the MPF. Scale bars: 0.752 mm in A, 100 mm in BeD and FeH, and 1.03 mm in E. P, pulp; ND, normal dentin.
disease. There is also agreement regarding the symmetric location involving the permanent first molars. In addition, Lee et al.24 analyzed multiple cases and concluded that the lower permanent first molars are always involved and that in 50% of cases, there are associated symptoms in the deciduous second molars and upper central incisors. Both Witt et al.23 and the present study found hypermineralized tissues in a thickened pulpal floor and observed the tissues using SEM and transmission electron microscopy. SEM in the present study revealed unique crystals that are thought to be formed by precipitation in a supersaturated solution, rather than by cell-controlled deposition. Using transmission electron microscopy, Witt et al. concluded that the mineralized globules in CMD consisted of hydroxyapatite. Witt et al.23 named the hypermineralized tissues cervical mineralized diaphragm and noted that it has a density between that of enamel and dentin and that it has the same constituents, including hydroxyapatite.
Those authors asserted that the CMD has a soft-tissue canal that originates from the dental follicle and forms collagen bundle like the PDL. However, in contrast to the study by Witt et al.,23 the present study revealed that the thickened pulpal floor comprises three layersdUPF, MPF, and LPFdand that the MPF comprises the CMD. Moreover, the MPF originates mainly from the apical pulp and partially from the dental follicle, and the MPF and LPF seem to communicate via blood vessels, as evidenced in the present immunohistochemical study. The internal cells of the MPF expressed DSP and OC, and the blood vessels of the MPF and LPF were stained by collagen XII; together, these results suggest that dentinal DSP facilitates initiation of hydroxyapatite formation along or inside the collagen fibril, leading to conversion of predentin to dentin at the mineralization front. The expression of DSP strongly indicates the mean active mineralization of dentin.25 Osteoblasts and osteocytes were immunostained for OC but not for DSP;
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Fig. 5. Immunohistochemical staining. A and B, Dentin sialoprotein (DSP) immunostaining in the upper and lower right permanent molars. DSP is strongly expressed in the middle layer of the pulpal floor (MPF) but very weakly expressed in the upper layer of the pulpal floor (UPF) and lower layer of the pulpal floor (LPF). C and D, Higher-magnification views of the labeled insets in B. DSP expression is weaker in the UPF than in normal dentin and is very weak in the LPF. E and F, Osteocalcin (OC) immunostaining in the upper and lower right permanent molars. OC is strongly expressed in the MPF, LPF, and cellular cementum but very weakly expressed in the UPF and normal dentin. G and H, Higher-magnification views of the labeled insets in F. OC is expressed in the MPF, but barely in the UPF. OC is strongly expressed in the LPF and MPF. I and J, Collagen XII immunostaining in the upper and lower right permanent molars. Collagen XII is strongly expressed in the periodontal ligament but is very weakly expressed in dentin, cementum, and pulp tissue. K and L, Higher-magnification views of the labeled insets in J. Collagen XII is not expressed in the UPF and is weakly expressed in the blood vessels of the MPF and LPF. Scale bars: 0.85 mm in A and E, 0.711 mm in B and F, 0.897 mm in I, 0.742 mm in J, and 100 mm in C, D, G, H, K, and L.
accordingly, the synthesis of OC can be regarded as the expression of a cell with an osteoblast-like phenotype. Cellular cementum and its associated cells express OC but not DSP.26 Collagen XII is expressed mainly in the dense connective tissues of tendons, ligaments, dermis, cornea, blood vessel walls, meninges, and developing membranous bones.27 The expression of collagen XII has been compared with that of type I collagen, which is the most abundant collagen in periodontal tissues.28 The presence of two origins was suspected on the basis of the proximity of the apical pulp and dental follicle. It is known that DSP and OC are expressed in
locations of active mineralization in teeth,29 whereas collagen XII is distributed around the PDL.30,31 In our study, DSP was seen to be strongly expressed in odontoblastic layer and OC in the cellular cementum (Supplemental Figure 3). Thus, the internal cell tissues of the MPF appear to comprise mostly odontoblast-like cells that originate from the apical pulp, and the adjacent hard tissues are thought to be dysplastic or amorphous dentin, the so-called osteodentin. The UPF comprises dysplastic and amorphous dentin, and the LPF is considered to be cellular cementum, as reported by Witt et al.23
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It is suspected that the etiology of the MPF is abnormal differentiation in the apical pulp and is caused by external factors, such as neurologic systemic disease or medication, at an age of 1 to 2 years. The apical pulp is composed of loose cellular tissues located under a developing tooth, including mesenchymal stem cells from the apical papillae (SCAP).28 SCAP is known to be associated with root development because they differentiate into odontoblasts.29 The apical pulp is segregated from the dental papilla by an apical cellerich zone and has a location and shape similar to those of the MPF. In addition, the SCAP are activated by external stimulation and rapidly differentiate into replacement or tertiary dentin, forming odontoblast-like cells; this is the hypothetical cause of excessive MPF mineralization.32,33 Further study is needed into whether systemic disease or medication can affect the root development in permanent first molars or deciduous second molars and whether the internal cell tissues in the MPF are identical to the odontoblast-like cells derived from the SCAP. Root malformation might arise from the intervention of epigenetic factorsdsuch as systemic disease or medicationdin the signaling pathways involved in normal root development. Recent animal testing revealed that a specific gene variation in root odontoblasts is able to affect root formation.34 The genes Nfic,35 Msx2,36 Shh,37 Sp6,38 Noggin,39 b-catenin,40 smadd4,41 Ntl,42 and Rankl43,44 are related to the shape, length, and number of roots. Among these, the most relevant to the present study is the Wnt/b-catenin signaling associated with root and cementum formation.45 Depending on the expression rate of the signaling, MIM-like symptoms, such as rootless molars, thin dentin, hyperplastic cementum, and incisor disruption, may occur.42,43 Further studies on signaling pathways and genes are warranted. Clinically, MIM teeth have various characteristics and are only extracted when absolutely necessary. Teeth are exfoliated in cases of apical abscess not related with dental caries in the permanent first molars and of spontaneous mobility during orthodontic treatment, as in the patient in the present study. Witt et al.23 had extracted a tooth with severe caries in dentin. DD I is associated with apical abscess without dental caries. Although the etiology has not yet been identified unequivocally, such abscesses are thought to be caused by pulp necrosis induced by pulp chamber obliteration or by pulp exposure induced by dentinal defects.46 Conventional treatments include exfoliation, follow-up, and apicoectomy, although a recent case report demonstrated successful endodontic treatment with a conventional endodontic procedure.47 Successful orthodontic treatment for DD I has also been reported.48 The malformed roots of MIM teeth render it difficult to
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apply endodontic treatments, but conservative pulp treatment can be applied with detailed and accurate information regarding the shape of the root and crown shape obtained using radiographic tools, such as computed tomography.
CONCLUSION MIM-affected molars are characterized by a normal crown, a malformed root, and a thickened pulpal floor in the permanent first molars. The thickened pulpal floor can be divided into three layers. The most distinctive layer, the MPF, comprises hard tissueelike amorphous dentin and odontoblast-like cells that originate mainly from the apical pulp and partially from the dental follicle, whereas the UPF comprises dysplastic and amorphous dentin derived from the dental papilla, and the LPF is considered to be cellular cementum from the dental follicle. Although the differentiation anomaly of the apical pulp and dental follicle as a result of external stimulation is thought to be the main cause of MIM, this needs to be confirmed in further studies. REFERENCES 1. Bei M. Molecular genetics of tooth development. Curr Opin Genet Dev. 2009;19:504-510. 2. Huang XF, Chai Y. Molecular regulatory mechanism of tooth root development. Int J Oral Sci. 2012;4:177-181. 3. O Carroll MK, Duncan WK, Perkins TM. Dentin dysplasia: review of the literature and a proposed subclassification based on radiographic findings. Oral Surg Oral Med Oral Pathol. 1991;72: 119-125. 4. Sauk JJ Jr, Lyon HW, Trowbridge HO, Witkop CJ Jr. An electron optic analysis and explanation for the etiology of dentinal dysplasia. Oral Surg Oral Med Oral Pathol. 1972;33: 763-771. 5. Tidwell E, Cummingham CJ. Dentinal dysplasia: endodontic treatment, with case report. J Endod. 1979;5:372-376. 6. Kim JW, Simmer JP. Hereditary dentin defects. J Dent Res. 2007;86:392-399. 7. Fanibunda KB, Soames JV. Odontodysplasia, gingival manifestations, and accompanying abnormalities. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1996;81:84-88. 8. Crawford PJ, Aldred MJ. Regional odontodysplasia: a bibliography. J Oral Pathol Med. 1989;18:251-263. 9. Azevedo RS, da Silveira LJ, Moliterno LF, Miranda AM, de Almeida OP, Pires FR. Segmental odontomaxillary dysplasia: report of a case emphasizing histopathological, immunohistochemical and scanning electron microscopic features. J Oral Sci. 2013;55:259-262. 10. Goodman JR, Gelbier MJ, Bennett JH, Winter GB. Dental problems associated with hypophosphataemic vitamin D resistant rickets. Int J Paediatr Dent. 1998;8:19-28. 11. Chaussain-Miller C, Sinding C, Wolikow M, Lasfargues JJ, Godeau G, Garabedian M. Dental abnormalities in patients with familial hypophosphatemic vitamin D-resistant rickets: prevention by early treatment with 1-hydroxyvitamin D. J Pediatr. 2003;142:324-331. 12. Kamarthi N, Venkatraman S, Patil PB. Dental findings in the diagnosis of idiopathic hypoparathyroidism. Ann Saudi Med. 2013;33:411-413.
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SUPPLEMENTARY DATA Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.oooo.2014. 10.013